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Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press STUDIES ON METABOLISM CONTROLLING OF MECHANISMS VIRUS-INFECTED IN BACTERIA THE 1 SEYMOUR S. COHEN The Children's Hospital of Philadelphia (Department of Pediatrics), and the School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania The work which I wish to present today extends the observations reported six years ago (Cohen, 1947) at the Cold Spring Harbor Symposium on Nucleic Acids and Nucleoproteins. Most of my studies since then represent an effort to test certain hypotheses formulated at that time concerning the control of the metabolism of E. coli, strain B, infected by T2 bacteriophage. In an effort to strengthen my biological equipment for the studies ahead, I had the good fortune to spend some months of 1947 and 1948 in the laboratory of M. Andre Lwoff at the Pasteur Institute, where problems of bacterial variability, involving studies of enzymatic adaptation and mutation, were being actively pursued. The practical advantages accruing from the experimental use of genetic and environmental variability as a tool in chemical virology will be apparent in this paper, and I take great pleasure in presenting this work at this session under the chairmanship of Dr. Lwoff. His laboratory has been a major institution of higher learning for workers from all over the world. While still on the subject of the advantages of biological equipment for the chemist, I would comment that, with the exception of work on bacterial viruses and their host cells, the study of the preparation and properties of host cells in other fields of virology has been sorely neglected. Indeed, it cannot be anticipated that chemical studies on the multiplication of plant and animal viruses can be seriously developed as long as the experimenter is constrained to the use of a biologically heterogeneous assortment of cells made available by the exigencies of embryological development, rather than by the requirements of the investigator. This follows from the fact that the most valuable technique until now in chemical virology has involved the analysis of events in a population of infected cells in which the proportion of uninfected cells is very low. By means of this technique it was possible to show that cells infected with T2 continued to respire and synthesize nucleic acid and protein, but no longer appeared to synthesize polymeric substances characteristic of structural constituents of the bacteria (Cohen, 1947). The polymers produced after infection could largely be isolated in virus under certain favorable conditions. This apparent redirection of the products of synthesis was particularly dramatic in the case of phosphorus 1 The work described in the paper was conducted u n d ~ a grant from the CommonwealthFund. utilization and nucleic acid synthesis. In growing bacteria about 80 per cent of the cell P is nucleic acid P and of this ribose nucleic acid (RNA) P is three to five times that of deoxyribose nucleic acid (DNA) P. After infection the P which would have normally gone into RNA is now routed into DNA while the RNA does not increase in amount. 2 It was proposed (Cohen, 1948a) that there was a common precursor to both the ribose phosphate of RNA and the deoxyribose phosphate of DNA, and that an inhibition of the formation of this ribose phosphate could account for the availability of more P for deoxyribose phosphate and DNA. A key portion of the hypothesis stated that the inability to make RNA, an important component of metabolically active structures, might limit the synthesis of more of these structures. We see no reason at present to change this particular formulation. Therefore, we undertook to determine the path of formation of ribose phosphate in E. cell, and to see the effect of virus infection upon this metabolic system. Although these working hypotheses will be seen to be incomplete and perhaps somewhat misleading, they permitted a type of experimentation which eventually tested and even supported some of these ideas. PATHS OF PENTOSE AND DEOXYPENTOSE FORMATION In the past six years, a schema of carbohydrate metabolism in E. coli has been elaborated in my laboratory~ as presented in Figure 1. It was shown initially by the use of yeast enzymes that pentose phosphates including ribose-5-phosphate were produced following the oxidative decarboxylation of 2 Hershey (1953b) has recently pointed to the existence of an acid-soluble, alkali-soluble component, distinguish. able from the bulk of the RNA, which has a rapid turnover in the infected cells as measured with pa2. Although he calls this metabolically active fraction RNA he has not demonstrated the existence of p82 in all or indeed any of the nucleotides of RNA, a procedure which recent experi. ments (Davidson and Smellie, 1952) would suggest to be important. It is especially critical to know if cytosine ribose nucleotides are synthesizedin virus infected cells. It is conceivable that this interesting material may prove to be an intermediate in the synthesis of DNA, a role excluded for the bulk of the RNA (Cohen, 1947; Manson, 1953). 8My collaborators in these studies of carbohydrate metabolism have included Dr. D. B. McNair Scott, and Miss Mary Lanning, Mrs. Ruth Raft, and Mrs. Lorraine Roth. [221] Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press 222 SEYMOUR S. COHEN FIGURE 1. glucose ATP ; glucose-6-phosphate. "fructose 1, 6-diphosphate 'I[ TPN 6-phospho~luconolactone ATP gluconate ~ ~ H20 6-phosphogluconate TPN ATP ribulose ~ ribulose.5.phosphate ,triose phosphate D-arabinose D-ribose ~ CH3CHO ATP ribose-5-phosphate deoxyribose-5-phosphate rihose-l-phosphate deoxyribose.l-phosphate +tt8P04 [ base rihose nueleoside 1 +H~P04 ] [ base deoxyrihose nucleoside 1 RNA DNA F~cuRr- l. Paths of ribose and deoxyribose phosphateformation in E. coli. 6.phosphogluconate (Cohen and Scott, 1950). The existence of ribose among the reaction products was demonstrated chromatographically (Scott and Cohen, 1951), and by means of a manometric assay (Cohen and Raft, 1951) using specifically adapted bacteria, which in this instance were ribose-utilizing mutants of strain B. Another pentose phosphate was found among the reaction products in largest amount, and this substance was subsequently identified as ribulose-5-phosphate (Horecker, Smyrniotis and Seegmiller, 1951). Shortly after these findings, the enzymatic formation of deoxyribose-5-phosphate from glyceraldehyde-3-phosphate and acetaldehyde was demonstrated (Racker, 1951). The reactions linking ribulose-5-phosphate and triose phosphate form an area of active study in several laboratories at the present time. These reactions also have been demonstrated in E. coll and this organism also possesses the enzymes to convert glucose to glucose-6-phosphate and the latter to phosphogluconate or to triose phosphate via the Embden-Meyerhof scheme. Thus E. coli has at least two alternative paths of glucose-6-phosphate utilization. At the present time, there is no reason to believe that other major pathways of gtucose-6phosphate utilization exist. The organism does not form the ketohexonie acids and under most conditions of growth the enzymes for the anaerobic cleavage of phosphogluconate (Entner and Doudoroff, 1952) are present in very slight amounts (Scott, unpub.). These reactions had been studied in cell-free extracts, but in order to approach these systems in the intact bacteria, it is necessary to supply unphosphorylated substrates. The ability of strain B or appropriate mutants to metabolize various carbohydrates is largely adaptive, and in order to demonstrate the insertion of substrates into the pathways in intact cells, it becomes necessary to clarify the steps involved. We were able to demonstrate the existence of the reactions: a. gluconate + ATP ~ 6-phosphogluconate q- ADP, in the presence of an adaptive gluconokinase (Cohen, 1951a). b. D-ribose q- ATP > D-ribose phosphate qADP, in the presence of an adaptive ribokinase (Cohen, Scott and Lanning, 1951). c. D-arabinose ~ D-ribulose, in the presence of Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press METABOLISM OF VIRUS INFECTED BACTERIA adaptive pentose isomerase (Cohen, 1953). d. D-ribulose -4- ATP .