THE DNA
DNA is a long polymer of deoxyribonucleotides.
The length of the DNA depends on the number of nucleotide pairs present in it.
Bacteriophage lambda has 48,502 base pairs.
Structure of Polynucleotide Chain
(1) A nucleotide has three components:
(a) A nitrogen base
(b) A pentose sugar (ribose in RNA and deoxyribose in DNA)
(c) A phosphoric acid.
(2) There are two types of nitrogen bases:
(a) Purines (Adenine and Guanine)
(b) Pyrimidines (Cytosine, Uracil and Thymine)
(3) Adenine, Guanine and Cytosine are common in RNA and DNA.
(4) Uracil is present in RNA and in DNA in place of Uracil, Thymine is present.
(5) In RNA, Pentose sugar is ribose and in DNA, it is Deoxyribose.
(6) Based on the nature of pentose sugar, two types of nucleosides are formed - ribonucleoside and deoxyribonucleotides.
(7) Two nucleotides are joined by 3’-5’ Phosphodiester linkage to form dinucleotide.
(8) More than two nucleotides join to form polynucleotide chain.
(9) The two strands of DNA (called DNA duplex) are antiparallel and complementary,
i.e., one in 5’->3’ direction and the other in 3”->5” direction.
THE SEARCH FOR GENETIC MATERIAL
Transforming Principle
In 1928, Frederick Griffith, in a series of experiments with Streptococcus pneumoniae (bacterium responsible for pneumonia),
witnessed a miraculous transformation in the bacteria.
During the course of his experiment, a living organism (bacteria) had changed in physical form.
When Streptococcus pneumoniae (pneumococcus) bacteria are grown
on a culture plate, some produce smooth shiny colonies (S) while others produce rough colonies (R).
This is because the S strain bacteria have a mucous (polysaccharide) coat, while R strain does not.
Mice infected with the S strain (virulent) die from pneumonia infection but mice infected
with the R strain do not develop pneumonia
S Strain → injected into mice → Mice die
R starin → injected into mice → Mice live
Griffith was able to kill bacteria by heating them.
When he injected a mixture of heat-killed S and live R bacteria, the mice died.
S Strain(heat-killed) → injected into mice → Mice live
S starin (heat killed + R strain(live) → injected into mice → M ice die
He concluded that the R strain bacteria had somehow been
transformed by the heat-killed S strain bacteria.
Some ‘transforming principle’, transferred from the heat-killed S strain,
had enabled the R strain to synthesise a smooth polysaccharide coat and become virulent.
The Genetic Material is DNA
The proof that DNA is the genetic material came from the experiments of Alfred Hershey and Martha Chase (1952).
They worked with viruses that infect bacteria called bacteriophages.
The bacteriophage attaches to the bacteria and its genetic material then enters the bacterial cell.
The bacterial cell treats the viral genetic material as if it was its own
and subsequently manufactures more virus particles.
Viruses grown in the presence of radioactive phosphorus contained radioactive DNA
but not radioactive protein because DNA contains phosphorus but protein does not.
Similarly, viruses grown on radioactive sulfur contained radioactive protein
but not radioactive DNA because DNA does not contain sulfur.
Radioactive phage were allowed to attach to E. coli bacteria.
The virus particles were separated from the bacteria by spinning them in a centrifuge.
Bacteria which was infected with viruses that had radioactive DNA were radioactive,
indicating that DNA was the material that passed from the virus to the bacteria.
Bacteria that were infected with viruses that had radioactive proteins were not radioactive.
This indicates that proteins did not enter the bacteria from the viruses.
DNA is hence the genetic material that is passed from virus to bacteria.
Genetic Material (DNA versus RNA)
A molecule as a genetic material must fulfill the following criteria:
(i) It should be able to generate its replica (Replication).
(ii) It should chemically and structurally be stable.
(iii) It should provide the scope for slow changes (mutation) that are required for evolution.
(iv) It should be able to express itself in the form of 'Mendelian Characters’.
