UBC Theses and Dissertations

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UBC Theses and Dissertations

Nucleic acid metabolism in rat intestinal mucosa Flanagan, Mary Louise 1969

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N U C L E I C ACID METABOLISM I N ' H A T I N T E S T I N A L MUCOSA  by Mary L o u i s e F l a n a g a n B.Sc.", U n i v e r s i t y o f B r i t i s h  C o l u m b i a , 1967  A T H E S I S SUBMITTED I N P A R T I A L FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  In t h e Department of Biochemistry  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e required standard f o rthe degree.of MASTER OF SCIENCE  The U n i v e r s i t y  of British  December 1969  Columbia  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the L i b r a r y s h a l l I  make i t f r e e l y a v a i l a b l e  f u r t h e r agree tha  permission  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f t h i s  that  study. thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s of  this  written  representatives. thesis  It  i s understood that copying o r p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  permission.  Department The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Columbia  not be allowed without my  i  ;  ABSTRACT  The  i n vivo  synthesis  p r e c u r s o r s was s t u d i e d to e l u c i d a t e  .  of. d e o x y r i b o n u c l e i c a c i d  i n the r a t i n t e s t i n a l  from  labelled  m u c o s a i n an a t t e m p t  t h e c o m p l e x p r o c e s s o f DNA r e p l i c a t i o n .  I n one s e t  3 of experiments, starved  t h e r a t s were i n j e c t e d  f o r 24 h o u r s ,  i n which  time  with  H-thymidine  the stable  and t h e n  DNA became  14 labelled with tritium.  C-T:hymidine was t h e n a d m i n i s t e r e d a n d  the  a n i m a l s were s a c r i f i c e d  5 minutes  the  n e w l y s y n t h e s i z e d DNA was l a b e l l e d  later.  By t h i s  procedure.  14  The  DNA, was f r a c t i o n a t e d  albumin k i e s e l g u h r w i t h a sodium  column.  chloride  with  C.  by chromatography  Only  solution  one m a i n p e a k o f DNA was  was  from t h i s  calculated:,  graphy, fraction  In  and t h e G + C c o n t e n t  W i t h i n t h e DNA p e a k o b t a i n e d f r o m MAK o f t h e DNA d e c r e a s e d w i t h  addition  t o these differences i n metabolic a c t i v i t y  were i n d i c a t e d  by t h e i r  o f t h e DNA f r a c t i o n s  f r o m MAK  fraction  f o r t h e DNA i n  chromato-  increasing  number.  were d i f f e r e n c e s which  p e a k was d e t e r m i n e d  the G + C'content  eluted  r a n g i n g i n c o n c e n t r a t i o n from  0 . 5 - 0 . 6 ? .M.The t h e r m a l d e n a t u r a t i o n t e m p e r a t u r e each.fraction  on a m e t h y l a t e d -  3  H/  i n base  composition, there  between t h e f r a c t i o n s ,  14  C ratios.  chromatography  number t o a maximum a t f r a c t i o n  The  3  H/  14  increased  C  ratio  with  4 o r 5 and t h e n  decreased.  3 I t was f o u n d t h a t constant,  thus  distributed between t h e  the  H/O.D. r a t i o  o f t h e f r a c t i o n s was n o t  s u g g e s t i n g • t h a t the t r i t i u m might  throughout  the fractions.  and ^ C - t h y m i d i n e  be u n e v e n l y  I f the time  injections  interval  was r e d u c e d  to 3  1/2  3 H/O.D. r a t i o became c o n s t a n t w h i l e t h e p a t t e r n o f  hours, the  •^H/ C r a t i o s  remained unchanged.  14  I f ^ C - t h y m i d i n e was  administered  3 20  minutes before  of  the  DNA  the  f r a c t i o n s f r o m MAK  increasing  fraction  number.  that' s m a l l m o l e c u l a r highly the  labelled with  high molecular  with  3  3  these  From t h e s e  C,  was  weight,  C  being  DNA,  incorporated  s t a b l e DNA  ratio  with  r e s u l t s i t was  weight, newly s y n t h e s i z e d 1 4  H/  chromatography i n c r e a s e d  concluded  which  with  was  time  f r a c t i o n , which i s  experiments  i t was  observed  that  14  the  •  H/  C ratio  versus  treatment given radioactive to  the  14  into labelled  H. During  of  a n i m a l s were s a c r i f i c e d ,  the  obtain  fraction  t o t h e DNA  counting.  its  I f the  v a l u e , and  water, s m a l l m o l e c u l a r  .  number v a r i e d a c c o r d i n g  sample p r i o r  pattern to  the  to the'preparation  for  s a m p l e was  d e n a t u r e d by  then/dialyzed". a g a i n s t  weight nucleotides  heating  distifled '  passed  into  the  dialysate. The the  d e n a t u r e d DNA  n a t i v e DNA  terase.  sample  s a m p l e on  On' the  denatured  a l s o gave d i f f e r e n t  digestion with sample, the  14 and  s n a k e venom p h o s p h o d i e s -  pattern  of r e l e a s e of  3  H  4  C  labelled material  indicated  that both these  along the DNA •' DNA  r e s u l t s from  chain.  s a m p l e s , the  i n t o the  l a b e l s were u n i f o r m l y  that  C labelled  expected  ^ C - p r e f e r e x i t i a l l y i n t o the The s e p a r a t i o n ' o f t h e  '  distributed  On t h e o t h e r h a n d , w i t h t h e n a t i v e 5 m i n . 14  r e l e a s e of  s o l u b l e f r a c t i o n was  acid soluble material,  material  f o r DNA  i n t o the  w h i c h had  incorporated  3* t e r m i n a l p o s i t i o n s . p y r i m i d i n e c l u s t e r s o f DNA  that t h o s e were n o t u n i f o r m l y  labelled  with  14  C and  acid  3  indicated H.  iii  "ACKNOWLEDGEMENTS  The  author wishes  appreciation  t o express her sincere  t o D r . S. H. Z b a r s k y  thanks and  for his continual  a d v i c e and  encouragement.  It  i s a p l e a s u r e t o acknowledge t h e p e r s o n a l a s s i s t a n c e  of t h e N a t i o n a l Council  Research C o u n c i l  and t h e M e d i c a l  i n t h e form o f S t u d e n t s h i p s .  cO  Research  iv TABLE OF CONTENTS Page INTRODUCTION  1  E a r l y Experiments  1  DNA R e p l i c a t i o n  5  Terminal  8  Incorporation of Nucleotides  I s o l a t i o n o f DNA The  12  P h y s i c a l and C h e m i c a l  Heterogeneity  o f DNA  ......  Metabolic Heterogeneity The  Present  15 •  Investigation  23,  MATERIALS AND METHODS Administration  . of Labelled Material  I s o l a t i o n o f DNA  from  solution  on  Albumin K i e s e l g u h r  26  Methylated . ..  28 •  Thermal D e n a t u r a t i o n  Curves  30  Radioactive Counting  Procedures  31  EXPERIMENTAL .  26 26  R a t I n t e s t i n a l Mucosa  C h r o m a t o g r a p h y o f t h e DNA  19  '  36 <P  Administration  of L a b e l l e d Thymidine P r e c u r s o r s  Characterization  of the Isolated  Chromatography o f t h e I s o l a t e d Methylated-Albumin  DNA  36 37  DNA o n  Kieselguhr  Thermal D e n a t u r a t i o n  .....  Curves  R a d i o a c t i v e C o u n t i n g o f D o u b l y - L a b e l l e d DNA D e g r a d a t i o n o f t h e I s o l a t e d DNA.by Snake Venom Phosphodiesterase The P u r i f i c a t i o n and C o u n t i n g o f t h e I n t e r p h a s e Layers o f the Tissue Extracts  38 39 44 64 70  V  • I s o l a t i o n and C h a r a c t e r i z a t i o n Pyrimidine Isostichs  SUMMARY  BIBLIOGRAPHY  Page of the ••••  •  «...  •••  70  7  9  83  vi  TABLES Page  I.  II.  III.  IV.  V.  VI.  VII.  T v a l u e s and G-C c o n t e n t o f s e p a r a t e o r t h e m a i n peak a f t e r c h r o m a t o g r a p h y DNA f r o m i n t e s t i n a l mucosa  fractions of 44  The r a t i o o f r a d i o a c t i v i t y due t o " ^ H / O p t i c a l D e n s i t y o f the DNA f r a c t i o n s o b t a i n e d from^ MAK C h r o m a t o g r a p h y i n e x p e r i m e n t s i n w h i c h Ht h y m i d i n e was a d m i n i s t e r e d t o r a t s 24 h o u r s b e f o r e the i n j e c t i o n o f C-thymidine  ^6  E f f e c t o f v a r i o u s t r e a t m e n t s on t h e r a d i o a c t i v i t y and a b s o r b a n c e o f a s o l u t i o n o f d o u b l y - l a b e l l e d r a t i n t e s t i n e DNA i n s a l i n e c i t r a t e  57  Radioactivity and a b s o r b a n c y a f t e r d i a l y s i s a g a i n s t d i s t i l l e d water o f f r a c t i o n s o f d o u b l y - l a b e l l e d D N A o b t a i n e d by chromatography on M A K . . .  58  Radioactivity and a b s o r b a n c y a f t e r h e a t i n g and then d i a l y s i s a g a i n s t d i s t i l l e d water o f • f r a c t i o n s o f d o u b l e — l a b e l l e d rDNA o b t a i n e d by c h r o m a t o g r a p h y on M A K  59  Radioactivity and a b s o r b a n c y o f t h e p y r i m i d i n e i s o s t i c h s i s o l a t e d by c h r o m a t o g r a p h y on . DEAE-Cellulose of the formic acid-diphenylamine h y d r o l y s a t e o f m u c o s a l D N A , w h i c h was a l l o w e d to.incorporate H f o r 3 1 / 2 h o u r s and C for 5 minutes Radioactivity and a b s o r b a n c y o f t h e p y r i m i d i n e i s o s t i c h s i s o l a t e d by c h r o m a t o g r a p h y on DEAE-Cellulose of the formic a c i d - diphenylamine h y d r o l y s a t e o f m u c o s a l D N A w h i c h was a l l o w e d to incorporate H f o r 3 1 / 2 h o u r s and C for 10 m i n u t e s . •  ..  74-  .  ?7  vii  FIGURES Page  1. '.L--: KornBerg*s .hodel of DNA.' replication ...•.».<•<• 2.  Suggested mechanism f o r t e r m i n a l o f n u c l e o t i d e s I n t o DNA  3. .  4.  5.  6.  8.  9.  incorporation  9 11  Integral discriminator bias curves f o r s t a n d a r d H ( H" 0) and C (Na„ C 0 ) obtained from the r e d s c a l e r o f the Packard T r i Carb 314 AX L i q u i d S c i n t i l l a t o r S p e c t r o p h o t o m e t e r 2  3  Integral discriminator bias curves for standard H and C d e t e r m i n e d on t h e g r e e n s c a l e r o f t h e P a c k a r d T r i C a r b 3 1 4 AX L i q u i d S c i n t i l l a t o r Spectrophotometer. The c u r v e s r e p r e s e n t t h e a v e r a g e o f t h e d e t e r m i n a t i o n o f s i x d i f f e r e n t window s e t t i n g s .  ..  C h r o m a t o g r a p h y o f DNA f r o m r a t i n t e s t i n a l - mucosa on a MAK c o l u m n . E l u t i o n was c a r r i e d out w i t h a g r a d i e n t o f N a C l i n 0.05 M p h o s p h a t e b u f f e r , pH 6 . 7  40  E f f e c t o f i n c r e a s i n g t e m p e r a t u r e on t h e o p t i c a l density of doubly-labelled r a t i n t e s t i n a l m u c o s a l DNA.  43  3 7.  ?••  14  Ratio of H cpm/ C cpm i n f r a c t i o n s c o m p r i s i n g t h e DNA p e a k e l u t e d by c h r o m a t o g r a p h y o f d o u b l y l a b e l l e d m u c o s a l DNA, w h i c h h a d b e e n a l l o w e d t o incorporate H f o r 24 h o u r s , on MAK c o l u m n s C h r o m a t o g r a p h y o f m u c o s a l DNA, J a b e l l e d w i t h - t h y m i d i n e f o r 3 1 / 2 h o u r s and C-thymidine f o r 5 m i n u t e s , on an MAK c o l u m n Chromatography o f mucosal D N A , ^ a b e l l e d wi t h t h y m i d i n e f o r 3 1 / 2 h o u r s and C-thymidine for 10 m i n u t e s , on an MAK c o l u m n  45  3  H ... 3  48  H4-9  3 •10.  11.  The r a t i o o f r a d i o a c t i v i t y due t o H/optical d e n s i t y o f t h e DNA f r a c t i o n s o b t a i n e d f r o m MAK c h r o m a t o g r a p h y i n e x p e r i m e n t s i n w h i c h H - t h y m i d i n e was a d m i n i s t e r e d t o r a t s 3 1 / 2 h o u r s b e f o r e i n j e c t i o n o f """^C-thymidine. The DNA was  ratio of H/ C i n f r a c t i o n s c o m p r i s i n g the p e a k e l u t e d b y ^ c h r o m a t o g r a p h y o f DNA w h i c h labelled with H-thymidine f o r 3 1/2 hours.  50  Vlll Page 12.  3  14  The r a t i o o f H/ C i n f r a c t i o n s comprising the DNA peak e l u t e d b y ^ c h r o m a t o g r a p h y - o f DIjIA w h i c h was l a b e l l e d w i t h H f o r 24 h o u r s and C for 20 m i n u t e s . The c o m p a r i s o n i s made between ;p f r a c t i o n s t h a t a r e d e n a t u r e d by h e a t i n g ( c h r o m a t o g r a p h y I b e f o r e r a d i a t i o n c o u n t i n g and t h o s e w h i c h h a d no t r e a t m e n t p r i o r t o c o u n t i n g <;  13.  C h r o m a t o g r a p h y on D E A E - C e l l u l o s e o f t h e d i s t i l l e d w a t e r d i a l y s a t e o f t h e m u c o s a l DNA t h a t h a d b e e n d e n a t u r e d by h e a t i n g p r i o r t o dialysis. E l u t i o n was c a r r i e d o u t w i t h a g r a d i e n t o f N a C l i n 7M u r e a - T r i s HC1.  pH 7. 8 14.  1'5.  16.  55  t  .  62  Release of u l t r a v i o l e t absorbing material and r a d i o a c t i v i t y by d i g e s t i o n o f d o u b l y l a b e l l e d m u c o s a l DNA w i t h s n a k e venom phosphodiesterase  67 (a)  C h r o m a t o g r a p h y on D E A E - c e l l u l o s e o f the diphenylamine-formic a c i d hydrolysate o f DNA, w h i c h had b ^ | n l a b e l l e d w i t h H for 3 1/2 h o u r s and C f o r 5 minutes. E l u t i o n was c a r r i e d o u t w i t h a g r a d i e n t o f L i C l i n l i t h u i m a c e t a t e - pH .5.3  75  C h r o m a t o g r a p h y on D E A E - c e l l u l o s e o f t h e diphenylamine-formic acid hydrolysatepf DNA, w h i c h h a d begn l a b e l l e d w i t h H for 3 1/2 h o u r s and C f o r 10 m i n u t e s . E l u t i o n was c a r r i e d o u t w i t h a g r a d i e n t of L i C l i n l i t h u i m a c e t a t e - pH 5.3 .  76  ix • L I S T OF ABBREVIATIONS DNA  deoxyribonucleic  acid  5 min.DNA  DNA w h i c h was a l l o w e d thymidine f o r 5 min.  to.incorporate  ^ C~  10 m i n . DNA  DNA w h i c h was a l l o w e d t o i n c o r p o r a t e t h y m i d i n e f o r 10 m i n .  ^" C~  20 m i n , DNA  DNA w h i c h was a l l o w e d t o i n c o r p o r a t e "*"Ct h y m i d i n e f o r 20 m i n .  A  adenine  G  guanine  C  cytosine  T  thymine a c h r o m a t o g r a p h i c column albumin kieselguhr.  mCi  millicurie  umole.  micromole  nm  nanometer  O.D. p p  O  -dimethyl  4  4  MAK  cpm  4  of  methylated-  " c o u n t s per. m i n u t e optical 2\6^d'iphehyl POPOP  CME-carbodiimide  density oxazole  _-  l,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene N-cyclohexyl-'N-[4-methylmorpholinum) •ethylcarbodiimide  1  INTRODUCTION The  l a s t twenty y e a r s have seen f u l l  r e a l i z a t i o n o f the  great b i o l o g i c a l importance o f the n u c l e i c acids^, f i r s t i s o l a t e d by Miescher  (1) i n t h e 1870's and i n c r e a s i n g knowledge o f the  chemical and p h y s i c a l p r o p e r t i e s o f d e o x y r i b o n u c l e i c  a c i d has  strengthened the e a r l y b e l i e f t h a t DNA was the t r a n s m i t t e r o f genetic information.  The u n r a v e l l i n g o f the complex process o f  h e r e d i t y has been made p o s s i b l e as a r e s u l t o f the i d e n t i f i c a t i o n of the s t r u c t u r e and f u n c t i o n s o f DNA. The  s t r u c t u r e s o f the n u c l e o t i d e  determined by chemical means postulated  components had been  (2) when Watson and Crick,  a double h e l i c a l s t r u c t u r e f o r DNA i n 1952 ( 3 ) .  From t h i s model, a s e m i - c o n s e r v a t i v e  mechanism o f r e p l i c a t i o n  was proposed i n which each s t r a n d o f the double h e l i x a c t s as a template f o r the formation  o f a new s t r a n d .  developed, f u r t h e r i n f o r m a t i o n  While  this  was a v a i l a b l e which was p o i n t i n g  t o the r o l e o f DNA i n h e r e d i t y , which had been suspected because o f the m e t a b o l i c s t a b i l i t y and c e l l u l a r l o c a t i o n o f t h i s nucleic acid.  Three types o f experiments  f u n c t i o n o f DNA i n h e r e d i t y .  First/  confirmed.this  the h i g h e s t r a t e o f  mutations i n b a c t e r i a induced by u l t r a v i o l e t l i g h t o c c u r r e d a t wave lengths acids capsule  (4).  c o r r e s p o n d i n g t o the a b s o r p t i o n Second/ Avery e t a l .  forming s t r a i n  (5) e x t r a c t e d DNA from a  Cs) o f pneumococcus, added i t t o a  c u l t u r e o f a non-capsule forming s t r a i n l a t t e r had a c q u i r e d  maxima o f n u c l e i c  (R) and found t h a t the  the a b i l i t y t o form c a p s u l e s .  Third,  2  H e r s h e y and. C h a s e protein and  ( 6 ) i n f e c t e d b a c t e r i a w i t h phage,  o f w h i c h was  studied  concluded  the  d i s t r i b u t i o n of  that  only  35  l a b e l l e d with  the  DNA  of  the the  S and  the  isotope  DNA  the 32  with  in bacteria.  phage e n t e r e d  P, They  the b a c t e r i a l  cell. E x p e r i m e n t s - i n DNA 1940  by 32 ">P  of  von  Euler  and  m e t a b o l i s m were done as  von  (7),  Hevesy  who  early  measured the  as uptake  i n t o DNA  in vivo. T h e r e was a p o s i t i v e c o r r e l a t i o n 32 between the uptake o f P by a t i s s u e and i t s m i t o t i c a c t i v i t y , 32 suggesting t h a t the uptake of P o c c u r r e d as a r e s u l t o f • 32 synthesis  of  DNA  which  uptake obtained,  however, i n d i c a t e d t h a t  b e i n g . - p r o d u c e d as mass, and  This  i s -a v e r y 32  stable  P due  w o u l d be  therefore  turnover.  i n DNA  was  accompanied m i t o s i s .  DNA  was  cell  t o the  the  As 32  large  terminal  t o have a  P u p t a k e was  (8)  that  thought  as w e l l  Schoenheimer  v/as  in tissue  the.total activity  nucleotides  P  continuous  b i o s y n t h e t i c mechanism can  e x p e r i m e n t s o f B a r n e s and  of  much DNA  increase  f i n d i n g of Brues  constituent.  constant,  i n the  the  values  t w i c e , as  from the  considered  contradicted  t o exchanges i n the  synthesis. Interest  expected  The  be  (9).  as  DNA of  the  to  be  to  traced They  net  back  found  15 ' that  N  i n ammonium c i t r a t e  urea,, h i s t i d i n e and purine  synthesis.  