~ D.-ribulose phosphate + ADP, in the presence of ribulokinase (Lanning and Cohen, unpub.). Thus, as shown in Figure 1, these substrates may be inserted at various levels of the phosphogluconate pathway and would appear to traverse this path alone, since, under conditions of growth and infection, the conversion of glucose-6-phosphate to 6-phosphogluconate appears essentially irreversible in contrast to most of the other steps presented. This was demonstrated by means of gluconate-l-C 14 in experiments in which the isotope content of the CO2 liberated was found to be 85 to 95 per cent of the isotope in the gluconate fed. Thus, the C1 was converted Io CO2 in the conversion of phosphogluconate to ribulose-5-phosphate, whereas formation of glucose-6-phosphate and degradation to triose via the Embden-Meyerhof scheme would have resulted in the conservation of isotope in the methyl carbon of pyruvate (Cohen, 1951b). Test of the role of the phosphogluconate pathway by the same technique during growth of E. coli on glucose-l-C 14 revealed that only a part of the glucose, estimated to be in the range of 16 to 38 per cent, was handled in this way (Cohen, 1951b), whereas the remainder was presumably degraded via the Embden-Meyerhof scheme. Under conditions of virus infection when RNA synthesis was inhibited and replaced by stimulated DNA synthesis, the utilization of the phosphogluconate path was reduced markedly, although not eliminated, thereby suggesting that the ribose of RNA is synthesized predominantly via this path. Although the rate of glucose utilization or of respiration was not significantly affected by infection, the isotope experiment revealed that the relative balance of the two paths was affected, and that the common precursor, glucose-6-phosphate, plays a pivotal role in this phenomenon. The pattern revealed by the isotope experiments has been checked in two ways. First, it has been shown that E. coli contains sufficient of the dehydrogenases of the phosphogluconate pathway to permit the utilization of 40 per cent of the glucose by this route (Scott and Cohen, 1953a and b). Second, analysis of the ribose of RNA of bacteria grown on glucose-l-C14 reveals that most of the ribose came from a pathway such as the phosphogluconate pathway in which the Ca of glucose was lost. However, the isotope content of the ribose was sufficiently high as to indicate a partial formation from fragments derived from the EmbdenMeyerhof scheme (Lanning and Cohen, 1952). THE INDIRECT EFFECT OF INFECTION ON CARBOHYDRATEMETABOLISM How does virus change the balance of the pathway? Are we merely shifting equilibria by removing deoxyribose phosphate in virus DNA? If so, RNA synthesis should proceed under conditions of 223 infection in which DNA synthesis is inhibited, as in infection with ghosts or irradiated phage. However, RNA synthesis is inhibited under all conditions of infection, and although this hypothesis of the linking of equilibrated reactions can explain the greater availability of P for increased DNA synthesis, it does not explain the apparently complete inhibition of RNA synthesis. The possible inhibition of the enzymes of the phosphogluconate pathway has been tested in the iollowing way: Strain B or the appropriate mutants, adapted to substrates which must be handled via the phosphogluconate pathway, were infected with T2r+ or T4r in the presence of these substrates as the sole carbon source. These substrates included gluconate, ribose, D-arabinose, and the purine ribosides. The production of DNA and virus on these substrates indicated the operation of all the known steps of carbohydrate metabolism leading to ribose nucleoside formation (Cohen and Roth, 1953). Isotope experiments with glucose-l-C 14 under conditions of infection had indicated that glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were still operative (Cohen, 1951b). We have concluded, therefore, that the lack of RNA synthesis is not due to an effect of virus on known steps of ribose phosphate formation and utilization. The prevention of this synthesis could affect carbohydrate metabolism in the manner described, but this would appear to be an indirect effect. It is concluded that the control of RNA synthesis is effected at other levels of metabolism. THE BASE COMPOSITIONOF THE T-EVEN PHAGES AND HYDROXYMETI-IYLCYTOSINE A clue as to the critical level of metabolic control was discovered in the course of quite another approach to the phages. It appeared of interest from several points of view to compare the constitution of the DNA of the three mutant pairs of related phages with which we have worked for several years, namely, T2r + and T2r, T4r+ and T4r, and T6r+ and T6r. a. The rigorous chemical description of the phages had been neglected. It could be anticipated that satisfactory interpretation of physiological and genetic data would not be possible without these data. b. In view of the hypothesis that variations of the structure of DNA may be responsible for genetic differences, organisms such as viruses with small numbers of DNA molecules may provide material in which changes of DNA composition may be detectable. A collaboration was developed with Dr. G. R. Wyatt of the Department of Agriculture of Canada to study this problem. Earlier efforts to characterize the bases had failed to account for more than 70 per cent of the bases of the phages or to find cytosine (Marshak, 1951). It is obvious that in- Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press SEYMOUR S. COHEN 224 complete recovery of the bases does not permit the report of the absence of a constituent, but merely suggested the inadequacy of a method, in this case the use of concentrated perchloric acid in hydrolysis. However, it was equatly evident that the phage nucleic acid possessed certain peculiarities warranting more careful study. As a result of the trial of many hydrolytic procedures, the previously missing base came to light. It proved not to be cytosine, but a compound whose spectrophotometric properties and chromatographic properties suggested that it was a derivative of cytosine (Wyatt and Cohen, 1953a). At our suggestion, 5hydroxymethyl cytosine was synthesized by Dr. Charles Miller of Sharp and Dohme, Inc. and the synthetic material was compared with the natural compound isolated in crystalline form from T6r+ phage. The comparison and elementary analyses became available within one week, and the natural compound proved to be 5-hydroxymethyl cytosine (HMC) (Wyatt and Cohen, 1952). Dr. Miller has also kindly made available 5-hydroxymethyl uracil (HMU). HMC is labile in perchloric acid and in other acids under various oxidizing conditions. A method has now been devised in which, in analysis of phage or of phage nucleic acid, molar base recovery approaches 100 per cent of the moles of P. The details of the method are in press (Wyatt and Cohen, 1953b) as are analyses of the T-even phages and some other viruses. In Table 1 are presented some of these analyses on intact phages. The mean ratio of estimated ba,~es/P is improved to 0.99 when the material analyzed is isolated phage DNA. It can be seen that the base compositions of the six phages are substantially identical, within