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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  ii  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 temperature.  In this case, protein synthesis occurred but  of DNA synthesis could not take place.  initiation  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. inhibitor.  This suggested a secondary effect of the  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 exponential 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.  Nucleotide  3  Biosynthesis  Pyrimidine Purine  3  Biosynthesis  Biosynthesis  Reduction  .  5 6  to Deoxyribonucleotides  TTP B i o s y n t h e s i s B.  8 9  DNA R e p l i c a t i o n a n d 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 Initiation  a n d C o m p l e t i o n o f DNA Rounds  of Repl i c a t i o n . C.  Cell  D.  Nucleotide  10 13  Division B i o s y n t h e t i c Enzymes  i n the Cell  Cycle  16  s t r a i n s and c u l t u r e methods  Chemicals  . . . . .  Synchronization  triphosphate  of acid-soluble pools  17 17  of cells  Measurement o f growth Determination  14 16  MATERIALS AND METHODS Bacterial  .  18 nucleoside 18  V  Table of Contents  (Continued)  Page  A.  Preparation of samples  . . .  B.  Chromatography  19  C.  Autoradiography  20  DNA Pulse label 1 ing  18  21  RESULTS  22  Growth curves in low phosphate concentrations  22  32 Determination of specific activity  Pi/PO^  23  Levels of nucleotide pools in exponential c e l l s . . . . .  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  V I  LIST OF FIGURES  Page  F igure Figure  1. 2.  Figure 3-  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  Figure  k.  Effect of varying specific activities on E_. col i B/r/1 .  27  Figure  5.  Nucleotide pool levels in exponential cultures maintained in a turbidostat.  31  Figure  6.  Synchronized cell growth of E_. col i B/r/1.  33  Figure  7.  DNA H-thymidine pulse of E. coli B/r/1 maintained in a turbidostat after cold concentration treatment.  35  Figure  8.  3  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  Figure 11.  Ribopurine triphosphates as a function of age in synchronous coli B/r/1 .  kQ  Figure 12.  Ribopyrimidine triphosphates as a function of age in synchronous E^. col i B/r/1.  kl  Figure 13.  Deoxyribopurine triphosphates as a function of age in synchronous E. col? B/r/1.  k2  Fjgure  9.  Figure 10a. b.  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 temperatures.  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. c o 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;;  ix  ABBREVIATIONS USED  DNA . . . . . . . . . . . . . . D e o x y r i b o n u c l e i c 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 PEI  TCA  ..chloramphenicol . . .poly(ethyleneLimine)  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 preparation 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. has not yet been fully elucidated.  The mechanism for  initiation  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  denote This in  continuous  type of  change  d i s c o n t i n u o u s enzyme a c t i v i t y  nucleotide synthetic The  in chemical composition of  purpose  for  enzymes  this  (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 age,  since claims of  no f l u c t u a t i o n s  data  is  (2) t o  l a c k i n g ; and  observed, to  the  to  the  regulation  relate  r e g u l a t i o n of of  cell  has been  (Literature  undertaking  DNA  division.  the  cell.  witnessed  review).  project  h a s been  two-fold:  l e v e l s as a f u n c t i o n h a v e been made b u t  the changes  in pool  r e p l i c a t i o n and  of  published levels  if  subsequently  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 regulation (0'Donovan, 1970; Blakeley f  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  Aspartate  -d|>R e press ion  ATP Glutamine HCCU  Glutamine  JUL  T/  ^Carbamyl ^ Aspartate v.  UMP.  JSJ  U T R j t >CTP  Carbamyl Phosphate  N-Acetyl Glutamate  Ornithine  Citrulline-*-* Arginine i  i  Ornithine, being the positive allosteric effector however, compensates 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 d i f f i c u l t 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 a l l  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 subsequent 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 .  Catalytic activity  Nucleotide  Specificity for base  ATP(2 x 10~ )  st imulation  pyrimid ines  dATP(lO~ )  stimulation  pyrimid ines  dATPClO )  inhibition  purines and pyrimidines  dGTP(lO~ )  st imulat ion  pur ines  dTTPQO )  st imulation  purines and pyrimidines  dATP(10 ) S ATP(2 x 10~3)  st imulation  pyr imid ines  dATP(lO~ ) & dTTP(lO )  inhibition  purines and pyrimidines  dGTP(lO X  inhibition  purines and pyrimidines  ATP(10" ) S dTTP(5 x 10 )  i nhi bi t ion  purines and pyrimidines  J  6  -4  5  -5  6  _2t  dATP(lO~h & -i,  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. to TTP inhibition.  