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236 BIOLOGY

CHAPTER 14

NATURE OF GENE : ITS EXPRESSION AND REGULATION

By now, you know that parents transmit the variety of proteins formed. For a very long time
heritable characters to the offspring. This forms thus, protein remained the strongest candidate
the basis of maintaining a sort of continuity as the chemical form of gene, even in the
amongst the members of a species. Also, factors absence of any direct experimental proof.
or genes responsible for these characters are 14.1 NATURE OF GENETIC MATERIAL
located on the chromosomes, which through the
gametes enter a diploid zygote cell composition While such experimental proofs were being
that explains the process of transmission. This sought, eukaryotic nuclei from a number of
process is common to all sexually reproducing sources containing another biomolecule,
organisms. Thus, each offspring receives 50 per deoxyribose nucleic acid or DNA, first described
cent component of chromosomes and thus the by Friedrich Miescher in 1868, became known.
genes, from male parent and 50 per cent The role of cell nucleus was already known in
component from female parent. In this Chapter, hereditary process. Such information led to a
you will study more about genes, their structure fresh search for unravelling the biochemical
and how their expressions are regulated. nature of gene. But the answer came from an
By the time these facts were discovered, the unexpected source.
cell was getting better understood in In 1928, Frederick Griffith, a British scientist
biochemical terms. This led to one important working on the pathogenicity of a bacterial
question as to which biomolecule contributes strain, Diplococcus pneumoniae now called
to the chemical form of gene, and whether streptococcus, discovered the process of
structure of that biomolecule can explain all the transformation. This strain causes bacterial
functions assigned to a gene. On the basis of pneumonia in mammals including humans.
the genetic knowledge, a gene is expected to The disease-causing or S-strain has cells
have following features: surrounded by a capsule, and forms smooth
(i) a gene should be able to store and express glistening colonies when grown on agar medium.
infor mation to confer the heritable Some mutant strains form rough colonies or R
characters of the living organisms; strains. This strain does not cause pneumonia.
(ii) a gene should have the capability to make Also, when S strains are heat-killed and injected
its own replica so that a copy can be into mouse, no disease symptom appears.
transmitted to the offspring; Surprisingly, however, when a mixture of heat-
(iii) a gene should also have a mechanism to killed S type (inactivated) and live R bacteria
undergo mutations that will generate the (nonpathogenic) were mixed and then injected,
biological variation so essential for disease reappeared. Live S type cells could be
evolution. recovered from the blood of dead mice. Griffith
If we consider the major biomolecules of a proposed that a ‘transforming principle’, a
cell and correlate their variety with the variety chemical substance was released by the killed
expected amongst different genes, proteins will S cells which transformed the R bacteria
attract our attention. While we expect each into S type. This was a permanent genetic
living organism to have different genes for change as S type bacteria continued to
corresponding phenotypes, we also have a whole produce similar cells (Fig. 14.1).
NATURE OF GENE : ITS EXPRESSION AND REGULATION 237

Experiment Result

Mouse dies

Living S strain

Mouse lives

Living R strain

Mouse lives

Heat-killed
S strain

Mouse dies

Living R strain Mixture of Living cells of S strain


heat-killed S strain isolated from dead mouse
and living R strain

Fig 14.1 Griffith’s experiment demonstrating transformation in Diplococcus bacterium

In 1944, Oswald T Avery, Colin MacLeod and Another definitive evidence came from
Maclyn McCarthy revealed the chemical nature experiments on T2 bacteriophage by Alfred D.
of the transforming substance to be DNA. They Hershey and Martha Chase in 1952. A
showed that DNA isolated from S bacteria could bacteriophage is simple in molecular constitution
by itself confer the pathogenic properties to R as it consists of a capsid that is made up of
cells. This fact suggested that DNA has the protein, and DNA that is contained within the
genetic properties. head portion of the capsid. Hershey and Chase
238 BIOLOGY

based their experiment on the fact that DNA but and the two were separated by centrifugation.
not the proteins contains phosphorus, and Hershey and Chase showed that when 32P was
similarly sulfur is present in proteins but not in used, all radioactivity was associated with
DNA. They then incorporated radioactive isotope bacterial cells and if followed, appeared in the
of phosphorus (32P) into phage DNA and that of progeny phage. However, when 35S was used,
sulfur (35S) into proteins of separate phage all radioactive material was limited to phage
cultures. These phage types were used ‘ghosts’. The conclusion was simple and
independently to infect the bacterium straightforward: DNA alone is able to impart all
Escherichia coli. After sometime, this mixture was the characteristics to the phage progeny and
agitated on a blender to separate the empty phage proteins did not play any role in this process
capsids or ‘ghosts’ of the bacterial cells (Fig. 14.2).

Experiment
32 35
P-containing DNA S-containing phage coats
Bacteria Bacteria

32 35
Little P Most S
in supernatant in supernatant

Interpretation

DNA is Protein is not


the hereditary material the hereditary material
Fig 14.2 Hershey and Chase’s experiment demonstrating that only phage DNA injected in the host cell determines
all the characteristics of the progeny phage
NATURE OF GENE : ITS EXPRESSION AND REGULATION 239

