Heredity and Development: Second Edition (1972)

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HEREDITY AND DEVELOPMENT: SECOND EDITION 152 7 The Substance of Inheritance Pneumonia in man is commonly caused by the bacterium Diplococcus pneu- moniae, known also by an older name—pneumococcus. Before the days of sulfa drugs and antibiotics, it was one of our most serious diseases. Diplococ- cus also produces disease in monkeys, rabbits, and mice. Horses, swine, sheep, dogs, cats, guinea pigs, chickens, and pigeons are resistant. Diplococcus pneumoniae shows a large amount of variability, much of which is now known to be genetic. For example, there are many dozen differ- ent types, usually designated by roman numerals. In the United States Type I and Type II are the most common. Types III and IV, as well as all the others, are less common. The cells of the various types seem to be identical so far as their general structure is concerned. Their specificity is due to the chemical composition of the capsule that surrounds them. The capsule is a thick, slimy, polysaccharide (a complex carbohydrate resembling the starch of plants). The types are identified by their reaction to antibodies. Thus, if Type II cells are injected into a rabbit, the rabbit will form Type II antibodies. These antibodies will react with the polysaccharide capsules of Type II cells but not with those of the other types. When capsulated cells are grown on culture plates they form colonies that are smooth and shiny in appearance. Mutations that give rise to cells lacking capsules are infrequent but, since the bacteria are in such tremendous num- bers, even rare mutations can easily be observed. Cells lacking the polysac- charide capsules form colonies that are rough in 

HEREDITY AND DEVELOPMENT: SECOND EDITION 153 appearance. The reverse mutation, of cells lacking capsules giving rise to cells that possess them, occurs rarely. There is an important biological property based on the presence or absence of the capsule. When capsules are present, the cells produce disease; when capsules are lacking the cells are harmless. Transformation. An experiment on these bacteria that was eventually to usher in a new era in genetics was reported by F.Griffith in 1928. Griffith was a Medical Officer working for the British Ministry of Health. His interests in Diplococcus, as judged by his publications, were entirely medical. He gave no suggestion of the tremendous implications that his work was to have for genetics. This is easy to understand, since in 1928 it was not believed that the variations observed in bacteria were comparable to the genetically controlled variations of higher organisms. Furthermore, the medical profession was nearly wholly ignorant of genetics, and geneticists were yet to begin a study of inheritance in bacteria. Griffith began one of his experiments with a culture of Type II bacteria that possessed capsules. Mice died if injected with these cells. As was usual, when these virulent bacteria were grown on culture plates, they produced the smooth colonies characteristic of capsulated cells. After repeated culturing, a few rough colonies appeared, which lacked the ability to synthesize the polysaccharide capsule. Thirty mice were injected with these capsuleless bacteria but no bacteremia occurred. The bacteria were harmless. Griffith observed, as had others before him, that only living capsulated cells would produce bacteremia in mice. If, for example, he killed the capsu- lated bacteria with heat, they could be injected into mice and no disease would result. It was established, therefore, that the capsular material itself would not produce disease. Next Griffith made a double injection: his mice received living capsuleless Type II bacteria plus heat-killed capsulated Type II. On the basis of the data given so far, we might predict that the mice would remain healthy. After all, they were receiving the harmless variant of living bacteria and dead cells of the virulent strain. Yet all of the four mice injected died after five days. Upon examination, their blood was found to be rich with Type II capsulated bacte- ria! So far as could be determined, they were the same as other strains of cap- sulated Type II cells. This was an almost unbelievable result. It appeared that the ability to syn- thesize a capsule had been transferred from the dead cells to the living cells. Any geneticist of 1928, who might have known of these 

