MOLECULAR BASIS OF INHERITANCE

4/6 M
Can you Recall???
• What is Nucleic acid??
• What are the types of
nucleic Acid?
• Functions of nucleic
acid.
• Difference between
prokaryotic and
eukaryotic DNA
• THE DISCOVERY OF DNA:

- Friedrich Miescher (1869)- isolation of nucleic

acid from pus cells
- 2 types of nucleic acids:
i. DNA ( deoxyribonucleic acid)
ii. RNA ( ribonucleic acid)
• DNA as a genetic material:

Griffith’s Experiment:

- Frederick Griffith (1928) worked with bacterium

Streptococcus pneumoniae that causes pneumonia in
humans and other mammals
- He used two strains of Streptococcus:
i. virulent, smooth, pathogenic, and encapsulated S
type
ii. Non virulent, rough, non- pathogenic and non
capsulated R type
• Exp.1: He injected living ‘R’ strain into
the mice and found that this strain
was non- pathogenic and the mice
survived

• Exp.2 : He injected living ‘S’ strain into

the mice and found that this strain
was pathogenic. Mice showed
symptoms of Pneumonia and died
eventually

• Exp. 3: He subjected living ‘S’ strain to

high temperature and killed them and
this was injected in the mice. Mice
were not infected

• Exp.4: He injected a mixture of living

‘R’ strain and heat killed ‘S’ strain.
Mice developed symptoms of
pneumonia and eventually died
 Observation
• After analyzing the blood samples of dead mice in
experiment 4 he found colonies of living ‘S’ .

• Somehow living ‘R’ were converted to living ‘s’ in the

presence of dead material of ‘s”

• Phenomenon is called transformation

• Transformation :
• Change in one strain of bacterium into another strain
by uptake of DNA from surrounding medium
• Avery, McCarty and MacLeod’s
Experiment:

- In 1944, Avery, Mc Carty and MacLeod

proved the DNA to be the genetic
material.
- They mixed purified DNA, RNA,
proteins and other materials from cell
free extract of S strain cells with R strain
bacteria
- Only DNA could transform the harmless
strain R into virulent strain S
- They further added protease, DNAse
and RNAse to see if transformation take
place, thus transforming substance was
neither a protein nor RNA
- However the DNAse inhibited the
transformation. Suggesting that DNA is
the main factor for transfroming
principle
Hershy and Chase Experiment:
• Hershy and Chase worked on bacteriohages
which are compose of DNA and protein
• They used radioactive phosphorous 32P in
the medium for some viruses and radioactive
35sulphur for some others
• Viruses grown in the presence of 32P
contained radioactive DNA but not
radioactive proteins as DNA contains
phosphorous but proteins do not
• Similarly viruses grown 35S contained
radioactive protein but they did not contain
radioactive DNA because DNA does not
contain sulphur
• Radioactive phages were allowed to infect E
coli bacteria .Ecoli bacteria grown on the
medium with normal ‘P’ and ‘S’ followed by
centrifugation
• Bacteria which were infected by 32 P virus
were radioactive indicating that DNA was the
material that passed from the viruses to the
bacteria
DNA packaging:

• A typical mammalian cell has 2.2 meters long

DNA double helix while a prokaryotic cell like
E.coli, it is 1.1 mm long.
Packaging in Prokaryotes:
• Prokaryotes do not have well organized nucleus. The DNA is in the form of a
small, Circular, highly folded, naked ring.
• The negatively charged DNA is supercoiled due to extensive looping with the
help of RNA connectors to form 40-50 loops. Each loop is further coiled with
the help of RNA connectors to form 40-50 loops. Each loop is further coiled
with the help of positively charged Histone like DNA binding proteins and
enzymes like DNA gyrase and DNA topoisomerase I
Packaging in Eukaryotes:

• A eukaryotic cell has well organized nucleus

containing nuclear membrane, nucleolus and
thread like material in the form of chromosomes
• In the chromosomes DNA is associated with
histone and non-histone proteins
• Histones are positively charged basic proteins rich
in basic amino acids as lysine and arginine . They
organize to form histone octamer of 8 histone
molecules
• A histone octamer consists of four types of histone
proteins viz. H2A, H2B, H3 and H4.
• The negatively charged DNA helix is coiled around
the positively charged histone octamer to form
nucleosome.
• H1 protein binds the DNA at its entry and exit in
the octamer
• The length of DNA in a nucleosome =
approximately 200 base pairs
• Six nucleosome coil to form solenoid
• The solenoid structure further supercoils to from a
chromatin fiber which further coils and condense
to form chromosome
• Non –histone chromosomal proteins are necessary
for the packaging of chromatin at higher levels.
Heterochromatin:
• Darkly stained
• Condensed chromatin in eukaryotic cells
• This DNA rich chromatin is located near centromere
and telomeres
• Genetically inactive

Euchromatin:
• Light stained
• Noncondensed chromatin in eukaryotic cells
• Fast replicating
• Genetically active
Functions of DNA:

• Carrier of genetic information

• Does the synthesis of chemical molecules
other than itself such as Synthesis of RNA,
Synthesis of protein
• It undergoes replication
DNA REPLICATION
• Sugar+ nitrogenous bases= nucleosides
• Sugar+ nitrogenous bases+ phosphate group=
nucleotides.
A=T
G= C
AT, GC
5’ ATGCATGC 3’
3’ TACGTACG 5’

If there are 60 purines in a Given DNA sequence

then how many pyrimidines would be there?

5’ ATGCATGC 3’
3’ UACGUACG 5’
Purines Pyrimidines
DNA Replication:

• It is a process by which DNA duplicates itself

to form two identical copies
• It is essential when the cell reproduces so as
to have an equal distribution of DNA to
daughter cells
• In eukaryotic organisms replication of DNA
occurs in the in the S-Phase of interphase in
the cell cycle
Steps involved in DNA Replication
• Process of replication starts at a specific point
of DNA molecule called as origin
• At the origin the DNA strand breaks because of
an INCISION OR NICK by an enzyme
ENDONUCLEASE
• The hydrogen bonds joining two strands are
broken
• Two strands start UNWINDING by enzyme DNA
HELICASE
• The point where the strands separate appears
like a fork hence termed as REPLICATION FORK
• A new strand is constructed on each old strand
and old strand is referred as TEMPLATE STRAND
• As DNA is a polynucleotide , the new strand has
to be formed by ADDITION of nucleotides with
each other which is done by enzyme DNA
POLYMERASE III
• DNA polymerase III add nucleotides to a pre-
existing chain and at 3’OH end
• As DNA polymerase III can not initiate DNA
synthesis a small segment of RNA called RNA
PRIMER complementary to DNA template is
formed by enzyme PRIMASE
• It is to this primer, the DNA polymerase III adds 5’ de-oxy ribonucleotides
and extends DNA
• DNA polymerase III can function only in 5’-----3’ direction
• As two strands run antiparallel to each other, one strand is formed
continuously in 5’-3’ called as LEADING STRAND
• Synthesis of other complementary strand called LAGGING STRAND is more
complex
• As DNA polymerase III can add new nucleotides to a free 3’ OH a short RNA
primer is synthesized on template strand
• To this RNA primer , DNA polymerase –III adds new nucleotides at 3’end
• DNA polymerase I replace it with DNA
• Finally the enzyme DNA LIGASES forms phosphodiester bond between 2
nucleotides
• As the DNA is synthesized in small fragments they are called as OKAZAKI
FRAGMENTS
• IN 2 NEW DNA MOLECULES FORMED ONE STRAND IS OLD AND THE OTHER
IS NEW HENCE TERMED AS SEMICONSERVATIVE REPLICATION
Experimental confirmation for
semiconservative replication:
• Semiconservative Replication was experimentally was experimentally
proved by Meselson and Stahl(1958) by using equilibrium density gradient
centrifugation technique
• They used cultures of E.Coli bacteria grown on 14N and then transferred
to 15 N medium and allowed to replicate for several generations
• The position of bands was recorded at every generation which proved that
DNA replication is semiconservative
Protein Synthesis:

• Proteins are very important biomolecules as they serve as

structural components, enzymes and hormones
• The process of protein synthesis includes transcription and
translation
• Transcription is the process of copying of genetic information
from one strand of DNA into a single stranded RNA transcript
• Translation is the synthesis of polypeptide chain as per
genetic code
• Central Dogma is the flow of information from DNA to RNA
to proteins
DNA------transcription-----m RNA---translation----proteins
1. Transcription :
o Formation of m-RNA from DNA inside nucleus
o Enzyme- RNA Polyemerase
o In this process genetic information from DNA is copied into RNA
o For transcription a part of DNA works called transcription unit
o Transcription unit consists of:
o Promoter b. the structural gene c. terminator
a. Promoter : a small DNA sequence which provide binding site for
RNA polymerase
b. Structural gene : gene which is to be transcribed
c. Terminator : a small sequence of DNA which terminates the
transcription process
Process:
• The DNA strand which is used for RNA
synthesis is called TEMPLATE STRAND
oriented in 3’- 5’
• The DNA strand not involved in RNA
synthesis called CODING STRAND
oriented in 5’- 3’
• During transcription the enzyme RNA
polymerase binds to promoter site and
bring about initiation
• The two strands of DNA separate from
each other
• According to DNA sequence, RNA
nucleotides are selected and joined one
after other to form m-RNA strand
• A small part of RNA is attached to
enzyme
• As the enzyme reaches the terminator
region both enzyme and newly
constructed RNA fall off
• In this way termination occurs
m- RNA processing:
• Prokaryotic m-RNA does not have
introns thus do not require any
processing
• Eukaryotic genes possess non-coding
sequences called introns. The coding
sequences are called exons .
• In eukaryotes newly formed RNA
undergoes following processes:
• Splicing : introns are removed form hn
RNA
• Capping : at 5’end of hn RNA METHYL
GTP is added
• Tailing : at 3’end ADENYLATE residues
are added
• This RNA can now function as m-RNA
• After transcription the m-RNA is
transported out of nucleus to
ribosomes in cytoplasm for
translation.
Genetic Code:
• During transcription the genetic information in
DNA is copied to m-RNA and then transferred
to a chain of amino acids during translation
• The Genetic code consists of triplet codons
each specifying one amino acid.
• It directs the sequence of amino acid during
synthesis of proteins
• With 4 nitrogen bases A,C,G,U there would be
64 different triplet codons
Characteristics of Genetic code:

•The code is universal. All prokaryotic and eukaryotic

organisms use the same codon to specify each amino acid

•The code is triplet. Three nucleotides make one

codon.61of them code for amino acids and 3viz.,UAA,UAG
and UGA are nonsense codons or chain termination
codons

•The code is degenerate. For a particular amino acid more

than one word can be used
• The code is non overlapping. A base in mRNA
is not used for two different codons

• The code is commaless. There is no special

signal or commas between codons.

• The code is non ambiguous. A particular

codon will always code for the same amino
acid, wherever it is found.
Mutations of Genetic Code:

• Mutations is the sudden change in the DNA sequence that

results in changed phenotype
• Mutations provide raw material for evolution
• During mutation there may be deletion or insertion of a DNA
segment
• Deletion or insertion of base pairs of DNA causes frame shift
mutations or deletion mutation
• There may be change in a single base pair of DNA causing
point mutation e.g. sickle cell anaemia or delete mutation
• Mutations may be due to insertion or deletion of entire codon
there by causing an extra or less amino acid in polypeptide
chain
t- RNA adapter molecule:
• Clover leaf structure of t- RNA
has s codon loop with anticodon
• The 3’ end of the amino acid
acceptor end with unpaired CCA
• For every amino acid there is
specific t-RNA
• There are no t-RNA’s for stop
codons
• The t-RNA molecule is said to be
an adapter molecule as it has a
unique ability to read the codon
and to bind with the amino acid.
• Translation:

Process of transfer of amino acids by t-RNA on ribosomes for

protein synthesis
Takes place as follows:
i) Activation of amino acids
Inactivated amino acids in the cell pool are activated by ATP in
the presence of “ Amino Acyl Synthease”
ii) Formation of Amino Acyl t- RNA complex
• Now each activated amino acid gets attached to its specific t
RNA molecule at end to form the Amino Acyl t-RNA complex
• There is a separate specific t-RNA for each of amino acid
• Activated Amino Acids + t-RNA= Amino Acyl t- RNA complex
Formation of polypeptide chain

Process requires following:

a) Ribosomes : each ribosome has 2 parts with 3 sites
Smaller 30’s’ subunit has ‘E’ site
Larger 70’s’ subunit has ‘P’ site and ‘A’ site
b) ATP and GTP as an energy source
c) AA-t RNA complex
Steps :
i) Initiation
ii) Elongation
iii) Termination
i) Initiation :
• The m-RNA binds to smaller ribosome subunit (30-s)
• The start codon ‘AUG’ lies at ‘P’ site of ribosome
• The AA1 – t RNA complex is carried towards it and attaches itself with
anticodon UAC of t-RNA at P-site (P- site AUG
t-rna UAC
ii) Elongation :
• On the ‘A’ site of ribosome 2nd codon is present
• AA2- t RNA complex with complementary anticodon now carried towards ‘A’
site and attaches itself
• Between AA1 and AA2 a peptide bond is formed . Enzyme involved: Peptidyl
transferase
• Ribosome moves along ‘m-RNA’ in step wise manner from one codon distance ,
called as translocation
• During translocation, the 1st t RNA is released from ‘P’ site and moves back in
the cytoplasm
• AA1+ AA2 t RNA complex is now at ‘P’ site due to movement of ribosome. As a
result ‘A’ site is vacant
• AA3 – t RNA complex with proper anticodon is carried towards ‘A’ site and
attaches itself
• Again a peptide bond is formed between AA2 and AA3
• Once again ribosome moves one codon distance and the
process continues , forming a long chain of amino acids

iii) Termination:

• Elongation continues until the ribosome adds the last amino

acid , coded by m- RNA
• If m-RNA has UAA, UAG and UGA , it acts as termination
codon and protein synthesis stops
• Termination factor R1, R2 and S help in identification of
termination codons and also release polypeptide chain from
ribosomes
• After termination ribosomal subunits separate from each other
• ATP/ GTP used as a source of energy
Regulation of gene Expression:

• Gene expression in eukaryotes is regulated at different levels like

-Transcriptional level (formation of primary transcript)
- Processing level (regulation of splicing)
- Transport of m-RNA from nucleus to the cytoplasm
- Translational level
- For example, an enzyme beta galactocidase is synthesized by E.coli
to hydrolyze lactose into glucose and galactose
- In absence of lactose in the surrounding medium, E.coli bacteria do
not produce the enzyme beta galactocidase
- In presence of lactose the enzyme is synthesized.
- Such adaptive enzymes are called inducible enzymes, the
phenomenon is called induction and molecule responsible for this
is called inducer.
Operon concept:

• It is transcriptional control mechanism of gene regulation

explained by Jacob and Monod
• In E. coli lactose sugar in the culture medium induces
production of three enzymes necessary for digestion of lactose
i. Beta Galactocidase : digests lactose into galactose and
glucose
ii. Beta galactocidase permease: permits lactose molecule to
enter into the cell
iii. Transacetylase : transfers the acetyl group from acetyl
Co-A to galactoside
• Synthesis of these enzymes is controlled by a long segment of
DNA known as operon
• The operon consist of an operator site O and three structural
genes Z, Y and A. The action of structural genes is regulated by
operator site with the help of repressor protein
• Repressor protein is produced by the action of gene I known as
regualtor gene
• In absence of inducer the repressor protein attaches to the
operator and switches it off and structural genes are not
expressed
• The operator is switched on by inducer as it binds with the
repressor protein, the operator is set free and the three genes
z,y and a transcribed by RNA polymerase.
Lac Operon components:
1. Regulator gene( repressor gene)
2. Promoter gene
3. Operator gene
4. Structural genes(z, y and a)
5. Inducer( lactose)-not a component of operon
Genomics:
• The total genetic constitution of an organism is
called the genome
Or
• It is complete copy of genetic information
Or
• One complete set of chromosomes of an
organism
• Genomics is the study of genomes through
analysis, sequencing and mapping of genes
along with the study of their functions
• Genomics may be:

a. Structural Genomics: that involves mapping,

sequencing and analysis of genome

b. Functional Genomics: that deals with the

study of functions of all gene sequences and
their expressions in organisms
• Application of Genomics:

- Improvement of crop plant, human health and live stock

- The information acquired from genomics research can be used in

medicine, biotechnology and social sciences

- It may be useful in the treatment of genetic disorders through gene

therapy

- In agriculture to develop transgenic crops

- Genetic markers may be used in forensic analysis

- To introduce new gene in microbes to produce enzymes, therapeutic

proteins and even biofuels
HUMAN GENOME PROJECT:

• The human genome project was co-ordinated by the US department of

Energy and National Institute of Health
• It is a multinational research project to determine the genomic structure of
humans
AIMS:
- Mapping the entire human genome at the level of nucleotide sequences
- To store collected information in databases
- To provide a complete and accurate sequence of the 3 billion DNA base
pairs that make up the human genome
- To find out the estimated number of human genes
- To develop tools and techniques for data analysis
- To transfer the related technologies to private sectors such as industries and
to take care of the legal, ethical and social issues arising from the project
• organisms studied under HGP are E. coli, Caenorhabaditis elegans,
Saccharomyces cerevisiae, Drosophila , Mus musculus etc.
DNA Fingerprinting :

• Genes present on chromosomes are responsible for determining characters of

organisms as well as for inheritance of character

• Every individual has its unique genetic make up which may be called as fingerprint

• The technique developed to identify a person with the help of DNA restriction
analysis is known as DNA fingerprinting

• Technique is based on identification of nucleotide sequence present on the

wonder molecule

• 99% nucleotide sequence in all person is same. In a population every person

shows unusual sequences of 20-100 base pairs repeated several times called a
VNTR’s

• Length of VNTR different in each individual hence key factor DNA fingerprinting
Steps in DNA fingerprinting :

1. DNA isolation : DNA is obtained from cells available like

blood, hair roots, skin through host

2. DNA amplification : If the DNA quantity is small it is

subjected to in vitro replication by a technique called
PCR to obtain more copies of DNA

3. DNA fragmentation : DNA sample then subjected to

restriction digestion by an enzyme endonuclease which
cuts DNA at specific locations making many pieces of
DNA having variable length called as RFLP
4. Electrophoresis: DNA samples are loaded for agarose gel
electrophoresis. Charge is applied where the DNA fragment with
negative charge move towards positive pole. movement of
fragments depend upon length Which results in formation of bands

5. Southern blotting : the bands are now blotted on nylon membrane

6. Hybridization : bands are flooded with single stranded radioactive

DNA probe. Sample DNA and probe DNA form double stranded
structure which remains on nylon membrane and single stranded
fragments are washed off.

7. Photography : This nylon membrane is kept in contact with X-ray

film. DNA bands due to radioactive probe give photographic image
for documentation
Applications of DNA fingerprinting:

• In forensic science, DNA fingerprinting is used to

solve rape and complicated murder cases

• DNA fingerprinting is used to find out the

biological father or mother or both of the child in
case of disputed parentage

• DNA fingerprinting is used in pedigree analysis in

cats, dogs, horses and humans