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The biosynthesis of deoxyribonucleic acid in vivo in the intestinal mucosa of rat Mezei, Catherine 1964

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THE BIOSYNTHESIS OF DEOXYRIBONUCLEIC  ACID  IN VIVO IN THE INTESTINAL MUCOSA OF RAT  by Catherine  Mezei  D i p l . Pharm*, U n i v e r s i t y o f M e d i c a l S c i e n c e s , B u d a p e s t 1954 M . S c , 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 , 1960  A THESIS SUBMITTED  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  In t h e D e p a r t m e n t 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 r e q u i r e d standard f o r t h e degree o f DOCTOR OF PHILOSOPHY  The U n i v e r s i t y o f B r i t i s h A p r i l 1964  Columbia  In the  presenting  r e q u i r e m e n t s f o r an advanced  British  Columbia, I agree  available mission  for reference  for extensive  representatives.  cation  Department o f  May 7,  1964.  the L i b r a r y  and s t u d y .  shall  I further  agree for  for  that  f i n a n c i a l gain  permission-  Biochemistry Columbia,  of •  make i t f r e e l y that  per-  scholarly  by t h e Head o f my Department  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada Date  fulfilment of  degree a t the U n i v e r s i t y  I t i s understood  of this thesis  w i t h o u t my w r i t t e n  that  i npartial  copying of t h i s thesis  p u r p o s e s may be g r a n t e d his  this thesis  o r by  copying or p u b l i -  shall  n o t be a l l o w e d  The U n i v e r s i t y o f B r i t i s h Columbia FACULTY OF GRADUATE STUDIES  PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE  DEGREE OF  DOCTOR. OF PHILOSOPHY  \  of  CATHERINE MEZEI  D i p l . Pharm. U n i v e r s i t y o f M e d i c a l Sciences Budapest, Hungary, 1954 . M.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1960  THURSDAY, MAY 7, 1964, a t 9:30 A.M. IN ROOM 231,  MEDICAL SCIENCES BUILDING, BLOCK A  COMMITTEE IN CHARGE Chairman: F.H. Soward J.J.R. Campbell A.W. Matthews M. D a r r a c h Ross Stewart P.H. J e l l i n c k G.M. Tener S.H.' Zbarsky External  Examiner:  Department  R.O. Hurst  of Biochemistry  Queen's U n i v e r s i t y  THE BIOSYNTHESIS OF DEOXYRIBONUCLEIC ACID IN VIVO IN THE INTESTINAL MUCOSA OF RAT ABSTRACT  The i n v i v o b i o s y n t h e s i s o f d e o x y r i b o n u c l e i c a c i d (DNA) from l a b e l l e d thymidine has been i n v e s t i g a t e d i n r a t i n t e s t i n a l mucosa. The DNA p r e p a r a t i o n s were f r a c t i o n a t e d by column chromatography and .the f r a c t i o n s were assayed f o r r a d i o a c t i v i t y by l i q u i d s c i n t i l l a t i o n c o u n t i n g methods. In t h e f i r s t experiments t h e DNA was i s o l a t e d from the i n t e s t i n a l mucosa o f r a t s which had r e c e i v e d H3thymidine 5, 10 or 20 minutes or 24 hours b e f o r e sacrifice. When the macromolecules were f r a c t i o n a t e d on E C T E O L A - c e l l u l o s e t h e r e s u l t s o b t a i n e d were i n c o n c l u s i v e because no d e f i n i t s p a t t e r n o f i n c o r p o r a t i o n of r a d i o a c t i v i t y was observed i n t h e f r a c t i o n s . Chromatography on E C T E O L A - c e l l u l o s e was c o n s i d e r e d u n s a t i s f a c t o r y , because o f t h e v a r i a t i o n s i n t h e e l u t i o n p a t t e r n s o f DNA p r e p a r a t i o n s from, experiment t o e x p e r i ment and e v i d e n c e i n d i c a t i n g d e g r a d a t i o n o f DNA d u r i n g the f r a c t i o n a t i o n p r o c e d u r e . In subsequent experiments f r a c t i o n a t i o n on m e t h y l a t e d a l b u m i n - k i e s e l g u h r (MAK) columns was employed and double l a b e l l i n g experiments were c a r r i e d out. The animals were i n j e c t e d i n t r a v e n o u s l y w i t h H - t h y m i d i n e and 24 hours l a t e r w i t h C ^ - t h y m i d i n e . The r a t s were k i l l e d 20 or 40 minutes a f t e r t h e second i n j e c t i o n and the double l a b e l l e d DNA was i s o l a t e d from t h e i n t e s t i nal mucosa. On f r a c t i o n a t i o n by MAK columns reproduc i b l e e l u t i o n p a t t e r n s were o b t a i n e d even a f t e r storage of t h e DNA s o l u t i o n s . The main DNA peak was always e l u t e d at t h e same range of sodium c h l o r i d e c o n c e n t r a t i o n and 95-97 percent o f the r a d i o a c t i v i t y was e l u t e d i n t h i s peak. Each s u b f r a c t i o n c o m p r i s i n g the.main peak was examined f o r H and C ^ a c t i v i t y . By s t u d y i n g the H^/cl^ r a t i o s o f t h e f r a c t i o n s newly s y n t h e s i z e d m a t e r i a l c o u l d be compared w i t h o l d e r , presumably s t a b i l i z e d DNA. When t h e animals were exposed t o t h e C ^l a b e l l e d thymidine f o r 40 minutes t h e H / C ^ r a t i o s of the s u b f r a c t i o n s were c o n s t a n t , i n d i c a t i n g no m e t a b o l i c d i f f e r e n c e s between t h e newly s y n t h e s i z e d DNA ( C ^ l a b e l l e d and the. " o l d " ( H - l a b e l l e d ) DNA. However, 3  3  L  3  3  1  when the time of exposure to the C ^ - l a b e l l e d -precursor • i h v i v o was 20 minutes, the '&?/G^ r a t i o s of s u b t r a c t i o n s i n c r e a s e d as the sodium c h l o r i d e c o n c e n t r a t i o n of the e l u a n t i n c r e a s e d . These r e s u l t s i n d i c a t e d some m e t a b o l i c d i f f e r e n c e s amongst these f r a c t i o n s . Stepwise enzymatic d e g r a d a t i o n by snake venom phosphodiesterase of the double l a b e l l e d DNA p r e p a r a t i o n s , and the main peak o b t a i n e d a f t e r MAK chromatography, i n d i c a t e d the i n c o r p o r a t i o n of thymidine i n t o newly s y n t h e s i z e d and " o l d " DNA o c c u r r e d w e l l w i t h i n the c h a i n .  GRADUATE STUDIES  Field  of Study:  Biochemistry V . J . O'Donnell  Endocrinology  W.J.  Enzymol. ogy  Polglase  Metabolism  S t a f f of the Department of B i o c h e m i s t r y  M o l e c u l a r S t r u c t u r e and B i o l o g i c a l Function  S t a f f of the Department of B i o c h e m i s t r y  Intermediary  Related  Studies:  Advanced Mammalian Physiology Immunochemistry I n t r o d u c t i o n to V i r u s e s Neurochemistry  S t a f f of the Department of P h y s i o l o g y D.C.B. Duff J.E. P.L.  Bismanis McGeer  PUBLICATION A Comparison  of Methods f o r the I s o l a t i o n o f D e o x y r i -  b o n u c l e i c A c i d from Small Amounts o f Rat T i s s u e . C. Mezei and S. H. Zbarsky. Physiol.  40, 1167  (1962).  Can. J . Biochem. &  i ABSTRACT The  ir> v i v o b i o s y n t h e s i s o f d e o x y r i b o n u c l e i c a c i d  has been i n v e s t i g a t e d i n r a t i n t e s t i n a l mucosa. c h e m i c a l and m e t a b o l i c h e t e r o g e n e i t y o f DNA were f r a c t i o n a t e d by column c h r o m a t o g r a p h y  (DNA)  from l a b e l l e d  thymidine  Because o f t h e suggested p h y s i c o -  o f mammalian o r i g i n , t h e DNA  preparations  and t h e f r a c t i o n s t h u s o b t a i n e d were  a s s a y e d f o r r a d i o a c t i v i t y by l i q u i d s c i n t i l l a t i o n c o u n t i n g methods. In t h e f i r s t  e x p e r i m e n t s t h e DNA  was  i s o l a t e d f r o m t h e i n t e s t i n a l mucosa o f  3 r a t s w h i c h had r e c e i v e d H - t h y m i d i n e 5, 10 o r 20 m i n u t e s o r 24 h o u r s b e f o r e The method o f e x t r a c t i o n o f t h e DNA  was  sacrifice.  b a s e d on t h e d i s i n t e g r a t i o n o f t i s s u e s by  h i g h f r e q u e n c y s o n i c o s c i l l a t i o n s , e x t r a c t i o n o f n u c l e o p r o t e i n from t h e n u c l e a r f r a gments w i t h s t r o n g s a l t s o l u t i o n s and d e p r o t e i n i z a t i o n o f DNA alcohol mixtures.  When t h e m a c r o m o l e c u l e s  with chloroform-amyl  were f r a c t i o n a t e d on  ECTEOLA-cellulose,  and t h e f r a c t i o n s were a s s a y e d f o r r a d i o a c t i v i t y , t h e r e s u l t s o b t a i n e d were i n c o n c l u s i v e b e c a u s e no d e f i n i t e p a t t e r n o f r a d i o a c t i v i t y was  observed i n the  fractions.  F u r t h e r , the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the f r a c t i o n s v a r i e d from to experiment.  B e c a u s e o f t h e s e f i n d i n g s i t was  C h r o m a t o g r a p h y on E C T E O L A - c e l l u l o s e was e l u t i o n p a t t e r n s o f DNA  d i f f i c u l t t o draw any c o n c l u s i o n s .  c o n s i d e r e d u n s a t i s f a c t o r y , because of the  p r e p a r a t i o n s were n o t r e p r o d u c i b l e f r o m e x p e r i m e n t t o e x -  periment, the chromatographic  p r o f i l e s changed on s t o r a g e o f t h e DNA  and b e c a u s e o f e v i d e n c e i n d i c a t i n g d e g r a d a t i o n o f DNA fractionation  experiment  solutions,  preparations during the  procedure.  B e c a u s e o f t h e i n c o n c l u s i v e r e s u l t s o b t a i n e d by ECTEQLA-chromatography, f r a c t i o n a t i o n on m e t h y l a t e d a l b u m i n - k i e s e l g u h r (MAK) subsequent  experiments.  c o l u m n s was  employed i n t h e  A l s o , a s a more s e n s i t i v e means t o w a r d s d e t e c t i n g m e t a b o l i c  d i f f e r e n c e s amongst t h e components i s o l a t e d f r o m t h e DNA  o f i n t e s t i n a l mucosa, d o u b l e  l a b e l l i n g e x p e r i m e n t s were c a r r i e d o u t . The a n i m a l s were i n j e c t e d i n t r a v e n o u s l y w i t h 3 14 H - t h y m i d i n e and 24 h o u r s l a t e r were i n j e c t e d w i t h C - t h y m i d i n e . The r a t s were  XX  k i l l e d 20 o r 40 m i n u t e s a f t e r t h e s e c o n d i n j e c t i o n , and t h e d o u b l e l a b e l l e d DNA was i s o l a t e d f r o m t h e i n t e s t i n a l mucosa by t h e p h e n o l t r e a t m e n t o f C o l t e r _et a l . (147).  On f r a c t i o n a t i o n by MAK c o l u m n s r e p r o d u c i b l e  t a i n e d even a f t e r s t o r a g e  o f t h e DNA s o l u t i o n s .  e l u t i o n p a t t e r n s were o b -  The main DNA peak was a l w a y s  e l u t e d a t t h e same r a n g 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 t was f o u n d  95 - 97 p e r c e n t o f t h e r a d i o a c t i v i t y was e l u t e d i n t h e main DNA peak. t h i s , each s u b f r a c t i o n c o m p r i s i n g vity.  By s t u d y i n g  t h e main peak was examined f o r H  14  synthesized  DNA  r a t i o s of the subfractions increased eluant  increased.  fractions.  d i f f e r e n c e s between t h e n e w l y 3  time o f exposure t o the C ^ - l a b e l l e d precursor  However, when t h e  in_ v i v o was 20 m i n u t e s , t h e  as t h e sodium c h l o r i d e c o n c e n t r a t i o n  These r e s u l t s i n d i c a t e d some m e t a b o l i c  H^/C^  of the  d i f f e r e n c e s amongst t h e s e  The r e l a t i o n o f t h e s e f i n d i n g s t o t h e b i o s y n t h e s i s and r e p l i c a t i o n o f  DNA i n t h e i n t e s t i n a l mucosa o f r a t has been Stepwise enzymatic degradation d o u b l e l a b e l l e d DNA p r e p a r a t i o n s ,  discussed.  by snake venom p h o s p h o d i e s t e r a s e o f t h e  and t h e main peak o b t a i n e d  a f t e r MAK c h r o m a t o -  graphy, i n d i c a t e d t h e i n c o r p o r a t i o n o f t h y m i d i n e i n t o newly s y n t h e s i z e d DNA o c c u r r e d  material  - r a t i o s o f the sub-  ( C - l a b e l l e d ) and t h e " o l d " ( H - l a b e l l e d ) DNA. 1 4  acti-  14  - l a b e l l e d t h y m i d i n e f o r 40 m i n u t e s t h e H /C i n d i c a t i n g no m e t a b o l i c  14  When t h e a n i m a l s were e x 3  f r a c t i o n s were c o n s t a n t ,  Because o f  and C  3 14 t h e H /C r a t i o s o f t h e i f r a c t i o n s newly s y n t h e s i z e d  c o u l d be compared w i t h o l d e r , p r e s u m a b l y s t a b i l i z e d DNA.  posed t o t h e C  3  that  w e l l within the chain.  S. H. Zbarsky  and " o l d "  iii  ACKNOWLEDGEMENTS  The ciation ment.  author  wishes t o express  h e r s i n c e r e t h a n k s and a p p r e -  t o D r . S. H. Z b a r s k y f o r h i s c o n t i n u a l a d v i c e and e n c o u r a g e The many h e l p f u l d i s c u s s i o n s w i t h D r . G. M. T e n e r , D r . P.  H. J e l l i n c k  and D r . G. G. J a c o l i a r e g r e a t l y a p p r e c i a t e d .  Thanks a r e due a l s o t o D r . M. S m i t h o f t h e T e c h n o l o g i c a l Research Laboratory,  F i s h e r i e s Research Board, f o r h i s kind c o -  o p e r a t i o n i n t h e i n i t i a l s t a g e s o f t h e MAK c h r o m a t o g r a p h y . Dr.  I . A. S r e t e r o f t h e D e p a r t m e n t o f Anatomy, 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 , and D r . Wm. L. Dunn k i n d l y p e r f o r m e d injection  of the radioactive precursors a t various  Thanks a r e due a l s o t o Mrs. for their  intravenous  times.  B. E. S t e w a r t and M i s s B. F o r d  a s s i s t a n c e i n c e r t a i n p a r t s o f t h e work.  I t 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 o f the N a t i o n a l Research C o u n c i l i n t h e form o f S t u d e n t s h i p s .  iv TABLE DF CONTENTS Page.  INTRODUCTION  1  The e a r l y I n v e s t i g a t i o n s o f DNA The C o n v e r s i o n  Biosynthesis  1  o f T h y m i d i n e t o Thymine N u c l e o t i d e s and  DNA  3  The E n z y m a t i c 5 y n t h e s i s o f DNA  4  The C e l l u l a r L o c a t i o n o f DNA  9  Synthesis  The R e p l i c a t i o n o f DNA  10  The P h y s i c a l and C h e m i c a l H e t e r o g e n e i t y The C h r o m a t o g r a p h y o f DNA (ECTEOLA) Columns The C h r o m a t o g r a p h y o f DNA  o f DNA  on S u b s t i t u t e d C e l l u l o s e 18 on  Methylated-Albumin-Kieselguhr  Columns  22  The M e t a b o l i c H e t e r o g e n e i t y The P r e s e n t  17  o f DNA  23  Investigation  27  EXPERIMENTAL I.  Materials  i  30  1.  Experimental  2.  Radioactive materials  I I . P r e p a r a t i o n o f DNA 1.  animals  30 30  f r o m S m a l l I n t e s t i n a l Mucosa o f R a t  Method o f Medawar and Zubay  (159)  30  2. .Method o f C o l t e r e t a l . (147) I I I . F r a c t i o n a t i o n o f t h e DNA 1.  32  Preparations  F r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e  30  33 exchanger  33  V  Page 2.  F r a c t i o n a t i o n on MAK c o l u m n s  34  3.  D e s a l t i n g o f t h e DNA f r a c t i o n s a f t e r C h r o m a t o g r a p h y ..  38  Gel  filtration  40  Dialysis  41  IV. E n z y m a t i c D e g r a d a t i o n o f DNA 1.  2.  v.  42  The p u r i f i c a t i o n o f snake venom p h o s p h o d i e s t e r a s e f r o m t h e c r u d e venom  42  T e s t f o r D e o x y r i b o n u c l e a s e a c t i v i t y i n t h e snake venom p h o s p h o d i e s t e r a s e p r e p a r a t i o n s  44  3.  A s s a y o f t h e snake venom p h o s p h o d i e s t e r a s e a c t i v i t y . . .  45  4.  S t e p w i s e d e g r a d a t i o n o f DNA by t h e p u r i f i e d venom p h o s p h o d i e s t e r a s e  47  snake  R a d i o a c t i v e Counting Procedures  49 3  1.  Liquid s c i n t i l l a t i o n counting of H DNA p r e p a r a t i o n s  2.  L i q u i d s c i n t i l l a t i o n counting o f the double (C and H ) l a b e l l e d DNA p r e p a r a t i o n s  52  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 l a y e r s o f the t i s s u e e x t r a c t s  67  4.  The p u r i f i c a t i o n  69  5.  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 a c i d - i n s o l u b l e m a t e r i a l obtained d u r i n g the enzymatic degradation o f DNA s o l u t i o n s  1 4  3.  6.  -labelled  3  and c o u n t i n g o f t h e p h e n o l l a y e r s ...  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 a c i d - s o l u b l e f r a c t i o n obtained during the enzymatic degradation . o f DNA s o l u t i o n s  49  69  71  RE5ULT5 AND DISCUSSION I.  E x p e r i m e n t s w i t h T r i t i u m L a b e l l e d DNA 1.  D e s i g n o f t h e i n v i v o e x p e r i m e n t s and p r e p a r a t i o n of t h e t r i t i u m l a b e l l e d t i s s u e  73  73  vi Page 2.  Fractionation  3.  Chromatography on Sephadex columns o f t h e f r a c t i o n s o f DNA o b t a i n e d by E C T E O L A - c e l l u l o s e chromatography  83  Removal o f i n o r g a n i c c o n s t i t u e n t s f r o m t h e DNA f r a c t i o n s by d i a l y s i s  87  3 Time-course of the i n c o r p o r a t i o n of H -thymidine i n t o t h e DNA o f s u b c u t a n e o u s l y i n j e c t e d r a t s  89  E f f e c t o f t h e r o u t e o f i n j e c t i o n on t h e s p e c i f i c a c t i v i t i e s o f t h e DNA p r e p a r a t i o n s  91  Specific activity-time interrelationships i n t t h e DNA f r a c t i o n s o b t a i n e d by E C T E O L A - c e l l u l o s e chromatography  93  4.  5. 6.  7.  on E C T E O L A - c e l l u l o s e  I I . E x p e r i m e n t s w i t h DNA and T r i t i u m 1.  2.  3.  4.  74  L a b e l l e d w i t h B o t h Carbon-14 98  D e s i g n o f t h e i n v i v o e x p e r i m e n t s and of the double l a b e l l e d t i s s u e The r e p r o d u c i b i l i t y o f t h e MAK method  preparation 98  fractionation 99  C o m p a r i s o n o f t h e MAK c h r o m a t o g r a p h i c p r o f i l e s o f t h e DNA p r e p a r a t i o n s o b t a i n e d by t h e methods o f Medawar and Zubay (159) and C o l t e r e t a l . (147) f r o m t h e same e x p e r i m e n t a l a n i m a l s The C"^ and e l u t i o n p r o f i l e s of the double • l a b e l l e d DNA p r e p a r a t i o n s , and t h e c o m p a r i s o n of the H3/C r a t i o s i n the d i f f e r e n t f r a c t i o n s o b t a i n e d a f t e r MAK c h r o m a t o g r a p h y  106  1 4  I I I . Measurement o f R a d i o a c t i v i t y i n t h e P r o t e i n - R N A I n t e r phases o f t h e T i s s u e E x t r a c t s O b t a i n e d by t h e Methods o f C o l t e r e t a l . (147) and Medawar and Zubay (159)  IV.  Enzymatic D e g r a d a t i o n o f the Double L a b e l l e d Preparations  113  126  DNA 129  SUMMARY  136  BIBLIOGRAPHY  139  vii TABLES Page I.  II. III.  E l u t i o n S c h e d u l e f o r DNA C h r o m a t o g r a p h y on ECTEQLAC e l l u l o s e Anion-Exchanger  35  B u f f e r S o l u t i o n s f o r Chromatography on MAK  37  Composition of Standards Containing the S i n g l e Isotope H or C Used f o r t h e D e t e r m i n a t i o n o f t h e I n t e g r a l D i s c r i m i n a t o r B i s s Curbes i n the Toluene  54  Composition of Standards Containing the S i n g l e Isotope H o r C-*-, Used f o r t h e D e t e r m i n a t i o n o f t h e I n t e g r a l D i s c r i m i n a t o r B i s s Curbes i n the Naphthalene/Dioxane S c i n t i l l a t o r System  54  f  IV.  3  V.  VI.  VII.  VIII.  IX.  X.  XI.  4  E f f e c t o f V o l t a g e D i s c r i m i n a t o r S e t t i n g s on t h e T r i t i u m and Carbon 14 A s s a y s o f 4 D i f f e r e n t S t a n d a r d s R e c o v e r y o f C o u n t s i n DNA and P u r i f i c a t i o n  XIII.  XIV.  a f t e r TCA P r e c i p i t a t i o n 72  Comparison o f t h e Percentage o f t h e 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 i n t h e Main F r a c t i o n s O b t a i n e d f r o m ECTEOLA-Chromatography o f t h e DNA p r e p a r a t i o n s  84  3 I n c o r p o r a t i o n of H -Thymidine DNA Samples I s o l a t e d  92  i n t o the D i f f e r e n t  Comparison o f the Percentage o f the R a d i o a c t i v i t y i n t h e Main F r a c t i o n s O b t a i n e d f r o m ECTEOLA-Chromatography DNA P r e p a r a t i o n s C o m p a r i s o n o f F r a c t i o n a t i o n on MAK DNA P r e p a r a t i o n  95  Column o f A l i q u o t s o f  Comparison o f the Percentage o f the 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 i n t h e F r a c t i o n s O b t a i n e d f r o m MAK-Chromatography of C and H L a b e l l e d DNA P r e p a r a t i o n s 1 4  XII.  64-65  3  14 3 Comparison o f t h e % D i s t r i b u t i o n o f C and H activities i n t h e Main and M i n o r F r a c t i o n s o f DNAs E l u t B d f r o m MAK Columns 3 14 C o m p a r i s o n o f t h e H /C A c t i v i t i e s i n t h e DNA F r a c t i o n s o b t a i n e d by MAK Chromatography 3 14 The R a t i o s o f H /C i n t h e I n t e r p h a s e and P h e n o l L a y e r s O b t a i n e d D u r i n g t h e P r e p a r a t i o n o f DNA  '.101  108  119 122  128  viii FIGURES Page 1.  P o s t u l a t e d mechanism f o r e x t e n d i n g a DNA  2.  R e p l i c a t i o n o f DNA, Watson and C r i c k  3.  4.  5.  6.  7.  B.  9.  10.  Ill  12.  13.  14.  15.  16.  chain  8  a c c o r d i n g t o t h e mechanism o f 12  S e m i c o n s e r v a t i v e and c o n s e r v a t i v e d i s t r i b u t i o n o f t h e two p a r e n t a l p o l y n u c l e o t i d e c h a i n s as p o s s i b l e a l t e r n a t i v e s i n t h e r e p l i c a t i o n o f DNA  13  A diagrammatic r e p r e s e n t a t i o n of the r e p l i c a t i o n of a c i r c l e o f DNA. The b l a c k t r i a n g l e marks t h e p o s i t i o n o f t h e h y p o t h e t i c a l s p i n n i n g mechanism  15  C a l i b r a t i o n c u r v e f o r measurement o f N a C l c o n c e n t r a t i o n by r e f r a c t o m e t r y  39  The DNA  46  e f f e c t o f e n z y m a t i c d e g r a d a t i o n on t h e v i s c o s i t y solutions  of  Time c o u r s e o f t h e h y d r o l y s i s o f p - n i t r o p h e n y l - t h y m i d i n e - 5 p h o s p h a t e by snake venom p h o s p h o d i e s t e r a s e  48  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 sample 1 f r o m I I I d e t e r m i n e d on t h e r e d s c a l e r  Table 55  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 sample 2 f r o m I I I d e t e r m i n e d on t h e r e d s c a l e r  Table  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 sample 3 f r o m I I I d e t e r m i n e d on t h e r e d s c a l e r  Table  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 sample 4 f r o m I I I d e t e r m i n e d on t h e r e d s c a l e r  Table  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 sample 5 f r o m IV d e t e r m i n e d on t h e r e d s c a l e r  Table  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 sample 6 f r o m IV d e t e r m i n e d on t h e r e d s c a l e r  Table  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 sample 1 f r o m I I I 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  Table  56  5?  