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

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PLEIOTROPIC EFFECT OF DnaA GENE ON INITIATION OF DNA REPLICATION AND CELL DIVISION ESCHERICHIA COLI by GEORGE KHACHATOURIANS B.A. San Franc isco State C o l l e g e , 1966 M.A. San Franc isco State 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 iology We accept th is thes is as. conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1971 In present ing t h i s thes is in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I fu r ther agree that permission for extensive copying of th is t h e s i s fo r s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It i s understood that copying or p u b l i c a t i o n o f th is thes is f o r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion . Department of Microbio logy The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada Date 12th J u l y , 1971 ABSTRACT C e l l d u p l i c a t i o n in Escher ich ia col i involves complex events , coordinated with chromosome r e p l i c a t i o n . Because of the importance of chromosomes in perpetuating the normal c e l l cyc le the i n i t i a t i o n of the i r r e p l i c a t i o n must be coordinated with c e l l u l a r d i v i s i o n . Fol lowing i n i t i a t i o n , the c e l l must r e p l i c a t e and segregate i ts chromosomes, create a s i t e necessary for septat ion and d i v i d e . These events could be coordinated by e i t h e r ; (1) biochemical react ions involv ing d i f f u s i b l e enzymes, or (2) 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 d i v i s i o n . In the l a t t e r case , the c y c l i c events of r e p l i c a t i o n , segregation and c e l l d i v i s i o n may be coordinated by physica1-chemica1 or biochemical means. In any case , physica l a s s o c i a t i o n implies p l e i o t r o p i c e f f e c t s . To test th is hypothes is , c e l l d i v i s i o n of the i n i t i a t o r mutant of E^ . c o l i , i so la ted by Kohiyama (1968) was s tud ied . The temperature-s e n s i t i v e i n i t i a t o r mutant E_. col ? CR 3**T83 (ts DnaA) grew normally at 30 C, and at the r e s t r i c t i v e temperature (42 C) . The DNA r e p l i c a t i o n as measured by rad ioac t ive precursor uptake, stopped a f te r approximately kO minutes and was equivalent to completion of rounds of r e p l i c a t i o n s t a r t e d . Measurement of r i b o - and deoxyr ibonucleot ide tr iphosphate pools by t h i n - l a y e r chromatography at 30 C and hi C indicated res idual DNA synthesis was not due to a l i m i t a t i o n in the DNA precursors . Using a combination of densi ty and d i f f e r e n t i a l rad ioac t ive l a b e l l i n g f o r the s t a r t s and ends of chromosomes, a pre fer red place fo r re-i n i t i a t i o n of new r e p l i c a t i o n cyc les was shown. It was shown that DNA r e p l i c a t i o n at Ml C terminated at a f ixed region of the chromosome, and was i d e n t i c a l to the 150 ug/ml chloramphenicol s e n s i t i v e step involved in the process of i n i t i a t i o n of chromosome r e p l i c a t i o n in E_. col i . A cessat ion of c e l l u l a r d i v i s i o n was noted by measurement of c e l l growth by Coul ter Counter, at a s h i f t from 30 C to hi C, r e s u l t i n g in f i lamentous growth. Upon a return to 30 C, the c e l l s resume d i v i s i o n a f t e r approximately 15 - 20 min. The p l e i o t r o p i c behaviour, that i s , the cessa t ion of c e l l d i v i s i o n and i n i t i a t i o n of DNA r e p l i c a t i o n was a resu l t of a point mutation in the gene DnaA, coding for a membrane bound prote in involved in i n i t i a t i o n . This mutation was mapped by t ransduct ion and was located at the i s o l e u c i n e - v a l i n e region of the EL c o l i map. When th is gene was transduced to d i f f e r e n t s t r a i n s of E_. col i K.J2 the same p le io t ropy was observed. This p le io t ropy could be uncoupled, however, at 30 C by i n h i b i t o r s of DNA synthesis or in i t i a t i o n . During recovery at 30 C from growth under kl C, expression of c e l l d i v i s i o n was propor t iona l to c e l l equivalents generated at the r e s t r i c t i v e temperature. RNA and prote in s y n t h e s i s , for 10 minutes during the recovery p e r i o d , was ob l iga tory for i n i t i a t i o n of new rounds of r e p l i c a t i o n , but not fo r the expression of c e l l d i v i s i o n . A c e l l d i v i s i o n " p o t e n t i a l " prote in was present under the r e s t r i c t i v e growth c o n d i t i o n . This " p o t e n t i a l " was made at a derepressed rate and underwent a rapid degradation i f kept at kl C. At any given time, when returning from kl C to 30 C, th is " p o t e n t i a l " allowed expression c e l l d i v i s i o n based on DNA/mass or normal c e l l equivalents generated at kl C. The h a l f - l i f e for decay of the d i v i s i o n " p o t e n t i a l " was estimated to be 1.k minutes. The resu l ts were in te rpre ted , in terms of an enzyme complex, which is common to the i n i t i a t i o n of DNA r e p l i c a t i o n and c e l l u l a r d i v i s ion . TABLE OF CONTENTS Page LITERATURE REVIEW. . . 1 I. The DNA r e p l i c a t i o n c y c l e in E_. col ? 2 A. The membrane attachment of DNA in E_. col ? 2 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 in E_. col ? k C. The r e p l i c a t i o n of the E_. col i chromosome 6 D. Separation of daughter chromosomes: Segregat ion . . 13 II. Ce l l d i v i s i o n of E_. col i 13 A. RNA and prote in synthesis in the normal d i v i s i o n c y c l e of E_. col ? 13 B. Phys io log ica l d i v i s i o n and septat ion 15 III. Regulat ion of the c e l l d u p l i c a t i o n c y c l e in E_. col i . . 15 A. Control of the DNA r e p l i c a t i o n in E_. col i 15 B. Regulat ion of c e l l d i v i s i o n in E_. col i 19 MATERIALS AND METHODS 21 I. Bac te r ia l and Phage s t r a i n s 21 A. Bacter ia l s t ra ins 21 B. Bacteriophage s t r a i n s 21 II. Media and Chemicals 21 A. Media 21 V Table of Contents (continued) Page B. Chemicals 23 III. Cul ture methods 23 A. Growth condi t ions for l i q u i d cu l tu res 23 B. P la t ing methods 23 C. Temperature s h i f t condi t ions T 2k IV. Measurement of macromolecular synthesis and c e l l growth. . 2k A. Measurement of c e l l numbers 2k B. Measurement of c e l l mass 25 C. Measurement oft tryptophanase a c t i v i t y 25 D. Measurement of DNA synthesis 25 1. Total DNA synthesis 25 2. Measurement of the rate of DNA synthesis 26 E. Meaisurement of ac id so lub le nucleoside t r i -phosphate p o o l s . . . . . 27 1. Preparat ion of the samples 27 2. Chromatography. 27 3. Autoradiography 28 V. Density gradient sedimentation a n a l y s i s . . . . 29 A. Measurement of BrLIra incorporat ion in the D N A . . . . 29 B. Density l a b e l l i n g 30 C. Preparat ion and a n a l y s i s of DNA samples by densi ty gradient centr i fugat ion 30 Table of Contents (continued) Page 1 . Ex t rac t ion of DNA 30 2. Cent r i fuga t ion and f r a c t i o n a t i o n . . . . 31 3. Measurement of r a d i o a c t i v i t y . . . 32 VI . Genetic ana lys is of the mutants. 32 A. Iso la t ion of the temperature res is tan t revertants 32 B. Transduct ion experiments . . 32 1. Bacteriophage donor lysate preparat ion 32 2. Transduct ion experiments 33 RESULTS AND GENERAL DISCUSSION 35 I. Proper t ies of CR34T83 • • • • 35 A. Ana lys is of macromolecular synthesis and c e l l . . . . d i v i s ion 35 1. Temperature s h i f t condi t ions 35 2. Ce 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 to non-permissive t e m p e r a t u r e s . . . . 37 a . Ce l l d i v i s i o n 37 b. Studies on DNA r e p l i c a t i o n in CR34T83 40 i . Total uptake of TdR 40 i i . Rate of DNA r e p l i c a t i o n 40 II. Recovery of CR3/tT83 at 30 C a f t e r growth, at 42 C 43 A. S ing le s h i f t experiments 43 v i i T a b l e of C o n t e n t s ( c o n t i n u e d ) Page 1. C e l l d i v i s i o n d u r i n g r e c o v e r y from a 42 C p u l s e 44 2. C e l l volume d i s t r i b u t i o n s f o r T83 a t r e c o v e r y 49 3. Rate o f DNA s y n t h e s i s 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 49 32 4. Measurement o f - P - l a b e l l e d n u c l e o s i d e t r i p h o s p h a t e s i n CR34T83 53 B. M u l t i p l e s h i f t e x p e r i m e n t s 55 C. P h y s i o l o g i c a l r e q u i r e m e n t s o f t h e r e c o v e r y from.. a s i n g l e s h i f t 58 1. R o l e o f DNA s y n t h e s i s i n r e c o v e r y 58 2. R o l e o f RNA s y n t h e s i s i n r e c o v e r y 62 3. R o l e o f p r o t e i n s y n t h e s i s on 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 d u r i n g the r e c o v e r y p e r i o d . . . . 65 a. C e l l d i v i s i o n .... 68 b. DNA s y n t h e s i s 74 D. A t t e m p t s t o u n c o u p l e 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 a t 30 C 79 1. I n h i b i t i o n o f i n i t i a t i o n o f new rounds by.... p h e n e t h y l a l c o h o l . . . . 79 2. U n c o u p l i n g 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 Daunomycin 83 v i i i Table of Contents (continued) Page 3. Uncoupling of cel l division from DNA replication by Nalidixic acid 85 III. Control of DNA synthesis in T 8 3 87 A. Identification of the place of resumption of DNA synthesis at recovery 87 B. Comparison of the effect of inhibition of protein synthesis and the T 8 3 mutation on the ini t iat ion of DNA repl ication 95 C. Studies on s tab i l i ty of the growing point and the replication complex at the non-permissive condition 99 IV. Synthesis and decay of the division potential in CR3i»T83 studied by in vivo kinet ics. 1 0 2 A. Time course of appearance of the division potential 1 0 2 B. The wave of expression of ce l l division at recovery in the absence of translation or transcription 10k V. Genetic analysis of the mutant CR3^T83 • 1 1 0 A. Study of temperature resistant (tr) revertants. . . 1 1 1 B. Construction and ana lysis of CR3.4T83 Hv ts DnaA strains 1 1 1 Table of Contents (continued) ix Page C. Introduction of the T83 gene into E_. col 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 114 DISCUSSION , , 117 BIBLIOGRAPHY 127 LIST OF TABLES x Page Table I. Bacter ia l s t r a i n s 22 Table II. C h a r a c t e r i s t i c s of l abe l l ed DNA in T83: Buoyant d e n s i t i e s and percentage of to ta l DNA 3k Table III. The frequency of j o i n t t ransduct ion of the I lv and DnaA l o c i 113 Table IV. Frequency of j o i n t t ransduct ions of the II v and DnaA l o c i from E. c o l i T83 115 LIST OF FIGURES x I Page F i g u r e 1. F i g u r e 2. F i g u r e 3. F i g u r e 4. F i g u r e 5-F i g u r e 6. F i g u r e 7-F i g u r e 8. F i g u r e 3. F i g u r e 10. F i g u r e 11. F i g u r e 12. F i g u r e 13. Response o f T83 grown a t 30 C t o a change i n t e m p e r a t u r e Growth o f T83 under p e r m i s s i v e and non-p e r m i s s i v e c o n d i t i o n s Uptake o f C - l 4 t h y m i d i n e i n t o CRT-83 growing a t 30 C and 42 C. Rate o f DNA s y n t h e s i s i n C R 3 4 T 8 3 a f t e r a s h i f t t o 42 C. Recovery o f CR34T83 a t 30 C f o l l o w i n g growth a t 42 C. 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 kept a t 30 C. The r e l a t i o n s h i p between t h e l a g i n r e c o v e r y t o t he p e r i o d o f growth a t kl C. A n a l y s i s o f 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 a t kl C. 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 32 D i s t r i b u t i o n o f P - l a b e l l e d d e o x y r i b o -n u c l e i s d d e t r i p h o s p h a t e s i n C R 3 4 T 8 3 . 32-r D i s t r i b u t i o n o f P - l a b e l l e d 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 i n CR34T83. I n h i b i t i o n o f c e l l d i v i s i o n d u r i n g the r e c o v e r y p e r i o d by s h i f t i n g t o t h e non-p e r m i s s i v e t e m p e r a t u r e . 36 39 41 kl 45 46 48 50 51 54 56 57 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 L i s t of Figures (continued) X I I Page Figure 14. E f fec t of i n h i b i t i o n of DNA synthesis on c e l l d i v i s i o n at recovery at 30 C. 61 Figure 15. The e f f e c t 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 o n 64 Figure 16. E f fec t of i n h i b i t i o n of tota l RNA synthesis on recovery 66 Figure 17- Measurement of the accumulation of d i v i s i o n potent ia l in T83- 67 Figure 18. E f fec t of i n h i b i t i o n of prote in synthesis on recovery of T83. 69 Figure 19- E f fec t of i n h i b i t i o n of prote in synthesis during and a f t e r a pulse at kl C , on recovery 71 Figure 20. E f fec t of i n h i b i t i o n of prote in synthesis on the recovery of c e l l s at 30 C. 73 Figure 21. Changes in the rate of c e l l d i v i s i o n during recovery from a k l C block in the presence of chloramphenicol . 75 Figure 22. The e f f e c t of i n h i b i t i o n of prote in synthesis at k l C on the DNA r e p l i c a t i o n at recovery 77 Figure 23. Role of i n h i b i t i o n of prote in synthesis on i n i t i a t i o n of new rounds during recovery 78 Figure l k . The e f f e c t of chloramphenicol on the rate of DNA synthesis during the recovery pe r iod . 80 Figure 25- Uncoupling of c e l l d i v i s i o n from DNA r e p l i -ca t ion by phenethyl a lcohol 82 Figure 26. Uncoupling of c e l l d i v i s i o n and DNA synthesis by Daunomycin. 84 Figure 27. Re la t ionsh ip of i n h i b i t i o n of DNA synthesis and c e l l d i v i s i o n during temperature s h i f t s in T83. 86 L i s t of Figures (continued) X I I I Page Figure 28. Construct ion of the model fo r DNA r e p l i c a t i o n in CR34T83. 88 Figure 29. Protocol for the experiments in Figures 30 and 3.1 . 90 Figure 30. CsCl gradient ana lys is of the place of the r e i n i t i a t i o n at recovery from growth at kl C. 92 Figure 31. CaCl gradient ana lys is of the place of the r e i n i t i a t i o n at recovery from growth in the presence of CAM. 93 Figure 32. Comparison of the kl C e f f e c t to that of add i t ion of 150 yg per ml CAM. 97 Figure 33. E f f e c t of a low level of CAM on DNA r e p l i -ca t ion in T83. 98 Figure 3k. DNA synthesis in T83 at kl C fo l lowing an exposure to N a l i d i x i c a c i d . 101 Figure 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 ac id treatment at 30 C. 103 Figure 36. The wave of c e l l d i v i s i o n during a t r a n s i -t ion from kl to 30 C. 106 Figure 37. Decay of c e l l d i v i s i o n potent ia l of CR3*»T83 at kl C. 108 Figure 38. Decay of c e l l d i v i s i o n potent ia l of CR3^T83 at kl C. 109 ACKNOWLEDGEMENTS To the Facul ty of the Department of Microbiology and to the Head of the Department, Dr. Jack J . R . Campbell , I extend my thanks for the environment and support given me during my development as a graduate student at the U n i v e r s i t y of B r i t i s h Columbia. My thanks and apprec ia t ion go to Dr. D. Joseph Clark for superv is ing th is thes is work. My thanks go to Drs. J . Levy, C. 0. Person and R . A . J . Warren for serving on my committee. i would l i k e to express my apprec ia t ion to my external examiner, Dr. W.L. Fangman, of the U n i v e r s i t y of Washington, for accept ing the evaluat ion of th is work. I am e s p e c i a l l y indebted to Professors K.G. Lark , and P .C . Hanawalt, and Drs . Lucien Caro, Roy C u r t i s s III, and T . J . Leighton for the i r he lpfu l advice in c e r t a i n phases of th is work. L a s t l y , I thank Mrs. Lydia Huzyk (Mychajlowska) for helping me with the nucleot ide t r iphosphate a n a l y s i s . 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 tr iphosphate dGTP Deoxyguanosine t r iphosphate ATP Adenosine tr iphosphate CTP Cyt id ine tr iphosphate GTP Guanos i ne t r i phosphate UTP Ur id ine t r iphosphate TCA T r i c h l o r o a c e t i c ac id LITERATURE REVIEW The cel l cycle of bacteria is a complex, but well-coordinated process. This process, which allows the cel 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 repl icat ion; and (2) the cel l division cycle. The separate aspects of the DNA replication cycle have the following expressions: (i) attachment; ( i i ) in i t ia t ion (an event); ( i i i ) the origin (a s i te ) ; (iv) polymerization (chain elongation); and (v) segregation. The separate aspects of cel l division are: (i) formation of division proteins; ( i i ) physiological division and physical sep-aration of the two c e l l s . Much of the ear l ier 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 Biology of 1968. Since that time, the subject of DNA replication has been reviewed by Bonhoeffer and 2 Messer (1969) and Lark (1969b),and the c e l l d i v i s i o n cyc le by Helmstetter (1969a, b ) , Donachie and Masters (1969) and a Symposia of the Society for General Microbio logy (1969). Since the appearance of these reviews, some observat ions have been made which should be added to the present reviews. The fo l lowing l i t e r a t u r e survey is an attempt to make the information cur ren t . I. The DNA r e p l i c a t i o n cyc le in E_. c o l i The model accepted fo r r e p l i c a t i o n supposes the Watson-Crick s t ruc ture of hydrogen-bonded assembly of four nucleot ides to make up the E_. col i ch romosome and r e p l i c a t i o n by a semiconservative mechanism. A. The membrane attachment of DNA in E_. col i Jacob, Brenner and Cuzin (1963) suggested a chromosome attachment s i t e on the c e l l membrane. This uni t sys temat ica l l y could hold the r e p l i c a s e u n i t , through which the chromosome c i r c u l a t e d , and could a c t , as w e l l , as a v e h i c l e for moving daughter chromosomes apart . Several groups have provided add i t iona l evidence and extensions of th is hypothesis . These studies are e i t h e r d i r e c t l y e lec t ron microscop ic , or f r a c t i o n a t i o n a l separat ion and biochemical s t u d i e s . The f r a c t i o n a -t iona l separat ion s tudies are v a r i a t i o n s of the M-band method (Tremblay et aj_. 1969). E s s e n t i a l l y , the method cons is ts of lys ing the c e l l s with sarkosyl and banding the lysate in a sucrose gradient contain ing M g + + ions . The magnesium ions induce the c r y s t a l i z a t i o n of sarkosyl 3 in s i t u . Ten to t h i r t y percent of the c e l l membrane, 75% of RNA in the c e l l and 30% of the DNA are found assoc ia ted with the c r y s t a l s . Because n u c l e i c acids alone have no a f f i n i t y for s a r k o s y l , the s implest inference is that the complexed DNA and RNA is attached to fragments of the membrane conta in ing the chromosome attachment s i t e and the RNA in tu rn , is presumably attached to the DNA from which i t was being t r a n s c r i b e d . Recent ly , Shachtele e_t aj_. (1970) and Daniels ( 1 9 7 0 using the M-band method have shown s p e c i f i c attachment of E_. col i DNA. F i e l d i n g and Fox (1970) provided evidence for a s tab le attachment at the r e p l i c a t i n g o r i g i n in E. co 1 i . The consensus from these studies is that the DNA at the attachment region is protected from son ic i s c i l . lat ion and represents approximately 0.6 percent of the to ta l DNA of the c e l l which would correspond to a length of 6 u. Fuchs and Hanawa 11 (1970), using a 5 -• 20% l i n e a r sucrose gradient layered over a 60% sucrose " s h e l f " , have succeeded in i s o l a t i n g a "growing point" complex from E. co 1 i . The l a t t e r contained 0 .5 - 1% of the en t i re E_. col i genome; that i s , at least 5 u of DNA. D i rect e lec t ron microscopic s tudies involv ing th in sec t ion ing of E_. coj_i_ s t r a i n s and subsequent s t a i n i n g , has indicated a s s o c i a t i o n of the nuclear regions to the membranes (Altenburg et a 1. 1970; Pontefract and Thatcher , 1970). In genera l , these studies do not permit an adequate demonstration of the attachment o rgane l l e . Furthermore, in one case , the s t r a i n 0111 - a of E. c o l i was used which is known to form " e x t r a membranes" (Weigand e t aj_. 1970), and c o u l d thus i n v a l i d a t e t h e o b s e r v a t i o n s of A l t e n b u r g and c o - w o r k e r s . B. The i n i t i a t i o n o f normal chromosome r e p l i c a t i o n i n E_. c o l i . The r e p l i c o n t h e o r y ( J a c o b , B r e n n e r and C u z i n , 1963) i m p l i c a t e d t h a t t h e 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 was c o n t r o l l e d by a gene whose p r o d u c t was " t h e i n i t i a t o r " . The i n i t i a t i o n was d e f i n e d as t h e i n t e r a c t i o n o f the i n i t i a t o r w i t h a p a r t i c u l a r s i t e o f the r e p l i c o n , t h e r e p l i c a t o r . Under normal growth c o n d i t i o n s , t h e " i n i t i a t o r " s y n t h e s i s i s c o n t i n u o u s and t h e m a t e r i a l i n c l u d e s p r o t e i n . However, i t i n v o l v e s more tha n one s t e p ( L a r k and Ranger, 1969; Ward and G l a s e r , 1969). The s t e p s a r e d i s s o c i a b l e by d i f f e r e n t l e v e l s o f c h l o r a m p h e n i c o l . One s t e p o c c u r i n g a t the t i m e o f , o r s l i g h t l y b e f o r e , i n i t i a t i o n , i s s e n s i t i v e t o h i g h (150 ug/ml) and r e s i s t a n t t o low (25 ug/ml), c o n c e n t r a t i o n s o f c h l o r a m p h e n i c o l , and i s the same as t h e amino a c i d s e n s i t i v e s t e p o f i n i t i a t i o n . A d i f f e r e n t s t e p , o c c u r r i n g much e a r l i e r i s s e n s i t i v e t o low l e v e l s o f CAM. The low l e v e l s o f CAM a r e 2 ug per ml and 25 ug per ml f o r E_. c o l i B/r and TAU r e s p e c t i v e l y . Cooper and W e u s t h o f f (1971). however, s u g g e s t t h a t t h e r e a r e e i t h e r more than two s t e p s i n v o l v e d i n CAM s e n s i t i v i t y o f i n i t i a t i o n , each d e f i n e d by i t s s e n s i t i v i t y t o a d i f f e r e n t (1 t o 20 ug per ml) con-c e n t r a t i o n o f CAM, o r as s u g g e s t e d by L a r k (1969b!,there a r e o n l y two s t e p s , w i t h a s p r e a d o f " p r o b a b i l i t y " t h a t the s t e p w i l l be i n h i b i t e d by CAM i n a p a r t i c u l a r c e l l . T here no l o n g e r can be much doubt t h a t amino a c i d s t a r v a t i o n can b r i n g t h e DNA r e p l i c a t i o n t o a 5 f i xed end p o i n t , and, on res tora t ion of amino a c i d s , synthesis w i l l take up again from t h i s , the natural s t a r t i n g point (Bird and Lark, 1968; Kohiyama, 1968). The s t a r t i n g point fo r the chromosome r e p l i c a t i o n , the o r i g i n , is not a point but a reg ion. The o r i g i n of r e p l i c a t i o n has been def ined on the genet ic map of E_. c o l i Kl2 and B/r/1, and is located between arg G and xy lose loc i (7 and 8 o ' c l o c k ) . This conclus ion is backed by three d i f f e r e n t experimental approaches: '(1)transduction (Abe and Tomizawa, 1967; Caro and Berg, 1968; Masters, 1970); (2) enzyme induct ion (Donachie and Masters, 1969; Wolf et_ al_. 