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UBC Theses and Dissertations

Cross reactivation and partial replication of bacteriophage T7 DNA Burck, Kathy Louise Bauman 1979

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CROSS REACTIVATION AND PARTIAL REPLICATION OF BACTERIOPHAGE T7 DNA by KATHY LOUISE BAUMAN BURCK B.Sc. (Hon) M i c h i g a n S t a t e U n i v e r s i t y , 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Genetics Programme) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA (c) K a t h y L o u i s e Bauman B u r c k , 1979 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l lowed w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f G e n e t i c s  The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date July 3. 1979 i i ABSTRACT Cross r e a c t i v a t i o n i s a process whereby genetic markers are rescued from a U V - i r r a d i a t e d phage by a c o i n f e c t i n g mutant phage. The p r o b a b i l i t y of rescue of a s p e c i f i c marker during T7 i n f e c t i o n depends on the map p o s i t i o n of the marker. This phenomenon i s observed a l s o w i t h b a c t e r i o -phage T4. During T7 i n f e c t i o n , markers from the l e f t end of the genome are rescued w i t h high e f f i c i e n c y . Recombination i s necessary f o r marker rescue. P h o t o r e a c t i v a t i o n and the E . c o l i p y r imidine dimer s p e c i f i c endonuclease enhance rescue e f f i c i e n c y . I t i s found that markers that are rescued e f f i c i e n t l y are i n segments of the DNA which r e p l i c a t e 32 e f f i c i e n t l y , i e P-labeled progeny DNA synthesized a f t e r i n f e c t i o n by a U V - i r r a d i a t e d T7 phage h y b r i d i z e s predominantly w i t h r e s t r i c t i o n f r a g -ments of T7 DNA from the l e f t end of the genome. The e f f e c t i s dependent on the UV dose: the higher the dose to the T7 phage, the fewer are the markers which are rescued e f f i c i e n t l y and the smaller i s the area of the genome which r e p l i c a t e s . Under the e l e c t r o n microscope, p a r t i a l l y r e p l i c a t e d , U V - i r r a d i a t e d T7 DNA has double stranded i n t e r n a l d u p l i c a t i o n s (bubbles) or branches. The d i s t r i b u t i o n of p a r t i a l l y r e p l i c a t e d regions determined by EM c o r r e l a t e s very w e l l w i t h regions which have r e p l i c a t e d as determined by h y b r i d i z a t i o n a n a l y s i s and both r e s u l t s c o r r e l a t e w i t h the d i s t r i b u t i o n of marker rescue frequencies along the T7 genome. The r e s u l t s are c o n s i s t e n t w i t h the b a s i c idea of the p a r t i a l r e p l i c a hypothesis which p o s t u l a t e s that r e p l i c a t i o n of a U V - i r r a d i a t e d genome s t a r t s from a s p e c i f i c o r i g i n and proceeds b i d i r e c t i o n a l l y to a UV l e s i o n . UV l e s i o n s block f u r t h e r r e p l i c a t i o n and those regions which are r e p l i c a t e d e f f i c i e n t l y are rescued e f f i c i e n t l y by a c o i n f e c t i n g , g e n e t i c a l l y marked phage. During the course of t h i s i n v e s t i g a t i o n , r e s u l t s were obtained which i n d i c a t e d that during T7 i n f e c t i o n , primary i n f e c t i n g phage prevent i n f e c t i o n o f t h e same c e l l by s e c o n d a r y o r s u p e r i n f e c t i n g phage. T h i s p r o c e s s i s termed s u p e r i n f e c t i o n e x c l u s i o n . P r e i n c u b a t i o n o f c e l l s i n c h l o r a m -p h e n i c o l (CM) a t 100 yg/ml f o r 5 m i n u t e s p r i o r t o p r i m a r y i n f e c t i o n a l l o w s p r o d u c t i o n o f i n f e c t i v e c e n t e r s by s u p e r i n f e c t i n g phage. W i t h o u t CM, r e c o m b i n a t i o n between a p r i m a r y and s e c o n d a r y phage d e c r e a s e s as t h e tim e between p r i m a r y and s e c o n d a r y i n f e c t i o n i s i n c r e a s e d . A f t e r a 5 mi n u t e p r e i n c u b a t i o n i n CM, s e c o n d a r y phage added as l a t e as 8 m i n u t e s a f t e r p r i m a r y i n f e c t i o n a r e a b l e t o recombine w i t h p r i m a r y phage a t a f r e q u e n c y n o t s i g n i f i c a n t l y d i f f e r e n t from t h a t found a f t e r s i m u l t a n e o u s i n f e c t i o n . S u p e r i n f e c t i o n e x c l u s i o n o c c u r s a t t h e i n j e c t i o n s t a g e . I n the absence o f s u p e r i n f e c t i o n e x c l u s i o n , C s C l g r a d i e n t a n a l y s i s r e v e a l s t h a t many p a r e n t a l phage a r e a b l e t o i n j e c t t h e i r DNA i n t o a s i n g l e c e l l . However, o n l y v e r y s m a l l amounts of p a r e n t a l ( c o n s e r v a t i v e ) DNA a r e found among t h e progeny phage i f u n a t t a c h e d p a r e n t a l phage a r e removed by d i f f e r e n t i a l s e d i m e n t a t i o n . i v TABLE OF CONTENTS Page A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i Acknowledgement. v i i i I . I n t r o d u c t i o n 1 C r o s s R e a c t i v a t i o n 1 M u l t i p l i c i t y R e a c t i v a t i o n . . . . 1 The P a r t i a l R e p l i c a H y p o t h e s i s 4 E v i d e n c e w i t h B a c t e r i o p h a g e T4 7 B a c t e r i o p h a g e T7 8 O b j e c t i v e s o f t h e R e s e a r c h 9 S u p e r i n f e c t i o n E x c l u s i o n 10 I I . M a t e r i a l s and Methods 11 S t r a i n s 11 1. B a c t e r i a 11 2. B a c t e r i o p h a g e 11 C h e m i c a l s and I s o t o p e s 12 M e d i a and B u f f e r s 12 U l t r a v i o l e t I r r a d i a t i o n o f Phage 13 Rescue o f T7 Amber M a r k e r s by U V - i r r a d i a t e d T7 Phage 14 P h o t o r e a c t i v a t i o n o f I n f e c t i v e C e n t e r s A f t e r C r o s s R e a c t i v a t i o n 14 P r e p a r a t i o n o f T7 Double M u t a n t s 14 P r e p a r a t i o n o f P a r t i a l l y R e p l i c a t e d T7 DNA f o r H y b r i d i z a t i o n 15 R e s t r i c t i o n Enzyme Fnu C D i g e s t s o f T7 DNA 15 P r e p a r a t i o n o f H y b r i d i z a t i o n F i l t e r C o n t a i n i n g Endo R.Fnu C c u t T 7 + DNA f r a g m e n t s s e p a r a t e d by A g a r o s e G e l E l e c t r o p h o r e s i s . . 16 H y b r i d i z a t i o n of 32p DNA t o N i t r o c e l l u l o s e F i l t e r Charged w i t h Endo R.Fnu C c u t T7 DNA 17 E l e c t r o n M i c r o s c o p y 17 Other Methods , 17 Page I I I . R e s u l t s 19 A. S u p e r i n f e c t i o n E x c l u s i o n and L a c k o f V C o n s e r v a t i o n - T r a n s f e r o f B a c t e r i o p h a g e T7 DNA 19 1. C h l o r a m p h e n i c o l i n h i b i t s s u p e r i n f e c t i o n e x c l u s i o n . 19 2. T7 DNA i s n o t c o n s e r v a t i v e l y t r a n s f e r r e d 23 B. C r o s s R e a c t i v a t i o n o f B a c t e r i o p h a g e T7 DNA 29 1. Dose r e s p o n s e o f T 7 + t o u l t r a v i o l e t i r r a d i a t i o n 29 2. Marker r e s c u e w i t h T 7 + 31 3. Marker r e s c u e w i t h T7VLG-3 38 4. Marker r e s c u e w i t h amber mutants 41 5. E f f e c t o f t h e b a c t e r i a l u v r A e n d o n u c l e a s e on marker r e s c u e '45 6. E f f e c t o f p h o t o r e a c t i v a t i o n on marker r e s c u e . . . . 49 7. E f f e c t o f T7 gene p r o d u c t s on marker r e s c u e 53 8. Marker r e s c u e : summary of r e s u l t s 55 C. P a r t i a l R e p l i c a t i o n o f B a c t e r i o p h a g e T7 DNA: H y b r i d i z a t i o n A n a l y s i s 56 1. R e s t r i c t i o n enzyme F n u C map o f b a c t e r i o p h a g e • T7 56 2. P r e p a r a t i o n o f p a r t i a l l y r e p l i c a t e d T7 DNA f o r h y b r i d i z a t i o n 59 3. H y b r i d i z a t i o n o f p a r t i a l l y r e p l i c a t e d T 7 + DNA w i t h r e s t r i c t i o n enzyme Fnu C c u t T-7 DNA. . . 59 D. S i z e o f I n t r a c e l l u l a r U V - i r r a d i a t e d P a r e n t a l T7 DNA 66 E. P a r t i a l R e p l i c a t i o n o f B a c t e r i o p h a g e T7 DNA: A n a l y s i s by E l e c t r o n M i c r o s c o p y 70 1. I s o l a t i o n o f p a r t i a l l y r e p l i c a t e d m o l e c u l e f o r e x a m i n a t i o n by e l e c t r o n m i c r o s c o p y 70 2. E l e c t r o n m i c r o s c o p y p a r t i a l l y r e p l i c a t e d T7+ DNA 74 IV. D i s c u s s i o n 81 A. S u p e r i n f e c t i o n E x c l u s i o n and L a c k o f C o n s e r v a t i v e T r a n s f e r o f B a c t e r i o p h a g e T7 DNA 83 B. Ma r k e r Rescue and P a r t i a l R e p l i c a t i o n o f B a c t e r i o p h a g e T7 DNA. 85 C. Some P r o c e s s e s I n v o l v e d i n C r o s s R e a c t i v a t i o n 95 D. C r o s s R e a c t i v a t i o n and t h e Requirement o f R e p l i c a t i o n f o r Normal R e c o m b i n a t i o n . . 100 V. B i b l i o g r a p h y 105 v i : L I S T OF TABLES Page T a b l e 1. E x p r e s s i o n of T7' genes a f t e r p r e v i o u s i n f e c t i o n by T7 5 am 28 20 T a b l e 2. Amber r e v e r t a n t s under r e s c u e c o n d i t i o n s 35 T a b l e 3. M u l t i p l i c i t y r e a c t i v a t i o n o f U V - i r r a d i a t e d T 7 + . . 37 T a b l e 4. Rescue o f markers from d o u b l e mutants compared t o s i n g l e mutants w i t h U V - i r r a d i a t e d T7VLG-3 40 T a b l e 5. Rescue o f markers from U V - i r r a d i a t e d amber phage 44 32 T a b l e 6. F r a c t i o n a l c o n t r i b u t i o n o f P - l a b e l e d phage DNA a f t e r a n n e a l i n g t o Endo R.Fnu C c u t T7 f r a g m e n t s 63 T a b l e 7. Dose r e s p o n s ^ o f t h e f r a c t i o n a l c o n t r i b u t i o n o f h y b r i d i z e d P-DNA p e r fragment and n o r m a l i z a t i o n t o phage c o n t r o l 64 T a b l e 8. D i s t r i b u t i o n o f c e n t e r s o f eye forms 78 v i i L I ST OF FIGURES Page F i g u r e 1. C r o s s r e a c t i v a t i o n 2 F i g u r e 2. The p a r t i a l r e p l i c a h y p h o t h e s i s 5-6 F i g u r e 3. R e c o m b i n a t i o n w i t h d e l a y e d i n f e c t i o n and e f f e c t o f c h l o r a m p h e n i c o l . 22 32 F i g u r e 4. R e p l i c a t i o n o f P - l a b e l e d p a r e n t a l T7 DNA a f t e r h i g h m u l t i p l i c i t y of i n f e c t i o n i n t h e p r e s e n c e and absence of CM 24-25 F i g u r e 5. C s C l d e n s i t y g r a d i e n t of progeny phage a f t e r h i g h m u l t i p l i c i t y o f i n f e c t i o n ± CM. 27-28 F i g u r e 6. Dose r e s p o n s e of T 7 + phage t o u l t r a v i o l e t i r r a d i a t i o n . . . 30 F i g u r e 7. Rescue o f T7 amber phage w i t h U V - i r r a d i a t e d T 7 + phage... 32 F i g u r e 8. Rescue o f T7 amber phage w i t h U V - i r r a d i a t e d T7VLG-3 phage 39 F i g u r e 9. L o c a t i o n s o f T7 amber and d e l e t i o n mutants 42 F i g u r e 10. E f f e c t o f h o s t u v r A gene on marker r e s c u e 48 F i g u r e 11. E f f e c t o f p h o t o r e a c t i v a t i o n on marker r e s c u e 51-52 F i g u r e 12. R e s t r i c t i o n map o f Endo R.Fnu C T 7 + f r a g m e n t s 57-58 F i g u r e 13. P r e p a r a t i o n o f p a r t i a l l y r e p l i c a t e d T7 DNA f o r h y b r i d i c a t i o n : C s C l p u r i f i c a t i o n 60 F i g u r e 14. H y b r i d i z a t i o n of p a r t i a l l y r e p l i c a t e d DNA w i t h Endo R.Fnu C r e s t r i c t i o n f r a g m e n t s 62 F i g u r e 15. S i z e of U V - i r r a d i a t e d T 7 + DNA. 67-68 F i g u r e 16. I s o l a t i o n o f p a r t i a l l y r e p l i c a t e d T7 DNA f o r e x a m i n a t i o n by e l e c t r o n m i c r o s c o p y 72-73 F i g u r e 17. P a r t i a l l y r e p l i c a t e d T7 DNA m o l e c u l e s 75 F i g u r e 18. N o r m a l i z e d l i n e d i a g r a m s o f p a r t i a l l y r e p l i c a t e d T7 m o l e c u l e s . . . . . 77 F i g u r e 19. H i s t o g r a m o f p a r t i a l l y r e p l i c a t e d r e g i o n s from U V - i r r a d i a t e d T7 DNA 79 ACKNOWLEDGEMENT I would l i k e t o t h a n k my a d v i s o r , Dr. R.C. M i l l e r , J r . . f o r h i s i n s p i r a t i o n and g u i d a n c e d u r i n g t h e c o u r s e o f t h i s r e s e a r c h . Deb T a y l o r and H e l e n Smith p r o v i d e d e x c e l l e n t t e c h n i c a l a s s i s t a n c e . I am g r a t e f u l t o Dr. Doug S c r a b a f o r t e a c h i n g me e l e c t r o n m i c r o s c o p y and f o r h i s a s s i s t a n c e w i t h t h e EM p o r t i o n o f t h i s work. I t h a n k D r s . A.H. Doermann and R.A.J. Warren f o r p r o v i d i n g many v a l u a b l e d i s c u s s i o n s and s u g g e s t i o n s . Dr. M i c h a e l S m ith and Ms. Ann L u i g e n e r o u s l y p r o v i d e d t h e r e s t r i c t i o n enzyme Fnu C and d e t e r m i n e d i t s s p e c i f i c i t y . I a p p r e c i a t e Dr. C l y d e H u t c h i n s o n ' s a s s i s t a n c e w i t h t h e p r o c e d u r e f o r t r a n s f e r of r e s t r i c t i o n f r a g m e n t s s e p a r a t e d by a g a r o s e g e l s t o n i t r o c e l l u l a s e f i l t e r s . I am g r a t e f u l t o t h e K i l l a m F o u n d a t i o n and t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada f o r p r o v i d i n g f i n a n c i a l a s s i s t a n c e . 1 I . INTRODUCTION C r o s s r e a c t i v a t i o n r e f e r s t o t h e a b i l i t y o f a g e n e t i c a l l y marked phage t o r e s c u e a p a r t i c u l a r l o c u s from a c o i n f e c t i n g , U V - i r r a d i a t e d phage. The p r o c e s s can be i l l u s t r a t e d b e s t by a g e n e t i c e x p e r i m e n t ( F i g u r e 1 ) . Upon s i n g l e i n f e c t i o n o f s e n s i t i v e b a c t e r i a e i t h e r by a U V - i r r a d i a t e d phage c o n t a i n i n g a t l e a s t one l e t h a l h i t (column 1) o r by g e n e t i c a l l y marked mutant phage, e.g., an amber mutant (column 3 ) , no w i l d t y p e progeny a r e p r o d u c e d . However i f t h e UV-damaged and amber phage c o i n f e c t a c e l l under c o n d i t i o n s p e r m i s s i v e f o r the mutant, w i l d t y p e progeny a r e p r o -duced (column 2 ) ; t h e s e progeny a r e s e l e c t i v e l y o b s e r v e d under non-p e r m i s s i v e c o n d i t i o n s . Marker r e s c u e p r o b a b l y o c c u r s by a complex p r o c e s s i n v o l v i n g r e c o m b i n a t i o n (see b e l o w ) . H i s t o r i c a l l y , t h e c l o s e l y r e l a t e d phenomenon of m u l t i p l i c i t y r e a c t -i v a t i o n was d e s c r i b e d by L u r i a i n 1947 ( 4 9 ) . I t was found t h a t b a c t e r i o -phage i n a c t i v a t e d by u l t r a v i o l e t i r r a d i a t i o n were s t i l l a d s o r b e d t o s e n s i t i v e b a c t e r i a b u t b a c t e r i a i n f e c t e d by o n l y one i r r a d i a t e d p a r t i c l e d i d n o t l y s e and no progeny were p r o d u c e d ( 4 9 , 5 1 ) . However, upon m u l t i p l e i n f e c t i o n o f a s i n g l e b a c t e r i u m by U V - i r r a d i a t e d T2, T4, T5, o r T6 phage, l y s i s o f t h e c e l l d i d o c c u r and progeny phage were pr o d u c e d . M u l t i p l i c i t y r e a c t i v a t i o n o f t h e s m a l l e r phages T l , T3, and T7 was n o t o b s e r v e d . L u r i a p o s t u l a t e d t h a t UV i r r a d i a t i o n damaged d e s c r e t e ' u n i t s ' i n t h e p a r e n t a l phage. These u n i t s were assumed c a p a b l e of i n d e p e n d e n t r e p r o d u c t i o n . Then, r e a c t i v a t i o n o c c u r r e d as a r e s u l t of r e c o m b i n a t i o n between a c t i v e , undamaged, i n d e p e n d e n t l y r e p r o d u c e d ' u n i t s ' . The f r a c t i o n of r e a c t i v a t e d phage d i m i n i s h e d w i t h i n c r e a s i n g doses of UV i r r a d i a t i o n and i n c r e a s e d w i t h i n c r e a s i n g m u l t i p l i c i t y of i n f e c t i o n . The i m p l i c a t i o n was t h a t m u l t i p l y i n f e c t e d b a c t e r i a p r o duced a c t i v e progeny o n l y i f a l l of t h e n e c e s s a r y phage ' u n i t s ' were p r e s e n t i n undamaged form among th e p a r e n t a l F i g u r e 1. C r o s s R e a c t i v a t i o n . A g e n e t i c e x p e r i m e n t w h i c h i l l u s t r a t e s c r o s s r e a c t i v a t i o n . N e i t h e r t h e U V - i r r a d i a t e d phage a l o n e (1) n o r the amber phage a l o n e (3) a r e a b l e to produce w i l d type progeny. However when th e UV-damaged and mutant phage c o i n f e c t a c e l l under p e r m i s s i v e c o n d i t i o n s f o r the mutant ( 2 ) , w i l d t y p e progeny a r e produced which can be s e l e c t i v e l y a s s a y e d under n o n - p e r m i s s i v e c o n d i t i o n s . The amber phage has succeeded i n r e s c u i n g the w i l d type a l l e l e from the i r r a d i a t e d p a r e n t by a complex p r o c e s s w h i c h i n v o l v e s r e c o m b i n a t i o n ( s e e t e x t ) . LM C 2 ] [33 phage. The o r i g i n a l m u l t i p l i c i t y r e a c t i v a t i o n d a t a (49,51) o b t a i n e d f o r low r a d i a t i o n doses f i t t h e t h e o r e t i c a l c u r v e s v e r y w e l l ( 5 1 ) . L a t e r e x p e r i m e n t s p e r f o r m e d a t h i g h e r UV doses r e v e a l e d d i f f e r e n c e s between t h e e x p e r i m e n t a l s u r v i v a l c u r v e s o f m u l t i p l y - i n f e c t e d b a c t e r i a ( " m u l t i c o m p l e x e s " and p r e d i c t i o n s based on a r e c o m b i n a t i o n t h e o r y of r e a c t i v a t i o n (21) so s u c h a t h e o r y was abandoned as e a r l y as 1952 ( 5 0 ) . I n 1955, Doermann, Chase, and S t a h l p u b l i s h e d e x t e n s i v e d a t a on c r o s s r e a c t i v a t i o n of b a c t e r i o p h a g e T4 u s i n g U V - i r r a d i a t e d w i l d t y p e and v a r i o u s mutant c a r r i e r phage ( 1 7 ) . T h e i r r e s u l t s i n d i c a t e d t h a t UV damages were l o c a l i z e d i n t h e g e n e t i c s t r u c t u r e of t h e phage and t h a t r e s c u e of a p a r t i c u l a r w i l d t y p e marker by a mutant c a r r i e r phage was dependent on t h e map p o s i t i o n o f t h e marker. From a n a l y s i s of s i n g l e b u r s t s , t h e y found t h a t a g i v e n marker from t h e U V - i r r a d i a t e d p a r e n t m i g h t be a b s e n t i n t h e progeny o f a b u r s t a l t h o u g h o t h e r m arkers were p r e s e n t i n a p p r e c i a b l e f r e q u e n c y . The f r e q u e n c y o f marker absences was d i r e c t l y r e l a t e d t o t h e i r r a d i a t i o n dose. C l o s e l y l i n k e d m a rkers were found t o be s i m u l t a n e o u s l y i n a c t i v a t e d more f r e q u e n t l y t h a n a c c o u n t a b l e by chance w h i l e u n l i n k e d m a rkers were i n a c t i v a t e d i n d e p e n d e n t l y . S i m i l a r c o n c l u s i o n s on marker absence ("knockout") were r e a c h e d by E p s t e i n f r o m a s t u d y of T4 m u l t i p l i c i t y r e a c t i v a t i o n ( 2 5 ) . These e a r l y e x p e r i m e n t s were h i n d e r e d by l a c k of s u i t a b l e g e n e t i c markers d i s t r i b u t e d o ver t h e e n t i r e T4 map. W i t h t h e i s o l a t i o n o f c o n d i t i o n a l l e t h a l amber mutants i n T4 ( 2 6 ) , a more compre-h e n s i v e g e n e t i c a n a l y s i s o f c r o s s r e a c t i v a t i o n became p o s s i b l e . Such an a n a l y s i s , p r e s e n t e d by Womack i n 1965 (85),. c o n f i r m e d and extended t h e c o n c l u s i o n t h a t r e s c u e e f f i c e i n c y was dependent on t h e map p o s i t i o n o f the, marker (see b e l o w ) . The most l i k e l y e x p l a n a t i o n f o r t h e c r o s s r e a c t i v a t i o n d a t a was t h a t w i l d t y p e markers were b e i n g r e s c u e d from t h e U V - i r r a d i a t e d phage by r e c o m b i n a t i o n w i t h t h e c a r r i e r phage. These r e s u l t s l e d Doermann e t . a l . t o q u e s t i o n whether t h e h y p o t h e s i s o f e l i m i n a t i o n o f l e t h a l s by g e n e t i c r e c o m b i n a t i o n d u r i n g m u l t i p l i c i t y r e a c t i v a t i o n had been abandoned p r e -m a t u r e l y . They s u g g e s t e d t h a t i t was p o s s i b l e t h a t the undamaged p a r t o f t h e i r r a d i a t e d phage m u l t i p l i e d and t h a t s u c h p a r t i a l r e p l i c a s o f t h e genome were a v a i l a b l e f o r r e c o m b i n a t i o n w i t h t h e c a r r i e r phage. The above and a d d i t i o n a l g e n e t i c d a t a (16) i n d i c a t e d t h a t i t was h i g h l y u n l i k e l y t h a t t h e l e t h a l damages t h e m s e l v e s were r e p l i c a t e d : m u l t i p l i c i t y r e a c t i v a t i o n o c c u r r e d a t a h i g h e f f i c i e n c y b u t l e t h a l s were n o t found i n t h e r e a c t i v a t e d progeny. I n a d d i t i o n i t was f o u n d t h a t t h e a v e r a g e b u r s t s i z e d u r i n g c r o s s r e a c t i v a t i o n was n o t d e p r e s s e d by heavy i r r a d i a t i o n o f t h e w i l d t y p e p a r e n t as i t s h o u l d have been i f damaged a r e a s were r e p l i c a t e d and a v a i l a b l e f o r i n c o r p o r a t i o n i n t o progeny. W i t h an e a r l y v e r s i o n o f t h e p a r t i a l r e p l i c a h y p o t h e s i s , e s s e n t i a l l y a m o d i f i c a t i o n of L u r i a ' s o r i g i n a l recom-b i n a t i o n t h e o r y , B a r r a c e l l i (4) was a b l e t o o b t a i n e x c e l l e n t agreement between q u a n t i t a t i v e p r e d i c t i o n s and D u l b e c c o ' s h i g h UV dose e x p e r i m e n t s ( 2 1 ) . F u r t h e r t h e o r e t i c a l development of p a r t i a l r e p l i c a h y p o t h e s e s was u n d e r t a k e n by B a r r a c e l l i (5) and B a r r a c e l l i and Doermann (6) t o a c c o u n t f o r o b s e r v e d p a t t e r n s o f m u l t i p l i c i t y and c r o s s r e a c t i v a t i o n . E s s e n t i a l l y , p a r t i a l r e p l i c a t h e o r i e s p o s t u l a t e t h a t p a r t i a l r e p l i c a s o f U V - i r r a d i a t e d phage genomes a r i s e as a r e s u l t o f t h e i n a b i l i t y o f t h e r e p l i c a t i o n p r o c e s s td i'proceed t h r o u g h a UV damaged a r e a . R e p l i c a t i o n b l o c k s c o u l d a r i s e as a r e s u l t o f c h e m i c a l m o d i f i c a t i o n s t o t h e DNA, f o r example, p y r i m i d i n e d i m e r s (66,67,69) but t h e e x a c t n a t u r e o f t h e b l o c k s i s n o t i m p o r t a n t t o t h e t h e o r y . The p r i n c i p l e i s i l l u s t r a t e d s c h e m a t i c a l l y i n F i g u r e 2 f o r t h e example of a h y p o t h e t i c a l genome w i t h one o r i g i n of r e p l i c a t i o n . (The X's i n d i c a t e UV i n d u c e d " b l o c k s " i n the DNA and a r e n o t meant as a s u g g e s t i o n o f the n a t u r e of t h e UV l e s i o n s . ) I n t h e n o n - i r r a d i a t e d 5 Figure 2. The p a r t i a l r e p l i c a hypothesis I. Non-irradiated genome. I n i t i a t i o n i s from the indicated o r i g i n . R e p l i c a t i o n i s assumed to proceed b i d i r e c t i o n a l l y . Because there are no blocks to DNA synthesis, the ent i r e molecule i s r e p l i c a t e d . I I . UV-irradiated genome. Again, i n i t i a t i o n i s from the indic a t e d o r i g i n and proceeds b i d i r e c t i o n a l l y but UV lesions prevent DNA synthesis past the nearest points marked with X's, The r e s u l t i s p a r t i a l r e p l i c a t i o n . R eplication of a DNA segment f a c i l i t a t e s rescue of markers i n that segment; thus am 2 w i l l rescue i t s wild type a l l e l e with high e f f i c i e n c y while the wild type l o c i corresponding to am's 1 and 3 w i l l be rescued with low e f f i c i e n c y or not at a l l . parental DNA newly r e p l i c a t e d DNA rescue X UV l e s i o n + o r i g i n wild^i'type a l l e l e am mutant a l l e l e I. N O N - I R R A D I A T E D G E N O M E (or igin I I . UV- IRRADIATED G E N O M E + + X am 1 am 2 j i 3= wild type al leles mutant a l le les am 3 7 m o l e c u l e , r e p l i c a t i o n i s presumed t o i n i t i a t e a t a s p e c i f i c o r i g i n ( w hich may be d e f i n e d p h y s i c a l l y , e.g. by e l e c t r o n m i c r o s c o p y , o r g e n e t i c a l l y — see below) and t o p r o c e e d b i d i r e c t i o n a l l y ; t h i s l e a d s t o t o t a l r e p l i c a t i o n o f t h e genome. I n t h e i r r a d i a t e d m o l e c u l e , however, UV l e s i o n s b l o c k s y n t h -e s i s p a s t c e r t a i n p o i n t s ; t h e r e s u l t i s p a r t i a l r e p l i c a t i o n . R e p l i c a t i o n of a segment of t h e genome f a c i l i t a t e s t h e r e s c u e o f m a rkers i n t h a t segment. T h e r e f o r e , t h e f a r t h e r a segment i s from an o r i g i n of r e p l i c a t i o n , t h e h i g h e r i s t h e l i k e l i h o o d of an i n t e r v e n i n g a r e a of UV damage and the l o w e r i s t h e p r o b a b i l i t y of b e i n g r e p l i c a t e d . C o n s e q u e n t l y t h e e f f i c i e n c y of r e s c u e o f a marker f a r f r o m an o r i g i n would be l o w e r a l s o . A number o f e x p e r i m e n t s w i t h T4 a r e c o n s i s t e n t w i t h a c o r r e l a t i o n o f p a r t i a l r e p l i c a t i o n w i t h o r i g i n s of DNA r e p l i c a t i o n . Womack's c r o s s r e a c t i v a t i o n e x p e r i m e n t s s c o r e d f o r w i l d t y p e r e c o m b i n a n t s a f t e r c o i n f e c t i o n of s e v e r a l E . c o l i s t r a i n s w i t h U V - i r r a d i a t e d T4D+ and one of a d e f i n e d s e t o f r l l or amber mutants ( 8 5 ) . F o r each s t r a i n t e s t e d , a p l o t of t h e phage f r a c t i o n c r o s s r e a c t i v a t e d vs t h e map p o s i t i o n of t h e marker r e v e a l e d 4 d i s t i n c t peaks i n t h e marker r e s c u e e f f i c i e n c y . T h i s number i s c o n s i s t e n t w i t h t h e number of o r i g i n s t h o ught t o o p e r a t e d u r i n g T4 r e p l i c a t i o n : an e l e c t r o n m i c r o s c o p i c a n a l y s i s of t h e T4 genome a t an e a r l y s t a g e o f r e p l i -c a t i o n showed m o l e c u l e s w i t h s e v e r a l i n t e r n a l b u b b l e s (14) w h i l e a n a l y s i s o f p a r t i a l l y r e p l i c a t e d T4 DNA by d e n s i t y t r a n s f e r and s h e a r i n g e x p e r i m e n t s i n d i c a t e d t h a t 3-6 r e p l i c a t i v e s i t e s p e r m o l e c u l e were p r e s e n t ( 4 0 ) . I n a d d i t i o n , one of t h e c r o s s r e a c t i v a t i o n peaks was n e a r gene 43, an a r e a of t h e T4 map r e p o r t e d t o c o n t a i n an o r i g i n of r e p l i c a t i o n ( 5 7 ) . More r e c e n t l y , R a y s s i g u i e r and V i g i e r have c o m p l e t e d an a n a l y s i s of t h e r e c o m b i n a n t c l o n e s i z e d i s t r i b u t i o n of progeny phage p r o d u c e d by m u l t i p l i c i t y r e a c t i v a t i o n of g e n e t i c a l l y marked, U V - i r r a d i a t e d p a r e n t a l phage (6 0 , 6 1 ) . T h e i r a n a l y s i s was c o n s i s t e n t w i t h t h e i d e a t h a t p a r t i a l r e p l i c a s of t h e damaged genomes 8 r e a s s o c i a t e by r e c o m b i n i n g p r i m a r i l y a t t h e i r e x t r e m i t i e s . Study of b a c t e r i o p h a g e T4 has r e s u l t e d i n t h e a c c u m u l a t i o n o f e v i d e n c e c o n s i s t e n t w i t h t h e p a r t i a l r e p l i c a h y p o t h e s i s . S t i l l , no T4 s t u d y has p o s i t i v e l y c o r r e l a t e d p a r t i a l r e p l i c a t i o n , o r i g i n s of DNA s y n t h e s i s , and t h e e f f i c i e n c y o f marker r e s c u e d u r i n g c r o s s o r m u l t i p l i c i t y r e a c t i v a t i o n . S e v e r a l f e a t u r e s o f t h e s m a l l e r c o l i p h a g e T7 make i t an a t t r a c t i v e o r g a n i s m f o r s u c h an a n a l y s i s . A l t h o u g h m u l t i p l i c i t y r e a c t i v a t i o n i s n o t o b s e r v e d w i t h T7 (see above and a l s o R e s u l t s , s e c t i o n B . 2 ) , c r o s s r e a c t i v a t i o n does o c c u r . The s i m i l a r i t y o f t h e two p r o c e s s e s i m p l i e s t h a t an e l u c i -d a t i o n o f t h e l a t t e r w i l l f a c i l i t a t e u n d e r s t a n d i n g o f t h e f o r m e r . T7 i s a d o u b l e s t r a n d e d DNA phage w i t h a l i n e a r genome o n l y about one-quarter t h e s i z e o f t h a t o f T4. The l e n g t h of t h e DNA, d e t e r m i n e d by e l e c t r o n m i c r o -scopy t o be a p p r o x i m a t e l y 12 ym, c o r r e s p o n d s t o a m o l e c u l a r w e i g h t of about 25X10 ( 3 5 ) . The G + C c o n t e n t i s s i m i l a r t o t h a t of E . c o l i , however t h e r e i s no s i g n i f i c a n t sequence homology between phage and h o s t . T7 DNA i s t e r m i n a l l y redundant ( e s t i m a t e s o f t h e redundancy range f r o m 70 t o 200 base p a i r s ) b u t n o t c i r c u l a r l y permuted, as i s T4 DNA ( 3 5 ) . N i n t e e n e s s e n t i a l and a number o f n o n - e s s e n t i a l genes have been mapped, p r i m a r i l y by F.W. S t u d i e r (75,77,78) and t h e i r p r o d u c t s have been a n a l y z e d by g e l e l e c t r o -p h o r e s i s ( 7 6 ) . The t i m e c o u r s e o f t r a n s c r i p t i o n and t r a n s l a t i o n has been e l u c i d a t e d . The e a r l y genes a r e t r a n s c r i b e d by t h e h o s t RNA p o l y m e r a s e . One o f t h e s e e a r l y genes codes f o r a T7 RNA p o l y m e r a s e , w h i c h t r a n s c r i b e s t h e m i d d l e and l a t e genes. I n a d d i t i o n t o c o d i n g f o r c o a t and assembly p r o t e i n s , T7 s y n t h e s i z e s a DNA p o l y m e r a s e , l i g a s e , and an e x o n u c l e a s e and e n d o n u c l e a s e e s s e n t i a l f o r g e n e r a l r e c o m b i n a t i o n of T7 DNA ( 3 5 , 7 6 ) . A n a l y s i s of a c o r r e l a t i o n between p a r t i a l r e p l i c a t i o n and marker r e s c u e of U V - i r r a d i a t e d T7 DNA was f a c i l i t a t e d by two r e s u l t s from o t h e r l a b o r a t o r i e s . I n 1972, an o r i g i n o f r e p l i c a t i o n of T7 DNA was mapped by e l e c t r o n m i c r o s c o p i c a n a l y s i s of p a r t i a l l y r e p l i c a t e d , n o n - i r r a d i a t e d T7 DNA ( 1 8 ) . I t s l o c a t i o n , 17% from t h e g e n e t i c l e f t end of t h e m o l e c u l e , makes i t p o s s i b l e t o examine the c o r r e l a t i o n between t h e p r o x i m i t y of a marker to t h i s d e f i n e d r e p l i c a t i o n o r i g i n and t h e r e s c u e e f f i c i e n c y o f t h a t marker. I n a d d i t i o n , r e s t r i c t i o n f r a g m e n t s of t h e T7 genome were r e c e n t l y a l i g n e d w i t h s p e c i f i c r e g i o n s of the g e n e t i c map ( 5 2 ) . T h i s r e s u l t p o t e n t i a l l y p r o v i d e s a p h y s i c a l a s s a y f o r p a r t i a l r e p l i c a t i o n ; i . e . , s p e c i f i c a l l y l a b e l e d progeny DNA s y n t h e s i z e d by U V - i r r a d i a t e d p a r e n t a l phage may be a n n e a l e d t o s u c h r e s t r i c t i o n f r a g m e n t s s e p a r a t e d by a g a r o s e g e l e l e c t r o -p h o r e s i s and t r a n s f e r e d t o n i t r o c e l l u l o s e f i l t e r s . The p r i m a r y o b j e c t i v e s of t h e r e s e a r c h u n d e r t a k e n f o r t h i s t h e s i s were to e l u c i d a t e t h e p a t t e r n s o f c r o s s r e a c t i v a t i o n of b a c t e r i o p h a g e T7 and t o c o r r e l a t e t h e o b s e r v e d marker r e s c u e e f f i c i e n c i e s ' w i t h p a r t i a l r e p l i c a t i o n of U V - i r r a d i a t e d T7 DNA. To t h i s end, marker r e s c u e e x p e r i m e n t s w i t h T7 s i m i l a r t o t h o s e of Womack w i t h T4 were p e r f o r m e d : w i l d t y p e i n f e c t i v e c e n t e r s or progeny were s c o r e d a f t e r c o i n f e c t i o n of E . c o l i w i t h UV-i r r a d i a t e d T7+ and one of a d e f i n e d s e t of amber mutants.