6.

MOLECULAR BASIS OF
INHERITANCE
 DEOXYRIBONUCLEIC ACID
(DNA)
• DNA is the long polymer of deoxyribonucleotide.
• Its length is defined as the number of nucleotide or base pairs.
• The number of base pairs is the characteristics of every organisms.
• Ex.Ф 174 ----5386 bp
• Lambda phage----48502 bp
6
• Escherichia coli ----4.6X1O bp
9
• Human being------------3.3x 10 bp(haploid cell)
• DNA was discovered by FREDERICH MEISCHER(1869) as an acidic
substance in the nucleus , he called it nuclein.
FREDERICH MEISCHER.
 Structure of polynucleotide chain of
DNA.
• DNA is the largest macromolecule made of helically
twisted, two, antiparallel polydeoxyribonucleotide
chains held together by hydrogen bonds.
• X-ray diffraction pattern of DNA by Rosalind Franklin
showed DNA a helix.
• Components of DNA are (i) deoxyribose sugar, (ii) a
phosphate, and (iii) nitrogen containing organic bases.
• DNA contains four different bases called adenine (A),
guanine (G) cytosine (C), and thymine (T).
• These are grouped into two classes on the basis of
their chemical structure: (i) Purines (with a double ring
structure) and (ii) Pyrimidines (with a single ring
structure)
• 1953.James Watson and Francis Crick
proposed three dimensional structure of DNA
• DNA double helix with sugar phosphate back
bone on outside and paired bases inside.
• Planes of the bases perpendicular to helix axis.
• Each turn has ten base pairs.( 34 A0)(3.4nm)
• Diameter of helix 20 A0
• Ten base pairs in each turn with 0.34nm between two base pairs.
• The length of DNA in E.coli-----------1.36 mm
• The length of DNA in Man -----------2.2m
• Two strands of DNA antiparallel.
• DNA found both in nucleus and cytoplasm.
• Extranuclear DNA found in mitochondria and
chloroplasts.
• Two chains complementary
• Two chains held together by hydrogen bond.
• Adenine-Thymine pair has two hydrogen bonds.
• Guanine-Cytosine pair has three hydrogen bonds.
• Upon heating at temperature above 80-90 degree two
strands uncoil and separate (Denaturation)
• On cooling two strands join together (renaturation
/annealing)
• DNA is mostly right handed and B form.
• Bacterial nucleoid consists of a single circular DNA
molecule .
•
 PACKAGING OF DNA HELIX
• DNA of eukaryotes is wrapped around positively
charged histone proteins to form nucleosome.
• # Nucleosome contains 200 base pairs of DNA
helix.
• # Histone octamer =2(H2a+H2b+H3+H4)
• # Linker DNA bears H1 protein
• # Chromatin fibres formed by repeated units of
nucleosomes.
• # Non histone proteins required for packaging.
• # Regions of chromatin, loosely packed and stains
lightly called euchromatin.
• # Regions of chromatin, densely packed and
stains darkly is called heterochromatin.
 DNA AS THE GENETIC MATERIAL
• Transformation experiment or Griffith effect.
• • Griffith performed his experiments on Mice using
Diplococcus pneumoniae.
• • Two strains of bacteria are S-type and R-type cells.
• • Experiments
• Living S-strain Injected into mice →Mice killed
• Living R-strain Injected into mice → Mice lived
• Heat Killed S-strain Injected into mice → Mice lived
• Living R-strain + Heat Killed S-strain Injected into
mice→ Mice killed
• # Griffith concluded that R type bacteria is transformed
into virulent form.
• # Transformation is the change in the genetic
constitution of an organism by picking up genes
present in the remains of its relatives.
F.GRIFFITH
• BIOCHEMICAL CHARACTERISATION OF
TRANSFORMING PRINCIPLE
• # Proved by Oswarld Avery, Colin Macleod,
Maclyn Mc Carty
• From this we conclude that DNA is the
genetic material.
 Alfred Hershey and Martha Chase
Experiment(1952)
• They made two different preparation of bacteriophage , in
one the DNA was made radioactive with 32P AND the
other the protein coat was made radioactive with 35S.
