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2013

H2 Biology 9648
REVISION NOTES FOR H2 A LEVEL BIOLOGY
LOH ZHENG YI 12S74

Version 2.5

CONTENTS
A. Core Themes
1. Cellular Functions

2

2. DNA & Genomics

37

3. Genetics of Viruses and Bacteria

59

4. Organization & Control of the Prokaryotic & Eukaryotic Genome

82

5. Genetic Basis for Variation

102

6. Cellular Physiology & Biochemistry

121

7. Diversity (Basic) & Evolution

185

B. Application Themes
1. Isolating, Cloning & Sequencing DNA

208

2. Applications of molecular and cell biology

225

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1. CELLULAR FUNCTIONS
1A – DESCRIBE AND INTERPRET DRAWINGS AND PHOTOGRAPHS OF TYPICAL ANIMAL
AND PLANT CELLS AS SEEN UNDER THE ELECTRON MICROSCOPE, RECOGNISING THE
FOLLOWING MEMBRANE SYSTEMS AND ORGANELLES: ROUGH AND SMOOTH ER,
GOLGI BODY, MITOCHONDRIA, RIBOSOMES, LYSOSOMES, CHLOROPLASTS, CELL
SURFACE MEMBRANE, NUCLEAR ENVELOPE, CENTRIOLES, NUCLEUS AND NUCLEOLUS
 Cytology (study of cells)
 Characteristics of cells
 Cells must come from pre-existing cells by division
 Fundamental unit of structure and function in living organisms
 All living things are made up of cells
 Generally in micrometres
 Microscopy
 Light Microscope
 Passing of visible light through specimens
 View sections of tissues and entire cells
 Magnifications of 4X, 10X, 40X, 60X
 Eyepiece magnification = 10X
 Electron Microscope
 Uses high electron beams to produce images of high resolution
 Higher magnification power than LM
 Characteristics
 Magnification = The ratio of an object’s image size to its real size
 Resolution = The shortest distance that is found between two distinguished points
 Contrast = The amount of colour or grayscale differentiation that exists (cells usually stained to
give higher contrast)
 Cell Fractionation (Used to separate out a homogenous population of subcellular organelles from the
cytoplasm as a whole)
 Homogenisation
 To break tissues into small fragments, disrupt cells and release individual organelles by lysing the
cell surface membrane
 Maintenance of the integrity of cellular organelles
 Isotonic Medium to ensure that cells do not crenate/plasmolyze or undergo
haemolysis/become more turgid due to osmosis caused by a net movement of water
molecules from a less negative water potential to a more negative water potential.
 A buffer solution to maintain pH
 A low temperature of 4°C to inhibit protease activity and preserve the unique 3D
conformation of proteins in organelles
 Methods of mechanical rupturing of cell membrane
 Ultrasound
 Osmotic Lysis (rupture protoplasts)
 Pressure
 Homogeniser (abrasive equipment)
 Differential Centrifugation

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Used to separate organelles and cellular components as a function of their sedimentation
coefficient, dependent on size, shape and density (as well as viscosity of the medium) (note 
Mass not involved)
 Allows for the study of isolated organelles to study cell structure and function.
 Bottom of tube  Pellet, Remaining fluid  Supernatant
 The homogenate is subjected to progressively increasing speeds, which causes particles to
separate in descending order of size and desity.
 A series of pellets containing cell organelles of decreasing sizes can therefore be obtained
 Nuclei, Mitochondria/Chloroplasts/Peroxisomes & Lysosomes, Plasma
Membrane/Microsomes/Polyribosomes, Ribosome Subunits, Cytosol
 Autoradiography
 Used to identify the synthesis and cellular distribution sites of metabolic products by tagging
specimen molecules with radioisotopes such as 15N and 32P. This traces secreted products and their
intermediates from synthesis sites, which are analyzed on photographic films.
 Steps
 Tissue specimens exposed to radioactive label
 Tissues washed off to remove excess label and later fixed and sectioned
 Sections are placed on microscopic slides
 Slides dipped in melted photographic emulsion in the darkroom
 Slides are dried and stored in the dark. Crystals in the photographic emissions are exposed over
radioactive sites in the section
 Emulsion is developed by standard radioactive techniques and slides examined under a light
microscope. Grains exposed in the emulsion mark sites of inactivity

1B – OUTLINE THE FUNCTIONS OF THE MEMBRANE SYSTEM AND ORGANELLES LISTED
IN (1A)
 Prokaryotes
 Unicellular organisms
 No membrane-bound organelles
 Strands of DNA in the centre; usually has flagellum
 Small ribosomes, 3 RNA molecules; about 55 proteins
 Eukaryotes
 Multicelular organisms or unicellular
 Membrane-bound organelles
 Large ribosomes, 4 RNA molecules; about 78 proteins
 Cell size
 Limited by the minimum amount of space needed to contain the essential elements of its
functions, as well as the surface to volume ratio
 As the cell size increases, its surface to volume ratio decreases, causing the number of chemical
exchanges that can be performed with the extracellular environment to be inadequate to
maintain the cell.
 Cell size is thus kept small to increase rate of chemical exchange
 Organelles
 Membranous organelles
 Rough Endoplasmic Reticulum
 Golgi Apparatus
 Smooth Endoplasmic Reticulum
 Lysosomes
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5   Vesicles  Vacuoles  Mitochondria  Chloroplasts  Non-membranous organelles  Centrioles  Ribosomes  Flagella  Cilia Other parts  Cell Wall  Cytosol (cytoplasmic fluid)  Cytoplasm  Nucleus  Nucleolus  Cell Plasma Membrane 4|P a g e .Version 2.

5  Cytoplasm  Refers to all the organelles + cytosol (without nucleus)  Cytosol  Aqueous solute rich matrix that contains  Essential Ions & soluble organic molecules such as sugars and amino acids  Soluble proteins  Cytoskeleton  Bounded by plasma membrane  Various metabolic reactions needed to sustain life takes place here  Membranous organelles  Nucleus  Function  Contains the genetic material which carry the coded instructions controlling the activities and characteristics of the cell  Regulates all cellular activities  (DNA replication and mRNA transcription occurs here)  Description  Largest membranous organelle. and proteins to enter and exit the nucleus  Nucleoplasm  Aqueous matrix which contains proteins. Appears as dark-coloured patches  Nucleolus  Description: Dense mass in centre of nucleus  Function: Composes of DNA carrying rRNA genes to synthesize ribosomal RNA.Version 2. Outer membrane continuous with the membrane of the endoplasmic reticulum  Perforated by nuclear pores. Appears as light-coloured patches  Tightly coiled chromatin known as heterochromatin (not being transcribed). which is involved in the assembly of ribosomes required for translation  Endomembrane system  Description: Inter-related membrane sacs. RNA and chromatin  Loosely coiled chromatin known as euchromatin (actively being transcribed). sacs or sheets called cisternae. spherical or oval in shape with a size of between 5-20µm. made up of a large protein complex which allows macromolecules such as mRNA and rRNA to exit the nucleus. region between them known as perinuclear space.  Contains  Nuclear envelope  Double membrane which separates the contents of the nucleus from the cytoplasm  Each of the two membranes is a lipid bilayer  Inner and outer membranes are continuous with each other. which is continuous with the endoplasmic reticulum lumen. related by either direct physical continuity or by the transfer of vesicles (membrane segments)  Routes  Exocytosis  Endocytosis (Use lysosome to digest)  Endoplasmic Reticulum  Description: Extensive network of membranous tubules. giving it its smooth appearance  Functions 5|P a g e . Internal space known as lumen  Smooth endoplasmic reticulum (SER)  Description: A network of tubules which lack ribosomes.

leading to a drop in pH level in the lumen. which fuses with a lysosome to form an autographic vacuole. where it folds into its functional conformation  Proteins synthesizes are destined for export or are targeted to various cell organelles  Proteins leave the rER in transport vesicles Golgi Apparatus (GA)  Description: A stack of flattened. breaking down the structure. Rough appearances as Ribosomes stud the cystolic face of the rough ER (AKA Cisternae) ER lumen continuous with perinuclear space  Function (As a site of protein synthesis and folding)  Ribosomes bound to rER produces polypeptides  Polypeptides are threaded into the ER lumen through a protein channel. membrane-bound sacs called cisternae.Version 2. which activates hydrolytic enzymes.  Autolysis  Cells (injured and dying tissues) can self-destruct (apoptosis) through the mass release of lysosomal contents into the whole cell.  Description  Appear homogenously electron dense under EM  Acidic contents to provide optimum pH for hydrolytic enzymes 6|P a g e . breaking down solid particles to soluble products via hydrolysis  Can also be used as defence mechanism.5    Synthesis of lipids  Metabolism of carbohydrates  Storage of calcium ions for use in muscle contraction as well as cell signalling  Drug detoxification  Rough endoplasmic reticulum (RER)  Description: Extensive network of flattered sacs. Each GA has a cisface (where new cisternae is being formed from vesicles) and a trans-face  Main Function (Modification and packaging of proteins)  Cis-face receives transport vesicles from the ER  Vesicle fuses with the cis-face membrane and deposits contents into Golgi Cisternae space  Golgi vesicles transport materials between parts of GA  Secretory vesicles bud off from trans-face to transport substances out of the cell  Lysosomes can also bud off to digest incoming substances  Golgi vesicles can also bud off to transport substances to other organelles within the cell  (Thus cells that are active in any form of secretion have large amount of GA)  Other functions  Progressive modification and packaging of ER products  Glycosylation  Trimming Lysosomes (lysosyme)  Function  Digestion of materials taken in by endocytosis  Food vacuoles fuse with lysosomes to form endosomes  Fusion causes an intake of H+ ions. Sheet-like appearance with a single membrane. result of fusion known as phagocytic vacuole  Autography of worn-out organelles  Unwanted structures are enclosed in a vesicle.

rod shaped. double membranous organelle  Outer membrane is smooth.1µm to 1.Version 2. allows for the localisation of moe ATP synthase and electron transport chans  Impermeable inner membrane that allows for the formation of an electrochemical gradient  Narrow.5   Contains hydrolytic enzymes to digest most biological macromolecules Size ranges from 0. inner membrane is highly convoluted with cristae to increase surface area for various enzyme systems and proteins embedded in the inner membrane. mobile vacuoles in animal cells  Description: Structurally similar to vesicles.2µm Secretory Vesicle Lysosome    Small. RNA and ribosomes. only larger Large (central) permanent vacuole in plant cells  Solution within the tonoplast known as cell sap  Functions  Storage of organic compounds  Disposal site for toxic metabolic by-products  Containment of pigments  Plant protection by accumulating toxic compounds  Cell growth & elongation (water accumulation causes cytoplasmic contents to be pushed to the periphery) Mitochondria  Description  Spherical. fluid-filled space between two membranes known as inter-membrane space  Compartment enclosed by inner membrane is the mitochondrial matrix which houses circular DNA.  Function  Site of cellular respiration where ATP is synthesized (aka respiration)  Number of mitochondria per cell corresponds to level of metabolic activity (cells involved in active transport have more mitochondria) 7|P a g e .

5  Chloroplasts  Description  Lens-shaped  5µm . in the mitochondrial matrix or chloroplast stroma  About 30nm in diameter  Small subunit has an mRNA binding site and the large subunit has an aminoacyl-tRNA sie.  Stroma site of Calvin cycle  Thylakoids site of light-dependent photophosphorylation  Non-membranous organelles  Ribosomes  Description: Spherical  Consists of two subunits.10µm in length  Surrounded by double-membrane (chloroplast envelope)  Inner membrane houses a semi-fluid compartment (Stroma)  internal-membrane system (Thylakoids)  Enclose an area known as thylakoid lumen  Photosynthetic pigments and some proteins required for photosynthesis are embedded in thylakoid membrane  Thylakoid disc stacked on each other form a granum. the large ribosomal subunit and the small ribosomal subunit. each member of a pair consisting of nine triplets of microtubule arranged in a ring  Function  Acts as microtubule organizing centre for the spindle apparatus to assemble spindle fibres  Centrosomes (present in plant cells)  Contains centrioles  Cell Wall  Description  Rigid and inflexible structure that is mainly made up of cellulose  Supports and defines the shape of plant tissues  Freely permeable to all but very large molecules  Functions  Protecting the cell from mechanical injury and invasion  Withstanding the hydrostatic pressure exerted by the uptake of water by the cell 8|P a g e . which are connected by intergranal lamellae.ribosomes. a peptidyl-tRNA site and an exit site  Function  Synthesize proteins for export or targeted to various membrane-bound organelles  Free cytosolic ribosomes synthesize proteins that function within the cytosol  Centrioles (absent in plant cells)  Description:  Pair of hollow cylinders located near nucleus  Found in pairs at right angles to each other.  Contains circular DNA . free in the cytosol.Version 2. starch grains  Function  Sites of photosynthesis where solar energy is converted to chemical energy by absorbing sunlight and using it to drive the synthesis of organic compounds from CO 2 and H2O. which are made of proteins and ribosomal RNA  Found attached to RER.

Fructose .  Breakage  A water molecule is added to a glycosidic bond in hydrolysis. thus producing one carbohydrate and another molecule. aldo-sugars form 6-ring structures and keto-sugars form 5 ring structures  Types  Monosaccharides (take note n is 3-7)  Exists as single sugar units in linear or ring structures (which exist in dynamic equilibrium) (diagram below)  Ring form  Formation of anomeric carbon (Carbon joined to two O atoms)  Anomeric hydroxyl group has two configurations (α. breaking the glycosidic bond between the anomeric hydroxyl group of one sugar unit and another hydroxyl group of the other sugar.β)  Uses  Energy stores to produce ATP during cellular respiration  Building blocks for synthesis of disaccharides and polysaccharides  Raw material for synthesis of other organic molecules.5 1C – DESCRIBE FORMATION & BREAKAGE OF A GLYCOSIDIC BOND  Polymers  Monomers linked together by covalent bonds  Synthesis  Formation of glycosidic bonds in a condensation reaction  Breakdown  Breaking of glycosidic bonds in hydrolysis  Requires enzymes for both formation/breakdown  Carbohydrates  Structure (expected to draw and label)  Formula = Cn(H2O)n (Carbon-Hydrated)  Presence of C=O in aldehyde/ketone group  Can be oxidized to form carboxylic acid groups (only for aldo-sugars)  Where n = 6. Galactose  Glycosdic Bond  Formation  The hydroxyl group on the anomeric carbon of one glucose reacts with another hyroxyl group on carbon X to form a single molecule with the loss of a single water molecule in a condensation reaction.  Nomenclature  α/β(x. (R – C – O – C – R’) Usually involves enzymes.2) Non Reducing alpha on glucose. creating a glycosidic bond. such as nucleotide  Examples: Glucose. Usually involves enzymes.4) beta on galactose  Sucrose  Glucose + Fructose α(1.Version 2. beta on fructose 9|P a g e .y) (depending on anomeric hydroxyl group config) and number name of carbon involved  Disaccharides  Lactose  Glucose + Galactose β(1. however acids can also hydrolyze glycosidic bonds.

resulting in a hydration sphere. Fats. phospholipids  Simple lipids  One alcohol + one or more fatty acid linked via ester linkage  E.  Can dissolve a great variety of solutes  The ions of ionic compounds form electrostatic interactions with water molecules.2 LIPIDS  Properties of lipids  Non-polymers (no polymerization of lipids)  Insoluble in water  Non-polar and hydrophobic  High proportion of C and H as compared to O  Highly soluble in non-polar solvents (aka alkanes)  Similar Structure: Long hydrocarbon tails. the glucose cannot uncoil back into the linear form (aldehyde configuration) and thus cannot participate in the redox reaction)  All monosaccharides are reducing. sterols. (each oxygen atom can hydrogen bond to 2 hydrogen atoms) 1 water molecule can form hydrogen bonds with 4 other molecules.g.  E.  Polar Molecule  Forms hydrogen bonds between each other. Triglyceride (AKA fats)  Formation: Glycerol + 3 fatty acids linked via ester linkage group which is formed during a 10 | P a g e .5  Maltose  Glucose + Glucose α(1. waxes. (ion-dipole attraction)  Molecule structure of water  2 electropositive Hydrogen atoms covalently bonded to one electronegative oxygen atom at an angle of 104.g. Some disaccharides are reducing  Tested using benedicts’ test for reducing sugars.1 WATER AS A UNIVERSAL SOLVENT  Properties of water  Universal Solvent  Makes up 70% of the weight of cells  Creates an aqueous extracellular environment to allow for the survival or organisms and cells. (copper (II) ions reduced) 1D – ANALYZE THE MOLECULAR STRUCTURE OF A TRIGLYCERIDE AND A PHOSPHOLIPID AND RELATE THESE STRUCTURES TO THEIR FUNCTIONS IN LIVING ORGANISMS 1D.Version 2.4) alpha on glucose  Reducing Sugars  Reducing sugars can reduce other compounds and are oxidized in the process.5°  Hydrophilic molecules are solutes with an affinity for water (polar molecules/ionic compounds/charged molecules)  Hydrophobic molecules are solutes that cannot dissolve in water due to the inability to form hydrogen bonds with water (or lack of electrostatic interaction) (non-polar) 1D.  Due to presence of a free anomeric carbon which is oxidized to form a carbonyl group (without the h)  H on aldehyde group removed and replaced with O  If there is no free anomeric carbon.

thus allowing animals to keep their mass to a minimum Does not affect water potential of cells (thus allowing cells to store as much fat as possible as it does not affect osmosis)  Fat is a good thermal insulator. Oil is liquid fat. Fatty acids  Properties  Composed of a carboxylic acid group attached to a hydrocarbon chain  Possess long carbon skeletons (12-20C)  Abundance of non-polar C-H bonds give fats its hydrophobic property  Differ in terms of  Length of hydrocarbon chain  Location and number of C=C double bonds (Saturated = no double bonds)  Double bonds causes fatty acids to have kinks in their tails at the location of a double bond  Naming of fatty acids  X:Y(∆?.?2 ). Functions of triglycerides in animals Structure High proportion of C & H atoms to O atoms Large number of C-H bonds Hydrophobic nature due to nonpolar C-H bonds Functions Efficient energy store Upon oxidation and cellular respiration.  Melting point decreases as degree of unsaturation of fatty acid tails increase  Kinks prevent the molecules from packing closely enough. bonds within triglycerides are broken to release large amounts of energy to produce ATP (38kj/g) (as opposed to carbohydrates 17kj/g) Releases metabolic water during cellular respiration (impt to animals in arid conditions) No associated water molecules No extra weight due to water of hydration.Version 2. resulting in a higher melting point as more thermal energy is required to break these interactions. thus a lot of fat helps to insulate the body. thus causing the hydrophobic interactions to be weaker. the greater the hydrophobic interactions between the chains. thus less thermal energy is required to break enough of these interactions to liquefy the triglycerides. Adipose tissue helps to cushion and protect vital organs from physical impacts Aids buoyancy of aquatic animals Weak hydrophobic interactions allow fat to slide under pressure Less dense than water due to lower molecular weight than water per unit volume  Compound lipids  Esters of fatty acids + Alcohol + Other chemical groups 11 | P a g e .?1. and thus melting point decreases. Henceforth most animal fats (saturated) are solid at room temperature and most plant fats (unsaturated) are liquid at room temperatures. where X is the total number of C.5      condensation reaction between the hydroxyl group of glycerol and carboxyl group of fatty acids. Y is the number of C=C bonds and n is the location of the carbon with the C=C bond Formed in a condensation reaction where a carboxyl group reacts with a hydroxyl group (losing a water molecule) to form an ester linkage (R-COO-R) Properties  Melting point increases as hydrocarbon chain length increases  As the hydrocarbon chain length increases.

g.  Liposome/Vesicle  Formed when a phospholipid bilayer folds back on itself. Found at cell membrane facing exterior env. 12 | P a g e .5  E.Version 2.g. a neurotransmitter E. thus achieving maximal stability  Creates a separate aqueous environment  Functions of phospholipids in animals/cells Structure Amphipathic molecules with a hydrophilic phosphate head and hydrophobic fatty acid tails Hydrophobic interactions between fatty acid tails Phospholipids containing choline  Functions Allows for compartmentalization of the cell by forming bilayer membranes Forms a selectively permeable cell membrane with a phospholipid bilayer.  Do not expose hydrophobic edge regions. thus acting as a barrier between the cell and its external environment to maintain cellular homeostasis Forms liposome/vesicle for the storage and transport of cellular products as well as digestion of waste and drug delivery Forms micelles used in the transport of fats Maintains the integrity of the lipid aggregates due to a large number of interactions Weak hydrophobic interacts permit lateral movement of phospholipids for membrane fluidity Stores Choline for the synthesis of acetycholine. Phospholipid  One glycerol + 2 fatty acids (linked via ester linkage) + negatively charged phosphate group (connected to 3rd OH of glycerol by phosphoester linkage) (may contain a charged nitrogencontaining base at the top)  Properties  Amphipathic  Hydrocarbon tails are non-polar and hydrophobic  Phosphate group and attachments are polar and hydrophilic  Lipid Aggregates (to shield the hydrophobic tails from water/aq environment)  Micelle  A small spherical droplet consisting of a phospholipid monolayer with phosphate heads on the outside and hydrocarbon tails on the water-free interior  Bilayer (two dimensional sheet)  Two layers of phospholipid with hydrophilic heads exposed to the polar exterior and hydrophilic tails being in contact with neighbouring molecules but excluded from water in the non-polar interior of the bilayer. Glycolipids  Glycerol + 2 Fatty acid tails/hydrocarbon tails (linked via ester) + Carbohydrate chain (linked via glycosidic bond to 3rd OH of glycerol) (Amphipathic)  Functions of glycolipids in animals/cells Structure Carbohydrate chain attached to lipids Functions For cell specificity. Serves as marker and binding site for cell-cell recognition.

thus affecting physical and chemical properties of proteins. polarity or hydrophobicity of R-groups will affect the type and location of bonds present at tertiary and quaternary structures. Disulfide Bonds (resistant to heat). charge. Hydroxyproline. able to act as buffers (due to presence of amine and carboxyl which makes them amphoteric)  Ionized form of carboxyl group can take in hydrogen atoms to reduce [H +]  Ionized form of amine groups can lose hydrogen atoms to reduce [OH-]  Affected by R-group (which can be classified as) 13 | P a g e . not soluble in organic solvents)  Ability to form zwitter ions (electrically neutral but dipolar)  Amine and Carboxyl group can readily ionize to form NH3+ and COO Thus. non-polar  Affects types of bonds formed i. the unique 3-dimensional conformation and eventually the function of protein.  Properties  Generally hydrophilic (soluble in water. Carbon.g. Hydrogen Bonds. Hydroxylycine)  Structure  Consists of a central carbon attached to four parts  Amine group (-NH2)  Carboxylic Acid group (-COOH)  Hydrogen atom  Variant group AKA R-group  Have unique physical & chemical properties  Chemical Property: polar uncharged. etc  Cholesterol  Serves to modulate membrane fluidity  Precursor for synthesis of bile acids. steroid hormones and vitamin D.5 Hydrocarbon tails - Used in cell-cell adhesion Hydrophobic interactions between fatty acid tails serve to anchor the glycolipid at the cell surface membrane (as part of bilayer  Lipid derivatives  E. Hydrogen and Sulfur*some cases  Forms peptide bonds with other amino acids to form polymers called polypeptides (Hence they are monomers of proteins)  20 amino acids in total (= 20 different R groups)  Classified into essential amino acids (cannot be synthesized by the body) and non-essential (can be synthesized by the body)  Rare amino acids include derivatives of fundamental amino acids (e.g. polar charged. Oxygen. fatty alcohols. 1E – DESCRIBE STRUCTURE OF AMINO ACID AND FORMATION AND BREAKAGE OF A PEPTIDE BOND  Amino Acid  Consists of Nitrogen. ketone bodies. Hydrophobic Interactions  Physical Property: Bulky R-groups disrupts formation of secondary structure by causing steric hindrance  Affects location of amino acid residues  Affects location of bonds formed  Size. Ionic Bonds.e.Version 2. Steroid hormones.

5  Non-polar (hydrophobic)  Usually hydrocarbon in nature  Causes amino acid to be hydrophobic and unreactive  Amino acids having these R-groups tend to be located in the interior of a protein shielded from the aqueous medium. The amine group of one amino acid reacts with the carboxyl group of another amino acid.  Polar. stable.  Breakage (hydrolysis)  Polypeptide  Many amino acids joined together by peptide bonds  Contains a free Amine group at the beginning of the polypeptide called an N-Terminus  Contains a free carboxyl group at the end of the polypeptide called a C-Terminus  Properties  Buffering capacity reduced (enhanced by polar charged R-groups)  Affected by  Length  Amino Acid Sequence  Test for peptide bonds (biuret test)  Purple colouration indicates positive result (nitrogen atoms form complexes with Cu2+ ions to give purple colouration. 3D-shape and is biologically functional  Classified based on shape  Fibrous  Shape: Elongated straight polypeptide chains wound around each other to form a rope-like structure  1o .Repetitive regular sequence of amino acids 14 | P a g e . charged (hydrophilic.groups  Net positive/negative charge  Peptide Bond  Formation  Two amino acids react in a condensation reaction involving the loss of a water molecule to form a peptide bond. uncharged (hydrophilic)  Presence of N-H. buffering capacity)  Presence of NH3+ or COO. causing the formation of a peptide bond (C=ONH). O-H (S-H)  No net charge  S-H can form disulfide bonds with another S-H (AKA Sulfhydryl Group)  Polar.Version 2. 1F – EXPLAIN AND DEFINE THE FOUR STRUCTURES OF PROTEINS AND THE TYPE OF BONDING HOLDING THEM IN PLACE  Proteins  Consists of one or more polypeptide chains folded into a unique 3D conformation which affects the functioning of proteins (not all proteins have all four structures)  Molecules that contain one or more polypeptide chains that has attained a unique.

perpendicular to the man axis  15 | P a g e .  Most common (doesn’t mean only) types of secondary structure at α-helix and βpleated sheet  α-helix  Structure  Takes the form of an extended spiral spring  One complete turn every 3.g.Fixed specific non-repetitive sequence of amino acids  3o . e. ionic and hydrogen bonds  Physical Property: Easily soluble in aqueous medium  Function: Performs metabolic functions. Physically tough. Haemoglobin  Four Levels of organization  Levels are interdependent on each other  Primary Structure  Definition: The unique number and linear sequence of amino acids that constitute the polypeptide chain  Proteins are synthesized in vivo by the stepwise polymerization of amino acids in the order specified by the sequence of nucleotides in a gene  Determines the secondary.Stable quaternary structure (if applicable) due to presence of strong. extensive network of hydrogen bonds and covalent cross-links between the polypeptide chains  Physical Property: Insoluble in water  Function: Performs structural functions in cells.Little or no tertiary structure  4o .6 amino acids  R groups project outwards.Secondary structure most important.g. collagen  Globular  Shape: Highly folded/bent/twisted polypeptides form a compact and spherical shape that has a specific 3-dimensional conformation  1o .Version 2.5  2o . e. tertiary and quaternary structures  The sequence influences the characteristics of a protein (R-groups determine the type and location of bonds present at higher levels of the protein) (refer to R-groups)  Secondary Structure  Definition: The regular coiling and folding of regions of the polypeptide chain which gives rise to repeated patterns  Maintained by regularly spaced hydrogen bonds formed at the polypeptide backbone between the amine group of one amino acid and the carbonyl group of another.Tertiary structure most important (spherical shape)  4o – Quaternary structure unstable due to presence of weak inter-chain hydrophobic interactions. long parallel polypeptide chains cross-linked at intervals forming long fibres or sheets  3o .

Hydrophobic Interactions.Version 2. Ionic Bonds and Disulfide Bonds (covalent)  Disulfide  Oxidation of sulfhydryl groups of any two Cystein (CYS) residues  Hydrogen bonds  Formed between hydroxyl groups or amine groups with another hydroxyl/amine group  Large number of such bonds increase the influence of hydrogen bonds on the overall shape and stability of the protein  Ionic Bonds  Formed between positively charged amine groups (NH3+) and negatively charged carboxyl groups (COO-)  Can be broken by changes in pH. sheet-like conformation  Anti-parallel (neighbouring hydrogen-bonded polypeptide chain runs in opposite direction)  Parallel (neighbouring hydrogen-bonded polypeptide chain runes in the same direction  Bulky R-groups interfere with the formation of β-pleated sheet by causing steric hindrance  R-Groups project above and below the plane in an alternate manner  Bonding  Stabilized by inter/intra chain hydrogen bonds between carbonyl and amine groups of the polypeptide backbone/different polypeptide backbone Tertiary Structure  Definition: The further bending.  Hydrophobic interactions  Interactions between hydrophobic side chains (AKA R-Group) 16 | P a g e .5  Therefore bulky R-groups do not cause steric hindrance  Bonding  Stabilized by intra-chain hydrogen bonds (parallel to the main axis) formed between the carbonyl and amine groups of the polypeptide backbone  The groups are four amino acid residues apart  All carbonyl and amine groups participate in the bonding  β-pleated sheet  Structure  Extended zigzag.  Structure: Depends on bonding  Bonding  Involves interaction between R-groups of amino acid residues which allows for the interaction of different regions of the polypeptide  Hydrogen Bonds. twisting and folding of the polypeptide chain with the secondary structures to give a precise overall unique three-dimensional conformation of a protein.

strong reducing agents (break disulfide bonds). pH changes (breaks ionic bonds). disulfide bonds. β-pleated sheet. hydrophobic interactions  Structure: Varied Structure Level Primary Structure Amino acid sequence Bonds involved Peptide Secondary α-helix.5   Causes polypeptide to fold to shield hydrophobic side chains from aqueous environment. Hydrophobic Interactions Groups Involved Amine/Carboxyl group Amine/Carbonyl of amino acid polypeptide backbone R-Group (and functional grps present)  Denaturation of proteins  Loss of specific three dimensional shape (bonds maintaining 3D conformation is broken)  Permanent in most cases but primary structure remains intact (generally)  Denaturation caused by heating (breaks bonds). etc Hydrogen Bonds Tertiary Quaternary Unique 3D conformation Association of multiple polypeptide chains to form a multimeric protein Hydrogen Bonds.Version 2. or hydrolytic agents (break peptide bonds) 1G – ANALYSE THE MOLECULAR STRUCTURE OF HAEMOGLOBIN AND COLLAGEN AND RELATE STRUCTURE TO FUNCTION  Haemoglobin  Found within red blood cells  Shape  Polypeptide chains are highly folded/bent/twisted to form a compact and spheroidal structure  Function  To transport oxygen to the lungs via the blood to other tissues in the body  Structure (Globular Protein)  Primary  α-(globin)-chain (141 amino acids  β-(globin)-chain (146) amino acids 17 | P a g e . Ionic Bonds. Disulfide Bond. Quaternary Structure  Definition: The overall protein structure that results from the association of two or more polypeptide chains to form a functional protein.  Each individual polypeptide (a protein subunit) has a tertiary structure (generally)  Proteins with quaternary structure also known as multimeric proteins  Bonding involved  Hydrogen bonds. ionic bonds.

 Structure (*unbranched)  Primary  Repeating Tripeptide sequence (Glycine-X-Y).  Each haem group can bind to one molecule of oxygen (Haem group can only interact with hydrophobic cleft)  Cooperative binding: Binding of one oxygen molecule to one of the 4 subunits causes the iron ion to be pulled closer. which causes a change in the conformation of those subunits. but ionic and hydrogen bonds also occur  Forms a spherical shaped unit held by many non-covalent interactions Function – Structure Provide a hydrophobic environment for the haem group to function Iron (II) ion is able to bind reversibly to account for the oxygen transporting ability of haemoglobin A soluble globular protein ideal for the transport of oxygen in blood Cooperative oxygen binding Eight α-helices connected by non-helical segments. allowing them to bind to oxygen more readily by increasing their affinity for oxygen  Folded such that amino acid residues located at the surface are mostly hydrophilic and those buried in the interior are mostly hydrophobic Quaternary  Tetramer made up of 2 identical dimers which is made of two types of polypeptide chains  Held together primarily by hydrophobic interactions. stabilized by intra-chain hydrogen bonds giving rise to the formation of a hydrophobic cleft containing a haem binding site Each subunit bears 1 haem prosthetic group with an Iron (II) ion within a porphyrin ring Polypeptide chains folded such that amino acid residues located at the surface are mostly hydrophilic and those buried in the interior are mostly hydrophobic Binding of one oxygen molecule to one of the 4 subunits causes the iron ion to be pulled closer.Version 2. creating a strain on other haemoglobin subunits. where X is often proline and Y hydroxylycine/hydroxyproline  Forms a polypeptide chain approximately 1000 amino acids long 18 | P a g e . allowing them to bind to oxygen more readily by increasing their affinity for oxygen  Collagen  Function  To provide the necessary support required for connective tissues  Great Tensile Strength  Shape  Elongated straight polypeptide chains wound around each other to form a rope-like structure. creating a strain on other haemoglobin subunits.5     Large variety of amino acids  Non repetitive amino acid sequence Secondary  Eight α-helices connected by non-helical segments. which causes a change in the conformation of those subunits. stabilized by intra-chain hydrogen bonds Tertiary  Each polypeptide chain consists of a haem prosthetic group that binds to the hydrophobic cleft.

associate side by side and are linked to each other by covalent crosslinks formed between the carboxyl end of one molecule and the amine end of another. where X is often proline and Y hydroxylycine or hydroxyproline Every third residue (Glycine) of the α-chain passes through the centre of the triple helix. allowing for the helical α-chains to pack tightly Stable structure with high tensile strength A quaternary structure – Tropocollagen – A right handed triple helix which has 3 parallel & identical α-chains wound around each other with a gentle-right handed twist to form a rope-like structure. allowing for the helical α-chains to pack tightly  Held together by an extensive network of hydrogen bonds between amine group of Gly in one α-chain and the carbonyl group of another amino acid residue in a neighbouring polypeptide  Hydroxy groups of hydroxyproline and hydroxylycine also participate in inter-chain hydrogen bonding  Covalent crosslinks between α-chains Formation of collagen fibres  Tropocollagen molecules possess an elongated conformation. forming a collagen fibril. associate side by side and are linked to each other by covalent crosslinks formed between the carboxyl end of one molecule and the amine end of another.  Arranged in a staggered manner. Proline’s ring structure stabilizes tropocollagen Held together by an extensive network of hydrogen bonds between amine group of Gly in one α-chain and the carbonyl group of another amino acid residue in a neighbouring polypeptide Covalent crosslinks between α-chains Tropocollagen molecules possess an elongated conformation.Version 2. thus allowing for stabilization by hydrophobic interactions  Lateral assembly of fibrils to form a fibre Function Structure Tight packing of helical αchains Repetitive tripeptide sequence consisting of Glycine-X-Y.  Residues of X and Y are located outside of tropocollagen where there is room for bulky R groups  Every third residue (Glycine) of the αchain passes through the centre of the triple helix.5   Small variety of amino acids  Secondary  α-chain (Left handed helical conformation AKA Collagen helix (3 residues per turn))  Tertiary  No tertiary structure  Quaternary  Tropocollagen – A right handed triple helix which has 3 parallel & identical α-chains wound around each other with a gentle-right handed twist to form a rope-like structure. Hydrophobic interactions between tropocollagen molecules due to staggered arragement Fibrils assemble laterally to form a fibre 19 | P a g e . forming a collagen fibril.

 Activation energy  Activation energy is the initial investment of energy required to start a reaction  Enzymes speed up the rate of reaction by lowering the activation energy required for a chemical reaction to take place  Enzyme characteristics  Enzymes are effective in minute amounts with a high turnover number.  Enzymes lower the activation energy required to activate the reactants. Amylose).  Enzyme activity is regulated by other organic and inorganic factors.5 1H – EXPLAIN THE MODE OF ACTION OF ENZYMES IN TERMS OF AN ACTIVE SITE. ES COMPLEX.  Enzymes are sensitive to pH changes and have an optimum pH at which they function most efficiently. concentration of substrate. can be inorganic metal ions or organic molecules (coenzymes)  Conjugated  Inhibitors  Importance of enzymes  Without enzymes. Hydrolases.  Catalytic site made up of catalytic amino acid residues  Catalytic amino acid residues directly involved in the making or breaking of chemical bonds  R groups of catalytic amino acid residues facilitate the reaction by acting as donators or acceptors of H+ or other groups on the substrate  Binding site  Binding amino acids residues hold the substrate in position while catalysis takes place through the formation of non-covalent bonds between the R-groups and the substrate  Structural amino acids 20 | P a g e . thereby enabling metabolic reactions to proceed quickly but at a low temperature.  Enzymes are specific in nature due to the unique 3D conformation of the active site and their ability to catalyse only one type of substrate/one group of substrate and one type of reaction (due to R groups)  Enzymes are sensitive to temperature and have an optimum temperature at which they function most efficiently. reactions in living cells would be too slow  Enzymes allow for the control/regulation of biological reactions  Nomenclature of enzymes  Prefix related to nature of reactant molecule (e. but can be denatured or inactivated depending on the temperature and pH level. the temperature and pH level. etc)  Enzyme structure  Globular proteins (with a tertiary or quaternary structure)  Active Site  Made up of a catalytic site where the reaction takes place and a binding site which holds the substrate in position. the latter is determined by the concentration of enzymes. and can thus be reused.Version 2. LOWERING OF ACTIVATION ENERGY AND ENZYME SPECIFICITY  Enzymes are biological catalysts which speed up chemical reactions in organisms by lowering the activation energy of the reactants. (operates at a narrow pH range)  Enzymes do not determine the rate of reactions. allowing more substrate molecules to attain the transition state.  Enzymes speed up the rate at which chemical equilibrium is attained but does not alter the chemical equilibrium.  AKA Cofactors.g. Suffix is –ase  Enzymes are classified to the types of reactions they catalyse (Isomerases. which is defined as the number of substrate molecules which one molecule of enzyme can turn into products per unit time  Enzymes remain chemically unchanged at the end of the reaction as they are not altered.

Version 2. allowing the substrate to attain the transition state configuration  Providing a microenvironment that favours the reactions  Following an effective collision between an enzyme and substrate  The Enzyme-substrate complex is formed  Substrate molecule held in active site by weak residue interactions such as hydrogen and ionic bonds between R-groups and substrate molecule  R groups of the catalytic amino acid residues catalyze the conversion of the substrate to the product  Alteration in chemical conformation causes the product molecule to be released from the enzyme as it is no longer complementary to the active site. causing the rate of formation of product to slow down due to a decrease in the frequency of effective collisions.5  Involved in maintaining the specific three dimensional conformation of the active site and the enzyme.  The amino acids which constitute the active site are moulded into a precise conformation and position such that reacting groups on substrates are brought together in close proximity in the correct orientation.  Products released after catalysis and active site returns to original state  Allows for a variety of substrate molecules that share similar structural or chemical properties as long as they are able to induce a conformation change to align the catalytic groups for reaction to occur (Allowing for group specificity) 1I – FOLLOW THE TIME COURSE OF AN ENZYME CATALYZED REACTION BY MEASURING RATES OF FORMATION OF PRODUCTS OR RATE OF DISAPPEARANCE OF SUBSTRATE  Rate of formation of product  Initially. when all substrate has been converted to products.  Induced fit hypothesis  **Active site of enzyme is flexible and not necessarily complementary to substrate  Substrate binds to active site. thus forming the ES complex. 21 | P a g e . the concentration of substrate molecules decrease as more are turned into products.  Thus facilitating catalysis by providing a favourable microenvironment for the reaction and lowering the activation energy of the reaction. which form products.  Eventually.  This stretches critical bonds and allows for the proper positioning of chemical R-groups of the catalytic amino acid residues in the active site close to the chemical bonds in the substrate. the frequency of effective collisions decreases to zero and the graph reaches a plateau. leading to a high frequency of effective collisions between enzyme and substrate molecules to form ES complexes. there is a high concentration of substrate molecules.  This causes the active site to fit more snugly around the substrate.  Over time. held by weak bonds to R-groups of binding amino acid residues and induces a conformation change in the enzyme’s active site. stabilizing the transition state.  Non-essential amino acid residues  No specific function  Mode of enzyme  Enzyme-Substrate Complex  Formed when enzyme and substrates are in close proximity and in the correct orientation during an effective collision  Lowers the activation energy by  Orientating the substrates in the correct orientation in proximity  Straining critical bonds in the substrate molecules.

all the substrates will be converted to products and the graph reaches zero.  At higher substrate concentration. An increase in temperature also raises the energy level of the enzyme and substrate molecules so that they are more likely to overcome the activation energy barrier and form products. Both effects increases the rate of reaction until optimal temperature is reached. hence the graph plateaus off due to the insufficient amount of molecules competing for available active sites.  At an even higher substrate concentration. which form products. which results in a slower increase in the rate of formation of products. thus increasing the rate of formation of the enzyme substrate complex. the enzyme concentration is the limiting factor. thus increasing the frequency of effective collisions between the substrate and the enzyme. The accumulation of end products causes the first enzyme in the metabolic pathway to be inhibited. more active sites are occupied by the substrate.  At high enzyme concentration. 22 | P a g e .Version 2. hence an increase in enzyme concentration will result in a proportional increase in the rate of reaction as more active sites are available for the formation of the enzyme-substrate complex at any given time.  Temperature (dual effect)  Reaction rate doubles for every 10 degrees in temperature from 10 degrees to optimal temperature  An increase in temperature increases the kinetic energy of the substrate and the enzyme molecules. further increase in enzymes do not increase the rate of reaction. hence an increase in substrate concentration will no longer increase the rate of reaction as the substrate concentration is no longer the limiting factor owning to the fact that enzymes are now working at the maximum rate and enzyme concentration is the limiting factor now. all the active sites are fully used up at any one time.  Enzyme concentration (E)  At low enzyme concentration conditions. as all the active sites of the enzyme are saturated at any one time. hence the rate of reaction increases proportionately to the increase in substrate concentration. This increases the rate of enzyme-substrate molecules formed. leading to the substrate having to wait for the products to leave the enzyme before they can bind to the active site.5  ***Allosteric end product inhibition. Substrate concentration is now the limiting factor. resulting in a proportional increase in the rate of reaction.  Over time. there is a high concentration of substrate molecules. the concentration of substrate molecules decreases as more substrate molecules are turned into products. leading to a high frequency of effective collisions between enzyme and substrate molecules to form ES complexes. thus the rate of formation of products decreases. hence the number of available active site decreases. which causes the enzyme-substrate complex to be formed a faster rate. 1J – FACTORS AFFECTING RATE OF ENZYME CATALYZED REACTIONS  Expected to describe by quoting values with units on top of explaining. hence enzyme concentration is no longer the limiting factor. as there are a lot of active sites available and an increase in the substrate concentration will result in a proportional increase in the frequency of effective collisions between enzyme and substrate molecules.  Substrate concentration (S)  At low substrate concentration conditions. causing the enzymes to be fully saturated.  Eventually. thus the rate of reaction increases slowly. leading to a decrease in the probability of the substrate finding a free active site as they must now compete among themselves. which increases the rate of product formation.  Rate of disappearance of substrate over time  Initially. the substrate concentration is the limiting factor. achieving the maximum rate of reaction. This increases the frequency of effective collisions between the enzymes and substrate molecules.

thus denaturing the enzyme.  R-groups of Catalytic Amino acids no longer possess the correct ionization to catalyze the required reaction  At optimum pH. ES complexes can no longer be formed as the active site is no longer complementary to the substrate. this type of inhibition can be overcome with a high substrate concentration.5  An increase in temperature above the optimal temperature causes the reaction rate to decrease drastically.  A change in pH level results in an alteration in the ionisation of amino acids. preventing the formation of the enzyme-substrate complex and hence preventing the formation of products which leads to the rate of reaction being reduced. which disrupts the ionic and hydrogen bonds responsible for maintaining the 3D conformation of the enzyme and active site. the rate of reaction equivalent to V Max can be obtained.  Enzymes exposed to high temperatures for a larger amount of time gain more thermal energy. as the substrate and inhibitor are in direct competition for the active site.  Binding amino acids can no longer hold the substrate in its correct orientation. at high concentrations of substrate. The enzyme is now denatured. due to the thermal agitation of the enzyme molecule which disrupts hydrogen and ionic bonds which are responsible for maintaining the precise 3D conformation of the enzyme. RoR is maximum.  Michaelis Constant Km The affinity of the enzyme for its substrate (measured in substrate concentration)  Measured as the substrate concentration that allows an enzyme-catalyzed reaction to proceed at half the maximum velocity  Low Km = Enzyme reaches its maximum catalytic rate at low concentration of substrate (high affinity)  High Km = Low affinity  Inhibitors  Molecules which reduce the rate of an enzyme-catalyzed reaction  Classified into  Competitive Reversible/Non-Reversible  Non-competitive Reversible/Non-Reversible  Competitive inhibition (KM)S < (KM)I. even beyond the optimum temperature. Vmax S > Vmax I 23 | P a g e . (Final amount of product formed is the same)  Competitive inhibitor lowers the affinity of the enzyme for the substrate  Non-competitive inhibition (KM)S=(KM)I. The greater the proportion of substrate molecules. thus optimum temperature is lower as a greater number of enzymes are denatured as … (basically enzymes cannot take high temperatures for long periods)  pH Level  Every enzyme has a pH range. hence causing no enzyme-substrate molecules to be formed. 1K – EFFECTS OF COMPETITIVE AND NON-COMPEITITVE INHIBITORS  VMax  The maximum rate at which an enzyme is able to perform the reaction in the presence of a specific concentration of enzyme and excess substrate. it competes with the substrate for the same active site. hence reducing the rate of reaction by preventing the substrate from binding to the active site.  However. causing the 3D active sites to be no longer complementary to the substrate. Thus. the greater the chance a substrate can outcompete the inhibitor to enter the active site.Version 2.  **Note that kinetic energy of enzyme and substrate increases as temperature increases. substrate can no longer bind in the correct orientation to the active site. and will not work outside this pH range. which leads to reaction rate dropping to zero. Effects of pH reversible as long as pH does not extend beyond the range. Vmax S = Vmax I  Due to the inhibitor having a structure similar to that of the substrate.

acting as an inhibitor on one of the enzymes controlling the previous step of the pathway  Alters conformation of active site and lowers its affinity for its substrate  Blocks the entry or the substrate into the active site  Prevents cells from wasting resources  Advantages  No accumulation of products in the cells as products become substrates for subsequent reactions  Reactants may be modified in a series of small steps.Version 2. Ionic/Hydrogen/Hydrophobic Interactions)  Irreversible  Covalent bonds Control and regulation of metabolic pathways  End-product inhibition  End-product of a metabolic pathway accumulates. altering the 3D conformation of the enzyme molecule and the active site such that the substrate is no longer complementary to the active site. an increase in substrate concentration has no effect on inhibition.  In the cytosol.  Since the substrate and inhibitor does not compete for the same site. attached to plasma membranes/membranes of organelles Extracellular Enzymes  Work outside of cells within an acidic/alkaline pH level  Synthesized in an inactive precursor form such that the enzyme is only activated at the right pH level  E. it does not compete with the substrate for the active site. No substrate binds to the active site and no ES complexes are formed.g. This arrangement orders the sequence of reactions and greatly increases the efficiency of the enzyme pathway Intercellular Enzymes  E. membrane bound organelles. which changes the enzyme to active or inactive mode. Pepsinogen (digestive enzymes) 24 | P a g e . enabling energy to be released in controlled amounts or minor adjustments to be made to the structure of molecules  Each step catalyzed by a specific enzyme. inhibitors stabilize inactive form. but instead binds to a site other than the active site. allowing for more points of control  Steps may be spatially arranged so that the product of one reaction is ideally located to become the substrate of the next enzyme. Reversible and irreversible inhibition  Reversible  Weak bonds (e. Allosteric regulation  Allosteric regulation refers to the regulation of an enzyme (usually made up of 2 or more polypeptides) by the binding of molecules (activators/inhibitors) at an allosteric site. decrease affinity of enzymes for its substrates.g.  Initial rate of reaction increases more slowly in the presence of non-competitive inhibitors.  Activators stabilize active form. increase affinity of enzymes for its substrates. permitting the build up of high local concentrations of substrate molecules so that biochemical reactions can proceed rapidly  Multienzyme complexes can be formed that consists of a team of enzymes for several steps of a metabolic pathway.g.5       Due to the inhibitor having a structure dissimilar to that of the substrate. This causes a certain proportion of enzyme molecules to be inactivated.

 If a species is successful in colonizing a particular niche.5 1I – EXPLAIN THE IMPORTANCE OF MITOSIS IN GROWTH.g. where X is the ploidy level and n refers to the number of chromosomes in one set  Reason for the production of genetically identical cells and fine control of replication  Changes in the genetic material may lead to cancerous cells.  Chromosomes align singly at the metaphase plate (there is no pairing of homologous chromosomes)  Separation of sister chromatids towards opposite poles of the cell by the shortening of kinetochore spindle fibres  Daughter chromosomes reach the opposite poles of cell before cytokinesis 25 | P a g e . (r-selection. thin chromatin into thick. there is little advantage in producing offspring that are different from the parents as these may be less successful. REPAIR AND ASEXUAL REPRODUCTION  For Unicellular Eukaryotes  Mitosis is important for asexual reproduction to produce genetically identical daughter cells. thus preventing the entanglement of chromatin. (No variation of genetic information)  Coiling of long.Version 2. New cells formed must be genetically identical to existing cells to carry out the same functions  Repair & Renewal of cells  Allows for the replacement of cells that die due to the normal wear and tear.  Regeneration  Mitosis allows for the regeneration of missing parts.  Maintain the diploid condition in further generations for genetic stability  DNA replication occurs prior to the distribution of genetic material to the two daughter cells. thus restoring a tissue to its original condition as new cells are genetically identical. condensed and distinct chromosomes during Prophase. e. thus it is better to quickly establish a colony of individuals similar to that of the parents. ecology)  Preservation of favourable traits from generation to generation  For multicellular eukaryotes  Growth  Enables sexually reproducing organisms to grow and fully develop from a single cell. arms of a starfish  Asexual reproduction  Vegetative propagation in plants 1M – EXPLAIN THE NEED FOR THE PRODUCTION OF GENETICALLY IDENTICAL CELLS AND FINE CONTROL OF REPLICATION  Ploidy level – The number of sets of chromosomes within the nucleus of a cell  Haploid cells have only one set of chromosomes  All sex cells and gametes are haploid and are generated by meiosis  Diploid cells have two sets of chromosomes  All somatic cells are diploid and are generated by mitosis  Polyploid  Organisms with more than 2 sets of chromosomes  Written as Xn. or the breaking of DNA during the separation of genetic material  Semi-conservative replication of DNA ensures that the parental DNA is used as a template for the synthesis of daughter DNA strands  DNA replication occurs before the disintegration of the protective nuclear membrane  Preservation of genetic stability across generations as the two daughter cells have the same number of chromosomes.

invade surrounding tissues and metastasize at distant sites Systemic treatment (radiation. 26 | P a g e . LOSS OF IMMUNITY) WHICH CAN INCREASE THE CHANCES OF CANCEROUS GROWTH. BUT DETAIL OF THE MECHANISM IS NOT REQUIRED. does not resemble parent cells Poorly defined tumour boundary. forming disordered. chemotherapy) is required along with surgery to ensure complete cure. GENETIC. Tumour cells remain clustered in a single mass at a localized region. Tumour cells do not cluster together.  Resisting cell death by apoptosis  Avoid apoptosis and live for an indefinite number of cell divisions  Enabling replicative immortality  Avoiding replicative cell senescence with telomerase (refer to organization of eukaryotic genome)  Inducing angiogenesis (Refer to multistep model of cancer)  Activating invasion and metastasis (refer to multistep model of cancer)  Avoiding immune destruction  Deregulating cellular energetics  Express an abnormally large amount of glucose transporters to consume large amounts of glucose to produce the ATP required for high rates of cellular division Benign tumour Low nuclear to cytoplasmic volume ratio Low rate of mitosis Well differentiated. (KNOWLEDGE THAT DYSREGULATION OF CHECKPOINTS OF CELL DIVISION CAN RESULT IN UNCONTROLLED CELL DIVISION AND CANCER IS REQUIRED.G. resembles parent cells Well defined tumour boundary. RADIATION.5 1N – EXPLAIN HOW UNCONTROLLED CELL DIVISION CAN RESULT IN CANCER.Version 2. May be surrounded by a layer of connective tissue Can be removed surgically Cancerous Tumour High nuclear to cytoplasmic volume ratio Very high rate of mitosis Poorly differentiated. invading tissues and metastasizing. CHEMICAL CARCINOGENS.  Hallmarks of cancer cells  Genome instability and mutation  Accelerated rate of chromosomal mutations (KIV Genetic basis of variation)  Genomic instability  Faulty DNA repair mechanisms  Gene mutations  Sustained proliferative signaling  Cells able to proliferate in the absence of growth factors  Evading growth suppressors  Cells are non-responsive to growth-inhibitory signals  Loss of anchorage dependence  Cells are able to proliferate in suspension cultures without attachment to a surface  Loss of contact inhibition and density dependent inhibition  Cells continue to proliferate after making contact with neighbouring cells. AND IDENTIFY CAUSATIVE FACTORS (E. multilayered patterns.)  Cancer results when cells divide excessively and invade other tissues  Uncontrolled cell division (Rate of cell division >>> rate of cell death) causes damaged genes to be passed on to altered daughter cells  Cells accumulate mutations and escape growth restrains.

