Molecular Basis of Inheritance Mr.

Pravin Bhosale

The Discovery of DNA:

• Modern understanding of DNA has evolved from the discovery of nucleic acid to the
development of the double-helix model.
• In 1869, Friedrich Miescher began working with white blood cells which are the major
component of pus from infections.
• He collected a lot of pus from bandages at the local hospital.
• He used a salt solution to wash the pus off the bandages.
• When he added a weak alkaline solution to the cells, the cells lysed and nuclei precipitated
out of the solution.
• From the cell nuclei, he isolated a unique chemical substance to which he called nuclein.
• Chemically, nuclein has high phosphorus content.
• Moreover it showed acidic properties. Hence it was named as nucleic acid.
• By the early 1900s, we knew that Miescher's nuclein was a mix (mixture) of proteins and
nucleic acids.
• There are two kinds of nucleic acids. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
The Genetic Material is a DNA:
• By the early 1900s, geneticist knew that genes control the inheritance of traits, that genes
are located on chromosome and that chemically chromosomes are mainly composed of DNA
and proteins.
• Initially, most geneticists thought that protein are large, complex molecules and store
information needed to govern cell metabolism.
• Hence it was assumed that proteins caused the variations observed within species.
Molecular Basis of Inheritance
• On the other hand DNA thought to be small, simple molecule whose composition varied
little among species.
• Over the time, these ideas about DNA were shown to be wrong.
• In fact DNA molecules are large and vary tremendously within and among species.
• Variations in the DNA molecules are different than the variation in shape, electrical charge
and function shown by proteins so it is not surprising that most researchers initially favoured
proteins as the genetic material.
• Over a period of roughly 25 years (1928-1952), geneticists became convinced that DNA and
not protein, was the genetic material.
• Let us study three important contributions that helped cause this shift of opinion.

Griffith’s experiments :

In 1928, a British medical officer Frederick Griffith performed an experiment on

bacterium Streptococcus pneumoniae that causes pneumonia in humans and other mammals.
Griffith used two strains or two genetic varieties of Streptococcus to find a cure for pneumonia,
which was a common cause of death at that time.
The two strains used were :

i. Virulent, smooth, pathogenic and encapsulated S type.

ii. Non-virulent, rough, non-pathogenic and non-capsulated R type.
Griffith conducted four experiments on these bacteria.
1.First, when he injected bacteria of strain R to mice, the mice survived because it did not develop
pneumonia.

2.Second, when he injected bacteria of strain S to mice, the mice developed pneumonia and died.

3.In the third experiment, he injected heat-killed strain S bacteria to mice, once again the mice
survived.

4.In fourth experiment, he mixed heat-killed S bacteria with live bacteria of strain R and injected to
mice.

The mice died and Griffith recovered large numbers of live strain S bacteria from the blood of the
dead mice.
In these four experiments, something had caused harmless strain R bacterium to change into deadly
S strain bacterium.
Griffith showed that the change was genetic.
 • He suggested that genetic material from heat-killed strain S bacterium had somehow
changed the living strain R bacterium into strain S bacterium.
• Griffith concluded that the R-strain bacterium must have taken up, to what he called a
"transforming principle" from the heat killed S bacterium, which allowed R strain to get
transformed into smooth-coated bacterium and become virulent.
Avery, McCarty and MacLeod’s experiment:
• In 1944, after some 10 years of research and experimentation, U. S. microbiologists Oswald
• T. Avery, Colin M. MacLeod and Maclyn McCarty (all at Rockefeller University in New York)
first evidenced to prove the DNA is a genetic material (transforming principle), through the
experiments.
• They purified DNA, RNA, Proteins (enzymes) and other materials from cell free extract of S
cells/ strain and mixed with heat killed S strain and R cells separately to confirm which one
could transform living R cells into S cells.
• Only DNA was able to transform harmless strain R into deadly strain S.
• They also discovered that protein –digesting enzymes (proteases), RNA-digesting enzyme
(RNAases) did not affect transformation, so the transforming substance was neither a
protein nor RNA.
• DNA digested with DNAse did inhibit the transformation, suggesting that DNA caused the
transformation.

• These experiments proved that the transforming principle is DNA but all biologists were not
convinced
Finally, Alfred Hershey and Martha Chase (1952) proved that DNA is the genetic material and not
proteins, by using bacteriophages.
Hershey - Chase Experiment:
1. Hershey and Chase worked with radioactive DNA (labelled DNA), but
viruses that infect bacteria i.e. not radioactive proteins because DNA
bacteriophages, which are composed contains phosphorus (labelled DNA)
of DNA and protein. but proteins do not. Similarly, viruses
2. They used radioactive phosphorous grown on radioactive sulphur
32P in the medium for some viruses contained radioactive protein but not
and radioactive sulphur 35S for some radioactive DNA because DNA does
others. not contain sulphur. Radioactive
3. They grew some viruses on a medium phages were allowed to infect E.coli
that contained radioactive bacteria grown on the medium
phosphorus and some others on containing normal ‘P’ and ‘S’.
medium that contained radioactive 5. Then, as the infection proceeded, the
sulphur. viral coats were removed with the
4. Viruses grown in the presence of help of centrifuge.
radioactive phosphorus contained
 6. Bacteria which were infected by
viruses with radioactive DNA, were
radioactive, indicating that DNA was
the material that passed from the
viruses to the bacteria.
7. Bacteria which were infected by
viruses having radioactive sulphur
(protein) were not radioactive. This
indicates that proteins from the
viruses, did not enter the bacteria.
8. DNA is, therefore, the genetic
material that is passed from virus to
bacteria
9. In other words, sometime after
infection, radioactivity for ‘P’ and ‘S’
was tested.
10. Only radioactive ‘P’ was found inside
the bacterial cell, indicating that DNA
is the genetic material.
4.3 DNA packaging :
Length of DNA double helix molecule, in a typical mammalian cell is approximately 2.2 meters.
Approximate size of a typical nucleus is 10-6 m. How this long DNA molecule can be then
accommodated in such a small nucleus? It, therefore, must be condensed, coiled and super coiled to
fit inside such small nucleus.
Packaging in Prokaryotes:

• In prokaryotes like E. coli, cell size is almost 2-3m long.

