Dissociation of Cellular Functions in Bacillus Cereus by 5-Fluorouracil

Home , Thymidine

MELVIN REICH1 AND H. GEORGE MANDEL Department of Pharmacology, The George Washington Uniiversity School of Medicine, Washington, D.C. Received for publication 20 July 1965 ABSTRACT REICH, MELVIN (The George Washington University School of Medicine, Wash- ington, D.C.), AND H. GEORGE MANDEL. Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil. J. Bacteriol. 91:517-523. 1966.-5-Fluorouracil (FU) produced a marked inhibition of growth and deoxyribonucleic acid (DNA) synthesis in Bacillus cereus 569H. Protein and ribonucleic acid (RNA) synthesis were not specifically inhibited, and proceeded at the rate of turbidometric increase of the cells. Cell-wall synthesis, respiration, and penicillinase production continued in the presence of FU at essentially the control rate. The addition of equimolar con- centrations of uracil and FU prevented growth inhibition but did not restore DNA synthesis. The addition of thymidine with FU did not relieve growth inhibition but did restore the DNA content to normal. Thymidine supplementation also increased the quantity of FU, but not uracil, incorporated into RNA and the acid-soluble fraction. The data indicate that inhibition of growth can be dissociated from inhibition of DNA synthesis and that more DNA is present in normal cells than is needed for growth and reproduction.

The metabolism and mechanism of action of preliminary report of this work has been pre- 5-fluorouracil (FU) have been studied in bacteria. sented (Reich and Mandel, Federation Proc.21: In Escherichia coli the drug was incorporated 176, 1962). into the acid-soluble fraction and into ribonucleic acid (RNA) in place of uracil, forming the cor- MATERIALS AND METHODS responding metabolic derivatives containing FU Growth. B. cereus was grown at 37 C in a Gyrotory (5). The most active form of the drug appears Shaker (New Brunswick Scientific Co., New Bruns- to be wick, N.J.) in media containing 2.5 X 10-2 M KH2PO4, 5-fluoro-2-deoxyuridine-5'-monophosphate, 2.0 X 10- M MgSO4c7H20, 1.2 X 10-5 M Fe(NH4)2- which inhibited the methylating enzyme, thy- (SO4)2-6H20, and 1% Casamino Acids (Difco) at midylate synthetase, and prevented the conversion pH 7.0. Growth was measured turbidimetrically at of deoxyuridine monophosphate to thymidine 540 m, in a Bausch & Lomb Spectronic-20 colorim- monophosphate. The result was a reduced de- eter. Radioactive compounds or pyrimidines were oxyribonucleic acid (DNA) content, unbalanced added during the early stage of logarithmic growth at growth, and thymineless death (7). an optical density (OD) of 0.3. One flask received 20 Additional observations include alterations in ,ug/ml of FU (0.16 jsmole/ml) dissolved in 0.5% ribosome metabolism (3), changes in amino acid Na2CO3, and others, serving as controls, received into the soluble and into Na2CO3 only. Zero-time samples were removed for incorporation pool (10) analysis immediately after addition of the drug and cellular protein (8), decreased enzyme synthesis at absorbancy increments (tOD) of 0.1 up to 0.6 or (9), formation of altered or inactive enzymes (8), at 0.6 only. At this point both the control and the and interference with cell-wall synthesis (25). inhibited culture contained 0.3 mg (dry weight) and This paper deals with the effect of FU on the about 2 X 108 cells per ml. synthesis of protein, nucleic acid, and cell wall, Cell fractionation. After growth in the presence of and on the respiration of Bacillus cereus. The FU-2-C'4, the cells were fractionated by the method effects of uracil and thymidine upon DNA syn- of Brockman et al. (5) or Mandel and Markham (14) thesis and FU incorporation is also described. A to establish the sites of drug incorporation. Analytical methods. Total protein was determined 1 Present address: Department of Microbiology, by the procedure of Oyama and Eagle (17), RNA by The George Washington University School of Medi- the orcinol method (27), DNA by the diphenylamine cine, Washington, D.C. reaction (6), and RNA stability by the method of 517 518 REICH AND MANDEL J. BAcr-ERioL.

and glycine-2-C'4 were purchased from Tracerlab, Waltham, Mass., and uracil, thymidine, pyridoxine hydrochloride, and penicillin G from Nutritional Biochemicals Corp., Cleveland, Ohio. Tritiated di- aminopimeic acid was furnished by J. L. Strominger of the University of Wisconsin, Madison.

