Std 12 chapter 6

STD 12
CHAPTER 6
MOLECULAR BASIS
OF INHERITANCE
SANJAY SIDDHAPURA
M.Sc., B.Ed., GSET, GPSC
(Ph.D. continue)

DNA (DEOXYRIBONUCLEIC ACID)
• J.D. Watson and F.H.C. Crick (1953) proposed double helical structure of DNA based on the
results of M.H.F.Wilkins and co-workers. All these three persons were awarded Nobel Prize in
1962 for this work.
• DNA is a long polymer of deoxyribonucleotides.
• The length of DNA is usually defined as number of nucleotides (or a pair of nucleotide referred
to as base pairs) present in it.
• Bacteriophage known as φ 174 has 5386 nucleotides
• Bacteriophage lambda has 48502 base pairs (bp),
• Escherichia coli has 4.6 × 106 bp
• Haploid content of human DNA is 3.3 × 109 bp.

STRUCTURAL AND FUNCTIONAL UNIT OF DNA

STRUCTURE AND FEATURE OF DNA
(POLYNUCLEOTIDE CHAIN)
• J.D. Watson and F.H.C. Crick (1953) proposed double helical structure of DNA.
• The following are some of the characteristic features of double helical structure of
DNA.
• (1) Each nucleotide consists of sugar, phosphate and a nitrogenous base. Many
such nucleotides are linked by phosphodiester bonds to form a polynucleotide chain or
strand.
• (2) Phospho diester bonds are formed between 5’carbon of sugar of one nucleotide
and 3' carbon of sugar of the next nucleotide.
• (3) Nitrogenous base is attached to 1’ carbon of sugar.
• (4) Polynucleotide strand is made of backbone of sugar and phosphate forming its long
axis and bases at right angles to it.

• (5) Chargaffs rule states that in natural DNAs the base ratio AT is always close to unity
and the GC ratio also to always close to unity indicated that A always pairs with T and G
pairs with C. A and T, G and C, therefore, are complementary base pairs.
• (6) Thus, if one DNA strand has A, the other would have T and if it has G, the other,
would have C. Therefore, if the base sequence of one strand is CAT TAG GAC, the base
sequence of other strand would be GTA ATC CTG. Hence, the two poly nucleotide
strands are called complementary to one another.
• (7) Two such complementary strands are joined with one another by hydrogen bonds
between their complementary nitrogenous bases. There are three hydrogen bonds
between cytosine and guanine and two hydrogen bonds between adenine and thymine.

• (8) The two polynucleotide chains are helically coiled around the same axis in such a way
that these can separate from one another only by uncoiling. Helical coiling is supposed
to be right handed. Such a form of DNA is now called B-DNA
• (9) The two chains or strands are antiparallel, i.e., they run in opposite directions in
relation to their sugar molecules. Their 5’P - 3' OH phosphodiester linkages are in
opposite directions
• (10) Double standed DNA molecule has a diameter of 20Aº.
• (11) The helix makes one complete turn every 34 Aº along its length.
• (12) There are 10 nucleotides per turn of helix. Thus the distance between two
neighbouring base pairs is 3.4 Aº.

CENTRAL DOGMA
• Francis Crick proposed the Central dogma in molecular biology, which states that the
genetic information flows from DNA  RNA  Protein.
• In some viruses the flow of information is in reverse direction, that is, from RNA to
DNA.

PACKAGING OF DNA HELIX
• .
• A length that is far greater than the dimension of a typical nucleus (approximately 10–6 m).
• DNA Packing in Prokaryotes
• In prokaryotes, such as, E. coli, though they do not have a defined nucleus,
the DNA is not scattered throughout the cell. DNA (being negatively
charged) is held with some proteins (that have positive charges)
• in a region known as ‘nucleoid’. The DNA in nucleoid is organised in large
loops held by proteins.

DNA Packing in Eukaryotes
• In eukaryotes, this organisation is much more complex.
• There is a set of positively charged, basic proteins called histones.
• A protein acquires charge depending upon the abundance of amino acids
residues with charged side chains.
• Histones are rich in the basic amino acid residues lysines and arginines. Both the
amino acid residues carry positive charges in their side chains.
• Histones are organised to form a unit of eight molecules called as histone
octamer.
• The negatively charged DNA is wrapped around the positively charged histone
octamer to form a structure called nucleosome.
• A typical nucleosome contains 200 bp of DNA helix.
• Nucleosomes constitute the repeating unit of a structure in nucleus called
chromatin, thread-like stained (coloured) bodies seen in nucleus.
• The nucleosomes in chromatin are seen as ‘beads-on-string’structure when
viewed under electron microscope (EM).

• Nucleosomes give beaded appearance to chromatin like
string on a bead.
• H1 Protein helps in grouping of nucleosomes while
nucleosomes help in packing of DNA.
• A chain of nucleosomes is once again coiled with 6
nucleosomes per turn to form solenoid (Klug 1982).
• Each solenoid has 1200 bp of DNA and 300 Å size.
• H1 is most species specific, most divergent and act as
marker protein.
• It is called plugging protein. It is rich in lysine and is non
conservative.
• It is not found in pairs and not form nucleosome.
• H3 and H4 are most conserved proteins.