• They do not have well organized nucleus. It is without nuclear membrane and nucleolus.
• The nucleoid is small, circular, highly folded, naked ring of DNA which is 1100m long in
perimeter, containing about 4.6 million base pairs.
• The 1100m long (approximately 1.1 mm, if cut and stretched out) nucleoid is to be fitted or
packaged into a cell which is hardly 2-3m long.
• Hence the negatively charged DNA becomes circular, reducing the size to 350mm in
diameter.
• This is further reduced to 30mm in diameter because of folding/ looping. 40-50 domains
(loops) are formed.
• Formation of loops is assisted by RNA connectors.
• Each domain is further coiled and supercoiled, thereby reducing the size down to 2m in
diameter.
• This coiling (packaging) is assisted by positively charged HU (Histone like DNA binding
proteins) proteins and enzymes like DNA gyrase and DNA topoisomerase I, for maintaining
super coiled state.
Packaging in Eukaryotes:
1. Eukaryotes show well organized nucleus containing nuclear membrane, nucleolus and
thread-like material in the form of chromosomes.
2. In the chromosomes, DNA is associated with histone and non-histone proteins as was
reported by R. Kornberg in 1974.
3. The organization of DNA is much more complex in eukaryotes.
4. Depending upon the abundance of amino acid residues with charged side chains, a protein
acquires its charge.
5. Histones are the proteins that are rich in lysine and arginine residues.
6. Both these amino acid residues are basic amino acids and carry positive charges with them.
7. So, histones are a set of positively charged, basic proteins (histones + protamine).
• These histones organize themselves to make a unit of 8 molecules known as histone
octamer.
• The negatively charged helical DNA is wrapped around the positively charged histone
octamer, forming a structure known as nucleosome.

• The nucleosome core is made up of two molecules of each of four types of histone proteins
viz. H2A, H2B, H3 & H4
• H1 protein binds the DNA thread where it enters (arrives) and leaves the nucleosome.
One nucleosome approximately contains 200 base pair long DNA helix wound around it (fig.
4.5). About 146 base pair long segment of DNA remains present in each nucleosome.
• Nucleosomes are the repeating units of chromatin, which are thread-like, stained (coloured)
bodies present in nucleus.
• These look like ‘beads-on-string’, when observed under an electron microscope.
• DNA helix of 200 bps wraps around the histone octamer by 1¾ turns.
Six such nucleosomes get coiled and then form solenoid that looks like coiled telephone wire.
The chromatin is packed to form a solenoid structure of 30 nm diameter (300A0) and further
supercoiling tends to form a looped structure called chromatin fibre, which further coils and
condense at metaphase stage to form the chromosomes.
The packaging of chromatin at higher levels, need additional set of proteins that are called Non-
Histone Chromosomal Protein (NHC)

Non-Histone Chromosomal Proteins (NHC) :

These are additional sets of proteins that contribute to the packaging of chromatin at a higher level.
Heterochromatin and Euchromatin :

1.Heterochromatin:
• In eukaryotic cells, some segments of chromonema/ chromosome during interphase and
early prophase remain in a condensed state.
• These region constitute heterochromatin.
• This term was proposed by Heitz.
• These regions are localized near centromere, telomeres and are also intercalated.
• It is genetically mostly inactive.
• It stains strongly and appears dark.
• Heterochromatin is 2 to 3 times more rich in DNA than in the euchromatin.
2. Euchromatin:

• The regions of chromonema which are in non-condensed state, constitute euchromatin.

Euchromatic regions stain light.
• Euchromatin is genetically very much active and fast replicating.
• Euchromatin is transcriptionally active, while heterochromatin is transcriptionally almost
inactive.
4.4 DNA Replication :
• The DNA molecule regulates and controls all the activities of the cell.
• Because of its unique structure, it is able to control the synthesis of other molecules of the
cell.
• At the same time when the cell reproduces, the DNA also should duplicate itself to distribute
equally to the daughter cells.
• As a carrier of genetic information,
DNA has to perform two important functions :
a.Heterocatalytic function :
1. When DNA directs the synthesis of chemical molecules other than itself, then such functions
of DNA are called heterocatalytic functions.
2. Eg. Synthesis of RNA (transcription), synthesis of protein (Translation), etc.
b. Autocatalytic function :
1. When DNA directs the synthesis of DNA itself, then such function of DNA is called
autocatalytic function.
2. Eg. Replication.
• The process by which DNA duplicates itself is called replication.

