BIOLOGY 4 Marks Questions

1. DNA is made up of two polynucleotide chains, where the backbone is made up of sugar and phosphate groups and the nitrogenous bases project towards the centre.
2. There is complementary base pairing between the two strands of DNA.
3. The two strands are coiled in right-handed fashion and are anti-parallel in orientation. One chain has a 5′ → 3′ polarity while the other has 3′ → 5′ polarity.
4. The diameter of the strand is always constant due to pairing of purine and pyrimidine, i.e., adenine is complementary to thymine while guanine is complementary to cytosine.
5. The distance between two base pairs in a helix is 0.34nm and a complete turn contains approximately ten base pairs. The pitch of the helix is 3.4nm and the two strands are righthanded coiled.

1. Deoxyribonucleic acid (DNA) is composed of two polynucleotide strands which form a ladder.
2. The nitrogenous bases in DNA store the information for the synthesis of polypeptide chains, essentially coding for every feature of the entire organism.
3. The two polynucleotide strands run 'antiparallel' (in opposite directions, parallel to one another) to each other, with nitrogenous bases projecting inwards.
4. The antiparallel strands twist in a complete DNA structure, forming a double helix.
5. The strands are held together by hydrogen bonds between the nitrogenous bases which lie opposite to each other.
6. Bases bonded together are termed 'paired' and are very specific as to which base they will join.
7. A purine will only pair with a pyrimidine. The purine adenine will always pair with the pyrimidine thymine (A-T). The purine guanine will only pair with the pyrimidine cytosine (G-C). These base pairings are termed complementary base pairings.
8. Purines will always bond with pyrimidines because purines are larger molecules composed of a double ring structure. So, in order to ensure that the polynucleotide strands are equally spaced apart, the larger bases must pair with the smaller bases.
9. The root for specific complementary base pairings is the number of hydrogen bonding sites available. Adenine and thymine have two sites each, whereas guanine and cytosine have three sites each. 

They also discovered that protein-digesting enzymes (proteases) and RNA digesting enzymes (RNases) did not affect transformation, so the transforming substance was not a protein or RNA. Digestion with DNase did inhibit transformation, suggesting that the DNA caused the transformation. They concluded that DNA is the hereditary material, but not all biologists were convinced.

• Viruses were grown on two media. One medium contained radioactive phosphorus and another contained radioactive Sulphur.
• Viruses grown on radioactive phosphorus contained radioactive DNA but no such protein because protein does not contain phosphorus.
• Viruses grown on radioactive Sulphur contained radioactive protein but no such DNA because DNA does not contain Sulphur.
• Radioactive phages were allowed to attach to E.coli bacteria. One the infection proceeded, the viral coat was removed from bacteria and then viral particles were separated from bacteria for further analysis.

Observation:

• Radioactive DNA was seen in only those bacteria which were infected with phages grown on radioactive phosphorus.
• Radioactive DNA was not seen in those bacteria which were infected with phages grown on radioactive Sulphur.

Conclusion: DNA was found to be the genetic material.

If both the DNA and proteins contained Sulphur and phosphorus, it would have not been possible to pinpoint the exact genetic material, i.e. DNA or proteins.

1. Frederick Griffith (1928) performed the experiments on Bacterial transformation with Streptococcus pneumoniae, the bacterium that causes pneumonia.

1. He observed two strains of this bacterium, one forming smooth shiny colonies with capsule (S-type) and the other forming rough colonies without capsule (R-type).
2. The S-type cells are virulent while the R-type cells are non-virulent.
3. When live S-type cells were injected into the mice, they suffered from pneumonia and died.

S strain → Injected into mice → Mice died.

4. When live R-type cells were injected into the mice, the disease did not appear and the mice survived.

R strain → Injected into mice → Mice lived

5. When heat-killed S-type cells were injected, the disease did not appear.

S strain → Injected into mice → Mice lived (Heat-killed)

6. When heat-killed S-type cells were mixed with live R-type cells and injected into the mice, the mice died of pneumonia and live S-type cells were isolated from the body of the mice.

S strain (Heat-killed) + R strain (live)→ Injected into mice → Mice died.

7. He concluded that the R-strain had somehow been transformed by the heat-killed S-strain bacteria into virulent S-strain, which must be due to the transfer of genetic material, the transforming principle.

2. ​​​​​​​

1. Avery, MacLeod and McCarty purified biochemicals like proteins, DNA and RNA from the heat-killed S-cells.
2. When these fractions were added individually to the culture of live R-cells, DNA was able to cause transformation of R-cells into S-cells.
3. They also found that protein-digesting enzymes and RNA-digesting enzymes did not affect transformation, indicating that transforming substance is not a protein or RNA.
4. Digestion with DNase did inhibit transformation; this suggests that the DNA caused transformation.