• The beads-on-string structure in chromatin is packaged to form chromatin fibers that
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 that
collectively are referred to as Non-histone Chromosomal (NHC) proteins.
• In a typical nucleus, some region of chromatin are loosely packed (and stains light) and
are referred to as euchromatin. Euchromatin is said to be transcriptionally active
chromatin.
• The chromatin that is more densely packed and stains dark are called as
Heterochromatin. heterochromatin is transcriptionally inactive.
EUCHROMATIN AND HETEROCHROMATIN

Genetic Material
• It is the substance that controls the inheritance of traits from one generation to the next
generation and is also able to express its effect through the formation and functioning of the
traits.
• Properties of Genetic Material
• (i) Hereditary information must be present in the coded form in the genetic material.
• (ii) The genetic material should be able to replicate and then transmitted faithfully to the next generation.
• (iii) The genetic material should also be capable of variations, i.e., mutations and recombinations. These
variations should be stable and inheritable.
• (iv) The genetic material should be able to generate its own kind and also new kinds of molecules.
• (v) Genetic material must be able to express its effect in the form of Mendelian characters.
• These requirements are found in DNA thus, DNA is now recognized as genetic material.

DNA as genetic material
(1) Direct evidences (2) Indirect evidences
• (1) Direct evidences :
• (i) Transformation :
• It is the conversion in the genetic constitution of an
organism by picking up genes present in the remains
of its dead relatives.
• Frederick Griffith in 1928
• Two strains of bacterium Diplococcus pneumoniae
(Streptococcus pneumoniae)
• (a) Smooth (S) or capsulated type- virulent
• (b) Rough (R) or non-capsulated- non virulent

• (a) When S- type bacteria injected into mice. The latter died as a result of pneumonia
caused by bacteria.
• (b) When R- type bacteria injected into mice. The latter lived and pneumonia was not
produced.
• (c) S- type bacteria which normally cause disease were heat killed and then injected into
the mice. The mice lived and pneumonia was not caused.
• (d) The mixed solution of Rough type bacteria (living) and smooth type heat-killed
bacteria (both known not to cause disease) injected into mice.
• Some mice died due to pneumonia and virulent smooth type living bacteria could also be
recovered from their bodies.
• The fourth part of the experiment indicates that some R-type bacteria (non-virulent) were
transformed into
• S- type of bacteria (virulent). The phenomenon is called Griffith effect or
transformation.

• Avery, Macleod and McCarty (l944) repeated the experiment in vitro to identify transforming
substance.
• They proved that this substance is in fact DNA.
• They purified biochemicals from the killed S-type bacteria into three components – DNA,
carbohydrate and protein.
• DNA fraction was further divided into two parts: one with deoxyribonuclease or DNase and the
other without it.
• The four components were then added to separate culture tubes containing R-type bacteria. After
some time they were then analysed for bacteria.
• Only DNA of S-type can changed R-type of bacteria into S-type. Therefore, the character or gene
of virulence is located in DNA.
• Thus they proved that the chemical to be inherited is DNA and it forms the chemical or
molecular basis of heredity.
Biochemical Characterisation of Transforming Principle

(ii) Multiplication of Bacteriophage (Transduction)
• The transfer of genetic material
from one bacterium to another
through bacteriophage is called
transduction.
• T2 is a Bacteriophage which
infects E. coli.
• Hershey and Chase (1952) used
radioactive phosphorus 32P &
radio-active sulphur 35S for their
experiment and proved that DNA
is a genetic matarial.

• They worked with viruses that infect bacteria called bacteriophages.
• The bacteriophage attaches to the bacteria and its genetic material then enters the
bacterial cell.
• The bacterial cell treats the viral genetic material as if it was its own and subsequently
manufactures more virus particles.
• Hershey and Chase worked to discover whether it was protein or DNA from the viruses
that entered the bacteria.
• They grew some viruses on a medium that contained radioactive phosphorus and some
others on medium that contained radioactive sulfur.
• Viruses grown in the presence of radioactive phosphorus contained radioactive DNA but
not radioactive protein because DNA contains phosphorus but protein does not.
• Similarly, viruses grown on radioactive sulfur contained radioactive protein but not
radioactive DNA because DNA does not contain sulfur.

• Radioactive phages were allowed to attach to E. coli bacteria.
• Then, as the infection proceeded, the viral coats were removed from the bacteria by
agitating them in a blender.
• The virus particles were separated from the bacteria by spinning them in a centrifuge.
• Bacteria which was infected with viruses that had radioactive DNA were radioactive,
indicating that DNA was the material that passed from the virus to the bacteria.
• Bacteria that were infected with viruses that had radioactive proteins were not radioactive.
• This indicates that proteins did not enter the bacteria from the viruses.
• DNA is therefore the genetic material that is passed from virus to bacteria

PROPERTIES OF GENETIC MATERIAL
(DNA VERSUS RNA)
• DNA that acts as genetic material in eukaryotic and prokaryotic organism.
• In some viruses, RNA is the genetic material (for example, Tobacco Mosaic viruses, QB
bacteriophage, etc.)
• A molecule that can act as a genetic material must fulfill the following criteria:
• (i) It should be able to generate its replica (Replication).
• (ii) It should chemically and structurally be stable.
• (iii) It should provide the scope for slow changes (mutation) that are required for evolution.
• (iv) It should be able to express itself in the form of ‘Mendelian Characters’.

Why DNA is more stable than RNA ?
• The genetic material should be stable enough not to change with different stages of life
cycle, age or with change in physiology of the organism.
• Stability as one of the properties of genetic material was very evident in Griffith’s
‘transforming principle’ itself that heat, which killed the bacteria, at least did not destroy
some of the properties of genetic material.
• This now can easily be explained in light of the DNA that the two strands being
complementary if separated by heating come together, when appropriate conditions are
provided.
• Further, 2'-OH group present at every nucleotide in RNA is a reactive group and makes
RNA labile and easily degradable. RNA is also now known to be catalytic, hence reactive.
• Therefore, DNA chemically is less reactive and structurally more stable when compared
to RNA. Therefore, among the two nucleic acids, the DNA is a better genetic material.

