CHANGES IN THE LEVEL OP FREE NUCLEOTIDES OP VACCINIA INFECTED CHORIOALLANTOIC MEMBRANE OP THE CHICK EMBRYO IN VIVO . y b VIVIAN WYLIE B.Sc. University of B r i t i s h Columbia 1962 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Bacteriology and Immunology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1964 In the presenting this thesis i n partial r e q u i r e m e n t s f o r an advanced British Columbia, I agree available mission f o r reference f o r extensive that fulfilment of degree a t the U n i v e r s i t y the L i b r a r y and s t u d y * shall I further copying of t h i s thesis make i t f r e e l y agree representatives. cation I t i s understood of t h i s thesis w i t h o u t my w r i t t e n Department of Date shall and Immunology Columbia, 23 September 1964 o r by n o t be a l l o w e d permission-. The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada per- that, c o p y i n g or p u b l i - f o r f i n a n c i a l gain Bacteriology that f o r scholarly p u r p o s e s may be g r a n t e d by the Head o f my Department his of • A B S T R A C T The ribonucleotides i n the chorioallantoic membrane of 12-day-old chick embryos have been isolated by ion-exchange chromatography and characterized by their spectrophotometric and paper chromatographic properties. tides were i d e n t i f i e d : dine-5 1 phosphate The following nucleo- adenosine-5 8 phosphate (AMP), u r i - (UMP), c y t i d i n e - 5 1 phosphate (CMP), uridine~5' diphosphate galactose (UDPGal), uridine-5' diphosphate N-acetyl hexosamine (UDPNAHexosamine), guanosine-5' phosphate (GMP), cytidine-5' diphosphate (CDP), uridine-5' diphosphate (UDP), adenosine-5 1 diphosphate (ADP), guanosine-5' diphosphate (GDP), cytidine-5' triphosphate (CTP), uridine-5' triphosphate (DTP), adenosine-5' triphosphate (ATP), and guanosine-5' triphosphate (GTP). Quantitative determinations of these nucleotides were made on the basis of their u l t r a v i o l e t absorption at 260 m «. S i m i l a r l y , concentrations of these nucleotides were / estimated i n 12-day-old chorioallantoic membranes a f t e r i n f e c t i o n with vaccinia v i r u s . Larger amounts of ribonucleoside-f? phosphates were present i n the infected tissue at 4 and 12 hours after i n f e c t i o n . The amounts of ribonucleoside-5' triphosphates were decreased. In tissues where, i t i s believed, synchronous i n f e c t i o n occurred, the amounts of ribonucleoside-5' diphosphates and t r i phosphates were markedly lower than i n controls after 12 hours of i n f e c t i o n . ii I n f e c t i o n i n the presence of t r i t i u m = l a b e l l e d thymidine showed t h a t the amount of l a b e l l e d mono-, d i ~ , and t r i p h o s p h a t e s thymidine-5* had i n c r e a s e d a f t e r 4 hours and t h a t the amounts of these n u c l e o t i d e s subsequently de creased. vii ACKNOWLEDGEMENTS The author would l i k e to thank Dr. J . E. Bismanis, Department of Bacteriology and Immunology, f o r supervising this study. Grateful thanks are also extended to Dr. M. Smith, Fisheries Research Board of Canada and Dr. G. M. Tener, Department of Biochemistry, f o r their helpful suggestions and use of their f a c i l i t i e s i n conducting many of the tests i n t h i s i n v e s t i gation. iii TABLE OP CONTENTS INTRODUCTION . l a 1 REVIEW OP LITERATURE MATERIALS AND METHODS 12 I Vaccinia Virus 12 a. Pock-Forming U n i t Determinations 12 . .12 b. V i a b i l i t y T e s t s II III IV V VI VII Fertile Chick Eggs 12 . . . . . I n f e c t i o n Procedures 13 Incubation Conditions 15 Membrane Removal and C o l l e c t i o n 15 Preparation of Nucleotide Extracts 15 A n a l y t i c a l Methods 16 a. P r e p a r a t i o n of Column f o r Ion-exchange Chromatography b. F r a c t i o n a t i o n of N u c l e o t i d e s by Column Chromatography c. Spectrophotometric D e t e c t i o n and Measurement o f N u c l e o t i d e s d. F r e e z e - d r y i n g of Samples f o r 16 . .17 Paper Chromatography 17 17 .18 e. Paper Chromatography o f F r a c t i o n s . . . f. I d e n t i f i c a t i o n o f . N u c l e o t i d e Sugars . . .19 g. T h i n Layer Chromatography .19 h. I d e n t i f i c a t i o n of N-Acetyl Hexosamine . . .' S p e c t r a l Measurements 20 20 i. iv j. 21 Radioactive Measurements i. ii. 21 Calibration Graph Radioactive Counting . . . . . .21 22 EXPERIMENTAL RESULTS I II Nucleotides of the chorioallantoic membrane of the Chick embryo .22 Changes i n the l e v e l s of Ribonucleotides due to vaccinia i n f e c t i o n a. Whole Membranes ( 0 . 2 ml) 25 b. III Limited Area chorioallantoic membrane ( 0 . 2 ml) c. Limited Area chorioallantoic membrane ( 0 . 4 ml) Changes i n the l e v e l s of thymidine 25 nucleotides due to vaccinia i n f e c t i o n GENERAL CONCLUSIONS AND DISCUSSION BIBLIOGRAPHY 27 31 . .35 .38 ^5 V L I S T OF T A B L E S Rj. Values of Paper and Thin Layer Chromatography , 24 Table I I Amount of Nucleotides Recovered (muM) from 2 5 . 0 gm, Wet-Weight, of CAM Tissue, 26 Table I I I Amount of Nucleotides Recovered (muM) from 8 . 5 gm, Wet-Weight, CAM Tissue . . 28 Table IV Amount of Nucleotides Recovered (muM) from 8 . 5 gm, Wet-Weight, of CAM Tissue . 30 Table V Amount of Nucleotides Recovered (muM) from 8 . 0 gm, Wet-Weight, CAM Tissue . 32 Table VI Amount of Nucleotides Recovered (mpM) from 8 . 0 gm, Wet-Weight, of CAM Tissue . . 34 Table VII Amount of Nucleotides Recovered (counts per minute - c.p.m.) i n 8 . 0 gm, WetWeight, of CAM Tissue 36 Amount of Nucleotides Recovered (counts per minute - c.p.m.) i n 8 . 0 gm, WetWeight, of CAM Tissue . . . . . . . . 37 Table I Table VIII vi L I S T OF F I G U R E S Fig. 1 Pathways of Nucleic Acid Synthesis 7 Fig. 2 E l u t i o n p r o f i l e of Nucleotides of the Chorioallantoic membrane of the Chick embryo 23 Effects on the l e v e l s of ribonucleotides due to vaccinia i n f e c t i o n (0.2 ml) 29 E f f e c t s on the l e v e l s of ribonucleotides and thymidine nucleotides due to vaccinia virus i n f e c t i o n (0.4 ml) 33 Fig. 3 Fig. 4 I N T R O D U C T I O N It i s now established that the nucleotides occurr i n g i n l i v i n g tissue function as nucleic acid precursors and coenzymes ( 8 , 1 4 ) . Viruses which are protein-coated nucleic acid molecules depend on the nucleotides of the invaded host c e l l f o r their replication (11, 20). Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) viruses lack the a b i l i t y to maintain l i f e outside the cytoplasm of the c e l l since only here can they f i n d ribosomes, a large pool of nucleotides, abundant amino acids, and the energy and enzymatic processes necessary for t h e i r r e p l i c a t i o n . The existence then of an i n f e c t i v e virus would be expected to t e r minate with the exhaustion of the host's metabolic reserves The present (11). study was undertaken to determine whether expropriation of nucleotides on the part of viruses upon i n vasion of a host c e l l was detectable i n vivo using ion-exchange chromatography, spectrophotometric analysis and radioactive tracing. REVIEW OF L I T E R A T U R E Up to the time of writing there are no known systems possessing v i r a l - l i k e q u a l i t i e s which have the a b i l i t y to r e p l i c a t e themselves without the intermediacy of a host The Cytopathogenic (11). effects of viruses depend l a r g e l y on a disruption of the metabolism of the host c e l l with a concomittant expropriation of nucleotides for their own r e p l i cation. This l i t t l e understood phenomenon of v i r a l monopoli- zation of host c e l l metabolism i s the key to v i r a l infection and disease. Interest In the biochemistry of vaccinia virus, a member of the pox group, has very markedly increased recently as i s evident from the extensive l i t e r a t u r e on i t s effects on host c e l l metabolism i n v i t r o ( 2 , 6 , 15, 17, 3 0 ) . The virus contains DNA i n i t s core surrounded by protein. I t lacks RNA. Noteworthy i s the f a c t that although the virus i s a DNA type, m u l t i p l i c a t i o n takes place i n the cytoplasm, (instead of the nucleus as i s the case with host DNA). This has been shown with the a i d of c l a s s i c a l and f l u o r - escent stains, electron microscopy and autoradiography about four and one half hours after i n f e c t i o n . Extensive investigation of metabolic changes i n the host c e l l following v i r a l i n f e c t i o n has helped to elucidate some of the many puzzling questions t i e d up i n t h i s i n t r i c a t e system ( l , 2 , 5 , 1 0 , 15, 16, 3 0 , 3 2 , 3 4 ) . 