With these two evidences and information molecule consisting of two similar strands
linking DNA and nuclei and transmission running in parallel and helical manner. The
through gametes involving principally nuclear helix makes one complete spiral turn every
material led to a fresh look at the structure of 3.4 nm and has a diameter of 2 nm
DNA with special reference to its function as (Fig.14.3a). Watson and Crick worked on these
genetic material. clues in the light of the functions assigned to
a gene and developed their famous model
14.2 DNA AND ITS STRUCTURE ‘Double Helix’. In this model, they proposed
By the time DNA structure was proposed by that DNA consists of two strands, which are
James Watson and Francis Crick in 1953, the helically coiled. Each strand consists of a
chemical composition of DNA was already backbone, made up of alternating deoxyribose
known. The deoxyribose nucleic acid (DNA) sugar and phosphate with phosphate joining
consists of four types of basic units called the two sugars through a phosphodiester
nucleotides (See Chapters 2 and 10). Each bond (phosphate group forming a bridge
nucleotide is made up of a pentose sugar between – OH groups of two adjacent sugars).
(deoxyribose type), a phosphate group, and a The bases are stacked inside and pair with
nitrogenous base. A subunit composed of only the base of the opposite strand through
sugar and the nitrogen base is known as hydrogen bonds. Keeping in mind Chargaff’s
nucleoside. The four nucleosides differ from rule, they proposed that the pairing will always
each other in the type of the base which could be between A and T, and G and C. The two
be adenine (A), guanine (G), thymine (T), or strand runs antiparallel, i.e., one in 5'→3'
cytosine (C). The Adenine and Guanine are the direction and the other 3'→5' direction
purines and Thymine and Cytosine are the (Fig 14.3b).
pyrimidines. In chemical terms, each nucleotide The specific pairing between purine and
is a deoxy-5 -monophosphate, for example dAMP, pyrimidine bases proposed by Watson and
dGMP, dTMP, or dCMP. So, DNA is a Crick not only fitted very well with the radius
polynucleotide. Erwin Chargaff in 1949 had of DNA determined from X-ray diffraction
shown from his studies on DNA derived from a pictures but also had necessary
number of sources that there are certain complementary ‘lock and key’ shape for efficient
empirical rules regarding the composition of hydrogen bonding, which happens to be two
bases in DNA known as Chargaff ’s rules. These between A and T (A=T) and three between G
are: and C (G=C). The stacking of bases creates
(i) Total amount of purine nucleotides always two types of grooves called major and minor
equals the total amount of pyrimidine grooves. Each turn accommodates 10 bases.
nucleotides i.e., For this work on DNA structure, Watson and
[A]+[G] = [T]+[C]. Crick along with Wilkins received Nobel Prize
(Medicine and Physiology) in 1962.
(ii) The proportion of A is equal to T and so
also of G is equal to C, Initially, two different forms of right-handed
DNA helix A and B were identified. Of these, B
but amount of [A]+[T] is not necessarily
is more hydrated and most frequently found
equal to [G]+[C].
in living cells. Subsequently, an additional
Therefore, [A]= [T]; [G] =[C] form of DNA was detected. This was a left-
handed form and based on its zigzag-like
backbone called Z DNA. The right-handed
DNA can acquire a left-handed structure, at
In 1953, Maurice H.F. Wilkins and Rosalind least over a short distance and temporarily,
E. Franklin took X-ray diffraction pictures of and through this process can influence the
crystalline DNA. They concluded that it is a long gene expression.
240 BIOLOGY

(a)

2 nm
(b)

Sugar

Base

Phosphate

0.34 nm

3.4 nm

0.34 nm

Major groove
Minor groove

Fig 14.3 (a) General structure of a DNA double helix showing the position of sugar(S) phosphate(P) and bases,
(b) The anti-parallel nature of the two strands. One strand starts with 5’ and ends at 3’ whereas the
other beginning at 3’ ends at 5’

14.3 RNA AND ITS STRUCTURE At least three major classes of cellular RNAs,
RNA or ribonucleic acid is another nucleic acid all functioning during gene expression, are
type. Like DNA, it is a polynucleotide but a number known. These are ribosomal RNA (rRNA),
of differences are found in their structure. In RNA, messenger RNA (mRNA), and transfer RNA
the pentose sugar is ribose and not deoxyribose; (tRNA). All the three types of molecules originate
also it contains uracil in place of thymine. Most as complementary copies of one of the two
RNAs are usually single stranded with partial strands of a DNA segment that constitutes a
double-stranded regions due to folding back of and gene, during the process of transcription. This
pairing within its single chain. RNA serves as the would mean that RNA will have the same
genetic material in many viruses, some of which sequence as the other strand of DNA, except
have double stranded RNA. that uracil will replace thymine against adenine.
NATURE OF GENE : ITS EXPRESSION AND REGULATION 241

These RNA types can be differentiated on the


basis of their size, sedimentation behaviour
and genetic functions.
Ribosomal RNA is generally not only the
largest but also the most prevalent of the Parent DNA
cellular RNA species. This is an important
structural component for protein synthesis.
Messenger RNA, as the name suggests, carries
the genetic message from DNA. Their length
and sequence vary depending upon the gene
which is being transcribed into mRNA.
Transfer RNA, the smallest of the three types,
carries amino acids to the ribosomes during
translation. This species of RNA contains
number of modified bases.
14.4 DNA AND GENE
Now that we know the structure of DNA, we
shall correlate this structure with the possible
functions of a gene. Watson and Crick had
believed that the specific pairing of the bases
in the helix will play a key role in the
transmission of the genetic character, both
from one cell to its successors and also in gene
expression.
DNA Replication
One of the prime functions of a gene is to make
its copies, which could be transmitted to the
daughter cells. This would help in maintaining
the uniform genetic composition. Watson and
Crick proposed that each DNA strand of a
double helix can act as template for the Daughter DNA Strands
synthesis of daughter strand in which bases
are incorporated by specific hydrogen-bonded
pairing (A with T, and G with C). The resulting Fig 14.4 (a) Semi-conservative mode of DNA
replication. The two resulting helices are
DNA strand will be complementary to the
exact replica of the parentals duplex
template strand and identical to the other
strand. As seen in the Fig.14.4a, the two
daughter helices are exact replicas of the heavy isotope in all nitrogen-containing
original double helix. The copying of DNA to compounds including bases.
make more DNA is known as DNA replication. Such cells were then shifted to normal 14N-
In the process outlined above, since the nitrogen for one or two generations. DNA was
daughter DNA consists of one old and one new isolated from the cells at each generation and
strand, the mode of replication is called the DNA molecules containing 15 N and 14N
semiconservative. were separated on CsCl-equilibrium density
The semiconservative DNA replication was gradient centrifugation. When a solution of
confirmed by an elegant experiment conducted Cesium chloride (CsCl) is spun in a centrifuge
by M. Meselson and F.W. Stahl in 1958. They at a very high speed (50,000 revolutions per
grew E. coli cells in the presence of heavy minute) for many hours, the salt tends to settle
isotope 15N (in the form of 15NH4Cl) for several down in the centrifuge tube creating a density
generations. This led to the incorporation of gradient.In this gradient, the highest ion
242 BIOLOGY

concentration will be encountered at the explains the two corresponding bands. Just a
bottom. When DNA is mixed with CsCl, it will year before Meselson and Stahl’s experiment,
finally settle at some point in the tube where Taylor had shown semiconservative mode of
the centrifugal force balances the buoyancy of replication at chromosomal level in bean root tip
DNA. cells.
DNA containing heavy isotope 15 N has
Mechanism of DNA Replication
greater density and is called heavy DNA. Such
a DNA settles at the heavier range of the Though the mode of replication looks simple,
gradient. After one generation, in 14 N the whole process is much complicated. A whole
medium, all DNA acquired an intermediate range of enzymes are required to take care of
density whereas after two generations two not only the various steps, and the structural
bands were seen, one at intermediate and the and mechanistic constraints, but also to
other at ‘light’ DNA density. If we follow Fig14.4b, maintain the accuracy of the process. The DNA
we can explain these results on the basis of replication in most bacteria starts at a single
semiconservative mode of replication. After one point, Origin of Replication or Ori, and moves
generation in 14N medium, both the daughter bidirectionally. In eukaryotes, there are several
DNAs will have 15N/14N composition instead of points of origin on the length of the DNA per
15N/15N of the original and thus will have an chromosome. The first requirement before any
intermediate density. After two generations, synthesis can take place, is to unwind the
from a 15 N/ 14 N DNA two daughter helices double helix, so that the two stands are free to
formed will be 15 N/ 14 N and 14 N/ 14 N. This act as templates. This function is carried out by