-- Rescue e f f i c i e n c i e s were p l o t t e d as a f u n c t i o n o f t h e map p o s i t i o n s of t h e m a r k e r s . P a r t i a l r e p l i c a t i o n of UV-damaged T7 DNA was examined as a f u n c t i o n of t h e i r r a d i a t i o n 32 dose by h y b r i d i z a t i o n of P - l a b e l e d DNA s y n t h e s i z e d by U V - i r r a d i a t e d phage to o r d e r e d r e s t r i c t i o n f r a g m e n t s of t h e T7 genome. I n o r d e r t o c o n f i r m t h e s e r e s u l t s , p a r t i a l l y r e p l i c a t e d m o l e c u l e s were v i s u a l i z e d d i r e c t l y by e l e c t r o n m i c r o s c o p y . R e c o m b i n a t i o n has been c o n s i d e r e d an e s s e n t i a l s t e p i n t h e marker r e s c u e p r o c e s s . E x p e r i m e n t s were d e s i g n e d t o examine t h e r o l e of r e c o m b i n a t i o n d u r i n g c r o s s r e a c t i v a t i o n and t o d e f i n e whether h o s t e x c i s i o n - r e p a i r enzymes c o u l d be i n v o l v e d . P h o t o r e a c t i v a t i o n o f i n f e c t i v e c e n t e r s by h o s t c e l l enzymes a l s o was examined. T7 codes f o r an e x o n u c l e a s e (gene 6 p r o d u c t ) and an e n d o n u c l e a s e (gene 3 p r o d u c t ) w h i c h have been i m p l i c a t e d i n general recombination of bacteriophage T7 DNA (41,46,47,48, 56,59). The p o s s i b l e i m p l i c a t i o n of these enzymes i n T7 marker rescue was considered. T7 phage have been shown to p r e f e r e n t i a l l y i n j e c t the l e f t end of t h e i r genomes f i r s t during i n f e c t i o n (58). I f UV i r r a d i a t i o n at the doses u s u a l l y employed l e d to breakage of the DNA, both cross r e a c t i v a t i o n and p a r t i a l r e p l i c a t i o n data could be explained p l a u s i b l y by p r e f e r e n t i a l i n j e c t i o n . Therefore, the e f f e c t of UV i r r a d i a t i o n on the i n t e g r i t y of T7 DNA was examined. During the course of t h i s work, r e s u l t s were obtained i n our l a b o r a t o r y which i n d i c a t e d that primary i n f e c t i n g T7 phage prevented i n f e c t i o n of the same c e l l by secondary, or s u p e r i n f e c t i n g , phage. Experiments published by French e t _ . a l . i n 1951(27) and 1952 (28) had shown that primary i n f e c t i o n of E. coll-'by the l a r g e r coliphages T2, T4, T5, and T6 l e d to breakdown of DNA c o n t r i b u t e d by s u p e r i n f e c t i n g phage. Breakdown was measured as an increase 32 i n the a c i d s o l u b i l i t y of P - l a b e l from the DNA of the secondary phage. I t was observed at that time that the smaller phages T l , T3, and T7 d i d not 32 induce s u p e r i n f e c t i o n breakdown of P-labeled DNA. French e_t.al_. (28) con-32 eluded, from attempts to t r a n s f e r P l a b e l from s u p e r i n f e c t i n g phage to progeny of the, primary i n f e c t i n g phage, that " l i t t l e of the phosphorus of T2 phage i s incorporated i n t o T l or T7 progeny when T2 i s excluded from the c e l l by these phages. Breakdown of s u p e r i n f e c t i n g v i r u s n u c l e i c a c i d thus i s 32 not necessary f o r e x c l u s i o n of i t s P from the progeny of an unrelated phage;". T7 phage were shown to exclude s u p e r i n f e c t i n g T7 phage by preventing i n j e c t i o n of t h e i r DNA i n t o the c e l l (7). Conditions which would prevent super-i n f e c t i o n e x c l u s i o n were defined. I I . MATERIALS AND METHODS 11 Strains 1. Bacteria: The following s t r a i n s were u t i l i z e d during the course of thi s research: E . c o l i B23 (non permissive host f o r phage carrying amber mutations i n e s s e n t i a l genes (sup°)); E . c o l i O i l ' ( E . c o l i B de r i v a t i v e , t h i , contains an amber suppressor (sup E); used generally as permissive host f o r phage carrying amber mutations (75)); E . c o l i BR3-4 (single colony i s o l a t e of E . c o l i BR3 (77); non permissive host f o r amber mutants i n e s s e n t i a l and non-- s + es s e n t i a l genes); E . c o l i AB2497 (F , \ , uvrA , leu , thr, t h i , arg, pro, h i s , thy, l a c , ara, g a l , mtl, x y l , s t r , T6 , sup E); E . c o l i AB2500 (isogenic with E . c o l i AB2497 except uvrA6)(39). Strains AB2497 and AB2500 were obtained from W. Masker. The o r i g i n a l s t r a i n E . c o l i BR3 was found to give inconsistent p l a t i n g r e s u l t s and a high proportion of plaques r e s u l t i n g from leaky i n f e c t i o n s when p l a t i n g mutants i n early genes. A sing l e colony i s o l a t e of BR3, BR3-4, was found to give consistent p l a t i n g r e s u l t s and low reversion frequencies for mutants i n the early genes 0.3 and 0.7 when grown d a i l y from a frozen culture. Stocks of BR3-4 transfered on a day to day basis eventually l o s t t h e i r "non-permissiveness" f o r amber mutants i n the non-essential genes 0.3 and 0.7. E . c o l i BR3-4 was found to plate T 7 + with an e f f i c i e n c y of 70-80%, compared to E . c o l i B23 when plates were incubated at 37°. The p l a t i n g e f f i c i e n c y —6 was less than 10 at room temperature. 2. Bacteriophage: A l l bacteriophage s t r a i n s were generously provided by E..W. Studier. NamX i s the general designation f o r amber mutants where N i s the gene number and X i s the s p e c i f i c mutant. NtsX i s the general designation for temperature s e n s i t i v e mutants. The various s t r a i n s used i n t h i s study were T 7 + (wild type), laml93, lam323, lam342a,2am64,3am29, 4am208, 5am28, 6am233, 8amll, llam37, 14aml40, 16am9, 16aml94, 17am290, 19am 10 (75), 0.7amJs62a (77), 0.3amCrl0b (78), 6tsl36, and T7 deletion LG-3 (designated T7VLG-3). The deletion i s from 15.2-19.2% on the genetic map (77). Amber-deletion and amber-temperature s e n s i t i v e double mutants were constructed i n the laboratory. Chemicals and Isotopes L-chloramphenicol (CM), 5-fluorodeoxyuridine (FdU), U r a c i l (U), and cytochrome c (type V) were purchased from the Sigma Chemical Company. Thymidine (dT), pronase, DNase, and RNase were from the Worthington Company. 5-Bromo-deoxyuridine (5-BrdU) was acquired from the Calbiochem Company. Radioactive 3 14- 32 32 isotopes, [methyl- H]thymidine, [methyl- C]thymidine, and P as PO^, were purchased from New England Nuclear Corporation. The density isotopes 15 2 NH^Cl, l^O, and deuterated a l g a l whole hydrolysate were from Merck, Sharp, and Dohme. Formamide, obtained from Matheson, Coleman, and B e l l , was deionized before use by treatment with Bio-Rad AG501-X8 mixed bed r e s i n . R e s t r i c t i o n enzyme Fnu C (Endo R.Fnu C) was generously provided by Ann L u i and Michael Smith. Media and Buffers TCG medium contained 0.1M T r i s . H C l , 0.16M Na2SC>4, l.OmM MgSO^, 0.05% NaCl, 0.1% glucose, 3yM F e C l 3 > 0.05% vitamin-free casamino acids (Di f c o ) , O.lmM CaCl 2 , 40 yg/ml of tryptophan, f i n a l pH 7.4 (43). TCG medium was suppl emented with phosphate by the addition of 0.1M KI^PO^ as 0.64ml/100ml for normal PO.-TCG; 0.128ml/100ml for 1/5 PO.-TCG; and 6.4ml/100ml for 10X 4 4 PO^-TCG. The 1/5 PO^-TCG contains 7yg of PO^/ml (7). Radioactively labeled phage were prepared and most radioactive experiments performed i n TCG medium. BrdU-TCG medium contained lOOyg/ml 5-BrdU. 9 Density medium contained per l i t e r of R^O, 7.0g Na^O^, 3.0g KI^PO^, l.Og 1 5NH 4C1, 0.5g NaCl, 0.03 ml of 0.1M F e C l 3 . A f t e r autoclaving, the following were added: 1.0ml of IM MgSO^, 0.1ml of IM C a C l 2 , 12.5 ml of 13 2 20% glucose i n H^ O and 1.12 ml of deuterated a l g a l whole hydrolysate (84). H-broth contained 5g of peptone (Difco), l g of glucose (Bacto-dextrose, D i f c o ) , 5g of NaCl, and 8g of nutri e n t broth (Difco) per l i t e r of d i s t i l l e d H 20, f i n a l pH 7;.0. L-broth contained lOg of tryptone (Di f c o ) , l g of glucose, 5g of yeast extract (Difco), and 5g of NaCl per l i t e r d i s t i l l e d H 20, f i n a l pH 7.2. L-broth plates contained, i n addition l l g of agar (D i f c o ) . L-broth top layer contained, i n addition, 7-8g agar. T-broth contained lOg of tryptone, l g of glucose, and 5g of NaCl per l i t e r d i s t i l l e d I^O. T-broth plates and top layer contained agar as indicated for L-broth plates and top lay e r . Tris/NaCl/EDTA buffer (TNE) contained 0.01M T r i s . H C l , 0.15M NaCl, 0.015M EDTA (pH 7.4). Tris/EDTA buffer contained 0.5M T r i s . H C l , 0.05M EDTA (pH 7.0). Lys i s buffer contained 0.1M NaCl, 0.02M EDTA, 0.01M KCN, 0.01M iodo-acetate, 0.1M Tris.HCl (pH 7.4) (84). DNA d i a l y s i s b u f f e r contained 0.15M NaCl, 0.05M Tri s . H C l , 0.002M EDTA (pH 7.4). T7- t r i s - s a l t was IM NaCl, 0.05M Tris.HCl (pH 7.4) Standard s a l i n e c i t r a t e (SSC) i s 0.15M NaCl, 0.015M Na c i t r a t e fcH 7.0). Preincubation Mixture (PM) contained 0.02% each of F i c o l l , p o l y v i n y l -pyrrolidone, and bovine serum albumin; 0.1% SDS; 5yg thymidine/ml; 5tig deoxyadenosine/ml i n 3X'SSC (15). U l t r a v i o l e t I r r a d i a t i o n of Phage Phage to be i r r a d i a t e d were d i l u t e d to 1X10"'"''" plaque forming units/ml i n T7 t r i s s a l t and placed i n a p l a s t i c p e t r i dish on i c e . I r r a d i a t i o n was for a defined time (generally 10, 20, 30, or 40 seconds) from a distance of 30 cm by a General E l e c t r i c G15T8 15 watt germicidal lamp. Lethal events 14 were quantitated by p l o t t i n g s u r v i v a l curves of the i r r a d i a t e d phage f o r each experiment. The dose, i n l e t h a l h i t s , i s given by - l n ( s u r v i v i n g phage fr a c t i o n ) (17,49). Rescue of T7 Amber Markers by UV-irradiated T7 Phage E . c o l i O i l ' were grown to 2X10 8 bacteria/ml i n H-broth at 30°. CM was added at lOOug/ml to the culture to i n h i b i t superinfection exclusion (see Results, section A . l ) . At one minute i n t e r v a l s aliquots of the c e l l s were coinfected with UV-irradiated T7 phage and one of a serie s of T7 amber phage. The m u l t i p l i c i t y of each phage type was close to 5 phage/bacterium and was monitored by p l a t i n g the surviving b a c t e r i a . Ten minutes a f t e r i n f e c t i o n , an a l i q u o t of the infe c t e d c e l l s was d i l u t e d into T7 antiserum, incubated 5 minutes at 37°, d i l u t e d further, and plated f o r T 7 + i n f e c t i v e centers on a non-permissive host. A l t e r n a t i v e l y , i n f e c t e d c e l l s were d i l u t e d out of a n t i -serum, incubated u n t i l l y s i s , and plated f o r T 7 + progeny phage. A l l of the amber phage represented on any p a r t i c u l a r graph were examined i n a sin g l e experiment. Inf e c t i v e center production i s not s i g n i f i c a n t l y influenced by incubation i n CM of up to 30 minutes. Photoreactivation of Inf e c t i v e Centers A f t e r Cross Reactivation Cross r e a c t i v a t i o n experiments normally are performed i n the dark. When photoreactivation was desired, rescue experiments were performed as descr-ibed i n the preceeding section except room l i g h t s (fluorescent) remained on. Infective centers were plated on E . c o l i B23; plates then were incubated approximately 30 cm beneath fluorescent lamps f o r 15 hours at room temperature. Preparation of T_7 Double Mutants 1. T7 amber-deletion mutants: T7VLG-3 was crossed with the following amber sin g l e mutants: laml93, lam323, lam342a, 2am64, 3am29, and 4am208. E . c o l i O i l ' was grown to 2X10^ bacteria/ml i n H-broth at 37°. CM then was added at lOOyg/ml to i n h i b i t superinfection exclusion (Results, s e c t i o n . A l ) . A f t e r incubation f o r 6 minutes, prediluted portions of T7VLG-3 and one of the amber mutants were mixed with an equal volume of host c e l l s . Phage were allowed to adsorb f o r 5 minutes at room temperature, d i l u t e d 10,000 f o l d i n T-broth with no CM and incubated without aeration at 30° u n t i l l y s i s . The burst s i z e was monitored on E . c o l i O i l ' while wild type recombinants were assayed on E . c o l i BR3-4, which i s non permissive f o r T7VLG-3 as well as for amber mutants. Plaques a r i s i n g on O i l ' were picked and analyzed f o r t h e i r a b i l i t y to grow on the following i n d i c a t o r s : O i l ' , BR3-4, B23, and BR3-4 plus T7VLG-3. The double mutants were indicated by plaques a r i s i n g on O i l ' but on none of the other i n d i c a t o r s . Such plaques were checked by complemen-tat i o n to confirm that they were of the required type and t h e i r reversion frequencies on the various i n d i c a t o r s were assayed. 2. T7 amber-temperature s e n s i t i v e mutant. The double mutant lam!93-6tsl36 was constructed previously by M. Lee (46). Preparation of P a r t i a l l y Replicated T7 DNA for Hybridization E . c o l i B23 was grown to 3X10 8 bacteria/ml i n 1/5 P0 4 TCG at 30° C and infected with T 7 + at an M0I of 5. Eight minutes a f t e r i n f e c t i o n , 500liCi of 32 P was added to the 2ml cul t u r e . The culture was c h i l l e d by the addition of 3 volumes of i c e cold TNE 17 minutes l a t e r . The infe c t e d c e l l s were sedimented and resuspended i n 1/2 volume of cold TNE. The c e l l s were lysed with SDS, treated with pronase, and extracted with phenol. Samples were sed-imented to equilibrium i n CsCl gradients. Approximately 50 f r a c t i o n s were c o l l e c t e d dropwise from the bottom of the tube; aliquots were TCA p r e c i p i t a t e d and assayed f o r r a d i o a c t i v i t y . Fractions containing the DNA were pooled, dialyzed vs DNA d i a l y s i s buffer (1-2 l i t e r s , flow d i a l y s i s apparatus), and 32 tested f o r resistance to a l k a l i . The P material was greater than 98% r e s i s t a n t to digestion with 1.0N KOH for 18 hours at 37° C. R e s t r i c t i o n Enzyme Fnu C Digests of T7 DNA The reaction mixture contained 6mM T r i s . H C l , pH 7.9, 6mM MgCl„, 6mM 2-mercaptoethanol, 1-2.5yg T7 DNA and 2ul (0.4 units) Endo R.Fnu C i n a f i n a l volume of 25til. (One unit i s the amount of enzyme necessary to digest lug of A DNA i n 1 hour at 37° C.) Mixtures were incubated overnight at 37°. Reactions were stopped by the addition of 5ul of stop mix containing 40% sucrose, 0.25M EDTA pH 7.4, and 0.02% bromophenol blue. The s p e c i f i c i t y of Endo R.Fnu C r e s t r i c t i o n enzyme was determined to be the same as Endo R. Mbo I and Endo R.Dpn II by Ann L u i and Michael Smith (personal communication). Preparation of Hybridization F i l t e r s containing Endo R.Fnu C cut T 7 + DNA  Fragments separated by Agarose Gel Electrophoresis T7 DNA was digested overnight with Endo R.Fnu C as described and the fragments were electrophoresed at 70-80V f o r 7-9 hours i n 15 cm 1% agarose gels containing 0.09M T r i s , 0.09M b o r i c a c i d , 0.025M EDTA (TBE b u f f e r ) , and lug/ml ethidium bromide. Only fragments A-E are retained on the gel a f t e r electrophoresis under these conditions. The procedure for t r a n s f e r r i n g the fragments to n i t r o c e l l u l o s e was modified from Southern (70) and C. Hutchinson (personal communication). DNA i n the gels was denatured f or 30-60 minutes i n a buf f e r containing 0.2M NaOH, 0.6M NaCl, and thymol blue as a pH i n d i c a t o r , rinsed b r i e f l y i n water, and neut r a l i z e d by soaking 30-60 minutes i n IM Tri s . H C l pH 7.4, 1.5M NaCl u n t i l the pH i n d i c a t o r turned green. Two Whatman 3MM f i l t e r s t r i p s were l a i d on a glass plate across a Pyrex dish and wet with 10X SSC (1.5M NaCl, 0.15M Na c i t r a t e ) . The gel was placed on these f i l t e r s , covered with a n i t r o c e l l -ulose f i l t e r , another piece of 3MM paper and a stack of paper towels. The towels were weighted down and the transfer was allowed to proceed overnight i n most cases. (Eight hours i s s u f f i c i e n t ) . The f i l t e r s t r i p s then were dried 1 hour at room temperature and baked i n vacuo 3-4 hours at 80-85° C. The f i l t e r s were stored under vacuum at room temperature u n t i l used f o r hybrid-i z a t i o n . Hybridization of P DNA' to N i t r o c e l l u l o s e f i l t e r s charged with Endo R. Fnu C cut T7 DNA The n i t r o c e l l u l o s e f i l t e r s containing the T7 r e s t r i c t i o n fragments were preincubated 6 hours i n 40 ml PM at 65°. ["^P]DNA was sonicated, heat denat-ured and i c e c h i l l e d , then added to the h y b r i d i z a t i o n mixture. Hybridization proceeded overnight (12-16 hours) at 65°. F i l t e r s then were washed i n 2X SSC four times and exposed f or autoradiography using Kodak X-Omat R XR2 f i l m . Developed films were scanned using a Quick Scan J r . densitometer, Helena Laboratories Corporation. Electron Microscopy P a r t i a l l y ^ r e p l i c a t e d T 7 + molecules were i s o l a t e d from CsCl gradients (Results, section E.l) and dialyzed overnight (4°) against 0.1M T r i s , 0.01M EDTA (pH 7.0) before being mounted for electron microscopic examination by the 40%-10% formamide spreading procedure of Davis e_t .al^. (13) . The spreading sol u t i o n contained l O y l Tris/EDTA buffer, 5 yl cytochrome c, 20yl deionized formamide, and 15-20yl of DNA. This s o l u t i o n was spread immediately onto a fresh hypophase containing lOmM T r i s , ImM EDTA, pH 7.2 and 10% formamide. The DNA-protein f i l m was picked up on parlodion-coated 150-mesh copper grids , stained with uranyl acetate, then rotary shadowed wi th 15-201 of Pt/C. Par-t i a l l y r e p l i c a t e d molecules were photographed at a'-magnif i c a t i o n of 44,000X (c a l i b r a t e d with a d i f f r a c t i o n grating r e p l i c a ) with a P h i l l i p s EM 300 operated at 60kV and using a 30ym objective aperture. Molecular lengths were determined with a map measuring device from tracings of 3X enlarged p r i n t s . A l l measurements were normalized to unit T7 length (found to be 12.0 + 0.5ym). Other Methods 32 3 Preparation of P- and H-labeled bacteriophage, neutral and a l k a l i n e sucrose gradient c e n t r i f u g a t i o n , p u r i f i c a t i o n of i n t r a c e l l u l a r and phage DNA, 18 CsCl equilibrium gradient c e n t r i f u g a t i o n , and uptake of radioactive l a b e l into a c i d insoluble material have been described (46,56). I I I . RESULTS A. S u p e r i n f e c t i o n E x c l u s i o n and L a c k of C o n s e r v a t i v e T r a n s f e r o f B a c t e r i o - phage T7 DNA 1. C h l o r a m p h e n i c o l i n h i b i t s s u p e r i n f e c t i o n e x c l u s i o n . S u p e r i n f e c t i o n e x c l u s i o n r e f e r s t o a p r o c e s s whereby t h e p r i m a r y phage i n f e c t i n g a b a c t e r i a l c e l l i s a b l e t o p r e v e n t p r o d u c t i o n o f progeny phage f r o m s e c o n d a r y , o r s u p e r i n f e c t i n g , phage i n an asynchronous i n f e c t i o n . D u r i n g T i n f e c t i o n t h i s t y p e of e x c l u s i o n i s a r e s u l t o f d e g r a d a t i o n o f t h e even & s e c o n d a r y phage DNA (2 7 , 2 8 ) . J . Benbasat (7) was a b l e to show t h a t s u p e r -i n f e c t i n g T7 phage were b l o c k e d a t t h e i n j e c t i o n s t a g e by p e r f o r m i n g a b l e n d e r e x p e r i m e n t s i m i l a r t o t h a t o f Hershey and Chase ( 3 7 ) . I n a c c o r d w i t h t h i s r e s u l t , she showed t h a t o n l y a l i m i t e d number of p r o s p e c t i v e p a r e n t a l phage were a b l e t o r e p l i c a t e . t h e i r DNA i n each c e l l (see b e l o w ) . Marker r e s c u e e x p e r i m e n t s ( s e c t i o n B) c r i t i c a l l y depend upon s i m u l t a n -eous i n f e c t i o n and DNA r e p l i c a t i o n by c o i n f e c t i n g p a r e n t a l phage. T h e r e -f o r e a s e a r c h was u n d e r t a k e n f o r c o n d i t i o n s w h i c h would i n h i b i t s u p e r i n f e c t -i o n e x c l u s i o n i n T7. I t was found t h a t 5-10 m i n u t e p r e i n c u b a t i o n i n t h e p r o t e i n s y n t h e s i s i n h i b i t o r c h l o r a m p h e n i c o l (CM) a t lOOyg/ml of c u l t u r e would b l o c k s u p e r i n f e c t i o n e x c l u s i o n upon subsequent i n f e c t i o n . T a b l e 1 i l l u s t r a t e s t he r e s u l t s o f a s i m p l e e x p e r i m e n t showing t h a t CM b l o c k s s u p e r i n f e c t i o n e x c l u s i o n . A c u l t u r e o f E . c o l i B23 (sup°) was 8 grown t o 3X10 b a c t e r i a / m l i n 10X PO^ TCG, t h e n s p l i t i n t o two p a r t s . CM a t lOOyg/ml was added t o one p a r t . Then each c u l t u r e was s p l i t i n t o 3 p a r t s and i n f e c t e d w i t h one o f the f o l l o w i n g : T 7 + (M0I=2); T7 5am28 (MOI=20); o r T7 5am28 (M0I=20) f o l l o w e d by T 7 + (M0I=2) 5 m i n u t e s l a t e r . I n f e c t i v e c e n t e r s were d i l u t e d 10 m i n u t e s a f t e r t h e l a t e s t i n f e c t i o n , i n c u b a t e d 5 m i n u t e s i n T7 a n t i s e r u m a t 37°, and p l a t e d on E . c o l i B23. I t can be seen t h a t i n ca s e 3, s u p e r i n f e c t i n g T 7 + a t a low MOI can be e x p r e s s e d i n t h e p r e s e n c e of CM. w h i l e T a b l e 1. E x p r e s s i o n o f T7 Genes A f t e r P r e v i o u s I n f e c t i o n by T7 5 am 28 I n f e c t i n g Phage I n f e c t i v e C e n t e r s ( x l O ^ ) / m l - CM + CM 1 T7 moi = 2 183 103 2 5 am 28 moi = 20 3 5 am 28 moi = 20 f o l l o w e d by T 7 + moi = 2 5 min l a t e r 107 E_. c o l i B23 (sup°) were grown t o 3 x 10 /ml i n 10 x PO^TCG. The c u l t u r e was s p l i t i n t o two p a r t s . To one p a r t , CM was added a t 100 Ug/ml 5 min p r i o r t o i n f e c t i o n . A t 0 min b o t h c u l t u r e were s p l i t i n t o 3 p a r t s and i n f e c t e d w i t h phage as i n d i c a t e d . S u p e r i n f e c t i o n was 5 min a f t e r p r i m a r y i n f e c t i o n i n c a s e 3. I n f e c t i v e c e n t e r s were d i l u t e d 10 min a f t e r t h e l a t e s t i n f e c t i o n , i n c u b a t e d 5 min i n T7 a n t i s e r a a t 37° and p l a t e d on B23. 21 w i t h o u t CM, t h e e x p r e s s i o n i s b l o c k e d . The d a t a i n F i g u r e 3 d e p i c t t h e r e s u l t s of r e c o m b i n a t i o n w i t h d e l a y e d i n f e c t i o n i n t h e p r e s e n c e and absence of CM. E . c o l i O i l 1 (sup E) was grown f o r 2 g e n e r a t i o n s a t 30° i n H - b r o t h . The c u l t u r e t h e n was s p l i t i n t o two p a r t s and CM a t lOOyg/ml was added t o p a r t A. F i v e m i n u t e s l a t e r , p a r t A was s p l i t a g a i n i n t o two p o r t i o n s : A - l was i n f e c t e d s i m u l t a n e o u s l y w i t h T7 5am28 and T7 4am208 a t an MOI o f 10 each. T h i s sample was i n c u b a t e d f o r 5 m i n u t e s ; t h e n an a l i q u o t was t r a n s f e r e d t o T7 a n t i s e r u m , i n c u b a t e d 5 add-i t i o n a l m i n u t e s , d i l u t e d 10,000 f o l d and i n c u b a t e d u n t i l l y s i s . The r e m a i n d e r of t h e c u l t u r e , p a r t A-2, was i n f e c t e d w i t h T7 5am28 (M01=10), t h e n a l i q u o t s were s u p e r i n f e c t e d w i t h T7 4am208 a t 1.0, 2.5, 4.0, 6.5, and 8.5 m i n u t e s a f t e r p r i m a r y i n f e c t i o n . Each sample was i n c u b a t e d f o r 5 m i n u t e s a f t e r s u p e r -i n f e c t i o n , t h e n a l i q u o t s were t r a n s f e r e d t o T7 a n t i s e r u m f o r 5 m i n u t e s . The samples th e n were t r e a t e d as d e s c r i b e d f o r p a r t A - l . The second p o r t i o n of the o r i g i n a l c u l t u r e , p a r t B, was t r e a t e d i d e n t i c a l l y , e x c e p t t h a t no CM was added p r i o r t o p r i m a r y i n f e c t i o n . Recombinants were a s s a y e d on E . c o l i B23 (sup°) and t o t a l progeny on E . c o l i 011'. The p e r c e n t a g e r e c o m b i n a t i o n i s (2X p l a q u e - f o r m i n g u n i t s on E . c o l i B 2 3 ) / ( p l a q u e f o r m i n g u n i t s on E . c o l i O i l ' ) . The f i g u r e c l e a r l y shows t h a t t h e r e c o m b i n a t i o n f r e q u e n c y d e c r e a s e s as a f u n c t i o n of t h e t i m e b e f o r e s u p e r i n f e c t i o n f o r t h e u n t r e a t e d c u l t u r e . A t t h e same t i m e , when c e l l s f i r s t a r e p r e i n c u b a t e d i n CM, t h e r e c o m b i n a t i o n f r e q u e n c y r e m a i n s e f f e c t i v e l y t h e same up to 8 m i n u t e s a f t e r p r i m a r y i n f e c t i o n . These d a t a p r o v i d e a d d i t i o n a l e v i d e n c e t h a t CM b l o c k s s u p e r i n f e c t i o n e x c l u s i o n . S i n c e CM a l l o w s t h e s i m u l t a n e o u s i n f e c t i o n o f a s i n g l e c e l l by s e v e r a l T7 p a r e n t a l phage, r e p l i c a t i o n o f many of t h e s e p a r e n t a l DNA m o l e c u l e s w i t h i n t h e c e l l may be p o s s i b l e . The r e s u l t s of t h e f o l l o w i n g e x p e r i m e n t show t h a t t h i s i s , i n f a c t , t h e c a s e . E . c o l i B23 was grown i n 5-BrdU-TCG medium supplemented w i t h 5yg FdU/ml and 25yg U/ml f o r 90 m i n u t e s a t 37° 22 Figure 3. Recombination with delayed i n f e c t i o n and e f f e c t of chloramphenicol. E . c o l i O i l ' was grown two generations i n H-broth and p o r t i o n s were i n f e c t e d as described i n the t e x t . Recombinants were assayed on E . c o l i B23 (sup ) and t o t a l progeny on E . c o l i O i l ' (sup E). Percent recombination=(2X plaque forming u n i t s on E . c o l i B23)/(plaque forming u n i t s on E . c o l i O i l ' ) . 0- -0- -0: I n f e c t i o n i n the presence of CM. @ 1 ® •' I n f e c t i o n i n the absence of CM. i n t h e d a r k , s h i f t e d t o 30° f o r 15 m i n u t e s , and i n c u b a t e d w i t h CM a t lOOug/ml 32 + f o r 5 m i n u t e s . The c u l t u r e was i n f e c t e d w i t h P - l a b e l e d T7 a t an MOI of 24. A t 7 m i n u t e s a f t e r i n f e c t i o n , t h e CM was removed by two c o n s e c u t i v e s e d i m e n t a t i o n s and t h e i n f e c t e d c e l l s r e s u s p e n d e d i n f r e s h 5-BrdU medium. 32 A l i q u o t s were p l a t e d f o r i n f e c t i v e c e n t e r s and P - l a b e l e d phage a t t a c h e d were d e t e r m i n e d by r a d i o a c t i v i t y . A t 30 m i n u t e s a f t e r i n f e c t i o n DNA was e x t r a c t e d from t h e sample and t h e d i s t r i b u t i o n of r a d i o a c t i v i t y i n a C s C l d e n s i t y g r a d i e n t was d e t e r m i n e d . U n r e p l i c a t e d p a r e n t a l DNA c o n t a i n i n g no 5-BrdU d e n s i t y l a b e l i n e i t h e r s t r a n d bands a t t h e LL l o c a t i o n w h i l e r e p l i c a t e d DNA w i l l band between t h e f u l l y l i g h t (LL) and f u l l y heavy (HH) l o c a t i o n s depending on t h e e x t e n t of r e p l i c a t i o n . F i g u r e 4B shows t h a t a l a r g e p e r c e n t a g e o f the p a r -32 e n t a l P bands a t l o c a t i o n s h e a v i e r t h a n L L . F i g u r e 4A shows th e r e s u l t s o f a s i m i l a r e x p e r i m e n t but w i t h o u t p r e i n c u b a t i o n i n CM p r i o r t o i n f e c t i o n . The l a t t e r e x p e r i m e n t was p e r f o r m e d i n c o n j u n c t i o n w i t h J . B e nbasat. The MOI i n t h i s case was 18. Comparison of p a r t s A and B of F i g u r e 4 r e v e a l s t h a t i n t h e absence of CM, o n l y a v e r y low p e r c e n t a g e o f i n p u t phage a t a h i g h MOI a r e a b l e t o r e p l i c a t e t h e i r DNA ( F i g u r e 4A) w h i l e a f t e r a 5 m i n u t e p r e -i n c u b a t i o n i n CM a t lOOug/ml, a much l a r g e r p r o p o r t i o n of t h e i n p u t DNA i s r e p l i c a t e d ( F i g u r e 4B). The c o n c l u s i o n f r o m t h e s e t h r e e e x p e r i m e n t s i s t h a t 5 m i n u t e i n c u b a t i o n of a c u l t u r e w i t h lOOyg CM/ml p r i o r t o T7 i n f e c t i o n i s a b l e t o b l o c k s u p e r -i n f e c t i o n e x c l u s i o n and a l l o w many p a r e n t a l genomes t o p a r t i c i p a t e i n r e p l i -c a t i o n , r e c o m b i n a t i o n , and e x p r e s s i o n . 2. T7_ DNA i s n o t c o n s e r v a t i v e l y t r a n s f e r e d . C o n s e r v a t i v e t r a n s f e r of DNA d u r i n g b a c t e r i o p h a g e i n f e c t i o n r e f e r s t o i n j e c t e d , p a r e n t a l DNA w h i c h i s n o t r e p l i c a t e d and y e t i s packaged i n t o a new progeny phage. J . Benbasat showed t h a t i n t h e absence of CM, when s u p e r -i n f e c t i o n e x c l u s i o n was o p e r a t i v e , o n l y v e r y s m a l l amounts of c o n s e r v a t i v e Figure 4. Rep l i c a t i o n of P-labeled parental T7 DNA a f t e r high m u l t i p l i c i t y  of i n f e c t i o n i n the presence and absence of CM. 32 + E . c o l i B23 was grown i n 5-BrdU medium and infe c t e d with P-labeled T7 at a high m u l t i p l i c i t y of i n f e c t i o n . DNA was extracted from samples a f t e r 30 minutes of i n f e c t i o n and sedimented to equilibrium i n CsCl. D e t a i l s of the experiments are given i n the text. Bottoms of the gradients are to the l e f t . A. No preincubation i n CM, m u l t i p l i c i t y of infection=18. B. 5 minute preincubation i n 100 yg/ml CM, m u l t i p l i c i t y of infection=24. 