Saturday 17 October 2015

DNA as hereditary Material

                     DNA as hereditary Material
Griffith experiment:The evidences of hereditary nature of DNA was provided by British Microbiologist Fredrick Griffith.

  • Types of bacteria used by Griffith:
1:S-type bacteria:The normal pathogenic form of this bacterium is referred as S-type bacterium or normal or wild type because it form smooth colonies on culture dish and contain POLYSACcHaRIDE coat.
2:R-type bacteria:The mutant form which lack the enzyme needed for the manufacture of polysaccharide coat,it is called mutant/R-type because it form rough colonies.

Experiment number 1:
  • Griffith infected the mice with virulent strain S-type of streptococcus pneumoniae  bacteria,the mice died of blood poisoning.
  • However when he infected the similar mice with mutant strain R-type of Pneumococcus that lack the plysaccharide coat.
  • The mice show no illness
  • The coat was apparently necessary for virulence.
Experiment number 2:
  • To determine whether the plysaccharide coat had itself toxic substance or not?
  • Griffith injected dead bacteria of virulent form S strain into ice the mice remain perfectly healthy.
  • As a control he injected the mixture of dead S and live R or coatless bacteria.
  • Unexpectedly the mice developed the disease and died of blood poisoning.
Conclusion:The blood of died mice was found high level of live virulent strains of S-type bacteria that was impossible.
Somehow information shows that coat was genetically transferred from Virulent S to Coatless R bacteria,this activity was not possible that the bacteria contain coat were dead.then how it was trasferred?




Three scientists, Oswald Avery, Colin MacLeod, and Maclyn McCarty, managed to show that Frederick Griffith’s transforming factor was in fact DNA, i.e. DNA is the heritable substance.

At first, Avery refused to believe Griffith’s results that actually challenged his own research on pneumococcal capsules - how could a rough capsule be converted into a smooth one? However, he soon confirmed Griffith’s results and set about trying to purify this mysterious “transforming principle” - a substance that could cause a heritable change of bacterial cells.
They extracted from Streptococcus pneumoniae S (containing a capsule) bacteria purified DNA, proteins and other materials and mixed R bacteria (lacking a capsule) with these different materials, and only those mixed with DNA were transformed into S bacteria. Therefore, DNA is the “transforming factor” and not proteins or other materials.
Amazingly, not everyone was convinced by the experiments of Avery's, MacLeod's and McCarty's as it was still widely assumed that genetic information was carried in protein. Firstly, due to Levene's influential "tetranucleotide hypothesis", many condsidered DNA to be a “stupid molecule,” made up of a repeat of the four chemical bases without any variations. Secondly, few biologists thought that genetics could be applied to bacteria, since they lacked chromosomes and sexual reproduction. Although the experimental findings of their experiment were quickly and independently a number of scientists still considered that protein contaminants were responsible the results. It was not be until the experiments of Hershey and Chase (1952) that DNA was finally proved and accepted to be the genetic material.

Hershey and Chase experiment

Historical background


In the early twentieth century, biologists thought that proteins carried genetic information.This was based on the belief that proteins were more complex than DNA.Phoebus Levene's influential "tetranucleotide hypothesis", which incorrectly proposed that DNA was a repeating set of identical nucleotides, supported this conclusion.The results of theAvery–MacLeod–McCarty experiment, published in 1944, suggested that DNA was the genetic material, but there was still some hesitation within the general scientific community to accept this, which set the stage for the Hershey–Chase experiment.
Hershey and Chase, along with others who had done related experiments, confirmed that DNA was the biomolecule that carried genetic information. Before that, Oswald Avery,Colin MacLeod, and Maclyn McCarty had shown that DNA led to the transformation of one strain of Streptococcus pneumoniae to another that was more virulent. The results of these experiments provided evidence that DNA was the biomolecule that carried genetic information.