1968; Helmstet ter , 1968); and (3) synchronized mutagenesis (Cerda-Olmeda e_t aj_. 1968; Wolf et_ a_l_. 1968). E s s e n t i a l l y , a l l experiments are based on gene-dosage e f f e c t s assuming doubling of a gene, or i t s product , upon r e p l i c a t i o n of that gene. Or ig ina l s tudies by Nagata (1963) and recent ly those of V i e l -metter et a l . (1968), d isagree with the idea of f ixed chromosome r e p l i c a t i o n o r i g i n . This con t rad ic t ion arose from studies of Hfr s t r a i n s , where i t was shown that the s i t e of in tegra t ion of the IF 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 of the i n s e r t i o n determined the d i r e c t i o n of r e p l i c a t i o n (Nagata, 1963). V ie lmet ter et a l . (1968) a l s o obtained resu l ts favouring th is c o n c l u s i o n . in view of these f ind ings i t remains confusing as to the s i t e of the o r i g i n fo r r e p l i c a t i o n in E_. c o l i . The idea that once i n i t i a t e d , the c i r c u l a r molecule of DNA r e p l i c a t e s sequent ia l l y along i t s length 6 has been re examined by Nagata and Meselsohn (1968). In t h e i r 3 experiments, a pulse of H-TdR was given to exponent ia l ly growing c e l l s . Subsequently, i f c e l l s were pulsed with 5~bromouracf1, i t was found that the t r i t i u m label was associa ted with the 5 -bromo-u r a c i l only when the interval between add i t ion of the rad ioac t ive and densi ty label pulse equal led a mul t ip le of the generat ion time. Thus, the o r i g i n a l observat ions of Cairns (1963) and Lark et a l . (1963) were confirmed on the sequential r e p l i c a t i o n . C. The r e p l i c a t i o n of the E_. col ? chromosome. Two important questions are to be asked here: ( l) how does the r e p l i c a t i o n s t a r t ; and (2) how does the chain elongate in E_. col i ? Several models for the l a t t e r have been proposed s ince 1968, by G i l b e r t and Dress ier (I968) and by Okazaki et^ al_. (1968); by Haskel and Davern (1969); by Richardson (1969); by Morgan (1970); and by Werner, (1971). Many of the current models are v a r i a t i o n s on the simple model proposed by Watson and Cr ick (1953) suggesting a separat ion for the strands at the growing p o i n t , add i t ion of nucleo-t ides by Watson-Crick base p a i r i n g , and j o i n i n g by enzymatic means ( G u i l d , 1968; Hurwitz e_t_ aj_. 1968; Okazaki et_ aj_. 1968; Kornberg, 1969; Richardson, 1969)• G i l b e r t and Dress ier have proposed a general model, the " r o l l i n g c i r c l e " to account for the feature of both E_. col i and other bacter ia or bacteriophage r e p l i c a t i o n . B r i e f l y , the model proposes that synthesis begins by opening one strand of the o r i g i n a l c i r c l e at a s p e c i f i c point by introducing a s i n g l e - s t r a n d break 7 d i s p l a y i n g a 3 l _ h y d r o x y l and a 5 1 -phosphory l end-group. In order to prevent l i gase from repa i r ing t h i s break, the 5 '~end is t rans -fer red to some s i t e , perhaps on a membrane. DNA polymerase then i n i -t i a t e s synthesis by e longat ion of the 3 1 - end and uses the intact c i r c u l a r strand as a template. The o ld p o s i t i v e strand is peeled o f f the c i r c u l a r template for DNA polymerase to synthesize the new negative s t rand . As the growing point continues around the c i r c l e , a daughter molecule is peeled o f f e n d l e s s l y . Kubitschek and Henderson (1966) and Morgan (1970) have proposed mechanisms in which nucleot ide precursors are paired in Watson-Crick base pa i rs before being incorporated into the daughter s t rands. Although the two models d i f f e r in many respects regarding the react ion mechanism, they both have the important fundamental property that only one parental DNA strand is copied in the event of a mismatch. To resolve some of the paradoxes created by a l l of the above models, Werner (1971) has suggested a new mechanism for DNA r e p l i c a t i o n in E_. col i , and has introduced a reasonable doubt about what has been accepted as p l a u s i b l e mechanisms for DNA r e p l i c a t i o n . In b r i e f , using short pulses of t r i t i a t e d thymidine, he found that high molecular weight DNA is formed p r i o r to the appearance of low molecular weight DNA, suggesting that large DNA is the precursor of DNA fragments, and implying that Okazaki pieces are not the resu l t of d iscont inuous s y n t h e s i s . During short p u l s e s , the r e l a t i v e amount of label found in Okazaki pieces var ied with the nature of the precursor used. 8 Twenty percent of the incorporated H-thymfne was found in the p i e c e s , and, in the presence of unlabeU.ed thymidine, the incorporat ion of 3 H-thymine into Okazaki pieces was e n t i r e l y suppressed. He proposed that the pieces arose from s i n g l e strand nicks in both parental and newly synthesized DNA to act as swivel points for the ro ta t ion of the DNA h e l i c s during 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 . He a l s o suggests 3 that the labe l ing of the pieces during short pulses of H-thymidine represents repa i r s y n t h e s i s , whi le thymine is used for DNA r e p l i c a t i o n . Haskel and Davern (1969) have presented the "p re - fo rk synthesis model for DNA r e p l i c a t i o n " . In summary, DNA synthesis is cont inuously i n i t i a t e d from parental strand nicks and occurs ahead of the fo rk . The nicks thus act as i n i t i a t i o n s i t e s for chain synthesis and small c h a i n s , l i k e Okazaki fragments, are synthesized v ia a polymerase. The fork serves as a locus for unwinding and separat ing the a lready rep l i ca ted strands of the two double h e l i c e s . The pred ic t ions of the models proposed above have been tested in E_. col i , and, in every c a s e , c e r t a i n drawbacks are present . Lark (1969a)and Caro (1970) have looked at the question of symmetry with respect to the G i l b e r t and Dress ier model (1968). It was concluded that in E_. col i , the DNA is rep l i ca ted by a symmetrical process and i n i t i a t i o n takes place on both daughter chromosomes at once (Caro, 1970; Lark , 1969a). Lark , in a very soph is t i ca ted way, excluded both the age and the p o l a r i t y of the template as a source for s e l e c t i v e asymmetry and thus absence of inherent asymmetry in the DNA molecule, which could r e s t r i c t the s e l e c t i o n of template for r e p l i c a t i o n . F i n a l l y the " r o l l i n g c i r c l e " model was tested in a n u t r i t i o n a l s h i f t -up of synchronous B/r/1 c e l l s . Results did not f i t with the asymmetrical model predicted (C. Bagwell , unpublished r e s u l t s ) . Thus, the " r o l l i n g c i r c l e " quest ion remains open for examination. The Haskell and Davern (1969) model has not been tested ex-per imenta l ly yet and probably should be c l a s s i f i e d under other schemes that have been compiled in the Journal of Theoret ica l Biology by Erhan (1969) and P h i l l i p s (1969)-The Werner (1971) model has already been chal lenged (Lark, personal commun.), p a r t i c u l a r l y when his proposed recogni t ion of thymine for r e p l i c a t i o n and thymidine for repai r could not be sub-s tant ia ted by the known sequence of the pyrimidine b iosynthe t ic pathway (0 1Donovan and Neuhard, 1970; 0'Donovan, personal commun.). F i n a l l y , the s t r a i n used in th is study (15 TAMT, Cairns and Denhardt, 1968) was grown at 14 C to e s t a b l i s h a generat ion time long enough (400 minutes) to al low for short pulse l a b e l l i n g . It has been well documented by Ingraham's group that macromolecular synthesis and growth of E_. col i at low temperatures is severely a f fec ted (Ng, Marr and Ingraham, 1962; Shaw and Ingraham, 1967; Shaw, 1968; Ng, 1969)- Thus, Werner's observat ions could well be an a r t i f a c t of growth at \k C. The remaining models which f i t into the o r i g i n a l suggestions of Watson and Cr ick (1953) are v i a b l e and d i f f e r oh:ly in the mechan-i s t i c sense (Okazaki et a K 1968; Richardson, 1969; Kornberg, 1969) -The f i r s t q u e s t i o n , "how does t h e r e p l i c a t i o n s t a r t " ? s t i l l r emains as a g r e y , r a t h e r than t h e b l a c k box o f m y s t e r y f o r m o l e c u l a r b i o l o g i s t s . A l t h o u g h the o r i g i n and t e r m i n u s o f a c i r c u l a r chromosome s t r u c t u r e a r e the ends o f a l i n e a r d o u b l e s t r a n d , u n t w i s t i n g the mole-c u l e , when i t i s i n h e l i c a l f o r m , would be g r e a t l y enhanced by o c c a s i o n a l b r e a k s i n t h e c h a i n s (Wang and D a v i s o n , 1 9 6 8 ) . However, t h e mechanism f o r t h e c r e a t i o n o f a s w i v e l , w h i c h would be the growing p o i n t o r the f o r k , i s s t i l l o b s c u r e . In o r d e r f o r the r e p l i c a t i o n t o s t a r t , s p e c i a l enzyme(s) o r r e p l i c a s e s a r e needed. These have been polymerase I (Kornberg p o l y -merase) I I , ( C a i r n s p o l y m e r a s e ) and I I I ( G e f t e r polymerase) (de L u c i a and C a i r n s , 1 9 6 9 ; T. Kornberg and G e f t e r , 1 9 7 0 ) , but the Kornberg p o l y m e r a s e no l o n g e r i s q u a l i f i e d i n r e p l i c a t i o n and seems t o be a r e p a i r r e p l i c a s e ( K e l l y e t a l . 1 9 6 9 ) . The chromosomal r e p l i c a s e s c o u l d , 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 c h a i n s . S i n c e a l l DNA p o l y m e r a s e s have been shown t o have an a b s o l u t e r e -q u i r e m e n t f o r a d d i n g n u c l e o t i d e s t o a f r e e 3 1-OH group o f p r e -e x i s t i n g p r i m e r ( R i c h a r d s o n , 1 9 6 9 ) , t h e newly formed c h a i n s a t the f o r k must e x t e n d t h e m s e l v e s i n an a n t i p a r a l l e l f a s h i o n by s y n t h e s i z i n g s h o r t segments o f DNA a t a g i v e n t i m e . The s y n t h e s i s would be r e -s t r i c t e d t o t h e f o r k r e g i o n , and t h e segments formed would s u b s e q u e n t l y be j o i n e d t o g e t h e r . Recent e v i d e n c e s u p p o r t s t h i s system and w i l l be g i v e n h e r e . In v ivo studies by Yudelevich et_ aj_. (1968) , with E_. col ? CR34, and Iyer and Lark (1970), with E_. c o l i 555~7 (TAMT~) and E_. col i Pol A have indicated a p re fe ren t i a l locat ion of newly synthesized DNA at the 3'~0H end of a large deoxynucleotide s t rand . Thus, re -gardless of the polymerase system used, the asymmetry in s t a r t i n g the r e p l i c a t i o n by the r e p l i c a s e mimics that of the in v i t r o synthesis of DNA by the Kornberg polymerase. Studies i n i t i a t e d by several workers on E_. col i (Okazaki et a 1. 1968; Yudelevich e_t a_j_. 1968; Sadowski et^ a]_. 1968; Bird and Lark, 1969; Iyer and Lark, 1970), has supported the discont inuous mode of r e p l i c a t i o n . C o l l e c t i v e l y , the fo l lowing conclusions have been reached: (1) with pulses of thymidine (or thymine, Lark, personal commun.), incorporated label is mainly found as small (1 micron) and large (3 to 300 microns) s i n g l e strand pieces ex t rac tab le by a l k a l i ; (2) at any ins tan t , there are about 5 short pieces in cu l tures with chromosome r e p l i c a t i o n time of k0 minutes and 3 with r e p l i c a t i o n time of 80 minutes; (3) the time required for the synthesis of a 1 u piece at 37 C is 2 seconds, and th is is the same for f as t and slow growth ra tes ; (4) s tudies with exonuclease I, which s p e c i f i c a l l y hydrolyses s i n g l e stranded DNA from the 3 l - e n d , confirms the extension of the chain from the 3 '~end. As pointed out by Iyer and Lark (1970), two explanat ions could v a l i d a t e the observed occurence of the short and long p i e c e s ; (1) synthesis occurs by the continuous extension from the 3'~end and, for short p i e c e s , in the 5' and 3' d i r e c t i o n ; (2) synthesis occurs symmetrical ly from 3 1 to 5' on both strands but a symmetrical fragmentation occurs only on the 5 , _ s t r a n d , such that i t contains the s i n g l e strand breaks. These points are not c l e a r in the E_. col i system. in EL s u b t i l i s , pieces are complementary to only one strand of parental DNA (Kainuma and Okazaki , 1970) whi le in Jk and Lambda phages, the short pieces anneal equa l ly to both strands (Ginsberg and Hurwitz, 1970; Okazaki et_ a_j_. 1970). Recent studies on to luenized E_. col? eel 1s (Moses and Richarson, 1970; Mordoh et_ aj_. 1970; Kohiyama and Kolber , 1970) have shown r e p l i c a t i v e synthesis of DNA under in v ivo c o n d i t i o n s , the presence of a l l four deoxyr ibonucleot ide t r iphosphates , and have indicated a s t imula t ion in synthesis by ATP a lone . Furthermore, the r e p l i c a t i v e synthesis could be abol ished in DNA temperature-sensi t ive mutants. Moses and Richardson (1970) have character i zed fur ther the newly-made DNA by sedimentation and pyenographic a n a l y s i s , and there is l i t t l e doubt that th is incorporat ion does correspond to chromosomal DNA. This system has not been explored enough to al low ana lys is of the steps involved in the r e p l i c a t i o n nor of the question of discont inuous r e p l i cat ion. The model of d iscont inuous synthesis impl icates the need for the DNA l igase to cova len t ly j o i n the fragments. In v i t r o (Modrich and Lehman, 1971; Sadowski et_ aj_. 1968) or in v ivo (Pauling and Hamm, 1969) studies on DNA l igase c l e a r l y document the presence of th is enzyme and purported f u n c t i o n s . 13 Prel iminary approaches to the d i s s e c t i o n of the a c t i v e r e p l i c a t i n g machinery, which would c l a r i f y whether or not the polymerase-1 igase-nuclease complex e x i s t s in E_. col i , are promising (Smith et a 1 . 1970; Knippers et_ aj_. 1970; Kohiyama and Kober, 1970). Hopefu l ly , these systems should demonstrate exact ly how the 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_. col i is s t a r t e d . D. Separation of daughter chromosomes: Segregat ion. Repl icated DNA and chromosomes eventual ly become evenly par t i t ioned between daughter c e l l s . Morphological s tudies coupled with autoradiography have shown that the E_. col 1 chromosome is indeed associa ted with the c e l l membrane (Ryter, 1968; Rubenstein et a l . 1970). Studies of Ryter , H i r o t a , and Jacob (1968) suggested that segregat ion is random, that i s , at each c e l l d i v i s i o n , each of the o ld strands has the chance of d i s t r i b u t i o n into e i ther progeny. Lark (1966) had obtained d i f f e r e n t r e s u l t s which suggested that a d e f i n i t e pattern of segregat ion e x i s t e d . More recent ly however, Chai and Lark (1970), upon reexamination of the previous model, support Ryter 's random segregat ion model. II. C e l l d i v i s i o n c y c l e of E_. col i A. RNA and prote in synthesis in the normal d i v i s i o n c y c l e of E_. col i During i ts d u p l i c a t i o n c y c l e , RNA and prote ins are synthesized simultaneously with r e p l i c a t i o n of the chromosome and thus the c e l l increases in s i z e , mass and c e l l u l a r c o n s t i t u e n t s . In the case of general RNA s y n t h e s i s , the rate is apparently proport ional to the amount of template DNA a v a i l a b l e (Cu l t l e r and Evans, 1967; Helmstet ter , 1969b). The RNA precursor p o o l s , on the other hand, ind icate f l u c t -uating patterns during the c e l l c y c l e (Mychalowska, 1970). Although a requirement for prote in synthesis has been demonstrated (Mathison, 1968) in c e l l d i v i s i o n , no s p e c i f i c prote ins have been i d e n t i f i e d unique to c e l l d i v i s i o n . However, changes in the " d i v i s i o n p ro te ins" have been impl icated by several groups (inouye and Guthr ie , 1969; Green et_ aj_. 1969; Inouye and Pardee, 1970; Reeve et_ aj_. 1970; Smith and Pardee, 1970; Inouye, 1971) but the work suf fe rs two c r i t i c i s m s : (1) i f these prote ins are enzymes, the i r substrates are not known; and (2) in no case have those prote ins been shown to be the gene product of the genet ic l e s i o n . A ro le in septat ion has been postulated for the " d i v i s i o n p ro te ins" in the above l i s t e d works in general . In the absence of prote in s y n t h e s i s , l imi ted d i v i s i o n has been observed (P ierucci and Helmstet ter , 1969)- When prote in synthesis was e n t i r e l y blocked during DNA r e p l i c a t i o n , in s p i t e of the com-p l e t i o n of rounds, no d i v i s i o n was observed. However, i f prote in synthesis was inh ib i ted subsequent to the completion of a round, d i v i s i o n was observed. These resu l ts were cons is tent with the " t r i g g e r " for d i v i s i o n idea prev ious ly suggested by Clark (1968) and Helmstetter and P ie rucc i C968). This concept is probably c o r r e c t , but i t does not e n t a i l 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 d i v i -s i o n (Donachie e_t_ aj_. 1 9 6 8 ) , t h a t i s , the c r i t i c a l mass-to-DNA r a t i o . Changes i n the p a t t e r n s o f t h e s e d i v i s i o n p r o t e i n s d u r i n g the normal c y c l e remains t o be s t u d i e d . B. 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 . The i m p o r t a n c e o f s e p a r a t i n g the d u p l i c a t e d c e l l , by compart-m e n t a l i z a t i o n , i n t o two p h y s i o l o g i c a 1 1 y s e p a r a t e e n t i t i e s has been emphasized by C l a r k ( 1 9 6 8 ) . A f t e r t h e end o f a round o f r e p l i c a t i o n , two c e l l s a r e p h y s i o l o g i c a l l y s e p a r a t e d by t h e f o r m a t i o n o f a weak septum and then by a s t r o n g c r o s s - w a l l ( C l a r k , 1 9 6 8 ) . E v i d e n c e has i m p l i c a t e d a b u r s t i n t h e s y n t h e s i s of membranes i n E_. c o l i a t t h e p o i n t o f t h e p h y s i o l o g i c a l d i v i s i o n ( D a n i e l s , 1 9 6 9 ) . I t 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 b u i l d i n g b l o c k s need membrane-proteins complexes f o r t h e i r a t t a c h m e n t and p r i m i n g b e f o r e t h e y can e r e c t t h e septum. In such a c a s e , p h y s i o l o g i c a l d i v i s i o n would a l l o w f o r m a t i o n o f a l i g h t b a r r i e r w h i c h would a c t as an a nchorage s i t e f o r d e p o s i t i o n o f b i n d i n g p r o t e i n s f o r septum enzymes. R e c e n t l y , a membrane p r o t e i n has been i s o l a t e d i n S_. f a e c a l i s whose f u n c t i o n i s o r g a n i z a t i o n a l r a t h e r than c a t a l y t i c i n t h a t i t s e r v e s as a b i n d i n g s i t e f o r ATPase (Baron and Abrams, 1 9 7 1 ) . I I I . R e g u l a t i o n o f t h e c e l l d u p l i c a t i o n c y c l e i n E. c o l i . A. C o n t r o l o f the DNA r e p l i c a t i o n i n E_. c o l i , H i r o t a e t a l . ( 1 9 6 8 ) s u g g e s t e d two modes o f r e g u l a t i o n f o r the i n i t i a t i o n of the chromosome r e p l i c a t i o n in E_. col i . The f i r s t is a negative system, that i s , the presence of a repressor which would block the r e p l i c a t o r (Jacob, Brenner, and Cuz in , 1963) from being a c c e s s i b l e to r e p l i c a t i n g enzymes. The derepression could occur by sudden c r i t i c a l v a r i a t i o n s in the concentrat ion of some molecule, the regulat ion 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 growth. The second is a p o s i t i v e regula t ion which impl icates that a molecule is produced in each DNA c y c l e which could d i r e c t l y i n i t i a t e new rounds. No f i rm experimental data in support of e i ther hypothesis is a v a i l a b l e . Studies of r e p l i c a t i o n under condi t ions in which gross prote in synthesis is inh ib i ted (Lark and Ranger, 1969 ) , or in which synthesis of s p e c i f i c i n i t i a t o r prote ins (Levine and Sinsheimer, 1968) is i n -h ib i ted have indicated that such i n i t i a t o r prote ins are present in s to ich iomet r i c amounts and that add i t iona l prote in synthesis is re -quired in order to i n i t i a t e new rounds of r e p l i c a t i o n . Rosenberg et a l . (1969) by s tarv ing E_. col i 55 -7 (a 15 T ) for T , obtained accumulation of i n i t i a t i o n p o t e n t i a l . . If amino ac ids were withdrawn, fo l lowing the T s t a r v a t i o n , net prote in synthesis was ha l ted . Under amino ac id s t a r v a t i o n , however, i f T was added back, DNA r e p l i c a t i o n continued for a round and then stopped. When CAM (300 yg per ml) was added to the l a t t e r c u l t u r e , "uncont ro l led" DNA r e p l i c a t i o n was observed over several hours. It was concluded that once a new c y c l e had been i n i t i a t e d , s t i l l other unregulated i n i t i a t i o n s could occur a n d , . t o prevent t h i s , small amounts of prote in was needed. S imi lar conclusions have been reached by Kogoma and L a r k (1970) where, f o l l o w i n g a p e r i o d o f i n h i b i t i o n o f r e p l i c a t i o n w i t h NAL, t e m p e r a t u r e s e n s i t i v e m u t a t i o n s , o r T s t a r v a t i o n , DNA s y n t h e s i s can proceed f o r many hours i n s p i t e o f the 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 . P r i t c h a r d , B a r t h and C o l l i n s (1969) have proposed an a l t e r n a t i v e model f o r the c o n t r o l o f DNA r e p l i c a t i o n . T h e i r model s u g g e s t s t h a t an i n i t i a t o r p r o t e i n i s made i n t h e c e l l c o n s t i t u i t i v e l y w hich con-s t i t u t e s a c o n s t a n t f r a c t i o n o f t o t a l p r o t e i n of t h e c e l l a t a l l growth r a t e s . An i n h i b i t o r p r o t e i n i s coded by a gene l o c a t e d a d j a c e n t t o t h e chromosome o r i g i n , o r p a r t o f the o r i g i n i t s e l f , and i s t r a n s -c r i b e d o n l y d u r i n g the r e p l i c a t i o n o f t h a t gene. Each i n h i b i t o r gene, and c o n s e q u e n t l y each chromosome o r i g i n , i s r e s p o n s i b l e f o r t h e s y n t h e s i s o f a f i x e d number o f the i n h i b i t o r p r o t e i n s a t a l l growth r a t e s . There i s a c o o p e r a t i v e i n t e r a c t i o n between t h e i n h i b i t o r p r o t e i n and e i t h e r the i n i t i a t o r o r chromosome o r i g i n . T h i s i n t e r -a c t i o n i s such t h a t a t w o - f o l d change i n i t s c o n c e n t r a t i o n e f f e c t s an i n h i b i t i o n o f i n i t i a t i o n o v e r a range from c o m p l e t e t o z e r o . Thus, when t h e c y t o p l a s m i c volume i n c r e a s e s by growth of the c e l l , the c o n c e n t r a t i o n o f the i n h i b i t o r w i l l be d i l u t e d p r o g r e s s i v e l y . When the l e v e l o f i n h i b i t o r f a l l s below t h e c r i t i c a l l e v e l , r e i n i t i a t i o n t a k e s p l a c e , w h i c h w i l l be a s s o c i a t e d w i t h t t h e p r o d u c t i o n o f a new p u l s e o f i n h i b i t o r . The f r e q u e n c y o f t h e i n i t i a t i o n w i l l be d e t e r -mined by the d i l u t i o n r a t e o f t h e i n h i b i t o r , t h a t i s , the r e c i p r o c a l o f t h e growth r a t e . An i m p o r t a n t f e a t u r e o f t h i s model i s t h a t (1) i t is s e l f - r e g u l a t o r y and (2) assumes a negative contro l of r e p l i c a t i o n . Simon (1968) proposed a semiquant i tat ive model for the c e l l c y c l e in E_. col ?, where i t was assumed that the DNA r e p l i c a t i o n s t a r t s when a threshold dTTP concentrat ion was reached. This model was not tested by the author . The dTTP leve ls during the c e l l c y c l e (Mychajlowska, 1970) however, could substant ia te th is model. The two models of regula t ion of DNA r e p l i c a t i o n are not without precedent. The p o s i t i v e model could ex is t with a negative contro l operat ing on the synthesis of the i n i t i a t o r prote in (H i ro ta , et a 1 . 