• Translation is the process of synthesis of protein from mRNA with the help of ribosome.
• A translational unit in mRNA from 5'→3' comprises of a start codon, region coding for a polypeptide, a stop codon and untranslated regions (UTRs) at both 5'-end and 3'-end for efficient process.

There are three stages of protein synthesis:

1. Initiation:

• Assembly of ribosome on mRNA.
• Activation of amino acids and its delivery to tRNA.

2. Elongation:

• Repeated cycle of amino acid delivery.
• Peptide bond formation and movement along the mRNA called translocation.

3. Termination:

• The release of a polypeptide chain.

1. Initiation:

• In prokaryotes, initiation requires the large and small ribosome subunits, the mRNA, initiation tRNA and three initiation factors (IFs).

• Activation of amino acid: Amino acids become activated by binding with aminoacyl tRNA synthetase enzyme in the presence of ATP.

• Transfer of amino acid to tRNA: The AA - AMP - Enzyme complex formed reacts with specific tRNA to form aminoacyl-tRNA complex. AA - AMP - Enzyme complex + tRNA → AA - tRNA + AMP + Enzyme.

• The cap region of mRNA binds to the smaller subunit of ribosome.

• The ribosome has two sites, A-site and P-site.

• The smaller subunit first binds to the initiator mRNA and then binds to the larger subunit so that initiation codon (AUG) lies on the P-site.

• The initiation tRNA, i.e., methionyl tRNA then binds to the P-site.

2. Elongation of polypeptide chain:

• Another charged aminoacyl tRNA complex binds to the A-site of the ribosome at the second codon.
• 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 mRNA from codon to codon in the 5′ → 3′ direction.
• According to the sequence of codons, amino acids are attached to one another by peptide bonds and a polypeptide chain is formed.

3. Termination of polypeptide:

• When the A-site of ribosome reaches a termination codon which does not code for any amino acid, no charged tRNA binds to the A-site.
• Dissociation of polypeptide from ribosome takes place, which is catalysed by a 'release factor'.
• There are three termination codons namely UGA, UAG and UAA.

1. Maurice Wilkins and Rosalind Franklin provided the X-ray diffraction data of DNA, that helped Watson and Crick to propose the double helical model of DNA.
2. Erwin Chargaff observed that for a double stranded DNA, the ratios between adenine and thymine and between guanine and cytosine are constant and equal to one; based on this, the base pairing between the two strands of DNA was proposed by Watson and Crick. 

1. The three types of RNA are:

1. Messenger RNA (mRNA).
2. Transfer RNA (tRNA).
3. Ribosomal RNA (rRNA).

2. tRNA.
3. The genetic information in DNA is transcribed into messenger RNA (mRNA). It stores the genetic information from DNA and decides the sequence of amino acid in a polypeptide.

Transfer RNA (tRNA) acts as an adaptor molecule that at one end reads the code on mRNA and accordingly bind to amino acid on the other end. It recognises the codon on mRNA by its anticodon and leaves amino acid at the site of protein synthesis.

Ribosomal RNA (rRNA) constitutes the ribosomal structure and helps to form peptide bond.

2. Satellite DNA is separated from the genomic DNA. by density gradient centrifugation; the satellite DNA forms smaller peaks, while the genomic DNA forms a major peak.
3.  

1. They form very useful tools in identification of criminals.
2. It is the basis of paternity testing, in case of disputes.

1. If both strands of DNA function as template, then these can code for the RNA having various sequences. If coded for polypeptides; they must have different amino acid sequences which will make the protein synthesis complex. When two RNA molecules are produced they will be complementary to one another; consequently dsRNA would result and to prevent RNA from being translated into protein.
2. In prokaryotes, the mRNA synthesis does not require any processing to become active and since both transcription and translation occur in the same cytosol; translation can start much before the mRNA is fully transcribed. Consequently, transcription and translation can be coupled.

1. An operon is a part of DNA that functions as a gene regulatory unit in transcription. 

1. Regulatory gene: This gene controls the operator gene. This produces a protein substance known as represser which combines with the operator gene to stop its function. 

2. Operator: It controls the functioning of structural genes which are expressed when operate, gene is turned on by inducer and not expressed when operates is turned off by repressor. 

3. Promoter: It is the site at which the RNA polymerase binds and reaches the structural genes for the transcription of mRNA to start.

4. Structural genes: These genes produce mRNA which synthesize the specific proteins such as enzyme regained for metabolism of Lactose.

2. If Lactose is withdrawn from the culture medium the operon is not induced or expressed.

If lactose is withdrawn from the culture medium the operon is not expressed. The repressor units with the operator gene and turns it off with the result the structural genes are inactivated and therefore the transcription and protein (enzyme) synthesis stops.