• RNA is unstable because of the presence of Uracil instead of Thymine.
• RNA has ribose sugar and DNA has deoxy-ribose sugar in their monomers.
• The presence of one free -OH group at 2nd Carbon in ribose sugar makes it more
prone to oxidation.
• The smaller grooves in DNA make it less prone to enzyme degradation and the larger
grooves in RNA makes it more prone to enzymes attack.
• The other reasons why RNA was eliminated as genetic material during the course of
evolution is because of the high chances of mutations that occur during replication.
• DNA has less chances of mutation , because the coding parts (exons, 2% of human
genome) and interrupted by non coding parts (introns, 98% of human genome), hence
in case of a mutation , there are high chances that they occur in non-coding region.
• One more advantage of DNA is the proof reading during the replication process,
which ensures that no mutations are inserted.

Replication of DNA
• The synthesis of DNA from DNA is called Replication.
• It usually occurs during S-phase of cell cycle.
• DNA performs two types of functions
• (A) Autocatalytic : DNA synthesizes new DNA by replication,
• (B) Heterocatalyic : DNA helps in the synthesis of other substances like RNA, protein.
• Semiconservative : One half is parent structure and one half new structure in each
replica. It was firstly suggested by watson and crick (1953).

Experiment of Meselson & Stahl
• Taylor (1957) discovered semiconservative nature of DNA replication by the use H3
radioactive thymidine in broad been (Vicia faba).
• Meselson and stahl (1958) proved that DNA replication is semiconservative. They
performed an experiment on E. coli.
• They cultured E. coli bacteria in culture medium containing N15H4Cl (Heavy isotope of N
as N15) for several generations.
• Now they introduced labelled Bacteria in another culture medium contain N14 H4 Cl
(Normal N14).
• Now they used density gradient centrifugation method with CsCl2 (ceasium chloride)
to examine DNA of its offsprings.
• They found that DNA was intermediate type in first generation in which one strand was
heavy (containing N15) and other strand was light (containing N14).

• Second generation of bacteria contained two types of DNA, 50% light (N14N14) and
50% intermediate (N15N14).
• In third generation of bacteria contained 25% intermediate (N15N14) and 75% light
(N14N14) in 1 : 3 ratio and fourth generation bacteria contained 12.5% N15N14 and 87.5%
N14N14 DNA in 1 : 7 ratio.

MECHANISM OF DNA REPLICATION
(1) Origin of Replication
• It starts at a particular place called origin of replication or Ori.
• In prokaryotes replication starts at one point & entire DNA strand takes part in replication
thus it contains single replicon while in Eukaryotes several replicons present.
• DNA replicaton is bidirectional, semi discontinuous and semiconservative In eucaryotes .
(2) Activation of Deoxyribonucleotides
• The phosphorylated nucleotides (deAMP, deGMP, deCMP , deTMP) are found in inactivated
form.
• They react with ATP in the presence of phosphorylase enzyme & converted in to active
deATP, deGTP, deCTP , deTTP.

• (3) Exposure of DNA helix :
• Helicase enzyme acts over the ori site of DNA
template and unwinds the two strands of DNA.
• SSB (single stranded binding) Protein
prevents the recoiling of uncoiled DNA strands.
• Topoisomerase cause nicking of one strand of
DNA (for removing coils) and resealing the
same.
• Along with Topoisomerase, bacteria possess
another enzyme called DNA Gyrase which can
introduce negative supercoils.
• Whole of the DNA does not open in one stretch
due to very high energy requirement but the
point of separation proceeds slowly from one
end to other .
• It gives the appearance of Y-shaped structure
called replication fork.

• (4) RNA Primer :
• It is small strand of RNA (5–10 nucleotide). It is synthesized at 5’end of new strand with
help of enzyme Primase.
• Formation of RNA primer constitutes the initiation phase of synthesis because without the
presence of RNA primer, DNA polymerase can not add nucleotides.
• (5) DNA Polymerase :
• Prokaryotes possess three types of DNA synthesising enzymes called DNA polymerases
III, II and I.
• They add nucleotides in 5’ 3’ direction on 3’ 5’ strand.
• DNA replication is mainly performed by DNA polymerase III.
• DNA polymerase I is major repair enzyme where as polymerase II is minor repair enzyme.

• (6) Base Pairing :
• Two separated strands of DNA in the replication fork function as template.
• (7) Chain formation
• It requires DNA polymerase III in prokaryotes
• In the presence of Mg++,ATP/ GTP, TPP and DNA polymerase -III, the adjacent
nucleotides attached to nitrogen bases of each template DNA strand establish
phosphodiester bonds and get linked to form

• Replication on one DNA template is
continuous in 5’ 3’ direction due to opening
of its 3’ end this newly formed strand is called
leading strand.
• On the second DNA template the replication
of DNA is discontinuous due to opening of
small stretch of fork at a time.
• Small fragments deposite with the help of
RNA primer. these fragements are called
okazaki fragements (1000 - 2000 nucleotides
in prokaryotes and 100–200 in eukaryotes).
• After deposition of each okazaki fragment
RNA primer is released and gap is filled by the
activity of DNA polymerase thus the new
strand is formed called Lagging strand.
• After deposition of bases DNA Ligase
enzyme seals these bases.
• Thus one strand grows continuously while the
other strand is formed discontinuously hence
DNA replication is semidiscontiuous.