arg i i i n e Since  appeared are  not  then, the  has  been w o r k e d o u t  for purines  by  and  for pyrimidines  by  and  Reichard  i n nucleic acids d i r e c t precursors  and in  complete b i o s y n t h e t i c B u c h a n a n and co-workers  scheme  colleagues (10).  that  This  (10) has  a l s o been f o l l o w e d by s t u d i e s on the i n c o r p o r a t i o n of and n u c l e o t i d e s i n t o p o l y n u c l e o t i d e s t r u c t u r e s . i n v e s t i g a t i o n s , thymidine i t occurs i n DNA  1,  3 -  great extent i n  RNA.  d e t e c t e d a s m a l l i n c o r p o r a t i o n of thymine  i n t o the p o l y n u c l e o t i d e thymine component of  regenerating (12).  these  has been s t u d i e d e x t e n s i v e l y because  and not t o any  Brown (11)  In  nucleosides  On  rat liver.  T h i s r e s u l t was  the o t h e r hand, P l e n t l and  w i t h 'other workers found acid precursor.  a l s o o b t a i n e d by Adams'  Schoenheimer  (13)  along  thymine to be i n e f f e c t i v e as a n u c l e i c  JiThyihine.- i s degraded to R-amino i s o b u t y r i c  a c i d which i s n o t i n c o r p o r a t e d i n t o DNA  (10)..  U n l i k e thymine, the n u c l e o s i d e thymidine  can be  utilized  because thymine i s degraded b e f o r e i t can be used and t i s s u e s have the n e c e s s a r y thymidine  The  enzymes f o r phosphoryla.ting thymidine  t r i p h o s p h a t e which i s the p r e c u r s o r f o r DNA.  Enzymatic S y n t h e s i s of  to (10)/  DNA  A f t e r the n u c l e i c a c i d p r e c u r s o r s were i d e n t i f i e d r a d i o a c t i v e t r a c e r s , the next s y n t h e s i s was  s t e p i n the e l u c i d a t i o n o f  t h e i s o l a t i o n of the enzymes i n v o l v e d .  P u r i f i e d enzymes, the r e a c t i o n was  determined  by DNA  Using  (15) as:  a)  the. b i o s y n t h e s i s o f p u r i n e and p y r i m i d i n e r i b o n u c l e o s i d e monophosphates b)  the p h o s p h o r y l a t i o n of these monophosphates  t o the diphosphate  stage and the c o n v e r s i o n o f these r i b o n u c l e o -  t i d e s t o the c o r r e s p o n d i n g  deoxyribonucleotides.  p h o s p h o r y l a t i o n o f the d e o x y r i b o n u c l e o s i d e  c)  the  diphosphates  to the  4 triphosphate stage and d) the polymerization of the deoxyribonucleoside tides.  triphosphates to give  deoxyribopolynucleo-  This l a s t polymerization can only take place i n the  presence of an appropriate DNA primer. The complex processes involved i n t h i s l a s t step have been c l a r i f i e d by the i s o l a t i o n of DNA polymerase from E_. c o l i by Kornberg (15). deoxyribonucleoside  He noted that, i n the presence of the four triphosphates, a p u r i f i e d enzyme preparation  catalyzes the r e p l i c a t i o n of a DNA primer.  The following  reaction summarizes t h i s process: n dTP'PP m dG PPP n dA PPP  dTP Mg  +  +  +  D  N  A  P  r i m e r  m dC PPP  dGP  ++  >  D  N  polymerase  A  -  dAP  + 2 (m+n)  dCP  m+n  The r a t i o of the amount of adenine plus thymine to A +T guanine plus cytosine (^  +  ^) i n the newly synthesized DNA i s  equal t o that of the primer.  Varying the r e l a t i v e q u a n t i t i e s  of the four precursors does not influence the composition of the newly formed DNA (16).  Unnatural bases can be incorporated  i n t o the new DNA e.g. 5-hydroxymethyl cytosine f o r cytosine, u r a c i l or 5-bromouracil  f o r thymine and hypoxanthine f o r  guanine, provided these are i n the system as deoxyribose triphosphates in.the presence of the other nucleoside t r i p h o sphates.  Therefore, the DNA polymerase catalyzes the b i o -  s;yrthesis of DNA, but the s p e c i f i c i t y , with regards to the base sequence o f the newly formed DNA molecule,  i s determined  5  by the DNA  primer.  phosphorylate  S p e c i f i c k i n a s e s are p r e s e n t which  o n l y the c o r r e c t bases to the  deoxyriboside  t r i p h o s p h a t e l e v e l and they determine' t h a t the newly-formed DNA  always has the i d e n t i c a l base sequence as the primer  The DNA  polymerase can r e c o g n i z e the p o s i t i o n and s t r u c t u r e of  t h e pentose phosphate m o i e t i e s of the t r i p h o s p h a t e s , but not the v a r i o u s ^  (16).  I f n a t i v e h e l i c a l DNA  p u r i f i e d enzyme, the product f o r i t s unusual denaturation  deoxyribonucleoside  bases.  i s used as a template  with  i s s i m i l a r t o n a t i v e DNA  except  c a p a c i t y to resume a h e l i c a l conformation  (16).  The product  a l s o has  after  a branched s t r u c t u r e  when examined w i t h an e l e c t r o n microscope. C o v a l e n t l y bound to the template  the  New  s t r a n d s are not  and can i n t e r n a l l y hydrogen-  bond t o form a h a i r p i n or p l e a t e d s t r u c t u r e (16).  DNA R e p l i c a t i o n B a s i c a l l y t h e r e are t h r e e p o s s i b l e methods o f r e p l i c a t i o n : a) c o n s e r v a t i v e , i n which the parent molecule  remains i n t a c t  and one e n t i r e newly-formed daughter molecule  i s formed,  s e m i - c o n s e r v a t i v e , i n which one f r o m t h e parent molecule  b)  s t r a n d o f each c h a i n o r i g i n a t e s  and the o t h e r i s newly s y n t h e s i z e d ,  c) d i s p e r s i v e i n which p a r e n t a l and o f f s p r i n g components a r i s e f r o m p a r t s o f a l l DNA  molecules.  The s e m i - c o n s e r v a t i v e method was Crick t h e two  (3).  suggested  by Watson and  R e p l i c a t i o n c o u l d be achieved by the unwinding of  s t r a n d s w i t h c o n c u r r e n t attachment o f the complementary  6 deoxynucleoside one  cycle  of  identical of t h e  replication,  composition  original  Supported t h i s were  isolated and  there  and sequence,  theory.  ^ N.  DNA  After  molecules  DNA  containing  each  until  twice  cycle  in  of  was p l a c e d  further  labelled  a culture  in a  the  DNA  s o l u t i o n of  CsCl  15  reached.  down t h e  with  medium  replication,  e q u i l i b r i u m was  travelled  was  DNA  (17)  The  • N  centrifuge  -  tube  than  14 the and  14 N - labelled  o r h y b r i d DNA  DNA  Thus t h e  ^N.  identified  three  because  types  of  DNA  of d i f f e r e n c e s  which contained c o u l d be  in their  they c a l c u l a t e d t h a t molecules  after  contained  two c y c l e s  15 50%  of  N a n d 50%  both  separated  buoyant  From t h e d a t a o b t a i n e d f r o m t h e d e n s i t y g r a d i e n t  DNA  of  strand  one  and Stahl  of Meselson  replicate  from these b a c t e r i a  labelled  two  each  B a c t e r i a , whose  After  4  centrifuged  strand.  parental  w o u l d be  experiment  allowed to  containing  to each  DNA.  classical  The  ^N,  triphosphates  equilibration, half  while the  N,  and  densities.  replication,  14  N  the  other  14 half' c o n t a i n e d o n l y  N.  These  a semi-conservative method  of  results  would  be  DNA  found i n bacteria,  viruses,  replicate  and e l e c t r o n  in a medium c o n t a i n i n g  photoradiographic tritium electron  microscopy.  reacted  to  emulsion. produce  micrographs  of  The dark  this  3  H  -  spots  to  the  autoradiography  Bacteria were  allowed  thymidine i n contact  0 particles  process  theory  a n d r a t ' . l i v e r - • m i t o c h o n d r i a (IE  Cairns (19) a p p r o a c h e d t h e p r o b l e m b y u s i n g techniques  for  replication.  There i s a d i f f i c u l t y i n a p p l y i n g t h i s circular  predicted  i n the  emitted  from  emulsion.  indicated  that  to with the  The  replication  7 o f the molecule s t a r t e d a t one p o i n t i n the c i r c l e . formation  The  o f 2 new DNA s t r a n d s r e s u l t e d i n a f o r k , w i t h  o f the f o r k b e i n g  joined together.  the ends  R e p l i c a t i o n proceeded  around the molecule u n t i l two daughter DNA m o l e c u l e s had been formed. Dounce  (20) c a l c u l a t e d the r a t e a t which the DNA molecule  must unwind i n o r d e r f o r the e n t i r e molecule t o r e p l i c a t e w i t h i n the time i n t e r v a l o f DNA r e p l i c a t i o n . e n e r g e t i c a l l y unfavourable a l t e r n a t e mechanism.  T h i s r a t e seemed  and he t h e r e f o r e proposed an  He suggested t h a t o n l y one s t r a n d o f the  molecule i s c o p i e d , the other  s t r a n d being prevented  from  doing  so by a h i s t o n e , a b a s i c p r o t e i n which i s known t o b i n d t o DNA i n the n u c l e u s .  Butler  (21) had e a r l i e r proposed a mechanism  t o account f o r the unwinding o f the DNA h e l i x .  He p i c t u r e d the  DNA polymerase as a d i s k w i t h two s l o t s , one f o r each o f the DNA primer s t r a n d s . end  The d i s k would a t t a c h i t s e l f t o a l o o s e  o f t h e molecule w i t h  the end o f one s t r a n d i n each h o l e .  As t h e complementary n u c l e o t i d e a t t a c h e s  t o t h e growing c h a i n ,  the d i s k moves along the DNA h e l i x , r o t a t i n g as i t p r o g r e s s e s , c a u s i n g the double h e l i x t o unwind. I n c o n t r a s t t o these  t h e o r i e s , C a v a l i e r i and Rosenberg  (22) proposed a mechanism/ which i s not w i d e l y t h e r e i s no s t r a n d  h e l d , i n which  separation.  Kornberg and h i s c o l l e a g u e s  (23) r e c e n t l y have suggested  a model f o r DNA polymerase by which i t i s e x p l a i n e d how the enzyme can achieve  r e p l i c a t i o n o f both s t r a n d s even though the  8  enzyme operates  o n l y i n the 5 '  > 3  1  direction.  a d d i t i o n to i t s r e p l i c a t i v e a c t i v i t y , the DNA  polymerase i s  b e l i e v e d to have a degree of h y d r o l y t i c a c t i v i t y . DNA  In  Thus the  polymerase c o u l d c a t a l y z e the i n t r o d u c t i o n of a n i c k i n  the DNA  c h a i n , which would i n i t i a t e r e p l i c a t i o n .  would then proceed by c o v a l e n t e x t e n s i o n 5 ' end may  of the 3'OH  be degraded to some e x t e n t by a 5  a c t i o n or may  Replication  >3'  1  a t t a c h t o some membrane s i t e .  end.  The  nuclease  After replication  has proceeded f o r a c e r t a i n d i s t a n c e , i t switches  to  the  complementary s t r a n d as a template to form a fork., which i s then c l e a v e d by an endonuclease.  A r e p e t i t i o n o f t h i s sequence l e a d s  to i n t e r r u p t i o n s or s m a l l p i e c e s of DNA fork.  L i g a s e c o u l d then s e a l these  near the . r e p l i c a t i n g  interruptions.  Rather  than a simultaneous s e q u e n t i a l r e p l i c a t i o n o f b o t h s t r a n d s ,  this  scheme i m p l i e s a r e p l i c a t i o n which i s s t a g g e r e d , a l t e r n a t i n g from one  s t r a n d t o the other as i l l u s t r a t e d  .Terminal  I n c o r p o r a t i o n of  t h e r e was  t h a t i f one  of  the product  t h e r e was  polymerase a c t i o n , A d l e r e t a l .  of the d e o x y r i b o n i i c l e o t i d e s was  o n l y l i m i t e d i n c o r p o r a t i o n of the r e m a i n i n g  i n t o the primer.  Degradation  by snake venom  lacking, nucleotides  phosphodiesterase;  formed under these c o n d i t i o n s suggested t h a t  a l i m i t e d i n c o r p o r a t i o n of one  i n t o the 3'OH  1.  Nucleotides  In e a r l y s t u d i e s of DNA (24).noted  i n Figure  or two  nucleotides  end.  Krakow e t a l . (25)  i s o l a t e d an enzyme from c a l f thymus  n u c l e i which c a t a l y z e s the t e r m i n a l i n c o r p o r a t i o n of n u c l e o t i d e s  9.  Nicked reckon  Covalent extension  Formation oP Pork  & nuclease cleavage F i g . 1. The mechanism of a c t i o n of DNA polymerase i n DNA r.epli6atIon according t o Korriberg (23).  10 i n t o the p r i m e r .  T h i s enzyme has been c a l l e d t e r m i n a l  nucleo-  t i d y l t r a n s f e r a s e and r e q u i r e s heat denatured DNA as a primer, Mg  i o n s and c y s t e i n e .  Keir  enzyme i s a c a t a l y t i c subunit  (26) suggested t h a t the t e r m i n a l o f the DNA polymerase because  of f i n d i n g s t h a t the t e r m i n a l enzyme has a lower weight and t h a t there are two o p t i m a l  Mg  + +  molecular  concentrations f o r  t e r m i n a l a d d i t i o n c a t a l y z e d by a p r e p a r a t i o n which  contains  both r e p l i c a t i v e and t e r m i n a l n u c l e o t i d y l t r a n s f e r a s e activities.  I t was suggested  d i f f e r e d from the s u b u n i t s  (26) t h a t the parent  enzyme  i n terms o f c o n f i g u r a t i o n and  d i s p o s i t i o n o f c o f a c t o r s and s u b s t r a t e s  a t the a c t i v e s i t e .  32 Studies  o f the d i s t r i b u t i o n o f  .following incubation with t o A d l e r e t a l . (24)  P i n nucleotides  the l a b e l l e d t r i p h o s p h a t e s  t h a t hydrogen bonding i s necessary f o r  t e r m i n a l i n c o r p o r a t i o n i f the two s t r a n d s As  suggested  I l l u s t r a t e d i n Figure  are o f d i f f e r e n t  lengths  2, the f i r s t n u c l e o t i d e i s c o v a l e n t l y  bonded t o the 3'OH end o f the s h o r t e r c h a i n a t a p o i n t which permits i t t o be hydrogen bonded t o i t s a p p r o p r i a t e t i d e i n the o p p o s i t e pairing principle.  strand according  nucleo-  t o the complementary base  Only one n u c l e o t i d e i s added i f the base  i n the p a i r i n g p o s i t i o n w i l l not hydrogen bond w i t h the base i n the o p p o s i t e  strand.  The f u n c t i o n o f t h i s l i m i t e d  incorporation  is.unknown, but i t may be t o r e p a i r the s h o r t e r s t r a n d o f a double h e l i x i n which the two s t r a n d s (24) .  '  are o f unequal  lengths  nucleotides into PNA.  Isolation  of  DNA  DNA  i s l o c a t e d mainly, i n the  i s bound t o p r o t e i n . nucleic acid  a c i d s by  and  then  shaking  the  pus  w h i c h c o u l d be isolated  in d i l u t e  insoluble  but  methods, p r o t e i n  cells  strength.  (27), which i s f o l l o w e d i n t h i s deoxycholate phenol  as  a detergent  t o denature  characterized  One  by  of  types of DNA  the  s e p a r a t i o n was and  The  saline  procedure,  (32)  a limited  separated  by  a e t al_.  utilizes  sodium  of which i s  be  purified  solubility (28)  and  techniques.  to separate  different  o f v a r i o u s DNA  or i n a l k a l i  differential  been used  (31).  (29).  s e p a r a t i o n o f DNA  Some  centrifugation Cesium  mainly  species i n  as  an  (30)  chloride a n a l y t i c a l :'  molecules  with  (17).  Fractionation had  can  h i s t o n e columns  since i t allows  different d e n s i t i e s  DNA  methods u s e d  has  day  alcohol/  method o f C o l t e r  investigation,  a l s o o b t a i n e d by  centrifugation  the  t o d i s r u p t membrane s t r u c t u r e s and  a t 85°C  c h r o m a t o g r a p h y on  gradient  has  the  give  In present  isoamyl  of chromatographic  earliest  From  i s precipitated  The  isolated  a variety  utilized  Physiological  acid.  p r o t e i n , most i m p o r t a n t  deoxyribonuclease.  ether to  off.  detergents,  solution  isolated  a c i d s which are s o l u b l e  in dilute  R i b o s o m a l RNA  first  with  filtered  chloroform or phenol.  where i t  with pepsin-hydrochloric  nucleic  i s r e m o v e d by  o f 1.0, bl i o n i c  (1)  d i g e s t e d mixture  n u c l e a r m a t e r i a l , he alkali  of cells.,  I n 1868-69, M i e s c h e r  digesting  a nuclear fraction,  nucleus  o f DNA  use.  single  by  counter  I n 1956, stranded  current  using this  from  double  distribution  procedure, stranded  DNA  Albertsson with  13 an e q u i r a o l a r N a H P 0 2  4  and N a H P 0 2  4  phosphate s o l u t i o n pH6.8-7.0  c o n t a i n i n g 4.4% p o l y e t h y l e n e g l y c o l and 7.0% d e x t r a n . Solution  With a  o f 0.7% sodium d e x t r a n s u l f a t e and 2% m e t h y l c e l l u l o s e '  i n O.B'M N a C l o r pH 7.0, F r i c k and L i f (33) o b s e r v e d  that the  p a r t i t i o n c o e f f i c i e n t between t h e phases o f a sodium d e x t r a n s u l f a t e - m e t h y l c e l l u l o s e aqueous system v a r i e d molecular  size of the nucleic acid.  w i t h the  The n a t u r e o f t h e s e p a r a t i o n •  i s n o t c l e a r , b u t DNA c o n t a i n i n g ,a h i g h p e r c e n t a g e o f adenine and thymine %s -'ca-rri-e'd in--' thev o r g a n i c pliase. 'Bendich- e t • al.".'- (: 4 8.?/  separated  the transforming  a c t i v i t i e s o f pneumococcal DNA on columns o f ECTEOLA c e l l u l o s e . The  t r a n s f o r m i n g a c t i v i t i e s were n o t r e s t r i c t e d t o any one  c h r o m a t o g r a p h i c peak o r r e g i o n , b u t were p r e s e n t i n s e v e r a l o f the DNA f r a c t i o n s , w h i c h had been o b t a i n e d by e l u t i o n w i t h solutions  o f w i d e l y v a r y i n g i o n i c s t r e n g t h and pH.  Rechromato-  graphy d i d n o t . g i v e r e p r o d u c i b l e r e s u l t s . F r a c t i o n a t i o n o f DNA on h y d r o x y a p a t i t e been v e r y s u c c e s s f u l .  