UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Pleiotropic effect of DnaA gene on initiation of DNA replication and cell division in Escherichia coli Khachatourians, George 1971

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1971_A1 K33.pdf [ 5.7MB ]
Metadata
JSON: 831-1.0101930.json
JSON-LD: 831-1.0101930-ld.json
RDF/XML (Pretty): 831-1.0101930-rdf.xml
RDF/JSON: 831-1.0101930-rdf.json
Turtle: 831-1.0101930-turtle.txt
N-Triples: 831-1.0101930-rdf-ntriples.txt
Original Record: 831-1.0101930-source.json
Full Text
831-1.0101930-fulltext.txt
Citation
831-1.0101930.ris

Full Text

PLEIOTROPIC EFFECT OF DnaA GENE ON INITIATION OF DNA REPLICATION AND CELL DIVISION ESCHERICHIA COLI  by  GEORGE KHACHATOURIANS  B.A.  San F r a n c i s c o S t a t e C o l l e g e ,  1966  M.A.  San F r a n c i s c o S t a t e C o l l e g e ,  1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  In the Department of Microb i o l o g y  We accept  this  t h e s i s as. conforming to the  required  THE UNIVERSITY  standard  OF BRITISH COLUMBIA  July,  1971  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  agree  fulfilment  the U n i v e r s i t y of  the L i b r a r y s h a l l make i t I  in p a r t i a l  freely  of  the  requirements  B r i t i s h Columbia, I agree  available  for  that permission for extensive copying of  of  this  representatives. thesis for  It  financial  this  thesis  of  gain shall  Microbiology  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  12th J u l y ,  1971  or  i s understood that c o p y i n g o r p u b l i c a t i o n  written permission.  Department  that  r e f e r e n c e and s t u d y .  f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Department by h i s  for  not  be allowed without my  ABSTRACT  Cell  duplication  in E s c h e r i c h i a c o l i  c o o r d i n a t e d with chromosome r e p l i c a t i o n . of chromosomes in p e r p e t u a t i n g of  their  i n v o l v e s complex e v e n t s , Because of the  the normal c e l l  importance  c y c l e the  initiation  r e p l i c a t i o n must be c o o r d i n a t e d with c e l l u l a r d i v i s i o n .  Following  initiation,  the c e l l must r e p l i c a t e  and segregate  its  chromosomes, c r e a t e a s i t e necessary f o r s e p t a t i o n and d i v i d e . These events c o u l d be c o o r d i n a t e d by e i t h e r ; i n v o l v i n g d i f f u s i b l e enzymes, or  (2)  (1)  biochemical  multienzyme complexes which  are  l o c a l i z e d at  the s i t e of DNA r e p l i c a t i o n and c e l l  the  latter case,  the c y c l i c events of  cell  replication,  division.  In  s e g r e g a t i o n and  d i v i s i o n may be c o o r d i n a t e d by physica1-chemica1 or biochemical  means.  In any c a s e ,  To t e s t of E^. c o l i , sensitive at  reactions  physical a s s o c i a t i o n implies p l e i o t r o p i c  this hypothesis, c e l l  d i v i s i o n of the  i s o l a t e d by Kohiyama (1968) was s t u d i e d .  initiator  30 C , and at  mutant E_. c o l ? CR 3**T83 (ts  the r e s t r i c t i v e  temperature  as measured by r a d i o a c t i v e p r e c u r s o r uptake, kO minutes and was e q u i v a l e n t started.  initiator  Measurement of  pools by t h i n - l a y e r  The  rounds of  r i b o - and d e o x y r i b o n u c l e o t i d e  chromatography at  30 C and hi  DNA s y n t h e s i s was not due to a l i m i t a t i o n  normally  The DNA r e p l i c a t i o n  stopped a f t e r  to completion of  mutant  temperature-  DnaA) grew  (42 C ) .  effects.  approximately  replication  triphosphate  C indicated  residual  in the DNA p r e c u r s o r s .  Using a combination of d e n s i t y and d i f f e r e n t i a l for  the s t a r t s  initiation  and ends of chromosomes, a p r e f e r r e d  of new r e p l i c a t i o n  replication  radioactive  at Ml  and was i d e n t i c a l  c y c l e s was shown.  C terminated 150  to the  i n v o l v e d in the process o f  at  a fixed  It  place for  re-  was shown t h a t DNA  r e g i o n of the chromosome,  ug/ml chloramphenicol s e n s i t i v e  initiation  labelling  step  o f chromosome r e p l i c a t i o n  in  E_. c o l i . A c e s s a t i o n of c e l l u l a r  d i v i s i o n was noted by measurement o f  growth by C o u l t e r C o u n t e r , at a s h i f t filamentous  a f t e r approximately  15 - 20 min.  the c e s s a t i o n of c e l l  i n v o l v e d in  When t h i s  initiation. the  T h i s mutation was mapped by i s o l e u c i n e - v a l i n e region of  the  gene was transduced to d i f f e r e n t s t r a i n s  at  is,  o f DNA r e p l i c a t i o n was a  E_. c o l i K.J2 the same p l e i o t r o p y was o b s e r v e d . be u n c o u p l e d , however,  that  in the gene DnaA, coding f o r a membrane  t r a n s d u c t i o n and was l o c a t e d at EL c o l i map.  in  resume d i v i s i o n  The p l e i o t r o p i c b e h a v i o u r ,  d i v i s i o n and i n i t i a t i o n  r e s u l t o f a p o i n t mutation bound p r o t e i n  from 30 C to hi C, r e s u l t i n g  Upon a r e t u r n to 30 C, the c e l l s  growth.  cell  30 C by i n h i b i t o r s o f  This pleiotropy  of  could  DNA s y n t h e s i s or  ini t i a t i o n . During recovery at cell  d i v i s i o n was p r o p o r t i o n a l  restrictive d u r i n g the of  30 C from growth  temperature.  to c e l l  under kl C, e x p r e s s i o n o f  equivalents  RNA and p r o t e i n  recovery p e r i o d , was o b l i g a t o r y  replication,  but not f o r  generated at  synthesis, for for  initiation  the e x p r e s s i o n of c e l l  10  the  minutes  o f new rounds  division.  A cell  d i v i s i o n " p o t e n t i a l " p r o t e i n was present under the r e s t r i c t i v e condition.  T h i s " p o t e n t i a l " was made at a d e r e p r e s s e d r a t e and  underwent a r a p i d d e g r a d a t i o n when r e t u r n i n g cell  if  kept at kl C.  from kl C to 30 C, t h i s  The h a l f - l i f e  f o r decay of  estimated  to be 1.k m i n u t e s .  The r e s u l t s were i n t e r p r e t e d , is common to the  d ivis ion.  initiation  At any g i v e n  time,  " p o t e n t i a l " allowed e x p r e s s i o n  d i v i s i o n based on DNA/mass or normal  at kl C.  which  growth  cell  equivalents  generated  the d i v i s i o n " p o t e n t i a l " was  in terms of an enzyme complex, of DNA r e p l i c a t i o n and c e l l u l a r  TABLE OF CONTENTS  Page  LITERATURE REVIEW. . . I.  1  The DNA r e p l i c a t i o n  c y c l e in E_. c o l ?  2  A.  The membrane attachment of DNA in E_. c o l ?  B.  The i n i t i a t i o n of normal chromosome r e p l i c a t i o n  k  in E_. c o l ?  II.  C.  The r e p l i c a t i o n of  D.  S e p a r a t i o n of daughter chromosomes: S e g r e g a t i o n . .  C e l l d i v i s i o n of A.  III.  the E_. c o l i chromosome  E_. c o l i  RNA and p r o t e i n  E_. c o l ?  C o n t r o l of the DNA r e p l i c a t i o n  B.  Regulation of c e l l  II.  Bacterial  in E_. c o l i  d i v i s i o n in E_. c o l i  MATERIALS AND METHODS I.  13  d u p l i c a t i o n c y c l e in E_. c o l i . .  A.  and Phage s t r a i n s  A.  Bacterial  strains  B.  Bacteriophage s t r a i n s  13  normal  P h y s i o l o g i c a l d i v i s i o n and s e p t a t i o n  R e g u l a t i o n of the c e l l  6  13  s y n t h e s i s in the  d i v i s i o n c y c l e of B.  2  15 15 15 19 21 21 21 21  Media and Chemicals  21  A.  21  Media  V  T a b l e o f Contents  (continued) Page  B. III.  IV.  23  C u l t u r e methods A.  Growth c o n d i t i o n s f o r  B.  P l a t i n g methods  C.  Temperature s h i f t  23  liquid cultures  23 conditions  2k  T  Measurement of macromolecular s y n t h e s i s and cell  growth..  2k  A.  Measurement of c e l l  B.  Measurement of c e l l mass  25  C.  Measurement o f t t r y p t o p h a n a s e a c t i v i t y  25  D.  Measurement of DNA s y n t h e s i s  25  1.  Total  25  2.  Measurement of  E.  V.  23  Chemicals  numbers  2k  DNA s y n t h e s i s the r a t e of DNA s y n t h e s i s  Meaisurement of a c i d s o l u b l e n u c l e o s i d e  26  tri-  phosphate p o o l s . . . . .  27  1.  P r e p a r a t i o n of the samples  27  2.  Chromatography.  27  3.  Autoradiography  28  Density gradient  sedimentation a n a l y s i s . . . .  A.  Measurement of BrLIra  i n c o r p o r a t i o n in the D N A . . . .  B.  Density  C.  P r e p a r a t i o n and a n a l y s i s of  29 30  labelling  density gradient  29  DNA samples by  c e n t r i fugat ion  30  T a b l e of Contents  (continued)  Page  VI.  1.  E x t r a c t i o n of DNA  30  2.  C e n t r i f u g a t i o n and f r a c t i o n a t i o n . . . .  31  3.  Measurement o f  32  radioactivity...  G e n e t i c a n a l y s i s of the mutants. A.  I s o l a t i o n of  the temperature  32 resistant  revertants B.  32  T r a n s d u c t i o n experiments  ..  1.  B a c t e r i o p h a g e donor l y s a t e p r e p a r a t i o n  32  2.  T r a n s d u c t i o n experiments  33  RESULTS AND GENERAL DISCUSSION I.  35  P r o p e r t i e s o f CR34T83 A.  ••••  A n a l y s i s o f macromolecular s y n t h e s i s and  35  1.  Temperature s h i f t  2.  C e l l d i v i s i o n and DNA r e p l i c a t i o n in a s h i f t  conditions  Cell division  b.  S t u d i e s on DNA r e p l i c a t i o n Total  ii.  Recovery o f CR3 tT83 at Single shift  in CR34T83  DNA r e p l i c a t i o n 30 C a f t e r  experiments  37 37  uptake of TdR  Rate of /  35  to n o n - p e r m i s s i v e t e m p e r a t u r e s . . . .  a.  i.  A.  35  cell....  d i v i s ion  II.  32  growth, at 42 C  40 40 40 43 43  vi i  Table of Contents  (continued) Page  1.  Cell  division  d u r i n g r e c o v e r y f r o m a 42 C p u l s e  2.  Cell  volume d i s t r i b u t i o n s  3.  R a t e o f DNA  f o r T83 a t r e c o v e r y  synthesis during recovery  44 49  from...  a 42 C p u l s e  49 32  4.  Measurement o f triphosphates  B.  Multiple  C.  Physiological a single  -P-labelled  nucleoside  i n CR34T83  53  s h i f t experiments requirements  55 of the recovery  from..  shift  58  1.  R o l e o f DNA  synthesis in recovery  58  2.  R o l e o f RNA  synthesis i n recovery  62  3.  Role of protein and  DNA  s y n t h e s i s on c e l l  replication  division...  during the recovery  period....  D.  a.  Cell  b.  DNA  65  division synthesis  Attempts  t o uncouple  division  a t 30 C  1.  Inhibition phenethyl  2.  ....  74 DNA  replication  and  cell.... 79  o f i n i t i a t i o n o f new  rounds  by....  alcohol....  Uncoupling c e l l by D a u n o m y c i n  68  division  79 from  DNA  replication 83  vi i i  Table of Contents  (continued) Page  3.  Uncoupling of c e l l d i v i s i o n from DNA 85  replication by N a l i d i x i c acid III.  Control of DNA synthesis in T 8 3 A.  87  Identification of the place of resumption of 87  DNA synthesis at recovery B.  Comparison of the effect of inhibition of synthesis and the T 8 3 mutation on the  protein  initiation 95  of DNA repl ication C.  Studies on s t a b i l i t y  of the growing point and  the replication complex at the non-permissive 99  condition IV.  Synthesis and decay of the d i v i s i o n potential  in  CR3 »T83 studied by in vivo k i n e t i c s .  102  i  A.  Time course of appearance of the d i v i s i o n 102  potential B.  The wave of expression of c e l l d i v i s i o n at recovery in the absence of translation  or  transcription V.  10k •  Genetic analysis of the mutant CR3^T83 A.  Study of temperature resistant  B.  Construction and ana l y s i s of CR3.4T83 Hv DnaA strains  (tr)  revertants...  110 111  ts 111  ix  T a b l e of Contents  (continued)  Page  C.  Introduction  of  the T83  gene i n t o E_. c o l i K12  s t r a i n s and the a n l y s i s of the p l e i t r o p y DISCUSSION BIBLIOGRAPHY  ,  ,  114 117 127  x  LIST OF TABLES  Page  Table  I.  Bacterial  Table  II.  C h a r a c t e r i s t i c s of l a b e l l e d DNA in T83: Buoyant d e n s i t i e s and percentage of t o t a l DNA  Table  Table  III.  IV.  strains  The frequency of j o i n t I l v and DnaA l o c i  22  t r a n s d u c t i o n of  Frequency of j o i n t t r a n s d u c t i o n s of II v and DnaA l o c i from E. c o l i T83  3k the 113  the 115  xI  L I S T OF FIGURES  Page  Figure  Figure  Figure  Figure  Figure  Figure  Figure  Figure  Figure  1.  2.  3.  4.  5-  6.  7-  8.  3.  F i g u r e 10.  F i g u r e 11.  Figure  Figure  12.  13.  R e s p o n s e o f T83 grown a t 30 C t o a c h a n g e in temperature  36  G r o w t h o f T83 u n d e r p e r m i s s i v e permissive conditions  39  and n o n -  U p t a k e o f C - l 4 t h y m i d i n e i n t o CRT-83 a t 30 C and 42 C. R a t e o f DNA s y n t h e s i s s h i f t t o 42 C.  growing 41  in CR34T83 after a  kl  R e c o v e r y o f CR34T83 a t 30 C f o l l o w i n g g r o w t h a t 42 C.  45  R e l a t i o n s h i p between c e l l s r e c o v e r i n g from kl C t o t h e i r c o n t r o l c u l t u r e s k e p t a t 30 C.  46  The r e l a t i o n s h i p b e t w e e n t h e l a g i n r e c o v e r y t o t h e p e r i o d o f g r o w t h a t kl C.  48  Analysis of the c e l l s i z e during f r o m g r o w t h a t kl C.  50  recovery  Rate of l a b e l l e d t h y m i d i n e i n c o r p o r a t i o n i n t o T83 d u r i n g r e c o v e r y  51  32 Distribution of P - l a b e l l e d deoxyribonucleisdde triphosphates in C R 3 4 T 8 3 . 32Distribution of P-labelled ribonucleos i d e t r i p h o s p h a t e s i n CR34T83.  56  Inhibition of c e l l d i v i s i o n r e c o v e r y p e r i o d by s h i f t i n g permissive temperature.  57  54  r  during the t o t h e non-  I n h i b i t i o n o f c e l l d i v i s i o n by p e r i o d i c e x p o s u r e to t h e n o n - p e r m i s s i v e t e m p e r a t u r e 59  XI I  L i s t of  Figures  (continued) Page  F i g u r e 14.  F i g u r e 15.  E f f e c t o f i n h i b i t i o n of DNA s y n t h e s i s on c e l l d i v i s i o n at recovery at 30 C . The e f f e c t of at  F i g u r e 16.  F i g u r e 17-  F i g u r e 18.  F i g u r e 19-  F i g u r e 20.  F i g u r e 22.  F i g u r e 23.  Figure l k .  F i g u r e 25-  F i g u r e 26.  F i g u r e 27.  of RNA s y n t h e s i s  recovery on the e x p r e s s i o n of d i v i s o n  E f f e c t of i n h i b i t i o n on recovery  of  total  E f f e c t of i n h i b i t i o n on recovery of T83.  66  of  protein  division  inhibition  67  synthesis 69  E f f e c t of i n h i b i t i o n o f p r o t e i n s y n t h e s i s d u r i n g and a f t e r a p u l s e at kl C , on r e c o v e r y E f f e c t of  64  RNA s y n t h e s i s  Measurement of the accumulation of p o t e n t i a l in T83-  the F i g u r e 21.  inhibition  61  of  recovery of c e l l s at  protein  71  s y n t h e s i s on  30 C.  73  Changes in the r a t e of c e l l d i v i s i o n during recovery from a k l C b l o c k in the presence of chloramphenicol.  75  The e f f e c t of i n h i b i t i o n of p r o t e i n s y n t h e s i s at k l C on the DNA r e p l i c a t i o n at recovery  77  Role of i n h i b i t i o n of p r o t e i n s y n t h e s i s on i n i t i a t i o n of new rounds d u r i n g recovery  78  The e f f e c t of chloramphenicol on the r a t e of DNA s y n t h e s i s d u r i n g the recovery p e r i o d .  80  Uncoupling of c e l l d i v i s i o n from DNA r e p l i c a t i o n by phenethyl a l c o h o l  82  Uncoupling o f c e l l  d i v i s i o n and DNA s y n t h e s i s  by Daunomycin.  84  R e l a t i o n s h i p of i n h i b i t i o n of DNA s y n t h e s i s and c e l l d i v i s i o n d u r i n g temperature s h i f t s in T83.  86  XI  L i s t of  Figures  (continued) Page  F i g u r e 28.  F i g u r e 29.  F i g u r e 30.  F i g u r e 31.  F i g u r e 32.  F i g u r e 33.  F i g u r e 3k.  F i g u r e 35-  F i g u r e 36.  F i g u r e 37.  F i g u r e 38.  C o n s t r u c t i o n of in CR34T83. Protocol for 30 and 3.1 .  the model f o r DNA  replication 88  the experiments  in F i g u r e s 90  CsCl g r a d i e n t a n a l y s i s of the p l a c e of the r e i n i t i a t i o n at recovery from growth at kl C.  92  CaCl g r a d i e n t a n a l y s i s of the p l a c e of the r e i n i t i a t i o n at r e c o v e r y from growth in the presence of CAM.  93  Comparison of the kl C e f f e c t to that of a d d i t i o n of 150 yg per ml CAM.  97  E f f e c t of a low l e v e l c a t i o n in T83.  98  of CAM on DNA r e p l i -  DNA s y n t h e s i s in T83 at kl C f o l l o w i n g exposure to N a l i d i x i c a c i d .  an 101  DNA r e p l i c a t i o n in T83 at kl C.subsequent to a 30 minute N a l i d i x i c a c i d treatment at 30 C .  103  The wave of c e l l d i v i s i o n d u r i n g a t i o n from kl to 30 C.  106  Decay of c e l l at kl C.  d i v i s i o n potential  Decay of c e l l at kl C.  d i v i s i o n potential  transi-  of CR3*»T83 108 of  CR3^T83 109  I I  ACKNOWLEDGEMENTS  To the F a c u l t y of the Department of M i c r o b i o l o g y and to Head of for  the Department,  Dr.  the  Jack J . R . C a m p b e l l , I extend my thanks  the environment and support g i v e n me d u r i n g my development  as a graduate student at  the U n i v e r s i t y of  My thanks and a p p r e c i a t i o n go to Dr. supervising this  t h e s i s work.  C. 0. Person and R . A . J .  B r i t i s h Columbia. D. Joseph C l a r k  for  My thanks go to D r s . J . L e v y ,  Warren f o r  s e r v i n g on my committee.  i would l i k e to express my a p p r e c i a t i o n to my e x t e r n a l examiner,  Dr. W.L. Fangman, of  a c c e p t i n g the e v a l u a t i o n of  the U n i v e r s i t y of Washington, f o r  this  work.  I am e s p e c i a l l y indebted to P r o f e s s o r s K . G . L a r k , and P . C . Hanawalt,  and D r s . L u c i e n C a r o , Roy C u r t i s s  for  helpful  their  III,  and T . J . L e i g h t o n  a d v i c e in c e r t a i n phases of t h i s work.  I thank Mrs. L y d i a Huzyk (Mychajlowska) nucleotide triphosphate a n a l y s i s .  Lastly,  f o r h e l p i n g me with  the  ABBREVIATIONS  (IUPAC-IUB-CBN  1970  Recommendations)  T  Thymi ne  dThd  Thymidine  BrdUrd  Bromodeoxyuri di ne  BrUra  Bromouraci1  dTTP  Deoxythymi di ne t r i phosphate  dATP  Deoxyadenos i ne t r i phosphate  dCTP  Deoxycytidine  dGTP  Deoxyguanosine  ATP  Adenosine  CTP  Cytidine  GTP  Guanos i ne t r i phosphate  UTP  Uridine  TCA  Trichloroacetic  triphosphate triphosphate  triphosphate triphosphate  triphosphate acid  LITERATURE REVIEW  The c e l l cycle of bacteria is a complex, but well-coordinated process.  This process, which allows the c e l l to generate its progeny,  is an accumulative one.  Thus, sequentially, starting with chromosome  replication and ending with physical separation of the daughter c e l l s , we see a number of events which, under normal conditions aim at one ultimate goal - the "duplication" of the c e l l . The duplication of E_. col i is a sum of two well controlled events: (l)  the DNA r e p l i c a t i o n ; and (2) the c e l l d i v i s i o n c y c l e .  The  separate aspects of the DNA replication cycle have the following expressions:  (i)  o r i g i n (a s i t e ) ; segregation.  attachment; (iv)  (ii)  initiation  (an event);  (iii)  the  polymerization (chain elongation); and (v)  The separate aspects of c e l l d i v i s i o n are:  of d i v i s i o n proteins; ( i i )  (i)  formation  physiological d i v i s i o n and physical sep-  aration of the two c e l l s . Much of the e a r l i e r work on the regulation of bacterial  growth  and duplication has been reviewed in the extensive treatise by Maalrfe and Kjeldgard  (1966).  The state of current knowledge concerning a l l aspects of  replication  of DNA in microorganisms has been amply discussed  in the Cold Spring  Harbour Symposia of Quantitative  Since that time,  Biology of 1968.  the subject of DNA replication has been reviewed by Bonhoeffer and  2  Messer  (1969) and Lark  Helmstetter  (1969b),and  the c e l l  (1969a, b ) , Donachie and Masters  of the S o c i e t y f o r General M i c r o b i o l o g y of these r e v i e w s ,  reviews.  (1969).  The f o l l o w i n g  attempt to make the information  The DNA r e p l i c a t i o n  by a s e m i c o n s e r v a t i v e mechanism.  Brenner and Cuzin (1963) suggested a chromosome  attachment s i t e on the c e l l hold the r e p l i c a s e u n i t , c o u l d a c t , as w e l l ,  membrane.  This unit systematically  hypothesis.  or f r a c t i o n a t i o n a l  as a v e h i c l e f o r moving daughter chromosomes a p a r t .  electron microscopic,  s e p a r a t i o n and b i o c h e m i c a l s t u d i e s .  Essentially,  The f r a c t i o n a -  o f the M-band method  (Tremblay  the method c o n s i s t s o f l y s i n g the c e l l s  w i t h s a r k o s y l and banding the l y s a t e ions.  evidence and e x t e n s i o n s o f  These s t u d i e s are e i t h e r d i r e c t l y  s e p a r a t i o n s t u d i e s are v a r i a t i o n s  et aj_. 1969).  could  through which the chromosome c i r c u l a t e d , and  Several groups have p r o v i d e d a d d i t i o n a l  + +  to make up  The membrane attachment o f DNA in E_. c o l i  Jacob,  Mg  is an  supposes the W a t s o n - C r i c k  o f hydrogen-bonded assembly o f f o u r n u c l e o t i d e s  A.  tional  l i t e r a t u r e survey  c y c l e i n E_. c o l i  the E_. c o l i ch romosome and r e p l i c a t i o n  this  S i n c e the appearance  current.  The model accepted f o r r e p l i c a t i o n structure  (1969) and a Symposia  some o b s e r v a t i o n s have been made which s h o u l d be  added to the present  I.  d i v i s i o n c y c l e by  in a s u c r o s e g r a d i e n t  The magnesium ions induce the c r y s t a l i z a t i o n  containing  of sarkosyl  3  Ten to t h i r t y p e r c e n t of the c e l l membrane, 75%  in s i t u . the c e l l  and 30% of  the DNA are found a s s o c i a t e d w i t h the c r y s t a l s .  Because n u c l e i c a c i d s alone have no a f f i n i t y simplest  inference  of RNA in  f o r s a r k o s y l , the  is t h a t the complexed DNA and RNA is  attached  to fragments of the membrane c o n t a i n i n g the chromosome attachment s i t e and the RNA in t u r n ,  is presumably a t t a c h e d to the DNA from which  i t was being t r a n s c r i b e d . Recently,  S h a c h t e l e e_t aj_.  ( 1 9 7 0 ) and D a n i e l s  M-band method have shown s p e c i f i c attachment and Fox  ( 1 9 7 0 u s i n g the  o f E_. c o l i DNA.  Fielding  (1970) p r o v i d e d e v i d e n c e f o r a s t a b l e attachment at the  replicating origin that the DNA at  in E. c o 1 i .  the attachment  The consensus from these s t u d i e s  is  region is p r o t e c t e d from s o n i c  i s c i l . l a t i o n and r e p r e s e n t s approximately 0.6  percent of the  total  DNA of the c e l l which would c o r r e s p o n d to a length o f 6 u. Fuchs and Hanawa 11  (1970), u s i n g a 5 -• 20%  l a y e r e d over a 60% s u c r o s e " s h e l f " ,  have succeeded in  "growing p o i n t " complex from E. c o 1 i . of the e n t i r e E_. c o l i genome; that  l i n e a r sucrose gradient isolating a  The l a t t e r c o n t a i n e d 0.5  i s , at  Direct electron microscopic studies  -  1%  l e a s t 5 u of DNA. involving thin sectioning of  E_. coj_i_ s t r a i n s and subsequent s t a i n i n g , has i n d i c a t e d a s s o c i a t i o n of the n u c l e a r regions to the membranes (Altenburg et a 1. and T h a t c h e r ,  1970).  In g e n e r a l ,  adequate demonstration of one c a s e ,  the s t r a i n 0111  these s t u d i e s do not permit  the attachment - a of  1970;  organelle.  E. c o l i was used which  Pontefract an  Furthermore,  in  is known to form  " e x t r a membranes" ( W e i g a n d e t a j _ .  1970),  t h e o b s e r v a t i o n s o f A l t e n b u r g and B.  The  The  replicon  implicated  initiation  t h a t the  as  the  interaction  of the  replicon,  the  synthesis  i s continuous  it  One  The  steps are  to high  initiator".  Under normal  and  step of  is s e n s i t i v e p e r ml  and  C o o p e r and more t h a n defined  by  to  one  step  25 ug  (150  two  steps  two  inhibited  by  CAM  (25  f o r E_. c o l i B / r  and  TAU  however, suggest  Lark  of " p r o b a b i l i t y "  in a particular starvation  cell. can  chloramphenicol.  ug/ml),  are 2  acid earlier ug  respectively.  that there are  sensitivity  by  Glaser,  s t e p , o c c u r r i n g much l e v e l s o f CAM  to a d i f f e r e n t  However,  the amino  low  o r as s u g g e s t e d  doubt t h a t amino a c i d  i s t h e same as  A different  i n v o l v e d i n CAM  steps, w i t h a spread  t o low  "initiator"  initiation,  The  its sensitivity  c e n t r a t i o n o f CAM,  before,  defined  the  Ward and  levels of  resistant  and  controlled  i n i t i a t i o n was  R a n g e r , 1969;  different  ug/ml) and  (1971).  Weusthoff  The  of, or s l i g h t l y  l e v e l s o f CAM.  p e r ml  chromosome was  includes protein.  ( L a r k and  d i s s o c i a b l e by  initiation.  low  1963)  growth c o n d i t i o n s , the  the m a t e r i a l  concentrations of chloramphenicol, sensitive  bacterial  Cuzin,  i n E_. c o l i .  i n i t i a t o r with a p a r t i c u l a r s i t e of  step o c c u r i n g a t the time  is s e n s i t i v e  of the  "the  replicator.  i n v o l v e s more t h a n  1969).  was  invalidate  chromosome r e p l i c a t i o n  ( J a c o b , B r e n n e r and  replication  by a g e n e w h o s e p r o d u c t  c o u l d thus  co-workers.  o f normal  theory  and  of  (1 t o 20  initiation, ug  are  t h a t the step w i l l  b r i n g t h e DNA  each  p e r ml)  (1969b!,there  T h e r e no  either  l o n g e r can  cononly be  be much  replication  to a  5  f i x e d end p o i n t ,  and, on r e s t o r a t i o n o f amino a c i d s ,  take up again from t h i s , 1968;  the n a t u r a l  starting  point  synthesis w i l l ( B i r d and L a r k ,  Kohiyama, 1968). The s t a r t i n g  p o i n t f o r the chromosome r e p l i c a t i o n ,  is not a p o i n t but a r e g i o n .  The o r i g i n o f r e p l i c a t i o n has been  d e f i n e d on the g e n e t i c map o f E_. c o l i between arg G and x y l o s e l o c i backed by three d i f f e r e n t (Abe and Tomizawa, enzyme i n d u c t i o n Helmstetter,  (7 and 8 o ' c l o c k ) .  experimental  1967; Caro and B e r g ,  This conclusion is  approaches: '(1)transduction 1968; M a s t e r s ,  based on gene-dosage e f f e c t s  1970); (2)  1969; Wolf et_ al_. 1968;  and (3) s y n c h r o n i z e d mutagenesis  e_t aj_. 1968; Wolf et_ a_l_. 1968).  product,  Kl2 and B/r/1, and i s l o c a t e d  (Donachie and M a s t e r s ,  1968);  the o r i g i n ,  (Cerda-Olmeda  E s s e n t i a l l y , a l l experiments a r e  assuming d o u b l i n g of a gene, o r i t s  upon r e p l i c a t i o n o f that gene.  Original  s t u d i e s by Nagata  (1963) and r e c e n t l y  those of V i e l -  metter e t a l . (1968), d i s a g r e e w i t h the idea of f i x e d chromosome replication origin. s t r a i n s , where  T h i s c o n t r a d i c t i o n arose from s t u d i e s o f H f r  i t was shown t h a t the s i t e o f i n t e g r a t i o n  f a c t o r served as the o r i g i n and the p o l a r i t y determined the d i r e c t i o n o f r e p l i c a t i o n et a l . (1968) a l s o o b t a i n e d  o f the i n s e r t i o n  (Nagata,  1963).  results favouring this  view o f these f i n d i n g s  it  origin for replication  in E_. c o l i .  o f the IF  Vielmetter  conclusion.  in  remains c o n f u s i n g as to the s i t e o f the The idea that once  initiated,  the c i r c u l a r molecule o f DNA r e p l i c a t e s s e q u e n t i a l l y along i t s  length  6  has been re examined by Nagata and Meselsohn (1968).  In  their  3 e x p e r i m e n t s , a p u l s e of  H-TdR was given to e x p o n e n t i a l l y  cells.  c e l l s were pulsed with 5~bromouracf1,  Subsequently, i f  was found that the t r i t i u m l a b e l was a s s o c i a t e d with the u r a c i l o n l y when the and d e n s i t y  label  interval  between a d d i t i o n of the  pulse equalled a multiple  Thus, the o r i g i n a l  o b s e r v a t i o n s of C a i r n s  (1963) were confirmed on the s e q u e n t i a l C.  E_. c o l i ?  it  5 bromo-  radioactive  of the g e n e r a t i o n  (1963) and Lark et  time. al.  replication.  The r e p l i c a t i o n o f the E_. c o l ? chromosome.  Two important does the  growing  replication  q u e s t i o n s are to be asked h e r e :  1968, by G i l b e r t and D r e s s i e r  and by Werner,  how  s t a r t ; and (2) how does the c h a i n e l o n g a t e  Several models f o r  by Haskel and Davern  (l)  the  in  l a t t e r have been proposed s i n c e  (I968) and by Okazaki et^ al_.  (1968);  (1969); by Richardson (1969); by Morgan (1970);  (1971).  Many of the c u r r e n t models are  variations  on the simple model proposed by Watson and C r i c k (1953) s u g g e s t i n g a separation for  the s t r a n d s at  the growing p o i n t , a d d i t i o n of n u c l e o -  t i d e s by W a t s o n - C r i c k base p a i r i n g , and j o i n i n g (Guild,  1968;  Richardson,  Hurwitz e_t_ aj_.  1969)•  1968;  by enzymatic means  Okazaki et_ aj_. 1968;  Kornberg,  G i l b e r t and D r e s s i e r have proposed a general  the " r o l l i n g c i r c l e " to account f o r  1969; model,  the f e a t u r e of both E_. c o l i and  o t h e r b a c t e r i a or b a c t e r i o p h a g e r e p l i c a t i o n .  Briefly,  the model  proposes that s y n t h e s i s begins by opening one s t r a n d of the c i r c l e at a s p e c i f i c p o i n t by i n t r o d u c i n g a s i n g l e - s t r a n d  original  break  7  d i s p l a y i n g a 3 h y d r o x y l and a 5 - p h o s p h o r y l e n d - g r o u p . l _  to prevent ferred  In order  1  l i g a s e from r e p a i r i n g  to some s i t e ,  t h i s break,  perhaps on a membrane.  the 5'~end is  DNA polymerase then  t i a t e s s y n t h e s i s by e l o n g a t i o n of the 3 - e n d and uses the 1  circular off  s t r a n d as a t e m p l a t e .  trans-  The o l d p o s i t i v e s t r a n d  ini-  intact  is peeled  the c i r c u l a r template f o r DNA polymerase to s y n t h e s i z e the new  negative s t r a n d .  As the growing p o i n t c o n t i n u e s around the c i r c l e ,  a daughter molecule is peeled o f f  endlessly.  