• These two preparation were allowed to infect the bacterial
cell separately.
• Soon after the infection the cultures were gently agitated in
a blender to separate the adhering protein coats of the
virus from the bacterial cell.
• The culture centrifuged to separate the viral coat and the
bacterial cells
• It was found that when the phage containing radioactive
DNA was used to infect bacteria, its radioactivity was found
in the bacterial cells(in the sediment) indicating that the
DNA has been injected into the bacterial cell
• So DNA is the genetic material not proteins.
Characteristics of genetic material
• Genetic material must have the following
properties.
• It should be able to generate its own replica.
• It should be chemically and structurally stable.
• It should provide the scope for slow
changes(mutation) that is necessary for
evolution.
• It should be able to express itself in the form
of Mendelian characters
• Nucleic acid can replicate but not protein.
• Major genetic material is DNA, but viruses
like TMV have RNA as the genetic material.
• The 2’ –OH in the nucleotide of RNA is a
reactive group and make the RNA liable and
easily degradable, RNA is more reactive and
hence DNA has the property to be the genetic
material.
 Semi conservative nature of DNA
Mathew Messelson and Franklin stahl
• E.coli
• Grown on 15 NH4Cl culture medium
• Both strands of DNA have 15N (N15 N 15)
• Shifted to 14NH4Cl culture medium
• DNA extracted subjected to CSCl density gradient
centrifugations
• After 20 minutes- Hybrid/ Intermediate type of DNA
(N15 N14)
• After 40 minutes -Equal amount of light DNA (N14 N14)
and hybrid DNA (N15 N14)
 Replication of DNA In Eukaryotes
• Definition: "Process by which DNA produces daughter
DNA molecules which are exact copies of the original
DNA.“
• In eukaryotes, DNA is double stranded. The two
strands are complementary to each other because of
their base sequences.
• Semi-conservative method of DNA replication
Important points:
• i) Most common method of DNA replication.
• (ii) Takes place in the nucleus where the DNA is present
in the chromosomes.
• (iii) Replication takes place in the S-phase (synthesis
phase) of the interphase nucleus.
• (iv) Deoxyribose nucleotides needed for formation of
new DNA strands are present in nucleoplasm
• At the time of replication, the two strands of DNA
first separate.
• Each strand then acts as a template for the
formation of a new strand.
• A new strand is constructed on each old strand,
and two exactly identical double stranded DNA
molecules are formed.
• In each new DNA molecule, one strand is old
(original) while the other is newly formed.
Hence, Watson and Crick described this method
as semi-conservative replication.
• An overall process of DNA replication showing
replication fork and formation of new strands
template and lagging template.
• The various steps involved in this process are
summarized as follows:
• i. Mechanism of replication starts at a specific point of
the DNA molecule, called origin.
• ii. At origin, DNA strand breaks because of an incision
(nick). This is made by an enzyme called incision
enzyme (endonuclease).
• iii. The hydrogen bonds joining the two strands are
broken by the enzyme.
• iv. The two strands start unwinding. This takes place
with the help of a DNA unwinding enzyme Helicases.
Two polynucleotide strands are thus separated.
• v. The point where the two strands separate appears
like a fork or a Y-shape. This is described as a
replicating fork.
• vi. A new strand is constructed on each old
strand.
This takes place with the help of a small RNA
primer molecule which is complimentary to the
DNA at that point.
• vii. Each old DNA strand acts as a template (site)
for the construction of new strand.
The RNA primer attaches itself to the old strand
and attracts the enzymes(DNA polymerase III)
which add new nucleotides through base
complementation.
• The deoxyribose nucleotides are present in the
surrounding nucleoplasm. New DNA strand is
thus constructed opposite to each old strand
• viii. Formation of new complementary strand
always begins at the 3' end of the template
strand (original strand) and progresses
towards the 5' end (i.e in 3' - 5' direction).
Since the new strand is antiparallel to the
template strand, it is obvious that the new
strand itself is always developed in the, 5'-3'
direction.
