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CHAPTER-5 : MOLECULAR BASIS OF INHERITANCE

CHAPTER AT A GLANCE
INTRODUCTION
5.1. THE DNA
5.2. THE SEARCH FOR GENETIC MATERIAL
5.3. RNA WORLD
5.4. REPLICATION
5.5. TRANSCRIPTION
5.6. GENETIC CODE

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5.7. TRANSLATION
5.8. REGULATION OF GENE EXPRESSION
5.9. DNA FINGERPRINTING
5.10. HUMAN GENOME PROJECT

THEORY
INTRODUCTION :
 DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are two types of nucleic acids found
in living system.
 DNA acts as the genetic material in most of the organism. 
 RNA also act as a genetic material in some viruses and also has additional role as well.
NUCLEIC ACID
Discovered by :- Friedrich Meischer
In nucleus of pus cell called it "nuclein’’
Polymer of Nucleotides
Nucleoside :- Nitrogen base + Pentose sugar.
Nucleotide :- Nitrogen Base + Pentose Sugar +Phosphate
1. NITROGEN BASES: Heterocyclic Nitrogenous compounds
Purines Pyrimidines

Adenine Cytosine

Guanine Thymine [DNA]

Uracil [RNA]

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2. PENTOSE SUGAR

Oxygen present Oxygen absent

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More reactive
Less reactive

Less stable More stable

3. PHOSPHATE
Due to phosphate both DNA and RNA show

1. Acidic properties and

2. Have Negative charge
NUCLEOTIDE FORMATION

H H
N
N 6
7 5 1N
H 8
9 43 2
N N H

O Phosphoester bond
O P O 5'
CH2 O
O 4' 1'
H H
Phosphate H H
3' 2'
OH H
Deoxyribose
Nucleoside

Nucleotide

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POLYNUCLEOTIDE CHAIN FORMATION
RIBONUCLEOTIDES (RNA)
N - base Nucleosides = N base + Ribose sugar Nucleotides = Nucleoside + P

Adenine Adenosine Adenylic acid

Guanine Guanosine Guanylic acid
Cytosine Cytidine Cytidylic acid
Uracil Uridine Uridylic acid

DEOXYRIBONUCLEOTIDES (DNA)

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N - base Nucleosides = N base + Deoxy Ribose Sugar Nucleotides
Adenine Deoxyadenosine Deoxyadenylic acid
Guanine Deoxyguanosine Deoxyguanylic acid
Cytosine Deoxycytidine Deoxycytidylic acid
Thymine Deoxy thymidine Deoxythymidylic acid

5.1. THE DNA

 DNA is a long polymer of deoxyribonucleotides.

Organisms Number of Nucleotides

 × 174 bacteriophage 5386 Nucleotides

λ bacteriophage 48502 base pairs

E.coli 4.6 × 106 base pairs

Human (Haploid cell) 3.3 × 109 base pairs

STRUCTURE OF DNA
1. DNA stores genetic information in the form of nucleotides and it is expressed in the form of
protein.
2. Wilkins and Franklin :- Studied DNA molecule with the help of X-ray crystallography
(diffraction experiment.)
3. Watson and Crick :- Gave Double Helix Model (1953).
4. Watson Crick and Wilkins were awarded by Noble Prize in 1962 for double helix model of
DNA.

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DOUBLE HELIX MODEL OF DNA

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 Watson and Crick (1953) proposed a double helix molel for DNA. The salient features of DNA
are as following :-
 One main hallmark (main point) of double helix model is complementary base pairing between
purine and pyrimidine.
 According to this model, DNA is composed of two polynucleotide chains.
 Both polynucleotide chains are complementary and antiparallel to each other.
 In both strandof DNA direction of phosphodiester bond is opposite.
 Both strand of DNA are held together by hydrogen bonds. These hydrogen bonds are present
between nitrogen bases of both the strand.
 Adenine binds to thymine by two hydrogen bonds and cytosine binds to guanine by three
hydrogen bonds.
 In a DNA molecule one purine always pairs with a pyrimidine. This generates approximately
uniform distance between the two strands of DNA Helix.
 In DNA plane of one base pair stacks over the other in double helix. This, in addition to
H-bonds, confers stability of the helical structure of DNA.
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H
H
5' P C H H
P C
H P C 3'
H P C
H OH
H
A T
G C

Hydrogen
T A bonds
C G
H
HO H
C P H
3' C P H
H C P P 5'
H C
H
H
Double Stranded Polynucleotide Chain