1968; 1970). The mutant, CR34T46, is assumed to have an a l t e r a t i o n in the p r o t e i n , which in some way, acts on the chromosome to al low r e p l i -ca t ion to s t a r t . Furthermore, th is component would act only when required for i n i t i a t i n g a new c y c l e . Hi rota et^ al_. (1970) have a l s o considered the regulat ion of the i n i t i a t o r mutant CR3^T46 by the negative c o n t r o l , the synthesis of a repressor during growth (Pr i tchard et_ aj_. 1969), or of the an t i repressor (Rosenberg et_'aj_. 1969) - In th is case , i t is argued that the Tk6 mutation is not an a l t e r a t i o n in the regulatory mechanism, s ince only 10 minutes at 30 C is enough fo r r e i n i t i a t i o n to occur , but would not be enough time for the d i l u t i o n of the repressor to occur . If the an t i repressor is the element a f f e c t e d , i t would be compatible with a model where the an t i repressor is made immediately a f te r DNA r e p l i c a t i o n has stopped. It should be p o s s i b l e to resolve these questions by study of several " i n i t i a t o r " mutants. In the pas t , several such mutants have been iso la ted in E_. col i (Kohiyama et a l . 1966; Fangman and Novick, 1968; Kuempel, 1969; C a r l , 1970). Phenotyp ica l l y , these mutants are character ized by the i r capac i ty for res idual DNA s y n t h e s i s . Geho-t y p i c a l l y , they do not f a l l into the DnaA c l a s s (Fangman and Novick, 1968; C a r l , 1970). It is a l s o poss ib le that the .cond i t iona l le thal mutants i s o l a t e d , which a f f e c t i n i t i a t i o n , deal s p e c i f i c a l l y with d i f f e r e n t stages of i n i t i a t i o n and the regula t ion of i n i t i a t i o n . To date t h i s is not c l e a r . B. Regulat ion of c e l l d i v i s i o n in £_. col i The only fact known for cer° ta in about normal E_. col ? eel 1 d i v i s i o n is that i t is coupled to DNA r e p l i c a t i o n (Clark , 1968b; Helm-s t e t t e r and P i e r u c c i , 1968). Inh ib i t ion of DNA r e p l i c a t i o n normally al lows for 25 percent res idual d i v i s i o n (C lark , 1968b; Helmstetter and P i e r u c c i , 1968), or causes an immediate cessat ion of c e l l d i v i s i o n (Donachie, 1969; Inouye, 1969), and DNA-less c e l l s are not produced. Even production of DNA-less m i n i c e l l s by a mutant of E_. col i was blocked upon stopping of DNA r e p l i c a t i o n (Clark , 1968b). However, i t is poss ib le to get temperature s e n s i t i v e mutants that could produce DNA-less c e l l s at kO C when they have lost the i r a b i l i t y to synthesize DNA (Hi rota et al_. 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 introducing a d e f e c t i v e recA gene, which is involved in recombinat ion, into E_. colj^ when DNA r e p l i c a t i o n was blocked. Hence DNA-less c e l l s r e s u l t . The product of the recA gene has been shown to reduce the amounts of nucleases produced by the recB and recC genes (Wi l le ts and C l a r k , 1969; Barbour et_ aj_. 1970), but the c o r r e l a t i o n between the recA gene product and the septum is not yet understood. In terest ing ly enough, whenever the 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 are uncoupled by mutations so that DNA-less bacter ia are produced, these mutants behave as i f rounds of r e p l i c a t i o n are completed at regular i n t e r v a l s . A r igorous a n a l y s i s of the regula t ion of E_. col i eel 1 d i v i s i o n was reported by Donachie and Begg, (1970), in which the coord inat ion between DNA segregat ion and c e l l d i v i s i o n were explained in terms of a unit c e l l concept . However, the mechanism for c e l l d i v i s i o n in E^. col ? , and the steps involved in the regula t ion of expression of d i v i s i o n , remain specu la t ive (Pardee, 1968; P r e v i c , 1970). MATERIALS AND METHODS I. Bac te r ia l and Phage St ra ins A. Bacter ia l s t r a i n s Bacter ia l s t r a i n s of Escher ich ia c o l i used in th is study are g iven i n Tab 1e I . B. Bacteriophage s t ra ins For the t ransduct ion experiments, the general ized t rans -ducing bacteriophage Plkc-L4 (Caro and Berg. 1971) was used. 1 I. Media and chemicals A. Media E_. col I CR34T83 was grown in a v /v mixture of tryptone broth medium A of Kaiser (1955) and the minimal s a l t s medium E of Vogel and Bonner ( 1 9 5 6 ) . The fo l lowing supplements were added: g lucose , 0.5 ug per ml; thymidine, 20 yg per ml; deoxyadenosine, 50 yg per ml; v i tamin B j , 7-5 yg per ml . These media w i l l be referred to as A and E, r e s p e c t i v e l y . When grown under minimal c o n d i t i o n s , the required amino ac ids were added at 20 yg per ml to minimal s a l t s medium E of Vogel and Bonner (1956). E_. col I B/r/1 was grown in 007 minimal s a l t s medium with 0.5% glucose added (Clark and Maal«5e, 1967). Table I. Bacter ia l s t r a i n s . Stra i n Number Mat ing Type Relevant Genetype Der ivat ion and source KG 5 5 KG 71 KG 7 1 - 2 KG 7 k KG 77 KG 78 (1-40) (141-216) KG 99 KG 110 KG 142 KG 146 KG 162 KG 163 KG 173 th r" , leu , pro , h is , thi , arg , lac , AB 1 1 5 7 from - - - - r r gal , ara , xyi , man , T^ , s t r thr , leu , thi , i l v , thyA , DnaA Same as KG 71 but low thymine requ i rer thr , leu , thi , i l v , thyA . thr , leu , thi , i l v + , thyA , KG 146, Ilv"*", DnaA" KG 163, i l v , DnaA Auxotroph thyA , arg , ura DnaA DnaA* Prototroph. T^ , , s t r , Tg , P1kc ind ica tor thi , arg , i l v ^ , metB trp , lac_ , tha , s t r t rp , l a c 2 , tha , i l v , s t r arg E.A. Adelberg CR34T83 from M. Kohiyama This work This work This work This work B/r/1 from D . J . Clark 15 TAU" from P. Hanawalt X289 from R. C u r t i s s I I I JC-533 from John Clark #BE235 from N. Otsuj i #BE269 from N. Otsuj i JE. col 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 , the media used were as s p e c i f i e d by Caro and Berg (1971). B. C h e m i c a l s . 14 Thymidine 2- C, Schwarz B i o r e s e a r c h i n c . , Orangeburg, 3 14 New Y o r k ; t h y m i d i n e m e t h y l - H, C - 5 - b r o m o - 2 - d e o x y u r i d i n e , Amersham/ 32 S e a r l e Co., Des P l a i n e s , I l l i n o i s ; P i was o b t a i n e d from T r a c e r i L a b o r a t o r i e s . C h l o r a m p h e n i c o l , Sigma Chemical C o r p o r a t i o n , S t . L o u i s , 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 s e a r c h F o u n d a t i o n , R e n s s e l a e r , New Y o r k ; Daunomycin-HC1, C a l b i o c h e m , Los A n a g e l e s , C a l i f o r n i a ; R i f a m p i n , C a l b i o c h e m , Los A n g e l e s , C a l i f o r n i a . I I I . C u l t u r e Methods A. Growth c o n d i t i o n s f o r l i q u i d c u l t u r e s . S t o c k c u l t u r e s o f the s t r a i n s used were m a i n t a i n e d on agar s l a n t s . 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 i n o c u l a t i o n i n t o g A and E medium t o a d e n s i t y o f 10 c e l l s per ml. These c u l t u r e s were kept a t 4 C u n t i l 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 the d e s i r e d t e m p e r a t u r e . B. P l a t i n g methods. V i a b i l i t y a s s a y s as w e l l as t r a n s d u c t i o n a s s a y s were c a r r i e d out on 1.5% agar p la tes which were incubated at 30 C to dry the p lates and check for contaminat ion. The p lates were stored at h C u n t i l needed. C. Temperature s h i f t c o n d i t i o n s . A l l temperature s h i f t s were accomplished by t r a n s f e r r i n g c u l t u r e s into f l a s k s prewarmed at the new temperature. If the pre -warmed f l a s k had a volume of ten times that of the volume of medium being t r a n s f e r r e d , temperature e q u i l i b r a t i o n was reached tn less than two minutes. IV. Measurement of macromolecular synthes is and c e l l growth. A. Measurement of c e l l numbers. The c e l l numbers were determined by a Coulter Counter (Nuclear Data, P a l a t i n e , I l l i n o i s ) and the i r s i z e d i s t r i b u t i o n was shown by an attached Nuclear Data 512 channel pulse height analyser (model 2200). C e l l s passed through a 30 u o r i f i c e for a time Interval of ten seconds. This system allows monitoring of the q u a l i t y of the growth by measuring the c e l l numbers and the d i s t r i -bution of the c e l l volumes wi th in the populat ion of c e l l s (Clark and Maaltfe, 1967; Painter and Marr, 1968). B. Measurement of c e l l mass. Spectrophotometric measurement of growth was performed using e i ther a Klett-Summerson p h o t o e l e c t r i c color imeter 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 spectrophotometer at 500 nm using a cuvette with a 1 cm l i g h t path. C. Measurement of tryptophanase a c t i v i t y . Tryptophanase assay was performed for tes t ing t rans -ductants with the unselected tna marker. i l v + t ransductants were p u r i f i e d on a s e l e c t i v e medium and then inoculated into 3 mis of l i q u i d tryptone broth . Tryptophanase a c t i v i t y was assayed by formation of indole in an overnight cu l tu re of a s t r a i n grown in tryptone bro th , by adding 2 ml of E h r l i c h so lu t ion and over -laying the mixture with 1 ml of x y l o l (Di fco Manual, 9th e d . , p. 53). D. Measurement of DNA s y n t h e s i s . 1 . Total DNA s y n t h e s i s . DNA synthesis was measured by the incorporat ion of rad ioac t i ve thymidine into the TCA inso lub le f r a c t i o n of the c e l l s . A t y p i c a l uptake experiment involved growing the c e l l s in r a d i o -a c t i v e l y l abe l l ed medium conta in ing the label at the desi red s p e c i f i c a c t i v i t y with unlabel led c a r r i e r at a f i n a l concentrat ion of 20 pg per ml . Deoxyadenosine was present in a l l cases at a concentrat ion of 50 ug per ml . At regular i n t e r v a l s , one ml samples were removed and added to 3 ml of 7-5% TCA with 200 ug per ml unlabel led thymidine. Each sample was f i l t e r e d through a 0.45 u M i l l i p o r e f i l t e r and washed four times with 5% TCA conta in ing 200 ug per ml thymidine. The f i l t e r s were washed two times with hot water, fo l lowing the TCA washes (Lark, Repko, and Hoffman, 1963). This method gave lower backgrounds than the use of TCA a lone . A f t e r heat d r y i n g , the f i l t e r s were placed into polyethylene s p e c t r a v i a l s (Amersham/Searle Corp.) conta in ing 10 ml of l i q u i d s c i n t i l l a t i o n counting so lu t ion (L iqu i f 1uor: toluene; 40 m l : l i t r e , Amersham/Searle C o r p . ) . Samples were counted for ten minutes in a Unilux S c i n t i l l a t i o n Counter (Nuclear Chicago Corp.) and counts were corrected for background. In some experiments, for the to ta l incorporat ion of the rad lo labe l chemica ls , the samples were processed by the batch method descr ibed by B y f i e l d and Scherbaum (1966). 2. Measurement of the rate of DNA syn thes is . The rate of synthesis of DNA was estimated by pu lse -14 3 l a b e l l i n g with C- or H-thymidine, again in the presence of 50 ug per ml deoxyadenosine. A t y p i c a l uptake experiment involved t rans -f e r r i n g one ml samples of c e l l s to 13 x 100 mm tubes conta in ing 100 u l of the l abe l l ed thymidine. A f te r three minutes the incorpora-t ion was stopped with three ml of 7-5% TCA conta in ing unlabel led thymidine. Each sample was washed and processed as above. 27 E. Measurement of ac id so lub le nucleoside tr iphosphate  poo ls . The chromatographic ana lys is of the tr iphosphates was based on the chromatographfc separat ion of the nucleoside t r i -phosphates by Randerath and Randerath (1967) as modif ied by L. Mychajlowska (1970 M.Sc. t h e s i s , UBC). 1. Preparat ion of the samples. -6 Cel ls .were grown in low phosphate buffer (10 M phosphate) and were allowed to e q u i l i b r a t e for the pools at least 32 one generat ion (40 minutes at 30 C) a f t e r add i t ion of the P . . To measure the nucleoside tr iphosphate p o o l s , 250 y l c e l l samples were mixed with 100 y l of 2N formic a c i d . The contents were then mixed and allowed to stand in an ice-bath for ten to f i f t e e n minutes for the l y s i s and re lease of the pool mater ia1. These samples were cent r i fuged in a Beckman Microfuge and the supernatants were saved for spot t ing on th in layer chromatographic p l a t e s . 2. Chromatography. Polyethyleneimine-impregnated c e l l u l o s e plates were made according to Randerath and Randerath (1967) by Miss Mychajlowska. These p lates were used wi th in a week a f te r prepara t ion . The sample, supernatant of the ac id h y d r o l y s i s , was added in 5 x 20 y l quan t i t i es to the plates in a slow manner to avoid f lood ing the surface of the p l a t e s . A f te r the spot t ing was completed, the plates were dr ied o f f and then washed with methanol (anhydrous) for 20 to 30 minutes to remove unnecessary s a l t s from the medium and contaminating l a b e l . Two dimensional chromatography was performed according to the method of Irr and Gal lant ( 1969) - The f i r s t dimension was 1N Acetate: lM L i C l (1:1, v /v) , u n t i l about one and one-ha l f inches from the top. A second run was performed immediately a f te r th is in a 1N Aceta te : 1.5M L i C l (1:1, v /v) solvent and allowed to run in the same d i r e c t i o n for f i v e and one-ha l f hours. To al low for a better separat ion of the deoxyr ibonucleoside t r i -phosphates from the r ibonuc1eoside t r iphosphates , wicks were made out of Whatman No. 3 f i l t e r paper which allowed the solvent to "run over" the p l a t e . At the end of the fiirst run, the p lates were taken out and dr ied and washed with methanol to remove the L i C l of the f i r s t run. The second solvent system was made of 3M NH^Acetate and h.3% Borate, pH 7.0, and the p la tes were allowed to run for four and one-ha l f hours in thfs system. At the end of th is run, the plates were dr ied and prepared for autoradiography. 3 . Autorad iography. 32 The dr ied plates conta in ing the P . - l a b e l l e d tr iphosphates were placed 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 for 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 ixer . The films were then rinsed again with water and dried. The areas which showed radioactivity were clearly seen as dark (exposed) spots on the f i lm. These films were then placed over their corresponding plates and with a pin the exposed spots were punched out on the plate. The radioactive area was then scraped off the plate, 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 sc in t i l l a t ion v i a l s . These via ls were properly marked as to the nature of the spot (ATP,.etc) and ten mi l l i i r t res of sc in t i l l a t ion f lu id was added. The radtoactivity was counted in a Unilux Scint i l la t ion Counter for ten minutes. Counts per minute were corrected for backgrounds. V. Density Gradient Sedimentation Analysis. 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 a density of 5 x 10^ ce l ls per ml was obtained. The ce l ls were then f i l tered and washed free of thymine and resuspended in ^C-BrUra (Schwartz-Bioresearch Co.) with a f inal specif ic act iv i ty of 0.033 yc per ml (6 yg per ml f inal concentration). These ce l ls 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 analysis, 10 mis of ce l ls were rprocessed as descr ibed below in Sect ion VI .D. B. Density label 1ing. Procedures for the l a b e l l i n g of the s t a r t s and ends of the chromosomes with rad ioac t ive thymidine and subsequent BU densi ty p ick-up of the desi red sec t ion of the chromosome was done according to Lark , Repko and Hoffman (1963). Overnight grown c e l l s of KG-71-2 3 were grown in the presence of H-thymidine, s p e c i f i c a c t i v i t y of 20.6 Ci per mM, f i n a l concentrat ion of 1 mCi per ml , at 30 C in a 50 ml f l a s k . The c e l l s were sh i f t ed to kl C for an hour in the 3 3 H-thymidine medium. Then, these c e l l s were f i l t e r e d f ree of H-thymidine, washed twice with prewarmed A and E medium, and resus-pended in f resh medium with supplements and C-thymidine, s p e c i f i c a c t i v i t y 52.8 mCi per mM, f i n a l concentrat ion 0.5 uCi per ml (Schwartz Bioresearch C o . ) , in 20 ml medium for 25 minutes. The c e l l s were f i l t e r e d and allowed to randomize for f i v e generat ions. A f te r an hour at kl C, the c e l l s were f i l t e r e d and put into 50 mis of medium conta in ing 10 yg per ml BrUra and allowed to grow for f i f t y minutes. At the end of th is time the c e l l s were c o l l e c t e d for lys i s . C. Preparat ion and ana lys is of DNA samples by densi ty gradient  centr i fugat ion . 1. Ex t rac t ion of DNA C e l l s c o l l e c t e d for the densi ty gradient technique were put d i r e c t l y onto frozen A and E medium and saved u n t i l needed. Frozen c e l l s were thawed at room temperature and then spun down at 8000 rpm for f i v e minutes. This p e l l e t was resuspended in one ml of Tr is -EDTA buffer (0.01 M each, at pH 8.2). Lysozyme (Calbiochem) was added at a f i n a l concentrat ion of 200 ug per ml and the mixture was incubated at 37 C for -20 minutes. A f te r 20 minutes, 1 ml of a 0.1% so lu t ion of sodium laury l s u l f a t e was added to the c e l l suspension. Immediately, the lysates became v i s c i o u s . The lysates were brought to a tota l of 3-5 ml with d i s t i l l e d water and were treated with 50 y l of pronase (Worthington), at f i n a l concentrat ion of 200 yg per ml , to d igest the bac te r ia l p r o t e i n s . 2. Cent r i fuga t ion and F r a c t i o n a t i o n . To 3-26 ml of the l y s a t e , 4.36 gm of CsCl (Harshaw, o p t i c a l grade) was added. The mixture was incubated at 37 C to d i s s o l v e the C s C l . This combination gave a f i n a l densi ty of I.76 •3 gm per cm (p = 1.405). The mixture was poured into c e l l u l o s e n i t r a t e cen t r i fuge tubes and o v e r l a i d with mineral o i l . C e n t r i -fugat ion was performed in a Beckman L2-65B u l t r a c e n t r i f u g e at 37,000 rpm for 40 hours at 20 C. Fol lowing c e n t r i f u g a t i o n , the base of the c e l l u l o s e n i t r a t e tube was punctured in a Beckman f r a c t i o n c o l l e c t o r . Ten drop samples were c o l l e c t e d per f r a c t i o n into 13 x 100 mm tubes. From every f i f t h f r a c t i o n a drop was removed for densitometry analys by an Abbe refractometer at 23 to 25 C (room temperature). 3. Measurement of r a d i o a c t i v i t y . Samples were d i l u t e d with 5% TCA, ice c o l d , and f i l -tered through 0.45 u HA M i l l i p o r e or Reeve Angel F iber Glass f i l t e r s . They were dr ied and counted as descr ibed p r e v i o u s l y . Samples were corrected for background and adjusted for the double label counts. VI . Genetic ana lys is of the mutants. A. Iso la t ion of the temperature r e s i s t a n t rever tan ts . The standard technique for i s o l a t i n g the temperature r e s i s t a n t revertants cons is ted of inocula t ing 0.1 ml of an overnight c u l t u r e d i l u t e d 1:100 to contain 1 x 10^ c e l l s per ml , d i r e c t l y into A and E broth or onto supplemented agar p lates prewarmed to 42 C. A f te r incubation overnight at 42 C, co lon ies were picked up and examined for the i r genet ic background and for the DnaA mutation phenotype. Growth in the tubes was checked m i c r o s c o p i c a l l y fo r snake format ion. B. Transduct ion experiments. 1. Bacteriophage donor lysate prepara t ion . Desired bac te r i a l donor s t r a i n s were grown from an overnight cu l tu re in 9 mis of Lurta broth with 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 s t ra Two to four hours incubation was necessary to give a dens i ty of g about 2 x 10 c e l l s per ml . Phage P1Kc was d i l u t e d to about 6 - 8 x 10^ pfu (plaque-forming uni ts) per ml , and 0.1 ml of th is was added to 1.9 ml of bacter ia in a s t e r i l e Wasserman tube at 37 C. A f te r twenty minutes of preadsorpt ion , 0.2 ml of th is adsorpt ion mixture was added to 2.5 rrnl of Lur ia sof t agar and poured onto Lur ia agar p l a t e s . A f te r f i v e minutes required for sol id i f feat ion of the o v e r l a y , the p lates were incubated at 37 C for s ix hours. To harvest the donor phage, 5 mis of Lur ia broth (without CaCl) was added per p la te and the plates were allowed to s i t overnight in the r e f r i g e r a t o r . Next morning, the sof t agar layer was broken up with a g lass spreader and was scraped into a cent r i fuge tube. About 0.1 ml of chloroform was added for every 5 mis of lysate and the mixture was vortexed for 1 minute to burst any un-lysed c e l l s . This mixture was centr i fuged at 5 ~ 6000 x g_ for 15 minutes to p e l l e t the c e l l debr is and the supernatant was saved for phage t i t r a t i o n . 