3 0 o- -0 H-labeled reference DNA • B 1 P-labeled DNA DNA were found among progeny phage i f u n a t t a c h e d p a r e n t a l phage were removed by d i f f e r e n t i a l s e d i m e n t a t i o n ( F i g u r e 5A). I t was p o s s i b l e t h a t c o n s e r v a t i v e t r a n s f e r was n o t o c c u r r i n g , even a t a h i g h i n p u t m u l t i p l i c i t y of i n f e c t i o n , due t o s u p e r i n f e c t i o n e x c l u s i o n . The f o l l o w i n g d e n s i t y t r a n s f e r e x p e r i m e n t was p e r f o r m e d t o examine c o n s e r v a t i v e t r a n s f e r i n t h e absence of s u p e r i n f e c t -i o n e x c l u s i o n . E . c o l i B23 was grown i n 5-BrdU medium as d e s c r i b e d above, p r e i n c u b a t e d 5 m i n u t e s b e f o r e i n f e c t i o n w i t h CM a t lOOug/ml, t h e n i n f e c t e d 32 + w i t h P - l a b e l e d T7 a t an MOI of 24. CM was removed by two c o n s e c u t i v e s e d i m e n t a t i o n s a t 7 m i n u t e s p o s t i n f e c t i o n and i n f e c t i v e c e n t e r s and a t t a c h e d phage were m o n i t o r e d . The c u l t u r e was i n c u b a t e d u n t i l l y s i s , t h e n progeny phage were p u r i f i e d by d i f f e r e n t i a l s e d i m e n t a t i o n and a n a l y z e d by C s C l d e n s i t y g r a d i e n t s e d i m e n t a t i o n . The r e s u l t s , p r e s e n t e d i n F i g u r e 5B, a r e v e r y s i m i l a r t o t h o s e o b t a i n e d w i t h o u t i n h i b i t i o n o f s u p e r i n f e c t i o n e x c l u s i o n ( F i g u r e 5A) and i n d i c a t e t h a t d e s p i t e t h e i n j e c t i o n o f many p a r e n t a l DNA m o l e c u l e s i n t o t h e c e l l , v e r y l i t t l e u n r e p l i c a t e d (LL) DNA i s packaged i n t o progeny phage p a r t i c l e s . Figure 5. CsCl density gradient of progeny phage a f t e r high m u l t i p l i c i t y  of i n f e c t i o n with and without CM. Aliquots of the cultures described i n the legend to Figure 4 were incubated u n t i l l y s i s and progeny phage p u r i f i e d by CsCl c e n t r i f u g a t i o n . D e t a i l s of the experiment are described i n the text. Bottoms of the grad-ients are to the l e f t . A. No;preincubation i n CM, m u l t i p l i c i t y of infection=18. B. 5 minute preincubation i n 100 Ltg/ml CM, m u l t i p l i c i t y of infection=24. 3 0 0 0 H-labeled reference DNA P-labeled DNA B. C r o s s R e a c t i v a t i o n of B a c t e r i o p h a g e T7 DNA The e x p e r i m e n t s t o be d e s c r i b e d i n t h i s s e c t i o n examine t h e p a t t e r n s of c r o s s r e a c t i v a t i o n (marker r e s c u e ) w h i c h a r e o b t a i n e d f o r b a c t e r i o p h a g e T7 under a v a r i e t y o f c o n d i t i o n s . The o b j e c t i v e s were t o c o r r e l a t e t h e iu marker r e s c u e p a t t e r n s w i t h the d e f i n e d o r i g i n o f T7 r e p l i c a t i o n (18) and w i t h p a r t i a l l y r e p l i c a t e d segments of t h e T7 genome ( s e c t i o n s C and E ) . To r e d u c e e x p e r i m e n t a l i n c o n s i s t e n c i e s . o c c a s i o n e d by a s y n c h r o n o u s i n f e c t i o n of c e l l s i n t h e p o p u l a t i o n by t h e c o i n f e c t i n g U V - i r r a d i a t e d and mutant p a r e n t a l phage, CM was used t o overcome s u p e r i n f e c t i o n e x c l u s i o n . As d e s c r i b e d i n s e c t i o n A, use of CM a l l o w s s i m u l t a n e o u s i n f e c t i o n by and r e p l i c a t i o n o f c o i n f e c t i n g p a r e n t a l m o l e c u l e s . R e c o m b i n a t i o n was shown t o be an e s s e n t i a l s t e p i n t h e c r o s s r e a c t i v a t i o n p r o c e s s ( s e c t i o n B.4). An a t t e m p t was made t o d e f i n e a r o l e f o r h o s t uvrA and p h o t o r e a c t i v a t i o n systems and f o r phage (gene 6) gene p r o d u c t s ( s e c t i o n s B.5, B.6, and B.7). 1. Dose r e s p o n s e of T7~*~ t o u l t r a v i o l e t i r r a d i a t i o n . Phage to be i r r a d i a t e d were d i l u t e d t o 1 X 1 0 ^ p l a q u e f o r m i n g u n i t s / m l i n T7 t r i s s a l t and p l a c e d i n a p l a s t i c p e t r i d i s h on i c e . I r r a d i a t i o n was f rom a d i s t a n c e of 30 cm by a g e r m i c i d a l lamp as d e s c r i b e d i n M a t e r i a l s and Methods. The s u r v i v a l c u r v e of U V - i r r a d i a t e d T 7 + i s b i p h a s i c ( F i g u r e 6 ) . T h i s phenomenon had been o b s e r v e d by o t h e r s (20,24,44). The maximum phage f r a c t i o n showing a l o w e r s e n s i t i v i t y t o UV c o m p r i s e s o n l y about 3% of t h e p o p u l a t i o n and does n o t i n t e r f e r e w i t h subsequent a n a l y s i s . A l l w i l d t y p e E . c o l i s t r a i n s used as i n d i c a t o r s gave s u r v i v a l c u r v e s o f t h i s t y p e . The c u r v e f o r a uvrA s t r a i n was monophasic (see s e c t i o n B.5). A f t e r p h o t o -r e a c t i v a t i o n , t h e s u r v i v a l c u r v e was a g a i n b i p h a s i c but s h i f t e d t o s h a l l o w e r s l o p e (see s e c t i o n B.6). L e t h a l e v e n t s f o r each e x p e r i m e n t were d e t e r m i n e d by p l o t t i n g s u r v i v a l c u r v e s such as F i g u r e 6. The dose was d e t e r m i n e d by c a l c u l a t i n g - l n ( s u r v i v i n g 30 F i g u r e 6. Dose r e s p o n s e o f 17**" phage to u l t r a v i o l e t  i r r a d i a t i o n . T 7 + phage were d i l u t e d t o 1X10"'"''" phage/ ml i n T7 t r i s s a l t and p l a c e d i n a p l a s t i c p e t r i d i s h on i c e . I r r a d i a t i o n was f o r t h e d e s i g n a t e d t i m e s from a d i s t a n c e o f 30 cm by a G e n e r a l E l e c t r i c G15T8 g e r m i c i d a l lamp. A l i q u o t s o f i r r a d i a t e d phage were d i l u t e d and p l a t e d on E . c o l i B23. S u r v i v a l c u r v e s a r e s i m i l a r i f phage a r e p l a t e d on the E . c o l i s t r a i n s O i l ' , BR3-4, and AB2497 ( u v r A + s t r a i n s ) . 0 10 20 IRRADIATION TIME (sec f r a c t i o n ) a c c o r d i n g t o t h e P o i s s o n d i s t r i b u t i o n i f h i t s a r e d i s t r i b u t e d a t random among t h e p o p u l a t i o n o f DNA m o l e c u l e s . 2. M a r k e r r e s c u e w i t h T7"1". C r o s s r e a c t i v a t i o n e x p e r i m e n t s w i t h T7 s i m i l a r t o t h o s e o f Womack w i t h T4 were con d u c t e d i n t h e f o l l o w i n g manner. P e r m i s s i v e E . c o l i O i l ' were c o -i n f e c t e d w i t h U V - i r r a d i a t e d T 7 + b a c t e r i o p h a g e and one of a s e t of d e f i n e d amber mutants. The i n f e c t e d c e l l s were s c o r e d f o r t h e p r o d u c t i o n o f w i l d t y p e i n f e c t i v e c e n t e r s from t h e mixed i n f e c t i o n . F i g u r e 7 shows t h e marker r e s c u e p a t t e r n s o b t a i n e d f o r v a r i o u s doses of UV i r r a d i a t i o n . F i g u r e 7B i l l u s t r a t e s t h e e f f i c i e n c y o f r e s c u e o f U V - i r r a d i a t e d T 7 + DNA c a r r y i n g 6.5 l e t h a l h i t s by a p a r t i c u l a r mutant. A t t h i s UV dose, r e s c u e o f m a r k e r s from 0 t o 30% on t h e T7 genome o c c u r s w i t h a t l e a s t 8 t o 11 t i m e s t h e f r e q u e n c y of marker r e s c u e from t h e r e g i o n o f 50 t o 100%. M a r k e r s i n t h e r e g i o n 50 to 100% of t h e genome a r e r e s c u e d a t a f r e q u e n c y n o t s i g n i f i c a n t l y above t h a t of background i n f e c t i v e c e n t e r p r o d u c t i o n by U V - i r r a d i a t e d phage a l o n e ( d o t -t e d l i n e , F i g u r e 7B). The-maximum marker r e s c u e o b s e r v e d r e p r e s e n t e d about 20% o f t h e i n f e c t i v e c e n t e r s p l a t e d on t h e sup E h o s t , O i l ' ; i . e . , t h e max-imum marker r e s c u e was c l o s e t o 4X10^ IC/ m l . The c u r v e s were v e r y s i m i l a r i f t h e i n f e c t i v e c e n t e r s were d i l u t e d , i n c u b a t e d u n t i l l y s i s , and t i t e r e d f o r y i e l d of T 7 + progeny phage. These d a t a a r e i n a c c o r d w i t h t h e h y p o t h e s i s t h a t DNA r e p l i c a t i o n i n i t i a t e s a t t h e 17% o r i g i n r e g i o n and p r o c e e d s b i d i r e c t -i o n a l l y t o a UV l e s i o n on t h e r i g h t and a UV l e s i o n ( o r t h e end o f t h e m o l e c u l e ) on t h e l e f t . R e p l i c a t i o n f a c i l i t a t e s r e s c u e o f t h e p a r t i a l l y r e p l i -c a t e d r e g i o n . N o n - r e p l i c a t e d r e g i o n s a r e n o t r e s c u e d t o any a p p r e c i a b l e e x t e n t . I f a l o w e r dose, e.g., 4.1 l e t h a l e v e n t s , i s d e l i v e r e d t o T 7 + , the r e s c u e p a t t e r n becomes b r o a d e r w i t h a g r a d u a l d e c r e a s e o f r e s c u e e f f i c i e n c y f r o m t h e g e n e t i c l e f t towards the r i g h t ( F i g u r e 7A). Rescue o f a l l m a r k e r s i s a t l e a s t F R A C T I O N O F M A X I M U M M A R K E R R E S C U E F i g u r e 7. Rescue o f T7 amber phage w i t h U V - i r r a d i a t e d T7 phage. The e f f i c i e n c y of marker r e s c u e f o r a s e t o f amber mutants was d e t e r m i n e d as o u t l i n e d i n M a t e r i a l s and Methods. The p o s i t i o n s o f i n d i v i d u a l amber m u t a t i o n s a r e denoted by t h e gene number o f the v a r i o u s ambers. l a , l b , and l c r e p r e s e n t l a m l 9 3 , lam323, and lam342a r e s p e c t i v e l y . I n f e c t i v e c e n t e r s were p l a t e d on BR3-4. A. 4.1 phage l e t h a l e v e n t s ; B. 6.5 phage l e t h a l e v e n t s ; C. 7.5 phage l e t h a l e v e n t s ; D. 9.5 phage l e t h a l e v e n t s . w somewhat above t h e background l e v e l . Rescue of t h e two r i g h t m o s t m arkers i s s l i g h t l y enhanced; t h i s r e s u l t may be c o r r e l a t e d w i t h enhanced r e p l i c a t i o n o f t h e r i g h t end due t o f o r m a t i o n of head t o t a i l concatemers d u r i n g n o r m a l phage growth (45,64,83; see s e c t i o n C.3). T h i s c u r v e i s a l s o i n a c c o r d w i t h r e p l i c a t i o n i n i t i a t i o n a t 17% w i t h b i d i r e c t i o n a l r e p l i c a t i o n t o s i t e s of UV damage. However, i f t h e r a d i a t i o n dose t o T 7 + i s i n c r e a s e d t o 9.5 l e t h a l h i t s p e r phage p a r t i c l e , m a rkers a l o n g t h e l e f t m o s t 10% of t h e m o l e c u l e a r e r e s c u e d w i t h an e f f i c i e n c y a t l e a s t 8 t o 13 t i m e s g r e a t e r t h a n markers on t h e r i g h t 50 t o 100% and 4 t o 6 t i m e s g r e a t e r t h a n t h a t of m a r k e r s i n t h e l e f t 10 Co 40% of t h e m o l e c u l e ( F i g u r e 7D). I n t e r m e d i a t e d o s e s , e.g., 7.5 l e t h a l e v e n t s ( F i g u r e 7C), g i v e i n t e r m e d i a t e c u r v e s . Thus, t h e dose r e s p o n s e of c r o s s r e a c t i v a t i o n t o UV i r r a d i a t i o n i s c l e a r l y i n d i c a t e d by t h i s f i g u r e : t h e h i g h e r t h e dose, t h e fewer t h e m a r k e r s t h a t a r e e f f i c i e n t l y r e s c u e d . C o n t r a r y t o t h e o r i g i n a l e x p e c t a t i o n , ' m arkers around t h e 17% r e g i o n a r e n o t r e s c u e d w i t h g r e a t e r e f f i c i e n c y as t h e UV dose i s i n c r e a s e d . I n s t e a d , m a r k e r s to t h e l e f t of, 17% c o n s i s t e n t l y a r e r e s c u e d w i t h g r e a t e r e f f i c i e n c y . S e v e r a l i n t e r p r e t a t i o n s of t h e s e r e s u l t s a r e p o s s i b l e . F i r s t , r e p l i -c a t i o n may i n i t i a t e f rom t h e l e f t end o r f r o m a n o t h e r i n t e r n a l o r i g i n l o c a t e d around 2-10% from t h e l e f t end. I n t h i s c a s e , t h e e f f i c i e n c y of r e s c u e i s assumed t o be t h e same f o r any p a r t i a l l y r e p l i c a t e d p i e c e . A n o t h e r p o s s i -b i l i t y i s t h a t , i n f a c t , t h e l e f t end i s r e s c u e d a t a g r e a t e r e f f i c i e n c y w h i l e i n i t i a t i o n s t i l l p r o c e e d s from t h e 17% o r i g i n . A t h i r d e x p l a n a t i o n i s t h a t UV h i t s a r e n o t d i s t r i b u t e d randomly a l o n g t h e genome and t h a t t h e p r o b a b i l i t y of a UV l e s i o n t o t h e r i g h t o f 17% i s g r e a t e r t h a n t o t h e l e f t of 17%, p e r h a p s due "to g r e a t e r A + T c o n t e n t i n t h a t r e g i o n . These i n t e r p r e t a t i o n s w i l l be d i s c u s s e d a t g r e a t e r l e n g t h below ( D i s c u s s i o n ; see a l s o s e c t i o n s C and E ) . I t was i m p o r t a n t t o d e t e r m i n e t h a t p a t t e r n s of marker r e s c u e were n o t due t o a h i g h e r r e v e r s i o n f r e q u e n c y o f amber mutants i n genes on t h e l e f t end 34 of the T7 map. Therefore the r e v e r s i o n frequency of amber mutants under r e s -cue c o n d i t i o n s was measured i n the f o l l o w i n g manner. E . c o l i O i l ' was grown 8 o to 2X10 b a c t e r i a / m l i n H-broth at 30 , preincubated 5 minutes i n CM, then a l i q u o t s were i n f e c t e d w i t h one of the set of amber mutants at an MOI of 5 but without U V - i r r a d i a t e d T 7 + phage. I n f e c t i v e centers then were p l a t e d on a non-permissive host, E . c o l i BR3-4, and scored f o r w i l d type plaques. C o l -umn 2 of Table 2 gives the number of r e v e r t a n t s a f t e r i n f e c t i o n of E . c o l i O i l ' by; amber phage alone. The t a b l e shows that such r e v e r t a n t s are g e n e r a l l y two to three orders of magnitude l e s s than w i l d type i n f e c t i v e centers ob-tained i n the presence of U V - i r r a d i a t e d T 7 + phage (columns 3 and 4) and that the r e v e r s i o n frequency has no p a r t i c u l a r r e l a t i o n to the order of genes on the genetic map. Consequently, the patterns of cross r e a c t i v a t i o n cannot be a t t r i b u t e d to s e l e c t i v e r e v e r s i o n of mutants i n genes on the.-left end of the T7 genome. Cross r e a c t i v a t i o n patterns a l s o are not explained by a higher e f f i c -iency of p l a t i n g of w i l d type markers rescued by amber mutants i n e a r l y genes r e l a t i v e to l a t e genes. When n o n - i r r a d i a t e d T 7 + was used to c o i n f e c t E . c o l i O i l 1 w i t h the defined set of amber mutants and i n f e c t i v e centers were p l a t e d on BR3-4 and O i l ' , there was no v a r i a t i o n among the i n f e c t i v e centers' w i t h the map p o s i t i o n of the marker, (data not shown.) The input m u l t i p l i c i t y of amber phage during cross r e a c t i v a t i o n i s not a c r i t i c a l f a c t o r . Rescue frequencies were e s s e n t i a l l y the same over a range of 2 to 10 amber phage per c e l l given a constant input of U V - i r r a d i a t e d phage. No attempt was made to vary the input of U V - i r r a d i a t e d phage at a constant MOI of amber phage; si n c e the maximum marker rescue e f f i c i e n c y i s only 0.2 (with equal inputs of U V - i r r a d i a t e d and amber phage), meaningful i n t e r p r e t a t i o n of such data would be d i f f i c u l t . U V - i r r a d i a t e d T phage undergo m u l t i p l i c i t y r e a c t i v a t i o n when the 35 T a b l e 2. Amber R e v e r t a n t s Under Rescue C o n d i t i o n s W i l d Type I.C. on BR3-4 Gene Amber Phage A l o n e (x 1 0 " 4 ) Amber Phage + UV T 7 + (x 1 0 ~ 4 ) Amber A l o n e Amber w i t h UV T 7 + 0.3 2 2160 .001 0.7 11 2440 .004 1-193 16 2820 .006 1-323 29 1700 .017 l - 3 4 2 a 8 2140 .004 2 50 1340 .037 3 5 1130 .004 4 <1 700 <.001 5 2 620 .003 6 <1 334 <.003 - 8 2 470 .004 11 3 162 .018 14 1 116 .009 16-194 1 82 .012 17 1 85 .012 19 <1 121 <.008 The r e v e r s i o n f r e q u e n c y of amber mutants under marker r e s c u e c o n d i t i o n s was d e t e r m i n e d by i n f e c t i n g E_. c o l i 011' w i t h each of t h e mutants i n t h e s e t a l o n e , w i t h o u t t h e a d d i t i o n o f U V - i r r a d i a t e d phage. W i l d t y p e i n f e c t i v e c e n t e r were a s s a y e d on E_. c o l i BR3-4. Ambers a r e d e p i c t e d by gene number a l o n e u n l e s s more t h a n one mutant i n a p a r t i c u l a r gene was used or a v a i l a b l e ; t h e n t h e mutant number a l s o i s g i v e n . i n p u t m u l t i p l i c i t y i s g r e a t e r t h a n 2. F o r example, a t an MOI o f 2.8, a p p r o x -i m a t e l y 77% of t h e b a c t e r i a w i l l be m u l t i p l y i n f e c t e d a c c o r d i n g t o t h e P o i s s o n d i s t r i b u t i o n . T6 phage c o n t a i n i n g a p p r o x i m a t e l y 7.9 l e t h a l e v e n t s p r o d u c e a c t i v e T 6 + progeny i n 37% of m u l t i p l y i n f e c t e d c e l l s a t an i n p u t m u l t i p l i c i t y o f 2.8 ( 5 1 ) . The i n p u t m u l t i p l i c i t y o f U V - i r r a d i a t e d T 7 + phage i n marker r e s c u e e x p e r i m e n t s was g e n e r a l l y 5. The e x t e n t t o w h i c h m u l t i p l i -c i t y r e a c t i v a t i o n o f T 7 + c o n t r i b u t e d t o t h e o b s e r v e d c r o s s r e a c t i v a t i o n f r e -q u e n c i e s was d e t e r m i n e d i n t h e f o l l o w i n g manner. E . c o l i B23 was grown t o 2X10^ c e l l s / m l i n H - b r o t h a t 30°, p r e i n c u b a t e d 5 m i n u t e s i n CM as f o r marker r e s c u e e x p e r i m e n t s , and i n f e c t e d a t v a r i o u s MOIs w i t h U V - i r r a d i a t e d T 7 + r e c e i v i n g 7.8 phage l e t h a l h i t s . The m u l t i p l i c i t y o f i n f e c t i o n was d e t e r -mined from t h e f r a c t i o n of s u r v i v i n g b a c t e r i a . Only c e l l s r e c e i v i n g 2 o r more U V - i r r a d i a t e d phage a r e e x p e c t e d t o y i e l d n o r m a l progeny i f m u l t i p l i c i t y r e a c t i v a t i o n o c c u r s . A t each MOI examined, a p p r o x i m a t e l y 1% of m u l t i p l y i n f e c t e d b a c t e r i a y i e l d e d n o r m a l i n f e c t i v e c e n t e r s ( T a b l e 3). There i s no s i g n i f i c a n t d i f f e r e n c e i n t h e o b s e r v e d r e a c t i v a t i o n f r e q u e n c y between v e r y low MOIs (e.g . 0.5) and 1 0 - f o l d h i g h e r MOIs (e.g. 5.3). T h i s f r e q u e n c y may be compared t o 37% f o r b a c t e r i a m u l t i p l y i n f e c t e d w i t h T6 (see above) and 20% maximum r e s c u e i n T 7 + c r o s s r e a c t i v a t i o n e x p e r i m e n t s . Thus ' m u l t i p l i c i t y r e a c t i v a t i o n ' i n b a c t e r i o p h a g e T7 o c c u r s a t a low b a c k g r o u n d l e v e l w h i c h might be due t o a low l e v e l o f i n f e c t i o n by phage s u r v i v i n g UV i r r a d i a t i o n . T h i s r e s u l t a l s o i s o b s e r v e d i n F i g u r e 7B where t h e background IC p r o d u c t i o n by U V - i r r a d i a t e d phage a l o n e i s due t o ' m u l t i p l i c i t y r e a c t i v a t i o n ' . The l o w e r IC p r o d u c t i o n o b s e r v e d i n t h e c a s e of t h e h i g h e s t MOI ( T a b l e 3,row 3) i s p r o b a b l y due t o l y s i s f rom w i t h o u t . M u l t i p l i c i t y r e a c t i v a t i o n p r e s umably i s e f f e c t i v e f o r T4 because t h i s phage p o s s e s s e s m u l t i p l e o r i g i n s of DNA r e p -l i c a t i o n d i s t r i b u t e d i n s e v e r a l r e g i o n s of t h e genome. The o r i g i n ( o r o r i g i n s ) o f T7 r e p l i c a t i o n o c c u r ( s ) , on t h e o t h e r hand, i n o n l y one r e g i o n o f i t s T a b l e 3. M u l t i p l i c i t y R e a c t i v a t i o n o f U V - i r r a d i a t e d T7 M u l t i p l i c i t y o f I n f e c t i o n % B a c t e r i a M u l t i p l y I n f e c t e d 2 p h a g e / c e l l ) IC on B23 (x 1 0 " 6 ) % r e a c t i v a t i o n I c V f r a c t i o n of multiply infected b a c t e r i a 0.5 3.5 4.0 5.3 6.2 9.0 86.4 90.8 96.8 98.5 Q.12 1.18 1.29 1.10 0.75 1.33 1.36 1.42 1.14 0.76 E_. c o l i B23 was grown t o 2 x 10 b a c t e r i a / m l and i n f e c t e d a t v a r i o u s m u l t i p l i c i t i e s o f i n f e c t i o n w i t h U V - i r r a d i a t e d T 7 + r e c e i v i n g 7.8 phage l e t h a l e v e n t s . The e f f e c t i v e moi was d e t e r m i n e d from t h e f r a c t i o n o f s u r v i v i n g b a c t e r i a . I t was assumed t h a t e v e n t s l e a d i n g t o d e a t h o f t h e c e l l s and m u l t i p l e i n f e c t i o n s f o l l o w e d t h e P o i s s o n d i s t r i b u t i o n . C e l l s were i n c u b a t e d i n 100 yg CM/ml t o overcome s u p e r i n f e c t i o n e x c l u s i o n . I n f e c t e d c e l l s were d i l u t e d i n t o T7 a n t i s e r u m 10 m i n u t e s a f t e r i n f e c t i o n , i n c u b a t e d 5 min a t 37°, and p l a t e d f o r i n f e c t i v e , c e n t e r s on B23. 38 genome ( D i s c u s s i o n ; see a l s o s e c t i o n E . 3 ) . 3. Marker r e s c u e w i t h T7VLG-3. The d e l e t i o n mutant T7VLG-3 i s m i s s i n g 15.2 t o 19.2% on t h e T7. map; t h i s i s t h e r e g i o n c o n t a i n i n g a presumed o r i g i n o f r e p l i c a t i o n ( 1 8 , 5 2 ) . T7VLG-3 U V - i r r a d i a t e d w i t h 6.9 phage l e t h a l e v e n t s was c o i n f e c t e d w i t h a s e t o f amber mutants i n an e x p e r i m e n t s i m i l a r t o t h o s e d e s c r i b e d i n Figure.'.,7. Rescue of markers on t h e l e f t 40% of t h e genome i s s t i l l a p p r e c i a b l e ( F i g -u r e 8 ) ; however, r e s c u e o f markers of t h e l e f t m o s t gene 1 mutants i s 3 t i m e s g r e a t e r t h a n markers i n mutants i n t h e 10-40% r e g i o n g i v i n g t h e c u r v e t h e o v e r a l l appearance o f F i g u r e 7D i n s t e a d of 7B w i t h w h i c h t h e UV dose most c l o s e l y c o r r e s p o n d s . M u t a t i o n s i n genes 0.3 and 0.7 were n o t i n c l u d e d i n t h i s a n a l y s i s because T7VLG-3 w i l l n o t p l a t e on t h e n o n - p e r m i s s i v e h o s t f o r t h e s e genes; i . e . , E . c o l i B23 was used as t h e n o n - p e r m i s s i v e h o s t o r g a n i s m because T7VLG-3 w i l l n o t p l a t e n o r m a l l y on E . c o l i BR3-4. Thus, when th e 17% o r i g i n r e g i o n i s d e l e t e d , r e s c u e of m a r k e r s from the l e f t end i s v e r y h i g h , r e c o m b i n a t i o n of genes i n t h e a r e a of t h e d e l e t e d o r i g i n i s d e p r e s s e d , and r i g h t hand markers p l a t e a t t h e background l e v e l . These d a t a i m p l y t h a t t h e absence o f an o r i g i n o f r e p l i c a t i o n a t 17% d e p r e s s e s marker r e s c u e i n t h i s a r e a because p a r t i a l r e p l i c a t i o n o f t h e s e segments i s . d e c r e a s e d . However, i t i s p o s s i b l e t h a t t h e d e l e t i o n i n t e r f e r e s d i r e c t l y w i t h r e c o m b i n a t i o n e v e n t s e s s e n t i a l f o r marker r e s c u e i n t h i s a r e a . I n o r d e r t o examine t h i s p o s s i b i l i t y , a s e t of T7 a m b e r - d e l e t i o n mutants was c o n s t r u c t e d by c r o s s i n g T7 NamX w i t h T7VLG-3 as d e s c r i b e d i n M a t e r i a l s and Methods. Only markers most l i k e l y t o be i n f l u e n c e d by t h e d e l e t i o n were i n c l u d e d i n t h e a n a l y s i s , i . e . , mutants i n genes 1,2,3, and 4. C r o s s r e a c t i v a t i o n s t h e n were p e r f o r m e d . E . c o l i O i l ' was g grown t o 2X10 b a c t e r i a / m l and i n f e c t e d w i t h U V - i r r a d i a t e d T7VLG-3 p l u s a s i n g l e mutant o r p l u s t h e c o r r e s p o n d i n g d o u b l e mutant. Rescue was s c o r e d by p l a t i n g i n f e c t i v e c e n t e r s on E . c o l i B23. The r e s u l t s p r e s e n t e d i n T a b l e 4 F i g u r e 8. Rescue o f T7 amber phage w i t h U V - i r r a d i a t e d T7VLG-3  phage. T7VLG-3 was i r r a d i a t e d t o 6.9 phage l e t h a l e v e n t s . Mar-k e r r e s c u e e f f i c i e n c i e s were d e t e r m i n e d as d e s c r i b e d i n M a t e r i a l s and Methods. The p o s i t i o n s o f i n d i v i d u a l amber mutants a r e d e s -i g n a t e d by the gene numbers of the v a r i o u s ambers. l a , l b , and 1c r e p r e s e n t l a m l 9 3 , lam323, and lam342a r e s p e c t i v e l y . I n f e c t i v e c e n t e r s were p l a t e d on B23. I I 1 I I I 1c 3 j 5 8 ) 2 4 MAP POSITION 2 0 4 0 6 0 8 0 100 P E R C E N T LENGTH OF G E N O M E Table 4. Rescue of Markers from Double Mutants Compared to Single Mutants with UV-irradiated T7 VLG-3 -7, IC on B23 (x 10 ') Gene Amber mutant Amber-deletion double mutant 1-193 4.92 3.53 1-323 2.74 2.58 l-342a 2.08 4.42 2 1.96 2.17 3 1.86 1.65 4 2.00 1.83 E_. c o l i O i l ' was grown to 2 x 10 /ml and infected with UV-i r r a d i a t e d T7 VLG-3 plus a sing l e amber mutant or plus the corresponding amber-deletion double mutant, each at an moi of 5. Marker rescue e f f i c i e n c i e s were determined as described i n Materials and Methods. The data presented i n the table are the average of two experiments where T7 VLG-3 received 5.8 and 6.6 phage l e t h a l events. Wild type i n f e c t i v e centers were assayed on B23. a r e t h e a v e r a g e of two e x p e r i m e n t s where T7VLG-3 r e c e i v e d a r o u n d 6.2 phage l e t h a l h i t s . The marker r e s c u e e f f i c i e n c i e s a r e v e r y s i m i l a r f o r t h e s i n g l e and d o u b l e mutants. Only w i t h t h e r i g h t m o s t gene 1 mutant (342a) i s t h e r e s c u e e f f i c i e n c y s i g n i f i c a n t l y i n c r e a s e d w i t h t h e d o u b l e mutant compared t o . t h e s i n -g l e mutant. The lam342a m u t a t i o n i s c l o s e l y l i n k e d t o t h e l e f t s i d e of t h e d e l e t e d r e g i o n ( F i g u r e 9) and t h e o b s e r v e d i n c r e a s e i s o n l y t w o - f o l d . T h e r e -f o r e , a l o w e r marker r e s c u e e f f i c i e n c y i n t h e v i c i n i t y o f t h e d e l e t e d o r i g i n i s n o t due t o d i r e c t i n t e r f e r e n c e of t h e d e l e t i o n w i t h t h e r e s c u e p r o c e s s . The e f f i c i e n t marker r e s c u e a t t h e l e f t end of t h e U V - i r r a d i a t e d m o l e c u l e i m p l i e s t h a t r e p l i c a t i o n may o r i g i n a t e a t o r near t h e l e f t end of U V - i r r a d -i a t e d T7 DNA when t h e r e g i o n around 17% i s d e l e t e d . 4. M a r k e r r e s c u e w i t h amber mutants. The m o l e c u l a r e v e n t s i n v o l v e d i n t h e c r o s s r e a c t i v a t i o n p r o c e s s a r e unknown. R e c o m b i n a t i o n has been c o n s i d e r e d an e s s e n t i a l s t e p i n t h e p r o c e s s because l e t h a l event c o n t a i n i n g , U V - i r r a d i a t e d phage a l o n e a r e u n a b l e t o c o n -d u c t p r o d u c t i v e i n f e c t i o n s and mutant phage a l o n e a r e u n a b l e t o p r o d u c e w i l d t y p e i n f e c t i v e c e n t e r s . Upon c o i n f e c t i o n of a c e l l w i t h UV-damaged and mu-t a n t phage, r e s c u e of the w i l d t y p e l o c u s by t h e mutant can o c c u r r e s u l t i n g i n t h e p r o d u c t i o n of w i l d t y p e i n f e c t i v e c e n t e r s . S i n c e n o r m a l progeny a r e produced as the r e s u l t o f c o i n f e c t i o n of a c e l l by two s u c h d e f e c t i v e phage, t r a n s f e r of g e n e t i c i n f o r m a t i o n a p p a r e n t l y o c c u r s ( 1 6 , 1 7 ) . The e x p e r i m e n t s d e s c r i b e d i n t h i s s e c t i o n were d e s i g n e d t o d e m o n s t r a t e t h a t r e c o m b i n a t i o n i n f a c t i s n e c e s s a r y f o r marker r e s c u e . I n a d d i t i o n t h e d a t a i n d i c a t e t h a t t h e r e s c u e e f f i c i e n c y i s always g r e a t e r f o r markers on t h e l e f t end o f t h e T7 genome. C r o s s r e a c t i v a t i o n e x p e r i m e n t s s i m i l a r t o t h o s e d e s c r i b e d i n s e c t i o n B.2 were p e r f o r m e d . Here t h e c o i n f e c t i o n s were w i t h two d i f f e r e n t amber phage, one o f w h i c h was UV i r r a d i a t e d . R e c i p r o c a l c r o s s e s w i t h t h e f o l l o w i n g mutants were examined: l a m l 9 3 and lam342a, l a m l 9 3 and 3am29, Figure 9. Genetic map of bacteriophage T_7 Map of T7 genes. The posit i o n s of amber mutations used i n marker rescue experiments are denoted by the small t r i a n g l e s . The l e t t e r s a,b,c,d, and e refer to mutants laml93, lam323, lam342a, 16am9, and 16aml94 respectively. The po s i t i o n of the deletion T7VLG-3 i s i n d i c a t e d by the broken l i n e . The arrow points to the 17% region containing the presumed o r i g i n s i t e . ho 16am9, and 16aml94, i . e . U V - i r r a d i a t e d l a m l 9 3 X lam342a and U V - i r r a d i a t e d lam 342a X l a m l 9 3 , e t c . C r o s s e s were s c o r e d f o r w i l d t y p e i n f e c t i v e c e n -t e r s on E . c o l i B23 and f o r t o t a l I C 's on E . c o l i O i l ' . I n each c a s e a c o n t r o l was i n c l u d e d where n e i t h e r p a r e n t was i r r a d i a t e d ; t h i s c r o s s gave t h e n o r m a l r e c o m b i n a t i o n f r e q u e n c y between t h e two m a r k e r s . The U V - i r r a d i a t e d phage may be c o n s i d e r e d t h e 'donor' o f t h e w i l d t y p e marker r e s c u e d by the non-i r r a d i a t e d phage t o produce a w i l d t y p e i n f e c t i v e c e n t e r , i . e . , t h e r e s c u e d w i l d t y p e marker c o r r e s p o n d s t o t h e mutant marker o f the n o n - i r r a d i a t e d p a r e n t . The r e s u l t s ( T a b l e 5) may be summarized as f o l l o w s . W i l d t y p e i n f e c t i v e c e n t e r s a r e o b s e r v e d i n each c a s e ; t h e r e f o r e r e c o m b i n a t i o n i s i n v o l v e d i n t h e marker r e s c u e p r o c e s s . M a r k e r s i n gene 1 ( c r o s s 1) a r e r e s c u e d a t an a p p r e c i a b l e f r e q u e n c y , t h e e f f i c i e n c y o f r e s c u e o f gene 16 markers ( c r o s s 3) i s l o w e r , w h i l e r e s c u e o f t h e gene 3 marker ( c r o s s 2) i s i n t e r m e d i a t e . The r e s c u e e f f i c i e n c y i n each o f t h e s e c a s e s i s comparable to t h e r e s c u e o f markers from U V - i r r a d i a t e d T 7 + r e c e i v i n g a s i m i l a r UV dose. Note t h a t each r e s u l t i s p r e s e n t e d as a r e c o m b i n a t i o n i n d e x w h i c h i s d e f i n e d as t w i c e t h e marker r e s c u e e f f i c i e n c y (see l e g e n d t o T a b l e 5 ) . I t can be seen ( T a b l e 5) t h a t t h e r e i s no s i g n i f i c a n t d i f f e r e n c e between t h e e f f i c i e n -c i e s o f r e s c u e o f t h e two gene 16 m a r k e r s ( c r o s s 3 ) . Gene 16 i s i n t h e r i g h t h a l f o f t h e T7 genome ( F i g u r e 9) and t h e r e s c u e e f f i c i e n c y o f markers i n gene 16 i s low as e x p e c t e d from t h e e a r l i e r r e s u l t s w i t h U V - i r r a d i a t e d T 7 + ( s e c t i o n B.2). On t h e o t h e r hand, r e s c u e o f markers t o t h e l e f t o f t h e 17% o r i g i n ( g e n e ! ) o c c u r s w i t h g r e a t e r e f f i c i e n c y t h a n r e s c u e o f markers t o t h e r i g h t of 17% (gene 3) even though the m u t a t i o n s l i e a l m o s t e q u i d i s t a n t f r o m the o r i g i n ( F i g u r e 9 ) . Rescue o f t h e w i l d - t y p e gene 1 marker f r o m U V - i r r a d -i a t e d 3am29 by c o i n f e c t i n g l a m l 9 3 i s 3 t i m e s g r e a t e r t h a n r e s c u e o f t h e w i l d - t y p e gene 3 marker i n t h e r e c i p r o c a l case ( c r o s s 2 ) . F u r t h e r m o r e , t h e c l o s e r t o t h e l e f t end a marker l i e s , t he g r e a t e r i s t h e e f f i c i e n c y w i t h w h i c h i t i s r e s c u e d : the l a m l 9 3 marker i s r e s c u e d a t n e a r l y t w i c e t h e e f f i c i e n c y Table 5. Rescue of Markers from UV-irradiated T7 Amber Phage Cross 1st Parent U V d o ^ e . t o ^ s t P a r e n t 2nd Parent . IC on B23 IC on O i l ' Recombination i f i r r a d i a t e d ( l e t h a l h i t s ) Index 1 am 193 - 1 am 342a 8;.3 x 10 ? 