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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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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.  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