the experimental errors of the analyses. However, the conclusion can not be drawn from these analyses that the nucleic acids of these phages do not differ since position isomerism without change in composition is possible. Furthermore, a real difference of one per cent in the content of a particular base, which is not a significant difference by current analytical methods, would reflect a difference of hundreds of residues. In the course of the analyses, no significant amounts of cytosine were found in these viruses, TABLE1. DNA BASECOMPOSITIONOF PHAGEST2, T4, A N D T6 Mean Moles/100 MolesEstimatedTotal Bases Hydroxymethyl Total Virus Adenine Thymine Guanine Cytosine Bases/P T2r+ T2r T4r+ T4r T6r+ T6r 32.0 32.3 32.3 32.2 32.5 32.3 33.3 33.4 33.1 33.5 33.5 33.4 18.0 17.6 18.3 18.0 17.8 17.7 16.8 16.7 16.3 16.3 16.3 16.6 0.99 0.95 0.96 0.94 0.99 0.88 and it was estimated that the maximal content of cytosine in T6r+, for instance, was 0.2 per cent of its content of HMC. Conversely, analysis of the bacterial host and of its DNA did not detect HMC and the maximal HMC contents of these ~aterials were estimated to be 0.2 and 0.6 per cent respectively of their cytosine content. It should be noted that cytosine is a constituent of both the RNA and DNA of E. coll. Analyses of the DNA of various strains of E. coli have revealed the four bases, adenine, thymine, guanine, and cytosine to be in approximately equimolecular proportions (Gandelman, Zameuhof and Chargaff, 1952). 5-methyl cytosine has not yet been found in any microorganism or virus. Thus a new pyrimidine base has been found in several viruses which has not yet been found in the bacterial host or its nucleic acids. It is conceivable that HMC might be present in trace amounts in E. coli, but the substance has not yet been demonstrated. If HMC is not a product of normal bacterial activity, this would be the first instance of a low molecular building block in a virus different from those of ;is host. In any case, the absence of cytosine in the virus and the shifting to HMC synthesis suggests that the metabolic systems involving these compounds may account for the inhibition of RNA synthesis, and now to extend the problem to its proper scope, for the inhibition of host DNA synthesis, as well. If an infected cell were compelled to synthesize HMC and were nnable to make or utilize cytosine such a cell would be unable to synthesize host nucleic acid, and might synthesize materials linked to HMC such as virus nucleic acid and virus proteins. The case of the lost kingdom and the horseshoe nail is herein postulated in modem dress. If the hypothesis were true it would be expected that virus infections in which host nucleic acids continue to be synthesized, as in lysogenic systems, would not involve the synthesis of HMC. This prediction has been confirmed by Simonovitch and Smith (personal communication) at least with respect to the absence of HMC in temperate phages. What situation prevails with other phages of the T series? Analysis of T7 and T54 has not revealed detectable amounts of HMC in these viruses. Of the odd numbered phages of the T set, nucleic acid metabolism has only been studied in T7 and T3 infected cells (Putnam et al., 1952; Pardee and Williams, 1953). Careful examination of the data presented in these papers reveal significant incorporation of paz into the RNA fraction in the first instance, and pentose increment in the second. The significance of these data has not yet been evaluated. We conclude that the data on the T-odd viruses can not yet be considered an argument against our hypothesis. 4 We are indebted to Dr. Kozloff and Dr. Lark, respectively, for these preparations. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press METABOLISM OF VIRUS INFECTED BACTERIA HMC has not been found in any other DNA so far examined for this substance. These include mammalian DNA and some animal virus preparations, including a polyhedral virus, vaccinia virus, and meningo-pneumonitis virus, which all contain the usual DNA bases, including cytosine. The latter virus is of particular interest since it is a member of the psittacosis group of viruses, in the multiplication of which folic acid has been found to play an important role. ORIGIN OF THE RING STRUCTURE OF HYDROXYMETHYL CYTOSINE Weed and I have published data indicating the transfer of the pyrimidine nucleotides of host DNA to become pyrimidine nucleotides of virus DNA (Weed and Cohen, 1951). In the course of that work, one of the pyrimidine nucleotides of virus DNA was stated to be deoxycytidylic acid. The incorrect identification of this material arose in the following manner: DNA was extracted with hot trichloroacetic acid from various tissues or organisms from which RNA had been previously removed by one of various methods. RNA was not present when the extraction was made from isolated virus. The DNA extract was concentrated with N HC1 and hydrolyzed in the acid solution. The hydrolysate was chromatogrammed on papers using a tertiary butyl alcohol--HC1 mixture (Smith and Markham, 1950), and two bands were obtained containing the pyrimidine nucleotides. Following the hydrolysis of thymus DNA, for instance, the band containing the cytosine nucleotide has little other base. The band containing thymine nucleotide is also fairly homogeneous. Nevertheless, it has been customary to purify the nucleotides further by adsorption and elution with an acid of appropriate strength on an anion exchange resin (Weed and Wilson, 1951). In the experiments with virus small amounts of the nucleotides were available, and were separated on paper only. Although the band in the thymidylic nucleotide region obtained in this way from a virus DNA hydrolysate is substantially free of bases other than thymine, the band in the "cytidylic" region has now been found to contain two components of approximately equal amount separable on the resin. The first, eluted with 0.01 N HC1, contains HMC as the sole base, and has an absorption maximum at 282 to 283 m~. The second, eluted with 0.01 N HC1, contains equimolecular amounts of HMC and thymine and has an absorption maximum of 274 rag. The mixture isolated from paper had a maximum of 278 m~, or that of deoxycytidylic acid. It is evident that it is neces. sarv to separate these components on both paper and resin in experiments in which the pyrimidines of host DNA are labeled by growth of the bacteria in labeled orotic acid. These experiments have now been repeated in this way in collaboration with Dr. Weed of the Army Medical School. Bacteria were grown to 3 X 225 10s per cc in a mineral medium containing glucose and orotic acid-2-C14, generously given us by Dr. D. W. Wilson. The cells were centrifuged and washed. One half was precipitated in two per cent perchloric acid, washed and dried. The remainder was resuspended at the same concentration in the medium free of orotic acid, and infected at a six fold multiplicity of T6r+ in the presence of 50 7 tryptophan per cc. The culture was aerated until lysis and virus was isolated following two cycles of differential centrifugation. The pyrimidine nucleotides of host DNA and virus DNA were isolated as described above and their isotope contents were estimated. The results are presented in Table 2, TABLE 2. T h E PYRIMIDINE NUCLEOTIDES OF THE D N A o r BACTERIA GROWN IN RADIOACTIVE OROTIC ACID AND OF VIRUS PRODUCED IN THE LABELED HOST Base Host Nucleotides Cts./Min./gMol. Thymine Cytosine 123 160 Virus Nucleotides Base Cts./Min./gMol. Thymine HMC 24, 25.4, 26.8 24 and show that the ring structure of a pyrimidine can be converted to the ring of HMC. The extensive dilution of the pyrimidine ring derived from host in virus HMC and thymine is to be expected in