(BOTANY)

Contents
TOPIC

Molecular Basis of Inheritance

Plot No. 46, Rajeev Gandhi Nagar, Kota, (Rajasthan) Pin code : 324005.
Help Desk : 09214233303 | 09214233343 |
 Molecular Basis of Inheritance 2
Nucleic acids :
* Friedrich Meisher discovered Nucleic acid in Nucleus of Pus cells (1869) and named it ‘Nuclein’
• Altmann found its acidic nature and termed ‘ Nucleic acid’
• DNA term was given by Zarich
* 2types of nucleic acid- 1. DeoxyRibonucleic Acid (DNA) - Genetic material in most organisms 2.
Ribonucleic Acid (RNA)-
* Genetic material in some viruses * Messenger molecule * Adapter molecule
* Catalytic (Ribozyme) * Structural component

DNA
DNA is a long polymer of deoxyribonucleotides.
• The length of DNA is usually defined as number of nucleotides (or a pair of nucleotide referred to as
base pairs , bp) present in it. This also is the characteristic of an organism.
i). Bacteriophage  174 -5386 nucleotides . ssDNA
ii). Bacteriophage lambda - 48502 base pairs (bp) - dsDNA
iii). Escherichia coli - 4.6 × 106 bp- dsDNA
iv). Haploid content (Genome) of human DNA . 3.3 × 109 bp. - dsDNA

There are two types of nitrogenous bases -

1.Purines (Adenine and Guanine)
2.Pyrimidines (Cytosine, Uracil and Thymine).
* Cytosine is common for both DNA and RNA and Thymine is present in DNA.
* Uracil is present in RNA at the place of Thymine.
Structure of Purines and Pyrimidines :
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 3
1.Adenine (= 6 Amino purine) 2.Guanine (= 2 Amino ,6 Oxy purine)
3.Cytosine (= 2 Oxy , 4 Amino pyrimidine) 4.Uracil (2,4 dioxy pyrimidine)
5.Thymine (=5 methyl Uracil )
Formation of Nucleotide :-
* PO4 attached to -OH group of C5 of sugar by Ester bond.
* Nitrogen base attaches to C1 of pentose sugar by N-glycosidic linkage/bond to form Nucleoside (N9 of
Purine & N1 of Pyrimidine.
Nucleosides are Basic in nature.
Nucleosides :
1.adenosine or deoxyadenosine 2.guanosine or deoxyguanosine
3.cytidine or deoxycytidine 4.Uridine
5.deoxythymidine.
When a phosphate group is linked to 5'-OH of a nucleoside through phosphoester linkage, a corre-
sponding nucleotide (or deoxynucleotide depending upon the type of sugar present) is formed

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005

STRUCTURE OF POLYNUCLEOTIDE CHAIN
* Two nucleotides are linked through 3'-5'
phosphodiester linkage to form a dinucleotide.
* A polymer thus formed has at one end a free
phosphate moiety at 5'-end of ribose sugar,
which is referred to as 5’-end of polynucleotide
chain.
* Similarly, at the other end of the polymer the
ribose has a free 3'-OH group which is referred
to as 3' -end of the polynucleotide chain.
* The backbone in a polynucleotide chain is
formed due to sugar and phosphates.
* The nitrogenous bases linked to sugar moiety
project from the backbone.
* Formula for No. of phosphodiester bonds
* In ss =
no. of nucleotides/no. of nitrogen bases-1
* In ds
= total no. of nucleotides/nitrogen bases - 2
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 4
* In RNA, every nucleotide residue has an
additional –VOH group present at 2' -position in
the ribose.
* Also, in RNA the uracil is found at the place of
thymine (5-methyl uracil).
* In Polynucleotide :- 2 adjacent nucleotides are
joined by:
* 3, 5 phosphodiester bond (2 Ester bonds on
either side)
* Links –OH(hydroxyl) group at C3 of pentose
sugar of one nucleotide to –PO4 (phosphate) at
C5 of pentose sugar of succeeding nucleotide
* Bond between – PO4 & –OH group of sugar
(ester bond)
* Polynucleotide chain has 2 ends-3’ and 5’ prime
 Molecular Basis of Inheritance 5

NUCLEIC ACIDS (DNA/RNA)

* DNA is in form of helix
* Polynucleotides
* DNA shows 3.4 angstrom periodicity
* Bond : Phospho-di-ester bond
2. Chargaff’s rule:
* Unit:Nucleotide (microbiomolecules)
* In ds – DNA , No.of Purines = No. of Pyrim-
* In 1953 that James Watson and Francis Crick,
idines
based on the X-ray diffraction data produced by
A+G=T+C
Maurice Wilkins and Rosalind Franklin, pro-
i.e A+G/T+C=1
posed a very simple but famous Double Helix
Base Ratio = A + T/G + C = Specific for a Spe-
model for the structure of DNA.
cies = unique identity of a species
* Noble prize was given in 1962 to Watson ,
In Human = 1.55
Crick and Wilkins
* In E.coli = 0.93 (< 1)
* > 1 eukaryotes (A+T>G+C)
WATSON – CRICK/ DOUBLE HELIX
* < 1 prokaryotes (A+T<G+C)
MODEL FOR B-DNA :
Double Stranded structure(Secondary
based on
structure)
1. X-ray diffraction data produced by Maurice
Wilkins and Rosalind Franklin –

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 6
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) Complimentary & Specific base pairing

* A = T (2 Hydrogen bonds), C „k G(3 Hydrogen bonds)
* This is the Hallmark of this structure , based on Chargaaf’s study as sequence of bases on one strand
can help in prediction of base sequence on other strand
* So , both strands are joined by H- bonds.

(iv) The bases in two strands are paired through hydrogen bond (H-bonds) forming base pairs (bp).
* This generates approximately uniform distance between the two strands of the helix.

(v) The two chains are coiled in a right-handed fashion.

(vi) The pitch of the helix is 3.4 nm (10-9 m) and there are roughly 10 bp in each turn. Consequently, the
distance between a bp in a helix is 0.34 nm (3.4 Angstrom) – base on study of Wilkins and Franklin
* One Complete turn (360°) has 34°A length (Pitch of helix) & 10 base pairs
* Distance between 2 adjacent base pairs 3.4 Ao & Rotation angle 360°
* (vii) 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.