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CHARGAFF’S RULE
In a ds-DNA
Total Purine = Total Pyrimidine
A+G=C+T
A+G/C+T = 1
or C + T/A + G = 1
A = T Or A/T = 1 Or T/A = 1
G = C Or G/C = 1 Or C/G = 1
This rule is not apply on ssDNA and ssRNA
PACKAGING OF DNA HELIX
Question :- Calculate the length of DNA in a diploid cell of human(in meters)?
Solution :- Length of DNA = Total bp × 3.4Å
= 6.6 × 109bp × 0.34 × 10-9 = 2.2 metres
The length is far greater then dimension of a typical nucleus, so to fit this DNA we require
packaging.
DNA packaging in Prokaryotes
 DNA is not scattered throughout the cell.
 DNA is packaged with the help of polyamines protein.
 Complex structure which is formed is known as nucleoid.
DNA packaging in Eukaryotes :
 It is carried out with the help of lysine and ariginine rich basic proteins called histone.
 There are five types of histone proteins H1, H2A, H2B, H3 and H4.
 Four of them occur in pairs to produce histone octamer (2 copies of each - H2A, H2B, H3 and H4).
called nubody or core of Nucleosome.
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 Their nubody positively charged ends are directed outside.
 The negatively charged DNA is wrapped around the positively charged histone octamer to form a
structure called nucleosome.
 The unit of compaction is nucleosome
 A typical nucleosome contains 200 bp of DNA Helix.

Packed by Histone Protein

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Five types
(H1, H2A, H2B, H3, H4)

Linker
DNA
H1 known as sealing protein

Negatively charged DNA Nucleosome

 DNA present between two adjacent nucleosomes is called linker DNA.

 Nucleosome chain gives a beads on string appearance under electron microscope.
 Nucleosomes constitute the repeating unit of a structure in nucleus called chromatin.
 The beads on string structure in chromatin is packaged to form chromatin fibres 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 (acidic) that
collectively are referred to as non-histone chromosomal (NHC) proteins.

Loosely packed
Euchromatin Lightly stain
Transcriptionally
active

Densely packed
Hetero
Dark stain
chromatin
Transcriptionally
inactive

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5.2. THE SEARCH FOR GENETIC MATERIAL
1. TRANSFORMATION EXPERIMENT
 Performed by Frederick Griffith in 1928.
 Worked on Streptococcus pneumoniae (bacterium responsible for pneumonia).
 He worked on 2 strain of this bacteria.

R type- S type-
(rough type) (smooth shiny type)
Mucous coat absent Mucous coat present
Non virulent Virulent

 The experiment can be described in following four steps :

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Living Living Heat-killed Living R-Cells +
R-Cells S-Cells S-Cells Heat-killed
S-Cells

Mice alive Mice dead Mice alive Mice dead

Bacterial transfomation experiments conducted by Griffith

CONCLUSION
 He concluded that R-strain bacteria had somehow been transformed by the heat killed
S-strain.
 Some ‘transforming principle’, transferred from the heat killed S strain, had enabled the R-strain
to synthesise a smooth polysaccharide coat and become virulent.

Act as genetic material

Transforming
principle
Biochemical
Nature (not known)

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Biochemical characterisation of Transforming Principle :
 Later, Avery, Macleod and McCarty (1933 - 1944) repeated the experiment in vitro to identify
the biochemical nature of transforming substance. They proved that this substance is DNA.
 Prior to the work, the genetic material was thought to be a protein.
 They purified biochemicals (proteins, DNA, RNA, etc.) from the heat-killed S cells to see which
one could transform live R cells into S cells.
 They discovered that DNA alone from S bacteria caused R bacteria to become transformed.

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Conclusion:-
1. Protease – Did not affect
transformation.
2. RNase - Did not affect transformation.
3. DNase - Did inhibit transformation.
(This proves that DNA is genetic
material). But all biologists are not
convinced.
Difference between DNAs and DNase
DNA’s DNase

A nucleic acid An enzyme

Monomers are DNA nucleotides Monomers are amino acids

Act as genetic material in most of the organisms. Cleaves the DNA

2. HERSHEY AND CHASE EXPERIMENT(1952)

 This experiment gave unequivocal proof that DNA is the genetic material.
 They worked with viruses that infects 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.
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Structure of bacteriophage
 Hershey and Chase worked to discover whether it was protein or DNA from the viruses that
entered the bacteria.

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 Use radioactive isotope of S(35S) and P(32P).
 They grew some viruses on a medium that contained radioactive phosphorus and some others
on medium that contained radioactive sulfur.
This experiment consist of three steps.
1. Infection : Both type of labelled phages were allowed to infect normally cultured bacteria in
separate experiments.
2. Blending : These bacteria cells were agitated in a blender to break the contact between virus and
bacteria.
3. Centrifugation: The virus particles were separated from the bacterium by spinning them in a
centrifuge.

The Hershey-Chase experiment

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Conclusion
1. After the centrifugation the bacterial cells showed the presence of radioactive DNA labelled with
P32 while radioactive protein labelled with S35 appeared on the outside of bacteria cells (i.e., in
the medium).
2. Labelled DNA was also found in the next generation of phage. This clearly showed that only
DNA enters the bacterial host and not the protein.
3. DNA, therefore, is the infective part of virus and also carries all the genetic information. This
provided the unequivocal proof that DNA is the genetic material.
Properties of genetic material
A molecule that can act as a genetic material must fulfill the following criteria:
(1) It should be able to generate its replica (Replication).