1. Minisatellites and microsatellites.

They are classified on the basis of:

1. Base composition, i.e. A-T rich or G-C rich.
2. Length of the segment.
3. Number of repetitive units.

1. The discovery of nuclein by Miescher and the proposition of principal of inheritance by Mendel were almost at the same time, i.e. 1869 and 1866, respectively. But the fact that DNA acts as a genetic material took a long time to be discovered and proven. By 1926, the quest to determine the mechanism for genetic inheritance had reached the molecular level and gradually the question, what molecule acts as genetic material got answered.

Transforming Principle

Frederick Griffith in 1928, carried out a series of experiments with Streptococcus pneumoniae (a bacterium that causes pneumonia). He observed that when these bacteria (Streptococcus pneumoniae) were grown on a culture plate, some of them produced smooth, shiny colonies (S-type), whereas the others produced rough colonies (R-type). This difference in appearance of colonies (smooth/rough) is due to the presence or absence of mucus (polysaccharide) coat on S-strains, but not on R-strains. In his experiments, he first infected two separate groups of mice. The mice that were infected with the S-strain died from pneumonia as S-strains are the virulent strains causing pneumonia. S-strain (virulent strain) → Injected into mice → Mice died. The mice that were infected with the R-strain did not develop pneumonia and they lived. R-strain (non-virulent strain) → Injected into mice → Mice lived.

In the next set of experiments, Griffith killed the bacteria by heating them. The mice that were injected with heat-killed S-strain bacteria did not die and lived. S-strain (heat killed) → Injected into mice → Mice lived. Whereas, on injecting a mixture of heat-killed S-strain and live R-strain bacteria, the mice died. Moreover, living S-bacteria was recovered from the dead mice. S-strain (heat killed) + R-strain (live) → Injected into mice + Mice died.

From all these observations Griffith concluded that the live R-strain bacteria, had been transformed by the heat-killed S-strain bacteria, i.e. some 'transforming principle' had transferred from the heat-killed S-strain, which helped the R-strain bacteria to synthesise a smooth polysaccharide coat and thus, become virulent. This must be due to the transfer of the genetic material. However, he was not able to define the biochemical nature of genetic material from his experiments.

2. Griffith was not able to find the chemical nature of transforming agent. It was finally Hershey and Chase who proved that DNA is the genetic material. They prov ed it by using radio labelled elements, such as ³⁵S and ³²P. Radioactivity was detected in the cell which was due to the presence of 32 Pin DNA.

1. Promoter: A nucleotide sequence that enables a gene to be transcribed is called promoter. It is recognized by RNA polymerase, which then initiates transcription.
2. Operator: A segment of DNA to which a repressor binds is called operator.
3. Structural genes: The genes that are co-regulated by the operon are called structural genes.

Inducible Operon: When the operon is regulated by an inducer, it is called inducible operon. An inducer can switch on or off the operon. Lac operon is an example of inducible operon. Lactose is a substrate of enzyme beta-galactosidase and is the inducer of lac operon.

The given diagram shows the working of lac operon. In the absence of an inducer the repressor binds to the operator region and prevents transcription.

In the presence of an inducer, repressor becomes inactive. This allows transcription in the operator region which results in release of mRNA. Subsequently, mRNA promotes translation and protein synthesis is accomplished.

• RAM HAS RED CAP.
• RAM HAS BRE DCA P.
• RAM HAS BIR EDC AP.
• RAM HAS BIG RED CAP.

1. Mathew Meselson and Franklin Stahl performed the experiment.
2. They grew E.coli, in a medium containing ¹⁵NH₄CI, until ¹⁵N was incorporated in the two strands of newly formed E.coli cells; this 'heavy' DNA can be separated from the normal ¹⁴N-DNA by centrifugation in cesium chloride (CsCl) density gradient.
3. Then they transferred the cells into a medium with normal ¹⁴NH₄Cl and took out samples at various time intervals.
4. They extracted the DNA and centrifuged it to measure the densities.
5. The DNA extracted from cells after one generation after transfer from the ¹⁵N medium to ¹⁴N medium (i.e. after about 20 minutes), had an intermediate/ hybrid density.
6. The DNA extracted after two generations (ie. after 40 minutes) consisted of equal amounts of 'light' DNA and 'hybrid' DNA.
7. This proves that after replication, each DNA molecule has one parental strand and one newly-synthesised strand, i.e. replication is semiconservative.

Initiation:

• In prokaryotes, initiation requires the large and small ribosome subunits, the mRNA, initiation tRNA and three initiation factors (IFs).
• Activation of amino acid: Amino acids become activated by binding with aminoacyl tRNA synthetase enzyme in the presence of ATP.'
• Transfer of amino acid to tRNA: The AA - AMP - Enzyme complex formed reacts with specific tRNA to form aminoacyl-tRNA complex. AA - AMP - Enzyme complex + tRNA → AA - tRNA + AMP + Enzyme.
• The cap region of mRNA binds to the smaller subunit of ribosome.
• The ribosome has two sites, A-site and P-site.
• The smaller subunit first binds to the initiator mRNA and then binds to the larger subunit so that initiation codon (AUG) lies on the P-site.
• The initiation tRNA, i.e., methionyl tRNA then binds to the P-site.