columns has n o t  However, by v a r y i n g t h e c o n d i t i o n s o f •  f r a c t i o n a t i o n , McCallum r e p o r t e d in fractionation. resulted  (35) c o r r e s p o n d i n g d i f f e r e n c e s  S e p a r a t i o n a c c o r d i n g t o base  composition  f r o m t h e " t h e r m a l " chromatography o f n a t i v e DNA w i t h  an e l u t i n g . s o l u t i o n o f c o n s t a n t phosphate c o n c e n t r a t i o n . R e n a t u r e d DNA c o u l d be i s o l a t e d f r o m d e n a t u r e d  DNA by chromato-  graphy a t h i g h t e m p e r a t u r e s and u s i n g a s a l t g r a d i e n t i n t h e eluting solution. separated  A t low t e m p e r a t u r e s ,  denatured  DNA i s  f r o m n a t i v e o r r e n a t u r e d DNA due t o i t s a b i l i t y t o  14 form i n t r a m o l e c u l a r secondary s t r u c t u r e s . a b l e to f u r t h e r f r a c t i o n a t e n a t i v e DNA  on  Bernardi  (36) was  hydroxyapatite  Columns a c c o r d i n g t o d i f f e r e n c e s i n base composition  or b i o -  logical properties.  on  b a s i s o f molecular The  Some f r a c t i o n a t i o n was  achieved  b i n d i n g of DNA  to n i t r o c e l l u l o s e membrane f i l t e r s f o r the i s o l a t i o n of DNA-RNA h y b r i d s .  Oberg e t a l . (37) have r e p o r t e d the s e p a r a t i o n o f double f r o m s i n g l e stranded  can  DNA  by g e l f i l t r a t i o n  on  on columns o f s i l k  r e v e r s i b l e b i n d i n g o f DNA f r a c t i o n a t e DNA  fibroin  pearl-condensed  (38).  to polymethacrylate  a c c o r d i n g to base composition  DEAE-Cellulose  has  The been used to  (39).  i s used e x t e n s i v e l y f o r s e p a r a t i n g  o l i g o n u c l e o t i d e s r a t h e r than h i g h e r m o l e c u l a r More r e c e n t l y , Cerny e t a l . (40)  weight  separated  DNA pyrimidine  isostichs  on a D E A E - c e l l u l o s e column a c c o r d i n g to c h a i n  a t pH 5.3  and  a t pH  stranded  A s i m i l a r s e p a r a t i o n f o r l a r g e q u a n t i t i e s of m a t e r i a l  be o b t a i n e d  molecules.  the  size.  has been used p a r t i c u l a r l y  agarose.  not  a c c o r d i n g to base composition  length  on another column  3.1. The most w i d e l y  used column f o r s e p a r a t i n g n u c l e i c a c i d s  i s t h e methylated albumin - K i e s e l g u h r p r e p a r a t i o n and by Lerman (41).  use  of t h i s m a t e r i a l was  By m o d i f y i n g  the column, M a n d e l l and  (M.A.K.) column.  f i r s t described i n  1955  the m a t e r i a l s and p r e p a r a t i o n o f  Hershey  (42) were a b l e to f r a c t i o n a t e  n u c l e i c a c i d s a c c o r d i n g to t h e i r m o l e c u l a r shown t o c o n s i s t o f one  The  size.  or more l a r g e m o l e c u l e s o f  T_  DNA  was  identical  15 l e n g t h which  c o u l d be e x t r a c t e d by p h e n o l w i t h o u t  Other workers l a r g e r and  (43,44) h a d  s m a l l e r p i e c e s , but Hershey  s h e a r i n g t o t h e T' fragments  reported that T  DNA  and  DNA  2  breakage.  was  a mixture  (45) a p p l i e d a  i s o l a t e d w i t h an MAK  of  controlled  column,  w h i c h were a f r a c t i o n o f t h e s i z e of the  original  molecules. S u e o k a and o n MAK  Yamane  columns, and  and C h e n g  (50)  (46) h a v e f r a c t i o n a t e d t R N A m o l e c u l e s  demonstrated  i t s heterogeneity.  found t h a t t h e r m a l l y denatured  Sueoka  DNA-was e l u t e d  With s o l u t i o n s of a higher s a l t c o n c e n t r a t i o n t h a t those f o r native D N A .  They a l s o o b s e r v e d  G + C content tended  t o be e l u t e d  t h e s e p a r a t i o n o f s o l u b l e and 23S  r i b o s o m a l RNA  t h a t DNA  (47).  molecules with a higher  first.  MAK  r i b o s o m a l RNA  columns a l s o a l l o w  and  e v e n 16S  and  Sueoka  (47)  From t h e s e e x p e r i m e n t s ,  postulated that the o v e r a l l e l u t i o n pattern i s a r e s u l t of interplay of three e f f e c t s :  the extent of hydrogen  b a s e c o m p o s i t i o n and m o l e c u l a r  The  P h y s i c a l and C h e m i c a l  i s o l a t i o n o f DNA t i o n and The  relatively  bonding,  size.  Heterogeneity of  These chromatographic  DNA  t e c h n i q u e s have e n a b l e d f r e e f r o m p r o t e i n and  f r a c t i o n a t i o n o f t h i s DNA  into different  the  RNA  contamina-  components.  m o l e c u l a r s i z e and b a s e c o m p o s i t i o n o f t h e s e f r a c t i o n s  been determined, o f homogenous DNA d i s t i n c t DNA  the  t o i n d i c a t e whether the f r a c t i o n s  are  have  fragments  m o l e c u l e s , or whether they are i n f a c t  molecules.  In bacteriophage.:and  some b a c t e r i a l  DNA  16 p r e p a r a t i o n s , t h e r e appears t o be one molecule o f DNA per c e l l and  the DNA can be e x t r a c t e d from p r e p a r a t i o n s  molecules o f uniform  l e n g t h and composition  as s m a l l e r  (45).  On the other  hand, p l a n t o r animal DNA c o n t a i n s molecules o f d i f f e r e n t s i z e s and  composition  (48,49).  D e n s i t y g r a d i e n t e q u i l i b r a t i o n has been used t o separate DNA molecules a c c o r d i n g t o s i z e and t o the percentage o f guanine plus cytosine  (G + C) .  S c h i l d k r a u t e t al« (50) observed a d i r e c t  r e l a t i o n s h i p between the buoyant d e n s i t y G + C content  (p) o f the DNA and i t s  as i n d i c a t e d i n the f o l l o w i n g p  =  equation:  1.660 + 0.098 (G + C)  Takahash'i and Marmur  (51) used t h i s e q u a t i o n  to calculate  the percentage o f G + C o f DNA from a t r a n s d u c i n g phage o f B.. subtilis.  These authors  found t h a t t h i s DNA had a G + C content  o f 67%, whereas o t h e r methods had g i v e n a v a l u e o f 17.5%. anomoly was a t t r i b u t e d t o the f a c t t h a t the phage  This  contained  u r a c i l i n s t e a d o f thymine. T h i s procedure has proven t o be a v a l u a b l e method t o study  t h e p h y s i c a l o r chemical  heterogeneity  o f DNA.  DNA from  most v e r t e b r a t e s p e c i e s e x i s t s as a s i n g l e band on c e n t r i f u g a t i o n . With mouse and guinea p i g DNA, however, a s a t e l l i t e appears.  T h i s minor band i n mouse DNA has a lower d e n s i t y than  the main band, w h i l e (52).  band  the r e v e r s e i s t r u e f o r t h e guinea p i g DNA  In most h a l o b a c t e r i a , t h e r e i s o n l y a s i n g l e band o f DNA  w i t h a G + C content  o f 55 - 64%.  In one s t r a i n o f h a l o b a c t e r i a ,  17 Joshi of  (53)  observed  the t o t a l  t w o DNA  Intracellular  whole  by t h i s  cell  DNA  chloroplast, DNA.  contains  DNA,  but contains  (54)  that  is  also  algae/  there  1.708,  which  is  a density  DNA  buoyant  (light)  solution  green  that  had p r o p e r t i e s  cellular  nonchromosomal  that  i n  a buoyant  algae,  is  also  This  aplastidic density  there  is  and a s a t e l l i t e  band  proposed  division.  a  band  of  found i n a  SPg h a s two  t h e H (heavy) strand  t h e RNA  denaturation  indicated  chloroplast  (55) .  i t s G + C content. (10)  chloroplast  the  Sager  DNA, to  been  i n the  the t o t a l  satellite  1.708  densities;  the L  thermal  of  have  2% o f t h e t o t a l  of bacteriophage  hybrids with  Jordan  39% o f  satellite  strain  which  reflects  whereas  DNA,  only  influence  than  The  is  DNA  A lighter  20% other  Chlamydomonas,  DNA m a y b e r e l a t e d  Pyrimidines forms  is  i n corresponding  aplastidic  different  band  composition  o n l y o n e m a i n DNA b a n d w i t h  whereas  Denatured of  58% a n d t h e  supported by the o b s e r v a t i o n  1.688.  mutant  In the plant  25% o f t h e t o t a l  m a i n DNA b a n d w i t h density  i n DNA  6% s a t e l l i t e  satellite  factors  theory  of  method.  of  comprises  67%.  differences  As the c h l o r o p l a s t  heredity  of  the s a t e l l i t e  this  one w h i c h  and has a G + C content  DNA  which has a G + C content  indicated  species,  and i s  synthesized temperature,  Early  studies  DNA h e a t e d  differing  components  strand  has  the only  more  strand  by the host T ^ o f DNA  (56). also  by Cosgrave and  i n a neutral  from those  of  salt  native  DNA.  18 Thomas (10) found t h a t the u l t r a v i o l e t a b s o r p t i o n i n c r e a s e s above a c e r t a i n temperature.  A t temperatures  g r e a t e r than 80°C  /  the v i s c o s i t y and a c t i v i t y o f t r a n s f o r m i n g DNA decreases as well. The temperature  at which the s t r a n d s o f DNA s e p a r a t e  was found t o depend upon the i o n i c s t r e n g t h o f the s o l v e n t and the r a t i o o f the amount o f adenine p l u s thymine t o quanine p l u s c y t o s i n e (the base r a t i o ) o f the DNA  (-51).  Heavy m e t a l s ,  diamines and h i s t o n e s , which b i n d t o DNA, a l s o a f f e c t t h e T . N a t u r a l l y the T^ a l s o depends upon the n a t u r e o f the DNA  itself.  P o l y d i s p e r s i t y l e a d s t o a broadening o f the temperature t r a n s i t i o n range  (58) .  Marmur and Doty have expressed  (59) t h e r e l a t i o n s h i p  between the T and the base c o m p o s i t i o n a s : M w  T where the T  M  M  = 69.3 + 0.4l(G + c)  i s the temperature  o f the m i d p o i n t o f the t r a n s i t i o n  from double s t r a n d e d t o s i n g l e stranded DNA as measured by hyperchromicity. in  T h i s e q u a t i o n a p p l i e s t o measurements taken  standard s a l i n e c i t r a t e  As hydrogen  (0.15 M NaCl, 0.015 M c i t r a t e pH 7.0).  bonding between G and C i s much s t r o n g e r t h a n . t h a t  between A and T, the T^ i s thus h i g h e r i n DNA molecules w i t h a h i g h e r p r o p o r t i o n o f G + C. A t temperatures  approximately 5°C above the T ,'the two  s t r a n d s o f the DNA h e l i x come a p a r t through Brownian movement. I f the s o l u t i o n i s c o o l e d r a p i d l y , the two s t r a n d s remain  19 separate,  but i f  recombination double the  helix.  buoyant  cooling of  of  cooled  the two strands  may o c c u r  This  density  (60).  1.700.  the s o l u t i o n i s  was  slowly cooled,  form with  with  m a m m a l i a n - DNA a s  been  metabolic  tends —  Bendich  i n  form has a  1.716,  to give does  density  cooled,  b u t when a  helical  not  occur  t o be heterogenous .  i t  i n  .  Heterogeneity a d d i t i o n t o p h y s i c a l and chemical experiments activity  the uptake  indicated  of  Recombination i t  changes  and r a p i d l y  recombined  1.704.  the  DNA u p o n h e a t i n g a n d  by heat  (60).  In  which  of  by o b s e r v i n g  had a d e n s i t y  the strands  a density  Metabolic  have  material  specific  to restore  DNA i n i t s n a t i v e  When i t w a s d e n a t u r e d  single-strand  nature  of pneumococcal  Pneumococcal  the  readily  was d e m o n s t r a t e d  slowly,  t o show t h a t  and f u n c t i o n o f DNA.  of radioactive  a preferential  et  a l . (28)  there  fractionated  there  may b e d i f f e r e n c e s E a r l y experiments  precursors  labelling of  differences,  some  into  in in  DNA w a s m e a s u r e d  o f t h e DNA f r a c t i o n s .  DNA o f v a r i o u s  rat tissues  into  •  a  soluble  and i n s o l u b l e  activities differences between  were  fraction  different  varied with  i n the two f r a c t i o n s  each  the two f r a c t i o n s  i n 0.8% N a C l .  tissue.  was a l s o  The  specific  and these  The d i f f e r e n c e  indicative of  i n  turnover  metabolic  heterogeneity. Frankel of  and Crampton  DNA i n E h r l i c h  ascites  (39)  studied the metabolic  tumor c e l l s .  T h e DNA w a s  activity  labelled  20 with  14  either  The  isolated  column.  * C-formate  DNA w a s  After  approximately  the  total  recovered  in  a brief  as  lysates  which sediments  sonic  i n newly synthesized sonic  d i s t r i b u t i o n of  predominantly synthesized  degradation,  DNA i s  the  interphase  i t  after  nucleoprotein. times  is  The  greater  DNA c a n bound to  be  than  the  the  experiment  the  activity  while  of  This  the  remaining  "nascent" "native"  DNA a l s o  DNA.  With  of  the a  separated  the  the  soluble does  not  therefore and  the  sonic  protein. from the  alcohol  of  DNA i s  not  and t h e r e f o r e  incubation of  to  found  small  fraction  bound to  DNA ( 6 2 ) .  thymidine'to  that  is  of  centrifugal  treatment  resistant  protein  unlabelled  specific  low  DNA, i s  This  activity  bulk  mainly  although  chloroform/isoamyl specific  at  particulate  it  11%  the  DNA s y n t h e s i s ,  especially  because  •Newly s y n t h e s i z e d DNA b e c a u s e  treatment.  i n a heavy  possibly  the  supernatant,  RNA ( 6 1 ) .  of  is  o s c i l l a t i o n , , most  rich  extensive  up t o  which  well.  which is  the  from the  activity  speed  'alter  13  a DNA f r a c t i o n ,  specific  untreated  fraction  1RC-50  DNA, c o n t a i n e d  greatly  DNA o f  on an A m b e r l i t e  high  after  newly  the  vitro.  periods,  total  The  and i n  the  even  occurs  coli,  After  amount,  the  varied  particulate  forces.  labelling  3% o f  fractions  I n __ the  fractionated  radioactivity.  remaining  in  then  short  was  3 or ' H-thymidine i n vivo  bulk  remains  extraction  interphase  After  mixture  of  interphase  of  DNA i s  addition in a  iri  of  chase  •  DNA d e c r e a s e s  DNA i n c r e a s e s . differs cesium  in  its  chloride  physical  state  centrifugation  of  21 a  labelled E.  coli  DNA p r e p a r a t i o n ,  DNA m o l e c u l e s  i n the  in  one  two b a n d s ,  heavier  DNA b a n d .  The  structure,  i n which  light  the  b a n d was v e r y  infected  particles  with  had a h i g h e r properties  was  able  to  by  the  that  the  found  principal  band had a  dissociated,  isolate  by p r e v e n t i n g  i n h i b i t o r of  molecular weight  of  than  heavier  were  detected  r e p l i c a t i o n were  lighter  the  subunits  bacteria  an i n c o m p l e t e  From p l a n t fractions  of  of  (63)  collapsed  whereas  the  unstable.  (64)  an  stages  and one  DNA m o l e c u l e s  Frankel phage  earliest  Rolfe  tissues  normal phage  chromatography  et  T  2  phage  synthesis.  double h e l i c a l  Sampson  DNA f r o m  formation of  protein than  replicative  T h i s DNA  DNA a n d  had  structure.  a l . (65)  obtained  o n a n MAK c o l u m n .  t w o DNA  One h a d  a  5 molecular weight other,  higher  turnover.  composition  10  and a r a p i d  molecular  The  composition,  of  weight  the  in CsCl.  DNA a s  was  i t  be  the  may be  genetic the  DNA h a d t h e  same  weight  DNA v a r i e d .  of  The  synthesis  which  differed  lighter  heavier  "The of  phosphate  major  fraction  function of  of  in their  minor species  non-genetic  processes  origin,  of  base  but  the  C h l o r e l l a , Iwamura and Kuwashima  labelled with  The  The  low rate  low molecular  DNA.  energy-producing  had a v e r y  of  rapidly  n o DNA s y n t h e s i s .  turnover.  tissue  t w o DNA s p e c i e s  densities  of  of  the  From c h l o r o p l a s t s isolated  fraction  high molecular weight  irrespective of  rate  the  this  buoyant  was  the  DNA r e l a t e d  was  postulated  metabolic  organelles"  metabolic  when t h e r e is  (66)  to  species  vigorous  (66).  to .  Recent experiments are  simultaneously  the  other.  indicate that  replicating  The p r o c e s s  i s now  termination  the  chromosome  which  of replication ( 6 7 ) .  This  c a n be d e t e c t e d  thought  o f e a r l y and l a t e  cultures  o f He L a c e l l s .  They c o n c l u d e d  discrete  DNA  a r e made a t d i f f e r e n t  labelling  replicating  non-randomly along  u n i t s , DNA  replicates  o f b o t h new RNA DNA. are  This  DNA  two d i f f e r e n t  to Mueller  complexes  that  in  synthesis times  sync:(  no t h y m i d i n e .  and a i ,  The  t o the  synthesis  replicating  ( 6 9 ) , that  p h y s i c a l a s s o c i a t i o n s o f DNA  c  In e a r l y  for late  and K a j i w a r a  i r o n  involves  on a d d i t i o n o f t h y m i d i n e  and p r o t e i n a r e n e c e s s a r y  suggested  and c u l t u i o .  t h e chromosomes.  system which p r e v i o u s l y contained  n  T h e number o.f  presence  distributed  o  ( 6 9 ) reported the  and K a j i w a r a  u n i t s , which  sites  t o a number o f f o r k s ,  by e l e c t r o n m i c r o s c o p y .  ( 6 8 ) , Mueller  Initiation  at well-defined  rise  Q  semi-conservative  t h e chromosome.  occurs  gives  t o be a  f o r k s p e r chromosome d e p e n d s on t h e g r o w t h medium conditions.  molecule-  f r o m one end o f t h e m o l e c u l e L  mechanism which i s s e q u e n t i a l a l o n g and  n o t a l l DNA  thei,  G  w i t h i n the  c o m p l e x s t r u c t u r e o f mammalian chromosomes.  The  new DNA  sedimentation and  rate  has a h i g h e r  The a b i l i t y  synthesized molecules  DNA  to alkaline  contain denatured DNA  regions.  a lower  sucrose  degradation  o f CME-carbodiimide  indicated to Salganik  o f newly s y n t h e s i z e d bacteria  f o r MAK,  i n neutral or alkaline  a higher-sensitivity  DNA. ( 7 0 ) .  affinity  gradients than  to bind  small  a c c u m u l a t e on n i a c i n  (72). A f t e r a d d i t i o n o f n i a c i n ,  }  to the ru  e t a l . , (71) t h a t These  nativ<  ! W  ther.t.  molecules  starvation i n  they"are  integrated  iy  ou  s t e p w i s e i n t o l a r g e r DNA m o l e c u l e s .  I n summary, t h e new DNA,  s y n t h e s i z e d by p o l y m e r a s e s from p a r e n t a l DNA a n d n u c l e o t i d e p r e c u r s o r s , appears i n t r a n s i e n t b u t m e t a b o l i c a l l y  stable  r e p l i c a t i v e forms w h i c h a r e p r o g r e s s i v e l y j o i n e d t o t h e l o n g e r chains  (73) . I t i s n o t known how t h e p r o c e s s o f DNA r e p l i c a t i o n i s  linked to c e l l division.  