The enzyme is sensitive  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 (1965) 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,  1967).  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 pathways, 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 hydroxyl end of the growing polypeptide chain (Kornberg, 1 9 6 1 ) .  I  _  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_. ( 1 9 6 8 ) 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 temperaturesensitive 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 polymerase, 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 c e 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 1968, 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, . period.  C-thymine was added for the remainder of the starvation  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 f i r s t 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 restoration 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, 1968),  and deoxyribonucleoside triphosphates for initiation of multiple  growing points.  Rasmussen  (1964),  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. reviewed in depth by Lark  (1969).  These topics have recently been  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 k  r  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 c e l l s .  Helmstetter  and Cooper  (1968)  desagree with the results of Lark  and Maaltfe  (1967)  as to the exact timing of the replication terminus  (1966)  and Clark  in E_. col i growing in poor carbon sources, although they agree about the 20 minute period in glucose grown c e l l s .  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,  1969).  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 . ( 1 9 6 8 ) , working with L cells, found a strict parallelism 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  12407),  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, 1968).  It is unable to synthesize DNA or divide at the non-  permissive temperature and requires thymine, deoxyadenosine, isoleucine, valine, leucine, and Vitamin By E_. col i B/r/1 was maintained on nutrient agar slants and in liquid cultures of  0.05M  TRIS-glucose mineral salts media. A  growth medium that was.low in phosphate was used to provide efficient incorporation of  32  Pi into the bacteria.  had the following composition:  0.05M  The media  TRIS (trishydroxymethyl-  _c  _Zi  -7  amino methane); 2 x 1 0 M ^HPO^; 1 x 1 0 M CaCl ; 2 x 1 0 M P  J  2  MgC1 ; 3 x 10~ M F e C l ^ ^ O ; 5 . 2 x 1 0 M NaCl; 3 x 10~ M (NH^SO^ 2  6  _2  2  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 t e r , 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 c e l l s .  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 distilled 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 a l l 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 to number of exponential c e l l s .  Pi in any nucleoside triphosphate 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 c e l l s .  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 c e l l s .  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 f i r s t direction, the IN Acetate; 1M L i C l , (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 i r s t 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 developer, rinsed rapidly in water and fixed for 10 minutes in Kodak X-ray fixer. water and dried.  The films were then rinsed in cold running 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 minutes.  H thymidine (lOuCi/0.5 ug/ml) for 5  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 t e r , which was rinsed with 9 volumes of 5% TCA and 2 volumes of hot (90 C) distilled water.  After drying  thoroughly,  the samples were placed in liquid scintiallation vials containing 10 mis toluene-PPO-POPOP. oxazoilyl-benzene)  (2,5-diphenyloxazole, p-Bis 2-(5-phenyl-  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 main8  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~ M PO^ and 2 x 10 M PO^.  Since 2 x 10~ M PO^ showed g no change in balanced growth until 2 x 10 cells/ml (Figure 3), 5  _5  5  this  concentration of PO^ was chosen as the lower limit for subsequent experiments. o  Exponential increase of cell numbers continued almost  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.  Determination of specific activity  32 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~ M PO^ 5  0 , 2 x 10 M PO^ -5  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 yCr Pi/ymole PO^; b) 250 y C i P i / 32  ymole PO^; c)  500 yCi  32  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 of time for cultures containing 100 yC i  32 32 Pi/ymoles P0^ and 250 yCi Pi/  ymoles P0^. The doubling time (k5 minutes) is identical to the 1  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. In an effort'to alleviate the suicide effects of 33 32 experiments were attempted with Pi instead of Pi. 32 has a lower energy level (0.25 Mev cf.1.7 Mev for h a l f - l i f e (25 days cf. 14.3 days for  32  Pi)  32  P i , similar This isotope  Pi), and a longer  (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 on E_. co 1 i. B/r/1. Symbols:  • ,  control  0 ,  100 yCi Pi/umole PO^  © ,  250 u C i P i / umole PO^  32  32  0 , 500 yCi Pi/umole P0, 32  Pi/PO  Viable  J  cell number  1 — l — i — i  i i ii  x 10  1 — i  i  i  i  i • i.  specific activities used for  Pi showed surprising results in that  cells at 100 uCi Pi/umole PO^ showed loss of viability after only J  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  JJ  Pi.  