1. There are 3164.7 million nucleotide bases in the human genome.
2. In an average gene, there are 3000 bases. The largest known human gene is Dystrophin (2.4 million bases).
3. Total number of genes in human genome are estimated at 30,000. Almost, all (99.9%) of the nucleotide bases are exactly same in every human individual.

1. Its knowledge is helpful in research involving biological systems including human biology.
2. With the whole genome sequences and newer technologies, we can be systematic in our approach to various medical questions on a broader scale.
3. All the genes in a genome, e.g. all the transcripts in a particular tissue/ organ/ tumour can be studied.

1. The human genome contains 3164.7 million nucleotides (base pairs).
2. The size of the genes varies; an average gene consits of 3000 bases, while the largest gene, dystrophin consists of 2.4 million bases.
3. The total number of genes is estimated to be 30000 and 99.9% of the nucleotides are the same in all humans.
4. The functions of over 50% of the discovered genes are not known
5. Only less than 2% of the genome codes for proteins.
6. Repetitive segments make up a large portion of the human genome.
7. Repetitive sequences throw light on chromosome structure and dynamics and evolution, though they are thought to have no direct coding functions.
8. Chromosome 1 has 2968 genes and Y-chromosome has the least number (231 genes).
9. Scientists have identified about 1.4 million locations, where DNA differs in single base in human beings; these are called single nucleotide polymorphisms (SNPs).

1. AUG - AUU
2. AUG - AUC
3. AUG - AUA

• Polymerase II facilitates transcription of hnRNA into mature mRNA.
• Primary transcripts cotain both the introns and exons and these are non-functional. Splicing takes place which results in removal of introns and joining of exons in define order.
• Capping and tailing happens in hnRNA. It acquires a cap of methyl guanosine and a tail of poly adenylate. Cap is added at 5’ end and Poly-A tail is added at 3’ end of hnRNA.
• Now, the hnRNA changes into mature mRNA.

1. The methods involve two major approaches:

1. One approach called Expressed Sequence Tags (ESTs), focuses on identifying all the genes that are expressed as RNAs.
2. Second approach called Sequence Annotation, is to simply sequence the whole set of genome, that includes all the coding and non-coding sequences and then assigning functions to different regions in the sequence.

2. The total DNA from the cell is isolated and converted into random fragments of relatively smaller sizes.
3. These fragments are then cloned in suitable hosts using specialised vectors; the commonly used hosts are bacteria and yeast and the vectors are bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
4. The fragments are then sequenced using automated DNA sequences.
5. The sequences are then arranged on the basis of certain overlapping regions present in them; this requires the generation of overlapping fragments for sequencing.
6. Specialised computer programmes are developed for alignment of the sequences.
7. These sequences are annotated and assigned to the respective chromosomes.

1. Total DNA from a cell is isolated and converted into random fragments of smaller sizes.
2. These fragments are cloned in a suitable host by using specialized vectors. The cloning results into amplification of each fragment, and makes it easy to sequence the fragment. Bacteria and yeast are the commonly used hosts for this purpose. The vectors were called as BAC (bacterial artificial chromosomes) and YAC (yeast artificial chromosomes).
3. Automated DNA sequencers were used to sequence the fragments. Then these sequences were arranged on the basis of some overlapping regions present in them.
4. For generating overlapping fragments in these sequences; help of computer programmes was taken because it was not possible for humans to do so.
5. Then the sequences were annotated and assigned to each chromosome.
6. Genetic physical mapping of genome was done on the basis of polymorphism in some segments of the DNA.

1. It should be able to generate its replica (Replication). Because of rule of base pairing and complementarity, both the nucleic acids (DNA and RNA) have the ability to direct their duplications. The other molecules in the living system, such as proteins fail to fulfill first criteria itself.
2. It should chemically and structurally be stable. 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.

3. It should provide the scope for slow changes (mutation) that are required for evolution. Both DNA and RNA are able to mutate. In fact, RNA being unstable, mutate at a faster rate. Consequently, viruses having RNA genome and having shorter life span mutate and evolve faster.
4. It should be able to express itself in the form of ‘Mendelian Characters’. RNA can directly code for the synthesis of proteins, hence can easily express the characters. DNA, however, is dependent on RNA for synthesis of proteins. The protein synthesising machinery has evolved around RNA. The above discussion indicate that both RNA and DNA can function as genetic material, but DNA being more stable is preferred for storage of genetic information. For the transmission of genetic information, RNA is better. RNA being a catalyst was reactive and hence unstable. Therefore, DNA has evolved from RNA with chemical modifications that make it more stable.

