Molecular Basis of Inheritance-1

2020
Molecular Basis of
Inheritance
Mr. Nandhan HD
Assistant Professor of Biosciences
+91-9535547049
IIPUC/NEET/AIIMS
12/7/2020
PU-II-BIOLOGY: MOLECULAR BASIS OF INHERITANCE
PU-II-BIOLOGY CHAPTER-6
MOLECULAR BASIS OF INHERITANCE
DNA AS GENETIC MATERIAL

In 1869, Johann Friedrich Miescher, a young Swiss Medical student, became fascinated
with an acidic substance that he isolated from pus cells obtained from bandages used to dress
human wounds. Miescher recovered that acidic substance and called as “nuclein’ which
contained a large amount of both Nitrogen and Phosphorous.
The importance of the substance that Miescher called nuclein was not documented until
1940. The role of nucleic acid in storing and transmitting genetic information was not
established until 1944 and the double helix structure of DNA was not discovered until 1953.
Even in 1953 many geneticists were reluctant to accept the idea that nucleic acids, rather than
proteins carried the genetic information, because nucleic acids exhibited less structural
variability than proteins.
When it became evident that the chromosomes were the organs of heredity, further
attempts to discover the chemical basis of heredity focused on the molecules present in
chromosomes. Extensive chemical analysis of chromosomes revealed that chromosomes contain
proteins and nucleic acids (DNA and RNA).
Early molecular geneticists have assigned the informational roles of genes to chromosomal
proteins, because they found nucleic acids too simple to carry out genetic information.
Around 1953, it was universally accepted that DNA is the genetic substance of most
microorganisms. According to molecular biologists certain requirements must be met by a
molecule if it is to be qualified as the substance that transmits genetic information from one
generation to the next.
 The genetic material must store genetic information and accurately transmit that
information from parents to the offsprings, generation after generation.
 The genetic material must contain biologically useful information that is maintained in a
stable form.
 The genetic material must control the development of the phenotype of the organisms, i.e.,
the genetic material must dictate the growth and differentiation of the organism from the
single celled zygote to the mature adult.
 The genetic material must undergo changes so that the organisms can adapt to
modifications in the environment without such changes, evolution could not occur.
Several lines of indirect evidences suggested that DNA harbors the genetic information of living
organisms:
1. Most of the DNA of cells is located in the chromosomes whereas RNA and proteins are
abundant in cytoplasm.
Mr.Nandhan HD, Assistant Professor of Biosciences Page 1

2. A precise correlation exists between the amount of DNA per cell and the number of sets of
chromosomes per cell.
3. The molecular composition of the DNA is the same (with rare exceptions) in all the
different cells of an organism, whereas the composition of both RNA and proteins is highly
variable from one cell type to another.
4. DNA is more stable than RNA or proteins, which are synthesized and degenerated quite
rapidly in living organisms. Since the genetic material must store and transmit
information from parents to offsprings, we might expect it to be stable, like DNA.
Although these correlations strongly suggest that DNA is the genetic material, they by no
means prove it.
DIRECT EVIDENCES FOR DNA AS THE GENETIC MATERIAL
The most conclusive evidences in support of DNA as the genetic material came from the
following avenues of approach on microorganisms:
THE TRANSFORMATION EXPERIMENT:
While chemists were working out the structure of DNA, biologists were attempting to
identify the source of genetic information. Two sets of experiments, one conducted on bacteria
and the other on viruses, provided evidence that DNA rather than protein was the genetic
material.
The discovery of the transformation principle:
 The first clue that DNA was the carrier of hereditary information came with the
demonstration that DNA was responsible for a phenomenon called Transformation.
 The phenomenon was first observed in 1928 by Frederick Griffith, English Physician
whose special interest was the bacterium that causes pneumonia, Streptococcus
pneumoniae or pneumococcus.
 Griffith had succeeded in isolating several different strains of S. pneumoniae (type I, II, III
and so forth).
 In the virulent forms (disease causing) of strain each bacterium is surrounded by a
polysaccharide coat which makes the bacterial colony appear smooth when grown on agar
plates; these forms are referred as ‘S’, for smooth.
 The avirulent form (non- pathogenic) of strains on the other hand lacks the capsule/coat
of polysaccharide and produces a rough-appearing colony on agar plate; these forms are
referred to as ‘R’, for rough.
 Both R and S forms occur in several types such as, R-I, R-II, R-III, etc and S-I, S-II, S-III,
etc respectively.

Colonies of rough (R, the small colonies) and smooth (S, the large colonies) strains of
Streptococcus pneumoniae. The S colonies are larger because of the gelatinous capsule on the S
cells.
 All these sub types of S and R bacteria differ with each other in the type of antigen they
produce.
 In the course of his work Griffith injected the laboratory mice with live R-II pneumococci,
the mice suffered no illness because R-II strain was avirulent.
 When the mice were injected with virulent S-III pneumococci, the mice suffered from
pneumonia and died.
 However when he injected the heat killed S-III bacteria in mice, they did not suffer from
pneumonia.
 Griffith knew that boiling killed all the bacteria and destroyed their virulence hence heat
killed S-III bacteria did not cause pneumonia in mice.
 The results of these experiments were not unusual. However, Griffith got a surprise when
he infected his mice with a small amount of living type II R bacteria along with a large
amount of heat killed type III S bacteria.
 Because both II R bacteria and heat-killed type III S bacteria were non-virulent, he
expected the mice to live.
 Surprisingly, 5 days after the injections, the mice suffered from pneumonia and died.
 When Griffith examined blood from the heart of these mice, he observed live type III S
bacteria; further these bacteria retained their type III S characteristics through several
generations; so the infectivity was heritable.

 Griffith results had several possible interpretations, all of which he considered:

 First, it could have been the case that he had not sufficiently sterilized the type III S
bacteria and thus a few live bacteria remained in the culture. Any live bacteria injected
into the mice would have multiplied and caused pneumonia.
 Griffith knew that this possibility is unlikely, because he had used only heat-killed type III
S type bacteria in the control experiment, and they never produced pneumonia.
 Second interpretation was that the live type II R bacteria had mutated to virulent S form.
 Such a mutation would cause pneumonia in mice, but it would produce type II S bacteria,
not the type III S that Griffith found in the dead mice.
 Many mutations would be required for type II bacteria to mutate to type III bacteria and
the chance of all the mutations occurring simultaneously was impossibly low.
 Griffith finally concluded that the type II R bacteria had somehow been transformed,
acquiring the genetic virulence of the dead type III S bacteria.
 Griffith did not understand the nature of transformation he theorized that some
substance in the polysaccharide coat of the dead bacteria might be responsible. He called
this substance as “Transforming Principle”.
IDENTIFICATION OF THE TRANSFORMING PRINCIPLE:
(Avery-MacLeod-McCarty experiment)
The first direct evidence showing that the genetic material is DNA rather than protein or
RNA was published by Oswald Theodore Avery, Colin Munro MacLeod and Maclyn Mc Carty in
1944.

They showed that the component of the cell responsible for transformation (the ‘transforming
principle’) in Streptococcus pneumoniae is DNA.
Proving the complete purity of any macromolecule (DNA/RNA/Protein) substance was extremely
difficult in those days. May be DNA preparation contained few molecules of protein which were
responsible for he observed transformation.
[Purified DNA extracted from heat-killed S cells can convert some living R cells into S cells, but the
material may still contain undetectable traces of protein and/or RNA.]
But the most definitive experiment was performed by Avery, McLeod and Mc Carty to prove that
DNA was the transforming principle.
They succeeded in isolating and purifying the transforming substance, which showed a chemical
composition similar to that of DNA and quite different from that of proteins.
Different enzymes were used which had no effect on the transforming substance.
In separate experiments, the highly purified DNA from type III S cells was treated with the
enzymes-
1. Deoxyribonuclease (DNase), which degrades DNA.

2. Ribonuclease (RNase), which degrades RNA.
3. Proteases, which degrades proteins.

The DNA was then tested for its ability to transform the type II R cells to type III S. Only DNase
treatment showed the loss of transforming activity of DNA.
Although the molecular mechanism by which transformation occurs remained unknown, the
results of Avery and co-workers clearly established that the genetic information in
pneumococcus is present in DNA.
Geneticists now know that the segment of DNA in the chromosome of pneumococcus that carries
the genetic information specifying the synthesis of type III capsule is physically inserted into the
chromosome of the type II R recipient cell during the Transformation process.
THE HERSHEY-CHASE EXPERIMENT:

(The Blender Experiment)
 The second evidence implicating DNA as the genetic material resulted from a study of T2
virus conducted by Alfred Hershey and Martha Chase.
 T2 bacteriophage is a phage that infects the bacterium Escherichia coli.

