Hartl, Jones - Genetics. Principlers and analysis - 1998, страница 10

However, it was discovered in 1928 that mice often died of pneumonia when injected witha mixture containing a small number of living R cells and a large number of dead S cells. Bacteriaisolated from blood samples of the mice infected with the mixture produced S cultures with acapsule typical of the injected S cells, even though the injected S cells had been killed by heat.Therefore, the material containing the dead S cells that was injected must have included a substancethat could convert, or transform, otherwise harmless cells of the R bacterial strain into S strain cellswith the ability to resist the immunological system of the mouse, multiply, and cause pneumonia.

Inother words, there was a genetic transformation of an R cell into an S cell. Furthermore, the newgenetic characteristics were inherited by descendants of the transformed bacteria.Figure 1.1Colonies of rough (R, the small colonies) and smooth (S, the large colonies) strains of Streptococcuspneumoniae. The S colonies are larger because of the capsule on the S cells.[Photograph from O.T. Avery, C.M. MacLeod, and M. McCarty.

1944. J. Exp. Med. 79: 137.]Page 4Connection It's the DNA!Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty 1944The Rockefeller University, New York, New York Studies on the Chemical Nature of the Substance InducingTransformation of Pneumococcal TypesThis paper is one of the milestones of molecular biology. Genetics and biochemistry were at last united throughthe finding that DNA was the chemical substance of the genetic material. There is very little biology in the paperbeyond the use of Streptococcus (then called Pneumococcus) to ascertain whether a particular batch of extract,or an extract treated in some manner, contained the active substance able to transform type R into type S cells.The thrust of the paper is biochemistry: purifying the substance, showing that no known macromolecules otherthan DNA could be found in the extract, and demonstrating that the transforming activity could be destroyed byenzymes that attack DNA but not by protease or RNase enzymes.Biologists have long attempted by chemical means to induce in higher organisms predictable and specific changeswhich thereafter could be transmitted as hereditary characters.

Among microorganisms the most striking exampleof inheritable and specific alterations in cell structure and function that can be experimentally induced is thetransformation of specific types of Pneumococcus. This phenomenon was first described by Griffith, whosucceeded in transforming an attenuated [nonvirulent] and nonencapsulated (R) variant into fully encapsulatedand virulent (S) cells. . . . The present paper is concernedWithin the limit of the analytical methods, the active fraction contains no demonstrable protein,lipid, or polysaccharide and consists principally, if not solely, of a highly polymerized form ofdeoxyribonucleic acid.with a more detailed analysis of the phenomenon of transformation of specific types of Pneumococcus. The majorinterest has centered in attempts to isolate the active principle from crude extracts and to identify its chemicalnature, or at least to characterize it sufficiently to place it in a general group of known chemical substances.

. . . Abiologically active fraction has been isolated in highly purified form which in exceedingly minute amounts iscapable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants intofully encapsulated forms of the same specific type as that of the heat-killed microorganisms from which theinducing material was recovered. . . . Within the limit of the analytical methods, the active fraction contains nodemonstrable protein, lipid, or polysaccharide and consists principally, if not solely, of a highly polymerized formof deoxyribonucleic acid. .

. . Various enzymes have been tested for their capacity to destroy the transformingactivity. Extracts to which were added crystalline trypsin and chymotrypsin [proteases], or combinations of both,suffered no loss in activity. . . . Prolonged treatment with crystalline ribonuclease under optimal conditions causedno demonstrable decrease in transforming activity. . . .

The blood serum of several mammalian species containsan enzyme which causes the depolymerization of deoxyribonucleic acid; fresh dog and rabbit serum are capableof completely destroying transforming activity. . . . The evidence presented supports the belief that a nucleic acidof the deoxyribose type is the fundamental unit of the transforming principle.Source: Journal of Experimental Medicine 79: 137-158What substance was present in the dead S cells that made transformation possible? In the early 1940s, componentsof dead S cells were extracted and added to R cell cultures. The key experiment was one in which DNA wasextracted from dead S cells and added to growing cultures of R cells and the resulting mixture spread onto an agarsurface (Figure 1.2A). Among the R colonies, a few of type S appeared! Although the DNA preparations may stillhave contained traces of protein and RNA, the addition of an enzyme that destroys proteins (a protease enzyme) orone that destroys RNA (an RNase enzyme) did not eliminate the transforming activity (Figure 1.2B).

