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Nucleoside triphosphate pools in cultures of Escherichia coli. Mychajlowska, Lydia 1970

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NUCLEOSIDE TRIPHOSPHATE POOLS IN CULTURES OF ESCHERICHIA COL I by LYDIA MYCHAJLOWSKA B.Sc. (Hons. Microbiology) McGill University, 1968 A thesis submitted in partial fulfilment of the requirement for the degree of Master of Science in the Department of Microb iology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada i i ABSTRACT Nucleotide pools in a' synchronized culture of Escherichia coli B/r/1 osci1 late as a function of age. Purine nucleotides showed a gradual increase from zero age to the time of subsequent division, with a maximal 50% increase immediately prior to division. In contrast, pyrimidine nucleotides underwent a diamatic increase of about 50% in the first half of.the generation cycle, declining at a time coincident with the termination of a round of DNA replication. A second'50 - 70% increase started at the time of the onset of DNA replication and continued'towards cell division, as did the purine. The fluctuation of pyrimidines between zero age and the middle of the division cycle suggests a functional relationship between pyrimidine pool fluctuations and the regulation of DNA replicat ion. Nucleotide pools decrease immediately in the presence of chloramphenicol to 10% of the control concentrations, and overshoot 50 - 70% in restoration of protein synthesis. Feedback inhibition of carbomyl phosphate synthesis (which is required for pyrimidine biosynthesis) by excess arginine may explain the fluctuations of nucleotide pools in the presence of chloramphenicol. Immediate depletion of nucleotide pools could be du to a very rapid turnover of nucleotide biosynthetic enzymes. The depletion of precursor pools, may explain the inability of a cell to reinitiate DNA replication in the absence of protein synthesis. In a comparison experiment, however, nucleotide pools in a temperature-sensitive initiator mutant were seen to accumulate at non-permissive temp-erature. In this case, protein synthesis occurred but initiation of DNA synthesis could not take place. This confirms the current hypothesis that a functional initiator protein is required for reinitation. Nucleotide pools in the presence of nalidixic acid dropped slightly and although no DNA synthesis occurred, pools showed no accumulation. This suggested a secondary effect of the inhibitor. Experiments prior to the pool analyses showed the importance of balanced'growth in such studies. The cell size distribution was seen to be a more valid criterion than exponen-tial increase in numbers. Exposure to cold temperatures was seen to upset balanced growth for at least one generation. TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 A. N u c l e o t i d e B i o s y n t h e s i s 3 P y r i m i d i n e B i o s y n t h e s i s 3 P u r i n e B i o s y n t h e s i s . 5 R e d u c t i o n t o D e o x y r i b o n u c l e o t i d e s 6 TTP B i o s y n t h e s i s 8 B. DNA R e p l i c a t i o n and R e g u l a t i o n 9 DNA p o l y m e r i z a t i o n 9 I n i t i a t i o n and C o m p l e t i o n of DNA Rounds o f Repl i c a t i o n . 10 C. C e l l D i v i s i o n 13 D. N u c l e o t i d e B i o s y n t h e t i c Enzymes i n the C e l l C y c l e . 14 MATERIALS AND METHODS 16 B a c t e r i a l s t r a i n s and c u l t u r e methods 16 C h e m i c a l s . . . . . 17 S y n c h r o n i z a t i o n o f c e l l s 17 Measurement o f growth 18 D e t e r m i n a t i o n o f a c i d - s o l u b l e n u c l e o s i d e t r i p h o s p h a t e p o o l s 18 V Table of Contents (Continued) Page A. Preparation of samples . . . 18 B. Chromatography 19 C. Autoradiography 20 DNA Pulse label 1 ing 21 RESULTS 22 Growth curves in low phosphate concentrations 22 32 Determination of specific activity Pi/PO^ 23 Levels of nucleotide pools in exponential cells. . . . . 28 Concentration of synchronized cells 29 Nucleoside triphosphates as a function of age. . . . . 37 Nucleotide pools in absence of protein synthesis . . . hh Nucleotide pools in an initiator mutant hS Inhibition of DNA synthesis by nalidixic acid. . . . . 52 DISCUSSION 55 LITERATURE CITED. 65 LIST OF FIGURES V I Page F igure 1. Figure 2. Figure 3-Figure k. Figure 5. Figure 6. Figure 7. Figure 8. Fjgure 9. Figure 10a. b. Figure 11. Figure 12. Figure 13. Biosynthetic pathways of pyrimidines and a rg i n i ne k Effect of varying concentrations of phosphate on E_. col i B/r/1. 2k Shift in size distribution of E_. col i B/r/1 growing ih 2 x 10~5M P O ^ 25 Effect of varying specific activities on E_. col i B/r/1 . 27 Nucleotide pool levels in exponential cultures maintained in a turbidostat. 31 Synchronized cell growth of E_. col i B/r/1. 33 DNA 3H-thymidine pulse of E. coli B/r/1 maintained in a turbidostat after cold concentration treatment. 35 3 H-thymidine incorporation during synchronous growth of E_. col i B/r/1. 36 Synchronous growth of E.. col ' B/r/1 , and mass increase as a function of age 38 Size distribution patterns of E_. coli B/r/1 during a cell division cycle 39 Ribopurine triphosphates as a function of age in synchronous coli B/r/1 . kQ Ribopyrimidine triphosphates as a function of age in synchronous E^ . col i B/r/1. kl Deoxyribopurine triphosphates as a function of age in synchronous E. col? B/r/1. k2 List of Tables (Continued) Figure ]k. Deoxypyrimidine triphosphates as a function of age in synchronous E_. col i B/r/1. Figure 15. Growth curves of E_. col i B/r/1 during chloramphenicol inhibition Figure 16. Fluctuations of dCTP and UTP during chloramphenicol inhibition and removal. Figure 17. Growth curves of E_. coli CRT83. Figure 18. Fluctuations in ribopyrimidine triphosphates and deoxyribopyrimidine triphosphates in E_. col ? CRT83 at permissive and non-permissive temperatures. Figure 19. Fluctuations in ribopurine triphosphates and deoxyribopurine triphosphates in E_. col i CRT83 at permissive and non-permissive temp-eratures. Figure 20. Nucleoside triphosphate pool levels in col i B/r/1 with nalidixic acid i nh i bi t ion. LIST OF TABLES Effect of nucleoside triphosphates on the activity of ribonucleosTde diphosphate reductase of E. co l i . Nucleoside triphosphate pool levels in exponentially growing E_. coH. B/r/1. Nucleoside triphosphate pool fluctuations in E_. col i B/r/1 in the absence of protein synthesis dues to chloramphenicol inhibition;; i x ABBREVIATIONS USED DNA . . . . . . . . . . . . . .Deoxyr ibonucleic acid RNA Ribonucleic acid IMP Inosine monophosphate ATP Adenosine triphosphate ' GTP Guanosihe triphosphate UTP Uridine triphosphate CTP Cytidine triphosphate dATP . .deoxyadenosirie triphosphate dGTP deoxyguanos ine triphosphate dCTP deoxycytidine triphosphate TTP deoxythymidine triphosphate dUMP deoxyuridine monophosphate CAM ..chloramphenicol PEI . . .poly(ethyleneLimine) TCA trichloroacetic acid X ACKNOWLEDGEMENTS I extend sincere thanks and appreciation to Dr. D. Joseph Clark for excellent supervision of the research, and for the encouragement and constructive criticizm offered in the prepara-tion of this thesis. I would like to thank Dr; Jim Hudson for editing the thesis and for his constructive remarks. Gratitude is extended to Dr. Jonathan Gallant for helpful discussions at the onset of the project; to Dr. G.A. 0'Donovan for the preprint of his review manuscript; and to Clayton R. Bagwell and George Khachatourians for helpful discussions and experimental assistance during the course of the project. Lastly, I thank Miss Jeanette Bellamy for the typing of the thesis. INTRODUCTION tn a biosynthetic system, it is reasonable to assume a regulatory relationship between precursors and end products. In studies of DNA, regulation is understood in a functional or constructive manner such as the process of polymerization, the attachment of DNA to the membrane and the necessity of protein synthesis -for continual replication. The mechanism for initiation has not yet been fully elucidated. It is not unreasonable to suppose that initiation of DNA replication could be associated with fluctuations of nucleotide triphosphate pools. Since a dependence of cell division on DNA replication has been established (Clark and Maaloe, 1967), a better understanding of DNA regulation might serve to further elucidate the control of cell division. Fluctuations of enzyme activities for nucleotide biosynthesis have been examined in animal and plant cell cultures (Johnson, 1966; Turner, I968) . There is convincing evidence of definite correlations of increased enzyme activity and initiation of DNA synthesis, and corresponding declines at the end of the replicating period (Cleaver, 1967)- In general, most enzymes are synthesized discontinuously at periods in the cell cycle which are characteristic for each enzyme (Mitchison, 1969) - Such patterns suggest an ordered sequence of synthesis within a cell cycle, which in turn 2 d e n o t e c o n t i n u o u s c h a n g e i n c h e m i c a l c o m p o s i t i o n o f t h e c e l l . T h i s t y p e o f d i s c o n t i n u o u s enzyme a c t i v i t y h a s b e e n w i t n e s s e d i n n u c l e o t i d e s y n t h e t i c e n z y m e s ( L i t e r a t u r e r e v i e w ) . The p u r p o s e f o r u n d e r t a k i n g t h i s p r o j e c t h a s b e e n t w o - f o l d : (1) t o a s s e s s n u c l e o s i d e t r i p h o s p h a t e l e v e l s a s a f u n c t i o n o f a g e , s i n c e c l a i m s o f no f l u c t u a t i o n s h a v e been made b u t p u b l i s h e d d a t a i s l a c k i n g ; and (2) t o r e l a t e t h e c h a n g e s i n p o o l l e v e l s i f o b s e r v e d , t o t h e r e g u l a t i o n o f DNA r e p l i c a t i o n a n d s u b s e q u e n t l y t o t h e r e g u l a t i o n o f c e l l d i v i s i o n . 