 Phage reproduces by attaching to the outer wall of a bacterial cell and injecting its DNA
into the cell, where it replicates and directs the cell to synthesize phage protein.
 At the time of Hershey-Chase study Biologist did not understand the exact process of
reproduction in phage.
 Hershey and Chase designed a series of experiments to determine whether the phage
protein or the phage DNA is transmitted to in phage reproduction.
 To follow the fate of protein and DNA, they used radioactive isotopes of phosphorous and
sulphur.
 A radio isotope can be used as a tracer to identify the location of a specific molecule.
 DNA contains phosphorous but not sulphur, so Hershey and Chase used 32P to follow
phage DNA during reproduction.
 Protein contains sulphur (cysteine and methionine) but not phosphorous so they used 35S
to follow the protein.

 Hershey and Chase grew one batch of E.coli in a medium containing 32P and infected the
bacteria with T2 so that all the new phages would have DNA labelled with 32P.

 They grew a second batch of E.coli in a medium containing 35S and infected these bacteria
with T2 so that all these new phages would have protein labelled with 35S.
 They then infected separate batches of unlabelled E.coli with the 35S and 32P labelled
phages.
 After allowing time for the phages to infect the cells, they placed the E.coli cells in a
blender and sheared off the empty protein coats (ghosts) from the cell walls.
 They separated out the protein coats and cultured the infected bacterial cells. Eventually,
the cells burst and new phage particles emerged.
 When phage labelled with 35S infected the bacteria, most of the radioactivity was detected
in the protein ghosts.
 When new phages emerged from the cell, they contained almost no 35S.
 This result indicates that although the protein component of a phage is necessary for
infection, it does not enter the cell and is not transmitted to progeny phages.
 In contrast, when they infected bacteria with 32P labelled phages and removed protein
ghosts, the bacteria were still radioactive.
 After the cells lysed and new progeny phages emerged many of these phages emitted
radioactivity from 32P demonstrating that DNA from the infecting phages has been passed
on to the progeny.
 These results confirmed that DNA, not protein, is the genetic material of phages.

NUCLEIC ACIDS
Nucleic acids are the large complex biological molecules present in the nuclei of cells of all
living organisms. They are most important components of all living organisms and responsible
for the transfer of genetic information from generation to generation and also take part in the
synthesis of proteins or enzymes.
They were first discovered by a Swiss biochemist ‘F. Meischer’ in nucleus of pus cell and
he called it "nuclein". The term nucleic acid was coined by ‘Altman’.
Based on the nature of pentose sugar, ‘P.A. Levene’ classified the nucleic acids into 2
types, such as Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA).
Occurrence:
 DNA is found in all organisms except a few viruses.
 In the cell, greatest amount of DNA is concentrated in the nucleus.
 DNA also occurs in cell organelles like mitochondria and plastids.
 RNA is found in all living cells. In most plant viruses and few animal viruses it is the
genetic material.
Chemical composition:
 Nucleic acids are polymer of nucleotides. Each nucleotide contains three components.
 Nucleotides = Pentose sugar + Nitrogen base + Phosphate
Pentose sugar (S):

Two types of sugars are present in nucleic acids. RNA contains ribose sugar with chemical
empirical formula C5H10O5 and DNA contains deoxyribose sugar with general empirical formula
C5H10O4.
Nitrogenous Base (N.B):

There are 2 categories of nitrogenous bases purines and pyrimidines.
1. Pyrimidines – Consist of one pyrimidine ring. Skeleton of ring composed of two nitrogen
and four Carbon atoms. Ex: Cytosine (C), Thymine (T) and Uracil (U).
2. Purines - Consist of two rings i.e. one pyrimidine ring (2N + 4C) and one imidazole ring
(2N + 3C). Ex: Adenine (A) and Guanine (G).
Phoshoric Acid (P):

Each nucleotide contains a phosphate group (H 3PO4). It combines two nucleotides
together by formation of phosphodiester bond.
Nucleosides and Nucleotides:

 The nitrogenous base and pentose sugar together constitutes Nucleoside. They are linked
by covalent or glycosidic bond.
Ex: Adenosine, Guanosine, Cytidine, Uridine. (Ribose+Base).
Deoxy adenosine, Deoxy thymidine, Deoxy guanosine, Deoxy cytidine (Deoxyribose+Base).
 The combination of nucleoside and phosphate is nucleotide.
 A Nucleotide is a fundamental unit of nucleic acids formed by a combination of a pentose
sugar, a nitrogenous base and a phosphate molecule.
Note:
 Nitrogen base forms bond with first carbon of pentose sugar to form a nucleoside.
 Nitrogen of third place (N3) forms bond with sugar in case of pyrimidines while in purines
nitrogen of ninth place (N9) forms bond with sugar.
 Phosphate forms ester bond (covalent bond) with fifth Carbon of sugar to form a complete
nucleotide.
Kinds of nucleotides in DNA:

Deoxy adenylic acid (d-AMP - Deoxy adenosine monophosphate)
Deoxy guanylic acid (d-GMP - Deoxy guanosine monophosphate)

Deoxy cytidilic acid (d-CMP - Deoxy cytidine monophosphate)

Deoxy thymidylic acid (d-TMP - Deoxy thymidine monophosphate)
Kinds of nucleotides in RNA:

Adenylic acid (AMP – Adenosine monophosphate)
Guanylic acid (GMP – Guanosine monophosphate)
Cytidilic acid (CMP – Cytidine monophosphate)
Uridylic acid (UMP – Uridine monophosphate)
DNA
 Discovered by - Meischer
 DNA term was given by - Zacharis
 In DNA pentose sugar is deoxyribose sugar and four types of nitrogen bases A, T, G, C.
 Maurice Wilkins and Rosalind Franklin studied DNA molecule with the help of X-Ray
crystallography technique and indicated the possible helical shape of DNA molecule.
 With the help of this study, Watson and Crick (1953) proposed a double helix model for
DNA. For this model Watson, Crick and Wilkins were awarded by Noble Prize in 1962.

Important features of DNA double helix model are:

 DNA is composed of two polynucleotide chains that form a double helix like a spiral
staircase.
 The sugar-phosphate units of nucleotides form the backbone or the uprights while
nitrogen bases form the cross rungs or steps of the helix.
 The adjacent nucleotides in a strand are joined together by phosphodiester bonds.
 Both polynucleotide chains are complementary and antiparallel to each other.
 In both strand of DNA direction of phosphodiester bond is opposite. i.e. If direction of
phosphodiester bond in one strand is 3'→5' then it is 5'→3' in another strand.
 The diameter of the DNA molecule is 20Aº.
 One full turn of helix is called Gyre and it measures 34Aº.
 The distance between two base pairs is 3.4Aº. Thus each gyre accommodates 10 base
pairs.
 The two strands are held together regularly at their nitrogen bases by weak hydrogen
bonds.
 Adenine always pairs with Thymine by two weak hydrogen bonds. Guanine always pairs
with Cytosine by three weak hydrogen bonds. This type of specific base pairing is called as
complementary base pairing.
 Chargaff's rule of base equivalence states that in a double stranded DNA molecule the
amount of adenine is always equal to the amount of thymine and the amount of guanine
is always equal to the amount of cytosine. i.e., the number of purines is equal to the
number of pyrimidines.
Purine = Pyrimidine
[A] + [G] = [T] + [C]
 A   G  1
 T   C
 Watson and Crick described the structure of B-DNA molecule. It is a right handed helix.
Note:
1. Base ratio =
 A   G  1= constant for a given species.
 T   C
2. In a DNA, A + T > G + C  A – T type DNA. Base ratio of A – T type of DNA is more than
one. Ex: Eukaryotic DNA.
3. In a DNA, G + C > A + T  G – C type DNA. Base ratio of G – C type of DNA is less than
one. Ex: Prokaryotic DNA.
4. Melting point of DNA depends on G - C contents.
5. More G - C contents means more melting point.
6. Tm = Temperature of melting. Tm of prokaryotic DNA > Tm of Eukaryotic DNA

7. Out of two strand of DNA only one strand participates in transcription, it is called
Antisense strand/Non coding strand/Template strand.
8. Other strand of DNA which does not participate in transcription is called Sense strand/
Coding strand/Non template strand.
9. Denaturation and renaturation of DNA:
If a normal DNA molecule is placed at high temperature (80-90°C) then both
strands of DNA will separate from each other due to breaking of hydrogen bonds. It is
called denaturation of DNA.
When denatured DNA molecule is placed at normal temperature then both strand
of DNA attaches and recoils to each other. It is called renaturation of DNA.
10. Hyperchromicity - When a double stranded DNA is denatured by heating then denatured
DNA molecule absorbs more amount of light, this phenomenon is called hyperchromicity.
11. Hypochromicity - When denatured DNA molecule cools slowly then it becomes double
stranded and it absorb less amount of light. This phenomenon is called hypochromicity.
12. Configuration of DNA Molecule:
o Distance between sugars of two strands is 11.1Aº.
o Length of hydrogen bonds between nitrogen bases is 2.8-3.0Aº. Angle between
nitrogen base and C1 Carbon of pentose is 51º.
o Molecular weight of DNA is 106 to 109 dalton.
o In nucleus of eukaryotes the DNA is associated with histone protein to form
nucleoprotein. Histone occupies major groove of DNA at 30º angle.
o Bond between DNA and Histone is salt linkage (Mg+2).
13. DNA in chromosomes is linear while in prokaryotes, mitochondria and chloroplast it is
circular.
14. In  x 174 bacteriophage the DNA is single stranded and circular.
15. DNA molecule is Dextrorotatory while RNA molecule is Laevorotatory.