On the otherhand, the addition of an enzyme that destroys DNA completely eliminated the transforming activity (Figure 1.2C).These experiments were carried out by Oswald Avery, Colin MacLeod, and Maclyn McCarty at the RockefellerUniversity. They concluded their landmark report by noting that "the evidence presented supports the belief that anucleic acid of the deoxyribose type is fundamentalPage 5Figure 1.2A diagram of the experiment that demonstrated that DNA is the active material in bacterialtransformation.(A) Purified DNA extracted from heat-killed S cells can convert some living R cells intoS cells, butthe material maystill containundetectable traces of protein and/or RNA.

(B) Thetransforming activityis not destroyed byeither protease or RNase. (C) The transformingactivity is destroyed byDNase and so probably consists of DNA.Page 6unit of the transforming principle." In other words, DNA seems to be the genetic material.Genetic Role of DNA in BacteriophageA second important finding concerned a type of virus that infects bacterial cells.

The virus, T2 by name, is knownas a bacteriophage, or phage for short, because it infects bacterial cells. Bacteriophage means "bacteria-eater." T2infects cells of the intestinal bacterium Escherichia coli. A T2 particle is illustrated in Figure 1.3. It is exceedinglysmall, yet it has a complex structure composed of head (which contains the phage DNA), collar, tail, and tail fibers.(For comparison, consider that the head of a human sperm is about 30 to 50 times larger in both length and widththan the T2 head.) T2 infection begins with attachment of a phage particle by the tip of its tail to the bacterial cellwall, entry of phage material into the cell, multiplication of this material to form a hundred or more progeny phage,and release of progeny by disruption of the bacterial host cell.Because DNA contains phosphorus but no sulfur, and proteins usually contain some sulfur but no phosphorus, theDNA and proteins in a phage particle can be labeled differentially by the use of radioactive isotopes of the twoelements.

This difference was put to use by Alfred Hershey and Martha Chase in 1952, working at the Cold SpringHarbor Laboratories. By that time it was already known that T2 particles are composed of DNA and protein inapproximately equal amounts. Hershey and Chase produced particles with radioactive DNA by infecting E. colicells that had been grown for several generations in a medium containing 32P (a radioactive isotope of phosphorus)and then collecting the phage progeny.

Other particles with labeled proteins were obtained in the same way, using amedium that contained 35S (a radioactive isotope of sulfur).The experiments are summarized in Figure 1.4. Nonradioactive E. coli cells were infected with phage labeled witheither 32P (Figure 1.4A) or 35S (Figure 1.4B) in order to follow the proteins and DNA separately. Infected cellswere concentrated by centrifugation, resuspended in fresh medium, and then agitated in a kitchen blender to shearattached phage material from the cell surfaces.

The blending was found to have no effect on the subsequent courseof the infection, which implies that the genetic material must enter the infected cells very soon after phageattachment. When intact bacteria were separated from the material removed by blending, most of the radioFigure 1.3(A) Drawing of E. coli phage T2, showing various components. The DNA isconfined to the interior of the head. (B) An electron micrograph of phageT4, a closely related phage.[Electron micrograph courtesy of Robley Williams.]Page 7Figure 1.4The Hershey-Chase (''blender") experiment, which demonstrated that DNA, not protein, is responsiblefor directing the reproduction of phage T2 in infected E. coli cells.

(A) Radioactive DNA is transmittedto progeny phage in substantial amounts. (B) Radioactive protein is transmitted to progeny phagein negligible amounts.Page 8Connection Shear MadnessAlfred D. Hershey and Martha Chase 1952Cold Spring Harbor Laboratories,Cold Spring Harbor, New YorkIndependent Functions of Viral Protein and Nucleic Acid in Growth of BacteriophagePublished a full eight years after the paper of Avery, MacLeod and McCarty, the experiments of Hershey andChase get equal billing. Why? Some historians of science suggest that the Avery et al.

experiments were "aheadof their time." Others suggest that Hershey had special standing because he was a member of the "in group" ofphage molecular geneticists. Max Delbrück was the acknowledged leader of this group, with Salvador Luriaclose behind. (Delbrück, Luria and Hershey shared a 1969 Nobel Prize.) Another possible reason is that whereasthe experiments of Avery et al. were feats of strength in biochemistry, those of Hershey and Chase werequintessentially genetic. Which macromolecule gets into the hereditary action, and which does not? Buried in themiddle of this paper, and retained in the excerpt, is a sentence admitting that an earlier publication by theresearchers was a misinterpretation of their preliminary results. This shows that even first-rate scientists, thenand now, are sometimes misled by their preliminary data. Hershey later explained, "We tried various grindingarrangements, with results that weren't very encouraging. When Margaret McDonald loaned us her kitchenblender the experiment promptly succeeded."The work [of others] has shown that bacteriophages T2, T3, and T4 multiply in the bacterial cell in a noninfective [immature] form.