Kubitschek and Henderson (1966) and Morgan (1970) have proposed mechanisms in which n u c l e o t i d e p r e c u r s o r s are p a i r e d base p a i r s before being i n c o r p o r a t e d Although the two models d i f f e r mechanism, they both have the one p a r e n t a l  DNA s t r a n d  i n t o the daughter  important  fundamental  reaction  p r o p e r t y that o n l y  is c o p i e d in the event o f a mismatch.  To  of the above models,  (1971) has suggested a new mechanism f o r  E_. c o l i ,  strands.  in many r e s p e c t s r e g a r d i n g the  r e s o l v e some of the paradoxes c r e a t e d by a l l Werner  in W a t s o n - C r i c k  DNA r e p l i c a t i o n  in  and has introduced a reasonable doubt about what has been  accepted as p l a u s i b l e mechanisms f o r DNA r e p l i c a t i o n . short pulses of t r i t i a t e d thymidine, weight DNA is formed p r i o r DNA, s u g g e s t i n g that  In b r i e f ,  using  he found that high m o l e c u l a r  to the appearance of  low m o l e c u l a r weight  l a r g e DNA is the p r e c u r s o r of DNA f r a g m e n t s ,  and implying that Okazaki p i e c e s are not the r e s u l t of d i s c o n t i n u o u s synthesis.  During s h o r t p u l s e s , the r e l a t i v e  amount of  label  found  in Okazaki p i e c e s v a r i e d with the nature of the p r e c u r s o r used.  8  Twenty and,  percent of  the  incorporated  H-thymfne was found  in the presence of unlabeU.ed t h y m i d i n e ,  the  in the p i e c e s ,  incorporation  of  3 H-thymine i n t o Okazaki  p i e c e s was e n t i r e l y s u p p r e s s e d .  t h a t the p i e c e s a r o s e from s i n g l e s t r a n d n i c k s newly s y n t h e s i z e d DNA to a c t as swivel DNA h e l i c s d u r i n g r e p l i c a t i o n  He proposed  in both parental  points for  and t r a n s c r i p t i o n .  the  and  r o t a t i o n of  the  He a l s o suggests  3 that the  l a b e l i n g of  represents  repair  the p i e c e s d u r i n g short p u l s e s of  s y n t h e s i s , w h i l e thymine  Haskel and Davern  H-thymidine  is used f o r DNA r e p l i c a t i o n .  (1969) have presented the " p r e - f o r k  model f o r DNA r e p l i c a t i o n " . i n i t i a t e d from p a r e n t a l  In summary, DNA s y n t h e s i s  synthesis  is c o n t i n u o u s l y  s t r a n d n i c k s and o c c u r s ahead o f the  fork.  The n i c k s thus a c t as i n i t i a t i o n s i t e s f o r c h a i n s y n t h e s i s and small chains,  l i k e Okazaki f r a g m e n t s ,  are s y n t h e s i z e d v i a a polymerase.  The f o r k serves as a locus f o r unwinding and s e p a r a t i n g replicated  the  s t r a n d s of the two double h e l i c e s .  The p r e d i c t ions of the models proposed above have been in E_. c o l i ,  and,  in every c a s e , c e r t a i n  (1969a)and Caro (1970) have looked at r e s p e c t to the G i l b e r t that  Lark,  1969a). L a r k ,  the age and the p o l a r i t y  Lark  the q u e s t i o n of symmetry with  and D r e s s i e r model  in E_. c o l i , the DNA is r e p l i c a t e d  tested  drawbacks are p r e s e n t .  (1968).  It  was concluded  by a symmetrical  process and  i n i t i a t i o n takes p l a c e on both daughter chromosomes at once 1970;  already  (Caro,  in a very s o p h i s t i c a t e d way, excluded both of the template as a source f o r  asymmetry and thus absence of  inherent  asymmetry  selective  in the DNA m o l e c u l e ,  which c o u l d r e s t r i c t Finally  the s e l e c t i o n of template f o r  the " r o l l i n g c i r c l e " model was t e s t e d  up of synchronous B/r/1 asymmetrical  cells.  model p r e d i c t e d  in a n u t r i t i o n a l  R e s u l t s d i d not f i t  perimentally  The Werner  (1969) model has not been t e s t e d  Finally, 1968)  (1971) model has a l r e a d y  replication  used in t h i s  for  repair  E_. c o l i at  to a l l o w f o r  (15 TAMT, C a i r n s and  Denhardt,  time long enough It  is s e v e r e l y a f f e c t e d  Werner's o b s e r v a t i o n s c o u l d well  has been well  1967;  Shaw, 1968; Ng, 1969)-  into the o r i g i n a l  (1953) are v i a b l e and d i f f e r et a K  growth  (Ng, Marr and  be an a r t i f a c t of growth at  The remaining models which f i t  i s t i c sense (Okazaki  commun.).  group that macromolecular s y n t h e s i s and  Shaw and Ingraham,  of Watson and C r i c k  biosynthetic  short p u l s e l a b e l l i n g .  low temperatures  1962;  study  of  c o u l d not be sub-  0'Donovan, personal  14 C to e s t a b l i s h a g e n e r a t i o n  documented by Ingraham's  Ingraham,  and thymidine  1  was grown at  (Lark,  when h i s proposed r e c o g n i t i o n  (0 Donovan and Neuhard, 1970; the s t r a i n  B i o l o g y by  been c h a l l e n g e d  by the known sequence of the p y r i m i d i n e  (400 minutes)  of  ex-  (1969)-  personal commun.), p a r t i c u l a r l y  pathway  Thus,  examination.  in the Journal of T h e o r e t i c a l  Erhan (1969) and P h i l l i p s  stantiated  the  yet and probably should be c l a s s i f i e d under other schemes  t h a t have been compiled  thymine f o r  with  shift-  (C. B a g w e l l , unpublished r e s u l t s ) .  the " r o l l i n g c i r c l e " q u e s t i o n remains open f o r The H a s k e l l and Davern  replication.  Thus,  \k C.  suggestions  oh:ly in the mechan-  1968; R i c h a r d s o n , 1969;  Kornberg, 1969) -  The  first  q u e s t i o n , "how  does t h e r e p l i c a t i o n  start"?  r e m a i n s a s a g r e y , r a t h e r t h a n t h e b l a c k box o f m y s t e r y biologists.  Although the o r i g i n  and  still  for molecular  terminus of a c i r c u l a r  chromosome  s t r u c t u r e a r e t h e ends o f a  l i n e a r double strand, untwisting  c u l e , when i t i s i n h e l i c a l  form, would  breaks for  i n t h e c h a i n s (Wang a n d  Davison, 1968).  the c r e a t i o n o f a s w i v e l , which would  fork,  is s t i l l  r e p l i c a s e s a r e needed. merase)  polymerase  1969; no  be t h e g r o w i n g p o i n t o r  These have been p o l y m e r a s e  T.  K o r n b e r g and  longer i s q u a l i f i e d  III (Gefter polymerase)  Gefter,  1970),  in replication  et a l . 1969).  and  s e g m e n t s o f DNA  stricted be j o i n e d  to the fork together.  be g i v e n h e r e .  chromosomal  t o a f r e e 3 -OH 1  (Richardson, 1969),  f o r k must e x t e n d t h e m s e l v e s short  (de  Lucia  Kornberg  replicases chains.  h a v e b e e n shown t o h a v e an a b s o l u t e r e -  f o r adding nucleotides primer  poly-  seems t o be a  upon t h e f o r m a t i o n o f t h e f o r k , s y n t h e s i z e t h e new polymerases  The  but the  could,  existing  (Kelly  I (Kornberg  replicase  quirement  the  enzyme(s) o r  repair  S i n c e a l l DNA  occasional  However, t h e mechanism  to start, special  I I , ( C a i r n s p o l y m e r a s e ) and  Cairns,  by  mole-  obscure.  In o r d e r f o r t h e r e p l i c a t i o n  and  be g r e a t l y e n h a n c e d  the  the newly  i n an a n t i p a r a l l e l  at a given time. r e g i o n , and Recent  The  group o f p r e -  formed  c h a i n s at the  f a s h i o n by  synthesizing  s y n t h e s i s would  t h e segments formed  evidence supports this  would  be r e subsequently  s y s t e m and  will  In v i v o s t u d i e s by Y u d e l e v i c h et_ aj_. and  Iyer and Lark  Pol A  have  DNA at  the 3'~0H  indicated a preferential  g a r d l e s s of the  (1970), with E_. c o l i  555~7 (TAMT~) and E_. c o l i  l o c a t i o n of newly  synthesized  end of a l a r g e d e o x y n u c l e o t i d e s t r a n d .  the polymerase system u s e d ,  replication  (1968) , with E_. c o l ? CR34,  by the  the asymmetry  r e p l i c a s e mimics that of  the  in  Thus,  re-  starting  in v i t r o  synthesis  of DNA by the Kornberg polymerase. Studies  initiated  1968;  Y u d e l e v i c h e_t a_j_.  1969;  Iyer and L a r k ,  replication. reached:  Collectively,  of 80 m i n u t e s ;  thymidine  thymine,  Lark,  5 short pieces  personal  (1 micron)  s i n g l e strand pieces extractable  by  and  alkali;  in c u l t u r e s  with  time of k0 minutes and 3 with r e p l i c a t i o n  (3) the time r e q u i r e d f o r  I,  l -  time  the s y n t h e s i s of a 1 u p i e c e  is the same f o r  s i n g l e stranded DNA from the 3 e n d ,  f a s t and slow  growth  which s p e c i f i c a l l y h y d r o l y s e s  c o n f i r m s the e x t e n s i o n of  the  (1970), two e x p l a n a t i o n s  could  3'~end.  As pointed out by Iyer and Lark validate  (or  is mainly found as small  (4) s t u d i e s with exonuclease  c h a i n from the  B i r d and L a r k ,  c o n c l u s i o n s have been  37 C is 2 s e c o n d s , and t h i s  rates;  et a 1.  the f o l l o w i n g  there are about  chromosome r e p l i c a t i o n  at  label  (3 to 300 microns) instant,  1968;  (Okazaki  1970), has supported the d i s c o n t i n u o u s mode of  incorporated  (2) at any  Sadowski et^ a]_.  1968;  (1) with p u l s e s of  commun.), large  by s e v e r a l workers on E_. c o l i  the observed occurence of  the s h o r t and long p i e c e s ;  (1)  s y n t h e s i s occurs by the continuous e x t e n s i o n from the 3'~end a n d ,  for  s h o r t p i e c e s , in the 5' and 3' d i r e c t i o n ; symmetrically  from 3  1  (2) s y n t h e s i s o c c u r s  to 5' on both s t r a n d s but a symmetrical  f r a g m e n t a t i o n o c c u r s o n l y on the 5 s t r a n d , , _  the s i n g l e s t r a n d b r e a k s . system. of  These p o i n t s a r e not c l e a r  DNA (Kainuma and O k a z a k i , 1970) w h i l e  the s h o r t p i e c e s anneal  and H u r w i t z ,  contains  in the E_. c o l i  (Ginsberg  1970; Okazaki et_ a_j_. 1970).  1970; Mordoh et_ aj_. replicative  in Jk and Lambda  e q u a l l y to both s t r a n d s  Recent s t u d i e s on t o l u e n i z e d  of a l l  it  in EL s u b t i l i s , p i e c e s a r e complementary to o n l y one s t r a n d  parental  phages,  such that  1970;  s y n t h e s i s of  E_. c o l ?  eel 1s (Moses and R i c h a r s o n ,  Kohiyama and K o l b e r , DNA under  1970) have shown  in v i v o c o n d i t i o n s , the presence  f o u r d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e s , and have i n d i c a t e d a  stimulation  in s y n t h e s i s by ATP a l o n e .  s y n t h e s i s c o u l d be a b o l i s h e d  Furthermore,  the  in DNA t e m p e r a t u r e - s e n s i t i v e  Moses and Richardson (1970) have c h a r a c t e r i z e d f u r t h e r  the  DNA by s e d i m e n t a t i o n and pyenographic a n a l y s i s , and there doubt that t h i s  replicative mutants. newly-made is  i n c o r p o r a t i o n does correspond to chromosomal DNA.  T h i s system has not been e x p l o r e d enough to a l l o w a n a l y s i s of steps  involved  little  in the  replication  nor of  the  the q u e s t i o n of d i s c o n t i n u o u s  r e p l i cat i o n . The model of d i s c o n t i n u o u s s y n t h e s i s i m p l i c a t e s DNA l i g a s e to c o v a l e n t l y j o i n the fragments. Lehman, 1971;  Sadowski et_ aj_.  1968) or  in v i v o  the need f o r  In v i t r o  purported  functions.  (Modrich and  ( P a u l i n g and Hamm,  s t u d i e s on DNA l i g a s e c l e a r l y document the presence of  the  1969)  t h i s enzyme and  13  Preliminary  approaches to the d i s s e c t i o n of the a c t i v e  machinery, which would c l a r i f y n u c l e a s e complex e x i s t s Knippers et_ aj_.  whether  in E_. c o l i , are promising  Kohiyama and Kober, 1970).  1970;  systems should demonstrate e x a c t l y in E_. c o l i  is D.  or not the  how the  polymerase-1igase(Smith et a 1 . 1970; Hopefully,  initiation  S e p a r a t i o n of daughter  between daughter c e l l s .  with a u t o r a d i o g r a p h y  of DNA r e p l i c a t i o n  chromosomes: S e g r e g a t i o n .  Ryter,  is random, t h a t  Hirota,  i s , at  s t u d i e s coupled  (Ryter,  1968;  each c e l l  d i v i s i o n , each of  into e i t h e r  progeny.  the o l d Lark  r e s u l t s which suggested that a d e f i n i t e  segregation e x i s t e d .  More r e c e n t l y  upon reexamination  the p r e v i o u s model, support R y t e r ' s  of  however,  indeed  Rubenstein et a l .  and Jacob (1968) suggested t h a t  has the chance o f d i s t r i b u t i o n obtained d i f f e r e n t  Morphological  become evenly  have shown that the E_. c o l 1 chromosome is  a s s o c i a t e d w i t h the c e l l membrane S t u d i e s of  these  started.  R e p l i c a t e d DNA and chromosomes e v e n t u a l l y partitioned  replicating  Chai and Lark  1970).  segregation strands (1966) had p a t t e r n of (1970), random  s e g r e g a t i o n model.  II.  C e l l d i v i s i o n c y c l e of A.  RNA and p r o t e i n  During  E_. c o l i  s y n t h e s i s in the normal d i v i s i o n c y c l e of E_. c o l i  i t s d u p l i c a t i o n c y c l e , RNA and p r o t e i n s are  s i m u l t a n e o u s l y with r e p l i c a t i o n of  synthesized  the chromosome and thus the  cell  increases general  in s i z e , mass and c e l l u l a r  RNA s y n t h e s i s , the  amount o f  rate  constituents.  is a p p a r e n t l y  template DNA a v a i l a b l e  (Cultler  proportional  patterns  d u r i n g the c e l l  Although a requirement (Mathison,  1968)  in c e l l  identified  unique to c e l l  Smith and Pardee, criticisms:  (1) i f  not known; and gene product of postulated for  1969; 1970;  protein  division.  1970).  1970;  Reeve et_ aj_.  but the work s u f f e r s  these p r o t e i n s a r e enzymes, t h e i r  (2) in no case have those p r o t e i n s lesion.  A  the " d i v i s i o n p r o t e i n s "  "division  groups (inouye and G u t h r i e ,  Inouye and Pardee,  the g e n e t i c  have been  However, changes in the  1971)  fluct-  s y n t h e s i s has been demonstrated  by s e v e r a l  Inouye,  the  Helmstetter,  indicate  d i v i s i o n , no s p e c i f i c p r o t e i n s  p r o t e i n s " have been i m p l i c a t e d 1969; Green et_ aj_.  hand,  c y c l e (Mychalowska,  for  to  and Evans, 1967;  1969b). The RNA p r e c u r s o r p o o l s , on the o t h e r uating  In the case of  role  1970;  two  substrates  are  been shown to be the  in s e p t a t i o n  has been  in the above l i s t e d works  in  general . In the absence of p r o t e i n observed  (Pierucci  was e n t i r e l y p l e t i o n of  synthesis, limited  and H e l m s t e t t e r ,  1969)-  blocked d u r i n g DNA r e p l i c a t i o n ,  d i v i s i o n has been  When p r o t e i n in s p i t e of  r o u n d s , no d i v i s i o n was o b s e r v e d .  However,  synthesis the com-  if  protein  s y n t h e s i s was i n h i b i t e d  subsequent to the completion of a round,  d i v i s i o n was o b s e r v e d .  These r e s u l t s were c o n s i s t e n t with  "trigger"  f o r d i v i s i o n idea p r e v i o u s l y suggested by C l a r k  Helmstetter  and P i e r u c c i  C968).  T h i s concept  is probably  the (1968) and correct,  but  i t does not  sion  entail  ( D o n a c h i e e_t_ aj_.  Changes cycle  i n the  t h e a d d i t i o n a l r e q u i r e m e n t s needed f o r 1968),  patterns  r e m a i n s t o be  of  that  i s , the  these d i v i s i o n  Physiological division  The  importance of i n t o two  and  separating  (1968).  Clark  two  p h y s i o l o g i c a l l y separated  are  t h e n by  a strong  implicated  a burst  in the  It  the  duplicated c e l l ,  end by  physiological division  of a the  round o f formation  (Clark, 1968).  cross-wall  synthesis  entities  o f membranes  erect  the  septum.  would a l l o w formation anchorage s i t e  of a  In s u c h a c a s e , light  f o r d e p o s i t i o n of  Recently,  a membrane p r o t e i n has  function  is organizational rather  a  binding  III. A.  site  f o r ATPase  Regulation Control Hirota  of  of  the  been  the DNA  cell  isolated  o f a weak  suggested  has the  priming  need before  division an  f o r septum enzymes.  i n S_. f a e c a l i s whose in that  i t serves  as  1971).  duplication cycle  replication  et a l . (1968)  replication,  i n E_. c o l i a t  physiological  proteins  Abrams,  been  building blocks  than c a t a l y t i c  ( B a r o n and  compart-  has  b a r r i e r w h i c h w o u l d a c t as binding  by  1969).  (Daniels,  i s p o s s i b l e t h a t s e p t a t i o n enzymes and  can  normal  Evidence  m e m b r a n e - p r o t e i n s c o m p l e x e s f o r t h e i r a t t a c h m e n t and they  the  ratio.  septation.  the  A f t e r the  s e p t u m and  point of  proteins during  physiologica11y separate  e m p h a s i z e d by cells  mass-to-DNA  studied.  B.  mentalization,  critical  divi-  i n E.  coli.  i n E_. c o l i , two  modes o f  regulation for  the  initiation  is a n e g a t i v e  of  the chromosome r e p l i c a t i o n  system, that  b l o c k the r e p l i c a t o r  the  enzymes.  variations  1963) from being  in the c o n c e n t r a t i o n of  some m o l e c u l e ,  r e g u l a t i o n of which was through DNA r e p l i c a t i o n or c e l l u l a r implicates  is produced in each DNA c y c l e which c o u l d d i r e c t l y No f i r m experimental S t u d i e s of synthesis  is  data  in support of e i t h e r  r e p l i c a t i o n under c o n d i t i o n s  inhibited  specific initiator  hibited  (Lark and Ranger, proteins  have i n d i c a t e d  quired  in o r d e r  to  (1969)  by s t a r v i n g  i n i t i a t e new rounds of  55 7 -  (a  15  following  the  Under amino a c i d  When CAM  (300 yg per ml)  was added to  several  was concluded that once a new c y c l e had been i n i t i a t e d , initiations  p r o t e i n was needed.  c o u l d o c c u r a n d , . t o prevent  this,  for  the  " u n c o n t r o l l e d " DNA r e p l i c a t i o n was observed over  unregulated  amounts o f  amino a c i d s were withdrawn,  al.  accumulation  T was added back, DNA r e p l i c a t i o n c o n t i n u e d  a round and then s t o p p e d .  other  in-  Rosenberg et  T ) f o r T , obtained  s y n t h e s i s was h a l t e d .  It  synthesis is  replication.  net p r o t e i n  hours.  is  re-  T starvation,  latter culture,  1968)  protein  If  if  protein  in  potential..  however,  available.  p r o t e i n s are present  initiation  starvation,  is  in which s y n t h e s i s  (Levine and S i n s h e i m e r ,  that such i n i t i a t o r  E_. c o l i  i n i t i a t e new rounds.  hypothesis  or  growth.  that a molecule  in which gross  1969),  s t o i c h i o m e t r i c amounts and that a d d i t i o n a l  of  first  The d e r e p r e s s i o n c o u l d occur by  The second is a p o s i t i v e r e g u l a t i o n which  of  The  i s , the presence of a r e p r e s s o r which would  ( J a c o b , B r e n n e r , and C u z i n ,  a c c e s s i b l e to r e p l i c a t i n g sudden c r i t i c a l  in E_. c o l i .  still  small  S i m i l a r c o n c l u s i o n s have been reached  by  Kogoma and  replication DNA of  with  synthesis protein  an  NAL,  can  initiator  rates.  proceed  Barth  protein  a constant An  f o r many h o u r s  and  (1969)  Collins DNA  replication.  i s made i n t h e c e l l fraction  inhibitor  of  protein  total  i s coded  t h e chromosome o r i g i n ,  or  cribed  replication  only during  g e n e , and the  the  There  p r o t e i n and action  part of  is a cooperative  e i t h e r the  of  initiation  concentration  inhibitor  of  of  the  inhibitor  of  inhibitor.  m i n e d by  the d i l u t i o n  the growth  rate.  The  h a v e p r o p o s e d an a l t e r n a t i v e Their  model  suggests  constituitively  p r o t e i n of  by a g e n e l o c a t e d a d j a c e n t origin  i t s e l f , and  t h a t gene.  Each  inhibitor for  p r o t e i n s at a l l growth  by g r o w t h o f  inhibitor This  inter-  effects  frequency of  important  the c e l l ,  the  level,  an  Thus, the  be d i l u t e d p r o g r e s s i v e l y .  withtthe  to  is trans-  is responsible  inhibitor  below the c r i t i c a l  the  con-  at a l l growth  change i n i t s c o n c e n t r a t i o n  will  that  which  the c e l l  o r chromosome o r i g i n .  be a s s o c i a t e d  r a t e of An  the  increases  falls  place, which w i l l  pulse of  of  of  inhibition  over a range from complete to z e r o .  volume  takes  inhibition  the  i n t e r a c t i o n between the  initiator  when t h e c y t o p l a s m i c  level  the  number o f  i s such t h a t a t w o - f o l d  inhibition  the  in s p i t e of  c o n s e q u e n t l y e a c h chromosome o r i g i n ,  synthesis of a fixed  rates.  of  temperature s e n s i t i v e mutations, or T s t a r v a t i o n ,  f o r the c o n t r o l of  stitutes  where, f o l l o w i n g a p e r i o d  synthesis.  Pritchard, model  (1970)  Lark  When  reinitiation  production  initiation will  of a be  new  deter-  inhibitor,  that  i s , the r e c i p r o c a l  feature of  this  model  is that  (1)  it  is s e l f - r e g u l a t o r y  (1968)  Simon cycle  and  (2)  assumes a n e g a t i v e c o n t r o l of  proposed a s e m i q u a n t i t a t i v e  in E_. c o l ?, where  it  model f o r  1970)  by the a u t h o r .  precedent.  The mutant,  the p r o t e i n , which c a t i o n to s t a r t . required for Hirota initiator  t h i s model.  initiating  mutant  protein  (Hirota,  et a 1 .  i s assumed to have an a l t e r a t i o n  repli-  t h i s component would a c t o n l y when  of  the  CR3^T46 by the n e g a t i v e c o n t r o l , the s y n t h e s i s of a (Pritchard  et_ aj_.  In t h i s c a s e , in the  30 C i s enough f o r  be enough time f o r  it  1969), o r o f the a n t i r e p r e s s o r is argued that the Tk6  regulatory  reinitiation  the d i l u t i o n of  mechanism, s i n c e o n l y  to o c c u r , but would not  the r e p r e s s o r to o c c u r .  is the element a f f e c t e d ,  model where the a n t i r e p r e s s o r  it  If  the  would be c o m p a t i b l e with a  is made immediately a f t e r  DNA r e p l i c a t i o n  has s t o p p e d . It  in  a new c y c l e .  is not an a l t e r a t i o n  antirepressor  initiator  control  (1970) have a l s o c o n s i d e r e d the r e g u l a t i o n  (Rosenberg et_'aj_. 1969) -  10 minutes at  the  CR34T46,  Furthermore,  et^ al_.  (Mychajlowska,  in some way, a c t s on the chromosome to a l l o w  r e p r e s s o r d u r i n g growth  mutation  cycle  The p o s i t i v e model c o u l d e x i s t with a n e g a t i v e  1970).  starts  r e g u l a t i o n of DNA r e p l i c a t i o n a r e not without  o p e r a t i n g on the s y n t h e s i s of 1968;  cell  T h i s model was not  The dTTP l e v e l s d u r i n g the c e l l  however, c o u l d s u b s t a n t i a t e The two models of  the  was assumed that the DNA r e p l i c a t i o n  when a t h r e s h o l d dTTP c o n c e n t r a t i o n was r e a c h e d . tested  replication.  should be p o s s i b l e to r e s o l v e these q u e s t i o n s by study of  several  "initiator"  been i s o l a t e d 1968;  mutants.  in E_. c o l i  Kuempel, 1969;  characterized typically,  mutants  It  date t h i s  is not B.  initiation  initiation, and the  R e g u l a t i o n of c e l l  DNA s y n t h e s i s .  deal  Geho-  lethal  s p e c i f i c a l l y with  r e g u l a t i o n of  initiation.  To  it  d i v i s i o n in £_. c o l i  known f o r c e r ° t a i n about normal  is coupled to DNA r e p l i c a t i o n  and P i e r u c c i , 1968).  a l l o w s f o r 25 percent  (Donachie,  residual  are  clear.  i s that  Pierucci,  have  Fangman and N o v i c k ,  is a l s o p o s s i b l e t h a t t h e . c o n d i t i o n a l  The o n l y f a c t division  1966;  such mutants  i n t o the DnaA c l a s s (Fangman and N o v i c k ,  i s o l a t e d , which a f f e c t stages of  several  P h e n o t y p i c a l l y , these mutants  capacity for  they do not f a l l  different  stetter  (Kohiyama et a l .  C a r l , 1970).  by t h e i r  1968; C a r l , 1970).  In the p a s t ,  residual  E_. c o l ? eel 1  (Clark,  I n h i b i t i o n of DNA r e p l i c a t i o n division  (Clark,  Inouye,  Even p r o d u c t i o n of  DNA-less c e l l s at DNA (Hi r o t a et al_.  and  division  1969), and DNA-less c e l l s a r e not produced.  DNA-less m i n i c e l l s by a mutant  blocked upon s t o p p i n g of DNA r e p l i c a t i o n is p o s s i b l e t o get  normally  1968b; H e l m s t e t t e r  1968), or causes an immediate c e s s a t i o n of c e l l 1969;  1968b; Helm-  temperature  (Clark,  s e n s i t i v e mutants  kO C when they have l o s t  their  of E_. c o l i was 1968b).  However,  it  that c o u l d produce ability  to s y n t h e s i z e  1968).  Inouye (1971) has shown that eel 1 d i v i s i o n can be uncoupled from DNA r e p l i c a t i o n  by i n t r o d u c i n g a d e f e c t i v e  in r e c o m b i n a t i o n ,  recA gene, which i s  involved  i n t o E_. colj^ when DNA r e p l i c a t i o n was b l o c k e d .  Hence DNA-less c e l l s  result.  The product of  the recA gene has been  shown to reduce the amounts of n u c l e a s e s produced by the genes  ( W i l l e t s and C l a r k ,  between the  1969;  1970),  but the  correlation  recA gene product and the septum i s not yet u n d e r s t o o d .  I n t e r e s t i n g l y enough, whenever uncoupled by mutations mutants  Barbour et_ aj_.  recB and recC  behave as i f  the DNA r e p l i c a t i o n and c e l l  so t h a t DNA-less b a c t e r i a  rounds of  d i v i s i o n are  are p r o d u c e d , these  r e p l i c a t i o n a r e completed at  regular  intervals. A r i g o r o u s a n a l y s i s of  r e g u l a t i o n of E_. c o l i eel 1 d i v i s i o n  the  was r e p o r t e d by Donachie and Begg, between DNA s e g r e g a t i o n and c e l l a unit c e l l  concept.  However,  E^. c o l ? , and the steps division,  in which the  d i v i s i o n were e x p l a i n e d  the mechanism f o r c e l l  involved  remain s p e c u l a t i v e  (1970),  coordination in terms of  division  in  in the r e g u l a t i o n of e x p r e s s i o n of  (Pardee,  1968;  Previc,  1970).  MATERIALS AND METHODS  I.  Bacterial A.  and Phage S t r a i n s  Bacterial  strains  Bacterial are g iven  B.  s t r a i n s of  Escherichia coli  study  i n Tab 1e I .  Bacteriophage s t r a ins For  the t r a n s d u c t i o n e x p e r i m e n t s ,  ducing b a c t e r i o p h a g e P l k c - L 4 (Caro and B e r g .  1 I.  used in t h i s  the g e n e r a l i z e d  trans-  1 9 7 1 ) was u s e d .  Media and c h e m i c a l s A.  Media E_. c o l I  broth medium A of  CR34T83 was grown in a v / v mixture of t r y p t o n e Kaiser  Vogel and Bonner ( 1 9 5 6 ) .  ( 1 9 5 5 ) and the minimal The f o l l o w i n g  g l u c o s e , 0.5 ug per m l ; t h y m i d i n e ,  supplements were added:  20 yg per m l ; deoxyadenosine,  50 yg per m l ; v i t a m i n B j , 7-5 yg per m l . referred  to as A and E , r e s p e c t i v e l y .  conditions, minimal  the  s a l t s medium E of  These media w i l l  When grown under minimal  r e q u i r e d amino a c i d s were added at  s a l t s medium E of Vogel and Bonner  E_. c o l I B/r/1 was grown in 007 minimal g l u c o s e added ( C l a r k and Maal«5e, 1 9 6 7 ) .  be  20 yg per ml  to  (1956). s a l t s medium w i t h 0.5%  Table  I.  Stra i n Number KG  55  KG 71  Bacterial  strains.  Mat ing Type  Relevant  Derivation and source  Genetype  t h r " , leu , pro , h i s , t h i , arg , l a c , AB 1 1 5 7 from r r E . A . Adelberg gal , a r a , x y i , man , T^ , s t r thr  , leu , t h i  , i l v , thyA , DnaA  CR34T83 from M. Kohiyama  KG 7 1 - 2  Same as KG 7 1 but low thymine requ i r e r  KG 7 k  thr  , leu , t h i  , i l v , thyA .  DnaA  T h i s work  KG 77  thr  , leu , t h i  , ilv ,  DnaA*  T h i s work  KG 78 (1-40)  KG 146, Ilv"*", DnaA"  (141-216)  +  thyA ,  T h i s work  T h i s work  KG 163, i l v , DnaA Auxotroph  B/r/1 from D.J. Clark  KG 110  thyA  15 TAU" from P. Hanawalt  KG 142  P r o t o t r o p h . T^ ,  KG 99  P1kc  , a r g , ura  , s t r , Tg ,  X289 from R. C u r t i s s  indicator  JC-533 from John C l a r k  KG 146  thi  , arg , i l v ^  KG 162  trp  , lac_ , tha , s t r  #BE235 from N. O t s u j i  KG 163  trp  , lac  #BE269 from N. O t s u j i  KG 173  arg  2  , metB  , tha , i l v , s t r  I II  JE. c o l i C-122 from L. Katz  For the t r a n s d u c t i o n e x p e r i m e n t s , specified  B.  by C a r o and  Berg  the media used  were  as  (1971).  Chemicals. 14 Thymidine  New  York;  2-  C,  Schwarz B i o r e s e a r c h i n c . ,  3 t h y m i d i n e m e t h y l - H,  S e a r l e Co.,  Des  Plaines,  Orangeburg,  14 C-5 bromo-2-deoxyuridine, 32 Illinois; P i was o b t a i n e d f r o m  Amersham/  -  i  Tracer  Laborator ies. C h l o r a m p h e n i c o l , Sigma C h e m i c a l  Corporation, St.  M i s s o u r i ; Na1 i d i x i c a c i d , S t e r l i n g W i n t h r o p R e n s s e l a e r , New California;  III.  York;  Daunomycin-HC1, C a l b i o c h e m ,  Rifampin, Calbiochem,  Los A n g e l e s ,  Los  Foundation, Anageles,  California.  C u l t u r e Methods A.  Growth c o n d i t i o n s  for liquid  cultures.  Stock c u l t u r e s of the s t r a i n s agar  Research  Louis,  slants.  used were m a i n t a i n e d  S t a r t e r c u l t u r e s were p r e p a r e d  by  inoculation  on into  g A and  E medium t o a d e n s i t y o f 10  were kept a t 4 C u n t i l  cells  per ml.  These  cultures  needed.  Batch c u l t u r e s were  i n c u b a t e d by s h a k i n g a t t h e  desired  temperature.  B.  Plating  methods.  Viability  a s s a y s as w e l l  as  transduction assays  were  c a r r i e d out on 1.5%  agar p l a t e s which were incubated at  the p l a t e s and check f o r c o n t a m i n a t i o n . h C until  C.  30 C to dry  The p l a t e s were s t o r e d  at  needed.  Temperature s h i f t c o n d i t i o n s . All  cultures  temperature  s h i f t s were accomplished by  i n t o f l a s k s prewarmed at  warmed f l a s k had a volume of being t r a n s f e r r e d ,  the new temperature.  ten times  temperature  transferring If  the  pre-  that of the volume of medium  e q u i l i b r a t i o n was reached tn l e s s than  two m i n u t e s .  IV.  Measurement of macromolecular s y n t h e s i s and c e l l  A.  