Experiment

Bacteria transferred to
14 N (light) medium;
Growing bacteria growth continues
in 15 N (heavy)
medium

Sample at Sample after Sample after


0 minutes 20 minutes 40 minutes
Centrifugation
Results DNA in Cesium chloride solution

First Second
Parental Parental generation generation
Interpretation

Fig 14.4 (b) Meselson and Stahl’s experiment to prove the semi-conservative replication of DNA
NATURE OF GENE : ITS EXPRESSION AND REGULATION 243

the enzyme Helicase, which unzips the two


strands beginning at the Ori site. As Leading strand
unwinding proceeds, other proteins called DNA polymerase III
single-stranded binding proteins, associate
with single strands and stabilise this
condition. Unwinding also creates a coiling
Helicase
tension ahead of the moving Replication fork,
a structure that will be formed when DNA
replication begins. This tension is reduced
by Topoisomerases. Figure 14.5 depicts
these and the subsequent steps in DNA
replication.
Single-strand
The most important DNA synthesising DNA-binding protein
enzyme is DNA polymerase III. It along with
other DNA polymerases (I and II) has the
capability to elongate an existing DNA strand
but cannot initiate the synthesis. All the three
DNA polymerases function in 5'→ 3' direction RNA primer
only for DNA polymerisation and have 3'→ 5'
Primase
exonuclease activity. Thus, to initiate DNA
synthesis, a small segment of RNA, called an
RNA primer complementary to the template
DNA is synthesised by an unique RNA
polymerase known as primase. It is to this
primer, that DNA polymerase III adds 5'-
deoxyribonucleotides and extends the DNA
(Fig. 14.5). A problem will be encountered, as
we know that two strands of DNA run
antiparallel to each other and DNA
polymerase III can function only in 5' → 3'
direction. This problem is solved in the
following manner. While on the one strand, the Lagging strand
DNA synthesis is continuous and in 5'→ 3' Okazaki fragments
direction, on the other strand, DNA is
synthesised in small stretches resulting in
discontinuous DNA synthesis. This happens
in the opposite direction to the first strand but
maintains the overall 5' → 3' direction as
required. Such a process is also referred to
as semi-discontinuous replication. The short
stretches of DNA, each primed by RNA are
called Okazaki fragments; named after the
Japanese scientist who discovered them.
RNA primers are then removed, and the gap
is filled by DNA nucleotide. Both steps are
performed by DNA polymerase I. These
DNA ligase
fragments are then sealed by the enzyme
Ligase. The strand which supports the
continuous DNA nucleotide is the leading
strand and the one which is replicated in short Fig 14.5 The molecular mechanism of semi-
stretches is called the lagging strand. discontinuous DNA replication
244 BIOLOGY

The process of replication also ensures the described which fell into the category of inherited
accuracy so as to maintain the nucleotide metabolic defects. It thus led to the idea that
sequence of the original DNA. Firstly, the DNA many disorders may be inherited as any other
polymerase action itself is very accurate. If, gene and that genes function by controlling the
however, a wrong nucleotide is inserted, both metabolism.
DNA polymerases I and III are able to detect and The direct proof for this gene-metabolism
excise the wrong base. Even though DNA relationship came from the work of George Beadle
synthesis is slower in eukaryotes as larger DNAs and Edward Tatum in the early 1940s on the
need to be replicated, the general steps of fungus Neurospora crassa. They irradiated the
replication are similar to those of prokaryotes. spores with X-rays and isolated a range of
nutritional mutants, which had requirement for
14.5. GENE EXPRESSION
an additional specific nutrient. Such mutants
The second important characteristic of the gene were called auxotrophs in comparison to the wild-
is to store and express the genetic information type (prototroph) which could grow on a simple
that will contribute towards the phenotype, and nutrient medium (minimal medium) containing
will be passed on to successive generations. In a few salts and a sugar. For example, a number
the following discussion, we shall explore as to of arginine-requiring mutants were isolated
how DNA can fit into this characteristic and how which could be classified into three types:
exactly it controls the gene expression. (i) some could grow on ornithine-, or citrulline-,
The idea that genes control the metabolism or arginine - containing medium,
was put forward as early as 1902 by Garrod. (ii) some could grow on citrulline- or arginine-
He studied several human disorders, which containing medium, and
seemed to be inherited. He called them inborn (iii) some could grow only on arginine
errors of metabolism. In one such disorder, supplemented medium (Table 14.1).
alkaptonuria, afflicted individuals cannot It means that all mutants could grow on
metabolise homogentisic acid, as a result it gets arginine-supplemented medium suggesting that
accumulated and is excreted in the urine. The arginine is the final product of this pathway,
oxidation products of this molecule are black and since some could not grow on ornithine,
and thus can be easily detected by the blackening this compound must be synthesised earlier than
of the urine upon exposure to air. The products citrulline and/or arginine. Beadle and Tatum
also tend to accumulate in the cartilaginous taking clue from this growth behaviour of
areas causing darkening of ear and nose, and Neurospora and other experiments proposed the
deposition in joints lead to mild arthritis. arginine biosynthetic pathway (Fig.14.6).
Interestingly, Garrod found that this disorder The precursor compound first gives rise to
is inherited as a recessive trait and reasoned ornithine, ornithine then gives rise to citrulline
that normal individuals must be metabolising and the latter is finally converted to arginine;
homogentisic acid. In affected individuals, each of these steps is mediated by an enzyme.
however, the metabolic pathway is blocked The mutants of class (i) could grow on ornithine,
resulting into alkaptonuria. Subsequently, a citrulline, or arginine suggesting that they
number of human diseases were identified and lacked the capacity to synthesise ornithine but

Table 14.1 Effect of Medium Supplements on the Growth of


Mutants of Neurospora crassa
Growth on medium supplemented with
Mutant types
Ornithine Citrulline Arginine

(i) + + +

(ii) – + +

(iii) – – +

+ growth; - no growth
NATURE OF GENE : ITS EXPRESSION AND REGULATION 245

Supplements to minimal medium


None Ornithine Citrulline Arginine

Strain

Wild
type

(iii)

(ii)

(i)

