Iit-Jam: Biotechnology (BT)

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IIT-JAM
Biotechnology (BT)
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Biotechnology (Sample Theory)
CELL BIOLOGY
1. CELL STRUCTURE AND FUNCTION
 The cell was discovered by Robert Hooke in 1665. Calls the chambers he see "cells".
Cells emerged on Earth at least 3.5 billion years ago. 1665 - 75 Anton van Leeuwenhoek,
the person incorrectly given credit for the invention of the microscope (actually, he was
just damn good at making and using them, and his scopes soon became the standard,
and history has just given him credit as the inventor of the microscope), studies organisms
living in pond water. He calls them "Animalcules."
 1830 - German scientists Schleiden and Schawann summarize the findings of many
scientists and conclude that all living organisms are made of cells. This forms the basis
of the Cell Theory of Biology.
a. The Cell Theory of Biology
 All organisms are composed of cells
 The cell is the structural unit of life - units smaller than cells are not alive
 Cells arise by division of preexisting cells - spontaneous generation does not exist
 Cells can be cultured to produce more cells
 in vitro = outside organism or cell
 in vivo = inside organism or cell
b. Properties of Cells
1. Cells are complex and highly organized
 They contain numerous internal structures
 Some are membrane bound (organelles) while others do not
2. Cells contain a genetic blueprint and machinery to use it
 All cells use the same types of information
 The genetic code is universal
 The machinery used for synthesis is interchangeable. However, for this to
function properly, information transfer must be error free
 Errors are called mutations
3. Cells arise from the division of other cells
 Binary fission - cell division in bacteria
 Mitosis - the genetic complement of each daughter cell is identical to the other and to the
mother cell. This is asexual reproduction
 Meiosis - the genetic complement of each daughter cell is reduced by half and
each daughter cell is genetically unique. This is used in sexual reproduction
 Daughter cells inherit cytoplasm and organelles from the mother cells
Asexual - organelles from mother cell
 Sexual - organelles predominately from one parent
 In eukaryotes, the chloroplasts and mitochondria come from the
egg cell
 This can be used to trace the evolutionary origin of the organism
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4. Cells can perform a variety of chemical reactions

 Transform simple organic molecules into complex molecules (anabolism)
 Breakdown complex molecules to release energy (catabolism)
 Metabolism = all reactions performed by cells
5. Cells can engage in mechanical activities
 Cells can move
 Organelles can move
 Cells can respond to stimuli
 Chemotaxis - movement towards chemicals
 Phototaxis - movement towards light
 Hormone responses
 Touch responses
6. Cells can regulate activities
 Cells control DNA synthesis and cell division
 Gene regulation - cells make specific proteins only when needed
 Turn on and off metabolic pathways
7. Cells all contain the following structures:
 Plasma membrane - separates the cell from the external environment
 Cytoplasm - fluid-filled cell interior
 Nuclear material - genetic information stored as DNA
Anatomy: There are two types of cells, eukaryotes, which contain a nucleus, and prokaryotes,
which do not. Prokaryotic cells are usually single-celled organisms, while eukaryotic cells can be either
single-celled or part of multicellular organisms.
c. Types of Cells
i. Features of Prokaryotic Cells
 Pro = before; karyon = nucleus, earliest cell type
 Relatively small - 5 to 10 µm, lack membrane-bound organelles
 Capsule - outer sticky protective layer
 Cell Wall - rigid structure which helps the bacterium maintain its shape. This is in no way
 the same as the cell wall of a plant cell
 Plasma membrane - separates the cell from the environment
 Mesosome - infolding of plasma membrane to aid in compartmentalization
 Nucleoid - region where naked DNA is found
 Cytoplasm
 semi-fluid cell interior
 no membrane-bound organelles
 location for metabolic enzymes
 location of ribosomes for protein synthesis
Archaea
 Originally thought to be prokaryotes
 relatively small - 5 to 10 µm
 lack membrane-bound organelles
 Usually live in extreme environments (thermophiles, halophiles, etc)

ii. Features of Eukaryotic Cells

 Eu = true; karyon = nucleus, contain membrane-bound organelles
 Evolved from prokaryotes by endosymbiotic association of two or more prokaryotes
 Include Protists, Fungi, Animals, and Plants
 Features shared with Prokaryotic cells
 Rigid cell wall
 Plant cells, some Fungi, some Protists
 Animal cells lack cell wall
 Plasma membrane
 Cytoplasm with ribosomes
 Nuclear material
 Cytoskeleton - flexible tubular scaffold of microfilaments
 maintains cell shape and provides support, anchors organelles & enzymes to
specific regions of the cell, contractility and movement (amoeboid movement)
 intracellular transport - tracks for vesicle and organelle movement by motor
proteins
 Cytoskeleton components
 Microfilaments
 solid protein (actin) which is assembled at one end and disassembled at
the other end
 Intermediate filaments-rope-like fibrous proteins
 provide structural reinforcement
 anchor organelles
 keep nucleus in place
 Microtubules - hollow tubes of tubulin (a globular protein)
 maintains cell shape
 anchor organelles
 movement of organelles
 track for motor proteins
 Cilia and Flagella - involved in cellular movement
 composed of microtubules
 cilia - short, numerous, complex
 flagella - longer, fewer, less complex
 both arranged in a 9+2 pattern with dynein arms projecting outward
 Nucleus
 Double membrane with pores
 Outer membrane continuous with ER
 Nuclear matrix - protein-containing fibrillar network
 Nucleoplasm - the fluid substance in which the solutes of the nucleus are dissolved
 Chromosomes - protein and DNA complexes
 Nucleolus - involved in the synthesis and assembly of ribosomes

 Endomembrane System
 Endoplasmic Reticulum - an extensive membranous network continuous with the
outer nuclear membrane.
 Rough ER - has ribosomes and is involved in secreted protein synthesis
 Smooth ER - lacks ribosomes and is involved in membrane lipid synthesis
 Golgi Apparatus
 Lysosome
 found only in animal cells
 contain enzymes for use in the hydrolytic breakdown of macromolecules
 Peroxisome
 Eukaryotic organelle that degrades fatty acids and amino acids
 Also degrades the resulting hydrogen peroxide
 Plant Central Vacuole - major storage space in center of plant cell with many functions
 Digestive - break down of macromolecules
 Storage - ions, sugars, amino acids, toxic waste
 Maintain cell rigidity - high ionic concentration generates high water potential
 Mitochondria
 Chloroplasts
 Ribosomes
 Technically not an organelle, since there is no membrane, but they are prominent
cellular structures and usually lumped in with the organelles
 The "factories" of the cell - involved in protein synthesis
 Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein
synthesis
 May either be free or bound to ER Made up of two subunits, the large and the small
subunit
 Both subunits are constructed out of protein and RNA (called rRNA)
 The ribosomes of prokaryotes and eukaryotes vary slightly with regard to size and
shape
Fig. Features of Eukaryotic Cells

Table: Comparison of features of prokaryotic and eukaryotic cells
Prokaryotes Eukaryotes
Typical organisms bacteria, archaea protists, fungi, plants, animals
Typical size ~ 1–5 µm ~ 10–100 µm
true nucleus with double
Type of nucleus nucleoid region; no true nucleus
membrane
linear molecules (chromosomes)
DNA circular (usually)
with histone proteins
RNA synthesis in the nucleus
RNA/protein synthesis coupled in the cytoplasm
protein synthesis in the cytoplasm
Ribosomes 50S and 30S 60S and 40S
highly structured by
Cytoplasmic structure very few structures endomembranes and
a cytoskeleton
Flagella and cilia containing
Cell movement flagella made of flagellin microtubules; lamellipodia and
filopodia containing actin
one to several thousand (though
Mitochondria none
some lack mitochondria)
Chloroplasts none in algae and plants
single cells, colonies, higher
Organization usually single cells multicellular organisms with
specialized cells
Mitosis (fission or budding)
Cell division Binary fission (simple division)
Meiosis
chromosomes single chromosome more than one chromosome
membranes none has membrane bound organelles
1.2 Transport across plasma membrane
The internal composition of the cell is maintained because the plasma membrane is a electively
permeable structure. The plasma membrane forms a barrier that blocks the free exchange of molecules
between the cytoplasm and the external environment of the cell.
Most biological molecules are unable to diffuse through the protein-free phospholipid bilayer (or
synthetic lipid bilayer). Small non-polar molecules, such as O2 and CO2, readily dissolve in synthetic lipid
bilayers and, therefore, diffuse rapidly across them. Small uncharged polar molecules, such as water or
urea, also diffuse across a synthetic lipid bilayer. The characteristics of lipid-phase diffusion and protein
mediated transport are basically different. Transport across the plasma membrane is of two types:
i. Passive transport
Passive transport occurs along the concentration gradient and without the use of metabolic
energy. If a transported substance carries a net charge, its movement is influenced by both its concentration
gradient and the membrane potential, the electric potential across the membrane. The combination of
these two forces, called the electrochemical gradient, determines the direction of transport of a charged
molecule across a membrane.
a. Simple diffusion : During simple diffusion, a molecule simply dissolves in the phospholipid
bilayer, diffuses across it. No membrane proteins are involved and the direction of transport
is determined simply by the relative concentrations of the molecule inside and outside of
the cell. The relative diffusion rate of any substance across a pure phospholipid bilayer is
proportional to its concentration gradient across the layer and to its hydrophobicity and
size. Movement of solutes by diffusion is always from a higher to a lower concentration,
and the rate is described by Fick's Law of Diffusion.
 Äc 
J = -D 
 Äx 
Where J is the flux per unit area, D is the diffusion coefficient (usually expressed as cm2/
sec), and c is the difference in concentration between two regions separated by a
distance x (membrane thickness in case of membrane transport). The negative sign
accounts for the fact that diffusion is towards the lower concentration.
b. Facilitated diffusion : Like simple diffusion, facilitated diffusion involves the movement of
solutes along the concentrations gradient. However, the passage is mediated by transport
protein (carriers and channels) and is selective in nature. Facilitated diffusion may be
carrier proteins or channel proteins mediated, The rate of transport of the molecule across
the membrane is far greater in facilitated diffusion as compared to simple diffusion.
Facilitated diffusion allows polar and charged molecules, such as carbohydrates, amino
acids, nucleosides and ions to cross the plasma membrane.
ii. Active transport
Active transport occurs against the concentration gradient and is mediated by carrier proteins.
Metabolic energy is used to move ions or molecules against a concentration gradient. Active transport
results in the accumulation of solute on one side of membrane. Active transport is different from carrier
proteins mediated facilitated diffusion. Active transport is of two types: Primary active transport and
secondary active transport
a. Primary active transport
Primary (direct) active transport is coupled directly with a metabolic source of energy,
such as ATP hydrolysis, or absorption of light by a carrier protein (in halobacteria). Transport
of Na+ and K + by carrier protein, Na+-K+ ATPase, is the most common example of primary
active transport. Virtually every animal cell maintains lower Na+ concentration and high
K + concentration than they are found in its surrounding. This imbalance is established and
maintained by an active transport system in the plasma membrane, involves the carrier
protein Na+-K+ ATPase coupled with breakdown of ATP (discovered by Jens Skou in 1957).
 Transport ATPase : Transport AT Pase are the ATP powered pumps, which transport
ions and various small molecules against their concentration gradients. Four major types
of ATPases are associated with membranes that couple ATP hydrolysis with the
translocation of ions and various small molecules across a membrane: P-, V-, F-ATPases
and ABC transporter.
 P-ATPases : have a simple polypeptide composition and are phosphorylated during
catalysis. Examples of P-ATPases are the Na+ K+-ATPase of the plasma membrane of
animal cells; the H+-ATPase of the plasma membranes of fungi and plants; and the Ca2+
-ATPase of the sarcoplasmic reticulum. The plasma membrane Na+-K+ ATPase and H+-
ATPase function to generate and maintain the plasma membrane electrical potential
difference (inside negative).
 V-ATPases : couple ATP hydrolysis to transport of protons against a concentration gradient.
These transport ATPase are found on the vacuolar membranes (from which the 'V' is
derived) in plant cells, endosomal and lysosomal membranes in animal cells and plasma
membrane of osteoclasts. V-class pumps generally function to maintain the low pH of
plant vacuoles and of lysosomes and other acidic vesicles in animal cells by pumping
protons from the cytosolic to the exoplasmic face of the membrane against a proton
electrochemical gradient.