HEREDITY AND DEVELOPMENT: SECOND EDITION 154 experiments, would have shuddered and rededicated himself to Drosophila melanogaster. Whatever the nature of the change, it seemed to have nothing to do with the rules that governed inheritance in peas, fruit flies, man, and every other species that had been studied. How could the results be explained? Possibly the first explanation that comes to mind would be the occurrence of a mutation of a gene lacking the ability to synthesize a capsule to an allele which could. Though possible, this seems unlikely. There had been thirty control mice, which were injected with capsuleless cells. They did not die. Yet all four mice receiving living caspule- less cells plus heat-killed caspulated cells had died. Another experiment showed that mutation was not the explanation, and also shed new light on the problem. Once again the experiment consisted of giving the mice a double injection: living capsuleless bacteria plus dead cap- sulated bacteria. This time, however, the cells were of different types: the living capsuleless cells were of Type II, but the dead capsulated cells were of Type I. Eight mice were injected. Two died, one on the third and one on the fifth day. Numerous bacteria were in the blood of these two famous mice and, when cultured and tested, they were found to be Type I capsulated cells. Somehow the Type II capsuleless cells had been converted into Type I capsu- lated cells. This was not a temporary change: the cells were cultured genera- tion after generation, and they remained Type I. The change was permanent and hence, in the broad sense, genetic. Griffith did not use such terms, but we would say today that one specific genetic type had been converted into another specific genetic type. The change was not a mutation but was appar- ently due to some influence of the dead cells—strange genetics indeed. Variations of the experiment gave similar results. Thus, Type II non- capsulated cells were converted to Type III capsulated cells, and Type I non- capsulated cells were converted to Type III capsulated cells. The changes occurred only when living non-capsulated cells plus dead capsulated cells were injected into mice. Griffith was unable to observe the same change when the experiments were carried out in test tubes. The transformation occurred in a living mouse but not in vitro. If we consider the tremendous medical problems caused by Diplococcus pneumoniae, it is not surprising that bacteriologists throughout the world were studying its biology. M.H.Dawson, of The Rockefeller Institute for Medical Research in New York, was one of these. In fact, he was doing exper- iments similar to those of Griffith in England. In 1927 Dawson, together with Oswald T.Avery (1877–1955), confirmed the even earlier observations that, when non-capsulated cells were in- 

HEREDITY AND DEVELOPMENT: SECOND EDITION 155 jected into mice, capsulated cells would usually be produced. One of their observations was: ‘In all cases in which transformation has been effected, reversion has invariably been toward the specific form from which the [cap- suleless] form was originally derived.’ This was 1927, a year before Griffith had published the results of his experiments using living and dead cells of different Types. In 1930, Dawson reported that he had confirmed Griffith’s experiments of 1928. He refined the experiments in important ways in order to ensure that the strange observations, though unexplainable, were true. One of his improvements was to begin the strains of bacteria from single cells, rather than use many cells as Griffith had done. A single cell was allowed to repro- duce and form a large population. Since this entire population had a single ancestor, and reproduction was by asexual means, it should have a high degree of genetic uniformity. With this precaution, one could rule out the possibility that the change from one type to another was not real but due to the use of mixed cultures. Using the strains obtained from single cells, Daw- son did the following: Non-capsulated cells derived from a Type II strain were injected into mice together with heat-killed capsulated cells of Type I, Type III, or Group IV. In each case the non-capsulated cells were transformed into capsulated cells of the type represented by the heat-killed cells. In later experiments, Dawson and his associates were able to produce the transformations in vitro. This was a most important discovery, making it far easier to find out what it was in the preparation containing the heat-killed cells that led to the transformation. In 1932 and 1933 J.L.Alloway reported that a crude extract of the capsulated cells would cause the transformation. The Chemistry of Transformation. The evidence was becoming quite con- vincing that a chemical substance was responsible for the transformations. It is probable that most workers expected the polysaccharide of the capsule to be the active agent. After all, the polysaccharide was responsible for the Type specificity but, wrote Alloway,* the polysaccharide ‘when added in chemi- cally purified form, has not been found effective in causing transformation of non-capsulated organisms derived from Diplococcus of one Type into capsu- lated forms of the other Type. When non-capsulated cells change into the capsulated form they always acquire the property of producing the specific capsular substance. The immunological specificity of the encapsulated cell depends upon the * What follows is not a direct quotation; I have modernized some of the technical terms. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 156 chemical constitution of the particular polysaccharide in the capsule. The synthesis of this specific polysaccharide is a function peculiar to cells with capsules. However, since the non-capsulated cells under suitable conditions have been found to develop again the capacity of elaborating the specific material, it appears in them this function is potentially present, but that it remains latent until activated by special environmental conditions. The fact that a non-capsulated strain derived from one Type of Diplococcus, under the conditions defined in this paper, may be caused to acquire the specific charac- ters of the capsulated forms of a Type other than that from which it was origi- nally derived implies that the activating stimulus is of a specific nature.’ There is nothing in this long quotation, or in any other writing of this period, to suggest that transformation might be a genetic phenomenon. Alloway and others seemed to regard the phenomenon as some sort of a phys- iological modification—a perfectly reasonable hypothesis on the basis of the available data. Dawson’s discovery that transformation could occur in vitro and Alloway’s discovery that a substance causing transformation could be extracted from the bacterial cells, suggested additional experiments. What was the chemical nature of the transforming substance? Alloway had demon- strated that it was not the polysaccharide of the capsule surrounding the cells. A likely guess was that it was a protein, for, in the 1930s, it seemed that nearly every important event that occurred in a living system involved or was controlled by proteins. The answer, however, lay elsewhere. DNA and Transformation. Work on the problem continued slowly at The Rockefeller Institute and, in 1944, a most important announcement was made. Avery, MacLeod, and McCarty reported that they had obtained the transforming substance in a highly purified state and had established its chem- ical nature beyond a reasonable doubt. They began with huge amounts of Type III cells, using in some experiments the cells from as many as 75 liters of culture medium. The cells passed through a procedure that involved extrac- tion, washing, precipitation, dissolving, and so on. In the end they had no more than 10 to 25 mg. of the active transforming substance. At frequent steps in the procedure they tested the preparation by seeing if Type II non- capsulated cells could be transformed into Type III capsulated cells. Their final extract, though small in amount, was highly active. In fact it was far more active, per unit of weight, than the original mass of cells. What was its chemical nature? The methods used in purifying the extract should have removed all 

HEREDITY AND DEVELOPMENT: SECOND EDITION 157 protein and all fat. As a check, however, the extract was tested for protein and none was found. Numerous other tests were made, including one for the pres- ence of deoxyribonucleic acid (DNA). The extract was found to be exceed- ingly rich in this substance. Tests for the closely similar compound, ribonucleic acid (RNA), gave only weakly positive results. In an effort to check on the reliability of the method, some purified DNA from animal cells was tested for RNA. This animal DNA gave the same weak test for RNA as did the purified transforming substance. These results suggested that the extract contained a large amount of DNA and possibly some RNA. One could not conclude, definitely, however, that the transforming substance was either compound. After all, other substances might be present and one or several of these be the active principle. Neverthe- less a good working hypothesis was: the transforming substance is DNA. The next step was to test the hypothesis. First a comparison was made of the elemental composition of the purified extract and the elemental composition of DNA. The percentage composition for the extract was: 34.88 carbon; 3.82 hydrogen; 14.72 nitrogen; and 8.79 phosphorus. In 1944 the structure of DNA was not accurately known, but the theoretical percentages of these elements were thought to be: 34.20 carbon; 3.21 hydrogen; 15.32 