3 LITERATURE REVIEW A. Nucleotide Biosynthesis Most of our knowledge on the regulatory mechanisms of nucleotide biosynthesis has been derived from studies of the enzymes involved. Two excel lent reviews are available defining the complexities of their regulationf(0'Donovan, 1970; Blakeley and Vitols, 19&8). Pyrimidine Biosynthesis Carbamyl phosphate is a precursor not only of'UTP and CTP but arginine as well. Carbamyl phosphate synthetase is an allosteric enzyme which is activated by ornithine and the purine IMP, and feedback inhibited by UMP. Various intermediates of both the p/rimidine and arginine pathways modulate the enzyme's activity so that there appears to be a critical ornithine UMP ratio within the cell (Figure 1). The lack of any regulation in the arginine pathway immediately following the uti1ization of carbamyl phosphate and the presence of rigid regulation in the pyrimidine pathway between carbamyl phosphate and carbamyl aspartate, results in preferential utilization of carbamyl phosphate for the synthesis of pyrimidines. Arginine synthesis occurs only if there is excess carbamyl phosphate for the synthesis of pyrimidines. Biosynthetic pattern of pyrimidines and arginine. This data is from O'Donovan, 1970, Review. Legend > Activation Inhibition -d|>R e press ion ATP Glutamine HCCU J U L Aspartate Carbamyl Phosphate ^Carbamyl ^ Aspartate v. UMP. JSJ UTRjt >CTP Glutamine N-Acetyl T/ Glutamate Ornithine Citrul l ine-*-* Arginine i i Ornithine, being the positive allosteric effector however, com-pensates for this inequality. CTP synthetase catalyzes the conversion of UTP to CTP. Product and substrate act as competitive inhibitors ,for the same site; an accumulation of one causes feedback inhibition of its own synthesis, resulting in an increase in the accumulation of the other. CTP synthetase is activated when either UTP or ATP is in excess. Since both these nucleotides have an affinity for the same sites, at a high concentration of one, the nucleotide will saturate all four sites (two per subunit), creating an active conformation of CTP synthetase. This results in an increase of CTP production. In low concentrations of an amino source (glutamine), CTP can behave as activator for its own synthesis. The regulation of CTP synthetase has been constructed from in vitro experiments, thus it is diff icult to assign a regulation pattern to the in vivo system. It must be recognized that they may not be the same: Purine Biosynthesis The de novo pathway of the purine ribonucleotide starts with an allosteric enzyme, phosphoribosylpyrophosphate amtdotransferase which is inhibited by IMP and AMP. IMP may be the main regulatory substance ih purine biosynthesis, analogous to CTP of the pyrimidine pathway. The other enzymes in purine biosynthesis do not appear to be controlled by feedback inhibition, though several such as IMP 6 transformylase seem to be sensitive to end-product repression. WHile purines (IMP and AMP) exhibit a regulatory control over pyrimidine biosynthesis, perhaps to maintain a balance between purines and pyrimidines, the reverse has not been observed. Reduction to Deoxyribonucleotides In the biosynthesis of deosyribonucleotides, the reduction of ribonucleotides to deoxyribonucleotides is catalyzed by ribonucleoside diphosphate reductase. The factors influencing it are so complex, howeVer, that predictions about in vivo regulation from the in vitro results are uncertain. The enzyme catalyzes the reduction of all four ribonucleoside diphosphates. The specificity of the enzyme for a substrate is determined by nucleoside triphosphates as shown ih Table I. The enzyme is also subject to derepression by thymine starvation, which results in an immediate increase iri the dATP pool, and a sub-sequent increase in the dCTP pool. Addition of TMP to a thymine pro-totroph results in an increase iri the thymidine nucleotide pool, a decrease in both the uracil and cytosine nucleotide pools, arid no significant change in the purine nucleotide pools. This type of evidence implicates TTP in an end-product control mechanism (Cannon and Breitman, 1967, 1968; Neuhard, 1966, 1966). Curiously, the affinity of ribonucleoside diphosphate reductase is 10-fold lower for UDP than for CDP, GDP, and ADP. This is, however, 7 Table I. Effect of nucleoside triphosphates on the activity of ribonucleoside diphosphate reductase of Escherichia coli . Nucleotide Catalytic activity Specificity for base ATP(2 x 10~J) dATP(lO~6) dATPClO-4) dGTP(lO~5) dTTPQO -5) st imulation stimulation inhibition st imulat ion st imulation pyrimid ines pyrimid ines purines and pyrimidines pur ines purines and pyrimidines dATP(10 ) S ATP(2 x 10~3) dATP(lO~6) & dTTP(lO_2t) dATP(lO~h & dGTP(lO-i,X ATP(10" ) S dTTP(5 x 10 ) st imulation inhibition inhibition i nhi bi t ion pyr imid ines purines and pyrimidines purines and pyrimidines purines and pyrimidines The data are from Brown and Reichard (1969). The concentrations given are those at which the effect is fully developed. 8 compensated for by one, and perhaps two.pathways in the biosynthesis of TTP, the nucleotide unique to DNA. TTP Biosynthesis Thymidylate synthetase catalyzes the conversion of dUMP to TTP, the dUMP usually being a product of the reduction of UDP. In studies of thymine mutants under conditions of thymine limitation, a dCTP deaminase pathway has been found which does not involve ribose uracil intermediates, but instead allows the production of dUTP which is subsequently converted to dUMP, and to TMP. The enzyme is sensitive to TTP inhibition. In addition, there is mounting evidence for the existence of a dCTP methylating enzyme which permits the formation of TTP without'the use of thymidylate synthetase. Forster and Hoildorf ( 1 9 6 5 ) have reported two classes of phenotypically similar thymine mutants, one lacking thymidylate synthetase, but maintaining dCTP methylating activity, the other with reverse characteristics. In addition, cytidine deaminase negative mutants of !E_. coli B derived 75 - 80% of their TMP from the dCTP pool without equilibration with the uridine nucleotide pools (Karlstrom and Larsson, 1 9 6 7 ) . The genetics and regulation of TTP production are currently being studied, but the general implication is that TTP, being unique to DNA, regulates all DNA precursor synthesis, and that its own production is safeguarded by the possible conversion from other biosynthetic path-ways, either via thymidylate synthetase from dUMP and UDP, or via 9 thymidylate synthetase and cytidine deaminase from CdR to UdR to dUMP, or via dCTP methylating enzyme from dCTP to TTP. B. DNA Replication and Regulation DNA Polymerization According to current theory, DNA is replicated by DNA polymerase which catalyzes the addition of nucleotide triphosphates to the 3 I _ hydroxyl end of the growing polypeptide chain (Kornberg, 1 9 6 1 ) . However, replication of DNA is both'sequential and unidirectional, and chain growth occurs from the 3'-hydroxy1 to the 5 ' end of the adjacent nucleotide, in each chain. Okazaki et_ aj_. (1968) demonstrated the synthesis of DNA in short segments which are later joined together into a continuous structure. Short radioactive thymidine pulses and subsequent isolation of DNA revealed label in pieces of DNA about' 1000 nucleotides long, which upon further incubation were found in high molecular weight DNA. The joining enzyme was found to be a polynucleotide 1igase (Fareed, 1 9 6 7 ; Weiss^ 1967) since temperature-sensitive mutants of this enzyme produced sjiort/segments of DNA, until restored to permissive conditions. Recently, Smith e_t^  aj_. , Knippers and Strati ing, Boyle et_ aJL (1970) have demonstrated an in vitro DNA- membrane complex free of DNA poly-merase, which synthesizes DNA. Working with cell-free membrane complexes imbedded in agar, and washed free of DNA polymerase, or with