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Replication and plasmid-bacteriophage recombination Smith, Richard Dana 1980

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REPLICATION AND PLASMID-BACTERIOPHAGE RECOMBINATION by RICHARD DANA SMITH A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the re q u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA December, 1980 © Richard Dana Smith, 1980 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it fr e e l y available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for f i n a n c i a l gain shall not be allowed without my written permission. Department of The University of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 ABSTRACT Marker rescue between a plasmid carrying T^' DNA and a mutant bacteriophage was used to study the r o l e of r e p l i c a t i o n i n genetic recom-bination. The r e p l i c a t i o n of plasmid and phage DNA's could be c o n t r o l l e d independently by a mutation i n the host bacterium and mutations i n T^ gene 5 (DNA polymerase), r e s p e c t i v e l y . Recombination was monitored by the production of wild-type phage. Results indicated that when only one molecule, i . e . plasmid or phage, could r e p l i c a t e , recombination was decreased s l i g h t l y . However, i f r e p l i c a t i o n was blocked on both mole-cules, no recombination was detected. 3 Agarose gel electrophoresis, H-thymidine incorporation, and copy number analysis showed that plasmid DNA was not degraded a f t e r T^ i n f e c t i o n . Density transfer experiments examined recombination between 3 a heavy labeled phage and a H-labeled plasmid molecule. Analysis of such experiments by CsCl density gradients showed that r e p l i c a t i o n was e s s e n t i a l for j o i n t molecule formation. R e p l i c a t i o n was responsible for the displacement of parental DNA which then was assimilated into a r e c i p i e n t DNA molecule. The e s s e n t i a l r o l e of the T^ gene 6 protein (5' exonuclease) during T^ recombination was determined. This exo-nuclease creates gaps on T^ molecules to provide regions where homologous donor DNA can base p a i r . Recombinant T^ molecules were found to con-t a i n a s i n g l e stranded i n s e r t i o n of homologous donor DNA. The r e s u l t s are discussed i n r e l a t i o n to various models which contain rol e s f or DNA synthesis during recombination. A model for early events during plasmid-phage recombination i s presented. i i i TABLE OF CONTENTS Page Ab s t r a c t i i L i s t of Figures v i L i s t of Tables . v i i i Acknowledgements i x I n t r o d u c t i o n A. Bacteriophage Lambda 1 B. Bacteriophages T^ and T^ 2 C. T^ Recombination i n v i t r o 3 D. Plasmid-Phage Recombination 4 M a t e r i a l s and Methods A. B a c t e r i a l S t r a i n s 7 B. Bacteriophage S t r a i n s 7 C. Chemicals and Isotopes 10 D. Media and Buf f e r s 10 E. I s o l a t i o n and P u r i f i c a t i o n of Plasmid DNA's 13 F. S i n g l e Step Marker Rescue Experiment 13 G. Marker Rescue Spot Tests 14 H. Copy Number A n a l y s i s 14 1. P u r i f i c a t i o n of T o t a l I n t r a c e l l u l a r DNA 14 2. L a b e l i n g of I n t r a c e l l u l a r DNA 15 3. P r e p a r a t i o n of F i l t e r s and H y b r i d i z a t i o n C o n d i t i o n s . 15 4. Copy Number Determination 16 5. Copy Number A n a l y s i s i n T 7 I n f e c t e d C e l l s 19 i v Page I. Density Transfer i n the M a x i c e l l System 20 1. Continuous L a b e l i n g 20 2. Pre-Labeling 21 3. CsC l E q u i l i b r i u m C e n t r i f u g a t i o n 21 Re s u l t s Sect i o n I . Plasmid-Phage Marker Rescue 22 A. C h a r a c t e r i z a t i o n of Clones Ca r r y i n g T^ Gene 5 22 B. C h a r a c t e r i z a t i o n of Clones C a r r y i n g T^ Gene 4 23 C. The Plasmid-Phage Recombination System 23 1. T 7 R e p l i c a t i o n 23 2. Plasmid R e p l i c a t i o n 26 D. Marker Rescue Experiments 26 1. Marker Rescue when Both Phage and Plasmid Can R e p l i c a t e 29 2. Marker Rescue when Only Bacteriophage DNA Can R e p l i c a t e 29 3. Marker Rescue when Only Plasmid DNA Can R e p l i c a t e ... 30 4. Marker Rescue i n the Absence of R e p l i c a t i o n 32 Sec t i o n I I . The Fate of Plasmid DNA a f t e r T ? I n f e c t i o n 34 A. A n a l y s i s of Plasmid DNA a f t e r I n f e c t i o n 35 B. H y b r i d i z a t i o n A n a l y s i s of Plasmid Copy Numbers 40 C. E f f e c t of Phage I n f e c t i o n on Plasmid DNA 40 D. A n a l y s i s of Tn DNA a f t e r I n f e c t i o n 42 Section I I I . The Role of the T 5' Exonuclease (gene 6) i n Plasmid-Phage Recombination 46 A. Marker Rescue and the E f f e c t of the T-, Exonuclease 46 V Page Sectio n IV. P h y s i c a l A n a l y s i s of Plasmid-Phage Recombination .... 52 A. Density Transfer during Plasmid-Phage Recombination 52 B. R e s u l t s 58 1. UV Dose Determination 58 2. Density Transfer w i t h Continuous L a b e l i n g a f t e r I n f e c t i o n 58 3 3. The Fate of H-LL DNA from Plasmids Labeled before I n f e c t i o n 63 4. The Lack of Density Transfer i n the Absence of Plasmid R e p l i c a t i o n 68 3 5. The F a i l u r e of H-Labeled DNA to be Transfered i n the Absence of the T^ 5' Exonuclease 71 3 6. A n a l y s i s of H-Labeled M a t e r i a l from the HH L o c a t i o n i n CsCl Gradients 71 D i s c u s s i o n A. R e p l i c a t i o n and Marker Rescue of Bacteriophage T^ DNA ... 84 B. Strand Transfer during Plasmid-Phage Recombination 87 C. The S t r u c t u r e of Recombinant Molecules 88 D. The Role of the T ? 5' Exonuclease (gene 6) and Strand Uptake 90 E. E a r l y Events during Plasmid-Phage Recombination 93 1. Enzymes 93 2. R e c i p r o c a l vs Non-Reciprocal Recombination 95 3. Comparison of Plasmid-Phage Recombination w i t h i n v i t r o T^ Recombination 96 4. A P o s s i b l e Mechanism f o r Plasmid-Phage Recombination. 97 Appendix I. Two Models f o r Genetic Recombination 101 L i t e r a t u r e C i t e d 103 v i LIST OF FIGURES Figure T i t l e Page 1 CsCl A n a l y s i s of HH Phage DNA 11 2 Standard Curve f o r the A n a l y s i s of H y b r i d i z a t i o n R a t i o s 17 3 Map of Hpa I Fragments Released from T^ + DNA 24 4 T o t a l DNA Synthesis i n E279 and E279-pRS202 27 5 E l e c t r o p h o r e t i c A n a l y s i s of Plasmid DNA's a f t e r I n f e c t i o n 36 6 Autoradiogram of Southern B l o t Containing Plasmid DNA's 38 7 Sucrose Gradient A n a l y s i s of T^ DNA at 43°C i n dnaB + and dnaB - Hosts 44 8 E f f e c t of T^ 5' Exonuclease on Plasmid-Phage Recombination 48 9 Strand Displacement 53 10 The Basic Molecular Events during a Cross between Heavy Labeled Phage and 3 H - l a b e l e d Plasmid DNA .... 56 11 Rate of DNA Synthesis and Plasmid/Chromosome R a t i o s a f t e r UV Exposure 59 12 a Survivors a f t e r UV Exposure 61 12 b Burst S i z e Determined a f t e r 25 Seconds of UV Exposure 61 3 13 D i s t r i b u t i o n of H-labeled DNA i n CsCl Gradients 64 14 A n a l y s i s of P r e l a b e l e d 3 H - l a b e l e d DNA i n CsCl Gradients 66 3 15 The Fate of P r e l a b e l e d H-LL DNA w i t h and without Plasmid R e p l i c a t i o n 69 3 16 D i s t r i b u t i o n of H-LL DNA i n the Presence and Absence of the T^ 5' Exonuclease 72 17 A P r e p a r a t i v e CsCl Gradient 76 3 18 . Rebanding and the E f f e c t of S o n i c a t i o n on H-HH DNA .. 78 v i i F i g u r e T i t l e Page 19 Sucrose Gradient A n a l y s i s of Sonicated T^ DNA 80 20 Sucrose Gradient A n a l y s i s of HH M a t e r i a l 82 21 Two P o s s i b l e Mechanisms f o r Strand Uptake 91 22 E a r l y Events i n Plasmid-Phage Recombination 99 v i i i LIST OF TABLES Table T i t l e Page 1 B a c t e r i a l S t r a i n s 8 2 Plasmid DNA's 9 3 E f f e c t of DNA R e p l i c a t i o n on T^ Marker Rescue 31 4 R e p l i c a t i o n and Marker Rescue of T^ Gene 4 Mutants ... 33 5 Copy Number A n a l y s i s i n Non-Infected C e l l s 41 6 H y b r i d i z a t i o n A n a l y s i s of Plasmid DNA a f t e r I n f e c t i o n . 43 7 The E f f e c t of the T ? 5' Exonuclease on Plasmid-Phage Recombination 47 LIST OF ABBREVIATIONS LB L u r i a Broth am amber ts temperature s e n s i t i v e FdU 5-fluordeoxyuridine Tc t e t r a c y c l i n e I.e. Infective center 0 phage p a r t i c l e W.T. Wild-type TCA T r i c h l o r o a c e t i c Acid X ACKNOWLEDGEMENTS I am very g r a t e f u l to Dr. R. C. M i l l e r , J r . , f o r h i s valuable i n s t r u c t i o n , guidance, and enthusiasm throughout the course of my research. I thank Drs. R. A. J . Warren, G. Spiegelman, and K. Maltman for very useful discussions of t h i s work. I am very g r a t e f u l to my wife, Katherine, f o r her love, constant moral support, and for a l l the time and energy she contributed by drawing figures and typing t h i s t h e s i s . By f a i t h we understand that the world was created by the word of GOD, so that what i s seen was made out of things which do not appear. Hebrews 11:3 INTRODUCTION REPLICATION AND RECOMBINATION During bacteriophage i n f e c t i o n , the processes of DNA r e p l i c a t i o n and recombination are i n t i m a t e l y r e l a t e d . H i s t o r i c a l l y , many systems have been the focus of st u d i e s concerning t h i s r e l a t i o n -ship. Several of these systems are examined here to review the nature and complexity of the problem addressed i n t h i s t h e s i s , i . e . the r o l e of r e p l i c a t i o n i n genetic recombination. A. Bacteriophage Lambda (A). . x_ In phage A,, a unique r e l a t i o n s h i p e x i s t s between DNA d u p l i -c a t i o n and recombination. Both processes are a v a i l a b l e to A i n order to produce concatameric DNA which i s e s s e n t i a l f o r packaging (74). F i r s t , A: can r e p l i c a t e by a r o l l i n g c i r c l e mechanism to provide con-catamers (19). However, when t h i s mode of r e p l i c a t i o n i s blocked, a second mechanism, recombination, can provide dimeric c i r c l e s ( f i g u r e -e i g h t s ) which can be packaged (48, 81). Therefore, when r e p l i c a t i o n i s blocked, recombination serves as a mechanism f o r the phage to comple i t s l i f e c y c l e . Normally, i n the absence of r e p l i c a t i o n , cross-overs are uniformly d i s t r i b u t e d across the length of the genome (71). However, the frequency of recombination events under the c o n d i t i o n s of blocked DNA synth e s i s are g r e a t l y decreased (F. S t a h l , personal communication). This i n d i c a t e s that normal l e v e l s of recombination are a s s o c i a t e d w i t h the r e p l i c a t i o n process. This i s supported by S t a h l and S t a h l (69) 2 who found extensive DNA s y n t h e s i s to be a s s o c i a t e d w i t h recombination events (70). B. Bacteriophages and Several l i n e s of evidence from these e x t e n s i v e l y s t u d i e d phages i n d i c a t e a complex r e l a t i o n s i p between r e p l i c a t i o n and recom-b i n a t i o n . F i r s t , enzymes which provide n u c l e o t i d e precursors f o r r e p l i c a t i o n by the degradation of host DNA are a l s o r e s p o n s i b l e f o r generating recombination intermediates,ae.g., ( i ) the products of genes 46 and 47 are d i r e c t l y i m p l i c a t e d i n gap formation during i n f e c t i o n (5, 8, 40), and mutations i n genes 46 and 47 decrease genetic recombination frequencies (5, 40); ( i i ) the product of genes 3 and 6, an endonuclease and a 5' exonuclease, r e s p e c t i v e l y (62), are r e s p o n s i b l e f o r the formation of gaps. Under gene 3 , gene 6 , or gene 3 6 c o n d i t i o n s , recombination frequencies are reduced (42, 57). Mutations i n genes 46, 47, and genes 3, 6, decrease recombination frequencies by decreasing the formation of j o i n t molecules and a l s o decrease the formation of concatamers (39, 43, 51). Second, s e v e r a l enzymes serve dual f u n c t i o n s , i . e . i n r e p l i c a t i o n and recombination. Examples of t h i s i n c l u d e : ( i ) the DNA polymerase (gene 43) pre-sumably f i l l s gaps created during j o i n t molecule formation which are subsequently sealed by l i g a s e (5, 20, 85); ( i i ) the DNA b i n d i n g p r o t e i n (gene 32) which may be i n v o l v e d i n s t a b i l i z i n g r e p l i c a t i o n f o r k s (1, 82). T h i r d , the expression of gene products necessary to form and r e p a i r recombination intermediates during T^ i n f e c t i o n i s coordinated w i t h the sequence of r e p l i c a t i o n - recombination events (38). Fourth, the generation of concatameric DNA during T A i n f e c t i o n s 3 i n v o l v e s both the r e p l i c a t i o n and recombination processes (21, 39, 54); and during i n f e c t i o n , e l i m i n a t i o n of the gene 6 product (5' exonu-clease) leads to concatamer i n s t a b i l i t y (22, 52). F i f t h , d e n s i t y t r a n s f e r experiments, under the c o n d i t i o n s of no DNA s y n t h e s i s (D.O.), show a very i n e f f i c i e n t production of j o i n t molecules when compared to the number of matings during normal or i n f e c t i o n s (2, 83, 84). However, when r e p l i c a t i o n i s g r e a t l y decreased but not t o t a l l y e l i m -i n a t e d , recombination frequencies approach normal l e v e l s (5, 35, 52). Sixth, and l a s t , during cross r e a c t i v a t i o n experiments, markers which r e p l i c a t e most e f f i c i e n t l y are those which recombine most e f f i c i e n t l y (10). A l l of the above i n f o r m a t i o n i n d i c a t e s that some DNA s y n t h e s i s i s a s s o c i a t e d w i t h normal l e v e l s of genetic recombination. C. Recombination i n v i t r o A unique approach to understanding the molecular aspects of recombination i s found i n Sadowski's i n v i t r o recombination system (61, 63). An important f e a t u r e of t h i s system i s that packaging can be separated from i n i t i a l recombination events. While concatamers package more e f f i c i e n t l y , i t i s s t i l l p o s s i b l e to use s i z e one DNA as a packaging s u b s t r a t e (36). Two pathways of recombination have been r e c e n t l y reported (61). One pathway r e l i e s on the T^ gene 6 product (5' exonuclease). The second pathway i n v o l v e s genes 3 (endonuclease) and 5 (DNA polymerase) and can be i n h i b i t e d by the presence of the T^ gene 4 product. How a c c u r a t e l y these pathways resemble i n v i v o c o n d i t i o n s , and to what extent each plays during normal T^ i n f e c t i o n s a r e - d i f f i c u l t ; . t o determine. Elements from each of the above systems s t r o n g l y i m p l i c a t e the a c t i v e p a r t i c i p a t i o n of DNA 4 synthesis i n normal genetic