0 REsuLTs Growth. FU caused a 75% reduction in the growth rate of B. cereus, as measured by the time required to double the initial OD of 0.3 0*3 (Fig. 1). This growth rate was intermediate between that of the control and that of completely 0 5s0o 00 150 200 250 300 inhibited cells at 0.8 ,mole of FU per ml. Micro- TIME (MIN) scopic examination of the inhibited cells revealed Fig. 1. Effect ofFU and pyrimidines on the growth long chains as opposed to the normal independent rate of Bacillus cereus. Each pyrimidine (0.16 4umole/ cellular morphology. Individual cells also ap- ml) was added to portions of an exponentially growing peared to be elongated. culture at an OD of 0.3. Thymidine addition was re- Growth inhibition was almost completely pre- peated every 15 mini. Symbols: 0, control; 0, FU; vented by the simultaneous addition of an equi- A, FU plus uracil; A, FU plus thymidine. molar concentration of uracil. An equimolar concentration of thymidine, added at zero-time Mandel (13). Incorporation studies of radioactive and at 15-min intervals, to compensate for its drugs and precursors into the various cellular fractions rapid cleavage to thymine (2), did not reverse were carried out by the membrane filtration technique The rate of the control (21). growth inhibition. growth In this last method, 2-ml samples of the bacterial culture was not affected by the addition of either suspension were mixed with equal volumes of 0.9% of the natural pyrimidines alone. NaCl, 10% cold trichloroacetic acid, or 10% tri- It has been reported that pyridoxine acted as a chloroacetic acid and heated at 100 C for 15 min, or noncompetitive antagonist of FU in Candida samples were mixed with 1 N KOH and incubated at albicans (11). No such effect was observed on 37 C overnight, followed by neutralization with 1 N FU-treated B. cereus with up to 4.9 ,umoles of HCl. Solutions were filtered through coarse membrane pyridoxine per ml. filters (Schleicher & Schuell Co., Keene, N.H.), Protein synthesis. In most of the present experi- attached to planchets, and counted in a Nuclear- ments, biosynthesis was measured in cultures at Chicago gas-flow proportional counter. The counts thus obtained represented incorporation into the similar bacterial turbidities. In this manner, an following fractions: (i) intact cells, (ii) cold acid- allowance was made for the slower rate of growth insoluble, (iii) hot acid-insoluble, and (iv) KOH-in- of the FU-treated cells, and the differences due to soluble (DNA). The difference between (i) and (ii) effects beyond that due to growth inhibition be- was the cold acid-soluble fraction; between (ii) and come more apparent. Turbidometric increases (iv), the KOH-soluble (RNA) fraction. are related to increases in cell dry weight. Table Penicillinase assays were performed manometri- 1 compared the incorporation of several amino cally at 37 C under air (18) with penicillin G as acids into the hot trichloroacetic acid-insoluble substrate and cell-free media from cells harvested fraction of cells grown with and without FU to at an OD of 0.6 as a source of the constitutive exo- similar turbidities. This fraction has been shown enzyme. Carbon dioxide production was measured 15 min after addition of the substrate. Oxygen con- to contain all the bacterial protein as well as sumption and CO2 production were measured at 37 C cell-wall constituents (21). FU did not inhibit in a Warburg apparatus (26) either over the entire the uptake of those amino acids which act solely growth period or over OD increments of 0.1. The as protein precursors [i.e., tyrosine, proline, ar- growth rate in the Warburg apparatus was essentially ginine (22)], but those amino acids which con- the same as that in the shaker. tribute carbon for the formation of cell wall (i.e., Chemicals. FU (NSC-19893) was supplied by the glycine, serine, aspartic acid) were all used more Cancer Chemotherapy National Service Center, extensively. It should be clear, however, that, Bethesda, Md. FU-2-C14 and proline-carboxyl-C14 since FU inhibited rate were purchased from Calbiochem. Adenine-2-C'4, growth, the of protein adenine-8-C'4, uracil-2-C14, orotic acid-2-C'4, hy- synthesis, in accord with that of growth, was poxanthine-8-C14, arginine (guanido-C'4), tyrosine- similarly inhibited. Colorimetric analysis of the 2-C'4, phenylalanine-3-C'4, methionine-2-C14, valine- total protein content revealed no specific inhibi- 1-C14, and serine-i-C14 were obtained from Isotopes tion of protein synthesis in the FU-treated cul- Specialties Co., Burbank, Calif. Aspartic acid-4-C14 ture (Table 2). VOL. 91, 1966 CELLULAR FUNCTIONS AND 5-FLUOROURACIL 519 TABLE 1. Incorporation of radioactive amino acids into the hot trichloroacetic acid-insoluble (protein plus cell wall) fraction ofBacillus 0 cereus during growth inhibition by FU cz

