UNIT I

DNA AS A GENETIC MATERIAL

The genetic material of any organism is the substance that carries information for
properties of organism and transferring genetic information from parent to progeny.

i. The Transforming Principle

It was done by Frederich Griffith in 1928, who was studying the bacterium responsible
for human pneumonia, Streptococcus pneumoniae. These capsulated bacteria produce smooth
edged colonies on sugar surface and it was lethal (virulent). Griffith isolated a rough edged
colonies which was non capsulated and non lethal (avirulent), but the mixture of both is lethal.
 A mouse was injected with a sample of virulent, smooth bacteria; as expected the
mouse contracted pneumonia.
 A second mouse was injected with avirulent, rough bacteria; again the expected results
were obtained and the mouse remained healthy.
 Next a sample of virulent, smooth bacteria were killed by heating at 60° for 3 hours and
then injected into the mouse, the results were the animal remained healthy. Again this was the
expected result because; only the live bacteria can cause the disease.
 Finally a sample of heat killed smooth bacteria incapable of causing the disease, were
mixed with a live avirulent rough bacteria and the mixture was injected into a mouse, this
time a wholly unexpected results was obtained. Rather than remaining healthy, the mouse
contracted pneumonia. In addition from this animal large numbers of living virulent, smooth
bacteria were eventually isolated, even though the only living bacteria in the original
inoculumn were avirulent, rough forms.

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 From this experiment he concluded that a component of the heat killed smooth bacteria
transformed the avirulent rough cells, this new bacteria then caused pneumonia after injection
into the mouse. The component responsible for the transformation could not be the capsular
polysaccharide itself because the heat killed smooth cells would not yield enough material to
coat all the living cells subsequently isolated from the mouse. Instead the transforming
principle, when introduced into the rough bacteria, confers on these cells a new, permanent,
heritable characteristic - the ability to synthesize the capsular polysaccharides characteristic of
the smooth serotype. This transforming principle must therefore be genetic material.
Identifying the transforming principle and the nature of the genetic material would be known.

ii. Experiment of Oswald Avery, Colin MacLeod and Maclyn McCarty

Griffith did not himself attempt to identify the transforming principle. Instead, the work
was carried out by Oswald Avery, Colin MacLeod and Maclyn McCarty.

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There strategy was to prepare a filtrate from heat killed smooth cells, containing the
transforming principle, and then to digest the individual composition of this filtrate with
specific degradative enzymes. For instance, a protease could be used degrade all the protein in
the filtrate, and a ribonuclease to degrade the ribonucleic acid (RNA), the second type of
nucleic acid present in living cells. After enzymatic digestion of one or more components, the
filtrate was tested for retention of its transforming ability. Surprisingly treatment with
protease had no effect on the ability of the filtrate to transform rough cells into the smooth
form. Similarly ribonuclease treatment had no effect on the transforming activity. However,
digestion of the DNA with deoxyribonuclease totally destroyed the transforming ability so
that the filtrate was no longer able to convert one cell type into the other.

Gradually it becomes clear that the transforming principle must be DNA, but they
needed more evidence. McCarty therefore set out to purify the DNA from heat killed smooth
cells, and by the use of techniques such as electrophoresis, ultracentrifugation and ultraviolet
spectroscopy confirmed that the transforming principle.

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iii. The Hershey – Chase experiment or The Blender experiment

This carried out by Alfered Hershey and Martha Chase, they prepared a radioactive
sample of T2 phage, one in which the protein was labeled with 35S and the DNA with 32P, from
a T2 infected culture of E.coli that had been grown with 35S and 32P labeled nutrients. The
labeled phages produced by this culture were used to infect a new, non-radioactive culture of
E.coli. However this time the infection process was interrupted a few minutes after
inoculation by agitating the cells in a Waring blender. These few minutes were long enough
for the genes to enter the bacteria but not enough time for new bacteriophages to be
synthesized and the bacteria killed. They believed that agitation would remove the phage
material attached to the outside of the cell so that the only component retained by the bacteria
would be the injected genes, the phage genes.

The culture was then centrifuged so that the relatively heavy bacterial cells, containing
the phage genes, collected at the bottom of the tube, leaving the empty phage particles in
suspension. They discovered that over 80% of the 32P was present in the bacterial pellet. The
bacteria were then allowed to continue through the infection process and produce new phages.
Almost half of the 32P, but less than 1% of the 35S, was present in these new phage particles.
Clearly the genetic material was DNA and not protein.

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Modern Concept of Gene organization

Introduction

The concept of gene first came from the work of Mendel. Although the term ‘gene’ was not used
at that time, Mendel from his experiments on pea plants, suggested that the characters of an
organism are controlled by factors located within its body.

Flemming recorded certain deeply stained bodies within the cell, which he termed
‘chromosomes’. Meischer on the other hand, extracted ‘nuclein’ a nucleoprotein constituent from
the sperm. Afterwards, due to researches by subsequent workers (Kossel and others), an idea
gradually developed on the chemical structure of chromosome.

After the chemical analysis of chromosome carried out by Meischer and his successors,
chromosomes were found to consist chiefly of protein and nucleic acid. It was regarded as
continuous frame-work of basic protein or histone, having active regions located on DNA the
genes, the hereditary material.

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Concept of Gene – Classical Vs. Molecular:

The term gene was coined by Johannsen (1909) for the hereditary factors of Mendel. The
classical concept of gene is that it is a unit of function occupying a definite position on the
chromosome (not sub-divisible by recombination) and is responsible for a particular phenotypic
character.

According to Watson and Crick (1953), Wilkins (1962), gene was defined as a macromolecule
attached to undifferentiated nucleoprotein thread (chromonema) which can pass from cell to cell
and from one generation to another generation. T. H. Morgan discussed about the nature of gene,
its characters, function which leads to classical concept of gene.

Further research on gene at molecular level have revolutionized the gene concept. Our current
molecular concept is, a gene is the unit of inheritance . Gene is the unit of function coding for
one polypeptide; operationally defined by the complementation test; may be divisible by
recombination test.

The changing concept of gene from classical to molecular level may be summarized as
follows:

i. Gene governs the inheritance which is bi-parental, i.e., both male and female parents contribute
equally in the inheritance of characters to next generation (exception cytoplasmic inheritance or
maternal inheritance).

ii. Characters of an individual are determined by paired genes, situated in definite number of
chromosome pairs or linkage groups.

iii. Many genes are present on each chromosome and are inherited together, called linked genes.

iv. Each gene-has a particular position on a particular chromosome being called as locus.
Chromosomal aberration of translocation or inversion type may change the position of a locus.

v. During gamete formation each pair of genes gets segregated and gametes possess only one
gene of that kind.

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Fine Structure of Gene:

The concept of gene controlling a single character must involve the expression of several
reactions and in its constituents involves a number of proteins. The work of Beadle and Tatum
demonstrated that the relationship between gene and enzyme is 1:1.

Evidently, if a gene is responsible for the synthesis of a single polypeptide then the gene as
visualized by Mendel is certainly not the ultimate unit of inheritance. The concept of ultimate
indivisible unit of gene underwent complete change following the work of different scientists.

Concept of Cistron, Recon, Muton (Rll Locus in T4 Phage):

Benzer carried out most refined analysis of rll locus in T4 bacteriophage. A mutant at this locus
is responsible for the formation of rough plaques or colonies on B strain of E. coli, but unable to
produce any plaques on K strain.

Complementation Test:

This test was carried out by Benzer in order to find out complementation relations between
different rll mutant alleles. He allowed mixed infection of K strain of E. coli by two rll mutants.
In most cases it did not result in plaque formation, but in some cases plaque formation occurred.

If two mutants did not form plaques on mixed infection, they were placed in the same
complementation group, but if plaques were produced, the two mutants involved in mixed
infection were placed in two different groups. In this manner, two groups A and B could be
established in rll region.

All mutants with the help of complementation test could be classified in these two groups, in
such a manner that two mutants from group A or two mutants from group B could not cause
plaque formation but mixed infection by one mutant of group A and another of group B, could
cause plaque-formation.

Since groups A and B are distinguished on the basis of cis-trans test, these were termed as cistron
A and cistron B. Two mutants from different cistrons (A and B) would give wild type (plaque
formation) even in trans configuration, which in other words is called complementation (Fig.
14.9).

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From the complementation test, it is obvious that in rll region, two cistrons A and B (Fig. 14.10)
of 2500 and 1500 nucleotide pairs respectively are independent functionally and must be respon-
sible for sequential synthesis of two separate products, which presumably are polypeptide chains.

Therefore, all mutants belonging to one cistron share a common deficiency, which is different
from the deficiency due to mutations belonging to the second cistron.

When two mutants belong to same cistron, both are deficient for same product and, therefore,
they cannot complement, but when two mutants belong to two different cistrons, they, being
deficient for different products, can complement, and may express wild phenotype, i.e., lysis and
plaque formation.

Recombination Test:

After rll mutants were classified in cistron A and cistron B, Benzer was interested in analysing
mutants, belonging to same cistron. It was, therefore, necessary to subject them to recombination

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test to find out whether they are located on same site or different sites separable by
recombination.

Deletion mutation of rll locus were arranged in a sets of overlapping deletion representing
segments of different sizes.

The principle involved in this technique was that if a particular point mutant lies in the region of
a deletion represented by a rIl mutant, then on mixed infection with this deletion mutant, the
point mutant will not be able to give rise to a wild type, but if it falls outside the deletion region,
it will be able to give wild type recombinant.

Using deletion mutants having successive overlapping deletions of smaller lengths, one could
locate a mutant to a fairly small region. All point mutants located in this particular small segment
of rll region, could then be subjected to recombination test.

For this purpose, two mutants at a time were used for mixed infection on E. coli B strain and the
lysate produced on plaques formed on B strain was used for infection on K strain to find out the
frequency of wild type phage particles produced.

Benzer eventually estimated 400-500 mutational sites in rll region and called each of them a unit
of mutation or a muton. Thus Benzer was not only able to divide rll region into cistrons A and B,
but was able to classify mutants belonging to same cistron into hundreds of mutational sites
separable due to recombination.

Cistron, Recon, Muton:

Thus a gene is not a unit of either function or recombination or mutation. Benzer, in view of his
work, coined the terms cistron (unit of function), recon (unit of recombination) and muton (unit
of mutation). Cistron was defined as a unit, the elements (alleles) of which exhibit cis-trans
phenomenon.

The smallest unit capable of undergoing recombination is called recon. A recon is further sub-
divisible into units of mutations called mutons, and several mutons in a recon will not be

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separable due to recombination. Therefore, cistron, recon and muton are the units, in the
descending order of size and structurally the gene of classical authors is comparable to cistron of
Benzer.

Overlapping Genes:

Overlapping genes are those genes which can be read or translated in two different ways to
produce two different proteins. From the information about proteins coded by the genome of
φx174, an estimate could be made of the number of nucleotides required (number of bp should
be 3 times the number of amino acids in protein).

This estimate of number of nucleotides exceeds 6000 which is much higher than the actual
number of nucleotides present in single stranded DNA of φx174 (the actual number of nucleotide
is 5400). Therefore, it was difficult to explain how these proteins could be coded from a DNA
segment which is not long enough to code the required number of amino acids.

On detailed study of the system, it was discovered that a single sequence can be utilized by two
different cistrons coding different proteins. Such overlapping of cistrons will be theoretically
possible if the two cistrons have to function at different times and their nucleotide sequences are
translated in two different reading frames.

Mobile Genetic Elements:

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The discovery of mobile sequences in chromosome causing genetic instability is an important
event in genetics. The mobile genetic elements are specific DNA sequences having the capacity
of movement from one location to another in the chromosome. Such sequences, often referred to
as mobile sequences or transposable sequences or jumping genes, were first identified in maize
by Barbara McClintock.

Central dogma of molecular biology

 The central dogma of molecular biology was first articulated by Francis Crick in 1958

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 DNA REPLICATION
Introduction
Genetic information is transferred from parent to progeny organisms by a faithful
replication of the parental DNA molecules. Usually the information resides in one or more
double stranded DNA molecules. However many kind of organisms differs in their genetic
material, they may contain single stranded DNA or ss RNA or ds RNA. The mode of
replication of each of these type of molecules differ in detail though certain fundamental
features are common to each mode.
Definition
Replication is a process in which DNA copies itself to produce identical daughter
molecules of DNA. The synthesis of a new DNA molecule is a complex process involving a
series of steps.
Replication Strategy:-
Models of Replication
There are three models of replication
1. Conservative Model
2. Semi conservative model
3. Dispersive Model
Semi conservative replication
Both the strands of the product DNA undergo simultaneous replication to produce two
daughter molecules, each one of the newly synthesized DNA has one half of the parental
DNA and one half of the newly synthesized DNA. This type of DNA replication is known as
semi conservative, and was first given by Meselson and Stahl.
Conservative replication
Both the strands of parent double helix would be conserved and the new DNA
molecule would consist of two newly synthesized strands.
Dispersive replication
In certain cases DNA synthesis is continuous on one strand and discontinuous on the
other strand called lagging strand. These small fragments are called Okazaki fragments.

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Eukaryotic replication
Basic requirements for dna replication
i. Substrate
ii. Template
iii. Primer
iv. Enzymes
Substrate
For DNA replication, the 4 deoxyribonucleotide triphosphate (d NTP)s such as
(dATP), (dCTP), (dGTP), (dTTP) are needed as substrates.
Template
A template is always required to direct the addition of the appropriate complementary
nucleotide to the newly synthesized DNA strand.
Primer
DNA polymerase cannot initiate synthesis of a complementary strand of DNA on a
totally single stranded template; rather they require a primer (i.e.) a short double stranded
region with a free hydroxyl group on the 3’ end of the shorter strand. This hydroxyl group
serves as the first acceptor of a nucleotide by the action of DNA polymerase. In DNA
synthesis a short stretch of RNA serves as a primer. It is synthesized by a specific RNA

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 polymerase called primase. This RNA primer is always complementary and antiparallel to the
DNA template and is later removed by DNA polymerase I.
Replication of a DNA molecule can be divided into three stages:
i. Initiation
ii. Elongation
iii. Termination
Overview of DNA replication
 As DNA pol III moves forward, the double helix is continuously unwinding ahead of the
enzyme to expose further lengths of single DNA strands that will act as templates.
 DNA pol III acts at the replication fork, the zone where the double helix is unwinding.
However, because DNA polymerase always adds nucleotides at the 3_ growing tip, only
one of the two antiparallel strands can serve as a template for replication in the direction
of the replication fork. For this strand, synthesis can take place in a smooth continuous
manner in the direction of the fork; the new strand synthesized on this template is called
the leading strand.
 Synthesis on the other template also takes place at 3_ growing tips, but this synthesis is in
the “wrong” direction, because, for this strand, the 5_-to-3_ direction of synthesis is away
from the replication.
 As we will see, the nature of the replication machinery requires that synthesis of both
strands take place in the region of the replication fork. Therefore, synthesis moving away
from the growing fork cannot go on for long.
 It must be in short segments: polymerase synthesizes a segment, then moves back to the
segment’s 5_end, where the growing fork has exposed new template, and begins the
process again. These short (1000–2000nucleotides) stretches of newly synthesized DNA
are called Okazaki fragments.
 Another problem in DNA replication arises because DNA polymerase can extend a chain
but cannot start a chain. Therefore, synthesis of both the leading strand and each Okazaki
fragment must be initiated by a primer, or short chain of nucleotides, that binds with the
template strand to form a segment of duplex DNA.
 The primers are synthesized by a set of proteins called a primo some, of which a central
component is an enzyme called primase, a type of RNA polymerase.

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 Primase synthesizes a short (_8–12 nucleotides) stretch of RNA complementary to a
specific region of the chromosome.
 On the leading strand, only one initial primer is needed because, after the initial priming,
the growing DNA strand serves as the primer for continuous addition. However, on the
lagging strand, every Okazaki fragment needs its own primer. The RNA chain composing
the primer is then extended as a DNA chain by DNA pol III.

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 A different DNA polymerase, pol I, removes the RNA primers and fills in the resulting
gaps with DNA.As mentioned earlier, pol I is the enzyme originally purified by
Kornberg. Another enzyme, DNA ligase, joins the 3_ end of the gap-filling DNA to the
5_ end of the downstream Okazaki fragment.
 The new strand thus formed is called the lagging strand. DNA ligase joins broken pieces
of DNA by catalyzing the formation of a phosphodiester bond between the 5_-phosphate
end of one fragment and the adjacent 3_-OH group of another fragment.
 Thus coordinating the synthesis of the leading and lagging strands. The lagging strand is
shown looping around so that the replisome can coordinate the synthesis of both strands
and move in the direction of the replication fork.
 Also shown is an important accessory protein called the sliding clamp, which encircles
the DNA like a donut. Its association with the clamp protein keeps pol III attached to the
DNA molecule.
 Thus, pol III is transformed from an enzyme that can add only 10 nucleotides before
falling off the template (termed a distributive enzyme) to an enzyme that stays at the
moving fork and adds tens of thousands of nucleotides (a processive enzyme).
 In sum, through the action of accessory proteins, synthesis of both the leading and the
lagging strands is rapid and highly coordinated. Note also that primase, the enzyme that
synthesizes the RNA primer, is not touching the clamp protein.
 Therefore, primase will act as a distributive enzyme—it adds only a few ribonucleotides
before dissociating from the template.

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 PROKARYOTIC DNA REPLICATION
Direction of Replication
In case of unidirectional replication one of the two ends would remain stationary
whereas the other end serves as replication fork. In case of bidirectional replication both the
ends are moving and serve as replication forks.
Types:
1.Replication of a DNA molecule can be divided into three stages:
iv. Initiation
v. Elongation
vi. Termination
Initiation

In prokaryotes DNA replication begins at a single unique nucleotide sequence called

the origin of replication. In E.coli replication origin is called “ oric” consists of 245 base
pairs. Many of which are highly conserved among bacteria. The oric is attached to the inner
surface of the cytoplasmic membrane. The key sequences are two series of short repeats:
i. 3 repeats of 13 base pair sequence
ii. 4 repeats of 9 base pair sequence
 At which the base pairs of the parent molecule are broken and the new polynucleotides
are synthesized, is called as ‘replication fork’.
 At least 8 different enzymes or protein participate in the initiation stage of replication
they open the DNA helix at the origin and establish a ‘prepriming complex’ that sets the
stage for subsequence reactions.
 The key component in the initiation process is DNA protein A complex of about 20 or
more molecules binds to the 4 repeats of 9 base pair sequence.
 In the next step DNA protein recognizes and successfully denature DNA in the region
of 3 repeats of 13 base pair sequence which is rich in A=T base pairs this step is facilitated
by the energy from ATP and by bacterial histone like protein, thus forming an initial
complex.
 Helicase binds to this region and unwinds the DNA bidirectionaly create two potential
replication force. This step completing the formation of ‘open complex’.