I f i n i t i a t i o n and t e r m i n a t i o n o f c e l l  d i v i s i o n i s t r i g g e r e d by i n i t i a t i o n and t e r m i n a t i o n o f DNA r e p l i c a t i o n , t h e h e t e r o g e n e i t y o f g e n e r a t i o n t i m e s may r e f l e c t a heterogeneous r a t e o f DNA r e p l i c a t i o n ( 6 8 ) .  The P r e s e n t I n v e s t i g a t i o n •Previous i n v e s t i g a t i o n s i n t h i s l a b o r a t o r y  (14) have  i n d i c a t e d t h a t t h e DNA. w h i c h i s i s o l a t e d from r a t i n t e s t i n a l mucosa y i e l d s f r a c t i o n s on chromatography  on an MAK column  w h i c h d i f f e r i n base c o m p o s i t i o n and i n c o r p o r a t i o n o f a . l a b e l l e d precusor.  The m e t a b o l i c a c t i v i t y v/as d e t e r m i n e d by  m e a s u r i n g t h e u p t a k e o f r a d i o a c t i v e t h y m i d i n e i n vivo., because t h y m i d i n e i s s p e c i f i c a l l y i n c o r p o r a t e d i n t o DNA w i t h o u t p r i o r degradation.  The a n i m a l s were i n j e c t e d w.ith H - t h y m i d i n e and 3  14  t w e n t y - f o u r h o u r s l a t e r , t h e y were i n j e c t e d w i t h  •  C-thymidine.  Twenty o r f o r t y m i n u t e s a f t e r t h i s second i n j e c t i o n , t h e a n i m a l s were s a c r i f i c e d and t h e DNA was i s o l a t e d from t h e i n t e s t i n a l 3  14  mucosa. The r a t i o o f H/ C was t a k e n as a measure' o f t h e m e t a b o l i c a c t i v i t y o f t h e DNA f r a c t i o n s . A t 40 m i n u t e s , t h e r e 3  was no d i f f e r e n c e i n t h e  14 •  H/  C among t h e DNA f r a c t i o n s from MAK  chromatography, whereas i n t h e 20 minute e x p e r i m e n t , t h e  3  14 "  H/  C  increased  i n the l a t e r  DNA f r a c t i o n s  from t h e chromatography  column.  I t was f e l t  that  t h e m e t a b o l i c d i f f e r e n c e s would  apparent i n experiments  i n which  p r e c u r s o r was p e r f o r m e d  shortly before s a c r i f i c e .  studies  similar  injection  be more  o f t h e second Accordingly,  t o t h o s e above were done i n w h i c h  the animals  14 were e x p o s e d  t o the.  C-thymidine  c o m p o s i t i o n o f t h e DNA i n e a c h determined f r o m ' i t s T  value  f o r 5 o r 10 m i n u t e s .  fraction  (59).  The base  o f t h e DNA peak was  Samples o f e a c h DNA  fraction  3 were a l s o a s s a y e d f o r r a d i o a c t i v i t y r a t i o s were  and from t h i s ,  14  t h e H/  C  calculated.  During the course of the i n v e s t i g a t i o n ,  3 apparent that t h e  14 H/  had been p r e v i o u s l y t h a t h a d riot b e e n out i n d i c a t e d  i t became  C ratios  calculated  from f r a c t i o n s  d e n a t u r e d by h e a t i n g d i f f e r e d  exposed  to this  treatment.  from  Studies  that  those carried  t h a t when t h e DNA s o l u t i o n was h e a t e d a n d c o o l e d ,  some n u c l e o t i d e m a t e r i a l p a s s e d t h r o u g h t h e d i a l y s i s b a g .  3 It had been various  assumed i n c o m p a r i n g  fractions,  with, t r i t i u m .  that  14  t h e H/  C ratios  t h e DNA m o l e c u l e s a r e . u n i f o r m l y  From t h i s ,  i t would  be p r e d i c a t e d  that  of the labelled  the r a t i o  3 of t r i t i u m to o p t i c a l each f r a c t i o n .  This  The ^H/O.D. r a t i o vary i r r e g u l a r l y .  density  ( H/O.D.) s h o u l d be c o n s t a n t f o r .  expectation  d i d not prove  t o be t h e c a s e .  was c a l c u l a t e d f o r e a c h f r a c t i o n a n d f o u n d t o I t i spossible, therefore, that during the  3 twenty-four hour is  exposure o f t h e t i s s u e  a t u r n o v e r i n t h e DNA, w h i c h w o u l d  distribution of tritium  to  H-thymidine.,  result  i n t h e DNA m o l e c u l e s .  i n an uneven  there  25 G r o s s and no  Rabinowitz.(72) reported that i n r a t l i v e r  further  after set  uptake of t r i t i a t e d  three hours of exposure  o f e x p e r i m e n t s was  thymidine to the  performed  into  there  t h e DNA  precursor.  i n which the  3  was  fraction  Therefore,  H-thymidine  a was  14 injected  into  thymidine  the  rats  injection.  sacrificed ratio of  f i v e or  three  10  experiments,  minutes a f t e r fraction  hours before  this  o f the  fractionation  were  second i n j e c t i o n .  The  chromatography  DNA is and  disadvantages  is a result  o f MAK  o f the  Thus each f r a c t i o n molecules.  chromatography i s t h a t  interplay  could consist  A more p r e c i s e way  to fractionate then  of three  the  DNA  composition,  as  p r e p a r a t i o n was  of widely  hydrolyzed  according  Cerny e t a l , ( 4 0 ) .  with  chromatographed  on  column.  to  The  f o r each i s o s t i c h .  radioactivity  due  to  3  H- was  DNA acid  Six to eight pyrimidine  radioactivity  due  In the  to  length  base  The  and  length  isostichs  of the  3 from the column.  population,  according to chain  i n e a c h e x p e r i m e n t upon e l u t i o n  size  different  diphenylamine-formic  t h e h y d r o l y s a t e was  were o b t a i n e d  molecular  preparation according to chain  d e s c r i b e d by  a DEAE-cellulose  and  factors:  t o compare t h e DNA  s u b f r a c t i o n a t e each f r a c t i o n  determined  was  c o n d i t i o n s , t h e r a t i o t e n d e d t o be 3 14 the p a t t e r n of H/ C . d i d not change.  d e g r e e .of h y d r o g e n b o n d i n g , b a s e c o m p o s i t i o n (47).  C-  animals  f r o m MAK  the  the  Under t h e s e  more, c o n s t a n t , b u t One  a half  In separate  H/O.D. i n e a c h  calculated.  and  hydrolysate  14 H and  C  was  5 minute experiment  -• l o c a t e d i n the p y r i m i d i n e  the  isostichs  14 V I and  V I I I ; than  due  I I and  V I I I . . In the  to 10  C was  present  mainly  m i n u t e e x p e r i m e n t on  the  i n the other  isostichs hand,  26 3 most o f t h e r a d i o a c t i v i t y in  the f i f t h The  the base  to both  i n d e t e r m i n i n g t o which  is preferentially sequence  attached  of nucleic  Administration . The  C was  recovered  e t a_l. ( 4 0 )  e x p e r i m e n t a l technique o f Cerny  of Labelled  labelled  base  as w e l l  great  radioactive  as i n d e t e r m i n i n g  MATERIALS  Material  precursor materials  were m e t h y l - ^ H - t h y m i d i n e ,  the  has  acids.  METHODS AND 1.  H and  isostich.  possibilities percursor  due  14  used  p u r c h a s e d f r o m New  i n these experiments England Nuclear  1 4  Corporation  and  2-  C-thymidine, obtained from the Radiochemical  C e n t r e , Amersham, E n g l a n d .  I n e a c h e x p e r i m e n t , two  r a t s w e i g h i n g a p p r o x i m a t e l y 200 was  mg.  a n a e s t h e t i z e d w i t h e t h e r and 0.5  with  ml o f a p h y s i o l o g i c a l  m C i o f ^H-thymidine Twenty-four hours  after  t h e same manner 0 . 5  8.2  and  this  e a c h were u s e d .  injected  saline  jjaaoles  Each  through the t a i l  solution  containing  of unlabelled  injection,  ml o f s a l i n e  male W i s t a r animal vein 0.1  precursor.  each animal r e c e i v e d i n  solution  containing  0.03  mCi  1 4  of  C-thymidine  Five  or t e n minutes  s t u n n e d by  and  8.2 after  jumoles o f t h e u n l a b e l l e d the second i n j e c t i o n ,  a b l o w on t h e head  was  removed f o r i s o l a t i o n  2.  Isolation  o f DNA  Colter et a l (27). %  as d e s c r i b e d  f r o m Rat I n t e s t i n a l  T h i s p r o c e d u r e was After  the animal  and d e c a p i t a t e d .  o f DNA  nucleoside.  The  was  intestine  below.  Mucosa  b a s e d on t h a t o r i g i n a l l y removal o f the s m a l l  described  intestine,  by  i t was  27 cut  into  small  contents with  segments. chilled  Each  segment was f l u s h e d  saline-Versene  KH P0  4  were  c u t l o n g i t u d i n a l l y and placed  2  glass  and 0.01 M.EDTA),  plate.  which i n h i b i t s  The mucosa  microscope  slide  nitrogen.  To 5 g o f  saline-Versene  solution  was removed  and placed  the frozen  solution,  2.5  tissue  m l . of  a n d 50 m l . o f w a t e r - s a t u r a t e d  prepared  b y a d d i n g 25 m l . o f  redistilled Omni-mixer  phenol.  f o r 20 m i n u t e s  transformer. minutes upper  The m i x t u r e  at  aqueous  layer  solution  and the m i x t u r e  the a  deoxycholate The l a t t e r  Servall  20 v o l t  of. t h e V a r i a c  setting  refrigerated  for  centrifuge.  The  removed. t o t h e DNA  f o r 5 minutes.  The p h e n o l  by c e n t r i f u g i n g  at  6000 r p m  refrigerated  and e x t r a c t e d  a second  centrifuge.  time  with  were  removed  removed  by repeated  extractions  by b u b b l i n g n i t r o g e n  solution  (O.Ol'.M s o d i u m c i t r a t e ,  phosphate.)  to i t s use, the d i a l y s i s  pH 6.3  and then  t u b i n g was washed  0.05 M stored with  with  through  T h e DNA s o l u t i o n w a s d i a l y z e d e x t e n s i v e l y  and 0.05M  10  p h e n o l was added  i n the S e r v a l l  w h i c h was i n t u r n  saline-citrate  was  i n a  t h e DNA w a s c a r e f u l l y  separated  the  above.  of phenol  solution.  Prior  were  was removed  as d e s c r i b e d  chloride  phenol.  45 m l . o f  was h o m o g e n i z e d  was s h a k e n  10 m i n u t e s  layer  Traces ether,  added  liquid  75 g o f  of water-saturated  phases  (4340 x g ^ ) f o r  phenol  with  a  to  the  containing  volume  upper  t h e edge o f  d i s t i l l e d water  x g i n a Servall  An equal  The  were  O.02M  on a  The e m u l s i o n w h i c h f o r m e d was c e n t r i f u g e d 27,000  and aqueous  at  filled  its  segments  upwards  10% s o d i u m  solution  glass  side  The  by s c r a p i n g  i n a beaker  of  (l.OMNaCl,  DNAse. I .  mucosal  free  against sodium  at  -15°C.  successive  28  c y c l e s o f d i s t i l l e d w a t e r and 0 . 5 N NaOH. washed f r e e o f a l k a l i  The t u b i n g was t h e n -~  and s t o r e d i n d i s t i l l e d water a t - 4 0 ° C .  T h i s p r o c e d u r e removes u l t r a v i o l e t a b s o r b i n g  m a t e r i a l from t h e  tubing. 3.  Chromatography o f t h e DNA S o l u t i o n on M e t h y l a t e d - A l b u m i n Kieselguhr ; T h i s t y p e o f chromatography  (41)  was f i r s t d e s c r i b e d  and i t s use "was d e v e l o p e d by M a n d e j l and Hershey  by Lerman (42). In  t h i s method, a 'column c o n s i s t i n g o f l a y e r s o f m e t h y l a t e d - a l b u m i n a r e used.  The k i e s e l g u h r used was t h e m a t e r i a l s o l d as C e l i t e  by J o h n s - M a n s v i l l e  Company.  The m e t h y l a t e d - a l b u m i n had been  p r e v i o u s l y p r e p a r e d i n t h i s l a b o r a t o r y by C. M e z e i by t h e method o f M a n d e l l and Hershey  (42) .  The b u f f e r s used i n p r e p a r i n g  t h e columns a r e l i s t e d as  follows: B u f f e r No. 1. 2. 3. 4.  a)  0.05.M sodium 0.1IW sodium 0.4.M sodium  c h l o r i d e i n 0.051M phosphate b u f f e r pH 6 . c h l o r i d e i n 0 . 0 5 7 4 phosphate b u f f e r pH 6 . 7 c h l o r i d e i n 0 . 0 5 M phosphate b u f f e r pH 6 . 7 1.5.Msodium c h l o r i d e i n 0 : 0 5 J H phosphate b u f f e r pH 6 . 7  Preparation  o f t h e P r o t e i n .Coated  Celite  A s u s p e n s i o n o f 20 g o f C e l i t e i n 1 0 0 m l . o f b u f f e r 2 was b o i l e d t o e x p e l a i r and t h e n c o o l e d erature.  F i v e m l . o f a 1% s o l u t i o n o f m e t h y l a t e d - a l b u m i n  i n waterwereb u f f e r 2.  t o room temp-  added, w i t h s t i r r i n g ,  f o l l o w e d by 20 m l . o f  The c h r o m a t o g r a p h i c column was p r e p a r e d i n a  2 x 2 5 cm. column and was s u p p o r t e d by a s i n t e r e d g l a s s  29  disk  a t the d e l i v e r y  small  layer  sintered  Before  disk.  The  column  under  washed w i t h  was  a pressure  300  b)  buffer  3 and  stored  P a c k i n g o f t h e MAK  For in  the f i r s t  Two  added w i t h final cm.  Any  at  2 and  suspended  stirring  and t h e n 15 m l . o f b u f f e r added  i n 10 m l . p o r t i o n s  of 3 p.s.i.  after  The  c o l u m n was  each a d d i t i o n  1 to avoid  suspended  2.  and were  The  in a 2 x  packed  25  under  of s l u r r y . was  contaminating the  C e l i t e were  a s u s p e n s i o n o f 12 g C e l i t e i n 80 m l . and c o o l e d .  the  from t h i s  c o l u m n was  manner t o t h a t  formed  f o r the f i r s t  from a b o i l e d  ml. b u f f e r  until  ml.  layer.  w h i c h had been b o i l e d  ; .10  i n 125  o f t h e column  Twenty ml. o f the p r o t e i n - c o a t e d  "  the  the suspension-was b o i l e d  s u s p e n s i o n r e m a i n i n g on t h e s i d e s  formed  of  column  m l . o f 1% m e t h y l a t e d a l b u m i n s o l u t i o n  washed down w i t h b u f f e r  to  The  8 g . o f C e l i t e were  c h r o m a t o g r a p h i c column.  second  addition  4, a f t e r w h i c h  removed,  over the  -40°C.  layer,  s u s p e n s i o n was  a pressure  of 3 p . s . i .  layered  Column  40 m l . o f b u f f e r  cooled.  packed a f t e r  ml. of b u f f e r  c o n t e n t s o f t h e . c o l u m n was" of  adding the s l u r r y , a  o f W h a t m a n . c e l l u l o s e powder was  each p o r t i o n was  end.  3.  The  layer.  3,  second l a y e r  of  The  final  layer  was  s l u r r y o f 1 g. C e l i t e i n  c o l u m n was nm  of buffer  slurry i n a similar .  and c o o l e d  t h e a b s o r b a n c e a t 260  The  added  washed'with'buffer 1  o f t h e e f f l u e n t was  *  -zero.•  30 and  the c o n d u c t i v i t y o f the e f f l u e n t was  t h a t o f the  the same as  eluent.  A l l s p e c t r o p h o t o m e t r i c measurements were done on G i l f o r d model 2000.  Recording Spectrophotometer  equipped w i t h a t h e r m o s t a t a b l e c e l l compartment. c o n d u c t i v i t i e s were measured w i t h a Radiometer meter.  a  The  conductivity  A s t a n d a r d c u r v e o f sodium c h l o r i d e c o n c e n t r a t i o n  i n the phosphate b u f f e r v e r s u s c o n d u c t i v i t y was from t h i s c u r v e , the c o n c e n t r a t i o n s o l u t i o n c o u l d be  drawn  .  and  o f N a C l i n a-n unknown  determined. l  c)  F r a c t i o n a t i o n o f the DNA  S o l u t i o n by MAK  - A p o r t i o n o f the DNA 40 absorbance u n i t s * was  Chromatography  solution containing  a p p l i e d t o the, column under a  p r e s s u r e o f 3 p . s . i . A f t e r the sample, had on the column, e l u t i o n was gradient 400  approximately  been absorbed  c a r r i e d out w i t h a l i n e a r  s o l u t i o n o f sodium c h l o r i d e e s t a b l i s h e d by u s i n g .  ml. o f b u f f e r 1 i n the m i x i n g chamber and  b u f f e r 4 i n the r e s e r v o i r .  400  A B u c h l e r micro-pump was  t o o b t a i n a f l o w r a t e o f . a b o u t 25-30 ml. per hour. ml.  f r a c t i o n s o f e f f l u e n t were c o l l e c t e d and  absorbance o f each a t 260 chloride concentration  ml.  nm was  measured.  o f each f r a c t i o n was  of used  Five  the The  sodium  also  determined.  4.  Thermal D e n a t u r a t i o n Curves  An absorbance u n i t i s the. amount o f n u c l e i c a c i d per m i l l i l i t e r of s o l u t i o n w h i c h has an o p t i c a l d e n s i t y o f 1 f o r a l i g h t p a t h of 1 cm (14). ;  31  The base c o n t e n t o f each f r a c t i o n was d e t e r m i n e d by t h e method o f Marmur and Doty (59) . f i n d i n g t h a t t h e temperature  T h i s method i s based on t h e i r  a t which'the  two s t r a n d s o f a DNA  m o l e c u l e s e p a r a t e depends on t h e amount o f guanine p l u s c y t o s i n e i n the molecule.  The t h e r m a l d e n a t u r a t i o n c u r v e s were  determined  as d e s c r i b e d by Marmur and Doty ( 5 9 ) . In order t o study the thermal d e n a t u r a t i o n curves o f the DNA f r a c t i o n s e l u t e d from t h e MAK column, t h e s e were f i r s t d i a l y z e d against standard s a l i n e - c i t r a t e s o l u t i o n 0.0I'5.:M Na c i t r a t e pH 7.0). temperature  (0.15 >M N a C l -  The i n c r e a s e i n absorbance  with  o f t h e d i a l y z e d DNA f r a c t i o n s was r e c o r d e d a u t o -  m a t i c a l l y u s i n g a G i l f o r d Model 2000 r e c o r d i n g . s p e c t r o p h o t o m e t e r , which.was equipped temperature.  t o r e c o r d changes i n o p t i c a l d e n s i t y w i t h  The t e m p e r a t u r e  i n t h e c u v e t t e chamber c o u l d be  r a i s e d a t a u n i f o r m r a t e w h i l e b e i n g r e c o r d e d and t h e o p t i c a l d e n s i t y c o u l d be r e c o r d e d a u t o m a t i c a l l y . The t e m p e r a t u r e corresponding t o t h e mid-point o f t h e hyperchromic  s h i f t was  t a k e n t o be t h e T„ v a l u e . M 5.  R a d i o a c t i v e Counting Procedures • The method was counted i n a P a c k a r d model 314A T r i - C a r b  li-quid-scintillator was  spectrophotometer.  i n " s p l i t " and number "2" p o s i t i o n  The a n a l y s i s mode s w i t c h (75).  Counting of the  d o u b l y - l a b e l l e d sample was c a r r i e d o u t i n a system used by C. M e z e i (14.) made up as f o l l o w s .  O n e - h a l f m l . o f t h e DNA s o l u t i o n was  p l a c e d i n a c o u n t i n g v i a l and t o t h i s was added 0.5 m l . o f hyamine h y d r o x i d e p r e p a r e d by t h e method o f ..Eisenberg •."( 76 )•,: .  '• _ 4-. 0 m l .  32  of absolute latter  ethanol'and  5.0  ml. o f a s c i n t i l l a t o r  c o n s i s t e d o f 0 . 6 3 % P P O and 0 . 0 2 6 2 % POPOP  solution.  