Amino acid Percentagecontrol* of

Glycine-2-C'4...... 187 z Serine-3-C4 ...... 165 Aspartic acid4-C'4 ...... 148 Valine-l-C'4 ...... 112 Tyrosine-2-C14 ...... 103 0 50 100 150 'C Proline-carboxy-C'4 ...... 102 Z IWINUTES X Arginine-guanido-C4 ...... 100 Methionine-2-C'4 ...... 97 z 20,000_ Phenylalanine-3-C14...... 94 0 0 * Radioactive amino acids were added to portions of an exponentially growing culture at OD x 0.3 with or without FU (0.16,4mole/ml). At OD 0.6, samples were removed and analyzed by the '10,000 _ ? membrane filtration technique as described in Materials and Methods. Control values (counts per minute) were arbitrarily assigned values of 100.

TABLE 2. Protein, RNA, and DNA contents of o 01 0-2 0 3 Bacillus cereus during growth inhibition by FU 0 D540 in the presence and absence of added pyrimidines* FIG. 2. Incorporation of DAP-H3 into the cold trichloroacetic acid-insoluble fraction of Bacillus Addition AOD Protein RNA DNA cereus during growth inhibition by FU. DAP-H3 was added to portions of an exponentially growing culture FU 0 94 105 96 at an OD of0.3 (zero-time) with or without FU (0.16 0.1 103 94 ,umole/ml). Samples were removed at the points in- 0.2 91 105 dicated and treated as described in Materials and 0.3 96 99 55 Methods. (top) Expressed as a function of time; (bottom) expressed as a function of turbidimetric FU plus uracil 0 102 104 96 inicrease. A correction for self-absorption was made by 0.1 104 102 multiplying counts per minute by the corresponiding OD. 0.2 96 103 Symbols: 0, control; 0, FU. 0.3 102 106 49 cells at the same OD. This result corresponds to FU plus thymidine 0 100 102 96 the increased incorporation of these amino acids 0.1 100 106 which are also cell-wall precursors. The rate of 0.2 91 107 cell-wall synthesis, however, was not affected 0.3 100 92 103 (Fig. 2, top). * Compounds were added as in Fig. 1. Results Nucleic acid synthesis. The RNA and DNA are expressed as a percentage of the control value contents, measured colorimetrically, of control (obtained from a control culture without added and inhibited cultures grown to identical tur- pyrimidines) at each OD increment of 0.1 from bidities are presented in Table 2. At the end of 0.3 to 0.6. Actual initial values for the control one generation time, the RNA content of the culture were (per milliliter): 30.0 .yg of protein, inhibited cells was normal, whereas the DNA 17.0 jug of RNA, 2.7 jAg of DNA. content was reduced to a value 55 % that of the control. By subtracting the DNA content present Cell-wall synthesis. The effect of FU on cell- at zero-time, it was calculated that the DNA wall synthesis was determined by measurement content of the newly formed cells was only 25%Vo of the incorporation of tritiated diaminopimelic of the control in the FU culture and 12% in the acid (DAP-H3) into the cold trichloroacetic FU plus uracil culture. acid-insoluble residue. As shown in Fig. 2 (bot- Subcultures of cells grown with or without the tom), the drug caused an increased uptake of this drug and with adenine-8-C14, when regrown in cell-wall precursor when compared with control drug-free media containing added nonlabeled 520 REICH AND MANDEL J. BACrzuoL. TABLE 3. Effect of thymidine on the incorporation ofFU-2-C14 and uracil-2-C4 into cell fractions of Bacillus cereus* FU-2 C14 Uracil 2 C4