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  SSB (single stranded DNA binding protein) or helix destabilizing protein that binds
only to single stranded DNA, stabilizing the separated DNA strands and preventing their
renaturation.
 They bind co-operatively that is the binding of molecules of SSB protein facilitates the
binding of addition molecules of SSB protein to the DNA strand. Thus forming the
prepriming complex.

Termination
In E.coli the replication terminus is large region flanked by a nearly identical non
palindromic termination sites. The arrest of replication fork movement by the action of TUS
protein. This protein is a 309 residue monomer which is encoded by the TUS gene
(termination utilization substrate). TUS protein specifically finds to termination sites and
prevents strand separation of DNA helicase, there by arresting the replication fork movement.

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2.BIDIRECTIONAL REPLICATION OR θ MODEL OF REPLICATION
The synthesis of new daughter strands proceeds on both directions (bi- directionally)
around bacterial chromosome the two replication forks move in opposite direction from
origin, meets each other completes the replication process.
 Replication is initiated by binding of DNA A protein to origin of replication sequence (oric).
The oric has three types of DNA sequence: i. A/T rich region, ii. DNA A box sequence, iii. GATC
metylation sites.
 DNA A box sequence particularly act as recognition site for DNA A protein and this protein
binds specifically to the sequence.
 Then DNA Aand DNA C will make DNA helicase to bind to the site and begins strand
separation within oric.
 DNA helicase binds to single strand DNA and moves the replication fork in 5’-3’ direction. The
two replication forks thus moves in opposite direction from origin.
 Then the primase binds and the RNA primers are synthesised.
 After the initiation of synthesis of leading strand at the origin, with the help of DNA polymerase
the first precursor fragment is synthesised.
 Replication is terminated when the replication forks meet at termination sequence; termination
binding protein binds to termination sequence and stops the movement of replication forks.
 Finally two double stranded DNA molecules are formed that are interwined. They are separated
by topisomerase.

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ROLLING CIRCLE OR σ REPLICATION
 It takes place in circular DNA molecule or bacterial plasmids and phages. The first cut is made
in double stranded circle producing free 3’ OH end and 5’ phosphate end.
 DNA is synthesized by the addition of dNTPs at 3’ end forming leading strand. The leading
strand is covalently linked to the parental template for the lagging strand. At the same time 5’
end strand displaces from the circle as the tail.
 When the tail is extended, complementary strand is synthesized as short segments that results
in double stranded tail. Rolling circle replication continues unabated, generating a long, linear
concatemer. The base sequence of the circular DNA is repeated many times, forming a
concatemer.
 This mode of replication requires no primer synthesis and resembles like Greek letter sigma
(σ).

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 ENZYMES AND ACCESSORY PROTEINS INVOLVED IN DNA REPLICATION
DNA polymerase

DNA polymerase are those enzymes which catalyses the synthesis of DNA in both
prokaryotic and eukaryotic organisms. These enzymes add deoxyribonucleotide monomers to
the growing end of the DNA strand. The DNA polymerase requires Mn2+ as the cofactors.
Prokaryotic DNA polymerase
The prokaryotic species have three different DNA polymerase
i. DNA polymerase I
ii. DNA polymerase II
iii. DNA polymerase III
DNA polymerase I
The DNA polymerase I was first isolated from E.coli cells by Kornberg in 1960.
Hence it is otherwise called Kornberg enzyme. It is a single polypeptide chain having
molecular weight 109000 Daltons. It has three important activities:
a. 5’-3’ polymerization activity
b. 5’-3’ exonuclease activity
c. 3’-5’ exonuclease activity
a. 5’-3’ polymerization activity

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 With this activity the DNA polymerase I can catalyze the addition of a precursor
deoxyribonucleotide to the 3’-OH end of the growing DNA strand.
b.5’-3’ exonuclease activity
With this activity the DNA polymerase I assures of great replication facility. As each
nucleotide is added to the chain DNA polymerase I checks to move some nucleotides added
instant correctly added to the complementary bases on the template strand.
For e.g.: If the template base is adenine, the enzyme mistakenly inserts cytosine instead of the
thymine into the new chain. The DNA polymerase I hydrolytically removes the cytosine.
c. 3’-5’ exonuclease activity
DNA polymerase I by this activity removes RNA primer, it located the nick between the
3’ end of newly synthesized DNA by DNA polymerase and 5’ end of adjacent RNA primer.
Next it removes RNA nucleoside moving in 5’-3’ direction. As it removes the RNA, DNA
polymerase I replaces it with deoxyribonucleotide.
DNA polymerase II
This enzyme mainly involves in DNA repair mechanisms. It has both 5’-3’
polymerization and 5’-3’ exonuclease activities.
DNA polymerase III
DNA polymerase III plays an essential role in DNA replication. It is a multimeric
enzyme or holo enzyme having 10 subunits.
α, β, θ, Ʃ, σ, γ, δ, δ’, ψ, χ
α, β, θ subunits are core enzymes, the remaining acts as proof reading enzymes. It lacks 5’-3’
exonuclease activity.
Eukaryotic DNA polymerase
In eukaryotes the DNA polymerase enzymes are about 5 types
a. DNA polymerase α
b. DNA polymerase β
c. DNA polymerase γ
d. DNA polymerase δ
e. DNA polymerase ε
a. DNA polymerase α

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 It is otherwise called cytoplasmic polymerase or large polymerase. It is the
predominant DNA polymerase in eukaryotic DNA replication. It is encoded by the gene pol-I.
b.DNA polymerase β
It is otherwise called nuclear polymerase or small polymerase. It serves a DNA repair
function.
c. DNA polymerase γ
It is present in mitochondria and hence it is called as mitochondrial enzyme. It
catalyses the mitochondrial replication and also involves in proof reading activity.
d.DNA polymerase δ
It is encoded by gene pol-II. It can use both RNA and DNA primers for the initiation
of DNA synthesis and also involves in proof reading activity.
e. DNA polymerase ε
This enzyme is encoded by the gene pol-III. It also involves in proof reading activity.
All these 5 forms of enzymes having 5’-3’ polymerization activity. But it is not clear
either it have 5’-3’ exonuclease activity or not.
DNA Helicase :-
The enzyme involved in the denticulation process.
Rep Protein:-
The helices enzyme active in E. coli DNA replication is an enzyme called Rep protein.
SSB Protein:-
Single strand binding protein involved in denaturation process

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Preprimosome Complex:-
The first step in primer synthesis is the formation of a complex preprimosome which
contains proteins namely n, n’, n” , I, Dna B, Dnac Proteins,
dnaA
 dnaA is a replication initiation factor which promotes the unwinding or denaturation of
DNA at oriC (around 240bp in Escherichia coli), during DNA replication in prokaryotes.
 The formation of the oriC/DnaA complex and DNA unwinding requires ATP
hydrolysis[1].
 The oriC site in E. coli has three AT rich 13 base pair regions (DUE's elements) followed
by five 9 bp regions.
 Around 10 dnaA molecules bind to the 9 bp regions, which wrap around the proteins
causing the DNA at the AT-rich region to unwind.
 The denatured AT-rich region allows for the recruitment of DnaB (helicase) complexes
with DnaC (helicase loader).
 DnaC helps the helicase to bind to and to properly accommodate the ssDNA at the 13 bp
region, this is accomplished by ATP hydrolysis after which DnaC is released.
 Single-strand binding protein's (SSBs) stabilize the single DNA strands in order to
maintain the replication bubble. DnaB is a 5'->3' helicase, so it travels on the lagging
strand.
 It associates with DnaG (a primase) to form the only primer for the leading strand and to
add RNA primers on the lagging strand.
 The interaction between DnaG and DnaB is necessary to control the longitude of
Okazaki fragments on the lagging strand.
 DNA polymerase III is then able to start DNA replication.
DNA gyrase
 DNA gyrase, often referred to simply as gyrase, is an enzyme that relieves strain while
double-stranded DNA is being unwound by helicase.
 This causes supercoiling of the DNA. Many antibiotics work by attacking bacterial DNA
gyrase.DNA gyrase is a type II topoisomerase that introduces negative supercoils (or
relaxes positive supercoils) into DNA by looping the template so as to form a crossing,

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 then cutting one of the double helices and passing the other through it before releasing
the break, changing the linking number by two in each enzymatic step.
 This process occurs in prokaryotes (in particular, in bacteria), whose single circular
DNA is cut by DNA gyrase and the two ends are then twisted around each other to form
supercoils.
 Very recently, gyrase has been reported from the apicoplast of malarial parasite
Plasmodium falciparum (unicellular eukaryote).
 The unique ability of gyrase to introduce negative supercoils into DNA is what allows
bacterial DNA to have free negative supercoils.
 The ability of gyrase to relax positive supercoils comes into play during DNA
replication.
 The right-handed nature of the DNA double helix causes positive supercoils to
accumulate ahead of a translocating enzyme, in the case of DNA replication, a DNA
polymerase.
 The ability of gyrase (and topoisomerase IV) to relax positive supercoils allows
superhelical tension ahead of the polymerase to be released so that replication can
continue.
Primase
 DNA primase is an RNAP enzyme involved in the replication of DNA.
 Primase catalyzes the synthesis of a short RNA segment (called a primer) complementary
to a ssDNA template.
 Primase is of key importance in DNA replication because no known DNA polymerases
can initiate the synthesis of a DNA strand without an initial RNA or DNA primer (for
temporary DNA elongation).
 In bacteria, primase binds to the DNA helicase forming a complex called the primosome.
 Primase is activated by DNA helicase where it then synthesizes a short RNA primer
approximately 11 ±1 nucleotides long, to which new nucleotides can be added by DNA
polymerase.
 The RNA segments are first elongated by DNA polymerase and then synthesized by
primase.

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  Then the DNA polymerase forms a protein complex with two primase subunits to form
the alpha DNA Polymerase primase complex.
 Primase is one of the most error prone and slow polymerases.
RNA polymerase
 RNA polymerase (RNAP or RNApol) is an enzyme that produces RNA.
 In cells, RNAP is needed for constructing RNA chains from DNA genes as templates, a
process called transcription.
 RNA polymerase enzymes are essential to life and are found in all organisms and many
viruses.
 In chemical terms, RNAP is a nucleotidyl transferase that polymerizes ribonucleotides at
the 3' end of an RNA transcript RNAP can initiate transcription at specific DNA
sequences known as promoters.
 It then produces an RNA chain, which is complementary to the template DNA strand.
 The process of adding nucleotides to the RNA strand is known as elongation;
 In eukaryotes, RNAP can build chains as long as 2.4 million nucleosides (the full length
of the dystrophin gene).
 RNAP will preferentially release its RNA transcript at specific DNA sequences encoded
at the end of genes known as terminators.
Products of RNAP include:
 Messenger RNA (mRNA)—template for the synthesis of proteins by ribosomes.
 Non-coding RNA or "RNA genes"—a broad class of genes that encode RNA that is not
translated into protein. The most prominent examples of RNA genes are transfer RNA
(tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of
translation. However, since the late 1990s, many new RNA genes have been found, and
thus RNA genes may play a much more significant role than previously thought.
o Transfer RNA (tRNA)—transfers specific amino acids to growing polypeptide
chains at the ribosomal site of protein synthesis during translation
o Ribosomal RNA (rRNA)—a component of ribosomes
o Micro RNA—regulates gene activity
o Catalytic RNA (Ribozyme)—enzymatically active RNA molecules

26
In contrast to DNA polymerase, RNAP includes helicase activity, therefore no separate enzyme
is needed to unwind DNA.
RNA polymerase in eukaryotes
Structure of eukaryotic RNA polymerase II (light blue) in complex with α-amanitin (red), a
strong poison found in death cap mushrooms that targets this vital enzyme
Eukaryotes have several types of RNAP, characterized by the type of RNA they synthesize:
 RNA polymerase I synthesizes a pre-rRNA 45S (35S in yeast), which matures into 28S,
18S and 5.8S rRNAs which will form the major RNA sections of the ribosome.
 RNA polymerase II synthesizes precursors of mRNAs and most snRNA and
microRNAs.This is the most studied type, and due to the high level of control required
over transcription a range of transcription factors are required for its binding to
promoters.
 RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the
nucleus and cytosol.
 RNA polymerase IV synthesizes siRNA in plants.
 RNA polymerase V synthesizes RNAs involved in siRNA-directed heterochromatin
formation in plants.
There are other RNA polymerase types in mitochondria and chloroplasts. And there are RNA-
dependent RNA polymerases involved in RNA interference.
Enzymes for Replication:-
Different prokaryotic and eukaryotic cells have been found it contain three kinds of
nuclear enzymes that act on DNA namely.
1. Nuclease
2. Polymerase
3. Ligases.
Nucleases:
Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link one
nucleotide to the next in a DNA strand. There are two different kinds of nuclease, such as:
1. Exonucleases remove nucleotides one at a time from the end of a DNA molecule.
2. Endonucleases are able to break the internal phosphodiester bonds within a DNA
molecule.

27
The main distinction between different exonucleases lies in the number of strands that are
degraded when a double stranded molecule is attacked. The enzyme called Bal31 (purified from
the bacterium Alteromonas espejiana) is an example of an exonuclease that removes nucleotides
from both strands of a double stranded molecule. The greater the length of time that Bal31 is
allowed to act on a group of DNA molecules, the shorter the resulting DNA fragments will be. In
contrast, enzymes such as E.coli exonuclease III degrade just one strand of a double stranded
molecule, leaving single stranded DNA as the product.

The same criterion can be used to classify endonucleases. S1 endonuclease (from the fungus
Aspergillus oryzae) only cleaves single strands, whereas deoxyribonuclease I (DNase I), which
is prepared from cow pancreas, cuts both single and double stranded molecules.
Uses of S1 nuclease:
1. S1 nuclease is used to degrade the hairpin loop formed while making a duplex DNA from
complementary DNA strand (cDNA).
2. It is used to remove the extra adenine base from DNAs prepared by polymerase chain
reaction.
3. It is used to remove the unwanted tail sequences from DNA fragments to make blunt
ends.
4. It can also be used to determine the degree of complementary base pairing between DNA
strands during hybridization.
DNA Ligases:
Although cutting the DNA precisely is very useful for DNA analysis its full potential is
revealed only when the fragments produced are joined together to give a new structure known as
recombinant DNA (r DNA). This joining or ligation is achieved by the use of a DNA ligase
enzyme. Source: All living cells produce DNA ligases but enzyme used in genetic engineering is
usually purified from E.Coli bacteria that have been infected with T4 phage.
Mechanism and action of DNA ligase:
With in the cell the enzyme carriers out the very important function of repairing any
discontinuities that may arise in one of the strands of a double stranded molecule.
A discontinuity is simply a position where a phosphodiaster bond between adjacent
nucleotides is missing. Although discontinuous may arise by chance breakage of cells DNA

28
molecules, they are also a result of natural process such as DNA replication and recombination
ligases therefore play several vital roles in the cell.
In the test tube purified DNA ligases as well as repairing single stranded discontinuous will
also join together individual DNA molecules or the two ends of the same molecule.
The chemical reaction involved in ligating 2 molecules is exactly the same as discontinuity
repair, except that two phosphodiester bonds must be made one for each strand.
Based on sources DNA ligases are of two types.
Mt.wt co factor
a) E.Coli DNA ligase ---------- 75,000 Daltons NAD+
b) T4 DNA ligase----------------- 65,000 Daltons ATP

E.Coli DNA ligase: - used for ligating only the DNA fragments with sticky ends.
T4 DNA ligase: - used for ligating DNA fragments with both sticky end and blunt ends.
DNA polymerase:-
All DNA polymerase of nucleotide catalyze the polymerization of nucleotide precursors
(Deoxyribonucleoties) into a DNA chain.
DNA REPAIR MECHANISM
Both prokaryotic and eukaryotic cells have a number of repair system to correct the damage
in DNA. All the use enzymes to make the corrections. If the repair system is unable to correct
the damage, the result is the mutant cell or too many mutation results in death of the cell. DNA
repair is possible because DNA damage in one strand can be removed and correctly replaced
by instructions of undamaged complementary strand.
Repair by DNA polymerase proof reading
DNA polymerase of E.coli have3’-5’ exonuclease activity which remove nucleotides from 3’
end of DNA chain. This checks the accuracy of assembled base pairs. This activity is a proof
reading mechanism that reduces the DNA replication errors.
i. Photoreactivation
 By the process of photoreactivation as light repair, the dimers are reverted back to the
original form by exposing to the visible light in the wave length of 320-370 nm.

29
  The photoreactivation is catalysed by an enzyme called photolyase encoded by protein
gene which is activated by a photon of light which splits the dimers, photolyase is found in
prokaryotes and lower eukaryotes but not in humans.
 The enzyme is inactive unless exposed to visible light, a folic acid cofactor associated
with the enzyme absorbs the light, then the enzyme uses the energy of the absorbed light to
perform the cleavage of the dimers.

Repair of alkylation damage

The alkylated base pairs are formed by the alkylating agents. In this repair system a enzyme
called O6 – methylguanine methyl transferase recognizes O6 – methylguanine in methylated
guanine and removes the methyl group their by changing it back to its original form.

30
 ii.Dark repair mechanism
 UV radiation exhibited higher rate of induced mutation in dark in E.coli. These mutants
were called UV radiation mutants. This can be repaired by dark repair or excision repair
system.
 The excision repair system not only correct pyrimidine dimmers but also other series
damage induced breakage of DNA helix.
 The DNA helix damage is recognized by UV radiation ABC endonuclease encoded by
3 genes uv r A, uv r B, uv r C.
 The enzymes make one cut in the damaged DNA strand 8 nucleotides to the 5’ side of
the dimer and 4 nucleotides to the 3’ side of the damage.
 The act release the 12 nucleotide of single strand DNA containing the dimmers. The 12
nucleotide gap is filled by 5’-3’ polymerization activity of DNA polymerase I and sealed
by DNA ligase.

Mismatch repair
 Many mismatched base pairs are corrected by mismatch repair mechanism. The
mismatch repair mechanism of E.coli contains 3 genes mut H, mut S, mut L.
 Mut H recognizes methylates DNA sequence, mut S locates mismatched, mut L joints
mut S and mut H.