58  59  60  61  3 14 L i n e a r r e l a t i o n s h i p between H /C r a t i o i n the standard s a m p l e s and t h e r a t i o o f t h e c o u n t s o b t a i n e d on r e d t o green s c a l e r  68  F r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e o f t r i t i a t e d DNA t h e i n t e s t i n a l mucosa o f r a t  75  from  Ix  17.  18.  19.  20.  21.  22.  F r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e o f t r i t i a t e d DNA i s o l a t e d f r o m r a t i n t e s t i n a l mucosa e x p o s e d t o s o n i c l a t i o n f o r , 1.25 min F r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e o f t r i t i a t e d f r o m r a t i n t e s t i n a l mucosa  oscil79  DNA 80  (A) F r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e o f t r i t i a t e d DNA o b t a i n e d f r o m t h e i n t e s t i n a l mucosa o f r a t s i n j e c t e d w i t h t h e p r e c u r s o r 10 m i n . p r i o r t o s a c r i f i c e (B) Sample o f an a l i q u o t o f t h e s t o c k s o l u t i o n o f DNA i l l u s t r a t e d i n A, b u t f r a c t i o n a t i o n p e r f o r m e d 2 weeks l a t e r  82  C h r o m a t o g r a p h y on 5ephadex o f DNA ECTEOLA-cellulose  85  82  f r a c t i o n s e l u t e d from  E l u t i o n o f r a d i o a c t i v i t y o f t r i t i a t e d DNA c h r o m a t o g r a p h e d on E C T E O L A - c e l l u l o s e column  88  S p e c i f i c a c t i v i t i e s o f DNA o b t a i n e d f r o m r a t s subcutaneously with H -thymidine  90  injected  3  23.  Fractionation  o f DNA  by c h r o m a t o g r a p h y on MAK  102  24.  Fractionation  o f DNA  by c h r o m a t o g r a p h y on MAK  103  25.  C h r o m a t o g r a p h y o f DNA  on a m o d i f i e d MAK column  105  26.  C h r o m a t o g r a p h y o f DNA  on a m o d i f i e d MAK  107  27.  C h r o m a t o g r a p h y on MAK column o f d o u b l e l a b e l l e d DNA p r e p a r e d f r o m i n t e s t i n a l mucosa o f r a t by method o f C o l t e r e t a l  28.  29.  30.  31.  32.  33.  column  C h r o m a t o g r a p h y on MAK column o f d o u b l e l a b e l l e d DNA o b t a i n e d f r o m t h e i n t e s t i n a l mucosa o f r a t by method o f C o l t e r e t a l . . C h r o m a t o g r a p h y on MAK column o f DNA f r o m i n t e s t i n a l o f r a t p r e p a r e d by method o f Medawar and Zubay  110  I l l  mucosa 112  C h r o m a t o g r a p h y on MAK column o f d o u b l e l a b e l l e d DNA o b t a i n e d f r o m i n t e s t i n a l mucosa o f r a t by method o f C o l t e r e t . a_l  115  C h r o m a t o g r a p h y on MAK o f d o u b l e l a b e l l e d DNA o b t a i n e d f r o m i n t e s t i n a l mucosa o f r a t by method o f C o l t e r e_t a l  116  E l u t i o n o f r a d i o a c t i v i t y o f d o u b l e l a b e l l e d DNA t o g r a p h e d on MAK column  chroma117  E l u t i o n o f r a d i o a c t i v i t y o f d o u b l e l a b e l l e d DNA t o g r a p h e d on MAK column  chroma118  X  34.  35.  36.  H /C by MAK  r a t i o s of the f r a c t i o n s chromatography  o f t h e DNA  peak o b t a i n e d 123  E n z y m a t i c d e g r a d a t i o n o f d o u b l e l a b e l l e d DNA o b t a i n e d f r o m t h e i n t e s t i n a l mucosa o f r a t by method o f C o l t e r e t a l .  132  E n z y m a t i c d e g r a d a t i o n o f t h e main DNA c h r o m a t o g r a p h y o f d o u b l e l a b e l l e d DNA mucosa o f r a t s  133  peak f r o m MAK from the i n t e s t i n a l  xi LIST OF ABBREVIATIONS  DNA  deoxyribonulceic acid  RNA  ribonucleic  s-RNA  soluble ribonucleic  DNase  deoxyribonuclease  TMP  t h e 5'-phosphate o f 2 ' - d e o x y r i b o s y l t h y m i n e  TDP  t h e 5' ( p y r o - ) d i p h o s p h a t e o f t h y m i d i n e  TTP  t h e 5' ( p y r o - ) t r i p h o s p h a t e o f t h y m i d i n e  dGPPP  t h e 5' ( p y r o - ) t r i p h o s p h a t e o f  deoxyuanosine  dAPPP  t h e 5' ( p y r o - ) t r i p h o s p h a t e o f  deoxyadenosine  dCPPP  t h e 5' ( p y r o - ) t r i p h o s p h a t e o f d e o x y c y t i d i n e  PP  acid acid  pyrophosphate  dAT  a copolymer  o f d e o x y a d e n y l a t e and d e o x y t h y m i d y l a t e  dGdC  a polymer c o n s i t i n g o f homopolymers o f and d e o x y c y t i d y l a t e  deoxyguanylate  poly  (AU)  a polymer c o n s i s t i n g o f homopolymers o f a d e n y l a t e and uridyld-te  poly  (dAT)  a polymer c o n s i s t i n g o f homopolymers o f . d e o x y a d e n y l a t e and t h y m i d y l a t e  MAK  m e t h y l a t e d a l b u m i n k i e s e l g u h r column f o r c h r o m a t o g r a p h y  mc  millicurie  g  microgram  m  micromole  m  millimicron  Cpm  counts per minute  Bg  background  TCA  trichloro-acetic  counts per minute acid  1 INTRODUCTION  The  E a r l y I n v e s t i g a t i o n s o f DNA Although  Biosynthesis  c l a s s i c a l n u t r i t i o n a l and  balance  s t u d i e s l e a d to the  t h a t n u c l e i c a c i d s can be s y n t h e s i z e d b i o l o g i c a l l y f r o m s i m p l e gave no  substances,  i n d i c a t i o n o f m a t e r i a l s u s e d o r t h e mechanisms i n v o l v e d .  e t u s f o r the development of our present as t r a c e r s i n b i o l o g i c a l e x p e r i m e n t s . phosphate l a b e l l e d w i t h P  32  was  knowledge was In 1940  conclusion  The  they  m a j o r imp-  p r o v i d e d by t h e use o f  isotopes  Hevesy (1) d e m o n s t r a t e d t h a t i n o r g a n i c  i n c o r p o r a t e d r a p i d l y i n t o t h e DNA  when  administered  15 i n vivo to animals.  Using  N  l a b e l l e d ammonium c i t r a t e , B a r n e s and  (2) showed t h a t ammonia i s a p r e c u r s o r o f n u c l e i c a c i d p u r i n e s and t h e r a t and  the pigeon.  substances,  as w e l l as n u c l e i c a c i d b a s e s , n u c l e o s i d e s and  utilized  Schoenheimer  pyrimidines i n  O t h e r i n v e s t i g a t i o n s (3) have d e m o n s t r a t e d t h a t many n u c l e o t i d e s can  simple  be  i n the b i o s y n t h e s i s o f v a r i o u s p o l y n u c l e o t i d e components.  R e s u l t s of i n v e s t i g a t i o n s w i t h r a d i o a c t i v e phosphorus f u r t h e r i n d i c a t e d t h a t the animal  t i s s u e s f a l l i n t o two  main c l a s s e s w i t h r e s p e c t t o i n c o r p o r a t i o n o f  32 P  i n t o t h e DNA:  w h i c h may and  those  be r e g a r d e d  s u c h as bone marrow, s p l e e n , thymus, and  as p r o l i f e r a t i n g t i s s u e s i n w h i c h t h e u p t a k e o f i s o t o p e i s ; h i g h ,  t h e r e m a i n d e r t y p i f i e d by l i v e r , k i d n e y  and  b r a i n , i n which i s o t o p e content  s m a l l and c e l l d i v i s i o n m i n i m a l i n t h e n o r m a l a n i m a l . t h e r e i s a d e c r e a s e w i t h age  lymph nodes and  s p l e e n , and  i n the r a t e of formation 32 the uptake of P  more r a p i d t h a n i n t h e a d u l t a n i m a l  (6).  the uptake of r a d i o a c t i v e p r e c u r s o r i n t o o f a d d i t i o n a l DNA  I t has been f o u n d o f l a b e l l e d DNA  by t h e DNA  Therefore, DNA  of weanling  (4,5)  is that  i n thymus,  rat liver i s  i t i s g e n e r a l l y accepted  o c c u r s as a r e s u l t o f t h e  that  synthesis  which accompanies m i t o s i s .  Nucleic acid synthesis i n regenerating techniques  i n t e s t i n a l mucosa,  by B r u e s e t a l . (7) who  e x t e n s i v e l y i n t o t h e DNA  and  l i v e r was  first  studied with tracer  f o u n d t h a t r a d i o a c t i v e p h o s p h a t e was  t h i s l a b e l was  retained for a considerable  incorporated time.  2 T h i s was cursors.  confirmed The  by s e v e r a l o t h e r w o r k e r s (8-11) u s i n g d i f f e r e n t l a b e l l e d  bulk of experimental  cells i s metabolically stable. l u l a r c o n s t i t u t e n t s , even RNA  evidence  i n d i c a t e d t h a t DNA  These o b s e r v a t i o n s s e t DNA  and  p r o t e i n may  i n non-dividing  a p a r t , s i n c e most c e l -  incorporate recently administered  p r e c u r s o r s i n n o n - d i v i d i n g as w e l l as d i v i d i n g c e l l s . t h e f a t e o f t h e p h o s p h o r u s o f DNA  Attempts to e l u c i d a t e  have l e d t o c o n f l i c t i n g c o n c l u s i o n s  (12-16) b u t  r e c e n t l y I v e s and Barnum (17,18) d e m o n s t r a t e d q u i t e c l e a r l y t h e m e t a b o l i c o f DNA  pre-  p h o s p h o r u s d u r i n g t h e r e p l i c a t i o n o f t h i s m a t e r i a l in_ v i v o .  stability  Moreover  P o t t e r e t a l . (19-23) p o i n t e d o u t t h e c l o s e c o r r e l a t i o n between l a b e l l i n g o f i n v i v o and  DNA  in_ v i t r o .  As can be s e e n , t h e e x p e r i m e n t s d e s c r i b e d above have r a t h e r l i m i t e d use i n i n v e s t i g a t i o n s concerning  b i o s y n t h e t i c processes.  One  has t o w o r k on t h e  enzymic  l e v e l t o o b t a i n d e t a i l e d i n f o r m a t i o n a b o u t t h e p o s s i b l e mechanisms i n v o l v e d i n these events.  At present i t i s c l e a r l y e s t a b l i s h e d , t h a t the f o r m a t i o n of  n u c l e i c acid molecules The  i s a c u l m i n a t i o n o f l o n g and complex c h a i n o f r e a c t i o n s .  o v e r a l l p i c t u r e concerning  t h e b i o s y n t h e s i s o f DNA  (a) t h e b i o s y n t h e s i s o f p u r i n e and  c a n be o u t l i n e d as f o l l o w s :  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,  t h e p h o s p h o r y l a t i o n o f t h e s e monophosphates t o t h e d i p h o s p h a t e version of these r i b o n u c l e o t i d e s to the corresponding the phosphorylation of the deoxyribonucleoside s t a g e and  s t a g e and  diphosphates  t o the  (c)  triphosphate  (d) t h e p o l y m e r i z a t i o n o f t h e d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e t o  Since the present  i n v e s t i g a t i o n i s l i m i t e d t o DNA  primer.  biosynthesis at  p o l y m e r a s e l e v e l , t h e f i r s t t h r e e s t e p s o u t l i n e d above a r e n o t d i s c u s s e d i n Because l a b e l l e d thymidine  was  l a t i o n s h i p between t h i s compound and DNA section.  (b)  the con-  deoxyribonucleotides,  y i e l d d e o x y r i b o p o l y n u c l e o t i d e i n t h e p r e s e n c e o f an a p p r o p r i a t e DNA  detail.  new  the any  u s e d as a r a d i o a c t i v e p r e c u r s o r , t h e r e i s considered b r i e f l y i n the f a l l o w i n g  3 The  Conversion  o f T h y m i d i n e t o Thymine N u c l e o t i d e s and  DNA  A c c o r d i n g t o P o t t e r (24) t h e u t i l i z a t i o n o f t h e p r e f o r m e d b u i l d i n g (e.g. bases,  blacks  n u c l e o s i d e s , n u c l e o t i d e s ) w i l l be p r e f e r r e d by n e a r l y a l l c e l l s  whenever t h e s e compounds a r e a v a i l a b l e , w h i l e t h e de novo, pathways w i l l be  used  o n l y when t h e b a s e s a r e n o t a v a i l a b l e .  and  " p r e f o r m e d " pathways f o r b o t h p u r i n e and  H i g h e r o r g a n i s m s have b o t h jde novo  pyrimidine b i o s y n t h e s i s present i n vary-  i n g r a t i o s i n d i f f e r e n t t i s s u e s and i n a d d i t i o n have e x t r e m e l y f o r b r e a k i n g down t h e p u r i n e s and p y r i m i d i n e s , c h i e f l y i n t h e  v i g o r o u s enzymes liver.  Thymine i s a p o o r p r e c u r s o r and no d a t a on i t s i n c o r p o r a t i o n i n t o i n whole a n i m a l experiments converted to thymidine The  DNA  have been r e p o r t e d , a l t h o u g h t h y m i n e i s v i g o r o u s l y  r i b o s i d e i n the presence  o f u r i d i n e and d e o x y u r . i d i n e  (25).  c o n c e n t r a t i o n o f t h e s e compounds i s a p p a r e n t l y q u i t e l o w i n v i v o , w h i l e  c a p a c i t y o f l i v e r t o c a t a b o l i z e thymine i s very h i g h . thymidine  i s an e f f e c t i v e p r e c u r s o r o f DNA  However, t h e  thymidylic acid  The  or other c e l l  under-  con-  compound i s r a p i d l y c o n v e r t e d t o t h y m i n e , w h i c h i s c a t a b o l i z e d  by t h e l i v e r w i t h i n 1 - 2 a l s o c o n v e r t e d t o TMP f o r DNA  deoxynucleoside,  (26-28) w i t h o u t  g o i n g any a p p r e c i a b l e c o n v e r s i o n o r i n c o r p o r a t i o n i n t o RNA stituents.  the  hours a f t e r injsection  by t h y m i d i n e  k i n a s e and  (24).  thence  During t h i s time i t i s t o TTP  which i s u t i l i z e d  synthesis. 14 The  p a t t e r n of phosphorylation r e a c t i o n s in. v i t r o of C  m i d i n e , has been s t u d i e d by B i a n c h i e t a l ( 2 9 ) . s p l e e n and  lymph nodes o f m i c e .  m e d i a t e i n t h e f o r m a t i o n o f TTP, a d d i t i o n t o TMP.  These w o r k e r s u s e d  T h e i r r e s u l t s i n d i c a t e d t h a t TDP  However, t h e o b s e r v a t i o n s o f B o l l u m  o c c u r s so r a p i d l y , t h a t t h e c o n c e n t r a t i o n o f TDP  cells  and  thy-  leukemic  i s n o t an  t h e l a t t e r compound b e i n g f o r m e d by  c o m p a t i b l e w i t h the sequence t h y m i d i n e — » T M P — » T D P  be d e t e c t e d .  - labelled  inter-  pyrophosphate  P o t t e r (23) w o u l d  >TTP i f t h e l a s t  be  step  i s at a l l times i n s u f f i c i e n t  R e s u l t s of f r a c t i o n a t i o n experiments  on e x t r a c t s o f E h r l i c h  (30) have shown t h a t t h r e e enzymes a r e i n v o l v e d i n t h e f o r m a t i o n o f  to  ascites TTP  4 from thymidine.  I t has  p r e v i o u s l y been shown t h a t TTP  m i d i n e by p r e p a r a t i o n s f r o m r e g e n e r a t i n g  rat liver  normal l i v e r i n possessing  kinases  the necessary  may  be farmed f r o m  thy-  (23,31,32) w h i c h d i f f e r s  (32-34).  Weissman e t a l  from (30)  demonstrated t h a t the appearance of the k i n a s e s a f t e r p a r t i a l hepatectomy tends t o be  s e q u e n t i a l , thymidine  i n c r e a s e and i n DNA  and TMP  kinases appearing  b e f o r e TDP  kinase.  decrease i n the a c t i v i t y of kinases i s c o i n c i d e n t with the  s y n t h e s i s i n the r e g e n e r a t i n g  c u l t u r e s of L c e l l s . sion of thymidine  liver.  A s i m i l a r f i n d i n g was  Potter (23).  Thus, t h e a v a i l a b i l i t y o f t h e d i s t i n c t k i n a s e s f o r c o n v e r -  t o TTP  and  P o t t e r and  sequence i s a l s o c o m p a t i b l e  the s e q u e n t i a l appearance of these  enzymes d u r i n g  two  with i n vivo f i n d i n g s . and TTP  compounds c o u l d n o t be a s s e s s e d  As t h e r e s u l t o f a v e r y r a p i d the metabolic  interrelation-  f r o m t h e i r d a t a and, f u r t h e r -  more f r o m t h e p o i n t o f v i e w o f t r a c e r k i n e t i c s , e i t h e r compound c o u l d w e l l s a t i s f y t h e r e q u i r e m e n t s f o r an i m m e d i a t e p r e c u r s o r o f DNA. t h e r e f o r e , permit  equally  Their  data,  no c o n c l u s i o n as t o t h e p o s s i b l e i d e n t i t y o f t h e mechanisms o f  DNA  s y n t h e s i s from thymidine  The  Enzymatic Synthesis of  i n _ v i v o and  in vitro.  DNA  S i n c e t h e p i o n e e r work o f K o r n b e r g and i n DNA  Bollum  N y g a a r d (35) t r i e d t o e s t a b l i s h w h e t h e r t h i s r e a c t i o n  e q u i l i b r a t i o n o f r a d i o a c t i v i t y between TDP ships of these  increase  observed i n  i n c r e a s e d m i t o t i c a c t i v i t y i s c o n s i s t e n t w i t h t h e p o s t u l a t e d scheme o f and  The  his collegues  (36) t h e l a s t  stage  b i o s y n t h e s i s , namely t h e p o l y m e r i z a t i o n and r e p l i c a t i o n o f t h i s m a c r a m b l a -  c u l e , has  a t t r a c t e d t h e a t t e n t i o n o f many s c i e n t i s t s .  p u r i f i e d f r o m E. c o l i  The  (36-38) c a t a l y z e s e x t e n s i v e f o r m a t i o n  t h a t i n v o l v e s r e p l i c a t i o n o f a DNA  primer.  According  enzyme DNA o f DNA  i z e d as f o l l o w s :  and  t h e DNA  itself.  The  by a mechanism  t o K o r n b e r g (36) t h i s r e -  p l i c a t i o n r e q u i r e s the presence of the f o u r d e o x y r i b o n u c l e o s i d e commonly f o u n d i n DNA,  polymerase,  triphosphates  o v e r a l l r e a c t i o n can be  summar-  5 n TPPP n dGPPP n dAPPP n dCPPP  +  enzyme DNA  TP dGP dAP dCP n  DNA  +  4(n)PP  The b a s i c a c t i o n o f t h i s enzyme i s t h a t i t c a t a l y z e s t h e s y n t h e s i s o f a new DNA c h a i n i n r e s p o n s e t o d i r e c t i o n s f r o m a DNA t e m p l a t e ; t e d by t h e h y d r o g e n - b o n d i n g cytosine.  these d i r e c t i o n s are d i c t a -  r e l a t i o n s h i p o f a d e n i n e t o t h y m i n e and o f g u a n i n e t o  There a r e f i v e major l i n e s o f evidence t h a t support t h i s  thesis.  The f i r s t l i n e o f e v i d e n c e i s d e r i v e d f r o m s t u d i e s o f t h e p h y s i c a l n a t u r e o f t h e DNA p r o d u c e d by t h e enzyme.  I n t h e e n z y m a t i c r e a c t i o n 90 - 95% o f t h e DNA  sample comes f r o m s u b s t r a t e s u s e d i n t h e r e a c t i o n .  The e n z y m a t i c p r o d u c t i s i n d i s t i n -  g u i s h a b l e from h i g h - m o l e c u l a r w e i g h t d o u b l e - s t r a n d e d DNA i s o l a t e d from n a t u r a l s o u r c e s (39).  I t has s e d i m e n t a t i o n c o e f f i c i e n t s i n t h e n e i g h b o r h o o d o f 25 and r e d u c e d  c o s i t y o f 40 d l / g w i t h an a v e r a g e m o l e c u l a r w e i g h t o f 6 m i l l i o n .  When t h e DNA i s  h e a t e d , i t s r o d - l i k e shape c o l l a p s e s and t h e m o l e c u l e becomes a c o m p a c t , coiled structure.  vis-  randomly  The d i g e s t i o n p r o d u c t s o b t a i n e d when s y n t h e t i c DNA i s c l e a v e d  by p a n c r e a t i c DNase a r e v e r y s i m i l a r t o t h o s e o b t a i n e d when n a t u r a l DNA i s u s e d . * Second, on t h e b a s i s o f o b s e r v a t i o n s (40,41) t h a t p y r i m i d i n e and p u r i n e a n a l o g s s u c h a s b r o m o u r a c i l and a z a g u a n i n e c a n be i n c o r p o r a t e d i n t o b a c t e r i a l and v i r a l DNA, i t was e x p e c t e d t h a t some l a t i t u d e i n t h e s t r u c t u r e o f t h e b a s e s c a n be t o l e r a t e d p r o v i d e d t h e r e i s no i n t e r f e r e n c e w i t h t h e i r h y d r o g e n Deoxyuridine t r i p h o s p h a t e o r 5-bromodeoxyuridine  bondings.  t r i p h o s p h a t e s u p p o r t e d DNA  s y n t h e s i s when u s e d i n p l a c e o f t h y m i d i n e t r i p h o s p h a t e b u t n o t when f o r t h e t r i p h o s p h a t e s o f deoxyadenosine, deoxyguanosine  substituted  or deoxycytidine.  has a l s o been n o t e d (42) t h a t 5-methyl and 5 - b r o m o c y t o s i n e  specifically  It  replaced  c y t o s i n e , hypoxanthine s u b s t i t u t e d o n l y f o r guanine and, u r a c i l f o r thymine. T h e s e f i n d i n g a r e b e s t i n t e r p r e t e d on t h e b a s i s o f hydrogen b o n d i n g o f t h e a d e n i n e - t h y m i n e and g u a n i n e - c y t o s i n e t y p e . * U n p u b l i s h e d e v i d e n c e b a s e d on e l e c t r o n - m i c r o g r a p h s and a n n e a l i n g b e h a v i o u r s u g g e s t s n e w l y s y n t h e s i z e d DNA d i f f e r s f r o m t h e n a t u r a l m a t e r i a l .  6 The  t h i r d l i n e o f e v i d e n c e comes f r o m a n a l y s i s o f t h e b a s e r a t i o s o f  . e n z y m a t i c a l l y s y n t h e s i z e d DNA.  In e x p e r i m e n t s u s i n g DNA  as p r i m e r s , t h e e n z y m a t i c a l l y s y n t h e s i z e d p r o d u c t the base c o m p o s i t i o n The (44).  of primers  from d i f f e r e n t  replicated rather  sources  faithfully  (43).  f o u r t h t y p e o f e v i d e n c e i s p r o v i d e d by t h e e x p e r i m e n t s o f J o s s e  He d e s c r i b e d a t e c h n i q u e  the  w h i c h e s t a b l i s h e d t h a t DNA  d i r e c t s the s y n t h e s i s of a product  from a given  e_t a l  source  i n w h i c h t h e f o u r b a s e s o c c u r n e x t t o one  another  i n t h e 16 p o s s i b l e a r r a n g e m e n t s n o t a t random b u t i n a p a t t e r n o f  frequencies  u n i q u e f o r t h a t DNA.  showed c o m p l e -  Moreover, the e n z y m a t i c a l l y synthezided  m e n t a r y p a i r i n g o f a d e n i n e t o t h y m i n e and of opposite  side triphosphates. and w i t h o u t  s y n t h e s i s r e q u i r e s t h e DNA W i t h o u t added DNA  C r i c k model  primer  and  t e d t h a t h e a t d e n a t u r e d DNA i s o l a t e d from  CpX 174  a b r u p t l y because of  of the bases i n the template  o f t h e p r i m e r DNA  (36).  i s c o n c e r n e d , i t has been d e m o n s t r a -  can s e r v e a s an e x c e l l e n t p r i m e r  v i r u s a s i n g l e s t r a n d e d DNA  f o r the polymerase system ( 4 4 ) .  