• (8) Proof reading and DNA repair
• DNA polymerase I removes the wrong base and attaches the correct base in the strand
in Prokaryotes where as DNA polymerase in eukaryotes.
• One gene–One enzyme hypothesis
• Beadle and Tatum, (1948) Proposed One gene–One enzyme hypothesis. They
conducted experiments on the nutrition of pink mould (Neurospora crassa).

Transcription Unit
• Synthesis of RNA from DNA is called transcription.
• One of the two strands of DNA takes part in transcription.
• According to Lewin (2000) transcription takes place at anti sense strand or – strand.
• The part of DNA which takes part in transcription is called transcription unit.
• The transcription unit has three components.
• (i) Promoter (ii) Structural gene (iii) Terminator

• (i) Promoter is situated upstream of structural gene at 5’ end of coding strand.
• In most of the cases, the promoter contains AT rich regions called TATA box.
• The latter has groove for the attachment of specific protein. TATA box is also called Pribnow
box.
• Promoter bears different parts for attachment to various transcription factors.
• It is a DNA sequence that provides binding site for RNA polymerase, and it is the presence of a
promoter in a transcription unit that also defines the template and coding strands.

• (ii)Structural gene is a part of strand of DNA having 3’5’ polarity. This strand of
DNA is called template strand or master strand or antisense, or (–) strand.
• DNA-dependent RNA polymerase also catalyse the polymerisation in only one direction,
that is 5'→3' .
• The other strand is non template strand that does not take part in transcription is also also
called sense or coding strand or plus (+) strand.
• (iii) Terminator at downstream of structural gene at 3’ end of coding strand.
• It usually defines the end of the process of transcription

Transcription Unit and the Gene
• A gene is defined as the functional unit of inheritance.
• The DNA sequence coding for tRNA or rRNA molecule also define a gene.
• Cistron as a segment of DNA coding for a polypeptide of RNA.
• Cistron : Prokaryotes bear polycistronic RNA and Eukaryotes bear monocistronic
RNA.
• In eukaryotes, the monocistronic structural genes have interrupted coding sequences – the
genes in eukaryotes are split.
• The coding sequences or expressed sequences are defined as exons.
• The non coding sequences or un expressed sequences are defined as introns.
• Introns or intervening sequences do not appear in mature or processed RNA.

RNA WORLD
• RNA was the first genetic material.
• There is now enough evidence to suggest that essential life processes (such as metabolism,
translation, splicing, etc.), evolved around RNA.
• RNA used to act as a genetic material as well as a catalyst (there are some important
biochemical reactions in living systems that are catalysed by RNA catalysts and not by
protein enzymes).
• But, RNA being a catalyst was reactive and hence unstable.
• Therefore, DNA has evolved from RNA with chemical modifications that make it more
stable.
• DNA being double stranded and having complementary strand further resists changes by
evolving a process of repair.

Structure of RNA (Ribonucleic acid)
• RNA or ribonucleic acid is present in all the living cells.
• It is found in the cytoplasm as well as nucleus.
• Sugar in RNA is ribose sugar.
• Phosphoric acid is similar to that present in DNA.
• Purine bases are adenine and guanine but pyrimidine bases are cytosine and uracil
(thymine being replaced by uracil).

TYPES OF RNA
• RNA is generally involved in protein synthesis but in some viruses, it also serves as a
genetic material.
• Therefore two major types of RNA are as follows.
• (a) Genetic RNA (b) Non-genetic RNA.
• (a) Genetic RNA : H. Frankle-Conrat (1957) showed that RNA present in TMV
(Tabacco Mosaic Virus) is genetic material. RNA acts as a genetic material in most plant
viruses.
• (b) Non-genetic RNA : This type of RNA is present in cells where DNA is genetic
material. Non-genetic RNA is synthesized on DNA template. It is of following three
types. (i) Messenger RNA (mRNA) (ii) Ribosomal RNA (rRNA) (iii) Transfer RNA (tRNA)

• (i) Messenger RNA (mRNA) :
• It carries genetic information present in DNA.
• mRNA constitutes about 5- 10% of the total RNA present in the cell.
• The molecular weight varies from 25,000 to 1,00,000.
• (ii) Ribosomal RNA (rRNA) :
• It is most stable type of RNA and is found associated with ribosomes.
• It forms about 80% of the total cell RNA.
• The molecular weight varies from 35,000 to 10,00,000.
• (iii) Transfer RNA (tRNA) : It is also known as soluble RNA (sRNA).
• These are the smallest molecules which carry amino acids to the site of protein synthesis.
• There are approximately 80 bases.
• These constitute about 10-15% of the total cell RNA.
• The molecular weight of tRNA varies from 23,000 to 30,000.