2. Transduct ion experiments. Desired r e c i p i e n t s were grown as descr ibed for the ind icator s t r a i n s above. Donor P1kc was d i l u t e d to give an M0I ( m u l t i p l i c i t y of in fec t ion) of 1 - 3 phages per bacterium. A f te r 20 minutes of preadsorpt ion , the contents of 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 plated on s e l e c t i v e p l a t e s . The supernatant was assayed for unadsorbed phage. The phage suspension was always checked for contaminating b a c t e r i a , and the rec ip ien t s t r a i n s were checked for revers ion as c o n t r o l s . RESULTS AND GENERAL DISCUSSION I . Propert ies of CR34T83 A. Ana lys is of macromolecular synthesis 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 is working with temperature s e n s i t i v e mutants, the d e f i n i t i o n of the s e n s i t i v e temperature, as well as the p h y s i o l o g i -cal temperature range at which the behaviour of the mutant could be examined, becomes one of utmost importance. The response of the T83 c e l l s to a temperature t r a n s i t i o n from 30 C to a range of temperature from 27 to kS C was tested in a temperature gradient b lock . In a typ ica l temperature s h i f t experiment, T83 c e l l s grown for several generat ions at 30 C in K le t t tubes were t ransfered to the temperature b lock . At timed i n t e r v a l s , c e l l numbers were determined using the Coulter Counter. F igure 1 represents the r e s u l t s of such an experiment. The control c e l l s grown at 30 C, :i:n th is system, go through two doublings during the 85 minute incubation per iod . At temperatures above kl C, there was no c e l l u l a r d i v i s i o n and, hence no net increase in the c e l l numbers for sh i f t ed c u l t u r e . At temperatures between 30 and 33-5 C, when compared to the c e l l s growing at 30 C, a s i g n i f i c a n t increase in the f i n a l c e l l numbers was observed. The l a t t e r changes were accounted f o r , by the fac t that between the 30 and 33-5 C, a fas te r growth rate was ach ieved, without a f f e c t i n g the T83, grown for several generat ions at 30 C, was d i s t r i b u t e d into 16 tubes (18 x 150 mm) at a ee l ] dens i ty of 5 x 10? c e l l s per m l , and was placed in a temperature gradient b lock . C e l l counts were monitored by a Coul ter Counter for the next 8 5 minutes (two genera t ions ) . F ina l c e l l numbers ( r e l a t i v e c e l l counts) are p lo t ted against the tempera-ture of incubat ion . temperature s e n s i t i v e c o n d i t i o n . However, at temperatures above 33-5 C, there was a general re tardat ion of growth and a complete cessa t ion of c e l l d i v i s i o n between 41 and 45 C. Forty-two degrees was thus chosen as the u l t imate non-permissive temperature. When T83, at d i f f e r e n t c e l l d e n s i t i e s between 1.5 and 10 x 10^ c e l l s per ml , were sh i f ted from 30 to 42 C, the response of the c e l l s was the same, that i s , there was no s i g n i f i c a n t res idual d i v i s i o n at 42 C. Thus, regardless of the c e l l d e n s i t y , the response was the same. 2. Ce 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 to non- permissive temperature, a . Cel1 d iv i s ion . The p o s s i b i l i t y of measuring bac te r i a l growth e l e c t r o n i c a l l y with a Coulter counter and the accuracy of th is method for monitoring the q u a l i t y of the bac te r ia l growth has been demonstrated by several l abora tor ies over the past decade (Kubit-schek, 1969a). In p r i n c i p l e , the c e l l volume d i s t r i b u t i o n s for cu l tu res of E_. col and bacter ia in g e n e r a l , growing under steady state condi t ions permit the determination of the c e l l volume change during the c e l l c y c l e , (Harvey and Marr, 1966) . Harvey et_ aij_. (1967) , and Kubitschek (1968a), gave a more r igorous d e r i v a t i o n of the c e l l 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 r a t i o s . The instrumental reso lu t ion of the Coulter counter is such that one could detect the smal lest changes in the q u a l i t y and quant i ty of bac te r ia l growth p o s s i b l e . In steady s t a t e , exponential growth c o n d i t i o n s , in A and E medium at 30 C , CR34T83 d iv ides with a generat ion time of kl minutes which corresponds to a s p e c i f i c growth rate of 1.k Hour \ Under such c o n d i t i o n s , when c e l l numbers increased in an exponential manner, the c e l l s i z e , as indicated by the peak channel p o s i t i o n , for 30 C grown c e l l s , remain constant at channel 75 (Figure 2b). Upon a s h i f t to kl C, there is a cessa t ion in c e l l d i v i s i o n (Figure 2a). Results obtained by Clark (1968a) and Helmstetter et_ aj_. (1968) indicated that c e l l d i v i s i o n depends upon the completion of a round of DNA r e p l i c a t i o n . In an exponential popu la t ion , broth grown cu l tu res of E_. col ?, about 18% of the c e l l s which have completed t h e i r DNA . r e p l i c a t i o n c y c l e but have not separated yet (Kubitschek, 1969b), are in doublet forms. These c e l l s have been shown to undergo d i v i s i o n when t h e i r DNA r e p l i c a t i o n has been blocked (Clark , 1968b;Helmstetter, 1968). It is c l e a r that th is event does not take place in CR34T83. Coincident with the cessat ion of the c e l l d i v i s i o n there is an increase in the c e l l mass which corresponds to mass increase expected of an exponential cu l tu re at kl C (Figure 2b). The c e l l s increase in s i z e , as viewed m i c r o s c o p i c a l l y , and f i lamentous forms r e s u l t . V i a b i l i t y counts for T83 c e l l s at kl C ind icate no loss in v i a b i l -i ty for the f i r s t f i f t y minutes. Upon longer incubat ion , however, there is a gradual d e c l i n e in the percent of surv ivors to f i f t y percent by 90 minutes, and to 100 percent by 180 minutes of incubation at kl C. 0 Figure 2. Growth of T83 under permissive and non-permissive 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 ind icated by the v e r t i c a l arrow. Ce l l numbers (panel A) and c e l l s i z e (panel B) were monitored as a func t ion of time during growth at the permissfve (30 C) and non-permissive (kl C) temperatures. 40 b. S t u d i e s on DNA r e p l i c a t i o n i n CR34T83. i . T o t a l u p t a k e o f TdR. S h i f t i n g a c u l t u r e o f CR34T83 from 30 t o 42 C r e s u l t s i n r e s i d u a l amounts o f DNA b e i n g s y n t h e s i z e d under n o n - p e r m i s s i v e c o n d i t i o n s ( F i g u r e 3)- T h i s r e s i d u a l DNA s y n t h e s i s proceeds a t a g r a d u a l l y d e c r e a s i n g r a t e f o r a p p r o x i m a t e l y f o r t y t o s i x t y m i n u t e s . No f u r t h e r d e t e c t a b l e s y n t h e s i s o c c u r s upon l o n g e r i n c u b a t i o n a t 42 C. The p e r c e n t i n c r e a s e i n the amount o f r e s i d u a l DNA s y n t h e s i z e d a t 42 C i n r i c h medium i s 40 t o 60%. In m i n i m a l medium, under i d e n t i c a l c o n d i t i o n s , t h e r e s i d u a l s y n t h e s i s i s 20 t o 30%. The same o b s e r v a t i o n s have been made f o r the o t h e r i n i t i a t o r mutant CR34T46 ( H i r o t a e t a 1. 1970). These r e s u l t s f i t n i c e l y w i t h t h e H e l m s t e t t e r - C o o p e r model o f DNA 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 ( H e l m s t e t t e r and Cooper, 1968). The model i n d i c a t e s t h a t c e l l s grown i n r i c h medium c o n t a i n m u l t i p l e f o r k s whereas, i n minimal medium, c e l l s have s i n g l e f o r k s . A c c o r d i n g l y , CR34T83 w i l l show a h i g h e r p e r c e n t a g e o f r e s i d u a l DNA s y n t h e s i s when grown i n b r o t h as compared t o t h e m i n i m a l c o n d i t i o n s . i i . Rate o f DNA r e p l i c a t i o n . A t a g i v e n i n s t a n t o f t i m e , t h e r a t e o f i n c o r p o r a t i o n 14 o f C - t h y m i d i n e i n t o DNA by a growing c u l t u r e i s a f u n c t i o n o f t h e number o f r e p l i c a t i n g f o r k s i n t h e c e l l s and t h e r e p l i c a t i o n v e l o c i t y . Under s t e a d y s t a t e c o n d i t i o n s i n an e x p o n e n t i a l p o p u l a t i o n , t h e r a t i o o f r a t e of DNA r e p l i c a t i o n / c e l l numbers s h o u l d remain c o n s t a n t (Maal^e and K j e d g a r d , 1966). I f any d i s t u r b a n c e o f anomaly from t h i s r u l e was MINUTES Figure 3. Uptake of C-1A thymidine into CRT-83 growing at 30 C and kl C. T83 growing in the presence of C-14 thymidine at a f i n a l concent ra t ion of 20 yg per ml and a s p e c i f i c a c t i v i t y of .3 yc/uM was s h i f t e d from 30 C to kl C. The c e l l dens i ty at the time of s h i f t was 1.5 x l O ^ / m l . The i n -corpora t ion of Z-\k thymidine in to the c o l d TCA inso lub le 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 (•) is given as a func t ion of t ime. 1 1 I 1 I 1 I 1 I 1 I r 4 2 ° M I N U T E S F i g u r e k. Rate o f DNA s y n t h e s i s i n CR3*tT83 a f t e r a s h i f t 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 p er m l , 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 samples 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 yg per m l , and w i t h 50 yg per ml AdR. Counts per m i n u t e o f ^ 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 t i m e 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) and c o n t r o l a t 30 C (•). to take p l a c e , one should see for example, an increase in the DNA per eel 1 r a t i o , that i s , a mul t inuc leate snake, or a decrease, as in a temperature s e n s i t i v e DNA r e p l i c a t i o n mutant. At 30 C and at kl C, the rate 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 incorporat ion of C-thymidine into cold TCA insolub le material of the c e l l . The r e s u l t s are shown in Figure k. The DNA per c e l l r a t i o ca lcu la ted from the counts per minute of Ik C-TdR incorporated per ml over the c e l l number per ml . For kl C grown c e l l s , the r a t i o of rate per c e l l decreased u n t i l a plateau value was reached, due to a cont inual but l imi ted DNA synthesis in the absence of c e l l d i v i s i o n at kl C. The 30 C c e l l s maintain a constant DNA per c e l l r a t i o . The conclus ion that DNA synthesis comes to a hal t a f t e r s i x t y minutes at kl C could be reached from the pulse exper iments. I I. Recovery of C R 3 4 T 8 3 at 3 0 C a f t e r growth at kl C. A. S ingle s h i f t experiments. The design of the experiments: Exponent ia l ly grown c e l l s at 3 0 C were t ransfer red to kl C, as descr ibed in sect ion (It . C. A por t ion of the o r i g i n a l c u l t u r e (1/5 the s t a r t i n g volume) was main-tained at 3 0 C throughout the rest of the experiment as the control reference c u l t u r e . A f te r appropr ia te incubation time at kl C , about 3/5 of th is was sh i f t ed down to 3 0 C and the remaining 1/5 was kept at kl C throughout the rest of the experiment. The terminology used in descr ib ing these resu l ts are as f o l l o w s : s h i f t up, a s h i f t from 30 C to kl C; s h i f t down, the reverse case; pu lse , incubation of a c u l t u r e at kl C for a short time as given by the length of the pu lse ; and recovery, the resumption of c e l l growth at 30 C a f t e r a pulse at kl C. During recovery from a pulse at kl C , the c e l l d i v i s i o n pattern shows three d i s t i n c t phases. These a re : (phase i ) , a lag in c e l l d i v i s i o n ; ( i i ) an acce lera ted d i v i s i o n for a short t ime; and ( i i i ) continued d i v i s i o n , corresponding to that of the normal 30 C c u l t u r e . In analys ing such r e s u l t s , the lag re fers to the length of time needed for the c u l t u r e to s ta r t d i v i d i n g , and the time needed for a 50% i n -crease in the c e l l numbers from the base l i n e , or the lag is re fer red to as the ha 1 f -s tep time. 1 . Ce l l d i v i s i o n during recovery from a kl C pu lse . Figure 5 descr ibes the response of exponential c e l l s of CR34T83 to varying pulses of growth at kl C. In every case , the c e l l s stopped d i v i d i n g a f te r a s h i f t s u p , and recovery in the s h i f t down contained (1) a lag (2) acce lera ted d i v i s i o n , and (3) normal d i v i s i o n phases. A comparison was made between the recovering cu l tu res pulsed for d i f f e r e n t periods at kl C and the contro l c u l t u r e s . The time during recovery for kl C grown c e l l s , when the accelerated d i v i s i o n curve changed i t s rate to normal r a t e , was used as the re ference . At th is t ime, the value for the number of c e l l s for the contro l c u l t u r e was assumed to be 100% d i v i s i o n , and the res idual d i v i s i o n at the i n f l e c t i o n point was ca lcu la ted a c c o r d i n g l y . As shown in Figure 5, for a short o • v • 0 • 20 40 60 80 100 MINUTES Figure 5- Recovery of CR34T83 at 30 C fo l lowing growth at kl C. A c u l t u r e of T83 growing exponent ia l l y at 30 C, was s h i f t e d to kl C at a dens i ty of 1.5 x 10 7 c e l l s per ml . At 10 (o) , 20 (•) , 30 ( v ) , k5 ( • ) , 60 ( • ) , and, 70 ( • ) minutes as indicated by v e r t i c a l arrows, subcul tures from kl C were t ransfered to 30 C and allowed to recover . C e l l counts are p lo t ted as a funct ion of t ime. The cont ro l c u l t u r e , maintained at 30 C, is represented by the dashed l i n e . Figure 6. Re la t tonship between c e l l s recover ing from kl C to t h e i r cont ro l c u l t u r e s kept at 30 C. Data from Figure 5 and numerous other s i m i l a r experiments were used to const ruct t h i s graph. The ord ina te represents the r a t i o of c e l l counts for c e l l s recover ing from a temperature block at kl C , to the cont ro 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 recover ing 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 , that i s , the i n f l e c t i o n points of the recovery curves . The a b s c i s s a represents the durat ion of the temperature block at kl C. pulse (up to 10 minutes) , the c e l l s showed a 100% recovery in numbers to that of the contro l 30 C grown c e l l s . However, th is f i g u r e dec l ines r a p i d l y , for pulses up to 50 minutes, to the 35% l e v e l . For pulses longer than 50 minutes, the same 35% level was maintained. The r e s u l t s from v i a b i l i t y data c l e a r l y ruled out the p o s s i b i l i t y of a loss in v i a b l e c e l l s during the f i r s t 50 minutes. Thus, the c e l l s at 30 C, which had continued the regular i n i t i a t i o n cyc les for the i r DNA r e p l i c a t i o n , gained a p r i o r i t y over those at kl C. In other words, poss ib le c e l l s at kl C, by staying at the non-permissive c o n d i t i o n , lost the nuclear equiva lents d i r e c t l y proport ional to the length of the b lock , whereas the c e l l s at 30 C i n i t i a t e d regu la r ly and gained nuclear equ iva len ts . In the f i n a l a n a l y s i s , it appeared that there had been a loss in res idual c e l l d i v i s i o n , o r , expressed in terms of DNA c y c l e s , a loss in i n i t i a t i o n . Furthermore, the DNA synthesis between kO and 60 minutes maintained the number of d i v i s i o n s obtained during the recovery to a constant , that i s , c e l l s which had a complete nuclear equiva lence . Ana lys is of the r e l a t i o n s h i p between the pulse in kl C and the length of the lag and time for 50% increase in the c e l l numbers a f te r recovery are given in Figure 7- The d i s t r i b u t i o n of lag for pulse length between 5 and 30 minutes was maintained at a r e l a t i v e l y constant length , averaging 14 ± 3 minutes. The corresponding h a l f - s t e p was IS ± k minutes. With longer than 30 minute p u l s e s , t h i s r e l a t i o n s h i p became protracted to larger v a l u e s . It appeared , . thus , that the length 15 20 25 30 PULSE LENGTH (Min.) 35 40 Figure 7. The r e l a t i o n s h i p between the lag in recovery to the per iod of growth at 42 C. Resul ts from Figure 5 and s i m i l a r experiments were used to construct t h i s graph. The lag (• ) , and the ha l f step (0), that is the time required fo r a 50 percent increase in c e l l numbers a f t e r the l a g , are p lo t ted as a func t ion of the pulse of non-permissive growth. of shorter temperature pulse was unimportant in decid ing the behaviour of the c e l l s in recovery. 1 2. Ce l l volume d i s t r i b u t i o n s for T83 at recovery. The change in s i z e d i s t r i b u t i o n of T83 during a s h i f t up and recovery is i l l u s t r a t e d in Figure 8. Under balanced growth c o n d i t i o n s , at 30 C T83 has a s i z e d i s t r i b u t i o n t y p i c a l to exponential cu l tu res of E_. col ? ( P a i n t e r and Marr, 1966). This d i s t r i b u t i o n moved toward higher channel numbers or larger c e l l volume upon s h i f t to kl C. The general shape of the s i z e d i s t r i b u t i o n curve changed to a f l a t t e r curve and there was a cons iderab le s h i f t in the mean c e l l volume. The f i laments resul ted in phase I which, upon a s h i f t back to the permissive c o n d i t i o n , fragmented to a populat ion of heterogeneous s izes during phase II, the acce lera ted d i v i s i o n to give three peaks at 105 minutes. Even tua l l y , the smaller normal s i z e gained dominance as the c e l l s were in t h e i r normal growth phase III, from 1^ 5 to 160 m i nutes. 3. Rate of DNA synthesis during recovery from a kl C pu lse . The rate of thymidine incorporat ion into c e l l s of T83 recover-ing from pulses of d i f f e r e n t length at kl C was determined by pulse Ik l a b e l l i n g the c e l l s with C-thymidine and counting the r a d i o a c t i v i t y incorporated into the cold TCA inso lub le m a t e r i a l . F igure 9 shows the resu l t of such experiments. Upon s h i f t i n g T 8 3 from 30 to kl C , the rate of thymidine incorporat ion into DNA decreased gradual ly and stopped. During the course of th is drop, the c e l l s went through the i r Figure 8. A n a l y s i s of the c e l l s i z e dur ing recovery from growth at kl C. T83, 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 minutes. C e l l volume d i s t r i b u t i o n s were obtained from p l o t s of the pulse height a n a l y s i s of the c e l l s . The ord ina te represents the c e l l number, and the a b s c i s s a , the pulse height ana lyser channel number (0-511). Each curve is numbered as to the sampling t ime. 51 2l I I I I I I I I I 1 L 20 40 60 80 100 M I N U T E S Figure 9. Rate of l a b e l l e d thymidine incorpora t ion into T83 during recovery . C e l l s grown at 30 C in A and E medium at a densi ty of 5 - 8 x 10^ c e l l s per ml (zero t ime) . A f te r 5 (V), 10 ( • ) and 15 (0) minutes (upper panel) or 30 ( T ) , 40 ( • ), and 60 (•) minutes (lower pane l ) , c e l l s were s h i f t e d back to 30 C as ind icated by v e r t i c a l l i n e s . C e l l s were pulsed with 0.025 uc per ml ^ C - T d R or 2.5 yc per ml 3|H-TdR, at a f i n a l concent ra t ion of 20 yg per ml TdR, and with 50 yg per ml AdR, for 3 minutes. Re la t i ve rate of r ad ioac t i ve thymidine incorporated (CPM) is p lo t ted aga inst t ime. The curves represent the best smooth l i n e s f i t t e d through the experimental values of two separate experiments. The broken l i n e represents the uptake for c e l l s grown and maintained at 42 C s ince zero t ime. res idual DNA r e p l i c a t i o n between kS and 60 minutes. When the cel l 's were sh i f t ed to 30 C, the permissive temperature, a f te r 60 minutes of incubation at kl C , the rate of incorporat ion of l abe l l ed thymidine into DNA rose rap id ly for 20 minutes and then continued at a rate compatible with that of the 30 C control c u l t u r e . The c e l l s returning from a kO minute nncubation at kl C , on the other hand, d isplayed a lag of approximately seven minutes before resuming the uptake at an increasing ra te . The 15 and 30 minute pulsed c e l l s , when returned to 30 C, went through a drop in the rate of incorporat ion of l abe l l ed thymidine into DNA un t i l kO to k5 minutes. A f te r th is p o i n t , the rate of uptake increased. For c e l l s incubated for f i v e and ten minutes, s i m i l a r continued drops in the rate were observed which, a f te r a short w h i l e , picked up to the normal rate of the 30 C c e l l s . Several points were noteworthy. F i r s t l y , for short temperature a pulses of f i v e arid ten minutes, the rate of incorporat ion at 30 C fo l lowing a s h i f t to the kl C temperature, did not get to normal for approximately seven to twelve minutes a f t e r the s h i f t down, and in f a c t , kept dropping l i k e that of the kl C c o n t r o l s . S i m i l a r l y , fo l lowing 15, 30, and kO minute incubations at kl C , the rate of uptake dropped despi te the c e l l s being incubated at 30 C. These c e l l s star ted a new rate of uptake at r e s p e c t i v e l y , 30, 15 and 5 minutes a f te r s h i f t down. In teres t ing ly enough, once sh i f ted to permissive temperature, the c e l l s had two places for s ta r t of r e p l i -ca t ion from the zero time o r i g i n at kl C. The f i r s t one occurred 53 at 22.5 minutes and the second at kS minutes. The r e l a t i o n s h i p of pulse and the s t a r t point seemed to be 22.5 minutes s tar t point for pulses shorter than ten minutes, and kS minutes for pulses 15 to kO minutes. The c e l l s with 60 minutes of incubation at kl C, when re -turned to 30 C, however, i n i t i a t e d the i r r e p l i c a t i o n within 3 minutes. 32 k. Measurement of - P - l a b e l l e d nucleoside tr iphosphates  in CR34T83. In a mutant, where there is a l imi ted a b i l i t y for synthesis of DNA, i t is poss ib le for the l i m i t a t i o n to reside in the a v a i l a b i l i t y or conversion of the r ibonuc leot ides to the deoxyr ibonucleot ide t r i -phosphates. The DNA-RNA precursor pools of T83 were measured according to 32 the presence of P - l a b e l l e d P. in the r i b o - and deoxyr ibonucleoside t r iphosphates . Figure 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 - l abe l l ed r i b o - and deoxyr ibonucleoside tr iphosphates under permissive (30 C) c o n d i t i o n s , at a s h i f t up (kl C ) , and during the recovery stage. Ana lys is of the deoxyr ibonucleoside tr iphosphate pools in T83, under the permissive growth c o n d i t i o n s , indicated in decreasing l e v e l s , the fo l lowing order : dATP; dCTP; dGTP; and dTTP. There was no de tec t -able increase in the dTTP l e v e l s . S imi la r order is es tab l ished for exponential cu l tu res of E_. col i B/r/1 when grown in glucose minimal medium at 37 C, that is in decreasing l eve ls of dATP, dCTP, dGTP and dTTP. During the 60 minutes of incubation 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 P - l a b e l l e d deoxyribonucleoside triphosphates in CR34T83. T83 was grown in 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 of 1.75 x 10 M. P. was added at 100 yc per ymole of PO^ and 60 minutes were allowed f o r complete e q u i l i b r a t i o n of the l a b e l i n t o 32 the pools p r i o r to sampling. D i s t r i b u t i o n of P - l a b e l l e d deoxy-nuc l e o s i d e t r i p h o s p h a t e s during recovery from growth at kl C was measured as described in M a t e r i a l s and Methods. The o r d i n a t e r e -presents the percent increase in the CPM over the i n i t i a l CPM observed, and the a b s c i s s a represents time. 