PRINCIPLE OF GENE

MANIPULATION
Assoc. Prof. Dr. Asmah Awal
Faculty of Plantation & Agrotechnology
asmah138@uitm.edu.my
Principles of Gene Manipulation
1. Biology of Cell – Structure and Organization
2. Basic Molecular Biology – DNA, RNA,
Proteins, Enzymes – Structure and Function
3. DNA Replication, Mitosis and Meiosis
4. Gene Expression – Transcription,
Translation

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 Cells : The foundation of life

 The cell is the smallest form of life.

 All life forms are made from one or more cells.
 Cells only arise from pre-existing cells.

Types of Cells

Prokaryotic cell
(genetic material is not
Eukaryotic cell
confined to nucleus)
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 Tissue

 Composed of cells

 Serves a particular function

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 Prokaryotic Cell
 appeared 3.5 billion years ago
 one cell organisms; smallest of all cells
 genetic material (DNA, deoxyribonucleic acid) is not confined to a
nucleus; in contact with the cytoplasm; have circular DNA (plasmid)
 Kingdom : Monera (simple bacteria or Eubacteria) and Archaea
 surrounded by a cell membrane (proteins and lipids), some have cell wall
 may have photosynthetic pigments, e.g. cyanobacteria (blue green algae)
 Some have external whip-like flagella for locomotion or hair like pili for
adhesion
 multiple shapes: cocci (round), baccilli (rods), and spirilla or spirochetes
(helical cells)
 important in biotechnology

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 Eukaryotic Cell

 evolved 1.5 billion years ago

 has a nucleus : serves as the control center for all activities;
contains genetic material, DNA
 Kingdom : Protista, Plantae, Fungi, Animalia
 plasma membrane (proteins and lipids), some have cell wall
 glycocalyx (components external to the plasma membrane)
 cytoskeleton - microfilaments and microtubules that suspend
organelles, give shape, and allow motion
 presence of membrane enclosed subcellular organelles that
perform specialized functions within the cell

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Eukaryotic Cell Composition

Cell membrane/plasma membrane

& cell wall (e.g. plant and fungi cell)

Organelles

Cytoplasm : watery cellular substance

outside the nucleus in which the cell's
organelles are suspended

Cellular molecules

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 Cell membrane allows diffusion and osmosis processes

diffusion
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 Cell wall enclosing a
plant cell

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 Organelles in a plant cell

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  Mitochondria : for respiration/production of ATP
 Vacuole : store water, nutrients, enzymes and waste materials
Organelles in eukaryotic cell
 Microtubules : act as the bones of the cell, assist the movement
of chromosomes during cell division
 Microfilaments : help the cell to move by waving back and forth
 Ribosomes : synthesis of protein molecules
 Golgi apparatus : remove water from the protein and prepare
them for export from the cell
 Endoplasmic reticulum : for transporting materials throughout the
cell
 Chloroplasts : photosynthesis
 Leucoplasts : storage of starches, proteins or lipids; abundant in
seeds
 Chromoplasts : manufacture pigments that give fruits their colour
and give leaves their colour in fall

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 Cellular molecule

Small cellular molecules Macromolecules

( synthesized from the small molecules)

Simple Fatty Amino polysaccharides proteins Nucleic

sugars acids acids acids
nucleotides
lipids

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 Protein
 three-dimentional structure
 20 monomers that can be polymerized in any order, size and
shape, and consequently, in function
 part of cellular component, substrates in cellular metabolism,
enzyme, may regulate the expression of other proteins
 a ligand (molecule) must fit exactly into a specific region on the
protein called its binding site
 enzymes are special proteins that catalyze chemical reactions;
form enzyme-substrate complex (lock-and-key model)

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 Protein Structure

primary structure : polymers of amino acids are synthesized through

repeated dehydration reactions (carboxyl group of one amino acid is
linked to the amino group of the next as peptide bond, with the loss of
water)

secondary structure : undergone folding; make up of most globular

protein

tertiary structure : results from the folding of secondary structures

quaternary structure ; consists of aggregates of tertiary structures

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 Nucleotide
(macro molecules : Nucleic Acids)

pentose sugar
nucleoside
nitrogenous base

Purines
(adenine, A & guanine, G)