Measurement of c e l l The  (Nuclear  Data,  cell  numbers.  numbers were determined  Palatine,  growth.  Illinois)  by a C o u l t e r Counter  and t h e i r  s i z e d i s t r i b u t i o n was  shown by an a t t a c h e d Nuclear Data 512 channel p u l s e h e i g h t a n a l y s e r (model  2200).  Interval  of  q u a l i t y of b u t i o n of and  C e l l s passed through a 30 u o r i f i c e f o r a time  ten seconds.  T h i s system a l l o w s m o n i t o r i n g of  the growth by measuring the c e l l the c e l l  Maaltfe,  numbers and the  volumes w i t h i n the p o p u l a t i o n of c e l l s  1 9 6 7 ; P a i n t e r and M a r r ,  1968).  the distri-  (Clark  B.  Measurement of c e l l  mass.  S p e c t r o p h o t o m e t r i c measurement of growth was using e i t h e r  a Klett-Summerson p h o t o e l e c t r i c c o l o r i m e t e r with a  No. 65 f i l t e r ,  or a P e r k i n - H i t t a c h i  using a c u v e t t e with a 1 cm l i g h t  C.  Measurement of  spectrophotometer  tryptophanase  on a s e l e c t i v e medium and then  tryptone  f o r m a t i o n of tryptone  broth.  indole  broth,  in an o v e r n i g h t  +  testing  t r a n s d u c t a n t s were  inoculated  A typical  c u l t u r e of a s t r a i n grown  Total  DNA s y n t h e s i s .  DNA s y n t h e s i s .  thymidine  i n c o r p o r a t i o n of  i n t o the TCA i n s o l u b l e f r a c t i o n of  uptake experiment  i n v o l v e d growing the c e l l s  l a b e l l e d medium c o n t a i n i n g the  activity  with u n l a b e l led c a r r i e r  Deoxyadenosine was present  50 ug per m l .  in  ( D i f c o Manual, 9 t h e d . , p. 5 3 ) .  actively  ml.  i n t o 3 mis of  was assayed by  DNA s y n t h e s i s was measured by the radioactive  trans-  by adding 2 ml of E h r l i c h s o l u t i o n and o v e r -  Measurement of 1.  ilv  Tryptophanase a c t i v i t y  l a y i n g the mixture with 1 ml of x y l o l  D.  500 nm  activity.  d u c t a n t s with the u n s e l e c t e d tna marker.  liquid  at  path.  Tryptophanase assay was performed f o r  purified  performed  At r e g u l a r  label  at a f i n a l in a l l  intervals,  at  in  the  cells.  radio-  the d e s i r e d s p e c i f i c  c o n c e n t r a t i o n of 20 pg per  cases at a c o n c e n t r a t i o n of  one ml samples were removed and  added to 3 ml of  7-5% TCA w i t h 200 ug  Each sample was f i l t e r e d four  per ml unlabel led  through a 0.45 u M i l l i p o r e  thymidine.  filter  and washed  times w i t h 5% TCA c o n t a i n i n g 200 ug per ml t h y m i d i n e .  were washed two times with hot w a t e r , f o l l o w i n g (Lark,  Repko, and Hoffman,  1963).  than the use of TCA a l o n e .  After  placed  into polyethylene  10 ml of  liquid  40 m l : l i t r e , minutes  heat  the TCA washes  d r y i n g , the f i l t e r s  Amersham/Searle C o r p . ) .  were  (Amersham/Searle C o r p . )  counting s o l u t i o n  in a U n i l u x S c i n t i l l a t i o n  filters  T h i s method gave lower backgrounds  spectravials  scintillation  The  containing  (Liquif 1uor:toluene;  Samples were counted f o r  Counter  (Nuclear  ten  Chicago C o r p . )  and counts were c o r r e c t e d f o r background. In some e x p e r i m e n t s , the  radlolabel  for  the t o t a l  i n c o r p o r a t i o n of  c h e m i c a l s , the samples were processed by the  batch  method d e s c r i b e d by B y f i e l d and Scherbaum (1966).  2.  Measurement of the  r a t e of  DNA s y n t h e s i s .  The r a t e of s y n t h e s i s of DNA was estimated 14 l a b e l l i n g with  3 C - or  per ml deoxyadenosine. ferring 100 u l  H-thymidine, A typical  again  in the presence of 50 ug  uptake experiment  one ml samples of c e l l s to 13 x 100 mm tubes of  the  by p u l s e -  labelled  thymidine.  After  involved  trans-  containing  three minutes  the  incorpora-  t i o n was stopped w i t h three ml of 7-5% TCA c o n t a i n i n g unlabel led thymidine.  Each sample was washed and processed as above.  27  E.  Measurement of a c i d s o l u b l e n u c l e o s i d e  triphosphate  pools. The chromatographic a n a l y s i s of the t r i p h o s p h a t e s was based on the chromatographfc s e p a r a t i o n of the n u c l e o s i d e phosphates by Randerath and Randerath Mychajlowska  tri-  (1967) as m o d i f i e d by L.  (1970 M . S c . t h e s i s , UBC).  1.  P r e p a r a t i o n of the samples.  -6 C e l l s . w e r e grown phosphate)  and were allowed  in low phosphate b u f f e r  to e q u i l i b r a t e  f o r the pools at  (10  M  least  32 one g e n e r a t i o n  (40 minutes at 30 C) a f t e r a d d i t i o n of the  To measure the n u c l e o s i d e t r i p h o s p h a t e  pools,  were mixed w i t h 100 y l of 2N f o r m i c a c i d . mixed and allowed for  in an i c e - b a t h f o r ten to f i f t e e n  minutes  These samples were  in a Beckman M i c r o f u g e and the supernatants were saved  s p o t t i n g on t h i n  2.  l a y e r chromatographic  plates.  Chromatography. Polyethyleneimine-impregnated  were made a c c o r d i n g to Randerath and Randerath Mychajlowska.  cellulose  to the p l a t e s  plates  (1967) by Miss  These p l a t e s were used w i t h i n a week a f t e r  The sample, supernatant quantities  samples  The c o n t e n t s were then  the l y s i s and r e l e a s e o f the pool m a t e r i a 1 .  centrifuged for  t o stand  250 y l c e l l  P..  preparation.  o f the a c i d h y d r o l y s i s , was added in 5 x 20 y l in a slow manner to avoid f l o o d i n g the s u r f a c e  of  the p l a t e s .  dried off  After  the s p o t t i n g was c o m p l e t e d , the p l a t e s were  and then washed w i t h methanol  (anhydrous)  f o r 20 to 30  minutes to remove unnecessary s a l t s from the medium and contaminating label. Two dimensional chromatography was performed a c c o r d i n g to the method of  Irr  and G a l l a n t  dimension was 1N A c e t a t e : l M L i C l one-half after  inches from the t o p .  this  (1:1,  The  v/v) , until  first  about one and  A second run was performed  in a 1N A c e t a t e : 1.5M L i C l  (1:1,  allowed to run in the same d i r e c t i o n f o r To a l l o w f o r a b e t t e r  (1969)-  v/v)  immediately  s o l v e n t and  f i v e and o n e - h a l f h o u r s .  s e p a r a t i o n of the d e o x y r i b o n u c l e o s i d e t r i -  phosphates from the r i b o n u c 1 e o s i d e t r i p h o s p h a t e s , wicks were made out of Whatman No. 3 f i l t e r "run o v e r " the p l a t e .  paper which allowed the s o l v e n t  At the end of the  fiirst  taken out and d r i e d and washed with methanol the f i r s t  run.  to  r u n , the p l a t e s were to remove the L i C l  The second s o l v e n t system was made of 3M NH^Acetate  and h.3% B o r a t e , pH 7.0, and the p l a t e s were allowed to run f o r and o n e - h a l f  of  hours in t h f s  system.  p l a t e s were d r i e d and prepared f o r  3.  At the end of  this  run,  four  the  autoradiography.  Autorad i o g r a p h y .  32 The d r i e d p l a t e s c o n t a i n i n g the  P.-labelled  t r i p h o s p h a t e s were p l a c e d over Kodak Royal Blue medical X-Ray f i l m s , placed  in a l i g h t - p r o o f  box, and allowed to process f o r  three  days.  29  These films were developed for 3-5 minutes in Kodak X-Ray developer, rinsed in water, and fixed ten minutes in Kodak X-Ray f i x e r .  The  films were then rinsed again with water and d r i e d . The areas which showed r a d i o a c t i v i t y were c l e a r l y seen as dark (exposed) placed over their  spots on the f i l m .  These films were then  corresponding plates and with a pin the exposed  spots were punched out on the plate.  The radioactive area was then  scraped off the p l a t e , with the aid of a razor blade, and the scraped an ion-exchange resin with the absorbed nucleotides were then collected from the plates and put in s c i n t i l l a t i o n v i a l s .  These v i a l s were  properly marked as to the nature of the spot (ATP,.etc) and ten milliirtres  of s c i n t i l l a t i o n f l u i d was added.  The radtoactivity was  counted in a Unilux S c i n t i l l a t i o n Counter for ten minutes.  Counts  per minute were corrected for backgrounds.  V.  Density Gradient Sedimentation A n a l y s i s . A.  Measurement of Brllra incorporation in the DNA. Strains 15-TAU and KG-71-2 were grown in the presence of  thymidine and deoxyadenosine until ml was obtained.  a density of 5 x 1 0 ^ c e l l s per  The c e l l s were then f i l t e r e d  and washed free of  thymine and resuspended in ^ C - B r U r a (Schwartz-Bioresearch Co.) with a final  s p e c i f i c a c t i v i t y of 0.033 yc per ml (6 yg per ml f i n a l  concentration).  These c e l l s were incubated at 30 C and, at  timed  Intervals, one ml samples were taken arid processed as described in Section V . A .  For the gradient a n a l y s i s , 10 mis of c e l l s were rprocessed  as d e s c r i b e d below  B.  Density  in S e c t i o n  VI.D.  label 1ing.  Procedures f o r  the  l a b e l l i n g of  the chromosomes with r a d i o a c t i v e p i c k - u p of to L a r k ,  and ends of  thymidine and subsequent BU d e n s i t y  the d e s i r e d s e c t i o n of  Repko and Hoffman  the s t a r t s  the chromosome was done a c c o r d i n g  (1963).  Overnight grown c e l l s of  KG-71-2  3 were grown in the presence of 20.6  Ci per mM, f i n a l  50 ml f l a s k .  3  H-thymidine  H-thymidine,  c o n c e n t r a t i o n of  The c e l l s were s h i f t e d medium.  specific activity  1 mCi per m l , at  30 C in a  to kl C f o r an hour  T h e n , these c e l l s were f i l t e r e d  of  in  free  the of  3  H-  t h y m i d i n e , washed twice w i t h prewarmed A and E medium, and r e s u s pended in f r e s h medium with supplements and activity (Schwartz  52.8  mCi per mM, f i n a l  Bioresearch C o . ) ,  c e l l s were f i l t e r e d After  an hour at  C-thymidine,  c o n c e n t r a t i o n 0.5 uCi per ml  in 20 ml medium f o r 25 m i n u t e s .  and allowed  t o randomize f o r f i v e  kl C , the c e l l s were f i l t e r e d  At the end of  this  The  generations.  and put  of medium c o n t a i n i n g 10 yg per ml BrUra and allowed f i f t y minutes.  specific  i n t o 50 mis  to grow f o r  time the c e l l s were c o l l e c t e d  for  lys i s .  C.  P r e p a r a t i o n and a n a l y s i s of DNA samples by d e n s i t y  gradient  centr ifugat ion. 1.  E x t r a c t i o n of DNA Cells collected for  the d e n s i t y g r a d i e n t  technique were  put d i r e c t l y  onto f r o z e n A and E medium and saved u n t i l  Frozen c e l l s were thawed at at  8000 rpm f o r  f i v e minutes.  one ml of T r i s - E D T A b u f f e r (Calbiochem)  was added at  and the mixture was minutes,  This p e l l e t  (0.01 a final  to a t o t a l  and were t r e a t e d with 50 y l  of  Centrifugation To 3-26  optical  grade)  ml of  was added.  d i s s o l v e the C s C l .  200 ug per ml  C f o r - 2 0 minutes. sodium l a u r y l  in  Lysozyme  After  20  s u l f a t e was added  l y s a t e s became v i s c i o u s .  of 3-5  ml with d i s t i l l e d  pronase ( W o r t h i n g t o n ) ,  c o n c e n t r a t i o n of 200 yg per m l ,  2.  37  Immediately, the  The l y s a t e s were brought  pH 8.2).  c o n c e n t r a t i o n of  s o l u t i o n of  suspension.  and then spun down  was resuspended  M e a c h , at  incubated at  1 ml of a 0.1%  to the c e l l  room temperature  needed.  to d i g e s t  at  the b a c t e r i a l  and  Fractionation.  the  lysate,  4.36  The mixture was  final proteins.  gm of CsCl  (Harshaw,  incubated at 37  T h i s combination gave a f i n a l  water  C to  d e n s i t y of  I.76  •3  gm per cm  (p = 1.405).  nitrate centrifuge  tubes and o v e r l a i d w i t h mineral  f u g a t i o n was performed rpm f o r  40 hours at  The mixture was poured i n t o  oil.  Centri-  in a Beckman L2-65B u l t r a c e n t r i f u g e  at  37,000  20 C.  Following c e n t r i f u g a t i o n , n i t r a t e tube was punctured  fraction  the base of the  in a Beckman f r a c t i o n  drop samples were c o l l e c t e d per f r a c t i o n From every f i f t h  cellulose  into  cellulose  collector.  Ten  13 x 100 mm t u b e s .  a drop was removed f o r  densitometry  analys  by an Abbe r e f r a c t o m e t e r  3.  at  Measurement of  23 to 25 C (room  temperature).  radioactivity.  Samples were d i l u t e d with 5% TCA, i c e c o l d , tered  through 0.45  u HA M i l l i p o r e or Reeve Angel F i b e r G l a s s  They were d r i e d and counted as d e s c r i b e d p r e v i o u s l y . corrected for  VI .  and  background and a d j u s t e d f o r  filters.  Samples were  the double l a b e l c o u n t s .  G e n e t i c a n a l y s i s of the mutants. A.  Isolation  of the  temperature  The standard t e c h n i q u e f o r resistant  revertants  culture diluted  c o n s i s t e d of  1:100  resistant  revertants.  i s o l a t i n g the  i n o c u l a t i n g 0.1  temperature ml of an o v e r n i g h t  to c o n t a i n 1 x 10^ c e l l s per m l ,  directly  i n t o A and E broth or onto supplemented agar p l a t e s prewarmed 42 C.  After  mutation  to  i n c u b a t i o n o v e r n i g h t at 42 C , c o l o n i e s were p i c k e d  up and examined f o r  for  fil-  phenotype.  their  g e n e t i c background and f o r  the DnaA  Growth in the tubes was checked m i c r o s c o p i c a l l y  snake f o r m a t i o n .  B.  Transduction experiments. 1.  B a c t e r i o p h a g e donor l y s a t e Desired b a c t e r i a l  overnight culture  preparation.  donor s t r a i n s were grown from an  in 9 mis of L u r t a b r o t h w i t h supplements and  2.5  _3 x 10  M CaCl with a e r a t i o n ,  and at 30 and 37 C, depending on the  stra  Two to f o u r hours i n c u b a t i o n was necessary to g i v e a d e n s i t y of g about 2 x 10 x 10^ pfu  c e l l s per m l .  (plaque-forming  units)  added to 1.9 ml of b a c t e r i a After  Phage P1Kc was d i l u t e d per m l , and 0.1  to about  6-8  ml of t h i s was  in a s t e r i l e Wasserman tube at 37 C.  twenty minutes of p r e a d s o r p t i o n , 0.2  ml of t h i s a d s o r p t i o n  mixture was added to 2.5 rrnl of L u r i a s o f t agar and poured onto L u r i a agar p l a t e s . the o v e r l a y ,  After  f i v e minutes r e q u i r e d f o r  s o l id i f feat ion of  the p l a t e s were incubated at 37 C f o r  h a r v e s t the donor phage, 5 mis of L u r i a b r o t h  six hours.  (without CaCl) was  added per p l a t e and the p l a t e s were allowed to s i t o v e r n i g h t the r e f r i g e r a t o r .  Next morning, the s o f t agar  phage  1 minute to b u r s t any u n -  T h i s mixture was c e n t r i f u g e d at  minutes to p e l l e t  tube.  ml of c h l o r o f o r m was added f o r every 5 mis of  l y s a t e and the mixture was vortexed f o r lysed c e l l s .  in  l a y e r was broken up  with a g l a s s spreader and was scraped i n t o a c e n t r i f u g e About 0.1  To  the c e l l  5 ~ 6000 x g_ f o r 15  d e b r i s and the supernatant was saved f o r  titration.  2.  Transduction experiments. D e s i r e d r e c i p i e n t s were grown as d e s c r i b e d f o r  indicator  s t r a i n s above.  (multiplicity  of  Donor P1kc was d i l u t e d  infection)  of  to g i v e an M0I  1 - 3 phages per b a c t e r i u m .  20 minutes of p r e a d s o r p t i o n , the c o n t e n t s of  the  After  the tube were spun down  and the c e l l s were resuspended in 10 mis of phage 0.1  or 0.05 mis of  the suspension were p l a t e d on s e l e c t i v e was assayed f o r  unadsorbed phage.  checked f o r  contaminating  bacteria,  checked f o r  r e v e r s i o n as c o n t r o l s .  plates.  The  supernatant  The phage suspension was and the  recipient  always  s t r a i n s were  RESULTS AND GENERAL DISCUSSION  I .  P r o p e r t i e s of  A.  CR34T83  A n a l y s i s of macromolecular s y n t h e s i s and c e l l d i v i s i o n . 1.  Temperature s h i f t c o n d i t i o n s .  Whenever one i s working w i t h temperature the d e f i n i t i o n of cal  temperature  the s e n s i t i v e temperature,  to a temperature  In a t y p i c a l  temperature  block.  from 30 C to a range of  shift  At timed  gradient  experiment,  30 C in K l e t t  u s i n g the C o u l t e r C o u n t e r . an experiment.  as the p h y s i o l o g i -  cell  the T83  temperature  block.  T83 c e l l s grown f o r  tubes were t r a n s f e r e d  intervals,  c o u l d be  The response of  in a temperature  temperature  g e n e r a t i o n s at  importance.  transition  from 27 to kS C was t e s t e d  several  as well  range at which the behaviour of the mutant  examined, becomes one of utmost cells  s e n s i t i v e mutants,  numbers were  to  determined  F i g u r e 1 r e p r e s e n t s the r e s u l t s of  The c o n t r o l c e l l s grown at  through two d o u b l i n g s d u r i n g the 85 minute  30 C , :i:n t h i s  the  such  system, go  incubation p e r i o d .  At  temperatures above kl C , there was no c e l l u l a r d i v i s i o n a n d , hence no net  i n c r e a s e in the c e l l  numbers f o r  shifted culture.  At  temperatures  between 30 and 33-5 C , when compared to the c e l l s growing at 30 C, a significant  i n c r e a s e in the f i n a l  l a t t e r changes were accounted f o r , and 33-5  C, a f a s t e r  cell  numbers was o b s e r v e d .  by the f a c t  The  that between the 30  growth r a t e was a c h i e v e d , without  affecting  the  T 8 3 , grown f o r s e v e r a l g e n e r a t i o n s at 30 C , was d i s t r i b u t e d i n t o 16 tubes (18 x 150 mm) at a e e l ] d e n s i t y of 5 x 10? c e l l s per m l , and was p l a c e d in a temperature g r a d i e n t b l o c k . C e l l counts were monitored by a C o u l t e r Counter f o r the next 8 5 minutes (two g e n e r a t i o n s ) . Final c e l l numbers ( r e l a t i v e c e l l counts) a r e p l o t t e d a g a i n s t the temperat u r e of i n c u b a t i o n .  temperature 33-5  sensitive condition.  C , t h e r e was a general  c e s s a t i o n of c e l l  However, at  retardation  temperatures  of growth and a complete  d i v i s i o n between 41 and 45 C.  was thus chosen as the u l t i m a t e n o n - p e r m i s s i v e When T83, at d i f f e r e n t cells  per m l , were s h i f t e d  was the same, that 42 C.  d e n s i t i e s between 1.5  the c e l l  r e s i d u a l d i v i s i o n at  in a s h i f t  to non-  temperature,  Cel1 d iv i s i o n .  The p o s s i b i l i t y of measuring  growth e l e c t r o n i c a l l y with a C o u l t e r counter and the a c c u r a c y  t h i s method f o r m o n i t o r i n g the q u a l i t y o f  been demonstrated by s e v e r a l schek,  and 10 x 10^  d e n s i t y , the response was the same.  C e l l d i v i s i o n and DNA r e p l i c a t i o n  a.  degrees  temperature.  i s , t h e r e was no s i g n i f i c a n t  permissive  bacterial  Forty-two  from 30 to 42 C , the response of the c e l l s  T h u s , r e g a r d l e s s of 2.  of  cell  above  the b a c t e r i a l  growth has  l a b o r a t o r i e s over the past decade  (Kubit-  1969a).  In p r i n c i p l e , the c e l l and b a c t e r i a  volume d i s t r i b u t i o n s  f o r c u l t u r e s of E_. c o l  in g e n e r a l , growing under steady s t a t e c o n d i t i o n s  the d e t e r m i n a t i o n  of  the c e l l  (Harvey and M a r r ,  1966) .  volume change d u r i n g the c e l l  Harvey et_ aij_.  gave a more r i g o r o u s d e r i v a t i o n of  permit  cycle,  (1967) , and Kubitschek (1968a), volume to c e l l  growth  r e l a t i o n s h i p by the e l e c t r o n i c measurement of average c e l l  growth  ratios.  The instrumental  t h a t one c o u l d d e t e c t of b a c t e r i a l  growth  the c e l l  r e s o l u t i o n of the C o u l t e r counter  is such  the s m a l l e s t changes in the q u a l i t y and  possible.  quantity  In steady s t a t e , medium at  exponential  growth c o n d i t i o n s ,  30 C , CR34T83 d i v i d e s with a g e n e r a t i o n  which corresponds to a s p e c i f i c growth such c o n d i t i o n s , when c e l l the c e l l  s i z e , as i n d i c a t e d  grown c e l l s ,  that c e l l  by C l a r k  DNA r e p l i c a t i o n .  of  E_. c o l ?, about 18% of  when t h e i r  DNA r e p l i c a t i o n  has been blocked  that t h i s  in the c e l l  is a gradual  cultures DNA .  1969b), are  (Clark,  c u l t u r e at  the c e l l  kl C ( F i g u r e  f i f t y minutes.  decline  1968b;Helmstetter, in  CR34T83.  d i v i s i o n t h e r e i s an  mass which corresponds to mass i n c r e a s e expected  counts f o r T83 c e l l s at  the f i r s t  (1968)  broth grown  (Kubitschek,  The c e l l s  2b).  s i z e , as viewed m i c r o s c o p i c a l l y , and f i l a m e n t o u s  for  et_ aj_.  event does not take p l a c e  C o i n c i d e n t with the c e s s a t i o n of  ity  shift  These c e l l s have been shown to undergo d i v i s i o n  is c l e a r  Viability  Upon a  2b).  ( F i g u r e 2a).  population,  c y c l e but have not separated yet  of an e x p o n e n t i a l  30 C  the c e l l s which have completed t h e i r  forms.  increase  division  manner,  p o s i t i o n , for  (1968a) and H e l m s t e t t e r  In an exponential  in doublet  It  in an e x p o n e n t i a l  (Figure  75  Under  d i v i s i o n depends upon the completion of a round  of  replication  channel  is a c e s s a t i o n in c e l l  Results obtained indicated  increased  time of kl minutes  1.k Hour \  by the peak channel  remain c o n s t a n t at  to kl C, t h e r e  1968).  numbers  r a t e of  in A and E  90 m i n u t e s , and to 100 percent  in  viabil-  i n c u b a t i o n , however,  s u r v i v o r s to f i f t y  by 180 minutes of  in  result.  kl C i n d i c a t e no l o s s  Upon longer  in the percent of  forms  increase  percent  i n c u b a t i o n at  there by  kl C.  0  F i g u r e 2.  Growth of T83  under p e r m i s s i v e and n o n - p e r m i s s i v e c o n d i t i o n s .  T83 grown in A and E medium at 30 C (•) was s h i f t e d to kl C (0) as i n d i c a t e d by the v e r t i c a l arrow. C e l l numbers (panel A) and c e l l s i z e (panel B) were monitored as a f u n c t i o n o f time d u r i n g growth at the p e r m i s s f v e (30 C) and n o n - p e r m i s s i v e (kl C) t e m p e r a t u r e s .  40  b.  Studies i.  on  Total  Shifting results  gradually  3)-  decreasing  percent C  i n r i c h medium  of  DNA  synthesized  i n the i s 40  amount o f  t o 60%.  other  r e s i d u a l DNA  In m i n i m a l i s 20  initiator  These r e s u l t s f i t n i c e l y w i t h  The  model  m u l t i p l e f o r k s whereas,  forks.  Accordingly,  the  when g r o w n ii. At  in broth  incubation  a t 42  synthesized  at  The  Rate of a given  grown  DNA  same  C.  identical observations  (Helmstetter in r i c h  medium, c e l l s  show a h i g h e r as  minutes.  H e l m s t e t t e r - C o o p e r model  in minimal  CR34T83 w i l l  a  m u t a n t CR34T46 ( H i r o t a e t a 1.  indicates that c e l l s  contain  non-permissive  medium, u n d e r  t o 30%.  C  proceeds at  r e p l i c a t i o n under d i f f e r e n t growth c o n d i t i o n s  synthesis  t o 42  under  synthesis  o c c u r s upon l o n g e r  residual synthesis  Cooper, 1968).  DNA  being  r e s i d u a l DNA  synthesis  h a v e b e e n made f o r t h e 1970).  DNA  CR34T83 f r o m 30  rate f o r approximately f o r t y to s i x t y  increase  c o n d i t i o n s , the  TdR.  a c u l t u r e of  This  further detectable  The 42  (Figure  r e p l i c a t i o n i n CR34T83.  uptake of  i n r e s i d u a l amounts o f  conditions  No  DNA  medium  have s i n g l e  percentage of  compared t o the  and  minimal  residual conditions.  replication.  instant of  time,  the  rate of  incorporation  14 of  C-thymidine  number o f  i n t o DNA  replicating  by  forks  a growing c u l t u r e in the  Under s t e a d y s t a t e c o n d i t i o n s of and  r a t e of  DNA  Kjedgard,  i n an  replication/cell  1966).  I f any  cells  and  the  exponential  numbers s h o u l d  disturbance  i s a f u n c t i o n of  the  replicationvelocity. population,  the  remain constant  of anomaly from t h i s  ratio (Maal^e  rule  was  MINUTES F i g u r e 3.  Uptake o f C-1A t h y m i d i n e  i n t o CRT-83 growing at  30 C and kl C.  T83 growing in the p r e s e n c e o f C-14 thymidine at a f i n a l c o n c e n t r a t i o n of 20 yg per ml and a s p e c i f i c a c t i v i t y o f .3 yc/uM was s h i f t e d from 30 C to kl C . The c e l l d e n s i t y at the time o f s h i f t was 1.5 x l O ^ / m l . The i n c o r p o r a t i o n of Z-\k thymidine i n t o the c o l d TCA i n s o l u b l e f r a c t i o n of the 30 C c u l t u r e (0) and kl C c u l t u r e (•) i s g i v e n as a f u n c t i o n o f t i m e .  1  1  I  1  I  1  I  1  I  1  I  r  42°  MINUTES  Figure  k.  R a t e o f DNA  synthesis  i n CR3*tT83 a f t e r  a shift  t o kl  C.  T83, g r o w i n g a t 30 C a t a c e l l d e n s i t y o f 2.2 x 10 c e l l s per ml, was s h i f t e d t o kl C a t z e r o t i m e . The r a t e s o f i n c o r p o r a t i o n o f -TdR i n t o c o l d TCA i n s o l u b l e f r a c t i o n was d e t e r m i n e d Tn a . s e r i e s o f 3 m i n u t e p u l s e s , u s i n g 1 ml c e l l s a m p l e s i n c u b a t e d w i t h ^C-TdR a t a f i n a l c o n c e n t r a t i o n o f 20 y g p e r m l , and w i t h 50 y g p e r ml AdR. Counts per minute of ^ C - l a b e l per c e l l i s p l o t t e d a g a i n s t time f o r t h e c u l t u r e s h i f t e d t o kl C (0) a n d c o n t r o l a t 30 C ( • ) .  to take p l a c e , one should see f o r example, an i n c r e a s e in the DNA per eel 1 r a t i o , in a temperature  that  i s , a multinucleate  snake, or a d e c r e a s e , as  s e n s i t i v e DNA r e p l i c a t i o n  At 3 0 C and at kl C , the  mutant.  r a t e of DNA r e p l i c a t i o n  in C R 3 4 T 8 3 was  ]k measured by the material  of  i n c o r p o r a t i o n of  the c e l l .  The DNA per c e l l  C-thymidine  i n t o c o l d TCA i n s o l u b l e  The r e s u l t s a r e shown in F i g u r e k. ratio  c a l c u l a t e d from the counts per minute  of  Ik C-TdR i n c o r p o r a t e d per ml over the c e l l grown c e l l s ,  the r a t i o  of  r a t e per c e l l  v a l u e was r e a c h e d , due to a c o n t i n u a l the absence of c e l l  d i v i s i o n at  c o n s t a n t DNA per c e l l to a h a l t a f t e r  ratio.  decreased u n t i l  but  kl C.  number per m l .  limited  For kl C a  plateau  DNA s y n t h e s i s  in  The 30 C c e l l s m a i n t a i n a  The c o n c l u s i o n that DNA s y n t h e s i s comes  s i x t y minutes at  kl C c o u l d be reached from the  pulse  exper iments. I I.  Recovery o f C R 3 4 T 8 3 at A.  Single shift  the e x p e r i m e n t s :  3 0 C were t r a n s f e r r e d  p o r t i o n of t a i n e d at  the o r i g i n a l  E x p o n e n t i a l l y grown c e l l s  to kl C , as d e s c r i b e d in s e c t i o n culture  3 0 C throughout  reference c u l t u r e .  After  the  (1/5 the s t a r t i n g  r e s t of  appropriate  kl C throughout  the r e s t of  volume)  the experiment  (It.  C.  A  was main-  as the  control  i n c u b a t i o n time at kl C , about  3/5 of t h i s was s h i f t e d down to 3 0 C and the at  kl C .  experiments.  The d e s i g n of at  3 0 C a f t e r growth at  remaining  the experiment.  1/5 was kept  The terminology used  in d e s c r i b i n g these r e s u l t s a r e as f o l l o w s : to kl C; s h i f t  down, the  kl C f o r a short the  During  growth at  (ii)  phases.  (phase  the c u l t u r e  ha 1 f - s t e p 1.  the  lag r e f e r s  numbers from the base l i n e ,  C e l l d i v i s i o n during  recovery  stopped d i v i d i n g a f t e r (1) a lag  recovery for changed i t s time,  a shift up, s  a lag  cell  time;  in and  (iii)  30 C c u l t u r e .  length of  or the  time needed  (2) a c c e l e r a t e d  p e r i o d s at  kl C.  is  referred  in the s h i f t  d i v i s i o n , and the  (3) normal  cultures.  r a t e , was used as the  the number of c e l l s f o r  assumed to be 100% d i v i s i o n , and the p o i n t was c a l c u l a t e d a c c o r d i n g l y .  of  division pulsed  The time d u r i n g  d i v i s i o n curve  reference.  the c o n t r o l  residual  cells  down  recovering cultures  kl C and the c o n t r o l  r a t e to normal  cells  In every c a s e , the  kl C grown c e l l s , when the a c c e l e r a t e d  the v a l u e f o r  lag  from a kl C p u l s e .  and recovery  A comparison was made between  for different  pattern  response of e x p o n e n t i a l  CR34T83 to v a r y i n g p u l s e s of growth at  phases.  division  time.  F i g u r e 5 d e s c r i b e s the  contained  recovery,  kl C .  the normal  to the  at  to s t a r t d i v i d i n g , and the time needed f o r a 50% i n -  in the c e l l  to as the  i),  an a c c e l e r a t e d d i v i s i o n f o r a short  In a n a l y s i n g such r e s u l t s ,  crease  the p u l s e ; and  kl C , the c e l l  These a r e :  from 30 C  i n c u b a t i o n of a c u l t u r e  length of  c o n t i n u e d d i v i s i o n , c o r r e s p o n d i n g to that of  for  up, a s h i f t  30 C a f t e r a p u l s e at  recovery from a p u l s e at  shows t h r e e d i s t i n c t division;  reverse case; p u l s e ,  time as given by the  resumption of c e l l  shift  At  this  c u l t u r e was  d i v i s i o n at  the  inflection  As shown in F i g u r e 5, f o r a short  o  •  v  20  • 40  0  60  •  80  100  MINUTES  F i g u r e 5-  Recovery of CR34T83 at 30 C f o l l o w i n g growth at  kl  C.  A c u l t u r e of T83 growing e x p o n e n t i a l l y at 30 C , was s h i f t e d to kl C at a d e n s i t y of 1.5 x 1 0 c e l l s per m l . At 10 (o), 20 ( • ) , 30 ( v ) , k5 ( • ) , 60 ( • ) , and, 70 ( • ) minutes as i n d i c a t e d by v e r t i c a l a r r o w s , s u b c u l t u r e s from kl C were t r a n s f e r e d to 30 C and a l l o w e d to r e c o v e r . C e l l counts are p l o t t e d as a f u n c t i o n of t i m e . The c o n t r o l c u l t u r e , m a i n t a i n e d at 30 C , is r e p r e s e n t e d by the dashed l i n e . 7  F i g u r e 6.  Relattonship control  between  cultures  cells  kept at  r e c o v e r i n g from kl C to  their  30 C.  