Molecular

basis
Of
Inheritance
 DNA

A bacteriophage known as φ×174 has 5386

nucleotides, Bacteriophage lambda has 48502
base pairs (bp), Escherichia coli has 4.6 × 106
bp, and haploid content of human DNA is 3.3 ×
-
109 bp

109
*b
Structure of DNA

The backbone of a
polynucleotide chain is
formed due to sugar and
phosphates.
DNA as an acidic substance present in nucleus was first
identified by Friedrich Meischer in 1869. He named it as
‘Nuclein’.

In 1953 that James Watson and Francis Crick, based on

the X-ray diffraction data produced by Maurice Wilkins and
Rosalind Franklin, proposed a very simple but famous
Double Helix model for the structure of DNA.

Erwin Chargaff observed that for a double stranded DNA,

the ratios between Adenine and Thymine and Guanine
and Cytosine are constant and equals one.
The salient features of the Double-helix structure of DNA

(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'-3', the other has 3'-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 and Guanine is bonded with Cytosine with
three H-bonds.
(iv) The two chains are coiled in a right-handed fashion.
The pitch of the helix is 3.4 nm and there are roughly 10
bp in each turn. Consequently, the distance between a
bp in a helix is approximately 0.34 nm.

(v) The plane of one base pair stacks over the other in
double helix. This, in addition to H-bonds, confers
stability of the helical structure
 Central Dogma

Francis Crick proposed the Central dogma in

molecular biology, which states that the genetic
information flows from DNA-RNA-Protein.
 Packaging of DNA

In prokaryotes 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.
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. 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
Hik S-stour
Sain
=
Transforming Principle
-> In 1928, Frederick Griffith, in a series of
Retrain experiments with Streptococcus
e
Transforming V pneumoniae (bacterium responsible for
princip @°
pneumonia)

.
.
Oswald Avery, Colin MacLeod and Maclyn
McCarty (1933-44)
They worked to determine the biochemical
nature of ‘transforming principle’ in Griffith's
experiment.

They also discovered that protein-digesting

enzymes (proteases) and RNA-digesting
enzymes (RNases) did not affect
transformation
Hershey-Chase experiment
 Characteristics of genetic material
(i) Itshouldbeabletogenerateitsreplica(Replication).
(ii) Itshouldbestablechemicallyandstructurally.
(iii) It should provide the scope for slow changes
(mutation) that are required for evolution.
(iv) It should be able to express itself in the form of
'Mendelian Characters’.
 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). But, RNA being a catalyst was reactive and hence
unstable.
Semi conservative model of DNA replication
Matthew Meselson and Franklin Stahl
Fin my i w

En
Very similar experiments involving use of radioactive
thymidine to detect distribution of newly synthesised DNA
in the chromosomes was performed on Vicia faba (faba
beans) by Taylor and colleagues in 1958. The
experiments proved that the DNA in chromosomes also
replicate semiconservatively.
Semi conservative model of DNA replication
TRANSCRIPTION
A transcription unit in DNA is defined primarily by the
three regions in the DNA :
(i) A Promoter
(ii) TheStructuralgene
(iii) ATerminator
Templ 3ATTEEEEGG si
-

coding sMAGCTTHAGGGCC
i
31

MRNA S'UKAGCUVAAGGGCC zi
craavuccgab
 Cistron

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).

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. Introns or intervening sequences do not appear in
mature or processed RNA.
Transcription in prokaryotes
Transcription in eukaryotes
There are at least three RNA polymerases in the nucleus.

The RNA polymerase I transcribes rRNAs (28S, 18S, and

5.8S)

RNA polymerase III is responsible for transcription of

tRNA, 5srRNA, and snRNAs (small nuclear RNAs).

RNA polymerase II transcribes precursor of mRNA, the

heterogeneous nuclear RNA (hnRNA).
The primary transcripts contain both the exons and the
introns and are non-functional. Hence, it is subjected to a
process called splicing where the introns are removed
and exons are joined in a defined order. 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.
GENETIC CODE
George Gamow
The salient features of genetic code are as follows:
(i)The codon is triplet. 61codons code for amino acid and 3
codons 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 reading mRNA in a contiguous
fashion.Thereare no punctuations.
(iv)The code is nearly universal
(v) AUG has dual functions. It codes for Methionine (met) ,
and it also act as initiator codon.
(vi) UAA,UAG,UGAarestopterminatorcodons.
 Mutations and Genetic Code
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 frameshift
insertion or deletion mutations.
 Translation
tRNA
For initiation, there is another specific tRNA that is referred to as
initiator tRNA. There are no tRNAs for stop codons.
 Translation

In the first phase itself amino acids are activated in the

presence of ATP and linked to their cognate tRNA – a
process commonly called as charging of tRNA or
aminoacylation of tRNA to be more specific.
 UTR

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.
 Lac Operon
Jacob and Monod
 HGP

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;
(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.
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).

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).
Salient Features of Human Genome

(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 1 has 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.
DNA fingerprinting

It 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.
As polymorphism in DNA sequence is the basis of
genetic mapping of human genome as well as of DNA
fingerprinting.
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. It was called as Variable Number
of Tandem Repeats (VNTR). 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.