2 It i s a well established fact that the inception of a new class of metabolic events within the c e l l Is i n d i c a t i v e of v i r a l invasion and i n f e c t i o n ( 3 4 ) . The q u a l i t a t i v e changes which take place seem to be unique to v i r a l i n f e c t i o n while at the same time quantitative changes occur i n c e l l u l a r nucleic acid, nucleotides, protein synthesis and the processes on which viruses depend f o r their r e p l i c a t i o n (34)} As synthesis i n the infected c e l l i s increased, nucleotide concentration may also increase. In a system i n - fected by Herpes simplex virus an increase i n the levels of nucleotides preceded v i r a l DNA synthesis ( 1 6 ) . I t was assumed that the precursors f o r this v i r a l DNA synthesis were drawn from this free nucleotide pool. 1. METABOLIC CHANGES FOLLOWING VIRAL INFECTION a. Nucleotide and Nucleic a c i d changes. Work with HeLa c e l l s as early as 1957 showed large increases i n cytoplasmic RNA following p o l i o virus i n f e c t i o n and i n the DNA a f t e r Herpes simplex i n f e c t i o n ( 3 4 ) . These increases were f a r i n excess of accountable v i r a l nucleic acid, besides which this excess nucleic acid was c h a r a c t e r i s t i c of the host and not of the v i r u s . Numerous investigations have been made on the r o l e of nucleic acid precursors i n an infected c e l l u l a r system (10, 16, 30, 3 2 ) . Sekiguchi and his co-workers investigated the Incorpor- 3 a t i o n of l a b e l l e d p r e c u r s o r i n t o RNA necessary f o r the s y n t h e s i s of v i r a l DNA ( 3 0 ) . A c i d - s o l u b l e and p h o s p h o l i p i d f r a c t i o n s showed i n c r e a s e d i n c o r p o r a t i o n o f ^ P-phosphate 2 t o 4 hours a f t e r 2 infection. T r i t i a t e d thymidine was a c t i v e l y i n c o r p o r a t e d i n t o the DNA of t h e u n i n f e c t e d c e l l , and maintained s t a r t i n g a f t e r 1 t o 2 hours a steady s t a t e f o r 24 hours. On the other hand i n c o r p o r a t i o n i n t o the i n f e c t e d c e l l s rose s t e a d i l y f o r 6 hours, then decreased, producing a lower v a l u e than f o r n o n - i n f e c t e d Becker and J o k l i k found cells. evidence o f the f o l l o w i n g stages o c c u r r i n g a f t e r v a c c i n i a v i r u s i n v a s i o n o f HeLa c e l l s ( 2 ) . V i r u s s p e c i f i c messenger RNA (mRNA) appeared e a r l y i n the cytoplasm and the g e n e t i c a c t i v i t y o f host c e l l DNA was d e f i n - itely affected. was Thus, d u r i n g the f i r s t 3 hours o f i n f e c t i o n there a steady s y n t h e s i s of host c e l l RNA which subsequently de- creased and ceased a t around 7 hours. Synthesis o f mRNA ceased a f t e r seven hours o f i n f e c t i o n . Recent work by M e r r e t t and Sunderland i n c r e a s e d c o n c e n t r a t i o n of adenosine b u i l d up o f reduced demonstrated an n u c l e o t i d e s and a d e f i n i t e n i c o t i n a m i d e adenine d e n u c l e o t i d e s (NADH . 2 and NADPH^) i n N i c o t i a n a g l u t i n o s a leaves i n f e c t e d w i t h TMV and showing n e c r o t i c l o c a l l e s i o n s ( 3 2 ) . On the other hand tobacco c a l l u s t i s s u e s y s t e m i c a l l y i n f e c t e d w i t h TMV showed i n c r e a s e d c o n c e n t r a t i o n s of NAD and an i n c r e a s e i n adenosine nucleotides. The authors proposed the theory t h a t the development o f l o c a l l e s i o n s might have stimul a t e d a recorded i n c r e a s e d r a t e o f r e s p i r a t i o n w h i l e a decreased 4 r a t e of r e s p i r a t i o n may have been due t o t i s s u e s s y s t e m i c a l l y infected. Increased c o n c e n t r a t i o n s o f NADH^ and NADPH 2 was I n - t e r p r e t e d as i n d i c a t i v e o f v i r a l n u c l e i c a c i d and p r o t e i n synt h e s i s demanding an i n c r e a s e d u t i l i z a t i o n o f ATP. T h i s meant an i n c r e a s e d supply o f ADP necessary f o r r e s p i r a t o r y chain phosphorylation. b. Enzyme Changes Recent data have i n d i c a t e d t h a t i n c r e a s e d enzyme a c t i v - i t y a s s o c i a t e d with DNA s y n t h e s i s precedes metabolism. any changes i n DNA Romberg found the f o l l o w i n g b i o c h e m i c a l changes o c c u r r i n g i n e x t r a c t s of E. c o l i a f t e r i n f e c t i o n with b a c t e r i o phages of the T-even s e r i e s (10). Immediately on entrance of the v i r a l DNA, s y n t h e s i s o f host c e l l DNA, RNA and p r o t e i n stops. Before the second minute, an RNA s p e c i f i c a l l y r e l a t e d t o the phage appears. Within four minutes a f t e r i n f e c t i o n new enzymic r e a c t i o n s , and s y n t h e s i s of new p r o t e i n s are observed. By s i x minutes, phage DNA s y n t h e s i s has i n c r e a s e d t o 5 times the r a t e of host c e l l DNA formation, f o l l o w e d In 20 t o 40 minutes by a r e l e a s e of 100 t o 200 phages per c e l l . I n f e c t i o n of c e l l s with T5 phage caused crease i n d e o x y c y t i d y l a t e kinase a c t i v i t y . a tenfold In- On the other hand, I n f e c t i o n with T2 phage demonstrated no such a c t i v i t y . This phenomenon was r e c o g n i z e d as the i n d u c t i o n of a new enzyme i n the T2 i n f e c t e d system, d e o x y c y t i d i n e - 5 which c l e a v e d d e o x y c y t i d i n e - 5 1 1 triphosphatase (dCTPase) t r i p h o s p h a t e (dCTP), the product 5 of kinase a c t i v i t y . T h i s enzyme was absent i n u n i n f e c t e d Deoxycytidylate (dCMP), the product i s acted on by hydroxymethylase v i a of dCTP cells. cleavage, deoxyhydroxymethylcytidine^ monophosphate k i n a s e t o give deoxyhydroxymethylcytidine-5 1 tri- phosphate (dHMCTP), t h i s b e i n g i n s u s c e p t i b l e t o dCTPase a t t a c k . T h i s . l a t t e r nucleotide phage DNA (dHMCTP) i s i n c o r p o r a t e d i n t o the T-even i n s t e a d of OTP (as i n T5 phage )(5, 1 0 ) . A second enzyme, g l u c o s y l t r a n s f e r a s e , was observed i n T2-infected c e l l s . glucose T h i s enzyme was i n v o l v e d i n t r a n s f e r r i n g from u r i d i n e - 5 diphosphate glucose 1 c y t o s i n e (HMC) r e s i d u e s i n DNA. t o hydroxymethyl- S i m i l a r enzymes were observed d u r i n g T4 and T6 p h a g e i n f e c t i o n s . The i n c r e a s e d l e v e l s of kinases and polymerase and the i n d u c t i o n of new enzymes i n the i n f e c t e d system was thought t o be d i r e c t e d by the i n v a d i n g phage DNA. The r o l e s of the new enzymes i n c l u d e d c o u n t e r a c t i n g the a c t i o n of host enzymes opposing v i r a l DNA s y s t h e s i s , and c a r r y i n g . o u t f u n c t i o n s unique i n the r e p l i c a t i v e processes. The s y n t h e s i s of p o l y n u c l e o t i d e s or n u c l e i c a c i d molecules r e s u l t s from a l o n g and complex chain o f c o o r d i n a t i n g enzymatic processes (7). The mechanism by which these r e g u l a t e the r a t e of t h i s s y n t h e s i s i s unknown. an i n c r e a s e i n the enzyme necessary enzymes For example, f o r phosphorylation thymidine i s an i n d i c a t i o n of i n c r e a s e d DNA s y n t h e s i s . of Feed- back mechanisms, p o s i t i v e and n e g a t i v e , are known t o take some part i n t h i s i n t r i c a t e system. 1 6 The f o l l o w i n g scheme ( F i g u r e l ) , m o d i f i e d r e c e n t l y by the work of Reichard and Larsson, r e p r e s e n t s p o s s i b l e pathways of n u c l e i c a c i d s y n t h e s i s (3, 1, 12, 24). Reduction of the diphos- phates of c y t i d i n e , adenosine and guanosine y i e l d s the c o r r e s ponding deoxynucleotides (3, 12, 24). t i o n from u r i d i n e to thymidine In the case of the transi- n u c l e o t i d e s , u r i d i n e - 5 ' mono- phosphate, the key i n t e r m e d i a t e , i s converted t o d e o x y u r i d i n e 5* monophosphate by an unknown mechanism, f o l l o w e d by r e a c t i o n with formaldehyde and t e t r a h y d r o f o l i c a c i d to y i e l d thymidine- s' phosphate (14). The t r i p h o s p h a t e s of each of f o u r n u c l e o t i d e s are necessary f o r the s y n t h e s i s of both RNA (PP) being s p l i t o f f i n the r e a c t i o n . and DNA, pyrophosphate The n u c l e i c a c i d molecule i s t h e r e f o r e b u i l t up of polymers of 4 n u c l e o t i d e monophosphates (14). The s t r u c t u r e of two n u c l e o t i d e s i s i l l u s t r a t e d i n the f o l l o w i n g examples ( 7 ) . o C-CH OH L H H H Thymidine-5 phosphate (TMP) (a d e o x y r i b o n u c l e o t i d e ) 1 cCH Adenosine-5 phosphate (a r i b o n u c l e o t i d e ) 1 (AMP) 7 F i g u r e 1. AMP 1 RNA • OTP * GDP dGDP dGTP Key t o F i g u r e 1. AMP — Adenosine-5' monophosphate GMP - Guanosine-5' monophosphate CMP - Cytidine-5* UMP - U r i d i n e - 5 ' monophosphate ADP - Adenosine-5 GDP - Guanosine-5 CDP - C y t i d i n e - 5 * diphosphate ATP - Adenosine-5 GTP - Guanosine-5 CTP - Cytidine-5 UTP - Uridine-5 dADP - deoxyadenosine-5* monophosphate dGDP - deoxyguanosine-5 monophosphate dCDP - deoxycytidine-5' dUMP - d e o x y u r i d i n e - 5 ' monophosphate TMP - Thymidine-5' monophosphate TTP - Thymidine-5' 1 monophosphate diphosphate 1 1 diphosphate triphosphate 1 1 triphosphate triphosphate 1 triphosphate 1 monophosphate triphosphate DNA 8 In 1962 McAuslan and h i s c o l l e a g u e s s t u d i e d enzyme metabolism i n HeLa c e l l s i n f e c t e d by pox v i r u s e s and found t h a t the a c t i v i t y of thymidine k i n a s e (dT) had between 4 to 6 hours a f t e r i n f e c t i o n , and 9 hours (15). Deoxythymidylic i n c r e a s e d 10-to l e v e l l e d o f f i n about a c i d k i n a s e and deoxythymidine diphosphate k i n a s e were not a f f e c t e d by i n f e c t i o n . (dT) was found to be p a r t i c u l a r l y i n s t r u m e n t a l wise p h o s p h o r y l a t i o n of deoxythymidylic thymidine diphosphate (TDP) T h i s enzyme i n increased a c i d (TMP) t o deoxythymidine t r i p h o s p h a t e as a b u i l d i n g b l o c k i n DNA Thus, - - - * TTP ~ - - * TDP step- through deoxy- t h i s l a s t b e i n g important TMP 15-fold (TTP), synthesis. Thus dT seems to e x h i b i t a p o s s i b l e r e g u l a t o r y c o n t r o l over DNA c. formation. P r o t e i n Changes Levy and co-workers demonstrated t h a t a HeLa c e l l i n f e c t e d w i t h adenovirus, a c i d s though there was line i n c r e a s e d i t s uptake of l a b e l l e d amino no r a p i d i n c o r p o r a t i o n i n t o p r o t e i n (34). L a t e r , Ackermann found t h a t the p r o t e i n c o n c e n t r a t i o n of HeLa c e l l s increased f o l l o w i n g i n f e c t i o n with p o l i o v i r u s . I n f e c t i o n of e x t r a c t s of E. c o l i with T~even phages i s c h a r a c t e r i z e d by the c o n t i n u i n g p r o d u c t i o n of p r o t e i n , i n c r e a s e d DNA s y n t h e s i s and c e s s a t i o n of accumulation of RNA (5). The e a r l i e s t p r o t e i n s s y n t h e s i z e d do not appear to be r e l a t e d to phage, but they are necessary f o r subsequent DNA synthesis. P r o t e i n s s y n t h e s i z e d l a t e r however, a r e e l i c i t e d by the phage. 