Data from F i g u r e 5 and numerous o t h e r s i m i l a r experiments were used to construct t h i s graph. The o r d i n a t e r e p r e s e n t s the r a t i o of c e l l counts f o r c e l l s r e c o v e r i n g from a temperature b l o c k at kl C , to the c o n t r o l c e l l s l e f t at 30 C , as expressed in percent v a l u e s . The r a t i o s used were taken at the time when the r e c o v e r i n g c e l l s entered t h e i r normal d i v i s i o n p e r i o d , t h a t i s , the i n f l e c t i o n p o i n t s of the recovery c u r v e s . The a b s c i s s a r e p r e s e n t s the d u r a t i o n of the temperature b l o c k at kl C.  pulse  (up to 10 m i n u t e s ) ,  to that of rapidly,  the c o n t r o l  for  the c e l l s showed a 100% r e c o v e r y  30 C grown c e l l s .  p u l s e s up to 50 m i n u t e s ,  longer than 50 m i n u t e s ,  at  30 C , which had c o n t i n u e d the r e g u l a r gained a p r i o r i t y  p o s s i b l e c e l l s at  the n u c l e a r e q u i v a l e n t s d i r e c t l y whereas the c e l l s at equivalents.  In the f i n a l  a loss  in r e s i d u a l  a loss  in  cell  initiation.  60 minutes maintained  50 m i n u t e s .  initiation  it  division, or, Furthermore,  T h u s , the  cycles for  kl C.  to the  that  their  In other  words, lost block,  nuclear  appeared that t h e r e had been  expressed in terms of DNA c y c l e s ,  the DNA s y n t h e s i s between kO and  the number of d i v i s i o n s obtained d u r i n g  r e c o v e r y to a c o n s t a n t ,  cells  length of the  r e g u l a r l y and gained  analysis,  possibility  the n o n - p e r m i s s i v e c o n d i t i o n ,  proportional  30 C i n i t i a t e d  declines  For p u l s e s  r u l e d out the  over those at  kl C , by s t a y i n g at  figure  was m a i n t a i n e d .  data c l e a r l y  in v i a b l e c e l l s d u r i n g the f i r s t  DNA r e p l i c a t i o n ,  this  to the 35% l e v e l .  the same 35% l e v e l  The r e s u l t s from v i a b i l i t y of a l o s s  However,  in numbers  the  i s , c e l l s which had a complete n u c l e a r  equivalence. A n a l y s i s of l e n g t h of  the  the  relationship  lag and time f o r  recovery are given  between  the p u l s e in kl C and  50% i n c r e a s e in the c e l l  in F i g u r e 7-  The d i s t r i b u t i o n  l e n g t h between 5 and 30 minutes was maintained l e n g t h , a v e r a g i n g 14 ± 3 m i n u t e s . IS ± k m i n u t e s . became p r o t r a c t e d  of  larger  values.  numbers a f t e r  lag f o r  pulse  at a r e l a t i v e l y  constant  The c o r r e s p o n d i n g h a l f - s t e p was  With longer than 30 minute p u l s e s , to  the  It  this  appeared,.thus,  relationship that the  length  15  20  25  30  35  40  PULSE L E N G T H (Min.)  F i g u r e 7.  The r e l a t i o n s h i p  between  p e r i o d o f growth at  the  lag  in r e c o v e r y to  the  42 C .  R e s u l t s from F i g u r e 5 and s i m i l a r experiments were used to c o n s t r u c t this graph. The lag ( • ) , and the h a l f s t e p (0), t h a t is the time r e q u i r e d f o r a 50 p e r c e n t i n c r e a s e in c e l l numbers a f t e r the l a g , a r e p l o t t e d as a f u n c t i o n o f the p u l s e of n o n - p e r m i s s i v e growth.  of  shorter  temperature  of  the c e l l s 2.  p u l s e was unimportant  in r e c o v e r y .  in d e c i d i n g the  1  C e l l volume d i s t r i b u t i o n s  f o r T83 at  The change in s i z e d i s t r i b u t i o n of T83 recovery at  is  in F i g u r e 8.  illustrated  30 C T 8 3 has a s i z e d i s t r i b u t i o n  E_. c o l ? ( P a i n t e r  shape of  1966).  and M a r r ,  higher channel numbers or general  larger  typical  cell  at  105 m i n u t e s .  to e x p o n e n t i a l  volume upon s h i f t  cultures  as the c e l l s were in t h e i r  The  curve changed to a f l a t t e r in the mean c e l l  to a p o p u l a t i o n of  the s m a l l e r normal  normal  of  toward  volume. back to  the  heterogeneous  the a c c e l e r a t e d d i v i s i o n to g i v e three  Eventually,  up and  to kl C .  in phase I w h i c h , upon a s h i f t  p e r m i s s i v e c o n d i t i o n , fragmented s i z e s d u r i n g phase II,  during a s h i f t  T h i s d i s t r i b u t i o n moved  curve and t h e r e was a c o n s i d e r a b l e s h i f t resulted  recovery.  Under balanced growth c o n d i t i o n s ,  the s i z e d i s t r i b u t i o n  The f i l a m e n t s  behaviour  peaks  s i z e gained dominance  growth phase III,  from 1^5  to  160  m i nutes. 3.  Rate of DNA s y n t h e s i s d u r i n g r e c o v e r y from a kl C p u l s e .  The r a t e of thymidine ing from p u l s e s o f d i f f e r e n t  incorporation  i n t o c e l l s of T83  length at kl C was determined  recover-  by p u l s e  Ik labelling  the c e l l s w i t h  incorporated the  C-thymidine and c o u n t i n g the  into the c o l d TCA i n s o l u b l e m a t e r i a l .  r e s u l t o f such e x p e r i m e n t s .  the r a t e of thymidine stopped.  F i g u r e 9 shows  Upon s h i f t i n g T 8 3 from 30 to kl C ,  incorporation  During the c o u r s e of  radioactivity  i n t o DNA decreased g r a d u a l l y and  t h i s d r o p , the c e l l s went through  their  F i g u r e 8.  A n a l y s i s of at  the c e l l  s i z e d u r i n g r e c o v e r y from  growth  kl C.  T 8 3 , growing at 30 C was s h i f t e d to kl C at 35 minutes and returned to 30 C at 90 m i n u t e s . C e l l volume d i s t r i b u t i o n s were o b t a i n e d from p l o t s of the p u l s e h e i g h t a n a l y s i s o f the c e l l s . The o r d i n a t e r e p r e s e n t s the c e l l number, and the a b s c i s s a , the p u l s e h e i g h t a n a l y s e r channel number (0-511). Each c u r v e is numbered as to the sampling t i m e .  51  2l  I  I 20  I  I  I  40  I  I  60  I 80  I  1 L 100  MINUTES  F i g u r e 9.  Rate o f during  l a b e l l e d thymidine  i n c o r p o r a t i o n i n t o T83  recovery.  C e l l s grown at 30 C in A and E medium at a d e n s i t y of 5 8 x 10^ c e l l s per ml (zero t i m e ) . A f t e r 5 ( V ) , 10 ( • ) and 15 (0) minutes (upper panel) o r 30 ( T ) , 40 ( • ) , and 60 (•) minutes (lower p a n e l ) , c e l l s were s h i f t e d back to 30 C as i n d i c a t e d by v e r t i c a l l i n e s . C e l l s were p u l s e d w i t h 0.025 uc per ml ^ C - T d R or 2.5 y c per ml 3|H-TdR, at a f i n a l c o n c e n t r a t i o n of 20 yg per ml TdR, and w i t h 50 yg per ml AdR, f o r 3 m i n u t e s . R e l a t i v e r a t e of r a d i o a c t i v e thymidine i n c o r p o r a t e d (CPM) i s p l o t t e d a g a i n s t t i m e . The curves r e p r e s e n t the best smooth l i n e s f i t t e d through the e x p e r i m e n t a l v a l u e s of two s e p a r a t e e x p e r i m e n t s . The broken l i n e r e p r e s e n t s the uptake f o r c e l l s grown and m a i n t a i n e d at 42 C s i n c e zero time. -  residual  DNA r e p l i c a t i o n  were s h i f t e d of  between kS and 60 m i n u t e s .  to 30 C, the p e r m i s s i v e temperature,  i n c u b a t i o n at  kl C , the r a t e  i n t o DNA rose r a p i d l y f o r  of  culture.  kl C , on the other  seven minutes  labelled  The c e l l s  of uptake  increased.  After  incubated f o r  an  when returned  incorporation of  kO to k5 m i n u t e s .  For c e l l s  returning  b e f o r e resuming the uptake at  30 C , went through a drop in the r a t e o f i n t o DNA u n t i l  thymidine  hand, d i s p l a y e d a  The 15 and 30 minute pulsed c e l l s ,  thymidine  60 minutes  20 minutes and then continued at a r a t e  from a kO minute nncubation at  increasing rate.  after  i n c o r p o r a t i o n of  c o m p a t i b l e w i t h t h a t of the 30 C c o n t r o l  lag o f a p p r o x i m a t e l y  When the c e l l ' s  this  to  labelled  point,  the  rate  f i v e and ten m i n u t e s ,  s i m i l a r c o n t i n u e d drops in the  r a t e were observed w h i c h , a f t e r a s h o r t  while,  r a t e of  picked up to the normal  Several  p o i n t s were noteworthy.  the 30 C c e l l s . Firstly,  for  short  temperature a  p u l s e s o f f i v e arid ten m i n u t e s , following a shift approximately fact,  to the kl C temperature,  seven to twelve minutes a f t e r  kept dropping l i k e  following  the r a t e of  t h a t of  15, 30, and kO minute  started  minutes a f t e r  down.  permissive temperature,  the s h i f t  i n c u b a t i o n s at  a new r a t e of uptake at shift  d i d not get  the kl C c o n t r o l s .  uptake dropped d e s p i t e the c e l l s being cells  i n c o r p o r a t i o n at 30 C  respectively,  kl C .  in  Similarly,  30 C.  These  30, 15 and 5  enough, once s h i f t e d  the c e l l s had two p l a c e s f o r  c a t i o n from the zero time o r i g i n at  down, and  for  kl C , the r a t e of  incubated at  Interestingly  to normal  The f i r s t  start  of  to repli-  one o c c u r r e d  53  at  22.5 minutes and the second at  p u l s e and the s t a r t p o i n t pulses shorter minutes. turned  kS m i n u t e s .  seemed to be 22.5  The r e l a t i o n s h i p  minutes  than ten m i n u t e s , and kS minutes  The c e l l s w i t h 60 minutes of  to 30 C , however,  for  start  point  for  p u l s e s 15 to kO  i n c u b a t i o n at  initiated their  of  kl C , when  replication  re-  within 3 minutes.  32 Measurement  k.  of  -P-labelled  nucleoside  triphosphates  in CR34T83. In a mutant, where t h e r e of  DNA, i t  is p o s s i b l e f o r  o r c o n v e r s i o n of  the  the  is a l i m i t e d  limitation  ribonucleotides  ability  to r e s i d e  for  in the  synthesis availability  to the d e o x y r i b o n u c l e o t i d e  tri-  phosphates. The DNA-RNA p r e c u r s o r pools of T83 were measured a c c o r d i n g  to  32 the presence of triphosphates.  P-labelled  P.  in the  r i b o - and d e o x y r i b o n u c l e o s i d e  F i g u r e 10 summarizes the r e s u l t s of d i s t r i b u t i o n  of  32 four  P-labelled  permissive recovery  r i b o - and d e o x y r i b o n u c l e o s i d e t r i p h o s p h a t e s  (30 C) c o n d i t i o n s , at a s h i f t  the d e o x y r i b o n u c l e o s i d e t r i p h o s p h a t e  under the p e r m i s s i v e growth c o n d i t i o n s ,  able  following order:  exponential  c u l t u r e s of  37 C , that  indicated  dATP; dCTP; dGTP; and dTTP.  i n c r e a s e in the dTTP l e v e l s .  medium at  up (kl C ) , and d u r i n g  the  stage.  A n a l y s i s of  the  under  is  Similar  E_. c o l i B/r/1  order  when grown  pools in  T83,  in d e c r e a s i n g  levels,  There was no d e t e c t is established  for  in g l u c o s e minimal  in d e c r e a s i n g l e v e l s o f dATP, dCTP, dGTP and dTTP.  During the 60 minutes of  i n c u b a t i o n under a s h i f t  up (kl C)  500 400 —  300 — 200  100  MINUTES  32 F i g u r e 10.  D i s t r i b u t i o n of triphosphates  P-labelled  deoxyribonucleoside  i n CR34T83.  T83  was grown i n A and E medium w i t h a f i n a l phosphate c o n c e n t r a t i o n -6 32 o f 1.75 x 10 M. P. was added a t 100 y c per ymole o f PO^ and 60 m i n u t e s were a l l o w e d f o r complete e q u i l i b r a t i o n o f t h e l a b e l i n t o 32 the p o o l s p r i o r t o s a m p l i n g .  D i s t r i b u t i o n of  nucleoside triphosphates during recovery measured as d e s c r i b e d presents and  the p e r c e n t  P - l a b e l l e d deoxy-  from growth a t kl C was  i n M a t e r i a l s and Methods.  The o r d i n a t e r e -  i n c r e a s e i n t h e CPM o v e r the i n i t i a l  the a b s c i s s a represents  time.  CPM o b s e r v e d ,  condition in T 8 3 , the pool levels showed a gradual  increase.  The  order of the pools matched with that of B/r/1 grown in the presence of 150 yg per ml chloramphenicol  (Mychajlowska,  1970).  The d i s t r i b u t i o n  32 of  P-labelled B.  ribonuc1eoside triphosphates  Multiple s h i f t  in T83 is shown in Figure 11  experiments.  The observation that T83 stopped dividing when shifted from 30 to kl C indicated that a d i v i s i o n substance was temperature  sensi-  t i v e and was required for the expression of c e l l d i v i s i o n until  the  moment before c e l l separation.  Since this effect was an immediate  one, one might expect that c e l l d i v i s i o n could be blocked  immediately  during the recovery period (at 30 C) when such recovering c e l l s were returned to kl C.  However,  c e l l s to be altered  it could be equally possible for recovering  so that they may be i r r e v e r s i b l y committed to c e l l  d i v i s i o n , that i s , during the rapid d i v i s i o n period, in which case they would be immune to the non-permissive temperature were designed to test these p o s s i b i l i t i e s . ment are shown in Figure 12.  effect.  Experiments  Results of such an experi-  When recovering c e l l s were returned  to  non-permissive conditions at 7-5 minutes after the s h i f t down, there was approximately  5% residual d i v i s i o n .  For c e l l s returned to kl C,  15 and 22.5 minutes after s h i f t down, that i s , c e l l s at their  rapid  d i v i s i o n phase, 22 and 35 percent residual d i v i s i o n was observed respectively. to their  F i n a l l y , upon s h i f t i n g the recovering c e l l s just  prior  return to normal d i v i s i o n phase to kl C, approximately 18  percent residual d i v i s i o n was observed.  -20  0  20  40  60  80  100  MINUTES  F i g u r e 11.  D i s t r i b u t i o n of  in CR34T83.  P-labelled  ribonucleoside triphosphates  ©  •  v  •  •  °  42< .V  10 8  V_  .a  20  40  60  80  100  MINUTES  Figure 12.  Inhibition of c e l l d i v i s i o n during the recovery period by s h i f t i n g to the non-permissive temperature.  A culture of T83 growing exponentially at 30 C was shifted to k2 C at zero time. After 15 minutes, the culture was returned to 30 C ( © ) . At 7.5 (•), 15 (v), 22.5 ( • ), and 30 ( • ) minutes, as shown by v e r t i c a l arrows, samples were removed from the 30 C f l a s k (•»-), and shifted to kl C. C e l l counts were followed by Coulter Counter (ordinate) for the next 100 minutes. Controls are 30 C grown c e l l s (o), and kl C grown c e l l s (o). I n i t i a l c e l l density was approximately 2 x 107 eel 1s per ml.  It  was concluded that d u r i n g the  the c e l l s were most l a b i l e On the c o n t r a r y ,  c e l l s at  recovery p e r i o d at  to the s h i f t their  up when at  accelerated  their  effect  escape than when they  d i v i s i o n phase.  to the e x p r e s s i o n of at kl C , a l l Apparently,  the s i i g h t  kl C .  to an experiment  d i v i s i o n was  lations  between  temperature division  (12 minutes)  There was a s l i g h t  showed a longer  d i v i s i o n phase of at  the C.  their  r a t e compatible  Role of  As long as the s h i f t than  the  the  the c e l l in c e l l  The c e l l s , at  than normal  to t h a t of  DNA s y n t h e s i s  to the  led  that oscil-  non-permissive  the e x p r e s s i o n of  d i v i s i o n was  inhibited.  numbers d u r i n g  the  the  down,  last  lag and had l o s t  Instead,  at  temperature  indicated  lag needed f o r  increase  recovery.  The c o o r d i n a t e d cell  The r e s u l t s  13).  they  shift the  rapid  resumed d i v i s i o n  the 30 C grown c e l l s .  P h y s i o l o g i c a l requirements 1.  for  systems were as expected  during recovery,  amount of  block  when the c e l l s were passed through  c o u r s e of these o s c i a l l a t i o n s . however,  30 C was e s s e n t i a l  that a l l  30 and kl C.  occurred e a r l i e r  Subsequent  b l o c k and the e x p r e s s i o n of d i v i s i o n  (Figure  inhibited  greater  division.  in which the response of T83 to m u l t i p l e  e s c i l l a t i o n was examined cell  stopped f u r t h e r  the minimum lag p e r i o d at  A rigorous prediction  and showed a  escape from the temperature  c l a s s e s of c e l l s c l e a r l y  escape from the temperature  lag phase.  d i v i s i o n phase were more  immune to the n o n - p e r m i s s i v e temperature resumed the normal  30 C  of in  relationship  the  recovery from a s i n g l e  shift.  recovery.. between DNA r e p l i c a t i o n  d i v i s i o n has been w e l l e s t a b l i s h e d  (Lark,  1969b;  and  Helmstetter,  1969b).  20  40  60  80  100  120  140  MINUTES  F i g u r e 13-  I n h i b i t i o n of c e l l the n o n - p e r m i s s i v e  d i v i s i o n by p e r i o d i c exposure to temperature.  A c u l t u r e of T83 growing e x p o n e n t i a l l y at 30 C was s h i f t e d to kl C. At times i n d i c a t e d by v e r t i c a l a r r o w s , t h i s c u l t u r e was o s c i l l a t e d between 30 C and kl C c y c l e s . A f t e r the l a s t s h i f t to 30 C , at 105 m f n u t e s , c e l l s were kept a t 30 C to a l l o w e x p r e s s i o n of c e l l d i v i s i o n . Growth o f the c o n t r o l (•) and e x p e r i m e n t a l (0) c u l t u r e s i s p l o t t e d as a f u n c t i o n of t i m e .  T e r m i n a t i o n of a round o f cell  division  (Clark,  filaments  (Bilen,  in chromosome r e p l i c a t i o n  1969;  coordinate regulation involved  0970)  in c e l l u l a r  and r e s u l t s  in c e l l  Donachie,  between  1969;  for  1968).  resulted  in  It  formation  1967).  Boyle e_t_ a K  is  This  DNA r e p l i c a t i o n and the v a r i o u s p r o c e s s e s  d i v i s i o n has been examined by Inouye and Pardee  i n d i c a t e d t h a t b l o c k i n g of DNA r e p l i c a t i o n  agents or by mutations involved  is a n e c e s s a r y c o n d i t i o n  1968b; P i e r r u c i and H e l m s t e t t e r ,  known that any a r r e s t of  replication  caused a change in membrane p r o t e i n s  by chemica  that were  division.  N a l i d i x i c a c i d has been shown to stop s e m i c o n s e r v a t t v e DNA r e p l i cation  immediately  ( D i e t z et al . 1966; Goss et a l .  repair  replication  ( E b e r l e and Masker,  Nalidixic acid, showed that  it  Clark  0968  d i d not a f f e c t  which had completed t h e i r  in E_. c o l i .  and H e l m s t e t t e r  the c e l l u l a r  and P i e r r u c i  the c o o r d i n a t i o n between DNA r e p l i c a t i o n  d i v i s i o n was examined  S i n c e d u r i n g the  residual  in T83.  i n c u b a t i o n at  N a l i d i x i c a c i d , at a  for c e l l u l a r  amount of DNA r e p l i c a t i o n represents added at  the  final  incubation  at  kl C, c e l l s c o n t i n u e to make a  amount of DNA, the b l o c k i n g of DNA r e p l i c a t i o n  a c i d should have allowed  (1968)  d i v i s i o n of those c e l l s  c o n c e n t r a t i o n of 10 ug per m l , was added to T83 d u r i n g kl C.  Using  DNA r e p l i c a t i o n .  The v a r i o u s a s p e c t s of and c e l l  a,b)  1971)  1965)as w e l l as  by N a l i d i x i c  d i v i s i o n proportional  t h a t had a l r e a d y o c c u r r e d .  to  the  F i g u r e 14A  r e s u l t s of an experiment where N a l i d i x i c a c i d was  the time of s h i f t  up and at  f i f t e e n minute  intervals  during  s z  • v  0 0  + NAL  u  9 F8  >  7  I—  <  6  UJ  "  5  -X—x—x-xoorv^  J  L  80  120  160  200  MINUTES  F i g u r e 1 4 . E f f e c t o f i n h i b i t i o n o f DNA s y n t h e s i s on c e l l  division  a t r e c o v e r y a t 30 C.  T 8 3 , grown a t 30 C, a t a c e l l d e n s i t y o f 3 x 10 /ml, was s h i f t e d t o 42 C a t z e r o t i m e . N a l i d i x i c a c i d was added t o subsamples d u r i n g t h e 45 m i n u t e i n c u b a t i o n a t n o n - p e r m i s s i v e t e m p e r a t u r e a t the time o f s h i f t up ( 0 ) , o r 15 ( 0 ) , 30 ( t ) , and 45 (V) minutes t h e r e a f t e r (panel A ) , o r a f t e r r e t u r n t o t h e p e r m i s s v e temperat u r e f o r r e c o v e r y a t z e r o ( 0 ) , 5 ( 0 ) , 10 ( • ) , 15 ( V ) , 2 0 ( T ) , 30 ( • ) , and 4 0 ( • ) m i n u t e s a f t e r s h i f t donw. V e r t i c a l arrows i n d i c a t e t h e times o f a d d i t i o n o f N a l i d i x i c a c i d t o a f i n a l c o n c e n t r a t i o n o f 10 y g p e r mi 11 M i t r e .  the n o n - p e r m i s s i v e t r e a t m e n t . cellular  d i v i s i o n resumed at  was a d i r e c t  correlation  the same time as the c o n t r o l  between  and the d i v i s i o n observed at the s h i f t If  up time  residual  the amount of  3 0 C.  (zero minutes)  f i f t e e n minutes o f  d i v i s i o n at  When the c e l l s were returned  resulted  Nalidixic  acid,  resulted  a d d i t i o n at  2.  initiation  rose to 140 and 1 6 0 percent in  of  to the  new ones  of  numbers  down.  For  residual  respectively.  b i n d i n g to RNA polymerase, R i f a m p i c i n s p e c i f i c a l l y b l o c k s transcription  initiation  but not  the completion of  of new c y c l e s of  i n t e r f e r e w i t h completion of of  during  recovery.  (Mizuno et_ al_. 1 9 6 8 ; Burgess et_ al_. 1 9 6 9 ) . prior  h5 m i n u t e s ,  in c e l l  down, however,  of  Nalidixic  between 0 and 2 0 m i n u t e s . s h i f t post-shift  residual  number  When  recovery p e r i o d , no i n c r e a s e  Role of RNA s y n t h e s i s  By  for  in c e l l  division.  at  t h i r t y minutes  3 0 C , when given the same amount  3 0 and hO minutes  levels  increase  in 3 0 % r e s i d u a l  was observed f o r a d d i t i o n s  hi C  division.  hi C , the  When DNA s y n t h e s i s was allowed  a c i d was added d u r i n g the  division  in 2 7 % r e s i d u a l  3 0 C went up to hl% and reached 5 9 % f o r  C o n t r o l c e l l s at  There  DNA s y n t h e s i z e d at  was allowed at  t h e r e was n e a r l y a one hundred percent recovery.  cells.  C e l l s that were t r e a t e d r i g h t  DNA r e p l i c a t i o n  DNA s y n t h e s i s .  to 3 0 C ,  (Silverstein  When the  but  prevents  in p r o g r e s s  Rifampin,  just  does not the  initiation  1970).  response of e x p o n e n t i a l l y  and T83 were t e s t e d  A d d i t i o n of  DNA r e p l i c a t i o n ,  rounds s t a r t e d  and B i l l e n ,  those  growing c e l l s of  E_. c o l i  f o r d i f f e r e n t c o n c e n t r a t i o n of R i f a m p i c i n ,  it  B/r/1 was  found  that  inhibition of  relatively  slowly.  H-uracil  when h i g h c o n c e n t r a t i o n s  B/r/1,  arid 1.2  a suitable concentration  CR34T83, ml  any i l l  lengths  g a v e 30,  13,  concentrations,  6.5, of  less  T h u s , 10 y g p e r ml was s e l e c t e d a s inhibited  RNA  i n medium A and E a t 30 C w i t h  t h e B/r/1 d a t a .  synthesis  the c u l t u r e at t h e recovery  When T83 was s h i f t e d t o kl  time,  (Figure  10 y g p e r  r e s i d u a l d i v i s i o n w h i c h was c o n s i s -  f r o m 10 t o 60 m i n u t e s , and R i f a m p i c i n  went r e s i d u a l d i v i s i o n  noticed  e f f e c t s on t h e c e l l s .  when grown  o f time,  higher  which e f f e c t i v e l y  R i f a m p i c i n , g a v e 50 p e r c e n t  tent with  i t was  division  respectively, f o r concentrations  At s t i l l  d i v i s i o n was o b t a i n e d .  having  of Rifampicin,  m i n i m a l medium, r e p r o d u c i b l y  10, 25, 50 and 100 y g p e r m l .  without  percent  H o w e v e r , when t h e p e r c e n t r e s i d u a l  percent residual d i v i s i o n  residual  f o r 50  (50 t o 500 y g p e r m l ) , e f f e c t i v e  the concentration  i n glucose  occurred  o f 1 t o 25 y g p e r ml o f R i f a m p i c i n , b u t  were used  i n h i b i t i o n was o b s e r v e d .  that  into the c e l l s  Up t o f i v e m i n u t e s was r e q u i r e d  inhibition at concentrations  was c o m p a r e d w i t h  uptake  a l l the c e l l s  15).  C f o r varying was a d d e d t o  recovered  I t was c o n c l u d e d  and u n d e r -  t h a t RNA  synthesis  s u b s e q u e n t t o t h e s h i f t down was o f l i t t l e  consequence t o c e l l  at  and d i v i s i o n m e s s e n g e r s a t  the  recovery. shift  P r e s u m a b l y , mRNA i n g e n e r a l ,  division  down t i m e , w e r e u n d e r g r a d e d and a v a i l a b l e f o r t r a n s l a t i o n .  Once s u c h t r a n s l a t i o n s w e r e c o m p l e t e d , r e s i d u a l d i v i s i o n , b a s e d on t h e amount o f DNA a n d c e l l cubation  a t kl  C, c e l l s  equivalents,  resulted.  A f t e r a kO m i n u t e i n -  t h a t w e r e s h i f t e d down t o 30 C i n t h e p r e s e n c e  6*4  5  z  MINUTES  Figure 1 5 . The effect of i n h i b i t i o n of RNA synthesis at  recovery  on the expression of d i v i s i o n .  Exponentially grown c e l l s of T83, at a density of 2 . 5 x 1 0 c e l l s per ml, were shifted from 3 0 to kl C. After incubation at kl C for 1 0 ( 0 ) , 1 8 (C), 2 5 (9), 3 2 (•), kO (V), k5 (?), and 6 0 (X) minutes, they were returned to 3 0 C. Return to 3 0 C was taken as the zero time for recovery. Rifampicin ( 1 0 ug per ml) was added to recovering c e l l s at zero time. Cell numbers were followed as a function of time.  of  Rifampicin  resulted  of approximately 40 minutes  at  different  during  20 percent  resulted  Similar  numbers.  hi C v i r t u a l l y  30 C.  gave i n c r e a s i n g amounts of  Shorter  residual  RNA s y n t h e s i s at temperature residual  hi C was needed.  15 minutes a f t e r If  the s h i f t  if  b l o c k s of  However,  potential  Between  in order  equivalents  once s h i f t e d  R i f a m p i c i n was added at  42 C , presumably, w i t h longer  to  i n c u b a t i o n at  in r e s i d u a l  d i v i s i o n were o b s e r v e d .  between  45 and 60 minutes was n e g l i g i b l e .  the time of 3•  shift Role of  at  recovery,  permissive in  42 C ,  However, It  to  the  incubation,  the  is  more not  significant change  was a l s o noted  42 C , f o r as s h o r t as 10 minutes  r e c o v e r y , was s u f f i c i e n t  for  5, 10 or  F i g u r e 17 shows t h a t t h i s  15 and 45 minutes  treatment at  of  RNA s y n t h e s i s  42 C , no g r e a t change  changes  Rifampicin  the  down to 30 C .  should be accumulated.  the c a s e .  at  total  Thus,  cell  in  the c a p a c i t y  the accumulation type of behaviour was p r o p o r t i o n a l  d u r a t i o n of growth at  that  prior  to  to reduce the maximum d i v i s i o n p o t e n t i a l  down by 50 p e r c e n t . protein  replication  than  42 C or  incubation  destroyed  their  from a 45 minute growth at  d i v i s i o n was observed  p u l s e at  division.  the c e l l s to express the d i v i s i o n of  incubations  in which R i f a m p i c i n was added  A 45 minute  16).  increase  division.  d u r i n g a 45 minute  30 C ( F i g u r e  R i f a m p i c i n at  Longer  in r e s i d u a l  were conducted  time i n t e r v a l s  the c e l l s to recover at  d i v i s i o n t h a t produced an  in c e l l  in r e d u c t i o n  experiments  recovery at  presence of  in r e s i d u a l  s y n t h e s i s on c e l l  d u r i n g the  recovery  d i v i s i o n and DNA  period.  seen  bb  6  —  5  z u  • 1 I > <  -  I I I 1 1 I  I I  1•  B.  •  - / ro-i-x—i  y  _l  0  I  20  I  1  1  40  1  60  I.I.I  L  80  100  120  Minutes  Figure  16.  Effect of inhibition  of total  RNA s y n t h e s i s o n  recovery.  S u b c u l t u r e s o f T 8 3 , a t a d e n s i t y o f 2.3 x 10 c e l l s per m l , were s h i f t e d t o kl C f o r k$ m i n u t e s a n d R i f a m p i c i n was a d d e d a t v a r i o u s times during the pulse. R e c o v e r y f r o m t h e t r e a t m e n t was e x a m i n e d a t 30 C. R i f a m p i c i n was a d d e d (10 u g p e r m l ) a t 0 (0), 10 (0), 18 ( 0 ) , 25 ( 0 ) , 32 ( • ) , 40 ( V ) , a n d k5 ( ? ) m i n u t e s . A l l of the s u b c u l t u r e s w e r e k e p t a t kl C u n t i l 45 m i n u t e s , a t w h i c h t i m e t h e y w e r e r e t u r n e d t o 30 C ( z e r o t i m e ) . T h i s i s shown i n p a n e l A. P a n e l B i s t h e same e x p e r i m e n t e x c e p t t h a t R i f a m p i c i n was a d d e d a t 0 (?), 5 ( •), 10 ( • ) a n d 15 (X) m i n u t e s a f t e r a kS m i n u t e p u l s e a t kl C. (  0  20  40  60  TIME  F i g u r e 17.  Measurement of in  CR34T83,  120  (min.)  the a c c u m u l a t i o n of d i v i s i o n  potential  T83.  at a d e n s i t y o f  30 C t o kl C and kept In each c a s e , at down (zero  100  80  2.3  there  10 ( t ) ,  x for  5 ( •  10  7  c e l l s per m l , was s h i f t e d  15 ( A ) ,  k5 (B) and 60 (C)  ) or 0 (?)  minutes b e f o r e  t i m e ) , R i f a m p i c i n was added t o a l l  counts were f o l l o w e d d u r i n g r e c o v e r y  time.  the c e l l s .  from  minutes shift Cell  temperature division  a.  Cel1 d iv i s ion.  The  d a t a p r e s e n t e d so f a r i n d i c a t e s  sensitive  and  block affected  chromosomal  o r s u b s t a n c e ( s ) needed is  involved  in this condition.  could  i n v o l v e a c o m p l e t e and  ature  l a b i l e c o m p o n e n t s and  t h e r e s y n t h e s i s o f t h e new s t r u c t u r e , o r perhaps The  following  I t seems t h a t  i n T83,  the  structure  f u n c t i o n s of the c e l l ' s  cycle  A c c o r d i n g l y , the temperature  p a r t s , the r e o r i e n t a t i o n of the  f o r the r e n a t u r a t i o n of the p r o t e i n be e n t e r t a i n e d  (a) t h e t e m p e r a t u r e  f o r such  sensitive  in question.  temperature  product or  d e n a t u r e d and  i n a c t i v e at the non-permissive temperature; or  inactive,  though remains  permissive temperature.  The  sensitive  following  e x p e r i m e n t s were  temperature p u l s e f o r ten minutes. chloramphenicol, at final f i v e minutes treated. turned  after  shift  As  (b) i t  as  likely  non-  undertaken candidates for  To one  was  s u b j e c t e d t o a kl  h a l f o f t h e kl  C  cells,  c o n c e n t r a t i o n o f 150 y g p e r m l , was t o kl  C.  A t t h e end o f 10 m i n u t e s  t o 30 C.  i s rendered  condition.  s e r i e s o f e x p e r i m e n t s , T83  In t h e f i r s t  structure  in the n a t i v e form a t the  to e x p l o r e the above d i s c u s s e d p o s s i b i l i t i e s the temperature  temper-  affected  a c t i v e a t the p e r m i s s i v e t e m p e r a t u r e , but  rendered  effect  t h e l a g thus r e p r e s e n t s t i m e needed f o r  i s n a t i v e and  is  the  in c e l l u l a r  i r r e v e r s i b l e d e n a t u r a t i o n of the  transitions could  sensitive condition:  a step required  replication.  