5  Cell cycle checkpoints  G1  Checks for the presence of growth factors  Checks for DNA damage and cell size  Initiates apoptosis by activating suicide genes if DNA damage is irreparable  G2  Checks if DNA is replicated without damage  Initiates apoptosis by activating suicide genes if DNA damage is irreparable  M  Checks for successful formation of spindle fibres and attachment of spindle fibres to the kinetochores of chromosomes  Ensures that there is successful separation of DNA  Arrests mitosis if conditions above not met. X-Rays) can produce free radicals within the cell.Version 2. leading to chromosomal mutations. humans accumulate mutations which increase the risk of cancer.  Loss of immunity  Allows cancerous cells to escape destruction and proliferate  Viral Infections  Viruses can integrate their genetic material into the DNA of their host cells and transform a normal cell into a tumourigenic cell by  Activating a proto-oncogene via a gain of function mutation  Deactivating a tumour suppressor gene via a loss of function mutation  Introducing an oncogene into a normal cell  Preventing the expression of p53 and other tumour suppressor proteins (by dictating the host cell to produce viral proteins) 27 | P a g e . insertions or deletions. This causes the DNA to be mutated. which interact with cellular DNA and produce double stranded breaks. (e.g. This causes tumour suppressor genes to be mutated via loss of function mutation. especially at p53 sites  Model of Cancer Development  Radiation Exposure  Ionizing radiation (Gamma. forming DNA adducts which cannot be repaired by cellular DNA repair mechanisms.  Causative factors of cancer  Lifestyle and Diet  Smoking/ Exposure to carcinogens  Carcinogens bind irreversibly to cellular DNA. breast cancer)  Individuals inherit a mutant copy of tumour suppressor genes from their parents and have an increased chance of developing cancer.  Genetic Predisposition (hereditary)  Certain types of cancer run in families.  UV rays causes thymine dimers to be formed and results in point mutation evens such as basepair substitution.  Age  As time passes. This causes cancer critical genes to be mutated. resulting in decreased expression of proteins that prevent cell growth.

it produces two genetically identical daughter cells. THE BEHAVIOUR OF CHROMOSOMES DURING THE MITOTIC CELL CYCLE AND THE ASSOCIATED BEHAVIOUR OF THE NUCLEAR ENVELOPE.g. CELL MEMBRANE AND CENTRIOLES  Mitosis – The nuclear division of one nucleus into two genetically identical nuclei.5 1O – DESCRIBE WITH AID OF DIAGRAMS.  Cytokinesis – The division of the cytoplasm to produce two completely separated daughter cells. to ensure equal distribution of the cell organelles  This forms two genetically identical cells. Colchicine which interfers with spindle function) 28 | P a g e .  Occurs simultaneously with telophase  Characteristics of mitotic cells  Nuclear envelope has disintegrated and nucleolus no longer present  Transcriptionally in active  DNA condenses into chromosomes  Drugs can be used to arrest cells at metaphase (e. Along with cytokinesis.  Cell organelles evenly distributed towards the two poles of the parent cell.Version 2.

11) Cell is elongated as the poles of the cell move further apart as polar spindle fibres slide past each other. 2) Two chromatids are joined at the centromere. along with astral spinder fibres.5 Cell Cycle Interphase Prophase Metaphase Anaphase Telophase  G1 Phase  Cells increase in size and acquire ATP. forming a chromosome 3) Nucleolus disappears and nuclear envelope disintegrates 4) Centrioles migrate to opposite ends of the cells and develop polar spindle fibres which span the cell from pole to pole. and migrate to the ends of each cell centromere first. (plant cells do not have centrioles but still assemble spindle fibres) 5) Kinetochore spindle fibres begin to assemble 6) Chromosomes shorten and thicken to their maximum limit 7) Chromosomes migrate and align singly at the metaphase plate 8) Kinetochores at centromeres of chromosomes attach to the kinetochore spindle fibres 9) Centromeres separate 10) Further shortening of kinetochore spindle fibres causes sister chromatides of each chromosome to separate. Chrom - - DNA 2x 4x 4x 2n 4x 2n 4x 4n 4x 4n 4x 29 | P a g e . forming daughter chromosomes. observable chromosomes. uncoil and lengthen to form chromatin threads 14) Spindle fibres disassemble and nucleolus reforms.Version 2.  Intensive cellular gene expression and synthesis of appropriate organelles and proteins  Building up of protoplasm  S (Synthesis) Phase  Replication of DNA  Synthesis of histone proteins  G2 Phase  Acquisition of ATP  Centrioles replicate  Mitotic spindles form 1) Chromatins condense by supercoiling to be folded into discrete. involving special motor proteins 12) Daughter chromosomes reach respective poles and a new nuclear envelope forms around each group 13) Chromosomes decondense.

 Kinetochore 30 | P a g e . folded with the aid of scaffolding proteins. which carries information from one generation to the next. causing the cleavage furrow to deepen until the parent cell pinches into two daughter cells. 2n 2x 1P – EXPLAIN WHAT IS MEANT BY HOMOLOGOUS PAIRS OF CHROMOSOMES  Chromatin  A complex of nucleic acids and associated histone and non-histone proteins that exist as an uncoiled and diffused state present during interphase of the cell cycle.  Cytokinesis in plant cells  Growth of a cell wall during telophase across the metaphase plate of the plant cell  Vesicles from the Golgi Apparatus containing materials to construct the primary cell wall and a middle lamella move to the middle of the cell where they fuse and form a cell plate  The cell plate enlarges until its surrounding membrane fuses with the cell surface membrane of the parent cell.5 Cytokinesis  Animal Cells  Cytoplasmic Cleavage  Formation of cleavage furrow which pinches the cell into two  A contractile ring of microfilaments on the cytoplasmic side of the furrow contracts. shape. forming two daughter cells with its own protoplasm. centromere position and sequence of gene loci. shape.  Exists as heterochromatin or euchromatin  Chromosome  The condensed form of chromatin. (One chromosome contains the paternal alleles. the other contains the maternal alleles)  Each chromosome of such a pair is called a homolog.  Cellulose laid down between the two membranes of the cell plate to form a cell wall. each with a complete nucleus and share of protoplasm. or in non-dividing cells. but genetically unidentical due to different alleles. due to the semi conservative nature of DNA replication. located at specific physical locations along each chromosome  Contains the information needed to synthesize one polypeptide chain or RNA in the form of a specific sequence of nucleotides  Gene locus (plu: loci)  The specific location of a gene  Homologous pairs of chromosomes  A pair of chromosomes which are structurally identical in size. resulting in equal distribution of genetic material.  Centromere  The specialized region where two sister chromatids join. centromere position and sequence of gene loci.  Allele  An alternative form of a gene at a particular gene locus  Sister chromatids  Replicated forms of a single chromosome joined together at the centromere  Genetically identical as they have the same alleles at each gene locus and structurally identical in size.  Gene  The fundamental physical and functional unit of heredity.  Allows for the proper alignment of chromosomes at the metaphase plate and segregation of sister chromatids to the poles.Version 2. associated with kinetochores for the attachment of spindle fibres. required for genetic stability in daughter cells.

Maintains a constant chromosome number in every generation of a species. forming a tetrad. 1Q – EXPLAIN THE NEED FOR REDUCTION DIVISION PRIOR TO FERTILIZATION IN SEXUAL REPRODUCTION  This is to maintain a constant number of chromosomes (by restoring the diploid number) and prevent chromosomal doubling in the next generation. causing them to exchange genetic material. bridged by the synaptonemal complex  Homologous chromosomes are joined at the chiasmata. attached to kinetochore spindle fibres  Plays an active part in the movement of chromosomes to the opposite poles  Centrioles  Barrel-shaped organelle found only in animal cells  Each centriole composed of nine triplets of microtubules  Pair of centrioles located perpendicular to each other  Located at the centrosome  Centrosome  Microtubule Organizing Centre  Includes a pair of centrioles and the surrounding cytoplasm. 1R – EXPLAIN HOW MEIOSIS AND RANDOM FERTILIZA TION CAN LEAD TO GENETIC VARIATION  Crossing over of homologous chromosomes during prophase I  Synapsis occurs as homologous chromosomes come together and pair along their entire length. forming recombinant chromatids. which contains proteins that aid in the assembly of spindle fibres  Assembles spindle fibres  Spindle fibres  An organized system of microtubules that attaches to the cetromeric regions of duplicated chromosomes and draws them to opposite poles during cell division  Astral spindle fibres  Radiate from the centriole towards peripheral regions of the cell  Serves as a brace for the functioning of the spindle fibres  Kinetochore spindle fibres  Pull the sister chromatids towards the opposite poles of a cell during anaphase  Polar spindle fibres  Fibres running from pole to pole overlapping at the equator of the spindle  Responsible for elongating the entire cell along the polar axis during anaphase. Crossover points are called chiasma 31 | P a g e .  DNA only replicates once in Interphase before it is immediately followed by two successive nuclear divisions to produce haploid cells  Alignment of bivalents at the metaphase plate ensures equal distribution of chromosomes to each daughter cell in subsequent anaphase I  Segregation of homologous chromosomes during metaphase I ensures the equal distribution of chromosomes to each daughter cell as the chromosome number in the daughter cells are halved and ploidy level is haploid  Chromosomes align singly at metaphase II to ensure equal distribution of chromosomes to each gamete such that the ploidy level of each gamete is haploid.  Crossing over occurs when chromatids twist and wrap around each other.Version 2.5  A structure formed by proteins on specific sections of the chromosome.

Recombinant chromatids carry alleles different from those carried on the non-recombinant sister chromatids.Version 2.  Independent assortment of bivalents at the metaphase plate during metaphase I  Random orientation of bivalents with respect to the poles at metaphase I and chromosomes at metaphase II results in an independent segregation of paternal and maternal chromosomes into gametes at anaphase I and II. The gametes get a new combination of alleles where the number of possible permutations is 2n (n is the haploid number of chromosomes)  Random fertilization  Fusion between male and female gametes is random due to random selection of mates There is but one error in both diagrams  Equator of 2nd processes are perpendicular to equator of 1st processes 32 | P a g e .5  This gives rise to new combinations of paternal and maternal alleles in each chromatid.

which separate. 4) Synapsis occurs as homologous chromosomes come together and pair along their entire length.5 1S – DESCRIBE. Crossing Over. along with astral spinder fibres. CELL M EMBRANE AND CENTRIOLES  Meiosis – Involves two successive nuclear divisions following a single replication of the chromosomes. producing four haploid daughter cells  Meiosis I – Reduction Division  Amount of DNA is the same as that of parent cell before replication  Chromosome number and ploidy level is reduced by half  Synapsis. (plant cells do not have centrioles but still assemble spindle fibres) 3) Kinetochore spindle fibres begin to assemble.  Intensive cellular gene expression and synthesis of appropriate organelles and proteins  Building up of protoplasm  S (Synthesis) Phase  Replication of DNA  Synthesis of histone proteins  G2 Phase  Acquisition of ATP  Centrioles replicate  Mitotic spindles form 1) Nucleolus disappears and nuclear envelope disintegrates 2) Centrioles migrate to opposite ends of the cells and develop polar spindle fibres which span the cell from pole to pole. THE BEHAVIOUR OF CHROMOSOMES DURING MEIOSIS. bridged by the synaptonemal complex 5) Homologous chromosomes are joined at the chiasmata. to ensure equal distribution of the cell organelles  This forms two cells. forming a tetrad.  Occurs simultaneously with telophase Cell Cycle Interphase Prophase I Metaphase I Anaphase I  G1 Phase  Cells increase in size and acquire ATP. Independent assortment take place  Meiosis II – Equational Division  Chromosome number does not change  Amount of DNA is half that of the parent cell before replication  Chromatids in meiosis II are not genetically identical to each other  Cytokinesis – The division of the cytoplasm to produce two completely separated daughter cells. independent of any other bivalent. causing them to exchange genetic material. - - DNA 2x 4x 4x 2n 4x 2n 4x 2n 4x 33 | P a g e .  Cell organelles evenly distributed towards the two poles of the parent cell. 9) Further shortening of kinetochore spindle fibres causes bivalents to split to form two homologous chromosomes . 8) Bivalents arrange themselves on the metaphase plate so that each bivalent is oriented to opposite poles in a completely random arrangement. in a process called independent assortment. forming recombinant chromatids 7) Kinetochore spindle fibres attach to the kinetochores of centromeres of chromosomes. AND THE ASSOCIATED BEHAVIOUR OF THE NUCL EAR ENVELOPE.Version 2. WITH THE A ID OF DIAGRAMS. Chrom. 6) Crossing over occurs when non-sister chromatids twist and wrap around each other.

(Disjunction) 11) Cell is elongated as the poles of the cell move further apart as polar spindle fibres slide past each other.Version 2. 18) Two chromatids are joined at the centromere. uncoil and lengthen to form chromatin threads 12) Spindle fibres disassemble and nucleolus reforms. involving special motor proteins 15) Homologous chromosomes reach respective poles and a new nuclear envelope forms around each set of chromosomes 16) Chromosomes decondense. and migrate to the ends of each cell centromere first. forming a chromosome 19) Nucleolus disappears and nuclear envelope disintegrates 20) Centrioles migrate to opposite ends of the cells and develop polar spindle fibres which span the cell from pole to pole at right angles to the spindle of metaphase plate of meiosis I. uncoil and lengthen to form chromatin threads 30) Spindle fibres disassemble and nucleolus reforms. 27) Cell is elongated as the poles of the cell move further apart as polar spindle fibres slide past each other. forming daughter chromosomes. 21) Kinetochore spindle fibres begin to assemble 22) Chromosomes shorten and thicken to their maximum limit 23) Chromosomes migrate and align singly at the metaphase plate (which is perpendicular to the metaphase plate in Metaphase I) 24) Kinetochores at centromeres of chromosomes attach to the kinetochore spindle fibres 25) Centromere separates 26) Further shortening of kinetochore spindle fibres causes sister chromatides of each chromosome to separate. observable chromosomes. 31) Cell Division *See mitosis 2n 4x n n 2x 2x n 2x 2n 2x 2n 2x n x 34 | P a g e . involving special motor proteins 28) Daughter chromosomes reach respective poles and a new nuclear envelope forms around each group 29) Chromosomes decondense. 13) Cell Division *See mitosis 17) Chromatins condense by supercoiling to be folded into discrete.5 Telophase I Cytokinesis I Prophase II Metaphase II Anaphase II Telophase II Cytokinesis II 10) Homologous chromosomes segregate and migrate towards the ends of each cell centromere first. along with astral spinder fibres.

5 35 | P a g e .Version 2.

5 APPENDIX 1A: PROTEIN R GROUPS 36 | P a g e .Version 2.

This maintains the integrity of the stored genetic information.Version 2. fused five and six member ring structure  Two ends  Free 5’ carbon containing a phosphate group  Free 3’ carbon containing a hydroxyl group  Secondary structure: Double Helix  Characteristics  Diameter .  Capable of genetic variation to allow for evolution to take place  Locations: Nucleus. mitochondria.1 – STRUCTURE AND ROLE OF DNA  Role of DNA: As hereditary material  Stable: DNA is chemically stable and will not change easily due to age. Guanine (A. T)  Six-membered ring structure  Purines  Adenine.2nm 37 | P a g e . Damaged strands are automatically repaired using the other stand as a template through complementary base pairing. exposure to environment only influences sugar phosphate backbone.  Replicates accurately  Redundancy of genetic information in DNA (due to present of two strands).  Folding and condensing DNA protects DNA from thermal and physical damage. chloroplasts  Structure of DNA  Primary Structure: Formed up of deoxyribonucleotides (basic monomers)  Made up of deoxyribonucleosides and phosphate groups N-Glycosidic Bond  Deoxyribonucleoside consists of a nitrogenous base + a deoxyribose sugar (pentose sugar)  Deoxyribose  Hydrogen attached to 2’ carbon in replace of a reactive hydroxyl group  Allows DNA to be more chemically stable and unreactive  Allows the DNA to coil in a tight double helix due to the absence of repulsion between the hydrogen atom and phosphate group Phosphodiester  Nitrogenous bases  Pyrimidines bond  Cytosine & Thymine (C. G)  Larger in size.5 2. nutrition or environment  Extensive hydrogen bonding between base pairs  Hydrophobic interactions between stacked base pairs  Nitrogenous bases safely tucked inside the double helix. DNA & GENOMICS 2A – DESCRIBE THE STRUCTURE AND ROLES OF DNA AND RNA 2A.

Version 2.5

2 strands
Coiled in the form of a helix, one complete twist every 3.4 nm (pitch length); 10 bases for
each complete turn of the helix
 Anti-parallel nucleotide strands (5’  3’ & 3’  5’) (shows opposite polarity)
 Phosphate group is negatively charged
 Nitrogenous bases stacked 0.34 nm apart
 Minor and major grooves allows proteins & enzymes to gain access to the nitrogenous bases
 Nitrogenous bases orientate inwards into the core of the double helix (hydrophobic) while
sugar phosphate backbone projects outside the double helix (hydrophilic)
 Complementary base pairing
 Occurs between Adenine and Thymine (2 hydrogen bonds) and Guanine and Cytosine (3
hydrogen bonds)
 Reasons
 Steric restrictions
 Sugar phosphate backbone of each DNA strand has a regular helical structure
with a uniform diameter of 2nm. Only pairing of one pyrimidine with one
purine can result in a correct fit of the physical dimensions of the double helix.
 Hydrogen bond factors
 Chemical groups which can form hydrogen bonds have well defined positions in
purines and pyrimidines. Adenine can form 2 hydrogen bonds with T, while
Guanine can form 3 hydrogen bonds with cytosine.
 Significance
 Nitrogenous base sequence of the template strand determines the nitrogenous
base sequence in the complementary strand  One strand can act as a template for
DNA replication and the subsequent transmission of stored genetic information
 Weak hydrogen bonds make it relatively easy to separate the two strands of DNA by
heating.
 Extensive hydrogen bonding makes the DNA molecule structurally stable
 Variation of linear base sequence: Linear sequence of the four bases is varied in
countless ways. Each gene has a unique order of bases.
 NOT EXIBITED by SINGLE-STRANDED DNA
 Chargaff’s rules of complementary base pairing
 In all the double stranded DNA, the molar amount of purines is equal to the molar amount of
pyrimidines (Pyrimidine to Purine ratio  1:1)
 The amount of Guanine is always equal to the amount of cytosine (Guanine: Cytosine  1:1)
 The amount of Adenine is always equal to the amount of Thymine (Thymine: Adenine  1:1)
 The base composition of DNA varies greatly from one organism to another, expressed in the
dissymmetry ratio (A+T)/(C+G).
 Base composition of DNA is constant throughout all somatic cells of the organism and is
characteristic for a given species
Bonds (all covalent bonds formed via condensation reaction)
 Phosphodiester bonds
 A phosphodiester bond is formed between the 5’-phosphate group of one end of the
deoxynucleotide and the 3’-hydroxyl group of the other end of the deoxyribonucleotide in a
condensation reaction.
 This process repeats many times to form a polynucleotide. (aka one stand of DNA)
 Results in a linear, unbranched sugar phosphate backbone.
 As a strong covalent bond, phosphodiester bonds confer strength and stability on the
polynucleotide chain.
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Version 2.5



Phosphoester bond
 Formed between the 5’ carbon of the deoxyribose sugar and the phosphate group, forming a
deoxyribonucleotide
Glycosidic bonds
 Nitrogenous base joins to the deoxyribose sugar via the 1’ carbon as an N-glycosidic bond,
forming a deoxyribonucleoside
Hydrophobic interactions
 Hydrophobic interactions between stacked base pairs
Hydrogen bonds
 Between complementary nitrogenous bases of opposite strands
Ionic bonds
 Between histone proteins and sugar phosphate backbone

 Packing of DNA in Eukaryotic Chromosomes
 Steps are highly precise and specific
 Function: Organises and packs the giant DNA
molecules of eukaryotic chromosomes into
structures that will facilitate their segregation into
daughter DNA.
 First Level: Nucleosome core + Linker DNA (10nm
chromatin fibre)
 DNA packed into nucleosomes to obtain 10-nm
chromatin fibres (aka nucleosome fibre)
 Involves an octamer of histone proteins, two of
each H2A, H2, H3 and H4. Histones are proteins
with a high concentration of positively-charged
residues which form ionic bonds with the
negatively charged sugar-phosphate backbone.
Double-stranded DNA coils around the octamer
to form a nucleosome core, joined to other
nucleosome cores via linker DNA.
 (Histones are removed during DNA replication
for a brief period of time)
 Second level: Solenoid (30nm chromatin fibre)
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Version 2.5

Histone H1 protein binds to the linker DNA and pulls the nucleosomes to associate into rings of
six nucleosomes which further coil into a nucleosome fibre to produce the 30nm chromatin fibre
(solenoid)
Third Level: Chromosomes
 Non-histone chromosomal proteins form a chromosome scaffold involved in condensing the
solenoids to form looped domains. Looped domains further fold and compact to form a
metaphase chromosome, usually 1400 nm in width.

2A.2 – STRUCTURE AND ROLE OF RNA
 Roles of Ribonucleic Acid
 Messenger RNA (mRNA)
 Carries information from DNA, which codes for amino acid sequences of proteins and ribosomes.
 Transfer RNA (tRNA)
 Serves as an adaptor molecule in protein synthesis
 Translates mRNA nucleotide sequence into an amino acid sequence by carrying a specific amino
acid for base pairing with mRNA codon
 Ribosomal RNA (rRNA)
 Catalyzes the formation of peptide bonds and is a component of large and small ribosomal
subunits.
 Small Nuclear RNA (snRNA)
 Plays structural and catalytic roles in spliceosomes
 Small Interfering RNA (siRNA) & micro RNA (miRNA)
 Involved in the regulation of gene expression
 Structure of RNA
 Made up of ribonucleotides
 Made up of ribonucleosides and
phosphate groups
 Ribonucleoside consists of a
nitrogenous base + a ribose sugar
(pentose sugar)
 Ribose
 Hydroxyl group attached to
2’ carbon
 Less stable than DNA
 Nitrogenous bases
 Pyrimidines
 Cytosine & Uracil (C, U)
 Six-membered ring
structure
 Purines
 Adenine, Guanine (A, G)
 Larger in size, fused five
and six member ring
structure
 Two ends
 Free 5’ carbon containing a phosphate group
 Free 3’ carbon containing a hydroxyl group
 Forms a single stranded helical molecule which can be folded into a complex tertiary structure
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Version 2.5



No complementary base pairing
Basic forms
 Messenger RNA
 Transfer RNA
 Ribosomal RNA
 Small Nuclear RNA
 Small interfering RNA
Location: Found throughout the cell, synthesized in the nucleus
Amount varies from cell to cell (and within cells)

2B – DESCRIBE THE PROCESS OF DNA REPLICATION AND THE EXPERIMENTAL
EVIDENCE FOR SEMI-CONSERVATIVE REPLICATION
2B.1 MODELS OF DNA REPLICATION
 General similarity across all models: Nucleotides are ordered into a new, complementary strand based on
an existing template strand through the process of base pairing
 Conservative Replication (Wrong Model)
 Parental DNA molecule is conserved during the replication
 Dispersive model (Wrong Model)
 All the four strands of DNA following one cycle of replication have a mixture of old and new DNA
 Semiconservative model (Right Model) (Watson and Crick’s model)
 Each new daughter DNA molecule has one old strand derived from the parent DNA molecule and one
newly synthesized strand
 Meselson-Stahl Experimental Evidence for Semiconservative Replication of DNA
 E. Coli bacteria nurtured in culture containing only heavy nitrogen 15N
 Parental DNA thus contains only 15N
heavy nitrogen.
 Use of E. Coli due to short generation
time as E. Coli multiplies very fast.
 E. Coli bacteria transferred over to
culture containing only light nitrogen 14N
 Daughter DNA strands would contain
light nitrogen 14N
 DNA samples extracted from the bacteria
after 20 mins and centrifuged
 First cycle of replication (eliminates
conservative DNA model)
 DNA samples extracted again after 40
mins and centrifuged.
 Second cycle of replication (eliminates dispersive DNA model)
 Watson’s and Crick’s hypothesis
 When a double helix DNA molecule replicates, the two parental DNA strands separate due to the
breakage of hydrogen bonds
 Each parental DNA strand serves as a template for the synthesis of a new complementary
daughter strand so that each of the two daughter DNA molecules formed consists of one parental
DNA strand and one newly synthesized daughter DNA strand.
 Nucleotides line up singly along the template of the DNA strand according to complementary
base pairing.
41 | P a g e

so new deoxyribonucleotides are only added to the 3’ end.  Requires a RNA primer to initiate the synthesis of daughter DNA strand (DNA cannot be synthesized de novo)  DNA Polymerase I  Replaces the RNA primer with DNA  DNA Ligase  Joins the Okazaki Fragments together  RNA Primase  Synthesizes RNA primers for leading strand and Okazaki fragments. 42 | P a g e .  Shows the polarity of the parental DNA strands  Anti-parallel DNA double helix strands ensures that the continuous synthesis of both DNA strands at a replication fork is not possible.Version 2.2 MECHANISM OF DNA REPLICATION  DNA Replication is Semi-Conservative & Semi-Discontinuous  Proteins/Enzymes involved + General Function  Topoisomerase  Unwinds the DNA double helix  DNA Helicase  Separates parental DNA strands  Single Strand DNA binding proteins (SSB Proteins) (spell out)  Stabilizes replication fork and ensures DNA is readable by DNA polymerase.5  DNA polymerase links the nucleotides together at their sugar-phosphate moieties 2B.  DNA Polymerase III  The specific 3D conformation of the active site limits the synthesis of DNA only from the 5’ to 3’ direction.

Synthesis of daughter DNA strands 11.5 DNA Replication Location of Origins of Replication Enzyme/Proteins Involved 1. Replication fork stability is maintained by SSB proteins which bind tightly to single-stranded regions of DNA. 10. starting from the RNA primer. 12. thus keeping the strands apart.Version 2. via semiconservative replication. 7. Parental DNA acts as templates along which deoxyribonucleotides align themselves by complementary base pairing. 4. Separation of Parental DNA strands 6. Energy Requirement (Not required to know) Topoisomerase DNA Helicase Yes (In the form of ATP) Single Stranded DNA binding Proteins (SSB proteins) RNA Primase DNA Polymerase III Yes (In the form of dNTP bond breakage) 43 | P a g e . A portion of the parental DNA serves as the complementary base sequence. The breakage of the pyrophosphate bond releases energy which is coupled to phosphodiester bond formation between the free 3' hydroxyl group of the terminal nucleotide of the growing polynucleotide chain and the free 5' phosphate group of the incoming deoxyribonucleotide. where new strands of DNA are synthesized Replication of DNA then proceeds in both directions from the origin of replication. attach to the DNA and separates the two strands. (supercoiled structure broken by nicking a single strand of DNA) DNA Helicase facilitates the separation of parental DNA strands by breaking the hydrogen bonds between the strands by using energy in the form of ATP. which speeds up the replication of DNA. In eukaryotic chromosomes. forming a replication bubble At one end of the replication bubble is the replication fork. 5. Replication of DNA begins at specific sites called the origins of replication. which RNA primase attaches on to make the RNA primer by joining ribonucleotides. 2. DNA polymerase III catalyzes the formation of phosphodiester bonds between the growing daughter DNA strand. while straightening out the single stranded DNA template and prevent it from reannealing. Synthesis of RNA Primers 9. (Multiple replication bubbles form and fuse) Topoisomerase facilitates the unwinding of the DNA double helix by creating a transient break. 3. many replication origins are present. preventing single stranded DNA from being degraded. Incoming dNTPs lose a pyrophosphate group. This provides a free 3' hydroxyl group for DNA polymerase III to synthesize the daughter DNA strand as DNA synthesis cannot occur de novo. Initiator Proteins recognize the specific nucleotide sequence at the origins of replication. 13. 8. thus relieving stress on the DNA molecule by allowing free rotation around the single strand to allow for the unwinding of the double helix ahead of the replication fork for initiation of replication. and the incoming dNTPs.

1 TRANSCRIPTION IN PROKARYOTIC CELLS  Prokaryotes do not possess a nuclear membrane. in which a termination factor called the ρ (rho) factor binds to a rho recognition site and moves along the mRNA towards the RNA polymerase.Version 2. it destabilizes the mRNA-DNA hybrid and releases the newly synthesized mRNA from the elongation complex. Each Okazaki fragment is then linked via a phosphodiester bond catalyzed by DNA ligase from the 3’ end of one Okazaki fragment to the 5’ end of the next Okazaki fragment of the growing daughter DNA strand. Prokaryotic Transcription Initiation 1. The leading strand is continuously synthesized as a single polymer along the template strand. 3. DNA polymerase III also has a 3' to 5' exonuclease activity to excise incorrect base pairs in a process called proof readng. causing RNA polymerase to dissociate. DNA Ligase DNA Polymerase I Yes (In the form of dNTP bond breakage) DNA Polymerase III 2C – DESCRIBE HOW THE INFORMATION ON DNA IS USED TO SYNTHESIZE POLYPEPTIDES IN PROKARYOTES AND EUKARYOTES. The lagging strand is synthesized discontinuously as a series of Okazaki Fragments. 2. 18. therefore transcription and translation take place simultaneously  There is thus no post-transcriptional modification  Termination achieved via either  Intrinsic termination. FORMATION OF MRNA FROM PRE-RNA AND TRANSLATION IS REQUIRED) 2C. DNA polymerase I replaces all RNA primers with deoxyribonucleotides. Synthesis of leading daughter DNA strand Synthesis of lagging daughter DNA strand Replacing RNA Primers Proof Reading 19. Components The σ (sigma) factor on RNA polymerase recognizes the RNA polymerase recognition sequence and binds at the Pribnow box RNA polymerase binds to the promoter and causes the two DNA strands to unwind by breaking hydrogen bonds between complementary base-pairs This forms a transcription bubble which exposes a short stretch of nucleotides on each strand σ factor RNA polymerase Promoter 44 | P a g e . polymerized in the 5’ to 3’ manner against the overall direction of the replication fork 17. When it catches up. in which a palindromic GC-rich sequence on the RNA forms a hairpin loop structure. polymerised in a 5’ to 3’ manner towards the replication fork 16.5 14. Uracil residues present in the DNA-RNA hybrid region is unable to hold the structure together. (DESCRIPTION OF THE PROCESSE S OF TRANSCRIPTION. Anti-parallel parental DNA strands and the limitation of DNA polymerase III ensures that the daughter strand has to be synthesized semidiscontinuously 15.  Rho dependent termination.

Elongation Termination One of the exposed DNA strands act as a template for complementary base pairing to direct the assembly of incoming ribonucleoside triphosphates (NTPs) RNA polymerase catalyzes the formation of the first phosphodiester bond. catalysed by RNA polymerase. RNA polymerase carries out proofreading functions and removes incorrectly inserted ribonucleotides NTPs 6.5 4. In intrinsic termination. This structure is followed by uracil residues which is unable to hold together the DNA-RNA hybrid. Termination occurs either via the intrinsic termination or the rhodependent termination 14. In rho dependent termination. 10. mRNA is thus synthesized in the 5’ to 3’ direction 11. causing RNA polymerase to dissociate. NTPs mRNA Palindromic GCrich sequence DNA-RNA hybrid ρ factor DNA-RNA hybrid 45 | P a g e . Ribonucleotides are added via complementary base pairing with the DNA template strand 9. a palindromic GC-rich sequence on the RNA forms a hairpin loop structure. 15. RNA polymerase moves along the template DNA in the 3’ to 5’ direction 7. When it catches up. RNA polymerase reanneals the unwound DNA behind it.Version 2. Incoming NTPs lose a pyrophosphate group. dissociating the growing RNA chain from the template 12. The breakage of the pyrophosphate bond releases energy which is coupled to phosphodiester bond formation between the free 3' hydroxyl group of the terminal nucleotide of the growing mRNA and the free 5' phosphate group of the incoming ribonucleotide. 5. it destabilizes the mRNA-DNA hybrid and releases the newly synthesized mRNA from the elongation complex. a termination factor called the ρ (rho) factor binds to a rho recognition site and moves along the mRNA towards the RNA polymerase. DNA double helix transiently unwinds at the same time 8. 13.

4. at 70S (50S + 30S) as compared to eukaryotic ribosomes (80S)  Initiator tRNA carries N-formyl methionine (an additional aldehyde group attached to the N-terminus) instead of methionine  Polycistronic mRNAs – Each mRNA contains different segments which give rise to different proteins Prokaryotic Translation Amino Acid 1.3 Translation)  Ribosomes  Smaller in size. tRNA 3. Activation of 2. all other components are similar to eukaryotic cells (refer to 2C.5 2C. 5. Amino acid and ATP enters the active site of the enzyme Adenosine Monophosphate is joined to the amino acid using energy through the breaking down of the pyrophosphate bond AMP is displaced by the incoming tRNA Aminoacyl-tRNA synthetase catalyzes the formation of a ester linkage between the amino acid and the 3’ CCA stem of the tRNA. forming an aminoacyl-tRNA The aminoacyl-tRNA is released from aminoacyl synthetase for translation Components Amino Acid ATP tRNA Aminoacyl-tRNA Synthetase 46 | P a g e .2 TRANSLATION IN PROKARYOTIC CELLS  Other than ribosomes and the initiator tRNA.Version 2.

19. 7. freeing the carboxyl end of the polypeptide The polypeptide is released through the exit tunnel of the large ribosomal subunit The ribosome releases the mRNA and dissociates into the small and large ribosomal subunits tRNA molecules are recycled and used to form new aminoacyl-tRNA Translation Initiation Factors RNA Small Ribosomal Subunit (30S) Initiator tRNAfMet Large Ribosomal Subunit (50S) Elongation Factors Aminoacyl-tRNA Peptidtyl-tRNA Deacylated tRNA Release Factors  Comparison of Prokaryotic Gene Expression vs Eukaryotic Gene Expression Enzymes Prokaryotes Eukaryotes 1 type of RNA polymerase Absence of GTF (sigma factor used instead) RNA polymerase contains 5 subunits and a sigma factor (6th) 3 types of RNA polymerases Presence of GTFs Absence of sigma factor. 11. with the aid of another tRNA. 14. Translocation 16. 20. 13. 12 subunits 47 | P a g e . allowing methionine to be transferred to the 2nd amino acid carried by the aminoacyl-tRNA at the A site. The small ribosomal subunit moves downstream in the 5’ to 3’ direction along the mRNA in search of the start codon. 22. The aminoacyl-tRNA in the A site now becomes a peptidyl-tRNA The ribosome is translocated one codon at a time in the 5’ to 3’ direction.5 Initiation 6. Elongation 12.Version 2. Peptidyl transferase in the large ribosomal subunit catalyzes the formation of a peptide bond between the carboxyl end of the methionine and the amino group of the 2nd amino acid The ester bond between methionine and the initiator tRNA is broken. 15. which signals the start of translation Initiator tRNA’s anticodon associates with the start codon on mRNA through complementary base pairing. 23. 18. guided by elongation factors using energy from the hydrolysis of GTP This relocates the deacylated tRNA to the E site where it diffuses out of the ribosome and repositions the peptidyl-tRNA at the P site The next codon on mRNA is exposed at the A site Steps 8 to 15 are repeated with new incoming aminoacyl-tRNAs until a stop codon (UAG. Termination 21. 25. UAA. Translation Initiation Factors aid the small ribosomal subunit in binding to the Shine-Dalgarno sequence in the 5’ UTR of the mRNA The Shine-Dalgarno sequence is recognized by the 16S rRNA of the small ribosomal subunit. Large ribosomal sub unit binds with the aid of another translation initiation factor. 10. 24. UGA) is encountered at the A site One of the 3 release factor binds to the stop codon in the A site. adding a water molecule to the C-terminus of the polypeptide chain This hydrolyzes the ester bond between the tRNA and the completed polypeptide in the P site. forming a prokaryotic 70S translation initiation complex Initiator tRNA sites in the P site of the ribosome and the initial methionine forms the N-terminus of the polypeptide Aminocyl-tRNAs are brought in by elongation factors using energy from the hydrolysis of GTP An aminoacyl-tRNA carrying the 2nd amino acid in the chain binds to the ribosomal A site via complementary base pairing between its anti-codon and the codon in the mRNA expose at the A site via hydrogen bonds. 8. 9. 17.

Version 2. or at an Internal Ribosome Entry Site Structure of mRNA Polycistronic Monocistronic Posttranslational modifications Limited Extensive (refer to 2C.5 Important Sequences in Transcription RNA polymerase recognition site Pribnow Box TATA box PostTranscriptional Modification Absent Present Ribosomal Stucture 70S (50S + 30S) 80S (60S + 40S) Binding of Ribosome Small Ribosomal Subunit binds to the Shine-Dalgarno sequence Small Ribosomal Subunit recognizes the 5’ cap Initiation of translation First start codon First or subsequent start codon.3 TRANSCRIPTION IN EUKARYOTIC CELLS  The process in which a complementary RNA copy is made under the direction of the template strand of a specific region of the DNA molecule.  Read in 3’ to 5’ direction  RNA synthesized in 5’ to 3’ direction  Template strand DNA sequence complementary to that of RNA  Termination Sequence  Codes for a polyadenylation signal sequence (AAUAAA)  Results in transcription terminaton  Is transcribed  RNA Polymerase  Enzyme comprising of several protein subunits  Found in the nucleoplasm  Synthesizes RNA using Ribonucleoside triphosphates as its substrate  Reads DNA template from the 3’ to 5’ direction 48 | P a g e . located 25bp upstream from the transcription start site  Serves as a binding site for a general transcription factor (TFIID)  Facilitates the binding of RNA polymerase  Determines which of the two strands of the DNA helix is used as the template strand  Not transcribed  Transcription Unit  The segment of DNA that is transcribed into a single-stranded RNA molecule  Only 1 of 2 strands of the DNA serves as the template strand for transcription. catalysed by RNA polymerase.4) 2C.  Gene  A section of the DNA that contains the information in the form of a specific sequence or nucleotides to direct the synthesis of one polypeptide chain or RNA  A unit of inheritances located on a locus on the chromosome  Promoter  RNA polymerase binding side  Transcription start site (whatever is upstream is not transcribed)  TATA box  TATAAAA sequence.

5  Catalyzes formation of phosphodiester bonds between the free 5’ phosphate group of the incoming ribonucleotide and the free 3’ hydroxyl group of the growing RNA polynucleotide chain  RNA synthesized from the 5’ to 3’ direction via complementary base pairing with the DNA template  Simultaneous transcription occurs  General Transcription Factors  A protein that is required for RNA polymerase molecules to bind to the promoter and initiate transcription  Assist in  Positioning RNA polymerase correctly at the promoter and assists it in binding to the promoter  Separate the 2 strands of DNA to allow transcription to begin  Release RNA polymerase from the promoter Eukaryotic mRNA Transcription Initiations 1. 13. dissociating the growing RNA chain from the template RNA polymerase carries out proofreading functions and removes incorrectly inserted ribonucleotides RNA polymerase transcribes a terminator sequence in the DNA. RNA polymerase continues transcription until about 10 to 35 nucleotides downstream of the polyadenylation signal sequence. Proteins bind to cut and free the pre-mRNA from RNA polymerase at this point. 8. 12. 2. 6. Enzyme/Proteins Involved General Transcription Factors. 10. 4. In eukaryotic cells. 3. 9. releasing the RNA chain and causes RNA polymerase to dissociate from the DNA. 15. The terminator sequence codes for a polyadenylation signal. Elongation 7. mRNA is thus synthesized in the 5’ to 3’ direction RNA polymerase reanneals the unwound DNA behind it. RNA polymerase moves along the template DNA in the 3’ to 5’ direction DNA double helix transiently unwinds at the same time Ribonucleotides are added via complementary base pairing with the DNA template strand Incoming NTPs lose a pyrophosphate group. 11. forming a transcription initiation complex. incl TFIID RNA Polymerase II Energy Requirement RNA polymerase In the form of NTP bond breakage Ribonucleoside Triphosphates (NTPs) RNA Polymerase Proteins to cleave mRNA at cleavage site 49 | P a g e . 16. Termination 14. TFIID binds to the TATA box RNA polymerase binds to the promoter and causes the two DNA strands to unwind by breaking hydrogen bonds between complementary base-pairs This forms a transcription bubble which exposes a short stretch of nucleotides on each strand One of the exposed DNA strands act as a template for complementary base pairing to direct the assembly of incoming ribonucleoside triphosphates (NTPs) RNA polymerase catalyzes the formation of the first phosphodiester bond. catalysed by RNA polymerase. The breakage of the pyrophosphate bond releases energy which is coupled to phosphodiester bond formation between the free 3' hydroxyl group of the terminal nucleotide of the growing mRNA and the free 5' phosphate group of the incoming ribonucleotide. General transcription factors assemble along the promoter and mediates the binding of RNA polymerase to the promoter. 5.Version 2.

Version 2.5 2C. Enzyme/Proteins Involved Spliceosome Energy Requirement Energy from the hydrolysis of ATP 50 | P a g e .4 POST-TRANSCRIPTIONAL MODIFICATION IN EUKARYOTIC CELLS  5' methylguanosine cap  Protects mRNA from degradation by nucleases (which cleave phosphodiester bonds) and phosphatases (which remove phosphate groups) from the 5’ end during its transport from the nucleus to the cytoplasm. 5' end of the new RNA molecule is attached to 7-methylguanosine triphosphate to form a 5’cap 2.  Signals the 5’ end of the mRNA serving as the assembly point to recruit the small subunit of the ribosome for translation to begin  Distinguishes mRNA from other types of RNA  3’ Poly(A) tail  Protects the mRNA from degradation by nucleases. mRNA is cleaved at the 5’ splice site and the intron joined to a branch point within the intron. thus making mRNA a more stable template for translation in the cytoplasm  Facilitates the export of mRNA out of the nucleus via nuclear pores  Exons  Protein-coding sequences in the gene  Introns  Non-coding sequences in the gene  Spliceosomes  Large complex consisting of several small nuclear ribonucleoproteins (snRNPs)  Each snRNP contains small nuclear RNAs (snRNAs) and asset of proteins Eukaryotic Post Transcriptional Modification Addition of 5' Methylguanosine Cap RNA Splicing 1.

known as the 3’ UTR (contains the polyadenylation signal) 51 | P a g e . introns are removed and remaining exons are spliced together to form mature mRNA The 3' end of the pre-mRNA is modified by adding ~200 adenine (A) nucleotides (AKA poly(A) tail) Mature mRNA is transported to the cytoplasm via nuclear pores. determine start and termination of translation using start (AUG) and stop codons. 4. The process repeats. recruit small ribosomal subunit.5 TRANSLATION IN EUKARYOTIC CELLS  The process in which a polypeptide chain is synthesized by ribosomes using genetic information encoded in an mRNA template  Mature Messenger RNA (mRNA)  Function: Acts as a template for translation. mRNA is cleaved at the 3’ splice site and exons are ligated together. Addition of 3' poly(A) tail 5.5 3. 6.  Protein-coding Region (sandwiched between start and stop codon)  Consists of a series of codons representing the amino acid sequence of the polypeptide  Untranslated regions  Additional sequence at the 5’ end known as the 5’ UTR and downstream of the stop codon. causing the intron to be excised as a lariat-like structure. Adenine Nucleotides Poly(A)-Polymerase 2C.Version 2.

held by complementary base-pairing within the single-stranded molecule  3 loops  On one of the loops. containing about 80 nucleotides  Ability of the anti-codon to base pair with an mRNA codon  Specific base pairing with ribosomes  Binds to a single specific amino acid determined by the anticodon sequence  A tRNA with an amino acid bounded to it is called an aminoacyl-tRNA  Secondary structure  2D Cloverleaf shape. catalyzes the formation of peptide bonds between amino acids  Translation factors  Initiations factors  Elongation Factors  Release factors 52 | P a g e .Version 2.  Ribosome  Characteristics  A large ribonucleoprotein complex comprised of ribosomal proteins and ribosomal RNA (rRNA)  Bacteria ribosomes are 70S while eukaryotic ribosomes are 80S  Structure  Small Subunit 40S  Contains an mRNA binding site  Large subunit 60S  A site (aminoacyl-tRNA site)  Holds the incoming tRNA carrying the next amino acid to be added  P site (Peptidyl-tRNA site)  Holds the tRNA carrying the growing polypeptide chain  E site (exit site)  Site of release of deacylated tRNA  Function  Provides an environment for the specific recognition between a codon of mRNA and an anticodon of tRNA  Holds the tRNA and mRNA in close proximity and positions the new amino acid for addition to the growing polypeptide  rRNA in the large ribosomal subunit has peptidyl transferase activity. the triplet base that binds to a specific mRNA codon via complementary base pairing  3’ End (CCA stem) of a tRNA molecule attached to a specific amino acid via an ester linkage  Tertiary Structure  Twisting and folding into a compact 3D L-shaped structure maintained by hydrogen bonds  Aminoacyl-tRNA synthetase  Responsible for linking amino acids to their corresponding tRNAs  About 20 different types.5  Transfer RNA (tRNA)  Serves as an adaptor molecule in the translation of an mRNA nucleotide sequence into the amino acid sequence in a polypeptide. one for each amino acid  Each type is able to recognize the specific anticodon sequence on the tRNA molecule as a well as a specific amino acid  Active site is complementary to the 3D conformation of the specific amino acid and anticodon sequence of the tRNA. 3 unpaired bases form an anticodon. brings in specific amino acid sequences  Properties  Small RNA molecules.

complexes with ribosomal proteins  Reads mRNA in the 5’ to 3’ direction.5  Polyribosomes  Ribosomes that aggregate in clusters  Simultaneous translation of polypeptides from the same mRNA strand  Increases the rate of translation  rRNA  Peptidyl transferase enzymatic activity which forms peptide bonds  Component of large and small ribosomal subunits.Version 2. searches for start codon  Aligns mRNA such that it pairs with tRNA in close proximity Initiation Elongation Termination 53 | P a g e .

The initiator tRNA has a unique anticodon loop that is distinct from that of tRNA and normally carries methionine 10. The small ribosomal subunit binds to the mRNA by recognizing it 5’ methylguanosine cap 8. Initiator tRNA’s anticodon associates with the start codon on mRNA through complementary base pairing. which signals the start of translation 9. Amino acid and ATP enters the active site of the enzyme Activation of 2. Peptidyl transferase in the large ribosomal subunit catalyzes the formation of a peptide bond between the carboxyl end of the methionine and the amino group of the 2nd amino acid 16.5 Eukaryotic mRNA Translation Amino Acid 1. Eukaryotic initiation factors dissociate using energy in the form of GTP 11. This relocates the deacylated tRNA to the E site where it diffuses out of the ribosome and repositions the peptidyl-tRNA at the P site 20. forming an aminoacyl-tRNA 5. The next codon on mRNA is exposed at the A site Enzyme/Proteins Involved Amino Acid Energy Requirement Energy in the form of ATP ATP tRNA Aminoacyl-tRNA Synthetase Eukaryotic Initiation Factors (eIF) Mature mRNA Small Ribosomal Subunit (40S) Initiator tRNA Energy in the form of GTP Large Ribosomal Subunit (60S) Elongation Factors Energy in the form of GTP Aminoacyl-tRNA Peptidtyl-tRNA Energy in the form of GTP Deacylated tRNA 54 | P a g e . The small ribosomal subunit moves downstream in the 5’ to 3’ direction along the mRNA in search of the start codon. Large ribosomal sub unit binds.Version 2. An aminoacyl-tRNA carrying the 2nd amino acid in the chain binds to the ribosomal A site via complementary base pairing between its anti-codon and the codon in the mRNA expose at the A site via hydrogen bonds. Aminoacyl-tRNA synthetase catalyzes the formation of a ester linkage between the amino acid and the 3’ CCA stem of the tRNA. Eukaryotic Initiation Factors bind to the small ribosomal subunit and position the initiator tRNA. to the P site 7. 15. The ribosome is translocated one codon at a time in the 5’ to 3’ direction. Initiator tRNA sites in the P site of the ribosome and the initial methionine forms the N-terminus of the polypeptide Elongation 13. The aminoacyl-tRNA is released from aminoacyl synthetase for translation Initiation 6. 17. Aminocyl-tRNAs are brought in by elongation factors using energy from the hydrolysis of GTP 14. guided by elongation factors using energy from the hydrolysis of GTP 19. The aminoacyl-tRNA in the A site now becomes a peptidyl-tRNA Translocation 18. allowing methionine to be transferred to the 2nd amino acid carried by the aminoacyl-tRNA at the A site. The ester bond between methionine and the initiator tRNA is broken. which carries a methionine. AMP is displaced by the incoming tRNA 4. Adenosine Monophosphate is joined to the amino acid tRNA using energy through the breaking down of the pyrophosphate bond 3. forming an eukaryotic 80S translation initiation complex 12.

each codon corresponding to an amino acid in the encoded protein. serine and tyrosine  Using Kinases and phosphatases  Attaching uniquitin (protein molecule)  Marks protein for proteolysis by the proteasome  At least 4 are required before proteolysis can take place  Addition of signal peptides  Structural changes  E. A release factor binds to the stop codon in the A site. Steps 8 to 15 are repeated with new incoming aminoacyl-tRNAs until a stop codon (UAG. Insulin 2C. lipids. The polypeptide is released through the exit tunnel of the large ribosomal subunit 25.5 21.g.  Consists of information in the form of 3 nucleotide bases (codons of mRNA). so that the protein can function.g. UAA. 55 | P a g e .Version 2. The ribosome releases the mRNA and dissociates into the small and large ribosomal subunits 26. This hydrolyzes the ester bond between the tRNA and the completed polypeptide in the P site.  Attaching to it biochemical functional groups (acetate. adding a water molecule to the C-terminus of the polypeptide chain 23. phosphate. methyl.7 THE GENETIC CODE  Is a list of codons of mRNA/ sequence of triplet bases in the non-template strand of DNA. carbohydrates.6 POST-TRANSLATIONAL MODIFICATION IN EUKARYOTIC CELLS  Function: To allow the newly translated polypeptide chain to coil and fold in a precise manner to assume the unique 3D conformation of a protein in the lumen of the rER or in the cytoplasm. Disulfide bonds  Proteolytic Cleavage  Cleaving a sequence of amino acids from the protein  E. tRNA molecules are recycled and used to form new aminoacyl-tRNA Release Factors 2C. UGA) is encountered at the A site Termination 22. etc)  Glycosylation  Addition of oligosaccharides to the protein to form glycoproteins  Reversible phosphorylation of threonine. freeing the carboxyl end of the polypeptide 24.

5  Only a triplet code contains enough combinations to code for 20 different amino acids. and the reading frame  Stop codon  UAA. same polypeptide has the same amino acid sequence)  Punctuation codons  Start codon  AUG. AUG and 3 serve as termination signals of polypeptide synthesis. UAG. thus the code is unambiguous  Most amino acids are encoded by degenerate codons that differ in the 3 rd position of the codon. codes for methionine  Defines the first amino acid. UAG.Version 2. UGA (stop codons)  Features  Triplet code  Each mRNA codon that specifies an amino acid in a polypeptide chain consists of 3 nucleotide bases  Linear code  Each mRNA codon is always read in the 5’ to 3’ direction  Universal code  Same code used by almost all organisms  Continuous and non-overlapping  Codon read as a triplet in the 5’ to 3’ direction  Nucleotides in the mRNA are read continuously as successive groups of 3 nucleotides without skipping any nucleotides  Degenerate. but unambiguous  A single amino acid is coded by more than 1 different codon  Only methionine (AUG) and typtophan (UGG) are coded for by a single codon each  Every codon codes for only 1 amino acid. Mutations can arise in this position of the codon without altering amino acid sequences (silent mutations)  Wobble base phenomenon  A single tRNA can recognize 2 or more degenerate codons in some situations  Wobble  Violation of the usual rules of base-pairing at the 3rd nucleotide of a codon is called a wobble  Base pairing at the 3rd base is not so specific (due to degenerate codons. UAA. 4 3 = 64 different codons  61 codons code for amino acids. inclusive of the start codon. UGA mark the end of protein synthesis  Not translated and do not code for any amino acid 56 | P a g e .

)  Classification by location  Germline Mutation  Occurs in germline cells  Hereditary. (KNOWLEDGE OF SUBSTITUTION. DELETION AND FRAMESHIFT MUTATION MAY BE REQUIRED. non-functional Mode Type of mutation In multiples of 3? Change in reading frame? Effects on mRNA Nucleotide Insertion & Deletion Missense Nonsense (adds a stop codon) Yes mRNA codes for extra amino acids mRNA codes for stop codon Effects on primary structure Addition of an amino acid Premature termination of polypeptide chain Effects on tertiary structure Effects on phenotype Alters 3D conformation Alters 3D conformation Alters function of protein Truncated protein. AND HENCE THE PHENOTYPE OF THE ORGANISM E. can be transmitted to the offspring  Somatic Mutation  Occurs in somatic cells  Non-hereditary Mode Nucleotide Substitution Type of mutation Substitutes Effects on mRNA Missense Effects on primary structure Changes an amino acid Effects on tertiary structure Effects on phenotype Alters 3D conformation Alters function of protein mRNA codes for a different amino acid Nonsense Silent A singe nucleotide mRNA codes for a mRNA codes for the stop codon same amino acid due to degeneracy of genetic code Premature termination of polypeptide chain Alters 3D conformation Truncated protein.G. premature termination of polypeptide chain Alters 3D Alters 3D conformation conformation Non-functional proteins Non-functional proteins 57 | P a g e . nonfunctional - Neutral mRNA codes for a different amino acid - Changes an amino acid to one with similar physical and chemical properties - - - Extensive missense Nonsense No Yes (Frameshift mutationv as mRNA read in series of non-overlapping codons) Downstream of Downstream of insertion site.Version 2. mRNA mRNA codes for codes for totally totally different different amino acids. insertion site. ADDITI ON. amino acids Generation of premature stop codon Changes totally Changes totally downstream of downstream of insertion insertion. SICKLE CELL ANAEMIA AND CYS TIC FIBROSIS.5 2D –EXPLAIN HOW A CHANGE IN THE SEQUENCE OF T HE DNA NUCLEOTIDE (GENE MUTATION) MAY AFFECT THE AMINO ACID SEQUENCE IN A PROTEIN.