The  in redistilled  toluene. It in  and c a r b o n -  i s p o s s i b l e t o assay f o r both t r i t i u m  a d o u b l e - l a b e l l e d compound due t o t h e e f f e c t i v e n e s s o f  scintillators difference  f o r counting  i n the energies  (77).  isotopes  0.155  for  operation settings  The maximum b e t a  All  pulses  H i s 0.018  voltage  Mev and  and d i s c r i m i n a t o r  f o r pulse-height  1  t o B are recorded  from B t o C a r e recorded  rate of a given voltage  standard  settings.  discrimination  44,000  cpm  was d e t e r m i n e d a t t h e  settings.  solutions consisted  cpm and a s t a n d a r d  (78).  solution  To o b t a i n t h e o p t i m a l  a s e r i e s o f g r a p h s was c o n s t r u c t e d  as a f u n c t i o n o f t h e h i g h  on t h e r e d  on t h e g r e e n s c a l e r .  The s t a n d a r d  98,000  water c o n t a i n i n g  o f Na^CO^ c o n t a i n i n g  discriminator  o f t h e two  1  d i f f e r e n t high  rate  and t h e  b y means o f d i s c r i m i n a t o r c o n t r o l s A A ; B and C .  s c a l e r and .pulses  of t r i t i a t e d  particles 3  energy f o r  T h e two c h a n n e l s  o f e n e r g y between A A  counting  isotopes  liquid  Mev a n d t h e two i s o t o p e s may be c o u n t e d i n one  (76) .  obtained  setting,  of the beta  by p r o p e r s e l e c t i o n o f h i g h  are  The  weak b e t a - e m i t t i n g  14  voltage  showing t h e c o u n t i n g  setting at different  T h e s e g r a p h s a r e shown i n F i g u r e s  3  and. 4 . 14 A standard  3 and  E^O  settings.  (98,000  s o l u t i o n containing both Na dpm)  was p r e p a r e d  The r a d i o a c t i v i t y  2  and c o u n t e d 3 14  due t o  H or  dpm)  at the various  C was c a l c u l a t e d  according  t o t h e d i s c r i m i n a t o r - r a t i o method.  described  by O k i t a  e t a l , ( 7 7 ) , who d e r i v e d  CO^ (44,000  T h i s method i s  the following  3* CaJ  3 F i g . 3. I n t e g r a l d i s c r i m i n a t o r b i a s c u r v e s f o r standard and  ( N^^COj  H (  ) o b t a i n e d from the red s c a l e r o f a  Packard 314- AX L i q u i d S c i n t i l l a t o r  Spectrophotometer.  3 H,jO)  33 (a)  !4  3  F i g . 4. I n t e g r a l d i s c r i m i n a t o r bias curves f o r standard determined  H and  C  on the green s c a l e r . The curves represent the  average of the determination of s i x d i f f e r e n t window s e t t i n g s .  3U<  1  2  3  High- Voltage  4  Sett  35 3 equations' f o r obtaining  14  H and  C cpm  from  doubly-labelled  compounds:  b  3„ J H dpm  N  l  = red  scaler  -  2 b - a j H efficiency factor  b(N 14^, , C dpm  where  N  -  2  =  0.034  a^)  b - a =  ^4  C efficiency factor  green s c a l e r  i s 0.236  = n e t cpm on r e d s c a l e r  N  = n e t cpm o n g r e e n  2  scaler  3 -_ n e t cpm o f _ H on green .  a  :  s c a l e _r  H on r e d s c a l e r  n e t cpm o f  14 ,  C on g r e e n  _ n e t cpm o f 14 net  cpm o f  C on r e d s c a l e r  3 and 4 ,  From F i g u r e s  scaler  the following  constants  were  calculated: =  a  .04,  b  =  1.1/  the red scaler  3  H efficiency  14 =  factor is  0.34  and t h e g r e e n s c a l e r  The  percent  recoveries  solution  were c a l c u l a t e d  recovery  f o r both  and  factor  .236. 3  that  C efficiency  simultaneous  to find  isotopes.  a high voltage discriminator  of  setting  settings  14 H and  C i n the standard  which s e t t i n g  These c a l c u l a t i o n s of 6,  corresponding  of 10:50:90  counting of both  isotopes.  gave t h e h i g h e s t indicated to 1025  were t h e b e s t f o r  volts,  36 EXPERIMENTAL 1. ' A d m i n i s t r a t i o n o f L a b e l l e d T h y m i d i n e Earlier  investigations  b y C.  Mezei  Precursors (14) h a d  the  p o s s i b i l i t y t h a t r a t i n t e s t i n a l m u c o s a may  DNA  fractions  The  3  was  contain d i s t i n c t  which d i f f e r i n t h e i r metabolic a c t i v i t y .  incorporation f o r DNA,  suggested  of ^H-thymidine, which i s a s p e c i f i c u s e d a s an i n d i c a t i o n .  H - l a b e l l e d DNA  was  The  precursor  of metabolic a c t i v i t y .  fractionated  on an MAK  c o l u m n and  s p e c i f i c a c t i v i t i e s o f .each f r a c t i o n w e r e d e t e r m i n e d .  the  One  of s i n g l e - l a b e l l i n g experiments i s the  >  of  the  difficulties  due  t o i s o l a t i o n b e c a u s e i d e n t i c a l - f r a c t i o n s - can-not'-'be. i s o i - a t e d i n  every experiment. of v a r i a t i o n  The  i n DNA  difficulty  isolated  overcome by t h e s e n s i t i v e  method o f d o u b l e l a b e l l i n g .  • t h y m i d i n e and t h e n i n j e c t e d assumed  as a  result  from experiment to experiment  t h i s t e c h n i q u e , r a t s were i n j e c t e d  I t was  i n interpretation  error  f i r s t with  use  tritiated  14 C-labelled  with the  To  was  precursor.  (14) t h a t d u r i n g t h e 24 h o u r s t h e DNA  would  be  14 u n i f o r m l y l a b e l l e d w i t h t r i t i u m and t h e u s e o f would  l a b e l newly  f o r m e d DNA.  C-thymidine  The DNA.was f r a c t i o n a t e d  and 3  the  r a d i o a c t i v i t y o f e a c h f r a c t i o n was  r a t i o was  determined.  The  constant i n the experiment i n which the r a t s  14 H/  C  were 14  s t u n n e d and k i l l e d thymidine.  f o r t y minutes a f t e r the i n j e c t i o n o f  This finding  s u g g e s t e d t h a t t h e o l d and  C-  new  14 molecules are u n i f o r m l y l a b e l l e d w i t h  C.  A t a s h o r t e r time 3 14  i n t e r v a l of twenty minutes, though, d i f f e r e n c e s r a t i o i n the f r a c t i o n s fractions  eluted  f r o m t h e MAK  i n the  column d i d . appear.  w i t h an e l u e n t a t a h i g h c o n c e n t r a t i o n  H/ The of  "C  37  sodium c h l o r i d e had h i g h e r  3  H/  1 4  C  ratios.  These l a t e r  fractions  were shown by T ^ m e a s u r e m e n t s and C s C l b u o y a n t d e n s i t y ugation  t o c o n t a i n a lower G + C  In t h e p r e s e n t experiments  was  content.  investigation,  carried  centrif-  a series  out except  of  similar  that the animals  were  14 sacrificed  5 minutes- a f t e r  m i n u t e e x p e r i m e n t was obtained 2.  injection  also carried  of  cells,  DNA  deoxyribonucleic  form o f a DNA-histone complex  acids  located within  be " i s o l a t e d b y m e c h a n i c a l l y  are i n the  the n u c l e i ,  isoamyl  a l c o h o l / c h l o r o f o r m (79) .  in i t s native state i s very  to smaller molecules  processes.  For this  by c h e m i c a l  reason,.it i s d i f f i c u l t  o f p u r e , u n d e r g r a d e d DNA  Because o f t h e success  i n this  or  DNA  or  shearing a  l a b o r a t o r y b y C. M e z e i t h i s method  (7 8) was  f o r the following investigations.  ,A sample o f t h e i s o l a t e d  agreed  weight  molecules.  f  9.6%  phenol  to obtain  t h e i s o l a t i o n m e t h o d o f C o l t e r e t al_. (27)  selected  and  High molecular  and  susceptible to denaturation  degradation  quantitative yield  DNA  d i s r u p t i n g the c e l l u l a r  n u c l e a r membranes a n d r e m o v i n g t h e b o u n d p r o t e i n w i t h  with  20  p r e v i o u s l y (14).  In mammalian  or  A  o u t t o check t h e r e s u l t s  C h a r a c t e r i z a t i o n of the I s o l a t e d  can  C-thymidme.  DNA  contained  p h o s p h o r u s , g i v i n g an a t o m i c N/P  with  values  high molecular  r e p o r t e d by C h a r g a f f  weight  DNA s. 1  14.4% n i t r o g e n ,  ratio  o f 3.32  which  (80) f o r a s e r i e s o f  38 3.  Chromatography of the K i e s e l g u h r Columns A prerequisite  a DNA p o p u l a t i o n i s fractionation Sueoka the  a n f  j  of  size,  0.5 . ;  a  which to  is  0.6M.  single  similar  a' s h a r p  peak  at  the  other  of  of  experiment  to  of  of  (78) ,  this  the  the  Because  of  eluting  the  this  buffer. give  reproducibility  s a m e DNA f r o m  MAK c h r o m a t o g r a p h y  has  the  main peak the  been  a  peaks  of  ultraviolet-absorbing material  deoxyribose just  before  it  is  present  is  applied to  the  deoxyribose  MAK c o l u m n ,  measured  concentrations.  total  from the  as  lower s a l t  of  eluted  eluate  not  again  s a m e DNA s o l u t i o n  in  eluted  containing  concentration  of  95%  contain  -  material  approximately  smaller  chromatography  widely  i n i n v e s t i g a t i n g DNA.  • Within  the  enable  base  deoxyribose  dealing with  experiment,  the  hydrogen bonding.  same c o n c e n t r a t i o n  profiles.  p o s s i b l i t y of  to  sodium c h l o r i d e  samples  and h i g h e r  permits  in  earlier,  according  Rechromatography  fractionation  tool  peak a  differences  MAK c h r o m a t o g r a p h y w i l l  and degree  eluted with  Chromatography of  used  which  As s t a t e d  DNA m o l e c u l e s  molecular  metabolic  technique  found that  DNA o n MAK y i e l d s  gives  of  ECTEOLA-cellulose chromatography  material of  study  suitable  (47)  fractionation  Unlike  a  the  DNA o n M e t h y l a t e d - A l b u m i n •  these molecules.  Cheng  composition,  of  of  for  Isolated  by t h e  Dische  material  test  These peaks,  radioactive  material.  The  the  DNA p e a k , i s  thought  to  be  treated  with  the  column  sample  is  (81).  The  other  peaks  Other  are  or  if  detected  (14).  however,  is  eluted  do  not  peak RNA  at  which  is  because:  RNAsebefore contain  some  it  39  unidentified t h e DNA  u l t r a v i o l e t - a b s o r b i n g m a t e r i a l , which remains i n  solution  even  Measurements and  composition single  Up  l a r g e peak  the s i n g l e  p a t t e r n was  Thermal D e n a t u r a t i o n  technique  behaviour  liberating  among t h e DNA  its  G + C  equation  the base r a t i o  than  For this  reason  this  p  =  •  by  direct the  separated  analysis  by  of the  will  give the  T h i s method r e q u i r e s  the s i m p l e r (50) was  obtained indirect used.  molecule  formulated  by  They  depends  on  the f o l l o w i n g  relationship.  1.660  t h e b u o y a n t d e n s i t y o f t h e DNA • v  A  i s to hydrolyze  those, i n e a c h f r a c t i o n  and t h e r e f o r e , t h e y  to express  when  obtained  vary with base c o m p o s i t i o n .  t h a t t h e b u o y a n t d e n s i t y o f a DNA content  altered  fractions  a p p r o a c h o f S . c h i l d k r a u t , Marmur and D o t y observed  column.  f o r s e v e r a l months.  amounts o f e a c h b a s e p r e s e n t .  chromatography.  on an MAK  s p o t s on t h e c h r o m a t o g r a m  l a r g e r amounts o f DNA  seen the  chromatography  not s i g n i f i c a n t l y  Spectrophotometric  ultraviolet-absorbing  MAK  i n base  t h e f r e e b a s e s w h i c h c a n be  paper, c h r o m a t o g r a p h y .  quantitative  differ  Curves  f o r determining  DNA sample  m u c o s a l DNA  (14)  shown t h a t t h e  I n F i g u r e 5 c a n be  s t o r e d a t -15°C  chromatography w i l l  o f DNA  m a t e r i a l obtained.by  rat intestinal  had been  'Chemical  peak  manner.  o f DNA  fractionation  t h e DNA  MAK  dialysis.  o f b u o y a n t d e n s i t i e s by o t h e r w o r k e r s  i n a graded  of the i s o l a t e d  4.  extensive  the< m e a s u r e m e n t s i n t h e p r e s e n t work h a v e  f r a c t i o n s making  The  after  + 0.098 sample  • .. •  •  ( G -F C)  where p i s  and i s d e t e r m i n e d "  by  to. 8  40.7  /  /  DNApeak [DM  L0.3 co  A  Si -0.1  ru •0.1  10  20  F i g . 5. Chromatography of DNA  30  HO  . . so  Tube  No.  + • 60  70 ,  from r a t i n t e s t i n a l mucosa on an MAK column. E l u t i o n was c a r r i e d out with a  eradient of NaCl i n 0 . 0 5 M. DhosDhate - DH 6.7  4J,.  reference density will  t o a DNA m o l e c u l e  o f known d e n s i t y . The b u o y a n t  a l s o d e p e n d s upon t h e m o l e c u l a r  vary  i f unnatural  The G+C denaturation  content  is  c a n a l s o be c a l c u l a t e d f r o m t h e t h e r m a l  temperature  ( T  ) a c c o r d i n g t o Marmur and D o t y ( 5 9 ) .  M  o n d e n a t u r a t i o n by h e a t i n g i n  a q u e o u s s o l u t i o n when i t u n d e r g o e s s t r a n d  related  t o i t s base composition.  Since  b o n d i n g ' b e t w e e n G.and.C i a : s t r o n g e r t h a n and  o f t h e DNA and  bases are present (50).  --The-behaviour .of....a-DNA ..molecule a dilute  size  T, t h e T  M  i s higher  i n DNA's w i t h  separation  the hydrogen t h a t between A  a higher proportion of  G a n d C. At  temperatures  the two s t r a n d s  o f t h e DNA h e l i x  -movement and c h a r g e rapidly,  approaching  remain separate,  recombination  restore the double h e l i x .  biological  i s cooled  but i f they  are cooled  f o r s t r a n d s e p a r a t i o n on  by p h y s i c a l ,  chemical  and  experiments (82).  A combination sedimentation molecular  Brownian  o f t h e 2 s t r a n d s "may o c c u r t o  Evidence  d e n a t u r a t i o n has been p r o v i d e d  temperature,  come a p a r t t h r o u g h  r e p u l s i o n . I f the s o l u t i o n  t h e two s t r a n d s  slowly, s p e c i f i c  the melting  of light  scattering,  c o e f f i c i e n t measurements i n d i c a t e d t h a t t h e  w e i g h t o f t h e DNA p a r t i c l e s  following denaturation labelled with  v i s c o s i t y , and  i 4  N  and r a p i d  were m i x e d w i t h  decreased  by h a l f  c o o l i n g . I f t h e DNA ^  5  N - l a b e l l e d DNA  molecules molecules  42 of  the  same o r i g i n , h e a t e d  and s l o w l y c o o l e d ,  hybrid  DNA  14 molecules,  i n w h i c h one  strand  is  labelled  with  N and  the  15 other  with  c o u l d be  N,  Biological double  stranded  certain other  activity,  the  denaturation  as  Strand at  solution.  The  of  this  the  7 or.8  residues  the  present  shown, t h e obtained  table  i t  content  can were  be  of  detected  is  is  observed the  that  seem t o  be  DNA  the  (10).  DNA in  DNA  this  as  a result  increase  the  a valid  regions  of  on  characteristic  the  proportional to  the  increase  during  in native  assumed  On  stranded  the  of  of  increases  in viscosity  shift  helical  property  DNA  by  presence  (60).  single  d i s r u p t i o n of  it  thermal  for  helix  of  in content,  measure  of  which contain  denaturation curve -  shown i n F i g u r e 6 . the  fractions  chromatography  While the  experiment,  of  centrifugation.  the  at  least  (10).  T^ v a l u e s  experiments. to  If  work i s  b y MAK  on h e a t i n g  bases maintained  d i s r u p t i o n of  A typical  lost  be  on the  transforming  decrease  hyperchromicity would of  in  can  from the  absorption  extent  the  a property  nm o r  hydrogen bonding. ultraviolet  as is  is  density  w h i c h depends  hyperchromic  the  by C s C l  immunological reaction  260  results  stacking  DNA's,  separation  absorption  process  such  DNA,  bacterial  hand,  detected  they seen  eluted  T s M  of  first  isolated  in  from  same p a t t e r n .  fractions  from the  DNA  In Table  varied slightly  the  the  I  comprising the  DNA  a l l f o l l o w e d the that  of  MAK  with  isolated are DNA  peak  these experiment From  a higher  G +  the C  c o l u m n " a n d t h e r e was  a  4^.  lr  s  tuu 0 9 2 IB '0*0  s  I  F i g . 6. E f f e c t of increasing temperature on the o p t i c a l density of doubly-labelled r ? t . i n t e s t i n a l mucosal  DNA.  1  44 gradual  decrease  i n the G + C content  number  These  Sueoka  and Cheng  findings differ  suggest  s i m i l a r to  that  there  Change in O.D.  0.312 0.432 0.641 1.160 0.667 0.791 0.661 0.352 0.243  0.365 0.577 0 . 853 1.414 0.927 1.056 0.780 0.428 0.261  0.053 0.14.5 0.212 0.254 0.260 0.265 0.119 0.076 0.022  7  8 9  Table fractions  I  - T., M  intestinal  after  radioactive  precursor  a difference  between  % (G + C )  91.4 91.0 89.6 8 8.6 88.0 87.4 86.8 86.7 86.7  method due t o  the difference  53.9 52.9 49.5 47.0 45.6 44.1 42.6 42.4 42.4 of  separate o f DNA f r o m  with  for detecting  i n uptake  t w o DNA f r a c t i o n s  i n metabolic  activity.  two i s o t o p e s these  at  (77) .  a  might  The  technique  provides  differences.  H and  C was c a l c u l a t e d  f o r each  f r o m t h e MAK c o l u m n b y t h e d i s c r i m a t o r r a t i o . m e t h o d et  of  a  very  The r a d i o -  14  3 activity  (83)  C o u n t i n g o f D o u b l y - L a b e l l e d DNA  l a b e l l i n g the molecules  sensitive  Sueoka  MAK c h r o m a t o g r a p h y  As p o i n t e d o u t e a r l i e r ,  bf  which  mucosa.  Radioactive  indicate  T  _M  v a l u e s and G - C c o n t e n t  o f main peak  by  'I h e s e  (14) .  <_:heng a n d  fraction  density centrifugation.  by  Final O.D.  1  reported  a r e s e v e r a l DNA s p e c i e s  composition.  these findings  increasing  those  and M e z e i and Zbarsky  Initial O.D.  2 3 4 5 6  5.  are  (47)  t h e i r base  in  confirmed  Fraction No.  findings  with  DNA f r a c t i o n of  Okita-.  44 [a)  Fig. 7. R a t i o o f ^ H/  i n f r a c t i o n s comprising the DNA pe^k eluted -by  .chromatography of d o u b l y - l a b e l l e d mucosal PNA, which h?d been 3 pllowed t o incorporate H f o r 24- hours, on MAK columns. Each  fraction  peak i s c a l l e d  is,one  tube  fraction  . The  no.  1.  first  tube  i n the  DNA  46 Figure  7 indicates  . • .  