Biochemical pathway :
Precursor Ornithine Citrulline Arginine

Encoded
enzyme
Location of
genes on
chromosome

Fig 14.6 Beadle and Tatum’s experiment on Neurospora crassa revealed the control of metabolism by genes
246 BIOLOGY

beyond that they could complete the pathway. 14.6 CENTRAL DOGMA OF MOLECULAR BI-
Similarly, mutants of class (ii) could not convert OLOGY
ornithine to citrulline but if supplied citrulline The expression of the genetic material occurs
could synthesise arginine. The last class of generally through the production of proteins.
mutant (iii) could not convert citrulline to This involves two consecutive steps. These are
arginine and had to be supplied the latter for transcription and translation. In transcription,
growth. They reasoned that these defects could the genetic information, stored in DNA, is
arise due to defective enzymes in each case. transferred to an RNA, which in turn uses this
Since such changes were mutational, they information to direct the synthesis of proteins
opined that one gene controls one enzyme in a during translation. This unidirectional flow of
pathway leading to their famous one gene-one information was described by F.H.C. Crick in
enzyme hypothesis. 1958 as the ‘Central dogma’ of Molecular Biology
Two ideas soon modified the one gene-one (Fig. 14.7).
enzyme hypothesis. Firstly, while nearly all
enzymes are proteins, all proteins are not
enzymes. Also, many proteins are made up of
Replication
subunits called polypeptides, with each distinct
polypeptide under the control of a gene.
The adult human haemoglobin, for example, DNA
consists of 4 polypeptides-2α and 2β and a
separate gene codes each type. This led to re- Reverse
evaluation of Beadle and Tatum’s hypothesis Transcription Transcription
into one gene-one polypeptide. Further
modification came when gene was identified as
a functional unit or cistron and the same was
called as one cistron-one polypeptide. RNA Replication
Basically, all the three tenets suggest that the
genes control the polypeptides.
Gene and Protein Translation
In the previous section, we have established
that DNA is the genetic material and we have
also seen that genes function by exerting their Protein
control over proteins or enzymes. Now, we shall
see as to how is the genetic information stored
in DNA and then how this information coded in
Fig 14.7 The flow of genetic information
DNA is transmitted in the process of protein
synthesis.
The work done in the late 1950s and early You have already known that DNA is a self-
1960s established that a specific sequence of replicating molecule. In 1970, however, an
4 bases in the DNA serves as the store house of important modification of this information flow
all genetic information or in essence is the basis was brought to light by the work of H.M. Temin
of all life on Earth. The unique way in which and D. Baltimore. Many tumor viruses contain
these bases are arranged serve as what is known RNA as genetic material and replicate by first
as genetic code, which determines the basic synthesising a complementary DNA. This
structure and therefore function of the whole process is called reverse transcription. It
variety of proteins. Thus, genetic code can be is carried out by an RNA-dependent DNA
equated with Morse code used in telegraphic polymerase called reverse transcriptase.
signal transmission. We shall describe in detail These viruses are known as retroviruses and
how gene expression occurs and how the genetic include Human Immunodeficiency Virus (HIV)
code was deciphered. that causes AIDS.
NATURE OF GENE : ITS EXPRESSION AND REGULATION 247

How the coded information of DNA is decoded that gets physically associated with DNA. Only
into proteins? We will now describe the two major one type of such an enzyme is found in
steps of the process. prokaryotes in contrast to eukaryotes where three
different forms of RNA polymerase are found. RNA
Transcription polymerase I, II and III catalyse the synthesis of
As described earlier, transcription generates a rRNA, mRNA and tRNA, respectively.
single-stranded RNA identical in sequence with RNA polymerase binds to a region of DNA
one of the strands of the DNA. All the three called promoter which is recognised by sigma
species of RNA are produced through (σ) subunit of the enzyme RNA polymerase in
transcription. The strand of DNA that directs prokaryotes and many transcription factors in
the synthesis of the mRNA via complementary eukaryotes. RNA polymerase not only initiates
base pairing is called coding or sense strand but also extends the RNA (chain elongation)
and acts as template for mRNA synthesis. The and functions always in 5' to 3' direction. The
other strand is known as the non-coding strand process terminates once the enzyme completes
or antisense strand. Transcription is the transcription of entire DNA segment and a
accomplished by an enzyme RNA polymerase termination sequence is reached (Fig. 14.8).

Initiation site Complementary strand Termination site


(5’______ 3’)

Template strand
(3’______ 5’)

Rewinding Unwinding
of DNA of DNA

RNA
polymerase

Nucleoside triphosphates

Direction of transcription

RNA transcript

sigma factor

RNA

Fig 14.8 Transcription in prokaryotes-Enzymatic synthesis of RNA by RNA polymerase


248 BIOLOGY

In many bacteria, genes of related functions


Start codon Coding sequences Stop codon
are grouped together and are called operons. Exon1 Exon2 Exon3
Eukaryotic
An operon acts as a single transcription unit DNA
and thus produces several mRNA strands or Intron1 Intron2
polycistronic mRNA. In eukaryotes, only Non-coding sequences
monocistronic mRNAs are generally produced.
Transcription
Unlike the situation in prokaryotic genes,
transcription in eukaryotes occurs within the pre-mRNA
nucleus and mRNA moves out of the nucleus
into the cytoplasm for translation. The initiation 5′ capping
and regulation of transcription is more extensive G-Cap
than prokaryotes. Another major difference
between prokaryotes and eukaryotes lies in the
fact that in eukaryotes the primary RNA Polyadenylation
transcript is further processed to form mRNA,
a process called maturation. Initially at the 5' Poly A tail
end a cap (consisting of 7-methyl guanosine or
7 mG) and a tail of poly A at the 3' end are
present (Fig.14.9). The cap is a chemically Splicing
modified molecule of guanosine triphosphate
(GTP). The primary eukaryotic mRNA transcript
is much longer and localised in the nucleus,
when it is also called heterogenous nuclear RNA
(hnRNA) or pre-mRNA. The eukaryotic primary Endonucleolytic Cleavage
mRNAs are made up of two types of segments; at splice junctions
non-coding introns and the coding exons. The
introns are removed by a process called RNA
SnRNP
splicing. Of a pair of small nuclear
ribonucleoprotein (SnRNPs pronounced
Spliceosome
“snurps”), one binds to 5' splice site and the
other to 3' splice site. A spliceosome forms
because of interaction between SnRNPs and
other proteins. This spliceosome uses energy of
ATP to cut the RNA, releases the introns and Sealing by
joins two adjacent exons to produce mature Ligase

mRNA. Besides, these two post-transcriptional Mature


modifications, RNA editing may also take place mRNA
before translation begins.
Fig 14.9 Transcription in eukaryotes
Translation : Biosynthesis of Proteins
characterised as 70S and 80S, respectively,
Translation is the process in which the genetic both consisting of a small and a large subunit
message carried by mRNA from the DNA is (Fig. 14.10). In prokaryotes, the large or 50S
converted in the form of a polypeptide chain subunit consists of 23S rRNA and a 5S rRNA
having a specific sequence of amino acids. + 32 different proteins and the 30S small
Besides mRNA, the three key players in this subunit is made up of 16S rRNA and 21
process are: ribosomes, tRNA, and amino acids. different proteins. The eukaryotic large
Ribosomes are the ribonucleoprotein particles subunit (60S), on the other hand, is composed
that provide the sites for protein synthesis. On of 28S rRNA, 5S rRNA and 5.8S rRNA and 50
the basis of their sedimentation coefficients, proteins and the 40S small subunit consists
prokaryotic and eukaryotic ribosomes are of 18S rRNA and 33 different proteins .
NATURE OF GENE : ITS EXPRESSION AND REGULATION 249