16. C - value = Total amount of DNA in a haploid genome of organism.
PACKAGING OF DNA HELIX: [Ultrastructure of Chromosomes: Nucleosome Model]

The human DNA in a cell contains 6.6 x 109 base pairs and its length is about 2.2 meters
(6.6 x 109 x 0.34 x 10-9m/bp). It is greater than the dimension of the nucleus (10-6). The long
polymer DNA is present in highly folded or packed form in the nucleus.
In prokaryotes the negatively charged DNA held with some positively charged proteins in a
region called nucleoid. Thus DNA forms large loops held by proteins.
In eukaryotes a positively charged proteins called histones held with DNA. Histone
contains amino acids lysine and arginine residues that carry positive charges in their side
chains. Eight histone molecules (two molecules each of H2A, H2B, H3 and H4) are organized to

form a structure called histone octamer. The negatively charged DNA is wrapped around the
positively charged histone octamer to form a complex called nucleosome. Each nucleosome is
wrapped by a DNA twice; the DNA that connects two nucleosomes is called linker DNA. H1
protein holds each nucleosomes tightly.
A typical nucleosome contains 200 base pairs of DNA helix. The polynucleosome thread
like stained structure in the nucleus is called chromatin. It appears like beads-on-string under
electron microscope. Thus nucleosomes are the structural and functional unit of chromatin
fibre. This nucleosome model was proposed by Roger Kornberg.
The chromatin fibers are further coiled and condensed at metaphase stage of cell division
to form chromosomes. The packaging of chromatin at higher level requires additional set of
proteins called Non-histone chromosomal (NHC) proteins. In a typical nucleus, some regions of
chromatin are loosely packed and lightly stained called euchromatin. The highly coiled and
darkly stained regions of chromatin are called heterochromatin. Euchromatin is said to be
transcriptionally active chromatin, whereas heterochromatin is inactive.

TYPES OF DNA:
On the basis of direction of twisting, there are two types of DNA.
1. Right Handed DNA- Clockwise twisting. Ex: The DNA for which Watson and Crick
proposed model was 'B' DNA.
DNA Helix Length No. of base pairs Distance between two pairs Diameter
'A' 28 A0 11 pairs 2.56 A0 23 A0
‘B' 34 A0 10 pairs 3.4 A0 20 A0
'C' 31 A0 9.33 pairs 3.32 A0 19 A0
'D' 24.24 A0 8 pairs 3.03 A0 19 A0
2. Left Handed DNA- Anticlockwise twisting. Ex: Z-DNA

It was discovered by Rich.
Phosphate and sugar backbone is zigzag.
Units of Z-DNA are dinucleotides (purine and pyrimidine are in alternate order)
Helix length – 45.6 A0
Diameter – 18.4 A0
No. of base pairs – 12 (6 dimers)
Distance between 2 base pairs – 3.75 A0
3. Palindromic DNA- Sequence of nucleotides same from both ends.
It was discovered by Wilson and Thomas

CC GG TA CC GG
GG CC AT GG CC

CENTRAL DOGMA OF MOLECULAR BIOLOGY
The unidirectional flow of genetic information from the DNA to the proteins through mRNA
is called Central Dogma of Molecular Biology.
It was proposed by Francis Crick.
D.N.A. REPLICATION
 The process of formation of new DNA molecule from the parental DNA is called replication
or duplication.
 DNA is the only molecule capable of self-duplication so it is termed as a "Living molecule".
 All living beings have the capacity to reproduce because of this characteristic of DNA.
 At the time of cell division, it divides in equal parts in the daughter cells.
 DNA replication helps to maintain same amount of DNA in daughter cells as in mother
cell and to maintain genetic continuity from generation to generation.
 Delbruck suggested three theoretical methods of DNA–replication. i.e.,

(1) Dispersive (2) Conservative (3) Semi – conservative
Experimental proof for Semi conservative mode of DNA Replication:

 Semi conservative mode of DNA replication was first proposed by Watson & Crick.
 It was first experimentally proved by Matthew Meselson and Franklin Stahl (1958) in
E.coli by using radioactive isotopes of nitrogen.
 They cultured E.coli in a nutrient medium containing ammonium chloride (NH4Cl) with
heavy isotope of nitrogen (15N). The 15N was incorporated into newly synthesized DNA. This
heavy DNA molecule could be distinguished from the normal DNA by density gradient
centrifugation in Cesium chloride (CsCl). The heavy isotope of nitrogen ( 15N) is separated
from normal nitrogen (14N) based on density gradient centrifugation.
 They transferred the E.coli cells with heavy DNA into a medium containing normal
14NH4Cl for many generations (the generative period of E.coli is 20 minutes). After the cells
were allowed to grow varying period of time, they extracted double stranded DNA and
analysed in cesium chloride (CsCl) gradients to measure the density of DNA.
 The DNA extracted from the culture after one generation i.e., 20 minutes, has
intermediate density termed as hybrid.
 DNA extracted after two generations or 40 minutes contains 50% of hybrid DNA and 50%
of light DNA.
 These results are precisely predicted by Watson and Crick in the semi conservative type of
DNA replication.
 In each generation out of two strands of DNA, one was obtained from parent DNA and
other was formed fresh from nucleotides. This proves semi conservative type of DNA
replication in E.coli.
 Taylor and colleagues performed similar experiments by using radioactive thymidine to
detect the newly synthesized DNA in the chromosomes of Vicia faba (faba beans). All these
experiments proved semi conservative type of replication in DNA.

MECHANISM OF D.N.A. REPLICATION
The process of formation of replica or identical copies of DNA is called DNA replication (or)
The process of formation of two identical daughter DNA molecules from a parent DNA molecule
is called DNA replication.
DNA replication takes place regularly at S-phase of inter phase during cell division. The
semi-conservative theory of DNA replication was first proposed by Watson and Crick and it was
proved qualitatively by J. Herbet Tayler in 1954 and quantitatively by Meselson and Stahl in
1958 by conducting experiments on E. coli bacterium by using radioactive isotopes of nitrogen.
Requirements:
1. Four types of nucleotides of DNA
2. Energy source (ATP)
3. Inorganic ions: Mg2+
4. Single Strand Binding Proteins (SSBP’s): avoids the rewinding of unwound DNA.
5. Enzymes:
Helicase: unwinds the DNA helix by breaking H-bonds [also known as unwindase].
Topoisomerases: type of endonuclease which breaks (induces a cut or nicking) and reseals
the DNA strands to release the stress in parental DNA (i.e., prevents supercoiling) [also
known as Gyrases in prokaryotes].
RNA primase: synthesizes RNA primer [short 8-10 nucleotide base pairs of RNA].
DNA polymerase-III: catalyses the synthesis of DNA [5'→3' polymerizing activity].
DNA polymerase-II: correct the wrong nucleotides in the newly synthesized strand [3'→5'
exonuclease activity- Proof reading].
DNA polymerase-I: remove RNA primers [5'→3' exonuclease activity- DNA repairing].
DNA ligase: join DNA fragments. (Khorana)
The mechanism of DNA replication involves the following steps:

1. Activation of nucleotides:
The nucleotides of DNA such as d-AMP, d-TMP, d-GMP and d-CMP are activated
and phosphorylated by ATP.
2. Unwinding of DNA helix:
DNA replication starts at a specific point called origin of replication or ORI site. In
prokaryotes there is only one ORI site, whereas in eukaryotes there are many ORI sites.
From ORI site the DNA helix starts unwinding/ unzipping and gets separated with the
help of helicase or unwindase enzyme. Topoisomerase breaks and reseals the parental
strand to release the stress on parental DNA during unwinding process. The separation of
two strands results in the formation of Y-shaped structure called replication fork. The two
separated strands act as templates and capable of synthesizing new complementary

strand of DNA. These strands are hold by SSBPs to avoid the rewinding of DNA. So that
other enzymes come and act on it.
3. Formation of RNA primer:
RNA primer is a short segment of RNA (with 8-10 nucleotide base pairs) which is
complementary to DNA template synthesized by RNA polymerase or RNA primase. RNA
primer possesses 3'-OH group which helps to initiate the synthesis of complementary DNA
strands with the help of DNA polymerase-III.
4. Initiation and elongation of DNA strand:
The DNA nucleotides are now added to exposed bases of parental DNA strand from
the end of RNA primer. This process is catalyzed by DNA Polymerase III and Mg2+ ions.
The addition of nucleotides of DNA proceeds only in 5'→3' direction on the 3'→5' template
DNA strand. The replication proceeds on both the parental template strands in opposite or
antiparallel direction, hence called bidirectional replication.
In one strand the synthesis of new DNA strand goes on continuously in 5'→3'
direction and the parental template which synthesis this continuous DNA strand is called
leading strand.
In the opposite strand, the addition of nucleotides proceeds as short segments
away from the replication fork. The parental template which synthesis these short
segments is called lagging strand. Each short single stranded fragment initiates from RNA
primer and these fragments were first discovered by R.Okazaki in 1968. Hence they are
also called as Okazaki fragments.
5. Termination of replication: Formation of daughter DNA molecule:
The termination of replication is signalled by specific sequence of DNA nucleotides.
After replication the DNA polymerase II takes an editing role to remove abnormal nitrogen
bases and incorporate the normal bases called Proof reading.
6. Formation of daughter DNA molecule:
RNA primer in the lagging strand is removed with the help of DNA polymerase-I and
later Okazaki fragments are joined to produce continuous strand with the help of DNA
ligase enzyme. Soon after this the formed strands gets binds with parental strand and
twist in a right-handed direction to form two daughter DNA molecules.
Replication of DNA is bi-directional because replication takes place towards ori site
and also opposite to ori site. According to semi-conservative theory during the replication
in the daughter DNA molecules, one of the parental strands (old strand) is conserved and
other strand is newly formed i.e. 50% of DNA is parental in daughter DNA molecules.

Note:
 The process of D.N.A. replication takes a few minutes in prokaryotes and a few hours in
Eukaryotes.
 During replication, the 2 phosphate groups of all Nucleotides are separated. In this
process energy is yielded which is consumed in DNA replication. So, it is clear that DNA
does not depend on mitochondria for its energy requirements.
 DNA Polymerase-I was discovered by Kornberg (1957). So it is also called as 'Kornberg's
enzyme'.
 DNA Polymerase-II is least reactive in replication process. It is also helpful in DNA-
repairing in absence of DNA polymerase-I and DNA polymerase-III.
 DNA Polymerase-III is the main enzyme in replication. It was discovered by Delucia and
Cairns. The larger chains are formed by this enzyme. This is also known as Replicase.
Functions of DNA:
1. DNA acts as genetic material and transmits characters from parents to offsprings in most
of the living organisms except in some plant and animal viruses.
2. DNA regulates all metabolic activities of a cell hence called master molecule.
3. DNA is capable of self replication and produces its own identical copies during cell
division and equal distribution to the daughter cells.
4. DNA produces different types of RNA and directs the synthesis of proteins.
5. DNA undergoes recombination and mutations, which brings about variations and leads to
evolution.
RIBO NUCLEIC ACID (RNA)
Structure of RNA is fundamentally the same as DNA, but there are some differences. The
differences are as follows:-
 In place of Deoxyribose sugar in DNA, there is a Ribose sugar in RNA.
 In place of nitrogen base Thymine in DNA, there is a Uracil in RNA.
 RNA is made up of only one polynucleotide chain i.e. R.N.A. is single stranded. [Exception-
RNA found in Reo-virus is double stranded, i.e. it has two polynucleotide chains].
TYPES OF RNA:
I. Genetic RNA or Genomic RNA:
In the absence of DNA, sometime RNA works as genetic material and it transfers
information from one generation to next generation. Ex: Reo virus, TMV, HIV, influenza
virus, QB bacteriophage, polio virus, etc.
II. Non-genetic RNA: (3 types)
1. Ribosomal RNA (rRNA):
 This RNA is 80% of the cell's total RNA.
 rRNA was discovered by Kuntze.
 It is found in ribosomes and it is produced in nucleolus.
 It is the most stable form of RNA.
 Eukaryotic cells have 80s type of ribosomes. Their subunits are 60s and 40s. In 60s
subunit of ribosome three types of rRNA are found- 5s, 5.8s, 28s. 40s subunit of
ribosome has only one type of rRNA i.e. 18s. So 80s ribosome has total 4 types of
rRNA.
 Prokaryotic cells have 70s type of ribosomes and its subunits are 50s and 30s. 50s
subunit of ribosome contains 2 types of rRNA i.e. 5s and 23s. 30s subunit of ribosome
has 16s type of r–RNA. So 70s RNA has total 3 types of r–RNA.
 Functions:
o rRNA is associated with ribosomal protein to become functional unit of ribosome
for protein synthesis.
o At the time of protein synthesis rRNA provides attachment site to tRNA and
mRNA and attaches them on the ribosome. The bonds formed between them are
known as Salt linkages. It attaches tRNA to the larger subunit of ribosome and
mRNA to smaller subunit of ribosome.
o It helps in the formation of peptide bond between amino acids during protein
synthesis.
2. Transfer RNA (tRNA):
 It is 10-15% of total RNA.
 It is synthesized in the nucleus by DNA.
 It is also known as soluble RNA (sRNA).
 It is also known as Adapter RNA.
 It is the smallest RNA.

 Function: At the time of protein synthesis it acts as a carrier of amino-acids.
 Discovery: tRNA was discovered by Hogland, Zemecknike and Stephenson.
 Structure: The structure of tRNA is most complicated. A scientist named Holley
presented Clover leaf model of its structure. In two dimensional structures the t–RNA
appears clover leaf like but in three dimensional structures (by Kim) it appears L–
shaped.
 The molecule of tRNA is of single stranded.

 There are three nucleotides present in a particular sequence at 3' end of tRNA and that
sequence is CCA.
 All the 5' ends are having G (guanine).
 3' end is known as Acceptor end.
 tRNA accepts amino acids at acceptor points. Amino acid binds to 3' end by its -COOH
group.
 The molecule of tRNA is folded and due to folding some complementary nitrogenous
bases come across with each other and form hydrogen bonds.
 There are some places where hydrogen bonds are not formed; these places are known
as loops.
 Loops: There are some abnormal nitrogenous bases in the loops, that is why hydrogen
bonds are not formed. Ex: Inosine (I), Pseudouracil (), Dihydrouridine (DHU).
i. TC Loop or Attachment loop:
This loop connects tRNA to the larger subunit of ribosome.
ii. Recognition Loop (Anticodon loop):
This is the most specific loop of tRNA and different types of tRNA are
different due to this loop. There is a specific sequence of three nucleotides called
Anticodon, present at the end of this loop.
On the basis of Anticodon, there are total 61 types of tRNA or we can also
say that there are 61 types of Anticodon.
tRNA recognizes its place on mRNA with the help of Anticodon.
The anticodon of tRNA recognizes its complimentary sequence on mRNA.
This complimentary sequence is known as codon.
iii. DHU Loop: It is also known as Amino-acyl synthetase recognition loop. Amino-acyl
synthetase is a specific type of enzyme. The function of this enzyme is to activate a
specific type of amino acid. After activation this enzyme attaches the aminoacid to
the 3' end of tRNA.
There are 20 types of enzymes for 20 types of amino acids.
The function of DHU loop is to recognize this specific Amino-acyl synthetase
enzyme.
3. Messenger RNA (mRNA):
 The mRNA is 1-5% of the cell's total RNA.
 Discovery: Messenger RNA was discovered by Huxley, Volkin and Astrachan. The term
mRNA was given by Jacob and Monad.
 The mRNA is produced by genetic DNA in the nucleus. This process is known as
Transcription.
 It is least stable RNA.
 Structurally mRNA is a single stranded polynucleotide chain contains the following 7

regions.
 It runs from 5'→3' direction. The 5' end of eukaryotic mRNA have a specialized
structure called CAP, made up of 7' methyl guanosine nucleotide (methylated
guanosine triphosphate). The cap protects the mRNA from degradation by nucleases
and may help in the recognition of specific ribosome. CAP is absent in prokaryotic
mRNA.
 Next to the CAP there is a non-coding region called leader sequence made up of about
200 nucleotides.