how t h e r a t i o s  14  i n which  C-  3 thymidine  was g i v e n 24 h o u r s  experiments tion in  after  H-thymidine vary.  w h e r e DNA was i s o l a t e d  o f ^ C-thymidine  to rats,  4  the l a t e r  fractions  20 m i n u t e s a f t e r  administra-  (A) t h e H / ^ C r a t i o 3  increased  4  i n agreement w i t h  results  In  obtained by  Mezei and Zbarsky ( 1 4 ) . In after  experiment  a d m i n i s t r a t i o n o f ^"^C-thymidine,  r a t i o versus of  (B) where DNA was i s o l a t e d  fraction  fraction 4 o r 5.  number  consistently  This finding  a r e two m e t a b o l i c a l l y a c t i v e beginning as  curve  fractions,  I I i n the l a t e r  due t o H d e c r e a s e d 3  o f the H/^ C 3  that  there  one e l u t e d a t t h e a t t h e end.  fractions,  considerably, while  4  showed a maximum  might suggest  o f t h e DNA p e a k a n d t h e o t h e r  shown i n T a b l e  5 minutes  However,  the radioactivity  the counts  due t o  14 C and t h e o p t i c a l only  slightly.  d e n s i t y o f t h e DNA m o l e c u l e s  T h i s o b s e r v a t i o n suggested  decreased  t h a t perhaps the 3  DNA m o l e c u l e s to  test  this  are not uniformly labelled possibility,  with  the r a t i o of the H 3  optical  d e n s i t y was c a l c u l a t e d  results  are l i s t e d  H.  In order  radioactivity/  f o r each f r a c t i o n .  The  i n Table I I . 3  Table density in  I I - The r a t i o o f r a d i o a c t i v i t y  o f t h e DNA f r a c t i o n s  experiments  obtained  from  due t o MAK  chromatography  i n w h i c h H - t h y m i d i n e was a d m i n i s t e r e d 3  14 24 h o u r s b e f o r e  H/optical  the injection  of  C-thymidine.  to rats  47 DNA Fraction Number 1 2 3 4 5 ' 6 7 8 9 10 11 12  3  3  H c/m  P.P./ml.  76.8 9.41 2396.5 4359 4638 4274 4185 4144 4074 4062 4035 4038  H/O.D.  1.910 2.960 2.711 1.845 2.274 1.917 1.594 1.678 1.080 1.222 1.158 .660  I t i s obvious from these c a l c u l a t i o n s , that  40.2 3.1 884 2362 2040 2660 2546 2347 3772 3324 3847 6118 H i s not  uniformly d i s t r i b u t e d throughout the DNA molecules. v a r i a t i o n was not regular.  The  A possible explanation f o r t h i s  f i n d i n g i s that during the 24 hour incubation period, some of the DNA may be degraded as the rate of turnover of c e l l s i n ..the , i n t e s t i n a l mucosa i s very high (84).  Furthermore, the  experiments of Gross and Rabinowitz (74) indicated t h a t no further uptake in...vivo of t r i t i a t e d thymidine into DNA occurred a f t e r 3 hours exposure to the radioactive precursor due to degradati Because of these considerations, another series o f 14 experiments was c a r r i e d out i n which the rats were given  C-  thymidine 3 1/2 hours a f t e r r e c e i v i n g H - l a b e l l e d thymidine. 3  The animals were s a c r i f i c e d 5 or 10 minutes a f t e r the second injection.  The f r a c t i o n s of mucosal DNA from the MAK column  were assayed f o r r a d i o a c t i v i t y and u l t r a v i b l e t absorbance as before.  The data from these experiments are presented i n  Figures 8•to 11.  The.elution p r o f i l e s of the DNA i s o l a t e d i n  the 5 minute experiment, as shown i n Figure 8," and 10 minute  to  1  20  HO  Fig.9. Chromatography of mucosal DNA, minutes, on  MAK  column.  so  t,o-  —1— 70  80  -f-  l a b e l l e d with »H? thynSdine f o r 3 i hours and C - thymidine f o r 10 H  90  H  5 min.'c i n c o r p o r a t i o n  A.1Qmin. G i n c o r p o r a t i o n .  e  $5-  50!  —  ZS)  '  '— —  n  —  j-—  ±  j-—  2,  3  4  —i  5  i n experiments i n which Each  fraction  :  i  f-  7  b  Fraction  3  F i g . ID. Ratio of r a d i o a c t i v i t y due to •  —f  r°-  :  i  1  4  8  ^  fraations  obtained from MAK  chromatography  H- thymidine was administered to rats 3g- hours before i n j e c t i o n of -tube-. F r a c t i o n  I  No.  H/ o p t i c a l density of the DNA  c o m p r i s e s one  1  no.  1 i s the f i r s t  tube  Q  f t h e DNA  C-thymidine. peak.  I  A 5 min. C incorporation ©10 m i n t incorporation  ©  z  -4-  H H  3  3 /IA F i g . 11, Ratio of  H  /^  l a b e l l e d with  1—  5-  Fraction  1— 6  C i n f r a c t i o n s comprising the DNA H- .thymidine f o r 3g- hours.  No.  8  id  pe»k eluted by chromatography of DNA /  which was  52 experiment, in  as shown i n F i g u r e  e a r l i e r experiments  As ratio  9, a r e s i m i l a r  i n t h e p r e s e n t works.  i l l u s t r a t e d i n Figure  of H/ 3  1 4  C among  t h e DNA  i n the 5 minute  reached  a maximum a t f r a c t i o n  and 10 m i n u t e  i n the experiments  tritiated-thymidine distribution  11, the d i s t r i b u t i o n  fractions  pattern  obtained  i n which  f o r 24 h o u r s .  of  distribution  these experiments  minutes  after  i n which  administration  3 minutes,  the r a t i o  of  a n d a t 40 m i n u t e s , fractions It  the  72,  73).  This  which  As -.shown, i n . F i g u r e t h e DNA  ratio  was  were e x p o s e d  also  to 10, the  fractions  I t s seems  therefore  14  H/  C ratios  is-characteristic  a n i m a l s were k i l l e d  5 o r 10  o f the second p r e c u r s o r .  H/  A t 20  C increases with f r a c t i o n  number  14  H/  C ratio  i s constant throughout  (14) .  i s possible  that  these r e s u l t s  metabolic heterogeneity within expected  similar  14 3  the  rats  experiments.  of the  a  of the  experiments.  o f t h e r a t i o o f ^H/O.D. among  3 this  followed  4 o r 5, a r e s u l t  was c o n s t a n t i n t h e 10 m i n u t e  that  to that obtained  from r e c e n t  theories  t h e DNA  are i n d i c a t i v e  of a  m o l e c u l e s , as c o u l d  o f chromosome r e p l i c a t i o n  S m a l l m o l e c u l e s o f newly  s y n t h e s i z e d DNA  be  (67,  would  14 c o n t a i n t h e g r e a t e s t amount o f " C r e l a t i v e t o t h e amount o f o l d , o r ^ H - l a b e l l e d DNA. As small molecules are eluted a t the. b e g i n n i n g o f t h e DNA  3 H/  peak on MAK  chromatography  (£42,,  53."•'•)•  the  14 C ratio of the early.fractions  observation  found  i n this  s h o u l d be l o w - a n  experiment.  After  some o f t h e s e s m a l l m o l e c u l e s may h a v e f o r m e d  5 o r 10 m i n u t e s , loose  aggregates  53 (67, 73) w h i c h a r e h e l d  t o g e t h e r b y weak h y d r o g e n  bonds a n d  c o n t a i n l a r g e r e g i o n s o f d e n a t u r a t i o n as p o s t u l a t e d by Salganik  e t a l . (71).  to v a r i o u s degrees the r e s u l t s  These molecules  will  t e n d t o be e l u t e d  o f Sueoka and Cheng  are aggregates  which are denatured  (47) .  last  S i n c e such  of the smaller metabolically  will  begin to decrease  i n the l a t e r  s m a l l newly s y n t h e s i z e d m o l e c u l e s aggregates stable.  C, t h e  on  incorporation  into  C  ratio  Between t h e  partially-denatured  i s metabolically  Sueoka's  e x p l a i n the curves obtained i n those  molecules  14  H/  fractions.  i s t h e b u l k o f t h e DNA, w h i c h  T h i s h y p o t h e s i s based  thymidine  3  and t h e i r  molecules  active  14 and h e n c e h a v e a h i g h p r o p o r t i o n o f  according to  suggestion could 14  experiments  i n which  . C  DNA was p e r m i t t e d f o r 5 o r 10  minutes. A f t e r a 20 m i n u t e i n t e r v a l  o f DNA m e t a b o l i s m  i n the  14 presence  of  aggregated  C-thymidine,  DNA m o l e c u l e s  some o f t h e s e s m a l l o r l o o s e l y  c a n be e x p e c t e d  t o be i n c o r p o r a t e d  i n t o t h e m a t u r e DNA  fraction  ( 6 7 , 7 3 ) . Thus some o f t h e H 14 l a b e l l e d DNA w i l l b e l a b e l l e d w i t h C, but. t h i s w i l l be a m i n o r f r a c t i o n c o m p a r e d t o t h e t o t a l ' DNA w h i c h i s l a b e l l e d 3 3 14 with H. T h e n e t r e s u l t s h o u l d be an i n c r e a s i n g H/ C ratio with f r a c t i o n  a c c o r d i n g t o i n c r e a s i n g m o l e c u l a r s i z e and-  metabolic-stability  o f t h e DNA  3  fractions  eluted  from  the :  column. 14 After available  40 m i n u t e s ,  very l i t t l e  f o r incorporation,  C-thymidine  and, a s a r e s u l t : ,  w o u l d be  the incorporation  14 of the  C-thymidine  i n t o t h e newly formed  material  would  54 decrease.  A f t e r '40 m i n u t e s ,  t h e r e f o r e , t h e DNA  w i t h respectAasotope i n c o r p o r a t i o n . synthesized molecules incorporation  into  will  The b u l k  the bulk  the isotope r a t i o .  w o u l d be e x p e c t e d Any  o f t h e DNA w i l l  The p l o t  of  14 H/  C v e r s u s DNA  to give a straight horizontal  c o n c l u s i o n s deduced  from  i n the krypts of Lieberkuhn  migrate and  to the tips  of the v i l l i ,  are e v e n t u a l l y sloughed  variability  of results  very probably  off.  from  as w e l l as b y s e c r e t i o n s f r o m the  small It  line.  F o r example,  d i v i d e most r a p i d l y and where t h e y  no l o n g e r  T h i s might  introduce  one e x p e r i m e n t  i s contaminated  fraction  t h e above r e s u l t s must be  t h a t the mucosal t i s s u e  -small i n t e s t i n e  their  h a v e no e f f e c t  t e m p e r e d by c o n s i d e r a t i o n o f o t h e r f a c t o r s . cells  o f t h e newly  be u n l a b e l l e d and t h e r e f o r e  3 on  i s stabilized  t o another.  isolated  It is  from t h e  w i t h muscle and b l o o d  glands  divide  and o r g a n s t h a t  cells serve  intestine. i s n o t known w h e t h e r o r n o t a l l t h e m a t u r e DNA i s  l a b e l l e d with  3  H and t h e n e w l y s y n t h e s i z e d m o l e c u l e s  with  14 C  3 and 3  H o r whether the l a b e l l i n g  i s incomplete.  H/O.D. i n t h e 3 1/2 h o u r e x p e r i m e n t  uniform on  labelling  pattern.  S i n c e MAK  only a broadly,  chromatography  three f a c t o r s working together, i t i s d i f f i c u l t  what e f f e c t m o l e c u l a r  size  its  A factor  in  indicates  The r a t i o o f  overall elution.  alone  t h e 20 m i n u t e e x p e r i m e n t s  mature  DNA.  depends  t o know  o f t h e DNA m o l e c u l e  h a s on  i n t h e 40 m i n u t e and p o s s i b l y  i s the rate of turnover of the  ndenatured o native  F i g . 12. R^tio of ' H/ C i i i f r a c t i o n s comprising the DNA perk eluted by chromatography of DNA which w?s l a b e l l e d with H f o r 2U hours and '"C f o r 20 minutes. The comparison i s made between f r a c t i o n s that are denatured by heating following chromatography before.radioactive counting and those-which had no treatment p r i o r to counting. 3  3  i h  56  An i n t e r e s t i n g  o b s e r v a t i o n was f o u n d  i n counting the  3 DNA f r a c t i o n s . fractions active  The d i s t r i b u t i o n  d e p e n d e d on t h e t r e a t m e n t  counting.  radioactive  of  assay.  C ratio  As a r e s u l t ,  a 20 m i n u t e e x p e r i m e n t 3 14  an i n c r e a s i n g  H/  curve  t h e DNA  f o rcounting.  typically  observed.  samples  denatured I n t h e case  o u t under these  C r a t i o was a s t r a i g h t  radio-  used f o r  t h e DNA was h e a t  carried  among t h e  o f t h e samples b e f o r e  were made were t h e n  cooled before being processed  the p a t t e r n o f t h e of  H/  I n some o f t h e e x p e r i m e n t s  upon w h i c h T ' d e t e r m i n a t i o n s  and  of the  14  circumstances,  line  instead  These r e s u l t s a r e  shown i n F i g u r e 12. In leading  order t o account  f o r these  as f o l l o w s .  The DNA f r a c t i o n s  c h r o m a t o g r a p h y were d i v i d e d o f each  distilled  two e q u a l p o r t i o n s .  One  separately against  f o rradioactivity.  dialyzed  E a c h d i a l y s a t e was  d e n s i t y a t 260 nm a n d r a d i o a c t i v i t y  and t h e  o f each  dialysate  T h e o t h e r p o r t i o n o f e a c h f r a c t i o n was  against standard  were e v a p o r a t e d ,  of  MAK .  t o s m a l l volume i n a f l a s h ^evaporator  were m e a s u r e d .  counting  from  water and d u p l i c a t e samples o f each  evaporated  dialyzed  into  f r a c t i o n was d i a l y z e d  f r a c t i o n were a n a l y z e d  optical  the steps  t o t h e c o u n t i n g o f t h e DNA f r a c t i o n s were e x a m i n e d i n  more d e t a i l  portion  differences,  saline  the resulting  impractical.  citrate.  high s a l t  The t h e r m a l  t h e DNA was d e t e r m i n e d  dialysates  c o n c e n t r a t i o n made  denaturation  i n each f r a c t i o n  s o l u t i o n w h i c h now c o n t a i n e d d e n a t u r e d against d i s t i l l e d  When t h e s e  and t h e r e s u l t i n g  DNA was  w a t e r and t h e u l t r a v i o l e t  temperature  dialyzed  absorbance  a t 260 nm  57 and t h e r a d i o a c t i v i t y dialysate  was  radioactivity were s i m i l a r l y  were measured.  The d i s t i l l e d  water  e v a p o r a t e d t o s m a l l volume and a s s a y e d f o r and a b s o r b a n c y . treated.  Samples o f u n f r a c t i o n a t e d  The r e s u l t s  from t h i s  DNA  experiment are  p r e s e n t e d i n T a b l e s I I I , I V and V.  Treatment  O.D.  3  14 .  H  c  V c 1 4  no t r e a t m e n t  16 139,665  13,289 10.5  dialyzed against d i s t i l l e d water  15 130,735  13^152  T„ & dialyzed against d i s t . water o f a s o l u t i o n cont a i n i n g 1.3 O.D. units.  .881  8422.2  10.4  O.D.  3  H  14  c  3  H/ C : 14  no d i a l j s a t e  -  .770  645 13.10 1.478  1675  -  -  E F F E C T OF VARIOUS TREATMENTS ON THE RADIOACTIVITY  I N T E S T I N E DNA  OF DOUBLY-LABELLED  IN SALINE C I T R A T E .  No X max 260 nm  79.1 21.1 X max a t 260 nm  TABLE I I I  ABSORBANCE OF A SOLUTION  UV Spectrum  RAT  AND  a  DNA S o l u t i o n Fraction No.  O.D.  3  H  14  c  Distilled  3  H/  1 4  C  Total O.D.  3  H  1 4  c  Water  3  H/  1 4  C  Dialysate  UV  Spectrum  X max  1  1.910  76.8  65  1.16  1.521  -  -  2  2.960  9.41  32  .18  1.197  -  -  3  2.711  2396  509  4.71  0.823  4  1.845  4359  445  9.79  0.907  - •  -  -  5  2.275  4638  484  9.57  1.007  -  -  -  JX  6  1.917  4274  320  13.34  0.967  -  7  1.594  4185  185  22.65  0.672  -  -  -  it  8  2.678  4144  220  18.81  0.786  -  -  No  a t 260 nm  -  -  *  36.30  1.287  -  -  - .  »  92 •  44.39  1.108  -  -  -  *  4035  56  71.67  0.993  -  -  -  4038  59  68.09  0.937  -  -  -  9  1.080  4074  10  1.222  4062  11  1.158  12  0.660  112  1  JL  TABLE I V RADIOACTIVITY AND ABSORBANCY AFTER D I A L Y S I S AGAINST D I S T I L L E D OF FRACTIONS OF DOUBLY-LABELLED DNA OBTAINED BY CHROMATOGRAPHY  WATER  ON MAK.  CO  DNA S o l u t i o n Traction No. O.D.  3  H  1 4  c  C  H/  49.2  1  1.552  2  1.502  -  3  1.338  786  151  4  1.678  4192  415  5  1.317  3624  377  6  1.165  3030  7  1.513  8  D i s t i l l e d Water  -  T M  -  Total O.D.  3  H  1 4  c  3  4  Dialysate  H/  1 4  C  Spectrum  X max a t 260 nm  1.958  196  2.210  214  41  5.0  II  89.2  0.956  224  42  5.36  H  88.3  1.040  920  80  9.6  87.4  1.297  194  34  307  9.8  86.8  1.010  305  16  18.73  II  990  112  8.45  86.2  1.192  233  16  14.15  II  0.927  651  105  6.2  85.6  0.920  839  57  14.79  II  9  1.015  767  81  9.5  85.6  1.412  453  52  10  1.024  615  64  9.5  1.349  647  51  12.69  It  11  1.065  291  70  4.1  1.606  553  •30  18.48  II  12  1.826  367  68  5.3  0.617  292  36  56.7  5.2 10.0  -  55.0  UV  11.51 3.74.  8.65  8.19  II  II  II  •I  T A B L E V RADIOACTIVITY AND ABSORBANCY AFTER HEATING AND THEN D I A L Y S I S AGAINST D I S T I L L E D WATER OF FRACTIONS OF DOUBLY-LABELLED DNA OBTAINED BY CHROMATOGRAPHY ON MAK.  ui  bU The  data i n Tables  ultraviolet  absorbing  IV and V i n d i c a t e  material  been d e n a t u r e d by h e a t i n g of  t h e DNA s o l u t i o n s  aneous i n c r e a s e recovered  not treated  i s l e s s than  t h i s way. T h e r e  is a  water d i a l y s a t e s  shows no maximum a r o u n d measured  i s probably  material  o f the denatured  T h e uv s p e c t r u m o f t h e d i a l y s a t e s  o p t i c a l density  that  simult-  i n t h e amount o f u l t r a v i o l e t a b s o r b i n g  n a t i v e .DNA s o l u t i o n s the  t h e amount o f  i n t h e DNA s o l u t i o n w h i c h h a s  p r i o r to counting  i n the d i s t i l l e d  DNA s o l u t i o n s .  that  from t h e  260 nm and h e n c e  due t o t h e m a t e r i a l 1  released  from t h e d i a l y s i s t u b i n g .  spectrum o f t h e d i s t i l l e d DNA s o l u t i o n s indicates The  water d i a l y s a t e s  show an a b s o r p t i o n  the presence  On t h e o t h e r h a n d , t h e uv  maximum a t 260 nm, w h i c h  of nucleotide  r a d i o a c t i v i t y of the dialysates  also  indicates  release  that  o f small In  o f the denatured  denaturation  material  o f t h e d e n a t u r e d DNA  of nucleic  acids  3  the experiment  H / 14c i s r e l a t i v e l y  When t h e DNA s o l u t i o n s against  distilled  fraction  led to •'  i n w h i c h t h e DNA s o l u t i o n s  constant  received  throughout  were  distilled  water-,  the fractions.  no t r e a t m e n t p r i o r t o d i a l y s i s  water, the ^H/ 14  c  increased  with  increasing  number.  In t h e d i a l y s a t e s ^H/  solutions  molecular weight p a r t i c l e s .  denatured by h e a t i n g p r i o r t o d i a l y s i s a g a i n s t the  i n the dialysates.  r a t i o was h i g h e r  remaining  inside  o f t h e d e n a t u r e d DNA s o l u t i o n s , t h e t h a n i n t h e c o r r e s p o n d i n g DNA  the d i a l y s i s tubing.  This  could  solutions  be a r e s u l t o f  61 preferential  loss  of  3  H-labelled material  through  the d i a l y s i s  14 tubing  or retention An  16.0  aliquot  O.D.  of  C-labelled  o f t h e DMA  material  inside  s o l u t i o n which contained approximately  u n i t s was c o u n t e d w i t h o u t a n y p r i o r  treatment.  