Replication of DNA In Eukaryotes
Definition:
Process by which DNA produces daughter DNA molecules which are exact copies of the original DNA.
In eukaryotes, DNA is double stranded. The two strands are complementary to each other because of their base sequences.
Semi-conservative method of DNA replication Important points:
(i) Most common method of DNA replication.
(ii) Takes place in the nucleus where the DNA is present in the chromosomes.
(iii) Replication takes place in the S-phase (synthesis phase) of the interphase nucleus.
(iv) Deoxyribose nucleotides needed for formation of new DNA strands are present in nucleoplasm.
At the time of replication, the two strands of DNA first separate.
Each strand then acts as a template for the formation of a new strand.
A new strand is constructed on each old strand, and two exactly identical double stranded DNA molecules are formed.
In each new DNA molecule, one strand is old (original) while the other is newly formed.
Hence, Watson and Crick described this method as semi-conservative replication.
An overall process of DNA replication showing replication fork and formation of new strands template and lagging template.
The various steps involved in this process are summarized as follows:
1. Mechanism of replication starts at a specific point of the DNA molecule, called origin.
2. At origin, DNA strand breaks because of an incision (nick). This is made by an enzyme called incision enzyme (endonuclease).
3. The hydrogen bonds joining the two strands are broken by the enzyme.
4. The two strands start unwinding. This takes place with the help of a DNA unwinding enzyme Helicases.
Two polynucleotide strands are thus separated.
5. The point where the two strands separate appears like a fork or a Y-shape.
This is described as a replicating fork.
6. A new strand is constructed on each old strand.
This takes place with the help of a small RNA primer molecule which is complimentary to the DNA at that point.
7. Each old DNA strand acts as a template (site) for the construction of new strand.
The RNA primer attaches itself to the old strand and attracts the enzymes (DNA polymerase III) which add new nucleotides through base complementation.
The deoxyribose nucleotides are present in the surrounding nucleoplasm.
New DNA strand is thus constructed opposite to each old strand.
8. Formation of new complementary strand always begins at the 3' end of the template strand (original strand)
and progresses towards the 5' end (ie in 3' - 5' direction).
Since the new strand is antiparallel to the template strand, it is obvious that the new strand itself is always developed in the, 5'-3' direction.
For this reason when the two original strands separate (then with respect to the origin of separation),
one acts as 3'-5' template while the other acts as 5'- 3' template.
9. Of the two, the replication of 3'-5' template begins first.
Hence the new strand formed on it is called the leading strand.
The other template (5'-3') must begin replication at the fork and progress back toward the previously transcribed fragment.
The new strand formed on it is called the lagging strand.
10. Replication of the lagging strand takes place in small fragments called Okazaki fragments.
These are then connected together by the enzyme ligase.
11. Replication may take place in only one direction on the DNA helix (unidirectional) or in two directions (bidirectional).
12. At the end of the process, two double stranded DNA molecules are formed from the original DNA molecule.
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Types of RNA:
1. Messenger RNA or mRNA- has the information to make a protein.
It is very unstable and comprises ~5% of total RNA polymer. Its length is highly variable, of the range 7503000 nucleotides.
2. Transfer RNA or tRNA- small molecule, about 90 nucleotides long.
It is highly folded into an elaborate 3-d structure and comprises about 15% of total RNA.
3. Ribosomal RNA or rRNA- 80% of the total RNA, is associated with subcellular structures called ribosomes
in which the polymer length varies from 120-3000 nucleotides and is folded into an elaborate structure which give ribosomes their shape.
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Application of Human genome project
a.Identification of defective genes.
b. Opportunity to offer early treatment.
c. Identification of genes that confer susceptibility to certain disease.
d. Prediction of protein that the genes produce.
e. Drug designing to enhance or inhibit the activities of the proteins.
TECHNIQUE FOR DNA FINGER PRINTING
Technique developed by Dr.Alec Jeffreys.
♦ Process is also known as DNA typing/DNA profiling.
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