recombination events. Several possible explanations e x i s t f o r t h i s conclusion. The absence of DNA synthesis could lead to a pool of DNA molecules too small to i n t e r a c t e f f i c i e n t l y during the recombination process. A l t e r -n a t i v e l y , the conditions used to block DNA d u p l i c a t i o n (D.O.) might also block recombination events. For example, D.O. conditions are frequently established by i n f e c t i n g an amber phage into a sup° host. If the en-zyme made defective by the amber mutation i s also required for recom-bination, then both processes w i l l be eliminated, e.g., T^ gene 32 product. Next, i t i s possible that a r e p l i c a t i v e intermediate i s normally used to i n i t i a t e recombination events. This may he viewed as a "passive" r o l e f or DNA synthesis i n that r e p l i c a t i v e enzymes are not involved per se; rather, the ac t u a l r e p l i c a t i v e structure i s required; e.g., the r e p l i c a t i o n fork could provide a denatured s i n g l e -stranded region where homologous single-strand DNA could invade to form a t r i p l e x (59, 60, 66). Last, r e p l i c a t i o n could resolve intermediates to y i e l d stable recombinant molecules. In order to obtain d i r e c t evidence for a r o l e f o r r e p l i c a -t i o n i n the recombination process, a system was developed where DNA synthesis of the two recombining molecules could be c o n t r o l l e d independently. D. Plasmid - Phage Recombination The plasmid-phage recombination system was designed i n order to study the r e l a t i o n s h i p between r e p l i c a t i o n and recombination. In t h i s system, marker rescue of a g e n e t i c a l l y marked bacteriophage by a recombinant DNA plasmid carrying the corresponding wild-type 5 a l l e l e was used to quantitateirecombination events. The r e p l i c a t i o n of the two recombining molecules (plasmid and phage DNA) could be con-t r o l l e d independently by mutations i n the host and phage molecules re s p e c t i v e l y . For example, the product of T^ gene 5 i s the T ^ - s p e c i f i c DNA polymerase which i s absolutely required f o r phage DNA r e p l i c a t i o n (26, 54). Therefore, amber (am) or temperature s e n s i t i v e (ts) muta-tions i n gene 5 under non-permissive conditions prevent phage DNA syn^ . thesis (76)- Under these non-permissive conditions such a phage could not r e p l i c a t e u n t i l i t had recombined with a wild-type a l l e l e on a plasmid. Recombination between an am5 or ts5 phage and a plasmid carrying a wild-type gene 5 could be monitored by scoring f or the production of wild-type phage during the i n f e c t i o n process. Plasmid r e p l i c a t i o n could be co n t r o l l e d by a temperature s e n s i t i v e mutation i n the host bacterium (see r e s u l t s ) . By manipulating the temperature, the suppressor character of the host, and the type of i n f e c t i n g phage, various r e p l i c a t i o n conditions could be established within the system and recombination values determined. The r e s u l t s of experiments i n such a system are reported i n t h i s thesis. Several major conclusions a r i s e from t h i s work. F i r s t , at l e a s t one of the i n t e r a c t i n g molecules, plasmid or phage, must r e p l i c a t e i n order for marker rescue'.to occur. When both molecules are prevented from r e p l i c a t i n g , no rescue i s observed. Second, t h i s r e s u l t does not r e f l e c t the need f o r a c r i t i c a l amount of i n t r a -c e l l u l a r DNA, i . e . , does not r e f l e c t the need for a minimal number of molecules with the p o t e n t i a l f o r recombination. Third, the marker rescue r e s u l t does not r e f l e c t the absence of an enzyme common to the processes of DNA synthesis and recombination. These fac t s suggest that 6 DNA synthesis plays an a c t i v e r o l e i n the i n i t i a t i o n of recombination events. DNA synthesis has been postulated to i n i t i a t e strand d i s -placement by Meselson and Radding (47). A l t e r n a t i v e l y , Stahl (72) has postulated that newly r e p l i c a t e d progeny DNA i s d i r e c t l y trans-f e r r e d to a r e c i p i e n t DNA duplex. Several other parameters of recombination i n t h i s system have been investigated. The e f f e c t s of mutations i n T^. gene 6 have been analyzed. F i n a l l y , the e f f e c t of phage i n f e c t i o n on plasmid DNA has been determined. This thesis then, describes the properties of a novel genetic recombination system with the long term goal of e s t a b l i s h i n g the r o l e of r e p l i c a t i o n i n genetic recombination. 7 MATERIALS AND METHODS A. B a c t e r i a l Strains JE. c o l i B22 (sup°) was used as the non-permissive host for T^ phage amber mutant. E. c o l i O i l ' , an E. c o l i B d e r i v a t i v e , c a r r i e d s u p E ^ and served as the permissive host for amber phage mutants. A l l b a c t e r i a l s t r a i n s used i n t h i s work are l i s t e d i n Table 1. Bacteria carrying plasmids are indicated as follows: X-pN, when pN represents the type of plasmid c a r r i e d by b a c t e r i a l s t r a i n X. Strains carrying plasmids were constructed by the methods of Wensink et a l . (88) or Cohen et a l (16). B a c t e r i a l plasmids are described i n Table 2. E_. c o l i CSR603 (recAl, uvrAl, phr-1, supE^) was used as the host bacterium for density s h i f t studies. This s t r a i n was trans-formed by the procedure of Wensink et a l (88) with: (i) pBR322 DNA, and ( i i ) pRS142 DNA which c a r r i e s Tn genes 13, 14 (68). B. Bacteriophage Strains A l l bacteriophage s t r a i n s were provided by F. W. Studier. The general designation for phage mutants i s as follows: amN-X or tsN-X where am stands for amber, ts stands for temperature se n s i -t i v e , N represents the gene number, and X represents the s p e c i f i c mutant. Amber mutants are described i n Studier (75). Density labeled phage were grown i n E. c o l i B^^ previously adapted to density media and prepared e s s e n t i a l l y by the method of Wolfson at al_ (91). 8 Table 1. B a c t e r i a l S t r a i n s STRAIN GENOTYPE REFERENCE F. W. St u d i e r (75) F. W. St u d i e r (77) B. Bachman (3) J . Wechsler (87) J . Campbell et a l . (11) A. J . C l a r k (15) D. Rupp (65) B23 O i l ' CR34 E279 HMS174 AB1157 CSR603 t h i , t h r , thyA, l e u , l a c Y , supE 44 CR34 dnaB ts + _R c rK12' ^ 1 2 ' r e c A 1 ' r i f » s u p t h i , t h r , thy, l e u , lacY, h i s , supE 44 recA, uvrA, phr, supE 44 Table 2. Plasmid DNA's PLASMID DESCRIPTION MOLECULAR WEIGHT* REFERENCE pBR322 r R cl o n i n g v e c t o r Tc , Amp 2.8 x 10 6 B o l i v a r e_t a l (6) pRS88 rescues T 7 amber 4-208 and ts4 phage 3.5 x 10 6 This paper PRS142 rescues T 7 ambers i n genes 13 and 14 (see Table ) 3.95 x 10 6 R. D. Smith (68) pRS202 rescues T ? amber 5-28 and ts5 phage 3.85 x 10 6 This paper Molecular Weights were determined by agarose g e l e l c t r o p h o r e s i s a gainst known molecular weight markers. Figure 1 demonstrates the banding l o c a t i o n of HH ts5 DNA r e l a t i v e to a LL phage DNA reference. C. Chemicals and Isotopes 5-fluordeoxyuridine (FdU), riboadenosine, thiamine, u r a c i l , and t e t r a c y c l i n e (Tc) were purchased from Sigma Chemical Company. Pronase and thymidine were purchased from Worthington Company. Agarose and Agarose A50M were purchased from Bio-Rad Company. Sephadex G100 3 was purchased from Pharmacia. Radioactive isotopes, [methyl- H ]-thy-32 32 midine, H^ PO^ and a , P-dNTP's were purchased from New England Nuclear Corporation. R e s t r i c t i o n enzymes were purchased from New England Bio Labs. D2O and deuterated a l g a l hydrolysate were obtained from Merck, Sharp, and Dohme. D. Media and Buffers Radioactively labeled phage were prepared i n TCG media (4). 32 3 P-labeled phage were prepared i n 1/5 PO^-TCG and H-labeled phage i n TCG containing 100 ug riboadenosine per ml, 20 ug u r a c i l per ml, 10 Ug FdU per ml, and 1 ug cold thymidine per ml. L-Broth contained 10 g of tryptone (Difco), 1 g of glucose, 5 g yeast extract (Difco) and 5 g NaCl per l i t e r with a f i n a l pH of 7.4. L-Broth plates and overlays contained 12 g and 7 g Difco agar, respectively. L-Broth was supplemented with 20 ug thiamine per ml and 10ugthymidine per ml where necessary. Density media contained per l i t e r of D^O: 7.0 g Na^HPO^, 3.0 g KH 2P0 4, 1.0 g 1 5NH 4C1, 0.5 g NaCl, 0.3 ml of 0.1M F e C l 3 , 1.0 ml of 1M MgS07, 0.1 ml of 1M C a C l 9 , 12.5 ml of 20% glucose, and 1.12 ml Figure 1. CsCl Analysis of HH Phage DNA. HH ts5 phage DNA was prepared i n density media containing 14 C-thymidine. Labeled phage were p u r i f i e d by a previous method (75) and DNA extracted with phenol followed by extensive ether washing. LL DNA was prepared as previously described. Samples of each DNA were analyzed i n a CsCl density gradient centrifuged i n a Beckman Type 65 rotor at 44,000 rpm for 48 hours. 60 seven-drop f r a c t i o n s were c o l l e c t e d , washed i n cold 5% TCA, r i n s e d . i n ethanol, dried, and radio-a c t i v i t y determined by s c i n t i l l a t i o n counting. t - § •= 1 4C-HH ts5 DNA A- • = 3H-LL T ? + DNA 12 P E R C E N T L E N G T H O F G R A D I E N T of deuterated a l g a l hydrolosate. TNE contained 0.01M T r i s - H C l pH 7.4, 0.15M NaCl, and 0.015M EDTA pH 7.4. T ? T r i s - s a l t contained 1.0M NaCl and 0.05M T r i s - H C l pH 7.4. Standard s a l i n e c i t r a t e (SSC) was 0.15M NaCl, 0.015M Na-c i t r a t e pH 7.0. Pr e - i n c u b a t i o n Mixture (PM) contained 0.02% F i c o l l , 0.02% BSA, 0.02% p o l y v i n y l p y r o l i d o n e , 0.1% SDS, and 5 yg thymidine per ml i n 3 x SSC (17). E. I s o l a t i o n and P u r i f i c a t i o n of Plasmid DNA's E. c o l i s t r a i n s c a r r y i n g plasmid molecules were grown i n LB supplemented w i t h 10 jig T e t r a c y c l i n e per ml at 30°C to a de n s i t y of 9 10 b a c t e r i a per ml. Chloramphenicol was added to a f i n a l c o n c e n t r a t i o n of 200 yg/ml. Cul t u r e s then were incubated an a d d i t i o n a l 12 hours. C e l l s were harvested by c e n t r i f u g a t i o n i n a Beckman Type 15 r o t o r at 8000 rpm f o r 20 minutes. C e l l s were gently l y s e d w i t h l y s o z y m e - t r i t o n . Cleared l y s a t e s were placed i n CsCl-ethidium bromide g r a d i e n t s and ce n t r i f u g e d f o r 72 hours at 33,000 rpm. DNA was removed w i t h a s y r i n g e , e x t r a c t e d w i t h n-butanol, and d i a l y z e d against 20 mM T r i s - H C l ph 7.4, 20 mM NaCl, 1 mM EDTA. F. S i n g l e Step Marker Rescue Experiment Plasmid c o n t a i n i n g b a c t e r i a were grown from a 1:40 d i l u t i o n of a f r e s h overnight c u l t u r e i n LB supplemented w i t h 10 yg thiamine per ml and 50 yg thymidine per ml to a d e n s i t y of 2 x 10 c e l l s / m l a t 30°C. I n f e c t i n g phage were added to a l i q u o t s of the growing c u l t u r e at an M.O.I, of 5.0. At 6.5 minutes p o s t - i n f e c t i o n , i n f e c t i v e centres and s u r v i v o r s were p l a t e d through T 7 a n t i s e r a . o n v a r i o u s i n d i c a t o r b a c t e r i a . A l s o , at 6.5 minutes i n f e c t e d c e l l s were d i l u t e d through a n t i s e r a and incubated to l y s i s . Phage were t i t r e d under permissive and non-permissive c o n d i t i o n s . Recombination frequencies., were c a l -c u l a t e d as the number of wi l d - t y p e phage d i v i d e d by the t o t a l phage y i e l d . In order to compare l e v e l s of marker rescue that were inde-pendent of bu r s t s i z e , the percentage of c e l l s y i e l d i n g w i l d type i n f e c t i v e centres was measured by p l a t i n g i n f e c t i v e centres a t 6.5 minutes under permissive and non-permissive c o n d i t i o n s . G. Marker Rescue Spot Tests A marker rescue t e s t on p l a t e s was developed as a f a s t screening method f o r d e t e c t i n g the genetic markers c a r r i e d by recom-binant plasmid molecules. LB agar p l a t e s were seeded w i t h a lawn of non-permissive b a c t e r i a . 2 ul of va r i o u s d i l u t i o n s of plasmid c a r r y -ing b a c t e r i a were overspotted w i t h 2 ul of amber phage. The t e s t was considered p o s i t i v e when plaques arose from spots c o n t a i n i n g recom-binant plasmid s t r a i n s and no plaques appeared on the c o n t r o l p l a t e s which contained c e l l s w i t h pBR322 alone at an equivalent d i l u t i o n . H. Copy Number A n a l y s i s ( l ) P u r i f i c a t i o n of T o t a l I n t r a c e l l u l a r DNA C e l l s c o n t a i n i n g plasmid DNA were grown.in supplemented LB at 30°C to a d e n s i t y of 2 x 10^/ml. At v a r i o u s times, 10 ml samples were withdrawn, c e n t r i f u g e d , washed i n 20mM T r i s - H l pH 7.4, 50 mM NaCl, 1 mM EDTA, and concentrated 10-fold. C e l l s were l y s e d w i t h 0.1% SDS plus 1 mg pronase per ml at 37°C f o r 12 hours. Lysates were e x t r a c t e d w i t h an equal volume of phenol and e x t e n s i v e l y ether washed. (2) L a b e l i n g of I n t r a c e l l u l a r DNA An a l i q u o t of p u r i f i e d i n t r a c e l l u l a r DNA, equivalent to 1 y g of A-,_ m a t e r i a l , was l a b e l e d i n v i t r o by n i c k t r a n s l a t i o n (4 5). zoU Labeled DNA was separated from r a d i o a c t i v e n u c l e o t i d e s by passing the r e a c t i o n mixture over a G100 Sephadex column and c o l l e c t i n g the appropriate m a t e r i a l . (3) P r e p a r a t i o n of F i l t e r s and H y b r i d i z a t i o n Conditions P u r i f i e d plasmid and chromosomal DNAs were bound to n i t r o -c e l l u l o s e membranes by the method of Denhardt (17). H y b r i d i z a t i o n s were performed i n PM b u f f e r (0.2 mg/ml F i c o l l , 0.2 mg/ml polyvinylpyro± l i d o n e , 0.2 mg/ml BSA, 0.01 mg/ml thymidine, 0.1% SDS i n 3 x SSC). F i l t e r s were incubated 4 hours at 65°C i n PM p r i o r to a d d i t i o n of the la b e l e d probe. Labeled DNAs were heat denatured and were added d i r e c t l y to the pre-incubated f i l t e r s . H y b r i d i z a t i o n s proceeded 15 hours at 65°C. F i l t e r s were then e x t e n s i v e l y washed i n 3 x SSC, d r i e d , and the r a d i o a c t i v i t y bound was determined by s c i n t i l l a t i o n counting. Under our c o n d i t i o n s plasmid f i l t e r s contained 2.5yg of bound DNA which was 100 times greater than the amount of:.:input DNA h y b r i d i z e d to f i l t e r s w i t h an e f f i c i e n c y of 35%. Hyb r i d i z e d f i l t e r s contained at l e a s t 50 times the amount of r a d i o a c t i v i t y compared to background f i l t e r s ; e.g., plasmid f i l t e r s g e n e r a l l y contained over 10,000 cpm whi l e background f i l t e r s contained 100 to 200 cpm. (;4 ) Copy Number Determination 32 Copy number was determined by h y b r i d i z i n g P-labeled t o t a l i n t r a c e l l u l a r DNA w i t h p u r i f i e d host and plasmid DNA f i x e d to separate n i t r o c e l l u l o s e f i l t e r s . The r a t i o of the r a d i o a c t i v i t y on the plasmid and host genome f i l t e r s r e f l e c t e d the copy number of the plasmid. In order f o r the r a t i o of r a d i o a c t i v i t y to a c c u r a t e l y r e f l e c t the copy number i t was necessary that plasmid and host DNA molecules be l a b e l e d w i t h the same e f f i c i e n c y during n i c k t r a n s l a t i o n . P u r i f i e d samples of host genome and plasmid DNA were prepared by banding each i n two successive CsCl-ethidium bromide g r a d i e n t s , e x t r a c t i n g w i t h n-butanol to remove ethidium bromide and d i a l y z i n g against 20 mM T r i s pH 7.4, 20 mM NaClj.lmM EDTA. These samples then were t r e a t e d w i t h RNase at a f i n a l c o ncentration of 20 yg per ml. RNase t r e a t e d samples were phenol e x t r a c t e d , ether washed, ethanol p r e c i p i t a t e d and resuspended i n b u f f e r . Equal A 0,~ amounts of each sample were l a b e l e d i n v i t r o zou 32 w i t h a P-dATP by n i c k t r a n s l a t i o n . 