Cold trichloroace- Cold trichloroace- Cold trichloroace- AOD tic acid-insoluble tic acid-soluble tic acid-insoluble

Control Plus t Control Plus thy- Control Plus thymidine midine midinei 0.1 192t 405 178 342 904 892 0.2 453 1,228 440 1,184 2,048 2,050 0.3 695 2,127 750 2,068 3,583 3,601 * FU-2-C14 or uracil-2-C04 (0.16 ;umole/ml) in FIG. 3. Incorporation of adenine-2-C14, orotic the presence or absence of thymidine (0.16,umole/ acid-2-C'4, and hypoxanthine-8-C'4 into the KOH- ml) was added at an OD of 0.3, and thymidine, soluble (RNA) and KOH-insoluble (DNA) fractions of every 15 min thereafter. At OD increments of 0.1, Bacillus cereus. A 0.16-inole amount of each py- samples were removed, treated with cold tri- rimidine was added at an OD of 0.3 to portions of an chloroacetic acid, and filtered as described in exponentially growing culture. All thymidine additions Materials and Methods. were repeated every 15 min. Cells were analyzed at an t Results expressed as counts per minute. OD of 0.6 as described in Materials and Methods. Control radioactivity values were set equal to 100. A, control; B, FU; C, FU plus uracil; D, FU plus thy- The acid-soluble fraction of the cells contained midine; E, FU plus uracil plus thymidine. the fluorinated analogues of the corresponding uracil-containing cofactors. adenine, did not lose any radioactivity from the The addition of an equimolar amount of KOH-insoluble (DNA) residue. Thus, the ob- uracil simultaneously with FU almost completely served reduction in DNA content was not due prevented the incorporation of FU-2-C'4 into B. to its instability or turnover after synthesis. cereus (19). Likewise, the RNA synthesized in the presence of Since Sells (23) reported that thymidine can FU was no less stable than that of the control reduce the incorporation of uracil into B. cereus, cells. it was thought that it might also antagonize FU As reported previously (19), the addition of incorporation, thereby permitting the observed uracil did not produce an increase in the DNA normal rate of DNA synthesis. No such reduction content, although it acted to restore the growth was observed. On the contrary, it was found rate to normal. The present experiments show (Table 3) that thymidine stimulated the incor- (Table 2) that thymidine, which did not relieve poration of FU into RNA and the acid-soluble growth inhibition, was effective in preventing the fraction by 206 and 176%, respectively, after one decrease in DNA. These results were confirmed generation time when compared with a control by a study of the incorporation of radioactive culture. The incorporation of uracil-2-C14 nucleic acid precursors (Fig. 3). FU did not in- remained unaffected by thymidine addition. The hibit the utilization of adenine-2-C'4 or orotic addition of thymine (0.16 ,umole/ml) every 15 acid-2-C'4 for RNA synthesis but markedly min stimulated the incorporation of FU-2-C'4 reduced their incorporation into DNA. This into RNA by only 9%. reduction was prevented by thymidine or thymi- Thymidine continued to stimulate FU incor- dine plus uracil. Thus, uracil and thymidine must poration into RNA for at least 90 min after sup- be added together for the cells to both grow and plementation was stopped (Table 4) and for at synthesize DNA at a normal rate. Wachsman et least 160 min (time required to reach OD 0.6 al. (28) reported that one partially FU-resistant upon subculturing) after its removal by centrifu- strain of B. megaterium required only uridine and gation (Table 4). In both experiments, the con- another required only thymidine to reverse FU- tinued addition of thymidine stimulated drug induced inhibition. uptake still further. Again, the incorporation of Incorporation of FU-2-C'4. After growth in the uracil-2-C4 remained unaffected by thymidine. presence of FU-2-C14, radioactivity was isolated Respiration and enzyme synthesis. FU produced from RNA as fluorouridylic acid. No activity up to a 100% increase in both 02 and CO pro- was recovered in the area corresponding to duction when comparisons were made between fluorocytidylic acid or in the DNA hydrolysate. growing cultures over equal OD increments. The VOL. 91, 1966 CELLULAR FUNCTIONS AND 5-FLUOROURACIL 521