Pyrimidines
(cytosine, C, thymine, T & uracil, U)

phosphate group

 adenosine triphosphate (ATP) & guanosine triphosphate (GTP) are

important in cellular bioenergetics; when hydrolyzed, inorganic phosphate
is produced accompanied by the release of energy
 2 types : deoxyribonucleic acid (DNA) & Ribonucleic acid (RNA)
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 DNA
 pentose sugar : deoxyribose

 nitrogenous base : purines (adenine, A & guanine, G) &

pyrimidines (cytosine, C & thymine, T)

 A pairs with T, G pairs with C

 hereditary / genetic material

 the structure of the DNA molecule is a double helix : 2

polynucleotide chains coil around a central axis in a spiral fashion,
the nucleotide chains are antiparallel with one chain runs in the 5’ to
3’ orientation and the other 3’ to 5’

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 Nitrogenous bases

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 RNA

 pentose sugar : ribose

 nitrogenous base : purines (adenine, A & guanine, G) &
pyrimidines (cytosine, C & uracil, U)
 most RNA is predominantly single-stranded
 certain animal and plant viruses use RNA as their
genetic material
 functions : expression of the genetic
information

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 RNA
messenger RNA (mRNA) :
molecular carrier of genetic information
from the DNA to ribosomes; serves
as a template for protein synthesis (transcription)

ribosomal RNA (rRNA) :

RNA found in the ribosomes of cells
transfer RNA (tRNA) :
have structures with triplet nucleotide sequences (anticodon)
that are complementary to the triplet nucleotide
coding sequences of mRNA (codon). The role of tRNAs
in protein synthesis is to bond with amino acids
and transfer them to the ribosomes, where proteins
are assembled according to the genetic code
carried by mRNA (translation)
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Taxonomy of Living Things (5 Major Kingdoms)

 Monera

 Protista

 Fungi

 Plantae

 Animalia

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 Taxonomy of Living Things – cont.

Monera
 prokaryotic cell
 unicellular organisms
 bacteria belong here
 bacteria are critical to genetic engineering technologies
as they provide enzymes (restriction endonucleases)
and plasmids (circular DNA molecules) for cloning
 bacteria as biorectors for making pharmaceutical
products

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 Bacteria come in a
wide variety of sizes
and shapes

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 Taxonomy of Living Things – cont.

Protista
 eukaryotes that do not fit other kingdoms
 most are unicellular organisms
 slime molds (left), algae (right)

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 Taxonomy of Living Things – cont.

Fungi
 filamentous eukaryotes that lack plastids
and photosynthetic pigments

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 Taxonomy of Living Things – cont.

Plantae
 Photosynthetic organisms
 Multicellular organisms
 have cell walls

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 Taxonomy of Living Things – cont.

Animalia
 Multicellular organisms
 non-photosynthetic organisms
 have complex sensory and neuromotor systems

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Cells are also defined according the need for
energy :

 Autotrophs : "self feeders" that use light or chemical

energy to make food, e.g. plants
 Heterotrophs : “other feeders”, obtain energy from other
autotrophs or heterotrophs. Many bacteria and animals
are heterotrophs.

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 Cell Reproduction

 Mitosis : growth & cell replacement

 Meiosis : division into cells that contain one-half of
chromosomes needed for the formation of the new life /
sexual propagation; generates genetic diversity

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 Mitosis

 starts with DNA replication : constitutes the fundamental condition

for biological growth (cell division).

 DNA replication enables each daughter cell receives an exact copy

of the dividing cell's DNA.

 cells have evolved mechanisms to correct mistakes that sometimes

occur during DNA replication, a DNA repair system. Abnormalities in
these processes, copying and repairing, result in a failure of
accurate replication and maintenance of DNA - a failure that can
have disastrous consequences, such as the development of cancer.