nitrogen; and 9.05 phosphorus. The parallel was strik- ing. Furthermore, the ratio of nitrogen to phosphorus, which was theoreti- cally 1.69 in DNA, was found to be 1.67 in the extract. If we assume that the extract consisted largely of the transforming substance, then the results sug- gested that the transforming substance could be DNA and that it could not be protein, nor fat, nor carbohydrate. Further tests substantiated this view. If we assume that the transforming substance is protein, then its activity should be destroyed by the enzymes that digest protein. Two protein-digesting enzymes of pancreatic juice, trypsin and chymotrypsin, were added to an extract containing the transforming sub- stance. There was no loss of activity. Another experiment indicated that the transforming substance was not RNA. Other workers had discovered an enzyme, ribonuclease, which destroys RNA. When this enzyme was added to the extract, there was no loss of activity. Avery, MacLeod, and McCarty concluded, The fact that trypsin, chymotrypsin, and ribonuclease had no effect on the transforming principle is further evidence that the substance is not ribonucleic acid or a protein sus- ceptible to the action of tryptic enzymes.’ Thus they were reasonably sure of some of the substances that the active principle could not be, but how could they prove the hypothesis 

HEREDITY AND DEVELOPMENT: SECOND EDITION 158 that it was DNA? Convincing evidence would be obtained if they could use some agent that specifically destroyed DNA. If such an agent destroyed the ability of the extract to transform cells, one could conclude that the transform- ing substance was DNA. Four years earlier, two investigators had reported that tissue extracts and blood serum contain an enzyme that breaks down the large molecules of DNA. The enzyme, which was obtained in a crude form, is known today as deoxyribonuclease (DNase). Avery, MacLeod, and McCarty prepared some of this enzyme and added it to their active extract of transforming substance. There was a complete loss of ability to transform cells. This was most con- vincing. In addition, many other experiments were carried out and the results were all explainable on the basis of the hypothesis that the transforming sub- stance was DNA. Preliminary observations suggested that the molecular weight of this DNA was very large—about 500,000. Its biological activity was also quite impres- sive: transformation could be induced when the DNA was present in a con- centration of one part in 600 million. The following quotation shows how the authors explained their observa- tions that an extract of the DNA of Type III capsulated cells could cause Type II non-capsulated cells to start producing capsules that were specific to Type III. In the present state of knowledge any interpretation of the mechanism involved in transformation must of necessity be purely theoretical. The biochemical events underlying the phenomenon suggest that the transforming principle interacts with the [non-capsulated]* cell giving rise to a coordinated series of enzymatic reactions that culminate in the synthesis of the Type III capsular antigen. The experimental findings have clearly demonstrated that the induced alterations are not random changes but are predictable, always corresponding in type specificity to that of the encapsulated cells from which the transforming substance was isolated. Once transformation has occurred, the newly acquired characteristics are thereafter transmitted in series through innumerable trans- fers in artificial media without any further addition of the transforming agent. Moreover, from the transformed cells themselves, a substance of identical activ- ity can again be recovered in amounts far in excess of that originally added to induce the change. It is evident, therefore, that not only is the capsular material reproduced in successive generations but that the primary factor, which con- trols the occurrence and specificity of capsular development, is also redupli- cated in the daughter cells. The induced changes are not temporary modifica- tions but are permanent alterations which persist provided * The terms in brackets are substitutions for older terms. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 159 the cultural conditions are favorable for the maintenance of capsule forma- tion. The transformed cells can be readily distinguished from the parent [non- capsulated] forms not alone by serological reactions but by the presence of a newly formed and visible capsule which is the immunological unit of type specificity and the accessory structure essential in determining the infective capacity of the microorganism in the animal body. It is particularly significant in the case of [the bacterial cells] that the experi- mentally induced alterations are definitely correlated with the development of a new morphological structure and the consequent acquisition of new antigenic and invasive properties. Equally if not more significant is the fact that these changes are predictable, type-specific, and heritable. Various hypotheses have been advanced in explanation of the nature of the changes induced. In his original description of the phenomenon Griffith sug- gested that the dead bacteria in the inoculum might furnish some specific pro- tein that serves as a ‘pabulum’ and enables the [non-capsulated] form to manu- facture a capsular carbohydrate. More recently the phenomenon has been interpreted from a genetic point of view. The inducing substance has been likened to a gene, and the capsular anti- gen which is produced in response to it has been regarded as a gene product. In discussing the phenomenon of transformation Dobzhansky has stated that “If this transformation is described as a genetic mutation—and it is difficult to avoid so describing it—we are dealing with authentic cases of induction of spe- cific mutations by specific treatments….” It is, of course, possible that the biological activity of the substance described is not an inherent property of the nucleic acid but is due to minute amounts of some other substance adsorbed to it or so intimately associated with it as to escape detection. If, however, the biologically active substance isolated in highly purified form as the sodium salt of deoxyribonucleic acid actually proves to be the transforming principle, as the available evidence strongly sug- gests, then nucleic acids of this type must be regarded not merely as structurally important but as functionally active in determining the biochemical activities and specific characteristics of [the bacterial] cells. Assuming that the sodium deoxyribonucleate and the active principle are one and the same substance, then the transformation described represents a change that is chemically induced and specifically directed by a known chemical compound. If the results of the present study on the chemical nature of the transforming principle are confirmed, then nucleic acids must be regarded as possessing biological specificity the chemical basis of which is as yet undetermined. Dobzhansky’s belief that the DNA was inducing mutations was probably shared by most geneticists. After all, it was clearly established that mutations could be produced experimentally. Muller had demonstrated this, in 1927, by using X rays (page 125). Other investigators sub- 

HEREDITY AND DEVELOPMENT: SECOND EDITION 160 sequently discovered that ultraviolet light and some chemical substances would cause mutations. Stimulus or Substance. There is a difference, of fundamental importance, between the effects of X rays and the phenomenon of transformation in bacte- ria. X rays do not produce specific mutations. One could not use X rays to produce mutations only at the white-eye locus in Drosophila. Instead, the X rays would produce many kinds of mutations. Possibly one would be at the white-eye locus, possibly not. The DNA extracted from capsulated bacteria, in contrast, could produce specific changes in non-capsulated cells. Since these specific changes were inheritable, there were grounds for calling them mutations. One could imagine the mechanism to work somewhat as follows: the bacteria contain a gene, which can be either in the capsule-producing or non-capsule-producing state. We can call the alleles c+ and c−, respectively. We would assume that DNA extracted from c+ cells has the ability to cause the mutation c− → c+. The implications of this hypothesis, if true, were enormous. Man could control inheritance to a degree never before possible. He might mold his own species and others of importance to him. To be sure, he could do this for only a single gene and in only one species of bacteria, but this limitation might be overcome with further research. Of course, there was no theoretical reason to suppose that the mutation process could not be controlled. Mutation, whatever it was, could only be a physical or chemical event—a scientist can conceive of no other possibility. Therefore any mutation, such as c− → c+, would become controllable once the necessary information about the biology of cells was at hand. Prior to 1940 a geneticist might have predicted that the cell biologist should be able to supply him with the necessary information about the year 2000. It was all the more remarkable, therefore, that the feat was accomplished in 1944. But the true explanation of transformation in bacteria by DNA lay elsewhere. Bacteriophage. Bacteria have their health problems too. Microorganisms known variously as the bacterial viruses, bacteriophages, or phages can attack bacterial cells and so disrupt the cell’s metabolism that death results. In recent years, one bacterium and its many phage parasites have given important new insights into genetic mechanisms. The bacterium, Escherichia coli, is a harmless inhabitant of the large intes- tine of man. It can be grown readily in the laboratory. Large populations are easy to obtain since cell division occurs about once every 20 minutes. Thus a geneticist, who would have to wait about 75 years 