Diameter of B-DNA = 2 nm/20A°

* Histone proteins bind at deep groove (major groove) at 30 degree angle
Laws Governing the DNA Structure
1.Antiparallelism
2.Complementarity
3.Chargaff’s Rule
* Base Ratio

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 7

PACKAGING OF DNA HELIX

* Histone proteins bind at deep groove (major
groove) at 30 degree angle Laws Governing the
DNA Structure
1. Antiparallelism
2.Complementarity
3.Chargaff’s Rule
* Base Ratio

* Distance between 2 bps=0.34 nm (0.34× 10-9 m)

* No. of bps in human= 6.6 - 109 bp
* Length of human DNA = 2.2 meter
* Size of human nucleus = 10-6 meter

* no. Of bp in Escherichia coli = 4.6 ×106 bp

* Distance between 2 bps=0.34 nm (0.34X 10-9 m)
* Length of E.coli DNA = 1.3 X 10-3 meter or 1.3
mm
* Size of cell (E.coli) = 1 micron

DNA packaging in Prokaryotes :

* DNA is present in cytoplasm in super coiled state .
* Non-Histone Basic protein called Polyamines
(positive charge) coils with negatively charged
DNA.
* The negatively charged DNA is wrapped around
* Packaged DNA is called Nucleoid/Genophore
the positively charged histone octamer to form a
* DNA packaging in Eukaryotes :
structure called nucleosome.
* In eukaryotes, packaging of chromosomes is
* A typical nucleosome contains 200 bp of DNA
much complex.
helix.
* Histone Basic protein (positive charge) binds
* Nucleosomes constitute the repeating unit of a
with negatively charged DNA.
structure in nucleus called chromatin, thread-like
* The packaging of chromatin at higher level
stained (coloured) bodies seen in nucleus.
requires additional set of proteins that collectively
* The nucleosomes in chromatin are seen as
are referred to as Non-histone Chromosomal
“beads-on-string” structure when viewed under
(NHC) proteins.
electron microscope.
* There is a set of positively charged, basic
* The beads-on-string structure in chromatin is
proteins called histones.
packaged to form chromatin fibers that are
* Histones are rich in the basic amino acid residues
further coiled and condensed at metaphase stage
lysine and arginine.
of cell division to form chromosomes.
* Both the amino acid residues carry positive
* In a typical nucleus, some region of chromatin
charges in their side chains.
are loosely packed (and stains light) and are
* Histones are organised to form a unit of eight
referred to as euchromatin.
molecules called as histone octamer/Nu body.
* The chromatin that is more densely packed and
(H2A/2B/H3/H4)
stains dark are called as Heterochromatin.
* Euchromatin is said to be transcriptionally active
chromatin, whereas heterochromatin is inactive.

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 8
Types of DNA :
1) Circular and Linear DNA
2) Repititive DNA – Sequence of N-bases is repeated more than once inDNA , present in centromere and
satellite region of chromosome
3) Palindromic DNA- Segment of DNA which reads the same when orientation of reading is kept same. ,
Restriction site for RE

C-Value of DNA :
* Total amount of DNA in genome
* Measured in pigogram (pg)

THE SEARCH FOR GENETIC MATERIAL

Transforming Principle
* In 1928, Frederick Griffith, in a series of experiments with Streptococcus pneumoniae (bacterium respon-
sible for pneumonia), witnessed a miraculous transformation in the bacteria , Host-Mice

He used RII and SIII strains of bacteria.

* He recovered live S strain from dead mice.
* He concluded that the R strain bacteria had somehow been transformed by the heat-killed S strain
bacteria.
* Some “transforming principle”, transferred from the heat-killed S strain, had enabled the R strain to
synthesize a smooth polysaccharide coat and become virulent.
* This must be due to the transfer of the genetic material. However, the biochemical nature of genetic
material was not defined from his experiments.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 9

Biochemical Characterization of Transforming Principle :

* Prior to the work of Oswald Avery, Colin MacLeod and Maclyn McCarty (1933-44), the genetic material
was thought to be a protein.
* They worked to determine the biochemical nature of “transforming principle” in Griffith's experiment.
They purified biochemicals (proteins, DNA, RNA, etc.) from the heat-killed S cells to see which ones
could transform live R cells into S cells.
* They discovered that DNA alone from S bacteria caused R bacteria to become transformed.
* They also discovered that protein-digesting enzymes (proteases) and RNA-digesting enzymes (RNases)
did not affect transformation, so the transforming substance was not a protein or RNA.
* Digestion with DNAse did inhibit transformation, suggesting that the DNA caused the transformation.
They concluded that DNA is the hereditary material
* TRANSDUCTION EXPERIMENT
* The unequivocal proof that DNA is the genetic material came from the experiments of Alfred Hershey
and Martha Chase (1952).
* They worked with viruses that infect bacteria called bacteriophages.

* Bacteria which was infected with viruses that had radioactive DNA were radioactive, indicating that
DNA was the material that passed from the virus to the bacteria.
* Bacteria that were infected with viruses that had radioactive proteins were not radioactive.
* This indicates that proteins did not enter the bacteria from the viruses.
* DNA is therefore the genetic material that is passed from virus to bacteria