Prokaryote ribosome Eukaryote ribosome

60S

50S

40S
70S 30S 80S

23S rRNA 16S rRNA 28S rRNA 18S rRNA

32 proteins 21 proteins 50 proteins 33 proteins

5S rRNA 5S rRNA 5.8S rRNA

Fig 14.10 Generalised structure of ribosome in prokaryotes and eukaryotes

The tRNA, smallest of the three RNA species tRNA contains the sequence for amino acid
known displays nearly identical structure in attachment. The anticodon loop carries an
pro- and eukaryotes. In 1965, R.W. Holley anticodon complementary to the known
proposed a clover-leaf model of tRNA which amino acid code for which it is specific.
results from intra-strand base pairing creating In three dimensions, tRNA looks like an
paired stems and unpaired loops. The 3' end of L-shaped molecule (Fig.14.11).

Amino acid
attachment site
(always CCA)

Hydrogen
bonds between
paired bases

D-loop TψU loop

anticodon

3D- computer generated 3D- L shaped structure 2D- clover leaf model
space-filling structure of a t RNA of a t RNA of a t RNA

Fig 14.11 Structure of tRNA


250 BIOLOGY

Steps in Translation 50S subunit joins the initiation complex, the


Twenty naturally occurring amino acids initiation factors are released and complete 70S
participate in protein synthesis- Translation ribosome is formed. The ribosome has binding
demands that a tRNA is specifically linked sites for two charged tRNAs labelled as P
to an amino acid, a pr ocess known as (peptidyl site), and A (aminoacyl site). The
charging. This is brought about under the
initiator tRNA is positioned at P site leaving the
direction of an enzyme, aminoacyl tRNA
synthetase which is highly specific, i.e. triplet (three bases of mRNA or codon) at the A
each recognizes only one amino acid. site unoccupied to allow the entry of another
Charging is a two-step process: charged tRNA (Fig. 14.12).

Step 1
Aminoacyl tRNA Synthetase*
Amino acid* + ATP → Aminoacyl adenylic acid + P-P
Step 2
Aminoacyl adenylic acid + tRNA* → Charged tRNA (tRNA - Amino acid*)
= P-A + aminoacyl tRNA synthetase
* Represents specific reaction between these components.

In the first step an amino acid reacts with Elongation


ATP in the presence of a specific aminoacyl tRNA Ribosome moves along the mRNA in the 5' to 3'
synthetase to produce the activated form or an direction. A charged tRNA whose anticodon is
aminoacyl adenylic acid. While still associated complementary to the second codon enters the
with the enzyme, the complex now reacts with empty A site. Once this happens, the peptidyl
a specific tRNA and the amino acid is transferred transferase, links the two amino acids through
to the adenine of 3' end of tRNA, and the enzyme a peptide bond. The 23S rRNA of large subunit
leaves the complex. has been assigned this catalytic function. The
The process of translation involves three initiator tRNA now delinks, and the dipeptide
steps, viz. initiation, elongation, and termination so formed is translocated to the P site. The
movement of entire mRNA - tRNA dipeptide
(Fig.14.12).
shifts by a codon exposing the next codon at
Initiation A site in a process called translocation. All
The translation of mRNA begins with the these steps are assisted by a number of
formation of initiation complex. For this, small proteins called elongation factors (EFs) and
subunit of ribosome, a mRNA, a specifically the energy from the hydrolysis of GTP (Fig.
charged initiator tRNA, GTP, Mg 2+ and 14.12). In prokaryotes, elongation factors are
proteinaceous initiation factors are assembled. called EF-Tu, EF-Ts and EF-G. Eukaryotes
These initiation factors are designated as IFs in require a more complex set of accessory factors.
prokaryotes and eIFs in eukaryotes. The
Termination
initiation factors bind to the smaller ribosome
subunit and then this complex binds to a The sequence of elongation continues till the
sequence of mRNA, preceding the initiator AUG whole of mRNA is translated and a signal in the
codon. The initiator AUG in prokaryotes codes form of termination codon (UAG, UAA, or UGA) is
for for mylmethionine (for mylmet) but in reached. These do not code for any amino acid
eukaryotes, this code is for methionine. and, therefore, the translation stops. They
also signal a GTP-dependent release factor
Binding of charged formyl-met tRNA to all the (RF 1 , RF 2 and RF 3 ) which cleaves the
components assembled at 30S subunit forms polypeptide from the terminal tRNA, releasing it
the initiation complex. Later, when the large from the translation complex (Fig. 14.12).
NATURE OF GENE : ITS EXPRESSION AND REGULATION 251

Initiation
Initiation factor

m RNA

Anticodon Initiation codon

t-RNA

Elongation

Peptidyltransferase
Elongation factor Translocation

P site A site
Peptide bond
formation

Incoming
tRNA

Termina-
tion

Met Pro Tyr Ala Leu Release


factor

Met Pro Tyr Ala Leu

Polypeptide

Fig 14.12 Stages in translation – Initiation, elongation and termination. The net result is the synthesis of a peptide
in which amino acids are aligned according to the message received from the gene
The polypeptide formed under the guidance of called polyribosomes or polysomes.
the mRNA thus consists of a specific sequence Some antibiotics inhibit bacterial protein
of amino acids. Once the initial portion of mRNA synthesis. This forms the basis of inhibiting the
is translated, it is free to engage into another bacterial invaders and stemming the
round of translation. This process can be infection without harming the human host
repeated several times creating a structure (Table 14.2 ).