 F-ATPases (also known as F-F ATPase) have a complex polypeptide composition very
similar to V-ATPases. All known V and F-ATPases transport only protons, in a process that
does not involve phosphorylation during catalysis.
In nonphotosynthetic eukaryotes, the F-ATPase is found exclusively on the inner membrane
of mitochondria, whereas in green plants and algae there are two distinct F-ATPases; one
in mitochondria and the other on the thylakoid membrane of chloroplasts. In bacteria, F-
ATPase is present in the plasma membrane. These ATPases couple the flow of protons
with ATP hydrolysis and synthesis.
b. Secondary active transport
Secondary active (indirect) transport occurs when endergonic (uphill) transport of one
solute is coupled with the exergonic (downhill) flow of a different solute that was originally
pumped uphill by primary active transport. Secondary active transport is either symport or
antiport, depending on whether the two solutes move in the same or opposite directions.
A common example of secondary active transport is the symport of Na+ and glucose, The
transmembrane protein Na+-glucose transporter (also known as Na+-glucose co-transporters
or symporters) allows Na+ and glucose to enter the cell together. Na+-glucose symporters
are a family of glucose transporter present on the apical surface (facing the lumen) of the
epithelium cell of the small intestine and actively transports glucose molecules into the cell
from the gut. The Na+ flows down their concentration gradient while the glucose molecules
are transported against their concentration gradient into the cell. Later, the Na+ is pumped
back out of the cell by the Na+-K+ ATPase and thus maintaining the inward Na+ gradient.
The GLUT confined to the basolateral (basal and lateral) surfaces of the cell allows the
same molecules to leave the cell by facilitated diffusion into the extracellular fluid on the
other side of the epithelium.
1.3 Thermodynamics of transport
The amount of energy needed for the transport of a solute against a concentration gradient can
be calculated from the initial concentration gradient. When there is transport of one mole of a solute
(uncharged) from a region in which its concentration is C1 to a place where its concentration is C2 and
the standard free energy change (Go) is zero, then free energy change (G) is given by
C2
ÄG =RT ln
C1
According to this equation, if C2 (Cin) is less than C1 (Cout), G is negative, and the process is
thermodynamically favourable. As more and more substance is transferred, C1 decreases and C2
increases, until C2 = C1. At this point G = 0, and the system is in equilibrium.
However, if the solute is an ion of charge Z, then the free energy change for transport across a
cell membrane involves two contributors: the normal concentration term, as given in equation (1), plus
a second term describing the energy change involved in moving a mole of ions across the potential
difference. If we consider a process in which ions are transported from outside to inside of a cell, then
G is given by:
Cin
ÄG =RTln + ZF Ä
Cout
Here F is the Faraday constant (96.5 kJ mole–I V–1) or 23,062 cal/(mol V)and  is the trans-
membrane electrical potential (in volts). Eukaryotic cells typically have electrical potentials across their
plasma membranes of about 0.05 to 0.1 V (with the inside negative relative to the outside).

1.4 Membrane potential

Electrical character of ion transport may be electroneutral i.e. electrically silent either by symport
of the oppositely charged ions or antiport of similarly charged ions or electrogenic i.e. result in charge
separation across the membrane. Electrogenic transport affects and can be affected by the membrane
potential. For example, the Na+- K+ pump imports 2K+ and simultaneously exports 3Na+; that is, it moves
1 positive charge out of the cell. Its electrogenic operation directly contributes to the negative inside
membrane potential, which is evidenced by the fact that stopping the pump using an alkaloid inhibitor,
ouabain, causes an immediate and slight depolarization of the cell membrane.
All cells have an electrical potential difference, or membrane potential, across their plasma
membrane, Electrical potential across cell membranes is a function of the electrolyte concentrations in
the intracellular and extracellular solutions and of the selective permeabilities of the ions. Active transport
of ions by ATP-driven ion pumps, generate and maintain ionic gradients. In addition to ion pumps, which
transport ions against concentration gradients, plasma membrane contains channel protein that allows
ions to move through it at different rates down their concentration gradient, Ion concentration gradients
and selective movements of ions create a difference in electric potential or voltage across the plasma
membrane. This is called membrane potential.
At equilibrium, the electric potential across the membrane equals the potassium equilibrium
potential in volts, EKS The magnitude of EK is given by the Nernst equation
Ek =
RT
log
KR 
ZF KL 
Where R (the gas constant) is 1.987 cal/(degree mol);
T (the absolute temperature in degrees Kelvin) is 293 K at 20oC;
Z (the charge, also called the valency) equals to 1;
F (the Faraday constant) is 23,062 cal/(mol V), or 96,000 coulombs/(mol V); and
[KR] and [KL] are the K+ concentrations on the right and left sides, respectively, at equilibrium.
The calculation for the charge of an ion across a membrane, The Nernst Potential, is relatively
easy to calculate. The equation is as follows: (RT/zF) log([X]out/[X]in). RT/F is approximately 61, therefore
the equation can be written as :
= (61/z) x log ([X]out/[X]in)

2. CYTOSKELETAL ELEMENTS: CYTOSKELETON & MOTOR PROTEINS
The cytoskeleton is a energetic 3-dimensional structure that fills the cytoplasm. It is present in
both eukaryotic and prokaryotic cells. The cytoskeleton acts as both muscle and skeleton, and plays a
role in cell protection, cell motility (migration), cytokinesis, intracellular transport, cell division and the
organization of the organelles within the cell.
Table : Three Major Filamentous Systems of the Cytoskeleton
Cytoskeletal Element Description (diameter) Protein Composition

microfilaments thin filaments (6-7 nm) actin + associated proteins
microtubules tubular structures (25 nm) tubulin + associated proteins
Intermediate filaments rope-like fibers (~10 nm) IF proteins
The cytoskeleton in the eukaryotic cell is made up of three kinds of protein filaments:
a. Actin filaments (also called microfilaments): Actin and Microfilaments
 Exhibits polarity (+ and - ends),(barbed and pointed ends)
 Filaments stabilized by other proteins Monomers of the protein actin polymerize to form
long, thin fibers that are about 8 nm in diameter. They provide mechanical strength to the
cell, link transmembrane and cytoplasmic proteins, anchor centrosomes during mitosis,
generate locomotion in cells and interact with myosin to provide the force of muscular
contraction
Actin Polymerization
G-actin-ATP
polymerization
F-actin-ATP
Pi
F-actin-ADP
depolymerization
G-actin-ADP
ADP/ATP exchange
G-actin-ATP
Fig. : Actin Polymerization
Actin can hydrolyze its bound ATP to ADP + Pi, releasing Pi. The actin monomer can exchange
bound ADP for ATP. The conformation of actin is different, depending on whether there is ATP or ADP
in the nucleotide-binding site. G-actin (globular actin) with bound ATP can polymerize, to form F-actin
(filamentous actin). F-actin may hydrolyze its bound ATP to ADP + Pi and release Pi. ADP release from
the filament does not occur because the cleft opening is blocked.
ADP/ATP exchange: G-actin can release ADP and bind ATP, which is usually present in the
cytosol at higher concentration than ADP.
Actin filaments have polarity. The actin monomers all orient with their cleft toward the same end
of the filament (designated the minus end). The diagram at right is very oversimplified. Actin monomers
spiral around the axis of the filament, with a structure resembling a double helix. The polarity of actin
filaments may be visualized by decorating the filaments with globular heads (designated S1) cleaved off
of myosin by proteases. Bound myosin heads cause an appearance of arrowheads in electron
micrographs. Actin filaments may undergo treadmilling, in which filament length remains approximately
constant, while actin monomers add at the (+) end and dissociate from the (-) end. This has been
monitored using brief exposure to labeled actin monomers (pulse labeling).
Capping proteins bind at the ends of actin filaments. Different capping proteins may either
stabilize an actin filament or promote disassembly. They may have a role in determining filament length.
For example:
 Tropomodulins cap the minus end, preventing dissociation of actin monomers.
 CapZ capping protein binds to the plus end, inhibiting polymerization. If actin monomers
continue to dissociate from the minus end, the actin filament will shrink.
Two toxins that have been useful experimentally:
 Cytochalasins (from fungi) bind to the (+) end of F-actin and block subunit addition.
Depolymerization at the (-) end may cause loss of the filament.
 Phalloidin (from Amanita mushroom) binds along the sides of actin filaments, stabilizing
them. Phalloidin labeled with a fluorescent chromophore is often used for visualization of
actin filaments by fluorescence microscopy
b. Myosin
Structure: Skeletal muscle contains 70 - 100 mg of myosin per gram of fresh muscle weight;
this corresponds to 40-50% of the total muscle proteins. Myosin is a globulin, soluble at a high salt
concentration, e.g. 0.6 M KCl, and insoluble at a low salt concentration, e.g. 0.03 M KCl. Thus, myosin
can be extracted from the muscle with 0.6 M KCl solution and purified by dilution of the extract with 20
volumes of distilled water. However, under these conditions, some actin remains bound to the myosin
and special procedures are required to prepare myosin free of actin.
Fig. : Structure myosin

Size and shape of the myosin molecule: Myosin is a large asymmetric molecule, it has a long
tail and two globular heads (Fig.). The tail is about 1,600 Å long, 65 Å wide and 40 Å deep at its thickest
part. The molecular weight of myosin is about 500,000. In strong denaturing solutions, such as 5 M
guanidine-HCl or 8 M urea, myosin dissociates into six polypeptide chains: two heavy chains (molecular
weight of each heavy chain is about 200,000) and four light chains (two with a molecular weight of 20,000
one with 15,000 and another with 25,000). The two heavy chains are wound around each other to form
a double helical structure. At one end both chains are folded into separate globular structures to form
the two heads. In the muscle, the long tail portion forms the backbone of the thick filament and the heads
protrude as crossbridges toward the thin filaments. Each head contains two light chains.