• Through replication, it forms two copies that are identical to it. In eukaryotic organisms,
replication of DNA takes place only once in the cell cycle.
• It occurs in the S- phase of interphase in the cell cycle.
• DNA replicates through Semiconservative mode of replication.
• The model for Semiconservative replication was proposed by Watson and Crick, on the basis
of antiparallel and complementary nature of DNA strands.
The process of Semiconservative replication is as below:
1.Activation of Nucleotides:
I. The four types of nucleotides of DNA i.e. dAMP, dGMP, dCMP and dTMP are present in the
nucleoplasm.
II. They are activated by ATP in presence of an enzyme phosphorylase.
III. This results in the formation of deoxyribonucleotide triphosphates i.e. dATP, dGTP, dCTP and
dTTP. The process is known as Phosphorylation.
2. Point of Origin or Initiation point:
I. It begins at specific point ‘O’ -origin and terminates at point ‘T’. Origin is flanked by ‘T’
sites.
II. The unit of DNA in which replication occurs, is called replicon.
III. In prokaryotes, there is only one replicon At the point ‘O’, enzyme endonuclease nicks
one of the strands of DNA, temporarily.
IV. The nick occurs in the sugar-phosphate back bone or the phosphodiester bond.
3. Unwinding of DNA molecule:
I. Now enzyme DNA helicase operates by breaking weak hydrogen bonds in the vicinity of ‘O.
II. The strands of DNA separate and unwind.
III. This unwinding is bidirectional and continues as ‘Y’ shaped replication fork.
IV. Each separated strand acts as template.
 V. The two separated strands are prevented from recoiling (rejoining) by SSBP (Single strand
binding proteins).
VI. SSB proteins remain attached to both the separated strands so as to facilitate synthesis of
new polynucleotide strands.

4. Replicating fork:
I. The point formed due to unwinding and separation of two strands appear like a Y-shaped
fork, called replicating/ replication fork.
II. The unwinding of strands imposes strain which is relieved by super-helix relaxing enzyme.

5. Synthesis of new strands:

I. Each separated strand acts as mould or template for the synthesis of new complementary
strand.
II. It begins with the help of a small RNA molecule, called RNA primer.
III. RNA primer get associated with the 3’ end of template strand and attracts complementary
nucleotides from surrounding nucleoplasm.
IV. These nucleotides molecules bind to the complementary nucleotides on the template strand
by forming hydrogen bonds (i.e. A=T or T=A; G = C or C = G).
V. The newly bound nucleotides get interconnected by phosphodiester bonds, forming a
polynucleotide strand.
VI. The synthesis of new complementary strand is catalyzed by enzyme DNA polymerase.
VII. The new complementary strand is always formed in 5’3’ direction.
6. Leading and Lagging strand:
I. The template strand with free 3’ end is called leading template and with free 5’ end is called
lagging template.
 II. The process of replication always starts at C-3 end of template strand and proceeds towards
C-5 end. As both the strands of the parental DNA are antiparallel, new strands are always
formed in 5’ → 3’ direction.
III. One of the newly synthesized strand develops continously towards replicating fork is called
leading strand.
IV. Another new strand develop discontinuously away from the replicating fork is called lagging
strand.
V. Maturation of Okazaki fragments :
VI. DNA synthesis on lagging template takes place in the form of small fragments, called Okazaki
fragments (named after scientist Okazaki).
VII. Okazaki fragments are joined by enzyme DNA ligase.
VIII. RNA primers are removed by DNA polymerase and replaced by DNA sequence with the help
of DNA polymerase-I in prokaryotes and DNA polymerase-α in eukaryotes.
IX. Finally, DNA gyrase (topoisomerase) enzyme forms double helix to form daughter DNA
molecules.
7. Formation of daughter DNA molecules:
I. At the end of the replication, two daughter DNA molecules are formed. In each daughter
DNA, one strand is parental and the other one is totally newly synthesized.
II. Thus, 50% is contributed by mother DNA.
III. Hence, it is described as semiconservative replication.
Experimental confirmation :
Semiconservative Replication : In newly formed DNA molecule, one strand is old (i.e. conserved) and
other strand is newly synthesized.
Thus, it is called Semiconservative mode of replication.
It is experimentally proved by Matthew Meselson and Franklin Stahl (1958) by using equilibrium -
density - gradient - centrifugation technique.

1. Meselson and Stahl in 1958 performed an experiment to prove semiconservative nature (mode)
of replication.
2. They cultured bacteria E.coli in the medium containing 14N (light nitrogen) and obtained
equilibrium density gradient band by using 6M CsCl2. The position of this band is recorded.
3.E. coli cells were then tranferred to 15N medium (heavy isotopic nitrogen) and allowed to replicate
for several generations.
At equilibrium point density gradient band was obtained, by using 6M CsCl2. The position of this
band is recorded.
4. The heavy DNA (15N) molecule can be distinguished from normal DNA by centrifugation in a 6M
Cesium chloride (CsCl2) density gradient.
The density gradient value of 6M CsCl2 and 15N DNA is almost same.
Therefore, at the equilibrium point 15N DNA will form a band.
In this both the strands of DNA are labelled with 15N.
5. Such E. coli cells were they transferred to another medium containing 14N i.e. normal (light)
nitrogen.
After first generation, the density gradient band for 14N 15N was obtained and its position was
recorded.
After second generation, two density gradient bands were obtained - one at 14N 15N position and
other at 14N position.
6. The position of bands after two generations clearly proved that DNA replication is
Semiconservative.
4.5 Protein synthesis :
1. Proteins are very important biomolecules.
2. They serve as structural components, enzymes and hormones.
3. The cell needs to synsthesize new protein molecules.
4. The process of protein synthesis includes transcription and translation.
5. The process of copying of genetic information from one (template) strand of DNA into a
single stranded RNA transcript, is termed as transcription.
6. During this process, synthesis of complementary strand of RNA takes place (Except that the
Adenine nitrogen base pairs with the Uracil base instead of Thymine).
Central Dogma

• Double stranded DNA molecule gives rise to mRNA which acts as a messenger to programme
the synthesis of a polypeptide chain (protein).
• This type of unidirectional flow of information from DNA to RNA to protein/ proteins is
referred as central dogma of molecular biology.
• It was postulated by F.H.C. Crick in 1958.