Mutant haemoglobin subunits stick to each other when oxygen concentration is low. deletions  Base Analogues  Substances with molecular structures similar to bases  May be incorporated into DNA. resulting in mispairing between the parental template strand and daughter DNA strand  Parts of the DNA that are folded back are copied more than once (may result in gene duplication)  Induced mutations  Application of mutagens  X-Rays  Production of free radicals that interact with DNA.Version 2. producing double stranded breaks leading to chromosomal rearrangements and deletions.  DNA slippage  DNA strands can slip and fold back during DNA replication. or base pair substitutions. causing severe pain and cell death in the surrounding tissue Fragile and easily destroyed  Causes of mutagenesis  Spontaneous mutations  Occurs during DNA replication. resulting in thymine dimers that can block transcription and DNA replication. producing base substitutions  Base-modifying agents  Modify the chemical structure and property of bases. forming fibre like structures (within red blood cells) This causes red blood cells to lose their normal morphology and become sickle-shaped Less able to move through capillaries Blocks blood flow. recombination or repair  Causes: DNA polymerase inserting the wrong nucleotide  Processes that reduce the effects of this problem  Proofreading  Mismatch repair  Incorrectly paired nucleotides causes deformities in the secondary structure of the final DNA moleculre  Enzymes recognize and fix these deformities  Replication errors can fail to be recognized and passed down to the next cellular generation. leading to mispairing during DNA replication  base substitution  Intercalating agents  Inserts themselves between adjacent bases. creating a hydrophobic spot on the external surface of the protein structure which sticks to a hydrophobic region of another beta chain. resulting in a missense mutation Sixth amino acid residue changed from glutamate (hydrophilic) to valine (hydrophobic) Alters specific 3D conformation and function of the protein. leading to insertions or deletions during DNA replication 58 | P a g e . insertions.  UV Ray  Absorbed by deoxyribonucleotides.5  Sickle-Cell Anaemia Effects on mRNA Effects on primary structure Effects on tertiary structure Effects on phenotype Physiological effects Substitution of thymine for adenine in the haemoglobin gene.

a new cell is synthesized from an existing cell. but a new virion is never synthesized from existing virions  Replication in viruses involves the synthesis of a large number of viral components in host cells before those are assembled into virions  Viruses can direct metabolic processes  Viruses in their intracellular state can direct the metabolic life processes of cells. viral replication occurs  Viruses are thought to be degenerate cells that have retained the minima genetic information essential for reproduction 3A.  Virus genomes can evolve  Viruses differ greatly in their structural and genetic complexity and no single gene is shared by all viruses 59 | P a g e .1 WHY VIRUSES ARE OBLIGATE PARASITES  Viruses do not have  Enzymes for most metabolic processes  Protein synthesizing machinery  Energy in the form of ATP to drive protein and nucleic acid synthesis and cellular metabolism.  Building blocks such as nucleic acids and amino acids 3A.  Cells that viruses infect are called host cells  In their intracellular states.5 3 – GENETICS OF VIRUSES AND BACTERIA 3A – DISCUSS WHETHER VIRUSES ARE LIVING OR NON-LIVING ORGANISMS AND EXPLAIN WHY VIRUSES ARE OBLIGATE PARASITES  Viruses are termed virions in their inactive extracellular states  Virions are metabolically inert  Structure by which the virus genome is carried from one cell to another.2 DISCUSS WHETHER VIRUSES ARE LIVING OR NON-LIVING ORGANISMS  Characteristics of living things  Movement  Excretion  Respiration  Irritability (response)  Growth  Reproduction  Adaptability  Nutrition (process of absorbing nutrients)  Arguments for viruses being living organisms  Viruses can reproduce  Viruses can reproduce In the intracellular state only  In cells. they can parasitize them from host cells and are thus dependent on their host cells.  Although viruses are unable to obtain nutrients by themselves.Version 2.

helical) depending on the way the protein subunits are arranged  Envelope (only found in envelope viruses.  Can be double stranded (ds) or single stranded (ss)  Capsid  Protein capsid which surrounds/encloses the viral genome  Each capsid is constructed from capsomeres (identical protein subunits)  Capsids have various shapes (icosahedral. evidently showing that viruses evolved polyphyletically and have many evolutionary origins. RNA Replicase 60 | P a g e .5  Viral genomes can be either double (ds) or single stranded (ss) RNA or DNA. do not require nutrition. are not irritable. which mostly infect animal cells)  The envelope is derived from the host cell surface membrane when the virus buds off  The envelope contains viral glycoproteins which help the virus attach itself to the host cell  The envelope protects the viral genome from the effects of various genomes and chemicals  Naked viruses  Viruses without an envelope  Other proteins (vary among viruses)  E. all the DNA in the virions is linear.g. and neither grow nor excrete  Conclusion  Viruses are infection particles which are active in their intracellular state but inactive in the virion state 3B – DESCRIBE THE STRUCTURAL COMPONENTS OF VIRUSES  Basic structure of a virus (20-300nm)  Viral Genome  Contains either RNA or DNA  Can be circular or linear (for the purposes of the viruses named below.Version 2.  Viruses evolve with their host and acquire their metabolic and translational genes from their host cells  Genetic recombination/mutations can cause the viral genome to evolve further  Arguments for viruses being non-living organisms  Viruses are not cells  Viruses lack a protoplasm or cell organelles. They generally contain a protein capside surrounding a nucleic acid core. Reverse Transcriptase. enveloped viruses. Some viruses. thus they do not share a common ancestor. have a membranous envelope surrounding the capsid  Viruses lack other characteristic of living organisms  Viruses are unable to carry out metabolism.

II. 4. T4 PHAGE. 3C. AN ENVELOPED VIRUS E. IV.5 3C – DESCRIBE THE REPRODUCTIVE CYCLES BACTERIOPHAGES THAT REPRODUCE VIA A BACTERIOPHAGES THAT REPRODUCE VIA A III. INFLUENZA. RETROVIRUSES E. contained in the head of the phage  Long Tail & Tail Sheath  Consists of a tail sheath which surrounds a central tube  The tail sheath contracts during penetration to thrust the central tube through the host membrane  Multiple Tail Fibres  Allows the phage to adsorb onto the surface of the bacterial cell by binding to the appropriate receptor site found on the cell surface. OF THE FOLLOWING VIRUS TYPES:I.G. HIV. LYSOGENIC CYCLE. E.Version 2. 5. LAMBDA PHAGE. through the central tube and into the host cells.  Reproductive Cycle – Lytic Cycle 61 | P a g e . enabling the base plate to come into contact with the surface of the cell  Base Plate  Comes into contact with the host cell surface and undergoes a conformational change to allow DNA to be extruded from the head. Adsorption Penetration Synthesis and Replication Assembly Release 3C.g.2 LYTIC REPRODUCTIVE CYCLE OF T4 PHAGE (A BACTERIOPHAGE)  Bacteriophages are viruses that infect bacteria (ALL bacteriophages have dsDNA)  E. T4 Phage – A virulent phage (undergoes lytic cycle)  Structure of T4 Phage  Viral Genome  Linear dsDNA  Capsid  Capsomeres surround the nucleic acid. 2. E.1 GENERAL REPRODUCTIVE CYCLES OF VIRUSES 1.G.G.G. 3. LYTIC CYCLE.

6.5  The lytic cycle results in the death of the host cell and the release of new T4 phages Lytic Cycle of T4 Phage Adsorption 1. 8. 15. The base plate comes into contact with the surface of the bacterial host cell. The multiple tail fibres of the T4 phage attach to specific receptor sites on the surface of a bacterial host cell. 4. 7. tRNAs and translation factors) into viral proteins. tails and tail fibres. Assembly 11. Synthesis and Replication 5. 2. 10. causing it to contract and thrust the central tube through the host membrane DNA is extruded from the head through the central tube and injected into the host cell passing through the cell wall and cell membrane (The capsid is left on the outside of the bacterial cell wall) Synthesis of host DNA. New T4 phages are released Proteins Involved T4 Phage tail fibres Base Plate Tail sheath Translational Machinery RNA Polymerase DNA Polymerase Viral Proteins Lysozyme 62 | P a g e . enzymes and inhibitory factors that stop host cell RNA and protein synthesis Three separate sets of viral proteins are assembled to form phage heads. 9. Penetration 3.Version 2. The tail sheath undergoes a conformational change. 13. RNA and proteins is halted The host cell machinery is taken over for nucleic acid and viral protein synthesis The host DNA is degraded into nucleic acids which are used to for phage DNA replication Host RNA polymerase synthesizes T4 phage mRNAs via transcription using T4 phage DNA The phage mRNA are translated by translational machinery (ribosomes. The different components are assembled into the bacteriophage The T4 phage lyses the host cell using the enzyme lysozyme. 14. which digests the bacteria cell wall Water enters the bacteria by osmosis causing the cell to swell and burst. Release 12.

In this integrated state. DNA is extruded from the head through the central tube and injected into the host cell passing through the cell wall and cell membrane 5.5 3C.Version 2. enzymes and inhibitory factors that stop host cell RNA and protein synthesis Assembly 15. RNA and proteins is halted Replication 11.  Reproductive cycle – Lysogenic Cycle  Lamda phage is capable of using both the lysogenic and lytic modes of reproductive within a bacterium. An environmental trigger causes the virus to switch from the Induction lysogenic cycle to the lytic cycle 9. allowing the lamda phage to exit from the bacterial chromosome Lamda phage then transits to the lytic cycle Lytic Cycle of λ Phage Synthesis and 10.A temperate phage  Structure of Lambda Phage  Viral Genome  Linear dsDNA  Capsid  Capsomeres surround the nucleic acid.  An environmental trigger switches the phage from the lysogenic cycle to the lytic cycle Lysogenic Cycle of λ Phage Adsorption 1.3 LYSOGENIC (& LYTIC) REPRODUCTIVE CYCLE OF LAMDA PHAGE  Lamda Phage . Spontaneous 8. The lamda phage genome circularizes and inserts itself into a Formation prophage insertion site on the bacterial chromosome via genetic recombination. (The capsid is left on the outside of the bacterial cell wall) Prophage 6. The different components are assembled into the bacteriophage Proteins Involved λ Phage tail fibres Base Plate Tail sheath Translational Machinery RNA Polymerase DNA Polymerase Viral Proteins 63 | P a g e . and is passed on to generations of daughter cells. Lysis genes repressed during lysogeny are activated. 7. Two separate sets of viral proteins are assembled to form phage heads and tail fibres. contained in the head of the phage  A single tail fibre  Allows the phage to adsorb onto the surface of the bacterial cell by binding to the appropriate receptor site found on the cell surface. Host RNA polymerase synthesizes λ phage mRNAs via transcription using λ phage DNA 14. The host cell machinery is taken over for nucleic acid and viral protein synthesis 12. 16. tRNAs and translation factors) into viral proteins. Synthesis of host DNA. 2. Penetration 3. The tail sheath undergoes a conformational change. The base plate comes into contact with the surface of the bacterial host cell. the viral DNA is replicated along with the chromosome each time the host cell divides. causing it to contract and thrust the central tube through the host membrane 4. The phage mRNA are translated by translational machinery (ribosomes. The tail fibre of the lada phage attaches to specific receptor sites on the surface of a bacterial host cell. The host DNA is degraded into nucleic acids which are used to for phage DNA replication 13.

4 REPRODUCTIVE CYCLE OF AN ENVELOPED VIRUS (INFLUENZA VIRUS)  Influenza virus – an enveloped virus in which the viral ssRNA is present in the virion in 8 separate pieces  The nucleocapsides are surrounded by membranous envelope  The ssRNA is the (-) sense type. thus it has to be translated into the (+) sense before it can be used as an mRNA  Influenza virus structure  Viral Genome 64 | P a g e . which digests the bacteria cell wall 18. The lamda phage lyses the host cell using the enzyme lysozyme. Water enters the bacteria by osmosis causing the cell to swell and burst. New lamda phages are released Lysozyme 3C.5 Release 17. 19.Version 2.

PB2. Rough Endoplasmic Reticulum bound ribosomes synthesize viral transmembrane surface glycoproteins which are glycosylated in the golgi apparatus and then incorporated into the host cell membrane via vesicles. Assembly 10. The (+) sense RNA is translated in the cytoplasm by host protein synthesizing machinery. lowering the pH of the vesicle. PA  Forms RNA replicase responsible for transcribing the (+) sense ssRNA from (-) sense ssRNA  RNA Replicase not found in host cells  NS1  Regulates viral replication mechanisms and cell signalling pathways Reproductive Cycle of Influenza Virus Adsorption 1. The hemagglutinin protein changes its conformation and causes the viral envelope and the endosome membrane to fuse. Synthesis and 5. 3. releasing the 8 viral segments of the influenza genome into the host cell cytoplasm. The (+) sense RNA is used as a template by RNA replicase to transcribe more full-length (-) sense viral ssRNAs to be packaged into new viral particles 7.5       8 different segments of (-) sense ssRNA Capsid  Nucleoproteins form a nucleocapsid which associates with the nucleic acid Viral Envelope  Phospholipid bilayer obtained from the host cell upon budding Surface glycoproteins  Hemagglutinin (HA)  Binds to sialic-acid containing receptors  Helps to attach the virus to the receptor on the host cell membrane  Neuraminidase  Hydrolyses mucus allowing the virus to enter cells of the respiratory tract  Facilitates budding by cleaving sialic-acid containing receptors Protein Envelope (made up of matrix protein M1 and M2)  M1 – Monomers of the protein envelope  M2 – acts as ion channel to lower or maintain the pH of the endosome Enzymes  PB1.Version 2. The virus is taken in by receptor mediated endocytosis. forming an endosome within the host cell with the influenza virus attached to its inner surface. PB2 and PA are packaged together. Influenza virus adsorb onto epithelial cells of the respiratory tract when hemagglutinin molecules on the viral membrane binds to sialic acid containing receptors. The endosome fuses with a lysosome. 8. 4. All 8 nucleocapsides ((-) sense viral ssRNAs aassociated with nucleoproteins) and enzymes PB1. Viral RNA replicase transcribes a (+) sense RNA using the (-) sense Replication RNA as a template 6. Free cytoplasmic ribosomes are used to synthesize enzymes. Penetration 2. matrix proteins and capsomeres which are eventually folded into their precise 3D conformation in the cytoplasm and packaged into the virion 9. Proteins Involved Hemagglutinin Sialic-acid containing receptors Hemagglutinin M2 Ion Channel RNA-dependent RNA polymerase Translational Machinery 65 | P a g e .

HA binds to sialic acid containing cellular receptors on the host cell membrane. NA and M2 studded membrane envelope at the same time. 12. **RNA replicase = RNA dependent RNA polymerase Hemagglutinin Sialic-acid containing receptors Neuraminidase *Note that the stage that uses (+) ssRNA which is used as a template to synthesize more (-) ssRNA is known as replication 66 | P a g e . releasing the virion. The virus is released from the host cell by budding.Version 2.5 Release 11. Neuraminidase cleaves sialic-acid residues on the cellular receptor. acquiring the HA. Host cell membrane with the inserted glycoproteins surround viral RNA and viral proteins. 13. causing the new viral particle to remain attached to the host cell 14.

formed from matrix proteins Enzymes  Reverse transcriptase  2 ribonucleoproteins  Reverse transcribes viral RNA into DNA  Integrase  Incorporates the dsDNA obtained from viral RNA into the host genome 67 | P a g e .5 *The stage where the (-) viral ssRNA is used as a template to transcribe (+) viral ssRNA is known as transcription 3C. retrovirus that replicates using a DNA intermediate synthesized by the enzyme reverse transcriptase  HIV Virus structure       Genome  Two identical ssRNA Capsid  Capsid surrounds the nucleic acids Viral envelope  Phospholipid bilayer obtained from host cell upon budding Surface glycoproteins  gp120  Binds to CD4 receptors on white blood cells (macrophage & T helper cells)  gp41  Aids in the fusion of the HIV envelope and the host cell membrane Protein Coat  A second layer of protein envelope enclosing the capsid.Version 2.5 REPRODUCTIVE CYCLE OF A RETROVIRUS (HUMAN IMMUNODEFICIENCY VIRUS)  HIV virus – An enveloped.

Proteins Involved Glycoprotein gp120 CD4 receptor Glycoprotein gp120 Co-receptor CXCR-4 Co-receptor CCR-5 Reverse Transcriptase Integrase Host cell RNA polymerase Ribosomes Viral glycoproteins Capsomeres HIV Protease 68 | P a g e . Glycoprotein gp120 binds to CD4. 5. gp120 undergoes a conformational change. Integrase catalyzes the incorporation of viral dsDNA into the host genome. matrix proteins and capsomeres which are eventually folded into their precise 3D conformation in the cytoplasm and packaged into the virion 12. Capsomeres surround two ssRNA along with enzymes. forming a mature HIV virion. releasing the viral contents (consisting of nucleic acids and enzymes) into the cells Latency Stage 4. The HIV envelope fuses with the host cell membrane. 11. A signal is received by the host cell. 9. allowing it to bind to a co-receptor CXCR-4 on the surface of T helper cells or CCR-5 on the surface of macrophages 3. a receptor found on the surface of T helper cells and macrophages (of the host immune system) Penetration 2. causing the provirus to be Replication transcribed by the host cell RNA polymerase into viral mRNA and genomic viral RNA. forming a provirus. forming a dsDNA 7. and assemble to form a viral capsid. The provirus remains in a latent state for several years and does not produce HIV. Free cytoplasmic ribosomes are used to synthesize enzymes. Copies of viral proteins and genomic viral RNA assembles near the host cell membrane 14. The remaining DNA strand is then used by reverse transcriptase as a template to synthesize the complementary DNA strand. Reverse transcriptase reverse transcribes a DNA strand using the viral RNA as a complementary template. Rough Endoplasmic Reticulum bound ribosomes synthesize viral transmembrane surface glycoproteins which are glycosylated in the golgi apparatus and then incorporated into the host cell membrane via vesicles. Release 15. Assembly 13. The viral capsid buds off from the host cell and obtains the viral glycoprotein studded membrane envelope. 17. integrase and protease. Host cell membrane with the inserted glycoproteins surround viral RNA and viral proteins.5  Protease  Cleaves viral polypeptides into functional proteins during viral maturation Reproductive Cycle of Human Immunodeficiency Virus Adsorption 1. 16. HIV protease cleaves the long chains of HIV proteins into smaller functional proteins. The RNA strand of the RNA-DNA hybrid is broken down by reverse transcriptase 6. The dsDNA passes through the nuclear pore into the nucleus 8. such as reverse transcriptase. Synthesis and 10.Version 2.

This causes the epithelial lining to be lost 9. 3.5 3D – EXPLAIN HOW VIRAL INFECTIONS CAUSE DISEASE IN ANIMALS. shortness of breath occurs 69 | P a g e . HIV AND T HELPER CELLS [DETAILS OF THE IMMUNE SYSTEM ARE NOT REQUIRED].G. INFLUENZA AND EPITHELIAL CELLS OF THE RESPIRATORY TRACT).Version 2. Excessive budding of new virions deplete the host cell of its cell membrane 7. Excessive mucus production and swelling due to the irritation 10. influenza 2. Symptoms such as sore throat. THROUGH THE DISRUPTION OF HOST TISSUE AND FUNCTIONS (E. ATP 6.G. 3D. 5.2 PATHOGENESIS OF INFLUENZA Pathogenesis of influenza Pathogenesis of 1. Immune system recognizes the viral glycoproteins and destroys the epithelial cells 8. 4. Influenza virus spreads to a new host via respiratory droplets Virus adsorbs on the surface of epithelial cells in the respiratory tract The viral genome penetrates into the host cell Viral replication begins (peaks at 48hrs after infection) Production of virions deplete host cell of essential raw materials such as nucleic acids. amino acids. E. MAMMALS. high fever.

E6 binds to the p53 and causes it to be degraded  HPV infection thus leads to genetic instability  HPV infection is a lifelong infection as integrated viral genomes are not cleared 3D. genetic recombination between the viral genome and host genome may occur. Host immune system targets and destroys infected T cells Latency Phase 5. Host immune system targets and destroys infected T cells 9.Version 2.  Human Papilloma Virus can cause cervical cancer  HPV proteins such as E6 and E7 act as oncogenes and promote tumour growth.5 3D.  During the integration of the viral genome into the host genome. the provirus is activated and viral replication occurs 8. 70 | P a g e .4 VIRAL STRAINS  A viral strain: A virus with a similar but non-identical genome as compared to the original strain of viruses. Integration of the virus into the host genome allows infected T cells to evade the immune system 6. Upon the stimulation of an immune response. Unaware host continues to pass on the virus to other hosts via sexual contact Symptomatic Phase 7. Destruction of the T cells leads to a compromised immune system 10. causing them to produce new viral particles 4. HIV spreads through sexual contact Virus travels through the blood stream and adsorbs on the surface of T helper cells Acute Phase 3.3 VIRUSES AND CANCER  Viruses can contribute to cancer by disrupting genomic sequences when the viral genome is integrated into the host genome. The host is unable to initiate a proper immune response against common infections **T helper cells do not divide.  During the integration of the viral genome into the host genome.  Causes  Point mutations (single nucleotide substitution) that occurs during the synthesis of new viral genomes results in different subtypes of various viral proteins (such as hemagglutinin and neuraminidase in influenze)  When two viral strains infect a cell at the same time. 2. resulting in new recombinant viral genomes that code of subtypes of viral proteins. leading to cancer. insertional mutagenesis may occur. The virus penetrates into the T helper cells.2 PATHOGENESIS OF HIV Pathogenesis of HIV Spreading 1. unlike in the lysogenic cycle 3D. Cancer critical genes can become mutated. random packaging viral (-) ssRNA from into new virions can result in the assembly of new subtypes of viral proteins.

 Organization  Genes grouped as operons. vicious layer formed using polysaccharides  Allows bacteria to cause diseases  Cell Wall  Composed of peptidoglycan (complex molecule comprising of amino acids and polysaccharides  Outer membrane present in gram-negative bacteria  Cell membrane  Flagellum  Pilus  Long. chloroplast/mitochondria. gene transfer and gene expression  Circular bacteria chromosome  Only 1 per bacteria cell  Contains essential genes  Compaction  The chromosome associates with positively charged histone proteins that compact the DNA into looped domains. 3E. centromeres  Plasmids  A few per bacteria cell  Small.  Genetic material is used in replication. thin appendages that a bacteria uses to transfer plasmids to another bacteria during conjugation  Cytoplasmic Structures  Ribosomes  70s ribosomes  No membrane bound organelles (no nucleus. and can be stored in either circular bacteria chromosome or plasmids. endoplasmic reticulum.1 BACTERIA CELL MORPHOLOGY  Shape  Coccus (sphere)  Bacilli (rod)  Spirochetes (spiral)  Cell Structure  Cell surface  Capsule  Thick. transformation and transduction 71 | P a g e .2 PROKARYOTIC GENOME  Genetic material is concentrated in the nucleoid. golgi apparatus) 3E.Version 2. telomeres. multiple genes under the control of 1 operator  Genes have no introns. circular extrachromosomal DNA  Can be passed to other bacteria cells via conjugation.5 3E – DESCRIBE THE STRUCTURE OF A BACTERIAL CHROMOSOME INCLUDING THE ARRANGEMENT OF DNA WITHIN BACTERIAL CELLS.  Looped domains are then supercoiled to achieve further compaction.

transduction and conjugation 72 | P a g e . 3F – DESCRIBE THE PROCESS OF BINARY FISSION. TRANSFORMATION.Version 2.1 BINARY FISSION  A form of asexual reproduction  Produces 2 genetically identical daughter cells  Includes replication of the bacteria chromosome  Does not give rise to genetic variation Binary Fission in Prokaryotes DNA Replication 1. 3. ability to ue a new metabolite. 2. TRANSDUCTION AND CONJUGATION INBACTERIA AND EXPLAIN THE ROLE OF F PLASMIDS IN BACTERIAL CONJUGATION. (KNOWLEDGE OF HFR IS NOT REQUIRED. Each bacterial chromosome now attaches to the cell membrane. The cell elongates. causing the 2 chromosomes to be moved apart. Cell Division 5.2 HORIZONTAL GENE TRANSFER  Involves a donor cell and a recipient cell  Allows for DNA to be transferred from one cell to another via transformation.5   Possess their own origin of replication Contain beneficial genes which confer a selective advantage to the bacteria such as antibiotic resistance. The bi-directional DNA replication begins at the origin of replication. etc. enzyme production. forming a replication bubble when the 2 DNA strands separate. The septal ring directs the assembly of the division septum 8. Each parental strand is used as a template for the synthesis of the daughter strand in a semi-conservative manner. This causes the bacteria to be split into 2 genetically identical cells each containing a chromosome via cytokinesis Molecules Involved Bacterial Chromosome Septal ring Septum 3F. 7. Cell wall and cell membrane materials are added to the septum. The bacterial chromosome attaches to the plasma membrane. This causes the replication bubble to grow bidirectionally away from the origin of replication until the entire bacterial chromosome is replicated.) 3F. 4. causing the cell membrane to invaginate. 6. toxin synthesis. 9.

5. homologous recombination takes place where the transferred part of the donor chromosome will be exchanged with a portion of the chromosome of the recipient cell that is very similar in sequence  Successful gene transfer thus takes place between bacteria of closely related species with similar gene sequences. A competent recipient cell takes up one or more donor DNA fragments into its cytoplasm via its competence factor. The donor bacterial cell lyses and releases naked DNA fragments. Homologous recombination of the donor DNA fragment takes place with a homologous section of the recipient cell’s chromosome The homologous segment of the donor cell’s DNA is integrated into the recipient cell’s chromosome. 4. caused by aberrations in the phage reproductive cycle  2 forms: Generalized transduction and specialized transduction  Generalized Transduction  The incorporation of a random fragment of the bacterial genome into another cell via a virulent bacteriophage  Occurs due to the accidental incorporation of a random fragment of DNA from its host cell  Each portion of the bacterial genome (any bacterial gene) has approximately the same probability of being transferred from donor to recipient. 3. homologous recombination does not take place as only a plasmid is transferred.5  In transduction and transformations.  Transformation – The process by which a recipient cell takes up small fragments of naked DNA from the surrounding environment  The DNA either arises from artificially constructed plasmids or a donor bacterial cell which lyses and releases its DNA into the surrounding environment  Only competent bacterial cell are able to naturally undergo transformation  Depends on competence factors. The recipient cell is now a recombinant cell Molecules Involved Donor DNA fragments Competence factor  Transduction – The transfer of genetic material via bacteriophages.  Recombinant cells  Cells that have undergone homologous recombination  In conjugation.Version 2.  Involves a virulent phage that carries out the lytic cycle 73 | P a g e . 2. cell surface proteins that bind to DNA fragments and aid in their uptake  Artificial transformation involves the addition of bacteria and artificial plasmids followed by electroporation/heat shock + CaCl2 Transformation Natural Transformation 1.

It also uses the host cell’s protein synthesizing machinery to make phage proteins 3. 7.5 Transduction Generalized Transduction  1. and new phages are released into the environment. 5. The donor DNA is incorporated into the second host cell’s genome by either homologous recombination or prophage integration (if viral genes contain the genes required to enter prophage).Version 2. the phage genome is excised from the first host cell’s chromosome. Components Involved Phage DNA Prophage Genes near the prophage integration site Phage Virion 74 | P a g e . During induction. forming a defective phage. a small piece of the first host cell’s degraded DNA is accidentally packaged within a phage capsid in place of the phage genome during the assembly stage of the lytic cycle. 2. Transduction Specialized Transduction 1. Due to imprecise excision. segments of the phage DNA that lack part of the normal phage genome and contain part of the bacterial chromosome located adjacent to the prophage attachment site get excised as well. forming a recombinant cell Specialized Transduction  The incorporation of a genes near the prophage insertion site into another cell via a temperate bacteriophage  Occurs due to the imprecise excision of the prophage. Components Involved Phage DNA First host cell’s DNA Phage Virion The virulent phage injects its DNA into its first host bacterial cell and the first host cell’s chromosome is degraded into DNA fragments. 6. The phage makes use of the host’s DNA replication machinery to synthesize more DNA. The phage makes use of the host’s DNA replication machinery to synthesize more phage DNA. and new phages are released into the environment. 6. The phage genome is integrated as prophage at the prophage insertion site. 2. The first host bacterium is lysed. 5. the phage DNA sometimes takes with it a small region of the bacterial DNA that was adjacent to the prophage excision site. The temperate phage injects its DNA into its first host bacterial cell.  All the released virions contain part of the bacteria DNA. 4. It also uses the host cell’s protein synthesizing machinery to make phage proteins Each newly formed phage now contains part of the first host cell’s DNA. 4. The first host bacterium is lysed. Occasionally. The donor DNA is incorporated into the second host cell’s genome via homologous recombination. The defective phage which contains the first host cell’s DNA infects a second host cell and injects the first host cell’s DNA fragment into it. forming a recombinant cell. 8. 3. The defective phage which contains the first host cell’s DNA infects a second host cell and injects the first host cell’s DNA fragment into it.

 The F factor replicates as it is transferred allowing both the donor and recipient cell to have an F factor after conjugation 75 | P a g e . converts to lytic upon induction Transfer of donor DNA Any donor bacterial gene Only donor bacterial genes near the prophage insertion site  Conjugation – The transfer of genetic material from the donor to the recipient cell via a cytoplasmic bridge established by direct contact between the 2 cells. the F plasmid has its own origin of replication.Version 2.5  Comparison between specialized and generalized transduction Generalized Transduction Specialized Transduction Type of Phage Virulent phage Temperate phage Phage reproductive cycle Lytic Lysogenic.  As a plasmid.  The presence of an F plasmid containing the F factor allows the bacteria to synthesize the sex pilus and transfer the F factor from a donor cell to a recipient cell.

4.recipient cell. but excludes lacI). 6. Each parental strand serves as a template for the synthesis of a complementary daughter strand in the respective cell via semiconservative rep0lication. 5.cell Mode of transfer Competent factors required to uptake DNA Via phage virions Sex pilus followed by cytoplasmic mating bridge 3G – DISTINGUISH BETWEEN STRUCTURAL AND REGULATORY GENES. 2. lacZ.Version 2. 3. 9.5 Conjugation Conjugation 1. which it uses to attach to an F. The sex pilus retracts.g. lacA. The nicked strand separates from its complementary strand and moves to the F. 8. 76 | P a g e . 7. catalyzed by DNA polymerase. From the syllabus – A structural gene is a region of DNA that codes for a protein or RNA molecule that forms part of a structure or has an enzymatic function (e.g. The sugar-phosphate backbone of one strand of the F plasmid in the F+ donor cell is nicked by an endonuclease. DNA ligase ligates the 2 ends of the daughter strand together. Upon completion. The F+ donor cell synthesizes a sex pilus.recipient cell via the cytoplasmic mating bridge. forming 2 bacterial cells that are both F+ Components Involved F factor in F plasmid Sex Pilus Cytoplasmic Mating Bridge Endonuclease DNA Polymerase DNA Ligase  Comparison of the 3 processes Transformation Transduction Conjugation DNA that is transferred Degraded naked DNA fragments Degraded fragments or excised portions of bacterial DNA F plasmid Donor Cells Dead/lysed cells Bacteria infected with bacteriophages F+ cell Recipient Cell Competent cells Competent Cells susceptible to the same bacteriophage F. pulling the 2 cells together. providing a route for DNA transfer. lacI). lacY. A cytoplasmic mating bridge is formed between the 2 cells. Each bacterial cell now has a full copy of the F plasmid The cells move apart. A regulatory gene codes for a specific protein product that regulates the expression of the structural genes (e.

lacY and lacA gene  lacZ codes for β-galactosidase which breaks down lactose to glucose and galactose  lacY codes for galactoside permease. which acts as an inducer  Code for enzymes involved in catabolic pathways  E. non-template strand)  Operator – Allows for the binding of regulatory proteins  Leader sequence (“5’ UTR”)  Contains the Shine-Dalgarno sequence used for ribosomal binding  Coding Region  Begins with the start codon AUG and ends with the stop codon  NO introns  Trailer sequence (“3’UTR”)  Terminator sequence  Dislodges RNA polymerase from the template DNA  Operon – The grouping of bacterial structural genes with related functions such that they are located adjacent to each other and are placed under the control of the same promoter and regulatory regions  Ensures the required proteins are produced in sync 3H – DISTINGUISH BETWEEN THE CONCEPT OF REPRESSIBLE AND INDUCIBLE SYSTEMS OF GENE REGULATION USING TRP AND LAC OPERON AS EXAMPLES RESPECTIVELY (ATTENUATION OF TRP OPERON IS NOT REQUIRED). which transports lactose into the bacteria  lacA codes for transacetylase (unknown function) 77 | P a g e . lac operon  Contains the lacZ. 3H.1 INDUCIBLE OPERONS  Characteristics  Normally inactive  Turned on by the substrate of the enzyme.g. non-template strand)  Pribnow box/RNA Polymerase binding site (5’-TATAAT-3’.Version 2.1 STRUCTURE OF PROKARYOTIC GENES  Parts  Promoter – Allows for the recognition and binding of RNA polymerase  RNA Polymerase recognition site (5’-TTGAGC-3’.5 3G.

thus inhibiting transcription. The repressor protein is synthesized by transcribing the lacI gene 2. This allows RNA polymerase to bind to the promoter and transcribe the lac operon 5. In the absence of lactose. In the presence of lactose.Version 2. Components lacI lac repressr protein lac operator RNA polymerase lacI lac repressr protein lac operator Allolactose RNA polymerase β-galactosidase Galactoside permease Transacetylase 78 | P a g e . the lac repressor protein assumes an active conformation and binds to the operator sequence of the operon as it is complementary in conformation to the lac operator sequence. The repressor protein is synthesized by transcribing the lacI gene lactose 2. 4. allolactose acts as an inducer and binds to the repressor protein. 3. This prevents RNA polymerase from binding to the promoter. changing its conformation to an inactive state. The operon is switched on and enzymes are produced to hydrolyze lactose. The operon is switched off and no enzymes are produced. The lac repressor protein assumes an inactive conformation and does not binds to the operator sequence of the operon as it is not complementary in conformation to the lac operator sequence. Presence of 1.5  Negative regulation by the lac repressor  Allows the enzymes to be produced only when necessary (in the presence of their substrate)  The lac repressor is coded by the lacI gene (a regulatory gene with its own promoter) Negative Regulation of the lac operon Absence of lactose 1. 3. 4.

presence 2. CAP assumes an active conformation and detaches from its binding site in the lac operon as it is not complementary in conformation to the binding site sequence.5  Positive regulation by the lac repressor (catabolite repression)  Prevents the enzymes from being produced when glucose is present. Absence of 1. 3. Components cAMP CAP RNA polymerase cAMP CAP RNA polymerase 3H. cAMP concentration increases in the absence of glucose glucose. This downregulates the rate of transcription.g. cAMP concentration falls in the presence of glucose glucose.Version 2. which functions as a co-repressor  Code for enzymes involved in anabolic pathways  E. 3. Due to a lack of binding between the catabolite activator protein of lactose and cAMP.2 REPRESSIBLE OPERONS  Characteristics  Normally active  Turned off by the end product of the metabolic pathway. presence 2. cAMP binds to the catabolite activator protein causes CAP to of lactose assume an inactive conformation and bind to its binding site in the lac operon as it is complementary in conformation to the binding site sequence. This enhances the binding of RNA polymerase to the promoter and upregulates the rate of transcription. even if lactose is present along with glucose  Glucose is preferentially utilized over lactose  Uses a catabolite activator protein (CAP) to increase the production of the 3 enzymes in the absence of glucose Positive Regulation of the lac operon Presence of 1. trp operon 79 | P a g e .

The repressor protein is synthesized by transcribing the trpR gene Tryptophan 2. the tryptophan acts as a corepressor ad binds to the trp repressor protein 3.5   Contains the trpA. 5. trpD and trpE gene  Produces enzymes that are involved in the synthesis of tryptophan Under the control of the repressor protein  The end product represses the synthesis of enzymes in this pathway (in the presence of their substrate)  Allows the bacteria to allocate its organic precursors and energy for other uses  The trp repressor is coded by the trpR gene (a regulatory gene with its own promoter) Regulation of the trp operon Absence of 1. In the absence of tryptophan. This prevents RNA polymerase from binding to the promoter and transcribing the trp operon. This allows RNA polymerase to bind to the promoter and transcribe the trp operon. trpC. The trp repressor protein assumes an active conformation and binds to the operator sequence of the operon as it is complementary in conformation to the lac operator sequence. trpB. The repressor protein is synthesized by transcribing the trpR gene tryptophan 2.Version 2. Presence of 1. Components trpR trp repressr protein trp operator RNA polymerase trpR Tryptophan trp repressr protein trp operator RNA polymerase 80 | P a g e . The operon is switched off and synthesis of tryptophan does not takes place. 4. 3. The operon is switched on and synthesis of tryptophan takes place. the trp repressor protein assumes an inactive conformation and does not bind to the operator sequence of the operon as it is not complementary in conformation to the lac operator sequence. 4. In the presence of tryptophan.

binds to the operator. breakdown of lactose Anabolic. allows transcription initiation when RNA polymerase binds to the promoter region Molecule bound to repressor protein Inducer which binds to the repressor and inactivates it. thus conserving available energy and resources 3I – DESCRIBE THE CONCEPT OF A SIMPLE OPERON (USING LAC OPERON AS AN EXAMPLE). e.g.5 3H. allows transcription initiation when RNA polymerase binds to the promoter region Co-repressor which binds to the repressor and activates it by changing it to its active conformation The repressor can now bind to the operator. synthesis of tryptophan Usual Condition Switched off Switched on Example lac operon trp operon Usual state of repressor protein Active. preventing the initiation of transcription by blocking RNA polymerase from binding to the promoter Conditions under which transcription occurs Availability of Substrate Insufficient amounts of end product Significance Ensures that enzymes are only synthesized when needed. thus conserving available energy and resources Ensures that resources are not used to synthesize products already present in sufficient quantities. Structural genes code for protein or RNA molecules that form part of a cellular structure or have an enzymatic function. e. Regulates the rate of transcription for the structural genes.  Operons – clustered arrangements of functionally related structural genes that come under the control of the same promoter and regulatory elements  Generates polycistronic mRNAs that encode for multiple proteins that are functionally related  Purpose  Prevents wastage of resources  Allows bacterial cells to adapt and survive in current environmental conditions  Structure  Promoter  Operator – A binding site for a repressor protein.  Regulatory genes – Genes that code for regulatory proteins whose sole function is to regulate the transcription of structural genes  Have their own promoters  Not part of the operon 81 | P a g e . does not bind to operator. The repressor can no longer bind to the operator.g.  Structural gene – A region of DNA that codes for any RNA or protein product other than a regulatory protein.3 COMPARING BETWEEN INDUCIBLE AND REPRESSIBLE OPERONS Inducible Operon Repressible Operon Type of metabolic pathway under control Catabolic.Version 2. preventing the initiation of transcription by blocking RNA polymerase from binding to the promoter Inactive.

thus conserving available energy and resources. confering a selective advantage to the bacteria  Also allows for the bacteria to use a variety of sugars/subtrates  Allows the bacteria to use a wide variety of sugars/substrates 82 | P a g e .Version 2.5  Structure  Coordinated Regulation of a cluster of functionally related genes using a single “on-off” switch  Ensures that enzymes are only synthesized when needed.

LINEARITY/CIRCULARITY. enhancer. operator and binding sites for regulatory molecules Promoter. with multiple genes clustered under the control of the same promoter and regulatory region Each gene has its own promoter and regulatory regions Proportion of structural genes High Low Proportion of intergenic. PRESENCE OF INTRONS AND TYPE OF REGULATORY SEQUENCES. IN TERMS OF SIZE. proximal control elements  Organization 83 | P a g e .  Comparison of the prokaryotic and eukaryotic genome  Structure Prokaryotes Eukaryotes Size of chromosome Small Large No. silencer.5 4.Version 2. non-coding regions Low High Presence of introns Absent Present Number of regulatory gene sequences Small Large Types of regulatory sequences Promoter. PACKING OF DNA. of chromosomes One per cell More than 1 per cell/1 per cell Structure of chromosomes Circular dsDNA Linear dsDNA Location Aggregated in a nucleoid Enclosed by the nuclear membrane Association with proteins Would around histone-like proteins Wound around histones Degree of compaction High Very high Number of Ori 1 Many Prokaryotes Eukaryotes Number of genes Few Many Gene density High Low Arrangement of genes Arranged in operons. ORGANISATION AND CONTROL OF PROKARYOTIC AND EUKARYOTIC GENOME 4A – COMPARE THE STRUCTURE AND ORGANISATION OF PROKARYOTIC AND EUKARYOTIC CHROMOSOMES.

Version 2.5  Control of gene expression in eukaryotes  All somatic cells have the same genome  Differential gene expression results from  Spatial Control  Temporal Control (Developmentally Regulated)  + External/Internal signals  Affects  Quantitative (Number of gene products)  Qualitative (Type of gene products)  Affects Function  Levels  Genomic Control  Transcriptional Control  Post-Transcriptional Control  Translational Control  Post-Translational Control  Epigenetic inheritance – Changes in the genome that do not involve changing the DNA code that can be passed on to further generations (of cells) 84 | P a g e .

 In larger organisms. PROMOTERS. WITH REFERENCE TO INTRONS. ENHANCER AND SILENCERS  Genome  The entire set of genetic material for all the proteins and RNA that the organism will ever synthesize needed to direct the development and maintenance of that organism  Eukaryotic Genome  Def of Gene. may be found within introns (Regions of DNA where gene regulatory proteins bind to control the rate of assembly of protein complexes required for gene expression)  Distal control elements (act at a distance >200bp upstream of the promoter or downstream from the final exon)  Enhancers . non coding sequences  Repetitive DNA – Sequences present in multiple copies in the genome  Tandemly repeated DNA – DNA sequences repeated multiple times and arranged adjacent to one another in a head-to-tail fashion 85 | P a g e . CENTROMERES.Activators bind to them  Silencers .5 4B – DESCRIBE THE STRUCTURE AND FUNCTION OF THE PORTIONS OF EUKARYOTIC DNA THAT DO NOT ENCODE FOR PROTEINS OR RNA. gene size is also larger due to an increase in regulatory sequences  No correlation between complexity and number of genes  No correlation between genome size and number of genes. 1P  Carried on chromosomes (usually linear)  Every eukaryotic cell has a complete copy of the nuclear genome  Also consists of the mitochondrial genome and the chloroplast genome  Complexity  More complex organisms tend to have larger genome sizes (base pairs per haploid genome)  More complex organisms tend to have lower gene densities due to the large proportion of intergenic DNA.Version 2.Repressors bind to them  Proximal control elements  Found 100-200 bp upstream of the transcription initiation start site  Serves as binding sites for GTFs  Promoter (Transcription Start site ≠ Start codon!!)  Resides 25-30 bp upstream of the transcription initiation start site  RNA polymerase and GTFs assemble here to form a transcription initiation complex  3’ UTR  Contains DNA sequence that codes for polyadenylation signal on mRNA  3’UTR contain binding sites for specific proteins that increase or decrease the rate of poly(A) tail shortening  5’ UTR  Contains Ribosome binding site on mRNA  eIFs bind here  Intergenic. Refer to Cellular Structure. TELOMERES.  Elements of DNA  Gene  Transcription Unit  Coding Sequences (Exons)  Non-coding (Introns)  Regulatory sequences.

5  Satellite DNA – Short sequences repeated many times in tandem to form a long array or cluster in a localized area of the genome. usually located in heterochromatin  Plays a role as regulators of gene expression  Types (based on length)  Regular Satellites (Centromeres) (14-500bp)  Mini Satellites (Telomeres) (10-100bp)  Micro Satellites  Prokaryotic genomes have much higher gene densities than eukaryotes  Human genome  ~3 x 109 nucleotide base pairs  22 different autosomes + two sex chromosomes  Gene types  Solitary genes  One copy per haploid set of chromosomes  Multigene Families (multiple copies of an identical DNA gene sequence)  Tandem Gene Families  Genes are clustered together in tandem to form a tandemly repeated array  Encode for nearly identical proteins or functional RNAs  Products usually in heavy demand (gene amplification)  Allows for RNAs and some proteins to be produced in large quantities.  Dispersed Gene Families  Genes with similar but non-identical DNA sequences  Can form a cluster on the same chromosome or be dispersed on different chromosomes  Encodes proteins with close but non-identical amino acid sequences. with related or identical functions.Version 2. 4C – DESCRIBE THE ROLES OF TELOMERES AND CENTROMERES 4C.1 – TELOMERES  Structure  Contains specialized nucleoproteins (telomeric DNA bound by proteins)  3’ single-stranded overhang  The 3’ end of the G-rich strand extends 12-16 nucleotides beyond the 5’ end of the complementary C-rich strand  Overhang folds back on itself to form a hairpin loop (telomere-loop) (t-loop)  Shelterin protein complexes responsible for stabilizing the t-loop 86 | P a g e .  Proteins encoded tend to be a protein family.

 Without telomeres. stem and cancer cells are able to lengthen their telomeres by adding telomeric repeat sequences to the ends of the chromosomes using telomerase. a small section at the extreme 3’ end of the parental DNA strand does not get replicated due to the lack of an upstream 3’OH group that DNA polymerase I that can use to add deoxyribonucleotides to fill the gap generated by the removal of the final primer. Regulating replicative The length of the telomere determines how many more times the cell can divide cell senescence before the length of telomeres become critically short. The unique end structure of a chromosome protects it from being recognized as a damaged DNA molecule (by the cell repair machinery) Maintaining Stability T-loops prevent chromosomal tips from joining to other chromosomes.  Replicative Cell Senescence  Def: The period in which a cell permanently withdraws from the cell cycle and stops dividing after reaching the Hayflick limit.Version 2.5  Characteristics  Telomeric repeat sequence varies between organisms  Telomeric DNA in vertebrates consists of long stretches of hundreds/thousands of a short nucleotide sequence with a high G content. the newly-synthesized daughter strands are shorter than the template strand.  Human telomeres contain hundreds – 2000 tandem repeats of the sequence 5’-TTAGGG-3’  Functions Function Protection Details Telomeric DNA forms t-loops with protein complexes. (~25-50 divisions)  Telomere length limits the number of times cells can divide and regulates a cell’s life span  Shortening telomeres linked to ageing process  Complete loss of telomeres triggers apoptosis  Germ. ensuring that the ends of homologous chromosomes do not spontaneously fuse.  Each time the chromosomes divide.  After the first round of replication. Preventing loss of Stretch of non-coding telomeric DNA at the tips of linear chromosomes protects genes the organisms’ genes from being eroded as the chromosomal ends shorten with each round of DNA replication due to the end-replication problem.  Telomeres shrink by ~100bp with every cell division  Repeated rounds of replication produce progressively shorter DNA molecules. vital genetic information that is needed to sustain a cell’s activities will be lost. protecting the 5’ends and 3’ overhang from degradation by cellular exonucleases. causing the cell to reach the Hayflick limit and enter replicative cell senescence (G0 stage)  End-replication problem  Standard DNA replication machinery is incapable of completely replicating all the way to the ends of the linear chromosomes. 87 | P a g e .

The breakage of the pyrophosphate bond releases energy which is coupled to phosphodiester bond formation between the free 3' hydroxyl group of the terminal nucleotide of the 3’ overhang and the free 5' phosphate group of the incoming deoxyribonucleotide. 3’AAUCCC 5’  Guides the insertion of the deoxyribonucleotide telomere repeat sequence onto the existing 3’ overhangs (lengthening the 3’ overhang)  Telomere Reverse Transcriptase (TERT)  Catalyzes the synthesis of DNA from an RNA template Mode of action of Telomerase Elongation 1. Incoming dNTPs lose a pyrophosphate group.Version 2. (not compulsory to write) Telomerase is translocated to the end of the extended overhang Repeated cycles of elongation and translocation cause the 3’ overhang to extend in the 5’ to 3’ direction. Enzyme/Proteins Involved dNTPs Telomerase DNA Polymerase III 88 | P a g e . 2. leaving a 3’ overhang. Telomerase recognizes and binds to the G-rich telomere sequence at the 3’ overhang on the parental strand. 3. cancer cells  Structure  Ribonucleoprotein Complex  RNA Sequence Template  Complementary to telomere repeat sequence. telomerase adds deoxyribonucleotides to the free 3’ end of the overhang using the RNA template for complementary base pairing.5 4C.2 TELOMERASE  Function: Lengthens telomeres. germline cells. 3’ nucleotides base pair with UAA in the RNA template Using its reverse transcriptase activity. extending the 3’ overhang. thus maintaining chromosomal ends (after end replication problem occurs)  Location: Stem cells. Synthesis of lagging strand 7. 6. 4. Replication of the lagging daughter strand is completed using the extensions as a template for the synthesis of a complementary strand by a DNA polymerase. Translocation 5.

Stem Cells  Genes encoding the telomerase protein and telomerase-associated RNA templates are active. leading to loss or duplication of chromosomes in the daughter cells. replicated chromosomes segregate randomly. AT rich sequences repeated thousands of time in tandem  NO centromere specific DNA sequence  Length of each repeat .  In cancer cells  Same genes are reactivated.5  Activity of Telomeres  In Germ. Centromeres ensure that the sister chromatids segregate properly so that one copy of the daughter chromosome goes to each of the daughter cell formed. 89 | P a g e .Version 2.3 CENTROMERES  Structure  Position is unique for each chromosome  Alpha satellite DNA (Centromeric DNA) for humans only  Short. so telomere lengths are maintained  Cancer cells and divide indefinitely  Avoid apoptosis 4C. The presence of only one centromere is critical as in the absence of a centromere.171bp  Centromeric DNA vary greatly in length (among eukaryotes)  Embedded in a large stretch of centric heterochromatin  Centromeric DNA in centric heterochromatin is bound to specialized nucleosomes containing a centromere-specific histone as well as other centromere-specific proteins (that compact the nucleosomes into dense arrangement)  DNA folded into specialized nucleosomes facilitates the assembly of other centromere-binding proteins to form kinetochores (that associate the centromere to the mitotic spindle) Function Sister chromatid adhension Kinetochore Formation Proper chromosome segregation Details Joins sister chromatids together to form a linear chromosome Provides a site for the assembly of kinetochores.

REPRESSORS.5 4D – DESCRIBE THE PROCESS AND SIGNIFICANCE OF GENE AMPLIFICATION WITHIN CELLS. TRANSCRIPTION FACTORS.  Histone tails contain various chemical groups that influence the tightness of DNA wound around the histones. SILENCER AND ENHANCERS) AND OTHER FACTORS (E. FOR EXAMPLE IN AMPLIFICATION OF THE GENES THAT CODE FOR RIBOSOMES EARLY IN CELL DEVELOPMENT. TO ENSURE THAT SUFFICIENT RIBOSOMES CAN BE MADE TO SYNTHESISE THE PROTEINS THAT MAKE UP THE CELL. serving as DNA templates for transcription of rRNA genes.2  Alternative Splicing  Use of different splice sites. PROMOTER.Version 2.  For 5’capping. which are positively charged and interact strongly with the negativelycharged phosphate groups of the DNA backbone. 2C.  The histone tails are accessible to enzymes which can catalyse the addition or removal of specific chemical groups. HISTONE MODIFICATION AND DNA METHYLATION) INFLUENCE TRANSCRIPTION. generating different polypeptides from the same pre-mRNA. 4F.G. RNA splicing and Polyadenylation.G. allowing different exons to be joined together in different combinations. Each contains about 1-20 copies of circular rRNA genes  Significance  Gene amplification results in a large amount of templates necessary to accommodate an enormous amount of ribosome biosynthesis that takes place during oogenesis.  Histone Acetylation (Catalyzed by Histone Transferase (HAT) ) 90 | P a g e . increasing the affinity of DNA for the nucleosome surface. in order to sustain the high rate of protein synthesis required for early embryonic development upon fertilization.  Gene amplification is the production of multiple copies of a specific gene to amplify the quantity of the gene product.1 – HISTONE MODIFICATION  Structure of a histone  Proteins rich in lysine residues.  One gene can thus code for different proteins  Some exons are excised in the process 4F – DEFINE CONTROL ELEMENTS AND EXPLAIN HOW CONTROL ELEMENTS (E. refer to DNA & Genomics.  Example: Ribosomal RNA gene amplification in Xenopus Laevis (A frog)  Observation  500 copies of rRNA genes are amplified by DNA replication to about 2 million copies  Formation of circular DNAmolecules known as minichromosomes. POLYADENYLATION AND 5’CAPPING.  Developmentally regulated (only happens on one stage in the life cycle) 4E – DESCRIBE THE EUKARYOTIC PROCESSING OF PRE-MRNA IN TERMS OF INTRON SPLICING.

reducing the affinity of the histone complex for the negatively-charged DNA molecule. preventing access of DNA regions to transcription factors and RNA polymerase.  Many CpG dinucleotides cluster together to form CpG islands. exposing DNA regions to transcription factors and RNA polymerase. 91 | P a g e . increasing the affinity of the histone complex for the negatively-charged DNA molecule. catalysed by DNA Methyltranferase.  This causes the chromatin structure to become more compact. usually found in the promoter.  This causes the chromatin structure to become less compact.5  Acetylation causes positively charged lysine residues to be neutralized and become hydrophobic.2 – DNA METHYLATION  DNA can be modified by the addition of a methyl group to a cytosine nucleotide in the sequence 5’-CG-3’ (CpG dinucleotide).  This prevents the transcription of the Gene  DNA methylation changes the 3D conformation of the DNA and prevents the binding of transcription factors to the promoter for transcription  Methylated DNA serves as recognition signals for methyl-CpG-binding proteins (MeCPs) which recruit other proteins such as histone deacetylases which causes histone deacetylation and prevents access of DNA regions to transcription factors and RNA polymerase. 4F.Version 2.  Histone deacetylation (Catalyzed by Histone deactlyases (HDACs) )  Deccetylation causes neutralized lysine residues to be positive and hydrophilic.

proximal control elements. one for DNA and the other for proteins of the transcription machinery  Types  Activators  Bind to enhancers and triggers interactions that increases the rate of transcription  Repressors  Bind to silencers and triggers interactions that decreases the rate of transcription  Modes  Prevents binding of the activator  Deactivates the bound activator  Interferes with the formation of the transcription initiation complex (zero transcription) 92 | P a g e . distal control elements)  Specific Transcription Factors  Function  Mediate the production of a response to a stimulus  Characteristics  Are able to enter the nucleus  Bind to distal control elements  Interact with components of the transcription machinery  Contains 2 binding domains.3 – CONTROL ELEMENTS  Control elements are segments of regulatory DNA sequences that help regulate transcription upon binding of gene regulatory proteins. (Promoter.5 4F.Version 2.

4.5 Transcriptional Control – Distal Control Elements Mode of action of activators and enhancers 1. the precise control of transcription depends on a particular combination of enhancers and their respective activators to be available at a precise timing during development in specific cells which are able to activate transcription of particular genes. Some activators bind to mediator proteins. DNA bending protein causes the looping of DNA. bringing activators bound to enhancers close to the promoter The activator interacts with components of the transcription machinery. 93 | P a g e . 2. causing activators to mediate a response and bind to their respective enhancers. increasing the rate of transcription.  A stimulus triggers a signal transduction pathway. 3. Enzyme/Proteins Involved Activators DNA Bending protein GTFs RNA Polymerase Mediator Proteins GTFs RNA Polymerase Notes  Transcription is able to proceed in the absence of these factors  In eukaryotes. It also facilitates the proper positioning of the transcription initiation complex on the promoter. improving the recruitment of GTFs and RNA polymerase to the promoter to form a stable transcription initiation complex.Version 2. which interact with proteins at the promoter and serve as adaptor molecules to help integrate signals from activators.