Structure of t-RNA
• Two dimentional clover leaf model of t-
RNA was proposed by Holley, (1965).
tRNA molecule appears like a clover leaf
being folded with three or more double helical
regions, each having loop.
• (i) Anticodons loop : It has 7 bases out of
which three bases form anticodon (nodoc) for
recognising and attaching to the codon of
mRNA.
• (ii) AA-Binding Site : It is amino acid
binding site. The site lies at the 3’ end
opposite the anticodon and has CCA–OH
group. The 5’ end bears G. Amino acid or
AA-binding site and anticodon are the two
recognition sites of tRNA . (i)
(ii)
(iii)
(iv)
(v)

• (iii) T C Loop : It has 7 bases out of
which (Pseudouridine) and rT
(ribothymidine) are unusual bases.The loop
is the site for attaching to ribosome.
• (iv) DHU Loop : The loop contains 8– 12
bases. It is largest loop and has
dihydroxyuridine. It is binding site for
aminoacyl synthetase enzyme.
• (v) Extra Arm : It is a variable side arm or
loop which lies between T C loop and
anticodon. It is not present in all tRNAs.
The exact role of arm is not known. The
three dimensional structure of this
tRNA was, however, found to be
characteristic L-shaped by Kim (1973).
(iii)
(iv)
(v)
(i)
(ii)

• The mRNA provides the template, tRNA brings aminoacids and reads the genetic code.
• rRNAs play structural and catalytic role during translation.
• There is single DNA-dependent RNA polymerase that catalyses transcription of all types
of RNA in bacteria.
• RNA polymerase binds to promoter and initiates transcription (Initiation).
• It uses nucleoside triphosphates as substrateand polymerises in a template depended
fashion following the rule of complementarity.
• It somehow also facilitates opening of the helix and continues elongation.
• Only a short stretch of RNA remains bound to the enzyme.
• Once the polymerases reaches the terminator region, the nascent RNA falls off, so also the
RNA polymerase.
• This results in termination of transcription.

Mechanism of Transcription in Prokaryotes

Transcription
• It takes place in G1 and G2 phases of interphase of cell cycle in the prokaryotes it occurs in the
cytoplasm.
• It takes place in G1 and G2 phases of interphase of cell cycle in the nucleus in eukaryotes.
• Initiation:
• There is single DNA-dependent RNA polymerase that catalyses transcription of all types of RNA in
bacteria.
• RNA polymerase binds to promoter and initiates transcription (Initiation).
• (sigma) factor recognizes the site of transcription on the promoter region of DNA template and
resting part of enzyme is called core enzyme.
• In Eukaryotes, Three types of RNA polymerase involve in the synthesis of three different types of RNA.
• (i) RNA polymerase : I - synthesizes - r - RNA (28S, 18S, 5·8S).
• (ii) RNA polymerase : II - synthesizes - Precursor of m – RNA that is the heterogeneous nuclear
RNA (hnRNA) and Sn RNA(small nuclear RNAs).
• (iii) RNA polymerase : III - synthesizes - t - RNA, 5S RNA & Sn RNA.

• Sigma factor binds on TATA box on the promoter region of master strand.
• Elongation :- Core enzyme proceeds transcription from promotor region towards
terminator region. Sigma factor is released.
• Termination:- When the chain of RNA reaches at terminator region, rho factor
factor) prevents its synthesis by ATPase activity resulting newly synthesized RNA
becomes separated. Which is called Primary transcript or Hn RNA (Heterogenous
RNA).
• In bacteria, since the mRNA does not require any processing to become active, and also
since transcription and translation take place in the same compartment (there is no
separation of cytosol and nucleus in bacteria), many times the translation can begin much
before the mRNA is fully transcribed.

• In eukaryotes, the primary transcripts hnRNA contain both the exons and the introns
and are non-functional.
• Hence, it is subjected to a process called splicing where the introns are removed and
exons are joined in a defined order.
• hnRNA undergo two additional processing called as capping and tailing.
• In capping an unusual nucleotide (methyl guanosine triphosphate) is added to the 5'-
end of hnRNA.
• In tailing, adenylate residues (200-300) are added at 3'-end in a template independent
manner.
• It is the fully processed hnRNA, now called mRNA, that is transported out of the
nucleus for translation.

GENETIC CODE
• It represents relationship of sequence of Amino acids in polypeptide and sequences of
nucleotides of mRNA/DNA.
• Genetic code was discovered by Nirenberg and Matthaei.
• Crick (1961) stated that deletion or addition of one or two bases in DNA disturbs the
DNA functioning.
• Nirenberg and Matthaei argued that single codon can specify four aminoacids (41 = 4)
Double codon can specify 42 = 16 aminoacids that are not sufficient for the coding of
essential 20 amino acid. Triplet codon can specify 43 = 64 aminoacids. That are sufficient
for 20 amino acids.
• George gamow gave concept of Triplet codon. He also coined the term Genetic code.

• The chemical method developed by Har Gobind Khorana was instrumental in
synthesising RNA molecules with defined combinations of bases (homopolymers and
copolymers).
• Marshall Nirenberg’s cell-free system for protein synthesis finally helped the code to be
deciphered.
• Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful in polymerising
RNA with defined sequences in a template independent manner (enzymatic synthesis of
RNA).

FEATURES OF GENETIC CODE
• (i) Triplet codon : Genetic code is Triplet codon composed of three adjacent nitrogen
bases. Codon - A sequence of three nucleotides specifying an amino acid
• (ii) Start signal or Initiation codon : It is mostly AUG (Methionine codon). But in
prokaryotes it can be GUG and UUG (Lewin 2000), In all cases they specify Methionine.
GUG and UUG specify different amino acids inside the polypeptide chain (GUG -
Valine, UUG- Leucine).
• (iii) Stop signal or Termination codon : Polypeptide chain termination is signalled by
three termination codon UAA (ochre), UAG (Amber) and UGA (opal). they do not
specify any amino acid and hence are called non sense codons.
• (iv) Non ambiguous codon : Normally one codon specifies only one amino acid and
not any other.

