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DNA synthesis and modification in ØW-14-infected Pseudomonas acidovorans Maltman, Kirk Lee 1981

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. DNA SYNTHESIS AND MODIFICATION IN 0W-14-INFECTED PSEUDOMONAS ACIDOVORANS by v KIRK LEE MALTMAN B.Sc. U n i v e r s i t y of Calgary, 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA /'. J u l y , 1981 (c) 1981,Kirk Lee Maltrnan In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M<cgtrntoinG-y The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date. ABSTRACT Experiments w i t h 0W-14-infected, t h y m i d i n e - r e q u i r i n g mutants of P_. acidovorans s t r a i n 29 demonstrated that deoxyuridine but not thymidine was a precursor of thymine i n 0W-14 DNA. Deoxyuridine was a l s o a precursor of the a-putrescinylthymine found i n 0W-14 DNA. The b i o s y n t h e s i s of a-putrescinylthymine and thymine was mediated by enzyme a c t i v i t i e s appearing a f t e r i n f e c t i o n . 0W-14 DNA s y n t h e s i s and DNA m o d i f i c a t i o n was r e s i s t a n t to the a n t i b i o t i c s trimethoprim and 5-f l u o r o d e o x y u r i d i n e . This i n d i c a t e d that endogenous thymidine b i o s y n -t h e s i s was u n l i k e that observed i n the uninfected host or i n other b i o l o g i c a l systems. These observations helped demonstrate that hydroxy-m e t h y l u r a c i l - c o n t a i n i n g n u c l e o t i d e s were precursors of thymine and a-putrescinylthymine-containing n u c l e o t i d e s (Neuhard et a l . , 1980). The absence of a - p u t r e s c i n y l thymine and thymine n u c l e o t i d e s i n 0W-14-i n f e c t e d c e l l n u c l e o t i d e pools suggested that these n u c l e o t i d e s might be synthesized from hydroxymethyluracil at the p o l y n u c l e o t i d e l e v e l . Degradative a n a l y s i s of nascent 0W-14 DNA demonstrated the presence of hydroxymethyluracil. Enzymatic degradation of p u l s e - l a b e l l e d , nascent 0W-14 DNA followed by TLC suggested the presence of three or more novel n u c l e o t i d e s not found i n uniformly l a b e l l e d DNA samples. These obser-v a t i o n s were c o n s i s t e n t w i t h n e u t r a l CsCl a n a l y s i s of p u l s e - l a b e l l e d 0W-14 DNA. This DNA contained unusual heavy d e n s i t y components. 0W-14 ts and amber mutants were screened f o r defects i n DNA r e p l i c a t i o n or DNA m o d i f i c a t i o n by CsCl gradient and/or degradative a n a l y s i s . Some DO mutants were i d e n t i f i e d . In a d d i t i o n , two DNA mod-i f i c a t i o n mutants were found. Am 42 made 0W-14 DNA c o n t a i n i n g lower-than-normal l e v e l s of a-putrescinylthymine and increased l e v e l s of thymine. Am 37 accumulated intermediates i n a-putrescinylthymine b i o -s y n t h e s i s . The c o n d i t i o n a l l y l e t h a l nature of the DNA m o d i f i c a t i o n l e s i o n was demonstrated. DNA sy n t h e s i s was adversely a f f e c t e d by t h i s mutation but DNA precursor s u p p l i e s were not impaired. Two a t y p i c a l mononucleotides were p u r i f i e d from am 37 DNA. One was i d e n t i f i e d as hydroxymethyldeoxyuridylate. The second nucleo-t i d e was an a c i d - l a b i l e d e r i v a t i v e of hydroxymethyldeoxyuridylate. 3 32 A n a l y s i s of [6- H ] - u r a c i l and PO^ l a b e l l i n g r a t i o s , chemical and enzymatic degradation and chromatographic a n a l y s i s of t h i s n u c l e o t i d e demonstrated that i t was the novel compound 5-(hydroxymethyl-0-pyro-phosphoryl)-deoxyuridylate (abbreviated to hmPPdUMP). 5-(hydroxymethyl-O-pyrophosphoryl)-uracil was shown to be a precursor of a-putrescinylthymine by i n v i t r o m o d i f i c a t i o n of am 37 DNA w i t h 0W-14 w i l d - t y p e i n f e c t e d P_. acidovorans c e l l - f r e e e x t r a c t s . In v i t r o m o d i f i c a t i o n confirmed that a-putrescinylthymine was formed at the p o l y n u c l e o t i d e l e v e l . 0W-14 DNA m o d i f i c a t i o n was not necessar-i l y coupled to r e p l i c a t i o n . The presence of hydroxymethyluracil i n am 37 DNA agreed w i t h the suggestion that hmPPura was formed by pyrophos-p h o r y l a t i o n of hydroxymethyluracil i n nascent DNA. HmPPdUMP had chro-matographic p r o p e r t i e s s i m i l a r to one of the compounds detected i n p u l s e - l a b e l l e d 0W-14 wi l d - t y p e DNA. iv TABLE OF CONTENTS Page INTRODUCTION 1 I (a) T-even bacteriophage 9 (b) T-even DNA glucosyl a t i o n 12 II B a c i l l u s s u b t i l i s bacteriophages 15 (a) 0e 15 (b) PBS 18 (c) SP 15 2 1 (d) SP 10 2 3 III D i n o f l a g e l l a t e DNAs 2 4 MATERIALS AND METHODS 2 7 I Organisms 2 7 II Media 2 7 I I I Buffers • • 2 9 IV Growth of Bacter i a 2 9 V Bacteriophage 2 9 (a) Preparation of h i g h - t i t r e phage lysates 30 (b) Phage t i t r a t i o n 31 VI Conditioned medium . 31 VII Testing for a n t i b i o t i c s e n s i t i v i t y 31 . V Page V I I I Phage mutagenesis 32 IX I s o l a t i o n of 0W-14 _ts mutants 32 X Complementation of 0W-14 ts_ mutants 32 XI Screening _ts_ and am mutants 32 XII DNA p u r i f i c a t i o n 33 X I I I A c i d - h y d r o l y s i s of DNA samples 33 XIV TLC of acid-hydrolyzed DNA samples 34 XV P u l s e - l a b e l l i n g procedures 34 XVI Nucleotide pool p r e p a r a t i o n 35 XVII Chromatography of n u c l e o t i d e pool preparations on P E I - c e l l u l o s e 36 XVIII CsCl d e n s i t y gradient a n a l y s i s of DNA 36 XIX S o n i c a t i o n of DNA 37 XX Heat treatment of DNA 37 XXI Measurement of DNA accumulation 37 (a) [ 6 - 3 H ] - u r a c i l 37 (b) [ 3H] - o r n i t h i n e 37 (c) [methyl- H]-thymidine 38 3 XXII Measurement of t r i t i u m r e l e a s e from [5- H ] - u r a c i l . . 38 XXIII Enzymatic d i g e s t i o n of DNA 38 (a) Sl-snake venom phosphodiesterase (SVPD) . . . . 38 (b) DNase I-SVPD 39 v i Page XXIV (a) Thin l a y e r chromatography of enzymatic DNA d i g e s t s 39 (b) Solvents f o r TLC on unmodified c e l l u l o s e . . . 39 (c) D e t e c t i o n of r a d i o a c t i v i t y on t h i n - l a y e r sheets 40 ( i ) Fluorography 40 ( i i ) Autoradiography 40 XXV B a c t e r i a l a l k a l i n e phosphatase (BAP) d i g e s t i o n . . . 40 XXVI Column chromatography 40 (a) B i o g e l P 2 40 (b) DEAE-Sephadex 40 (c) DEAE-Sephadex-7 M urea 41 XXVII P u r i f i c a t i o n of the unknown n u c l e o t i d e 41 XXVIII P r e p a r a t i o n of c e l l - f r e e e x t r a c t s 42 ) (a) I n v i t r o s y n t h e s i s of a-putrescinylthymine (putThy) 42 XXIX Chemicals 43 XXX Enzymes 43 XXXI Radiochemicals 43 RESULTS AND DISCUSSION 44 I Nucleotide poo Is 44 I I Deoxyuridine but not deoxythymidine was a 0W-14 DNA precursor 50 v i i Page (a) Deoxyuridine i n c o r p o r a t i o n 51 (b) Deoxythymidine i n c o r p o r a t i o n 59 (c) BUdR does not d e n s i t y l a b e l 0W-14 DNA . . . . 66 I I I Endogenous thymidine u t i l i z a t i o n i n 0W-14-infected P_. acidovorans 3L 66 (a) Resistance to FUdR 66 3 ^IV [5- H ] - u r a c i l r e l e a s e i n 0W-14-i n f e c t e d P_. acidovorans 78 V Trimethoprim and trimethoprim-sulfonamide e f f e c t s upon 0W-14 reproduction 82 3 VI T r i t i u m i s not l o s t from [6- H ] - u r a c i l during 0W-14 DNA syn t h e s i s 9 3 VII R e p l i c a t i n g DNA . 9 4 (a) T h i n - l a y e r chromatography of DNA components . 107 ( i ) Bases 1 1 2 ( i i ) N ucleotides 115 (b) P u l s e - l a b e l l e d DNA i n 0W-14-i n f e c t e d P_. acidovorans 119 3 ( i ) [6- H ] - u r a c i l bases 122 32 ( i i ) P - l a b e l l e d n u c l e o t i d e s 123 ( i i i ) CsCl gradients 124 ( i v ) T h i n - l a y e r chromatography of DNA components 130 (c) Tests f o r i n h i b i t o r s of DNA synt h e s i s and m o d i f i c a t i o n 1^0 v i i i Page (d) Screening f o r 0W-14 _ts and am mutants 148 (e) 0W-14 _ts_ 19a i n f e c t i o n of P_. acidovorans . . . 163 (f ) Am 42-infected P_. acidovorans accumulated DNA w i t h a d e n s i t y greater than that of w i l d - t y p e phage reference DNA 170 V I I I 0W-14 am 37 177 (a) The c o n d i t i o n a l l y l e t h a l nature of the DNA m o d i f i c a t i o n l e s i o n 177 (b) Am 37 DNA contains a novel n u c l e o t i d e 181 (c) DNA synt h e s i s i n 0W-14 am 37-infected c e l l s . . 200 3 (d) T r i t i u m r e l e a s e from [5- H ] - u r a c i l i n am 37 - i n f e c t e d 3L 200 (e) The s t a b i l i t y of am 37 DNA 203 (f) CsCl gradient a n a l y s i s of am 37 DNA 206 (g) P a r e n t a l l y l a b e l l e d am 37 DNA 210 (h) P u r i f i c a t i o n of the novel n u c l e o t i d e found i n am 37 DNA 210 ( i ) I d e n t i f i c a t i o n of the s t r u c t u r e of the novel n u c l e o t i d e 219 ( j ) Am 37 i n v i t r o DNA m o d i f i c a t i o n 230 LITERATURE CITED 240 i x LIST OF TABLES Table Page 1. The p r o p e r t i e s of phage DNAs c o n t a i n i n g modified bases ... . ............. 2 2. B a c t e r i a l s t r a i n s used i n t h i s study 28 3. The deoxynucleoside triphosphate pools of 0W-14-infected P_. acidovorans s t r a i n 29 49 4. Deoxyuridine l a b e l l i n g of bases i n 0W-14-i n f e c t e d 3L/FUR_2 56 5. The e f f e c t of trimethoprim or FUdR upon the buoyant d e n s i t y of 0W-14 DNA synthesized i n P_. acidovorans 75 6. The base composition of 0W-14 DNA prepared i n the presence of trimethoprim or FUdR 76 7. 0W-14 DNA m o d i f i c a t i o n i s independent of the host s t r a i n 77 3 8. T r i t i u m i s not l o s t from [6- H ] - u r a c i l during putThy or Thy b i o s y n t h e s i s . . . . . 95 9. The f a t e of putThy and Thy i n p a r e n t a l l y l a b e l l e d 0W-14 DNA 101 10. The base composition of P_. acidovorans s t r a i n 29 DNA 114 11. The n u c l e o t i d e composition of p u l s e -l a b e l l e d 0W-14 DNA 116 12. The base composition of r e p l i c a t i n g 0W-14 DNA . . . . . . 121 13. The r e l a t i v e chemical l a b i l i t y of p u l s e -l a b e l l e d 0W-14 DNA 128 X Table ' -Page 14. The base composition of 0W-14 DNA made i n ne t r o p s i n - t r e a t e d c e l l s 151 15. Complementation a n a l y s i s and screening of 0W-14 _ts mutants 153 16. The p r o p e r t i e s of DNA ex t r a c t e d from 0W-14 am-infected P_. acidovorans 160 17. The base composition of DNA made i n 0W-14 ts 19a-inf ected _P. acidovorans . 173 18. The n u c l e o t i d e composition of 0W-14 am 42 DNA 176 19. The p l a t i n g e f f i c i e n c y of 0W-14 am 37 on P_. acidovorans s t r a i n s 179 20. Base compositions of 0W-14 and am 37 DNAs 180 21. Nucleotide composition of 0W-14 and am 37 DNAs 190 22. S t a b i l i t y of 0W-14 am 37 DNA 199 23. The n u c l e o t i d e composition of 0W-14 am 37 DNA prepared at va r i o u s times a f t e r the i n f e c t i o n of P_. acidovorans s t r a i n 29 209 24. A c i d h y d r o l y s i s of n u c l e o t i d e s p u r i f i e d from 0W-14 am 37 DNA 220 25. P r o p e r t i e s of the unknown n u c l e o t i d e . . . . . . . . . . 221 26. Conversion of the unknown n u c l e o t i d e to i t s nucleoside 222 x i LIST OF FIGURES Figure Page 1. The s t r u c t u r e of modified bases found i n bacteriophage DNAs 5 14 2. The r e s o l u t i o n of [2- C ] - u r a c i l - l a b e l l e d n u c l e o t i d e s i n uninfected and i n f e c t e d c e l l s 45 3. The i n c o r p o r a t i o n of u r a c i l i n 3L/FU _2 52 4. The i n c o r p o r a t i o n of deoxyuridine i n 0W-14-i n f e c t e d 3L/FUR_2 54 5. The accumulation of Thy and putThy i n 0W-14 DNA . . . 57 6. Thymidine i n c o r p o r a t i o n i n s t r a i n 3L and i n 0W-14-infected s t r a i n 3L 60 7. Thymidine was not re q u i r e d f o r phage production i n 0W-14-infected s t r a i n 3L 62 8. N o n s p e c i f i c l a b e l l i n g of 0W-14 DNA by high s p e c i f i c a c t i v i t y thymidine 64 9. BUdR has no e f f e c t upon the buoyant de n s i t y of 0W-14 DNA synthesized i n i t s presence 67 10. The e f f e c t of trimethoprim and FUdR upon phage production i n 0W-14-infected s t r a i n 3L 69 11. The e f f e c t of f l u o r o d e o x y u r i d i n e upon DNA synthesis i n 0W-14-infected P_. acidovorans 3L 71 12. The accumulation of 0W-14 DNA i n the presence of FUdR 73 x i i Figure Page 13. DNA synthesis and tritium release in 0W-14-infected P_. acidovorans 79 14. The effects of trimethoprim or trimethoprim and sulfonamide upon the growth of P_. acidovorans strains 29 and 3L 83 15. The effect of trimethoprim upon 0W-14 DNA synthesis . 89 16. The effect of trimethoprim and sulfonamide upon 0W-14 DNA synthesis 91 17. Two-dimensional thin-layer chromatography of the pyrimidine products released from 0W-14 DNA by acid hydrolysis 96 32 18. CsCl buoyant density gradient analysis of P-labelled 0W-14 parental DNA 98 19. The effect of heat treatment upon the buoyant density profile of 0W-14 intracellular or virion DNA 102 20. CsCl buoyant density analysis of 0W-14 virion and P_. acidovorans DNA 105 21. The uniform distribution of putThy in 0W-14 DNA . . . 108 22. The effect of heat treatment and shearing upon the distribution of label in 0W-14 DNA extracted from infected cells 110 23. Two-dimensional thin-layer chromatography of acid-hydrolyzed, [2-14c]-uracil-labelled P. acidovorans DNA 113 x i i i F igure Page 32 24. Two-dimensional t h i n - l a y e r chromatography of P-l a b e l l e d 0W-14 DNA digested w i t h nuclease SI and snake venom phosphodiesterase (SVPD) 118 32 25. Two-dimensional t h i n - l a y e r chromatography of P-l a b e l l e d n u c l e o t i d e s present i n 0W-14 DNA digested w i t h DNase I and SVPD 120 32 26. CsC l buoyant d e n s i t y a n a l y s i s of P p u l s e -l a b e l l e d 0W-14 DNA 125 27. The enzymatic degradation of p u l s e - l a b e l l e d 0W-14 DNA 131 32 28. Two-dimensional t h i n - l a y e r chromatography of P p u l s e - l a b e l l e d 0W-14 DNA d i g e s t s 133 29. The e f f e c t of chloramphenicol upon the i n c o r p o r a t i o n of u r a c i l i n t o 0W-14 DNA 142 30. The e f f e c t of n e t r o p s i n upon the growth of P_. acidovorans s t r a i n 29 144 31. Phage production i n n e t r o p s i n t r e a t e d P_. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14 146 32. CsCl buoyant d e n s i t y gradient a n a l y s i s of 0W-14 DNA e x t r a c t e d from P_. acidovorans s t r a i n 29 c e l l s t r e a t e d w i t h n e t r o p s i n 149 33. DNA synt h e s i s i n 0W-14-infected P_. acidovorans s t r a i n 29 at 20°C 155 34. CsCl buoyant.density gradients of DNA from P. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14 am mutants 158 x i v Figure Page 35. The i n c o r p o r a t i o n of [ H ] - o r n i t h i n e i n 0W-14 _ts_ 19a-inf ected J?. acidovorans s t r a i n 29 164 36. DNA s y n t h e s i s i n 0W-14 ts_ 19a-infected P_. acidovorans s t r a i n 3L 166 37. The buoyant d e n s i t y of DNA made i n 0W-14 ts 19a-infected P_. acidovorans s t r a i n 29 168 38. 0W-14 _ts_ 19a DNA s y n t h e s i s can be rescued by a temperature s h i f t from the nonpermissive to the permissive temperature 171 39. DNA s y n t h e s i s i n 0W-14 am 42-infected P. acidovorans 174 40. Two-dimensional t h i n - l a y e r chromatography of [ 6 - 3 H ] - u r a c i l - l a b e l l e d n u c l e o t i d e s present i n am 42 DNA prepared i n P_. acidovorans s t r a i n 29 178 41. The reproduction of 0W-14 am 37 i n P_. acidovorans s t r a i n 29 or sup 2 182 42. Buoyant d e n s i t y of DNA synthesized by phage-i n f e c t e d c e l l s 184 43. Nucleotides i n am 37 DNA 187 44. Two-dimensional t h i n - l a y e r chromatography of [8--^C]-adenine-labelled n u c l e o t i d e s present i n Sl-SVPD d i g e s t s of am 37 DNA prepared i n ]?. acidovorans s t r a i n 29 194 X V Figure Page 45. Two-dimensional thin-layer chromatography of [8-14c]-adenine-labelled nucleotides present i n DNase I-SVPD digests of am 37 prepared in P_. acidovorans strain 29 195 46. Two-dimensional thin-layer chromatography of 32p-]_abelled nucleotides present in DNase I-SVPD digests of am 37 DNA prepared in P_. acidovorans strain 29 197 47. DNA synthesis by phage-infected cells 201 48. DNA synthesis and tritium release in infected cultures 204 49. CsCl buoyant density analysis of 0W-14 am 37 DNA prepared at various times after infection of P_. acidovorans strain 29 207 50. CsCl buoyant density analysis of parentally labelled 0W-14 am 37 DNA 211 51. The purification of the novel nucleotide by paper chromatography 213 52. Two-dimensional thin-layer chromatography of the purified novel nucleotide 215 53. DEAE-Sephadex column chromatography of the novel nucleotide 217 54. Alkaline phosphatase treatment of the novel nucleotide 223 x v i Figure Page 55. Proposed s t r u c t u r e of the novel pyrimidine base . . . . 227 56. DEAE-Sephadex-urea column chromatography of a n u c l e o t i d e mixture from am 37 DNA 228 32 57. Release of r a d i o a c t i v i t y from P - l a b e l l e d am 37 DNA 231 58. Conversion of hmPPura i n am 37 DNA i n t o putThy . . . . 234 59. Summary of the pathways of 0W-14 DNA precursor sy n t h e s i s and m o d i f i c a t i o n 238 x v i i ACKNOWLEDGEMENTS I am especially indebted to Dr. R.A.J. Warren for his concern, advice, enthusiasm and friendship throughout my time in his laboratory. I also wish to thank Dr. R.C. Miller for his encouragement and advice. Special thanks to P. Miller and H. Lewis who provided excel-lent technical assistance, as well as moral support. I also wish to thank my wife, Jessyca, for her patience, understanding and support, as well as for her assistance in the preparation of this thesis. 1 INTRODUCTION 0W-14 i s a l y t i c bacteriophage of some Pseudomonas acidovorans s t r a i n s (Kropinski and Warren, 1970). The bacteriophage contains double-stranded DNA with a moi % G + C content of 51.4 (M. Mandel, per-sonal communication). The native v i r i o n DNA has a lower-than-expected buoyant density i n neutral CsCl density gradients, and a higher-than-expected melting temperature (Tm) (Kropinski et a l . , 1973). These aber-rant properties are due to a hypermodified pyrimidine which replaces approximately 50 percent of the t o t a l expected thymine f r a c t i o n i n the bacteriophage DNA. The base i s a C-5 modified pyrimidine, 5-(4-amino-butylaminomethyl)uracil, also c a l l e d a-putrescinylthymine or putThy. The aminomethyl group of the putThy molecule i s at the same oxidation l e v e l as the hydroxymethyl function of hydroxymethyluracil (hmUra). The structure of the acid-hydrolyzed base i s assumed to accurately r e f l e c t the structure of the putThy nucleotide. This assumption i s consistent with the ph y s i c a l and chemical properties of 0W-14 DNA. The p o s i t i v e charges c a r r i e d by the p u t r e s c i n y l function could t h e o r e t i c a l l y n e u t r a l -i z e one quarter of the t o t a l negative charges on the DNA molecule at ph y s i o l o g i c a l pH (Kropinski and Warren, 1973). These p o s i t i v e charges s t a b i l i z e the DNA h e l i x since 0W-14 DNA has a very high melting temper-ature (Tm = 99.3° i n lxSSC) (Table 1). The low buoyant density ( p ) of 0W-14 DNA i s also a r e f l e c t i o n of the presence of the p u t r e s c i n y l side chains. P o s i t i v e l y charged amines exclude cesium from the cesium-DNA 2 TABLE 1 . — P r o p e r t i e s of phage DNAs co n t a i n i n g modified bases Phage Host DNA molecular weight (x 10-6) Moles phage % G+C Host Base change T4 E s c h e r i c h i a c o l i 110 34 50 5-hmCyt f o r Cyt 0e B a c i l l u s s u b t i l i s 100 39 43 5-hmUra f o r Thy PBS2 B. s u b t i l i s 150 28 43 Ura f o r Thy yP12 yanthomonas oryzae 30 67 64 5-mCyt f o r Cyt S-2L Synechococcus elongatus 28 69 70 2-nAde f o r Ade SP15 B. s u b t i l i s 250 42 43 5-dhpUra f o r Thy SP10 B. s u b t i l i s 59 f 43 43 a-gluThy f o r Thy 0W-14 Pseudomonas acidovorans 92 5 1 e 67 a-putThy f o r Thy a The extent to which the modified base replaces the normal base. b Thermal t r a n s i t i o n temperatures are extrapolated to the value i n 0.15 M NaCl. c Buoyant den s i t i e s i n neutral CsCl, assuming a value of 1,710 g ml ^ for E_. c o l i DNA. d These are the values expected f o r a DNA of the same moles % G + C and of normal composition. e M. Mandel, personal communication. f Not reported, but may be the value given (K. Bott, personal communica-ti o n i n r e f . 6). SOURCE: R. A. J. Warren, 1980, Modified bases i n bacteriophage DNAs, Ann. Rev. Mi c r o b i o l . 34, pp. 137-56. 3 TABLE 1.—Continued Extent of change 3 (%) observed T b m expected Buoyant observed d e n s i t y expected 100 84 84 1.700 1.694 100 77.5 85.3 1.742 1.703 100 76.5 81.5 1.722 1.690 100 101.5 95.4 1.710 1.726 100 101.9 98.3 1.731 1.728 41 61.7 86.2 1.761 1.702 15-20 81.5 86.9 1.723 1.703 50 99.3 90.3 1.666 1.716 4 complex (p = 1.666 gcc "S . The low buoyant d e n s i t y of the 0W-14 DNA can a l s o be p a r t i a l l y a t t r i b u t e d to the presence of the four methylene f u n c t i o n s i n the amino-butyl group (Warren, 1980). The b i o s y n t h e t i c o r i g i n s of the s t r u c t u r a l components of the putThy base are known. U r a c i l i s a precursor of the p y r i m i d i n e r i n g p o r t i o n of the molecule ( K e l l n , Ph.D. Thesis, 1973). Serine l a b e l s putThy and Thy residues i n 0W-14 DNA. The l a b e l l i n g of t h i s methylene 14 3 f u n c t i o n w i t h [2- C]-serine or [2,3- H]-serine i s c o n s i s t e n t w i t h the t r a n s f e r of a one carbon fragment from N^-N^n m e t h y l e n e - t e t r a h y d r o f o l i c a c i d (THFA) (K a r r e r , M.Sc. Thesis, 1973) and w i t h thymine not being a precursor of putThy. O r n i t h i n e i s a precursor of the putThy s i d e chain, s i n c e o r n i t h i n e l a b e l s putThy residues i n 0W-14 DNA ( Q u a i l et a l . , 1976). P_. acidovorans i s impermeable to p u t r e s c i n e under normal c o n d i t i o n s (Karrer and Warren, 1974). Many other organisms and bacteriophages w i t h modified bases i n t h e i r DNA have been i d e n t i f i e d , and many of the bases have been char-a c t e r i z e d . Most procaryotes and eucaryotes c o n t a i n v a r i a b l e amounts of the base 5-methylcytosine. In mammalian c e l l s , between 2 and 8 per-cent of the t o t a l d e o x y c y t i d y l a t e residues are found i n t h i s form ( H a l l , 1966). P r o c a r y o t i c DNA a l s o contains s m a l l amounts of 5-methyl-d e o x y c y t i d y l a t e and N^-methyldeoxyadenylate. Methylated bases i n s p e c i f i c DNA sequences can p r o t e c t DNA from r e s t r i c t i o n endonucleases r e c o g n i z i n g those sequences. The mechanism of s y n t h e s i s of methylated bases i s w e l l understood but i s not r e l e v a n t to t h i s d i s c u s s i o n . Bacteriophages are the r i c h e s t sources of modified bases i n DNA (Table 1) (Figure 1). The T-even bacteriophage group provided the f i r s t 5 FIGURE 1.—The structure of modified bases found in bacteriophage DNA. o K J X J o K j CH2OH COOH CH NHCH COOH 8 Figure 1 Structures of modified bases in phage DNAs: 1. 5-hydroxymethylcytosinc (hmCyt); 2. 5-methy!cytosine (ruCyt); 3. uracil (Ura); 4. 5-hydroxymethyluracil (hmUra); 5. a-putre-scinylthymine (putThy); 6. 5-dihydroxypentyluracil (dhpUra); 7. a-glutamylthymine (gluThy); 8. 2-aminoadenine (nAde). 6 examples of a modified base in bacteriophage DNA (Wyatt and Cohen, 1953). T-even phage included T2, T4 and T6 which are similar but not identical in many respects. They a l l contain the base 5-hydroxymethyl-cytosine (hmCyt) which completely replaces cytosine in the bacterio-phage DNA. 5-hydroxymethylcytosine base pairs with guanine in the same manner as cytosine. 5-hydroxymethylcytosine residues are further modi-fied by the covalent and stereospecific addition of d-glucose molecules. The glucosylation patterns are specific for each bacteriophage type (Revel and Luria, 1970). The cases of the T-even bacteriophage and other bacteriophage containing modified bases in their DNA w i l l be considered in more detail. The presence of modified bases in DNA raises many intriguing p o s s i b i l i t i e s . The biosynthetic origins and functions of modified bases w i l l be examined. The structures of modified bases include a variety of permuta-tions upon the four normal bases found in DNA. The best studied examples are bases which are modified pyrimidines. Modification takes place in a manner which does not alter the base-pairing properties of the base. Modification occurs after the formation of the complete pyrimidine ring structure. Therefore, uridine monophosphate (UMP), is a precursor of a l l modified pyrimidine bases. The C-5 hydrogen bond is a favoured site for the substitution reactions generating modified bases. This is probably due to the high intrinsic reactivity of this site. Modifications occurring here can also extend out and away from the base-pairing regions of the DNA helix. For example, the methyl group of 7 thymine l i e s i n the major groove of the DNA double h e l i x (Mahler and Cordes, 1971). Although base modification,voccurs ' i n a way which does not a l t e r base-pairing properties, modification often a l t e r s appreciably the phys-i c a l properties of the DNA. A catalogue of e f f e c t s i s shown i n Table 1 (Warren, 1980). The equations used to pr e d i c t moi % G + C content of DNA are derived from studies of normal DNA molecules. Properties of DNA such as melting temperature or buoyant density are dependent upon the GC content of the DNA. A l t e r a t i o n s i n the basic components of the DNA have e f f e c t s upon melting temperature and buoyant density. Differences i n moi % G + C contents predicted by melting temperature analysis or from buoyant density determinations are usually i n d i c a t i v e of the pre-sence of a modified base i n DNA. Degradative analysis of DNA suspected of containing a modified base i s used to confirm suspicions; i n addition, the normal base substituted i s i d e n t i f i e d by i t s absence or p a r t i a l replacement. DNA may be degraded by acid hydrolysis to bases or by enzymatic dig e s t i o n to nucleotides or nucleosides. I f acid hydrolysis of DNA i s used to demonstrate the presence of a modified base i t i s also necessary to prove that the hydro l y s i s procedure does not a l t e r the base, e.g. hmCyt residues i n T4 DNA are glycosylated but acid hydro-l y s i s of DNA removes the glucose. Unusual bases which are stable under conditions of acid hydrolysis are c a l l e d hypermodified bases. Enzymatic dige s t i o n of DNA-containing a c i d - l a b i l e structures generally releases bases i n t h e i r unaltered form. Glucosylated T4 DNA i s r e s i s t a n t to r e s t r i c t i o n endonucleases (Revel and L u r i a , 1970). SP 15 DNA i s not 8 completely degraded to mononucleotides by sequential DNase I and snake venom phosphodiesterase (SVPD) treatments.(Brandon, Ph.D. Thesis, 1973). The presence of a modified base in bacteriophage DNA provides opportunities for the dynamic analysis of phage nucleotide metabolism and biochemistry. If infection increases the rate of DNA synthesis over that seen in an uninfected c e l l then the rate of synthesis or supply for the precursors of DNA must also increase. If the mol % G + C content of the infecting phage DNA is different from that of the uninfected host, then the phage must encode functions to reallocate precursors to reflect the changes in DNA base composition. These changes may appear as altera-tions in the size of the nucleotide pool. However, the size of the nucleotide pool is not likely to be as important as the rate of flow of nucleotides through the pool. If the phage DNA contains a modified nucleotide which is not found in uninfected cells then the phage must encode functions that allow the de_ novo synthesis of the modified nucleo-tide. In addition, the phage must also inhibit the synthesis and/or prevent the incorporation of the normal nucleotide into phage DNA. Bacteriophages which partially replace a normal base must regulate the levels of substitution of each base. Modified bases or normal bases may also be altered after polymerization and these modification functions must also be regulated. The manner by which various phages accomplish the synthesis of DNA containing unusual components w i l l be considered with respect to the preceding points. The biological consequences of base substitution w i l l also be considered. 9 T-even bacteriophage (T2, T4, T6) In E_. c o l i infected with T-even bacteriophage, many of the points made above are i l l u s t r a t e d . T-even phage make hydroxymethylcytosine at the mononucleotide l e v e l and use i t to completely replace cytosine i n t h e i r DNA (Wyatt and Cohen, 1953). Glucosylation of T-even phage DNA provides a model for p o s t - r e p l i c a t i o n a l modification of DNA. The reprogramming of the c e l l u l a r biosynthetic machinery i n T4-infected c e l l s begins with the i n h i b i t i o n of host DNA synthesis and other c e l l u l a r biosynthetic processes. Cytosine-containing host DNA i s degraded. The deoxyribonucleases which mediate these processes are encoded i n the T4 genome (Mathews, 1977). The i n i t i a l steps i n the degradation sequence are due to nucleases which s p e c i f i c a l l y recognize and cleave cytosine-containing DNA. Hydroxymethylcytosine-containing DNA i s not a substrate for these enzymes (Kutter and Wiberg, 1969). Degradation of host DNA not only destroys a l l host-encoded information but also generates a pool of precursors which can be used for T-even phage DNA synthesis (Price and Warner, 1969). dCMP, formed by host DNA degradation and by de novo synthesis, i s a precursor of hmdCMP (Cohen, 1968). The synthesis of hmdCMP involves the N^-N^Q-methylene-tetrahydrofolate-mediated reaction catalyzed by the T4 enzyme deoxycytidylate hydroxymethylase. The reaction i s analogous to the N^-N^Q-methylene-THFA-mediated C - l transfer occurring i n thymine biosynthesis; however, transfer at the oxidation l e v e l of the hydroxy-methyl group does not r e s u l t i n the oxidation of THFA to DHFA (Flaks and Cohen, 1957; Cohen, 1968). In addition to converting dCMP to hmdCMP, T4-infected c e l l s induce a kinase capable of making hmdCTP (Mathews, 1977). 