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In this article we will discuss about the evidence that DNA is the genetic material in viruses.
Is DNA
The conclusion reached by Avery, Macleod and McCarty was not immediately accepted. Some investigators suggested that the DNA preparations contained a mutagen which caused mutation to the S form. Others pointed out that transformation was due to traces of some specific protein remaining in the DNA samples used.
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One apparent reason why the significance of this work was only gradually recognised was the absence of knowledge about bacterial genetics at that time. The existence of a chromosome capable of exchanging genes was not fully known in bacteria.
Moreover doubts continued as to whether DNA or protein was functioning as the genetic material. DNA was mostly ruled out because its only variable components were the four nitrogenous bases (the sugar and phosphate being identical in all nucleotides).
On the other hand the molecular complexity of a protein, due to twenty different amino acids in various sequences could better account for the diversity required in a genetic material for performing the varied functions in an organism.
Looking back it seems that it was perhaps due to lack of knowledge that the great achievement of Avery, Macleod and McCarty was not rewarded with a Nobel Prize. Their discovery however, did have the impact of initiating studies in molecular genetics with the use of micro-organisms.
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One of the finest demonstrations that DNA is the genetic material came in 1950’s through investigations on bacterial viruses by A. D. Hershey and Martha C. Chase. They were studying the mechanism by which the virus infects E. coli.
Bacterial viruses known as bacteriophages are obligate parasites which must infect a host cell in order to reproduce. The bacterial viruses most studied are those that infect E. coli bacteria particularly those classified as T2. It was known at that time that T2 consists exclusively of DNA and protein.
In EM the T2 virus has a hexagonal body and a protruding tail. When a suspension of these viruses is mixed with a suspension of E. coli and left at 37°C, the viruses infect the host cells by becoming attached by their tails to the bacterial cell wall. After about 30 minutes of this attachment, the bacteria burst (lyse) releasing several hundred newly synthesised viruses.
The noteworthy point in the above experiment is that the original infecting viruses can still be seen attached to the bacterial membrane. Hershey and Chase became interested to find out what molecular event occurred between the time of T2 attachment to E. coli and the release of new virus progeny.
Obviously, some material contained in the infecting viruses must have passed into the host bacterium where it caused the formation of new viruses. This must be the genetic material of the virus. Hershey and Chase now prepared bacterial viruses in which either the phosphorus or sulphur were radioactively labelled.
By doing so they could distinguish between viral DNA and viral protein and trace them during the process of infection. They did this by first growing E. coli cells in a nutrient medium containing radioactive phosphate (32P) and sulphur (35S).
The labelled E. coli cells were used as hosts for un-labelled viruses. The resulting virus progeny labelled with 32P and 35S was used to infect un-labelled E. coli cells in a series of experiments popularly known as the waring blender experiments (Fig. 13.2).
Since sulphur is present only in protein and not in DNA and phosphorus only in DNA but not in protein, it was clear that in the labelled viruses, the protein component was selectively labelled with 35S and the DNA with 32P. When a suspension of labelled viruses was mixed with un-labelled E. coli cells and left for a few minutes, the viruses were observed in EM attached to the bacteria.
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By stirring the mixture in a warring blender, the attached viruses were broken away from the host cell. The suspension was divided into two parts. One portion was incubated for some more minutes and the bacteria burst to liberate the newly formed progeny virus. The remaining suspension was cooled and centrifuged to separate the bacteria from the remains of attached viruses.
The amounts of radioactive sulfur and phosphorus were assayed in each. They found that 85% of 32P of the viruses had passed into the bacteria whereas 80 % of 35S had remained in the viruses. It was apparent therefore that most of the 32P containing DNA had entered the host bacterium whereas bulk of the proteins did not.
Experiments performed in a more refined way showed that almost all the virus DNA enters the infected E. coli cell and only about 3 per cent of the protein. Evidently the genetic material of the virus is contained in the DNA rather than in the trace of protein. With conclusions drawn from Hershey and Chase experiments, DNA was indisputably recognised as the hereditary material.
