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Molecular Events in the Reproduction of Animal Cells I . The Effect of Puromycin on the Duplication of DNA* GERALD C. MUELLER, KAZUTO AND K@&jiwu@, ROLAND R. ELTON STUBBLEFIELD, RUECKERT (McArdZe Memorial Laboratory, University of Wisconsin Medical *SCFiOO4 Madison, Wiaxnwin) SUMMARY The initiation of DNA synthesis was studied in cultures of HeLa cells which were synchronized by the induction and timely reversal of a thymidineless state. It was . @ observed that the synthesis of DNA was initiated whereas its effects on protein synthesis were a immediate. that the synthesis of DNA in the mammalian localized changes involving protein synthesis process. slow rate but was more than (1@) it was demonstrated that DNA replication occurs only after a specific triggering event and that the formation of new DNA is a nonlinear process within a single nucleus. Under the condi tions of these experiments levels of puromycin which inhibit protein synthesis prevented the ac celeration of DNA synthesis without inhibiting the established rate of DNA synthesis. On the basis of the results of these studies and the studies on chromosome labeling in the second paper of this series, it is proposed that the DNA of the triggered IleLa cell exists in two physiologi cal states : a fraction which is competent for repli cation, and another which becomes competent for a This work was supported United States Public Health by grant CY-1897-C7 from the Service and by funds from the Alexander and Margaret Stewart Trust. The data support the concept cell is a focalized phenomenon and that are required to initiate the replication replication through a puromycin-sensitive presumed to be protein synthesis. The replication of mammalian cells can be visu alized as a cyclic process in which the cell prog resses through a sequence of molecular events in volving the timely synthesis or activation of spe cific macromolecules. The present study concerns the mechanisms underlying the initiation of DNA synthesis. With use of synchronized cultures of HeLa @ at doubled after hours. This acceleration of DNA synthesis could be prevented with amounts of puromycin which simultaneously inhibited protein synthesis. Puromycin, however, did not inhibit DNA synthesis which was in progress at the time of addition, process MATERIALS AND METHODS The origin and maintenance of the HeLa cell strain used in these experiments has been described in detail previously (1@). Stock cultures were grown in spinner flasks using Eagle's HeLa medi um (7) from which the calcium and magnesium were omitted. This medium was supplemented further with 0.1 m@& glycine, 0.1 m@ serine, 0.01 mM inositol, 0.1 per cent pluronic F-681 (14), and 10 per cent bovine serum (medium is referred to as BEHM) . Stock culture suspensions were di luted daily with fresh medium to an initial cell con centration of 1.5 X 1O@cells per milliliter. Cells were thus maintained in logarithmic growth with the growth potential of the medium approximate ly half expended at the time of refeeding. The gen eration time under these conditions averaged around hours. For the experimental studies replicate mono layer cultures were prepared by inoculating 3—5X 10@ cells in scription Received for publication May 11, 1962. 1 Wyandotte 10 milliliters bottles. Chemical After Co., BEHM @4hours' Wyandotte, into 3-oz. incubation pre at Michigan. 1084 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research. MUELLER ci @2l.