view of the extensive net synthesis of DNA in this system. However, whether the DNA base, cytosine, is an. obligate intermediate of HMC is in no way answered by these experiments. When the DNA was: analyzed from virus produced in ceils infected in the presence of radioactive orotic acid, the radioactivities of HMC and thymine were essentially similar suggesting that, despite the extensive dilution of the orotic acid by considerable de nova synthesis of the pyrimidine ring, the two bases might have a common precursor not far removed from the end products of the reaction sequences. ON THE ORIGIN OF THE HYDROXYMETHYL GROUP The fl-carhou of serine is known to he a source of one carbon fragments such as the methyl group of thymine and the C2 and Cs of purines. Formate has also been found to be a source of these atoms in the nucleic acid bases in many organisms. However, the oxidation level at which these atoms are added to the molecule has not yet been determined. Serine is considered to be cleaved to glycin and a moiety containing the fl-carbon at the oxidation level of formaldehyde or that of the hydroxymethyl group. We, therefore, undertook to see if the fl-carbon of serine could become the carbon atom of the hydroxymethyl group of HMC. These experiments 5 were facilitated by the fact that the C2 of the pyrimidine ring, unlike that of 5 We are indebted to Dr. J. Stekol for a sample of DL~ serine-3-C14. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press SEYMOUR S. COHEN 226 TABLE 3. THE LABELtNC OF THE DNA BASES OF BACTERIA GROWN IN SERINE-3-C14, AND OF VIRUS PRODUCED IN THESE BACTERIA IN THE PRESENCE AND ABSENCE OF LABELED SERINE5 Bacteria Substance Adenine Guanine Thymine Cytosine Cts./Min.//zMol. 2730 2840 1450 157 T6r+ Grown in Absence of Serine-3-C14 T6r+ Grown in Presence of Serine-3-C14 Substance Cts./Min./gMol. Substance Adenine Guanine Thymine HMC Adenine Guanine Thymine HMC the purine ring, is not derived from this type of one-carbon precursor. Therefore, it could be anticipated that growth of E. coli in the presence of serine-3-C14 would give rise to bases in the DNA in which the purines, thymine, and cytosine should contain two, one, and 0 labeled atoms respectively. The isolation of the bases was carried out by hydrolysis of the DNA fractions of the bacteria in formic acid and chromatography on paper in isopropanol-HCl (Wyatt, 1951). To free the bases from possibly contaminating amino acids, the individual bands were eluted, adsorbed on an anion exchange resin and eluted, and rechromatographed on paper using butanol-HzO. Typical analyses are presented in Table 3, as are analyses of the bases of T6r+ reproduced in the same bacteria in the presence or absence of radioactive serine. Three experiments have yielded consistent results. It can be seen that the ratios of radioactivity in purines, thymine, and cytosine of bacterial DNA are 2, 1, and 0.1. In virus produced in labeled bacteria in the absence of labeled serine, the dilution of thymine was somewhat greater than that accounted for by net DNA synthesis, whereas that of the others was not far from the expected dilution to about one-eighth the initial activity. The low activity of the tIMC implies that the methyl group of thymine is not converted in large amounts, if at all, to hCH2OH, and that the --CHzOH in this experiment was derived from essentially unlabeled precursors, rather than serine or hydroxymethyl donors stored in the host. This also implies that the cytosine of host DNA can supply the pyrimidine ring for HMC. Virus grown in the presence of serine-3-C14 contained highly active HMC, which in one experiment not presented in the table slightly exceeded the activity of thymine. Since the ring structure of cytosine formed in the presence of serine contains small amounts of C14, and HMC, whose ring is derived from cytosine or cytosine precursor, contains large amounts of C14, it is concluded that the /3carbon of serine is a potentially important precursor of the hydroxymethyl group. We have not studied other possible precursors of this carbon atom. 310 502 55 23 Cts./Min.//zMol. 1920 2090 952 700 THE ATTEMPTED ISOLATIONOF THE HMC DEOXYRIBOSIDE FOLLOWINGENZYMATIC DEGRADATION In Figure 2 are presented the structures of the pyrimidines with which we are concerned as well as some of their possible metabolic relationships. Three of these, cytosine, uracil, and thymine or 5methyl uracil have been found in the host. HMC and thymine are known in the virus. 5-methyl cytosine and 5-hydroxymethyl uracil have not been found in either. Very little is known of the metabolism of these compounds, and one must be prepared to cope with the possibility that their major metabolic routes involve the deoxyribosides. Methods for the isolation of deoxyribosides generally begin with the enzymatic degradation of DNA by deoxyribonuclease followed by dephosphorylation with a suitable phosphatase. We have developed this procedure using pancreatic deoxyribonuclease followed by purified intestinal alkaline phosphatase for small amounts of thymus DNA, cuhninating in the separation of the deoxyribosides by paper chromatography in butanol-NH4OH. Following elution of the bands with H.~O, we have been able to obtain solutions of deoxycytidine and thymidine which possess satisfactory spectrophotometric characteristics and are free of contamination by the other pyrimidine deoxyriboside, as determined with thymineless or cytosine-uracilless mutants of E. coll. Under short treatments with our sample of alkaline phosphatase, it is possible to obtain deoxyguanosine and deoxyadenosine. With N~C-NH~ I I N=C-NH z CH, O o<1 IICH RN--CN CYTOSINE N=C-OH III URACIL .o:cI C-C.,OH It N~C-NH 2 1 I ? ,o:~ I-CH~ RN--CH RN--C 5-HYDROXYMETHYL CYTOSINE 5-METHYL CYTOSINE II c.,o O,C C H RN-CH I I N=C-OH I I -O=C C-CH2OH Ill RN-CH ~' 5"HYDROXYMETHYL URACIL N=C-OH II I ! RN--C :O~C C-CH~, THYMINE FmURE2. Structures and possible metabolic relations of pyrimidines. R -" a hydrogenatom or a glycosylmoiety. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press METABOLISM OF VIRUS INFECTED BACTERIA prolonged treatment, an impurity of adenosine deaminase produces hypoxanthine deoxyriboside which is less readily separable from the deoxy. guanosine. Crystalline thymidine was also prepared by Dr. D. B. McNair Scott (Brady, 1942 and personal communication). Polymeric virus DNA, prepared by the urea method (Cohen, 1947), was subjected to the same procedure. Whereas the P of thymus DNA was liberated to the extent of 92 to 98 per cent, various preparations of ~:irus DNA liberated only 65 to 82 per cent of their P after prolonged treatment. Chromatography of the digest revealed large amounts of thymidine which was further purified and crystallized by Dr. Scott. It had the same melting point and spectrophotometric properties as the thymidine of thymus DNA. No band containing the HMC nucleoside was detectable. A band containing guanine and hypoxanthine in the position characteristic of their nucleosides was found. This band has also been found on occasion to contain very small amounts of HMC and thymine. Hydrolysis of the purine nucleosides from this band in 0.01 N HC1 and paper chromatography in a butanol-ethanol-H20 mixture (4:1:5), revealed a deoxypentose having the Rr value of deoxvpentose derived from deoxyribosides of thymus DNA. The bulk of the HMC was found in the nucleotide fraction non-mobile in butanol-NH4OH. This material was eluted in water and showed a broad spectrum whose maximum was shifted from 260 mg to 265 mg. Hydrolysis of the enzymaticaUy resistant non-mobile nucleotides derived from three preparations of T6r+-DNA had the compositions presented in Table 4. In each case, the concentraTABLE 4. BASE COMPOSITION