Transforming Principle:

• Frederick Griffith (1928) conducted experiments with Streptococcus pneumoniae (bacterium causing pneumonia).
• He observed two strains of this bacterium-one forming smooth shiny colonies (S-type) with capsule, while other forming rough colonies (R-type) without capsule.
• When live S-type cells were injected into mice, they died due to pneumonia.
• When live R-type cells were injected into mice, they survived.
• When heat-killed S-type cells were injected into mice, they survived and there were no symptoms of pnuemonia.
• When heat-killed S-type cells were mixed with live R-type cells and injected into mice, they died due to unexpected symptoms of pneumonia and live S-type cells were obtained from mice.
• He concluded that heat-killed S-type bacteria caused a transformation of the R-type bacteria into S-type bacteria but he was not able to understand the cause of this bacterial transformation.

1. The regulatory (i) gene codes for repressor protein, which is synthesised constitutively all the time.
2. It binds to the operator and prevents the RNA polymerase from transcribing the operon when lactose is absent; so the operon is 'switched off'.
3. The promoter is the sequence of DNA where RNA polymerase binds for transcription.
4. There are three structural genes in the lac operon of E.coli.

1. Gene z codes for B-galactosidase, that catalyses the hydrolysis of lactose into glucose and galactose.
2. Gene y codes for permease, which increases permeability of lactose into the cell.
3. Gene a codes for transacetylase, that makes the lactose into its active form.

5. When lactose is present in the cell, it functions as inducer.
6. When it binds to the repressor and inactivates it, the repressor does not bind to the operator.
7. This allows RNA polymerase access to the promoter and transcription proceeds, i.e. the operon is 'switched on'.

1. If two such charged tRNAs are brought close enough, the formation of peptide bond between them would be favoured energetically. The presence of a catalyst would enhance the rate of peptide bond formation. The cellular factory responsible for synthesising proteins is the ribosome. The ribosome consists of structural RNAs and about 80 different proteins. In its inactive state, it exists as two subunits; a large subunit and a small subunit.
2. When the small subunit encounters an mRNA, the process of translation of the mRNA to protein begins. There are two sites in the large subunit, for subsequent amino acids to bind to and thus, be close enough to each other for the formation of a peptide bond. The ribosome also acts as a catalyst (23S rRNA in bacteria is the enzyme- ribozyme) for the formation of peptide bond or polyribonucleotides.
3. A translational unit in mRNA is the sequence of RNA that is flanked by the start codon (AUG) and the stop codon and codes for a polypeptide. 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.
4. For initiation, the ribosome binds to the mRNA at the start codon (AUG) that is recognised only by the initiator tRNA. The ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon. The ribosome moves from codon to codon along the mRNA.
5. Amino acids are added one by one, translated into olypeptide sequences dictated by DNA and represented by mRNA. At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome.

1. The gene p is defective.
2. It indicates that one gene controls the synthesis of one enzyme.
3. An operon has the following components.

1. Structural gene(s) which transcribes the mRNA for the proteins synthesis.
2. An operator gene, which controls the structural gene(s) as a unit.
3. A promoter, where the RNA polymerase binds for transcription.

4. Each one has a role to play and even if one fails, normal progress is not possible.

1. The students repeated the experiment performed by Oswald Avery, Colin MacLeod and Maclyn McCarty (1933-44), who worked to determine the biochemical nature of transforming principle' in Griffith's experiment.
2. Hershey and Chase experimented to find our whether it was protein or DNA from the viruses= that cnrered into the bacreria. They took rwo separate media- for gro*ing the; bacteriophages. Out of the rwo, one medium fontain-ed radioacrive phosphorus and the other medium contained radioactive sulPhur.
3. Frederick Griffith in 1928 performed the transformation experiment.
4. Scientific attitude, awareness and love for animals and respect towards government policies.

1. The promoter: It is the binding site for RNA polymerase for initiation of transcription.

2. The structural gene: It codes for enzyme or protein for structural functions.

3. The terminator: It is the region where transcription ends.

1. An amino acyl tRNA complex reaches the A site and attaches to mRNA codon next to initiation codon with the help of its anticodon. The step requires GTP and an elongation factor.
2. A peptide bond (-CO-NH-) is established between the carboxylic gp of (-COOH) amino acid attached to tRNA at P site and amino gp (NH) of amino acid attached to tRNA at A-site. The reaction is catalysed by enzyme peptidyl. transferase.
3. In the process the connection between tRNA and amino acid at the P-site breaks. The free t-RNA of the P-site slips away and the A site carries peptidyl tRNA complex.
4. Now the ribosome or mRNA rotates slightly. The process is called translocation.
5. As a result of translocation, the A site codon along with peptidyl-tRNA complex reaches the P site. A new codon is exposed at the A site. It attracts a new amino-acyl tRNA complex.
6. The process of bond formation and translocation is repeated. One by one all the codons of mRNA are exposed at the A' site and get decoded through incorporation of amino acids in the peptide chain. The peptide chain elongates.