HEREDITY AND DEVELOPMENT: SECOND EDITION 161 for three generations in man, or six weeks in Drosophila, could observe three generations in E. coli in one hour. If E. coli is infected with a phage, such as one called T2, the bacterial cell is killed in about 20 minutes. The main steps are as follows. The phage attacks the cell and produces a profound change in the cell’s metabolism. Before the phage appeared, the cell was synthesizing its own specific molecules: bacterial proteins, bacterial nucleic acids, and on on. The phage changes all this. In some manner it assumes control of the cell’s synthetic machinery and directs it to produce phage molecules instead of E. coli molecules. In about 20 minutes, the bacterial cell will have been forced to make about 100 phage particles. It is at this point that the bacterial cell ruptures and liberates the newly formed phage. Each new phage can infect another E. coli cell and repeat the cycle. Phage particles have a specific identity that is shown in many ways: the T2 phages can grow only in living E. coli cells; they have a characteristic struc- ture which is revealed when they are photographed with an electron micro- scope; chemically they are comparatively simple, being composed of an outer coat of protein and a core of DNA. The phage particles that are released by the bursting bacterial cell are, in the vast majority of cases, the same as the particle that first entered. Thus the phages exhibit genetic continuity. It follows, therefore, that a biological sys- tem consisting of no more than a protein coat and a DNA core contains all the genetic information needed to direct a bacterial cell to make more T2 phage. These phage particles are only about one-fifth of a micron in length. They are, therefore, far smaller than the familiar carriers of genetic information— the chromosomes. In this genetic system, so simple that it consists of only two parts, it might be possible to determine which part is the molecular basis of inheritance. Does inheritance in the T2 phage depend on the protein coat, on the DNA core, or on the interaction of both? A partial answer to this ques- tion was provided in 1952 by A.D.Hershey and Martha Chase. Core or Coat. Hershey and Chase used radioactive substances to tag sepa- rately the protein coat and the DNA core of the phage. This is possible because of a fundamental chemical difference between the protein and DNA. DNA is rich in phosphorus, but it contains no sulfur. On the other hand, the protein of the phage coat contains sulfur, but little or no phosphorus. In 1952 radioactive isotopes of both phosphorus, such as P32, and sulfur, such as S35, were available. It should be possible, therefore, to obtain phage with its pro- tein containing radioactive sulfur and its DNA containing radioactive phosphorus. Since the phage reproduced only in the living cells of E. coli, ma- 

HEREDITY AND DEVELOPMENT: SECOND EDITION 162 terials in the cell of coli must be the source of the newly synthesized phage. It was necessary, therefore, to introduce the radioactive markers into the T2 phage by way of the bacterial cell. The procedure was as follows. One group of bacteria was grown in a medium that contained small amounts of P32. The phosphorus entered the bacterial cells and became part of the cell’s molecules. Another group of bac- teria was grown in a medium containing S35. Each group of bacteria was allowed to grow for about four hours and was then infected with phage. The phage entered the bacterial cells and reproduced. The new phage was pro- duced, of course, from the materials in the bacterial cells, which contained the radioactive markers. Thus in one group of phage the protein coats became marked with S35 and in the other group the DNA was marked with P32. It would then be possible to trace the movements of the radioactively labelled phage protein in one case and the DNA in the other during the infection of bacteria. At this point we must digress to mention an important observation 7–1 The T2 phage as photographed with the electron microscope at a magnification of 37,000× (photo by J.S.Murphy. Journal of General Physiology 36:28). 