31
  In E.coli methylation occurs in GATC sequence in which adenine is methylated by
methylase enzyme.
 Mut S forms a complex with mut L, this complex when locates a mismatch it interacts
with mut H by looping mechanism.
 This stimulates mut H to make a cut in non methylated DNA strand after the strand is
cut and exonuclease digests the non methylated DNA strand in the direction of mismatch
and proceeds beyond the mismatch.
 This leaves a gap in the daughter strand repaired by DNA polymerase and DNA ligase.
Recombination repair
 A thymine dimer inhibit DNA replication, because it prevents base pairing in new
daughter strand. Thus a gap will be created in the region that cannot be replicated.
 DNA gaps are harmful and causes chromosomal breaks. This is repaired by genetic
recombination.
 Thymine dimers in parental strand leaves a gap in new daughter strand. First the gap is
replaced with same region in original DNA strand, a short segment of original strand
replaces the corresponding segment of new strand. This removes the gap in new strand but
creates a gap in original strand.
 The next step is to fill the gap in original strand using the complementary strand as
template with the help of DNA polymerase. Thus no gap is found in any of the replicated
DNA strands.
SOS repair
 SOS means ‘Save Our Soul’. It is an error prone process, even though intact the DNA
strands contained correct bases.
 The functional gene involved in this repair is rec A gene. The rec A protein bind tightly
to ss DNA containing pyrimidine dimer.
 When DNA polymerase III comes in contact with a dimer to which rec A is bound and
interacts with ε subunit of polymerase and stops the editing function of polymerase.
 Thus allowing the replication fork to move. The DNA polymerase in use the residual
base pairing ability of thymine dimer and add adenine in the daughter strand.
 Thus the mispaired base remains in the daughter strand as mutation.

32
33
 QUESTION BANK
UNIT-I
1 MARK QUESTIONS

1. Replication.
2. Okazaki fragments.
3. Primer.
4. Template.
5. Replication fork.
6. Photoreaction.
7. Concept of gene

5 MARK QUESTIONS

8. Explain about the central dogma of molecular biology.

9. Write a short note on models of replication.
10. Discuss in θ model of replication.
11. Explain about the rolling circle model.
12. Comment on DNA polymerase.
13. Explain excision repair.
14. Modern concept of gene organization

10 MARK QUESTIONS

15. Explain in detail about DNA damage & repair mechanism.

16. Trace the mechanism of DNA replication.
17. Describe in detail the role of enzymes in DNA replication.
18. DNA as a genetic material

34
UNIT II
PROKARYOTIC TRANSCRIPTION
Introduction
Gene expression is accomplished by the transfer of genetic information from DNA to RNA
molecules and then from RNA to protein molecules. This is the central dogma of protein
synthesis is given by Watson and Crick. RNA molecules are synthesized by using a portion of
one strand of DNA as a template in a polymerization reaction that is catalyzed by enzymes
called RNA polymerase.
Definition
Transcription is the transfer of information from a double strand template DNA molecule to a
single strand RNA. This process is also called as first stage of gene expression.
Properties of RNA polymerase
RNA polymerase is multi subunit enzyme that recognises nucleotide sequence in DNA. Then it
makes a complementary RNA copy of DNA template and also recognize the end of DNA
sequence. RNA is synthesized from 5’-3’ end antiparallel to its DNA template. A transcription
unit extends from promoter to termination region and the product of the process of transcription
by RNA polymerase is termed as primary transcript.
RNA polymerase
Core enzyme: it consists of 5 subunits, they are two identical α subunits, β, β’ and ω subunits.
These are responsible for 5’ – 3’ RNA polymerase activity and are reffered to as core enzyme.
However it lacks specificity and cannot recognize promoter region on DNA template.
Holo enzyme: the σ subunit enables the polymerase to recognize promoter region on the DNA.
The σ subunit combines with core enzyme is known as holo enzymes. It is essential to initiate
transcription, after the synthesis of few nucleotides the σ factor release out and the core enzyme
continues the elongation process. The released σ factor activates other core enzyme and thus
the cycle repeats.
Steps involved in transcription
The process by which the gene is transcribed is conveniently divided into three phases
i. Initiation
ii. Elongation
iii. Termination

35
Initiation
During the process of transcription the components required to initiate RNA chain synthesis are
i. Template (DNA strand)
ii. Divalent metal ion (Mg 2+ or Mn2+)
iii. RNA polymerase
iv. Nucleotide triphosphate (ATP, GTP, CTP, UTP)
 Initiation of RNA polymerase involves binding of RNA polymerase to a region on DNA that
determines the specificity of transcription of that particular gene. That region is known as promoter
region (20 to 200 bp).
 The characteristic nucleotide sequence of prokaryotic promoter that are recognized by RNA
polymerase include Pribnow box (-10 region) and -35 region.
Pribnow box: This is a sequence of 6 nucleotide (TATAAT) centered about 8 – 10 nucleotide to the left
of transcription starting site (upstream) that codes for initial base for RNA.
-35 sequence: A second nucleotide sequence (TTGACA) located about 35 bases upstream to the
transcription start site is also recognized by RNA polymerase.
 The holoenzyme first binds the DNA and migrates to -35 regions forming closed complex.
 The DNA is then unwound for about 17 base pair beginning at -10 regions exposing the DNA
template at initiation site and thus forms open complex.
 RNA polymerase bind more tightly to this unwound region and the RNA synthesis begins.

36
Elongation
 Chain elongation occurs by core enzyme that moves along the DNA template, σ subunit is
required only to ensure specific recognition of promoter. Once a few phospho diester bonds are formed,
the σ subunits dissociates leaving the core polymerase to complete the synthesis of RNA.
 After beginning of elongation, transcription goes on a rate of 30 – 60 nucleotides/ second. The
released σ factor can combine with any of the other core enzymes and that is reused for initiation of new
chain.
 Moreover it is believed that after the release of σ factor the nucleotide become associated with
core enzyme and modulates the rate of elongation.
 Unlike DNA polymerase, RNA polymerase does not require a primer and has no exonuclease
activity and therefore error correction is not performed in RNA.
 The first base of RNA synthesized is always in the form of purine (i.e) either GTP or ATP.
 RNA polymerase utilizes ribonucleoside triphosphate and releases Ppi each time when
nucleotide is added to growing chain.

37
Termination
The process of elongation of RNA chain continuous until a termination is reached. RNA
polymerase, in some cases recognize termination region on DNA template (ρ independent
termination). Alternatively an addition of protein ρ may be required for the release of RNA
product (ρ dependent termination)
ρ independent termination
It requires two important structural features in newly synthesized RNA.
First the RNA transcript must be able to form a stable hairpin turn that slow down the progress
of RNA polymerase and causes it to pause. The hairpin turn of RNA is complementary to the
region of DNA template near termination region that exhibit two fold symmetry. In the hairpin
sequence there is rich of G and C sequence. This stabilizes the structure of hairpin.
Following the hairpin turn the RNA transcript contains of strings of U. the bonding of U
corresponding to DNA template A is weak. This facilitates the separation of newly synthesized
RNA from DNA template.

38
ρ dependent termination
 It requires ρ protein which is an hexamer. ρ requires single stranded RNA as binding region ρ
factor moves along RNA from its binding site to the enzyme RNA polymerase. The movement is aided
by hydrolysis of ATP that provides energy.
 RNA polymerase wraps around outside of the ρ factor consequently ρ protein comes in contact
of core enzyme of RNA polymerase. This wrapping distrubs non-covalent bonds that hold between
RNA and DNA. Thus ρ protein – RNA complex is set free from RNA polymerase DNA complex.
 Finally core RNA polymerase is released and interacts with σ factor that is used for initiation of
second round of transcription.
EUKARYOTIC TRANSCRIPTION:
 Transcription involves synthesis of RNA on DNA template in such a way that the m-RNA
resembles one of the two DNA strands.
 This DNA strand which is identical to m-RNA is called as coding strand and the other
complementary DNA strand is called the template strand.
 The transcription is carried out by an enzyme called RNA polymerase or transcriptase and a
transcription unit may include more than one gene.
 The mechanisms of transcription and RNA polymerase used for transcription differ in
prokaryotes and eukaryotes.
Transcription in Eukaryotes:
 Eukaryotes have three distinct types of RNA polymerases which transcribe three different classes
of genes.

39
  In addition RNA polymerase does not bind to the promoter by itself.
 It binds to certain transcription factors which must bind to appropriate promoter sequences.
Classes of genes:
 Class I gene—encodes for 28s, 5.8s, 18s r-RNA.
 Class II gene—encodes m-RNA.
 Class III gene—5s r-RNA and t-RNA molecules.
RNA polymerases:
All eukaryotes posses 3 distinct classes of RNA polymerases called RNA pol I, II, III. In
addition, mitochondria and plastids in eukaryotes have a separate class of enzymes i.e., similar to
bacterial enzyme.
RNA polymerase I:
 Contain 2+ < 10 subunits.
 Large subunit is homogenous to β’ subunit of E.coli.
 Located in nucleolus.
 Responsible for the synthesis of ribosomal RNA.
 Not sensitive to α amanitin Abic.
 Promoter is located just upstream of the start point. Constitute 50-70% RNA pol activity.
 Carboxy terminal domain is absent (CTD).
 Bind with upstream binding factor 1, selectivity factor1 (UBF1, SL1). It is bicyclic octapeptide.
RNA polymerase II:
 Contain 2+ < 10 subunits. One smaller subunit is similar to α subunit of E.coli.
 Responsible for the transcription of m-RNA genes.
 Located in nucleoplasm.
 Very sensitive to α amanitin.
 Recognize promoter located upstream of the start point.
 Constitute 20-40% of total RNA pol activity.
 Carboxy terminal domain is present.
 Binds with general, specific, upstream transcriptional factors.
 It transcribes heterogenous nuclear RNA (hn RNA) a precursor for m- RNA molecules.
RNA polymerase III:
 Contain 2+ < 10 subunits.
 Responsible to transcribe the 5s r-RNA and t-RNA molecules.

40
  Recognize the promoter located to downstream of the start point.
 Constitute~10% of total RNA pol activity.
 CTD is absent. Binds with 3 ancillary factors. Use sensitive to α amanitin.
The 3 polymerases are large protein of 500,000 da or more. Each of them has 2 large &
10-12 small subunits. The largest subunit has carboxyterminal domain (CTD) which contain
multiple repeats of consensus sequences try-ser-pro-thr-ser-pro-ser amino acids.
CTD is highly phosphorylated which is involved in transcription initiation and release of
transcription factors from RNA polymerase. Each of eukaryotic RNA polymerases catalyzes
transcription in a 5’ to 3’ direction and synthesizes RNA complementary to the antisense or
coding strands.
It requires precursor nucleotides like ATP, GTP, UTP and CTP (no Primer) for initiation.
Rna polymerases require general and specific transcriptional factor to bind on their respective
promoters.
Transcription of class I genes: (Initiation)
 Class I genes carry pre r RNA transcription unit. It contain 3 sequences that encode the 28s, 5.8s,
18s r-RNA.
 Pre r RNA units are arranged in cluster in the genome as long tandem repeats. RNA polymerases
are responsible for the continuous synthesis of r RNA during interphase.
 Class I genes exist as 5’ cluster of around 40 copies of r RNA gene.
 Each r RNA gene produces 45s r RNA transcripts.
 This transcripts is cleaved to get 28s RNA (5000 nltds), 18s RNA (2000 nltds) and 5.8s RNA
(160 nltds).
Elongation:
 Certain accessory proteins involved in transcription elongation is called the elongation factors
(EF).
 It enhances the overall activity of RNA polymerases leading to the increase in elongation rate.
 Transcription factors like TFIIS helps in elongation by relieving the obstructions in the path. It is
also involved in hydrolytic cleavage at 3’ ends of RNA chain.
Termination:
 In eukaryotes, the actual termination may takes place through termination site.
 It is achieved in association with “snurps” (small nuclear RNA- protein complex). Termination
signals or sequences are 5’AAUAAA3’.

41
TATA box

In molecular biology, the TATA box (also called the Goldberg-Hogness box)[1] is

a sequence of DNA found in the core promoter region of genes in archaea and eukaryotes.
[2]
 The prokaryotic homolog of the TATA box is called the Pribnow boxwhich has a
shorter consensus sequence.

The TATA box is considered a non-coding DNA sequence (also known as a cis-regulatory

element). It was termed the "TATA box" as it contains a consensus sequence characterized by
repeating T and A base pairs.[3] How the term "box" originated is unclear. In the 1980s, while
investigating nucleotide sequences in mouse genome loci, the Hogness box sequence was found
and "boxed in" at the -31 position.When consensus nucleotides and alternative ones were
compared, homologous regions were "boxed" by the researchers.[4] The boxing in of sequences
sheds light on the origin of the term "box".
The TATA box was first identified in 1978 as a component of eukaryotic
promoters. Transcription is initiated at the TATA box in TATA-containing genes. The TATA
box is the binding site of the TATA-binding protein (TBP) and transcription factors in some
eukaryotic genes. Gene transcription by RNA polymerase II depends on the regulation of the
core promoter by long-range regulatory elements such as enhancers and silencers.[5] Without
proper regulation of transcription, eukaryotic organisms would not be able to properly respond to
their environment.
Based on the sequence and mechanism of TATA box initiation, mutations such
as insertions, deletions, and point mutations to this consensus sequence can result
in phenotypic changes. These phenotypic changes can then turn into a diseasephenotype.
Some diseases associated with mutations in the TATA box include gastric
cancer, spinocerebellar ataxia, Huntington's disease, blindness, β-
thalassemia, immunosuppression, Gilbert's syndrome, and HIV-1. The TATA-binding protein

(TBP) could also be targeted by viruses as a means of viral transcription.

Discovery
The TATA box was the first eukaryotic core promoter motif to be identified in 1978 by
American biochemist David Hogness
Location
Promoter sequences vary between prokaryotes and eukaryotes. In eukaryotes, the TATA box is
located 25 base pairs upstream of the start site that Rpb4/Rbp7 use to initiate transcription.
In metazoans, the TATA box is located 30 base pairs upstream of the transcription start
site. While in yeast, S. cerevisiae, the TATA box has a variable position which can range from

42
40 to 100 bp upstream of the start site. The TATA box is also found in 40% of the core
promoters of genes that code for the actin cytoskeleton and contractile apparatus in cells.[5]
The type of core promoter affects the level of transcription and expression of a gene. TATA-
binding protein (TBP) can be recruited in two ways, by SAGA, a cofactor for RNA polymerase
II, or by TFIID.When promoters use the SAGA/TATA box complex to recruit RNA polymerase
II, they are more highly regulated and display higher expression levels than promoters using the
TFIID/TBP mode of recruitment.
In prokaryotes, promoter regions may contain a Pribnow box, which serves an analogous
purpose to the eukaryotic TATA box. The Pribnow box has a 6 bp region centered around the
-10 position and a 8-12 bp sequence around the -35 region that are both conserved.

Structure

Figure 2. Mechanism for transcription initiation at the TATA box. Transcription factors, TATA binding protein (TBP),
and RNA polymerase II are all recruited to begin transcription.

Diseases
Mutations in the TATA box region affects the binding of the TATA-binding protein(TBP) for
transcription initiation, which may cause carriers to have a diseasephenotype.
Gastric cancer is correlated with TATA box polymorphism.[38] The TATA box has a binding site
for the transcription factor of the PG2 gene. This gene produces PG2 serum, which is used as
a biomarker for tumours in gastric cancer. Longer TATA box sequences correlates with higher
levels of PG2 serum indicating gastric cancer conditions. Carriers with shorter TATA box
sequences may produce lower levels of PG2 serum.

43
Hogness box
The Hogness box, also known as the Goldberg–Hogness box, the ATA box, and, on most
occasions, the TATA box, is the binding site in protists, metazoans, fungi, and eukaryotic viruses
for the TATA-binding protein (TBP), a subunit of transcription factor II D (TFIID). In the gene
transcribed by RNA polymerase II, the first base of the Hogness box, the consensus sequence
TATA(t/a)A(t/a), is located approximately 30 bp upstream of the transcription start sites.

The affinity of TBP for the Hogness box is an important characteristic of gene promoters,
because its predicted values correlate with the values of many moleculargenetic characteristics
such as the transcription reinitiation rates, levels of transcriptional activity, gene expression
levels, and norms of reaction. Hogness box polymorphisms have been associated with hereditary
diseases, plant and animals traits valuable in breeding practices, and trends in the current
acquired immunodeficiency syndrome (AIDS) pandemic.

Eukaryotic promoters are commonly classed into TATA-containing promoters and TATA-less
promoters depending on the Hogness box score in Bucher’s position-weight matrix. In mammals,
switchovers from TATA-containing promoters to TATA-less promoters in the Gli1 and Gli3
genes correlate with switchovers from r-selection to K-selection in the course of evolution.

CAAT Box

A CAAT box (also CAT box) is a region of nucleotides with the following consensus sequence:
5’ GGCCAATCT 3’. The CAAT box is located about 75-80 bases upstream of the transcription
initiation site and about 150 bases upstream of the TATA box. It binds transcription
factors (CAAT TF or CTFs) and thereby stabilizes the nearby preinitiation complex for easier
binding of RNA polymerases. CAAT boxes are rarely found in genes that express proteins
ubiquitous in all cell types.

In molecular biology, a CCAAT box (also sometimes abbreviated a CAAT box or CAT box) is a

distinct pattern of nucleotides with GGCCAATCT consensus sequencethat occur upstream by
60–100 bases to the initial transcription site. The CAAT box signals the binding site for
the RNA transcription factor, and is typically accompanied by a conserved consensus sequence.
It is an invariant DNAsequence at about minus 70 base pairs from the origin of transcription in
many eukaryotic promoters.

Genes that have this element seem to require it for the gene to be transcribed in sufficient
quantities. It is frequently absent from genes that encode proteins used in virtually all cells. This
box along with the GC box is known for binding general transcription factors. Both of these
consensus sequences belong to the regulatory promoter. Full gene expression occurs when
transcription activator proteins bind to each module within the regulatory promoter. Protein
specific binding is required for the CCAAT box activation. These proteins are known as CCAAT
box binding proteins/CCAAT box binding factors.

44
A CCAAT box is a feature frequently found before eukaryote coding regions, but is not found in
prokaryotes.

Enhancers:

 They are the control elements located, many 1000 of bp away from the transcription start
site.
 They are characteristically 100-200 bp long.
 It contains multiple sequence elements which contribute to the total activity of the
enhancers.
 They may be universal or cell type specific.
 Characteristics of enhancers are,
 Activation of transcription from the correct start site.
 Activate transcription when placed in either orientation to linked genes.
 Able to function over long distances of more than 1kb whether from an upstream or
downstream to the start point.
 Exert stimulation of the promoter.

Upstream regulatory elements (URE):

 Activity of basal promoters can be increased by the presence of other URE.

 “CCAAT” box is called as upstream regulatory elements.
 It is present in multiple copies.
 Located within 100-200 bp of upstream from the promoter.
 It ensures the efficiency of transcription by the promoter.