BIOCHEMICAL MODIFICATION AND PROTEIN DEGRADATION). 5’CAPPING. 1. thus increasing the stability of mRNA and increases the quantity of transferrin receptors produced. 5’ and 3’ UTR)  Interferes with the initiation of translation by blocking the attachment of ribosomes and other translation initiation factors. (However such a change has an effect on all the translational activity and is thus not specific)  Translational Repressors  Bind to various regions (e.1 – MODIFYING MRNA STABILITY  mRNA stability determines the duration for which translation can occur (use terms like up/down regulate)  Determined by the length of the poly(A) tails  3’UTR contain binding sites for specific proteins that increase or decrease the rate of poly(A) tail shortening  E.G. HALF LIFE OF RNA. thus decreasing the stability of mRNA and decreases the quantity of transferrin receptors produced. 2. The 3’UTR and endonucleolytic cleavage site is exposed.g.  mRNA in eukaryotic cells have longer ½ lives than mRNA in prokaryotic cells  Half-life  The time taken for ½ the initial amount of RNA to be degraded.Version 2.g. thus affecting the rate of translational initiation. 4G. aconitase binds to the 3’UTR and blocks the endonucleolytic cleavage site of the mRNA. INITIATION OF TRANSLATION) AND POST-TRANSLATIONAL LEVEL (E. Exonuclease shortens the poly(A) tail to a critical length The 5’cap is removed and the exposed mRNA is rapidly degraded from the 5’ end mRNA continues to be degraded from the 3’ end An endonuclease cleaves the mRNA internally at the endonucleolytic cleavage site and removes the Poly(A) tail in one step The 5’cap is removed and the exposed mRNA is rapidly degraded from the 5’ end mRNA continues to be degraded from the 3’ end Enzyme/Proteins Involved Exonuclease mRNA Endonuclease mRNA 4G. 3. Translational Control – mRNA degradation pathways Poly(A) tail shortening Internal Cleavage of mRNA 1.5 4G – STATE THE VARIOUS WAYS IN WHICH GENE EXPRESSION MAY BE CONTROLLED AT TRANSLATIONAL (E. 2.G. 3.3 – ALTERNATIVE TRANSLATION INITIATON SITES  Use of 2nd or subsequent start codon (AUG) for translation initiation 94 | P a g e . 4G.  Poly(A) tails can also be lengthened via cytoplasmic poly(A) tail addition to increase the stability of mRNA. iron binds to aconitase.2 – CONTROL AT INITIATION OF TRANSLATION  Eukaryotic Initiation Factors can be varied in abundance and activity. in Transferrin  Iron is taken up by receptor mediated endocytosis (receptors are transferrin)  In iron deficient environments.  In iron rich environments.

 Independent of the 5’cap  A protein with a different primary structure is produced. *Difference between miRNA and siRNA is that siRNA is made from a linear RNA transcript and is thus straight. The miRNA-protein complex then inhibits translation by blocking the formation of the translation initiation complex or degrades the mRNA. One strand of the RNA fragment is degraded by RNAmRNA induced silencing complex (RISC). Enzyme/Proteins Involved RNA Precursor RNA precursors synthesized by transcribing miRNAencoding genes fold back on themselves.  Resulting in proteins that vary in their N-terminus sequence  Initiation of translation in the middle of the mRNA  The Internal Ribosome Entry Site (IRES) allows for translation initiation in the middle of an mRNA sequence.4 – RNA INTERFERENCE Translational Control – RNA Interference RNA Interference 1. The remaining strand binds to RISC to form a miRNA-protein complex.5  Leaky scanning causes the small ribosomal subunit to initiate translation at the next AUG. The enzyme Dicer trims the loop and cuts the double RISC stranded RNA transcripts into smaller fragments. 4. 6. 5. 3. 4G. 95 | P a g e . forming one or more hairpin structures held together by hydrogen Dicer bonds. Each hairpin is then cut away from the precursor by an miRNA enzyme. This complex binds to an mRNA molecule that has a complementary sequence. 2.Version 2.

etc)  Glycosylation  Addition of oligosaccharides to the protein to form glycoproteins  Reversible phosphorylation of threonine.g. so that the protein can function. Insulin SUMMARY Category Genomic Control Method of control Gene amplification by DNA Replication Histone Modification Location Nucleus DNA Methylation Nucleus Transcriptional Control Enhancers and Silencers Nucleus Post – Transcriptional Control Alternative Splicing mRNA Stability management Repressors and eIFs Nucleus Nucleus Alternative Translation Initiation sites RNA Interference Cytoplasm Pre-Transcriptional Control Translational Control Chromatin in Nucleus Cytoplasm Cytoplasm Involves DNA (Genes) DNA replication machinery Histones Histone Transferase (HAT) Histone Deacetylases (HDAC) CpG islands DNA Methyltransferase methyl-CpG-binding proteins (MeCPs) Activators and Repressors Distal Control Elements Proximate Control Elements Spliceosome Exonuclease Endonuclease Repressors Eukaryotic Initiation Factors Start Codons IRES Site miRNA 96 | P a g e .5 4G. lipids.5 – POST-TRANSLATIONAL MODIFICATION  Function: To allow the newly translated polypeptide chain to coil and fold in a precise manner to assume the unique 3D conformation of a protein in the lumen of the rER or in the cytoplasm.g. carbohydrates. Disulfide bonds  Proteolytic Cleavage  Cleaving a sequence of amino acids from the protein  E.  Attaching to it biochemical functional groups (acetate. phosphate. methyl.Version 2. serine and tyrosine  Using Kinases and phosphatases  Attaching uniquitin (protein molecule)  Marks protein for proteolysis by the proteasome  At least 4 are required before proteolysis can take place  Addition of signal peptides  Structural changes  E.

Version 2.5 Post-Translational Control Biochemical Modification Selective degradation of proteins Cytoplasm Cytoplasm Dicer RNA-induced silencing complex (RISC) Enzymes Proteasome Ubiquitin 97 | P a g e .

g.Version 2. selective degradation. biochemical modifications) Transcriptional Control Genomic Control 98 | P a g e .  Prokaryotes have different controls due to the lack of post-transcriptional modification  Major modes in prokaryotes  Transcriptional control  Post-translational control Prokaryotes Eukaryotes DNA methylation DNA methylation Histone Acetylation Level of control Limited regulation due to small numbers of regulatory sequences Extensive regulation of transcription due to large numbers of regulator sequences Down-regulation of gene expression Binding of a repressor protein to the operator Binding of repressors to silencers Up-regulation of gene expression Binding of activators to specific sequences near the promoter Binding of activators to enhancers Post-transcriptional control None Alternative Splicing mRNA degradation Translational Control None RNA interference Alternative translation initiation sites Translational Repressors Post-Translational Control Limited to simple modifications (e.5 4H – OUTLINE THE DIFFERENCES BETWEEN PROKARYOTIC CONTROL OF GENE EXPRESSION WITH THE EUKARYOTIC MODEL. glycoslation) Extensive (e.g.

1 – TUMOUR SUPPRESSOR GENES  Function of tumour suppressor genes  Code for proteins that prevent inappropriate cell cycle progression by suppressing cell growth and proliferation  Mutation of tumour suppressor gene by loss of function mutation  A mutation that results in a reduced/abolished protein function  Both alleles must be mutated for tumour suppressor genes to act in a recessive manner  Quantitative: Mutation at control region causing less p53 proteins to be expressed.Version 2. p21 inhibits the cell cycle by binding to cyclin dependent kinases  Consequences of mutation  Mutant cell able to proliferate uncontrollably  Evade apoptosis  Accumulate cancerous mutations Proteins involved p53 p21 Cyclin dependent kinases 4I. 2.2 – PROTO-ONCOGENES  Function of proto-oncogenes  Code for gene products that promote normal cell growth. apoptosis and maintenance of genetic stability  p53 tumour suppressor protein (specific transcription factor)  Cell Cycle Control  Apoptosis  Triggers apoptosis if DNA is irreparable  Maintenance of genetic stability  Stimulates DNA repair mechanisms to rectify DNA damage Cell Cycle Control by p53 protein Cell Cycle Control 1. promoting the transcription of genes such as p21 3.5 4I – DESCRIBE THE FUNCTIONS OF COMMON PROTO-ONCOGENES AND TUMOUR SUPPRESSOR GENES (LIMITED TO RAS AND P53) AND EXPLAIN HOW LOSS OF FUNCTION MUTATION AND GAIN OF FUNCTION MUTATION CAN CONTRIBUTE TO CANCER.  Functions of tumour suppressor proteins  Repression of genes that are essential for the continuation of the cell cycle  Take part in cell-signalling pathways to inhibit the cell cycle  Halt cell division if DNA is damaged  Triggers DNA repair mechanisms  Initiate apoptosis if DNA is irreparable  Maintains cell adhesion  p53 tumour suppressor gene  Codes for p53 protein involved in cell cycle control. DNA damage prompts the activation of a series of protein kinases. activating the p53 protein. Activated form of p53 binds to specific DNA control elements. 4I.  Qualitative: Mutation of the gene causing faulty p53 proteins to be expressed.  Function of oncogenes 99 | P a g e .

The transcription factor binds to a control region of a gene and promotes the expression of proteins that stimulate the cell cycle. Leading to excessive rate of cell division and ultimately cancer.  Functions of proto-oncogene proteins  Growth Factors – Stimulate cellular division  Growth Factor Receptors – Proteins that bind to growth factors  Protein Kinases – Enzymes that attach a phosphate group to a protein  Inhibitors of apoptosis  Transcription factors  ras proto-oncogene  Codes for a ras protein (G Protein) involved in signal transduction  ras protein  Relays signals from a growth factor receptor to a series of protein kinases. causing a transcription factor to be activated 3. causing the ras protein to be constantly activated 2. Proteins involved ras protein Protein kinases Transcription Factor 100 | P a g e .  Quantitative: Mutation at control region causing more ras proteins to be expressed.Version 2.g. GTP remains infinitely bound to ras protein.  Activated by binding of GTP. and the conversion of the cell to a malignant state  Mutation of proto-oncogene gene into oncogene by gain of function mutation  A mutation that confers an abnormal new or enhanced activity to a protein  Only one allele needs to be mutated to cause to oncogene to act in a dominant manner. Gene Amplification  Increasing the number of copies and templates of the proto-oncogene)  Qualitative: Mutation of the gene causing mutated hyperactive ras proteins to be expressed. (e. 4. deactivated when it hydrolyses GTP to form GDP + P i  Mode of action of mutant ras protein Cell Cycle Stimulation by mutant ras protein 1.  Consequences of mutation  Mutant cell proliferates uncontrollably in the absence of a growth factor.5  Codes for a protein that promotes the loss of growth control. This causes a phosphorylation cascade.

3. at distant sites. Cancer cells are transported by the circulatory system tissues and blood throughout the body. Proto-oncogenes are activated to oncogenes via gain of function mutations. Proteins involved p53 ras Angiogenesis-activating proteins Telomerase 101 | P a g e . Activation of 9. Genes controlling apoptosis are also mutated. process where 11. Genes regulating angiogenesis are mutated Process by which 7. These blood vessels supply nutrients and oxygen while removing toxic waste products. Tumour suppressor genes are deactivated via loss of function mutations. 2. A growing tumour stimulates the formation of new blood new blood vessels vessels. 4. are formed 8. allowing cells to become immortal Angiogenesis – 6. where they establish new secondary tumours secondary tumours (metastases) at distant sites.5 4J – DESCRIBE THE DEVELOPMENT OF CANCER AS A MULTI-STEP PROCESS  Characteristics of cancer development  Multistep model. by releasing angiogenesis-activating proteins. The cancerous growth is now a malignant tumour.Version 2. progression of permanent alterations in a single cell lineage  Selection among tumour cells for those that are able to proliferate more aggressively Tumorigenesis Multistep Model of cancer progression Accumulation of 1. causing the cell to proliferate and normal cell checkpoints to become dysfunctional. Metastasis . 13. 5. Requires several independent mutations in cancer-critical Mutations genes in the lineage of a single cell. This causes an increased expression of oncogenic proteins and a decreased expression of tumour suppressor proteins. Genes regulating metastasis are mutated.A 10. 14. vessels. Cancer cells leave the bloodstream and enter particular establishing organs. cells invade local 12. Reactivation of telomeric genes causes the telomeres of Telomerase cancer cells to be maintained indefinitely. Cancer cells invade surrounding tissues and penetrate the primary tumour walls of the lymphatic and blood vessels.

5  Case Study: Hereditary Colorectal Cancer (Familial Adenomatous Polyposis) Refer to lecture notes for more info  Mutations  Tumour Suppressor Genes  APC  p53  DCC  Activation of proto-oncogenes  ras  Progression 1. Large benign growth – Adenoma 3. Malignant Tumour 102 | P a g e . Small benign growth – polyp 2.Version 2.

(AKA the orientation of homologous chromosomes at the metaphase plant in metaphase I is independent of each other)  Thus during gamete formation.5 5.  Homozygote  An individual having a homozygous gene pair  Heterozygous gene pair  Alleles of a gene pair in a diploid condition are different. HETEROZYGOUS.  Gene – A unit of inheritance that determines the phenotype (character) of an individual.  Allele – An alternative form of a gene at a particular gene locus. HOMOZYGOUS. which segregate randomly such that each gamete has the same chance of receiving either allele. located at a particular locus of a chromosome  Genotype  The specific allelic composition of a cell  Phenotype  The physical manifestation of a genetic trait that results from a specific genotype and its interaction with the environment. GENETIC BASIS FOR VARIATION 5A – EXPLAIN THE TERMS.Version 2. members of one allelic pair segregate independently from members of the other allelic pair  Gametes have the same chance of receiving either alleles 103 | P a g e .  Mandel’s second law of independent assortment (Basis for dihybrid crosses)  The segregation of one pair of alleles is independent of the segregation of other pairs. RECESSIVE. LOCUS. CODOMINANT.  Heterozygote  An individual having a heterozygous gene pair  Heredity  Transmission of genetic characteristics  Variation  Differences between individuals of the same species 5B – EXPLAIN HOW GENOTYPE IS LINKED TO PHENOTYPE AND HOW GENES ARE INHERITED FROM ONE GENERATION TO THE NEXT VIA THE GERM CELLS OR GAMETES. ALLELE.  Central Dogma of Molecular Biology  Gene  mRNA  Polypeptide Chain (Gene Expression) Phenotype  Genes  Meiosis  Fertilization (transmission to next generation)  Mandel’s first law of segregation (Basis for monohybrid crosses)  Each somatic cell of a diploid individual carries two alleles at any one gene locus. DOMINANT. responsible for determining contrasting traits of the same character  Dominant Allele  An allele that expresses its phenotypic effect in both homozygous and heterozygous conditions  Recessive Allele  An allele that only expresses its phenotypic effect in the homozygous condition  Locus(loci) – Specific location of a gene on a chromosome (containing alternate forms of a gene)  Homozygous gene pair  Alleles of a gene pair in a diploid condition are identical. PHENOTYPE AND GENOTYPE.

thus they do not mature to become queens. yarrow plants grew tall  At mid-altitudes.  Effect of Temperature  Coat Colour In Himalayan Rabbits  All Himalayan rabbits are homozygous for the ch allele which codes for a heat-sensitive tyrosinase.  The workers are fed honey and pollen. thus flowers are blue.  Effect of Diet  In honey bees. the queen and the workers have exactly the same amount of genetic material. thus only at parts of the body which are cool enough does black fur grow. aluminium is unavailable.  Effect of Soil Acidity on Hydrangea Macrophylla  Soil acidity affects the availability of aluminium to plants  In acidic soils.Version 2. which produces melanin.  This enzyme is only active in areas where the air temperature is below 33°C.  In basic soils. resulting in black fur. WITH EXAMPLES.  Effect of Elevation/Altitude  Height of Yarrow Plants  At high and low altitudes. 5D – USE GENETIC DIAGRAMS TO SOLVE PROBLEMS IN DIHYBRID CROSSES. but are phenotypically different. aluminium is readily taken up by the plant. yarrow plants remained short. causing the plant flowers to be pink. INCLUDING THOSE INVOLVING SEX LINKAGE. HOW THE ENVIRONMENT MAY AFFECT THE PHENOTYPE.  The queen is fed royal jelly. CODOMINANCE AND MULTIPLE ALLELES.1 TWO-GENERATION MONOHYBRID CROSSES (FOUNDATION FOR DYHYBRID CROSS) Let T represent the dominant allele for tall plants Let t represent the recessive allele for dwarf plants Parental Phenotype Tall Plants Parental Genotype TT Parental Gametes T X X X ○ F1 Genotype F1 Phenotypes F1 Phenotype F1 Genotype F1 Gametes t ○ All Tt All Tall Plants X X Tall Plants Tt T ○ Dwarf Plants tt t ○ T ○ Random Fertilization F1 Female Gametes F2 Genotypic Ratio F2 Phenotypic Ratio 1 TT : 3 Tall Plants Tall Plant Tt t ○ F1 Male Gametes T ○ t ○ T ○ TT Tt t ○ Tt tt 2 Tt : : 1 tt 1 Dwarf Plant 104 | P a g e . EPISTASIS. AUTOSOMAL LINKAGE. 5D. stimulating the maturation of the female reproductive system due to its high protein content.5 5C – EXPLAIN.

105 | P a g e .3 TWO-GENERATION MONOHYBRID CO-DOMINANCE  Codominance of alleles happen when both alleles present at a particular gene locus are expressed simultaneously in the phenotype of the heterozygote. Let CR represent the allele for red coat in cattle Let CW represent the allele for white coat in cattle Parental Phenotype Red coated cattle X White coated Cattle Parental Genotype CRCR X CWCW R Parental Gametes C X CW F1 Genotype All CRCW F1 Phenotypes All Red and White (roan) coated cattle F1 Phenotype Roan Coated cattle X Roan Coated cattle F1 Genotype CRCW X CRCW R W R F1 Gametes C C C CW CR F1 Male Gametes CR CW R R CC CRCW CW CRCW Random Fertilization F1 Female Gametes F2 Genotypic Ratio 1 CRCR F2 Phenotypic Ratio 1 Red coated cattle **No dominant/recessive allele in this context : : 2 CRCW 2 Roan Coated cattle CWCW : : 1 CWCW 1 White coated cattle  This is due to both gene products being equally expressed.5 5D.Version 2.2 TWO-GENERATION MONOHYBRID INCOMPLETE DOMINANCE Let CR represent the allele for red snapdragon flowers Let CW represent the allele for white snapdragon flowers Parental Phenotype Red Snapdragon Flowers X White Snapdragon flowers R R Parental Genotype CC X CWCW R Parental Gametes C X CW R W F1 Genotype All C C F1 Phenotypes All Pink Snapdragon Flowers F1 Phenotype Pink Snapdragon Flowers X Pink Snapdragon Flowers F1 Genotype CRCW X CRCW R W R F1 Gametes C C C CW CR F1 Male Gametes CR CW CRCR CRCW CW CRCW Random Fertilization F1 Female Gametes F2 Genotypic Ratio 1 CRCR F2 Phenotypic Ratio 1 Red Flower **No dominant/recessive allele in this context 2 CRCW 2 Pink Flowers : : CWCW : : 1 CWCW 1 White Flower  This is due to insufficient enzymes being produced to create a “100% intense” red colour 5D.

In individuals with the genotype IAIB.Version 2.5 TWO-GENERATION DIHYBRID CROSS Let R represent the dominant allele for round seeds Let r represent the recessive allele for wrinkled seeds Let Y represent the dominant allele for yellow seeds Let y represent the recessive allele for yellow seeds Parental Phenotype Round and Yellow seeds Parental Genotype RRYY Parental Gametes RY ○ F1 Genotype F1 Phenotypes F1 Phenotype F1 Genotype F1 Gametes X X X Wrinkled and green seeds rryy ry ○ All RrYy All Round and Yellow Seeds Round and Yellow Seeds X Round and Yellow Seeds RrYy X RrYy RY ○ Ry ○ rY ○ ry ○ RY ○ Ry ○ rY ○ ry ○ 106 | P a g e . both surface antigen A and B are expressed simultaneously on the surface of red blood cells. Let IA represent the allele for the production of type A antigen Let IB represent the allele for the production of type B antigen Let IO represent the allele for the production of no antigen Parental Phenotype Blood Group AB Parental Genotype IAIB A Parental Gametes I IB Random Fertilization F1 Female Gametes IO 1 IAIO 1 Blood Group A 1 Blood Group A IAIO A I IO F1 Genotype F1 Phenotypes F1 Phenotypes F1 Genotype F1 Gametes X X X Blood Group O IO IO IO F1 Male Gametes IA IB IAIO IBIO 1 IBIO 1 Blood Group B 1 Blood Group B IBIO : : X X IB F1 Female Gametes 1 IAIB 1 Blood Group AB IB IO IAIO 1 IAIO 1 Blood Group A : : IO F1 Male Gametes IA IO IAIB IBIO Random Fertilization F2 Genotypic Ratio F2 Phenotypic Ratio IO : : IO IO 1 IBIO 1 Blood Group B : : 1 I O IO 1 Blood Group O 5D.4 TWO-GENERATION MONOHYBRID MULTIPLE ALLELES/CO-DOMINANCE  IA and IB give rise to distinct functional products.5 5D.

short stems stems d.Version 2. Hairy stems X Yellow tomatoes. X Red tomatoes with scattered. fruits. SINGLE CO-DOMINANCE Let R represent the dominant allele for red fruits Let r represent the recessive allele for yellow fruits Let SH represent the allele for hairy stems Let SL represent the allele for hairless stems Parental Phenotype Red tomatoes. short hairy Hairless hairs on hairs on stems stems stem stem *The presence of codominant alleles or incomplete dominance will cause more phenotypes to be produced F2 Genotypic Ratio F2 Phenotypic Ratio 107 | P a g e . fruits. fruits. Yellow Yellow scattere hairy hairless scattere fruits.6 DIHYBRID.5 Random Fertilization F1 Female Gametes F1 Male Gametes RY ○ Ry ○ rY ○ ry ○ RY ○ RRYY RRYy RrYY RrYy Ry ○ rY ○ ry ○ RRYy RRyy RrYy Rryy RrYY RrYy rrYY rrYy RrYy Rryy rrYy rryy F2 Genotypic Ratio F2 Phenotypic Ratio 9 R_Y_ : 3 R_yy : 3 rrY_ 9 Round & : 3 Round & : 3 Wrinkled Yellow Green and Yellow **Genotypic Ratio for dihybrid crosses always 9:3:3:1 (heterozygous * heterozygous) : : 1 rryy 1 Wrinkled and Green 5D. fruits. short hairs on stem F1 Phenotype Red tomatoes with scattered. d. short hairs short hairs on stem on stem H L F1 Genotype RrS S X RrSHSL H L H L H L F1 Gametes RS RS rS rS RS RS rSH rSL Random Fertilization F1 Male Gametes RSH F1 Female Gametes RSL rSH rSL RSH RRSHSH RRSHSL RrSHSH RrSHSL RSL RRSHSL RRSLSL RrSHSL RrSLSL rSH RrSHSH RrSHSL rrSHSH rrSHSL rSL RrSHSL RrSLSL rrSHSL rrSLSL 6 R_SHSL : 3 R_SHSH : 3 R_SLSL : 2 rrSHSL : 1 rrSHSH : 1 rrSLSL 7 Red : 7 Red : 3 Red : 2 Yellow : 1 : 1 fruits. Hairless stems H H Parental Genotype RRS S X rrSLSL H Parental Gametes RS X rSL H L F1 Genotype All RrS S F1 Phenotypes All red tomatoes with scattered.

whereas Y chromosome only contains a few genes (as it is shorter)  Certain characteristics are linked with the sex chromosomes  Colour blindness  Duchenne Muscular Dystrophy  Haemophilia  Characteristics of sex-linked disorders  They tend to affect males  Males are always hemizygous for every sex-linked locus  As males possess only one x chromosome.Version 2. every allele on the x chromosome is expressed regardless of whether it is recessive or dominant  Females with heterozygous genotypes are carriers who do not show the condition but carry a recessive allele  Worked Example (Haemophilia – Recessive Sex linked)  Haemophilia causes a reduced ability of the blood to clot Let XH represent the x chromosome carrying the dominant allele for normal blood clotting Let Xh represent the x chromosome carrying the recessive allele for haemophilia Parental Phenotype Female Carrier X Normal Male Parental Genotype XHXh X XHY H h H Parental Gametes X X X X Random Fertilization F1 Female Gametes XH Xh Y F1 Male Gametes XH Y H H X X XHY XHXh XhY : 1 XHY : 1 Normal Male Offspring Genotypic Ratio 1 XHXH : 1 XHXh Offspring Phenotypic 1 Normal : 1 Female Ratio Female Carrier  Worked Example (Coffin–Lowry syndrome – Dominant Sex Linked) : : 1 XhY 1 Haemophiliac Male Let XL represent the x chromosome carrying the dominant allele for normal cell signalling Let Xl represent the x chromosome carrying the recessive allele for Coffin–Lowry syndrome Parental Phenotype Female Carrier X Normal Male Parental Genotype XLXl X XlY L l l Parental Gametes X X X X Y 108 | P a g e .5 5D. the sex chromosomes exist as one single long X chromosome and a single short Y chromosome  Males produce up to 2 types of gametes (sperm cells either carry the X or Y chromosome)  Males are thus heterogametic  Sex-linked inheritance  Sex-linkage refers to the carrying of genes on the sex chromosome  X chromosome contains the loci of many genes that are required for both sexes.7 SEX LINKED INHERITANCE  Humans have 22 pairs of autosomes and 1 pair of sex chromosomes (one X chromosome and one Y chromosome)  Sex is determined by the pair of sex chromosomes (the 23rd pair)  In diploid female cells. the sex chromosomes exist as a homologous pair of two X chromosomes which are identical in length  Females only produce one type of gamete (All egg cells carry the X chromosome)  Females are thus homogametic  In diploid male cells.

A homozygous dominant child is typically born as a stillbirth due to the link with lethal genes.8 LETHAL GENES  Sometimes. Let A represent the dominant allele for archondroplasia Let a represent the recessive allele for normal people Parental Phenotype Affected Individual Parental Genotype Aa Parental Gametes A a ○ X X ○ a ○ Random Fertilization F1 Female Gametes Offspring Genotypic Ratio Offspring Phenotypic Ratio Affected Individual Aa A ○ F1 Male Gametes A ○ a ○ A ○ AA Aa a ○ Aa aa 1 AA (dead) : 2 Aa 2 affected individuals : : 1 aa 1 Normal Individual 109 | P a g e . Males more commonly affected Skip Generations Affected homozygous mothers definitely produce affected sons Affected fathers definitely produce affected daughters Affected Parents definitely produce affected individuals Unaffected parents will not produce affected individuals Inheritance Pattern Autosomal Autosomal Recessive Dominant No No X-linked Recessive X-linked Dominant Yes Yes Yes No No No Yes Yes No Yes No No No Yes Yes No Yes No No Yes No Yes 5D. certain genes are linked to lethal genes.  E.5 XL F1 Male Gametes Xl Y L l XX XLY Xl XlXl Random Fertilization F1 Female Gametes F2 Genotypic Ratio F2 Phenotypic Ratio l l 1 XX 1 Normal Female : : L l 1XX 1 affected female : : XlY 1 XlY 1 Normal Male : : 1 XL Y 1 affected male  Pedigree Analysis  Only one copy of the recessive allele needs to be present in any male for it to be expressed as males are hemizygous  Females must possess 2 copies of the recessive alleles for the recessive allele to be expressed as females have 2 X chromosomes. Achondroplasia.Version 2. a disorder in which a dominant allele causes a person to be extremely short. so a certain allelic combination of the gene will trigger the lethal gene and cause death in some individuals.g.

RECESSIVE Gene Locus E: Controls the deposition of pigment Gene Locus B: Controls the colour of the pigment deposited Let E represent the dominant allele for the deposition of pigment Let e represent the recessive allele preventing deposition of pigment Let B represent the dominant allele for black pigment Let b represent the recessive allele for a brown pigment Parental Phenotype Black Coat Dog X Parental Genotype EEBB X Parental Gametes X EB Yellow Coat Dog eebb ○ F1 Genotype F1 Phenotypes F1 Phenotype F1 Genotype F1 Gametes All EeBb All Black Coat Dogs X X Black Coat Dog EeBb EB ○ Eb ○ eB ○ eb ○ eb ○ EB ○ Black Coat Dog EeBb Eb ○ eB ○ eb ○ 110 | P a g e .5 5D.Version 2.9 NON-EPISTATIC GENE INTERACTION Let R represent the dominant allele for Pea-shaped comb Let r represent the recessive allele for a single comb Let P represent the dominant allele for rose comb Let p represent the recessive allele for a single comb Parental Phenotype Pea-shaped Comb Chicken Parental Genotype RRpp Parental Gametes Rp X X X ○ F1 Genotype F1 Phenotypes F1 Phenotype F1 Genotype F1 Gametes rP ○ All RrPp All Walnut Comb chickens Walnut Comb chickens X Walnut Comb chickens RrPp X RrPp RP ○ Rp ○ rP ○ rp ○ Random Fertilization F1 Female Gametes F2 Genotypic Ratio F2 Phenotypic Ratio Rose Comb Chicken rrPP 9 R_P_ 9 Walnut Comb chickens : : RP ○ Rp ○ rP ○ rp ○ F1 Male Gametes RP ○ Rp ○ rP ○ rp ○ RP ○ RRPP RRPp RrPP RrPp Rp ○ rP ○ rp ○ RRPp RRpp RrPp Rrpp RrPP RrPp rrPP rrPp RrPp Rryy rrPp rrpp 3 R_pp 3 Pea-shaped Comb Chicken : : 3 rrP_ 3 Rose Comb Chicken : : 1 rrpp 1 Single Comb Chicken 5D.10 EPISTATIS.

Version 2. RECESSIVE Gene Locus E: Controls production of intermediate pigment from the precursor Gene Locus Y: Controls production of anthocyanin from the intermediate pigment Let C represent the dominant allele for the production of the intermediate pigment in sweet peas Let c represent the recessive allele preventing the production of the intermediate pigment in sweet peas Let P represent the dominant allele for the production of anthocyanin in sweet peas 111 | P a g e .11 EPISTATIS. DOMINANT Gene Locus E: Controls pigment production Gene Locus Y: Controls the colour of the pigment produced Let E represent the dominant allele inhibiting pigment production in summer squash Let e represent the recessive allele for pigment production in summer squash Let Y represent the dominant allele for yellow pigment production Let y represent the recessive allele for a green pigment production Parental Phenotype White Fruit X Green Fruit Parental Genotype EEYY X eeyy Parental Gametes X EY ey ○ F1 Genotype F1 Phenotypes F1 Phenotype F1 Genotype F1 Gametes ○ All EeYy All White Fruits X X White Fruit EeYy EY ○ Ey ○ eY ○ ey ○ Random Fertilization F1 Female Gametes F2 Genotypic Ratio F2 Phenotypic Ratio White Fruit EeYy EY ○ Ey ○ eY ○ ey ○ F1 Male Gametes EY ○ Ey ○ eY ○ ey ○ EY ○ EEYY EEYy EeYY EeYy Ey ○ eY ○ ey ○ EEYy EEyy EeYy Eeyy EeYY EeYy eeYY eeYy EeYy Eeyy eeYy eeyy 12 (9 E_Y_ + 3 E_yy) 12 White Fruits : : 3 eeY_ 3 Yellow Fruits : : 1 eeyy 1 Green Fruit 5D.5 Random Fertilization F1 Female Gametes F2 Genotypic Ratio F2 Phenotypic Ratio 9 E_B_ 9 Black Coat Dogs : : F1 Male Gametes EB ○ Eb ○ eB ○ eb ○ EB ○ EEBB EEBb EeBB EeBb Eb ○ eB ○ eb ○ EEBb EEbb EeBb Eebb EeBB EeBb eeBB eeBb EeBb Eebb eeBb eebb 3 E_bb 3 Brown coat Dogs : : 4 (3 eeB_ + 1 eebb) 4 yellow coat dogs 5D.12 DUPLICATE EPISTASIS.

Random Fertilization F1 Female Gametes F1 Male Gametes T ○ T ○ t ○ Tt Tt t ○ Tt Tt Random Fertilization F1 Female Gametes F1 Male Gametes T ○ t ○ t ○ Tt tt t ○ Tt Tt For the first cross. 112 | P a g e . all offspring will display the dominant trait.Version 2.5 Let p represent the recessive allele preventing the production of anthocyanin in sweet peas Parental Phenotype White X White Parental Genotype CCpp X ccPP Parental Gametes X Cp cP ○ F1 Genotype F1 Phenotypes F1 Phenotype F1 Genotype F1 Gametes ○ All CcPp All Purple Plants X X Purple Plants CcPp CP ○ Cp ○ cP ○ cp ○ Random Fertilization F1 Female Gametes F2 Genotypic Ratio F2 Phenotypic Ratio 9 C_P_ 9 Purple Plants Purple Plants CcPp CP ○ Cp ○ cP ○ cp ○ F1 Male Gametes CP ○ Cp ○ cP ○ cp ○ CP ○ CCPP CCPp CcPP CcPp Cp ○ cP ○ cp ○ CCPp CCpp CcPp Ccpp CcPP CcPp ccPP ccPp CcPp Ccpp ccPp ccpp : : 7 (3 C_pp + 3 ccP_ + 1 ccpp) White Plants 5E – USE GENETIC DIAGRAMS TO SOLVE PROBLEMS INVOLVING TEST CROSSES  Test Cross = Cross with a homozygous recessive specimen and then using the results to justify the test organism’s genotype  Genetic diagrams are drawn in the same format as the above ones 5E.1 MONOHYBRID TEST CROSS When crossing a plant showing the dominant trait (aka the plant is either homozygous dominant or heterozygous) with a homozygous recessive plant. there are two possibilities. whereas for the second cross half of the offspring will display the recessive trait.

only two phenotypes will be exhibited  If the genes are incompletely linked. four phenotypes will be exhibited  Percentage of parental phenotypes are approximately equal and percentage of recombinant phenotypes are approximated equal  No fixed ratio  If the expected phenotypic ratio is obtained.5 5E.Version 2.3 SEX-LINKED RECIPROCAL CROSS  Conducted to check if the trait is sex-linked recessive  Done by performing two crosses  Homozygous Dominant with Hemizygous Recessive  Homozygous Recessive with Hemizygous Dominant  If trait is not x-linked recessive. there is no linkage present 5F – EXPLAIN THE MEANING OF THE TERMS LINKAGE AND CROSSING-OVER AND EXPLAIN THE EFFECT OF LINKAGE AND CROSSING-OVER ON THE PHENOTYPIC RATIOS FROM DIHYBRID CROSSES.2 DIHYBRID TEST CROSS Random Fertilization F1 Male Gametes (RrYy) RY ○ F1 Female Gametes (Homozygous Recessive) ry ○ Random Fertilization Ry ○ RrYy Rryy F1 Male Gametes (RRYY) rY ○ ry ○ rrYy RY ○ rryy RrYy F1 Male Gametes (RRYy) RY ○ F1 Male Gametes (RrYY) Ry ○ RY ○ rY ○ F1 Female Gametes RrYy Rryy RrYy rrYy ry ○ (Homozygous Recessive) The above table demonstrates the results obtained by test crossing a plant that is homozygous recessive for both gene loci with one that expresses both dominant traits (out of which there are 4 genotypic combinations) 5E.  Linkage  Genes located on the same chromosome are linked 113 | P a g e .4 TEST CROSS FOR LINKAGE  A test cross with a homozygous recessive will determine if the two genes are linked  If the genes are completely linked. the phenotypic results of the two crosses should be the same Random Fertilization F1 Male Gametes X Xh XHXh Y XHY XH XHXh XHY H F1 Female Gametes Random Fertilization F1 Male Gametes X XH XHXh Y XhY Xh XHXh XhY h F1 Female Gametes 5E.

Grey Body Long Wings.Version 2. Grey Body LG lg F1 Male Gametes LG lg LG LG LG lg LG lg lg lg : : 1 lg lg 1 Short Wings. can result in the genetic recombination of alleles  1 pair of non-sister chromatids exchange genetic information. Ebony Body 5F. l represent the recessive allele for short wings Let G represent the dominant allele for grey bodies. Ebony Body Parental Genotype X LG lg LG lg Parental Gametes X LG lg F1 Genotype LG lg All Long Wings.  Recombinant gametes can fuse with other gametes to form recombinant offspring  However. if the two gene loci are far away from each other on the same chromosome (incomplete linkage)  Crossing over during prophase I. g represent the recessive allele for ebony bodies Parental Phenotype Long Wings. Colour Blind X Normal Blood clotting and Vision Male Female Parental Genotype X hb HB hb ⇁ Parental Gametes X HB ⇁ hb 114 | P a g e . the two alleles will be inherited as one unit (will not be segregated) (Complete Linkage)  However. h represent x-linked the recessive allele for haemophilia Let B represent the x-linked dominant allele for normal vision. Grey Body X LG LG lg lg lg LG lg LG F1 Phenotypes F1 Phenotype F1 Genotype F1 Gametes Random Fertilization LG F1 Female Gametes F2 Genotypic Ratio F2 Phenotypic Ratio 1 : LG LG lg 2 3 Long Wings.2 – X-LINKED INCOMPLETE LINKAGE Let H represent the x-linked dominant allele for normal blood clotting. creating recombinant gametes.1 – COMPLETE LINKAGE Let L represent the dominant allele for long wings. causing chiasmata to be formed. the dihybrid ratios will not be observed  This is due to the absence of independent assortment  Thus. Grey Body X Short Wings. resulting in two new allelic combinations (as compared to complete linkage). the ratio is not fixed and offspring produced shot a majority of parental allele combinations and a minority of recombinant allele combination 5F.5  Genes located on different chromosomes are not linked  If the two gene loci are linked. b represent the x-linked recessive allele for colour blindness Parental Phenotype Haemophiliac. Grey Body X Long Wings.

but not a complete set (2n+1.5 Random Fertilization F1 Genotypic Ratio F1 Phenotypic Ratio F1 Phenotype F1 Genotype F1 Gametes F1 Female Gametes HB 1 hb 1 Normal (Carrier) Female Normal Female HB hb HB hb Hb hB Random Fertilization F1 Female Gametes F1 Male Gametes hb : ⇁ HB HB hb hb ⇁ hb ⇁ 1 Haemophiliac. Colour Blind Male Haemophiliac.  Gene Mutation: A change in the nucleotide sequence of DNA.2n-1. the exact order of various genes can be determined and be used to generate a chromosomal map. refer to gene mutations. Colour Blind Male hb ⇁ ⇁ hb 1 : X X F1 Male Gametes hb ⇁ HB hb Hb hB HB HB HB ⇁ hb hb hb ⇁ Hb hb Hb ⇁ hB hb hB ⇁  Note that 8 different phenotypes (not in all cases) are produced with no explicit ratio  **Grey colour  Recombinant gametes/genotypes/  ⇁ represents the Y chromosome 5F. and thus the greater the proportion of recombinants formed.3 – CHROMOSOME MAPPING  Patterns of linkage can be used to map chromosomes on a wide variety of organisms  The chance of crossing over between two linked genes is proportional to the distance between them  The further apart the linked genes. FOR CHROMOSOMAL ABERRATION. etc) 115 | P a g e . 5G – EXPLAIN WHAT IS MEANT BY THE TERMS GENE MUTATION AND CHROMOSOME ABERRATION. resulting in the formation of new alleles. DELETION] IS REQUIRED. KNOWLEDGE OF NUMERICAL [ANEUPLOIDY] AND STRUCTURAL [TRANSLOCATION. INVERSION. DUPLICATION. (brought about by insertion and substitution.  Change in Chromosomal Number  Aneuploidy: A condition in which an organism loses or gains one or more chromosomes.  Thus.Version 2.  Cross over Value = % of recombinant offspring (1% COV = a distance of 1 Centimorgan (cM) on a chromosome)  By analyzing the relative distance between different pairs of genes. which occurs at a single gene locus on a chromosome. chapter 2D)  Chromosomal Aberration: A mutation which causes a change in the chromosomal structure or in the number of chromosomes. the greater the chance that crossing over will separate them. proportion of determinants  Distance apart between genes.

and a portion of it is lost.  Change in chromosomal structure  Deletion  A chromosome breaks in one or more places. or a few Continuous Variation – Variation in characters of degree Little/none Phenotypes can be modified by the cumulative effect of varying environmental factors acting on different genotypes Histograms showing the frequency distribution of the different traits of the character (Normal distribution curve formed by phenotypic measurements) Bar Graphs Range of phenotypes observed.Version 2. DNA slippage)  Inversion  Happens when a chromosome is broken at two points.  Variation: Recognizable differences in characteristics (phenotype) between organisms of the same natural population or species Observable Phenotype No. and then middle portion is then joined back but inverted. 5H – DESCRIBE THE DIFFERENCES BETWEEN CONTINUOUS AND DISCONTINUOUS VARIATION AND EXPLAIN THE GENETIC BASIS OF CONTINUOUS VARIATION (MANY. which is joined to another chromosome.  Occurs due to non-disjunction of all homologous chromosomes.5  Occurs due to non-disjunction of either homologous chromosomes or sister chromatids during anaphase I or anaphase II (failure of homologous chromosomes/sister chromatids separating and moving to opposite poles during anaphase of meiosis I/II)  Non-disjunction can happen on any number of homologous chromosomes/sister chromatids  Polypoloidy: A condition in which more than two copies of the haploid chromosome set are found (Xn)  Autopolypoloidy: Addition of one or more sets of chromosomes identical to the haploid component of the same species.  Diploid hybrids formed from haploid gametes of different species are sterile.g. ADDITIVE. (This rearranges the linear sequence)  Translocation  Breakage of one segment of a chromosome. producing a diploid gamete  Two sperms may fertilize an ovum. GENES CONTROL A CHARACTERISTIC) AND DISCONTINUOUS VARIATION (ONE OR FEW GENES CONTROL A CHARACTERISTIC). including intermediates Multiple Additive Genes. (can occur in the middle or at the end) (deletion at one loci only may not be lethal)  Duplication  Happens when one part of the genetic material is repeated more than once in the genome  May occur due to unequal crossing over or DNA replication error (e. Polygenic Inheritance *Effects of single genes too slight to be detected 116 | P a g e . of Genes controlling phenotypic variation Effect of environment on phenotype Mode of phenotypic measurement Discontinuous Variation – Variation in characters of type Discrete phenotypic classes (no intermediates) One. producing a triploid zygote  Plants are able to reproduce as long as the haploid set is a whole number  Allopolypoloidy: Addition of one or more sets of chromosomes identical to the haploid component of different species.

117 | P a g e .Version 2. mean) Examples ABO Blood Group Height of Humans  Polygenic Inheritance – A single phenotypic character affected by 2 or more genes (no interaction) E. Mutations that occur in reproductive organs are inheritable  Gene Mutation and Chromosomal Aberrations  May give rise to new alleles  Environmental Factors (Refer to 5C)  The ultimate factor for determining the full potential of the phenotype expression is the genotype. the environment may affect the subsequent expression of this genetic potential but will never be able to cause an effect greater than what is allowed by the genotype. Continuous Variation in skin tone 5I –DESCRIBE THE CAUSES OF GENETIC VARIATION IN A POPULATION  Allelic variation is the basis for hereditary variation  Genetic Factors  Meiosis  Crossing over in prophase I where a chromatid of a bivalent break and joint to a non-sister chromatid causes an exchange of alleles between maternal and paternal chromologous chromosomes. The gametes get a new combination of alleles where the number of possible permutations is 2 n (n is the haploid number of chromosomes)  Random mating between individuals  Random fusion of gametes  Mutation  Sudden. resulting in the formation of new linkage groups and different combination of alleles. Furthermore the chiasmata is formed at a random position  Random alignment of bivalent at metaphase I and chromosomes at metaphase II results in an independent assortment of paternal and maternal chromosomes into gametes at anaphase I and II.  However. spontaneous random change in genome.5 Nature of Qualitative Quantitative – Statistical analyses give estimates of measurement population parameters (SD.g.

the homozygous recessive condition (bb) for the epistatic gene results in the suppression of alleles of the hypostatic gene at a different locus (A/a) by inhibiting the conversion of white to black pigment. water and favourable soil conditions. causing the dog to be yellow. (different locus)  Due to both genes playing a role in the biochemical pathway  Different from complete dominance (the masking of alleles at the same gene locus)  The effects of the epistatic gene can be  Recessive (meaning a homozygous recessive condition for the epistatic gene is required for epistasis)  The homozygous recessive condition for the epistatic gene (aa) results in the suppression of alleles of the hypostatic gene (B/b) at a different locus. there would be no substrate for the 2nd enzyme to convert to the final product. the homozygous recessive condition (ee) for the epistatic gene results in the suppression of alleles of the hypostatic gene at a different locus (B/b) prevents the deposition of pigment.2 – GENE INTERACTION.g. (KNOWLEDGE OF THE EXPECTED RATIO FOR VARIOUS TYPES OF EPISTASIS IS NOT REQUIRED.  E. only if given ample light. Alleles for tallness determine your potential to grow tall. as long as the individual has a homozygous recessive condition for the allele a. but only one characteristic is studied in non-epistatic gene interaction  A new phenotype will be formed that is completely different from that of the parents (resulted from an interaction between the two genes product at the biochemical or cellular level) 5J. A reduction in the supply of any one of these will prevent the gene for height from exerting its full effect. but whether that happens depends on your diet and environment.Version 2. FOCUS OF THIS SECTION IS ON PROBLEM SOLVING. in Labrador Retrievers. thus causing the mouse to have a white fur coat.5   Mendel’s tall variety plants normally attain a height of 6 feet. in Mice. NON-EPISTATIC  Two Independently assorting genes may interact to influence a single character  Same phenotypic and genotypic ratio as in dihybrid inheritance. thus all individuals with genotypes aaB_ and aabb will exhibit the <recessive epistasis trait>  E.g. **Example of discontinuous variation being affected by the environment Human height.  Both gene loci are in the same biochemical pathway whereby the dominant allele A codes for an enzyme that converts the <initial product> to an <intermediate product> while the dominant allele B codes for an enzyme that converts the <intermediate product> to the <final product>. 5J – DESCRIBE THE INTERACTION BETWEEN LOCI (EPISTASIS) AND PREDICT PHENOTYPIC RATIOS IN PROBLEMS INVOLVING EPISTASIS..  Dominant (meaning one dominant epistatic allele is sufficient for epistasis)  The presence of a single dominant allele for the epistatic gene results in the suppression of both alleles of the hypostatic gene at a different locus by coding for a protein that inhibits the pathway for <the phenotypic effect expressed by the hypostatic gene> 118 | P a g e . no intermediate products will be synthesized.  Even with the genotype B_.)  Gene interaction takes place when two or more unliked genes influence one particular character 5J. EPISTATIC  Epistatic gene interaction occurs when the expression of an allele at one gene inhibits the expression of alleles at another gene.1 – GENE INTERACTION.  Thus.

05 unless otherwise stated  Steps to the χ2 test 1. the allele W codes for the inhibition of pigment production. that means that the observed experimental results do not coincide with the expected results and is thus the expected ratios are wrong. or due to monohybrid inheritance. State the hypotheses (both null and alternate hypotheses) 2.5  E. in this context) must be discrete/discontinuous  χ2 test assumptions  Fertilization is random  Equal opportunity of survival among offspring  A statistically significant number of offspring are produced  χ2 test objective  To check whether the deviation of observed experimental results from expected results is statistically significant (If derived value is larger. 2 – GUIDED EXAMPLE (PRIMULA PLANTS) Two heterozygous breeding lines of Primula plants are crossed to produce a variety of flowers. (Both genes are epistatic to each other)  *Not necessary to state the gene locus for genetic diagrams 5K – USE THE CHI-SQUARED TEST TO TEST THE SIGNIFICANCE OF DIFFERENCES BETWEEN OBSERVED AND EXPECTED RESULTS  χ2 test characteristics  Variables (phenotypes.  Calculating χ2 value  χ2 = ∑ (Observed−Expected)2 ????????  Degree of freedom = number of phenotypes – 1  Level of significance . CASE 1: MONOHYBRID INHERITANCE Let W represent the dominant allele for white flowers Let w represent the recessive allele for blue flowers Parental Phenotype White Flowers Parental Genotype Ww Parental Gametes W w ○ ○ X X White Flowers Ww W ○ w ○ 119 | P a g e . The presence of one W is sufficient to cause the fruit to be white  Duplicate recessive  Similar to recessive. the observed experimental results coincide with the expected results.  If the deviation is not statistically significant. Calculate the χ2 value 4. Compare the calculated χ2 value with the χ2 probability table (find critical χ2 value) 5. In summer squash. Calculate the expected number of individuals per phenotype 3. It is hypothesized that this could be either due to dominant and recessive epistasis. except that the homozygous recessive conditions of both genes are epistatic to their dominant counterparts. Write a conclusion 8K.take 0. the deviation is statistically significant)  If the deviation is statistically significant. Use the χ2 test to check which hypothesis is correct. thus the expected ratios are correct. 380 plants were found to have white flowers while 100 plants were found to have blue flowers.g.Version 2.

84. Calculate χ2 value F1 Male Gametes WK ○ Wk ○ wK ○ wk ○ WK ○ Wk ○ WWKK WWKk WwKK WwKk WWKk WWkk WwKk Wwkk wK ○ WwKK WwKk wwKK wwKk wk ○ WwKk Wwkk wwKk wwkk 13 (9 W_K_ + 3 W_kk + 1 wwkk) 13 White Flowers : : 3 wwK_ 3 Blue Flowers Classical Mandelian Monohybrid Inheritance Dominant and Recessive Epistasis H0: There is no difference from the 3:1 ratio HA: There is a difference from the 3:1 ratio Expected number of White Flowers = 3/4 x 480 = 360 flowers Expected number of Blue Flowers = 1/4 x 480 = 120 flowers H0: There is no difference from the 13:3 ratio HA: There is a difference from the 13:3 ratio Expected number of White Flowers = 13/16 x 480 = 390 flowers Expected number of Blue Flowers = 1/4 x 480 = 90 flowers χ2 = ∑ (O − E)2 ? = (380 − 360)2 360 + (100 − 120)2 120 χ2 = ∑ (O − E)2 = 4. thus the probability that chance alone is the reason for the difference in the observed results from the expected results is less At a degree of freedom of 1.84 χ2 crit = 3.44 is higher than that of the critical χ2 value at 3. Conclude wk ○ ○ ○ ○ ? = (380 − 390)2 390 + (100 − 90)2 90 = 1.37 is lower than that of the critical χ2 value at 3.Version 2. Calculate Degree of Freedom 5. Find out χ2 crit value 6.44 4. the calculated χ2 value at 4. the calculated χ2 value at 1. State Hypotheses 2 Calculate expected number of individuals for each phenotypic class 3.5 Random Fertilization F1 Male Gametes W ○ F1 Female Gametes F1 Genotypic Ratio F1 Phenotypic Ratio 1 WW w ○ W ○ WW Ww w ○ Ww ww : 3 White Flowers 2 Ww : : 1 ww 1 Blue Flower CASE 2: DOMINANT & RECESSIVE EPISTASIS Let K represent the dominant allele for the synthesis of blue pigment Let k represent the recessive allele preventing the synthesis of the blue pigment Let W represent the dominant allele preventing the synthesis of the blue pigment Let w be the recessive allele allowing the synthesis of blue pigment Parental Phenotype White Flowers X White Flowers Parental Genotype WwKk X WwKk Parental Gametes wk wK wK ○ Wk Wk WK WK ○ ○ ○ Random Fertilization F1 Female Gametes F1 Genotypic Ratio F1 Phenotypic Ratio 1.84. thus the probability that chance alone is the reason for the difference in the observed results from the expected results is more than 5%.37 df = n -1 = 2-1 = 1 df = n -1 = 2-1 = 1 χ2 crit = 3. The 120 | P a g e .84 At a degree of freedom of 1.

the second hypothesis is correct. expected 13:3 ratio is not significant. it can be concluded that the results are caused by dominant and recessive epistasis. Thus. *Answer to the question 121 | P a g e . Thus.5 than 5%.Version 2. accept reject Ho in favour of HA H0 From the results of the two χ2 tests. Thus. The deviation of the observed results deviation of the observed results from the from the expected 3:1 ratio is significant.

NADPH and carbon dioxide  Carbon Fixation 122 | P a g e . EXPLAIN THE LI GHT DEPENDENT REACTIONS OF PHOTOSY NTHESIS (NO BIOCHEMICAL DETAILS ARE NEEDED BUT WILL INCLUDE THE OUTLINE OF CYCLIC AND NON-CYCLIC LIGHT DEPENDENT REACTIONS. Light-independent reaction/ Dark Reaction  Production of glyceraldehyde-3-phosphate using ATP. producing oxygen. producing ATP (and NADPH)  Photolysis of water. Light harvesting  Capturing of light energy by a mixture of pigments (including chlorophyll) 2.5 6. 6A. CELLULAR PHYSIOLOGY AND BIOCHEMISTRY 6A – WITH REFERENCE TO T HE CHLOROPLAST STRUC TURE.1 AUTOTROPHIC NUTRITION  Nutrition – The process of acquiring energy and material for cell metabolism including the maintenance and repair of cells. AND TH E TRANSFER OF ENERGY FOR THE SUBSEQUENT MANUFACTURING OF CARBOHYDRATES FROM CARBON DIOXIDE). Light-dependent reaction/ Photophosphorylation/ Light Reaction  Excitation of a special chlorophyll a molecule light energy  Flow of electrons via ETC. electrons and hydrogen atoms 3. which is then used to provide energy for the Calvin Cycle.  Divided into 3 stages: 1.Version 2.  A form of nutrition used by autotrophs (organisms that make their own food)  Utilizes either chemical energy or light energy to make their own food via photosynthesis and chemosynthesis  Photosynthesis  Utilized by photoautotrophs  Provides a source of complex organic molecules for heterotrophic organisms  Releases oxygen as a by-product  Overall Equation: 6CO2 + 12H2O  C6H12O6 + 6O2 + 6H2O  Uses light energy to form ATP from ADP and Pi via photophosphorylation.