FEATURES OF GENETIC CODE
• (v) Non overlapping code : A nitrogen base is a constituent of only one codon.
• (vi) Universal code : A codon specifies the same amino acid in all organisms from virus
to human.
• (vii) Commaless : There are no pauses so that genetic code reads continuously. If a
nucleotide is deleted or added, the whole genetic code will read differently.
• (viii) Colinearity : The sequence of codons of DNA/mRNA correspond to the sequence
of amino acids ina polypeptide.
• (ix) Related codons : Amino acids with similar properties have related codons Ex:
aromatic amino acids tryptophan (UGG), Phenylalanine (UUC, UUU), and tyrosine (UAC,
UAU).

• (x) Degeneracy of codons : Since there are 64 triplet codons and only 20 amino acids,
Tryptophan (UGG) and Methionine (AUG) are specified by single codons.
• All other amino acids are specified by 2–6 codons. The latter are called degenerated
codons.
• Wobble hypothesis (crick, 1966) : In degenerated codons the first two nitrogen bases
are similar while the third one is different. The third nitrogen base has no effect on
coding actually 5’ end base of t-RNA anticodon is able to wobble and get paired with
even noncomplementary base of m-RNA
• Ex: CCA, CCC, CCG, and CCU all specify amino acid proline.

CENTRAL DOGMA
• It is the unidirectional flow of information that proceeds from DNA to mRNA and then
decoding information present in m-RNA in the formation of polypetptide chain or protein
(translation).
• The concept of central dogma was proposed by crick in 1958.
• Commoner (1968) propounded concept of circular flow of information (from DNA
RNA  Protein RNA DNA).

• Temin and Baltimore (1970) reported that retroviruses operate a central dogma
reverse (Inverse flow of information). RNA of these viruses first synthesises DNA
through reverse transcriptase or RNA dependent DNA polymerase.
• This DNA synthesized on RNA template is called c-DNA or Retroposon.
RNA  c-DNA  RNA Protein
REVERSE TRANSCRIPTION

Mutations and Genetic Code
• Sudden inheritable change in an organism is called mutation.
• Mutation was discovered by Hugo de vries in Oenothera lamarckiana (Evening primerose).
• Gene Mutations: Sudden stable changes in the structure of gene or cistron due to change in
nucleotide sequence and nucleotide type are called gene Mutations.
• Usually Gene mutations appear during replication of DNA therefore It is called copy error
mutation.
• Most of the gene mutations include change in single nucleotide. These are called point mutations.
• Seth Wright (1791) firstly recorded point mutation. He observed short legged lamb (ancon
sheep). Another example is sickle cell anemia.
• If mutations takes place in more than one base pair is called gross mutation.

• Gene mutations involve three types
• (i) Substitution: It includes replacement of one type of nitrogenonus base by other.
• It is of two types
• (a) Transition : In this type, one purine is replaced by another purine while one pyrimidine by another pyrimidine.
• (b) Transversion : Purine base is replaced by a pyrimidine base or vice versa.
• (ii) Inversion : A deterioration of DNA by mutagen can change the base sequence of a cistron
in the reverse order.
• (iii) Frame-shift Mutations : In this types the entire reading frame of base sequence shifts
laterally either in the forward direction due to insertion of one or more nucleotides or in the
backward direction due to deletion of one or more nucleotides. It is also called gibberish
mutations. It is of two types
• 1. Deletion : One more nucleotides are eliminated from segment of DNA or gene.
• 2. Insertion : Addition of one or more nucleotides in the segment of DNA or gene.
TYPE OF GENE MUTATIONS

• Insertion or deletion of three or its multiple bases insert or delete one or multiple
codon hence one or multiple amino acids, and reading frame remains unaltered from
that point onwards. Such mutations are referred to as frame-shift insertion or
deletion mutations.
• This forms the genetic basis of proof that codon is a triplet and it is read in a
contiguous manner.

TRANSLATION ( Protein synthesis)
• Translation refers to the process of polymerisation of amino acids to form a polypeptide.
• The order and sequence of amino acids are defined by the sequence of bases in the mRNA.
• The amino acids are joined by a bond which is known as a peptide bond.
• It involves following steps
• (1) Activation of Amino acid
• (2) Synthesis of polypeptide chain
• (i) Initiation of polypeptide chain
• (ii) Elongation of Polypeptide chain
• (iii) Termination of polypeptide chain

(1) Activation of Amino acid
• Formation of a peptide bond requires energy. Therefore, in the first phase itself amino
acids are activated in the presence of ATP and linked to their cognate tRNA– a process
commonly called as charging of tRNA or aminoacylation of tRNA.
• Amino acid + ATP + t-RNA AA-t RNA + AMP + E.
+ AMP + E.
amino acyl tRNA
synthetase

(2) Synthesis of polypeptide chain
• (i) Initiation of polypeptide chain
• Initiation factors are required for initiation of polypeptide
chain
• m-RNA is fused with P-site of small subunit 40S of ribosome
(30 S in procaryote) in the presence Initiation factors to form
40 S - mRNA complex.
• Now 40 S - mRNA complex attracts Amino acyl t- RNA
Complex both fuse and form new complex.
• Now this complex is fused with large subunit 60 S of ribosome.
• At this time P site and complex are covered by Ribosome now
its A site is exposed at the front of next codon of mRNA.
40s ribosome- mRNA
complex
60s ribosome

(ii) Elongation of Polypeptide chain
• Amino acyl t-RNA complex reaches on A side.
• Peptidyl transferase (Ribozyme) enzyme stimulates the fusion of Amino
acids.
• In this process - COOH (carboxylic group) group of amino acid of
complex of P-site and – NH2 group (Amino group) of amino acid of
complex of A-site are fused to form CO – NH bond or peptide bond in
the presence of peptidyl transferase enzyme.
• After this process t- RNA of P site breaks and slips away.
• The dipeptide complex moves on A- site due to translocase enzyme.
• Dipeptide complex is shifted on p-site and the A-site is again exposed at the
front of next codon of mRNA.
• This process repeats again & again as result a long polypeptide chain is
formed.