Upstream transcriptional factors:

 Factors which recognize specific short sequences located upstream of the start point is
called “upstream transcriptional factors”. e.g. SP1, CTF family, CP1 family.
POST TRANSCRIPTIONAL MODIFICATIONS:
The RNAs produced during transcription are called primary transcripts. They undergo
many alterations – terminal base additions, base modifications, splicing etc., which are
collectively referred to as post transcriptional modifications. This process is required to connect
the RNAs into the active forms. A group of enzymes namely ribonucleases, are responsible for
the processing of tRNAs and rRNAs of both prokaryotes and eukaryotes.
The prokaryotic mRNA synthesized in transcription is almost similar to the functional mRNA. In
contrast, eukaryotic mRNA (ie,hnRNA) undergoes extensive post transcriptional changes.
MESSENGER RNA

45
 The primary transcript of the mRNA is the hnRNA in eukaryotes, which is subjected to many
changes before functional mRNA is produced.
1. The 5’Capping:
The 5’ end of mRNA is capped with 7-methylguanosine by an unusual 5’ 5’triphosphate
linkage. 5-Adenosylmethionine is the donor of methyl group. This cap is required for translation,
besides stabilizing the structure of mRNA.
Cap function:
The cap provides,
i. Protection of the m RNA from degradation
ii. Enhancement of the m RNA translatability.
iii. Transport of the m RNA out of the nucleus.
iv. Proper splicing of the pre m RNA.
2. Poly-A tail:
A large number of eukaryotic mRNAs possess an adenine nucleotide chain at the 3’end. This
poly-A tail, as such, is not produced during transcription. It is later added to stabilize mRNA.
However poly-A chain gets reduced as the mRNA enters cytosol.
Splicing:
The excision of introns and joining together of all exons of a gene in a proper sequence to
yields mature m RNA is called splicing. The two boundaries between the two exons and the
intron is called splicing junction.
i. Intron--- non coding sequence that does not encodes for a ptn.
ii. Exons ---coding sequences.
During splicing the phosphodiester bond at the 2 splicing junction are cleaved and a new
phosphodiester bond is formed between the 2 exons.
Spliceosome mediated splicing:
The processing of bulk of mRNA precursors is mediated by particles called as
spliceosomes. Spliceosomes are assembly of 4-5types of ribo nucleo protein (RNP particles).
Splicing is independent of transcription and RNA modification like capping and polyadenylation.
RNA editing:
 The term RNA editing describes those molecular processes in which the information
content in an RNA molecule is altered through a chemical change in the base makeup.

46
 These changes have been observed in tRNA, rRNA, mRNA and microRNA molecules of
eukaryotes but not prokaryotes.
 RNA editing occurs in the cell nucleus and cytosol, as well as in mitochondria and
plastids, which are thought to have evolved from prokaryotic-like endosymbionts.
 The diversity of RNA editing mechanisms includes nucleoside modifications such as
cytidine (C) to uridine (U) and adenosine (A) to inosine (I) deaminations, as well as non-
templated nucleotide additions and insertions.
 RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein
so that it differs from that predicted by the genomic DNA sequence.
Editing by insertion or deletion:
 RNA editing through the addition and deletion of uracil has been found in kinetoplasts
from the mitochondria of Trypanosoma brucei. Because this may involve a large fraction
of the sites in a gene, it is sometimes called "pan-editing" to distinguish it from topical
editing of one or a few sites.
 Pan-editing starts with the base-pairing of the unedited primary transcript with a guide
RNA (gRNA), which contains complementary sequences to the regions around the
insertion/deletion points.
 The newly formed double-stranded region is then enveloped by an editosome, a large
multi-protein complex that catalyzes the editing .
 The editosome opens the transcript at the first mismatched nucleotide and starts inserting
uridines. The inserted uridines will base-pair with the guide RNA, and insertion will
continue as long as A or G is present in the guide RNA and will stop when a C or U is
encountered.
 The inserted nucleotides cause a frameshift and result in a translated protein that differs
from its gene.

47
Nuclear export of m RNA:
 Eukaryotic pre m RNA synthesis and processing takes place in an orderly fashion within
the cell nucleus. But the translation occurs only in cytosol. So the m RNA molecules had
to be exported from nucleus to cytoplasm.
 After pre mRNA is synthesized and processed, it is bound by a variety of protein
including the cap binding protein, SR protein, poly A binding ptn.
 Only if the proper sets of proteins are bound to the m RNA is guided through nuclear
pore complex into the cytosol.
Nuclear pore complex:
 They are aqueous channel in the nuclear membrane that directly connects the
nucleoplasm and cytoplasm.
 Small molecules less that 50,000da can diffuse freely through them.
 Most of the macromolecules including m RNA are complexes with ptns are far too large
to pass through pores. So they must be exported by active transport (occurs in both
direction).
 Nuclear pore complex is composed of 50 different protein called nucleoporins that are
arranged with a striking octagonal symmetry.
 Nuclear envelope contains 3000-4000 pore complexes. Pore complex contain 4 structural
building blocks.
a. Column subunit:- forms the bulk of pore wall.
b. Annular subunit:- which extend spokes towards the centre of the pore.

48
 c. Luminal subunit:- contain transmembrane protein and anchor the complex to the
membrane.
d. Ring subunit:- forms the cytosolic and nuclear faces of the complex.
Transport of m RNA from nucleus to cytosol:
 Mature m RNA carry signals in the form of bound proteins that specifies their fate.
 Not all hn RNP ptns remain in the nucleus, some are known to remain bound to fully
processed m RNA and accompany them to the cytoplasm.
 Mature m RNA are often bound with cap binding complex, splicing related protein SR,
RNA export factors, hn RNP ptns and poly (A) binding ptns.
 Splicing related protein, that are bound at exon-exon boundaries, RNA export factors, hn
RNP after providing signals for export, reside in nucleus itself.
 CBP and PBP guide m RNA to export from nucleus to cytoplasm and enter the cytoplasm
along with m RNA.
 In cytoplasm CBP is replaced by eIF-4G and 4E.
QUESTION BANK UNIT-II
1 MARK
1. Transcription
2. RNA polymerase.
3. 5’ capping.
4. Splicing.
5. RNA editing.
6. Promotor.
5 MARK
7. Write short note on RNA splicing.
8. Describe the exportation of mRNA from nucleus.
9. Explain about the TAATA,CAAT & Hognes box.
10. Explain about Post transcriptional modification of eukaryotic mRNA
10 MARK
11. Explain in detail the modifications in RNA in the process of transcription.
12. Explain the different stages in transcription. Add a note on regulation of transcription.
13. Discuss about the prokaryotic transcription..

49
UNIT III
PROKARYOTIC TRANSLATION
Definition
Protein is a polymer of amino acid joined together by peptide bonds. The mechanism of protein
synthesis from mRNA template is called translation.
The synthesis of every protein molecule in a cell is directly by an mRNA intermediate, which is
copied from DNA. Synthesis of proteins from mRNA is called translation. The translation
system consists of four major components:
i. Ribosomes
ii. Transfer RNA
iii. Aminoacyl tRNA synthetases
iv. Initiation, elongation and release factors

i. Ribosomes
These are particles on which the mechanics of protein synthesis is carried out. They contain the
enzyme needed to form a peptide bond between aminoacids, a site for binding one mRNA
molecule, and sites for bringing in and aligning the amino acids in preparation for assembly
into the finished polypeptide chain.
ii. Transfer RNA
Amino acids do not bind to mRNA, but the order of amino acids in a particular protein is
determined by the base sequence in the mRNA molecule. This ordering is accomplished by a
set of adaptor molecules called transfer RNA. At tRNA molecule “reads” the base sequence of
mRNA. Each type of tRNA specifically recognizes three adjacent bases on an mRNA molecule
and is charged with a specific amino acid. The amino acid that corresponds to each specific
three base sequence in the mRNA defines the genetic code.

i. Aminoacyl tRNA synthetases

This set of enzymes catalyzes the attachment of the specific amino acids to its corresponding
tRNA.
ii. Initiation, elongation and release factors
These molecules are proteins needed at particular stages of polypeptide synthesis.

50
Mechanism of translation
The process of protein synthesis can be divided in following steps
iii. Activation of amino acid
iv. Initiation
v. Elongation
vi. Termination
i. Activation of amino acid
 The amino acid need to be activated before they can be incorporated into the peptide
chain. The key enzyme in this process is amino acyl t RNA synthetase.
 These are specific for a particular amino acid and also for tRNA. These are atleast 20
different tRNAs and 20 different amino acyl tRNA synthetase in a protein synthesizing
system.
 The enzyme varies in molecular size, subunit and amino acid composition. These is at
least one and sometimes two specific enzyme for each amino acid.
 Very high specificty is the most significant feature, because once amino acid is attached
to tRNA, recognition of a specific codon on mRNA is entirely to the tRNA, not to the
amino acid.
 Very high specificty of amino acyl tRNA synthetases is due to high selectivity in
acceptance of the amino acid to be activated and high selectivity toward the tRNA to
which the activated amino acid will be transferred.
Steps of activation of amino acid
 The reaction requires amino acid, tRNA and ATP, first an intermediate is formed.
Amino acid +ATP → Amino acyl AMP + Ppi
 Transfer of aminoacyl group to tRNA
Amino acyl AMP + tRNA → Amino acyl tRNA + AMP

Overall reaction
Amino acid + ATP + tRNA → Amino acyl tRNA + AMP + Ppi
 In the amino acyl tRNA, the α carboxyl group of amino acid remains esterified with the
3’ OH of 3’ terminal Adenosine on acceptor arm of tRNA.

51
 The hydrolysis of pyrophosphate which renders the reaction virtually irreversible drives
the reaction to completion.
 Thus two high energy bonds of ATP are used in the formation of an amino acyl tRNA.
ii. Initiation
The initiation complex requires
i. 30S subunit
ii. f-met tRNA
iii. initiation factors – IF1, IF2, IF3
iv. GTP

v. mRNA (with initiation codon AUG)

The process of initiation can be divided in three stages
i. Formation of 30 S subunit
ii. Formation of 30 S initiation complex
iii. Formation of 70 S complex
i. Formation of 30 S subunit
 The 70S particle is made up of 50S and 30S subunit and they are in equilibrium.
30S + 50S ↔ 70S
 The Mg2+ ions present in physiological concentrations keep the ribosomes as tight 70S.
Since the initiation requires 30S particle, the dissociation of 70S particle is brought about by IF1
and IF3.
K1 IF3
70S ↔ 50S + 30S → 30S IF3 + 50S
K2 IF1
IF3 acts primarily as anti association factor while IF1 acts directly to increase K1
ii. Formation of 30 S initiation complex
 In the second stage, it involves f – met tRNA, GTP and 30S. the specific tRNA
molecule called as the initiator tRNA brings to the 30S subunit. It is AUG codon which acts as
the initiation point.
 The same enzyme aminoacyl transferase tRNA synthetase links both of these tRNA to
met, but a specific enzyme formyl transferase, adds a formyl group to the amino group of
methionine that is attached to tRNA to form N – fmet – tRNA.

52
 The formyl group is transferred from N10 – formyl tetrahydrofolate

tRNA N10 – formyl tetrahydrofolate

Methionine → met tRNA → N – fmet – tRNA
f transformylase

 Aminoacyl tRNA synthetase

 In bacterial mRNA being poly cistronic, bears as many initiation sites as the number of
genes present. Each mRNA possesses a ribosome binding site, a short sequence of bases of
purine rich nucleotides on the 5’ prime of the mRNA molecule called Shine Dalgarno sequence
which interacts with the 3’ end of 16 S rRNA.
 The consequence of purine rich region is 5’-AGGAAAU-3’. Following the binding of
three IF to the 30S ribosomal particle, mRNA and the initiator f-met tRNA bind to 30S
IF1, IF2, IF3 particle.
 IF2 is the factor responsible for the binding of initiator tRNA to the 30S particle. It
stablize the interaction of formylated initiator tRNA with 30 S particle.
 GTP is required in initiation complex formation. This allows the stable association of
IF2 with the 30S particle at a relatively low concentration of the factor.
iii. Formation of 70 S complex
The 30S initiation complex reacts with the 50S subunit to form the 70S initiation complex.
During the formation of 70S particle,GTP is hydrolyzed to GTP and Pi. IF 1 and IF2, GDP and Pi
are all released from the ribosomal complex.
30S.mRNA. IF1, IF2-f-met-tRNA.GTP + 50S →
mRNA.70S-f-met-tRNA.GTP + IF1 + IF2 + GDP + Pi
The hydrolysis of GTP is necessary to convert one steric effector, GTP to another GDP. Thus
GDP has low affinity for the ribosomal complex and dissociates from it, allowing IF 1 to effect
the release of IF2. The P site is occupied by f-met tRNA hydrogen bonded to AUG.

53
Elongation
The elongation cycle in protein synthesis begins with the insertion of an aminoacyl
tRNA into the empty site on the ribosome.
Stages in elongation
i. Transfer of aminoacyl tRNA to A site
ii. Formation of peptide bond
iii. Translocation

i. Transfer of aminoacyl tRNA to A site

 The delivery of an aminoacyl tRNA to the empty A site on the ribosome is carried with
the help of EF-TU. EF-TU contains GTP.
 After EF-TU has positioned the aminoacyl tRNA at A site, GTP is hydrolysed to GDP-
EF-TU that dissociated from the ribosome.
 EF-TS the second elongation factof joins the EF-TU complex and induces the release of
GDP. Finally GTP binds to EF-TU and EF-TS is released.
 EF-TU-GTP complex is now ready to pick up and deliver another aminoacyl t RNA.
ii. Formation of peptide bond
 Once the initiator f-met tRNA occupies the P site and the next amino acyl tRNA
occupies the A site, a peptide bond is formed by an enzyme peptidyl transferase belonging to
the 50S subunit.
 Activate f-met at P site is transferred to amino group of amino acyl tRNA at the A site
to form a dipeptidyl tRNA.
 The unchanged tRNA occupies the P site and the dipeptide bond formed is attached to
the second tRNA occupying the A site.
iii. Translocation
 This is the last step of the elongation cycle. The naked tRNA leaves the P site, the
peptidyl tRNA moves from the A site to the P site , mRNA moves nucleotides a head towords
3’ direction.
 This results in positioning of the next codon for reading by aminoacyl tRNA. It is
brought about by elongation factor G which is also called as translocase. EF-G bound to GTP
that derives the translocation step.

54
 Hydrolysis of GTP enables EF-G to be released from ribosomes. A site becomes vacnt
ready to accept next aminoacyl tRNA.

Termination
 This is the final step during the process of protein synthesis is stopped. Termination
codons (i.e) UAA, UGA, UAG and release factors RF 1, RF2 and RF3 are required for this
process.
 RF1 recognizes UAA and UAG, RF2 recognizes UAA and UGA. The third factor RF 3
does not posses any release activity of its own, but it binds to GTP and stimulates the binding
of RF1 and RF2 with the ribosomes.
 Polypeptide chain leaves the ribosome followed by tRNA and mRNA. 70S ribosome
dissociates into its constituent subunit.

Eukaryotic translation:
Translation is a process by which the sequence of nucleotides in a m rna molecule directs the
incorporation of amino acids in proteins. It occurs on ribosome.
Eukaryotic m rna:
 All eukaryotic m rna are monocistronic, coding for a single polypeptide specified by a
single cistron.
 It contain 5 region like leader and cap region, initiation codon, coding region, termination
codon, trailer and poly(A) tail.
 Eukaryotic m rna seems to be present in two forms:
a. Active form- present as polysomes and actively support translation.
b. Inactive form- remain associated with rna paticles and does not support
translation.
 5’cap is often associated with cap binding proteins where as 3’poly(A) with poly(A)
binding protein.
 Makes up only 3% of the total cellular rna.
Initiation sites:
 7 base length of conserved sequence called kozak sequence serves as initiation sites.

55
  Kozak sequences are 5’ACCATGG3’.
 This sequence is scanned by 40s ribosomal subunit to ignition the translation process.
Ribosomes and r rna:
 80s ribosomes are present in eukaryotic cytoplasm but mitochondria and chloroplast have
70s.
 Ribosomes contain 2 subunit, large subunit – 60s and smaller subunit- 40s.
 60s large subunit contain one copy each of 28s, 5s, 5.8s r rna with 49- l proteins.
 40s small subunits have 18s r rna with 33s protein
Mechanism of translation:
It occurs in 3 step mechanism,
i. Initiation
ii. Elongation
iii. Termination.
Initiation:
Scanning model of initiation:
 Eukaryotic ribosomes together with the intiator t rna(t rna met
) generally locate the
appropriate start codon by binding to the 5’ cap of the m rna.
 It scans downstream until they find the 1st AUG codon.
 The above complex finds the kozak sequence ACCATGG and binds tightly.
 the ribosomes by pass the 1st AUG and continue to scan for a most favourable one.
 Such bypass is inhibited by stem loop structure formed between cap and as initiation site.
 Eukaryotic initiation factor 4 F (e IF 4 F) is a cap binding protein composed of 3 parts
(4A, 4E, 4G).
 e IF 4 F recognise cap and initiate the translation.
 e IF 4A has helicase activity that unwinds the hairpins found in the 5’ leaders of m rna.
 This task requires the help of e IF4B and ATP.
 e IF 4G is an adaptor protein that is capaple to bind with e IF 4E and e IF 3 (40s
ribosomal binding protein).
 e IF 4G recruits 40s subunits to the m rna and stimulate translation initiation.

56
Elongation:
It proceeds in 3 steps, namely
a. Binding an aminoacyl t rna to the A site of the ribosomes.
b. Peptide bond formation.
c. Translocation.
Step I:
 A ternary complex formed by e EF-TU, eIF 4A and 2, transfers amino acyl t rna to
ribosomes A site without the hydrolysis of GTP.
 GTP is hydrolysed by a ribosome dependent GTPase activity of EF-TU and EF-TU+GTP
complex dissociate from the ribosomes.
 eF-TS generates an EF-TU GTP complex by exchanging GTP for GDP.
Step II:
 peptide bonds are formed by a ribosomal enzyme called peptidyl transferase.
 This activity residue on the 60s subunit.
 The r rna plays a major role in this activity and probably contains the catalytic centre.
Step III:
 Each translocation event moves the m rna one codon length, 3 nucleotide through the
ribosome.
 It requires GTP hydrolysis.
Termination:
 Termination describes the release of nascent polypeptide chain and separation of
complete ribosomes from the m rna.
 It is signalled by 3 codons UAG, UAA and UGA, is mediated by certain proteins called
ribosomal release factors (RRF).
Post translational modifications:
 In the final stage of protein synthesis, the nascent polypeptide chain is folded and
processed into its biologically active form.
 During or after its synthesis, the polypeptide progressively assumes its native
conformation, with the formation of appropriate hydrogen bonds and van der Waals,
ionic, and hydrophobic interactions.