10 The phage must ensure that dCTP i s eliminated from the nucleotide pools since incorporation of cytosine i n t o T4 DNA would r e s u l t i n the destruc-t i o n of the DNA by cytosine-recognizing nucleases. dCTP i s eliminated from i n f e c t e d - c e l l nucleotide pools by the synthesis of a T4-coded dCTPase (Price and Warner, 1969). dCTP i s converted to dCMP and pyro-phosphate. dCMP i s a substrate for dCMP hydroxymethylase (Cohen, 1968). dCMP i s also a substrate for the T4 enzyme dCMP deaminase. The product of dCMP deaminase i s dUMP, which, i n turn, i s a substrate for another T4 enzyme, thymidylate synthase. T h i r t y - f i v e percent of the dCMP a r i s i n g from mononucleotides released from host DNA i s ultim a t e l y reincorporated into T4 DNA. The rate of DNA synthesis i n T4-infected c e l l s increases ten f o l d a f t e r i n f e c t i o n but the deoxyribonucleoside pools remain at a constant s i z e (Mathews, 1972). This r e f l e c t s the increased rate of flow of precursors through the active pool. Pool s i z e s i n T4-infected c e l l s r e f l e c t the GC content of the DNA synthesized. dCMP deaminase i s a T4 enzyme which helps reroute pyrimidines from the c y t i d y l a t e pathways i n the uninfected host (mol % G + C = 50) to the thymidylate pathway i n the inf e c t e d host (mol % G + C = 34). In T4-infected E. c o l i the r a t i o of Thy and hmCyt deoxyribonucleotides i n the pools i s 2.0 to 1.0 (Flanegan and Greenberg, 1977; Chiu et a l . , 1977). This i s the same r a t i o that i s found f o r these nucleotides i n T4 DNA. The maintenance of a precise r a t i o l i n k i n g the supply of precursors to DNA polymerization suggests a complex regulatory mechanism (Mathews et a l . , 1979). The regulation of pyrimidine deoxyribonucleotides involves a multienzyme complex. There i s some controversy over the components of th i s complex. Mathews (1979) has proposed that the complex contains the T4 enzymes dCMP hydromethylase, 11 thymidylate synthase, dCTP/dUTPase, ribonucleotide diphosphate reductase, deoxynucleoside monophosphate kinase, dCMP deaminase and the host-coded enzyme nucleoside diphosphate kinase. Experiments performed i n Greenberg's lab have suggested the complex contains dCMP hydroxymethyl-ase, thymidylate synthase, dCTP/dUTPase, ribonucleotide diphosphate reductase, T4 DNA polymerase, 3-glucosyltransferase and gene products 32 (unwinding protein) or 44 (DO) and 45 (DO) (Tomich and Greenberg, 1973; Wovcha et a l . , 1973; Wovcha et a l . , 1977; Tomich et a l . , 1974; Chiu et a l . , 1977; Flanegan et a l . , 1977; Greenberg and Chiu, 1978). He believes the complex channels the deoxynucleoside precursors d i r e c t l y to the r e p l i c a t i n g forks of T4 DNA. The T4 complex proposed by Mathews contains enzymes involved i n the generation of pyrimidine precursors but not any proteins involved d i r e c t l y i n DNA r e p l i c a t i o n (Reddy et a l . , 1977). The evidence upon which these conclusions are based i s derived p a r t i a l l y from the analysis of c a r e f u l l y f r a c t i o n a t e d c e l l - f r e e extracts, and plasmolyzed mutant-infected c e l l s . C e l l s infected with gene 1 (T4 deoxyribonucleotide kinase) or gene 42 (dCMP hydroxymethylase) mutants cannot synthesize DNA i n v i t r o , even when the metabolic block i s thwarted by the addition of deoxynucleoside 5'-triphosphates. These data were interpreted as evidence f o r p r o t e i n complexes. Assembly of enzymes in t o multienzyme complexes channeled DNA precursors into DNA and activated enzymes involved i n precursor synthesis. Evidence supporting these con-clusions was obtained i n vivo. dCMP hydroxymethylase and thymidylate synthase do not function ±n vivo u n t i l they are assembled into m u l t i -enzyme complexes (Tomich et a l . , 1974). These a c t i v i t i e s sediment quickly i n v i t r o as one peak of enzyme a c t i v i t y (Reddy et a l . , 1977). It should be noted that T4 DNA polymerase mutants accumulate DNA precur-sors when DNA synthesis i s blocked (Chiu et a l . , 1976). However, gene 42 mutants have been found which can accumulate hmdCMP iji vitro but which w i l l not make T4 DNA (Tomich et a l . , 1974). These are probably enzyme complex assembly mutants. dCMP hydroxymethylase and dTMP syn-thase can be assayed in vivo using Greenberg's tritium release assay. 3 3 [5- H]-uracil enters nucleotide pools and some is converted to [5- H]-3 dCMP or [5- H]-dUMP. These nucleotides are substrates for dCMP hydroxy-methylase and thymidylate synthase. Enzymatic transfer of one carbon fragments from THFA to the C-5 position of the pyrimidine ring results 3 in the formation of hmdCMP or dTMP. Tritium i s released as ^O. 3 Radioactivity in H^ O can be recovered by f i l t e r i n g samples through a PCA and charcoal slurry. Labelled nucleotides are retained in the char-coal but the tritiated water is not. The amount of tritium detected in water is proportional to the level of tritium-releasing enzyme activity. Although the data are incomplete, i t can be concluded that T4 DNA synthesis i s s t r i c t l y regulated. DNA precursors are made in the correct ratios and fed to the polymerizing complex at a precise rate which determines the rate of DNA synthesis. It i s useful for an organ-ism to ensure delivery of an adequate supply of a precursor to i t s intracellular site of uti l i z a t i o n . Compartmentalization of related functions by formation of enzyme complexes performs this function in T4-infected c e l l s . T-even DNA glucosylation Glucosylation of hmCyt residues in T4 DNA is a model system for post replicational modification of DNA (Revel and Luria, 1970). T2, T4 and T6 a l l have d i f f e r e n t DNA g l u c o s y l a t i o n patterns which are d i s t i n c t and r e p r o d u c i b l e . T4 has two kinds of g l u c o s y l a t i n g enzyme: a - g l u c o s y l t r a n s f e r a s e and 3-glucosyl t r a n s f e r a s e . T2 and T6 have only a - g l u c o s y l t r a n s f e r a s e s . In T4 70 percent of the g l u c o s y l a t e d hmCyt molecules are a - g l u c o s y l a t e d , w h i l e 30 percent of the l i n k a g e s are 3-glucosylated (Lehman and P r a t t , 1960). U r i d i n e diphosphoglucose (UDPG), which i s synthesized by host enzymes, t r a n s f e r s glucose to hmCyt i n T4 and to hmCyt and the 6 p o s i t i o n of glucose i n T2 and T6 (Romberg et a l . , 1961; Josse and Kornberg, 1963). T6 has 72 percent of i t s hmCyt r e s -idues d i g l u c o s y l a t e d w h i l e only 5 percent of T2 hmCyt i s d i g l u c o s y l -ated. T4 a - g l u c o s y l t r a n s f e r a s e mutants produce DNA w i t h a l l the hydroxymethylcytosine residues 3-glucosylated. T4 3-glucosyl t r a n s -f e r a s e mutants produce DNA w i t h a l l the hmCyt a-g l u c o s y l a t e d . Twenty-f i v e percent of T2 and T6 hmCyt i s not g l u c o s y l a t e d i n v i v o . Non g l u c o s y l a t e d T-even DNA may be produced i n UDPG hosts. However, phage w i t h non g l u c o s y l a t e d DNA i s subject to r e s t r i c t i o n i n some b a c t e r i a l s t r a i n s (Georgopoulus, 1967). R e s t r i c t i o n of non g l u c o s y l a t e d T-even DNA i s governed by two host genes which can destroy hmCyt-containing DNA (Fleischman and Richardson, 1971). I n T4-infected JE. c o l i the DNA-r e s t r i c t i n g a b i l i t y i s l o s t . T4 must code f o r a product which i n h i b i t s T4 r e s t r i c t i o n . The f u n c t i o n of T4 DNA g l u c o s y l a t i o n appears to be p r o t e c t i o n from r e s t r i c t i o n (Hewlett and Mathews, 1975). A l l the T-even a - g l u c o s y l t r a n s f e r a s e show some sequence s p e c i -f i c i t y . T2 DNA g l u c o s y l a t e d i n v i v o cannot serve as a s u b s t r a t e f o r i n v i t r o T2-mediated g l u c o s y l a t i o n , but w i l l a l l o w g l u c o s y l a t i o n by T6 a - g l u c o s y l t r a n s f e r a s e . In T4 DNA purine residues l i n k e d to the 3' 14 p o s i t i o n of a hmCyt residue appear to hinder g l u c o s y l a t i o n (Lunt et a l . , 1964). T4 a - g l u c o s y l t r a n s f e r a s e incubated w i t h non g l u c o s y l a t e d T6 DNA could g l u c o s y l a t e a l l hmCyt residues except those adjacent to other hmCyt residues (de Waard et a l . , 1967). They d i d not f i n d any B-glucosyl t r a n s f e r a s e s p e c i f i c i t y . However, a (or g ) - g l u c o s y l t r a n s f e r a s e w i l l g l u c o s y l a t e s i t e s i n v i t r o that are normally 8 (or a ) - g l u c o s y l a t e d (Josse and Kornberg, 1962). The s t r u c t u r e of the DNA h e l i x i s important f o r m o d i f i c a t i o n ; n a t i v e hmCyt-containing DNA i s a b e t t e r s u b s t r a t e f o r g l u c o s y l a t i o n than heat-denatured hmCyt-containing DNA (Josse and Kornberg, 1962). The reasons f o r non random d i s t r i b u t i o n of g l u c o s y l -ated hmCyt residues are not understood. G l u c o s y l a t i o n i s not r e q u i r e d f o r phage v i a b i l i t y . Wild-type mutants of T4 grow poorly on a r i f a m p i c i n - r e s i s t a n t mutant of E. c o l i . They form " i n d i s t i n c t plaques". I n d i s t i n c t plaque formers can be converted to d i s t i n c t plaque formers by a mutation mapping i n the (3-glucosyl t r a n s f e r a s e gene. Mathews (1977) has i n t e r p r e t e d t h i s experiment as evidence f o r the involvement of g l u c o s y l a t e d hmCyt i n RNA polymerase template i n t e r a c t i o n s . T r a n s c r i p -t i o n i n T 4 - i n f e c t e d c e l l s i s c a r r i e d out by host RNA polymerase. S p e c i -f i c phage-encoded m o d i f i c a t i o n s of RNA polymerase occur at s p e c i f i e d times i n T4 i n f e c t i o n . These m o d i f i c a t i o n s a l l o w t r a n s c r i p t i o n of pre-v i o u s l y p r o s c r i b e d domains on the bacteriophage genome. In normal T4 DNA a l l hmCyt residues are g l u c o s y l a t e d . However, non g l u c o s y l a t e d T4 phage r e p l i c a t e s normally. The s u b s t i t u t i o n of c y t o s i n e f o r hmCyt i n T4 DNA blocks l a t e gene expression. There seems to be a requirement f o r hmCyt-containing DNA f o r l a t e gene expression (Kutter et a l . , 1975; K u t t e r and Wiberg, 1968). There i s no corresponding requirement f o r 15 glucosylated hmCyt residues. Mathews' conclusions regarding g-gluco-syltransferase do not bear upon the s i t u a t i o n pertaining to wild-type RNA polymerase i n t e r a c t i o n with glucosylated hmCyt-containing template. T4 DNA glucosylation i s coupled to r e p l i c a t i o n (McNichol and Goldberg, 1973). Glucosylated T-even DNA i s immunogenic. Antisera with s p e c i f i c i t y f o r a or 3 or a and 3-glucosylated T4 DNA can be pre-pared. Nori'glucosylated T4 DNA i s not immunologically detectable i n T4-infected E. c o l i . 3-glucosyl transferase i s supposed to be a component of a multienzyme DNA precursor synthesizing complex (Mathews, 1979). In T6-infected E. c o l i newly synthesized non glucosylated DNA was demon-str a b l e (Erikson and Szybalski, 1964). In vivo, T6 DNA synthesis begins 2 minutes before • the onset of DNA glucosylation. After the synthesis of glucosylating enzyme begins, a l l the T6 DNA formed was glucosylated. B a c i l l u s s u b t i l i s bacteriophage B a c i l l u s s u b t i l i s bacteriophages with hmUra-containing DNA are common. They include 0e, SP8, SP01, SP82, SP5 and 2C. The presence of hmUra i n DNA causes the DNA to band at a heavier density than expected i n neutral CsCl density gradients. 0e-infected B_. s u b t i l i s reprogram c e l l u l a r processes to stop host biosynthesis of Thy-containing nucleo-tides and to eliminate Thy-containing nucleotides. HmdUTP completely replaces dTTP i n the nucleotide pools of infected c e l l s (Roscoe, 1969). 0e accomplishes t h i s i n a manner which i s understood. dTMP cannot be converted to HmdUMP. dTTP i s destroyed by the action of a nucleotidase which converts dTTP to dTMP and pyrophosphate (Price et a l . , 1972). A multiple gene phage mutant with a defective dTTPase and a temperature-s e n s i t i v e hydroxymethylase has thymine i n i t s DNA replacing up to 20 16 percent of the hmUra. I t can grow and t r a n s f e r Thy-containing DNA to progeny phage (Marcus and Newlon, 1971). HmUra i n 0e DNA comes from the i n d u c t i o n of a phag e - s p e c i f i c dUMP hydroxymethylase enzyme ( P r i c e et a l . , 1972). A l l the hmUra i n the 0e DNA i s synthesized from de novo n u c l e o t i d e p r e c u r s o r s . 0e i n h i b i t s host DNA sy n t h e s i s but the host DNA i s not degraded to mononucleotides (Roscoe, 1969). 0e-infected c e l l s induce an i n h i b i t o r of thymidylate synthase. This leaves dUMP a v a i l a b l e as a substrate f o r the phage coded hydroxymethylase (Roscoe and Tucker, 1969; Haslam e t a l . , 1967). In the 0 e - r e l a t e d phage SP5, a new enzyme, thymidine phosphatase, i s induced. I t cleaves dTMP to thymidine and orthophosphate (Aposhian and Tremblay, 1966). A phage-encoded dCMP deaminase i s induced. This p i v o t a l enzyme routes the c o r r e c t amount of dCMP to dCTP or hmdUTP pathways. Such r e r o u t i n g of precursors i s re q u i r e d whenever the host GC content i s d i f -f e r e n t from the phage GC content. This enzyme would be a good t a r g e t f o r a l l o s t e r i c r e g u l a t i o n , but i s the only known dCMP deaminase not subjec t to feedback r e g u l a t i o n by dNTPs ( N i s h i h a r a et a l . , 1967). Phage w i t h hmUra-containing DNA are s u s c e p t i b l e to host r e s t r i c -t i o n . 0e DNA i s s u s c e p t i b l e to s e v e r a l r e s t r i c t i o n endonucleases (Berkner and Folk, 1979). The hydroxymethyluracil groups i n 0e DNA are not modified by the a d d i t i o n of sugars or any other m o i t i e s ( A l l e g r i a and Kahan, 1968). The hmUra i n SP01 or 0e DNA i s not req u i r e d f o r r e p l i c a t i o n or t r a n s c r i p t i o n . SP01 induces a pha g e - s p e c i f i c DNA polymerase. This enzyme w i l l use e i t h e r hmdUTP or dTTP as a substrate in v i v o or in v i t r o (Yehle and Ganesan, 1973). 0e DNA re q u i r e s host DNA polymerase I I I s i n c e a J^ s_ DNA p o l I I I J3. s u b t i l - i s - 0 e i n f e c t e d c e l l shuts o f f 0e DNA syn t h e s i s when i t i s s h i f t e d from the permissive to the non permissive temperature ( L a v i et a l . , 1974). I t i s b e l i e v e d that a phage-encoded p o l I I I modifying p r o t e i n i s synthesized which increases the a f f i n i t y of the polymerase f o r the 0e template ( L a v i et a l . , 1974). This cannot be an absolute requirement f o r hmUra i n the 0e DNA s i n c e 0e DNA con-t a i n i n g thymine r e p l i c a t e s normally. DNA r e p l i c a t i o n i n J3_. s u b t i l i s i s i n h i b i t e d by the a n t i f o l a t e drugs trimethoprim and aminopterin. DNA r e p l i c a t i o n i n 0 e - i n f e c t e d B_. s u b t i l i s i s r e s i s t a n t to these drugs. Thymidine b i o s y n t h e s i s i s i n h i b i t e d i n c e l l s exposed to a n t i f o l a t e drugs. D i h y d r o f o l a t e reductase i s the ta r g e t enzyme f o r trimethoprim and aminopterin i n h i b i t i o n . Thymidine b i o s y n t h e s i s i s i n h i b i t e d because, during the formation of dTMP from dUMP and N^-N^Q-methylene-tetrahydrofolic a c i d , a C - l t r a n s f e r i s c a t a l y z e d by thymidylate synthase. This t r a n s f e r r e s u l t s i n the o x i d a t i o n of THFA to DHFA. Normally, THFA i s regenerated from DHFA and NADPH by the a c t i o n of the enzyme d i h y d r o f o l a t e reductase (Mahler and Cordes, 1971). When d i h y d r o f o l a t e reductase i s i n h i b i t e d , the s t o i c h i o -metric requirement f o r THFA i n thymidylate b i o s y n t h e s i s e v e n t u a l l y depletes the c e l l u l a r supply of THFA. THFA i s re q u i r e d f o r many c e l l u -l a r b i o s y n t h e t i c processes such as the formation of pu r i n e s , p y r i m i d i n e s and the amino acids methionine, s e r i n e and g l y c i n e . This requirement f o r THFA i s c a t a l y t i c but not s t o i c h i o m e t r i c . The a c t i o n of a n t i f o l a t e drugs i s antagonized by the a d d i t i o n of thymidine s i n c e t h i s reduces the endogenous requirement f o r the regeneration of THFA. C e l l s i n f e c t e d by bacteriophage 0e synthesize hmdUMP ins t e a d of dTMP. h.mdUMP b i o s y n t h e s i s 18 req u i r e s the c o f a c t o r N^-N^g methylene t e t r a h y d r o f o l i c a c i d , the dUMP hydroxymethylase r e a c t i o n does not r e s u l t i n the o x i d a t i o n of THFA. Therefore, d i h y d r o f o l a t e reductase a c t i v i t y i s not re q u i r e d to regen-erate l a r g e q u a n t i t i e s of DHFA. In a l l c e l l s C - l t r a n s f e r to THFA i s cata l y z e d by the enzyme s e r i n e transhydroxymethylase (Mahler and Cordes, 1971). Caution should be ex e r c i s e d i n i n t e r p r e t i n g trimethoprim r e s i s -tance as demonstrating a requirement f o r dTMP b i o s y n t h e s i s . Drug r e s i s -tance f a c t o r s , encoding r e s i s t a n c e to a n t i f o l a t e drugs, act by i n c r e a s i n g the number of d i h y d r o f o l a t e reductase molecules. Plasmid or phage-coded d i h y d r o f o l a t e reductase may be i n t r i n s i c a l l y more r e s i s t a n t to a n t i f o l a t e drugs (Meynell, 1973). PBS1 i s a pseudolysogenic bacteriophage of B_. s u b t i l i . s . The DNA of PBS1 contains u r a c i l which completely replaces thymine i n b a c t e r i o -phage DNA. PBS2 i s a c l e a r plaque d e r i v a t i v e of PBS1 (Takahashi and Marmur, 1963). As expected, PBS2 DNA has a higher-than-predicted buoyant d e n s i t y i n CsCl and a lower-than-predicted Tm (Table 1). I n f e c t i o n of B_. sub t i l l s by PBS2 r e q u i r e s the e x c l u s i o n of dTTP and i t s replacement by dUTP i n n u c l e o t i d e pools. The phage must a l s o i n h i b i t host-encoded enzymes which destroy u r a c i l - c o n t a i n i n g DNA. M i s i n c o r p o r a t i o n of u r a c i l or the deamination of cytosine residues i n DNA occurs at a low frequency i n a l l c e l l s (Tye et a l . , 1977). Mechanisms to reduce the frequency of t r a n s i t i o n mutations that deamination of cytosine would cause have evolved. C e l l s possess the enzyme uracil-DNA g l y c o s y l a s e which cleaves the N - g l y c o s i d i c bond between u r a c i l and deoxyribose moieties i n DNA. This i s the i n i t i a l step i n a process which then e x c i s e s the deoxyribose sugar and r e p a i r s the molecule using the complimentary DNA stran d 19 (Frledberg et a l . , 1975; Duncan et a l . , 1976). PBS2 codes f o r a s m a l l , heat s t a b l e i n h i b i t o r of the enzyme uracil-DNA g l y c o s y l a s e (Tomita and Takahashi, 1976; Friedberg e t a l . , 1975). PBS2 i n f e c t i o n a l s o induces thymidine phosphorohydrolase and dUMP kinase a c t i v i t i e s (Kahan, 1963). Both enzymes serve to l i m i t the supply of dTMP. dOMP kinase a l s o increases the supply of dUTP. Thymidylate synthase i s not i n h i b i t e d i n PBS2 i n f e c t e d c e l l s (Hitzeman et a l . , 1978). PBS2-infected c e l l s a l s o c o n t a i n a dCTP deaminase a c t i v i t y (Tomita and Takahashi, 1969). Coupled w i t h host dCMP deaminase these enzymes increase the a v a i l a b i l i t y of dUTP or i t s p r e c u r s o r s . These enzymes route precursors to dUTP precursor pathways ( P r i c e and F r a t o , 1975; Rima and Takahashi, 1973). T h i r t y to -50 percent of the dUTP used f o r PBS2 DNA s y n t h e s i s i s generated by the a c t i o n of dCTP deaminase. The r e s t comes from de_ novo sources v i a dCMP deaminase and r i b o n u c l e o t i d e reductase (Rima and Takahashi, 1979; P r i c e , 1980). Host DNA s y n t h e s i s i s i n h i b i t e d by PBS2 but host DNA i s not degraded, t h e r e f o r e , a l l deoxynucleotides come from de novo synthe-s i s . PBS2 i s a transducing phage (Takahashi, 1963). I t i s obvious that Thy-containing DNA i s not d i s c r i m i n a t e d against during packaging. The b i o l o g i c a l consequences which are due to the presence of u r a c i l i n PBS DNA are c l e a r . U r a c i l - c o n t a i n i n g DNA i s s u s c e p t i b l e to nuclease or r e s t r i c t i o n endonuclease cleavage although r a t e s may d i f f e r from normal substrates (Berkner and F o l k , 1979). PBS2-infected c e l l s have a RNA polymerase a c t i v i t y which i s r e s i s t a n t to r i f a m p i c i n or s t r e p t o l y d i g i n . T r a n s c r i p t i o n occurs even i f the a n t i b i o t i c s are added p r i o r to i n f e c -t i o n . U r a c i l DNA has l i t t l e or no template a c t i v i t y w i t h host RNA 20 'polymerase w h i l e the v i r a l RNA polymerase shows a p p r e c i a b l e t e m p l a t e a c t i v i t y o n l y w i t h PBS2 DNA o r p o l y (dA-dT) ( C l a r k e t a l . , 1974; P r i c e and F r a b o t t a , 1972; P r i c e e t a l . , 1974; Rima and T a k a h a s h i , 1973). The n a t u r e of e a r l y PBS2 t r a n s c r i p t i o n i s o b s c u r e . P B S 2 - s p e c i f i e d RNA p o l y -merase does not appear u n t i l the m i d d l e of the l y t i c c y c l e , but e a r l y phage t r a n s c r i p t i o n i s r i f a m p i c i n r e s i s t a n t ( C l a r k , 1978). T h i s sug-g e s t s t h a t PBS2 v i r i o n s c a r r y a RNA p o l y m e r a s e - m o d i f y i n g enzyme which c o u l d r e n d e r t r a n s c r i p t i o n drug r e s i s t a n t . DNA r e p l i c a t i o n i s a c t i v e w i t h p u r i f i e d PBS2 polymerase u s i n g e i t h e r dTTP or dUTP as a s u b s t r a t e ( P r i c e , 1980). S m a l l amounts of d T T P - c o n t a i n i n g DNA can be made jLn v i v o by growing c e l l s a t a h i g h pH. At t h i s pH the i n h i b i t i o n of h o s t dUTPase i s i n a c t i v a t e d and enough dUMP to a l l o w the f o r m a t i o n of dTMP accumulates. dTMP p h o s p h o r o h y d r o l a s e must a l s o be i n h i b i t e d a t t h i s h i g h pH ( P r i c e and F o g t , 1973; P r i c e and F r a t o , 1975). The PBS2-induced DNA polymerase does not r e q u i r e dUTP i n  v i v o f o r p o l y m e r i z a t i o n . L i n d a h l has p o i n t e d out t h a t the low moi % G + C c o n t e n t of PBS2 DNA c o u l d be a b i o l o g i c a l consequence of the p r e s e n c e of u r a c i l i n PBS2 DNA ( L i n d a h l , 1979). He p roposes t h a t PBS2 DNA has undergone a l l th e a l l o w a b l e GC to AU t r a n s i t i o n s and t h a t t h e r e would be a s t r o n g s e l e c t i v e p r e s s u r e a g a i n s t f u r t h e r t r a n s i t i o n m u t a t i o n s . T h i s h y p o t h e s i s s h o u l d be t e s t a b l e . I t p r e d i c t s t h a t PBS2 phage would be e x t r e m e l y s u s c e p t i b l e t o k i l l i n g by a t r a n s i t i o n - i n d u c i n g mutagen such as n i t r o u s a c i d and t h a t the t h i r d base i n DNA codons s h o u l d be some o t h e r base than u r a c i l i n a l l o r most a l l o w a b l e c a s e s . A q u e s t i o n about PBS1, the p s e u d o l y s o g e n i c p a r e n t of PBS2, a l s o 21 a r i s e s . Does ly s o g e n i c PBS1 DNA co n t a i n Thy instead of Ura? I f not, how does PBS1 p r o t e c t i t s DNA against host u r a c i l N-glycosidase? U r a c i l i n PBS2 i s not modified a f t e r i n c o r p o r a t i o n i n t o DNA. 5-FU i s incorporated i n t o PBS2 DNA (Lozeron and S z y b a l s k i , 1967). J3_. 14 s u b t i l i s i n f e c t e d i n the presence of [2- C]-FUdR make DNA i n which u r a c i l i s replaced by FUdR. The buoyant d e n s i t y of the DNA i n CsCl d e n s i t y gradients increases from 1.722 to 1.737-1.745 gcc Label i s q u a n t i t a t i v e l y recovered from the heavy DNA f r a c t i o n and a c i d h y d r o l y s i s and TLC of the DNA samples shows that the l a b e l remains a s s o c i a t e d w i t h 5-FU. A replacement of one mol % of u r a c i l by 5-FU i s accompanied by an increase i n buoyant d e n s i t y of 0.34 mg cc ^. This paper proves that 5-FdUTP must be a sub s t r a t e f o r PBS2 DNA polymerase. PBS2 phage formed w i t h 5-FU i n DNA r e p l i c a t e but are more UV-sensitive than normal PBS2 phage. In other phage systems i n c o r p o r a t i o n of 5-FU i n t o DNA would i n d i c a t e that the phage used dUTP as a p o l y m e r i z a t i o n s u b s t r a t e . XP 12 i s a bacteriophage of Xanthomonas oryzae (Kuo et a l . , 1968). I t has a mol % G + C content of 67 compared to 64 f o r i t s host. The observed m e l t i n g temperature value f o r the phage DNA i s 101.5°, higher than expected. The buoyant d e n s i t y of the DNA i s 1.710 gcc \ lower than expected. 5-methylcytosine completely replaces c y t o s i n e i n the DNA of the phage. 5-methylcy t o s i n e i s synthesized from dCMP by a N^-N^-methylene-THFA-dependant enzyme. XP 12 a l s o encodes i t s own thymidylate synthase. Host DNA r e p l i c a t i o n i s i n h i b i t e d a f t e r i n f e c t i o n but DNA i s not degraded ( E r l i c h et a l . , 1977). SP 10 and SP 15 are examples of bacteriophages i n which thymine DNA residues are p a r t i a l l y replaced by another hypermodified p y r i m i d i n e . 22 a-glutamylthymine (gluThy) replaces thymine i n SP 10 DNA and 5-dihydroxy-p e n t y l u r a c i l (dhpUra) replaces thymine i n SP 15 DNA. Both DNA types are si m i l a r to 0W-14 DNA i n that they contain f i v e major DNA bases. The biosynthetic o r i g i n s of these bases have been investigated. SP 15 DNA contains dhpUra, and the nucleotide dhpUTP has been found i n infected c e l l nucleotide pools (Walker and Mandel, 1978a). dhpUra i s derived from u r a c i l and ribose (Walker and Mandel, 1978b). SP 15 DNA synthesis i s i n h i b i t e d by FUdR. FUdR i n h i b i t i o n of DNA syn-thesis can be reversed by the addition of TdR to cultures. SP 15 phage DNA which i s made i n the presence of FUdR and TdR has lower-than-normal l e v e l s of dhpUra. SP 15 phage with lower l e v e l s of dhpUra and increased l e v e l s of thymine are v i a b l e . Exogenously supplied TdR and BUdR are incorporated into phage DNA. dTTP i s present i n the nucleotide pools of infected c e l l s . The formation of dhpUra nucleotides i s also i n h i b i t e d by the presence of FUdR. This i s obvious since r e v e r s a l of FUdR i n h i b i -t i o n of DNA synthesis r e s u l t s i n abnormally high l e v e l s of Thy i n DNA. However, i n h i b i t i o n of DNA synthesis with aminopterin, followed by rever-s a l of the i n h i b i t ion by TdR r e s u l t s i n the synthesis of SP 15 DNA with normal amounts of dhpUra and Thy. dUMP but not dTMP i s a dhpUra precur-sor (Walker and Mandel, 1978a; Walker and Mandel, 1978b; Walker, Ph.D. Thesis, 1977). Host DNA i s not degraded by SP 15 i n f e c t i o n . Since SP 15 i s a generalized transducing phage of B_. sub t i l l s (Taylor and Thome, 1963) , dhpUra-containing DNA i s not required f o r DNA packaging. dhpUra i n SP 15 DNA i s t r i g l u c o s y l a t e d with one sugar linked i n a phosphodiester bond. The sugars are added to DNA a f t e r r e p l i c a t i o n i n 23 UDPG-mediated r e a c t i o n s which may be coupled to r e p l i c a t i o n . A s t r u c -ture f o r the sugar-phosphate modifying f u n c t i o n s has been proposed (Brandon, Ph.D. Thesis, 1973). Only dhpUra i s i s o l a t e d from a c i d hydro-l y s a t e s of SP 15 DNA. There i s only one chromatographically r e s o l v a b l e dhpUra-glucosylated and phosphorylated n u c l e o t i d e i n Sl-snake venom phosphodiesterase d i g e s t s of SP 15 DNA. The most remarkable f e a t u r e about SP 15 i s that DNA r e p l i c a t i o n n a t u r a l l y proceeds using f i v e n u c l e o t i d e triphosphate precursors. Under normal c o n d i t i o n s the r a t i o of dhpUra to Thy i s c o n t r o l l e d . Forty-two percent of the p o t e n t i a l DNA thymine content i s a c t u a l l y dhpUra (Marmur et a l . , 1972). The mechanism used to r e g u l a t e the l e v e l of dhpUra sub-s t i t u t i o n has not been i n v e s t i g a t e d . I t i s a l s o not known i f the d i s -t r i b u t i o n of dhpUra and Thy i n SP 15 i s random. SP 15 r e p l i c a t i o n i s s e n s i t i v e to i n h i b i t o r s of RNA polymerase (Dosmar et a l . , 1977). Therefore, i t i s l i k e l y that SP 1 5 - i n f e c t e d c e l l s use host RNA polymerase or phage-modified forms of host-modified polymerase. The presence of g l u c o s y l a t e d dhpUra residues has no e f f e c t upon t r a n s c r i p t i o n of SP 15 DNA. Bacteriophage SP 10 i s another IS. s u b t i l i s g e n e r a l i z e d t r a n s -ducing phage (Taylor and Thorne, 1963). a-glutamylthymine replaces 15 to 20 percent of the thymine residues i n SP 10 DNA. The gamma c a r -boxyls of the glutamyl s i d e c h a i n carry an u n i d e n t i f i e d h y d r o p h i l i c s u b s t i t u e n t c a r r y i n g a primary amine (Warren, 1980). L i k e other DNA species w i t h modified bases, SP 10 DNA d i s p l a y s unusual behaviour i n n e u t r a l cesium c h l o r i d e d e n s i t y gradients (p = 1.723). The m e l t i n g temperature of SP 10 DNA i s 81.5°, lower than p r e d i c t e d from i t s mol % G + C content. 