Fig. : Size and shape of the myosin molecule

Fig. (A) Different components of the myosin molecule. Various proteolytic enzymes cleave the
molecule into heavy meromyosin (HMM) and light meromyosin (LMM). The point of cleavage of flexible
in the intact molecule (hinge region) and serves to bring the cross-bridge to the surface of the myosin
filament. The HMM moiety comprises the remainder of the -helical rod (S2 fragment) and the two
globular heads (S1 fragments), to which the light chains are attached. The two globular heads form a
cross-bridge. (B) Enlargement of one globular myosin head to show the three components (segments).
The 50-kD segment is divided into upper and lower domains by a cleft, the size of which is regulated
by the interaction of ATP with its “pocket.” Based on Vibert and Cohen (1988) and Rayment et al. (1993).
2.1 Function
i. Myosin-actin binding : One of the biologically important properties of myosin is its ability
to combine with actin. The complex formed is called actomyosin. The actin binding by
myosin is highly specific; no other protein can substitute actin. Physiologically, when actin
and myosin combine the muscle produces force.
ii. Intermediate filaments : Cytoplasmic fibers averaging 10 nm in diameter, which is between
that of 7 nm actin (microfilaments), and that of 25 nm microtubules. Intermediate filaments
(IFs) are cytoskeletal components found in animal cells. Although they were initially
designated 'intermediate' because their average diameter is between those of narrower

microfilaments (actin) and wider myosin filaments found in muscle cells. Intermediate
filaments contribute to cellular structural elements and are often crucial in holding together
tissues like skin. There are several types each constructed from one or more protein (e.g.
keratins, nuclear lamins, neurofilaments, vimentins). All types of intermediate filaments
provide a supporting framework within the cell.
 keratins are found in epithelial cells and also form hair and nails;
 nuclear lamins form a meshwork that stabilizes the inner membrane of the nuclear
envelope;
 neurofilaments strengthen the long axons of neurons;
 vimentins provide mechanical strength to muscle (and other) cells.
iii. Microtubules
Fig. Microtubules
Straight, hollow cylinders averaging 25 nm in diameter, built of -tubulin and -tubulin dimers, two
globular proteins. The cytoskeleton is the framework of the cell which forms the structural supporting
component. Microtubules are the largest element of the cytoskeleton. They participate in a wide variety
of cellular activities with most involving motion. Motion is provided by protein 'motors' that use the energy
of ATP hydrolysis to move along the microtubule. Like microfilaments, microtubules can dissolve and
reform quickly. They are also the structural elements of flagella, cilia, and centrioles (the latter are the
two perpendicular bodies of the centrosome). In animal cells, the centrosome is the microtubule-organizing
center.
Microtubule motors
There are two major groups of microtubule motors:
a. Kinesins (most of these move toward the plus end of the microtubules)
b. Dyneins (which move toward the minus end)
c. Cilia & Flagella
a. Kinesins are a large family of proteins with diverse structures. Mammalian cells have at least
40 different kinesin genes. The best studied is referred to as conventional kinesin, kinesin I, or simply
kinesin. Some are referred to as kinesin-related proteins (KRPs).
Kinesin I (conventional type) has a structure somewhat analogous to but distinct from that of
myosin. There are 2 copies each of a heavy chain and a light chain. Each heavy chain includes a
globular ATP-binding motor domain at the N-terminus.
Stalk domains of the heavy chains interact in an a-helical coiled coil that extends from the heavy
chain neck to the tail. The coiled coil is interrupted by a few hinge regions that give flexibility to the
otherwise stiff stalk domain.
Fig. Kinesin structure

 N-termini of the two light chains associate with the two heavy chains near the tail. The
diagram above is over simplified. Light chains at the N-terminus include a series of
hydrophobic heptad repeats that are predicted to interact with similar repeats in heavy
chains near the tail region, in a 4-helix coiled coil.
 C-terminal tail domains of kinesin light chains include several "tetratrico peptide repeats"
(TPRs). The 34 amino acid TPRs mediate protein-protein interactions. Kinesin light chain
TPR repeats are involved in binding of kinesins to cargo. C terminal domains of heavy
chains may also participate in binding some kinesins to cargo. Cargo proteins bound by
kinesins are diverse.
 Some organelle membranes contain transmembrane receptor proteins that bind kinesins.
Kinectin is an endoplasmic reticulum membrane receptor for kinesin-I.
 Scaffolding proteins, first identified as being involved in assembling signal protein complexes,
mediate binding of kinesin light chains to some cargo proteins or receptors.
 Some membrane-associated Rab GTPases, that provide specificity for vesicle transport
and fusion, are known to bind particular kinesins.
In the absence of cargo, the kinesin heavy chain stalk folds at hinge regions, bringing heavy chain
tail domains into contact with the motor domains. In this folded over state, kinesin exhibits decreased
ATPase activity and diminished binding to microtubules. This may prevent wasteful hydrolysis of ATP by

kinesin when it is not transporting cargo. Unfolding of kinesin into its more extended active conformation
is promoted by phosphorylation of kinesin light chains, catalyzed by a specific kinase, or binding of cargo.
Some examples:
 The rapid transport of organelles, like vesicles and mitochondria, along the axons of
neurons takes place along microtubules with their plus ends pointed toward the end of the
axon. The motors are kinesins.
 The migration of chromosomes in mitosis and meiosis takes place on microtubules that
make up the spindle fibers. Both kinesins and dyneins are used as motors.
 Vincristine, a drug found in the Madagascar periwinkle (a wildflower), binds to
tubulin dimers preventing the assembly of microtubules. This halts cells in
metaphase of mitosis.
 Taxol, a drug found in the bark of the Pacific yew, prevents depolymerization of
the microtubules of the spindle fiber. This, in turn, stops chromosome movement,
and thus prevents the completion of mitosis.
b. Dyneins: They are minus end-directed motor proteins. Dynein is large and complex. Cytoplasmic
dynein has a molecular weight exceeding one million.
 Dyneins were first studied in cilia & flagella.
 Many cytoplasmic dyneins have now been discovered. Cytoplasmic dyneins mediate ATP-
dependent retrograde movements of vesicles and organelles along microtubules toward
the centrosome (MTOC - microtubule organizing center).
Dynein includes 2 or 3 heavy chains. Each is about 4600 amino acid residues long and includes
a globular motor domain.
Extending out from each motor domain is a narrow stalk that ends in a small globular domain.
It is this domain at the end of the stalk that interacts with microtubules. The stalk may help avoid steric
interference when multiple dyneins interact with a microtubule. The stalk is an intra-molecular coiled coil,
formed by interaction of a-helical segments on either side of the microtubule-binding segment of the
dynein heavy chain.
Each heavy chain motor domain of dynein includes 6 repeats of an ATPase of the AAA gene
family. High resolution electron microscopy with image averaging indicates a heptameric wheel-like
structure of the dynein motor domain, with the six AAA domains plus an additional C-terminal domain.
One of the AAA domains is postulated to be the functional ATPase that drives movement. A stalk
(assumed to be the microtubule-binding segment) protrudes out from between two of the six AAA
domains.
Dynactin is a large complex that mediates binding of dynein to membranes or other cargo. Dynein
may bind to some cargo proteins directly via its light chains, but interactions with cargo are often
mediated by dynactin.

Dynactin includes:
 Glued, an elongated 150 kDa dimeric protein, has at its distal end two microtubule-binding
globular heads. Binding of glued to microtubules may increase processivity of dynein
movement along microtubules.
Arp1, an actin-related protein forms a short filament (rod) of constant length (8-10 subunits),
capped at one end by the actin-capping protein CapZ, and at the other end by proteins
unique to dynactin.
 Dynamitin, with another small protein, links the Arp1 rod to glued.
Dynein and dynactin are associated with golgi membranes, which also have a spectrin network
on their surface. Location of the golgi apparatus near the centrosome is thought to be due to its being
drawn along microtubules toward their minus ends by dynein.
c. Cilia & flagella
 Cilia and flagella are bounded by the plasma membrane.
 A basal body, which is a single centriole cylinder, is at the base of each cilium or flagellum.
 Cilia and flagella have a core axoneme, a complex of microtubules and associated proteins.
 Some distinctions between cilia and flagella:
 Flagella are usually 1 or 2 per cell. They tend to have a rotary or sinusoidal
movement. They may have additional structures outside of the core axoneme.
 Cilia are usually many per cell. They tend to have a whip-like movement.
The axoneme of cilia or flagella includes:
 Nine doublet microtubules around the periphery. The A tubule of each doublet has attached
dynein arms.
 Two singlet central microtubules, surrounded by a sheath.
 Nexin links & radial spokes. These provide elastic connections between microtubule
doublets and between the A tubule of each doublet and the central sheath.
Fig. structure of Cilia & flagella

Bending of a cilium involves ATP-dependent walking of motor domains of A-tubule dynein arms
along adjacent B tubules, toward the minus end. This causes the sliding of microtubule doublets. Minus
ends are anchored in the basal body, and flexible links between doublets (radial spokes and nexin links)
limit sliding. The result is bending of the cilium.
Several lines of evidence support this mechanism:
1. ATP is required for bending.
2. Inactivating dynein mutations eliminate ciliary bending

3. If isolated axonemes, with their membrane removed, are treated with mild protease, the
radial spokes and nexin links are degraded. ATP addition then causes the microtubule
doublets to slide apart.
4. If a bent cilium is examined in cross-section by electron microscopy, fewer than 9 doublet
microtubules are seen at the tip.
Few mammalian cell types have motile cilia or flagella, including some respiratory epithelial cells
and sperm cells. Many mammalian cells have a single short non-motile primary cilium. The photoreceptor
structure of each retinal rod and cone cell develops from a non-motile cilium.
2.2 Initiation of Muscle Contraction
Step 1) Neuromuscular Control
The axons of the nerve cells of the spinal cord branch and attach to each muscle fiber forming
a neuromuscular junction.
i. An action potential passes down the nerve.
ii. The nerve releases Ca++ that results in the release of Acetylcholine (ACh)
Step 2) ACh binds with the sarcolemma.
Step 3) Muscle Fiber Action Potential
i. ACh binds with receptors and opens Na+ channels
Na+ Channels open and Na+ in
There is a decrease in the resting potential
ii. Na+ rushes in and the sarcolemma depolarizes.
iii. The regional depolarization spreads rapidly.
The positive patch in the membrane changes the adjacent patch of the membrane.
Thus depolarization spreads.
iv. The K+ channels open and the region repolarizes
Immediately after the action potential passes the membrane permeability changes again.
Na+ channels close and K+ channels open.
K+ rushes out of the cell.
Cell reploraizes
++
Step 4) Ca is released from the sarcoplasmic reticulum.
i. Ca++ is stored in the sarcoplasmic reticulum.
ii. Depolarization releases the Ca++.
iii. The Ca++ clears the actin binding sites.

Fig. Ca++ is released from the sarcoplasmic reticulum.

Step 5) Sliding Filament Theory of Contraction
During muscle contraction the thin actin filaments slide over the thick myosin filament.
When Calcium is present the blocked active site of the actin clears.
Step A: Myosin head attaches to actin. (High energy ADP + P configuration)
Step B: Power stroke: myosin head pivots pulling the actin filament toward the center.
Step C: The cross bridge detaches when a new ATP binds with the myosin.
Step D: Cocking of the myosin head occurs when ATP a ADP + P. Another cross bridge can
form.

Fig. Sliding Filament Theory of Contraction

The end result is a shortening of the sarcomere.
 The distance between the Z discs shortens
 The H zone disappears
 The dark A band increases because the actin & the myosin overlap more
 The light I band shortens.

Component of the sarcomere:
Component Description
Z-discs Narrow, plate –shaped regions that separate one sarcomere from the next
The dark, middle part of the sarcomere that extends the entire length of the thick
A-band filaments and also concludes those parts of the thin filaments that overlap with
the thick filaments
The lighter region of the sarcomere that contains the thin filaments but no thick
I-band
filament. A Z-disc passes through the center of each I-band
An arrow region in the center of the sarcomere that contains thick filaments but no
H-zone
thin filaments.
A region in the center of the H-zone that contains protein, myomesin,that holds the
M-line
thick filaments together at the center of the sarcomere.
Step 6) Ca++ is removed from the cytoplasm
Step 7) Tropomysin blocks the actin site
Muscle fiber contraction and relaxation
Muscle Fiber Contraction Muscle Fiber Relaxation

1. The distal end of a motor neutron releases 1. Acetylcholinesterase decomposes acetylcholine
acetylcholine. and the muscle fiber membrane is no longer
stimulated.
2. Acetylcholine diffuses across the gap at the 2. Calcium ions are actively tansported into the
neuromuscular junction. sarcoplasmic reticulum.
3. The sarcolemma is stimulated, and a muscle 3. ATP causes linkages between action and
impulse travels over the surface of the muscle fiber myosin filaments to break without ATP breakdown.
and deep into the fiber through the transverse
tubules and reaches the sarcoplasmic reticulum.
4. Calcium ions diffuse from the sarcoplasmic 4. Cross-bridges recock.
iculum into the sarcoplasm and bind to troponin
molecules.
5. Tropomyosin molecules move and expose 5. Troponin and tropomyosin molecules inhibit the
specific sites on action filaments. interaction between myosin and action filaments.
6. Actin and myosin filaments for linkages. 6. Muscle fiber remains relaxed, yet ready until
stimulated again.
7. Actin filaments are pulled inward by myosin cross-
bridges.
8. Muscle fiber shortens as a contraction occurs.