DNA polymerase-negative mutants, the system demonstrated seroi-conservattve replication at a rate comparable to that of in vivo replication. Although present efforts are directed at purifying the crude extract and further elucidating its mode of action, the system stands in support of recent theories of a correlation between the membrane and the DNA replicating apparatus of the ce l l . If does however, oppose the function assigned to Kornberg's DNA polymerase, whose in vitro action results in a distorted DNA produced at a rate several orders of magnitude below that of in vivo replication. , This group of workers' (1970) have contributed to the accumulating evidence suggesting a repair function for the DNA polymerase. Initiation and Completion of DNA Rounds of Replication The DNA replication process was found to be subject to a very complex regulatory mechanism. Maal«5e and Hanawalt (1961) proposed a relationship between protein synthesis and DNA replication. High concentrations of chloramphenicol (150 ug/ml) or amino acid starvation allowed only a limited synthesis of DNA. In 1 9 6 8 , Bird and Lark proved that amino acid starvation allows completion of a round of replication but inhibits initiation until amino acids are restored. Transferring a succinate culture of E_. col i 15T- to a similar medium with thymine, but lacking amino acids, cells were allowed to replicate DNA for 100 minutes. Amino acids and ^H-thymine were then added simultaneously and the culture was incubated for 28 minutes. Cells were again starved 11 for amino acids in the presence of unlabel led thymine and after kS' minutes, . C-thymine was added for the remainder of the starvation period. The culture was then shifted to a complete, non-radioactive medium and pulsed with a density label to trace the labels in a complete 3 round of replication.- The H-thymine was found only at the beginning of the cycle, incorporated after the first starvation period iri the presence of amino acids. This demonstrated the necessity of protein 14 synthesis for DNA replication. The, C label.was found at the end of the second starvation period, thus indicating the ability of a cell to complete a round of replication during amino acid starvation, but rot to initiate a new orie. Measuring residual DNA synthesis of synchronized cells at various ages; in the presence of chloramphenicol, Clark and MaalfSe (1967) found that cells at later stages of replication synthesized less DNA than cells just starting rounds. This suggested that protein synthesis was essential for initiation of new rounds. Lark and Renger (1969) in experiments involving inhibition and restora-tion of protein synthesis, showed that the cell aquires the potential to re-initiate DNA replication almost 15 minutes before re-initiation actually occurs. This potential was seen to be amino acid requiring and chloramphicol resistant (25ug/ml) and was necessarily preceeded by a chloramphenicol sensitive synthetic step 30 minutes prior to re-initiation. Both products of these reactions regulate initiation stoichiometrically and thus potential for initiation is not available until both processes are completed. A third process, the lag between potential for initiation and actual initiation is presently being stud ied. If DNA synthesis is inhibited but protein synthesis continues new growing points will be initiated upon removal of the DNA inhibitor. Presumably, protein synthesis must continue during the DNA inhibition to allow accumulation of initiator protein, (Helmstetter and Cooper, 1 9 6 8 ) , and deoxyribonucleoside triphosphates for initiation of multiple growing points. Rasmussen ( 1 9 6 4 ) , working with a thymine negative mutant, observeda thymine incorporation pattern which he could not explain at the time. Restoring a high thymine concentration after partial starvation, the rate of DNA synthesis increased several fold that of the control until it reached the level of the control reaction. It is now evident that an accumulation of initiator protein occurred at low thymine concentration, and that upon restoration of sufficient thymine, multiple growing points were initiated to catch up to control level! Multiple growing points also explain a AO min DNA polymerase travel time in an enriched culture with a 20 min generation time (Yoshikawa et^  aj_. 1964; Clark, 1968; and Helmstetter and Cooper, 1968) . The control of DNA synthesis has been extensively studied with the aid of temperature-sensitive DNA mutants, metabolic inhibitors, phage DNA and episomal replication. These topics have recently been reviewed in depth by Lark ( 1 9 6 9 ) . C. Cell Division In most cell systems, cell division cannot take place until a round of DNA replication Is terminated, thus ensuring a complete set of DNA material to each daughter cell Clark (1967) established, and Helmstetter (1967) confirmed that a necessary 20 minute interval occurs between the end of a round of replication and eel 1 divis ion, and that the signal for cell division is given by the end of a round of DNA replication. Thus in a kr minute generation, by 25 minutes the cell contains two complete genomes, and concurrently has synthesized the initiator protein. Additional findings showed the presence of a 10 minute interval between end of round of DNA replication and cell division in glycerol, succinate and acetate grown cells. Helmstetter and Cooper (1968) desagree with the results of Lark (1966) and Clark and Maaltfe (1967) as to the exact timing of the replication terminus in E_. col i growing in poor carbon sources, although they agree about the 20 minute period in glucose grown cells. In 1968, Clark offered convincing evidence by inhibition of DNA synthesis in synchronous' cultures, that the end of a round of DNA replication triggers cell division, and that the subsequent eel 1 division cycle was independent of the start of a new round of DNA replication. The blocking of DNA synthesis by thymine starvation or Tri temperature sensitive DNA synthesis mutants supported this conclusion in that the amount of residual division correlated with the amount of completed chromosome' (Fangman and Novick, 1968; Inouye, 1 9 6 9 ) . D. Nucleotide Biosynthetic Enzymes in the Cell Cycle The extensive study of DNA replication patterns have-not been extended to a study of DNA precursors during a growth cycle. Numerous assumptions have been made on the lack of nucleotide fluctuations during a growth cycle without actual presentation of published data. Johnson and Schmidt (1966) witnessed periodic changes ih thymidylate kinase activity in synchronous cultures of Chlorella pyrenoidosa which reached maximal activity prior to the maximal level of DNA synthesis. Similar results have been found' with nucleotide biosynthetie enzymes of various mammalian tissues. Turner et ?a1 . (1968) , working with L cells, found a strict paral-lelism between ribonucleotide reductase levels and the portion of cells replicating DNA. The enzyme was seen to lose activity rapidly to undetectable levels after the DNA synthetic period ended. Inhibition of protein synthesis exhibited rapid decay of the enzyme. In Chinese hamster fibroblast cells (Stubblefield and Murphree, 1967), thymidine kinase levels increased continuously shortly after DNA replication and decreased at or shortly after cell division. Eker (1965) suggested that the fluctuations of enzymes involved in deoxythymidine triphosphate formation may play an important role in the control of DNA synthesis. Cleaver (1967) in an excellent account of thymidine metabolism, describes the increase of thymidine enzyme activities at the onset of DNA replication and a decrease ih activity 15 as DNA synthesis ceases. Cleaver formulates the idea that the close relationship of these enzymes and DNA synthesis may be at the level of control of initiation. MATERIALS AND METHODS Bacterial strains and culture methods The bacterium used for this study was Escherichia coli B/r/1, (ATCC 1 2 4 0 7 ) , a prototroph, used for the synchronous culture technique since it does not form chains. A temperature-sensitive initiator mutant, Escherichia col? CRT83, was also used (Kohiyama, 1 9 6 8 ) . It is unable to synthesize DNA or divide at the non-permissive temperature and requires thymine, deoxyadenosine, iso-leucine, valine, leucine, and Vitamin By E_. col i B/r/1 was maintained on nutrient agar slants and in liquid cultures of 0 . 0 5 M TRIS-glucose mineral salts media. A growth medium that was.low in phosphate was used to provide 3 2 efficient incorporation of Pi into the bacteria. The media had the following composition: 0 . 0 5 M TRIS (trishydroxymethyl-_c _Zi -7 amino methane); 2 x 1 0 PM ^HPO^; 1 x 1 0 M CaCl 2; 2 x 1 0 JM MgC12; 3 x 10~6M F e C l ^ ^ O ; 5 . 2 x 1 0 _ 2 M NaCl; 3 x 10~2M (NH^SO^ and 2% carbon source (glucose or acetate); pH was adjusted to 7 . 