1 0 y 1 a l i q u o t s of l a b e l e d 32 m a t e r i a l were e x t e n s i v e l y washed i n co l d 5% TCA and P i n c o r p o r a t i o n determined by s c i n t i l l a t i o n counting. The i n c o r p o r a t i o n of l a b e l i n t o each species was found to be i d e n t i c a l . Therefore, i n experimental samples where t o t a l i n t r a c e l l u l a r DNA was l a b e l e d , the r a t i o s obtained could be i n t e r p r e t e d d i r e c t l y . Plasmid to chromosome r a d i o a c t i v i t y r a t i o s a f t e r h y b r i d i z a -t i o n were converted i n t o copy numbers v i a a standard curve. Standard curves were constructed by mixing known r a t i o s of l a b e l e d plasmid and chromosome molecules, h y b r i d i z i n g these samples w i t h n i t r o c e l l u l o s e membranes c a r r y i n g E. c o l i DNA, plasmid DNA, and no DNA at 65°C f o r 15 hours,(Figure 2). The reconstructed r a t i o s represented copy numbers Figure 2. Standard Curve for the Analysis of Hybridization Ratios. A standard curve was constructed by mixing known r a t i o s of 32 P-labeled plasmid and host chromosomal DNA. Ratios were constructed to represent copy numbers of 10, 20, 30, 40, and 50. These mixtures of DNA were hybridized i n v i a l s containing a set of 3 f i l t e r s : (1) plasmid DNA, (2) host DNA, (3) no DNA. Two concentrations of input DNA were hybridized i n PM at 65°C for 12 hours. The amount of input DNA was at l e a s t 100 X le s s than the amount of DNA on each plasmid f i l t e r . The above f i g u r e represents a t y p i c a l standard curve. The 32 h y b r i d i z a t i o n r a t i o can be defined as: cpm P plasmid DNA hybridized 32 cpm P host DNA hybridized Both concentrations of input DNA yielded l i n e a r and very s i m i l a r l i n e s , showing that the DNA on the f i l t e r was i n adequate excess. A = plasmid DNA bound to f i l t e r i s at 200 f o l d excess at copy number of 50 • = plasmid DNA bound to f i l t e r i s at 100 f o l d excess at copy number of 50 18 based on molecular weights of 2 x 10 f o r the E. c o l i genome and 3.85 x 10 f o r pRS202 DNA. The experimental h y b r i d i z a t i o n r a t i o s obtained were compared to the constructed r a t i o of molecules; then these values were used to c a l c u l a t e the copy numbers obtained i n experimental samples. ( 5 ) Copy Number A n a l y s i s i n T^ Inf e c t e d C e l l s E279-pRS202 was grown i n LB at 30°C to a den s i t y of 5 x 10 ? c e l l s per ml. C e l l s were p e l l e t e d by c e n t r i f u g a t i o n , washed i n TCG media, c e n t r i f u g e d , and resuspended i n TCG co n t a i n i n g 250 yg KH^PO^ per ml, 10 yg thiamine per ml, 5 yg thymidine per ml, 100 yg adenosine per ml, 10 yg 5-fluordeoxyuridine per ml, and 20 yg u r a c i l per ml. 3 H-thymidine was added to a f i n a l c o n c e n t r a t i o n of 50 y d per ml. The c u l t u r e then was incubated at 30°C and 42°C to a den s i t y of 3x10^ c e l l s per ml. At t h i s time, the c u l t u r e was s p l i t and incubated a t 30°C and 42°C f o r 15 minutes. C e l l s were i n f e c t e d w i t h [ P ] - T 7 am5 phage ( s p e c i f i c a c t i v i t y = 2.0 mCi/mg P) at an M.O.I, of 5.0. 2.0 ml samples were removed at va r i o u s times, i c e c h i l l e d , c e n t r i f u g e d , and resuspended i n 0.02 M T r i s , 0.15M NaCl pH 7.4. 50 y i a l i q u o t s were removed and p l a t e d f o r i n f e c t i v e centres. 10 y l a l i q u o t s were removed and the c o l d TCA p r e c i p i t a b l e r a d i o a c t i v i t y was determined. The remainder of each sample was incubated w i t h 0.01M EDTA,.0.1% SDS, and pronase. A f t e r l y s i s , phenoland ether washing, equal volume a l i q u o t s were removed and h y b r i d i z e d a t 65°C i n PM b u f f e r (Denhardt, (17) ) to f i l t e r s c o n t a i n i n g pBR322 DNA. H y b r i d i z a t i o n s were performed at s e v e r a l input DNA concentrations w i t h the f i l t e r bound DNA being at l e a s t 100 f o l d excess. A f t e r h y b r i d i z a t i o n , f i l t e r s were washed i n 20 3 x SSC, dried, and the bound r a d i o a c t i v i t y determined. A l l f i l t e r s contained at l e a s t 1,000 cpm, and background h y b r i d i z a t i o n was l e s s 3 than 50 cpm. Bound H-labeled material was l i n e a r with respect to input. Values of annealed r a d i o a c t i v i t y were divided by recovered 32 i n f e c t i v e centres or recovered P-labeled i n t r a c e l l u l a r DNA. These d i v i s i o n s yielded i d e n t i c a l r e s u l t s and c a l i b r a t e d a l l samples for recovery of i n t r a c e l l u l a r DNA. Copy numbers before i n f e c t i o n were determined as outlined above and calculated a f t e r i n f e c t i o n i n r e l a t i o n 3 to the normalized H-thymidine-labeled material annealed to f i l t e r bound plasmid DNA. I. Density Transfer Experiments i n the M a x i c e l l System A fresh stationary phase culture of CSR603-pRS142 was g d i l u t e d 1:20 i n L-Broth, and grown to a density of 2 x 10 c e l l s per ml at 35°C. C e l l s were p e l l e t e d and resuspended i n TCG media con-ta i n i n g 1 yg Thd per ml. The culture was exposed to UV l i g h t at a -2 -1 distance of 8.8 cm for 25 seconds at a fluence rate of 0.5 - J m s . The culture was then incubated at 35°C for 7.5 hours i n the dark. (1) Continuous Labeling The incubated culture was s h i f t e d to 43°C and infected with HH ts5 phage at an M.O.I. = 12.0. At 6 minutes p o s t - i n f e c t i o n , an a l i q u o t was removed and plated through T ? antisera f o r i n f e c t i v e 3 centres. At 10 minutes p o s t - i n f e c t i o n , H-thymidine was added at 30 y C i per ml. Samples were removed at various times for a n a l y s i s . (2) Pre-Labeling 3 A f t e r 7.5 hours of i n c u b a t i o n , H-thymidine was added at 30 y d per ml f o r 45 minutes. The c u l t u r e was washed w i t h l a r g e volumes of c o l d TCG, c e n t r i f u g e d , and the c e l l s resuspended i n TCG co n t a i n i n g 10 yg thymidine per ml. The c u l t u r e was s h i f t e d to 43°C and i n f e c t e d w i t h HH ts5 phage at an M.O.I. = 12.0. I n f e c t i v e centres were p l a t e d as i n d i c a t e d above and samples were removed f o r a n a l y s i s . (3) CsCl E q u i l i b r i u m C e n t r i f u g a t i o n Samples to be analyzed were l y s e d by SDS-pronase, phenol e x t r a c t e d , and washed w i t h ether. Samples were mixed w i t h a s o l u t i o n of saturated CsCl i n a r a t i o of 1:3, r e s p e c t i v e l y . A n a l y t i c a l g r a dients contained 1.5 ml sample plus 4.5 ml saturated CsCl. C e n t r i -f u g a t i o n was c a r r i e d out i n a Beckman Type 65 r o t o r f o r 48 hours at 44,000 rpm. P r e p a r a t i v e g r a d i e n t s were run i n a Beckman Type 50.2 r o t o r f o r 72 hours at 36,000 rpm. Gradients were c o l l e c t e d from the bottom of each tube. F r a c t i o n s were c o l l e c t e d onto 3mm paper, washed i n c o l d 5% TCA, washed i n 95% ethanol, d r i e d , and the r a d i o a c t i v i t y determined by s c i n t i l l a t i o n counting. RESULTS Sec t i o n I. Plasmid-Phage Marker Rescue A. C h a r a c t e r i z a t i o n of Clones C a r r y i n g T 7 Gene 5 Four hundred a m p i c i l l i n s e n s i t i v e , t e t r a c y c l i n e r e s i s t a n t clones were test e d f o r i n s e r t e d T 7 DNA by a f f i x i n g the DNA of each clone onto n i t r o c e l l u l o s e membranes by the method of Grunstein and 32 Hogness (27). These membranes then were h y b r i d i z e d w i t h P-labeled T 7 + prepared by n i c k t r a n s l a t i o n . 380 out of the 400 clones h y b r i d -i z e d the T ? DNA probe. In order to i s o l a t e clones c o n t a i n i n g T 7 gene 5, 2 yg of T 7 Hpa I fragment D (Figure 3) were i s o l a t e d from a 1.5% agarose g e l . By removing the p o r t i o n of the g e l c o n t a i n i n g the D fragment, e l e c t r o -e l u t i n g the DNA i n t o a d i a l y s i s membrane, phenol e x t r a c t i n g and ether washing the e l u a t e , p u r i f i e d Hpa I fragment D was obtained. This DNA was l a b e l e d i n v i t r o by n i c k t r a n s l a t i o n and used as a h y b r i d i z a t i o n probe on Grunstein-Hogness type colony f i l t e r s c a r r y i n g 200 recombinant clones. F i f t e e n clones h y b r i d i z e d the fragment D probe and four were s e l e c t e d f o r f u r t h e r a n a l y s i s . Marker rescue t e s t s i n d i c a t e d that a t l e a s t one clone, CR34-pRS202, contained T ? gene 5. CR34-pRS202 rescued T ? am.5-28 and T ? -2 -2 ts5 w i t h frequencies of 1.42 x 10 and 2.1 x 10 , r e s p e c t i v e l y . D i f f e r e n c e s i n rescue frequencies a r i s e due to the p o s i t i o n of mutations r e l a t i v e to the inserted T 7 DNA and the v e c t o r DNA. The e f f i c i e n c y of rescue i s decreased f o r genetic markers l o c a t e d near ve c t o r DNA because there i s l e s s homologous f l a n k i n g DNA f o r crossing-over. This e f f e c t was a l s o seen by Campbell et a l (11). B. C h a r a c t e r i z a t i o n of Clones C a r r y i n g Gene 4 gene 4 i s a l s o l o c a t e d on Hpa I fragment D,(Figure 3). Therefore, the f i f t e e n clones which were p o s i t i v e f o r h y b r i d i z a t i o n w i t h the fragment D probe were t e s t e d f o r t h e i r a b i l i t y to rescue T^ mutants i n gene 4. One clone, CR34-pRS88 was found to rescue T^ -3 -3 am4-208 and T^ ts4 w i t h frequencies of 4 x 10 and 6:xl0 , r e s p e c t i v e l y . C. '.-The Plasmid-Phage Recombination System S a t i s f i e d that pRS202 would rescue amber dr ts mutants w i t h i n gene 5 and that pRS88 would rescue amber or ts mutants i n gene 4, a system was d e s i r e d such that the r e p l i c a t i o n of the two i n t e r -a c t i n g DNA s p e c i e s , i . e . T^ and plasmid, could be manipulated inde-pendently during the marker rescue experiments. (1) T-, R e p l i c a t i o n I t has been shown p r e v i o u s l y that amber or ts mutations i n genes 4 and 5 are D.O., i . e . they are d e f e c t i v e i n DNA s y n t h e s i s under non-permissive c o n d i t i o n s (71). The product of gene 5 i s the Ty s p e c i f i c DNA polymerase (26, 54). Density t r a n s f e r experiments have shown that not even one round of DNA r e p l i c a t i o n occurs i n a :gene 5 mutant under non-permissive c o n d i t i o n s (R. C. M i l l e r , J r . , unpublished o b s e r v a t i o n s ) . 24 Figure 3. Map of Hpa I fragments re l e a s e d from DNA. (A) Approximate p o s i t i o n of genes (46). (B) Hpa I r e s t r i c t i o n fragments of DNA (46). (C) T 7 map u n i t s . 25 to 10 -31 ro -35 —140 -89 -3-—149 co 65 __—3 co r-CD IO -^28 ro cvj in ro' CD L U "CO a o o o CD O "CO O co IO o "ro O cvj < CO o (2) Plasmid R e p l i c a t i o n pBR322 i s a composite plasmid constructed by Bolivar e_t a l -(6). from C o l E l and pSClOl. The nucleotide sequence for pBR322 i s known (80). The copy number of pBR322 i s amplifiable i n the presence of chloramphenicol as i s the plasmid ColEl which suggests that t h e i r mode of r e p l i c a t i o n may be s i m i l a r . Several authors have determined the e f f e c t s of various E_. c o l i DNA synthesis mutants on Col E l replica-^:, t i o n (24, 25, 39). The e f f e c t of several DNA synthesis mutations on pSClOl r e p l i c a t i o n has also been documented (28, 29). The e f f e c t s of the E_. c o l i dnaB mutation on pRS202 r e p l i -cation was examined. The .E. c o l i s t r a i n E279 was transformed with 3 pRS202. H-labeled thymidine incorporation was used to follow the t o t a l DNA synthesis i n these mutants. Figure 4 shows the r e s u l t s of t h i s a n a l ysis. DNA synthesis i n the dnaB mutant with and without the plasmid ceases immediately upon s h i f t to 42°C. I t follows that plasmid r e p l i c a t i o n ceases at 42°C i n a dnaB mutant. This conclusion i s confirmed by copy number experiments described later i n t h i s paper. Therefore, by varying the parameters of temperature, the suppressor character of the host and the type of i n f e c t i n g phage, independent con t r o l of phage and plasmid r e p l i c a t i o n was possible during marker rescue experiments. D. Marker Rescue Experiments In the experiments descibed here, recombinant plasmid mole-cules carrying a wild-type T 7 gene 5 a l l e l e rescue i n f e c t i n g amber 5 or ts5 phage by recombination to produce wild-type phage. The experiments described below investigate the r o l e of r e p l i c a t i o n i n the marker rescue Figure 4. T o t a l DNA synt h e s i s i n E279 and E279-pRS202. C e l l s were grown to 5 x 10^ per ml i n LB at 30°C, c e n t r i -fuged, washed, and resuspended i n TCG media c o n t a i n i n g 10 yg thiamine per ml, 5 yg thymidine per ml, and 100 yg riboadenosine per ml. C u l -o 8 3 tures were incubated at 30 C to a de n s i t y of 1 x 10 per ml. H-thymidine was added to a f i n a l c o ncentration of 10 y C i per ml. 100 y l samples were withdrawn at va r i o u s times. The c u l t u r e then was s p l i t and one-half was s h i f t e d to 42°C ( i n d i c a t e d by arrows) and samples were withdrawn. The TCA i n s o l u b l e m a t e r i a l was monitored by s c i n t i l -l a t i o n counting. (a) E279 at 