For this reason when the two original strands
separate (then with respect to the origin of
separation), one acts as 3'-5' template while
the other acts as 5'- 3' template.
• ix. Of the two, the replication of 3'-5' template begins first.
• Hence the new strand formed on it is called the leading
strand.
• The other template (5'-3') must begin replication at the
fork and progress back toward the previously transcribed
fragment.
• The new strand formed on it is called the lagging strand.
• x. Replication of the lagging strand takes place in small
fragments called Okazaki fragments.
• These are then connected together by the enzyme ligase.
• xi. Replication may take place in only one direction on the
DNA helix (unidirectional) or in two directions
(bidirectional).
• xii. At the end of the process, two double stranded DNA
molecules are formed from the original DNA molecule.
 Central dogma of molecular biology
Transcription
Translation
DNA RNA PROTEIN.
RNA WORLD .
RNA was the first genetic material.
There are three types of RNA
1. Messenger RNA(m RNA ). –It bring the genetic information
of DNA transcribed on it for protein synthesis. It is single
stranded.
2. Transfer RNA/soluble RNA. It act as an adaptor molecule
that reads the code on one hand and bind to the specific
amino acid on the other hand.
3. tRNA has a clover leaf like secondary structure
It has an amino acid acceptor end at 3’ and an “
anticodon-loop,where the three bases are complimentary
to the bases of the codon of the purticular aminoacid.
4. Ribosomal RNA (r RNA ) –It forms the structure of ribosomes.
Clover leaf model of t RNA
• ANTICODON :- The sequence of nitrogenous
bases on RNA that is complementary to the
codon for particular amino acid.
• CODON :- It is a sequence of three
nitrogenous bases on m-RNA that code for a
particular amino acid.
Transcription unit
• TEMPLATE STRAND
• 1. The DNA strand that has the polarity 3‘→5‘ acts as
template during transcription is called as template
strand.
• 2. It is also called as master strand or (-) or sense
strand.
• 3. This takes part in transcription.

• CODING STRAND
• 1. The strand which has polarity of 5‘→3‘ is called as
codon strand.
• 2. It is called (+) because genetic code present in this
strand is similar to genetic code (based on mRNA).
Thymine is replaced by uracil in m RNA.
• 3. This does not take part in transcription.
 Transcription in Prokaryotes
• In prokaryotes the structural genes are polycistronic and continuous.
• In prokaryotes there is a single DNAdependent RNA polymerase,that catalyse the
transcription of all the three types of RNA( m RNA, t RNA , r RNA ).
Initiation.
• RNA polymerase binds to the promoter and initiates the process along with certain
initiating factors.
• It uses ribonucleoside triphosphate for polymerisation on a DNA.
Elongation.
• The enzyme facilitates the opening of the DNA-helix and elongation continues.
Termination.
• Once the RNA polymerase reaches the terminator , the RNA polymerase falls
off and the nascent RNA separates. It is called termination of transcription.
• It is facilitated by certain termination factors
• In prokaryotes m RNA SYNTHESISED does not require any processing to become
active.
• Both transcription and translation occur in the same cytosol.
• Transcription and translation can be coupled(translation can start much before the
m RNA is fully transcribed.)
Transcription in Prokaryotes
 Transcription in Eukaryotes
• In eukaryote the structural genes are monocistronic
and split.
• They have coding sequences called EXONS that forms
part of a m RNA and non coding sequences called
INTRONS, that do not form the part of m RNA and are
removed during splicing.
• In Eukaryotes three different RNA polymerases.
• 1.RNA polymerase-I – Transcribes r RNAs
• 2.RNA polymerase-II– Transcribes the precursor of m
RNA (HETEROGENOUS NUCLEAR RNA(hn RNA).
• 3.RNA polymerase –III- It catalyses the transcription of
t RNA.
• SPLICING :- The process in eukaryotic genes by
which the introns are removed and the exons
are joined together to form m-RNA.
• The hn RNA undergoes two additional processes
called capping and tailing
• In capping ,methyl guanosine triphosphate is
added to the 5’ end of hn RNA .