9 2. ASPECTS OF HOST METABOLISM I t has been suggested deoxyribonucleotides t h a t the i n c e p t i o n of s p e c i f i c i n t r a c e l l u l a r l y , or changes i n t h e i r pool s i z e , may be i n d i c a t i v e o f development and r e p l i c a t i o n o f t h a t cell (28). In a system such as the d e v e l o p i n g c h i c k embryo the importance o f d e o x y r i b o n u c l e o t i d e metabolism cannot be overemphasized. The c o n t r o l and balance between anabolism and c a t a - b o l i s m o f these n u c l e o t i d e s may be the r e g u l a t o r y mechanism o f deoxyribonucleotide supply towards DNA s y n t h e s i s (and hence s y n t h e s i s ) and c e l l m u l t i p l i c a t i o n . of deoxyribonucleotides viral Because o f the low l e v e l s o c c u r r i n g i n most t i s s u e (approxi- mately 0 . 8 t o \% o f t o t a l n u c l e o t i d e c o n t e n t ) , s t u d i e s on poss i b l e c o n t r o l mechanisms o f DNA s y n t h e s i s by these nucleotides are r e l a t i v e l y l a c k i n g . I n v e s t i g a t i o n o f deamina§e a c t i v i t y on d e o x y c y t i d y l a t e (dCMP), deoxyguanylate (dGMP) and deoxyadenylate (dAMP), p l u s t h e i r corresponding n u c l e o s i d e s i n the d e v e l o p i n g c h i c k embryo, showed t h a t c a t a b o l i c a c t i v i t y on each n u c l e o t i d e v a r i e s w i t h r e s p e c t t o p e r i o d s o f development. The deaminases are mainly c a t a b o l i c enzymes i n v o l v e d i n b r e a k i n g down n u c l e o s i d e s by s p l i t t i n g o f f t h e i r amino groups, producing xanthine d e r i v a t i v e s , these b e i n g f u r t h e r degraded t o u r i c a c i d and e x c r e t e d . D e o x y c y t i d y l a t e deaminase a c t i v i t y was found t o be h i g h i n the t h r e e - d a y - o l d c h i c k embryo, d e c r e a s i n g g r a d u a l l y t o the 1 5 t h 10 day. On the other hand dAMP deaminase a c t i v i t y was not d e t e c t e d u n t i l about the 7 t h day and then maintained a steady s t a t e . A c t i v i t y on dGMP or deoxyguanosine was d e t e c t a b l e a f t e r hatching. In the 4_to 8-day-old c h i c k , dGMP phosphatase a c t i v i t y was high, whereas dCMP phosphatase a c t i v i t y was low f o r the same p e r i o d of development. An i n c r e a s e was noted however, w i t h l a t e r de- velopment of the embryo. 3. NUCLEOTIDE CHANGES IN ABNORMAL TISSUE Comparative i n v e s t i g a t i o n s o f the f r e e n u c l e o t i d e s of A s c i t i c Hepatoma, normal and r e g e n e r a t i n g l i v e r o f the r a t , showed lower l e v e l s o f AMP, n i c o t i n a m i d e adenine d i n u c l e o t i d e s and u r i d i n e d i p h o s p h a t e - s a c c h a r i d e s (13). i n the a s c i t i c hepatoma ATP was not s i g n i f i c a n t l y higher i n the hepatoma normal l i v e r . regenerating The cells than However, i t was much higher when compared w i t h liver. t r i p h o s p h a t e s of guanosine, u r i d i n e and c y t o s i n e were much higher i n the hepatoma than i n normal or r e g e n e r a t i n g liver. The authors concluded that t h i s increase i n triphosphates was due t o the m u l t i p l e f u n c t i o n s of these compounds; t h a t i s , the s y n t h e s i s o f n u c l e i c a c i d s , p r o t e i n s , p o l y s a c c h a r i d e s and phosphatides 4. and f o r m a t i o n o f s u b c e l l u l a r s t r u c t u r e s . INFECTION PROCEDURES In the i n f e c t i o n o f a c e l l c u l t u r e w i t h v a c c i n i a v i r u s , s e v e r a l methods may be used (35). I n t r o d u c t i o n o f a small dose w i l l cause p r o g r e s s i v e spread o f i n f e c t i o n from a d i s c r e t e focus 11 throughout the c e l l c u l t u r e u n t i l a l l s u s c e p t i b l e c e l l s are destroyed. O n t h e other hand, i n f e c t i o n with a massive dose w i l l cause simultaneous i n f e c t i o n o f most c e l l s of the c u l t u r e . Under these c o n d i t i o n s f o c a l l e s i o n s develop and can u s u a l l y be v i s u a l i z e d w i t h the naked eye a f t e r 24 hours. I n f e c t i o n of the c h o r i o a l l a n t o i c membrane w i t h v a c c i n i a v i r u s under i n v i v o and i i i v i t r o an i n c r e a s e there c o n d i t i o n s has i n d i c a t e d t h a t i n v i r u s occurs at about 10 hours ( 1 7 ) . i s a logarithmic production After t h i s of v i r u s , accompanied between the 24th t o 48th hour by gross changes i n the membrane, c o n s i s t i n g of hypertrophy and h y p e r p l a s i a . There are l i m i t a t i o n s t o these I n f e c t i o n procedures however, and i n case of i n f e c t i o n o f a l l c e l l s o f a c u l t u r e , the resultant information i s only i n d i c a t i v e o f the average behav- i o u r of the c e l l c u l t u r e . population are e q u a l l y s u s c e p t i b l e t o v i r a l i n f e c t i o n u s i n g mas- s i v e dose techniques, fected. Moreover, not a l l c e l l s of a given and only a c e r t a i n p r o p o r t i o n w i l l be i n - 12 M A T E R I A L S I AND M E T H O D S VACCINIA VIRUS Unexpired g l y c e r i n a t e d s m a l l pox v a c c i n e was obtained from stocks of the Student H e a l t h S e r v i c e s - U n i v e r s i t y of B r i t i s h Columbia. A new l o t of ten s e a l e d lymph c a p i l l a r i e s was opened under s t e r i l e c o n d i t i o n s and d i l u t e d w i t h p h y s i o l o g i c a l s a l i n e i n p r o p o r t i o n 1:25, and t h i s used f o r each experiment. D i l u t i o n s of t h i s o r i g i n a l suspension on c h o r i o a l l a n t o i c mem- 5 brane (CAM) y i e l d e d a c o n c e n t r a t i o n of approximately 3.0 pock-forming u n i t s a. (PPU) /ml. PFU Determinations S e v e r a l d i l u t i o n s , 1 i n 10, 1 i n 100, 1 i n 1,000 I i n 10,000, were made of the v i r u s suspension and each duced onto the CAM 48 hours. of 3 f e r t i l e eggs. and intro- These were incubated f o r Countable pocks were noted and the average concentra- t i o n of v i r u s c a l c u l a t e d b. x 10 (27, 29). V i a b i l i t y Tests In each experiment, an a d d i t i o n a l two membranes were i n f e c t e d w i t h each b a t c h . Two eggs were chosen a t random t h e r e - a f t e r , and f u r t h e r incubated f o r 48 hours. Growth of v i r u s , as evidenced by the appearance of plaques, v e r i f i e d the v i a b i l i t y of the o r i g i n a l suspension. II FERTILE CHICK EGGS Lots of f i v e dozen f e r t i l e white Leghorn c h i c k eggs were obtained from the P o u l t r y Farm - U n i v e r s i t y of B r i t i s h umbia, and i n c u b a t e d a t 36.5 Col- - 37°C. i n a F a v o r i t e Incubator f o r 13 a p e r i o d o f 12 days ( 2 6 ) . They were turned d a i l y t o ensure maximal s u r v i v a l o f embryos. A t t h e end of the i n c u b a t i o n p e r i o d , the eggs were candled. That i s , w i t h the a i d o f a m i c r o s c o p i c lamp, the eggs were examined f o r f e r t i l i t y and embryos which showed up as a b l a c k spot (due t o eyes) were l o c a t e d and marked. In A i r sacs were d e a l t w i t h i n l i k e manner. e a r l i e r experiments, 20$ of the eggs were i n f e r t i l e . f i g u r e i n c r e a s e d t o 30$ i n l a t e r Ill This experiments. INFECTION PROCEDURES The candled f e r t i l e eggs were d i v i d e d up i n t o 4 batches o f 12 eggs each. One dozen was t o serve as c o n t r o l and the other three dozens f o r i n f e c t i o n a t v a r i o u s time P r e l i m i n a r y experiments intervals. demonstrated no s i g n i f i c a n t d i f f e r e n c e s i n the c o n c e n t r a t i o n s o f n u c l e o t i d e s i n c o n t r o l s run w i t h each i n f e c t i o n , t h e r e f o r e a s i n g l e c o n t r o l was ext r a c t e d w i t h the 12-hour i n c u b a t i o n b a t c h . Each b a t c h of eggs was s t e r i l i z e d w i t h a 2% i o d i n e s o l u t i o n i n the areas of l o c a t e d embryo and a i r s a c . T r i a n g u l a r windows ( l cm. e q u i l a t e r a l ) were made by means o f a r o t o r e l e c t r i c d r i l l , care being taken t o ensure that only the egg s h e l l was c u t , the s h e l l membrane and CAM which a r e immediately A slit air below the s h e l l feeing l e f t i n t a c t ( 2 6 ) . was made w i t h the e l e c r i c d r i l l i n the r e g i o n of the sac and a hole made w i t h the a i d o f a s t e r i l e probe. 14 The t r i a n g u l a r window was then removed u s i n g a probe and v e r y c a r e f u l l y a s l i g h t puncture was s h e l l membrane. CAM CAM made i n the egg T h i s ensured the e n t r y of a i r on top of the when s u c t i o n was The dropped sterile now a p p l i e d a t the a i r sac w i t h a rubber b u l b . r e s t i n g on top of the embryo was ready f o r infection. In p r e l i m i n a r y experiments, 0 . 