TABLE 4. Effect of thymidine pretreatment on incorporation of FU-2-C14 and uracil-2-C'4 by Bacillus cereus*

FU-2-C"t Uracil-2-CU4 Expt Control Thymidine Thymidine Control Thymidine Thymidine (no thymidine addition addition (no thymidine addition addition addition) discontinued continued addition) discontinued continued A 5,051t 8,197 11,142 11,396 10,050 10,256 B 2,893 6,130 9,299 3,585 3,998 3,795 * Cells were grown from OD 0.3 to 0.6 in the absence or presence of thymidine (0.16 ,mole/ml) added every 15 min. In experiment A at OD 0.6 (zero-time), FU-2-C14 or uracil-2-C"4 (0.16 Amole/ml) was added, and growth continued for 90 min with or without thymidine (0.16 ,mole/ml added every 15 min). In experiment B at OD 0.6, the cells were centrifuged and washed with, and suspended in, fresh media at OD 0.3. FU-2-C4 or uracil-2-C'4 (0.16 Mmole/ml) was added, and the cells were permitted to grow to OD 0.6 with or without the addition of thymidine (0.16 /Amole/ml) every 15 min. At OD 0.6, samples of the culture were removed, treated with cold trichloroacetic acid and filtered as described in Ma- terials and Methods. t Results expressed as counts per minute. rate of respiration, however, was slightly reduced growth. Indeed, similar experiments have shown by FU. that growth was not inhibited until after three The penicillinase activity assayed in the cell- doublings of the initial OD despite a marked free media of the inhibited cells was essentially reduction in the DNA content (19). equal to that of the control. It should be stressed, however, that the RNA which is formed, although present in normal DIscussIoN amounts, as well as the acid-soluble components, The results presented here suggest that FU is atypical in that it contains FU. The synthesis of exerts at least two effects on B. cereus. One effect, messenger RNA containing FU may explain the the inhibition of DNA synthesis, is prevented by report of Gros and Naono (8) regarding the thymidine but not by uracil. Thus, the addition formation of bacterial protein with a reduced of thymidine permits the synthesis of normal content of tyrosine and proline and an elevated amounts of DNA despite the increased incorpora- arginine level. In the present experiments, no tion of FU, without relieving the inhibition of changes in the pattern of amino acid incorpora- growth. Gros and Naono (8) also reported that tion were observed, nor was the enzymatic thymidine could restore DNA synthesis in E. coli activity of penicillinase affected. but did not report its effect on growth. In the time interval between the inhibition of A second effect, the incorporation of FU into DNA synthesis and the inhibition of growth, the RNA and the soluble uracil-containing cofactors, small amount of newly synthesized DNA might is prevented by uracil but not by thymidine. With be sufficient to permit normal growth, protein, the addition of uracil, the growth rate remains and RNA synthesis provided, as is true only in normal while DNA synthesis is reduced to a value the uracil-supplemented culture, that little or no only 12% of the control. In several instances, the FU is incorporated into RNA or the soluble added uracil, although preventing FU incorpora- cofactors. This would seem to indicate that most tion, actually reduced the DNA content below of the cellular complement of DNA normally that of the FU-inhibited culture. Although this formed during growth is not needed for growth. effect was small (6% in Table 2, see also 19), it If true, this situation could be analogous to the was verified by isotope incorporation (Fig. 3, finding by Allfrey, Littau, and Mirsky (1) that up columns B and C) and seems to be real. The to 80% of the DNA in thymus nuclei could be ability of uracil to reverse FU-induced growth removed without affecting protein or RNA inhibition in E. coli has been demonstrated by synthesis. They postulated that most of the DNA Cohen et al. (7) but not by Gros and Naono (8). was inactive or repressed since it was not synthe- The ability of B. cereus to grow at a control sizing messenger RNA. In a multinucleate cell rate in cultures containing FU plus uracil and to such as B. cereus, this excess DNA may be synthesize protein and RNA in normal amounts associated with the four chromatin bodies con- while forming only 12% of the normal quantity of taining DNA (16). DNA suggests that the inhibition of DNA An alternative explanation might be the synthesis precedes the resulting inhibition of utilization of pre-existing DNA formed prior to 522 REICH AND MANDEL J. BACmmIOL. the addition of FU and pyrimidines. This does Institute and by Public Health Service grant AI-04264 not appear likely, because the thymidine-sup- from the National Institute of Allergy and Infectious plemented culture has the same quantity of Diseases. pre-existing DNA, as well as a normal content LITERATURE CrrD of newly synthisized DNA, yet its growth rate 1. ALLFREY, V. G., V. C. LrrrAU, AND A. E. MIRSKY. remains inhibited. It is possible, however, that 1963. On the role of histones in regulating the DNA made during growth inhibition by FU, ribonucleic acid snythesis in the cell nucleus. in the presence or absence of thymidine, is not Proc. Natl. Acad. Sci. U.S. 49:414-421. functional. 