2.28 x 10 8 0.73 1 1 am 193 7.3 1 am 342a 1.62 x 10 ? o1.98 x 10 8 0.16 1 am 342a 6.9 1 am 193 3.02 x 10 7 2.20 x 10 8 0.27 1 am 193 - 3 am 29 1.87 x 10 3.10 x 10 1.20 2 1 am 193 7.6 3 am 29 1.32 x 10 7 2.98 x 10 8 0.09 3 am 29 7.5 1 am 193 4.18 x 10 ? 3.01 x 10 8 0.28 16 am 9 - 16 am 194 8.8 x .107 2.52 x 10 8 0.7 3 16 am 9 4.8 16 am 194 3.34 x 10 6 2.22 x 10 8 0.03 16 am 194 5.8 16 am 9 3.32 x 10 6 2.46 x 10 8 0.02 E_. c o l i O i l ' was grown to 3 x 10 bacteria/ml and coinfected with UV-irradiated and non-irradiated amber phage as outlined i n the table. In each experiment, the moi of each parental type was close to 5. Cross r e a c t i v a t i o n e f f i c i e n c i e s were determined by p l a t i n g i n f e c t i v e centers on 011' (total) and B23 (wild type). The marker rescue e f f i c i e n c y i s defined as (IC on B23)/(IC on 011'). The recombination index i s twice the marker rescue e f f i c i e n c y . Note that when neither parent i s i r r a d i a t e d , the recombination index .is the recombination frequency between the two markers. of t h e lam342a marker ( c r o s s 1 ) . These r e s u l t s thus s u p p o r t t h e c o n c l u s i o n s d e r i v e d f r o m c r o s s r e a c t i v a t i o n ' d a t a o b t a i n e d w i t h U V - i r r a d i a t e d T 7 + and T7VLG-3: markers on t h e l e f t end of t h e T7 m o l e c u l e c o n s i s t e n t l y a r e r e s c u e d w i t h t h e h i g h e s t e f f i c i e n c y . 5. E f f e c t o f t h e b a c t e r i a l uvrA e n d o n u c l e a s e on marker r e s c u e . R e c o m b i n a t i o n i s n e c e s s a r y f o r c r o s s r e a c t i v a t i o n b u t i t i s n o t known w h i c h h o s t and/or phage enzymes may be i m p l i c a t e d i n t h i s r e c o m b i n a t i o n , n o r i s i t c l e a r t h a t r e c o m b i n a t i o n i s the o n l y p r o c e s s ^ i n v o l v e d i n t h e c r o s s r e a c t i v a t i o n phenomenon. T h i s s e c t i o n examines t h e p o s s i b l e r o l e of t h e h o s t uvrA gene p r o d u c t d u r i n g marker r e s c u e . The uvrA and uvrB genes o f E . c o l i code f o r an e n d o n u c l e a s e w h i c h has been i m p l i c a t e d i n t h e e x c i s i o n of t h y -m i d i n e d i m e r s d u r i n g d a r k r e p a i r of UV-damaged DNA (9,38,39,65). Howard-F l a n d e r s j a t . a l . (39) d e m o n s t r a t e d e x c i s i o n o f p y r i m i d i n e d i m e r s i n v i v o + - 3 by u v r s t r a i n s b u t n o t by u v r s t r a i n s by l a b e l i n g b a c t e r i a l DNA w i t h H-t h y m i d i n e , U V - i r r a d i a t i n g t h e c e l l s , i n c u b a t i n g them f o r 2 hours a t 37° t o a l l o w e x p r e s s i o n of r e p a i r enzymes, th e n t r e a t i n g the c e l l s w i t h t r i c h l o r o -a c e t i c a c i d . S o l u b l e and i n s o l u b l e f r a c t i o n s were h y d r o l y s e d s e p a r a t e l y i n t r i f l u o r o a c e t i c a c i d and s u b j e c t e d t o d e s c e n d i n g paper chromatography f o r d e t e c t i o n of the d i m e r s . The uvrA and B s t r a i n s t e s t e d were u n a b l e t o e x c i s e UV-induced d i m e r s . D e t a i l s of t h e e n z y m a t i c s t e p s i n t h e e x c i s i o n r e p a i r p r o c e s s were p r e v i o u s l y examined u s i n g h i g h l y p u r i f i e d enzymes ( 3 8 ) . The uvrAB e n d o n u c l e a s e i s s p e c i f i c f o r U V - i r r a d i a t e d DNA i n v i t r o . C r o s s r e a c t i v a t i o n e x p e r i m e n t s n o r m a l l y a r e p e r f o r m e d i n the d a r k so t h a t p h o t o r e a c t i v a t i o n of t h e U V - i r r a d i a t e d phage does n o t o c c u r (6,19,33 58,66). However d a r k r e p a i r o f U V - i r r a d i a t e d p a r e n t a l DNA may f a c i l i t a t e r e s c u e . One mechanism by w h i c h p r o m o t i o n of r e s c u e m i g h t o c c u r i s by r e m o v a l o f b l o c k s t o DNA r e p l i c a t i o n ; t h e n more segments o f DNA c o u l d r e p l i c a t e and, a c c o r d i n g t o the p a r t i a l r e p l i c a h y p o t h e s i s , markers i n t h e s e segments would be r e s c u e d w i t h a h i g h e r e f f i c i e n c y . On t h e b a s i s of t h i s model, one would p r e d i c t marker r e s c u e c u r v e s t o peak s h a r p l y around the o r i g i n o f r e p l i c a t i o n i n t h e absence o f t h e uvrA gene p r o d u c t . I t a l s o i s c o n c e i v a b l e t h a t marker r e s c u e i s an a r t i f a c t of t h e r e p a i r p r o c e s s . I f e x c i s i o n o f t h y m i d i n e dimers o c c u r r e d p r e f e r e n t i a l l y a t the l e f t end of t h e T7 genome, ' r e s c u e ' of markers from t h i s r e g i o n would appear t o be enhanced because n o r m a l w i l d t y p e i n f e c -t i v e c e n t e r s would be produced once t h e DNA was r e p a i r e d . A t t h e same t i m e , genomes c o n t a i n i n g UV l e s i o n s on t h e r i g h t h a l f o f t h e m o l e c u l e , w h i c h p r e -sumably^ a r e r e p a i r e d a t a much lo w e r r a t e , would p r o d u c e n o r m a l i n f e c t i v e c e n t e r s a t a much l o w e r f r e q u e n c y . I f marker r e s c u e i s an a r t i f a c t o f r e p a i r , the absence o f t h e uvrA e n d o n u c l e a s e and t h e r e s u l t i n g -.absence of e x c i s i o n r e p a i r would r e s u l t i n background c r o s s r e a c t i v a t i o n f r e q u e n c i e s f o r m a r k e r s on t h e l e f t , as w e l l as on t h e r i g h t , h a l v e s of the m o l e c u l e . The i s o g e n i c E . c o l i s t r a i n s AB2497 ( u v r A + , sup E) and AB2500 (uvrA6, sup E) (see M a t e r i a l s and Methods) were used i n t h i s a n a l y s i s . Sup E s t r a i n s a r e n e c e s s a r y i n o r d e r t o o b t a i n c o n s i s t e n t r e c o m b i n a t i o n f r e q u e n c i e s between c o i n f e c t i n g p a r e n t a l phage. I n o r d e r t o q u a n t i t a t e e f f e c t i v e l e t h a l e v e n t s t o phage grown i n each of t h e s e s t r a i n s , UV s u r v i v a l c u r v e s o f T 7 + were p l o t t e d u s i n g the u v r A — s t r a i n s as i n d i c a t o r s . The c u r v e o b t a i n e d w i t h AB2497 was e s s e n t i a l l y i d e n t i c a l t o t h o s e o b t a i n e d f o r O i l ' or B23 ( F i g u r e 6 ) . W i t h AB2500, however, t h e c u r v e was monophasic and of a s t e e p e r s l o p e t h a n any p a r t of t h e AB2497 c u r v e . Thus T 7 + phage e f f e c t i v e l y c o n t a i n 3 t o 5 a d d i t i o n a l l e t h a l e v e n t s when p l a t e d on t h e uvrA s t r a i n compared t o t h e u v r A + s t r a i n . C r o s s r e a c t i v a t i o n e x p e r i m e n t s w i t h the u v r A - s t r a i n s were p e r f o r m e d i n t h e f o l l o w i n g manner. P a r a l l e l c u l t u r e s of AB2497 and AB2500 were grown t o 8 a p p r o x i m a t e l y 3X10 b a c t e r i a / m l and c o i n f e c t e d w i t h U V - i r r a d i a t e d T7 and one of a s e t of amber mutants. The m u t a t i o n s used were p r i m a r i l y i n genes l y i n g on t h e l e f t 40% of t h e T7 map; one mutant i n gene 16 s e r v e d as c o n t r o l f o r t h e r i g h t h a n d p p r t i o n of the genome. I n f e c t i v e c e n t e r s were a s s a y e d on 47 E . c o l i BR3-4 ( w i l d t y p e ) and O i l ' ( t o t a l progeny) t o p r e c l u d e s y s t e m a t i c e r r o r due t o p o s s i b l e d i f f e r e n c e s i n t h e e f f i c i e n c y o f p l a t i n g o r o f IC p r o d u c t i o n on t h e u v r A - s t r a i n s . The r e s u l t s , w i t h T 7 + r e c e i v i n g 5.5 phage l e t h a l h i t s , a r e p l o t t e d as (IC on BR3-4/IC on O i l ' ) f o r each u v r A - s t r a i n i n o r d e r t o a c c o u n t f o r ^ d i f f e r e n c e s i n t h e a c t u a l number of c o n t r o l b a c t e r i a between AB2497 and AB2500. F i g u r e 10 shows t h a t t h e shape of t h e r e s c u e c u r v e i s the same i n t h e absence as i n t h e p r e s e n c e of the u v r A gene p r o d u c t . Thus c r o s s r e a c t i v a t i o n i s n o t an a r t i f a c t o f e x c i s i o n r e p a i r . The i m p l i -c a t i o n of t h i s r e s u l t i s t h a t e i t h e r t h e u v r A + e n d o n u c l e a s e does n o t remove b l o c k s t o DNA r e p l i c a t i o n as p o s t u l a t e d above o r t h a t DNA r e p l i c a t i o n i s i n i t i a t i n g a t an a l t e r n a t i v e s i t e o r s i t e s a l o n g the l e f t end of t h e m o l e c u l e (see s e c t i o n s B.2, E, and D i s c u s s i o n ) . A t t h e same t i m e , t h e e f f i c i e n c y o f r e s c u e i s l e s s t h a n 50% as g r e a t w i t h AB2500 compared t o AB2497. T h e r e f o r e , t h e u v r A gene p r o d u c t i s i n v o l v e d , i n some way, d u r i n g c r o s s r e a c t i v a t i o n . One e x p l a n a t i o n t h a t c o u l d a c c o u n t f o r t h i s r e s u l t i s t h a t t h e e f f i c i e n c y of g e n e r a l r e c o m b i n a t i o n i s r e d u c e d i n AB2500 compared t o AB2497. T h i s p o s s i b i l -i t y was t e s t e d by comparing the r e c o m b i n a t i o n f r e q u e n c y between 4am208 and 5am28 i n t h e two s t r a i n s . W i l d t y p e progeny were a s s a y e d on B23 and t o t a l p rogeny on O i l ' . A r e c o m b i n a t i o n f r e q u e n c y between the^phage mutants of 3.5% was f o u n d u s i n g AB2497 w h i l e w i t h AB2500, t h e f r e q u e n c y was 3.0%. So g e n e r a l r e c o m b i n a t i o n i n t h e uvrA s t r a i n i s a t l e a s t 86% as e f f e c t i v e as i n t h e u v r A + s t r a i n and, a t most, o n l y 14% of t h e d i f f e r e n c e i n r e s c u e e f f i c -i e n c i e s between t h e two s t r a i n s can be a c c o u n t e d f o r by d i f f e r e n c e s i n g e n e r a l r e c o m b i n a t i o n . S i n g l e s t r a n d n i c k i n g has been i m p l i c a t e d i n m o l e c u l a r recom-b i n a t i o n ( s e e , e.g., 5 5 ) . D i f f e r e n c e s between t h e u v r A - s t r a i n s i n t h e e x t e n t of n i c k i n g o f T7 DNA were examined by a l k a l i n e s u c r o s e g r a d i e n t s e d -i m e n t a t i o n (see s e c t i o n D). T7 DNA r e c o v e r e d from i n f e c t e d AB2500 c e l l s had fewer s i n g l e s t r a n d s c i s s i o n s t h a n DNA r e c o v e r e d from AB2497. Fewer s i n g l e s t r a n d b r e a k s may a c c o u n t f o r some l o w e r i n g of t h e r e c o m b i n a t i o n f r e q u e n c y Figure 10. E f f e c t of host uvrA gene on marker rescue. T7+ was i r r a d i a t e d to 6.1 phage l e t h a l events (assayed on BR3-4) and used with a set of amber mutants to coinfect p a r a l l e l cultures of AB2497 (uvrA+) and AB2500 (uvrA-). Marker rescue e f f i c i e n c i e s were determined as described i n Materials and Methods. The positions, of i n d i v i d u a l amber mutants are designated by the gene numbers of the various ambers, l a and l c represent laml93 and lam342a re s p e c t i v e l y . I n f e c t i v e centers were monitored on BR3-4 and O i l ' . For each s t r a i n , the data are presented as (IC on BR3-4)/ (IC on O i l ' ) . D e t a i l s of the experiment are in the text. ® © o Rescue e f f i c i e n c y a f t e r i n f e c t i o n of AB249 7 (uvrA+) Ch -O--O Rescue e f f i c i e n c y a f t e r i n f e c t i o n of AB2500 (uvrA-) 1C I 32 4 16 M A P POSIT ION PERCENT LENGTH OF GENOME 49 but a g a i n n o t enough to a c c o u n t f o r t h e d i f f e r e n c e i n r e s c u e e f f i c i e n c i e s . I t i s p o s s i b l e t h a t t h e d i f f e r e n c e i s due t o r e d u c e d v i a b i l i t y of U V - i r r a d -i a t e d DNA i n t h e uvrA h o s t due t o h i g h e r e f f e c t i v e r a d i a t i o n doses. I f DNA c o n t a i n i n g UV damage t h a t r e n d e r s a gene u n a b l e t o f u n c t i o n i s f o u n d among progeny m o l e c u l e s ( 2 9 ) , s u c h DNA might be " r e s c u e d " , i . e . , recombine i n t o t h e amber genome, but n o t be o b s e r v e d because i t i s u n a b l e t o g e n e r a t e w i l d t y p e progeny. Hence, a c l e a r l y d e f i n e d r o l e f o r t h e u v r A e n d o n u c l e a s e cannot be deduced f r o m t h e s e r e s u l t s , however, i n t h e absence of t h i s enzyme, the e f f i c i e n c y of r e s c u e i s r e d u c e d . 6. E f f e c t of p h o t o r e a c t i v a t i o n on marker r e s c u e . P h o t o r e a c t i v a t i o n i s a n o t h e r h o s t - m e d i a t e d p r o c e s s f o r r e p a i r o f UV-damaged DNA ( 6 6 , 6 8 ) . P h o t o r e a c t i v a t i o n - c o m p e t e n t c e l l s show a h i g h e r s u r v i v a l r a t e when i n c u b a t e d a f t e r i r r a d i a t i o n i n t h e p r e s e n c e o f v i s a b l e l i g h t t h a n when i n c u b a t e d i n t h e d a r k . Maximum p h o t o r e a c t i v a t i o n o c c u r s a t w a v e l e n g t h s between 320 and 450nm. P h o t o r e a c t i v a b l e c e l l s have been shown t o code f o r an enzyme w h i c h monomerizes UV-induced p y r i m i d i n e d i m e r s i n v i t r o ( 6 6 , 6 8 ) . I n v i v o , i l l u m i n a t i o n of U V - i r r a d i a t e d b a c t e r i a w i t h p h o t o r e a c t i v a t i n g wave-l e n g t h s r e s u l t s i n t h e d e s t r u c t i o n o f p y r i m i d i n e d i m e r s ' ( i . e . , t h e i r l o s s f rom t h e DNA); presumably dimer " d e s t r u c t i o n " r e p r e s e n t s t h e m o n o m e r i z a t i o n of d i m e r s ( 6 8 ) . When th e s u r v i v a l of U V - i r r a d i a t e d T 7 + i s m o n i t o r e d under p h o t o r e a c t i -v a t i o n c o n d i t i o n s , i t i s found t h a t t h e shape o f t h e s u r v i v a l c u r v e r e m a i n s b i p h a s i c ( F i g u r e 6) i n d i c a t i n g t h a t " d a r k r e p a i r " o c c u r s e i t h e r i n t h e p r e -sence o r absence of p h o t o r e a c t i v a t i n g l i g h t (see s e c t i o n B.5). However t h e s l o p e s of b o t h p o r t i o n s of t h e s u r v i v a l c u r v e a r e l e s s s t e e p a f t e r p h o t o -r e a c t i v a t i o n . P h o t o r e a c t i v a t e d T7 phage e f f e c t i v e l y c o n t a i n fewer l e t h a l e v e n t s ; t h e number of l e t h a l e v e n t s removed depends on t h e o r i g i n a l UV dose ( t h e h i g h e r t h e dose, i n g e n e r a l t h e more h i t s t h a t a r e removed. The a c t u a l number depends on the c o n d i t i o n s of t h e e x p e r i m e n t ) . S i n c e p h o t o r e a c t i v a t i o n i s known t o remove p y r i m i d i n e d i m e r s (see a b o v e ) , a t l e a s t some of t h e l e t h a l e v e n t s a r e due t o d i m e r s . I f p y r i m i d i n e d i m e r s i n f a c t a c t as b l o c k s t o DNA r e p l i c a t i o n and DNA r e p l i c a t i o n i s r e q u i r e d f o r marker r e s c u e , p h o t o r e a c t i v a t i o n d u r i n g c r o s s r e a c t i v a t i o n s h o u l d i n c r e a s e t h e e f f i c i e n c y of marker r e s c u e i n r e g i o n s o f t h e genome w h i c h , a t a g i v e n UV dose, n o r m a l l y show l i t t l e r e s c u e . To i n -g v e s t i g a t e t h i s p o s s i b i l i t y , E . c o l i O i l ' was grown t o 3X10 c e l l s / m l and c o -i n f e c t e d w i t h U V - i r r a d i a t e d T 7 + c o n t a i n i n g 10.1 phage l e t h a l e v e n t s and one of a s e t of amber mutants. Two c r o s s r e a c t i v a t i o n e x p e r i m e n t s were r u n s e q u e n t i a l l y ; b o t h employed t h e same U V - i r r a d i a t e d phage s t o c k . I n one, i n f e c t i o n s were p e r f o r m e d and t h e r e s u l t i n g p l a t e s i n c u b a t e d i n t h e d a r k . I n t h e o t h e r , i n f e c t i o n s were p e r f o r m e d w i t h t h e f l u o r e s c e n t room l i g h t s on and p l a t e s were i n c u b a t e d a p p r o x i m a t e l y 30cm b e n e a t h f l u o r e s c e n t l i g h t s . The s e t of mutants i n c l u d e d m a rkers i n genes •Sjcovering t h e whole of t h e T7 map w i t h the e x c e p t i o n o f 0.3 and 0.7; due t o t h e t e c h n i c a l n e c e s s i t y of i n c u b a t i n g p l a t e s a t room t e m p e r a t u r e d u r i n g p h o t o r e a c t i v a t i o n , B23 was used as t h e n o n - p e r m i s s i v e h o s t i n s t e a d of BR3-4 (see M a t e r i a l s and M e t h o d s ) . A f t e r p h o t o r e a c t i v a t i o n , t h e U V - i r r a d i a t e d phage c o n t a i n e d 7.5 e f f e c t i v e l e t h a l e v e n t s . I n t h e absence of p h o t o r e a c t i v a t i o n , o n l y m a rkers i n t h e l e f t 10% of t h e T 7 + genome a r e r e s c u e d w i t h a p p r e c i a b l e f r e q u e n c y ; t h e e f f i c i e n c y o f r e s c u e i s a t t e n u a t e d s h a r p l y t o t h e r i g h t of t h e l a m l 9 3 marker ( F i g u r e 1 1 ) . T h i s r e s u l t i s i n a c c o r d w i t h t h e e f f i c i e n c y o f r e s c u e o b s e r v e d a f t e r c r o s s r e a c t i v a t i o n o f T 7 + r e c e i v i n g 9.5 phage l e t h a l e v e n t s ( F i g u r e 7D, s e c t i o n B.2). However i f c r o s s r e a c t i v a t i o n o c c u r s i n t h e p r e s e n c e of p h o t o r e a c t i v a t i n g l i g h t , a v e r y d i f f e r e n t p a t t e r n i s o b t a i n e d ( F i g u r e 1 1 ) . D e s p i t e heavy UV i r r a d i a t i o n of the T 7 + p a r e n t a l phage, c o i n f e c t i n g amber phage a r e a b l e t o r e s c u e markers a t a s i g n i f i c a n t f r e q u e n c y f r o m the l e f t 40-50% o f t h e genome. Figure 11. E f f e c t of photoreactivation on marker rescue. E . c o l i O i l 1 was grown to 3X10 bacteria/ml and coinfected with UV-i r r a d i a t e d T 7 + (10.1 phage l e t h a l events) and one of a defined set of amber mutants. Marker rescue e f f i c i e n c i e s were determined as described i n Materials and Methods. Infective centers were plated on B23. Two sequential experiments using the same UV-irradiated phage were performed; one was performed as usual i n the dark and plates were incubated i n the dark. The second was performed with the room (fluorescent) l i g h t s on and plates were incubated under fluorescent l i g h t s (distance approximately 30 cm). At the given UV dose, photoreactivated phage contained only 6.5 l e t h a l events as monitered by s u r v i v a l curve. The positions of i n d i v i d u a l amber mutants are designated by the gene numbers of the various ambers, l a , l b , and l c represent laml93, lam323, and lam342a r e s p e c t i v e l y . 0- -0- -0 IC plates incubated i n the dark I 1 • IC plates incubated under photo-r e a c t i v a t i n g l i g h t . 52 0 20 40 60 80 100 PERCENT LENGTH OF GENOME The c u r v e now more c l o s e l y r e s e m b l e s t h a t f o r r e s c u e a f t e r a l o w e r UV dose ( e . g . , F i g u r e 7B). T h i s r e s u l t i s c o n s i s t e n t w i t h t h e r e m o v a l o f s e v e r a l l e t h a l h i t s f r o m t h e h e a v i l y i r r a d i a t e d T 7 + phage as j u d g e d by p l o t t i n g a s u r v i v a l c u r v e ; see a b o v e ) . A c c o r d i n g t o t h e p a r t i a l r e p l i c a h y p o t h e s i s , b l o c k s t o DNA r e p l i c a t i o n p r e v e n t e f f i c i e n t r e s c u e of m arkers f r o m noh-r e p l i c a t e d segments of the DNA. The e f f e c t of p h o t o r e a c t i v a t i o n i s t o remove p y r i m i d i n e d i m e r s from UV-damaged DNA ( 6 6 , 6 8 ) . The f a c t t h a t e f f i c i e n t r e s c u e o f m a rkers from t h e l e f t 40% of a h e a v i l y i r r a d i a t e d T 7 + m o l e c u l e o c c u r s a f t e r p h o t o r e a c t i v a t i o n i s c o m p a t i b l e w i t h p y r i m i d i n e d i m e r s c o n s t i t u t i n g b l o c k s t o DNA r e p l i c a t i o n . Dimers a p p a r e n t l y a r e removed a t random i n a p r o c e s s w h i c h i s n o t 100% e f f i c i e n t . The r e s u l t s d e p i c t e d i n F i g u r e 11 a r e c o n s i s t e n t w i t h r e p l i c a t i o n i n i t i a t i n g f rom a s i t e around o r t o t h e l e f t of 17%, p r o c e e d i n g b i d i r e c t i o n a l l y p a s t r e p a i r e d p y r i m i d i n e d i m e r s and h a l t i n g a t t h e f i r s t u n r e p a i r e d l e s i o n . A g a i n , e f f i c i e n t marker r e s c u e i s f a c i l i t a t e d by r e p l i c a t i o n o f DNA segments c o n t a i n i n g t h e m a r k e r s . 7. E f f e c t of T7 gene p r o d u c t s on marker r e s c u e . The T7 gene 6 e x o n u c l e a s e and gene 3 e n d o n u c l e a s e a r e n e c e s s a r y f o r m o l e c u l a r and g e n e t i c r e c o m b i n a t i o n of b a c t e r i o p h a g e T7 DNA (41,46,47,48,56, 59). T h e r e f o r e i t i s c o n c e i v a b l e t h a t e i t h e r o r b o t h of t h e s e enzymes a r e i n v o l v e d i n t h e T7 c r o s s r e a c t i v a t i o n p r o c e s s . I f a gene p r o d u c t i s i n v o l v e d i n marker r e s c u e , i t s h o u l d be p o s s i b l e t o d e m o n s t r a t e a r e d u c t i o n o f r e s c u e e f f i c i e n c y f o r c r o s s r e a c t i v a t i o n e x p e r i m e n t s p e r f o r m e d under s e m i p e r m i s s i v e c o n d i t i o n s f o r the gene p r o d u c t . Comparisons of the r e s c u e e f f i c i e n c i e s of a w i l d t y p e marker f r o m a U V - i r r a d i a t e d phage c a r r y i n g a t e m p e r a t u r e s e n s i t i v e m u t a t i o n i n gene 3 or gene 6 by an amber c a r r i e r phage c o u l d be made fr o m c r o s s e s w h i c h were homozygous o r h e t e r o z y g o u s f o r t h e t e m p e r a t u r e s e n s i t i v e m u t a t i o n under p e r m i s s i v e or s e m i p e r m i s s i v e c o n d i t i o n s f o r t h e t e m p e r a t u r e s e n s i t i v e enzyme. S e m i p e r m i s s i v e c o n d i t i o n s f o r t e m p e r a t u r e s e n s i t i v e mutants 54 o f t e n a r e found a t t e m p e r a t u r e s i n t e r m e d i a t e between t h e p e r m i s s i v e and non-p e r m i s s i v e t e m p e r a t u r e s . F o r example, t h e t e m p e r a t u r e s e n s i t i v e , e x o n u c l e a s e of T7 6 t s l 3 6 i s i n a c t i v a t e d a t 42° b u t i t r e t a i n s some a c t i v i t y a t 37° ( 5 6 ) . So a p o t e n t i a l e x p e r i m e n t would be t o c r o s s U V - i r r a d i a t e d 6 t s l 3 6 w i t h l a m l 9 3 ( h e t e r o z y g o u s case) o r w i t h I a m l 9 3 - 6 t s l 3 6 (homozygous c a s e ) a t 30° ( p e r m i s s i v e t e m p e r a t u r e ) and a t 37° ( s e m i p e r m i s s i v e t e m p e r a t u r e ) . I t i s n e c e s s a r y t o measure progeny phage r a t h e r t h a n i n f e c t i v e c e n t e r s i n such an ex p e r i m e n t t o i n s u r e t h a t s e m i p e r m i s s i v e c o n d i t i o n s a r e m a i n t a i n e d . T h i s p r o c e d u r e s h o u l d n o t a f f e c t t h e r e s u l t s as marker r e s c u e e f f i c i e n c i e s a r e s i m i l a r whether i n f e c t i v e c e n t e r s o r progeny phage a r e a s s a y e d (see s e c t i o n B.2). However, one p o t e n t i a l p r o b l e m w i t h s u c h e x p e r i m e n t s i s e n t a i l e d by t h e f a c t t h a t b u r s t s i z e s i n marker r e s c u e e x p e r i m e n t s a r e t y p i c a l l y around 20 phage p e r IC. I n a d d i t i o n , b u r s t s under c o n d i t i o n s d e s i g n e d t o d e m o n s t r a t e t h e i n v o l v e -ment of t h e gene 3 e n d o n u c l e a s e o r gene 6 e x o n u c l e a s e d u r i n g r e c o m b i n a t i o n a r e t y p i c a l l y l e s s t h a n 2 phage p e r i n f e c t e d c e l l ( 4 1 , 5 6 ) . Thus i t m i g h t be e x p e c t e d t h a t t h e v e r y low b u r s t s i z e s a n t i c i p a t e d i n an e x p e r i m e n t i n -v o l v i n g b o t h c r o s s r e a c t i v a t i o n and s e m i p e r m i s s i v e c o n d i t i o n s f o r e i t h e r T7 n u c l e a s e would make m e a n i n g f u l i n t e r p r e t a t i o n of r e s u l t i n g d a t a i m p o s s i b l e . P r e l i m i n a r y r e s u l t s from e x p e r i m e n t s p e r f o r m e d as o u t l i n e d above w i t h t h e t e m p e r a t u r e s e n s i t i v e gene 6 mutant have i n d i c a t e d t h a t , i n f a c t , b u r s t s i z e s under s e m i p e r m i s s i v e c o n d i t i o n s f o r the homozygous c r o s s a r e e x t r e m e l y low. The t o t a l b u r s t measured on O i l 1 i n one s u c h e x p e r i m e n t was 0.01 phage p e r i n f e c t i v e c e n t e r . No w i l d t y p e progeny were d e t e c t e d a t t h e l o w e s t p o s s i b l e d i l u t i o n i n t h e e x p e r i m e n t ( l e s s t h a n 0.0001 phage p e r I C ) . The T7 gene 6 e x o n u c l e a s e and gene 3 e n d o n u c l e a s e a r e i n v o l v e d i n many a s p e c t s o f i n t r a -c e l l u l a r phage DNA m e t a b o l i s m ( e . g . g e n e r a l r e c o m b i n a t i o n , f o r m a t i o n o f con-catemers, 41,46,56); because o f t h i s , i t i s v e r y d i f f i c u l t t o e l u c i d a t e a r o l e f o r them i n marker r e s c u e w i t h o u t s e r i o u s l y a f f e c t i n g t h e i r o t h e r a c t i v i t i e s w h i c h a r e e s s e n t i a l f o r n o r m a l phage development. I n a d d i t i o n , t h e low b u r s t 55 s i z e s i n h e r e n t i n c r o s s r e a c t i v a t i o n e x p e r i m e n t s t h e m s e l v e s a p p a r e n t l y make the c l e a r d e m o n s t r a t i o n o f i n v o l v e m e n t of t h e s e T7 gene p r o d u c t s i n t h e marker r e s c u e p r o c e s s i m p o s s i b l e . 