30°C and 42°C; (b) E279 + pRS202 at 30°C and 42°C. IO b 1 0 X 10 CL O M lO o » X X 10 Q_ O CM 0 20 40 60 80 100 120 140 160 180 200 TIME (min) process by comparing l e v e l s of marker rescue when one or both of the i n t e r a c t i n g DNA molecules cannot r e p l i c a t e . (1) Marker Rescue When Both Phage and Plasmid Can Replicate Maximum marker rescue frequencies were established i n exper-iments where both phage and plasmid could r e p l i c a t e . CR34-pRS202, CR34-g pBR322, E279-pRS202, and E279-pBR322 were grown to 2 x 10 c e l l s per ml and infected with either am5 or ts5 at an M.O.I, of 5.0. I n f e c t i v e centres and progeny phage were plated under conditions per-missive f o r the parental phage. The frequency of marker rescue was quantitated by several procedures. F i r s t , the number of wild-type phage produced during i n f e c t i o n was divided by the t o t a l number of progeny phage l i b e r a t e d . A second method of expressing rescue f r e -quencies was to divide the number of wild-type phage produced by the number of i n f e c t i v e centres (under permissive conditions the i n f e c t i v e centres were always greater than 90% of the c e l l s ) . A t h i r d approach was to divide the number of i n f e c t i v e centres detected under non-permissive conditions by the number of c e l l s infected. The r e s u l t s as expressed by methods two and three appear i n Table 3. Interpretations of marker rescue frequencies generated by a l l three procedures are identical.. Measuring marker rescue frequencies by i n f e c t i v e centres or progeny phage also yielded i d e n t i c a l r e s u l t s during c r o s s - r e a c t i v a t i o n studies (10). (2) Marker Rescue When Only Bacteriophage DNA Can Replicate Cultures of E279-pBR322 and E279-pRS202 were grown to a g density of 2 x 10 c e l l s per ml. One-half of each culture was s h i f t e d to 42°C and incubated f o r 20 minutes before i n f e c t i o n w i t h T 7 am5-28 phage. This 20 minute p e r i o d was s u f f i c i e n t time f o r DNA s y n t h e s i s i n the host c e l l s at 42°C to stop completely due to the dnaB mutation. The a n a l y s i s of marker rescue experiments showed a marked decrease i n the production of w i l d - t y p e phages at 42°C (Table 3, l i n e s d, f ) . Therefore, under c o n d i t i o n s when only phage DNA can r e p l i c a t e , fewer w i l d - t y p e phage are produced than when both plasmid and phage are r e p l i c a t i n g . (3) Marker Rescue When Only Plasmid DNA Can R e p l i c a t e To create the c o n d i t i o n where plasmid DNA was allowed to r e p l i c a t e but T 7 DNA was not, the s t r a i n s CR34-pBR322 and CR34-pRS202 g were grown to a d e n s i t y of 2 x 10 c e l l s per ml at which time one-h a l f of each c u l t u r e was s h i f t e d to 42°C. The s t r a i n s were incubated 20 minutes at 42°C p r i o r to i n f e c t i o n w i t h T 7 ts5 phage. Comparison of the rescue frequencies at 30°C and 42°C (Table 3, l i n e s j , 1) i n d i c a t e d that the absence of T 7 r e p l i c a t i o n d i d reduce the number of w i l d - t y p e phage produced; but the amount of marker rescue was s t i l l 45 times the background l e v e l . A s i m i l a r r e s u l t was obtained when the sup° s t r a i n s HMS174-pBR322 and HMS174-pRS202 were i n f e c t e d w i t h am5 phage. Under these c o n d i t i o n s , rescue frequencies are decreased s i g -n i f i c a n t l y (compare Table 3, l i n e s h and b ) , but are s t i l l more than 25 times the background (compare Table 3, l i n e s g and h). When only plasmid DNA can r e p l i c a t e , the production of w i l d type phage was reduced more than when only T 7 DNA was allowed to r e p l i cate. This e f f e c t could be a t t r i b u t e d to at l e a s t two phenomena. F i r s t , the r e p l i c a t i n g T 7 molecules may i n i t i a t e strand t r a n s f e r more Table 3. EFFECT OF DNA REPLICATION ON T^ MARKER RESCUE S t r a i n Phage Temper-ature R e p l i c a t i o n Phage/Plasmid Burst wt. 0 c e l l wt I.C. c e l l a. CR34-pBR322 am5 30°C + + • 135 .003 .0005 b. ,CR34-pRS202 am 5 30° + + 120 .86 .628 c. E279-pBR322 am5 30° + + 125 <.019 <.0008 d. E279-pRS202 am 5 30° + + 142 .55 .178 e. E279-pBR322 am5 42° + 48 <.016 .0003 f. E279-pRS202 am5 42° + 20 .19 .066 g- HMS174-pBR322 am 5 30° + 1.0 .004 <.0007 h. HMS174-pRS202 am5 30° + 4.4 . 166 .069 i . CR34-pBR322 ts5 30° + + 150 .014 j • CR34-pRS202 ts5 30° + + 195 .383 k. CR34-pBR322 ts5 42° + 16 .0065 1. CR34-pRS202 ts5 42° + 10 . 190 m. E279-pBR322 ts5 30° + + 95 .065 n. E279-pRS202 ts5 30° + + 144 .83 o. E279-pBR322 ts5. 42° - .07 .017 P- E279-pRS202 ts5 42° - . 14 .007 Table 3. g S t r a i n s of E_. c o l i were grown to 2 x 10 c e l l s / m l i n L broth. C e l l numbers were q u a n t i t a t e d by p l a t i n g and Petroff-Hausser counting. The c e l l s were i n f e c t e d w i t h the i n d i c a t e d 1n mutants at an M.O.I. = 5, incubated seven minutes at 30° or 42° and d i l u t e d .through a n t i s e r a . The i n f e c t e d c e l l s then were e i t h e r p l a t e d f o r i n f e c t i v e centres or incubated u n t i l l y s i s and p l a t e d f o r phage. I n f e c t i v e centres and progeny phage were t i t r e d under permissive and non-permissive c o n d i t i o n s . Phage and plasmid r e p l i c a t i o n were regulated by temperature and the presence of an amber suppressor. S t r a i n s are described i n Table-1. e f f i c i e n t l y than r e p l i c a t i n g plasmid molecules; and second, the T 7 gene product may be r e q u i r e d to r e p a i r j o i n t molecules i n forming covalent recombinants. For example, i n bacteriophage T^, the DNA polymerase (gene .43) i s r e q u i r e d to form covalent molecules. In the absence of covalent r e p a i r , some recombinants may not synthesize.the T 7 DNA polymerase (gene 5), which would be necessary f o r subsequent steps i n the r e p l i c a t i o n process. (4) Marker Rescue i n the Absence of R e p l i c a t i o n Since i t appeared that only one of the p a r t i c i p a t i n g mole-cules must r e p l i c a t e i n order to recombine DNA, i t was p o s s i b l e that the complete l a c k of r e p l i c a t i o n would stop the marker rescue process a l t o g e t h e r . This was t e s t e d by a n a l y z i n g w i l d - t y p e phage per c e l l from E279-pBR322 and E279-pRS202 at 42°C i n f e c t e d w i t h T ? ts5 phage. C e l l s were i n f e c t e d v20".minutes a f t e r s h i f t to 42°C. Under these c o n d i t i o n s a l l r e p l i c a t i o n was s h u t - o f f during the time course of i n f e c t i o n . Table 3, l i n e s n, p i n d i c a t e that no marker rescue i s d e t e c t a b l e above background^ when there i s no DNA s y n t h e s i s . S i m i l a r r e s u l t s are obtained when marker rescue i s measured i n a dnaB mutant c a r r y i n g plasmid pRS88, which normally r e s -cues T 7 ts4-101; at 42°C, there i s no rescue (Table 4, l i n e s e, f ) . Therefore, r e p l i c a t i o n plays a major r o l e i n the recombination pro-cess between the plasmid and phage DNA molecules. ^"Background i s defined i n these experiments as the amount of marker rescue obtained from c o - i n f e c t i o n s of E^ _ c o l l s t r a i n s c a r r y i n g pBR322 DNA. Table 4. REPLICATION AND MARKER RESCUE OF T-. GENE 4 MUTANTS S t r a i n Phage Temperature R e p l i c a t i o n wt 0 / c e l l Phage/.Plasmid a. CR34-pBR322 ts4 30° + + .005 b. CR34-pRS88 ts4 30° + + .050 c. E279-pBR322 ts4 30° + + .0065 d. E279-pRS88 ts4 30° + + .046 e. E279-pBR322 ts4 42° - - <.0018 f. E279-pRS88 ts4 42° - - <.0010 Table 4. g S t r a i n s of E_. c o l i were grown to 2 x 10 c e l l s / m l i n L broth. C e l l numbers were q u a n t i t a t e d by p l a t i n g and Petroff-Hausser counting. The c e l l s were i n f e c t e d w i t h t h e ^ n d i c a t e d T 7 mutants at an M.O.I, of 5, incubated seven minutes at 30 or 42 and d i l u t e d through a n t i s e r a . The i n f e c t e d c e l l s then were e i t h e r p l a t e d f o r i n f e c t i v e centres or incubated u n t i l l y s i s and p l a t e d f o r phage. I n f e c t i v e centres and progeny phage were t i t r e d under permissive and non-permissive c o n d i t i o n s . Phage and plasmid r e p l i c a t i o n were regulated by temperature and the presence of an amber suppressor. S t r a i n s are described i n Table 1. 34 Section I I . The Fate of Plasmid DNA A f t e r I n f e c t i o n . Introduction Experiments described above suggest a r o l e for DNA d u p l i -cation i n genetic recombination. Recombination was monitored by quantitating wild-type phage produced during marker rescue experiments. In the absence of p r i o r phage r e p l i c a t i o n there i s a burst of phage i f the plasmid can r e p l i c a t e ; there i s no burst i f the plasmid cannot r e p l i c a t e . I t follows that there must be a cross-over event between the plasmid (gene 5 +) and the phage ( gene 5 ) before the phage can r e p l i c a t e . If there i s no cross-over, then there i s no T 7 DNA poly-merase produced and hence, there i s no phage burst. The r e s u l t s i n d i c a t e that when either plasmid or phage DNA i s prevented from r e p l i -cating, recombination occurs but at a decreased l e v e l . However, when there i s no DNA synthesis there i s no detectable recombination. Due to the complex nature of T 7 i n f e c t i o n s , several a l t e r n a t i v e r o l e s for r e p l i c a t i o n must be examined. F i r s t , the number of DNA molecules i n the c e l l a v a i l a b l e f or recombination could be important i n determining rescue frequencies and t h i s would be af f e c t e d by r e p l i c a t i o n (or the lack of i t ) . Second, plasmid DNA or phage DNA could be degraded when prevented from r e p l i c a t i n g by endonuclease damage which f a i l s to be repaired. This would mean fewer DNA molecules would be a v a i l a b l e f or recombination and might cause lowered frequencies. Third, plasmid DNA could be cured from c e l l s i n the absence of r e p l i c a t i o n , again reducing the number of a v a i l a b l e molecules for recombination. Fourth, the appa-rent dependence of recombination on DNA synthesis could r e f l e c t a need for e i t h e r dnaB protein or the T 7 DNA polymerase (gene 5) for some d i r e c t r o l e i n recombination per se. Last, DNA r e p l i c a t i o n may play a d i r e c t r o l e i n recombination by providing a recombinogenic structure or re s o l v i n g a recombinogenic structure. The experiments described below are designed to descriminate among these p o s s i b i l i t i e s . A. Analysis of Plasmid DNA A f t e r Infection Infection by bacteriophage T 7 i s accompanied by the degra-dation of the host genome. T 7 genes 3 and 6 code for an endonuclease and exonuclease responsible for t h i s breakdown (12,34). The l i b e r a t e d host nucleotides are u t i l i z e d i n phage r e p l i c a t i o n . In order to e s t a b l i s h the i n t e g r i t y of plasmid DNA molecules a f t e r i n f e c t i o n , HMS174-pRS202 (sup°) was infected with amber 5 phage at 30°C and the t o t a l i n t r a c e l l u l a r DNA was analyzed on agarose gels at various times a f t e r i n f e c t i o n . Degradation of host DNA was detecta-ble 20 minutes a f t e r i n f e c t i o n . This i s demonstrated by the conversion of the high molecular weight DNA near the o r i g i n of the gel to lower molecular weight fragments (Figure 5). A Southern b l o t (67) was made 32 from t h i s gel and hybridized with [ . P]rpBR322 DNA. The autoradiogram (Figure 6) showed that plasmid DNA escaped the degradative process since the plasmid molecules formed d i s c r e t e bands even 30 minutes a f t e r i n f e c t i o n . Densitometer tracings of the autoradiogram indicated that there was no loss of plasmid DNA up to 30 minutes a f t e r i n f e c t i o n . These r e s u l t s were confirmed by h y b r i d i z a t i o n analysis to be described. Figure 5. Electrophoretic analysis of plasmid DNAs a f t e r i n f e c t i o n . P u r i f i e d t o t a l i n t r a c e l l u l a r DNA was prepared from HMSl74-pRS202 infected with am5 phage. Equal aliquots were removed from the infected culture at: (A) 0 minutes; (B) 5 minutes; (C) 10 minutes; (D) 20 minutes: (E) 30 minutes p o s t - i n f e c t i o n . Samples of equal volume were analyzed on a 0.5% agarose gel containing 1 yg e t h i -dium bromide per ml. The arrow indicates the o r i g i n of the g e l . Figure 6. Autoradiogram of Southern b l o t containing plasmid DNAs. The DNAs from the agarose g e l i n Figure 5 were transferred by the method of Southern (67) to n i t r o c e l l u l o s e paper. This bound DNA was hybridized with [ 3 2P]-pBR322 DNA for 15 hours at 65°C. This method v i s u a l i z e s plasmid DNA molecules from samples removed at: (A) 0 minutes; (B) 5 minutes; (C) 10 minutes; (D) 20 minutes; (E) 30 minutes p o s t - i n f e c t i o n . The arrow indicates the o r i g i n of the g e l . 40 B. H y b r i d i z a t i o n A n a l y s i s of Plasmid Copy Numbers An a n a l y s i s of plasmid copy number i n CR34-pRS202 and E279-pRS202 was performed i n order to e s t a b l i s h the number of i n t e r a c t i n g plasmid molecules present during the ;temperature s h i f t s of a marker 32 rescue experiment. The copy number was determined by h y b r i d i z i n g P-l a b e l e d t o t a l i n t r a c e l l u l a r DNA w i t h p u r i f i e d host and plasmid DNA f i x e d to separate n i t r o c e l l u l o s e f i l t e r s . The r a t i o of the r a d i o -a c t i v i t y on the plasmid and host genome f i l t e r s r e f l e c t e d the copy number of the plasmid. Table 5 shows the copy numbers obtained w i t h the a n a l y s i s of CR34-pRS202 and E279-pRS202 during a temperature s h i f t regime. Since the i n f e c t i o n c y c l e of bacteriophage T 7 i s completed by 7 25 minutes at 42 C, these r e s u l t s i n d i c a t e that there i s no s i g n i f i c a n t decrease i n the number of plasmid molecules during the course of marker rescue experiments due to s h i f t i n g the. temperature from 30° to 42°C, i . e . t u r n i n g o f f r e p l i c a t i o n . C. E f f e c t of Phage I n f e c t i o n on Plasmid DNA The f a t e of plasmid DNA a f t e r i n f e c t i o n was examined by 3 l a b e l i n g plasmids continuously w i t h H-thymidine and q u a n t i t a t i n g the r a d i o a c t i v e m a t e r i a l annealing to plasmid DNA bound to n i t r o c e l l u -l o s e f i l t e r s . This procedure determined the t o t a l amount of plasmid DNA at any time a f t e r i n f e c t i o n ; the procedure had the ca p a c i t y to 32 measure both increases and decreases of plasmid DNA. P-labeled T 7 phage were used to i n f e c t the plasmid c a r r y i n g c e l l s so that an i n t e r n a l standard was a v a i l a b l e to monitor the recovery of i n t r a c e l l u -l a r DNA at each stage of the experiment. This approach e l i m i n a t e d s e v e r a l problems w i t h copy number a n a l y s i s a f t e r i n f e c t i o n . Copy Table 5. COPY NUMBER ANALYSIS IN NON-INFECTED CELLS Temperature Time CR34-pRS202 E279-pRS202 Copy # Copy # 30°C -15' 38 20 30° - 42°C 0' 38 20 42°C 40' 30 21 Table 5. CR34-pRS202 and E279-pRS202 were grown exactly as i n marker rescue experiments. Samgles were removed at the indicated times r e l a t i v e to the 30 C to 42 C temperature s h i f t . Samples were prepared as described i n the text. The copy number i s expressed as the number of plasmid molecules per genome. 