—Effectof Puromycin 370 C. in an atmosphere of 5 per cent CO2 in air to permit attachment of the cells to the glass, a thymidine deficiency was produced by the addition of 1.1 ml. of BEHM containing 10@ M amethop term and 5 X 10@ M adenosine. At the indicated time, usually 16 hours after the addition of ame thopterin, the cultures were rescued by the addi tion of thymidine in the amounts described. Allhandling of the cells was carried out in a 37°C. room, and all media were prewarmed to 37°C. in a thermostated water bath prior to use in order to avoid any cold shock synchronization of the cul tures. Experimental cultures were harvested dicated times by rinsing cell sheets 0.9 cent per saline followed by at the in successive @.0ml. of 88 per cent cell residue was dissolved formic acid, RESULTS and suitable in au quots taken for analysis. Isotopic determinations were carried out on aliquots plated on clean aiumi num planchets and counted for radioactivity in a gas-flow proportional counter. DNA was deter mined by the modified fluorometric procedure of Kissane and Robins (8), with salmon-sperm DNA used as the reference standard (California Corp. for Biochemical Research). Protein determinations were made with the Oyama and Eagle modification of the Lowry procedure (11). For the autoradiographic studies with tritium labeled thymidine the cells were planted on cover slips (10 X 50 mm.) The culture 1.0 ml.; inserted into Leighton size was reduced all other conditions tubes. proportionately were maintained to iden tical to those of the 10-mi. cultures in prescription bottles. At the indicated times the cells were la beled with tritiated thymidine (@curies per milli mole). At the conclusion of the labeling period the cover slip with its adherent cells was removed from the Leighton tube, rinsed with water, and fixed in methanol—acetic acid (3 : 1) at room temperature for 30 minutes. The cover slip was then rinsed in a second change of fixative and was rapidly air dried. This procedure flattened the nuclei and fa cilitated grain counting subsequently. The cover slip was mounted to a microscope slide and coy ered with stripping film according to the procedure of Pelc (5). After the proper exposure the auto graphs were developed in Kodak of the emulsion with dilute HC1. The preparations were then air-dried, and a second cover slip was mounted with Permount. Localization and count ing of the silver granules were carried out with phase-contrast microscope. Thymidine @-C'@and leucine-1-C'4 were oh tamed from New England Nuclear Corporation; tritium-labeled thymidine was obtained from the Schwartz Biochemical Company. The puromycin was a generous gift of Dr. Nestor Bohonas and the Lederie Company. These materials were dis solved in double-distilled water and added as a @5or 50-microliter aliquot to the culture medium as indicated. washes to dry. The dry, defatted 1085 @2times with with two 1O-ml. volumes of 4 per cent perchioric acid, 80 per cent ethanol, and absolute ethanol. The cell sheets were then rinsed with ether and permitted on DNA D-19 developer for 7 minutes, rinsed in distilled water, and fixed in Kodak acid fixer. The nuclei were stained through the stripping film with Harris' hematoxy un for 30 minutes, followed by a brief de-staining Synchronization of the culturea.—The induc tion of a thymidineless state with amethopterin in HeLa cultures was described previously (1@). This state, which is analogous to that obtained in bac terial cultures with a thymidine deficiency (3, 4), is characterized by the immediate cessation of DNA synthesis and cell divisions while RNA and protein continue to accumulate. After an optimal exposure to amethopterin (16 hours) the addition of exogenous thymidine results in a synchronized wave of DNA synthesis which is followed by a similar wave of cell divisions. The wave of cell divisions, however, takes place only after the cell's complete complement of DNA has been synthe sized. The typical response of the basic thymidine rescue experiment is depicted in Charts 1 and %. In all experiments it was observed that DNA syn thesis was resumed on addition of exogenous thy midine at a rate one-third to one-half the maxi mum rate; the maximal rate was attained in the cultures only after an initial s-hour period during which the acceleration took place. In order to elucidate further the molecular events taking place in this system it was first nec essary to establish the manner in which the popu lation of cells was responding. For this purpose preliminary kinetic experiments were carried out on the ability of cultures to incorporate thymidine @-C'4 after various periods of pretreatment with amethopterin. Table 1 illustrates that a maximal rate of DNA synthesis was obtained only after 16 hours of pretreatment; this is in accord with earlier studies which showed that 16 hours was the opti mum time for reversal of the thymidineless state so as to obtain a maximal synchronization of cell divisions with a minimum of thymidineless death (1@). Susceptibility of the incorporated thymidine-2-C'4 to the action of DNa@e.