the s i n g l e stranded  deoxyribonucleo-  f o r hydrogen bonding  a l l f o u r t r i p h o s p h a t e s s y n t h e s i s s t o p s e a r l y and  As f a r as t h e n a t u r e  strands  (45).  a l l four  t h e r e i s no t e m p l a t e  l a c k o f a h y d r o g e n - b o n d i n g mate f o r one  primer  g u a n i n e t o c y t o s i n e between t h e  p o l a r i t y , as p r o p o s e d by t h e Watson and  F i n a l l y , DNA  DNA  (47) and  (46).  Sinsheimer  has  i t i s a l s o an e x c e l l e n t  I t s h o u l d be m e n t i o n e d , t h a t t h e DNA  in  c o n d i t i o n i s not o n l y a s u i t a b l e primer but i s the o n l y a c t i v e  f o r m when t h e most p u r i f i e d enzyme p r e p a r a t i o n s a r e u s e d ( 3 6 , 4 8 ) . p a r a t i o n s the n a t i v e , double stranded  DNA  treated s l i g h t l y with deoxyribonuclease.  i s i n e r t unless i t i s heated or Recently Richardson  t h e f r a c t i o n a t i o n o f E. c o l i p o l y m e r a s e on h y d r o x y l a p a t i t e . p e a k s were o b t a i n e d , one  m a j o r peak, w h i c h c o n t a i n e d  a c t i v i t y , and  a m i n o r peak.  o f n a t i v e DNA  by 2 t o 10 f o l d .  W i t h such p r e -  90%  e_t a l (49)  new  described  On c h r o m a t o g r a p h y 2  o f t h e added p o l y m e r a s e  T h i s l a t t e r component i n c r e a s e s t h e p r i m i n g The  pre-  activity  component has been shown t o be a p h o s p h a t a s e  w h i c h s p l i t s 3 ' - p h o s p h o r y l e s t e r s a t t h e ends o f DNA the priming  a c t i v i t y o f n a t i v e DNA.  p o n e n t i n c o n t r o l l i n g DNA DNA  i n t o the primer  c h a i n s and  b i o s y n t h e s i s i n v i v o and  primer.  transforming n a t i v e non-primer  a few  s i d e ends o f some o f t h e DNA the missing n u c l e o t i d e .  of the  deoxyribonucleotides  a r e added t o t h e  c h a i n s , but f u r t h e r s y n t h e s i s i s blocked  In t h i s mechanism t h e d e o x y r i b o c l e o s i d e g r o u p a t t h e g r o w i n g end  i n o r g a n i c p y r o p h o s p h a t e i s e l i m i n a t e d , and  nucleotide  c a l l e d the " l i m i t e d r e a c t i o n " ,  A p o s t u l a t e d mechanism f o r e x t e n d i n g  a t t a c k e d by t h e 3 ' - h y d r o x y l  deoxyribonucleotide  small yet s i g n i f i c a n t quantity of  T h i s s y n t h e s i s was  under these c i r c u m s t a n c e s  shown i n F i g u r e 1.  com-  state.  s u b s t r a t e s i s l a c k i n g an e x t r e m e l y  and  increases  T h i s enzyme m i g h t be a v e r y i m p o r t a n t  A d l e r et_ a l (50) have d e s c r i b e d , t h a t when one  i s l i n k e d t o t h e DNA  thereby  t h e DNA  nucleo-  f o r lack of chain i s  5'-triphosphate  is  of a polydeoxynucleotide  the c h a i n i s lengthened  by one  chain  unit.  Krakow _et _ a l (51-53) r e p o r t e d t h e p u r i f i c a t i o n o f an enzyme f r a c t i o n  from  c a l f thymus n u e j l e i c a t a l y z i n g a l i m i t e d i n c o r p o r a t i o n o f d e o x y r i b o n u c l e o t i d e s r i b o n u c l e o t i d e s i n t o t h e t e r m i n a l p o s i t i o n s o f DNA. d i s t i n c t from the polymerase.  I t a l s o r e q u i r e s Mg^  The +  enzyme a p p e a r s t o  and DNA,  and  be  but i n c o n t r a s t t o  p o l y m e r a s e , t h e r e a c t i o n i s i n h i b i t e d by t h e p r e s e n c e o f a f u l l complement o f the deoxyribonucleoside  triphosphates;  one  triphosphate i s present  deoxyribonucleoside  o p t i m a l i n c o r p o r a t i o n p r o c e e d s when o n l y during the i n c o r p o r a t i o n .  The  r e a c t i o n c a t a l y z e d by t h i s n u c l e a r enzyme i s s i m i l a r t o t h e l i m i t e d r e a c t i o n  y m e d i a t e d c b y p o l y m e r a s e and may suggested  a l s o p l a y a r o l e i n t h e s y n t h e s i s o f DNA.  (36) t h a t t h e s e r e a c t i o n s r e p r e s e n t  It  was  the r e p a i r of the s h o r t e r s t r a n d  of  a double H e l i x i n which the s t r a n d s are of unequal l e n g t h s . Two occurs  r e a c t i o n s have been d i s c o v e r e d  i n t h e a b s e n c e o f added DNA  (54-56) i n w h i c h p o l y m e r s y n t h e s i s  after lag periods.  a c o p o l y m e r , composed e x c l u s i v e l y o f d e o x y a d e n y l a t e and a c t l y a l t e r n a t i n g sequence.  In one  case the product  deoxythymidylate  In t h e s e c o n d c a s e , t h e p r o d u c t  contains  in  only  is  ex-  8  Figure  1.  P o s t u l a t e d mechanism f o r extending a DNA c h a i n . (According to r e f . ( 3 6 ) ) .  9 d e o x y g u a n y l a t e and d e o x y c y t i d y l a t e a r r a n g e d as hydrogen  bonded homopolymers.  When e i t h e r p o l y m e r , dAT o r dGdC i s i s o l a t e d and u s e d as a p r i m e r f o r t h e  enzy-  matic r e a c t i o n , a shortened l a g p e r i o d o c c u r s , which i s r e l a t e d i n v e r s e l y t o t h e amount o f p r i m e r added, and e x t e n s i v e s y n t h e s i s o f an i d e n t i c a l p o l y m e r o c c u r s . Thus K o r n b e r g e t a l (54-56) c o n c l u d e d , t h a t t h e p o l y m e r s a r e s y n t h e s i z e d de novo d u r i n g t h e l a g p e r i o d and t h a t t h e f i r s t few m o l e c u l e s o f p o l y m e r s s e r v e as a primer f o r r e p l i c a t i o n . s i g n i f i c a n c e o f DNA  A d d i t i o n a l e x p e r i m e n t s h a v i n g a b e a r i n g on t h e b i o l o g i c a l  p o l y m e r a s e have been r e p o r t e d .  L i t m a n and S z y b a l s k i r e p o r t e d  (57,58) t h a t E. c o l i p o l y m e r a s e i n t h e p r e s e n c e o f d e n s i t y - l a b e l l e d t r a n s f o r m i n g DNA  denatured  primer c a t a l y z e d the synthesis of primer-free b i o l o g i c a l l y  active  molecules. The C e l l u l a r L o c a t i o n o f DNA S y n t h e s i s . In mammalian t i s s u e s DNA in  p o l y m e r a s e a c t i v i t y can be r e a d i l y  demonstrated  t h e s u p e r n a t a n t f r a c t i o n s o b t a i n e d by h i g h speed c e n t r i f u g a t i o n o f d i s r u p t e d  cell was  suspensions (22,59-61).  T h i s f i n d i n g was  u n e x p e c t e d , p a r t i c u l a r l y when i t  f o u n d t h a t h i g h a c t i v i t i e s o f t h e enzyme c o u l d n o t be d e m o n s t r a t e d  c o r r e s p o n d i n g sediment f r a c t i o n s which c o n t a i n e d the c e l l n u c l e i . ulated  ( 6 2 ) , t h a t t h e enzyme was  i n the  I t was  so h i g h l y s o l u b l e , t h a t most o f i t was  leached  o u t i n t o t h e e x t r a c t i o n medium f r o m t h e n u c l e i a n d / o r c y t o p l a s m under t h e c t i o n c o n d i t i o n s used.  in  t h e y c o n t a i n e d an a c t i v e p o l y m e r a s e  (62,64-66).  n u c l e i i s o l a t e d f r o m s e a u r c h i n embryos was  gardner  (67).  extra-  A c c o r d i n g l y , when n u c l e i o f r e g e n e r a t i n g r a t l i v e r o r c a l f  thymus were p r e p a r e d i n nonaqueous medium by m o d i f i c a t i o n s o f t h e B e h r e n s (63),  spec-  presence of  polymerase  a l s o d e s c r i b e d by M a z i a and  The e x a c t l o c a t i o n o f t h e s i t e o f DNA  u n s e t t l e d at the present.  The  procedure  Hine-  b i o s y n t h e s i s i n the c e l l i s  S m i t h and K e i r (62) t e n d t o b e l i e v e t h a t t h e r e i s an  e q u a l d i s t r i b u t i o n o f t h e p o l y m e r a s e between n u c l e u s and c y t o p l a s m o f t h e thymus cell. merase?  The q u e s t i o n t h e n a r i s e s :  What i s t h e f u n c t i o n o f t h e c y t o p l a s m i c p o l y -  I f t h e t o t a l p o l y m e r a s e were c o n c e n t r a t e d i n t h e v i c i n i t y o f t h e n u c l e a r  10 membrane, o u t s i d e and for  i n s i d e , a s a t i s f a c t o r y e x p l a n a t i o n w o u l d t h e n be a v a i l a b l e  t h e e x i s t i n g knowledge o f t h e l o c a t i o n and  not d i f f i c u l t  s y n t h e s i s o f DNA  t o i m a g i n e a p r o c e s s whereby s u i t a b l y s m a l l and  in vivo, for i t i s  d i f f u s i b l e u n i t s of  DNA  m i g h t be r e p l i c a t e d a t t h e n u c l e a r membrane by t h e c o n c e r t e d  and  cytoplasmic  and  Sadron (68) have d i s c o v e r e d a DNase f r o m c h i c k e n e r y t h r o c y t e s w h i c h d e g r a d e s  polymerase.  action of  nuclear  In t h i s c o n n e c t i o n , i t i s o f i n t e r e s t t h a t B e r n a r d i  5 DNA to  to double h e l i c a l sub-units of molecular  p r i m e t h e p o l y m e r a s e r e a c t i o n i s c o n f e r r e d on DNA  m i l d l y a l k a l i n e pH v a l u e s .  pH r e g u l a t i o n .  The  chromosomal a p p a r a t u s  s o l u t i o n s a f t e r exposure to  I t a p p e a r s t h e r e f o r e t h a t t h e r e a r e mechanisms a v a i l -  a b l e f o r t h e c o n v e r s i o n o f DNA and  w e i g h t 5-6x10 ; f u r t h e r , t h e c a p a c i t y  t o d i f f u s i b l e p r i m i n g s u b - u n i t s by e n z y m i c a c t i o n  more c o m p l e x o p e r a t i o n o f r e a s s e m b l y o f t h e m a s s i v e  c o u l d t h e n be c o n f i n e d t o t h e n u c l e u s  and t h u s t h e c o n -  c e p t u a l p r o b l e m s o f passage o f v e r y l a r g e s t r u c t u r a l u n i t s a c r o s s t h e membrane w o u l d no l o n g e r e x i s t The  R e p l i c a t i o n of In  (62).  DNA  s p i t e o f t h e a d v a n c e s i n o u r knowledge i n DNA  s t i l l considerable controversy. molecule served  one  The  (a) c o n s e r v a t i v e , t h e p a r e n t m o l e c u l e  e n t i r e l y newly-formed daughter molecule  c o n s e r v a t i v e , each m o l e c u l e p a r e n t a l and  r e p l i c a t i o n there i s  S e v e r a l schemes f o r t h e r e p l i c a t i o n o f  have been p r o p o s e d ( 6 9 ) :  i n t a c t and  nuclear  c o n s i s t s o f one  o l d and one  new  DNA i s pre-  i s added, (b)  half,  semi-  (c) d i s p e r s i v e ,  o f f s p r i n g components f o r m p a r t o f a l l m o l e c u l e s . c o m p l e m e n t a r y a r r a n g e m e n t o f p u r i n e and  p o l y n u c l e o t i d e c h a i n s o f DNA replicate itself  suggested  d i r e c t l y by h a v i n g  p y r i m i d i n e bases of the  t o Watson and C r i c k (70)  each c h a i n o f DNA  the f o r m a t i o n o f i t s complementary c h a i n . o f t h e o r i g i n a l d o u b l e h e l i x s e p a r a t e , and  t h a t DNA  s e r v e as a t e m p l a t e  They h y p o t h e s i z e d e a c h p u r i n e and  t h a t t h e two  two  might for strands  p y r i m i d i n e base a t t r a c t s  and h o l d s i n p l a c e by s p e c i f i c h y d r o g e n bonds a c o m p l e m e n t a r y f r e e n u c l e o t i d e  11 a v a i l a b l e f o r p o l y m e r i z a t i o n w i t h i n the c e l l . of deoxyribonucleotide-5'-triphosphates for polyesterification (36), a f t e r having new  and  These f r e e n u c l e o t i d e s i n t h e  a l r e a d y possess the n e c e s s a r y f r e e energy  a r e j o i n e d t o one  a n o t h e r by t h e DNA  o f complementary r e p l i c a s  has  present  taken place along  two  m o l e c u l e s a r e now  and  hence i n f o r m a t i o n a l c o n t e n t ,  one  c y c l e of r e p l i c a t i o n i s complete. The  p o l y m e r a s e enzyme  been h e l d i n p l a c e on t h e p a r e n t a l t e m p l a t e c h a i n , t o f o r m a  p o l y n u c l e o t i d e molecule of the r e q u i r e d sequence.  (71).  form  T h u s , a f t e r g r o w t h _de novo  both p a r e n t a l p o l y n u c l e o t i d e  t h a t are i d e n t i c a l i n base c o m p o s i t i o n with the p a r e n t a l double h e l i x .  and  sequences,  At t h i s  These e v e n t s a r e r e p r e s e n t e d  chains,  point,  i n Figure  W a t s o n - C r i c k r e p l i c a t i o n mechanism (70) makes an i m p o r t a n t  prediction  about the d i s t r i b u t i o n of the substance o f the p a r e n t a l m o l e c u l e over the  replica  d u p l e x e s , n a m e l y , t h a t t h e p a r e n t a l d o u b l e h e l i x becomes e q u a l l y d i s t r i b u t e d t h e two  daughter molecules of the f i r s t  replication cycle.  intact.  s i n c e t h e o r i g i n a l p o l y n u c l e o t i d e c h a i n s o f t h e p a r e n t a l DNA  atoms  p a r e n t a l p o l y n u c l e o t i d e c h a i n and  two m o l e c u l e s w h i c h each c o n t a i n one  two  m o l e c u l e s w h i c h c o n t a i n no  parental  second of  o f t h e p a r e n t a l DNA  i n the r e p l i c a t i o n  t h e r e i s among t h e d a u g h t e r DNA c y c l e s , a l w a y s one  d i s t r i b u t i o n which e n t i r e l y conserves the process.  Under c o n s e r v a t i v e  m o l e c u l e s g e n e r a t e d by one  in integrity  replication,  o r more r e p l i c a t i o n  i n d i v i d u a l whose atoms a r e e n t i r e l y p a r e n t a l w h i l e t h e  ce -of a l l o t h e r i n d i v i d u a l s  i s e n t i r e l y de n o v o .  s e r v a t i v e d i s t r i b u t i o n o f t h e two e r a t i o n c y c l e s are represented  The  semi-conservative  parental polynucleotide chains  i n Figure 3  (71).  the  substance.  T h i s mode o f d i s t r i b u t i o n o f t h e p a r e n t a l atom i s c a l l e d s e m i - c o n s e r v a t i v e , contradiction to a conservative  should  molecule romain  F o r i n s t a n c e , among t h e f o u r d a u g h t e r m o l e c u l e s g e n e r a t e d by t h e  r e p l i c a t i o n c y c l e , there are present  over  In s u b s e q u e n t r e p l i c a -  t i o n c y c l e s , however, no f u r t h e r d i s p e r s a l o f t h e o r i g i n a l p a r e n t a l DNA occur,  2  substan-  and  con-  d u r i n g two  gen-  12  Figure 2.  R e p l i c a t i o n o f DNA, a c c o r d i n g t o mechanism o f Watson and C r i c k . (According to |ref. (71)).  the  13  Original parvnt moLtcuit  First gerura.tt.ort. daughter molectU&s  F i g u r e 3.  Semiconservative and c o n s e r v a t i v e d i s t r i b u t i o n o f the two p a r e n t a l p o l y n u c l e o t i d e c h a i n s as p o s s i b l e a l t e r n a t i v e s i n the r e p l i c a t i o n o f DNA. (According t o r e f . ( 7 1 ) ) .  14 The main s t u m b l i n g b l o c k t o mechanisms c o n s i d e r i n g c o n s e r v a t i v e r e p l i c a t i o n i s the c l a s s i c experiment  o f M e s e l s o n and 5 t a h l ( 7 2 ) . By t h e use o f t h e i r  of density gradient e q u i l i b r i u m sedimentation t i o n o f t h e DNA  these workers proved  technique  that the r e p l i c a -  o f E. c o l i does i n d e e d r e s u l t i n t h e s e m i - c o n s e r v a t i v e  distribution  p r e d i c t e d by t h e mechanism o f Watson and C r i c k ( 7 0 ) ; a f t e r E. c o l i w h i c h have been l a b e l l e d by g r o w i n g  i n a medium c o n t a i n i n g N^""\ a r e a l l o w e d t o grow f o r 2 more gen14  e r a t i o n s i n a c u l t u r e medium c o n t a i n i n g t h e n o r m a l i s o t o p e N DNA m o l e c u l e s  are of a density corresponding  while the other h a l f of the molecules The M e s e l s o n - S t a h l  experiment  o t h e r t h a n E. c o l i  (73-7B).  , h a l f of the b a c t e r i a l  t o t h e i s o t o p e r a t i o 5 0 % N"^ and 50%  have a d e n s i t y c o r r e s p o n d i n g  has been amply c o n f i r m e d  t o 100%  R e c e n t l y C a i r n s ( 7 9 ) d e m o n s t r a t e d by t h e use o f a u t o -  c a t e s i n a s e m i - c o n s e r v a t i v e manner. 3 o r g a n i s m s i n t h e presence o f H  o f E. c o l i  - t h y m i d i n e , and e x t r a c t e d t h e DNA  by v e r y  o f a b o u t 2000 m i l l i o n , a l e n g t h o f a b o u t 1mm  corresponded  t o t h e e n t i r e b a c t e r i a l chromosome.  n i q u e some o f t h e DNA m o l e c u l e s t h a t d u p l i c a t i o n of the molecule  the  gentle  Electronmicro-  g r a p h s o f t h i s p r e p a r a t i o n o c c a s i o n a l l y showed t h a t t h e b a c t e r i a l DNA was  these dimensions  repli-  He l a b e l l e d b a c t e r i a l DNA by g r o w i n g  methods t h a t a v o i d e d , as f a r a s p o s s i b l e , t u r b u l a n c e and s h e a r .  had a m o l e c u l a r w e i g h t  N^.  u s i n g c e l l u l a r systems  r a d i o g r a p h y and e l e c t r o n m i c r o s c o p y t h a t t h e c i r c u l a r chromosome  and  N^  circular,  l o n g , and  By t h i s  tech-  were c a p t u r e d i n t h e r e p l i c a t i v e s t a g e , and showed s t a r t e d a t one p o i n t i n t h e - c i r c l e , t h e two s t r a n d s :  s e p a r a t i n g i n t o two l i m b s , t h e t e r m i n a l ends o f t h e f o r k b e i n g j o i n e d t o g e t h e r . S i n c e t h e b a c t e r i a were l a b e l l e d f o r s l i g h t l y l e s s t h a n two d i v i s i o n c y c l e s , most o f t h e p a r e n t a l DNA m o l e c u l e s  had one l a b e l l e d and one u n l a b e l l e d s t r a n d .  During  t h e r e p l i c a t i o n o f t h e s e h y b r i d m o l e c u l e s , t h e main body o f t h e DNA m o l e c u l e  was  l a b e l l e d i n o n l y one s t r a n d , and a t t h e b r a n c h i n g  label-  led  p a i n t ane o f t h e l i m b s was  i n b o t h s t r a n d s , and t h e o t h e r was l a b e l l e d i n o n l y one s t r a n d .  r e p r e s e n t a t i o n o f t h e r e p l i c a t i o n o f a c i r c l e o f DNA Because o f o b j e c t i o n s c o n c e r n i n g double  helices  the f e a s i b i l i t y  i s presented  o f unwinding  A  diagrammatic  i n F i g u r e 4.  the very long  ( 8 0 - 8 2 ) , a l t e r n a t i v e h y p o t h e s e s have been s u g g e s t e d .  DNA  Dounce has  15  F i g u r e 4.  A diagrammatic r e p r e s e n t a t i o n o f the r e p l i c a t i o n o f a c i r c l e o f DNA. The b l a c k t r i a n g l e marks the p o s i t i o n o f the h y p o t h e t i c a l s p i n n i n g mechanism. (According to r e f . ( 7 9 ) ) .  16 p r o p o s e d a mechanism (83) i n w h i c h one  s t r a n d o f t h e DNA  e x a c t l y while the other s t r a n d i s prevented w h i c h may  be h i s t o n e .  o f d i s k w i t h two  With  t h e end  B u t l e r (84) e n v i s a g e d  The  by one  t h e DNA  o f one  and  RNA  s t r a n d i n each h o l e .  The  accommodate one  the d i f f i c u l t i e s  end  of the  strands  o f t h e DNA  fibre  complementary n u c l e o t i d e f o r the  t a k e i t s p l a c e , and  t o t h e g r o w i n g c h a i n t h e d i s k w i l l move f o r w a r d  r o t a t i n g as i t goes and t h e r e b y  factor,  p o l y m e r a s e as a k i n d  d i s k w o u l d f i r s t a t t a c h i t s e l f t o one  g i v e n p o s i t i o n i n t h e s e q u e n c e w i l l now one  f r o m f u n c t i o n i n g by some o t h e r  h o l e s o r s l o t s each o f w h i c h can  o f t h e p r i m e r DNA.  double h e l i x i s copied  as t h e s e  are  attached  a l o n g t h e DNA  unwinding the double stranded  helix.  fibre,  This  avoids  w h i c h have been s u g g e s t e d o f f i n d i n g a mechanism f o r u n w i n d i n g  the  helix. C a v a l i e r i and  R o s e n b e r g (85-88) s u g g e s t e d t h a t t h e c o n s e r v e d u n i t o f  r e p l i c a t i o n i s the double h e l i x r a t h e r than the s i n g l e c h a i n of double Each d a u g h t e r c e l l , t h e r e f o r e , r e c e i v e s one h e l i x , joined together  as a p a i r .  bonds o f w h i c h a r e c o n s i d e r e d  The  experimental  The  mechanism may  w e l l be  (87) x - r a y  kinetics  (90) and  that  template  (89).  the s y n t h e s i s of homopolymeric p o l y  dependent s y s t e m s .  can  (dAT).  was  obtain-  e l e c t r o n microscopy  R e c e n t r e s u l t s o f C a v a l i e r i ' s g r o u p a l s o seem t o s u p p o r t p o l y m e r a s e can u t i l i z e  the  s i m i l a r to  e v i d e n c e f o r t h e e x i s t e n c e o f t h e s e b i u n i a l molecules-  have f o u n d t h a t c r o s s l i n k e d DNA  double  Thus, r e p l i c a t i o n  i s s y n t h e s i z e d on an u n d e n a t u r e d DNA  I t has been shown ( 9 2 ) , t h a t DNA for  newly s y n t h e s i z e d  as a weak t y p e o f c o n n e c t i o n .  ed by means o f enzyme k i n e t i c s (91).  one  helix.  These a r e c a l l e d " b i u n i a l m o l e c u l e s " ,  w o u l d not i n v o l v e s t r a n d s e p a r a t i o n . o c c u r i n g when messenger RNA  o l d and  DNA  poly  Zimmerman and  s e r v e as a p r i m e r f o r RNA  this  (AU)  hypothesis.  as a  template  Geiduschek and DNA  in  (93) DNA  Thus, i t a p p e a r s p o s s i b l e t h a t t h e b a s e s o f t h e d o u b l e h e l i x  became a v a i l a b l e f o r " t e m p l a t i n g " w i t h o u t  strand separation.  C a v a l i e r i ' s group  (91) a l s o c o n s t r u c t e d a model i n w h i c h t h e c o n f i g u r a t i o n o f t h e t e m p l a t e h e l i c a l during the r e p l i c a t i o n of the  DNA.  remains  17 In o r d e r t o t e s t C a v a l i e r i ' s m o d e l , DNA  samples o f b a c t e r i a l o r i g i n  been s t u d i e d by x - r a y s c a t t e r i n g t e c h n i q u e s ( 9 4 ) . i n e x c e l l e n t agreement w i t h t h e W a t s o n - C r i c k  The  have  experimental results  m o d e l ( 7 0 ) , and f u r t h e r  are  demonstrated  t h a t t h e m o l e c u l e s a r e i n d e p e n d e n t l y d i s t r i b u t e d i n s o l u t i o n , w i t h o u t any sid» by side associations.  The r e s u l t s were i d e n t i c a l f o r b o t h u n i t a r y and b i u n i a l m o l e -  c u l e s and a p p a r e n t l y d i d n o t s u p p o r t C a v a l i e r i ' s s u g g e s t i o n s .  