The below mentioned article provides a note on DNA:- 1. DNA Chemistry 2. DNA Replication 3. Carrier of Genetic Information.
DNA Chemistry:
DNA is found to consist of thread-like particles of high molecular weight in isolated condition.
A number of units called nucleotides become linked together and form the nucleic acid. A nucleotide is composed of a molecule of Pentose (5-carbon sugar), a nitrogen base (Purine and Pyrimidine) and phosphoric acid.
The purine bases are adenine and guanine and the pyrimidine bases are cytosine and thymine. The difference in the chemical composition of DNA is due mainly to the difference in arrangement of the nitrogen bases.
In an intact DNA molecule, base remains attached to a 5-carbon sugar (Deoxyribose) to form a deoxynucleoside. On analysis of DNA samples it has been found that the bases do not occur at a random fashion and that the sum of the purines is equal to the sum of the pyrimidines. Further, it is seen that the content of adenine equals the content of thymine and the content of guanine equals the content of cytosine.
Model of Watson and Crick:
Watson and Crick (1953) have constructed a most satisfying three dimensional model of DNA. According to this model, a DNA molecule consists of two long chains.
The chains run in opposite direction and are coiled round each other forming a double helix (Fig. 2.7). Each chain consists of a nitrogen base attached to pentose and the pentose in its turn is attached to the phosphoric acid. The bases of one chain are joined with those of the other chain by hydrogen bond. But the bondage between the bases is not at random and their sequences form a regular pattern.
Adenine is always linked with Thymine and Cytosine is always linked with Guanine. Thus the sequence of the bases of one strand determines the sequence of the bases of the other strand and this arrangement makes the strands complementary.
It is believed that the base pairs carry all the genetic information in an, organism. Thus the four alphabets A, T, C and G (A=adenine, T=thymine, C=cytosine, G=guanine) carry all the codes for the genetical specifications of all organisms. However, it is not known how many base pairs make up a gene.
DNA Replication:
In 1950, Chargaff showed that in any DNA, the amount of adenine equals to the amount of thymine and the amount of guanine equals to the amount of cytosine. Using X-ray crystallography technique, Watson and Crick in 1953 suggested that the DNA molecule consists of two polynucleotide strands inter-wined to form a double helix. The two strands are linked together by weak hydrogen bonds between the paired bases.
They further advocated that:
(1) the two polynucleotide strands run in opposite directions to give the observed symmetry and
(2) that pairing takes place between one Purine base and one Pyrimidine base.
Otherwise the links would become either too small or too large. This theory has well been substantiated from studies from X-ray diffraction, electron micrograph and enzymatic studies. It is now accepted as a working hypothesis.
The strong point in favour of the hypothesis is that it offers an explanation of how replication is affected when the chromosomes appear as double structures in the prophase stage of mitosis and meiosis.
To quote Watson and Crick:
“Our model of deoxyribose nucleic acid is, in effect a pair of templates, each of which is complementary to the other. We imagine that prior to duplication the hydrogen bonds are broken, and the two chains unwind and separate. Each chain then-acts as a template for the formation on itself a new companion chain, so that eventually we shall have two pairs of chains where we had only one before. Moreover, the sequences of the pairs of bases will have been duplicated exactly” (Figs. 2.8 and 2.9).
The experimental support to it:
A good experimental support for the correctness of the explanation came from the work of Meselson and Staht in 1958 on Escherichia coli. The bacterium when grown in a medium with a nitrogen source containing only 15N, this heavy isotope’ becomes incorporated in the new DNA formed.
This can be located because the DNA is now denser than 14N and it can be separated by centrifugation and its sedimentation rate measured.
The bacteria are then transferred to a medium containing only14N and the DNA is analysed at each successive doubling of the chromosomes. In such analysis three kinds of DNA were recognised—15N DNA, 14N DNA and an intermediate form 15N/14N DNA (Fig. 2.10). The result thus shows complete agreement to the model of Watson and Crick.
DNA as Carrier of Genetic Information:
The chromosomes consist of proteins, DNA and RNA. In the search for likely genetic material, the proteins were first of all regarded as likely candidates. However, in 1944 work on Diplococcus pneumoniae, a bacterium causing pneumonia, was carried out in America by Avery and his collaborators and their work showed that DNA must carry the genetic information.