3. MITOCHONDRIA
The first observations of intracellular structures that probably represent mitochondria were published
in the 1840s. In 1894, Richard Altmann was established them as cell organelles and called them
"bioblasts". The term "mitochondria" itself was coined by Carl Benda in 1898.
Mitochondria are structures within cells that convert the energy from food into a form that cells
can use. Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have
a small amount of their own DNA. This genetic material is known as mitochondrial DNA or mtDNA. In
humans, mitochondrial DNA spans about 16,500 DNA building blocks (base pairs), representing a small
fraction of the total DNA in cells. Mitochondria are typically round to oval in shape and range in size from
0.5 to 10 m. A mitochondrion contains outer and inner membranes composed of phospholipid bilayersand
proteins. There are five distinct parts to a mitochondrion. They are:
1. The outer mitochondrial membrane,
2. The intermembrane space (the space between the outer and inner membranes),
3. The inner mitochondrial membrane,
4. The cristae space (formed by infoldings of the inner membrane), and
5. The matrix (space within the inner membrane).
Fig. Structure of mitochondria

a. Outer mitochondrial membrane : It contains large numbers of integral proteins called
porins. These porins form channels that allow molecules 5000 daltons or less in molecular
weight to freely diffuse from one side of the membrane to the other. The mitochondrial
outer membrane can associate with the endoplasmic reticulum (ER) membrane, in a
structure called MAM (mitochondria-associated ER-membrane). This is important in the
ER-mitochondria calcium signaling and involved in the transfer of lipids between the ER
and mitochondria.
b. Intermembrane space : It is the space between the outer membrane and the inner
membrane. It is also known as perimitochondrial space. Large proteins must have a
specific signaling sequence to be transported across the outer membrane, so the protein
composition of this space is different from the protein composition of the cytosol. One
protein that is localized to the intermembrane space in this way is cytochrome c.
c. Inner membrane : The inner mitochondrial membrane contains proteins with five types
of functions:

1. Those that perform the redox reactions of oxidative phosphorylation

2. ATP synthase, which generates ATP in the matrix
3. Specific transport proteins that regulate metabolite passage into and out of the
matrix
4. Protein import machinery.
5. Mitochondria fusion and fission protein.
The inner membrane is also loaded with proteins involved in electron transport and ATP
synthesis. This membrane surrounds the mitochondrial matrix, where the citric acid
cycle produces the electrons that travel from one protein complex to the next in the inner
membrane. At the end of this electron transport chain, the final electron acceptor is
oxygen, and this ultimately forms water (H2O). At the same time, the electron transport
chain produces ATP. (This is why the process is called oxidative phosphorylation.) During
electron transport, the participating protein complexes push protons from the matrix out
to the intermembrane space. This creates a concentration gradient of protons that another
protein complex, called ATP synthase, uses to power synthesis of the energy carrier
molecule ATP.
Fig. : The electrochemical proton gradient and ATP synthase

At the inner mitochondrial membrane, a high energy electron is passed along an electron
transport chain. The energy released pumps hydrogen out of the matrix space. The
gradient created by this drives hydrogen back through the membrane, through ATP synthase.
As this happens, the enzymatic activity of ATP synthase synthesiszes ATP from ADP.
d. Cristae : The inner mitochondrial membrane is compartmentalized into numerous cristae,
which expand the surface area of the inner mitochondrial membrane, enhancing its ability
to produce ATP. These folds are studded with small round bodies known as F1 particles
or oxysomes. These are not simple random folds but rather invaginations of the inner
membrane, which can affect overall chemiosmotic function.
e. Matrix : The matrix is the space enclosed by the inner membrane. It contains about 2/
3 of the total protein in a mitochondrion. Mitochondria have their own genetic material, and
the machinery to manufacture their own RNAs and proteins.
3.1 Targeting of Mitochondrial proteins
The mitochondrial membranes contain specific machineries (translocases) for recognition,
translocation, and membrane insertion of precursor proteins.
Mitochondrial precursor proteins can be separated into two main classes. Preproteins that are
destined for the mitochondrial matrix, as well as a number of proteins of the inner membrane and

intermembrane space, carry N-terminal cleavable extensions, termed presequences. These positively
charged extensions function as targeting signals that interact with the mitochondrial import receptors and
direct the preproteins across both outer and inner membranes. The second class of precursor proteins,
carrying various internal targeting signals, includes all outer membrane proteins along with many
intermembrane space and inner membrane proteins.
The mitochondrial preproteins are supposed to be maintained in a translocation competent state
by cytosolic Chaperones, in particular members of the HSP70 (Heat shock protein family of 70 kDa)
protein family and by binding factors specific for presequences. The translocation across the mitochondrial
membranes is then mediated by the import machineries of the Outer (TOM (Translocase of Outer
Mitochondrial membrane) Complex) and the inner membrane (TIM (Translocase of Inner Mitochondrial
Membrane) Complex) at sites of close contact between both membranes. Receptors on the outer
surface of the mitochondrial outer membrane specifically recognize and bind the precursors prior to their
translocation.
TOM Complex represents the central entry gate for practically all nuclear-encoded mitochondrial
proteins. The TOM complex consists of seven different subunits that can be grouped into three categories:
 The receptors TOM20, TOM22, and TOM70; the channel-forming protein TOM40; and
three small TOMproteins, TOM5, TOM6, and TOM7.
 TOM20 is the first receptor involved in recognizing the presequence of a preprotein. A
typical presequence has a length of about 10-30 amino acid residues and forms an
amphipathic Alphahelix. One half of the helix possesses a hydrophobic surface that is
recognized by a binding groove within TOM20, whereas the other half is positively charged
and recognized by the receptor TOM22.
 With the help of the small protein TOM5, the preprotein is then transported to the general
import pore formed by the essential Beta-barrel protein TOM40. After translocation through
theTOM40 pore, the presequence binds to the intermembrane space domain of the receptor
TOM22.
 TOM40 itself does not simply form a passive pore but rather interacts with the preproteins
in transit. The other small TOM proteins, TOM6 andTOM7, do not directly interact with
precursor proteins but are required for the assembly and stability of the TOM complex
After passing through the TOM complex, the precursor proteins can follow one of three
major pathways. Preproteins with a presequence are transferred to the presequence
translocase of the inner membrane, also termed the TIM23 complex (translocase of the
inner membrane.
 The presequence translocase of the inner membrane consists of three integral and essential
membrane proteins: TIM50, TIM23, and TIM17.
 Insertion of preproteins into the TIM23 channel strictly depends on the presence of the
membrane potential across the inner membrane. TIM23 complex remains associated with
presequence translocase-Associated Motor(PAM), which is a member of Hsp70 family
chaperone. It binds to a translocation polypeptide chain and drives its movement into the
matrix in a reaction cycle that requires hydrolysis of ATP.
 Translocation of protein across mitochondrial membranes is an active process. The
membrane potential activates the channel-forming protein TIM 23.

Fig. : Import of presequence proteins into the mitochondrial matrix

After passing through the channel of the TOM complex, presequence-carrying preproteins are
recognized by an intermembrane space (IMS) domain of Tom22. From here, they are brought into
contact with Tim50, which then assists in guiding them to the presequence translocase (TIM23 complex).
Their subsequent insertion into the channel, formed by Tim23, requires the membrane potential ()
across the inner membrane (IM). A further driving force is provided by the PAM in the matrix. The central
component of this motor, mtHsp70, is transiently anchored at the translocase by Tim44 and requires
assistance from Pam18, Pam16, and Mge1 for promotion of the reaction cycle. The presequences of
the precursor proteins are cleaved off by the MPP in the matrix. OM, outer membrane.
After transport through the TOM machinery, the SAM (Sorting and Assembly Machinery complex)
Complex is required for the insertion of Beta-barrel Outer membrane proteins into the Outer membrane.
The precursors of the hydrophobic Carrier proteins of the inner membrane, e.g. the ADP/ATP carrier or
the Phosphate carrier, are initially recognized by a different receptor at the mitochondrial surface, TOM70.
These precursors are usually synthesized without a presequence but contain multiple internal targeting
signals. After recognition by TOM70 and translocation by the TOM pore across the outer membrane,
these Carrier proteins are bound by a complex of small TIM proteins in the intermembrane space, the
TIM9-TIM10 complex.
Besides the essential TIM9-TIM10 complex, the homologous yet non-essential TIM8-TIM13 complex
also supports the transfer of selected inner membrane proteins. The TIM9-TIM10 complex delivers the
precursor proteins to the membrane-integrated insertion machinery of the inner membrane by directly
docking to it. This insertion machinery is termed the TIM22 complex or the carrier translocase. The
peripheral membrane protein TIM12 serves as the docking site for the TIM9-TIM10 complex. The precursor
is then translocated to the core of the insertion machinery, the channel-forming protein TIM22.
Thus, mitochondrial import requires the action of a variety of components in the cytosol to
maintain an import-competent conformation, in the outer mitochondrial membrane for recognition of the
precursor, in both outer and inner membrane for protein translocation, and in the matrix for proteolytic
processing and folding to the active conformation.

4. ENDOPLASMIC RETICULUM
The lacey membranes of the endoplasmic reticulum were first seen by Keith R. Porter, Albert
Claude, and Ernest F. Fullam in 1945.
The endoplasmic reticulum is a network of tubules and flattened sacs that serve a variety of
functions in the cell. There are two regions of the ER that differ in both structure and function. One region
is called rough ER because it has ribosomes attached to the cytoplasmic side of the membrane. The
other region is called smooth ER because it lacks attached ribosomes. Typically, the smooth ER is a
tubule network and the rough ER is a series of flattened sacs. The space inside of the ER is called the
lumen. The ER is very extensive extending from the cell membrane through the cytoplasm and forming
a continuous connection with the nuclear envelope. Since the ER is connected with the nuclear envelope,
the lumen of the ER and the space inside the nuclear envelope are part of the same compartment.
Rough endoplasmic reticulum synthesize proteins, while smooth endoplasmic reticulum synthesize
lipids and steroids, metabolize carbohydrates and steroids, and regulate calcium concentration, drug
detoxification, and attachment of receptors on cell membrane proteins. Sarcoplasmic reticulum solely
regulate calcium levels.
i. Rough endoplasmic reticulum
The binding site of the ribosome on the rough endoplasmic reticulum is the translocon. A ribosome
only binds to the RER once a specific protein-nucleic acid complex forms in the cytosol. This special
complex forms when a free ribosome begins translating the mRNA of a protein destined for the secretory
pathway. The first 5-30 amino acids polymerized encode a signal peptide, a molecular message that is
recognized and bound by a signal recognition particle (SRP). Translation pauses and the ribosome
complex binds to the RER translocon where translation continues with the nascent protein forming into
the RER lumen and/or membrane. The protein is processed in the ER lumen by an enzyme (a signal
peptidase), which removes the signal peptide. Ribosomes at this point may be released back into the
cytosol; though, non-translating ribosomes are also known to stay associated with translocons.
The membrane of the rough endoplasmic reticulum forms large double membrane sheets that
are located near, and continuous with, the outer layer of the nuclear envelope. Although there is no
continuous membrane between the endoplasmic reticulum and the Golgi apparatus, membrane-bound
vesicles shuttle proteins between these two compartments. Vesicles are surrounded by coating proteins
called COPI and COPII. COPII targets vesicles to the Golgi apparatus and COPI marks them to be
brought back to the rough endoplasmic reticulum. The rough endoplasmic reticulum works in concert
with the Golgi complex to target new proteins to their proper destinations. A second method of transport
out of the endoplasmic reticulum involves areas called membrane contact sites, where the membranes
of the endoplasmic reticulum and other organelles are held closely together, allowing the transfer of lipids
and other small molecules.
The rough endoplasmic reticulum is key in multiple functions:
 Manufacture of lysosomal enzymes with a mannose-6-phosphate marker added in the
cis-Golgi network
 Manufacture of secreted proteins, either secreted constitutively with no tag or secreted in
a regulatory manner involving clathrin and paired basic amino acids in the signal peptide.
 Integral membrane proteins that stay embedded in the membrane as vesicles exit and
bind to new membranes. Rab proteins are key in targeting the membrane; SNAP and
SNARE proteins are key in the fusion event.
 Initial glycosylation as assembly continues. This is N-linked (O-linking occurs in the Golgi).
 N-linked glycosylation: If the protein is properly folded, Oligosaccharyl transferase recognizes
the AA sequence NXS or NXT (with the S/T residue phosphorylated) and adds a 14-sugar
backbone (2-N-acetylglucosamine, 9-branching mannose, and 3-glucose at the end) to
the side-chain nitrogen of Asn.
ii. Smooth endoplasmic reticulum