—Inorder to assure that the incorporated thymidine-@-C'4 resided in DNA, Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research. Cancer Research 1086 experiments @ were carried out to correlate the loss of label from the nucleoprotein residue with the hy drolysis of DNA by DNase. In Table it is dem onstrated that the release of label parallels com pletely the solubilization of the DNA by the spe cific enzyme. The triggering of individual ce& for DNA syn thesis.—While the kinetic studies in whole cultures suggested strongly that individual cells were ac 0 Ui M (1) Ui 1.9 DNA by autoradiography. C) that 1 MENT ON ABILITY OF HELA CUL TURES To INCORPORATE THYMI. DINE.2.C― U) >- U) 0) Pretreatment with amethopterin 1.3 0 8 illustrates EmcT OFAMETHOPTERIN PRETREAT. I., 0 Chart TABLE l-7I@ z 196@ cumulated by the thymidine deficiency in a state of readiness for DNA synthesis, it was necessary to verify this picture at the level of individual cells. For this purpose cultures were permitted to incorporate tritium-labeled thymidine for a 10minute period at indicated times after the start of the amethopterin treatment; the individual cells were then examined for the amount of isotope in P1 z @,October no more than 30 per cent of the cells at any one time in a logarithmically growing HeLa culture are in the process of synthesizing their DNA; the rest are in the interphase or post-DNA synthesis 12.1 II Vol. incorporation (counts/minXlO')4 (hr.)Thymidine.5-C1' I. I 6.37 7.65 7.79 7.42 8 12 16 184.58 HOURS AFTER THYMIDINE ADDITION Cultures of HeLa pared and pretreated Ciwrr 1.—Theaccumulation of DNA and cells in cultures cells were pre with amethopterin 10-' M. At the indicated times 10 pg. following thymidine rescue. Cultures were prepared as under “Methods―and treated with amethopterin for 16 hours prior to the addition of 10 pig. of thymidine-2-04 per culture. DNA synthesis was measured from the amount of thymidine-2-C'4 incorporated into DNA. thymidine.2-C― were added to the cal tures and the incorporation into DNA was measured over a 4-hour period. TABLE 2 PARALLEL REMOVAL BY DNASE OF DNA AND II 2.32j@/HR, § 0 8 RADIOACTIVITY FROM THYMIDINE. 2-C'4LABELEDCELLS N.J •@Gi• 10 Houss £Pm 7. TUTMIDDII 5-C― aaa&zanioR.4DI0AcTITITT aa@[email protected] ia DNA5EDNA centCounts/ sascusHocus cent6 z@ / 0@3- 2 4 80 @°Ij I I mm/flaskPer I Ill I 2 3 4 5 6 7 8 9 10 II 12 HOURS AFTER THYMID1NE-2-@d4' ADDITION 0 2 414.2 6.4 2200 45 48 2.4 17 800 16 100 6140 100 15.6 3.8 1600 26 25 1.6100 10.55180 600100 10.8 Replicate monolayer cultures were sacrificed 6 or 8 hours after rescue with thymidine-2.C'4. The cell sheets were washed 8 times with saline, precipitated on the glass with 4 per cent perchioric acid for 2 minutes at room temperature, washed with distilled water, and incubated at 87°C. with DNaae (Nutritional Biochemicals Corporation 1 X recrystallized) 250 CHART rescue 2.—The acceleration of thymidine-deficient of DNA cultures synthesis following as measured accumulation of thymidine-2-C―in DNA. the by the pg/mi in 0.1 M tris buffer containing 10' M MgSo4. Monolay. ers were prepared and analyzed for DNA as described under “Materials and Methods.― Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research. MUELLER ci al.