CHAPTER l2083CH06

5
MOLECULARBASIS
I OF
NHER ITANCE

5.1 TheDNA
5.2 The Searchjor Genetic
Material In the previous chapter, you have learnt the inheritance
patterns and the genetic basis of such patterns. At the
5.3 RNA World
time of Mendel, the nature of those 'factors' regulating
5.4 Replication the pattern of inheritance was not clear. Over the next
5.5 Transcription hundred years, the nature of the putative genetic
material was investigated culminating in the
5.6 Genetic Code realisation that DNA - deoxyribonucleic acid - is the
5.7 Translation genetic material, at least for the majority of organisms.
In class XI you have learnt that nucleic acids are
5.8 Regulation of polymers of nucleotides.
Gene Expression
Deoxyribonucleic acid (DNA) and ribonucleic acid
5.9 Human Genome Project (RNA) are the two types of nucleic acids found in
living systems. DNA acts as the genetic material in
5.10 DNA Fingerprinting
most of the organisms. RNA though it also acts as a
genetic material in some viruses, mostly functions as a
messenger . RNA has additional roles as well. It
functions as adapter, structural, and in some cases as a
catalytic molecule . In Class XI you have already
learnt the structures of nucleotides and the way these
monomer units are linked to form nucleic acid
polymers. In this chapter we are going to discuss the
structure of DNA, its replication, the process of making
RNA from DNA (transcription), the genetic code that
determines the sequences of amino acids in proteins,
the process of protein synthesis (translation) and

2024-25
elementary basis of their
regulation. The
determination

2024-25
 BIOLOGY

of complete nucleotide sequence of human genome during last decade

has set in a new era of genomics. In the last section, the essentials of
human genome sequencing and its consequences will also be
discussed.
Let us begin our discussion by first understanding the structure of
the most interesting molecule in the living system, that is, the DNA. In
subsequent sections, we will understand that why it is the most
abundant genetic material, and what its relationship is with RNA.

5.1 THE DNA

DNA is a long polymer of deoxyribonucleotides. The length of DNA is
usually defined as number of nucleotides (or a pair of nucleotide referred
to as base pairs) present in it. This also is the characteristic of an
organism. For example, a bacteriophage known as cj> x l 74 has 5386
nucleotides, Bacteriophage lambda has 48502 base pairs (bp),
Escherichia coli has
4.6 x 106 bp, and haploid content of human DNA is 3.3 x 109 bp. Let
us discuss the structure of such a long polymer.

5. 1. 1 Structure of Polynucleotide Chain

Let us recapitulate the chemical structure of a polynucleotide chain (DNA
or RNA). A nucleotide has three components - a nitrogenous base, a
pentose sugar (ribose in case of RNA, and deoxyribose for DNA), and a
phosphate group. There are two types of nitrogenous bases - Purines
(Adenine and Guanine), and Pyrimidines (Cytosine, Uracil and Thymine).
Cytosine is common for both DNA and RNA and Thymine is present in
DNA. Uracil is present in RNA at the place of Thymine. A nitrogenous
base is linked to the OH of I'C pentose sugar through a N-glycosidic
linkage to form a nucleoside, such as adenosine or deoxyadenosine,
guanosine or deoxyguanosine, cytidine or deoxycytidine and uridine or
deoxythymidine. When a phosphate group is linked to OH of 5' C of a
nucleoside through phosphoester linkage, a corresponding nucleotide
(or deoxynucleotide depending upon the type of sugar present) is formed.
Two nucleotides are linked through 3'_5' phosphodiester linkage to form
a dinucleotide. More nucleotides can be joined in such a manner to form
a polynucleotide chain. A polymer thus formed has at one end a free
5' phosphate

I 6
CV-H p 3'hytxy'
H
c

Figure 5.1 A Polynucleotide chain

2024-25
MOLECULAR BASIS OF INHERITANCE

phosphate moiety at 5'-end of sugar, which is referred to as 5'-end of

polynucleotide chain. Similarly, at the other end of the polymer the sugar
has a free OH of 3'C group which is referred to as 3'- end of the
polynucleotide chain. The backbone of a polynucleotide chain is formed
due to sugar and phosphates. The nitrogenous bases linked to sugar
moiety project from the backbone (Figure 5.1).
In RNA, eve:ry nucleotide residue has an additional -OH group
present at 2'-position in the ribose. Also, in RNA the uracil is found at the
place of thymine (5-methyl uracil, another chemical name for thymine).
DNA as an acidic substance present in nucleus was first identified by
Friedrich Meischer in 1869. He named it as 'Nuclein'. However, due to
technical limitation in isolating such a long polymer intact, the
elucidation of structure of DNA remained elusive for a ve:ry long period
of time. Itwas only in 1953 that James Watson and Francis Crick,
based on the X-ray diffraction data produced by Maurice Wilkins and
Rosalind Franklin, proposed a ve:ry simple but famous Double Helix
model for the structure of DNA. One of the hallmarks of their proposition
was base pairing between the two strands of polynucleotide chains.
However, this proposition was also based on the obseivation of EIWin
Chargaff that for a double stranded DNA, the ratios between Adenine and
Thymine and Guanine and Cytosine are constant and equals one.
The base pairing confers a ve:ry unique property to the polynucleotide
chains. They are said to be complementa:ry to each other, and therefore if
the sequence of bases in one strand is known then the sequence in other
strand can be predicted. Also, if each strand from a DNA (let us call it as
a parental DNA) acts as a template for synthesis of a new strand, the
two double stranded DNA (let us call them as daughter DNA) thus,
produced would be identical to the parental DNA molecule. Because of
this, the genetic implications of the structure of DNA became ve:ry clear.
The salient features of the Double-helix structure of DNA are as follows:
(i) It is made of two polynucleotide chains, where the backbone
is constituted by sugar-phosphate, and the bases project
inside.
(ii) The two chains have anti-parallel polarity. It means, if
one chain has the polarity 5'-7 3', the other has 3'-7 5'.
(iii) The bases in two strands are paired through hydrogen bond
(H-bonds) forming base pairs (bp). Adenine forms two
hydrogen bonds with Thymine from opposite strand and
vice-versa.
Similarly, Guanine is bonded with Cytosine with three H-bonds. .
As a result, always a purine comes opposite to a pyrimidine. This •
generates approximately uniform distance between the two
strands of the helix (Figure 5.2).
(iv) The two chains are coiled in a right-handed fashion. The
pitch of the helix is 3.4 nm (a nanometre is one billionth
of a metre, that is 10-9 m) and there are roughly 10 bp in
each

2024-25
 BIOLOGY

5' ®-cH H

H H
c H
C OH3'
I

T
''
''
c
''' :: ',,'''
' :: :: ',,'''
''' !,,!!' ..hydrogen
'
:

HOG
'''
'
' ''
'
:: !, bonds
:
!, !'

H
H
3'

c lj l ---®
< ( p 5'

H
H

Figure 5.2 Double stranded polynucleotide chain

turn. Consequently, the distance

be twe en a b p in a h elix is

( < ) approximately 0.34 nm.

(v) The plane of one base pair stacks
Adenine Thymine over the other in double helix.
This, in addition to H-bonds,
confers stability of the helical
structure (Figure 5.3).
Guanine Cytosine Compare the structure of purines
and py rimidines. Can y ou find out
why the d is tance betw een tw o pol y
- - ,, -+---- Sugar phosphate
nucleotide chains in DNA remains
backbone
almost constant?
The proposition of a double helix
structure for DNA and its simplicity in
explaining the genetic implication became
revolutionary. Very soon, Francis Crick
proposed the Central dogma in molecular
Figure 5.3 DNA double helix biology, which states that the genetic
information flows from DNA-7 RNA-
replication 7Protein.

transcription translation
mRNA ----- protein

2024-25
 Central dogma

2024-25
MOLECULAR BASIS OF INHERITANCE

In some viruses the flow of information is in reverse direction, that is,

from RNA to DNA. Can you suggest a simple name to the process?