in the v i t a l  that  The  r e s t o f t h e c u l t u r e was  a t kl  shown i n F i g u r e 18,  given  C,  not  both c u l t u r e s were r e -  the c e l l s  that  received  C  MINUTES  Figure  18.  Effect of  of  inhibition  of  protein  s y n t h e s i s on  recovery  T83.  P a r t of an e x p o n e n t i a l c u l t u r e of CR34T83 growing at 30 C (t) was s h i f t e d to kl C ( 0 ) . At 5 minutes p r i o r ( • ) and post (9) shift down, CAM ( 1 5 0 yg per ml) was added to s u b c u l t u r e s ( 0 ) . C e l l counts a r e p l o t t e d as a f u n c t i o n of t i m e .  chloramphenicol d i d not To t e s t  for  recover.  the n e c e s s i t y of new p r o t e i n  p r e s s i o n of subsequent c e l l  r e s u l t s are seen in F i g u r e 18. kl C , when c e l l s were returned  if  After  amount of division  the s h i f t  down.  the 10 minute  lag p e r i o d .  p r o t e i n s y n t h e s i s at  was e s s e n t i a l  for  30 C  The  to undergo p r o t e i n  at  synthesis However,  s y n t h e s i s was b l o c k e d , such t h a t t h e r e was o n l y a  in r e c o v e r y .  ex-  a s h o r t p u l s e of 10 minutes  and allowed  d i v i s i o n was expressed a f t e r  protein  the  d i v i s i o n during the recovery p e r i o d at  chloramphenicol was added f i v e minutes a f t e r  full  synthesis for  limited  30 C, t h e r e was a decreased amount of  A p p a r e n t l y , a short p e r i o d o f p r o t e i n  the e x p r e s s i o n of  synthesis  the d i v i s i o n a f t e r a 10 minute  growth at kl C. Similar  t e s t s were performed, except w i t h an extended  kS minutes at  kl C (Figure  19).  a t . 3 0 C and was d i s t r i b u t e d to kl C f o r f l a s k s at  into e i g h t  10, 18, 25, 32, kO, and kS minutes at shift  was  for  inhibited  down.  It  shifted  kl C, and at  in T83 grown at  if  was of  the time o f s h i f t  protein  the  5 or  synthesis  However,'  if  down (kS m i n u t e s ) ,  back to 30 C , e x a c t l y one d o u b l i n g was o b s e r v e d . interest  d i v i s i o n potential  to f i n d out the mode of a c q u i s a t i o n of  and see i f  10  kl C f o r kS m i n u t e s ,  d i v i s i o n was observed d u r i n g recovery p e r i o d .  the'shift It  generations  f l a s k s and  was concluded t h a t ,  5 to 35 m i n u t e s ,  chloramphenicol was added at after  identical  several  kS minutes a f t e r which chloramphenicol was added to  minutes a f t e r  ho c e l l  T83 was grown f o r  p u l s e of  i t was an a c c u m u l a t i v e one.  If  the so,  or  71  MINUTES  Figure 19-  E f f e c t of  i n h i b i t i o n of  a f t e r a p u l s e at  protein  s y n t h e s i s d u r i n g and  kl C , on r e c o v e r y  T83 grown at 30 C in A and E medium to a d e n s i t y of 1 . 5 x 10 cells per m l , was s h i f t e d to kl C f o r k5 m i n u t e s . CAM ( 1 5 0 ug per ml) was added to s u b c u l t u r e s , as i n d i c a t e d by v e r t i c a l a r r o w s , a f t e r 1.8 (X) , 25 (©) , 32 (o) , o r kO (©) minutes i n c u b a t i o n at kl C , or at 0 ( • ) , $ ( • ) , 10 ( • ) , 15 (v), 25 ( • ) , and 3 5 (o) minutes a f t e r r e t u r n t o 30 C. The r e t u r n to 30 C a f t e r a kS minute p u l s e at kl C was taken as z e r o time f o r r e c o v e r y . R e l a t i v e c e l l counts a r e p l o t t e d a g a i n s t t i m e . The dashed l i n e r e p r e s e n t s the u n t r e a t e d c o n t r o l .  gradually longer  i n c r e a s i n g l e v e l s of  i n c u b a t i o n at  experiment of  shift  time.  shift  protein  observed at  67,  kO,  such experiments to kl  kl  are presented  i n c u b a t i o n at  32 and kO minutes of  87 and 100 percent  kl  at  kO mjnutes.  made at data  kl  inhibition  in c e l l  kl  C,  a parallel  kl  C to that of c e l l  of  these data was that  was that of a n a t i v e  at  at  related  kl  the time of  C , and shift  increase  to the p u l s e at  residual  relationship division. (a)  For  and a c t i v e its  reactivation  at  in the c e l l  r e c o v e r y based on the amount of  rose.  Thus,  respectively,  percent  in the c e l l  kl  In-  C , which  numbers plateaued  r e s i d u a l DNA  the amount of  for c e l l  DNA made  implication  division  potential  30 C going to n a t i v e  but  upon r e t u r n to 30 C; and  (b)  down from any length  e x i s t e d enough p o t e n t i a l  cell  division  a 110  T h u s , the general  protein  any  DNA s y n t h e s i s and d i v i s i o n  between  the t r a n s i t i o n  in  of  number was o b s e r v e d .  When compared with the amounts of  at  inactive  The percent  C , the p l o t s o f percent  revealed  lengths  increase  C , the amount of  i n c u b a t i o n at  increase  counts.  was found to be l i n e a r l y off  time  in F i g u r e 20.  c u b a t i o n s of kS to 60 minutes y i e l d e d a p p r o x i m a t e l y in the c e l l  the  C , and r e t u r n to 30 C in the absence of  r e c o v e r y under chloramphenicol  25,  increase  led to an  C for varying  s y n t h e s i s , t h e r e was a 33 percent  With longer  with 18,  The accumulation h y p o t h e s i s  down to the T 8 3 c e l l s grown at  a 10 minute  counts.  C.  d i v i s i o n was produceds 'upon  where 150 ug per ml chloramphenicol was added at  R e s u l t s of  further  kl  residual  temperature  block,  to express d i v i s i o n or  DNA per c e l l  equivalents.  there cellular  I  1  I  I  1  1  -0at  v  ui co  -0-i —f-  ft  Z  V  V  t  Q  0 0  0_  IU > <—j  Ul  I  I  20  I  1  40  I  1  60  I  80  I  100  •  1  120  •  140  MINUTES  Figure 20.  Effect of  inhibition  recovery of c e l l s  of protein  at 30  s y n t h e s i s on  the  C.  C R 3 4 T 8 3 c e l l s w e r e p u l s e d a t kl C f o r v a r i a b l e l e n g t h s o f t i m e a n d , a t t h e t i m e o f s h i f t down t o 3 0 C ( z e r o t i m e ) , 1 5 0 u g p e r ml c h l o r a m p h e n i c o l was a d d e d t o t h e c u l t u r e . Cell counts are plotted a g a i n s t t i m e s i n c e t h e r e t u r n t o 3 0 C. Symbols r e p r e s e n t i n g p u l s e s a t kl C a r e : ( 0 ) , 1 0 m i n u t e s ; ( 0 ) , 1 8 m i n u t e s ; ( • ) , 2 5 m i n u t e s ; (V) , 3 2 m i n u t e s (T) , kO m i n u t e s ; ( • ) , kS m i n u t e s ; and ( • ) 6 0 mi n u t e s .  lk  The d i v i s i o n r a t e s at which the underwent,  were compared with c o n t r o l  c h l o r a m p h e n i c o l at r a t e s of c e l l and a small  shift  i n c r e a s e in the r a t e s  longer  showed a g r a d u a l l y  i n c u b a t i o n at kl C.  of d i v i s i o n between c e l l s r e c o v e r i n g at It at  There was a change in  recovery phase II  (rapid  the  division)  between 2 0 and kS minutes which  r a t e upon longer  the c e l l s which had l o s t t h e i r recovery,  c e l l s and w i t h those given  down ( F i g u r e 2 1 ) .  d i v i s i o n d u r i n g the  d e c l i n e d to a slower  at  recovery c e l l s from a kl C b l o c k  incubation.  capacity for  On the  s y n t h e s i s of  contrary, proteins  i n c r e a s i n g r a t e of d i v i s i o n upon  There was a f o u r - f o l d incubated at  kl C f o r  change in the  rate  k5 to 6 0 minutes when  3 0 C in the presence of chloramphenicol  (Figure 2 1 ) .  was obvious t h a t , a s i d e from the f o r m a t i o n of DNA e q u i v a l e n t s  kl C , with the  division,  longer  i n c u b a t i o n s , the stage was r e a d i e d f o r  presumably by a l l o w i n g accumulation of other  which had enough of a p o t e n t i a l  division  rapid proteins  to a l l o w e x p r e s s i o n of d i v i s i o n even  in the absence of any p r o t e i n s y n t h e s i s . b.  DNA s y n t h e s i s .  From the c e l l concluded that p r o t e i n  d i v i s i o n data d u r i n g r e c o v e r y ,  s y n t h e s i s at  kl C was not needed f o r  based on c y c l e s of DNA r e p l i c a t i o n  completed at  the time of  from kl to 3 0 C .  to c o r r e l a t e  the e f f e c t s  b i t i o n of  It  the p r o t e i n  c a t i o n to that of  was  important  s y n t h e s i s needed f o r  new rounds of  it  was  division shift of  inhi-  DNA r e p l i -  recovery from the growth under n o n - p e r m i s s i v e c o n d i t i o n s .  ol  1  0  I  I  20  1  I  i  40  60  i  80  PULSE (min.)  Figure  21.  Changes  in the  f r o m a kl  r a t e of c e l l  C block  in the  dtvtsion  presence  of  during  recovery  chloramphenicol.  The r a t e o f c e l l d i v i s i o n ( d y / d x ) was c a l c u l a t e d d u r i n g t h e r a p i d d i v i s i o n p h a s e o f r e c o v e r y i n t h e p r e s e n c e (V) and a b s e n c e (T) o f p r o t e i n s y n t h e s i s . T 8 3 c e l l s , g r o w n a t kl C, w e r e s h i f t e d t o 30 C for recovery. To one h a l f o f t h e c u l t u r e 150 y g p e r ml CAM was a d d e d a t t h e s h i f t down t i m e ( ? ) . The a b s c i s s a r e p r e s e n t s t h e l e n g t h o f t i m e a t kl C.  The f a c t  that the mutant was d e f e c t i v e  of DNA c y c l e s at hi net  hi  C should r e s u l t  in f u r t h e r  initiation  C implied t h a t b l o c k i n g p r o t e i n s y n t h e s i s at in no net DNA s y n t h e s i s at  recovery.  However,  s y n t h e s i s subsequent to a d d i t i o n of CAM would have implied a r e -  c y c l i n g of  the o l d  initiated cycle.  a r e shown in F i g u r e 22. minutes a f t e r  shift  The r e s u l t s of such experiments  No new DNA s y n t h e s i s was observed f o r  down to 30 C .  Meanwhile,  the c o n t r o l  culture  showed resumption of new s y n t h e s i s in a p p r o x i m a t e l y 8 minutes shift  down.  There was no r e l a t i o n s h i p between the d u r a t i o n  c o n t i n u e d p r o t e i n s y n t h e s i s at hi synthesized.  C and the amount of  T h u s , u n l i k e the d i v i s i o n at  after  of  r e s i d u a l DNA  r e c o v e r y , the  initiation  of new rounds of  r e p l i c a t i o n needed new p r o t e i n s y n t h e s i s at  r e c o v e r y time as  i n d i c a t e d by the c o n t r o l  the  cells.  The r e l a t i o n s h i p between the r o l e of p r o t e i n s y n t h e s i s at 30 C r e c o v e r y p e r i o d and of the forupulses of 23.  initiation  10 and 30 minutes at  hi  C.  C e l l s r e c o v e r i n g from 10 minutes of  60  the  of new rounds was examined R e s u l t s a r e shown in F i g u r e  i n c u b a t i o n at  more than 5 minutes of p r o t e i n s y n t h e s i s f o r  C required  hi  resumption of DNA s y n t h e s i s ,  s i n c e a d d i t i o n o f c h l o r a m p h e n i c o l at 0 to 5 minutes subsequent to shift  down r e s u l t e d  90 minutes at their  30 C.  in a 0 to 5 i n c r e a s e in r e s i d u a l During t h i s  time,  were returned held t r u e .  DNA s y n t h e s i s over  the c o n t r o l c e l l s  l e v e l s of DNA s y n t h e s i s to over 100%.  Similarly,  to 30 C from a 30 minute b l o c k at  hi  the  increased if  the c e l l s  C, the same r u l e  N o t i c e a b l e l e v e l s of new r e p l i c a t i o n took p l a c e o n l y when  UJ  M  l  '  i 42<  O 'o  +CAM ^  r  5  I  1  I  i/ I  '  30°  0  l  V  X  X i  l  I 60  -45  MINUTES  F i g u r e 22. The e f f e c t  of  i n h i b i t i o n of p r o t e i n  on the DNA r e p l i c a t i o n at  s y n t h e s i s at  42 C  recovery.  T83 grown in the presence of 0.5 y c per ml H-TdR, 20 yg per ml c o l d TdR, and 50 yg per ml AdR, was s h i f t e d to 42 C f o r kS m i n u t e s . To s u b c u l t u r e s at 42 C , CAM (150 yg per ml) was added at the times i n d i c a t e d by the v e r t i c a l a r r o w s . A f t e r 45 m i n u t e s , the c e l l s were r e t u r n e d to 30 C, and a l l o w e d to r e c o v e r . The CPM o f 3n-TdR i n c o r p o r a t e d f o r the c o n t r o l (X) and the experimental c u l t u r e s at 5 ( 0 ) , 25 ( V ) , and 35 ( • ) minutes w i t h CAM at 42 C are p l o t t e d as a f u n c t i o n of t i m e .  i  1  I +CAM  1  I  1  I  1  I  1  /  MINUTES  F i g u r e 23.  Role o f  i n h i b i t i o n of protein  s y n t h e s i s on  initiation  o f new rounds d u r i n g r e c o v e r y .  T83 was grown in the presence o f H-thymidine (0.5 uc per ml) 20 yg per ml c o l d t h y m i d i n e , and 50 yg per ml d e o x y a d e n o s i n e , a t 30 C . The c e l l s were s h i f t e d to hi C and were kept t h e r e (upper p a n e l ) . Ten minutes l a t e r , a p o r t i o n o f t h e s e c e l l s was t r a n s f e r r e d back to 30 C ( • ) , a n d , 5 minutes a f t e r s h i f t down, CAM (150 yg per ml) was added to h a l f o f the 30 C c e l l s (•).  A f t e r 30 m i n u t e s , a p a r t was r e t u r n e d to 30 C (lower p a n e l ) . Five (0) and 10 (?) minutes a f t e r s h i f t down, CAM (150 yg per ml) was added to s u b c u l t u r e s , and the i n c o r p o r a t i o n of thymidine (^H-methylt h y m i d i n e , 0.5 y c per ml and 20 yg per ml c o l d TdR) i n t o the TCA i n s o l u b l e f r a c t i o n was f o l l o w e d . CPM o f r a d i o a c t i v i t y i s p l o t t e d against time.  approximately  10 minutes of p r o t e i n s y n t h e s i s was a l l o w e d .  The nature o f DNA made subsequent ot 5 minutes of s y n t h e s i s , was determined If,  by l o o k i n g at  d u r i n g the 5 m i n u t e s , new i n i t i a t o r  the r a t e of  protein  replication.  molecules were  inaugurated,  the r a t e of DNA r e p l i c a t i o n should have shown an i n c r e a s e . the r a t e of  the DNA r e p l i c a t i o n was determined  When  in a s e r i e s of 3 minute  14 pulses using  C-thymidine, a burst  noticed approximately  18 minutes a f t e r  b u r s t had r e s u l t e d from the p r o t e i n at  shift  down.  r e p l i c a t i o n was Presumably,  s y n t h e s i s t h a t had taken  this  place  r e c o v e r y s i n c e b l o c k i n g p r o t e i n s y n t h e s i s at 42 C immediately  prior  to a s h i f t  down did not produce a b u r s t  was concluded that the p r o t e i n for  in the r a t e o f  the  initiation D..  To t e s t  of new rounds of DNA r e p l i c a t i o n  (Figure  24). division  30 C . the p o s s i b i l i t y of u n c o u p l i n g the DNA r e p l i c a t i o n  d i v i s i o n , t h r e e experiments were c a r r i e d 1.  It  r e c o v e r y was n e c e s s a r y  Attempts to uncouple DNA r e p l i c a t i o n and c e l l at  from c e l l  s y n t h e s i z e d at  in DNA s y n t h e s i s .  I n h i b i t i o n of  initiation  out.  of new rounds by phenethyl  alcohol. Phenethyl a l c o h o l shown to a l l o w 1962)  residual  by i n t e r f e r r i n g  ( T r e i c h and Konetzka,  (PEA), at a 0.25%  DNA s y n t h e s i s in E_. c o l i  with the  1964).  c a t e d that PEA had m u l t i p l e  initiation  l e v e l , has been (Berrah and Konetzka,  of new c y c l e s of  R e s u l t s from s e v e r a l  replication  laboratories  immediate e f f e c t s on the c e l l  indi-  such as on  z Q >•  x — i IJ CN I  O  20  40  80  MINUTES  F i g u r e 24.  The e f f e c t  of chloramphenicol  during  recovery  the  on the  r a t e of  DNA s y n t h e s i s  period.  An e x p o n e n t i a l c u l t u r e o f T83 growing at 30 C ( t ) was s h i f t e d t o 42 C (0). A p a r t of the c u l t u r e was returned to 30 C (9) a n d , a f t e r 5 minutes at 30 C , CAM (150 yg per ml) was added to a p o r t i o n of the recovering c e l l s (0). To determine the r a t e of DNA s y n t h e s i s , c e l l s were p u l s e d f o r 3 minutes w i t h ^ C - T d R at 20 yg per ml in the presence o f 50 yg per ml AdR. CPM of C - T d R i n c o r p o r a t e d a r e p l o t t e d as a f u n c t i o n of time. 1Zf  the a c t i v i t y  of c e l l  membrane  (Silver  r e a c t i o n s and n u c l e o t i d e pools and o x i d a t i v e  phosphorylation  (Plagemann,  (1966)  (1964)  results ascertained  the DNA r e p l i c a t i o n acid  at  1970),  confirmed the  and on  transport  respiration  1970).  Studies  that,  Konetzka  in E_. c o l ?, PEA blocked  the same p o s i t i o n on the chromosome as amino  starvation.  at  30 C in A and E medium, approximately  occurred  (Figure  25A).  s y n t h e s i z e d by kl C grown c e l l s .  for  the c o m p l e t i o n of  the  residual  a f t e r which time no f u r t h e r evidence,  the  temperature  ing c o m p l e t i o n o f  started  Examination of c e l l revealed  residual  rate.  mately  50%.  cell tions  effect  DNA s y n t h e s i s residual  the time needed  DNA s y n t h e s i s was kO to 50 m i n u t e s , Based on  and PEA had s i n g u l a r a c t i o n  d i v i s i o n data  this in  allow-  subsequent to a d d i t i o n of 0.25%  As shown in F i g u r e 25 B, d u r i n g 25  PEA, c e l l u l a r residual  d i v i s i o n continued at  the  d i v i s i o n c a l c u l a t e d was a p p r o x i -  was p o s s i b l e to uncouple the two p r o c e s s e s ,  d i v i s i o n and DNA r e p l i c a t i o n , by b l o c k i n g one p r o c e s s ,  under p e r m i s s i v e temperature  s i n c e in the absence of any new  the c e l l s c o u l d express o n l y r e s i d u a l d i v i s i o n observed was  Furthermore,  of  rounds.  The amount of Thus, it  55% r e s i d u a l  i n c r e a s e was o b s e r v e d .  division.  minutes a f t e r a d d i t i o n of normal  growing c u l t u r e s  T h i s f i g u r e matches n i c e l y with the  DNA  PEA  on  r e s u l t s of T r e i c h and  When 0.25% PEA was added to e x p o n e n t i a l l y T83  1967),  (Cosgrove and T r e i c h ,  by Lark and Lark and t h e i r  and Wendt,  division.  increased by an a d d i t i o n a l  The amount of  10%  if  NaCl  condiinitiation, residual  (0.65%  CO I  o  U c  E  O u  u LU  >  -20  0  20  40  MINUTES Figure  2  5  .  U n c o u p l i n g of phenethyl  cell  divi-jiuii  from DNA r e p l i c a t i o n  by  alcohol.  T 8 3 , growing at 3 0 C f o r s e v e r a l g e n e r a t i o n s in the presence of 2 0 yg per ml of c o l d and 0 . 5 y c per ml ^H-thymidine and 5 0 yg per ml deoxya d e n o s i n e , was t r e a t e d w i t h phenethyl a l c o h o l at 0.25% f i n a l c o n c e n t r a t i o n , as shown by the v e r t i c a l arrow. Uptake of r a d i o a c t i v e TdR ( t r i a n g l e s ) and c e l l counts (squares) were f o l l o w e d f o r c o n t r o l (empty symbols) and PEA t r e a t e d c e l l s ( f u l l s y m b o l s ) . The c e l l d e n s i t y at the time of PEA treatment was 4 x 1 0 ' c e l l s per m l .  83  final  concentration)  a d d i t i o n of  salt  was added s i m u l t a n e o u s l y with PEA.  had no s t i m u l a t o r y  2.  Uncoupling c e l l  effect  However,  on the r e s i d u a l  DNA r e p l i c a t i o n .  d i v i s i o n from DNA r e p l i c a t i o n  by  Daunomyci n. Daunomycin  (DM),  an a n t i b i o t i c  in the a n t h r a c y c l i n e  i s o l a t e d from a c u l t u r e of Streptomyces p e u c e t i u s , was shown to both DNA-depehdent RNA-polymerase and DNA polymerase in E_. c o l i (Hartman et a l . thesis  1964).  (thymidine  DNA polymerase) The i n h i b i t o r y ability 1966)  replicating of c e l l  in HeLa c e l l s were not a f f e c t e d a c t i o n of  the normal of T83 at of  enzymes.  rate.  on DNA r e p l i c a t i o n  (Calendi  et a 1.  1965;  lies  in  its  K e r s t e i n et a 1.  of the template to  the  the u n c o u p l i n g  in T83 was e x p l o r e d . to DNA s y n t h e s i s  in  RNA s y n t h e s i s c o n t i n u e d , however f o r 30 minutes  at  inhibitory  When DM was added"to an e x p o n e n t i a l l y  30 C , c e l l  residual  vitro  1968) .  by DM (Kim £t_ aj_.  The p o s s i b i 1 i t y of using DM f o r  20 yg per m l , was found  d i v i s i o n stopped a f t e r  40 m i n u t e s .  growing  culture  The amount  d i v i s i o n o b t a i n e d a f t e r a d d i t i o n was 25 percent as shown  in F i g u r e 26B. at  this antibiotic  i n t e r f e r e with the a v a i l a b i l i t y  E_. c o l i B/r/1 .  in  k i n a s e , d e o x y c y t i d i n e monophosphate deaminase, and  d i v i s i o n and DNA r e p l i c a t i o n  DM, at  inhibit  The in v i v o enzymes a s s o c i a t e d with DNA s y n -  to bind to the DNA template  and to  group  There was a gradual  30 C in the presence of DM.  decline  In the f i r s t  in the  r a t e of DNA r e p l i c a t i o n .  10 minutes of  incubation,  14 t h e r e was a 50 percent into  the DNA.  reduction  in the r a t e of  C-TdR  The r a t e of DNA s y n t h e s i s was promptly  incorporation  reduced to 17  84  F i g u r e 26.  Uncoupling of c e l l  d i v i s i o n and DNA s y n t h e s i s by  Daunomycin.  Daunomycin (DM) at  30 C , at  was added to p a r t of a c u l t u r e of C R 3 4 T 8 3  a final  c o n c e n t r a t i o n of 20 yg per m l . (•)  DM i s p l o t t e d  growing  Growth of  the  c u l t u r e , with  (0)  and without  as a f u n c t i o n of  time in panel  B.  The r a t e of DNA s y n t h e s i s was measured by the 14  i n c o r p o r a t i o n of  C-thymidine at a f i n a l  per ml and a s p e c i f i c a c t i v i t y material.  yc per yM i n t o TCA i n s o l u b l e  The r a t e of DNA s y n t h e s i s per c e l l  c u l t u r e with in panel A .  of 0.61  c o n c e n t r a t i o n of 20 yg  (0)  and without  ( t ) DM is p l o t t e d  for  the  experimental  as a f u n c t i o n of  time  percent of the c o n t r o l r e s u l t s of  w i t h i n an hour a f t e r drug t r e a t m e n t .  t h i s experiment  are shown in F i g u r e 26A.  enough, the k i n e t i c s of DM i n h i b i t i o n effect  in  The  Interestingly  resembled t h a t of the kl C  T83.  T h i s study showed t h a t , (and RNA)  by i n h i b i t i o n  of p o l y m e r i z a t i o n of DNA  in T83, one c o u l d observe r e s i d u a l  d i v i s i o n at  30 C.  For  the d u r a t i o n of DM t r e a t m e n t , c e l l s remained v i a b l e , and recovered immediately when the drug was removed. 3•  Uncoupling of eel 1 d i v i s i o n from DNA r e p l i c a t i o n NaT i d i x i c  acid.  The c o u p l i n g between DNA r e p l i c a t i o n and c e l l examined  in another  events was t e s t e d  experiment,  in r e l a t i o n  the N a l i d i x i c a c i d e f f e c t . N a l i d i x i c acid for Ten minutes a f t e r  to a temperature  T83  d i v i s i o n was  d u r i n g which c o o r d i n a t i o n of  c e l l s grown at  i n c u b a t i o n at  interfered,  in c o n j u n c t i o n w i t h temperature,  30 C were t r e a t e d with  block.  If  is shown in F i g u r e 27.  with the c e l l  20 minutes  prior  residual  d i v i s i o n , showed no net  to 30 C acid  d i v i s i o n by committed  i n d e e d , t h i s was not the case as  C u l t u r e A , that had been t r e a t e d w i t h  acid for  down.  kl C.  Nalidixic  any means, then the c e l l s should not have d i v i d e d to t h e i r The r e s u l t s show t h a t  two  b l o c k imposed between  kl C , the c e l l s were returned  to r e c o v e r from the temperature  level.  the  20, 15 and 10 minutes b e f o r e being put at  and allowed  30%  by  to s h i f t  u p , by e x p r e s s i n g i t s  i n c r e a s e in the c e l l  C u l t u r e B c o u l d express o n l y 27 percent  30  Nalidixic  percent  number a f t e r  shift  before s t o p p i n g d i v i s i o n  MINUTES  F i g u r e 27.  R e l a t i o n s h i p of  i n h i b i t i o n of  d i v i s i o n d u r i n g temperature  DNA s y n t h e s i s and c e l l  shifts  in  T83.  To subsamples of T83 grown at 30 C , N a l i d i x i c a c i d was added at 20 ( A ) , 15 ( B ) , and 10 (C) minutes p r i o r to and at the time of a s h i f t to Ul C. C e l l s were r e t u r n e d to 30 C and c e l l d i v i s i o n was a s s a y e d . Recovery of the c o n t r o l without a d d i t i o n of N a l i d i x i c a c i d i s r e p r e s e n t e d by X.  6ue  to the kl C c o n d i t i o n .  before  shift  and another  D expressed a l l  of  The obvious  C u l t u r e C expressed 20 percent of 30 C  12 percent at  its division after  interpretation  of  r e c o v e r y , and f i n a l l y , being returned  Furthermore,  the p o t e n t i a l  recall  reduced.  of  the c e l l u l a r  tempera-  d i v i s i o n at  s u c c e s s f u l l y to the  level  o f the d i v i s i o n owed to be expressed at  T h i s d i v i s i o n , however,  chloramphenicol was added at  kl C  those c e l l s that c o u l d d i v i d e .  those c e l l s t h a t had the necessary c o n d i t i o n f o r  d i v i s i o n would have d i v i d e d their  to 30 C .  these r e s u l t s was t h a t the  t u r e s e n s i t i v e c o n d i t i o n that a f f e c t e d d i d not o b l i t e r a t e  culture  needed p r o t e i n  cellular  e x p e c t e d , and r e c o v e r y was not  synthesis s i n c e ,  the same time with N a l i d i x i c  acid,  if  there  was no d i v i s i o n .  III.  Control of A.  DNA s y n t h e s i s  I d e n t i f i c a t i o n of at  the p l a c e of  a fixed  i s now well  between  that  1968;  Wolf et_al_.  1968;  the arg G and x y l o s e l o c i .  replication  the " r e p l i c a t i o n  Replication  in a c l o c k w i s e  in T83 at  1968a),  Helmstetter,  Experiments were designed to determine rounds of  replication origin"  (Lark et a 1. 1963; Abe and Timizawa, 1967;  here and c o n t i n u e s s e q u e n t i a l l y  of  DNA s y n t h e s i s  rounds of chromosome  p o i n t on the genome c a l l e d  established  Caro and B e r g , located  resumption of  recovery.  In E_. c o l i the f a c t s t a r t at  in T83•  recovery.  and  starts  is from  direction.  the p l a c e of (a)  If  the  T83 was  resumption tempera-  bb  Figure 2 8 .  C o n s t r u c t i o n of  the model f o r  DNA r e p l i c a t i o n  in  CR34T83.  The c o n s t r u c t i o n DNA r e p l i c a t i o n Time  (t)  on the  is based on the H e l m s t e t t e r f o r c e l l s grown under minimal  needed f o r a growing p o i n t  and Cooper Model  and broth c o n d i t i o n s .  to t r a v e r s e  the genome is shown  l e f t and is expressed as a f u n c t i o n of chromosome  Open c i r c l e s represent most r e c e n t l y represent  first  f o r k s and f i 1 led c i r c l e s  e s t a b l i s h e d f o r k s f o r b r o t h medium.  attachment  point  for  for  length.  represent  Triangles  DNA to the membrane  (dots).  The sequence begins with random p o p u l a t i o n of chromosomes at various  stages of  they w i l l patterns during  replication  complete the predicted  for  at  initiated  3 0 C.  Upon a s h i f t  rounds.  to 3 0 C s h i f t  are commenced in a synchronous f a s h i o n .  C,  Three p o s s i b l e s e g r e g a t i o n  b r o t h grown c e i l s are shown.  the recovery at a kl  to kl  down, new  Finally, initiations  ture s e n s i t i v e for the p l a c e of  initiation  reinitiation  chromosome and initiation  the  at  (b)  if  of DNA r e p l i c a t i o n ,  the temperature  s e n s i t i v e step was b l o c k i n g  the same p l a c e as the 150 ug per ml CAM s t e p , by a CAM b l o c k should  the  then identify  origin. The p r e d i c t i o n of a s h i f t  up on an exponential  to kl C is shown in F i g u r e 28. on r e s i d u a l  C,  Chromosomes a f t e r  p o p u l a t i o n from 30  kS m i n u t e s ,  DNA s y n t h e s i s d a t a , a l i g n themselves a n d , at  new rounds a r e kl  recovery,  would be at a s p e c i f i c s i t e on the  recovery from a kl C p u l s e f o l l o w e d the  at  initiated.  If,  at  the time o f a s h i f t  H-TdR was added, the completion of  be l a b e l l e d  ( F i g u r e 29A).  During the  rounds of  based  recovery,  from 30 to  replication  r e c o v e r y , one c o u l d add a  would different  \k label,  for  example  C-TdR, f o r a s h o r t  chase with c o l d TdR ( F i g u r e labelled. several  If  29B).  time,  T h u s , the s t a r t s  c e l l s were grown e x p o n e n t i a l l y  generations  and randomized,  to  identify  the unique o r i g i n of  starts  and ends  recovery  by a new temperature  recovery,  label,  time  (Figure  kl  DNA.  C pulse was at  (Figure  If  block  {kl C) a n d ,  the  at  f o r example 5 B r d U r d f o r a short -  (1.70 gm per c c )  the p l a c e of  29D).  in DNA c y c l e s ,  The r e s u l t s of d e n s i t y c e n t r i f u g a t i o n  29E).  DNA would y i e l d a l i g h t gm per cc)  rounds would be  in u n l a b e l l e d TdR f o r  the c e l l s c o u l d be r e a l i g n e d be g i v e n a d e n s i t y  of  l a b e l , and  t h e r e would be a f i x e d , number of  c e l l s whose chromosomes were l a b e l l e d at In o r d e r  withdraw the  o f such  DNA and h y b r i d BrUra-T (1.76  reinitiation  after  the second  the same r e g i o n of the chromosome as the f i r s t  one,  CHROMOSOME  KEY SYMBOLS  3  |4  NORMAL D N A H-THYM1DINE C-THYMIDINE  5 - BROMODEOXYURIDINE  HYBRID 1.76  LIGHT 1.70  •••  fl * If II  ii ii II  /;  then the o n l y l a b e l  a s s o c i a t e d with the h y b r i d d e n s i t y  material  14 would be the l a b e l  used at  the o r i g i n  (  C-TdR).  Similarly,  c e l l s were exposed to 150 ug per ml CAM at 30 C a f t e r z a t i o n from the f i r s t chromosomes.  If  if  the  the randomi-  42 C p u l s e , again they should a l i g n  their  CAM was removed and c e l l s were resuspended in f r e s h  medium w i t h 5 B r d U r d to a l l o w new c y c l e s to r e i n i t i a t e , -  the  density  Ik label  should be a s s o c i a t e d with the  C-TdR l a b e l .  The r a t e of DNA s y n t h e s i s decreases when BrUra is for T. in B/r/1  1.80  Approximately 120 minutes  density  labelled  is r e q u i r e d f o r a round of  1969)-  at 37 C ( P i e r u c c i , 14  substituted  As d e t e c t e d by the appearance of  C - 5 - B r U r a , a round of r e p l i c a t i o n  mately 240 minutes at  30 C f o r  E_. c o l i 15 TAU or T83.  takes a p p r o x i -  For the  ments d e s c r i b e d above, a 50 minute BrdUrd p u l s e was used f o r density  l a b e l l i n g of the s t a r t s .  the maximum d e n s i t y  labelled  replication  experi-  the  Thus 20 percent of the DNA would be  region.  