42 number experiments normally measure r a t i o s of plasmid DNA to host DNA; however, host DNA i s degraded by enzymes coded f o r by T 7, so t r a d i t i o n a l copy numbers are not u s e f u l measurements a f t e r i n f e c t i o n . The d e t e r -3 mination of H-thymidine l a b e l e d DNA annealed to f i l t e r bound plasmids circumvents t h i s problem. Copy numbers before i n f e c t i o n can be r e l a t e d to plasmid molecules per c e l l a f t e r i n f e c t i o n by c o r r e l a t i n g the amount 3 of H-labeled m a t e r i a l annealed to f i l t e r bound plasmid DNA at any p a r t i c u l a r time. The r e s u l t s (Table 6) show that there i s no dra-matic change i n the amount of plasmid DNA per c e l l a f t e r i n f e c t i o n and are i n agreement w i t h the i n d i c a t o r s from the l e s s q u a n t i t a t i v e approach of h y b r i d i z a t i o n according to the procedure of Southern (67), which was used to examine the s t r u c t u r e of the plasmids a f t e r i n f e c t i o n . They confirm that the plasmid can r e p l i c a t e at 30°C and does not r e p l i -cate at 42°C i n a dnaB host. D. A n a l y s i s of T 7 DNA a f t e r I n f e c t i o n To ensure that n o n - r e p l i c a t i n g T 7 DNA was not degraded during marker rescue experiments, CR34-pRS202 and E279-pRS202 were 32 o i n f e c t e d w i t h P-labeled phage at 43 C. The DNA was analyzed by sucrose gradients at various times a f t e r i n f e c t i o n (Figure 7). This ana-l y s i s confirms that T 7 DNA molecules are not p r e f e r e n t i a l l y degraded i n E279 vs. CR34 at 43°C. Therefore, lower or non-existant recombination f r e -quencies are not due to the l o s s of T DNA under these c o n d i t i o n s . Table 6. HYBRIDIZATION ANALYSIS OF PLASMID DNA AFTER INFECTION Time A f t e r Temperature Plasmid Hybridization Copy Number Infec t i o n 0 min 30° 4.21 X i o " 2 20 5 min 30° 4.23 X i o " 2 21 15 min 30° 4.73 X i o ' 2 22 20 min 30° 5.34 X i o " 2 24 0 min 42° 3.2 X i o " 2 20 5 min 42° 3.28 X i o ' 2 21 10 min 42° 3.20 X i o " 2 20 15 min 42° 3.57 X i o " 2 22 20 min 42° 3.4 X i o " 2 21 Table 6. 3 E_. coli^were grown i n TCG containing H-thymidine and were infected with P-labeled phage; 2 ml aliquots were removed from the c u l -tures at various times, the DNA was extracted, and plasmid DNA was annealed to f i l t e r bound DNA. The amount of annealed ' plasmidJDNA was divided^by the recovered i n f e c t i v e centres or the amount of recovered P-labeled DNA. This c a l c u l a t i o n corrected values of the plasmid DNA as determined by h y b r i d i z a t i o n for recovery of i n t r a c e l l u l a r DNA (described i n Materials and Methods). 1 3 Plasmid h y b r i d i z a t i o n i s defined as the H-labeled plasmid radio-a c t i v i t y annealed to plasmid bound DNA divided by the recovered i n f e c t i v e centres or P-labeled DNA (cpm/cell). 2 The copy number i s calculated by multiplying the copy number deter-mined at 0 minute by the r a t i o of the plasmid h y b r i d i z a t i o n at 0 minute.; to the plasmid h y b r i d i z a t i o n at any other time. Figure 7. Sucrose gradient analysis of T 7 DNA at 43°C i n dnaB + and dnaB hosts. CR34-pRS202 and E279-pRS202 were grown i n LB to 3 x 10 8 c e l l s per ml, s h i f t e d to 43 C, and i n f ected with P—labeled T 7 ts5 phage (M.O.I. = 5.0). Samples were withdrawn at 10, 20, and 30 minutes p o s t - i n f e c t i o n . C e l l s were lysed with 0.1% SDS and pronase, extracted with phenol and washed with ether. DNA was analyzed on 5-20% neutral sucrose gradients. a = 10 minute sample from CR34-pRS202; b = 20 minute sample from CR34-pRS202; c = 30 minute sample from CR34-pRS202; d = 10 minute sample from E279-pRS202; e = 20 minute sample from E279-pRS202; f = 30 minute sample from E279-pRS202; 32 where t • = P-T 7 ts5, and 0 o = 3H-T-,+ reference DNA. P E R C E N T R E C O V E R Y 46 Section I I I . The Role of the T-, 5' Exonuclease (gene 6) i n Plasmid- Phage Recombination Introduction It has been shown that the T 7 gene 6 product i s necessary for genetic recombination of bacteriophage T 7 DNA (35, 57). Also, mutations i n gene 6 i n t e r f e r e with the normal processing of T 7 con-catemers (52). This data implicates the T 7 exonuclease i n several d i f f e r e n t facets of T 7 recombination. F i n a l l y , i t has been shown that i n v i t r o recombination can occur i n the presence of the gene 6 product alone, i . e . T 7 genes 3 , 4 , 5 (61). To determine i f t h i s exonuclease was required for plasmid-phage recombination, marker rescue experiments were conducted under conditions r e s t r i c t i v e for the gene 6 product. Since an amber 6 mutation under non-permissive conditions would prevent the processing of concatemers and thereby prevent a burst of phage, temperature s e n s i -t i v e mutants of gene 6 were used at various temperatures, ranging from permissive (30°C) to non-permissive (42°C). A. Marker Rescue and the E f f e c t of the T? Exonuclease The frequency of recombination was determined by the number of wild-type phage produced. Table 7 presents the data from such ex-periments and Figure 8 i s graphic representation of these r e s u l t s . As expected, the bursts decrease with increasing temperature. The back-ground l e v e l of recombination (Table 7, l i n e s a to d) i s s i m i l a r to r e s u l t s presented i n Section I. The e f f e c t of decreasing amounts of gene .6:;product. on ..phage-phage "recombination.: i s , presented ihrTable .7, 47 Table 7. THE EFFECT OF THE T 7 5' EXONUCLEASE ON PLASMID-PHAGE RECOMBINATION S t r a i n Phage Temper-ature R e p l i c a t i o n Phage/Plasmid Burst Frequency a. CR34-pBR322 am5ts6 30° + + 80 .0006 b. II II 37° + + 10 .001 c. i i II 40° + + 2 < .001 d. ii II 42° + + 0.2 < .001 e. CR34-pRS202 am5ts6 30° + + 200 .030 f. II II 37° + + 57 .043 g. II II 40° + " + 40 .063 h. II II II 42° + + 30 .073 i . CR34 am5ts6 x amlts6 30° + + 240 .120 j • II II 37° + + 50 .050 k. II II 40° + + 3 .050 1. II II 42° + + 0.8 .011 m. HMS174-pRS202 am5ts6 30° - + 4.0 .035 n. II II 37° - + 0.40 .015 o. II II 40° - + 0.25 .0008 P- II II 42° - + 0.15 .0009 q. CR34-pRS202 am5 30° + + 95 .040 r. II II 37° + • + 102 .080 s. II M 40° + + 110 .110 t. II II 42° + + 140 .202 Table 8. B a c t e r i a l s t r a i n s were i n f e c t e d w i t h T 7 mutant phage, d i l u t e d through T 7 a n t i -s era, incubated to l y s i s , and p l a t e d f o r phage. S t r a i n s are described xn Table 1. Frequencies are expressed as: Wild-type progeny . T o t a l progeny Figure 8. E f f e c t of T 7 5' exonuclease on plasmid-phage recombination. This f i g u r e i s a graphic r e p r e s e n t a t i o n of the values; presented i n Table 8. . I = am5 x CR34-pRS202 A"""A= am5ts6.;x CR34-pBR322 • ' • = am5ts6 x CR34-pRS202 CD~""~0= am5ts6 x amlts6; s u + host am5ts6 x HMS174-pRS202 T E M P E R A T U R E (*C) l i n e s i to 1; and demonstrates the dependence of th i s type of recom-bination on the exonuclease. In contrast however, plasmid-phage recombination does not show th i s same dependence (Table 7, l i n e s e to h). However, comparison of l i n e s h and t shows that i n the absence of the gene 6 product plasmid-phage_recombination i s decreased. Under conditions where plasmid DNA i s allowed to r e p l i c a t e and the phage i s prevented from doing so, the plasmid acts as the donor during recombination (under the constraints of models which include DNA synthesis as e s s e n t i a l . See Appendix). Table 7, l i n e s m to p show the r e s u l t s of such an experiment. This r e s u l t suggests that: ( i ) as the amount of exonuclease decreases, fewer gaps are made: i n the T 7 molecule; ( i i ) fewer gaps decrease the chance of invasion by a plasmid DNA si n g l e strand and therefore, ( i i i ) when no gaps are made on the T 7 molecules, no donor DNA can be assimilated. This argument suggests that T7DNA recombination does not proceed v i a "D" loop invasion (recA mediated) but rather by the invasion of a gap. This proposal i s supported by the f a c t that 1^-1^ recombination i s not affected by mutations i n the host recA gene (57). Plasmid-phage recombination does not appear to be hampered by a recA condition (see Section IV). To explain the increasing frequency values i n the absence of the gene 6 p r o t e i n when both molecules can r e p l i c a t e , the following i s proposed: ( i ) T 7 gene 6 protein creates gaps i n T 7 molecules; ( i i ) when both plasmid and phage can r e p l i c a t e (both could act as donors), the gene 6 product would not be required because T 7 DNA could i n i t i a t e events by invading the plasmid molecule. This could occur v i a a recA mediated path or gap formation by a host nuclease. ( i i i ) I t follows that the increasing frequencies seen i n t h i s case .reflect .the normal r e l a t i o n s h i p between temperature and recombination frequency (Table 7, l i n e s q to t ) . The f i n a l proof of such an argument would be to demonstrate that no recombination occured i n a density transfer experiment under gene 6 conditions. 52 Section IV. P h y s i c a l Analysis of Plasmid-Phage Recombination  Introduction In Sections I and II of t h i s t h e s i s , the requirement of r e p l i c a t i o n during marker rescue was demonstrated. Marker rescue i s a process d i r e c t l y dependent on genetic recombination. This section of the thesis w i l l explore the r o l e (s) that r e p l i c a t i o n plays during recombination events. DNA synthesis could be an e s s e n t i a l factor i n genetic recombination i n at le a s t four ways. F i r s t , DNA synthesis could pro-mote displacement of a parental s i n g l e strand which would then be assimilated into the r e c i p i e n t duplex. This mode of action has been proposed by Messelson and Radding (47). Second, newly synthesized progeny DNA could be displaced from a r e p l i c a t i o n fork and d i r e c t l y assimilated as proposed by Stahl (72). Third, a branch of the r e p l i -cation fork could be cut by an endonuclease which would be free to base p a i r i n a gap on the r e c i p i e n t duplex (see Figure 9). Fourth, r e p l i c a t i o n could be a factor i n r e s o l u t i o n of recombinant i n t e r -mediates. Density transfer experiments are described next, i n order to discriminate among these p o s s i b i l i t i e s . A. Density Transfer During Plasmid-Phage Recombination E_. c o l i CSR603 was chosen as the host bacterium i n these experiments f or the following reasons. Upon UV i r r a d i a t i o n , .E. c o l i recA uvrA c e l l s stop r e p l i c a t i o n and f a i l to r e p a i r damaged DNA (33). Also, the phr-1 mutation of CSR603 prevents photo-reactivation of thymine dimers. The net r e s u l t of exposing these c e l l s to UV followed . Figure 9. Strand Displacement. Representation of three ways i n which r e p l i c a t i o n could i n i t i a t e recombination events by displacement of a s i n g l e strand: (a) Progeny DNA could be displaced from the r e p l i c a t i o n fork by branch migration. (b) The action of an endonuclease ( ) could attack the s e n s i t i v e r e p l i c a t i o n fork and create a free parental strand, (c) DNA synthesis could dislodge parental DNA located adjacent to the r e p l i c a t i o n fork. 54 (a) 1 \ \ (b) (c) by incubation f or several hours i s the degradation of the host genome to nucleotides (65). I t was important i n these experiments that a minimal amount of l a b e l be incorporated into the host chromosome because a f t e r T 7 i n f e c t i o n , the host i s normally degraded to nucleo-tides which could be u t i l i z e d by the phage for ( i ) r e p l i c a t i o n and ( i i ) repair synthesis. Any l a b e l found incorporated into HH f u l l y labeled phage DNA by these two processes would mask l a b e l transferred by recombination. In addition, T 7 ts5 phage were used i n these i n -fections and the experiments were performed at a non-permissive tem-perature (43°C). T 7 gene 5 codes for the T 7 s p e c i f i c DNA polymerase and the previous density transfer experiments have shown that less than one round of r e p l i c a t i o n occurs under these conditions. There-fore, when a density labeled ts5 phage i n f e c t s a previously UV i r r a d i a t e d CSR603 c e l l , recombination between homologous regions of DNA on the phage and plasmid can be detected as r a d i o l a b e l at the HH locus i n CsCl gradients with a very minimal contribution of l a b e l from repair or r e p l i c a t i o n of the T 7 molecules. Figure 10 repre-sents the basic molecular event i n these experiments. Two a d d i t i o n a l f a c t s should be mentioned about t h i s system. (1) The dose of UV i r r a d i a t i o n was adjusted to allow degradation of the host genome, but leaving plasmid molecules i n t a c t (see Results). (2) Plasmid molecules are not degraded a f t e r phage i n f e c t i o n (see Results, Section I ) . Figure 10. The basic molecular events during a cross between heavy 3 labeled phage and H-labeled plasmid DNA's. Under the constraints of a density transfer experiment, the heavy labeled phage never r e p l i c a t e s because of the absence of the gene 5 product. The plasmid pRS142 contains T 7 genes 13 and 14, and recombination occurs i n t h i s area of the T 7 genome. The plasmid 3 molecules are labeled with H. Therefore, recombination between plasmid 3 and phage molecules manifests i t s e l f as H-label at the heavy l o c a t i o n i n a CsCl density gradient of DNA extracted from.cells a f t e r i n f e c t i o n . Two types of recombinants are shown. A j o i n t molecule consists of base paired inserted homologous DNA that i s not covalently attached to the r e c i p i e n t duplex. The covalent recombinant i s a completely repaired, i n t a c t duplex. covalent recombinant B. R e s u l t s (1) UV Dose Determination In order to determine a UV dose which would render chro-mosomal DNA degraded and leave plasmid DNA i n t a c t , two experiments were performed. F i r s t , the amount of DNA s y n t h e s i s was measured a f t e r exposure to UV and second, the amount of plasmid DNA a f t e r UV was determined by a h y b r i d i z a t i o n technique. Figure 11 shows the r e s u l t s of such an a n a l y s i s . A f t e r 10 seconds of UV exposure and 8 hours i n c u b a t i o n , the r a t e of DNA s y n t h s i s dropped by 95%. The maximum p l a s m i d / c o l i r a t i o was determined to be between 20 and 40 seconds of exposure time. The time chosen f o r d e n s i t y s h i f t e x p e r i -ments was 25 seconds. This time maximized the number of s u r v i v i n g plasmids and minimizeid the l a b e l i n g p o t e n t i a l of the host genome. A f t e r 25 seconds of UV exposure and 7/5 hours of i n c u -b a t i o n , there were: ( i ) 0.015% s u r v i v i n g v i a b l e c e l l s (Figure 12a); ( i i ) 60% i n f e c t i v e centres, and ( i i i ) b u rst s i z e s of 30 to 40 phage per i n f e c t i v e centre when c e l l s were i n f e c t e d w i t h T 7 w i l d - t y p e phage (Figure 12b). (2) Density Transfer w i t h Continuous L a b e l i n g A f t e r I n f e c t i o n To maximize the amount of l a b e l t r a n s f e r r e d from plasmid 3 DNA to the phage DNA, continuous l a b e l i n g w i t h H-thymidine was used during the . i n f e c t i o n process. The amount of t r a n s f e r p o s s i b l e can be estimated given the s i z e of the T 7 i n s e r t w i t h i n pRS142 and the rescue frequency of markers i n gene 14 and pRS142. The T 7 i n s e r t i s equivalent to 33% of pRS142. The rescue frequency, determined w i t h T ? 