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005

PROPERTIES OF AS A GENETIC MATERIAL
A molecule that can act as a genetic material
must fulfill the following criteria:
(i) It should be able to generate its replica (Replica-
tion).
(ii) It should chemically and structurally be stable.
(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’.
The two strands of DNA being complementary if
separated by heating come together, when
appropriate conditions are provided.
* Further, 2'-OH group present at every nucleotide
in RNA is a reactive group and makes RNA
labile and easily degradable.
* RNA is also now known to be catalytic, hence
reactive.
Therefore, DNA chemically is less reactive and
structurally more stable when compared to RNA.
* Therefore, among the two nucleic acids, the
DNA is a better genetic material.
* In fact, the presence of thymine at the place of
uracil also confers additional stability to DNA.
* RNA being unstable, mutate at a faster rate
CENTRAL DOGMA OF MOLECULAR
BIOLOGY
* Francis Crick proposed the Central dogma in
molecular biology, which states that the genetic
information flows from DNA  RNA Protein.
In some viruses the flow of information is in
reverse direction, that is, from RNA to DNA.
For storage of Genetic information , DNA is
preferred
* For transmission of Genetic information , RNA is
preferred
* RNA as genetic material is present in some
viruses. (TMV , QB Bacteriophage)
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 10
RNA WORLD
RNA was the first genetic material.
RNA used to act as a genetic material as well as a
catalyst (there are some important biochemical
reactions in living systems that are catalysed by
RNA catalysts and not by protein enzymes).
But, RNA being a catalyst was reactive and
hence unstable.
* Therefore, DNA has evolved from RNA with
chemical modifications that make it more stable.
* DNA being double stranded and having comple-
mentary strand further resists changes by
evolving a process of repair.
DNA REPLICATION
It is Synthesis of new DNA strand from parent
DNA or on existing DNA strand.
* It occurs in S-phase of cell cycle
* It occurs in cytoplasm in prokaryotes , nucleus/
Mt/chloroplast in Eukaryotes.
* While giving double helical structure of B-DNA ,
Watson & Crick gave the scheme for DNA
Replication.
“The parent DNA strands break and each strand
acts as template for synthesis of new DNA
strand’
* 3 modes of DNA replication :
1. Semi-conservative – accepted mode , experimen-
tally proven by Messelson and Stahl
2.Conservative 3.Dispersive
 Molecular Basis of Inheritance 11
DNA REPLICATION
Messelson and Stahl’s Experiment (1958)
* Experimental model-E.coli * He used -
1. Heavy isotope of N2- N15 2. Light isotope of N2- N14
* He used Density gradient centrifugation by CsCl

Very similar experiments involving

* use of radioactive Tritiated thymidine (H3 in place of H1)
* to detect distribution of newly synthesized 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.

Mechanism of DNA Replication - The Machinery and the Enzymes

* It is enzyme mediated.
* Energetically replication is a very expensive process.
* It occurs in parts due to high energy requirement.
DNA dependent DNA Polymerase (main enzyme)
* adds 2000 bps per second * very fast
* very accurate because Any mistake during replication would result into mutations.
* E.coli – 4.6 X106 bp , replication in 38 min.

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 12
1) ORI = origin of replication 2 types of new strands :
* DNA replication starts at ori 1.Continuous/Leading strand :
* 1 ori (prokaryotes) , >1 ori (eukaryotes) * Synthesized continuously
* If desired DNA is to be propagated through * Single primer required
recombinant DNA technology , then vector is * Okazaki DNA fragments absent
required because vector has Ori. * Faster formation
2) Activation of Nucleotides (DEEP) : * Formed on parent / Template strand 3' 5'
* Deoxyribonucleotide,Phosphorylase Enzyme ,
Energy, two Pi required. 2.Lagging strand :
* Deoxyribonucleoside triphosphates serve dual * Synthesized discontinuously
purposes - * >1 primer required
1. In addition to acting as substrates * Okazaki fragments present (joined by DNA
2. they provide energy for polymerisation reaction ligase)
(the two terminal phosphates in a * Slower
deoxynucleoside triphosphates are high-energy * Formed on parent / Template strand 5'3'
phosphates, same as in case of ATP).

3) Unwinding of DNA helix :

a) Helicase – separates 2 strands of DNA , by
breaking H-bonds.This creates Y-shaped struc-
ture called ‘Replication Fork’
b) SSBP (single stranded binding proteins)-bind to ss
of DNA (as tetramer) to prevent renaturation of
DNA i.e reformation of H-bonds. It provides
stability to single strands.

c)Topoisomerase -
* It release tension due to super coiling at opposite
end of replication fork.
* It nicks , release tension &then re-seals.
* In prokaryotes , this function is performed by
DNA Gyrase. * In eukaryotes, the replication of DNA takes
place at S-phase of the cell-cycle.
4) Primer formation : * The replication of DNA and cell division cycle
* DNA-P cannot initiate replication on its own. should be highly coordinated.
* So , RNA Primer (small sequence of ss RNA , * A failure in cell division after DNA replication
complimentary to parent DNA strand) is formed results into polyploidy(a chromosomal anomaly).
at 3’ end of parent strand Proof reading :
* using enzyme DNA dependent RNA Poly- * Done mainly by DNA-P I
merase/RNA Primase * In 3'5' direction
* After completion of new strand , primer is * Exonuclease activity
removed(5'÷3') & gap is filled by DNA-P I
DNA replication is
5) Elongation of DNA strand * Fast
* After primer addition , DNA-P III attaches * Semi-conservative
¡¥activated nucleotides¡¦ to the primer  two * Semi-continuous
terminal pyrophosphate high energy are removed * Bidirectional
releasing energy  which is used in forming * Almost accurate
phospho-di-ester bonds * Energy consuming
* DNA-P III adds about 2000 bps/second
* DNA-P forms new strand in 5' 3' direction

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 13
RNA :
* RIBONUCLEIC ACID
* Single stranded polynucleotide * Parts of RNA:
1.Ribose sugar 2.Phosphate 3.Nitrogen bases- A , G, C, U

TYPES OF RNA :
1.mRNA 2.tRNA 3.rRNA

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 14
t-RNA : 2 models 4.sc RNA – small cytoplasmic RNA
1.Inverted L model – * 7-s scRNA + Proteins  signal recognition
* by Kim & Klug peptide (SRP)*
* 3-D , Difficult to study * They help in binding ribosome & nascent peptide
2.Clover leaf model - to ER for formation of regulatory protein.
* By Holley * All 3 types of RNA are required in protein
* 2-D / Secondary structure , easy to study synthesis i.e mRNA, tRNA and rRNA

CENTRAL DOGMA OF MOLECULAR

BIOLOGY
•Unidirectional flow of genetic information
•from master copy (DNA)
•to working copy(RNA)
•to trait expressing molecule (polypeptide)
•proposed by Francis Crick