57
  In this way the linear, or one-dimensional, genetic message in the mRNA is converted
into the threedimensional structure of the protein.
 Some newly made proteins, both prokaryotic and eukaryotic, do not attain their final
biologically active conformation until they have been altered by one or more processing
reactions called pos ttranslational modifications.
Amino-Terminal and Carboxyl-Terminal Modifications:
 The first residue inserted in all polypeptides is N-formylmethionine (in bacteria) or
methionine (in eukaryotes).
 However, the formyl group, the amino-terminal Met residue, and often additional amino-
terminal (and, in some cases, carboxyl-terminal) residues may be removed enzymatically
in formation of the final functional protein.
 In as many as 50% of eukaryotic proteins, the amino group of the amino-terminal residue
is N-acetylated after translation.
 Carboxyl-terminal residues are also sometimes modified.
Loss of Signal Sequences:
 Polysomes can occur either free in the cytoplasm or bound to membranes forming rough
ER.
 The membrane bound polysomes are synthesizing proteins which are either secreted from
the cell on form part of membrane or packaged into specialized organelles.
 Synthsized polypeptide itself carries a signal sequence (amino acid sequence) at or near
N terminals which mediates attachment to membranes.
 About 20 amino acids termed as signal peptide binds to a receptor of the membranes.
 Specific membrane associated peptidases remove signals peptides and imports the
proteins into the organelles.
Modification of Individual Amino Acids:
 The hydroxyl groups of certain Ser, Thr, and Tyr residues of some proteins are
enzymatically phosphorylated by ATP the phosphate groups add negative charges to
these polypeptides.
 The functional significance of this modification varies from one protein to the next. For
example, the milk protein casein has many phosphoserine groups that bind Ca 2+. Calcium,

58
 phosphate, and amino acids are all valuable to suckling young, so casein efficiently
provides three essential nutrients.
 Extra carboxyl groups may be added to Glu residues of some proteins. For example, the
blood-clotting protein prothrombin contains a number of carboxyglutamate residues in
its amino-terminal region, introduced by an enzyme that requires vitamin K.
Attachment of Carbohydrate Side Chains:
 The carbohydrate side chains of glycoproteins are attached covalently during or after
synthesis of the polypeptide. In some glycoproteins, the carbohydrate side chain is
attached enzymatically to Asn residues (N-linked oligosaccharides), in others to Ser or
Thr residues (O-linked oligosaccharides).
 Many proteins that function extracellularly, as well as the lubricating proteoglycans that
coat mucous membranes, contain oligosaccharide side chains.
Addition of Isoprenyl Groups:
 A number of eukaryotic proteins are modified by the addition of groups derived from
isoprene (isoprenyl groups).
 A thioether bond is formed between the isoprenyl group and a Cys residue of the protein.
 The isoprenyl groups are derived from pyrophosphorylated intermediates of the
cholesterol biosynthetic pathway, such as farnesyl pyrophosphate.
 Proteins modified in this way include the Ras proteins, products of the ras oncogenes and
proto-oncogenes, and G proteins, and lamins, proproteins found in the nuclear matrix.
The isoprenyl group helps to anchor the protein in a membrane
 The transforming (carcinogenic) activity of the ras oncogene is lost when isoprenylation
of the Ras protein is blocked, a finding that has stimulated interest in identifying
inhibitors of this posttranslational modification pathway for use in cancer chemotherapy.
Addition of Prosthetic Groups:
 Many prokaryotic and eukaryotic proteins require for their activity covalently bound
prosthetic groups.
 Two examples are the biotin molecule of acetyl-CoA carboxylase and the heme group of
hemoglobin or cytochrome c.

59
Proteolytic Processing:
 Many proteins are initially synthesized as large, inactive precursor polypeptides that are
proteolytically trimmed to form their smaller, active forms.
 Examples include proinsulin, some viral proteins, and proteases such as
chymotrypsinogen and trypsinogen.
Formation of Disulfide Cross-Links:
 After folding into their native conformations, some proteins form intrachain or interchain
disulfide bridges between Cys residues.
 In eukaryotes, disulfide bonds are common in proteins to be exported from cells.
 The cross-links formed in this way help to protect the native conformation of the protein
molecule from denaturation in the extracellular environment, which can differ greatly
from intracellular conditions and is generally oxidizing.

Phosphorylation:
 Other typ of processing involve the addition the addition of moieties to the amino acids
residue.
 Moieties such as lipids, complex co-factors and po4 groups may be added to proteins.
 Phosphorylation in which a po4 is transferred from ATP to a serine, threonine or tyrosine
residues by protein kinase.
 It plays an important role in biochemical processes as glycogen metabolism, glycolysis ,
gluconeogenesis and protein synthesis.
Carboxy methylation:
 Monomethyl and dimethyl lysine residues occur in some protein and in cytochrome c.
 Calmodulin involved in signal transduction contains tri methyl lysine.
Protein folding:
Polypeptide begins to fold as they enter ER lumen; folding of a 50 kDa protein is completed
within <3-4 minutes where its synthesis requires only 1 minutes.
 Folding occurs independently in a series of domains from N-terminus to C terminus.
 Protein folding is facialtes by the action of specialized protein called molecular
chaperones.

60
 a. Formation of disulphide cross links:
 After folding into their native conformations, some proteins from intra chain or
inter chain disulphide bridges between cytosine residues.
b. Protein acylation:
 Covalent attachment of lipids moieties to protein is called acylation.
 3 different modes of acylation have been characterized in plant cells.
i. Linkage between coo- gp and N gp of glycine.
ii. Formation of a thioester or ester bond between a.acids.
iii. Covalent attachment of a phosphatidyl inositol gp to coo- terminus.
Protein localization:
Proteins perform structural, hormonal, enzymatic or regulatory functions at specific sites within
or outside the cells. Therefore ptn have to be transported or localized at their respective sites.
Sites for protein functions:
The various sites of protein function may be summarised as follows,
1. Cytosol
2. Membranes
3. Organelles
4. Extracellular sites.
Cytosol:
 Proteins that function in cytosol are synthesized in the cytosol itself and remain there
either in a soluble form or as component of macromolecular structures like centrioles.
 No specific transport is needed for such proteins in the cytosol.
Membrane:
 Cytoplasm of eukaryotic cells contain numerous membranous bodies like ER, Golgi
apparatus, endosomes lysomes together called as vacuolar apparatus.
 In addition membranes enclose mitochondria, chloroplast and nuclei.
 Proteins become integrated into different membranes by specific interactions between
specific sequences or signals present in the protein molecules and receptor present with
membranes. This is known as protein trafficking or protein sorting.
Organelles:

61
  Cells contain several membrane enclosed spaces called organelles; to enter which protein
must enter through the membranes enclosing them is called protein translocation.
Extracellular sites:
 Some proteins may be located outside the cells where it is synthesised.
 These proteins enter the ER, Golgi apparatus and pass through the plasma membrane.
Such proteins are called secretary proteins.
Import of protein into mitochondria:
 Mitochondrial compartment contain its own genetic material, most of the proteins with
this semi autonomous structure are synthesized in the cytosol.
 Proteins that are destined for these organelles are synthesized as large precursors with an
amino terminal extension that is not present in the mature protein.
 This extra sequence is called as signal peptide or transit peptide.
Features of signal peptides:
 Size varies from 20-100 amino acids residues.
 There is usually an abundance of serine threonine in signal peptides.
 Small hydrophobic a.acids such as valine and alanine are quite often found.
 Overall charge of the transit peptide is usually basic with very few acidic amino acids
residues.
 Regions in transits peptide can form amphiphilic α helices or β sheets.
 Signal peptide binding protein was discovered by kregstra et al 1989.
 ATP is required to transport the ptns in organelles.
 Precursor’s ptns destined for mitochondria have amino terminal signal sequences which
are often bound by cytosolic chaperone proteins.
 The precursors are delivered to receptors on the exterior surface of the target organelle
then to to protein channel that usually span the inner and oute membrane of the
organelles.
 Translocation is facilitated by hydrolysis of ATP or GTP and in some cases trans
membrane electro chemical potentials.
 Mitochondrial signal sequences are generally 20-35 a.acids residues in length and are rich
in serine, threonine and basic residues.

62
  Protein precursors become bound by either HSP 70 or mitochondrial import stimulation
factors (MSTF).
 These chaperones stabilize unfolded conformation of the protein precursors.
 MSTF is specific to mitochondria; Hsp 70 belongs to a common class of chaperones.
 TOM and TIM are porins molecules encoded by nuclear genes with 4-6kda.
 Bound mitochondrial protein precursors is delivered to a ptn complex called TOM
(transport across mitochondrial outer membrane) and it provides a channel through the
outer membrane.
 Another ptn complex called TIM (transport across mitochondrial inner membrane),
translocates the precursors in to the mitochondrial matrix.
 Translocation is fuelled by interactions with a mitochondrial HSP 70 ptn coupled to ATP
hydrolysis and electro chemical gradient across the inner membrane.
 Within the matrix folding of the mt protein is facilitated by a number of chaperons and
signals sequences is removed by a processing (protease) in matrix.

e.g. alcohol dehydrogenase------matrix

subunit IV of cytochrome C oxidase----- inner membrane
cytochrome c ---- inner membrane space
10000 molecular weight protein----- outer membrane.
 After cleavage of signal peptides, proteins are folded and turned to be functional.
Import of protein into chloroplast:
 All nuclear encoded genes for chloroplast proteins have 5’ cap and 3’ poly (A) sequence
in m rna.
 They are translated on 80s ribosomes in the cytosol.
 They direct the synthesis of pre-proteins that contains additional aminoacids attached to
n-terminal of the mature ptns. Such sequences are called signal peptides.
 The signal sequences direct the protein into chloroplast and they are present in two
numbers. They differ from ER, Nucleus, mitochondria signals.
 These signals contain information for recognition and transport of the protein by the
chloroplast double envelope membranes.

63
  This was 1st demonstrated in the case of “RUBISCO” small subunits by Ellis in 1978.
Synthesis, transport and assembly of RUBISCO in chloroplast:
 Ribulose biphosphate carboxlase is an enzyme involved in photosynthesis reaction in
chloroplast.
 It is composed of 2 subunits, one is larger subunit encoded by chloroplast DNA other
smaller subunit is encoded by nuclear genes.
 Transit peptide is 20-100 amino acid length in RUBISCO enzyme.
 Smaller subunits synthesized in nucleus have to be imported into chloroplast (site of their
function).
 Precursor subunit carries 2 signal sequences which direct their transport in to thylacoid of
chloroplast.
 Signals peptide I id proteolytically cleaved before their entry into chloroplast membrane.
 Signal peptide II is removed before their entry into thylacoid membrane.
Import of protein into nucleus:
 Molecular communication between the nucleus and the cytosol requires the movement of
macromoles through nuclear pores.
 RNA molecules are synthesized in nucleus and are exported to the cytoplasm.
 Ribosomal proteins synthesised in cytosolic ribosomes are imported into the nucleus and
assembled into 60s and 40s ribosomal subunits in the molecules.
 A variety of nuclear proteins like RNA, DNA, polymerases, histones, topoisomerases are
synthesized in the cytosol and impoted into the nucleus.
 This traffic is modulated by a complex system of molecular signals and transport the
proteins.
 In most eukaryotes, the nuclear envelope breaks down at each cell division.
 Once cell division is completed and the nuclear envelope is e established, the dispersed
nuclear ptns must be re imported.
 To allow repeated nuclear importation the signal sequence (nuclear localization
sequences(NLS) is not removed.
 Nuclear importation is mediated by a number of proteins that cycle between the cytosol
and the nucleus called importin α and β.

64
  Another ptn called “Ran”, a GTP ase enzyme.
 A hetrodimer of importin α and β acts as a soluble receptor for proteins targeted to the
nucleus with α subunit binding NLS bearing proteins in the cytosol.
 The complex docks at a nuclear pore and is translocated through the pore by an energy
dependent mechanisms.
 This mechanism requires RAN GTP ase and separates the β importin from the complex.
 Finally NLS bearing protein dissociates from importin α inside the nucleus.
Synthesis of secretary and membrane protein:
 All eukaryotic cells use essentially the same secretory pathwayfor synthesizing and
sorting secreted proteins and soluble luminal proteins in the ER, Golgi, and lysosomes.
 For simplicity, we refer to these proteins collectively as secretory proteins.
 Although all cells secrete a variety of proteins (e.g., extracellular matrix proteins), certain
types of cells are specialized for secretion of large amounts of specific proteins.
 Pancreatic acinar cells, for instance, synthesize large quantities of several digestive
enzymes that are secreted into ductules that lead to the intestine.
 Because such secretory cells contain the organelles of the secretory pathway (e.g., ER
and Golgi) in great abundance, they have been widely used in studying this pathway.
Early pulse-labeling experiments with pancreatic acinar cells showed that radioactively
labeled amino acids are incorporated primarily into newly synthesized secretory proteins.
 The ribosomes synthesizing these proteins are actually bound to the surface of the ER. As
a consequence, the portion of the ER that receives proteins entering the secretory
pathway is known as the rough ER because these membranes are densely studded with
ribosomes .
 When cells are homogenized, the rough ER breaks up into small closed vesicles, termed
rough microsomes, with the same orientation (ribosomes on the outside) as that found in
the intact cell.
 Secretory proteins are synthesized on ribosomes bound to the cytosolic face of the ER
membrane, they become localized in the lumen of ER vesicles during their synthesis.
Synthesis of secretory proteins and their cotranslational translocation across the ER
membrane.

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  Once the ER signal sequence emerges from the the translocon, hydrolyze their bound
GTP and then are ready to ribosome, it is bound by a signal-recognition particle (SRP).
 The SRP delivers the ribosome/nascent polypeptide complex to the SRP receptor in the
ER membrane. This interaction is strengthened by binding of GTP to both the SRP and
its receptor.
 Transfer of the ribosome/nascent polypeptide to the translocon leads to opening of this
translocation channel and insertion of the signal sequence and adjacent segment of the
growing polypeptide into the central pore. Both the SRP and SRP receptor, once
dissociated from initiate the insertion of another polypeptide chain.

GENETIC CODE
Introduction
Genetic information is encoded in the base sequence of DNA molecule as a series of
genes. Gene expression is the term used to describe how cell decode the information to
synthesis proteins required for cell function. The expression of genes involves the synthesis of
complementary sequences of RNA molecule determine the sequence of amino acid of a
polypeptide.

Genetic code
It is a set of triplet code words in DNA or mRNA coding for the specific sequence of
amino acids of the protein.

Codon
A sequence of three adjacent nucleotides in a nucleic acid that codes for specific amino
acid.

Triplet code
There are 20 essential amino acids, each amino acids has to be coded by a codon. There
are four bases present in DNA (A,T,G,C) and RNA (A,U,G,C). When three bases combine to
form a codon, then there is a possibility of 43= 64 codons.

66
Characters of Genetic code
 The genetic code is a triplet code.
 The genetic code is comma free (i.e) continous.
 The genetic code is non overlapping. The mRNA nucleotide are read as lysine-lysine-
lysine.
 The genetic code is almost universal. All micro organism shares some genetic language.
Eg lysine is coded by AAA or AAG in many organisms. However it is not completely
universal.
 The genetic code is degenerate (i.e) one amino acid coded by more than one codon.
Eg phenyl alanine – UUU & UUC, lysine – AAA & AAG.
 The genetic code has start and stop codons. In both prokaryotes and eukaryotes the
AUG (codes for methionine) is usually the start codon. In some cases, GUG (codes for valine)
is used. Only 61 of 64 codons are sense codons which codes amino acid. The stop codons do
not specify any amino acid. They just specify the end of translation. They are UAG –amber,
UAA – ochre, UGA – opal. This three codons are non sense codon or termination codon.

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 Wobble hypothesis: it was proposed by Francis crick; nearly 61 tRNA molecules are
required for 61 codons. But actually less than 61 tRNAs are required. This is due to wobble in
anticodon (i.e) the nucleotide at the 5’ end of anticodon in tRNA can pair without any
complementry rule to 3’ end of codon in mRNA. Eg Inosine in anticodon 5’ end can pair with
A,U or C in codon 3’ end.

Nucleotide at 5’ end of anticodon Nucleotide at 3’ end of codon

G can pair with U/C
C can pair with G G
A can pair with U U
U can pair with A/G A/G
I can pair with A,U/G

 The genetic code exhibits contex effects in codon translation (i.e) site specific
variation.eg UGA is a stop codon. But when UGA is located inside, it codes for
selenocystiene. This site specific variation is called contex effect.

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 QUESTION BANK
UNIT III
1 MARK
1. Translation.
2. Ribosomes.
3. Phosphorylation.
4. Genetic code.
5. Signal peptides.

5 MARK
6. Analyze the events in translation process.
7. How are proteins imported into mitochondria?
8.Genetic code.
9.How are proteins imported into nucleus?
10.How are proteins imported into chloroplast?
10 MARK

9. Explain the details about translation in prokaryotes.

10. Describe about post translational modifications.
11. How are proteins imported into mitochondria and chloroplast?

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UNIT IV
Operon concepts in prokaryotes
Francois Jacob and Jacques Monod in 1961 proposed a model in order to induction
and repression of enzyme synthesis. This model is popularly known as operon model. They
studied the induction of β-galactosidase activity in E.coli.
Operon is a set of structural genes arranged together in a regulating unit under the control of a
single promoter.
An operon is defined as a unit of coordinated control of protein synthesis which consists
i. An operator gene which controlled the activity. It was controlled by a repreesor
molecule synthesized by a regulator gene which is not a part of operon. It is present on
either side of promoter.
i. A number of structural gene which took part in synthesis of proteins. It will synthesize
mRNA under the control of an operator gene.
Thus the members of an operon are transcribed coordinately a single, long, polycistronic
mRNA molecule, one such operon in E.coli is called the lactose/ lac operon.
Lac operon of E.coli
Lactose is a disaccharide composed of a single glucose unit attached to a single
galactose unit. It is used as carbon and energy source. Lactose is broken down into glucose
and galactose by the action of the enzyme β-galactosidase which has a tetrameric structure of
identical proteins.
Regulation of lactose utilization
The molecular and genetic relationship between enzyme induction and enzyme repression.
Induction
 There are three different loci in the genetic map of the E.coli that influence the
formation of β-galactosidase designed as Z, I & O.
 The Z locus specifies the amino acid sequence of the β-galactosidase molecule.
Mutations in this locus lead to synthesis of a faulty / an inactive enzyme molecule. This
gene is called a structural gene.
 The I locus functions as an inhibitory gene and determine whether the structural gene
for β-galactosidase will be transcribed. Such gene are called regulatory gene. This

70
 undergoes mutation and become defective, they can no longer inhibit the transcription of
the structural genes.
 If the bacteria, grown in a medium containing lactose as the sole carbon and energy
source there are about 3000 copies of this enzyme is present. This process is known as
induction. Then the substrates (lactose) ars called as inducers and the enzymes are called as
inducible enzymes.
Repression
 The opposite phenomenon to induction is commonly known as repression. The
regulatory gene codes for an amino acid sequence of a specific protein are called repressor.
 The repressor protein physically binds a specific site on the DNA of the cell next or
near the structural gene whose product it controls.
 When the repressor is bound in this manner, it prevents the trnscription of the structural
gene coding for the enzyme.
 The site at which the repressor binds is called operator or the O locus. It is located close
to the structural gene for β-galactosidase.
Operon model
In the absence of an inducer (lactose)
In the absence of an inducer, the repressor (allolactose) occur in its active site and
combines with the operator (O) locus, thus preventing the transcription of structural gene for
β-galactosidase.
In the presence of an inducer (lactose)
 Inducer combines with the repressor protein to form a repressor inducer complex,
rendering the repressor incapable of binding to the O locus.
 The structural gene for enzyme becomes free for transcription to yield mRNA and the
enzyme is then synthesized.
 The interaction between the repressor and the inducer is reversible and when the
inducer is removed from the medium or used up by the enzyme, the repressor reverts to its
inhibitory form and binds to the operator, prevents the synthesis of enzyme.
 It induces a set of three enzymes namely, β-galactosidase, permease and acetyl
transferase which are coded by three genes Z, Y and A respectively. It is regulated by I and
O locus.