• In tailing ,adenylate residues(200-300) are
added at the 3’ end of hn RNA.
• The fully processed hn RNA is called m RNA and
is released from the nucleus into the cytoplasm.
Transcription in Eukaryotes
 GENETIC CODE
• Salient features of genetic code.
• Universal –the codons code for any aminoacid in any organism,be it
a bacterium or a human being.
• Non ambiguous-each codon codes for only one aminoacid, so the
genetic code is unambiguous and specific.
• Comma less-the codons are read in 5’3’ direction and no
punctuation.
• Degenerate – some amino acids are coded by more than one
codon , the genetic code is said to be degenerate.
• Non overlapping-3 successive nitrogen bases code for only one
aminoacid.
• Nonsense codon (UAA,UAG,UGA)-do not code for any amino acids,
but act as terminating/stop codons of protein synthesis.
• Linear-the sequence of amino acids present in a polypeptide chain
corresponds to sequence of nitrogen bases of DNA with 3
successive nitrogen bases forming a single codon.
• Triplet-there are 64(4x4x4) codons,61 codons code for 20
aminoacids.
• Initiation Codon AUG
 Protein synthesis.
• 1.Activation of amino acids
AA+ATP+E Mg+2 AA-AMP-E+ PPi
AA-AMP-E+tRNA AA-tRNA+AMP+E
• 2. Initiation
Small subunit (40s) of ribosome binds with mRNA.
Charged t RNA specific for initiation codon reaches P site
Larger subunit (60s) of ribosome now combines with 40s-m RNA—t
RNA met complex in the presence of Mg+2
• 3. Elongation
• Second t-RNA charged with amino acid occupies A site of ribosome.
• Peptide bond formation between methionine and second amino acids
with the help of enzyme peptide transferase.
• Ribosomes moves over m RNA in 5’3’
• 4. Termination
• Translation stops when non sense codons (Stop codons) reached.
• No t RNA for stop codons (UAA,UAG,UGA)
• Synthesized polypeptide is released with the help of release factor.
•
• * AA—Amino acid
• *ATP—Adenosine Triphosphate
• *E—Pyrophosphate
• AA—AMP-E-Amino acid adenylate enzyme
complex
• AA—t RNA—Amino acyl-t RNA complex
•
 OPERON
• OPERON :- All the genes controlling a
metabolic process constitute an operon.
• Discovered by Jacob and Manod.
• *Experimented on E.coli.
 HUMAN GENOME PROJECT (HGP)
• STEPS
• Sequence annotation (Sequence the whole set of genome)
• -Isolation of total DNA from the cell
• -Fragmentation by restriction endonuclease
• Fragments cloned in suitable host BAC/YAC
• Fragments sequenced using automated DNA sequences.
• Sequences arranged on the basis of overlapping regions.
• Alignment of the sequences by specialized computer based
programmes
• Expressed sequence Tags (EST) (Identifying all the genes
Expressed as RNA)
 Salient features of Human Genome

1. Functions of 50% discoursed genes unknown

2.Repetitive sequences contribute large portion
3.Largest gene dystrophic
4.3164.7 millions Nucleotides
5.Average gene consists of 3000 bases
6.Total genes 30,000
7.<2% gene codes protein
8.Chromosome-1 has 2968 genes
9.Y chromosome has 231 genes
Application of Human genome project
• Identification of defective genes.
• -: Opportunity to offer early treatment.
• -: Identification of genes that confer
susceptibility to certain disease.
• -: Prediction of protein that the genes
produce.
• -: Drug designing to enhance or inhibit the
activities of the proteins.
 TECHNIQUE FOR DNA FINGER PRINTING

• Technique developed by Dr.Alec Jeffreys.

• Process is also known as DNA typing/DNA profiling.
• DNA extraction from the cells in high speed refrigerated centrifuge.