Since then a good number of evidences have emerged out to prove that DNA is the carrier of genetic information.
They are discussed below:
Transformation of bacteria:
Certain strains of Diplococcus pneumoniae are avirulent and if injected into an animal would not cause the disease. Similarly if a virulent strain is first killed by heat treatment and then injected it would not produce the disease.
But if the animal is inoculated with a mixture of these two types (Avirulent and heat killed), the animal shows symptoms of the disease and the avirulent type has now been transformed into virulent type.
This genetic change is known as ‘Transformation’ when carried out in test-tube condition. The other known evidence in favour of DNA’s role as hereditary determinant in metabolism comes from the studies of the transformation of Pneumococci bacteria. Experiments have established that DNA from one strain of this bacteria can alter the inherited metabolic properties of a second strain of bacteria.
By the application of mutation technique a strain of Pneumococcus can be produced which is incapable of utilising Mannitol. These bacteria lack in the enzyme, Mannitol-phosphate-dehydrogenase and are designated as M– cells.
If DNA is isolated from M+ cells and is introduced in a culture of M–cells, many M– cells become transformed into M+ cells and the progeny of the transformed cells becomes capable of producing the enzyme.
The result proves in a convincing way that the hereditary capacity of the cells can be permanently altered. Further, the experiment makes it clear that at least a part of the genetical material in DNA is taken up by the chromosomes of the host and in certain percentage of the cases this incorporated material is replicated in subsequent generation.
However, the mechanism by which the DNA is incorporated into a permanent functional structure is not known.
Certain strains of bacteria are resistant to antibiotics such as Penicillin. If purified DNA, extracted from a Penicillin resistant strain of Pneumococcus is added to a culture medium in which Penicillin sensitive Pneumococcus are being cultured, transformation takes place and the whole culture becomes Penicillin resistant.
Transformation has also been studied in the bacterium Bacillus subtilis. A strain of this bacterium is unable to synthesize Tryptophan and as such cannot thrive in a culture medium lacking the amino acid Tryptophan. If such a culture is treated with DNA from a normal strain, a number of ‘Transformants’ appear in the Tryptophan-free medium. From these, many colonies of the new strain arise since the trait is heritable.
Many such experiments involving the transformation of genetic character have been made. These experiments have proved that the transforming agent is DNA and that DNA is the major chemical that controls the synthesis and operation of enzymatic machinery from generation to generation.
Bacteriophage infection:
Identification of DNA as the genetic messenger is also proved by experiments with virus. Bacteriophage is a virus on bacteria and is popularly called phage. The phage is made up of two parts —a head and a tail. The DNA of the phage lies in the head.
Both the head and tail remain capsulated in a protein coat. During infection the phage becomes attached to the bacterial cell wall and injects its DNA into the bacteria. Inside the bacterial cell, the DNA of the phage takes over the metabolic machinery and starts producing phage DNA and phage protein like its own kind.
Finally, the protein combines with the DNA to form complete phages. After some time the bacteria lyse and break releasing a large number of new phages.
In an experiment the DNA of the phage was lebelled with 35P and these were used to infect the bacteria. The result showed that the host DNA became radio-active. This proves that the DNA of the phage enters the DNA of the bacteria forming new phages at the expense of the host bacteria. The life cycle of the phage is presented in Fig. 2.11.
The evidence proves beyond doubt that a foreign DNA is capable of influencing the synthetic capacities of the host cell. This has led us to believe that an altered DNA or virus DNA is responsible for many diseases specially cancer.
Transduction:
Transduction is an experimental device by which genetic characteristics of one bacterium is transferred to another bacterium by using a third party. This third party in transduction is a phage. Thus when a bacterium sensitive to Streptomycin is injected with phages grown previously on bacterial cells resistant to Streptomycin, a small portion of the sensitive cells becomes Streptomycin resistant.
The resistant cells here are donors and sensitive cells are recipients and the third party is phage. While multiplying, in the donor cell the phage incorporated itself with some of the donor’s DNA which is Streptomycin resistant. The phage when transduced, this resistant and incorporated portion of the host DNA into the recipients DNA made some of the recipients DNA Streptomycin resistant.
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