Version 2. FAD. ribosomes. serving as carriers of electrons or small functional groups  E. addition of hydrogen)  Decarboxylation  Removal of carbon atoms from a compound to form carbon dioxide  Coenzymes  Small molecules that function along with enzymes. addition of oxygen. the opposite can occur  Can be produced via  Oxidative phosphorylation  Substrate level phosphorylation  Photophosphorylation  Biological Reactions  Redox reactions  Oxidation (removal of electrons. which are connected by intergranal lamellae. removal of hydrogen)  Reduction (addition of electrons. removal of oxygen.5  Adenosine Triphosphate  Universal energy currency for all living organisms  Consists of an adenine nucleoside (adenosine) attached to 3 phosphate groups  Phosphate groups attached to each other via phosphoanhydride bonds  The breakage of one phosphoanhydride bond converts ATP to ADP + Pi and releases large amount of energy  Conversely. NAD.g.  Contains circular DNA.2 CHLOROPLAST STRUCTURE  Description  Lens-shaped membranous organelles  5µm . starch grains  Function 123 | P a g e .10µm in length  Surrounded by double-membrane (chloroplast envelope)  Double membrane  Outer membrane contains porins (proteins with large channels)  Inner membrane houses a semi-fluid compartment (Stroma) and only allows substances through it via transport proteins  Internal-membrane system (Thylakoids)  Enclose an area known as thylakoid lumen  Photosynthetic pigments and some proteins required for photosynthesis are embedded in thylakoid membrane  Thylakoid disc stacked on each other form a granum. NADP 6A.

 Carotenoids absorb excessive light energy and dissipate them. 124 | P a g e . depending on the type of chlorophyll  Types  Chlorophyll a  Major pigment that absorbs blue and red light  Participates directly in the photophosphorylation  Chlorophyll b  Accessory pigment that reflects green and blue light and pass on the light energy to chlorophyll a molecules  Associated with specific proteins to form light-harvesting complexes  Carotenoids  Accessory pigments that pass on the light energy to chlorophyll a molecules  Absorb strongly in the blue-violet range  Two main types: Carotenes and Xanthophylls  Functions  Broadening the spectrum of light of photosynthesis  Accessory pigments are able to absorb the intermediate wavelengths of light which chlorophyll cannot.Version 2. light absorbing porphyrin ring comprising of a magnesium atom in the centre  Helps to absorb red and blue-violet light  Magnesium deficiency causes less chlorophyll to be produced.3 PHOTOSYNTHETIC PIGMENTS. Carotenoids are inefficient as photosynthetic pigments and only transfer about 10% of their absorbed energy  Photoprotection  Carotenoids are required to absorb excessive light and prevent the auto-oxidation of chlorophyll which results in photobleaching as the chlorophyll molecules are destroyed. Stroma site of Calvin cycle Thylakoids site of light-dependent photophosphorylation 6A. ADSORPTION SPECTRUM AND ACTION SPECTRUM  Pigments – Compounds that reflect and absorb only certain wavelengths of light (thus appearing in certain colours)  Act as energy receiving antennas in photosynthesis  Two kinds: Chlorophyll and Carotenoids  Chlorophyll  Pigments that absorb mainly red and blue-violet light and reflect green light  Structure  One flat. resulting in yellowing of plants  A hydrophobic phytol tail (hydrocarbon tail) that keeps the chlorophyll molecule embedded in the thylakoid lipid bilayer membrane  Varying side chains on the chlorophyll head.5    Sites of photosynthesis where solar energy is converted to chemical energy by absorbing sunlight and using it to drive the synthesis of organic compounds from CO2. thus broadening the spectrum of colours that drive photosynthesis  But.

Version 2.5  Adsorption spectrum (phototrophs only absorb light in the visible light range)  A graph that shows the amount of light adsorbed at different wavelengths by a pigment  Action spectrum  A graph that shows the effectiveness of different wavelengths of light driving photosynthesis (adsorption rates of all pigments)  Light at wavelengths 450nm and 650nm are most effective (blue and red) 6A.  The energy then excites the chlorophyll a molecule causing one of its electrons to be excited to a higher energy level.5 PHOTOPHOSPHORYLATION (LIGHT REACTION)  Photosystems  Large protein complexes that catalyse the conversion of captured light energy in excited chlorophyll a molecules into useful forms  Embedded in the thylakoid membrane  Consists of  An antenna complex (light harvesting complex)  An reaction centre  Contains a pair of special chlorophyll a molecule that serves as an irreversible trap for energy as its excited electron is immediately passed on to a chain of electron acceptors in the same protein complex  Primary electron acceptor 125 | P a g e .  An electron donor (water) then donates electrons and reduces the chlorophyll a molecule back to its original oxidation state. causing it to be transferred to the primary electron acceptor.4 LIGHT HARVESTING STAGE  Takes place in the light harvesting complex of photosystems  Light energy is absorbed by pigments in the light harvesting complex and then transferred to neighbouring pigments via resonance energy transfer until the energy is transferred to the special chlorophyll a molecule. 6A.

activating catalytic sites on F1 at the same time. Cytochrome b6f complex and Plastocyanin (PC) (in this order)  The cascade of electrons down the ETC drives the pumping of hydrogen protons into the thylakoid lumen. Chemiosmosis: The diffusion of hydrogen protons down the concentration gradient from the thylakoid space acrss the thylakoid membrane into the stroma through ATP synthase.  Each protein electron carrier is reduced (receives and electron) and is then oxidized (donates an electron)  Coenzyme NADP works with NADP+ reductase to reduce hydrogen protons  First Electron Transport Chain  Consists of Plastoquinone (PQ). thus leading to the transfer of electrons from one protein to another. electrons are transferred from ferrodoxin to the first ETC.Version 2.  The impermeability of the thylakoid membrane to charged hydrogen protons causes the hydrogen protons to accumulate in the thylakoid lumen and not diffuse back into the stroma.  Involves the photolysis of water using light energy to form hydrogen protons. facing the stroma  Function: To act as a light-dependent system to generate ATP via cyclic photophosphorylation or transfer the electrons to ferrodoxin to reduce NADP+ Reductase to produce NADPH via non-cyclic photophosphorylation  Photosystem II  Contains a P680 reaction centre (its special chlorophyll a molecule has a maximum absorption of 680nm light (orange-red light))  Located only in the stacked regions of the granum  Function: To use light energy to reduce NADP to its fully reduced form NADPH with the assistance of PSI Electron Transport Chain  Consists of electron carrier proteins that are arranged in order of increasing electron affinity. electrons and hydrogen protons  In cyclic photophosphorylation. through the cytochrome b 6f complex  Generates the proton gradient  Second Electron Transport Chain  Consists of Ferrodoxin (Fd) and NADP+ reductase  NADP+ reductase catalyses the formation of NADPH from NADP+. they provide energy for ATP synthase to catalyse the formation of ATP from ADP and Pi (endergonic phosphorylation of ADP)  Consists of 2 components: a F0 stalk and the F1 particle (which catalyzes ATP formation)  Hydrogen atoms diffusing through the membrane passes through a small channel on the F0 stalk and rotates it. electrons and oxygen (by-product)  Divided into 3 stages: Charge Separation. thus maintaining the proton gradient Photophosphorylation  Primary Goal: To synthesize NADPH (Reduced nicotinamide adenine dinucleotide phosphate) and ATP to provide reducing power and energy respectively in the Calvin Cycle to synthesize glucose by fixing carbon dioxide. Electron Transport and Chemiosmosis 126 | P a g e .5      Two types: Photosystem I and Photosystem II  Photosystem I  Contains a P700 reaction centre (its special chlorophyll a molecule has a maximum absorption of 700nm light (red light))  Located exclusively in the stroma thylakoids and unstacked regions of the granum. ATP Synthase  A transmembrane enzyme that acts as a channel protein for hydrogen protons to diffuse out of the thylakoid lumen  As hydrogen protons diffuse out (exergonic passage of H+).

Version 2.5  2 possible routes: Cyclic and Non-cyclic Z-SCHEME OF PHOTOSYNTHESIS (NON-CYCLIC PHOTOPHOSPHORYLATION) 127 | P a g e .

The exergonic diffusion of hydrogen protons out of the thylakoid lumen into the stroma via chemiosmosis provides energy for the endergonic phosphorylation of ATP using ADP and Pi by ATP synthase. causing it to be captured by a primary electron acceptor. 2 electrons and an oxygen atom 5. The active pumping of hydrogen protons into the thylakoid lumen and the photolysis of water in the thylakoid lumen generates an electrochemical proton gradient between the thylakoid lumen and the stroma. This relay continues until the energy is captured by one of the special P700 chlorophyll a molecules in the PSII reaction centre 11. 16. producing 2 hydrogen proton. energy is released from the sequential process of reduction and oxidation of cytochromes and coupled to the active transport of hydrogen protons across the thylakoid membrane from the stroma into the thylakoid lumen. The photoexcited electron is passed from the primary electron acceptor down the second electron transport chain through ferrodoxin.5 Z-Scheme of Photosynthesis Light Harvesting 1. NADP+ reductase transfers electrons from ferrodoxin to NADP+. The electron from the electron transport chain is used to reduce the oxidized P700 special chlorophyll a molecule back to its original oxidation state. This excites one of the electrons of the P680 special chlorophyll a molecule to a higher energy state. One electron is used to reduce the oxidized P680 special chlorophyll a molecule back to its original oxidation state. This forms a proton motive force that causes protons to diffuse back into the stroma via ATP Synthase. 13. generating a proton gradient. A photon of light strikes a pigment molecule in the light harvesting complex of PSII and the energy is relayed to other pigment molecules via resonance energy transfer. causing it to be captured by a primary electron acceptor. 4. As the electron is passed down the electron transport chain. 7. 10. A photon of light strikes a pigment molecule in the light harvesting complex of PSI and the energy is relayed to other pigment molecules via resonance energy transfer. This excites one of the electrons of the P700 special chlorophyll a molecule to a higher energy state. This relay continues until the energy is captured by one of the special P680 chlorophyll a molecules in the PSII reaction centre Charge Separation + Photolysis of water Electron Transport Light Harvesting Charge Separation Electron Transport Chemiosmosis 3. 12. The oxygen atom combines with another oxygen atom to form oxygen molecules as a by-product. 6. 2. Molecules Involved Photosystem II LHC Pigment compounds P680 Chlorophyll A molecule P680 Chlorophyll A molecule Primary Electron Acceptor in PSII Plastoquinone Cytochrome b6f pump Plastocyanin Photosystem I LHC Pigment compounds P700 Chlorophyll A molecule P700 Chlorophyll A molecule Primary Electron Acceptor in PSI Ferrodoxin NADP+ Reductase ATP Synthase Hydrogen Protons ADP + Pi 128 | P a g e . 9. Light energy is used for the photolysis of water in the thylakoid lumen. reducing it adding a hydrogen proton to form NADPH 15. 14. 8. The photoexcited electron is passed from the primary electron acceptor down the electron transport chain until it reaches PSI.Version 2.

causing it to be captured by a primary electron acceptor. A photon of light strikes a pigment molecule in the light harvesting complex of PSI and the energy is relayed to other pigment molecules via resonance energy transfer. This relay continues until the energy is captured by one of the special P700 chlorophyll a molecules in the PSII reaction centre Charge Separation 3. 2. This excites one of the electrons of the P700 special chlorophyll a molecule to a higher energy state.5 CYCLIC PHOTOPHOSPHORYLATION Cyclic Photophosphorylation Light Harvesting 1.Version 2. Molecules Involved Photosystem I Pigment compounds P700 Chlorophyll A molecule P700 Chlorophyll A molecule 129 | P a g e .

10.5 4. Primary Electron Acceptor in PSI Ferrodoxin Plastoquinone Cytochrome b6f pump Plastocyanin ATP Synthase Hydrogen Protons ADP + Pi  Comparing Cyclic vs Non-cyclic Photophosphorylation Involves Water requirement Products By products Involvement of NADP+ reductase Formation of oxygen Involvement of plastoquinone First electron donator Electrons returned to photosystem Final Electron Acceptor Cyclic Photophosphorylation Photosystem I No ATP No Non-Cyclic Photophosphorylation Photosystems I and II Yes NADPH and ATP Oxygen Yes No No Yes Yes PSI (P700 molecules) No Water Yes NADP P700 in PSI  NADPH and ATP are generated in the stroma and used in the Calvin cycle 6B – OUTLINE THE THREE PHASES OF THE CALVIN CYCLE: (I) CO 2 UPTAKE (II) CARBON REDUCTION AND (III) RIBULOSE BISPHOSPHAT E (RUBP) REGENERATIO N AND INDICATE THE ROLES OF ATP AND NADP IN THE PROCESS.1 CALVIN CYCLE (DARK REACTION)  Location: Stroma of chloroplasts  Goal: To reduce carbon dioxide using ATP and NADPH  Molecules involved  Ribulose Biphosphate (RuBP) 130 | P a g e . 6. 8. 5. generating a proton gradient. Electron Transport Chemiosmosis The electron from the electron transport chain is used to reduce the oxidized P700 special chlorophyll a molecule back to its original oxidation state. The photoexcited electron is passed from the primary electron acceptor down the electron transport chain through ferrodoxin. The exergonic diffusion of hydrogen protons out of the thylakoid lumen into the stroma via chemiosmosis provides energy for the endergonic phosphorylation of ATP using ADP and Pi by ATP synthase. 9.Version 2. The electron is passed from ferrodoxin to plastoquinone 7. This forms a proton motive force that causes protons to diffuse back into the stroma via ATP Synthase. 6B. As the electron is passed down the first electron transport chain. The active pumping of hydrogen protons into the thylakoid lumen and the photolysis of water in the thylakoid lumen generates an electrochemical proton gradient between the thylakoid lumen and the stroma. energy is released from the sequential process of reduction and oxidation of cytochromes and coupled to the active transport of hydrogen protons across the thylakoid membrane from the stroma into the thylakoid lumen. The electron is then passed back to PSI. where it is used to reduce the oxidized P700 special chlorophyll a molecule back to its original oxidation state.

3-biphosphoglycerate Glyceraldehyde-3-phosphate (G3P) Ribulose Biphosphate Carboxylase Oxygenase (RuBisCo) Calvin Cycle Carbon Dioxide Uptake 1.3biphosphoglycerate 6 NADPH spent 6G3P 131 | P a g e . Carbon Dioxide diffuses through stomata into the cytoplasm of cells and into the chloroplasts Carbon dioxide combines with ribulose biphosphate (RuBP) to form an unstable 6-carbon intermediate. 3.Version 2. 1 phosphate group from each molecule is removed in this step Molecules Involved 3 Carbon Dioxide 3 RuBP RuBisCo 6 GP 6 ATP Spent 6 1. 5. fixing the carbon dioxide. to form 6 1. 2.5     3-Phosphoglycerate (GP) 1. catalysed by Ribulose Biphosphate Carboxylase Oxygenase (RuBisCo) The unstable 6-carbon intermediate breaks down into 2 3phosphoglycerate molecules Each 3-phosphoglycerate molecule is phosphorylated by 1 ATP.3-biphosphoglycerate is then reduced by NADPH to form 6 glyceraldehyde-3-phosphate.3-biphosphoglycerate Each 1. Carbon Reduction 4.

requiring 3 ATP in the process. generated with 6 turns of the cycle.  Photorespiration consumes ATP and does not produce sugar. RuBisCo tends to add oxygen to the Calvin Cycle  This produces 2 2-carbon product which is subsequently rearranged and split by peroxisomes and mitochondria. used to form other sugar molecules such as glucose  Problems with the Calvin Cycle  RuBisCo has an affinity for oxygen as well as carbon dioxide. It only acts to reduce the number of harmful substances generated by the light reaction on hot days. 7. 8. causing carbon dioxide concentrations to drop and oxygen concentrations to rise. Product Synthesis 9. The rest of the 5 glyceraldehyde-3-phosphates are used to form 3 ribulose biphosphate molecules.2 C 4 PLANTS  C4 plants use 2 types of cells for photosynthesis – bundle sheath cells and mesophyll cells  The Calvin cycle takes place exclusively in the bundle sheath cells (which surround the vascular tissue)  C4 plants first fix carbon dioxide into a 4-carbon molecule which breaks down into a 3-carbon molecule  This is catalysed by Phosphophenolpyruvate (PEP) carboxylase which adds carbon dioxide to PEP to form oxaloacetate  PEP Carboxylase is able to fix carbon dioxide efficiently as it has no affinity for oxygen unlike RuBisCo  This process requires additional ATP as carbon dioxide has to be fixed twice 132 | P a g e .Version 2. are combined to form a hexose sugar. This decreases the photosynthetic output of photosynthesis  Photorespiration is thus seen as a wasteful process 6B. thus on hot days when stomata is closed. One glyceraldehyde-3-phosphate is then exported as a product. 1 G3P Exported 3 RuBP Hexose sugar  Net Consumption  3 Carbon Dioxide Molecules  9 ATP molecules  6 NADPH molecules  Net Production  1 G3P sugar molecule.5 Ribulose Biphoshate Regeneration 6. The ribulose biphosphate molecules are then used in carbon fixation and the cycle repeats 2 G3P molecules. releasing carbon dioxide in a process called photorespiration. used to form other sugar molecules.

Version 2.5

C4 Carbon Fixation
C4 carbon fixation
in mesophyll cells

1.
2.

Bundle Sheath
Cells

3.
4.

Regeneration of
PEP

5.

PEP carboxylase catalyses the formation of Oxaloacetate from PEP
and Carbon dioxide
Oxaloacetate is rearranged into malate, which is then exported into
the bundle sheath cells
In the bundle sheath cells, malate is decarboxylated to form carbon
dioxide and pyruvate.
Carbon dioxide enters the Calvin cycle and pyruvate is transported
back to the mesophyll cells
Pyruvate is converted to PEP using ATP

Molecules Involved
Carbon Dioxide
PEP Carboxylase
PEP
Oxaloacetate
Malate
Carbon Dioxide
Pyruvate
(Calvin Cycle
Components)
Pyruvate
ATP
PEP

6B.3 CRASSULACEAN ACID METABOLISM (CAM) PLANTS
 Uses very similar mechanisms to C4 plants, but rather than separating the steps into 2 cells, CAM plants
separate the steps temporally.
 In the night, the stomata open to let carbon dioxide in, which is fixed and stored into 4-carbon
intermediates.
 In the day, the stomata close to conserve water. The 4-carbon intermediates are decarboxylated to
generate carbon dioxide. In the presence of light, photophosphorylation takes place and supplies ATP
and NADPH for the Calvin cycle.
 All this happens in the same cell.

6B.4 USE OF PHOTOSYNTHETIC PRODUCTS
 A large proportion is converted into hexose sugars, such as glucose and fructose. These molecules are
often stored as starch or polymerised to form cellulose.
 They can also be converted into lipids (enter glycolysis) and proteins

6B.5 ROLE OF COENZYME NADPH
 Final electron acceptor in non-cyclic photophosphorylation, allows for electrons to continuously move
down the ETC in a unidirectional manner. The energy released is coupled to the generation of a proton
gradient which is harnassed in ATP synthesis.
 NADP accepts 2 electrons and 1 hydroge ion to form NADPH, a source of reducing power in the Calvin
cycle. NADPH reduces 1,3-biphosphoglycerate to G3P which is used for the synthesis of other organic
compounds.

6B.6 ROLE OF MEMBRANES
 Thylakoid membrane holds the photosystems and the electron transport chain in a fixed position; allows
protein complexes to be placed near each other to maximize the efficiency of electron flow and reduce
energy lost when the electron is transported. (from enzymes)
 Thylakoid membrane maintains the proton gradient since the membrane is impermeable to hydrogen
protons and does not allow hydrogen protons to simply diffuse back into the stroma (photosynthesis)
 Organization and Localization of Function – Double membrane of chloroplast is selectively permeable to
solutes and allows for the compartmentalization of enzsmes in the Calvin Cycle. (from cell membrane)

133 | P a g e

Version 2.5

6C – DISCUSS LIMITING FACTORS IN PHOTOSYNTHESIS AND CARRY OUT
INVESTIGATIONS ON THE EFFECTS OF LIMITING FACTORS, SUCH AS LIGHT INTENSITY,
CO 2 CONCENTRATION AND TEMPERATURE, ON THE RATE OF PHOTOSYNTHESIS.
6C.1 – LIMITING FACTORS OF PHOTOSYNTHESIS
 A limiting factor is a factor that will directly affect the rate of reaction if its quantity is changed. Usually the
factor is at its minimal value and the rate of reaction is affected by several factors.
 (Recall factors affecting enzyme catalyzed reactions 1J)
 Light as a limiting factor
 At low light intensity, photosynthetic rates increases linearly as light intensity increases, thus light is
the limiting factor.
 However, once light saturation point is reached, all the available chloroplasts are fully occupied in
light absorption, thus light is no longer the limiting factor although light intensity increases.
 Compensation Point  When the rate of photosynthesis is equivalent to the rate of respiration, thus
there is no gaseous exchange between the plant and the environment.
 Light intensity is unlikely to be a limiting factor in most natural environments except at early
moning/rainy days/deep shade/etc.
 Role of light in photosynthesis: Photolysis and the excitation of an electron to a higher energy level,
which is then passed along a series of electron acceptors. The energy released is coupled to ATP
synthesis.
 Under natural conditions, carbon dioxide concentration is the major limiting factor
 As the rate of carbon dioxide increases, the rate of photosynthesis increases and tails off as carbon
dioxide is no longer the limiting factor.
 Temperature can also be a limiting factor as the photosynthesis is an enzyme controlled reaction and is
therefore temperature sensitive.
 Chlorophyll concentration can also be a limiting factor due to a lack of iron, magnesium and nitrogen,
which causes leaves to turn pale yellow and eventually stop photosynthesis due to the inability to
synthesize chlorophyll molecules (chlorosis)
 Can also be caused by senescence, lack of light, diseases
 Light wavelength
 Affects adsorption and action spectrum
 Use of inhibitors (e.g. DCMU)
 Inhibitors can be used to block electron flow between photosystems
 Poisons that bind irreversibly to plant photosynthetic enzymes such as ozone/sulphur
dioxide/superoxides
 Water
 Plants close their stomata to prevent water loss, resulting in restricted access to carbon dioxide
 Forces plants to switch over to cyclic photophosphorylation, causing NADPH to be in limited supply.
 Affects the rate at which Calvin Cycle can proceed
 Pollution
 Low levels of certain pollutive gases such as ozone and sulfur dioxide can damge the leaves of the
plants.
 Soot can block stomata and reduce the transparency of the leaf epidermis.
 Oxygen (competitive inhibitor)
 Inhibits photosynthesis by competing with carbon dioxide for the active site in RuBisCo
 Results in photorespiration and a decrease in the overall rate of photosynthesis

134 | P a g e

Version 2.5

6C.2 – INVESTIGATE THE FACTORS AFFECTING PHOTOSYNTHESIS
 Test for starch
 Varying light intensity
 Change the distance of the light bulb to the beaker + Use heat buffer (such as water to absorb the
heat)
 Sodium Bicarbonate concentration, temperature, light bulb power should remain constant
 Varying carbon dioxide concentration
 Change the concentration of sodium bicarbonate (can also use sodium hydroxide and potassium
hydroxide to remove carbon dioxide)
 Power of light bulb, distance of light bulb to plant, temperature should remain constant
 Varying temperature
 Change the temperature of the different water baths.
 The power of the light bulb, the sodium bicarbonate concentration and the distance of the bulb to the
plant should remain constant

6C.3 – SUN AND SHADE LEAVES
 Sun plants are adapted to grow in full sunlight while shade plants are adapted to grow in permanently
shaded positions.
 Shade plants have
 Thinner leaves with fewer palisade mesophyll layers
 Thus, they reach light compensation points at lower rates of photosynthesis as fewer cells require less
maintenance
 However, they are also unable to achieve high rates of photosynthesis due to a lack of photosynthetic
cells
 Light compensation point is the point where the rate of photosynthesis is equivalent to the rate of
respiration, thus the net gaseous exchange is 0.
 In this graph, the shade leaves are seen to require a lower light intensity to reach light compensation
point. This is due to the fact that shade leaves have a lower rate of respiration than sun leaves and will
require a lower light intensity to get their rate of photosynthesis to be higher than the rate of respiration,
thus shade leaves have a higher photosynthetic rate than sun leaves in shady areas, as sun leaves often
have a higher respiratory rate and would a higher intensity of light to get their photosynthetic rates above
their respiratory rates. However, in sunny areas, at the light saturation point, sun leaves have a higher
photosynthetic rate than shade leaves, as sun leaves are able to make full use of the strong light intensity
to increase their photosynthetic rates while shade leaves are unable to do so. Thus, this shows that shade
leaves are adapted to low light intensity while sun leaves are adapted to high light intensity.

Net CO2 uptake

Sun growth
Higher rate of respiration

Max photosynthetic rate

Shade growth

Light Intensity

Light compensation point
Shady areas

Light saturation point
135 | P a g e
Sunny areas

Version 2.5

6D – LIST AND GIVE AN OVERVIEW OF THE 4 STAGES OF AEROBIC RESPIRATION AND
INDICATE WHERE EACH STAGE TAKES PLACE IN AN EUKARYOTIC CELL AND
MITOCHONDRIA, AND ADD UP THE ENERGY CAPTURED (AS ATP, REDUCED NAD AND
FAD) IN EACH STAGE.
6D.1 OVERVIEW OF METABOLISM
 Metabolism includes many specific metabolic pathways
 Catabolism  Degradation/Breakdown of absorbed food substances into simpler, smaller molecules
 Used to release energy required to drive cellular functions
 Formation of building blocks for biosynthesis
 Anabolism  Biosynthesis of complex molecules from simpler compounds
 Synthesize polymers for the growth and repair of the cell
 Methods of forming ATP
 Substrate-Level phosphorylation
 Involves an enzyme catalysed reaction where a phosphate group is transferred from a substrate
to ADP to form ATP
 Produces only a small amount of ATP in glycolysis and the Krebs Cycle
 Oxidative phosphorylation
 Coupling of energy released from the exergonic transport of electrons down the ETC to the final
electron acceptor molecule to the enzymatic endergonic phosphorylation of ADP to form ATP
 Produces large amounts of ATP in oxidative phosphorylation
 Photophosphorylation (photosynthesis light reaction)
 Coenzymes used in respiration – serves as resevoirs of electrons and protons to form ATP via oxidative
phosphorylation [Each reduced coenzyme carries 1 pair of electrons]
 Nicotinamide adenine dinucleotide (NAD)
 Flavin adenine nucleotide (FAD)
 Organic Reactions
 Redox Reactions
 Oxidation – Addition of Oxygen/Removal of Hydrogen
 Reduction – Addition of Hydrogen/Removal of Oxygen
 Decarboxylation – The Removal of carbon atoms from a compound to form carbon dioxide
 Dehydrogenation – Removal of hydrogen atoms

6D.2 RESPIRATION
 Can be anaerobic and aerobic
 Aerobic respiration involves 4 stages
 Glycolysis – Cytoplasm
 Link Reaction – Mitochondrial
matrix
 Krebs Cycle (Citric Acid
Cycle/Tricarboxylic Acid Cycle) –
Mitochondrial Matrix
 Oxidative Phosphorylation –
Mitochondrial Inner Memb
 Anaerobic Respiration = Fermentation
 Involves lactic acid fermentation or
ethanol fermentation
136 | P a g e

3 GLYCOLYSIS  Location: Cytoplasm of all cells  Metabolism  10 step catabolic pathway that splits glucose into 2 pyruvate molecules  Involves the reduction (dehydrogenation) of NAD into NADH and substrate level phosphorylation of ADP into ATP  Overall Substrates  1 Glucose molecule  ADP + Pi  NAD+ (oxidizing agent)  Net output  2 pyruvate molecules  2 ATP (net +)  2 NADH  Water  Major phases (10 step reaction)  Energy-investment phase  1-3: Activation of glucose  4-5: Cleavage/Lysis  Energy-payoff phase  6: Oxidation/Dehydrogenation  7-10: Substrate Level phosphorylation 137 | P a g e .Version 2.5 6D.

stimulated by AMP. [Step 3 is the rate-limiting step] Step 4-5: Lysis.  Rate Limiting Enzyme – Phosphofructokinase [Alosterically inhibited by ATP. 5.6-biphosphate.4 ADP.6-biphosphate  Dihydroxyacetone Phosphate  Glyceraldehyde-3-phosphate (G3P)  Hexokinase  Phosphogluco-isomerase  Phospho-fructokinase  Aldolase  Isomerase  .Version 2. 4. + 4 ATP  G3P  1. Step 7 & 10: Substrate-level phosphorylation of ADP occurs. it is their only mode of ATP formation  Glycolysis rates increase when muscle cells require more energy but lack the oxygen to produce it  Supplies cells with essential biosynthetic precursors  Liver cells require glycolysis to provide it with precursors for a wide range of molecules it synthesize  Red Blood cells with no mitochondria produce energy solely from glycolysis  Microorganisms such as yeast and E. Each G3P is oxidized and NAD is reduced to form NADH.2 NAD+. 2 pyruvate molecules are produced at the end of glycolysis Molecules Involved  Glucose  Glucose 6-phosphate  Fructose 6-phosphate  Fructose 1. There is a net gain of 2 ATP per glucose molecule.6-bisphosphate is cleaved to form 2 G3P. . syncrhonizes the rates of glycolysis and the Krebs Cycle] 6D. There is a net production of NADH per glucose.coli also require energy and biosynthetic precursors from glycolysis  Role of Glycolysis  Hydrolysis of 1 molecule of glucose to form 2 molecules of pyruvate which then enter the mitochondria to be completely oxidized  Production of 2 net ATP by substrate level phosphorylation and 2 NADH to supply high energy electrons for oxidative phosphorylation. Energy Payoff Phase 3. Step 6: Oxidation/Dehydrogenation.2 Pi. catalysed by Pyruvate Dehydrogenase  Occurs in the mitochondria matrix 138 | P a g e . 2.3-biphosphoglycerate  3-phosphoglycerate  2-phosphoglycerate  Phosphoenol-pyruvate (PEP)  Triose Phosphate Dehydrogenase  Phospho-glycerokinase  Phospho-glyceromutase Enolase  Pyruvate Kinase  +2 Pyruvate  Importance of glycolysis  Glycolysis is the only catabolic reaction that can be completed without oxygen  Thus for mammalian red blood cells (with no mitochondria). + 2 NADH  . Energy is provided by hydrolysing 2 ATP molecules.4 LINK REACTION/PYRUVATE OXIDATION  Conversion of pyruvate into Acetyl CoA which enters the Krebs Cycle via oxidative decarboxylation. Fructose 1. Each NADH supplies 2 energised electrons for ATP production via oxidative phosphorylation. producing 4 ATP molecules. Step 1-3: Activation of Glucose Glucose is activated by phosphorylation to fructose 1.2 ATP  .5 Glycolysis Energy Investment Phase 1.

5  1 of the carbons of pyruvate (carboxylic acid group) is removed as carbon dioxide and the 2 carbon intermediate is added to coenzyme A to form acetyl-CoA  2 electrons and a hydrogen proton are removed from the 2 carbon intermediate and added to NAD+ to form NADH  2 pyruvate + 2 NAD + 2 CoA  2 Acetyl CoA + 2 NaDH + 2 CO2 6D. NAD+ 4. Succinate dehydrogenase 7.4 KREBS CYCLE  Location: Mitochondrial Matrix  Metabolism  8 step catabolic pathway that occurs twice for every glucose molecule broken down  Known as a cycle as oxaloacetate which is added to incoming acetyl CoA is regenerated at the end of one turn of the cycle. 2 FADH2 2. FAD 5. 139 | P a g e . Aconitase 3.8  Involves substrate level phosphorylation of ADP into ATP at step 5  Involves the reduction (hydrogenation) of FAD into FADH 2 at step 6  Main goal: To produce large amounts of high energy electron carries to drive chemiosmosis via the exergonic passage of electrons down the electron transport chain. Malate dehydrogenase  Overall Substrates 1. 2 ATP Waste product – 4 CO2 4. H2O  Net output (per 1 glucose) 1. ADP + Pi 3.4.Version 2.  Involves the reduction (hydrogenation) of NAD into NADH at steps 3. α-ketoglutarate dehydrogenase 5. 6 NADH 3. 1 Acetyl-CoA 2.  Enzymes involved in TCA steps 1. Isocitrate dehydrogenase 4. Citrate Synthase 2. Fumarate hydratase 8. Succinyl CoA synthetase 6.

+2 NADH  + 2 CO2  Citrate Synthase  Aconitase  Isocitrate dehydrogenase  α-ketoglutarate dehydrogenase  Succinyl CoA  GTP 140 | P a g e . 4. Citrate is converted to isocitrate Oxidative decarboxylation of isocitrate results in the removal of one carbon atom as a CO2 molecule. This converts isocitrate to αketoglutarate Oxidative decarboxylation of α-ketoglutarate results in the removal of one carbon atom as a CO2 molecule.Version 2. Coenzyme A is then added. reducing 1 NAD+ molecule to form 1 NADH. reducing 1 NAD+ molecule to form 1 NADH. NAD+ reduction 1 &2 1.5 Krebs Cycle Oxidative decarboxylation. 2. Formation of ATP 5. 3. converting α-ketoglutarate to Succinyl CoA Exergonic hydrolysis of Succinyl CoA to form coenzyme A and succinate provides energy for the substrate-level Molecules Involved  Acetyl-CoA  Oxaloacetate  Citrate  Isocitrate  α-ketoglutarate  -2 NAD+. Acetyl CoA is added to oxaloacetate to form citrate.

NAD+ reduction 3 6. + 1NADH 6D.5 OXIDATIVE PHOSPHORYLATION  Location: Inner mitochondrial membrane  Functions: To release the energy stored in high energy electrons in FADH2 and NADH to drive chemiosmosis via the exergonic passage of electrons down the electron transport chain. III and IV are responsible for pumping protons into the intermembrane space. 7. (and oxygen)  Flavoproteins such as FMN (located in protein complex I)  Ubiquinone/Coenzyme Q that transports electrons between protein complexes I/II and III  Cytochrome C that transports electrons between protein complexes III and IV  Cytochromes that form the rest of the electron transport chain  Cytochromes have a heme group that are able to receive and donate electrons to subsequent electron carrier proteins. . GTP then donates a phosphate group to ADP to form ATP and reform GDP. causing FAD to be reduced to FADH2 Fumarate is converted to malate Malate is oxidized to form oxaloacetate.  ATP Synthase  A transmembrane enzyme that acts as a channel protein for hydrogen protons to diffuse out of the thylakoid lumen  As hydrogen protons diffuse out (exergonic passage of H+). 141 | P a g e . thus leading to the one-way transfer of electrons from one protein to another. causing NAD+ to be reduced to NADH. & To regenerate FAD and NAD.  Electron Transport Chain  Consists of electron carrier proteins that are arranged in order of increasing electron affinity. they provide energy for ATP synthase to catalyse the formation of ATP from ADP and Pi (endergonic phosphorylation of ADP)  Consists of 2 components: a F0 stalk and the F1 particle (which catalyzes ATP formation)  Hydrogen atoms diffusing through the membrane passes through a small channel on the F0 stalk and rotates it. forming GTP. + 1 FADH2 .Version 2.1 Pi Succinate .1 FAD.1 H2O Fumarate Malate Oxaloacetate -1 NAD+. Succinate is oxidized to fumarate.1 ADP. . 8.          GDP + 1 ATP. activating catalytic sites on F1 at the same time.5 Formation of FADH2 Regeneration of Malate. phosphorylation of GDP.  Role of oxygen  Oxygen is the final electron acceptor and reacts with protons to form water [Maintains unidirectional electron flow]  Allows for FADH2 and NADH to be oxidized to FAD and NAD  Each protein electron carrier is reduced (receives and electron) and is then oxidized (donates an electron)  Protein complexes I.

6 SHUTTLE SYSTEMS  NADH produced from glycolysis is unable to directly penetrate into the mitochondria due to the lack of a transport protein. 3. 6. [Results in low pH] 5. 7. The exergonic passage of hydrogen protons out of the intermembrane space into the mitochondrial matrix via chemiosmosis provides energy for the endergonic phosphorylation of ATP using ADP and Pi by ATP synthase. 142 | P a g e . NADH and FADH2 shuttle high-energy electrons extracted during glycolysis and the Krebs cycle into an electron transport chain build in the inner mitochondrial membrane. FADH2 donates its electrons to protein complex II and NADH donates its electrons to protein complex I. FAD and NAD+ are thus regenerated to be reused in glycolysis and the Krebs Cycle 4. The exergonic cascade of electrons down the electron transport chain releases energy for the active transport of protons across the inner mitochondria membrane into the inter-membrane space.5  Chemiosmosis: An energy-coupling mechanism that uses energy stored in the form of a proton gradient across a membrane to drive cellular work Oxidative Phosphorylation 1. Molecules Involved  FADH  NADH  Protons  Electron Transport Chain protein complexes  Ubiquinone  Cytochrome C  Oxygen  ATP synthase  + Water *Cascade of electrons from complexes I to IV pumps 3 protons while the cascade of electrons from complexes II to IV pumps 2 protons Note: Inner mitochondrial membrane not permeable to NADH and H + 6D. Oxygen acts as the final electron acceptor and is reduced to form water. resulting in a proton motive force that causes protons to diffuse back into the mitochondrial matrix via ATP Synthase. 2.Version 2. The continuous pumping of protons into the intermembrane space creates an electrochemical proton gradient.

5  The final amount of ATP generated depends on which shuttle system is used  Shuttle systems  Glycerophosphate shuttle  Electrons are stripped from cytosolic NADH and then transferred to FAD via an intermediate.5 ATP is formed for every cytosolic NADH oxidized  Found in liver and heart muscle cells  Account for the total number of ATP produced (per molecule of glucose)  Yield of oxidative phosphorylation 2. glycerol-3-phosphate  1. malate and asparate  2.Version 2.5 ATP/NADH and 1.5 ATP formed for every cytosolic NADH oxidized  Found in muscle and nerve cells  Malate-Asparate shuttle  Electrons are stripped from cytosolic NADH and then transferred to matrix NAD + via intermediates.10 0 FADH2 0 +2 -2 + 36/38 ATP +2 0 +2 + 32/34 143 | P a g e .5 ATP/FADH2 Glycolysis Link Reaction Krebs Cycle Oxidative Phoshorylation Total 0 NADH +2 +2 +6 .

7 OXIDATION OF FATS AND PROTEINS  Carbohydrates need to be broken down into monomers such as glucose before they can be fed to the glycolysis pathway for catabolism  Proteins need to be broken down into amino acids.5 6D. AcetylCoA produced from the beta oxidation pathway enter the Krebs Cycle  The beta oxidation pathway also produces FADH2 and NADH which deliver electrons for oxidative phosphorylation 144 | P a g e . which are then subsequently deanimated (waste removed as urea or other nitrogenous waste) and then used to form intermediates in the Krebs Cycle or glycolysis  Fats are first broken down into glycerol and fatty acids  Glycerol is converted to G3P which enters glycolysis  Fatty acids enter the beta oxidation pathway until they are completely broken down.Version 2.

 Fermentation  Involves mainly glycolysis. Pyruvate is converted into acetaldehyde via oxidative  2 Acetaldehyde of NAD+ decarboxylation which removes 1 of the carbons as molecules carbon dioxide  + 2 NAD+  + 2 Ethanol 145 | P a g e . generating 2 molecules of ATP and  2 Pyruvate 2 molecules of NADH. fungi and in animals  Lactic acid is toxic but can be reconverted to pyruvate which is then undergoes aerobic respiration  Alcoholic Fermentation  Occurs in yeast and in most plant tissues  The energy in ethanol remains locked and cannot be used to form pyruvate  Occurs when the rate of oxygen supply cannot meet the oxygen demand to generate sufficient ATP via aerobic respiration.Version 2. Alcoholic Fermentation Molecules Involved Glycolysis 1. 1 Glucose molecule undergoes glycolysis to produce 2  1 Glucose molecules of pyruvate.5 6E – EXPLAIN THE PRODUCTION OF A SMALL YIELD OF ATP FROM ANAEROBIC RESPIRATION AND THE FORMATION OF ETHANOL IN YEAST AND LACTATE IN MAMMALS. no acetyl-CoA is formed and no additional ATP can be generated  Uses organic molecules as final electron acceptors  Oxygen independent process  Happens in the cytoplasm  Two types  Lactic Acid Fermentation  Occurs in some bacteria. along with a chemical process that regenerates the NAD+ required for glycolysis  No electron transport chain is used  Electrons are transferred from NADH to pyruvate to oxidize NADH to NAD+  No further oxidation of pyruvate occurs.  + 2 ATP  + 2 NADH  (intermediates + enzymes) Regeneration 2.

forming lactate/ethanol - Present in Prokaryotes & Eukaryotes Prokaryotes & Eukaryotes ATP yield Oxygen Full oxidation of glucose Very high (36~38) Required Yes Very low (2) Not required No Location Cytoplasm + Mitochondria Cytoplasm  Respiratory Quotient  The volume of carbon dioxide produced divided by the volume of the oxygen used during respiration  When RQ = 1.7. anaerobic respiration is taking place (may occur in the shortage of oxygen) 146 | P a g e .  When RQ = 0. forming lactate and  2 Pyruvate of NAD+ NAD+  + 2 NAD+  + 2 Lactate  Comparing between the 3 different forms of respiration Glycolysis Krebs Cycle Electron Transport Chain/ ATP Synthase Utilizes Chemiosmosis Regeneration of NAD+/ FAD Aerobic Present Present Present Fermentation Present Absent Absent Yes Via oxidative phosphorylation Final electron acceptor molecule Oxygen No Via reduction of sugars. Acetaldehyde is used to oxidize NADH.99. aerobic respiration is taking place and carbohydrates are used as the respiratory substrate.5 3.Version 2. forming ethanol and NAD+ Lactic Acid Fermentation Molecules Involved Glycolysis 1. 1 Glucose molecule undergoes glycolysis to produce 2  1 Glucose molecules of pyruvate. Pyruvate is used to oxidize NADH.  When RQ =0.  + 2 ATP  + 2 NADH  (intermediates + enzymes) Regeneration 2. generating 2 molecules of ATP and  2 Pyruvate 2 molecules of NADH. aerobic respiration is taking place and proteins are used as the respiratory substrate.  When RQ > 1. aerobic respiration is taking place and fats are used as the respiratory substrate.

4) glycosidic bonds Amylose is coiled into a helical shape due to bond angle of α(1.4) glycosidic bonbds Amylopectin is highly branched due to presence of α(1. Insoluble Large store of carbon and energy (as a respiratory substrate to produce ATP) Easily hydrolyzed by enzymes present in animals and plants Compact shape Compact Shape for easier storage Easily hydrolyzed due to presence of many branches.Version 2. thus allowing more enzymes to act on it at any one time Starch is chemically unreactive and chemically stable. Functions/Properties Ideal as storage material as it is insoluble – Does not affect water potential within cells and living organisms.4) + α(1.4) α-glucose Straight chain of glucose coiled into a more compact helical shape Poorly Soluble in water (does not exert osmotic influence) α(1. thus allowing more enzymes to act on it at any one time Iodine Test Centre of helix is hydrophobic.6) glycosidic bonds Most anomeric carbons are used in glycosidic bond formation leaving behind few free anomeric hydroxyl groups  Highly Compact and can contain up to twice as many glucose units as amylose Red violet colouration when iodine in potassium iodide is added to amylopectin in solution.6) α-glucose Branched chain of glucose Branch occurs every 12-30 residues **Also helical coiling Easily hydrolyzed due to presence of many branches. ideal as storage compound Glycogen (very very similar to amylopectin) 147 | P a g e . allowing iodine in potassium iodide solution to pack within to give rise to a blue-black colouration  Function Structure Macromolecule Contains several hundreds to thousands of glucose monomers Linked by α(1. GLYCOGEN AND CELLULOSE AND THEIR ROLE IN PLANTS/ANIMALS  Polysaccharides  Purpose  Structural and Storage materials  Starch  Repeating units  α-Glucose  Compound units Percentage Composition of Starch Glycosidic Bonds Monomers Structure Properties Tests Amylose 10-30% Amylopectin 70-90% α(1.5 6F – COMPARE THE STORAGE AND STRUCTURAL FORMS OF STARCH.

 Long unbranched straight chain. thus allowing more enzymes to act on it at any one time Glycogen is chemically unreactive and chemically stable. with hydroxyl groups projected outwards  Hydrogen bonds form between protruding hydroxyl groups of neighbouring chains.6) Structure: Extensive branched chain of glucose. which are laid down in the cell wall.  Properties  High tensile strength due to cross linking gives cellulose stability as a structural material  Permits the cell wall to withstand forces exerted in all directions as a result of cellulose fibres being laid down in different orientations in different layers of the cell wall  Full permeability to water and solutes to ensure proper functioning of cell wall.4) results in a helical shape due to bond angle so that the structure is more compact and ideal for storage Compact Shape for more efficient storage Easily hydrolyzed due to presence of many branches.4)  Structure  Alternate monomers rotated 180° to obtain β(1. which further associates with other non-cellulose polysaccharides to form macrofibril. Branch occurs every 8-12 residues (more compact than amylopectin) Test: Iodine tests gives a red-violet colour Function Structure Macromolecule Contains several hundreds to thousands of glucose monomers Glucose units linked by α(1.6) glycosidic bonds Most anomeric carbons are used in glycosidic bond formation leaving behind few free anomeric hydroxyl groups  Functions/Properties Ideal as storage material as it is insoluble – Does not affect water potential within cells Insoluble Large store of carbon and energy (as a respiratory substrate to produce ATP) α(1.4) can be hydrolyzed by glycogen phosphorylase α(1.4) glycosidic bonds (also helical coil) Glycogen is highly branched due to presence of α(1. a fuel source to generate ATP for muscle contraction Composed of : α-Glucose Glycosidic Bonds: α(1.4) glycosidic bonds. unbranched.4) + α(1.  Function Structure Alternately inverted β-Glucose units linked by β(1.Version 2.4) glycosidic bonds Functions/Properties Long.5       Found in  Liver as a source of glucose to maintain blood sugar levels  Skeletal Muscle in the form of cytoplasmic granules. establishing rigid cross-links between parallel chains  These parallel cross-linked chains associate in groups to form microfibril. parallel chains 148 | P a g e . ideal as storage compound Cellulose  Found in  Cell wall. where it makes up 50%  Purpose: Provide Structural Support  Composed of: β-Glucose  Glycosidic Bonds: β(1.

Localization of electron carrier complexes on the thylakoid membranes of chloroplasts (ETC) & Location of lysozymes within lysosomes  Signal transduction – Detects specific signals (chemical) that bind to specific protein receptors. PROTEINS & GLYCOPROTEINS 6G. 149 | P a g e . Selectively permeable cell membrane allows for desirable substances to be kept within and undesired substances to be kept out.e.4) glycosidic bonds Allows extensive hydrogen bonds to form between parallel chains resulting in the formation of cross-links which are grouped into microfibrils. E. define boundaries of organelles). resulting in high tensile strength for structural support. INCLUDING AN OUTLINE OF THE ROLES OF PHOSPHOLIPIDS. ions.1 FUNCTION OF CELL PLASMA MEMBRANE  Define the boundaries of the cell  Keeps the interior of the cell physically separated from the surrounding environment.Version 2.g.5 Long and unbranched with hydroxyl groups projecting outwards from cellulose chain (as a result of β(1. Higher tensile strength for structural support Insoluble 6G – DESCRIBE AND EXPLAIN THE FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE.  thus maintaining cellular homeostasis for optimum chemical reactions  Regulation of cell’s contents (allows for control of substances entering and exiting the cell)  To take up useful substances such as water. glucose. etc. GLYCOLIPIDS. thus triggering specific responses  Cell-Cell communication – Membrane proteins that bind to the extracellular matrix or cell surface constituents that control cell-cell adhesion (glycolipid) & cell-cell communication.4) glycosdic bonds) Microfibril associated in bundles to form macrofibril Macromolecule Not easily hydrolyzed by acid or digested by enzymes as few organisms have cellulose required to breakdown β(1.  Removal of metabolic waste products  Confinement of certain chemicals to certain parts of the cell  Organization and localization of functions (i.

support for cell lost  Low temperatures will inactivate proteins and cause ice crystals to form 6G.  Dynamic yet stable  Due to molecular interactions  Hydrophilic portions of proteins and phosphate groups of phospholipids face outwards  Carbohydrate antennae face outwards  Hydrophobic portions of proteins and hydrocarbon tails of phospholipids face into the middle of the membrane. 8nm)  Allows small and hydrophobic molecules to diffuse across the phospholipid bilayer freely (such as N2.3 COMPONENTS OF THE CELL SURFACE MEMBRANE  Lipids  Phospholipids (refer to Cellular Functions 1D: Lipids)  Description  Hydrophilic phosphate heads that will associate with the aqueous environment  Hydrophobic hydrocarbon tail  Function  Fatty acid tails form an effective hydrophobic barrier against polar and charged solutes. while hydrophobic tails face inwards and form a hydrophobic core.5 6G.  Acts as a selectively permeable membrane due to the presence of a hydrophobic barrier in the form consisting of fatty acid tails of phospholipids.2 FLUID MOSIAC MODEL  Two layers of phospholipids = lipid bilayer  Phospholipids can move laterally within the bilayer membrane => fluid  Implication: Membrane proteins are able to move laterally and be repositioned at diferent parts of the membrane  Implication: Allows for the invagination of the membrane which pinches off to form a transport vesicle  Collage of proteins that are randomly distributed and loosely attached => mosaic  Implication: Large number of proteins attached to the phospholipid bilayer that is able to perform different functions. polar molecules and ions such as glucose and sodium ions  Factors affecting the fluidity of the cell membrane  Temperature 150 | P a g e . O2)  Blocks large molecules.  The movement of solutes across the membrane depends on their chemical property (whether hydrophobic or hydrophilic) and their physical property (size).Version 2.  Self-repair  Ability to form specialised organelles (such as vacuoles during phagocytosis)  Alcohol will dissolve the phospholipid layer  Outward movement of cell content  Less support for cell content  Intense heat will denature the proteins  Outward movement of cell content.  Characteristics  Membrane is asymmetrical – Two layers differ in composition of proteins & lipids  Membrane is amphipathic – Hydrophilic phosphate head point outwards into aqueous environments inside and outside the cell. (Their length determines the thickness of the membrane.

restricting the movement of phospholipids. thus decreasing the permeability of a phospholipid bilayer to ions and small polar molecules.  There is more laterally mobility than transverse mobility  Cholesterol (Refer to Cellular Functions 1D – Lipids)  Description  Polar head group (OH-) with a planar steroid ring structure consisting of 4 fused steroid rings Bulky molecule.  Length of fatty acid chains  As the length of fatty acid chains increase.Version 2. thus causing phospholipids to become tightly packed. causing the fluidity of the membrane bilayer to increase and the membrane to exist in a fluid state.  Decreases membrane permeability – Controls uncontrolled leakage of polar molecules and ions  Cholesterol fills in the spaces between hydrocarbon chains of phospholipids. the phospholipids are able to pack more closely together.  Degree of saturation of fatty acid chains  As the degree of saturation of fatty acid chains increase. the degree of hydrophobic interactions between phospholipid fatty acid tails decrease.  As the length of fatty acid chains decrease.  Regulates membrane fluidity (Dual effect on membrane fluidity)  At high temperatures. thus plugging transient gaps through which polar molecules might otherwise pass through.5  As temperature increases. cholesterol restrains the movements of phospholipids by interfering with the motions of the hydrocarbon chains. the phospholipids are able to pack less closely together due to the presence of kinks. Non-polar hydrophobic tail. thus increasing the degree of hydrophobic interactions.  Membrane stability  Hydrophobic fatty acid tails of phospholipids interact with hydrophobic amino acid residues of integral proteins to anchor them. the degree of hydrophobic interactions between phospholipid fatty acid tails increase. resulting in increased membrane fluidity.  As the degree of saturation of fatty acid chains decrease.  As temperature decreases. cholesterol prevents the hydrocarbon chains from packing closely together. causing membrane fluidity to decrease. causing membrane fluidity to decrease. thus increasing the motion of the phospholipids. thus enhancing the mechanical stability of the membrane. the kinetic energy of the phospholipids increases. thus decreasing the degree of hydrophobic interactions. This result in increased lateral movements.  Glycolipids  Description 151 | P a g e . the kinetic energy of the phospholipids decreases.  Functions  For mechanical stability of the membrane  Its rigid steroid ring structure interferes with the motions of the hydrocarbon chains of phospholipids. thus decreasing the tendency of the membrane to freeze. causing the fluidity of the membrane bilayer to decrease and the membrane to exist in a semi-solid state. (These proteins function as …)  Mobility  Exhibit lateral and transverse mobility  Weak hydrophobic interactions allow phospholipids to move laterally. causing membrane fluidity to increase. resulting in increased hydrophobic interactions. thus overcoming the hydrophobic interactions between phospholipids. resulting in decreased membrane fluidity (counters effect of phospholipids)  At low temperatures. causing membrane fluidity to increase.

Sodium/Potassium Pump  Channel proteins (facilitated diffusion only)  Channel proteins contain a hydrophilic channel which permits the movement of water.g.  Held in place by cytoskeleton filaments in the cytosolic side  Functions  Anchorage  Anchoring proteins attach the cell membrane to microfilaments of the cytoskeleton on the cytoplasmic side and fibres of the extracellular matrix on the exterior side. must be dislodged using non-polar solvents e.5  Lipids with oligosaccharide which project towards the extra cellular matrix attached to them via a glycosidic bond.  Function  Involved in cell-to-cell attachment. Diffuse vs active T. hexane  Glycoproteins are integral proteins with oligosaccharides attached to them. Transmembrane proteins have two hydrophilic heads and 1 hydrophobic body  Insoluble in aqueous medium.g. voltage gated Na+ channels)  Enzymes – Catalyze reactions in the extracellular fluid or within the cytosol  Several enzymes can be anchored together to carry out sequential steps in a metabolic pathway. ATP Synthase 152 | P a g e . held in place by extensive hydrophobic interactions with the hydrophobic core.g. and the binding to extracellular signal molecules  Confers an asymmetry between the two surfaces of the membrane and maintains the membrane orientation through their interaction with the extracellular matrix  Proteins  Description  Extrinsic/peripheral proteins  Loosely bound to the polar surfaces (surface of cell membrane) through hydrogen bonds/ionic bonds between hydrophilic amino acids and hydrophilic phosphate head. ions and other small hydrophilic solutes. cell-cell recognition.  Leak channels – Permit movement of polar solutes (charged/uncharged) at all times (aquaporins.  Intrinsic/Integral proteins  Partially embedded (unilateral) or span the membrane entirely (transmembrane)  Contains both hydrophobic and hydrophilic regions.  Can be found on both inside and outside of cell  Proteins found on cytoplasmic side held by cytoskeleton  Proteins found on exterior side may be attached to fibres of the extracellular matrix. thus speeding up the rate of reactions in a pathway by reducing the chance of unstable intermediates breaking down before it can enter the subsequent reaction in the metabolic pathway  E.Version 2. stabilizing the position of the cell membrane.  Soluble in aqueous medium  Can be easily dislodged by adjusting pH or ionic strength of surrounding medium.  Hydrophilic channel lined with hydrophilic amino acid residues that are able to interact with the aqueous environment. induces and conformational change and causes the solute to be transported across the membrane.  Transport  Carrier Proteins (facilitated diffusion & active transport)  Solutes bind to carrier proteins.)  E.  ATP requirement depends on mode of transport (fac.