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(2) It should be stable chemically and structurally.
(3) It should provide the scope for slow changes (mutation) that are required for evolution.
(4) It should be able to express itself in the form of 'Mendelian Characters’.
Difference between DNA & RNA

DNA RNA
1. Genetic material in most of the organism 1. Act as a genetic material in some
viruses .
Eg .TMV, QB-bacteriophage
2. Deoxyribose sugar is present 2. Ribose sugar is present

3. Thymine is present (it provide additional 3. Uracil is present

stability)

4. It is generally double stranded 4. It is generally single stranded

Exception:-Φ x 174 bacteriophage Exception:- reovirus.

5. More stable & less reactive. 5. Less stable & more reactive.

6. DNA being more stable is preferred for 6. For the transmission of genetic
storage for information. information RNA is better genetic
material.
7. Can’t code for the protein directly 7. Can directly code for the synthesis of
protein.

8. Can’t play catalytic role 8. Can act as catalyst (ribozyme

activity)
9. Slow mutations 9. Faster mutations

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5.3. RNA WORLD
 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).
 RNA being a catalyst was reactive and hence unstable. Therefore, DNA has evolved from RNA
with chemical modifications that make it more stable.
RNA DNA

Uracil Thymine
Ribose Deoxyribose
Single strand Double stranded

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 DNA being double stranded and having complementary strand further resists changes by
evolving a process of repair.
CENTRAL DOGMA
 Central dogma was given by Crick.
 The formation (production) of mRNA from DNA and then synthesis of protein from it is known
as Central Dogma.

Reverse Transcription :
 The formation of DNA from RNA is known as Reverse - transcription.
5.4. REPLICATION
 Formation of identical copies of DNA is known as replication.
G
C
A
T AG
T T
C
A
C
A
G G
T CT
A
C
AG T
T A
G C
T A
5' A T 3
C G
G G '
T
C A T
A C
A C
T CT A
GA G
A
GT G
T T T
GA C C G
A
C C
Watson–Crick Model for semiconservative
DNA replication
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 DNA capable of self-duplication.
 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 replication results into polyploidy (a chromosomal anomaly).
 The scheme suggested that the two strands would separate and act as a template for the synthesis
of new complementary strands.
SEMI CONSERVATIVE MODE OF DNA REPLICATION
 First proposed by Watson & Crick.
 Later on it was experimentally proved by Matthew Meselson & Frankllin Stahl (1958) in E -
Coli and Taylor in Vicia faba (1958).
 Taylor used Radiotracer Technique in which Radioisotopes (tritiated thymidine = 1H3) were

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used.
 Meselson and Stahl used heavy isotope of nitrogen (15N).
 Note:- 15N is not a radioactive isotope, and it can be separated from 14N only based on densities.
Meselson and Stahl’s experiment
 They grew E. coli in a medium containing 15NH4Cl (15N is the heavy isotope of nitrogen) as the
only nitrogen source for many generations.
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 The result was that N was incorporated into newly synthesised DNA. This heavy DNA
molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride
(CsCl) density gradient.
 Then they transferred the cells into a medium with normal 14NH4Cl and took samples at various
definite time intervals.

Generation I Generation II
15
N-DNA 14N-DNA 14
N-DNA
15
N-DNA 15
N-DNA
20 min 40 min 14
N-DNA
Gravitational force 14
N-DNA

15 15 14 15 14 14 14 15
N N N N N N N N
Heavy Hybrid Light Hybrid

Separation of DNA by Centrifugation

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The Machinery and the Enzymes
 In living cells, such as E. coli, the process of replication requires a set of catalysts (enzymes).
 The following steps are included in DNA replication :-
(1) Unzipping (Unwinding) :
 The separation of 2 chains of DNA is termed as unzipping. And it takes place due to the breaking
of H-bonds.
 The process of unzipping starts at a certain specific point which is termed as initiation point or
origin of replication.
 At the place of origin the enzyme responsible for unzipping (breaking the hydrogen bonds) is
Helicase.
 SSB (single stranded DNA binding protein) prevents the reformation of H-bonds.
 Topoisomerase (in prokaryotes also called as DNA gyrase) release the tension arises due to

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supercoiling.
 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.
 Note : The process of DNA replication takes a few minutes in prokaryotes and a few hours in
Eukaryotes.

