IINDEPENDENT FUNCTIONS OF VIRAL PROTEIN AND NUCLEIC ACID IN GROWTH OF BACTERIOPHAGE
By A. D. HERSHEY AND MARTHA CHASE
The viruses that infect bacteria are called bacteriophages. Phages have often a head and a tail. The tail structure allows them to penetrate to the host cell membranes and cell walls to inject their DNA into the cell. Their heads are containing protein. And their nucleic acid can be DNA or RNA depending on the type of phage wrapped in a protein coat for protection. They cannot multiply without benefit of a host cell. To start the infection the phage binds to the receptors on the cell surface and absorbs the bacterial cell. In general speaking; the next step is, the phage injects its entire DNA into the cells where transcription of RNA begins almost immediately.
Bacteriophages may have a lytic cycle or a lysogenic cycle, and a few viruses are capable of carrying both. With lytic phages such as the T4 phage, bacterial cells are lysed and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. In contrast, the lysogenic cycle does not result in immediate lysis of the host cell. Those phages able to undergo lysogeny are known as temperate phages.
Hershey and Chase Experiment
The Hersey- Chase experiment was based on the biology of the bacteriophage T2 (a virus of bacteria) which attacks to a bacterium. Viruses were known to be composed of a protein shell and DNA. A Part but not all of the virus enters to the bacterial cell and in about 20 min later, the cell bursts and releases dozens of the DNA which are belong to the virus from that virus infected the cell. They were curious about which part of that bacteriophage were staying outside and which part were being injected. Was it the DNA or the protein?
Back at that time from previous researches what they knew was that; the phages attach to bacteria by their tails, osmotic shock ruptures the phage producing an empty- headed phage ‘ghost’ that is non-infectious and agitation of phage and bacteria in a Waring blender prevents the infection.
They ran set of experiments by gathering these information. Basically; they chose to uniquely label each DNA and protein of the phage with a different elemental isotope. This method allowed them to observe and analyze the protein and the DNA separately. They used P32 to label for the DNA. The two strands of DNA have a sugar- phosphate backbone that contains phosphorus atoms. Phosphorus is not present in most of the proteins. They used S35 to label the proteins which contain sulfur. Because the sulphur is found in amino acids cysteine and methionine and not present in the DNA.
So, they grew the virus in two different media to label the bacteriophage. One was containing labelled sulphur whereas the other media was containing the labeled phosphorus. After labeling them by growing in these media, they added these phage into a fresh culture where E. coli hosts were present. They gave enough time to the virus to infect these cells and after that, they dethatched these virus particles from the cell via vigorous shaking with a blendor. Samples then place into the tubes and centrifuged to force the bacterial cell to move to the bottom of the tube whereas the virus would stay in the supernatant. Centrifugation allowed for the separation of the phage coats from the bacteria. For each of these samples, the labelled components would me in different fractions, either in the pellet or the supernatant.
They found that most of the labeled phosphorus were in the pellet whereas the most of the labeled sulphur were in the supernatant fluid with the viruses. So, they conclude that the DNA enters to the host cell and multiply itself not the protein.
Hershey and Chase also showed that the introduction of deoxyribonuclease (referred to as DNase), an enzyme that breaks down DNA, into a solution containing the labeled bacteriophages did not introduce any P32 into the solution. This demonstrated that the phage is resistant to the enzyme while intact. Additionally, they were able to plasmolyze the bacteriophages so that they went into osmotic shock, which effectively created a solution containing most of the P32 and a heavier solution containing structures called “ghosts” that contained the S35 and the protein coat of the virus. It was found that these “ghosts” could adsorb to bacteria that were susceptible to T2, although they contained no DNA and were simply the remains of the original bacterial capsule. They concluded that the protein protected the DNA from DNAse, but that once the two were separated and the phage was inactivated, the DNAse could hydrolyze the phage DNA. At the end they concluded that T2 phage were consist of ~50% protein and 50% DNA, the phage tail was the part that binds to E. coli cells, the progeny viruses produced in the infected cell and the bacterium lysed to release progeny virus.
As a result, Hershey and Chase concluded that protein was not likely to be the hereditary genetic material. However, they did not make any conclusions regarding the specific function of DNA as hereditary material and only said that it must have some undefined role.