5. 1.2 Packaging of DNA Helix

Taken the distance between two consecutive base pairs
as 0.34 nm (0.34x10-9 m), if the length of DNA double DNA H l histone
helix in a typical mammalian cell is calculated (simply
by multiplying the total number of bp with distance
between two consecutive bp, that is, 6.6 x 109 bp x
0.34 x I0-9 m/ bp), it comes out to be approximately
2.2 metres. A length that is far greater than the
dimension of a typical nucleus (approximately 10-6
m). How is such a long polymer packaged in a cell?
If the length of E. coli DNA is 1.36 mm, can you
calculate the number of base pairs in E.coli?
Core of histone molecules
In prokaryotes, such as, E. coli, though they do
not have a defined nucleus, the DNA is not Figure 5.4a Nucleosome
scattered throughout the cell. DNA (being negatively
charged)
is held with some proteins (that have positive
charges) in a region termed as 'nucleoid'. The
DNA in nucleoid is organised in large loops
held by proteins.
In eukaryotes, this organisation is much more
complex. There is a set of positively charged,
basic proteins called histones. A protein acquires
charge depending upon the abundance of amino
acids residues with charged side chains. Histones
are rich in the basic amino acid residues lysine
and arginine.
Both the amino acid residues carry positive charges Figure 5.4b EM picture _ 'Beads-on-String'
in their side chains. Histones are organised to form
a unit of eight molecules called histone octamer.
The negatively charged DNA is wrapped around the positively
charged histone octamer to form a structure called nucleosome (Figure
5.4 a). A typical nucleosome contains 200 bp of DNA helix.
Nucleosomes constitute the repeating unit of a structure in nucleus
called chromatin, thread like stained (coloured) bodies seen in
nucleus. The nucleosomes in chromatin are seen as 'beads-on-string'
structure when viewed under
electron microscope (EM) (Figure 5.4 b).
.
Theoretically , how many such beads (nucleosomes) do you imagine
•
are present in a mammalian cell?
The beads-on-string structure in chromatin is packaged to form
chromatin fibers that are further coiled and condensed at metaphase stage
of cell division to form chromosomes. The packaging of chromatin at
higher level requires additional set of proteins that collectively are
referred to as
2024-25
 BIOLOGY

Non-histone Chromosomal (NHC) proteins. In a typical nucleus, some

region of chromatin are loosely packed (and stains light) and are referred
to as euchromatin. The chromatin that is more densely packed and
stains dark are called as Heterochromatin. Euchromatin is said to be
transcriptionally active chromatin, whereas heterochromatin is inactive.

5.2 THE SEARCH FOR GENETIC MATERIAL

Even though the discovery of nuclein by Meischer and the proposition

for principles of inheritance by Mendel were almost at the same time,
but that the DNA acts as a genetic material took long to be discovered
and proven. By 1926, the quest to determine the mechanism for
genetic inheritance had reached the molecular level. Previous
discoveries by Gregor Mendel, Walter Sutton, Thomas Hunt Morgan
and numerous other scientists had narrowed the search to the
chromosomes located in the nucleus of most cells. But the question of
what molecule was actually the genetic material, had not been
answered.

Transforming Principle
In 1928, Frederick Griffith, in a series of experiments with
Streptococcus pneumoniae (bacterium responsible for pneumonia) ,
witnessed a miraculous transformation in the bacteria. During the
course of his experiment, a living organism (bacteria) had changed in
physical form.
When Streptococcus pneumoniae (pneumococcus) bacteria are grown
on a culture plate, some produce smooth shiny colonies (S) while others
produce rough colonies (R). This is because the S strain bacteria have a
mucous (polysaccharide) coat, while R strain does not. Mice infected
with the S strain (virulent) die from pneumonia infection but mice
infected with the R strain do not develop pneumonia.

S strain - -) Inject into mice - -) Mice die

R strain - -) Inject into mice - -) Mice live

Griffith was able to kill bacteria by heating them. He observed that

heat-killed Sstrain bacteria injected into mice did not kill them. When he

S strain - -) Inject into mice --) Mice live

(heat-killed)

S strain
(heat-killed)
+ - -) Inject into mice --) Mice die
R strain
(live)

2024-25
MOLECULAR BASIS OF INHERITANCE

injected a mixture of heat-killed S and live R bacteria, the mice died.

Moreover, he recovered living Sbacteria from the dead mice.
He concluded that the R strain bacteria had somehow been
transformed by the heat-killed S strain bacteria. Some 'transforming
principle', transferred from the heat-killed S strain, had enabled the
R strain to synthesise a smooth polysaccharide coat and become
virulent. This must be due to the transfer of the genetic material.
However, the biochemical nature of genetic material was not defined
from his experiments.

Biochemical Characterisation of Transforming Principle

Prior to the work of Oswald Avery, Colin MacLeod and Maclyn McCarty
( 1933-44), the genetic material was thought to be a protein. They
worked to determine the biochemical nature of 'transforming principle' in
Griffith's experiment.
They purified biochemicals (proteins, DNA, RNA, etc.) from the
heat-killed S cells to see which ones could transform live R cells into
Scells. They discovered that DNA alone from Sbacteria caused R
bacteria to become transformed.
They also discovered that protein-digesting enzymes (proteases) and
RNA-digesting enzymes (RNases) did not affect transformation, so the
transforming substance was not a protein or RNA. Digestion with DNase
did inhibit transformation, suggesting that the DNA caused the
transformation. They concluded that DNA is the hereditary material, but
not all biologists were convinced.
Can you think of any difference between DNAs and DNase?

5.2. 1 The Genetic Material is DNA

The unequivocal proof that DNA is the genetic material came from the
experiments of Alfred Hershey and Martha Chase (1952). They
worked with viruses that infect bacteria called bacteriophages.
The bacteriophage attaches to the bacteria and its genetic material
then enters the bacterial cell. The bacterial cell treats the viral genetic
material as if it was its own and subsequently manufactures more
virus particles. Hershey and Chase worked to discover whether it was
protein or DNA from the viruses that entered the bacteria.
They grew some viruses on a medium that contained radioactive
phosphorus and some others on medium that contained radioactive sulfur. .
•
Viruses grown in the presence of radioactive phosphorus contained
radioactive DNA but not radioactive protein because DNA contains
phosphorus but protein does not. Similarly, viruses grown on radioactive
sulfur contained radioactive protein but not radioactive DNA because
DNA does not contain sulfur.

2024-25
 BIOLOGY

Radioactive phages were allowed to attach to E. coli bacteria. Then, as

the infection proceeded, the viral coats were removed from the bacteria
by agitating them in a blender. The virus particles were separated from
the bacteria by spinning them in a centrifuge.
Bacteria which was infected with viruses that had radioactive DNA
were radioactive, indicating that DNA was the material that passed from
the virus to the bacteria. Bacteria that were infected with viruses that had
radioactive proteins were not radioactive. This indicates that proteins did
not enter the bacteria from the viruses. DNA is therefore the genetic
material that is passed from virus to bacteria (Figure 5.5).

rm\----Bacteriophage----ff!i1 32
35
-..... -Radioactive ( P)
Radioactive ( S) labelled labelled DNA
protein capsule

C\ p 1. Infection

C\ p 2. Blending

No Radioactive
35
( S) detected in 3. Centrifugation
cells

C't1
Radioactive
32
35 ( P) detected in
Radioac ve ( S) cells
detected in supernatant
No Rad oactivity

PP
detected in supernatant

Figure 5.5 The Hershey-Chase experiment

5.2.2 Properties of Genetic Material (DNA versus RNA)

From the foregoing discussion, it is clear that the debate between proteins
versus DNA as the genetic material was unequivocally resolved from
2024-25
Hershey-Chase experiment. It became an established fact that it is DNA
that acts as genetic material. However, it subsequently became clear that