The r e s u l t s from CsCl d e n s i t y g r a d i e n t  centrifugation  o f DNA  r e p l i c a t e d d u r i n g recovery from a 42 C p u l s e or growth under 150 yg per ml CAM at 30 C are presented The d e n s i t y g r a d i e n t  in F i g u r e s 30 and 31,  p r o f i l e s have two main bands of hybrid and  d e n s i t y c o r r e s p o n d i n g to 1.76 The c h a r a c t e r i s t i c s of the  and 1.70  gm per c c buoyant d e n s i t i e s .  in T a b l e  was c l e a r that 5 B r U r a became p r e f e r e n t i a l l y -  3  light  l a b e l l e d DNA found under these bands were  analysed and the r e s u l t s are g i v e n  l a b e l l e d DNA and not  respectively.  H-labelled  DNA.  II.  In e i t h e r  case, 14  a s s o c i a t e d with  The bulk of the  C-  ^H-labelled  it  FRACTION NUMBER  Figure 2 9 .  Protocol  for  the experiments  TREATMENT  in F i g u r e s 30 and  31.  PURPOSE  A.  kl C + H-TdR  Label chromosome completions  B.  30 C -  Label chromosome o r i g i n s  3  H-TdR \k + C-TdR  14 30 C -  Replicate  chromosomes  (randomizat  C-TdR  + c o l d TdR  D.  1)  Complete chromosomes  30 C + 150 yg per ml CAM + c o l d TdR or  2)  kl C - CAM + c o l d TdR  E.  1)  Reinitiate  30 C - CAM -  replication  TdR + 5-BrdUrd 2)  30 C - TdR + 5-BrdUrd  TEST REPLICATION OF H - OR \ - D N A 3  DE0XYURIDINE  1  BY INCORPORATION OF 5"BR0M0-2-  I  I  I  1  1  1  I  1  I  5  10  15  20  25  30  35  40  45  FRACTION NUMBER  Figure 31.  CaCl g r a d i e n t at  a n a l y s i s of  r e c o v e r y from growth  the p l a c e of  the  reinitiation  in the presence of CAM.  The d e t a i l e d e x p e r i m e n t a l procedure is d e s c r i b e d under M a t e r i a l s and Methods, and in the t e x t . The a b s c i s s a r e p r e s e n t s the f r a c t i o n s collected. The o r d i n a t e i s CPM per f r a c t i o n represented as the p e r c e n t of the t o t a l r a d i o a c t i v i t y (10,420 CPM C - and 6, 140 CPM 3rl) f o r » C - ( i ) and 3H- (O) thymine. X X r e p r e s e n t s buoyant dens i t y . 1 Z f  1 /  F i g u r e 30.  CsCl at  The d e t a i l e d  gradient  a n a l y s i s o f the p l a c e o f the  recovery from growth at kl C.  experimental  procedure i s d e s c r i b e d under  and Methods, and in the t e x t . yc  per.ml)  residual  reinitiation  DNA was l a b e l l e d w i t h  Materials H-TdR (0.0^5  with 2 yg per ml unlabel led TdR and 50 yg per ml AdR, d u r i n g  r e p l i c a t i o n at kl C .  C e l l s were returned  After  60 m i n u t e s ,  t o 30 C and given ^ C - T d R  H-TdR was removed.  (0.5 y c per ml)  in the  presence of 2 yg per ml unlabel led TdR and 50 yg per ml AdR, f o r lk  \k minutes.  C-TdR was removed and c e l l s were grown f o r 5 g e n e r a t i o n s  in c o l d TdR medium, f o r r a n d o m i z a t i o n .  C e l l s were then pulsed with  a second kl C i n c u b a t i o n f o r 60 m i n u t e s . filtered  f r e e of TdR and resuspended in f r e s h medium c o n t a i n i n g 10 yg  per ml B r d U r d , f o r 50 m i n u t e s . at  35,000 rpm in a CsCl  fractions  collected.  H-thymine)  buoyant  for  density.  1k  DNA was e x t r a c t e d  density gradient.  The o r d i n a t e  as a percent o f the t o t a l  3  S u b s e q u e n t l y , c e l l s were  C - (•)  3  The a b s c i s s a  i s CPM per f r a c t i o n  radioactivity and  and run f o r 41 hours represents represented  (9,800 CPM * C - and 5,900 CPM  H- (0) thymine.  1 2  X  X represented  Table  II.  C h a r a c t e r i s t i c s of and percentage of  Conditions for reinitiation following chromosome alignment at 42 C  A.  Recovery from hi  DNA  labelled total  C  H-thymi ne  C.  Recovery from CAM at  H-thymine  30 C. C-thymine  Numbers represent  percent of  Hybrid band = f r a c t i o n s Light  densities  % t o t a l label between f r a c t i o n s 18 to 35  Sum of r a d i o a c t i v i t y in d e n s i t y band Hybrid  C-thymine  B.  Buoyant  DNA.  labelled  as •'H or  DNA in T83:  band = f r a c t i o n s  76.66  6.08  70.58  (100%)  (8.0%)  (92.0%)  67.45  12.60  54.85  (100%)  (18.6%)  (81.4%)  67-95  5-05  62.90  (100%)  (7.5%)  (92-5%)  71.43  17.43  54.00  (100%)  (24.4%)  (75.6%)  t o t a l CPM per f r a c t i o n  between between  Light  1.780 and  in the d e n s i t y  band,  1.7344 d e n s i t y , peak at  1.760  1.7344 and 1.670 d e n s i t y , peak at 1.706.  DNA, on the c o n t r a r y ,  remained at  the  light  density region.  It  must  be borne in mind t h a t , d u r i n g 5-BrdUrd l a b e l l i n g of the DNA o n l y 50_^ minutes w o r t h , or a p p r o x i m a t e l y 20 percent of  the o r i g i n were d e n s i t y  14 labelled.  The  C - l a b e l l i n g of  20_minutes, or 33 p e r c e n t , of 60 -label t h a t 17.5  would be present  initiation  the DNA.  r e g i o n was a p p r o x i m a t e l y  Thus 20 percent of  in the h y b r i d d e n s i t y band.  percent from the kl C i n i t i a t i o n ,  CAM i n i t i a t i o n It  the  banded in t h i s  and 12.6  It  of  recovery from kl C, T83  the r e i n i t i a t i o n  the  reinitiated  new  B.  the DNA.  subsequent to c o m p l e t i o n  rounds from 150 yg per ml CAM (Lark et a l .  t h a t of the temperature  was e v i d e n t  percent from  rounds of chromosome r e p l i c a t i o n at a s p e c i f i c r e g i o n of the p l a c e of  total  region.  was concluded that at  Furthermore,  the  1963)  was. the same as  sensitive step.  Comparison o f the e f f e c t  of  and the T83 m u t a t i o n , on the  i n h i b i t i o n of p r o t e i n s y n t h e s i s initiation  of DNA r e p l i c a t i o n .  S t u d i e s on DNA r e p l i c a t i o n under c o n d i t i o n s in which gross protein  s y n t h e s i s is i n h i b i t e d  a r e present synthesis  in a c e l l  initiator  proteins  in s t o i c h i o m e t r i c amounts, and a d d i t i o n a l  is needed f o r normal  The r e s u l t s presented temperature  has i n d i c a t e d t h a t  reinitiation  of new rounds ( L a r k ,  the p r o p e r t i e s of a p r o t e i n ,  the requirements of which were evidenced by kl C i n c u b a t i o n .  temperature  1969b).  in the p r e v i o u s s e c t i o n p o i n t e d out that  s e n s i t i v e mutation a f f e c t e d  CAM d u p l i c a t e d the e f f e c t  protein  of  the kl C, i t  s e n s i t i v e mutation  involved  was concluded t h a t  initiator  protein(s).  Since the Thus,  the  it  was d e s i r a b l e to examine the s e p a r a t e e f f e c t s of  l e v e l s of CAM on the process of  replication  high and low  in T 8 3 and compare that  to the 42 C e f f e c t .  •3 The i n c o r p o r a t i o n of  H-TdR i n t o DNA at  30 C and.kl C in  presence or absence of CAM was examined and the in F i g u r e 32.  C e l l s at  DNA r e p l i c a t i o n at is linear kl  for  C effect  30 C t r e a t e d  r e s u l t s are  kO minutes a f t e r a d d i t i o n of CAM.  ( F i g u r e 32B),  the r a t e of  50 m i n u t e s .  When compared with  and the p l a t e a u was  l e v e l , and was compar-  a b l e to the r e s u l t s of Maal^e and Hanawa 11 (1961). l e v e l was s l i g h t l y  When T83 was t r e a t e d c o n t i n u e d f o r at observed f o r  least  treated  cells.  ( F i g u r e 33).  to a kl  percent  plateau value  DNA s y n t h e s i s .  in r e s i d u a l reached at  the  i n c r e a s e in the  When the c e l l s were s h i f t e d  lasted for approximately  was a second burst  30 C , DNA r e p l i c a t i o n rate  A p l a t e a u was reached by 110 minutes  w i t h CAM, DNA r e p l i c a t i o n came to a h a l t  This plateau  Final  less.  with 25 ug per ml CAM at  post t r e a t m e n t , which was equal DNA s y n t h e s i s  In the presence  two hours but at about 25 percent o f  the c o n t r o l  the  However, the amount of DNA  r e p l i c a t e d was kO to 50 percent of the r e s i d u a l  of CAM, t h i s  The r a t e  r e p l i c a t i o n was a c c e l e r a t e d ,  most probably due to the change in temperature, reached in a p p r o x i m a t e l y  presented  w i t h 150 ug per ml CAM continued  the r a t e of the 30 C ( F i g u r e 32A).  4/10  the  after  to kl  C and  20 m i n u t e s .  50 m i n u t e s , a f t e r which  s y n t h e s i s amounting to .19 kl  residual  there  percent.  C thus added up to 46 percent  residual  MINUTES  Figure 32.  Comparison o f  150  the kl C e f f e c t  t o that o f a d d i t i o n o f  yg per ml CAM.  Uptake o f H-methyl-TdR at and 50 TJg per ml AdR, i n t o the p r e s e n c e of 150 yg per CAM (empty s y m b o l s ) , at 30  1 y c per m l , 20 yg per ml u n l a b e l led TdR, the c o l d TCA i n s o l u b l e f r a c t i o n in T 8 3 , in ml CAM ( f i l l e d symbols) and absence o f C i s shown in panel A and at kl C in panel  Figure 33.  E f f e c t of a low l e v e l  of  CAM on DNA r e p l i c a t i o n  In T83-  A c u l t u r e o f T83 grown In A and E medium In the presence o f 0.5 y c per ml of ^H-TdR, 20 yg per ml c o l d TdR, and 50 yg per ml AdR, was d i v i d e d t h r e e ways. CAM (25 yg per ml) was added to s u b c u l t u r e s at 30 C (0) and hi C ( • ) . Uptake o f l a b e l l e d TdR i n t o c o l d TCA p r e c i p l t a b l e m a t e r i a l was f o l l o w e d in the p r e s e n c e of CAM f o r 30 C (0) and hi C (•)• The d i a g o n a l s o l i d l i n e r e p r e s e n t s the 30 C c o n t r o l without CAM. CPM a r e plotted versus time.  99  C.  S t u d i e s on s t a b i l i t y complex at  et a l .  1965).  the growing p o i n t and the  chromosome r e p l i c a t i o n c o u l d be i n h i b i t e d  (Pritchard  and L a r k ,  induce new rounds of  P r i t c h a r d and Lark  Premature  replication  a d d i t i o n of (premature  initiations  initiated  to c o m p l e t i o n .  were a l s o induced f o l l o w i n g  reversal  N a l i d i x i c a c i d - i n d u c e d i n h i b i t i o n of chromosome r e p l i c a t i o n 1970;  P r i t c h a r d et a l .  It  1969)-  As to the e f f e c t  stopped  (Hoi lorn and P r i t c h a r d ,  of N a l i d i x i c a c i d on t r a n s f e r ,  been reached which d i s a g r e e with one a n o t h e r . have been i n t e r p r e t e d  as a r e v e r s i b l e  r e s u l t s and c o n c l u s i o n s of  bacterial  mating  in E_. c o l i ,  1965;  Barbour,  The r e s u l t s of  i n h i b i t i o n of t r a n s f e r ,  Bouck and A d e l b e r g  Pritchard whereas  from the  origin.  to those of  Bouck and A d e l b e r g , 1970), s i n c e they  1971;  concluded t h a t t t h e r e p l i c a t i n g E_. c o l i w i t h N a l i d i x i c a c i d f o r  f o r k was d e s t r o y e d by treatment  df  30 m i n u t e s .  The Ward and G l a s e r model p r e d i c t s t h a t , when N a l i d i x i c a c i d added to a c u l t u r e of T83 at  1967).  (1970) and Hane (1971)  (1970) o b s e r v a t i o n s were s i m i l a r  (Hane,  (Ward et a 1.  two c o n c l u s i o n s have  i n d i c a t e d commencement of a new round of t r a n s f e r The Ward and G l a s e r  of  has a l s o been observed t h a t , when a  p u l s e of N a l i d i x i c a c i d was a p p l i e d d u r i n g c o n j u g a t i o n chromosome t r a n s f e r  thymine initiations).  (1964) provided e v i d e n c e t h a t p r e v i o u s l y  chromosomes c o n t i n u e d r e p l i c a t i o n  by  1964) or N a l i d i x i c a c i d (Goss  In the case of thymine s t a r v a t i o n ,  caused c e l l s to  replication  the n o n - p e r m i s s i v e c o n d i t i o n .  In E_. co 1 i , thymine s t a r v a t i o n  of  42 C f o r s u f f i c i e n t  time to  alldW'  is  complete  inactivation  residual  DNA s y n t h e s i s subsequent to  such experiments  of  the f o r k ,  However,  c a t i o n went to the  same s a t u r a t i o n  NAL added at  shortly after  times  DNA than those t r e a t e d a f t e r  shift  its  removal.  are shown in F i g u r e 3^.  blocked DNA r e p l i c a t i o n .  60% r e s i d u a l  one should not observe any  replication  shift  longer  resulted  NAL, when added,  upon r e m o v a l , level  The r e s u l t  the  effectively  residual  repli-  as the c o n t r o l s .  C e l l s with  up y i e l d e d much more  residual  i n c u b a t i o n at  for c e l l s  kl C .  For  example,  t r e a t e d 5 minutes  after  up as compared to 10 to lk% f o r c e l l s t r e a t e d a f t e r kS minutes  at  the n o n - p e r m i s s i v e temperature.  This  kl  C approached completion of  rounds w i t h time which  in very  little  that T83  residual  reinitiated  prematurely  the s h i f t  initiator  20 to 30 percent  and general  of  case.  The f i n a l In B_.  the c e l l  1969, 1970).  detectable  removal  of DNA was observed  o f NAL  protein  presence  synthesis.  i n t o f r e s h medium  residual  DNA s y n t h e s i s  replicating  3-5 hours with NAL  i n c u b a t i o n was used and NAL at  degradation  The p o s s i b i l i t y  s u b t i 1 is s t r a i n s ,  In these e x p e r i m e n t s ,  however, the  at  resulted  kl C in the  DNA in the  region occured when c e l l s were t r e a t e d f o r  than 35 minutes  down.  30 m i n u t e s , c e l l s were r e l e a s e d  was the same in e i t h e r  and R e i t e r ,  that c e l l s held  subsequent to the  l a c k i n g NAL but with or without CAM.  d a t i o n of  indicated  C e l l s were incubated at  of NAL and CAM to prevent Subsequently, a f t e r  their  d i v i s i o n at  has a l s o been examined.  level  of  level  (Cook et_ aj_.  a degrapoint (Ramareddy  not more u s e d , no  1966).  Thus  3 residual  DNA s y n t h e s i s  in a t o t a l  uptake of  H-TdR  i n t o p r e l a b e l l e d DNA  a. CL  MINUTES  F i g u r e 34.  DNA s y n t h e s i s i n T83 a t 42 C f o l l o w i n g an e x p o s u r e t o Na1 i d i x i c a c i d .  A c u l t u r e o f T83 grown a t 30 C i n A and E medium w i t h 5 uc per ml 3H -TdR, 20 y g per ml u n l a b e l l e d t h y m i d i n e ( f i n a l c o n c e n t r a t i o n ) , and 50 y g per ml AdR, were s h i f t e d t o kl C a t z e r o t i m e ( • ) . A t 5 (• ) , 10 ( f ) , 25 ( 0 ) , 35 ( 0 ) , and kS (•) m i n u t e s , as i n d i c a t e d by v e r t i c a l a r r o w s , p o r t i o n s from t h e c o n t r o l f l a s k were removed and t h e c e l l s were p u l s e d i n NAL (10 y g per m l ) . A f t e r 30 minutes ( d o t t e d l i n e ) they were washed f r e e o f NAL w i t h kl C prewarmed medium and immediately resuspended i n f r e s h N A L - f r e e medium. R e s i d u a l i n c o r p o r a t i o n o f r a d i o a c t i v e l a b e l i n t o t h e TCA i n s o l u b l e f r a c t i o n was f o l l o w e d d u r i n g i n c u b a t i o n a t kl C, and i s r e p r e s e n t e d as t h e p e r c e n t i n c r e a s e i n r a d i o a c t i v i t y (CPM). Open t r i a n g l e s (V) r e p r e s e n t r e s u l t s from a s i m i l a r e x p e r i m e n t , w i t h a 5 m i n u t e p u l s e i n NAL 10 m i n u t e s a f t e r the s h i f t t o kl C.  c o u l d not be due to turnover o f degraded The Ward and G l a s e r model should undergo 100  percent  NAL f o r 30 minutes at such an e v e n t ,  p r e d i c t s that the  residual  conditions.  model  is  in e r r o r ,  s i n c e the  level  c o m p a t i b l e w i t h the n o t i o n of  resumption of  temprarily  blocked  the c o n t r o l .  in CR34T83  studied  kinetics.  A c d o s e r examination of  of  DNA s y n t h e s i z e d remains at a  Time c o u r s e o f appearance of the d i v i s i o n  r e c o v e r y showed t h a t ,  potential  DNA r e p l i -  the Ward and G l a s e r  S y n t h e s i s and decay of the d i v i s i o n p o t e n t i a l  A.  for  residual  In  The r e s u l t s from such  Clearly,  kl C and which approximates  by in v i v o  mutant  to kl C without NAL.  T83 would be a l i g n e d f o r o n l y one round of  experiments a r e shown in F i g u r e 35.  IV.  initiator  DNA s y n t h e s i s when t r e a t e d with  30 C and then s h i f t e d  c a t i o n under the r e s t r i c t i v e  c y c l e s at  label.  potential.  r e s u l t s from p h y s i o l o g i c a l  f o r c e l l s kept at  requirements  kl C , there e x i s t e d a  f o r d i v i s i o n which accumulated c o i n c i d e n t with the rounds  DNA completed. It  was assumed t h a t the e x p r e s s i o n of d i v i s i o n p o t e n t i a l  available  at  r e c o v e r y , when net  be p r o p o r t i o n a l and o t h e r  protein  already  s y n t h e s i s was i n h i b i t e d ,  to the amount of completed DNA c y c l e s ,  cellular  r u l e s governing the r e p l i c a t i o n - s e g r e g a t i o n complex.  must growth, Ex-  periments were set to measure the amounts and k i n e t i c s of decay and s y n t h e s i s of  this division potential.  In c o n n e c t i o n with t h i s  question,  IS  20  40  60  100  80  120  140  MINUTES  F i g u r e 35.  DNA r e p l i c a t i o n  in T83  at  kl C subsequent to a 30  minute  N a l i d i x i c a c i d treatment at 30 C.  A c u l t u r e of T83, grown at 30 C in the presence o f 0.8 y c per ml ^H-TdR, 20 yg per ml u n l a b e l led TdR, and 50 yg per ml AdR, was s p l i t into three p a r t s . At z e r o t i m e , one p o r t i o n (•) was t r e a t e d w i t h NAL f o r 30 minutes at 30 C , washed f r e e o f NAL, and s h i f t e d to kl C. A second p o r t i o n (0) was t r e a t e d w i t h NAL at 5 minutes (25 minutes at 30 C ) , s h i f t e d t o kl C, and l e f t in NAL f o r 5 minutes at kl C. The NAL was removed and the i n c u b a t i o n at kl C was c o n t i n u e d . A third p o r t i o n ( • ) which served as c o n t r o l , was s h i f t e d to kl C at the same time as the o t h e r s but was not t r e a t e d w i t h NAL. R e s i d u a l i n c o r p o r a t i o n o f r a d i o a c t i v e l a b e l i n t o the c o l d TCA i n s o l u b l e f r a c t i o n was f o l l o w e d d u r i n g i n c u b a t i o n at kl C and i s r e p r e s e n t e d as the p e r c e n t i n c r e a s e in r a d i o a c t i v i t y (CPM).  experiments of (b)  were designed to d e t e r m i n e :  the d i v i s i o n p o t e n t i a l the  of  its  (c)  inhibition and  (e)  the measurement  sensitive condition. p u l s e s at defined  the k i n e t i c s  of RNA s y n t h e s i s ; of  (d)  of  the s u r v i v a l  from  messenger  using  Rifampicin  the two  effects;  o f mRNA in the  temperature  inhibitors  the process of gene e x p r e s s i o n .  of  T h u s , not  the e l o n g a t i o n  polypeptide  a c t i n g on w e l l only  process of  c h a i n s and the  half  the coding c a p a c i t y o f the preformed mRNA.  B.  The wave of e x p r e s s i o n of c e l l absence of t r a n s l a t i o n  F i g u r e 36 d e s c r i b e s the  d i v i s i o n potential  at  shift  the time c o u r s e of  d i v i s i o n at  from experiments  up and r e c o v e r y .  in  the  where the  net  If  15 to 60 minutes  at  the appearance of  to the l e n g t h of the kl C b l o c k ,  build-up.  the appearance of  each c u l t u r e was d i v i d e d  recovery  tnanscription.  of T83, p u l s e d f o r  was p r o p o r t i o n a l  then one should see a gradual  shift-up,  or  results  synthesis of a c u l t u r e  kl C , was i n h i b i t e d  of  30 C ,  These q u e s t i o n s c o u l d be s t u d i e d by means of  mRNA, but a l s o the f o r m a t i o n  protein  transcription  the comparison of  c o u l d one measure the time needed f o r  l i f e of  growth at  the d i v i s i o n p o t e n t i a l  kl C in c o n c e r t with a number of  steps o f  potential  (kl C ) , and d u r i n g r e c o v e r y  the measurement of  decay by the study of  the d i v i s i o n  synthesis during exponential  the n o n - p e r m i s s i v e temperature  a kl C p u l s e ;  synthesis  was a continuous or d i s c o n t i n u o u s one;  r a t e of the appearance and decay of  and r e g u l a t i o n at  (a)whether the  into  To have an a c c u r a t e the d i v i s i o n p o t e n t i a l  estimate before  30 s u b c u l t u r e s which were t r e a t e d  at one minute shift at  intervals  down, w i t h CAM at  at kl C , f o r  15 minutes b e f o r e and a f t e r  150 yg per m l .  During the next 200 minutes  30 C , the c e l l s went through some r e s i d u a l d i v i s i o n , p r o p o r t i o n a l  the p o t e n t i a l  they had f o r  such d i v i s i o n , and came to a h a l t .  p l a t e a u v a l u e s f o r each c u l t u r e were then p l o t t e d a g a i n s t when CAM was added. h i b i t i o n of p r o t e i n if  It  c o i n c i d e n t with the r e s i d u a l  a f t e r k5 minutes of  In f a c t ,  numbers i n t e r c e p t  such an  the  greater  such was  numbers o c c u r e d .  c o u p l i n g between c e l l  the  temperature  the g e n e r a t i o n time of the c e l l .  i n c u b a t i o n , d e s p i t e the  more i n c r e a s e in c e l l a stringent  time  i n t e r v a l s of p r o t e i n s y n t h e s i s ,  e q u i v a l e n t s were g e n e r a t e d .  l i n e s c l o s e l y followed  substrates for  DNA s y n t h e s i s , was a l l o w e d ,  case s i n c e the p l a c e where c e l l shift  the  s y n t h e s i s , the c e l l s c o u l d express d i v i s i o n o n l y  O b v i o u s l y at kl C , as longer  numbers o f c e l l  The  must be remembered t h a t , d u r i n g the CAM i n -  they had the necessary p r o t e i n s and o t h e r  event.  to  However,  i n c r e a s e in c e l l mass, no  C l e a r l y , t h e r e must have been  d i v i s i o n and DNA r e p l i c a t i o n which  determined the number of c e l l s generated under such c o n d i t i o n s .  The  experiment  in  every c a s e ,  represented  in F i g u r e 36 shows c l e a r l y t h a t v i r t u a l l y  upon i n c u b a t i o n o f c e l l s at  kl C longer than 10 m i n u t e s ,  in the absence of p r o t e i n s y n t h e s i s , one o b t a i n e d a complete l o s s of d i v i s i o n potential The l o s s  of the  cells.  in the d i v i s i o n p o t e n t i a l  at  kl C was c l e a r l y not due  to a d e c r e a s i n g amount of messengers which remained t r a n s l a t a b l e . fact,  when mRNA s y n t h e s i s was i n h i b i t e d  by R i f a m p i c i n b l o c k ,  (see  In the  20  0  40  60  80  MINUTES F i g u r e 36.  The wave o f c e l l  kl t o  30  d i v i s i o n during a t r a n s i t i o n  from  C.  grown at 30 C to a d e n s i t y o f 3-0 x 10' c e l l s per ml was s h i f t e d t o kl C . A f t e r 15 (•), 30 ( o ) , kS ( • ) , and 60 (V) m i n u t e s , c e l l s were r e t u r n e d to 30 C. F i f t e e n minutes b e f o r e and a f t e r the s h i f t down to 30 C , a l i q u o t s were removed from the main f l a s k t r e a t e d w i t h CAM (150 yg per ml) and were d i s p e n s e d i n t o new f l a s k s . The f i n a l c e l l numbers a r r i v e d at a f t e r 200 minutes f o l l o w i n g the s h i f t down a r e p l o t t e d a g a i n s t the time o f a d d i t i o n of CAM. The v e r t i c a l d o t t e d l i n e s r e p r e s e n t the z e r o time f o r s h i f t down to 30 C f o r each c u l t u r e . The e f f e c t o f R i f a m p i c i n at 10 yg per ml under i d e n t i c a l c o n d i t i o n s f o r the 15 minute p u l s e i s r e p r e s e n t e d by squares.  CR34T83  r e s u l t s of f i n a l  cell  numbers in the absence of t r a n s c r i p t i o n )  the number of c e l l s generated was g r e a t e r  36),  The two a n t i b i o t i c s ,  though a f f e c t i n g  (Figure  from that of CAM a l o n e .  macromolecular s y n t h e s i s in a  unique f a s h i o n , c o u l d have had d i f f e r e n t  effects  this'  in the presence of CAM,  1  case.  protein  When E_. c o l i were incubated  s y n t h e s i s was a r r e s t e d  was continuous  (Nomura and Watson, 1959)  ( S e l l s and S a y l e r ,  the RNA polymerase, b l o c k s the the data  here,  i s meager.  seems l e s s  inactivated  at  However,  likely  1971).  initiation  s u p p o r t i n g the e x i s t e n c e  half-lives  this  of new mRNA c h a i n s .  If,  in f a c t ,  The h a l f  i n c u b a t i o n at  minutes.  In f a c t ,  showed mRNA h a l f  Currently,  precluded  messenger RNA was exponential  residual  f u n c t i o n , - , The  d i v i s i o n as a  kl C , in the absence of  protein  shows that t h i s was the case indeed.  in the presence of R i f a m p i c i n F i g u r e 37,  of percent  l i f e o f the d i v i s i o n p o t e n t i a l  c l o s e to 1.4  to  of any s u r v i v i n g mRNA, assumed  to be c o n s t a n t , should a l s o have been an exponential  s y n t h e s i s used in F i g u r e 36,  in  RNA synthes  R i f a m p i c i n , by b i n d i n g  curves c o u l d be  r a t e of t r a n s l a t i o n  f u n c t i o n of p u l s e of  while  p o s s i b i l i t y , although not  to be the c a s e .  semi 1ogarithmic r e p r e s e n t a t i o n  division  in E_. c o l ? of mRNA with widespread  random, mRNA s u r v i v a l  f u n c t i o n s and the  on c e l l  c a l c u l a t e d from t h i s was very  the measurement of the  in experiments  l i f e of  1.3  similar  minutes.  concentration  used i n h i b i t e d well over 90  incorporation  into RNA.  residual  division  to those of  The R i f a m p i c i n  percent of  radioactive  uracil  F i g u r e 37-  Decay of c e l l  d i v i s i o n potential  of CR34T83 at  T h i s graph r e p r e s e n t s the semi l o g a r i t h m i c p l o t of the experiment p l a c e of  the  represented  in F i g u r e 36 before z e r o time.  i n t e r c e p t of wave curves with the s h i f t  taken to be 100 percent r e s i d u a l d i v i s i o n . in c e l l value  number  from The  a x i s was  The percent  in the CAM t r e a t e d samples from the  is p l o t t e d  recovery  the data  kl C.  increase  intercept  a g a i n s t the time of a d d i t i o n of CAM p r i o r  to  MINUTES  Figure  38.  Decay of c e l l  T h i s graph r e p r e s e n t s experiment  represented  d i v i s i o n potential  the s e m i l o g a r i t h m i c in F i g u r e 36.  wave curves w i t h the s h i f t division.  The p e r c e n t  of a d d i t i o n of  a x i s was taken  intercept  Rifampicin prior  to  CR34T83  p l o t of  value  to be 100  number  the  kl  C.  from  intercept  percent  in the  is p l o t t e d  recovery.  at  the data  The p l a c e of  i n c r e a s e in c e l l  t r e a t e d samples from the  of  the of  residual  Rifampicin  against  the  time  V.  G e n e t i c a n a l y s i s of  P l e i o t r o p i c mutants m i g h t ,  in t h e o r y ,  as a r e s u l t o f a s i n g l e mutation is  involved  Several  the former  Holland and T h r e f a l l , fore,  (2)  in a complex; or  examples of  CR34T83.  the mutant  arise  in a s t r u c t u r a l  mutations..  type are known (Jones-Mortinner, It  1965).  d e s i r a b l e to e s t a b l i s h the g e n e t i c b a s i s f o r  s e n s i t i v e mutation and the  (1)  gene whose product  from two independent  Demoss and Wagmar,  1969;  in two ways:  the  1968;  was,  there-  temperature  pleiotropy.  P r e l i m i n a r y mapping of T83 by c o n j u g a t i o n had l o c a t e d the mutation  in the  llv  region  ( H i r o t a et a 1.  observed between temperature  1968).  sensitive  However,  initiation  the  and c e l l  correlation division  led to a c l o s e r examination of the g e n e t i c s of T83 and a more p r e c i s e mapping by t r a n s d u c t i o n . two m u t a t i o n s , properties  It  one a f f e c t i n g  involved  was q u i t e p o s s i b l e that there  existed  DNA i n i t i a t i o n  membrane  in d i v i s i o n .  and the o t h e r ,  The e x i s t e n c e o f m u l t i p l e  mutation  was p o s s i b l e s i n c e NTG, which was known to s p e c i f i c a l l y produce s e v e r a l mutations at  the f o r k  been used in p r e p a r i n g t h i s  region  strain  (Cereda-Olmedo et a 1 .'  (Kohiyama et al . 1966).  types of experiments were c a r r i e d o u t : resistant  revertants  DnaA l l v  genotype; and  +  (tr  DnaA);  (2)  (l)  the study of  (3) the c o n s t r u c t i o n of other  the  llv  the one minute  DnaA segment.  transduced p o r t i o n of  » had  -Three  temperature  the study of T83 s t r a i n s with  F£. c o l ? K12 which were i s o g e n i c f o r more than 38% of except f o r  H9£8)  strains  ts  of  t h e i r chromosome,  t h e i r chromosome,  111  A.  Study of  if  temperature  resistant  (tr)  revertants.  the T 8 3 mutation was to be a p o i n t m u t a t i o n ,  frequency to temperature  r e s i s t a n c e in the range of  should have been o b s e r v e d . due to a double m u t a t i o n ,  On the o t h e r hand, i f the frequency of  two markers would have been much lower.  then a r e v e r s i o n 10 "* to 10 ^  the p l e i o t r o p y was  the back mutation  The nature of  for  the c o u p l i n g  o f DNA to c e l l u l a r growth needed f o r c o l o n y f o r m a t i o n allowed examination of  0.1  ml of  plates.  these two p o s s i b i l i t i e s .  was grown o v e r n i g h t  T83  t h i s was i n o c u l a t e d After  the  2k hours of  in A and E medium, d i l u t e d i n t o a number of t e s t  i n c u b a t i o n at  1:100 and then  tubes or onto agar  kl C , growth was observed  in the tubes and 20 to Ik c o l o n i e s were formed on the p l a t e s . of  these c o l o n i e s were i n o c u l a t e d  further  for cell  into  l i q u i d medium and were t e s t e d  d i v i s i o n and DNA r e p l i c a t i o n .  r e p l i c a t i o n was found to be normal and c e l l kl  C.  The d o u b l i n g time f o r  was r e t a r d e d  to a p p r o x i m a t e l y  tr  Each  In g e n e r a l , the DNA  d i v i s i o n c o n t i n u e d at  s t r a i n s , under kl C growth c o n d i t i o n s ,  50 m i n u t e s .  The r e v e r s i o n frequency  -6 c a l c u l a t e d was a p p r o x i m a t e l y  2 x 10  , which is  w i t h a s i n g l e p o i n t mutation  (Hayes,  1968).  B.  C o n s t r u c t i o n and a n a l y s i s of C R 3 4 T 8 3  E_. c o l ? C R 3 4 T 8 3  requires  in good agreement  llv  ts DnaA s t r a i n s .  i s o l e u c i n e and v a l i n e f o r growth.  o r d e r of  the markers that a r e known to be in t h i s  are tna,  the r e g u l a t o r y gene f o r  Riley,  +  r e g i o n of  The  the map  tryptophanase p r o d u c t i o n (Gartner  1965) , phoS, the r e g u l a t o r y  gene f o r  and  phosphatase (Echols et_ al_.  1961),  11v, t h e i s o l e u c i n e - v a l i n e g e n e c l u s t e r  1965),  a n d DnaA  (Hirota eta l .  O  ( D C  JQ  73 The llv the  1968; 1970).  