—Effect of Puromycin @ state. Since the generation time of the cells was about hours in monolayers, these data corre spond to an over-all DNA synthesis time of 6.6 hours; this agrees with observations on other ani mal cells in culture. While the addition of amethopterin blocks the corporation 10 ag/mi permitted synthesis attached to the glass in a morphologically of thymidine deficiency process state and trated @ hours' 90 per cent and nearly all the cells to remain intact synthesis, With this concentration of puromycin the in hibition of protein synthesis could be reversed readily by removing the puromycin medium, washing the cell sheet once with 10 ml. of fresh chart, this the synthesis continues results of ribo (1@). As illus in a progressively greater number of cells becoming triggered for or achieving the basic competency to initiate the syn thesis of their DNA: by 16 hours after the addition of amethopterin, over 90 per cent of the cells are able to synthesize DNA if provided with a source of exogenous thymidine. Accordingly, the acceler ation of DNA synthesis observed during the first hours of the thymidine rescue must take place in cells already in the process of DNA synthesis. Counting the number of silver grains in the au toradiographs permitted a comparison of the rela tive abilities of the individual cells to incorporate tritiated thymidine into DNA during a short ex posure. In the case of the logarithmically growing cultures, 30 per cent of those cells which were in the process of DNA synthesis were labeled ap proximately twice as heavily as the rest of the cells which were making DNA. A similar examination of cells from cultures which were synchronized by 16 @ a thymidine by all incorporation (Chart 4). concentrations of puromycin proportion of the cells to de a puromycin concentration of form over a period of DNA in the produces of leucine-1-C'4 @5j@g/ml blocked Whereas the higher caused a significant tach from the glass, in those cells which are in the nucleic acid and protein @ 1087 on DNA pretreatment with amethopterin 8I0 I4 HOURS AFTER AMETHOPTERIN ADDITION re vealed that the actual number of heavily labeled cells remained the same while the percentage dropped to 8 per cent. This indicates that the newly triggered cells entered into a state of light labeling when first rescued from the thymidine de ficiency. Two hours later and after the acceleration had taken place, almost all cells could be classified as labeled heavily. This proved further that the observed acceleration of DNA synthesis was tak ing place in a large majority of the cell population. The influence of puromycin on the accelera2ion of DNA synthesis.—A number of investigators have demonstrated that puromycin is a highly effective inhibitor of protein synthesis in both intact ani mals and cell-free systems (@, 9, 10, 15, 16). This inhibition results from the blocking of the trans fer of activated amino acids from soluble RNA into the formation of peptide bonds in ribosomes (16). Since the observed acceleration of DNA synthesis in thymidine rescued cultures might well depend on protein synthesis, experiments with puromycin were done. In the initial studies it was demon strated that puromycin was indeed highly effective in blocking protein synthesis in HeLa cultures and acted immediately; 10 pg/mi inhibited the in of 8—1@hours. CHART 8.—The accumulation synthesis following the induction of nudei capable of DNA of a thymidine deficiency. Populations of HeLa cells grown for the indicated times in amethopterin medium were permitted thymidine for a 10-minute period, to incorporate tritiated and the percentage of labeled nuclei was determined (upper curve). The lower curve depicts the expected cell-number increase from control cultures supplemented with thymidine throughout triggering of nuclei are plotted The data for the as N plus 100 per cent to show the relationship of the triggering event to the expected cell number increase. BEHM, and replacing this wash with 10 ml. of puromycin-free medium. Although the resumption of protein synthesis was almost immediate, the rate of leucine incorporation was slightly slower than the controls after hours of puromycin treat ment (Chart 5). This effect was exaggerated some what by an additional hours of puromycin treat ment. The viability of the puromycin-treated cells was further tested by the ability of individual cells to give rise to clones. Such studies demonstrated that cloning efficiency declined only 15 per cent after 3 hours in puromycin