The present concept of central dogma in retroviruses or riboviruses is given by Temin (1970) and
Baltimore (1970):

Accordingly enzyme RNA dependent DNA polymerase, synthesizes DNA from RNA
A.Transcription:
1. During transcription, information of only one strand of DNA is copied into RNA.

2. This strand of DNA acts as template.

3. Enzyme RNA polymerase catalyses the formation of RNA transcript.
 4. DNA is located in the nucleoid of Prokaryotes and in nucleus of Eukaryotes.

5. DNA transcription takes place in nucleus in eukaryotes whereas translation occurs in

cytoplasm.
6. DNA transfers information to m-RNA which then moves to ribosomes.
7. Transcription occurs in the nucleus during G1 and G2 phases of cell cycle.

8. DNA has promotor and terminator sites.

9. Transcription starts at promotor site and stops at terminator site.
10. Actually the process of transcription, in both Prokaryotes and Eukaryotes, involves three
stages viz. Initiation, Elongation and Termination.

Transcription Unit:

1. Each transcribed segment of DNA is called transcription unit.

2. It consists of a. Promotor, b. The structural gene, c. A terminator.
3. Two strands of DNA in the structural gene show following features :
a.Promotor
• Promotor is located towards 5’ end of structural gene i.e. upstream.
• It is a DNA sequence that provides binding site for enzyme RNA polymerase.
• RNA polymerase binds to specific Promotor.
• In prokaryotes, the enzyme recognizes the promotor by its sigma factor sub unit.
b. Structural genes
• Two strands of DNA have opposite polarity.
• DNA dependent RNA polymerase catalyses polymerisation in 5’→3’ direction.
• So the DNA strand having 3’→5’ polarity acts as template strand.
• The other strand of DNA having 5’→3’ polarity is complementary to template strand.
• The sequence of bases in this strand, is same as in RNA (where Thymine is replaced by
Uracil).
• It is the actual coding strand.
 • The information on this strand of DNA is copied on mRNA.
• This is called sense strand.
c. The terminator
Terminator is located at 3’ end of coding strand i.e. downstream. It defines the end of the
transcription process.

4.After binding to promoter, RNA polymerase moves along the DNA and causes local unwinding of
DNA duplex into two chains in the region of the gene.

5.Exposed ATCG bases project into nucleoplasm.

6.Only one strand functions as template (antisense strand) and the other strand is complementary
which is actually a coding strand (sense strand).
7.The ribonucleoside tri phosphates join to bases of DNA template chain.

8.As transcription proceeds, the hybrid DNA-RNA molecule dissociates and makes mRNA molecule
free.

9.When RNA polymerase reaches the terminator signal on the DNA, it leaves DNA and fully formed
mRNA (primary transcript) is released.
10.As the mRNA grows, the transcribed region of DNA molecule becomes spirally coiled and attains
(regains) double helical form.
11.In bacteria, m-RNA does not require any processing because it has no introns.
12.Prokaryotes posses only one type of RNA polymerase.
13.In eukaryotes, there are three types RNA polymerases.
14.RNA polymerase-I transcribes r-RNA.
15.RNA polymerase-II transcribes m-RNA (primary transcript) and heterogeneous nuclear RNA (or
hnRNA).
16.RNA polymerase-III is responsible for transcription of t-RNA and small nuclear RNA (snRNA).
Transcription unit and the gene:
1. The DNA sequence coding for m-RNA/ t-RNA or r- RNA is defined as a gene.
2. Cistron is a segment of DNA coding for a polypeptide.
3. A single structural gene in transcription unit is said to be monocistronic
4. where as a long segment of DNA having set of various structural genes in one transcription
unit is referred as polycistronic.
5. Structural genes in eukaryotes have interrupted non-coding sequences (introns).
6. The coding sequences or express- sequences are defined are exons.
7. Only exons appear in processed mRNA in Eukaryotes.
Processing of hnRNA:
1. In eukaryotes, forms of RNA transcribed from DNA are called primary transcripts.
2. Such transcripts undergo changes called processing or maturation before becoming
functional.
3. Primary transcript is non functional and contains both exons and introns.
4. During processing only introns are removed by the process called splicing.
5. Exons are joined in a definite sequence (order) by DNA ligase enzyme.
6. Heterogeneous nuclear RNA, undergoes the process of capping and tailing.
7. In capping, methylated guanosine tri phosphate is added to 5’ end of hnRNA.
8. In tailing, polyadenylation take place at 3’end.
9. It is the fully processed hnRNA, now called m-RNA.
10. For translation m-RNA is transported out of the nucleus through nuclear pore.
Genetic Code:
1. It is already known that DNA is a master molecule of a cell that initiates, guides, regulates
and controls the process of protein synthesis.
2. To perform this complicated function, it must carry the requisite information for the
synthesis of proteins.
3. Obviously this information has to be verily located in the DNA itself.
4. The site for storing this information lies in the sequence of nucleotides (i.e. nitrogen bases),
as evidenced by Yanofski and Sarabhai (1964).
5. About, 20 different types of amino acids are involved in the process of synthesis of proteins.
6. DNA molecule has 4 types of nitrogen bases to identify these 20 different types of amino
acids. Question arises then, how is it possible that 20 types of amino acids are encoded by 4
types of nitrogen bases?
7. According to F.H.C. Crick, this information is stored in the form of coded language
(cryptogram) called genetic code, that contains code words (codons) each one specifying
(representing) specific amino acid.
8. Genetic code, therefore, is a collection of base sequences that correspond to each amino
acid.
• A single nitrogen base in a codon (singlet codon) will encode for only four different
types of amino acids.
• A combination of two nitrogen bases (doublet codon) will specify only 16 different
types of amino acids.
• A combination of three nitrogen bases (triplet codon) will specify 64 different types
of amino acids. Hence G. Gamov (1954) suggested that in a codon, there must be
combination of three consecutive nitrogen bases that will be sufficient to specify 20
different types of amino acids.
9. Thus, there would be 64 different codons (code words) in the dictionary of genetic code and
that each code word has to be a triplet codon.
10. Every three consecutive nucleotides in DNA will constitute a triplet codon.
11. Genetic code is a triplet code, was evidenced first by Crick (1961) using “frame- shift
mutation”. However, M. Nirenberg and Matthaei were able to synthesize artificial m-RNA
which contained only one type nitrogenous base i.e. Uracil (Homopolymer).
12. This synthetic poly-U sequence was transferred to protein synthesizing enzymes.
13. A small polypeptide molecule was produced/ formed by the linking of phenylalanine
molecules.
14. This explains that UUU codes for phenyl alanine.
15. Later different homopolymer codons were deciphered.
16. Codons formed by two or more bases were also tried.
Dr. Har Gobind Khorana :