The x - r a y o b s e r v a -  t i o n c o u l d be r e c o n c i l e d w i t h C a v a l i e r i ' s e x p e r i m e n t s , however, on t h e  assumption  t h a t t h e two u n i t a r y m o l e c u l e s were l o o s e l y a s s o c i a t e d i n t h e b i u n n i a l m o l e c u l e , t h e a v e r a g e d i s t a n c e between them b e i n g l a r g e compared t o t h e d i a m e t e r . p e r i m e n t s o f S h o o t e r and B a l d w i n C a v a l i e r i model o f DNA  complete  first  replication.  I n a s t u d y o f a l k a l i n e d e g r a d a t i o n o f heavy these workers demonstrated  showed t h a t i n c r e m e n t i n b u o y a n t  t h a t at the  pH  density corresponding to  d e n a t u r a t i o n , t h e d e n s i t y o f t h e h y b r i d i n c r e a s e d by l e s s t h a n  of t h i s increment.  ex-  (95) a r e a l s o i n a p p a r e n t c o n t r a d i c t i o n t o t h e  and h y b r i d b r o m o u r a c i ! l a b e l l e d DNA, where heavy DNA  The  one-fifth  I f t h e h y b r i d had been composed o f a b i u n i a l m o l e c u l e  (one  heavy u n i t a r y d o u b l e h e l i x and one l i g h t u n i t a r y d o u b l e h e l i x i n l o o s e a s s o c i a t i o n ) a l i g h t u n i t a r y band s h o u l d have a p p e a r e d  a t a pH a t w h i c h t h e heavy u n i t a r y  molecule  denatured.  The  P h y s i c a l and C h e m i c a l H e t e r o g e n e i t y o f  DNA  Whatever t h e a c t u a l mechanism o f DNA p u z z l i n g problem  d u p l i c a t i o n may  i n b i o c h e m i c a l s t u d i e s o f DNA  be, t h e r e i s s t i l l  p r e p a r a t i o n s . A l t h o u g h i t has been  shown t h a t i n t h e T-even b a c t e r i o p h a g e s , t h e r e i s a s i n g l e m o l e c u l e o f DNA infectious particle  (96,97) and t h e DNA  l e n g t h and c o m p o s i t i o n , DNA  tion  p r e p a r a t i o n s o f a n i m a l and b a c t e r i a l o r i g i n c o n t a i n  t h i s inhomogeneity  The t e c h n i q u e s w h i c h have been  a r e as f o l l o w s :  differential  (98), d e t e r m i n a t i o n of the u l t r a v i o l e t a b s o r p t i o n spectrum  denaturation  per  c a n be e x t r a c t e d as m o l e c u l e s o f u n i f o r m  m o l e c u l e s o f d i f f e r e n t s i z e and c o m p o s i t i o n . ployed to demonstrate  a  ( 9 9 ) , chromatography  on v a r i o u s t y p e s o f c o l u m n s ,  em-  centrifuga-  during thermal (100-103)  18  and  through  technique guinea (105).  t h e use o f 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 .  By t h e l a t t e r v e r y m i l d  b i m o d a l d i s t r i b u t i o n s have been d e t e c t e d f o r DNAs f r o m mouse (104,105)  p i g (104) o r m a r i n e c r a b s , s u c h a s C a n c e r b o r e a l i s and C a n c e r  ictitatus  J o s h i et_ a l (106) d e s c r i b e d t h e p r e s e n c e o f two s p e c i e s o f DNA i n some  h a l o b a c t e r i a and s e v e r a l i n v e s t i g a t o r s (107-109) d e m o n s t r a t e d t h e p r e s e n c e o f s a t e l l i t e bands t o t h e main DNA band i n h i g h e r p l a n t s .  Very r e c e n t l y K i r b y (110)  has used c o u n t e r c u r r e n t d i s t r i b u t i o n f o r s t u d y i n g h e t e r o g e n e i t y o f n a t i v e and denatured  DNAs.  The it  p o l y a n i o n i c c h a r a c t e r o f DNA a t n e u t r a l and a l k a l i n e pH v a l u e s makes  s u i t a b l e f o r f r a c t i o n a t i o n on a n i o n e x c h a n g e r s .  103)  e x p l o r e d t h i s p o s s i b i l i t y , and t h e r e s u l t h a s been t h e a d a p t a t i o n o f column;  chromatographic techniques  s u c h a s f r a c t i o n a t i o n on t h e s u b s t i t u t e d c e l l u l o s e  exchanger, ECTEOLA-cellulose The  and on m e t h y l a t e d - a l b u m i n - k i e s e l g u h r  g e n e r a l p r o p e r t i e s and t h e e x p e r i m e n t a l  niques The  Several i n v e s t i g a t o r s (101-  f i n d i n g s concerning  (MAK) c o l u m n s . t h e s e two t e c h -  are considered i n the following s e c t i o n .  C h r o m a t o g r a p h y o f DNA on S u b s t i t u t e d C e l l u l o s e - ( E C T E O L A ) Columns. ECTEOLA-cellulose  was o r i g i n a l l y p r e p a r e d  who r e a c t e d sodium c e l l u l o s e w i t h a m i x t u r e  by P e t e r s o n  and S o b e r  (111),  o f e p i c h l o r o h y d r i n and t r i e t h a n o l a m i n e .  By v a r i a t i o n o f t h e q u a n t i t y o f t r i e t h a n o l a m i n e i n p r o p o r t i o n t o t h e o t h e r s t i t u e n t s ECTEOLA p r e p a r a t i o n s w i t h v a r i a b l e n i t r o g e n c o n t e n t s be o b t a i n e d  (112-114).  Bendich  con-  and c a p a c i t i e s c a n  (112) compared t h e a d a p t a b i l i t y and e f f i c i e n c y o f  v a r i o u s i o n e x c h a n g e r s t o DNA f r a c t i o n a t i o n and d e m o n s t r a t e d t h a t ECTEOLA was t h e most s u i t a b l e f o r t h i s p u r p o s e . and  On t h e b a s i s o f s e d i m e n t a t i o n  l i g h t s c a t t e r i n g measurements (101) i t was c o n c l u d e d  s t u d i e s (115,116)  that the fractionation  p r o c e d u r e d i s c r i m i n a t e d among DNA s p e c i e s a c c o r d i n g t o t h e i r s i z e d i f f e r e n c e s . M o n o n u c l e o t i d e s were n o t r e t a i n e d on t h e column and a m i x t u r e o f o l i g o n u c l e o t i d e s , c o n t a i n i n g c o n s i d e r a b l e amounts o f h e p t a and o c t a n u c l e o t i d e s were e l u t e d by l o w salt concentrations  (101).  On t h e b a s i s o f t h e s e e x p e r i m e n t s B e n d i c h  concluded  19 (101) t h a t t h e DNA  f r a c t i o n s e l u t e d a t N a C l c o n c e n t r a t i o n s h i g h e r t h a n 0.2M  be l a r g e r t h a n o c t a n u c l e o t i d e s .  According to Bendich  would  (101) t h e f r a c t i o n s o b t a i n e d  f r o m ECTEOLA-chromatography showed s i g n i f i c a n t v a r i a t i o n s i n b a s e c o m p o s i t i o n . is felt  It  however, t h a t t h e s e m i g h t r e p r e s e n t e x p e r i m e n t a l f l u c t u a t i o n s r a t h e r t h a n  f r a c t i o n a t i o n a c c o r d i n g t o t h e b a s e c o n t e n t o f t h e DNA.  An i n t e r e s t i n g f e a t u r e o f  t h e a n a l y t i c a l d a t a i s t h e f a i l u r e i n many c a s e s t o o b t a i n u n i t y r a t i o s f o r t h e adenine/thymine  and g u a n i n e / c y t o s i n e p a i r s ( 1 0 1 ) , and on t h e b a s i s o f t h i s  Bendich  q u e s t i o n e d t h e adequacy o f t h e s p e c i f i c b a s e - p a i r i n g h y p o t h e s i s o f Watson and (70) as a c o m p l e t e The  r e d u p l i c a t i o n i n chromosomes.  ECTEOLA f r a c t i o n a t i o n t e c h n i q u e was  chromatographic and DNA  e x p l a n a t i o n f o r DNA  p r o f i l e s o f DNA  reported to give reproducible  p r e p a r a t i o n s from i d e n t i c a l t i s s u e s o u r c e s  o b t a i n e d f r o m t h e same s o u r c e (human g r a n u l o c y t e ) by two d i f f e r e n t  c e d u r e s gave e s s e n t i a l l y t h e same p a t t e r n s ( 1 0 1 ) . t h e chromatograms o f DNA  k i d n e y , b r a i n and i n t e s t i n e .  I t s h o u l d be m e n t i o n e d  (117) t h a t DNA  was  r e s o l v i n g power o f E C T E O L A - c e l l u l o s e was w i t h n o r m a l E. c o l i  in  e.g.  t h a t most o f t h e p r e v i o u s  Bendich c l a i m e d the  shown t o be s a t i s f a c t o r y i n e x p e r i m e n t s  f r o m c a l f thymus f o l l o w i n g t r e a t m e n t w i t h m u t a g e n i c profiles  coli,.  s p e c i e s s p e c i f i c and t h a t p r e p a r a t i o n s  and 5 - b r o m o u r a c i l l a b e l l e d E. c o l i ,  the chromatographic  (101)  L a r g e d i f f e r e n c e s were o b s e r v e d  f r o m d i f f e r e n t o r g a n s o f a g i v e n s p e c i e s were i d e n t i c a l .  DNA  c a l f thymus, E.  t h e p r o f i l e s o f DNAs d e r i v e d f r o m d i f f e r e n t o r g a n s o f t h e same s p e c i e s ,  i n v e s t i g a t o r s concluded  pro-  f r o m d i f f e r e n t s o u r c e s a r e d i f f e r e n t , and each o f t h e  Wi c e r e u s , pneumoccus, T 6 r phage and T 6 r t phage.  rat  (101),  According to Bendich et a l  f o l l o w i n g p r e p a r a t i o n s can be d i s t i n g u i s h e d by t h e p r o f i l e :  in  Crick  and a l t e r a t i o n s i n t h e a g e n t s were a l s o  reflected  (101).  I n I 9 6 0 d i M a y o r c a e t a l (118) r e p o r t e d  t h a t s a m p l e s o f n o r m a l and l e u k e m i c DNA  d i f f e r e d s i g n i f i c a n t l y when examined by  ECTEOLA-anion-exchange c h r o m a t o g r a p h y .  Moreover,  t h e amount o f  a b s o r b i n g m a t e r i a l e l u t e d w i t h l o w e r s a l t c o n c e n t r a t i o n s was DNA  samples o b t a i n e d f r o m l e u k e m i c p a t i e n t s a f t e r t r e a t m e n t  ultraviolet  increased i n case of v i v o w i t h the  20 a l k y l a t i n g agent m e t h a n e s u l f o n i c a c i d t e t r a m e t h y l e n e e s t e r . The  DNA  f r a c t i o n s obtained  t o r e t a i n t h e i r i n t e g r i t y as was and  p r e c i p i t a t i o n with ethanol  r e s u l t e d i n the production  f r o m t h e e x c h a n g e r a t n e u t r a l pH were f o u n d  i n d i c a t e d by t h e i r e s s e n t i a l n o n - d i a l y z a b i l i t y (119).  R a i s i n g t h e pH o f t h e f r a c t i o n s t o  o f t h e h y p e r c h r o m i c e f f e c t a t 260  n i z e d as c h a r a c t e r i s t i c o f n a t i v e DNA.  m/A,  B i o l o g i c a l a c t i v i t y was  f r a c t i o n s of pneumococcal t r a n s f o r m i n g  DNA,  which i s  f o u n d (101)  w h i c h showed b i o l o g i c a l  t h a t , i f a p a r t i c u l a r column f r a c t i o n was  t h e m a j o r p o r t i o n o f t h e m a t e r i a l was  again  recog-  retained i n  the  activities  comparable or g r e a t e r than t h a t present i n the o r i g i n a l p r e p a r a t i o n was  pH13  (101).  It  put back upon t h e  eluted at p r e c i s e l y that  column,  concentration  o f s o l u t e w h i c h i n i t i a l l y c a u s e d t h e f r a c t i o n t o be removed f r o m t h e c o l u m n . a d d i t i o n , however, s m a l l amounts o f m a t e r i a l were f o u n d b o t h a t l o w e r and concentrations p e r i m e n t was  of solute.  again  B e n d i c h i n t e r p r e t e d t h e s e f i n d i n g s i n d i c a t i n g t h a t each peak i n t h e was  h e t e r o g e n e o u s , and  higher  I f t h e main peak, f r o m t h e f i r s t r e c h r o m a t o g r a p h y  p l a c e d back on t h e c o l u m n , t h e s@me b e h a v i o r was  In  ex-  observed. chromatogram  s u b s e q u e n t r e c h r o m a t o g r a p h y o f s u c h peak e f f e c t s f u r t h e r  purification. On  t h e b a s i s o f t h e e x p e r i m e n t s c a r r i e d o u t by B e n d i c h and  seemed p o s s i b l e t h a t i n v e s t i g a t i o n s o f t h e e x t e n t provide  a basis f o r demonstrating genetic  ical level.  (121-122) and  Contrary  d i f f e r e n c e s between t i s s u e s a t t h e  S m i t h and  p r o f i l e s o f DNA  t o B e n d i c h e t a l , (101)  Kaplan  (125)  preparations  chromatographic e l u t i o n patterns and  nature of heterogeneity  could chem-  However, f u r t h e r i n v e s t i g a t i o n s o f t h i s k i n d have been i n c o n c l u s i v e  or c o n t r a d i c t o r y .  graphic  and  co-workers, i t  Qsawa ( 1 1 5 ) ,  have n o t e d no d i f f e r e n c e s i n t h e f r o m m a l i g n a n t and  (121-124) and  K i t f e l t that these discrepancies  i n t h e p r o c e d u r e s used f o r t h e p r e p a r a t i o n  o f DNA  S.  Kit  chromato-  normal t i s s u e s .  o f l u n g , thymus, k i d n e y and  k i d n e y o f t h e r a t , have a l s o been s t u d i e d  sere observed.  Kondo and  The  l i v e r o f t h e mouse no marked d i f f e r e n c e s  were p r o b a b l y due  to  differences  samples, t o d i f f e r e n c e s i n  the  21 p r o p e r t i e s o f t h e e x c h a n g e r s used by t h e v a r i o u s l a b o r a t o r i e s , and t o d i f f e r e n c e s i n the e l u t i o n schedules.  To i n v e s t i g a t e t h i s p o s s i b i l i t y he s e t up e x p e r i m e n t s  f o r t h e c r i t i c a l e v a l u a t i o n o f t h e number o f f a c t o r s p e r t i n e n t t o t h e c h r o m a t o g r a p h y o f DNA p r e p a r a t i o n s on ECTEOLA- c e l l u l o s e e x c h a n g e r s  (112).  U s i n g a d i s c o n t i n u o u s e l u t i o n scheme K i t f o u n d t h a t s o l o n g a s t h e same c h r o m a t o g r a p h i c p r o c e d u r e s were v i g o r o u s l y a d h e r e d t o , t h e e l u t i o n p r o f i l e s were reproducible.  The p e r c e n t  o f DNA e l u t e d by 0.6M sodium c h l o r i d e ( w h i c h  supposedly  r e p r e s e n t s l o w m o l e c u l a r w e i g h t DNA s p e c i e s (101) ) depended on t h e f o l l o w i n g factors: (a) The n i t r o g e n c o n t e n t o f t h e c e l l u l o s e e x c h a n g e r .  The l o w e r t h e n i t r o g e n c o n -  t e n t , t h e more m a t e r i a l was e l u t e d by 0.6M sodium c h l o r i d e . (b) The p r o c e d u r e u s e d i n p r e p a r i n g t h e DNA.  When t h e DNA was p r e p a r e d  Hammarsten method (126) most o f t h e DNA was e l u t e d i n t h e r e g i o n o f 0.6M  by t h e NaCl,  whereas p r e p a r a t i o n s b y t h e methods o f K i r b y (127) and M i r s k y and P o l l i s t e r ( 1 2 8 ) r e q u i r e d 2M N a C l and a l k a l i n e s o l u t i o n s f o r e l u t i o n . ( c ) The e x t e n t o f d e o x y r i b o n u c l e a s e When a u t o l y s i s t o o k  a c t i v i t y during the preparation o f the t i s s u e .  p l a c e t h e amount o f DNA e l u t e d i n t h e r e g i o n o f l o w s a l t  con-  c e n t r a t i o n was m a g n i f i e d . (d) The manner i n w h i c h t h e t i s s u e was h o m o g e n i z e d .  When t h e t i s s u e was b l e n d e d  a t h i g h speed t h e f r a c t i o n e l u t e d by 0.6M N a C l was i n c r e a s e d . (e) The " c o n f i g u r a t i o n " o f t h e DNA. by l o w s a l t c o n c e n t r a t i o n .  Heat d e n a t u r e d  p r e p a r a t i o n s were e l u t e d  mostly  The same was f o u n d f o r RNA.  ( f ) The amount o f p r o t e i n c o n t a m i n a t i o n  i n t h e DNA p r e p a r a t i o n s .  The c h r o m a t o -  g r a p h i c p r o f i l e s were n o t s i g n i f i c a n t l y m o d i f i e d when t h e DNA p r e p a r a t i o n s were treated with chymotrypsin, creased.  o r when t h e p r o t e i n c o n t e n t o f t h e p r e p a r a t i o n was i n -  22 On t h e b a s i s o f t h e above f i n d i n g s , K i t s u g g e s t e d s a l t c o n c e n t r a t i o n s from ECTEOLA-cellulose artefact.  exchangers  t h a t DNA  e l u t e d by  i s p r o b a b l y an e x p e r i m e n t a l  O t h e r p o s s i b i l i t i e s , however, t h a t a f r a c t i o n o f t h e t i s s u e DNA  p a r t l y degraded  low  is  d u r i n g p h y s i o l o g i c a l a c t i v i t y , t h a t t i s s u e s n o r m a l l y c o n t a i n some  v e r y low m o l e c u l a r w e i g h t DNA.  o r d u r i n g c e r t a i n phases o f the d i v i s i o n  a n i m a l c e l l s c o n t a i n some s i n g l e s t r a n d e d DNA,  cycle  c o u l d n o t be e x c l u d e d by t h e s e  ex-  periments. The  C h r o m a t o g r a p h y o f DNA In 1955  absorbed  on M e t h y l a t e d - A l b u m i n - K i e s e l q u h r Columns.  Lerman (102) i n t r o d u c e d t h e use o f c o l u m n s o f m e t h y l a t e d  on k i e s e l g u h r f o r t h e f r a c t i o n a t i o n o f DNA.  M a n d e l l and H e r s h e y  d e v e l o p e d t h i s method t o t h e p o i n t w h i c h p e r m i t t e d s e p a r a t i o n o f DNA t h e i r molecular s i z e .  s h e a r i n g t o t h e DNA  they c o u l d i d e n t i f y fragments  t h e o r i g i n a l m o l e c u l e s on t h e c o l u m n . ed t h a t t h e DNA  can  by be  by a p p l y i n g a c o n t r o l l e d  o f h a l f and q u a r t e r t h e s i z e o f  On t h e b a s i s o f t h i s f i n d i n g , t h e y c o n c l u d -  o f phage T2 e x i s t s i n t h e phage p a r t i c l e i n t h e f o r m o f one o r more  molecules of i d e n t i c a l The  Furthermore,  (129)  and RNA  These w o r k e r s have shown (97) t h a t phage T2 DNA  i s o l a t e d as r a t h e r u n i f o r m l a r g e m o l e c u l e s .  albumin  experiments  size. o f Sueoka and Cheng (130) r e v e a l e d more p r o p e r t i e s o f t h e  c o l u m n , such a s r e c o g n i t i o n o f b a s e c o m p o s i t i o n and o f h y d r o g e n bond c o n t e n t o f DNA.  I t was  f o u n d t h a t DNA  w i t h g r e a t e r g u a n i n e - c y t o s i n e c o n t e n t was  l o w & r s a l t c o n c e n t r a t i o n t h a n i s n a t i v e DNA. ing "deoxypolyadenine-thymine"in t h e main DNA and i t was  of the animal.  U s i n g t h i s method, a n a t u r a l l y  a m a r i n e c r a b C a n c e r b o r e a l i s was  isolated  but  existfrom  The method has a l s o been a p p l i e d t o f r a c t i o n a t e  f o u n d t h a t i t n o t o n l y s e p a r a t e s s o l u b l e and r i b o s o m a l RNA,  16 S and 23 5 r i b o s o m a l  eluted at  RNA,  also  RNA.  Sueoka and Yamane (131) were a b l e t o r e s o l v e t h e amino-acyl-RNA o f amino a c i d s , and a c h a r a c t e r i s t i c p r o f i l e was  o b t a i n e d f o r each o f t h e 16  different amino-  23 a c y l - s RNAs examined.  Using t h i s technique, Hayashi ejt al;(132) could detect i n  RNA from induced c e l l s the presence o f components not detectable i n noninduced controls. two  I t was a l s o found by 5ueoka and 5piegelman (133) t h a t , by the use o f  i s o t o p i c l a b e l s to i d e n t i f y RNA synthesized during various periods i n the  T2-E.  c o l i system, the two RNAs were always e a s i l y d i s t i n g u i s h a b l e on MAK columns  i f they were synthesized at d i f f e r e n t times and i d e n t i c a l i f they came from the same p e r i o d .  The molecular f a t e of DNA i n genetic transformation of pneumococcus  (139) was a l s o followed by t h i s technique.  F r a c t i o n a t i o n on MAK showed that very  soon a f t e r , or during i t s i n t r o d u c t i o n i n t o the c e l l , DNA i s converted  partly into  low-molecular weight fragments and p a r t l y i n t o s i n g l e stranded p o l y n u c l e o t i d e s . A portion of t h i s l a t t e r component was p a r t l y incorporated i n t o the host DNA. The  same technique was used by Pouwels et_ al-(135) who used chromatography  on MAK to i n v e s t i g a t e the molecular f a t e of DNA of bacteriophage of E. c o l i B .  T4 a f t e r i n f e c t i o n  He was able to separate p u r i f i e d phage DNA from the host DNA and RNA,  and showed that the host DNA was g r a d u a l l y degraded to o l i g o n u c l e o t i d e s , whereas the phage DNA remained i n double stranded form.  Others (136,137) have c a r r i e d out  s i m i l a r experiments. MAK chromatography proved to be i n some respects even superior to cesium c h l o r i d e density gradient c e n t r i f u g a t i o n . For example, Hayashi et al.(13B)  pre-  pared adequate amount of the r e p l i c a t i v e form of <J>X 174 v i r u s by employing chromatography on MAK.  The DNA of bacteriophage  SPB,, which when denatured, y i e l d s  two components d i f f e r i n g i n buoyant density i n cesium c h l o r i d e gradients was separable i n t o these components on an MAK column  The Metabolic Heterogeneity  (139).  of DNA  If the heterogeneity of DNA preparations i s indeed an i n vivo phenomenon, one would a u t o m a t i c a l l y r a i s e the question - What i s the b i o l o g i c a l s i g n i f i c a n c e of t h i s compositional heterogeneity? gators , attempted the demonstration -  During the l a s t 10-15 years s e v e r a l i n v e s t i -  o f metabolic heterogeneity o f DNA preparations.  24 One  of the e a r l i e s t r e p o r t s  d i f f e r e n t DNA  f r a c t i o n s obtained  describing a p r e f e r e n t i a l l a b e l l i n g of  f r o m r a t t i s s u e s was  t h a t o f B e n d i c h e_t a l . ( 1 4 0 ) .  These w o r k e r s f r a c t i o n a t e d DNA  of r a t small i n t e s t i n e , spleen,  b r a i n , t e s t i s and  l i v e r i n t o two  i n 0.87  regenerating  percent NaCl.  The  pancreas, kidney,  fractions, differing in  solubility  r a t i o o f i n s o l u b l e t o s o l u b l e f r a c t i o n was  vary from organ t o organ.  the  When t h e m e t a b o l i s m o f t h e s e DNA  found  f r a c t i o n s was  to  studied  14 using C  l a b e l l e d f o r m a t e as t h e n u c l e i c a c i d p r e c u r s o r ,  s p e c i f i c a c t i v i t i e s o f t h e n u c l e i c a c i d b a s e s i n t h e two  b o l i c d i f f e r e n c e s between t h e  two  DNA  and  o f some o f t h e  The  f r a c t i o n s were o b t a i n e d  extracted  Three s u c c e s s i v e found t h a t the  (142).  base  possible metabolic heterogeneity  was  On  Wood (143)  incubated C chicken  samples  to  the  with  f r a c t i o n a t i o n of the t r a n s f o r m i n g  of  hours i n -  heterogeneous with respect  S i m i l a r r e s u l t s have been o b t a i n e d  r a b b i t t i s s u e s and  trans-  principle,  activities.  l a b e l l e d thymidine i n  bone marrow.  e x t r a c t i o n s y i e l d e d t h r e e d i f f e r e n t DNA  the  After incubation  f r o m t h e t i s s u e s by t h e d e t e r g e n t p r o c e d u r e o f Kay  et  preparations.  s p e c i f i c a c t i v i t i e s o f t h e s e DNAs d i f f e r e d w i d e l y .  when t h e s e i n d i v i d u a l DNA  the  al.