REVERSE CENTRAL DOGMA / Teminism -

* An exception to central dogma
* Proposed by Temin and Baltimore in 1970 (Nobel
prize)
* In some viruses the flow of information is in
reverse direction, that is, from RNA (genetic
RNA) to DNA.
Some important terms :
1.Gene : Viral genetic RNA (v RNA) DNA  RNA
* a segment of DNA which codes for protein , t-  PROTEIN
RNA , r-RNA * Reverse transcription  synthesis of DNA from
* It is functional unit of inheritance 2.Cistron – a RNA by Reverse transcriptase (RNA dependent
segment of DNA which codes for a specific DNA polymerase)enzyme
polypeptide Autocatalytic DNA 
* Monocistronic m-RNA in Eukaryotes * DNA  DNA
* Polycistronic m-RNA in Prokaryotes 3.Recon- * DNA replication
recombinant cistron 4.Muton- mutant cistron * Heterocatalytic DNA 
* DNA  RNA
1.Genetic/genomic RNA – in some viruses * Transcription
* HIV , Hepatitis A/C virus, TMV (ss RNA)
* Reovirus (ds RNA) 2.Catalytic RNA- RNA acting TRANSCRIPTION
as enzymes (Peptidyl transferase) called * The process of copying genetic information from
Ribozymes one strand of the DNA into RNA
* 23-s r-RNA (Prokaryotes) * DNA dependent RNA Polymerase
* 28-s r-RNA (Eukaryotes)
3.sn RNA –small nuclear RNA Like DNA replication , The principle of
* small RNA freely dispersed in nucleus complementarity governs the process of tran-
* These attaches with 7-8 proteins , forming small scription,
nuclear Ribonuclear proteins(sn RNP/snurps) * except the adenosine , now forms base pair with
* Have Role in Splicing uracil instead of thymine.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 15

* Why both the strands are not copied during transcription?

* First, if both strands act a template, they would code for RNA molecule with different sequences,
* and in turn, if they code for proteins, the sequence of amino acids in the proteins would be different.
* Hence, one segment of the DNA would be coding for two different proteins,
* and this would complicate the genetic information transfer machinery.

* Second, the two RNA molecules if produced simultaneously would be complementary to each other,
* hence would form a double stranded RNA.
* This would prevent RNA from being translated into protein
* and the exercise of transcription would become a futile one.
* Template/Non coding/Anti-sense strand of DNA –
* Strand with polarity - 3'5'
* RNA is synthesized on this strand in 5'3'as RNA Polymerase works in 5'3'¦
* Coding /Non template/ Sense strand of DNA –
* Strand with polarity - 5'3'
* RNA formed is similar to coding strand , but formed on template strand

Transcription Unit
* It is segment of DNA involved in transcription.
* 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

Structural gene is flanked on both sides by promoter (5' of coding strand) and terminator( 3' of coding
strand)
* Promoter –
* provides binding site for RNA-Polymerase
* present at 5' of coding strand (upstream) or 3' of template strand
* The presence of promoter defines coding and template strand
*By switching promoter with terminator , the definition of coding and template strand reverses.

Recognition/conserve sequences –
* present in promoter region , recognised by RNA-Polymerase (sigma factor)
* may be present further upstream or downstream to the promoter
* In prokaryotes-

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005

* at -10 bp( 10 bp before/upstream to start point)
* TATAAT (Pribnow box)
* In eukaryotes –
* at -20 bp (20 bp before/upstream to start point)
* TATAAAT / TATATAT (Hogness/TATA box)
* -70 to -80 bp CAAT box
RNA polymerase
* Forms RNA in 5'3' on template strand
* In prokaryotes – only one type which forms all
types of RNA
* In eukaryotes – 3 types of RNA Polymerase
1. RNA polymerase I (nucloelus) transcribes
rRNAs (28S, 18S, and 5.8S)
2. RNA polymerase II (nucleoplasm)transcribes
mRNA – the heterogeneous RNA (hnRNA).
3. RNA polymerase III (nucleoplasm) is transcribes
tRNA , 5s rRNA, scRNAs and snRNAs
RNA Polymerase has 5 subunits :
* Core enzyme = 2 + 1+1 + 1 = role in
elongation of RNA
* Sigma factor (£m ) = role in initiation of tran-
scription
* Rho factor (£l )= role in termination of transcrip-
tion
* Holoenzyme = core enzyme + Sigma factor
Terminator –
* Defines end of transcription
* present at 3' of coding strand (downstream) or 5' of
template strand
Structural gene –
* In eukaryotes – monocistronic genes
* In eukaryotes , Split gene present =
1. Functional Exons /coding/expressed sequences
(appear in mature or processed RNA)
2. with intervening Introns (junk DNA/ intermediate
DNA/non-functional / non-coding/don't appear in
mature or processed RNA)
* In Prokaryotes– Polycistronic genes
* In Prokaryotes,split gene absent-only exons
* Split gene discovered by -Roberts and Sharp
* Exon undergo transcription and translation
* Introns undergo transcription but not translation
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 16
TRANSCRIPTION IN PROKARYOTES
* There is single DNA-dependent RNA poly-
merase that catalyse transcription of all types of
RNA in bacteria.
* It occurs in cytoplasm.
* 3 steps –
1.Initiation 2.Elongation 3.Termination
1.Initiation –
* Sigma factor of RNA Polymerase recognise
conserved sequences in promoter region.
* Now RNA polymerase binds to promoter and
initiates transcription.
2. Elongation –
* RNA Polymerase uses nucleoside triphosphates
as substrate and polymerises in a template
depended fashion following the rule of
complementarity.
* It somehow also facilitates opening of the helix
and continues elongation till it reaches terminator
site.
* Only a short stretch of RNA remains bound to
the enzyme.
3. Termination –
* Once the polymerases reaches the terminator
region , Rho factor helps in release of RNA
Polymerase,
* the nascent RNA falls off, so also the RNA
polymerase.
* This results in termination of transcription.
* In bacteria, since mRNA does not require any
processing to become active,
* and also since transcription & translation take
place in the same compartment (there is no
separation of cytosol & nucleus in bacteria),
* many times translation can begin much before
the mRNA is fully transcribed.
* The transcription and translation can be coupled
in bacteria.