(iii) Termination of polypeptide chain
• When termination codons UAA or UAG or UGA is exposed on m-RNA.
• It has no information about AA t- RNA complex therefore elongation of polypeptide chain
is stopped.
• GTP based releasing factors require for the activation of termination codons.
• An mRNA also has some additional sequences that are not translated and are referred as
untranslated regions (UTR).
• The UTRs are present at both 5' -end (before start codon) and at 3' -end (after stop codon).
• They are required for efficient translation process.

REGULATION OF GENE EXPRESSION
• Regulation over the functioning of genes is called regulation of gene expression.
• In eukaryotes, it can be exerted at four levels.
• (i) Transcriptional level during formation of primary transcript.
• (ii) Processing like splicing, terminal additions or modifications.
• (iii) Transport of RNAs from nucleus to cytoplasm.
• (iv) Translation level.

• Genes expression is of three types (1) Inducible (2) constitutive (3) repressible.
• (1) Inducible : In this types the Gene is switched on in response to the
presence of substrate (Inducer).
• (2) Constitutive : Genes and their enzymes remain operational throughout.
• (3) Repressible : It is of two types
• (a) Positive Control : The product of regulatory gene initiates expression of genes
under of its control.
• (b) Negative control : The product of a regulatory gene switches off the expression of
genes under its control.

OPERON MODEL
• An operon is a segment of DNA that functions as single regulated unit comprising a
regulator gene, a promoter gene, an operator gene, one or more structural genes.
• Repressor and an inducer or corepressor, these systems are common in prokaryotes.
• First operon lac–Operon was discovered by Jacob and Monod (1961) in E.coli.
• Operons involve two types.
• (1) Inducible operon model
• (2) Repressible operon model.

INDUCIBLE OPERON MODEL
• It is found in catabolic pathway Ex: Lactose operon or Lac operon.
• Lac operon consists of following components.
• (i) Structural genes : They actually control the synthesis of m-RNA through
transcription.
• They determine primary structure of polypeptide chain.
• In Lac operon three structural gene z, y, a take part in the formation of polycistronic
mRNA.
• Structural gene z, y, a that regulates the synthesis of galactosidase, permease and
transacetylase enzymes respectively.
• Galactosidase hydrolyses lactose in glucose & galactose.
• Permease allows the entry of Lactose in the cell.
• Transacetylase performs metabolism of toxic thiogalactosides.

• (ii) Operator gene : It controls the activity of structural genes. When repressor of regulator
gene binds to the operator gene. the operator gene becomes switched off.
• (iii) Promoter gene : It acts as initiation signal. It bears RNA polymerase enzyme. When
operator gene is functional, its RNA polymerase travels on structural gene and perform
transcription.
• (iv) Regulator gene : It regulates the synthesis of repressor . It is also called inhibitor gene
or i gene.
• (v) Repressor : It is proteinaceous substance formed by Regulator gene. It has two allosteric
sites, one for the attachment of operator gene and other for the attachment of inducer.
• (vi) Inducer : It is chemical substance (Hormone, enzyme etc). When inducer is present in
the medium, inducer combines with repressor resulting some conformational changes occur in
the repressor in such a way that it becomes unable to bind on operator gene. therefore the
latter continuously operative. when inducer is completely consumed. Repressor is again
activated. In lac-operon lactose acts as inducer (Actual allolactose acts as inducer) &
substrate.

• Repressible operon System is found in anabolic pathways. Ex:Tryptophan or trp operon of E.coli.
• Trp operon consists of the following components.
• (i) Structural Genes : The genes are connected to transcription of mRNAs. Tryptophan operon has
five structural genes trp E, D, C, B, A. They form enzymes for five steps of tryptophan synthesis.
• (ii) Operator Gene : It regulates the activity of structural genes usually. Aporepressor produced by
regulator gene is unable to completely block operator gene.
• (iii) Promoter Gene : It acts as initiation signal. It bears RNA polymerase enzyme. When operator
gene is functional, its RNA polymerase travels on structural gene and perform transcription.
• (iv) Other Regulatory Components : It involves two components that lie between operator gene
and structural gene E.
• (a) Leader sequence (L) : It is controller of attenuator.
• (b) Attenuator (A) : It helps in reducing tryptophan synthesis when it is available in sufficient amount without
switching off the operon.
REPRESSIBLE OPERON MODEL

• (v) Regulator Gene : It produces proteinaceous component (such as Aporepressor) for
possible blocking the activity of operator gene.
• (vi) Aporepressor : It is a proteinaceous substance formed by regulator gene. Independently
it is unable to block the activity of operator gene. for this purpose It requires a corepressor.
• (vii) Corepressor : It is a nonproteinaceous component of repressor that may be an end
product of reactions.
• In trp operon, when end product Tryptophan is accumulated in sufficient amount its some
molecules act as corepressor. the latter combines with aporepressor. and forms repressor that
block the operator gene resulting structural genes become switched off. The phenomenon is
called feed-back repression that exerts a negative control.