24 The b i o s y n t h e s i s of SP 10 DNA i s not i n h i b i t e d by FUdR. Thy-midine i n c o r p o r a t i o n stops permanently a f t e r i n f e c t i o n of a Thy host (Markewych et a l . , 1977). Thymidylate synthase a c t i v i t y d e c l i n e s and a dTTPase a c t i v i t y appears i n i n f e c t e d c e l l s . dTTP l e v e l s f a l l to l e s s than 5 percent of p r e i n f e c t i o n l e v e l s (Markewych et a l . , 1979). Although host DNA i s degraded i n SP 10-infected c e l l s , none of the Thy released i s re i n c o r p o r a t e d i n t o phage DNA (Markewych et a l . , 1977). These r e s u l t s suggest that dTTP i s not an i n v i v o s u b s t r a t e f o r SP 10 DNA polymerase. I n f e c t e d c e l l s induce a dUMP hydroxymethylase a c t i v i t y and a novel kinase a c t i v i t y capable of generating hmdUTP from hmdUDP (Witmer and Dosmar, 1978). SP 10- i n f e c t e d c e l l s c o n t a i n hmdUTP but not dUTP. hmdUTP i s l i k e l y a precursor of both gluThy arid Thy i n SP 10 DNA. Soluble gluThy n u c l e o t i d e s are not found i n i n f e c t e d c e l l s . hmUra-con t a i n i n g SP 10 DNA does not accumulate under normal c o n d i t i o n s during DNA r e p l i c a t i o n . M o d i f i c a t i o n of hmUra to Thy and gluThy i s probably coupled to DNA r e p l i c a t i o n . I t i s not known i f DNA m o d i f i -c a t i o n i n SP 10-infected c e l l s i s sequence s p e c i f i c . SP 10 dUMP hydroxymethylase must be r e s i s t a n t to FUdR. Sus-c e p t i b i l i t y of the enzyme to 5FdUMP, i n v i t r o , has not been t e s t e d . The p a r t i a l s u b s t i t u t i o n of hmUra f o r thymine has been reported i n Gyrodinium c o h n i i , a d i n o f l a g e l l a t e (Rae, 1973). Rae has used den-s i t y and melting temperature p r o f i l e s to screen d i n o f l a g e l l a t e DNA f o r the presence of other unusual bases. Cryptothecum (Gyrodinium) c o h n i i contains 11 percent of i t s DNA nuc l e o t i d e s i n hydroxymethyldeoxyuri-d y l a t e . C\_ c o h n i i a l s o replaces 3 percent of i t s c y t o s i n e residues 25 w i t h 5-methylcytosine. The simultaneous presence of hmUra, Thy, Cyt and 5-MeCyt i s common i n d i n o f l a g e l l a t e s . E x u v i a e l l a cassebia contains hmUra, Thy, Cyt and 5-MeCyt i n i t s DNA. The counteracting e f f e c t s due to the presence of hmUra and 5-MeCyt i n DNA average out the e f f e c t s upon buoyant d e n s i t y and melti n g temperature. Buoyant d e n s i t y and meltin g temperature values a c c u r a t e l y p r e d i c t e d the Tm and p values f o r E. cassebia DNA. This misleading r e s u l t suggested that E_. cassebia DNA contained only the four normal DNA bases and demonstrated the wisdom of coupling CsCl gradient and Tm a n a l y s i s w i t h base or n u c l e o t i d e a n a l y s i s of unknown DNA samples when screening f o r unusual n u c l e o t i d e s . The d i s t r i b u t i o n of hmUra and Thy i n the DNA of _C. c o h n i i i s not random. CsSO^-AgCl d e n s i t y gradients demonstrate the presence of three d i s t i n c t d e n s i t y populations of DNA (Rae, 1973). hmUra i s p r e f e r e n t i a l l y l o c a t e d i n the d i n u c l e o t i d e s 5'-hmUra-Ade-3' and hmUra-Cyt. hmUra i s al s o enriched i n t r i n u c l e o t i d e sequences 5'-purine-hmUra-purine-3'-Methylcytosine occurs predominantly i n the sequence 5'-methylCyt-Gua-3' (Steele and Rae, 1980). The b i o s y n t h e t i c pathways f o r the d i n o f l a g e l l a t e DNA n u c l e o t i d e s have not been i n v e s t i g a t e d . To a c e r t a i n extent the p r o p e r t i e s of c e l l s i n f e c t e d by phage c a r r y i n g modified bases can be g e n e r a l i z e d . I n a l l cases but one, a major s u b s t i t u t i o n of a modified base leads to the complete replacement of the s u b s t i t u t e d base i n the n u c l e o t i d e pools. For a l l the phage, except SP 15, t h i s ensures that DNA r e p l i c a t i o n proceeds us i n g only four n u c l e o t i d e s . Replacement of a base i n v o l v e s steps to remove any r e s i -dual base. The flow of precursors i s rerouted to r e f l e c t d i f f e r e n c e s between the GC content of the phage and that of the host. Post-repli-cational modification occurs rapidly after the synthesis of unmodified DNA and unmodified DNA does not accumulate. The question of the biological significance of modified signi-fiance of modified bases in DNA remains largely unanswered. In some phage DNA modification prevents restriction of DNA. Generally speaking, the modified base is not required for phage v i a b i l i t y . Modified bases may aid phage enzymes in recognizing templates for transcription or replication. The absence of a modified base is sometimes enough to ensure the degradation of host DNA. This thesis presents results which show that most of these generalizations apply to the replication and modification of 0W-14 DNA. MATERIALS AND METHODS Organisms The b a c t e r i a l s t r a i n s employed were a l l Pseudomonas acidovorans s t r a i n s (Table 2). Working c e l l stocks were stored at -20° i n 40 per-cent g l y c e r o l and Casamino acids-mannitol (CAA-M) medium. Working stocks were prepared from permanent stocks i n the f o l l o w i n g manner: 25 ml of c e l l s were grown overnight at 30° i n a water bath shaking at 200 rpm. The c e l l s were spun down and resuspended i n 4 ml of s t e r i l e CAA-M. One ml of the c e l l suspension was added to 4 ml of 50 percent glycerol-CAA-M. The suspension was stored at —70°, u n t i l needed. Working stocks were stored at -20°, and were s t a b l e at t h i s temperature f o r three to s i x months. Media CAA-M was used as a complete, undefined medium. I t c o n s i s t s of 10 g CAA ( D i f c o ) , t e c h n i c a l grade; 5 g yeast e x t r a c t ; 5 g mannitol and 0.05 g tryptophan per l i t r e of medium. Medium was adjusted to pH 7.0 w i t h concentrated NaOH. TCS 1 x P_^  was used f o r experimental purposes r e q u i r i n g defined medium. TCS 1/5 x P^ i s the same as TCS 1 x P except that i t has one-32 f i f t h the amount of orthophosphate. I t was used i n P O ^ - l a b e l l i n g experiments. TCS (g/1) i s tris(hydroxymethyl)aminomethane ( T r i s ) 12.1; KC1, 5.0; Na 2S0 4, 0.0227; FeCl 3'6H 20, 0.0008; C a C l 2 , 0.017; KH 2P0 4, 0.0174. Medium i s adjusted to pH 7.0. 28 TABLE 2 . — B a c t e r i a l s t r a i n s used i n t h i s study. Bacterium S t r a i n Source Pseudomonas 29 p r o t o t r o p h i c R.Y. S t a n i e r acidovorans 3L derived from s t r a i n 29; r e q u i r e s 250 yg TdR m l - 1 f o r growth. derived from 3L; r e s i s t a n t to 5-FU: r e q u i r e s 250 yg TdR m l _ l f o r growth. sup 1 derived from s t r a i n 29 sup 2 derived from s t r a i n 29 sup 3 derived from s t r a i n 29 sup 4 derived from s t r a i n 29 sup 5 derived from s t r a i n 29 JE 1 p r o t o t r o p h i c ; s o i l i s o l a t e . JE 1 sup 1 derived from JE 1 JE 1 sup 2 derived from JE 1 JE 1 sup 3 de r i v e d from JE 1 R.A. K e l l n This study R.A.J. Warren R.A.J. Warren R.A.J. Warren R.A.J. Warren R.A.J. Warren J . E t h i e r P. M i l l e r P. M i l l e r P. M i l l e r 29 TCS was supplemented w i t h 2 ml 10 percent succinate and 0.5 ml 10 percent c h a r c o a l - f i l t e r e d CAA per 100 ml of medium. S o l i d media were prepared by the a d d i t i o n of 15 g agar ml ^ . Soft agar overlays were prepared by adding 7.5 g agar-1 to CAA-M. Phage were maintained i n a one-to-four mixture of L u r i a Broth (LB) and TCS, or i n 3XD b u f f e r at 4°. Other B u f f e r s and Media TN - 10 mM T r i s - H C l pH 7.4, 0.15 M NaCl. TNE - 10 mM T r i s - H C l pH 7.4, 0.15 M NaCl, 0.01 M EDTA. SSC - 0.15 M NaCl, 0.015 M Na 3 c i t r a t e . LB - 2.5 percent n u t r i e n t b r o t h , 0.5 percent yeast e x t r a c t . Growth of B a c t e r i a Cultures were u s u a l l y grown at 30°. L i q u i d c u l t u r e s i n TCS were grown overnight i n one-tenth the normal amount of succinate and CAA i n a gyrotory water bath shaking at 200 rpm. In the morning the normal con-c e n t r a t i o n s of suc c i n a t e (0.2 percent) and CAA (0.05 percent) were added and the shaking r a t e of the water bath was increased to 250 rpm. C e l l d e n s i t y was determined w i t h a Klett-Summerson c o l o r i m e t e r , equipped w i t h a No. 54 f i l t e r . K l e t t numbers were converted to c e l l numbers by r e f e r -ence to a standard curve. P l a t i n g c u l t u r e s f o r plaque assays were prepared by growing c e l l s i n CAA-M medium or i n a four-to-one mixture of CAA-M and TCS. Bacteriophage Bacteriophage 0W-14 w + was prepared from stocks derived from K r o p i n s k i and Warren's (1970) o r i g i n a l i s o l a t e . Working stocks were always prepared from the o r i g i n a l l y s a t e s . 30 0W-14 ts_ mutants were i s o l a t e d as described i n Results s e c t i o n ; am mutants were i s o l a t e d by P. M i l l e r . P r e p a r a t i o n of h i g h - t i t r e phage l y s a t e s 0W-14 w + - 200 ml of P_. acidovorans were grown to a c e l l d e n s i t y of 3.0 x 10 c e l l s per ml. 0W-14, f r e s h l y prepared from a s m a l l volume of l y s a t e , was added (approximate moi = 0.1). The c u l t u r e was incubated u n t i l l y s i s was complete. A few drops of chloroform were added to the l y s a t e and i n c u b a t i o n was continued f o r 30 minutes. At t h i s p o i n t l y s a t e s were g e n e r a l l y stored overnight at 4°. In the morning, l y s a t e s were warmed to 30°. DNAse I (^5 yg ml "S was added and the l y s a t e was gen t l y a g i t a t e d f o r 60 minutes. The l y s a t e was digested w i t h 10 yg ml of pronase ( s e l f - d i g e s t e d 30 minutes at 37°) f o r two hours or u n t i l most of the v i s i b l e c e l l d e b r i s were dig e s t e d . The l y s a t e was c e n t r i f u g e d at 4° f o r 5 minutes and 5,000 rpm; the supernatant was t r a n s f e r r e d to p o l y -carbonate c e n t r i f u g e tubes and spun at 13,000 rpm f o r 40 minutes. The - p e l l e t was gently resuspended i n a four-to-one mixture of TCS and LB (TCS/LB). A f t e r resuspension, the l y s a t e was subjected to another c y c l e of d i f f e r e n t i a l c e n t r i f u g a t i o n . The high-speed p e l l e t was resuspended i n 20 ml of TCS/LB and stored at 4° over CHC1 3. The p u r i f i c a t i o n procedures f o r am and ts_ l y s a t e s were s i m i l a r ; however, h i g h - t i t r e l y s a t e s could only be obtained w i t h c e l l s grown on TCS/CAA-M at four-to-one v/v (TCS/CAA-M). Ts l y s a t e s were prepared at 20-22°. Twenty-five ml of P_. acidovorans s t r a i n 29 c e l l s were grown to s a t u r a -t i o n on TCS/CAA-M. Two ml of t h i s c u l t u r e were d i l u t e d i n t o 25 ml of 8 f r e s h medium and incubated at 20-22° u n t i l c e l l d e n s i t y reached 2-3 x 10 31 per ml. Several i n d i v i d u a l plaques were picked w i t h a Pasteur p i p e t t e and added to the c u l t u r e s . Cultures were incubated u n t i l l y s i s then + t r e a t e d i n the same manner as w l y s a t e s . The l y s a t e s were t i t e r e d at 20° and 30°. Am 0W-14 l y s a t e s were prepared i n the same way on s t r a i n 29 sup 2 c e l l s at 30°. The l y s a t e s were t i t e r e d on s t r a i n 29 and s t r a i n sup 2. Phage t i t r a t i o n 0W-14 was t i t e r e d on CAA-M using the s o f t agar overlay technique (Adams, 1959). Conditioned medium Conditioned medium was prepared u s i n g s t r a i n 29 or 3L c e l l s grown 8 —1 on TCS to a c e l l d e n s i t y of 3 x 10 ml . C e l l s were c e n t r i f u g e d down and the supernatant was used immediately or f i l t e r e d through 0.45 u M i l l i p o r e membranes i n t o s t e r i l e glass b o t t l e s and s t o r e d at 4°. Media conditioned w i t h 3L o r i g i n a l l y contained 500 yg ml 1 thymidine. T e s t i n g f o r a n t i b i o t i c s e n s i t i v i t y A n t i o b i o t i c s were added at the appropriate c o n c e n t r a t i o n to e x p o n e n t i a l l y growing c u l t u r e s of ]?. acidovorans. Growth was monitored w i t h the K l e t t . The e f f e c t of v a r i o u s a n t i b i o t i c s on phage growth was monitored by p l a t i n g f o r pfu throughout i n f e c t i o n i n the presence or absence of a n t i b i o t i c s . For a n t i b i o t i c s which d i d not a f f e c t the growth of the host c e l l , a n t i b i o t i c s e n s i t i v i t y was a l s o measured by t e s t i n g f o r pfu on p l a t e s . 32 Phage mutagenesis 0W-14 w + stocks were mutagenized to 0.1" percent s u r v i v i a l w i t h n i t r o u s a c i d . They were prepared according to the methods of Roth. The i s o l a t i o n of 0W-14 t s mutants Mutagenized 0W-14 were d i l u t e d and p l a t e d so that each p l a t e con-tained 100 to 200 plaques at 20°. Plaques were picked w i t h s t e r i l e t o o t h -p i c k s and r e p l i c a t e d onto lawns of P_. acidovorans s t r a i n 29. R e p l i c a s were incubated at 30° and 20°. Phage which l y s e d lawns of s t r a i n 29 at 20° but not at 30° were plaque p u r i f i e d f o r f u r t h e r study. I s o l a t i o n of 0W-14 am mutants 0W-14 am mutants were i s o l a t e d from mutagenized stocks of 0W-14 by P. M i l l e r . P_. acidovorans s t r a i n 29 (sup 2) was the permissive host and P_. acidovorans s t r a i n s 29 or 3L were the non permissive hosts. Complementation of ts 0W-14 Ts stock l y s a t e s were d i l u t e d to approximately 10^ to 10^ pfu ml-"*". Samples were spotted onto a p l a t e backed by a numbered g r i d and c r o s s -t e s t e d against other 0W-14 ts_ mutants. P l a t e s were incubated at 30°. Lawn c l e a r i n g at 30° by two separate _ts_ mutants was scored as +. The method was not s a t i s f a c t o r y f o r leaky _ts_ mutants. Screening _ts_ and am mutants Ten ml c u l t u r e s of P_. acidovorans s t r a i n 29, growing at 30°, were i n f e c t e d at m u l t i p l i c i t i e s of i n f e c t i o n of 20. At 20 minutes a f t e r i n f e c t i o n , samples f o r s u r v i v o r s were p l a t e d on CAA-M agar. At 25 min-• -1 3 utes a f t e r i n f e c t i o n , 1.0 uCi ml of [6- H ] - u r a c i l without c a r r i e r was 33 added and i n c o r p o r a t i o n was continued u n t i l 45 minutes p o s t - i n f e c t i o n f o r am mutants and l a t e r f o r _ts_ mutants. DNA was p u r i f i e d from the phage-i n f e c t e d c e l l s as described below. DNA p u r i f i c a t i o n Samples were poured i n t o an equal volume of i c e - c o l d TNE and c e n t r i f u g e d at 8,000 rpm f o r 5 minutes at 4°. The supernatant was d i s -carded. ( I n f e c t e d c e l l s s t i c k to the s i d e of the c e n t r i f u g e tube.) Ice-cold TNE was added to the tube and the c e l l s scraped from the s i d e . Two mgml "cjf s e l f - d i g e s t e d pronase was added. Then an equal volume lxSSC and SDS to a f i n a l c o n c e n t r a t i o n of 0. 5 percent was added. The v i s c o u s l y s a t e was incubated overnight at 37°. In the morning, SSC-washed, r e d i s t i l l e d phenol was added and the DNA was e x t r a c t e d by r o l l i n g at low speed f o r 2 hours. The mixture was spun at 5,000 rpm f o r 5 minutes and the phenol l a y e r and the i n t e r f a c e was removed. The aqueous phase was r e e x t r a c t e d once w i t h phenol as described above. The aqueous l a y e r was washed three times w i t h water-saturated ether. The DNA was p r e c i p i t a t e d w i t h two v o l -umes of 95 percent ethanol. The p r e c i p i t a t e d DNA was resuspended overnight i n TNE. The next day 50 yg ml 1 of h e a t - t r e a t e d p a n c r e a t i c RNase was added. D i g e s t i o n w i t h RNase was c a r r i e d our f o r 1 hour at 37°. The DNA was r e e x t r a c t e d once w i t h phenol and then ether washed. The DNA was pre-c i p i t a t e d w i t h 95 percent ethanol and resuspended i n TNE. DNA s o l u t i o n s were kept f r o z e n at -20° u n t i l ready f o r use. A c i d h y d r o l y s i s of DNA samples A DNA sample was e t h a n o l - p r e c i p i t a t e d , washed three times w i t h 95 percent ethanol and twice w i t h ether. The DNA was a i r - d r i e d and resuspended 34 i n 0.2 ml of 6N HCI. The sample was incubated u n t i l i t had dissolved and was transferred to a hydrolysis v i a l . The hydrolysis v i a l s were sealed under reduced pressure and then placed at 100° for 2 hours. This treatment converted 80 to 90 percent of the pyrimidine nucleotides to free bases. Although conversion was greater than 90 percent for a longer hydrolysis time, the deamination of cytosine was increased also. The other pyrimidine bases were stable. After h y d r o l y s i s , v i a l s were opened and the HCI was removed from samples by evaporation i n a vacuum dessicator over NaOH. TLC of acid-hydrolyzed DNA The samples were resuspended i n 25 y l of 0.2 N HCI and 25 y l of a standard base mixture containing 1 mg ml ^ each of putThy, Cyt, Ura, hmUra and Thy. The t o t a l hydrolysates were spotted as small (<0.5 cm) diameter spots on c e l l u l o s e t h i n - l a y e r sheets. Bases were resolved by two-dimensional development with solvents B and D. The thin-layers were thoroughly a i r - d r i e d a f t e r chromatography i n each solvent. P u l s e - l a b e l l i n g procedures The p u l s e - l a b e l l i n g protocol was s i m i l a r f o r a l l the isotopes. 8 —1 Cultures were grown to a density of 3 x 10 ml and infected at a mul-t i p l i c i t y of i n f e c t i o n (moi) of 10. At 35 to 40 minutes a f t e r i n f e c t i o n , 32 3 3 the c e l l s were l a b e l l e d with [6- H ] - u r a c i l or [5- H ] - u r a c i l f o r 32 10 seconds. C e l l s f o r PO^ p u l s e - l a b e l l i n g were grown i n TCS 1/5 x P. and the pulse was stopped by pouring the culture over one volume of par-t i a l l y frozen 0.1 M sodium phosphate buffer pH 7.0, containing EDTA (0.01 M) and 0.02M KCN. 35 C e l l s f o r t r i t i a t e d u r a c i l p u l s e - l a b e l l i n g were grown i n TCS 1 x P^ and the pulse was stopped by pouring the c u l t u r e over one volume of p a r t i a l l y f r o z en TNE b u f f e r c o n t a i n i n g 1 rag ml 1 u r a c i l and 0.02 M KCN. The c e l l s were spun down at 5,000 rpm f o r 5 minutes, resuspended i n i c e - c o l d TNE and r e p e l l e t e d . Once again the c e l l s were resuspended i n TNE and processed, as described p r e v i o u s l y , f o r the e x t r a c t i o n of i n t r a c e l l u l a r DNA. A l l pulse experiments were c a r r i e d out at 30°. Nucleotide pool a n a l y s i s 14 32 [2- C ] - u r a c i l and PO ^ - l a b e l l e d n u c l e o t i d e pools were prepared according to published procedures. 32 PO^ was added one to two c e l l doublings p r i o r to i n f e c t i o n i n order to all o w e q u i l i b r i u m l a b e l l i n g of the n u c l e o t i d e pools. At a den-8 —1 s i t y of 3 x 10 c e l l s ml the c u l t u r e was i n f e c t e d at a m u l t i p l i c i t y of i n f e c t i o n of 10. Fiv e ml samples were taken a t the i n d i c a t e d times and passed through 0.45 p M i l l i p o r e HA membranes. The f i l t e r s were washed w i t h 2.5 ml each of i c e - c o l d TCS 1 x P. and t r a n s f e r r e d to a beaker con-l t a i n i n g 2.0 ml of 0.3 N HCOOH. The formic a c i d - e x t r a c t e d c e l l s on the f i l t e r were kept on i c e . A f t e r 30 minutes the c e l l s were resuspended i n the formic a c i d by scraping them o f f the f i l t e r s . The f i l t e r s were r e -extr a c t e d w i t h 0.5 ml of 0.3 N HCOOH. The two f r a c t i o n s were combined and c e n t r i f u g e d at 8,000 rpm f o r 10 minutes at 4°. The supernatant was removed t a k i n g care not to d i s t u r b the c e l l p e l l e t , which was discarde d . The formic a c i d e x t r a c t was l y o p h i l i z e d , and the residue resuspended i n a measured volume of dH^O. The n u c l e o t i d e pools were stored at -20° u n t i l chromatography. 36 Chromatography of n u c l e o t i d e pool  preparations of P E I - c e l l u l o s e Chromatography procedures were described by Randerath and Randerath (1966). The PEI sheets used were prepared according to the procedures of Randerath (1966). CsCl d e n s i t y gradient a n a l y s i s of DNA Fi v e g of a saturate d s o l u t i o n of CsCl i n dR^O was added to a polyallomer or n i t r o c e l l u l o s e u l t r a c e n t r i f u g e tube. DNA samples and TNE were added u n t i l the t o t a l gradient weight was 6.0 g. The grad-i e n t s were mixed, overlayed w i t h p a r a f f i n o i l and c e n t r i f u g e d f o r 72 hours at 30,000 rpm i n a SW 50.1 or SW 39 r o t o r . The bottoms of the tubes were punctured and the gradients dripped onto glass f i b e r f i l t e r s or Whatman 3MM paper squares. The glass f i b r e f i l t e r s were d r i e d at 80°, overlayed w i t h s c i n t i l l a n t and counted. 3 14 Gradients c o n t a i n i n g H and C l a b e l were dripped onto num-bered squares of paper. The squares were washed three times i n i c e -c o l d 5 percent TCA, three times i n 95 percent ethanol and twice i n ether. The DNA samples were el u t e d from the paper i n v i a l s overnight w i t h 0.5 ml of 0.1 N HCI. In the morning, 0.5 ml of 0.1 N NaOH was added to n e u t r a l i z e the a c i d , 10 ml of TritonrToluene or Bray's s c i n -t i l l a n t (1960) was added and the samples were mixed, allowed to stab-i l i z e , and counted. Cross channel overlap was estimated by preparing standards c o n t a i n i n g known r a t i o s of isotopes and t r e a t i n g them i n an i d e n t i c a l manner to authe n t i c samples. Quenching was assumed to be i d e n t i c a l f o r a l l s i m i l a r l y processed samples and standards. 37 So n i c a t i o n of DNA DNA samples were d i l u t e d to the same conc e n t r a t i o n before son-i c a t i o n . S o n i c a t i o n was f o r a t o t a l of 1 minute (4 x 15 second b u r s t s at a s e t t i n g of 50 on a B r o n w i l l s o n i c o s c i l l a t o r ) . Samples were kept on i c e p r i o r to and during s o n i c a t i o n . Heat treatment of DNA DNA samples i n the TNE or 1 x SSC were b o i l e d f o r 5 minutes i n a capped tube and then quick-cooled by p l a c i n g the tube i n an i c e buc-ket. Measurement of DNA accumulation 3 -1 [6- H ] - u r a c i l - Cultures contained 1.0 uCi ml of l a b e l and 10 yg ml 1 of c o l d u r a c i l . 0.1 ml samples were taken d i r e c t l y i n t o 1.0 ml of 0.3 NaOH and incubated overnight at 37°. In the morning 1.0 ml of 0.3 N HCI and 0.25 ml of 50 percent TCA were added to the samples. A f t e r thorough mixing the samples were c h i l l e d on i c e f o r at l e a s t 60 minutes. The p r e c i p i t a t e d m a t e r i a l was trapped on 0.45 y M l l l i p o r e membranes. The tubes were washed three times w i t h i c e - c o l d 5 percent TCA and then three times w i t h 95 percent e t h a n o l , each washing being passed through the membrane. The f i l t e r s were placed i n s c i n t i l l a t i o n v i a l s , d r i e d at 80°, overlayed w i t h Toluene-based s c i n t i l l a n t and counted. 3 -1 [ H ] - o r n i t h i n e - Cultures contained 1.0 y C i ml of l a b e l and 10 yg ml 1 of o r n i t h i n e and 100 yg ml 1 of a r g i n i n e . 0.1 ml samples were taken i n t o 1.0 ml of TNE c o n t a i n i n g 0.5 percent SDS and 100 yg ml 1 each of a r g i n i n e and o r n i t h i n e . Pronase was added to a f i n a l concentra-t i o n of 2 yg ml 1 and d i g e s t i o n was c a r r i e d out overnight at 37°. I n the 38 morning, TCA was added to a f i n a l c o ncentration of 5 percent. The samples were c o l l e c t e d , washed, d r i e d and counted as described above. 3 3 [methyl- H]-TdR - Cultures contained 10 y C i [ H]-TdR per ml of c u l -t ure and 250 yg TdR per ml. 0.1 ml samples were taken i n t o 1 ml of 5 percent TCA. Samples were c h i l l e d and processed as described above. 3 Measurement of t r i t i u m r e l e a s e from [5- H"|-uracil 3 [5- H ] - u r a c i l was added to the c u l t u r e s c o n t a i n i n g 10 yg c o l d u r a c i l ml \ 0.5 ml samples were taken at i n t e r v a l s i n t o 1.0 ml of a PCA-Norit s l u r r y (20 g c h a r c o a l per 100 ml of 4 percent PCA). The sam-p l e s were f i l t e r e d through a GF/F f i l t e r and the f i l t r a t e was r e t a i n e d . An a l i q u o t of the f i l t r a t e was counted i n Bray's s o l u t i o n or i n S c i n t i -verse. Enzymatic d i g e s t i o n of DNA The concentration of the DNA sample was determined spectrophoto-m e t r i c a l l y and the r e q u i r e d amount of DNA was p r e c i p i t a t e d w i t h two volumes of 95 percent e t h a n o l , washed s e v e r a l times w i t h 70 percent e t h a n o l , sev-e r a l times w i t h 95 percent e t h a n o l , then w i t h ether and a i r - d r i e d . The sample was r e d i s s o l v e d i n the minimum volume of s t e r i l e d i s t i l l e d , d eion-i z e d water, then b o i l e d f o r 5 minutes to i n a c t i v a t e r e s i d u a l nucleases and to ensure denaturation of DNA. Ammonium acet a t e , pH 5.0 was added to give a f i n a l c o n c e n t r a t i o n of 50 mM, ZnSO^ was added to give a f i n a l concentra-t i o n of 0.1 mM and 10 u n i t s of S, nuclease were added per yg of DNA. The sample was incubated f o r 4 hours at 55°, then l y o p h i l i z e d . The r e s i d u e was resuspended i n d e i o n i z e d , d i s t i l l e d water and l y o p h i l i z e d again. The residue was suspended i n the minimum volume of d e i o n i z e d , d i s t i l l e d water and NH.C0„, pH 8.4, and MgCl„ were added to f i n a l concentrations 39 of 100 mM and. 15 mM, r e s p e c t i v e l y . Snake venom phosphodiesterase was -1 added to a f i n a l c o n c e n t r a t i o n of 20 yg ml and the sample was i n c u -bated at 37° f o r 2 hours. L i m i t d i g e s t i o n of DNA w i t h DNase I and snake venom phospho-d i e s t e r a s e was performed as f o l l o w s . The DNA was t r e a t e d as described f o r S^ d i g e s t i o n . The d r i e d sample was d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water. T r i s - H C l , pH 8.2 and MgC^ were added to give f i n a l concentra-t i o n s of 50 mM and 15 mM, r e s p e c t i v e l y . Then 20 yg of each enzyme were added per ml of DNA s o l u t i o n and the mixture incubated at 37° f o r 4 hours. The n u c l e o t i d e s released were separated by t h i n - l a y e r chroma-tography and detected by fluorography f o r t r i t i u m - l a b e l l e d samples or by 32 autoradiography f o r P - l a b e l l e d samples, (see below). T h i n - l a y e r chromatography of enzymatic DNA d i g e s t s The enzymatic d i g e s t s were spotted d i r e c t l y on sheets of unmod-i f i e d c e l l u l o s e (Eastman chromogram, 6064, without f l u o r e s c e n t i n d i c a t o r ) . The sheets were washed twice by ascending development w i t h 95 percent ethanol to remove i n o r g a n i c s a l t s . Mononucleotides remained at the o r i g i n . Mononucleotides were separated by development w i t h solvent E and then w i t h s o l v e n t A. The d i r e c t i o n of s e p a r a t i o n i n the f i r s t dimension was perpendicular to the d i r e c t i o n of washing. Chromatography sheets were d r i e d i n between f i r s t dimension and second dimension chromatography. Solvents f o r TLC on unmodified c e l l u l o s e Solvent A - saturated (NH^^SO^:! M Na a c e t a t e : i s o p r o p a n o l (80:12:2 v / v ) . 40 Solvent B - t-butanol:2-butanone:cone. HCI-rH^O (40:30:10:20 v / v ) . Solvent C - n-butanol:H 20 (86:14 v / v ) . Solvent D - n-butanol:H 20:cone. NH^OH (86 :9:5 v / v ) . Solvent E - i s o b u t y r i c acid:H 20:cone. NH^OH (66:20:1 v / v ) . Detection of r a d i o a c t i v i t y on t h i n - l a y e r sheets T r i t i u m - l a b e l l e d n u c l e o t i d e s were detected by impregnating sheets w i t h a 7 percent s o l u t i o n of PPO i n ether. The ether was evaporated and the chromatogram was exposed to f i l m at -70° f o r an appropriate l e n g t h of time p r i o r to development (Randerath, 1969). 32 Chromatograms c o n t a i n i n g P - l a b e l l e d n u c l e o t i d e s were exposed to f i l m and the f i l m was developed a f t e r an appropriate length of time. B a c t e r i a l a l k a l i n e phosphate d i g e s t i o n 0.03 u n i t s of BAP-F ( M i l l i p o r e Corp.) was added to the sample i n 50 mM T r i s - H C l , pH 8.4 co n t a i n i n g 15 mM MgCl 2. D i g e s t i o n was c a r r i e d out at room temperature f o r one hour. The enzymatic d i g e s t s were spotted d i r e c t l y on t h i n - l a y e r sheets. Column chromatography B i o g e l P 2 chromatography of S^-snake venom phosphodiesterase d i g e s t of am 37 DNA. c o l U m n dimensions were 45 x 1 cm. The e l u -t i o n b u f f e r was 0.1 M ammonium acetate, pH 7.0.. The sample was loaded i n a volume of 0.8 ml of e l u t i o n b u f f e r , and 2.5 ml f r a c t i o n s were c o l -l e c t e d . DEAE - Sephadex chromatography. The column was 8 x 1 cm. •The. sample-was a p p l i e d i n 20 mM. T r i s - H C l , pH.8,.0.. The column was 41 e l u t e d w i t h a l i n e a r gradient of NaCl from 0 to 0.4 M, t o t a l volume 160 ml, and 3 ml f r a c t i o n s were c o l l e c t e d . DEAE-Sephadex-7 M urea chromatography. Twenty mM T r i s - H C l pH 32 7.5 P - l a b e l l e d am 37 DNA was digested to mononucleotides w i t h nuclease and snake venom phosphodiesterase. The d i g e s t was d i l u t e d to 3.0 ml w i t h 20 mM T r i s - H C l pH 7.5, 7 M urea. dTTP and dTDP were added to give f i n a l concentrations of 75 yg ml 1 . The sample was a p p l i e d to a DEAE-Sephadex A-25 column (1 x 40 cm). The r e s i n was washed w i t h one bed volume of 20 mM T r i s - H C l , 7 M urea and the n u c l e o t i d e s were eluted w i t h a l i n e a r gradient on NaCl (0 to 0.4M) i n t h i s b u f f e r . The t o t a l volume of e l u a t e was 500 ml. I t was pumped through the column at a constant r a t e of 1 ml min 1 . F r a c t i o n volume was 5 ml. Samples were assayed f o r Cerenkov r a d i a t i o n and f o r absorbance at 267 nm. DEAE-Sephadex columns were i n the c h l o r i d e form. P u r i f i c a t i o n of the unknown n u c l e o t i d e 32 P - l a b e l l e d S^-snake venom phosphodiesterase d i g e s t s of am 37 r e p l i c a t i n g DNA were a p p l i e d as a band 9 cm from the top of a sheet of Whatman SFC-40 f i l t e r paper (40 x 20 cm). The sheet was washed by descending development i n 95 percent ethanol. I t was then d r i e d and developed i n one dimension by descending development w i t h Solvent E. The bottom of the sheet had been s e r r a t e d to ensure the uniform flow of solvent as i t dripped o f f the paper. A f t e r autoradiography, to l o c a t e the n u c l e o t i d e s , s t r i p s c o n t a i n i n g them were cut from the sheets and washed f r e e of i s o b u t y r a t e w i t h 95 percent ethanol by descending chromatography. The s a l t - f r e e n u c l e o t i d e s were recovered from paper s t r i p s by descending development w i t h d i s t i l l e d water. 42 P r e p a r a t i o n of c e l l - f r e e e x t r a c t s 8 —1 S t r a i n 29 was grown to a d e n s i t y of 3 x 10 c e l l s ml then i n f e c t e d w i t h phage at a m u l t i p l i c i t y of i n f e c t i o n of 20. The c e l l s were c o l l e c t e d by c e n t r i f u g a t i o n at 35 minutes a f t e r i n f e c t i o n (3,000 g x 5 minutes at ambient temperature). The c e l l p e l l e t was resuspended i n one growth volume of b u f f e r (50 mM T r i s - H C l , pH 7.5, 10 mM 3-mercap-toetha n o l , 10 mM MgC^ and 50 mM KCl) and r e p e l l e t e d . The c e l l s were 9 -1 resuspended i n the T r i s b u f f e r at a d e n s i t y of 5 x 10 ml . Lysozyme was added to a f i n a l c o ncentration of 1 mg ml ^ and the suspension was incubated at 30° f o r 30 minutes. The suspension was q u i c k - f r o z e n i n an a l c o h o l - d r y - i c e bath and q u i c k l y thawed at 30°. The freeze-thaw step was repeated three times. The r e s u l t i n g e x t r a c t was passed through a 26 gauge needle to reduce the v i s c o s i t y . In v i t r o s y n t h e s i s of putThy Reaction mixtures contained 30 y l p u t r e s c i n e (10 mM), 30 y l 32 -1 crude c e l l e x t r a c t , 30 y l of P - l a b e l l e d am 37 DNA (400 yg ml ) and 210 y l of Reaction b u f f e r (see above). The am 37 DNA substrate contained an unknown p r o p o r t i o n of s t r a i n 29 DNA and p a r e n t a l am 37 DNA. The e f f e c t of these substrates on the r e a c t i o n i s not known. At i n t e r v a l s during i n c u b a t i o n at 30°, 50 y l samples were removed to 0.5 ml of i c e -c o l d T r i s - H C l (10 mM), 0.15 M NaCl, 0.01 M EDTA, pH 7.5, c o n t a i n i n g 100 yg ml of u n l a b e l l e d 0W-14 DNA. Two volumes of i c e - c o l d 95 percent ethanol were then added to the samples. A f t e r standing on i c e f o r 30 minutes the DNA was c o l l e c t e d by c e n t r i f u g a t i o n and 500 y l of the e t h a n o l - b u f f e r supernatant was removed f o r the determination of released r a d i o a c t i v i t y . The DNA was repurified and digested with SI and snake venom phospho-diesterase as described earlier. The mononucleotides were separated by 2-dimensional*thin-layer-chromatography on cellulose thin-layer sheets. Chemicals Chemicals were of analytical grade and were used without fur-ther purification. Enzymes Pancreatic RNase was purchased from Calbiochem. It was dis-solved in TNE at a concentration of 1 mg ml 1 and boiled at 100° for 5 minutes prior to use. DNase I and snake venom phosphodiesterase were purchased from Calbiochem and were used without further treat-ment or purification. Nuclease SI was obtained from Miles Laboratories Inc. Bacterial alkaline phosphatase F (BAP-F) was purchased from the Millipore Corporation. Radiochemicals A l l radiochemicals were purchased from New England Nuclear and were used without further purification. RESULTS AND DISCUSSION Nucleot i d e pools K e l l n (1973) detected putThy n u c l e o t i d e s i n a c i d - s o l u b l e e x t r a c t s of 0 W-14-infected P_. acidovorans s t r a i n 29. This was pre-sumptive evidence that putdTTP was a precursor of putThy i n 0W-14 DNA. Seve r a l attempts to repeat t h i s experiment were un s u c c e s s f u l . 