TRANSCRIPTION IN EUKARYOTES
* It occurs in Nucleus (also in mitochondria ,
plastids )
* There are at least three DNA dependent RNA
polymerases in the nucleus. There is a clear cut
division of labour.
1. RNA polymerase I transcribes rRNAs (28S, 18S,
and 5.8S)
2. RNA polymerase II transcribes precursor mRNA
– the heterogeneous RNA (hnRNA).
3. RNA polymerase III is transcribes
tRNA,5srRNA, and snRNAs
* The primary transcripts contain both the exons
and the introns & are non-functional.
* Hence, it is subjected to a process called Splicing
* where the introns are removed & exons are
joined in a defined order.
* hnRNA undergo two additional processing called
as capping and tailing.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 17
Post transcriptional modification
in Eukaryotes –
1. Capping (5') –
* addition of unusual nucleotide (methyl guanosine
triphosphate , 7 methyl Gppp (GTP) ) to 5¡¬-end
of hnRNA. (substrate : GTP , Enzyme:Guanyl
transferase)
* Provide stability to m-RNA
* Helps to form ribosome-m RNA complex (cap
recognition by 18-s r RNA of smaller subunit of
ribosome)
2. Tailing (3) /Polyadenylation–
* addition of 200-300 adenylate residues (Poly A
tail) at 3'-end in a template independent manner
* substrate : ATP
* Enzyme : Poly-A Polymerase (Template inde-
pendent enzyme)
* Provides stability to m-RNA 3. Splicing - introns
are removed by Splicosome/snRNP/Snurps &
exons joined (by RNA ligase) in a defined order.
 Molecular Basis of Inheritance 18

* He suggested that in order to code for all the 20

* It is the fully processed hnRNA, now called
amino acids,
mRNA, that is transported out of the nucleus into
* the code should be made up of three nucleotides.
cytoplasm for translation
* Codons are triplet in nature
* Difference between transcription in prokaryotes
* This was a very bold proposition, because a
and eukaryotes
permutation combination of 43 (4 × 4 × 4) would
* The split gene arrangements represent probably
generate 64 codons.
an ancient feature of genome.
* NO. OF CODONS = 4n , where n= no. of
* The presence of introns is reminiscent of antiq-
nitrogen bases in each codon
uity,
Experimental proof of Triplet codon – given by
* and the process of splicing represents the
1.Har Gobind Khorana :
dominance of RNA-world.
* The chemical method developed by him was
instrumental in synthesising RNA molecules with
GENETIC CODE
defined combinations of bases (homopolymers
* Genetic code - It is a sequence of nitrogen bases/
and copolymers).
nucleotides on m-RNA , which have coded
2. Marshall Nirenberg :
information for amino acid synthesis.
* He helped in deciphering codon by providing cell-
* Direct sequence of amino acids during synthesis
free system for protein synthesis.
of proteins.
3. Severo Ochoa:
* Codon – it is a sequence of nitrogen bases which
* He synthesised Severo Ochoa enzyme (Poly-
codes for a specific amino acid synthesis.
nucleotide phosphorylase),
* helpful in polymerising RNA with defined se-
* George Gamow, a physicist, found that since
quences
there are only 4 bases and if they have to code
* in a Template/DNA Independent manner (enzy-
for 20 amino acids, the code should constitute a
matic synthesis of RNA).
combination of bases. (Term-Genetic code)
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 19
* Har Gobind Khorana , Marshall Nirenberg ,
Severo Ochoa received Nobel prize in 1968

* Salient features of Genetic Code:

(i) The codon is triplet. 61 codons code for amino
acids and 3 codons do not code for any amino
acids, hence they function as stop codons.
(ii) One codon codes for only one amino acid,
hence, it is unambiguous and specific (except –
GUG-Valine/methionine at initiation point)

(iii) Some amino acids are coded by more than

one codon , hence the code is Degenerate.
* 3 aa – 6 codons(SAL-Serine , Alanine, Lysine)
* 5 aa – 4 codons
* 1 aa – 3 codons
* 9 aa – 2 codons
* Total 18 aa show degeneracy
* 2 aa are coded by only 1 codon(Exception to Wobble Hypothesis –
Degeneracy) eg- Methionine by AUG & Tryp- * proposed by Francis Crick
tophan by UGG * The specificity of codon is determined by first
* 3 Codons are Stop codons two nitrogen bases
* The third nitrogen base does not affect the
(iv) The codon is read in mRNA in a contiguous expression of codon
fashion. There are no punctuations. * This third position is called Wobbly position
(v) The code is nearly universal: * But all codons don't follow this hypothesis
* for example, from bacteria to human UUU would * This hypothesis allows economy of t RNA during
code for Phenylalanine (phe). protein synthesis
* Some Exceptions to this rule have been found in * i.e one tRNA (anti-codon ) recognise more than
mitochondrial codons, and in some protozoans. one codon
(vi) AUG has dual functions.
* It codes for Methionine (met) , Mutations and Genetic Code
* and it also act as initiator codon. * A classical example of point mutation is a change
of single base pair in the gene for beta globin
Stop/Terminator/ Non-sense codons: chain that results in the change of amino acid
1.UAA – Ochre (Ullu Aya Aya) residue glutamate to valine. It results into a
2.UAG – Amber(Ullu Aya Gaya) diseased condition called as Sickle cell anemia.
3.UGA – Opal (Ullu Gaya Aya) * Insertion or deletion of three or its multiple bases
* Initiation/Start codon – insert or delete one or multiple codon hence one
1.AUG or multiple amino acids, and reading frame
2.GUG(only if at 1st position) remains unaltered from that point onwards. Such
mutations are referred to as Frame-shift insertion
* First discovered codon – UUU , codes for or deletion mutations.
Phenylalanine
* Punctuation codon = Start codon & Stop codons 1.Frame shift insertion or deletion mutation –
* Insertion or deletion of one or two bases changes
the reading frame from the point of insertion or
deletion.