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 distr ibution 32 of P-labelled ribonuc1eoside triphosphates in T83 is shown in Figure 11 B. Multiple shift experiments. The observation that T83 stopped dividing when shifted from 30 to kl C indicated that a division substance was temperature sensi-t ive and was required for the expression of ce l l division until the moment before ce l l separation. Since this effect was an immediate one, one might expect that ce l l division could be blocked immediately during the recovery period (at 30 C) when such recovering ce l ls were returned to kl C. However, it could be equally possible for recovering ce l ls to be altered so that they may be irreversibly committed to ce l l d iv is ion , that i s , during the rapid division period, in which case they would be immune to the non-permissive temperature effect. Experiments were designed to test these poss ib i l i t i es . Results of such an experi-ment are shown in Figure 12. When recovering ce l ls were returned to non-permissive conditions at 7-5 minutes after the shift down, there was approximately 5% residual d iv is ion . For ce l ls returned to kl C, 15 and 22.5 minutes after shift down, that i s , ce l ls at their rapid division phase, 22 and 35 percent residual division was observed respectively. F ina l ly , upon shift ing the recovering ce l ls just prior to their return to normal division phase to kl C, approximately 18 percent residual division was observed. -20 0 20 40 60 80 100 MINUTES F igure 11. D i s t r i b u t i o n of P - l a b e l l e d r ibonuc leos ide t r iphosphates in CR34T83. 10 8 42< © • v • • ° .V V_ . a 20 40 60 80 100 MINUTES Figure 12. Inhibition of ce l l d iv is ion during the recovery period by shif t ing 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 vert ical arrows, samples were removed from the 30 C f lask (•»-), and shifted to kl C. Cell counts were followed by Coulter Counter (ordinate) for the next 100 minutes. Controls are 30 C grown ce l l s (o), and kl C grown ce l ls (o). Ini t ia l ce l l density was approximately 2 x 107 eel 1s per ml. It was concluded that during the recovery period at 30 C the c e l l s were most l a b i l e to the s h i f t up when at the i r lag phase. On the con t ra ry , c e l l s at the i r acce lera ted d i v i s i o n phase were more immune to the non-permissive temperature e f f e c t and showed a greater escape than when they resumed the normal d i v i s i o n phase. Subsequent to the expression of the s i i g h t escape from the temperature block at kl C, a l l c l a s s e s of c e l l s c l e a r l y stopped fur ther d i v i s i o n . Apparent ly , the minimum lag period at 30 C was essent ia l for the escape from the temperature block and the expression of d i v i s i o n at kl C. A r igorous p r e d i c t i o n that a l l systems were as expected led to an experiment in which the response of T83 to mul t ip le temperature e s c i l l a t i o n was examined (Figure 13). The resu l ts indicated that c e l l d i v i s i o n was inh ib i ted when the c e l l s were passed through o s c i l -l a t ions between 30 and kl C. As long as the s h i f t to the non-permissive temperature occurred e a r l i e r than the lag needed for the expression of d i v i s i o n (12 minutes) during recovery , the c e l l d i v i s i o n was i n h i b i t e d . There was a s l i g h t amount of increase in c e l l numbers during the course of these o s c i a l l a t i o n s . The c e l l s , at the las t s h i f t down, however, showed a longer than normal lag and had los t the rapid d i v i s i o n phase of the i r recovery. Instead, they resumed d i v i s i o n at the rate compatible to that of the 30 C grown c e l l s . C. P h y s i o l o g i c a l requirements of the recovery from a s i n g l e s h i f t . 1 . Role of DNA synthesis in recovery . . The coordinated r e l a t i o n s h i p between DNA r e p l i c a t i o n and c e l l d i v i s i o n has been well es tab l ished (Lark, 1969b; Helmstet ter , 1969b). 20 40 60 80 100 120 140 MINUTES Figure 13- I nh ib i t ion of c e l l d i v i s i o n by p e r i o d i c exposure to the non-permissive temperature. A c u l t u r e of T83 growing exponent ia l l y at 30 C was s h i f t e d to kl C. At times ind icated by v e r t i c a l arrows, 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 las t s h i f t to 30 C, at 105 mfnutes, c e l l s were kept at 30 C to a l low expression of c e l l d i v i s i o n . Growth of the cont ro l (•) and experimental (0) c u l t u r e s is p lo t ted as a func t ion of t ime. Termination of a round of r e p l i c a t i o n is a necessary condi t ion for c e l l d i v i s i o n (C lark , 1968b; P i e r r u c i and Helmstet ter , 1968). It is known that any a r res t in chromosome r e p l i c a t i o n resul ted in formation of f i laments ( B i l e n , 1969; Donachie, 1969; Boyle e_t_ a K 1967). This coordinate regula t ion between DNA r e p l i c a t i o n and the var ious processes involved in c e l l u l a r d i v i s i o n has been examined by Inouye and Pardee 0970) and r e s u l t s indicated that b locking of DNA r e p l i c a t i o n by chemica agents or by mutations caused a change in membrane prote ins that were involved in c e l l d i v i s i o n . N a l i d i x i c ac id has been shown to stop semiconservattve DNA r e p l i -ca t ion immediately (Dietz et al . 1966; Goss et a l . 1965)as well as repai r r e p l i c a t i o n (Eberle and Masker, 1971) in E_. col i . Using N a l i d i x i c a c i d , C lark 0968 a,b) and Helmstetter and P i e r r u c i (1968) showed that i t d id not a f f e c t the c e l l u l a r d i v i s i o n of those c e l l s which had completed the i r DNA r e p l i c a t i o n . The var ious aspects of the coord inat ion between 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 was examined in T83. N a l i d i x i c ac id , at a f i n a l concentrat ion of 10 ug per ml , was added to T83 during incubation at kl C. Since during the incubation at kl C, c e l l s continue to make a res idual amount of DNA, the blocking of DNA r e p l i c a t i o n by N a l i d i x i c ac id should have allowed for c e l l u l a r d i v i s i o n proport ional to the amount of DNA r e p l i c a t i o n that had a l ready occurred . F igure 14A represents the r e s u l t s of an experiment where N a l i d i x i c ac id was added at the time of s h i f t up and at f i f t e e n minute i n t e r v a l s during s z 0 0 • v + N A L u 9 F -8 > 7 I— < 6 U J " 5 -X—x—x-xoorv^  J L 80 120 MINUTES 160 200 F i g u r e 14. E f f e c t of i n h i b i t i o n of DNA sy 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. T83, grown at 30 C, at a c e l l d e n s i t y of 3 x 10 /ml, was s h i f t e d to 42 C at zero time. N a l i d i x i c a c i d was added to subsamples during the 45 minute incubation at non-permissive temperature at the time of s h i f t up ( 0 ) , or 15 ( 0 ) , 30 ( t ) , and 45 (V) minutes t h e r e a f t e r (panel A ) , or a f t e r r e t u r n to the permissve tempera-ture f o r recovery at zero ( 0 ) , 5 ( 0 ) , 10 (•), 15 (V), 20 ( T ) , 30 ( • ) , and 40 ( • ) minutes 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 the times of a d d i t i o n of N a l i d i x i c a c i d to a f i n a l c o n c e n t r a t i o n of 10 yg per mi 11 M i t r e . the non-permissive treatment. When the c e l l s were returned to 3 0 C , c e l l u l a r d i v i s i o n resumed at the same time as the contro l c e l l s . There was a d i r e c t c o r r e l a t i o n between the amount of DNA synthesized at hi C and the d i v i s i o n observed at 3 0 C. C e l l s that were treated r ight at the s h i f t up time (zero minutes) resul ted in 2 7 % res idual d i v i s i o n . If f i f t e e n minutes of DNA r e p l i c a t i o n was allowed at hi C, the residual d i v i s i o n at 3 0 C went up to hl% and reached 5 9 % for t h i r t y minutes of res idual DNA s y n t h e s i s . When DNA synthesis was allowed for h5 minutes, there was near ly a one hundred percent increase in c e l l number during recovery. Control c e l l s at 3 0 C , when given the same amount of N a l i d i x i c a c i d , resul ted in 3 0 % res idual d i v i s i o n . When N a l i d i x i c ac id was added during the recovery p e r i o d , no increase in c e l l numbers was observed for add i t ions between 0 and 2 0 m i n u t e s . s h i f t down. For add i t ion at 3 0 and hO minutes p o s t - s h i f t down, however, residual d i v i s i o n leve ls rose to 140 and 1 6 0 percent r e s p e c t i v e l y . 2 . Role of RNA synthesis in recovery. By binding to RNA polymerase, Ri fampicin s p e c i f i c a l l y blocks i n i t i a t i o n of t r a n s c r i p t i o n but not the completion of those in progress (Mizuno et_ al_. 1 9 6 8 ; Burgess et_ al_. 1 9 6 9 ) . Addi t ion of Ri fampin, jus t p r i o r to the i n i t i a t i o n of new cyc les of DNA r e p l i c a t i o n , does not i n t e r f e r e with completion of rounds star ted but prevents the i n i t i a t i o n of new ones ( S i l v e r s t e i n and B i l l e n , 1 9 7 0 ) . When the response of exponent ia l ly growing c e l l s of E_. co l i B / r / 1 and T83 were tested for d i f f e r e n t concentrat ion of R i fampic in , i t was found t h a t i n h i b i t i o n o f H - u r a c i l u ptake i n t o the c e l l s o c c u r r e d r e l a t i v e l y s l o w l y . Up t o f i v e m i n u t e s was r e q u i r e d f o r 50 p e r c e n t i n h i b i t i o n a t c o n c e n t r a t i o n s o f 1 t o 25 yg per ml o f R i f a m p i c i n , but when h i g h c o n c e n t r a t i o n s were used (50 t o 500 yg per m l ) , e f f e c t i v e i n h i b i t i o n was o b s e r v e d . However, when t h e p e r c e n t r e s i d u a l d i v i s i o n was compared w i t h the c o n c e n t r a t i o n o f R i f a m p i c i n , i t was n o t i c e d t h a t B/r/1, i n g l u c o s e m i n i m a l medium, r e p r o d u c i b l y gave 30, 13, 6.5, arid 1.2 p e r c e n t r e s i d u a l d i v i s i o n r e s p e c t i v e l y , f o r c o n c e n t r a t i o n s o f 10, 25, 50 and 100 yg per ml. At s t i l l h i g h e r c o n c e n t r a t i o n s , l e s s r e s i d u a l d i v i s i o n was o b t a i n e d . Thus, 10 yg per ml was s e l e c t e d as a s u i t a b l e c o n c e n t r a t i o n w h i c h e f f e c t i v e l y i n h i b i t e d RNA s y n t h e s i s w i t h o u t h a v i n g any i l l e f f e c t s on t h e c e l l s . C R 3 4 T 8 3 , when grown i n medium A and E a t 30 C w i t h 10 yg per ml R i f a m p i c i n , gave 50 p e r c e n t 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 -t e n t w i t h t h e B/r/1 d a t a . When T83 was s h i f t e d t o kl C f o r v a r y i n g l e n g t h s o f t i m e , from 10 t o 60 m i n u t e s , and R i f a m p i c i n was added t o t h e c u l t u r e a t t h e r e c o v e r y t i m e , a l l t h e c e l l s r e c o v e r e d and under-went r e s i d u a l d i v i s i o n ( F i g u r e 15). I t was c o n c l u d e d t h a t RNA s y n t h e s i s subsequent t o t h e s h i f t down was of l i t t l e consequence t o c e l l d i v i s i o n a t r e c o v e r y . P r e s u m a b l y , mRNA i n g e n e r a l , and d i v i s i o n messengers a t t h e s h i f t down t i m e , were undergraded 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 such t r a n s l a t i o n s were 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 , based on t h e amount o f DNA and c e l l e q u i v a l e n t s , r e s u l t e d . A f t e r a kO m i n u t e i n -c u b a t i o n a t kl C, c e l l s t h a t were 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 M I N U T E S Figure 1 5 . The effect of inhibit ion of RNA synthesis at recovery on the expression of d iv is ion . Exponentially grown ce l ls of T83, at a density of 2 . 5 x 1 0 ce l ls 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 ce l ls at zero time. Cell numbers were followed as a function of time. of Ri fampicin resul ted in res idual d i v i s i o n that produced an increase of approximately 20 percent in c e l l numbers. Longer incubations than 40 minutes resul ted in reduct ion in residual d i v i s i o n . S imi la r experiments were conducted in which Rifampicin was added at d i f f e r e n t time in te rva ls during a 45 minute pulse at 42 C or during recovery at 30 C (Figure 16). A 45 minute incubation in the presence of Ri fampicin at hi C v i r t u a l l y destroyed the capaci ty of the c e l l s to recover at 30 C. Shorter blocks of to ta l RNA synthesis gave increasing amounts of residual d i v i s i o n . Thus, in order for the c e l l s to express the d i v i s i o n of the i r c e l l equivalents at recovery, RNA synthesis at hi C was needed. However, once sh i f ted to permissive temperature from a 45 minute growth at 42 C, no great change in res idual d i v i s i o n was observed i f Ri fampicin was added at 5, 10 or 15 minutes a f t e r the s h i f t down to 30 C. If the accumulation type of behaviour was proport ional to the durat ion of growth at 42 C, presumably, with longer incubat ion , more potent ia l should be accumulated. Figure 17 shows that th is is not the case . Between 15 and 45 minutes incubation at 42 C , s i g n i f i c a n t changes in residual d i v i s i o n were observed. However, the change between 45 and 60 minutes was n e g l i g i b l e . It was a l s o noted that Ri fampic in treatment at 42 C, for as short as 10 minutes p r io r to recovery , was s u f f i c i e n t to reduce the maximum d i v i s i o n potent ia l seen at the time of s h i f t down by 50 percent . 3• Role of prote in synthesis on 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 during the recovery pe r iod . bb 5 z u > < 6 — - B. • 1 I I I I 1 1 I I I 1 • • - / ro-i-x—iy _ l I I 1 1 1 L I . I . I 0 20 40 6 0 80 100 120 Minutes F i g u r e 16. E f f e c t o f i n h i b i t i o n o f t o t a l RNA s y n t h e s i s on r e c o v e r y . 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 and R i f a m p i c i n was added a t v a r i o u s t i m e s d u r i n g t h e p u l s e . R e c o v e r y from t h e t r e a t m e n t was examined a t 30 C. R i f a m p i c i n was added (10 ug per ml) a t 0 (0), 10 (0), 18 ( 0 ) , 25 ( 0 ) , 32 (•), 40 (V), and k5 (?) m i n u t e s . A l l o f t h e s u b c u l t u r e s were ke 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 were 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 panel 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 added a t 0 (?), 5 ( •), 10 (•) ( and 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 4 0 60 80 100 120 TIME (min.) Figure 17. Measurement of the accumulation of d i v i s i o n poten t ia l in T83. CR34T83, at a dens i ty of 2.3 x 10 7 c e l l s per ml , was s h i f t e d from 30 C to kl C and kept there fo r 15 (A) , k5 (B) and 60 (C) minutes In each c a s e , at 10 ( t ) , 5 ( • ) or 0 (?) minutes before s h i f t down (zero t ime) , R i fampic in was added to a l l the c e l l s . C e l l counts were fol lowed during recovery t ime. a. C e l 1 d i v i s i o n . 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 t h a t i n T83, the t e m p e r a t u r e s e n s i t i v e b l o c k a f f e c t e d a s t e p r e q u i r e d i n c e l l u l a r d i v i s i o n and chromosomal r e p l i c a t i o n . I t seems t h a t t h e s t r u c t u r e o r s u b s t a n c e ( s ) needed i n t h e v i t a l f u n c t i o n s o f the c e l l ' s c y c l e i s i n v o l v e d i n t h i s c o n d i t i o n . A c c o r d i n g l y , t h e t e m p e r a t u r e e f f e c t c o u l d i n v o l v e a c o m p l e t e and i r r e v e r s i b l e d e n a t u r a t i o n o f the temper-a t u r e l a b i l e components and the l a g thus r e p r e s e n t s time needed f o r t h e r e s y n t h e s i s o f t h e new p a r t s , t h e r e o r i e n t a t i o n o f the a f f e c t e d s t r u c t u r e , o r perhaps f o r t h e r e n a t u r a t i o n o f the p r o t e i n i n q u e s t i o n . The f o l l o w i n g t r a n s i t i o n s c o u l d be e n t e r t a i n e d f o r such t e m p e r a t u r e s e n s i t i v e c o n d i t i o n : (a) t h e t e m p e r a t u r e s e n s i t i v e p r o d u c t o r s t r u c t u r e i s n a t i v e and a c t i v e a t t h e p e r m i s s i v e t e m p e r a t u r e , but i s rendered d e n a t u r e d and i n a c t i v e a t the n o n - p e r m i s s i v e t e m p e r a t u r e ; o r (b) i t i s r e n d ered i n a c t i v e , though remains i n the n a t i v e form a t the non-p e r m i s s i v e t e m p e r a t u r e . The f o l l o w i n g e x p e r i m e n t s were u n d e r t a k e n t o 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 as l i k e l y c a n d i d a t e s f o r t h e t e m p e r a t u r e s e n s i t i v e c o n d i t i o n . In t h e f i r s t s e r i e s o f e x p e r i m e n t s , T83 was s u b j e c t e d t o a kl C t e m p e r a t u r e p u l s e f o r t e n m i n u t e s . To one h a l f of the kl C c e l l s , c h l o r a m p h e n i c o l , a t f i n a l c o n c e n t r a t i o n o f 150 yg per m l , was g i v e n f i v e m i n u t e s a f t e r s h i f t t o kl C. The r e s t o f t h e c u l t u r e was not t r e a t e d . At t h e end o f 10 m i n u t e s a t kl C, both c u l t u r e s were r e -t u r n e d t o 30 C. As shown i n F i g u r e 18, the c e l l s t h a t r e c e i v e d MINUTES Figure 18. E f f e c t of i n h i b i t i o n of p ro te in synthesis on recovery of T83. Part of an exponential 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) s h i f t down, CAM (150 yg per ml) was added to subcul tures ( 0 ) . C e l l counts are p lo t ted as a func t ion of t ime. chloramphenicol did not recover . To test for the necess i ty of new prote in synthesis for the ex-pression of subsequent c e l l d i v i s i o n during the recovery period at 30 C chloramphenicol was added f i v e minutes a f t e r the s h i f t down. The r e s u l t s are seen in Figure 18. A f te r a short pulse of 10 minutes at kl C , when c e l l s were returned and allowed to undergo prote in synthesis f u l l d i v i s i o n was expressed a f t e r the 10 minute lag per iod . However, i f prote in synthesis was b locked, such that there was only a l imi ted amount of prote in synthesis at 30 C, there was a decreased amount of d i v i s i o n in recovery. Apparent ly , a short period of prote in synthesis was essent ia l for the expression of the d i v i s i o n a f t e r a 10 minute growth at kl C. S imi lar tests were performed, except with an extended pulse of kS minutes at kl C (Figure 19). T83 was grown for several generations at.30 C and was d i s t r i b u t e d into eight ident ica l f l asks and sh i f ted to kl C for kS minutes a f t e r which chloramphenicol was added to the f l a s k s at 10, 18, 25, 32, kO, and kS minutes at kl C, and at 5 or 10 minutes a f t e r s h i f t down. It was concluded tha t , i f prote in synthesis was inh ib i ted for 5 to 35 minutes, in T83 grown at kl C for kS minutes, ho c e l l d i v i s i o n was observed during recovery pe r iod . However,' i f chloramphenicol was added at the time of s h i f t down (kS minutes) , or a f t e r t h e ' s h i f t back to 30 C , exact ly one doubling was observed. It was of in terest to f ind out the mode of acqu isa t ion of the d i v i s i o n potent ia l and see i f i t was an accumulative one. If s o , 71 M I N U T E S F igure 19- E f f e c t of i n h i b i t i o n of p ro te in synthes is dur ing and a f t e r a pulse at kl C , on recovery T83 grown at 30 C in A and E medium to a dens i ty of 1 .5 x 10 c e l l s per ml , was s h i f t e d to kl C for k5 minutes. CAM (150 ug per ml) was added to s u b c u l t u r e s , as ind icated by v e r t i c a l arrows, a f t e r 1.8 (X) , 25 (©) , 32 (o) , or kO (©) minutes incubat ion at kl C , or at 0 ( • ) , $ ( • ) , 10 ( • ) , 15 (v), 25 ( • ) , and 35 (o) minutes a f t e r return to 30 C. The return to 30 C a f t e r a kS minute pulse at kl C was taken as zero time f o r recovery . Re la t i ve c e l l counts are p lo t ted against t ime. The dashed l i n e represents the untreated c o n t r o l . gradual ly increasing leve ls of residual d i v i s i o n was produceds 'upon longer incubation at kl C. The accumulation hypothesis led to an experiment where 150 ug per ml chloramphenicol was added at the time of s h i f t down to the T 8 3 c e l l s grown at kl C for varying lengths of t ime. Results of such experiments are presented in Figure 20. For a 10 minute s h i f t to kl C , and return to 30 C in the absence of any fur ther prote in s y n t h e s i s , there was a 33 percent increase in c e l l counts . With longer incubation at kl C , the amount of d i v i s i o n observed at recovery under chloramphenicol i n h i b i t i o n rose . Thus, with 18, 25, 32 and kO minutes of incubation at kl C, r e s p e c t i v e l y , kO, 67, 87 and 100 percent increase in c e l l number was observed. In-cubations of kS to 60 minutes y ie lded approximately a 110 percent increase in the c e l l counts . The percent increase in the c e l l numbers was found to be l i n e a r l y re lated to the pulse at kl C, which plateaued o f f at kO mjnutes. When compared with the amounts of res idual DNA made at kl C, the p lots of percent res idual DNA synthesis and d i v i s i o n data revealed a p a r a l l e l r e l a t i o n s h i p between the amount of DNA made at kl C to that of c e l l d i v i s i o n . Thus, the general impl ica t ion of these data was that (a) the t r a n s i t i o n for c e l l d i v i s i o n potent ia l was that of a nat ive and a c t i v e prote in at 30 C going to nat ive but inac t ive at kl C , and i ts r e a c t i v a t i o n upon return to 30 C; and (b) at the time of s h i f t down from any length temperature b lock , there ex is ted enough potent ia l in the c e l l to express d i v i s i o n or c e l l u l a r recovery based on the amount of DNA per c e l l equ iva len ts . at ui co Z IU > < —j Ul I 1 I 1 I 1 -0- - 0 - i — f -v V ft t V 0 Q 0_ 0 I I I 1 I 1 I 20 40 60 80 MINUTES I • 1 • 100 120 140 F i g u r e 2 0 . E f f e c t o f 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 on t h e r e c o v e r y o f c e l l s a t 3 0 C. C R 3 4 T 8 3 c e l l s were 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 and, 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 ug per ml c h l o r a m p h e n i c o l was added t o t h e c u l t u r e . C e l l c o u n t s a r e p l o t t e d 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 rates at which the recovery c e l l s from a kl C block underwent, were compared with contro l c e l l s and with those given chloramphenicol at s h i f t down (Figure 2 1 ) . There was a change in the rates of c e l l d i v i s i o n during the recovery phase II (rapid d i v i s i o n ) and a small increase in the rates between 20 and kS minutes which dec l ined to a slower rate upon longer incubat ion. On the cont ra ry , the c e l l s which had los t the i r capac i ty for synthesis of prote ins at recovery, showed a gradual ly increasing rate of d i v i s i o n upon longer incubation at kl C. There was a f o u r - f o l d change in the rate of d i v