2024-25
MOLECULAR BASIS OF INHERITANCE

in some viruses, RNA is the genetic material (for example, Tobacco

Mosaic viruses, QB bacteriophage, etc.). Answer to some of the questions
such as, why DNA is the predominant genetic material, whereas RNA
performs dynamic functions of messenger and adapter has to be found
from the differences between chemical structures of the two nucleic acid
molecules.
Can you recall the two chemical dijferences between DNA and RNA?
A molecule that can act as a genetic material must fulfill the
following criteria:
(i) It should be able to generate its replica (Replication).
(ii) It should be stable chemically and structurally.
should provide the scope for slow changes (mutation) that
(iii) It
are required for evolution.
(iv) It should be able to express itself in the form of 'Mendelian
Characters'.
Ifone examines each requirement one by one, because of rule of base
pairing and complementarity, both the nucleic acids (DNA and RNA)
have the ability to direct their duplications. The other molecules in the
living system, such as proteins fail to fulfill first criteria itself.
The genetic material should be stable enough not to change with
different stages of life cycle, age or with change in physiology of the
organism. Stability as one of the properties of genetic material was
very evident in Griffith's 'transforming principle' itself that heat, which
killed the bacteria, at least did not destroy some of the properties of
genetic material. This now can easily be explained in light of the DNA
that the two strands being complementary if separated by heating come
together, when appropriate conditions are provided. Further, 2'-OH
group present at every nucleotide in RNA is a reactive group and
makes RNA labile and easily degradable. RNA is also now known to
be catalytic, hence reactive. Therefore, DNA chemically is less
reactive and structurally more stable when compared to RNA.
Therefore, among the two nucleic acids, the DNA is a better genetic
material.
In fact, the presence of thymine at the place of uracil also confers
additional stability to DNA. (Detailed discussion about this requires
understanding of the process of repair in DNA, and you will study these
processes in higher classes.)
Both DNA and RNA are able to mutate. In fact, RNA being unstable,
mutate at a faster rate. Consequently, viruses having RNA genome and .
•
having shorter life span mutate and evolve faster.
RNA can directly code for the synthesis of proteins, hence can easily
express the characters. DNA, however, is dependent on RNA for
synthesis of proteins. The protein synthesising machinery has evolved
around RNA. The above discussion indicate that both RNA and DNA
can function as

2024-25
 BIOLOGY

genetic material, but DNA being more stable is preferred for storage of
genetic information. For the transmission of genetic information, RNA
is better.

5.3 RNA WORLD

From foregoing discussion, an immediate question becomes evident -

which is the first genetic material? It shall be discussed in detail in the
chapter on chemical evolution, but briefly, we shall highlight some of
the facts and points.
RNA was the first genetic material. There is now enough evidence to
suggest that essential life processes (such as metabolism, translation,
splicing, etc.), evolved around RNA. RNA used to act as
a genetic material as well as a catalyst (there are some
important biochemical reactions in living systems that
are catalysed by RNA catalysts and not by protein
enzymes). But, RNA being a catalyst was reactive and
hence unstable. Therefore, DNA has evolved from
RNA with chemical modifications that make it more
stable. DNA being double stranded and having
complementary strand further resists changes by
evolving a process of repair.

5.4 REPLICATION

While proposing the double helical structure for DNA,

5' Watson and Crick had immediately proposed a scheme
for replication of DNA. To quote their original
statement that is as follows:
"It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material" (Watson and Crick, 1953).
The scheme suggested that the two strands would
separate and act as a template for the synthesis of
Figure 5.6 Watson- Crick model for new complementary strands. After the
completion of
s
emiconse rvative DNA replication, each DNA molecule would have one
replication parental and one newly synthesised strand. This
scheme was termed as semiconservative DNA
replication (Figure 5.6).
5.4. 1 The Experimental Proof
Itis now proven that DNA replicates semiconservatively. Itwas shown first in
Escherichia coli and subsequently in higher organisms, such as plants

2024-25
MOLECULAR BASIS OF INHERITANCE

and human cells. Matthew Meselson and Franklin Stahl performed the
following experiment in 1958:
(i) They grew E. coli in a medium containing 15NH Cl (15N is the
4
heavy isotope of nitrogen) as the only nitrogen source for
many generations. The result was that 15N was incorporated into
newly synthesised DNA (as well as other nitrogen containing
compounds). This heavy DNA molecule could be distinguished
from the normal DNA by centrifugation in a cesium chloride
(CsCl) density gradient (Please note that 15N is not a
radioactive isotope, and it can be separated from 14N only
based on densities).
(ii) Then they transferred the cells into a medium with normal
14
NH Cl and took samples at various definite time intervals
4
as the cells multiplied, and extracted the DNA that remained
as double-stranded helices. The various samples were
separated independently on CsCl gradients to measure the
densities of DNA (Figure 5.7).
Can you recall what centrifugal force is, and think why a
molecule with higher mass/ density would sediment jaster?

The results are shown in Figure 5.7.

Generation I
15 14
N-DNA N-DNA
15
N-DNA

l\YAYAY_ =:>
20 min

Gravitational force [:>

14 N1sN

Hybrid Light Hybrid

Heavy

(Separation of DNA by Centrifugation)

•• •
Figure 5.7 Meselson and Stahl's Experiment

(iii) Thus, the DNA that was extracted from the culture one
generation after the transfer from 15N to 14N medium [that is
after 20 minutes; E. coli divides in 20 minutes] had a hybrid
or intermediate density. DNA extracted from the culture
after another generation [that is after 40 minutes, II
generation] was

2024-25
 BIOLOGY

composed of equal amounts of this hybrid DNA and of 'light'

DNA.
If E. coli was allowed to grow for 80 minutes then what would be
the proportions of light and hy brid densities DNA molecule?
Very similar experiments involving use of radioactive thymidine to
detect distribution of newly synthesised DNA in the chromosomes was
performed on Viciafaba (faba beans) by Taylor and colleagues in
1958. The experiments proved that the DNA in chromosomes also
replicate semiconservatively.

5.4.2 The Machinery and the Enzymes

In living cells, such as E. coli, the process of replication requires a set of
catalysts (enzymes). The main enzyme is referred to as DNA-dependent
DNA polymerase, since it uses a DNA template to catalyse the
polymerisation of deoxynucleotides. These enzymes are highly efficient
enzymes as they have to catalyse polymerisation of a large number of
nucleotides in avery short time. E. coli that has only 4.6 x 106 bp
(compare it with human whose diploid content is 6.6 x 109 bp),
completes the process of replication within 18 minutes; that means the
average rate of polymerisation has to be approximately 2000 bp per
second. Not only do these polymerases have to be fast, but they also have
to catalyse the reaction with high degree of accuracy. Any mistake during
replication would result into mutations. Furthermore, energetically
replication is avery expensive process. Deoxyribonucleoside
triphosphates serve dual purposes. In addition to acting as substrates,
they provide energy for polymerisation reaction (the two terminal
phosphates in a deoxynucleoside triphosphates are high-energy
phosphates, same as in case of ATP).
In addition to DNA-dependent DNA polymerases, many additional
enzymes are required to complete the process of replication with high
degree of accuracy. For long DNA molecules, since the two strands of
DNA cannot be separated in its entire length (due to very high energy
requirement), the replication occur within a small opening of the DNA
helix, referred to as replication fork . The DNA-dependent DNA
polymerases catalyse polymerisation only in one direction, that is 5'-73'.
This creates some additional complications at the replicating fork.
Consequently, on one strand (the template with polarity 3'-75'), the
replication is continuous, while on the other (the template with
polarity 5'-73'), it is discontinuous. The discontinuously synthesised
fragments are later joined by the enzyme DNA ligase (Figure 5.8).
The DNA polymerases on their own cannot initiate the process
of replication. Also the replication does not initiate randomly at any
place in DNA. There is a definite region in E. coli DNA where the
replication originates. Such regions are termed as origin of
replication. It is

2024-25
MOLECULAR BASIS OF INHERITANCE

because of the requirement of the origin of 5' 3'

replication that a piece of DNA if needed to be
propagated during recombinant DNA procedures,
requires a vector. The vectors provide the origin
of replication.
Further, not every detail of replication is Template DNA
(parental strands)
understood well. In eukaryotes, the replication of
DNA takes place at S-phase of the cell-cycle. The
replication of DNA and cell division cycle should
be highly coordinated. A failure in cell division
after DNA r eplication r esu lts into poly ploidy(a
chromosomal anomaly). You will learn the detailed
nature of origin and the processes occurring at this
site, in higher classes.