dicate, however, medium. Other that puromycin-treated studies in cells re Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research. @ Cancer Research 1088 tamthecapacity tosynthesize poliovirus2 onre versal of the inhibition. Accordingly the puromy cm effects are largely and readily reversible within the exposure periods used. In Chart 6 the effect of puromycin treatment on the acceleration of DNA synthesis in cultures res cued with thymidine is illustrated. It is evident that puromycin does not inhibit DNA synthesis which is in process, but acts to prevent the accelera Vol. October 196@ demonstrated in Chart 7, where the removal of puromycin resulted in an immediate acceleration of DNA synthesis in the cultures. It was thus demonstrated that the cultures were still compe tent to respond. It is of interest that the kinetics of the acceleration of DNA synthesis were similar whether the puromycin was removed after @,4, or 8 hours. Of additional interest in these studies was the observation that cells blocked with puromycm 4 hours after the reversal of the thymidineless state failed to divide until the puromycin was removed from the cultures. The possibility that the syn thesis of a particular protein necessary for mitosis occurs at this time is being investigated. However, in accord with the findings of DNA it is also pos sibie the puromycin sensitive reaction was affect ing the duplication of the last few DNA molecules 30 LEUCINE-C14 LNCORPORATLO@4 /Jc3 PUROMYCIN PER ML z [email protected] 4.—Suppression by puromycin of leucine-1-C―incor poration into HeLa protein. Replicate monolayer cultures in the logarithmic phase of growth, prepared as described under “Methods,―were overlaid with S ml. prewarmed BERM con taming puromycin at the indicated concentrations and 10 pg. DL-leuclne-1-C― (sp. act. 8.75 mc/m.mole). After an incorpora tion period described of 4 hours under at 87°C., monolayers “Methods―and counted were sacrificed as for radioactivity in 0 20 0. @1 0 0 the acid-insolubledefatted monolayer. Data are expressedas per cent of incorporation occurring mycin (6,820 counth/min/culture). in controls without 10 pure 0. 0 @ tion of DNA synthesis. Thus the addition of puro mycin 4 hours after the addition of exogenous thymidine had little influence on the established maximal rate of DNA synthesis. However, when added hours after thymidine rescue, labeling proceeded at almost control rates for the first few hours, then decelerated as the amount of DNA synthesized @ in the culture tended to plateau. When added at zero time, however, puromycin permitted a steady linear rate of labeling but completely oh literated the acceleration of labeling noted in the controls. A similar result was obtained when puro mycin was added hours prior to thymidine rescue. Of particular interest in these experiments was the observation that the amount of DNA syn thesized under the influence of puromycin tended to plateau at different levels depending at which point in the wave of DNA synthesis the puromycin was added. The reversibility of this puromycin effect is 2 J@ Olson and G. C. Mueller (unpublished data). 2 4 6 8 10 “OURS CHART 5.—Reversal synthesis. Cultures of the puromycin inhibition of protein of HeLa cells were exposed to puromycin 10 pg/mi for 2 or 4 hours, at which time the monolayers were washed with BEHM and the medium was replaced with puromycin-free medium. Leucine-1-C― was present throughout the experiment. Data are expressed as counts/mm in total protein of the culture. of the cell's complement of genetic material. In any case puromycin appears also to offer a tool for studying the specific events associated with mitosis. DISCUSSION The data on the molecular events in the repro duction of mammalian cells in this and the subse Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research. MUELLER et al.