1. It represents the parental or template DNA strands, showing the replication fork.
2.  
3. 

1. The deoxyribonucleoside triphosphates provide energy.
2. DNA dependent DNA polymerase and DNA ligases are the enzymes involved;

1. On the template strand with 3' → 5' polarity, DNA synthesis is continuous, in the 5' → 3' direction.
2. On the template strand with 5' → 3' polarity, DNA synthesis is discontinuous; DNA strand is synthesised as short segments, also in the 5' → 3' direction and the discontinuously synthesised segments become joined by DNA ligase.

1. Transcription unit in a bacterium consists of a promoter, structural genes, terminator and the enzyme DNA-dependent RNA-Polymerase.
2. In bacteria, there is a single RNA polymerase, which catalyses transcription of all the three types of RNAs (mRNA, tRNA and rRNA).
3. It binds transiently with the initiation factor (sigma factor), binds to the promoter and initiates transcription.
4. It also facilitates opening of the double helical DNA and only the DNA strand with 3' → 5' polarity is transcribed, as the enzyme can polymerise the nucleotides only in 5' → 3' direction; it catalyses elongation using the ribonucleotides.
5. Once the polymerase reaches the terminator sequence, it associates with the termination factor and the nascent RNA falls off and termination of transcription occurs.

1. The bead-like structures are the nucleosomes:

1. There is a set of positively charged proteins, called histones, rich in basic amino acid residues, lysine and arginine.
2. Histones are organised to form a unit of eight molecules, called histone octamer.
3. The negatively charged DNA is wrapped around the positively charged histone octamer, to form a nucleosome; a nucleosome contains 200 bp of DNA helix.

1. The nucleosomes form the repeating units of the chromatin fibre in the nucleus; these nucleosomes of a chromatin are seen as the beads-on-string structure under electron microscope.
2. The chromatin fibres are further coiled and condensed at metaphase stage to form the chromosomes.
3. The packaging of chromatin at higher levels requires another set of proteins, called non histone chromosomal (NHC) proteins.

1. Repressor will attach at the operator (o) region.
2. If an inducer (like lactose) binds to the repressor and inactivates it, it cannot bind to the operator.
3. If repressor is not bound to the operator, the operon is 'on' and transcription occurs.
4. Rwgulation of lac operon by repressor is referred to as negative regulation. Lac operon can work under the control of positive regulation also.

1. Streptococcus pneumoniae:

1. R-cells are those cells that form rough colonies, without a capsule and are non-virulent, i.e., they did not cause pneumonia.
2. S-cells are those cells that form smooth colonies, protected by a capsule and are virulent, i.e., they caused the disease, pneumonia.
3. He showed that DNA is the transforming principle.

2. A. Mice died of pneumonia. B. Mice lived and did not suffer from pneumonia.
3. Frederick Griffith performed the steps (a), (b) and (c).
4. This indicates that DNA is the transforming principle.

When DNase is added to the medium, the DNA of the heat-killed cells gets digested and is unable to carry out the transformation.

1. The shaded portions are introns and unshaded portions are exons.
2. The primary RNA contains both introns and exons. By the mechanism of splicing, introns are removed and exons are joined to form functional mRNA after capping and tailing.

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.
• 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 mRNA by the process called splicing.

3. In prokaryotes, the structural gene is continuous and is not differentiated into exons and introns unlike eukaryotes. In prokaryotes, transcription is followed by translation without RNA splicing mechanism.

1. When the inducer is present, it combines with the repressor, coded by i gene. After reacting with repressor it inactivates the repressor The repressor now cannot bind to the operator; hence the pathway for RNA polymerase is open. The structural genes (z, y, a) are transcribed and the metabolism continues.
2. Lactose is inducer.

The methods involve two major approaches:

1. Expressed sequence tags (ESTs): This method focusses on identifying all the genes that are expressed as RNA.

2. Sequence annotation: It is an approach of simply sequencing the whole set of genome that contains all the coding and non-coding sequences, and later assigning different regions in the sequence with functions.