5.5 TRANSCRIPTION

The process of copying genetic information from

Figure 5.8 Replicating Fork
one strand of th e DNA into RNA is ter m ed as
transcription . He re also , the princi ple
of
complementarity governs the process of transcription, except the
adenosine complements now forms base pair with uracil instead of
thymine. However, unlike in the process of replication, which once set in,
the total DNA of an organism gets duplicated, in transcription only a
segment of DNA and only one of the strands is copied into RNA. This
necessitates defining the boundaries that would demarcate the region
and the strand of DNA that would be transcribed.
Why both the strands are not copied during transcription has the
simple answer. First, if both strands act as a template, they would code
for RNA molecule with different sequences (Remember complementarity
does not mean identical), and in tum, ifthey code for proteins, the
sequence of amino acids in the proteins would be different. Hence, one
segment of the DNA would be coding for two different proteins, and
this would complicate the genetic information transfer machinery.
Second, the two RNA molecules if produced simultaneously would be
complementary to each other, hence would form a double stranded RNA.
This would prevent RNA from being translated into protein and the
exercise of transcription would become a futile one.

5.5. 1 Transcription Unit •

A transcription unit in DNA is defined primarily by the three regions in
the DNA:
(i) A Promoter
(ii) The Structural gene
(iii) A Terminator

2024-25
 BIOLOGY

There is a convention in defining the two strands of the DNA in the

structural gene of a transcription unit. Since the two strands have
opposite polarity and the DNA-dependent RNA polymerase also
catalyse the polymerisation in only one direction, that is, 5'_g', the
strand that has the polarity 3'-D' acts as a template, and is also
referred to as template strand. The other strand which has the polarity
(5'-3') and the sequence same as RNA (except thymine at the place of
uracil}, is displaced during transcription. Strangely, this strand (which
does not code for anything) is referred to as coding strand. All the
reference point while defining a transcription unit is made with
coding strand. To explain the point, a hypothetical sequence from a
transcription unit is represented below:
3'-ATGCATGCATGCATGCATGCATGC-5' Template Strand
5'-TACGTACGTACGTACGTACGTACG-3' Coding Strand
Can you now write the sequence of RNA transcribed.from the above DNA?

Transcription start site

Promoter Terminator
3, 1- --1 S-tru_c_tural g_e_n_e T-e_mpla_te strand +- --1i--
5,

5· 1- --1 .... -t---1•3·

Coding strand

Figure 5.9 Schematic structure of a transcription unit

The promoter and terminator flank the structural gene in a

transcription unit. The promoter is said to be located towards 5'-end
(upstream) of the structural gene (the reference is made with respect to
the polarity of coding strand). Itis a DNA sequence that provides
binding site for RNA polymerase, and it is the presence of a
promoter in a transcription unit that also defines the template and
coding strands. By switching its position with terminator, the definition
of coding and template strands could be reversed. The terminator is
located towards 3'-end (downstream) of the coding strand and it
usually defines the end of the process of transcription (Figure 5.9).
There are additional regulatory sequences that may be present further
upstream or downstream to the promoter. Some of the properties of
these sequences shall be discussed while dealing with regulation of
gene expression.

5.5.2 Transcription Unit and the Gene

A gene is defined as the functional unit of inheritance. Though there is no
ambiguity that the genes are located on the DNA, it is difficult to literally

2024-25
MOLECULAR BASIS OF INHERITANCE

define a gene in terms of DNA sequence. The DNA sequence coding

for tRNA or rRNA molecule also define a gene. However by defining
a cistron as a segment of DNA coding for a polypeptide, the structural
gene in a transcription unit could be said as monocistronic (mostly in
eukaryotes) or polycistronic (mostly in bacteria or prokaryotes). In
eukaryotes, the monocistronic structural genes have interrupted coding
sequences -the genes in eukaryotes are split. The coding sequences
or expressed sequences are defined as exons. Exons are said to be
those sequence that appear in mature or processed RNA. The exons
are interrupted by introns. Intrans or intervening sequences do not
appear in mature or processed RNA. The split-gene arrangement
further complicates the definition of a gene in terms of a DNA
segment.
Inheritance of a character is also affected by promoter and regulatory
sequences of a structural gene. Hence, sometime the regulatory
sequences are loosely defined as regulatory genes, even though these
sequences do not code for any RNA or protein.

5.5.3 Types of RNA and the process of Transcription

In bacteria, there are three major types of RNAs: mRNA (messenger
RNA), tRNA (transfer RNA), and rRNA (ribosomal RNA). All three
RNAs are needed to synthesise a protein in a cell. The mRNA provides
the template, tRNA brings aminoacids and reads the genetic code, and
rRNAs play structural and catalytic role during translation. There is
single DNA-dependent RNA polymerase that catalyses transcription of
all types of RNA in bacteria. RNA polymerase binds to promoter and
initiates transcription (Initiation). Ituses nucleoside triphosphates as
substrate

3' 3'

5'
DNA helix

5'

3'

Elongation

3' 5'
5' 3' •
RNA
Termination
Polymera
Rho factor

Figure 5.10 Process of Transcription in Bacteria

2024-25
 BIOLOGY

and polymerises in a template depended fashion following the rule of

complementarity. It somehow also facilitates opening of the helix and
continues elongation. Only a short stretch of RNA remains bound to
the enzyme. Once the polymerases reaches the terminator region, the
nascent RNA falls off, so also the RNA polymerase. This results in
termination of transcription.
An intriguing question is that how is the RNA polymerases able
to catalyse all the three steps, which are initiation, elongation and
termination. The RNA polymerase is only capable of catalysing the
process of elongation. It associates transiently with initiation-factor
(cr) and termination-factor (p) to initiate and terminate the
transcription, respectively. Association with these factors alter the
specificity of the RNA polymerase to either initiate or terminate
(Figure 5.10).
In bacteria, since the mRNA does not require any processing to
become active, and also since transcription and translation take place
inthe same compartment (there is no separation of cytosol and nucleus
in bacteria), many times the translation can begin much before the
mRNA is fully transcribed. Consequently, the transcription and
translation can be coupled inbacteria.
In eukaryotes, there are two additional complexities -
(i) There are at least three RNA polymerases in the nucleus (in addition
to the RNA polymerase found in the organelles). There is a clear
cut division of labour. The RNA polymerase I transcribes rRNAs

5'

3'

3' mRNA

Capping Cap
m
5' GPPP

RNA splicing Polyadenylation

m
5' GPPP

3'

Messenger RNA

Figure 5.11 Process of Transcription in Eukaryotes

2024-25
MOLECULAR BASIS OF INHERITANCE

(28S, 185, and 5.8S}, whereas the RNA polymerase III is

responsible for transcription of tRNA, 5srRNA, and snRNAs
(small nuclear RNAs). The RNA polymerase II transcribes precursor
of mRNA, the heterogeneous nuclear RNA (hnRNA).
(ii) The second complexity is that the primary transcripts contain both
the exons and the intrans and are non-functional. Hence, it is
subjected to a process called splicing where the intrans are
removed and exons are joined in a defined order. hnRNA
u ndergoes additional processing called as capping and tailing. In
capping an unusual nucleotide (methyl guanosine triphosphate)
is added to the 5'-end of hnRNA. In tailing, adenylate residues
(200-300) are added at 3'-end in a template independent manner.
It is the fully processed hnRNA, now called mRNA, that is
transported out of the nucleus for translation (Figure 5.11).
The significance of such complexities is now beginning to be
understood. The split-gene arrangements represent probably an ancient
feature of the genome. The presence of intrans is reminiscent of
antiquity, and the process of splicing represents the dominance of RNA-
world. In recent times, the understanding of RNA and RNA-dependent
processes in the living system have assumed more importance.