OF ENZYME-REsISTANT NON-MOBILE NUCLEOTIOES %P UnhydroPreparation lyzed Adenine Thymine Intact T6r+ DNA A B C -25 33 37 32.5 19.1 26.5 28.0 Guanine HMC 17.8 16.6 12.1 11.9 16.3 47.1 35.5 34.6 33.5 17.3 24.6 25.5 tion of HMC in an enzyme-resistant fraction may be observed. It is tempting to suppose that the survival of injected virus DNA into a bacterium containing enzymes capable of degrading host DNA to the nucleoside level is associated with the existence of HMC in enzyme-resistant units. One might therefore, imagine that survivability, specificity, and the parasitic function may all be conferred on ,. virus DNA in the replacement of cytosine by HMC. ISOLATION AND PROPERTIES OF DEOXYRIBOSIDE HMC Several procedures involving acid hydrolysis of virus DNA were then employed. Hydrolysis in N 227 HC1 was followed by separation of the nucleotides on paper as described previously. The HMC nucleotides were then hydrolyzed with phosphatase and the HMC deoxyriboside was separated in butanolNH4OH. The product so obtained is spectrophotometrically satisfactory, but is low in over-all yield. Direct chromatography in butanol-NH~OH of the product of acid hydrolysis and treatment by phosphatase yieIds a material slightly contaminated by guanine. The procedure of choice involved hydrolysis of 20 mg. DNA per cc. 1.5 N HC104 at 100 ~ for one hour. Neutralization to pH 7.5 with KOH and centrifugation following chilling removed most of the KC104 and guanine. The pH was adjusted to 8.8 with NH4OH and the Mg++ concentration was adjusted to 0.25 per cent. Alkaline phosphatase was added (30 units per cc.) and the mixture incubated for three hours at 37 ~ The hydrolysate was centrifuged to remove MgNH4P04, concentrated, and chromatographed on paper in hutanol-HzO. Three bands were discernible. The first, at Rt <0.1 contained about 10 per cent of the HMC of the preparation as the free base. The second, at R~ .13 to .17 when applied in small amounts on paper, and somewhat more diffuse in concentrated solutions, contained the HMC deoxyriboside and a slight contaminant of guanine ( < 5% of the preparation). The third, at R~ .45 to .55 contained both adenine and thymidine. The band containing the HMC deoxyriboside has been further purified by paper chromatography at about 25 ~ in ethanol-acetate pH 3.5 (Cohen and Scott, 1950), in which both guanine (Rt .36), and free HMC (Rt .47) may be separated from the deoxyriboside (Rf .57). The HMC deoxyriboside has its maximum absorption at 282 m/~ in 0.1 N HC1 in contrast to that of HMC at 278 to 279 rag. The compound isolated in this way is devoid of phosphorus. The maximum of the deoxyriboside is the same at pH 7 and 13 in contrast to free HMC. Owing to the small amount of deoxyriboside available it has been used until now for metabolic experiments, and has not been consumed in various elementary analyses. As a consequence of this, the molecular extinction coefficient for the deoxyriboside has not been determined, and the values obtained by Fox and Shugar (1952) for deoxycytidine have been adopted as a close approximation. As an additional difficulty, it may be noted that following storage of frozen solutions for a month, 30 to 40 per cent liberation of free HMC from the deoxyriboside was revealed. In the initial chromatography in butanol-H20 in three of five isolations the HMC deoxyriboside band has been split. When the contents of each of the split bands were rechromatographed in dilute solution in butanol-H20 and ethanol-acetate, they were then apparently identical. They possessed the same spectrophotometric properties at pH 1, 7, Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press SEYMOUR S. COHEN 228 pH .80 .70 and 13, and in tests to be discussed below appeared essentially identical with respect to enzymatic deamination and microbiological assay. The nature of this phenomennon is not clear at present. In Figures 3A and 3B are presented spectra of the deoxyribosides of cytosine and HMC as prepared by these methods. It is a very difficult matter to reduce end absorption below 245 m/~, when ~eparation procedures are confined to paper chromatography. Nevertheless, it would appear that an effect of the hydroxymethyl group becomes spectrophotometrically visible at pH 13. 07 x 13 THE UTILIZATION OF PYRIMIDINE DERIVATIVESe >- tz b.I 0 240 250 2s 2"}0 28o 26o 36o m~ 45- pl-I 9 I 07 x 13 .35- >- z ILl t:3 .10- '~ .05 o 36o m~ Fzcu•E 3. (a) Spectra of deoxycytidine isolated from thymus nucleic acid, as described in text, (b) Spectra of deoxyriboside of HMC isolated from T6r-I" nucleic acid, as described in text. It appeared of interest to know whether 5-hydroxymethyl derivatives of the pyrimidines were used by various microorganisms. Pyrimidine-requiring mutants of E. coli are rare, and those available at the present time possess numerous complicating quirks, as we shall see. The organisms, besides strain B, on which we have concentrated include a uracil or cytosine-requiring mutant of strain W, We- obtained from Dr. B. Davis, and a thyminerequiring strain of strain 15, 15T- obtained from Dr. J. Gots. These requirements may be replaced by the pyrimidine nucleosides. We- was grown overnight in a mineral medium containing 0.7 mg glucose, and 4 -/cytosine per ml. Growth was limited to ca 7 )< 10 s cells per cc. Aliquots were then inoculated to the medium containing glucose and test pyrimidine or deoxyribose at 5 • 107 to 10s cells per cc and growth was followed turbidimetrically over a six-hour interval. The compounds permitting growth and multiplication are presented in Table 5. The mass doubling time was about 20 per cent faster with uracil than with cytosine. Similar studies were done with strain 15,~-. In overnight growth about 0.4 ,/thymine was limiting at about 8 X 10s cells per cc in mineral medium containing one mg glucose per cc. The tests of the ability of a compound to support growth were run on cells grown in the glucose-thymine medium which were inoculated at about 10 s per cc of glucose-mineral medium. In the growth of strain 15Ton thymine, uracil or uracil derivatives accumulated and were excreted in the medium, as determined by support of the growth of We-. HMC, HMU, or the deoxyribosides of these compounds at 5 ~, base per ml did not significantly affect the growth rate of B nor DNA synthesis of B infected with T2 when the bacterial concentration was at 5 X 107 to 3 X l0 s per cc. Dr. Helen Skeggs of Sharp and Dohme, Inc. has tested preparations of the HMC deoxyriboside in microbiological assay with Lactobacillus bi~dus,. Thermobacterium acidophilus R 26, and Lactobacib lus leishrnaniL Since these organisms react nonspecifically to the deoxyribosides, the test is presum6 Miss Hazel Barner was a most active collaborator in this phase of the work. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press METABOLISM OF VIRUS INFECTED B~4CTERIA o 50' + HMC x 0 rr 3 0 HMC DEOXYRIBOStDE DEOXYRiBOStDE ~,, Ld 13- o o o < 20 Z o ,-, I0 o g 3o 6'o -g io MINUTES FIGURE4. The lack of effect of HMC deoxyriboside on the DNA content of T2r+-infected E. coli, W strain. ably a measure of the availability of deoxyribose phosphate rather than the utilizability of the base. All three organisms grow equally well on HMC deoxyriboside, cytosine deoxyriboside, and thymidine. However, the deoxyriboside of HMU was unable to support the growth of L. bifidus, the only organism of the three tested on this compound. PHAGE INFECTION OF STRAIN W It has been. observed that W strains of E. coli will adsorb T2 and T6 , but do not support their multiplication. The possibility arose that W was an HMC-requiring strain. W was grown to 3 X 10s cells per cc in glucose-mineral medium, and infected with T2r+ at a virus to cell ratio of 4.6 in the absence and presence of HMC and HMC deoxyriboside. No significant differences were noted in these cultures. As can be seen in Figure 4, the DNA content of the culture rapidly fell about one-half, a frequently observed phenomenon with this organism after infection. No evidence has yet been obtained to account for the block to the multiplication of T-even phage in this strain. It may be noted that W has been reported effective in the hydroxymethylation of ketovaline to ketopantoic acid (Maas and Vogel, 1953). PHAGE INFECTION OF SULFANILAMIDE-INHIBITED STRAIN B It had been reported that sulfanilamide-inhibited B could support the growth of T1, T3, and T7 in the presence of the metabolites, methionine, xanthine, valine, and thymine, but not the growth of T2, T4, and T6 (Rutten, Winkler and deHaan, 1950). It seemed possible that the action of this inhibitor was to prevent the formation of HMC. In order to duplicate the resultsof Rutten et al.,it was 229 found necessary to grow B in the sulfanilamidemetabolite media and to infect in this media. It is important to note that the metabolites provided do not restore growth rate to more than 30 per cent of that in the absence of sulfanilamide. Further, if cells are grown in this fashion, and infected in glucose-mineral medium without sulfanilamide, T4r for instance, may multiply, albeit with a longer lag in the inception of net DNA synthesis and a lower rate for this function. When these cells are infected in the complete medium containing sulfanilamide and metabolites, T4r does not multiply, and the DNA content of the cells falls to about 60 per cent of their original value. The addition of HMC, HMU, or HMC deoxyriboside at 3 T base per ce still does not permit virus multiplication, as can be seen in Figure 5. THE ASSIMILATION OF PYRIMIDINE DERIVATIVES When cytosine deoxyriboside or cytosine is incubated with strain B during growth or during infection with T2, a marked shift and reduction in the spectra of these substances may be observed. The spectra were determined in two per cent perchloric acid extracts of the cultures. The shifts may be seen after correction for the spectral changes in control cultures prepared in the absence of added pyrimidine in the medium. The amounts of supplement added to the medium are limited by the optical densities of the acid extracts from unsupplemented cultures, and by the amounts of substance whose assimilation can be detected under the physi- 70t 6o 20. ,o. o _: 9 NO ,sMf: L: INHIBITOR(SM) o ox , os,o "--.. 2b 4:o 6b 8'0 16o MINUTES FIGURE5. The lack of effect of hydroxymethylated derivatives on T4r-infected cultures of B, in media containing SM (sulfanilamide and metabolites--see text). Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press 230 SEYMOUR S. COHEN ological conditions of our experiments. Typical transformations of these compounds by infected cells are presented in Figure 6. It appears that the compounds are both deaminated and assimilated in these systems. Under comparable conditions, uracil is assimilated, but significant uptake of HMC, HMU, 5-methyl cytosine, and thymine cannot be detected. Although the latter is surprising, it appears that the amounts incorporated into DNA under the conditions used, are at about the limits of detection by this procedure. In any case, neither HMC nor 5-methyl cytosine are deaminated by infected or growing B. The deoxyriboside of HMC does appear to be assimilated and deaminated with the accumulation of a compound with the absorption of the HMU or 4 HMC .3 .2 .1 i Z hi t3 :3- HMC -0 DEOXYRIBOSIDE .2 CYTOSINE 9 Oo, 0 30' x 60' o .3 230 2 .0 250 260 2- 0 .2 FIGURE 7. The fate of HMC and the deoxyriboside of HMC in cultures of B infected by T2r+. B = 3 X 108 per cc; T r + = 1.5 X 109 per cc. .1" :t- metrically as described by Wang, Sable, and Lampen (1950). A typical result is presented in Figure 8. It was found that HMC and 5-methyl cytosine DEOXYCYTIDI NE z bJ O .2 .1' ~ 290 3oo m~ o~ _ .9. ~ 81 / \OEAM,NAO CYTOSINE 230 240 2.% 260 2?0 28o L:xjo 300 m~. F[CURE 6. The deamination and assimilation of cytosine and deoxycytidine by B infected by T2r+. B = 3 X 10s per cc; T r + _-- 1.5 X 109 per cc. /J its deoxyriboside. These curves are presented in Figur~ 7. I >.- I.- "o. _.d(~YTOS I N E STUDIES ON THE ENZYMATIC DEAMINATION OF H M C AND THE HMC DEOXYRIBOSIDE Cytoxine deaminase has been reported in yeast, deoxycytidine deaminase in E. coli strain W. Since We- grows better on uracil than cytosine, it appeared likely that the utilization of cytosine was effected throuzh a deamination to uracil. We- was grown in media containing cytosine and cell-free extracts were obtained by the alumina procedure from organisms harvested during exponential growth. The extracts proved to have both deaminases, of which the cytosine deaminase was quite labile. In tests of the latter enzvme 5 g moles of base in 0.8 cc of .05 M tris pH 7.0 were incubated with 0.2 cc of extract for 90 minutes at 37 ~ and the reaction product was examined spectrophoto- W r~ 9 I \ FIGURE 8. Speetrophotometrie analysis of the deamination of cytosine by deaminase of extracts of We-. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press METABOLISM OF VIRUS INFECTED BACTERIA TABLE 5. COMPOUNDSPERMITTINGGROWTHOF We" AND 15,~Compound Strain Wr Uracil .................................................. Dihydrouracil .................................... Orotic acid ...................................... Uracil deoxyriboside .................. 5-hydroxymethyl uracil (HMU) .... HMU deoxyriboside ......................... Cytosine ............................................ Cytosine deoxyriboside ................... HMC ................................................ HMC deoxyriboside ...................... Thymine ......................................... Thymidine ....................................... 5-methyl cytosine .............................. Strain 15~- + --+ --+ + ------ -4"4- were inert to the cytosine deaminase of E. coli. It had been reported (Kream and Chargaff) that the cytosine deaminase of baker's yeast will deaminate both cytosine and 5-methyl cytosine. This enzyme was, therefore, prepared in cell-free extract and tested as described above. In Figure 9, it can be seen that cytosine had been converted to uracil, 5methyl cytosine to thymine, whereas the spectrum of HMC did not reveal appreciable deamination. The deoxycytidine deaminase of E. colt is normally a very active enzyme in this organism and is about two and a half times more active on this substrate than on cytidine. The enzyme has a broad 91 230 240 250 260 270 280 290 300 m~t FIGURE 9. Spectrophotometrie analysis of the products of incubation of cytosine, 5-methyl cytosine, and HMC with the cytosine deaminase of baker's yeast. 