It synthesizes lipids, phospholipids, and steroids. Cells which secrete these products, such as
those in the testes, ovaries, and skin oil glands have a great amount of smooth endoplasmic reticulum.
It also carries out the metabolism of carbohydrates, drug detoxification, attachment of receptors on cell
membrane proteins, and steroid metabolism.
iii. Sarcoplasmic reticulum
The sarcoplasmic reticulum (SR) is smooth ER found in myocytes. The only structural difference
between this organelle and the smooth endoplasmic reticulum is the medley of proteins they have, both
bound to their membranes and drifting within the confines of their lumens.
This fundamental difference is indicative of their functions: The endoplasmic reticulum synthesizes
molecules, while the sarcoplasmic reticulum stores and pumps calcium ions. The sarcoplasmic reticulum
contains large stores of calcium, which it sequesters and then releases when the muscle cell is stimulated.
It plays a major role in excitation-contraction coupling.
4.1 Protein targeting via the secretory pathway
Protein targeting, or protein trafficking, is the moving of proteins from their site of synthesis to the
place where they are needed.
Most proteins are synthesised in the cytoplasm of cells, where the ribosomes are located, but
this is not always their place of function. There are two basic targeting pathways for proteins in eukaryotic
cells: Post-translational and co-translational. The signals involved are called sorting signals. These are
usually short sequences of amino acids, found either at the N-terminus of the protein, or integrated into
its structure. They bind to specific receptors; either on the target organelle surface or on intermediate
carrier proteins.
i. Post-translational targeting: e.g. proteins targeted for nucleus, mitochondria, chloroplasts.
The signal is recognised after transcription is complete.
ii. Co-translational targeting (secretory pathway): e.g. ER, Golgi, lysosomes, plasma
membrane and secreted proteins. When the signal sequence is transcribed, the signal
recognition particle (SRP) pauses transcription and redirects the mRNA, ribosome and
partially translated peptide to a membrane. The SRP then docks into a receptor on a
membrane surface and the protein is threaded through the membrane as it is translated.
The endoplasmic reticulum can bind ribosomes. A protein can be inserted into or through the ER
membrane as it is being translated (co-translational). This is initiated with a signal sequence at the N-
terminus of the emerging peptide chain. The signal sequence binds to an SRP (signal recognition
particle), and translation is paused. The ribosome-complex then binds to the ER via a receptor and the
signal sequence crosses the ER membrane. Translation then continues, with the peptide chain being
pulled into the ER lumen. Whilst in the ER many proteins start to undergo glycosylation. The majority
are packaged into vesicles, and enter the cis-phase of the Golgi- where they are further packaged and
processed.
From the ER, proteins are dispatched to the Golgi and on to a range of locations. They may be
packaged into vesicles for secretion or fused into the membranes of vesicles which then merge with the
plasma membrane. Post-translational modification in the Golgi, such as the addition of mannose-6-
phosphate may target proteins to lysosomes. Resident ER proteins contain a KDEL sequence which
direct them back to the ER in COPI vesicles if they are transported to the Golgi.

Fig. Translational targeting (secretory pathway)

5. GOLGI APPARATUS
It was discovered in 1898 by Italian physician Camillo Golgi during an investigation of the
nervous system. Golgi apparatus, also called Golgi complex or Golgi body, membrane bound organelle
of eukaryotic cells (cells with clearly defined nuclei) that is made up of a series of flattened, stacked
pouches called cisternae. The Golgi apparatus is responsible for transporting, modifying, and packaging
proteins and lipids into vesicles for delivery to targeted destinations. It is located in the cytoplasm next
to the endoplasmic reticulum and near the cell nucleus.
5.1 Structure
1. Cis Golgi network (Goods inwards) : Also called the cis Golgi reticulum it is the entry
area to the Golgi apparatus. It follows the 'transitional elements' which are smooth areas
of the RER that are also known as the 'endoplasmic reticulum Golgi intermediate
compartments' (ERGIC).
2. Golgi stack (Main processing area) : This section is composed of a variable number,
typically 3-6, of flattened sacs called cisternae (sing. cisterna). The cisternae of the Golgi
stack are divided into three working areas: cis cisternae, medial cisternae and trans
cisternae.
3. Trans Golgi network (Goods outwards) : This section is directly connected to the trans
cisternae and it is here that final reactions and sorting takes place. The concentrated
biochemicals are packed into sealed droplets or vesicles that form by budding off from the
trans Golgi surface. The vesicles are then transported away for use in the cell and
beyond.
Fig. : Golgi Apparatus

a. The Outbound Path
 Transition vesicles pinch off from the surface of the endoplasmic reticulum carrying
 integral membrane proteins
 soluble proteins awaiting processing
 processing enzymes
 Pinching off requires that the vesicle be coated with COPII (Coat Protein II)
 The transition vesicles move toward the cis Golgi on microtubules.
 As they do so, their COPII coat is removed and they may fuse together forming larger
vesicles.
 These fuse with the cis Golgi

 Sugars are added to proteins in small packets so many glycoproteins have to undergo a
large number of sequential steps of glycosylation, each requiring its own enzymes.
 These steps take place as shuttle vesicles carry the proteins from cis to medial to the
trans Golgi compartments.
 At the outer face of the trans Golgi, vesicles pinch off and carry their completed products
to their various destinations.
b. The Inbound Path
The movement of cisternal contents through the stack means that essential processing enzymes
are also moving away from their proper site of action.
Using a variety of signals, the Golgi separates the products from the processing enzymes that
made them and returns the enzymes back to the endoplasmic reticulum.
This transport is also done by pinching off vesicles, but the inbound vesicles are coated with
COPI (coat protein I)
Fig. The Out/in bound Path

5.2 Vesicular transport
Those proteins destined for areas of the cell other than either the endoplasmic reticulum or Golgi
apparatus are moved towards the trans face, to a complex network of membranes and associated
vesicles known as the trans-Golgi network (TGN). This area of the Golgi is the point at which proteins
are sorted and shipped to their intended destinations by their placement into one of at least three different
types of vesicles, depending upon the molecular marker they carry.

Types Description Example

Vesicle contains proteins destined for extracellular release. After Antibody
Exocytotic
packaging, the vesicles bud off and immediately move towards release by
vesicles
the plasma membrane, where they fuse and release the contents into activated
(continuous)
the extracellular space in a process known as constitutive secretion. plasma B cells
Vesicle contains proteins destined for extracellular release. After Neurotransmitter
Secretory packaging, the vesicles bud off and are stored in the cell until a signal release from
vesicles is given for their release. When the appropriate signal is received they neurons
(regulated) move towards the membrane and fuse to release their contents. This
process is known as regulated secretion.
Vesicle contains proteins and ribosomes destined for the lysosome, an Digestive
organelle of degradation containing many acid hydrolases, or to proteases
Lysosomal lysosome-like storage organelles. These proteins include both destined for the
vesicles digestive enzymes and membrane proteins. The vesicle first fuses with lysosome
the late endosome, and the contents are then transferred to the
lysosome via unknown mechanisms.
5.3 Protein glycosylation within the protein:
Protein processing within the Golgi involves the modification and synthesis of the carbohydrate
portions of glycoproteins. One of the major aspects of this processing is the modification of the N-linked
oligosaccharides that were added to proteins in the ER. Three glucose residues and one mannose are
then removed while the polypeptides are still in the ER. Following transport to the Golgi apparatus, the
N-linked oligosaccharides of these glycoproteins are subject to extensive further modifications.
N-linked oligosaccharides are processed within the Golgi apparatus in an ordered sequence of
reactions (Fig.). The first modification of proteins destined for secretion or for the plasma membrane is
the removal of three additional mannose residues. This is followed by the sequential addition of an N-
acetyl glucosamine, the removal of two more mannoses, and the addition of a fucose and two more N-
acetyl glucosamines. Finally, three galactose and three sialic acid residues are added.
Different glycoproteins are modified to different extents during their passage through the Golgi,
depending on both the structure of the protein and on the amount of processing enzymes that are
present within the Golgi complexes of different types of cells. Consequently, proteins can emerge from
the Golgi with a variety of different N-linked oligosaccharides.
Fig. : Processing of N-linked oligosaccharides in the Golgi

The processing of the N-linked oligosaccharide of lysosomal proteins differs from that of secreted
and plasma membrane proteins. Rather than the initial removal of three mannose residues, proteins
destined for incorporation into lysosomes are modified by mannose phosphorylation.
In the first step of this reaction, N-acetyl glucosamine phosphates are added to specific mannose
residues, probably while the protein is still in the cis Golgi network (Figure). This is followed by removal
of the N-acetylglucosamine group, leaving mannose-6-phosphate residues on the N-linked oligosaccharide.
Because of this modification, these residues are not removed during further processing. Instead, these
phosphorylated mannose residues are specifically recognized by a mannose-6-phosphate receptor in
the trans Golgi network, which directs the transport of these proteins to lysosomes.
Fig. : Targeting of lysosomal proteins by phosphorylation of mannose residues

5.4 Protein sorting and export from the golgi apparatus
 Proteins, as well as lipids and polysaccharides, are transported from the Golgi apparatus
to their final destinations through the secretory pathway.
 This involves the sorting of proteins into different kinds of transport vesicles, which bud
from the trans Golgi network and deliver their contents to the appropriate cellular locations.
 Some proteins are carried from the Golgi to the plasma membrane by a constitutive
secretory pathway, which accounts for the incorporation of new proteins and lipids into the
plasma membrane, as well as for the continuous secretion of proteins from the cell.
 Other proteins are transported to the cell surface by a distinct pathway of regulated
secretion or are specifically targeted to other intracellular destinations, such as lysosomes
in animal cells or vacuoles in yeast.
 Proteins that function within the Golgi apparatus must be retained within that organelle,
rather than being transported along the secretory pathway.
 In contrast to the ER, all of the proteins retained within the Golgi complex are associated
with the Golgi membrane rather than being soluble proteins within the lumen.
 The signals responsible for retention of some proteins within the Golgi have been localized
to their transmembrane domains, which retain proteins within the Golgi apparatus by
preventing them from being packaged in the transport vesicles that leave the trans Golgi
network. In addition, like the KKXX sequences of resident ER membrane proteins, signals
in the cytoplasmic tails of some Golgi proteins mediate the retrieval of these proteins from
subsequent compartments along the secretory pathway.