5 with 6N HC1. Cultures were incubated at 3 7 C in a New Brunswick Gyrotory Shaker for volumes of 1 l i ter , and in a New Brunswick Metabolyte Water Bath Shaker for volumes of 1 0 - 1 5 mis. C hemica1s Pi (orthophosphate-free) was obtained from Tracer Lab; H thymidine from Schwartz Biochemical Research; chloramphenicol from Sigma; nalidixic acid from Sterling Winthrop Research; unlabel led marker nucleotides from Calbiochem; PEI (polyethylineimine) from Chemirad Corp.; and Cellulose MN 300HR from Canlab. Synchronization of cells. Synchronous cu1tures of bacteria were obtained by a method developed by Helmstetter and Cummings Cl965)- This method employed an 0.22u Millipore filter* 16 cm ih diameter., through which about 300 ml exponentially growing cells (2 x 10 /^ml) were passed under 2 approximately 5 lbs./in. pressure. The membrane allowed binding since size of pores was smaller than that of cel ls. The membrane was inverted and clamped into position ih a 37 C incubator. Conditioned media (fi1trate:from exponential culture) was allowed to pass through at 10 mls/min. Bound cells grew and divided on the surface; one daughter cell was released with the effluent while the other remained bound; A sample, which was collected over a 2 or 3 minute period exhibited the step-wise growth pattern of a synchronous culture. In the case of a culture growing on a poor carbon source (acetate) the membrane had been boiled three times In distil led water to remove the glycerol from the surface and thus prevent a shift-up condition. Measurement of growth Cells were counted with a Coulter Counter (Nuclear Data, Palatine, 111.) and size distribution was shown by an attached Nuclear Data (model 2200-512) channel pulse-height analyzer. Cells passed through a 30u orifice via constant pressure for 10 seconds. Determination of acid-soluble nucleoside triphosphate pools A. Preparation of samples 32 In all Pi experiments, samples were not removed earlier than 30 m|n. (0.75 generation) after addition of isotope to medium; complete equilibration of the triphosphate pools and 32 exogenous Pi was attained within about half a generation, as 32 judged by a constant ratio of Pi in any nucleoside triphosphate to number of exponential cells. In addition, a 20 yl TCA-precipitate of the labelled culture was always counted to ensure that a minimum of 25,000 cpm were present, showing sufficient isotope incorporation,for statistical results. This ensured a minimal amount of radioactive label in the very low concentrations 19 of deoxyribonucleotide pools. To measure the triphosphate pools, 250ul samples were acidified with lOOul of 2N formic acid containing unlabel led carrier cel ls. After extensive mixing with a Vortex, the samples were left in an ice bath for a minimum of 15 minutes to allow complete lysis of cells. B. Chromatography After centrifugation in a Beckman Microfuge, the super-natant from sample described above was spotted on a thin layer of PEl-impregnated cellulose. These high an ion-exchanger plates were prepared by the procedure of Randerath and Randerath (1967) and used within a week for quantitative results. The sample was added slowly enough to avoid flooding the surface of the plate, but no precaution was taken to restrict the size of the moistened area, as the nucleotides rapidly absorb to a small central spot of the ion exchange resin (Randerath and Randerath, 1967)- About 20 umoles of each carrier deoxy- and ribo-nucleotide were added at the point of origin. Prior to running the sample in each direction,.the plate was subjected to a 20 min anhydrous methanol wash, to remove the salts of the medium. Failure to do so, results in extensive streaking of nucleoside triphosphates. Plates were chromatographed, following the method of Irr and Gallant (1969). To ensure more distinct separation of deoxynucleotides from their corresponding ribonucleotides, wicks were made out of Whatman No. 3 paper. Thus in the first direction, the IN Acetate; 1M LiCl , (1/1)(v/v) solvent was run 3/4 of the distance up the plate and immediately immersed in the IN Acetate; 1.5M LiCl (l/l)(v/v) solvent and allowed to run in the same direction for 5 1/2 hours with the attached wick. After drying and a methanol wash to remove the LiCl of the f irst solvent, the plate was run in the second direction in 3M NH^Acetate +4.3% Borate, pH 7 for 4 1/2 hours, again with a fresh wick. Dried plates were then ready for autoradiography. C. Autoradiography Royal blue medical X-ray films (Kodak) were placed over the plates which were then stored for 3 days in light-proof Kodak X-ray exposure holders, between layers of lead sheets. The films were developed for 3 1/2 minutes in Kodak X-ray devel-oper, rinsed rapidly in water and fixed for 10 minutes in Kodak X-ray fixer. The films were then rinsed in cold running water and dried. Autoradiograms were superimposed over their corresponding chromatog rams, and each exposed spot was punched through the film onto the plate, the marked area then scraped off and put into scintillation vials. Autoradiography was found essential since it was easier to distinguish borders between spots of quantitatively different labelling than between U.V. spots of equal intensity. Counts per minute were corrected for isotope decay, counting efficiency, volume spotted, conversion factor for yCi to dpm, specif 32 activity, and rate of mass increase to give mumoles Pi per ml per nucleotide. DNA pulse label 1ing Thymidine incorporation was measured by pulse labelling lml. samples of cultures with H thymidine (lOuCi/0.5 ug/ml) for 5 minutes. The reaction was stopped with 3 volumes of cold TCA, final concentration of 5%, containing 200 ug unlabel led thymidine/ml. The samples were kept in an ice bath prior to filtering onto an 0.22u Millipore f i l ter , which was rinsed with 9 volumes of 5% TCA and 2 volumes of hot (90 C) disti l led water. After drying thoroughly, the samples were placed in liquid scintiallation vials containing 10 mis toluene-PPO-POPOP. (2,5-diphenyloxazole, p-Bis 2-(5-phenyl-oxazoilyl-benzene) and radioactivity was counted. RESULTS Growth curves in low phosphate concentrations The concentration of deoxy and ribonucleotide precursors in bacteria is extremely low (Randerath and Randerath, 1964). Since 32 pool levels are measured using Pi to label the nucleotides, a 32 high specific activity of Pi is required to obtain sufficient 32 labelling of the precursors. On the other hand, Pi releases gamma rays which are deleterious for the biological system. 32 Hence a balance must be maintained; a specific activity of Pi high enough to measure the pools,yet low enough to maintain the balanced growth of the culture. In all experiments, cells were allowed to grow exponential ly for at least six generations prior to the experiment. Preliminary results in our laboratory and Gallant's (personal communication) -k showed that at 10 MPO^, E_. col ? B/r/1 grew exponentially to levels 8 we11 over 1 x 10 cells/ml, the cell concentration necessary for our experiments. An exponential culture of k x 10^  cells/ml was diluted -k appropriately to yield final P0^ concentrations of 1 x 10 M, -5 -5 5 x 10 M, and 2 x 10 M. The cultures were monitored for five hours for rate of growth and size distribution. In steady state exponential growth the size distribution of a population does not fluctuate (Painter, Marr, 1968) . A shift in the size distribution of a population is indicated by a change in the peak position of the size distribution curve. This in turn reflects metabolic changes. In the measurement of very rigidly regulated metabolic precursors, such fluctuations cannot be tolerated. -k As shown in Figure 2, a PO^  concentration of 1 x 10 M main-8 tained exponential cell number increase until 7.5 x 10 cells/ml whereas a shift in the size distribution pattern occurred at g 5 x 10 cells/ml. Similar analysis was applied to cultures growing at 5 x 10~5M PO^  and 2 x 10_5M PO .