 Next to leader sequence initiator codon is present. It is always AUG and very rarely
GUG.
 Initiator codon is followed by coding region (sensible range). The triplet codons
present in this region are capable of selecting amino acids during protein synthesis.
 Coding region is followed by a terminator codon. It may be UAA or UAG or UGA. This
codon stops the process of protein synthesis.
 Next to terminator codon a few nucleotides forms a pseudo helix. It is non-coding
region called trailer and perhaps provide stop signal for cleavage.
 At 3' end, in eukaryotic mRNA there is a Poly-A tail with only Adenine nucleotides.
The Poly-A tail is absent in prokaryotic mRNA.
 Functions of mRNA: mRNA carry genetic message from DNA to the ribosomal complex
in the form of triplet codon for protein synthesis.
 Types of mRNA: there are 2 types of mRNA, they are:
i. Monocistronic mRNA: the mRNA containing one structural gene and carries
information for the formation of single polypeptide is called monocistronic
mRNA. This mRNA is present in eukaryotes and generally it is long lived.
ii. Polycistronic mRNA: the mRNA containing many structural genes and carries
information for the formation of several polypeptides is called polycistronic
mRNA. This mRNA is present in prokaryotes and generally it is short lived.
Monocistronic mRNA Polycistronic mRNA

1 Found in all eukaryotes Found in all prokaryotes
2 Codes for only one protein Codes for many proteins
3 It has cap and tail It does not possess cap and tail
4 Life span is long (4 hours) Life span is short (2 minutes)
5 It undergo post transcriptional process It do not undergo post transcriptional process
DNA RNA
1 It is double stranded. It is single stranded.
2 Most of the DNA is present inside the
Most of the RNA is present in cytoplasm.
nucleus.
3 It contains deoxy ribose sugar. It contains ribose sugar.
4 Nitrogen bases are A,G, T, C. Nitrogenous bases are A, G, U, C.
5 It is made up of more than 4 million
It is made up of about 12,000 nucleotides.
nucleotides.
6 Molecular weight is more. Molecular weight is less.
7 It undergoes self replication. It cannot undergo self replication.
8 DNA is of only one type. RNA is of 3 types.
9 It directs protein synthesis. It takes part in protein synthesis.
10 It acts as genetic material in most of the It acts as genetic material in some plant and
living organisms. animal viruses.

11 DNA is less reactive and more stable RNA is inactive and less stable.
12 DNA being stable, mutate slower. RNA being unstable, mutate faster.
13 DNA is preferred for storage of genetic RNA is preferred for transmission of genetic
information. information.
TRANSCRIPTION
The process of copying genetic information from one strand of the DNA into RNA is called
transcription. (The synthesis of mRNA from one of the DNA template strand with the help of RNA
polymerase enzyme is called transcription). It takes place inside the nucleus.
The segment of DNA involved in transcription is ‘Cistron’.
The synthesized RNA has base sequence complementary to template strand. But similar
to sense or coding strand of DNA with the exception that T is replaced by U.
Both the strands of DNA are not transcribed because,
 They may form two kinds of proteins with opposite sequence of amino acids that
may cause dysfunctional proteins or other defects.
 The complementary RNAs can pair and form dsRNA which may not be functional
and also prevent translation of proteins.
Transcription Unit:
The part of DNA that takes part in transcription is called Transcription unit. It consists of
three regions such as- a promoter site or initiator site (where transcription starts), the structural
gene and a terminator (where transcription stops).
1. A promoter: A promoter is located towards 5' end (upstream) of the structural gene. It
provides site for attachment of transcription factors and DNA dependent RNA polymerase.
The promoter consists of a specific base sequence called TATAA-box. It was discovered by
Pribnow hence called Pribnow box.
2. The structural gene: The structural gene is the area of template strand that is involved
in transcription of RNA. The 3'→5' strand of DNA acts as template for the synthesis of
RNA and is referred to as template strand. The 5'→3' strand has the sequence same as
RNA is referred to as coding strand.
3. A terminator: A terminator is located towards 3' end (downstream) of the coding strand
and defines the end of the process of transcription. It has either palindromic sequence or
Poly-A sequence.

Mechanism of Transcription:
The DNA dependent RNA polymerase enzyme along with the sigma factor (σ) slides along
the length of DNA strand. Sigma factor recognizes the promoter site of DNA. Once it encounters
the promoter sequence RNA polymerase binds firmly and the uncoiling of DNA strands takes
place due to the breakage of hydrogen bonds. As a result two strands of DNA get separated. This
leads to the formation of transcription bubble.
One of the separated DNA strand acts as template strand to produce complementary
mRNA called antisense/non coding strand and the other opposite non template strand is called
sense/coding strand.
After the unwinding, RNA polymerase helps to assemble the ribonucleotides
complementary to the DNA template strand and this is followed by the establishment of
phophodiester bonds between successive nucleotides. Once a short 8-10 base pairs of RNA
molecule is formed the sigma factor dissociates from the enzyme, this marks an end of initiation.
Then, in elongation the core enzyme carries out the synthesis of mRNA strand in 5'→3'
direction and it is anti parallel to the template strand.

When the mRNA is completely formed, for termination there is a specific signal in DNA
template to stop the process. These signals are recognized by a terminator protein called Rho
factor (). When it is attached to the DNA, the RNA polymerase cannot move further and leads to
the termination of transcription process.
Finally the two strands are rewound back by RNA polymerase and the new RNA formed
gets detached from the DNA template.
POST TRANSCRIPTIONAL PROCESS:

In eukaryotes, the RNA synthesized are non-functional, to become functional it has to
undergo post transcriptional modification. This involves splicing process, capping and tailing.
1. Splicing: The process of removal of non-coding sequence, introns and joining the coding
sequence, exons to form a functional RNA.
2. Capping: The process of addition of unusual nucleotide, 7' methyl guanosine triphosphate
to the 5' end of hnRNA.
3. Tailing: The process of adding of repeated Adenine nucleotides of about 200-300 residues
[Poly-A tail] to the 3' end of hnRNA. (or Poly-adenylation).
After processing mRNA releases into cytoplasm through nuclear pore.
In prokaryotes, mRNA contains only exons. Hence RNA processing does not take place.

DIVISION OF LABOUR:
In eukaryotes, there are at least three RNA polymerases in the nucleus (in addition to the RNA
polymerase found in the organelles). There is a clear cut division of labour.
 RNA Polymerase I: [found in nucleolus] transcribes 3 molecules of rRNA (28s, 18s and
5.8s)
 RNA Polymerase II: [found in nucleoplasm] transcribes mRNA (in precursor form, hnRNA-
hetergenous nuclear RNA and snRNA- small nuclear RNA)
 RNA Polymerase III: [found in nucleoplasm] transcribes tRNA, 5s rRNA and some snRNA
(that are not synthesized by RNA Pol II)
REVERSE TRANSCRIPTION (Teminism):

In some viruses RNA is used as template for DNA synthesis, the synthesis of DNA from
RNA is called reverse transcription. This process is influenced by the enzyme RNA dependent
DNA polymerase or reverse transcriptase. This is discovered by Temin and Baltimore in 1972.
Ex:HIV.
Difference between prokaryotic gene and eukaryotic gene:

Prokaryotic gene Eukaryotic gene
1 Introns are absent Introns are present
2 Entire gene is transcribed into Introns are removed after transcription
mRNA to form mRNA
3 It is polycistronic It is monocistronic
4 Each gene contains a continous Genes are split genes
stretch of DNA
5 Short life span Long life span
NOTE:
 RNA polymerase of E.coli has five polypeptide chains: , ', ,  and .  polypeptide chain
is also known as  factor (sigma factor).
 Core enzyne + Sigma factor  RNA Polymerase

(, ', , ) ()