1 ml. of the 1:25 d i l u t i o n of v a c c i n i a v i r u s suspension was window onito the CAM 1 c c . B.D. i n t r o d u c e d through the of each embryo w i t h the a i d of a Y a l e s y r i n g e and needle (size 26). sterile This quantity and method of i n t r o d u c t i o n proved inadequate s i n c e maximal cont a c t o f v i r u s w i t h membrane r e q u i r e d an i n c r e a s e i n volume and . a l s o a method which produced a spray t o cover the t o t a l imate area o f 16 cm . approx- M o d i f i c a t i o n s i n the shape of the needle o u t l e t were made to t r y t o a c h i e v e a spray e f f e c t . A volume of 0 . 2 ml. of the 1:25 was vaccinia suspension i n t r o d u c e d onto CAMs w i t h the a i d of the m o d i f i e d needle and the egg t i l t e d s e v e r a l times to a l l o w maximum c o n t a c t . For r a d i o a c t i v i t y s t u d i e s , an i n c r e a s e d c o n c e n t r a t i o n of v i r u s ( 0 . 4 ml. of 1:25 of 9 embryos each. d i l u t i o n ) was used t o j n f e c t two One hour a f t e r v i r u s i n f e c t i o n , 0 . 2 ml. (10 micro c u r i e s , e q u i v a l e n t to about 2 . 2 x 10 per m i n u t e ) ( 2 3 ) batches of t r i t i a t e d l a n d Nuclear C o r p o r a t i o n ) was and needle onto the membrane. thymidine disintegrations (obtained from New Eng- introduced with s t e r i l e syringe C o n t r o l s run s i m u l t a n e o u s l y w i t h each b a t c h r e c e i v e d s i m i l a r c o n c e n t r a t i o n s of ^H-thymidine, but 15 no v i r u s . IV INCUBATION CONDITIONS A f t e r s e a l i n g the openings w i t h s t e r i l e tape, the eggs were p l a c e d i n a F a v o r i t e Incubator at 3 6 . 5 - 37°C. f o r the a l l o t t e d time o f i n c u b a t i o n and removed at p r e c i s e times f o r r e moval of the membrane. In the case of r a d i o a c t i v i t y s t u d i e s , 2 batches o f 9 eggs each (1 c o n t r o l , 1 i n f e c t e d ) were incubated at 36.5 - 3 7 ° 0 . f o r 4 hours, the c o n t r o l f o r 3 hours w i t h ?H-thy- midine, w h i l e another 2 batches were incubated f o r 12 hours. V MEMBRANE REMOVAL AND COLLECTION At p r e c i s e times, i . e . 3 hours, 4 hours, 6 hours and 12 hours, eggs were removed and u s i n g s t e r i l e f o r c e p s , the CAMs were removed as q u i c k l y as p o s s i b l e and dropped i n t o a f r e e z i n g mixture o f d r y - i c e and acetone. Owing t o the u n a v a i l a b i l i t y of d r y - i c e , l i q u i d n i t r o g e n was used i n r a d i o a c t i v i t y s t u d i e s . The membranes were e x t r a c t e d w i t h s t e r i l e f o r c e p s and dropped Into l i q u i d n i t r o g e n where they f r o z e VI immediately. PREPARATION OF NUCLEOTIDE EXTRACTS The weighed f r o z e n t i s s u e from each batch was p u l v e r - i z e d i n a c o l d mortar and added t o 4 volumes o f b o i l i n g l e d water w i t h s t i r r i n g ( 3 6 ) . distil- The mixture was maintained at ap- p r o x i m a t e l y 95°C. f o r 5 minutes w i t h constant s t i r r i n g . t h i s time, i t was homogenized i n a Waring Blendor at f a s t f o r one minute After speed and c e n t r i f u g e d at 12,100 x g i n a S e r v a l l Re- f r i g e r a t e d - Automatic C e n t r i f u g e f o r 2o minutes. A s i m i l a r ex- t r a c t i o n procedure was again c a r r i e d out on the r e s i d u e and the s u p e r n a t a n t s combined. Seven volumes of 95$ e t h y l a l c o h o l were 16 then added t o produce a 70% e t h a n o l e x t r a c t . The e x t r a c t was then evaporated on a r o t a r y evaporator o at 30 C. t o remove the a l c o h o l and any r e s i d u a l acetone. f i n a l e x t r a c t was clarified by c e n t r i f u g a t i o n at 1 7 , 3 0 0 x g(5°C.) for 15 minutes. for l o a d i n g on t o a chromatographic form was The pH was chromatographed a. adjusted to 8.6 column. and was ready One drop of c h l o r o - E x t r a c t s were s t o r e d at 0 C , and were a l l w i t h i n one week of p r e p a r a t i o n . ANALYTICAL METHODS P r e p a r a t i o n of Column f o r Ion-exchange Chromatography A g l a s s column measuring 70 x 1 cm. was anion-exchange packed w i t h d i e t h y l a m i n o e t h y l (DEAE) c e l l u l o s e i n 1 M monium carbonate s o l u t i o n was now added t o each e x t r a c t as a p r e c a u t i o n a r y measure a g a i n s t o b a c t e r i a l growth. VII The (33). am- Before use, the D E A E - c e l l u l o s e sedimented and decanted s e v e r a l times i n d i s t i l l e d water to remove f i n e p a r t i c l e s , then 2 M ammonium carbonate s o l u t i o n added t o i t . The D E A E - c e l l u l o s e now i n the carbonate form was added i n a t h i n s l u r r y t o the column h a l f f i l l e d w i t h 1 M c a r bonate and packed by i n c r e a s i n g a i r p r e s s u r e of 5 t o 10 l b s . per square i n c h . The h e i g h t of c e l l u l o s e was 50 cm. from the s i n t e r e d disc. About 500 ml. of 2 Manmonium carbonate was through the column, f o l l o w e d by 1,000 remove excess carbonate and l i g h t l y ml. of d i s t i l l e d water t o absorbed compounds. S t a r t i n g b u f f e r , 0 . 0 0 2 M ammonium b i c a r b o n a t e pH 8 . 6 , added u n t i l then passed the pH of the e f f l u e n t was was now the same as the i n f l u e n t 17 and the o p t i c a l d e n s i t y a t 260 tn yx was below 0 . 0 1 . The n u c l e o t i d e e x t r a c t a t pH 8 . 6 was added t o the column, then s t a r ting buffer ( 0 . 0 0 2 M ammonium b i c a r b o n a t e pH 8 . 6 ) added u n t i l the o p t i c a l d e n s i t y of the e f f l u e n t a t 260 m u was below 0 . 0 5 measured on a Beckman Model DU or DK Spectrophotometer. b. F r a c t i o n a t i o n of N u c l e o t i d e s by Column Chromatography G r a d i e n t e l u t i o n apparatus c o n s i s t e d of a r e s e r v o i r and mixer connected by a U-tube siphon ( 3 1 ) . The r e s e r v o i r h e l d 1500 ml. of 0 . 2 M ammonium b i c a r b o n a t e s o l u t i o n a t pH 8.6 and the mixer, 1500 ml. of 0 . 0 0 2 M ammonium b i c a r b o n a t e pH 8 . 6 . A magnetic s t i r r e r i n the mixer maintained a homogeneous conc e n t r a t i o n l e a v i n g t h i s c o n t a i n e r i n t o the column. were r u n a t room A l l columns temperature. A G i l s o n M e d i c a l F r a c t i o n C o l l e c t o r c o l l e c t e d 200 tubes w i t h f r a c t i o n s measuring approximately 12 ml. a t 8 t o 9 minute i n t e r v a l s . c. S p e c t r o p h o t o m e t r y D e t e c t i o n and Measurement of N u c l e o t i d e s E l u t e d n u c l e o t i d e s were d e t e c t e d and measured a t 260 m p. ( i n 1 cm. quartz c e l l s w i t h e l u t i n g s o l u t i o n as b l a n k ) on a Beckman Spectrophotometer. The r e l a t i o n s h i p , optical d e n s i t y a g a i n s t f r a c t i o n numbers, was p l o t t e d on a graph g i v i n g an e l u t i o n p r o f i l e d. picture. F r e e z e - d r y i n g of Samples f o r Paper Chromatography N u c l e o t i d e f r a c t i o n s c o l l e c t e d by column chromato- graphy were r e p e a t e d l y evaporated by means of a r o t a r y evap" 18 o r a t o r t o remove the e l u t i n g s a l t , ammonium b i c a r b o n a t e , before being used i n paper chromatography (31). F u r t h e r removal of r e s i d u a l e l u a t e was accomplished by f r e e z e - d r y i n g the samples. The evaporated m a t e r i a l i n a s m a l l volume (2 ml.) was made f r o z e n by immersion i n a d r y - i c e acetone bath and Immediately p l a c e d i n a d e s i c c a t o r i n the presence of magnesium p e r c h l o r a t e and potassium hydroxide. mm Hg and l e f t o v e r n i g h t . of water f o r paper e. The d e s i c c a t o r was evacuated t o 0 . 0 1 The r e s i d u e was d i s s o l v e d i n 0.01ml. chromatography. Paper Chromatography of F r a c t i o n s The descending technique o f paper chromatography was used i n a l l c a s e s . Samples of unknowns (approximately 0 . 1 pM) were s p o t t e d near one end of Whatman No. 40 paper t o g e t h e r with a p p r o p r i a t e standards o b t a i n e d from Pabst L a b o r a t o r i e s . Stan- dard sodium s a l t s of n u c l e o t i d e s were changed t o t h e ammonium form by treatment w i t h c a t i o n exchange (Rohm and Haas). r e i i n , Amberlite-IR 120 Papers were run i n the f o l l o w i n g systems v i z . I s o b u t y r i c a c i d /ammonium hydroxide /water 66/33/1 ( I ) , ( 1 8 ) , s a t u r a t e d ammonium sulphate / i s o p r o p a n o l / l M sodium a c e t a t e 80/2/18 ( I I ) (22) and e t h a n o l /ammonium a c e t a t e 5 / 2 , pH 3 . 8 (CV) (19) at 20°C. Time of r u n n i n g i n the case of the I s o b u t y r i c sys- tem was 16 t o 20 hours, and the ammonium sulphate and ammonium a c e t a t e about 10 hours. At the t e r m i n a t i o n of a r u n , t h e papers were removed, d r i e d , and n u c l e o t i d e s which showed up as dark spots when viewed d i r e c t l y i n u l t r a v i o l e t l i g h t were l o c a t e d and marked o f f . Rf v a l u e s were c a l c u l a t e d . 