Comment on the utility of variability in number of tandem repeats during DNA fingerprinting.
Tandemness in repeats provides many copies of the sequence for finger-printing and variability in nitrogen base sequences present in them. Being individual-specific, this proves to be useful in the process of DNA fingerprinting.
Give an account of Hershey and Chase experiment. What did it conclusively prove? If both DNA and proteins contained phosphorus and sulphur do you think the result would have been the same?
Hershey and Chase conducted experiments on bacteriophage to prove that DNA is the genetic material.
Hershey and Chase experiment
(i) Some bacteriophage virus were grown on a medium that contained radioactive phosphorus ( ${ }^{32} \mathrm{P}$ ) and some in another medium with radioactive sulphur ( ${ }^{35} \mathrm{~S}$ ).
(ii) Viruses grown in the presence of radioactive phosphorus ( ${ }^{32} \mathrm{P}$ ) contained radioactive DNA.
(iii) Similar viruses grown in presence of radioactive sulphur ( ${ }^{35} \mathrm{~S}$ ) contained radioactive protein.
(iv) Both the radioactive virus types were allowed to infect E. coli separately.
(v) Soon after infection, the bacterial cells were gently agitated in blender to remove viral coats from the bacteria.
(vi) The culture was also centrifuged to separate the viral particle from the bacterial cell.
Observations and Conclusions
(i) Only radioactive ${ }^{32} \mathrm{P}$ was found to be associated with the bacterial cell, whereas radioactive ${ }^{35} \mathrm{~S}$ was only found in surrounding medium and not in the bacterial cell.
(ii) This indicates that only DNA and not protein coat entered the bacterial cell.
(iii) This proves that DNA is the genetic material which is passed from virus to bacteria and not protein.
If both DNA and proteins contained phosphorus and sulphur, the result might change.
In case (i)
Radioactive ${ }^{35} \mathrm{~S}$ and + Bacteriophage ${ }^{32} \mathrm{P}$ labelled protein capsule $\longrightarrow$ No radioactive
${ }^{35} \mathrm{~S}$ and ${ }^{32} \mathrm{P}$ Detected in cells + Radioactivity ( ${ }^{35} \mathrm{~S}$ and ${ }^{32} \mathrm{P}$ ) detected in supernatant
In case (ii)
Radioactive ${ }^{35} \mathrm{~S}$ and ${ }^{32} \mathrm{P}$ lebelled DNA + Bacteriophage $\longrightarrow$ Radioactive ${ }^{32} \mathrm{P}$ and ${ }^{35} \mathrm{~S}$
Detected in cells + No radioactivity detected in supernatant
During the course of evolution why DNA was choosen over RNA as genetic material. Give reasons by first discussing the desired criteria in a molecule that can act as genetic material and in the light of biochemical differences between DNA and RNA.
A molecule that can act as a genetic material must fulfil the following
(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.
Biochemical differences between DNA and RNA
(i) Both nucleic acid (DNA and RNA) are able to direct their duplication proteins fails for the first criteria.
(ii) RNA is reactive, it also acts are catalyst, hence DNA is less reactive and structurally more stable than RNA.
(iii) Presence of thymine at the place of uracil also confers additional stability to DNA.
Give an account of post transcriptional modifications of a eukaryotic mRNA.
Post-transcriptional Modifications
The primary transcripts are non-functional, containing both the coding region, exon and non-coding region, intron in RNA and are called heterogenous RNA or hnRNA. In eukaryotes, three types of RNA polymerases are found in the nucleus
(i) RNA polymerase I transcribes rRNAs ( 28 S and 5.8 S ).
(ii) RNA polymerase II transcribes the precursor of mRNA (called heterogeneous nuclear RNA or hnRNA).
(iii) RNA polymerase III transcribes $t$ RNA, 5 S rRNA and snRNAs (small nuclear RNAs).
The hnRNA undergoes two additional processes called capping and tailing.
In capping, an unusual nucleotide, methyl guanosine triphosphate is added to the 5 '-end of hnRNA.
In tailing, adenylate residues (about 200-300) are added at 3'-end in a template independent manner.
Now the hnRNA undergoes a process where the introns are removed and exons are joined to form $m$ RNA by the process called splicing.
$$ \text { Diagram representation of a post transcriptional modification in eukaryotes } $$
$$ \text { Discuss the process of translation in detail. } $$
There are three-stages of protein synthesis
(i) Initiation
Assembly of Ribosomes on mRNA In prokaryotes, initiation requires the large and small ribosome subunits, the mRNA, initiation $t$ RNA and three Initiation Factors (IFs).
Activation of Amino Acid Amino acids become activated by binding with aminoacyl $t$ RNA synthetase enzyme in the presence of ATP.
$$ \text { Amino acid (AA) }+ \text { ATP } \xrightarrow[\text { synthetases }]{\text { AminoacytRNA }} \text { AA-AMP-Enzyme complex }+P_i $$
Transfer of Amino Acid to tRNA The AA-AMP-enzyme complex formed reacts with specific $t$ RNA to form aminoacyl $t$ RNA complex.
$$ \text { AA-AMP-Enzyme complex }+t \text { RNA } \longrightarrow \text { AA } t \text { RNA }+ \text { AMP }+ \text { Enzyme. } $$
The cap region of $m R N A$ binds to the smaller subunit of ribosome.
The ribosome has two sites, A-site and P-site.
The smaller subunit first binds the initiator $t$ RNA then and then binds to the larger subunit so, that initiation codon (AUG) lies on the P-site.
The initiation $t$ RNA, i.e., methionyl $t$ RNA then binds to the P -site.
(ii) Elongation
Another charged aminoacyltRNA complex binds to the A-site of the ribosome.
Peptide bond formation and movement along the mRNA called translocation. A peptide bond is formed between carboxyl group ( -COOH ) of amino acid at P -site and amino group ( -NH ) of amino acid at A-site by the enzyme peptidyl transferase.
The ribosome slides over $m R N A$ from codon to codon in the $5^{\prime} \rightarrow 3^{\prime}$ direction.
According to the sequence of codon, amino acids are attached to one another by peptide bonds and a polypeptide chain is formed.
(iii) Termination
When the A-site of ribosome reaches a termination codon which does not code for any amino acid, no charged $t$ RNA binds to the A-site.
Dissociation of polypeptide from ribosome takes place which is catalysed by a 'release factor'.
There are three termination codons, i.e., UGA, UAG and UAA.