 In prokaryotes before the 10nt base from ‘Promoter site’ a sequence of 6 base pairs
(TATAAT) is present on DNA, which is called ‘Pribnow box’.
 In eukaryotes before the 20nt base from ‘Promoter site’ a sequence of 7 base pairs
(TATAAAA) or (TATATAT) is present on DNA which is called ‘TATA box or Hogness box’.
 The ribonucleotides are present in the form of triphosphates ATP, GTP, UTP and CTP.
When they are used in transcription, pyrophosphatase hydrolyses two phosphates from
each activated nucleotide. This releases energy. This energy is used in the process of
transcription.
GENETIC CODE
A sequential arrangement of the Nucleotide bases in the DNA molecule (or mRNA) that
controls the sequence of amino acids in a protein is called Genetic code.
During protein synthesis DNA sends coded information in the form of mRNA by a process
of transcription that is later translated into a protein.
A polypeptide chain typically contains amino acids, and is formed by a specific
arrangement of 20 different amino acids. This arrangement is directed by the coded information
present in genes i.e. DNA. The DNA has 4 different types of nucleotide bases (ATGC) that specify
the sequence of 20 different amino acids of the protein. For all study purpose of genetic code, the
sequence of nucleotide bases present in the mRNA is taken into consideration.
The term ‘genetic code’ was given by George Gamow. Nirenberg, Robert Holley and Har
Gobind Khorana provided experimental proof of deciphering the genetic code and were awarded
Nobel Prize in 1968.
CODON SYSTEM:
To describe the relationship of nucleotides and the amino acids, different types of codon
systems have been proposed. They are:
1. Singlet codon system: According to this system, one nucleotide base code for one amino
acid. Since there are 4 different nucleotides bases A, U, G and C, they can code for only 4
amino acids. So this cannot be accepted as 20 different naturally occurring amino acids
are used in proteins synthesis.
2. Doublet codon system: According to this system, 2 nucleotide bases together code for
one amino acid. The 4 different nucleotide bases can form 16 different combinations and
thereby could specify a maximum of 16 different amino acids. It is again inadequate and
not accepted.
3. Triplet codon system: According to this system, 3 nucleotide bases together code for one
amino acid. The 4 nucleotide bases can form 64 triplet codons or combinations. This can
be sufficient to specify 20 different amino acids.

PROPERTIES OF GENETIC CODE:

1. Triplet codon: The fundamental unit of genetic code found in mRNA that code for a
specific amino acid during protein synthesis is called Codon. A codon is made up of
sequence of 3 nucleotides; hence the genetic code is called as Triplet codon.
2. Universal: The genetic code is called universal because the same codons code for the
same amino acid during protein synthesis in every form of life, whether it is bacteria or
humans. However, a few exceptions to this are known. For example, in yeast
mitochondria, the non-sense codon UGA codes for the tryptophan.
3. Non-overlapping: From initiation codon on mRNA, the sequence of nucleotide bases are
read in blocks of 3, correspond to the sequence of amino acids, without any overlapping of
bases i.e. any one base is a part of any one codon and not more than one codon.
4. Degenerative type: A code in which 2 or more different codons code for specific amino
acid is called degenerative type. Here one amino acid is coded by more than one codon
and such codons are called as degenerative codons or synonymous codons.
Ex: Leucine-CUU, CUC, CUA, CUG, UUA and UUG.
Note: 18 amino acids have degenerative codons. Only methionine coded by AUG and
tryptophan coded by UGG have single codons specifying it.
5. Commaless: The message is continuous without any breaks or pauses between codons
and such a code is called 'commaless'.
6. Non-ambiguous: A particular codon always codes for the same amino acid and never for a
different amino acid.
7. Non-sense codon: Also called terminator codon. Out of 64 triplet codons, 3 of them do not
represent any amino acid and these are called non-sense codons. Non-sense codons are
UAA (Ochre), UAG (Amber) and UGA (Opal). They provide signal to stop translation of
mRNA during protein synthesis.

8. Initiator codon: Protein synthesis is initiated by a particular codon 'AUG' which is

present towards 5' end of mRNA. Rarely GUG may be present as initiator codon. AUG
codes for methionine.
9. Principle of Colinearity: Genetic code works on the principle of colinearity i.e. it explains
specific relationship between DNA, RNA and Polypeptide chain. The linear order of
nucleotides in DNA determines the linear order of codons in mRNA which in turn
determines the linear order of amino acids in a polypeptide chain.
10. Missense mutation: Any alteration in a codon due to replacement of any one of the
nucleotide bases that result in specifying another amino acid is called missense mutation.
Ex: the abnormal haemoglobin in sickle cell anemia is a result of missense mutation. Here
glutamic acid (GAA, GAG) is replaced by valine (GUA, GUG). Here U replaces nucleotide
base A.
WOBBLE HYPOTHESIS:
It was proposed by Francis Crick. According to this hypothesis, in a degenerate type of
codons where an amino acid has more than one triplet codon specifying it the first two-
nucleotide bases in the triplet specify the amino acid, the third is less specific.
In a degenerative code, the position of the third base on the codon in mRNA is known as
wobble position. Change in this base may not alter the amino acid. Such mutations that do not
cause any visible effects are called Silent mutations.
This hypothesis explains how a tRNA recognizes more than one codon. The wobbling
favours economy of numbers of t-RNA.
Wobble base pairing:

Anticodon in tRNA Codon in mRNA
A → U
C → G
G → C/U
U → A/G
I → A/C/U
Derivative from
Inosine base
(hypoxanthine).
Found in RNA more
MUTATIONS
 Gene mutations are sudden heritable changes in the genes.

 It is caused by change in number, sequence and types of nucleotides.
 Mutation caused due to change in a single nucleotide is called point mutation.
 Mutation caused by change in more than one nucleotide pair is called gross mutation.
 Mutation may develop during replication and transcription due to copy error.

 Mutation may be spontaneous or induced.

 Frameshift mutation or Gibberish mutation:
The gene mutation where reading frame of codon is changed due to insertion or
deletion of one or more nucleotides.
It shifts forward if insertion occurs and it shifts backwards due to deletion.
Ex: AGGGUCCAA →insertion of U→ AUG, GGU, CCA, A
Met Gly Pro
AGGGUCCAA →deletion of G→ AGG, UCC, AA
Arg Ser
PROTEIN SYNTHESIS (TRANSLATION)
The process of decoding of coded message present on m-RNA to produce specific proteins
in the ribosomal complex is called translation. (The process of polymerization of amino acids to
form a polypeptide is called translation). Translation takes place in the cytoplasm and involves
the following 4 steps:
Activation and selection of amino acids:

The amino acids are present in an inactive form in the amino acid pool of cytoplasm. Each
amino acid is activated by ATP in the presence of Mg2+ to form aminoacyl-AMP complex.
Amino acid + ATP (Mg2+) Aminoacyl-AMP complex + Pi
The activated aminoacyl AMP complex attaches to the binding site of specific t-RNA to
form aminoacyl-t RNA complex in the presence of aminoacyl-tRNA synthetase enzyme.
Aminoacyl-AMP complex + t-RNA Aminoacyl-tRNA complex
There are twenty different types of t-RNAs to carry twenty different amino acids.
Initiation of polypeptide chain:

After transcription m-RNA attaches to smaller subunit of ribosome (30s subunit) and
ribosome subunit slides on m-RNA till initiator codon AUG occupies P-site of ribosome. The t-
RNA containing UAC anticodon carries methionine [N-formyl methionine] amino acid and binds
with the initiator codon AUG on m-RNA at P-site to form initiation complex (A-site (Amino acyl
site) is free or vacant), immediately larger subunit of ribosome (50s subunit) attaches to the
smaller subunit to form the functional ribosome (70s unit).
When the second amino acid carrying t-RNA enters into the ribosomal complex, it binds
with corresponding codon on m-RNA at A-site. Immediately a peptide bond is formed between
the first amino acid present at P-site and second amino acids present at A-site by peptidyl
transferase. The initiation process is catalyzed by GTP and initiation factors like IF1, IF2, and
IF3.

Elongation of polypeptide chain:

During this process amino acids are added one by one to elongate the polypeptide chain
after initiation. When dipeptide chain is formed, the first t-RNA releases outside through the E-
site of the complex. Then ribosome slides on m-RNA from 5'→3' direction by one codon length so
the dipeptide chain is shifted from A-site to P-site. Then the A-site becomes vacant. Immediately
third amino acid carrying t-RNA enters into ribosome and binds to the corresponding codon on
m-RNA, at A-site. Then a peptide bond is formed between second and third amino acid with the
help of peptidyl transferase enzyme. In the same way different amino acids correspond to all
codons on m-RNA are linked together with the help of peptide bonds to form long polypeptide
chain. This process is influenced by many elongation factors.
Termination of polypeptide chain:

The elongation of polypeptide chain is continued till one of the termination codons is
reached. The presence of one termination codon (UAA, UAG, UGA) at the A-site gives a stop
signal as there is no t-RNA to carry any corresponding amino acid. The releasing factors like
RF1, RF2 and RF3 binds to the termination codon and it helps to release the polypeptide chain
from the ribosomal complex into cytoplasm. Mean while the two sub units of ribosome gets
separated and m-RNA is degraded rapidly to produce nucleotides.