2. ALPEN, E. L., AND H. G. MANDEL. 1960. A rapid assay method for tritium in bacterial cells. The selective stimulation by thymidine (or a Biochim. Biophys. Acta 43:317-321. derivative) of FU incorporation into RNA and 3. ARONSON, A. I. 1961. The effect of 5-fluorouracil the acid-soluble fraction without influencing on bacterial protein and ribonucleic acid uracil incorporation is surprising. Thymidine may synthesis. Biochim. Biophys. Acta 49:98-107. act by affecting the anabolism or catabolism of 4. BODMER, W. F., AND S. GRETHER. 1965. Uptake FU, its ribosides and ribotides. Maley and Maley and incorporation of thymine, thymidine, (12) have shown that added thymidine (acting uracil, uridine, and 5-fluorouracil into the as thymidine triphosphate) stimulated the in- nucleic acids of Bacillus subtilis. J. Bacteriol. corporation of deoxycytidine into 89:1011-1014. chick-embryo 5. BROCKMAN, R. W., J. M. DAvIs, AND P. STurrs. DNA by blocking the conversion of cytidine to 1960. Metabolism of uracil and 5-fluorouracil uridine and uridine monophosphate. On the by drug-sensitive and drug-resistant bacteria. other hand, Mukerjee and Heidelberger (15) Biochim. Biophys. Acta 40:22-32. found that thymine reduced the catabolism and 6. BURTON, K. 1955. The relation between the increased the toxicity of FU in mice by competing synthesis of deoxyribonucleic acid and the for the pyrimidine catabolizing enzymes. synthesis of protein in the multiplication of If thymidine did act by increased anabolism or bacteriophage T2. Biochem. J. 71:473-483. decreased catabolism of FU in B. cereus, one 7. COHEN, S. S., J. G. FLAKS, H. D. BARNER, M. R. might expect an increase in con- LOEB, AND J. LIcHsTENsTEN. 1958. The mode intracellular of action of 5-fluorouracil and its derivatives. centration of 5-fluoro-2-deoxyuridine-5'-mono- Proc. Natl. Acad. Sci. U.S. 44:1004-1012. phosphate, with a resultant increase in growth 8. GROS, F., AND S. NAONO. 1961. Bacterial synthesis inhibition in those cultures receiving thymidine. of "modified" enzymes in the presence of a This was not observed. Further, to explain the pyrimidine analog, p. 195-205. In T. J. C. selective action of thymidine by this mechanism Harris [ed.], Protein biosynthesis. Academic would require the existence of separate sets of Press, Inc., New York. anabolic and catabolic enzymes for FU and 9. HoRowrrz, J., J. J. SAUKKONEN, AND E. uracil, yet both pyrimidines are apparently acted CHARGAFF. 1960. Effects of fluoropyrimidines upon by identical enzymes (15, 24). in the synthesis of bacterial proteins and nucleic The of to acids. J. Biol. Chem. 235:3266-3272. inability thymine stimulate EU 10. KEMPNER, E. S., AND J. H. MILLER. 1962. Alter- incorporation (9% increase into RNA versus ation of carbon metabolism by a base analog. 206% for thymidine in Table 3) indicates that Biophys. J. 2:327-337. it is not the active derivative. If FU inhibits 11. L[TMAN, L., AND T. MIWATANI. 1961. Reversal thymidine phosphorylase in B. cereus, as has of toxicity of 5-fluorouracil and 5-fluoro- been shown in B. subtilis (4), then even less deoxyuridine for Candida albicans by pyridoxine thymine (or deoxyribose-1-phosphate) would be and pyridoxamine. Nature 192:1155-1159. formed. Indeed, the presence of an intracellular 12. MALEY, G. F., AND F. MALEY. 1963. Nucleotide pool of thymidine would explain the continuing interconversions. VIII. Thymidine sparing action of effect on the utilization of de-cytidine by thymidine long after its removal. chick-embryo muscle. Biochim. Biophys. Acta The continued synthesis of cell wall by cells 68:293-301. grown in the presence of FU suggests that the 13. MANDEL, H. G. 1961. Further studies on the fluorouridine analogue of the cell-wall precursor, modifications of nucleic acid synthesis of B. uridine-diphosphate-N-acetylglucosamine, may cereus by 8-azaguanine. J. Pharmacol. Exptl. be readily utilized and may function similarly to Therap. 133:141-150. the normal metabolite. This is not the case in 14. MANDEL, H. G., AND R. MARKHAM. 1958. The Staphylococcus aureus, where FU causes the effects of 8-azaguanine on the biosynthesis of accumulation of cell-wall precursors ribonucleic acid in Bacillus cereus. Biochem. (20). J. 79:297-306. ACKNOWLEDGMENTS 15. MUKHERJEE, K. L., AND C. HEIDELmBRGER. 1960. Studies on fluorinated pyrimidines. IX. The This investigation was supported by Public Health degradation of 5-fluorouracil-6-C4. J. Biol. Service Grant CA-02978 from the National Cancer Chem. 235:433-437. VOL. 91, 1966 CELLULAR FUNCTIONS iAND 5-FLUOROURACIL 523

