MOLECULAR BASIS OF

INHERITANCE
THE DNA:

⚫DNA is a long polymer of

deoxyribonucleotides.
⚫The length of the DNA depends on,
number of nucleotide pair present in it.
⚫Characteristics of the organism depend on
the length of the DNA.
⚫Bacteriophage ø174 has 5386 nucleotides.
⚫Bacteriophage lambda has 48502 base
pairs.
⚫Escherichia coli have 4.6 X 106 base pairs.
⚫Human genome (haploid) is 3.3 X 109 bp.
Structure of polynucleotide chain:

⚫ A nucleotide has three component:-

⚫ A nitrogen base
⚫ A pentose sugar ( ribose in RNA and deoxyribose
in DNA)
⚫ A phosphoric acid.

⚫ There are two types of nitrogen bases:

⚫ Purines ( Adenine and Guanine)
⚫ Pyrimidines ( Cytosine, Uracil and Thymine)

⚫ Adenine, Guanine and Cytosine is common in

RNA and DNA.
⚫ Uracil is present in RNA and Thymine is
present in DNA in place of Uracil.
⚫ Pentose sugar is ribose in RNA
and Deoxyribose in DNA.
⚫ A nitrogen base attached to the pentose sugar
at C1 of pentose sugar by
⚫Phosphoric acid attached to the 5’ OH of a nucleoside
by Phosphodiester linkage a corresponding nucleotide is
formed. (Ribonucleotide or deoxyribonucleotides
depending on the sugar unit).
⚫Two nucleotides are joined by 3’-5’ Phosphodiester linkage
to form dinucleotide.
⚫More than two nucleotides joined to form polynucleotide
chain.
⚫Polynucleotide chain has a free phosphate moiety at 5’ end
of sugar, is referred to as 5’ end
⚫In the other end of the polymer with 3’-OH group called 3’
end.
⚫The backbone of the polynucleotide chain is sugar and
phosphate.
⚫Nitrogen bases linked to the sugar moiety project from the
backbone.
⚫In RNA every nucleotide has an additional –OH group at
History of DNA:

⚫DNA is an acidic substance in the nucleus was

first identified by Friedrich Meischer in 1869.
He named it as ‘Nuclein”
⚫1953 double helix structure of DNA was given
by James Watson and Francis Crick, based on
X-ray defraction data produced Maurice
Wilkins and Rosalind Franklin.
⚫Hallmark of their proposition was base
pairing between two strands of polynucleotide
chains. This was based on observation
of Erwin Chargaff.
⚫Chargaff’s observation was that for a double
stranded DNA, the ratio between Adenine
and Thymine, and Guanine and Cytosine are
 Salient features of Double helix
structure of DNA:
⚫Made of two polynucleotide chains.
⚫Sugar and phosphate forms the backbone and bases projected
to inside.
⚫Two chains have anti-parallel polarity.
⚫Two strands are held together by hydrogen bond present in
between bases.
⚫Adenine of one strand pairs with Thymine of another strand by
two hydrogen bonds and vice versa.
⚫Guanine of one strand pairs with Cytosine of another strand by
three hydrogen bonds and vice versa.
⚫A purine comes opposite to a pyrimidine. This generates
approximately uniform distance between the two strands of the
helix.
⚫The two chains are coiled in a right – handed fashion.
⚫The pitch of the helix is 3.4 nm or 34 A0
⚫There are roughly 10 bp in turn.
⚫The distance between the bp in a helix is 0.34nm or 3.4 A0.
⚫The plane of one base pair stacks over the other in double helix.
Packaging of DNA Helix:

⚫Distance between two conjugative base pairs

is 0.34nm, the length of the DNA in a typical
mammalian cell will be 6.6 X109 bp X 0.34
X10-9 /bp, it comes about 2.2 meters.
⚫The length of DNA is more than the
dimension of a typical nucleus (10-6m), how is
such a long polymer packaged in a cell?
⚫Packaging in prokaryotes:
⚫They do not have definite nucleus.
⚫The DNA is not scattered throughout the cell.
⚫DNA is held together with some proteins in a
region is called ‘nucleoid’.
⚫The DNA in nucleoid is organized in large
 Packaging in Eukaryotes:

⚫ In eukaryotes the packaging is more complex.