Ori site

DNA
SSB(SINGLE Strand DNA Helicase
Binding) Protein

Topoisomerase :- Prevent Topoisomerase :- Prevent

supercoiling supercoiling

(2) Formation of New Chain :

 To start the synthesis of new chain, special type of RNA is required which is termed as RNA
Primer.
 The formation of RNA primer is catalysed by an enzyme - RNA Polymerase (primase).
 Synthesis of RNA primer takes place in 5'  3' direction.
 The formation of new chains is catalysed by an enzyme DNA Polymerase. In prokaryotes it is of
3 types :

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1. DNA - Polymerase I :- This was discovered by ARTHUR KORNBERG (1957). So it is also
called as 'Kornberg's enzyme'. It separates RNA - primer from DNA and also fills the gap. It is
also known as DNA-repair enzyme.
2. DNA - Polymerase II :- It is least reactive in replication process.
3. DNA - Polymerase III :- This is the main enzyme in DNA Replication. It is most important.
This is also known as Replicase.
 All DNA polymerase I, II and III enzymes have 5'3" polymerisation activity and 3'5"
exonuclease activity.
 The average rate of polymerisation has to be approximately 2000 bp per second.
 For the formation of new chain nucleotides are obtained from Nuceloplasm.
 In the nucleoplasm, Nucleotides are present in the form of triphosphates like dATP, dGTP,
dCTP, dTTP etc.

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 Deoxyribonucleoside triphosphate serve dual purposes :-
1. Acting as substrates for enzyme.
2. They provide energy for polymerisation.
 The formation of new chain always takes place in 5'  3' direction.
 As a result of this, one chain of DNA is continuously formed and it is termed as Leading strand.
 The formation of second chain begins from the centre and not from the terminal points, so this
chain is discontinuous and is made up of small segments called Okazaki Fragments.
 This discontinuous chain is termed as Lagging strand.
 The Okazaki fragments are joined together by an enzyme DNA Ligase and formed a
complete new chain.

5' 3'

Template DNA
(Parental strands)

5'
Continous 3'
Discontinuous
synthesis synthesis

3' Newly synthesised 5'

5' 3'
strands
Replicating Fork
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Types of RNA :
(A) Genetic RNA or Genomic RNA - In some viruses RNA works as genetic material eg. TMV, QB
bacteriophage.
(B) Non-genetic RNA - mainly of 3 types -
(1) rRNA (2) tRNA (3) mRNA
RNA functions as adapter, structural and in some cases as a catalyst (Ribozyme)
(1) Ribosomal RNA (r-RNA) :-
 This RNA is 80% of the cell's total RNA.
 It is found in ribosomes and it is produced in nucleolus. (Except 5s)
 It is the most stable form of RNA.

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 rRNAs play structural and catalytic role during translation.
 At the time of protein synthesis, r-RNA provides attachement site to t-RNA and m-RNA and
attaches them on the ribosome.
(2) Messenger RNA (mRNA) :
 The m - RNA is 1 - 5% of the cell's total RNA.
 The name m-RNA was given by Jacob and Monod.
 The mRNA is produced by genetic DNA in the nucleus.
 It is least stable RNA.
 The mRNA provides the template for protein synthesis.
(3) Transfer RNA (tRNA) :
 It is 10-15% of total RNA.
 It is synthesized in the nucleus by DNA.
 It is also known as soluble RNA (sRNA)
 It is also known as Adapter RNA.
 It is the smallest RNA (4s).
Function : At the time of protein synthesis it acts as a carrier of amino-acids.
STRUCTURE OF tRNA
 The structure of tRNA is most complicated.
 It looks like a clover leaf but in actual structure the tRNA is a compact molecule which looks like
inverted 'L'.
 There are present three nucleotides are present in a particular sequence at 3' end of tRNA and that
sequence is CCA.

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 3' end is known as Acceptor end.
 There are some abnormal nitrogenous bases in the loops, that is why hydrogen bonds are not

formed. e.g. (i) Inosine (I) (ii) Pseudouracil () (iii) Dihydrouridine (DHU)

5' 5'
Ser Tyr
3' 3'
tRNA tRNA

U C A Anticodon U C A
A G U Codon U A C mRNA
5'

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3'
tRNA - the adapter molecule

t-RNA have following loops :-

(A) TΨC Loop or Attachment loop :-
This loop connects tRNA to the larger subunit of ribosome.
(B) Recognition Loop (Anticodon loop) :-
 tRNA recognizes its place on mRNA with the help of Anticodon.
 The anticodon of tRNA recognises its complementary sequence on mRNA.
 This complementary sequence is known as codon (mRNA)
(C) DHU Loop :
 It is also known as Aminoacyl synthetase recognition loop.

5.5. TRANSCRIPTION
 The process of copying genetic information from one strand of the DNA into RNA is termed
as transcription.
 Out of two strand of DNA only one strand participates in transcription and is called " Antisense
strand" or "Template strand".
 Here also, the principle 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 but in transcription only a segment of DNA and only one of the strands is
copied into RNA.
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Why both the strands of DNA are not copying during transcription?
Ans. (1) If both strands act as a template during transcription they would code for RNA molecule with
different sequences and If they code for proteins the sequence of amino acids in the protein
would be different.
(2) If the two RNA molecules produced simultaneously they would be complementary to
each other hence would form a dsRNA which prevent translation of RNA.
GENE AND CISTRON
A gene is defined as the functional unit of inheritance.
 Segment of DNA which can be transcribed is known as gene.
 The segment of DNA which coding for a polypeptide is known as Cistron.