19 f. I d e n t i f i c a t i o n of N u c l e o t i d e Sugars Samples c o n t a i n i n g suspected n u c l e o t i d e sugars reason of t h e i r R f (by v a l u e s i n a p p r o p r i a t e s o l v e n t s ) were a d j u s t e d to pH 2 w i t h f o r m i c a c i d and heated a t 100°C. f o r 15 minutes t o r e l e a s e the sugar moeity (36). After cooling rapidly, samples were f r e e z e - d r i e d immediately i n the presence of po- A s m a l l q u a n t i t y of water (0.01 tassium h y d r o x i d e . the ml.) was added to each r e s i d u e and these s o l u t i o n s used f o r t h i n l a y e r chromatography. §• T h i n Layer Chromatography A suspension of MN-cellulose powder 3 0 0 , from Macherey, Nagel and Co., was u r i n g 20 x 10 cm. chromatography obtained spread on g l a s s p l a t e s meas- i n the p r e s c r i b e d manner f o r t h i n l a y e r (21). Samples (0.01 ml.) of h y d r o l y s e d n u c l e o t i d e sugars together w i t h other sugar standards, were s p o t t e d on one end of the p l a t e s and p l a c e d i n a system water 5/4/17 ( I I I ) (21) of running was c o n t a i n i n g n-butanol/acetone/ f o r ascending chromatography. The time about I f hours. The p l a t e s were removed, d r i e d , and.sprayed 0 . 0 0 5 $ s a t u r a t e d aqueous s i l v e r n i t r a t e i n acetone. d r y i n g , the p l a t e s were a g a i n sprayed w i t h 0 . 5 i d e i n ethanol s o l u t i o n (21). with After N sodium hydrox- 20 h. I d e n t i f i c a t i o n o f N - A c e t y l Hexosamine The h y d r o l y s e d sample was ( 0 . 5 ml.) o f n u c l e o t i d e sugar added t o 0 . 1 ml. o f potassium t e t r a b o r a t e pH 9-1 and heated i n a b o i l i n g water bath f o r 3 minutes ( 2 5 ) . After cooling, 3 ml. o f p-dimethylaminobenzaldehyde was added and the mixture incubated f o r 20 minutes at 37 ° C i a t e l y at 5^4 m u. The sample was r e a d immed- A p p r o p r i a t e c o n t r o l s and blanks were a l s o run s i m u l t a n e o u s l y . i. S p e c t r a l Measurements Located n u c l e o t i d e s on paper chromatograms were e l u t e d from the paper i n the f o l l o w i n g manner. The a r e a was c u t out i n the shape o f a r e c t a n g l e , a p o i n t made a t one o f i t s l o n g e r ends. T h i s s t r i p of paper, p o i n t up, was suspended i n a s m a l l q u a n t i t y of d i s t i l l e d water, i t s end j u s t t o u c h i n g the water. When the water f r o n t had t r a v e l l e d the t o t a l d i s t a n c e of the paper i t was removed and p l a c e d p o i n t downwards on a s t r i p o f aluminum foil. T h i s was then f o l d e d inwards and p l a c e d i n a c e n t r i f u g e tube and c e n t r i f u g e d a t 2 , 3 0 0 x g (24°C.) f o r 5 minutes. The r e s u l t i n g n u c l e o t i d e s o l u t i o n was used f o r s p e c t r a l a n a l y s i s ( 1 8 ) . A spectrum o f the s o l u t i o n at pH 7 was made on a Beckman Model DK Recording Spectrophotometer from 300 m u t o 220 m u (18). The s o l u t i o n was then a d j u s t e d t o pH 11 w i t h 2 M sodium hydroxide and a spectrum again r e c o r d e d , superimposed on the f i r s t (pH 7 ) . After adjusting this alkaline solution t o pH 2 w i t h h y d r o c h l o r i c a c i d , a t h i r d spectrum was run, superimposed on the l a s t two. A l l s p e c t r a l measurements were c a r r i e d out u s i n g t h i s modus o p e r a n d i • 21 J. R a d i o a c t i v e Measurements i. Calibration Graph I t was n e c e s s a r y t o a s c e r t a i n where thymidine nucleotides ( o c c u r r i n g i n low c o n c e n t r a t i o n s i n t h e CAM and t h e r e f o r e not u l t r a v i o l e t d e t e c t a b l e ) would emerge i n the e l u t i o n under the u s u a l c o n d i t i o n s o f column chromatography. b r a t i o n graph c o n s i s t i n g o f known u l t r a v i o l e t profile A cali- (UV) d e t e c t a b l e q u a n t i t i e s o f thymidine n u c l e o t i d e s , thymidine-5' monophosphate (TMP), thymidine-5 phosphate 1 diphosphate (TDP) arid thymidine-5* tri- (TTP) and adenosine n u c l e o t i d e s , n i c o t i n a m i d e adenine d i n u c l e o t i d e , adenosine-5' monophosphate, adenosine-5' diphosphate, adenosine-5' t r i p h o s p h a t e , was made. The p o s i t i o n s where these n u c l e o t i d e s emerged on the graph were noted f o r further reference. ii. R a d i o a c t i v e Counting One ml. samples chromatography of each f r a c t i o n c o l l e c t e d by column i n the t h r e e r e g i o n s o f thymidine n u c l e o t i d e e l u t i o n were mixed w i t h 10 ml. o f Bray's S c i n t i l l a t o r solution i n p l a s t i c c o n t a i n e r s (4). They were read i n a Packard T r i Carb L i q u i d S c i n t i l l a t i o n Spectrometer, Model 314EX. Counts were made f o r p e r i o d s o f t h i r t y minutes on each sample. counting e f f i c i e n c y was about 5.8$. The 22 E X P E R I M E N T A L I NUCLEOTIDES OP THE CAM A t o t a l of 12 recovered R E S U L T S OF THE CHICK EMBRYO 2 n u c l e o t i d e sugars were n u c l e o t i d e s and by column chromatography from an aqueous-ethanol ex- t r a c t i o n of the c h o r i o a l l a n t o i c membrane of the 12-day-old c h i c k embryo. F i g u r e 2 i l l u s t r a t e s such an e l u t i o n p r o f i l e of n u c l e o t i d e s obtained from 8 . 5 When dry i c e was was a t 260 mu) recovered tissue, (wet-weight). used to p u l v e r i z e t h i s t i s s u e , a lower c o n c e n t r a t i o n of n u c l e o t i d e s 260 mu) gm, these (55 o p t i c a l d e n s i t y (O.D.) u n i t s a t compared w i t h a higher y i e l d when l i q u i d n i t r o g e n was used. The (85 reason O.D. units for this presumably r e s i d e s i n the f a c t t h a t more e f f i c i e n t g r i n d i n g of t i s s u e and hence g r e a t e r r u p t u r e of c e l l s i s achieved when the t i s s u e i s completely The frozen. n u c l e o t i d e s were i d e n t i f i e d by t h e i r e l u t i o n p o s i - t i o n on column chromatography, R f values i n paper and chromatography (Table I) compared w i t h standard sugars, and thin layer nucleotides and spectral characteristics. They were i d e n t i f i e d as f o l l o w s : Peak A uridine-5 1 2 contained a mixture of n u c l e o t i d e s - AMP, diphosphate g a l a c t o s e (UDPGal.), u r i d i n e * 5 diphosphate N-acetylhexosamine (UDPN-AHexosamine), and i n f e c t i o n ; Peak B - GMP; ADP; Peak C - CDP; ?§ak..?F - a mixture of GDP UMP, and CMP with Peak D - UDP; CTP; synchronous Peak E - Peak G - DTP; . 23 F R A C T I O N NO. FIGURE 2 -\ E l u t i o n p r o f i l e of N u c l e o t i d e s of the CAM of the Chick Embryo. S e p a r a t i o n of an aqueous-ethanol e x t r a c t ( 8 . 0 gm t i s s u e ) on DEAEcellulose. Column 50 x 1 cm., Gradient e l u t i o n 0 . 0 0 2 M t o 0 . 2 M ammonium b i c a r b o n a t e pH 8 . 6 . T o t a l Volume 3 1. Fractions 12 ml. See pages 22 and 25 f o r peak i d e n t i f i c a t i o n . 24 T A B L E R I Values on Paper and T h i n Layer Chromatography. S Y S T E M I NUCLEOTIDES R f II R CDP* Rf .34 UMP .25 AMP .51 UDPGal. .12 .79 UDPNAHexosamlne .12 .79 GMP .22 CDP .21 .34 .81 1.6 .73 .35 .36 .28 .60 .12 OTP .07 ATP .18 .50 GTP .06 .63 .80 .25 N-Acetyl Hexosamine .32 Galactose .19 R s Rf 1.4 CTP * CDP IV 1.0 .62 GDP Rf ' .79 .62 ADP RAMP i n .44 CMP UDP ' M o b i l i t y w i t h r e f e r e n c e t o CDP. 25 Peak H - ATP and Peak I - GTP. Peak A, d i d not y i e l d any known nucleotides. C y t i d i n e - 5 * monophosphate was not n o r m a l l y present i n u l t r a v i o l e t d e t e c t a b l e c o n c e n t r a t i o n s and only became evident d u r i n g an enormous b u i l d - u p of monophosphates a f t e r apparent syn chronous i n f e c t i o n ( T a b l e V I I ) . II CHANGES IN THE LEVELS OF RIBONUCLEOTIDES DUE TO VACCINIA INFECTION a. Whole Membranes (0.2 ml) Use of whole CAM t i s s u e (Table I I ) i n these a f t e r r e l a t i v e l y m i l d i n f e c t i o n w i t h 0.2 ml of v i r u s did studies, suspension, not produce as marked r e s u l t s as those obtained when l i m i t e d area CAM was s u b j e c t e d t o massive i n f e c t i o n . Compared w i t h the c o n t r o l , I n f e c t i o n seemed t o have caused a g e n e r a l l o w e r i n g of most of the n u c l e o t i d e s i n 3 hours of i n f e c t i o n , f o l l o w e d by an Increase i n t r i p h o s p h a t e s a f t e r 6 hours, DTP e s p e c i a l l y marked i n t h i s r e s p e c t (98$), and then a decrease a g a i n i n 12 hours. On the whole, the t r i p h o s p h a t e s seemed t o have been most a f f e c t e d by the i n f e c t i o n , r i s i n g t o t h e i r h i g h e s t value a t 6 hours and then d e c r e a s i n g i n 12 hours to l e v e l s below the cont r o l - i n the cases of ATP and GTP,-28$ and -42$ r e s p e c t i v e l y . A d e n o s i n e - 5 ' diphosphate underwent a d i m i n u t i o n i n v a l u e a f t e r i n f e c t i o n w h i l e GDP s u f f e r e d no s i g n i f i c a n t change. No cant changes were recorded i n the monophosphates between signifi3-12 hours, but compared w i t h the c o n t r o l , a g e n e r a l l o w e r i n g i n 4T A B L E II Amount o f N u c l e o t i d e s Recovered (muM) from 25.0' gm, Wet-Weight, o f CAM T i s s u e . I N F E C T E D NUCLEOTIDES CONTROL (c) muM 3 HOUR (3H) INCUBATION muM <& CHANGE . 1490 1205 muM 605 273 640 1060 2430 229 724 657 3002 445 + NSC mpM = : = - 30 1005 - 16 505 427 520 613 1520 - 16 + 56 186 NSC - 19 587 820 1430 510 % CHANGE FROM i (c) 1040 - 18 - 45 - 37 + 25 - 50 NSC % increase % decrease no s i g n i f i c a n t change m i l l i micro-mole 12 HOUR (12H) INCUBATION 6 HOUR ,(6H) INCUBATION i AMP UMP UDPGal. UDPNAHexosamine GMP CDP UDP ADP GDP CTP UTP ATP GTP M E M B R A N E S muM (3H) | i (c) 910 1005 - 39 - 16 NSC NSC 1079 1210 505 190 574 613 1905 322 1015 1302 3501 679 - 16 - 30 NSC 605 256 320 455 2080 251 775 669 2160 256 NSC - 45 - 22 + 40 + 40 + 98 + 16 + 52 " 55 NSC NSC -25 + 73 + 73 + 59 +144 + 33 . % CHANGE FROM (3H) (6H) - 29 ^NSC + 18 NSC + 19 + 19 NSC + 21 NSC - 40 - 50 - 39 - 57 - 26 - 14 + 37 NSC + 35 NSC •^32 NSC - 18 - 28 + 51 - 42 - 50 + 21 + 35 - 49 - 26 - 19 - 22 - 23 - 48 - 38 - 62 27 values o c c u r r e d at 6 hours, p o s s i b l e caused by a concomitant i n crease i n t r i p h o s p h a t e s . b. L i m i t e d Area CAM ( 0 . 