NOTE:
 In prokaryotes with the help of ‘SD sequence’ (Shine-Delgarno sequence) m-RNA
recognizes the smaller sub unit of ribosome. A sequence of 8nt base is present before the
4-12nt base of initiation codon on mRNA, called ‘SD sequence’. In smaller subunit of
ribosome, a complementary sequence of ‘SD sequence’ is present on 16s r-RNA, which is
called ‘Anti Shine-Delgarno sequence’ (ASD sequence). With the help of 'SD' and 'ASD
sequence' mRNA recognises the smaller sub unit of ribosome.
 While in eukaryotes, smaller sub unit of ribosome is recognized by ‘7mG cap’. 18s r-RNA
of smaller sub unit has a complementary sequence of ‘7mG cap’.
 In larger sub unit of ribosome there are three sites for t-RNA:
‘P’ site = Peptidyl site
‘A’ site = Amino acyl site
‘E’ site = Exit site
 Peptide bond forms between -COOH group of P-site amino acid and -NH2 group of A-site
amino acid.
 Peptidyl transferase enzyme induces the formation of peptide bond. In peptide bond
formation, 23s r-RNA is also helpful. This r-RNA acts as an enzyme so it is also called
‘Ribozyme’.
 Translocase enzyme is helpful in movement of ribosome (translocation). GTP provides
energy for sliding of ribosome.
 In prokaryotes three elongation factors are present: EF-Tu, EF-Ts and EF-G.
 In eukaryotes two elongation factors are present: eEF1 and eEF2.
 In eukaryotes only one release factor is known: eRF1.
REGULATION OF GENE EXPRESSION
The gene expression results in the formation of a polypeptide chain, it can be regulated at
several levels in eukaryotes as follows.
1. During transcriptional level (formation of primary transcript)
2. Processing level (regulation of splicing)
3. Transport of m-RNA from nucleus to cytoplasm
4. Translational level

LAC-OPERON CONCEPT (Gene structure and action): [Example for Inducible operon]
The Lac operon concept was proposed by Jacob and Monad in 1961 to explain the gene
action or regulation of protein synthesis in E. coli bacteria. For this significant work they were
awarded noble prize in 1965.
Jacob and Monod explained the activation and inactivation of genes that control lactose
metabolism in E.coli. The group of highly co-ordinated structural and control genes that regulate
lactose metabolism in E.coli is called Lac-operon. Lac-operon consists of two types of genes
namely: 1. Structural genes 2. Control genes
Structural genes:
These are the cistrons (segments of DNA coding for polypeptides) responsible for
synthesizing m-RNA and later proteins and enzymes. There are 3 types of structural genes
namely Lac-z, Lac-y and Lac-a. These genes produce a polycistronic m-RNA to synthesize 3
enzymes necessary for lactose metabolism in E.coli bacteria.
a) Lac z gene - β-galactosidase (hydrolyse lactose into glucose & galactose)
b) Lac y gene - permease (increases permeability of the cell to β-galactosidase)
c) Lac a gene - transacetylase (transfers acetyl group from acetyl CoA to galactosidase)
Control genes:
These genes control the activities of structural genes. There are 3 types of control gene,
namely:
1. Operator gene (O): This gene is located adjacent to the structural gene. It controls the
formation of m-RNA by structural genes. It acts as a switch by blocking or allowing the
structural genes to perform the function.
2. Promoter gene (P): This gene is located adjacent to the operator gene to which RNA
polymerase enzyme binds to initiate m-RNA synthesis.
3. Regulator gene (i): This gene is located adjacent to the promoter gene and regulates the
activity of gene operator by producing a protein called repressor protein.
Mechanism of gene action:

1. Lac-operon switched off:
When lactose or inducer is absent in the medium, the repressor protein produced by
the regulator gene binds to the operator gene and switches off the operon system.
As a result, it cannot initiate RNA polymerase and prevents the synthesis of mRNA by
structural genes. There will be no synthesis of proteins and enzymes. This is because E.coli
does not require enzymes since lactose is absent.

2. Lac-operon switched on:

When lactose is present in the medium, a few molecules of lactose diffused into the
bacterium cell and binds with the repressor protein produced by the regulator gene. So the
repressor protein undergoes structural changes and become inactive called inducer-repressor
complex. This complex is unable to bind the operator. As the result, the operator is set free
and switches on the operon system. RNA polymerase binds to promoter and transcribes m-
RNA by the structural genes. The m-RNA produces proteins to synthesize the enzymes β-
galactisidase β-galactoside permease & β-galactoside transacetylase required for lactose
metabolism. Here lactose acts as inducer in the synthesis of enzymes. Hence operon concept
in E.coli is called Lac-operon concept.
NOTE:
1. Induction: The mechanism in which inducer inactivates the repressor protein to facilitate
m-RNA and protein synthesis.
2. Repression: The mechanism by which repressor protein binds the operator gene to stop
the m-RNA and protein synthesis.
3. Certain genes which are in their switched on state continue to synthesize a metabolite
till it is produced in amount more than required. In other words genes continue to
express themselves till the end product inhibits or repress their expression. Inhibition by
end product is known as ‘feedback repression’.
4. An operon is a part of genetic material or DNA, which acts as a single regulated unit
having one or more structural genes- an operator gene, a promoter gene and a regulator
gene. (or) Operon is unit of Transcription. (or) Operon is a cluster of genes which
synthesizes its mRNA and proteins together.
5. Operons are of two types: (i) Inducible [Ex: lac-operon] (ii) Repressible [Ex:trp-operon]
6. Allolactose is an isomer of lactose; it is a real or true inducer of lac-operon.

GENOMICS
Genomics is the study of genomes and genes based on DNA sequencing. Genome is the
total gene complement of a haploid set of chromosomes and inherited as a unit from one parent
through the gamete. A haploid (such as prokaryotic) cell contains a single genome; a diploid
(such as a cell of higher plant or animal) has two genomes, one paternal and other maternal.
Additional DNA is also present in mitochondrion which is inherited from one’s mother.
HUMAN GENOME PROJECT

The human genome project is an international project to determine the base sequences of
DNA molecules of 23 chromosomes (haploid set).
HGP was launched in 1990 by National Institute of Health (NIH) and US Department of
Energy. Wellcome Trust (UK), Japan, France, Germany, China also collaborated in the project.
HGP was 13 years project and was completed in 2003. It was an international
collaboration of people with over 20 centres involving several thousand scientists.
HGP is considered as a mega project because of the following reasons-

1. Due to huge cost, estimated to be nine billion dollars.
2. 2.3 x 109 billion bp to be identified and sequenced.
3. Involving a large number of scientists, technicians and supporting staff.
4. Information is stored in high speed computational devices and bioinformatics help the
project.
[If the 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]
Objectives/Goals of HGP:
1. To sequence the whole genome and to find out all base pairs.
2. Identify all the genes about 20,000-25,000 protein coding genes and determine their
functions.
3. Identify various gene that causes genetic disorders.
4. To store information in data bases to develop computer based analysis.
5. To transfer technologies developed during HGP to industry, agriculture and forestry.
6. To solve ethical, legal and social issues (ELSI) that may arise during the project.
Methodologies:
HGP followed two methods for sequencing human genome.
i. Identifying all the genes which are expressed as RNAs known as Expressed Sequence
Tags (ESTs) .
ii. Sequencing the whole genome both coding and non-coding and later assigning functions
to different regions. This is known as Sequence Annotation (SA).
The whole sequence of genome of the cell is isolated and broken randomly into smaller
fragments, they are separated and are inserted into specialized vectors like BAC (Bacterial Artificial
Chromosome) and YAC (Yeast Artificial Chromosome) the fragments are cloned in bacterial and
yeast cells. PCR (Polymerase Chain Reaction) can also be used for cloning or making multiple
copies of DNA fragments.
The fragments were sequenced using automated DNA sequencers that worked on the
principle of a method developed by Frederick Sanger. The sequences were then arranged based
on some overlapping regions present in them. Hence overlapping fragments were generated.
The sequences were then aligned with the help of computer based programmers. The
sequences were then annotated and assigned to each chromosome.
All the human chromosomes have been sequenced; 22 autosomes, X and Y (a total of
24 chromosomes). The chromosome-I was last to be sequenced in May 2006.
Salient Features of Human Genome:

i. Human genome contains 3164.7 million nucleotide bases.
ii. The average gene consists of 3000 bases, but sizes vary greatly, with the largest known
human gene being dystrophin at 2.4 million bases.
iii. The total number of genes is estimated at 30,000 much lower than previous estimates of
80,000 to 1,40,000 genes. 99.9% o f D NA i n hu man po pul ation is si mil ar; the
di fferences occu r on ly 0.1% o f hu man geno me .
iv. The functions are unknown for over 50% of the discovered genes.
v. Less than 2% of the genome codes for proteins.
vi. Repeated sequences make up very large portion of the human genome.
vii. Repetitive sequences are stretches of DNA sequences that are repeated many times,
sometimes hundred to thousand times. They are thought to have no direct coding
functions, but they shed light on chromosome structure, dynamics and evolution.
viii. Chromosome- 1 has most genes (2968) and the Y has the fewest (231).
ix. Scientists have identified about 1.4 million locations where single base DNA differences
(SNPs - single nucleotide polymorphism, pronounced as ‘snips’) occur in humans. This
information promises to revolutionise the process of finding chromosomal locations for
disease-associated sequences and tracing human history.
NOTE:
1. First prokaryote in which complete genome was sequenced is Haemophilus influenzae.
2. First Eukaryote in which complete genome was sequenced is Saccharomyces cerviceae.