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 Every cistron is a gene but every gene is not a cistron.
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
 The strand that has the polarity 3'→5' 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 known as coding strand (which does not code for anything)
 All the reference point while defining a transcription unit is made with coding strand.

Transcription start site

Promoter Terminator
Structural gene Template strand
3' 5'

5' 3'
Coding strand
Schematic structure of a transcription unit

 The promoter is said to be located towards 5'-end (upstream) of the structural gene (coding strand).
 It 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.
 The terminator is located towards 3'-end (downstream) of the coding strand and it usually defines
the end of the process of transcription.
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Prokaryotes Eukaryotes

1 Transcription and translation take place in the  In eukaryotes transcription occur

1. same compartment. in nucleus and translation occur in
cytoplasm.

2. Single type of RNA polymerase is present.  In eukaryotes there are three types
of RNA polymerases that
synthesised different RNA.

3. It can form all the three types of RNA.  RNA polymerase-I for 28s rRNA,
18s rRNA, 5.8s rRNA synthesis.

4. No formation of pre cursor of mRNA.  RNA polymerase-II for hnRNA

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synthesis (Precursor of m-RNA)

 RNA polymerase-III for tRNA, 5s

rRNA, snRNA synthesis.

5. The structural gene in prokaryotes is mostly  The structural gene in eukaryotes

polycistronic. is mostly monocistronic.

6. The genes in prokaryotes are not splited. The gene is splited and have exons

and introns.

PROCESS OF TRANSCRIPTION

 There is single DNA dependent RNA polymerase that catalyses transcription of all types of RNA

in bacteria.

Following steps are present in transcription.

(1) INITIATION:

 DNA has a Promoter site where RNA polymerase binds and a terminator site where

transcription stops.

 Sigma factor recognises the promoter site of DNA.

 With the help of sigma factor RNA polymerase enzyme is attached to a specific site of DNA

calledPromoter site.

(2) ELONGATION :

 After initiation, sigma factor separates.

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 At start point RNA polymerase enzyme breaks hydrogen bonds between two DNA strands and

separates them.

 One of them strand takes part in transcription.

 Transcription proceeds in 5‘ to 3' direction.

 Ribonucleoside triphosphate come to lie opposite complementary nitrogen bases of anti sense

strand.

 These Ribonucleotides are present in the form of triphosphate ATP, GTP, UTP and CTP

(perform dual role).

 RNA polymerase enzyme establishes phosphodiester bond between adjacent ribonucleotides.

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(3) TERMINATION:

 When RNA polymerase enzyme reaches at terminator site, it separates from DNA template.

 In most cases RNA polymerase enzyme can recognise the ‘Terminator site’ and stop the

synthesis of RNA chain, but in prokaryotes, it recognizes the terminator site with the help of

Rho factor (ρ factor).

 Rho (ρ) factor is a specific protein which helps RNA polymerase enzyme to recognize the

terminator site.

3' 3'
5' 5'
Promoter DNA helix

RNA polymerase Sigma factor

Intitiation
3' 5'

5' 3'
Terminator
RNA
Elongation

3' 5'

5' 3'

Termination RNA
P polymerase
RNA

Rho factor
Process of Transcription in Bacteria

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SPLIT GENE

The process of splicing occur in nucleus of eukaryotic cell.

 Mostly eukaryotic genes are example of split gene.
 Mostly prokaryotic genes are example of non split gene.

 Exception:- Archaebacteria.
 The split gene represent an ancient (primitive) feature of gene.
 The presence of introns is probaly reminiscent of antiquity (remember of past).

 The splicing process represents the dominance of RNA world.

5' 5'

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3' 3'

3' hnRNA
Capping Cap Intron
m
G
5' ppp
Exon Polyadenylation
RNA splicing
m 3'
5' Gppp
Poly A tail
m
5' Gppp
3'
m
5' Gpp Messenger RNA
Process of Transcription in Eukaryotes

5.6. GENETIC CODE

 Term Given by George Gamow.

 Discovered by Nirenberg, Matthaei and Khorana.
 Codon :- Is the nucleotide sequence on mRNA which codes for particular amino acid.

 Genetic code:- It is the sequence of nucleotides on mRNA which codes for whole polypeptide
chain.

TRIPLET NATURE OF CODON

 Gamow (1954) pointed out the possibility of three letters code (Triplet code).
 The main problem of genetic code was to determine the exact number of nucleotide in a codon
which codes for one amino acid.
 There are four types of Nitrogen bases in mRNA (A, U, G, C) for 20 types of amino acids.