2 ml) T a b l e s I I I and IV a t t e s t the r e s u l t s when only the a r e a i n d i r e c t c o n t a c t w i t h i n t r o d u c e d v i r u s was removed. A volume of 0 . 2 ml w i l l a p p a r e n t l y only s t i m u l a t e a m i l d i n f e c t i o n d u r i n g the time a l l o t t e d f o r the t e s t s . In the f i r s t o f these experiments (Table I I I ) monophos- phates s l o w l y i n c r e a s e d a f t e r 6 hours of i n f e c t i o n and reached t h e i r h i g h e s t value i n 12 hours. The second experiment (Table IV and F i g u r e 3) showed a more r a p i d i n c r e a s e i n monophosphates a f t e r 6 hours of i n f e c t i o n ( v i s i b l e i n F i g u r e 3 from (a) t o ( c ) ) and de- creased ( F i g u r e 3 (d)) at 12 hours, s t i l l above c o n t r o l l e v e l how- ever. In both experiments the diphosphate response t o i n f e c t i o n was borne by ADP; levels. ADP, UDP and GDP showed no s i g n i f i c a n t changes i n i n both experiments showed i n c r e a s e d l e v e l s at 6 and 12 hours compared with the control. Triphosphate a c t i v i t y was ments. not constant i n both e x p e r i - Table I I I shows an i n c r e a s e d l e v e l i n ATP a f t e r 6 hours of i n f e c t i o n , which decreased a f t e r 12 hours, whereas Table IV shows a marked decrease from 3 t o 6 hours of i n f e c t i o n and t h i s climbed again t o a value j u s t i n excess of the c o n t r o l i n c r e a s e d g r a d u a l l y t o 200$ i n 12 hours. i n f e c t i o n w h i l e UTP decreased as d i d ATP. i n 12 hours. OTP was GTP u n a f f e c t e d by J A B L E III (muM) from 8 . 5 gm, Wet-Weight, CAM T i s s u e . Amount o f N u c l e o t i d e s Recovered I N F E C T E D NUCLEOTIDES CONTROL 'v. (c) muM 6 HOUR (6H) INCUBATION mpM ^-CHANGE. M E M B R A N E S 12 HOUR INCUBATION mpM- % CHANGE_FROM. . (0) km? UMP UDPGal. UDPNAHexosamine GMP CDP UDP ADP GDP CTP UTP ATP GTP 510 304 650 315 + 27 106 120 NSC - - - - 202 127 751 88 640 64 + NSC muM 780 710 + 53 + 134 t- 20 + 127 305 128 + 180 + 150- -• - 154 1-128 + 154 NSC • 128 + 154 + 25 NSC NSC NSC NSC NSC NSC - 37 + 17 + 37 + 35 205 800 77 203 275 404 425 NSC 240 340 1715 187 + 33 • 15 1165 195 1290 162 = = = (6H) % increase % decrease no s i g n i f i c a n t change m i l l i micro-mole - 15 NSC NSC + 61 - 20 " 32 NSC 29 0.3 H — A i E 7F 0.2 0.20 0.15 0.1 - * |1 L-j-»_JLj\ l r 0.5 1 0.4 (fo- 11 'U • 0.10 0.05 • i 0.3 JI 0.2 £ O to • _ r- 0.15.i • < 0.10 o cr C <O 0.05 o CQ 0.1 CvJ r > 0.4 co 2j 0.3 Q. • J 0.20 0.2 g 0. 0.20 UJ 0.15 "•••/ 0.05 >H CC < 0.20 1 M0L •• A 0.3 Tj" 0.2 f 0.15 (1 50 < 0.10 o a. o 0.1 h z o 1 100 FRACTION J\jl -A NO. 1 150 0.10 " 0.05 200 FIGURE 3 - E f f e c t s on the l e v e l s of r i b o n u c l e o t i d e s due to v a c c i n i a i n f e c t i o n ( 0 . 2 ml) (a) u n i n f e c t e d c e l l s (b) 3 hour i n f e c t e d c e l l s (c) 6 hour i n f e c t e d c e l l s (d) 12 hour i n f e c t e d c e l l s . • T A B L E Amount o f N u c l e o t i d e s Recovered IV (muM) from 8 . 5 gm, Wet-Weight, of CAM T i s s u e . I N F E C T E D ME MB R A N E S 3L2 HOUR (12H) 3 HOUR (3H) ^ 6 HOUR (6H) INCUBATION INfCUBATION CONTROL INCUBATION (c) r mjuM | % CHANGE muM % CHANGE FROM mjuM ! $ ci•iANGE FROM muM I (3H) (c) 1(c) (3H) ,(6H) .„ ..J NUCLEOTIDES t AMP UMP UDPGal. UDPNAHexosamine GMP CDP UDP ADP GDP CTP OTP ATP GTP 'SO +•100 + 70 700 705 560 +• 140 570 *l48 NSC 440 180 128 150 + 430 240 158 220 + 670 + 88 + 33 *23 NSC + 21 + 44 NSC - 61 + 46 141 + 13 770 76 1190 600 . 230 34 84 200 670 50 170 310 1030 72 +NSC muM 710 •+ 100 1140 f 18 1210 600 750 72 210 130 810 130 +- 52 - 25 NSC + 44 + 23 - 140 - 27 + 80 = % increase - % decrease = s i g n i f i c a n t change = m i l l i micro-mole n o 850 72 215 120 650 120 NSC NSC NSC NSC - 37 - 20 NSC NSC 72 91 240 280 1100 220 - 16 - 38 NSC - 42 +•91 - 21 111 - 60 NSC - 29 - 29 NSC - 23 - 70 +17 - 42 - 36 NSC + 15 NSC + 52 NSC NSC + 41 NSC NSC NSC +115 + 132 NSC + 36 + 70 205 4-69 + 83 L 31 c. L i m i t e d Area CAM ( 0 . 4 ml) The use of 0 . 2 ml of v i r u s suspension a p p a r e n t l y produced a slow p r o g r e s s i v e spread o f i n f e c t i o n w i t h r e l a t i v e l y slow changes i n the l e v e l s o f n u c l e o t i d e s . Complete dispersal of t h i s volume on the membrane c o u l d probably produce as d r a s t i c an i n f e c t i o n as a doubled volume o f 0 . 4 ml. T a b l e s V and V I exemplify p o s s i b l e simultaneous infec- t i o n o f s u s c e p t i b l e c e l l s on the membrane w i t h an i n f e c t i v e dose of 0 . 4 ml. R e s u l t s i n T a b l e V demonstrate a marked i n c r e a s e i n monophosphates, a f t e r 4 hours o f i n f e c t i o n , which reached enormous p r o p o r t i o n s a f t e r 12 hours. The b u i l d up on monophosphates a t t h i s p o i n t r e v e a l e d the presence o f CMP f o r the f i r s t time. AMP i n c r e a s e d t o 234$ and GMP t o 324$. At 4 hours. the diphosphate l e v e l no change was observed After after 12 hours o f i n f e c t i o n , however, ADP d i d decrease considerably. Both experiments bore out a d e f i n i t e decrease i n t r i phosphates a f t e r 4 and 12 hours of i n f e c t i o n , the v a l u e s dropping to exhaustive p r o p o r t i o n s i n one experiment. (Table V and F i g u r e 4) ATP and GTP decreased 90$ a f t e r 12 hours. Though the a c t u a l c o n c e n t r a t i o n s o f n u c l e o t i d e s were d i s s i m i l a r i n both o f these experiments, the t r e n d due t o i n f e c t i o n was n e v e r t h e l e s s analogous. A p o s s i b l e reason f o r t h i s d i f f e r e n c e l a y i n the r a p i d i t y o f i n f e c t i o n of the s u s c e p t i b l e c e l l population. In the f i r s t o f these experiments, T a b l e V, T.ABLE V Amount o f N u c l e o t i d e s Recovered (muM) from 8 . 0 gm, Wet-Weight, CAM T i s s u e . 12 Hour I n c u b a t i o n 4 Hour Incubation CONTROL INFECTED CONTROL NUCLEOTIDES AMP UMP UDPGal. UDPNAHexosamine CMP GMP CDP UDP ADP GDP CTP OTP &TP GTP muM muM 840 800 1240 525 0.0 153 125 430 910 ' 103 325 393 1330 290 •*- " = NSC = muM = 1005 % CHANGE miuM muM + 47 840 2800 1210 * 24 NSC 540 0.0 256 160 333 961 76 240 267 790 230 - * 67 + 28 - 22 NSC NSC - 26 " 32 - 40 - 20 % increase % decrease no s i g n i f i c a n t change m i l l i micro-mole 808 424 0.0 123 146 202 961 103 320 474 1325 256 505 137 521 160 444 409 44 160 101 88 21 I N/ F E C T E[ D '% CHANGE FROM 4 HR % CHANGE INFECTED ' + 234 * 50 + 126 + 19 19 * 137 + 324 NSC 4- 1-37 + 103 NSC + 119 - 57.5 - 57 " 50 - 79 - 93 - 92 NSC * 33 - 57.5 - 42 - 33 62 91 91 33 1.0 0.9 H < Z TMP s 0.8 DPN 0.7 E 0.6 O 0.5 <c > (- a: < o CD TDP 0.4 AMP 0.3. 0.2 i n TTP Id < 0.20s s co z a z ADP Pi 0.1 - ATP o i- a. o 50 ~ - ' 50 100 150 FRACTION NO. u 100 FRACTION 200 150 NO. 50 0.15 "* 0.10 v -0.05 * 200 100 200 150 FRACTION NO. Ribonucleotides thymidine n u c l e o t i d e s FIGURE 4 - E f f e c t s on the l e v e l s o f r i b o n u c l e o t i d e s and thymidine n u c l e o t i d e s due t o v a c c i n i a v i r u s i n f e c t i o n (0.4 ml.) (a) C a l i b r a t i o n graph o f thymidine n u c l e o t i d e s (b) u n i n f e c t e d c e l l s i n c u b a t e d w i t h ^H-thymidine, f o r 3 hours ( c ) 4 hour i n f e c t e d c e l l s i n c u b a t e d w i t h 3H~thymidine f o r 3 hours (d) u n i n f e c t e d c e l l s incubated w i t h 3 H - t h y m i d i n e f o r 11 hours (ej 12 hour i n f e c t e d c e l l s i n c u b a t e d w i t h 3 f o r 11 hours. H t n y m i d i n e T A B L E VI Amount o f N u c l e o t i d e s Recovered (mpM) from 8 . 0 gm, Wet-Weight, o f CAM T i s s u e . 4 Hour Incubation CONTROL NUCLEOTIDES 1 muM muM AMP IMP UDPGal. UDPNAHexosamine GMP CDP UDP ADP GDP CTP UTP ATP GTP I N F E C T E D CONTROL ! INFECTED ! 12 Hour I n c u b a t i o n ' 1100 1112 1230 1210 505 251 174 498 960 34 106 221 801 205 540 + NSC mjuM = = 230 96 241 930 51 187 152 670 119 muM % CHANGE muM + 11.8 - 8.8 970 1005 " 1560 1105 405 405 197 147 271 812 67 212 289 805 282 160 283 715 59 187 105 455 59 NSC NSC - 45 " 51 NSC + 50 76 - 31 + - 16 - 42 % increase % decrease no s i g n i f i c a n t change m i l l i micro-mole 196 % CHANGE + 61 " " + 9.7 NSC + 43 NSC NSC - 12 NSC NSC - 64 - 44 - 70 % "CHANGE" FROM 4 HR INFECTED + 27 - 8.7 - 25 - 22 * 66 NSC - 23 NSC NSC ~ 31 " 32 - 50 35 a needle w i t h a r e l a t i v e l y e f f i c i e n t spray was used, producing p o s s i b l e synchronous infection. In the second, T a b l e V I , owing to unforeseen circumstances, a l e s s e f f i c i e n t spray was used, probably p r o d u c i n g p r o g r e s s i v e s p r e a d i n g of i n f e c t i o n w i t h i n the a l l o t t e d time of t e s t s . Ill CHANGES IN THE LEVELS OP THYMIDINE NUCLEOTIDES DUE TO VACCINIA INFECTION The use of t r i t i a t e d thymidine c o n t r i b u t e d towards elucidation o f p h o s p h o r y l a t i o n i n the i n f e c t e d system i n vivo. Increased i n c o r p o r a t i o n of the l a b e l l e d n u c l e o s i d e , T a b l e V, i n t o monophosphate t r i p h o s p h a t e (57$) fell (130$), diphosphate (106$) and o c c u r r e d a f t e r 4 hours of i n f e c t i o n , then o f f g r a d u a l l y i n the monophosphates and diphosphates a f t e r 12 hours, but r a p i d l y decreased i n the case of t r i p h o s p h a t e s (-90$). A d u p l i c a t e experiment T a b l e V I I I , showed s i m i l a r i n c r e a s i n g trends of i n c o r p o r a t i o n i n t o monophosphates (100$), especially and diphosphates a f t e r 4 hours of i n f e c t i o n but a de- crease i n t r i p h o s p h a t e s . After 12 hours the i n c o r p o r a t i o n f o l l o w e d the same p a t t e r n as r e s u l t s i n T a b l e V I I , though not as a c t i v e l y . T A B L E Amount o f N u c l e o t i d e s Recovered CAM T i s s u e . VII (counts per minute-c.p.m) i n 8 gm, Wet-Weight, of 4 Hour Incubation NUCLEOTIDES 12 Hour I n c u b a t i o n CONTROL INFECTED % CHANGE _c_.p.m. . _ c.p.m. 1 Thymidine 5 Phosphate (TMP) and Thymidine 5 ' d i phosphate- sugar (TDPX) 2894.4 6566.4 H-130 Thymidine 5» d i phosphate (TDP) 3324.0 6854.4 4-106 Thymidine 5* t r i phosphate (TTP) 3513.6 5529.6 CONTROL INFECTED % CHANGE 1 c.p.m. 1c.p.m. % CHANGE FROM 4 HR INFECTED 1 •r - % i n c r e a s e - - % decrease 4- 57 3775.2 816 129.6 5280 2764.8 576 •+ 40 " 19 240 - 59 * 340 - 90 •4- J A B L E VIII -Amount o f N u c l e o t i d e s Recovered (counts per minute-c.p.m.) I n 8 . 