5.6 GENETIC CODE

During replication and transcription a nucleic acid was copied to form

another nucleic acid. Hence, these processes are easy to conceptualise
on the basis of complementarity. The process of translation requires
transfer of genetic information from a polymer of nucleotides to
synthesise a polymer of amino acids. Neither does any complementarity
exist between nucleotides and amino acids, nor could any be drawn
theoretically. There existed ample evidences, though, to support the
notion that change in nucleic acids (genetic material) were responsible
for change in amino acids in proteins. This led to the proposition of a
genetic code that could direct the sequence of amino acids during
synthesis of proteins.
If determining the biochemical nature of genetic material and the
structure of DNA was very exciting, the proposition and deciphering
of genetic code were most challenging. In a very true sense, it
required involvement of scientists from several disciplines -
physicists, organic chemists, biochemists and geneticists. Itwas
George Gamow, a physicist, who argued that since there are only 4
bases and if they have to code for
20 amino acids, the code should constitute a combination of bases. He •
suggested that in order to code for all the 20 amino acids, the code
should be made up of three nucleotides. This was a very bold
proposition, because a permutation combination of 43 (4 x 4 x 4)
would generate 64 codons; generating many more codons than
required.
Providing proof that the codon was a triplet, was a more daunting
task. The chemical method developed by Har Gobind Khorana was
2024-25
 BIOLOGY

instrumental in synthesising RNA molecules with defined combinations

of bases (homopolymers and copolymers). Marshall Nirenberg's cell-free
system for protein synthesis finally helped the code to be deciphered.
Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful
in polymerising RNA with defined sequences in a template independent
manner (enzymatic synthesis of RNA). Finally a checker-board for
genetic code was prepared which is given in Table 5.1.
Table 5. 1: The Codons for the Various Amino Acids
Third
position

po r
Second position

l
F irst u
C A G
u
UUU Phe UCU Ser UAU Tyr UGU Cys
u UUC Phe UCC Ser UAC Tyr UGC Cys c
UUA Leu UCA Ser UAA Stop UGA A
UUG Leu UCG Ser UAG Stop Stop UGG G
Trp u
CUU Leu CCU Pro CAU His CGU Arg
c CUC Leu CCC Pro CAC His CGC Arg c
CUA Leu CCA Pro CAA Gln CGA Arg A
CUG Leu CCG Pro CAG Gln CGG G

AAU Asn
Arg
AGU Ser
u
AUU Ile ACU Thr
A AUC Ile ACC Thr AAC Asn AGC Ser c
AAA Lys AGA Arg A
AUA Ile ACA Thr
AUG Met ACG Thr AAG Lys AGG G
Arg u
GUU Val GCU Ala GAU Asp GGU Gly
G GUC Val GCC Ala GAC Asp GGC Gly c
GGA Gly A
GUA Val GCA Ala GAA Glu
GUG Val GCG Ala GAG Glu GGG G

The salient features of genetic code are as follows:

(i) The codon is triplet. 61 codons code for amino acids and
3codons do not code for any amino acids, hence they function
as stop codons.
(ii) Some amino acids are coded by more than one codon, hence
the code is degenerate.
(iii) The codon is read in mRNA in a contiguous fashion. There
are no punctuations.
(iv) The code is nearly universal: for example, from bacteria to
human UUU would code for Phenylalanine (phe). Some
exceptions to this rule have been found in mitochondrial
codons, and in some protozoans.
(v) AUG has dual functions. It codes for Methionine (met) , and it
also act as initiator codon.
(vi) UAA, UAG, UGA are stop terminator codons.
If following is the sequence of nucleotides in mRNA, predict the
sequence of amino acid coded by it (take help of the checkerboard ):
-AUG UUU UUC UUC UUU UUU
UUC-

2024-25
MOLECULAR BASIS OF INHERITANCE

Now try the opposite. Follow ing is the sequence of amino acids
coded by an mRNA. Predict the nucleotide sequence in the RNA:

Met-Phe-Phe-Phe-Phe-Phe-Phe

Do you face any difficulty in predicting the opposite?

Can you now correlate which two properties of genetic code you
have lear nt?

5.6. 1 Mutations and Genetic Code

The relationships between genes and DNA are best understood by
mutation studies. You have studied about mutation and its effect in
Chapter 4. Effects of large deletions and rearrangements in a segment
of DNA are easy to comprehend. Itmay result in loss or gain of a gene
and so a function. The effect of point mutations will be explained
here. A classical example of point mutation is a change of single base
pair in the gene for beta globin chain that results in the change of
amino acid residue glutamate to valine. Itresults into a diseased
condition called as sickle cell anemia. Effect of point mutations that
inserts or deletes a base in structural gene can be better understood by
following simple example.
Consider a statement that is made up of the following words each
having three letters like genetic code.
RAM HAS RED CAP

If we insert a letter B in between HAS and RED and rearrange

the statement, it would read as follows:
RAM HAS BRE DCA P

Similarly, if we now insert two letters at the same place, say BI'. Now
it would read,
RAM HAS BIR EDC AP

Now we insert three letters together, say BIG, the statement would read
RAM HAS BIG RED CAP

The same exercise can be repeated, by deleting the letters R, E and

D, one by one and rearranging the statement to make a triplet word.
RAM HAS EDC AP
•
RAM HAS DCA P

RAM HAS CAP

The conclusion from the above exercise is very obvious. Insertion or

deletion of one or two bases changes the reading frame from the point of
insertion or deletion. However, such mutations are referred to as

2024-25
 BIOLOGY

frameshift insertion or deletion mutations. Insertion or deletion of

three or its multiple bases insert or delete in one or multiple codon
hence one or multiple amino acids, and reading frame remains
unaltered from that point onwards.

5.6.2 tRNA- the Adapter Molecule

From the ve:ry beginning of the proposition of code, it was clear to
Francis Crick that there has to be a mechanism to read the code and also
to link it to the amino acids, because amino acids have no structural
specialities to read the code uniquely . He postulated the presence of an
adapter molecule that would on one hand read the code and on other
hand would bind to specific amino acids. The tRNA, then called
sRNA (soluble RNA}, was known before the genetic code was
postulated. However, its role as an adapter molecule was assigned
much later.
tRNA has an
anticodon loop
that has bases
complementary to
tRNA - tRNA the code, and it
- also has an
amino acid
acceptor end to
which it binds to
amino acid
s.
A U A C tRNAs are specific
5' - - - - 1111111'1 llllllllilll•C•o• d• o• n- - - - - - - •' 1111111'1 1111 1'- m• R•
N• A-3' for each amino acid
(Figure 5.12). For
Figure 5.12 tRNA - the adapter molecule initiation, there is
another specific tRNA that is referred to as initiator tRNA. There are
no tRNAs for stop codons. In figure 5.12, the seconda:ry structure of
tRNA has been depicted that looks like a clover-leaf. In actual
structure, the tRNA is a compact molecule which looks like inverted
L.