231 pH range for optimal activity. It is convenient to follow the course of a deamination by following the decrease in density at 282 m/z in tris buffer at pH 7.0. However, the addition of a cell free extract of E. coli, rich in nucleic acid and protein, to limited amounts of the deoxyribosides of cytidine and HMC obscures such a measurement. Furthermore, the extract increases its density in the absence of substrate, perhaps as a function of nuclease activity on the nucleic acids of the extract. Also it has been shown (Wang, Sable and Lampen, 1950) that the deamination product may be cleaved to the free pyrimidine. It is, therefore, desirable to purify the nucleoside deaminase. The following procedure permitted the isolation of an active stable preparation of deoxycytidine deaminase low in absorption at 282 mg and virtually free of a hydrolytic activity under the conditions of our measurement. A wet pellet of cells harvested during exponential growth was ground in the cold with alumina (Cohen, 1951a) and extracted with a 10 fold volume of 0.05 M tris buffer at pH 7. The extract was sedimented at 5000 RPM for 30 minutes and 40,000 RPM for two hours. To the supernatant fluid was added one tenth volume of 0.25 M MnC12, and after 30 minutes Mn nu. cleates were removed by centrifugation. The supernatant fluid was made 89 saturated with (NH4)2SO4 at pH 7.6 and the precipitate was discarded. The precipitate formed between 50 and 75 per cent saturation, (NH4)zS04 at pH 7.6 was separated by centrifugation, and was dissolved in one seventh the original volume. It possessed one tenth of the original nucleic acid content and contained about 85 per cent of the activity of the extract. The enzyme preparation could be stored in the frozen state for months without apparent loss of activity. Complete deamination of 0.1 micromole of deoxycytidine per cc 0.05 M tris pH 7.0 may be effected by 0.01 cc of the deaminase in ten minutes. After three hours, the reaction product still behaves as deoxyuridine, since the position of the maximum at pH 7 and 12 are essentially identical. The deamination of the deoxyriboside of HMC by the same amount of enzyme proceeded at a rate only two to four per cent of the rate on deoxycytidine. This was measured spectrophotometrically, and corrected for differences in density changes per mole at 282 mg, assuming ~ma~ X 10 -4 - - 0.9 at pH 7.0 for both substances. The coefficients determined from the spectra were then ~2s2 X 10 -4 - 0.60 for deoxycytidine, and c2s2 X 10 -4 = 0.72 for the deoxyriboside of HMC. It was found that the deamination was complete in four hours, and the reaction products behaved as the deoxyribo. side of HMU, as presented in Figure 10. If these materials are deaminated at five- to tenfold concentrations with proportionately larger Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press 232 SEYMOUR S. COHEN DF'AMINATION PP,ODUCT \~.,. o,/ / "~.'~DEOXYRIBOSIDE 42 >. 3 Z2 u.l 23,0 240 250 260 2~/0 28,0 290 360 m.u, FIGURE 10. Spectrophotometrlc analysis of the deamination product of the deoxyriboside of HMC, effected by a purified deoxycytidine deaminase of E. coll. amounts of enzyme for three-hour intervals at 37 ~ they may then be readily separated by paper chromatography with butanol-HzO. The Rt values of the nucleosides in this solvent are: deoxycytidine --0.23, deoxyuridine--0.36, HMC deoxyriboside --0.14, and HMU deoxyriboside --0.24. Starting materials and end products showed only a single spot in these experiments. Thus the enzymatic technique permits the preparation of deoxyuridine and the deoxyriboside of HMU, products whose formation was indicated during experiments on the assimilation of the aminated pyrimidine deoxyribosides. POSSIBLE SIGNIFICANCEOF THE METABOLIC DATA Although the isotope experiments suggest the conversion of host cytosine to virus HMC, the metabolic data presented suggest that a. the conversion is effected as the deoxyribosides, whereas HMC and HMU are inert, and b. two routes are theoretically available for the conversion, either CH20 1. deoxycytidine ) deoxyriboside of HMC, or -NH3 CH~O 2. deoxycytidine > deoxyuridine > +NH3 deoxyriboside of HMU ~ deoxyriboside of HMC. It is not considered likely that isotope experiments alone will clarify this situation, and the study of the mechanism of hydroxymethylation in cellfree extracts would appear to be required. Although the activity of the deoxycytidine deaminase of B in intact cells suggests that deoxyuridine is readily formed from deoxycytidine, and may, therefore, be an active intermediary in the formation of HMC, it is not at all clear that deoxyuridine is the actual substrate for the hydroxyrnethylation. If a competition for amination of the uracil and HMU nucleosides (route 2) were established in a system containing deoxycytidine deaminase, it can he seen that, other factors being equal, the deoxyriboside of HMC would tend to accumulate, owing to the resistance of this compound to the deaminase. Indeed, if this compound were then organized into the enzyme resistant nucleotides characteristic of phage DNA, it is evident that all the known relations of HMC facilitate the winning out of phage products, without requiring any additional inhibitory action on the cytosine metabolism of the host. In addition, it will be recalled that the deoxyribosides of hydroxymethyl derivatives do not fill uracil or cytosine requirements in We-. The hydroxyrnethylation would seem, therefore, to be essentially irreversible and this would magnify the selection for hydroxymethyl compounds in the postulated competitive relations. However, since the irreversibility of the hydroxymethylation appears so complete, how are we to explain the winning out of cytosine and uracil derivatives in normal metabolism? It is possible that the deoxyriboside of HMU is not a normal metabolic intermediate, even though on paper it has the additional advantage of also providing a possible precursor for thymidine as well. In this connection, it should be noted that the task of removing the oxygen from the hydroxymethyl group at this type of carbon atom has no known biochemical analogies. However, a deoxygenation must occur somewhere, although it is known that thyrnidine may be generated by transglycosidation from thymine and deoxyuridine. The conversion of hydroxymethyl to methvl may occur at some other metabolic system and an intact methyl group may be placed on uracil or deoxyuridine. The problem of the formation of thymidine is thus seen to be tied up with the problem of a possible physiological role for the deoxyriboside of HMU. If the formation of a hydroxyrnethylated pyrimidine deoxyriboside is to be explained as an abnormal product, and the relative inertness of HMC and HMU deoxyriboside as contrasted to deoxycytidine and deoxyuridine is taken to mean that these hy. droxymethylated compounds do not serve as inter. rnediary rnetabolites, two additional possibilities may he mentioned: a. The enzymes responsible for the hydroxymethylation of pyrimidine nucleosides are evolutionary vestiges in E. coli. It may be supposed that these enzymes are normally not active in the life of the bacterium, but are activated by infection. b. The virus supplies the enzyme or other com. portent essential for these particular hydroxymethylations. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press METABOLISM OF VIRUS INFECTED BACTERIA ON THE GENERATION OF " N E w " FUNCTIONS BY VIRUS INFECTION Having confronted the possibility that a virus may confer a new metabolic activity upon the infected cell, we then proceeded, quite by chance, to find such an activity in an apparently unrelated system. Dr. Zinder will dicuss "infective heredity" or "transduction" as it was known in the ancient literature (Zinder and Lederberg, 1952). The production of toxin by diphtheria bacillus as a function of infection has recently been reported (Groman, 1953). The systems used by Zinder and Groman are lysogenic, permitting genetic analysis of the infected progeny. This is not possible in the phenomenon I will briefly report. The thymine or thymidine requirements of strain 15T- were described earlier. Strain 15x- will support the multiplication of T2, but not T4 or T6. When grown on broth, it absorbs T2 much better than when grown on glucose, amino acids, and thymine. This is not an adsorption cofactor effect; the organism appears to have nutritional requirements for the formation of receptors. When 15~- is grown in broth, suspended in a glucose-amino acid mixture in which it is multiply infected, and subsequently, transferred to media containing thymine or free of thymine, it was found that the infected ceils produced almost equivalent amounts of DNA whether thymine were present in the medium or not (Figure 11). T2r+ virus was isolated from lysates of these cells infected in the absence of thymine, and it was found that the isolated DNA of this virus preparation was comparable to other preparations of T2r+-DNA, possessing thymine and HMC. In experiments comparable to those reported by Hershey on base s?nthesis by infected cells, it I00. o U u3 80 rr 60" W n o Z 40200 NO THYMINE IO~/ML THYMINE o 2'o 4:o sb MINUTES 16o 6o FI6UR~ 11. The synthesis of DNA by the thymine requiring strain of E. coli 15T-, when infected by T2r+ in the presence or absence of thymine. 