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Possible Number of Total Coded Remaining
Nitrogen Bases in a Codon codon amino acids amino acids

Singlet (one N base code one 41 = 4 4 16

amino acid)

Doublet (two N bases code 42 = 16 16 4

one amino acid)

Triplet (three N bases code 43 = 64 20 0

one amino acid)
Deciphering of genetic code :-
 Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerising RNA

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with defined sequences in a template independent manner (enzymatic synthesis of RNA).
 Marshall Nirenberg’s developed cell-free system for protein synthesis finally helped the code to
be deciphered.
 The chemical method developed by Har Gobind Khorana.
 He was instrumental synthesising RNA molecules with defined combinations of bases
(homopolymers and copolymers).
Finally a checker-board for genetic code was prepared.

Characteristics of Genetic Code :

(i) Triplet in Nature :
 A codon is composed of three adjacent nitrogen bases which code on specifies one amino acid
in polypeptide chain.
For e.g. : In mRNA if there are total 90 Nitrogen bases.
Then this mRNA determines 30 amino acids in polypeptide chain.

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In above example, number of nitrogen bases are 90 so codons are 30 and 30 codons decide
30 amino acids in polypeptide chain.
(ii) Universality :
 The genetic code is nearly universally. The same genetic code is present in all kinds of living
organism including viruses, bacteria, unicellular and multicellular organisms.
 For e.g. in Bacteria :- UUU code Phenylalanine
Plants :- UUU code Phenylalanine
Animals :- UUU code Phenylalanine
 Exception :- Mitochondrial codon and some Protozoan codon.
(iii) Unambigous :
 Genetic code is unambiguous i.e. one codon specifies only one amino acid and not any other.
 In this case one codon never code for two different amino acids.

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 Exception GUG codon which codes both Valine and Methionine amino acids.
(iv) Non-Overlapping :
A nitrogen base is a constituent of only one codon.

AUG CUA GUC

m-RNA
(v) Commaless :
 There is no punctuation (comma) between the adjacent codon i.e. each codon is immediately
followed by the next codon.
 If a nucleotide is deleted or added, then whole genetic code read differently from the point of
insertion or deletion.
 A polypeptide chain having 50 amino acids shall be specified by a linear sequence of
150 nucleotides.
 If a nucleotide is added in the middle of this sequence, the first 25 amino acids of polypeptide
will be same but next 25 amino acids will be different.
(vi) Degeneracy of genetic code :- Single amino acid coded by more than one codon.
GGG
GGC
(4 codon)
e.g. :- Glycine GGA
(One Amino Acid)
GGU
Leucine :- 6 codon Exception : Methionine :- 1 codon
Serine :- 6 codon Tryptophan :- 1 codon
Arginine :- 6 codon
(vii) Chain Initiation and Chain Termination Codon :-
 AUG has dual functions. It codes for Methionine (met), and it also act as initiator codon..
 AUG codes methionine amino acid in eukaryotes and in prokaryotes AUG codes N formyl
methionine.
 Out of 64 codons 3 codons are stop or nonsense or termination Codon .
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 Nonsense codons do not specify any amino acid.
UAA (Ochre)
UAG (Amber)
UGA (Opal)
 So only 61 codons are sense codons which specify 20 amino acid.
5.7. TRANSLATION
 Translation refers to the process of polymerisation of amino acids to form a polypeptide.
 The order and sequence of amino acids are defined by the sequence of bases in the mRNA.

Fig :- Translation
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Translation
1. Activation of amino acid :-

+ PPi(Pyrophosphate)
2. Charging of t-RNA

+Amp + enzyme

3. Translation :- 3 steps
(A) Initiation (B) Elongation (C) Termination
(A) Initiation :-
Requirment :-
1. m-RNA
2. Charged t-RNA,
3. 30s and 50s sub units of ribosome
4. GTP
5. Mg+2
6. Initiation factors - In prokaryotes 3 factors:- IF1, IF2, IF3
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(A) Initiation

AUG

m-RNA
5’ Shine Delgarno 3’
(SD)
Sequence
Attachment of
smaller subunit of
ribosome(30s)

SD AUG

m-RNA
5’ 3’

30s 16s r-RNA

Anti SD sequence

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Attachment of First
AA1 charged t-RNA by
IF2 and GTP

AA1

UAC

Initiator t-RNA by IF2 and GTP

UAC

AUG
m-RNA
5’ 3’
30s 30S Ribosome m-RNA
t-RNA complex

Attachment of larger
subunit of ribosomes(50s) Peptidyl site Amino acyl site
by Mg+2
AA1
P A

P A 50s E

50s E AUG GGG

m-RNA
5’ 3’

30s

Initiation complex

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(B) Elongation:- By elongation factors (prokaryotes) :- 3 factors EF-Tu, EF-Ts, EF-G)

AA2

AA1 AA2 By EF-Tu, EF-Ts

and GTP

P A

50s E AUG GGG

m-RNA
5’
3’

30s

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Formed by
Peptidyl transferase
Peptide bond
enzyme(23s r-RNA);-
Ribozyme
AA A
1 A2