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 DNA Replication

 a portion of the double helix is unwound by a helicase.

 enzymes & proteins catalyze the replication of DNA. A molecule of

DNA polymerase binds to one strand of the DNA and begins moving
along it in the 3' to 5' direction, using it as a template for assembling a
leading strand of nucleotides and reforming a double helix. In
eukaryotes, this molecule is called DNA polymerase delta (δ).

 as DNA synthesis can only occur 3' to 5', a molecule of a second type
of DNA polymerase (epsilon, ε, in eukaryotes) binds to the other
template strand as the double helix opens. This molecule must
synthesize discontinuous segments of polynucleotides (called
Okazaki fragments). Another enzyme, DNA ligase then stitches
these together into the lagging strand.

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DNA
replication

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 Interphase
 DNA replication precedes the start
of mitosis.

 The cell is engaged in metabolic

activity and performing its prepare
for mitosis.

 Chromosomes are not clearly

discerned in the nucleus.
 The cell may contain a pair of
centrioles (or microtubule
organizing centers in plants).

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 Prophase

 Chromatin in the nucleus begins to

condense and becomes visible in the
light microscope as chromosomes.

 The nucleolus disappears.

 Centrioles begin moving to opposite

ends of the cell and fibers extend
from the centromeres.

 Some fibers cross the cell to form the

mitotic spindle.

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 Prometaphase

 The nuclear membrane dissolves,

marking the beginning of
prometaphase.
 Proteins attach to the centromeres
creating the kinetochores.
 Microtubules attach to the
kinetochores
 The chromosomes begin moving.

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 Metaphase

 Spindle fibers align the chromosomes

along the middle of the cell nucleus.

 This line is referred to as the

metaphase plate/equator.

 This organization helps to ensure that

in the next phase, when the
chromosomes are separated, each
new nucleus will receive one copy of
each chromosome.

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 Anaphase

 The paired chromosomes separate at

the kinetochores and move to
opposite sides of the cell.

 Motion results from a combination of

kinetochore movement along the
spindle microtubules and through the
physical interaction of polar
microtubules.

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 Telophase
 Chromatids arrive at opposite poles
of cell

 New membranes form around the

daughter nuclei.

 The chromosomes disperse and are

no longer visible under the light
microscope.

 The spindle fibers disperse

 Cytokinesis or the partitioning of the

cell may also begin during this stage.

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 Cytokinesis

 In animal cells, cytokinesis results

when a fiber ring composed of a
protein called actin around the center
of the cell contracts pinching the cell
into two daughter cells, each with one
nucleus.

 In plant cells, the rigid wall requires

that a cell plate be synthesized
between the two daughter cells.

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 Meiosis

meiosis 1 Meiosis 2
(same as mitosis)

(In meiosis 1, chromosomes in a diploid cell resegregate,

producing four haploid daughter cells. It is this step in
meiosis that generates genetic diversity)

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 Haploid cell

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 Prophase 1  DNA replication precedes the start
of meiosis I.

 During prophase I, homologous

chromosomes (chromatid) pair and
form synapses, a step unique to
meiosis.

 The paired chromosomes are

called bivalents

 Formation of chiasmata caused by

genetic recombination.

 Chromosomal condensation can be

viewed in the microscope.
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Prometaphase 1

 The nuclear membrane disappears.

 The chromosomes attach to
spindle fibers begin to move.

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 Metaphase 1

 Bivalents, each composed of two

chromosomes (four chromatids)
align at the metaphase
plate/equator.

 The orientation is random, with

either parental homologue on a
side.

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 Anaphase 1

 Chromosomes, each with two

chromatids, move to separate
poles.

 Each of the daughter cells is now

haploid, but each chromosome has
two chromatids.

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 Telophase 1

 Nuclear envelopes may reform, or

the cell may quickly start meiosis 2.

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 Meiosis 2

 analogous to mitosis

 last phase of meiosis 2 is Cytokinesis

 four haploid daughter cells are formed.