⚫ There is a set of positively charged, basic protein calledHistones.
⚫ Histones are positively charged due to rich in basic amino acids
like Lysines and arginines.
⚫ Histones are organized to form a unit of eight molecules calledhistone
octamere.
⚫ Negatively charged DNA wrapped around positively charged histone
octamere to form a structure callednucleosome.
⚫ A typical nucleosome contains 200 bp of DNA helix.
⚫ Nucleosome constitutes the repeating unit of a structure in nucleus
called chromatin, thread like stained bodies seen in the nucleus.
⚫ The nucleosomes are seen as ‘beads-on-string’ structure when viewed
under electron microscope.
⚫ The chromatin is packaged to form chromatin fibers that are further
coiled and condensed at metaphase stage to form chromosome.
⚫ Packaging at higher level required additional set of proteins called Non-
histone Chromosomal (NHC) proteins.
⚫ In a typical nucleus some loosely coiled regions of chromatin (light
stained) is called euchromatin.
⚫ The chromatin that more densely packed and stains dark are
THE SEARCH OF GENETIC MATERIAL:
Transforming principle:

⚫ Given by Frederick Griffith in 1928.

⚫ His experiment based on Streptococcus
pneumoniae (caused pneumonia).
⚫ There is change in physical form of bacteria.
⚫ There are two colonies of bacteria:
⚫ Smooth shiny colonies called S strain.
⚫ Rough colonies called R strain.

⚫ S-strain bacteria have a mucous (polysaccharide) coat.

⚫ R-strain does not have mucous coat.
⚫ S-strain is virulent and caused pneumonia in mice and
died when infected.
⚫ R-strain is non-virulent and does not caused
pneumonia in mice when infected.
⚫ Heat killed S-Strain is non-virulent and does not causes
pneumonia.
⚫ The heat killed S-Strain mixed with live R-Strain
injected into mice; the mice developed pneumonia and
died.
Biochemical characterization of transforming
principle:

⚫Biochemical nature of transforming principle

was discovered by Oswald Avery, Colin
Macleod and Maclyn McCarty. (1933-44)
⚫Prior to their work genetic material was
thought to be protein.
⚫They worked to determine the biochemical
nature of the ‘transforming principle’ of
Griffith’s experiment.
⚫They purified biomolecules (proteins, DNA and
RNA) from the heat killed S cells to see which
one could transform live R cells to S cells.
⚫Heat killed S-Strain + protease + Live R-Strain
→ transformation.
Conclusion of the experiments:

⚫Protein of heat killed S-Strain is not the

genetic material
⚫RNA of heat killed S-Strain is not the
genetic material.
⚫DNA of heat killed S-Strain is the genetic
material, because DNA digested with
DNase mixed with R-strain unable to
transform R-Strain to S-Strain.
Conclusion of experiment:

⚫R – Strain bacteria had somehow

been transformed by the heat killed S-
Strain bacteria.
⚫Some ‘transforming principle’,
transferred from heat killed S-Strain
bacteria, had enabled the R-Strain to
synthesize smooth polysaccharide coat and
become virulent (S Strain).
⚫The transformation of R-Strain to S-
Strain is due to transfer
of Genetic material.
⚫However the biochemical nature of
genetic material was not defined from his
The Genetic Material is DNA:

⚫‘DNA is the genetic material’ is proved

by Alfred Hershey and Martha Chase (1952).
⚫They worked on the virus that infects bacteria
called bacteriophage.
⚫During normal infection the bacteriophage
first attaches the bacteria cell wall and then
inserts its genetic material into the bacterial
cell.
⚫The viral genetic material became integral
part of the bacterial genome and subsequently
manufactures more virus particle using host
machinery.
⚫Hershey and Chase worked to discover
whether it was protein or DNA from the
viruses that entered the bacteria.
Experiment :( blenders
experiment)