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
 Molecular Basis of Inheritance 20
* Insertion or deletion of three or its multiple bases TRANSLATION
insert or delete one or multiple codon hence one * The process of polymerisation of amino acids to
or multiple amino acids, and reading frame form a polypeptide.
remains unaltered from that point onwards * It occurs in cytoplasm in both prokaryotes and
eukaryotes.
RAM HAS RED CAP * Cellular factory for protein synthesis is Ribo-
RAM HAS BRE DCA P some.
RAM HAS BIR EDC AP * The order and sequence of amino acids are
RAM HAS BIG RED CAP defined by the sequence of bases in the mRNA.
RAM HAS EDC AP (R removed)
RAM HAS DCA P (RE removed) Machinery for translation :
RAM HAS CAP (RED removed) 1. r RNA ( as structural component of ribosome)
* This forms genetic basis of proof that codon is a 2. t RNA
triplet & it is read in a contiguous manner 3. m RNA
4.Ribosome
2. Substitution / Point mutation  complete 5.Mg2+ ions
reading of frame is not altered 6.Amino acids (raw material)
* THE FAT CAT ATE THE RAT (BAT) 7.Aminoacyl t RNA synthetase enzyme
8.Initiation factors , elongation factors , release
a)Transition – factors
* If Pu is mutated to Pu or Py to Py When 2 subunits of ribosome combines ,
1.AUG (methionine)  GUG(valine) there is formation of 3 sites
2.AUG (methionine)  ACG (threonine) 1.P site (Peptidyl site)
2.A site ( Aminoacyl site)
b)Transversion – 3.E site ( Exit site)
* If Pu is mutated to Py or Py is mutated to Pu
1.AUG (methionine)  CUG(Leucine) Process of translation :
2.AUG (methionine)  AAG(Lysine) 1.Activation of amino acid :
* In the first phase itself amino acids are activated
1. Mis-sense mutation –if by change of 1 nitrogen in the presence of ATP
base , aa is changed , may be lethal, A min o acyl
* Sickel cell anemia * Amino acid + ATP t  RNA synthetase
* AUG (methionine)  GUG(valine)
2. Same-Sense / Silent mutation – if by change of 1 Amino acyl AMP- enzyme complex + PP
nitrogen base , aa is not changed , not lethal , role
in evolution AA + ATP + Aminoacyl synthetase enzyme (E) +
* CCC (proline)  CCG (proline) Mg2+
3. Non -Sense mutation - if by change of 1 nitrogen  AA-AMP-E Complex + Ppi
base , there is appearance of any stop codon , There is a separate gAmino acyl t-RNA
may be lethal , the protein synthesis stops after synthetase enzyme for each kind of amino acid
this point.
* UGG (Tryptophan)  UGA (Stop codon) 2. Charging of tRNA or Aminoacylation of tRNA
Q) Assume there are – :
a) 5 types of Nitrogen bases in RNA and there are * Activated Amino acids linked to their cognate
2 bases in each codon , then calculate no. of tRNA AA-AMP-E Complex + specific t RNA
codons possible –  tRNA –VAA (Charged tRNA) + AMP + Enzyme
b) 7 types of Nitrogen bases in RNA and there are
2500 amino acids, then calculate i) No. of 3. Formation of polypeptide chain :
nitrogen bases in codon ii) No. of codons possible * The amino acids are joined by a bond which is
known as a peptide bond.
* Formation of a peptide bond requires energy.

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005

* The Ribosome also acts as a catalyst (23S rRNA
in bacteria is enzyme- Ribozyme) for the forma-
tion of peptide
A) Binding of mRNA with smaller subunit of
ribosome
B) Binding of charged tRNA with m RNA-Ribosome
complex
C) Chain elongation –
i) Peptide bond formation
* After formation of initiation complex , A-site is
established next to P-site (where 1st charged
tRNA is attached)
Now next charged t RNA binds to A-site (codon-
anticodon binding)
* When 2 charged t RNA are brought in close
contact, then peptide bond formation is favoured
energetically
* Catalyst Peptidyl transferase (Ribozyme) helps in
this bond formation
* Peptide bond is formed between COOH of one
amino acid and –NH2 of next amino acid.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 21
* This t-RNA of P site released from ribosome via
E-site and dipeptide attaches with A site.
* Now t-RNA of A-site is transferred to P-site and
hence A-site becomes empty.
ii) Translocation – moving of ribosome on m RNA ,
from one codon to other.
* Now , process of translocation and peptide bond
formation will continue till at A-site , stop codon
appears.
* The amino acids are getting added one after the
other , leading to polypeptide sequence , dictated
by DNA
iii ) Chain – Termination :-
* Due to sliding of ribosome over m-RNA when
any Non-sense codon (UAA, UAG, UGA)
available at A site of ribosome , then polypeptide
chain terminates
* The linkage between the last tRNA and the
polypeptide chain is broken by three release
facctor called RF1, RF2, RF3 with the help of
GTP.

A translational unit in mRNA
* The sequence of RNA that is flanked by the start
codon (AUG) and the stop codon
* and codes for a polypeptide
Untranslated regions (UTR) –
* An mRNA also have some additional sequences
that are not translated
* 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)
* For initiation, the ribosome binds to the mRNA at
the start codon (AUG) that is recognised only by
the initiator tRNA.
* The ribosome proceeds to the elongation phase
of protein synthesis.
* During this stage, complexes composed of an
amino acid linked to tRNA, sequentially bind to
the appropriate codon in mRNA by forming
complementary base pairs with the tRNA
anticodon.
* The ribosome moves from codon to codon along
the mRNA.
* Amino acids are added one by one, translated
into Polypeptide sequences
* dictated by DNA and represented by mRNA.
* At the end, a release factor binds to the stop
codon, terminating translation and releasing the
complete polypeptide from the ribosome.
Summary of Translation –
I.Amino acid activation – 1 ATP required
II.Charging of t-RNA – No ATP required (X)
III.Polypeptide chain formation -
1.Chain initiation :-
a)m-RNA ribosome (smaller subunit) complex- (X)
b)Addition of charged t-RNA – 1 GTP required
c)Larger subunit of ribosome attaches 2. Chain –
elongation :-
a)Peptide bond formation –X
b)Translocation- 1 GTP required 3. Chain termination
- 1 GTP required
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 22
Regulation of Gene Expression :
* Regulation over the functioning of genes is called
regulation of gene expression. It can be exerted
at four levels.
(i) Transcriptional level during formation of primary
transcript.
(ii) Processing level (regulation of splicing)
(iii)Transport of mRNAs from nucleus to cytoplasm
(iv) Translational level
Operon Model :
•An operon is a segment of DNA that functions as
single regulated unit comprising
1.a regulator gene
2.a promoter gene
3.an operator gene
4.one or more structural genes
Operons involve two types –
(1) Inducible operon model
(2) Repressible operon model.
First operon lac–Operon was
* discovered by Jacob and Monod (1961) in E.coli.
The genes in a cell are expressed to perform a
particular function or a set of functions.
* For example, if an enzyme called beta-galactosi-
dase is synthesised by E. coli, it is used to
catalyse the hydrolysis of a disaccharide, lactose
into galactose and glucose;
* the bacteria use them as a source of energy.
* Hence, if the bacteria do not have lactose around
them to be utilised for energy source, they would
no longer require the synthesis of the enzyme
beta-galactosidase.
* In simple terms, it is metabolic, physiological or
environmental conditions that regulate expression
of genes.
* The development and differentiation of embryo
into adult organisms are also a result of the
coordinated regulation of expression of several
sets of genes.
* In prokaryotes, control of the rate of transcrip-
tional 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 regu-
lated by interaction with accessory proteins,
which affect its ability to recognise start sites.
* These regulatory proteins can act both positively
(activators) and negatively (repressors).
 Molecular Basis of Inheritance 23
* The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of
proteins with sequences termed operators.
* The operator region is adjacent to the promoter elements in most operons and in most cases the se-
quences of the operator bind a repressor protein.
* Each operon has its specific operator and specific repressor.
* For example, lac operator is present only in the lac operon and it interacts specifically with lac repressor
only.
Inducible Operon Model :
• It is found in catabolic pathway
• Ex: Lactose operon or Lac operon.
The Lac operon
* Geneticist , Francois Jacob and a biochemist, Jacque Monod
* a transcriptionally regulated system
* In lac operon (here lac referes to lactose),
* a polycistronic structural gene is regulated by a common promoter and regulatory genes.
* Such arrangement is very common in bacteria and is referred to as operon.