^ Since 2 x 10~5M PO^  showed g no change in balanced growth until 2 x 10 cells/ml (Figure 3), this concentration of PO^  was chosen as the lower limit for subsequent experiments. Exponential increase of cell numbers continued almost o to 3 x 10° cells/ml. Figure 3 shows a shift in the size distribution of a population limited for P0^. Since magnitude of size change was not important, but rather the fact of the change itself, tedious calibrations for absolute size were not carried out. 32 Determination of specific activity Pi/P0. To establish a working level of specific activity, viable growth 32 curves were carried out with various concentrations of Pi . An exponentiallygrowing culture of 1 x 10^  cells/ml was diluted back Effect of varying concentrations of phosphate of E_. col i B/r/1 . -4 Symbols: 0 , 1 x 10 M PO^  • , 5 x 10~5M PO^  0 , 2 x 10-5M PO^  shift in size distribution Figure 3. Shift in size distribution of E_. col i B/r/1 at 2 x 10_^M PO, at 20k rain, and 257 min. into four flasks which had: a) 100 yCr32Pi/ymole PO ;^ b) 250 yC i 3 2 Pi/ ymole PO ;^ c) 500 yCi Pi/ymole P0^; d) a control. At regular time intervals 100 ul samples were withdrawn from each flasks, diluted and plated on nutrient agar plates. For each sample, a clean disposable 100 ul pipette was used. Plates were incubated 18 hours in a 30 C incubator, and viable cell numbers were counted. Figure k shows the growth rate obtained by plotting viable cells as a function 32 32 of time for cultures containing 100 yC i Pi/ymoles P0^ and 250 yCi Pi/ ymoles P0^. The doubling time (k5 minutes) is1 identical to the 32 generation time of the control. The culture containing 500 yC i Pi/ ymoles P0^ exhibited loss of viable cells after 60 minutes incubation. This experiment was repeated using optical density readings at 660 my over a 3 hour period, and results supported the viable counts. This 32 ruled out the possibility that cell death at 500 yC i Pi/ymoles P0^ occurred through secondary radiation effects only after several 32 hours of incubation oh the plates. Thus 250 yCi Pi/ymoles P0^ 32 became the upper limit of specific activity, while 100 yCi Pi/ymoles P0 was chosen as the safe working level. 32 In an effort'to alleviate the suicide effects of Pi , similar 33 32 experiments were attempted with Pi instead of Pi. This isotope 32 has a lower energy level (0.25 Mev cf.1.7 Mev for Pi), and a longer half - l i fe (25 days cf. 14.3 days for 3 2 Pi ) (New England Nuclear). This isotope is presumably safer to handle and would allow longer 33 term experiments. Viability experiments with Pi at the same Figure A. Effect of varying specific activities Pi/PO on E_. co 1 i. B/r/1. Symbols: • , control 0 , 100 yCi32Pi/umole PO^  © , 250 uCi 3 2 Pi/ umole PO^  0 , 500 yCi32Pi/umole P0, V i a b l e ce l l number x 10 J 1 — l — i — i i i i i 1 — i i i i i • i . specific activities used for Pi showed surprising results in that cells at 100 uCi JPi/umole PO^  showed loss of viability after only a few minutes, and cells at higher specific activities showed no viability at a l l . We can only attribute this result to secondary radiation effects of JJPi. Levels of nucleotide pools in exponential cells Prior to analyzing nucleotide pools in synchronous cultures, control levels had to be determined in exponentially growing cultures. In order to obtain the greatest incorporation of label, compatible with cell survival over a generation, a manual turbidostat was set up which ensured a high constant cell number and continual replenishment and depletion of radioactive medium at a rate identical to the maximal growth rate. Calculations for the turbidostat were based on the following equations describing growth in continuous cultures; (1) ^ = kx - Dx In a steady state: (2) 5 f = 0 and k = D dt (k = specific growth rate constant; x = cell number; t = time; D = f_ ; f = flow rate in mis/hour; v = volume in mis.- D = dilution v dilution rate). In a turbidostat, the dilution rate must equal the maximum growth rate, thus determining the k and choosing the desired flow rate, the functional volume of the turbidostat can be calculated. - ^ U k , max t 2 (ti = generation time) Thus, at a 45 minute generation time, and a desired flow rate of 15 mis./hour, km a v = - 6 9 = .91 = ,f>=. 15 mls/hr. max ——— , r— (—) : : .75 hr. hr. v volume therefore, v = 17.5 mis. g In this manner, an exponential culture was maintained at 1 x 10 . 32 cells/ml, containing 100 uCi Pi/umole PO .^ After a 30 minute equilibration period, samples were withdrawn every 30 minutes over a duration of 2 hours and analyzed as described before. A balanced steady state was evidenced by radioactive cpm per nucleotide which were almost identical in all samples. The exponential pool levels are listed in Table II, and represented in Figure 5 by TTP and UTP. Concentration of synchronized cells Following the synchronization technique of Helmstetter and Cooper (1965), about 2 - 5 x 10^  cells/ml were obtained in the effluent, at a medium flow rate of 10 mls/min. Since such low cell 32 numbers would not incorporate detectable amounts of.. Pi , a necessary 30 Table II. Nucleoside triphosphate pool levels in exponentially growing E. coli B/r/1. 32 Nucleoside triphosphate mumoles Pi/nucleotide/ 1 x 108 cells/ml GTP 0.212 ATP 0.559 CTP 0.151 UTP 0.153 dGTP 0.022 dATP 0.0H8 dCTP 0.020 TTP 0.019 Figure 5. Nucleotide pool levels in exponential cultures maintained in a turbidostat. 20 30 40 50 6 0 M inutes concentration of the cells was attempted. The desired concentration 8 was 1 x 10 cells/ml which would not be exceeded when maintained by the turbidostat technique. Two to five hundred mis of synchronized cells were collected in a flask placed in an ice-water bath. After filtering in the cold through an 0.22y Millipore f i l ter 2.5 cm in diameter, the synchronized cells were resuspended in warm medium to yield approxi-g mately 1 x 10 cells/ml. Zero time was the time of addition of warm medium. In every experiment, synchrony was excellent and remained so for at least one generation beyond a culture which was taken directly from the membrane without the cold treatment. The turbidostat technique produced a step-wise curve where constant numbers showed a decline in cell number due to dilution and doubling cell numbers at division showed an incline (Figure 6). As a control, a diluted cold sample was followed without the turbidostat method, and compared to a warm sample directly off the membrane. A 5-minute lage in division was evidenced in the cold concentrated sample. This observation suggested a change in the steady state, and a second parameter of DNA replication needed to be verified. The DNA replication cycle was chosen as an indication of balance or imbalance of growth of a synchronous culture resulting from a temperature shift. A thymidine pulse on a synchronous culture maintained at a maximum g of 1 x 10 cells/ml exhibited a definite shift in the start of rounds of replication, although the second division cycle showed no lag Synchronized cell growth of E_. col i B/r/1 . Symbols: 9 , culture directly off membrane 0 , from cold concentrated cells 0 , from cold cells maintained in turbidostat. 33 1 1 1 1 40 6 0 8 0 Ce I I a ge (mi n utes) 100 120 (compare Figure 7 and 8 ) . A DNA thymidine pulse was carried out'on a warm control culture as well (Figure 8 ) ; since cell numbers were low, the turbidostat method could not be employed. Since no concentration had occurred, cell numbers were too low to undergo further dilution by the turbidostat technique. Had it been used, it might have reduced the amount of radioisotope incorporation below that of statistical validity. To partially compensate for the low cell numbers, sampling was not begun until a division cycle had occurred, allowing a doubling of cell number and thus isotope incorporation. In this case, the mean time for start of round of replication (doubling burst) was 2 0 - 2 5 minutes prior to cell division. In the cold culture, the mean doubling time occurred about 2 7 - 3 0 minutes prior to division, and accumulation continued until almost 3 5 minutes as compared to a plateau at 2 5 min, in the warm control. The 5 minute lag in cell division and the somewhat more extended lag in DNA synthesis forced us to abondon the cold' concentration technique on the suspicion that nucleotide pools,might be unbalanced, and that fluctuations under such conditions might be strictly artefactual. Concentration of cells was then confined to decreasing the rate of flow of media over the membrane to 3 mls/min, thus effecting a 3 _ 4 - fo ld concentration. Experiments were not performed until one division cycle had occurred, thus final concentra-tion was 3 - 5 x 1 0 ^ cells/ml. Untampered metabolism had to com-pensate for lower cell number and isotope incorporation. r Figure 7 . DNA H thymidine pulse of E_. col i B/r/1 maintained in a turbidostat after cold concentration treatment. M i n u t e s Figure 8. H thymidine incorporation during synchronous growth of E. coli B/r/1 . 