⚫They grew some viruses on a medium

having radioactive phosphorus and some others
on medium havingradioactive sulfur.
⚫Viruses grown in radioactive
Phosphorus have radioactive DNA but not
radioactive protein because Phosphorus present in
DNA not in protein.
⚫Viruses grown in radioactive
sulfur have radioactive protein not radioactive
DNA because sulfur present in protein but not in
DNA.
⚫Infection: radioactive phages were allowed to
attach to E.coli bacteria; the phages transfer the
genetic material to the bacteria.
⚫Blending: the viral coats were separated from the
bacteria surface by agitating them in a blender.
⚫Observation:
⚫Bacteria infected with viruses that had
radioactive DNA were radioactive and no
radioactivity in the supernatant.
⚫Bacteria infected with viruses that had
radioactive protein were not radioactive, but
radioactivity found in the supernatant.
⚫Conclusion of Experiment:
⚫DNA is the infecting agent that made the
bacteria radioactive hence DNA is the genetic
material not the protein.
PROPOERTIES OF GENETIC MATERIAL (DNA
VERSUS RNA):

⚫Criteria for genetic material:

⚫It should be able to generate its replica
(replication)
⚫It should be chemically and structurally
stable.
⚫It should provide the scope for slow changes
(mutation) that required for evolution.
⚫It should be able to express itself in the form
of ‘Mendelian Character’.
⚫Protein dose not fulfill the criteria hence it is
not the genetic material.
⚫RNA and DNA fulfill the criteria.
⚫RNA is unstable:
⚫2’-OH group present at every nucleotide (ribose sugar)
in RNA is a reactive group and makes RNA liable and
easily degradable.
⚫RNA is also now known as catalyst, hence reactive.
⚫RNA is unstable and mutates faster. Consequently the
viruses having RNA genome and having shorter life
span mutate and evolve faster.
⚫DNA is more stable:
⚫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.
⚫Two strands being complementary if separated by
heating come together, when appropriate conditions
are provided.
⚫Presence of Thymine in place of uracil confers
additional stability to DNA
Better genetic material (DNA or
RNA)

⚫Presence of thymine at the place of uracil

confers more stability to DNA.
⚫Both DNA and RNA are able to mutate.
⚫In fact RNA being unstable mutate at a faster
rate.
⚫RNA can directly code for the synthesis of
proteins, hence easily express.
⚫DNA however depends on RNA for protein
synthesis.
⚫The protein synthesis machinery has evolved
around RNA.
⚫Both RNA and DNA can functions as genetic
material, but DNA being more stable is
preferred for storage of genetic information.
RNA WORLD:

⚫RNA is the first genetic material.

⚫Essential life processes evolved around
RNA.
⚫RNA used to act as a genetic material as
well as catalyst.
⚫But RNA being catalyst was reactive and
hence unstable.
⚫Hence DNA has evolved from RNA with
chemical modifications that make it more
stable.
⚫DNA being double stranded and having
complementary strand further resists
Types of RNA:

⚫In prokaryotes there are three major types

of RNAs: mRNA
(messenger), tRNA (transfer),
and rRNA(ribosomal).
⚫All three RNAs are required to synthesize
protein in a cell.
⚫The mRNA provides the template and
having genetic information in the form of
genetic code.
⚫The tRNA brings the amino acids and
read the genetic code of mRNA.
⚫The rRNA is the structural part of the
REPLICATION: THE PROCESS:

⚫Watson and Crick proposed a scheme for

replication of DNA.
⚫The Original statement that “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 template for the
synthesis of new complementary strands.
⚫New DNA molecule must have one parental
strand and one new strand.
⚫This scheme of replication is
called Semiconservative type of replication.
 Experimental Proof of semiconservative
nature of replication:

⚫It is now proved experimentally that replication

is semiconservative type.
⚫It was first shown in Escherichia coli and
subsequently in higher organism.
⚫Mathew Messelson and Franklin
Stahl performed the following experiment in
1958.
STEPS OF THE EXPERIMENTS:
⚫They grew E.coli in 15NH4Cl medium for many
generations. (15N is heavy nitrogen not radioactive
element)
⚫The result was that 15N was incorporated into
newly synthesized DNA and other nitrogen
containing compound as
well.
⚫This heavy DNA molecule could be distinguished
from normal DNA by centrifugation in a cesium
chloride (CsCl) density gradient.
⚫Then they transferred the E.coli into a medium
with normal 14NH4Cl and let them grow.(E.coli
divides in 20 minutes)
⚫They took samples at definite time intervals as the
cells multiplied, and extracted the DNA that
remained as double-stranded helices.
⚫Various samples were separated independently
on CsCl gradients to measure the densities of DNA.
Experiment by Taylor and
colleagues:

⚫Used radioactive thymidine to detect

distribution of newly synthesized DNA in
the chromosomes.
⚫They performed the experiment on Vicia
faba (faba beans) in 1958.
⚫They proved the semiconservative nature
of DNA replication in eukaryotes
⚫Replication Machinery and Enzymes:
⚫In all living cells such as E.coli replication requires a set
of enzymes.
⚫E.coli completes the replication of its DNA in within 38
min.
⚫The average rate of polymerization has to be
approx. 2000 bp per sec.
⚫The polymerization process must be accurate; any
mistake during replication would result into mutation.
⚫Deoxyribonucleoside triphosphates (dATP, dGTP,
dCTP, dTTP) serve dual purposes:
⚫Provide energy for polymerization.
⚫Acts as substrates for polymerization.
⚫The replication process occurs within a small opening
of the DNA helix called replication fork.
⚫The region where, replication fork formed is
called origin of replication.
⚫The replication fork is formed by an enzyme
called helicase.
⚫Two separated strand is called template strands.
⚫Main enzyme is DNA-dependent DNA
polymerase, since it uses a DNA template to
catalyze the polymerization of
deoxyribonucleotides.
⚫DNA polymerase catalyses polymerization only in
one direction i.e. 5’→3’.
⚫On one strand (template with 3’→5’ polarity) the
replication is continuous hence called leading
strand.
⚫In another strand (template with 5’→3’ polarity)
the polymerization takes place in the form of short
fragment called Okazaki fragment.
⚫The short fragments are joined by DNA
ligase, hence called lagging strand.
TRANSCRIPTION:

⚫‘The process of copying genetic information

from one strand of the DNA into RNA is
termed as transcription’.
Transcription vs. Replication:
⚫Principle of complementarity governs the
process of transcription except Adenosine of
DNA forms base pair with theUracil instead of
thymine. During replication Adenine pairs
with thymine instead of uracil.
⚫During replication once started the whole
DNA is duplicated, where as transcription
takes place only a segment of DNA.
⚫In replication both strand acts as template,
where as in transcription only one strand is
acts as template to synthesize RNA.
⚫In replication DNA copied from a DNA,
where as in transcription RNA copied from
 Why both strands of DNA not
copied during transcription:
⚫If both strand of DNA acts as template, they
would translated into two RNA of different
sequences and in turn if they code for
proteins, the sequence of amino acids in the
protein would be different. Hence one
segment of DNA would be coding for two
different proteins.
⚫The two RNA molecules if produced from
simultaneously would be complementary to
each other, hence will form double stranded
RNA. This would prevent RNA translation
into protein.
⚫Transcription unit:
⚫A transcription unit in DNA consists of three
regions:
⚫A promoter
⚫The structural gene
⚫A terminator.
⚫DNA dependent RNA polymerase catalyses the
polymerization in only one direction that is
5’→3’.
Structural gene:

⚫The DNA strand having polarity 3’→5’ is

called template strand for transcription.
⚫The other strand of DNA having
polarity 5’→3’ is called coding strand.
⚫The sequences of nitrogen base in the
RNA transcribed from the template strand
are same as the coding strand of DNA
except having Thymine in place of Uracil.
⚫All the reference point defining a
transcription unit is made with the coding
strand only, not the template strand.
⚫Promoter and Terminator present on either side of
structural gene.
⚫The promoter located towards 5’ end (upstream) of
the structural gene.
⚫It is a short sequence of DNA that provides binding
site for RNA polymerase. (mostly TATA , Commonly
called TATA box)
⚫Presence of the promoter defines the template and
coding strands.
⚫If the position of promoter is changed with
terminator the definition of coding and template
strand will be reversed.
⚫Terminator:
⚫The terminator located towards 3’ end (down
stream) of coding strand.
⚫It terminates the process of transcription.
⚫It is also a short segment of DNA which recognizes
the termination factor. (ρ-factor)
Transcription unit and the gene:

⚫ Gene is defined as the functional unit of inheritance.