0 gm, Wet-Weight, o f CAM T i s s u e . 12 Hour I n c u b a t i o n 4 Hour Incubation CONTROL c.p.m. NUCLEOTIDES INFECTED c.p.m. Thymidine-5' Phosphate (TMP) and Thymidine-5 d i phosphate- sugar (TDPX) 3908.3 7842.0 Thymidine-5 d i phosphate (TDP) 4595.4 5506 Thymidine-5* t r i phosphate (TTP) 4400 3400.8 % CHANGE % CHANGE % CHANGE FROM 4 HR INFECTED CONTROL c.p.m. INFECTED c.p.m. + 100 1617.6 2839.2 + 75 - 64 + 20 1012.8 1680.0 + 66 - 89 " 23 1142.4 1389.6 1 1 + - % increase - = % decrease .+ 2 2 - 59 38 G E N E R A L C O N C L U S I O N S & D I S C U S S I O N An understanding of the mechanism of the cyto- pathogenic e f f e c t s of v i r u s i n f e c t i o n must be d e r i v e d basically from a knowledge of i t s e f f e c t on host c e l l metabolism i n v i t r o and especially in vivo. Moreover, i t i s necessary t o be f a m i l - i a r with normal host c e l l metabolism, t h i s s e r v i n g as background f o r subsequent changes due t o impairment by v i r u s e s . In t h i s i n v e s t i g a t i o n a l l experiments were c a r r i e d out with host c e l l s growing e x p o n e n t i a l l y i n v i v o , and contained maximal c o n c e n t r a t i o n s of n u c l e o t i d e s . Any therefore increase i n the l e v e l s of n u c l e o t i d e s formed as a r e s u l t of the presence of i n f e c t i v e v a c c i n i a DNA ground n u c l e o t i d e had t o be d e t e c t e d a g a i n s t high back- concentrations. Ribonucleotides occur i n l a r g e q u a n t i t i e s i n the cytoplasm of the c e l l , and most t i s s u e e x t r a c t s y i e l d them In UV detectable are present amounts. On the other hand, deoxyribonucleotides mostly i n the nucleus of the c e l l in concentrations below the t h r e s h o l d of UV d e t e c t a b i l i t y , except where l a r g e nucleated c e l l s are s t u d i e d or s p e c i a l c o n d i t i o n s are met l e e c h them out of the to nucleus. Spectrophotometric d e t e c t i o n of n u c l e o t i d e s y i e l d e d only r i b o n u c l e o t i d e s i n these s t u d i e s , whereas the use of more s e n s i t i v e r a d i o a c t i v e t r a c i n g system d e t e c t e d concentrations of thymidine n u c l e o t i d e s i n the the diminutive system. 39 E f f e c t s of V a c c i n i a I n f e c t i o n on R i b o n u c l e o t i d e Metabolism R i b o n u c l e o t i d e s are empowered with m u l t i p l e funct i o n s i n the c e l l . They must act as p a r t s of coenzymes, which are i n v o l v e d i n p o l y s a c c h a r i d e s y n t h e s i s , l i p i d s y n t h e s i s , and p r o t e i n s y n t h e s i s as w e l l as s e r v i n g as p r e c u r s o r s f o r deoxyribonucleotide synthesis. In a d d i t i o n they serve as monomeric u n i t t o form the backbone of the RNA molecule. I t would seem l i k e l y t h e r e f o r e to expect great . changes i n t h e i r l e v e l s f o l l o w i n g v i r a l i n f e c t i o n due creased a c t i v i t y of enzymatic and energy the to i n - processes. However, owing to the h i g h background l e v e l s of n a t u r a l occurrence, not l a r g e except the changes seen i n the present s t u d i e s were under c o n d i t i o n s of r e l a t i v e l y high and r a p i d infectivity. F i g u r e 4 and Table VI e x e m p l i f i e d such a s i t u a t i o n . A f t e r 4 hours of apparent r a p i d i n f e c t i o n , monophosphates i n creased as the p h o s p h o r y l a t i v e mechanism was hyperactivated reached probably due enormous p r o p o r t i o n s a f t e r 12 hours, and to a b u i l d - u p as s y n t h e s i z e d v i r a l p a r t i c l e s terminated most processes. There was a d e f i n i t e e x p r o p r i a t i o n of n u c l e o t i d e s by v i r u s as evidenced decrease by an i n c r e a s e i n monophosphates and i n triphosphates, a f t e r 4 hours of i n f e c t i o n . a This t r e n d continued t o 12 hours a f t e r i n f e c t i o n , where an a d d i t i o n a l c o n c e n t r a t i o n of monophosphates e l i c i t e d by v i r a l DNA towards 40 increased triphosphate was noted. l e v e l s f o r i n f e c t i v e v i r u s production, In f a c t , the diphosphate and t r i p h o s p h a t e a f t e r 12 hours were almost levels non-existent. In experiments conducted with an i n f e c t i v e dose of 0.2 ml., the r e s u l t s seem t o i n d i c a t e a slow p r o g r e s s i v e spread of v i r a l i n v a s i o n . levels The o b s e r v a t i o n t h a t most n u c l e o t i d e never rose f a r above or dropped below the c o n t r o l s u b s t a n t i a t e d t h i s claim. Furthermore, i n f e c t i o n of the exposed s e c t i o n of the CAM with subsequent removal o f the t o t a l CAM g i v e s slightly d i f f e r e n t r e s u l t s than when only the exposed p o r t i o n i s removed. The e n t r y of v a c c i n i a DNA i n t o CAM c e l l s seemed t o cause an e x t r a u t i l i z a t i o n o f most n u c l e o t i d e s a f t e r 3 hours, an i n d i c a t e d by a decrease i n l e v e l s . T h i s was f o l l o w e d by an i n c r e a s e of a l l n u c l e o t i d e s a f t e r 6 hours, presumably t o take care of the i n c r e a s e d needs o f the v i r u s f o r the r e p l i c a t i v e processes. V i r a l s y n t h e s i s was probably complete by 12 hours, as i n d i c a t e d by a marked r e d u c t i o n i n the t r i p h o s p h a t e levels. One might e n v i s i o n from these experiments then, c o n t r o l of the metabolic that system of the c e l l occurs before the t h i r d hours and a p p r o p r i a t i o n o f n u c l e o t i d e s reaches a maximum around 6 hours as v i r a l p a r t i c l e s are s y n t h e s i z e d . t h e s i s i s probably Viral syn- complete by 12 hours as the n u c l e o t i d e pool i s markedly reduced. 41 E f f e c t s of V a c c i n i a DNA on Thymidine N u c l e o t i d e s The a c t i v i t y of t r i t i a t e d thymidine CAM i n the a t once r a i s e s the q u e s t i o n as to whether t h i s infected activity c o u l d be used as an index of d e o x y r i b o n u c l e o t i d e metabolism i n the i n f e c t e d system. T h i s i s h a r d l y l i k e l y s i n c e the r e g u l a t o r y mechanism f o r the r a t e of thymidine nucleotide s y n t h e s i s coupled w i t h feedback mechanisms, i s probably f e r e n t f o r other d e o x y r i b o n u c l e o t i d e s . dif- However, i t c o u l d be assumed t h a t the t r e n d shown by thymidine nucleotide levels i n the i n f e c t e d system w i l l probably be s i m i l a r to other deoxynucleotides. A f t e r 4 hours of synchronous i n f e c t i o n by DNA t h e r e i s a marked i n c r e a s e of thymidine n u c l e o t i d e s , as evidenced by an i n c r e a s e d i n c o r p o r a t i o n of t r i t i a t e d d i n e i n t o monophosphates and T h i s e l e v a t i o n may thymi- diphosphates. be a t t r i b u t e d to an i n c r e a s e d a c t i v i t y of the p h o s p h o r y l a t i v e machinery i n the c e l l u l a r system. viral infected In other words, the i n t r o d u c t i o n of l a b e l l e d n u c l e o s i d e w i t h i t s subsequent d e t e c t i o n i n n u c l e o t i d e f r a c t i o n s i n d i c a t e d t h a t phosphate m o i e t i e s had been added. As v i r a l DNA t h e r e was a decrease thymidine-5' s y n t h e s i s progressed i n thymidine cell, n u c l e o t i d e s a f t e r 12 t r i p h o s p h a t e most n o t a b l y . phates were exhausted i n the Apparently b e f o r e e i t h e r the diphosphates hours, triphosor the monophosphates, these l a s t m a i n t a i n i n g a r e l a t i v e l y h i g h f o r replenishment of exhausted reserves. level 42 The v a l u e s obtained f o r the c o n t r o l s a f t e r 12 hours of i n c u b a t i o n i n d i c a t e d t h a t the r a p i d l y p r o l i f e r a t i n g of the c h i c k embryo were a c t i v e l y i n c o r p o r a t i n g thymidine nuc- l e o t i d e s i n t o c e l l u l a r DNA. cells C o n t r o l s i n both experiments. T a b l e s V I I and V I I I r e v e a l e d s i m i l a r l e v e l s of i n c o r p o r a t i o n at 4 hours i n c u b a t i o n , f o l l o w e d by a d i m i n u t i o n i n l e v e l s a f t e r 12 hours. On