(144). It  was  Furthermore,  f r a c t i o n s were f r a c t i o n a t e d on E C T E O L A - c e l l u l o s e c o l u m n ,  the d i f f e r e n t f r a c t i o n s obtained geneity  dis-  i n a number o f t h e  w h i c h were d i f f e r e n t i n t r a n s f o r m i n g 14  F r i e d k i n and  presence of various was  A d d i t i o n a l meta-  p r e s e n c e o f 5 - b r o m o - u r a c i l f o r 18-24  population  of t h i s pyrimidine.  f o r m i n g p n e u m o c o c c a l DNA  DNA  so  a p p l i c a t i o n o f t h e ECTEOLA f r a c t i o n a t i o n t e c h n i q u e t o DNA  d i c a t e d t o them t h a t t h e DNA  In 1956  isotope  the  organs.  i s o l a t e d f r o m E_. c o l i grown i n t h e  incorporation  not  f r a c t i o n s were i n d i c a t e d a l s o by t h e  Bendich et al.(141) also s t u d i e d the E. c o l i DNA.  k i d n e y , and  small i n t e s t i n e .  s i m i l a r e x t e n t of the apparent r e t e n t i o n of the constituents  found t h a t  f r a c t i o n s were d i s s i m i l a r .  L a r g e d i f f e r e n c e s c o u l d be s e e n i n t h e c a s e o f t e s t i s and s t r i k i n g d i f f e r e n c e s i n pancreas, spleen  i t was  in specific activities.  a f t e r d e v e l o p i n g t h e column a l s o showed  hetero-  25 F r a n k e l and acid  ( A m b e r l i t e IRC-50) i n t h e  eluted the  Crampton (145)  from the  nucleic  t o C"^  magnesium f o r m .  a c i d i s o l a t e d from E h r l i c h a s c i t e s  l a b e l l e d formate or  on c o l u m n s o f  I t was  o b s e r v e d t h a t DNA  tumor c e l l s t h a t had  l a b e l l e d t h y m i d i n e b o t h in_ v i v o and  i n base c o m p o s i t i o n .  The  g r e a t e r f o r m o l e c u l e s r i c h i n a d e n i n e and p r o p e r t i e s o f DNA  a f f i n i t y of the  thymine.  could  of t h i s p o r t i o n ,  been e x p o s e d  in_ v i t r o ,  the  DNA  c o u l d be  of the  so as  investigated  the  was  specific found  First  to e x h i b i t  Second,  to two  after  mare t h a n 3% o f  total radioactivity.  fractionated  in  r e s i n was  synthesis in actively dividing c e l l s .  c o n t a i n e d as much as 1 1 %  be  When  These s t u d i e s r e v e a l e d  s h o r t l a b e l l i n g p e r i o d s , a s m a l l f r a c t i o n , a m o u n t i n g t o not t o t a l DNA,  polymethacrylic  t h i s t e c h n i q u e , f r a c t i o n s were o b t a i n e d t h a t v a r i e d  a c t i v i t i e s as w e l l as  interesting  DNA  r e s i n as f r a c t i o n s w i t h g r a d u a l l y v a r y i n g b a s e c o m p o s i t i o n .  c h r o m a t o g r a p h e d by  be  fractionated  the  exclusive  almost a  twofold  variation in specific activity. K u s a k a r i e t a l . ( 1 4 6 ) who  i n t r a c e l l u l a r transfer  of  nucleic  14 a c i d s i n v i v o , found t h a t a f t e r a d m i n i s t r a t i o n  of C  labelled  4-amino-5-imidozole-  14 carboxamide or C  l a b e l l e d o r o t i c a c i d , t h e DNA  phenol e x t r a c t i o n the  method o f C o l t e r  fraction eluted  tained  no  by  a l l of the  0.06M NaCL f r o m ECTEOLA c o l u m n s .  been shown t h a t newly synthesized DNA  the  b u l k o f c e l l u l a r DNA  The  DNA  by  of lysozyme l y s a t e s  fractions  s e d i m e n t i n g a t low  most o f t h i s m a t e r i a l  ory  had  The  n u c l e i by  the  radioactivity in  other fractions  con-  radioactivity. I t has  o f DNA  et al.(147),  i s o l a t e d from the  was  not  material  i t s r e l a t i v e resistance o f E_. c o l i c e l l s was centrifugal  i n t o the  solubilized_even  was  forces.  distinguished  to sonic v i b r a t i o n  found l a r g e l y i n the  i n p r e p a r a t i o n s o f E_. c o l i .  particulate shifted  However, a s m a l l amount  a f t e r e x t e n s i v e s o n i c t r e a t m e n t , and  v e r y r i c h i n n e w l y synthesiBed  from  (148).  B r i e f sonic treatment  h i g h speed s u p e r n a t a n t .  B e n - P o r a t et. a l . ( 1 4 9 ) d e s c r i b e d t h e " n a s c e n t " DNA  c o u l d be  this  refract-  DNA.  e x i s t e n c e o f a more r a p i d l y  This material  seemed t o be  labelled  somehow  26 associated  with  interphase  l a y e r a f t e r d e p r o t e i n i z a t i o n o f DNA  procedure.  p r o t e i n , b e c a u s e i t c o u l d be  Bach (151)  .  and  the microsomes c o n t a i n e d ,  during  preparations  DNA  i n a d d i t i o n t o RNA  polymerase a c t i v i t y .  during  by R o l f e  decreased buoyant d e n s i t y  nucleic acid preparations  growing b a c t e r i a at sedimentation e q u i l i b r i u m i n C s C l .  a r e f o u n d i n a t l e a s t two  molecules i n the  extracted  b n d and a  an u n u s u a l DNA  s p e c i f i c density regions.  corresponds to the f r o m T2  was  on MAK  shown t o p o s s e s s an u n u s u a l l y  This  b u o y a n t d e n s i t y and  Pulse  from  one  label-  One  of these regions  corres-  w i t h i n the r e g i o n  l i g h t s a t e l l i t e band.  Frankel  of  by 5ampson e_t a l . ( 1 5 4 ) .  o f g r o w i n g p l a n t t i s s u e s on a MAK  the  (153)  terminal  an i n h i b i t o r o f p r o t e i n s y n t h e s i s . i t s strong  This binding  extreme f r a g i l i t y under hydrodynamic s h e a r .  P r o b a b l y t h e c l e a r e s t d e m o n s t r a t i o n o f a m e t a b o l i c a l l y d i f f e r e n t DNA reported  of  exponentially  radioactive  h i g h m o l e c u l a r w e i g h t , by  c o l u m n , r a p i d s e d i m e n t a t i o n , and  DNA  (152).  phage i n f e c t e d b a c t e r i a i n w h i c h t h e  s t a g e s o f phage g r o w t h were p r e v e n t e d by DNA  exposures,  e a r l i e s t stages of r e p l i c a t i o n  ponds t o t h e d e n s e r s a t e l l i t e b a n d , a n o t h e r i s c o n t a i n e d p r i n c i p a l DNA  This  Changes i n t h e p h y s i c a l s t a t e o f  band o f i n c r e a s e d  s t u d i e s have shown t h a t DNA  3  H  polymerase, a s i g n i f i c a n t part  t h e r e p l i c a t i o n c y c l e i n b a c t e r i a have been r e p o r t e d  i n studying  (150)  La c e l l s .  short-time  i n v e s t i g a t o r o b s e r v e d a s a t e l l i t e DNA  ling  rich  by t h e Sevag  o f a m i c r o s o m a l f r a c t i o n f r o m He  became l a b e l l e d p r i o r t o n u c l e a r  o f t h e c e l l u l a r DNA  from the p r o t e i n  presented evidence f o r the r a p i d i n c o r p o r a t i o n of  l a b e l l e d t h y m i d i n e i n t o t h e DNA m i c r o s o m a l DNA  extracted  These i n v e s t i g a t o r s r e s o l v e d DNA column i n t o two  was  from a v a r i e t y  distinct fractions.  One  of  these  5 has  an a v e r a g e m o l e c u l a r w e i g h t o f 10  r a t e of turnover.  The  and  i s characterized  b a s e c o m p o s i t i o n o f t h i s DNA  high molecular weight counter  part.  by  a relatively  rapid  appears t o d i f f e r from i t s  27 The  Present I n v e s t i g a t i o n On t h e b a s i s o f e x p e r i m e n t a l  h e t e r o g e n e i t y o f DNA,  an i n v e s t i g a t i o n was  i n mammalian s y s t e m s . rat,  evidence  The  the p o s s i b l e metabolic  undertaken to explore t h i s  c h o i c e o f t i s s u e was  had  t o be f o u n d .  r e l a t i v e l y m i l d and n o n - d e g r a d a t i v e .  p r o c e d u r e were q u i t e h i g h , c o n t a m i n a t i n g s t r u c t u r e o f DNA lations.  Previous i n v e s t i g a t i o n s i n t h i s  (157,158) i n d i c a t e d t h a t t h e i s o l a t i o n p r o c e d u r e o f Medawar and  (159) was  was  RNA  The was  y i e l d s o f DNA  n o t e f f e c t e d a p p r e c i a b l y by t h e h i g h f r e q u e n c y  but i n the l a t t e r stages of the experiments,  the technique  p h e n o l e x t r a c t i o n and t h e a v o i d a n c e o f s o n i c d e g r a d a t i o n , and  Colter's  the p o s s i b i l i t y of degradation  and  lab-  Zubay  the  this  secondary  sonic  oscil-  t h e r e f o r e adopted,  o f C o l t e r e t a l . (147)  used because o f the r e l a t i v e l y g r e a t e r ease o f o p e r a t i o n s .  t i o n o f t h e DNA,  and  o b t a i n e d by  g r e a t l y r e d u c e d , and  F o r most o f t h e p r e s e n t i n v e s t i g a t i o n , t h i s method was  of  a rapid replace-  (13,155,156). To a p p r o a c h t h e p r o b l e m , s u i t a b l e i s o l a t i o n  f r a c t i o n a t i o n techniques oratory  possibility  t h e mucosa o f s m a l l i n t e s t i n e  w h i c h has been shown t o p o s s e s s a h i g h m i t o t i c a c t i v i t y and  ment t i m e f o r DNA  was  concerning  By t h e use ethanol  d e n a t u r a t i o n was  of  precipitaminimized  technique. As had been i n d i c a t e d , t h e s u b s t i t u t e d a n i o n e x c h a n g e r ,  ECTEOLA-cellulose  has been a d a p t e d by s e v e r a l i n v e s t i g a t o r s (101,121-124) f o r f r a c t i o n a t i o n o f  DNA.  T h i s method seemed t o o f f e r p r o m i s e f o r e x p l o r i n g t h e p o s s i b l e h e t e r o g e n e i t y DNA  p r e p a r a t i o n s f r o m r a t i n t e s t i n a l mucosa.  t i v e i n d i c a t o r of a metabolic active precursor.  The  I t was  (26-28).  The  h e t e r o g e n e i t y w o u l d be t h e i n c o r p o r a t i o n o f a r a d i o -  c h o i c e o f r a d i o a c t i v e p r e c u r s o r f o r t h i s p u r p o s e was  s p e c i a l metabolic  of time  thymi-  incorporated  into  d i f f e r e n c e s or precursor r e l a t i o n s h i p s of  t h e d i f f e r e n t f r a c t i o n s ought t o be more d i s t i n c t l y d e m o n s t r a t e d i n where t h e a n i m a l s  of  f e l t further, that a sensi-  d i n e , s i n c e i t has been shown t h a t t h i s m a t e r i a l i s s p e c i f i c a l l y t h e DNA  in  a r e e x p o s e d t o t h e s p e c i f i c DNA  (5,10,20 m i n u t e s b e f o r e s a c r i f i c e ) .  The  experiments,  precursor only f o r a short period experiments which are  described  28 t h e r e i n , however, t e n d e d  t o g i v e r a t h e r i n c o n c l u s i v e and c o n t r a d i c t a r y r e s u l t s .  M o r e o v e r , f u r t h e r d i f f i c u l t i e s were e n c o u n t e r e d o f DNA was  on E C T E O L A - c e l l u l o s e  when i t was  that fractionation  d i d not y i e l d r e p r o d u c i b l e chromatographic  c l a i m e d by o t h e r s ( 1 0 1 , 1 2 1 - 1 2 4 ) .  During the time of the present  s i m i l a r f i n d i n g s were r e p o r t e d by Klouwen and W e i f f e n b a c h In v i e w o f t h e d i f f i c u l t i e s e n c o u n t e r e d , o f DNA  found  on E C T E O L A - c e l l u l o s e ,  i t was  o t h e r f r a c t i o n a t i o n methods.  profiles  investigation,  (160).  e s p e c i a l l y w i t h the  fractionation  f e l t t o be i m p o r t a n t and n e c e s s a r y  Chromatography on MAK  as  to look f o r  c o l u m n s seemed t o be  promising,  s i n c e t h e c o n d i t i o n s o f use a r e m i l d , and no l o s s o f b i o l o g i c a l a c t i v i t y o f n u c l e i c a c i d p r e p a r a t i o n s a f t e r f r a c t i o n a t i o n has been r e p o r t e d ( 1 2 9 ) . s e v e r a l experiments  Accordingly,  were c a r r i e d o u t w i t h t h i s m a t e r i a l and i t was  reproducible elution patterns.  The  e l u t i o n p a t t e r n s o f t h e DNA  found t o g i v e  preparations  w e r e , however, d i f f e r e n t f r o m t h o s e o b t a i n e d on E C T E O L A - c e l l u l o s e .  To a s s u r e  even more s e n s i t i v e d e t e c t i o n o f p o s s i b l e m e t a b o l i c d i f f e r e n c e s amongst DNA use was  made o f t h e d o u b l e  l a b e l l i n g technique.  The  a n i m a l s were f i r s t  an  fractions,  given  tri-  14 t i u m l a b e l l e d t h y m i d i n e , and The  24 h o u r s l a t e r t h e y were g i v e n C  t i m e o f e x p o s u r e t o t h i s l a t t e r p r e c u r s o r was  minutes).  Thus DNA  molecules  c o m p a r i s o n o f t h e H^/C^  d i f f e r e n c e s . The  i n d i c a t e d , t h a t t h e d i f f e r e n c e s w h i c h were d e t e d t e d  be  label.  preparations  r e s u l t s have  a f t e r 20 m i n u t e s were no  longer  minutes.  As an e s s e n t i a l p a r t o f i n v e s t i g a t i o n s o f DNA l a b e l l e d p r e c u r s o r s , i t was the r a d i o a c t i v e thymidine DNA  40  which would c a r r y the t r i t i u m  r a t i o s o f t h e d i f f e r e n t f r a c t i o n s o f DNA  c o u l d t h e n be u s e d as an i n d i c a t o r o f m e t a b o l i c  p r e s e n t a f t e r 40  s h o r t (20 o r  thymidine.  s y n t h e s i z e d during the s h o r t exposure times c o u l d  d i s t i n g u i s h e d f r o m t h e b u l k o f t h e c e l l u l a r DNA The  relatively  -labelled  of importance  b i o s y n t h e s i s in_ v i v o f r o m  t o know w h e t h e r t h e i n c o r p o r a t i o n o f  i s a measure o f t h e f o r m a t i o n o f new  and  characteristic  or represents the a d d i t i o n of the deoxyribonucleoside to the p r e - e x i s i t n g  of the p o l y d e o x y r i b o n u c l e o t i d e c h a i n s .  To i n v e s t i g a t e t h i s , t h e t i m e c o u r s e  ends of  29 the enzymatic degradation l e a s e o f r a d i o a c t i v i t y and  o f t h e DNA  1  m a t e r i a l i n the a c i d  re-  soluble  F o r t h i s p u r p o s e , a p u r i f i e d snake venom  u s e d , w h i c h has been shown (161)  to l i b e r a t e  stepwise  i  ,1  5 ' - m o n o n u c l e o t i d e s f r o m t h e 3 -OH r e s u l t s obtained occurred  f o l l o w e d by t h e measurement o f t h e  u l t r a v i o l e t absorbing  f r a c t i o n of the i n c u b a t i o n m i x t u r e . p h o s p h o d i e s t e r a s e was  was  ends o f p o l y n u c l e o t i d e s .  The  experimental  i n d i c a t e d t h a t the i n c o r p o r a t i o n of the r a d i o a c t i v e  w e l l w i t h i n the p o l y n u c l e o t i d e  chains  of  DNA.  precursor  30 EXPERIMENTAL I  Materials 1.  Experimental animals. M a l e W i s t a r r a t s w e i g h i n g 180-200g e a c h , f r o m t h e c o l o n y o f t h e 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 were u s e d i n t h e e x p e r i m e n t s . 2.  Radioactive materials. T r i t i a t e d t h y m i d i n e was o b t a i n e d f r o m S c h w a r t z B i o r e s e a r c h , I n c . The  l a b e l was i n t h e t h y m i n e \ m o i e t y a t p o s i t i o n s n o t d e f i n e d by t h e s u p p l i e r .  Two  14 s a m p l e s o f 2-C  - t h y m i d i n e were u s e d .  One was p u r c h a s e d f r o m t h e R a d i o c h e m i c a l  C e n t r e , Amersham, E n g l a n d , a n d t h e s e c o n d was p r e p a r e d b y D r . 5. H. Z b a r s k y a c c o r d i n g t o F r i e d k i n and R o b e r t s ( 1 6 2 ) .  T h i s l a t t e r m a t e r i a l was p u r i f i e d  f u r t h e r by a d s o r p t i o n and e l u t i o n f r o m c h a r c o a l c o l u m n s (163) and b y chromat o g r a p h y on Whatman No. 40 p a p e r u s i n g a s o l v e n t c o n t a i n i n g e t h y l a c e t a t e : w a t e r : formic acid  (66:35:5). 14  N u c l e a r C o r p . , and C  T r i t i a t e d w a t e r s t a n d a r d was o b t a i n e d f r o m New E n g l a n d  - l a b e l l e d sodium c a r b o n a t e was s u p p l i e d by t h e N u c l e a r  Chicago Corp. Other reagents a r e d e s c r i b e d i n t h e a p p r o p r i a t e s e c t i o n s . II P r e p a r a t i o n o f DNA f r o m S m a l l I n t e s t i n a l Mucosa o f R a t . 1.  Method o f Medawar and Zubay (159) Reagents:  Neutral saline-Versene solution: —  8.78g o f sodium c h l o r i d e and 3.72g o f  e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d sodium were d i s s o l v e d i n g l a s s d i s t i l l e d The  water.  pH o f t h e s o l u t i o n was a d j u s t e d t o 7 w i t h 0.1N sodium h y d r o x i d e s o l u t i o n  and t h e f i n a l volume o f t h e s o l u t i o n 3M Sodium c h l o r i d e s o l u t i o n : —  was made up t o 1000 m l .  175.35g o f N a C l was d i s s o l v e d i n d i s t i l l e d  w a t e r t o o b t a i n a f i n a l volume o f 1000 m l . Chloroform-amyl a l c o h o l mixture: —  3 volumes o f c h l o r o f o r m were mixed w i t h 1 Q  volume o f a m y l a l c o h o l .  These s o l u t i o n s were s t o r e d a t 4 C.  r e m a i n i n g a f t e r 4 weeks were d i s c a r d e d .  Any s o l u t i o n s  31 Procedure: A f t e r t h e r e q u i r e d t i m e each e x p e r i m e n t a l a n i m a l was on t h e head and d e c a p i t a t e d . 10 cm.  The  s m a l l i n t e s t i n e was  k i l l e d by a b l o w  removed and c u t  into  segments w h i c h were f l u s h e d f r e e o f c o n t e n t s w i t h c o l d n e u t r a l s a l i n e -  Versene s o l u t i o n . plate.  The  The  segments were s p l i t open and  m u c o s a l e p i t h e l i u m was  applied to a c h i l l e d  then scraped from the m u s c u l a r i s w i t h  edge o f a m i c r o s c o p e  s l i d e and  t h o r o u g h l y m i x e d and  f r o m t h e p o o l e d mucosa an a l i q u o t was  f o r t h e e x t r a c t i o n o f t h e DNA.  glass  placed i n l i q u i d nitrogen.  The  s c r a p i n g s were  immediately  3g o f i n t e s t i n a l mucosa was  the  used  homogenized  briefly  (10 - 20 s t r o k e s ) w i t h a T e f l o n h o m o g e n i z e r i n a f i n a l volume o f a b o u t 60 o f the n e u t r a l s a l i n e - V e r s e n e s o l u t i o n .  The  c e l l suspension  s o n i c i r r a d i a t i o n i n a Raytheon s o n i c o s c i l l a t o r min.  exposed t o  (9 K c y c l e s / s e c . ) f o r  0.7-1  In o r d e r t o a s s u r e c o m p l e t e breakdown o f c e l l and n u c l e a r membranes, b u t  p r e v e n t d e g r a d a t i o n o f t h e DNA,  the sonic treatment  p o i n t , when m i c r o s c o p i c e x a m i n a t i o n (164)  was  ml  was  c o n t i n u e d up t o  o f t h e sample s t a i n e d w i t h t o l u i d i n e  showed no i n t a c t c e l l s o r n u c l e i i n t h e homogenate.  were f r e e d o f t h e o t h e r d e b r i s by w a s h i n g 6-B Versene s o l u t i o n .  The  n u c l e a r m a t e r i a l was  w a s h i n g was  the  The  blue  nuclear fragments  t i m e s w i t h 60 ml o f n e u t r a l s a l i n e -  done by a l t e r n a t e c e n t r i f u g a t i o n .  The  c o l l e c t e d each t i m e by c e n t r i f u g i n g f o r 8 m i n u t e s a t  xg i n an I n t e r n a t i o n a l r e f r i g e r a t e d c e n t r i f u g e .  The  1448  washed n u c l e a r f r a g m e n t s  were r e s u s p e n d e d i n 45 ml o f c o l d n e u t r a l s a l i n e - V e r s e n e s o l u t i o n i n a cup a S e r v a l l Omni M i x e r and homogenized f o r 1 m i n . p e r m i n u t e a t 3°C. i n g was  N i n e t y m l o f 3M.  a t a s p e e d o f 3000 r e v o l u t i o n s  N a C l s o l u t i o n was  c o n t i n u e d f o r a n o t h e r 20 m i n u t e s .  The  a t once added and  o p a l e s c e n t s o l u t i o n was  blend-  emul-  s i f i e d by s h a k i n g w i t h 30-40 m l o f C H C l ^ - a m y l a l c o h o l m i x t u r e f o r 10 m i n . a v i b r a t o r y shaker.  The  emulsion  was  The  on  s e p a r a t e d by c e n t r i f u g a t i o n a t 1448xg on  t h e I n t e r n a t i o n a l r e f r i g e r a t e d c e n t r i f u g e and t h e aqueous and were r e t a i n e d s e p a r a t e l y .  of  e m u l s i f i c a t i o n p r o c e s s was  interphase layers  repeated with  the  32 aqueous l a y e r up t o 9 t i m e s u n t i l no f u r t h e r i n t e r f a c i a l f i l m o f p r o t e i n produced  by f u r t h e r e m u l s i f i c a t i o n .  The DNA  was  p r e c i p i t a t e d f r o m t h e aqueous  l a y e r by a d d i t i o n o f an e q u a l volume o f e t h a n o l .  The  p r e c i p i t a t e d DNA  was  d i s s o l v e d a g a i n i n t h e minimum amount o f n e u t r a l s a l i n e - V e r s e n e s o l u t i o n centrifuged i f necessary to obtain a c r y s t a l c l e a r s o l u t i o n . c i p i t a t e d from t h e s u p e r n a t a n t w i t h e t h a n o l as p r e v i o u s l y . was  washed s e v e r a l t i m e s w i t h 5-10  DNA The  was  and  pre-  fibrous material  ml o f 7 5 % e t h a n o l t h e n d r i e d i n vacuo a t room  t e m p e r a t u r e o v e r p h o s p h o r u s p e n t o x i d e f o r 24 h o u r s .  The d r y p r e p a r a t i o n was  w e i g h e d , and d i s s o l v e d i n e i t h e r n e u t r a l s a l i n e - V e r s e n e s o l u t i o n o r i n a u t i o n c o n t a i n i n g 0.05M N a C l , Q.01M  was  sol-  sodium c i t r a t e i n 0.05M p h o s p h a t e pH 7.  f i n a l c o n c e n t r a t i o n o f t h e s e s o l u t i o n s was  1 mg DNA/ml.  The  The  s o l u t i o n s were s t o r e d  a t -15°C. 2.  Method o f C o l t e r e t a l . (147) Reagents:  Sodium c h l o r i d e - V e r s e n e - p h o s p h a t e b u f f e r pH 7.3:  —  58.45g N a C l , 2.72g p o t a s s i u m  d i h y d r o g e n p h o s p h a t e and 3.72g e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d sodium were d i s solved i n d i s t i l l e d water.  The  pH o f t h e s o l u t i o n was  NaOH, and t h e volume made up t o 1 l i t r e w i t h d i s t i l l e d 10%  sodium d e o x y c h o l a t e s o l u t i o n : —  x y c h o l a t e i n 100  The  a d j u s t e d t o 7.3  w i t h IN  water.  s o l u t i o n c o n t a i n e d : 10g o f sodium  deo-  ml.  Water s a t u r a t e d p h e n o l : — 25 m l o f d i s t i l l e d  T h i s s o l u t i o n c o n t a i n e d 75g o f r e d i s t i l l e d  phenol  water. Procedure:  The t i s s u e s were p r e p a r e d as d e s c r i b e d u n d e r method 1.  