Table 14.2 Some Inhibitors of Bacterial Protein Synthesis


252 BIOLOGY

14.7 GENETIC CODE and J.H. Matthei in early 1960s. For this work,
The process by which the information coded Har Gobind Khorana shared Nobel Prize in 1968
in RNA (mRNA) is decoded into a polypeptide with Nirenberg and Holley. They used
is one of the exciting discoveries of biology. It synthetic RNA homopolymers such as poly U,
is often referred to as deciphering the genetic poly C, poly A, and poly G or copolymers such
as poly UG, AC, etc. This RNA was used in a
code. With only four biochemical letters
test tube protein synthesis system with one of
(A,G,C,U) a one-letter code could not
20 amino acids in labelled form. Thus the first
unambiguously encode 20 amino acids. A two-
code UUU coding for amino acid phenylalanine
letter code could encode only 4 x 4 = 16 amino
was deciphered. All the 64 possible codons can
acids and is still not enough. So, a triplet be represented through what is known as
code, based on three biochemical letters or genetic code dictionary (Fig. 14.13). This clearly
nucleotide bases could make up to 4 x 4 x 4 = shows that the first and second biochemical
64 codons. This is the minimal that will be letters remain the same for a particular amino
required to code for 20 or so different amino acid, the third biochemical base can be
acids. different. Subsequent experiments established
The discovery of the genetic code became that genetic code is nonoverlapping,
possible through the significant contributions unambiguous, degenerate (one amino acid
of Francis H.C. Crick, Severo Ochoa, coded by more than one codon), and commaless
Marshell W. Nirenberg, Har Gobind Khorana (continuous).

Fig 14.13 A code dictionary depicting all the 64 possible codons.


Note the initiation and 3 termination codons
NATURE OF GENE : ITS EXPRESSION AND REGULATION 253

14.8 MOLECULAR MECHANISM OF MUTA- sentence shifts the reading frame of that
TION message, producing the message THE ATC ATA
With the identification of biochemical nature of TET HER AT which is meaningless. A specific
gene as a defined segment of deoxyribose nucleic base sequence within the gene is crucial for it
acid or DNA, mutation mechanisms were to express a particular phenotype. Any change
worked out at molecular level. We know that in in the base sequence may change the codon
DNA,specific sequence of bases forms the and thus leads to altered expression or
reading frame in the form of genetic code for a mutation. Base substitutions can also create
gene. Mutations are brought about by two main mismatches. Each living cell possesses repair
mechanisms that alter the reading frame: mechanisms to rectify such mistakes. Only
(i) Substitution and when these mechanisms are defective, then
(ii) frame-shift mutations. mutations are effected.
In substitution, one base is replaced by 14.9 REGULATION OF GENE EXPRESSION
another base. Addition or deletion of few bases
changes the reading frame and results in frame- In the last section, you have seen as to how the
shift mutations (Fig. 14.14). For example, the genetic information stored in DNA or gene is
expressed through the process of protein
synthesis. However, if we analyse the
concentration of different proteins, even in a
Normal DNA strand
simple bacterial cell like E.coli, vast differences
SUBSTITUTION can be seen. While some proteins may be
Base substitution synthesised in as few as 5 to 10 molecules,
at position 2 :
others may be produced in as many as 100,000
copies per cell. This suggests that gene
expression is regulated. It was known since
SUBSTITUTION 1900 that lactose-metabolising enzymes are
Base substitution synthesised by yeast only when it is grown in
at position 7 :
the presence of this sugar. Soon, bacteria were
shown to adapt to their chemical environment
by synthesising certain enzymes depending
upon the substrate present. Such enzymes are
called inducible enzymes. In contrast, some
enzyme types are synthesised all the times
irrespective of the chemical constitution of the
Normal DNA strand environment; these are described as
constitutive. Another type of regulation is
revealed when the end product of a biosynthetic
FRAME SHIFT
addition at position 10 :
pathway such as an amino acid is provided in
the medium. Under such a condition, the
internal biosynthesis of the said amino acid is
stopped. This regulation is referred to as
FRAME SHIFT repressible. Regulation can also be described
deletion at position 9 :
as negative or positive control. Under negative
control, the product of the regulatory gene shuts
off the expression of the said gene(s). Under
positive control, on the other hand, the product
Fig 14.14 Molecular mechanisms of gene mutation of the regulatory gene activates transcription
of the concerned gene(s). The regulatory
addition of T in the sentence THE FAT CAT ATE mechanisms play a very important role in the
THE RAT may read as THE TFA TCA TAT ETH life of a cell as they prevent any unwanted gene
ERA T changing the meaning altogether. expression thus ensuring efficient energy
Similarly, the deletion of letter F from the same management.
254 BIOLOGY

(a) Lac operon and its regulator


Regulator Operator Structural genes
gene Promotor
i p o z y a

β-galactosidase β-galactoside permease β- galactoside transacetylase

Repressor

(b) Lactose absent


RNA polymerase

Transcription is blocked

Repressor
(c) Lactose present

Transcription of lac mRNA


mRNA

Inactive repressor
can not bind to operator

Lactose

Fig 14.15 Inducible control regulation of lac operon in E.coli. (a) the genetic organisation of lac operon and its
regulatory elements, (b) the situation in the absence of lactose, (c) the situation in the presence of
lactose
Note that the genes are expressed only when required, i.e., when lactose is present

Inducible Control original proposal, another region ahead of


The classical example of regulation of gene activity operator was identified as the promoter. You have
was first proposed by Francois Jacob and read about the promoter in the earlier section. A
Jacques Monod in 1961 while studying lactose regulator gene lac i also exists which controls
metabolism in E. coli. By analysing a number of the transcription from the operon by producing
mutants, all defective in lactose metabolism, they a repressor molecule.
came to the conclusion that a group of genes are The regulatory mechanism works on the basis
expressed and regulated together as a unit that of whether lactose is absent or present. When
they called operon. In this case, the operon cells are grown in absence of lactose, the
consists of three structural genes Z, Y, and A, repressor molecule produced by lacI binds with
as well as the adjacent sequence referred to as the operator region switching off the operon. This
operator region (fig 14.15) . Subsequent to their is logical because when lactose is not present,
NATURE OF GENE : ITS EXPRESSION AND REGULATION 255

(a) Tryptophan operon and its regulator

Regulator Promotor Operator Structural genes


gene
r p o E D C B A

enzymes for tryptophan pathway

(b) Tryptophan absent RNA polymerase

Transcription of trp mRNA


mRNA

Repressor
(inactive)

(c) Tryptophan present

Activated repressor binds to operator


Transcription is blocked

Tryptophan (corepressor)