i s i o n between c e l l s incubated at kl C for k5 to 60 minutes when recovering at 30 C in the presence of chloramphenicol (Figure 2 1 ) . It was obvious tha t , as ide from the formation of DNA equivalents at kl C , with the longer incubat ions , the stage was readied for rapid d i v i s i o n , presumably by a l lowing accumulation of other d i v i s i o n prote ins which had enough of a potent ia l to a l low expression of d i v i s i o n even in the absence of any prote in syn thes is . b. DNA syn thes is . From the c e l l d i v i s i o n data during recovery, i t was concluded that prote in synthesis at kl C was not needed for d i v i s i o n 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 s h i f t from kl to 30 C. It was important to c o r r e l a t e the e f f e c t s of i n h i -b i t i o n of the prote in synthesis needed for new rounds of DNA r e p l i -ca t ion to that of recovery from the growth under non-permissive c o n d i t i o n s . ol 1 I I 1 i I i 0 20 40 60 80 PULSE (min.) F i g u r e 21. Changes i n t h e r a t e o f c e l l d t v t s i o n d u r i n g r e c o v e r y from a kl C b l o c k i n t h e p r e s e n c e o f c h l o r a m p h e n i c o l . The r a t e o f c e l l d i v i s i o n (dy/dx) was c a l c u l a t e d d u r i n g the r a p i d d i v i s i o n phase o f r e c o v e r y i n the p r e s e n c e (V) and absence (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 , grown a t kl C, were s h i f t e d t o 30 C f o r r e c o v e r y . To one h a l f o f t h e c u l t u r e 150 yg per ml CAM was added a t t h e s h i f t down ti 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 fac t that the mutant was de fec t i ve in fur ther i n i t i a t i o n of DNA c y c l e s at hi C implied that b locking prote in synthesis at hi C should resu l t in no net DNA synthesis at recovery. However, net synthesis subsequent to add i t ion of CAM would have implied a re -c y c l i n g of the o ld i n i t i a t e d c y c l e . The r e s u l t s of such experiments are shown in Figure 22. No new DNA synthesis was observed for 60 minutes a f t e r s h i f t down to 30 C. Meanwhile, the contro l c u l t u r e showed resumption of new synthesis in approximately 8 minutes a f t e r s h i f t down. There was no r e l a t i o n s h i p between the durat ion of continued prote in synthesis at hi C and the amount of residual DNA synthes ized . Thus, un l ike the d i v i s i o n at recovery, the i n i t i a t i o n of new rounds of r e p l i c a t i o n needed new prote in synthesis at the recovery time as indicated by the contro l c e l l s . The r e l a t i o n s h i p between the ro le of prote in synthesis at the 30 C recovery period and of the i n i t i a t i o n of new rounds was examined forupulses of 10 and 30 minutes at hi C. Results are shown in Figure 23. C e l l s recovering from 10 minutes of incubation at hi C required more than 5 minutes of prote in synthesis for resumption of DNA s y n t h e s i s , s ince add i t ion of chloramphenicol at 0 to 5 minutes subsequent to the s h i f t down resul ted in a 0 to 5 increase in res idual DNA synthesis over 90 minutes at 30 C. During th is t ime, the contro l c e l l s increased t h e i r l eve ls of DNA synthesis to over 100%. S i m i l a r l y , i f the c e l l s were returned to 30 C from a 30 minute block at hi C, the same ru le held t rue . Not iceable l eve ls of new r e p l i c a t i o n took place only when UJ O 'o r X ^ 5 i M l ' i I 1 I i / I ' +CAM V 42< 0 l 3 0 ° X l I i - 4 5 6 0 MINUTES Figure 22. The e f f e c t of i n h i b i t i o n of p ro te in synthes is at 42 C on the DNA r e p l i c a t i o n at recovery . T83 grown in the presence of 0.5 yc per ml H-TdR, 20 yg per ml co ld TdR, and 50 yg per ml AdR, was s h i f t e d to 42 C for kS minutes. To subcul tures at 42 C, CAM (150 yg per ml) was added at the times ind icated by the v e r t i c a l arrows. A f t e r 45 minutes, the c e l l s were returned to 30 C, and allowed to recover . The CPM of 3n-TdR incorporated for the cont ro l (X) and the experimental c u l t u r e s at 5 (0), 25 (V) , and 35 ( • ) minutes with CAM at 42 C are p lo t ted as a f unc t ion of t ime. 1 I 1 I 1 I 1 I 1 + C A M / MINUTES Figure 23. Role of i n h i b i t i o n of prote in synthes is on i n i t i a t i o n of new rounds dur ing recovery . T83 was grown in the presence of H-thymidine (0.5 uc per ml) 20 yg per ml co ld thymidine, and 50 yg per ml deoxyadenosine, at 30 C. The c e l l s were s h i f t e d to hi C and were kept there (upper pane l ) . Ten minutes l a t e r , a por t ion of these c e l l s was t rans fer red back to 30 C ( • ) , and, 5 minutes a f t e r s h i f t down, CAM (150 yg per ml) was added to ha l f of the 30 C c e l l s ( • ) . A f t e r 30 minutes, a part was returned to 30 C (lower pane l ) . F ive (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 incorpora t ion of thymidine (^H-methyl-thymidine, 0.5 yc per ml and 20 yg per ml co ld TdR) into the TCA inso lub le f r a c t i o n was fo l lowed . CPM of r a d i o a c t i v i t y is p lo t ted against t ime. approximately 10 minutes of prote in synthesis was a l lowed. The nature of DNA made subsequent ot 5 minutes of prote in s y n t h e s i s , was determined by looking at the rate of r e p l i c a t i o n . If, during the 5 minutes, new i n i t i a t o r molecules were inaugurated, the rate of DNA r e p l i c a t i o n should have shown an increase. When the rate of the DNA r e p l i c a t i o n was determined in a se r ies of 3 minute 14 pulses using C-thymidine, a burst in the rate of r e p l i c a t i o n was not iced approximately 18 minutes a f t e r s h i f t down. Presumably, th is burst had resul ted from the prote in synthesis that had taken place at recovery s ince blocking prote in synthesis at 42 C immediately p r i o r to a s h i f t down did not produce a burst in DNA s y n t h e s i s . It was concluded that the prote in synthesized at recovery was necessary for the i n i t i a t i o n of new rounds of DNA r e p l i c a t i o n (Figure 24). D.. Attempts to uncouple 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  at 30 C. To test the p o s s i b i l i t y of uncoupling the DNA r e p l i c a t i o n from c e l l d i v i s i o n , three experiments were c a r r i e d out . 1. Inh ib i t ion of i n i t i a t i o n of new rounds by phenethyl  a l c o h o l . Phenethyl a lcohol (PEA), at a 0.25% l e v e l , has been shown to al low res idual DNA synthesis in E_. col i (Berrah and Konetzka, 1962) by i n t e r f e r r i n g with the i n i t i a t i o n of new c y c l e s of r e p l i c a t i o n (Treich and Konetzka, 1964). Results from several l abora tor ies i n d i -cated that PEA had mul t ip le immediate e f f e c t s on the c e l l such as on z Q >• x i— IJ CN I O 20 40 MINUTES 80 F igure 24. The e f f e c t of chloramphenicol on the rate of DNA synthes is dur ing the recovery p e r i o d . An exponential c u l t u r e of T83 growing at 30 C ( t ) was s h i f t e d to 42 C (0) . A part of the c u l t u r e was returned to 30 C (9) and, a f t e r 5 minutes at 30 C, CAM (150 yg per ml) was added to a por t ion of the recover ing c e l l s (0) . To determine the rate of DNA s y n t h e s i s , c e l l s were pulsed f o r 3 minutes with ^ C - T d R at 20 yg per ml in the presence of 50 yg per ml AdR. CPM of 1 Z f C-TdR incorporated are p lo t ted as a funct ion of t ime. the a c t i v i t y of c e l l membrane (S i l ve r and Wendt, 1967), on transport react ions and nucleot ide pools (Plagemann, 1970), and on r e s p i r a t i o n and o x i d a t i v e phosphorylat ion (Cosgrove and T r e i c h , 1970). Studies by Lark and Lark (1966) confirmed the r e s u l t s of T re ich and Konetzka (1964) and the i r r e s u l t s ascer ta ined tha t , in E_. col ?, PEA blocked the DNA r e p l i c a t i o n at the same p o s i t i o n on the chromosome as amino ac id s t a r v a t i o n . When 0.25% PEA was added to exponent ia l ly growing cu l tu res of T 8 3 at 30 C in A and E medium, approximately 55% res idual DNA synthesis occurred (Figure 25A) . This f i g u r e matches n i c e l y with the residual DNA synthesized by kl C grown c e l l s . Furthermore, the time needed for the completion of the res idual DNA synthesis was kO to 50 minutes, a f t e r which time no fur ther increase was observed. Based on th is ev idence, the temperature e f f e c t and PEA had s ingu lar ac t ion in a l low-ing completion of s tar ted rounds. Examination of c e l l d i v i s i o n data subsequent to add i t ion of 0.25% PEA revealed res idual d i v i s i o n . As shown in Figure 25 B, during 25 minutes a f te r add i t ion of PEA, c e l l u l a r d i v i s i o n continued at the normal ra te . The amount of res idual d i v i s i o n ca lcu la ted was approx i -mately 50%. Thus, i t was poss ib le to uncouple the two processes , 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 , under permissive temperature c o n d i -t ions by blocking one process , s ince in the absence of any new i n i t i a t i o n , the c e l l s could express only res idual d i v i s i o n . The amount of res idual d i v i s i o n observed was increased by an add i t iona l 10% i f NaCl (0.65% CO I o U c E O u u LU > -20 0 20 40 MINUTES Figure 2 5 . Uncoupling of c e l l divi-jiuii from DNA r e p l i c a t i o n by phenethyl a l c o h o l . T 8 3 , growing at 3 0 C for several generat ions in the presence of 2 0 yg per ml of co ld and 0 . 5 yc per ml ^H-thymidine and 50 yg per ml deoxy-adenosine , was treated with phenethyl a lcohol 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 rad ioac t ive TdR ( t r i ang les ) and c e l l counts (squares) were fol lowed for cont ro l (empty symbols) and PEA treated c e l l s ( f u l l symbols) . The c e l l dens i ty at the time of PEA treatment was 4 x 1 0 ' c e l l s per ml . 83 f i n a l concentrat ion) was added simultaneously with PEA. However, add i t ion of s a l t had no st imulatory e f fec t on the res idual DNA r e p l i c a t i o n . 2. Uncoupling 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  Daunomyci n. Daunomycin (DM), an a n t i b i o t i c in the an thracyc l ine group iso la ted from a c u l t u r e of Streptomyces peucet ius , was shown to i n h i b i t both DNA-depehdent RNA-polymerase and DNA polymerase in E_. col i in v i t r o (Hartman et a l . 1964). The in v ivo enzymes associa ted with DNA syn-thes is (thymidine k inase , deoxycyt id ine monophosphate deaminase, and DNA polymerase) in HeLa c e l l s were not a f fec ted by DM (Kim £t_ aj_. 1968) . The i n h i b i t o r y ac t ion of th is a n t i b i o t i c on DNA r e p l i c a t i o n l i e s in i t s a b i l i t y to bind to the DNA template (Calendi et a 1. 1965; Kerste in et a 1. 1966) and to i n t e r f e r e with the a v a i l a b i l i t y of the template to the r e p l i c a t i n g enzymes. The p o s s i b i 1 i t y of using DM for the uncoupling of 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 T83 was explored. DM, at 20 yg per ml , was found i n h i b i t o r y to DNA synthesis in E_. col i B/r/1 . RNA synthesis cont inued, however for 30 minutes at the normal ra te . When DM was added"to an exponent ia l ly growing c u l t u r e of T83 at 30 C, c e l l d i v i s i o n stopped a f t e r 40 minutes. The amount of residual d i v i s i o n obtained a f te r add i t ion was 25 percent as shown in Figure 26B. There was a gradual d e c l i n e in the rate of DNA r e p l i c a t i o n . at 30 C in the presence of DM. In the f i r s t 10 minutes of incubat ion , 14 there was a 50 percent reduct ion in the rate of C-TdR incorporat ion into the DNA. The rate of DNA synthesis was promptly reduced to 17 84 Figure 26. Uncoupling of c e l l d i v i s i o n and DNA synthesis by Daunomycin. Daunomycin (DM) was added to part of a c u l t u r e of CR34T83 growing at 30 C, at a f i n a l concentrat ion of 20 yg per ml . Growth of the c u l t u r e , with (0) and without (•) DM is p lot ted as a funct ion of time in panel B. The rate of DNA synthesis was measured by the 14 incorporat ion of C-thymidine at a f i n a l concentrat ion of 20 yg per ml and a s p e c i f i c a c t i v i t y of 0.61 yc per yM into TCA insolub le m a t e r i a l . The rate of DNA synthesis per c e l l for the experimental c u l t u r e with (0) and without ( t ) DM is p lot ted as a funct ion of time in panel A. percent of the contro l wi thin an hour a f t e r drug treatment. The r e s u l t s of t h i s experiment are shown in Figure 26A. In terest ing ly enough, the k i n e t i c s of DM i n h i b i t i o n resembled that of the kl C e f f e c t in T83. This study showed tha t , by i n h i b i t i o n of polymer izat ion of DNA (and RNA) in T83, one could observe residual d i v i s i o n at 30 C. For the durat ion of DM treatment, 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 by  NaT i d i x i c a c i d . The coupl ing between 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 was examined in another experiment, during which coord inat ion of the two events was tested in r e l a t i o n to a temperature block imposed between the N a l i d i x i c ac id e f f e c t . T83 c e l l s grown at 30 C were treated with N a l i d i x i c ac id for 20, 15 and 10 minutes before being put at kl C. Ten minutes a f t e r incubation at kl C, the c e l l s were returned to 30 C and allowed to recover from the temperature b lock . If N a l i d i x i c ac id i n t e r f e r e d , in conjunct ion with temperature, with the c e l l d i v i s i o n by any means, then the c e l l s should not have d iv ided to the i r committed 30% l e v e l . The r e s u l t s show that indeed, t h i s was not the case as is shown in Figure 27. Cul ture A , that had been treated with N a l i d i x i c ac id for 20 minutes p r io r to s h i f t up, by expressing i ts 30 percent res idual d i v i s i o n , showed no net increase in the c e l l number a f te r s h i f t down. Cul ture B could express only 27 percent before stopping d i v i s i o n M I N U T E S Figure 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 DNA synthes is and c e l l d i v i s i o n during temperature s h i f t s in T83. To subsamples of T83 grown at 30 C , N a l i d i x i c ac id 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 returned to 30 C and c e l l d i v i s i o n was assayed. Recovery of the cont ro l without a d d i t i o n of N a l i d i x i c ac id is represented by X. 6ue to the kl C c o n d i t i o n . Cul ture C expressed 20 percent of 30 C before s h i f t and another 12 percent at recovery, and f i n a l l y , cu l tu re D expressed a l l of i t s d i v i s i o n a f te r being returned to 30 C. The obvious in te rpre ta t ion of these r e s u l t s was that the tempera-ture s e n s i t i v e cond i t ion that a f fec ted the c e l l u l a r d i v i s i o n at kl C did not o b l i t e r a t e the potent ia l of those c e l l s that could d i v i d e . Furthermore, those c e l l s that had the necessary cond i t ion for c e l l u l a r d i v i s i o n would have d iv ided s u c c e s s f u l l y to the level expected, and the i r r e c a l l of the d i v i s i o n owed to be expressed at recovery was not reduced. This d i v i s i o n , however, needed prote in synthesis s i n c e , i f chloramphenicol was added at the same time with N a l i d i x i c a c i d , there was no d i v i s i o n . III. Control of DNA synthesis in T83• A. I d e n t i f i c a t i o n of the place of resumption of DNA synthesis  at recovery. In E_. col i the fac t that rounds of chromosome r e p l i c a t i o n s t a r t at a f ixed point on the genome c a l l e d the " r e p l i c a t i o n o r i g i n " is now well es tab l ished (Lark et a 1. 1963; Abe and Timizawa, 1967; Caro and Berg, 1968; Wolf et_al_. 1968; Helmstet ter , 1968a), and is located between the arg G and xy lose l o c i . Rep l i ca t ion s t a r t s from here and continues sequent ia l l y in a c lockwise d i r e c t i o n . Experiments were designed to determine the place of the resumption of rounds of r e p l i c a t i o n in T83 at recovery. (a) If T83 was tempera-bb Figure 2 8 . Construct ion of the model for DNA r e p l i c a t i o n in C R 3 4 T 8 3 . The const ruc t ion is based on the Helmstetter and Cooper Model for DNA r e p l i c a t i o n for c e l l s grown under minimal and broth c o n d i t i o n s . Time (t) needed for a growing point to t raverse the genome is shown on the l e f t and is expressed as a funct ion of chromosome length. Open c i r c l e s represent f i r s t forks and f i 1 led c i r c l e s represent most recent ly es tab l ished forks fo r broth medium. T r iang les represent attachment point for DNA to the membrane (dots) . The sequence begins with random populat ion of chromosomes at var ious stages of r e p l i c a t i o n at 3 0 C. Upon a s h i f t to kl C, they w i l l complete the i n i t i a t e d rounds. Three poss ib le segregation patterns predicted for broth grown c e i l s are shown. F i n a l l y , during the recovery at a kl to 3 0 C s h i f t down, new i n i t i a t i o n s are commenced in a synchronous f a s h i o n . ture s e n s i t i v e fo r the i n i t i a t i o n of DNA r e p l i c a t i o n , at recovery , the place of r e i n i t i a t i o n would be at a s p e c i f i c s i t e on the chromosome and (b) i f the temperature s e n s i t i v e step was blocking the i n i t i a t i o n at the same place as the 150 ug per ml CAM s tep , then recovery from a kl C pulse fol lowed by a CAM block should iden t i f y the o r i g i n . The p red ic t ion of a s h i f t up on an exponential populat ion from 30 to kl C is shown in Figure 28. Chromosomes a f t e r kS minutes, based on res idual DNA synthesis da ta , a l i g n themselves and, at recovery, new rounds are i n i t i a t e d . If, at the time of a s h i f t from 30 to kl C , H-TdR was added, the completion of rounds of r e p l i c a t i o n would be l abe l l ed (Figure 29A). During the recovery , one could add a d i f f e r e n t \k l a b e l , for example C-TdR, for a short t ime, withdraw the l a b e l , and chase with cold TdR (Figure 2 9 B ) . Thus, the s ta r ts of rounds would be l a b e l l e d . If c e l l s were grown exponent ia l ly in unlabel led TdR for several generations and randomized, there would be a f i x e d , number of c e l l s whose chromosomes were l abe l l ed at s t a r t s and ends (Figure 29D). In order to iden t i fy the unique o r i g i n of recovery in DNA c y c l e s , the c e l l s could be real igned by a new temperature block {kl C) and, at recovery , be given a densi ty l a b e l , for example 5 - BrdUrd for a short time (Figure 29E). The resu l ts of dens i ty c e n t r i f u g a t i o n of such DNA would y i e l d a l i g h t (1.70 gm per cc) DNA and hybrid BrUra-T (1.76 gm per cc) DNA. If the place of the r e i n i t i a t i o n a f t e r the second kl C pulse was at the same region of the chromosome as the f i r s t one, CHROMOSOME KEY SYMBOLS NORMAL D N A 3H-THYM1DINE | 4C-THYMIDINE 5 - BROMODEOXYURIDINE HYBRID LIGHT 1.76 1.70 ••• If II fl * ii ii II / ; then the only label associa ted with the hybrid densi ty mater ia l 1 4 would be the label used at the o r i g i n ( C-TdR). S i m i l a r l y , i f the c e l l s were exposed to 150 ug per ml CAM at 30 C a f t e r the randomi-zat ion from the f i r s t 42 C p u l s e , again they should a l i g n the i r chromosomes. If CAM was removed and c e l l s were resuspended in f resh medium with 5 - BrdUrd to al low new cyc les to r e i n i t i a t e , the densi ty Ik label should be associa ted with the C-TdR l a b e l . The rate of DNA synthesis decreases when BrUra is subst i tu ted for T. Approximately 120 minutes is required for a round of r e p l i c a t i o n in B/r/1 at 37 C ( P i e r u c c i , 1969)- As detected by the appearance of 14 1.80 densi ty l abe l l ed C-5-BrUra , a round of r e p l i c a t i o n takes approx i -mately 240 minutes at 30 C for E_. col i 15 TAU or T83. For the e x p e r i -ments descr ibed above, a 50 minute BrdUrd pulse was used for the densi ty l a b e l l i n g of the s t a r t s . Thus 20 percent of the DNA would be the maximum dens i ty l abe l led reg ion . The r e s u l t s from CsCl densi ty gradient c e n t r i f u g a t i o n of DNA r e p l i c a t e d during recovery from a 42 C pulse or growth under 150 yg per ml CAM at 30 C are presented in Figures 30 and 31, r e s p e c t i v e l y . The densi ty gradient p r o f i l e s have two main bands of hybrid and l igh t densi ty corresponding to 1.76 and 1.70 gm per cc buoyant d e n s i t i e s . The c h a r a c t e r i s t i c s of the l abe l l ed DNA found under these bands were analysed and the r e s u l t s are given in Table II. In e i ther c a s e , i t 14 was c l e a r that 5 - BrUra became p r e f e r e n t i a l l y associated with C-labe l l ed DNA and not 3 H - l a b e l l e d DNA. The bulk of the ^H- labe l led FRACTION NUMBER Figure 2 9 . Protocol for the experiments in Figures 30 and 31. TREATMENT PURPOSE A. kl C + 3H-TdR Label chromosome completions B. 30 C - H-TdR \k + C-TdR Label chromosome o r i g i n s 14 30 C - C-TdR + cold TdR Repl ica te chromosomes (randomizat D. 1) 30 C + 150 yg per ml CAM + cold TdR or 2) kl C - CAM + cold TdR Complete chromosomes E. 1) 30 C - CAM -TdR + 5-BrdUrd 2) 30 C - TdR + 5-BrdUrd R e i n i t i a t e r e p l i c a t i o n TEST REPLICATION OF 3 H - OR 1 \ - D N A BY INCORPORATION OF 5"BR0M0-2-DE0XYURIDINE I I I 1 1 1 I 1 I 5 10 15 20 25 30 35 40 45 FRACTION NUMBER F igure 31. CaCl gradient a n a l y s i s of the p lace of the r e i n i t i a t i o n at recovery from growth in the presence of CAM. The d e t a i l e d experimental procedure is descr ibed under Mate r ia ls and Methods, and in the tex t . The a b s c i s s a represents the f r a c t i o n s c o l l e c t e d . The ord ina te is CPM per f r a c t i o n represented as the percent of the t o t a l r a d i o a c t i v i t y (10,420 CPM 1 Z f C - and 6, 140 CPM 3rl) fo r 1 / » C - ( i ) and 3H- (O) thymine. X X represents buoyant dens i t y . Figure 30. CsCl gradient a n a l y s i s of the place of the r e i n i t i a t i o n at recovery from growth at kl C. The d e t a i l e d experimental procedure is descr ibed under Mater ia ls and Methods, and in the text . DNA was labe l l ed with H-TdR (0.0^5 yc per.ml) with 2 yg per ml unlabel led TdR and 50 yg per ml AdR, during res idual r e p l i c a t i o n at kl C. A f t e r 60 minutes, H-TdR was removed. C e l l s were returned to 30 C and given ^ C - T d R (0.5 yc per ml) in the presence of 2 yg per ml unlabel led TdR and 50 yg per ml AdR, for lk \k minutes. C-TdR was removed and c e l l s were grown for 5 generations in cold TdR medium, fo r randomizat ion. C e l l s were then pulsed with a second kl C incubation for 60 minutes. Subsequently, c e l l s were f i l t e r e d f ree of TdR and resuspended in f resh medium conta in ing 10 yg per ml BrdUrd, fo r 50 minutes. DNA was extracted and run for 41 hours at 35,000 rpm in a CsCl densi ty grad ient . The absc issa represents f r a c t i o n s c o l l e c t e d . The ord inate is CPM per f r a c t i o n represented as a percent of the to ta l r a d i o a c t i v i t y (9,800 CPM 1 2 * C - and 5,900 CPM 3 1 k 3 H-thymine) for C- (•) and H- (0) thymine. X X represented buoyant d e n s i t y . Table II. C h a r a c t e r i s t i c s of l abe l led DNA in T83: Buoyant d e n s i t i e s and percentage of tota l DNA. Condit ions for re -i n i t i a t i o n fo l lowing chromosome alignment at 42 C DNA labe l led as •'H or C % to ta 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 densi ty band Hybrid Light A. Recovery from hi C. H-thymi ne C-thymine 76.66 (100%) 67.45 (100%) 6.08 (8.0%) 12.60 (18.6%) 70.58 (92.0%) 54.85 (81.4%) B. Recovery from H-thymine CAM at 30 C. C-thymine 67-95 5-05 62.90 (100%) (7.5%) (92-5%) 71.43 17.43 54.00 (100%) (24.4%) (75.6%) Numbers represent percent of to ta l CPM Hybrid band = f r a c t i o n s between 1.780 Light band = f r a c t i o n s between 1.7344 per f r a c t i o n in the densi ty band, and 1.7344 d e n s i t y , peak at 1.760 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 l igh t densi ty reg ion . It must be borne in mind tha t , during 5-BrdUrd l a b e l l i n g of the DNA only 50_^  minutes worth, or approximately 20 percent of the o r i g i n were densi ty 14 l a b e l l e d . The C - l a b e l l i n g of the i n i t i a t i o n region was approximately 20_minutes, or 33 percent , of the DNA. Thus 20 percent of the to ta l 60 - l a b e l would be present in the hybrid dens i ty band. It was evident that 17.5 percent from the kl C i n i t i a t i o n , and 12.6 percent from the CAM i n i t i a t i o n banded in th is reg ion . It was concluded that at recovery from kl C, T83 r e i n i t i a t e d new rounds of chromosome r e p l i c a t i o n at a s p e c i f i c region of the DNA. Furthermore, the place of the r e i n i t i a t i o n subsequent to completion of rounds from 150 yg per ml CAM (Lark et a l . 