3 S p e c i f i c a c t i v i t i e s of [ H ] - o r n i t h i n e ten times greater than used i n normal DNA accumulation p r o t o c o l s d i d not l a b e l any n u c l e o t i d e s r e s o l v a b l e by 2D-PEI c e l l u l o s e t h i n - l a y e r chromatography. 14 [2- C ] - u r a c i l l a b e l l i n g of n u c l e o t i d e pools i n i n f e c t e d and u n i n f e c t e d P_. acidovorans s t r a i n 29 was a l s o undertaken. A l l u r a c i l - l a b e l l e d compounds separable u s i n g the t o t a l pool s e p a r a t i o n p r o t o c o l appeared i n both i n f e c t e d and uninfe c t e d c e l l e x t r a c t s (Figure 2). Micromole q u a n t i t i e s of l a b e l l e d n u c l e o t i d e s could have been detected and res o l v e d . Thy, hmUra or Ura n u c l e o t i d e s con-t a i n i n g the same number of phosphate groups were not res o l v e d from each other. W i t h i n the l i m i t s of d e t e c t a b i l i t y there were no putThy n u c l e o t i d e s i n the s o l u b l e pools of i n f e c t e d c e l l s . Sub-sequently Neuhard and Warren (1980) were able to demonstrate the presence of hmdUTP i n the n u c l e o t i d e pools of 0 W-14-infected c e l l s . dTTP i s excluded from the n u c l e o t i d e pools of i n f e c t e d c e l l s . dTTP, dUTP and hmdUTP were not resolved i n the normal P E I - c e l l u l o s e system used f o r the r e s o l u t i o n of n u c l e o t i d e triphosphates. A FIGURE 2 . — R e s o l u t i o n of [2- C ] - u r a c i l - l a b e l l e d n u c l e o t i d e s i n unin f e c t e d and i n f e c t e d c e l l s . Samples were c o l l e c t e d from A) Uninfected P_. acidovorans s t r a i n 29, and B) P_. acidovorans s t r a i n 29 at 35 minutes a f t e r i n f e c t i o n w i t h 0W-14. The l a b e l l i n g , e x t r a c t i o n and chromatography of the n u c l e o t i d e pools are as described i n the M a t e r i a l s and Methods s e c t i o n . 46 47 B three-dimensional system employed by Neuhard re s o l v e s dTTP and hmUTP from each other (Neuhard, personal communication). I n v i t r o , the s o l e d e t e c t a b l e product of THFA-mediated 14 14 [ C]-formaldehyde l a b e l l i n g of dUMP was [ C]-hmdUMP (Neuhard et a l . , 1980). Kinases capable of generating hmdUTP from hmdUMP were a l s o detected. d!TTP i s excluded from i n f e c t e d c e l l nucleo-t i d e pools by 30 minutes a f t e r i n f e c t i o n . 0W-14 i n f e c t i o n i n -duces an a c t i v i t y capable of i n h i b i t i n g host thymidylate synthase as w e l l as a potent dUTP/dTTPase (Neuhard et a l . , 1980). Measurements of n u c l e o t i d e l e v e l s i n pools extracted from c e l l s at various times a f t e r i n f e c t i o n were performed (Table 3). 32 I n f e c t e d c e l l pools uniformly l a b e l l e d w i t h PO^ were separated by 2D-TLC on P E I - c e l l u l o s e . In 0W-14-infected c e l l s the l e v e l s of a l l measured n u c l e o t i d e s dropped d e s p i t e the f a c t that DNA syn t h e s i s rates 1 a f t e r i n f e c t i o n are greater than r a t e s i n u n i n -f e c t e d c e l l s ( K e l l n and Warren, 1973). This means that the flow of precursors through n u c l e o t i d e pools was i n e q u i l i b r i u m w i t h the demands of DNA synth e s i s f o r precursors. More meaningful determinations of n u c l e o t i d e pool a c t i v i t i e s could probably be obtained u s i n g procedures to measure the d i f f e r e n t i a l s y n t h e s i s r a t e of each p o o l n u c l e o t i d e . The dCTP and hmdUTP pools d e c l i n e d i n s i z e a f t e r i n f e c t i o n u n t i l they reached an e q u i l i b r i u m l e v e l (Table 3). This c o n t r a s t s w i t h the data of Neuhard (personal communication), who observed i n c r e a s e s i n pool s i z e s f o r hmdUTP and dCTP a f t e r an i n i t i a l d e c l i n e . The d i f f e r e n c e s between 49 TABLE 3.—The deoxynucleoside triphosphate pools of 0W-14-infected P. acidovorans s t r a i n 29. Nucleot i d e 0 min 10 min Time a f t e r 20 min i n f e c t i o n 30 min 40 min 50 min dGTP 1318 601 366 274 266 218 dATP 429 338 362 - 169 271 dCTP 5555 1281 1376 1961 1225 1181 dUTP/dTTP/hmdUTP 1704 750 414 421 408 236 Nuc l e o t i d e pools were l a b e l l e d , prepared and separated as described i n the M a t e r i a l s and Methods. The values given are cpm i n the area cut from the chromatogram. dUTP, dTTP and hmdUTP were not separated from one and other i n t h i s experiment. r e s u l t s can probably be explained by the use of d i f f e r e n t media, growth temperature and a e r a t i o n c o n d i t i o n s . In our l a b o r a t o r y , accumulation of nuc l e o t i d e s i n i n f e c t e d c e l l s i s seen only i n DO amber mutants grown under nonpermissive c o n d i t i o n s (P. M i l l e r , unpublished o b s e r v a t i o n s ) . These mutants accumulate n u c l e o t i d e s a f t e r i n f e c t i o n because they are unable to make phage DNA. Un-l i k e T4, i n 0W-14 DNA hmdUTP and dCTP are not found i n the s o l -uble n u c l e o t i d e pools at the same r a t i o s that they are found i n phage DNA. The s i z e of the dCTP pool i n i n f e c t e d c e l l s was approx-imately four times greater than the s i z e of the hmdUTP pool. This d i f f e r e n c e d i d not appear to have any s i g n i f i c a n c e . The s i z e of the i n f e c t e d c e l l s ' dCTP and hmdUTP pools was l e s s important than the r a t e of flow of precursors through the po o l . The flow of dCTP, hmdUTP, dGTP and dATP flow through the n u c l e o t i d e pools was balanced. I n v i t r o assays and pool s t u d i e s by Neuhard and Warren suggested that hmdUTP and not dTTP i s the precursor of putThy and Thy i n 0W-14 DNA. Deoxyuridine but not deoxythymidine  was a 0W-14 DNA precursor Before r a d i o a c t i v e deoxyuridine could be used to l a b e l 0W-14 DNA, i t was necessary to i s o l a t e a d e r i v a t i v e of s t r a i n 3L which would not incor p o r a t e exogenous u r a c i l i n t o DNA. 3 This was necessary because commercial preparations of [6- H]-dUdR 14 and [2- C]-dUdR contain! appreciable q u a n t i t i e s of r a d i o a c t i v e 51 u r a c i l . U r a c i l phosphorylase mutants are r e s i s t a n t to 5-fluor-o u r a c i l because they cannot make 5-fluoro UMP (O'Donovan and Neuhard, 1970). Therefore host mutants without u r a c i l phosphor-ylase a c t i v i t y are r e a d i l y i s o l a t e d by growing c e l l s i n the pre-sence of 5 - f l u o r o u r a c i l . 5 - f l u o r o u r a c i l d e r i v a t i v e s of 3L were i s o l a t e d from plates spread with 3L and seeded with c r y s t a l s of 5-FU. Colonies appearing i n s i d e zones of growth i n h i b i t i o n a f t e r 2 to 3 days were picked, restreaked and r e i s o l a t e d as described above. Five 3L/FU s t r a i n s were obtained t h i s way. They required TdR for growth and were s e n s i t i v e to 0W-14 i n f e c t i o n . The i s o l a t e s were tested for the a b i l i t y to incorporate [6- H ] - u r a c i l (Figure 3 ) . Of the f i v e clones, only one, 3L/FU _2, was found which did not 3 incorporate [ H ] - u r a c i l i n t o TCA i n s o l u b l e counts i n uninfected or infected c e l l s (Figure 3) . 3L/FU 2_ w a s tested for the a b i l i t y 3 to incorporate [6- H]-dUdR into uninfected 0W-14 infected c e l l DNA. 3 Only infected c e l l s accumulated [6- H]-dUdR i n . a l k a l i - r e s i s t a n t , TCA-precipitable material (Figure 4). Acid hydrolysis and t h i n -3 layer chromatography of 0W-14 DNA l a b e l l e d with [6- H]-dUdR showed that i t contained l a b e l l e d Thy and putThy (Table 4). Cytosine 3 was not l a b e l l e d . As a c o n t r o l , [ H ] - o r n i t h i n e - l a b e l l e d 0W-14 DNA grown i n 3L/FU 2_ was prepared and subjected to acid hydrolysis and t h i n - l a y e r chromatography. Ornithine l a b e l s this DNA, and i t was assumed that the compound detected i n hydrolysates was putThy. In 0W-14 infected 3L/FUR2_ l a b e l l e d with [6-3H]-dUdR, putThy and Thy accumulated i n the DNA at equal rates throughout i n f e c t i o n (Figure 5). FIGURE 3.—The i n c o r p o r a t i o n of u r a c i l by 3L/FU _2. 3 - 1 - 1 At 0 minutes [6- H ] - u r a c i l (1.0 uCi ml , 10 pg ml ) was added to c u l t u r e s of 3 L , 0 ; 3L/FUR_2,# ; and 0W-14 i n f e c t e d 3L/FUR2_, k. I n c o r p o r a t i o n of the l a b e l i n t o TCA p r e c i p i t a b l e m a t e r i a l was foll o w e d . 0 10 20 30 40 50 MINUTES 54 FIGURE 4.—The i n c o r p o r a t i o n of deoxyuridine i n 0W-14 i n f e c t e d 3L/FU _2. A growing c u l t u r e of 3L/FU j2 was s p l i t i n t o two halves. One h a l f was 3 -1 i n f e c t e d w i t h 0W-14. [6- H]-UdR (4.0 y C i ml , without c a r r i e r ) was added at 10 minutes a f t e r i n f e c t i o n . The i n c o r p o r a t i o n of r a d i o a c t i -v i t y i n t o a l k a l i r e s i s t a n t , TCA p r e c i p i t a b l e m a t e r i a l was fol l o w e d . 0W-14 i n f e c t e d 3L/¥lSR2,m ; 3L/FUR_2, O . The arrow i n d i c a t e s the begin-ning of c e l l l y s i s i n the i n f e c t e d c u l t u r e . 400 i 10 20 30 40 50 60 70 80 MINUTES TABLE 4.—Deoxyuridine l a b e l l i n g of bases i n 0W-14 i n f e c t e d 3L/FU 2_. Base [ 3H]-orn [6- 3H]-Ura putThy Cyt Thy 1593 ( 0 . 9 5 ) a 37 (0.02) 55 (0.03) 1853 (0.48) 88 (0.02) 1903 (0.50) 0W-14 i n f e c t e d c u l t u r e s of 3L/FU"^2 were l a b e l l e d w i t h [ JH]-orn (0.1 y - 1 3 - 1 C i yg ) or [6- H]-dTJdR (4.0 y C i ml ). The c u l t u r e s were allowed to l y s e and the DNA was p u r i f i e d from the phage p a r t i c l e s . The DNA was acid-hydrolyzed and the bases were separated by one dimensional t h i n -l a y e r chromatography i n solvent E. a The values shown are cpm i n areas cut from the chromatogram. The values i n parentheses are the f r a c t i o n s of the r a d i o a c t i v i t y recov-ered i n l a b e l l e d bases. FIGURE 5.—The accumulation of Thy and putThy i n 0W-14 DNA. R 3 3L/FU _2 was i n f e c t e d w i t h 0W-14 and l a b e l l e d w i t h [6- H]-UdR as des-c r i b e d i n Figure 4. A l i q u o t s of the i n f e c t e d c u l t u r e were withdrawn at i n t e r v a l s a f t e r i n f e c t i o n and DNA was p u r i f i e d from the c e l l s . The DNA was hydrolyzed i n 6N HCI and Thy and putThy were separated by 1D--TLC. 3 H - l a b e l i n Thy,0 ; 3 H - l a b e l i n putThy,* . K e l l n had reported that [methy1- JH]-TdR would l a b e l 0W-14 DNA i n P_. acidovorans 3L. This r e s u l t could not be reproduced. The i n c o r p o r a t i o n of thymidine i n u n i n f e c t e d and i n f e c t e d 3L was measured. Only uninfected 3L accumulated a l k a l i - r e s i s t a n t , T CA-precipitated l a b e l (Figure 6). Warren has obtained s i m i l a r r e s u l t s , and he has a l s o shown that host DNA p r e l a b e l l e d w i t h 3 [ H]-TdR i s not degraded and r e i n c o r p o r a t e d i n t o 0W-14 DNA (Maltman et a l . , 1980). Although the data s t r o n g l y suggested t h a t 0 W-14-infected c e l l s could not i n c o r p o r a t e deoxythymidine, i t was p o s s i b l e that t h i s was due to an overwhelming preference f o r endogenously sup-p l i e d deoxythymidine n u c l e o t i d e s . 0 W-14-infected s t r a i n 3L pro-duced v i a b l e phage bursts a f t e r exogenous thymidine was removed by c e n t r i f u g a t i o n and washing (Figure 7). 0 W-14-infected 3L was spun down, washed and resuspended i n conditioned TCS medium -1 3 without c o l d thymidine but c o n t a i n i n g 25 y C i ml [methyl- H]-TdR. The i n f e c t e d c e l l s were allowed to l y s e and DNA was e x t r a c -ted from the p u r i f i e d phage. A small amount of t r i t i u m - l a b e l l e d 0W-14 DNA was obtained which banded w i t h reference 0W-14 DNA i n n e u t r a l CsCl gradients (Figure 8). A c i d - h y d r o l y s i s and TLC of t h i s DNA showed that none of the l a b e l was i n thymine; most of the l a b e l i n bases ran i n the guanine, adenine region of the chromatogram. Only 6 percent of the l a b e l was recovered in. bases. Therefore, even s p e c i f i c a c t i v i t i e s of l a b e l great enough to cause "garbage l a b e l l i n g " of 0W-14 DNA, f a i l e d to l a b e l the thy-mine bases of 0W-14 DNA. FIGURE 6.—Thymidine i n c o r p o r a t i o n i n s t r a i n 3L and i n 0W-14-i n f e c t e d s t r a i n 3L. 3 -1 -1 [Methyl- H]-thymidine (0.04 uCi yg , 250 yg ml ) was added to a c u l t u r e of 0W-14 i n f e c t e d 3L,#; and to an u n i n f e c t e d c u l t u r e of 3L,0 . The i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o TCA p r e c i p i -t a b l e r a d i o a c t i v i t y was determined. 61 FIGURE 7.—Thymidine was not req u i r e d f o r plaque production i n 0W-14 i n f e c t e d s t r a i n 3L. At 10 minutes a f t e r i n f e c t i o n of s t r a i n 3L w i t h 0W-14, the i n f e c t e d c u l t u r e was spun down and resuspended i n one volume of thymidine f r e e conditioned TCS medium. A l i q u o t s of the i n f e c t e d c u l t u r e were pla t e d through CHCL„ at i n t e r v a l s . 25 35 45 MINUTES A F T E R RESUSPENSION FIGURE 8 . — N o n s p e c i f i c l a b e l l i n g of 0W-14 DNA by high s p e c i f i c a c t i v i t y thymidine. P. acidovorans 3L was i n f e c t e d w i t h 0W-14 and at 10 minutes a f t e r i n f e c t i o n the c e l l s were spun down and resuspended i n conditioned -1 3 medium c o n t a i n i n g 25.0 uCi ml of [methyl- H]-TdR (without c a r -r i e r ) . The c u l t u r e s were allowed to l y s e and the DNA was p u r i f i e d from phage p a r t i c l e s . P a r t of the DNA was a c i d hydrolyzed and the bases were separated by 1D-TLC i n solvent B. The remaining DNA 3 was analyzed on a n e u t r a l CsCl d e n s i t y gradient. [ H]-TdR,0 ; 3 2 P - l a b e l l e d 0W-14. reference DNA, • . 65 8.01 ro O 6.0 Q. O 4.0 H 3= 2-°i A 30.0 20.0 [ 10.0 10 20 30 40 FRACTION NUMBER CM I o 2 CL O Q . CvJ ro 66 Attempts were also made to d e n s i t y l a b e l 0W-14 DNA w i t h the thymidine analogue, BUdR. 3L grown i n TdR was spun down and resu s --1 3 pended i n 500 yg ml of BUdR. The 0W-14 DNA, a l s o l a b e l l e d w i t h [ H]-o r n i t h i n e , was ext r a c t e d from phage and analyzed on n e u t r a l CsCl d e n s i t y gradients (Figure 9 ). Reference DNA and 0W-14 DNA from phage grown i n the presence of BUdR banded a t the same d e n s i t y , showing that BUdR was not incorporated i n t o DNA i n 0 W-14-infected c e l l s (Figure 9 ). I t was concluded that exogenously s u p p l i e d TdR was not a precur-3 sor of thymine i n 0W-14 DNA. The a b i l i t y of [6- H]-UdR to l a b e l putThy and Thy bases demonstrated that the enzyme thymidine kinase was a c t i v e i n 0 W-14-infected P_. acidovorans (Table 4) . Endogenous thymidine u t i l i z a t i o n i n 0 W-14-infected P_. acidovorans 3L Exogenously s u p p l i e d TdR was not a precursor of thymine i n 0W-14 DNA. This d i d not exclude the p o s s i b i l i t y that endogenously s u p p l i e d TdR was the source of the thymine residues i n DNA. The methods f o r demonstrating endogenous requirements f o r TdR are w e l l e s t a b l i s h e d but i n d i r e c t , and r e l y upon the use of i n h i b i t o r s w i t h known mechanisms of a c t i o n . FUdR was found to i n h i b i t the growth of P_. acidovorans s t r a i n 29 at concentrations greater than 250 yg ml ^ ( K e l l n and Warren, 1973). P_. acidovorans 3L, a d e r i v a t i v e of s t r a i n 29, i s r e s i s t a n t to high con-c e n t r a t i o n s of FUdR. 3L i s a thymidylate synthase mutant which r e q u i r e s at l e a s t 250 yg m l " 1 TdR f o r c e l l growth ( K e l l n , Ph.D. Thesis, 1973). C e l l growth was measured i n these experiments by p l a t i n g on s o l i d CAA-M medium i n the presence of the drug. 500 yg ml ^ FUdR d i d not prevent FIGURE 9.—Buoyant d e n s i t y of 0W-14 DNA synthesized i n the presence of BUdR. 0 W-14-infected J?. acidovorans s t r a i n 3L was spun down and resuspended i n conditioned TCS medium without thymidine. BUdR (500 yg ml 1 ) , - 1 3 -1 -1 adenosine (100 yg ml ) and H-ornithine (1.0 y C i ml , 10 yg ml ) were added and the c e l l s were allowed to l y s e . DNA was p u r i f i e d from phage p a r t i c l e s and analyzed on n e u t r a l CsCl buoyant d e n s i t y gradients 3 32 + H-orn l a b e l , O ; P - l a b e l l e d 0W-14 w phage reference DNA,* . ro i O CL O X ro 12.01 8,0 4.0 B 60.0 40.0 20.0 10 20 30 40 FRACTION NUMBER ro i O Q_ O Q_ CVJ ro 68 the normal accumulation of plaque-forming u n i t s i n 0 W-14-infected s t r a i n 3L (Figure 10). 0W-14 a l s o formed plaques on 3L growing on CAA-M p l a t e s c o n t a i n i n g TdR and FUdR. The f a i l u r e of FUdR to i n h i b i t phage formation i n 0 W-14-infected c e l l s d i d not exclude the p o s s i b i l i t y that FUdR could a f f e c t DNA r e p l i -c a t i o n or m o d i f i c a t i o n . FUdR had l i t t l e or no e f f e c t upon the accumula-14 3 t i o n of DNA measured using [2- C ] - u r a c i l or [ H ] - o r n i t h i n e (Figure 11). The d i f f e r e n c e s i n l a b e l accumulated by i n f e c t e d c u l t u r e s w i t h and w i t h -out added FUdR were not s i g n i f i c a n t . When the data p o i n t s were r e p l o t t e d as a percentage of the f i n a l i n c o r p o r a t i o n v a l u e , the r e l a t i v e r a t i o s of accumulation f o r a l l c e l l c u l t u r e s were i d e n t i c a l (Figure 12). I d e n t i c a l 14 3 accumulation r a t e s f o r [2- C]-Ura and [ H]-0rn l a b e l suggested that DNA r e p l i c a t i o n and DNA m o d i f i c a t i o n proceeded normally i n the presence of FUdR. P e r m e a b i l i t y of P_. acidovorans to FUdR was evident. Growth of s t r a i n 29 was i n h i b i t e d by FUdR and r a d i o a c t i v e deoxyuridine l a b e l l e d 0W-14 DNA i n 3L/FU 2. FUdR could e x e r t a non l e t h a l e f f e c t upon DNA m o d i f i c a t i o n i n 0 W-14-infected c e l l s . FUdR might be incorporated i n t o phage DNA or FUdR 3 might i n h i b i t the accumulation of putThy or_ Thy res i d u e s . [6- H]-Ura-l a b e l l e d 0W-14 DNA prepared i n the presence of FUdR was examined. FUdR added t o c e l l s 30 minutes p r i o r t o , or at the onset of phage i n f e c t i o n had no e f f e c t upon the buoyant de n s i t y of the DNA synthesized (Table 5). A f t e r a c i d h y d r o l y s i s and TLC, phage DNA prepared i n the presence of FUdR showed normal l a b e l l i n g r a t i o s f o r putThy, Cyt and Thy bases (Table 6). Gradient and h y d r o l y s i s data were s i m i l a r whether s t r a i n s 29 or 3L were used (Table 7.) . FIGURE 10.—The e f f e c t of trimethoprim and FUdR upon plaque production i n 0 W-14-infected s t r a i n 3L. Cultures of s t r a i n 3L growing i n TCS medium w i t h 250 yg ml 1 TdR were supplemented w i t h 500 yg ml 1 FUdR, A; or w i t h 100 yg ml 1 Tp,0 ; no g a d d i t i o n , * . The c u l t u r e s were then grown to a d e n s i t y of 3.0 x 10 c e l l s ml 1 and i n f e c t e d w i t h 0W-14. The number of plaque forming u n i t s i n each c u l t u r e was determined at i n t e r v a l s a f t e r i n f e c t i o n . 70 I0 9 0 15 30 45 60 75 MINUTES FIGURE 11.—The e f f e c t of fl u o r o d e o x y u r i d i n e upon DNA sy n t h e s i s i n 0 W-14-infected P. acidovorans 3L. S t r a i n 3L was i n f e c t e d w i t h 0W-14 and l a b e l was added to c u l t u r e s 20 minutes l a t e r . O r n i t h i n e l a b e l l i n g of 0W-14 DNA was followed by 3 measuring the i n c o r p o r a t i o n of [ H]-orn i n t o pronase r e s i s t a n t , TCA 14 p r e c i p i t a b l e m a t e r i a l . The i n c o r p o r a t i o n of [2- C ] - u r a c i l was followed by measuring the accumulation of a l k a l i r e s i s t a n t , TCA 3 p r e c i p i t a b l e m a t e r i a l . I n f e c t e d c e l l s l a b e l l e d w i t h [ H]-orn,.#; -1 3 i n f e c t e d c e l l s grown i n FUdR (500 yg ml ) and l a b e l l e d w i t h [ H]-14 o r n , 0 ; i n f e c t e d c e l l s l a b e l l e d w i t h [2- C ] - u r a c i l , A; i n f e c t e d -1 14 c e l l s grown i n FUdR (500 yg ml ) and l a b e l l e d w i t h [2- C ] - u r a c i l , 73 FIGURE 12.—The accumulation of 0W-14 DNA i n the presence of FUdR. The data was taken from the previous f i g u r e and r e p l o t t e d as a per-centage of the f i n a l i n c o r p o r a t i o n value. The symbols are defined i n the legend of Figure 11. ZD I| X < -z. o OF _ATI NT ZD LU O IT o LU o Q_ < 100 80 60 40 20 20 25 30 35 40 MINUTES AFTER 45 50 55 NFECT I0N TABLE 5.—The e f f e c t of trimethoprim or FUdR upon the buoyant d e n s i t y of 0W-14 DNA synthesized i n P_. acidovorans 0 W-14-infected s t r a i n 29 s t r a i n 3L Time of a d d i t i o n of FUdR 0 min. + a + 30 min. before i n f e c t i o n 4- + Time of a d d i t i o n of Tp 0 min. + + 90 min. before i n f e c t i o n - + 3 The DNA used i n these experiments was l a b e l l e d w i t h [6- H ] - u r a c i l and ext r a c t e d from the i n f e c t e d c e l l s at 45 minutes a f t e r i n f e c t i o n . Thes procedures are described i n the M a t e r i a l s and Methods. a + i n d i c a t e s that the DNA made i n i n f e c t e d c e l l s had the same buoy-ant d e n s i t y as authe n t i c 0W-14 reference DNA i n n e u t r a l CsCl g r a d i e n t s . b S t r a i n 29 c e l l s w i t h an e s t a b l i s h e d i n h i b i t i o n by trimethoprim d i d not support 0W-14 plaque production or DNA s y n t h e s i s . TABLE 6.—The base composition of 0W-14 DNA prepared i n the presence of Trimethoprim or FUdR. Phage/Host/Inhibitor putThy Cyt Thy 0/W-14 w +/29/none 2931 (0 . 2 4 ) a 6027 (0.49) 3454 (0. 28) 0W-14 w +/3L/none 4113 (0.20) 10912 (0.52) 5798 (0. 28) 0W-14 w+/29/Tp (100 yg ml" 1) 1787 (0.21) 4027 (0.51) 2255 (0. 28) 0W-14 w+/3L/Tp (100 yg ml" 1) 10980 (0.21) 27315 (0.54) 12543 (0. 25) 0W-14 w+/29/FUdR (500 yg ml" 1) 27982 (0.22) 65100 (0.52) 31417 (0. 25) 0W-14 w+/3L/FUdR (500 yg ml" 1) 34670 (0.22) 81435 (0.53) 38126 (0. 25) The i n f e c t e d c e l l s were l a b e l l e d from 25 minutes a f t e r i n f e c t i o n w i t h 3 [6- H ] - u r a c i l . The a n t i b i o t i c s were added j u s t before i n f e c t i o n . The DNA was extracted from the i n f e c t e d c e l l s at 45 minutes a f t e r i n f e c t i o n . DNA samples were a c i d hydrolyzed and the bases were resolve d by 1D-TLC i n solvent B. a The values represent cpm i n the area cut from the chromatogram. The values i n parentheses represent the f r a c t i o n of the l a b e l found i n putThy, Thy and Cyt bases. TABLE 7.—0W'<-14- DNA m o d i f i c a t i o n was independent of the host s t r a i n . Base host s t r a i n 29 3L putThy 4972 ( 0 . 2 4 ) a 13981 (0.23) Cyt 10858 (0.51) 31661 (0.52) Thy 5285 (0.25) 15318 (0.25) DNA was i s o l a t e d from 0W-14 p a r t i c l e s a f t e r i n f e c t i o n and [6- 3H]-u r a c i l l a b e l l i n g of growing c u l t u r e s . The DNA samples were processed as described i n the M a t e r i a l s and Methods. a The values are cpm i n the area cut from the chromatogram. The values i n parenthesis are the f r a c t i o n of the l a b e l i n the pyrimidine bases. 78 I n c o r p o r a t i o n of 5-FUdR i n t o PBS2 DNA has been reported (Lozeron and S z y b a l s k i , 1967). Accumulation of FUdR i n the DNA made i t h e a v i e r . I t was u n l i k e l y 0 W-14-infected c e l l s used dUTP as a precursor of putThy 3 or Thy. FUdR i s an analogue of UdR. [6- H]-UdR l a b e l l e d putThy and Thy residues i n 0W-14 DNA, but as shown e a r l i e r , DNA prepared i n the pre-sence of FUdR had the same buoyant d e n s i t y as 0W-14 reference DNA. Therefore, the r e s i s t a n c e of 0W-14 DNA r e p l i c a t i o n and m o d i f i -c a t i o n to FUdR a l s o e f f e c t i v e l y excluded the p o s s i b i l i t y that u r a c i l might be a precursor of putThy i n 0W-14 DNA. Evidence of a c e l l ' s a b i l i t y to synthesize dTMP or hmdUMP from UdR can be obtained by i n v i v o measurement of t r i t i u m r e l e a s e from 3 [5- H ] - u r a c i l - l a b e l l e d c e l l s . 3 [5- H ] - u r a c i l r e l e a s e i n 0 W-14-infected P. acidovorans Thymidylate synthase or any enzyme c a t a l y z i n g the r e l e a s e of 3 t r i t i u m from [5- H ] - u r a c i l can be detected i n v i v o i n c e l l s capable of converting u r a c i l to dUMP. In v i v o , t r i t i u m r e l e a s i n g a c t i v i t y was pre-sent i n s t r a i n 29 but not i n s t r a i n 3L uninf e c t e d c e l l s (Figure 13). 3 Both s t r a i n s r e t a i n e d the a b i l i t y to accumulate [5- H ] - u r a c i l - l a b e l l e d 3 DNA. [5- H ] - u r a c i l l a b e l s c y t o s i n e bases i n DNA. 3L i s a thymidylate s y n t h a s e - d e f i c i e n t d e r i v a t i v e of s t r a i n 29 ( K e l l n , Ph.D. Thesis, 1973). 0 W-14-infected P_. acidovorans s t r a i n s 29 or 3L had almost i d e n t i c a l t r i t i u m r e l e a s e curves, i n d i c a t i n g that a t r i t i u m - r e l e a s i n g a c t i v i t y was induced by bacteriophage i n f e c t i o n (Figure 13). The jLn v i v o r e s u l t s agree w i t h the i n v i t r o r e s u l t s of K e l l n (1973). S i m i l a r t r i t i u m r e l e a s e curves f o r s t r a i n s 29 and 3L i n f e c t e d by 0W-14 a l s o suggested that the FIGURE 13.—DNA synthesis and tritium release in 0W-14-infected P_. acidovorans. 3 A) The incorporation of [5- H]-uracil into a l k a l i resistant, TCA precipitable material. P_. acidovorans strain 29, uninfected, O ; P_. acidovorans strain 3L, uninfected, • ; P_. acidovorans strain 29, 0W-14 infected, A; J?. acidovorans strain 3L, 0W-14 infected, A. 3 B) The release of tritium from [5- H]-uracil. Measurement of the in vivo activity of dUMP hydroxymethylase. P_. acidovorans strain 29, uninfected, O ; J?. acidovorans strain 3 L , uninfected,*.; P_. acidovorans strain 29, 0W-14 infected A; P_. acidovorans strain 3L, 0W-14 infected, A. 80 81 20 40 60 MINUTES 82 host thymidylate synthase activity was suppressed by bacteriophage infection, otherwise tritium release in 0W-14-infected strain 29 would have been greater than in 0W-14-infected strain 3L. 3 These results demonstrated the presence of [5- H] releasing activities in 0W-14-infected cells but they did not prove that tritium release occurred at the level of the nucleotide pool. Kelln detected 3 enzyme activities capable of releasing tritium from [5- H]-dUMP in vitro. Kelln claimed that the products of these reactions were dTMP and hmdUMP. Neuhard has shown that the sole product of this reaction i s hmdUMP (Neuhard et a l . , 1980). The hmdUMP-synthesizing enzyme is probably resistant to FUdR. Concentrations of 5-FdUMP ten times greater than those inhibiting the host thymidylate synthase do not inhibit the 0W-14 hydroxymethylase activity i n extracts (Neuhard and Warren, unpublished observations). The inabi l i t y of 0W-l4-infected cells to u t i l i z e endo-genous or exogenous TdR, coupled with the evidence of tritium releasing activity in infected cells supported the theory that hmdUMP was the only precursor of Thy and putThy in 0W-14 DNA. Trimethoprim (Tp) and trimethoprim and sulfonamide  (sulfa) effects on 0W-14 reproduction The minimal inhibitory concentration of Tp for P_. acidovorans strain 29 is between 2.5 and 5 ug ml 100 yg ml 1 of trimethoprim inhibits the growth of strain 29 but does not inhibit the growth of the thymidylate synthase minus strain, 3 L . Tp or Tp and sulfa-induced cul-tures of strain 29 stop growing by 4 hours after addition of drugs to exponential cultures (Figure 14). Additions of adenosine, thymidine, pantothenic acid and amino acids in attempts to prevent or reverse the FIGURE 14.—The e f f e c t s of trimethoprim or trimethoprim and sulfonamide upon the growth of P_. acidovorans s t r a i n s 29 and 3L. Cultures of s t r a i n s 29 and 3L were grown i n TCS medium supplemented w i t h succinate (2 mg ml "S or w i t h succinate and Casamino acids (1 mg ml ^ ) , adenosine (100 ug m l . 1 ) , thymidine (500 yg ml 1 ) , and pantothenic a c i d (50 yg ml 1 ) . At the time i n d i c a t e d by the arrow, Tp, (100 yg m l - 1 ) or Tp (100 yg m l - 1 ) and sulfonamide (500 yg ml" 1) were added to the c u l t u r e s . The increases i n t u r b i d i t y were followed w i t h a K l e t t c o l o r i m e t e r . A) S t r a i n 29 grown i n su c c i n a t e ; no a d d i t i o n s , •; Tp,0; Tp and S u l f a , A. B) S t r a i n 29 grown i n s u c c i n a t e , Casamino a c i d s , thymidine, adenosine, and pantothenic a c i d ; no a d d i t i o n s , O; Tp,#; Tp and S u l f a , A. C) S t r a i n 3L grown i n succinate and thymidine; no a d d i t i o n s , O ; Tp, • ; Tp and S u l f a , A. D) S t r a i n 3L grown i n s u c c i n a t e , Casamino a c i d s , thymidine, adenosine, and pantothenic a c i d ; no a d d i t i o n s , Q; Tp,#; Tp and S u l f a , A. c 88 i n h i b i t o r y e f f e c t s of the drugs were only p a r t i a l l y s u c c e s s f u l . Over-night i n c u b a t i o n of c u l t u r e s of Tp or Tp and s u l f a - t r e a t e d c u l t u r e s increased the t u r b i d i t y by 50 percent i f supplements were present. C u l -tures without supplements d i d not grow at a l l . Supplements i n c l u d e a l l products normally r e q u i r i n g a THFA c o f a c t o r f o r b i o s y n t h e s i s . Tp had no e f f e c t upon plaque production by 0 W-14-infected s t r a i n 3L c e l l s grown i n TCS medium or p l a t e d upon CAA-M plus TdR plus Tp p l a t e s (Figure 10). 3L i s r e s i s t a n t to Tp because i t does not deplete THFA and the r e f o r e does not need to regenerate l a r g e amounts of THFA from DHFA. Tp i n h i b i t s the enzyme d i h y d r o f o l a t e reductase which mediates the reduc-t i o n of DHFA to THFA (Mahler and Cordes, 1971). DHFA i s generated from THFA s t o i c h i o m e t r i c a l l y during thymidylate b i o s y n t h e s i s . I f thymidylate s y n t h e s i s proceeds w h i l e d i h y d r o f o l a t e reductase i s i n h i b i t e d , a l l c e l l -u l a r a c t i v i t i e s r e q u i r i n g THFA w i l l h a l t . Trimethoprim or Tp and s u l f a -t r e a t e d c e l l s synthesized 0W-14 DNA i n s t r a i n 29 (Figures 15 and 16) when the i n h i b i t o r was added at the onset of phage i n f e c t i o n . The buoy-ant d e n s i t y of 0W-14 DNA ex t r a c t e d from phage-infected s t r a i n 29 or 3L c e l l s was examined. The buoyant de n s i t y of DNA made i n 3L or 29 was the same as the buoyant d e n s i t y of 0W-14 reference DNA (Table 5). S t r a i n 29 c e l l s , p r e t r e a t e d f o r long periods of time w i t h Tp, do not support phage growth, presumably because Tp treatment causes the c e l l s to undergo thymine-less death p r i o r to i n f e c t i o n . Pretreatment of s t r a i n 3L c e l l s w i t h Tp f o r one doubling p r i o r to i n f e c t i o n had no e f f e c t upon the buoy-ant d e n s i t y of 0W-14 DNA synthesized (Table 5). 3 [6- H]-Ura l a b e l l i n g r a t i o s f o r 0W-14 DNA prepared i n the pre-sence of Tp were al s o normal (Table 6). FIGURE 15.—The e f f e c t of trimethoprim upon 0W-14 DNA s y n t h e s i s . Trimethoprim was added to one-half of an i n f e c t e d c u l t u r e at 20 3 minutes a f t e r i n f e c t i o n . The i n c o r p o r a t i o n of [6- H ] - u r a c i l i n t o a l k a l i r e s i s t a n t , TCA p r e c i p i t a b l e m a t e r i a l was fo l l o w e d . J?. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14, no a d d i t i o n s , * ; P_. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14, Tp (100 yg ml "*") , O . 90 20 25 30 35 40 45 50 MINUTES A F T E R INFECTION FIGURE 16.—The e f f e c t of trimethoprim and sulfonamide upon 0W-14 DNA s y n t h e s i s . Tp and S u l f a were added to one-half of a c u l t u r e at 20 minutes a f t e r 3 i n f e c t i o n . The i n c o r p o r a t i o n of [6- H ] - u r a c i l i n t o a l k a l i r e s i s t a n t , TCA p r e c i p i t a b l e m a t e r i a l was determined. P_. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14, no a d d i t i o n s , •; P_. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14, Tp (100 yg m l " 1 ) , S u l f a (500 yg m l " 1 ) , O. 92 2.8 4 20 25 30 35 40 45 50 MINUTES A F T E R INFECT ION The gradients contained DNA which was ex t r a c t e d from i n f e c t e d c e l l s at 45 minutes a f t e r i n f e c t i o n or from phage p a r t i c l e s a f t e r l y s i s The absence of heavy de n s i t y DNA excluded the p o s s i b i l i t y that i n f e c t e d c e l l s made unmodified DNA which was not packaged. C o l l e c t i v e l y , the Tp, Tp and s u l f a , and FUdR r e s u l t s suggested that endogenous thymidine was not a precursor of thymine i n 0W-14 DNA. Exogenous TdR i s a l s o not a precursor. putThy n u c l e o t i d e s were not found i n i n f e c t e d c e l l p o ols, but i n f e c t e d c e l l s induced enzymes which 3 c a t a l y z e d the r e l e a s e of t r i t i u m from [5- H ] - u r a c i l . This s i t u a t i o n i s s i m i l a r to systems where hmdUMP i s the product of t r i t i u m r e l e a s i n g enzymes. h'mdUMP b i o s y n t h e s i s r e q u i r e s a THFA co f a c t o r but the THFA requirement i s c a t a l y t i c . THFA i s not o x i d i z e d , t h e r e f o r e , hmdUMP syn-t h e s i s i s not s e n s i t i v e to the presence of trimethoprim. These con c l u -sions are supported by the dis c o v e r y of hmdUMP and hmdUTP i n the s o l u b l n u c l e o t i d e pools of i n f e c t e d c e l l s (Neuhard et a l . , 1980). Warren has al s o shown the i n d u c t i o n of a dUTP/dTTPase a c t i v i t y i n 0 W-14-infected c e l l s . I t was concluded that 0W-14 s y n t h e s i s r e q u i r e d the four deoxy-nucleoside triphosphates; dATP, dGTP, dCTP and hmdUTP, that dTTP was excluded, and that both Thy and putThy i n 0W-14 DNA were generated by p o s t - r e p l i c a t i o n a l m o d i f i c a t i o n of hmUra. These conclusions prompted a d e t a i l e d e v a l u a t i o n of the nature of r e p l i c a t i n g 0W-14 DNA. The goals of t h i s work were the demonstration of hmURA i n r e p l i c a t i n g 0W-14 DNA and the e l u c i d a t i o n of the b i o s y n t h e t i c pathways f o r putThy and Thy. 3 T r i t i u m i s not l o s t from [6- H ] - u r a c i l  during 0W-14 DNA syn t h e s i s 0W-14 DNA l a b e l l e d w i t h [ 2 - 1 4 C ] - u r a c i l or [ 6 - 3 H ] - u r a c i l was pre pared. A f t e r a c i d - h y d r o l y s i s and TLC of the bases, the d i s t r i b u t i o n of 94 both l a b e l s i n putThy, Cyt and Thy was the same (Table 8). B a s e - l a b e l l i n g r a t i o s f o r both l a b e l s r e f l e c t e d the moi % G + C values determined f o r 0W-14 DNA. The b i o s y n t h e s i s of putThy and Thy i n 0W-14 DNA preserved 3 3 the [6- H ] - u r a c i l l a b e l . E q u i l i b r i u m l a b e l l i n g of bases w i t h [6- H]-Ura a c c u r a t e l y r e f l e c t e d t h e i r r e l a t i v e l e v e l s i n 0W-14 DNA. R e p l i c a t i n g DNA hmdUTP i s the n u c l e o t i d e pool precursor of Thy and putThy i n 0W-14 DNA (Neuhard et a l . , 1980). A c i d h y d r o l y s i s of 0W-14 v i r i o n DNA followed by two-dimensional t h i n - l a y e r chromatography showed that hydroxy-m e t h y l u r a c i l was not present i n the DNA (Figure 17). 3 [6- H ] - u r a c i l - l a b e l l e d 0W-14 grown i n s t r a i n 29 or 3L had the same base composition (Table 7 ) . Changes i n growth temperature, growth medium or i n f e c t i o n of c e l l s at v a r y i n g c e l l d e n s i t i e s had no e f f e c t upon the thymine and a putrescinylthymine l e v e l s i n 0W-14 DNA (data not presented). R e p l i c a t i n g 0W-14 DNA was analyzed on n e u t r a l CsCl d e n s i t y grad-i e n t s . 0W-14 DNA i s o l a t e d from i n f e c t e d c e l l s had the same buoyant den-s i t y as reference marker 0W-14 prepared from p u r i f i e d v i r i o n DNA (Table 5). Buoyant d e n s i t y i s an accurate r e f l e c t i o n of m o d i f i c a t i o n because the low d e n s i t y of 0W-14 DNA i s a consequence of the putThy i n 0W-14 DNA (Warren, 1980). R e l a t i v e l y s m a l l changes i n putThy l e v e l s w i l l r e s u l t i n d e t e c t a b l e changes i n 0W-14 DNA buoyant de n s i t y . R e p l i c a t i n g 0W-14 DNA may be examined i n d i r e c t l y u s i n g p a r e n t a l l y l a b e l l e d 0W-14 DNA and f o l l o w i n g i t through i n f e c t i o n w i t h n e u t r a l CsCl gradient a n a l y s i s (Figure 18). Obviously, the putThy i n 0W-14 DNA was TABLE 8 . — T r i t i u m i s not l o s t from [6- H ] - u r a c i l during putThy or Thy b i o s y n t h e s i s . 0W-14 DNA l a b e l l e d w i t h : 3 14 Base [6- H ] - u r a c i l [2- C ] - u r a c i l putThy 25840 ( 0 . 2 2 ) a 19089 (0.22) Thy 31561 (0.26) 23717 (0.28) Cyt 62229 (0.52) 42260 (0.50) The DNA was l a b e l l e d w i t h r a d i o a c t i v e u r a c i l and the DNA was e x t r a c t e d from the i n f e c t e d c e l l s at 35 minutes a f t e r i n f e c t i o n . The bases i n ac i d hydrolyzed DNA samples were separated by 1D-TLC on c e l l u l o s e sheets. a The values represent cpm i n the area cut from the chromatogram. The values i n parentheses represent the f r a c t i o n of the l a b e l found i n putThy, Thy and Cyt bases. FIGURE 17.—TLC of the pyrimidine products released from 0W-14 DNA by a c i d h y d r o l y s i s . 14 [2- C ] - u r a c i l - l a b e l l e d 0W-14 v i r i o n DNA was prepared and hydrolyzed at 100° f o r 90 minutes. The sample was mixed w i t h a standard base mixture and bases were resolve d by 2D-TLC on c e l l u l o s e sheets. The procedures employed are described i n greater d e t a i l i n the M a t e r i a l s and Methods. 97 FIGURE 1 8 . — C s C l d e n s i t y gradient a n a l y s i s of P - l a b e l l e d 0W-14 p a r e n t a l DNA. 32 P - l a b e l l e d 0W-14 phage was prepared i n P_. acidovorans s t r a i n 29. The r a d i o a c t i v e phage prep a r a t i o n was used to i n f e c t a growing c u l t u r e . At i n t e r v a l s a f t e r i n f e c t i o n , a l i q u o t s of the i n f e c t e d c u l t u r e were removed and DNA was p u r i f i e d . The DNA was banded i n 32 3 n e u t r a l CsCl d e n s i t y g r a d i e n t s . P - l a b e l , O ; [6- H ] - u r a c i l l a b e l l e d phage reference and host reference DNA were added to the experimental samples,*. 99 i o 5-OL O X 10 4.0 Z.0 50 ' p i . <r 6.0 "? 6 o -K ^ 4.0 2 0-o X 2.0 to CL CM to 20 40 FRACTION NUMBER vir ion D N A m i O CL CM to 20 40 FRACT ION N U M B E R i O CL O X l O 4.0 2.0 10 p i . o to — i O x x -= 4.0 ^ o o_ o o_ CM IO 1 2.0 20 40 FRACT ION N U M B E R 20 p.i. I o 0_ o 0_ CM to I o 5 0_ o X 10 20 40 FRACTION NUMBER 4.0 2.0 35 ' p.i. a o o_ to 20 FRACTION 40 NO. 100 not removed or altered a f t e r i n f e c t i o n . There was no transfer of r e p l i c a t i n g parental DNA to heavier d e n s i t i e s . Hybrid unmodified or hmUra-containing DNA did not accumulate i n appreciable quantities i n infected c e l l s . Acid-hydrolysis and TLC of bases from parentally l a b e l l e d 0W-14 DNA extracted from infected c e l l s allowed s i m i l a r conclusions regarding the fate of Thy and putThy. Both were stable components of 0W-14 DNA (Table 9 ) . 0W-14 i n t r a c e l l u l a r parentally l a b e l l e d DNA heated to j u s t below the Tm of 0W-14 DNA banded with a broader density p r o f i l e than v i r i o n DNA (data not shown). Only short fragments of DNA would be denatured by such heat treatments and they would band at s l i g h t l y heavier de n s i t i e s than duplex 0W-14 DNA. The density s h i f t and peak broadening probably was a consequence of heat denaturation of nicked regions i n r e p l i c a t i n g DNA. Nicks are a consequence of the r e p l i c a t i o n , recombination and the d i s p e r s a l of the parental DNA into progeny DNA. This was not a s u r p r i s -ing r e s u l t but i t showed that the majority of parental l a b e l DNA recovered and banded i n these experiments was active i n these processes. V i r i o n DNA must have been l e s s extensively nicked than i n t r a -c e l l u l a r DNA since peak s p l i t t i n g or broadening i n heat-treated v i r i o n DNA samples was not observed (Figure 19). CsCl gradients of v i r i o n DNA 3 14 l a b e l l e d with [5- H ] - u r a c i l and [5- C]-ornithine formed a s i n g l e , homo-genous peak before and a f t e r heat treatment. 0W-14 i n t r a c e l l u l a r DNA o 14 prepared from infected c e l l s l a b e l l e d with [5—JH]-uracil and [5- C]-ornithine also formed a s i n g l e , homogenous peak when banded i n neutral CsCl density gradients (Figure 19). Heat treatment of these samples p r i o r TABLE , 9 .—The f a t e of putThy and Thy i n p a r e n t a l l y l a b e l l e d 0W-14 DNA. Sample taken (min. a f t e r i n f e c t i o n ) Base 10 min. 30 min. 50 min. phage (0 min.) putThy 3657 (0.23) 3 3051 (0.21) 2205 (0.21) 9475 (0.21) Cyt 8972 (0.55) 8220 (0.57) 5719 (0.55) 26949 (0.59) Thy 3578 (0.22) 3276 (0.22) 2399 (0.23) 9293 (0.20) [6- H ] - u r a c i l l a b e l l e d phage was prepared and was used to i n f e c t a growing c u l t u r e of P_. acidovorans s t r a i n 29. At v a r i o u s times a f t e r i n f e c t i o n a l i q u o t s of the c u l t u r e were removed and the i n t r a c e l l u l a r DNA was p u r i f i e d . The DNA was a c i d hydrolyzed and the bases were res o l v e d by 1D-TLC. a The values are cpm i n the area cut from the chromatogram. The values i n parentheses are the f r a c t i o n of the l a b e l recovered i n putThy, Thy and Cyt bases. FIGURE 19.—The e f f e c t of heat treatment upon the buoyant d e n s i t y p r o f i l e s of 0W-14 i n t r a c e l l u l a r and v i r i o n DNAs. 3 14 [5- H ] - u r a c i l and [5- C ] - o r n i t h i n e - l a b e l l e d DNA was p u r i f i e d from v i r i o n s and from i n f e c t e d c e l l s . L a b e l l i n g , e x t r a c t i o n and heat treatment procedures are described i n the M a t e r i a l s and Methods. A) n a t i v e 0W-14 i n t r a c e l l u l a r DNA B) heat t r e a t e d 0W-14 i n t r a c e l l u l a r DNA C) n a t i v e 0W-14 v i r i o n DNA D) heat tr e a t e d 0W-14 v i r i o n DNA 0W-14 i n t r a c e l l u l a r DNA was p u r i f i e d from i n f e c t e d c e l l s at 35 3 14 minutes a f t e r i n f e c t i o n . [5- H ] - u r a c i l , # ; [5- C ] - o r n i t h i n e , O . 2 0 . 0 10.0 8.0 4 .0 10 2 0 3 0 4 0 FRACT ION N U M B E R A CM 1 to | o O X X Q_ o_ O o O X * — — 8.0 4 .0 CU 4 . 0 2 2 .0 10 2 0 3 0 4 0 F R A C T I O N N U M B E R B 12.0 f&.O I 4.0 10 2 0 3 0 4 0 F R A C T I O N N U M B E R I o CL O o ro 6 0 . O X ro 3 0 . 0 20-0 10.0 8.0 4 .0 CM I O 0_ o 10 2 0 3 0 4 0 F R A C T I O N N U M B E R c D 104 to l o a d i n g on the gradient s p l i t the sample i n t o two peaks c o n t a i n i n g approximately equal r a t i o s of l a b e l . PutThy or Thy-rich regions of the DNA ( i f there are any) are not p r e f e r e n t i a l l y heat-denatured s i n c e o r n i -t h i n e or u r a c i l - l a b e l l e d DNA co n t r i b u t e d e q u a l l y to the s l i g h t l y denser s i n g l e - s t r a n d e d peak. The o r i g i n a l purpose of t h i s experiment was to determine whether or not nascent, unmodified DNA could be freed from non covalent attachment to a l a r g e r mass of f u l l y modified DNA. This might have aided i n the d e t e c t i o n and i s o l a t i o n of bases which were i n t e r -mediates i n the Thy or putThy b i o s y n t h e t i c pathways. These expectations were not r e a l i z e d because such heavy DNA components do not e x i s t i n amounts l a r g e enough to be detected. The absence of peak s p l i t t i n g i n v i r i o n DNA a f t e r p a r t i a l d enaturation i n d i c a t e s that i t i s not exten-s i v e l y n i c ked. Peak s p l i t t i n g i n the r e p l i c a t i n g DNA samples probably was a consequence of the presence of n i c k s i n the DNA of i n f e c t e d c e l l s . Short segments of duplex DNA bounded by s i n g l e - s t r a n d n i c k s on the same chain of the DNA duplex are completely heat-denatured from DNA. Short duplex DNA fragments have a lower Tm than longer duplex DNA fragments of the same moi % G + C content (Mahler and Cordes, 1971). Reconstruc-t i o n experiments u s i n g p u r i f i e d s t r a i n 29 DNA and 0W-14 DNA i n d i c a t e d that peak s p l i t t i n g was not due to 0W-14 DNA being c a r r i e d down the gradients by host DNA (Figure 20). DNA i s o l a t e d from i n f e c t e d c e l l s contained both host and phage DNA. V i r i o n DNA preparations contained only phage DNA. Completely heat denatured 0W-14 DNA banded as a s i n g l e peak of uniform d e n s i t y . The e f f e c t of shearing upon the buoyant d e n s i t y p r o f i l e of 0W-14 DNA was s t u d i e d . Peak broadness i n n e u t r a l CsCl d e n s i t y gradients was a FIGURE 2 0 . — C s C l buoyant d e n s i t y a n a l y s i s of 0W-14 v i r i o n and P_. acidovorans DNA. A) 3 2 P - l a b e l l e d 0W-14 DNA mixed w i t h 50 yg u n l a b e l l e d host DNA. B) 3 2 P - l a b e l l e d 0W-14 DNA mixed w i t h 50 yg u n l a b e l l e d host DNA and then heat denatured. C) 3 2 P - l a b e l l e d s t r a i n 29 DNA mixed w i t h 50 yg u n l a b e l l e d 0W-14 DNA. D) 3 2 P - l a b e l l e d s t r a i n 29 DNA mixed w i t h 50 yg u n l a b e l l e d 0W-14 DNA and then heat denatured. The samples were loaded on CsCl, c e n t r i f u g e d and f r a c t i o n a t e d as described i n the M a t e r i a l s and Methods. 106 IO O A O 40.0 G L C J 10 20.0 10 20 30 40 FRACTION NUMBER IO I O B 40.0 0. O C L CM lO 20.0 A 10 20 30 40 FRACTION NUMBER 60.0 40.0 C L o 0- 20.0 IO c 10 20 30 40 FRACTION NUMBER IO i O C L O C L CM IO 40.0 20.0 D 10 20 30 40 FRACTION NUMBER r e f l e c t i o n of the s i z e of the DNA molecules, s m a l l molecules having higher d i f f u s i o n c o e f f i c i e n t s than l a r g e r DNA molecules. Shearing DNA 14 reduces the average length of the DNA molecules. [2- C ] - u r a c i l and 3 [5- H ] - o r n i t h i n e - l a b e l l e d v i r i o n and i n t r a c e l l u l a r phage DNA were exam-ined a f t e r shearing (Figure 21). The d i s t r i b u t i o n of both l a b e l s was uniform f o r both v i r i o n and i n t r a c e l l u l a r DNA. Shearing i n t r a c e l l u l a r DNA d i d not s h i f t a s i g n i f i c a n t p r o p o r t i o n of the DNA to a heavier den-3 s i t y . The e f f e c t of s e q u e n t i a l shearing and heat treatment on [6- H]-u r a c i l l a b e l l e d 0W-14 i n t r a c e l l u l a r DNA was a l s o examined (Figure 22). DNA formed a s i n g l e homogenous peak i n CsCl before shearing or heat treatment. Shearing caused peak broadening w h i l e h e a t i n g caused peak s p l i t t i n g . Shearing and then heating a DNA sample r e s u l t e d i n a broad uniform peak. Heavy d e n s i t y components were not r e l e a s e d from 0W-14 i n t r a c e l l u l a r DNA even through the combined a c t i o n of shearing and heati n g . Therefore, there were no extensive putThy, GC nor unmodified b a s e - r i c h domains i n 0W-14 DNA. I f there was any unmodified DNA i n i n f e c t e d c e l l s i t was present i n very s m a l l amounts and i t d i d not accumulate during the course of the normal i n f e c t i o u s c y c l e . In the experiments described above, DNA was e x t r a c t e d from c e l l at 35 minutes a f t e r i n f e c t i o n . Normally, at t h i s time, phage DNA syn-t h e s i s was w e l l - e s t a b l i s h e d . I t was assumed that i n t r a c e l l u l a r DNA con tent at t h i s time would r e f l e c t the nature of 0W-14 i n t r a c e l l u l a r DNA e x t r a c t e d at any point during DNA r e p l i c a t i o n . T h i n - l a y e r chromatography of DNA components The degradative a n a l y s i s of newly formed r e p l i c a t i n g 0W-14 DNA was preceded by the e v a l u a t i o n of procedures used f o r the h y d r o l y s i s or 108 FIGURE 21.—The uniform d i s t r i b u t i o n of putThy i n 0W-14 DNA. 3 [ H ] - o r n i t h i n e - l a b e l l e d 0W-14 DNA was prepared from i n f e c t e d c e l l s and from p u r i f i e d v i r i o n s . [ 2 - ^ C ] - u r a c i l l a b e l l e d 0W-14 DNA was prepared from i n f e c t e d c e l l s and from p u r i f i e d v i r i o n s . The DNA was sheared as described i n the M a t e r i a l s and Methods. A) sheared v i r i o n DNA; B) sheared i n t r a c e l l u l a r DNA. [ 3 H ] - o r n i t h i n e , — * - ; [ 2 - 1 4 C ] - u r a c i l , — O — 109 FIGURE 22.—The e f f e c t of heat treatment and shearing upon the d i s t r i -b u t i o n of l a b e l i n 0W-14 DNA ex t r a c t e d from i n f e c t e d c e l l s , and banded on CsCl g r a d i e n t s . 3 [6- H ] - u r a c i l l a b e l l e d DNA was e x t r a c t e d from 0W-14 i n f e c t e d P_. acidovorans at 35 minutes a f t e r i n f e c t i o n . A) 0W-14 DNA. B) 0W-14 DNA, sheared. C) 0W-14 DNA, heat t r e a t e d . D) 0W-14 DNA, sheared and heat t r e a t e d . The procedure employed i s described i n the M a t e r i a l s and Methods. I l l B 16.0 i 40 50 60 70 40 50 60 70 F R A C T I O N N U M B E R F R A C T I O N N U M B E R c 40 50 60 70 40 50 60 70 F R A C T I O N N U M B E R F R A C T I O N N U M B E R 112 d i g e s t i o n and s e p a r a t i o n of 0W-14 DNA components. This was necessary because techniques p r e v i o u s l y employed d i d not give complete d i g e s t i o n and s e p a r a t i o n of the DNA components most l i k e l y to be precursors of Thy and putThy ( K r o p i n s k i , Ph.D. Thesis, 1971; K e l l n and Warren, 1973). With solvent B, one-dimensional s e p a r a t i o n of 6 N HCI hydro-l y s a t e s of 0W-14 DNA d i d not r e s u l t i n the complete r e s o l u t i o n of a l l p o s s i b l e p y r i m i d i n e products. Two-dimensional t h i n - l a y e r s e p a r a t i o n of hydrolysates i n s o l v e n t s B and D gave complete r e s o l u t i o n of a l l p y r i -14 midine bases from nucleosides and n u c l e o t i d e s . A sample of [2- C]-u r a c i l l a b e l l e d P_. acidovorans s t r a i n 29 DNA was hydrolyzed and separ-ated by two-dimensional t h i n - l a y e r chromatography i n s o l v e n t s B and D (Figure 23). The h y d r o l y s i s procedure d i d not r e s u l t i n the complete conversion of n u c l e o t i d e s to bases. However, a l l n u c l e o t i d e s had Rf values of zero i n solvent D. Cytosine was not e x t e n s i v e l y converted to u r a c i l during h y d r o l y s i s . Background counts f o r the areas of the chromatogram outside the authentic standard spots were very low and independent of the amount of r a d i o a c t i v i t y loaded. The procedure was s e n s i t i v e enough to detect the presence of 5-methylcytosine i n the DNA of s t r a i n 29. 5-methylcytosine i s present as approximately 0.5 percent of the p y r i m i d i n e bases l a b e l l e d (Table 10) . 14 The h y d r o l y s i s and TLC of [2- C ] - u r a c i l - l a b e l l e d 0W-14 v i r i o n DNA i s a l s o shown (Figure 17). Although h y d r o l y s i s was not complete, the only major pyrimidine bases re l e a s e d by a c i d h y d r o l y s i s were those reported by K r o p i n s k i and Warren (1973). hmUra and u r a c i l are not detec-t a b l e h y d r o l y s i s products of 0W-14 v i r i o n DNA (Figure 17). 113 FIGURE 23.—Two-dimensional t h i n - l a y e r chromatography of a c i d -hydrolyzed, [2-l^C] - u r a c i l - l a b e l l e d P. acidovorans DNA. I v |j14rAWHA. 1 o n . IS <6 I * 114 TABLE 10.—The base composition of P_. acidovorans s t r a i n 29 DNA. compound cpm f r a c t i o n of cpm i n compound spot #. Ade 110 <0.001 5 meAde 95 <0.001 6 CdR 700 . 0.004 7 Cyt 119126 0.617 8 meCyt 948 0.005 9 - 31 <0.001 10 hmUra 325 0.002 11 UdR 846 0.005 12 Ura 1087 0.006 13 Thy 51057 0.260 14 putThy 37 - 16 o r i g i n 307 0.002 15 Gua 301 0.002 17 [2- C ] - u r a c i l l a b e l l e d P_. acidovorans s t r a i n 29 DNA was a c i d hydro-lyz e d i n 6N HCI. The samples were mixed w i t h a standard base reference mixture and separated by 2D-TLC on c e l l u l o s e sheets. Spot number r e f e r s to the spots which were detected under UV l i g h t on the chromatographic sheet. Compounds 1 to 4 are unhydrolyzed n u c l e o t i d e products. There were 23718 cpm i n unhydrolyzed n u c l e o t i d e s . See Figure 23. 115 This combination of solvent systems had the i d e a l c h a r a c t e r i s -t i c s of r e s o l v i n g a l l the tested pyrimidine bases over f i v e to 80 per-cent of the run length i n the f i r s t dimension. The second dimension completely freed bases from other hydrolysis products l i b e r a t e d by t h i s 3 32 procedure. Doubly-labelled DNA ( H and P) samples could be acid 3 hydrolyzed and separated. H-labelled bases were completely free of 32 contaminating P background r a d i a t i o n . This two-dimensional system i s s i m i l a r i n separation c h a r a c t e r i s t i c s to isopropanol:HC1rH^O (70:10:20)/n-butanol:H 20:NH 3 vapour - ( 8 6 :5:saturated NHg) ( H a l l , 1971) but the r e s o l u t i o n of hmUra and u r a c i l was better when solvent B was used for the f i r s t dimension. Acid hydrolysis of DNA, e s p e c i a l l y DNA containing unusual bases was not a completely s a t i s f a c t o r y procedure since i t could have resulted i n the destruction of a c i d - l a b i l e bases. 0W-14 DNA was digested to \ mononucleotides by sequential SI and snake venom phosphodiesterase treatment. 0W-14 DNA was digested to f i v e components, one of these, putdTMP had the highest Rf i n both solvents A and E (Table 11). putdTMP runs at or near the solvent front i n most common PEI-cellulose TLC sys-tems. This i s a r e s u l t of i t s net p o s i t i v e charge at the pH of chrom-atography. The putdTMP spot i s l a b e l l e d by ornithine. On two-dimen-s i o n a l thin-layer chromatograms of 0W-14 DNA digests the presence of only one extra spot with the chromatographic properties which would be expected of a putThy-containing nucleotide suggested that the putres-c i n y l functions of 0W-14 DNA were not modified by acid hydrolyzable groups (Figure 24). 0W-14 DNA cannot be completely degraded to mononucleotides by 116 TABLE 11A.—The n u c l e o t i d e composition of pulse l a b e l l e d 0W-14 DNA dig e s t s r e s o l v e d on P E I - c e l l u l o s e . N ucleotide CPM ( f r a c t i o n of t o t a l l a b e l ) 1 unknown 611 (0.15) 2 unknown 59 (0.01) 3 unknown 158 (0.04) 4 unknown 63 (0.01) 5 unknown 28 (0.01) 6 unknown 27 (0.01) 7 orthophosphate 1305 (0.31) 8 AMP and dAMP 977 (0.23) 9 UMP and dTMP 257 (0.06) 10 CMP and dCMP 309 (0.07) 11 GMP and dGMP 413 (0.10) The numbers correspond to the number of the n u c l e o t i d e or spot on the chromatographic sheet. R a d i o a c t i v i t y i n each compound was q u a n t i t a t e d by c u t t i n g the area c o n t a i n i n g the compound from the sheet and count-i n g i t i n a toluene-based s c i n t i l l a n t . DNA was digested w i t h nuclease SI and snake venom phosphodiesterase according to procedures described i n the M a t e r i a l s and Methods. The nu c l e o t i d e s were separated on PEI-c e l l u l o s e sheets according to the procedures of Randerath and Randerath. 117 TABLE 11B.—The n u c l e o t i d e composition of p u l s e - l a b e l l e d 0W-14 DNA di g e s t s r e s o l v e d on c e l l u l o s e . Nucleotide CPM ( f r a c t i o n of t o t a l l a b e l ) E Rf A 1 dAMP 1040 (0.12) 56 27 2 dGMP 489 (0.06) 26 39 3 dTMP 464 (0.05) 36 52 4 dCMP 714 (0.08) 43 70 5 putdTMP 360 (0.04) 55 79 6 hmdUMP 58 (<0.01) 23 62 7 unknown 266 (0.03) 9 65 8 rAMP 981 (0.11) 49 34 9 rGMP 292 (0.03) 15 51 10 rCMP 72 (<0.01) 30 77 11 rUMP 173 (0.02) 17 71 12 unknown 497 (0.06) - -13 orthophosphate 3282 (0.38) 23 100 The numbers correspond to the number of the n u c l e o t i d e or spot on the chromatographic sheet. The procedures employed are described i n part A of t h i s Table except that the nu c l e o t i d e s were separated according to the procedures described by Dawid et a l . , 1970. 118 FIGURE 24.—Two-dimensional t h i n - l a y e r chromatography of P - l a b e l l e d 0W-14 DNA digested w i t h nuclease SI and snake venom phosphodiesterase (SVPD). 119 simultaneous or s e q u e n t i a l treatments w i t h DNase I and snake venom phosphodiesterase (Figure 25). L i m i t d i g e s t s appeared to accumulate dimers c o n t a i n i n g putThy and one other base; dAMP, dGMP, dCMP and dTMP, but not putdTMP, were l i b e r a t e d i n l a r g e q u a n t i t i e s . The d e f i c i e n c y 32 i n putdTMP and the appearance of only three other P - l a b e l l e d spots running as dimers suggested that the d i s t r i b u t i o n of putThy i n 0W-14 DNA was not random s i n c e completely random d i s t r i b u t i o n of putThy i n t dimers would generate f i v e dimers. Lewis (unpublished observations) has subsequently pursued the problem of putThy d i s t r i b u t i o n i n 0W-14 DNA, showing that the r a t i o of putThy to Thy f o r s i n g l e p y rimidine n u c l e o t i d e s bounded by purine n u c l e o t i d e s i s 2.4 to 1.0, i n d i c a t i v e of an ordered d i s t r i b u t i o n of putThy i n 0W-14 DNA. The CsCl gradient r e s u l t s presented e a r l i e r merely suggested the absence of gross hetero-geneity i n the d i s t r i b u t i o n of putThy and Thy i n 0W-14 DNA. P u l s e - l a b e l l i n g of DNA i n 0 W - l 4 - i n f e c t e d P. acidovorans Of n e c e s s i t y , the development and a n a l y s i s of d i g e s t i o n and sep a r a t i o n procedures preceded the d e t a i l e d a n a l y s i s of nascent 0W-14 DNA. 32 The t r i t i a t e d u r a c i l and PO^ pulses were performed as des-c r i b e d i n the M a t e r i a l s and Methods. A c i d h y d r o l y s i s and two-dimen-3 s i o n a l t h i n - l a y e r chromatography of [6- H ] - u r a c i l - p u l s e d 0W-14 DNA i n d i c a t e d that putThy and Thy were not recovered i n normal r a t i o s (Table 12). L a b e l l e d hmUra and u r a c i l were found i n the hy d r o l y s a t e s . The u r a c i l could a r i s e from Cyt by deamination during a c i d h y d r o l y s i s , from RNA present i n the p u r i f i e d DNA or from the i n c o r p o r a t i o n of 120 32 FIGURE 25.—Two-dimensional t h i n - l a y e r chromatography of P - l a b e l l e d n u c l e o t i d e s present i n 0W-14 DNA digested w i t h DNase I and SVPD. TABLE 12.—Base composition of r e p l i c a t i n g 0W-14 DNA Base 6-(10 3 - H - u r a c i l sec pulse) L a b e l l e d Precursor 5-3H-uracil 6- H-(10 sec pulse) (20 min - u r a c i l l a b e l l i n g ) putThy c y t o s i n e hydroxymethyl-u r a c i l u r a c i l thymine unhydrolyzed n u c l e o t i d e s 2200 a (7.7) 15230 (53.2), 740 (2.6) 1830 (6.4) 5205 (18.2) 3410 (11.9) 531 (1.7) 27024 (86.5) 26 (>0.1) 28 (>0.1) 980 (3.1) 2653 (8.5) 14430 (18.2) 34585 (43.6) 157 (0.2) 180 (0.2) 21976 (27.7) 7987 (10.0) DNA was l a b e l l e d , e x t r a c t e d and hydrolyzed as described i n M a t e r i a l s and Methods. The bases were separated by two-dimensional t h i n - l a y e r chrom-atography on c e l l u l o s e sheets. a Cpm i n the area cut from the chromatogram. The f i g u r e s i n parenthe-ses represent the percentage of recovered r a d i o a c t i v i t y . T o t a l recovery of a p p l i e d r a d i o a c t i v i t y was >90 percent. u r a c i l i n t o DNA. The [ 5 - J H ] - u r a c i l pulse served as a c o n t r o l . A c i d 3 h y d r o l y s i s of [5- H ] - u r a c i l p u l s e - l a b e l l e d DNA d i d not r e l e a s e l a b e l l e d u r a c i l , t h e r e f o r e , p a n c r e a t i c RNase treatment of DNA e f f e c -t i v e l y removed s u s c e p t i b l e RNA and a c i d h y d r o l y s i s d i d not cause the deamination of c y t o s i n e to u r a c i l under the c o n d i t i o n s employed. U r a c i l was not incorporated i n t o nascent 0W-14 DNA. I t i s p o s s i b l e that the u r a c i l observed a r i s e s from the conversion of some a c i d - l a b i l C 5 modified base during h y d r o l y s i s . The presence of l a b e l i n putThy 3 and Thy i n the [5- H ] - u r a c i l p u l s e - l a b e l l e d DNA sample was probably a 3 consequence of the contamination of commercial [5- H ] - u r a c i l w i t h 3 [6- H ] - u r a c i l . 2.6 percent of the t o t a l p y r i m i d i n e l a b e l i n the 3 [6- H ] - u r a c i l pulse was found i n hmUra. This compared to 0.2 percent of the t o t a l l a b e l i n a uniformly l a b e l l e d 0W-14 i n t r a c e l l u l a r DNA h y d r o l y s a t e . This r e s u l t demonstrated that the hmdUTP found i n the a c i d - s o l u b l e pools of i n f e c t e d c e l l s was incorporated i n t o 0W-14 DNA. The s m a l l amount of hmUra r e l a t i v e to Thy and putThy demonstrated the r a p i d nature of hmUra conversion. Caution should be e x e r c i s e d i n e v a l u a t i n g the r e l a t i v e recovery of l a b e l i n Thy and putThy. I f both put-Thy and Thy were derived by p o s t - r e p l i c a t i o n a l m o d i f i c a t i o n of hmUra then the preponderance of Thy suggested e i t h e r that the Thy-forming r e a c t i o n ( s ) were f a s t e r or t h a t the Thy-forming r e a c t i o n ( s ) occurred before the putThy-forming r e a c t i o n ( s ) . I t should be remembered that the recovery of nascent DNA sequences r i c h i n putThy or Thy, or t h e i r p r e c u r s o r s , are not n e c e s s a r i l y equal. The p o s s i b i l i t y that putThy-c o n t a i n i n g sequences were p r e f e r e n t i a l l y l o s t during p u r i f i c a t i o n of DNA cannot be excluded. 123 The nascent DNA f r a c t i o n of DNA i n T7-infected E_. c o l i i s denatured during the phenol e x t r a c t i o n step of DNA p u r i f i c a t i o n . Son-i c a t i o n of l y s a t e s p r i o r to phenol p u r i f i c a t i o n s t a b i l i z e s the nascent DNA f r a c t i o n (Petkau et a l . , 1975). E x t r a c t i o n of p u l s e - l a b e l l e d DNA i n our l a b o r a t o r y i s performed using the SDS-pronase-phenol method (Lewis et a l . , 1976). This method, coupled w i t h ethanol p r e c i p i t a t i o n and winding out of DNA during p u r i f i c a t i o n , could have r e s u l t e d i n the p r e f e r e n t i a l l o s s of s i n g l e - s t r a n d e d DNA components. On CsCl gradients 0W-14 p u l s e - l a b e l l e d DNA oft e n had a heavy shoulder. This could have been due to the presence of s h o r t , s i n g l e - s t r a n d e d pieces of DNA, which were denatured during DNA p u r i f i c a t i o n . 