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Hershey–Chase experiment

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Overview of experiment and observations

The Hershey–Chase experiments were a series of experiments conducted in 1952[1] by Alfred Hershey and Martha Chase that helped to confirm that DNA is genetic material. While DNA had been known to biologists since 1869,[2] many scientists still assumed at the time that proteins carried the information for inheritance because DNA appeared simpler than proteins. In their experiments, Hershey and Chase showed that when bacteriophages, which are composed of DNA and protein, infect bacteria, their DNA enters the host bacterial cell, but most of their protein does not. Although the results were not conclusive, and Hershey and Chase were cautious in their interpretation, previous, contemporaneous, and subsequent discoveries all served to prove that DNA is the hereditary material.

Hershey shared the 1969 Nobel Prize in Physiology or Medicine with Max Delbrück and Salvador Luria for their “discoveries concerning the genetic structure of viruses.”[3]

Historical background

In the early twentieth century, biologists thought that proteins carried genetic information.[4] This was based on the belief that proteins were more complex than DNA.[4] Phoebus Levene's influential "tetranucleotide hypothesis", which incorrectly proposed that DNA was a repeating set of identical nucleotides, supported this conclusion.[5] The results of the Avery–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.[4][6]

Hershey and Chase, along with others who had done related experiments, confirmed that DNA was the biomolecule that carried genetic information.[1] 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.[7] 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.[1]

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 progeny contained the radioactive isotopes in their structures. This procedure was performed once for the sulfur-labeled phages and once for phosphorus-labeled phages.[1] The labeled progeny were then allowed to infect unlabeled bacteria. The phage coats remained on the outside of the bacteria, while genetic material entered. Disruption of phage from the bacteria by agitation in a blender followed by 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 RNA from RNAse, but that once the two were separated and the phage was inactivated, the DNAse could hydrolyze the phage DNA.[1] However, it subsequently became clear that in some viruses, RNA is the genetic material.

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.[1]

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 bacterium. 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.[1]

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.[1]

Discussion

Confirmation

Further information: DNA and DNA § History of DNA research

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.[1][4]

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.[8]

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.[9] 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.[10] 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.[11] 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.[12]

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.[4]

Legacy

The Hershey–Chase experiment, its predecessors, such as the Avery–MacLeod–McCarty experiment, and successors served to unequivocally establish that hereditary information was carried by DNA. This finding has numerous applications in forensic science, crime investigation and genealogy. It provided the background knowledge for further applications in DNA forensics, where DNA fingerprinting uses data originating from DNA, not protein sources, to deduce genetic variation.[13]

References

  1. ^ a b c d e f g h i Hershey A, Chase M; Chase (1952). "Independent functions of viral protein and nucleic acid in growth of bacteriophage" (PDF). J Gen Physiol. 36 (1): 39–56. doi:10.1085/jgp.36.1.39. PMC 2147348. PMID 12981234.
  2. ^ Dahm R (January 2008). "Discovering DNA: Friedrich Miescher and the early years of nucleic acid research". Hum. Genet. 122 (6): 565–81. doi:10.1007/s00439-007-0433-0. PMID 17901982.
  3. ^ "The Nobel Prize in Physiology or Medicine 1969". Nobel Foundation. Retrieved 6 April 2011.
  4. ^ a b c d e O'Connor, Clare (2008). "Isolating hereditary material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase". Scitable by Nature Education. Retrieved 20 March 2011.
  5. ^ Carr, Steven M. (2009). "Tetranucleotide Hypothesis". Retrieved 1 May 2011.
  6. ^ Matthew Cobb; Morange, Michel (1998). A history of molecular biology. Cambridge: Harvard University Press. pp. 37–38, 44. ISBN 0-674-00169-9.
  7. ^ Avery OT, Macleod CM, McCarty M; MacLeod; McCarty (February 1944). "Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III". J. Exp. Med. 79 (2): 137–58. doi:10.1084/jem.79.2.137. PMC 2135445. PMID 19871359.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ^ Pauling L, Corey RB; Corey (February 1953). "A Proposed Structure For The Nucleic Acids". Proc. Natl. Acad. Sci. U.S.A. 39 (2): 84–97. Bibcode:1953PNAS...39...84P. doi:10.1073/pnas.39.2.84. PMC 1063734. PMID 16578429.
  9. ^ "L.O. Rosalind Franklin and the Double Helix. Physics Today, March 2003". Physics Today. Retrieved 6 April 2011.
  10. ^ Crick, Francis (1988). "Chapter 8: The genetic code". What mad pursuit: a personal view of scientific discovery. New York: Basic Books. pp. 89–101. ISBN 0-465-09138-5.
  11. ^ Berk V, Cate JH; Cate (June 2007). "Insights into protein biosynthesis from structures of bacterial ribosomes". Curr. Opin. Struct. Biol. 17 (3): 302–9. doi:10.1016/j.sbi.2007.05.009. PMID 17574829.
  12. ^ Jackson DA, Symons RH, Berg P; Symons; Berg (October 1972). "Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli". Proc. Natl. Acad. Sci. U.S.A. 69 (10): 2904–9. Bibcode:1972PNAS...69.2904J. doi:10.1073/pnas.69.10.2904. PMC 389671. PMID 4342968.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  13. ^ Jobling MA, Gill P; Gill (October 2004). "Encoded evidence: DNA in forensic analysis" (PDF). Nat. Rev. Genet. 5 (10): 739–51. doi:10.1038/nrg1455. PMID 15510165.

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