HEREDITY AND DEVELOPMENT: SECOND EDITION 163 concerning the mechanics of infection. Other investigators had found that the T2 phages were elongate structures with a wide cylindrical ‘head’ and a nar- row cylindrical ‘tail’ (Fig. 7–1). Photos taken with an electron microscope showed the tail of the phage attached to the cell wall of the bacterium. To these observations, Hershey and Chase added others that seemed to indicate that the phage became attached to the bacterium and then injected its DNA into the cell. If only the DNA of the phage entered the cell, one could con- clude that the DNA alone carried genetic information. Having tagged either the protein or the DNA of the phage, Hershey and Chase were in a position to test this hypothesis. Bacteria were infected with phage that had their protein coats labelled with S35. A few minutes later the bacteria were put in a Waring blendor. The cells are too small to be injured by the whirling blades. But the solution was agi- tated so violently that the phage particles were torn loose from the bacterial cell walls. The cells were then separated from the fluid medium. Both frac- tions were tested for S35. It was found that 80 per cent of the S35 was in the fluid and only 20 per cent was associated with the cells. Nevertheless, the cells were infected: in 20 minutes they burst and liberated a new crop of phage. In a parallel experiment, other bacteria were infected with phage that had their DNA labelled with P32. In a few minutes the bacteria, plus phage, were put in a Waring blendor, the phage ripped from the bacterial cells, and then the cells and phage separated. Analysis of the cells and fraction containing the phage gave results precisely opposite to those observed with S35. This time it was found that about 70 per cent of the P32 was associated with the cells and only 30 per cent with the detached phage particles. Once again, the phage reproduced in the cells, in spite of the drastic treatment. Thus infection and phage reproduction occurred when most of the DNA entered the cell and most of the protein coat stayed on the outside. The results were somewhat equivocal, for all of the S35, which was the marker for pro- tein, had not remained on the outside. Nevertheless it was not unreasonable to advance this working hypothesis: the phage DNA carries all the genetic information needed for phage replication. Another way to test this hypothesis is based on the following argument. The hereditary substance is undoubtedly more stable than other substances in the organism. We should expect, therefore, that it would persist intact genera- tion after generation, while any non-hereditary materials would not. If the P32 is associated with the hereditary material but the S35 is not, one would predict different behaviors for them. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 164 Consider first the case of a phage particle with its DNA marked with P32 infecting a cell. If Hershey and Chase were correct, the DNA alone would enter the cell. Reproduction would begin, and the original DNA would be divided among the daughter phage particles and become diluted, so to speak, but not diminished in total amount. After the infection cycle was completed, and the cell ruptured, the 100 liberated phages should have among them the original DNA of the entering phage. Any hereditary substance should be expected to behave in this way. If the protein were a hereditary material, it should behave in the same way; if not, we might expect that only a small por- tion of the S35 that entered the cell would appear in the progeny. Hershey and Chase put these ideas to experimental test. They found less than one per cent of the S35 of the initial phage was recovered in the daughter phages. Other investigators (including James D.Watson of whom we shall hear more shortly) had just reported that about 50 per cent of the P32 that first entered the bacterial cells was recovered in the daughter phage particles. Hershey and Chase concluded, ‘Our experiments show clearly that a physi- cal separation of the phage T2 into genetic and non-genetic parts is possible…. The chemical identification of the genetic part must wait, how- ever, until some of the questions asked above have been answered.’ These questions were: ‘(1) Does any sulfur-free phage material other than DNA enter the cell? (2) If so, is it transferred to the phage progeny? (3) Is the trans- fer of phosphorus (or hypothetical other substance) to progeny direct—that is, does it remain at all times in a form specifically identifiable as phage sub- stance—or indirect?’ Hershey and Chase were showing necessary and commendable caution in interpreting their remarkable experiments. The implication of their experi- ments was clear, however: DNA is the substance of inheritance in T2 phage. When one remembers the near universality of genetic phenomena, it is not too difficult to extend the hypothesis to cover all organisms: DNA is the sub- stance of inheritance; the chemical compound of which the genes are composed. With this hypothesis in mind, we can reinterpret the experiments on trans- formation in Diplococcus. Avery and his co-workers had shown that DNA extracted from capsulated cells could cause non-capsulated cells to form cap- sules. They suggested that the DNA stimulates a specific gene mutation in the non-capsulated cells. In the light of the work of Hershey and Chase, an entirely different mechanism for transformation might be proposed: The extracted DNA consists, in part, of genes that have the ability to cause the cell to synthesize capsules; these genes enter the non-capsulated cells and become part of the genetic machinery 