Adelberg-, i s shown  (marker)  75  (map l o c a t i o n ,  l l v a n d t s DnaA a l l o w e d  minutes)  f o r introduction o f the  marker by t r a n s d u c t i o n , and a n a l y s i s o f such t r a n s d u c t a n t s f o r  +  proximal  t s DnaA  marker.  In a t r a n s d u c t i o n e x p e r i m e n t , a l y s a t e o f g e n e r a l i z e d phage P i k e T83  (Lk) w a s made o n EM c o l ?  was i n f e c t e d w i t h  tested  under  o f T83 l l v Table  transductants  +  typical  r e c i p i e n t s had l o s t resulted +  1970).  was a b o u t  2.2  llv,  transductants  Fifty-six  percent  o f the l l v This  could  T83  +  have  a w i l d type a l l e l e  f o r DnaA  t h a t was u s e d h e r e c o u l d  distance f o r cotransduction  f o r cotransduction  t oone another.  trans-  ( C a r o and o f two  m i n u t e s o f t h e map ( T a y l o r a n d T r o t t e r , 1967)  which were a d j a c e n t  trans-  +  a g a r p l a t e s a t kl C.  3 m i n u t e s w o r t h o f E_. c o l i chromosome  the p o s s i b i l i t y  were  l a c k i n g l l v ; a n d (b) g r o w t h  phage l y s a t e c a r r i e d  The l o n g e s t  r e s u l t s agreed with DnaA a n d  results.  +  ( a ) g r o w t h o f T83 M v  supplemented  T h e Lk s t r a i n o f p h a g e P i k e  duce approximately  +  the temperature s e n s i t i v i t y .  i f the donor  llv .  on f u l l y  transducing  w h i c h was l l v .  phage and t h e l l v  two s e l e c t i v e c o n d i t i o n s :  IV shows  Schnos,  the donor  K12 AB 1157,  a t 30 C o n m i n i m a l a g a r p l a t e s  ductants  and  below.  I  74  l i n k a g e between  Their order  4-1O  3  .-  Q  (Ramakrichnan and  markers  • The  o f two m a r k e r s ,  +  Table  III.  Donor  AB  1157  (DnaA +)  +  | l v  The frequency of j o i n t  . . Recipient n  r  CR34T83 (ts DnaA |  l  v  )  t r a n s d u c t i o n s o f the I l v and DnaA  Genotype _ , [ , Selected  Mv  +  DnaA  T r a n s d u c t a n t s were scored on minimal requirements  Numbers o f . Transductants scored  supplements  Marker  T  Type  70 (100%)  DnaA  32 (100%)  Ilv  Number  39 (56%)  +  32 (100%)  medium supplemented with a l l the  but f o r i s o l e u c i n e and v a l i n e  30 C , o r with f u l l  Unselected  loci.  (selection  (selection f o r DnaA ) +  Experiments were performed as d e s c r i b e d in M a t e r i a l s  for l l v ) +  at  at kl C . and Methods.  114  C.  Introduction  of the T83 gene i n t o E_. c o l i K12 s t r a i n s  and the a n a l y s i s of One of the p a r e n t a l used as donor  the  pleiotropy.  strains  isolated  in the experiments  listed  was ts DnaA and had r e c e i v e d the  llv  Pi phage l y s a t e s were made on t h i s  +  was l i n k e d with the  below.  marker  Table  The r e s u l t s c l e a r l y  l l v and tna markers.  that were i s o l a t e d , a n a l y s i s of growth at In 15 of  This s t r a i n ,  from E_. c o l i K12 AB 1175-  IV shows the  H-thymidine  30 C and 42 C was performed.  fraction, Thus,  resembled t h a t of  minutes  into  llv  1 to 2 minutes of  +  transductants  DNA r e p l i c a t i o n  i n c o r p o r a t i o n of  the c o l d TCA i n s o l u b l e  c o n s t r u c t e d , which  the T83 chromosome from the 73 to 74  r e g i o n of the map, the p l e i o t r o p y d i s p l a y e d must have been  a c q u i r e d v i a the t r a n s d u c i n g phage p a r t i c l e . formed f o r  these e x p e r i m e n t s ,  s e p a r a t e mutations It  30 C .  division  T83.  in the E_. c o l i Kl2  had i n h e r i t e d  in c e l l u l a r  the r a t e of  ( s i n c e the s t r a i n s were thy )  results  Of the ts DnaA t r a n s d u c t a n t s  r e s u l t e d upon i n c u b a t i o n at kl C from growth at as examined by measurement o f  strains  K12  i n d i c a t e d t h a t the t s DnaA  the kl ts DnaA s t r a i n s c h e c k e d , a h a l t  patterns  KG 776,  s t r a i n and c e l l s of other  were i n f e c t e d w i t h t h i s donor phage. of these e x p e r i m e n t s .  in the p r e v i o u s s e c t i o n was  the p o s s i b i l i t y of  responsible for  remains to be seen whether  ment of the chromosome a r e  With the c o n t r o l s  the e x i s t e n c e of  the p l e i o t r o p y , were r u l e d  o n l y one or more c i s t r o n s  involved  in the  and in the o t h e r membrane p r o p e r t i e s .  regulation  However,  per-  recent  out.  in t h i s  of DNA  two  seg-  initiation  s t u d i e s of  115  Table  IV.  Frequency from  of j o i n t  E. c o l i  Reci i e n t ecipien  Donor  t r a n s d u c t i o n s o f t h e I l v and DnaA  T83.  Genotype Selected  Unselected  Number o f Transductants scored  KG  776  KG 146  loci  Ilv  60  Marker Numbers  Type  (100%)  DnaA  (Hv")  ts  23 ( 4 0 % )  DnaA"*  34 ( 6 0 % )  ts DnaA ) t  n  a  KG 163  Ulv",  Ilv  67  (100%)  tna  tna")  tna  KG 166  llv  80  +  (100%)  (Nv", tna  , phoS  T r a n s d u c t a n t s w e r e s c o r e d on m i n i m a l  medium s u p p l e m e n t e d  b u t f o r i s o l e u c i n e and v a l i n e ( s e l e c t i o n  or w i t h  supplements  (selection  was t e s t e d by t h e t r y p t o p h a n a s e a s s a y . described  i n M a t e r i a l s and M e t h o d s .  10 ( 1 5 % )  t n a DnaA  +  52  tna"DnaA  +  19 ( 2 4 % )  +  Experiments  18 ( 2 7 % )  0 (0%)  (65%)  tna DnaA  t S  9 (11%)  tna"DnaA  t S  0 (0%)  with a l l the  for llv )  f o r D n a A ) a t 42 C.  (58%)  DnaA +„ „ts t n a DnaA ts t n a DnaA +  +  requirements full  39  DnaA  +  a t 30 C  The t n a m a r k e r  were performed  as  H i r o t a et_ a K  (1970) on the g e n e t i c s of both TkS and T83 have  d i c a t e d that such p o s s i b i l i t i e s a r e very  unlikely.  DISCUSSION  The  t s DnaA g e n e was  t i n u a t i o n of  DNA  temperature. by  shown t o a f f e c t  replication  i n E_. c o l i  Support f o r t h i s  the g e n e t i c  studies.  since w i l d type  The  cellular  CR34T83  pleiotropic  l e s i o n was  revertants which could  division  at the  and  con-  non-permissive  r e l a t i o n s h i p was  shown t o be  a point  grow n o r m a l l y  a t kl  obtained mutation, C were  -6 isolated  at a frequency  o f 2 x 10  r a t e of a point mutation.  The  i s o l e u c i n e - v a l i n e c l u s t e r and t h e enzyme t r y p t o p h a n a s e . a n a l y s i s of ductional t h e T83  H i r o t a and  when t h e  1 min  transduced  g e n e was was  IV)  which could segment o f  the  and  the  gene f o r  extended  1968; 1970).  t h e r e was  r e s p o n s i b l e f o r the  the Trans-  nothing  but  pleiotropy, since  chromosome c a r r y i n g t h e  E_. c o l i K12  reversion  structural  ( H i r o t a et a l .  indicated that be  the  co-transducible with  l i n k e d to the  co-workers  to w i l d type  with  These r e s u l t s confirmed  a n a l y s i s (Table  mutation  , compatible  strains, a similar  DnaA g e n e  was  response  was  obtained. The  DnaA m u t a t i o n  replication normal  a t kl  (results  t i m e a t kl  C.  C.  not  s p e c i f i c a l l y a f f e c t e d c o n t i n u a t i o n of Uptake o f exogenous thymidine  s h o w n ) , y e t DNA  tions  ( K o h i y a m a et_ aj_.  1966)  .  a t kl  C was  with  completion  exponential  compatible  The  population, according  i n t o the c e l l  s y n t h e s i s ceased a f t e r a  These r e s u l t s agree w i t h  Kohiyama's o r i g i n a l  amount o f of  to the  the  r e s i d u a l DNA  rounds of Helmstetter  DNA  short observasynthesized  replication and  was  i n an  Cooper model.  The gradual  decline  side triphosphate  in the DNA s y n t h e s i s was not due to  pools.  In f a c t ,  pools accumulated d u r i n g the However,  i n c u b a t i o n at  the n u c l e o s i d e kl C ( F i g u r e  the dTTP pool d i d not expand s i g n i f i c a n t l y .  Pritchard's strains  most of  of  (1971)  finding  that dTTP pool was normally  E_. c o l i , supports t h i s o b s e r v a t i o n .  low dTTP l e v e l  in T83 at  precursor for  thymine,  triphosphate  10 and 1 1 ) .  Beacham and low in  thy  The presence of a  kl C , c o u l d a l s o be taken as  evidence f o r Werner's arguments  low n u c l e o -  supportive  (Werner, 1971) that dTTP is not  in DNA r e p l i c a t i o n .  the  T h i s would e x p l a i n why  o n l y dATP, dCTP and dGTP pools accumulated with the c e s s a t i o n of DNA synthesis; no o t h e r  they were the  DNA s y n t h e s i s .  resolve this question.  S i n c e the  bacteriophages lk and lambda were u n a f f e c t e d  (Kohiyama, all  four  1968),  it  replication  growth c o n d i t i o n s at  (Lark and Ranger,  DNA tion  kl C , d i d not a l t e r However,  1969).  at a l e v e l  clear.  of  resulted  kl C .  the mode of  kl C in  (Figure  of CAM at  new rounds of DNA in r e s i d u a l  DNA  A d d i t i o n of CAM at residual  of 25 yg per ml of  s y n t h e s i s curve r e s u l t e d is not  30 C, a d d i t i o n  known to b l o c k r e i n i t i a t i o n  compatible with t h a t of growth at  32).  in T 8 3 grown at  precursors.  a concentration  at  replication  c o u l d be argued t h a t t h e r e was no l i m i t a t i o n  Under e x p o n e n t i a l  ml,  However,  thymine c o n t a i n i n g p r e c u r s o r s of DNA s y n t h e s i s was measured  in t h i s work to f u r t h e r of  t r u e p r e c u r s o r s of  33).  replication  150 ug per  DNA s y n t h e s i s  CAM, a b i p h a s i c  The reason f o r  (Figure residual  this  devia-  119  I n i t i a t e d DNA r e p l i c a t i o n at  kl C, to the e f f e c t s  forks  in T83 were shown to be s t a b l e ,  of N a l i d i x i c a c i d .  Ward and G l a s e r  (1970)  have shown that treatment o f E_. c o l i B / r with N a l i d i x i c a c i d f o r 30 minutes prevents the resumption o f the r e p l i c a t i o n o f the a f f e c t e d forks.  A c c o r d i n g l y , in T 8 3 , r e s i d u a l  DNA s y n t h e s i s a f t e r  from a N a l i d i x i c a c i d b l o c k c o u l d occur o n l y As shown in F i g u r e 3k,  residual  i f o l d f o r k s were s t a b l e .  DNA s y n t h e s i s at kl C d i d occur  sequent to removal o f the N a l i d i x i c a c i d b l o c k . p e r s . commun.)  release  Lark  sub-  ( J . Urban,  has used a d e n s i t y l a b e l 1?ng technique to f o l l o w DNA  replication after  1  N a l i d i x i c a c i d treatment f o r 50 m i n u t e s .  Their  r e s u l t s agreed with t h i s work i n d i c a t i n g completion o f p r e v i o u s l y initiated cycles.  The evidence of Ward and G l a s e r , which  on g e n e t i c a p p r o a c h e s , cannot be r e f u t e d ,  yet i t  is based  i s not c o m p a t i b l e  w i t h these f i n d i n g s . In the study of an i n i t i a t o r  type mutant,  e s t a b l i s h the p l a c e of r e i n i t i a t i o n  after  t h e s i s at the n o n - p e r m i s s i v e temperature.  to at  i n h i b i t i o n of DNA s y n -  I d e a l l y , one should a l s o  between the p l a c e f o r r e i n i t i a t i o n and  the o r i g i n o f DNA r e p l i c a t i o n .  O968)  is c r i t i c a l  of rounds of DNA r e p l i c a t i o n  recovery at the p e r m i s s i v e temperature  demonstrate the r e l a t i o n s h i p  it  In the c h a r a c t e r i z a t i o n  examined the p l a c e f o r the t e r m i n a t i o n  o f T 8 3 , Kohiyama  of chromosome r e p l i c a t i o n  14 at  kl C .  T h i s was shown by  a c i d s t a r v e d c e l l s a t 30 C .  C-TdR l a b e l l i n g o f the DNA in amino By r e t u r n i n g  the c e l l s to amino a c i d  3 supplemented media at 30 C and s u b s e q u e n t l y p u l s i n g with  H-TdR, the  s t a r t s of  rounds of  r e p l i c a t i o n were l a b e l l e d .  was chased w i t h 600 yg per ml c o l d t h y m i d i n e , allowed  to randomize t h e i r  DNA c y c l e s .  g i v e n 5-BrdUrd in the p l a c e of a n a l y s i s of  the e x t r a c t e d  c o n t a i n e d 50% o f  the  14  thymidine.  41 C , and the s h i f t  r e p l i c a t i o n at  The d e n s i t y  to 41 C and  gradient  DNA showed t h a t the h y b r i d d e n s i t y band 3 the "^H-labelled DNA.  C- and 25% of  initiation  Thus,  preferential  rounds as d e f i n e d by amino a c i d  of chromosomes.  However,  if  it  of DNA c y c l e s  to 41 C with 5-BrdUrd allowed  the terminus of  t i o n and alignment  H-TdR,  and the c e l l s were  C e l l s were s h i f t e d  was concluded that T83 was blocked in the at  The p u l s e of  starva-  t h i s argument were to  3 be t r u e ,  no  H-TdR should have banded in the h y b r i d d e n s i t y  There a r e s e v e r a l o t h e r o b j e c t i o n s to t h i s (1)  The use of amino a c i d s t a r v a t i o n  widespread, method  i s not very s a t i s f a c t o r y .  is t h a t the c a p a c i t y of  d u r i n g amino a c i d s t a r v a t i o n  chromosomes complete rounds o f (2) Kohiyama At t h i s  When l a b e l l i n g  97 minutes  the s t a r t s  of  chromosome at  and Z a r i t s k y ,  initiation  (Caro and B e r g ,  not  all  1969)'  rounds of DNA r e p l i c a t i o n , (0.5 yg per ml)  1970).  time at  of  thymidine.  37 C is equal  T h u s , very  l i t t l e of  to the  would have been l a b e l l e d d u r i n g the 8 minute  3 p u l s e of  H-thymidine  employed. 3  (3)  In o r d e r  this  DNA d e c l i n e s  1966) and t h e r e f o r e ,  the chromosome r e p l i c a t i o n  (Pritchard  to a l i g n chromosomes although  One problem a s s o c i a t e d with  replication  (1968) used low c o n c e n t r a t i o n s level,  experiment:  the c e l l s to r e p l i c a t e  (Doudney,  region.  to chase the  H-TdR, 600 yg per ml of u n l a b e l led  121  TdR was used f o r 90 m i n u t e s .  A d d i t i o n of  large quantities  of  thymidine  to c e l l s expands the d e o x y r i b o s e - 5 - p h o s p h a t e pools and r e t a r d s cellular  respiration  DNA r e p l i c a t i o n , and Denhardt, (4) to permit  1968).  (Lomax and Greenberg,  s i n c e the two processes are  the  T h i s could  interdependent  affect  (Cairns  1968).  The 90 minutes of  i n c u b a t i o n at 30 C was not  long enough  randomization of the p o p u l a t i o n of chromosomes a f t e r  alignment.  Thus, f o r t u i t o u s l y ,  picked up a f t e r  the s h i f t  In the experiments  the ends of  their  rounds c o u l d have been  into 5-BrdUrd medium.  d e s c r i b e d in F i g u r e 3 1 ,  sumption of DNA r e p l i c a t i o n  the p l a c e of  d u r i n g recovery was e s t a b l i s h e d .  reThe  3 chromosomes were l a b e l l e d at  ends with  H-TdR and the s t a r t s  with  14 C-TdR.  The d e n s i t y - l a b e l l e d DNA s y n t h e s i z e d at recovery was always 14 a s s o c i a t e d with the C-label. S i m i l a r l y , when chromosomes were a l i g n e d by chloramphenicol  treatment  (Figure 3 2 ) , t h e d e n s i t y  label  14 was always found to be a s s o c i a t e d with the  C - l a b e l , that  the s t a r t s o f r o u n d s , as d e f i n e d by the temperature the p l a c e of  resumption of DNA r e p l i c a t i o n  the same as the o r i g i n of DNA r e p l i c a t i o n —  i s , with  block.  Whether  in T83 d u r i n g recovery (7 - 8 o ' c l o c k )  is  on the  c o l i genet i c map, remains to be t e s t e d . I n h i b i t i o n of p r o t e i n  s y n t h e s i s d u r i n g the f i r s t  recovery o f the p e r m i s s i v e temperature, reinitiation  of  rounds of  replication  was shown to (Figures.22,  10 minutes  of  i n t e r f e r e with  23,  24).  If  protein  synthesis was permitted, resumed DNA replication temperature,  then c e l l s returned to 30 C from a kl C pulse (Figure 9) ••- Cells returned to the permissive  after a pulse at kl C, showed a drop in the rates of DNA  replication for some time. unknown.  One interpretation  The reason for this behaviour remains of the residual decline in the rate of  r e p l i c a t i o n at 30 C could be a need for the c e l l s to recover from temperature  inactivated  d i l u t i o n mechansim.  initiation  molecules by a "flush out" or  Such a mechanism would predict a.variable  the c e l l s to acquire a l l for  initiator  lag for  the functional and active molecules needed  replication of their chromosomes.  In f a c t ,  the DNA  r e p l i c a t i o n resumed with a variable lag which was dependent upon the length of pulse at kl C (Figure 9 ) .  However, with incubation at  kl C, longer than 15 minutes, the length of lag decreased, a result contrary to the predictions of the "flush out" mechanism. possible that complete inactivation of the i n i t i a t o r  It  is  molecules in an  exponential culture requires treatment longer than 10 minutes at kl C; for pulses of 10 minutes or l e s s , only a f r a c t i o n of the population is a f f e c t e d , whereas with longer pulses, the whole population is h i t . Figure 9 could be interpreted according to the model of DNA replication proposed by Rosenberg et a 1. ( 1 9 6 9 ) .  The enzyme needed  for i n i t i a t i o n of a round are always present in the c e l l , but a repressor of i n i t i a t i o n et a l . its  interferes with their  inauguration.  (1969) suggested that an antirepressor triggers  interaction with the repressor.  Rosenberg  i n i t i a t i o n by  The antirepressor, synthesized  at  t h e end  of every  replication. element  replication  It could  be a s s u m e d t h a t  i s the a n t i r e p r e s s o r .  t i m e , a l l chromosomes would i n i t i a t e a t 30  C  longer  delays  results  at  a t kl  recovery  I t was c e a s e d and  and  shown  C  in c e l l s  t h e end  would  be  f o r longer short  induces  ( F i g u r e 2)  other  divided recovery by  not  had  no  shown).  was in  of  long  would  the a n t i r e p r e s s o r . p r o d u c e d and  While t h i s explains  t h a n 10  minutes, there  lag observed with  strains  filamentous  a t kl  i n t o normal a t 30  C  acid  coli  of  e f f e c t on  C  (results  growth, the  sized cells  the  10  hence, the  is  minute  T83  d i v i s i o n was  Pantoyl  induction of  C,  (Adler  tempera1969)  Hirota,  shown).  r e p l i c a t e d chromosomes  ( F i g u r e 8).  division  filaments  ( R i c h a r d and  Cell  C,  segregated  filaments  division  during  i n chromosome  substantiated that  d e p e n d e n t upon c o m p l e t e d chromosome c y c l e s . residual cell  CR34T83.  upon a r e t u r n t o 30  a t kl  cell  g r o w i n g as  salt not  C,  E_. c o l i t o d i v i d e  i n c u l t u r e s t h a t were b l o c k e d ( F i g u r e 14)  t o kl  Similarly, division  ( r e s u l t s n o t shown) a n d ,  Nalidixic  of  C for a  r o u n d s , and  rounds a r e  r e s u l t e d i n £.  filamentous  i n T83  ineffective  normally  their  t h a t , once s h i f t e d  growth  1965), b u t  (results  During  cycles  temperature s e n s i t i v e  k e p t a t kl  required.  t u r e s e n s i t i v e f i l a m e n t s by a d d i t i o n o f was  of  f e w e r ends o f  f o r the  filamentous  Hardigree,  a t kl  in T 8 3 , the  new  9)•  (Figure  lactone  C,  trigger  upon t h e a c q u i s i t i o n  f o r temperature pulses  no o b v i o u s e x p l a n a t i o n pulse  Thus,  be a t  immediately  With s h o r t e r pulses  c y c l e , would  replication  division  Anr. a p p r e c i a b l e  o b s e r v e d when N a l i d i x i c  a c i d was  change  added  after  40 to 50 minutes  time a f t e r initiated  shift  r e c o v e r y at 30 C ( F i g u r e  14B).  from 42 C to 30 C , new c y c l e s o f  which went to c o m p l e t i o n a p p r o x i m a t e l y  T h u s , at  some  r e p l i c a t i o n were 50 minutes a f t e r  the  shift. If  s y n t h e s i s of RNA and p r o t e i n were blocked at  from kl C to 30 C , r e s i d u a l I n h i b i t i o n of t o t a l ute  i n c u b a t i o n at  cell  d i v i s i o n at  RNA s y n t h e s i s at  kl C , r e s u l t e d  numbers whereas a d o u b l i n g  chloramphenicol or N a l i d i x i c a c i d conditions  (Figures  15,  16),  d i v i s i o n at  cells"  number of  of a l l  normal c e l l  1970)  kS min-  i n c r e a s e in c e l l  1k, 19, 2 0 ) .  of  Under the same  to complete two d o u b l i n g s  a v a i l a b l e mRNAs in the  recovery was p r o p o r t i o n a l  (Donachie and Begg,  to 30 C a f t e r  number o c c u r r e d upon a d d i t i o n  the a b i l i t y  probably was due to t r a n s l a t i o n Cell  the s h i f t  (Figures  shift  recovery was o b s e r v e d .  in a f o u r - f o l d  in c e l l  the time of a  to the number of  generated at  kl C .  cells.  "unit  S i n c e the maximum  e q u i v a l e n t s generated at kl C , would be  limited  by the numbers of chromosomal c o p i e s a v a i l a b l e a f t e r c o m p l e t i o n of rounds of  replication,  final  d i v i s i o n should p l a t e a u f o r T h i s was demonstrated The a c t i v i t y recovery section  of  cell  p u l s e s at  in F i g u r e the  number produced by r e s i d u a l  kl C longer than 45 m i n u t e s .  21.  initiator  molecule was not regained  in the presence of c h l o r a m p h e n i c o l . II.  C) c l a s s i f y the behaviour o f  42 C as denatured and i n a c t i v e synthesis  is o b l i g a t o r y  for  cell  the  R e s u l t s presented initiator  (type a_ t r a n s i t i o n ) .  new a c t i v i t y  at  molecule  Thus new  (see at  protein  d u r i n g the 30 C recovery p e r i o d .  However, the t r a n s i t i o n f o r  the d i v i s i o n element  t h a t of n a t i v e and a c t i v e at (type b_ t r a n s i t i o n ) . r e c o v e r y at cell  30 C to n a t i v e but  The express ion of e e l l u l a r  30 C based on " p o t e n t i a l  mass, d i d not r e q u i r e a c t i v e p r o t e i n  the a d d i t i o n of chloramphenicol at to 30 C. is  cell  This  i n a c t i v e at 42 C d i v i s ion dur ing  equivalents",  ie.,  DNA to  s y n t h e s i s as was shown by  the time of the s h i f t  i n d i c a t e d t h a t the t r a n s i t i o n  r e v e r s i b l e once returned  in the complex was  from kl C  f o r the d i v i s i o n element  to the p e r m i s s i v e temperature,  and does  not r e q u i r e s y n t h e s i s o f new p r o t e i n s . The occurence of p l e i o t r o p i c e f f e c t s is not without  precedent.  in c o n d i t i o n a l  Chiu and Greenberg  lethal  (1968) d e s c r i b e d  mutants a  mutation  in the gene f o r dCMP-hydroxymethylase that rendered b a c t e r i o -  phage lk  DNA s y n t h e s i s i n o p e r a t i v e at kl  C but not at  HMase is one of the e a r l y enzymes in T.4-infected the t e t r a h y d r o f o l a t e - d e p e n d e n t dCMP, and is coded by the lk shown to  interact  in a double s h i f t  of the t h i r d  E_. c o l i and c o n t r o l s  c o n v e r s i o n of dCMP to 5~hydroxymethy1-  gene kl.  Ik  Gene kl  DNA polymerase (gene 43) was  experiment  and 43 p r o t e i n s were unable  (28-42-28-42) when  complex was an o b l i g a t o r y is q u i t e  likely  requirement  The i n t e g r i t y  to  after  of the new  f o r DNA r e p l i c a t i o n .  that the complex events that a r e known to be  a s s o c i a t e d w i t h DNA-membrane attachment and d i v i s i o n at region,  viral  the s y n t h e s i s  component, which was made between 5 and 8 minutes  r e c o v e r y , was blocked by c h l o r a m p h e n i c o l .  It  dCMP-  w i t h the dCMP-HMase and a t h i r d component b e f o r e  DNA s y n t h e s i s c o u l d commence. operate  28 C.  i n v o l v e protein-DNA and p r o t e i n - p r o t e i n  the septum  b i n d i n g r e a c t i o n s of  126  a cooperative nature.  In any e v e n t ,  served can best be e x p l a i n e d l o c a l i z e d at division.  the c e l l  the p l e i o t r o p i c  relation  in terms o f a multi-enzyme  membrane,  involved  ob-  complex  in DNA r e p l i c a t i o n and c e l l  BIBLIOGRAPHY  Abe,  M. a n d J . T o m i z a w a . 1967Replication of the Escherichia col? K12 c h r o m o s o m e . P r o c . N a t . A c a d . S c i . U.S. 5 8 : 1911-1918.  Adler,  H . I . a n d A.A. H a r d i g r e e . 1965. G r o w t h a n d d i v i s i o n o f f i l a m e n t o u s forms o f E s c h e r i c h i a c o l ? . J . Bacteriol. 9 £ : 223-226.  A l t e n b u r g , B.C., J . C . S u i t , B.R. B r i n k l e y . 1970. U l t r a s t r u c t u r e o f deoxyribonucleic acid-membrane a s s o c i a t i o n s in Escherichia coli. J . B a c t e r i o l . 104: 5 4 8 - 5 5 5 . B a r b o u r , S.D. 1967• E f f e c t o f n a l i d i x i c a c i d on c o n j u g a t i o n a l t r a n s f e r and e x p r e s s i o n o f e p i s o m a l l a c genes i n E s c h e r i c h i a c o l i K12. J . Mol. Biol. 2 8 : 373~376. B a r b o u r , S.D., H. N a g a i s h i , A. T e m p l i n a n d A . J . C l a r k . 1970. B i o c h e m i c a l and G e n e t i c S t u d i e s o f R e c o m b i n a t i o n P r o f i c i e n c y i n E s c h e r i c h i a c o l i , I I . R e c r e v e r t a n t s c a u s e d by i n d i r e c t suppression o f Rec" mutations. Proc. Nat. Acad. S c i . +  67_:  128-135.  B a r o n , C. a n d A. A b r a m s . 1971. I s o l a t i o n o f a b a c t e r i a l membrane p r o t e n e c t i n , e s s e n t i a l f o r the attachment o f adenosine t r i phosphatase. J . B i o l . Chem. 246: 1542-1544. Beacham, I.R. and R.H. P r i t c h a r d . 1 9 7 1 . The r o l e o f n u c l e o s i d e phosphorylases i n the degradation o f deoxyribonucleosides by t h y m i n e - r e q u i r i n g m u t a n t s o f E s c h e r i c h i a c o l i . Mol.  Gen. G e n e t i c s .  110: 289-298.  B a r r a h , G. and W.A. K o n e t z k a . 1 9 6 2 . S e l e c t i v e and r e v e r s i b l e i n h i b i t i o n o f t h e s y n t h e s i s o f b a c t e r i a l DNA by p h e n e t h y l alcohol. J . B a c t e r i o l . 83_: 738-744. Billen,  Bird,  D.  1 9 6 9 - R e p l i c a t i o n o f t h e B a c t e r i a l Chromosome: L o c a t i o n new initiation sites after irradiation. J. Bacteriol. 97: 1169-1175.  R. a n d K.G. L a r k . 1 9 6 8 . I n i t i a t i o n a n d t e r m i n a t i o n o f DNA r e p l i c a t i o n a f t e r amino a c i d s t a r v a t i o n o f E s c h e r i c h i a coli 15T". C o l d S p r i n g H a r b o r Symp. Q u a n t . B i o l . 33_: 799-808"!  of  Bird,  R. and K . G . L a r k . 1970. Chromosome r e p l i c a t i o n in E s c h e r i c h i a c o l ? 151 a t d i f f e r e n t growth r a t e s : Rate of r e p l i c a t i o n o f the chromosome and the r a t e f o r f o r m a t i o n o f small p i e c e s . J . Mol . B i o l . 49_: 343-366.  Blakley,  R . L . and E . V i t o l s . 1968. The c o n t r o l of n u c l e o t i d e synthesis. A n n . Rev. Biochem. 3_7_: 201-224.  bio-  B o n h o e f f e r , F. and W. M e s s e r . 19^9Replication of bacterial chromosome. A n n . Rev. G e n e t i c s . 3_: 233"247. Bouck, N . , and E . A . A d e l b e r g . 1970. Mechanism of a c t i o n of n a l i d i x i c a c i d on c o n j u g a t i n g b a c t e r i a . J . B a c t e r i o l . 102: 688-701. B o y l e , J . V . , T . M . Cook and W.A. G o s s . 1967. Induction of e x c e s s i v e d e o x y r i b o n u c l e i c a c i d s y n t h e s i s in Escher ich ia c o l ? by naladixic acid. J . Bacteriol. 9 _4_: 1664-1671 . B u r g e s s , R . P . , A . A . T r a v e r s , J . J . Dunn and E . K . F . Bautz. 1969. F a c t o r s t i m u l a t i n g t r a n s c r i p t i o n by RNA polymerase. Nature 221: 43-47. B y f i e l d , J . E . and O . H . Scherbaumv 1966. A r a p i d r a d i o a s s a y technique f o r c e l l u l a r s u s p e n s i o n s . A n a l y t . Biochem. J_7_: 434-443. C a i r n s , J . 1963 - The chromosome o f E s c h e r i c h i a c o l ? . Harbour Symp.'Quant. B i o l . 28~: 43-45.  Cold Spring  C a i r n s , J . and D . T . Denhardt. 1 9 6 8 . E f f e c t of c y a n i d e and carbon monoxide on the r e p l i c a t i o n of b a c t e r i a l DNA in v i v o . J . M o l . B i o l . 3_6_ 3 3 5 - 3 4 2 . :  Calendi,  E . , A . Di Marco, M. R e g g i a n i , B. S c a r p i n a t o and L. V a l e n t i n i . I965. Physicochemica1 i n t e r a c t i o n s between daunomycin and nucleic acids. B i o c h i m . B i o p h y s . Acta 1 0 3 : 2 5 - 4 9 .  Carl,  P . C . 1970. E s c h e r i c h i a c o l i mutants w i t h temperature s e n s i t i v e s y n t h e s i s o f DNA. M o l . Gen. G e n e t i c s . 109: 107-122.  Caro,  L.  Caro,  L . G . and C M .  1970. Chromosome r e p l i c a t i o n in Escher ich ia c o l i S e g r e g a t i o n o f chromosomal s t r a n d s in m u l t i - f o r k e d cation. J . M o l . B i o l . 48_: 329-338. Berg.  I968.  Chromosome r e p l i c a t i o n  s t r a i n s of Escherichia col? Quant. B i o l . 33: 559"573Caro,  III. repli-  in some  K12. Cold Spring Harbour  Symp.  L.and C M . B e r g . 1969Chromosome r e p l i c a t i o n in E s c h e r i c h i a c o l i . II. O r i g i n o f r e p l i c a t i o n in F~ and F strains. J . M o l . B i o l . 45: 325-336. +  129  Caro,  L. and C . B e r g . 1 9 7 1 . PI t r a n s d u c t i o n . Methods in Enzymology. V o l . XII, Part C (Methods in N u c l e i c A c i d s ) , Grossman and Moldave ( e d s . ) , in p r e s s .  Caro,  L . G . and M. Schnos. 1 9 6 6 . The attachment of the m o d e - s p e c i f i c b a c t e r i o p h a g e F1 to s e n s i t i v e s t r a i n s of E s c h e r i c h i a c o l i . P r o c . Nat. A c a d . S c i . 56.: 1 2 6 - 1 3 2 .  Cerda-Olmedo, E . , P . C . Hanawalt and N. Guerola . 1 9 6 8 . Mutagenesis o f the r e p l i c a t i o n p o i n t by n i t r o s o g u a n i d i n e : Map and p a t t e r n of r e p l i c a t i o n of E s c h e r i c h i a c o l I chromosome. J . Mol. B i o l . 33; 705-719. c  Chai,  N. and K.G. L a r k . 1 9 7 0 . C y t o l o g i c a l s t u d i e s of DNA r e p l i c a t i o n in E s c h e r i c h i a c o l ? 15~T~: R e p l i c a t i o n at slow growth r a t e s and a f t e r a s h i f t up i n t o r i c h medium. J . B a c t e r i o l . 104: 401-409.  Clark,  D.J. 1 9 6 8 a . The r e g u l a t i o n of DNA r e p l i c a t i o n and c e l l d i v i s i o n in E s c h e r i c h i a c o l ? B / r . Cold S p r i n g Harbor Symp. Quant. B i o l . 33_: 8 2 3 - 8 3 8 .  Clark,  D . J . 1968b. R e g u l a t i o n of DNA r e p l i c a t i o n in E s c h e r i c h i a c o l i B / r . J . Bacteriol.  Clark,  D . J . and 0. Maal^e. 1 9 6 7 . DNA r e p l i c a t i o n and the d i v i s i o n c y c l e in E s c h e r i c h i a c o l ? . J . Mol. B i o l . 