(KNOWLEDGE OF OSMOSIS.Version 2. making them ideal as receptor molecules for chemical signalling between cells. G protein-coupled receptors which recognize adrenaline and other molecules  Cell-Cell recognition (Usually involves glycoproteins)  Wide array of carbohydrate side chains  Allows cells to recognize other cells  Provides a means for foreign substances to be recognized and attacked by the immune system. ENDOCYTOSIS AND EXOCYTOSIS IS REQUIRED)  Roles of transport across membrane  To maintain a suitable pH and ionic concentration within the cell for enzyme specificity  To obtain food supplies for energy and raw materials  To excrete toxic substances and secrete useful substances  To generate the ionic gradients essential for nervous and muscular activity  Passive processes  Characteristics  Substance moves from a region of higher concentration to a region of lower concentration down a concentration gradient  No energy in the form of ATP required. ACTIVE TRANSPORT.5  Signal transduction  Proteins have specific shapes.g. branched chains of oligosaccharides (<15 units) that are attached to either glycoprotein or glycolipids through colvalent bonds (glycosidic bonds)  Functions  Maintain the orientation of proteins as carbohydrates are highly hydrophilic and thus associate with the aqueous environment and are unlikely to rotate towards the interior.  Act as recognition compounds  For cell specificity – recognition of same cell type for immune responses (hence certain cells can be rejected and targeted by the immune system)  Binding extracellular signal molecules in antibody-antigen reactions  For intercellular adhesion – for tissue formation and maintenance of integrity of tissue 6H – OUTLINE THE ROLES AND FUNCTIONS OF MEMBRANES WITHIN CELLS AND AT THE SURFACE OF CELLS. FACILITATED DIFFUSION.  E.  Diffusion  Def: Net movement of solute molecules from a region of higher concentration to a region of lower concentration down the concentration gradient until dynamic equilibrium is achieved  Characteristics  Passive Process 153 | P a g e .  Ligands (chemical signal) bind to the receptor protein.g. Binding of extacellular signal molecules in an antibody-antigen reaction  Intracellular joining  Membrane proteins of adjacent cells may adhere at intercellular junctions  Carbohydrates  Description  Short.  Usually exists as glycoproteins  E.  Various cells have differing types and numbers of receptor proteins depending on the cells’ function. activates intracellular signal transduction pathways and triggers a cellular response.

 Characteristics  Special type of diffusion 154 | P a g e .Version 2.  Channel proteins  Possesses a hydrophilic pore that allows for free movement of the transported substance across the membrane  Does not undergo conformational change  Carrier proteins  Molecule binds on the binding side.  And molecules (e. amino acids and ions.  Size and nature of diffusing molecules  The smaller the molecule. Oxygen. carrier protein undergoes conformational change to transport the substance across the membrane. the faster diffusion takes place  Distance over which diffusion takes place  Shorter distance means faster diffusion (Thin cell membranes  faster diffusion)  Surface area  The larger the ratio the higher the number of molecules that can diffuse across per unit time. the higher the rate of diffusion  Temperature  The higher the temperature.  Susceptible to poisons Two directions of movement   Osmosis  Def: Net movement of freely moving water molecules from a region of less negative water potential to a region of more negative water potential through a selectively permeable membrane. Nitrogen) which are hydrophobic and can pass through the membrane’s hydrophobic core easily  Can occur for either direction until dynamic equilibrium is achieved.g. the faster the rate of diffusion  Nature of the membrane across which diffusion occurs  The presence of transient gaps in the cell membrane enhances diffusion  The number and type of transport proteins present per unit surface area of the membrane also affects the diffusion rate. Simple diffusion  Occurs for molecules with small molecular weights that can pass through transient gaps in the membrane. the higher the rate of diffusion  The more soluble the substance is in the medium. where no net movement of substances occurs.5     No membrane/carrier proteins needed for simple diffusion Factors affecting rate of diffusion  Concentration gradient  The steeper the concentration gradient. Facilitated Diffusion  Def: A type of diffusion whereby a substance is allowed through the membrane by specific protein complexes such as channel or carrier proteins  Characteristics  Highly specific  Passive  (NO ENERGY IN THE FORM OF ATP REQUIRED)  Transport proteins used to enhance the rate of transport  Occurs for larger and hydrophilic substances such as glucose. the faster the diffusion process.

 Flaccidity in plant cells  Water potential of cell is less negative than solution.  Water leaves the cell by osmosis. Water is much less likely to enter the plant cell. More solutes present  More –ve solute and water potential  Pressure potential is the measure of the pressure exerted by the cell wall on its contents  Increases as the cell absorbs water and increases in volume  Always positive as it tends to move water out of the cell  Pressure potential increases as the cell absorbs water.5  Water always moves from less negative water potential to a more negative water potential (less freely moving water molecules due to interactions with solutes)  Passive process  Water potential  Measure of the tendency of water to move from one region to another  Pure water has a potential of 0 (highest)  Solute potential is the measure of the ability of a solute to make the water potential more negative  Dissolving solutes in water reduces the number of free water molecules due to interactions with solutes.  Uptake of nutrients into the cell (when concentration outside is low)  Removal of unwanted waste materials (when concentration outside is high)  Types  Uniport 155 | P a g e .  Always negative.  If too much water is lost.  Effects on Plant Cells  Due to water potential – expressed as a sum of the solute potential and pressure potential  Turgidity in plant cells  Caused by water moving into the cells by osmosis as water potential of cell is more negative than solution.  Requires energy  Movement occurs in 1 direction  Transport against concentration gradient  Carriers required (conformational change required to ensure that there is no leakage of solutes back across the membrane along the concentration gradient  Uses  Maintain internal concentrations of molecules that are much higher or lower than those in the extracellular environment. the water potential of the plant cell becomes less negative. thus making the water potential of a solution more negative. which exerts the same force back on the water molecules. (where 50% of the plant cells in a population are plasmolysed)  Plasmolysis occurs when the cell membrane pulls away from the cell wall  Vacuole shrinks  Animal cells  Lyses when too much water enters cell in a hypotonic solution  Cell crenates when water moves out of cell in a hypertonic solution  Active transport  Def: Movement of a molecule from a region of low concentration to a region of high concentration against the concentration gradient (by specific protein carriers). causing water molecules to exert a greater force on the cell wall. from the cytoplasm then the vacuole.  Cell swells and becomes turgid. As the plant cell becomes turgid. requiring energy in the form of ATP. incipient plasmolysis may occur where pressure potential reaches 0.Version 2.

antibodies.Version 2. forming clathrin-coated vesicles.5  Symport (two substances transported in the same direction)  Antiport (two substances transported in the opposite direction) (Sodium potassium pump)  Bulk transport  Def: Transport of large molecules and liquids in and out of the cell  Energy in the form of ATP required  Endocytosis  A process where the cell takes in macromolecules by the invagination of the plasma membrane to form vesicles  Process used to incorporate extracellular substances  Phagocytosis  Pseudopodia extends outwards from the cell surface membrane to take in solid/particulate substance by engulfing them in vesicles which then pinch off from the cell surface membrane.  Exocytosis  Secretion of hormones. Coated pits reinforced on the cytoplasmic side with clathrin.  Pinocytosis  Take in solution/liquid (non-specific)  Receptor-mediated endocytosis  Coated pits form vesicles with specific molecules bound to receptor proteins on the cell surface. enzymes. a selective form of endocytosis. cellular wastes through the fusion of secretory vesicles with the cell surface membrane. 156 | P a g e .

and continuously removing toxic substances.g.2 REFLEX CONTROL  Reflex Control – The use of long distance pathways to receive input about a change. EFFECTORS. Chemical constituency of ICF 6I.  Allows for the proper functioning of cells.5 6I – EXPLAIN THE NEED FOR CONTROL IN ORGANISED SYSTEMS AND EXPLAIN THE PRINCIPLES OF HOMEOSTASIS IN TERMS OF RECEPTORS. AND NEGATIVE FEEDBACK.1 PRINCIPLES OF HOMEOSTASIS  Homeostasis – The maintenance of a constant internal environment  Ensures a degree of independence from the external environment  A dynamic state of internal equilibrium  Set point fluctuates continuously within narrow limits  Involves  Processes that ensure the steady state physiological condition or variable (at the set point)  Constant monitoring of physiological conditions  Especially maintaining the immediate surroundings of cells (intracellular fluid (ICF) )  Coordinated responses to minimize any disturbance or deviation from the steady state  Types of control systems  Localized  Involves paracrine & autocrine signalling  Systemic  Involves reflex control pathways with an integrating centre (via the nervous & endocrine system)  Reasons for the need of control  Maintain a physiologically favourable internal environment to meet physiological needs and function smoothly. at certain points in life. 6I. Osmotic Pressure. Puberty  Due to acclimatization to the new environment  Variables to be regulated  Temperature  Glucose concentration  Pressure. set points may change (such changes are still regulated)  E.  Consists of response loops and feedback loops  Response loop  Input signal involving a stimulus and a receptor  Integration of the signal  Output signal involving the effector and response  Stimulus  Receptor  Afferent Pathway  Integrating Centre  Efferent Pathway  Effector  Response  Feedback loop 157 | P a g e .Version 2. organs and systems at optimal efficiency as most biochemical reactions are catalysed by enzymes that function optimally only over a narrow range of pH and temperature  To maintain the rate of cellular respiration by bringing in a constant supply of glucose and oxygen. integrate the information and react appropriately.  Allows cells to carry out vital cellular processes and functions  However. tissues. During the menstrual cycle.

1 COMMUNICATION SYSTEMS  Intercellular communication systems  Endocrine signalling  Uses hormones and the endocrine system to maintain homeostasis and trigger feedback mechanisms  Paracrine & Autocrine signalling  Local signalling molecules are secreted by certain cells and targeted at cells around them.  Endocrine System  Uses hormones that are transported by the circulatory systems  Hormones act as chemical messengers to trigger a sustained.5  Response acts as a stimulus to stop or trigger more responses  Efficiency judged by  The extent of deviation from the set point  The speed with which the set point is restored after a change has occurred.  Synaptic Signalling  Main mode of transmission of signals between nerve cells  Neuroendocrine Signalling  Neurosecretory cells secrete neurohormones which then travel through the circulatory system to target cells  Neurosecretory cells are activated by synaptic signalling.Version 2. allowing the organism to function as an integrated and coherent whole consisting of a multitude of cells in diverse 158 | P a g e .  In autocrine signalling.2 REASONS FOR THE NEED OF COMMUNICATION SYSTEMS  Maintenance of homeostatic bodily processes crucial to the survival and perpetuation of a complex multicellular organism  Allows for the reception and response of cells to information from the internal and exteral environment  Allows for the coordination and collaboration of activities such as growth and metabolism. 6J – EXPLAIN THE NEED FOR COMMUNICATION SYSTEMS WITHIN ORGANISMS. 6J.  Types of systems involved  Nervous System  Uses high-speed electrical impulses in the form of neurotransmitters carried across synapses to coordinate responses to sudden environmental changes. prolonged response to stress stimuli  Restricted to cells with specific receptors 6J. the signal molecules secreted by a cell is targeted back at the same cell.  The effects of responses tend to be short-lived and are restricted to specific target cells.

including Nissl bodies (groups of ribosomes involved in neurotransmitter synthesis)  Cytoskeleton that provides internal support  Consists of neurofilaments and neurotubules (microfilaments and microtubules)  Numerous mitochondria for ATP synthesis  Dendrites  Slender. which end in synaptic terminals  Converts the electrical signal to a chemical signal via the release of neurotransmitters  Usually wrapped in a myelin sheath (schwann cells that wraps itself around the axon)  Gaps between 2 myelinated regions is known as the Nodes of Ranvier  These nodes have high concentrations of voltage gated channels  Properties  Extreme longevity  Amitotic (unable to divide due to the lack of centrioles)  Extremely high metabolic rate  Excitable by changing membrane permeability 159 | P a g e . 6K – DESCRIBE AND EXPLAIN THE TRANSMISSION OF AN ACTION POTENTIAL ALONG A MYELINATED NEURONE. sensitive and highly branched  Carry information from the source of stimulus to the cell body in the form of graded potentials  Axons  A long cytoplasmic body that conducts signals away from the cell body in the form of action potentials  The axon hillock joins the axon to the main cell body and triggers the initiation of an action potential  The axon branches out into collaterals.Version 2.1 NEURON STRUCTURE AND FUNCTION  Glial Cells/Neuroglia – Cells that provide important physical and biochemical support to neurons  Schwann Cells – Myelinates motor neurons and increases speed of conduction  Astrocyte – Cells that provide physical and nutritional support for neurons  Neurons: Cells that convey information in the form of nerve impulses  Cell Body  Nucleus  Protein synthesizing machinery.) + + 6K. (THE IMPORTANCE OF NA AND K IONS IN THE IMPULSE TRANSMISSION SHOULD BE EMPHASISED.g. gene expresson and cell proliferation) such that the events occur in the right cells at the right time.5 tissues and organs that coordinate their cellular events (e.

potassium and calcium channels  Open in response to a change in membrane potential  Primarily found in the axon 6K. decreasing the potential of the cytoplasm  Chemically gated sodium.2 MEMBRANE PROTEINS [KIV CELL MEMBRANE]  Sodium/ Potassium ions leak channels  Allow for the movement of sodium and potassium ions down the electrochemical gradient  Sodium-Potassium ATPase pump  ATP pump that moves 3 Na+ out of the cell for every 2K+ in.Version 2. chloride channels  Open in response to a binding chemical compound  Primarily found at dendrites  Voltage gated sodium.3 MEMBRANE POTENTIAL  Membrane potential – Difference in electrical charge across a cell membrane  In physics terms. this is more similar to an electromotive force rather than voltage (although both measured in volts)  Resting membrane potential – The difference in electrical charge between the inside and outside of an unstimulated neuron (~-70 to 100 mV)  Unequal ion distribution across the membrane  Extracellular fluid contains a high concentration of Na+ and Cl.ions 160 | P a g e .5 Axons Carries information away from the cell body One axon per cell Absence of ribosomes Branches further from the cell body Smooth surface May be myelinated Transmit signals as action potential Dendrites Bring information to the cell body Many dendrites per cell Contains ribosomes Branches near the cell body Rough surface Not myelinated Transmit signals as graded potential 6K.

this results in a net loss of positive charge from the cytosol. which contribute to high internal negative charge as they are unable to diffuse across the cell membrane. resulting in a negatively charged cytosol  Changes in membrane potential  Polarization (membrane voltage is not 0)  Depolarization (a change in membrane potential that makes the membrane less negative than resting potential)  Hyperpolarization (a change in membrane potential that makes the membrane more negative than resting potential)  Repolarization (happens after depolarization) 6K.  Facilitated diffusion of K+  Rate of K+ diffusing across the membrane is high due to the presence of many potassium leak channels. resulting in a negatively charged cytosol  Active transport of sodium and potassium ions by the sodium potassium ATPase pump  This results in a net loss of positive charge from the cytosol.  The presence of a myelin sheath Type Location Type of channel proteins involved Type Strength Factor initiating the signal Changes in intensity of initiation factor Graded Potential Input Dendrites Chemically gated ion channels Action Potential Conduction Axon Voltage gated ion channels Depolarizing/Hyperpolarizing Variable Entry of ions (no threshold required) Depolarizing Fixed Above threshold graded potential at axon hillock Increases/Decrease frequency of action potential Increases/Decrease strength of graded potential 161 | P a g e .  Always depolarizations  All or none principle as they occur at a maximum depolarization or they do not occur  Amplitude of the graded potential determines the frequency of the action potential  Speed of conduction depends on  The diameter of the axon  The larger the diameter.4 GRADED & ACTION POTENTIAL TRANSMISSION  Graded Potential  Signals of variable strength that lose their strength as they travel through the cytoplasm  Can result in either hyperpolarization (influx of anions) or depolarization (influx of cations)  Action Potential  Large.  This intensifies the negative charges present in the cytosol  Facilitated diffusion of Na+  Few sodium leak channels restrict the speed of sodium ions diffusing into the cytosol  Coupled with the high rate of potassium ion diffusion.  The movement of potassium ions will eventually reach equilibrium as the accumulated positive charge results in an electrical gradient that opposes further movement of potassium ions.5  Cytosol contains a high concentration of K+ and negatively charged proteins.Version 2. strong depolarizations that travel rapidly for long distances throughout the axon without losing strength. the smaller the resistance to local current flow and the faster the speed of conduction.

 This is required to overcome the hyperpolarized state of the membrane potential to reach the threshold.  Saltatory Propagation  Continuous propagation is inhibited along a myelinated axon due to presence of a myelin sheath which acts as an electrical insulator and prevents the loss of current in the form of ions by restricting the flow of ions across the membrane  Between Schwann cells. Refractory period exists No effect  Refractory Period  The time when the membrane will not respond to any additional depolarizing stimuli when depolarization takes place  Prevents the backward transmission of the action potential.  Absolute refractory period  Time: From depolarization to resetting of voltage gated sodium channels  This is due to the inability of the inactivation gate to be opened by waves of depolarization (only the activation gate can respond to waves of depolarization)  Relative refractory period  Time: From the resetting of the voltage gated sodium channels to the time when the resting membrane potential is restored.5 Summable? Effect of increasing distance Yes Decreases signal strength No. *Large diameter of axon  lower resistance to flow of ions  faster transmission 162 | P a g e .Version 2. voltage gated sodium and potassium channels are concentrated at the Nodes of Ranvier.  The action potential thus jumps from node to node rather than move along the axon continuously  Advantages  Conserves Metabolic energy as ATP is not required to maintain the sodium potassium ATPase pump within myelinated regions  Faster action potential propagation as the action potential does not need to be regenerated at myelinated sections.  An action potential can be initiated if a very strong graded potential occurs.

5 163 | P a g e .Version 2.

16. generating a positive feedback loop (Hodgkins cycle) that causes more voltage-gated sodium channels to open. 12. A suprathreshold depolarizing stimulus triggers the opening Generation – of the activation gate of the voltage gated sodium channel Rising Phase 10. depolarization causes the membrane potential to have a reverse polarity. as well as the low concentration of sodium ions in the axoplasm. (Generating both a concentration and electrical gradient) 17. causing the net influx of sodium ions to stop 15.5 Signal Transmission in Neurons Graded Potential 1. Once the voltage gated potassium channels close. potassium ions diffuse back into the axoplasm via potassium leak channels. The graded potential is transmitted in this manner to the axon hillock. The graded potential begins as an influx of ions at a certain location 3. the voltage-gated potassium channel opens. (Generating both a concentration and electrical gradient) 11. The sodium potassium ATPase pump restores the original ionic concentration within the axoplasm Action Potential 21. Action Potential 13. Summation of excitatory post-synaptic potential (EPSP) and Hillock inhibitory post-synaptic potential (IPSP) at the axon hillock. 20. This causes the voltage-gated sodium channels to close using Generation – the inactivation gate. & Return 19. a net movement of electrical charge away from the point of origin 5. The potential spreads through the cytoplasm as a local current. resulting in overshoot. This results in depolarization/hyperpolarization of the local region 4. At the same time. the voltage gated sodium channels reset both inactivation and activation gates to the original positions Action Potential 18. an action potential is generated. This results in repolarization of the membrane potential. The positive current from the entry of sodium ions spread into Propagation in adjacent sections of the membrane due to the electrochemical gradient.Version 2. This results in an efflux of potassium ions via facilitated diffusion due to the positively charged interior of the axoplasm.55 mV). 8. A myelinated neuron at rest has a membrane potential of Transmission 70mV. as well as the low concentration of potassium ions in the extracellular matrix. Molecules Involved Chemically-gated sodium/chlorine pumps Sodium/Chloride ions Voltage-gated sodium channel Sodium ions Voltage-gated sodium channel Voltage-gated potassium channels Potassium ions Voltage-gated potassium channels Sodium potassium ATPase Pump Voltage-gated channels 164 | P a g e . If the sum is above the threshold (~. causing the membrane potential to increase until resting membrane potential is reached. or until it is diminished Encounter at Axon 7. Action Potential 9. 2. 6. Falling Phase 14. Eventually. This results in an influx of sodium ions via facilitated diffusion due to the negatively charged interior of the axoplasm. This causes the cell to depolarize further. At the same time. The slow closing of the voltage gated potassium channels Generation – results in hyperpolarization as the membrane potential falls Hyperpolarization below the resting membrane potential. The potential strength diminishes as it travels through the cell body as charge-carrying ions leak through the membrane via leak channels. Membrane permeability to sodium ions decrease to resting levels.

the presynaptic neuron passes the message on to the post synaptic neuron  Graded depolarization (excitatory) 165 | P a g e . 19 The positive current from the entry of sodium ions spread into adjacent sections of the membrane due to the electrochemical gradient. This results in a wave of depolarization that causes the voltage gated sodium channels to open in the adjacent region of the trigger zone.5 unmyelinated axons OR Saltatory Propogation 22. 22 The action potential jumps from one node of Ranvier to the next via salutatory conduction as myelin acts as an ninsulator. 20 The local current skips the internodes spreads outwards from the axon hillock. 21 This results in a wave of depolarization that causes the voltage gated sodium channels to open in the adjacent nodes of Ranvier of the trigger zone. generating an action potential in the adjacent region. 23. INCLUDING THE ROLE OF CA 2+ IONS. Sodium & Potassium Ions Sodium potassium ATPase pump Schwann Cells & Myelin Sheath Nodes of Ranvier 6L – DESCRIBE THE STRUCTURE OF A CHOLINERGIC SYNAPSE AND EXPLAIN HOW IT FUNCTIONS. This self-sustaining wave of depolarization moves unidirectionally from the axon hillock to the synapse due to the refractory period.  Synaptic Transmission – Propagating messages from one nerve cell to another via the synapse  Synapse – The point where the neuron meets the target cell  Electrical Synapse  Transmission of electrical impulses from one nerve cell to another via the passage of ion across gap junctions between cells  Chemical Synapse consisting of  Presynaptic terminal containing vesicles with neurotransmitters  Postsynaptic knob of the target cell containing specific receptor sites for neurotransmitters  Synaptic cleft (gap between presynaptic knob and postsynaptic knob)  At a synapse between 2 neurons. speeding up the rate of transmission 23 This self-sustaining wave of depolarization moves unidirectionally from the axon hillock to the synapse due to the refractory period.Version 2. generating an action potential in that node of Ranvier.

This opens voltage-gated calcium channels.Version 2. The arrival of an action potential depolarizes the presynaptic knob presynaptic knob Entry of calcium 2. depleting the supply of acetylcholine  The resynthesis and transport mechanisms are unable to keep up with the demand for acetylcholine  Synaptic delay of 0.  Involves the influx of cations (Na+)  Graded hyperpolarization (inhibitory)  Brings the membrane potential further away from the threshold and inhibits the formation of an action potential. Molecules Involved Voltage Gated Calcium Channels 166 | P a g e . resulting in an influx of ions calcium ions.  Involves the efflux of cations (K+) or the influx of anions (Cl-)  Example of a chemical synapse: Choligernic Synapse  Makes use of the neurotransmitter acetylcholine (ACh)  Choligernic synapses located at neuromuscular junctions always induce depolarization  The larger the amount of acetylcholine released. the larger the number of open channels and the greater the effect of the graded potential generated. releasing ACh into the synaptic cleft  Diffusion of ACh across the synaptic cleft and binding of ACh to receptor molecules on the postsynaptic membrane (main factor) Cholinergic Synapse Signal Transmission Depolarization of 1.5 ms exists due to the time required for  The entry of calcium ions into the presynaptic neurone  The fusion of secretory vesicles containing acetylcholine with the presynaptic membrane.  Synaptic fatigue may occur when the neuron is overstimulated.5  Brings the membrane potential closer to the threshold and favouring the formation of an action potential.2-0.

4. 8. This causes a change in the 3D conformation of chemically-gated ion channels. Choline is actively transported back into the presynaptic knob where it is used to synthesize more acetylcholine using acetate provided by coenzyme A (CoA) The remaining acetate diffuses away from the synaptic cleft and can be metabolized by cells. an excitatory post-synaptic graded potential is generated in the local region. Removal of acetylcholine 10. 167 | P a g e . Calcium ions are rapidly transported out of the cell or into the mitochondria by active transport using ATP Acetylcholine difuses across the synaptic cleft and binds reversibly to specific receptors on the postsynaptic membrane. Depolarization of postsynaptic knob 5. Synaptic fatigue may occur if the neuron is overstimulated and the supply of neurotransmitter exhausted. Calcium ions bind to secretory vesicles containing acetylcholine.  Temporal Summation  Occurs when a single synapse is excited repeatedly (when a second stimulus affects the same synapse before it is fully depolarized from the first stimulus)  Spatial Summation  Occurs when simultaneous stimuli at different synapses have a cumulative effect on the membrane potential. This results in the efflux of either cations (leading to depolarization) or anions (leading to hyperpolarization) In the case of depolarization. 6. 7. 13. 11. 9. Acetylcholinase then breaks down acetylcholine into acetate and choline. If the membrane potential reaches above threshold at the axon hillock. an action potential is generated.Version 2. 12. Secretory vesicles containing Acetylcholine Acetylcholine Chemically-gated ion channels Ions Acetylcholinase Choline & Acetate  Summation of excitatory and inhibitory postsynaptic potentials  Both excitatory (EPSP) and inhibitory postsynaptic potentials (IPSP) can combine their effects cumulatively.5 3. stimulating vesicles containing acetylcholine to fuse with the presynaptic membrane and release acetylcholine into the synaptic cleft via exocytosis.

 Achieved by  Stimulating the synthesis of an enzyme or structural protein  Increasing or decreasing the rate of synthesis of an enzyme of protein  Deactivating or activating an existing enzyme by altering its specific 3D conformation. 6M. activities and quantities of important enzymes and structural proteins. which transports hormones to target cells 6M. Oxytoxin  Mode of Hormone Action  Hormones work via cell signalling (Reception  Signal Transduction  Response)  Differing mechanisms depending on the chemical properties of the hormone (hydrophobic vs hydrophilic) 168 | P a g e .g. Estradiol.g.5 6M – EXPLAIN WHAT IS MEANT BY AN ENDOCRINE GLAND. Cortisol.  Types (chemical classes)  Polypeptides  Usually formed from the cleavage of polypeptide chains  Hydrophilic  E.g. WITH REFERENCE TO THE ISLETS OF LANGERHANS IN THE PANCREAS. Thyroxine.2 HORMONES  Description: Chemical Messengers secreted by endocrine glands and transport away to act on specific target cells away from where they are synthesized  Properties  Able to act on multiple tissues & cells  Some elicit more than 1 type of response in the body  Effective in low concentrations  Sustained and prolonged effects  Binds to specific protein or glycoprotein receptors on target cells  Needs to be terminated for signalling to stop  Function: Alter cellular operations by changing the types.1 ENDOCRINE SYSTEM  Endocrine system consists of endocrine glands (organs) and endocrine cells (tissue) which secrete hormones  Functions to maintain homeostasis and trigger body responses to certain stimuli  Primarily regulated by negative feedback control mechanisms  Endocrine Glands (“endo” = within/not exposed)  Ductless glands that secrete hormones directly into the blood stream. Epinephrine. Progesterone  Amines  Can be hydrophilic or hydrophobic  Derived from amino acids  E. Testosterone.Version 2. Insulin  Steroids  Derived from fat derivatives  Hydrophobic  E.

etc E. Water soluble hormones are secreted by endocrine cells and transported via the bloodstream to targeted cells. 7.Version 2. 2. This eventually changes cytoplasmic function or transcriptional activity in the nucleus. 4. Signal 3. This forms a hormone receptor complex. A phosphorylation cascade relays the signal onto an effector. Molecules Involved Hormone Cell Surface Receptor G Proteins Effector Protein Secondary Messengers Secondary Messengers Other Proteins Transcription Factors. Lipid soluble hormones are secreted by endocrine cells and transported via the bloodstream to targeted cells by binding to transport proteins. 5. Hormonal Signalling – Hydrophilic Hormones (A) Reception 1. Adrenaline Hormonal Signalling – Hydrophobic Hormones (B) Reception 1. which moves into the Transduction & nucleus and binds to certain DNA sequences to up regulate Response expression of certain genes.5  Water soluble proteins are effective at low concentrations due to their ability to initiate chain reactions a secondary messenger. The prolonged binding of the hormone to cell surface receptors stimulates the effector continuously. 2. Water soluble hormones then bind onto cell surface receptors Signal 3. This causes the concentration of the secondary messenger to increase Response 6. The secondary messenger relays information from the extracellular chemical signal and translates it into an intracellular response within the cell by activating inactive proteins/enzymes. This receptor acts as a specific transcription factor to promote the synthesis of vitellogenin mRNA which is translated into vitellogenin. Transduction which activates a secondary messenger. 169 | P a g e . Molecules Involved Lipid Soluble Hormones Transport Protein Intracellular Receptor Hormone-Receptor Complex  An example of such a hormone is estradiol. which amplifies the initial effect.g. Lipid soluble hormones then diffuse across the cell surface membrane and bind to intracellular receptors located within the cell. which binds to a receptor in the cytosol.

Effectors 5. Response 6.1 NEGATIVE FEEDBACK MECHANISM  A Homeostatic feedback mechanism that counteracts the corrective mechanism once the set point is restored. collectively known as the Islets of Langerhans)  β cells secrete insulin while α cells secrete glucagon. 4.5 6M. This change is detected by the β cells of the islets of Langerhans of the pancrease. delta and F cells. Things Involved β cells of Islets of Langerhans Insulin RTKs Liver. Insulin binds to tyrosine kinase receptors on the cell surface membrane of liver. beta.Version 2. resulting in the activation of RTKs. skeletal muscle and adipose cells. This also inhibits the secretion of glucagon by α cells of the islets of Langerhans. 6N.  Highly conserved amino acid sequence among vertebrates  Glucagon  A peptide hormone with 29 amino acids  Highly conserved amino acid sequence among vertebrates  Blood Glucose Concentration Regulation  Hormonal negative feedback mechanism involving antagonistic hormones Hormonal Signalling – Insulin Stimulus 1. This causes the β cells of the islets of Langerhans to secrete insulin into the bloodstream.4 PANCREAS & ISLETS OF LANGERHANS  Pancreas  An organ that consists of many exocrine and endocrine glands  Exocrine glands involved in the digestive system by secreting enzymes  Endocrine glands  4 types of cells – (alpha. 6N – EXPLAIN HOW THE BLOOD GLUCOSE CONCENTRATION IS REGULATED BY INSULIN AND GLUCAGON. Tyrosine Kinase Receptors start a phosphorylation cascade to bring about cellular responses. Integrating Centre 3. skeletal muscle and adipose cells.2 BLOOD GLUCOSE REGULATION & DIABETES  Insulin  A peptide hormone with 51 amino acids  Synthesized from a precursor consisting of 1 chain. Blood glucose concentration rises higher than the set point of 90mg/100ml of blood Receptor 2.  This ensures that the variable being regulated stays within its steady state range of values (oscillates around the set point)  Maintains dynamic equilibrium  The primary mechanism of homeostatic regulation. RTKs G-protein Relay ATP 170 | P a g e . provides long term control over internal conditions and systems  Associated with responses that eliminate/oppose the stimulus by reducing its intensity 6N. which is then cleaved into 2 chains joined by disulfide bonds.

10. 11. skeletal muscle and adipose cells. [Accelerate Glucose Utilization] Glycogen synthase and Glucokinase is activated.5 7. Response 7. Glucagon binds to glucagon receptors on the cell surface membrane of liver. 4. Lipogenesis is stimulated. skeletal muscle and adipose cells. stimulating glycogenesis from glucose. loop 14. [Increase Glucose Uptake] Vesicles with bound glucose transporters (GLUT4) fuse with the cell surface membrane to increase the rate of glucose uptake via facilitated diffusion. Less stimulation of β cells and no further decrease in blood glucose concentration Hormonal Signalling – Glucagon Stimulus 1. Effectors 5.Version 2. 12. Blood glucose concentration is lower than the set point Receptor 2. Negative Feedback 13. [Accelerate Glucose Utilization] Glycogenolysis is inhibited Amino acid absorption and protein synthesis is stimulated in skeletal muscle cells. Integrating Centre 3. This also inhibits the secretion of insulin by β cells of the islets of Langerhans. cAMP relays information from the glucagon and translates it into an intracellular response within the cell by activating protein kinase A Glucose Transporters Glucokinase Things Involved β cells of Islets of Langerhans Glucagon Glucagon Receptors Liver. converting excess glucose into triglycerides Once blood glucose concentration decreases to the set point. 6. This causes the α cells of the islets of Langerhans to secrete glucagon into the bloodstream. 8. a negative feedback loop prevents further release of insulin. 9. Adenyyl cyclase produces cyclic AMP from ATP 9. G-protein Relay Adenylyl Cyclase cAMP ATP Protein Kinase A 171 | P a g e . This change is detected by the α cells of the islets of Langerhans of the pancreas. Some of these receptors are GPCRs which become activated and in turn activate a specific G Protein. 8. Glucagon Receptors start a phosphorylation cascade and activate adenylyl cyclase. [Accelerate Glucose Utilization] The rate of glycolysis is increased to produce more ATP from breaking down glucose.

low blood pH  Coma in extreme cases  Glucose appears in the urine if the concentration of glucose exceeds 180 mg/100cm 3 due to the lack of available carrier proteins to transport the glucose into the cells lining the proximal convoluted tubules. Negative Feedback 15. overwhelming the reabsorption capabilities of the kidneys  Diabetes (Type I)  Caused by attack on β cells. 13.3 POSITIVE FEEDBACK (NON-HOMEOSTATIC) MECHANISM  A non-homeostatic feedback mechanism that amplifies a certain response  This leads to further deviation from the set point. a loop negative feedback loop prevents further release of glucagon 16. stimulatating glycogenolysis which stimulates the breakdown of glycogen to glucose.Version 2.  Symptoms of diabetes  Glucose not converted into glycogen  Blood glucose level rises and stays high  Slow Metabolism  Blood glucose levels drop very slowly  Kidney unable to absorb all the glucose thus some is passed out in urine + Extra water and salts are also lost  Person feels thirsty and hungry frequently  Uptake of glucose into cells is very slow despite high blood glucose level due to unresponsiveness of liver and muscle cells to insulin  Cells metabolize fats and proteins instead  Produces keto acids that lower blood pH  Dehydration. converting triglycerides to glucose. which phosphorylate glycogen synthase and deactivates it.5 10. 11.  Results in hypoglycaemia. destroying 90% of them (autoimmune disease) or lack of gene that codes for insulin production  Pancreas incapable of secreting enough insulin  Regular injections of insulin required  Diet needs to be carefully controlled (Can eat sugar as sugar is a direct source of glucose which is able to increase/restore the blood glucose level immediately as opposed to eating starchy food. 12.  Managed by diet control and exercise which uses up glucose to provide energy for aerobic respiration. 14. Glycogenesis in liver and muscle cells is inhibited. leading to an unstable situation and an even greater response  Have specialized functions within the body. Activated protein kinase A phosphorylates glycogen phosphorylase kinase. which activates it.)  Diabetes (Type II)  Pancreas produces insulin at high levels but cells are unresponsive to insulin  Reduced responsiveness of liver and muscle cells to insulin  May be caused by non-functional insulin receptors or defects in proteins involved in glucose uptake and metabolism. salt loss. Lipolysis is stimulated. 172 | P a g e . which requires time to be digested and then absorbed into the blood. 6N. Less stimulation of α cells and no further increase in blood glucose concentration Glycogen phosphorylase kinase Glycogen synthase Glycogen phsophorylase  Diabetes Mellitus  Characterised by a deficiency or absence of insulin/decreased response to insulin in target cells. Gluconeogenesis is stimulated where glucose is synthesized from proteins and lipids. Glycogen phosphorylase kinase also phosphorylates glycogen phsophorylase. Once blood glucose concentration increases to the set point.

Version 2.5

 Associated with responses that eliminate a potentially dangerous scenario or stressful situation
 Example – Blood Clotting
 Each step of the blood clotting pathway releases chemicals to attract more platelets and clotting
factors, leading to a rapid build-up of platelets at the site of injury.
 These form a blood clot that plus the vessel wall to stop bleeding.
 Example – Uterine Contractions during childbirth
 Estradiol activates oxytocin receptors on uterus
 Oxytocin secreted from the posterior pituitary gland stimulates the uterus to contract, as well as
stimulating the placenta to synthesize prostaglandins which further stimulate uterine contraction.
 Uterine contractions stimulate the production of more oxytocin (Positive feedback mechanism)

6O – DESCRIBE THE MAIN STAGES OF CELL SIGNALLING – LIGAND-RECEPTOR
INTERACTION, SIGNAL TRANSDUCTION (INCLUSIVE OF PHOSPHORYLATION AND
SIGNAL AMPLIFICATION) AND CELLULAR RESPONSES. (LIMITED TO AN OVERVIEW OF
INSULIN & RTK SIGNALLING AND GLUCAGON & G-PROTEIN SIGNALLING. CANDIDATES
WILL BE EXPECTED TO GENERALISE THEIR UNDERSTANDING OF THESE SYSTEMS IN
SOLVING PROBLEMS INVOLVING OTHER CELL SIGNALLING SYSTEMS.) ADVANTAGES
AND SIGNIFICANCE OF HAVING A CELL SIGNALLING SYSTEM MAY BE REQUIRED.
 Cell Signalling
 Divided into 3 stages – Reception, Signal Transduction, Cellular Response

6O.1 SIGNAL RECEPTION
 In signal reception, a signalling molecule (aka ligand) binds to a protein receptor (Detection of an
extracellular signalling molecule)
 In general, ligand refers to any molecule that specifically binds to another molecule (usually a larger
one)
 Protein Receptors
 Can be either be found in the cytosol (intracellular) or on the
plasma membrane (depending on the chemical property of the
ligand; whether it is hydrophobic or hydrophilic)
 Has a structure that is complementary to the sie on the target
cell receptor
 Change their conformation and is activated upon the binding of
a ligand, thus transmitting exteracellular signal information
within the cell.
 G Protein-Coupled Receptors (GPCRs)
 Cell surface transmembrane receptor

173 | P a g e

Version 2.5

Associated with G Proteins
 Proteins that bind to GTP in their activated form (or GDP in their inactivated form)
 Has GTPase activity to hydrolyse GTP into GDP and Pi
 Structure
 One single polypeptide chain with 7 transmembrane α-helices
 Loops connecting the α-helices form the intracellular G-Protein binding site & extracellular ligand
binding site binding sites for ligands.
 G proteins mediate the passage of the signal from the membrane surface into the cell interior [function
depends on whether GTP is attached]
Mode of GPCRs
Signal Reception
(GPCR)

1.

2.

3.

4.

A ligand binds to the G protein-coupled receptor on the
extracellular ligand binding site and causes the GPCR to
change its specific 3D conformation, activating the GPCR.
A G protein binds to the cytoplasmic binding site of the GPCR
and the activated GPCR displaces the bound GDP on the G
protein with a GTP, activating the G protein.
The activated G protein dissociates from the GPCR and binds
to a target protein activating the protein which elicits a
cellular response/signal transduction cascade.
The G protein uses its GTPase activity to hydrolyse the bound
GTP to a GDP and Pi, deactivating the G protein, shutting
down the transduction pathway

Molecules Involved
Ligand
GPCR
G Protein
GTP, GDP and Pi
Enzyme/Relay
proteins

 Receptor Tyrosine Kinases (RTKs)
 Cell surface transmembrane receptor
 Kinase  Enzyme that phosphorylates another protein
 Tyrosine Kinase  Catalyzes the transfer of a phosphate group from ATP to tyrosine
 Work in pairs
 Common cause of cancer [KIV RAS]
 Able to trigger multiple signal transduction pathways
 Structure
 Extracellular ligand binding site
 1 transmembrane α-helix

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Version 2.5

Intracellular tail containing multiple tyrosines and a tyrosine kinase domain that catalyzes the
transfer of a phosphate group from ATP to tyrosine residues

Mode of Receptor Tyrosine Kinases
Signal Reception
1. A ligand binds to the receptor tyrosine kinase on the extracellular
(RTKs)
binding site, causing the RTK to change its specific 3D conformation.
2. Two of such RTK monomers associate closely with each other,
forming a dimer.
3. Dimerization activates the tyrosine kinase region of each monomer,
causing tyrosine kinase to catalyse the transfer of a phosphate
group to a tyrosine at the end of each tail of the other RTK from ATP
4. The activated RTK can now be recognized by specific relay proteins
inside the cell.
5. Each relay protein binds to a phosphorylated tyrosine and
undergoes a conformational change that causes it to be activated.
6. Each activated protein elicits a cellular response/signal
transduction

Molecules Involved
Ligand
GPCR
G Protein
GTP, GDP and Pi
Enzyme/Relay
proteins

 Ion Channel Receptors
 A transmembrane transport protein with a gate on it.
 Binding of the ligand to the receptor causes the gate to undergo a conformational change to be
switched into its “open” state.
 This allows for the influx/efflux of specific ions.
 Crucial to the nervous system

175 | P a g e

Version 2.5

 Intracellular Receptors
 Associated with hydrophobic/small ligands (able to pass through the cell membrane)
 E.g. NO
 Commonly associated with steroid hormones (hydrophobic) such as testosterone/estradiol
 Refer to 6M.2 for more details.

6O.2 SIGNAL TRANSDUCTION
 Def: Process by which a target cell converts an extracellular signal into an intracellular signal that esults in
a specific cellular response. A multistep process involving a series of relay proteins and second
messengers.
 Activation of proteins usually caused by phosphorylation
 Protein Phosphorylation and Dephosphorylation
 Kinase  Enzyme that phosphorylates another protein
 Protein kinases act on other protein kinases, which then act on other protein kinases, leading to a
phosphorylation cascade

Mode of Protein Kinases
Signal Transduction 1.
(Phosphorylation
2.
Cascade)
3.

4.

The activated protein phosphorylates an inactive protein kinase
The inactive protein kinase undergoes a 3D conformational
change and is now activated.
This active protein kinase now phosphorylates another protein
kinase by transferring a phosphate group from ATP to the next
protein kinase.
This causes the next protein kinase to be activated.

Molecules Involved
Relay Protein
Protein Kinases
ATP, ADP

176 | P a g e

the ECM and the mitochondria. The activated G protein phosphorylates an inactive adenylyl Transduction cyclase such that it undergoes a 3D conformation and is now active. cytosolic calcium ion concentration is extremely low. inositol triphosphate (IP 3) and diacylglycerol (DAG) IP3 as secondary messengers Signal Transduction 1. The target protein undergoes a conformational change and is now activated. 2. This increases the cytosolic concentration of cAMP. Cyclic AMP. 3. DAG functions as a second messenger in other pathways Molecules Involved Relay Protein Phospholipase C PIP2 DAG IP3 177 | P a g e . The final protein kinase phosphorylates the target protein by transferring a phosphate group from ATP to the target protein. 7. cAMP Protein Kinase A  Calcium ions and inositol triphosphate IP3  Calcium ions usually used as a secondary messenger  Normally. Phospholipase C cleaves a phospholipid called PIP 2 into DAG and IP3.5 5. 2. coordinated response following stimulation by a single extracellular signal molecule  E. 6. The phosphorylation and activation of the next protein kinase continues under the last relay protein kinase is activated. Adenylyl cyclase catalyzes the synthesis of cAMP by removing a pyrophosphate group from ATP 3. which activates protein kinase A. Protein kinase A goes on to activate other proteins which eventually elicits a cellular response. eliciting a cellular response. Dephosphorylation of the protein kinases is carried out by protein phosphatases that catalyse the removal of phosphate groups from the kinases. and brief increases in cytosolic calcium ion concentration can trigger signal transduction pathways.Version 2.  Pathways that lead to calcium ion release from the smooth endoplasmic reticulum normally involve another intermediate messenger.g. Target Protein Protein Phosphatases  Second Messengers  Non-protein hydrophilic ligands that are unable to pass through the cell surface membrane rely on the use of a secondary messenger to pass on the signal. Calcium ions  Cyclic AMP (cAMP)  Synthesized from ATP by the enzyme adenylyl cyclase (which is indirectly stimulated by adrenaline)  Broken down by phosphodiesterase into AMP Cyclic AMP as secondary messengers Signal 1. 4. The activated protein G phosphorylates an inactive phospholipase C such that it changes its 3D conformation and is now active. Dephosphorylation 8. making them inactive and available for reuse.  Readily spread via diffusion  Result in a large scale. Molecules Involved Relay Protein Adenylyl Cyclase ATP.  Low cytosolic Ca2+ concentration maintained by calcium pumps that actively transport calcium ions into the sER lumen.

raising the cytosolic Ca2+ level and causing various proteins to be activated in 1 or more signalling pathways.  Due to the multistep pathway of signal transduction. IP3 diffuses through the cytosol and binds to an IP3 gated calcium channel in the sER membrane. every step increases the number of molecules that the signal is transferred to.g.3 CELLULAR RESPONSE  Types of cellular response (leads to the regulation of 1 or more cellular activities)  Nuclear response  Regulation of the rate of transcription of genes by activating specific transcription factors (activators/silencers)  E. thus amplifying the signal. allowing for fine tuning of the cellular response.g. IP3 gated calcium channel Calcium ions Other proteins 6O. signals must be terminable] 178 | P a g e .  A small amount of ligands can have a large impact on the cell  Provides opportunities for more coordination and regulation than simpler systems.Version 2. causing the gate to undergo a conformational change and open.  Proteins at one step often persist in the active form long enough for it to process numerous molecules of substrate before they become inactive again. Adrenaline eventually triggers the activation of glycogen phosphorylase which converts glycogen to glucose 1-phosphate  Cell wide responses  Apoptosis  Mating (in unicellular eukaryotes)  Advantages of cell signalling  Signal Amplification  Amplification of a signal occurs when some molecules in the pathway transmit the signal to numerous molecules at the next step in the series. 5. This causes calcium ions to diffuse out of the sER into the cytosol. [Each signalling molecule must last only for a short time.5 4. allows the cell to receive new signals. stimulation of genes promoting cell division by proto-oncogenic proteins  Cytoplasmic Response  Regulation of the activity of certain proteins  E.

different cells have different proteins and thus respond differently to ligands.  The specific constitution of receptors and relay molecules result in the different responses of cells to signals. triggering different cellular responses  One signalling pathway results in the inhibition/activation of relay proteins of other signalling pathways  Signalling efficiency  Relay proteins that are randomly distributed throughout the cytosol tend to relay the signal inefficiently. thus inactivating them. released upon extensive protein misfolding or irreparable DNA damage.  Cellular Apoptosis  Programmed cell death  Cell dies and forms lobes  Triggered by death signalling molecules that bind to cellular receptors.  Scaffolding proteins that bind different relay proteins together ensures that the signal transduction pathway is efficient.  There are generally 4 possible outcomes  Single.  Phosphodiesterase activity that catalyzes the formation of AMP from cAMP  Reversible binding of ligands to the cellular receptors. Signalling specificity  Due to differential gene expression.  Intrinsic GTPase activity of the G protein that hydrolyzes GTP to GDP. 179 | P a g e .5    Signal termination prevents target cell desensitization Protein phosphatases catalyse the dephosphorylation of activated kinase proteins.Version 2. linear pathway  Branched pathway. triggering specific signal transduction pathways that eventually leads to apoptosis  Can also be triggered by internal signals.

ECOLOGICAL. ORDER. each group is more exclusive. MORPHOLOGICAL. Bacteria. etc)  Taxonomic groups (from most inclusive to least inclusive. PHYLUM. there are less organisms in each taxon  Importance  Creates order in the study and referencing of organisms  Reveals natural evolutionary relationships and acts as a guide to the study of evolutionary pathways  Helps with the identification of newly discovered organisms  Forms the basis for the international system of nomenclature 7B – DESCRIBE THE CLASSIFICATION OF SPECIES INTO TAXONOMIC GROUPS (GENUS.  Specific epithet (start with a small letter)  Underline both names  Taxonomy: The science of naming. FAMILY. CLASS. genus. describing and classifying the diversity of organisms  Hierarchical Classification  A way of assigning species to a hierarchy of groups.  Binomial nomenclature  Species = Genus + Specific Epithet  Genus (Capitalize it)  The genus can be reduced to the first capital letter. PHYLOGENETIC CONCEPTS OF SPECIES IS REQUIRED. Eukarya)  Kingdom  Phylum  Class  Order  Family  Genus  (sub species) – morphs/breeds/strains/varieties. Bacteria)  Domain (3 domains: Archaea.5 7.)  Taxon  A formal grouping of organisms at any given level (specie. DIVERSITY (BASIC) & EVOLUTION  Evolution  The change in allele frequencies in a gene pool of a population over time  Microevolution  Evolution that occurs within a population over a few generations  Macroevolution  Broad pattern of evolution above the species level 7A – EXPLAIN THE BINOMIAL NOMENCLATURE OF A SPECIES AND HIERARCHICAL CLASSIFICATION. Eukarya. (KNOWLEDGE OF BIOLOGICAL.  Moving down the hierarchy. KINGDOM) AND EXPLAIN THE VARIOUS CONCEPTS OF THE SPECIES. provided that it has previously been named.Version 2. * not counted as a taxon 180 | P a g e . based on the 3 domain system: Archaea.

are reproductively isolated from oher species. thus blocking gene flow  Each species has a separate gene pool from that of other species  Advantages  Focuses on reproductive barriers and how speciation takes place via reproductive isolation  Limitations  No way to evaluate the reproductive isolation of fossils  Does not apply to organisms that reproduce asexually and self-fertilizing species  There are many species which are morphologically and ecologically distinct. but gene flow still occurs.Version 2. forming rare hybrids  Some individuals of the same species rarely interbreed  Definition: Ecological (*KIV ecology definitions. biochemistry and DNA sequences  Definition: Biological**  A species is a population or a group of populations whose members have the potential to interbreed in nature and produce fertile. physiological and behavioural features and a common gene pool  Differentiated from other species based on morphology. chapter 10)  Species: Groups of organisms sharing the same ecological niche (refer to ecology)  Advantages  Can be applied to all organisms regardless of their mode of reproduction  Disadvantages  Does not take into account morphology and reproductive compatibility  Definition: Morphological  Characterizes a species by the body shape and other structural features  Organisms are classified under the same species if their anatomical traits are similar  Advantages  Can be applied to all organisms regardless of their mode of reproduction  No information on gene flow required  Disadvantages  Difficult to decide how many morphological characters to consider when characterizing individuals  Difficult to analyse quantitative traits that vary in a continuous way  Members of the same species sometimes look differently from each other (different morphs)  Definition: Phylogenetic  Species: The smallest group of individuals that share a common ancestor  Obtained by comparing the morphological characteristics and biochemistry differences between organisms  Advantages  Distinguish groups of individuals that are sufficiently different to be considered separate species  Reveals the existence of “sibling species” (species that are morphological alike but are still separate species)  Disadvantages  Difficult to determine the degree of difference required to indicate separate species 181 | P a g e . physiology. fertile hybrids. share similar morphological. biological barriers that impede 2 members of different species from producing viable.5  Species (Smallest independently evolving unit)  Definition (overall): Group of orgaisms which can interbreed to produce viable fertile offspring. viable offspring  Exam species definition  Hinges on reproductive/genetic isolation.

[Identify Allopatric vs Sympatric Speciation] 6.1 SPECIATION  Speciation: The evolution of a new species when formerly interbreeding populations become reproductively/genetically isolated from one another. 8. 10. The 2 populations are now separate species. 7. Subgroups of the ancestral species become divided and are separated by a reproductive barrier. the two populations are now reproductively isolated and unable to produce viable and fertile offspring. This results in divergent evolution. Differentiation due to different selection pressures [Explain natural selection] IV. Each population lives in a different environment with different selection pressures which select for different adaptive traits.5 7C – EXPLAIN HOW SPECIES ARE FORMED WITH REFERENCE TO GEOGRAPHICAL ISOLATION.1 REPRODUCTIVE BARRIERS Prezygotic Barriers Reproductive isolating mechanisms Habitat Isolation (Geographic Isolation)  Two species that occupy different habitats within the same area may encounter each other rarely. leading to reproductive isolation (such as a geographical barrier or ecological barrier) [Identify the barrier in the question] 4.Version 2. The ancestral species exists as a single reproductive community. 9. Single population II. resulting in different favourable alleles being passed down to the subsequent generation due to differential reproduction. Due to the accumulation of sufficient reproductive isolating mechanisms and genetic diversity. This interrupts gene flow between the two populations and prevent interbreeding 5. 7C. causing them to be separated from each other Mating Attempt Mechanical Isolation  Morphological features such as size and incompatible genitalia prevents members of 2 species from successfully interbreeding 182 | P a g e . Reproductive Isolation 1. the founder effect may occur. Temporal Isolation  Species that breed during different times of the day/seasons/years cannot mix their gametes Behavioural Isolation  Sexual attraction between males and females of different species is limited due to differences in behaviour or physiology  Courtship behaviour attracts mates and other behaviours unique to a species by enabling mate recognition Physiological/ Ecological Isolation  Species diverge in ecological niches. and all its members interbreed and are reproductively isolated from other species. If only a few members of the species become separated from the rest. 2. Development of Barriers III. PHYSIOLOGICAL ISOLATION AND BEHAVIOURAL ISOLATION 7C.  Adaptive Radiation: The evolution of many diversely adapted species from a common ancestor over a relatively short amount of time. Variation in phenotypes and genotypes exist in the ancestral species 3. Speciation I. This changes the allelic frequencies in the two gene pools such that the two reproductively isolated populations become genetically distinct due to natural selection. if at all.