—Effect of Puromycin quent papers of this series can best be integrated in terms of a fundamental “cell cycle―such as de picted in Chart 8. As illustrated, the daughter cells formed by a previous mitosis enter the interphase state; in this condition they synthesize the macro molecular and micromolecular products which characterize them as specific cell types (i.e., skin, liver, kidney, etc.). Cells may remain in interphase for weeks, months, or even years depending on ex ternal stimulating factors and the reactive poten tial of the particular cell type to its environment. on DNA 1089 mulates the rest of the cells in the triggered state in which they are ready to initiate DNA synthesis, but are unable to do so because of the deficiency of thymidine. From these data it can be concluded that the triggering phenomenon in itself is inde pendent of the I0 presence of thymidine derivatives THVMIDINE-C14 -INTODNA Ui CONTROL a: PURO-2HRS Lii PURO 4HRS a: PURO 8HRS U a: Lii 0@ MEDIUM 0 (3 CHART synthesis. CHANGED I I 2 8HOURS 4 7.—Reversal of the Thymidine-rescued 6 puromycin cultures effect of HeLa on DNA cells were treated with puromycin 10 pg/mI for 2, 4, or 8 hours and then changed to puromycin-free medium. Thymidine-2-C'4 was present throughout the experiment. Data are expressed as HOURS AFTER THYMIDINE RESCUE CHART corporation 6.—Effect of puromycin on into DNA of synchronized the accumulated counts/zulu of thymidine-2-C― incorporated into the DNA of the culture. thymidine-2-C― in HeLa cells. Replicate monolayer cultures of HeLa were previously grown for 16 hours in amethopterin medium to induce the critical thymidine deficiency state as described under “Methods.―At the time L.:..@;' (DAUGHTER CEL.t.S) designated “zero,― cultures wererescuedby adding thymidine 2-C'4 (sp. act. 1.15 X 10 counts/min/pmole) to a final con centration of 10' is. Puromycin (10 pg/mi) was added at —2, 0, 2, and 4 hours with respect to the addition of thymidine. . @ =controls (no puromycin); J =puromycin added 2 hours prior to thymidine; ®=puromycin added at time of thymidine rescue; 0 =puromycin added 2 hours after thymi dine rescue; 0 =puromycin added 4 hours after thymidine rescue. However, through processes which require both protein and RNA synthesis (1@) they may over come the proposed barriers (A-E) and progress to the “triggered―state. In this state they are en dowed with a basic competency to initiate the duplication of their genetic material, DNA. In the logarithmically growing HeLa cultures used in these experiments only 80 per cent of the cells of the random population are engaged in DNA synthesis at any one time. However, growth in the amethopterin medium for 16 hours accu I A @.INTERPHASE DNA SYNTHESIS FUNCTIONS @t C NUCLEAR AND CELL. GROWTH TRIGGER CELL CHART 8.—A hypothetical CYCLE reproductive cycle of a mam malian cell. A—'E constitute stages or barriers within the interphase period which have to be overcome in the progression of a cell toward the initiation of a nuclear replication. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research. @ Cancer Research 1090 and that this state can be maintained for appre ciable periods of time in the absence of DNA syn thesis (i.e., at least 8—1@ hours). The reversal of the thymidineless state was at tended by the initiation of DNA synthesis in all cells simultaneously; however the newly synthe sized DNA was accumulated at a slow rate in the first hour of the reversal and only after the second hour was the maximal rate of accumulation at tamed. The observation that this acceleration could be prevented with puromycin under condi tions which blocked protein synthesis suggests that the synthesis of some specific proteins may be required for the acceleration. As shown by the autoradiographic studies of the subsequent paper (13), the DNA which is syn thesized during the first hour after reversal of the thymidineless state is distributed nonrandomly among the various chromosomes of the HeLa cell and is also distributed nonrandomly along the length of a specific chromosome. These studies also have demonstrated that the acceleration of DNA synthesis observed in the present study is correlated with the activation of many more sites of DNA synthesis along the chromosomes. Thus the synthesis of DNA in this mammalian cell nu Vol. October 196@ ACKNOWLEDGMENTS The authors are very grateful for the excellent and cc operative assistance of Mrs. Eleanor Erikson and Mrs. Kath leen Deighton. REFERENCES 1. BILLEN,D. Effects of Prior Alteration in Nucleic Acid and Protein Metabolism on Subsequent Macromolecular Syn thesis by Irradiated Bacteria. J. BacterioL, 80:86—OS,1960. 