the c o n t r a r y , the presence of v a c c i n i a i n the system e l i c i t e d and maintained much h i g h e r l e v e l s than the c o n t r o l s f o r the same p e r i o d s . That the c o n d i t i o n s of i n f e c t i o n w i l l a f f e c t outcome of i n c o r p o r a t i o n was second experiment the the evidenced by r e s u l t s o f the - Table V I I I . In the f i r s t experiment of s e r i e s , T a b l e V I I , apparent simultaneous i n f e c t i o n took place. R e s u l t s of T a b l e V I I I demonstrated a slower The t r e n d i n p h o s p h o r y l a t i v e a c t i v i t y however, was infection. i n good agreement, the f i r s t more a c t i v e than the second. It seems l o g i c a l t o suggest then t h a t i n order to d e r i v e meaningful r e s u l t s from m u l t i c e l l u l a r c u l t u r e s , the a c t i v i t i e s of a l l c e l l s i n t h a t system should be s y n c h r o n i z e d . T h i s may the b e s t be a c h i e v e d by i n f e c t i n g the whole p o p u l a t i o n i n s h o r t e s t time p o s s i b l e to a v o i d slow p r o g r e s s i v e spread, and t h e r e f o r e e l i m i n a t i n g i n d i v i d u a l My problem variation. i n t h i s r e g a r d l a y not o n l y i n concen- t r a t i o n of i n f e c t i o n but a l s o i n a c h i e v i n g synchronous c o n t a c t of was c e l l s with v i r u s . T i l t i n g of eggs unduly t o a c h i e v e t h i s avoided as much a s . p o s s i b l e t o e l i m i n a t e p o s s i b l e trauma 43 and d e a t h of i n f e c t e d embryos. Much of the work on changes i n n u c l e i c a c i d and r i b o n u c l e o t i d e metabolism of c e l l u l a r systems i n f e c t e d by a n i m a l v i r u s e s , has been c a r r i e d out on c e l l l i n e s (2, 17, 30, 15, 34). S e k i g u c h i and co-workers s t u d i e d e f f e c t s of v a c c i n i a on HeLa c e l l l i n e s and found t h a t t h e r e was an i n c r e a s e i n the l e v e l of r i b o n u c l e o t i d e s a f t e r 2 t o 4 hours of They d i d not r e p o r t subsequent changes (30). n o t e d t h a t t h e r e was infection. Newton a l s o an i n c r e a s e i n n u c l e o t i d e s system i n f e c t e d w i t h Herpes s i m p l e x v i r u s i n an i n v i t r o (16). I n v i v o s t u d i e s i n c l u d e i n v e s t i g a t i o n of b a c t e r i a l systems i n f e c t e d w i t h T~even phages by Kornberg (10), p l a n t systems i n f e c t e d w i t h TMV and by Sunderland and M e r r e t t (32). I n the p r e s e n t i n v i v o s t u d i e s , changes were r e c o r d e d i n b o t h r i b o n u c l e o t i d e s and deoxyribonucleotides by t h y m i d i n e n u c l e o t i d e s ) . The (represented changes i n r i b o n u c l e o t i d e s were s i m i l a r i n t r e n d t o those r e c o r d e d i n i n v i t r o s t u d i e s a f t e r 4 hours. R e s u l t s i n d i c a t e t h a t i n v i v o systems may i n g f u l r e s u l t s i f s p e c i a l c o n d i t i o n s a r e met. should y i e l d mean- These c o n d i t i o n s i n c l u d e synchronous i n f e c t i o n of the c e l l population, and removal of the i n f e c t e d p o r t i o n of t i s s u e f o r i s o l a t i o n n e c e s s a r y compounds. of 44 I t i s not CAM intended t h a t the r e s u l t s obtained w i t h t i s s u e i n f e c t e d by v a c c i n i a v i r u s i n v i v o should be p l i c a b l e to other h o s t - v i r u s systems, without adequate con- s i d e r a t i o n s b e i n g g i v e n to the p h y s i o l o g i c a l s t a t e of t i s s u e and the ap- s t r u c t u r e of the v i r u s . the 45 B I B L I O G R A P H Y 1. ACKERMANN, ¥. 2. BECKER, Y. AND JOKLIK, W. 1964 Messenger RNA i n C e l l s Infected with Vaccinia. V i r u s . Proc. of Nat. Academy of Sciences 51, 577"584. 3. BERTANI, E., HAGGMARK, A. AND REICHARD, P. 1963 Enzymatic S y n t h e s i s of D e o x y r i b o n u c l e o t i d e s . J . of B i o l . Chem. 238 22-23, (10) 223-231. 1958-59 C e l l - V i r u s Relationship - Bact. Rev. 3407-13. 4. BRAY, G. A. i 9 6 0 A Simple E f f i c i e n t L i q u i d S c i n t i l l a t o r f o r Counting Aqueous S o l u t i o n i n a L i q u i d S c i n t i l l a t i o n Counter. A n a l y t i c a l Biochem. 1, 2 7 9 - 2 8 5 . 5. COHEN, S. S. 1961 N u c l e i c A c i d S y n t h e s i s i n B a c t e r i a Inf e c t e d by T-even Bacteriophages. In d e o x y r i b o n u c l e i c A c i d S t r u c t u r e , S y n t h e s i s and F u n c t i o n . Proceedings of the 11th Annual Reunion of the S o c i e t e De Chimie Physique 102-109. 6. COHEN, S. 1963 of Biochem. 3 2 , 7. DAVIDSON, J . N. 1961 The B i o c h e m i s t r y of the N u c l e i c A c i d s . S i r Rudolph Peters and F. G. Young ( E d i t o r s ) 2 1 6 - 2 1 7 . 8. DAVIDSON, J . N. 1961 The B i o c h e m i s t r y of the N u c l e i c A c i d s . S i r Rudolph Peters and F. G. Young ( E d i t o r s ) 20. 9. HURLBERT, R. B., SCHMIDT, H., BRUMM, A. F. AND VAN POTTER, R. 1954 N u c l e o t i d e Metabolism - Chromatographic S e p a r a t i o n of A c i d - S o l u b l e N u c l e o t i d e s . J . of B i o l . Chem. 209, 2 3 " 5 3 . The B i o c h e m i s t r y of V i r u s e s . 83"l40. Annual Review 10. KORNBERG, A. I 9 6 I Enzymatic S y n t h e s i s of DNA. i n M i c r o b i a l Biochemistry. 69-102. 11. LINDEGREN, C. C. 12. LARSSON, A. 1963 Enzymatic S y n t h e s i s of Deoxyribonucleotides. J . of B i o l . Chem. 238 (10) 3414-19. 13. MANDEL, P., WINTZERITH, M., KLEIN-PETE, N. AND MANDEL, L. 1963 Comparative I n v e s t i g a t i o n of the f r e e n u c l e o t i d e s of an a s c i t i c hepatoma and of normal or r e g e n e r a t i n g l i v e r . Nature 198 (4884) 1000-1001. 14. MICHELSON, A. M. 1963 The Chemistry of Nucleosides N u c l e o t i d e s . Academic Press 2 5 1 3 0 0 . 197, 566-568. 1963 CIBA L e c t u r e s V i r u s e s Genes and C i s t r o n s . _ Nature and 46 15. McAUSLAN, B. R., and JOKLIK, W. K. 1962 S t i m u l a t i o n o f Thymidine Phosphorylating system i n HeLa c e l l s on I n f e c t i o n w i t h Pox V i r u s . Biochem. and Biophys. Res. Comm. 8 ( 6 ) 486-491. 16. NEWTON, A. A. 1963 R e p l i c a t i o n of DNA i n V i r u s I n f e c t e d Cells. Biochem. J . 87(2) 17P. 17. OVERMAN, J . R. and TAMM, I . 1957 M u l t i p l i c a t i o n of V a c c i n i a V i r u s i n CAM i n v i t r o . V i r o l o g y 3, 173"l84. 18. PABST LABORATORIES 1961 U. V. A b s o r p t i o n R i b o n u c l e o t i d e s - Paper Chromat. 19. PALADINI, A. C. and LELOIR, L. F. 1952 S t u d i e s on U r i d i n e Diphosphate Glucose. Biochem. J . 51, 426-430. 20. PIRIE, N. W. 566-568. 1963 Spectra V i r u s e s Genes and C i s t r o n s . of 5 ' " Nature 197, 21. RANDERATH, K. 1963 Press 200-204. T h i n - l a y e r Chromatography. Academic 22. RANDERATH, K. Press 188. 1963 T h i n - l a y e r Chromatography. Academic 23. RAUEN, H. M. Verlag. 720. 24. REICHARD, P. 1962 Enzymatic Synthesis o f Deoxyribonucleotides. J . o f B i o l . Chem. 237(11) 3513~19. 25. REISSIG, J . L., STROMINGER, J . L. and LELOIR, L. F. 1955 A m o d i f i e d C o l o r i m e t r i c Method f o r the e s t i m a t i o n o f NA c e t y l A m i n o Sugars. J . o f B i o l . Chem. V o l . 217, 959"966. 26. RHODES, A. 'J. and VAN R00YEN, C. E . 1962 Textbook of V i r o l o g y . F o u r t h E d i t i o n . The W i l l i a m s and W i l k i n s Company. 42-46. 27. RHODES, A. J . and VAN ROOYEN, C. E. 1962 Textbook o f V i r o l o g y , F o u r t h E d i t i o n . The W i l l i a m s and W i l k i n s Company. 1956 Biochemisches Taschenbuch - S p r i n g e r - 53-56. 28. ROTH, J . S. and BUCCIN0, G. 1963 Some Aspects o f Deoxyr i b o n u c l e o t i d e Metabolism i n the developing c h i c k embryo. A r c h i v e s of Biochem. and Biophys. 101, 5 1 6 - 5 2 2 . 29. ROIZMAN, B., HOGGAN, M. D. and CORNFIELD, J . i 9 6 0 Linear and P a r a b o l i c Estimates o f the t i t r e s o f Herpes simplex from Pock counts on the CAM o f embryonated eggs. V i r o l o g y 11, 5 7 2 . 47 30. SEKIGUCHI, T., KILAMURA, T. and SANO, Y. 1963 B i o c h e m i c a l S t u d i e s on V a c c i n i a V i r u s i n f e c t i o n i n HeLa c e l l s . J . of Biochem. 5 3 ( 6 ) 4 5 3 - 4 6 0 . 31. STAEHELIN, M. I 9 6 I S e p a r a t i o n o f O l i g o n u c l e o t i d e s on DEAE C e l l u l o s e . Biochim e t Biophys a c t a 4 9 , 11-19. 32. SUNDERLAND, D. N . , and MERRETT, M. J . 1964 Adenosine Diphosphate and Adenosine Triphosphate Concentrations i n l e a v e s showing N e c r o t i c l e s i o n s . V i r o l o g y 2 3 , 2 7 4 . 33. TOMLINSON, R. V. and TENER, G. M. 1963 The e f f e c t o f Urea, Pormamide and G l y c o l s on the secondary b i n d i n g f o r c e s i n Ion-Exchange Chromatograph o f P o l y n u c l e o t i d e s on DEAEC e l l u l o s e . Biochemistry 2, 697"702. 34. TYRELL, D. A. and KLEMPERER, H. G. 1959 p l i c a t i o n i n the l i g h t o f new techniques. B u l l e t i n 15 (3) 1 8 9 - 1 9 1 . 35. WESTWOOD, J . C. N. 1959 T i s s u e C u l t u r e i n R e l a t i o n t o V i r u s e s . B r i t i s h M e d i c a l B u l l e t i n 15 (3) 181-184. 36. WYLTE, V. and SMITH, M. 1964 N u c l e o t i d e s o f the P a c i f i c Oyster C r a s s o s t r e a Gigas (Thunberg) Canadian J . o f B i o chem. 4 2 ( 9 ) 1347-1351. Virus MultiB r i t i s h Medical
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UBC Theses and Dissertations
Changes in the level of free nucleotides of vaccinia infected chorioallantoic membrane of the chick embryo… Wylie, Vivian 1964
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