Five g of  f r o z e n t i s s u e were t r a n s f e r r e d t o t h e b l e n d e r cup o f a S e r v a l l Omni M i x e r 45 ml o f s o d i u m - c h l o r i d e - V e r s e n e - p h o s p h a t e  b u f f e r s o l u t i o n , 2.5  ml o f  and  10%  sodium d e o x y c h o l a t e and 50 ml o f w a t e r s a t u r a t e d p h e n o l s o l u t i o n were added. The b l e n d e r cup was  s e t i n an i c e b a t h and t h e m i x t u r e was  homogenized a t a  and  33 l o w speed c o r r e s p o n d i n g t o a P o w e r - s t a t s e t t i n g o f 2 0 v .  F o l l o w i n g homogeniza-  t i o n t h e e m u l s i o n was c e n t r i f u g e d f o r 10 m i n . a t 27.000xg i n a S e r v a l l r e f r i g e r a t e d c e n t r i f u g e , and t h e upper aqueous l a y e r c o n t a i n i n g t h e DNA was c a r e f u l l y removed f r o m t h e l o w e r p h e n o l phase and d e n a t u r e d p r o t e i n w h i c h at  the interface.  The DNA s o l u t i o n was s u b j e c t e d t o two a d d i t i o n a l  w i t h e q u a l volumes o f w a t e r s a t u r a t e d p h e n o l .  collected  extractions  These were o f 5 m i n u t e s d u r a t i o n  and were c a r r i e d o u t i n t h e c o l d on a m e c h a n i c a l s h a k e r .  A f t e r each  extraction,  t h e p h e n o l and aqueous p h a s e s were s e p a r a t e d by a b r i e f c e n t r i f u g a t i o n a t 4340xg in the Servall refrigerated  centrifuge.  T r a c e s o f p h e n o l were removed f r o m t h e DNA s o l u t i o n by 4 o r 5 b r i e f e t h e r e x t r a c t i o n s , and r e s i d u a l e t h e r was e l i m i n a t e d by b u b b l i n g N^ t h r o u g h t h e solution.  The s o l u t i o n was t h e n s t o r e d o v e r n i g h t a t 4°C, and was c l a r i f i e d by  c e n t r i f u g a t i o n f o r 10 m i n u t e s  a t 4340xg i n t h e S e r v a l l r e f r i g e r a t e d  centrifuge.  The DNA was p r e c i p i t a t e d by t h e a d d i t i o n o f an e q u a l volume o f e t h a n o l . p r e p a r a t i o n was t h e n t r e a t e d a s d e s c r i b e d i n method 1.  The  I n some e x p e r i m e n t s t h e  p r e c i p i t a t i o n o f DNA by e t h a n o l was o m i t t e d and t h e c l e a r e x t r a c t i n t h e sodium-chloride-Versene-phosphate  b u f f e r was d i a l y z e d e x t e n s i v e l y a g a i n s t a  b u f f e r c o n t a i n i n g 0.01M c i t r a t e 0.05M N a C l i n 0.05M p h o s p h a t e The Ill  b u f f e r , pH 6.3.  d i a l y z e d s o l u t i o n was t h e n k e p t a t -15°C. F r a c t i o n a t i o n o f t h e DNA P r e p a r a t i o n s 1.  F r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e  exchanger.  The E C T E O L A - c e l l u l o s e used i n t h i s s t u d y was o b t a i n e d f r o m Brown Company and had a c a p a c i t y o f 0.03 m i l l i e q u i v a l e n t / g .  The powder was suspended  washed s e v e r a l t i m e s t o remove f i n e s , t h e n w i t h 1.0N NaOH u n t i l gave z e r o o p t i c a l d e n s i t y r e a d i n g s a t 260m,**.  i n w a t e r , and  the supernatant  The ECTEOLA was g i v e n a f i n a l  wash w i t h 4N ammonium c a r b o n a t e , i n w h i c h i t was s t o r e d .  A s l u r r y o f 0.5g o f  ECTEOLA i n 8 m l o f 4N ammonium c a r b o n a t e was p o u r e d i n t o a column (0.8x10 cm) and was a l l o w e d t o s e t t l e by g r a v i t y .  The g l a s s column had a s i n t e r e d  glass  34 d i s c a t t h e l o w e r end and was e q u i p p e d w i t h s t o p c o c k t o r e g u l a t e t h e r a t e o f flow of the e f f l u e n t . phosphate  P r i o r t o chromatography  pH 7, u n t i l t h e e f f l u e n t was f r e e o f ammonium i o n , a s c h e c k e d by s p o t  t e s t i n g with Nessler's reagent. v i o l e t a b s o r p t i o n a t 260m/*. 50-70.000 a b s o r b a n c e pH7 were a p p l i e d  The e f f l u e n t was a l s o c h e c k e d A p p r o x i m a t e l y 2.5-3.5 mg DNA  for ultra-  (equivalent t o  u n i t s * a t 2 6 0 ™ ^ ) i n 0.05M N a C l 0.001M p h o s p h a t e  on t h e c o l u m n .  g r a v i t y through t h e exchanger it  t h e column was washed w i t h 0.001M  buffer  The s o l u t i o n was t h e n a l l o w e d t o p e r c o l a t e b y  a t a r a t e n o t e x c e e d i n g 2 t o 4 m l p e r h o u r , and  was washed i n w i t h 10-15 m l o f 0.001M p h o s p h a t e  buffer.  The column was  a l l o w e d t o r e m a i n o v e r n i g h t a t 3°C, a f t e r w h i c h i t was washed w i t h 30 m l o f 0.001M p h o s p h a t e The  buffer.  d i s c o n t i n u o u s e l u t i o n s c h e d u l e was i d e n t i c a l t o t h a t u s e d b y K i t  (122-124) and i s d e s c r i b e d i n T a b l e I .  F i v e m l f r a c t i o n s were c o l l e c t e d by  means o f a G i l s o n a u t o m a t i c f r a c t i o n c o l l e c t o r over a p e r i o d o f 2 days.  The s o l v e n t s p a s s e d t h r o u g h t h e c o l u m n u n d e r t h e  f o r c e o f g r a v i t y a t an a v e r a g e was  ( c o n s t a n t volume o p e r a t i o n )  r a t e o f 30 m l / h o u r .  The amount o f DNA  measured a t 260nry«by means o f a Beckman Dli). s p e c t r o p h o t o m e t e r .  eluted  In some  c a s e s t h e amount o f t h e DNA was a l s o measured by t h e d e t e r m i n a t i o n o f t h e d e o x y ribose content of the f r a c t i o n using the Dische 2.  F r a c t i o n a t i o n on MAK  (165) d i p h e n y l a m i n e  reaction.  columns.  M a t e r i a l s and r e a g e n t s : K i e s e l g u h r was p u r c h a s e d i n t h e g r a d e s o l d a s " C e l i t e " a n a l y t i c a l f i l t e r a i d by t h e J o h n s - M a n v i l l e P r o d . I n c . The e s t e r i f i e d a l b u m i n was a g i f t f r o m D r . M. Smith o f t h e T e c h n o l o g i c a l Research L a b o r a t o r y , F i s h e r i e s Research  Board.  * A b s o r b a n c e u n i t s a r e d e f i n e d a s t h e a b s o r b a n c e o b s e r v e d w i t h t h e Beckman DU s p e c t r o p h o t o m e t e r a t 260rv «for a 1 cm l i g h t p a t h x 10 x t h e volume, i n m l , o f t h e s o l u t i o n a p p l i e d t o t h e column V  35 TABLE I E l u t i o n S c h e d u l e f o r DNA Chromatography on E C T E O L A - C e l l u l o s e A n i o n - E x c h a n g e r  Fraction  Total* Volume i n ml  Composition  E  30  0.001M sodium p h o s p h a t e  I  30  0.05M N a C l , 0.001M sodium p h o s p h a t e pH7  II  30  0.2M N a C l , 0.001M sodium p h o s p h a t e pH7  III  30  0.6M N a C l , 0.001M sodium p h o s p h a t e pH7  IV  30  0.8M N a C l , 0.001M sodium p h o s p h a t e pH7  V  30  2..0M N a C l , 0.01M  pH7  sodium  p h o s p h a t e pHB VI  30  0.01M N H , 2M N a C l 0.1M N H , 2M N a C l  VII  30  VIII  60  IX  30  X  30  XI  30  XII  30  0.6M m ,  XIII  30  1.0M NH , 2M N a C l  XIV  30  0.5M NaOH  XV  3D  1.0M NaOH  3  3  0.2M N H , 2M N a C l 3  0.3M N H , 2M N a C l 3  0.4M NH , 2M N a C l 3  0.5M NH , 2M N a C l 3  3  2M N a E l  3  The amount o f t h e s e e l u a n t s were s l i g h t l y v a r i a b l e f r o m e x p e r i m e n t t o experiment A l l o f t h e s e e l u a n t s ( e x c e p t NaOH s o l u t i o n s ) were s a t u r a t e d w i t h f o r m t o p r e v e n t b a c t e r i a l and f u n g a l g r o w t h .  chloro-  36 if"  E s t e r i f i e d a l b u m i n s o l u t i o n : — A 1% s o l u t i o n o f t h i s was  s o l v i n g l g i n 100 for  ml o f d i s t i l l e d w a t e r .  w a s h i n g t h e c o l u m n s and  Protein coated C e l i t e : — described 100  by M a n d e l and  ml o f b u f f e r 2 was  e l u t i n g t h e DNA  the p r e p a r a t i o n Hershey (129).  i n t o 2 x 30 cm  composition of buffer  o f t h i s m a t e r i a l was  In t h i s p r o c e d u r e 20g  added, w i t h  poured i n t o the s l u r r y .  then cooled s t i r r i n g and The  c h r o m a t o g r a p h i c c o l u m n , w h i c h had  on t h e s i n t e r e d g l a s s d i s k .  The  column was  o f t h e s u s p e n s i o n i n t h e t u b e and  dis-  solutions  a r e shown i n T a b l e I I .  b o i l e d t o e x p e l a i r and  F i v e ml o f e s t e r i f i e d a l b u m i n was i t i o n a l b u f f e r 2 was  The  p r e p a r e d by  as  of C e l i t e i n  t o room t e m p e r a t u r e . t h e n 20 ml o f  s o l u t i o n was a pad  performed  add-  transferred  o f powdered c e l l u l o s e  f o r m e d by p l a c i n g 5-10  ml  portions  f o r c i n g t h e e x c e s s s a l i n e u n d e r an a i r p r e s -  2 sure^of with  3 lb/in  .  The  f i n i s h e d column w h i c h o c c u p i e s a b o u t 50 m l , was  200-300 ml o f b u f f e r 4 a t t h e p r e s s u r e  t h e column were s u s p e n d e d i n 125 as r e q u i r e d .  The  stated.  The  e n t i r e contents  ml o f b u f f e r 3, s a t u r a t e d  unused s u s p e n s i o n was  washed of  w i t h C H C l ^ and  s t o r e d i n the r e f r i g e r a t o r u n t i l  used re-  quired. Preparation The and  o f t h e column f o r c h r o m a t o g r a p h y preparation  Hershey (129).  The  chromatographic tube.  o f t h e column was column was  I t was  e s s e n t i a l l y as d e s c r i b e d  composed o f t h r e e l a y e r s i n a 2 x 30  p r e p a r e d as f o l l o w s :  Two  f i r s t l a y e r o f t h e column was as d e s c r i b e d  a b o v e , and  and  3,  added t o  t h e n 15 ml o f a d d i t i o n a l b u f f e r 2.  The  f o r m e d w i t h t h i s s u s p e n s i o n an c e l l u l o s e powder  t h e e x c e s s p r o t e i n was  washed down i n t o t h e column  b u f f e r 1 to a v o i d c o n t a m i n a t i o n of the second l a y e r . c o a t e d c e l i t e was  cm  6g i n 40 ml o f b u f f e r  ml o f 1% e s t e r i f i e d a l b u m i n was  t h e f i r s t b e a k e r w i t h s t i r r i n g , and  Mandel  S u s p e n s i o n s o f C e l i t e were  b o i l e d i n t h r e e b e a k e r s : (1) 8g i n 40 ml b u f f e r 2,(2) (3) l g i n 10 ml o f b u f f e r 3.  by  Then 10 ml o f p r o t e i n -  added t o t h e s u s p e n s i o n i n b e a k e r two.  Hershey, t h i s mixture should  n o t be  with  According  t o Mandel  p r e p a r e d i n advance b e c a u s e o f  the  37 TABLE I I B u f f e r S o l u t i o n s f o r Chromatography  B u f f e r number  *  on MAK  Composition of B u f f e r *  1.  D.05M sodium c h l o r i d e i n 0.Q5M p h o s p h a t e b u f f e r pH 6.7  2.  D.1M sodium c h l o r i d e i n 0.05M p h o s p h a t e b u f f e r pH 6.7  3.  0.4M sodium c h l o r i d e i n 0.05M p h o s p h a t e b u f f e r pH 6.7  4.  1.5M sodium c h l o r i d e i n 0.05M p h o s p h a t e b u f f e r pH 6.7  5.  D.05M sodium c h l o r i d e 0.01M sodium c i t r a t e i n 0.05M p h o s p h a t e b u f f e r pH 6.7  A l l o f t h e s e s o l u t i o n s were s a t u r a t e d w i t h c h l o r o f o r m t o p r e v e n t b a c t e r i a l and f u n g a l g r o w t h .  38 p o s s i b i l i t y o f t r a n s f e r o f p r o t e i n from the coated t o t h e uncoated C e l i t e .  The  s e c o n d l a y e r o f t h e column was b u i l t up w i t h t h i s s u s p e n s i o n i n t h e same manner as t h e f i r s t and was c o v e r e d w i t h t h e c o n t e n t s o f t h e t h i r d b e a k e r , w h i c h ved  as a m e c h a n i c a l b a r r i e r t o t h e working p o r t i o n s o f t h e column.  ser-  The c o l u m n  was washed p r o m p t l y w i t h b u f f e r 1 u n t i l t h e o p t i c a l d e n s i t y o f t h e e f f l u e n t was z e r o , and t h e r e f r a c t i v e i n d e x o f t h e e f f l u e n t was t h e same a s t h a t o f the e l u a n t .  When more t h a n 20.000 a b s o r b a n c e u n i t s o f DNA were a p p l i e d on  t h e c o l u m n , t h e amounts o f m a t e r i a l s i n t h e s e c o n d l a y e r were d o u b l e d . washed column was l o a d e d w i t h a n u c l e i c a c i d p r e p a r a t i o n 40.000 a b s o r b a n c e u n i t s i n 50 m l o f b u f f e r 1.  containing  The  15 -  A l i n e a r sodium c h l o r i d e  gra-  d i e n t was e s t a b l i s h e d u s i n g 400 m l o f b u f f e r 1 i n t h e m i x i n g chamber and 400 ml o f b u f f e r 4 i n t h e r e s e r v o i r . u n d e r an a i r p r e s s u r e hour.  of 3 l b / i n  The g r a d i e n t  . 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 m l /  In l a t e r experiments a B u c h l e r  pressure  system.  as d e s c r i b e d  e l u t i o n had t o be m a i n t a i n e d  Micro-pump was s u b s t i t u t e d f o r t h e a i r  E f f l u e n t f r a c t i o n s were c o l l e c t e d and a s s a y e d f o r DNA  on page  3^-.  The g r a d i e n t s  were m o n i t o r e d by r e f r a c t i v e i n d e x  measurements on s e l e c t e d e l u a n t f r a c t i o n s . 60 r e f r a c t o m e t e r  (Bellingham  and S t a n l e y  content  These were d e t e r m i n e d on an Abbe  Inc.).  The i n s t r u m e n t was  calibra-  t e d by m e a s u r i n g t h e r e f r a c t i v e i n d i c e s o f a s e r i e s o f N a C l s o l u t i o n s o f known concentration  i n 0.05M p h o s p h a t e b u f f e r .  shown i n F i g u r e reference  5 and t h e c o m p o s i t i o n o f an unknown s o l u t i o n was d e t e r m i n e d by  to this  3.  The c a l i b r a t i o n c u r v e o b t a i n e d i s  curve.  D e s a l t i n g o f t h e DNA f r a c t i o n s a f t e r c h r o m a t o g r a p h y .  In e x p e r i m e n t s where t h e 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 was t o be d e t e r m i n e d i t was n e c e s s a r y t o remove t h e i n o r g a n i c c o n s t i t u e n t s s i n c e i t has been f o u n d t h a t s a l t s s u c h as sodium c h l o r i d e and p h o s p h a t e m a r k e d l y r e d u c e d t h e c o u n t i n g r a t e because o f t h e i r quenching e f f e c t .  Two methods d e s c r i b e d  b e l o w were  39  F i g u r e 5.  C a l i b r a t i o n curve f o r measurement of NaCl c o n c e n t r a t i o n by r e f r a c t o m e t r y .  40 S t u d i e d t o d e t e r m i n e t h e i r u s e f u l n e s s and c o n v e n i e n c e f o r d e s a l t i n g t h e fractions. Gel  filtration  Gel f i l t r a t i o n  i s a method t h a t makes p o s s i b l e t h e s e p a r a t i o n o f d i m e n s i o n s (166)  with d i f f e r e n t molecular  u s e f u l f o r d e s a l t i n g high molecular The  use o f p a r t i c u l a r d e x t r a n  duced by P o r a t h decreasing  and  t o be  particularly  w e i g h t s u b s t a n c e s u c h as p r o t e i n  The  (167).  t o s i z e was  range o f s e p a r a t i o n i s i n c r e a s e d  with  p r e p a r a t i o n o f t h e Column:  Sweden.  The  was  u s e d and o b t a i n e d  d r y Sephadex powder was  r e d f o r a few m i n u t e s t o a l l o w i t t o s w e l l . water s e v e r a l times  t o remove f i n e s .  the r a t i o supernatant (2 x 30 cm)  was  t o s e d i m e n t was  mounted v e r t i c a l l y and  The  10:1.  the column.  The  C a r e was  suspension  Pharmacia,  suspended i n t a p - w a t e r and I t was  t h e n washed w i t h  Before being  distilled  c h o s e n so  litre  o f s o l u t i o n was  The  system was  mounted on  then f i l l e d  t a k e n t h a t no a i r b u b b l e s were p r e s e n t  o f 5ephadex was  added t o t h e f u n n e l and  b o t t o m o u t l e t f r o m t h e column was  opened t o a l l o w t h e s u s p e n s i o n  a r a t e o f 5 t o 20 m l p e r m i n u t e .  A r i s i n g h o r i z o n t a l boundary o f the  e v i d e n c e o f good p a c k i n g .  s e t t l e d , t h e f u n n e l and  connecting  with in  procedure. to flow  The at  packed  A f t e r a l l of the m a t e r i a l  t u b e were removed and  a filter  w i t h a d i a m e t e r s l i g h l y s m a l l e r t h a n t h e b o r e o f t h e column t u b e was on t h e h o r i z o n t a l even s u r f a c e o f t h e bed.  the  stirred  s t i r r e r d u r i n g the packing  considered  that  packed, the column  i n the f u n n e l w i t h a motor-driven  m a t e r i a l was  stir-  f i l l e d w i t h t a p w a t e r a t room t e m p e r a -  A f u n n e l l a r g e enough t o h o l d one  w a t e r up t o t h e f u n n e l .  from  volume o f w a t e r was  t o p o f t h e column t h r o u g h a r u b b e r s t o p p e r .  had  intro-  degree of c r o s s - l i n k a g e of the g e l m a t e r i a l .  In t h e s e e x p e r i m e n t s Sephadex G-50  ture.  i s claimed  gels f o r separation according  F l o d i n (168).  The  Upsala,  and  substances  To l e t t h e bed  paper placed  s t a b i l i z e , i t was  percolated f o r s e v e r a l hours, p r e f e r a b l y overnight, with g l a s s d i s t i l l e d  water.  41 A DNA  fraction  (5  ml volume) o b t a i n e d  a p p l i e d on t h e c o l u m n , and  f r o m ECTEOLA-chromatography was  t h e m a t e r i a l was  eluted with d i s t i l l e d water.  f r a c t i o n s were c o l l e c t e d as d e s c r i b e d p r e v i o u s l y and absorbing  m a t e r i a l a t 260  then The  assayed f o r u l t r a v i o l e t  nrj^and f o r c h l o r i d e i o n by t h e AgNQ^ p r e c i p i t a t i o n  test. Dialysis D i a l y s i s i s one  o f t h e most common methods f o r d e s a l t i n g l a r g e m o l e -  c u l a r weight substances. c e l l u l o s e t u b i n g , 3/4"  For the experiments d e s c r i b e d h e r e i n , seamless  d i a m e t e r was  used.  P u r i f i c a t i o n of the d i a l y s i s I t was  found t h a t the t u b i n g contained  m a t e r i a l s w h i c h had  tubing.  l a r g e amounts o f u l t r a v i o l e t  a peak a b s o r p t i o n a t  I t was  274IT1/*.  t r e a t t h i s t u b i n g t o remove t h e u l t r a v i o l e t a b s o r b i n g p u r i f i c a t i o n p r o c e d u r e s u g g e s t e d by D r . G.  G.  Jacoli  ate t h i s blank  absorption:  50  flushed through with d i s t i l l e d water.  cm  l o n g and  t h e d i a l y s i s t u b i n g was  were t h e n p l a c e d i n t o a b e a k e r , c o v e r e d o f t h e b e a k e r were b o i l e d . w i t h 0.5N  NaOH and  The  (169)  was  The  repeated.  The  the  covered was  repeated  until  n e g l i g i b l e a b s o r p t i o n a t 274m ««.  kept i n the c o l d u n t i l used.  by ECTEOLA o r MAK  c h r o m a t o g r a p h y were p l a c e d i n s m a l l bags p r e p a r e d  The  DNA  f r a c t i o n s obtained  c a r r i e d o u t i n t h e c o l d room a t 4°C,  tap water o r a g a i n s t s e v e r a l changes of g l a s s d i s t i l l e d  water.  c a s e t h e b a g s were a g i t a t e d by use o f a m a g n e t i c s t i r r e r . when t h e i n d i v i d u a l 5 ml f r a c t i o n s o b t a i n e d  The  /  washed t u b i n g was  d i a l y s i s was  contents  brownish supernatant  n e u t r a l and  The  tubing  d i s c a r d e d , t h e t u b i n g was  the supernatant  tubing.  following  used t o e l i m i n -  pieces of  w a s h i n g w i t h b o i l i n g d i s t i l l e d w a t e r was had  The  c u t i n t o segements a b o u t  a g a i n d i s c a r d e d and was  therefore to  materials.  w i t h d i s t i l l e d w a t e r and  w a t e r was  t h e b o i l i n g was  necessary  absorbing  from  against In t h e  In t h o s e  a f t e r c h r o m a t o g r a p h y had  this  running latter  cases, very  42 little  u l t r a v i o l e t absorbing material,  t h e f r a c t i o n s c o m p o s i n g one s m a l l peak  on t h e chromatogram were c o m b i n e d , d i a l y z e d in  a f l a s h evaporator.  0.5 m l a l i q u o t s  IV  and e v a p o r a t e d t o a l m o s t  dryness  The r e s i d u e was made up t o a s m a l l known v o l u m e , and  o f t h i s s o l u t i o n were c o u n t e d .  E n z y m a t i c D e g r a d a t i o n o f DNA 1.  The p u r i f i c a t i o n o f snake venom p h o s p h o d i e s t e r a s e f r o m t h e c r u d e  venom; Materials: Lyophilized Inst.  C r o t a l u s adamanteus venom was p u r c h a s e d f r o m Ross A l l e n  Reptile  C a r b o x y m e t h y l - c e l l u l o s e wps a p r o d u c t o f t h e Brown Co., w i t h a c a p a c i t y  o f 0.7 m i l l i e q u i v a l e n t / g . Reagents: 0.05M A c e t a t e b u f f e r pH4 - 30.Og o f g l a c i a l a c e t i c a c i d was added t o d i s t i l l e d w a t e r , t h e pH o f t h e s o l u t i o n was a d j u s t e d t o 4 w i t h NH^ s o l u t i o n and t h e volume was made up t o 1000 m l w i t h d i s t i l l e d  water.  0.05M A c e t a t e b u f f e r pH6 - 3.0g g l a c i a l a c e t i c a c i d i n 1000 m l H^Q, pH a d j u s t e d as d e s c r i b e d above. 0.2M A c e t a t e b u f f e r pH6'i~12.0q ted  glacial acetic  a c i d i n 1000 m l h^O, pH a d j u s -  as d e s c r i b e d above.  0.5M A c e t a t e b u f f e r pH6 - 30.Og g l a c i a l a c e t i c  a c i d i n 1000 m l H^O.  Procedure: ( a ) The p r e p a r a t i o n o f venom p h o s p h o d i e s t e r a s e was p e r f o r m e d  essentially  a c c o r d i n g t o t h e p r o c e d u r e o f K o e r n e r and S i n s h e i m e r ( 1 7 0 ) .  F i v e hundred  o f c r u d e venom was d i s s o l v e d was l e f t for  i n 30.0 m l o f d i s t i l l e d  a t room t e m p e r a t u r e f o r an h o u r .  15 m i n u t e s  mg  w a t e r , and t h e s o l u t i o n  The s o l u t i o n was t h e n  a t 3030xg on t h e S e r v a l l r e f r i g e r a t e d c e n t r i f j u g e .  centrifuged To 29.5  ml o f t h e c l e a r s u p e r n a t a n t s o l u t i o n 20.0 m l o f 0.5M A c e t a t e b u f f e r pH4 was  43 a d d e d , and t h e b u f f e r e d s o l u t i o n was t r a n s f e r r e d i n t o t h e c o l d room (4°C) where a l l t h e subsequent  p r o c e d u r e s were c a r r i e d o u t . The s o l u t i o n was t r a n s f e r r e d  i n t o p o l y e t h y l e n e t u b e s and 36.25 m l o f a c e t o n e a t - 2 0 ^ p i p e t t e d r a p i d l y on t o t h e b u f f e r e d venom.  The t u b e s were c o v e r e d t i g h t l y w i t h a l u m i n i u m  s w i r l e d r a p i d l y t o mix t h e r e a g e n t s .  