5.7 TRANSLATION
Translation refers to the process of polymerisation of amino acids to
form a polypeptide (Figure 5.13). The order and sequence of amino
acids are defined by the sequence of bases in the mRNA. The amino
acids are joined by a bond which is known as a peptide bond.
Formation of a peptide bond requires energy. Therefore, in the first
phase itself amino acids are activated in the presence of ATP and
linked to their cognate tRNA-a pr ocess com monly called as
charging of tRNA or aminoacylation of tRNA to be more specific.
Iftwo such charged tRNAs are brought close enough, the formation of

2024-25
peptide bond between them

2024-25
MOLECULAR BASIS OF INHERITANCE

would be favoured energetically. The

presence of a catalyst would enhance
the rate of peptide bond formation.
The cellular factory responsible
for synthesising proteins is the
ribosome. The ribosome consists of
structural RNAs and about 80
different proteins. In its inactive
state, it exists as two subunits; a
large subunit and a small subunit.
When the small subunit encounters
an mRNA, the process of translation
mRNA
of the mRNA to protein begins.
There are two sites in the large
subunit, for subsequent amino acids Figure 5.13 Translation
to bind to and thus, be close enough
to each other for the formation of a
peptide bond. The ribosome also acts as a catalyst (23SrRNA in bacteria
is the enzyme- ribozyme) for the formation of peptide bond.
A translational unit in mRNA is the sequence of RNA that is
flanked by the start codon (AUG) and the stop codon and codes for a
polypeptide. An mRNA also has some additional sequences that are
not translated and are referred as untranslated regions (UTR). The
UTRs are present at both 5'-end (before start codon) and at 3'-end
(after stop codon). They are required for efficient translation process.
For initiation, the ribosome binds to the mRNA at the start codon
(AUG) that is recognised only by the initiator tRNA. The ribosome
proceeds to the elongation phase of protein synthesis. During this stage,
complexes composed of an amino acid linked to tRNA, sequentially
bind to the appropriate codon in mRNA by forming complementary
base pairs with the tRNA anticodon. The ribosome moves from codon to
codon along the mRNA. Amino acids are added one by one, translated
into Polypeptide sequences dictated by DNA and represented by mRNA.
At the end, a release factor binds to the stop codon, terminating
translation and releasing the complete polypeptide from the ribosome.

5.8 REGULATION OF GENE EXPRESSION

Regulation of gene expression refers to a very broad term that may occur
at various levels. Considering that gene expression results in the
formation of a polypeptide, it can be regulated at several levels. In •
eukaryotes, the regulation could be exerted at
(i) transcriptional level (formation of primary transcript),
(ii) processing level (regulation of splicing),
(iii) transport of mRNA from nucleus to the cytoplasm,
(iv) translational level.

2024-25
 BIOLOGY

The genes in a cell are expressed to perform a particular function

or a set of functions. For example, if an enzyme called beta-
galactosidase is synthesised by E. coli, it is used to catalyse the
hydrolysis of a disaccharide, lactose into galactose and glucose; the
bacteria use them as a source of energy. Hence, if the bacteria do not
have lactose around them to be utilised for energy source, they would
no longer require the synthesis of the enzyme beta-galactosidase.
Therefore, in simple terms, it is the metabolic, physiological or
environmental conditions that regulate the expression of genes. The
development and differentiation of embryo into adult organisms are
also a result of the coordinated regulation of expression of several
sets of genes.
In prokaryotes, control of the rate of transcriptional initiation is the
predominant site for control of gene expression. In a transcription
unit, the activity of RNA polymerase at a given promoter is in turn
regulated by interaction with accessory proteins, which affect its
ability to recognise start sites. These regulatory proteins can act both
positively (activators) and negatively (repressors). The accessibility
of promoter regions of prokaryotic DNA is in many cases regulated
by the interaction of proteins with sequences termed operators. The
operator region is adjacent to the promoter elements in most operons
and in most cases the sequences of the operator bind a repressor
protein. Each operon has its specific operator and specific repressor.
For example, lac operator is present only in the lac operon and it
interacts specifically with lac repressor only.

5.8. 1 The Lac operon

The elucidation of the lac operon was also a result of a close association
between a geneticist, Francois Jacob and abiochemist, Jacque Monod.
They were the first to elucidate a transcriptionally regulated system. In
lac operon (here lac refers to lactose), a polycistronic structural gene is
regulated by a common promoter and regulatory genes. Such arrangement
is very common in bacteria and is referred to as operon. To name few
such examples, lac operon, trp operon, ara operon, his operon, val
operon, etc.
The lac operon consists of one regulatory gene (the i gene - here
the term i does not refer to inducer, rather it is derived from the word
inhibitor) and three structural genes (z, y, and a). The igene codes for
the repressor of the lac operon. The z gene codes for beta-galactosidase
({3-gal), which is primarily responsible for the hydrolysis of the
disaccharide, lactose into its monomeric units, galactose and glucose.
The y gene codes for permease, which increases permeability of the cell
to {3-galactosides. The a gene encodes a transacetylase. Hence, all the
three gene products in lac operon are required for metabolism oflactose.
In most other operons as well, the genes present in the operon are
needed together to function in the same or related metabolic pathway
(Figure 5.14).
2024-25
MOLECULAR BASIS OF INHERITANCE

In absence of inducer

1 Repressor binds to the operator

Repressor mRNA region(o) and prevents RNA polymerase
from transcribing the operon
1
C) ---'
Repressor

z y In presence of inducer

Transcription

Repressor
mRNA
1 1 1 1 Translation
C) -galactosidase permease transacetylase

Induce
0(InactiveC())
repressor)

Figure 5.14 The lac Operon

Lactose is the substrate for the enzyme beta-galactosidase and it

regulates switching on and off of the operon. Hence, it is termed as
inducer. In the absence of a preferred carbon source such as glucose,
iflactose is provided in the growth medium of the bacteria, the lactose
is transported into the cells through the action of permease
(Remember, a very low level of expression of lac operon has to be
present in the cell all the time, otherwise lactose cannot enter the
cells). The lactose then induces the operon in the following manner.
The repressor of the operon is synthesised (all-the-time -
constitutively) from the i gene. The repressor protein binds to the
operator region of the operon and prevents RNA polymerase from
transcribing the operon. In the presence of an inducer, such as lactose or
allolactose, the repressor is inactivated by interaction with the inducer.
This allows RNA polymerase access to the promoter and
transcription proceeds (Figure 5.14).
Essentially, regulation of lac operon can also be visualised as regulation
of enzyme synthesis by its substrate.
I
Remember, glucose or galactose cannot act as ind ucers for lac
operon. Can you thinkfor how long the lac operon would be expressed
in the presence of lactose?
Regulation of lac operon by repressor is referred to as negative
regulation. Lac operon is under control of positive regulation as well,
but it is beyond the scope of discussion at this level.

2024-25
 BIOLOGY

5.9 HUMAN GENOME

PROJECT

In the preceding sections you have learnt that it is the sequence of bases
in DNA that determines the genetic information of a given organism. In
other words, genetic make-up of an organism or an individual lies in the
DNA sequences. Iftwo individuals differ, then their DNA sequences
should also be different, at least at some places. These assumptions led to
the quest of finding out the complete DNA sequence of human
genome. With the establishment of genetic engineering techniques
where it was possible to isolate and clone any piece of DNA and
availability of simple and fast techniques for determining DNA
sequences, a very ambitious project of sequencing human genome was
launched in the year 1990.
Human Genome Project (HGP) was called a mega project. You can
imagine the magnitude and the requirements for the project if we simply
define the aims of the project as follows:
Human genome is said to have approximately 3 x 109 bp, and if the
cost of sequencing required is US $ 3 per bp (the estimated cost in the
beginning), the total estimated cost of the project would be
approximately 9 billion US dollars. Further, if the obtained sequences
were to be stored in typed form in books, and if each page of the
book contained 1000 letters and each book contained 1000 pages,
then 3300 such books would be required to store the information of
DNA sequence from a single human cell. The enormous amount of
data expected to be generated also necessitated the use of high speed
computational devices for data storage and retrieval, and analysis.
HGP was closely associated with the rapid development of a new area
in biology called Bioinformatics.