37 Nucleoside triphosphates as a function of age Nucleotide pool analysis was carried out on a synchronous culture obtained directly from the membrane as described above. Figure 9 shows a typical synchronous growth pattern, as well as three patterns of mass increase. Since nucleotides are expressed as concentrations, corrections were necessary on a per mass basis rather than per number. Cells were corrected for the Painter - Marr model for mass increase (Painter, Marr; 1968), in which cells immediately after cell division exhibit the slowest rate of.mass increase while those just beyond half a generation grow at the maximal rate. There is supporting evidence for this model in synchronous cell length studies of E_. col i by Adler et_ a_j_. (1969). The need for calibration to mass is shown in Figure 10 which follows size distribution of a synchronous popu-lation through one'division cycle. Figures 1 1 - 1 4 provide an account of ribonucleoside- and deoxyribonucleoside-triphosphate concentrations during a full generation of 42 minutes. Bearing in mind that the data have been corrected for mass increase, the purine triphosphate exhibited a gradual increase in concentration, reaching a maximum at 75 minutes and dropping sharply as cell division occurred (Figure 11 and 13). dATP showed an exaggerated effect but clearly demonstrated that the maximal amount of purine biosynthesis occurred between 60 and 75 minutes. In all cases, there was approximately a 50% increase in Figure 9 . Synchronous growth of E_. col i B/r/1 Symbols: 0 , Mass increase as a function of age , 1inear , exponential Painter-Marr 33 IX) O Figure 10a Figure 10b. Size distribution patterns of E_. col i B/r/1 at different times in a cell division cycle. 39a 33b Figure 1 1 . Ribopurine triphosphates as a function of age in synchronous E_. col i B/r/1 . C e l l age (m i nutes) Figure 12. Ribopyrimidtne triphosphates as a function of age in synchronous E. coli B/r/1. .2 " — i " £ .15 o * 0 CVI , ro ' 1 o. ** / Q </> <D o .05 mm >> E 0 o- U T P o . J O - 0 - . . 0 0..-0 b - 0 ' ' , . . - 0 - 0 " 0 x ..0 1 C T P JL 4 0 5 0 60 70 8 0 C e l l age (minutes) Figure 13. Deoxyribopurine triphosphates as a function of cell age in synchronous E_. col i B/r/1 . Figure ]h. Deoxyr ibopyr irhid ine tr iphosphates as a function of age in synchronous E_. col i B/r/1 . <*3 .025 .02 h \ O CM ro CL if) * .01 .015 e .005 o 40 o d C T P A , ° 0 B 0 - - ° ®N 6 \ @-T T P 50 60 70 80 C e l l ag e ( m i n u t e s ) concentration towards the time of cell division as compared to the levels at zero time (42 min). The pyrimidine nucleotides all exhibited a double peak pattern during the division cycle, between the time of cell division and termination of DNA replication, and again to the following cell division. The dedxyribose pyrimidine triphosphate concentrations showed almost parallel fluctuation patterns (Figure 14), whereas the ribopurines, CTP and UTP appeared to oscillate in opposing directions (Figure 12). The unusual kinetics observed in the pyrimidine pools posed the question of a possible relationship between nucleotide bio-synthesis and the end of a round of DNA replication. However, the fact that in a 42 minute generation starts and ends of rounds coincide, nucleotide pools were next analyzed in a system where no initiation of DNA replication occurred. Nucleotide pools in absence of protein synthesis 32 100 uCi Pi/umole P0^ were added to an exponential culture of IE. coli B/r/1 (zero time) and after 30 minutes equilibration, control samples were withdrawn and treated as described in Materials and Methods, except that final nucleotide concentrations were expressed g per 1 x 10 cells/ml, this being an exponential culture. At 39 min, the culture was spl it , one part to be maintained by the-turbi-dostat method as a control, the other given a final chloramphenicol concentration of 150 )ig/ml. The growth curve (Figure 15) shows 30% residual division after chloramphenicol addition. Within 6 minutes, each nucleotide had lost 60 - 70% of its accumulated pool, and within another 20 - 30 minutes, had reached a minimum of 10% of its control level (Figure 16). Chloramphenicol was removed at 106 minutes by washing with fresh medium over a 0.h5]X Millipore f i l ter . As evident in the drop of the growth curve (Figure 15), some eel Is adhered to the membrane and could not be resuspended,. but after a 30 minute lag, the culture continued growing at a doubling time of 20 minutes. Restoration of protein synthesis, effected an immediate increase in nucleoside triphosphate pools. By 115 minutes, a 50 - 70% overshoot occurred for each nucleotide and quickly dropped to control level. Figure 16 shows a typical pattern for deoxyribose and ribonucleoside triphosphates treated in this manner, while Table III summarizes the pool levels with 150 ug/ml chloramphenicol; immediately after removal of the inhibi-tor; and the control level. Nucleotide pools in an initiator mutant As a comparison to the chloramphenicol - inhibition experiment nucleotide pools were analyzed in E_. col i CRT 83, a temperature-sensitive initiator mutant. Figure 17 shows a typical growth curve where division ceased immediately at hi C, but when restored to the Growth curve of E_. col i B/r/1 during chloramphenicol inhibition (CAM+) and after removal of inhibition (CAM-) Symbols: 9 - 9 , control untreated turbidostat i 1 1 1 r i o x ro 8 6 3 C ^CAM* i 1 1 1 1 1 1 r ^ C A M " T 1 T— -0^0 / .<flcr° - 0 - 0 - 0 , -o ^ 0 > - o - o - o - o ^ ° 0> o 0 J 1 I I I I I ' I I I 1 20 4 0 60 80 100 120 J 1 l I L 140 160 M i n u te s Figure 16. Fluctuations of dCTP and UTP. Symbols: • , during chloramphenicol inhibition and removal. 0 , control untreated turbidostat M i n u t e s Table III. Nucleoside triphosphate pool fluctuations in E_. col ? B/r/1 in the presence of chloramphenicol. 32 rnumoles Pi incorporated Nucleos ide tr iphosphate control CAM+ at 80 min. CAM" at 115 min. GTP 0.179 0.025 0.248 ATP 0.528 0.052 0.716 CTP 0.176 0.019 0.307 UTP 0.1 44 0.011 0.178 dGTP 0.018 0.004 0.025 dATP 0.040 0.006 0.069 dCTP 0.015 0.003 0.026 TTP 0.016 0.002 0.030 Figure 17. "Growth curve of E^ . col i CRT 83 Symbols: 9 , control at 30 C. 0 , experimental culture at Ml C and 30 C. permissive temperature division continued at a rate greater than the control, sinee the cell division process has been left behind the rate of other metabolic processes. In this particular experiment, a 60 min incubation at non-permissive temperature showed a slight loss of viability characteristic of lengthy exposure to k2 C (Khachatourians, personal communication), thus, the control rate.was attained at the permissive temperature, but not control numbers. Residual DNA synthesis continued for 30 minutes (Kohiyama, 1968); no initiation of starts of rounds of replication occured until restored to the permissive temperature. Thus, like the chloramphenicol treated cel ls , starts of rounds of replication were not permitted, but in this case protein synthesis was allowed to continue. Figures 17 and 18 demonstrate DNA and RNA precursors at non-permissive.temperature, and then rapid depletion as replication and division resumed. Deoxyribonucleotides'appeared to accumulate with mass increase at non-permissive temperature whereas ribonucleotides seems to accumulate at a rate several fold greater than can be accounted for by mass increase alone. In both these experiments, it. was evident that protein biosynthesis is essential for nucleotide biosynthesis but neither indicated any regulatory pattern due to the completion of DNA synthesis. Figure 18. Fluctuations in ribopyrimidine triphosphates (0), and deoxyribopyrimidine triphosphates (t) , in E_. col i CRT 83 at permissive (30 C) and non-permissive (kl C) temperatures. 1 j h 1 1 I — ~ M i n u te s Figure 19. Fluctuations in rtbopurine triphosphate (0) , and deoxyribopurine triphosphates (•), in JL ' col ? CRT 83 at permissive (30 C) and non-permissive (42 C) temperatures. 52 00 I O to "D O <D O 13 C 0 6 h CO O 4 h 2h 0 T  1 r i r 0 30' 42' 0 \ ©( o ATP ©dATP J 1 1 I I I • « 30 p ©~ J L oGTP ©d GTP" 40 60 80 M i n u t e s 100 120 53 Inhibition of DNA synthesis by nalidixic acid Nalidixic acid (10 ug/ml) was added to a synchronized culture of E_. col? B/r/1 after 30 minutes (0.75 generation). This aborted the completion of a round of replication by immediately stopping DNA synthesis (Goss et_ al_. 1965; Boyle et_ al_. 