8. Marker r e s c u e : summary of r e s u l t s . The e f f i c i e n c y o f r e s c u e o f w i l d t y p e markers from U V - i r r a d i a t e d T7 phage by c o i n f e c t i n g amber phage depends on t h e map p o s i t i o n of t h e marker. M a r k e r s l o c a t e d on t h e l e f t p o r t i o n of t h e T7 genome a r e r e s c u e d w i t h t h e g r e a t e s t e f f i c i e n c y . The e f f e c t i s dose dependent: t h e h i g h e r t h e dose t o t h e U V - i r r a d i a t e d phage, t h e fewer t h e markers w h i c h a r e r e s c u e d . A t t h e h i g h e s t d o s e s , o n l y markers from t h e l e f t 10% o f t h e m o l e c u l e a r e r e s c u e d w i t h a p p r e c i a b l e f r e q u e n c y i n t h e p r e s e n c e as w e l l as i n t h e absence o f the presumed DNA r e p l i c a t i o n i n i t i a t i o n r e g i o n a t 17%. R e c o m b i n a t i o n i s n e c e s s a r y f o r c r o s s r e a c t i v a t i o n . Absence of t h e b a c t e r i a l uvrA e n d o n u c l e a s e d e c r e a s e s r e s c u e e f f i c i e n c y o v e r t h e map by a t l e a s t a f a c t o r o f 2 w h i l e p r e s e r v i n g t h e o v e r a l l shape of the c r o s s r e a c t i v a t i o n c u r v e . P h o t o r e a c t i v a t i o n o f i n f e c t i v e c e n t e r s d u r i n g c r o s s r e a c t i v a t i o n r e s u l t s i n a s u b s t a n t i a l i n c r e a s e i n t h e e f f i c i e n c y o f r e s c u e , commensurate w i t h t h e r e m o v a l of l e t h a l e v e n t s ( p y r i m i -d i n e d i m e r s ) by t h i s p r o c e s s . The T7 e x o n u c l e a s e (gene 6 p r o d u c t ) and/or T7 e n d o n u c l e a s e (gene 3 p r o d u c t ) a l s o may be i n v o l v e d i n marker r e s c u e but p r e l i m i n a r y e x p e r i m e n t s w i t h a t s gene 6 mutant gave such low b u r s t s i z e s under s e m i p e r m i s s i v e c o n d i t i o n s t h a t c l e a r i n t e r p r e t a t i o n o f t h e d a t a was i m p o s s i b l e . C. P a r t i a l R e p l i c a t i o n o f B a c t e r i o p h a g e T7 DNA: H y b r i d i z a t i o n A n a l y s i s The p a r t i a l r e p l i c a h y p o t h e s i s p r e d i c t s t h a t t h o s e markers w h i c h a r e e f f i c i e n t l y r e s c u e d f r o m a U V - i r r a d i a t e d phage genome l i e i n segments o f t h e DNA w h i c h a r e r e p l i c a t e d d u r i n g phage i n f e c t i o n . A t t h e same t i m e , m a r k e r s w h i c h a r e n o t r e s c u e d l i e i n u n r e p l i c a t e d r e g i o n s . The o b j e c t i v e o f C t h e e x p e r i m e n t s t o be r e p o r t e d i n t h i s s e c t i o n was t o p r o v i d e a p h y s i c a l c h a r a c t e r - i z a t i o n of t h e DNA s y n t h e s i z e d a f t e r i n f e c t i o n of E . c o l i w i t h U V - i r r a d i a t e d T 7 + phage. R e p l i c a t i o n of s p e c i f i c r e g i o n s of T7 DNA was a s s a y e d by h y b r i d -32 i z a t i o n o f P - l a b e l e d progeny DNA t o a l i g n e d r e s t r i c t i o n f r a g m e n t s of t h e T7 genome. The e x t e n t o f r e p l i c a t i o n a f t e r v a r i o u s doses o f UV i r r a d i a t i o n can be c o r r e l a t e d w i t h t h e d e f i n e d o r i g i n o f DNA r e p l i c a t i o n (18) and w i t h t h e p a t t e r n s o f marker r e s c u e d e s c r i b e d i n s e c t i o n B. 1. R e s t r i c t i o n enzyme Fnu map of b a c t e r i o p h a g e T7. The f i v e l a r g e r e s t r i c t i o n f r a g m e n t s g e n e r a t e d by Endo R.Fnu C d i g e s t i o n of T 7 + DNA form a c o n v e n i e n t group f o r the s t u d y o f p a r t i a l l y r e p l i c a t e d T7 DNA. Endo R.Fnu C was i s o l a t e d by A. L u i and M. S m ith and shown by them to have t h e same s p e c i f i c i t y as Endo R.Dpn I I ( 5 2 ) . The f r a g m e n t s g e n e r a t e d by Endo R.Dpn I I d i g e s t i o n of T 7 + DNA have been a l i g n e d by McDonnel e _ t . j i l . (52) w i t h t h e T7 g e n e t i c map ( F i g u r e 1 2 ) . When s e p a r a t e d by e l e c t r o p h o r e s i s on 1% a g a r o s e g e l s o n l y t h e f i v e l a r g e f r a g m e n t s , A-E, a r e r e t a i n e d . The amount of DNA i n each band i s p r o p o r t i o n a l t o t h e s i z e of t h e f r a g m e n t . T h i s means t h a t each band r e p r e s e n t s a u n i q u e fragment df t h e T7 genome; t h e r e f o r e , when t r a n s f e r e d from a g a r o s e g e l s t o n i t r o c e l l u l o s e f i l t e r s , s u c h s e p a r a t e d f r a g -32 ments can be used t o q u a n t i t a t e P - l a b e l e d progeny DNA from s p e c i f i c r e g i o n s of t h e T7 genome by h y b r i d i z a t i o n . The Endo R.Fnu C fragment l e n g t h s a r e compared i n t a b l e 6 w i t h the h y b r i d i z e d f r a c t i o n a l c o n t r i b u t i o n s t o each 32 fragment f r o m s o n i c a t e d , P - l a b e l e d DNA e x t r a c t e d from T7 phage. H y b r i d i -z a t i o n i s seen t o be random arid e q u a l l y e f f i c i e n t o v e r t h e l e n g t h of t h e g e l . Figure 12. R e s t r i c t i o n map of Endo R. Fnu C T7' fragments. Map of the fragments generated from T 7 + DNA by cutting with DpnII. (Taken from McDonnel e t . a l . (52)). Endo R. Fnu C has the same s p e c i f i c i t y as DpnII (M. Smith and A. L u i , personal communication). The p o s i t i o n of the deletion T7VLG-3 i s indicated by the broken l i n e . The arrow points to the 17% region containing the presumed o r i g i n s i t e . The small tria n g l e s mark the p o s i t i o n of the p a r t i c u l a r amber mutants used i n the marker rescue experiments. The l e t t e r s a,b,c,d, and e r e f e r to mutants laml93, lam323, lam342a, 16am9, and 16aml94 re s p e c t i v e l y . 2. P r e p a r a t i o n o f p a r t i a l l y r e p l i c a t e d T7 DNA f o r h y b r i d i z a t i o n . Progeny DNA from U V - i r r a d i a t e d T 7 + phage was s p e c i f i c a l l y l a b e l e d w i t h 32 8 P i n t h e f o l l o w i n g manner. E . c o l i B23 was grown t o 3X10 c e l l s / m l i n 1/5 + 32 PO^ TCG and i n f e c t e d w i t h U V - i r r a d i a t e d T7 a t an MOI o f 5. P a t a h i g h s p e c i f i c a c t i v i t y (50mCi/mg PO^) was added 8 m i n u t e s a f t e r i n f e c t i o n . A t t h a t t i m e , u p t a k e o f l a b e l i n t o h o s t DNA i s n e g l i g i b l e a f t e r i n f e c t i o n w i t h n o n - i r r a d i a t e d T 7 + . However t h e DNA-DNA h y b r i d i z a t i o n a s s a y does n o t depend on e x c l u s i v e i n c o r p o r a t i o n i n t o phage sequences i n t h e event t h a t U V - i r r a d i a t e d phage a r e n o t a b l e t o s h u t o f f h o s t DNA s y n t h e s i s . I n c o r p o r a t i o n was a l l o w e d t o p r o c e e d u n t i l 25-30 m i n u t e s p o s t i n f e c t i o n and t h e n t e r m i n a t e d by t h e add-i t i o n o f 3 volumes o f i c e c o l d TNE. By t h i s p r o c e d u r e , r e p l i c a t i o n o f t h e U V - i r r a d i a t e d DNA was a l l o w e d t o p r o c e e d t o b l o c k a g e p o i n t s a t UV l e s i o n s o r to t h e end of the m o l e c u l e ; t h i s p r o c e s s s i m u l a t e s c o n d i t i o n s d u r i n g d e v e l o p -ment of i n f e c t i v e c e n t e r s i n t h e c o u r s e o f c r o s s r e a c t i v a t i o n . (The f i r s t T7 progeny phage appear by 25-30 m i n u t e s a f t e r i n f e c t i o n w i t h n o n - i r r a d i a t e d T 7 + . ) I n t r a c e l l u l a r DNA was p h e n o l e x t r a c t e d from S D S - l y s e d and p r o n a s e -t r e a t e d c e l l s ; t h i s DNA t h e n was f u r t h e r p u r i f i e d by s e d i m e n t a t i o n t o e q u i l -i b r i u m i n C s C l g r a d i e n t s . The d i s t r i b u t i o n o f t r i c h l o r o a c e t i c a c i d p r e c i p i -t a b l e c o u n t s i n a t y p i c a l g r a d i e n t i s d e p i c t e d i n F i g u r e 13. The l a r g e peak c e n t e r e d a t f r a c t i o n 5 i s RNA. The s m a l l peak c e n t e r e d a t f r a c t i o n 35 i s DNA; f r a c t i o n s from t h e DNA peak were p o o l e d , d i a l y z e d , and t e s t e d f o r r e s i s -32 t a n c e t o a l k a l i . The P m a t e r i a l was fo u n d t o be g r e a t e r t h a n 98% r e s i s t a n t t o IM KOH f o r 18 ho u r s a t 37° v e r i f y i n g t h a t t h i s m a t e r i a l i s , i n d e e d , DNA. 3. H y b r i d i z a t i o n o f p a r t i a l l y r e p l i c a t e d T7 DNA w i t h r e s t r i c t i o n enzyme Fnu C  c u t T 7 + DNA. The h y b r i d i z a t i o n r e a c t i o n i s an a d a p t a t i o n o f t h e Denhardt DNA-DNA h y b r i d - i z a t i o n p r o c e d u r e ( 1 5 ) : n i t r o c e l l u l o s e f i l t e r s c o n t a i n i n g a g a r o s e g e l s e p a r -a t e d Endo R.Fnu C c u t T7 f r a g m e n t s were p r e i n c u b a t e d t o p r e v e n t n o n - s p e c i f i c F i g u r e 13. P r e p a r a t i o n of p a r t i a l l y  r e p l i c a t e d T7 DNA f o r h y b r i d i z a t i o n : C s C l p u r i f i c a t i o n . E . c o l i B23 were i n f e c t e d w i t h U V - i r r a d i a t e d T7+ as d e s c r i b e d i n M a t e r i a l s and Methods. 3 2 - P - l a b e l e d progeny DNA f r o m i n f e c t e d c e l l s was p h e n o l e x t r a c t e d p r i o r t o c e n t r i f u g a t i o n i n a C s C l g r a d i e n t . The s m a l l peak c e n t e r e d a t f r a c t i o n 35 i s t h e DNA; t h e l a r g e r . p e a k i s RNA. The b o t t o m o f the g r a d i e n t i s to t h e l e f t . A d d i t i o n a l d e t a i l s a r e d e s c r i b e d i n the t e x t . 32 b i n d i n g of DNA. The P - l a b e l e d h y b r i d i z a t i o n probe was s o n i c a t e d , h e a t de-n a t u r e d , and i c e c h i l l e d p r i o r t o a d d i t i o n t o t h e h y b r i d i z a t i o n m i x t u r e . A f t e r h y b r i d i z a t i o n ( o v e r n i g h t a t 65°), f i l t e r s were washed and exposed f o r a u t o r a d i o g r a p h y . A u t o r a d i o g r a m s were q u a n t i t a t e d b y . d e n s i o t o m e t e r s c a n n i n g . H y b r i d i z a t i o n p a t t e r n s o b t a i n e d f o r T 7 + phage r e c e i v i n g v a r i o u s doses of UV i r r a d i a t i o n a r e p r e s e n t e d i n F i g u r e 14. The p a t t e r n o b t a i n e d by a n n e a l -32 + + i n g P - l a b e l e d DNA e x t r a c t e d from T7 phage to T7 f r a g m e n t s on n i t r o c e l l u -l o s e f i l t e r s i s i l l u s t r a t e d i n F i g u r e 14A. Q u a n t i t a t i o n o f t h e bands on t h i s a u t o r a d i o g r a m ( F i g u r e 14A) r e v e a l s t h a t t h e f r a c t i o n a l c o n t r i b u t i o n o f h y b r i -32 d i z e d P-DNA i n each band i s c l o s e t o t h e f r a c t i o n a l c o n t r i b u t i o n by w e i g h t of t h e fragment ( T a b l e 6 ) . T h i s r e s u l t a l l o w s one t o examine t h e s p e c i f i c i t y 32 of P - l a b e l e d progeny DNA s y n t h e s i z e d by p a r e n t a l phage r e c e i v i n g v a r i o u s doses of UV i r r a d i a t i o n . 32 + H y b r i d i z a t i o n of i n t r a c e l l u l a r P - l a b e l e d DNA f r o m n o n - i r r a d i a t e d T7 phage i s p o r t r a y e d i r i F i g u r e 14B. The r e l a t i v e amounts of a n n e a l i n g i n t h e s e bands do not c o r r e s p o n d e x a c t l y w i t h t h e m o l e c u l a r w e i g h t s of t h e T7 f r a g -ments ( T a b l e 7 ) . The amount of fragment A i s s l i g h t l y d e p r e s s e d . T h i s r e s u l t s u g g e s t s t h a t i n i t i a t i o n a t t h e 17% r e g i o n p r o d u c e s s l i g h t l y more o f t h e B,D, and E segments i n v i v o d u r i n g n o r m a l w i l d t y p e i n f e c t i o n ; t h a t i s , a few mole-32 + c u l e s a t t h e t i m e of i s o l a t i o n o f P-T7 progeny DNA were p a r t i a l l y r e p l i c a t e d . The amount of l a b e l e d C fragment i s d e p r e s s e d l e s s t h a n t h e amount o f A f r a g -ment ( T a b l e 7) even though fragment C l i e s t o t h e r i g h t of fragment A. T h i s r e s u l t i s c o n s i s t e n t w i t h t h e f o r m a t i o n of head t o t a i l c oncatemers by p r o -geny DNA (45,64,83); t h e s e concatemers would r e p l i c a t e b i d i r e c t i o n a l l y . Fragment C t h e n would be r e p l i c a t e d by a g r o w i n g f o r k moving t o the l e f t on t h e T7 genome and would appear i n a h i g h e r r e l a t i v e amount i n a p a r t i a l l y r e p l i c a t e d m o l e c u l e . The a n n e a l i n g p a t t e r n f o r DNA e x t r a c t e d from c e l l s i n f e c t e d w i t h UV-i r r a d i a t e d T 7 + phage c o n t a i n i n g 4.6, 7.0, and 9.4 phage l e t h a l e v e n t s was D E N S I T O M E T E R A B S O R B A N C E F i g u r e 14. H y b r i d i z a t i o n o f p a r t i a l l y r e p l i c a t e d DNA w i t h Endo R.Fnu C r e s t r i c t i o n f r a g m e n t s . 3 2 - P - l a b e l e d DNA was s o n i c a t e d , h e a t d e n a t u r e d , i c e c h i l l e d , and added t o a n i t r o c e l l u l o s e f i l t e r c h a r g e d w i t h Endo R.Fnu C c u t T7 DNA t r a h s f e r e d from an ag a r o s e g e l . The f i l t e r was i n c u b a t e d w i t h the 32-P-DNA, washed, and exposed f o r a u t o r a d i o g r a p h y . Developed f i l m s were scanned by a Q u i c k Scan J r . d e n s i t o m e t e r . A. A n n e a l i n g o f 3 2 - P - l a b e l e d DNA e x t r a c t e d from T7 p a r t i c l e s . B. A n n e a l i n g of 32-P l a b e l e d r e p l i c a t i v e DNA e x t r a c t e d from c e l l s i n f e c t e d w i t h n o n - i r r a d i a t e d T7+. C. A n n e a l i n g of 3 2 - P - l a b e l e d r e p l i c a t i v e DNA e x t r a c t e d from c e l l s i n f e c t e d w i t h . U V - i r r a d i a t e d T7+ r e c e i v i n g 4.6 l e t h a l e v e n t s . D. A n n e a l i n g o f 3 2 - P - l a b e l e d r e p l i c a t i v e DNA e x t r a c t e d from c e l l s i n f e c t e d w i t h U V - i r r a d i a t e d T7+ r e c e i v i n g 7.0 l e t h a l e v e n t s . E. A n n e a l i n g o f 3 2 - P - l a b e l e d r e p l i c a t i v e DNA e x t r a c t e d from c e l l s i n f e c t e d w i t h U V - i r r a d i a t e d T7+ r e c e i v i n g 9.9 l e t h a l e v e n t s . 63 32 Table 6. Fr a c t i o n a l Contribution of P-labeled Phage DNA After Annealing to Endo R.Fnu C Cut T7 Fragments Fragment Endo R.Fnu C Length Hybridized F r a c t i o n a l ( f r a c t i o n T7 genome) Contribution  A 0.542 0.500 B 0.210 0.235 C 0.097 0.132 D 0.079 0.074 E 0.072 0.059 Tota l 1.000 1.000 Endo R.Fnu C lengths are given i n (52). Hybridization f r a c t i o n a l contributions were calculated by integr a t i n g the area under the densitometer peaks of Figure 14A and d i v i d i n g i n d i v i d u a l areas by the t o t a l area. T a b l e 7. Dose Response o f t h e F r a c t i o n a l C o n t r i b u t i o n o f H y b r i d i z e d 32 P-DNA per Fragment and N o r m a l i z a t i o n t o Phage C o n t r o l Endo R.Fnu C Fragment E x p e r i m e n t A B C D E Phage C o n t r o l F r a c t i o n a l C o n t r i b u t i o n N o r m a l i z a t i o n 0.500 1.000 0.235 1.000 0.132 1.000 0.074 1.000 0.059 1.000 0 PLH F r a c t i o n a l C o n t r i b u t i o n N o r m a l i z a t i o n 0.372 0.744 0.295 1.255 0.128 0.970 0.115 1.555 0.090 1.525 4.6 PLH F r a c t i o n a l C o n t r i b u t i o n N o r m a l i z a t i o n 0.308 0.616 0.302 1.285 0.086 0.652 0.173 2.338 0.125 2.119 7.0 PLH F r a c t i o n a l C o n t r i b u t i o n N o r m a l i z a t i o n 0.127 0.254 0.524 2.230 0.048 0.364 0.190 2.568 0.111 1.881 9.4 PLH F r a c t i o n a l C o n t r i b u t i o n N o r m a l i z a t i o n 0 0 0.724 3.080 0 0 0.172 2.324 0.103 1.746 H y b r i d i z a t i o n f r a c t i o n a l c o n t r i b u t i o n s were c a l c u l a t e d by i n t e g r a t i n g t h e a r e a under t h e d e n s i t o m e t e r s peaks o f F i g u r e 14 and d i v i d i n g t h e i n d i v i d u a l a r e a by t h e t o t a l a r e a . The f r a c t i o n a l c o n t r i b u t i o n o f each fragment was n o r m a l i z e d t o t h e c o r r e s p o n d i n g f r a c t i o n a l c o n t r i b u t i o n from t h e phage c o n t r o l . examined ( F i g u r e 1 4 ) . A t the l o w e s t dose ( F i g u r e 14C), t h e r e l a t i v e amounts 32 of P - l a b e l e d progeny DNA h y b r i d i z i n g i n bands B, D, and E i n c r e a s e compared t o t h e n o n - i r r a d i a t e d c o n t r o l ( T a b l e 7 ) . These segments a r e from th e l e f t end of t h e T7 genome. A n n e a l i n g i n bands C and A on t h e r i g h t end of t h e genome d e c r e a s e s . A t i n c r e a s i n g UV d o s e s , t h e s e e f f e c t s become even more pronounced. ( F i g u r e 14D,E). By 9.9 phage l e t h a l e v e n t s p e r phage p a r t i c l e ( F i g u r e 1 4 E ) , h y b r i d i z a t i o n of i n t r a c e l l u l a r DNA i s t o t h e B, D, and E f r a g m e n t s and n o t a t a l l t o A and C ( T a b l e 7 ) . There may appear t o be more r e p l i c a t i o n of t h e C fragment t h a n m i g h t be e x p e c t e d when t h e p a r e n t a l phage has r e c e i v e d 4.6 phage l e t h a l h i t s ( F i g u r e 14C). However, w i t h a m u l t i p l i c i t y o f i n f e c t i o n of 5, e s s e n t i a l l y a l l b a c t e r i a a r e i n f e c t e d by a t l e a s t one phage p a r t i c l e and 1% o f phage r e c e i v i n g 4.6 PLH a r e undamaged a c c o r d i n g t o t h e P o i s s o n d i s t r i b u t i o n i f l e t h a l h i t s and phage a d s o r b t i o n a r e random p r o c e s s e s . Such undamaged phage may r e p l i c a t e n o r m a l l y and p roduce head t o t a i l c oncatemers w h i c h would p r o v i d e a r e l a t i v e enhance-ment of t h e amount of C fragment (see a b o v e ) . The r e s u l t s p r e s e n t e d i n t h i s s e c t i o n show t h a t UV-damaged T7 phage r e p -l i c a t e t h e l e f t end of t h e DNA m o l e c u l e p r e f e r e n t i a l l y and t h a t t h e e x t e n t of r e p l i c a t i o n i s dependent on t h e d e n s i t y of UV damages. I n . o t h e r words, th e 17% r e p l i c a t i o n o r i g i n i s a p p a r e n t l y l o c a t e d n e a r th e r i g h t end of fragment B ( F i g u r e 1 2 ) ; t h i s i m p l i e s t h a t i f r e p l i c a t i o n p r o c e e d s i n two d i r e c t i o n s f r o m t h i s p o i n t t o t h e n e a r e s t s i t e of UV damage, t h e n t h e r e p l i c a t i o n o f segments B, D, and E s h o u l d be most r e s i s t a n t t o UV i r r a d i a t i o n . T h i s i s i n f a c t the c a s e . The r e s u l t s agree v e r y w e l l w i t h t h e c r o s s r e a c t i v a t i o n d a t a p r e s e n t e d i n s e c t i o n B; segments of U V - i r r a d i a t e d T7 DNA w h i c h r e p l i c a t e t h e most e f f i c i e n t l y c o n t a i n t h e m a rkers w h i c h a r e r e s c u e d most e f f i c i e n t l y . M a r k e r s w h i c h a r e n o t r e s c u e d a r e l o c a t e d i n u n r e p l i c a t e d p o r t i o n s of t h e genome. D. S i z e of I n t r a c e l l u l a r U V - l r r a d l a t e d P a r e n t a l T7 DNA I t has been r e p o r t e d t h a t T7 i n j e c t s t h e l e f t end of- t h e genome f i r s t ( 5 8 ) . I f UV i r r a d i a t i o n l e d t o bre a k a g e of t h e T7 DNA, i t m ight be argued t h a t o n l y t h e l e f t end of t h e m o l e c u l e c o u l d be r e s c u e d because o n l y t h e l e f t end c o u l d i n j e c t and r e p l i c a t e . The f o l l o w i n g e x p e r i m e n t was d e s i g n e d g t o examine t h i s p o s s i b i l i t y . E . c o l i B23 was grown t o 3X10 b a c t e r i a / m l i n 10X P0^ TCG a t 30°. The c u l t u r e t h e n was i n f e c t e d w i t h U V - i r r a d i a t e d , H-l a b e l e d T 7 + phage r e c e i v i n g 7.0 l e t h a l h i t s a t an MOI o f 5. A t 5 and 20 m i n u t e s p o s t i n f e c t i o n , a l i q u o t s were w i t h d r a w n i n t o 2.5 volumes o f i c e c o l d 8 TNE, s e d i m e n t e d , and r e s u s p e n d e d a t a c o n c e n t r a t i o n o f 6X10 c e l l s / m l . I n -f e c t e d c e l l s were l y s e d w i t h SDS, t r e a t e d w i t h p r o n a s e , and e x t r a c t e d w i t h 32 + p h e n o l . Samples were mixed w i t h P - r e f e r e n c e DNA e x t r a c t e d f r o m T7 phage and s e d imented t h r o u g h s u c r o s e d e n s i t y g r a d i e n t s a t n e u t r a l and a l k a l i n e pH. Samples on a l k a l i n e g r a d i e n t s were d e n a t u r e d i n 0.2N NaOH p r i o r t o c e n t r i -f u g a t i o n . A p p r o x i m a t e l y 30 f r a c t i o n s were c o l l e c t e d d r o p w i s e from t h e bottom o f the g r a d i e n t and a s s a y e d f o r r a d i o a c t i v i t y . 3 F i v e m i n u t e s a f t e r m i x i n g phage and b a c t e r i a , H - l a b e l e d , U V - i r r a d i a t e d + 32 + T7 DNA c o s e d i m e n t s w i t h P - l a b e l e d DNA e x t r a c t e d f r o m T7 phage a t b o t h n e u t r a l ( F i g u r e 15A) and a l k a l i n e ( F i g u r e 15C) pH. S i m i l a r r e s u l t s a r e o b t a i n e d f o r t h e n o n - i r r a d i a t e d c o n t r o l . I n a d d i t i o n , t h e g r a d i e n t p r o f i l e s a r e s i m i l a r when c u l t u r e s a r e i n c u b a t e d f o r 5 m i n u t e s i n 100yg/ml CM p r i o r t o i n f e c t i o n . T h e r e f o r e t h e r e s u l t s r e p o r t e d i n s e c t i o n s B and C cannot be e x p l a i n e d by p r e f e r e n t i a l i n j e c t i o n o f t h e l e f t end of UV-damaged T7 DNA. A f t e r 20 m i n u t e s , U V - i r r a d i a t e d T 7 + p a r e n t a l DNA rema i n s u n i t l e n g t h i n t h e d o u b l e s t r a n d e d s t a t e ( F i g u r e 1 5B), b u t s i n g l e - s t r a n d n i c k s have been i n t r o -duced ( F i g u r e 15D). I t m i g h t be e x p e c t e d t h a t p a r t i a l r e p l i c a t i o n w o u l d r e s u l t i n l a r g e r DNA m o l e c u l e s w h i c h w o u l d be v i s a b l e as a s h o u l d e r on t h e heavy s i d e o f t h e T7 peak 20 m i n u t e s a f t e r i n f e c t i o n . A s i g n i f i c a n t s h o u l d e r Figure 15. Size of UV-irradiated, parental T7' DNA. E . c o l i B23 was grown to 3X10 8 bacteria/ml i n 10X PO^ TCG at 30° C. The culture was i n f e c t e d at an MOI of 5 with UV-irradiated (7.0 phage l e t h a l + 3 events) T7 , labeled with H-dThd at a s p e c i f i c a c t i v i t y of 2.0 mCi/mg dThd. At 5 and 20 minutes post i n f e c t i o n , a l i q u o t s were withdrawn to 2.5 volumes of i c e cold TNE, sedimented, and resuspended at a concentration of 6X10 bacteria/ml. Infected c e l l s were lysed with SDS, treated with pronase, and 32 extracted with phenol. Samples were mixed with P-labeled reference DNA extracted from T 7 + p a r t i c l e s and sedimented through sucrose density gradients at n e utral and a l k a l i n e pH. A. 5 min i n t r a c e l l u l a r DNA, neutral pH B. 20 min i n t r a c e l l u l a r DNA, n e u t r a l pH C. 5 min i n t r a c e l l u l a r DNA, a l k a l i n e pH D. 20 min i n t r a c e l l u l a r DNA, a l k a l i n e pH 0 0 0 3H DNA •- -• • 32 P reference DNA P E R C E N T LENGTH OF GRADIENT i s n o t o b s e r v e d because under t h e c o n d i t i o n s employed, no more t h a n 20% of th e T7 m o l e c u l e s a r e p a r t i a l l y r e p l i c a t e d and t h e e x t e n t of p a r t i a l r e p l i -c a t i o n i s , on t h e a v e r a g e , l e s s t h a n 20% (see s e c t i o n E.2) F i g u r e 15 shows t h a t U V - i r r a d i a t e d p a r e n t a l phage DNA r e c o v e r e d from i n f e c t e d c e l l s i s n o t s i g n i f i c a n t l y d i f f e r e n t from u n i r r a d i a t e d DNA e x t r a c t e d from phage p a r t i c l e s . T here i s no e v i d e n c e t h a t U V - i r r a d i a t i o n a t t h e doses used i n t h e s e e x p e r i -ments b r e a k s DNA; on t h e c o n t r a r y , t h e e v i d e n c e i n d i c a t e s t h a t U V - i r r a d i a t e d p a r e n t a l DNA i s n o t b r o k e n . 70 E. P a r t i a l R e p l i c a t i o n of B a c t e r i o p h a g e T7 DNA: A n a l y s i s by E l e c t r o n  M i c r o s c o p y A n a l y s i s of t h e progeny DNA p r o d u c e d by U V - i r r a d i a t e d T 7 + p a r e n t a l phage by h y b r i d i z a t i o n i n d i c a t e s t h a t segments on t h e l e f t o f the T7 genome a r e r e p l i c a t e d w i t h g r e a t e r e f f i c i e n c y t h a n t h o s e on t h e r i g h t . The d a t a s u p p o r t t h e p a r t i a l r e p l i c a h y p o t h e s i s . However, t h e l o c a t i o n and s i z e o f t h e r e s t r i - c t i o n f r a g m e n t s g e n e r a t e d by Endo R.Fnu C d i g e s t i o n a r e s u c h t h a t a f i n e s t r u c t u r e mapping of t h e p a r t i a l r e p l i c a s was n o t p o s s i b l e . E l e c t r o n m i c r o -scopy was p o t e n t i a l l y v a l u a b l e f o r f i n e s t r u c t u r e a n a l y s i s o f p a r t i a l l y r e p -l i c a t e d r e g i o n s i n t h e l e f t end of U V - i r r a d i a t e d T7 DNA. A f t e r i s o l a t i o n o f p a r t i a l l y r e p l i c a t e d , n o n - i r r a d i a t e d m o l e c u l e s from E . c o l i a t e a r l y t i m e s a f t e r i n f e c t i o n , e l e c t r o n m i c r o s c o p y o r i g i n a l l y had been used by D r e s s i e r e t . a l . (18) t o map t h e 17% o r i g i n of T7 DNA r e p l i c a t i o n . Under n o r m a l c o n d i t i o n s , i t i s i m p o s s i b l e t o s e p a r a t e h o s t f r o m phage sequences by C s C l e q u i l i b r i u m s e d i m e n t a t i o n because T7 and E . c o l i have s i m i l a r G + C c o n t e n t s . S e p a r a t i o n may. be a c c o m p l i s h e d by u s i n g a d e n s i t y l a b e l i n t h e h o s t DNA and i n f e c t i n g 2 15 w i t h n o r m a l d e n s i t y T7 phage. The heavy i s o t o p e s H and N were chosen as d e n s i t y l a b e l s b e c a u s e , a l t h o u g h c e l l s grown i n t h e p r e s e n c e o f t h e s e i s o t o p e s have a l o n g e r g e n e r a t i o n t i m e t h a n n o r m a l d e n s i t y c e l l s (see b e l o w ) , t h e y a r e o t h e r w i s e n o r m a l and h e a l t h y . I n c o r p o r a t i o n of t h e a l t e r n a t i v e d e n s i t y l a b e l , 5-^BrdU, r e s u l t s i n l i g h t s e n s i t i v e and i n v i a b l e c e l l s w h i c h form l o n g snakes a f t e r l e s s t h a n 2 h o u r s of growth. P r e p a r a t i o n o f a s u f -f i c i e n t amount of p a r t i a l l y r e p l i c a t e d DNA f o r e l e c t r o n m i c r o s c o p i c a n a l y s i s r e q u i r e d a l a r g e s c a l e (50ml) c u l t u r e of e x p e n s i v e d e n s i t y i s o t o p e s . T h e r e -f o r e a s t u d y o f dose dependent p a r t i a l r e p l i c a t i o n by EM was n o t a t t e m p t e d . 1. I s o l a t i o n of p a r t i a l l y r e p l i c a t e d m o l e c u l e s f o r e x a m i n a t i o n by e l e c t r o n  m i c r o s c o p y . E . c o l i B23 was a d a p t e d f o r g r o w t h i n d e n s i t y medium by s e r i a l p a s s a g e t h r o u g h 20, 40, 60, 75, 90, 95, and 100% s u b s t i t u t e d medium ( 8 4 ) . The a d a p t e d c e l l s had a g e n e r a t i o n t i m e of 75-80 m i n u t e s a t 37° compared t o 35 m i n u t e s f o r n o r m a l d e n s i t y c e l l s . N o n - i r r a d i a t e d T 7 + phage f o l l o w e d a n o r m a l s i n g l e s t e p growth c u r v e a t 30° i n t h e d e n s i t y l a b e l e d c e l l s , g i v i n g a b u r s t o f 120 phage/ c e l l by 45 m i n u t e s a f t e r i n f e c t i o n . A 50 ml c u l t u r e o f d e n s i t y l a b e l e d E . c o l i B23 was grown t o a p p r o x i m a t e l y 3X10^ b a c t e r i a / m l a t 37° i n heavy medium c o n -3 t a i n i n g 5yg/ml dT, 5yg/ml FdU, 25yg/ml U and 5 0 0 y C i ( m e t h y l - H ) t h y m i d i n e , t h e n s h i f t e d t o 30° f o r 15 m i n u t e s . T h i s p r o c e d u r e a l l o w e d l a b e l i n g o f t h e b a c t e r -i a l c e l l s f o r HH r e f e r e n c e ( b o t h s t r a n d s l a b e l e d ) i n C s C l g r a d i e n t s . The c e l l s 32 t h e n were i n f e c t e d a t an MOI" l e s s t h a n o r e q u a l t o 2 w i t h U V - i r r a d i a t e d , P-l a b e l e d , LL (no d e n s i t y l a b e l i n e i t h e r s t r a n d ) T 7 + c o n t a i n i n g 7.3 l e t h a l 32 e v e n t s p e r genome. The P - l a b e l was a t a s p e c i f i c a c t i v i t y of 2mCi/mg PO^ 32 w h i c h l e a d s t o t h e i n c o r p o r a t i o n of a P atom i n 2/5 of the phage p a r t i c l e s . The MOI was d e t e r m i n e d by m o n i t o r i n g s u r v i v i n g b a c t e r i a . S i n c e no a t t e m p t was made i n t h i s e x p e r i m e n t t o overcome s u p e r i n f e c t i o n e x c l u s i o n (see s e c t i o n A ) , a c a l c u l a t e d MOI of 2 does n o t n e c e s s a r i l y mean t h a t any c e l l s r e c e i v e d i n -j e c t e d DNA f r o m more t h a n one phage. I n f e c t i v e c e n t e r s and background phage a l s o were m o n i t o r e d . U V - i r r a d i a t e d T7 phage a r e u n a b l e t o co n d u c t a p r o d u c t i v e i n f e c t i o n : i n f e c t i v e c e n t e r s a r e t y p i c a l l y o n l y 0.1-0.2% of t h e c o n t r o l b a c -t e r i a whereas n o n - i r r a d i a t e d T 7 + p r o d u c e i n f e c t i v e c e n t e r s on 90% o f the con-t r o l b a c t e r i a . A t 25 m i n u t e s p o s t i n f e c t i o n , the c u l t u r e was c h i l l e d by p i p e t t i n g i n t o 2 volumes of i c e c o l d TNE p l u s 1 volume o f l y s i s b u f f e r . The 9 i n f e c t e d c e l l s were s e d i m e n t e d and r e s u s p e n d e d a t a c o n c e n t r a t i o n o f 1.5X10 b a c t e r i a / m l i n l y s i s b u f f e r . C e l l s t h e n were l y s e d and t h e l y s a t e d e p r o t e i n -i z e d as d e s c r i b e d i n t h e l e g e n d t o F i g u r e L6. A l i q u o t s of t h e l y s a t e were c e n t r i f u g e d t o e q u i l i b r i u m i n C s C l . A p p r o x i m a t e l y 70 f r a c t i o n s were c o l l e c t e d 3 32 f r o m the bottom o f t h e tube and a s s a y e d f o r H (HH E . c o l i ) and P ( T 7 ) . As e x p e c t e d , T 7 + i r r a d i a t e d t o 7.3 phage l e t h a l e v e n t s d i d n o t undergo even one Figure 16. I s o l a t i o n of p a r t i a l l y r e p l i c a t e d T7 DNA f o r examination by  electron microscopy. g E . c o l i B23 was grown to 3X10 bacteria/ml i n density labeled media and infe c t e d with UV-irradiated T7 (7.3 phage l e t h a l events) at an MOI le s s than or equal to 2. At 25 min post i n f e c t i o n , the culture was c h i l l e d by pipeting into 2 volumes of i c e cold TNE plus 1 volume of l y s i s b u f f e r . The c e l l s were 9 sedimented and resuspended at 1.5X10 bacteria/ml i n l y s i s b u f f e r . C e l l s then were lysed with lysozyme (400 ug/ml, 45 min) followed by detergent (sodium l a u r y l sarconsinate, 0.1%, 65°, 20 min). The ly s a t e was deproteinized by treatment with s e l f digested pronase (1 mg/ml, 37°, 12 hours). Aliquots of the l y s a t e were mixed 1:4 with saturated CsCl ( i n d i s t i l l e d water) and centr-fuged to equilibrium i n an International SB 287 rotor (72 hours, 30,000rpm, 12° C). Approximately 70 f r a c t i o n s were c o l l e c t e d from the bottom of the tube. M a t e r i a l banding on the heavy side of the T7 peak was pooled and resedimented i n a second CsCl gradient (type 40 rotor, 72 hours, 32,000rpm, 12" C) as shown i n the f i g u r e . The material i n the f r a c t i o n s from the second gradient in d i c a t e d by the brackets was pooled for electron microscopy. 