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.  k  - ^ U  ,  max  t2  (ti = generation time) Thus, at a 45 minute generation time, and a desired flow rate of 15 mis./hour, k  m a v  max  =  - 9 6  = .91 = ,f>=.  , .75 hr. therefore, v = ———  r—  15 mls/hr.  (—)  hr. v 17.5 mis.  :  :  volume  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 a l l samples. are listed in Table II,  The exponential pool levels  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.. P i , a necessary  30  Table II.  Nucleoside triphosphate pool levels in exponentially growing E. coli B/r/1.  Nucleoside triphosphate  32 mumoles Pi/nucleotide/ 1 x 10 cells/ml 8  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  M inutes  60  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 t e r 2.5 cm in diameter, the synchronized cells were resuspended in warm medium to yield approxig mately 1 x 10 cells/ml. warm medium.  Zero time was the time of addition of  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 60 80 100 Ce I I a ge ( m i n u t e s )  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 - f o l d concentration.  Experiments were not  performed until one division cycle had occurred, thus final concentration 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.  Minutes  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 population 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 a l l 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 .  Cell  age  (m i n u t e s )  Figure 12.  Ribopyrimidtne triphosphates as a function of age in synchronous E. coli B/r/1.  .2  "  —  i  "  o-  £ .15  UTP  o 0 .JO-0-..  o*  CVI  ro o.  '  **  ,  /  1  0  0..-0  ,..-0-0"0  x  ..0  b-0''  Q  </>  <D  o .05 E  0  mm  40  CTP  >>  50 60 C e l l age  1  JL  70 80 (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  odCTP A  .025  \  .02 h  ,°0  O  CM ro  ® 6  .015  N  \  CL 0--°  B if)  *  @-T T P  .01  e .005 o  40  50  60  C e l l ag e  70  80  (minutes)  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 biosynthesis 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 s p l i t , one part to be maintained by the-turbidostat 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 filter.  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 inhibitor; 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 temperaturesensitive 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 i o  1  8  1  1  r  1  i  1  1  1  1  r  T  - 0 -  T—  /  x  ro  1  -0^0  CAM"  ^  ^CAM*  6  1  0-0  .<flcr° ,-o ^0  3  >-o-o-o-o^°  C  0>  o  J  0  1  20  I  I  40  I  I  I  60  '  80 M  i  I  I  100  n u te s  I  1  120  J  140  1  l  160  I  L  Figure 16.  Fluctuations of dCTP and UTP. Symbols:  • ,  during chloramphenicol and removal.  inhibition  0 ,  control untreated turbidostat  Minutes  Table III.  Nucleoside triphosphate pool fluctuations  in E_. col ?  B/r/1 in the presence of chloramphenicol.  32 Nucleos ide tr iphosphate  rnumoles control  Pi  incorporated  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 c e l l s , 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 nonpermissive (kl C) temperatures.  1  j  h  M i n u te s  1  1  I—~  Figure 19.  Fluctuations in rtbopurine triphosphate (0) , and deoxyribopurine triphosphates (•), JL' col ? CRT 83 at permissive (30 C) and non-permissive (42 C) temperatures.  in  52  T  1  r  i  r  o ATP ©dATP  00 I O  0\ © (  30'  42'  to "D  30p ©~  O <D O 13 C  0  J  1  1  I  I  I  •  «  J  L  oGTP ©d GTP"  6h CO O  4h  2h 0 0  40  60 M i n u tes  80  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 c e l l s .  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. (1968)  Stubblefield and Murphree (I967) and Turner et a l .  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 unreasonable 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 a l 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 ( 1 9 6 7 ) draw attention to the very complex relationship  involved  In t h e  o f CTP. t e n d  i n t e r c o n v e r s i o n o f UTP  to feedback  accumulation  o f UTP.  a c t i v a t e CTP  synthesis  shown t h a t  inhibit.  CTP  Conversely, (Figure  in pyrimidine  b e t w e e n UTP  The  of  evident  h a l f of  to  by  be  a l t e r n a t e theory  was  regulatory  t o the completion  regulatory  of a  Cleaver  replication.  the  possibility  system which would  t o be  data,  (1967),  of the  H o w e v e r , no  protein.  theory,  Should  i t Would not  conand being  molecule this  be s u r p r i s i n g However;  an  be e x p l a i n e d  by  r e q u i r e s the a c t u a l conclusion  un-  initiation  the a c t i v a t i n g molecule.  