• HGP is a mega project started by U.S. Department of Energy and national Institute of
Health for sequencing human genome in 1990.
• Welcome Trust (UK) joined the project as a major partner.
• Later on japan, France, Germany, China and some other countries also joined it.
• Aims of HGP :
• (i) To determine the sequence of 3.2 billion base pairs of human genome
• (ii) To Identify all the genes of human genome and determined their functions
• (iii) To Identify those genes that are responsible for genetic disorders.
• (iv) To Store this information in data bases.
• (v) The project may result in many ethical, legal and social issue which must be addressed and
solved.
Human Genome project (HGP)

• Methodology : Two approaches have been recognized for analysing the human genome.
• (i) ESTs or expressed sequence tags : To Identify all the genes that are expresed as RNA.
• (ii) Sequence annotation : Sequencing both coding and noncoding regions of whole
genome and assigning the different regions with functions. The project was completed for
sequencing in 2003.
• However, chromosome I was last to be sequenced in May 2006.
Human Genome project (HGP)

• (1) Human genome contains 30,000 genes which are much lower than previous estimate of
80,000 – 10,0000.
• (2) Human genome contains 3.1647 billion nucleotide base.
• (3) Less than 2% of the genome shows structural genes that code for proteins.
• (4) Average gene size is 3000 base pairs but size vary greatly, with the largest known human
gene being dystrophin at 2·4 million bases.
• (5) Chromosome I contains 2968 genes (maximum gene) while Y-chromosome bears 231
genes (minimum genes) in human chromosome.
• (6) 99.9% of the nucleotide bases are exactly similar in all human beings.
• (7) Repeated sequences makeup very large portion of the human genome.
Salient features of human Genome

• (8) About 1.4 million locations have been reported where single nucleotide differences or
SNPs(snips) or single nucleotide polymorphism are found.
• They have the potential to helping and finding chromosomal locations for disease associated
sequences and tracing human history.
• (9) Repeated sequences are stretches of DNA sequnces that are repeated hundred to thousand
times.
• They are thought to have no direct coding functions but they shed light on chromosome
structure dynamics & evolution.
• Many non human organisms like, Bacteria (E. coli), yeast, Caenorhabditis elegans, Drosophila,
plants (Rice and Arabidopsis) etc have also been sequenced.

• Sir Alec jeffreys (1984) discovered the DNA fingerprinting technique.
• Dr. K. kashyap and Dr. Lalji Singh started the fingerprinting technology in India.
• What is DNA-fingerprinting ?
• It is a technique to identify a person on the basis of his//her DNA specificity.
• Principle of DNA Fingerprinting : Jeffreys observed that DNA of each individual contains
some noncistronic hyper-variable repeat minisatellite sequences. These repeat minisatellite
sequences are called variable number of tandem repeats or VNTRs (These are also
called minisatellites). The numbers of repeat show very high degree of polymorphism. As a
result the size of VNTR varies in size from 0.1 to 20 kb.
• These sequence show high degree of polymorphism and form the basis of DNA
fingerprinting. Since DNA from every tissue (such as blood, hair-follicle, skin, bone, saliva,
sperm etc.), from an individual show the same degree of polymorphism.
DNA Fingerprinting (DNA Profiling)

• What is DNA polymorphism ?
• DNA Polymorphism (variation at genetic level) arises due to mutations OR if an inheritable
mutation is observed in a population at high frequency, it is referred to as DNA
polymorphism (frequency greater than0.01)
• The DNA polymorphism are similar only in monozygotic twins and vary in number from
person to person.
• These repeats are inherited in the offsprings by their parents.
• These are used as genetic markers in a personal identity test.
• One half of VNTR alleles of the offspring resemble that of the mother and other half that
of the father.
• * Inheritable mutation is observed in a population at high frequency, it is referred to as
DNA polymorphism.

• (i) The DNA is isolated from the nuclei of white blood cells or spermatozoa or the hair follicle cells.
• (ii) Restriction endonuclease enzyme performs digestion of DNA molecules. The Restriction
endonuclease enzyme cuts DNA in to fragements. The fragments of DNA also contain the VNTRs.
• (iii) Gel electrophoresis is used to separate these fragments according to their size.
• (iv) VNTRs are multiplied through PCR technique. The VNTRs are treated with alkaline chemicals
to split them into single stranded DNAs.
• (v) SS DNA fragments of the gel are shifted onto a nylon paper/nitrocellulose membrane by
southern blotting technique.
• (vi) Radioactive SS-DNA-probes are used for hybridization with VNTR on the nylon membrane.
• (vii) Now the nylon membrane is exposed at the front of X-ray film and mark the places where the
radioactive DNA probes have bound to the DNA fragements. These places are marked as dark bands
when X-ray film is developed. This is known as autoradiography.
• (viii) The dark bands on X-ray film show the DNA fingerprints.
Technique of DNA fingerprinting

• (i) It is very useful in the detection of crime and illegal pursuits.
• (ii) Paternity maternity disputes can be solved by DNA fingerprinting.
• (iii) It can be used to study the breeding patterns of animals facing the danger of extinction.
• (iv) It provides information about relationship of man with apes.
• (v) It determining population and genetic diversities.
Applications of DNA Fingerprinting

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

• (iii) Satellite DNA : It is a part of repetitive DNA that has long repetitive nucleotide
sequences in tanden that forms a separate fraction on density ultracentrifugation. satellite
DNA is of two types.
• (a) Microsatellite sequences 1– 6 bp repeat units flanked by conserved sequences.
• (b) Minisatellite sequences 11– 60 bp flanked by conserved restriction sites. these are
hypervariable and are specific for each individual. They are used in DNA finger printing.

Std 12 chapter 6

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