15~ = 3.2 X 108 per ce; average multiplicity of infection = 8.3 T2r+ per bacterium. 233 was also found that HMC and thymine were synthesized immediately following infection. Of course, it is not evident whether virus infection stimulated relatively inactive systems to normal activity (Hypothesis a.), or introduced the ability to make thymine (Hypothesis b.), but it seems that the possible validity of one of the hypotheses of this type must be taken more seriously as a result of this finding. This system bears some similarity to the stimulation of thymine synthesis in infection of strain B by T2. However, the apparently absolute initial requirement for thymine by 15a~ places a different aspect on the phenomenon. SUMMARY 1. The site of inhibition of synthesis of host nucleic acid in bacteria infected by T-even phages is being studied. Although the effect is manifested in pathways of carbohydrate metabolism leading to ribose formation, the enzymes of this pathway in infected cells are not directly inhibited by virus. 2. Base analyses for the r and r+ strains of the T-even phages are reported, and are seen to be indistinguishabIe. The existence and identification of a new pyrimidine, 5-hydroxymethyl cytosine (HMC) in these phages are described. The absence of cytosine in this material and of HMC in the DNA of the host and other virus DNA is noted. The possible correlation of the presence of HMC and the inhibition of synthesis of host nucleic acid is discussed. 3. The origin of HMC has been studied by isotope experiments. The t-carbon of serine is an active precursor of the -CH2OH group. 4. An enzyme-resistant nucleotide fraction was found to be particularly rich in HMC. 5. The isolation of HMC deoxyriboside is described. 6. The hydroxymethylated pyrimidines or their derivatives were unable to fill nutritional requirements of certain pyrimidineless strains of E. coli, or to permit synthesis of virus DNA in infected W, and in infected B, inhibited by sulfanilamide. 7. The deamination and utilization of certain pyrimidines and their derivatives by growing or virus infected bacteria are described. 8. The cytosine deaminases of E. coli and yeast were unable to deaminate HMC. 9. The enzymatic deamination of the deoxyriboside of HMC by the deoxycytidine deaminase of E. coli is described. The preparation and some properties of the deoxyriboside of HMU are presented. 10. The possible significance of these metabolic data in terms of the route of formation of HMC deoxyriboside is discussed, as are some hypotheses on the mechanism of control of pyrimidine metabolism. 11. The stimulation of thymine synthesis by T2 infection of the thymine-requiring strain 15r- is described. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press 234 S E Y M O U R S. C O H E N REFERENCES BRADY, T. G.,,1942, Adenosine deaminasc. Biochem. J. 36: 478-484. COHEN, S. S., 1947, The synthesis of bacterial viruses in infected cells. Cold Spr. Harb. Symp. Quant. Biol. 12: 35-49. 1948a., The synthesis of bacterial viruses. I. The synthesis of nucleic acid and protein in Escherichia coli B infected with T2r+ bacteriophage. J. Biol. Chem. 174:281-294. 1948b, The synthesis of bacterial viruses. II. The origin of the phosphorus found in the desoxyribonueleic acids of the T2 and T4 bacteriophages. J. Biol. 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Biochem. J. 46:509-513. WANe, T. P., SABLE, H. Z., and LAMPEN, J. O., 1950, Enzymatic deamination of cytosine nucleosides. J. Biol. Chem. 184:17-28. WEED, L. L., and COHEN, S. S, 1951, The utilization of host pyrimidines in the synthesis of bacteria viruses. J. Biol. Chem. 192:693-700. WEED, L. L., and WrLSON, D. W., 1951, The incorporation of C14-orotic acid into nucleic acid pyrimidines in vitro. J. Biol. Chem. 189:435-44.2. WYATT, G. R., and COHEN, S. S., 1952, A new pyrimidine base from bacteriophage nucleic acids. Nature, Lond. 170 : 1072. 1953a, The bases of the deoxyribonucleic acids of T2, T4, and T6 bacteriophages. Ann. Inst. Pasteur 84: 143-146. 1953b, The nucleic acid bases of some bacterial and animal viruses; the occurrence of 5-hydroxymethyl cytosine. Biochem. J. (in press). ZINDER, N. D., and LEDERBERG,J., 1952, Genetic exchange in Salmonella. J. Bact. 64:679-699. DISCUSSION MANSON: I have done experiments similar to those that Dr. Cohen has reported here today. I tested the ability of glucose-grown E. coli B cells to use intermediates in the oxidative pathway of glucose metabolism for their ability to act as the sole carbon source during multiplication of T4r. Cells were raised in a glucose-salts medium. When the cell concentration had reached 2 X 10 s p e r ml, they were centrifuged a n d washed in cold saline. They were then resuspended in media in wbich the sole c a r b o n source was glucose, gluco- Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press METABOLISM OF VIRUS INFECTED BACTERIA hate, ribose, phosphogluconate, or ribose-5-phosphate. One-step growth curves were carried out at 37 ~ with T4r. Burst sizes of gIucose-grown cells with glucose were about 75, with phosphogluconate about 50, and with ribose-5-phosphate about 30. No measurable bursts were obtained with either gluconate or ribose. With cells raised on gluconate, the burst size with glucose was about 80, with gluconate and phosphogluconate about 50, and with ribose-5-phosphate 25. There was no burst with ribose. This indicated that the intermediates of the oxidative pathway can be used for the synthesis of phage materials. Also, the cells had no difficulty in using phosphorylated intermediates as carbon sources, since in the experiments carried out with 235 glucose grown-cells they could not use the dephosphorylated product. GoTs: We have another example of a biochemically deficient mutant which is capable of phage synthesis in an environment which cannot support the growth of the non-infected bacteria. This is a purine requiring mutant of E. coli B. In a saltsglucose medium containing amino acids, growth of the bacteria will not occur unless a purine is added. Yet, this medium will allow the production of phage when the cells are infected with T1 and T5 coliphages, in a manner indistinguishable from the wild type. T2, T3 and T4 will not be produced unless a purine is added. T6 and T7 have not as yet been examined. Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press STUDIES ON CONTROLLING MECHANISMS IN THE METABOLISM OF VIRUS-INFECTED BACTERIA Seymour S. Cohen Cold Spring Harb Symp Quant Biol 1953 18: 221-235 Access the most recent version at doi:10.1101/SQB.1953.018.01.033 References This article cites 31 articles, 18 of which can be accessed free at: http://symposium.cshlp.org/content/18/221.refs.html Article cited in: http://symposium.cshlp.org/content/18/221#related-urls Email alerting service Receive free email alerts when new articles cite this article sign up in the box at the top right corner of the article or click here To subscribe to Cold Spring Harbor Symposia on Quantitative Biology go to: http://symposium.cshlp.org/subscriptions Copyright © 1953 Cold Spring Harbor Laboratory Press