• He devised a technique for artificially synthesizing m- RNA with repeated sequences of

known nucleotides.
• By using synthetic DNA, Dr. Khorana prepared chains of polyribonucleotides with known
repeated sequences of two or three nucleotides.
• eg. CUC UCU CUC UCU.
• This resulted in formation of polypeptide chain having two different amino acids placed
alternately (Leucine and Serine).
• Similarly polynucleotide chain with three- nitrogen base repeats gave polypeptide chain with
only one amino acids.
• Eg. CUA CUA CUA CUA (leucine).
 • Later, Severo Ochoa established that the enzyme (polynucleotide phosphorylase) was also
helpful in polymerising RNA with defined sequences in a template- independent manner (i.e.
enzymatic synthesis of RNA).
• Finally Nirenberg, Matthaei and Ochoa deciphered all the 64 codons in the dictionary of
genetic code.

• During replication and transcription, a nucleic acid is copied to form another nucleic acid.
• These two processes are based on complementarity principle.
• During translation, genetic information is transferred from a polymer of nucleotides to a
polymer of amino acids.
• Here, complementarity principle does not exist.
It is evident that change in nucleic acid (genetic material) results in the change in amino acids of
proteins.
This clearly explains that genetic code directs the sequence of amino acids during synthesis of
proteins.
Characterestic of Genetic code:
Genetic code of DNA has certain fundamental characteristics –
i.Genetic code is a triplet code:
• Sequence of three consecutive bases constitute codon, which specifies one particular amino
acid. Base sequence in a codon is always in 5’ 3’ direction.
• In every living organism genetic code is a triplet code.
ii. Genetic code has distinct polarity :

• Genetic code shows definite polarity i.e. direction.

• It, therefore, is always read in 5’ 3’ direction and not in 3’ 5’ direction.
• Otherwise message will change e.g. 5’ AUG 3’.
iii. Genetic code is non-overlapping :
• Code is non overlapping i.e. each single base is a part of only one codon.
• Adjacent codons do not overlap.
 • If non-overlapping, then with 6 consequtive bases only two amino acid molecules will be in
the chain.
• Had it been overlapping type, with 6 bases, there would be 4 amino acid molecules in a
chain. Experimental evidence is in favour of non-overlapping nature.
iv. Genetic code is commaless :

• There is no gap or punctuation mark between successive/ consecutive codons.

v. Genetic code has degeneracy :
• Usually single amino acid is encoded by single codon.
• However, some amino acids are encoded by more than one codons. e.g. Cysteine has two
codons, while isoleucin has three codons.
• This is called degeneracy of the code.
• Degeneracy of the code is explained by Wobble hypothesis.
• Here, the first two bases in different codons are identical but the third one, varies.
vi. Genetic code is universal :
• By and large in all living organisms the specific codon specifies same amino acid.
• e.g. codon AUG always specifies amino acid methionine in all organisms from bacteria up to
humans.
vii. Genetic code is non-ambiguous :
• Specific amino acid is encoded by a particular codon.
• Alternatively, two different amino acids will never be encoded by the same codon.
viii. Initiation codon and termination codon:
• AUG is always an initiation codon in any and every mRNA.
• AUG codes for amino acid methionine.
• Out of 64 codons, three codons viz. UAA, UAG and UGA are termination codons which
terminate/ stop the process of elongation of polypeptide chain, as they do not code for any
amino acid.
ix. Universal : Usually in all organisms the specific codon specifies same amino acid.
x. Codon and anticodon :
• Codon is a part of DNA e.g. AUG is codon. It is always represented as 5’ AUG 3’.
• Anticodon is a part of tRNA.
• It is always represented as 3’UAC 5’.
Mutations and Genetic Code:
1. Mutation is a phenomenon in which sudden change in the DNA sequence takes place.
2. It results in the change of genotype (i.e. character).
3. Along with recombination, mutation is raw material for evolution as it also results in
variations.
4. During mutation, possibility of loss (deletion) or gain (insertion/ duplication) of a segment of
DNA results in alteration in the chromosome.
5. Mutation can also occur due to change in a single base pair of DNA.
6. This is known as point mutation. Eg. Sickle cell anaemia (Refer to earlier chapter).
7. Deletion or insertion of base pairs of DNA causes frame – shift mutations or deletion
mutation.
 8. Insertion or deletion of one or two bases changes the reading frame from the point of
insertion or deletion.
9. Insertion or deletion of three or multiples of three bases (insert or delete) results in insertion
or deletion of amino acids and reading frame remains unaltered from that point onwards.
t-RNA- the adapter molecule:
Scientists considered that there has to be a mechanism in which t-RNA will read the codon and also
simultaniously binds with the amino acid as amino acid does not have any special capacity to read
the codon.
So t-RNA is considered as an adapter molecule. This role of tRNA was understood much later.