2. BOSCH, L, and Bi.or.xmiiAx@, H. The Effect of Puromycin on Nucleotide and Amino Acid Transfer from Soluble Ribonucleic Acid to the Microsomes. Biochim. Biophys. Acta,51:613—15, 1961. 3. COHEN,S. S., and B@uuiza, H. D. Studies on Unbalanced Growth in Eaclierichia coli. Proc. Nat. Acad. Sd., 40:885— 98,1954. 4. . Studies on the Induction of Thymine Deficiency and on Effects of Thymine and Thymidine Analogues in Escherkhia coli. J. Bacteriol., 71:588—97,1956. 5. DONIACH, I., and Psac, S. R. Autoradiograph Technique. Brit. J. Radiol., 23: 184—92, 1950. 6. DOTTDNEY, C. 0. Recovery of Deoxyribonucleic Acid Syn thesis in Bacteria after Amino Acid Starvation. Biochem. Biophys. Res. Comm., 5:405—9, 1961. 7. EAGLE, H. Nutrition Needs of Mammalian Cells in Tissue Culture. Science 122:501—4, 1955. 8. KISSANE, J. M., and RoBINs, E. The Fluorometric Meas urement of Deoxyribonucleic Acid in Animal Tissues, with Special Reference to the Central Nervous System. J. BioL Chem., 233: 184—88, 1958. dens should be regarded as a focalized phenomenon. 9. MUELLER,G. C. ; GonsKI, J. ; and AIZAWA,Y. The Role Accordingly, it is suggested that during the ac celeration of DNA synthesis more and more mac tive loci are progressively converted to a state 10. NATHANS, D. and LIPMANN, F. AminoAcid Transfer from competent is dependent for DNA synthesis by a process on protein synthesis. In the which case of the synchronized cells of these experiments, a fraction of the DNA is already in the competent state at the time of the thymidine rescue. Studies of Doudney (6) on DNA synthesis in amino acid-starved bacteria similarly point to a requirement for protein synthesis in the replica tion of E. coli DNA. Their work also suggests a re quirement for RNA synthesis. Similarly, Billen (1) has obtained data which suggest a stoichiometric relationship between DNA and protein synthesis. The present observations that the total amount of DNA synthesized in the presence of puromycin tends to plateau at different levels depending on the time at which the puromycin was added rela tive to the acceleration of DNA synthesis is com patible with the expressed concept. of Protein Synthesis in Early Estrogen Nat. Acad. Sci., 47:164—69, 1961. Action. Proc. Aminoacyl-ribonucleic Acids to Protein on Ribosomes of Eseherichia cdi. Proc. Nat. Acad. Sd., 47:497—504, 1961. 11. OYAMA,V. I., and EAGLE, H. Measurement of Cell Growth in Tissue Culture with a Phenol Reagent (Folin-Ciocal teau). Proc. Soc. Exper. Biol. & Med., 91:805—7, 1956. 12. RUECKERT, R. R., and MUELLER, G. C. Studies on Unbal anced Growth in Tissue Culture. I. Induction and Come quences of Thymidine Deficiency. Cancer Research, 20: 1584—91, 1960. 13. STUBBLEFIELD, E., and MuEu@zn, G. C. Molecular Events in the Reproduction of Animal Cells. II. The Focalized Synthesis of DNA in the Chromosomes of HeLa Cells. Cancer Research, 22: 1091-99, 1962. 14. Swms, H. E., and P@nxun, R. F. Effect of Pluronic P68 on Growth of Fibroblasts in Suspensionon Rotary Shaker. Proc. Soc. Exper. BioL & Med., 103:252-54, 1960. 15. TAKEDA, Y.; HAYASHI, S.; NAKAGAWA, H.; and Suzuxi, F. The Effect of Puromycin on RibonucleicAcid and Protein Synthesis. J. Biochem., 48: 169-77, 1960. 16. Y@uu&owisxy,M. B., and DRi@HABA,G. L. Inhibition by Puromycin of Amino Acid Incorporation Proc. Nat. Acad. Sci., 46: 1721—29,1959. into Protein. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research. Molecular Events in the Reproduction of Animal Cells: I. The Effect of Puromycin on the Duplication of DNA Gerald C. Mueller, Kazuto Kajiwara, Elton Stubblefield, et al. Cancer Res 1962;22:1084-1090. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/22/9/1084 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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