Avery-MacLeod-McCarty experiment

Avery-MacLeod-McCarty experiment

The Avery-MacLeod-McCarty experiment was an experimental demonstration, reported in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, that DNA is the substance that causes bacterial transformation. It was the culmination of research in the 1930s and early 1940s at the Rockefeller Institute for Medical Research to purify and characterize the "transforming principle" responsible for the transformation phenomenon first described in Griffith's experiment of 1928: killed "Streptococcus pneumoniae" of the virulent strain type III-S, when injected along with living but non-virulent type II-R pneumococci, resulted in a deadly infection of type III-S pneumococci. In their paper "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", published in the February 1944 issue of the "Journal of Experimental Medicine", Avery and his colleagues suggest that DNA, rather than protein as widely believed at the time, may be the hereditary material of bacteria, and could be analogous to genes and/or viruses in higher organisms.Cite journal| doi = 10.1084/jem.79.2.137| volume = 79| issue = 2| pages = 137–158| last = Avery| first = Oswald T.| coauthors = Colin M. MacLeod, Maclyn McCarty| title = 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| journal = Journal of Experimental Medicine| accessdate = 2008-09-29| date = 1944-02-01| url = http://www.jem.org/cgi/content/abstract/79/2/137] [Fruton (1999), pp. 438-440]

Experimental work

After Griffith's experiment of 1928, Rockefeller Institute researchers Martin Dawson and James Alloway worked to extend Griffith's findings, resulting in the extraction of aqueous solutions of the transforming principle by 1933. Colin MacLeod worked to purify such solutions from 1934 to 1937, and the work was continued in 1940 and completed by Maclyn McCarty. [Fruton (1999), p. 438]

For the Avery-MacLeod-McCarty experiment, the scientists prepared the purified transforming principle of several types of pneumococci, and used them to transform Type II, strain R36A into other types. The R strain, as in Griffith's experiment, was a non-virulent strain of Type II pneumococci that had lost its polysaccharide capsule and produced characteristically "rough"-looking colonies when grown in the lab. Normally, pneumococcus is characterized by "smooth" colonies and has a polysaccharide capsule that induces antibody formation; the different types are classified according to their immunological specificity.

The purification procedure consisted of first killing the bacteria with heat and extracting the saline-soluble components, then precipitating out the protein using chloroform and hydrolyzing the polysaccharide capsules with an enzyme, using an immunological precipitation caused by type-specific antibodies to verify the complete destruction of the capsules. Then, the active portion was precipitated out by alcohol fractionation, resulting in fibrous strands that could be removed with a stirring rod.

Chemical analysis showed that the proportions of carbon, hydrogen, nitrogen, and phosphorus in this active portion were consistent with DNA. To show that it was DNA rather than some small amount of RNA, protein, or some other cell component that was responsible for transformation, Avery and his colleagues used a number of biochemical tests. They found that trypsin, chymotrypsin and ribonuclease did not affect it, but an enzyme preparation of "desoxyribonucleodepolymerase" (a crude preparation, obtainable from a number of animal sources, that could break down DNA) destroyed the extract's transforming power.

Followup work in response to criticism and challenges included the purification and crystallization, by Moses Kunitz in 1948, of a DNA depolymerase (deoxyribonuclease I), and extremely precise work by Rollin Hotchkiss showing that virtually all the detected nitrogen in the purified DNA came from glycine, a breakdown product of the nucleotide base adenine, and that undetected protein contamination was at most .02% by Hotchkiss's estimation. [Fruton (1999), p. 439] [Cite journal| volume = 170| issue = 4| pages = 1443–1447| last = Witkin| first = Evelyn M.| title = Remembering Rollin Hotchkiss (1911–2004)| journal = Genetics| accessdate = 2008-09-26| date = 2005-08| url = http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1449782| pmid = 16144981]

Reception and legacy

The experimental findings of the Avery-MacLeod-McCarty experiment were quickly confirmed, and extended to a number of other bacterial species as well. However, there was considerable reluctance to accept the conclusion that DNA was the genetic material. According to Phoebus Levene's influential "tetranucleotide hypothesis", DNA consisted of repeating units of the four nucleotide bases and has little biological specificity. DNA was therefore thought to be the structural component of chromosomes, whereas the genes were thought likely to be made of the protein component of chromosomes. This line of thinking was reinforced by the 1935 crystallization of tobacco mosaic virus by Wendell Stanley, and the parallels among viruses, genes, and enzymes; many biologists thought genes might be a sort of "super-enzyme", and viruses were shown according to Stanley to be proteins and to share the property of autocatalysis with many enzymes. Furthermore, few biologists thought that genetics could be applied to bacteria, since they lacked chromosomes and sexual reproduction. In particular, many of the geneticists known as the phage group, which would become influential in the new discipline of molecular biology in the 1950s, were dismissive of DNA as the genetic material (and were inclined to avoid the "messy" biochemical approaches of Avery and his colleagues). Some biologists, including fellow Rockefeller Institute Fellow Alfred Mirsky, challenged Avery's finding that the transforming principle was pure DNA, suggesting that protein contaminants were instead responsible. [Morange (1998), pp. 30-39] [Fruton (1999), pp. 440-441]

By the 1952 Hershey-Chase experiment, geneticists were more inclined to consider DNA as the genetic material, and Alfred Hershey was an influential member of the phage group. Erwin Chargaff and Rollin Hotchkiss had shown that the base composition of DNA varies by species (contrary to the tetranucleotide hypothesis), and Hotckiss (working with Stephen Zamenhof) published his experimental evidence of the absence of protein in Avery's transforming principle in 1952. Furthermore, the field of bacterial genetics was quickly becoming established, and biologists were more inclined to think of heredity in the same terms for bacteria and higher organisms. After Hershey and Chase used radioactive isotopes to show that it was primarily DNA, rather than protein, that entered bacteria upon infection with bacteriophage, it was soon widely accepted that DNA was the genetic material. Despite the much less precise experimental results (they found a not-insignificant amount of protein entering the cells as well as DNA), the Hershey-Chase experiment was not subject to the same degree of challenge. Its influence was boosted by the growing network of the phage group and, the following year, by the publicity surrounding the DNA structure proposed by Watson and Crick (Watson was also a member of the phage group). Only in retrospect, however, did either experiment definitively prove that DNA is the genetic material. [Morange (1998), pp. 44-50] [Fruton (1999), pp. 440-442]

Notes

References

* Fruton, Joseph S. "Proteins, Enzymes, Genes: The Interplay of Chemistry and Biology". Yale University Press: New Haven, 1999. ISBN 0-300-07608-8
* Morange, Michel. "A History of Molecular Biology". Translated by Matthew Cobb. Harvard University Press: Cambridge, Massachusetts, 1998. ISBN 0-674-00169-9

Further reading

*Cite journal| volume = 34| issue = 2| pages = 207–232 | last = Deichmann| first = Ute| title = Early responses to Avery et al.'s paper on DNA as hereditary material| journal = Historical Studies in the Physical and Biological Sciences| accessdate = 2008-09-29| date = 2004-03| url = http://caliber.ucpress.net/doi/abs/10.1525/hsps.2004.34.2.207| doi = 10.1525/hsps.2004.34.2.207
*Cite journal| volume = 136| issue = 2| pages = 423–426 | last = Lederberg | first = Joshua| title = The Transformation of Genetics by DNA: An Anniversary Celebration of Avery, MacLeod and McCarty (1944)| journal = Genetics| accessdate = 2008-09-29| date = 1994-02| url = http://www.genetics.org/cgi/reprint/136/2/423


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