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Mechanism of enzyme repressor
 The enzyme whose amount is reduced by the presence of their end product is called
repressible enzymes.
 The end product metabolites when introduced into the growth medium specifically
decrease the amount of particular enzyme. These are known as co-repressor.
 The repressor molecule in such cases is inactive by itself, but binds the repressing
metabolite to form a repressor-co-repressor complex. This complex then binds to O locus
and prevents the transcription of the corresponding structural genes.
 There are two classes of repressor molecule, one operative in the induction of enzyme
activity and the other in repression of the end product.
Promoter
The promoter represents the short sequence of bases normally less than 100
nucleotides, which is recognised by a DNA dependent RNA polymerase. It lies in between the
regulatory gene and the operator. It is also the binding site for another specific type of protein,
the cyclic AMP receptor protein (CRP)/ catabolite activator protein (CAP).
In E.coli the glucose effect is brought about two molecule intermediates, namely CAP
and CAMP. CAP is sensitive to interacellular level of CAMP and has no influence on

72
 transcription until CAMP binds with it. It is the positive control element for all the glucose
sensitive operons. Its main purpose is to allow preferential catabolism of glucose.
Important features in lac regulation
The operon is under negative control, when it interacts with the lac repressor, operon
transcription is inhibited. The repressor is specified by lac I regulatory gene.
The operon is under positive control, when it interacts with a regulatory protein, such
as CAP, operon transcription is enhanced. A risk in glucose concentration causes CAP to
leave its controlling element and thus lac transcription drops significantly.
Structure of the lac operon

2. The lac operon consists of three structural genes, and a promoter, a terminator, regulator,
and an operator. The three structural genes are: lacZ, lacY, and lacA.
 lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the
disaccharide lactose into glucose and galactose.
 lacY encodes β-galactoside permease (LacY), a membrane-bound transport
protein that pumps lactose into the cell.
 lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an
acetyl group from acetyl-CoA to β-galactosides.
Only lacZ and lacY appear to be necessary for lactose catabolism.

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Trp operon:

Structure of the trp operon.

Trp operon is an operon - a group of genes that are used, or transcribed, together - that
codes for the components for production of tryptophan.
 The Trp operon is present in many bacteria, but was first characterized in Escherichia
coli. It is regulated so that when tryptophan is present in the environment, it is not used. It
was an important experimental system for learning about gene regulation, and is
commonly used to teach gene regulation.
 Discovered in 1953 by Jacques Monod and colleagues, the trp operon in E. coli was the
first repressible operon to be discovered.
 While the lac operon can be activated by a chemical (allolactose), the tryptophan (Trp)
operon is inhibited by a chemical (tryptophan).
 This operon contains five structural genes: trp E, trp D, trp C, trp B, and trp A, which
encodes tryptophan synthetase.
 It also contains a promoter which binds to RNA polymerase and an operator which
blocks transcription when bound to the protein synthesized by the repressor gene (trp R)
that binds to the operator.

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  In the lac operon, allolactose binds to the repressor protein, allowing gene transcription,
while in the trp operon, tryptophan binds to the repressor protein effectively blocking
gene transcription.
 In both situations, repression is that of RNA polymerase transcribing the genes in the
operon. Also unlike the lac operon, the trp operon contains a leader peptide and an
attenuator sequence which allows for graded regulation.
 It is an example of negative regulation of gene expression. Within the operon's regulatory
sequence, the operator is blocked by the repressor protein in the presence of tryptophan
(thereby preventing transcription) and is liberated in tryptophan's absence (thereby
allowing transcription). The process of attenuation (explained below) complements this
regulatory action.
Repression:
 This is a negative repressive feedback mechanism. The repressor for the trp operon is
produced upstream by the trpR gene, which is continually expressed at a low level. It
creates monomers, which associate into tetramers.
 These tetramers are inactive and are dissolved in the nucleoplasm. When tryptophan is
present, these tryptophan repressor tetramers bind to tryptophan, causing a change in
conformation (in the repressor), which allows the repressor to bind the operator.
 This prevents RNA polymerase from binding to and transcribing the operon, so
tryptophan is not produced from its precursor.
 When tryptophan is not present, the repressor is in its inactive conformation and cannot
bind the operator region, so transcription is not inhibited by the repressor.
Attenuation:
 Attenuation is a second mechanism of negative feedback in the trp operon. While the
TrpR repressor decreases transcription by a factor of 70, attenuation can further decrease
it by a factor of 10, thus allowing accumulated repression of about 700-fold.
 Attenuation is made possible by the fact that in prokaryotes (which have no nucleus), the
ribosomes begin translating the mRNA while RNA polymerase is still transcribing the
DNA sequence. This allows the process of translation to directly affect transcription of
the operon.

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 At the beginning of the transcribed genes of the trp operon is a sequence of 140
nucleotides termed the leader transcript (trpL). This transcript includes four short
sequences designated 1-4.

Mechanism of transcriptional attenuation of the trp operon.

 Sequence 1 is partially complementary to sequence 2, which is partially complementary
to sequence 3, which is partially complementary to sequence 4. Thus, three distinct
secondary structures (hairpins) can form: 1-2, 2-3 or 3-4.
 The hybridization of strands 1 and 2 to form the 1-2 structure prevents the formation of
the 2-3 structure, while the formation of 2-3 prevents the formation of 3-4.
 The 3-4 structure is a transcription termination sequence, once it forms RNA polymerase
will disassociate from the DNA and transcription of the structural genes of the operon
will not occur.
 Part of the leader transcript codes for a short polypeptide of 14 amino acids, termed the
leader peptide. This peptide contains two adjacent tryptophan residues, which is unusual,

76
 since tryptophan is a fairly uncommon amino acid (about one in a hundred residues in a
typical E. coli protein is tryptophan).
 If the ribosome attempts to translate this peptide while tryptophan levels in the cell are
low, it will stall at either of the two trp codons.
 While it is stalled, the ribosome physically shields sequence 1 of the transcript, thus
preventing it from forming the 1-2 secondary structure. Sequence 2 is then free to
hybridize with sequence 3 to form the 2-3 structure, which then prevents the formation of
the 3-4 termination hairpin, thus the 2-3 structure is called anti-termination hairpin.
 RNA polymerase is free to continue transcribing the entire operon. If tryptophan levels in
the cell are high, the ribosome will translate the entire leader peptide without interruption
and will only stall during translation termination at the stop codon.
 At this point the ribosome physically shields both sequences 1 and 2. Sequences 3 and 4
are thus free to form the 3-4 structure which terminates transcription.
 The end result is that the operon will be transcribed only when tryptophan is unavailable
for the ribosome, while the trpL transcript is constitutively expressed.
To ensure that the ribosome binds and begins translation of the leader transcript immediately
following its synthesis, a pause site exists in the trpL sequence. Upon reaching this site, RNA
polymerase pauses transcription and apparently waits for translation to begin. This mechanism
allows for synchronization of transcription and translation, a key element in attenuation.
A similar attenuation mechanism regulates the synthesis of histidine, phenylalanine and
threonine.
GENE SILENCING:
Control of gene expression by controlling when and how often the gene is transcribed. It
is called as gene silencing. It occurs by
a. DNA methylation
b. RNA editing
c. Regulated RNA transcript
d. Change in m RNA stability
e. Cytoplasmic poly(A) addition.
DNA methylation:
 Transcription can be regulated by methylation of cytosine residues at DNS level.

77
  An enzyme called maintenance methy transferase acts on those CG sequences on DNA.
 Addition of a methyl group to DNA.
 Methylation of cytosine base in CG sequences is used to keep the genes in an inactive
state.
RNA editing:
 Production of a functional m RNA by insertion or alteration of individual nucleotides in
an RNA molecule after it is synthesized.
 R RNA and t RNA are modified in ways transcriptionally.
Regulated RNA transcript:
 1/20 of the total mass of RNA synthesized ever leaves the nucleus.
 Such left over RNA are degraded in nucleus.
 Incompletely processed or damaged RNA are also eventually degraded as a part of the
quality control system of RNA production.
 This degradation is coded out by the exosomes.
Change in m RNA stability:
 M RNA in eukaryotic cells is more stable. E.g β-globin half lives of more than 10 hours.
 There are 2 major degradation pathway to change the m RNA stability namely
a. De adenylation dependent decay:
Once the mRNA enters the cytosol, its polyA tail (average about 200 adenine
in length) is gradually shortened by an exonucleae and 5’ cap is removed by
decapping. Finally the RNA is degraded rapidly.
b. De adenylation independent decay:
m RNA is degraded by the action of specific endonucleases which cleave
simply the poly A tail from the rest of the m RNA in one step.

Cytoplasmic poly (A) addition:

 The initials polyadenylation of an RNA molecule occurs in the nucleus for all eukaryotic
m RNA precursors.
 Then the poly (a) tails are gradually shortened in cytoplasm.
In some cases poly (a) tails of specific m RNA are lengthened in the cytosol for the
translational regulation.

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RECOMBINATION

Recombination basically involves the exchange of genetic information. There are mainly two
types of recombination.

1. Homologous recombination;

This is also called as general recombination, and occurs between identical or nearly identical
chromosomes ( DNA sequences). The best example is the recombination between the paternal
and maternal chromosomal pairs.

2. Non- Homologous recombination;

This is regarded as illegitimate recombination and does not require any special homologous
sequences. Transposition is a good example of non homologous recombination. Random
integration of outside genes into mammalian chromosome is another example.

Homologous recombination;

It is known fact that the chromosomes are not passed on intact from generation to generation.

Instead, they are inherited from both the parents. This is possible due to homologous
recombination. Three models have been put forth to explain homologous recombinations.

 Holliday model
 Meselson- Radding model
 Double-strand break model.

Holliday model;

Holliday model (proposed by Holliday in 1964) is the simplest among the homologous
recombination models. The two homologous chromosomes come closer, get properly aligned,
and form single-strand breaks. This results in two aligned DNA duplexes. Now the strands of

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each duplex partly unwind and invade in the opposite direction to form a two strands cross
between the DNA molecules.

There occurs simultaneous unwinding and rewinding of the duplexes in such a way that there is
not net change in the amount of base pairing, but the position of crossover moves. This
phenomenon referred to as branch migration, results in the formation of heteroduplex DNA.
The enzyme DNA ligase seals the nick. The two DNA duplexes (4 strands of DNA ), joined by a
single crossover point can rotate to create a four – strand Holliday junction. Now the DNA
molecules are subjected to symmetrical cuts in either of the two directions, and the cut ends are
resealed by ligase.

The DNA exchange is determined by the direction of the cuts which could be horizontal or
vertical. If the cross strands are cut horizontally the flanking genes remain intact, and no
recombination occurs on the other hand, if the parental strands are cut vertically, the flanking
genes get exchanged due to recombination.

Holliday model

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Non- Homologous recombination

The recombination process without any special homologous sequences of DNA is regarded as
non homologous recombination.

Transposition

Transposition primarily involves the movement of specific pieces of DNA in the genome.

The mobile segment of DNA are called transposons or transposed elements they were first
discovered by Barbara Macclintock (in 1950) in maize, and their significance was ignored for
about two decades by the other workers.

Retrotranspositions;

Transposition involving RNA intermediate represents retrotransposition. By the normal process

of transcription, a copy of RNA formed from a transposon(also called as retrotransposons). Then
by the enzyme reverse transcriptase, DNA is copied from the RNA. The newly formed DNA
which is a copy of the transposon gets integrated into the genome. This integration may occur
randomly on the same chromosome or, on a different chromosome. As a result of the
retrotransposition, there are now two copies transposons, at different points on the genome.

DNA transposition;

Some transposons are capable of direct transposition of DNA to DNA. This may occur either by
replicative transposition or conservative transposition. Both the mechanisms require enzymes
that are mostly coded by the genes with in the transposons.

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 QUESTION BANK
UNIT-IV
1 MARKS
1. Operon
2. Recombination
3. Gene silencing
4. Enhancer
5. Repressor
6. Holiday junction
7. Gene expression
5 MARK & 10 MARK
8. Describe about the lac operon
9. Explain in detail about the recombination
10. Comment on gene silencing
11. Discuss about structure & functions tryp operon

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UNIT V
ONCOGENES (CELLULAR & VIRAL ONCOGENES)
Cancer causing genes are called as oncogenes or transforming genes. Some
changes in cellular genes can cause normal cells to transform into neoplastic cells (tumor). This
change is called as epigenetic changes, e.g. mutation & viral infections. Proto oncogenes are wild
type allele of oncogenes. Upon activation the proto oncogenes are transformed into oncogene
that leads to cancer development.

SNO ONCOGENES PROTO ONCOGENES PROTEIN PRODUCTS

1 Sis c-sis PDGF B chain
2 erB c-erB,2,3 EGF receptor kinases
3 Ras c-ras GTP/GDP binding protein
4 Src c-src Membrane associated protein
5 Raf c-raf Protein serine kinases
6 Mos c-mos Protein serine kinases
7 Myc c-myc DNA binding protein
8 Myb c-myb Transcription factors

VIRAL AND CELLULAR ONCOGENES:

Oncogene is the genes that have the potential to cause cancer. In tumor cells
they are often mutated or expressed at high levels. Proto oncogenes are the normal gene that
controls the normal cell division and differentiation.
Oncogenes upon mutations or viral infections synthesize oncoprotein (regulate
cell growth and differentiation- involved in signal transduction). Upon activation the
protooncogenes are converted into tumor inducing agents e.g., RAS, WNT, MYC, ERK, TRK
Myc – Burkitts lymphoma- chromosomal translocation moves an enhancer sequence
HISTORY
1. First discovered oncogenes-src (sarcoma) – chicken retro virus1970, by G. Steve Martin, UC.
2. Wild of v-src-sequenced by A.P (Zernilofsky, 1980)
3. 1976, J.Micheal Bishop, Harold E.varmus won noble prize and postulated that oncogenes are
defective proto oncogenes.
ONCOGENE ACTIVATION:
There are 3 basic activation steps;

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1. Mutation within proto oncogenes can cause change in protein structure that leads to increased
protein activity & loss of regulation
2. An increased in protein concentration causes increased protein level, increased protein
stability (prolonged existence0 and gene duplication.
3. A chromosomal translocation leads to increased gene expression in the wrong cell type or at
wrong time and increased expression of active hybrid proteins

MECHANISM CONVERTING PROTO ONCOGENES TO ONCOGENES

Proto-oncogenes are termed also as celullar oncogenes (c-onc) with acronyme from
corresponding viral oncogene (e.g. c-src, c-myc, c-myb etc). They are not cancer genes. The term
”proto-oncogenes” is used to denote their ability to require an oncogenic potential.
In normal cells they have functions in cell growth and differentiation: They
• code for phosphorylate proteins
• influence DNA replication
• control mRNA production
• bind GTP
Since the retroviral oncogenes are mutants of normal proto-oncogenes , they have provided a
key to open a door to understand proto-oncogene – oncogene conversion. It was accepted that
following crucial events may be responsible for conversion of proto-oncogenes to”cancer”
genes:

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There are 5 mechanisms namely,
1. Point mutation
2. Proviral insertion
3. Deletion
4. Translocation
5. Amplification

1. POINT MUTATION (SINGLE NUCLEOTIDE SUBSTITUTION)

Substitution or deletion or addition of one or a few bases is called as point mutation.

Ras oncogenes family consists of 3 ras cellular oncogenes (H- ras, K-ras, N-ras). Ras oncogene
encodes 21 Kda (p21) membrane associated proteins binds to GTP-G proteins involved in signal
transduction. Wild type c-ras-glycine (12), glutamine (61) mutation bladder carcinoma
Proto oncogenes Hras 12glycine 61glutamine wild type protein
Oncogenes VH-ra 12 12 valine 61glutamine Bladder carcinoma
Oncogenes VH-ras 12 12 glycine 61leucine lung cancer
TYPES OF POINT MUTATION:

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Point mutations are single base changes, that do not affect the reading frame; that is, the mutation
only makes a single change in a single codon, and everything else is undisturbed.  There are
three types of point mutation:

 Silent Mutation: There is a base change, but the new codon means exactly the same
thing as the old one; this is due to the degeneracy of the codon -> amino acid conversion
code.  There is no phenotypic change.
 Missense Mutation: The mutation alters the meaning of the codon, so that the amino
acid coded for is not the one that is supposed to be there. This could have no phenotypic
effect, if the substituted amino acid was similar in character to the original; it might be a
nonfunctional protein, or it could be a conditional lethal, where the protein works under
normal conditions, but not in the same operating range as the original protein.
 Nonsense Mutation:  This mutation changes the codon to a stop codon, which
prematurely ends translation when the mRNA transcript is being read by the ribosomes. 
This almost always results in a nonfunctional protein, because the latter chunk of it will
be missing.

When talking about point mutations, it is important to remember which bases are purines (A/G)
and which are pyrimidines (C/T).  When a point mutation causes a purine to convert to another
purine (for example, C to T), this is known as a transition.  When a point mutation changes a
purine to a pyrimidine, or vice versa, (i.e., A to T), this is known as a transversion.

Frameshift mutations alter the reading frame of the DNA. They come in two flavors:

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  Insertion: This mutation inserts a base pair (or more) into the DNA, shifting everything
to the right (or left, depending on your point of view) by one base pair.
 Deletion: This mutation deletes a base pair (or more), shifting everything the opposite
direction of the insertion.