32 32 PO^ pulses were a l s o performed. Using ®^/^  t o p u l s e - l a b e l DNA f a c i l i t a t e d the d e t e c t i o n of novel n u c l e o t i d e s and lowered the costs i n c u r r e d i n the pulse experiments. 0W-14 DNA p u l s e - l a b e l l e d f o r 32 10 seconds w i t h PO^ was p u r i f i e d and digested to mononucleotides w i t h SI nuclease and snake venom phosphodiesterase. Digests were separated by two-dimensional t h i n - l a y e r chromatography on unmodified c e l l u l o s e or on P E l - c e l l u l o s e sheets. A f t e r autoradiography, r a d i o -a c t i v e areas of the chromatograms were cut out and counted. P a r t of the DNA sample was r e t a i n e d f o r a n a l y s i s of the s e n s i t i v i t y of the nascent DNA to SI nuclease and f o r CsCl d e n s i t y gradient a n a l y s i s . Uniformly l a b e l l e d i n t r a c e l l u l a r DNA had the same buoyant den s i t y as v i r i o n DNA. P u l s e - l a b e l l e d DNA was examined to see i f i t showed a d i f f e r e n t d e n s i t y p r o f i l e than uniformly l a b e l l e d DNA. DNA 3 l a b e l l e d w i t h [6- H ] - u r a c i l u n t i l 35 minutes a f t e r i n f e c t i o n and pulsed 32 f o r 10 seconds w i t h PO. was prepared as described i n the M a t e r i a l s 124 and Methods. The n a t i v e gradients showed that although almost 100 percent of the t r i t i u m l a b e l was found at 0W-14 or host d e n s i t y , only 48 percent of the pulse l a b e l banded at the l i g h t phage d e n s i t y (Figure 32 26). T h i r t y - e i g h t and 14 percent of the P l a b e l banded at heavy and intermediate d e n s i t i e s , r e s p e c t i v e l y . Heat treatment of the p u l s e -l a b e l l e d r e p l i c a t i n g DNA sample caused the s p l i t t i n g of the u n i f o r m l y and p u l s e - l a b e l l e d l i g h t peak i n t o a three-peak p a t t e r n (Figure 26). The p u l s e - l a b e l l e d DNA was s h i f t e d toward the heavy end of the grad-i e n t . F i f t y - t w o percent of the t o t a l pulse l a b e l banded i n the l i g h t peaks, w h i l e 11 and 37 percent of the m a t e r i a l banded at intermediate and heavy d e n s i t i e s , r e s p e c t i v e l y . Heat treatment of 0W-14 pulsed, r e p l i c a t i n g DNA d i d s l i g h t l y s h i f t the l i g h t peak DNA to a heavier d e n s i t y . This could be due to the presence of unmodified bases i n nascent DNA or i t could be due to the hyperdensity s h i f t of e a s i l y -denatured nascent DNA fragments. The l a t t e r p o s s i b i l i t y seemed u n l i k e l y s i n c e the u n i f o r m l y - l a b e l l e d peaks on the gradient marked the buoyant de n s i t y of s i n g l e - s t r a n d e d and double-stranded 0W-14 DNA. The DNA preparations were used to compare the chemical and p h y s i c a l s t a b i l i t y of the p u l s e - l a b e l l e d and u n i f o r m l y - l a b e l l e d DNA 32 samples (Table 13). P - l a b e l l e d 0W-14 DNA served as a c o n t r o l . Both 32 3 P and H l a b e l were r e s i s t a n t to the a c t i o n of p a n c r e a t i c RNase which was employed during the p r e p a r a t i o n of the DNA samples. The s t a b i l i t y of the DNA samples to heating i n d i s t i l l e d water and 0.3 N NaOH was examined. The p u l s e - l a b e l l e d m a t e r i a l appeared s l i g h t l y more l a b i l e than the u n i f o r m l y - l a b e l l e d DNA samples. I n a d d i t i o n , 30 percent of the p u l s e - l a b e l l e d m a t e r i a l was s o l u b l i z e d when incubated i n SI b u f f e r , FIGURE 2 6 . — C s C l d e n s i t y a n a l y s i s of P - p u l s e - l a b e l l e d 0W-14 DNA. A) Native DNA. B) Heat tre a t e d DNA. The DNA sample was p u r i f i e d from c e l l s 35 minutes a f t e r i n f e c t i o n 32 w i t h 0W-14. The length of the P-pulse was approximately 10 sec-3 -1 onds. C e l l s were l a b e l l e d w i t h [6- H ] - u r a c i l (0.1 y C i yg , 10 yg ml "*") from 20 minutes a f t e r i n f e c t i o n . [6- 3H']-uracil l a b e l l e d DNA, •; 3 2PO. pulse l a b e l l e d DNA, O . F R A C T I O N N U M B E R B 8.0 7.0 i FRACT ION N U M B E R TABLE 13.—The r e l a t i v e chemical l a b i l i t y of p u l s e - l a b e l l e d DNA. 3 [6- H ] - U r a - l a b e l l e d 0W-14 DNA was e x t r a c t e d from c e l l s 35 minutes 32 a f t e r i n f e c t i o n . PO^ was used to pulse l a b e l 0W-14 i n f e c t e d 32 P_. acidovorans f o r 10 seconds at 35 minutes a f t e r i n f e c t i o n . P-l a b e l l e d 0W-14 DNA was e x t r a c t e d from v i r i o n s as described i n the M a t e r i a l s and Methods. A f t e r treatment DNA samples were spotted on f i l t e r papers and subjected to TCA p r e c i p i t a t i o n as described i n the M a t e r i a l s and Methods. Each value i n the t a b l e represents the average of s i x determinations. 3 [6- H]-Ura unifor m l y 32 PO^ p u l s e - l a b e l l e d l a b e l l e d 0W-14 DNA 0W-14 DNA 32 PO^ uniformly l a b e l l e d 0W-14 v i r i o n DNA Treatment CPM ( 3H/ 3 2P) % recovery ( 3H/ 3 2P) CPM ( 3 2P) % recovery dH 20 (0 min.) 4008/478 1756 90/81 98 (30 min.) 3581/387 1707 0.3 N NaOH (0 min.) 3733/409 1816 95/91 95 (30 min.) 3535/370 1713 0.05 M Na acetate, 0.3 M NaCl, pH 4.5 (0 min.) 5548/923 100/70 (120 min.) 5567/642 130 without enzyme, f o r four hours. Uniform l a b e l i n i n t r a c e l l u l a r DNA 32 was no more l a b i l e to the various treatments than the P - l a b e l l e d phage v i r i o n DNA. There was no p r e f e r e n t i a l s o l u b l i z a t i o n of p y r i m i d i n e - c o n t a i n i n g sequences under the c o n d i t i o n s employed (Lewis et a l . , 1 9 7 6 ) . The d i f f e r e n c e s i n chemical s t a b i l i t y f o r pulse and uniform l a b e l s suggested that nascent 0W-14 DNA might cont a i n n o v e l , unstable n u c l e o t i d e s . 32 As was noted, 30 percent of P pulse l a b e l was released from DNA i n the absence of S i . Of the remaining DNA approximately 30 per-cent of the pulse l a b e l and 4 percent of the uniform l a b e l remained TCA i n s o l u b l e a f t e r l i m i t d i g e s t i o n w i t h SI i n high s a l t (Figure 27). 32 SI d i g e s t i o n i n high s a l t (0.3 M NaCl) of P - l a b e l l e d DNA was l e s s extensive than SI degradation of the same DNA sample performed at low s a l t concentrations(0.05 M ammonium a c e t a t e ) . This could i n d i c a t e incomplete denaturation of the nascent DNA f r a c t i o n or the presence of SI r e s i s t a n t sequences. A c o n t r o l mixture of 0W-14 DNA uniformly 32 3 l a b e l l e d w i t h PO^ and [6- H ] - u r a c i l was almost completely converted to TCA-soluble m a t e r i a l (Figure 27). Two-dimensional t h i n - l a y e r chrom-atography of a low s a l t S i d i g e s t on c e l l u l o s e t h i n l a y e r s resolved a complex n u c l e o t i d e mixture which contained r i b o s e as w e l l as deoxy-r i b o s e - c o n t a i n i n g n u c l e o t i d e s and o l i g o n u c l e o t i d e s (Figure 28). Sub-sequent snake venom phosphodiesterase treatment of the S i - t r e a t e d , p u l s e - l a b e l l e d m a t e r i a l converted i t almost completely to mononucleo-t i d e s (Figure 28). Between 5 and 6 percent of the t o t a l counts i n the d i g e s t s were r e t a i n e d at the o r i g i n during chromatography. This m a t e r i a l was assumed to be contaminating, l a b e l l e d m a t e r i a l s . The 131 FIGURE 27.—Nuclease SI degradation of p u l s e - l a b e l l e d 0W-14 DNA. 3 A) The DNA was l a b e l l e d uniformly w i t h [6- H ] - u r a c i l , # ; f o r 20 min-32 utes and then pulse l a b e l l e d w i t h P°^> O; f o r 10 seconds. 3 B) The v i r i o n DNA was uniformly l a b e l l e d w i t h [6- H ] - u r a c i l , •; and 32 w i t h PO^, O. D i g e s t i o n s were performed i n 0.05 M Na acetate pH 4.5, 0.3 M NaCl; -4 10 M ZnCl^. The samples were then TCA p r e c i p i t a t e d , washed, d r i e d and counted as described i n the M a t e r i a l s and Methods. The values are expressed as a percentage of the o r i g i n a l amount of TCA p r e c i p i t a b l e m a t e r i a l . 132 20 4 0 60 80 100 120 140 160 180 MINUTES AFTER Si 133 FIGURE 28.—Two dimensional t h i n - l a y e r chromatography of P-pulse-l a b e l l e d 0W-14 DNA d i g e s t s . 32 A) P E I - c e l l u l o s e t h i n - l a y e r chromatography of P - p u l s e - l a b e l l e d 0W-14 DNA digested s e q u e n t i a l l y w i t h nuclease SI and snake venom phospho-d i e s t e r a s e . 32 B) C e l l u l o s e t h i n - l a y e r chromatography of P - p u l s e - l a b e l l e d 0W-14 DNA digested s e q u e n t i a l l y w i t h nuclease SI and SVPD. 32 C) C e l l u l o s e t h i n - l a y e r chromatography of P - p u l s e - l a b e l l e d 0W-14 DNA digested w i t h nuclease SI. 32 D) C e l l u l o s e t h i n - l a y e r chromatography of P - u n i f o r m l y - l a b e l l e d 0W-14 and s t r a i n 29 DNA digested w i t h nuclease SI f o r 2 hours at 55°. The amount of DNA per sample was 50 yg ml 1 i n 0.05 M NH^-acetate pH 5.0, -4 10 M Z n C l 2 and w i t h 10 u n i t s of SI per yg of DNA. The d i g e s t i o n and chromatography procedures are described i n d e t a i l i n the M a t e r i a l s and Methods. The numbers on autoradiograms A and B r e f e r to Table 12. 134 137 138 s u s c e p t i b i l i t y of the remaining m a t e r i a l to s o l u b l i z a t i o n by nuclease treatment i n d i c a t e d that i t was n u c l e i c a c i d . Since a l l d i g e s t i o n steps were performed i n a s i n g l e tube, there was no p o s s i b i l i t y of p r e f e r -e n t i a l l o s s of any n u c l e o t i d e . 32 P a r t i a l SI d i g e s t i o n of P - l a b e l l e d s t r a i n 29 and 0W-14 DNA was performed (Figure 28). The products were separated by two-dimen-s i o n a l t h i n - l a y e r chromatography and compared to chromatograms of l i m i t 32 S i or S i and snake venom phosphodiesterase d i g e s t s of P p u l s e - l a b e l l e d 0W-14 DNA. Comparison of the unmodified c e l l u l o s e chromatograms helped mark m i g r a t i o n p o s i t i o n s of o l i g o n u c l e o t i d e d i g e s t i o n products. Migra-t i o n p o s i t i o n s of nu c l e o t i d e s on the commercial c e l l u l o s e l a y e r s were very r e p r o d u c i b l e . Rf values on P E I - c e l l u l o s e sheets were not repro-d u c i b l e . This was important s i n c e the samples were being analyzed f o r novel n u c l e o t i d e s w i t h unknown Rf values. These d i g e s t s were analyzed on c e l l u l o s e and P E I - c e l l u l o s e t h i n l a y e r s . Equal amounts of DNA incubated f o r 2 hours at 55° were analyzed. The y i e l d of smaller o l i g o n u c l e o t i d e s was greater f o r s t r a i n 29 DNA, i . e . the r a t e of c l e a v -age of 0W-14 DNA to s m a l l o l i g o n u c l e o t i d e s was slower than that f o r 32 s t r a i n 29 DNA. A comparison of S I l i m i t d i g e s t s of P u n i f o r m l y -32 l a b e l l e d and P p u l s e - l a b e l l e d DNA i d e n t i f i e d at l e a s t three compounds which were not found i n s t r a i n 29 or 0W-14 p a r t i a l DNA d i g e s t s . These products were s t i l l present i n d i g e s t s which were t r e a t e d w i t h snake venom phosphodiesterase and were probably novel n u c l e o t i d e s found only i n p u l s e - l a b e l l e d 0W-14 DNA preparations. A l l but three of the components found on the c e l l u l o s e t h i n l a y e r and s i x of the components on the P E I - c e l l u l o s e t h i n l a y e r were 139 i d e n t i f i a b l e . In the PEI system, r i b o s e and deoxyribose-containing n u c l e o t i d e s r an together w h i l e i n the c e l l u l o s e system they were re s o l v e d . Ribonucleotides made up about 15 percent of the t o t a l r a d i o -32 a c t i v i t y on the chromatograms. Free PO^ was a l s o released from DNA di g e s t s i n much l a r g e r amounts than from c o n t r o l S i and snake venom 32 phosphodiesterase d i g e s t s of 0W-14 DNA. The r e l e a s e of PO^ was a 32 consequence of the S1 h y d r o l y s i s c o n d i t i o n s s i n c e r e l e a s e of PO^ a l s o occurred from pulsed m a t e r i a l t r e a t e d only w i t h SI nuclease. One of the e x t r a components found i n the c e l l u l o s e system had the chromato-graphic p r o p e r t i e s of hmdUMP. The low Rf values f o r the e x t r a compounds on P E I - c e l l u l o s e suggested that they had net negative charges greater than two. Some of the e x t r a compounds were l i k e l y o l i g o n u c l e o t i d e s r e s u l t i n g from incomplete d i g e s t i o n of 0W-14 DNA. Approximately 22 percent of the t o t a l l a b e l on the P E I - c e l l u l o s e t h i n - l a y e r was found i n s i x u n i d e n t i f i a b l e compounds w h i l e approximately 10 percent of the l a b e l found on the c e l l u l o s e t h i n - l a y e r sheets were i n the three u n i d e n t i f i e d spots (four minor compounds ran i n areas on c e l l u l o s e t h i n - l a y e r sheets which corresponded to the m i g r a t i o n p o s i t i o n s f o r o l i g o n u c l e o t i d e s ; these compounds were not counted)(Table 1 1 ) . The presence of 10 to 20 percent of the pulse l a b e l i n novel compounds agreed w e l l w i t h the a n a l y s i s of pulse l a b e l l e d DNA on CsCl g r a d i e n t s . A maximum of 15 percent of the l a b e l was RNA, ther e f o r e only 15 percent of the l a b e l i n the heavy peak on the CsCl gradient was RNA. The remaining 35 to 40 percent of the p u l s e - l a b e l l e d m a t e r i a l banding at heavy and intermediate d e n s i t i e s on CsCl gradients must have contained some unmodified or p a r t i a l l y modified DNA. The y i e l d of hmdUMP from 140 the P-pulse l a b e l l e d DNA was lower than that p r e d i c t e d from the 3 [6- H ] - u r a c i l pulse. The r a t i o of recovery of putdTMP to dTMP f o r enzymatic d i g e s t s was approximately equal and was greater than the recovery r a t i o noted p r e v i o u s l y f o r the f r e e bases of these compounds 3 i n a [6- H ] - u r a c i l pulse l a b e l l e d 0W-14 DNA sample. Uniform l a b e l l i n g 32 of n u c l e o t i d e s i n the pulse experiments was u n l i k e l y . P O ^ - l a b e l l e d purine n u c l e o t i d e s more r a p i d l y than pyrimidine n u c l e o t i d e s . putdTMP and dTMP l e v e l s were comparable because they were both derived p o s t -r e p l i c a t i o n a l l y from hmdUMP. Sever a l conclusions can be made from the a n a l y s i s of pulse l a b e l l e d DNA. hmUra was found only i n nascent DNA i n i n f e c t e d c e l l s . Heavy d e n s i t y DNA d i d accumulate during DNA synth e s i s but DNA m o d i f i -c a t i o n occurs r a p i d l y a f t e r DNA s y n t h e s i s . There may be three or more precursors of putThy and Thy and these compounds probably have unusually low net negative charges. The s m a l l amount of nascent DNA a v a i l a b l e from p u l s e - l a b e l l e d i n f e c t e d c e l l s n e c e s s i t a t e d the development of d i f f e r e n t approaches to the putThy and Thy b i o s y n t h e s i s problem. Two approaches were considered, screening f o r i n h i b i t o r s of DNA m o d i f i c a -t i o n and the i s o l a t i o n of DNA m o d i f i c a t i o n mutants. Tests f o r i n h i b i t o r s of 0W-14 DNA  syn t h e s i s and m o d i f i c a t i o n The major purpose of the i n h i b i t o r s t u d i e s described so f a r i n t h i s t h e s i s was the attempt to demonstrate that 0W-14 does not u t i l i z e thymidine (dTTP) f o r DNA s y n t h e s i s . S e v e r a l other i n h i b i t o r s were teste d i n the hope that they might have s p e c i f i c e f f e c t s upon 0W-14 DNA r e p l i c a t i o n or m o d i f i c a t i o n . 141 Two i n h i b i t o r s of o r n i t h i n e decarboxylase were t e s t e d . O r n i -t h i n e i s a precursor of the p u t r e s c i n y l s i d e c h a i n of putThy (Karrer and Warren, 1973). Depriving c e l l s of o r n i t h i n e and consequently of p u t r e s c i n e might lead to the accumulation of unmodified or p a r t i a l l y modified 0W-14 DNA. Neither a-methylornithine nor d i f l u o r o m e t h y l o r n i -thine i n h i b i t e d the growth of P_. acidovorans or the a b i l i t y of the t r e a t e d c e l l s to support 0W-14 DNA m o d i f i c a t i o n or r e p l i c a t i o n or phage production (data not shown). Chloramphenicol i s an i n h i b i t o r of p r o c a r y o t i c p r o t e i n synthe-s i s . The minimal i n h i b i t o r y c oncentration of chloramphenicol f o r P_. acidovorans s t r a i n 29 grown i n TCS medium was 5 to 10 yg ml The e f f e c t of a d d i t i o n of chloramphenicol upon 0W-14 r e p r o d u c t i o n was examined. Plaque production and DNA s y n t h e s i s were i n h i b i t e d when chloramphenicol was added to c u l t u r e s 20 minutes a f t e r i n f e c t i o n (Figure 29). This demonstrated that i n 0 W-14-infected c e l l s p r o t e i n s y n t h e s i s was r e q u i r e d before phage DNA could be produced. These observations were not pursued. Ne t r o p s i n , a DNA-binding a n t i b i o t i c , was the only i n h i b i t o r found which appeared to have a s p e c i f i c e f f e c t upon phage reproduction. N e t r o p s i n i s known to bind to DNA and to s t e r i c a l l y b l ock the movement of polymerases along a DNA duplex ( W a r t e l l et a l . , 1974). N e t r o p s i n , at a concentration of 100 yg ml \ had no e f f e c t upon the growth r a t e of P_. acidovorans s t r a i n 29 (Figure 30). Netropsin delayed l y s i s of i n f e c t e d c e l l s (Figure 30) and plaque production was i n h i b i t e d (Figure 31). N e t r o p s i n - t r e a t e d c e l l s made DNA which had a heavier peak over-lapping a normal phage d e n s i t y peak i n CsCl buoyant d e n s i t y gradients 142 FIGURE 29.—The effect of chloramphenicol upon the incorporation of uracil into 0W-14. Chloramphenicol was added to one-half of a culture at 20 minutes after 3 infection. The incorporation of [6- H]-uracil into a l k a l i resistant, TCA precipitable material was followed. P_. acidovorans strain 29 infected with 0W-14, •; F_. acidovorans strain 29 infected with 0W-14 and treated with chloramphenicol (100 yg ml , O. The incorporation of radioactivity i s expressed as the percentage of input label which becomes a l k a l i resistant and TCA precipitable. MINUTES A F T E R I N F E C T I O N FIGURE 30.—The e f f e c t of Net r o p s i n upon the growth of P_. acidovorans s t r a i n 29. The c e l l s were grown i n TCS medium supplemented w i t h s u c c i n a t e and Casamino a c i d s . Netropsin (100 yg ml 1) was added at 125 minutes to an a l i q u o t of the growing c e l l s . The t u r b i d i t y of the i n f e c t e d and the u n i n f e c t e d c u l t u r e s was followed w i t h a K l e t t c o l o r i m e t e r . P_. acidovorans s t r a i n 29, no a d d i t i o n s , * ; P_. acidovorans s t r a i n 29, n e t r o p s i n t r e a t e d , O ; P_. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14 at 240 minutes, n e t r o p s i n t r e a t e d , A. 145 0 50 100 150 200 250 300 350 M I N U T E S FIGURE 31.—Plaque production i n n e t r o p s i n - t r e a t e d P_. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14. A c u l t u r e of growing c e l l s was i n f e c t e d w i t h 0W-14 and s p l i t i n t o two halves. One c u l t u r e was tre a t e d w i t h n e t r o p s i n . A l i q u o t s of both c u l t u r e s were p l a t e d through chloroform i n order to determine the number of plaque forming u n i t s . 0W-14 i n f e c t e d P_. acidovorans s t r a i n 29, no a d d i t i o n s , •; 0W-14 i n f e c t e d ! ? , acidovorans s t r a i n 29, w i t h n e t r o p s i n (100 ug ml ^ ) , O . MINUTES 148 (Figure 32). The aberrant density p r o f i l e was not due to the presence of unmodified DNA. Complete acid-hydrolysis and two-dimensional t h i n -layer chromatography of the DNA l i b e r a t e d only normal amounts of putThy, Thy and Cyt. hmUra did not accumulate (Table 14). The nature of netrop-sin-induced i n h i b i t i o n of phage with respect to reproduction was not established. The heavy density DNA seen on CsCl could have been s i n g l e -stranded. This p o s s i b i l i t y was not tested. 0W-14 _ts_ and am mutants In attempts to f a c i l i t a t e the i n v e s t i g a t i o n of DNA r e p l i c a t i o n and modification, a search for c o n d i t i o n a l l y l e t h a l DNA modification and r e p l i c a t i o n mutants was undertaken i n the b e l i e f that mutants f a i l i n g to make putThy or Thy would be unable to reproduce. This assumption was based upon the observation that putThy and Thy l e v e l s i n 0W-14 DNA were constant and could not be manipulated (K.L. Maltman, unpublished observations). Seventy temperature-sensitive mutants of 0W-14 were i s o l a t e d from nitrous acid mutagenized phage stocks. These phage were impaired i n the a b i l i t y to form plaques on P_. acidovorans s t r a i n 29 at 30° but not at 20° to 22°. T h i r t y - s i x of the mutants were leaky and were set aside. Thirty-four of the mutants were cross-tested against each other and the a b i l i t y to form plaques during mixed i n f e c t i o n s on b a c t e r i a l lawns was determined. Five of the t h i r t y - f o u r mutants gave poor or v a r i a b l e complementation r e s u l t s and could not be segregated into com-plementation groups. The remaining twenty-nine mutants were grouped into twenty-two separate complementation groups which define twenty-two 149 FIGURE 3 2 . — C s C l buoyant de n s i t y gradient a n a l y s i s of 0W-14 DNA e x t r a c t e d from P_. acidovorans s t r a i n 29 c e l l s t r e a t e d w i t h n e t r o p s i n . Netropsin (100 yg ml ^) was added to a growing c u l t u r e of c e l l s which were then i n f e c t e d w i t h 0W-14. At 25 minutes a f t e r i n f e c t i o n [6- H]-u r a c i l (1.0 y C i ml 10.0 yg ml was added and the c e l l s were i n c u -bated u n t i l 45 minutes a f t e r i n f e c t i o n . DNA was extracted from i n f e c t e d c e l l s and analyzed on a n e u t r a l CsCl buoyant d e n s i t y g r a d i e n t . 0W-14 32 DNA synthesized i n the presence of n e t r o p s i n , * ; P - l a b e l l e d 0W-14 reference DNA,O. 20.0 16.0 12.0 8.0] 4.0 10 20 30 40 FRACTION NUMBER TABLE 14.—The base composition of 0W-14 DNA made i n n e t r o p s i n -t r e a t e d c e l l s . Base cpm i n base f r a c t i o n i of t o t a l cpm putThy 25408 (0 .22) Cyt 55302 (0 .49) HmUra 233 (<0 .01) Ura 1947 (0 .02) Thy 30672 (0 • 27) unhydrolyzed n u c l e o t i d e s 86 (<o .01) DNA h y d r o l y s i s i n 6N HCI took place f o r 3 hours at 100°C. The bases were separated by two-dimensional TLC on c e l l u l o s e . The l a b e l l i n g and i s o l a t i o n of n e t r o p s i n t r e a t e d 0W-14 DNA was described i n Figure 32. 152 d i f f e r e n t 0W-14 genes. The complementation a n a l y s i s and grouping i s shown i n Table 15. P. M i l l e r has i s o l a t e d forty-two amber mutants from mutagenized 0W-14 phage st o c k s . These mutants w i l l form plaques on P_. acidovorans s t r a i n 29 (sup 2) but not on P_. acidovorans s t r a i n 29 w i l d - t y p e c e l l s . They have a l s o been assigned to complementation groups. The r e l a t i o n -ship between the fcs and amber complementation groups has not been e s t a b l i s h e d . Attempts were made to screen f o r phage r e p l i c a t i o n and m o d i f i -3 c a t i o n mutants us i n g [ H ] - o r n i t h i n e i n c o r p o r a t i o n at 30° and 20°. Phage-infected c e l l s which d i d not make putThy-containing DNA would not accumulate l a b e l i n DNA. I n w i l d - t y p e phage-infected c e l l s at 30°, DNA sy n t h e s i s begins at 20 to 25 minutes a f t e r i n f e c t i o n and continues u n t i l 60 to 70 minutes a f t e r i n f e c t i o n . At 20°, DNA sy n t h e s i s begins at 60 to 70 minutes a f t e r i n f e c t i o n and continues u n t i l 140 to 150 minutes a f t e r i n f e c t i o n (Figure 33). 0W-14 _ts l y s a t e s were used to i n f e c t s t r a i n 29 at 20° or 30° i n 3 TCS medium.[ H ] - o r n i t h i n e was added and the accumulation of l a b e l i n bacteriophage DNA measured. At 30 minutes, many mutants appear to e x h i b i t a l t e r e d time courses f o r i n i t i a t i o n of DNA s y n t h e s i s . The amount of DNA made a l s o v a r i e d g r e a t l y . I t was not p o s s i b l e to use o r n i t h i n e i n c o r p o r a t i o n to screen p o t e n t i a l DNA m o d i f i c a t i o n mutants i n the _ts_ system because l i t t l e DNA was being synthesized a t the non permissive temperature i n a l l mutants t e s t e d . The o r n i t h i n e l a b e l l i n g p r o t o c o l allowed l a b e l l i n g of DNA at the permissive temperature of 20°. Instead, both am and _ts_ mutants were test e d by preparing TABLE 15.—Complementation a n a l y s i s and screening of 0W-14 _ts_ mutants. Mutant Group s i z e Complemen-t a t i o n group ts index Bases putThy ; i n Thy DNA Hmu Ura % sur-v i v o r : l a 1 1 1.5 x 10 5 nt nt nt nt 33.0 l b 1 2 6.0 x 1 0 3 + + - - 21.0 2a - - 1.3 x 10 6 + + - 14.6 2b 1 3 2.5 x 10 6 + + + - 33.0 2c 4 4 8.5 x 10 4 + + - - 27.3 2e 1 5 6.4 x 10 4 + + - - <0.1 6a 2 6 >10 5 + + - - 14.0 6b 1 7 8.5 x 10 3 + + - - 12.3 9d 1 8 8.4 x 1 0 4 + + - - 10.7 11a 1 9 6.3 x 10 5 + + - - <0.1 12a 1 10 4.5 x 1 0 4 + + - - <0.1 19a 4 4 >10 5 + + - - 10.0 21b 1 11 4.1 x 10 5 + + - - <0.1 24a 1 12 7.2 x 10 6 + + - - <0.1 27a 1 13 leaky + + - - 1.3 29a - - leaky + + - - 5.9 30a - - leaky + + - - 16.6 34a 1 14 leaky + + - - 50.0 Alb 1 15 >10 4 + 4- - - 50.0 A4a 2 16 3.8 x 1 0 4 + + - - 2.8 A4c 2 16 5.1 x 10 6 + 4- - - 1.1 A4d 1 17 >10 5 nt nt nt nt nt A4f 1 18 9.5 x 10 5 + + - - 5.7 A5a 1 19 leaky + 4- - - 0.8 A l i a 2 6 >10 3 nt nt nt nt nt A12a 4 4 >10 3 nt nt nt nt nt A12c 1 20 nt nt nt nt nt nt A17b 4 4 5.0 x 10 5 + 4- - - 6.0 A18c 2 21 >10 5 + + _ — 2.3 154 TABLE 15.—Continued Bases i n DNA Group Complemen- jts_ % s u r -Mutant s i z e t a t i o n group index: putThy Thy Hmu Ura v i v o r s A18d i 22 >10 4 nt nt nt n't nt A20c 2 21 > i o 5 nt nt n t nt nt A21b 1 23 > i o 5 nt nt nt nt nt A22a - - >10 5 nt nt nt nt nt B5a — — > i o 4 + +_ - - 3.8 nt (not tested) High t i t r e l y s a t e s of phage mutants were prepared as described i n the M a t e r i a l s and Methods. P_. acidovorans s t r a i n 29 was i n f e c t e d at a 3 m u l t i p l i c i t y of 20; at 25 minutes a f t e r i n f e c t i o n [6- H ] - u r a c i l was added and the c u l t u r e s were incubated u n t i l the c e l l suspension began to clump. DNA was e x t r a c t e d and processed as described i n the M a t e r i a l s and Methods. Ts index i s the r a t i o of plaque forming u n i t s i n a l y s a t e at 20° com-pared to the number of plaque forming u n i t s i n the same l y s a t e at 30°. 3 [6- H ] - u r a c i l - l a b e l l e d bases detected i n a c i d hydrolysates of DNA were 3 scored (+), bases not l a b e l l e d by [6- H ] - u r a c i l were scored (-), bases present i n marginal amounts were scored as (±). Percent s u r v i v o r s i s the measure of colony forming u n i t s s u r v i v i n g the screening i n f e c t i o n . Complementation a n a l y s i s was performed as described i n the M a t e r i a l s and Methods. FIGURE 33.—DNA synthesis i n 0W-14-infected P. acidovorans s t r a i n 29 at 20°C. 3 The i n c o r p o r a t i o n of [6- H ] - u r a c i l i n t o a l k a l i r e s i s t a n t , TCA p r e c i -p i t a b l e m a t e r i a l was followed. The procedures used are described i n the M a t e r i a l s and Methods. 10 ALKALI RESISTANT e T C A PRECIPITABLE 6 C P M x IO-3 4 20 40 60 80 100 120 140 MINUTES POST INFECTION 157 [6- H ] - u r a c i l - l a b e l l e d DNA from c e l l s i n f e c t e d under non permissive c o n d i t i o n s . DNA from am-infected s t r a i n 29 c e l l s was harvested 45 to 50 minutes a f t e r i n f e c t i o n . DNA from t s - i n f e c t e d s t r a i n 29 c e l l s grown at 30° was harvested from c e l l s when c u l t u r e s took on a granular appear-ance. (Generally, i n f e c t e d c e l l s became s t i c k y , s t a r t e d to clump and had a grainy appearance 10 to 15 minutes p r i o r to l y s i s . In w i l d - t y p e c e l l s at t h i s stage, DNA s y n t h e s i s was w e l l - e s t a b l i s h e d . ) S u r v i v o r s of the screening i n f e c t i o n s were measured (Table 15). DNA extracted from 0W-14 am-infected c e l l s was analyzed on n e u t r a l CsCl d e n s i t y g r a d i e n t s . This screening procedure detected phage which d i d not shut o f f host DNA s y n t h e s i s e f f e c t i v e l y (ambers 36, 38), mutants which d i d not make 0W-14 w i l d - t y p e d e n s i t y DNA (ambers 6, 35, 45), and mutants making DNA of unusual d e n s i t y (ambers 37, 42). Representative examples of screening gradients are shown i n Figure 34 (see Table 16). 3 The [6- H ] - u r a c i l - l a b e l l e d amber DNA preparations were a c i d -hydrolyzed and the bases were separated by two-dimensional t h i n - l a y e r chromatography i n solvents B and D. Authentic base standards were run w i t h h y d r o l y s a t e s . The a u t h e n t i c base mixture contained putThy, Cyt, hmUra, Ura and Thy. The base spots were lo c a t e d under u l t r a v i o l e t l i g h t and cut out and counted. Base spots c o n t a i n i n g s i g n i f i c a n t amounts of a base were scored as p o s i t i v e (+) (Table 16). Using t h i s procedure, of a l l the amber mutants screened, only am 37 accumulated hmUra i n i t s DNA. Am 37 DNA contained t r i t i u m - l a b e l l e d Thy, Cyt and 3 hmUra. L i t t l e [6- H ] - u r a c i l - l a b e l l e d putThy was recovered from am 37 DNA. Am 42 DNA was a l s o analyzed by a c i d h y d r o l y s i s . The only l a b e l l e d products r e l e a s e d were putThy, Thy and Cyt (Table 18). 158 FIGURE 3 4 . — C s C l buoyant d e n s i t y gradients of DNA from P_. acidovorans s t r a i n 29 i n f e c t e d w i t h 0W-14 am mutants. The l a b e l l i n g and i s o l a t i o n procedures are described i n the M a t e r i a l s and Methods. A) am 37 DNA e x t r a c t e d 45 minutes a f t e r i n f e c t i o n ; B) am 42 DNA extracted 45 minutes a f t e r i n f e c t i o n ; C) am 35 DNA 3 e x t r a c t e d 60 minutes a f t e r i n f e c t i o n . [6- H ] - u r a c i l l a b e l l e d DNA, • ; 32 + PO, l a b e l l e d 0W-14 w phage reference DNA, O. A 6.0 4 .0 2.0 i O Q_ O 0_ CM m 10 20 30 FRACTION NUMBER ro , 12.0 O 8.0 Q. O X ro 4 .0 4 B 6.0 * O 4.0 2.0 CL O D L CM ro 10 20 30 FRACTION NUMBER 4 .0 2.0 [ 4.0 I 2.0 i O CL O 0-CM to 10 20 30 FRACTION NUMBER 160 TABLE 16.