Fig 14.16 Repressible control of tryptophan biosynthetic genes in E.coli. (a) Genetic organization of trp operon
and its regulatory gene, (b) Expression of trp genes in absence of external tryptophan, (c) Expression
of trp genes in the presence of tryptophan
Note that like in lac operon, trp genes are turned on only when required

the enzymes which process the lactose and are different. Jacob and Monod had shown that
coded by three structural genes are also not when tryptophan is present in the growth
required. However, when lactose is present, it medium, the wild-type E. coli cells repress the
acts as inducer. The inducer binds to repressor synthesis of enzymes of the tryptophan
and modifies its structure in such a way that biosynthesis pathway. Enzyme repression
repressor cannot bind to operator. The three again reflects upon the economy exerted by the
structural genes could now be transcribed and cell.
translated (Fig. 14.15). The RNA produced from Further investigations by them revealed that
the operon is polycistronic mRNA. The lac operon five genes situated close to each other on E.
is the best-understood regulatory mechanism coli chromosome are involved in tryptophan
both in genetic and biochemical terms. biosynthesis. They proposed that these five,
trpE, D, C, B and A are the structural genes,
Repressible Control and like lac operon, preceded by a regulatory
If we compare the regulation of genes involved region constitute trp operon (Fig.14.16). The
in biosynthesis of amino acids and other regulatory region here is divided into promoter,
essential macromolecules, the situation is very operator and a leader sequence in which the
256 BIOLOGY

latter is transcribed but not translated as part The role of nucleus in differentiation can be
of the trp structural gene products. The operon easily understood by nuclear transplantation
is regulated by a distantly placed regulatory experiments such as those carried out in the
gene trpR. In the absence of external creation of a clonal frog. In these experiments,
tryptophan, the repressor produced by trpR is diploid nuclei from differentiated cells were
inactive, which cannot bind to trpO (operator), transplanted into unfertilized eggs whose haploid
thus allowing the transcription of polycistronic nuclei were inactivated before hand. When such
mRNA. In the presence of tryptophan, however, genetically diploid eggs were artificially induced
tryptophan binds to repressor converting this to divide and grow, occasionally they formed adult
complex into active repressor that binds to frog whose chromosomal constitution was exactly
operator, stopping all transcriptional activities. like the donor. Although nuclei derived from larval
Such a repression of the operon, once again can intestinal cells could support clonal reproduction,
be classified as negative control. The trp operon those of adult frog do not. This suggests that
and in fact other amino acid biosynthesis pluripotency (ability to dedifferentiate completely)
operons are subjected to other types of control may be lost upon maturation. Similarly, if nerve
as well. cells are grown outside their normal cellular
environment, they produce similar cells. With
14.10 HOUSE-KEEPING GENES
higher plants, however, the situation is often
From the ongoing discussion it should become opposite. A complete plant can be regenerated even
clear that gene expression is extensively from the differentiated cells of the mature plant
regulated based on the environment. In (totipotency).
multicellular highly differentiated organisms, As in bacteria, in multicellular organisms,
there are regulations exerted even at tissue all genes do not function at all times.
level. For example, some genes may express in Mechanisms must exist therefore, to control
liver, some in kidney, and still others in which genes will act and when, and these
reproductive tissues. In contrast to such genes, controls are much more complex than what we
there are common sets of expressed gene have seen in bacteria.
functions that are needed in all cell types. Such A wealth of information has been collected by
genes are known as house-keeping genes and using the fruit fly, Drosophila, as a working model
their function is referred to as house-keeping and the developmental mutants that have been
or constitutive activity. isolated. It is very clear that during development,
a single fertilized egg gives rise to cells that have
14.11 DIFFERENTIATION AND DEVELOPMENT
different developmental fates. This asymmetry may
While talking of bacteria we usually refer to a be a part of the egg itself in terms of distribution of
single-celled organism with little or no its cytoplasmic components as in Drosophila or
differentiation. All higher plants and animals, may be initiated in the initial division cycles (as
on the other hand, are constructed from a large in mammals). It is logical to suppose that each
variety of cell types. You must be aware that cell type is characterised by its pattern of gene
such multicellular organisms also originate from expression or by the particular gene products that
a single cell, the zygote that forms as a result of it produces. In Drosophila, many types of genes
fertilization. Different cell types must arise from have been identified by mutation and analysing
this zygote through a highly ordered and their functions.
coordinated way, a process known as These genes not only regulate one another but
differentiation. The instructions for how a perhaps the target genes also that code for
fertilized egg will develop into an adult are all structural proteins. Many of these genes possess
written in the linear sequences of the bases in conserved motifs, the commonest of which are
the DNA or gene. This genetic information is known as homeobox. Homeoboxes are distributed
expressed in a regulated manner not only in in other eukaryotes also, such as worms, frogs
space and time but also allows cell-to-cell and mammals, suggesting that they may be
interactions to produce different body parts. characteristics of all animals.
NATURE OF GENE : ITS EXPRESSION AND REGULATION 257

14.12 CANCER AND ONCOGENES (iii) At some stage after transformation they
acquire the ability to metastasize, that is,
You have studied in the earlier Unit that each they become capable of moving and
somatic cell undergoes a precisely programmed
invading normal tissue and find a new place
cell cycle. This, in fact, controls the division
away from the original site to form cancers.
and growth of a cell. This is reflected in a defined
life span of not only the individual somatic cells When cells derived from a vertebrate are
but also the organism. One notable exception is grown in culture they require a solid support to
provided by cancer cells (Fig.14.17). Cancer is a attach and essential growth factors or serum.
disease of cells, in which the controls that Their growth is inhibited by cell to cell contact
and they show characteristic cytoskeletal
normally restrict cell proliferation do not operate.
organisation. Thus, in a culture these cells grow
as a monolayer. The tumor cells, show changes
Proto-oncogene in some or all of these properties. Besides these,
multiple genetic changes occur during the
(a)
transformation of a normal cell to cancer cell, a
process termed as oncogenesis.
Tumor suppressor gene Such a transformation could be caused
spontaneously or by the agents known as
Oncogene carcinogens that promote them. The
carcinogens include agents such as radiations,
(b)
some chemical compounds as well as certain
viruses. Currently two types of genes have been
identified, whose mutations result in cancerous
Tumor suppressor gene transformations.
(i) Oncogenes : Oncogenes act to stimulate
cell division. Interestingly, these genes
Fig 14.17 Regulation of normal cell divisions have normal cellular counterparts, called
(a) Cell division regulation by proto-oncogenes proto-oncogenes (c-onc) which are involved
and constraints imposed by tumor
in normal cell functions. Mutations of
suppressor genes
(b) Transformation of a normal cell into proto-oncogenes lead to oncogenes. Many
cancer cell if this regulation is upset tumor causing viruses may carry similar
onco gene (v-onc) sufficient to induce
carcinogensis.
There are three major differences between the (ii) Tumor Suppressor Genes : As the name
cancer cells and the normal cells. suggests they normally suppress the
tumors and the disease appears only when
(i) Unlike the normal cells, cancer cells are
the appropriate active gene is lacking or
immortalized and show indefinite growth.
both the alleles are lost. By function, they
(ii) Since they are derived from normal cells, usually impose some constraints on cell
they seem to arise through transformation cycle. Their absence leads to the release of
in which normal constraints of growth are these constraints, leading to induction of
bypassed. tumors.
258 BIOLOGY