1963) was. the same as that of the temperature s e n s i t i v e s tep . B. Comparison of the e f f e c t of i n h i b i t i o n of prote in synthesis  and the T83 mutat ion, on the i n i t i a t i o n of DNA r e p l i c a t i o n . Studies on DNA r e p l i c a t i o n under condi t ions in which gross prote in synthesis is inh ib i ted has indicated that i n i t i a t o r prote ins are present in a c e l l in s t o i c h i o m e t r i c amounts, and add i t iona l prote in synthesis is needed for normal r e i n i t i a t i o n of new rounds (Lark, 1969b). The r e s u l t s presented in the previous sect ion pointed out that the temperature s e n s i t i v e mutation a f fec ted the proper t ies of a p r o t e i n , the requirements of which were evidenced by kl C incubat ion . Since CAM dupl icated the e f f e c t of the kl C, i t was concluded that the temperature s e n s i t i v e mutation involved i n i t i a t o r p r o t e i n ( s ) . Thus, i t was d e s i r a b l e to examine the separate e f f e c t s of high and low leve ls of CAM on the process of r e p l i c a t i o n in T83 and compare that to the 42 C e f f e c t . •3 The incorporat ion of H-TdR into DNA at 30 C and.kl C in the presence or absence of CAM was examined and the resu l ts are presented in F igure 32. C e l l s at 30 C treated with 150 ug per ml CAM continued DNA r e p l i c a t i o n at 4/10 the rate of the 30 C (Figure 32A). The rate i s l i nea r for kO minutes a f t e r add i t ion of CAM. When compared with the kl C e f f e c t (Figure 32B), the rate of 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, and the plateau was reached in approximately 50 minutes. However, the amount of DNA rep l ica ted was kO to 50 percent of the residual l e v e l , and was compar-able to the r e s u l t s of Maal^e and Hanawa 11 (1961). In the presence of CAM, th is level was s l i g h t l y l e s s . When T83 was treated with 25 ug per ml CAM at 30 C, DNA r e p l i c a t i o n continued for at least two hours but at about 25 percent of the rate observed for the contro l c e l l s . A plateau was reached by 110 minutes post treatment, which was equal to a kl percent increase in the res idual DNA synthesis (Figure 33). When the c e l l s were sh i f ted to kl C and treated with CAM, DNA r e p l i c a t i o n came to a hal t a f te r 20 minutes. Th is plateau lasted for approximately 50 minutes, a f te r which there was a second burst in residual synthesis amounting to .19 percent . F ina l plateau value reached at kl C thus added up to 46 percent residual DNA s y n t h e s i s . M I N U T E S Figure 32. 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. Uptake of H-methyl-TdR at 1 yc per m l , 20 yg per ml unlabel led TdR, and 50 TJg per ml AdR, into the co ld TCA inso lub le f r a c t i o n in T83, in the presence of 150 yg per ml CAM ( f i l l e d symbols) and absence of CAM (empty symbols) , at 30 C is shown in panel A and at kl C in panel Figure 33. E f f e c t of a low leve 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 of T83 grown In A and E medium In the presence of 0.5 yc per ml of ^H-TdR, 20 yg per ml co ld TdR, and 50 yg per ml AdR, was d iv ided three ways. CAM (25 yg per ml) was added to subcul tures at 30 C (0) and hi C (•) . Uptake of l a b e l l e d TdR into co ld TCA p r e c i p l t a b l e mater ia l was fol lowed in the presence of CAM fo r 30 C (0) and hi C (•)• The diagonal s o l i d l i n e represents the 30 C cont ro l without CAM. CPM are p lo t ted versus t ime. 99 C. Studies on s t a b i l i t y of the growing point and the r e p l i c a t i o n  complex at the non-permissive c o n d i t i o n . In E_. co 1 i , chromosome r e p l i c a t i o n could be inh ib i ted by thymine s ta rva t ion (Pr i tchard and Lark , 1964) or N a l i d i x i c ac id (Goss et a l . 1965). In the case of thymine s t a r v a t i o n , add i t ion of thymine caused c e l l s to induce new rounds of r e p l i c a t i o n (premature i n i t i a t i o n s ) . Pr i tchard and Lark (1964) provided evidence that prev ious ly i n i t i a t e d chromosomes continued r e p l i c a t i o n to complet ion. Premature i n i t i a t i o n s were a l s o induced fo l lowing reversal of N a l i d i x i c ac id - induced i n h i b i t i o n of chromosome r e p l i c a t i o n (Ward et a 1. 1970; Pr i tchard et a l . 1969)- It has a l s o been observed tha t , when a pulse of N a l i d i x i c ac id was appl ied during conjugat ion in E_. col i , chromosome t ransfer stopped (Hoi lorn and P r i t c h a r d , 1965; Barbour, 1967). As to the e f f e c t of N a l i d i x i c ac id on t r a n s f e r , two conclus ions have been reached which disagree with one another. The r e s u l t s of Pr i tchard have been interpreted as a r e v e r s i b l e i n h i b i t i o n of t r a n s f e r , whereas r e s u l t s and conclus ions of Bouck and Adelberg (1970) and Hane (1971) indicated commencement of a new round of t ransfer from the o r i g i n . The Ward and Glaser (1970) observat ions were s i m i l a r to those of bac te r ia l mating (Hane, 1971; Bouck and Adelberg , 1970), s ince they concluded that t the r e p l i c a t i n g fork was destroyed by treatment df E_. col i with N a l i d i x i c ac id for 30 minutes. The Ward and Glaser model p red ic ts tha t , when N a l i d i x i c ac id is added to a c u l t u r e of T83 at 42 C for s u f f i c i e n t time to a l l d W ' complete i n a c t i v a t i o n of the f o r k , one should not observe any res idual DNA synthesis subsequent to i t s removal. The resu l t of such experiments are shown in Figure 3^. NAL, when added, e f f e c t i v e l y blocked DNA r e p l i c a t i o n . However, upon removal, the residual r e p l i -ca t ion went to the same satura t ion level as the c o n t r o l s . C e l l s with NAL added at times s h o r t l y a f t e r s h i f t up y ie lded much more residual DNA than those treated a f t e r longer incubation at kl C. For example, 60% res idual r e p l i c a t i o n resul ted for c e l l s treated 5 minutes a f te r s h i f t up as compared to 10 to lk% for c e l l s treated a f t e r kS minutes at the non-permissive temperature. This indicated that c e l l s held at kl C approached completion of the i r rounds with time which resul ted in very l i t t l e residual d i v i s i o n at the s h i f t down. The p o s s i b i l i t y that T83 r e i n i t i a t e d prematurely subsequent to the removal of NAL has a l s o been examined. C e l l s were incubated at kl C in the presence of NAL and CAM to prevent i n i t i a t o r and general prote in s y n t h e s i s . Subsequently, a f t e r 30 minutes, c e l l s were released into f resh medium lacking NAL but with or without CAM. The f i n a l res idual DNA synthesis level was the same in e i ther case . In B_. subti 1 is s t r a i n s , a degra-dat ion of 20 to 30 percent of the c e l l DNA in the r e p l i c a t i n g point region occured when c e l l s were treated for 3-5 hours with NAL (Ramareddy and R e i t e r , 1969, 1970). In these experiments, however, not more than 35 minutes incubation was used and NAL at the level used, no detectable degradation of DNA was observed (Cook et_ aj_. 1966). Thus 3 res idual DNA synthesis in a to ta l uptake of H-TdR into p re labe l led DNA a. CL MINUTES Figure 34. DNA sy n t h e s i s in T83 at 42 C f o l l o w i n g an exposure to Na1 i d i x i c a c i d . A c u l t u r e of T83 grown at 30 C in A and E medium w i t h 5 uc per ml 3H -TdR, 20 yg per ml unlabel led thymidine ( f i n a l c o n c e n t r a t i o n ) , and 50 yg per ml AdR, were s h i f t e d to kl C at zero time ( • ) . At 5 (• ) , 10 ( f ) , 25 ( 0 ) , 35 ( 0 ) , and kS (•) minutes, as i n d i c a t e d by v e r t i c a l arrows, p o r t i o n s from the c o n t r o l f l a s k were removed and the c e l l s were pulsed in NAL (10 yg per ml). A f t e r 30 minutes (dotted l i n e ) they were washed f r e e of NAL w i t h kl C prewarmed medium and immediately resuspended i n f r e s h NAL-free medium. Residual i n c o r p o r a t i o n of r a d i o a c t i v e l a b e l i n t o the TCA i n s o l u b l e f r a c t i o n was followed during incubation at kl C, and i s represented as the percent increase 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) represent r e s u l t s from a s i m i l a r experiment, with a 5 minute pulse i n NAL 10 minutes a f t e r the s h i f t to kl C. could not be due to turnover of degraded l a b e l . The Ward and Glaser model pred ic ts that the i n i t i a t o r mutant should undergo 100 percent res idual DNA synthesis when treated with NAL for 30 minutes at 30 C and then sh i f t ed to kl C without NAL. In such an event , T83 would be a l igned for only one round of DNA r e p l i -ca t ion under the r e s t r i c t i v e c o n d i t i o n s . The r e s u l t s from such experiments are shown in Figure 35. C l e a r l y , the Ward and Glaser model is in e r r o r , s ince the residual DNA synthesized remains at a level compatible with the notion of resumption of temprar i ly blocked c y c l e s at kl C and which approximates the c o n t r o l . IV. Synthesis and decay of the d i v i s i o n potent ia l in CR34T83 studied by in v ivo k i n e t i c s . A. Time course of appearance of the d i v i s i o n p o t e n t i a l . A cdoser examination of r e s u l t s from phys io log ica l requirements fo r recovery showed tha t , for c e l l s kept at kl C , there ex is ted a potent ia l for d i v i s i o n which accumulated co inc ident with the rounds of DNA completed. It was assumed that the expression of d i v i s i o n potent ia l a lready a v a i l a b l e at recovery , when net prote in synthesis was i n h i b i t e d , must be proport ional to the amount of completed DNA c y c l e s , c e l l u l a r growth, and other ru les 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. Ex-periments were set to measure the amounts and k i n e t i c s of decay and synthesis of t h i s d i v i s i o n p o t e n t i a l . In connection with th is ques t ion , IS 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 MINUTES Figure 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 ac id treatment at 30 C. A c u l t u r e of T83, grown at 30 C in the presence of 0.8 yc per ml ^H-TdR, 20 yg per ml unlabel led TdR, and 50 yg per ml AdR, was s p l i t in to three p a r t s . At zero t ime, one por t ion (•) was t reated with NAL f o r 30 minutes at 30 C , washed f ree of NAL, and s h i f t e d to kl C. A second por t ion (0) was t reated with NAL at 5 minutes (25 minutes at 30 C ) , s h i f t e d to kl C, and l e f t in NAL for 5 minutes at kl C. The NAL was removed and the incubat ion at kl C was cont inued. A t h i r d 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 others but was not t reated with NAL. Residual i n -c o r p o r a t i o n of r a d i o a c t i v e label into the cold TCA inso lub le f r a c t i o n was fol lowed dur ing incubat ion at kl C and is represented as the percent increase in r a d i o a c t i v i t y (CPM). experiments were designed to determine: (a)whether the synthesis of the d i v i s i o n potent ia l was a continuous or discont inuous one; (b) the rate of the appearance and decay of the d i v i s i o n potent ia l and regula t ion of i t s synthesis during exponential growth at 30 C, at the non-permissive temperature (kl C ) , and during recovery from a kl C pu lse ; (c) the measurement of the d i v i s i o n potent ia l messenger decay by the study of the k i n e t i c s of t r a n s c r i p t i o n using Ri fampicin i n h i b i t i o n of RNA s y n t h e s i s ; (d) the comparison of the two e f f e c t s ; and (e) the measurement of the surv iva l of mRNA in the temperature s e n s i t i v e c o n d i t i o n . These questions could be studied by means of pulses at kl C in concert with a number of i n h i b i t o r s ac t ing on w e l l -def ined steps of the process of gene express ion . Thus, not only could one measure the time needed for the e longat ion process of mRNA, but a lso the formation of p o l y p e p t i d e chains and the hal f l i f e of the coding capac i ty of the preformed mRNA. B. The wave of expression of c e l l d i v i s i o n at recovery in the  absence of t r a n s l a t i o n or t n a n s c r i p t i o n . Figure 36 descr ibes the r e s u l t s from experiments where the net prote in synthesis of a cu l tu re of T83, pulsed for 15 to 60 minutes at kl C , was inh ib i ted at s h i f t up and recovery. If the appearance of d i v i s i o n potent ia l was proport ional to the length of the kl C b lock , then one should see a gradual b u i l d - u p . To have an accurate estimate of the time course of the appearance of the d i v i s i o n potent ia l before s h i f t - u p , each cu l tu re was d iv ided into 30 subcultures which were treated at one minute in te rva ls at kl C , for 15 minutes before and a f t e r s h i f t down, with CAM at 150 yg per ml . During the next 200 minutes at 30 C, the c e l l s went through some res idual d i v i s i o n , proport ional to the potent ia l they had for such d i v i s i o n , and came to a h a l t . The plateau values for each c u l t u r e were then p lot ted against the time when CAM was added. It must be remembered tha t , during the CAM i n -h i b i t i o n of prote in s y n t h e s i s , the c e l l s could express d i v i s i o n only i f they had the necessary prote ins and other substrates for such an event. Obviously at kl C , as longer in te rva ls of prote in s y n t h e s i s , co inc ident with the res idual DNA s y n t h e s i s , was a l lowed, greater numbers of c e l l equiva lents were generated. In f a c t , such was the case s ince the place where c e l l numbers intercept the temperature s h i f t l i n e s c l o s e l y fol lowed the generat ion time of the c e l l . However, a f t e r k5 minutes of incubat ion , despi te the increase in c e l l mass, no more increase in c e l l numbers occured. C l e a r l y , there must have been a s t r ingent coupl ing between 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 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 represented in Figure 36 shows c l e a r l y that v i r t u a l l y in every case , upon incubation of c e l l s at kl C longer than 10 minutes, in the absence of prote in s y n t h e s i s , one obtained a complete loss of d i v i s i o n potent ia l of the c e l l s . The loss in the d i v i s i o n potent ia l at kl C was c l e a r l y not due to a decreasing amount of messengers which remained t r a n s l a t a b l e . In f a c t , when mRNA synthesis was inh ib i ted by Ri fampicin b lock , (see the 0 20 40 60 80 MINUTES Figure 36. The wave of c e l l d i v i s i o n during a t r a n s i t i o n from kl to 30 C. CR34T83 grown at 30 C to a dens i ty of 3-0 x 10' c e l l s per ml was s h i f t e d to kl C. A f t e r 15 (•), 30 ( o ) , kS ( • ) , and 60 (V) minutes, c e l l s were returned to 30 C. F i f t e e n minutes before 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 reated with CAM (150 yg per ml) and were dispensed into 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 fo l lowing the s h i f t down are p lo t ted against the time of a d d i t i o n of CAM. The v e r t i c a l dotted l i n e s represent the zero time for s h i f t down to 30 C fo r each c u l t u r e . The e f f e c t of R i fampic in 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 for the 15 minute pulse is represented by squares . r e s u l t s of f i n a l c e l l numbers in the absence of t r a n s c r i p t i o n ) (Figure 36), the number of c e l l s generated was greater from that of CAM a lone. The two a n t i b i o t i c s , though a f f e c t i n g macromolecular synthesis in a unique f a s h i o n , could have had d i f f e r e n t e f f e c t s on c e l l d i v i s i o n in t h i s ' 1 case . When E_. co l i were incubated in the presence of CAM, prote in synthesis was arrested (Nomura and Watson, 1959) while RNA synthes was continuous (Se l ls and S a y l e r , 1971). R i fampic in , by binding to the RNA polymerase, blocks the i n i t i a t i o n of new mRNA cha ins . C u r r e n t l y , the data support ing the ex is tence in E_. col ? of mRNA with widespread h a l f - l i v e s is meager. However, th is p o s s i b i l i t y , although not precluded here, seems less l i k e l y to be the case . If, in f a c t , messenger RNA was inact ivated at random, mRNA surv iva l curves could be exponential funct ions and the rate of t r a n s l a t i o n of any surv iv ing mRNA, assumed to be constant , should a l s o have been an exponential funct ion , - , The semi 1ogarithmic representat ion of percent res idual d i v i s i o n as a funct ion of pulse of incubation at kl C , in the absence of prote in synthesis used in Figure 36, shows that th is was the case indeed. The ha l f l i f e of the d i v i s i o n potent ia l ca lcu la ted from th is was very c l o s e to 1.4 minutes. In f a c t , the measurement of the residual d i v i s i o n in the presence of Ri fampicin in experiments s i m i l a r to those of Figure 37, showed mRNA hal f l i f e of 1.3 minutes. The Ri fampicin concentrat ion used inh ib i ted well over 90 percent of r ad ioac t i ve u r a c i l incorporat ion into RNA. Figure 37- Decay of c e l l d i v i s i o n potent ia l of CR34T83 at kl C. This graph represents the semi logar i thmic p lot of the data from the experiment represented in Figure 36 before zero time. The place of the intercept of wave curves with the s h i f t ax is was taken to be 100 percent res idual d i v i s i o n . The percent increase in c e l l number in the CAM treated samples from the intercept value is p lot ted against the time of add i t ion of CAM pr io r to recovery MINUTES Figure 38. Decay of c e l l d i v i s i o n po ten t ia l of CR34T83 at kl C. Th is graph represents the semi logar i thmic p lo t of the data from the experiment represented in F igure 36. The place of the in tercept of wave curves with the s h i f t ax is was taken to be 100 percent res idua l d i v i s i o n . The percent increase in c e l l number in the Ri fampic in t reated samples from the in tercept value is p lo t ted against the time of a d d i t i o n of R i fampic in p r i o r to recovery . V. Genetic a n a l y s i s of the mutant CR34T83. P l e i o t r o p i c mutants might, in theory, a r i s e in two ways: (1) as a resu l t of a s i n g l e mutation in a s t ruc tu ra l gene whose product is involved in a complex; or (2) from two independent mutat ions. . Several examples of the former type are known (Jones-Mortinner, 1968; Holland and T h r e f a l l , 1969; Demoss and Wagmar, 1965). It was, there-f o r e , d e s i r a b l e to e s t a b l i s h the genet ic basis for the temperature s e n s i t i v e mutation and the p l e i o t r o p y . Prel iminary mapping of T83 by conjugat ion had located the muta-t ion in the l l v region (Hirota et a 1. 1968). However, the c o r r e l a t i o n observed between temperature s e n s i t i v e i n i t i a t i o n and c e l l d i v i s i o n led to a c l o s e r examination of the genet ics of T83 and a more prec ise mapping by t ransduct ion . It was qu i te poss ib le that there existed two mutat ions, one a f f e c t i n g DNA i n i t i a t i o n and the o ther , membrane proper t ies involved in d i v i s i o n . The existence of mu l t ip le mutation was poss ib le s ince NTG, which was known to s p e c i f i c a l l y produce several mutations at the fork region (Cereda-Olmedo et a 1 .' H9£8) » had been used in preparing th is s t r a i n (Kohiyama et al . 1966). -Three types of experiments were c a r r i e d out : (l) the study of temperature r e s i s t a n t revertants (tr DnaA); (2) the study of T83 s t ra ins with ts DnaA l l v + genotype; and (3) the const ruc t ion of other s t r a i n s of F£. col ? K12 which were isogenic for more than 38% of the i r chromosome, except for the one minute transduced port ion of the i r chromosome, the l l v DnaA segment. 111 A. Study of temperature re s is ta n t (tr) rever tan ts . i f the T83 mutation was to be a point mutat ion, then a revers ion frequency to temperature res is tance in the range of 10 "* to 10 ^ should have been observed. On the other hand, i f the p le io t ropy was due to a double mutat ion, the frequency of the back mutation for two markers would have been much lower. The nature of the coupl ing of DNA to c e l l u l a r growth needed for colony formation allowed the examination of these two p o s s i b i l i t i e s . T83 was grown overnight in A and E medium, d i l u t e d 1:100 and then 0.1 ml of th is was inoculated into a number of test tubes or onto agar p l a t e s . A f te r 2k hours of incubation at kl C, growth was observed in the tubes and 20 to Ik co lon ies were formed on the p l a t e s . Each of these co lon ies were inoculated into l i q u i d medium and were tested fur ther for 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 genera l , the DNA r e p l i c a t i o n was found to be normal and c e l l d i v i s i o n continued at kl C. The doubling time for t r s t r a i n s , under kl C growth c o n d i t i o n s , was retarded to approximately 50 minutes. The reversion frequency -6 ca lcu la ted was approximately 2 x 10 , which is in good agreement with a s i n g l e point mutation (Hayes, 1968). B. Construct ion and ana lys is of C R 3 4 T 8 3 l l v + ts DnaA s t r a i n s . E_. col ? C R 3 4 T 8 3 requires iso leuc ine and v a l i n e for growth. The order of the markers that are known to be in th is region of the map are tna , the regulatory gene for tryptophanase production (Gartner and R i l e y , 1965) , phoS, the regulatory gene for phosphatase (Echols et_ al_. 