P A

50s E AUG GGG

m-RNA
5’ 3’

30s

AA3

AA AA2 AA2
AA1
1

Translocation by
P A EF-G and GTP P A

50s E AUG GGG UUU AU

50s E GGG
m-RNA UUU m-RNA
G
3’ 5’ 3’
5’
30s 30s

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(C) Termination :- by releasing factors (in prokaryotes RF1,RF2,RF3)

RF1 or RF2 and GTP

Polypeptide chain

AA1 AA2 AA3 AA4

P A

E UAG
m-RNA
5’ 3’

30s Stop codon

Untranslated regions :-

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 An mRNA also have 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).
 The UTR (untranslated regions) present on mRNA are required for efficient translation process
(by recognising the smaller subunit of ribosome by mRNA)
5.8. REGULATION OF GENE EXPRESSION
 It is the molecular mechanism by which expression of any gene is regulated as per requirement of
cell/organism.

Types of gene

Constitutive gene Non-Constitutive gene

(house keeping gene) (smart gene )

 Gene which always remain  Genes which are not always active.
active. (Always ON)  They require regulation.
 Need not to be regulation  Require ON & OFF mechanism.
 e.g- Gene of ATPase enzyme
Non-Constitutive gene (smart gene )

Inducible gene Repressible gene

The inducible genes are The repressible genes continue to

switched- on in presence of a express themselves till a chemical, often
chemical substance called an end product of the metabolism inhibits
inducer, required for the or represses their activity. Such type of
functioning of gene activity. inhibition is called feed back inhibition.

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Gene regulation in eukaryotes :-
In eukaryotes, it can be regulated at several levels
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.
Gene regulation in prokaryotes :-
 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).

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 In 1961, two French microbiologist Francois Jacob and Jacque Monod at the Pasteur
Institute in Paris, proposed a mechanism called operon model for the regulation of gene
action in E. coli.
 An operon is a segment of DNA, which acts as a single regulated unit having :-
I. One or more structural genes
II. An operator gene,
III. A promoter gene,
IV. A regulator gene.
Operon are of two types :-
1. Inducible:- An inducible operon system normally remains in switched off condition and begins
to work only when the substance to be metabolised by it is present in the cell. Inducible operon
system generally occurs in catabolic pathways. e.g. Lac operon of E. coli.
(2) Repressible:- A repressible operon system is normally in it's switch on state and continue to
synthesise a metabolise till the latter is produced in amount more than required, or else it
becomes available to the cell from outside. Repressible operon system is commonly found in
anabolic pathway. e.g. Tryptophan operon of E. coli.
THE LAC OPERON
 Lac means lactose, a disacchride which is made up of glucose and galactose.
 In lac operon, a polycistronic structural gene is regulated by a common promoter and regulatory
genes.
 GENES OF LAC OPERON :-
Structural genes -
 There are three genese.
(i) The z gene codes for beta-galactosidase.
Responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose
and glucose.
(ii) The y gene codes for permease, which increases permeability of the cell to -galactosides
(Lactose).
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(iii) The ‘a’ gene encodes a transacetylase (transfers an acetyl group from acetyl Co A to beta-
galactosidase).
 Hence, all the three gene products in lac operon are required for metabolism of lactose.
Operator gene - It lies adjacent to the structural genes and directly controls the synthesis of
mRNA over the structural genes.
Promoter gene - This gene is the site for initial binding of RNA polymerase.
Regulator gene(i) - It produces a repressor that binds to operator gene and stops the working of
the this gene.
 Repressor has two binding site (1) operator gene (2) effective molecule (inducer)
 Inducer - It is a chemical (substrate, hormone or some other metabolite) which after coming in
contact with the repressor, forms an inducer repressor complex.
 This complex cannot bind with the operator gene, which is thus switched on.

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 The inducer for lac operon of Escherichia coli is lactose.
 Allolactose is real inducer of lac operon.
Working of Lac operon :- Off conditions

p i p o z y a In absence of inducer

Repressor binds to the operator region(o)

Repressor mRNA and prevents RNA polymerase from
transcribe the operon

Repressor

p i p o z y a In presence of inducer
Transcription

Repressor mRNA lac mRNA

Translation

-galactosidase permease transacetylase

Inducer

(Inactive repressor)
The lac Operon

5.9. DNA FINGERPRINTING

 Also known as DNA profiling /DNA typing/DNA test.
 It is technique to identify a person on the basis of his/her DNA specificity.
 This technique was invented by sir Alec Jeffreys (1984).
 In India DNA Finger printing has been started by Dr. V.K. Kashyap & Dr. Lal Ji Singh at
(CCMB Hydrabad).
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Gel electrophoresis:-
 The cutting of DNA by restriction endonucleases results in the fragments of DNA. These
fragments can be separated by a technique known as gel electrophoresis.
 Since DNA fragments are negatively charged molecules they can be separated by forcing them to
move towards the anode under an electric field through a medium/matrix.
 The DNA fragments separate (resolve) according to their size through sieving effect provided by
the agarose gel. Hence, the smaller the fragment size, the farther it moves.
 In DNA fingerprinting gel - electrophoresis used for separation of DNA fragments.
PRINCIPLE :-
 DNA of human is almost (99.9%) same but very small amount (0.1%) that differs from person to
person.