Version 2. the hybrid zygote cannot undergo meiosis as odd number of chromosomes cannot pair up.  Can also be caused by the inability of male gametes to survive in the female reproductive tract Postzygotic Barriers Successful Fertilization Reduced Hybrid Viability  The fertilized egg fails to develop past its early embryonic stages  Genes may interact in such a way that causes the embryo to survive but as a frail organism (aka selected against) Reduced Hybrid Fertility [Includes physiological isolation via polyploidy]  Hybrids that are vigorous and able to compete with other organisms in the environment may be sterile and unable to produce offspring.  Modes  Polyloidy (multiple haploid sets of chromosomes)  Instantaneous speciation. Hybridization between 2 closely-related species  Autopolyploidy  Fusion of gametes from the same species  Non-disjunction of homologous chromosomes resulting in diploid gametes.5 Gametic Isolation  Fertilization fails to take place because the gametes are unable to fuse with each other due to incompatibility of surface proteins. fertile offspring 7C. When 2 diploid gametes from the same species fuse.3 SPECIATION TYPES  Allopatric speciation/Geographical Isolation  The formation of a new species as a result of geographical isolation and subsequently natural selection and/or genetic drift  Geographical barrier  Any physical barrier that results in 2 populations being reproductively isolated  The geographic barrier required to separate organisms depends on the organism’s ability to move about.  Gene flow interrupted when a population is divided into geographically isolated subpopulations  If the original isolated population is small  Founder effect likely to occur  Sympatric speciation/Physiological Isolation  Evolution of new species from individuals living in the same area  Can occur very rapidly in a very short amount of time  Due to the evolution of reproductive isolating mechanisms at the start of the speciation process.  Chromosomes in allopolyploids are not homologous and cannot pair during meiosis Hybrid Breakdown  Fertile and viable offspring that mate with each other produces generations that are increasingly frail and sterile Viable.  Autopolyploids poduce gametes with even sets of chromosomes.  This species is reproductively isolated from its parent species due to problems related to the fusion of a haploid gamete and a diploid gamete to form an organism with odd multiples of sets of chromosomes (which are sterile as meiosis is unable to produce proper gametes as bivalents cannot be formed)  Tetraploids can only mate with tetraploids  Allopolyploidy  Fusion of gametes from 2 different species 183 | P a g e . etc. a tetraploid species is formed. When such gametes fuse with other gamtes with oddsets of chromosomes.

As given by the syllabus  Classification – The act of systematically arranging organisms into groups based on particular characteristics and their similarities  May not take evolutionary relationships into consideration  Phylogeny – The organization of species which takes into account evolutionary relationships between spcies  Definition: A hypothesis about patterns of evolutionary relationships among organisms that have been systematically arranged into groups based on specific characteristics and similarities. .  Phylogenetic trees  An evolutionary tree which shows a visual representation of a phylogeny to illustrate lineages and their evolutionary relationships  Traces patterns of shared ancestry between lineages and shows which organisms are more closely or distantly related to one another  Measured in MYA (millions of years)  Constructed from a series of dichotomies  Components  Branch points (nodes) represent a common ancestor of the branched texa  Represents the divergence of 2 or more species from common ancestor  Ancestors are often hypothetical  Position of the node on the evolution timeline shows the rate of speciation  *If there are more than 2 branches leading from 1 node. 7D – EXPLAIN THE RELATIONSHIP BETWEEN CLASSIFICATION AND PHYLOGENY.) Classification is the organisation of species according to particular characteristics. that node is called a polytomy  Tips (end point of branches)  Taxonomic units  Branches  Represents evolution over time 184 | P a g e .Version 2.  Sterile hybrids (odd chromosomal sets) can regain their fertility by either  Producing unreduced gametes that fuse with a haploid gamete from one of the parental species that has an odd number of chromosomes  Multiplying their chromosomal sets (resulting in allotraploidy) due to non-disjunction Ecological Isolation  Occurs when part of the population moves to a new ecological niche and as a result. Classification may not take into consideration evolutionary relationship between the species.5    An organism that contains more than 2 sets of chromosomes from 2 or more species The fusion of 2 gametes usually results in a hybrid gamete where chromosomes are not homologous and thus cannot pair during meiosis  Allodiploid  An alloploid that has only one set of chromosome from 2 different species. get separated from the parental species via reproductive isolating mechanisms. (Evolutionary relationships between organisms should be reflected in systematic classification. Phylogeny is the organisation of species according to particular characteristics which takes into consideration the evolutionary relationship between the species. Speciation then takes place as both populations independently evolve.

WITH EXAMPLES. The branch only shows the most recent common ancestor of 2 texa  Does not show how much genetic change occurred  A taxon on a genetic tree does not evolve from the taxon preceding it  Grouping of ancestors and descendants  Monophyletic  A taxon that includes an ancestral species and all of its descendants (all the branches from a node)  Sister taxa share a recent common ancestor with each other than either taxon does with any other group shown on the phylogenetic tree  Polyphyletic  A taxon that consists of several evolutionary lines that do not share the same recent common ancestor  An erroneous grouping that is made when scientists do not have enough information  Paraphyletic  A taxon that includes an ancestral species and some of its descendants 7E – EXPLAIN WHY VARIATION IS IMPORTANT IN SELECTION.5  Left to right (most ancient to present)  Lineage  The descendants from a particular ancestor  Limitations of phylogeny  Does not show phenotypic similarity  The sequence of branching does not necessarily indicate the actual (absolute) age of the particular species. Genetic variation thus provides the raw material for evolutionary change and selection 7F – EXPLAIN.  Limited Resources  Directional Selection (Soapberry Bugs. resulting in random mating such that the genotypic frequency stays constant over time (Hardy-Weinberg equilibrium)  Only genetic variation can have evolutionary consequences as acquired traits cannot be passed on to the offspring.Version 2. without variation there is no selection as all the individuals will have the same fitness.  Variations is a pre-requisite for natural selection. HOW ENVIRONMENTAL FACTORS ACT AS FORCES OF NATURAL SELECTION. Jadera haemotoloma)  Selection Pressure  Limited food resource 185 | P a g e .

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Variable  Proboscis Length; continuous variation
Soapberry bugs (Jadera haemotoloma) usually use a proboscis to feed on the balloon vine’s seeds
(Cardiospermum corindum). However, soapberry bugs in central Florida face a lack of food due to
the scarcity of balloon vines and as a result have no choice but to feed on the goldenrain tree’s
fruits. The seeds of such fruits are located much closer to the surface than as compared to the
balloon vine’s fruits. Thus, there was no need for a long proboscis and soapberry bugs that had
long proboscis were selected against in central Florida. This eventually led to a drop in the average
proboscis length of soapberry bugs in central Florida due to natural selection.
 Disruptive Selection + Allopatric Speciation + Adaptive Radiation (Galapagos Finches)
 Selection Pressure  Different food resources on the various islands
 Variable  Beak size and shape; continuous variation
 The Galapagos finches have beaks which are specialized in helping the birds to feed. Each species
has a beak which is adapted to the particular food source on each respective island. This can be
attributed to allopatric speciation, in which one of the Galapagos Islands was first colonized by a
small population of finches from South America. Natural selection then resulted in adaptations to
the local environment. Coupled with the founder effect, this resulted in an accumulation of
reproductive isolating mechanisms. Gene flow between the island populations and the South
American continent population was also restricted due to geographical isolation. As a result,
allotropic speciation took place. Subsequent cycles of speciation eventually led to adaptive
radiation.
 Diseases
 Stabilizing Selection – Sickle cell anaemia
 Selection Pressure  Malaria infection by Plasmodium falciparum (an apicomplexan) + Death due
to sickle cell anaemia
 Variable  Type of red blood cell (discontinuous variation)
 In malaria-prone regions, individuals who are heterozygous at the gene locus coding for the β
polypeptide subunit of haemoglobin have a greater fitness than do both kinds of homozygotes due
to their body’s ability to destroy sickle-shaped red blood cells. This results in the death of the
parasite Plasmodium that inhabits these red blood cells, granting these individuals partial
protection from malaria.
 These heterozygotes thus have the heterozygote advantage in malaria prone areas. Natural
selection thus seeks to maintain both alleles in the population.
 Homozygotes either die to due malaria (homozygous dominant) or due to sickle cell disease
(homozygous recessive)
 Predation
 Directional selection – Industrial Melanism (Peppered Moth, Biston betularia)
 Selection Pressure  Predation by birds
 Variable  Moth colour; continuous variation
 Peppered moths usually come in 2 forms, black and white (wild type). Before industrialization,
almost 100% of these moths were pale as black moths were obvious when resting on light-coloured
lichen covered tree trunks and as a result experienced high rates of predation while pale moths
were able to camouflage well and survive.
 Following the industrial revolution in the 1940s, the soot and smoke from nearby factories killed
the lichens and made tree trunks black. White moths were now highly obvious among black tree
trunks and experienced higher rates of predation relative to black moths. Due to natural selection,
more black moths survived and reproduced themselves than white moths.
 Environmental programs put into place to limit pollution in the 1950s onwards caused some lichens
to make a comeback on tree trunks while reducing the amount of soot and smoke. This caused

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pale moths to be able to camouflage themselves again, resulting in increased numbers of pale
moths.
 Poisons/Drugs
 Directional Selection – DDT resistance allele on D. Melanogaster
 Selection Pressure  Insecticide DDT
 Variable  Resistance to DDT; discontinuous variation
 The insecticide DDT served as a powerful environmental selective force as it is a powerful poison
that killed off much of the population of D. Melanogaster (a fruit fly) that did not have resistance
to DDT. As a result, the allelic frequency of the allele that conferred to D Melanogaster resistance
towards DDT increased from 0% in the early 1930s to 37% in 1960 due to natural selection
 Directional Selection – Antibiotic Resistance on Straphylococcus aureas
 Selection Pressure  Antibiotic Penicillin & Methicillin
 Variable  Resistance to antibiotics; discontinuous variation
 The introduction of penicillin in 1943 resulted in the deaths of many S. aurea. However, it also
resulted in the directional selection of S. aurea that were resistant to penicillin. Mutations in these
strains resulted in strains that could produce penicillinase, an enzyme that destroys penicillin. As a
result, the S. aurea population became increasingly penicillin resistant.
 Following the introduction of methicillin, a more powerful antibiotic, these populations started to
decrease but new strains emerged that were methicillin resistant due to directional selection.
These methicillin resistant S. aurea strains (MRSA) were even able to cause fatal infections in
humans. The use of more antibiotics has only resulted in MRSA strains that are resistant to multiple
antibiotics due to directional selection.
 Disruptive Selection – Copper tolerance in grasses
 Selection Pressure  High concentrations of toxic metal copper
 Variable  Resistance to high concentrations of copper; continuous variation
 Mine residues often contain high concentrations of toxic metals such as copper. Although most
grasses are unable to grow on soil containing high copper concentrations, certain grasses are able
to. These grasses develop resistance to toxic metals and are able to take advantage of
contaminated soils. However, the increase energy expended by such grasses in dealing with the
copper contamination also means that they are outcompeted by other grasses in noncontaminated soils. As a result, higher death rates of resistant plants on non-contaminated soil and
higher death rates of non-resistant plants on contaminated soil will eventually lead to the
increasing divergence of the population into 2 subpopulations with the 2 extreme manifestations
of the character.
 Intermediate phenotypes are selected against as they are neither able to compete with resistant
plants in contaminated soils or non-resistant plants in non-contaminated soils.
 Others
 Stabilizing selection – Birth mass in Homo sapiens
 Selection Pressure  Survival rate of babies
 Variable  Birth mass; continuous variation
 Babies at both extremes tend to have lower survival rates

7G – EXPLAIN HOW NATURAL SELECTION MAY BRING ABOUT EVOLUTION.
7G.1 DARWIN-WALLACE’S THEORY OF NATURAL SELECTION
 Natural selection: The process by which the environment selects for those well-adapted individuals with
inherited traits that are best suited for the local environment. These selected individuals with the selection

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advantage are more likely to survive to maturity and reproduce and leave behind more offspring than those
less fit.
 Evolution acts as the selection pressure to determine the direction and results of selection
 Increases the frequency of favourable genotypes
 Brings about adaptive evolution
 Often compromises (e.g. Bats sacrificed a pair of limbs to fly)
 Acts directly on phenotypes (but still acts on genotypes indirectly)
 Only acts on existing variations (not creation/magic!)
 Limited by historical constraints (works on the ancestral anatomy, not a new one)
 Interacts with chance (genetic drift + gene flow) and the environment (thus limiting the supply of
alleles in the gene pool)
 Adaptation: Evolutionary modifications that improves the chances of survival and reproductive success in a
given environment
 Relative fitness: The contribution an individual makes to the gene pool of the next generation relative to
the contribution of other individuals
 Differential Reproduction: “Like” produces “Like”
Darwin-Wallace’s Theory of Natural Selection leading to Evolution
Overreproduction
1. All organisms will produce a large number of offspring that will lead to a geometric increase
of Offspring
in population size if all the offspring survived.
(Observation 1)
Consistency in
2. However, most populations maintain relative constant numbers due to the carrying capacity
numbers
of the environment, due to the limited available resources.
(Observation 2)
3. Majority of the offspring thus die before they reach sexual maturity
Struggle for
4. Competition is thus inevitable when there are more organisms than what the environment
existence
can support.
(Inference 1)
5. Only a few organisms will survive to reproduce in each generation.
Development of
6. No 2 offspring are identical due to variation.
variation within
7. Individuals that are better adapted to survive (by chance) happens to have higher fitness
the population
than the rest of the population
(Observation 3)
Survival of the
8. Selected individuals with higher rates of survival are more likely to survive long enough to
fittest by Natural
reach sexual maturity and reproduce due to natural selection.
Selection
(Inference 2)
Differential
9. These selected individuals produce offspring similar to them and pass on the selective
reproduction
advantage to the next generation.
(Observation 4)
10. This results in adaptation to the environment
Formation of new
11. Over time, the proportion of individuals possessing the advantageous traits increases while
species
the proportion of those lacking such traits decreases, leading to the evolution of the
population.
12. The accumulation of several characteristics and adaptive mechanisms in a population over
many generations may result in the formation of a new species.
**Green parts are related to evolution; blue & white to natural selection

7G.2 NEO-DARWINISM – THE NEW SYNTHESIS
 Neo-Darwinism  The Modern Theory of Evolution
 Incorporates Mandelian genetics and Molecular biology into Darwin’s theory of natural selection
 Emphasizes on natural selection as the main driving force of evolution (similar to Darwin)
 Takes into account sources of variation
 Mutations
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Meiosis (crossing over + independent assortment)
Environmental factors

Neo-Darwinism Theory of Natural Selection leading to Evolution
1. Genetic variation exists within a population due to spontaneous mutation; crossing
Development of
over and independent assortment in meiosis; the random fusion of gamtes and
variation within
random mating between individuals. Each individual thus has a unique genotype.
the population
2. Individuals that are better adapted to survive have higher fitness than the rest of the
population
Natural Selection
3. Selected individuals with genetic variations best adapted to the environment are
& Differential
more likely to survive long enough to reach sexual maturity and reproduce due to
reproduction
natural selection.
4. These selected individuals produce genetically similar offspring and pass on the
favourable alleles to the next generation.
5. Over time, the proportion of individuals possessing the advantageous alleles
increases while the proportion of those lacking such alleles decreases.
6. This changes the allelic frequencies within a population and leads to further
evolutionary change.
**MUST STATE SELECTION PRESSURE

7G.3 TYPES OF NATURAL SELECTION
 Directional Selection
 A form of natural selection where the environment favours one extreme of the phenotype range and
shifts the population mean for the selected character
 Aims to reduce the frequency of the other extreme genotype and the alleles coding for this extreme
 This is often in response to a changing environment, where organisms at one extreme end tends to
have lower fitness than organisms at the other end.
 Disruptive Selection
 A form of natural selection where the environment favours the 2 extreme ends of the range of
phenotypes over the intermediate phenotype
 May form new species by splitting the gene pool into 2 separate ones
 Stabilizing Selection
 A form of natural selection where the environment favours the existing mean and selects against both
extreme phenotypes
 Reduction in variance and favours intermediate phenotypes
 Often the case in an unchanging environment where competition is not severe

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the genotypic frequency and allelic frequency is constant generation after generation. and as a result the allelic frequency is at equilibrium over time.5 7H – EXPLAIN WHY THE POPULATION IS THE SMALLEST UNIT THAT CAN EVOLVE.1 GENE POOLS.  Significance: Used to determine if microevolution has occurred in a population  Takes into account  Mandelian genetics  Mandel’s first law of independent segregation of alleles  Mandel’s second law of independent assortment of bivalents  Does not take into account sources of genetic variation  Natural Selection  Gene flow (migration)  Genetic Drift  Sexual selection/Inbreeding  Mutations  Equations (assuming 2 alleles are present at gene loci A)  Allelic Frequency  ? + ? = 1  P = frequency of dominant allele A. populations do not evolve 190 | P a g e .Version 2. random fusion of gametes and random mating  All individuals are subjected to the same selective pressures  Individuals cannot evolve! Natural selection can only act on populations by selecting certain individuals with favourable traits.  Definition of Population: A group of individuals of the same species that live in a defined geographic area  Capable of interbreeding and producing fertile offspring  Share a common gene pool  Provides the genetic variation for natural selection via meiosis. q = frequency of recessive allele a   Genotypic Frequency  ?2 + 2?? + ?2 = 1  P2 = frequency of homozygous dominant genotypes (AA)  pq = frequency of heterozygous genotypes (Aa)  q2 = frequency of homozygous recessive genotypes (aa)  Calculations  Obtain the homozygous recessive/dominant genotypic frequency by dividing the number of a specific genotype over the total number of genotypes  Root the number to obtain the allelic frequency  Take 1 minus the previous number to obtain the allelic frequency for the other allele When the Hardy-Weinberg equilibrium is true. Individuals can only introduce new alleles via mutations and pass on these alleles to the next generation 7H. HARDY-WEINBERG EQUILIBRIUM  Gene Pool  Definition: The total collection of all genes  Allelic Frequency  The relative proportion of alleles of a gene in the population  Genotype Frequency  The relative proportion of a particular genotype in the population  Hardy-Weinberg equilibrium  Definition: Denotes the state of an ideal non-evolving population. where the frequency of alleles and genotypes in a population’s gene pool remains constant from generation to generations.

g.  Causes random change of allelic frequencies (does not lead to adaptive evolution)  Genetic drift can cause harmful alleles to become fixed simply by chance. threatening the survival of the population.3 GENETIC DRIFT  Genetic Drift: The random change of allelic frequencies as a result of chance alone. gene flow can result in 2 populations combining into a single population with a common gene pool.2 GENE FLOW  Gene flow: The transfer of alleles into and out of the population due to the movement of fertile individuals or their gametes.  If extensive enough. 7H. 191 | P a g e .  Alleles transferred by gene flow can affect how well organisms adapt to local conditions.  Characteristics  Gene flow reduces the genetic differences between populations. Differs from generation to generation in a small gene pool  Based on the effects of chance events on populations that result in the death/irreproducibility of certain individuals (e. Creates genetic divergence between populations  Significant within small populations where chance events have a disproportionately high influence on the allelic frequencies of the population’s gene pool. Cyclone)  Characteristics  Results in less genetic variation within populations as certain alleles that are only carried by certain individuals are lost when those individuals fail to reproduce.Version 2.5  Conditions for the Hardy-Weinberg Equilibrium to hold true  Large population  Changes in gene frequency less likely to be due to chance (AKA genetic drift)  Random mating  No sexual selection/inbreeding  No gene flow  Movement of individuals into a population can affect the overall allelic frequency  No mutations  No introduction/removal of alleles that affects the gene pool and overall allelic frequency  No natural selection  All genotypes have equal fitness 7H.

resulting in an increase in the allelic frequencies of such alleles at the expense of other alleles.  This results in the disproportionate representation of alleles in the new remote population 7H.Version 2. resulting in a small group of survivors whose gene pool is not representative of the composition of the original gene pool. forming hybrids  Advantages  Hybrids have traits which are superior to both parents  Increase in heterozygosity  Sexual selection  A form of selection in which individuals with certain inherited characteristics are more likely than other individuals to obtain mates  Can result in sexual dimorphism (difference between the 2 sexes in secondary sexual characteristics such as colour.4 NON-RANDOM MATING/ARTIFICIAL SELECTION & SEXUAL SELECTION  Artificial selection  Non-random selective breeding process where changes in allelic frequencies are determined by deliberate human actions.  Fast and rapid process  Only selected individuals with alleles favoured by humans are chosen to mate.5  Bottleneck Effect  A chance event somehow kills a large percentage of the population. etc)  Intrasexual selection  Individuals of one sex compete directly for mates of the opposite sex  Usually occurs among males  Intersexual selection  Individuals of one sex (usually females) are choosy in selecting their mates from the other sex  Selection usually depends on the male’s appearance or behaviour 192 | P a g e .  Types  Inbreeding: Breeding closely related individuals  Increases the proportion of individuals that are homozygous at many gene loci (Increase in genetic inbreeding)  Advantages  Maintains desirable characteristics  Disadvantages  Reduces genetic diversity  Reduced rate of fertility and disease resistance.  Certain alleles may be under-represented. size. easily eliminated by environmental changes  Outbreeding: Selective controlled reproduction between members of genetically distant populations. over-represented or eliminated  Reduces genetic variability of the population  Founder Effect  Occurs when a few individuals colonizes a habitat that is isolated from their place of origin  Alleles that they carry are only a small fraction of the total gene pool and are not representative of the total gene pool.

g. with modifications  Molecular Homology  The use of the genetic code by all organisms on earth. bright plumage in peacocks) 7I – EXPLAIN HOW HOMOLOGY (ANATOMICAL. but not common ancestry  Analogy  A characteristic that appears to be homologous superficially but is actually independently acquired due to adaptations to similar environments 193 | P a g e .2 CONVERGENT EVOLUTION  Convergent evolution  Involves organisms with independent evolutionary lineages such that they develop similar phenotypes as they share similar cological niches  Results in analogous structures  Features which share similar function.Version 2.1 HOMOLOGY & DIVERGENT EVOLUTION  Homology  Similarity in characteristics resulting from a shared ancestry  Homologous structures  Matching structures in animals with a shared ancestry (Features that share a common ancestry.5   Certain traits are usually correlated to beneficial alleles/overall male health May not lead to adaptive evolution. showing that all organisms originated from a common ancestor that used this code  Conserved sequences of nucleotides in DNA/RNA and amino acids in proteins  Some genes are highly conserved in many organisms (e.g. but not necessarily a similar function)  Anatomical Homology  Homologous structures that indicate a common ancestry  Homologous structures do not have a common function and are modified from the ancestral structure (descent with modification)  Allows the tracing of species back to a common ancestors  Indicates divergent evolution where different organisms have diversified and diverge to occupy different niches  Vestigial structures  Remnants of features in ancestors that served important functions but are now greatly reduced in current organisms as they have no apparent function  Indicates shared ancestry with organisms in which these structures are functional  Embryological homology  Developmental Homology: All early embryos of vertebrates possess a post anal tail and pharyngeal pouches  All such structures descent from a common ancestor. EMBRYOLOGICAL AND MOLECULAR) SUPPORTS DARWIN’S THEORY OF NATURAL SELECTION (WITH EMPHASIS ON DESCENT WITH MODIFICATION). 7I. ribosomal genes) 7I. some forms of showing off to the other sex may even result in heightened risk of predation (e.

while other organisms which shared this ancestral character are in the “in group”  Shared derived characters  synapomorphies (e. resulting in the diversity of life. especially when fossils are formed after landmasses separate  Species that appear phenotypically similar have arisen due to convergent evolution as they are in similar environments with similar selection pressures that select for the same adaptive traits. giving rise to new species  Continental Biogeography  Shows convergent evolution. resulting in analogous structures. the backbone)  Organisms which do not have the ancestral character are considered to be in the “out group”.Version 2.  Closely related species sharing similar characteristics can be found in the same geographic region 7J. speciation will take place due to the founder effect after years of natural selection and the accumulation of sufficient reproductive isolating mechanisms and genetic diversity. showsthat descent with modification produced increasingly large differences among related groups of organisms.  Presence of transitional fossils that link between ancient and modern organisms shows that life arose from a common ancestor  Biogeography  The geographic distribution of species based on the geologic timescale  Takes into account the movement of different continents around the world since the earth’s inception. With different environments on each island.  Confirms that Founder Effect of population evolution. thus limiting gene flow.5 7J – EXPLAIN HOW BIOGEOGRAPHY AND THE FOSSIL RECORD SUPPORT THE EVOLUTIONARY DEDUCTIONS BASED ON HOMOLOGIES.g. along with. feathers)  Traits that are more recent  Species that share derived characters form a clade  Cladogram  A phylogenetic tree inferred by synapomorphies  Character tables are formed by comparing which organisms have shared traits 194 | P a g e .2 RECONSTRUCTING PHYLOGENY USING MORPHOLOGICAL DATA  Steps in constructing phylogeny  Sort out the homologous features from analogous features  Inferring phylogeny using these homologous characters  Cladistics  Uses common descent as the primary criterion to classify organisms and place them into groups called clades (monophyletic grouping)  Uses shared ancestral characters and shared derived traits to derive common descent  Shared ancestral characters  Plesiomorphies (e. as stated by the plate tectonics theory  Island Biogeography  Shows divergent evolution  Islands play an important role in speciation as they are geographically isolated.  Fossil Record (Palaeonontological evidence)  Preserved remains of once-living organisms  Different fossils showing the appearance and disappearance of many species.g. which along with radiometric dating that determines the age of fossils indicates the timing of speciation  Progressive changes in the anatomies of organisms with a gradual increase in anatomical complexity.

with silent mutations being the dominant type.  Using molecular systematics (molecular homologies) in classifying organisms  Compares macromolecules which are functionally similar to see if they are homologous  Genetic sequences change over the course of many generations due to the accumulation of mutations.  The degree of homology in the primary sequence of macromolecules reflects how much time has passed since the groups diverged from a common ancestor  Basis of using Nucleic Acid sequences in molecular systematics  Choice of genes for analysis  Non-conserved. hence DNA sequences from closely related organisms are more similar than to sequences from distantly related species. genes are subjected to selection pressures thus unfavourable gene mutations will be eliminated from the gene pool.5 7K – EXPLAIN THE IMPORTANCE OF THE USE OF GENOME SEQUENCES IN RECONSTRUCTING PHYLOGENETIC RELATIONSHIPS AND STATE THE ADVANTAGES OF MOLECULAR (NUCLEOTIDE AND AMINO ACID SEQUENCES) METHODS IN CLASSIFYING ORGANISMS.  Since DNA is expressed as proteins. thus changes in base sequences are passed down to subsequent generations.g microsatellites) These regions are non-coding and not subjected to selection pressures by the environment.Version 2. The genes are passed down the maternal line. In contrast. mutations in coding DNA may lead to changes in amino acid sequences  Thus. the more evolutionarily related 2 species are. the greater the homology in the primary sequence of macromolecules between 2 species. coding regions – These regions are present in most species and is tus a good basis for comparison as the gene tends to mutate slowly but steadily.  Conserved. can be used as molecular clocks to determine time of divergence.  Basis of using Amino Acid sequences in molecular systematics  Conserved amino acid sequence among species indicates that such sequences are needed for the proper functioning of the protein  Natural selection selects against any organisms with mutations at such positions  Advantages  Universal application  Molecular methods can be used on all organisms due to the universality of the genetic code (the unifying biochemical homology among all living organisms!) 195 | P a g e . non-coding regions – (e.

 Different genes evolve at different rates. even in the same evolutionary lineage. thus maintaining genetic diversity 196 | P a g e .  Balancing Selection  Polymorphism  The existence of 2 or more forms/morphs that are genetically distinct from one another but contained within the same interbreeding population  Balancing selection  Occurs when natural selection maintains stable frequencies of 2 or more phenotypic forms in a population. etc) Allows scientists to determine the evolutionary timeline and the relative periods of time in which evolution took place.  Applicable to New species that evolve might not show obvious phenotypic differences but have different DNA. proteins and RNA Allows for the reconstruction of phylogenies among groups of present-day prokaryotes and other microorganisms for which fossil records are absent (thus limiting the uses of comparative anatomy. behavioural and physiological traits as well. 7L – EXPLAIN HOW GENETIC VARIATION (INCLUDING RECESSIVE ALLELES) MAY BE PRESERVED IN A NATURAL POPULATION.Version 2. such sequences contribute to anatomical. which continue to propagate them. the frequency of phenotypes will oscillate over time and balancing selection ensures that genetic diversity of maintained  Neutral variation **Refer to section 7M. and such individuals are rarer when the frequency of recessive alleles is smaller. Due to the central dogma of molecular biology. Applicable to a huge amount of genetic variation  An enormous amount of data can be accessed by molecular method Quantifiable  Nucleic acid and amino acid sequence data is precise.5         Foundational genetics  Comparison of amino acid/nucleic acid sequences are easy. thus facilitating the objective assessment of evolutionary relationships Provides information about the process of evolution Large database of molecular information Applicable to ambiguous cases where the phylogenetic relationships cannot be determined using comparative anatomy & other non-molecular methods alone.  Only homozygous recessive individuals may be selected against.1 below  No selective advantage  Allelic frequencies stay consistent. a state called Balanced polymorphism  Heterozygote advantage  Refer to 7F. depending on which genes are used.  Diploidy  Recessive alleles that contribute negatively to an organism’s fitness can be hidden in heterozygotes. molecular trees can represent short or long periods of time. diseases. As a result. accurate and easy to quantify. sickle-cell anaemia stabilizing selection  Maintains 2 or more alleles at a particular gene loci.  Maintains a huge pool of alleles which might not be favourable under present circumstances but which could be beneficial when the environment changes. thus maintaining genetic diversity  Frequency-dependent selection  The fitness of a phenotype depends on how common it is in the population  Thus.

) 7M.  Does not go against natural selection theories (some evolutionary changes are still influenced by natural selection)  Only subjected to genetic drift and neutral mutations  Features  The rate of evolution is approximately constant with regard to neutral substitution that do not affect protein structure or function  Linear plot on graph  Proteins that are less important to the survival of the organism tend to evolve faster than more important proteins or regions of a protein  Amino acid substitutions that do not disrupt the existing structure and function of a protein occur more frequently in evolution than disruptive amino acid changes  Selective elimination of definitely deleterious mutations and random fixation of neutral or slightly deleterious alleles by genetic drift occur far more frequently in evolution than natural selection 7M.5 7M – BRIEFLY DESCRIBE THE NEUTRAL THEORY OF MOLECULAR EVOLUTION IN TERMS OF MUTATIONS PRODUCING NEW MOLECULAR VARIANTS WHICH ARE SELECTIVELY NEUTRAL.1 NEUTRAL THEORY OF MOLECULAR EVOLUTION (NEUTRAL VARIATION)  Neutral Theory: The vast majority of evolutionary changes at the molecular level are caused by random genetic drift or selectively neutral mutations that do not affect the phenotype of the organism  Suggests that much evolutionary change in genes is not influenced by natural selection  Neutral Mutations  Mutations found in the non-coding or coding regions that do not confer any selective advantage or disadvantage to the organism. there may still be chance deviations above and below the average rate  The same gene may evolve at different rates in different groups of organisms  Some genes evolve at much higher rates than others (even if they accumulate mutations at linear rates) 197 | P a g e . (KNOWLEDGE OF GENETIC DRIFT AND MOLECULAR CLOCK IS REQUIRED. thus they are selectively neutral  Used to deduce how species evolve and to fix the date in which 2 species diverged in the evolutionary timeline  Number of nucleotide difference is directly proportionate to the age of the common ancestor  Assumption: Neutral mutations accumulate at a relatively constant rate  Thus serving as a molecular clock to help measure evolutionary time  However.2 MOLECULAR CLOCKS  The molecular clock measures the number of mutations which accumulate in the DNA sequence of different species over time.  Such mutations do not necessarily increase an organism’s fitness. molecular clocks are not perfectly linear even over millions of years  Reading a molecular clock  X-axis: Evolutionary time  Y-axis: Number of nucleotide sequences mutated  Difficulties of using molecular clocks  Some portions of the genome accumulate neutral mutations at irregular rates and thus do not fit into the general trend  Over time.Version 2.

E.Version 2. A pianist’s child still has to learn piano if he/she wants to play piano  Charles Darwin (1809-1882)  Sailed around the world in the HMS Beagle and visited prominent locations including the South American Coastline and the Galapagos Islands  Noted that  Organisms that lived in temperate areas of South America resembled organisms that lived in tropical areas of South America rather than organisms that lived in temperate areas of Europe (also true of fossils)  Observed finches that shared similar features except their beaks which was adapted to catching food in their home island (Adaptation)  Artificial selection (Genetic selection) by humans have produced crops that bear little resemblance to their ancestors (e.g. Allopatric Speciation of antelope squirrels on opposite rims of the Grand Canyon 198 | P a g e .g. Cabbage from Mustard)  Species always overreproduce with respect to the carrying capacity of the environment  Argues that  Individual with favoured traits will be able to survive better in the environment as compared to the rest of the population  Grants them the ability to better survive and reproduce in a given environment than other individuals  This unequal ability of individuals to survive and reproduce will lead to the accumulation of favourable traits in the population over generations  The main factor causing evolutionary change is natural selection  Published On the Origin of Species by means of natural selection in 1859  Did not directly state the word “evolution” but rather emphasized on descent with modifications  Believed that all organisms descended from an ancestor in the past. descent with modification eventually led to the present biodiversity  Alfred Russel Wallace (1823-1913)  Developed the theory of natural selection while working in the Malayan archipelago  First scientist to formally publish a paper on the theory of natural selection SPECIATION  Allopatric  E.5 APPENDIX 7.g. Reasoned that over long periods of time.0 EXAMPLES THAT HELP TO ILLUSTRATE EVOLUTION [NOT IN SYLLABUS] HISTORY OF THE THEOR IES OF EVOLUTION & MAIN ARCHITECTURES  Carolus Linnaeus (1707-1778)  Developed the binomial nomenclature of a species  Used the nested classification system  Advocated creationism over evolution  Jean-Baptiste de Larmark (1744-1829)  Hypothesized that evolution took place via 2 principles  Used and disuse principle  Idea that body parts which were not used degenerated and that those used extensively became larger and stronger  Inheritance of acquired traits  Stated that such modifications (above) could be passed on to its offspring  **Wrong! Only genetic traits can be passed on to the offspring.

only 2 populations remained with total numbers below 50. Gene flow between populations led to the further spread of these alleles.g.  E. Amish People  The Amish population fled from Switzerland to Pennsylvania (USA) due to religious prosecution.g. resulting in higher death rates. Greater Prairie Chickens (Tympanuchus cupido)  The Greater Prairie Chicken’s range used to have a large range in Illinois.  Sympatric. Research confirmed that these surviving birds had lost 9 alleles as compared to the original gene pool. Bottleneck Effect  E. Domestication from the wild mustard (Brassica oleracea) 199 | P a g e . Accumulation of sufficient RIMs and genetic diversity leads to the reproductive incompatibility of both populations.07 is much higher than that for the general population. Since apple matures earlier than hawthorn. It is theorized that the development of the Grand Canyon (stage 2) led to the geographical isolation of the ancestral species into 2 populations. some populations have moved to colonize apple trees. It was discovered that females born in the eastern population survive twice as well as females born in the central population.  Genetic Drift.001.Version 2. In the population of origin. as one couple that migrated to USA carried the allele for Ellis-van Crevald syndrome. even though there are high levels of gene flow between both populations. In the small Dutch island of Vlieland. This can be attributed to the founder effect. The surviving birds had low genetic variation and less than 50% of their edges hatched. The current wheat used for bread (T. Founder Effect  E. their offspring also had lower fitness. Further research showed that the reason behind this was due to large amounts of gene flow between the central population and the mainland.g.  Genetic Drift. both populations undergo separate adaptive evolution. Inbreeding among the population led to a rapid increase in population size in Lancastor County Amish.5  Harris’s antelope squirrel (Ammospermophilus harrisii) inhabits the current’s south rim while the closely-related white-tailed antelope squirrel (Ammopermophilus leucurus) inhabits the north rim.g. aestivum) is an an allohexaploid and consists of 6 sets of chromosomes from 3 different species. the frequency of these resistance alleles increased due to natural selection. Ecological  E. resulting in speciation.g. Further natural selection eventually leads to speciation. Bread Wheat  Wheat originates from the genus Triticum. the 2 populations in hawthorn trees and apple trees are now temporally separated and prevented from breeding. 0. there are 2 populations of songbirds (Paras Major) located on the central and eastern part of the island. Since mainland genotypes were poorly adapted to the island. Together with the founder effect and natural selection.  Artificial Selection  E.  Sympatric. suggesting that the bottleneck effect may have led to an increase in the frequency of harmful alleles. A scientific enquiry discovered that the allelic frequency for Ellis-van Crevald Syndrome at 0.g. the range of the greater prairie chicken dropped and thousands died. EVOLUTION  Gene Flow  E. resulting in the development of RIMs and genetic diversity.g. By 1993. The North American apple maggot fly (Rhagoletis pomonella) once occupied the hawthorn trees but since European colonization of the USA. Allopolyploid  E. Following the conversion of many prairies (grasslands) into farmlands. Gene flow has resulted in the worldwide spread of resistance alleles in Culex pipies (a mosquito).

g.  Convergent Evolution  E. kale.g. resulting in different breeds of dogs such as the greyhound and the sheepdog. Fins and Flippers in fishes and cetaceans  E. Pigs.g. Gliders in marsupial and eutherians  The Australian marsupial Sugar Glider is superficially similar to flying squirrels. kohlrabi. broccoli. Camels and Cows  Cross comparison of the astragalus (ankle bones) between Pakicetus (an early Cetacean) and the even-toed ungulates revealed this discovery as both groups had astragaluses with double humps on both ends  Biogeography  E. broad feet and small build  These horses eventually evolved into modern day horses that were better adapted at grass land.Version 2. Perissodus microlepis (scale eating fish) 200 | P a g e . namely cabbage.5  Selection for different parts of the plants resulted in the formation of new subspecies (variant/strains/breeds). birds and bats EVIDENCE FOR EVOLUTION  Fossil Record  Horses  Modern day horses belong to the Genus Equus  Evolved from ancestral horses (Genus Hyracotherium) found in forests with short legs. gliding eutherians in North America.g. Comparing the primary structure of cytochrome C (mobile electron carrier in the mitochondrion ETC)  Found in the mitochondria of every aerobic eukaryote PRESERVATION OF GENETIC VARIATION  Frequency Dependent Selection  E.g.g Domestication of dogs from the grey wolf. This is due to both mammals having adapted to the environment in similar ways. However.  E.  E. Sugar Gliders have many other characteristics which makes it a marsupial and not an eutherian. Brussels sprouts. resulting in modern da horses that:  Have a large build  Have less toes on their feet and the development of a bony hoof  Enabled the horses to run at high speeds across grasslands  Have larger teeth with a complex pattern of ridges on molars and premolars  Able to chew through gritty vegetation such as grasses which wears teeth down  Such evolutionary adaptations were in response to the natural selection of individuals that were able to withstand the climate change and adapt to grasslands  Cetaceans  Marine mammals  Fossil records suggest that cetaceans shared a common ancestor with even-toed ungulates such as Deer. Wings in insects.g. Endemic species on the Galapagos islands that closely resembled South American species but are yet totally new species on their own suggests that they evolved from populations of mainland species that colonized the islands  Amino Acid Sequencing  E.

one being leftmouthed and the other being rightmouthed. 201 | P a g e .Version 2. There are 2 phenotypes. allowing the other phenotype to gain more opportunities in attacking the prey. Thus.5   Selection Pressure  Prey (that guards against the most common species) These fishes eat the scales of other fishes by attaching themselves to the side of a fish. Prey species guard against whichever phenotype is more common. natural selection favours the least common mouth phenotype and balancing selection keeps the frequency of each phenotype close to 50%.

Components Required Gene of Interest Restriction Enzyme Plasmid Vector ATP DNA Ligase 202 | P a g e . Sticky ends only anneal to complementary sticky ends due to complementary base pairing while blunt ends may associate with any other blunt end.  Restriction sites  Sequences 4-8 nucleotides in length that can be cut to form restriction fragments  Palindromic  Bacteria DNA similar in sequence to restriction sites are methylated by DNA methyltransferase. which changes the 3D conformation of DNA and prevents restriction enzyme from cutting them. CLONING AND SEQUENCING DNA 1A – DESCRIBE THE NATURAL FUNCTION OF RESTRICTION ENZYMES  Restriction Enzyme (AKA Restriction Endonuclease)  An enzyme that binds to a dsDNA at a specific sequence (restriction site) within the DNA molecule and makes a double stranded cut at the sequence by hydrolysing phosphodiester bonds.2 PROCEDURE OF RECOMBINATION Formation of Recombinant DNA molecules Isolation of Gene of Interest 1. thus defined segments can be excised from a larger molecule.  The enzyme may cut restriction sites in the middle. ATP.  Advantage: Allows the gene of interest to be inserted into the vector. Foreign DNA containing the gene of interest is cleaved with a restriction enzyme to produce a DNA fragment containing the gene of interest A plasmid vector is cut using the same restriction enzyme. ISOLATING.1 COMPONENTS OF RECOMBINATION  DNA Ligase  Ligates together cut ends that are either blunt or sticky by forming phosphodiester bonds between cut ends.  Requires ATP  Plasmid/Cloning Vectors  DNA fragment containing the gene of interest 1B.Version 2. 3.  Function  Provide a protective mechanism to help bacteria resist attacks from bacteriophages  Helps in the removal of viral sequences by recognizing specific sequences in phage DNA and cleaving those sequences  This prevents the bacteriophage from succeeding in causing a full-blown bacterial infection  Highly specific and only cut certain restriction sites.  Types  The enzyme may cut restriction sites in a staggered manner. Recombination 2. forming blunt ends  Limitation: requires linker DNA which is cleaved to generate sticky ends/requires adaptor with sticky ends.5 B1. The cut plasmid. forming sticky ends with short singlestranded overhangs that can form hydrogen bonds via complementary base pairing. 1B – EXPLAIN THE FORMATION OF RECOMBINANT DNA MOLECULE 1B. DNA fragment containing the gene of interest and DNA ligase is added into a solution.

forming a recombinant DNA molecule Cut Fragment with GOI 1C – OUTLINE THE PROCEDURES FOR CLONING AN EUKARYOTIC GENE IN A BACTERIAL PLASMID AND DESCRIBE THE PROPERTIES OF PLASMIDS THAT ALLOW THEM TO BE USED AS DNA CLONING VECTORS 1C. Easiest method to eliminate host cells that did not take up recombinant plasmids.  Limitations  Size  Plasmids can only carry DNA fragments of up to 10kb in size  Incompatibility with eukaryotic gene of interests  Introns in eukaryote-protein coding genes are not compatible with bacteria protein synthesizing machinery due to the lack of RNA splicing mechanisms.  Fluorescent genes such as GFP (Green Fluorescent proteins) that allows the host cell to glow under a fluorescent microscope. Kanamycin.  Eukaryotic promoters are not compatible with prokaryotic promoters. Can be overcome by using a cDNA form of the gene of interest. depending on the size required (b = basepairs)  Plasmids (10 kb)  Bacteriophages (20 kb)  Cosmid (48 kb)  Bacterial artificial chromosomes (300 kb)  Yeast artificial chromosomes (> 1 mb)  Properties  Presence of an Origin of Replication  Allows the circular double stranded plasmid to replicate independently for numerous copies to be passed down to genetically identical daughter cells  Presence of a variety of unique restriction sites (Multiple Cloning Sites/Polylinkers)  Allows many restriction enzymes to be used  Each restriction site is only present once to prevent fragmentation of the plasmid when a restriction enzyme is used. Tetracyclin or Chloramphenicol.  Genes determining nutritional requirement such as laz Z which allows the host cell to survive on certain substrates such as lactose.  Selectable markers  Confers well-defined traits to the host cell that can be selected for such as antibiotic resistance genes.  Foreign DNA fragments cut with the same restriction enzyme can be inserted to form the recombinant plasmid.5 4. DNA ligase ligates the ends of the vector with the ends of the DNA fragment containing the gene of interest. or genes determining nutritional requirement to distinguish between transformed and non-transformed cells. The gene of interest can be genetically engineered to be placed before a prokaryotic promoter to solve this problem.Version 2.1 FEATURES OF A CLONING VECTOR  Cloning vector – A DNA molecule that carries foreign DNA into a host cell and is able to replicate in the host cell (Plasmids usually used)  Can be made from a variety of DNA fragments. Such 203 | P a g e .  Marker Types  Antibiotic resistance genes allow host cells to survive on LB with antibiotics such as Ampicillin.

4. 2. coli cells may be induced to take up foreign DNA via the heat shock method or electroporation. 3.2 PROCESS OF CLONING DNA USING E. In the heat shock method. 5. 4. 3. E. ATP. Foreign DNA containing the gene of interest is cleaved with a restriction enzyme to produce a DNA fragment containing the gene of interest A plasmid vector is cut using the same restriction enzyme.COLI Cloning DNA in vivo using E.1 FEATURES OF THE HOST CELL  Host cells are used for in vivo amplification of DNA molecules (PCR is used for ex vivo amplification)  Escherichia coli is often used because  It is safe and easy to handle  Easy to grow and does not require a large space or nutrition. Calcium ions adhere to the cell membrane to reduce the repulsion between negatively charged DNA and phospholipids. COLI CELLS TO PRODUCE EUKARYOTIC PROTEINS TO AVOID THE PROBLEMS ASSOCIATED WITH INTRONS. forming a recombinant DNA molecule.coli Isolation of Gene of Interest 1. coli cells are first suspended in ice-cold calcium chloride solution. Transformation 7.Version 2. At the same time.5 promoters may also have operators which then result in the GOI being transcribed only under certain conditions.  It has a very rapid replication time  It is easy to kill and dispose of  It can take up and tolerate foreign DNA segments  For eukaryotic GOIs. Can be grown on nutrient agar plates or in a liquid culture. Isolation of GOI Recombination of GOI and vector Transformation Selection Scaling Up 1D – EXPLAIN HOW EUKARYOTIC GENES ARE CLONED USING E. 6. baker’s yeast (Saccharomyces cerevisiae) is used as it has the necessary mechanisms required to carry out post-transcriptional and post-translational modification 1D. Components Required Gene of Interest Restriction Enzyme Plasmid Vector ATP DNA Ligase Cut Fragment with GOI E. 1C. 8. The cut plasmid. 1D. reannealed plasmids which do not contain the GOI may also be formed. 5. E. coli Foreign DNA CaCl2 (aq) 204 | P a g e . Recombination 2. DNA fragment containing the gene of interest and DNA ligase is added into a solution under conditions which favor annealing.2 PROCEDURE OF CLONING 1. Some recombinant plasmids containing other foreign DNA may also be formed. DNA ligase ligates the ends of the vector with the ends of the DNA fragment containing the gene of interest.

These are the desired colonies. A process of selection is carried out to determine which cells have taken up the plasmid with the GOI. The process differs based on the selection markers present on the plasmid. Cross comparison of the positions of the bacteria colonies on the 2 different plates will reveal missing colonies.  For nutritional requirement genes as selectable markers  The lacZ gene codes for β-galactosidase which cleaves the substrate X-Gal (white) to form a bluecoloured product. the E. E. transforming E. coli cells with the ligated products. The ligation products are then added to the solution. 11. 4 types of cells are now present: Cells with recombinant plasmids containing the GOI. the β-galactosidase produced will not be functional. As DNA & other charged molecules goes through the pores. 205 | P a g e . Scaling up is done whereby the cells containing the plasmid with the GOI are grown in nutrient broths and then harvested to obtain the gene products. 16. coli to recover from the heat shock. As a result. The ligation products are then added into the solution. As a result.  Transformed cells are plate directly onto a plate containing X-Gal. 10. a piece of sterile velvet is pressed onto the original plate. The cells are then incubated in non-selective growth media to allow E. coli is first suspended in deionized ice-cold water. the cell membrane discharges and the pores close. picking up colonies. which have taken up the recombinant plasmid and are unable to survive on the plate with antibiotics. E. the β-galactosidase produced will not be functional. coli is then subjected to a heat shock (42°C) which promotes the formation of transient pores in the membranes.  If the GOI is not inserted into the lacZ gene. 12. the bacteria is unable to cleave X-Gal and their colonies remain white. cells with recombinant plasmids. 13.  In replica plating. transforming E.3 SELECTION OF CLONES BASED ON SELECTABLE MARKERS  Differentiating between bacteria with recombinant plasmids against those which do not have plasmids or have reannealed plasmids  For antibiotic resistance genes as selectable markers  Selecting for bacteria with the antibiotic resistance gene  Grow the bacteria on a LB plate containing the antibiotic that the gene confers resistance towards.5 Selection Scaling Up 9. coli is then subjected to electroporation which promotes the formation of transient pores in the membranes.  If the GOI is inserted into the lacZ gene. 15. cells with reannealed plasmids and cells without plasmids.  Selecting for bacteria without the antibiotic resistance gene  This is often used to identify recombinant plasmids where the antibiotic resistance gene has been deactivated via insertional inactivation  The method used is called replica plating. coli cells with the ligated products. the bacteria is able to cleave X-Gal and their colonies turn blue. Bacteria colonies present will definitely contain the antibiotic resistance gene. 19. Ligation Products Water bath Non-selective growth media Di Water Ligation Products Electricity generator Antibiotics X-Gal Radioactive Probes Nutrient Broth 1D. In electroporation.Version 2. 18.  Adding antibiotics is usually the first step to eliminating cells without any plasmids in them. 17. 14. The same piece of cloth is then pressed onto a second plate containing the antibiotic.

Colonies that contain recombinant plasmids are collected and transferred onto a nitrocellulose filter.Version 2. Comparison of the positions of the hybridization signal on the autoradiograph with those of the colonies on the master plate will indicate which colonies contain the gene of interest. colonies with the GOI will give off fluorescence that can be seen using the fluorescent microscope. thus prokaryotic translational machinery cannot recognize the start site Insert the DNA form of the Shine-Dalgarno sequence in the 5’UTR of the eukaryotic gene Prokaryotes have no post translational modification. 4.) 1E.  The desired colony can then be scaled up. resulting in low rates of expression.  It is thus only used to synthesize short sequences 206 | P a g e . the GOI can be synthesized artificially by the stepwise addition of nucleotides to a DNA molecule which has the 5’ end attached to a support.5  Blue colonies thus contain bacteria with reannealed plasmids while white colonies contain bacteria with recombinant plasmids  Differentiating between bacteria with recombinant plasmids containing the recombinant plasmids with the GOI against those that have recombinant plasmids with no GOI. (OUTLINE OF THE PROCESS OF THE FORMATION OF THE LIBRARIES AND APPLICATIONS OF EACH OF THE TYPES OF LIBRARY IS REQUIRED.4 OVERCOMING PROBLEMS OF USING EUKARYOTIC GENES WITH PROKARYOTES  All in 1 solution: Use a simple eukaryotic organism such as yeast to express the genes Problem Solution Eukaryotic genes contain introns which are absent in prokaryotes as prokaryotes do not have RNA splicing machinery. 5. The cells can then be grown in a liquid culture or large tank  Using fluorescence  If the GOI contains a fluorescent gene. The filter is then subjected to autoradiography which detects the radioactivity. which binds to the nitrocellulose.  Using Nucleic Acid Hybridization (Colony Hybridization) 1.1 SOURCES OF THE GENE OF INTEREST  Artificial Synthesis  If the DNA sequence of the GOI is known. The cells are lyzed to release their DNA. 2. Pre-mRNA is directly expressed and additional amino acids are incorporated Reverse transcribe the mRNA of the gene of interest to form cDNA Bacteria transcriptional machinery does not recognize the eukaryotic promoter. thus proteins are not folded into their native conformation Purify and modify expressed proteins manually 1E – DISTINGUISH BETWEEN A GENOMIC DNA AND CDNA LIBRARY. Place the eukaryotic gene under the control of a strong prokaryotic promoter Eukaryotic mRNA lack a Shine-Dalgarno sequence. This ssDNA is bound to the filter and can be hybridised by a complementary RNA or DNA radioactive probe. this method produces too many inaccuracies in long sequences. 3.  However. The dsDNA is also degraded to form ssDNA. 1D.

 Allows a scientist to construct phylogenic relationships between organisms and discover the functions of genes by comparing the unknown gene to a similar one with known functions. 3. 2.5  DNA libraries  Two types: Genomic DNA & Complementary DNA.  Note  Reverse transcriptase is not used for the whole process as it is prone to errors (favours mutations)  Advantages  GOIs in this form can be easily inserted cloned and expressed in prokaryotic cells as there are no introns present. Each recombinant vector molecule undergoes transformation into a separate bacteria cell. Each individual fragment is inserted into a separate cloning vector.  Limitations  Genes that produce rRNA or tRNA cannot be isolated using this method as they do not have a poly(A) tail. although the sequence can still be determined when multiple pieces of a gene are cut to form overlapping sequences.  An abundance of a certain mRNA sequence would mean that the gene is highly expressed.  A cDNA library constitutes a much smaller portion of the entire genome as compared to a gDNA library 207 | P a g e .2 COMPLEMENTARY DNA LIBRARY  A complementary DNA library is a library containing the portion of an organism’s genome that is expressed as proteins. An organism’s entire genome is cleaved into a large number of restriction fragments. This allows researchers to study patterns in gene expression throughout an organism’s development. 1E.Version 2.2 GENOMIC DNA LIBRARY  A genomic DNA library is a collection of bacterial clones each containing a copy of a particular DNA fragments from an entire foreign genome  Advantages  Allows researchers to isolate and study specific genes including its regulatory sequences  Allows for the study of intergenic sequences  Limitations  Genes may be cut into multiple pieces and carried in multiple plasmids. The resulting population of bacteria which contain all the genomic DNA sequences of the organism is called a genomic DNA library Components Required Restriction Enzyme Host Genome Cloning Vector Transformation apparatus Bacteria host 1E.  The frequency of transcription on any one gene cannot be determined Synthesizing a gDNA library Whole-genome shotgun approach 1. 4.