shown i n T a b l e I I I , t h e u n f r a c t i o n a t e d m a t e r i a l high  Dialysis in  against  the o p t i c a l  distilled  density  water  T h e r e was some uv a b s o r b i n g m a t e r i a l radioactivity  c o u l d be d e t e c t e d .  d i a l y s a t e h a d no maximum the  distilled  contained both r a d i o a c t i v i t y •an - a b s o r p t i o n maximum  b u t no  of the  On t h e o t h e r h a n d ,  The  3 14 H/ C ratio  sample with  o f the  t h a t more H - l a b e l l e d 3  than  .  optical  density  labelled  indicate  dialysis  the d i a l y s i s solution  tubing.  dialysis These  of small molecular tubing.  that prolonged d i a l y s i s  t h e d e g r a d a t i o n o f DNA b y d i p h e n y l a m i n e  causes  before  The  that heat denaturation o f the doubly-  through the d i a l y s i s  (85) o b s e r v e d  tubing.  t h e O.D. was 0.881.  DNA r e l e a s e d n u c l e o t i d e m a t e r i a l  weight which passed Petersen  through  o f t h e d e n a t u r e d DNA  1.130, w h i l e a f t e r  observations  of  solution;  and uv a b s o r b i n g m a t e r i a l ,  a t 260 nm.  C - l a b e l l e d m a t e r i a l passed  was  The u v s p e c t r u m  decrease  o f t h e h e a t d e n a t u r e d DNA  d i a l y s a t e was 21.1 w h i c h s u g g e s t s  14  o f t h e DNA  i n the d i a l y s a t e  a r o u n d '260 nm.;  water d i a l y s a t e  o f 10.5..  ZLad.: t o o n l y a s l i g h t  and r a d i o a c t i v i t y  As  as i s o l a t e d had  3 14 3 14 H and C w i t h a H/ C ratio  counts o f both  the tubing.  shorter oligonucleotides  t o pass  S u t t o n and .  of the products  i n formic  through  acid  the d i a l y s i s  61 (a)  . 1 3 . Chromatography on DEAE - C e l l u l o s e of the d i s t i l l e d water d i a l y s a t e of the mucosal DNA that h?d been denatured by heating p r i o r t o d i a l y s i s . E l u t i o n was c a r r i e d out,with a .gradient of NaCl i n 7M ureaT r i s H C l - pH 7.8  63  An which DNA  e x p e r i m e n t .was  i s lost'-.on d i a l y s i s .  which  boiling  A  contained a t o t a l  and  .distilled 398  done t o t r y t o i d e n t i f y  allowed to cool  water,  O.D.  sample o f u n l a b e l l e d o f 532  O.D.  slowly.  t h e s o l u t i o n was  c o n t a i n e d 114  fractionated (86) w h i c h  by  O.D.  After  found  units.  mucosal  u n i t s was  heated  dialysis  a total  t h e same.  The  of  The  dialysate  was  a chromatographic procedure described  i s a modification  to  against  to contain  u n i t s w h i l e i t s volume r e m a i n e d  dialysate  the m a t e r i a l •  by  o f t h e method o f T o m l i n s o n  Lee and  Tener  (87) .  DEAE-cellulose chloride  (Whatman DE22) was  f o r m as i n s t r u c t e d by  the manufacturer,  column..was p a c k e d w i t h t h e e x c h a n g e r and HC1, the  equilibrated pH  7.8  under  with 7 M urea c o n t a i n i n g  per l i t e r .  The  t o p . o f t h e c o l u m n and  gradient  prepared i n the  of sodium  s a m p l e was  e l u t i o n was  c h l o r i d e w h i c h was  30 m l  carried formed  by  u r e a - 0 .I'M  sodium  As  a t 260  nm  and  shown i n F i g u r e  13,  f r a c t i o n was  chloride  f o u r peaks  of the d i a l y s a t e . are t y p i c a l  material..  were n o t f u r t h e r  following  The  peaks  reasons.  two  liters  Five  of  f o r short  '.  of  ml  assayed for-  ultraviolet chromatographic  chain  In order to determine  salt  nucleotide  analyzed f o r the t h e number  of  -  onto  liters  T h e y were e l u t e d w i t h  concentrations which  p.s.i.  concentration.  a b s o r b i n g m a t e r i a l ' w e r e o b t a i n e d from the fractionation  two  i n the r e s e r v o i r .  absorbancy  cm  o f 0.1 IM T r i s  out w i t h a  b u f f e r e d 7."M  and e a c h  90  loaded c a r e f u l l y  7 M u r e a i n t h e m i x i n g chamber and  f r a c t i o n s .were c o l l e c t e d  2 x  a pressure of 5  of b u f f e r e d  NaCl  A  v  64  molecules o f each chain  length present,  i t i s necessary  know two o f t h e f o l l o w i n g f a c t o r s ; c h a i n oligonucleotide, present,  total  or total  known, .  of the Cor  number o f m o l e s . o f b a s e  amount o f p h o s p h a t e  nucleotide material, provided phosphate  length  nucleotide)  c o v a l e n t l y bound t o t h e  t h e number o f m o l e s o f  (mole o f b a s e ) p r e s e n t  i n each o l i g o n u c l e o t i d e i s  Phosphate a n a l y s i s would  i n d i c a t e t h e amount o f  "organic" phosphate i . e . phosphate i n t h e n u c l e o t i d e present.  to  material,  However, i t i s n o t known how many m o l e s o f p h o s p h a t e  are p r e s e n t  a t the terminal  ends o f each o l i g o n u c l e o t i d e .  The  c h a i n l e n g t h o f e a c h o l i g o n u c l e o t i d e c a n o n l y be s u r m i s e d from i t s p o s i t i o n o f e l u t i o n r e l a t i v e o f known c h a i n  length  to that of oligonucleotides  i n t h e same c h r o m a t o g r a p h i c  Hydrolysis of the nucleotide material  followed  procedure.  by paper  chromatography and q u a n t i t a t i v e s p e c t r o p h o t o m e t r y o f t h e s p o t s would i n d i c a t e t h e t o t a l moles o f each base p r e s e n t . experiments a r e beyond t h e scope o f t h i s  DEGRADATION OF THE ISOLATED DNA  An  observation H/  C was h i g h e r  d e n a t u r e d DNA  solution.  Heating  together.  small molecules binding  together  the. two c h a i n s o f  A n u n b o n d e d e n d on one o f t h e  c h a i n s might be s u s c e p t i b l e t o c l e a v a g e Terminal  that  w o u l d d e s t r o y -hydrogen b o n d i n g  as w e l l as t h o s e  t h e DNA d o u b l e h e l i x  e x p e r i m e n t s was  i n the d i a l y s a t e than i n the  w h i c h m i g h t be i n v o l v e d i n h o l d i n g to form aggregates  thesis.  BY SNAKE VENOM PHOSPHODIESTERASE  from t h e d i a l y s i s  the r a t i o o f  These  during  denaturation.  i n c o r p o r a t i o n o f one o r two n u c l e o t i d e s  could  produce  65  t h e s e f r e e ends 3'OH  end  (24).  and  and  the  3  the  products of  ?  (24) .  As  terminal  incorporation  occurs  s n a k e venom p h o s p h o d i e s t e r a s e  y i e l d i n g 5'  nucleotides,  i t was  at  cleaves  decided  to  from  examine  s n a k e venom p h o s p h o d i e s t e r a s e d e g r a d a t i o n  radioactivity.  The  presence of  a higher proportion  the  of  3  for  H  14 relative  to  C  i n the  d i a l y s a t e m i g h t be  a r e s u l t of  terminal  3 • incorporation w o u l d be  H-labelled  released  through the studies  of  from the  dialysis  precursor,  DNA  tubing  were c a r r i e d o u t  chain  on  i n t o the  on  w h i c h i f unbonded, heating  and  dialysate.  samples of  pass  These  enzyme  doubly-labelled  DNA  3 isolated  f r o m r a t s w h i c h were e x p o s e d t o  H-thymidine  for  24  14 h o u r s and  C-thymidine  s o l u t i o n s were u s e d -solution of  the  15.7  O.D.  O.D.  units of  b)  the  the  f o r these studies:  the  the  material,  pH  8.9.  ml  solution, was  of  on  0.01M  MAK  incubated  at  O.D.  followed  by  of  of  Dr.  37°C. placed  optical density  of  One  a saline-citrate DNA  which  material  obtained  on  MAK  c)  the  units, of  the  R a z z e l l , was of  the  automatic recording  an  aliquot  peak of  before  dissolved  M Tris  buffer,  phosphodiesterase a d d e d and  the  s o l u t i o n was  i n a micro-curvette.  t h i s sample was  by  entire  s a m p l e s was i n 0.04  14.4  freeze-  heat denaturation  t h e s e DNA  ml.  contained  containing  a s n a k e venom W.  DNA  residue,  magnesium c h l o r i d e  i m m e d i a t e l y and  a Gilford  a)  following  chromatography o f  Each of  One-half ml. a gift  12.5  o b t a i n e d by  being freeze-dried. 4.5  i s o l a t e d DNA  containing  i s o l a t e d DNA  in  freeze-dried  e n t i r e peak o f DNA  chromatography of  o f DNA  The  isolated, unfractionated  units  dried residue,  for 5 minutes.  recorded  The  mixture  removed  increase  in  automatically  in  spectrophotometer with  the  cell  ",  compartment incubated  maintained  at  portions  37°C  of  the  centrifuge  and 0.5  ml of  tube  was  acid-soluble  to  fractions'.  ethanol-ether They were pH  6.5  -  of  trichloroacetic  acid  centrifuged  for  20  at  was  acid  were  solution  added t o  the  precipitates (1:1  v/v)  Each  2  then  ml of  30,000  were  g.  The  pipette  in  0.5  and  ml.  of  centrifugation. acid  soluble  washed w i t h  2 ml.  anhydrous  0 . 1 M ammonium  s o l u t i o n was  ml.  albumin  solution  twice  and 2 m l o f  12  resulting  followed by  were  ml  i n cold  a Pasteur  washed  was  1% s e r u m  corresponding  d i s s o l v e d i n 1.0  0.01M M g C l .  minutes  removed w i t h  fractions  mixture  then  Ten m i c r o l i t e r s  20%  0.5  and p l a c e d  The  were The  removed  intervals,  reaction.  fraction  washings  time  the  1% t r i c h l o r o a c e t i c The  remaining mixture  stop  acid-insoluble  the  The  various  tubes.  ice-cold  each  suspension  and at  37°C.  m i x t u r e were  Servall  added t o  at  then  of  ether.  acetate  assayed  for  three  times  radioactivity.  Each a c i d - s o l u b l e with  5 ml of  ether  to  remove  the r e s i d u a l e t h e r w a s the  solution.  at 260 are  extracted  trichloroacetic  assayed  for  nm a n d r a d i o a c t i v i t y .  The  results  of  of  through  optical this  and  density  experiment  14.  observation nucleotides  i n the o t h e r  acid  removed by b u b b l i n g n i t r o g e n were  shown i n F i g u r e  release  the  was  solutions  One  than  The  fraction  two  from these graphs is  greater  samples.  is  that  the  rate  i n t h e d e n a t u r e d DNA  of  sample  67,  The  r e s u l t s obtained  sample and  the  unfractionated  d i f f e r markedly The  rate  soluble  of  uv  measuring  the  O.D.  of  the  DNA  .similar  and  fractionated  material  automatically  the  very  denatured  absorbing  f r a c t i o n recorded by  fractionated native  sample are  from those of  of r e l e a s e  obtained the  f o r the  i n t o the  agreed with  acid-soluble  sample  acid  that portions  of  a l i q u o t s w h i c h were r e m o v e d a t s p e c i f i c i n t e r v a l s .  The labelled  rate  of r e l e a s e  nucleotides  calculated  from the  of each a l i q u o t .  3  of  i n t o the  H-labelled  acid soluble  r a d i o a c t i v i t y of  From t h e  nucleotides  the  denatured,  14  and  fractions  C-  was  acid soluble  portion  f r a c t i o n a t e d DNA  sample,  3 the  rate  of r e l e a s e -  than that  release  the  H-labelled  nucleotides  was  greater  14  of- t h e  curves are  of  C-labelled nucleotides .  s i m i l a r , there  i s no  Since  indication of  the  rate  a marked  o f one  w i t h r e s p e c t t o t h e o t h e r and t h e r e f o r e i t was 3 14 concluded that H and C are u n i f o r m l y d i s t r i b u t e d throughout. the c h a i n . From t h e f r a c t i o n a t e d ' n a t i v e and unfractionated 3 DNA s a m p l e s , t h e a low  level  material..  release  of  H-labelled  corresponding to This  observation  material  a slow r e l e a s e suggests, t h a t  of  the  occurs  acid 3  at  soluble  H-labelled  material  o c c u r s w e l l w i t h i n t h e DNA s t r a n d s . On t h e o t h e r 14 hand, the r e l e a s e o f C - l a b e l l e d n u c l e o t i d e s i n t o the a c i d s o l u b l e f r a c t i o n , e s p e c i a l l y from the u n f r a c t i o n a t e d DNA, occurs slowly  at  first  and  a f t e r 20  minutes there  is a  14 release  of  C  followed  by  expected r e s u l t i f t e r m i n a l precursor  has  occurred.  a tapering  off.  incorporation  This of  is  the  a labelled  Time  in  minutes  Fig. 14. Release of u l t r a v i o l e t absorbing material ( • ) and r a d i o a c t i v i t y 3 H (O ) and C (• ) - into the acid soluble f r a c t i o n upon digestic of doubly-labelled mucosal DNA with snake venom phosphodiesterase. The^E/^C r a t i o i n the acid-insoluble f r a c t i o n (A) i s also sho'/n. ,M  68  The  distribution  of  the  labelled  nucleotides  3 the  DNA  chain  can  acid-insoluble  a l s o be  i n d i c a t e d by  fraction.  the  d e n a t u r e d , f r a c t i o n a t e d DNA  nucleotides  are  chain. ratio with  On  the  could  probably other  H/  evenly  other.  as  of  in two  labelled  Concurrent with  the  or decrease i n  of  the  the  the  d i s t r i b u t e d throughout  hand, a sharp i n c r e a s e  to the  C ratio  sample, the  i n d i c a t e a m a r k e d l o s s o f one  respect  14  I f i t remains constant,  case of the  throughout  this  nucleotides  the  observed  14 release of  3 in  the  C-labelled acid soluble material  i s an  increase  14 H/  C ratio  of  the  a c i d - i n s o l u b l e f r a c t i o n s of  f r a c t i o n a t e d and u n f r a c t i o n a t e d DNA samples. The r e s u l t s o b t a i n e d by C. M e z e i (14) f o r DNA 14 for  20  or  40  minutes to  the  denatured  the  native  C-thymidine  5 m i n u t e DNA.  On  the  are  the  exposed  s i m i l a r to those  other  hand, the  native  data  with  from  5 m i n u t e DNA s u g g e s t t h a t t h e r e i s a t e r m i n a l 14 i n c o r p o r a t i o n of C - l a b e l l e d thymidine n u c l e o t i d e . A possible e x p l a n a t i o n f o r t h e s e d i f f e r e n c e s may be t h e f o l l o w i n g . After  3 t h e DNA  m o l e c u l e s have been r e p l i c a t i n g  i n the  presence of  H-  3 thymidine  f o r 24  throughout the  hours, the  chains.  .  H may  During  the  become u n i f o r m l y d i s t r i b u t e d 20  minute pr  40  minute  14  interval  i n which  newly s y n t h e s i z e d  C-thymidine DNA  molecules  w i l l become i n c o r p o r a t e d net  result will  molecules-.  be  be  f o r DNA,  (highly l a b e l l e d with  i n t o the  a group of  T h e r e may  i s a precursor  m a i n DNA  fairly  chains  uniformly  some t e r m i n a l  the 1 4  and  small,  C),  so  the  labelled  DNA  i n c o r p o r a t i o n of  the  14 C-labelled  nucleotide,  but  this  may  be  m a s k e d by  a  greater  14 proportion  of  C-labelled material well within  the  DNA  chain  as  69 a result  o f DNA  During the  clearly and  a short  degree of  terminal  replication.  DNA  interval  replication will  i n c o r p o r a t i o n of  1 4  f r a c t i o n a t e ' ! DNA  during  has  by  been r e p o r t e d  The of  a f t e r d e n a t u r e d DNA  3  as  the  C-thymidine,  great  native  r e s u l t s of  any  might  be  unfractionated  the  denatured incorporated  However, t h i s  observation  workers.  h o w e v e r , do  not  H-labelled material material  and  terminally  denaturation.  other  above r e s u l t s ,  l a r g e amounts o f  be  s u g g e s t that-fche  ^"^C-thymidine i s l o s t not  case of  s a m p l e s . The  sample  not  1 4  C - l a b e l l e d nucleotides  i n d i c a t e d as. i n t h e  fractionate-.?.' DNA  of exposure to  e x p l a i n the i n the  presence  dialysates  i s d i a l y z e d . I t may  be  that  the  3 H-labelled  material  occurs  An  experiment which could  of  the  DNA  by  to a great  degree a t the  indicate this  phosphodiesterase  5'  would i n v o l v e  I I , which cleaves  end. degradation  from the  5"  end.  The  Purification  Tissue  Extracts After  removed, the with  and  Counting of  Formed D u r i n g  the  DNA  p h e n o l was  as  o u t l i n e d by  in  the  the  containing  interphase  Interphase Layers of  I s o l a t i o n of  and  prepared  ( 7 8 ) . The  c e n t r i f u g e tube a f t e r the  the Mucosal  aqueous l a y e r had  l a y e r formed d u r i n g  purified C.Mezei  the  the  been  DNA  carefully  deproteinization  for radioactive  phenol  the  l a y e r , which  aqueous l a y e r and  counting remained  the  interphase  had b e e n r e m o v e d , was a l s o p u r i f i e d purified  and a l i q u o t s  of the  s o l u t i o n , were c o u n t e d ( 7 8 ) .  In d o u b l e - l a b e l l i n g  experiments  i n which  3  H-thymidine  14 was a d m i n i s t e r e d 24 h o u r s b e f o r e activity  C-thymidine,  some  radio-  due t o H was o b s e r v e d i n b o t h t h e i n t e r p h a s e a n d 14 phenol l a y e r s . When t h e t i m e i n t e r v a l o f . C - i n c o r p o r a t i o n 14 3  was 5 m i n u t e s ,  the recorded  i n t e r p h a s e and t h e p h e n o l  C cpm were v e r y h i g h i n b o t h t h e  layers.  This  finding  i s attributed  to the presence o f small molecular weight newly-synthesized 14 DNA m o l e c u l e s , h i g h l y  labelled with  C, m  t h e i n t e r p h a s e and  14 C-thymidine, which DNA,  i n the phenol  DNA m a t e r i a l  had n o t y e t been i n c o r p o r a t e d  layer.  Attempts  i n t o the  were made t o i s o l a t e t h e  from t h e i n t e r p h a s e b u t these d i d n o t prove  t o be  .successful. I s o l a t i o n and C h a r a c t e r i z a t i o n o f t h e P y r i m i d i n e I s o s t i c h s Other chromatographic to f i n d  t e c h n i q u e s were t r i e d  a method f o r s e p a r a t i n g  t h e DNA m o l e c u l e s a c c o r d i n g t o  o n l y one p a r a m e t e r  i . e . molecular size,  degree o f hydrogen  bonding.  DEAE-cellulose, a g i f t t h e DNA  samples.  With  different  means o f e l u c i d a t i n g  i n mind, b e h z o y l a t e d to fractionate  but the observed  to. f o l l o w any p a r t i c u l a r  A slightly  composition or  of u l t r a v i o l e t absorbing  m a t e r i a l were o b t a i n e d o n e l u t i o n ; d i d n o t appear  this  base  f r o m D r . T e n e r , was u s e d  S e v e r a l peaks  i n an attempt  fractionation  pattern.  