A f t e r i t s t o o d f o r 30 m i n u t e s , t h e m i x -  t u r e was c e n t r i f u g e d and t h e s u p e r n a t a n t l i q u i d was d e c a n t e d i n t o c e n t r i f u g e tube. stage.  f o i l and  another  A s l i g h t l y t u r b i d s u p e r n a t a n t l i q u i d was o b s e r v e d a t t h i s  Seven m l o f a c e t o n e were added, and t h e s o l u t i o n was s t o r e d f o r 12 h o u r s  i n stoppered tubes.  I t was t h e n c e n t r i f u g e d and t h e c l e a r s u p e r n a t a n t  decanted i n t o another c e n t r i f u g e tube.  S i x m l o f a c e t o n e were added and t h i s  s o l u t i o n was c e n t r i f u g e d a f t e r b e i n g a l l o w e d t o s t a n d f o r 30 m i n u t e s . f i n a l p r e c i p i t a t e was d i s s o l v e d i n 5.0 m l o f d i s t i l l e d  The  water.  (b) C h r o m a t o g r a p h y on C a r b o x y m e t h y l - c e l l u l o s e was p e r f o r m e d e_t a l . ( 1 7 1 ) .  solution  according to Felix  The a c e t o n e p r e c i p i t a t e i n 5.0 m l o f d i s t i l l e d w a t e r was d i a l y z e d  a g a i n s t 0.05 M a c e t a t e b u f f e r , pH6, and was p l a c e d on a column o f c a r b o x y m e t h y l - c e l l u l o s e , 2 x 18cm, p r e v i o u s l y e q u i l i b r a t e d w i t h t h e same b u f f e r .  The  p r o t e i n was e l u t e d s t e p w i s e u s i n g 90 m l o f D.05M a c e t a t e b u f f e r pH6, 90 m l o f 0.2M a c e t a t e b u f f e r pH6 order stated.  t  and f i n a l l y 90 m l o f 0.5M a c e t a t e b u f f e r pH6 i n t h e  F r a c t i o n s o f 3 m i s were c o l l e c t e d a t a f l o w r a t e o f 6 m l / h o u r  io and a t a t e m p e r a t u r e o f 3 C.  The f r a c t i o n s were a s s a y e d f o r 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 a t 280»v *«, and f o r p h o s p h o d i e s t e r a s e a c t i v i t y . v  The f o l l o w -  i n g p r o c e d u r e was d e v e l o p e d f o r q u a l i t a t i v e a s s a y o f p h o s p h o d i e s t e r a s e in the f r a c t i o n s .  One t e n t h m l o f t h e s u b s t r a t e s o l u t i o n  activity  (composition of the  s o l u t i o n d e s c r i b e d on page 4-5) was p l a c e d i n t o a m i c r o - t e s t t u b e and i n c u b a t e d i n a s h a k i n g w a t e r - b a t h a t 37°C.  One d r o p o f s o l u t i o n f r o m a g i v e n f r a c t i o n  was added i n t o t h e t e s t t u b e and t h e a p p e a r a n c e  o f y e l l o w c o l o u r , due t o t h e  l i b e r a t i o n o f p a r a - n i t r o - p h e n y l a t e , i n one m i n u t e was a c c e p t e d a s a p o s i t i v e reaction f o r phosphodiesterase  activity.  44 2.  T e s t f o r D e o x y r i b o n u c l e a s e a c t i v i t y i n t h e snake venom  phosphodies-  terase preparations. Reagents: B u f f e r e d DNA s o l u t i o n —  T h i s s o l u t i o n c o n t a i n e d 2 mg DNA d i s s o l v e d i n 10 m l  o f b u f f e r c o n s i s t i n g o f 0.1M ammonium a c e t a t e and 0.01M magnesium c h l o r i d e a t pH7. DNase s t o c k s o l u t i o n —  2 0 0 / t j o f DNase I ( W o r t h i n g t o n Co.) was d i s s o l v e d i n  1.0 m l o f 0.1M a c e t a t e , 0.01M M g C l  2  b u f f e r pH7.  F o r t h e s t e p w i s e d e g r a d a t i o n o f DNA by snake venom p h o s p h o d i e s t e r a s e , i t i s e s s e n t i a l t o have an enzyme p r e p a r a t i o n f r e e f r o m c o n t a m i n a t i n g I t i s known (172-174) t h a t a c t i o n o f an e n d o n u c l e a s e  endonucleases.  on DNA s o l u t i o n s i s  a c c o m p a n i e d by a marked d e c r e a s e i n v i s c o s i t y o f t h e s e s o l u t i o n s .  T h i s phen-  omenon s u p p l i e d a r e l a t i v e l y s e n s i t i v e a s s a y f o r t h e d e t e r m i n a t i o n o f DNase activities.  W i l l i a m s e t a l _ . (175) showed t h a t when t h e i n t r i n s i c  viscosities  o f a DNA s o l u t i o n were p l o t t e d a g a i n s t t h e number o f d i a s t e r bond c l e a v a g e s p e r DNA m o l e c u l e , t h e a c t i o n o f DNase r e s u l t e d i n a d r a s t i c d r o p i n t h e i n t r i n s i c v i s c o s i t y a t t h e i n i t i a l phase o f t h e r e a c t i o n , w h e r e a s venom phosphod i e s t e r a s e c a u s e d a d e c r e a s e i n v i s c o s i t y p r o p o r t i o n a l t o t h e number o f i n t e r n u c l e o t i d e bonds b l e a v e d .  T h e r e f o r e , i t was e x p e c t e d , t h a t d u r i n g t h e f i r s t  30 - 200 m i n u t e s o f i n c u b a t i o n a snake venom p h o s p h o d i e s t e r a s e p r e p a r a t i o n e s s e n t i a l l y f r e e f r o m DNase a c t i v i t y , w o u l d n o t e f f e c t a p p r e c i a b l y t h e v i s c o s i t y o f a DNA  solution. To t e s t t h i s , t h e f o l l o w i n g a s s a y method was d e v e l o p e d : 5.0 m l o f t h e  b u f f e r e d DNA s o l u t i o n were p l a c e d i n an O s t w a l d v i s c o s i m e t e r , w h i c h had an average  f l o w t i m e o f 0.77 m i n . f o r t h e b u f f e r a l o n e .  immersed i n a w a t e r - b a t h a t 37°C.  The v i s c o s i m e t e r was  The DNase o r venom p h o s p h o d i e s t e r a s e  u t i o n s were a l s o i n c u b a t e d a t t h e same t e m p e r a t u r e .  sol-  The t i m e o f f l o w o f t h e  45 DNA s o l u t i o n was d e t e r m i n e d by u s i n g a s t o p - w a t c h .  A t z e r o t i m e . 0*1 m l o f  enzyme s o l u t i o n c o n t a i n i n g e i t h e r 0.1/*t^of DNase o r snake venom d i e s t e r a s e was added t o t h e DNA s o l u t i o n , t h e m i x t u r e was t h o r o u g h l y m i x e d was m e a s u r e d . intervals. ted  and t h e f l o w - t i m e  D u r i n g t h e f o l l o w i n g 30 m i n u t e s t h e s e were r e p e a t e d a t 5 m i n .  F i g u r e 6 shows t h e r e s u l t s o b t a i n e d when DNA s o l u t i o n s were i n c u b a -  w i t h f o u r d i f f e r e n t enzyme p r e p a r a t i o n s , namely; w i t h c r y s t a l l i n e DNase, an  a l i q u o t from t h e f i r s t  p r o t e i n c o n t a i n i n g peak o b t a i n e d on c a r y b o x y m e h t y l - c e l l u l -  o s e f r a c t i o n a t i o n o f t h e a c e t o n e p r e c i p i t a t e d venom p h o s p h o d i e s t e r a s e , t h e a c e t o n e p r e c i p i t a t e d d i e s t e r a s e , and w i t h t h e c a r b o x y m e t h y l - c e l l u l o s e f r a c t i o n a t e d diesterase.  phospho-  From t h e e f f e c t on t h e f l o w t i m e shown i n F i g u r e 6, i t was c o n c l u d e d  t h a t t h e p h o s p h o d i e s t e r a s e p r e p a r a t i o n w h i c h was f r a c t i o n a t e d on c a r b o x y m e t h y l c e l l u l o s e had e s s e n t i a l l y no e n d o n u c l e a s e  a c t i v i t y and t h a t t h e endogeneous DNase  a c t i v i t y o f t h e c r u d e m a t e r i a l was e l u t e d p r o b a b l y i n t h e f i r s t c o n f i r m s t h e f i n d i n g s o f F e l i x et_ a l .  peak.  The r e s u l t  (171) who o r i g i n a l l y d e s c r i b e d t h e f r a c t i o n a -  t i o n o f snake venom p h o s p h o d i e s t e r a s e from B o t h r o p s a t r o x venom.  3.  A s s a y o f t h e snake  venom p h o s p h o d i e s t e r a s e  activity.  Reagents: 0.1M T r i s - 0.Q02M Magnesium a c e t a t e b u f f e r p H 8 . 9 - 1 2 . l l g o f T r i s  (hydroxy-  m e t h y l aminomethane) and 0.43g Mg (CH^COQ),, were d i s s o l v e d i n d i s t i l l e d  water,  and t h e pH o f t h e s o l u t i o n was a d j u s t e d t o 8.9 w i t h a c e t i c a c i d and t h e s o l u t i o n was made up t o 1 l i t r e . 5ubstrate solution —  13.6 mg o f p - n i t r o - p h e n y l - t h y m i d i n e - 5 ' - p h o s p h a t e  was d i s s o l v e d i n 25 m l o f t h e b u f f e r  solution.  Snake venom p h o s p h o d i e s t e r a s e s o l u t i o n —  One mg o f W o r t h i n g t o n venom phospho-  d i e s t e r a s e was d i s s o l v e d i n 1.0 m l o f b u f f e r  solution.  46  O DNase A Acetone p r e c i p i t a t e d snake venom d i e s t e r a s e O F i r s t peak from carboxymethyl c e l l u l o s e chromatography ^ Snake venom d i e s t e r a s e a f t e r carboxymethyl c e l l u l o s e chromatography I.IOT  'CO ©  1.00 -  -t-> 3  C  •H  s c E  o  E  0.90-  0.80  F i g u r e 6,  1 1 1 r30 10 15 20 25 R e a c t i o n Time i n Minutes The e f f e c t o f enzymatic d e g r a d a t i o n on the v i s c o s i t y o f DNA s o l u t i o n s .  47 Procedure: The  a s s a y f o r d i e s t e r a s e a c t i v i t y was  R a z z e l and 1.4  Khorana ( 1 7 6 ) .  One  and  p e r f o r m e d e s s e n t i a l l y as d e s c r i b e d  one  h a l f mis  o f s u b s t r a t e s o l u t i o n and  ml o f t h e b u f f e r s o l u t i o n were p i p e t t e d i n t o a Beckman q u a r t z  (1 cm  l i g h t path).  s o l u t i o n was  A t z e r o t i m e 0.1  mixed t h o r o u g h l y .  The  l i b e r a t i o n of paranitrophenylate  ml o f enzyme s o l u t i o n was  measured a t 400*ry*with  spectrophoto-meter equipped w i t h a thermostatable a t 37°C. of  An  Q.l/tmof  curvette  added and  i n c r e a s e i n o p t i c a l d e n s i t y due  was  i n c r e a s e i n o p t i c a l d e n s i t y o f 1.2  to  l i n e obtained  c e l l compartment  maintained  u n i t s i n d i c a t e s the h y d r o l y s i s  obtained.  The  plotted  slope of  the  f o r t h e W o r t h i n g t o n enzyme i n d i c a t e s an i n c r e a s e i n o p t i c a l  d e n s i t y o f 0.016/min. whereas t h a t o f t h e c a r b o x y m e t h y l - c e l l u l o s e phosphodisterase  i s 0.014/min.  0.078/am and 0 . 0 6 6 / t m o f  These v a l u e s c o r r e s p o n d  s u b s t r a t e p e r h o u r by t h e s e  a b o u t 6.6/*»viof  purified  to the h y d r o l y s i s of  enzyme p r e p a r a t i o n s .  f o u n d t h a t t h e p u r i f i e d s n a k e venom p h o s p h o d i e s t e r a s e  ml o f t h e  It  s o l u t i o n hydrolyzed  s u b s t r a t e / h o u r / O . l ml o f t h e o r i g i n a l enzyme s o l u t i o n .  v a l u e c o r r e s p o n d e d t o a b o u t 17/t^ o f t h e W o r t h i n g t o n venom  4.  the  substrate.  a g a i n s t t h e i n c u b a t i o n t i m e , a s t r a i g h t l i n e was  p e r 0.1  the  a Cary r e c o r d i n g  F i g u r e 7 shows t h a t , when t h e i n c r e a s e i n o p t i c a l d e n s i t y was  was  by  This  phosphodiesterase  solution.  Stepwise degradation  o f ONA  by t h e p u r i f i e d snake venom p h o s p h o d i e s -  terase. Reagents: 0.01M  magnesium c h l o r i d e , 0.04M T r i s b u f f e r pH8.9 —  2.09g o f M g C l ^ h ^ O and  o f T r i s were d i s s o l v e d i n d i s t i l l e d w a t e r , t h e pH o f t h e s o l u t i o n was to  8.9  w i t h HC1  and  t h e volume was  1%  serum a l b u m i n s o l u t i o n : —  made up t o 1000  ml w i t h d i s t i l l e d  c o n t a i n s l g a l b u m i n / 1 0 0 ml o f d i s t i l l e d  4.B5g  adjusted water. water  48  O  Worthington enzyme  ^  Carboxymethyl c e l l u l o s e p u r i f i e d enzyme  0.4  0.3  "  0.2  -  0.1  -  o o +•>  a  o v G  a  u  o <  Figure  7.  10 15 20 R e a c t i o n Time i n Minutes Time course o f the h y d r o l y s i s o f p - n i t r o p h e n y l thymidine-5-phosphate by snake venom phosphodiesterase.  49 2 0 % TCA 2% TCA  — —  c o n t a i n s 20g TCA/100 ml o f d i s t i l l e d c o n t a i n s 2g TCA/100 ml o f d i s t i l l e d  water.  water.  Procedure: R a d i o a c t i v / e DNA a f t e r MAK 8.9,  o r t h e f r e e z e - d r i e d r e s i d u e o b t a i n e d f r o m t h e main DNA  c h r o m a t o g r a p h y was  d i s s o l v e d i n 0.01M  MgCl  2  - 0.04M T r i s b u f f e r  t o o b t a i n a c o n c e n t r a t i o n o f a p p r o x i m a t e l y 1 - 2 mg DNA  and one h a l f ml o f t h e r e s u l t i n g DNA  phosphodiesterase.  The  r e a c t i o n was  The  i n a c t i v i t y t o 80 o r 160/^j o f  The m i x t u r e was  i n t e r v a l s a l i q u o t s were w i t h d r a w n  per 5 ml.  s o l u t i o n were i n c u b a t e d w i t h 0.5  t h e p u r i f i e d snake venom p h o s p h o d i e s t e r a s e s o l u t i o n a t 37°C. p u r i f i e d enzyme used c o r r e s p o n d e d  The  ml  section.  The  was measured i n t h e u l t r a v i o l e t r e g i o n .  Worthington  a d d i t i o n o f 0.5  ml o f c o l d 2 0 %  absorbance Due  by a  determined  tubes. TCA,  centrifuga-  procedure  of the a c i d - s o l u b l e  t o t h e h i g h amount o f TCA  i n the a c i d - s o l u b l e part, the u l t r a v i o l e t absorption curves of these The  of  and p l a c e d i n 12 m l S e r v a l l c e n t r i f u g e  r a d i o a c t i v i t y o f b o t h f r a c t i o n s were d e t e r m i n e d  were r a t h e r d i s t o r t e d .  Four  shaken o c c a s i o n a l l y and a f t e r v a r i o u s t i m e  s t o p p e d by t h e d r o p w i s e  d e s c r i b e d i n a subsequent  pH  amount o f t h e  and t h e a c i d - s o l u b l e and a c i d - i n s o l u b l e f r a c t i o n s were s e p a r a t e d by tion.  peak  fraction present  solutions  u l t r a v i o l e t a b s o r p t i o n c u r v e s were, t h e r e f o r e , a l s o  a f t e r e x t r a c t i o n three times with ether.  0.5  ml a l i q u o t s o f t h e o r i g i n a l DNA  V.  R a d i o a c t i v e Counting  As " z e r o t i m e  controls"  s o l u t i o n w i t h o u t added enzyme were  used.  Procedures 3  1.  L i q u i d s c i n t i l l a t i o n c o u n t i n g o f H - l a b e l l e d DNA p r e p a r a t i o n s .  In the present i n v e s t i g a t i o n the r a d i o a c t i v i t y o f the t r i t i u m - l a b e l l e d p r e p a r a t i o n s was spectrometer  determined  i n an a u t o m a t i c T r i - C a r b l i q u i d  ( P a c k a r d I n s t r u m e n t Co.,  314A).  The  DNA  scintillation  s a m p l e s were c h i l l e d a t  p r i o r t o c o u n t i n g and m a i n t a i n e d a t t h a t t e m p e r a t u r e  during counting.  The  -3°C  50 c o u n t i n g r a t e s on t h e r e d c h a n n e l were r e c o r d e d w i t h a d i s c r i m i n a t o r s e t t i n g o f 10:50 was  volts.  A h i g h v o l t a g e s e t t i n g , h e r e a f t e r r e f e r r e d as t a p  s e l e c t e d w h i c h gave maximum c o u n t i n g  setting,  efficiency.  Materials: 25 y  - diphenyloxazole  (PPO)  and  were o b t a i n e d f r o m t h e P a c k a r d and Adamson Co.  Hyamine 10-X  2,2'~paraphenylene-5-phenyloxazole I n s t . Co.  T o l u e n e was  purchased  ( D i - i s o b u t y l cresoxy ethoxy  b e n z y l ammonium c h l o r i d e monohydrate) was  ethyl  (POPQP) from Baker dimethyl  o b t a i n e d from Rohm and Haas, P h i l a -  delphia. S o l v e n t s and S o l u t i o n s : Hyamine H y d r o x i d e a s ; . f o l l o w s : ; 1.4  solution—  kg hyamine 10-X  and t h e s o l u t i o n was 4TJ o v e r n i g h t . colourless.  Hyamine 10-X  R e c r y s t a l l i z a t i o n was  The  suspension  was  t h e n 25.5  stirred  g Ag^O  The  supernatant  w i t h IN HC1  between 0.8 Scintillator 1000  and  added.  was  The  solution  The  The  stirrer  temperature.  i n t o a c l e a r g l a s s s t o p p e r e d b o t t l e , and  u s i n g p h e n o l p h t a l e i n as i n d i c a t o r .  ml  were added, w i t h v i g o r -  x g a t room  i l l u m i n a t e d i n s u n l i g h t u n t i l i t c l e a r e d up c o m p l e t e l y . ted  at  f o r 10 m i n u t e s w i t h a m a g n e t i c  q u i c k l y c e n t r i f u g e d f o r a b o u t 10 m i n u t e s a t 810 decanted  refrigerated  were d i s s o l v e d i n 100  m l d i s t i l l e d w a t e r was  and  was  toluene with heating,  s o l u t i o n was  96g o f t h e r e c r y s t a l l i z e d hyamine 10-X  The  toluene  r e p e a t e d u n t i l the mother l i q u i d  a l l o w e d t o r e a c h room t e m p e r a t u r e ,  ous s h a k i n g .  r e c r y s t a l l i z e d from  d i s s o l v e d i n 2.51  f i l t e r e d while hot.  g l a s s d i s t i l l e d m e t h a n o l and 1.8 was  was  was  was  s o l u t i o n was  titra-  c o n c e n t r a t i o n ranged  1.0N.  solution —  ml of r e d i s t i l l e d  6.3  g o f PPO  and 0.262 g o f PQP0P were d i s s o l v e d i n  toluene.  P r e p a r a t i o n o f t h e r a d i o a c t i v e sample f o r c o u n t i n g : P r e v i o u s o b s e r v a t i o n s f r o m t h i s l a b o r a t o r y (177)  i n d i c a t e d the f o l l o w i n g  counting  51 s y s t e m t o be t h e most s u i t a b l e f o r c o u n t i n g t r i t i u m l a b e l l e d  The  0.4 1.0 3.0  ml o f a s o l u t i o n o f t h y m i n e i n 0.1 ml o f hyamine h y d r o x i d e ml o f a b s o l u t e e t h a n o l  5.0  ml o f t o l u e n e c o n t a i n i n g 0.63%  s a m p l e s were made up i n 20 ml g l a s s v i a l s .  procedures  5.0  reasonable  t o a s s a y 0.5  PPO  NHC1  and 0.0262% P0P0P  Since d u r i n g the  ml f r a c t i o n s were r o u t i n e l y c o l l e c t e d , i t was  a c t i v i t y o f t h e DNA  ml a l i q u o t s o f t h e s e f r a c t i o n s .  s a m p l e s was  50  thought  t o be more  f e l t desirable to i n -  Under t h e s e c o n d i t i o n s , however,  t h e volume o f a b s o l u t e e t h a n o l had t o be i n c r e a s e d t o 4.0 s e p a r a t i o n when t h e s a m p l e s were c h i l l e d .  fractionation  Moreover, s i n c e the  low i n most c a s e s , i t was  c r e a s e t h e volume o f t h e sample t o be a s s a y e d .  thymine:  Each sample was  ml t o a v o i d phase counted  f o r 30  to  minutes. In e a r l y e x p e r i m e n t s  the chromatographic  f r a c t i o n s c o n t a i n i n g 'NaCl  formed c r y s t a l l i n e p r e c i p i t a t e which c l u n g t o t h e s i d e s o f the v i a l s .  It i s  a l s o known t h a t sodium c h l o r i d e and a l k a l i have an i n t r i n s i c q u e n c h i n g e f f e c t r a d i o a c t i v e s o l u t i o n s (178).  A l l r a d i o a c t i v e samples were, t h e r e f o r e , d e s a l t e d  p r i o r t o c o u n t i n g as d e s c r i b e d a b o v e . Under t h e s e c o n d i t i o n s t h e r e was  D u p l i c a t e s o f each sample were  The  r e l a t i v e counting e f f i c i e n c y  u s i n g an a p p r o x i m a t e s t a n d a r d o f t r i t i a t e d  thymidine.  o r d e r t o c o r r e c t f o r r a d i o a c t i v e decay, v a r i a t i o n s i n the c o u n t i n g of the instrument  yde,  d u p l i c a t e 0.5  w a t e r were c o u n t e d were made.  counted.  a l i n e a r r e l a t i o n s h i p between t h e amount o f  p r e s e n t and t h e r a d i o a c t i v i t y d e t e c t e d . t h i s s y s t e m i s 3.0%  on  w i t h each s e r i e s o f e x p e r i m e n t s  of In  efficiency  and f o r v a r i a t i o n s i n t h e c o n c e n t r a t i o n o f t h e hyamine 3 ml a l i q u o t s o f a s t a n d a r d H - t h y m i d i n e  DNA  hydrox-  solution in d i s t i l l e d  and t h e n e c e s s a r y c o r r e c t i o n s  52 14 2. DNA  L i q u i d s c i n t i l l a t i o n c o u n t i n g o f t h e double  (C  3 and H ) l a b e l l e d  preparations. The e f f e c t i v e n e s s o f l i q u i d  scintillators  f o r c o u n t i n g weak b e t a -  e m i t t i n g i s o t o p e s and t h e d i f f e r e n c e i n t h e e n e r g i e s o f t h e b e t a p a r t i c l e s o f 14 t r i t i u m and c a r b o n - C l a b e l l e d compound.  make i t p o s s i b l e t o a s s a y f o r b o t h i s o t o p e s i n a  The maximum b e t a e n e r g y f o r  0.155M v and t h e two i s o t o p e s may be c o u n t e d s c i n t i l l a t i o n spectrometer,  t a g e and d i s c r i m i n a t o r - s e t t i n g s .  i s 0.018Mev and f o r C"*"^  i n one o p e r a t i o n i n t h e P a c k a r d  e  Tri-Carb l i q u i d  by p r o p e r s e l e c t i o n o f h i g h  The two c h a n n e l s  i s fed into i t s respective scaler.  vol-  f o r pulse-height discrimina-  t i o n a r e o b t a i n e d by means o f d i s c r i m i n a t o r c o n t r o l s AA', B and C. o f each c h a n n e l  double-  The  output  A l l p u l s e s o f energy be-  tween AA' t o B a r e r e c o r d e d on t h e r e d s c a l e r and p u l s e s f r o m B t o C a r e r e g i s t e r e d by t h e g r e e n s c a l e r .  To o b t a i n optimum s e t t i n g s , t h e c o u n t i n g r a t e s  were p l o t t e d v e r s u s t h e h i g h v o l t a g e t a p s e t t i n g s f o r b o t h i s o t o p e s a t d i f f e r ent d i s c r i m i n a t o r s e t t i n g s .  The optimum c o n d i t i o n s were d e t e r m i n e d  on t h e  14 graph a t which C  and t r i t i u m c o u l d be c o u n t e d 14  e f f i c i e n c i e s , while the C  c o u l d be c o u n t e d  no t r i t i u m p u l s e s c o m i n g i n t o t h a t c h a n n e l .  i n the r e d s c a l e r with high  i n t h e g r e e n s c a l e r w i t h few o r The optimum d i s c r i m i n a t o r and h i g h  v o l t a g e t a p s e t t i n g s were f u r t h e r e v a l u a t e d by m a k i n g up a s e r i e s o f s t a n d a r d s o l u t i o n s c o n t a i n i n g b o t h o f t h e i s o t o p e s i n known and v a r i a b l e c o n c e n t r a t i o n s and c o u n t i n g them a t d i f f e r e n t d i s c r i m i n a t o r and t a p s e t t i n g s . the double tillation  The c o u n t i n g o f  l a b e l l e d compounds was p e r f o r m e d on a P a c k a r d T r i - C a r b l i q u i d spectrometer,  m o d e l 314 AX.  scin-  The a n a l y s i s mode s w i t c h was i n s p l i t  and number " 2 " p o s i t i o n ( 1 7 9 ) . The d e t e r m i n a t i o n o f optimum h i g h v o l t a g e t a p and d i s c r i m i n a t o r s e t t i n g s . In o r d e r t o o b t a i n optimum c o n d i t i o n s i t was n e c e s s a r y q u e n c h i n g e f f e c t o f hyamine and DNA  on t h e C  14  and H  3  t o know t h e  c o n t a i n i n g systems.  53 T h e r e f o r e , a s e r i e s o f s a m p l e s were p r e p a r e d .  