23: 99-112.  and c e l l d i v i s i o n S6_: 1214-1224.  Cook, T . M . , W.A. Goss and W.H. D e i t z . 1 9 6 6 . Mechanism o f a c t i o n of n a l i d i x i c a c i d on E s c h e r i c h i a c o l i '. V. P o s s i b l e mutagenic effect. J . B a c t e r i o l . 9J_: 780 R 78T. Cooper, S. and C E . H e l m s t e t t e r . eel 1 d i v i s i o n c y c l e of 3J_: 5 1 9 - 5 4 0 .  1 9 6 8 . Chromosome r e p l i c a t i o n Escherichia c o l i B/r. J . Mol.  and Biol.  Cooper, S. and G. Weusthoff. 1 9 7 1 . Comment on the use of c h l o r a m phenicol to study the i n i t i a t i o n of d e o x y r i b o n u c l e i c a c i d synthesis. J . B a c t e r i o l . 1 0 6 : 709-711 C o s g r o v e , E . V . and R.W. T r e i c k . 1 9 7 0 . Comparison of PEA with i n h i b i t o r s of r e s p i r a t i o n and uncoupjers o r o x i d a t i v e phorylation. B a c t e r i o l . P r o c . p. 1 3 9 . Cutler,  phos-  R . G . and J . E . Evans. 1 9 6 7 . R e l a t i v e t r a n s c r i p t i o n a c t i v i t y of d i f f e r e n t segments of the genome throughout the c e l l d i v i s i o n c y c l e of E s c h e r i c h i a c o l ? . The mapping of ribosomal and t r a n s f e r RNA and the d e t e r m i n a t i o n of the d i r e c t i o n of replication. J . M o l . B i o l . 26_: 9 1 - 1 0 5 .  130  Daniels, M.J. 1 9 6 9 - L i p i d s y n t h e s i s in r e l a t i o n to the c e l l c y c l e of B a c i l l u s megaterium KM and E s c h e r i c h i a c o l i . Biochem. J. 1 1 5 : 697-701. Daniels, M.J. 1 9 7 1 . Some f e a t u r e s o f the b a c t e r i a l membrane s t u d i e d w i t h the a i d of a new f r a c t i o n a t i o n t e c h n i q u e . Biochem. J . 122:  198-208.  de L u c i a , P. and J . C a i r n s . 1 9 6 9 - I s o l a t i o n of an E s c h e r i c h i a c o l ? s t r a i n with a mutation a f f e c t i n g DNA polymerase. Nature 224:  1164-1168.  DeMoss, J . A . and J . Wegman. 1 9 6 5 - An enzyme aggregate in the t r y p t o phan pathway of Neurospora c r a s s a . P r o c . Nat. A c a d . S c i .  U.S. 5 4 : 241-247.  Dietz,  W . H . , T . M . Cook and W.A. Goss. 1 9 6 6 . Mechanism of a c t i o n of n a l i d i x i c a c i d on Escher ich ia c o l i . III. Conditions required for l e t h a l i t y . J . B a c t e r i o l . 9J_: 7 6 8 - 7 7 3 .  Donachie, W.D. 1 9 6 9 - C o n t r o l o f c e l l d i v i s i o n in Escher ich ia c o 1 i : Experiments with thymine s t a r v a t i o n . J . Bacteriol. 1 0 0 : 2 6 0 - 2 6 8 . Donachie, W.D. and K . J . Begg. 1 9 7 0 . Growth of Nature 2 2 5 . : 1 2 2 0 - 1 2 2 4 .  the b a c t e r i a l  cell.  Donachie, W.D. and M. M a s t e r s . 1 9 6 9 - Temporal c o n t r o l o f gene exp r e s s i o n in b a c t e r i a . The C e l l C y c l e : Gene-enzyme i n t e r a c t i o n . G.M. P a d i l l a , G . L . Whitson and I.L. Cameron (eds.) Academic P r e s s , N.Y. pp. 3 7 ? 7 7 . Donachie, W . D . , D.G. Hobbs and M. M a s t e r s . 1 9 6 8 . Chromosome r e p l i c a t i o n and c e l l d i v i s i o n in E s c h e r i c h i a c o l ? 15T~ a f t e r growth in the absence of DNA s y n t h e s i s . Nature 2 1 9 : 1 0 7 9 - 1 0 8 0 . Doudney, C O . 1 9 6 6 . Requirement f o r r i b o n u c l e i c a c i d s y n t h e s i s . f o r d e o x y r i b o n u c l e i c a c i d r e p l i c a t i o n in b a c t e r i a . Nature 211 :  39-41  .  E b e r l e , H. and W. Master. 1 9 7 1 - E f f e c t of n a l i d i x i c a c i d on s e m i c o n s e r v a t i v e r e p l i c a t i o n and r e p a i r s y n t h e s i s . a f t e r u l t r a v i o l e t i r r a d i a t i o n in E s c h e r i c h i a c o l i . J . Bacteriol. 105:  908-912.  E c h o l s , H . , A . G a r e n , S. Garen and A . T o r r i a n . 1 9 6 1 . Genetic control of r e p r e s s i o n of a l k a l i n e phosphatase in E s c h e r i c h i a c o l i . J.  Mol.  Biol.  3:  425-438.  Erhan,  1969The unwinding o f the DNA molecule during J . T h e o r e t . B i o l . 23_: 339-341.  S.  replication.  Gangman, W.L. and A . N o v i c k . 1968. C h a r a c t e r i z a t i o n of two b a c t e r i a l mutants with t e m p e r a t u r e - s e h s i t i v e s y n t h e s i s of DNA. G e n e t i c s 6£: 1-17. Fielding,  Fuchs,  P. and C F . Fox. 1970. Evidence f o r s t a b l e attachment of DNA to membrane at the r e p l i c a t i o n o r i g i n of E s c h e r i c h i a c o l i . Biochem. B i o p h y s . Res. Commun. 4l_: 157-162.  E. and P. Hanawalt. 1970. I s o l a t i o n and c h a r a c t e r i z a t i o n of DNA r e p l i c a t i o n complex from E s c h e r i c h i a c o l i . J . Mol. B i o l . 52_: 301-323.  the  G a r t n e r , T . K . and M. R i l e y . 1964. G e n e t i c s t u d i e s on tryptophanase mutants o f E s c h e r i c h i a c o l i K12. B a c t e r i o l . P r o c . p. 18. Gilbert,  W. and D. D r e s s i e r . 1968. DNA r e p l i c a t i o n - The c i r c l e model. Symp. Quant. B i o l . 3_3_; 473-484.  Ginsberg,  rolling  B. and J . H u r w i t z . 1970. Unbiased s y n t h e s i s of p u l s e l a b e l l e d DNA fragments of b a c t e r i o p h a g e lambda and T4. J . M o l . B i o l . 52: 265-280.  G o s s , W . A . , W.H. D e i t z and T . M . Cook. 1964. Mechanism of a c t i o n of n a l i d i x i c a c i d on E s c h e r i c h i a c o l i . B a c t e r i o l . 88: 1112-1118. Green, M . H . L . , J . Greenberg and J . Donch. 1969E f f e c t of a recA gene on c e l l d i v i s i o n and c a p s u l a r p o l y s a c c h a r i d e p r o d u c t i o n in a Ion s t r a i n of E s c h e r i c h i a c o l i . Genet. Res. 14: 159-162. Grula,  E . A . and M.M. G r u l a . 1962. C e l l d i v i s i o n in a s p e c i e s of Erwi n i a , V . E f f e c t of m e t a b o l i c i n h i b i t o r s on t e r m i n a l d i v i s i o n and c o m p o s i t i o n of d i v i s i o n medium. J . Bacteriol. 84_: 492-498.  G u i l d , W.R. 1968. See the d i s c u s s i o n f o l l o w i n g Okazaki et a K Cold S p r i n g Harbor Symp. Quant. B i o l . 3_3_: 142-14X Hane, M.W.  1971Some e f f e c t s of n a l i d i x i c a c i d on c o n j u g a t i o n E s c h e r i c h i a c o l i K12. J . B a c t e r i o l . 105: 46-56.  Hartman, G . , H. G o l l e r , K. K o s c h e l , W.U. Kersten and H. K e r s t e n . Hemmung der DNA-Abhangingen RNA-und DNA synthese Durch A n t i b i o t i c a Biochem. Z . , 341: 125-128. Harvey,  (1968)  in  1964.  R . J . and A . G . Marr. 1966. Measurement of s i z e d i s t r i b u t i o n bacterial c e l l s . J . B a c t e r i o l . 92: 805-811.  of  Harvey,  R . F . , A . G . Marr and P.R. P a i n t e r . 1967- K i n e t i c s of growth of i n d i v i d u a l c e l l s of E s c h e r i c h i a c o l ? and A z o t o b a c t e r a g i 1 i J . B a c t e r i o l . 9 3 : 605-617-  Haskell,  E . H . and C . I . Davern. 1969. DNA r e p l i c a t i o n . P r o c . Nat.  Hayes, W.  P r e - f o r k s y n t h e s i s : A model f o r A c a d . S c i . U . S . 64: 1 0 6 5 - 1 0 7 1 .  1 9 6 8 . The g e n e t i c s of b a c t e r i a and t h e i r John Wiley S Sons, Inc. New York.  viruses.  Helmstetter, C E . 1 9 6 8 . O r i g i n and sequence of chromosome r e p l i c a t i o n in E s c h e r i c h i a c o l i B / r . J . B a c t e r i o l . 94_: 1 6 3 4 - 1 6 4 1 . Helmstetter, C E . 1969a. Sequence of b a c t e r i a l Ann. Rev. M i c r o b i o l . 23.: 2 2 3 - 2 3 8 .  reproduction.  Helmstetter, C E . 1969b. R e g u l a t i o n of chromosome r e p l i c a t i o n and c e l l d i v i s i o n in Escher ich ia c o l i : The C e l l C y c l e : Geneenzyme i n t e r a c t i o n . G.M. P a d i l l a , G . L . Whltson and I.L. Cameron (eds.) Academic P r e s s , N.Y. pp. 1 5 3 7 -  H e l m s t e t t e r , C E . and S. Cooper. 1 9 6 8 . DNA s y n t h e s i s duniing the d i v i s i o n c y c l e of r a p i d l y growing Escher ich ia c o l i B / r . J . Mol . B i o l . 3J_: 5 0 7 - 5 1 8 . H e l m s t e t t e r , C E . and 0. P i e r u c c i . 1 9 6 8 . Cell d i v i s i o n during i n h i b i t i o n of d e o x y r i b o n u c l e i c a c i d s y n t h e s i s in E s c h e r i c h i a col i . J . B a c t e r i o l . 95.: 1 6 2 7 - 1 6 3 3 . H e l m s t e t t e r , C , S. Cooper, 0. P i e r u c c i and E. R e v e l a s . 1 9 6 8 . On the bacterial l i f e cycle. Cold S p r i n g Harbour Symp. Quant. Biol . 33j 809-822. Hirota,  Y. , J . Mordoh and F. J a c o b . 1 9 7 0 . On the process of c e l l u l a r d i v i s i o n in E s c h e r i c h i a c o l i : I I I• T h e r m o s e n s i t i v e mutants of E s c h e r i c h i a c o l ? a l t e r e d in the process of DNA i n i t i a t i o n . J . Mol . B i o l . 53_: 3 6 9 - 3 8 7 .  Hirota,  Y . , F. J a c o b , A . R y t e r , G. B u t t i n and T . N a k a i . 1 9 6 8 . On the process of c e l l u l a r d i v i s i o n in E s c h e r i c h i a c o l i . I. Asymmetrical c e l l d i v i s i o n and p r o d u c t i o n of d e o x y r i b o n u c l e i c acid-less bacteria. J . M o l . B i o l . 35_: 1 7 5 - 1 9 2 .  Holland,  I.B. and J . T h r e l f a l l . 1969- I d e n t i f i c a t i o n of c l o s e l y l i n k e d l o c i c o n t r o l l i n g u l t r a v i o l e t s e n s i t i v i t y and r e f r a c t i v i t y to C o l i c i n E2 in E s c h e r i c h i a c o l ? . J . Bacteriol. 97: 91-96.  'HoMom, S. and R.H. P r i t c h a r d . 1965E f f e c t o f i n h i b i t i o n o f DNA s y n t h e s i s on mating in E s c h e r i c h i a c o l i K12. Genet. Res.  6: 479-483.  Inouye, M.  1969- U n l i n k i n g of c e l l d i v i s i o n from d e o x y r i b o n u c l e i c a c i d r e p l i c a t i o n in a t e m p e r a t u r e - s e n s i t i v e DNA s y n t h e s i s mutation o f E s c h e r i c h i a c o l i . J . B a c t e r i o l . 99_: 842-850.  Inouye, M.  1971. P l e i o t r o p i c e f f e c t of the recA gene of E s c h e r i c h i a c o l i : Uncoupling of c e l l d i v i s i o n from d e o x y r i b o n u c l e i c acid r e p l i c a t i o n . J . B a c t e r i o l . 106: 539"542.  Inouye, M. and J . P . G u t h r i e . membrane p r o t e i n o f  1969-  A mutation which changes a  Escherichia c o l i .  S c i . U.S. 64: 957-961.  P r o c . Nat.  Acad.  Inouye, M. and A . B . Pardee. 1970. Changes of membrane p r o t e i n s and t h e i r r e l a t i o n to d e o x y r i b o n u c l e i c a c i d s y n t h e s i s and c e l l d i v i s i o n of E s c h e r i c h i a c o l i . J . B i o l . Chem. 245: 5813-5819 Irr,  J . and J . G a l l a n t . 1969- The c o n t r o l of RNA s y n t h e s i s in E s c h e r i c h i a c o l i . II. S t r i n g e n t c o n t r o l of energy metabolism J . B i o l . Chem. 244: 2233-2239-  I y e r , V . N . and K . G . L a r k . 1970. DNA r e p l i c a t i o n in Escher ich ia c o l i : l o c a t i o n of r e c e n t l y i n c o r p o r a t e d thymidine w i t h molecules of high m o l e c u l a r weight DNA. P r o c . Nat. A c a d . S c i . 67:  629-636.  Jacob., F. , S. Brenner and F. C u z i n . 1963On the r e g u l a t i o n of DNA r e p l i c a t i o n in b a c t e r i a . Cold Spring Harbor Symp. Quant. B i o l . 28_: 329-348. J o n e s - M o r t i m e r , M.C. 1968. Escherichia c o l i .  P o s i t i v e c o n t r o l of s u l f a t e Biochem. J . 110: 583-602.  Kaiser, A.D. 1955. A g e n e t i c study of V i r o l o g y 1_: 424-443. Kerstein,  Kelly,  reduction  temperate c o l i p h a g e  in  lambda.  W., H. K e r s t e i n and W. S z y b a l s k i . 1966. Physicochemical p r o p e r t i e s of complexes between d e o x y r i b o n u c l e i c a c i d and a n t i b i o t i c s which a f f e c t r i b o n u c l e i c a c i d s y n t h e s i s ( A c t i n o m y c i n , daunomycin, c i n e r u b i n , n o g a l a m y c i n , chromomycin, methramycin and o l i v o m y c i n ) . B i o c h e m i s t r y 5} 236-244.  R . B . , M.R. A t k i n s o n , J . A . Huberman and A . Kornberg. 1969. E x c i s i o n o f thymine dimers and o t h e r mismatched sequences by DNA polymerase o f E s c h e r i c h i a c o l i . Nature 224: 495~501.  Kim,  J . H . , A . S . Gilbard, B. Djordjevic, S.H. Kim and A . G . Perez. 1968. Action of daunomycin on the nucleic acid metabolism and v i a b i l i t y of HeLa c e l l s . Cancer Research. 28: 2437-2442.  Knippers, R and W. S t r a t i i n g . 1970. The DNA replicating capacity of isolated Escherichia col? c e l l - w a l l membrane complexes. Nature 22G_: 713~717. Kogoma, T. and K.G. Lark. 1970. DNA replication in Escherichia c o l i : Replication in absence of protein synthesis after replication inhibition. J . Mol. B i o l . 52_: 143-165Kohiyama, M. 1968. DNA synthesis in temperature sensitive mutants of Escherichia c o l i . Cold Spring Harbor Symp. Quant. B i o l .  33_: 317-324.  Kohiyama, M. and A.R. Kolber. 1970. Temperature sensitive mutant of the DNA replication system in Escherichia c o l i . Nature  228: 1157-1160.  Kohiyama, M., H. Lanfrom, S. Brenner and F. Jacob. 1963• Modifications de fonctions indespensab1es chez des mutants termosensibles d E_. col i sur une mutation empechant la replication du chromosome bacterien. Comp. Rend. Acad. S c i . 257: 19791981. 1  Kornberg, A.  1969-  1410-1418.  Active center of DNA polymerase.  Science 163:  Kornberg, T. and M. Gefter. 1970. DNA synthesis in c e l l - f r e e extracts of a DNA polymerase-defective mutant. Biochem. Biophys. Res. Commun. 4p_: 1348-1356. Kubitschek, H.E. 1968a. Biophys. J . 8_:  Linear c e l l growth in Escherichia c o l i .  792-802.  Kubitschek, H.E. 1968b. Constancy of uptake during the c e l l in Escherichia col i . Biophys. J . 8_: 1401-1412.  cycle  Kubitschek, H.E. 1969a. in Methods in Microbiology. R.W. Ribbons and J . R . N o r r i s , (eds). Academic Press, Inc., London. Kubitschek, H.E.  1969b.  Analysis of c e l l  Growth during the bacterial size d i s t r i b u t i o n .  cell cycle:  Biophys. J .  9_: 792-809.  Kubitschek, H.E. and T.R. Henderson. 1966. DNA r e p l i c a t i o n . Proc. Nat. Acad. S c i . U.S. 55: 512-519-  Kuempel,.P. 1969- T e m p e r a t u r e - s e n s i t i v e i n i t i a t i o n of chromosome r e p l i c a t i o n in a mutant o f Escher ich ia c o l ? . J . Bacteriol. 100: 1302-1310. Lark,  K.G.  1 9 6 6 . R e g u l a t i o n of chromosome r e p l i c a t i o n and in b a c t e r i a . B a c t . Rev. 3_0j 1 — 3 5 .  segregation  Lark,  K.G.  1969a. Role o f d e o x y n u c l e o t i d e s t r a n d age or p o l a r i t y in d e t e r m i n i n g asymmetric chromosome r e p l i c a t i o n a f t e r thymine starvation. J . M o l . B i o l , 44_: 2 1 7 - 2 3 1 .  Lark,  K.G.  1969b. I n i t i a t i o n and c o n t r o l o f DNA s y n t h e s i s . Ann. Rev. Biochem. 3_8_: 5 6 9 - 6 0 4 .  Lark,  K . G . and C. L a r k . 1 9 6 6 . R e g u l a t i o n of chromosome r e p l i c a t i o n E s c h e r i c h i a c o l i : A comparison of the e f f e c t s of phenethyl a l c o h o l treatment with those of amino a c i d s t a r v a t i o n . J . Mol . B i o l . 20_: 9~19.  Lark,  K . G . and H. Ranger. 1 9 6 9 . I n i t i a t i o n of DNA r e p l i c a t i o n in E s c h e r i c h i a c o l i 15T~: C h r o n o l o g i c a l d i s s e c t i o n of t h r e e p h y s i o l o g i c a l processes required for initiation. J . Mol .' B i o l . 42_: 2 2 1 - 2 3 6 .  Lark,  K . G . , T . Repko a n - E . J . Hoffman. 1963• The e f f e c t of amino a c i d d e p r e i v a t i o n on subsequent DNA r e p l i c a t i o n . Biochim. B i o p h y s . Acta 76.: 9-24.  in  L e v i n e , A . J . and R . L . S i n s h e i m e r . 1 9 6 8 . The process of i n f e c t i o n with b a c t e r i o p h a g e 0 X 1 7 4 . XIX. I s o l a t i o n and c h a r a c t e r i z a t i o n of a c h l o r a m p h e n i c o l - r e s i s t a n t p r o t e i n from 0 X - i n f e c t e d cells. J . M o l . B i o l . 32.: 5 6 7 - 5 7 8 . Lomax, M.S. and G.R. Greenberg. 1 9 6 8 . C h a r a c t e r i s t i c s of the deo o p e r o n : R o l e in thymine u t i l i z a t i o n and s e n s i t i v i t y to deoxyribonucleosides. J . B a c t e r i o l . 96: 501-514. Maal^e,  0. and P . G . Hanawalt. 1 9 6 1 . Thymine d e f i c i e n c y and the normal DNA r e p l i c a t i o n . J . M o l . B i o l . 3} 144-155.  Maal^e,  0. and N.0. K j e l d g a r d . 1 9 6 6 . C o n t r o l of macromolecular synthesis, e d . by B.D. D a v i s , W.A. Benjamin, Inc. New Y o r k .  M a s t e r s , M. 1 9 7 0 . O r i g i n and d i r e c t i o n of r e p l i c a t i o n of the chromosome of E s c h e r i c h i a c o l ? B / r . P r o c . Nat. A c a d . S c i . U.S. 6 5 : 6 0 1 - 6 0 8 .  136  M a t h l s o n , G.E. 1968. K i n e t i c s o f d e a t h i n d u c e d by P e n i c i l l i n a n d chloramphenicol i n synchronous c u l t u r e s o f E s c h e r i c h i a c o l ? . N a t u r e 219: 405-407. M i z u n o , S., H. Y a m a z a k i , K. N i t t a a n d H. Umezawa. 1968. I n h i b i t i o n o f D N A - d e p e n d e n t RNA p o l y m e r a s e r e a c t i o n o f E s c h e r i c h i a c o l i by a n a n t i m i c r o b i a l a n t i b i o t i c , s t r e p t o v a r c i n . Biochim. B i o p h y s . A c t a 157: 322-332. M o d r i c h , P. a n d I.R. Lehman. 1971. E n z y m a t i c c h a r a c t e r i z a t i o n o f a m u t a n t o f E s c h e r i c h i a c o l i w i t h an a l t e r e d DNA l i g a s e . P r o c . N a t . A c a d . S c i . ' jJjfPl002-1005. M o r d o h , J . , Y. H i r o t a a n d F. J a c o b . 1970. On t h e p r o c e s s o f c e l l u l a r d i v i s i o n i n E s c h e r i c h i a c o l i . V. I n c o r p o r a t i o n o f d e o x y n u c l e o s i d e t r i p h o s p h a t e s by DNA t h e r m o s e n s i t i v e m u t a n t s o f E s c h e r i c h i a c o l ? a l s o l a c k i n g DNA p o l y m e r a s e a c t i v i t y . P r o c . N a t . A c a d . S c i . 6_7_: 773"778. M o r g a n , A.R. 1970. Model merase. Nature  f o r DNA r e p l i c a t i o n by K o r n b e r ' g DNA  227: 1310-1313-  M o s e s , R.E. a n d C C . R i c h a r d s o n .  1970.  R e p l i c a t i o n and r e p a i r  o f DNA i n c e l l s o f E s c h e r i c h i a c o l i P r o c . N a t . A c a d . S c i . 67: 671-681.  treated with  M y c h a j l o w s k a , L. 1970. 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 of Escher ich ia c o l i . M.Sc. T h e s i s , U.B.C N a g a t a , T.  poly-  toluene.  in cultures  1963- T h e m o l e c u l a r s y n c h r o n y and s e q u e n t i a l r e p l i c a t i o n o f DNA i n E s c h e r i c h i a c o l i . P r o c . N a t . A c a d . S c i . U.S. 49:  551-559-  N a g a t a , T. a n d M. M e s e l s o n .  1968.  Periodic  r e p l i c a t i o n o f DNA i n  s t e a d i l y g r o w i n g E s c h e r i c h i a c o l ? : The l o c a l i s e d o r i g i n o f replication. C o l d S p r i n g H a r b o r Symp. Q u a n t . B i o l . 33_: 553 558. Ng,  H.  1969E f f e c t o f d e c r e a s i n g g r o w t h t e m p e r a t u r e on c e l l of E s c h e r i c h i a c o l ? . J . B a c t e r i o l . 98: 232-237-  Ng,  H., J . L . Ingraham a n d A.G. M a r r .  1962.  Damage a n d  yield  derepression  in E s c h e r i c h i a c o l i r e s u l t i n g from growth a t low t e m p e r a t u r e s . J . B a c t e r i o l . 84: 331-339Nomura, M. a n d J.D. W a t s o n . 1959. chloromycetin-inhibited  204-217.  Ribonucleoprotein Escher ich i a col i.  particles within J . M o l . B i o l . 1_:  137  O'Donovan, G . A . and J . Neuhard. 1970. P y r i m i d i n e metabolism organisms. B a c t . Rev. 34: 278-343.  in m i c r o -  Okazaki,  R . , R. O k a z a k i , K. Sakabe and K. Sugimoto. 1968. In v i v o mechanism o f DNA c h a i n growth. Cold Spring Harbor Symp. Quant. B i o l . 33: 129-144.  Okazaki,  R . , K. Sugimoto, T . O k a z a k i ,  Y . Imae and A . S u g i n o . 1970.  DNA c h a i n growth: In v i v o and i n v v i t r o s y n t h e s i s p o l y m e r a s e - n e g a t i v e mutant o f E s c h e r i c h i a c o l i . Nature 228_: 223-226. Painter,  in a DNA  P . R . and A . G . Marr. 1968. Mathematics o f m i c r o b i a l lations. A n n . Rev. M i c r o b i o l . 22_: 519"548.  popu-  Pardee, A . B . 1968. C o n t r o l o f c e l l d i v i s i o n - m o d e l s from m i c r o organisms. Cancer Res. 28_: 1802-1809. P a t o , M . L . and D . A . G l a s e r . .196.8. The o r i g i n and d i r e c t i o n o f r e p l i c a t i o n o f the chromosome o f E s c h e r i c h i a c o l i B / r . P r o c . Nat. A c a d . S c i . U . S . 60: 1268-1274. C. and L. Hamm. 1969• P r o p e r t i e s o f a DNA l i g a s e mutant o f Escher ich ia c o l i . II':, Intermediates in DNA r e p l i c a t i o n . Biochem. B i o p h y s . R e s . Commun. 37.: 1015-1021.  Pauling,  P h i l l i p s , A . P . 1969. Unwinding models f o r double h e l i c a l DNA d u r i n g i t s r e p l i c a t i o n and t r a n s c r i p t i o n . J . T h e o r e t . B i o l . 24:  273-278.  Pierucci,  0. 1969.,Regulation o f c e l l B i o p h y s . J . 9_: 90-112.  division  in E s c h e r i c h i a c o l ? .  Pierucci,  0. and C E . H e l m s t e t t e r . 1969. Chromosome r e p l i c a t i o n , p r o t e i n s y n t h e s i s and c e l l d i v i s i o n i n E s c h e r ? c h i a c o l ? . Fed. P r o c . 28_: 1755-1760.  Plagemann, P.G.W. 1970. E f f e c t s o f phenethyl a l c o h o l on t r a n s p o r t r e a c t i o n s , n u c l e o t i d e pools and macromolecu l a r s y n t h e s i s in N a v i k o f f Rat Hepatoma c e l l s growing in suspension c u l t u r e s . J . C e l l P h y s i o l . 75: 315~328. P o n t e f r a c t , R . D . and F . S . T h a t c h e r . 1970. An e l e c t r o n microscope study o f mesosomes in i r r a d i a t i o n r e s i s t a n t mutants o f Escherichia c o l i . J . U l t r a s t r u c t . R e s . 3£: 78-86. Previc,  E . P . 1970. Biochemical d e t e r m i n a t i o n o f b a c t e r i a l morphology and the geometry o f c e l l d i v i s i o n . J . Theoret. B i o l .  27: 471-497.  138  Pritchard,  R.H. and K . G . L a r k . thymine s t a r v a t i o n at col i.  J.  Mol.  Biol.  1 9 6 4 . Induction o f r e p l i c a t i o n by the chromosome o r i g i n in E s c h e r i c h i a 9_:  288-307.  Pritchard,  R.H. and A . Z a r i t s k y . 1 9 7 0 . E f f e c t of thymine c o n c e n t r a t i o n on the r e p l i c a t i o n v e l o c i t y of DNA in a thymineless mutant of E s c h e r i c h i a c o l i . Nature 2 2 6 : 1 2 6 - 1 3 1 .  Pritchard,  R . H . , P.I. Booth and J . C o l l i n s . 1969C o n t r o l of DNA s y n t h e s i s in b a c t e r i a . Symp. Soc. Gen. M i c r o b i o l . 1_9_: 2 6 3 - 2 9 8 .  Ramakrichnan, T . and E . A . A d e l b e r g . 1 9 6 5 . Regulatory mechanisms in b i o s y n t h e s i s of i s o l e u c i n e and v a l i n e . III. Map order o f s t r u c t u r a l genes and o p e r a t o r genes. J . Bacteriol. 8 9 : 661-664. Ramareddy,  G. and H. R e i t e r . 1 9 6 9 . S p e c i f i c l o s s of newly r e p l i c a t e d d e o x y r i b o n u c l e i c a c i d in n a l i d i x i c a c i d t r e a t e d B a c i 1 l u s subtil is 1 6 8 . J . B a c t e r i o l . 1 0 0 : 7 2 4 - 7 2 9 .  Ramareddy,  G. and H. R e i t e r . 1970. Sequential s t a r v e d Baci 1 lus s u b t i 1 is 168 c e l l s .  Randerath,  K. and E. Randerath.. 1967. In S . P . Colowick and N . 0 . Kaplan ( e d s . ) Methods in Enzymology, V o l . 1 2 , Part A . Academic P r e s s , N.Y. P. 3 2 3 -  l o s s of J . Mol.  l o c i in thymineB i o l . 5_0: 5 2 5 - 5 3 2 .  Reeve, J . N . , D . J . Groves and D . J . C l a r k . 1970. R e g u l a t i o n of c e l l d i v i s i o n in E s c h e r i c h i a c o l i : C h a r a c t e r i z a t i o n of temperat u r e s e n s i t i v e d i v i s i o n mutants. J . Bacteriol. 104: 1052-1064. R i c h a r d , M. and Y. H i r o t a . 1969• E f f e c t s of s a l t s c e l l d i v i s i o n in E s c h e r i c h i a c o l i . ( F r e ) . (Paris). 268: 1335-1338. Richardson, C C 19693_8_: 7 9 5 - 8 4 0 . Rosenberg,  Enzymes  in DNA m e t a b o l i s m .  on the process of C R . A c a d . S c i . D.  Ann. Rev.  Biochem.  B . H . and L . F . C a v a l i e r i . 1968. Shear s e n s i t i v i t y o f the E s c h e r i c h i a c o l ? genome: M u l t i p l e membrane attachment p o i n t s of the E s c h e r i c h i a c o l i DNA. Symp. Quant. B i o l . 33_: 6 5 7 2 . _  Rosenberg, B . H . , L . F . C a v a l i e r i and C . Lingers. 1969. c o n t r o l mechanism f o r Escher i ch ia c o l i : DNA P r o c . Nat. A c a d . S c i . U . S . 6_3_: 1 4 1 0 - 1 4 1 7 .  The n e g a t i v e replication.  R u b e n s t e i n , K . E . , M.M. Noss and S . S . Cohen. 1970. Synthetic c a p a b i l i t i e s of plasmalysed c e l l s and s p h e r o p l a s t s of Escher ich ia c o l i . J . Bacteriol. 109: 443-452.  "Ryter,  A.  1968. bacteria:  A s s o c i a t i o n of the nucleus and the membrane of A morphological study. B a c t . Rev. 3 2 :  Ryter,  A . , Y , H i r o t a and F . J a c o b . 1 9 6 8 . DNA-membrane complex and n u c l e a r s e g r e g a t i o n in b a c t e r i a . Symp. Quant. B i o l . 33:  39 54.  669-676.  Sadowski,  P . , B. G i n s b e r g , A . Y u d e l e v i c h , L. F e i n e r and J . H u r w i t z . 1968. Enzymatic mechanisms of the r e p a i r and breakage o f DNA. Cold S p r i n g Harbor Symp. Quant. B i o l .  1  33.: 165-178.  Sells,  B . H . and J . S a y l e r . 1 9 7 1 . Messenger RNA accumulation E s c h e r i c h i a col? during chloramphenicol treatment. Biochim. Biophys. Acta  in  232: 421-425.  S c h a c h t e l e , C . F . , D . L . Anderson and P. Rogers. 1 9 7 0 . Isolation of a r a p i d l y sedimenting c a n a v a n y l - p r o t e i n - D N A membrane complex from E s c h e r i c h i a c o l i . J . M o l . B i o l . 4_9_: 2 5 5 - 2 6 1 . Shapiro,  B.M. , A . B . S i c c a r d i , Y . H i r o t a and F. J a c o b . 1970. On the process o f c e l l u l a r d i v i s i o n in E s c h e r i c h i a c o l ? . II. Membrane p r o t e i n a l t e r a t i o n s a s s o c i a t e d w i t h mutations a f f e c t i n g the i n i t i a t i o n of DNA s y n t h e s i s . J . Mol. B i o l . 5 2 : 7 5 - 8 9 -  Shaw, M . E . 1 9 6 8 . Formation of f i l a m e n t s and s y n t h e s i s of macromolecules at temperatures below the minimum f o r growth of E s c h e r i c h i a c o l ?.  J . Bacteriol.  95_: 2 2 1 - 2 3 0 .  Shaw, M.K. and J . L . Ingraham. 1 9 6 7 - S y n t h e s i s o f macromolecules by E s c h e r i c h i a c o l ? near the minimal temperature f o r growth. J.  Silver,  Bacteriol7"9¥:  157-164.  S . and L. Wendt. 1 9 6 7 . Mechanism o f a c t i o n o f phenethyl a l c o h o l : Breakdown o f the c e l l u l a r p e r m e a b i l i t y barrier. J . B a c t e r i o l . 93_: 5 6 O - 5 6 6 .  Silverstein, of Simon, Z .  S . J . and D. B i l l e n . 1970. RNA involvement DNA s y n t h e s i s . B a c t e r i o l . P r o c . p. 7 1 .  in  initiation  1 9 6 8 - S e m i q u a n t i t a t i v e eel 1 c y c l e model f o r slow growing Escherichia col i . C e l l T i s s u e K i n e t . 1_: 3 7 7 ~ 3 8 1 .  S m i t h , H . S . and A . B . Pardee. 1 9 7 0 . Accumulation o f a p r o t e i n r e q u i r e d f o r d i v i s i o n d u r i n g the c e l l c y c l e of Escher ich ia c o l ? . J.  Smith,  Bacterloi:  101: 901-909.  D.W., H . E . S c h a l l e r and F . J . B o n h o e f f e r . in v i v o . Nature 2 2 6 : 7 1 1 - 7 1 3 .  1 9 7 0 . DNA s y n t h e s i s  Sueoka,  N. a n d W.G. Quinn. I968. Membrane attachment of the chromosome r e p l i c a t i o n o r i g i n in B a c i 1 1 us s u b t i 1 ? s . Cold Spring Harbor Symp. Quant. B i o l . 3 3 : 6 9 5 ~ 7 0 5 .  Taylor,  A . L . and C D . T r o t t e r . 1 9 6 7 - Revised l i n k a g e map of Escherichia col i. B a c t e r i o l . Rev. 3J_: 3 3 2 - 3 5 3 -  Treick,  R.W. and W.A. Konetzka. 1 9 6 4 . P h y s i o l o g i c a l s t a t e of E s c h e r i c h i a c o l i and the i n h i b i t i o n of d e o x y r i b o n u c l e i c a c i d s y n t h e s i s by phenethyl a l c o h o l . J . Bacteriol. 88: 1580-1584.  Tremblay,  G . Y . , M . J . D a n i e l s and M. S c h a e c h t e r . 1 9 6 9 - Isolation a c e l l membrane-DNA-nascent RNA complex from b a c t e r i a . J . Mol . B i o l . 40_: 6 5 " 7 6 .  of  V i e l m e t t e r , W., W. Messer and A . S c h u t t e . 1 9 6 8 . Growth, d i r e c t i o n and s e g r e g a t i o n of the E s c h e r i c h i a c o l ? chromosome. Cold S p r i n g Harbor Symp. Quant. B i o l . 33.: 5 8 5 " 5 9 8 . V o g e l , M . J . and D. Bonner. 1 9 5 6 . A c e t y l o r n i t h i n a s e of E s c h e r i c h i a c o l i : P a r t i a l p u r i f i c a t i o n and some p r o p e r t i e s . J . Biol. Chem.  218^: 9 7 - 1 0 6 .  Wang, J . C . and N. D a v i s o n . 1 9 6 8 . C y c l i z a t i o n of phage DNA. Spring Harbor Symp. Quant. B i o l . 33_: 4 0 9 " 4 1 5 .  Cold  Ward,  C . B . and D.A. resistant coli B/r.  Glaser. 1 9 6 9 - A n a l y s i s of the chloramphenicol s t e p s : The i n i t i a t i o n of DNA s y n t h e s i s in E s c h e r i c h i a P r o c . Nat. A c a d . S c i . U . S . 6 4 : 9 0 5 - 9 1 2 .  Ward,  C . B . , M.W. Hane and D.A. G l a s e r . 1 9 7 0 . Synchronous r e - i n i t i a t i o n of chromosome r e p l i c a t i o n in Escher ich ia c o l i B/r a f t e r n a l a d i x i c acid treatment. P r o c . Nat. A c a d . S c i . U . S . 6 6 : 365-370.  Watson, J . D . and F . H . C . C r i c k . 1 9 5 3 - A structure nucleic a c i d . Nature 1 7 1 : 7 3 9 ~ 7 4 0 .  for  deoxyribose  Watson, J . D . and F . H . C . C r i c k . 1 9 5 3 - G e n e t i c a l i m p l i c a t i o n s of the s t r u c t u r e of d e o x y r i b o n u c l e i c a c i d . Nature 1 7 1 : 9 6 4 - 9 6 7 Weigand,  R . A . , J . M . S h i v e l y and J.W. Greenawalt. 1 9 7 0 . Formation and u 1 t r a s t r u c t u r e of e x t r a membranes in E s c h e r i c h i a c o l i . J . Bacteriol. 1 0 2 : 240-249-  Werner, R.  1971.  Mechanism of  DNA r e p l i c a t i o n .  Nature  230: 570-572.  Willets,  Wolf,  N . S . and A . J . C l a r k . 1969. C h a r a c t e r i s t i c s of some m u l t i p l e r e c o m b i n a t i o n - d e f i c i e n t s t r a i n s o f Escher ich ia c o l ? . J . B a c t e r i o l .' 100: 231-239-  B . M , M . L . P a t o , C . B . Ward and D.A. G l a s e r . 1968. On the o r i g i n and d i r e c t i o n o f r e p l i c a t i o n of the E s c h e r i c h i a col? chromosome. Cold Spring Harbor Symp. Quant. B i o l . 33: 57558T] 1  Y u d e l e v i c h , A . , B. Ginsberg and J . H u r w i t z . 1968. Discontinuous s y n t h e s i s o f DNA d u r i n g r e p l i c a t i o n . Proc. Nat. Acad. S c i .  U.S. 61: 1129-1136.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101930/manifest

Comment

Related Items