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 Comparing Meiosis and Mitosis
 Chromosome behavior
 Mitosis: Homologous chromosomes independent
 Meiosis: Homologous chromosomes pair forming bivalents until
anaphase I, random orientation of chromosomes at metaphase I
 Chromosome number
 mitosis- identical daughter cells
 meiosis- chromosome number of daughter cell is halved
 Genetic identity of progeny:
 Mitosis: identical daughter cells
 Meiosis: daughter cells have new assortment of parental
chromosomes; chromatids not identical due to crossing over (formation
of chiasmata)
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 Comparing Meiosis and Mitosis – cont.

 Mitosis can proceed independent of ploidy of cell, homologous

chromosomes behave independently
 Meiosis can only proceed if the nucleus contains an even number of
ploidy (diploid, tetraploid).

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 Central Dogma of Molecular Biology

 The DNA is situated in the nucleus, organized into chromosomes.

DNA is duplicated (DNA replication) before a cell divides / mitosis.
The transfer of DNA is accomplished by several types of ribonucleic
acid (RNA) that carries genetic information needed to form protein in
the cell. Proteins perform important tasks for the cell functions or
serve as building blocks.

 the flow of genetic information where DNA is transcribed into mRNA

and mRNA is translated into protein, and the protein then forms the
organism is known as the central dogma of molecular biology.

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 Central Dogma of Molecular Biology – cont.

 DNA carries the genetic information of a cell and consists of

thousands of genes. Each gene serves as a recipe on how to build
a protein molecule. When proteins are needed, a discrete segment
of DNA called a gene is a template for the synthesis of a
complementary RNA. The corresponding genes are transcribed into
RNA (transcription).

 The RNA is then processed so that non-coding parts are removed

(processing) and is then transported out of the nucleus (transport).

 Outside the nucleus, the proteins are built in ribosomes based upon
the code in the RNA (translation).

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 Processing

translation

Central Dogma of Molecular Biology

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 RNA Transcription

In all eucaryotic cells DNA never leaves the nucleus, instead the
genetic code (the genes) is copied into RNA which then in turn is
decoded (translated) into proteins in the cytoplasm. Why ?

 The cytoplasm is a dangerous environment for the DNA, which has

to stay intact in order to maintain life. Therefore, RNA works as a
sort of throw-away version of DNA - good for limited work but not
for long-term storage.
 Regulation the rate of protein synthesis.

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 RNA Processing
A newly made mRNA (pre-mRNA) must undergo three major
modifications before it can leave the nucleus and be translated into a
protein :
1) Capping - a special nucleotide is attached at the 5' end of the mRNA. It
is believed that this modification is necessary for efficient initiation of
protein synthesis and serves as stabilisation.
2) Poly(A)-tail - a special enzyme attaches a chain of 150-200 adenine
nucleotides to the 3' end of the pre-mRNA directly after transcription.
This is primarily believed to increase the stability and therefore prolong
the lifetime of an mRNA molecule.
3) Splicing - The removal of noncoding sequences, introns, from the pre-
mRNA to create mRNA, containing only the coding sequences, exons,
for a protein. This process is carried out by the splicosome.

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 Processing : non-coding sequences (introns) are removed

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 Translation

 Translation is the final step on the way from DNA to protein. It is the
synthesis of proteins directed by a mRNA template.
 The information contained in the nucleotide sequence of the mRNA
is read as three letter words (triplets), called codons. Each word
stands for one amino acid.
 the tRNA, a specialised RNA molecule that carries an amino acid at
one end and has a triplet of nucleotides, an anticodon, at the other
end. The anticodon of a tRNA molecule can basepair, i.e form
chemical bonds, with the mRNA's three letter codon. Thus the tRNA
acts as the translator between mRNA and protein by bringing the
specific amino acid coded for by the mRNA codon.

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 translation

mRNA

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 Protein synthesis

 Protein synthesis is carried out in the ribosome. Ribosome consists

of two different subunits, one small and one large and is built up
from rRNA and proteins.
 Inside the ribosome the amino acids are then linked together
through multiple biochemical reactions to form a polypeptide chain
which will later be folded into a protein.

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 ONLINE LECTURE

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