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 Human genome has 3.3 × 109 bp
 These differences are mainly due to – Repetitive DNA sequences.
 These repetitive DNA are separated from bulk genomic DNA as different peaks during density
gradient centrifugation.

bulk DNA

Repetitive DNA

Satellite DNA

 These sequences normally do not code for any proteins,

 These sequence show high degree of polymorphism and form the basis of DNA
fingerprinting.
Polymorphism:- (variation at genetic level)
 Differences in DNA sequences.
 Arises due to mutations.
 Allelic 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.
Depending on base composition (A : T rich or G:C rich), length of segment, and number of
repetitive units, the satellite DNA is classified into many categories,
(1) Minisatellites (2) Microsatellites

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Micro-satellites:-
1. This satellites has 1 to 6 bp repetition.
2. Also called SSR (Simple Sequence Repeat).
Mini-satellites :-
1. This satellites has 11 to 60 bp repetition.
2. Also called VNTR (Variable Number of Tandem Repeats ).
3. Size of VNTR varies in size from 0.1 to 20 kb.
4. Repeat number vary in different human even though their repeat number also vary from
chromosome to chromosome in an individual. Except: monozygotic twins
5. On both sides of VNTR pallindromic sequences are present(restriction site).
Individual A Individual B

ATA….CA ATA….CA ATA….CA ATA….CA ATA….CA ATA….CA ATA….CA ATA….CA

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Cut by Restriction Cut by Restriction
enzyme enzyme

Restriction fragment Restriction fragment

Restriction Fragment Length Polymorphism (RFLP)

Isolation of DNA DNA cut by Restriction

Sample endonuclease
taken
If amount of DNA is very
low than increase by PCR
VNTR Probe
Nylone membrane
Gel

Southern Hybridization using

Autoradio-
Blotting labelled VNTR Probe graphy

Transfer of DNA from gel to VNTR Probe :- short ss-DNA

nylone membrane or Have radioactivity
nitrocellulose membrane Complimentry to VNTR

Nylone membrane

Autoradio- Development
graphy of X-ray film

X- ray film
X- ray film

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DNA cut by
Isolation Separation of DNA
Restriction
of DNA fragments by gel
endonuclease
electrophoresis

Denaturation by
Alkaline medium

Southern Blotting

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Autoradiography Hybridization using labelled
(Use X-ray film) VNTR Probe

Application of DNA fingerprinting :-

(1) Identification of the criminal (forensic science)
(2) In determining population and genetic diversities
(3) Paternity Tests :- The RFLP pattern of child 50% match with father and 50% with mother

Schematic representation of DNA fingerprinting

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5.10. HUMAN GENOME PROJECT (HGP)
 Started in 1990.
 Completed in 2003.
 Chromosome 1st completed in MAY 2006.
 Studied 24 chromosomes = 22 Autosomes + 2 sex chromosome
Mega project :-
In human genome 3 × 109 bp present
Cost of sequencing required per bp is :- 3$ US dollars
Estimated cost of the project would be approximately:- 9 billion US dollars
 If the obtained sequences were to be stored in typed form in books, and if each page of the book

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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.
 HGP was closely associated with the rapid development of a new area in biology called as
Bioinformatics.
 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.
Goals of HGP
(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;
(iii) Store this information in databases;
(vi) Improve tools for data analysis;
(v) Transfer related technologies to other sectors, such as industries;
(iv) Address the ethical, legal, and social issues (ELSI) that may arise from the project.
Method :-
(1) ESTs (Expressed Sequence Tag):- Identifying all the genes that are expressed as RNA.
(2) Sequence Annotation:- Sequencing the whole set of genome that contained all the coding and
non - coding sequence.
Instrument:- Automated DNA sequencer based on SANGER principle
Vector:- BAC (bacterial artificial chromosomes)
YAC (yeast artificial chromosomes)

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Features :-
1. Human genome contain :- 3164.7 million bp.
2. Average gene consists of :- 3000 bases.
3. Largest gene :- Dystrophin at 2.4 million bases
4. Estimated genes :- 30000.
5. Function unknown :- Over 50 % genes.
6. Coding part :- Less than 2%.
7. Most genes :- Chromosome 1 (2968).
8. Fewest genes :- Chromosome Y (231).
9. SNPs- (single nucleotide polymorphism) :- 1.4 million locations.

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10. Repetitive sequence:- Large portion of human genome
Applications and Future Challenges:-
 One of the greatest impacts of having the Human genome 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 broader scale.

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