16. MURRAY, R. G. E. 1960. The internal structure 22. ROODYN, D. B., AND H. G. MANDEL. 1960. The of the cell, p. 35-96. In I. C. Gunsalus and R. Y. differential effect of 8-azaguanine on cell wall Stainer [ed.], The bacteria, vol. 1. Academic and protoplasmic protein synthesis in Bacillus Press, Inc., New York. cereus. J. Biol. Chem. 235:2036-2044. 17. OYAMA, V. I., AND H. EAGLE. 1956. Measurement 23. SELLS, B. H. 1960. The effects of thymidine upon of cell growth in tissue culture with a phenol the incorporation of uracil into Bacillus cereus. reagent. Proc. Soc. Exptl. Biol. Med. 91:305- Biochim. Biophys. Acta 40:548-549. 307. 24. SKOSLD, 0. 1960. Nucleoside and nucleotide 18. POLLOCK, M. R. 1950. Penicillinase adaptation derivatives of 5-fluorouracil. Arkiv Kemi in B. cereus: adaptive enzyme formation in the 17:59-71. absence of free substrate. Brit. J. Exptl. Pathol. 25. TomAsz, A., AND E. BOREK. 1962. The mechanism 31:739-753. of an osmotic instability induced in E. coli K-12 19. REICH, M., AND H. G. MANDEL. 1964. Uracil: by 5-fluorouracil. Biochemistry 1:543-552. failure to relieve DNA synthesis while relieving 26. UmBRErr, W. W., R. H. BURRIS, AND J. F. STAUF- 5-fluorouracil induced inhibition. Science FER. 1957. Manometric techniques, 3rd ed. 145:276-277. Burgess Publishing Co., Minneapolis. 20. ROGERS, H. J., AND H. R. PERKINS. 1960. 5-Flu- 27. VOLKIN, D., AND W. E. COHEN. 1954. orouracil and mucopeptide Estimation biosynthesis by of nucleic acids. Methods Biochem. Staphylococcus aureus. Biochem. J. 77:448-459. Analy. 21. ROODYN, D. B., AND H. G. MANDEL. 1960. A 2:287-305. simple membrane fractionation method for 28. WACHSMAN, J. T., S. KEMP, AND L. HOGG. 1964. determining the distribution of radioactivity Comparative effects of 5-fluorouracil on strains in chemical fractions of Bacillus cereus. Bio- of Bacillus megaterium. J. Bacteriol. 87:1011- chim. Biophys. Acta 41:80-88. 1018.