1961), 11v, th e i s o l e u c i n e - v a l i n e gene c l u s t e r (Ramakrichnan and Adelberg-, 1965), and DnaA ( H i r o t a e t a l . 1968; 1970). T h e i r o r d e r i s shown below. ( D C O J Q 3 4-1O Q .- I (marker) 73 74 75 (map l o c a t i o n , m i n u t e s ) The l i n k a g e between l l v and t s DnaA a l l o w e d f o r i n t r o d u c t i o n o f t h e l l v + 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 t h e p r o x i m a l 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 t r a n s d u c i n g phage P i k e (Lk) was made on EM c o l ? K12 AB 1157, w h i c h was l l v + . T83 was i n f e c t e d w i t h the donor phage and t h e l l v + t r a n s d u c t a n t s were t e s t e d under two s e l e c t i v e c o n d i t i o n s : (a) growth o f T83 M v + t r a n s -d u c t a n t s a t 30 C on m i n i m a l a g a r p l a t e s l a c k i n g l l v ; and (b) growth o f T83 l l v + t r a n s d u c t a n t s on f u l l y supplemented a g a r p l a t e s a t kl C. T a b l e IV shows t y p i c a l r e s u l t s . F i f t y - s i x p e r c e n t o f t h e l l v + T83 r e c i p i e n t s had l o s t t h e t e m p e r a t u r e s e n s i t i v i t y . T h i s c o u l d have r e s u l t e d i f t h e donor phage l y s a t e c a r r i e d a w i l d t y p e a l l e l e f o r DnaA + and l l v + . The Lk s t r a i n o f phage P i k e t h a t was used h e r e c o u l d t r a n s -d uce a p p r o x i m a t e l y 3 m i n u t e s w o r t h o f E_. c o l i chromosome (Caro and Schnos, 1970). The l o n g e s t d i s t a n c e f o r c o t r a n s d u c t i o n o f two markers was about 2.2 m i n u t e s o f the map ( T a y l o r and T r o t t e r , 1967) • The r e s u l t s a g r e e d w i t h t h e p o s s i b i l i t y f o r c o t r a n s d u c t i o n o f two m a r k e r s , DnaA and l l v , w h i c h were a d j a c e n t t o one a n o t h e r . Table III. The frequency of j o i n t t ransduct ions of the Ilv and DnaA l o c i . n . . Genotype Numbers of Unselected Marker Donor Recipient _ , [ , T . r Selected Transductants scored Type Number AB 1157 CR34T83 M v + 70 (100%) DnaA + 39 (56%) (DnaA+ (ts DnaA | l v + ) | l v - ) DnaA 32 (100%) Ilv 32 (100%) Transductants were scored on minimal medium supplemented with a l l the requirements but for iso leuc ine and v a l i n e (se lec t ion for l l v + ) at 30 C , or with f u l l supplements (se lec t ion for DnaA+) at kl C. Experiments were performed as descr ibed in Mater ia ls and Methods. 114 C. Introduction of the T83 gene into E_. col i K12 s t ra ins and the ana lys is of the p l e i o t r o p y . One of the parental s t r a i n s iso la ted in the previous sect ion was used as donor in the experiments l i s t e d below. This s t r a i n , KG 776, was ts DnaA and had received the l l v + marker from E_. col i K12 AB 1175-Pi phage lysates were made on th is s t r a i n and c e l l s of other K12 s t r a i n s were infected with th is donor phage. Table IV shows the r e s u l t s of these experiments. The resu l ts c l e a r l y indicated that the ts DnaA was l inked with the l l v and tna markers. Of the ts DnaA transductants that were i s o l a t e d , a n a l y s i s of growth at 30 C and 42 C was performed. In 15 of the kl ts DnaA s t r a i n s checked, a hal t in c e l l u l a r d i v i s i o n resul ted upon incubation at kl C from growth at 30 C. DNA r e p l i c a t i o n patterns as examined by measurement of the rate of incorporat ion of H-thymidine (since the s t r a i n s were thy ) into the cold TCA inso lub le f r a c t i o n , resembled that of T83. Thus, in the E_. col i Kl2 l l v + t ransductants const ruc ted , which had inher i ted 1 to 2 minutes of the T83 chromosome from the 73 to 74 minutes region of the map, the p le io t ropy d isplayed must have been acquired v ia the transducing phage p a r t i c l e . With the con t ro ls per-formed for these experiments, the p o s s i b i l i t y of the ex is tence of two separate mutations responsib le for the p l e i o t r o p y , were ruled out . It remains to be seen whether only one or more c i s t r o n s in th is seg-ment of the chromosome are involved in the regulat ion of DNA i n i t i a t i o n and in the other membrane p r o p e r t i e s . However, recent studies of 115 T a b l e IV. Frequency o f j o i n t t r a n s d u c t i o n s o f t h e I l v and DnaA l o c i from E. c o l i T83. Donor Re c i i e n t Genotype Number of e c i p i e n S e l e c t e d T r a n s d u c t a n t s s c o r e d U n s e l e c t e d Marker Type Numbers KG 776 t n a t s DnaA ) KG 146 ( H v " ) KG 163 U l v " , tna") I l v I l v 60 (100%) 67 (100%) DnaA DnaA"* t s t n a DnaA tna DnaA + +„ „ts tna DnaA tna DnaA t s 23 (40%) 34 (60%) 39 (58%) 18 (27%) 10 (15%) 0 (0%) KG 166 l l v + ( N v " , tna , phoS T r a n s d u c t a n t s were s c o r e d on minimal medium supplemented w i t h a l l the r e q u i r e m e n t s but 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 f o r l l v + ) a t 30 C or w i t h f u l l supplements ( s e l e c t i o n f o r DnaA +) a t 42 C. The tna marker was t e s t e d by the t r y p t o p h a n a s e a s s a y . E x p e r i m e n t s were performed as d e s c r i b e d i n M a t e r i a l s and Methods. 80 (100%) tna DnaA + 52 (65%) tna"DnaA + 19 (24%) t n a + D n a A t S 9 (11%) t n a " D n a A t S 0 (0%) Hirota et_ a K (1970) on the genet ics of both TkS and T83 have d icated that such p o s s i b i l i t i e s are very u n l i k e l y . DISCUSSION The t s DnaA gene was shown t o a f f e c t c e l l u l a r d i v i s i o n and c o n -t i n u a t i o n o f DNA r e p l i c a t i o n i n E_. c o l i CR34T83 a t 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 . Support f o r t h i s p l e i o t r o p i c r e l a t i o n s h i p was o b t a i n e d by the g e n e t i c s t u d i e s . The l e s i o n was shown t o be a p o i n t m u t a t i o n , s i n c e w i l d t y p e r e v e r t a n t s w h i c h c o u l d grow n o r m a l l y a t kl C were -6 i s o l a t e d a t a f r e q u e n c y o f 2 x 10 , c o m p a t i b l e w i t h t h e r e v e r s i o n r a t e o f a p o i n t m u t a t i o n . The gene was c o - t r a n s d u c i b l e w i t h 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 was l i n k e d t o t h e s t r u c t u r a l gene f o r t h e enzyme t r y p t o p h a n a s e . These r e s u l t s c o n f i r m e d and e xtended the a n a l y s i s o f H i r o t a and c o - w o r k e r s ( H i r o t a e t a l . 1968; 1970). T r a n s -d u c t i o n a l a n a l y s i s ( T a b l e IV) i n d i c a t e d t h a t t h e r e was n o t h i n g but t h e T83 m u t a t i o n w h i c h c o u l d be r e s p o n s i b l e f o r t h e p l e i o t r o p y , s i n c e when the 1 min segment o f the chromosome c a r r y i n g the DnaA gene was t r a n s d u c e d t o w i l d t y p e E_. c o l i K12 s t r a i n s , a s i m i l a r r e s p o n s e was o b t a i n e d . The DnaA m u t a t i o n 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 o f DNA r e p l i c a t i o n a t kl C. Uptake o f exogenous t h y m i d i n e i n t o t h e c e l l was normal ( r e s u l t s not shown), y e t DNA s y n t h e s i s ceased a f t e r a s h o r t t i m e a t kl C. These r e s u l t s a g r e e w i t h Kohiyama's o r i g i n a l o b s e r v a -t i o n s (Kohiyama et_ aj_. 1966) . The amount o f t h e r e s i d u a l DNA s y n t h e s i z e d a t kl C was c o m p a t i b l e w i t h c o m p l e t i o n o f rounds o f r e p l i c a t i o n i n an e x p o n e n t i a l p o p u l a t i o n , a c c o r d i n g t o t h e H e l m s t e t t e r and Cooper model. The gradual d e c l i n e in the DNA synthesis was not due to low nucleo-s ide t r iphosphate p o o l s . In f a c t , most of the nucleoside t r iphosphate pools accumulated during the incubation at kl C (Figure 10 and 11). However, the dTTP pool did not expand s i g n i f i c a n t l y . Beacham and P r i t c h a r d ' s (1971) f i nd ing that dTTP pool was normally low in thy s t ra ins of E_. col i , supports th is observa t ion . The presence of a low dTTP level in T83 at kl C, could a l s o be taken as support ive evidence for Werner's arguments (Werner, 1971) that dTTP is not the precursor for thymine, in DNA r e p l i c a t i o n . This would expla in why only dATP, dCTP and dGTP pools accumulated with the cessat ion of DNA s y n t h e s i s ; they were the true precursors of DNA syn thes is . However, no other thymine conta in ing precursors of DNA synthesis was measured in th is work to fur ther resolve th is ques t ion . Since the r e p l i c a t i o n of bacteriophages lk and lambda were unaffected in T 8 3 grown at kl C (Kohiyama, 1968), i t could be argued that there was no l i m i t a t i o n in a l l four p recursors . Under exponential growth condi t ions at 30 C, add i t ion of CAM at a concentrat ion known to block r e i n i t i a t i o n of new rounds of DNA r e p l i c a t i o n (Lark and Ranger, 1969). resul ted in residual DNA r e p l i c a t i o n compatible with that of growth at kl C. Add i t ion of CAM at 150 ug per ml , at kl C, did not a l t e r the mode of res idual DNA synthesis (Figure 32). However, at a level of 25 yg per ml of CAM, a b iphas ic residual DNA synthesis curve resul ted (Figure 33). The reason for th is d e v i a -t ion is not c l e a r . 1 1 9 In i t ia ted DNA r e p l i c a t i o n forks in T83 were shown to be s t a b l e , at kl C, to the e f f e c t s of N a l i d i x i c a c i d . Ward and Glaser (1970) have shown that treatment of E_. col i B/r with N a l i d i x i c ac id for 30 minutes prevents the resumption of the r e p l i c a t i o n of the a f fec ted f o r k s . A c c o r d i n g l y , in T83, res idual DNA synthesis a f t e r re lease from a N a l i d i x i c ac id block could occur only i f o ld forks were s t a b l e . As shown in Figure 3k, res idual DNA synthesis at kl C did occur sub-sequent to removal of the N a l i d i x i c ac id b lock . Lark ( J . Urban, pers . commun.) has used a dens i ty 1 label 1?ng technique to fo l low DNA r e p l i c a t i o n a f t e r N a l i d i x i c ac id treatment for 50 minutes. Their r e s u l t s agreed with th is work ind ica t ing completion of prev ious ly i n i t i a t e d c y c l e s . The evidence of Ward and G l a s e r , which is based on genet ic approaches, cannot be re fu ted , yet i t is not compatible with 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, i t is c r i t i c a l to e s t a b l i s h the place of r e i n i t i a t i o n of rounds of DNA r e p l i c a t i o n at recovery at the permissive temperature a f te r i n h i b i t i o n of DNA syn-thes is at the non-permissive temperature. Idea l ly , one should a lso demonstrate the r e l a t i o n s h i p between the place for r e i n i t i a t i o n and the o r i g i n of DNA r e p l i c a t i o n . In the c h a r a c t e r i z a t i o n of T 8 3 , Kohiyama O968) examined the place for the termination of chromosome r e p l i c a t i o n 14 at kl C. Th is was shown by C-TdR l a b e l l i n g of the DNA in amino ac id starved c e l l s at 30 C. By returning the c e l l s to amino acid 3 supplemented media at 30 C and subsequently puls ing 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 . The pulse of H-TdR, was chased with 600 yg per ml co ld thymidine, and the c e l l s were allowed to randomize the i r DNA c y c l e s . C e l l s were sh i f t ed to 41 C and given 5-BrdUrd in the place of thymidine. The densi ty gradient a n a l y s i s of the extracted DNA showed that the hybrid dens i ty band 14 3 contained 50% of the C- and 25% of the "^H-labelled DNA. Thus, i t was concluded that T83 was blocked in the i n i t i a t i o n of DNA cyc les at 41 C, and the s h i f t to 41 C with 5-BrdUrd allowed p r e f e r e n t i a l r e p l i c a t i o n at the terminus of rounds as def ined by amino ac id s t a r v a -t ion and alignment of chromosomes. However, i f th is argument were to 3 be t rue , no H-TdR should have banded in the hybrid densi ty reg ion. There are several other ob ject ions to th is experiment: (1) The use of amino ac id s ta rva t ion to a l i g n chromosomes although widespread, is not very s a t i s f a c t o r y . One problem associated with th is method is that the capaci ty of the c e l l s to r e p l i c a t e DNA dec l ines during amino ac id s ta rva t ion (Doudney, 1966) and there fo re , not a l l chromosomes complete rounds of r e p l i c a t i o n (Caro and Berg, 1969)' (2) When l a b e l l i n g the s t a r t s of rounds of DNA r e p l i c a t i o n , Kohiyama (1968) used low concentrat ions (0.5 yg per ml) of thymidine. At th is l e v e l , the chromosome r e p l i c a t i o n time at 37 C is equal to 97 minutes (Pr i tchard and Z a r i t s k y , 1970). Thus, very l i t t l e of the chromosome at i n i t i a t i o n would have been l abe l l ed during the 8 minute 3 pulse of H-thymidine employed. 3 (3) In order to chase the H-TdR, 600 yg per ml of unlabel led 121 TdR was used for 90 minutes. Addi t ion of large quant i t i es of thymidine to c e l l s expands the deoxyribose-5-phosphate pools and retards the c e l l u l a r r e s p i r a t i o n (Lomax and Greenberg, 1968) . Th is could a f f e c t DNA r e p l i c a t i o n , s ince the two processes are interdependent (Cairns and Denhardt, 1968). (4) The 90 minutes of incubation at 30 C was not long enough to permit randomization of the populat ion of chromosomes a f te r the i r al ignment. Thus, f o r t u i t o u s l y , the ends of rounds could have been picked up a f t e r the s h i f t into 5-BrdUrd medium. In the experiments descr ibed in Figure 31, the place of re -sumption of DNA r e p l i c a t i o n during recovery was e s t a b l i s h e d . The 3 chromosomes were l abe l l ed 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 synthesized at recovery was always 14 assoc ia ted with the C - l a b e l . S i m i l a r l y , when chromosomes were a l igned by chloramphenicol treatment (Figure 32) , the densi ty label 14 was always found to be associated with the C - l a b e l , that i s , with the s t a r t s of rounds, as def ined by the temperature b lock . Whether the place of resumption of DNA r e p l i c a t i o n in T83 during recovery is the same as the o r i g i n of DNA r e p l i c a t i o n (7 - 8 o ' c l o c k ) on the — c o l i genet ic map, remains to be t e s t e d . Inh ib i t ion of prote in synthesis during the f i r s t 10 minutes of recovery of the permissive temperature, was shown to in te r fe re with r e i n i t i a t i o n of rounds of r e p l i c a t i o n (F igures .22 , 23, 24). If prote in synthesis was permitted, then cel ls returned to 30 C from a kl C pulse resumed DNA replication (Figure 9) ••- Cells returned to the permissive temperature, after a pulse at kl C, showed a drop in the rates of DNA replication for some time. The reason for this behaviour remains unknown. One interpretation of the residual decline in the rate of replication at 30 C could be a need for the ce l ls to recover from temperature inactivated ini t iator molecules by a "flush out" or di lut ion mechansim. Such a mechanism would predict a.variable lag for the ce l ls to acquire a l l the functional and active molecules needed for in i t ia t ion replication of their chromosomes. In fact , the DNA replication 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. It is possible that complete inactivation of the ini t iator molecules in an exponential culture requires treatment longer than 10 minutes at kl C; for pulses of 10 minutes or less, only a fraction of the population is affected, 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. (1969). The enzyme needed for in i t ia t ion of a round are always present in the c e l l , but a re-pressor of in i t ia t ion interferes with their inauguration. Rosenberg et a l . (1969) suggested that an antirepressor triggers in i t ia t ion by its interaction with the repressor. The antirepressor, synthesized a t the end o f e v e r y r e p l i c a t i o n c y c l e , would t r i g g e r new c y c l e s o f r e p l i c a t i o n . I t c o u l d be assumed t h a t i n T 8 3 , t h e t e m p e r a t u r e s e n s i t i v e element i s t h e a n t i r e p r e s s o r . Thus, i n c e l l s kept a t kl C f o r a l o n g t i m e , a l l chromosomes would be a t t h e end o f t h e i r r o u n d s , and would i n i t i a t e a t 30 C i m m e d i a t e l y upon t h e a c q u i s i t i o n o f t h e a n t i r e p r e s s o r . W i t h s h o r t e r p u l s e s a t kl C, fewer ends o f rounds a r e produced and hence, l o n g e r d e l a y s a t r e c o v e r y would be r e q u i r e d . W h i l e t h i s e x p l a i n s t h e r e s u l t s f o r t e m p e r a t u r e p u l s e s f o r l o n g e r than 10 m i n u t e s , t h e r e i s no o b v i o u s e x p l a n a t i o n f o r t h e s h o r t l a g o b s e r v e d w i t h the 10 m i n u t e p u l s e ( F i g u r e 9)• I t was shown ( F i g u r e 2) t h a t , once s h i f t e d t o kl C, c e l l d i v i s i o n c eased and f i l a m e n t o u s growth r e s u l t e d i n £. c o l i CR34T83. P a n t o y l l a c t o n e i n d u c e s o t h e r f i l a m e n t o u s s t r a i n s o f E_. c o l i t o d i v i d e ( A d l e r and H a r d i g r e e , 1965), but had no e f f e c t on T83 growing as f i l a m e n t s a t kl C ( r e s u l t s not shown). S i m i l a r l y , d i v i s i o n i n d u c t i o n o f tempera-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 s a l t ( R i c h a r d and H i r o t a , 1969) was i n e f f e c t i v e i n T83 a t kl C ( r e s u l t s not shown). D u r i n g f i l a m e n t o u s g r o w t h , t h e r e p l i c a t e d chromosomes s e g r e g a t e d n o r m a l l y ( r e s u l t s n o t shown) and, upon a r e t u r n t o 30 C, f i l a m e n t s d i v i d e d i n t o normal s i z e d c e l l s ( F i g u r e 8). C e l l d i v i s i o n d u r i n g r e c o v e r y a t 30 C i n c u l t u r e s t h a t were b l o c k e d i n chromosome r e p l i c a t i o n by N a l i d i x i c a c i d ( F i g u r e 14) a t kl C, s u b s t a n t i a t e d t h a t d i v i s i o n was dependent upon completed chromosome c y c l e s . Anr. a p p r e c i a b l e change i n r e s i d u a l c e l l d i v i s i o n was o b s e r v e d when N a l i d i x i c a c i d was added a f t e r 40 to 50 minutes recovery at 30 C (Figure 14B). Thus, at some time a f t e r s h i f t from 42 C to 30 C , new c y c l e s of r e p l i c a t i o n were i n i t i a t e d which went to completion approximately 50 minutes a f t e r the s h i f t . If synthesis of RNA and prote in were blocked at the time of a s h i f t from kl C to 30 C, residual c e l l d i v i s i o n at recovery was observed. Inh ib i t ion of tota l RNA synthesis at the s h i f t to 30 C a f t e r kS min-ute incubation at kl C , resul ted in a f o u r - f o l d increase in c e l l numbers whereas a doubling in c e l l number occurred upon add i t ion of chloramphenicol or N a l i d i x i c ac id (Figures 1k, 19, 20). Under the same cond i t ions (Figures 15, 16), the a b i l i t y to complete two doublings probably was due to t r a n s l a t i o n of a l l a v a i l a b l e mRNAs in the c e l l s . Ce l l d i v i s i o n at recovery was proport ional to the number of "uni t c e l l s " (Donachie and Begg, 1970) generated at kl C. Since the maximum number of normal c e l l equiva lents generated at kl C , would be l imi ted by the numbers of chromosomal copies a v a i l a b l e a f te r completion of rounds of r e p l i c a t i o n , f i n a l c e l l number produced by residual c e l l d i v i s i o n should plateau for pulses at kl C longer than 45 minutes. This was demonstrated in Figure 21. The a c t i v i t y of the i n i t i a t o r molecule was not regained at recovery in the presence of chloramphenicol . Results presented (see sect ion II. C) c l a s s i f y the behaviour of the i n i t i a t o r molecule at 42 C as denatured and inac t ive (type a_ t r a n s i t i o n ) . Thus new prote in synthesis is ob l iga tory for new a c t i v i t y during the 30 C recovery per iod . However, the t r a n s i t i o n for the d i v i s i o n element in the complex was that of nat ive and a c t i v e at 30 C to nat ive but inac t ive at 42 C (type b_ t r a n s i t i o n ) . The express ion of e e l l u l a r d iv i s ion dur ing recovery at 30 C based on "potent ia l c e l l e q u i v a l e n t s " , i e . , DNA to c e l l mass, did not require a c t i v e prote in synthesis as was shown by the add i t ion of chloramphenicol at the time of the s h i f t from kl C to 30 C. This indicated that the t r a n s i t i o n for the d i v i s i o n element is r e v e r s i b l e once returned to the permissive temperature, and does not require synthesis of 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 in cond i t iona l le tha l mutants is not without precedent. Chiu and Greenberg (1968) descr ibed a mutation in the gene for dCMP-hydroxymethylase that rendered b a c t e r i o -phage lk DNA synthesis inoperat ive at kl C but not at 28 C. dCMP-HMase is one of the ear ly enzymes in T.4-infected E_. col i and con t ro ls the tetrahydrofolate-dependent conversion of dCMP to 5~hydroxymethy1-dCMP, and is coded by the lk gene kl. Ik DNA polymerase (gene 43) was shown to in teract with the dCMP-HMase and a t h i r d component before v i r a l DNA synthesis could commence. Gene kl and 43 prote ins were unable to operate in a double s h i f t experiment (28-42-28-42) when the synthesis of the t h i r d component, which was made between 5 and 8 minutes a f t e r recovery , was blocked by chloramphenicol . The i n t e g r i t y of the new complex was an ob l iga tory requirement for DNA r e p l i c a t i o n . It is qui te l i k e l y that the complex events that are known to be associa ted with DNA-membrane attachment and d i v i s i o n at the septum reg ion , involve protein-DNA and p r o t e i n - p r o t e i n binding react ions of 126 a cooperat ive nature. In any event , the p l e i o t r o p i c r e l a t i o n ob-served can best be explained in terms of a multi-enzyme complex l o c a l i z e d at the c e l l membrane, involved in 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 . BIBLIOGRAPHY Abe, M. and J . Tomizawa. 1967- R e p l i c a t i o n o f t h e E s c h e r i c h i a c o l ? K12 chromosome. P r o c . Nat. Acad. S c i . 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