In either case, it should be obvious that a shift in the reading frame will create a random mess of
a protein (much like reading a sentence will if you chop off the first letter of each word and stick
it to the end of the previous word). DNA’s coding is in such a way that an altered reading frame
will generate stop codons, to limit the amount of energy expended if such mutation occurs. That
way, the cell won’t waste energy building the new protein, if it absolutely makes no sense and
has no function.

2. PROVIRAL INSERTION:
A provirus is a virus genome that is integrated into the DNA of a host cell. A provirus does not
directly make new DNA copies of itself while integrated into a host genome in this way. Instead,
it is passively replicated along with the host genome and passed on to the original cell's
offspring; all descendants of the infected cell will also bear proviruses in their genomes.

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Figure: Proto-oncogene activation by insertional mutagenesis of MMTV. (a) Insertion of viral
genomic DNA into somatic cellular DNA in close proximity of a silent oncogene. (b) Inserted
proviral DNA induces the transcription of the oncogene.

E.g., Avian Leukemia virus (ALV)

ALV lacks oncogenes but can induce tumors by activating cellular proto
oncogenes. C myc contains 3 exons separated by 2 introns. First exons don’t encode a protein
which is invoved in regulatory mechanism and are untranslated. It contain 2 promotors namely
p1 & p2.
3. Deletion
Removal of a part of coding sequence is called as deletion. Crb B gene- epidermal
growth factor receptor glycoprotein which has an intrinsic kinase activity
4. Translocation
Change in the position of a chromosomal segment within the complement
chromosome is called as translocation. It results in the formation of fused elements.
 E.g., Burkitts Lymphoma 8:14 translocation.
Chronic Myelogenous Leukemia 9:22

88
5. Amplifications
Localized reduplication of DNA is called as amplification.
EG., K-ras gene- 30 fold amplified in the Y1DM mouse adrenal gland
A. sis oncogenes:
1. Proto oncogene of sis (promote growth) - human- platelet derived growth factor B
2. Contain unique homo purine & homo pyrimidine promoter sequence
3. Encodes PGDFB (activate receptor protein tyrosine kinase) which have mitogen and
chemotactic factor functions in connective tissue cells
4. V-sis oncogenes obtained from Simian sarcoma virus can cause Glioblastoma, fibro sarcoma,
Melanoma, lung and breast carcinoma
B. ras oncogenes ( RAT sarcoma):
1. Transforming oncogenes
2. 1st identified in Harvey and Kirsten sarcoma viruses
3. Named by Edward M. Scolnick of NIH
4. Viruses with ras oncogene were discovered by Jennifer Harvey and Wernee Kirsten 1960.
5. Its counterpart is H-ras, N-ras & X-ras proto oncogenes.
6. Ras genes encode small GTPases which are involved in cellular signal transduction, cell
growth, differentiation and cell survival.
7. Ras protein contain 6 beta sheets and 5 alpha helices

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8. G- domain contains 166 amino acids that binds to guanosine nucleotide and have a molecular
weight of 20kda.
9. Point mutation causes ras proto oncogenes into ras oncogene.
C. crb-B oncogenes:
1. Otherwise known as HER2 oncogenes (Human Epidermal Growth factor Receptor 2);
2. HER 2 protein is a cell membrane surface bound receptor tyrosine kinase
3. EGF is 6kda protein which is composed of 53 amino acids
4. Amplification of HER 2 that leads to over expression of protein products
5. It causes Ovarian cancers, stomach cancer.
6. V- crbB seen in avian erythroblastosis virus.
D. src oncogenes (sarcoma):
1. Discovered by Micheal Bishop, Harold E. Varmus, 1989
2. Encode membrane associated tyrosine kinases
3. Retro virus-v src (Rous Sarcoma Virus)
TUMOR SUPPRESSOR GENES OR ANTIONCOGENES
Genes that suppress the tumors caused by oncogenes is called as tumor
suppressor genes. It blocks the tumor development by regulating genes controlling cell growth.
Loss of function i.e., deletion or inactivating mutations in TSG leads to CANCER.

HISTORY OF TUMOR SUPPRESSOR GENES:

Cellular oncogenes causing cancer was discovered by Krontiris,
Cooper 1981. They concluded that dominantly acting genes are oncogenes. Harris, Klien 1969,
1971 performed Hybridization experiments to describe the tumor suppressor genes.

Functions of tumor suppressor genes:

Tumor suppressor genes encode tumor suppressor proteins
which shows repressive effects on the regulating of the cell cycle or promote apoptosis
1. Repression of genes - Cell cycle does not continue inhibits cell division
2. Coupling the cell cycle to DNA damage
3. Initiate apoptosis to remove the threat

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4. Block the loss of contact inhibition and inhibits metastasis. So called as metastasis inhibitors
or suppressors
5. DNA repair proteins are tumor suppressor proteins.
If mutation in such genes cause cancer
Characteristics of tumor suppressor genes:
Mutation in tumor suppressor genes leads to loss of function those results in
uncontrolled growth or cancer.
Tumor suppressor genes require 2 independent mutational events to cause
abnormal growth regulations.
Mutations in tumor suppressor genes cause sporadic form of cancer which is
not transmitted to the next generations. Tumor suppressor genes are tissue specific in causing the
tumors.

GENE PROTEIN FUNCTIONAL FEATURES

RB pRB Transcriptional regulation
P53 P53 Transcription factor
WT1 WT1 Transcriptional regulation interact
with splicing factors
VHL VHL Interacts with elongation factors
INK4A/MTS1 P16 Involved in cell cycle regulation;
inhibitors of cyclins-cdk complex
BRCA1 BRCA1 ? zinc finger proteins
BRCA2 BRCA2 ?
NF1 NF1 GTPase activating protein acts on
ras protein
NF2 NF2 Effect cytoskeletal organization
APC APC Biunds beta catenin of cuts in cell-
cell adhesion and signaling
PTC PTC Plasma membrane protein and
signaling
FNIT FNIT Functions in nucleotide metabolism
DPC4 DPC4 Transcriptional regulation

STRUCTURE, FUNCTION & ACTION OF PRB

(TS PROTEIN)

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 Retinoblastoma is a tumor of the eye- which is seen in the children of <5
years of age. RB gene is located in the long arm of chromosome 13. Mutation in RB gene leads
to bone cancer (Osteosarcoma), bladder, prostate, and breast and small cell lung carcinomas.
The gene is 200 kb in size and contains 27 exons (31-1899 nucleotides)
which are separated by introns (80 bp-60kbp). It contain promoter which is located 70bp
upstream region and are rich in G+C content
1st TSP discovered is Prb tumor suppressor protein. It is Tissue specific and species specific in
function.
STRUCTURE OF RB PROTEIN
RB gene encodes Prb a nuclear phospho protein binds
nonspecifically to ds DNA. Its molecular weight is 106kda and contains 928 amino acids.
PRB contain 4 protease resistant globular domains (N terminal, R, A, B
domains). N & R domains are responsible for oligomerization of the protein under invitro.
A & B domains are altered during tumor or growth suppression in cell
culture. During G1 phase of the cell cycle, it undergoes phosphorylation and binds to E2F2 and
oncoproteins of DNA tumor viruses (SV40). C-terminal proteins (300 amino acids) has non
specific DNA binding activity
TUMOR SUPPRESSION BY THE RB GENE:
RB gene inactivation leads to tumor formation and causes Retinoblastoma soft
tissue sarcoma, osteosarcoma, lung esophagus, breast, prostate and renal carcinomas
RB genes due to mutation acquire the loss of function that causes the oncogenic
transformation of retinal precursor cells.
RB PROTEINS OF CELL CYCLE REGULATION:
RB proteins interact with several proteins in two ways namely tissue dependent
manner and time dependent manner. Mutated oncoprotein binds to RB and form a complex that
causes inactivation of PRB proteins. Nearly 30 cellular proteins bind to PRB namely
transcriptional factors, growth regulators, protein kinase, protein phosphates and nuclear
proteins.

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1. PHOSPHORYLATION:
During G0 & G1 phases the PRB protein is unphosphorylated & poorly
phosphorylated which is in active state.
In the S1, G2, M phases, the PRB under goes increased level of
phosphorylation which is inactive state and the cell cycle will progress.
During late mitosis, the PRB gets dephosphorylated by enzyme protein Phosphatase I which
become active.
2. BINDING OF UNPHOSPHORYLATED PRB TO PROTEINS IN G1 PHASE:
In unphosphorylated state, the PRB proteins are bounded with nuclear associated protein
like p84, E2F & TF. P84 binds to N terminal portion of hypophosphorylated of PRB. During
G0/G1, the PRB binds to TF-E2F and form of E2F - RB complex. E2F is a transcription factor of
viral and cellular genes involved in DNA replication.
1. Late M phase – Dephosphorylation of PRB:
2. G1 phase – Unphosphorylated of PRB binds to proteins:
3. Mid G1 – PRB phosphorylation, E2F release & division signal:
Cyclin dependent kinases are cdc2 kinase enzyme which is a gate keeper of G1
to S and G2 to M phase transitions. CDK contain 2 subunits namely catalytic subunit (cdk) and a
regulatory subunit (Cyclin).

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 Viral oncoprotein like large T antigen SV 40, E1A of Adeno virus, E7 of
Papilloma virus binds to unphosphorylated PRB and inactivates PRB at G1 so that the cell cycle
will progress into S phase & leads to tumor.
4. S & G2 – Phosphorylation of PRB:
Phosphorylation of PRB at serine or threonine residues takes place at S
phase and near G2/Mtransition
5. Association of RB with proteins in the M phase:
H-Nuc is 90 kda proteins which is responsible for metaphase spindle
elongation. Mitosin is 350 kda which is called as PRB associated proteins. Over expression of
Mitosin causes delayed exit from G2 / M transition.
p53 Tumor suppressor gene:
The gene p53 encodes for a protein with a molecular weight 53 kilodaltons (hence the name). it
is believed that the protein produced by this gene helps DNA repair and suppress cancer
development. Certain damages that occur in DNA may lead to unlimited replication and
uncontrolled multiplication of cells. In such a situation, the protein encoded by p53 gene binds to
DNA and blocks replication. Further, it facilitates the faulty DNA to get repaired. The result is
that the cancerous cells are not allowed to establish and multiply. Thus, p53 is a cancer
suppressor gene and acts as a guardian of cellular DNA.
Any mutation in the p53 is likely to alter its tumor suppressor function that lead to cancer
development. And in fact, the altered forms of p53 recovered from the various tumor cells
(breast, bone, brain, colon, bladder, skin, lung) confirm the protective function against cancers.
It is believed that the environmental factors may cause mutations in p53 gene which may
ultimately lead to cancer. Some of the mutations of p53 gene may be inherited, which probably
explains the occurrence of certain cancers in some families.
STRUCTURE, FUNCTION & ACTION OF P53
P53 gene is10kb long and contain 11 exons. The 1 st exon is non coding
sequence located 6-10kb away from the other 10 exons. P53 gene keeps the cell normal. If P53
gene is absent / mutated / complexes with other molecule, it leads to cancer.
Vogel Stein, 1989 discovered the P53 gene which is responsible for
colorectal cancer. P53 gene encodes P53 protein which is a transcriptional regulator activated in
response to DNA damage. The C - terminal of the gene product binds to DNA.

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FUNCTION OF P53 PROTEIN:
1. It halts the cell cycle until the DNA damage is repaired
2. P53 acts as transcriptional factor and stimulates the synthesis of 21kda proteins which blocks
CDK from interacting with cyclin.
3. P53 trigger apoptosis
4. P53 stimulates the DNA repair mechanisms.
P53 PROTEIN:
The p53 Protein is a phospho protein which contain393 amino acid in it. It exists as
tetramer (4 subunits). The 3subunit forms structural domains.
1. N-terminal domain-1st 75-80 amino acids- very acidic
2. Amino acids 75-150 are praline rich, hydrophobic
3. C-terminal domain 319-393- basic
The p53 Protein remains stable for 20 minutes in the normal cell. But the
mutated form of p53 Protein remains several hour within the cell.

P53 PROTEIN AND CELL CYCLE:

Cell division cycle contains 4 phases like G1, S, G2 & M. the progression of
cell cycle depends on protein complex. The protein complex is formed by cyclin dependent
kinases & cyclin which is found only during cell cycle. CDK’s add phosphate group to the target
protein at serine & threonine residues. During phosphorylation cyclin binds to cdk and form
CDK protein complex.
DNA damage triggers P53 gene to synthesize P53 protein. It binds to the
promotors and activates the expression of other genes. E.g. WAF1 gene produce 21 kda proteins
called P21 protein and it’s over expression arrest the cell cycle in G1 phase. P21 inhibits the
CDK2-cyclin complex and inhibits the DNA polymerase delta enzyme which further stops the
replication of the cells that have entered the S phase.

95
MUTATION IN P53 GENE:
Mutation in the P53 gene leads to 2 phenomenon,
1. Loss of function – so that tumor suppression by TSP is lost.
2. Gain in function – TSP induce cancer (uncontrolled cell division).
MECHANISMS INACTIVATING THE P53:
There are 4 basic mechanisms to inactivate the p53 Protein namely,
1. Mutation in the p53 Protein:
Mutation in the P53 gene causes G to C and C to A transitions. E.g.
benzopyrene. UV rays cause the cells to commit suicide or induce thymine dimmer formations.
2. Degradation of p53 Protein:
Viral protein called E6 from papilloma virus degrades P53 protein that leads
to cervical cancer.
3. Trapping of p53 Protein by oncogenes protein:
MDM2 oncogene encodes MDM2 oncoprotein which is 90 kda
inactivates the P53 protein that leads to breast or liver cancer.
4. Cytoplasmic localization of p53 protein:
P53 proteins synthesized in cytoplasm are blocked by Cytoplasmic anchor
protein / undergo phosphorylation so that its translocation into the nucleus is ceased.

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Metastasis
Metastasis is the medical term for cancer that spreads to a different part of the body from where
it started. When this happens, doctors say the cancer has “metastasized.” Other names for
metastasis are “metastatic cancer” and “stage 4 cancer.” Sometimes the term “advanced cancer”
also describes metastatic disease, but this isn’t always true. For instance, “locally advanced”
cancer is not the same as metastatic cancer. It describes cancer that has spread to nearby tissues
or lymph nodes but not throughout the body.

How metastases develop

Metastases (the plural form of metastasis) most commonly develop when cancer cells break
away from the main tumor and enter the bloodstream or lymphatic system. These systems carry
fluids around the body. This means that the cancer cells can travel far from the original tumor
and form new tumors when they settle and grow in a different part of the body. Metastases can
also sometimes develop when cancer cells from the main tumor, typically in the abdominal
(belly) cavity, break off and directly “seed” other areas within the abdominal cavity.

Any type of cancer can metastasize (spread). Whether this happens depends on several factors.
These include:

 The type of cancer

 How aggressive (fast growing) it is
 How long you have it before treatment
 Other factors

Where metastases develop

Metastasis to the bones, brain, liver, lymph nodes, and lungs is common. Cancer cells can also
metastasize to the pleural space (the lining around the lungs) or the abdominal cavity. This may
cause excess fluid buildup in these areas (called malignant pleural effusions and
malignant ascites). Multiple tiny metastases in the abdominal cavity are referred to as peritoneal
carcinomatosis. Less frequently, cancer can also spread to the skin, muscle, or other organs
throughout the body.
Some cancers tend to spread to certain parts of the body. For example:

 Breast cancer tends to spread to the bones, liver, lungs, chest wall, and brain.
 Lung cancer tends to spread to the brain, bones, liver, and adrenal glands.
 Prostate cancer tends to spread to the bones.
 Colon and rectal cancers tend to spread to the liver and lungs.

Metastasis

Doctors give a metastasis the same name as the original cancer. So a breast cancer that spreads to
the liver, for example, is referred to as “metastatic breast cancer,” not liver cancer. This is
because the cancer started in breast cancer cells. However, even though the tumors in each of

97
these different locations represent breast cancer, doctors are learning more about how metastases
may differ from the primary (original) tumor at the molecular and genetic level. This is referred
to as intrapatient tumor heterogeneity.

Diagnosis
If you already had cancer treatment for non-metastatic cancer, you probably have a follow-up
care plan. You see your doctor for regular examinations and tests. Part of the reason for those
follow-up tests is to look for any evidence of metastases.
Alternatively, some individuals already have metastases at the time of their original cancer
diagnosis, and they are found during the initial staging evaluation.

Cancer may or may not cause symptoms, such as pain or shortness of breath. Sometimes these
symptoms lead a doctor to perform the necessary tests to diagnose the presence of metastases.

Treatment
Treatment depends on:

 The original cancer and where it started

 How much the cancer has spread and where it is located
 Your age and health
 Your personal treatment choices

Treatment for metastasis is often different from the treatment used for the original tumor. Most
commonly, doctors might try one type of chemotherapy, and then switch to another when the
first treatment no longer works. Or you might have a combination of treatments, such as
chemotherapy, radiation, and/or even surgery to remove the metastases (see below).
Treatment options

The main treatments for metastasis include:

 Treatment that affects your entire body – Doctors call this “systemic” therapy. It
includes chemotherapy and other medications, such as targeted therapy, hormone
therapy, and biologic treatment.
 Treatment for the area with cancer–Doctors call this “local” therapy. It includes
surgery, radiation therapy, and some other treatments.

When you choose treatment, talk with doctors who have experience treating a metastasis.
Doctors can have different opinions on the best treatment plan. Learn more about getting
a second opinion.

Does treatment cure metastatic cancer?

In some situations, metastatic cancer can be cured, but most commonly, treatment for metastases
is not curative. But doctors can treat it to slow its growth and reduce symptoms. It is possible to

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live for many months or even years with certain types of cancer, even after the development of
metastatic disease.

How well any treatment works depends on:

 The type of cancer

 How far the cancer has spread, and where it is located
 How much cancer there is
 If the cancer is growing quickly or slowly
 The specific treatment
 How the cancer responds to treatment

It is important to ask your doctor about the goals of treatment. These goals may change during
your care, depending on whether the cancer responds to the treatment. It is also important to
know that pain, nausea, and other side effects can be managed with the help of your health care
team. This is called palliative careand should be a part of any treatment plan.
Treatment in clinical trials

Clinical trials offer treatments that are not yet available to the public. A trial might be the main
treatment for metastases, or just one of the options. Just 3% to 5% of adults with cancer take part
in clinical trials. The trial treatment may or may not help. But even if it does not, it gives
researchers information that could help future patients. If you are interested in clinical trials, talk
with your doctor and health care team.