The lac operon consists of –

1. one regulatory gene (the i gene ¡V here the term i does not refer to inducer, rather it is derived from the
word inhibitor)
2. three structural genes (z, y, and a).
* i gene codes for repressor of lac operon

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005

1. z gene codes for beta-galactosidase (â-gal),
which is primarily responsible for the hydrolysis
of the disaccharide, lactose into its monomeric
units, galactose and glucose.
2. y gene codes for permease, which increases
permeability of the cell to â-galactosides.
3. a gene encodes a transacetylase
Hence, all the three gene products in lac operon
are required for metabolism of lactose.
* In most other operons as well, the genes present
in the operon are needed together to function in
the same or related metabolic pathway.
* Lactose is the substrate for the enzyme beta-
galactosidase and it regulates switching on and
off of the operon. Hence, it is termed as
inducer(Lactose).
* In the absence of a preferred carbon source such
as glucose,
* if lactose is provided in the growth medium of the
bacteria,
* the lactose is transported into the cells
* through the action of permease
(Remember, a very low level of expression of lac
operon has to be present in the cell all the time,
otherwise lactose cannot enter the cells).
* Lactose then induces the operon in the following
manner.
* The repressor of the operon is synthesised (all-
the-time – constitutively) from i gene.
* Repressor protein binds to the operator region of
the operon & prevents RNA polymerase from
transcribing operon.
* In presence of an inducer, like lactose or
allolactose,
* repressor is inactivated by interaction with
inducer
* This allows RNA polymerase access to the
promoter and transcription proceeds
* Essentially, regulation of lac operon can also be
visualised as regulation of enzyme synthesis by
its substrate.
* Glucose or galactose cannot act as inducers for
lac operon
* Regulation of lac operon by repressor is referred
to as negative regulation.
* Lac operon is under control of positive regulation
as well, but it is beyond the scope of discussion at
this level.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 24
Types of Genes :
(1) House Keeping Genes (Constitutive Genes)
:
* These genes are constantly expressing them-
selves in a cell because their products are
required for normal cellular activities
* Ex: genes for glycolysis , regulator genes
(2) Non-constitutive Genes (Luxury / Smart
Genes)
* The genes are not always expressing themselves
in a cell.
* They are switched on or off according to the
requirement of cellular activities
* lactose system in Escherichia coli (structural
genes)
If lactose absent , Switch Off
* If lactose present , Switch On
* Repressor protein (i-gene) + Operator gene =
Switch Off
* Repressor protein (i-gene) + Inducer (Lactose/
Allolactose) = Switch On
Human Genome project (HGP) :
* HGP is a mega project
* Started by U.S. Department of Energy and
National Institute of Health
* For sequencing human genome
* In 1990
* Welcome Trust (UK) joined project as a major
partner
* Later on Japan, France, Germany, China & some
other countries also joined it.
* Expected scheduled completion – 2005
* But completed in 2003 , in 13 years
* Till 2003 , all 23 (21 A + X +Y) chromosomes
except chromosome no. 1
* Last chromosome to be sequenced is chromo-
some 1 (May 2006)
* Why called Mega project ???
1. Human genome is said to have approximately
3 x 109 bp,
* and if the cost of sequencing required is US $ 3
per bp (the estimated cost in the beginning),
* total estimated cost of the project would be
approximately 9 billion US dollars
2. If obtained sequences were to be stored in
typed form in books,
* and if each page of the book contained 1000
letters
* and each book contained 1000 pages,

* then 3300 such books would be required to store
the information of DNA sequence from a single
human cell.
3. The enormous amount of data expected to be
generated also necessitated the use of high speed
computational devices for data storage and
retrieval, and analysis.
* HGP was closely associated with the rapid
development of a new area in biology called as
Bioinformatics (use of high speed computational
devices for data storage and retrieval, and
analysis)
Goals of HGP :
(i) Identify all 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.
Many non-human model organisms have also
been sequenced -
1.Bacteria
2.Yeast
3.Caenorhabditis elegans (a free living non-patho-
genic nematode)
4.Drosophila (the fruit fly)
5.Plants (rice and Arabidopsis)
Methodology :
* Two approaches have been recognized for
analysing human genome
(i) ESTs or Expressed Sequence Tags :
* To identify all the genes that are expressed as
RNA.
(ii) Sequence annotation :
* Sequencing both coding and noncoding regions of
whole genome and later assigning different
segions in the sequence with functions
* Blind approach
* For sequencing,
1.Total DNA from a cell is isolated
2.Then converted into random fragments of
relatively smaller sizes (recall DNA is a very long
polymer, and there are technical limitations in
sequencing very long pieces of DNA)
3. Then , Cloned in suitable host using specialised
vectors.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 25
* The cloning resulted into amplification of each
piece of DNA fragment so that it subsequently
could be sequenced with ease.
* Commonly used hosts- Bacteria & Yeast
* Vectors-
1. BAC (bacterial artificial chromosomes)
2. YAC (yeast artificial chromosomes
4. Fragments were sequenced using Auto-
mated DNA sequencers:-
* that worked on the principle of a method devel-
oped by Frederick Sanger.
* Sanger is also credited for developing method for
determination of amino acid sequences in pro-
teins.
5. These sequences were then arranged based on
some overlapping regions present in them.
* This required generation of overlapping frag-
ments for sequencing.
* Alignment of these sequences was humanly not
possible.
* Therefore, Specialised Computer Based Pro-
grams were developed . These sequences were
subsequently annotated and were assigned to
each chromosome
* Another challenging task was assigning the
genetic and physical maps on the genome.
* This was generated using information on poly-
morphism of restriction endonuclease recognition
sites & some repetitive DNA sequences known
as Microsatellites
* One of applications of polymorphism in repetitive
DNA sequences - DNA fingerprinting).