14 aml40, was found to be 0.12 at 43°C. The product of these Figure 11. Rate of DNA synthesis and plasmid/chromosome r a t i o s a f t e r UV exposure. The rate of DNA synthesis was determined by growing CSR603-g pBR322 to a density of 2 x 10 . c e l l s per ml, followed by exposure to UV l i g h t as indicated. Aliquots from each exposure time were pulse-3 labeled with 10 y£± H-thymidine per ml i n TCG media for 5 minutes. The amount of synthesis was measured by TCA i n s o l u b i l i t y . Ratios of Plasmid to Host Chromosome were determined by 3 extracting the DNA from c e l l s pulse-labeled with H-thymidine a f t e r UV exposure and h y b r i d i z i n g t h i s labeled DNA with n i t r o c e l l u l o s e contain-ing pBR322 DNA and E. c o l i chromosomal DNA. The h y b r i d i z a t i o n tech-nique i s outlined i n Materials and Methods. The a c t u a l cpms for plasmid DNA increased up to the 30-second point then began decreasing while chromosome cpms decreased constantly. • • = Rate of DNA synthesis 0-^  • = Hybridization Ratio R A T E O F D N A S Y N T H E S I S ( C P M X I O " 2 ) P L A S M I D / C H R O M O S O M E R A T I O o Figure 12. V...(a) Survivors a f t e r UV exposure. A culture of CSR603-pRS142 was grown to a density of g 2 x 10 c e l l s per ml, i c e c h i l l e d , and exposed to UV l i g h t (fluence -2 -1 rate of 0.5 J m sec ) for various lengths of time. Survivors were plated immediately on LB plates and incubated 12 hours at 35°C. (b) Burst s i z e determined a f t e r 25 seconds of UV exposure. g CSR603-pRS142 were grown to 2 x 10 c e l l s per ml, i c e c h i l l e d , and exposed to 25 seconds of UV l i g h t . The i r r a d i a t e d c e l l s were incubated for various times, infected with T 7 14 aml40 and the burst s i z e determined. Burst i s expressed as phage per .1 i n f e c t i v e centre. BURST SIZE (PHAGE/I.C.) values gave an estimated transfer of H to the HH l o c i as 4% of the recovered a c t i v i t y . The actual value could be higher because rescue frequency i s packaging dependent, where as the density transfer experi-ments are not. Figure 13 shows the CsCl gradient analysis of DNA a f t e r a continuous l a b e l i n g experiment. The d i s t r i b u t i o n of labeled material i n three separate experiments was i d e n t i c a l . The percentage of recovered material at the HH locus was found to be 1.85% at 20 minutes p o s t - i n f e c t i o n and 3.0% at 30 minutes p o s t - i n f e c t i o n . The amount of "HL" material at 20 and 30 minutes was found to be 1.8% and 1%, res p e c t i v e l y . With increasing time, the amount of HH material increased while the amount of "HL" material decreased. Control i n f e c t i o n s of CSR603-pBR322 determined the contribution of 3H at the HH and "HL" peaks due to ( i ) r e p l i c a t i o n of revertant ts5 phage, and ( i i ) repair 3 synthesis of the T 7 molecules. As shown, the amount of H at the HH and "HL" peaks i n the control i n f e c t i o n i s minimal.,Labeling was quanti-tated.. during the i n f e c t i o n by measuring the amount of TCA in s o l u b l e r a d i o a c t i v i t y and was found to s t e a d i l y increase i n t h i s type of experiment. 3 (3) The Fate of H-LL-DNA from Plasmids. Labeled Before In f e c t i o n CSR603-pBR322 and CSR603-pRS142 were exposed to UV, incubated 3 and labeled with H-thymidine. P r i o r to i n f e c t i o n , the c e l l s were washed thoroughly i n large volumes of TCG containing 10 yg cold thymidine per ml. The washed cultures were s h i f t e d to 43°C and infected with HH ts5 phage. TCA insoluble r a d i o a c t i v i t y was measured at various times a f t e r i n f e c t i o n and was found to remain constant. Figure.^14 shows the d i s -3 t r i b u t i o n of H-labeled DNA a f t e r CsCl density c e n t r i f u g a t i o n . Label Figure 13. D i s t r i b u t i o n of ~?H-labeled DNA i n CsCl g r a d i e n t s . CSR603-pBR322 and CSR603-pRS142 were exposed to UV l i g h t , incubated, s h i f t e d to 43°C, i n f e c t e d w i t h HH ts5 phage, and "^H-thy-midine added at 10 minutes p o s t - i n f e c t i o n . Samples were withdrawn at v a r i o u s times, l y s e d and c e n t r i f u g e d i n CsC l 48 hours at 44,000 rpm. 60 seven-drop f r a c t i o n s were c o l l e c t e d . 90% to 98% of the l a b e l was recovered from each gradient. Each gradient represents approximately 40,000 TCA p r e c i p i t a b l e counts per minute. (a) Sample at 20 minutes p o s t - i n f e c t i o n ; (b) sample at 30 minutes p o s t - i n f e c t i o n . • • = CSR603-pBR322 • = CSR603-pRS142 Figure 14. Analysis of prelabeled H-labeled DNA i n CsCl gradients. CSR603-pBR322 and CSR603-pRS142 were exposed to UV l i g h t , 3 incubated for 7.5 hours, labeled with H-thymidine for 45 minutes, washed thoroughly, s h i f t e d to 43°C and infe c t e d with HH ts5 phage. Samples were withdrawn at: (a) 15 minutes, (b) 20 minutes, (c) 30 minutes p o s t - i n f e c t i o n . 60 seven-drop f r a c t i o n s were c o l l e c t e d with a 95% recovery of a c t i v i t y . There were approximately 70,000 TCA p r e c i p i t a b l e cpm loaded onto each gradient A • = CSR603-pBR322 • • = CSR603-pRS142 P E R C E N T R E C O V E R Y — ro OJ * 6 6 6 6 _i i i - i at the HH locus decreased from 1.5% with time. The material at the "HL" locus decreased also. Control i n f e c t i o n s of CSR603-pBR322 showed minimal amounts of material at either l o c i . In contrast to a continuously labeled c u l t u r e , the majority 3 of TCA insoluble H i n t h i s type of experiment would be found i n parental plasmid DNA p r i o r to recombination. Label displaced to the HH and HL locations strongly suggested that parental DNA was being incorporated into the r e c i p i e n t T 7 molecules. To determine if. r e p l i -cation was responsible for the displacement of the parental DNA, the following experiment was performed. (4) The Lack of Density Transfer i n the Absence of Plasmid  R e p l i c a t i o n : Prelabeled CSR603-pRS142 c e l l s were s h i f t e d to 43°C and one-half of the culture was treated with 100 yg Novobiocin per ml p r i o r to i n f e c t i o n . Novobiocin has been shown to s p e c i f i c a l l y block r e p l i c a t i o n and not i n t e r f e r e with recombination (35). Therefore, a l l DNA synthesis i n t h i s system was halted p r i o r to i n f e c t i o n . The TCA insolu b l e radio-a c t i v i t y was constant during the i n f e c t i o n process. This experiment tested the hypothesis that DNA synthesis was required to i n i t i a t e recom-bination events. Figure 15 shows that without r e p l i c a t i o n , there was no l a b e l transferred to either the HH or HL l o c i , while the con t r o l (no 3 Novobiocin) experiment showed the t y p i c a l d i s t r i b u t i o n of H for a pre-la b e l i n g experiment. This r e s u l t shows that r e p l i c a t i o n i s absolutely required f o r the formation of j o i n t molecules. Figure 15. The fate of prelabeled JH-LL DNA with and without plasmid r e p l i c a t i o n . CSR603-PRS142 was exposed to UV l i g h t , incubated 7.5 hours, 3 labeled with H-thymidine for 45 minutes, washed thoroughly, s h i f t e d to 43°C, and infected with HH ts5 phage. P r i o r to i n f e c t i o n , one-half of the culture was treated with 100 yg Novobiocin per ml. Samples were removed at: (a) 15 minutes, (b) 20 minutes p o s t r i n f e c t i o n . 62 seven-drop f r a c t i o n s were c o l l e c t e d a f t e r c e n t r i f u g a t i o n . 45,000 TCA p r e c i p i t a b l e cpm were recovered, representing approximately 92% recovery from, each gradient. A • = 100 yg Novobiocin per ml. • • = No Novobiocin. (5) The F a i l u r e of H-Labeled DNA to be Tran s f e r r e d i n the  Absence of the T ? 5' Exonuclease Genetic r e s u l t s i n Secti o n I I I i n d i c a t e d that when only plasmid DNA could r e p l i c a t e , the T 7 gene 6 product was necessary f o r recombination. I t was post u l a t e d that the T 7 exonuclease was neces-sary to create a gap on the T ? molecule where a d i s p l a c e d plasmid s i n g l e strand could invade. I f t h i s were the case, then during a den s i t y t r a n s f e r experiment i n the absence of the T 7 exonuclease, no 3 H-LL DNA would be found at the HH or HL l o c i i n a CsCl gradient. CSR603-pRS142 was exposed to UV, incubated, s h i f t e d to 43°C and 3 i n f e c t e d w i t h e i t h e r HH ts5 or HH t s 5 t s 6 phage. H-thymidine was then added. Figure 16 shows the a n a l y s i s of molecules from such an 3 experiment i n a CsCl g r a d i e n t . As i n d i c a t e d , no H - l a b e l was t r a n s -f e r r e d to the HH or HI l o c i i n samples from t s 5 t s 6 phage-infected c e l l s . The c o n t r o l i n f e c t i o n (ts5) shows the normal d i s t r i b u t i o n of 3 H-labeled m a t e r i a l f o r a continuous l a b e l i n g experiment. In the absence of the gene 6 product, plasmid DNA f a i l s to be a s s i m i l a t e d i n t o the phage DNA. Therefore, the T 7 exonuclease i s r e q u i r e d to create gaps on the T 7 molecules which would be v u l n e r a b l e to a t t a c k by homologous s i n g l e stranded DNA. 3 (6) A n a l y s i s of H-Labeled M a t e r i a l from the HH L o c a t i o n i n  CsCl Gradients DNA was e x t r a c t e d from a l a r g e s c a l e p r e p a r a t i o n of con-t i n u o u s l y l a b e l e d CSR603-pRS142 i n f e c t e d w i t h HH ts5 phage at 43°C. Pr e p a r a t i v e CsCl gradients were run and f r a c t i o n s c o l l e c t e d i n t o micro-t i t r e t r a y s from the bottom of each tube. A 1/10 volume a l i q u o t of Figure 16. D i s t r i b u t i o n of H-LL DNA i n the presence and absence of the T 7 5' exonuclease. CSR603-pRS142 was exposed to UV l i g h t , incubated 7.5 hour s h i f t e d to 43°C, infe c t e d with phage and labeled with 3H-thymidine throughout i n f e c t i o n . Samples were withdrawn at: (a) 20 minutes, (b) 30 minutes p o s t - i n f e c t i o n , and analyzed i n CsCl density gradients 60 seven-drop f r a c t i o n s were c o l l e c t e d with 90% recovery. Each gradient represents 60,000 TCA p r e c i p i t a b l e cpm. t — • = HH ts5 • • = HH ts5ts6 each f r a c t i o n was assayed for H a f t e r a TCA/ethanol wash. Figure 17 shows a t y p i c a l preparative gradient pattern. Fractions from the HH lo c a t i o n were pooled and dialyzed against .TNE. Dialyzed HH material was rebanded i n a CsCl density gra-dient. Figure 18 shows the r e s u l t s of rebanding, and confirmed the 3 presence of H-labeled DNA at a l o c a t i o n heavier than the l i g h t p l a s -mid„DNA. Some material returned to the LL l o c a t i o n . This may be 3 due to the shearing of H plasmid DNA attached to HH phage DNA during handling of samples. 3 To determine the nature of the as s o c i a t i o n between H-3 l a b e l and the HH phage DNA, a sample of H-HH DNA was sonicated by a procedure which rendered s i z e one T 7 DNA to a molecular weight of approximately 1.5 x 10 d (Figure 19). The sonicated material was analyzed by ce n t r i f u g a t i o n i n a CsCl gradient. Figure 18b shows t h i s 3 analysis. Comparison of Figures 18 a and b indicated that the H-l a b e l which was stably associated with the HH phage DNA migrated to the HL l o c a t i o n upon sonication. This strongly suggests that the 3 heavy phage DNA contained a sing l e stranded H-LL DNA i n s e r t . To determine i f the suspected s i n g l e strand i n s e r t was 3 covalently attached to the phage duplex, H-HH DNA was analyzed i n sucrose gradients. Figure 20a shows that approximately 50% of the 3 recovered H l a b e l was associated with s i z e one T 7 DNA. Approxi-3 mately 30% of the H was located near the top of the gradient with the remainder; p o l y - d i s t r i b u t e d between these two regions. Treat-3 ment with 0.2N NaOH reduced the amount of s i z e one T 7 associated H 3 by 10% (Figure 20b) i n d i c a t i n g that approximately 40% of the H l a b e l i n the HH f r a c t i o n s was due to covalently sealed HH T 7 molecules con-3 tai n i n g a H-labeled s i n g l e stranded i n s e r t . I t was possible that the a l k a l i l a b i l e material (the 10% l o s t from the s i z e one T 7 asso-ciated peak) consisted of j o i n t molecules as defined i n Figure 10. The majority of the p o l y - d i s t r i b u t e d material was located near the top of the gradient a f t e r treatment with a l k a l i . Figure 17. A preparative CsCl gradient. Preparative gradients were centrifuged i n a Beckman Type 50.2 rotor f o r 72 hours at 36,000 rpm. 85 fifteen-drop f r a c t i o n s were c o l l e c t e d from the bottom of each tube. Aliquots were washed i n 5% TCA and ethanol, then dried. R a d i o a c t i v i t y was determined by s c i n t i l l a t i o n counting. Brackets i n d i c a t e the f r a c t i o n s pooled f o r further a n a l y s i s . 