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Direct Evidences for DNA As the Genetic Material |
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Direct Evidences for DNA As the Genetic Material
The most conclusive evidences in support of DNA as the genetic material comes from following three avenues of approach on micro-organisms: transformation of bacteria. mode of infection of bacteriophages and conjugation in bacteria.
1. Bacterial Transformation and Griffith EffectIn 1928, Frederick Griffith encountered a phenomenon now known as genetic transformation. Colonies of virulent strain (pathogenic) of pneumonia causing spherically-shaped bacterium, Diplococcus pneumoniae grown on nutrient agar, have a smooth (s) glistering appearance owing to the presence of a type specific, ploysaccharide (a polymer of glucose and glucuronic acid) capsule.
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The avirulant (non-pathogenic) strains of this bacterium, on the other hand, lack this capsule and they produce dull, rough (R) colonies and is unable to cause the pneumonia disease. Smooth (S) and rough (R) characters are directly related to the presence or absence of the capsule and this trait is known to be genetically determined. Both S and R forms occur in several types and are designed as S-1, S-II, S-III, etc., and R-I, R-II, R-III, etc., respectively. All these subtypes of S and R bacteria differ with each other in the type of antigens, they produce. The kind of antigens produced is likewise genetically determined. Smooth (S) forms sometimes mutate to rough (R) forms, but this change have not been found reversible.
In the course of his work, Griffith injected laboratory mice with live R-II pneumococci; the mice suffered no illness because R-II pneumococci was avirulent.
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But when the mice were injected with the mixture of living avirulent R-II and heat killed S-III virulent, the unexpected symptoms of pneumonia appeared and high mortality was resulted in them. By postmortoming the dead mice, it was found that their heart blood had both R-II and S-III pneumococci. From these results, Griffith concluded that the presence of the heat-killed S-IU bacteria must have caused a transformation of the living R-II bacteria, so as to restore to them the capacity for capsule formation they had earlier lost by gene mutation. This was called "Griffith effect" or more popularly as "bacterial transformation".
Identification of the "transforming substance"-Griffith could not understand the cause of bacterial transformation and that is first of all identified by O. T. Avery, C. M. McLeod and M McCarthy (1944). They tested a fraction of heat killed S-III bacteria for the transforming ability.
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They could remove proteins, lipids, polysaccharides and ribonucleic acids from the S-III extract by a variety of chemical and enzymatic methods without seriously diminishing its power to transfer R-II mutants into the S-III wild types. They ultimately concluded that because a cell free and highly purified DNA extract of S-III bacteria could bring about transformation of R-I1 bacteria into S-III, therefore, DNA is the genetic material of pneumococci. Later on, such a transforming substance, the DNA, was found in a variety of bacteria (i.e., Hemophilus influenzae. Bacillus subtilus, Shigelia paradysenteriae, etc.) and several other organisms.
| A. Protein labelled with S35 or DNA labelled with P32 |
B. T2 bacteriophage |
| C. DNase has no effect on DNA |
D. Attachment |
| E. Injection of DNA molecule |
F. Ghost |
| G. Replication of daughter DNA molecule |
H. Fully formed bacteriophage |
I. Lysis of Escherichia coli cell |
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2. Mode of Infection of Bacteriophages and Identification of DNA as their Genetic Material
By using radioactive tracers, A. Hershey and M. Chase (1952) provided further direct proof that DNA is the genetic material in certain bacterial viruses. These investigators were studying the bacteriophages that attack the bacterium, Escherichia coli. They prepared a chemically defined culture medium (for E. coli) containing phosphoric acid and sulphuric acid as the only source of phosphorus and sulphur.
Known quantities of radioactive isotopes of phosphorus (P32) and sulphur (S32) were added to the culture medium. E. coli grown on such a culture medium incorporated the P32 and S35 into their chemical constituents. When T2 bacteriophage particles infected the radioactive bacteria, they incorporated the labeled DNA.
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Phage capsid proteins did not contain appreciable amounts of phosphorus and only the DNA was labeled with P32. Similarly, the protein envelope around the phage was selectively labeled with S35. DNA contains virtually no sulphur and was not labeled with S35 By this method it was possible to differentially label the phage DNA and proteins of phage-capsid. After phage growth, the phage particles were separated from the host cell by centrifugation. The radioactive phages were next introduced to non-radioactive bacterial cultures where they attacked the bacteria. Subsequently the viruses were separated from the host cell by agitation and the content of P32 and S35 in the host (E. coli) and parasite (bacteriophage) was determined. The phosphorus label was found to be associated with the bacterial cells and the sulphur label was in the proteins coats (capsids) left in the medium. This indicated that the DNA had panetrated the cells but that the protein coat or capsid of the phage was left outside the wall of the bacterium. The labeled DNA was found to reproduced in the host cells. These experiments, thus, clearly demonstrated that genetic informations of these bacteriophages resided in their DNA molecules and not in the protein molecules of their capsids.
Later on Hershey-Chase experiment was repeated for other viruses and it was found that DNA is also the genetic material in a number of other viruses (see E. Rosenberg, 1971).
3. Bacterial Conjugation
Another conclusive evidence for DNA as the genetic material came from the phenomenon of conjugation of bacteria. Laderberg and Tatum (1946) found that when a F+ ('male') E. coli cell conjugated with a F- ('female') E. coli cell an unidirectional transfer of F+ factor of 'male' cell to F- or 'female' cell took place; so that the latter was converted into a F+ or 'male' strain. The F+ factor was found to be a fragment of DNA molecule which occurred in the cytoplasm of bactarial cell.
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