Salient features of Human Genome :
(i) The human genome contains 3164.7 million
nucleotide bases.(3.1647 Billion)
(ii) The average gene consists of 3000 bases,
* but sizes vary greatly
* largest known human gene being dystrophin at
2.4 million bases
* Smallest human gene-TDF (Testis Determining
Factor on Y-chromosome Y 14 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 % of
discovered genes
(v) < 2 % of 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, some-
times 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), & the Y
has the fewest (231)
(ix) Scientists have identified about 1.4 million
locations where single- base DNA differences
(SNPs – Single Nucleotide Polymorphism,
pronounced as “snips”) occur in humans
* This information helps in processes of finding
chromosomal locations for disease-associated
sequences
* and Tracing human history.
* DNAFingerprinting (DNA Profiling/ Typing/
Imprinting) Historical Aspect :
* Sir Alec jeffreys (1984) developed the DNA
fingerprinting technique.
* Dr.K.kashyap and Dr. Lalji Singh started the
fingerprinting technology in India.
* What is DNA-fingerprinting :
* It is a technique to identify a person on the basis
of his//her DNA specificity
* Technique of determining nucleotide sequences in
certain DNA segments , which is unique to an
individual.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 26
* DNA fingerprinting involves identifying differ-
ences 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.
* Bulk DNA forms a major peak and the other
small peaks are referred to as satellite DNA.
* Depending on base composition (A : T rich or
G:C rich), length of segment & number of
repetitive units , satellite DNA is classified into
many categories, such as micro-satellites, mini-
satellites etc.
* These sequences normally don¡¦t code for any
proteins, but they form a large portion of human
genome.
* These sequence show high degree of polymor-
phism & form basis of DNA fingerprinting.
* Since DNA from every tissue (such as blood,
hair -follicle, skin, bone, saliva, sperm etc.), from
an individual show the same degree of polymor-
phism, they become very useful identification tool
in forensic applications.
* Further, as polymorphisms are inheritable from
parents to children, DNA fingerprinting is the
basis of paternity testing, in case of disputes.
* 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.
* New mutations may arise in an individual either
in somatic cells or in the germ cells (cells that
generate gametes in sexually reproducing
organisms).
* If a germ cell mutation does not seriously impair
individual's ability to have offspring who can
transmit the mutation, it can spread to the other
members of population (through sexual reproduc-
tion).
* 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.

* The probability of such variation to be observed
in non- coding DNA sequence would be higher
as mutations in these sequences may not have
any immediate effect/impact in an individual¡¦s
reproductive ability.
* These mutations keep on accumulating genera-
tion after generation, and form one of the basis of
variability/polymorphism.
* There is a variety of different types of polymor-
phisms ranging from single nucleotide change to
very large scale changes.
* For evolution and speciation, such polymorphisms
play very important role.
* 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 11-60 bp long)/Minisatellites
* The technique, as used earlier, involved Southern
blot hybridisation using radiolabelled VNTR as a
probe.
(i) isolation of DNA
(ii) digestion of DNA by restriction endonucleases
(iii) separation of DNA fragments by electrophoresis
(iv) transferring (blotting) of separated DNA frag-
ments to synthetic membranes, such as nitrocel-
lulose or nylon
(v) hybridisation using labelled VNTR probe
(vi) detection of hybridised DNA fragments by
autoradiography.
* The VNTR belongs to a class of satellite DNA
eferred to as mini-satellite.
* A small DNA sequence is arranged tandemly in
many copy numbers.
* The copy number varies from chromosome to
chromosome in an individual.
* The numbers of repeat show very high degree of
polymorphism.
* As a result the size of VNTR varies in size from
0.1 to 20 kb.
* Consequently, after hybridisation with VNTR
probe, the autoradiogram gives many bands of
differing sizes.
* These bands give a characteristic pattern for an
individual DNA
* It differs from individual to individual in a popula-
tion except in the case of monozygotic (identical)
twins.
* The sensitivity of the technique has been in-
creased by use of polymerase chain reaction.
Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005
Molecular Basis of Inheritance 27
* Consequently, DNA from a single cell is enough
to perform DNA fingerprinting analysis.
* In addition to application in forensic science, it
has much wider application, such as in determin-
ing population and genetic diversities.
* Currently, many different probes are used to
generate DNA fingerprints.
Technique of DNA fingerprinting :
(i) The DNA is isolated from the nuclei of white
blood cells or spermatozoa or the hair follicle
cells.
(ii) Restriction endonuclease enzyme performs
digestion of DNA molecules. The former cuts
DNA in to fragments. The fragments of DNA
also contain the VNTRs.
(iii) Gel electrophoresis is used to separate these
fragments according to their size
(iv) VNTRs are multiplied through PCR technique.
The VNTRs are treated with alkaline chemicals
to split them into single stranded DNAs.
(v) SS DNA fragments of the gel are shifted onto a
nylon paper/nitrocellulose membrane by Southern
blotting technique.
(vi) Radioactive SS-DNA-probes are used for
hybridization with VNTR on the nylon mem-
brane.
(vii) Now the nylon membrane is exposed at the front
of X-ray film and mark the places where the
radioactive DNA probes have bound to the DNA
fragments. These places are marked as dark
bands when X-ray film is developed. This is
known as Autoradiography.
(viii) The dark bands on X-ray film show the DNA
fingerprints.
Applications of DNA Fingerprinting :
(i) It is very useful in the detection of crime and
illegal pursuits.
(ii) Paternity maternity disputes can be solved by
DNA fingerprinting.
(iii) It can be used to study the breeding patterns of
animals facing the danger of extinction.
(iv) It provides information about relationship of man
with apes.
(v) It determining population and genetic diversities.
 Molecular Basis of Inheritance 28

Schematic representation of DNA fingerprinting :

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

Head Office :- Plot No. 46, In front of Skyline Apartments, Corner Building, Rajeev Gandhi nagar, Kota (Raj.) Pin code : 324005