Goals of HGP
Some of the important goals of HGP were as follows:
(i) Identify all the approximately 20,000-25,000 genes in human DNA;
(ii) Determine the sequences of the 3 billion chemical base pairs that
make up human DNA;
(iiii) Store this information in databases;
(iv) Improve tools for data analysis;
(v) Transfer related technologies to other sectors, such as industries;
(vi) Address the ethical, legal, and social issues (ELSI) that may
arise from the project.
The Human Genome Project was a 13-year project coordinated by
the U.S. Department of Energy and the National Institute of Health.
During the early years of the HGP, the Wellcome Trust (U.K.) became
a major partner; additional contributions came from Japan, France,
Germany, China and others. The project was completed in 2003.
Knowledge about the effects of DNA variations among individuals can
lead to revolutionary new ways to diagnose, treat and someday
prevent the thousands of
2024-25
MOLECULAR BASIS OF INHERITANCE

disorders that affect hu man beings. Besides providing clues to

understanding human biology, learning about non-human organisms
DNA sequences can lead to an understanding of their natural
capabilities that can be applied toward solving challenges in health
care, agriculture, energy production, environmental remediation. Many
non-human model organisms, such as bacteria, yeast, Caenorhabditis
elegans (a free living non-pathogenic nematode), Drosophila (the fruit
fly), plants (rice and Arabidopsis), etc., have also been sequenced.
Methodologies : The methods involved two major approaches. One
approach focused on identifying all the genes that are expressed as
RNA (referred to as Expressed Sequence Tags (ESTs). The other took
the blind approach of simply sequencing the whole set of genome that
contained all the coding and non-coding sequence, and later assigning
different regions in the sequence with functions (a term referred to as
Sequence Annotation). For sequencing, the total DNA from a cell is
isolated and converted into random fragments of relatively smaller
sizes (recall DNA is a very long polymer, and there are technical
limitations in sequencing very long pieces of DNA) and cloned in
suitable host using specialised vectors. The cloning resulted into
amplification of each piece of DNA fragment so that it subsequently
could be sequenced with ease. The commonly used hosts were bacteria
and yeast, and the vectors were called as BAC (bacterial artificial
chromosomes), and YAC (yeast artificial chromosomes).
The fragments were sequenced using automated DNA sequencers
that worked on the principle of a method developed by Frederick
Sanger. (Remember, Sanger is also credited for developing
method for d etermination of amino acid
sequ ences in proteins) . These
sequences were then arranged
based on some overlapping r
egions present in them. This
required generation of overlapping
fragments for sequencing.
Alignment of these sequ ences
was h u manly not possible.
Therefore, specialised computer
based programs were developed
(Figu re 5.15) . These
sequ ences were subsequ ently
annotated and were assigned to
each chromosome. The sequ ence
of chromosome 1was completed
only in May 2006 (this was the
last of the
24 hu man chromosomes - Figure 5.15 A representative diagram of human
22 autosomes and X and Y - to genome project
be

2024-25
 BIOLOGY

sequenced). Another challenging task was assigning the genetic and

physical maps on the genome. This was generated using information on
polymorphism of restriction endonuclease recognition sites, and some
repetitive DNA sequences known as microsatellites (one of the
applications of polymorphism in repetitive DNA sequences shall be
explained in next section of DNA fingerprinting).

5.9. 1 Salient Features of Human Genome

Some of the salient observations drawn from human genome project
are as follows:
(i) The human genome contains 3164.7 million bp.
(ii) The average gene consists of 3000 bases, but sizes vary greatly,
with the largest known human gene being dystrophin at 2.4
million bases.
(iii) The total number of genes is estimated at 30,000-much lower
than previous estimates of 80,000 to 1,40,000 genes. Almost
all (99.9 per cent) nucleotide bases are exactly the same in all
people.
(iv) The functions are unknown for over 50 per cent of the discovered
genes.
(v) Less than 2 per cent of the genome codes for proteins.
(vi) Repeated sequences make up very large portion of the human genome.
(vii) Repetitive sequences are stretches of DNA sequences that are
repeated many times, sometimes hundred to thousand times. They
are thought to have no direct coding functions, but they shed light
on chromosome structure, dynamics and evolution.
(viii) Chromosome 1has most genes (2968), and the Y has the fewest (231).
(ix) Scientists have identified about 1.4 million locations where single
base DNA differences (SNPs - single nucleotide polymorphism,
pronounced as 'snips') occur in humans. This information
promises to revolutionise the processes of finding chromosomal
locations for disease-associated sequences and tracing human
history.

5.9.2 Applications and Future Challenges

Deriving meaningful knowledge from the DNA sequences will define
research through the coming decades leading to our understanding of
biological systems. This enormous task will require the expertise and
creativity of tens of thousands of scientists from varied disciplines in
both the public and private sectors worldwide. One of the greatest
impacts of having the HG sequence may well be enabling a radically
new approach to biological research. In the past, researchers studied
one or a few genes at a time. With whole-genome sequences and
new high-throughput technologies, we can approach questions
systematically and on a much
2024-25
MOLECULAR BASIS OF INHERITANCE

broader scale. They can study all the genes in a genome, for example, all
the transcripts in a particular tissue or organ or tumor, or how tens of
thousands of genes and proteins work together in interconnected networks
to orchestrate the chemistry oflife.

5.10 DNA FINGERPRINTING

As stated in the preceding section, 99.9 per cent of base sequence

among humans is the same. Assuming human genome as 3 x 109 bp,
in how many base sequences would there be dijferences? Itis these
differences in sequence of DNA which make every individual
unique in their phenotypic appearance. If one aims to find out
genetic differences between two individuals or among
individuals of a population,
sequencing the DNA every time would be a daunting 6
and expensive
task. Imagine trying to compare two sets of 3 x 10 base pairs. DNA
fingerprinting is a very quick way to compare the DNA sequences of any
two individuals.
DNA fingerprinting involves identifying differences in some
specific regions in DNA sequence called as repetitive DNA, because
in these sequences, a small stretch of DNA is repeated many times.
These repetitive DNA are separated from bulk genomic DNA as
different peaks during density gradient centrifugation. The bulk DNA
forms a major peak and the other small peaks are referred to as
satellite DNA. Depending on base composition (A : T rich or G:C
rich), length of segment, and number ofrepetitive units, the satellite
DNA is classified into many categories, such as micro-satellites, mini-
satellites etc. These sequences normally do not code for any proteins,
but they form a large portion of human genome. These sequence
show high degree of polymorphism and form the basis of DNA
fingerprinting. Since DNA from every tissue (such as blood, hair -
follicle, skin, bone, saliva, sperm etc.), from an individual show the
same degree of polymorphism, they become very useful
identification tool in forensic applications. Further, as the
polymorphisms are inheritable from parents to children, DNA
fingerprinting is the basis of paternity testing, in case of disputes.
As polymorphism in DNA sequence is the basis of genetic
mapping of human genome as well as of DNA fingerprinting, it is
essential that we u nderstand what DNA polymorphism means in
simple terms. Polymorphism (variation at genetic level) arises due to
mutations. (Recall different kind of mutations and their effects that
you have already studied in Chapter 4, and in the preceding
sections in this chapter.) New mutations may arise in an individual
either in somatic cells or in the germ cells (cells that generate
gametes in sexually reproducing organisms). If a germ cell mutation
does not seriously impair individual's ability to have offspring who can
transmit the mutation, it can spread to

2024-25
 BIOLOGY

the other members of population (through sexual reproduction).