6. CHLOROPLAST
Chloroplasts are organelles, specialized subunits, in plant and algal cells. Their main role is to
conduct photosynthesis, where the photosynthetic pigment chlorophyll captures the energy from sunlight,
and stores it in the energy storage molecules ATP and NADPH while freeing oxygen from water. This
origin of chloroplasts was first suggested by Russian biologist Konstantin Mereschkowski in 1905 after
Andreas Schimper observed that chloroplasts closely resemble cynobacteria in 1883. Chloroplasts are
only found in plants and algae.
The chloroplast is made up of 3 types of membrane:
a. A smooth outer membrane which is freely permeable to molecules.
b. A smooth inner membrane which contains many transporters: integral membrane proteins
that regulate the passage in an out of the chloroplast of
 small molecules like sugars
 proteins synthesized in the cytoplasm of the cell but used within the chloroplast
c. A system of thylakoid membranes
1. The outer membrane is permeable to small organic molecules, whereas the inner
membrane is less permeable and studded with transport proteins. The innermost matrix
of chloroplasts, called the stroma, contains metabolic enzymes and multiple copies of the
chloroplast genome.
2. Chloroplast .
Fig. Chloroplast
3. Thylakoids
 The thylakoid membranes enclose a lumen: a system of vesicles (that may all be
interconnected).
 At various places within the chloroplast these are stacked in arrays called grana
(resembling a stack of coins).
 Four types of protein assemblies are embedded in the thylakoid membranes:
1. Photosystem I which includes chlorophyll and carotenoid molecules
2. Photosystem II which also contains chlorophyll and carotenoid molecules
3. Cytochromes b and f
4. ATP synthase

These carry out the so-called light reactions of photosynthesis. The thylakoid membranes are
surrounded by a fluid stroma. The stroma contains:
 All the enzymes, e.g., RUBISCO, needed to carry out the "dark" reactions of photosynthesis;
that is, the conversion of CO2 into organic molecules like glucose.
A number of identical molecules of DNA, each of which carries the complete chloroplast genome.
The genes encode some but not all of the molecules needed for chloroplast function. The others are:
 Transcribed from genes in the nucleus of the cell
 Translated in the cytoplasm and
 Transported into the chloroplast
6.1 Chemiosmotic generation of ATP in chloroplasts and mitochondria
Fig. : In mitochondria
Electron transport generates a proton gradient across the inner membrane, which is then used
to drive ATP synthesis in the matrix. In chloroplasts, the proton gradient is generated across the thylakoid
membrane and used to drive ATP synthesis in the stroma.
The major difference between chloroplasts and mitochondria, in terms of both structure and
function, is the thylakoid membrane. This membrane is of central importance in chloroplasts, where it
fills the role of the inner mitochondrial membrane in electron transport and the chemiosmotic generation
of ATP. (Fig. Chemiosmotic generation of ATP in chloroplasts and mitochondria).
The inner membrane of the chloroplast envelope (which is not folded into cristae) does not
function in photosynthesis. Instead, the chloroplast electron transport system is located in the thylakoid
membrane, and protons are pumped across this membrane from the stroma to the thylakoid lumen. The
resulting electrochemical gradient then drives ATP synthesis as protons cross back into the stroma. In
terms of its role in generation of metabolic energy, the thylakoid membrane of chloroplasts is thus
equivalent to the inner membrane of mitochondria.
6.2 Import and sorting of chloroplast proteins
Protein import into chloroplasts generally resembles mitochondrial protein import (above Figure).
Proteins are targeted for import into chloroplasts by N-terminal sequences of 30 to 100 amino
acids, called transit peptides, which direct protein translocation across the two membranes of the

chloroplast envelope and are then removed by proteolytic cleavage. The transit peptides are recognized
by the translocation complex of the chloroplast outer member (the Toc complex), and proteins are
transported through this complex across the membrane. They are then transferred to the translocation
complex of the inner membrane (the Tic complex) and transported across the inner membrane to the
stroma. As in mitochondria, molecular chaperones on both the cytosolic and stromal sides of the
envelope are required for protein import, which requires energy in the form of ATP. In contrast to the
presequences of mitochondrial import, however, transit peptides are not positively charged and the
translocation of polypeptide chains into chloroplasts does not require an electric potential across the
membrane.
Fig. : a. Protein import into the chloroplast stroma.

Proteins are targeted for import into chloroplasts by a transit peptide at their amino terminus. The
transit peptide directs polypeptide translocation through the Toc complex in the chloroplast outer membrane
and the Tic complex in the chloroplast inner membrane. This peptide is then removed by proteolytic
cleavage within the stroma. Both cytosolic and chloroplast chaperones (Hsp60 and Hsp70) are required
for protein import.

Proteins incorporated into the thylakoid lumen are transported to their destination in two steps
(Figure b). They are first imported into the stroma, as already described, and are then targeted for
translocation across the thylakoid membrane by a second hydrophobic signal sequence, which is exposed
following cleavage of the transit peptide. The hydrophobic signal sequence directs translocation of the
polypeptide across the thylakoid membrane and is finally removed by a second proteolytic cleavage
within the lumen.
Fig b. Import of proteins into the thylakoid lumen

Proteins are imported into the thylakoid lumen in two steps. The first step is import into the
chloroplast stroma, as illustrated in Figure 10.15. Cleavage of the transit peptide then exposes a second
hydrophobic signal sequence, which directs protein translocation across the thylakoid membrane.

Biotechnology (MSP)
Biotechnology (BT)
Model Solved Paper
Duration : 180 minutes Maximum Marks : 100
Read the following instructions carefully.
1. This test paper has a total of 60 questions carrying 100 marks. The entire question paper
is divided into Three Sections A, B and C. All sections are compulsory. Questions in
each section are of different types.
2. Section – A contains Multiple Choice Questions (MCQ). Each MCQ type question has
four choices out of which only one choice is the correct answer. This section has 30
Questions and carry a total of 50 marks. Q.1 – Q.10 carry 1 mark each and Questions
Q.11 – Q.30 carry 2 marks each.
3. Section – B contains Multiple Select Questions (MSQ). Each MSQ type question is
similar to MCQ but with a difference that there may be one or more than one choice(s)
that are correct out of the four given choices. The candidate gets full credit if he/she
selects all the correct choices only and no wrong choices. This section has 10 Questions
and carry 2 marks each with a total of 20 marks.
4. Section – C contains Numerical Answer Type Questions (NAT). For these NAT type
questions, the answer is a real number which needs to be entered using the virtual
numerical keypad on the monitor. No choices will be shown for these type of questions.
This section has 20 Questions and carry a total of 30 marks. Q.1 – Q.10 carry 1 mark
each and Questions Q.11 – Q.20 carry 2 marks each.
5. In all sections, questions not attempted will result in zero mark. In Section – A (MCQ),
wrong answer will result in NEGATIVE marks. For all 1 mark questions, 1/3 marks will
be deducted for each wrong answer. For all 2 marks questions, 2/3 marks will be deducted
for each wrong answer. In Section – B (MSQ), there is NO NEGATIVE and NO
PARTIAL marking provisions. There is NO NEGATIVE marking in Section – C
(NAT) as well.

Biotechnology (MSP)
SECTION-(A) MULTIPLE CHOICE QUESTIONS (MCQ)
1. Select the correct statement from the following regarding cell membrane
(A) Lipids are arranged in a bilayer with polar heads towards the inner part
(B) Fluid mosaic model of cell membrane was proposed by Singer and Nicolson
(C) Na+ and K+ ions move across cell membrane by passive transport
(D) Proteins make up 60 to 70% of the cell membrane
2. Polysome is formed by
(A) Ribosomes attached to each other in a linear arrangement
(B) Several ribosomes attached to a single mRNA
(C) Many ribosomes attached to a strand of endoplasmic reticulum
(D) A ribosome with several subunits
3. Study the following lists and select the correct match from the option given below.
List-I List-II
(a) Initiation of spindle fibres (I) Anaphase - I
(b) Synthesis of RNA and Protein (II) Zygotene
(c) Action of endonuclease (III) G1 phase
(d) Movement of chromatids towards (IV) Pachytene
opposite poles (V) Anaphase - II
(A) a - I, b - III, c - V, d - IV (B) a - III, b - II, c - I, d - V
(C) a - II, b - III, c - IV, d - V (D) a - V, b - III, c - I, d - II
4. A person with unknown blood group under ABO system, has suffered much blood loss in an
accident and needs immediate blood transfusion. His one friend who has a valid certificate of his
own blood type, offers for blood donation without delay. What would have been the type of blood
group of the donor friend ?
(A) Type A (B) Type B
(C) Type AB (D) Type O
5. Excessive growth of hair on the pinna is a feature found only in males because
(A) The gene responsible for the character is recessive in females and dominant only in
males
(B) The character is induced in males as males produce testosterone
(C) The female sex hormone estrogen suppresses the character in females
(D) The gene responsible for the character is present on the Y chromosome only

Biotechnology (MSP)
6. Identify ‘Z’ in the following reaction series,

Aq. NaOH Al2 O3 HOCl
CH3·CH2CH2Br   (X) 
Heat
 (Y)   (Z) :
(A) Mixture of CH3—CH—CH2 and CH3—CH—CH2

| | | |
Cl Cl OH Cl
(B) CH3—CH—CH2
| |
OH Cl
(C) CH3—CH—CH2
| |
Cl OH
(D) CH3—CH—CH2
| |
Cl Cl
7. Identify the state quantity among the following:

(A) q (B) q – w
(C) q + w (D) q/2
8. In an oscillating LC-circuit, the maximum charge on the capacitor is Q. The charge on the
capacitor, when the energy is stored equally between the electric and magnetic fields is
(A) Q/2 (B) Q/ 3
(C) Q/ 2 (D) Q
9. In a refrigerator, heat from inside at 277 K is transferred to a room at 300 K. How many joules
of heat shall be delivered to the room for each joule of electrical energy consumed ideally?
(A) 10 J (B) 11 J
(C) 12 J (D) 13 J
x 3 y 6 z4
10. The plane which passes through the point (3, 2, 0) and the line   is
1 5 4
(A) x – y + z = 1 (B) x + y + z = 5
(C) x + 2 y – z = 1 (D) 2 x – y + z = 5
11. Calcium deficiency in the body can be found due to absence of

(A) Vitamin C (B) Vitamin B
(C) Vitamin D (D) Vitamin E
12. Which one of the following statements is false in respect of viability of mammalian sperm?
(A) Viability of sperm is determined by its motility
(B) Sperms must be concentrated in a thick suspension
(C) Sperm is viable for only up to 24 hours
(D) Survival of sperm depends on the pH of the medium and is more active in alkaline
medium

Biotechnology (MSP)
13. The sequence of events mentioned below are symbolized by alphabets. Choose the correct
answer where the alphabets are matched with the processes.
DNA  (A)  DNA  (B)  mRNA (C)  Polypeptide
(A) A = Transformation, B = Transcription, C = Translation
(B) A = Translation, B = Transcription, C = Replication
(C) A = Transcription, B = Translation, C = Transduction
(D) A = Replication, B = Transcription, C = Translation
14. Which stage of mitosis is most characterized by the shortening of kinetochore microtubules ?
(A) Prometaphase (B) Metaphase
(C) Anaphase I (D) Anaphase II
15. Which of the following immunoglobulins is present normally in plasma at the highest concentration
?
(A) IgG (B) IgM
(C) IgA (D) IgD
16. What type of cell are activated by antigen fragments complexed with MHC I proteins.
(A) CD8+ T cells (B) CD4+ T cells
(C) CD8+ B cells (D) CD4+ B cells
17. Porogamy is
(A) Fertilization in which pollen tube enters the ovule through integument
(B) Fertilization without pollen grain
(C) Fertilization in which pollen tube enters the ovule through chalaza
(D) Fertilization in which pollen tube enters the ovule through micropyle
18. Arrange the following electron acceptors in the proper order in which they participate in electron
transport.
1 = Cytochrome c
2 = Oxygen
3 = Cytochrome c oxidase
(A) 1, 2, 3 (B) 1, 3, 2
(C) 2, 3, 1 (D) 3, 1, 2
19. Which of the following is a true statement regarding gene regulation in prokaryotes ?
(A) Nuclear DNA may be modified prior to transcription
(B) RNA is modified in the nucleus before translation
(C) Repressors may act to turn off operons in both inducible and repressible operons
(D) Genes are likely “on” when histone proteins are methylated.
20. An obligatory association between two different species that is beneficial to both populations of
organisms is
(A) parasitic (B) predatory
(C) symbiotic (D) mutualistic
Biotechnology (MSP)
21. In the following compounds,