—The p r o p e r t i e s of DNA e x t r a c t e d from 0W-14 am-Infected P_. acidovorans. P_. acidovorans s t r a i n 29 was i n f e c t e d w i t h 0W-14 am mutants at a mul-3 t i p l i c i t y of i n f e c t i o n of twenty. The c e l l s were l a b e l l e d w i t h [6- H]-u r a c i l from 25 minutes to 45 minutes a f t e r i n f e c t i o n . DNA was e x t r a c -ted and processed as described i n the M a t e r i a l s and Methods. Bases detected a f t e r a c i d h y d r o l y s i s and two-dimensional t h i n - l a y e r chromatography of DNA were scored (4-) when the base was detected i n s i g n i f i c a n t q u a n t i t i e s . The absence of the base or i t s marginal pre-sence was scored as (-) or (±), r e s p e c t i v e l y . The DNA was a l s o analyzed on n e u t r a l CsCl d e n s i t y g r a d i e n t s . DNA peaks banding at host, phage or other buoyant d e n s i t y were scored (4-) . The absence of a peak banding at host, phage or other d e n s i t i e s was r e c o r -ded as (-). The suppressor index i s the r a t i o of plaque forming u n i t s i n a l y s a t e p l a t e d on P_. acidovorans sup 2 compared to the number of plaque forming u n i t s i n the same l y s a t e p l a t e d on P_. acidovorans s t r a i n 29. Percent s u r v i v o r s i s the measure of colony forming u n i t s s u r v i v i n g the screen-i n g i n f e c t i o n . 161 am mutant Bases putThy detected In Thy Hmu DNA Ura DNA detected i n CsCl host 0W-14 other sup index % s u r v i v o r s 2 + + - - + >10 6 1.3 3 + + - + + >105 <1.0 4 + + - - + >105 8.8 5 + + - - + >106 0.9 u 7 + + - - + >io5 0.9 8 + + - - + 1.6 x 10 3 0.6 10 + + - - + >10 5 0.4 11 + + - - + >io5 <1.0 12 + + - + + >io6 4.9 14 + + - - + >10 5 <1.0 15 + + - - + >10 5 <1.0 16 + + - - + >io6 0.2 17 + + - - + >10 5 <1.0 18 + + - - + >io6 0.5 19 + + - - + - >io6 0.4 20 + + - nt nt nt >10 6 0.6 21 + + - + + 1.5 x 10 5 19.0 22 + + - + + >10 5 7.0 23 + + - + + 1.4 x 10 5 9.0 24 + + - + + leaky >50 25 + + - + + leaky >50 162 am Bases detected in DNA DNA detected in CsCl sup mutant putThy Thy Hmu Ura host 0W-14 other index survivors 26 + 4- - - + ± - >io 6 14.0 27 + + - - + + - >io 6 9.0 28 + + - - - + - >105 1.0 29 + + - - + + - >106 9.0 30 + + - - - + . - >io 6 1.0 31 4- + - - - + >io 6 1.0 32 + + - - - + - >106 1.0 33 + + - - - + - 2.5 x 10 6 1.0 34 + + - - - + - 3.2 x 10 5 1.0 35 - + - - + - - >106 1.0 36 + + - - + + - leaky 1.0 37 - + + - + - + 2.3 x 10 5 1.0 38 4- + - - + + - >106 1.0 39 4- + - - + + - 1.8 x 10 6 8.0 40 4- + - - - + - >105 1.0 41 4- 4- - - - + - >104 1.0 42 + + - - - - + 3.9 x 10 4 1.0 43 4- + - - + + - 3.0 x 10 5 13.0 44 + + - - + + - 1.5 x 10 6 9.0 45 — + _ _ + _ _ 1.4 x 10 5 7.0 163 3 Twenty-five _ts_ mutants were analyzed by pr e p a r a t i o n of [6- H]-u r a c i l - l a b e l l e d DNA. Ts 19a made only s m a l l amounts of putThy-contain-i n g DNA (Table 15). Am 37, am 42 and _ts 19a were analyzed i n more d e t a i l . (N.B. The recovery of u r a c i l from _ts_ 2a DNA and of hmUra from _ts_ 2b DNA have not been confirmed.) 0W-14 _ts_ 19a-inf e c t i o n of P_. acidovorans Ts 19a di d not form plaques on s t r a i n 29 grown on CAA-M p l a t e s at 30°. Plaques on CAA-M medium at 20° were s m a l l . The burst s i z e of ts 19a i n l i q u i d TCS medium at 30° was l e s s than 1.0 pfu per c e l l . The burs t s i z e f o r _ts_ 19a grown on TCS medium at 20° was 16 pfu per c e l l . Ts 19a phage stocks are prepared on CAA-M medium where phage y i e l d s per i n f e c t e d c e l l are greater. 3 The accumulation of [ H ] - o r n i t h i n e l a b e l i n Jts_ 19a-infected c e l l s i s shown (Figure 35). O r n i t h i n e - l a b e l l e d DNA accumulated at the permissive temperature of 20° but not at the non permissive temperature of 30°. Measurement of DNA accumulation i n _ts_ 19a-infected c e l l s w i t h 3 [6- H ] - u r a c i l gave a d i f f e r e n t p a t t e r n (Figure 36). Label accumulated i n c e l l s i n f e c t e d at 30° u n t i l 70 or 80 minutes a f t e r i n f e c t i o n . The f i n a l amount of l a b e l accumulating i n DNA at 20° was more than four times the f i n a l amount of l a b e l i n DNA at 20°. N e u t r a l CsCl d e n s i t y gradient a n a l y s i s of DNA ext r a c t e d from _ts_ 19a-infected c e l l s incubated at 30° u n t i l 40 minutes a f t e r i n f e c t i o n demonstrated that a l l the l a b e l was i n ho s t - d e n s i t y DNA (Figure 37). An i d e n t i c a l c u l t u r e incubated u n t i l 70 minutes a f t e r i n f e c t i o n contained a sm a l l amount of 0W-14 FIGURE 35.—The i n c o r p o r a t i o n of [ H ] - o r n i t h i n e i n 0W-14 ts 19a-i n f e c t e d P_. acidovorans s t r a i n 29. 3 The i n c o r p o r a t i o n of [ H ] - o r n i t h i n e i n t o pronase r e s i s t a n t , TCA p r e c i p i t a b l e cpm was measured i n 0W-14 _ts_ 19a i n f e c t e d J_. a c i d o - vorans s t r a i n 29 at 30°, •; and at 20°, O. The procedures used are described i n the M a t e r i a l s and Methods. L y s i s of the 30° c u l t u r e occurred at 90 to 100 minutes a f t e r i n f e c t i o n . 165 7.0 0 40 80 120 160 200 MINUTES AFTER INFECTION FIGURE 36.—DNA synthesis i n 0W-14 _ts_ 19a-infected P_. acidovorans s t r a i n 3L. 3 The i n c o r p o r a t i o n of [6- H ] - u r a c i l i n t o a l k a l i r e s i s t a n t , TCA p r e c i -p i t a b l e cpm was determined f o r 0W-14 _ts_ 19a i n f e c t e d c e l l s at 30°, O 3 -1 -1 and at 20°, •. ( [ 6 - H ] - u r a c i l 0.5 y C i ml , 10 yg ml .) L y s i s of the 30° c u l t u r e occurred 90 to 100 minutes a f t e r i n f e c t i o n . 167 168 FIGURE 37.—The buoyant d e n s i t y of DNA made i n 0W-14 t s 19a-infected P_. acidovorans s t r a i n 29. 3 [6- H ] - u r a c i l - l a b e l l e d DNA was ext r a c t e d from c e l l s and banded on CsCl d e n s i t y g r a d i e n t s , •. A) The c e l l s were i n f e c t e d at 30° and DNA was e x t r a c t e d 40 minutes a f t e r i n f e c t i o n . B) The c e l l s were i n f e c t e d at 30° and DNA was ext r a c t e d 75 minutes a f t e r i n f e c t i o n . C) The c e l l s were i n f e c t e d at 30° and s h i f t e d to 20° at 40 minutes a f t e r i n f e c t i o n . The DNA was e x t r a c t e d at 75 minutes a f t e r i n f e c t i o n . D) , The c e l l s were i n f e c t e d at 30° and s h i f t e d to 20° at 40 minutes a f t e r i n f e c t i o n . The DNA was e x t r a c t e d at 85 minutes a f t e r i n f e c t i o n . E) The c e l l s were i n f e c t e d at 20° and were allowed to l y s e . The DNA was extracted from p u r i f i e d 0W-14 ts^ 19a v i r i o n s . 3 [6- H ] - u r a c i l was added to c u l t u r e s at 20 minutes a f t e r i n f e c t i o n . 32 Gradient A i n c l u d e s P - l a b e l l e d host reference DNA, O; Gradient E 32 inclu d e s P - l a b e l l e d phage reference DNA, Q . 169 2 o X to 8.0 4.0 8.0 4.0 10 2 0 3 0 4 0 IO t O Q. O O. CVJ IO FRACTION NUMBER A IO I O Cu o X IO e.o 4.0 10 2 0 3 0 4 0 FRACTION NUMBER B IO i O Q L X to 8.0 4 .0 10 2 0 3 0 4 0 FRACTION NUMBER C IO i O O X IO 8.0 4 . 0 10 2 0 3 0 4 0 FRACTION NUMBER D IO FRACTION NUMBER E 170 w i l d - t y p e - d e n s i t y DNA. When 30° c u l t u r e s are s h i f t e d down to 20° at 40 minutes a f t e r i n f e c t i o n , ts_ 19a DNA synth e s i s was rescued (Figure 38). 0W-14 w i l d - t y p e - d e n s i t y DNA appears i n GsCl gradients a f t e r s h i f t down (Figure 37). The shut o f f of host DNA synth e s i s was not e f f i c i e n t . S u r v i v o r s of i n f e c t i o n s were al s o high. Ts_ 19a DNA, which i s made at 30°, does c o n t a i n putThy. hmura and/or Ura d i d not accumulate i n DNA (Table 17). Ts 19a DNA made at 20° was normal. I t banded at the same de n s i t y as w i l d - t y p e phage reference DNA (Figure 37) and had normal l e v e l s of putThy and Thy i n i t s DNA. Ts 19a could be a DNA delay mutant or a leaky DO mutant. In a d d i t i o n , tj3 19a could be impaired i n i t s a b i l i t y to shut o f f host DNA s y n t h e s i s . This a n a l y s i s was hampered due to the high l e v e l s of s u r v i v o r s i n ts_ 19a i n f e c t i o n s . Am 42- i n f e c t e d F_. acidovorans s t r a i n 29 accumulated DNA w i t h a d e n s i t y greater than that of the w i l d - t y p e phage reference DNA 3 [6- H ] - u r a c i l accumulation i n am 42-infected s t r a i n 29 or sup 2 demonstrated that the same amount of l a b e l accumulated i n both hosts. The DNA synth e s i s program began 35 to 45 minutes a f t e r i n f e c t i o n and proceeded u n t i l about 105 minutes a f t e r i n f e c t i o n (Figure 39). Enzymatic d i g e s t i o n and two-dimensional t h i n - l a y e r chromato-3 graphy of [6- H ] - u r a c i l - l a b e l l e d DNA prepared i n the non permissive host showed that DNA was d e f i c i e n t i n putThy (Table 18). The d e f i -ciency i n putThy was compensated f o r by an increase i n l e v e l s of Thy. HmUra was not detected i n am 42 DNA. DNase I treatment of chloroform-treated am 42 phage l y s a t e s demonstrated that am 42 DNA was packaged a f t e r i n f e c t i o n of permissive and non permissive hosts (Figure 39). 171 FIGURE 38.—0 W-14 _ts_ 19a DNA s y n t h e s i s can be rescued by a temperature s h i f t from the nonpermissive to the permissive temperature. A growing c u l t u r e of P_. acidovorans s t r a i n 29 was i n f e c t e d w i t h 0W-14 t s 19a. The i n f e c t e d c u l t u r e was incubated at 30°. At 10 minutes 3 -1 -1 a f t e r i n f e c t i o n [6- H ] - u r a c i l (0.25 y C i ml , 10 yg ml ) was added to the c u l t u r e . At 35 minutes a f t e r i n f e c t i o n one-half of the c u l t u r e was s h i f t e d to 20°. DNA s y n t h e s i s was measured by f o l l o w i n g the i n c o r -p o r a t i o n of r a d i o a c t i v i t y i n t o a l k a l i r e s i s t a n t , TCA p r e c i p i t a b l e m a t e r i a l . 30°, •; 30° downshifted to 20°, O. 172 M I N U T E S A F T E R I N F E C T I O N TABLE 17.—The Base composition of DNA made i n 0W-14 _ts 19a-infected P. acidovorans. DNA sample 35 minutes/30 o s h i f t e d Base 35 minutes/30 o 90 minutes/20 o to 20° f o r 30 minutes putThy 407 (<0.01) a 32162 (0. 18) 10306 (0.05) Cyt 29799 (0.58) 104744 (0. 59) 115418 (0.57) Thy 21373 (0.41) 39802 (0. 23) 77772 (0.38) Hmu 310 (<0.01) - 519 (<0.01) Ura 221 (<0.01) — 715 (<0.01) The DNA was l a b e l l e d , e xtracted and hydrolyzed as described i n the M a t e r i a l s and Methods. The bases were separated by two-dimensional t h i n - l a y e r chromatography on c e l l u l o s e sheets. a The values are cpm i n the area cut from the chromatogram. The f i g u r e s i n parentheses represent the f r a c t i o n of the r a d i o a c t i v i t y recovered i n bases. The t o t a l recovery of r a d i o a c t i v i t y was greater than 90 percent. FIGURE 39.—DNA synthe s i s i n 0W-14 am 42- i n f e c t e d P. acidovorans. 3 -1 -1 The i n c o r p o r a t i o n of [6- H ] - u r a c i l (1.0 y C i ml , 10.0 yg ml ) i n t o a l k a l i r e s i s t a n t , TCA p r e c i p i t a b l e m a t e r i a l was measured f o r am 42 i n f e c t e d s t r a i n 29, O; and am 42 i n f e c t e d s t r a i n sup 2, •. The two po i n t s not connected to the curves represent DNase I r e s i s t a n t , a l k a l i r e s i s t a n t , TCA p r e c i p i t a b l e m a t e r i a l i n CHC1„ tr e a t e d l y s a t e s . ALKALI RESISTANT T C A PRECIPITABLE C P M x IO- 3 176 TABLE 18.—The n u c l e o t i d e composition of 0W-14 am 42 DNA. Nucleotide 0W-14 w + DNA 0W-14 am 42 DNA putdTMP dCMP dTMP hmdUMP putdTMP/dTMP 6904 (0.25) 14007 (0.50) 7219 (0.25) none 0.96 4001 (0.07) 29812 (0.52) 23639 (0.41) none 0.17 3 DNA was l a b e l l e d w i t h [6- H ] - u r a c i l and p u r i f i e d and processed as described i n the M a t e r i a l s and Methods. Nucleotides were separated by two-dimensional t h i n - l a y e r chromatography on c e l l u l o s e sheets. a The values are cpm i n the area cut from the chromatogram. The f i g u r e s i n parentheses represent the f r a c t i o n of the r a d i o -a c t i v i t y recovered i n the n u c l e o t i d e s . The recovery of r a d i o -a c t i v i t y was greater than 90 percent. 177 SI and snake venom phosphodiesterase d i g e s t s of a sample of 3 [6- H ] - u r a c i l - l a b e l l e d am 42 DNA prepared i n s t r a i n 29 were separated by two-dimensional t h i n - l a y e r chromatography on P E I - c e l l u l o s e and c e l -l u l o s e t h i n - l a y e r s (Figure 40). Am 42 DNA contained depressed l e v e l s of putdTMP and elevated l e v e l s of dTMP. The s i n g l e peak on the CsCl gradient r u l e d out the p o s s i b i l i t y that the i n c r e a s e i n dTMP l e v e l s detected was due to the presence of l a b e l l e d host DNA (Figure 32). The moi % G + C content suggested by the n u c l e o t i d e l a b e l l i n g was 52. The c o n d i t i o n a l l y l e t h a l nature of t h i s mutant has not been confirmed. P o t e n t i a l l y , am 42 could provide evidence that p r e c i s e l e v e l s of putThy and Thy i n 0W-14 DNA are r e q u i r e d f o r 0W-14 v i a b i l i t y . 0W-14 am 37 3 A [6- H ] - u r a c i l - l a b e l l e d sample of DNA prepared i n P_. acidovorans s t r a i n 29 was analyzed on a n e u t r a l CsCl d e n s i t y gradient (Figure 34). The l a b e l i n the DNA was found i n three d i s t i n c t peaks. The l i g h t e s t of these peaks i s b e l i e v e d to be l a b e l l e d host DNA (p,= 1.72 gcc ^ ) . 32 -1 PO^ phage reference DNA (p = 1.666 gcc ) marks the p o s i t i o n where normally-modified 0W-14 should have banded. Two peaks (p = 1.73 to 1.74 and 1.77 to 1.78) mark p o s i t i o n s of two new bands of heavy d e n s i t y DNA. A c i d h y d r o l y s i s and t h i n - l a y e r chromatography of the DNA sample revealed that Cyt, Thy, and hmUra but l i t t l e putThy were present i n the DNA (Table 20') . The a b i l i t y of am 37 and w i l d type phage to p l a t e on P_. acidovorans s t r a i n s 29 and JE1 and s e v e r a l of t h e i r sup d e r i v a t i v e s was determined (Table 19). 0W-14 w + p l a t e s w i t h high e f f i c i e n c y on 1 7 8 FIGURE 40.—Two-dimensional thin-layer chromatography of [ 6 - H]-uracil-labelled nucleotides present in am 42 DNA prepared in P_. acidovorans strain 29 . T t 1 7 9 TABLE 19.—The p l a t i n g e f f i c i e n c y of 0W-14 am 37 on P _ . acidovorans s t r a i n s . Host s t r a i n 0W--14 w + e.o.p. 0W-14 am 37 e. 0 . a P -29 3.8 X I O 1 1 1.0 1.1 X i o 5 2.3 X I O " 7 29 (sup 1) 4.2 X 1 0 1 1 1.1 4.7 X i o 1 1 1.0 29 (sup 2) 4.7 X i o 1 1 1.2 4.8 X i o 1 1 1.0 29 (sup 3) 5.1 X i o 1 1 1.3 5.0 X i o 1 1 1.1 29 (sup 4) 4.5 X i o 1 1 1.2 4.9 X i o 1 1 1.0 29 (sup 5) 4.5 X i o 1 1 1.2 5.0 X i o 1 1 1.1 JE1 3.8 X i o 1 1 1.0 3.0 X i o 5 6.4 X I O " 7 JE1 (sup 1) 5.2 X i o 1 1 1.4 3.5 X i o 5 7.4 X I O " 7 JE1 (sup 2) 4.9 X i o 1 1 1.3 2.6 X 10 5 5.5 X I O " 7 JE1 (sup 3) 5.4 X i o 1 1 1.4 3.1 X I O 5 6.6 X i o " 7 Lysates of 0 W - 1 4 w and am 37 phages were p l a t e d on v a r i o u s P _ . a c i d o - vorans s t r a i n s . The values are the numbers of plaque forming u n i t s i n one m i l l i l i t e r of a l y s a t e . Wild type 0 W - 1 4 l y s a t e s were prepared on P _ . acidovorans s t r a i n 29. 0 W - 1 4 am 37 l y s a t e s were prepared on P _ . acidovorans s t r a i n sup 2. a e.o.p. i s the e f f i c i e n c y of p l a t i n g . I t i s the r a t i o of plaques formed by a l y s a t e on the permissive host r e l a t i v e to the number of plaques formed on another host. P _ . acidovorans sup 2 was the per-m i s s i v e host f o r 0W-14 am 37. TABLE 20.—Base compositions of 0W-14 and am 37 DNAs. a Base am 3 7 / s t r a i n 29 Source of DNA 2 am 3 7 / s t r a i n sup 0W-14/strain 29 putThy 1074 b (2.7)° 5062 (19.9) 14430 (18.2) cy t o s i n e 18016 (44.5) 10539 (41.5) 34585 (43.6) hmUra 5903 (14.6) 1028 (4.0) 157 (0.2) u r a c i l 1209 (3.0) 176 (0.7) 180 (0.2) thymine 9353 (23.1) 6173 (24.3) 21976 (27.7) unhydrolyzed n u c l e o t i d e s 4964 (12.3) 2441 (9.6) 7987 (10.1) a R e p l i c a t i n g DNA was l a b e l l e d w i t h [ 6 - J H ] - u r a c i l . A f t e r h y d r o l y s i s , the bases were separated by t h i n - l a y e r chromatography, using s o l v e n t B i n the 1st and solvent D i n the 2nd dimension. b Cpm i n the area cut from the chromatogram. c The f i g u r e s i n parentheses are the percentages of the t o t a l r a d i o a c t i v i t y recovered from the chromatogram. Recoveries were r o u t i n e l y greater than 90 percent. 181 a l l the tested s t r a i n s . 0W-14 am 37 p l a t e d only on the f i v e sup d e r i v -a t i v e s of P_. acidovorans s t r a i n 29. The 0W-14 am 37 r e v e r s i o n frequency —6 i s l e s s than 10 wi l d - t y p e plaques formed per mutant phenotype plaque. The b u r s t s i z e of am 37 grown on s t r a i n s 29 and sup 2 was det e r -mined (Figure 41). A l i q u o t s of i n f e c t e d c u l t u r e s were p l a t e d through chloroform at time p o i n t s a f t e r i n f e c t i o n . A f t e r an e c l i p s e p e r i o d of 60 to 70 minutes phage began to appear i n sup 2 c u l t u r e s . The l y s i s time was v a r i a b l e but u s u a l l y occurred 140 to 180 minutes a f t e r i n f e c -t i o n . There was not any r e l e a s e of i n f e c t i o u s v i r u s from am 37 i n f e c t e d s t r a i n 29 c e l l s . The average burst s i z e f o r am 37 phage i n s t r a i n sup 2 grown on TCS medium was 50 plaque forming u n i t s per c e l l . The c o n d i t i o n a l nature of the DNA m o d i f i c a t i o n l e s i o n was dem-onstrated. I n the sup 2 host am 37 i n f e c t e d c e l l s accumulated some DNA w i t h the same den s i t y as 0W-14 w + reference DNA. S t r a i n 29 c e l l s i n f e c t e d w i t h am 37 accumulated heavy d e n s i t y DNA but not w i l d type, l i g h t d e n s i t y DNA. Suppression of the m o d i f i c a t i o n l e s i o n i n the sup-pressor host was incomplete. The am 37-sup 2 i n f e c t e d c e l l s contained DNA w i t h a d e n s i t y greater than normally seen i n sup 2 or s t r a i n 29 c e l l s i n f e c t e d w i t h w i l d - t y p e 0W-14 (Figure 42). These experiments suggested that a gene r e s p o n s i b l e f o r some aspect of 0W-14 DNA modi-f i c a t i o n was mutated i n am 37. Furthermore, t h i s mutation was respon-s i b l e f o r the c o n d i t i o n a l l y l e t h a l phenotype of am 37 phage reproduc-t i o n . Am 37 DNA contains a novel n u c l e o t i d e Q u a n t i t a t i v e a c i d h y d r o l y s i s and t h i n - l a y e r chromatography of 3 [6- H ] - u r a c i l l a b e l l e d DNA samples was performed (Table 20). DNA FIGURE 41.—The reproduction of 0W-14 am 37 i n P_. acidovorans s t r a i n s 29 or sup 2. At i n t e r v a l s a f t e r i n f e c t i o n of s t r a i n s 29 or sup 2 a l i q u o t s of the c u l t u r e s were removed and d i l u t e d through CAA-M over CHCl^. Plaque forming u n i t s were assayed by p l a t i n g on P_. acidovorans s t r a i n sup 2 P_. acidovorans was grown i n TCS medium supplemented w i t h s u c c i n a t e and Casamino a c i d s . Am 37 i n f e c t e d s t r a i n sup 2,0; am 37 i n f e c t e d s t r a i n 29, •-. M I N U T E S A F T E R I N F E C T I O N FIGURE 42.—Buoyant d e n s i t y of DNA synthesized by phage-infected c e l l s 3 Infected c e l l s were l a b e l l e d w i t h [6- H ] - u r a c i l and the DNA ext r a c t e d from the c e l l s 35 minutes a f t e r i n f e c t i o n . S t r a i n 29 i n f e c t e d w i t h w i l d - t y p e phage (A) and w i t h am 37 (C); s t r a i n sup 2 i n f e c t e d w i t h 3 w i l d - t y p e phage (B) and w i t h am 37. (D) [6- H ] - u r a c i l - l a b e l l e d 32 DNA; P reference DNA from phage p a r t i c l e s . The bottom of the gradient i s on the l e f t . 185 186 i s o l a t e d from s t r a i n 29 c e l l s i n f e c t e d w i t h am 37 had extremely low l e v e l s of putThy when compared to am 37 DNA prepared i n the sup 2 host or to w i l d - t y p e DNA i s o l a t e d from i n f e c t i o n of s t r a i n 29. Thymine l e v e l s i n am 37 DNA prepared i n e i t h e r the permissive or the non per-m i s s i v e host were normal. DNA samples from am 37-infected s t r a i n 29 contained 14.6 percent of the t o t a l p y r imidine l a b e l i n hmUra re s i d u e s . DNA samples from am 37 i n f e c t e d s t r a i n sup 2 contained 4.0 percent of the t o t a l pyrimidine l a b e l i n hmUra. In 0W-14 w + i n f e c t e d c e l l s 0.2 percent or l e s s of the t o t a l p y r i m i d i n e l a b e l was found i n hmUra r e s -idues . 3 2 P 0 4 and [ 6 - 3 H ] - u r a c i l l a b e l l e d am 37 DNA and 0W-14 w + DNA were prepared and analyzed by two-dimensional t h i n - l a y e r chromatography a f t e r nuclease SI and snake venom phosphodiesterase d i g e s t i o n (Figure 43) (Table 21). Am 37-infected s t r a i n 29 c e l l s contained DNA w i t h two e x t r a p yrimidine n u c l e o t i d e s not found i n uniformly l a b e l l e d prepara-t i o n s of w i l d - t y p e 0W-14 DNA d i g e s t s . The two n u c l e o t i d e s shared chrom-atographic p r o p e r t i e s w i t h two of the three e x t r a n u c l e o t i d e s detected 32 i n P-pulse l a b e l l e d 0W-14 DNA d i g e s t s . The Rf value f o r each nucleo-t i d e was measured i n s o l v e n t s E and A on c e l l u l o s e t h i n - l a y e r sheets (Table 21). One of the e x t r a components had chromatographic p r o p e r t i e s s i m i l a r to hmdUMP. The other u n i d e n t i f i e d compound was more h e a v i l y 32 3 l a b e l l e d w i t h PO^ than w i t h [6- H ] - u r a c i l . This suggested that t h i s compound contained more than one phosphate residue per pyrimidine base. I t was p o s s i b l e that t h i s compound was an o l i g o n u c l e o t i d e which r e s u l t e d from incomplete d i g e s t i o n of am 37 DNA. A r e l a t i v e l y l a r g e amount of 32 32 f r e e PO^ was a l s o released from P - l a b e l l e d am 37 DNA during FIGURE 4 3 . — N u c l e o t i d e s i n am 37 DNA. 3 C e l l s were l a b e l l e d w i t h [6- H ] - u r a c i l a f t e r i n f e c t i o n w i t h am 37. The DNA was ext r a c t e d from the c e l l s at 45 minutes a f t e r i n f e c t i o n and digested to mononucleotides which were separated by t h i n - l a y e r chromatography. A: s t r a i n sup 2 as host; B: s t r a i n 29 as host. 1: dTMP; 2: dCMP; 3: hmdUMP; 4: unknown n u c l e o t i d e ; 5: putdTMP TABLE 2 1 . — N u c l e o t i d e composition of 0W-14 and am 37 DNAs. 3 Nucleotide am 3 7 / s t r a i n 29 ( [ 6 - % ] - u r a c i l ) Source am 3 7 / s t r a i n 29 (3 ^ F orthophosphate) ot DNA am 3 7 / s t r a i n sup2 ( [ 6 - 3 H ] - u r a c i l ) 0 W-14/strain.29 (32p orthophosphate) E R f d A dGMP 40 b ( o . i ) c 3944 (16.1) 0 13355 (24.1) 26 39 dAMP 72 (0.2) 3012 (12.3) 0 13921 (25.1) 56 27 dTMP 8080 (27.6) 2972 (12.1) 41218 (27.0) 7219 (13.0) 36 52 dCMP 13252 (45.2) 5943 (24.2) 81876 (53.7) 14007 (25.2) 43 70 putdTMP 385 (1.3) 87 (0.4) 27835 (18.3) 6904 (12.4) 55 79 hmdUMP 2043 (7.0) 865 (3.5) 256 (0.2) 29 (<0.1) 23 62 unknown 5430 (18.5) 7750 (31.5) 1264 (0.8) 47 (<0.1) 9 65 a R e p l i c a t i n g DNA was l a b e l l e d w i t h [6-~"H]-uracil or J^P-orthophosphate. A f t e r d i g e s t i o n w i t h nuclease SI and snake venom phosphodiesterase, the mononucleotides were separated by t h i n - l a y e r chromatography. b Cpm i n the area cut from the chromatogram. c The f i g u r e s i n parentheses are the percentages of the t o t a l r a d i o a c t i v i t y recovered as n u c l e o t i d e s , d Rf values are given f o r n u c l e o t i d e s i n solvents E and A. o 191 enzymatic d i g e s t i o n w i t h nuclease SI and snake venom phosphodiesterase. putdTMP made up 1.3 percent or l e s s of the t o t a l p y r imidine l a b e l i n am 37 DNA e x t r a c t e d from s t r a i n 29 but was 18 percent of the t o t a l p y r i m i d i n e l a b e l i n am 37 DNA e x t r a c t e d from s t r a i n sup 2 c e l l s . dTMP was found i n a l l am 37 DNA samples i n normal q u a n t i t i e s . Am 37 was d e f i c i e n t i n i t s a b i l i t y to make putdTMP i n a non permissive host, but was the e x t r a r a d i o a c t i v e component a novel nucleo-t i d e or an o l i g o n u c l e o t i d e r e s u l t i n g from the incomplete d i g e s t i o n of hmdUMP co n t a i n i n g DNA? I t was p o s s i b l e to p r e d i c t the buoyant d e n s i t y of hmUra c o n t a i n i n g DNA of known mol % G + C content. The p r e d i c t e d buoyant d e n s i t y of n a t i v e duplex DNA c o n t a i n i n g guanine, adenine, c y t o -s i n e and thymine i s defined by the formula p = 0.098 (G + C) + 1.660. For DNA w i t h a mol % G + C of 52.0 the p r e d i c t e d buoyant d e n s i t y i s 1.711 gcc 1 . For the same mol % G + C v a l u e , DNA c o n t a i n i n g hmUra in s t e a d of thymine would have a buoyant de n s i t y of 1.751 gcc \ w h i l e DNA w i t h a mol % G + C value of 52.0 c o n t a i n i n g equal amounts of Thy and hmUra would have a buoyant d e n s i t y of 1.731 gcc These c a l c u l a -t i o n s were preformed according to formulae described by Rae (1973). The h e a v i e s t d e n s i t y am 37 DNA peak has a buoyant d e n s i t y between 1.77 and 1.78 gcc 1 . This value i s much greater than could be p r e d i c t e d , even f o r complete s u b s t i t u t i o n of putThy and Thy by hmUra. I t was l i k e l y that am 37 DNA contained a novel n u c l e o t i d e . This n u c l e o t i d e was probably an intermediate i n the b i o s y n t h e t i c pathway l e a d i n g to putdTMP. 3 [6- H ] - u r a c i l l a b e l l e d hmUra i n a c i d hydrolysates of am 37 DNA prepared i n s t r a i n 29 formed a greater percentage of the t o t a l a c i d hydrolyzed l a b e l than [6- H ] - u r a c i l l a b e l l e d hmdUMP i n enzymatic d i g e s t s of a l i q u o t s of the same DNA sample (Tables 20 and 21). This suggested that the novel n u c l e o t i d e broke down to form hmUra during a c i d h y d r o l y s i s . Since nuclease SI d i g e s t i o n employed an a c i d i c b u f f e r i t was p o s s i b l e that the hmdUMP i n samples was an a c i d degradation product of the novel n u c l e o t i d e . The novel pyrimidine n u c l e o t i d e was a l s o d e t e c t a b l e i n SI and snake venom phosphodiesterase d i g e s t s of am 37 DNA prepared i n s t r a i n sup 2 (Figure 43A) (Table 21). hmdUMP and the novel n u c l e o t i d e made up approximately 18.3 percent of the t o t a l p yrimidines l a b e l l e d . As mentioned e a r l i e r , suppression of the am 37 phenotype i n sup 2 was incomplete. I t i s not known i f the presence of the novel n u c l e o t i d e i n the am 37 DNA from the sup 2 host r e f l e c t s the mechanism of putThy formation. I f putThy formation i s normally coupled to r e p l i c a t i o n then i t i s l i k e l y that the mutant m o d i f i c a t i o n p r o t e i n could f a i l to convert a p o r t i o n of the novel n u c l e o t i d e to putdTMP. However, i f the modifying enzyme could convert the novel n u c l e o t i d e to putdTMP when-ever i t i s presented, then the DNA from the sup 2 s t r a i n i n f e c t i o n should not co n t a i n any intermediate i n putThy b i o s y n t h e s i s , unless the subst r a t e i s formed or removed by DNA packaging at a r a t e greater than the r a t e of formation of the product. The a n a l y s i s of the k i n e t i c s of formation of the novel n u c l e o t i d e as w e l l as the k i n e t i c s of i t s con-v e r s i o n to putThy might answer the question: i s putThy b i o s y n t h e s i s a b s o l u t e l y coupled to DNA r e p l i c a t i o n ? 14 Am 37 DNA was l a b e l l e d w i t h [8- C]-adenine i n s t r a i n 29. The DNA was digested w i t h SI and snake venom phosphodiesterase. Two^ -193 dimensional t h i n - l a y e r chromatography revealed that the only l a b e l l e d products were dAMP and dGMP (Figure 44). Therefore, the novel nucleo-t i d e was not a product of the incomplete d i g e s t i o n of am 37 DNA. Sam-32 + pi e s of P - l a b e l l e d 0W-14 w DNA were digested w i t h DNase I and snake venom phosphodiesterase. Two dimensional t h i n - l a y e r chromatography revealed that l i t t l e putdTMP was rele a s e d from 0W-14 w + DNA (Figure 25). 32 S i m i l a r l y , d i g e s t i o n of P - l a b e l l e d am 37 DNA prepared i n s t r a i n 29 i n f e c t e d c e l l s was not complete (Figure 46). L i m i t d i g e s t s of am 37 DNA contained dGMP, dAMP, dCMP and dTMP as w e l l as some of the novel n u c l e o t i d e . In a d d i t i o n there were three other r a d i o a c t i v e compounds (Figure 46). These were probably dimers c o n t a i n i n g the novel nucleo-t i d e and another pyrimidine n u c l e o t i d e . A f t e r two-dimensional t h i n -l a y e r chromatography of DNase I and snake venom phosphodiesterase 14 d i g e s t s of [8- C]-adenine l a b e l l e d am 37 DNA prepared i n s t r a i n 29 dAMP and dGMP were the only l a b e l l e d p r o d u c t s ( F i g u r e 45) . Therefore, the r a d i o -a c t i v e products accumulating i n incomplete DNA d i g e s t s were d i g e s t i o n r e s i s t a n t p y rimidine o l i g o n u c l e o t i d e s . Another fea t u r e of the DNase I and snake venom phosphodiesterase d i g e s t s of am 37 DNA was the absence of a hmdUMP spot. I t i s p o s s i b l e that the hmdUMP seen on chromatograms of SI d i g e s t s of am 37 DNA was an a r t i f a c t generated by the degradation of the novel n u c l e o t i d e i n the a c i d i c SI d i g e s t i o n b u f f e r . However, 32 am 37 DNA l a b e l l e d w i t h PO, does not r e l e a s e l a b e l when incubated i n — 4 SI b u f f e r i n the absence of enzyme (Table 22). A l t e r n a t i v e l y , hmdUMP residues might have been present i n sequences which were r e s i s t a n t to DNase I and snake venom phosphodiesterase d i g e s t i o n . 32 The q u a n t i t y of PO. rele a s e d from am 37 DNA during DNase I 14 FIGURE 44.—Two-dimensional thin-layer chromatography of [8- C]-adenine-labelled nucleotides present in Sl-SVPD digests of am 37 DNA prepared in P_. acidovorans strain 29. Nucleotides were detected by fluorography after 2D-TLC in solvents E and A. DNA was digested sequentially with nuclease SI and SVPD. 1) dAMP; 2) dGMP. FIGURE 45.—Two-dimensional t h i n - l a y e r chromatography of [ 8 - ^ 0 ] a d e n i n e - l a b e l l e d n u c l e o t i d e s present i n DNase I-SVPD d i -gests of am 37 DNA prepared i n P_. acidovorans s t r a i n 29. Nucleotides detected by fluorography a f t e r 2D-TLC i n so l v e n t s E and A. The DNA was digested w i t h DNase I and SVPD. 1) dAMP; 2) dGMP. 196 Obi* "T*»> fH t . * w FIGURE 46.—Two-dimensional t h i n - l a y e r chromatography of P - l a b e l l e d n u c l e o t i d e s present i n DNase I-SVPD d i g e s t s of am 37 DNA prepared i n P_. acidovorans s t r a i n 29. The n u c l e o t i d e s were detected by autoradiography a f t e r 2D-TLC i n solven t s E and A. The DNA sample was digested w i t h DNase I and SVPD. 1) dAMP 6) unknown mononucleotide 2) dGMP 7) unknown 3) dTMP 8) unknown 4) dCMP 9) unknown 5) P 0 4 198 TABLE 22.—0W-14 am 37 DNA was s t a b l e i n a l k a l i and SI b u f f e r . treatment cpm percent recovery none 16365 ddH 0, 37°/16 hours 15991 98 none 15993 0.3 N NaOH, 37°/16 hours 15319 96 none 9742 SI b u f f e r , 0.05 M NH -ac e t a t e , pH 5.0, 55°/4 hours 9508 98 J i P - l a b e l l e d 0W-14 am 37 DNA was ethanol p r e c i p i t a t e d and then resus-pended i n d e i o n i z e d , d i s t i l l e d water. A l i q u o t s of each sample were spotted onto Whatman 3MM paper squares and washed s e q u e n t i a l l y w i t h 95 percent ethanol and absolute ether. The remaining p o r t i o n of the sample was t r e a t e d as described. Each value represents the average of three determinations. A f t e r treatment the samples were subjected to TCA p r e c i p i t a t i o n . The papers were washed three times w i t h 95 per-cent ethanol and twice w i t h absolute ether and were then d r i e d and counted. 