.DNA extraction from the cells in high speed refrigerated centrifuge
• Amplification of DNA content by PCR (Polymerase chain reactions)
• DNA fragmentation by Restriction endonuclease
• Gel electrophoresis
• Double stranded DNA split into single stranded
• Southern blotting (Transferring separated DNA to nylon or
nitrocellulose sheet)
• Nylon sheet immerse in a bath having probes/marker*
(Hybridisation)
• Nylon membrane pressed on X-ray film (Autoradiography)
• Dark band develops at probe site
• *Probes/ Markers are radioactive synthetic DNA complementary to
VNTR
 DEFINITIONS
• ANTICODON :- The sequence of nitrogenous
bases on RNA that is complementary to the
codon for particular amino acid.
• BACTERIOPHAGE :- A virus that infects a
bacterium.
• CODON :- It is a sequence of three nitrogenous
bases on m-RNA that code for a particular amino
acid.
• CONSTITUTIVE GENES :- Constitutive genes are
those genes which are constantly expressed &
whose products are continuously needed for
cellular activity.
• DNA POLYMORPHORISM :- Refers to the
variations at genetic level where an
inheritable mutation is observed in a
population in a frequency greater than 0.01.
• EXON :- The regions of a gene which become
part of m-RNA & code for the different
regions of proteins.
• FRAME SHIFT MUTATION :- A type of
mutation where addition or deletion of one
or two bases changes the reading frame from
the site of mutation, resulting in a protein
with a different set of amino acids.
• GENE :- Segment of DNA that code for
RNA/functional unit of heredity.
• INTRONS :- The regions of a gene which do not
form part of m-RNA and are removed.
• NUCLEOSOME :- Structure formed when
negatively charged DNA is wrapped around the
positively charged histone octamer.
• OPERON :- All the genes controlling a metabolic
process constitute an operon.
• ORIGIN OF REPLICATION :- It is the definite
region of DNA where replication originates/
starts.
• REPLICATION FORK :- The Y- shaped structure
formed when the double standard DNA is
unwound up to a point during its replication
• SATELLITE DNA :- The repetitive DNA sequences which do
not code for any protein, but form a large portion of
human genome; and show high degree of
polymorphorism.
• SILENT MUTATION :- Mutation which do not cause any
change in protein.
• SPLICING :- The process in eukaryotic genes by which the
introns are removed are the exons are joined together to
form m-RNA.
• TRANSCRIPTION :- It is the process of formation of RNA
from DNA.
•
• TRANSFORMATION :- It is the phenomenon by which the
DNA isolated from one type of cell, when introduced into
another type is able to bestow some of the properties of
the former to later.
• TRANSLATION :- It is the process of polymerization of
amino acids to form a polypeptide dictated by mRNA
 Differences
• DNA
• 1. It usually occurs inside nucleus and some cell organelles.
• 2. DNA is a genetic material.
• 3. It is a double stranded with the exception of some viruses (rabies,
AIDS etc.)
• 4. DNA contains over a million nucleotides.
• 5. It contains deoxyribose sugar.
• 6. Nitrogen bases thymine occurs in DNA along with three others-
adenine, cytosine and three guanine.
• RNA
• 1. Very little RNA occurs inside nucleus. Most it is found in the cytoplasm.
• 2. RNA is not a genetic material except in certain viruses, e.g., Reovirus.
• 3. It is a single stranded with the exception of some viruses(e.g., double
stranded in Reovirus)
• 4. Depending upon the type, RNA contains 70-12000 nucleotides.
• 5. It contains ribose sugar.
• 6. Thymine is replaced by uracil in RNA. The other are similar – adenine,
cytosine and guanine.
•
• PROCARYOTIC TRANSCRIPTION

• 1. It occurs in contact with cytoplasm.

• 2. Products of transcription become effective in situ.
3. There is only one RNA polymerase.
4. RNA polymerase does not have separate transcription factors.
5. mRNA is generally polycistronic.
6. Splicing is generally not required.
• EUKARYOTIC TRANSCRIPTION
• 1. It occurs inside the cytoplasm.
• 2. Products of transcription come out of the nucleus for functioning
in cytoplasm.
• 3. There are three types of RNA polymerase.
• 4. Transcription factors are involved in recognition of promoter site.
• 5. mRNA is generally monocistronic.