1) Cloverleaf structure (2 dimentional) of t-RNA possess an anticodon loop that has bases
complementary to the codon.
2) It is called anticodon. It shows amino acid acceptor end (3’ end) having unpaired CCA bases
(i.e. amino acid binding site) to which amino acid binds.
3) For every amino acid, there is specific t- RNA.
4) Initiator t-RNA is specific for methionine.
5) There are no t-RNA’s for stop codons.
6) In the actual structure, the t-RNA molecule looks like inverted L (3 dimentional structure).
A.Translation - Protein synthesis :
1) Translation is the mechanism in which codons of mRNA are translated and specific amino
acids in a sequence form a polypeptide on ribosomes.
2) All types of proteins are synthesised by the cell, within itself (i.e. intracellularly).
3) Process of translation requires amino acids, mRNA, tRNA, ribosomes, ATP, Mg++ ions,
enzymes, elongation, translocation and release factors.

i.Amino acids form raw material for protein synthesis.

About 20 different types of amino acids are known to form proteins.
 These are available in the cytoplasm.
ii.DNA controls synthesis of proteins having amino acids in specific sequence.

This control is possible through transcription of m-RNA. Genetic code is specific for particular
amino acid.
iii. RNAs serve as intermediate molecules between DNA and protein.

iv. Ribosomes serve as site for protein synthesis.

Each ribosome consists of large and small subunits.
These subunits occur separately in cytoplasm.

Only during protein synthesis, these two subunits get associated together due to Mg++ ions.

4) A ribosome has one binding site for m-RNA and 3 binding sites for t-RNA.
5) They are P site (peptidy t-RNA site), A site (aminoacyl – t-RNA site) and E site (exit site).
6) Only first t- RNA- amino acid complex, directly enters P site of ribosome.
7) In Eukaryotes, a groove is present between two subunits of ribosomes.
8) It protects the Polypeptide chain from the action of cellular enzymes and also protects
mRNA from the action of nucleases.
Mechanism of translation (i.e. synthesis of polypeptide chain) :
It involves three steps : i. Initiation, ii. Elongation, iii. Termination
i.Initiation of Polypeptide chain :
a. Activation of amino acids is essential before translation initiates for which ATP is essential.
b. Small subunit of ribosome binds (attaches) to the m-RNA at 5’ end. Initiator codon, AUG is
present on m-RNA which initiates the process of protein synthesis (translation).
 c. Initiator t- RNA binds with initiation codon (AUG) by its anticodon (UAC) through hydrogen
bonds.
d. It carries activated amino acid methionine (in Eukaryotes) or formyl methionine (in
prokaryotes).
e. Now the large subunit of ribosome joins with the smaller subunit, that requires Mg++ ions. c.
Initiator charged t-RNA (with activated amino acid methionine) occupies the P- site of
ribosome and A- site is vacant.
2. Elongations of polypeptide chain :
a. During this process, activated amino acids are added one by one to first amino acid
(methionine). Amino acid is activated by utilising energy form ATP molecule.
b. This amino acid binds with amino acid binding site of t-RNA- This results in formation of t-
RNA- amino acid complex.
c. Addition of Amino acid occurs in 3 Step cycle
1.Condon recognition- Amino acyl t- RNA molecule enters the ribosome at A-site.
Anticodon binds with the codon by hydrogen bonds.
2.Amino acid on the first initiator t-RNA at P-site and amino acid on t-RNA at A-site
join by peptide bond.
Here enzyme Ribozyme acts as a catalyst.
At this time first tRNA at ‘P’ site is kicked off.
3.Translocation- The t- RNA at A-site carrying a dipeptide at A-site moves to the P-
site.
This process is called translocation.
d. In translocation, both the subunits of ribosome move along in relation to tRNA and mRNA.
e. Hence, tRNA carrying dipeptide now gets positioned at ‘P’ site of ribosome, making ‘A’ site
vacant. At this site, then next charged tRNA molecule carrying amino acid will be received.
f. During this process, first uncharged tRNA is discharged from E-site.
g. This process is repeated as amino acids are added to Polypeptide.
h. It takes less than 0.1 second for formation of peptide bond.
i. Third charged t-RNA with its amino acid, arrives at A-site of ribosome.
j. Anticodon and codon bind by hydrogen bond. Polypeptide bond is formed.
k. Second t-RNA is discharged from P-site to E-site and leaves the ribosome.
l. So the events like arrival of t-RNA- amino acid complex, formation of peptide bond,
ribosomal translocation and removal of previous tRNA, are repeated.
m. As ribosome move over the m- RNA, all the codons on m RNA are exposed one by one for
translation.
3.Termination and release of polypeptide:
a. At the end of m-RNA, there is a stop codon (UAA/ UAG/ UGA).
b. It is exposed at the A-site. It is not read and joined by anticodon of any t-RNA.
c. The release factor binds to the stop codon, thereby terminating the translation process.
d. The Polypeptide is now released in the cytoplasm.
e. Two subunits of Ribosome dissociate and last tRNA is set free in the cytoplasm.
f. m-RNA also has some additional sequences that are not translated and are referred as
untranslated regions (UTR).
g. The UTRs are present at both 5’-end (before start codon) and at 3’-end (after stop codon).
h. They are required for efficient translation process.
i. Finally mRNA is also released in the cytoplasm.
j. It gets denatured by nucleases immidiately.
k. Hence mRNA is short -lived.
4.6 Regulation of gene expression:
• It is the multistep process by which a gene is regulated and its product is synthesized.
• Thus, gene expression results in the formation of a Polypeptide.
• Gene expression process is regulated at different levels.
In eukaryotes, the regulation can be at different levels like
1. Transcriptional level (formation of primary transcript)
2. Processing level ( regulation of splicing)
3. Transport of m-RNA from nucleus to the cytoplasm.
4. Translational level.
Genes of a cell are expressed to perform different functions.
For eg. An enzyme beta galactosidase is synthesised by E-coli.
It is used for hydrolysis of lactose into galactose and glucose.