1969). One step division occured as a result of the previously completed round of replication, but no subsequent division was possible. Nucleo-tide pools were measured after 15, 30 and k5 minutes of DNA synthesis inhibition. As Figure 19 demonstrates, nalidixic acid has caused a drop in nucleotide pools below the level of control culture. Considering control level at 33 minutes as 100%, the inhi-bited cells showed a 10 - 30% lower level than their corresponding control points, however para 1 lei changes were noted between treated and untreated cells. Figure 2 0 . Nucleoside triphosphate pool levels in £. coli B/r/1 with nalidixic acid inhibition ( 0 ) ; and control levels ( C ) . i i 1 1 1 1 r DISCUSSION The fluctuations observed in nucleotides as a function of age are in support of enzymatic studies carried out with animal cells. The consistent increase in concentration halfway through the division cycle corresponds to the initiation of a new round of replication, and the subsequent decrease is always associated with the onset of cell division. Stubblefield and Murphree (I967) and Turner et a l . ( 1 9 6 8 ) found similar results in thymidine kinase and ribonucleo-tide reductase levels during the growth cycle of animal cells. In addition, Painter and Marr ( 1 9 6 8 ) have described the kinetics of mass increase (Figure 9 ) . There is a correlation between rate of mass increase and concentration of purines, and it is not un-reasonable to assume that their functions in energy metabolism and carbohydrate interconversions might be a factor for their fluctua-; tions. The involvement of all the nucleotides in carbohydrate intervoncersions (Mahler, Cordes: 1966) may at least in part account for the second peak i n al l the nucleotides, both purines and pyrimidines. The unusual kinetics of CTP and UTP suggest an interplay of regulation. The oscillations in opposing directions suggest that levels of one are affected by concentrations of the other. Long and Pardee (1967) draw attention to the very complex relationship i n v o l v e d In t h e i n t e r c o n v e r s i o n o f UTP t o CTP. High c o n c e n t r a t i o n s o f CTP. tend t o f e e d b a c k i n h i b i t . CTP s y n t h e s i s , t h u s a l l o w i n g some a c c u m u l a t i o n o f UTP. C o n v e r s e l y , h i g h UTP c o n c e n t r a t i o n s tend t o a c t i v a t e CTP s y n t h e s i s ( F i g u r e 1 ) . Dennis and Herman (1970) have shown t h a t i n p y r i m i d i n e 1 i m i t e d g r o w t h , the UTP pool d e c r e a s e s much more r a p i d l y than does t h e i n t r a c e l l u l a r CTP p o o l . They draw no c o n c l u s i o n about t h i s o b s e r v a t i o n but do m ention t h e r e g u l a t o r y a s p e c t s between UTP and CTP s y n t h e s i s . The d o u b l e peaks o f the p y r i m i d i n e s s u g g e s t a n o t h e r r e g u l a t o r y f u n c t i o n not e v i d e n t i n t h e p u r i n e s . T hat t h e y form a peak i n t h e f i r s t h a l f o f t h e d i v i s i o n c y c l e p r i o r t o the c o m p l e t i o n o f a round o f r e p l i c a t i o n i s t h u s f a r o n l y an o b s e r v a t i o n . C l e a v e r ( 1 9 6 7 ) , i n a s t u d y o f t h y m i d i n e s y n t h e t i c enzymes, does d e s c r i b e a d e c r e a s e i n enzyme a c t i v i t y a t t h e end o f DNA r e p l i c a t i o n . I t i s not un-r e a s o n a b l e t o assume t h a t a sudden c r i t i c a l v a r i a t i o n i h t h e con-c e n t r a t i o n o f , p e r h a p s , TTP t r i g g e r s i n i t i a t i o n . H i r o t a , R y t e r and J a c o b ( 1 9 6 8 ) , have d e s c r i b e d t h e p o s s i b i l i t y o f DNA i n i t i a t i o n b e i n g r e g u l a t e d by a n e g a t i v e system w h i c h would r e q u i r e a s m a l l m o l e c u l e t o a c t i v a t e an a l r e a d y - e x i s t i n g i n i t i a t o r - p r o t e i n . Should t h i s t h e o r y be s u p p o r t e d by e x p e r i m e n t a l d a t a , i t Would not be s u r p r i s i n g i f a n u c l e o t i d e proved t o be the a c t i v a t i n g m o l e c u l e . However; an a l t e r n a t e t h e o r y was a l s o proposed w h i c h c o u l d not be e x p l a i n e d by n u c l e o t i d e o s c i a l l a t i o n s . T h i s t h e o r y , r e q u i r e s t h e a c t u a l s y n t h e s i s o f t h e i n i t i a t o r p r o t e i n . However, no c o n c l u s i o n can be drawn about a possible dependence of the f irst pyrimidine peak with, the end or start of a round of'replication. The peak may be entirely coinciden-tal with the time of DNA completion and initiation, and may just be an unrelated function of the cell age. Manipulation of cells to change their patterns of starts and ends of rounds of replication might help elucidate this point. An attempt was made duringgthe course of the project .to .separate ends and starts of rounds of DNA replication. E. coli B/r/1 was grown and synchronized in acetate as the sole source of carbon, to give a step-wise growth pattern every 100 minutes. Helmstetter (1967) has shown that there exists at least a 20 min gap between ends of rounds of replication and actual initiation of new rounds. However, the slow growth rate, the low cell number (5 x 10 /^ml) and the reduced mass per eel 1 (1/4 .to 1/2 that of glucose grown cells) gave statistically in-valid rad ioact ive counts in that they were only 10 - 15% of those of glucose grown cells. There may be an alternative method for determining a functional relationship between nucleotide fluctuations and the DNA replication patterns. This would require cells with multiple growing points. A broth culture growing at a 20 minute generation time initiates new growing points every 20 minutes so that a baby cell from a broth synchronized culture contains two completed genomes. This type of experiment would be aimed at looking for c o r r e l a t i o n s of n u c l e o t i d e f l u c t u a t i o n s w i t h s h i f t in r e p l i c a t i o n p a t t e r n r e l a t i v e to d i v i s i o n , as opposed to look ing f o r a c o r r e l a t i o n between n u c l e o t i d e f l u c t u a t i o n s and s t a r t s and ends of rounds, as p o s s i b l e in the acetate -g rown c u l t u r e due to a DNA gap. In b r o t h , no s e p a r a t i o n of ends and s t a r t s of rounds o c c u r s . M o n i t o r i n g such a c u l t u r e m e a n i n g f u l l y would not p o s s i b l e , s i n c e the exper imental techniques cou ld not d i s c e r n the r a p i d and superimposed f l u c t u a t i o n s . On the other hand, i t might be p o s s i b l e to sub jec t b roth synchronized c e l l s to a lower temperature to extend the r e p l i c a t i o n and d i v i s i o n t imes at l e a s t t w o - f o l d . T h i s would prov ide a p o p u l a t i o n w i t h a d i f f e r e n t chromosomal p a t t e r n from that of c e l l s grown ih g l u c o s e -minimal s a l t s . Th is type of experiment would e n t a i l a m u l t i t u d e o f - c o n t r o l s , f i r s t to decrease m e t a b o l i c ra te s u f f i c i e n t l y w i t h o u t ' damaging the balance of m e t a b o l i c p r e c u r s o r s , then to e s t a b l i s h DNA polymerase t r a v e l t i m e , and to check ra te and time of i n i t i a t i o n of new growing p o i n t s . Th is type of experiment should be p o s s i b l e ; a s h i f t in the n u c l e o t i d e p a t t e r n s , e s p e c i a l l y of the f i r s t p y r i m i d i n e peak, might c o i n c i d e w i t h the change i i i r e p l i c a t i o n p a t t e r n r e l a t i v e to c e l l d i v i s i o n . If s o , one concludes a causual r e l a t i o n s h i p between DNA r e p l i c a t i o n and f l u c t u a t i o n of the p y r i m i d i n e p o o l s . The i n a b i l i t y of a c e l l to r e i n i t i a t e DNA s y n t h e s i s in the absence of p recursor p o o l s , seems a reasonable phenomenon r e g a r d l e s s of the absence of the i n i t i a t o r p r o t e i n . Our o b s e r v a t i o n of a 90% decrease of pool l e v e l s in the presence of ch loramphenico l supports a result of Ball (1970) in a study of energy metabolism in yeast. He found a dramatic fall of ATP concentration with chloramphenicol inhibition and attributed it to a reduced or suppressed level of respiratory chain enzymes. This explanation may account, at least in part, for the decrease of the purines. However feedback in-hibition may explain the decrease in pyrimidines in the presence of chloramphenicol. In the absence of protein synthesis, there is an accumulation of amino acids. In the de novo pathway of pyrimidine biosynthesis, an accumulation of arginine inhibits the production of ornithine, the activator for the synthesis of carbamyl phosphate (Figure 1). In addition, arginine represses the actual synthesis of carbamyl phosphate. Since there is no activator in the pyrimidine branch of this synthetic process, an accumulation of arginine could effect a dramatic decrease in UTP and CTP pools. That the pools remain at a level of ]0% suggests that SOZ were utilized for con-tinued repl ication of DNA, and that turnover must'be very rapid. Removal of this inhibition exhibits rapid derepression, in that the pools overshoot and then stabilize to control levels. It seems that inhibition of protein synthesis not only regulates the initiator protein but in addition the nucleotide biosynthetic enzymes. Turner et a l . C1968) have shown ribonucleoside d i -phosphate reductase to be subject to very rapid turnover in L-cells. This may also be the case in E_. co 1 i . Edlin and Neuhard (1967) have checked the effect of arginine starvation on nucleotide pools and found a decrease In the pyrtmidines as well. The study was aimed at stringency control, but an obvious explanation was not offered since it was felt that the stringency effect may have been complicated by a metabolic drain which would result when starving an auxotroph for an amino acid whose biosynthetic pathway consumes high energy phosphate. Stringency response in the absence of a required amino acid has been shown to affect protein, l ipid, and nucleotide synthesis in addition to the much studied effect on RNA synthesis (Sokawa et a l . 