O
N N N N
| | |
H H H
I II III IV
the order of basicity is:
(A) IV > I > III > II (B) III > I > IV > II
(C) II > I > III > IV (D) I > III > II > IV
22. Surfactants and detergents have the same common property of......in them.
(A) Detergency (B) Surface activity
(C) Viscosity (D) None of these
23. The correct de Broglie relationship is:

 h
(A) = p (B)  =
mv mv
h v
(C)  = (D)  m =
mp p
24. If K1 and K2 are the respective equilibrium constants for the two reactions,
XeF6 (g) + H2O(g)  XeOF4(g) + 2HF(g)
XeO4 (g) + XeF6(g)  XeOF4(g) + XeO3F2(g)
The equilibrium constant for the reaction, XeO4 (g) + 2HF(g)  XeO3 F2(g) + H2O(g) is:
(A) K1 K 2 (B) K1/K22
(C) K2/K1 (D) K1/K2
25. A railway engine and a car are moving on parallel tracks in opposite directions with speed of 144
kmh–1 and 72 kmh–1, respectively. The engine is continuously sounding a whistle of frequency 500
Hz. The velocity of sound is 340 ms–1. Calculate the frequency of sound heard in the car when
the car and the engine are approaching each other.
(A) 200 (B) 400
(C) 600 (D) 800
26. A semiconductor has equal electron and hole concentration of 6 × 108 m–3. On doping with certain
impurity, electron concentration increases to 9 × 1012 m–3. Calculate the new hole concentration.
(A) 4 × 104 m–3 (B) 2 × 102 m–3
(C) 1 × 101 m–3 (D) none
27. The radius of a wheel of a car is 0.4 m. The car is accelerated from rest by an angular
acceleration of 1.5 rad s–2 for 20 s. How much distance the wheel covers in this time interval and
what will be its linear velocity?
(A) 300 (B) 120
(C) 200 (D) 150
Biotechnology (MSP)
28. The harmonic mean of 3, 7, 8, 10, 14 is
3  7  8  10  14 1 1 1 1 1
(A) (B)    
5 3 7 8 10 14
1 1 1 1 1
    5
(C) 3 7 8 10 14 (D)
5 1 1 1 1 1
   
3 7 8 10 14
29. If In =  xn e-x dx for n  N, then In – n In–1 =

0
(A) e (B) 1/e

(C) – 1/e (D) None of these
30. In an arranged discrete series in which total number observations ‘n’ is even, median is
n
(A) th item
2
n 
(B)  2  1 th item
 
n n 
(C) the mean of th and   1 th item
2 2 
(D) none of these
SECTION-(B) MULTIPLE SELECT QUESTIONS (MSQ)
1. Choose the correct statement about G-proteins

(A) G proteins all stay active for the same amount of time
(B) G proteins bind to and change the conformation of target proteins
(C) G proteins change their own conformation when they bind GTP
(D) G proteins change their own conformation when they hydrolyze GTP
2. All of the following are true EXCEPT

(A) An epitope is a small portion of a macromolecule
(B) The variable region domains contain the antigen recognition site
(C) The class of an immunoglobulin is determined by its light chain
(D) An IgG antibody is trivalent
3. The culture fluid of 1000 to 5000 colonies of hybridoma are screened for monoclonal antibody by
(A) Antigen capture analysis (B) Western blot analysis
(C) Northern blot analysis (D) Antibody capture analysis

Biotechnology (MSP)
4. Gram positive cells____

(A) Have thin, homogeneous cell walls
(B) Glycerol teichoic acids present in cell wall
(C) The peptidoglycan (also known as murein) layer constitutes almost 95% of the cell wall
(D) None of these
5. 1 volt coulomb are :

(A) Equal to 1 joule (B) An unit of energy
(C) Equal to 1 joule/coulomb (D) Equal to 107 erg
6. For the reaction,

PCI5 (g)  PCI3 (g) + CI2 (g)
the forward reaction at constant temperature is favoured by:
(A) Introducing an inert gas at constant volume
(B) Introducing an inert gas at constant pressure
(C) Increasing the volume of the container
(D) Introducing PCI5 at constant volume
7. The moment of inertia of a thin square plate ABCD of uniform thickness about an axis passing
through the centre O and perpendicular to the plate is
4
A B
3
0
D C
2
(A) I1 + I2 (B) I3 + I4
(C) I1 + I3 (D) I1 + I2 + I3 + I4
8. A parallel plate capacitor is charged and the charging battery is then disconnected. If the plates
of the capacitor are moved farther apart by means of insulating handles,
(A) The charge on the capacitor increases
(B) The voltage across the plates increases
(C) The capacitance increases
(D) The electrostatic energy stored in the capacitor increases
9. If  and  are non-real cube roots of unity and x = a + b, y = a  + b , z = a  + b , then

(A) x + y + z = 1 (B) x + y + z = 0
3 3 3 3 3
(C) x + y + z = 3(a + b ) (D) None of these
10. If k R and the inequality

x2 – 2(4k – 1) x + 15 k2 – 2k – 7 > 0
holds true for all x  R, then
(A) 2 < k < 4 (B) (k – 2) (k – 4) < 0
(C) k < 2 or k > 4 (D) None of these

Biotechnology (MSP)
SECTION-(C) NUMERICAL ANSWER TYPE QUESTIONS (NAT)
1. The diploid no. of an organism is 45. How many chromosomes would be in trisomy condition_____
2. Phenotypes of wild type (RY) red flowers is 180 and 20 (ry) white flower. Calculate the frequency
of R (dominant )allele______%.
3. Increasing rate of substrate concentration to increases rate of reaction from 10 % of Vmax to 80

% of Vmax. Calculate the change in V will be_____
4. The number of E.coli increased from 102 cells/ml to108 cells/ml. Calculate the number of generation
_______
5. With how many molecules of acetic anhydride does one molecule of glucose react?
6. The number of S—S bonds present in trimer of SO3(S3O9) are :
7. Displacement is given by x = 1 + 2t + 3t2. Find the value of instantaneous acceleration.
8. A bend in a level road has a radius of 100 metres. Find the maximum speed which a car turning
this bend may have without skidding, if the coefficient of friction between the tyres and road is
0.8.
9. Three of the six vertices of a regular hexagon are chosen at random. The probability that the
triangle with these vertices is equilateral is ______.
10. Coefficient of x in the expansion of (1 + 3x + 8x2)10 is equal to _____.
11. You have 10 ng of a 2.0 kb plasmid. How many copies (106 copies) of your plasmid do you have?
12. The no. of germ line genes for heavy & light chain in an individual. Calculate the approximate
number of diverse IgG [kappa] molecules.
Germ line genes Heavy chain K chain
V 10 20
D 30 –
J 5 4
13. Enzyme and substrate interact each other if Km= 3.0 × 10–5 M, Kcat= 6.0 × 102 sec–1. Calculate
the specificity constants _____ 107 M–1 sec–1
14. What would be the equilibrium potential for the ion Na+ if [Na+]out = 10 mM and [Na+]in = 200mM
?

Biotechnology (MSP)
15. The interatomic distances in H2 and CI2 molecules are 74 and 198 pm respectively. The bond
length of HCI is :
16. For, 2C6H6(l) + 15O2 (g)  12CO2 (g) + 6H2O(g) the difference in H and E values are at 25°C
(kilojoule) :
17. Two small charged spheres A and B have charges 10 C and 40 C respectively, and are held
at a separation of 90 cm from each other. At what distance from A, electric instensity would be
zero?
18. A tuning fork of frequency 200 Hz is in unison with a sonometer wire. How many beats per
second will be heard if the tension of the wire were increased by 2%?
19. The 8th term of the sequence 1, 1, 2, 4, 7, 13, 24, ..... is ______.
20. The sum of an infinite number of G.P. is 20, and the sum of their squares is 100. The first term
of the G.P. is ______.

Biotechnology (MSP)
ANSWER KEY
1 2 3 4 5 6 7 8 9 10
B B C D D B C C D A
11 12 13 14 15 16 17 18 19 20
C C D C A A D B C D
21 22 23 24 25 26 27 28 29 30
D B B C C A B D C C

1 2 3 4 5 6 7 8 9 10
B,C,D C,D A,D B,C A,B,D B,C,D A,B,C B,D B,C A,B
1 2 3 4 5 6 7 8 9 10
46 90 70 19.93 5 0 6 28 0.1 30
11 12 13 14 15 16 17 18 19 20
4632 120,000 2.0 – 79.3 136 + 7.43 30 2 44 8

Biotechnology (MSP)
SOLUTION
1. (B) The fluid mosaic model was first proposed by S.J. Singer and Garth L. Nicolson in 1972
to explain the structure of the plasma membrane. Layers of the plasma membrane have
the hydrophilic heads pointing toward the outside; the hydrophobic tails form the inside of
the bilayer.
2. (B) Polysome, Polyribosomes (or polysomes) also known as ergosomes are a cluster of
ribosomes, bound to a mRNA molecule, first discovered and characterized by Jonathan
Warner, Paul Knopf, and Alex Rich in 1963.
3. (C) Formation of spindle fibres in zygotene, In G1 phase protein and RNA synthesis was done.
Action of endonuclease in pachytene phase and Movement of chromatids towards opposite
poles in Anaphase - II.
4. (D) Type O blood group is universal donor. Group O blood can be donated to anybody.
5. (D) Excessive growth of hair on the pinna is a feature found only in males because The gene
responsible for the character is present on the Y chromosome only.
NaOH(aq.) Al2O3
6. (B) CH3CH2CH2Br   CH3CH2CH2OH 
 CH3CH = CH2
HOCl
  CH3—CHCH2Cl
|
OH
7. (C)  qabs = U + (–w)
 U = q + w; U is state function.
8. (C) Initial energy stored in the capacitor,
Q2
UC 
2C
When the energy is stored equally between electric and magnetic fields,
1 Q 2 1 Q 2
U’C = U or 
2 C 2C 2 2C
Q
or Q’ = .
2
9. (D) Coefficient of performance of a refrigerator,
Q2 T2
 
W T1  T2
T2
 Q2  W 
T1  T2
But W = Energy consumed by the refrigerator = 1 J, T1 = 300 K, T2 = 277 K
277 277
 Q2 = 1 ×  = 12 J
300  277 23
Heat rejected by the refrigerator,
Q1 = W + Q2 = 1 + 12 = 13 J.

Biotechnology (MSP)
10. (A) Required plane is

x3 y6 z4
3  3 2  6 0  4  0.
1 5 4
11. (C) Calcium deficiency in the body can be found due to absence of vitamin D. Calcification
of bones before epiphyseal closure in immature mammals due to deficiency or impaired
metabolism of vitamin D. Lack of adequate calcium in the diet may also lead to rickets.
12. (C) If healthy sperm get beyond the cervix into the uterus and up to the fallopian tubes, they
can live as long as 5-7 days after ejaculation. On average, though, sperm live about
3-4 days once they’ve made it all the way to the fallopian tubes, where fertilization takes
place.
13. (D) Formation of DNA to DNA is called replication, then transcription is the first step of gene
expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA
polymerase. In translation, messenger RNA (mRNA) produced by transcription from DNA
is decoded by a ribosome to produce a specific amino acid chain, or polypeptide.
14. (C) Anaphase I begins when the two chromosomes of each bivalent (tetrad) separate and
start moving toward opposite poles of the cell as a result of the action of the spindle.
15. (A) IgG is the most common type of antibody found in the circulation. IgG molecules are
created and released by plasma B cells. IgG immunoglobulins is present normally in
plasma at the highest concentration.
16. (A) T cells cannot recognize, and therefore cannot respond to, 'free' antigen. T cells can only
'see' an antigen that has been processed and presented by cells via carrier molecules like
MHC and CD1 molecules. Most cells in the body can present antigen to CD8+ T cells via
MHC class I molecules and, thus, act as "APCs"; however, the term is often limited to
specialized cells that can prime T cells
17. (D) The usual route by which a pollen tube enters an ovule the micropyle in the fertilization
of angiospermous and gymnospermous plants. When the pollen tube penetrates the ovule
by a different route, the process is called a porogamy (for example, penetration of the
chalaza is known as chalazogamy).
18. (B) Electron acceptors in the proper order such cytochrome c than cytochrome c oxidase and
last is oxygen, they all participate in electron transport.
19. (C) Prokaryotes do not contain a nucleus and are generally thought to not contain histone
proteins. Both inducible and repressible operons can work with repressor proteins. Inducible
operons contain repressors that are active by default. Repressible operons contain
repressors that are inactive by default.