200 and snake venom phosphodiesterase d i g e s t i o n was l e s s than that released from am 37 DNA during d i g e s t i o n w i t h nuclease SI. The source of the 32 PO, released i s not known. 4 DNA synth e s i s i n 0W-14 am 37 i n f e c t e d - c e l l s 3 The i n c o r p o r a t i o n of [6- H ] - u r a c i l i n t o a l k a l i r e s i s t a n t , TCA p r e c i p i t a b l e m a t e r i a l was measured f o r w i l d - t y p e and am 37-infected s t r a i n s 29 and sup 2 (Figure 47). DNA accumulation i n w i l d - t y p e i n f e c t e d s t r a i n 29 c e l l s began at 25 minutes a f t e r i n f e c t i o n and proceeded u n t i l 60 to 70 minutes a f t e r i n f e c t i o n . Wild-type i n f e c t i o n of sup 2 followed a slower time course. DNA synth e s i s began around 40 minutes a f t e r i n f e c t i o n and continued u n t i l 100 minutes. The f i n a l l e v e l s of DNA accumulation f o r both w i l d - t y p e i n f e c t i o n s were s i m i l a r . Am 37- i n f e c t e d s t r a i n 29 or sup 2 c e l l s accumulated approximately equal q u a n t i t i e s of DNA and followed s i m i l a r time courses. Am 37 DNA synth e s i s began around 40 minutes a f t e r i n f e c t i o n and proceeded u n t i l 100 minutes. The amount of DNA synthesized i n am 37-infected c e l l s was l e s s than one-half the amount of DNA made i n the same s t r a i n s during w i l d - t y p e 0W-14 i n f e c t i o n . Neither am 37 nor w i l d - t y p e 0W-14 DNA were l a b e l l e d by [ 2 - ^ C ] -3 hmdU. Am 37 DNA was not l a b e l l e d by [ H ] - o r n i t h i n e . 3 T r i t i u m r e l e a s e from [5- H"|-uracil  i n amber 37- i n f e c t e d 3L T r i t i u m r e l e a s e was used to measure the i n v i v o a c t i v i t y of dUMP hydroxymethylase. C o r r e c t i o n of the data f o r d i f f e r e n c e s i n sample volumes and counting e f f i c i e n c i e s allowed a d i r e c t comparison FIGURE 47 .—DNA sy n t h e s i s by phage-infected c e l l s . 3 The i n c o r p o r a t i o n of [6- H ] - u r a c i l i n t o a l k a l i - r e s i s t a n t , TCA-i n s o l u b l e m a t e r i a l was determined f o r w i l d - t y p e phage i n s t r a i n 29 (O) and i n s t r a i n sup 2 (•); and f o r am 37 i n s t r a i n 29 (A) 3 -1 -1 and i n sup 2 (A). [6- H ] - u r a c i l ( 1 . 0 uCi ml , 10 yg ml u r a c i l ) . 25 45 65 85 MINUTES AFTER INFECTION of the accumulation of [ 5 - J H ] - u r a c i l l a b e l i n c y t o s i n e bases i n DNA 3 and the r e l e a s e of t r i t i u m from [5- H]-dUMP i n n u c l e o t i d e pools. In w i l d - t y p e 0 W-14-infected c e l l s the r a t i o of t r i t i u m r e l e a s e to t r i t i u m i n c o r p o r a t i o n was one-to-one (Figure 48). This was not s u r p r i s i n g s i n c e , i n a phage w i t h a DNA mol % G + C of approximately 50 percent, the requirements f o r c y t o s i n e and hmUra nucle o t i d e s would be equal. The one-to-one r a t i o i s destroyed i n 0W-14 DO mutants (P. M i l l e r , unpublished observations) and i n am 37-infected s t r a i n 3L (Figure 48). I n am 37 i n f e c t i o n the amount of t r i t i u m released was greater than the amount of t r i t i u m accumulated i n DNA. T r i t i u m r e l e a s e and DNA synthe-s i s began about 20 to 25 minutes a f t e r i n f e c t i o n . In am 37-infected 3L the r a t e of DNA i n c o r p o r a t i o n slowed r e l a t i v e to the r a t e of t r i t i u m r e l e a s e as the i n f e c t i o n progressed. The r a t e and absolute amount of t r i t i u m r e l e a s e i n am 37-infected 3L was l e s s than the r a t e and the amount of t r i t i u m r e l e a s e d i n w i l d - t y p e 0VJ-14 i n f e c t i o n s of 3L. I t i s p o s s i b l e that the nature of the DNA template had an e f f e c t upon t r i t i u m r e l e a s e . Since t r i t i u m r e l e a s e was greater than DNA s y n t h e s i s i t was u n l i k e l y that the lower r a t e of DNA s y n t h e s i s was due to a shortage i n the supply of DNA precursors. The s t a b i l i t y of am 37 DNA Measurements of DNA accumulation r e q u i r e d exposure of am 37 DNA 32 to extreme c o n d i t i o n s of b a s i c i t y and a c i d i t y . The s t a b i l i t y of P-l a b e l l e d components of am 37 DNA to treatment w i t h 0.3 N NaOH and 5 percent TCA were i n v e s t i g a t e d (Table 22). N i n e t y - e i g h t percent of input l a b e l was recovered from a TCA p r e c i p i t a t e d _am 37 DNA sample 204 FIGURE 48.—DNA synthesis and t r i t i u m r e l e a s e by i n f e c t e d c u l t u r e s . 3 I n f e c t e d c e l l s of s t r a i n 3L were l a b e l l e d w i t h [5- H ] - u r a c i l . Samples were assayed f o r the i n c o r p o r a t i o n of l a b e l i n t o a l k a l i - r e s i s t a n t , TCA-insoluble m a t e r i a l (•) and f o r the r e l e a s e of t r i t i u m (O). A: w i l d -type phage; B: am 37. 205 206 which was preincubated i n d e i o n i z e d - d i s t i l l e d water at 37° for 16 hours. 32 Ninety-six percent of the P l a b e l was recovered from am 37 DNA samples incubated for 16 hours i n 0.3 N NaOH at 37° and then TCA p r e c i p i t a t e d . 32 I t was concluded that the P l a b e l i n am 37 DNA was stable under alka-l i n e incubation conditions and was not affected by subsequent TCA p r e c i p i t a t i o n . The methods used to measure DNA accumulation i n am 37-infected c e l l s were v a l i d . CsCl gradient analysis at am 37 DNA 32 P-labelled am 37 DNA was extracted from s t r a i n 29 c e l l s at 45, 60 and 75 minutes a f t e r i n f e c t i o n and analyzed i n neutral CsCl density gradients. DNA extracted from c e l l s at 45 minutes a f t e r i n f e c -t i o n contained the two peaks of heavy and intermediate density described e a r l i e r (Figure 49A). DNA extracted from c e l l s at 60 or 75 minutes a f t e r i n f e c t i o n contained only one major peak of heavy density DNA (Figure 49, B and C). Two-dimensional thin-layer chromatography of SI 32 and snake venom phosphodiesterase digests of P-labelled DNA samples demonstrated that the percentages of novel nucleotide and hmdUMP were constant throughout the 30 minute time i n t e r v a l (Table 23). The i n t e r -mediate density am 37 DNA peak was probably a hybrid DNA duplex con-ta i n i n g "unmodified" DNA i n one strand and parental DNA i n the other strand. As r e p l i c a t i o n proceeded, parental DNA was s h i f t e d to hybrid density by DNA r e p l i c a t i o n . Eventually, parental DNA was dispersed by r e p l i c a t i o n and recombination and the hybrid density peak became a small proportion of the DNA. The constant nature of the am 37 DNA nucleotide composition coupled to the differences i n CsCl p r o f i l e s 207 FIGURE 49 . — C s C l buoyant density analysis of 0W-14 am 37 DNA prepared at various times a f t e r i n f e c t i o n of P_. acidovorans s t r a i n 29. The DNA was p u r i f i e d from infected c e l l s at 45, 60 and 75 minutes a f t e r i n f e c t i o n . Equal volumes of DNA extracted from 5 ml of infected c e l l s were loaded on a neutral CsCl gradient. The procedures employed are described i n d e t a i l i n the Materials and Methods. C 75' 208 16.0 14.0 12.0 10.0 2 8.0 a. 6.0 o w 4.0 ro 2.0 A 45' B 60' to • O 70.0 60.0 50.0 40.0 Cu 30.0 O C L £ 20.0 10.0 to 100.0 90.0 80.0 70.0 60.0 50.0 2 40.0 C L O Q. 30.0 ro 20.0 10.0 10 20 30 10 20 30 10 20 30 FRACTION NUMBER FRACTION NUMBER FRACTION NO. 209 TABLE 2 3 . — T h e n u c l e o t i d e c o m p o s i t i o n o f 0W-14 am 37 DNA p r e p a r e d a t v a r i o u s times a f t e r the i n f e c t i o n of P. a c i d o v o r a n s s t r a i n 29. N u c l e o t i d e 45 min. 60 min. 75 min. dTMP 2972 (0.17) 4195 (0.18) 4507 (0.17) dCMP 5943 (0.34) 8457 (0.36) 9333 (0.35) putdTMP 87 (<0.01) 117 (<0.01) 105 (<0.01) hmdUMP 865 (0.05) 1180 (0.05) 1244 (0.05) unknown 7750 (0.44) 9248 (0.40) 11160 (0.42) The DNA was p u r i f i e d from i n f e c t e d s t r a i n 29 c e l l s as d e s c r i b e d i n the M a t e r i a l s and Methods. N u c l e a s e SI and snake venom p h o s p h o d i e s t e r a s e d i g e s t i o n s were performed and the n u c l e o t i d e s were s e p a r a t e d by 2D-TLC on c e l l u l o s e . The DNA used was p a r t of the sample used f o r C s C l a n a l -y s i s i n F i g u r e 47. a The v a l u e s a r e cpm i n the a r e a c u t from the chromatogram. The v a l u e s i n p a r e n t h e s e s a r e the f r a c t i o n o f the t o t a l l a b e l l e d p y r i m i d i n e n u c l e o t i d e s . 210 proved that the l a b e l l e d DNA peaks did not d i f f e r i n t h e i r l a b e l l e d nucleotide composition. Parentally l a b e l l e d am 37 DNA The transfer of parental l a b e l to hybrid density i n am 37-infected s t r a i n 29 was demonstrated (Figure 50). Most of the parental am 37 DNA remained at parental density, i n d i c a t i n g that i t was i n a c t i v e . This probably was a r e f l e c t i o n of the high m u l t i p l i c i t i e s of i n f e c t i o n required i n am 37 experiments. P a r e n t a l l y - l a b e l l e d hybrid density DNA was only found a f t e r the onset of DNA r e p l i c a t i o n i n samples taken 45 minutes a f t e r i n f e c t i o n . This agreed with the r e s u l t s from the pro-geny l a b e l l e d gradients. There was no transfer of parental l a b e l to hybrid density i n am 37 infec t e d sup 2 c e l l s . The parentally l a b e l l e d DNA had a broad but uniform density p r o f i l e . P u r i f i c a t i o n of the novel nucleotide  found i n am 37 DNA In order to study the nature of the novel am 37 nucleotide, 32 3 large amounts of P and [6- H]-uracil-.labelled am 37 DNA were prepared i n infected s t r a i n 29 c e l l s . SI and snake venom phosphodiesterase digests of the p u r i f i e d DNA were loaded on Whatman 40 SFC paper and treated as described i n the Methods section of th i s t h e s i s . A f t e r chromatography the nucleotides were l o c a l i z e d by autoradiography (Figure 51). The novel nucleotide and the nucleotide t e n t a t i v e l y i d e n t i f i e d as hmdUMP were well separated from the o r i g i n and from other nucleotides. When the p u r i f i e d nucleotide was eluted from the paper and rechromatographed only one radioactive nucleotide was found on the FIGURE 5 0 . — C s C l buoyant d e n s i t y a n a l y s i s of p a r e n t a l l y l a b e l l e d 0W-14 am 37 DNA. 3 [6- H ] - u r a c i l - l a b e l l e d 0W-14 am 37 phage were prepared i n s t r a i n sup 2. Radioactive phage was used to i n f e c t c u l t u r e s of s t r a i n s 29 and sup 2. DNA was ext r a c t e d from the i n f e c t e d c e l l s at 45 minutes a f t e r i n f e c t i o n of s t r a i n 29, (A); and at 60 minutes a f t e r 32 4-i n f e c t i o n of s t r a i n sup 2, (B). P - l a b e l l e d 0W-14 w phage r e f -3 erence DNA, •; [6- H ] - u r a c i l l a b e l l e d sample DNA, O. 212 C L O I 10 36.0 32.0 28.0 24.0 20.0 16.0 12.0 8-0 4.0 IO 6 8.0 4.0 O C L CM IO 10 20 30 40 FRACTION NUMBER 32.0 28.0 24.0 20.0 16.0 0_ O X CM IO 12.0 1 8.0 1 4.0 H 16.0 12.0 8.0 [ 4.0 io i O 5 C L O CL CM ro 10 20 30 40 FRACTION NUMBER FIGURE 51.—The purification of the novel nucleotide by paper chromatography. 32 A P-labelled 0W-14 am 37 DNA sample was digested with nuclease SI and with SVPD. Descending paper chromatography and the recovery of the nucleotides are described in the Materials and Methods. chromatogram (Figure 52). The p u r i t y of the r a d i o a c t i v e product was a l s o demonstrated on a short DEAE-Sephadex column (Figure 53). The p u r i f i e d n u c l e o t i d e and an u n l a b e l l e d d i g e s t of am 37 DNA were loaded on the column. Chromatography of the sample revealed that the novel n u c l e o t i d e was more s t r o n g l y retarded on the column when compared to the f i v e other mononucleotides present i n the sample. Even on a short 8 cm column the novel n u c l e o t i d e was almost completely r e s o l v e d from the other mononucleotides. A l l of the r a d i o a c t i v i t y loaded on the column eluted as a s i n g l e peak i n d i c a t i n g that the compound prepared by paper chromatography was r a d i o c h e m i c a l l y pure. The chromatographic p r o p e r t i e s of the n u c l e o t i d e on DEAE-Sephadex suggested that i t had a net negative charge greater than the other mononucleotides. This was not s u r p r i s i n g s i n c e the chromatographic p r o p e r t i e s of the n u c l e o t i d e observed on PEI and unmodified c e l l u l o s e a l s o suggested the same t h i n g . 3 32 [6- H ] - u r a c i l and PO^ l a b e l l i n g r a t i o s demonstrated that the novel n u c l e o t i d e c a r r i e d e x t r a phosphate resi d u e s . The approximate s i z e of the novel n u c l e o t i d e was e s t a b l i s h e d by g e l f i l t r a t i o n chromatography on B i o g e l P^ columns. A l l the nucleo-t i d e s e l u t e d as a s i n g l e peak, although the e a r l y peak f r a c t i o n s con-tained more of the novel n u c l e o t i d e and dGMP el u t e d i n l a t e r peak f r a c t i o n s (data not shown). Attempts to recover the novel n u c l e o t i d e from the DEAE-Sephadex and B i o g e l columns passing the elu a t e f r a c t i o n s through N o r i t A were not s u c c e s s f u l , the novel n u c l e o t i d e was degraded. P u r i f i e d s a l t - f r e e samples of the novel n u c l e o t i d e and hmdUMP were r o u t i n e l y obtained by paper chromatography. FIGURE 52.—Two-dimensional thin-layer chromatography of the purified novel nucleotide. An aliquot of the novel nucleotide purified by descending paper chromatography was spotted on a cellulose sheet and subjected to 2D-TLC with solvents E and A. 217 FIGURE 53.—DEAE-Sephadex column chromatography of the novel n u c l e o t i d e . 32 A P - l a b e l l e d paper p u r i f i e d sample of the novel n u c l e o t i d e was loaded on a short (8 cm) column of DEAE-Sephadex, w i t h an u n l a b e l l e d d i g e s t of am 37 DNA. The n u c l e o t i d e s were e l u t e d w i t h a l i n e a r gradient of NaCl i n 20 mM T r i s - H C l , pH 8.0. Three ml f r a c t i o n s were c o l l e c t e d . Cerenkov r a d i a t i o n and kr,rn were measured f o r each f r a c t i o n . The pro-cedures employed are described i n d e t a i l i n the M a t e r i a l s and Methods. 32 P - l a b e l n u c l e o t i d e , O; A„, n, • . F R A C T I O N N U M B E R Samples of a [6- H ] - u r a c i l l a b e l l e d p r e p a r a t i o n of hmdUMP and the novel n u c l e o t i d e were hydrolyzed i n 6 N HCI. The f r e e bases were separated by two-dimensional t h i n - l a y e r chromatography. The only r a d i o a c t i v e product released by a c i d h y d r o l y s i s was hmUra. This r e s u l t confirmed the i d e n t i f i c a t i o n of hmdUMP and demonstrated that the novel n u c l e o t i d e was an a c i d - l a b i l e d e r i v a t i v e of hmdUMP (Table 24). The l a b i l i t y of the novel n u c l e o t i d e under a c i d i c and a l k a l i n e i n c u b a t i o n c o n d i t i o n s was examined. I t was f a i r l y s t a b l e to b o i l i n g i n d e i o n i z e d - d i s t i l l e d water or heating i n 0.3 N KOH at 37° (data not shown). I t decomposed upon treatment w i t h 1 N HCI f o r 30 minutes at 37° or 7 minutes a t 98°. The r e l e v a n t p r o p e r t i e s of the novel nucleo-t i d e are summarized i n Table 25. A c i d treatment releases hmdUMP and orthophosphate. The r a t i o of a c i d - l a b i l e to a c i d - s t a b l e phosphate 32 was two. The apparent r a t i o of t r i t i u m to P l a b e l i s approximately one to three. The a c i d l a b i l i t y experiments suggested that the novel n u c l e o t i d e was a diphosphorylated d e r i v a t i v e of hmdUMP. The s e n s i t i v i t y of the novel n u c l e o t i d e to the enzyme b a c t e r i a l a l k a l i n e phosphatase (BAP) was determined. Incubation w i t h BAP caused 3 the complete conversion of the n u c l e o t i d e to [ H]-hmdUra and PO^ (Table 26). The products were determined chromatographically (Figure 54). hmdUra was i d e n t i f i e d by comparing i t s chromatographic behaviour to auth e n t i c deoxynucleoside standards i n f i v e d i f f e r e n t s o l v e n t s (Table 26). A l l phosphate residues on the novel n u c l e o t i d e were s e n s i t i v e to BAP (Table 26). The novel n u c l e o t i d e was s t a b l e i n the i n c u b a t i o n b u f f e r employed (Figure 54). The in f o r m a t i o n obtained about the novel n u c l e o t i d e suggested TABLE 2 4 . — A c i d h y d r o l y s i s of n u c l e o t i d e s p u r i f i e d from 0W-14 am 37 DNA. Authentic base unknown n u c l e o t i d e hmdUMPb putThy 12 a .. 0 Cyt 0 17 hmUra 2673 1362 Ura 27 0 Thy 4 0 The l a b e l l i n g , e x t r a c t i o n and p u r i f i c a t i o n procedures are described i n the M a t e r i a l s and Methods. Bases were separated by 2D-TLC i n solv e n t s B and D. The recovery of ap p l i e d l a b e l was greater than 3 90 percent. The nu c l e o t i d e s were l a b e l l e d w i t h [6- H ] - u r a c i l . a The values are cpm i n the areas cut from the chromatogram. b I d e n t i f i e d as hmdUMP on the b a s i s of i t s chromatographic p r o p e r t i e s . TABLE 2 5 . — P r o p e r t i e s of the unknown n u c l e o t i d e . 3 Treatment^ 3H unknown n u c l e o t i d e 32 P 3 2 P / 3 H 3H hmdUMP 32 J P 3 2P/ 3H inor g a n i c phosphate hmdUra None° 2298 6759 2.9 1 N HCI 37°C 30 min 1903 2040 1.1 4626 1 N HCL 98°C 7 min 1978 2334 1.2 4550 a l k a l i n e phosphatase 6292 2018 a The DNA from which the nucl e o t i d e s were p u r i f i e d was l a b e l l e d w i t h [ 6 - 3 H ] - u r a c i l and 3 2 P orthophosphate. b The products were i d e n t i f i e d by t h i n - l a y e r chromatography w i t h known standards. N3 r-1 TABLE 26.—Conversion of the unknown n u c l e o t i d e to i t s nucleoside. -BAP +BAP Unknown n u c l e o t i d e 32 PO, 3747 138 19 3422 I d e n t i f i c a t i o n of the unknown nucleoside by TLC. Rf i n Solvent B C Authentic Standards hmUdR TdR UdR CdR Unknown -BAP +BAP 76 83 83 72 f r o n t 75 59 46 61 56 74 58 78 90 81 51 unstable 78 26 50 38 16 0 27 20 49 27 26 0 19 a A sample of P - l a b e l l e d n u c l e o t i d e was treated w i t h BAP. The e f f i -ciency of BAP cleavage was monitored by measuring the formation of -^po^. The nucleoside was i d e n t i f i e d by measuring Rf values i n 5 d i f f e r e n t s o l v e n t s and comparing them to those determined f o r authen t i c reference standards. Solvent A, dR^O; sol v e n t B, (NR^^SO^rNa acetate (IM):isopropanol, 80/12/2 v/v; s o l v e n t C, t-butanol:MEK:HCl: H2O, 40/30/10/20 v/v; so l v e n t D, n-butanol:H20, 86/14 v/v; s o l v e n t E n-butanol:H 20:NH40H, 86/9/5 v/v. FIGURE 5 4 . — A l k a l i n e phosphatase treatment of the novel n u c l e o t i d e . 32 A sample of P - l a b e l l e d novel n u c l e o t i d e was subjected to treatment w i t h B a c t e r i a l a l k a l i n e phosphatase (BAP). The sample was s p l i t i n t o two p a r t s . One part was incubated i n 50 mM T r i s - H C l pH 8.4, 15 mM MgCl 2 at room temperature f o r one hour. The remaining p o r t i o n of the sample was d i s s o l v e d i n the same b u f f e r and supplemented w i t h BAP (0.03 u n i t s ) . D i g e s t i o n was c a r r i e d out f o r one hour at room temper-at u r e . The products were resolve d by 1D-TLC on P E I - c e l l u l o s e sheets, A; or by 2D-TLC on c e l l u l o s e sheets, B. 224 226 that i t s base was 5(hydroxymethyl-0-pyrophosphoryl) u r a c i l (Figure 55). This s t r u c t u r e was c o n s i s t e n t w i t h a l l the f a c t s : 1) the novel n u c l e o t i d e i s a pyrimidine monophosphate n u c l e o t i d e derived from SI and snake venom phosphodiesterase d i g e s t i o n of am 37 DNA; 2) a c i d h y d r o l y s i s of the novel n u c l e o t i d e generates hmUra; 3) a c i d treatment of the novel n u c l e o t i d e l i b e r a t e s hmdUMP and PO^ i n a r a t i o of one to two; 4) BAP treatment of the novel n u c l e o t i d e generates f r e e hmdUra and PO^. The s t r u c t u r e proposed p r e d i c t e d a molecule w i t h a r e l a t i v e net nega-t i v e charge of f i v e . This p r e d i c t i o n was confirmed. A l a r g e batch of 32 crude P - l a b e l l e d am 37 DNA was prepared and digested to mononucleo-t i d e s . The sample was loaded on a DEAE-Sephadex-urea column along w i t h dTDP and dTTP as net negative charge markers. The column was washed wi t h s t a r t i n g b u f f e r and then e l u t e d w i t h a l i n e a r gradient of NaCl (Figure 56). The r a d i o a c t i v e mononucleotides e l u t e d as a l a r g e , s l i g h t l y asymmetrical peak. The mononucleotide peak contained two O.D^ components; however, the second s m a l l e r peak i s l i k e l y p r o t e i n present i n , or added t o , the samples during d i g e s t i o n . The other O.D.^^-j peaks mark the e l u t i o n p o i n t s of the charge marking standards, dTDP (-3) and dTTP (-4). The second, smaller r a d i o a c t i v e peak marks the e l u t i o n p o s i t i o n of the novel n u c l e o t i d e . Assuming a l i n e a r r e l a t i o n s h i p be-tween peak e l u t i o n p o s i t i o n and r e l a t i v e net negative charge, i t was determined that the novel n u c l e o t i d e had a net negative charge of f i v e . This experiment corroborated the p r e d i c t i o n made from the previous experiments. The novel n u c l e o t i d e was assigned the d e s i g n a t i o n of hmPPdUMP (base-hmPPUra). FIGURE 55.—Proposed structure of the novel pyrimidine base. 0 = 5-( hydroxymethyl-o- pyrophosphor uracil 228 FIGURE 56.—DEAE-sephadex-urea column chromatography of a n u c l e o t i d e mixture from am 37 DNA. 32 A sample of P - l a b e l l e d am 37 DNA was prepared.from i n f e c t e d c e l l s of s t r a i n 29. The DNA was digested to mononucleotides which were chromatographed on DEAE-Sephadex A25 (1 x 40 cm) column w i t h u n l a b e l l e d dTDP and dTTP as reference compounds. F r a c t i o n s of 5 ml were c o l l e c t e d and assayed f o r Cerenkov r a d i a t i o n (•) and absorbance at 260 nm ( — - ) . The r e l a t i v e charges are given f o r each peak. F R A C T I O N N U M B E R The a c i d l a b i l i t y of both phosphates i n the pyrophosphoryl group i s not s u r p r i s i n g . The hmUra group has b e n z y l i c character (Brown et a l . , 1968; S a n t i , 1967) and the r a t e of h y d r o l y s i s i n strong a c i d i s about 100 times greater f o r monobenzyl phosphate than f o r simple a l i p h a t i c phosphates (Kumamoto and Westheimer, 1955). F u r t h e r -more, allylpyrophosphates are q u i t e unstable below pH 5, c l e a v i n g i n t o a l l y l a l c o h o l s and i n o r g a n i c pyrophosphate (Goodman and Popjak, 1960). The a c i d treatments used w i l l s e l e c t i v e l y hydrolyze other phos-phate e s t e r s under c e r t a i n c o n d i t i o n s . Heating ATP at 98° i n 1 N HCI fo r 7 minutes removes 5f and g phosphate r e s i d u e s . Heating ppGpp at 98° f o r 7 minutes hydrolyzes a l l pyrophosphate bonds. Short exposure of ppGpp to 1 N HCI at 37° f o r 30 minutes converts i t to ppGp (Sy and Lippman, 1973). Amber 37 _in v i t r o DNA m o d i f i c a t i o n The presence of hmPPUra i n am 37 DNA suggested that i t was an a c t i v a t e d precursor of putThy. C e l l - f r e e e x t r a c t s of s t r a i n 29 c u l -32 tures i n f e c t e d w i t h w i l d - t y p e 0W-14 or am 37 were prepared. P-l a b e l l e d am 37 DNA was used as a sub s t r a t e i n an i n v i t r o experiment to t e s t f o r a precursor and product r e l a t i o n s h i p between hmPPUra and putThy. C e l l - f r e e e x t r a c t s of s t r a i n 29 c e l l s i n f e c t e d w i t h w i l d - t y p e 32 0W-14 were capable of r e l e a s i n g PO^ from am 37 DNA i n the presence of p u t r e s c i n e . C e l l - f r e e e x t r a c t s of am 37-infected s t r a i n 29 c e l l s were i n a c t i v e i n t h i s assay (Figure 57). DNA samples incubated w i t h 0W-14 w i l d - t y p e e x t r a c t s were p u r i -f i e d , digested to mononucleotides and separated by two-dimensional FIGURE 57.—Release of r a d i o a c t i v i t y from P - l a b e l l e d am 37 DNA. 32 A sample of P - l a b e l l e d am 37 DNA was prepared from i n f e c t e d c e l l s of s t r a i n 29. The DNA was incubated w i t h p u t r e s c i n e and a c e l l - f r e e e x t r a c t from i n f e c t e d c e l l s of s t r a i n 29. Samples were removed at i n t e r v a l s and assayed f o r a l c o h o l - s o l u b l e r a d i o a c t i v i t y (because of the a c i d - l a b i l i t y of hmPPUra). E x t r a c t s were from w i l d - t y p e - i n f e c t e d c e l l s (•) and am 37-infected c e l l s (O).-C P M R E L E A S E D x I 0 ' 3 CO 233 t h i n - l a y e r chromatography on c e l l u l o s e . Nucleotides were detected by autoradiography. A l l n u c l e o t i d e s were cut out and counted. The 32 r e s u l t s were corrected f o r the l o s s of PO. during i n v i t r o m o d i f i c a -4 ° t i o n and f o r other non s p e c i f i c losses during p u r i f i c a t i o n , d i g e s t i o n and chromatography. The r a d i o a c t i v i t y i n each n u c l e o t i d e was p l o t t e d as a percentage of the t o t a l r a d i o a c t i v i t y (Figure 58). As m o d i f i c a t i o n proceeded, the l e v e l of hmPPdUMP i n the DNA d e c l i n e d to about 50 percent of the pre i n c u b a t i o n l e v e l . hmdUMP d i d not accumulate, t h e r e f o r e , hmPPdUMP was not being converted to hmdUMP by the l o s s to two phosphates. The uniform l e v e l of hmdUMP i n the trea t e d DNA a l s o suggested that t h i s n u c l e o t i d e d i d not a r i s e from the a c i d - c a t a l y z e d d e s t r u c t i o n of hmPPdUMP during SI d i g e s t i o n of the DNA samples. DNA samples w i t h 50 percent l e s s hmPPdUMP would y i e l d 50 percent l e s s hmdUMP i f the SI d i g e s t i o n c o n d i t i o n s were re s p o n s i b l e f o r the formation of hmdUMP. The d e c l i n e i n hmPPdUMP l e v e l s i n am 37 DNA was accompanied by an increase i n putdTMP l e v e l s . PutdTMP increased from an o r i g i n a l 0.3 percent of the l a b e l to almost 6.0 percent of the l a b e l . The d e c l i n e i n the amount of hmPPdUMP and the increase i n the amount of putdTMP was con-s i s t e n t w i t h the l o s s of two phosphates or one pyrophosphate from hmPPdUMP during putdTMP formation. The amount of l a b e l detected i n dTMP i n am 37 DNA d i d not change. E x t r a c t s of 0W-14 w + i n f e c t e d s t r a i n 29 c e l l s were not capable of con-v e r t i n g hmdUMP or hmPPdUMP to dTMP under the in c u b a t i o n c o n d i t i o n s employed. Attempts to create a s u b s t r a t e f o r the hmdUMP phosphorylating enzyme by removing the phosphates from am 37 DNA wi t h BAP were not FIGURE 58.—Conversion of hmPPUra i n am 37 DNA i n t o putThy. Procedure e s s e n t i a l l y as described i n the legend to Figure 57. The DNA i n samples was digested to mononucleotides which were separated by t h i n - l a y e r chromatography. The nu c l e o t i d e s were detected by autoradiography , e x c i s e d from the sheets and t h e i r r a d i o a c t i v i t y determined. dTMP (X); hmdUMP ( A ) ; putdTMP (•) ; hmPPdUMP (O). 30 60 90 MINUTES INCUBATION 236 s u c c e s s f u l . Native and denatured 0 e DNA were t e s t e d as substrates i n 32 conju n c t i o n w i t h - P ATP and 0W-14 c e l l - f r e e e x t r a c t s . No a c t i v i t y was observed. The formation of putThy from hmPPUra may i n v o l v e the d i s p l a c e -ment of a pyrophosphoryl group by an incoming amino n i t r o g e n w i t h the formation of a carbon-nitrogen bond. This type of r e a c t i o n i s found i n the formation of p h o s p h o r i b o s y l a n t h r a n i l a t e from a n t h r a n i l a t e and phosphoribosylpyrophosphate, i n the formation of dihydroopterate from hydroxymethylpteridine pyrophosphate and p-aminobenzoate and i n the formation of thiamine from a C5-pyrophosphoryl-hydroxymethyl p y r i m i d i n e base and t h i a z o l e (Walsh, 1979). hmUra w i l l a l k y l a t e poorly n u c l e o p h i l i c aromatic amines i n aqueous a l k a l i n e medium ( S a n t i , 1967). The hydroxymethyl group i s sus-c e p t i b l e to n u c l e o p h i l i c a t t a c k . N u c l e o p h i l i c displacement could be enhanced by the presence of a pyrophosphoryl l e a v i n g group. Some other e s t e r s of hmUra are a l s o s e n s i t i v e to n u c l e o p h i l i c displacement ( S a n t i , 1971). Such r e a c t i o n s proceed by an i n i t i a l a t t a c k at the 6 p o s i t i o n of the pyrimidine r i n g ( P o g o l o t t i and S a n t i , 1977). Conversion of putThy could conceivably occur by d i r e c t displacement at the 5-methylene group or v i a an i n i t i a l n u c l e o p h i l i c a t t a c k of the enzyme or putre s c i n e at the 6 p o s i t i o n . Mechanisms r e q u i r i n g the s o l u b u l i z a t i o n of the 6-3 hydrogen are excluded s i n c e [6- H ] - u r a c i l l a b e l l e d putThy and Thy to 14 the same extent as [2- C ] - u r a c i l (Table 8). Trimethoprim d i d not i n h i b i t 0W-14 DNA synth e s i s or m o d i f i c a -t i o n . Therefore THFA i s not l i k e l y i n v o l v e d i n the formation of Thy at the p o l y n u c l e o t i d e l e v e l . This does not r u l e out the p o s s i b i l i t y that Thy i s derived from hmPPUra. There are several immediate problems which should be pursued. In vitro formation of putdTMP from hmPPdUMP was only 50 percent effec-tive. Reaction conditions should be optimized and the nature of the am 37 DNA substrate should be investigated. The reaction conditions and necessary substrates for hmPPdUMP biosynthesis should also be investigated. The requirements for dTMP synthesis are also unknown. The search for conditionally lethal DNA modification mutants should continue. 238 FIGURE 59.—Summary of the pathways of 0W-14 DNA precursor s y n t h e s i s and m o d i f i c a t i o n . This f i g u r e summarizes our knowledge of 0W-14 DNA metabolism. 0W-14 i n f e c t i o n of a c e l l r e s u l t s i n the i n h i b i t i o n of host DNA synth e s i s and the synth e s i s of products necessary f o r phage DNA s y n t h e s i s . The syn t h e s i s of dTMP i s i n h i b i t e d and dTTP i s destroyed by a phage coded dTTPase. Enzymes required f o r the sy n t h e s i s of hmdUMP are made. 0W-14 DNA i s synthesized w i t h four nucleoside triphosphates, dGTP, dATP, dCTP and hmdUTP. hmdUMP i s modified p o s t r e p l i c a t i o n a l l y to dTMP and putdTMP. hmPPdUMP i s an intermediate i n the b i o s y n t h e t i c pathway l e a d i n g to putdTMP. Nothing i s known about b i o s y n t h e t i c pathway, l e a d i n g from hmdUMP to dTMP. Host pathways ( ); 0W-14 pathways host DNA dTTP t (dATP, dGTP, dCTP) dTDP dTMP dUTP hmdUDP dCMP (TtputT)-DNA I putrescine r PP 1 "I |(T+limU)-DNAj j (XPP) f(T+ hmU)-DNA~| 1 J hmU-DNA (dATP, dGTP, dCTP) hmdUTP 240 LITERATURE CITED Adams, M.H. 1959. Bacteriophages. Interscience Publishers, New York. A l e g r i a , A.H., and Kahan, F.M. 1968. 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