Methods and results

Structural overview of T2 phage
Hershey and Chase needed to be able to examine different parts of the phages they were studying separately, so they needed to isolate the phage subsections. Viruses were known to be composed of a protein shell and DNA, so they chose to uniquely label each with a different elemental isotope. This allowed each to be observed and analyzed separately. Since phosphorus is contained in DNA but not amino acids, radioactive phosphorus-32 was used to label the DNA contained in the T2 phage. Radioactive sulfur-35 was used to label the protein sections of the T2 phage, because sulfur is contained in amino acids but not DNA.
Hershey and Chase inserted the radioactive elements into the bacteriophages by adding the isotopes to separate media within which bacteria were allowed to grow for 4 hours before bacteriophage introduction. When the bacteriophages infected the bacteria, the progenycontained the radioactive isotopes in their structures. This procedure was performed once for the sulfur-labeled phages and once for phosphorus-labeled phages.The labeled progeny were then allowed to infect unlabeled bacteria. The phage coats remained on the outside of the bacteria, while genetic material entered. Centrifugation allowed for the separation of the phage coats from the bacteria. These bacteria were lysed to release phage progeny. The progeny of the phages that were originally labeled with 32P remained labeled, while the progeny of the phages originally labeled with 35S were unlabeled. Thus, the Hershey–Chase experiment helped confirm that DNA, not protein, is the genetic material.
Hershey and Chase 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 32P 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 32P and a heavier solution containing structures called “ghosts” that contained the 35S 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.

Experiment and conclusions

Hershey and Chase were also able to prove that the DNA from the phage is inserted into the bacteria shortly after the virus attaches to its host. Using a high speed blender they were able to force the bacteriophages from the bacterial cells after adsorption. The lack of 32P labeled DNA remaining in the solution after the bacteriophages had been allowed to adsorb to the bacteria showed that the phage DNA was transferred into the bacterial cell. The presence of almost all the radioactive 35S in the solution showed that the protein coat that protects the DNA before adsorption stayed outside the cell.
Hershey and Chase concluded that DNA, not protein, was the genetic material. They determined that a protective protein coat was formed around the bacteriophage, but that the internal DNA is what conferred its ability to produce progeny inside a bacteria. They showed that, in growth, protein has no function, while DNA has some function. They determined this from the amount of radioactive material remaining outside of the cell. Only 20% of the 32P remained outside the cell, demonstrating that it was incorporated with DNA in the cell's genetic material. All of the 35S in the protein coats remained outside the cell, showing it was not incorporated into the cell, and that protein is not the genetic material.
Hershey and Chase's experiment concluded that little sulfur containing material entered the bacterial cell. However no specific conclusions can be made regarding whether material that is sulfur-free enters the bacterial cell after phage adsorption. Further research was necessary to conclude that it was solely bacteriophages' DNA that entered the cell and not a combination of protein and DNA where the protein did not contain any sulfur.

Discussion

Confirmation


Hershey and Chase concluded that protein was likely not 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.
Confirmation and clarity came a year later in 1953, when James D. Watson and Francis Crick correctly hypothesized, in their journal article "Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid", the double helix structure of DNA, and suggested the copying mechanism by which DNA functions as hereditary material. Furthermore, Watson and Crick suggested that DNA, the genetic material, is responsible for the synthesis of the thousands of proteins found in cells. They had made this proposal based on the structural similarity that exists between the two macromolecules, that is, both protein and DNA are linear sequences of amino acids and nucleotides respectively.

Other experiments

Once the Hershey–Chase experiment was published, the scientific community generally acknowledged that DNA was the genetic code material. This discovery led to a more detailed investigation of DNA to determine its composition as well as its 3D structure. Using X-ray crystallography, the structure of DNA was discovered by James Watson and Francis Crick with the help of previously documented experimental evidence by Maurice Wilkins and Rosalind Franklin. Knowledge of the structure of DNA led scientists to examine the nature of genetic coding and, in turn, understand the process of protein synthesis. George Gamow proposed that the genetic code was composed of sequences of three DNA base pairs known as triplets or codons which represent one of the twenty amino acids. Genetic coding helped researchers to understand the mechanism of gene expression, the process by which information from a gene is used in protein synthesis. Since then, much research has been conducted to modulate steps in the gene expression process. These steps include transcription, RNA splicing, translation, and post-translational modification which are used to control the chemical and structural nature of proteins.Moreover, genetic engineering gives engineers the ability to directly manipulate the genetic materials of organisms using recombinant DNA techniques. The first recombinant DNA molecule was created by Paul Berg in 1972 when he combined DNA from the monkey virus SV40 with that of the lambda virus.
Experiments on hereditary material during the time of the Hershey-Chase Experiment often used bacteriophages as a model organism. Bacteriophages lend themselves to experiments on hereditary material because they incorporate their genetic material into their host cell's genetic material (making them useful tools), they multiply quickly, and they are easily collected by researchers.


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