Allelic (again recall the definition of alleles from Chapter 4) sequence
variation has traditionally been described as a DNA polymorphism if
more than one variant (allele) at a locus occurs in human population
with a frequency greater than 0.01. In simple terms, if an inheritable
mutation is observed in a population at high frequency, it is referred
to as DNA polymorphism. The probability of such variation to be
observed in non coding DNA sequence would be higher as mutations
in these sequences may not have any immediate effect / impact in an
individual's reproductive ability. These mutations keep on
accumulating generation after generation, and form one of the basis of
variability /polymorphism. There is a variety of different types of
polymorphisms ranging from single nucleotide change to very large
scale changes. For evolution and speciation, such polymorphisms play
very important role, and you will study these in details at higher
classes.
The technique of DNA Fingerprinting was initially developed by
Alec Jeffreys. He used a satellite DNA as probe that shows very high
degree of polymorphism. Itwas called as Variable Number of
Tandem Repeats (VNTR) . The technique, as used earlier, involved
Southern blot hybridisation using radiolabelled VNTR as a probe. It
included
(i) isolation of DNA,
(ii) digestion of DNA by restriction endonucleases,
(iii) separation of DNA fragments by electrophoresis,
(iv) transferring (blotting) of separated DNA fragments to synthetic
membranes, such as nitrocellulose or nylon,
(v) hybridisation using labelled VNTR probe, and
(vi) detection of hybridised DNA fragments by autoradiography. A
schematic representation of DNA fingerprinting is shown in Figure
5.16.
The VNTR belongs to a class of satellite DNA referred to as mini-
satellite. A small DNA sequence is arranged tandemly in many copy
numbers. The copy number varies from chromosome to chromosome in
an individual. The numbers of repeat show very high degree of
polymorphism. As a result the size of VNTR varies in size from 0.1 to
20 kb. Consequently, after hybridisation with VNTR probe, the
autoradiogram gives many bands of differing sizes. These bands give a
characteristic pattern for an individual DNA (Figure 5.16). It differs from
individual to individual in a population except in the case of monozygotic
(identical) twins. The sensitivity of the technique has been increased by
use of polymerase chain reaction (PCR you will study about it in
Chapter 9). Consequently, DNA from a single cell is enough to perform
DNA fingerprinting analysis. In addition to application in forensic
science, it has much wider application, such as
2024-25
MOLECULAR BASIS OF INHERITANCE

Paternal
chromosome

Maternal
chromosome Chromosome 7 Chromosome 7

I I I I I I I I I I I I I I
I I I I I I I I I I I I I I
Chromosome 2 Chromosome 2

I I I I I I I I I I I I
I I I I I I
Chromosome 16 Chromosome 16

DNA from individual A I DNA from individual B

Number of short
tandem repeats

-
Q)
1 0..
0 11 11 Q)

c=J- -
.
[J 10
e
c : : ..
Q)
9 'O
8
Chromosome 7
I I I I I I I
7 3
t: :
I I I I I I I
Chromosome 2
6
.s
5 .....rn
4 0
I I I I I I .....
Q)
3
2
Chromosome 16 1
z
1
I DNA from ciiine scene (C)I Amplified repeats.separated by size
on a gel, give a DNA fingerprint

Figure 5.16 Schematic representation of DNA fingerprinting : Few representative chromosomes

have been shown to contain different copy number of VNTR. For the sake of
understanding different colour schemes have been used to trace the origin of each
band in the gel. The two alleles (paternal and maternal) of a chromosome also
contain different copy numbers of VNTR. It is clear that the banding pattern of DNA
from crime scene matches with individual B, and not with A.

in determining population and genetic diversities . Currently, many

different probes are used to generate DNA fingerprints .

2024-25
 BIOLOGY

SUMMARY
Nucleic acids are long polymers of nucleotides . While DNA stores
genetic information, RNA mostly helps in transfer and expression of
information . Though DNA and RNA both function as genetic material,
but DNA being chemically and structurally more stable is a better
genetic material. However, RNA is the first to evolve and DNA was
derived from RNA. The hallmark of the double stranded helical
structure of DNA is the hydrogen bonding between the bases from
opposite strands. The rule is that Adenine pairs with Thymine through
two H-bonds , and Guanine with Cytosine th rou gh three H -bond s.
This makes one strand complementary to the other. The DNA
replicates semiconservatively , the process is guided by the
complementary H-bonding . A segment of DNA that codes for RNA
may in a simplistic term can be referred as gene. During transcription
also, one of the strands of DNA acts a template to direct the synthesis
of complementary RNA. In bacteria , the transcribed mRNA is
functional , hence can directly be translated . In eukaryotes , the gene
is split. The coding sequences , exons, are interrupted by non-coding
sequences, introns . Introns are removed and exons are joined to
produce functional RNA by splicing . The messenger RNA contains
the base sequences that are read in a combination of three (to make
triplet genetic code) to code for an amino acid . The genetic code is read
again on the principle of complementarity by tRNA that acts as an
adapter molecule . There are specific tRNAs for every amino acid. The
tRNA binds to specific amino acid at one end and pairs through H-
bonding with codes on mRNA through its anticodons . The site of
translation (protein synthesis) is ribosomes , which bind to mRNA and
provide platform for joining of amino acids. One of the rRNA acts as a
catalyst for peptide bond formation, which is an example of RNA
enzyme (ribozyme) . Translation is a process that has evolved around
RNA, indicating that life began around RNA. Since, transcription and
translation are energetically very expensive processes , these have to
be tightly regulated . Regulation of transcription is the primary step for
regulation of gene expression . In bacteria , more than one gene is
arranged together and regulated in units called as operons. Lac operon
is the prototype operon in bacteria , which codes for genes responsible
for metabolism of lactose . The operon is regulated by the amount of
lactose in the medium where the bacteria are grown. Therefore , this
regulation can also be viewed as regulation of enzyme synthesis by
its substrate.
Human genome project was a mega project that aimed to sequence
every base in human genome . This project has yielded much new
information . Many new areas and avenues have opened up as a
consequence of the project. DNA Fingerprinting is a technique to find
out variations in individuals of a population at DNA level. It works on
the principle of polymorphism in DNA sequences. It has immense
applications in the field of forensic science, genetic biodiversity and
evolutionary biology .

2024-25
MOLECULAR BASIS OF INHERITANCE

EXER CISES
1 Group the following as nitrogenous bases and nucleosides:
Adenine, Cytidine, Thymine, Guanosine, Uracil and Cytosine.
2. If a double stranded DNA has 20 per cent of cytosine, calculate the per
cent of adenine in the DNA.
3. If the sequence of one strand of DNA is written as follows:
5'-ATGCATGCATGCATGCATGCATGCATGC-3'
Write down the sequence of complementary strand in 5'-s' direction.
4. If the sequence of the coding strand in a transcription unit is written
as follows:
5'-ATGCATGCATGCATGCATGCATGCATGC-3'
Write down the sequence of mRNA.
5. Which property of DNA double helix led Watson and Crick to hypothesise
semi-conservative mode of DNA replication? Explain.
6. Depending upon the chemical nature of the template (DNA or RNA)
and the nature of nucleic acids synthesised from it (DNA or RNA), list
the types of nucleic acid polymerases.
7. How did Hershey and Chase differentiate between DNA and protein in
their experiment while proving that DNA is the genetic material?
8. Differentiate between the followings:
(a) Repetitive DNA and Satellite DNA
(b) mRNA and tRNA
(c) Template strand and Coding strand
9. List two essential roles of ribosome during translation.
10. In the medium where E. coli was growing, lactose was added, which
induced the lac operon. Then, why does lac operon shut down some
time after addition of lactose in the medium?
11. Explain (in one or two lines) the function of the followings:
(a) Promoter

(b) tRNA
(c) Exons
12. Why is the Human Genome project called a mega project?
13. What is DNA fingerprinting? Mention its application.
14. Briefly describe the following:
(a) Transcription
(b) Polymorphism
(c) Translation
(d) Bioinformatics

2024-25