1970). Both the arginine repression of carbamyl phosphate synthesis and the effects of arginine starva-tion have been well documented. That they both produce a decrease in pyrimidine pools may be due to some preferential cessation of synthesis of arginine-rich protein, thus possibly inhibiting some enzymatic turnover. Both Turner (1968) and our observation suggest that nucleotide biosynthetic enzymes are subject to extremely rapid turnover in view of the very rapid osciallations on the removal and restoration of protein synthesis. Our observation (90% decrease of precursors)nevertheless helps to understand the inability of a cell to reinitiate a new round of replication in the absence of protein synthesis. It has been firmly established by Lark and Renger (1969) that the absence of protein synthesis allows for the completion of a round of replication but not for the reinitiation of a new one. Our results seem to be but an additive consequence of the lack of protein synthesis* but nevertheless an important factor in the synthesis of DNA. ^ In the presence of protein synthesis but absence of DNA re-plication (Figure 18, 19) nucleotide pool levels increased with increase in mass, and fel l sharply as DNA synthesis, multiple initiation, and cell division resumed. This is not surprising si rice Kohiyama (1968) has claimed CRT83 to be a mutant with a defective initiator protein at the non-permissive temperature, but with a normal complement of other DNA-synthesis-associated enzymes such as the Kornberg polymerase. That this mutant is truly an initiator mutant has been confirmed by Khachatourians (personal communication) through density labelling of ends and starts of • rounds of replication. Fangman and Novick (1968) have evidence to show considerable deoxynucleotide increase per cell in other temperature-sensitive replication and division mutants. Similarly, JE. col? CRT83 synthesizes DNA precursors in step with mass increase at non-permissive temperature. In comparison to the chloramphenicol inhibition experiment, this result was expected iii view of the presence of protein synthesis. The considerable increase in pools in our experiments and Fangman's, can be mostly accounted for by mass increase, temperature increase, and excess nucleotides which would normally be utilized ?n the polymerization of DNA molecules. Interpretation of nalidixic acid inhibition on nucleotide pools is hindered by a lack of knowledge of the mode of action of nalidixic acid. The decrease in pools with nalidixic acid may be due to secondary effects. Similar results of decrease in nucleotides have been reported by Cosgrove and Treick (1970). They found a drop in all four ribonucleoside triphosphates with phenethyl alco-hol (PEA) inhibition. This was attributed to PEA inhibition of electron transport on membrane bound respiratory chain enzymes. Khachatourians (personal communication) has recently shown an abnormal ESR (electron spin resonance) pattern of membrane .lipid fractions when subjected to nalidixic acid inhibition.. PEA inhibition shows membrane abnormalities as wel1. Although the gross effects of PEA and nalidixic acid are different, this correlation may at least suggest a secondary effect of nalidixic acid on the membrane, thus affecting nucleotide biosynthesis. Since protein synthesis and RNA synthesis are reportedly normal (Goss et a l . 1965) one would have expected an excess of at least deoxynucleotides in the absence of DNA synthesis as in the temperature sensitive initiator mutant. This however was not the result. Our decision to eliminate a cold concentrated synchronous turbidostat was not without regret. It is our firm belief that a turbidostat is the ideal method for carrying out an experiment involving a potentially limiting concentration of a metabolite. The shift observed in cell division indicated an imbalance of growth presumably from the decrease in metabolism due to cold exposure. The somewhat greater lag in DNA replication pattern however, was not anticipated. Cytological studies carried out by Fitz-James (1965) on mesosomal response to rapid chilling may in fact, make this observation less surprising. Ryter and Jacob (1964) provided convincing evidence for mesosomes acting as an anchoring apparatus for the DNA replicating complex. Fitz-James has shown that rapid shift to cold temperature disarranges the mesosomes in that some shift towards the cell periphery spilling their contents into the wal1-membrane space, others are drawn into the chromatin body and become vacuolated. Upon rewarming, regrowth of mesosomes occurs and the return of the balanced state of division depends on the recovery of mesosomal structure. This has been noted to require 40 minutes at 35 C in Bacillus species. In our experi-ment, a lag was observed during the f irst generation of 42 minutes. The analyses of nucleotide pools in the presence of various inhibitors have provided results which appear to be associated with general biosynthetic mechanisms, such as protein.synthesis. Chloramphenicol inhibition in particular has demonstrated the impossibility of DNA reinitiation in the absence of protein synthesis, due not only to the lack of an initiator protein, but also to the loss of 30% of precursor pools. Nucleotide pools of the temperature-sensitive initiator mutant are in accord'with the chloramphenicol results, and they confirm the necessity of an initiator protein for the initiation of DNA synthesis even with an excess of precursor pools. Nalidixic acid inhibition resulted in a phenome-non suggesting secondary effects, other than just direct action on DNA polymerization. The mode of action of this inhibitor, is s t i l l being explored in several laboratories. The most exciting results of this project have developed from the study of nucleotide fluctuations as a function of age. That there are oscillations is an intersting phenomenon in itself, since no similar studies have been reported in bacterial cultures, and the general assumption has been one to the contrary. Further-more, the double peak of the pyrimidines incites thought'of a regulatory function. That one should coincide with the time of chromosomal completion, creates the possibility of a regulatory relationship between nucleotide concentrations and the end of a round of replication. This in turn might involve the initiation of division. The second peak appears to be associated with the start of DNA replication and the actual onset of cell division. Although supporting evidence exists in animal cell studies, no regulatory hypothesis is available'to account for these fluctuations. It is our strong recommendation that this project be continued in an effort to establish the relationship of these fluctuations to other cell processes, be they regulatory or strictly metabolic. The suggestions we have offered in the discussion on shifting cell division patterns and replication patterns may serve as a starting base. 65 LITERATURE CITED 1. Adler, H.I., W.D. Fisher, A.A. Hardigree. 1969- Cell division in Escherichia col i . Transactions N.Y. Acad. Sci. p. 1059-1070. 2. Ball, A.J .S. , E.R. Tustanoff. 1970. Effect of D(-) and L(+) -threo-chloramphenicol on nucleotide and related activities in yeast undergoing metabolic repression and derepression. Biochim. Biophys. Acta 1 9 9 : 4 7 6 - 4 8 9 . 3 . Behki, R.M., W.S. Morgan. 1964. Studies on the phosphorylation of thymidine in regenerating rat liver. Arch. Biochem. Biophys. 107:427-433. 4 . 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N.Y. p. 323. 54. Ryter* A. , F. Jacob. 1964. Etude au microscope electronlque de la 1 iason entre noyau et mesosome chez B_. subti 1 is Ann. Inst. Pasteur. 107:384. 55. Sokawa, Y., E. Nokao-Sato, Y. Kaziro. 1970. RC gene control ih Escherichia coli is not restricted to RNA synthesis. Biochim. Biophys. Acta 199:256-264. 56. Smjth, D.W., H.E. Shaller, F.J. Bonhoeffer. 1970. DNA synthesis in vitro. Nature 226:711"713 • 57- Stubblefield, E., S. Murphree. 1967. Synchronized mammalian cell cultures. II. Thymidine kinase activity in calcemide synchronized fibroblasts. Exp. Cell Res. 48:652-656. 58. Turner, K., R.Abrams, 1. Lieberman. I968. Levels of ribonucleo-tide reductase activity during the division cycle of the L ce l l . J . Biol. Chem. 243:3725-3728. 7 0 5 9 . W e i s S j B., C C . Richardson. 1 9 6 7 . Enzymatic breakage and join-ing of DNA. 1 . Repair of single s t r a n d breaks in DNA by an enzyme system from Escherichia coli infected with Tk bacteriophage. Proc. Natl. Acad. Sci. 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