• For sequencing, the total DNA from cell is first isolated and broken down in relatively small sizes as fragments.
• These DNA fragments are cloned in suitable host using suitable vectors. When bacteria is used as vector, they are called bacterial artificial chromosomes (BAC) and when yeast is used as vector, they are called yeast artificial chromosomes (YAC).
• Frederick Sanger developed a principle according to which the fragments of DNA are sequenced by automated DNA sequences.
• On the basis of overlapping regions on DNA fragments, these sequences are arranged accordingly.
• For alignment of these sequences, specialised computer-based programmes were developed.
• Finally, the genetic and physical maps of the genome were constructed by collecting information about certain repetitive DNA sequences and DNA polymorphism, based on endonuclease recognition sites.

1. In prokaryotes only one type of RNA polymerase is involved whereas in eukaryotes three types of RNA polymerases are involved.
2. For Description of processing of hnRNA involving-introns/ exons/ splicing in eukaryotes and for Description of capping and tailing.

Translation:

• Translation is the process of synthesis of protein from mRNA with the help of ribosome.
• A translational unit in mRNA from 5' → 3' comprises of a start codon, region coding for a polypeptide, a stop codon and untranslated regions (UTRs) at both 5'-end and 3'-end for efficient process.

There are three stages of protein synthesis:

1. Initiation:

• Assembly of ribosome on mRNA.
• Activation of amino acids and its delivery to tRNA.

2. Elongation:

• Repeated cycle of amino acid delivery.
• Peptide bond formation and movement along the mRNA called translocation.

3. Termination:

• The release of a polypeptide chain.

1. Initiation:

• In prokaryotes, initiation requires the large and small ribosome subunits, the mRNA, initiation tRNA and three initiation factors (IFs).

• Activation of amino acid: Amino acids become activated by binding with aminoacyl tRNA synthetase enzyme in the presence of ATP.

• Aminoacid(AA) + ATP → synthetases Aminoacy lt RNA AA - AMP - Enzymecomplex + Pi

• Transfer of amino acid to tRNA: The AA - AMP - Enzyme complex formed reacts with specific tRNA to form aminoacyl-tRNA complex. AA - AMP - Enzyme complex + tRNA → AA - tRNA + AMP + Enzyme.

• The cap region of mRNA binds to the smaller subunit of ribosome.
• The ribosome has two sites, A-site and P-site.
• The smaller subunit first binds to the initiator mRNA and then binds to the larger subunit so that initiation codon (AUG) lies on the P-site.
• The initiation tRNA, i.e., methionyl tRNA then binds to the P-site.

2. Elongation of polypeptide chain:

• Another charged aminoacyl tRNA complex binds to the A-site of the ribosome at the second codon.
• 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 mRNA from codon to codon in the 5′ → 3′ direction.
• According to the sequence of codons, amino acids are attached to one another by peptide bonds and a polypeptide chain is formed.

1. DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA.
2. The technique has the following steps.

1. DNA Isolation DNA is ocracted from the cells in a high speed centrifuge.

2. Amplification Many copies of the extracted DNA can be made by the use of polymerase chain reaction.
3. Digestion of DNA by restriction endonucleases.
4. Separation of DNA fragments by gel electrophoresis.
5. Southern blotting Transfer of separated DNA fragments to synthetic membranes (like nylon or nitrocellulose).
6. Hybridisation with the help of a radio labelled VNTR probe (small segments of DNA which help to detect the presence of a gene in a long DNA sequence) which gets anached to single stranded VNTRs having complementary nucleotide sequences.
7. Autoradiographic exposure to X-ray films Detection of hytridised DNA fragments by autorediography. This shows many dark bands of different sizes.

2. Applications of DNA fingerprinting are-

1. As a tool in forensic investigations.
2. to settle paternity disputes.

3. The judge showed wisdom and intention to search for the truth.

1. DNA replication begins at a unique and fixed point called origin of replication or 'ori'. Initiation.
2. The complementary strands of DNA double helix are separated by two enzymes, DNA gyrase and DNA helicase. This is called unwinding of double-stranded DNA.
3. The separated strands tend to rewind, therefore these are stabilised by proteins called single strand binding proteins (ssBPs), which bind to the separated strands.
4. Unwinding of double-stranded DNA forms a Y-shaped configuration in the DNA duplex, which is called replication fork.Elongation.
5. An enzyme called primase initiates replication of the strand oriented in the 3′ (towards origin) → 5′ (towards fork) direction. This generates 10 - 60 nucleotides long primer RNA (replicated in 5′ → 3′ direction).
6. The free 3′-OH of this RNA primer provides the initiation point for DNA polymerase for sequential addition of deoxyribonucleotides.
7. DNA polymerase progressively adds deoxyribonucleotides to the free 3′-end of the growing polynucleotide chain so that replication of the 3′ → 5′ strand of the DNA molecule is continuous (growth of the new strand in 5′ → 3′ direction).
8. The replication of 3′ → 5′ strand is continuous and it is called leading strand, while the replication of second strand (5′ → 3′ strand) of the DNA molecules is discontinuous and it is known as the lagging strand.
9. The replication of lagging strand generates small polynucleotide fragments called 'Okazaki fragments' (after R. Okazaki, who first identified them).
10. These Okazaki fragments are then joined together by enzyme called DNA ligase.