14' Figure 18. Rebanding and the e f f e c t of sonication on H-HH DNA. (a) A dialyzed sample was rebanded i n CsCl. 15,000 (70%) TCA p r e c i p i t a b l e cpm were recovered. The arrow indicates the 32 p o s i t i o n of the LL P reference DNA. 3 (b) A dialyzed sample of H-HH DNA was sonicated as described i n the text, and banded i n CsCl. 17,500 (92%) TCA pre-c i p i t a b l e cpm were recovered. The arrow indicates the LL reference p o s i t i o n . I • • 1 « .25 .30 .35 .40 45 PERCENT LENGTH OF GRADIENT Figure 19. Sucrose gradient analysis of sonicated T 7 DNA. 32 P-labeled T ? phage DNA was d i l u t e d to a 1 ml Volume i n TNE and sonicated using a Biosonik microprobe f or 12 seconds, repeated four times. Size one T 7 and sonicated T 7 were analyzed on separate 5-20% neutral sucrose gradients. Centrifugation was for 3 hours at 34,000 rpm. The s i z e of the sonicated DNA was determined by the re l a t i o n s h i p developed by Hershey and Burgi (31): where D represents distance migrated, and M represents molecular weight. where (a) Non-sonicated J^P-T 7 DNA; (b) Sonicated 3 2 P - T ? DNA; • • = 3 2 P - l a b e l e d T ? DNA, and A- A = 1 4 C - l a b e l e d T, DNA. Figure 20. Sucrose gradient analysis of HH material. The pooled HH peaks from a preparative CsCl gradient were 3 dialyzed against TNE. H material to be analyzed i n neutral sucrose were loaded d i r e c t l y onto a 5 - 20% gradient. Samples to be analyzed i n a l k a l i were treated i n 0.2N NaOH at 25°C for 30 minutes p r i o r to 32 loading onto a 5 - 20% sucrose gradient. P-labeled s i z e one T 7 DNA was loaded onto each gradient as a reference (indicated by the arrow). Gradients were centrifuged at 34,000 rpm for 3.25 hours i n a Beckman Type SW50.1 rotor. 40 five-drop f r a c t i o n s were c o l l e c t e d , washed i n cold 5% TCA, followed by ethanol, dried, and the radio-a c t i v i t y determined by s c i n t i l l a t i o n counting. (a) Neutral sucrose analysis of HH peak material: 3 16,000 cpm H loaded = 92% recovery. (b) A l k a l i n e sucrose analysis of HH peak material: 3 15,000 cpm H loaded = 60% recovery. DISCUSSION A. R e p l i c a t i o n and Marker Rescue•of Bacteriophage DNA The objective of the experiments described here was to estab-l i s h a r o l e f o r r e p l i c a t i o n i n the process of genetic recombination. Marker rescue between a recombinant plasmid molecule and a mutant bac-teriophage was used to study t h i s r e l a t i o n s h i p because the r e p l i c a t i o n each DNA species could be c o n t r o l l e d independently. R e p l i c a t i o n of the plasmid was c o n t r o l l e d by a temperature s e n s i t i v e dnaB mutation i n the 3 host bacterium. H-thymidine incoporation into TCA insoluble material, copy number analysis and h y b r i d i z a t i o n studies confirmed that the p l a s -mids did not r e p l i c a t e i n a dnaB mutant at 42°C. Phage r e p l i c a t i o n was co n t r o l l e d by amber or temperature s e n s i t i v e mutations i n the DNA poly-merase (gene 5). Recombination between the recombinant plasmid and bacteriophage DNAs was monitored by quantitating wild type phage or i n f e c t i v e centres produced by the process. The r e s u l t s i n d i c a t e that r e p l i c a t i o n plays: an e s s e n t i a l r o l e i n determining normal l e v e l s of recombination i n t h i s system. When either plasmid or phage DNA i s prevented from r e p l i c a t i n g there i s a decrease i n the number of wild type phage produced; and under conditions where no DNA synthesis i s allowed, there i s no detectable recombination. R e p l i c a t i o n could influence recombination i n d i r e c t l y by several mechanisms. F i r s t , the number of DNA molecules i n the c e l l a v a i l a b l e f or recombination could be important i n determining recom-bination frequencies, and t h i s would be affected by r e p l i c a t i o n . How-ever, experiments reported here show that the number of i n t e r a c t i n g molecules i s not the l i m i t i n g f a c t o r . Marker rescue i s high even when the phage cannot r e p l i c a t e at a l l ; copy number determinations show that the amount of plasmid DNA does not change during the course of i n f e c t i o n at 42°C i n a dnaB mutant. Consequently, the plasmid molecules are not decreasing i n number under r e s t r i c t e d conditions. These r e s u l t s are consistent with the data from T 7 phage recombination experiments where the number of i n t r a c e l l u l a r T 7 DNA molecules was l i m i t e d either by n a l a d i x i c acid or a DNA polymerase mutation (35, 52); neither condition blocked recombination u n t i l r e p l i c a t i o n was eliminated. The number of i n t a c t plasmid molecules i s not decreasing a f t e r i n f e c t i o n e i t h e r , because agarose g e l electrophoresis shows that unlike the E_. c o l i genome, pBR322 d e r i v a t i v e plasmids are not degraded to nucleotides a f t e r i n f e c -t i o n . A second p o s s i b i l i t y i s that the apparent dependence of t h i s plasmid-phage recombination on r e p l i c a t i o n merely r e f l e c t s the need for either the dnaB p r o t e i n , or the T 7 DNA polymerase (gene 5) at some c r i -t i c a l stage of the recombination process. In other words, i t might be speculated that the dnaB protein complements the gene 5 product i n a recombination function. Both the T 7 am5-28 or T 7 ts5 mutations used here map within the gene known to code for the T 7 s p e c i f i c DNA poly-merase (26, 75), which i s e s s e n t i a l for phage r e p l i c a t i o n (76). Results presented here i n d i c a t e that the dnaB protein of E_. c o l i i s required for the r e p l i c a t i o n of pBR322 and recombinant plasmids where pBR322 serves as a vector. There i s no evidence that the dnaB protein, has any polymerase or^nuclease a c t i v i t y . Therefore, i t i s u n l i k e l y that the dnaB protein complements the T 7 DNA polymerase i n an e s s e n t i a l recombination function. Furthermore, complementation between the cloned T fragment ca r r i e d on pRS202 and the gene 5 mutants i s not l i k e l y to occur i n t h i s system because the cloned T 7 fragment does not contain the e n t i r e coding region for gene 5 (46); and there i s no T 7 promoter reported to be within the cloned segment (78). F i n a l l y , both gene 5 and gene 4. temperature s e n s i t i v e mutants f a i l to recombine with plasmids carrying homologous DNA i n a dnaB host at 42°C. Yet both gene 5 and gene 4 mutants are rescued under non-permissive conditions i n a wild-type E_. c o l i (11). I t i s extremely u n l i k e l y that the dnaB protein would complement both the gene 4 and gene 5 products i n an e s s e n t i a l recombination.function. Therefore, i t seems most l i k e l y then that under conditions non-permissive for the dnaB pr o t e i n and the T ? poly-merase, or the gene.4 product, recombination i s affected, not by the f a i l u r e of these proteins to perform a common task i n recombination per;;se, but by the absence of DNAcreplication. Several models for genetic recombination describe mechanisms which include DNA synthesis as an important element i n the process of j o i n t molecule formation (for review, see r e f . 68). Models which u t i l i z e r e p l i c a t i o n i n t h i s manner d i f f e r i n the proposed function that DNA synthesis performs (see Appendix I ) . In the Meselson and Radding Model (47), DNA synthesis leads to the displacement of a parental strand from one DNA duplex. The displaced strand then becomes assimilated into another duplex, forming a j o i n t molecule. In the u n i s e x - c i r c l e model (72) a newly synthesized (progeny) strand from a r e p l i c a t i o n loop invades another duplex at a denatured region or at a gap, and the r e c i p i e n t duplex then assimilates the donated strand. Either model makes use of an enzyme l i k e the T 7 exonuclease (gene 6) which i s important i n the formation of j o i n t molecules (42, 83) and genetic recombination (35, 52, 57). B. Strand Transfer During Plasmid-Phage Recombination Density s h i f t experiments reported here demonstrated the phys-3 i c a l transfer of H-labeled l i g h t DNA to heavy labeled T 7 molecules. 3 The system minimized the amount of extraneous H l a b e l associated with the heavy DNA due to repair and/or r e p l i c a t i o n of the T 7 phage. The l a b e l contributed by these processes was determined by. control i n f e c t i o n s of CSR603-pBR322 i n which no recombination occurs. DNA analyzed i n CsCl gradients from continuously labeled, infected c e l l s showed a s i g n i -f i c a n t amount of l a b e l present at the HH l o c a t i o n , which was w e l l above 3 background l e v e l s obtained i n control i n f e c t i o n . H l a b e l was also found at an intermediate l o c a t i o n i n CsCl gradients. A majority of the 3 H-HH material returned to the HH l o c a t i o n upon irebanding i n CsCl. This demonstrates a stable a s s o c i a t i o n of phage and plasmid DNA i n these molecules. During a continuous l a b e l i n g experiment, i t was not possible to determine i f parental or progeny DNA was being transferred to the 3 phage molecules. Therefore, c e l l s were labeled with H-thymidine, then thoroughly washed p r i o r to i n f e c t i o n . Since no further l a b e l was i n c o r -porated a f t e r i n f e c t i o n , i t was very u n l i k e l y that any l a b e l would be found at the HH l o c a t i o n i f newly r e p l i c a t e d (progeny) DNA were being transferred. However, DNA extracted from c e l l s a f t e r such an i n f e c t i o n 3 and analyzed i n CsCl density gradients showed s i g n i f i c a n t H l a b e l at the HH l o c a t i o n . In such a p r e - l a b e l i n g experiment, the majority of l a b e l would be present i n parental DNA. Thus, f i n d i n g l a b e l at the HH l o c a t i o n strongly suggests that parental DNA was displaced from a r e p l i -cating plasmid molecule and assimilated into the phage duplex. This experiment did not r u l e out the p o s s i b i l i t y that progeny DNA could also be transferred. I t should be noted that the percentage of ~"H l a b e l at the HH l o c a t i o n was greater i n continuous l a b e l i n g samples compared to p r e - l a b e l i n g at equivalent sampling times. This experiment did show parental DNA to be d i r e c t l y invovled i n recombination. Displacement of a parental strand could occur v i a several mechanisms (Figure 9, b, c ) . To determine i f r e p l i c a t i o n was a require-ment for strand displacement, pre-labeled c e l l s were i n f e c t e d i n the presence of novobiocin. Analysis of DNA i n CsCl gradients showed that without r e p l i c a t i o n , no transfer occurred. This r e s u l t suggests that r e p l i c a t i o n was necessary to promote displacement of parental DNA. C. The Structure of Recombinant Molecules 3 The structure of the H-HH DNA was examined by several methods. 3 F i r s t , rebanding i n CsCl confirmed that there was DNA containing H-l a b e l due to recombination at a density heavier than the LL l o c a t i o n . Second, sonication of t h i s material moved i t s l o c a t i o n i n CsCl density gradients from HH to HL, i n d i c a t i n g that the recombined DNA was pre-sent as a s i n g l e strand i n s e r t i o n and not as a double strand i n s e r t i o n . Since;only the r strand of T ? DNA i s used as a template for t r a n s c r i p -t i o n (79), recombination of wild-type DNA into t h i s strand could allow gene expression of normal proteins. Recombination of wild-type DNA into the 1 strand would require a round of semi-conservative r e p l i c a t i o n to achieve expression of normal proteins. Of course, mutations i n genes e s s e n t i a l for DNA synthesis would only be converted to wild-type by recombination i n the r strand. At t h i s time, i t i s assumed that strand s e l e c t i o n i s random during recombination. The nature of insertion..heterozygoses formed - .after recombination was examined. Neutral sucrose gradients indicated that 3 about 50% of the H-labeled DNA was associated with s i z e one T 7 mole-3 cules. This confirmed that much of the H was st a b l y present i n heavy phage DNA. Upon treatment i n a l k a l i , 10% of the l a b e l was l o s t from the s i z e one p o s i t i o n and relocated near the top of the gradient. This indicated that the inserted DNA was small i n s i z e and that approximately 3 40% of the H-label was covalently attached to s i z e one T 7 molecules. This r e s u l t suggests that the T 7 DNA polymerase (not expressed i n these experiments) i s not e s s e n t i a l for the f i l l i n g of gaps i n some l a t e ^ intermediates i n recombination. I t could be argued that the T 7 poly-2 merase i s required to resolve c e r t a i n early intermediates. Since density transfer experiments.detect the : formation, o f ' j o i n t molecules, an intermediate found early i n the recombination process, i t i s not possible to say i f the T 7 polymerase i s used i n a l a t e r process to resolve these intermediates. However, the formation of some stable covalently bonded recombinants indicates that some recombinant struc-tures are resolved without the gene 5 product. This may in d i c a t e the existence of two mechanisms a v a i l a b l e f o r recombinant molecule form-ation: one dependent on the gene 5 product; the other, not. F i n a l l y , i t could be argued that r e p l i c a t i o n of the donor (plasmid DNA) i s required to resolve intermediates. This p o s s i b i l i t y cannot be ruled out at t h i s time. A l a t e intermediate i s defined i n t h i s thesis as a recombined mole-cule found a f t e r endonucleotic s c i s s i o n of the cross connection. An early intermediate i s defined as two duplex molecules joined at a crossover point. 