⚫ Genes are located on the DNA.
⚫ The DNA sequence coding for tRNA and rRNA
molecule also define a gene.
⚫ Cistron: a segment of DNA (structural gene) coding for
a polypeptide.
⚫ Monocistronic: most of eukaryotic structural gene
codes for single polypeptide.
⚫ Polycistronic: Most prokaryotic structural gene code
for more than one polypeptides.
⚫ In eukaryotes the monocistronic structural gens have
interrupted coding sequences, the genes are said to
besplit gene:
⚫ The coding sequences or expressed sequences are
called Exons.
⚫ Exons are interrupted by Introns.

⚫Exons are said to be those sequences that appear in

mature or processed mRNA.
⚫Introns never appear in mature of processed mRNA.
 Process of transcription:
prokaryotes.
⚫There is a single DNA dependent RNA polymerase that
catalyses transcription or synthesis of all three types of RNAs in
prokaryotes.
⚫The process of transcription completed in three steps:
⚫Initiation:
⚫RNA polymerase binds to the specific site of DNA
called promoter.
⚫Promoter of the DNA is recognized by initiation factor or sigma
(σ).
⚫RNA polymerase along with initiation factor binds to the
promoter.
⚫ Elongation:
⚫RNA polymerase unzipped the DNA double helix and forms an
open loop.
⚫It uses ribonucleoside triphosphates as substrate and
polymerizes in a DNA template following the rule of
complementarity.
⚫Only a short stretch of polymerized RNA remains binds with the
enzyme.
⚫The process of polymerization continued till the enzyme
 Additional complexities in eukaryotes:
⚫ There are three different types of RNA polymerases
in the nucleus:
⚫ RNA polymerase I transcribes rRNA (28S, 18S, and 5.8S)
⚫ RNA polymerase II transcribes heterogeneous nuclear
RNA (hnRNA).
⚫ RNA polymerase III transcribes tRNA, 5srRNA and
snRNA.
⚫ Post transcriptional processing: (occurs inside the
nucleus)
⚫ (a) Splicing:

⚫ The primary transcript (hn RNA) contain

both exons and introns and required to be processed
before translationally active (mRNA).
⚫ The introns are removed and exons are joined in a
defined order.
⚫ This process is catalyzed by SnRNP, introns removed
as spliceosome.
⚫ (b) Capping: an unusual nucleotide called methyl
guanosine triphosphate is added to the 5’ end of
hnRNA.
 GENETIC CODE:
⚫Contribution to discovery:
⚫The process of replication and transcription based on
complementarity.
⚫The process of translation is the transfer of genetic information
form a polymer of nucleotides to a polymer of amino acids.
There is no complementarity exist between nucleotides and
amino acids.
⚫If there is change in the nucleic acid (genetic material) there is
change in amino acids in proteins.
⚫There must be a genetic code that could direct the sequence of
amino acids in proteins during translation.
⚫George Gamow proposed the code should be 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.
⚫Har Govind Khorana enables instrumental synthesizing RNA
molecules with desired combinations of bases(homopolymer
and copolymers).
⚫Marshall Nirenberg’s cell – free system for protein synthesis
finally helped the discovery of genetic code.
⚫Severo Ochoa enzyme (polynucleotide phosphorylase) was also
helpful in polymerizing RNA with desired sequences in a
 Salient features of genetic code:
⚫The codon is triplet. Three nitrogen base sequences
constitute one codon.
⚫There are 64 codon, 61 codes for amino acids and 3
codons are stop codon.
⚫One codon codes for only one amino acid, hence it
is unambiguous.
⚫Degeneracy: some amino acids are coded by more
than one codon.
⚫Comma less: the codon is read in mRNA in a
continuous fashion. There is no punctuation.
⚫Universal: From bacteria to human UUU codes for
phenyl alanine.
⚫Initiation codon: AUG is the first codon of all mRNA.
And also it codes for methionine (met), hence has dual
function.
⚫Non-overlapping: The genetic code reads linearly
⚫Direction: the code only read in 5’ → 3’ direction.
Mutation and Genetic code:
⚫Relationship between DNA and genes are best
understood by mutation.
⚫Point mutation:
⚫It occurs due to replacement nitrogen base
within the gene.
⚫It only affects the change of particular amino
acid.
⚫Best understood the cause of sickle cell
anemia.
⚫Frame shift mutation:
⚫It occurs due to insertion or deletion of one or
more nitrogen bases in the gene.
⚫There is change in whole sequence of amino
 tRNA-the Adaptor molecule:
⚫The tRNA is called sRNA (soluble
RNA)
⚫It acts as an adapter molecule.
⚫tRNA has an anticodon loop that
base complementary to the
codon.
⚫It has an amino acid
accepter end to which it binds
with amino acid.
⚫Each tRNA bind with specific
amino acid i.e 61 types of tRNA
found.
⚫One specific tRNA with anticodon
UAC called initiator tRNA.
⚫There is no tRNA for stop
codons. (UAA, UGA, UAG)
⚫The secondary structure is like
clover-leaf.
⚫The actual structure of tRNA is
TRANSLATION:

⚫ It refers to polymerization of amino acids to

form a polypeptide.
⚫ The number and sequence of amino acids are

defined by the sequence of bases in the

mRNA.
⚫ The amino acids are joined by peptide bond.

⚫ Amino acids are activated in the presence of

ATP and linked to their specific tRNA is

called charging of tRNAor aminoacylation of
tRNA.
⚫ Ribosome is the cellular factory for protein

synthesis.
⚫ Ribosome consists of structural rRNA and 80

different proteins.
⚫ In inactive state ribosome(70S) present in two
⚫Initiation:
⚫The process of translation or protein synthesis
begins with attachment of mRNA with small subunit
of ribosome.
⚫The ribosome binds to the mRNA at the start codon
(AUG).
⚫AUG is recognized by the initiator tRNA.
⚫Elongation:
⚫Larger subunit attached with the initiation complex.
⚫Larger subunit has two site ‘A’ site and ‘P’ site.
⚫Initiator tRNA accommodated in ‘P’ site of large
subunit, the subsequent amino-acyl-tRNA enters
into the ‘A’ site.
⚫The sub subsequent tRNA selected according to the
codon of the mRNA.
⚫Codon of mRNA and anticodon of tRNA are
complementary to each other.
⚫Formation of peptide bond between two amino
Termination:

⚫Elongation continues until a stop codon

arrives at ‘P’ site.
⚫There is no tRNA for stop codon.
⚫A release factor binds to the stop codon.
⚫Further shifting of ribosome leads to
separation of polypeptide.
⚫An mRNA also has some additional
sequences that are not translated
called untranslated regions (UTR).
REGULATION OF GENE
EXPRESSION:

⚫Regulation of gene expression in eukaryotes takes place

in different level:
⚫Transcriptional level (formation of primary transcript)
⚫Processing level (regulation of splicing)
⚫Transport of mRNA from nucleus to the cytoplasm.
⚫Translational level.
⚫In prokaryotes control of rate of transcriptional
initiation is the predominant site for control of gene
expression.
⚫The activity of RNA polymerase at the promoter is
regulated by accessory proteins, which affects its ability
to recognize the start site.
⚫The regulatory proteins can acts both positively
(activators) or negatively (repressor)
⚫The regulatory proteins interact with specific region of
DNA called operator, which regulate the accessibility of
RNA polymerase to promoter.
 Lac operon:
⚫ Francois Jacob and Jacque Monod first to describe a transcriptionally
regulated system of gene expression.
⚫ A polycistronic structural gene is regulated by common promoter and
regulatory genes. Such regulation system is common in bacteria and is
called operon.
⚫ Lac operon consists of:-

⚫ One regulator gene ( i-gene)

⚫ Three structural genes (z,y,a)

⚫ Operator. (binding site of repressor protein)

⚫ Promoter.(binding site of the RNA polymerase)

⚫ The i-gene codes for repressor of the lac operon.

⚫ The structural gene consist of three gene (z, y and a)

⚫ ‘z’-gene codes for beta-galactosidase, which hydrolyze lactose into

Galactose and glucose.
⚫ ‘y’ –gene codes for permease, which increases the permeability of
bacterial cell to lactose.
⚫ ‘a’-gene codes for transacetylase
⚫ All three genes are required for the metabolism of lactose in bacteria.
⚫ Inducer: lactose is the substrate for β- galactosidase and it regulates the
switching on and off of the lac operon. Hence it is called inducer.
⚫ In the absence of glucose, if lactose is added in the growth medium of
the bacteria, the lactose is transported into the cell by permease.
⚫ Very low level of expression of lac operon has to be present in the
Mechanism of regulation of lac
operon:

⚫The repressor protein is synthesized from i-

gene (all time constitutively)
⚫In the absence of the inducer i.e. lactose the
active repressor binds to the operator and
prevents RNA polymerase from transcribing
the structural gene
⚫In the presence of the inducer such
as lactose or allolactose, the repressor is
inactivated by interaction with inducer.
⚫This allows RNA polymerase access to the
promoter and transcription proceeds.
⚫The regulation of lac operon by repressor is
referred to as negative regulation.
HUMAN GENOMIC PROJECT:

⚫Genetic make-up of an organism or an individual

lies in the DNA sequences.
⚫Two individual differs in their DNA sequences at
least in some places.
⚫Finding out the complete DNA sequence of
human genome.
⚫Sequencing human genome was launched in 190.
⚫Goals of HGP:
⚫Identify all the approximately 20.000 – 25000
genes in human DNA.
⚫Determine the sequence of all 3 billion chemical
base pairs.
⚫Store this information in data bases.
⚫Improve tools for data analysis.
⚫Transfer related technologies to other sectors,
such as industries.
⚫Address the ethical, legal, and social issues (ELSI)
Methodology:

⚫To identify all the genes that expressed as

RNA referred asExpressed Sequence Tags
(ETSs).
⚫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 calledSequence Annotation.
⚫The commonly used hosts for sequencing
were bacteria and yeast and vectors were
called as BAC (bacterial artificial
chromosome) and YAC (yeast artificial
chromosome).
Salient features of Human
Genome:

⚫The human genome contains 3164.7

million nucleotide bases.
⚫The average gene consists of 3000 bases.
⚫The largest known human gene
being dystrophin at 2.4 million bases.
⚫The total number of gene is estimated at 30.000.
⚫99.9 percent nucleotide base sequences are same
in all peoples.
⚫The function of 50% genes discovered is unknown.
⚫Less than 2 percent of the genome codes
for proteins.
⚫Repeated sequences make up very large portion
of human genome.
⚫Chromosome I has most genes (2968) and
the Y has the fewest (231).
DNA FINGER PRINTING:

⚫ DNA finger printing is a very quick way to compare the DNA

sequences of any two individual.
⚫ DNA fingerprinting involves identifying differences in some specific
regions in DNA called repetitive DNA,because in these sequences, a
small stretch of DNA is repeated many times.
⚫ During centrifugation the bulk DNA forms major peak and the other
small peaks are called satellite DNA.
⚫ Depending on base composition (A:T rich or G:C rich), length of
segment, and number of repetitive units, the satellite DNA classified
into many types, such as mini –satellite and micro – satellite.
⚫ These sequences dose not code for any proteins.
⚫ These sequences show high degree of polymorphism and form basis
of DNA fingerprinting.
⚫ Polymorphism in DNA sequence is the basis of genetic mapping of
human genome as well as of DNA fingerprinting.
⚫ Polymorphism (variation at genetic level) arises due to mutations.
⚫ If an inheritable mutation is observed in a population at high
frequency it is referred as DNA polymorphism.
The process:

⚫ DNA fingerprinting was initially developed

by Alec Jeffreys.
⚫ He used satellite DNA as the basis of DNA
fingerprinting that shows very high degree of
polymorphism. It was called as Variable
Number Tandem Repeats.(VNTR)
⚫ Different steps of DNA fingerprinting are:-

⚫ Isolation of DNA.
⚫ Digestion of DNA by restriction endonucleases.

⚫ Separation of DNA fragments by gel

electrophoresis.
⚫ Transferring (blotting) of separated DNA
fragments to synthetic membranes, such as
nitrocellulose or nylon.
⚫ Double stranded DNA made single stranded.

⚫ Hybridization using labeled VNTR probe.

⚫ Detection of hybridized DNA fragments by

⚫The VNTR belongs to a class of satellite DNA
referred to as mini-satellite.
⚫The size of VNTR varies from 0.1 to 20 kb.
⚫After hybridization with VNTR probe the
autoradiogram gives many bands of different
sizes. These bands give a characteristic pattern for
an individual DNA. It differs from individual to
individual.
⚫The DNA from a single cell is enough to perform
DNA fingerprinting.
⚫Applications:
⚫Test of paternity.
⚫Identify the criminals.
⚫Population diversity determination.
⚫Determination of genetic diversity.
⚫
Thanking
you