• 6. In most of the cases splicing required for removing intervening
sequences
•
• LEADING STRAND
• 1. It is a replicated strand of DNA which grows continuously without any
gap.
• 2. It does not require DNA ligase for its growth.
• 3. The direction of growth of the leading strand is 5‘→3‘
• 4. Only a single RNA primer is required.
• 5. Its template opens in 3‘→5‘ direction.
• 6. Formation of leading strand begins immediately at the beginning of
replication.
• LAGGING STRAND
• 1. Lagging strand is a replicated strand of DNA which is formed in short
segment called discontinuous.
• 2. DNA ligase is required for joining Okazaki fragments.
• 3. The direction of growth of the lagging strand is 3‘→5‘though in each
Okazaki fragment it is 5‘→3‘.
• 4. Starting of each Okazaki fragment requires a new RNA.
• 5. Its template opens in 5‘→3‘ direction
• 6. Formation of lagging strand begins a bit later than that of leading
strand.
•
 QUESTIONS
• 1. There are proteins which are positively charged
and there are also negatively charged proteins.
What makes the protein get its charge
• 2. What is ESTs? 3. A particular human gene has
the largest number of bases. Identify it
• . 4. Why is mRNA of eukaryotic cells said to
monocistronic, while that of prokaryotic cell is
polycistronic?
• 5. A point mutation leads to adverse change in
the function of hemoglobin (B-globin chain).
Identify the disease that may occur due to this
mutation. Mention the change of amino acids in
the polypeptide due to mutation
• 6. Two persons filed a case against a lady
claiming to be the father of her only daughter.
How to find the real biological father
• 7. If a nucleosome contains 200bps, how
many nucleosome are there in a mammalian
cell? What changes occur to beads of strings
of DNA during metaphase?
• 8. Write the mRNA transcribed from the DNA
segment with the base sequence TAC TAG TCG
ACT. How many amino acids will there be in
the oligopeptide translated by the mRNA?
Why?
• 9. Lac operon is negatively regulated. What is
meant by this? Why is lactose called the
inducer of lac operon in E.coli?
• 10. (i) Describe the two major approaches to
sequencing of genomes? (ii) Expand SNPs.
What are they? (iii) Explain VNTR as the basis
of DNA fingerprinting?
• 1. The two strands of DNA have antiparallel
polarity. What does it mean?
• 2. DNA fingerprinting is a technique to find out
variations at DNA level among individuals of
population. What is the principle on which it
works?
• 3. What term is given to the flow of information
from RNA to DNA in certain viruses?
• 4. A criminal case is 10 years old was registered
for investigation. What samples they might have
tested?
• 5. Pick out the untransalated regions from the
given mRNA. 5‘ ACG UCG AUG GCG CCC UUU
UAG GAG GAA 3‘ Where are they normally
located
.

only ONE MARK QUESTION

• 1.Name the genetic material in TMV.
• 2.Write the scientific name of the plant on which
Taylor et al performed their experiment.
• 3.What would be the proportion of light and hybrid
density DNA molecules after 80 minutes of a
single cell of E. coli growth?
• 4.When does DNA replicate in the cell cycle ?
• 5.Name the amino acids having one codon.
TWO MARK QUESTION
1.What is meant by semiconservative nature of DNA
replication?
2. What are the functions of DNA polymerase?
3. What is frame shift mutation ?Name the type of
mutation that does not affect protein synthesis .
4.What are the untranslated regions (UTRs) ?
5.Briefly describe polymorphism
THREE MARK QUESTIONS
1.Describe the discontinuous synthesis of DNA.
2. How is Lac operon “switched on” in an E.coli cell ?
3.Name the three RNA Polymerases found in eukaryotes
and mention their functions.
4.Explain the two major approaches involved in the
sequencing of genomes.
FIVE MARKS QUESTIONS
1.Describe the salient features of the double helical model
of DNA.
2. Bring out the salient features of genetic code .
3.Describe in detail the steps in the technique of DNA
finger printing.
4.Describe the process of replication of DNA.
5. What is satellite DNA ?Name their types. Mention their
basis for the classification of satellite DNA