90 D. The Role of the T 7 5' Exonuclease (gene 6) and Strand Uptake Previous experiments have defined several functions f o r the T 7 5' exonuclease during normal T 7 i n f e c t i o n s . F i r s t , t h i s enzyme i s responsible for the breakdown of host DNA (34). Second, i t i s neces-sary for genetic recombination (35, 42, 57). Third, the gene 6 pro-duct i s necessary for the s t a b i l i t y of concatemers (22, 52). Marker rescue and density transfer experiments i n t h i s thesis i n d i c a t e that the gene 6 product i s e s s e n t i a l for plasmid-phage recom-bin a t i o n only under conditions where phage r e p l i c a t i o n i s blocked. When both plasmid and phage can r e p l i c a t e , i n the absence of gene 6, recombination frequencies are decreased compared to the gene 6 + con-d i t i o n . This r e l a t i o n s h i p can be explained by examining the early events i n plasmid-phage recombination. There are several structures i n recipient-.molecules- which could f a c i l i t a t e the uptake of donor DNA. F i r s t , Radding has proprosed D loop structures (Figure 21 a, b) which can take up a si n g l e homo-logous strand of DNA to form a t r i p l e x (66). Upon endonuclease digestion, the looped out strand may be l o s t and a stable base paired region established. The recA gene product has been shown to mediate these events(66). I t has been demonstrated for A DNA that loop forma-t i o n i s not dependent on recA (13). However, the recA protein could s t i l l function i n the formation or r e s o l u t i o n of the t r i p l e x . Second, there are other loop structures which could be considered recombino-genic. Two examples are the r e p l i c a t i o n fork and l o c a l l y unwound regions of DNA during t r a n s c r i p t i o n . Third, the ends of molecules have been shown to be recombinogenic i n X and T^ (8, 53, 70). F i n a l l y , the presence of gaps within DNA duplexes may stimulate the formation of Figure 21. Two possible mechanims for strand uptake. 1. (a) and (b) represent strand uptake v i a a D loop. (a) The free end of a displace s i n g l e strand i s taken up i n t o a looped-out region of the r e c i p i e n t duplex at an area of homology. The action of endonucleases A 1 and 2 cut the looped out r e c i p i e n t strand. (b) The base paired donor DNA can now be assimilated v i a branch migration. 2. (c) and (d) represent strand uptake into a p r e - e x i s t i n g gap on the r e c i p i e n t duplex. In (c) the action of an endonuclease (3) nicks the r e c i p i e n t DNA. (d) A gap i s formed by the a c t i o n of an exonuclease and the homologous donor s i n g l e strand can be taken up and assimilated. (a) (b) (c) (d) recombinant molecules. For example, mutations i n genes 46 and 47 , whose gene products are responsible for gap formation, decrease recom-bination frequencies (5, 8, 40). Also, mutations i n genes 3 and 6 decrease recombination frequencies (42, 57). The action of the T^ endonuclease (gene 3) and exonuclease (gene 6) could create gaps on T^ molecules. During recombination between a r e p l i c a t i n g plasmid and a non-replicating phage DNA, the plasmid w i l l be defined here as the donor, i . e . the phage can only receive DNA during early events i n recombination. The non-replicating T^ genome i s u n l i k e l y to contain any r e p l i c a t i o n forks. I t i s possible that the T^ DNA could contain recombinogenic structures mentioned above. However, i n the absence of gene 6, no recombination i s detected by density transfer. Since T^ recombination i s independent of the recA protein (57), i t i s u n l i k e l y that D loops, as proposed by Radding, play a s i g n i f i c a n t r o l e during the uptake of plasmid DNA by the T^ molecule. The dependence of recombination upon the gene 6 protein strongly suggests that T^ : molecules recombine v i a gaps treated by the T^ 5' exonuclease. Density transfer experiments i n :this thesis support t h i s conclusion. Under conditions where no gene 6 product i s present during recombination.events, no l a b e l i s transferred from the plasmid to the phage molecule.. Such p h y s i c a l examinations of molecules a f t e r recombination are independent of concatemer formation and packaging while rescue frequencies are not. Therefore, a r o l e for the T exo-nuclease can be established for recombination independent of any e f f e c t s on e i t h e r host degradation or concatemer s t a b i l i t y . Under conditions where both molecules can r e p l i c a t e , e i t h e r can serve as a donor. Through a cross-over event, the phage can s t i l l r e c e i v e w i l d - t y p e DNA from the plasmid and thus recombination i s inde-pendent of the gene 6 p r o t e i n . The frequency d i f f e r e n c e s between gene 6"*" and 6 c o n d i t i o n s when both molecules can r e p l i c a t e may r e f l e c t the unstable nature of concatameric T 7 DNA and the i n a b i l i t y of the ;phage molecule to act as a r e c i p i e n t i n e a r l y recombination events. E. E a r l y Events during Plasmid-Phage Recombination (1) Enzymes During plasmid-phage recombination, two recombination systems are u t i l i z e d which normally do not i n t e r a c t . C o l E l and C o l E l - d e r i v e d plasmids, such as pBR322, r e l y on host enzymes f o r cross-over events. The host rec system i s r e q u i r e d as are other DNA b i n d i n g p r o t e i n s , such as the.E.. c o l i HDP p r o t e i n (14, 18). On the other hand, bacteriophage T 7 r e l i e s on enzymes which are s e l f - c o d e d , such as the products of genes 3, 4, 5, and 6. T 7 does not appear to r e q u i r e any of the know rec f u n c t i o n s of the JS. c o l i host (57). I t i s not known i f T 7 i s completely independent of a l l host enzymes during recombination. Several host p r o t e i n s are known to be i n h i b i t e d by T 7 i n f e c t i o n . F i r s t , the product of T 7 gene 2 i n h i b i t s the E. c o l i RNA polymerase (32). Second, a c t i v i t y of the recBC nuclease i s i n h i b i t e d upon i n f e c t i o n (64). T h i r d , the product of T 7 gene 0.7 phosphorylates many host p r o t e i n s and shuts o f f host RNA syn-t h e s i s , (78). I t i s not known i f the recA p r o t e i n i s i n h i b i t e d by phage i n f e c t i o n . However, the recA p r o t e i n does not appear to be e s s e n t i a l f o r plasmid-phage recombination because d e n s i t y t r a n s f e r experiments i n recA b a c t e r i a and marker rescue i n E. c o l i HMS174 show the formation of recombinant T-, molecules and the production of w i l d -type phage, res p e c t i v e l y . The absence of the recA protein could e f f e c t the structure of plasmidrrecombinant molecules and/or the mechanism responsible f or t h e i r formation, but the formation of T^ recombinants i s l i k e l y to be independent of recA. (2) Reciprocal vs Non-reciprocal Recombination Genetic studies u t i l i z i n g 3-factor crosses have determined that recombination i n bacteriophages ^4' a n a ^ -*-s n o n - r e c l P r o c a l (44, 73, 86). Therefore, there i s good reason to assume that T^ recom-bin a t i o n would also be non-reciprocal. R e p l i c a t i o n and/or r e p l i c a t i o n enzymes may play a r o l e i n the asymetric conversion of recombinant intermediates (33, 58, 73, 90). For instance, r e p l i c a t i o n could drive branch migration, a process which can lead to the formation of non-r e c i p r o c a l products (58). However, f i n d i n g covalent recombinants i n the absence of the T^ polymerase would indi c a t e that i s not the case for a l l intermediates during plasmid-phage recombination. This i s supported by marker rescue experiments i n which phage, defective i n gene 5, can only r e p l i c a t e a f t e r they have recombined. I t i s possible that plasmid r e p l i c a t i o n could drive branch migration and thus f a c i l i t a t e a s s i m i l a t i o n and strand transfer. However, models have been proposed which account fo r non-reciprocity without mismatch co r r e c t i o n or r e p l i c a t i o n (73). A unique feature of the plasmid-phage recombination system i s that cross-over events and some products of T^ recombination can be studied without p r i o r phage r e p l i c a t i o n . In b a c t e r i a l c e l l s , some s p e c i a l i z e d cases of recombination are r e c i p r o c a l . Two examples are the i n s e r t i o n of the Lambda prophage and the i n t e g r a t i o n of F f a c t o r s i n t o the host genome (30, 41). The evidence f o r plasmid-plasmid recombination being r e c i p r o c a l i s l e s s s u b s t a n t i a l . E l e c t r o n micrographs of plasmid recombination interme-d i a t e s confirm the presence of intermediates p o s t u l a t e d by H o l l i d a y (56). These s t r u c t u r e s are not dependent oh r e p l i c a t i o n f o r t h e i r formation. However, the Meselson-Radding model (47) i n c l u d e s these s t r u c t u r e s p l u s two important f e a t u r e s . F i r s t , : t h e i r - f o r m a t i o n - i s dependent on r e p l i c a t i o n ; and second,the asymmetric i n i t i a t i o n of recombination events r e s u l t s i n the formation of n o n - r e c i p r o c a l recom-binant molecules. A l l data i n t h i s t h e s i s support models which u t i l i z e DNA s y n t h e s i s , such as the Meselson-Radding model. Since e a r l y events are asymmetric i n such models, i t f o l l o w s that recombinants formed during plasmid-phage recombination are probably n o n - r e c i p r o c a l . (3) Comparison of Plasmid-Phage Recombination w i t h In V i t r o T_, Recombination R e s u l t s from an i n v i t r o recombination system i n d i c a t e there are s e v e r a l pathways of recombination f o r bacteriophage T 7 (61). The f i r s t i s a " w i l d - t y p e " pathway, which u t i l i z e s the products of T 7 genes 3, 4, 5, and 6.^ The second i s the "endonuclease" pathway which can form recombinants using the products of T 7 genes 3 and 5 independent of the gene 6 product (5' exonuclease). And f i n a l l y , the "exonuclease" pathway i s reported which can form recombinants i n the absence of genes 3 and 5, but i s dependent on the gene 6 product. The plasmid-phage system, under c o n d i t i o n s when both molecules The products of T genes are as f o l l o w s : gene 3 (endonuclease), gene 4(primase), gene 5 (polymerase), and gene 6 (exonuclease). can r e p l i c a t e (gene 3 5 ) but i n the absence of the exonuclease (gene 6 ) , i s s i m i l a r to the conditions of the "endonuclease pathway,." Under these conditions, recombination i s not dependent on the exonuclease. Since, i n t h i s case, the exonuclease cannot create gaps on molecules, strand a s s i m i l a t i o n would, have to occur v i a some other structure, possibly i n the r e p l i c a t i o n fork. I t i s possible that another enzyme with an exonuclease a c t i v i t y could complement the gene 6 protein. An example of such an enzyme i s the DNA polymerase (61). Data from experiments under gene 5 6 conditions (which eliminates r e p l i c a t i o n forks on DNA, the exonuclease a c t i v i t y of the polymerase and the gene 6 product) indicates a s t r i c t dependence on the gene 6 protein for recombination. Therefore, when both molecules can r e p l i c a t e , plasmid-phage recombination may proceed v i a a pathway s i m i l a r to the "endonuclease pathway" proposed for the i n v i t r o system. When phage DNA cannot r e p l i c a t e (5 ) recombination i s dependent on gene 6, which i s s i m i l a r to conditions established f o r the "exonuclease pathway". I t i s possible therefore, that plasmid-phage recombination may pro-ceed by several pathways depending on the r e p l i c a t i o n status of the phage DNA. These proposed pathways are consistent with r e s u l t s that show r e p l i c a t i o n i s necessary for strand displacement. (4) A Possible Mechanism for Plasmid-Phage Recombination Postulated early events i n plasmid-phage recombination are summarized i n Figure 22. In vivo recombination may occur p r i m a r i l y according to the model proposed by Meselson and Radding (47). T^ genes 3 (endonuclease), 5 (DNA polymerase), and 6 (exonuclease) l i k e l y provide the necessary enzymes. In t h i s model, DNA synthesis displaces s i n g l e stranded parental DNA from a nick located j u s t ahead o a.J r e p l i c a t i o n fork. The displaced donor DNA i s taken up i n t o a gap on the r e c i p i e n t molecule created by the action of the T 7 5' exonucleas A cross-over event occurs and the r e s u l t i n g intermediates are resolved forming non-reciprocal recombinant structures. Figure 22. Early events i n plasmid-phage recombination. (a) A nick i s introduced at s i t e "a" j u s t ahead of the r e p l i c a t i o n fork on the donor molecule. On the r e c i p i e n t molecule, a nick i s i n t r o -duced '(possibly by the endonuclease) and a gap i s subsequently formed by the action of the 5' exonuclease (gene 6). (b) DNA synthesis ( d i r e c t i o n indicated by arrows) displaces parental DNA which i s then free to base p a i r with homologous DNA within the gap on the r e c i p i e n t duplex. (c) A s s i m i l a t i o n of donor DNA acts to displace r e c i p i e n t DNA at s i t e "b" which i s then a v a i l a b l e to cross-over and be taken up by the donor molecule. Recipient DNA could be assimilated at s i t e "c" either by d i r e c t l y base p a i r i n g with the parental DNA template or by forming a t r i p l e x structure i n v o l v i n g both parental and progeny DNA. Resolution of intermediates would y i e l d non-reciprocal recombinants. Resolution could include such processes as branch migration, isomerization, endonuclease cutting at s p e c i f i e d s i t e s and exonuclease digestion of non-assimilated s i n g l e strands.. (d) The structure depicted here i s known to e x i s t a f t e r plasmid-phage recombination. 100 APPENDIX I: TWO MODELS FOR GENETIC RECOMBINATION The Meselson-Radding Model (47) (a) DNA synthesis displaces a strand. (b) The free displaced strand i s taken up by. a homologous double stranded molecule. (c) The D loop i s cleaved by endonuclease. (d) The displaced strand i s assimilated. (e) A one-strand cross-over becomes two-^stranded a f t e r isomerization. (f) Branch migration. A Sex C i r c l e Model (67) In t h i s model, progeny DNA i s displaced by branch migration and taken up into a D loop structure. The loop i s cleaved and a s s i m i l a t i o n occurs, d i s p l a c i n g r e c i p i e n t DNA at s i t e m. The free end of strand m can now cross-over and be taken up by eith e r the upper or lower arc of the r e p l i c a t i o n fork. 102 LITERATURE CITED Al b e r t s , B.M. and L. Frey. 1970. "T4 bacteriophage gene 32: A s t r u c t u r a l p r o t e i n i n the r e p l i c a t i o n and recombination of DNA." Nature, 227, 1313-1318. Anraku, N. and J . Tomizawa. 1965. "Molecular mechanisms of genetic recombination i n bacteriophage. I I I . Joining of parental polynucleotides of phage T4 i n the presence of 5-fluordeoxyuridine." J . Mol. B i o l . , 12, 805-815. Bachman, B.J. 1972. "Pedigrees of some mutant s t r a i n s of Escher i c h i a c o l i K-12." B a c t e r i o l Rev., 36, 525-557. Benbasat, J . , K. Burck, R.C. M i l l e r , J r . 1978. "Superinfection exclusion and lack of conservative transfer of bacteriophage T7 DNA." Virology, 87, 164-171. Bernstein, H. 1968. "Repair and recombination i n phage T4: I. Genes a f f e c t i n g recombination." Cold Spring Harbor Symp. Quant. 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