This  ih the  r e q u i r e a small  not  decrease  H i r o t a , Ryter  o f DNA  a l s o proposed which could  the round  I t i s not  variation  triggers initiation.  nucleotide osciallations. initiator  no  form a peak i n  observation.  o f DNA  by e x p e r i m e n t a l  i f a n u c l e o t i d e proved  They draw  suggest another  already-existing initiator- protein.  supported  d e c r e a s e s much  s y n t h e t i c enzymes, does d e s c r i b e a  a t t h e end  a negative  pool.  That they  cycle prior  have d e s c r i b e d  a c t i v a t e an  theory  pyrimidines  t o assume t h a t a sudden c r i t i c a l  (1968),  regulated  CTP  to  (1970) have  pool  do m e n t i o n t h e  purines.  c e n t r a t i o n o f , p e r h a p s , TTP Jacob  but  i s t h u s f a r o n l y an  enzyme a c t i v i t y  reasonable  in the  of thymidine  Herman  tend  synthesis.  the  the d i v i s i o n  replication  in a study in  CTP  double peaks of  f u n c t i o n not first  and  concentrations  D e n n i s and  intracellular  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  concentrations  s y n t h e s i s , t h u s a l l o w i n g some  h i g h UTP  1).  High  1 i m i t e d g r o w t h , t h e UTP  more r a p i d l y t h a n d o e s t h e  aspects  t o CTP.  can  be  synthesis  drawn a b o u t  a possible dependence of the f i r s t 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 invalid rad ioact ive counts in that they were only 10 - 15% of those of glucose grown c e l l s .  There may be an alternative method for  determining a functional relationship between nucleotide fluctuations and the DNA replication patterns. multiple growing points.  This would require cells with  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  replication  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 l o o k i n g 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 r o u n d s , as p o s s i b l e in the a c e t a t e - g r o w n c u l t u r e due t o a DNA gap. no s e p a r a t i o n o f ends and s t a r t s of rounds o c c u r s .  In b r o t h ,  Monitoring  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 e x p e r i m e n t a l t e c h n i q u e s c o u l d not d i s c e r n the r a p i d and superimposed  fluctuations.  On the o t h e r hand, i t might be p o s s i b l e t o s u b j e c t b r o t h  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 times at l e a s t t w o - f o l d . different  chromosomal p a t t e r n from that of c e l l s grown i h g l u c o s e -  minimal s a l t s . of-  T h i s would p r o v i d e a p o p u l a t i o n w i t h a  controls,  T h i s type of experiment would e n t a i l a m u l t i t u d e first  to d e c r e a s e m e t a b o l i c r a t e s u f f i c i e n t l y  damaging the b a l a n c e o f m e t a b o l i c p r e c u r s o r s , polymerase t r a v e l  a shift  then to e s t a b l i s h  t i m e , and to check r a t e and time of  of new growing p o i n t s .  without'  initiation  T h i s type o f experiment should be p o s s i b l e ;  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 t h e f i r s t  pyrimidine  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 to c e l l d i v i s i o n .  If  The i n a b i l i t y of a c e l l  i n the  p o o l s , seems a r e a s o n a b l e phenomenon  levels  between  pools.  to r e i n i t i a t e DNA s y n t h e s i s  of the absence of the i n i t i a t o r p r o t e i n . d e c r e a s e of pool  relative  s o , one c o n c l u d e s a c a u s u a l r e l a t i o n s h i p  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  absence of p r e c u r s o r  DNA  regardless  Our o b s e r v a t i o n o f a 90%  in the presence of c h l o r a m p h e n i c o l  supports  a result of Ball (1970) in a study of energy metabolism in yeast. He found a dramatic f a l l 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 continued 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 Lcells.  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 i p i d , 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 starvation 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 replication (Figure 18, 19) nucleotide pool levels increased with increase in mass, and f e l 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 alcohol (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 mutant.  initiator  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 i r s t 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. of division.  This in turn might involve the initiation  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. 1 9 6 9 - Cell division in Escherichia c o l i . Transactions N.Y. Acad. Sci. p. 1059-1070.  2.  Ball, A . J . S . , E.R. Tustanoff. 1 9 7 0 . Effect of D(-) and L(+) threo-chloramphenicol on nucleotide and related activities in yeast undergoing metabolic repression and derepression. Biochim. Biophys. 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