Reverse transcriptase transcribes a strand of complementary DNA from the mRNA strand. Restriction enzymes cut DNA at specific restriction sites for both cDNA and vector. The oligo-dT polynucleotide binds to the poly(A) tail via complementary base pairing. Reverse transcriptase is removed and RNase H is added to hydrolyze the mRNA strand into nucleotides. serving as a primer for the 5’ to 3’ synthesis of DNA by reverse transcription. 8. 5. forming a DNARNA hybrid. 7. 6. S1 nuclease cleaves the hairpin loop at the end of the dsDNA. Recombinant vectors are transformed into host cells Components Required mRNA oligo-dT Reverse Transcriptase RNase H DNA Polymerase S1 nuclease Linker DNA DNA Ligase ATP Restriction Enzyme 1E. Mature mRNA is mixed in a solution containing a chemically synthesized oligo-dT or poly-dT nucleotide sequence. thus it is easier to find the gene of interest as cDNA only contains the exons and is enriched in certain species of mRNA  Intact genes are cloned as the splicing of exons is accurate and precise. 10. The double stranded DNA is then ligated with linkers that provide the necessary sticky ends that complementary base pair with the cut ends of the vector. 9. DNA polymerase synthesizes a complementary DNA strand to form a double-stranded DNA.5 Synthesizing a cDNA library Isolation of mRNA Synthesis of cDNA strand Preparation of cDNA for recombination Transformation 1. 2. 3. 4. Intergenic noncoding regions are stored Intergeic noncoding regions are not stored Sequences contain introns Sequences do not contain introns Most clones usually only have part of the coding sequence of a gene All clones have a continuous coding sequence Most genes are only present in 1 copy cDNA is enriched in certain mRNA sequences  Advantages of cDNA library  More compact as introns have been removed  Represents genes that are expressed.Version 2. 208 | P a g e . The 3’ end of the ssDNA folds back onto itself to form a hairpin loop which acts as a primer for the synthesis of complementary strand. DNA ligase ligates both cDNA and vector to form a recombinant vector 11.3 COMPARING GDNA AND CDNA LIBRARY Genomic DNA Library Complementary DNA Library The entire genome is represented Only the portion of the genome that is expressed as proteins is represented A larger number of clones is required to store the library A smaller number of clones is required to store the library The same gDNA is obtained from all the cells of the same individual Different cDNA libraries are obtained from different cells as genes are not expressed at the same rate across all cells.

4. Mature mRNA is obtained from the anterior pituitary gland and reverse transcribed to form a cDNA library The cDNA library is digested with HaeIII. Based on known amino acid sequences of A & B chains. The recombinant plasmids are transformed into the E. Transformation 4. The longer segment is retained for use in the construction of the recombinant plasmid. Scaling up and Production 8. HUMAN GROWTH HORMONE AND INSULIN). The smaller segment is replaced by an artificial DNA molecule that contains the start of the hGH gene and provides the correct signals for translation in E.5 1F – OUTLINE TWO IMPORTANT PROTEINS THAT CAN BE PRODUCED BY GENETIC ENGINEERING TECHNIQUE (E. Recombination 5. which cuts the hGH cDNA into 2 fragments.Version 2. 2. one representing the A chain and one the B chain. Transformation 6. 1F. trinucleotides representing all the codons are synthesized and joined together in a specific order. Selection 7.G.coli.  Characteristics of insulin that facilitates its production by recombinant DNA techniques  No glycosylation is required  Insulin is a small protein consisting of 51 amino acids Producing Insulin using Recombinant DNA techniques Isolation of Gene of Interest 1.coli cells via electroporation or heat shock E. 3.coli clones carrying the recombinant plasmids are selected for using selectable markers encoding well-defined traits such as antibiotic resistance. Lactose is used to induce production of hGH. Each artificial gene is placed under the control of a strong lac promoter and a part of the lacZ gene by ligating it to a plasmid containing the 2 components. 9. The modified gene is ligated into a plasmid containing the lac promoter Components Required mRNA cDNA components HaeIII Synthetic Leader Sequence DNA Ligase ATP Plasmid Vector Transformation Components Varies The recombinant plasmids are transformed into the E. Two artificial genes are constructed.1 HUMAN GROWTH HORMONE (SOMATOTROPHIN)  hGH is a peptide hormone produced by the anterior pituitary gland  Effects (Lack results in dwarfism)  Increases height in childhood  Increased muscle mass  Decreased fat mass  Growth of internal organs Producing Human Growth Hormones using Recombinant DNA techniques Isolation of Gene of Interest 1.coli cells via electroporation or heat shock Components Required Trinucleotides DNA Ligase ATP Plasmid Vector Transformation Components 209 | P a g e . hGH secreted is isolated and purified.2 INSULIN  Insulin is a protein consisting of 2 chains bound together by disulfide bonds. 2. Lactose 1F. Recombination 3.

It must be known.5 Selection 5. This is possible as the 2 portions are separated by a methionine residue. Lactose is used to induce production of the 2 fusion proteins of the first few amino acids of β-galactosidase and chain A or B.  Also contains buffering salts and detergent to maintain pH  Thermal Cycler 1G.  2 sets of ssDNA Primers  Chemically synthesized nucleotide sequences that flank the nucleotide sequence of interest and are complementary to the 3’ end of both template strands  Deoxyribonucleoside triphosphates  Must be present in excess to ensure that they anneal to the templates before the templates reanneal  Taq DNA Polymerase  Thermostable DNA polymerase that is not denatured by repeated heat treatments in the PCR cycle. Components Required dsDNA template strand 210 | P a g e . The mixture is briefly heated to 95°C to break down the hydrogen bonds between the 2 strands of the dsDNA template.  Reaction Buffer  Contains magnesium ions that function as cofactors required for DNA polymerase activity. 7.Version 2. 9. Varies Lactose Cyanogen Bromide 1G – DESCRIBE THE POLYMERASE CHAIN REACTION (PCR) AND EXPLAIN THE ADVANTAGES AND LIMITATIONS OF THIS PROCEDURE.1 COMPONENTS OF PCR  DNA Template  This double stranded DNA template contains the nucleotide sequence of interest. Polymerase Chain Reaction Cycle Denaturation of DNA templates 1. Addition of cyanogen bromide cleaves the fusion protein into the first few amino acids of β-galactosidase and chain A or B.coli clones carrying the recombinant plasmid with the GOI is identified.  Polymerase Chain Reaction – A technique used to selectively amplify a specific sequence of DNA from an original trace sample into many copies of identical DNA in vitro by using newly synthesized strands as templates for subsequent cycles 1G.2 PCR CYCLE  Characteristics  Within a few cycles. Selection for E. the dominant DNA is identical to the sequence flanked by the 2 DNA primers  The number of DNA molecules formed in n cycles is 2n  Only in the 3rd cycle does the targeted DNA sequence exists in a double-stranded DNA molecule  Specific – only the DNA between the 2 primers is amplified  Amplify – Make a large number of copies of a DNA sequence from a small amount of original sample in a short time. forming single-stranded DNA molecules. 8. The 2 chains are then isolated and mixed together. The 2 chains are reduced and oxidized to form disulfide bonds present in insulin. Scaling up and Production 6.

 Not applicable to proteins 1H – EXPLAIN HOW GEL ELECTROPHORESIS IS USED TO ANALYSE DNA.3 ADVANTAGES AND LIMITATIONS OF PCR  Advantages  Sensitive – PCR is able to amplify sequences from minute amounts of DNA  Speed & Ease – PCR is rapid and automated  Robust – PCR permits the amplification of specific sequences from material in which DNA is badly degraded or embedded in a medium where the isolation of DNA is difficult  Limitations  Easily contaminated – Due to the extreme sensitivity of PCR. 211 | P a g e . Primer extension 4. This entire process is then repeated 20-30 times DNA Primers Taq DNA polymerase dNTPs 1G. The mixture is cooled to 54°C in the presence of a large excess of the 2 DNA oligonucleotide primers. some prior sequence information is required. The primers hybridize to their complementary sequences at the 3’ ends of the two single-stranded DNA template strands via hydrogen bonding. 3.5 Annealing of Primers 2. DNA migrates towards the positively charged anode from wells near the negatively charged cathode. 5. The mixture is then heated to 72°C to allow Taq DNA polymerase to catalyze the synthesis of the complementary DNA sequence downstream of the primers in the 5’ to 3’ direction at its optimal temperature.Version 2.  Electrophoresis – A technique used to separate charged molecules based on their differential rates of migration through the gel matrix in an electric field  Basis  Charge  Due to the negatively charged sugar phosphate backbone of DNA. contamination of the reaction by nontemplate nucleic acids would cause these nucleic acids to be amplified as well (provided the primers attach to them)  Infidelity of DNA replication in vitro – Taq DNA polymerase lacks a 3’ to 5’ exonuclease activity  Short size and limiting amounts of PCR product – PCRs for products longer than a few thousand base pairs are less efficient due to enzyme activity loss and accumulation of inaccuracies  Need for target DNA sequence information – In order to construct specific oligonucleotide primers.

 Thus. a complex mixture of linear DNA fragments is size-fractionated into discrete bands. 11. Agarose powder is mixed with a buffer and heated until agarose dissolves. (DNA molecules have a constant charge density)  In the same period of time. The gel comb is removed. The gel is submerged in a DNA-binding dye such as ethidium bromide or methylene blue. Staining 12. The gel front formed can thus be used to track if DNA is at risk of migrating out of the gel.  At the same time. The gel tray is placed into the electrophoresis chamber such that the wells are nearer to the cathode and a buffer solution is loaded until it covers the gel. DNA Loading 6. The solution is poured into the gel tray (sealed with masking tape) and a gel comb is placed at one end to create wells in the gel.  The loading dye is a negatively charged compound that migrates independently of DNA and faster than DNA. The gel is allowed to cool and solidify. DNA is otherwise colourless. Equipment setup 3.5  Mass  The rate of migration is directly proportional to the length of the DNA molecule. The current is turned on The current is switched off when the gel front has reached the edge of the gel. 10. xylene cyanol.  Purpose  To separate/isolate/purify DNA fragments according to size.  Glycerol also makes the DNA heavier so that it sinks to the bottom of the wells. a longer piece of DNA would have travelled a shorter distance compared a shorter piece of DNA. gel buffer Gel tray. 8. electrophoresis buffer Loading dye (electricity) DNA Stain  Other components  The gel buffer is used to maintain DNA in a stable form during electrophoresis  The electrophoresis buffer is an ionic solution that allows an electric current to flow from the cathode to the anode. as shorter DNA molecules encounter less resistance from pores in the molecular sieve as compared to longer ones. 13. *Staining of bands can also be done via autoradiography DNA is radioactively labelled. 4. 2.  The gel needs to be stained with a DNA-binding dye at the end of the run (ethidium bromide or methylene blue) so that DNA can be visualized as a series of bands. The bands can then be visualized and their size determined by cross comparison with the DNA ladder. Electrophoresis 7.Version 2. Gel comb (masking tape) Electrophoresis chamber. 9. glycerol) Each sample is loaded into an individual well. DNA Gel Electrophoresis Gel Preparation 1. The DNA ladder is loaded into a separate well. or when it has moved a suitable distance. Components Required Agarose powder. this makes the DNA samples visible and easier to be loaded into the wells. The DNA samples are mixed with a loading dye (bromophenol blue. 5. 212 | P a g e . The safety cover is placed over the electrophoresis chamber.

generated from PCR or chemically synthesized  Advantages  Sensitive: Complementary sequences detected at very low concentrations  Selective: Only sequences complementary to the probe sequence will be hybridized  The incubation temperature can be varied to let partly-complementary sequences hybridize to the probe DNA as well  Applications  Detect.1 NUCLEIC ACID HYBRIDIZATION  Nucleic Acid Hybridization – The process by which 2 complementary. fluorescent or chemical markers  Can be cloned for cDNA library. etc.  Components  Target DNA sequence  Probe DNA  The probe is labelled with radioactive. single-stranded nucleic acid chains base-pair and form a double-stranded hybrid  2 step process  Denaturation: The target DNA and probe DNA are denatured into 2 single-stranded DNA molecules by disrupting the hydrogen bonds between complementary base pairs with heat/low salt concentration/high pH  Hybridization: The DNA probe will anneal to complementary base sequences on the target DNA via hydrogen bonding.  Study gene expression and changes in gene expression profiles  Screen libraries and identify clones carrying the gene of interest  Compare nucleotide sequences between 2 DNA samples 213 | P a g e . 1I. characterize and quantify specific nucleotide sequences or genes  Localise particular genes of interest/related genes in cells.Version 2. tissues.5 1I – OUTLINE THE PROCESS OF NUCLEIC ACID HYBRIDISATION AND EXPLAIN HOW IT CAN BE USED TO DETECT AND ANALYSE RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP).

followed by a stack of blotting paper. Gel electrophoresis is used to separate the mixture of restriction fragments based on their size (refer to 1H) A sheet of either nitrocellulose or nylon membrane is placed over the gel. 5. individuals may be either heterozygous or homozygous for a specific RFLP allele 1I. used to study patterns in gene expression RFLP analysis Isolation of RFLP allele and Restriction Digest 1. The alkaline buffer solution helps to denature the double-stranded DNA into single strands.  RFLPs help to create genetic markers – A DNA sequence that varies within a population (and is thus generally unique to an individual)  RFLPs are caused by genetic polymorphisms at a specific locus on a chromosome – Genetic variations at gene loci with alleles at frequencies significantly higher than expected of genetic mutation. Not necessarily required. 9.  Southern Blotting  Purpose: Detect the presence of a specific DNA sequence (restriction fragment in this context) **Northern blotting – uses RNA samples instead of DNA samples.Version 2. DNA containing the RFLP allele is isolated and cleaved with a restriction enzyme to produce a restriction fragments of various lengths. The membrane is removed and washed thoroughly so that all unbound probes are removed.  In the form of either differences in the sequence of nucleotides or number of tandem repeats  As humans are diploid. only needed if there is insufficient DNA samples. This cause the single-stranded DNA to be transferred to the nitrocellulose/nylon membrane. Visualization 8.5 1I. The nitrocellulose/nylon membrane consisting of the bound ssDNA is incubated in a sealed bag with a buffer solution and radioactively labelled ssDNA/ssRNA probes which are complementary to the sequences on the restriction fragments. Autoradiography is conducted so that the annealed probes show up as bands on the autoradiograph Components Required DNA containing the RFLP allele Restriction Enzyme PCR Components G.3 ROLE OF NUCLEIC ACID HYBRIDIZATION IN RFLP ANALYSIS  Main Role – Allow for the detection of specific restriction fragments of differing lengths via southern blotting and autoradiograhy  RFLP Analysis – A method used to detect individual differences in DNA  Due to RFLP. Southern Blotting 4. substitution and deletions of nucleotides. which can be detected by agarose gel electrophoresis. Nucleic Acid Hybridization 7. 6. Gel Electrophoresis 3. resulting in variations in DNA fragment length and number after digestion with the same restriction enzyme.2 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)  RFLP – Variations in DNA sequences due to nucleotide changes at a certain locus in the genome caused by insertion.  Directly results in a change in the number & length or only the length of restriction fragments. the pattern of restriction fragments will differ and that can be detected by nucleic acid hybridization after performing agarose gel electrophoresis. PCR 2. Electrophoresis Equipment Paper Wick Alkaline buffer Nitrocellulose/Nylon Membrane Blotting Paper Radioactively Labelled Probes Autoradiograph Equipment Cite the Yellow Area when asked about the role of nucleic acid hybridization in RFLP analysis 214 | P a g e .

) 1J.1 LINKAGE ANALYSIS  Linked genes – Genes found on the same chromosome  Linkage analysis – The use of known RFLP loci can as genetic markers to map the genetic loci of disease genes that are closely linked to the RFLP loci. PCR AND NUCLEIC HYBRIDISATION IN RFLP MAY BE REQUIRED. II. (Cosegregation)  However. (DETAILS OF APPLICATION OF GEL ELECTROPHORESIS. DISEASES DETECTION.Version 2. SICKLE CELL ANAEMIA. DNA FINGERPRINTING.  Note: there must be a way to identify the gene involved (either via the phenotype or genotype itself) 215 | P a g e .  Principle  The closer linked genes are to each other. this is not absolute. and the detection of a specific RFLP allele does not necessarily mean that the person has the corresponding allele of the disease-causing gene associated with this RFLP allele  This exception allows the distance between the 2 loci to be determined in centimorgans by carrying out pedigree analysis (patter of disease trait inheritance) across hundreds of families to determine the % of recombination alleles. the less likely they are to be separated via crossing over during meiosis I. a RFLP locus that is tightly linked to a disease-causing gene is usually inherited together with the other gene.G. E. done by studying patterns of inheritance of the disease trait with a linked RFLP allele within an affected family.  In the context of RFLP. III.5 Highlighted area – Use when question states “outline” 1J – EXPLAIN HOW RFLP ANALYSIS FACILITATED THE PROCESS OF: I. GENOMIC MAPPING IN TERMS OF LINKAGE MAPPING.

 Principle  The VNTR allele is flanked by restriction sites. there will be a match in banding patterns.  Application (pedigree analysis)  Wild type and captive populations have different DNA fingerprints.) 1K. 216 | P a g e . These are genetic markers as VNTR copies are highly variable and each VNTR allele is highly polymorphic.  RFLP analysis can then be used to differentiate between wild-type and disease alleles (note: humans are diploid) are present 1J.1 HUMAN GENOME PROJECT  A 13 year effort to sequence the entire human genome coordinated by the US Department of Energy and National Institutes of Health. the restriction fragments produced from such genes are different in length and number depending on which gene allele is present. multiple VNTR loci are analysed by RFLP analysis to generate a unique DNA fingerprint per individual.  Limitations of DNA fingerprinting in forensics  A large sample of DNA is required. INCLUDING THE BENEFITS AND DIFFICULT ETHICAL CONCERNS FOR HUMANS. 1K – DISCUSS THE GOALS AND IMPLICATIONS OF THE HUMAN GENOME PROJECT.  The length of the restriction fragment is then detected as different patterns of bands on a southern blot.5 1J.  Principle  The RFLP loci lies directly within the disease-causing gene.  The DNA fingerprints of captive organisms can obtained using the methods above.  The DNA obtained must be intact and non-degraded  The DNA obtained must be flanked by restriction sites of available restriction enzymes  DNA fingerprinting with VNTRs is thus used for paternity testing instead.  Due to RFLP. (KNOWLEDGE OF THE TECHNICAL PROCEDURE OF THE HUMAN GENOME PROJECT AND SEQUENCING IS NOT REQUIRED. creating or destroying restriction sites present in the wild type allele.  If the organisms are bred in captivity.  The number of tandem repeats is thus directly proportional to the length of restriction fragment produced. more than is typically available at a crime scene.2 DISEASE DETECTION  Function of RFLP Analysis: Used to distinguish between 2 different alleles of a disease-causing gene.Version 2.  In practice.3 DNA FINGERPRINTING  VNTRs – Variable Number Tandem Repeats (minisatellite and microsatellite DNA that is repeated many times in each individual).

ornl. 217 | P a g e .5  Apart from sequencing the human genome. eukaryotes. and familial breast cancer. including Alzheimer's disease. legal. 1K.2 GOALS **From Human Genome Project Website (http://www. Store this information in databases.000 genes in human DNA.shtml)       Identify all the approximately 20.  Provides new insights about relationships among the three kingdoms of life: archaebacteria. and Address the ethical. Determine the sequences of the 3 billion chemical base pairs that make up human DNA. 1K.  Promotes a new era of molecular medicine characterized less by treating symptoms and more by looking to the most fundamental causes of disease in the human genome)  Comparative Genomics  Comparative genomics between humans and other organisms such as mice already has led to similar genes associated with diseases and traits. and prokaryotes.  It was discovered that majority of the human genome was comprised of intergenic sequences  Repetitive DNA (Satellite DNA)  Transposable elements (DNA that can technically move around the genome)  Retrotransposons (code for reverse transcriptase)  Normal Transposon  Endogenous Viral sequences  LINE-1 elements  Alu elements (transcribed into RNA of an unknown function) *Crossover at transposable elements at 2 different loci can lead to genomic evolutions FYI: DNA sequencing is carried out by using ddNTPs then determining the length of each strand via lasers. Further comparative studies will help determine the yetunknown function of thousands of other genes. Improve tools for data analysis. such as E.3 BENEFITS **From Human Genome Project Website & Wikipedia  Pharmacogenomics (molecular medicine)  Increasingly detailed genome maps have aided researchers seeking genes associated with dozens of genetic conditions.Version 2. there were also minor efforts to sequence the genome of other organisms which served as lab models. Transfer related technologies to the private sector.  Collaboration between biologists  Researchers working on genomics can now refer to the human genome project database and check the views of other scientists on certain genes.000-25.gov/sci/techresources/Human_Genome/home.coli and the fruit fly Drosophilia. It is an automated process. and social issues (ELSI) that may arise from the project.

and social risks. adoption agencies. among others. copyrights. courts.  Clinical issues including the education of doctors and other health service providers. 218 | P a g e .g.4 ETHICAL CONCERNS **From Human Genome Project Website & Wikipedia  Fairness in the use of genetic information by insurers. heart disease) linked to multiple genes and gene-environment interactions.  Privacy and confidentiality of genetic information. patients.5 1K. and the general public in genetic capabilities. and reproductive rights.  Commercialization of products including property rights (patents. and the military. scientific limitations.. use of genetic information in reproductive decision making.  Reproductive issues including adequate informed consent for complex and potentially controversial procedures. and concepts of health and disease. employers. schools. and trade secrets) and accessibility of data and materials.  Uncertainties associated with gene tests for susceptibilities and complex conditions (e.  Conceptual and philosophical implications regarding human responsibility. and implementation of standards and quality-control measures in testing procedures.Version 2.  Psychological impact and stigmatization due to an individual's genetic differences. free will vs genetic determinism.

Telomeres are maintained and stem cells can overcome the Hayflick limit and continue mitosis indefinately  Ability to differentiate – Both internal and external signals can induce differentiation of stem cells  Internal signals are epigenetic signals that lead to differential gene expression. Endoderm]  Descended from totipotent stem cells  Do not have the ability to give rise to extra-embryonic membranes  Inner cell mass of the blastocyst  E. Directed differentiation can be done by manipulated stem cells in lab conditions  External signals can be chemicals secreted by other cells. Zygotic Stem Cells [Fertilized egg and the first few cells when the zygote divides rapidly via mitosis] with the ability to become any cell type in the adult body and the extra-embryonic membranes (Amnion + Placenta)/ largest possible number of developmental pathways  Pluripotent – Ability to give rise to cells that form the 3 germ layers [Ectoderm. Adult Stem Cells. physical contact. or molecules present in the microenvironment of the cell (such as nutrients. APPLICATIONS OF MOLECULAR AND CELL BIOLOGY 2A – DESCRIBE THE UNIQUE FEATURES OF ZYGOTIC STEM CELLS.g.5 B2. thus increasing stem cell numbers  Stem cells possess telomerase. foetus or adult that undergoes longterm self-renewal via mitosis and differentiate into specialized cells + Possess a large nuclear to cytoplasmic ratio  Characteristics of stem cells  Unspecialized – Does not have any tissue-specific structures that allow it to perform specialized functons  Capable of self-renewal – Stem cells can proliferate via mitosis in response to external signals. From the syllabus  Definition of Stem Cells: An unspecialized cell from the embryo.Version 2. Differentiating Division [2 Committed] 219 | P a g e .  Generates progenitor [precursor] cells that further divide and differentiate to give rise to differentiated tissue. growth factors)  Types of stem cells (based on Potency: range of cell types to which a stem cell can give rise to)  Totipotent – Ability to give rise to all cell types that make up an organism  E. Blood Stem Cells  Committed Stem Cells – Stem cells with a more limited pathway of development compared to pluripotent cells and are destined to produce a specific group of cells. Infant Stem Cells. pluripotency (embryonic stem cells which have ability to differentiate into almost any cell type to form any organ or type of cell and so are not totipotent but are multipotent) and multipotency (blood stem cells which have ability to differentiate into a limited range of cell type and so are not pluripotent or totipotent). Replicating and Differentiating division [1 Committed + 1 Stem]  Symmetric. EMBRYONIC STEM CELLS AND BLOOD STEM CELLS Using the terms totipotency (zygotic stem cells which have ability to differentiate into any cell type to form whole organisms and so are also pluripotent and multipotent). derived from pluripotent stem cells  E.  Progenitor cells are more limited in developmental pathways than a multipotent stem cell  Ways in which stem cells divide  Asymmetric. which adds repeats to the telomeres. Mesoderm.g. Fetal Stem Cells. Embryonic stem cells  Multipotent – Ability to differentiate into a limited range of cell types  More specialized that totipotent or pluripotent stem cells.g.

Spleen. dispersed in tissues throughout the animal  Low numbers  Most have unknown origins  Ability for long term self-renewal  Functions  Maintain and repair the tissue  Replace cells that die due to injury or disease  Gives rise to fully differentiated cells that have mature phenotypes. EMBRYONIC STEM CELLS AND BLOOD STEM CELLS). which give rise to platelets  Lymphoid Lineage  Derived from the common lymphoid progenitor cell 220 | P a g e . etc. and can thus form the inner cell mass and differentiate into the 3 germ layers that further differentiate into various organs  Ectoderm  Skin  Endoderm  Gastrovascular Cavity  Mesoderm  Heart.G.Version 2. capable of developing into all fetal tissues. Mesoderm. 1B. Replicating Division [2 Stem] Types of committed stem cells formed depends on the localization of regulatory molecules or differential segregation of cell membrane proteins Committed cells do not switch commitment halfway 2B – EXPLAIN THE NORMAL FUNCTIONS OF STEM CELLS IN A LIVING ORGANISM (E. 1B. Lungs.  Clonogenic – 1 cell can give rise to a colony of genetically identical cells  Blood Stem Cells (Hematopoietic Stem Cells) [Hematopoiesis = Blood Formation]  Function: Replace blood cells  Derivation: Mesoderm layer  Lineages  Erythroid [Red Blood Cell] Lineage  Derived from common myeloid progenitor cell  Yields erythrocytes [Red Blood Cells]  Yields megakaryocytes. Blood Vessels.2 ADULT STEM CELLS  Adult Stem Cells: Undifferentiated cells found among differentiated cells in a tissue with the ability for self-renewal and can differentiate to yield major specialized cell types  Characteristics  Rare.  Easy to obtain pure and can be cultivated in large numbers  Functions  Clonogenic – 1 cell can give rise to a colony of genetically identical cells. are fully integrated into the tissue and capable of specialized functions that are appropriate.5    Symmetric. Endoderm].1 EMBRYONIC STEM CELLS  Derivation: From inner cell mass in blastocyst from embryos  Properties  Can undergo an unlimited number of symmetric replicating divisions via mitosis  2n chromosomes  Gives rise to cell types that are derived from the 3 germ layers [Ectoderm.

1 GENE THERAPY  Gene Therapy: Introduction of a normal functional allele of a gene to the genome of a person carrying defective alleles of the gene [Important Considerations: Cell Targeting and safe integration & expression of the transgene]  Transgene: Gene that has been introduced into a cell or an organism [which is said to be transgenic]  Components of gene therapy  Targeted Cell  Methodology  Delivery Vector 1. after gene is transferred the cells are implanted back into the patient  Advantage: No fear of rejection  Limitation: Need for surgical removal of cells  In vivo gene therapy  Direct method  Cells located in the patient  Advantage: No need for surgical removal of cells 221 | P a g e . THE TREATMENT OF SCID AND CYSTIC FIBROSIS. (DETAILS OF MANIPULATION OF GENES AND FORMATION OF VECTORS CARRYING GENES ARE NOT REQUIRED.Version 2. II.G. Methods of gene transfer  Ex vivo gene therapy  Indirect method  Cells located outside of patient. USING VIRAL AND NON-VIRAL GENE DELIVERY SYSTEMS RESPECTIVELY. TWO TYPES OF GENETIC DISEASES E.) 2C. SCID (SEVERE COMBINED IMMUNODEFICIENCY) AND CYSTIC FIBROSIS.5   Yields B and T lymphocytes used in the adaptive immune system Myeloid Lineage  Derived from common myeloid progenitor cell  Yields the rest of the white blood cells (mobile units of the body’s immune system) 2C – DESCRIBE: I.

Might cause insertional mutagenesis  Adenovirus  In-vivo Therapy Efficiency: High  Duration of Expression: Transient  Advantages: Larger DNA sequences >30 000 bp can be introduced.  Advantages: Long Term expression due to integration of therapeutic gene with host genome. Only infect dividing cells. Do not invoke host immune system response  Limitations: Very inefficient at gene transfer. the transgene can be expressed in a large population of daughter cells. Stable and easily purified adenovirus. 3.Version 2. Do not invoke host immune system response  Limitations: Inefficient at gene transfer. includes stem cells  Advantages  Effects limited to one individual. Transient expression  Plasmids  In-vivo Therapy Efficiency: Poor  Duration of Expression: Transient  Advantages: Larger DNA sequences >20 000 bp can be introduced. Adenovirus can infect non-dividing cells  Limitations: Unstable and transient expression. Wide host cell range with high levels of gene expression.5 2. Unstable and easily denatured by exonucleases 222 | P a g e . Stimulation of host immune response. safeguards against the danger of transmitting negative effects of the therapy to future generations  Less risk  If stem cells are used.  Types  Retrovirus  In-vivo Therapy Efficiency: Poor  Duration of Expression: Long Term due to integration of transgene into host genome via integrase. which can potentially cure the patient Vectors  Definitions  Transfection: A technique used to transfer a gene into a patient in gene therapy  Vector: A carrier molecule or particle which can be viral or non-viral  Criteria for vectors  Efficiency: Therapeutic transgene must be expressed to form functional proteins  Safety: Body must not react adversely to gene product [due to immune system].  Limitations: Only short sequences <8000 bp can be inserted. Gene introduced must not cause insertional mutagenesis or have any deleterious effects.  Limitation: Need for cell targeting to deliver the gene only to intended tissues Type of cell used  Germ Cells  Targeted Cell: Early embryo or germ cell [Sperm/Egg]  Limitations: All descendants of the patient would obtain a new heritable mutation  Somatic Cells  Targeted Cells: Somatic cells. Adenoviruses contain genes involved in the process of malignant transformation  Liposome/Vesicle  In-vivo Therapy Efficiency: Variable  Duration of Expression: Transient  Advantages: Larger DNA sequences >20 000 bp can be introduced.

Blood stem cells from the bone marrow of the patient are isolated and grown in cell culture outside the body. Selection of successfully transformed cells. pancreatic inflammation etc.  This dilutes the mucus outside the ell to form a moist. which causes water to leave the epithelial cell by osmosis.Version 2. breathing difficulties and permanent lung damage  Others: Inflammation of nasal sinuses. bacterial infections. This causes deoxyadenosine to build up to toxic levels that kill immature lymphocytes  Treatment Options  Retroviral Gene Therapy  Targeted Cells: Blood Stem Cells  Methodology: Ex-Vivo gene therapy  Delivery Vector: Retrovirus Ex-Vivo gene therapy for SCID 1.  Components Blood Stem Cells Retroviral Vector Therapeutic Gene (Normal Allele) Others  Raise in a sterile environment  Bone marrow transplant  Injections of ADA at frequent intervals 2C. preventing B and T cells from developing and functioning normally  Note: More likely to affect males than females [Refer to GBV]  Autosomal Recessive SCID  Mutated Gene: ADA gene on chromosome 20  Mutated Phenotypic Effects: Adenosine Deminase (ADA) enzyme becomes defective. thin mucus  Mutated Phenotypic Effects 223 | P a g e . which are implanted back to the patient.2 SEVERE COMBINED IMMUNODEFICIENCY (SCID) & TREATMENT  SCID occurs when the B and T cells of the immune system are defective.  Cause: Autosomal Recessive disorder  Mutated Gene: Deletion of 3bp on the Cystic Fibrosis transmembrane conductance regulator (CFTR) gene on chromosome 7  Normal (Functional) Effects:  The CFTR channel opens and releases chloride ions to the outside of the cell.5 2C. 2. coughing of blood. leaving the patient with little or no immunity (~HIV)  Types  X-Linked SCID  Mutated Gene: Interleukin 2 Receptor Gamma (IL2RG) gene on the X-chromosome  Mutated Phenotypic Effects: A protein that is necessary for growth and maturation of lymphocytes is not produced. 3. The cells are subsequently transfected with a retroviral vector containing the normal functional therapeutic gene.3 CYSTIC FIBROSIS & TREATMENT  Cystic Fibrosis: A chronic & fatal disease that affects the epithelial cells of the respiratory track due to abnormalities in the glands that secrete sweat and mucus  Effects  Loss of excessive amounts of salt  Thick accumulation of mucus which leads to malnutrition. heart enlargement.

leading to a bacterial infection when bacteria become trapped in the thick mucous  The narrowing of airways causes breathing to be more difficult  Blocking of pancreatic ducts and ducts in reproductive organs due to thick mucous  Thick mucus in intestines causes poor absorption of nutrients  Treatment Options  Liposomal Gene Therapy  Targeted Cells: Epithelial cells of the respiratory tract  Methodology: In-Vivo gene therapy  Delivery Vector: Liposomes In-Vivo gene therapy for Cystic Fibrosis (~ICS) 1. requires multiple reapplications as DNA is not integrated into the chromosomes. DNA Ligase Liposome. resulting in low levels of gene expression  Problems with viral vectors  Toxicity – Occurs when viral genes are transcribed to form toxic metabolites  Immune and inflammatory responses  Insertional mutagenesis for retroviruses 224 | P a g e . Plasmid is cut with the same restriction enzyme 3. Restriction Enzyme.  Epigenetic factors can also suppress the expression of therapeutic genes. Components CFTR Gene. CFTR gene is isolated using a restriction enzyme 2. Liposomes fuse with targeted cells via receptor mediated endocytosis when embedded glycolipids are recognized by stel cells’ receptors.5  The CFTR non-functional protein causes chloride ions to accumulate inside the cell such that water moves into it via osmosis.Version 2. Epithelial cells  Advantages  Liposomes do not invoke host immune system response  Limitations  Expensive. Plasmid. resulting in transient expression of the gene  Liposomes do not delivery transgene to somatic cells efficiently enough to be effective  CFTR-containing liposomes are not take up by all the targeted cells 2D – EXPLAIN WHAT ARE THE FACTORS THAT KEEP GENE THERAPY FROM BECOMING AN EFFECTIVE TREATMENT FOR GENETIC DISEASES  Transient nature of gene therapy  The transgene needs to remain functional and the cells containing the transgene must be long-lived and stable  Problems  Rapidly dividing target cells  Difficulty of inserting new genes into target cells  Difficulty of inserting the transgene into the host genome  Sufficient amounts of functional therapeutic genes need to be delivery for the treatment to be effective  Immune response  Viral vectors and foreign gene constructs tend to induce an immune response that leads to rejection of the new genes by the host. Normal CFTR gene is inserted into the plasmid using DNA ligase 4. Liposomes taken as nasal spray 6.  This forms thick mucus in the lungs. Recombinant plasmid is then inserted into liposomes 5.

Whether gene therapy should be subsidized by the government  The nature of capitalism – the biopharmaceutical industry has to be trusted with issues of privacy and regulation of gene use  Vested Financial Interest  The biopharmaceutical industry needs to be trusted to do what is in the best interests for the patient in terms of both financial cost and effectiveness rather than choose the most profitable road. 2E.  Others argue that germline cell therapy facilitates eugenics and could also backfire by eliminating potentially useful alleles from the gene pool of the population.Version 2.  Clinical trials may be rushed to provide a large profit margin for the industry  Safety Concerns  E.g. Insertional Mutagenesis 2E.2 ETHICAL CONSIDERATIONS  Manipulation of human genes  Gene therapy is a grey area that threads a thin line on the limit of genetic engineering.  Transparency & Accountability  Nobody has yet to decide who is to be held accountable should gene therapy backfire and create disastrous side effects  The public also needs to be aware of what goes on during clinical trials so that they have sufficient information to gauge for themselves if they require gene therapy and its possible side effects 225 | P a g e .  The need to target multiple genes further reduces the effectiveness of gene therapy 2E – DISCUSS THE SOCIAL AND ETHICAL CONSIDERATIONS FOR THE USE OF GENE THERAPY. but it can potentially be abused to provide enhancement instead.5  Multigene disorders  Most disorders today such as cancer and Alzheimer’s disease are caused by multiple genes which have not been fully discovered. some might argue that germline gene therapy eliminates potential problems from future generations and is thus fulfilling a noble medical goal. and threatens to degenerate into widespread practices of eugenics and gene enhancement which are potentially more controversial than gene therapy  Objection of religious groups against “playing god” in altering the human genetic makeup  Therapy vs Enhancement  Gene therapy is morally acceptable because it serves the noble medical goal of disease prevention and treatment.1 SOCIAL CONSIDERATIONS  Cost and Commercialisation  Social access to therapies and the cost of therapies.  Gene enhancement could potentially lead to dangerous mutations and imbalance social hierarchies  Difficult to draw the line between enhancement and therapy  Somatic Cell vs Germline Cell therapy  Universal agreement that germline gene therapy is morally and ethically unacceptable  However.

Establishment – Choosing and disinfecting the explant before placing it into culture and allowing it to initiate microshoots Multiplication – Inducing multiple shoot production and the multiplication of microshoots by maintaining the culture in a stabilized state with a high ratio of cytokinin to auxin in the culture medium.5 2F – DISCUSS CLONING IN PLANTS IN TERMS OF PLANT TISSUE CULTURE TECHNIQUES. (GENE CLONING IN PLANTS USING AGROBACTERIUM SPP. 2. Acclimatization – Shifting the plantlet from a heterotrophic to an autotrophic condition and gradually moving plants to outdoor conditions 2F. 4. root or other cells)  Callus – A mass of disorganized and unspecialized cells that can be induced to form roots or shoots using growth regulators 2F. AND GENE GUN IS NOT REQUIRED. 3.) 1.1 TISSUE CULTURE  Establishment  Purpose: Place an explant into aseptic culture by avoiding contamination and then providing an in vitro environment that promotes stable shoot production. leaf. Root Formation – Initiating roots on microcuttings using a culture medium with a high auxin to cytokinin ratio and preparing the explants for transplanting out of the aseptic protected environment.  Explant Disinfection  Required to prevent the growth of undesirable microbes such as fungi and bacteria which can outcompete the plant tissues for nutrients  Culture Medium (should contain)  Carbon and Metabolite – Sucrose preferred 226 | P a g e .Version 2.1 DEFINITIONS  Tissue Culture – Act of establishing and maintaining plant organs and plant tissues in aseptic culture and grown in vitro using agar gel containing nutrients and growth regulators such as auxins and cytokinins  Explant – The piece of the plant used to initiate the micropropagation or tissue culture process (can be a portion of the shoot.

Multiplication 5. they are harvested and transferred to stage 3 for rooting Microcuttings are either rooted in vitro or ex vitro.  Cytokinins promote shoot growth  Auxins promote root growth  Stabilization  Multiplication  Purpose: Maintain the culture in a stabilized state and multiply the microshoots to the number required for rooting  Growth regulators such as cytokinins and auxins are required to direct the development of specific tissues. the whole process is carried out in a test tube whereas in ex vitro rooting. 4. Some plants require repeated subculturing to produce a uniform. Acclimatization 9.Version 2. The whole plantlet is then shifted to an autotrophic condition to allow it to acclimatize to the surroundings. nutrients as well as plant growth regulators such as cytokinins and auxin. 3. 8. 227 | P a g e . Greenhouse)  Methods  In vitro rooting (in a test tube)  Ex vitro rooting (in a container containing compost. The growth medium is altered such that there is a high cytokinin to auxin ratio to promote shoot proliferation Once the microshoots have reached an appropriate length. well-growing culture. 6. sealed containers which are then placed in growth rooms where environmental conditions are strictly controlled. Subsequent contamination is prevented by growing the tissue culture in special.g. A suitable explant is chosen and its surface disinfected with dilute bleach solution.5  Organic Nutrients – Vitamins for source of coenzymes and catalysts. The explant is stabilized once the main terminal shoot elongates with limited proliferation of axillary shoots. the culture medium is altered to have a high auxin to cytokinin ratio to promote root formation In in vitro rooting. not soil)  High auxin to cytokinin ratio promotes root formation  Acclimatization Tissue Culture Establishment 1. 2. the microcuttings are inserted directly in soil-less greenhouse rooting medium and placed under high humidity conditions. In both cases. Root Formation 7. Amino Acids as precursors to protein synthesis to stimulate cell growth  Inorganic Nutrients – Includes inorganic ions and mineral salts  Nitrogen – Major component of proteins and chlorophyll  Magnesium – Required for chlorophyll synthesis  Sulfur – Required for protein production and chlorophyll formation  Phosphorous/Phosphate – Required for nucleic acid synthesis  Plant Hormones – Includes growth regulators such as auxins and cytokinins at particular ratios. bark that is free of contaminants.  Shoot initiation is supported by increased cytokinin cncentraton (auxin usually absent) (not overly high)  Root Formation  Purpose: To root microcuttings and prepare them for transplanting out of the aseptic protected environment into an open area (e. The culture medium selected contains a suitable metabolite.

2. 2F. 4.5 10. A suitable plant organ is chosen and cut into pieces. sealed containers which are then placed in growth rooms where environmental conditions are strictly controlled. sugar & salts in a petri dish to digest the cell walls of the leafe cells.4 CALLUS CULTURE  Callus are produced on explants as a response to wounding and the ratio of plant hormones (either externally applied or internally present)  Calli contain an inner mass of undifferentiated older tissues and an outer mass of meristematic tissue (where cell division occurs) Callus Culture ~Tissue Culture Establishment 1. Subsequent contamination is prevented by growing the tissue culture in special. Protoplast Culture ~Tissue Culture Establishment 1. 3. which are soaked in a solution containing cellulose. proteins. Multiple subculturing is done to continue to preserve the calli. 2F. and other large molecules. since new genetic material can be incorporated directly into the cells of an organism. A suitable explant is chosen and its surface disinfected with dilute bleach solution.Version 2. The culture medium selected contains a suitable metabolite. Centrifuge the cells to remove the cell wall debris and obtain the protoplasts. High humidity is maintained for a short period of time before the plants are exposed to atmospheric humidity.5 PROTOPLAST CULTURE  Protoplast – Living parts of plant cells with the cell wall removed  Advantages of Protoplast Culture  Viruses can be more easily incorporated into protoplasts for gene delivery  Protoplasts of different genotypes can be fused during somatic hybridization  Protoplasts easily take up DNA. 2. 228 | P a g e .3 ORGAN CULTURES  4 Stages still apply  Type of Organ Cultures  Seed Culture (2n)  Increases the probability of germinating seeds that are difficult to germinate in vivo  Embryo Culture (2n)  Used to rescue embryos of genetic crosses that would not form any seeds  Ovary and Ovule Culture (n)  Anther Culture (n)  Can be manipulated to cause non-disjunction and restore normal chromosome number  Endosperm Culture (3n)  Creates seedless and infertile plants 2F. nutrients as well as a moderate ratio of cytokinins and auxin to induce calli formation The callus is stabilized once the main terminal shoot elongates with limited proliferation of axillary shoots. This is useful especially in genetic engineering.

taking up little space.  High start-up cost due to expensive equipment  Low success rate of transplanting plantlets from controlled conditions to soil conditions  High probability of random mutation that could lead to problems such as sterility in plants that only surface after a few years. 9. the whole process is carried out in a test tube whereas in ex vitro rooting. High humidity is maintained for a short period of time before the plants are exposed to atmospheric humidity.  Reliable Quality Control – Standardized growing controls and a controlled environment makes it easy for quality checks to be done. Transfer colonies after 2-3 weeks into agar medium to induce shoot development The growth medium is altered such that there is a high cytokinin to auxin ratio to promote shoot proliferation Once the microshoots have reached an appropriate length. the culture medium is altered to have a high auxin to cytokinin ratio to promote root formation In in vitro rooting. Heat treatment can be applied to kill bacteria. allowing for rapid genetic changes to take place in a short period of time. 4. 2F. Culture the protoplasts on a petri dish with feeder cells embedded in agar. Seedless plants (bananas) cannot be propagated conventionally.5 Multiplication 3.6 ADVANTAGES AND LIMITATION OF PLANT CLONING  Advantages of Plant Cloning  Many clones with desirable characteristics can be produced. out of season and even stored in cold storage until required. Root Formation 7. unlike conventional methods where it is difficult to produce true breeding lines since heterozygotes do not breed true.Version 2. 10.  Devastating consequences if environment is not sufficiently controlled or wrong conditions are used. The culture medium selected contains a suitable metabolite. the microcuttings are inserted directly in soil-less greenhouse rooting medium and placed under high humidity conditions. In both cases. 8.  High flexibility in production – Plants can be produced at any time during the year.  Advantages of Plant Tissue Culture  Rapid production of large numbers of plants from just one or a few stock plants  Plant diseases can be avoided. 6. Plants are also produced at a reliable rate. providing a sales advantage.  Limitations of Plant Tissue Culture  Labour intensive production requiring skilled labour that incurs high costs. The whole plantlet is then shifted to an autotrophic condition to allow it to acclimatize to the surroundings.  Convenient and practical method of propagation for some plants (orchids) compared to conventional methods where seeds may be difficult to germinate.  Plants can now be customized and designed according to the buyer’s wish. Acclimatization Microcuttings are either rooted in vitro or ex vitro. thus viral-free cells can be obtained from meristems.  Convenient transport – Micropropagated plants are light and small in size and can thus be air-freighted and transported easily and cheaply. 229 | P a g e . they are harvested and transferred to stage 3 for rooting 5. Viruses often infect vascular tissues.  Exotic plants that are hard to produce using conventional methods can be cloned in large numbers and sold  Genetic engineering can be carried out on cloned plants to form transgenic plants. nutrients as well as growth regulators.

spp  Special characteristics of genetically modified organism  The Bt-corn is able to produce Cry proteins that is cleaved by enzymes in the larva’s stomach to generate the toxic form of the Cry protein.2 SIGNIFICANCE OF GENETIC ENGINEERING  Reduce World Hunger and Alleviate Malnutrition  Plant biotechnology can be used to help increase yields on available land  Transgenic plants are more resistant towards herbivores. 2G. BT CORN. labour and environmental damage. plant diseases and adverse environmental conditions  Improve Yields  Increased growth rates 2G.Version 2. 2G.1 DEFINITIONS  Genetic Engineering/Modification – Special molecular biological techniques that alter the genetic makeup of living organisms [NOT cloning]  Genetically Modified/Transgenic Organism (GMO) – An organism that has artificially acquired one or more genes from the same or different species. GM SALMON). hence reducing their reliance on chemical insecticides. The Cry protein binds to the larva’s intestines and makes it leaky.3 APPLICATIONS OF GENETIC ENGINEERING  Bt-corn (Insect Resistance)  GMO: Corn (Possibly potatoes. broccoli)  Problem  The European corn borer’s larvae preys on corn and causes more than $1 billion in damages every year with up to 40 tonnes of losses.G.  Higher harvest yields  Lesser economic losses due to pest infestation  Reduces the cost in the food-production sector 230 | P a g e .  The larva eventually dies of blood poisoning (septicaemia)  Benefits  Farmers no longer need to worry about infestation. which reduces cost.5 2G – EXPLAIN THE SIGNIFICANCE OF GENETIC ENGINEERING IN IMPROVING THE QUALITY AND YIELD OF CROP PLANTS AND ANIMALS IN SOLVING THE DEMAND FOR FOOD IN THE WORLD (E. cotton. GOLDEN RICE.  Selected Organism  Bacillus thuringiensis (Bt)  Bt produces crystallized proteins (Bt toxin = Cry proteins) that act as insect stomach poisons that kill he European corn borer when they are ingested  Transgene & Transformation Procedure  Transgene Components  Marker Gene + Promoter  Bt Transgene + Promoter  Procedure  ICS Style  Using Agrobacterium .

with no consequences for humans and vertebrates Concerns  Insects may evolve genetic resistance towards the Bt protein in transgenic plants  Non-pest species could be harmed  Bt protein might cause allergies  Transgene could be transferred to closely-related weeds.Version 2.  Transgene Components  Promoter + Psy (Phytoene synthase) Gene that converts geranyl geranyl diphosphate (GGPP) to phytoene  Promoter + crtl (Phytoene desaturase) Gene that converts phytoene to lycopene  Special characteristics of genetically modified organism  New Biochemical Pathway  GGPP  Phytoene  Lycopene  β-Carotene  Last step is catalysed by β-cyclase which is produced in rice endosperm tissue  β-Carotene is now produced in the rice’s endosperm tissue  Benefits  Alleviates vitamin A deficiencies  Sustainable and cost-effective alternative to vitamin supplements  Concerns  Most children that suffer from vitamin A deficiency have intestinal infections that interfere with the absorption of β-Carotene. resulting in the potential wipeout of the wild population  GM salmon outpace wild salmon in obtaining a mate  231 | P a g e .5  Bt toxin is highly specific and very effective. which leads to toxicity  GM Salmon (Growth)  GMO: Transgenic Atlantic Salmon  Problem  Wild salmon does not grow fast enough nor is it large enough to be harvested  Transgene Components  Antifreeze Protein Promoter + Somatotrophin cDNA  (Antifreeze protein gene)  Special characteristics of genetically modified organism  Transgenic salmon has greatly accelerated growth rates compared to native strains in colder waters  Antifreeze Proteins can also be integrated into the salmon genome to protect salmon from cold temperatures and freezing  Economic Benefits  Increased overall production and profits due to shorter production cycles  Shorter growing period reduces the risk of diseases and feed requirements. thus lowering costs  Reduced pressure on wild fisheries  Less pollution from salmon farms  Concerns  GM salmon that escape into the environment could introduce their growth hormone genes to the gene pool of the wild population. Golden rice therefore does not solve the root problem of vitamin A deficiency – Low socioeconomic status & Health complications  There might have been unknown implications on the ecosphere  Could result in excessive intake of vitamin A. upsetting the ecological balance  Golden Rice (Nutrition)  GMO: Rice  Problem  Vitamin A deficiency in less developed countries is a leading cause of blindness in children.

forming super weeds that can be invasive  Reduced effectiveness of pesticides  Insects may slowly adapt to the production of toxins by GM crops.G. 2H.  Inadequate risk assessment 232 | P a g e . may outcompete and drive their wild counterparts to extinction. leading to increased resistance  Upsetting the ecological balance  GMOs possess a selective advantage compared to their wild counterparts and once released. companies will not find any incentive to develop GMOs  Increased inequality between DCs and LDCs  GMOs. being expensive. GOLDEN RICE AND GM SALMON).5  GM salmon are also much more aggressive and may displace wild salmon out of their native habitats 2H – DISCUSS THE ETHICAL AND SOCIAL IMPLICATIONS OF GENETICALLY MODIFIED CROP PLANTS AND ANIMALS (E.Version 2.2 ETHICAL IMPLICATIONS  Ethical consideration of the genetic manipulation of living organisms  Some individuals consider genetic engineering to be a violation of a natural organism’s intrinsic values.1 SOCIAL IMPLICATIONS  Human Safety Issues  New Pathogens  Genetic markers such as antibiotic resistance genes can be passed on to harmful bacteria thus causing them to be resistant to antibiotics  Transgenes can be transferred to harmful bacteria and increase their pathogenicity  Allergies  Transgenic food may be a source of allergies as gene products from other sources are incorporated into the GMO  Unknown effects  There is no complete certainty that GMOs pose no health risk to humans due to a lack of research  Environmental Safety Issues  GM crops might establish themselves as weeds  GM crops that are modified to withstand unfavourable environmental conditions may be carried to other places where they proliferate quickly  GM crops may increase the resistance of weeds due to gene transfer. 2H. and believe that science should not attempt to “play god”  Infringement on animal rights  The use of transgenes in certain animals have led to increased risks of certain diseases  Religious Concerns/Dietary Restrictions  Due to a lack of labelling of GMOs. some individuals worry that they could be consuming the genes of certain organisms that are forbidden by their religion/lifestyle choice  Patency of a GMO  Some individuals believe that organisms should not be patented as they are living things and not objects  However. others believe that without intellectual property protection. BT CORN. could cause richer countries to benefit at the expense of poorer countries as most GMOs are developed and patented by companies operating in richer countries.

5  Some believe that it is ethically inconsiderate for companies to market GMOs without conducting thorough research first  Consumer Rights  Consumers have the right to know what they are consuming  There is currently a lax oversight on the labelling of GMOs and the mixing of GMOs with other natural products 233 | P a g e .Version 2.

com/scienceGraphics.jameshedberg.blogspot.H.php o Darwin’s Finches – http://davidguilbault.com/such_is_life_by_david_gui/2009/02/darwins-finches. Organization of the Eukaryotic Genome Sun W.sg/2011/01/psychedelic-diatoms.A. Campbell Biology.3 Lee S.php o Virus – http://health. DNA and Genomics C.H. Molecular Basis of Cancer Low. Lury L.com/top-ten/2009/sea-creatures/sea-creatures-9.edu/academics/graduate/ms-compbio/index.S Biological Molecules .S.htm o Ribosome – http://rna. Linda M.W. Endocrine system Heng Y..html o Pea Plant – http://www.L. Tay Animal Physiology Lecture Notes Sets 1.V.ucsc.. Wasserman S.discovery.K.com/medicine/modern-technology/light-virus.com/factfile/5126/mendelian-genetics o ATP Synthase – http://www. Lim Genetics of Viruses J. Enzymes Toh H.S. Genetic Basis for Variation II I. Minorsky P.W.Carbohydrates Sun. Jackson R.qiagen.L.. Eukaryotic Cell Structure and Function Nah W.Version 2.howstuffworks. Cell Membrane and Transport Across Membrane Ong L.greenbusgroup. 8th Edition (International) 234 | P a g e .2.H.H.edu/rnacenter/ribosome.typepad.cosmosmagazine. Cellular Respiration Heng Y. Prescott’s Microbiology.B.S Biological Molecules . Reece J. Cain M. Gene and Chromosomal Mutations Sun W.L.B.N.com/?p=146 o Axolotl – http://science. Tay Cell and Nuclear Division C.html o Passion Flower – http://thepaperseed. 9th Edition (International) Joanne M.njit. W. DNA & Genomics: Eukaryotic Gene Expression Chan S.L. Heng Genetics of Bacteria PICTURE REFERENCES    Most pictures are taken from Pearson 's art for students Other pictures are taken from around the web Influenza virus replicative cycle picture taken from https://www.html OTHER REFERENCES     Mr Heng Yew Han's overviews Mr Heng Yew Han's tutorial scribbles Campbell N..html o DNA – http://math..5 LECTURE NOTE REFERENCES                     Chan S. Christopher J. J.W. Genetic Basis for Variation I Ong L.com/geneglobe/pathwayview.html o Ecology Background – http://www.W.A. K.aspx?pathwayID=247  Pictures from the cover page are taken from o Diatom – http://deepbluehome.Lipids Chan S... Biological Molecules .Proteins Low K.A..com/human-ecology.S Control of Eukaryotic Gene Expression Ildasolha.

These notes shall not be photocopied or mass distributed for commercial purposes without the permission of the author. 12S74's bio teacher : ) DISCLAIMER The author shall bear no responsibilities for any kind of biology grades achieved by simply reading these notes.Version 2. 235 | P a g e .5 Special Thanks to Mr Heng YH.