If E.coli bacteria do not have lactose in the surrounding medium as a source of energy, then enzyme
b-galactosidase is not synthesised.
So, it is the metabolic or physiological or environmental conditions that regulate expression of
genes.
The development and differentiation of embryo into an adult organism, is also a result of the
coordinated regulation or expression, of several sets of genes.
Now one has to understand and know the mechanism by which the organisms regulate gene
expression in response to changes in the environment.
If so, whether single mechanism exists for regulation of the expression of different genes/ sets of
genes or different genes are regulated by different mechanisms.
Certain bacteria like E.coli adapt to their chemical environment by synthesizing certain enzymes
depending upon the substrate present.
Such adaptive enzyme is called inducible enzymes.
A set of genes will be switched on when there is necessity to metabolise a new substrate.
This phenomenon is called induction and small molecule responsible for this, is known as inducer.
It is positive control.
4.7 Operon concept :
1. It is a transcriptional control mechanism of gene regulation.
2. Francois Jacob and Jacques Monod (1961) explained that metabolic pathways are regulated
as a unit.
3. For example in E.coli, when lactose sugar is provided to the culture medium, cell induces
production of three enzymes necessary for digestion of lactose.
4. The enzymes are :
i.b-galactosidase : Digests lactose into galactose and glucose.
 ii.b-galactoside permease : Permits lactose molecules to enter into the cell.
iii. Transacetylase (b-Galactoside acetyltransferease) :Transfers an acetyl group from acetyl
CO-A to galactoside.
5. Synthesis of these three enzymes, is controlled by a long segment of DNA known as Operon.
6. It consists of an operator site O and three structural genes Z, Y and A .
7. The action of structural genes is regulated by operator site with the help of a repressor
protein. Repressor protein is produced by the action of gene i (inhibitor) known as regulator
gene.
8. The gene expression depends on whether operator is switched on or switched off.
9. If the operator is switched on, the three genes z, y and a are transcribed by RNA Polymerase
into a single m-RNA.
10. Each structural gene is generally known as cistron and the transcribed long m-RNA covering
various cistrons is known as Polycistronic.
11. Switching on or switching off of the operator is achieved (acomplished) by a protein called
repressor. When this protein is attached to the operator and blocks it, the switch is turned
off and structural genes are not expressed.
Lac operon : Lactose or lac operon of E.coli is inducible operon.
The operon is switched on when a chemical inducer- lactose is present in the medium.
Lac operon consists of following components :
1. Regulator gene (repressor gene)
2. Promoter gene
3. Operator gene
4. Structural genes
5. Inducer - It is not a component of operon.
1. Regulator gene :
• This gene controls the operator gene in cooperation with an inducer present in the
cytoplasm. Regulator gene preceeds the promoter gene.
• It may not be present immidiately adjacent to operator gene.
• Regulator gene produces a protein called repressor protein. Repressor binds with operator
gene and represses (stops) its action.
• It is called regulator protein.
2. Promoter gene :
• This gene preceeds the operator gene. It is present adjacent to operator gene.
• The promoter gene marks the site at which the RNA Polymerase enzyme binds.
• When the operator gene is turned on, the enzyme moves over the operator gene and
transcription of structural genes starts.
• Promoter gene base sequence determines which strand of DNA acts a template.
3. Operator gene :
• It preceeds the structural genes.
• This controls the functioning of structural genes. It lies adjacent to the Structural genes.
• When operator gene is turned on by an inducer, the Structural genes produce m-RNA.
• Operator gene is turned off by a product of repressor gene.
4. Structural gene :
• When lactose is added to the E.coli culture, the structural genes catalyse (produce) m-RNA
which in turn produces polypeptides, on the ribosomes.
• The polypeptides formed, act as enzymes to caltalyse lactose in the cell.
• There are 3 structural genes in the sequence lac-Z, lac-Y and lac-A. Enzymes produced are b-
galactosidase, b-galactoside permease and transacetylase respectively.
5. Inducer :

• It is a chemical in the cytoplasm (allolactose) which inactivates the repressor.

• When lac operon is switched on, then inducer joins with repressor protein preventing the
binding of repressor to the operator gene.
• So the Operator gene is free and now enzyme RNA polymerase can move from promoter to
structural genes via operator gene.

Role of lactose :
1. A few molecules of lactose enter into the cell by an enzyme permease.
2. A small amount of this enzyme is present even when operon is switched off.
3. A few molecules of lactose, act as inducer and bind to repressor.
4. This repressor – inducer complex fails to join with the operator gene, which is then turned
on. Structural genes produce all enzymes.
5. Thus, lactose acts as an inducer of its own break down.
6. When the inducer level falls, the operator is blocked again by repressor.
7. So structural genes are repressed/ inactivated again.
8. This is negative feedback.
4.8 Genomics:
• The term Genome (introduced by H.Winkler in 1920) is the total genetic constitution of an
organism. Alternatively, it is a complete copy of genetic information (DNA) or one complete
set of chromosomes (monoploid or haploid) of an organism.
 • The term Genomics (term coined by T.H. Roderick in 1986) is the study of genomes through
analysis, sequencing and mapping of genes along with the study of their functions.
• The sequencing of yeast, Drosophila and mouse genome was done in order to facilitate
comparative studies between humans and other organisms commonly used for genetic
studies, in laboratory. Several additional genomes are now either actively being sequenced
or strongly considered for sequencing.
• These include several microbes, bee, tomato and other crops.
Genomics study may be classified into two types:
a.Structural genomics:
It involves mapping, sequencing and analysis of genome.
b. Functional genomics:
It deals with the study of functions of all gene sequences and their expression in organisms.