3. First plant in which complete genome was sequenced is Arab idopsis thaliana (small
mustard plant).
4. First animal in which complete genome was sequenced is Caenorhabditis elegans
(Nematode).
5. β-globin and insulin gene are less than 10 kilo base pair. T.D.F. gene is the smallest
gene (14 base pair) and Duchenne muscular Dystrophy gene is made up of 2400 kilo
base pair (Longest gene).
Applications of HGP and future challenges:

1. There are more than 1200 genes that cause common cardiovascular diseases, hormonal
diseases like diabetes, neurological disorders like Alzheimer, cancers, etc. it will be
possible to alter and cure disorders.
2. To determine the genes that change cancerous cells to normal cells.
3. To study interactions between thousands of genes, proteins formed in tissues, organs or
tumours.
4. To study changes that occur in different developmental stages helps us to achieve
healthier living.
5. To get designer drugs, genetically modified diets according to individual needs.
6. Identifying single gene defects, their locations, reasons for defects and methods of
correction can be found out.
7. It has given huge data on various aspects of human genome.
8. Along with HGP, a number of model organism’s genomes are sequenced. Ex: E.coli,
Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (free living nematode), Mus
musculus (mouse), Drosophila melanogaster (fruit fly), rice and Arabidopsis thaliana.
DNA/ GENETIC FINGERPRINTING/ DNA TYPING/ DNA PROFILING/ DNA TEST

 DNA fingerprint is an identity mark of a person just like finger print and represented by
identical base tandem between the genes in DNA.
 DNA finger printing was developed by Alec Jeffrey in 1985, using a technique called RFLP-
Restriction fragment length polymorphism (variation at genetic level).
 The non coding sequences of DNA with highly repetitive base sequences are called short
tandem repeats (STRs). Ex: ATTGATTGATTG……
 The STRs with less than 15 base pairs repeated in 10-100 times are called microsatellites.
There are about 2,00,000 microsatellites in human genome.
 The STRs with 15-50 base pairs repeated in thousands of times are called minisatellites.
There are about 30,000 minisatellites in human genome.
 The STRs vary from individual to individual; hence these are also called Variable number
of tandem repeats (VNTRs). They provide genetic markers in the individuals and can be
employed as tools to create a genetic finger print of an individual.
 The method of identification of variable number of tandem repeats in a DNA fragment is
called DNA fingerprinting.
Technique of DNA fingerprinting:

The steps involved in the DNA finger printing are as follows:
1. Extraction of DNA from (individuals) the sources like blood, semen, hair roots, bone,
saliva, etc.
2. The extracted DNA is made into small fragments by using restriction endonuclease
enzymes.
3. The DNA fragments are separated according to their length by passing through agarose
gel electrophoresis.
4. The double stranded DNA is made into single stranded DNA by using alkali.
5. The single stranded DNA are transferred on to the nylon membrane (nitrocellulose
sheet) by a technique called Southern blotting and mixed with radioactive DNA probes.
6. DNA probes pair with the ssVNTRs having complementary sequences.
7. The nitrocellulose membrane with paired DNA probes is radiographed. Dark bands are
developed called DNA fingerprints.
8. These bands are compared to the prints of all suspects.
APPLICATIONS OF DNA FINGERPRINTING:

1. It is used as a forensic tool for identification of criminals involved in crimes like rape and
murder.
2. It helps to solve the parental disputes.
3. It provides information about human lineage from apes.
4. It helps to trace the path of human evolution from Africa.
5. It is used in diagnosis of hereditary diseases like haemophilia, sickle cell anaemia,
thalassemia, cystic fibrosis and others.
6. It helps to identify antibiotic resistant bacteria.
7. It is used to study genetic variation in wild animals and to determine purity of species.
8. It helps to identify racial groups their origin and migration.
9. It is also used to identify an individual from the other, except identical twins.
Polymorphism: the variation at genetic level due to mutation is called polymorphism. They
are unique to specific sites of DNA forming satellite DNA in 500 nucleotides or 107 times per
genome. These are formed due to deletions, inversions, single base substitutions and occur in
non coding introns. These are used in DNA finger printing.
NOTE:
 In India DNA finger printing was started by Dr.V.K. Kashyap & Dr. Lal Ji
Singh.
 In India the centre for DNA finger printing and diagnosis (CDFD) is located at
Hyderabad.
-THE END-

Molecular Basis of Inheritance-1

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Molecular Basis of Inheritance-1

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2020

MOLECULAR BASIS OF INHERITANCE

DNA AS GENETIC MATERIAL

Mr.Nandhan HD, Assistant Professor of Biosciences Page 1

DIRECT EVIDENCES FOR DNA AS THE GENETIC MATERIAL

THE TRANSFORMATION EXPERIMENT:

The discovery of the transformation principle:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 2

Mr.Nandhan HD, Assistant Professor of Biosciences Page 3

 Griffith results had several possible interpretations, all of which he considered:

IDENTIFICATION OF THE TRANSFORMING PRINCIPLE:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 4

1. Deoxyribonuclease (DNase), which degrades DNA.

Mr.Nandhan HD, Assistant Professor of Biosciences Page 5

THE HERSHEY-CHASE EXPERIMENT:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 6

to follow the protein.

Mr.Nandhan HD, Assistant Professor of Biosciences Page 7

Mr.Nandhan HD, Assistant Professor of Biosciences Page 8

Pentose sugar (S):

Nitrogenous Base (N.B):

Phoshoric Acid (P):

Nucleosides and Nucleotides:

Kinds of nucleotides in DNA:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 10

Deoxy cytidilic acid (d-CMP - Deoxy cytidine monophosphate)

Kinds of nucleotides in RNA:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 11

Important features of DNA double helix model are:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 12

14. In  x 174 bacteriophage the DNA is single stranded and circular.

15. DNA molecule is Dextrorotatory while RNA molecule is Laevorotatory.

PACKAGING OF DNA HELIX: [Ultrastructure of Chromosomes: Nucleosome Model]

Mr.Nandhan HD, Assistant Professor of Biosciences Page 13

Mr.Nandhan HD, Assistant Professor of Biosciences Page 14

2. Left Handed DNA- Anticlockwise twisting. Ex: Z-DNA

CENTRAL DOGMA OF MOLECULAR BIOLOGY

 Delbruck suggested three theoretical methods of DNA–replication. i.e.,

Experimental proof for Semi conservative mode of DNA Replication:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 16

MECHANISM OF D.N.A. REPLICATION

The mechanism of DNA replication involves the following steps:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 17

Mr.Nandhan HD, Assistant Professor of Biosciences Page 18

RIBO NUCLEIC ACID (RNA)

 It is the smallest RNA.

 The molecule of tRNA is of single stranded.

 Structurally mRNA is a single stranded polynucleotide chain contains the following 7

Mr.Nandhan HD, Assistant Professor of Biosciences Page 22

Monocistronic mRNA Polycistronic mRNA

Mr.Nandhan HD, Assistant Professor of Biosciences Page 23

Mr.Nandhan HD, Assistant Professor of Biosciences Page 24

Mr.Nandhan HD, Assistant Professor of Biosciences Page 25

POST TRANSCRIPTIONAL PROCESS:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 26

REVERSE TRANSCRIPTION (Teminism):

Difference between prokaryotic gene and eukaryotic gene:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 27

(, ', , ) ()

Mr.Nandhan HD, Assistant Professor of Biosciences Page 28

PROPERTIES OF GENETIC CODE:

Mr.Nandhan HD, Assistant Professor of Biosciences Page 29

8. Initiator codon: Protein synthesis is initiated by a particular codon 'AUG' which is