3H cpm X 10 3 ( E . c o l i DNA) -•- 3 2 P cpm X 10 3 ( T7 DNA ) FRACTION NUMBER 74 complete round of r e p l i c a t i o n as no p a r e n t a l DNA banded a t t h e h y b r i d l o c a t i o n . M a t e r i a l b a n d i n g on t h e heavy s i d e o f t h e T7 peak i n t h e f i r s t g r a d i e n t was p o o l e d and r e s e d i m e n t e d i n a second C s C l g r a d i e n t t o f u t h e r . . s e g r e g a t e h o s t s p e c i e s and u n r e p l i c a t e d , u n i t l e n g t h T7 m o l e c u l e s ( F i g u r e 1 6 ) . The f r a c t i o n s of t h i s g r a d i e n t i n d i c a t e d by t h e b r a c k e t were p o o l e d f o r a n a l y s i s by e l e c t r o n m i c r o s c o p y . F i g u r e 16 shows t h a t the T7 DNA was w e l l s e p a r a t e d from contam-i n a t i n g E . c o l i DNA. 2. E l e c t r o n m i c r o s c o p y of p a r t i a l l y r e p l i c a t e d T 7 + DNA. P a r i t a l l y r e p l i c a t e d , U V - i r r a d i a t e d T 7 + DNA was i s o l a t e d from a C s C l g r a d i e n t a f t e r a d e n s i t y t r a n s f e r e x p e r i m e n t ( s e c t i o n E . l ) and examined by e l e c t r o n m i c r o s c o p y . Four c a t e g o r i e s o f p a r t i a l l y r e p l i c a t e d m o l e c u l e s o f T 7 - s i z e DNA were d i s t i n g u i s h e d : 1) m o l e c u l e s c o n t a i n i n g i n t e r n a l d u p l i c a t i o n s (eye f o r m s ) , 2) m o l e c u l e s c o n t a i n i n g f o r k s w i t h two e q u a l arms, 3) m o l e c u l e s c o n t a i n i n g b o t h eye forms and f o r k s , and 4) m o l e c u l e s c o n t a i n i n g f o r k s w i t h arms of u n e q u a l l e n g t h where two of t h e b r a n c h e s t o g e t h e r were o f T7 l e n g t h . M o l e c u l e s i n t h i s l a t t e r c l a s s were assumed t o be eye forms b r o k e n because of t h e f r a g i l i t y o f s i n g l e s t r a n d r e g i o n s a t t h e f o r k . Examples of s e v e r a l p a r t i a l l y r e p l i c a t e d m o l e c u l e s a r e shown i n F i g u r e 17. F i g u r e 17a shows a T7 l e n g t h m o l e c u l e w i t h an i n t e r n a l d u p l i c a t i o n o f 13.7%. I n t e r n a l l y r e p l i c a t e d r e g i o n s s p a n n i n g 1-25% o f u n i t T7 l e n g t h were o b s e r v e d . F i g u r e 17b i s a m o l e c u l e w i t h a l a r g e e q u a l armed f o r k c o v e r i n g 19.2% of t h e m o l e c u l e . I n a d d i t i o n t o l a r g e f o r k s s p a n n i n g up t o 38% of T7 l e n g t h , s m a l l f o r k s o c c u r r i n g a t one end of t h e m o l e c u l e were f o u n d ( F i g u r e 1 7 c ) . Such f o r k s e xtended o v e r as l i t t l e as 2% of t h e genome. A s m a l l number of m o l e c u l e s c o n t a i n i n g b o t h a f o r k a t one end and an i n t e r n a l l y dup-l i c a t e d r e g i o n a t t h e same end ( F i g u r e 17d) were o b s e r v e d . S i x t y f o u r p a r t i a l l y r e p l i c a t e d m o l e c u l e s were p h o t o g r a p h e d and measured. Only m o l e c u l e s m e a s u r i n g 12.0 + 0.5um (T7 u n i t l e n g t h under t h e s p r e a d i n g Figure 17. P a r t i a l l y r e p l i c a t e d T7 DNA molecules. a) T7 l e n g t h molecule c o n t a i n i n g an eye form b) Molecule c o n t a i n i n g a l a r g e branch. The sum of the long arm p l u s e i t h e r of the short arms i s u n i t length (12.0 + 0.5 ym). c) T7 length molecule c o n t a i n i n g a short branch. d) T7 l e n g t h molecule c o n t a i n i n g both a short branch and an eye form. / and o b s e r v a t i o n c o n d i t i o n s d e s c r i b e d i n M a t e r i a l s and Methods) were i n c l u d e d i n t h e a n a l y s i s . M o l e c u l e s were n o r m a l i z e d t o a s c a l e of 100 u n i t s and a l i g n e d such t h a t a l l p a r t i a l l y r e p l i c a t e d r e g i o n s o c c u r i n t h e same end ( F i g u r e 1 8 ) . I t was f e l t t h a t i t was r e a s o n a b l e t o assume t h a t t h e s e r e g i o n s a r e a l l l o c a t e d a t one end, t h e g e n e t i c l e f t end of t h e m o l e c u l e , f o r t h e f o l l o w i n g r e a s o n s : 1) progeny DNA pr o d u c e d by i n f e c t i o n w i t h U V - i r r a d i a t e d T 7 + phage c o n t a i n i n g 6-7 phage l e t h a l e v e n t s p e r genome h y b r i d i z e s t o r e s t r i c t i o n f r a g m e n t s from th e l e f t p o r t i o n o f t h e T 7 + m o l e c u l e b u t n o t t o f r a g m e n t s f r o m t h e r i g h t s i d e ( s e c t i o n C.3); 2) Wolf son ^ t _ . a l . (84) and D r e s s i e r e_t.al_. (18) examined p a r t i a l d e n a t u r a t i o n maps of p a r t i a l l y r e p l i c a t e d , n o n - i r r a d i a t e d T 7 + DNA and found t h a t f o r k s and eye forms were l o c a t e d on t h e l e f t s i d e of t h e m o l e c u l e ; 3) f o u r m o l e c u l e s c o n t a i n i n g b o t h a f o r k and an eye fo r m were o b s e r v e d and t h e s e r e g i o n s b o t h were l o c a t e d i n t h e same end o f the m o l e c u l e ; and 4) no m o l e c u l e s c o n t a i n i n g p a r t i a l l y r e p l i c a t e d r e g i o n s a t b o t h ends were seen. I t i s e v i d e n t from t h e d i s t r i b u t i o n o f eye forms o v e r t h e l e f t 20% o f the m o l e c u l e t h a t no s i n g l e o r i g i n o f r e p l i c a t i o n was u t i l i z e d ( F i g u r e 1 8 ) . I f r e p l i c a t i o n i s assumed t o p r o c e e d b i d i r e c t i o n a l l y a t t h e same r a t e from th e c e n t e r o f an eye form, t h e d i s t r i b u t i o n o f r e p l i c a t i o n o r i g i n s among the eye forms i n F i g u r e 18 ( i n c l u d i n g u n e q u a l f o r k s and eyes i n m u l t i p l e s t r u c t u r e s ) i s g i v e n i n T a b l e 8. I n a d d i t i o n , t h e r e a r e 8 m o l e c u l e s w i t h f o r k s s p a n n i n g 0-5% of the m o l e c u l e and an a d d i t i o n a l 6 s p a n n i n g 0-10% of the genome. T h e r e f o r e i n i t i a t i o n can p r o c e e d f r o m a p o i n t a t o r c l o s e t o the l e f t end of a U V - i r r a d i a t e d T7 DNA m o l e c u l e as w e l l as f r o m numerous l o c a t i o n s a l o n g t h e l e f t 30% of the genome. The summed t o t a l o f p a r t i a l l y r e p l i c a t e d r e g i o n s o b s e r v e d i n t h e mole-c u l e s d e p i c t e d i n F i g u r e 18 i s g i v e n by the h i s t o g r a m i n F i g u r e 19; 75% o f t h e p a r t i a l l y r e p l i c a t e d a r e a s l i e t o t h e l e f t of 17% w h i l e o n l y 25% a r e t o the r i g h t o f t h a t p o i n t . T h i s c u r v e c l o s e l y r e s e m b l e s t h a t o b t a i n e d f o r Figure 18. Normalized l i n e diagrams of p a r t i a l l y r e p l i c a t e d T7 mol er.nl es. T7 molecules of length 12.0 + 0.5um were measured, normalized, and aligned such that a l l p a r t i a l l y r e p l i c a t e d regions occur i n the l e f t end (see text). 3-3-I | I | I 50 100 I | I | 50 100 r 0 50 =1"**] 100 PERCENT LENGTH T 7 T a b l e 8. D i s t r i b u t i o n o f C e n t e r s of Eye Forms Segments o f T7 DNA (% l e n g t h ) 0 - 5 5 - 1 0 1 0 - 1 5 1 5 - 2 0 >20 Number of eye m i d p o i n t s 6 . 9 10 9 7 The m i d p o i n t s o f t h e eye forms r e p r e s e n t e d by t h e l i n e d r a w i n g s o f F i g u r e 18 were measured. T h i s t a b l e l i s t s t h e number of t i m e s an eye form m i d p o i n t f a l l s w i t h i n a p a r t i c u l a r segment of T7 DNA among t h e m o l e c u l e s diagrammed i n F i g u r e 18. Figure 19. Histogram of p a r t i a l l y r e p l i c a t e d regions from UV-irradiated  T7 DNA. Lengths of T7+ DNA in a 1% increments were scored f o r r e p l i c a t i o n as represented i n the l i n e drawings of Figure 18, The histogram describes the number of times a p a r t i c u l a r segment of DNA was r e p l i c a t e d i n the pool of molecules diagrammed in Figure 18. The open c i r c l e s represent marker rescue frequencies normalized to the maximum rescue and d i s t r i b u t e d along the genetic map of T7 as a function of percent length of genome. (Taken from Figure 7C) • 1 0 0 1 — 5 0 T 1 I 0 1 0 0 Percent T7 Length marker r e s c u e e x p e r i m e n t s u s i n g T7 i r r a d i a t e d w i t h a s i m i l a r dose ( s e c -t i o n B.2). The c l o s e f i t df t h e h i s t o g r a m w i t h t h e c r o s s r e a c t i v a t i o n c u r v e ( F i g u r e 19) i m p l i e s t h a t p a r t i a l r e p l i c a t i o n of t h e l e f t end of a U V - i r r a d -i a t e d T 7 + phage can a c c o u n t f o r t h e d i s t r i b u t i o n o f marker r e s c u e e f f i c i e n c y . I n a d d i t i o n , t h e e l e c t r o n m i c r o s c o p i c a n a l y s i s a g r e e s v e r y w e l l w i t h e s t i -mates of p a r t i a l r e p l i c a t i o n as d e t e r m i n e d by DNA h y b r i d i z a t i o n . IV. DISCUSSION The r e s u l t s p r e s e n t e d i n t h i s t h e s i s may be summarized as f o l l o w s . S u p e r i n f e c t i o n e x c l u s i o n by b a c t e r i o p h a g e T7 i s i n h i b i t e d by a d d i t i o n t o h o s t c e l l s o f lOOug c h l o r a m p h e n i c o l / m l 5 t o 10 m i n u t e s b e f o r e p r i m a r y i n -f e c t i o n . When s u p e r i n f e c t i o n e x c l u s i o n i s i n h i b i t e d , i n j e c t i o n of s e c o n d a r y phage DNA o c c u r s ; t h i s DNA may be. e x p r e s s e d , and i t i s a v a i l a b l e f o r recom-b i n a t i o n w i t h p r i m a r y DNA. I n j e c t i o n of a l a r g e amount of p a r e n t a l DNA i n t o t h e c e l l does n o t r e s u l t i n t h e p a c k a g i n g of s i g n i f i c a n t amounts of u n r e p l i c a t e d ( c o n s e r v a t i v e ) DNA. The r e s c u e e f f i c i e n c y o f m a r k e r s f r o m U V - i r r a d i a t e d T 7 + phage by c o -i n f e c t i n g mutant c a r r i e r phage i s dependent on t h e map p o s i t i o n of t h e marker Only markers f r o m t h e i . l e f t p o r t i o n of t h e T7 genome a r e r e s c u e d a t a p p r e -c i a b l e f r e q u e n c y i f the i r r a d i a t i o n dose i s g r e a t e r t h a n o r e q u a l t o 4 phage l e t h a l e v e n t s . A t i n c r e a s i n g UV d o s e s , fewer markers a r e r e s c u e d ; t h e r e s -cued markers c o n s i s t e n t l y a r e f r o m t h e l e f t 0 t o 10% of t h e genome i n s t e a d of f rom the r e g i o n around-17%. D e l e t i o n of t h e o r i g i n s h i f t s t h e c r o s s r e a c t i v a t i o n c u r v e t o t h e l e f t end of t h e genome a t l o w e r r a d i a t i o n d o s e s . Recom-b i n a t i o n i s a n e c e s s a r y s t e p of t h e c r o s s r e a c t i v a t i o n p r o c e s s . Absence of t h e h o s t uvrA e n d o n u c l e a s e d e c r e a s e s t h e r e s c u e e f f i c i e n c y by a t l e a s t a f a c t o r o f 2 b u t does n o t change t h e g e n e r a l shape o f the c r o s s r e a c t i v a t i o n c u r v e . P h o t o r e a c t i v a t i o n e f f e c t i v e l y d e c r e a s e s t h e number of l e t h a l e v e n t s w h i c h i n c r e a s e s t h e marker r e s c u e e f f i c i e n c y . P r e l i m i n a r y e x p e r i m e n t s t o e l u c i d a t e a r o l e f o r t h e T7 gene 6 e x o n u c l e a s e i n d i c a t e d t h a t b u r s t s i z e s were too low t o a l l o w m e a n i n g f u l i n t e r p r e t a t i o n o f the d a t a . 32 + P - l a b e l e d progeny DNA s y n t h e s i z e d by U V - i r r a d i a t e d T7 p a r e n t a l phage h y b r i d i z e s p r e d o m i n a n t l y t o r e s t r i c t i o n f r a g m e n t s f r o m t h e l e f t end o f t h e T7 genome. As t h e r a d i a t i o n dose i s i n c r e a s e d , fewer segments a r e r e p l i c a t e d A t t h e h i g h e s t UV doses u s e d , h y b r i d i z a t i o n i s o n l y t o r e s t r i c t i o n f r a g m e n t s f r o m t h e l e f t s i d e o f t h e T7 m o l e c u l e . Under t h e e l e c t r o n m i c r o s c o p e , p a r t i a l l y r e p l i c a t e d T7 DNA may c o n t a i n i n t e r n a l d u p l i c a t i o n s , f o r k s , o r b o t h . No m o l e c u l e s c o n t a i n i n g p a r t i a l l y r e p l i c a t e d r e g i o n s on b o t h ends of th e genome were f o u n d . The d a t a s u p p o r t t h e p a r t i a l r e p l i c a h y p o t h e s i s . B o t h r e p l i c a t i o n and marker r e s c u e e f f i c i e n c y a r e dependent on t h e UV d o s e % Segments o f t h e T7 genome w h i c h r e p l i c a t e e f f i c i e n t l y c o n t a i n m a r k e r s w h i c h a r e r e s c u e d e f f i c -i e n t l y d u r i n g c r o s s r e a c t i v a t i o n . U V - i r r a d i a t e d T7 p a r e n t a l DNA r e c o v e r e d f r o m i n f e c t e d c e l l s i s u n i t l e n g t h . T h e r e f o r e the r e s u l t s cannot be e x p l a i n e d by p r e f e r e n t i a l i n j e c t i o n o f t h e l e f t end of t h e T7 genome. 83 A. S u p e r i n f e c t i o n E x c l u s i o n and L a c k of C o n s e r v a t i v e T r a n s f e r of B a c t e r i o - phage T7 DNA D u r i n g b a c t e r i o p h a g e i n f e c t i o n o f E . c o l i , t h e f i r s t phage i n j e c t i n g t h e i r DNA i n t o a c e l l o f t e n a r e a b l e t o p r e v e n t n o r m a l r e p l i c a t i o n , recom-b i n a t i o n , or e x p r e s s i o n of DNA f r o m s e c o n d a r y or s u p e r i n f e c t i n g phage. T h i s p r o c e s s i s c a l l e d s u p e r i n f e c t i o n e x c l u s i o n . E a r l y e x p e r i m e n t s (27,28) dem-o n s t r a t e d t h a t s u p e r i n f e c t i n g T2 phage were u n a b l e t o t r a n s f e r t h e i r DNA t o progeny of t h e p r i m a r y phage. I f t h e p r i m a r y phage were T2, T4, T5, o r T6, 32 s u p e r i n f e c t i o n breakdown was o b s e r v e d , t h a t i s , P - l a b e l e d DNA from t h e s u p e r -i n f e c t i n g p a r e n t was r e n d e r e d a c i d s o l u b l e . I n t h e cas e of p r i m a r y i n f e c t i o n by T l , T3, or T7, s u p e r i n f e c t i o n breakdown was n o t o b s e r v e d b u t , a t t h e same 32 t i m e , no ' P - l a b e l from s u p e r i n f e c t i n g T2 phage was f o u n d i n t h e progeny of any o f t h e s e phage. T7 phage were d e m o n s t r a t e d t o e x c l u d e s u p e r i n f e c t i n g T7 phage a t t h e s t a g e of i n j e c t i o n ( 7 ) . The f a c t t h a t T7 e x h i b i t s s u p e r i n f e c t i o n e x c l u s i o n under n o r m a l c o n d i t i o n s o f a s y n c h r o n o u s i n f e c t i o n r a i s e d t h e q u e s t i o n o f whether i n c o n s i s t e n c i e s o b s e r v e d i n T7 r e c o m b i n a t i o n (75) and d u r i n g marker r e s c u e e x p e r i m e n t s ( t h i s work) c o u l d be due t o t h i s p r o c e s s . T h e r e f o r e , a s e a r c h was u n d e r t a k e n f o r c o n d i t i o n s w h i c h would i n h i b i t s u p e r i n f e c t i o n e x c l u s i o n by b a c t e r i o p h a g e T7. C o n d i t i o n s were d e s i r e d s u c h t h a t once s e c -ondary phage had i n j e c t e d t h e i r DNA, r e m o v a l of t h e c o n d i t i o n s would a l l o w the r e s u m p t i o n of n o r m a l i n t r a c e l l u l a r p r o c e s s e s . I t was f o u n d t h a t 5-10 m i n u t e p r e i n c u b a t i o n i n t h e p r o t e i n s y n t h e s i s i n h i b i t o r c h l o r a m p h e n i c o l (CM) a t 100yg/ml would b l o c k e x c l u s i o n o f s u p e r i n f e c t i n g phage. The e f f e c t s o f CM a r e e a s i l y r e v e r s i b l e upon r e m o v a l of t h e d r u g by c e n t r i f u g a t i o n o r by s i m p l e d i l u t i o n o f i n f e c t e d b a c t e r i a i n t o CM-free medium. Upon i n j e c t i o n i n t o t h e h o s t , s u p e r i n f e c t i n g T7 DNA may be e x p r e s s e d and p a r t i c i p a t e i n r e c o m b i n a t i o n and r e p l i c a t i o n . 84 Three c r i t e r i a were used t o d e m o n s t r a t e t h a t CM overcomes s u p e r i n f e c t i o n e x c l u s i o n and a l l o w s s u p e r i n f e c t i n g T7 phage t o i n j e c t , r e p l i c a t e , and e x p r e s s t h e i r genomes. D e n s i t y t r a n s f e r e x p e r i m e n t s ( F i g u r e 4) i n d i c a t e d t h a t i n t h e p r e s e n c e of CM, many p a r e n t a l phage may i n j e c t t h e i r DNA i n t o a s i n g l e c e l l and a good p e r c e n t a g e o f t h i s DNA p a r t i c i p a t e s i n r e p l i c a t i o n . S u p e r i n f e c t -i n g T 7 + produce w i l d t y p e i n f e c t i v e c e n t e r s i n t h e p r e s e n c e of a h i g h m u l t i -p l i c i t y o f amber phage a f t e r p r e i n c u b a t i o n of c e l l s i n CM ( T a b l e 1 ) . Second - a r y amber phage a r e a b l e t o recombine w i t h p r i m a r y amber phage t o p r o d u c e w i l d t y p e progeny i n t h e p r e s e n c e of CM b u t n o t i n i t s absence ( F i g u r e 3 ) . The i m p l i c a t i o n o f t h e s e r e s u l t s i s t h a t one o r more T7 p r o t e i n s s y n t h e s i z e d v e r y e a r l y a f t e r i n f e c t i o n a r e r e s p o n s i b l e f o r p r e v e n t i n g i n j e c t i o n by super - i n f e c t i n g T7 phage. I n h i b i t i o n of T7 p r o t e i n s y n t h e s i s a l l o w s s u p e r i n f e c t i o n because t h e i n h i b i t o r y p r o t e i n i s n o t a v a i l a b l e . The p r o t e i n ( s ) i n v o l v e d and t h e mechanism by w h i c h i n j e c t i o n i s i n h i b i t e d a r e p r e s e n t l y unknown. D e s p i t e t h e i n j e c t i o n of many p a r e n t a l genomes i n t o a h o s t c e l l , v e r y : l i t t l e o f t h e u n r e p l i c a t e d ( c o n s e r v a t i v e ) DNA i s f o u n d among th e progeny phage ( F i g u r e 5 ) . E a r l i e r d a t a from d e n s i t y t r a n s f e r e x p e r i m e n t s c o u l d have been i n t e r p r e t e d as e v i d e n c e t h a t c o n s e r v a t i v e t r a n s f e r o c c u r s d u r i n g n o r m a l •T7 i n f e c t i o n ( 1 2 , 5 4 ) . The r e s u l t s i n F i g u r e 5 d e m o n s t r a t e t h a t c o n s e r v a t i v e t r a n s f e r does n o t ' o c c u r w i t h s i g n i f i c a n t f r e q u e n c y d u r i n g T7 i n f e c t i o n even when many p a r e n t a l genomes a r e a v a i l a b l e f o r p a c k a g i n g . They s u g g e s t r e p l i -c a t i v e DNA i s p r e r e q u i s i t e f o r p a c k a g i n g . C r o s s r e a c t i v a t i o n e x p e r i m e n t s r e q u i r e c o i n f e c t i o n of a c e l l by UV-i r r a d i a t e d phage and mutant c a r r i e r phage. I f one p a r e n t a l t y p e i s e x c l u d e d f rom c e l l s , i n c o n s i s t e n c i e s i n marker r e s c u e d a t a w i l l i n e v i t a b l y r e s u l t . The o b s e r v a t i o n t h a t s u p e r i n f e c t i o n e x c l u s i o n can be i n h i b i t e d by a s h o r t p r e -i n c u b a t i o n i n c h l o r a m p h e n i c o l p r e c l u d e s i n c o n s i s t e n c i e s due t o e x c l u s i o n and a l l o w s r e l i a b l e r e p e t i t i o n of marker r e s c u e e x p e r i m e n t s and i n t e r p r e t a t i o n O o f r e s u l t s . 85 B. M a r k e r Rescue and P a r t i a l R e p l i c a t i o n of B a c t e r i o p h a g e T7 DNA The r e s u l t s p r e s e n t e d i n t h i s t h e s i s s u p p o r t t h e i d e a of t h e p a r t i a l r e p l i c a h y p o t h e s i s as an e x p l a n a t i o n f o r t h e p a t t e r n s of marker r e s c u e f r e -q u e n c i e s o b s e r v e d d u r i n g b a c t e r i o p h a g e c r o s s r e a c t i v a t i o n ( 6 , 1 7 ) . U V - i r r a d -i a t e d phage DNA m o l e c u l e s a r e presumed t o r e p l i c a t e b i d i r e c t i o n a l l y f rom an o r i g i n t o p o i n t s of UV damage, a r e b l o c k e d a t t h e s e p o i n t s f r o m f u r t h e r r e p l i c a t i o n and a r e r e s c u e d most e f f i c i e n t l y f o r m arkers i n t h e segments of t h e genome w h i c h have r e p l i c a t e d . P a r t i a l r e p l i c a s of b a c t e r i o p h a g e DNA o r i g i n a l l y were p o s t u l a t e d on g e n e t i c grounds t o a c c o u n t f o r t h e p a t t e r n s of c r o s s and m u l t i p l i c i t y r e a c t -i v a t i o n o b s e r v e d w i t h b a c t e r i o p h a g e T4 ( 6 , 1 6 , 1 7 ) . S e v e r a l l i n e s of e v i d e n c e were shown t o be c o n s i s t e n t w i t h t h e h y p o t h e s i s . I n mixed i n f e c t i o n by UV-i r r a d i a t e d T4D + phage and d i f f e r e n t members of a s e t of d e f i n e d amber and r l l m u t a n t s , t h e a b i l i t y o f a p a r t i c u l a r mutant t o r e s c u e t h e damaged T4D + genome was dependent on t h e map p o s i t i o n of t h e marker ( 8 5 ) . Four d i s t i n c t peaks were o b t a i n e d i n t h i s e x p e r i m e n t : t h e segments o f the genome w i t h a h i g h e f f i c i e n c y o f r e s c u e i n c l u d e genes 1-5, genes 25-27, genes 33-34, and genes 42-43. Four peaks a r e c o n s i s t e n t w i t h t h e number of o r i g i n s t h o u g h t to o p e r a t e d u r i n g T4 r e p l i c a t i o n ( 14,40). An o r i g i n of r e p l i c a t i o n was mapped i n t h e v i c i n i t y o f gene 43 ( 5 7 ) . The g e n e t i c l o c a t i o n s of t h e o t h e r o r i g i n s a r e u n c e r t a i n , however, i t may be n o t a b l e t h a t t h e e f f i c i e n t l y r e s c u e d r e g i o n s a r e c l o s e t o a q u a r t e r genome a p a r t ( 1 0 ) . F i n a l l y , an a n a l y s i s of t h e recom-b i n a n t c l o n e s i z e d i s t r i b u t i o n of progeny phage p r o d u c e d by m u l t i p l i c i t y r e a c t i v a t i o n of U V - i r r a d i a t e d , g e n e t i c a l l y marked p a r e n t a l y p h a g e was c o n s i s t e n t w i t h t h e i d e a t h a t p a r t i a l r e p l i c a s of t h e damaged genomes r e a s s o c i a t e d by r e c o m b i n i n g p r i m a r i l y a t t h e i r e x t r e m i t i e s ( 6 1 ) . These e x p e r i m e n t s s u g g e s t e d a c o r r e l a t i o n between o r i g i n s o f DNA r e p l i c a t i o n , and t h e a b i l i t y o f a p a r t -i c u l a r segment of the genome t o be r e s c u e d e f f i c i e n t l y . S e v e r a l f e a t u r e s o f b a c t e r i o p h a g e T7,have f a c i l i t a t e d an e x a m i n a t i o n o f t h e r e l a t i o n s h i p s among marker r e s c u e , p a r t i a l r e p l i c a t i o n o f U V - i r r a d i a t e d DNA, and o r i g i n s o f DNA r e p l i c a t i o n . T7 i s a r e l a t i v e l y s m a l l , d o u b l e s t r a n d e d DNA phage whose p h y s i c a l , g e n e t i c , and p h y s i o l o g i c a l a t t r i b u t e s have been w e l l c h a r a c t e r i z e d (35,75,76). I n p a r t i c u l a r , c o n d i t i o n a l l y l e t h a l amber m u t a t i o n s i n a l l e s s e n t i a l and s e v e r a l n o n - e s s e n t i a l T7 genes have been mapped. An o r i g i n o f r e p l i c a t i o n was mapped f o r T 7 + DNA by e l e c t r o n m i c r o s c o p i c ana-l y s i s o f p a r t i a l l y r e p l i c a t e d , n o n - i r r a d i a t e d T 7 + m o l e c u l e s ( 1 8 ) . F u r t h e r -more, r e s t r i c t i o n f r agments o f t h e T7 genome were a l i g n e d w i t h s p e c i f i c r e -g i o n s o f t h e g e n e t i c map ( 5 2 ) . C r o s s r e a c t i v a t i o n e x p e r i m e n t s w i t h T7 s i m i l a r t o t h o s e of Womack w i t h T4 were p e r f o r m e d u s i n g a d e f i n e d s e t o f amber mutants as c a r r i e r phage. P a r t i a l r e p l i c a s o f b a c t e r i o p h a g e T7 DNA were c h a r a c t e r i z e d by h y b r i d i z a t i o n a n a l y s i s and by e l e c t r o n m i c r o s c o p y . S u r v i v a l c u r v e s o f U V - i r r a d i a t e d T 7 + p l a t e d on E . c o l i s t r a i n s c o n t a i n i n g DNA r e p a i r enzymes ( e . g . , B23, O i l ' , AB2497) a r e b i p h a s i c ( F i g u r e 6 ) . T h i s phenomenon was r e p o r t e d as e a r l y as 1950 ( 2 0 ) . Each phage s u b p o p u l a t i o n i s d i s t i n g u i s h e d by a u n i q u e and s h a r p l y d e f i n e d e x p o n e n t i a l c u r v e . The s l o p e o f t h e c u r v e r e p r e s e n t i n g t h e more r e s i s t a n t f r a c t i o n i s about one-half t h a t o f t h e " s e n s i t i v e " f r a c t i o n . The maximum phage f r a c t i o n showing a l o w e r s e n s i t i v i t y t o UV c o m p r i s e s o n l y about 3% o f t h e p o p u l a t i o n . S i n c e marker r e s c u e f r e q u e n c i e s a r e v e r y h i g h compared t o t h i s maximum f r a c t i o n , t h e b i -p h a s i c n a t u r e o f t h e T7 s u r v i v a l c u r v e does n o t i n t e r f e r e w i t h i n t e r p r e t a t i o n o f t h e c r o s s r e a c t i v a t i o n r e s u l t s . A t t e m p t s t o i s o l a t e U V - r e s i s t a n t T7 phage from the r e s i s t a n t s u b p o p u l a t i o n a f t e r e x t e n s i v e UV i r r a d i a t i o n have p r o v e d n e g a t i v e (R.C. M i l l e r , J r . , p e r s o n a l c o m m u n i c a t i o n ) . The progeny o f such phage show th e same b i p h a s i c s u r v i v a l c u r v e as t h e o r i g i n a l p o p u l a t i o n . When U V - i r r a d i a t e d T7 a r e p l a t e d on r e p a i r d e f i c i e n t o r r a d i a t i o n s e n s i t i v e E . c o l i s t r a i n s ( e . g . AB2500), t h e s u r v i v a l c u r v e i s monophasic w i t h a s t e e p e r s l o p e t h a n any p a r t o f t h e n o r m a l b i p h a s i c c u r v e . T h i s e f f e c t a l s o had been r e p o r t e d 87 p r e v i o u s l y ( 2 4 ) . On t h e o t h e r hand, t h e UV s u r v i v a l c u r v e s o f phages T2, T4, and T5 a r e monophasic and s i m i l a r on n o r m a l o r r a d i a t i o n - s e n s i t i v e E_. c o l i s t r a i n s ( 2 4 ) . T7 phage p l a t e d on AB2500 e f f e c t i v e l y c o n t a i n 3-5 a d d i t i o n a l l e t h a l h i t s compared t o phage r e c e i v i n g an i d e n t i c a l UV dose grown on a r e p a i r p r o f i c i e n t s t r a i n . The i m p l i c a t i o n of t h e s e r e s u l t s i s t h a t h o s t r e p a i r enzymes a c t upon U V - i r r a d i a t e d T7 DNA b u t n o t upon T2, T4, or T5 DNA. Phage i n t h e more U V - r e s i s t a n t f r a c t i o n r e p r e s e n t a s u b s e t o f t h e p o p u l a t i o n w h i c h have been r e p a i r e d t o a g r e a t e r e x t e n t . These phage a r e n o t a_ p r i o r i more r e s i s t a n t t o UV i r r a d i a t i o n . The r e s u l t s may have i m p l i c a t i o n s f o r t h e enzymes i n v o l v e d i n marker r e s c u e : r e s c u e o f T7 mark-e r s may be more dependent on h o s t r e p a i r enzymes t h a n , f o r example, T4 w h i c h codes f o r i t s own dimer e x c i s i o n enzyme ( 1 0 ) . M a r k e r s i n t h e U V - i r r a d i a t e d T 7 + genome a r e r e s c u e d i n a s p e c i f i c p a t t e r n w i t h r e g a r d t o t h e g e n e t i c map ( F i g u r e 7 ) . A t most UV d o s e s , markers i n t h e l e f t end a r e r e s c u e d w i t h t h e g r e a t e s t e f f i c i e n c y and m arkers towards t h e r i g h t end a r e r e s c u e d l e s s e f f i c i e n t l y depending on how f a r t o t h e r i g h t t h e y a r e on t h e map. The e x c e p t i o n i s t h e l o w e s t UV d o s e , 4.1 phage l e t h a l h i t s ( F i g u r e 7A), where markers on t h e extreme r i g h t o f t h e map a r e r e s c u e d a t s l i g h t l y h i g h e r e f f i c i e n c y t h a n t h o s e i m m e d i a t e l y t o t h e l e f t . The shape of t h e marker r e s c u e c u r v e i s dependent on t h e dose o f UV i r r a d i a t i o n . A t i n c r e a s i n g d o s e s , fewer m arkers a r e r e s c u e d e f f i c i e n t l y . The most e f f i c i e n t l y r e s c u e d markers l i e a t t h e l e f t end o f t h e genome i n each c a s e . A t . t h e l a r g e s t UV dose employed, o n l y m arkers i n t h e l e f t 10% of t h e genome a r e r e s c u e d w i t h a p p r e c i a b l e f r e q u e n c y . M a r k e r r e s c u e p a t t e r n s cannot be e x p l a i n e d by p r e f e r e n t i a l r e v e r s i o n o f amber m u t a t i o n s i n genes on t h e l e f t end o f t h e T7 genome ( T a b l e 2) n o r by a h i g h e r e f f i c i e n c y o f p l a t i n g o f markers r e s c u e d from t h e l e f t hand s i d e . whereas t h e c u r v e s o b t a i n e d f o r l o w e r doses a r e i n a c c o r d w i t h t h e h y p o t h e s i s t h a t r e p l i c a t i o n i n i t i a t e s a t t h e 17% o r i g i n , p r o c e e d s b i d i r e c t i o n a l l y a t t h e same r a t e t o s i t e s o f UV damage ( o r t h e end 88 o f t h e m o l e c u l e ) l o c a t e d a t comparable d i s t a n c e s f r o m t h e o r i g i n , and t h a t r e s c u e e f f i c i e n c y i s t h e same f o r any p a r t i a l l y r e p l i c a t e d p i e c e , t h e d a t a f rom t h e h i g h e r doses i m p l y t h a t some m o d i f i c a t i o n o f t h e h y p o t h e s i s i s n e c -e s s a r y . A m u t a t i o n d e l e t i n g t h e r e g i o n c o n t a i n i n g t h e presumed o r i g i n s i t e d e c r e a s e s marker r e s c u e e f f i c i e n c y i n t h a t r e g i o n ( F i g u r e 8 ) . A t a UV dose (6.9 PLH) s i m i l a r t o t h a t where sym m e t r i c r e s c u e o f T 7 + markers around t h e 17% o r i g i n o c c u r s (6.5 PLH, F i g u r e 7 B ) , r e s c u e o f markers from U V - i r r a d i a t e d T7VLG-3 i s a p p r e c i a b l e o n l y a t t h e l e f t end of t h e genome, comparable t o t h e h i g h dose ca s e f o r T 7 + (9.5 PLH, F i g u r e 7D). The e f f e c t i s n o t due t o d i f f -i c u l t i e s o f i n t e r a c t i o n between t h e d e l e t i o n mutant and t h e n o r m a l l e n g t h c a r r i e r phage ( T a b l e 4 ) . A l s o t h e r e a r e no m a j o r e x p a n s i o n s o r c o n t r a c t i o n s o f t h e g e n e t i c map (75,77,78) as d e t e r m i n e d by a c o m p a r i s o n o f p h y s i c a l and g e n e t i c c h a r a c t e r i z a t i o n s o f t h e T7 genome. T h i s means t h a t d e l e t i o n o f t h e 17% o r i g i n most p r o b a b l y d e p r e s s e s marker r e s c u e i n t h a t r e g i o n because p a r t i a l r e p l i c a t i o n i s d e p r e s s e d . I t i s r e a s o n a b l e t o assume t h a t t h e marker r e s c u e e f f i c i e n c i e s r e f l e c t d i f f e r e n c e s i n t h e a b i l i t y o f s p e c i f i c segments of t h e genome t o r e p l i c a t e . A t t h e same t i m e , a d d i t i o n a l i n t e r p r e t a t i o n s a l s o a r e p o s s i b l e . I t i s con-c e i v a b l e t h a t r e p l i c a t i o n i n i t i a t e s a t t h e 17% o r i g i n , p r o c e e d s b i d i r e c t i o n a l l y a t t h e same r a t e t o more o r l e s s s y m m e t r i c a l l y l o c a t e d UV l e s i o n s , and t h a t markers from t h e l e f t end f o r some r e a s o n a r e r e s c u e d w i t h g r e a t e r e f f i c i e n c y . However, c o r r e l a t i o n o f p a r t i a l r e p l i c a t i o n d a t a w i t h marker r e s c u e e f f i c i e n c i e s (see below) makes t h e s u p p o s i t i o n o f d i f f e r e n c e s i n r e s c u e e f f i c i e n c y d e s p i t e s i m i l a r e x t e n t s o f r e p l i c a t i o n u n n e c e s s a r y . A n o t h e r i n t e r p r e t a t i o n o f t h e s e r e s u l t s i s t h a t r e p l i c a t i o n o f U V - i r r a d i a t e d T 7 + DNA i s i n i t i a t e d a t an o r i g i n o r o r i g i n s t o t h e l e f t o f 17%. T h i s p o s s i b i l i t y w i l l be d i s c u s s e d a t g r e a t e r l e n g t h below. I n a d d i t i o n , i t i s p o s s i b l e t h a t b l o c k s t o DNA r e p l i c a t i o n a r e n o t d i s t r i b u t e d a t random around t h e supposed o r i g i n b u t t h a t 89 t h e y l i e c l o s e r t o t h e r i g h t t h a n t o t h e l e f t o f 17%. The p r i m a r y mechanism o f u l t r a v i o l e t r a d i a t i o n damage t o DNA i s t h e f o r m a t i o n o f p y r i m i d i n e , and e s p e c i a l l y t h y m i d i n e , d i m e rs ( 6 9 ) . W i t h a G + C/ A + T r a t i o o f a p p r o x i m a t e l y 1 as i n T7 DNA, t h e e f f i c i e n c y o f