Application of genomics:
Structural and functional genomics is used for different purposes in the improvement of crop plant,
human health and livestock.
The knowledge and understanding acquired from genomics research can be applied in a number of
different sectors, including medicine, biotechnology and social sciences.
• It helps in the treatment of genetic disorders through gene therapy.
• Genomics is used in agriculture to develop transgenic crops having more desirable
characters.
• Genetic markers developed in genomics, have applications in forensic analysis.
• Genomics can lead to introduce new gene in microbes to produce enzymes, therapeutic
proteins and even biofuels.
4.9 Human Genome Project :
• The human genome project was initiated in 1990 under the International administration of
the Human Genome Organization (HUGO).
• This project was co-ordinated by the US department of Energy and National institute of
health. Additional contributors included universities across the United States and
international partners in the United Kingdom, France, Germany, Japan, India and China. The
• Human Genome Project formally began in 1990 and was completed in 2003.
• The human genome project is a multinational research project to determine the genomic
structure of humans.
The main aims of project are –
I.Mapping the entire human genome at the level of nucleotide sequences.
II. To store the information collected from the project in databases.
III. To develop tools and techniques for analysis of the data.
IV. Transfer of the related technologies to the private sectors, such as industries.
V. Taking care of the legal, ethical and social issues which may arise from project.
 A. HGP (Human Genome Project) was closely associated with rapid development of a new area
in biology, called Bioinformatics.
B. The work of human genome project has allowed researchers to begin to understand the
blueprint in building and constructing the human genome.
C. As researchers learn more about the functions of genes and proteins, this knowledge will
have a major impact in the fields like Medicine, Biotechnology and the Life sciences.
Therefore HGP is very important.
• Human Genome Project was to provide a complete and accurate sequence of the 3 billion
DNA base pairs that make up the human genome and to find out the estimated number of
human genes.
• Now about 33000 genes have been estimated to be present in humans.
• The project was also aimed to sequence the genomes of several other organisms such as
bacteia e.g. E.coli, Caenorhabditis elegans (a free living non-pathogenic nematode),
Saccharomyces cerevisiae (yeast), Drosophila (fruit fly), plants (rice and Arabidopsis), Mus
musculus (mouse), etc.
• Complete genome sequences of these model organisms will be useful for comparative
studies that will allow researchers to study gene functions in these organisms.
• The secret of our complexity may lie not in the number of our genes but how we use them.
• It will lead to the understanding of gene structure and function in other species.
• Since we possess many of the genes same as these of flies, round worms and mice, such
studies will lead to a greater understanding of human evolution.
• DNA fingerprinting technique is based on identification of nucleotide sequence present in
this wonder molecule.
• About 99.9% of nucleotide sequence in all persons, is same.
• Only some short sequences of nucleotides differ from person to person.
• In the population, every person shows unusual sequences of 20100 base pairs, which are
repeated several times.
• They are termed as Variable Number of Tandem Repeats (VNTRs).
• The length of the regions having VNTRs is different in each individual and hence is the key
factor in DNA profiling.

Steps involved in DNA finger printing are as follows:

1.Isolation of DNA:

• The DNA must be recovered from the cells or tissues of the body (host).
• Only small amount of tissue like blood, hair roots, skin, etc. is required.
2. Restriction digestion:

• The isolated DNA is treated with restriction enzymes.

• The restriction enzymes cut the DNA into small fragments having variable lengths.
• This phenomenon is called Restriction Fragment Length Polymorphism (RFLP).
3. Gel electrophoresis:
• The DNA samples are loaded for agarose gel electrophoresis under an electric influence.
• The DNA fragments, which are negatively charged move to the positive pole.
• The movement of these fragments depends on length of the fragments.
• This results in formation of bands. dsDNA splits into ssDNA by alkali treatment.
4. Southern blotting:

• The separated DNA fragments are transferred to a nylon membrane or a nitrocellulose filter
paper by placing it over the gel and soaking them with filter paper overnight.
5. Selection of DNA probe:

• A known sequence of single- stranded DNA is prepared. It is called DNA Probe.

• DNA Probe is obtained from organisms or prepared by cDNA preparation method.
• The DNA probe is labelled with radioactive isotopes.
6. Hybridization:
• Probe DNA is added to the nitrocellulose filter paper containing host DNA.
• The single-stranded DNA probe pairs with the complementary base sequence of the host
DNA strand.
• As a result DNA-DNA hybrids are formed on the nitrocellulose filter paper.
• Remaining single stranded DNA probe fragments are washed off.
7. Photography:
• The nitrocellulose filter paper is photographed on an X-ray film by autoradiography.
• The film is analysed to determine the presence of hybrid DNA.

Application of DNA fingerprinting

1. In forensic science, DNA finger printing is used to solve problems of rape and some complicated
murder cases.
2. DNA finger printing is used to find out the biological father or mother or both, of the child, in case
of disputed parentage.
3. DNA finger printing is used in pedigree analysis in cats, dogs, horses and humans.