HEREDITY AND DEVELOPMENT: SECOND EDITION 165 of the invaded cells; in their new environment they initiate the synthesis of capsules. Avery’s extract, therefore, could be thought of as containing func- tional genes. These genes could ‘infect’ bacterial cells in much the same way as phage infect E. coli. The experiments of Avery, MacLeod, and McCarty, and of Hershey and Chase, were outstanding examples in a large body of data that made it increas- ingly probable that the substance of inheritance is DNA. In a few viruses, such as the tobacco mosaic virus, the closely similar ribonucleic acid (RNA) appeared to be the hereditary material. Apart from these few exceptions, all of which are viruses, the most likely candidate for the hereditary molecule was DNA. Many important observations were made possible by a staining procedure known as the Feulgen reaction. This procedure, first developed in 1924, proved to be a specific stain for DNA. That is, when properly used, only the DNA of cells is stained. It is possible, therefore, to use this technique to local- ize the DNA in cells. Numerous observations revealed that the nuclei of ani- mal and plant cells are rich in DNA. The cytoplasm is never stained, or is at most, stained very feebly. During mitosis the chromosomes stain deeply whereas in non-dividing cells the nuclei are nearly uniformly stained. Thus the DNA was localized in precisely that part of the cell where the geneticists had unequivocally located the genes. Late in the 1940s, A.W.Pollister, of Columbia University, and others per- fected a photometric method for measuring the amount of DNA in a single nucleus. The Feulgen reaction can be used as a quantitative method for mea- suring DNA, that is, the amount of dye bound is proportional to the amount of DNA. Cells are stained and put under a microscope. Exceedingly sensitive photo-cells (working on the same principle as the familiar exposure meters of the photographer) are used to measure the amount of light that passes through the nucleus. The amount of light that passes depends on the amount of stain in the nucleus. If the nucleus contains much DNA, the stain is heavy, and lit- tle light will pass. This method allows one to measure quite accurately rela- tive amounts of DNA in different nuclei. Many investigators used this technique to measure the DNA in a wide vari- ety of tissues of animals and plants. The basic findings were these: in any species the diploid nuclei of the somatic cells appear to have the same amount of DNA; after meiosis, however, the nuclei of sperm and ova have only half as much DNA. An exact parallel is found, therefore, between the amounts of DNA and the number of chromosomes. The work surveyed in this chapter leads to this tremendous thought: the once mysterious gene, which could be mapped but not known, was 

HEREDITY AND DEVELOPMENT: SECOND EDITION 166 revealed as an identifiable molecule—deoxyribonucleic acid. Clearly we must learn more about the nature of this DNA, and we shall do so in the next chapter. Suggested Readings A few general references are given here but, since the questions raised in this chapter find their answers in the next, one should also refer to the general references listed there. The Readings include a fascinating letter written by Avery to his brother describing his experiments on transformation. DUNN, L.C. 1969. ‘Genetics in historical perspective.’ In Genetic Organization. Volume 1. Edited by Ernst W.Caspari and Arnold W.Ravin. New York: Academic Press. RAVIN, ARNOLD W. 1965. The Evolution of Genetics. New York: Academic Press. WHITEHOUSE, H.L.K. 1969. Towards an Understanding of the Mechanism of Heredity. New York: St. Martin’s Press. The original papers referred to most frequently in this chapter are: ALLOWAY, J.L. 1932. ‘The transformation in vitro of R pneumococci into S forms of different specific types by the use of filtered pneumococcus extracts.’ Journal of Experi- mental Medicine 55:91–9. AVERY, O.T., C.M.MACLEOD, and M.MCCARTY. 1944. ‘Studies on the chemical nature of the substance inducing transformation of pneumococcal types.’ Journal of Experimental Medicine 79:137–58. DAWSON, M.H. 1930. ‘The transformation of pneumococcal types.’ Journal of Experi- mental Medicine 51:99–147. GRIFFITH, FRED. 1928. ‘The significance of pneumococcal types.’ Journal of Hygiene 27:113–59. HERSHEY, A.D. and MARTHA CHASE. 1952. ‘Independent functions of viral protein and nucleic acid in growth of bacteriophage.’ Journal of General Physiology 36:39–56.