t h y m i d i n e dimer f o r m a t i o n i s g r e a t e r t h a n t h a t o f mixed o r c y t i d i n e d i m e r s . There a r e s e v e r a l l i n e s o f e v i d e n c e w h i c h i n d i c a t e t h a t p y r i m i d i n e dimers a r e b l o c k s t o DNA s y n t h e s i s and t h a t s u c h b l o c k s a r e c o r r e l a t e d w i t h l e t h a l h i t s t o b a c t e r i o p h a g e DNA (66,67)'. I n v i t r o , a d e c r e a s e i n t h e p r i m i n g a b i l i t y o f DNA i r r a d i a t e d w i t h 280nm r a d i a t i o n i s r e s t o r e d by subsequent i r r a d i a t i o n a t 240nm, a p r o c e s s known t o remove d i m e r s . The s e n s i t i v i t y o f d i f f e r e n t DNA's t o l o s s o f p r i m i n g a b i l i t y i n c r e a s e s w i t h A + T c o n t e n t . I n v i v o , i t i s o b s e r v e d t h a t DNA s y n t h e s i s a f t e r UV i r r a d i a t i o n s t o p s i n U V - s e n s i t i v e c e l l s a f t e r a s m a l l i n i t i a l s y n t h e s i s and n e v e r resumes. I n U V - r e s i s t a n t c e l l s , s y n t h e s i s s t o p s b u t l a t e r resumes. The r e s u m p t i o n o f s y n t h e s i s i s c o n c o m i t a n t w i t h t h e r e m o v a l o f d i m e r s . A l t h o u g h UV r a d i a t i o n has s e v e r a l a d d i t i o n a l e f f e c t s on DNA ( e . g . , i t may cause c h a i n b r e a k s , d e n a t u r a t i o n , DNA o r n u c l e i c a c i d - p r o t e i n c r o s s l i n k s , o r p r o d u c t i o n o f c y t o s i n e and u r a c i l h y d r a t e s ) , t h e r e i s no p r o o f t h a t changes i n DNA o t h e r t h a n d i m e r s can cause l e t h a l e f f e c t s , t h a t i s , b l o c k s t o DNA r e p l i c a t i o n ( 6 6 ) . E x a m i n a t i o n o f t h e p a r t i a l d e n a t u r a t i o n map o f T7 (31) shows t h a t A + T r i c h r e g i o n s a r e n o t u n i f o r m l y d i s t r i b u t e d a l o n g T7. m o l e c u l e s . The r e g i o n f r o m around 10-30% c o n t a i n i n g t h e presumed DNA r e p l i c a t i o n o r i g i n s i t e a t 17% i s v e r y r i c h i n A + T base p a i r s w h i l e t h e r e g i o n from around 5-10% i s p r a c t i c a l l y d e v o i d of s i t e s s u s c e p t i b l e t o p a r t i a l d e n a t u r a t i o n . W i t h t h i s c o n s i d e r a t i o n , t h e marker r e s c u e d a t a c o u l d be c o n s i s t e n t w i t h b i d i r e c t i o n a l r e p l i c a t i o n f r o m an o r i g i n l o c a t e d a t 17% from t h e l e f t end u n t i l a s i t e o f UV damage i s r e a c h e d . Such s i t e s w o u l d be more p r o b a b l e t o t h e r i g h t t h a n t o t h e l e f t o f t h e o r i g i n . I f t h y m i d i n e d i mers do p r e v e n t DNA r e p l i c a t i o n t o t h e r i g h t o f 17% a t h i g h UV d o s e s , r e m o v a l o f d i m e rs s h o u l d a l l o w r e p l i c a t i o n of a d d i t i o n a l s e g -ments o f t h e T7 genome. Removal w i l l enhance c r o s s r e a c t i v a t i o n e f f i c i e n c i e s o f markers l o c a t e d i n t h o s e segments a c c o r d i n g t o t h e p a r t i a l r e p l i c a hypo-t h e s i s . To t e s t t h i s p o s t u l a t e , p h o t o r e a c t i v a t i o n d u r i n g c r o s s r e a c t i v a t i o n was p e r f o r m e d . P h o t o r e a c t i v a t i o n i s a s p e c i f i c e n z y m a t i c p r o c e s s w h i c h mono-m e r i z e s t h y m i d i n e d i mers ( f o r r e v i e w - see 66,68). A p p r o x i m a t e l y 35% o f a U V - i r r a d i a t e d T7 p o p u l a t i o n i s p h o t o r e a c t i v a b l e compared t o 20% f o r T4 ( 2 0 , 2 3 ) . One would p r e d i c t t h a t a f t e r p h o t o r e a c t i v a t i o n , c r o s s r e a c t i v a t i o n c u r v e s f o r h i g h UV doses w o u l d b r o a d e n t o i n c l u d e r e s c u e o f markers t o t h e r i g h t of 17% as i s o b s e r v e d a t l o w e r UV d o s e s . T h i s i s i n d e e d t h e c a s e ( F i g u r e 1 1 ) . D i f f e r e n t segments o f U V - i r r a d i a t e d T 7 + DNA were shown t o r e p l i c a t e 32 w i t h d i f f e r e n t e f f i c i e n c i e s by h y b r i d i z a t i o n o f P - l a b e l e d progeny DNA s y n t h e s i z e d by U V - i r r a d i a t e d p a r e n t a l phage t o a l i g n e d r e s t r i c t i o n f r a g m e n t s o f t h e T7 genome. Segments i n t h e l e f t p o r t i o n o f t h e m o l e c u l e r e p l i c a t e most e f f i c i e n t l y whereas segments toward t h e r i g h t end r e p l i c a t e l e s s e f f i c -i e n t l y ( F i g u r e 1 4 ) . The d i f f e r e n c e s i n t h e a b i l i t i e s o f v a r i o u s segments t o r e p l i c a t e a r e a c c e n t u a t e d by i n c r e a s i n g t h e dose o f UV i r r a d i a t i o n . T h e r e f o r e , p a r t i a l r e p l i c a s o f U V - i r r a d i a t e d T 7 + DNA do e x i s t . T here i s a c l o s e c o r r e s p o n d e n c e between segments o f t h e genome w h i c h r e p l i c a t e most e f f i c i e n t l y and segments c o n t a i n i n g markers w h i c h a r e r e s c u e d most e f f i c i e n t l y . F o r example, i t was o b s e r v e d t h a t a t low UV doses (4.1 P L H ) , markers l y i n g on t h e extreme r i g h t o f t h e genome were r e s c u e d a t s l i g h t l y h i g h e r e f f i c i e n c y t h a n markers i m m e d i a t e l y t o t h e l e f t ( F i g u r e 7A, see a b o v e ) . S i m i l a r l y , e x a m i n a t i o n o f t h e h y b r i d i z a t i o n p a t t e r n f o r a low dose (4.6 PLH). r e v e a l s t h a t r e p l i c a t i o n o f t h e r i g h t m o s t Endo R.Fnu C f r a g -ment C i s s l i g h t l y enhanced compared t o t h e l a r g e fragment A i m m e d i a t e l y t o 91 the r i g h t . A numerical c o r r e l a t i o n between the e f f i c i e n c y with which a marker r e p l i c a t e s and i s rescued can be established i n the following manner. To determine the r e l a t i v e e f f i c i e n c i e s of the r e p l i c a t i o n of the fragments, one can divide the f r a c t i o n a l contribution of a UV-irradiated fragment by the f r a c t i o n a l contribution of the same fragment i n the non-irradiated c o n t r o l . The f r a c t i o n a l contribution: i s defined as the area under the peak on the densitometer tracing of the p a r t i c u l a r h y b r i d i z a t i o n band divided by the summed area of a l l the peaks (see Tables 6 and 7). For example, one divides the area of fragment A, panel A (Figure 14), by the t o t a l area of the curve of panel A. This value i s divided i n turn by the r a t i o of the area of fr a g -ment A, panel D, to the t o t a l area under the curve of panel D. A s i m i l a r c a l c u l a t i o n i s made for fragment B. Thus by d i v i d i n g the c a l c u l a t i o n f o r fragment A r e p l i c a t i o n by fragment B r e p l i c a t i o n , one can determine the r e l a - t i v e e f f i c i e n c y of r e p l i c a t i o n . At a dose of 7.0 phage l e t h a l events, the r e l a t i v e e f f i c i e n c y of r e p l i c a t i o n of fragment A compared to B i s 0.1 or 10% as e f f i c i e n t . If one compares the e f f i c i e n c y of rescue of a marker i n the middle of fragment A (14aml40) to a marker i n the middle of fragment B (laml93) at a s i m i l a r dose of UV i r r a d i a t i o n , the r a t i o i s about 0.0'5. S i m i l a r l y i f the r e l a t i v e e f f i c i e n c y of r e p l i c a t i o n of fragment E compared to fragment B i s calculated, then E r e p l i c a t e s about 84% as e f f i c i e n t l y as B; a marker i n E (5am28) i s rescued with an e f f i c i e n c y of 0.5 (50%) compared to a marker i n B (laml93). Therefore, the rescue of a marker i s correlated with the e f f i c i e n c y of r e p l i c a t i o n of the segment of the DNA containing that marker. The h y b r i d i z a t i o n data indi c a t e that segments of T7 DNA to the l e f t of 17% are r e p l i c a t e d with greater e f f i c i e n c y than those to the r i g h t . The re s u l t s are i n accord with r e p l i c a t i o n i n i t i a t i n g at 17% and proceeding b i d i r e c t i o n a l l y to UV-induced blocks with such blocks occurring closer to the r i g h t than to the l e f t of 17% due to higher A + T content i n the former 92 r e g i o n . I t a l s o ; i s p o s s i b l e t h a t r e p l i c a t i o n i n i t i a t e s a t s i t e s t o t h e l e f t o f 17%. The l o c a t i o n and s i z e o f t h e Endo R. Fnu C r e s t r i c t i o n f r a g -ments used i n t h i s a n a l y s i s p r e c l u d e a f i n e s t r u c t u r e mapping of t h e l e f t end o f p a r t i a l l y r e p l i c a t e d T7 m o l e c u l e s by t h i s method and t h u s p r e v e n t th e assessment o f t h e r e l a t i v e m e r i t s o f t h e s e two e x p l a n a t i o n s . However, i t does seem c l e a r t h a t segments w h i c h r e p l i c a t e p r o b a b l y a r e r e s c u e d w i t h s i m i l a r - e f f i c i e n c i e s . The d a t a c l e a r l y s u p p o r t p a r t i a l r e p l i c a h y p o t h e s e s . U V - i r r a d i a t e d T 7 + DNA can be r e c o v e r e d i n t a c t f rom i n f e c t e d c e l l s a s h o r t t i m e a f t e r i n f e c t i o n ( F i g u r e 1 5 ) . T h e r e f o r e , i t i s r e a s o n a b l e t o assume t h a t U V - i r r a d i a t e d phage i n j e c t t h e i r e n t i r e genomes b u t t h a t r e p -l i c a t i o n o f complete m o l e c u l e s i s b l o c k e d due t o UV damage. The c r o s s r e a c t -i v a t i o n and h y b r i d i z a t i o n d a t a cannot be e x p l a i n e d by p r e f e r e n t i a l i n j e c t i o n o f t h e l e f t end o f U V - i r r a d i a t e d DNA. A l t h o u g h h i g h energy i o n i z i n g r a d i a t i o n i s c a p a b l e o f b r e a k i n g d o u b l e s t r a n d e d DNA ( 5 8 ) , u l t r a v i o l e t r a d i a t i o n a t the doses used i n t h e s e e x p e r i m e n t s i s n o t . + F u r t h e r c h a r a c t e r i z a t i o n o f p a r t i a l l y r e p l i c a t e d , U V - i r r a d i a t e d T7 DNA was a c c o m p l i s h e d by e l e c t r o n m i c r o s c o p y . E l e c t r o n m i c r o s c o p i c a n a l y s i s o f p a r t i a l l y r e p l i c a t e d , n o n - i r r a d i a t e d T7 m o l e c u l e s i s o l a t e d a t e a r l y t i m e s a f t e r i n f e c t i o n had e s t a b l i s h e d t h e major o r i g i n o f DNA r e p l i c a t i o n a t a s i t e a p p r o x i m a t e l y 17% from t h e g e n e t i c l e f t end ( 1 8 ) . I t was hoped t h a t a s i m i l a r a n a l y s i s o f U V - i r r a d i a t e d DNA i s o l a t e d under c o n d i t i o n s where r e p l i c a t i o n c o u l d p r o c e e d as f a r as p o s s i b l e , i . e . , t o UV-induced b l o c k a g e p o i n t s (as i n t h e c r o s s r e a c t i v a t i o n and h y b r i d i z a t i o n a n a l y s e s ) w o u l d p r o -v i d e f i n e s t r u c t u r e mapping o f p a r t i a l l y r e p l i c a t e d r e g i o n s . T7 phage i r r a d i a t e d t o 7.3 phage l e t h a l e v e n t s were used t o i n f e c t E . c o l i grown i n d e n s i t y , medium. I t . was n e c e s s a r y t o i s o l a t e p a r t i a l l y r e p l i c a t e d T7 DNA from C s C l g r a d i e n t s a f t e r d e n s i t y t r a n s f e r e x p e r i m e n t s due t o t h e s i m i l a r G + C c o n t e n t o f E . c o l i and T7 DMA under n o r m a l c o n d i t i o n s . To faci3„itate i s o l a t i o n o f p a r t i a l l y r e p l i c a t e d s p e c i e s and f u r t h e r s e g r e g a t e h o s t and u n i t l e n g t h T7 DNA, f r a c t i o n s f r o m t h e heavy s i d e o f t h e T7 peak i n t h e f i r s t g r a d i e n t were s u b j e c t e d t o a second s e d i m e n t a t i o n t h r o u g h C s C l b e f o r e e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n ( F i g u r e 1 6 )• The e l e c t r o n m i c r o s c o p i c s t u d y p r o v i d e s d i r e c t v i s u a l e v i d e n c e f o r t h e e x i s t e n c e o f p a r t i a l r e p l i c a s o f U V - i r r a d i a t e d T 7 + DNA. From t h e m a t e r i a l p o o l e d a f t e r C s C l c e n t r i f u g a t i o n , s e v e r a l v a r i e t i e s o f p a r t i a l l y r e p l i c a t e d m o l e c u l e s were o b s e r v e d . As e x p e c t e d from t h e e a r l i e r s t u d y of n o n - i r r a d i a t e d m o l e c u l e s , T7 DNA c o n t a i n i n g l a r g e f o r k s and i n t e r n a l d u p l i c a t i o n s s p a n n i n g the 17% r e g i o n were o b s e r v e d ( F i g u r e 17a,b). However, m o l e c u l e s c o n t a i n i n g s m a l l f o r k s ( F i g u r e 17c) and b u b b l e s n o t c e n t e r e d a t 17% a l s o were o b s e r v e d . I n a d d i t i o n , s e v e r a l m o l e c u l e s c o n t a i n i n g two p a r t i a l l y r e p l i c a t e d r e g i o n s were seen ( F i g u r e 1 7d). A number of m o l e c u l e s c o n t a i n i n g d o u b l e s t r a n d e d , p a r t i a l l y r e p l i c a t e d s t r u c t u r e s were p h o t o g r a p h e d and measured. S i x t y - f o u r of t h e s e m o l e c u l e s w i t h l e n g t h s m e a s u r i n g 12.0 + 0.5ym were i n c l u d e d i n t h e a n a l y s i s . The d e n s i t y t r a n s f e r e x p e r i m e n t and the l e n g t h measurement c r i t e r i a i n s u r e d t h a t o n l y T7 DNA was b e i n g examined. The m o l e c u l e s were n o r m a l i z e d t o a s c a l e o f 100 u n i t s and a l i g n e d s u c h t h a t the p a r t i a l l y r e p l i c a t e d r e g i o n s o c c u r r e d i n t h e l e f t p o r t i o n o f t h e genome ( F i g u r e 1 8 ) . D e n a t u r a t i o n a n a l y s i s was n o t p e r f o r m e d on t h e s e m o l e c u l e s , however, p r e v i o u s d e n a t u r a t i o n s t u d i e s of p a r t i a l l y r e p l i c a t e d T7 DNA (18,84) as w e l l as t h e h y b r i d i z a t i o n a n a l y s i s d e s c r i b e d i n t h i s work i n d i c a t e d t h a t d u p l i c a t e d r e g i o n s i n p a r t i a l l y r e p l i -c a t e d m o l e c u l e s i n d e e d o c c u r on t h e l e f t hand s i d e . I t i s i m m e d i a t e l y a p p a r e n t t h a t U V - i r r a d i a t e d T7 DNA i s a b l e t o i n i t i a t e r e p l i c a t i o n a t a number of s i t e s w i t h i n t h e l e f t end of t h e m o l e c u l e ( F i g -u r e 1 8 ) . S i n c e o r i g i n s o c c u r o n l y i n one r e g i o n o f t h e genome, s i g n i f i c a n t m u l t i p l i c i t y r e a c t i v a t i o n i s n o t e x p e c t e d f o r T7 phage a c c o r d i n g t o t h e p a r -t i a l r e p l i c a h y p o t h e s i s and v e r y l i t t l e m u l t i p l i c i t y r e a c t i v a t i o n a c t u a l l y i s o b s e r v e d ( T a b l e 3 ) . M u l t i p l i c i t y r e a c t i v a t i o n i s presumed t o o c c u r o n l y 94 i f each p o r t i o n of the genome can be r e p r e s e n t e d i n undamaged f o r m , and t h u s can be r e p l i c a t e d , i n a t l e a s t one of t h e c o i n f e c t i n g U V - i r r a d i a t e d p a r e n t a l phage. I f r e p l i c a t i o n p r o c e e d s b i d i r e c t i o n a l l y f r o m an o r i g i n of r e p l i c a t i o n t o a s i t e of UV damage, b a c t e r i o p h a g e ( s u c h as T4) w i t h m u l t i p l e i n i t i a t i o n s i t e s d i s t r i b u t e d t h r o u g h o u t t h e genome have an i n c r e a s e d p r o b a b i l i t y f o r p a r t i a l r e p l i c a t i o n o f a l l segments of t h e m o l e c u l e among th e damaged i n p u t phage. I f o r i g i n s a r e l o c a t e d i n o n l y one r e g i o n o f the m o l e c u l e , s u c h as f o r T7, a f t e r UV i r r a d i a t i o n , o n l y one p o r t i o n of t h e m o l e c u l e w i l l be r e p -l i c a t e d . The u n r e p l i c a t e d p o r t i o n does n o t p a r t i c i p a t e i n r e a c t i v a t i o n so MR i s n o t o b s e r v e d f o r s u c h genomes. Most of t h e i n i t i a t i o n s i t e s p r o b a b l e a r e l o c a t e d t o t h e l e f t of 17% ( T a b l e 8 ) . The t i m i n g o f t h e e x p e r i m e n t a l l o w e d r e p l i c a t i o n t o p r o c e e d as f a r as p o s s i b l e so one c a n n o t say e x a c t l y where, a l o n g an eye f o r m , r e p l i c a t i o n s t a r t e d s i n c e r e p l i c a t i o n s t o p s a t a UV l e s i o n on e i t h e r s i d e of t h e i n i t i a t i o n s i t e ; t h a t i s , r e p l i c a t i o n d i d not n e c e s s a r i l y i n i t i a t e a t t h e ^center of the eye. I n a c c o r d w i t h t h e p a r t i a l r e p l i c a h y p o t h e s i s , t h e s e d a t a i n d i c a t e t h a t i t i s n o t s u r p r i s i n g t h a t m a rkers t o t h e l e f t of 17% c o n s i s t e n t l y a r e r e s c u e d w i t h g r e a t e s t e f f i c i e n c y . I n f a c t , 75% of t h e p a r t i a l l y r e p l i c a t e d r e g i o n s o b s e r v e d were t o t h e l e f t of 17% w h i l e o n l y 25% were t o t h e r i g h t ( F i g u r e 1 9 ) . The e l e c t r o n m i c r o s c o p i c a n a l y s i s c o n f i r m s and e x t e n d s t h e p r e v i o u s r e s u l t s ; There i s an e x c e l l e n t c o r r e l a t i o n between t h e a r e a s o f the genome w h i c h r e p l i c a t e e f f i c i e n t l y and t h o s e w h i c h a r e r e s c u e d e f f i c i e n t l y d u r i n g c r o s s r e a c t i v a t i o n . 95 C. Some P r o c e s s e s I n v o l v e d i n C r o s s R e a c t i v a t i o n The e a r l i e s t a c c o u n t s of m u l t i p l i c i t y and c r o s s r e a c t i v a t i o n supposed t h a t r e c o m b i n a t i o n was an e s s e n t i a l p a r t of t h e c r o s s r e a c t i v a t i o n p r o c e s s ( 1 7 , 4 9 ) . Indeed, i t i s d i f f i c u l t t o i m a g i n e a r e s c u e mechanism t h a t would not i n v o l v e r e c o m b i n a t i o n a t some p o i n t . U V - i r r a d i a t e d phage a l o n e a r e u n a b l e t o p r o d u c t i v e l y i n f e c t E . c o l i c e l l s and amber mutants a r e u n a b l e t o p r o d u c e w i l d t y p e progeny w i t h s i g n i f i c a n t f r e q u e n c y ; y e t n o r m a l , undamaged, w i l d t y p e progeny a r e produced as a r e s u l t of m u l t i p l i c i t y o r c r o s s r e a c t i v a t i o n . I n o r d e r t o d e m o n s t r a t e t h a t r e c o m b i n a t i o n i s n e c e s s a r y f o r marker r e s c u e , c r o s s r e a c t i v a t i o n e x p e r i m e n t s were p e r f o r m e d where t h e i r r a d i a t e d donor phage as w e l l as the n o n - i r r a d i a t e d c a r r i e r phage c a r r i e d an amber m u t a t i o n . I n each c a s e , p r o d u c t i o n of w i l d t y p e i n f e c t i v e c e n t e r s was o b s e r v e d showing t h a t r e c o m b i n a t i o n i s , a t l e a s t , p a r t of the r e s c u e p r o c e s s ( T a b l e 5 ) . I t i s n o t a b l e t h a t the r e s c u e e f f i c i e n c i e s i n each e x p e r i m e n t where i r r a d i a t e d amber mutants s e r v e d as t h e donor phage were s i m i l a r t o t h o s e o b s e r v e d when T 7 + i r r a d i a t e d t o a s i m i l a r dose was used. I t i s p r o b a b l e t h a t t h e r e s c u e event i n v o l v e s a d o u b l e c r o s s - o v e r between t h e c a r r i e r phage and t h e r e p l i -c a t e d p o r t i o n o f t h e donor phage. A l t h o u g h r e c o m b i n a t i o n p l a y s a r o l e i n marker r e s c u e , t h e s e e x p e r i m e n t s do n o t p r o v e t h a t r e c o m b i n a t i o n , i n a d d i t i o n t o p a r t i a l r e p l i c a t i o n of t h e r e s c u e d r e g i o n , i s t h e o n l y p r o c e s s i n v o l v e d i n c r o s s r e a c t i v a t i o n . P h o t o r e a c t i v a t i o n , an e n z y m a t i c p r o c e s s f o r t h e r e m o v a l of p y r i m i d i n e d i m e r s from UV-damaged DNA was shown t o i n c r e a s e t h e e f f i c i e n c y o f r e s c u e of m a rkers t o t h e r i g h t of 17% a f t e r h i g h UV doses t o T 7 + ( F i g u r e 1 1 ) . The e f f e c t most l i k e l y i s due t o r e m o v a l of b l o c k s t o DNA r e p l i c a t i o n caused by t h e d i m e r s . The p h o t o r e a c t i v a t i o n enzyme i t s e l f p r o b a b l y does n o t p l a y a r o l e i n t h e a c t u a l r e s c u e p r o c e s s . A n o t h e r s y s t e m w h i c h c o u l d be i m p l i c a t e d i n t h e marker r e s c u e p r o c e s s i s h o s t c e l l r e a c t i v a t i o n . Host c e l l r e a c t i v a t i o n (HCR) i s a p r o c e s s whereby h o s t b a c t e r i a can i n c r e a s e t h e p l a q u e f o r m i n g a b i l i t y o f some U V - i r r a d i a t e d b a c t e r i o p h a g e . HCR i s o b s e r v e d f o r p h a g e s . T l , T3, T7 ( 2 4 ) , P22 ( 3 0 ) , and A ( 8 ) , b u t n o t f o r T2, T4, o r T6 ( 6 8 ) . D i f f e r e n c e s i n t h e c a p a c i t y o f v a r i o u s E . c o l i s t r a i n s t o p e r f o r m HCR have been o b s e r v e d ( 2 4 , 4 4 ) ; t h e s e d i f f e r e n c e s i m p l y a g e n e t i c , and hence e n z y m a t i c , b a s i s f o r r e a c t i v a t i o n ( 3 3 ) . S i n c e p y r i m i d i n e d i m e r s p r o b a b l y c o n s t i t u t e t h e p r i m a r y b l o c k s t o DNA s y n t h e s i s and a r e i m p l i c a t e d as . ' l e t h a l e v e n t s ' , i t m ight be e x p e c t e d t h a t h o s t c e l l r e a c t i v a t i o n would i n v o l v e r e m o v a l of p y r i m i d i n e d i m e r s f r o m phage DNA by h o s t r e p a i r enzymes. E . c o l i c e l l s p o s s e s s two d a r k r e p a i r mechanisms i n a d d i t i o n t o t h e p h o t o r e a c t i v a t i o n mechanism r e q u i r i n g v i s a b l e l i g h t , by w h i c h p y r i m i d i n e d i m e r s a r e removed from the genome. These mechanisms r e n d e r w i l d t y p e b a c t -e r i a r e s i s t a n t t o r a d i a t i o n and a l s o t o some c h e m i c a l damages ( 6 3 ) . One mechanism, r e p l i c a t i o n - d e p e n d e n t r e p a i r r e c o m b i n a t i o n , i s dependent on recom-b i n a t i o n , as t h e name i m p l i e s , and a p p a r e n t l y i n v o l v e s t h e r e c genes (3 8 , 6 3 ) . The o t h e r , e x c i s i o n r e p a i r , i n v o l v e s the a c t i o n s of s p e c i f i c enzymes w h i c h e x c i s e o l i g o n u c l e o t i d e s w h i c h c o n t a i n dimers from t h e damaged s t r a n d of t h e d o u b l e h e l i x and r e p a i r r e s u l t i n g gaps u s i n g t h e complementary s t r a n d as t e m p l a t e (38,68). T h i s system does n o t depend on r e c o m b i n a t i o n (10) nor does i t s i g n i f i c a n t l y a l t e r ^ r e c o m b i n a t i o n f r e q u e n c i e s when a b s e n t (see below, R e s u l t s s e c t i o n B.5). The f i r s t s t e p of e x c i s i o n r e p a i r i n E . c o l i i s c o n t r o l l e d by a dimer s p e c i f i c e n d o n u c l e a s e coded f o r by t h e u v r A and uvrB genes (9,39, 65). M u t a n t s i n t h e s e genes a r e d e f e c t i v e i n dimer e x c i s i o n and show i n c r e a s e d s e n s i t i v i t y t o UV i r r a d i a t i o n ( 3 8 , 3 9 ) . I n a d d i t i o n , s u c h mutants a r e d e f i c i e n t i n h o s t c e l l r e a c t i v a t i o n ( 3 3 , 3 4 ) . The d i f f e r e n c e s i n HCR o b s e r v e d f o r T7 and T4 can p o s s i b l y be a t t r i b u t e d t o m o d i f i e d bases i n t h e DNA of t h e l a t t e r phage. Host r e p a i r enzymes would be c a p a b l e of a c t i n g on T7 DNA but n o t on T4. T h i s v i e w i s s u p p o r t e d by t h e f a c t t h a t T4 codes f o r i t s own dimer s p e c i f i c e n d o n u c l e a s e , t h e p r o d u c t of t h e v gene ( 1 0 , 7 9 ) , and by t h e o b s e r v a t i o n t h a t HCR o f b a c t e r i o p h a g e A i n v o l v e s e x c i s i o n o f p y r i m i d i n e d i m e r s by h o s t c e l l enzymes ( 8 ) . C r o s s r e a c t i v a t i o n e x p e r i m e n t s w i t h T 7 + p e r f o r m e d i n h o s t b a c t e r i a l a c k i n g t h e u v r A e n d o n u c l e a s e (AB2500 uvrA6) showed a 2 - f o l d d e c r e a s e i n marker r e s c u e e f f i c i e n c y compared t o t h e s t r a i n w i t h t h e e x c i s i o n - r e p a i r enzyme (AB2497 u v r A + ) ( F i g u r e 1 0 ) . However, t h e shape o f t h e r e s c u e c u r v e s , and t h u s the p a r t i c u l a r m a rkers w h i c h were r e s c u e d , was t h e same i n b o t h c a s e s . These r e s u l t s s u p p o r t t h e EM d a t a i n d i c a t i n g t h a t c r o s s r e a c t i v a t i o n i s n o t an a r t i f a c t o f e x c i s i o n r e p a i r o c c u r r i n g p r e f e r e n t i a l l y i n t h e l e f t p o r t i o n of the T7 genome. The e f f e c t a l s o i n n o t due t o a d e c r e a s e i n r e c o m b i n a t i o n e f f i c i e n c y i n t h e u v r A s t r a i n . One p o s s i b l e e x p l a n a t i o n of t h e d a t a i s t h a t r e p a i r competent u v r A + c e l l s e x c i s e p y r i m i d i n e dimers w h i c h b l o c k DNA r e p l i c a t i o n . The r e m o v a l of d i m e r s t h e n a l l o w s p a r t i a l r e p l i c a t i o n of more of t h e genome w i t h a s u b s e q u e n t l y h i g h e r r e s c u e e f f i c i e n c y . I f r e p l i c a t i o n o f U V - i r r a d i a t e d t e m p l a t e s i n i t i a t e d o n l y a t t h e 17% r e g i o n as o r i g i n a l l y p o s t u l a t e d , a peak of r e s c u e a r o u n d t h i s o r i g i n would be e x p e c t e d i n t h e absence of e x c i s i o n r e p a i r . The o b s e r v e d f a i l u r e t o f i n d s u c h a peak i n the uvrA c a s e c o u l d i n d i c a t e t h a t dimer e x c i s i o n and i n c r e a s e d p a r t i a l r e p l i c a t i o n were n o t o c c u r r i n g i n AB2497 compared t o AB2500. However, e l e c t r o n m i c r o s c o p i c a n a l y s i s of p a r t i a l l y r e p l i c a t e d T 7 + DNA showed t h a t m u l t i p l e i n i t i a t i o n s i t e s were employed by U V - i r r a d i a t e d T7 phage ( F i g u r e 1 7 ) . I n i t i a t i o n a t m u l t i p l e s i t e s w o u l d r e s u l t i n s i m i l a r e x t e n t s o f r e p l i c a t i o n i n b o t h uvrA-t s t r a i n s b u t more r e p l i c a t i o n would o c c u r i n t h e AB2497 i n f e c t e d p o p u l a t i o n . An a l t e r n a t i v e e x p l a n a t i o n of t h e r e s u l t s i s as f o l l o w s . I n t h e absence of e x c i s i o n r e p a i r , r e p l i c a t i o n - d e p e n d e n t r e p a i r r e c o m b i n a t i o n s t i l l i s o p e r a t i v e ( 2 9 , 6 2 ) . DNA s y n t h e s i z e d by b a c t e r i a c o n t a i n i n g a uvrA m u t a t i o n c o n t a i n s gaps and s e d i m e n t s s l o w l y i n a l k a l i n e s u c r o s e g r a d i e n t s 98 soon a f t e r e x p o s u r e t o UV i r r a d i a t i o n ( 6 2 , 6 3 ) . L a t e r dimers a r e removed f r o m t h e DNA g r a d u a l l y by p o s t r e p l i c a t i o n r e p a i r ; c o n c o m i t a n t l y d i m e r s appear i n d a u g h t e r s t r a n d s s y n t h e s i z e d a f t e r i r r a d i a t i o n ( 2 9 ) . I t i s p o s s i b l e t h a t a s i m i l a r mechanism o p e r a t e s i n uvrA c e l l s i n f e c t e d w i t h U V - i r r a d i a t e d T7 phage. I f d i m e r s o c c u r i n p a r t i a l l y r e p l i c a t e d r e g i o n s as a r e s u l t o f p o s t r e p l i c a t i o n r e p a i r , DNA c o n t a i n i n g t h e damage m i g h t be ' r e s c u e d ' by r e c o m b i n a t i o n i n t o a c o i n f e c t i n g amber genome b u t n o t c o n t r i b u t e t o t h e o b s e r v e d r e s c u e f r e q u e n c y s i n c e t r a n s c r i p t i o n , and t h u s e x p r e s s i o n , of damaged DNA i s b l o c k e d ( 6 8 ) . Rescued, r e p a i r e d DNA, however, w i l l be ex-p r e s s e d and o b s e r v e d s u b s e q u e n t l y as w i l d t y p e progeny o r i n f e c t i v e c e n t e r s . I n c o n j u n c t i o n w i t h t h e e l e c t r o n m i c r o s c o p e d a t a w h i c h p r o v e t h a t U V - i r r a d i a t e d m o l e c u l e s may i n i t i a t e DNA r e p l i c a t i o n a t numerous l o c a t i o n s , t h e s e c r o s s r e a c t i v a t i o n r e s u l t s i m p l y , i n e i t h e r c a s e , t h a t dimer e x c i s i o n by t h e h o s t HCR system can c o n t r i b u t e t o i n c r e a s e d r e s c u e e f f i c i e n c i e s f o r T 7 + m a r k e r s . R e p l i c a t i o n - d e p e n d e n t r e p a i r r e c o m b i n a t i o n i s t h o u g h t t o be t h e p r i m a r y mechanism by w h i c h m u l t i p l i c i t y r e a c t i v a t i o n i n T4 o c c u r s ( 1 0 , 3 8 ) . The p r o c e s s i n T4 r e q u i r e s t h e p r o d u c t s of T4 genes 58,w,x, and y i n a d d i t i o n t o n o r m a l phage r e p l i c a t i o n p r o t e i n s ( 1 0 ) . T4 gene 58 mutants o v e r p r o d u c e o r under r e p a i r s i n g l e s t r a n d e d r e g i o n s . M u t a n t s i n genes w, x, and y a r e d e f i c i e n t i n g e n e r a l r e c o m b i n a t i o n as w e l l as h a v i n g i n c r e a s e d s e n s i t i v i t y t o UV r a d i a t i o n (32,79) b u t none o f t h e s e genes a r e e s s e n t i a l t o phage growth. Gene w mutants a l s o a r e d e f e c t i v e i n t h e p r o d u c t i o n o r m a i n t e n a n c e o f concatemers d u r i n g n o r m a l T4 r e p l i c a t i o n ( 3 2 ) . The p r o d u c t s coded f o r by t h e s e genes a r e p r e s e n t l y unknown. I t i s l i k e l y , however, t h a t any gene p r o d u c t s i n v o l v e d d u r i n g m u l t i p l i c i t y r e a c t i v a t i o n p l a y a r o l e i n c r o s s r e a c t i v a t i o n as w e l l . The T7 gene 6 e x o n u c l e a s e has been i m p l i c a t e d i n g e n e t i c and m o l e c u l a r r e c o m b i n a t i o n of b a c t e r i o p h a g e T7 (41,46,47,56,59) and a l s o i n t h e m a i n -te n a n c e of concatemers i n t h e p r e s e n c e of t h e gene 3 e n d o n u c l e a s e ( 4 6 , 5 6 ) . I t was p o s s i b l e t h a t , a n a l o g o u s t o the T4 c a s e , impairment o f T7 g e n e r a l r e c o m b i n a t i o n m i g h t d e c r e a s e r e p l i c a t i o n - d e p e n d e n t r e p a i r r e c o m b i n a t i o n and t h e r e f o r e T7 marker r e s c u e e f f i c i e n c i e s . I n o r d e r t o t e s t t h i s h y p o t h e s i s , p r e l i m i n a r y e x p e r i m e n t s w i t h a t e m p e r a t u r e s e n s i t i v e gene 6 mutant were p e r f o r m e d . However, b u r s t s i z e s e i t h e r i n c r o s s r e a c t i v a t i o n e x p e r i m e n t s or w i t h gene 6 mutants a r e v e r y low ( 4 1 , 5 6 ) ; i n c o n j u n c t i o n t h e two e f f e c t s r e s u l t i n such low b u r s t s i z e s t h a t no m e a n i n g f u l i n t e r p r e t a t i o n of t h e d a t a can be made. The presumed r o l e o f t h e T7 e x o n u c l e a s e d u r i n g g e n e r a l recom-b i n a t i o n i s the p r o d u c t i o n o f gaps w h i c h f a c i l i t a t e p a i r i n g o f homologous DNA s t r a n d s ( 5 5 ) . U V - i r r a d i a t e d t e m p l a t e s a l r e a d y c o n t a i n gaps as a r e s u l t of t h e i n a b i l i t y o f t h e r e p l i c a t i o n p r o c e s s t o bypass p y r i m i d i n e d i m e r s (29,62,63). T h e r e f o r e , i t i s p o s s i b l e t h a t a d d i t i o n a l gaps c r e a t e d by t h e gene 6 p r o d u c t do n o t s i g n i f i c a n t l y enhance r e s c u e f r e q u e n c i e s . I t a l s o i s p o s s i b l e t h a t h o s t g e n e r a l r e c o m b i n a t i o n enzymes p l a y t h e major r o l e i n marker r e s c u e o f T7 DNA a l t h o u g h t h e p r o d u c t s of E . c o l i r e c A , B , and C genes a p p a r e n t l y a r e n o t r e q u i r e d f o r g e n e r a l r e c o m b i n a t i o n i n t h i s phage ( 4 1 , 5 9 ) . I n a d d i t i o n , t h e T7 e x o n u c l e a s e i s e s s e n t i a l f o r nor m a l phage m e t a b o l i s m u n l i k e t h e T4 p r o d u c t s o f genes w, x, and y. The r o l e o f t h e T7 gene 6 p r o d u c t i n o t h e r phage p r o c e s s e s would make a d e m o n s t r a t i o n o f i t s a c t i v i t y , i f any, i n t h e c r o s s r e a c t i v a t i o n p r o c e s s d i f f i c u l t . 