SUMMARY

Experimental work on biochemical and molecular nature of hereditary material conclusively


demonstrated that DNA (and not protein) is indeed the genetic material. Using information
gathered by many scientists, Watson and Crick worked out the biological implications and
suggested a double helix model of DNA. In this, two DNA strands are wound together
around each other and run in antiparallel fashion. The backbone of the molecule consists
of alternating deoxyribose sugar and phosphate, and the nitrogenous bases are stacked
inside. The two strands are held together by hydrogen bonding between adenine (A) and
thymine (T), and guanine (G) and cytosine (C). This model also suggested the way DNA
can be replicated, a prime requirement for genetic material. Replication takes place semi-
conservatively, in which the two strands act as templates to allow the synthesis of new
strands, which have a complementary base sequence. Replication is achieved with the aid
of several enzymes, and starts at a specific point, known as origin of replication. Since
the main DNA replicating enzyme acts only in 5' à 3' direction, one strand is continuously
replicated but in the other, DNA is synthesised in small fragments called Okazaki fragments.
The process is known as semi-discontinuous. The DNA polymerisation requires a small
RNA primer also synthesised by a special enzyme, primase. Primers are subsequently
removed and the gaps are filled by DNA polymerase I. Okazaki fragments are later joined
by the enzyme DNA ligase.
The idea that the genes control the metabolism was available by the early part of the
last century, and the same was confirmed by mid 1940s through the work of Beadle and
Tatum. This and several other information established that the control is brought about
by directing the synthesis of proteins which have a variety of functions. The protein synthesis
consists of two steps, transcription and translation, which constitute the fundamental
processes essential to the expression of genetic material. Transcription leads to the synthesis
of three species of single-standard RNA under the guidance of the enzyme RNA polymerase
from a DNA template. The three types of RNA have different functions in protein synthesis.
The mRNA carries the message from the gene or the DNA, rRNA along with some proteins
constitutes the ribosomes, the site at which translation takes place and tRNA transfers
the required amino acid to the ribosome during translation. Both, transcription and
translation can be subdivided into initation, elongation, and termination, and like
replication rely on complementary base pairing. The two processes are more complex in
eukaryotes where primary mRNA transcript is modified in various ways.
The genetic information is stored in the DNA in the form of a code, which is triplet,
degenerate, unambiguous, non-overlapping and commaless in nature. The complete code
dictionary consists of 64 possible codes, of which 61 codons code for 20 essential amino
acids including the initiation codon, and 3 are meant to terminate translation. Any
mutational change may result into a new codon coding for different amino acids. The
expression of genes is highly regulated both by the external and the internal environments.
This checks any wasteful synthesis of proteins and enables the cell to conserve energy.
The synthesis of inducible class is initiated only when their substrate is available, whereas
the synthesis of repressible class is stopped if their products are supplied. Also the
regulation can be classified as negative or positive control. Besides repression, many
biosynthetic enzymes are regulated by additional control. There are enzymes which are
required by the cell althrough and are called constitutive.
The regulation of gene expression in eukaryotes is more complex, which obviously is a
reflection of their multicellularity, larger genome size and other aspects of gene expression.
The process of development and differentiation and expression of specific sets of gene such
as oncogene have served as models for gene regulation in higher organisms. The whole
NATURE OF GENE : ITS EXPRESSION AND REGULATION 259

process of development and differentiation is based on differential gene expression, as the


basic genetic information carried by each somatic cell in a multicellular organism is same.
Though tissue specific gene expression is known, there are some common sets of expressed
genes also. Such genes are known as house-keeping genes.
An aberration in the normal plan of cellular growth and its regulation can lead to
carcinogenesis or induction of cancer. In this process, the normal cells are transformed to
a state where they lose normal constraints of growth. A cell possesses proto-oncogene or
c-onc in which alteration can lead to their transformation into cancerous cells. Many
tumor causing viruses may carry similar c-onc gene (v-onc) which may be sufficient for
carcinogenesis. Similarly, a normal cell may carry tumor suppressor genes and the loss
of the same may result in tumor induction.

EXERCISES

1. Which of the nucleotide compositions will be possible, if DNA is double stranded?


(a) All A; (b) Only A and T; (c) Only C and T; (d) Only A and G; (e) only A,G and T.
2. Given below is the transcribed strand of the DNA duplex:
3’− TAC CGA TCC GAG CTG −5’
(a)Draw the complementary DNA polynucleotide chain
(b)Construct the RNA molecule, which will be transcribed.
3. From the following DNA sequence representing a part of the gene, derive:
(a) the RNA transcript;
(b) the processed mRNA (assuming that all the codons containing a C represent
the intron DNA);
(c) the number of amino acids it can code for:
TACCCCCAC GAGTTATATATACGG GGGCATCATATG
4. Consider DNA molecules which contain most of its nitrogen in the form of 15N
(heavy isotope). Such molecules are allowed to replicate in an environment, where
the nitrogen source is 14N. What will be the proportion of the DNA molecules that
will contain some 15N after: (a) one round of replication; and (b) two rounds of
replication?
Provide experimental evidence for the semi-conservative mode of replication of DNA.
5. Given below is sequence of the processed mRNA ready for translation:
−3’
− AUG CUA UAC CUC CUU UAU CUG UGA−
5’−
(a) How many amino acid residues will make up the polypeptide corresponding to
this mRNA?
(b) How many different tRNA molecules would be necessary to translate this mRNA?
6. What was the rationale of using 32P and 35S by Hershey and Chase? Instead, if we use
radiolabelled C and N, will the results be any different?
7. List three main differences between DNA and RNA?
8. Which molecule bears codons and which molecule anticodons?
9. What are the three essential requirements of the genetic materials?
10. How did Hershey and Chase prove that DNA is the genetic material?
260 BIOLOGY

11. Does each of the two complementary strands of DNA carry the same biological message?
Explain.
12. Enlist the role of following in protein synthesis:
(i) mRNA
(ii) rRNA
(iii) tRNA
(iv) ribosomes
(v) Amino Acids
(vi) ATP
13. Describe an inducible operon and differentiate it from a repressible operon.
14. What changes are brought about in a cell when it is transformed into a cancer cell?
15. What do you understand by a leading strand and a lagging strand during DNA
replication?
16. Give the two terms used to describe the base triplets on the RNA molecules.
17. If one strand of double-stranded DNA has the following sequence:
5' … AGC ATTCG …. 3'
What would be the sequence of the opposite strand in its 5' to 3' direction
18. Consider the short message:
AUGGCAGUGCCA
Answer the following questions:
(a) How many codons would be represented in this oligonucleotide?
(b) If the second G were changed to a C, how many codons would be changed?
19. Give the exception to the general rule that DNA is the genetic material in all organisms.
Give evidences that support these exceptions.
20. All the cells in a multicellular organism have the same genetic constitution yet they
function differently. How do you explain this?
21. What is the inducer in the lac operon? How does it ensure the ‘switching on’ of genes?
22. How does an excess of tryptophan cause a ‘switching off ’ of the tryptophan operon?