Biotechnology (MSP)
20. (D) Mutualism is the way two organisms of different species exist in a relationship in which
each individual benefits from the activity of the other. Similar interactions within a species
are known as co-operation. Mutualism can be contrasted with interspecific competition.
21. (D) In (II) and (IV) lone pair is involved in resonance.
22. (B) Both surfactants and detergents possess the surface activity, i.e., the tendency to lower
surface tension of water. A surfactant also having cleansing action, i.e., detergency in
addition to surface activity is called detergent.
h
23. (B) de Broglie equation is  = .
mv
[XeOF4 ][HF]2
24. (C) K1 =
[XeF6 ][H2O]
[XeOF4 ][XeO3F2 ]
K2 = [XeO4 ][XeF6 ]
K 2 [XeO3F2 ][H2O]
By Equation (ii)/(i) we have  = Kc.
K1 [XeO4 ][HF]2
25. (C) Here vs = 144 km h–1
144  1000
= = 40 ms–1
3600
72  1000
and v0 = 72 km h–1 = = 20 ms–1
3600
v = 500 Hz, v = 340 ms–1
When the car and the engine approach each other,
vs = + 40 ms–1, v0 = –20 ms–1
+ve vs –ve v0
S O
Engine Car
v  v0 340  20
v’ = v  v  v  340  40 × 500
s
360
= × 500 = 600 Hz.
300
26. (A) As ne nh = ni2
ni2 (6  108 )2
 nh    4  104 m3 .
ne 9  1012
27. (B) Here r = 0.4 m, w0 = 0,

 = 1.5 rad s , –2
t = 20 s
Angular displacement,
1
 = 0 t +  t2
2
Biotechnology (MSP)
1
= 0 + × 1.5 × (20)2 = 300 rad
2
Distance covered by the wheel,
s = r = 0.4 × 300
= 120 m.
28. (D) Required harmonic mean
n 5
 .
 1 111 1  1
 
 xi  3 7 8 10 14
I I
n -x 1
29. (C) In =  x e dx = -x n e-x  + x  xn-1 e-x dx
0
I II  0 0
= – e-1 + n In-1
 In – n In-1 = –1/e.
30. (C) When n is even and data is arranged either in ascending or indecending order, then
1 n n  
median =  th item    1 th item 
2 2 2  
1. (B,C,D)
G-proteins are bind and change the conformation of target proteins. G proteins change
their own conformation when they bind GTP or hydrolyze GTP.
2. (C,D) An IgG antibody is bivalent not trivalent. The class of an immunoglobulin is determined by
its heavy chain.
3. (A,D) The culture fluid of 1000 to 5000 colonies of hybridoma are screened for monoclonal
antibody by Antigen capture analysis and antibody capture analysis. A capture antibody is
immobilized to often prove to be less than ideal for hybridoma screening. The screening
methods involved capturing antibodies from crude supernatants using Fc-specific antibody
surfaces and monitoring antigen binding at a single concentration. After normalizing the
antigen responses for the amount of antibody present, a simple interaction model was fit
to all of the binding responses simultaneously.
4. (B,C) Gram positive cell wall have thin, homogeneous cell walls with glycerol teichoic acids. The
peptidoglycan (also known as murein) layer constitutes almost 95% of the cell wall.
5. (A,B,D)
Passing through the cell. Since the EMF is measured in volts and if the quantity of current
is measured in coulomb, the practical unit of electrical energy is, therefore, defined as the
energy developed when one coulomb is passed through a circuit by an EMF of one volt;
7
this unit is called volt coulomb and absolute volt coulomb is equal to 10 ergs or one joule.
6. (B,C,D)
Dissociation of phosphorus pentachloride:
PCI5 (g)  PCI3 (g) + CI2 (g)
Favourable conditions for forward reaction are :
Biotechnology (MSP)
(i) low pressure

(ii) high temperature and
(iii) excess of PCl5
(iv) Introducing an inert gas at constant pressure
Continuous removal of gaseous product favors formation of more products.
7. (A,B,C)
According to theorem of perpendicular axis
I = I1 + I2 and I = I3 + I4
As I1 = I3       I = I1 + I3.
8. (B,D)
+q –q +q –q
d0 d > d0
charge on plate = q charge on plate = q
0 A 0 A
C0 = d C =  C < C0
0 d
q q
V0 = V=  V > V0
c c
1 1
U0 = qV0 U = qV  U > U0
2 2
 Option (b) and (d) are correct.
9. (B,C) Here, x + y + z
= a + b + a + b + a + b
= a(1 +  + ) + bc (1 +  + ) = 0
 x + y + z3 = 3 xyz
3 3
= 3(a + b) (a + b) (a + b)

= 3(a3 + b3)
10. (A,B) x2 – 2 (4k – 1) x + 15k2 – 2k – 7 > 0
for all x  R
 (2(4k – 1))2 – 4(15k2 – 2 k – 7) < 0
 (4k – 1)2 – (15k2 – 2k – 7) < 0
 k2 – 6k + 8 < 0
 (k – 2) (k – 4) < 0 2 < k < 4.
1. 46 In trisomy are those which have an extra chromosome than diploid & they are represented
as 2n + 1. So in trisomy condition 45 + 1 = 46 chromosomes will be there.
2. 90 20 of 200 chromosomes in this sample carry recessive allele r
q = 20/200 = 0.1 = 10% r allele
p = 1 – q = 1 – 0.1 = 0.9 or 90 % R alleles

Biotechnology (MSP)
Vm [s]
3. 70 V=
k m + [s]
Change in V will be (V’ – V)

v = 80/100 Vm, v = 10/100 Vm
Change in V will be = 70
4. 19.93 N = No2n
log N = log No + nlog2
n = (log N – logNo)/log2 = (8-2)/0.301 = 19.93 or 20 generations
5. 5 Glucose has penta hydroxy nature. CH3COCI attacks at —OH gp.
so one molecule of glucose react with 5 molecules of acetic anhydride.
6. 0 S3O9 has the structure,
O O
S O
O
O S
O
S O
O O
7. 6 Instantaneous velocity,
dx
v= = 0 + 2 + 6 t = 3 + 6 t
dt
dv
Instantaneous acceleration, a = = 6 unit.
dt
8. 28 Here r = 100 m,  = 0.8, g = 9.8 ms–2
The car will not skid if the force of friction provides the necessary centripetal force.
2
mv max
 f = mg =
r
or vmax = rg  0.8  100  9.8 = 28 ms–1.

9. 0.1 Three vertices can be selected in 6C3 = 20 ways and in only two cases, we get an
equilateral triangle.
E D
F C
A B
2 10
10. 30 Now, (1 + 3x + 8x )
= {1 + (3x + 8x2)}10
10 10
= C0 + C1 (3x + 8x2) + 10
C2 (3x + 8x2)2 + .... + 10
C10 (3x + 8x2)10
 Coefficient of
10
x= C13 = 10 × 3 = 30.
Biotechnology (MSP)
11. 4632 Number of copies = (ng * 6.022 × 1023)/ (Length *1 × 109 × 650)
 ng is the amount of DNA (plasmid, primer etc.) you have in nanograms
 6.022 × 1023 = Avogadro's number
 length is the length of your DNA fragment in base pairs. Just multiply by 1000 if
you are working in kb.
 We multiply by 1 × 109 to convert our answer to nanograms
Calculation : Number of copies = (10*6.022 × 1023)/(2,000*1 × 109*650)
= (6.022 × 1024) / (2000 × 109 × 650)
= (6.022 × 1024) / (130 × 1013)
= 4632 × 106
12. 120,000
Diverse heavy chain = V × D × J
= 10 × 30 × 5 = 1500
Diverse light chain = 20 × 4 = 80
Different IgG molecule = 1500 × 80 = 120,000
13. 2.0 Specificity constants = Kcat/Km (M–1 sec–1)
= 6.0 × 102 sec–1/3.0 × 10–5 M
= 2.0 × 107 M–1 sec–1
14. – 79.3 Ek = (61/z) × log ([X]out/[X]in) [z = 1]
= (61/1) log ([10mM] / [200mM])
= (61) × log [0.05]
= 61 × [–1.30] = – 79.3 mV
74 198
15. 136 Atomic radius of H + atomic radius of CI =  = 136 pm
2 2
16. + 7.43 H – E = nRT
Here; n = 18 – 15 = 3
= 3 × 0.00831 × 298
R = 0.00831 KJ mol–1
= 7.43 K joule
17. 30
EB EA
10C 40C
A P B
x 0.90 – x
At point P, EA = EB
1 10  10 6 1 40  106
or .  .
40 x2 40 (0.90  x)2
1 4
or 2

x (0.90  x)2
or 0.90 – x = 2x
or x = 0.30 m = 30 cm.

Biotechnology (MSP)
18. 2 Here v1 = 200 Hz

As the ension in the wire is increased by 2%, therefore if T1 = 100 units, then T2 = 102
units
1/ 2
v2 T2 102  2 
Now    1 
v1 T1 100  100 
1 2
 1 +  = 1.01
2 100
 v2 = 1.01 v1 = 1.01 × 200 = 202 Hz
Beat frequency = v2 – v1 = 202 – 200 = 2 Hz.
19. 44 Each term beginning with fourth is the sum of three previous terms i.e.
T4 = T1 + T2 + T3, T5 = T2 + T3 + T4,
T6 = T3 + T4 + T5, ....... etc.
so 7 + 13 + 24 = 44
20. 8 Let the G.P. be a + ar + ar2 + ...., | r | < 1
then a + ar + ar2 + .... = 20
a
 = 20 ...(1)
1- r
and a2 + a2r2 + a2r4 + .... = 100
a2
 = 100 ...(2)
1- r2
Dividing (1) by square of (2)
1- r 2
2 = 4
1- r 
1+ r 3
 = 4  r=
1- r 5
and then form (1),
2
a = 20 (1 – r) = 20 × = 8
5

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Eduncle.com For Counselling

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4. Cells can perform a variety of chemical reactions

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ii. Features of Eukaryotic Cells

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Fig. Features of Eukaryotic Cells

Table: Comparison of features of prokaryotic and eukaryotic cells

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1.4 Membrane potential

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2. CYTOSKELETAL ELEMENTS: CYTOSKELETON & MOTOR PROTEINS

Cytoskeletal Element Description (diameter) Protein Composition

Fig. : Structure myosin

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Fig. : Size and shape of the myosin molecule

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Fig. Kinesin structure

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Fig. structure of Cilia & flagella

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Fig. Ca++ is released from the sarcoplasmic reticulum.

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Fig. Sliding Filament Theory of Contraction

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Component of the sarcomere:

Muscle Fiber Contraction Muscle Fiber Relaxation

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Fig. Structure of mitochondria

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1. Those that perform the redox reactions of oxidative phosphorylation

Fig. : The electrochemical proton gradient and ATP synthase

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Fig. : Import of presequence proteins into the mitochondrial matrix

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ii. Smooth endoplasmic reticulum

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Fig. Translational targeting (secretory pathway)

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Fig. : Golgi Apparatus

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Fig. The Out/in bound Path

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Types Description Example

Fig. : Processing of N-linked oligosaccharides in the Golgi

Fig. : Targeting of lysosomal proteins by phosphorylation of mannose residues

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Fig. : a. Protein import into the chloroplast stroma.

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Fig b. Import of proteins into the thylakoid lumen

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Duration : 180 minutes Maximum Marks : 100

Read the following instructions carefully.

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SECTION-(A) MULTIPLE CHOICE QUESTIONS (MCQ)

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