p r o c e d u r e was u s e d  t h e c o m p l e x p r o c e s s o f DNA  as a p o s s i b l e metabolism-.  71 Cerny et  a l . (40)  according labelled  to  chain  be  of  identified, the  which remain with  formic  determined along  the  after  10  nucleotides  chain  Similar DNA 3  was  length  to  been  then  and base  isolated for  the.  isostichs  of  were  chain  lengths  from rats 3 1/2  in  this  w h i c h were  hours  and  14  sequence  be  separated  Isostichs et  these base  from'the  in  that  can  by Cerny  their  hydrolysis  are  shown  isostichs  of  (40).  of  by Thus,  identical  investigation to  to using  composition  heterogenous  allowed  up  al.(40)  pH 3.1  pyrimidine isostichs  tried  DNA c h a i n  existing  have  subfractionated  composition  the  lengths  al.(88)  to  a  which  been removed by  chain  isolated  according  the  thymidine,  know t o  pyrimidine bases  different  isolate  experiments  H-thymidine  have  Their  et  As  bound.  on a D E A E - c e l l u l o s e column at  able  length  to  isostichs  is  on a D E A E - c e l l u l o s e column.  They  chain  fractionation Were  of  l o n g have  procedure.  they  of  if  fragments  purine'bases  Spencer  isostichs  that  possible  the  pyrimidine  animals  t h y m i d i n e was are  of  composition.  the  thought  w o u l d be  number  DNA c h a i n .  chromatography  identical  the  base  to  acid-diphenylamine.  by  this  i t  was  isostichs  by the  pyrimidine  and then  i t  labelled  Pyrimidine  separation  DNA g i v e n  nucleoside,  pyrimidines  the  length  precursor  pyrimidine could  reported  •  mixture.  i n which  incorporate  C-thymidine for  5 or  10  minutes. . T h e DNA w a s and P e t e r s e n  (89).  diphenylamine were  hydrolyzed according F o r t y m g . DNA a n d incubated  at  30°C  25 for  to  the  ml. 18  method  formic hours  of  Burton  acid-  i n the  dark.  72  The  h y d r o l y s a t e was  w a t e r and  cooled.  then The  u n d e r vacuum t h r o u g h filter  was  trapped to  diluted  greenish-white  washed w i t h  on  a flash  40 ml  The  the  repeated  of the  t h e pH  The  total  final  removed by filter  filtrate  v o l u m e was  b u f f e r pH  The  was  obtained  filtration  t o 80 m l  NaOH and  and  it  in air.  dried  from  with  Whatman  The  The  0.1N 0.01M  The  was  diphenylamine  a fine-grade distilled  water.  NH^OH and  the  lithium  acetate  t h e pH  0.5IN/HC1 as recommended by rinsing  E q u i l i b r a t i o n was  t o 5.3  settled,  decanted.  One the  c o l u m n was  with  s l u r r y was  o f 0.01M  poured  washed w i t h t h i s  into  washed  acetic  lithium cm  buffer until  suspending  acid  and  After  c o n t a i n i n g the  a 25  with  the  a c h i e v e d by  0.1M  through  i n a b s o l u t e ethanol',  l i t h i u m hydroxide.  the supernatant  hundred ml.  the  (DE-32 M i c r o g r a n u l a r )  D E A E - c e l l u l o s e was  following a final  s l u r r y had  The  distilled  the m a t e r i a l which passed  f i v e grams o f t h e powder i n 30 m l _ o f  a d d e d and  20 m l .  the  evaporated  f o r chromatography o f  Only  used.  manufacturer  adjusting  ml  3.5.  through  washed w i t h  to use.  mesh s i e v e was  c y c l e s o f 0.5'N  was  200  was  subsequence e v a p o r a t i o n  neutralized with  made up  and any  re-evaporated.  s o l u t i o n was  D E A E - c e l l u l o s e used  sieved prior  a 200  filtered  5.3.  h y d r o l y s a t e was and  and  filtrate  Another  s o l u t i o n was  w a t e r and  sintered-glass  distilled  w a t e r t o remove  combined  addition of d i s t i l l e d  p r e c i p i t a t e was  with  p r e c i p i t a t e was  distilled  evaporator.  w a t e r were a d d e d and  until  ml.  a fine-grade sintered-glass f i l t e r  oligonucleotides.  20 m l .  t o 200  the  fines  a c e t a t e pH x 1.5 t h e pH  cm  was  5.3  were  column.  and  O.D.  at  73  260  run o f e f f l u e n t  neutralized  w e r e t h e same a s t h o s e  dilute hydrolysate  was  t h e n washed w i t h  was  free  1  liter  was a b s o r b e d  starting buffer  of ultraviolet  until  absorption.  o f 0.01 M l i t h i u m  of the eluent.  on t h e column, the effluent  A salt  acetate buffer  gradient,  pH 5.3, i n t h e r e s e r v o i r . ,  F i f t e e n ml.  fractions  assayed  absorbancy  for  The  fractions  within  The  a c e t a t e pH 5.3for  e a c h peak o b t a i n e d  solution  formed by  elution.  conductivity.  f r o m t h e above  The-pH o f t h e s o l u t i o n s  t o 5.0 a n d t h e v o l u m e o f e a c h  O.D-,,,. '} df e a c h  solution  a n d e a c h f r a c t i o n was  a t 260 nm a n d  c h r o m a t o g r a p h y were c o m b i n e d . adjusted  was u s e d  were c o l l e c t e d  which  pH 5.3 i n t h e m i x i n g  chamber a n d a h e q u a l v o l u m e o f . O.0|.':.M l i t h i u m O.HM L i C l  The  solution  was m e a s u r e d  was  was m e a s u r e d .  and t h e t o t a l  number  2.70 HON.  of was  moles o f t h y m i d y l i c calculated  from  acid  and c y t i d y l i c  the following  acid  i n each  solution  equation, according to  Spencer e t al.(98) zx-moles- b a s e =  oAn**  "  7<>  O.D. " •' x 10 J 27  8.68  c *  0  volume i n l i t e r s  x Volume  (liters)  270 where  ^ ^ for  thymidylic  acid  and d e o x y c y t i d y l i c  acid i s  3 8.68 since  x 10 .  This  equation  a t pH 5.0, b o t h  c a n be u s e d  thymidylic  have'the', same-.;•' a b s o r b a n c e  acid  a s 270 nm.  for  this  calculation  and d e o x y c y t i d y l i c  acid  74 Inorganic were d e t e r m i n e d length the be  and t o t a l phosphorus b y t h e method o f K i n g  of the i s o s t i c h  from  (91).  the formula  i n each  isostich  Py ."p.. n (n4=l) J  r  t h e number o f m o l e s o f p y r i m i d i n e p r e s e n t During  the formic  b a s e and s u g a r the  isostichs  a r e removed  The  results  Isostich No. I II III IV V VI VII VIII  leaving  i n each i s o s t i c h  radioactivity  a r e known,  f r a c t i o n can  w h e r e Py n  refers  to  J  i n each  hydrolysis,  isostich. the purine  a p h o s p h a t e on e a c h e n d o f  and hence t h e f o r m u l a  phosphate present The  acid-diphenylamine  fractions  I f the chain  and o r g a n i c phosphate p r e s e n t  t o t a l moles o f base p r e s e n t calculated  i n the i s o s t i c h  f o r t h e number o f m o l e s o f is(n>l]«  o f e a c h i s o s t i c h was a l s o  measured.  a r e shown i n F i g u r e s 15 and 16 a n d T a b l e s V I a n d V I I .  O.D.  Volume (ml)  270/ml.  umoles pyrimidine  26 56 97* 111 55 70 42 88  0.172 0.113 0.210 0.260 0.127 0.258 0.120 0 .118  520 736 2328 3330 770 2100 506 1014  TABLE V I - RADIOACTIVITY AND isolated  formic-acid-diphenylamine  14 C f o r5  minutes.  1 4  c  cmp/ml  40 112 2966 2112 3500 6828 3820 4960  6 96 72 74 72 94 74 91  -ABSORBANCY• * o f t h e p y r i m i d i n e  by chromatography  w h i c h was a l l o w e d  H cpm/ml. 3  on D E A E - C e l l u l o s e  of the  h y d r o l y s a t e o f mucosal  to incorporate H 3  isostichs  DNA,  f o r 3 1/2 h o u r s a n d  L  *  F i g . 15- Chromatography on DEAE - Cellulose of the diphenylamine - formic acid hydrolysate of DNA,  3 I  had been l a b e l l e d with  '  14  H f o r 3g- hours 'and  gradient of L i C l i n l i t h i u m  which  acetate - pH 5.3  C f o r 5 minutes. E l u t i o n was carried out with a '  '  •  '  40  T u b e  N o .  F i g . 16. Chromatography on DEAE - Cellulose of the diphenylamine 3 U been l a b e l l e d with  H f o r 3§- hours and  of L i C l i n l i t h i u m acetate - pH  5.3  C f°  r  - formic acid hydrolysate  of DNA,  which had  10-'aainutes. E l u t i o n was c a r r i e d out with a gradient  77 Isostich No.  Volume (ml)  O.D. 270/ml.  umoles Pyrimidine  42 41 174 56 117 90  2.153 0.851 0.740 0.692 0.143 1.510  10,500 4,018 14,964 4,480 1,872 15,570  I II III IV V VI  TABLE V I I - RADIOACTIVITY AND isolated  3  H  1 4  cpm/ml  172 512 850 800 72410 4932  48 96 108 98 2200 448  ABSORBANCY o f t h e P y r i m i d i n e I s o s t i c h s  b y c h r o m a t o g r a p h y on D E A E - C e l l u l o s e  formic acid-diphenylamine w h i c h was  allowed  c  cpm/ml  of the  h y d r o l y s a t e o f mucosal f o r 3 1/2  to incorporate H 3  DNA,  h o u r s and  14 C f o r 10 The  elution  i n Figure 15, fractions.  minutes. profile  f o r t h e 5 m i n . DNA,  shows t h e p r e s e n c e  according  m e t h o d o f S p e n c e r e t al» ( 9 0 ) The r a d i o a c t i v i t y  fractions of  of eight separate  isostich  The number o f m i c r o m o l e s o f b a s e p r e s e n t  i s o s t i c h was c a l c u l a t e d  VI.  as i l l u s t r a t e d .  VI and V I I I  t o the  as i n d i c a t e d  ;'yumoles o f p y r i m i d i n e .  spectrophotometric  and t h e v a l u e s  due t o t r i t i u m  i n each  are l i s t e d  i n Table  i s located mainly i n  by t h e r a t i o  o f H-cpm/no. 3  On t h e o t h e r h a n d , t h e r a t i o o f  14 C cpm/no o f and  pinoles o f pyrimidine  VIII contained  indicate that isostichs II 14 a greater proportion of C-^labelled m a t e r i a l .  In the experiment with were o b t a i n e d 3  Hand  a s shown i n F i g u r e 1 6 .  75% o f t h e  In order accordings isostichs or  t h e 10 m i n . DNA,  14 C were r e c o v e r e d  Approximately m  t o s u b f r a c t i o n a t e these  t o base  composition,  of i d e n t i c a l  only  the f i f t h  isostich  i t i s necessary  s i x peaks 90% o f t h e  isostich.  fractions to pool the  chain length which are obtained  two'other f r a c t i o n a t i o n s  i n order  f r o m one  t o o b t a i n enough m a t e r i a l "  78  to  be  d e t e c t e d on  experiment using isolated, and to  2 rats,  However i n e a c h  approximately  repeat  the  fractionation.  i n v e s t i g a t i o n s , suggest  homogeneous DNA activities.  molecules  These r e s u l t s  labelling  30 mg  a l l o f w h i c h i s r e q u i r e d f o r one  chromatography experiment.  these  be  elution.  o f DNA  are  that these  e x t e n d e d , i n w h i c h l a r g e amounts o f DNA  enough  are  DNA  obtained  sequences  which have d i f f e r e n t suggest  not  tentative results  that there  be  formic acid-hydrolysis  Hence t h e r e was The  can  in  of  metabolic experiments collected.  should  79  SUMMARY 3 1.  The p a t t e r n  of incorporation  thymidine i n t o  DNA  The l a b e l l e d  DNA/  of  H- and  C-labelled  o f r a t i n t e s t i n a l mucosa was u s e d  means o f e l u c i d a t i n g 2.  i n vivo  14  t h e c o m p l e x p r o c e s s o f DNA  isolated  f r o m t h e mucosa, was  metabolism. fractionated  on a column o f me'thylated-albumin k i e s e l g u h r , w h i c h t y p e s o f DNA  a c c o r d i n g to base  c o n t e n t o f t h e DNA, to  37.0% i n t h e l a s t  f r o m t h e MAK 3.  ranged  composition.  o f t h e DNA  separated  The G + C  f r o m 48.1% i n t h e f i r s t  fraction  as a  material  fraction eluted  column.  The l a b e l l i n g  e x p e r i m e n t s were a c o n t i n u a t i o n o f t h o s e  p r e v i o u s l y done i n t h i s  laboratory.  In the e a r l i e r  3 •experiments, 24 h o u r s  later  f o r t y minutes sacrificed experiment  the  was  C-thymidine after  this  injected was  second  into  the r a t s  administered. injection,  and  Twenty o r  t h e a n i m a l s were  a n d t h e i n t e s t i n a l DNA i s o l a t e d . I n t h e 40 m i n . 3 14 t h e H/ C r a t i o o f t h e DNA f r a c t i o n s f r o m MAK  chromatography 3  H-thymidine 14  was  c o n s t a n t , b u t i n the- 20 m i n .  experiment,  14 H/  C ratio  increased with increasing  fraction  This observation  suggested the p o s s i b i l i t y  of metabolic  heterogeneity occurring within  the mucosal  DNA.  number.  In the  p r e s e n t i n v e s t i g a t i o n , t h e a n i m a l s were s a c r i f i c e d 5 m i n . 14 after injection of C-thymidine. The r e s u l t s s u b s t a n t i a t e the e a r l i e r f i n d i n g o f m e t a b o l i c h e t e r o g e n e i t y i n t h e DNA.  3 In t h e 5 mm.  experiment,  maximum a t f r a c t i o n results  the  14  H/  C ratio  increased  4 o r 5 a n d then' d e c r e a s e d .  i n conjunction with e a r l i e r  to a  These  f i n d i n g s were  interpreted  80 to  indicate  that  newly synthesized,  small molecular  weight  14 DNA, w h i c h was beginning  of  the  C ratio  H/  c o u l d be are  highly labelled DNA p e a k  in  loose  the  C was  eluted  at  f r o m MAK c h r o m a t o g r a p h y .  later  fractions  aggregates  held together  with  of  these  of  the  The  5 min.  As the  low  experiment  small molecules  by hydrogen bonds.  the  which  interval  of  14 DNA s y n t h e s i s  i n the  presence  two p r o c e s s e s  occur.  First,  m o l e c u l e s - g r a d u a l l y become  of  C-thymidine  the  increased,  n e w l y s y n t h e s i z e d DNA  incorporated  into  the  high  m o l e c u l a r w e i g h t s t a b l e DNA f r a c t i o n , w h i c h i s l a b e l l e d w i t h 3 14 H. A f t e r 25 m i n u t e s , v e r y l i t t l e C-thymidine remains for i n c o r p o r a t i o n i n t o DNA a n d t h e DNA m o l e c u l e s n e w l y s y n t h e s i z e d 3 14 are  mostly  ratio It  is  was  unlabelled  constant  and t h u s  throughout  found that a f t e r  thymidine,  the  distributed  a  24  after  the  the  the  fractions.  hour  incorporation with  r a d i o a c t i v i t y due  throughout  40 m i n u t e s ,  to  3  H was  DNA f r a c t i o n s  not  H/  3  C  H -  uniformly  from the  MAK c o l u m n .  3 This  was  This  finding is  in- the similar to  shown b y  to  those  time  H-thymidine to  double  3 1/2  variation  attributed  intestinal  3  the  to  mucosa. above  was  hours  incorporation  the  hours.  d i d not  H to  absorbancy  high turnover  that  the  were  the  rate  carried  period of  Shortening 3 14  alter  H/  the  of  ratio. cells  out  exposure exposure  C pattern  in  the  experiments. 3  pattern  the  Experiments  except  3 .1/2  m  The  observed  of  the  manner  the  20 m i n . DNA f r a c t i o n s  i n which the  the  14 H/  DNA w a s were  C ratios treated  denatured  was  before  influenced counting.  by heating,  the  by If  81 3  14  H/  C r a t i o was  constant  throughout  the  fraction. 3  t r e a t m e n t was was  not  a rising  c a r r i e d out,  curve,  Quantitative  as  found  the  DNA  the  denaturation  against  distilled  through the 3 14 The  H/  of the was  cleaved  during  water, t h i s  dialysis  C ratio  of  s o l u t i o n of  higher  and  processes  indicated that  o l i g o n u c l e o t i d e m a t e r i a l was during  the  the  the  involved  from the  DNA  the  d e n a t u r e d DNA  d e n a t u r e d DNA. 3  DNA  of  H-labelled  have been  molecules  and  This  indicated that  the  H-labelled material  p o i n t was  not  took p l a c e  size  a means o f  only,  since  separating  S u e o k a and  chromatography allows according  out  dialysate  incorporated  released  during  heat  denatured  s n a k e venom  C-thymidine might  be  Another  preferential release  from the  5"* end.  This  tested.  Other chromatographic techniques to find  lost  finding  t e r m i n a l l y i n a p r e f e r e n t i a l manner.  p o s s i b i l i t y which remains i s t h a t the 3  step  material  m o l e c u l e s were d e g r a d e d w i t h 14  of  weight  sample  sample i n t o the  and  incorporated  after  m a t e r i a l which passed  However, e x p e r i m e n t s i n w h i c h t h e  phosphodiesterase,  ratio  m a t e r i a l was  denaturation. n a t i v e DNA  C  following dialysis  nucleotide  than t h a t of the  i n t o the  H/  tubing.  d i a l y s a t e , which could  terminally  of  14  small molecular  nucleotide  suggested a p r e f e r e n t i a l r e l e a s e into  pattern  this  in earlier investigations.  examination of  the i s o l a t i o n of  the  If  to three  the  were u t i l i z e d  DNA  molecules  Cheng had  separation  parameters.  No  attempt  according  reported o f DNA  i n an  that  to MAK  molecules  conclusive  r e s u l t s were  82  obtained with  fractionation 3  The  pattern of  w o u l d n o t be molecular 8.  DNA  H/  C ratio  o f t h e DNA  the  and  I I and  this  In the 3  activity While  due  these  to both  .  further  based  result on  the  5 and  due  to H  10  On.the  and  minute 5-minute  was  3  t h a t due  located mainly 14 to C was i n  10 m i n u t e e x p e r i m e n t  the  radio-  14 H and  f i n d i n g s may  n o t .occur i n t o  The  procedure.  VIII, while  VI.  this  formic acid-diphenylamine  radioactivity  i s o s t i c h s . VI  and  fractions.  i s o s t i c h s were i s o l a t e d .  experiment,  isostichs  irregular  i f c h r o m a t o g r a p h y was  s a m p l e s were a n a l y z e d by  in  was  s a m p l e s were t r e a t e d ! w i t h  pyrimidine  DEAE-cellulose.  14  expected  size  of benzoylated  C was  indicate  located i n isostich  t h a t i n c o r p o r a t i o n does  a l l pyrimidine clusters  confirmatory experiments  V.  to the  s h o u l d be  same  carried  extent, out.  the  83 BIBLIOGRAPHY  1.  Miescher, F., Hoppe-Seyler's Untersuchungen 441 ( 1 8 7 1 ) .  Medicinisch-chemische  2.  M u l l e r , W., i n " I n t r o d u c t i o n t o N u c l e i c A c i d s " e d . E . H a r b e r s R e i n h o l d Book C o r p o r a t i o n p g . 32-87 New Y o r k 1968.  3.  W a t s o n , J.D. a n d C r i c k , F.H.C., N a t u r e  4.  H o l l a e n d e r , A . a n d Emmons, C.W., C o l d S p r i n g H a r b o r Q u a n t . B i o l . 9, 179 ( 1 9 4 1 ) .  5.  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