100 D. Cross R e a c t i v a t i o n and the Requirement of R e p l i c a t i o n f o r Normal  Recombination P a r t i a l r e p l i c a t i o n of segments of U V - i r r a d i a t e d T7 genomes has been shown to c o r r e l a t e w i t h rescue of genetic markers l o c a t e d i n these.segments during cross r e a c t i v a t i o n experiments. Recombination i s an e s s e n t i a l p a r t of the rescue process. Therefore i t i s of i n t e r e s t to consider whether these r e s u l t s imply that r e p l i c a t i o n i s r e q u i r e d f o r recombination of b a c t e r i o -phage T7 DNA. Studies of recombination i n bacteriophage T4 have e l u c i d a t e d two sequen-t i a l steps i n the formation of molecular recombinants. F i r s t , j o i n t mole-cules are produced by breakage and reunion of the recombining parents; j o i n t molecules are he l d together only by hydrogen bonds and the pieces can be separated by a l k a l i n e sucrose d e n s i t y sedimentation. L a t e r , covalent j o i n i n g of the p a r e n t a l c o n t r i b u t i o n occurs and true recombinant molecules are formed (2,3,80,81). T4 j o i n t molecules can be observed during b i p a r e n t a l recom-b i n a t i o n i n the absence of DNA synt h e s i s (3), however the y i e l d s are low compared to normal molecular recombination and very long i n c u b a t i o n times must be employed before s i g n i f i c a n t y i e l d s are obtained. In these exper-iments, E . c o l i are i n f e c t e d with-'iBrdU d e n s i t y - l a b e l e d phage i n the m a j o r i t y 32 and P-labeled l i g h t phage i n the m i n o r i t y . Molecular recombination i s 32 assayed as a s h i f t i n the P - l a b e l to greater d e n s i t y upon e q u i l i b r i u m gradient c e n t r i f u g a t i o n . No mature phage are produced i n the system (80) so genetic a n a l y s i s i s not p o s s i b l e . During the transformation of j o i n t molecules to recombinant molecules, some DNA synthesis i s r e q u i r e d (1,80). The s y n t h e s i s apparently can be undertaken by the host DNA polymerase s i n c e T4 mutants d e f e c t i v e i n phage DNA synt h e s i s nevertheless can have t h e i r b i p a r e n t a l j o i n t molecules c o v a l e n t l y r e p a i r e d to recombinants. The con-c l u s i o n from these experiments i s that molecular recombinants c o n t a i n 101 s i n g l e s t r a n d e d gaps when f i r s t formed; t h e s e gaps t h e n a r e f i l l e d by r e p a i r s y n t h e s i s . F i n a l l y , DNA l i g a s e s e a l s r e m a i n i n g s i n g l e s t r a n d n i c k s to c o m p l ete the c o v a l e n t r e c o m b i n a n t m o l e c u l e ( 1 ) . I t i s a p p a r e n t t h a t one needs t o d i s t i n g u i s h DNA r e p a i r , s y n t h e s i s from 'normal' s e m i c o n s e r v a t i v e r e p l i c a t i o n w h i c h i s assumed t o f a c i l i t a t e recom-b i n a t i o n d u r i n g T7 marker r e s c u e . I t must be conceded . t h a t r e c o m b i n a t i o n o c c u r s i n t h e absence of n o r m a l r e p l i c a t i o n f o r b a c t e r i o p h a g e T4 but t h e p r o c e s s i s n o t e f f i c i e n t . A v e r y low number of T7 b i p a r e n t a l r e c o m b i n a n t s were d e t e c t e d i n an e l e c t r o n m i c r o s c o p i c a n a l y s i s of t h e i n t e r a c t i o n between r e p l i c a t i o n d e f i c i e n t T7 mutants ( 8 2 ) , however such r e c o m b i n a n t s c o u l d h o t be d e t e c t e d by d e n s i t y t r a n s f e r e x p e r i m e n t s even when s u p e r i n f e c t i o n e x c l u -s i o n was i n h i b i t e d (R.C. M i l l e r , J r . p e r s o n a l c o m m u n i c a t i o n ) . D u r i n g n o r m a l growth of E . c o l i (36) and presumably i t s phages, r e p -l i c a t i o n , r e p a i r , and r e c o m b i n a t i o n of DNA o c c u r by a complex, i n t e r c o n n e c t e d s e r i e s of m e t a b o l i c pathways. One t h e n i s l e d t o a s k whether r e p l i c a t i o n of DNA f a c i l i t a t e s r e c o m b i n a t i o n d u r i n g t h e u s u a l c o u r s e of e v e n t s . E a r l y e x p e r i m e n t s by K o z i n s k i and K o z i n s k i (42) l e d them t o c o n c l u d e t h a t s e m i -c o n s e r v a t i v e r e p l i c a t i o n was c l o s e l y a s s o c i a t e d w i t h m o l e c u l a r r e c o m b i n a t i o n . They found t h a t a r e d u c t i o n of t h e DNA p o o l a f t e r i n h i b i t i o n o f r e p l i c a t i o n by FdU a l l o w e d the d e m o n s t r a t i o n of T4 i n t e r p a r e n t a l r e c o m b i n a t i o n a f t e r p r o l o n g e d c o n t a c t between p a r e n t a l DNA m o l e c u l e s but t h a t r e p l i c a t i o n s t i l l needed t o be a c l o s e l y accompanying p r o c e s s . S i m i l a r l y , B r o k e r and Lehman ( 1 1 ) , s t u d y i n g T4 b i p a r e n t a l r e c o m b i n a n t s by e l e c t r o n m i c r o s c o p y , found t h a t a d e c r e a s e d r a t e of DNA s y n t h e s i s p r o l o n g e d the i n t e r v a l d u r i n g w h i c h r e c o m b i n a t i o n c o u l d o c c u r and i n c r e a s e d t h e number of b r a n c h e d r e c o m b i n a t i o n i n t e r m e d i a t e s w h i c h were o b s e r v e d . E l e g a n t e x p e r i m e n t s by S t a h l and h i s c o l l a b o r a t o r s (71,72,73,74) w i t h b a c t e r i o p h a g e X a l s o e s t a b l i s h e d a c l o s e a s s o c i a t i o n o f DNA s y n t h e s i s w i t h r e c o m b i n a t i o n . E s s e n t i a l l y , t h e e x p e r i m e n t s c o n s i s t e d of i n f e c t i n g l i g h t E . c o l i i n l i g h t medium w i t h g e n e t i c a l l y marked, d e n s i t y l a b e l e d phage. L y s a t e s were s u b j e c t e d t o e q u i l i b r i u m g r a d i e n t c e n t r i f u g a t i o n and t h e f r a c t i o n s a s s a y e d g e n e t i c a l l y f o r p r o d u c t i o n o f v a r i o u s r e c o m b i n a n t s . C r o s s e s c o v e r i n g the r i g h t , 'middle, and l e f t s e c t i o n s of t h e A genome were p e r f o r m e d under v a r i o u s r e c o m b i n a t i o n c o n d i t i o n s , i . e . e m p l o y i n g t h e phage m e d i a t e d I n t and Red systems and t h e E . c o l i Rec system. DNA s y n t h e s i s was r e d u c e d o r i n h i b i t e d c h e m i c a l l y or. by b a c t e r i a l and/or phage m u t a t i o n s . I t was f o u n d t h a t r e c o m b i n a t i o n i n some i n t e r v a l s o f t h e A map was a s s o c i a t e d w i t h more DNA s y n t h e s i s t h a n o t h e r s . B l o c k a g e of DNA s y n t h e s i s r e s u l t e d i n a r e l a t i v e r e d u c t i o n of r e c o m b i n a t i o n f r e q u e n -c i e s i n r e g i o n s h a v i n g l a r g e r amounts of r e c o m b i n a t i o n - a s s o c i a t e d s y n t h e s i s . T h e r e f o r e i t was c o n c l u d e d t h a t DNA s y n t h e s i s enhanced r e c o m b i n a n t f o r m a t i o n more i n some a r e a s t h a n i n o t h e r s . I t c o u l d n o t be e x c l u d e d t h a t i n t h o s e i n t e r v a l s where r e c o m b i n a t i o n was a s s o c i a t e d w i t h DNA s y n t h e s i s , t h e s y n -t h e s i s was e s s e n t i a l f o r f o r m a t i o n o f r e c o m b i n a n t s . A t t h e same t i m e , t h e r e l a t i o n o f DNA s y n t h e s i s t o p r o d u c t i o n o f r e c o m b i n a n t s was n o t e l u c i d a t e d . S e v e r a l p o s s i b i l i t i e s were c o n s i d e r e d : 1) r e c o m b i n a t i o n o c c u r s b e s t a t r e p l i c a t i n g f o r k s o r w i t h newly r e p l i c a t e d DNA; 2) r e c o m b i n a t i o n o c c u r s i n s u c h a way t h a t t h e A chromosome must r e p l i c a t e b e f o r e i t can mature; 3) r e c o m b i n a t i o n i s a s o u r c e o f r e p l i c a t i o n f o r k s ; and 4) r e p l i c a t i o n changes t h e t o p o l o g y o f t h e DNA so t h a t r e g i o n s a r e a c c e s s i b l e t o r e c o m b i n a t i o n systems. A r e c e n t s t u d y of A c o n c l u d e d t h a t u n r e p l i c a t e d DNA was a poor s u b s t r a t e f o r i n v i v o r e c o m b i n a t i o n u n l e s s i t was U V - i r r a d i a t e d ( 3 6 ) . The e x p e r i m e n t s employed a t r a n s f e c t i o n a s s a y w h i c h , i n t h e i r r a d i a t e d c a s e , was a l s o an a s s a y f o r r e p a i r . B o t h r e p a i r and U V - s t i m u l a t e d r e c o m b i n a t i o n were i n h i b i t e d by a n t a g o n i s t s of DNA g y r a s e . G e n e r a l r e c o m b i n a t i o n was low i n t h e absence of phage DNA r e p l i c a t i o n and r e p a i r . E a r l i e r A s t u d i e s w h i c h c o n c l u d e d r e p l i c a t i o n was n o t e s s e n t i a l f o r n o r m a l r e c o m b i n a t i o n 103 .employed a s s a y s w h i c h d i s c r i m i n a t e d a g a i n s t unrecombined DNA. I t t u r n s out t h a t m a t u r a t i o n o f A r e q u i r e s l a r g e r t h a n u n i t l e n g t h m o l e c u l e s and s u c h m o l e c u l e s may be g e n e r a t e d by t h e s i t e s p e c i f i c I n t s y s t e m i n t h e absence of r e p l i c a t i o n o r g e n e r a l i z e d r e c o m b i n a t i o n ( 5 3 ) . Two l a b o r a t o r i e s s t u d y i n g t h e genes i n v o l v e d i n T7 r e c o m b i n a t i o n a r r i v e d i n d i r e c t l y a t t h e c o n c l u s i o n t h a t r e p l i c a t i o n m i g h t be n e c e s s a r y f o r r e c o m b i n a t i o n of t h i s phage (4 1 , 5 9 ) . B o t h t h e gene 5 p o l y m e r a s e and the gene 4 p r o d u c t , e s s e n t i a l f o r T7 r e p l i c a t i o n ^ were found a l s o t o be n e c e s s a r y f o r r e c o m b i n a t i o n . D u r i n g marker r e s c u e , r e p l i c a t i o n c o u l d enhance . r e c o m b i n a t i o n i n two ways. The s t r u c t u r a l f e a t u r e s of t h e p a r t i a l l y r e p l i c a t e d m o l e c u l e c o u l d be n e c e s s a r y i n o r d e r f o r r e c o m b i n a t i o n t o o c c u r , o r , r e p e a t e d r e p l i c a t i o n o f a segment between UV l e s i o n s c o u l d i n c r e a s e t h e copy number o f t h e s e g -ment and hence t h e p r o b a b i l i t y t h a t r e c o m b i n a t i o n o f m a r k e r s i n t h a t s e g -ment would be o b s e r v e d . E l e c t r o n m i c r o s c o p i c a n a l y s i s of p a r t i a l l y r e p l i -c a t e d , U V - i r r a d i a t e d T7 DNA showed t h a t m u l t i p l e i n i t i a t i o n a l o n g a chromo-some was p o s s i b l e , however r e i n i t i a t i o n w i t h i n a p a r t i a l l y r e p l i c a t e d segment was n e v e r o b s e r v e d ( F i g u r e 1 8 ) . A n a l y s i s of p a r t i a l l y r e p l i c a t e d , non-i r r a d i a t e d T7 chromosomes r e v e a l e d t h a t r e i n i t i a t i o n c o u l d o c c u r b u t d i d so r a r e l y ( 8 4 ) . The a n a l y s i s c o u l d n o t e x c l u d e t h e p o s s i b i l i t y t h a t p a r t i a l l y r e p l i c a t e d p i e c e s do n o t r e m a i n a s s o c i a t e d w i t h t h e chromosome b u t a r e shunted i n t o t h e i n t r a c e l l u l a r r e p l i c a t i v e and r e c o m b i n i n g DNA p o o l . A t t h e same t i m e , the c l o s e f i t o f t h e summed p a r t i a l l y r e p l i c a t e d r e g i o n s d e t e r m i n e d by EM w i t h t h e marker r e s c u e c u r v e a t a s i m i l a r UV dose ( F i g u r e 19) i m p l i e s t h a t gene dose may a c c o u n t f o r o b s e r v e d marker r e s c u e f r e q u e n -c i e s w i t h o u t the n e c e s s i t y of p o s t u l a t i n g ' e x t r a c o p i e s ' of p a r t i a l l y r e p -l i c a t e d r e g i o n s . A l t h o u g h t h e r e s c u e of a marker i s c o r r e l a t e d w i t h t h e e f f i c i e n c y o f r e p l i c a t i o n of t h e segment of DNA c o n t a i n i n g t h a t m a r k e r , i t i s n o t p o s s i b l e t o say from t h e r e s u l t s p r e s e n t e d h e r e whether r e p l i c a t i o n of a segment i s r e q u i r e d a b s o l u t e l y b e f o r e a marker i n t h e segment can be r e s c u e d a t a l l . The r e s u l t s show t h a t the e f f i c i e n c y o f r e s c u e i s c o r r e -l a t e d w i t h the e f f i c i e n c y o f r e p l i c a t i o n b u t i t i s d i f f i c u l t t o d e t e r m i n e whether some r e s c u e o c c u r s i n t h e complete absence of r e p l i c a t i o n . The d i f f i c u l t y a r i s e s because of the low background l e v e l o f T 7 + i n f e c t i v e c e n t e r s p r oduced by U V - i r r a d i a t e d phage a l o n e and because UV i r r a d i a t i o n does n o t i n h i b i t r e p l i c a t i o n c o m p l e t e l y . F u t h e r . e l u c i d a t i o n of whether r e p l i c a t i o n i s r e q u i r e d f o r r e c o m b i n a t i o n may be a c h i e v e d by marker r e s c u e e x p e r i m e n t s e m p l o y i n g T7 p l a s m i d s under v a r i o u s h o s t and phage c o n d i t i o n s w h i c h a l l o w m a n i p u l a t i o n of r e p l i c a t i o n , r e c o m b i n a t i o n , and gene copy number 105 BIBLIOGRAPHY 1. Anraku, N. and I. Lehman. 1969. Enzymatic j o i n i n g of polynucleotides VII. The r o l e of the T4-induced l i g a s e i n the formation of recom-binant molecules. Journal of Molecular Biology 46: 467-479. 2. Anraku, N. and J . Tomizawa. 1965. Molecular mechanisms of genetic recombination i n bacteriophage I I I . Joining of parental poly-nucleotides of phage T4 i n the presence of 5-fluorodeoxyuridine. Journal of Molecular Biology 11:501-508. 3. Anraku, N. and J . Tomizawa. 1965. Molecular mechanisms of genetic recombination of bacteriophage V. Two kinds of j o i n i n g of par-ental DNA molecules. Journal of Molecular Biology 12:805-815. 4. B a r r a c e l l i , N. 1956. A "chromosomic" recombination theory f o r m u l t i -p l i c i t y r e a c t i v a t i o n i n phages. Acta B i o t h e r o r e t i c a 11:107-120. 5. B a r r a c e l l i , N. 1960. Anp.analytical approach to the problems of phage recombination and reproduction I. M u l t i p l i c i t y r e a c t i v a t i o n and the nature of r a d i a t i o n damages. Virology 11:99-135. 6. B a r r a c e l l i , N. and A.H. Doermann. 1961. An a n a l y t i c a l approach to the problems of phage recombination arid reproduction I I I . Cross r e a c t i v a t i o n . Virology 13:460-476. 7. Benbasat, J . ; K. Bauman 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. 8. Boyle, J . and R.B. Setlow. 1970. Correlations between host c e l l r e a c t i v a t i o n , u l t r a v i o l e t r e a c t i v a t i o n , and pyrimidine dimer excision i n the DNA of bacteriophage A. Journal of Molecular  Biology 51:131-144. 9. Braun, A. and L. Grossman. 1974. An endonuclease from Escherichia c o l i that acts p r e f e r e n t i a l l y on UV-irradiated DNA and i s absent from the uvrA and uvrB mutants. Proceedings of the National  Academy of Sciences USA 71:1838-1842. 10. Broker, T.R. and A.H. Doermann. 1975. Molecular and genetic recom-bination of bacteriophage T4. Annual Review of Genetics 9^:213-244. 11. Broker, T.R. and I. Lehman. 1971. Branched DNA molecules: intermed-iates i n T4 recombination. Journal of Molecular Biology 60: 131-149. 12. Carlson, K. 1968. I n t r a c e l l u l a r fate of deoxyribonucleic acid from T7 bacteriophages. Journal of Virology 2:1230-1233. 13. Davis, R.W.; M. Simon; N. Davidson. 1971. Electron microscope hetero-duplex methods for mapping regions of base sequence homology i n nu c l e i c acids. i n L. Grossman and K. Moldare (Eds.) Methods i n  Enzymology, Academic Press, New York. v o l . XXI. pp.413-428. 106 14. Delius, H.; C. Howe; A. Kozinski. 1971. Structure of the r e p l i c a t i n g DNA from bacteriophage T4. Proceedings of the National Academy  of Sciences, USA 68:3049-3053. 15. Denhardt, D.T. 1966. A membrane f i l t e r technique f o r the detection of complementary DNA. Biochemical and Biophysical Research Comm  linications 23:641-646. 16. Doermann, A.H. 1961. The analysis of u l t r a v i o l e t l e s i o n s i n b a c t e r i o -phage T4 by cross r e a c t i v a t i o n . Journal of C e l l u l a r and Comparative  Physiology 58f(suppl. 1): 79-9 3. 17. Doermann, A.H.; M. Chase; F. Stahl. 1955. Genetic recombination and r e p l i c a t i o n i n bacteriophage. Journal of C e l l u l a r and Comparative  Physiology 45 (suppl):51-74. 18. Dressier, D.; J . Wolfson; M. Magazin. 1972. I n i t i a t i o n and r e i n i t i a t i o n of DNA synthesis during r e p l i c a t i o n of bacteriophage T7. Proceedings  of the National Academy of Sciences, USA 69:998-1002. 19. Dulbecco, R. 1949. Reactivation of u l t r a v i o l e t l i g h t i n a c t i v a t e d bacteriophage by v i s a b l e l i g h t . Nature 163:949-950. 20. Dulbecco, R.J. 1950. Experiments on photoreactivation of bacteriophages inact i v a t e d with u l t r a v i o l e t r a d i a t i o n . Journal of Bacteriology 59.: 329-347. 21. Dulbecco, R. 1952. A c r i t i c a l test of the recombination theory of m u l t i p l i c i t y r e a c t i v a t i o n . Journal of Bacteriology 63:199-207. 22. Dulbecco, R. 1952. Experiments on photoreactivation of i n a c t i v e bacteriophages. Journal of C e l l u l a r and Comparative Physiology 39 (suppl. 1):125-128. 23. Dulbecco, R. 1955. Photoreactivation. i n A. Hollaender (Ed.) Radiation Biology, McGraw-Hill, New York, vol.11. PP.455-486. 24. E l l i s o n , S.; R. Feiner; R.F. H i l l . 1960. A host e f f e c t on b a c t e r i o -phage s u r v i v a l a f t e r u l t r a v i o l e t i r r a d i a t i o n . Virology 11:294-296. 25. Epstein, R.H. 1958. A study of m u l t i p l i c i t y - r e a c t i v a t i o n i n b a c t e r i o -phage T4 I. Genetic and functional analysis of T4D-K12(X) complexes. Virology 6_: 382-404. 26. Epstein, R.H.; A. B o l l e ; C. Steinberg; E. Kellenberger; E. Boy de l a Tour; R. Chevalley; R. Edgar; M. Susman; G. Denhardt; A. L i e l a u s i s . 1963. Ph y s i o l o g i c a l studies of co n d i t i o n a l l e t h a l mutants of bacteriophage T4. Cold Spring Harbor Symposium on Quantitative Biology 28: 375-392. 27. French, R. ; S. Lesley; A.Graham'; C. van Rooyen. 1951. Studies on the r e l a t i o n s h i p between vi r u s and host c e l l I I I . The breakdown of 32-P-lebeled T2r+ bacteriophage adsorbed to E . c o l i previously infected by other coliphages of the T group. Canadian Journal of Medical Science 29:144-148. 28. French, R.; A.Graham; S. Lesley; C. van Rooyen. 1952. The contribu-tion of phosphorus from T2r+ bacteriophage to progeny. Journal of Bacteriology 64:597-607. 29. Ganesan, A.K. 1974. Persistence of pyrimidine dimers during post-r e p l i c a t i o n repair i n u l t r a v i o l e t l i g h t - i r r a d i a t e d Escherichia  c o l i K12. Journal of Molecular Biology 87:103-119. 30. Garen, A. and N. Zinder. 1955. R a d i o l o g i c a l evidence f o r p a r t i a l genetic homology between bacteriophage and host b a c t e r i a . Virology 1:347-376. 31. Gomez, B. and D. Lang. 1972. Denaturation map of bacteriophage T7 DNA and d i r e c t i o n of DNA t r a n s c r i p t i o n . Journal of Molecular Biology 70:239-251. 32. Hamlett, N. and H. Berger. 1975. Mutations a l t e r i n g genetic recom-bination and repair of DNA i n bacteriophage T4. Virology 63: 539-567. 33. Harm, W. 1963. Repair of l e t h a l u l t r a v i o l e t damage i n phage DNA. i n Sobels: Repair from Genetic Radiation, Permagon Press, New York pp.107-124. 34. Harm, W. 1965. Suppression of phage T4 by c o - i n f e c t i o n with UV-in a c t i v a t e d homologous phage. Photochemistry and Photobiology 4^:569-573. 35. Hausmann, R. 1976. Bacteriophage T7 Genetics, i n Current Topics i n Microbiology and Immunology 75:77-110. 36. Hays, J . and S. Boehmer. 1978. Antagonists of DNA gyrase i n h i b i t repair and recombination of UV-irradiated phage A. Proceedings  of the National Academy of Sciences USA 7 5 _ : 4 1 2 5 ~ 4 1 2 9 . 37. Hershey, A.D. and M. Chase. 1952. Independent functions of v i r a l i n f e c t i o n and nucleic acid i n growth of bacteriophage. Journal  of General Physiology 36:39-56. 38. Howard-Flanders, P. 1973. DNA repair and recombination. B r i t i s h Medical B u l l e t i n 29:226-235. 39. Howard-Elanders, P.; R.P. Boyce; L. Theriot, 1966. Three l o c i i n Escherichia c o l i that control the excision of pyrimidine dimers and c e r t a i n other mutagen products from DNA. Genetics 53:1119-1136. 40. Howe, C.; P. Buckley; K. Carlson; A. Kozinski. 1973. Mu l t i p l e and s p e c i f i c i n i t i a t i o n of T4 DNA r e p l i c a t i o n . Journal of Virology 12:130-148. 41. Kerr, C. and P. Sadowski. 19 75. The involvement of genes 3,4,5, and 6 i n genetic recombination i n bacteriophage T7. Virology 65:281-285. 42. Kozinski, A. and P. Kozinski. 1964. R e p l i c a t i v e fragmentation i n T4 bacteriophage DNA I I . Biparental molecular recombination. Proceed- ings of the National Academy of Sciences USA 52:211-218. 107 108 43. Kozinski, A.W. and W. Szybalski. 1959. Dispersive transfer of the parental DNA molecule to the progeny of phage t()X174. Virology £:260-274. 44. Kuemmerle, N.B. and W. Masker. 1977. In v i t r o packaging of UV r a d i a t i o n damaged DNA from bacteriophage T7. Journal of Virology 23:509-516. 45. Langman, L.; V. Paetkau; D. Scraba; R.C. M i l l e r , J r . ; G.S. Roeder; P.D. Sadowski. 1978. The structure and maturation of intermediates i n bacteriophage T7 DNA r e p l i c a t i o n . Canadian Journal of Biochemistry 56:508-516. 46. Lee, M. 1976. The e s s e n t i a l roles of the T7 endonuclease (gene 3) and the T7 exonuclease (gene 6) i n recombination of bacteriophage DNA. PhD Thesis, The Uni v e r s i t y of B r i t i s h Columbia. 47. Lee, M. and R.C. M i l l e r , J r . 1974. T7 exonuclease (gene 6) i s necessary for molecular recombination of bacteriophage T7. Journal of  Virology 14:1040-1048. 48. Lee, M.; R.C. M i l l e r , J r . ; D. Scraba; V. Paetkau. 1976. The e s s e n t i a l r o l e of bacteriophage T7 endonuclease (gene 3) i n molecular recombination. Journal of Molecular Biology 104:883-888. 49. L u r i a , S.E. 1947. Reactivation of i r r a d i a t e d bacteriophage by transfer of s e l f reproducing u n i t s . Proceedings of the National Academy of  Sciences USA 33:253-264. 50. L u r i a , S.E. 1952. Reactivation of u l t r a v i o l e t - i r r a d i a t e d bacteriophage by multiple i n f e c t i o n . Journal of C e l l u l a r and Comparative Physio- logy 39(suppl 1):119-123. 51. L u r i a , S.E. and R. Dulbecco. 1949. Genetic recombinations leading to production of active bacteriophage from u l t r a v i o l e t i n a c t i v a t e d bacteriophage p a r t i c l e s . Genetics 34:93-125. 52. McDonnel, M.; M. Simon; F.W. Studier. 1977. Analysis of r e s t r i c t i o n fragments of T7 DNA and determination of molecular weights by electrophoresis i n neutral and a l k a l i n e gels. Journal of Molecular  Biology 110:119-146. 53. McMilin, K. and V. Russo. 1972. Maturation and recombination of bacteriophage X DNA molecules i n the absence of DNA d u p l i c a t i o n . Journal of Molecular Biology 68:49-55. 54. M i l l e r , R.C, J r . 1968. Parental to progeny molecular recombination with bacteriophage T7. Journal of Virology 2_: 157-159. 55. M i l l e r , R.C. J r . 1975. Replication and molecular recombination of T-phage. Annual Review of Microbiology 29:355-376. 56. M i l l e r , R.C. J r . ; M. Lee; D.G. Scraba; V. Paetkau. 1976. The ro l e of bacteriophage T7 exonuclease (gene 6) i n genetic recombination and production of concatemers. Journal of Molecular Biology 101:223-234. 109 57. Mosig, G. 1970. A preferred o r i g i n and d i r e c t i o n of bacteriophage T4 DNA r e p l i c a t i o n I. A gradient of a l l e l e frequencies i n crosses between normal and small T4 p a r t i c l e s . Journal of Molecular  Biology .53:503-514. 58. Pao, C.C. and J.F. Speyer. 1973. Order of i n j e c t i o n of T7 b a c t e r i o -phage DNA. Journal of Virology 11:1024-1026. 59. Powling, A. and R. Knippers. 1974. Some functions involved i n b a c t e r i o -phage T7 genetic recombination. Molecular and General Genetics 134:173-180. 60. Rayssiguier, C. and P. V i g i e r . 19 72. On the repair mechanism respon-s i b l e f o r m u l t i p l i c i t y r e a c t i v a t i o n i n bacteriophage T4. Molecular and General Genetics 115:140-145. 61. Rayssiguier, C. and P. V i g i e r . 1977. Genetic evidence f o r the existence of p a r t i a l r e p l i c a s of T4 genomes in a c t i v a t e d by i r r a d i a t i o n under u l t r a v i o l e t l i g h t . Virology 78:442-452. 62. Rupp, W. and P. Howard-Flanders. 1968. D i s c o n t i n u i t i e s i n the DNA synthesized i n an excision-defective s t r a i n of Esche r i c h i a c o l i following u l t r a v i o l e t i r r a d i a t i o n . Journal of Molecular Biology 61:291-304. 63. Rupp, W.; C. Wilde I I I ; D. Reno; P. Howard-Flanders. 1971. Exchanges between DNA strands i n u l t r a v i o l e t - i r r a d i a t e d Escherichia c o l i . Journal of Molecular Biology 61:25-44. 64. Schlegel, R.A. and CA. Thomas, J r . 1972. Some s p e c i a l s t r u c t u r a l features of i n t r a c e l l u l a r bacteriophage T7 concatemers. Journal  of Molecular Biology 68:319-345. 65. Seeberg, E.; J . Nissen-Meyer; P. S t r i k e . 1976. Inc i s i o n of u l t r a -v i o l e t - i r r a d i a t e d DNA by extracts of E . c o l i requires three d i f f e r e n t gene products. Nature 263:524-525. 66. Setlow, J.K. 1966. The molecular basis of b i o l o g i c a l e f f e c t s of u l t r a -v i o l e t r a d i a t i o n and photoreactivation. i n M. Ebert and A. Howard (Eds.) Current Topics i n Radiation Research, North Holland Publish-ing Co., Amsterdam, v o l 2. pp.195-248. 67. Setlow, R.B. 1964. Physical changes and mutagenesis. Journal of C e l l u l a r and Comparative Physiology 64 (suppl 1):51-68. 68. Setlow, R.B. 1968. The photochemistry, photobiology, and repair of polynucleotides. Progress i n Nucleic Acids Research and Molecular  Biology 8.:257-295. 69. Setlow, R.B. and W.L. C a r r i e r . 1966. Pyrimidine dimers i n u l t r a v i o l e t i r r a d i a t e d DNA. Journal of Molecular Biology 17:237-254. 70. Southern, E.M. 1975. Detection of s p e c i f i c sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98: 503-517. 110 71. Stahl, F.; K. McMilin; M. Stahl; R. Malone; Y. Nozu; V. Russo. 1972. A r o l e for recombination i n the production of "free loader" lambda bacteriophage p a r t i c l e s . Journal of Molecular Biology 68:57-67. 72. Stahl, F.; K. McMilin; M. Stahl; Y. Nozu. 1972. An enhancing role for DNA synthesis i n formation of bacteriophage A recombinants. Proceedings of the National Academy of Sciences, USA 69:3598-3601. 73. Stahl, F. and M. Stahl. 1971. DNA synthesis associated with recombin-ation I I . Recombination between repressed chromosomes. i n A.D. Hershey (Ed.) The Bacteriophage Lambda, Cold Spring Harbor Labor-atory, Cold Spring Harbor, New York. pp. 443-453. 74. Stahl, M. and F. Stahl. 1971. DNA synthesis associated with recom-bination I. Recombination i n a DNA-negative host. i n A.D. Hershey (Ed.) The Bacteriophage Lambda, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. pp.431-442. 75. Studier, F.W. 1969. The genetics and physiology of bacteriophage T7. Virology 39:562-574. 76. Studier, F.W. 1972. Bacteriophage T7. Science 176:367-376. 77. Studier, F.W. 1973. Genetic analysis of non-essential bacteriophage T7 genes. Journal of Molecular Biology 79:227-236. 78. Studier, F.W. 1975. Gene 0.3 of bacteriophage T7 acts to overcome the DNA r e s t r i c t i o n system of the host. Journal of Molecular Biology 94^:283-295. 79. Symonds, N.; H. Heindi; P. White. 1973. Radiation s e n s i t i v e mutants of phage T4. A comparative study.-, Molecular and General Genetics 120:253-259. 80. Tomizawa, J . 1967. Molecular mechanisms of genetic recombination i n bacteriophage: J o i n t molecules and t h e i r conversion to recom-binant molecules. Journal of C e l l u l a r Physiology 70 i(suppl 1): 201-213. 81. Tomizawa, J . and N. Anraku. 1964. Molecular mechanisms of genetic recombination i n bacteriophage I I . Joining of parental DNA molecules of phage T4. Journal of Molecular Biology _8:516-540. 82. Tsujimoto, Y. and H. Ogawa. 19 77. Intermediates i n genetic recom-bination of bacteriophage T7 DNA. Journal of Molecular Biology 109:423-436. 83. Watson, J.D. 1972. Or i g i n of concatemeric T7 DNA. Nature New Biology 239:197-201. 84. Wolfson, J . ; D. Dressier; M. Magazin. 1972. Bacteriophage T7 DNA r e p l i c a t i o n : A l i n e a r r e p l i c a t i n g intermediate. Proceedings of the National Academy of Sciences, USA 69:499-504 . 85. Womack, F.C. 1965. Cross r e a c t i v a t i o n differences i n bacteriophage T4D. Virology 26_: 758-761. 

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