1. Replication of DNA.
2. Replication of DNA occurs during S-phase of cell cycle.
3. During replication of DNA, the two strands unwind upto a point and a replication fork is formed.
4. Since the unwinding cannot take place for the entire length, replicaion starts from the replication fork.
5. Both the strands act as templates for DNA synthesis.
6. DNA polymerase is the enzyme that polymerises the bases/ nucleotides into a strand of DNA.
7. Since this enzyme polymerises the nucleotides in 5' → 3' direction only, on one of the template strands (with 3' → 5' polarity), the new strand is synthesised as a continuous stretch (leading strand); it is called continuous synthesis.
8. On the other template strand, (with 5' → 3' polarity), DNA strand is synthesised as short stretches (using primers), called Okazaki fragments; this is called discontinuous synthesis.
9. Later, these short stretches are joined by DNA-ligase (lagging strand).
10. 

1. DNA is made of two polynucleotide chains, where the backbone is constituted by sugar-phosphate and the nitrogen bases project inside.
2. The two chains have antiparallel polarity, i.e. one chain has 5' → 3' polarity, while the other has 3' → 5' polarity.
3. The nitrogen bases in the two strands are paired through weak hydrogen bonds, forming base pairs; a purine always pairs with a pyrimidine, i.e., adenine pairs with thymine through two hydrogen bonds and guanine with cytosine through three hydrogen bonds.
4. The two chains are coiled in a right-handed fashion;
5. the pitch of the helix is 3.4nm and there are 10 base pairs in each turn, with the distance between consecutive base pairs being 0.34nm.
6. The plane of one base pair stacks over the other in double helix; this confers stability of the helical structure.

1. First the total DNA from a cell is isolated and converted into random fragments of smaller sizes.
2. Then these fragments are cloned in a suitable host, (such as bacteria and yeast) using specialised vectors such as BAC (Bacterial Artificial Chromosome) and YAC (Yeast Artificial Chromosome).
3. The cloning of DNA fragments lead to the amplification of each fragment, which helps in easy sequencing process.
4. The DNA fragments were sequenced using automated DNA sequencers, which work on the principle developed by Frederick Sanger.
5. These sequences were then arranged on the basis of some overlapping regions present in them. This required generation of overlapping fragments for sequencing.
6. The specialised computer based programmes were developed for the alignment of these sequences.
7. These sequences were subsequently annotated and assigned to each chromosome. The sequence of chromosome I was completed only in May, 2006 (this was the last of the 24 human chromosomes to be sequenced).
8. The next challenging task was to assign the genetic and physical maps on the genome. This was generated using the information on polymorphism of restriction endonuclease recognition sites and certain repetitive DNA sequences called as microsatellites.

1. Regulatory gene in this operon is i gene. Role of regulatory gene in switching off operon is as follows:

2. It codes for the repressor protein of the operon, which is synthesised constitutively.
3. The repressor has affinity for the operator gene. It binds to operator and prevents the RNA polymerase from transcribing the structural genes.

2. When repressor binds to the operator, the operon is switched off and transcription is stopped. So, it is called a negative regulation.
3. Lactose is an inducer molecule.

1. Z gene codes for B-galactosidase (b-gal), that helps in the hydrolysis of disacchoride into its monomeric units, i.e. lactose into galactose and glucose.
2. Y gene codes for pwemease, that increases the permeability of the cell to B-galactosides.
3. a gene codes for a transacetylase.

The bulk DNA forms a major peak and the other small peaks are referred to as satellite DNA. Depending on base composition (A : T rich or G : C rich), length of segment, and number of repetitive units, the satellite DNA is classified into many categories, such as micro-satellites, mini-satellites etc. These sequences normally do not code for any proteins, but they form a large portion of human genome.

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, they become very useful identification tool in forensic applications. Further, as the polymorphisms are inheritable from parents to children, DNA fingerprinting is the basis of paternity testing, in case of disputes.

The technique of DNA fingerprinting was initially developed by Alec Jeffreys. Lalji Singh is called father of Indian DNA fingerprinting or DNA profiling or DNA typing. He used a satellite DNA as probe that shows very high degree of polymorphism. It was called as Variable Number of Tandem Repeats (VNTR).

The technique, as used earlier, involved Southern blot hybridisation using radiolabelled VNTR as a probe. It included,

1. Isolation of DNA.
2. Digestion of DNA by restriction endonucleases.
3. Separation of DNA fragments by electrophoresis.
4. Transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon.
5. Hybridisation using labelled VNTR probe.
6. Detectionof hybridised DNA fragments by autoradiography.