Interaction of Cancer cells with Normal cells

Cancer remains one of the most difficult diseases to treat. Despite the best efforts of physicians,
many deaths are caused because patients gradually cease to respond to their treatment. This
enables the cancer to spread throughout the body and establish additional tumors.  This
development of resistance to treatment and spread of the cancer is associated with increased
expression of signals that help cancer cells survive therapy and continue to grow. 

Understanding how these signals work and how to counteract them will provide more options for
treating cancer patients.  One of these survival signals comes from a compound called autotaxin.
Autotaxin is not properly regulated and its production increases during many cancer treatments. 
This results in the production of more survival signals, which makes cancers more difficult to
treat. 

It is commonly believed that autotaxin is overexpressed in the cancer cells themselves.  My

research is showing that most autotaxin expression comes from normal cells that surround the
cancer cells. These normal cells respond to damage produced by cancer treatments during
chemotherapy and radiation therapy.  As a result, these normal cells increase their production of
autotaxin to protect themselves and to repair the damage.

Equally, cancer cells interact with the normal cells that surround the tumor and increase the
production of autotaxin.  This allows cancer cells to acquire even more autotaxin from normal

99
cells and ensure their survival. These events are part of a vicious cycle that gradually decreases
the effectiveness of the treatments for the cancer patient. The purpose of my work is to
understand the interactions between normal cells and cancer cells within the tumor. By doing
this, I will discover how to prevent the production of autotaxin and therefore be able to improve
the effectiveness of treatment for cancer patients.

DNA Finger printing

DNA fingerprinting is a method used to identify an individual from a sample of DNA by looking
at unique patterns in their DNA.

Background

 Almost every cell? in our body contains our DNA?. 

 On average, about 99.9 per cent of the DNA between two humans is the same. 
 The remaining percentage is what makes us unique (unless you are an identical twin!). 
 Although this might sound like a small amount, it means that there are around three
million base pairs? that are different between two people. These differences can be compared and
used to help distinguish you from someone else.  
 Minisatellites are short sequences (10-60 base pairs long) of repetitive DNA that show
greater variation? from one person to the next than other parts of the genome?. This variation is exhibited
in the number of repeated units or ‘stutters’ in the minisatellite sequence.
 The first minisatellite was discovered in 1980. 

DNA fingerprinting

 DNA fingerprinting was invented in 1984 by Professor Sir Alec Jeffreys after he realised
you could detect variations in human DNA, in the form of these minisatellites. 
 DNA fingerprinting is a technique that simultaneously detects lots of minisatellites in the
genome to produce a pattern unique to an individual. This is a DNA fingerprint.
 The probability of having two people with the same DNA fingerprint that are not
identical twins is very small. 
 Just like your actual fingerprint, your DNA fingerprint is something you are born with, it
is unique to you.

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Steps:

1. The first step of DNA fingerprinting was to extract DNA from a sample of human
material, usually blood.
2. Molecular ‘scissors’, called restriction enzymes?, were used to cut the DNA. This resulted in
thousands of pieces of DNA with a variety of different lengths.
3. These pieces of DNA were then separated according to size by a process called gel
electrophoresis?
:

 The DNA was loaded into wells at one end of a porous gel, which acted a bit like a sieve. 
 An electric current was applied which pulled the negatively-charged DNA through the
gel.
 The shorter pieces of DNA moved through the gel easiest and therefore fastest. It is more
difficult for the longer pieces of DNA to move through the gel so they travelled slower. 
 As a result, by the time the electric current was switched off, the DNA pieces had been
separated in order of size. The smallest DNA molecules were furthest away from where
the original sample was loaded on to the gel.

4. Once the DNA had been sorted, the pieces of DNA were transferred or ‘blotted’ out of
the fragile gel on to a robust piece of nylon membrane and then ‘unzipped’ to produce single
strands of DNA. 
5. Next the nylon membrane was incubated with radioactive probes. 

 Probes are small fragments of minisatellite DNA tagged with radioactive phosphorous.
 The probes only attach to the pieces of DNA that they are complementary? to – in this case they
attach to the minisatellites in the genome.

6. The minisatellites that the probes have attached to were then visualised by exposing the
nylon membrane to X-ray film. 

 When exposed to radioactivity a pattern of more than 30 dark bands appeared on the film
where the labelled DNA was. This pattern was the DNA fingerprint. 

To compare two or more different DNA fingerprints the different DNA samples were run side-
by-side on the same electrophoresis gel.

101
DNA microarray

102
Summary of DNA Microarrays. Within the organisms, genes are transcribed and spliced to
produce mature mRNA transcripts (red). The mRNA is extracted from the organism and reverse
transcriptase is used to copy the mRNA into stable ds-cDNA (blue). In microarrays, the ds-
cDNA is fragmented and fluorescently labelled (orange). The labelled fragments bind to an
ordered array of complementary oligonucleotides, and measurement of fluorescent
intensity across the array indicates the abundance of a predetermined set of sequences. These
sequences are typically specifically chosen to report on genes of interest within the organism's
genome.[1]
A DNA microarray (also commonly known as DNA chip or biochip) is a collection of
microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure
the expression levels of large numbers of genes simultaneously or to genotype multiple regions
of a genome. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence,
known as probes (or reporters or oligos). These can be a short section of a gene or other DNA
element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample
(called target) under high-stringency conditions. Probe-target hybridization is usually detected
and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to
determine relative abundance of nucleic acid sequences in the target. The original nucleic acid
arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based
analysis was published in 1981.

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 Principle

The core principle behind microarrays is hybridization between two DNA strands, the property
of complementary nucleic acid sequences to specifically pair with each other by
forming hydrogen bonds between complementary nucleotide base pairs. A high number of
complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between
the two strands. After washing off non-specific bonding sequences, only strongly paired strands
will remain hybridized. Fluorescently labeled target sequences that bind to a probe sequence
generate a signal that depends on the hybridization conditions (such as temperature), and
washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the
amount of target sample binding to the probes present on that spot. Microarrays use relative
quantitation in which the intensity of a feature is compared to the intensity of the same feature
under a different condition, and the identity of the feature is known by its position.

The steps required in a microarray experiment

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Application or technology Synopsis

In an mRNA or gene expression profiling experiment

the expression levels of thousands of genes are simultaneously monitored
to study the effects of certain treatments, diseases, and developmental
Gene expression profiling stages on gene expression. For example, microarray-based gene
expression profiling can be used to identify genes whose expression is
changed in response to pathogens or other organisms by comparing gene
expression in infected to that in uninfected cells or tissues. [3]

Comparative genomic Assessing genome content in different cells or closely related organisms. [4]
[5]
hybridization

Small microarrays to check IDs of organisms in food and feed

GeneID (like GMO [1]), mycoplasms in cell culture, or pathogens for disease
detection, mostly combining PCRand microarray technology.

DNA sequences bound to a particular protein can be isolated

by immunoprecipitating that protein (ChIP), these fragments can be then
hybridized to a microarray (such as a tiling array) allowing the
Chromatin determination of protein binding site occupancy throughout the genome.
immunoprecipitation on Example protein to immunoprecipitate are histone modifications
Chip (H3K27me3, H3K4me2, H3K9me3, etc.), Polycomb-group
protein(PRC2:Suz12, PRC1:YY1) and trithorax-group protein(Ash1) to
study the epigenetic landscape or RNA Polymerase II to study
the transcription landscape.

Analogously to ChIP, genomic regions bound by a protein of interest can

be isolated and used to probe a microarray to determine binding site
occupancy. Unlike ChIP, DamID does not require antibodies but makes
DamID
use of adenine methylation near the protein's binding sites to selectively
amplify those regions, introduced by expressing minute amounts of
protein of interest fused to bacterial DNA adenine methyltransferase.

SNP detection Identifying single nucleotide polymorphism among alleleswithin or

between populations.[6] Several applications of microarrays make use of
SNP detection, including genotyping, forensic analysis,
measuring predisposition to disease, identifying drug-candidates,

105
 evaluating germlinemutations in individuals or somatic mutations in
cancers, assessing loss of heterozygosity, or genetic linkageanalysis.

An exon junction array design uses probes specific to the expected or

potential splice sites of predicted exons for a gene. It is of intermediate
density, or coverage, to a typical gene expression array (with 1–3 probes
per gene) and a genomic tiling array (with hundreds or thousands of
Alternative splicingdetection
probes per gene). It is used to assay the expression of alternative splice
forms of a gene. Exon arrays have a different design, employing probes
designed to detect each individual exon for known or predicted genes, and
can be used for detecting different splicing isoforms.

A Fusion gene microarray can detect fusion transcripts, e.g. from cancer

specimens. The principle behind this is building on the alternative
Fusion genesmicroarray splicing microarrays. The oligo design strategy enables combined
measurements of chimeric transcript junctions with exon-wise
measurements of individual fusion partners.

Genome tiling arrays consist of overlapping probes designed to densely

represent a genomic region of interest, sometimes as large as an entire
Tiling array human chromosome. The purpose is to empirically detect expression
of transcripts or alternatively spliced formswhich may not have been
previously known or predicted.

Right-handed double-stranded B-DNA microarrays can be used to

characterize novel drugs and biologicals that can be employed to bind
Double-stranded B-DNA specific regions of immobilized, intact, double-stranded DNA. This
microarrays approach can be used to inhibit gene expression.[7][8][9] They also allow for
characterization of their structure under different environmental
conditions.

Left-handed double-stranded Z-DNA microarrays can be used to identify

short sequences of the alternative Z-DNA structure located within longer
Double-stranded Z-DNA stretches of right-handed B-DNA genes (e.g., transcriptional enhancement,
microarrays recombination, RNA editing).[7][8][9][10] The microarrays also allow for
characterization of their structure under different environmental
conditions.

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 Multi-stranded DNA and RNA microarrays can be used to identify novel
Multi-stranded DNA drugs that bind to these multi-stranded nucleic acid sequences. This
microarrays (triplex-DNA approach can be used to discover new drugs and biologicals that have the
microarrays and quadruplex- ability to inhibit gene expression.[7][8][9][11][12] These microarrays also allow
DNA microarrays) for characterization of their structure under different environmental
conditions.

1. The two samples to be compared (pairwise comparison) are grown/acquired. In this

example treated sample (case) and untreated sample (control).
2. The nucleic acid of interest is purified: this can be all RNA for expression
profiling, DNA for comparative hybridization, or DNA/RNA bound to a
particular protein which is immunoprecipitated (ChIP-on-chip) for epigeneticor
regulation studies. In this example total RNA is isolated (total as it is nuclear
and cytoplasmic) by Guanidinium thiocyanate-phenol-chloroform extraction (e.g. Trizol)
which isolates most RNA (whereas column methods have a cut off of 200 nucleotides)
and if done correctly has a better purity.
3. The purified RNA is analysed for quality (by capillary electrophoresis) and quantity (for
example, by using a NanoDrop or NanoPhotometer spectrometer). If the material is of
acceptable quality and sufficient quantity is present (e.g., >1μg, although the required
amount varies by microarray platform), the experiment can proceed.
4. The labelled product is generated via reverse transcription and sometimes with an
optional PCR amplification. The RNA is reverse transcribed with either polyT primers
(which amplify only mRNA) or random primers (which amplify all RNA, most of which
is rRNA); miRNA microarrays ligate an oligonucleotide to the purified small RNA
(isolated with a fractionator), which is then reverse transcribed and amplified. The label
is added either during the reverse transcription step, or following amplification if it is
performed. The sense labelling is dependent on the microarray; e.g. if the label is added
with the RT mix, the cDNA is antisense and the microarray probe is sense, except in the
case of negative controls. The label is typically fluorescent; only one machine
uses radiolabels. The labelling can be direct (not used) or indirect (requires a coupling
stage). For two-channel arrays, the coupling stage occurs before hybridization,
using aminoallyl uridine triphosphate(aminoallyl-UTP, or aaUTP) and NHS amino-
reactive dyes (such as cyanine dyes); for single-channel arrays, the coupling stage occurs
after hybridization, using biotin and labelled streptavidin. The modified nucleotides
(usually in a ratio of 1 aaUTP: 4 TTP (thymidine triphosphate)) are added enzymatically
in a low ratio to normal nucleotides, typically resulting in 1 every 60 bases. The aaDNA
is then purified with a column(using a phosphate buffer solution, as Tris contains amine
groups). The aminoallyl group is an amine group on a long linker attached to the
nucleobase, which reacts with a reactive dye. A form of replicate known as a dye flip can

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 be performed to remove any dye effects in two-channel experiments; for a dye flip, a
second slide is used, with the labels swapped (the sample that was labeled with Cy3 in
the first slide is labeled with Cy5, and vice versa). In this example, aminoallyl-UTP is
present in the reverse-transcribed mixture.
5. The labeled samples are then mixed with a propriety hybridization solution which can
consist of SDS, SSC, dextran sulfate, a blocking agent (such as COT1 DNA, salmon
sperm DNA, calf thymus DNA, PolyA or PolyT), Denhardt's solution, or formamine.
6. The mixture is denatured and added to the pinholes of the microarray. The holes are
sealed and the microarray hybridized, either in a hyb oven, where the microarray is mixed
by rotation, or in a mixer, where the microarray is mixed by alternating pressure at the
pinholes.
7. After an overnight hybridization, all nonspecific binding is washed off (SDS and SSC).
8. The microarray is dried and scanned by a machine that uses a laser to excite the dye and
measures the emission levels with a detector.
9. The image is gridded with a template and the intensities of each feature (composed of
several pixels) is quantified.
10. The raw data is normalized; the simplest normalization method is to subtract background
intensity and scale so that the total intensities of the features of the two channels are
equal, or to use the intensity of a reference gene to calculate the t-value for all of the
intensities. More sophisticated methods include z-ratio, loess and lowess regression and
RMA (robust multichip analysis) for Affymetrix chips (single-channel, silicon chip, in
situ synthesised short oligonucleotides).

GENE MAPPING

Gene mapping, also called genome mapping, is the creation of a genetic map assigning DNA
fragments to chromosomes.
 When a genome is first investigated, this map is nonexistent. The map improves with the
scientific progress and is perfect when the genomic DNA sequencing of the species has
been completed.
 During this process, and for the investigation of differences in strain, the fragments are
identified by small tags. These may be genetic markers (PCR products) or the unique
sequence-dependent pattern of DNA-cutting enzymes. T
 The ordering is derived from genetic observations (recombinant frequency) for these
markers or in the second case from a computational integration of the fingerprinting data.
The term "mapping" is used in two different but related contexts.
Two different ways of mapping are distinguished. Genetic mapping uses classical genetic
techniques (e.g. pedigree analysis or breeding experiments) to determine sequence features

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within a genome. Using modern molecular biology techniques for the same purpose is usually
referred to as physical mapping.
Types of Genome Maps:
The most important objective of human genome project was to construct a series of maps
for each chromosome.
1. Cytogenic map:
This is a map of the chromosome in which the active genes respond to a chemical dye and
display themselves as bands on the chromosome.
2. Gene linkage map:
A chromosome map in which the active genes are identified by locating closely associated
marker genes. The most commonly used DNA markers are restriction fragment
polymorphism(RFLP), variable number tandem repeats (VNTRs) and short tandem repeats
(STRs). VNTRs are also called as minisatellites while STRs are microsatellites.
3. Restriction Fragment Map:
This consist of the random DNA fragments that have been sequenced.
4. Physical map:
This is the ultimate map of the chromosome with highest resolution base sequence.
Physical map depicts the location of the active genes and the number of bases between the
active genes.
Physical Mapping:
 In physical mapping, the DNA is cut by a restriction enzyme. Once cut, the DNA
fragments are separated by electrophoresis. The resulting pattern of DNA migration (i.e.,
its genetic fingerprint) is used to identify what stretch of DNA is in the clone.
 By analysing the fingerprints, contigs are assembled by automated (FPC) or manual
means (Pathfinders) into overlapping DNA stretches. Now a good choice of clones can be
made to efficiently sequence the clones to determine the DNA sequence of the organism
under study (seed picking).
 Macrorestriction is a type of physical mapping wherein the high molecular weight DNA
is digested with a restriction enzyme having a low number of restriction sites.
 There are alternative ways to determine how DNA in a group of clones overlap without
completely sequencing the clones.

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  Once the map is determined, the clones can be used as a resource to efficiently contain
large stretches of the genome. This type of mapping is more accurate than genetic maps.
Principles of Map-based or Positional Cloning
 The first step of map-based or positional cloning is to identify a molecular marker that
lies close to you gene of interest. This procedure typically is done my first finding a
marker in the vicinity of the gene (several cM away).
 For the initial screening smaller population sizes are used (60-150 individuals) The next
step is to saturate the region around that original molecular marker with other markers.
 At this point you are looking for a one that rarely shows recombination with your gene.
At this stage, the population size could increase to 300-600 individuals.
 The next step is to screen a large insert genomic library (BAC or YAC) with your marker
to isolate clones that hybridize to your molecular marker.
 Once you identify the initial markers that map are near (or better yet) flank your gene and
fournd a a clone to which the markers hybridize, you are on your way to determining
where that gene resides. The steps that follow are termed chromosomal walking.
 This procedure involves creating new markers (usually sequences at the end of the clone)
and screening your segregating population with these new markers. Often this population
is large (1000-3000 individuals).
 The goal is to find a set of markers that co-segegate (no recombination) with your gene
of interest.
 Co-segregation means that whenever one allele of your gene is expressed, the markers
associated with that allele are also present. In other words, recombination is not seen
between your gene and the markers.
 If these markers do not cosegregate, you select new large insert clones and repeat the
process until you have a clone whose markers co-segregates with your gene.
 To speed the cloning process, it is best to begin with a marker that is tightly linked to the
gene with which your are working. Therefore you will not have to do a lot of additional
screening.
 Because you have your gene flanked on a single clone between two markers, you now
know that the gene must be between those two markers.

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  DNA fragments between the flanking markers are cloned and introduced into a genotype
mutant for your gene by a genetic engineering technique called plant transformation. If
transgenic plant expresses the wild type phenotype, you then know the gene of interest
is on that fragment.
 At this point you must sequence the fragment to find a potential open reading frame
(ORF), sequences that most likely will encode a gene product.
 In the best situation, only a single ORF is found, but this often is not the case. Usually
several possible ORFs are found and new transgenic plants are created by transforming
with a single ORF.
 Once this ORF is shown to rescue the mutant phenotype, you then perform an in-depth
molecular and biochemical analysis of newly cloned gene.
QUESTION BANK
UNIT V
1 MARKS
1. Oncogene
2. P53
3. DNA finger print
4. Micro array
5. TSP genes
6. Cancer cells
7. metastasis
8. Gene mapping
5 MARK & 10 MARK
9. Describe about the oncogenes
10. Explain in detail about the functions of tumor suppressor gene
11. Comment on gene mapping
12. Microarray techniques
13. Explain about viral & cellular oncogenes.

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