E a c h sample c o n t a i n e d 0.5ml  14 o f t h e s i n g l e i s o t o p e ( e i t h e r 0.5 ing  44,400 dpm/min o r 0.5  ml o f C  - l a b e l l e d Na^CO^ s t a n d a r d c o n t a i n -  m l o f H - l a b e l l e d H^O  s t a n d a r d c o n t a i n i n g 98,000  dpm/min), t h e amounts o f e t h a n o l and s c i n t i l l a t o r v i o u s l y and v a r i a b l e amounts o f DNA III.  The  s o l u t i o n as d e s c r i b e d p r e -  and Hyamine H y d r o x i d e  as shown i n T a b l e  compounds were added t o t h e v i a l s i n t h e f o l l o w i n g o r d e r : r a d i o -  a c t i v e s o l u t i o n , h y a m i n e , e t h a n o l and s c i n t i l l a t o r t i o n s from another  l a b o r a t o r y (180)  indicated that a  system ( c o m p o s i t i o n of naphthalene/dioxane 5% n a p t h a l e n e  solution.  Previous  naphthalene/dioxane  s o l v e n t : 1% PPO,  0.05%  P0P0P,  i n d i o x a n e : e t h y l e n e g l y c o l m o n o e t h y l e t h e r 5 ; l ) gave a p p r o x -  i m a t e l y 1 0 % e f f i c i e n c y when t r i t i u m l a b e l l e d s a m p l e s were c o u n t e d . i n t e r e s t , t h e r e f o r e , t o compare t h i s s y s t e m w i t h t h e t o l u e n e Table  IV shows t h e c o m p o s i t i o n  dioxane  observa-  o f the samples prepared  I t was  of  scintillator.  i n the  napthalene/  system. The  ples 1 - 6  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 t h e r e d c h a n n e l o f sam-  a r e shown i n f i g u r e s 8 - 1 3 .  F i g u r e 14 shows a s e r i e s o f  curves  o b t a i n e d by p l o t t i n g t h e c o u n t i n g r a t e s on t h e g r e e n s c a l e r a g a i n s t t h e  high  v o l t a g e t a p s e t t i n g s a t d i f f e r e n t d i s c r i m i n a t o r s e t t i n g s u s i n g Sample 1.  On  i n s p e c t i o n , t h e s e d i a g r a m s r e s u l t e d i n t h e f o l l o w i n g o b s e r v a t i o n s : (a) i n c r e a s ing  t h e volume o f Hyamine H y d r o x i d e 14 3  on b o t h C  and  s a m p l e s 2, 4 and the r i g h t  H  counting r a t e s . 6, and  The  ml has a d e f i n i t e q u e n c h i n g  effect  counting r a t e s are g e n e r a l l y lower i n  t h e peaks o f maximum e f f i c i e n c i e s a r e s h i f t e d  ( 1 7 9 ) , (b) t h e p r e s e n c e  counting rates,  t o 1.0  o f added DNA  towards  has no a p p r e c i a b l e e f f e c t on 14  ( s e e F i g u r e 10, sample 3 ) , ( c ) t h e e f f i c i e n c i e s o f b o t h C  the and  3  H  l a b e l l e d s a m p l e s were h i g h e r i n t h e n a p t h a l e n e / d i o x a n e  Figure 12).  T h i s s y s t e m , however, had t h e b i g d i s a d v a n t a g e  days o f s t o r a g e , t h e s o l u t i o n s became d i s c o l o u r e d . scintillator  s y s t e m (Sample 5,  s y s t e m was  used f o r f u r t h e r s t u d i e s .  that after  For t h a t reason, the Diagrams 8 - 1 4  also  2-3 toluene  54 TABLE I I I C o m p o s i t i o n o f S t a n d a r d s C o n t a i n i n g t h e S i n g l e I s o t o p e H o r C , Used f o r the D e t e r m i n a t i o n o f t h e 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 Curves i n t h e Toluene S c i n t i l l a t o r System 3  Sample number  DNA i n n_g  1 4  hyamine h y d r o x i d e i n ml  1  -  0.5  2  -  1.0  3  0.5  0.5  4  0.5  1.0  t  TABLE IV  3 14 C o m p o s i t i o n o f S t a n d a r d s C o n t a i n i n g t h e S i n g l e I s o t o p e H o r C , Used f o r the Determination o f t h e 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 Curves i n t h e Naphthalene/ Dioxane S c i n t i l l a t o r System.  Sample number  DNA i n mg  ftyamine hydroxide i n ml  naphthalene/ dioxane s c i n t i l l a t o r i n ml  5  0.6  0.5  9.0  6  0.5  1.0  8.5  55  .14 IT  24-,  18  to X  12 A  s o  6i 10:100 «V10:70 '^10:50 /-10:40 «-10:30 ^10:20 r  T~  T T 1 r 3 4 5 6 7 8 2 High V o l a t a g e Tap S e t t i n g  F i g u r e 8,  i  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 sample 1 from T a b l e I I I determined on the r e d s c a l e r For d e t a i l s see t e x t .  56  High V o l t a g e Tap F i g u r e 9.  Setting  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 sample 2 from Table I I I determined on the r e d s c a l e r . For d e t a i l s see t e x t .  57  High V o l t a g e Tap F i g u r e 10.  Setting  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 curves f o r sample 3 from Table I I I determined on the r e d s c a l e r . For d e t a i l s see t e x t .  58  14 rf  24-r  18-  to o  s ft  12-  1 Figure  11,  2 3 4 5 6 High V o l a t g e Tap  7 8 Setting  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 sample 4 from T a b l e I I I determined on the r e d s c a l e r . For d e t a i l s see t e x t .  59  ,14  24  FT T  18 to  s o  12 -  6 ^10:100 10:70 <>V10:50 t<_/10:40 r  2  T  18118  i  4  5  6  8  High V o l t a g e Tap S e t t i n g Figure  12,  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 curves f o r sample 5 from Table IV determined on the r e d s c a l e r . For d e t a i l s see t e x t .  60  1 1 2  I  i  I  3  1  4  1  5  H i g h V o l t a g e Tap F i g u r e 13.  1  6  7  1  8  r  9  Setting  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 sample 6 from T a b l e I V d e t e r m i n e d on the r e d s c a l e r . F o r d e t a i l s see t e x t .  61  24 -  18 -  to o  6 -  High V o l t a g e Tap F i g u r e 14.  Setting  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 curves f o r sample 1 from Table I I I determined on the green s c a l e r . The curve r e p r e s e n t s the average of the d e t e r m i n a t i o n of 6 d i f f e r e n t window s e t t i n g s . For d e t a i l s see t e x t .  62 indicated  t h a t optimum h i g h v o l t a g e t a p s e t t i n g s f o r d o u b l e l a b e l l e d compounds  s h o u l d be e i t h e r 4 o r 5. containing  A t t h e s e h i g h v o l t a g e s e t t i n g s , b o t h t r i t i u m and C  14  s a m p l e s were c o u n t e d a t a r e l a t i v e l y h i g h e f f i c i e n c y on t h e r e d 14  s c a l e r , whereas C  was c o u n t e d w i t h  s c a l e r w i t h o u t t o o much i n t e r f e r e n c e To o b t a i n  optimum d i s c r i m i n a t o r  a r e a s o n a b l y good e f f i c i e n c y on t h e g r e e n by c o u n t s a r i s i n g f r o m t r i t i u m  pulses.  s e t t i n g s , 4 standard samples c o n t a i n i n g  _14 3 C and H i n known amounts were a n a l y z e d a t d i f f e r e n t window and h i g h  both voltage  14 tap  s e t t i n g s , and t h e p e r c e n t a g e r e c o v e r i e s  culated  o f both C  and t r i t i u m were  a c c o r d i n g t o t h e d i s c r i m i n a t o r - r a t i o method i n r e f . ( 1 8 1 , 1 8 2 ) .  calThis  method u s e s t h e c o u n t s / m i n . on t h e g r e e n s c a l e r t o t h o s e o f t h e r e d s c a l e r , w h i c h were o b t a i n e d f r o m s t a n d a r d s a m p l e s o f each o f t h e i s o t o p e s . al.  (181) d e r i v e d  the following  i n a sample c o n t a i n i n g  both b  .3 H dpm  red  and C'''  4  dpm  isotopes: l  N  -  b  =  equation f o r the c a l c u l a t i o n of  Okita _ t  scaler H  2  H  a  efficiency factor  and  b(N L  dpm  - al^)  2  =  j j  green s c a l e r C where  =  efficiency factor  n e t cpm on r e d s c a l e r  -  "  "  green  _  n e t cpm o f H  scaler  3 a  net h•  _  cpm o f H  on g r e e n 3  scaler  on r e d s c a l e r  n e t cpm ~^ on gar e e n s c a l e r c o f C"*j^_= n e t cpm o f C on r e d s c a l e r  The c o m p o s i t i o n o f t h e f o u r  d o u b l e (H^ and C"^)  l a b e l l e d standard solutions  as f o l l o w s :  2 0.5 0.5 4.0 5.0  ml ml ml ml  of of of of  standard (containing both H hyamine h y d r o x i d e absolute ethanol s c i n t i l l a t o r solution  j_4 and C isotopes)  was  63 T a b l e V shows t h e e f f e c t o f v o l t a g e and d i s c r i m i n a t o r  s e t t i n g s on t h e t r i t i u m  14 and C  assays of 4 d i f f e r e n t standards.  The f o l l o w i n g  i s a sample c a l c u l a t i o n  u s i n g t h e d a t a f o r s t a n d a r d 1 a t v o l t a g e t a p s e t t i n g 4 and d i s c r i m i n a t o r  setting  10:20:100 B a c k g r o u n d c o u n t s on  r e d s c a l e r = 17.5 cpm  B a c k g r o u n d c o u n t s on g r e e n s c a l e r = 33.8 cpm T o t a l c o u n t s on r e d s c a l e r = 1047.3 cpm T o t a l c o u n t s on g r e e n s c a l e r = 749.6 cpm l\l  = T o t a l c o u n t s on r e d s c a l e r - bg = 1047.3 -  17.5 1029.8 cpm = T o t a l c o u n t s on g r e e n s c a l e r - bg = 749.6 -33.8 715.8 cpm  N  a = 0  ( a and b were d e t e r m i n e d from F i g u r e s 8,14  respectively)  b = 0.87 3 red  scaler H  e f f i c i e n c y f a c t o r = 0.01685 I*  g r e e n s c a l e r C e f f i c i e n c y f a c t o r = 0.0786 ( e f f i c i e n c y f a c t o r s were d e t e r m i n e d from F i g u r e s 8,14  H  3  BN - N -i——±— b - a red s c a l e r e f f i c i e n c y factor 2  dpm =  b(N -14  C  .  z  - al\l )  b_=_e  dpm .  respectively)  (0.87x1029.B-K715.B 0.87-0 = 12,300 0.01685  0.87(715.8-0x1029.8) 0.87 - 0 =  9107  0 > 0 7 a 6  green s c a l e r e f f i c i e n c y factor From i n s p e c t i o n  o f t h e d a t a i n T a b l e V- i t was c o n c l u d e d t h a t  were most a c c u r a t e l y  assayed a t d i s c r i m i n a t o r  t a g e t a p s e t t i n g 5.  S t a n d a r d 4 was an e x c e p t i o n  standards  1-3  s e t t i n g s 10:30:100 and h i g h v o l and a l s o t h e w i d e s t  from t h e t h e o r e t i c a l c o u n t s were o b s e r v e d on a s s a y i n g t h i s , ^ s a m p l e .  variations  64 T a b l e V. E f f e c t o f V o l t a g e and D i s c r i m i n a t o r Assays o f 4 D i f f e r e n t Standards. Standard 1 Discriminator &. h i g h v o l t a g e tap s e t t i n g s 10:20:100 tap 4 10:20:100 tap 5 10:30:100 tap 4 10:30:100 tap 5 10:40:100 tap 4 10:40:100 tap 5 10:50:100 tap 4 10:50:100 tap 5  C o n t a i n s T h e o r e t i c a l l y 15•680 H  Cpm-bg Cpm-bg on r e d on g r e e n scaler scaler  10:.20sl00 tap 4 10:20:100 tap 5 10:30:100 tap 4 10:30:100 tap 5 10:40:100 tap 4 10:40:100 tap 5 10:50:100 tap 4 10:50:100 tap 5  H dpm recovered  3  ,14 C dpm recovered  dpm and 8880i.C  14  dpm.  % deviation % deviation from t h e o - from t h e o retical H r e t i c a l C^3  4  1029.8  715.8  12,300  9107  21.6  2.5  737.0  2064.7  17,130  8543  9.2  3.8  1863.6  677.4  13,742  8889  12.4  0.1  1318.2  2123.6  15,260  8885  2.7  0.1  2570.9  707.1  12,590  8332  19.7  6.2  1842.9  2102.1  16,450  8785  4.9  1.1  3153.3  69B.2  12,160  8794  22.5  1.0  2331.6  2135.5  15,406  8855  1.8  0.3  Standard 2 Discriminator &. h i g h v o l t a g e tap s e t t i n g s  S e t t i n g s on t h e T r i t i u m and Carbon 14  C o n t a i n s t h e o r e t i c a l l y 15,680 dpm  Cpm-bg Cpm-bg on r e d on g r e e n scaler scaler  ^ H dpm recovered  rC A dpm recovered 1 4  and 4440 C ^  dpm.  % deviation % deviation f r o m theo:? f r o m t h e o - , 3 14 retical H retical C  659.3  344.7  15,610  4386  0.5  1.2  493.2  1053.3  16,140  4321  2.9  2.7  1152.8  331.1  16,010  4345  2.1  2.1  879.8  1080.4  15,959  4440  1.8  0.0  1518.9  348.4  13,970  4399  11.0  0.9  1206.6  1069.3  16,590  4444  5.8  0.1  1889.0  351.5  14,940  4465  1477.1  1071.1  16,540  4345  4.7 5.5  0.6 2.1  65 T a b l e V. c o n t i n u e d Standard Discriminator &. h i g h v o l t a g e tap settinqs 10:20:100 tap 4 10:20:100 tap 5 10:30:100 tap 4 10:30:100 tap 5 10:40:100 tap 4 10:40:100 tap 5 10:50:100 tap 4 10:50:100 tap 5  3  Cpm-bg on g r e e n scaler  H dpm recovered  1330.3  677.0  32,740  9043  4.4  1.8  1008.5  2099.9  33,780  8600  7.7  3.2  2333.9  640.0  34,950  8398  11.4  5.4  1746.4  2171.8  31,308  8931  0.2  0.6  30,870  8729  1.6  1.7  691.3--.  3  C"^ dpm recovered  2083.9  34,866  8644  11.2  2.7  3787.6  679.2  32,890  8658  4.9  2.5  3018.8  2103.0  35,016  8572  11.6  3.5  Cpm-bg on r e d scaler  C o n t a i n s T h e o r e c t i c a l l y 7840 dpm H Cpm-bg on g r e e n scaler  dpm recovered  C dpm recovered  C^  % deviation % devia from t h e o - from t h i retical r e t i c a l -14  2432.3  Standard 4  10:20:100 tap 4 10:20:100 tap 5 10:30:100 tap 4 10:30:100 tap 5 10:40:100 tap 4 10:40:100 tap 5 10:50:100 tap 4 10:50:100 tap 5  and 8S80 dpm  Cpm-bg on r e d scaler  3116.7  Discriminator L high voltage tap s e t t i n q s  C o n t a i n s t h e o r e c t i c a l l y 31,360 dpm  3  and 8880 dpm  C^  % d e v i a t i o n % deviafrom t h e o ^ from t h i retical H retical  890.4  699.4  5136  8899  34.5  0.2  601.8  2120.2  7835  8828  0.1  0.6  1672.9  664.0  ,7569  8722  3.5  1.8  1099.8  2096.4  7112  8739  9.3  1.6  2315.0  701.3  5245  8855  33.1  0.3  1577.9  2070.4  8355  8678  6.6  2.3  2900.1  696.2  4969  8888  36.6  0.1  2015.7  2098.8  7584  8692  3.3  2.1  66 T h i s was e x p e c t e d , H"^ dpm/C^ Standard  s i n c e a c c o r d i n g t o O k i t a et. a l . (181) f o r a c c u r a t e  dpm s h o u l d be between 1-15.  assays  T h i s r a t i o , , was l e s s t h a n one f o r  4. 'On'the b a s i s o f t h e d a t a o f g r a p h s and T a b l e V i t was d e c i d e d t h a t ,  f o r t h e r o u t i n e assays o f double be  l a b e l l e d compounds t h e f o l l o w i n g s y s t e m w o u l d  adopted: 3 14 0.5 m l o f a s o l u t i o n o f H and C labelled materiali n d i s t i l l e d water 0.5 ml o f hyamine h y d r o x i d e 4.0 m l o f a b s o l u t e e t h a n o l 5.0 m l o f s c i n t i l l a t o r s o l u t i o n  The  s a m p l e s were c o u n t e d  f o r 10 t o 100 m i n u t e s ( d e p e n d i n g  on t h e a c t i v i t i e s )  a t d i s c r i m i n a t o r s e t t i n g s o f 10:30:100 and h i g h v o l t a g e t a p s e t t i n g o f 5 w i t h t h e " a n a l y s i s mode" s w i t c h i n s p l i t and number 2 p o s i t i o n .  These s e t t i n g s  were m o d i f i e d f r o m t i m e t o t i m e a c c o r d i n g t o c h a n g e s i n e f f i c i e n c y o f t h e apparatus, one  u s u a l l y observed  after servicing.  i s not counting a t the "balance  Since i n double  isotope counting  p o i n t " o r peak o f t h e v o l t a g e - e f f i c i e n c y  c u r v e , a s l i g h t f l u c t u a t i o n i n l i n e v o l t a g e may c a u s e a s i g n i f i c a n t change i n the counting r a t e .  T h e r e f o r e , a number o f s t a n d a r d s c o n t a i n i n g known b u t  v a r i a b l e r a t i o s o f H"^ dpm, ments and t h e n e c e s s a r y  dpm were c o u n t e d  w i t h each s e r i e s o f e x p e r i -  c o r r e c t i o n s f o r i n s t r u m e n t s e t t i n g s were made.  R e c e n t l y P r o c k o p and E b e r t  (183) have r e p o r t e d a s i m p l e method w h i c h  3 , 14 p e r m i t s t h e r a p i d and e f f i c i e n t d e t e r m i n a t i o n o f H /C ratios i n partially 3 purified biological materials.  I n t h i s method t h e known r a t i o s o f H  14 dprn/C  dpm o f a s e r i e s o f s t a n d a r d s were p l o t t e d a g a i n s t t h e r a t i o s o f cpm i n r e d scaler/cpm  i n green s c a l e r o b t a i n e d f o r these samples.  dicated a linear relationship.  Their results i n -  I t was a l s o f o u n d t h a t a change i n t h e s l o p e  o f t h e s t a n d a r d curve r e f l e c t s changes i n t h e s t a t e o f t h e i n s t r u m e n t .  67 In t h e c h e c k i n g the  present i n v e s t i g a t i o n , therefore,  instrument from experiment to experiment.  v e r y s u i t a b l e f o r r a p i d d e t e r m i n a t i o n o f H"Vc^ F i g u r e 15 red  shows t h e  also  r a t i o s i n unknown s a m p l e s . o b t a i n e d on  Knowing t h e c o u n t s on  the  known H"Vc^  the  two  dpm  scalers  14  H /C  r a t i o s o f an unknown sample can  F i g u r e 15  serviced.  The  determined from the  The  graph.  p u r i f i c a t i o n and  co n  3  u  c o u n t i n g of the  t i  In  o c c a s i o n when t h e  d o t t e d l i n e , o b t a i n e d a f t e r s e r v i c i n g has  i n d i t i n g . niche, efficiency for H 3.  be  i s i l l u s t r a t e d t h e change w h i c h o c c u r e d a t one  s t r u m e n t was siope,  adopted f o r  technique i s  g r e e n s c a l e r were p l o t t e d a g a i n s t t h e  of the v a r i o u s standard s o l u t i o n s .  the  The  s t a n d a r d c u r v e s o b t a i n e d , when t h e cpm  s c a l e r / c p m on t h e  3.  t h i s method was  a  in-  greater  „ . 9  i n t e r p h a s e l a y e r s of the  tissue  extracts. A f t e r c a r e f u l l y r e m o v i n g t h e DNA phase l a y e r s o b t a i n e d d u r i n g t h e t h e DNA  The  extracted  t i m e was was  with  two  more t i m e s w i t h  then e x t r a c t e d  s t e p s i n the  l i f t e d with  a spatula  chloroform-amyl alcohol l a y e r .  5 ml o f i c e c o l d 20%  centrifuged  aqueous l a y e r t h e  TCA  i s o l a t i o n of  The  The  frozen  suspension  was  20-30 ml e t h e r , f o l l o w e d The  was  s u s p e n s i o n w h i c h f o r m e d each  a t 926xg i n a r e f r i g e r a t e d c e n t r i f u g e .  each t i m e a t 926xg a t room t e m p e r a t u r e .  from the  u s i n g a Vortex J r . mixer, then i t  5 ml o f 2% TCA.  four times with  inter-  p r e p a r e d f o r c o u n t i n g o f the r a d i o a c t i v i t y  i n t e r p h a s e l a y e r was  "phenol l a y e r , o r f r o m t h e  treated  deproteinization  (147,159) were p u r i f i e d and  as f o l l o w s :  containing  r e s i d u e was  The by  precipitate centrifugation  d r i e d i r _ vacuo a t  o room t e m p e r a t u r e and The t o Brown and  was  f u r t h e r d r i e d a t 100  c o u n t i n g of the Badman ( 1 8 4 ) .  added and  d r i e d and  To  KOH  was  0.5  ml o f hyamine h y d r o x i d e was  10 mg  the m i x t u r e was  C i n t h e oven f o r 5-8  p u r i f i e d powder was  of dry  hours.  performed  powdered i n t e r p h a s e 0.5  b o i l e d i n a w a t e r - b a t h f o r 10  min.  added t o t h e c l e a r a l k a l i n e s o l u t i o n  according  ml o f  IN  After slowly,  that  68  October 1.8  December  1963 1963  n  4  6 Theoretical  Figure  15.  8  10  H /C 3  1 4  L i n e a r r e l a t i o n s h i p between H /C r a t i o i n the standard samples and the r a t i o o f the counts o b t a i n e d on r e d to green s c a l e r .  69 with s t i r r i n g . centrifuged  The  s o l u t i o n was t h e n n e u t r a l i z e d w i t h 0.5  a t 2500xg.  0.5  m l o f 2Ni HC1.and  m l a l i q u o t s o f t h e c l e a r s u p e r n a t a n t l a y e r were  u s e d f o r c o u n t i n g i n t h e system d e s c r i b e d on page  4.  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 p h e n o l l a y e r s  (147)  The p h e n o l l a y e r s , o b t a i n e d a f t e r e x t r a c t i o n o f t h e t i s s u e s a c c o r d i n g t o t h e method o f C o l t e r et_ a l (147) were combined and 1/4 w a t e r was  added.  The m i x t u r e was  shaken f o r 5 m i n u t e s and c e n t r i f u g e d  lO.OOOxg i n a S e r v a l l r e f r i g e r a t e d c e n t r i f u g e . the  p h e n o l l a y e r s was  o f t h e volume o f  A s e c o n d aqueous e x t r a c t i o n  a l s o p e r f o r m e d and t h e combined aqueous e x t r a c t s  shaken 4 t i m e s w i t h e q u a l volumes o f e t h e r .  at  The  s o l u t i o n was  of  were  evaporated almost  t o d r y n e s s i n a F l a s h - e v a p o r a t o r a t room t e m p e r a t u r e , t h e c o n c e n t r a t e t a k e n up i n 2.0 m l o f w a t e r and 0.5 m l a l i q u o t s o f t h i s s o l u t i o n were c o u n t e d i n a l i q u i d s c i n t i l l a t i o n c o u n t e r as d e s c r i b e d on page  5.  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 a c i d - i n s o l u b l e m a t e r i a l  ed d u r i n g t h e e n z y m a t i c d e g r a d a t i o n o f DNA  solutions.  To c o u n t t h e a c i d - i n s o l u b l e m a t e r i a l w h i c h t h e d o u b l e l a b e l l e d DNA was  was  obtain-  obtained i n the experiments i n  exposed t o snake venom p h o s p h o d i e s t e r a s e i t  important t o p u r i f y the m a t e r i a l .  P r e s e n c e o f any q u e n c h i n g m a t e r i a l ,  such  a s s a l t s , i n t h e r a d i o a c t i v e sample w o u l d i n f l u e n c e t h e r a t i o s o f t h e c o u n t s o b t a i n e d i n t h e two c h a n n e l s .  F o r t h a t r e a s o n v a r i o u s t e c h n i q u e s were  f o r the p u r i f i c a t i o n of the a c i d i n s o l u b l e  studied  material.  S e v e r a l a u t h o r s r e p o r t e d good c o u n t i n g e f f i c i e n c i e s and r e p r o d u c i b i l i t y when a c i d - i n s o l u b l e m a t e r i a l was c o l l e c t e d and c o u n t e d on M i l l i p o r e membranes (185-187).  T h i s t e c h n i q u e was  A s t o c k s o l u t i o n was 1 mg/ml) and d u p l i c a t e  tried  first.  p r e p a r e d f r o m t r i t i u m l a b e l l e d DNA  a l i q u o t s o f t h i s s o l u t i o n r a n g i n g from 0.1  (concentration t o 0.5  ml  70 Ten m i c r o l i t e r s o f 1% a l b u m i n s o l u t i o n  were p l a c e d i n m i c r o - t e s t t u b e s .  added t o each t u b e , t h e c o n t e n t s were c h i l l e d i n an i c e - b a t h p r o t e i n were p r e c i p i t a t e d  w i t h 0.5 m l o f c o l d  quantitatively transferred p l a i n 25 mm d i a m e t e r ) . ice cold  2% TCA and t h e n w i t h 5.0 m l t o l u e n e .  (HA 0.45  proportionality  white  o f o c c l u d e d TCA f r o m t h e membrane, t h u 9  o f the f i l t e r .  The membranes were a i r d r i e d f o r F i v e ml o f t o l u e n e  s c i n t i l l a t o r s o l u t i o n were added and t h e s a m p l e s were c o u n t e d . the duplicates  was  The t o l u e n e t r e a t m e n t was f o u n d  h o u r and p l a c e d h o r i z o n t a l l y i n t o t h e c o u n t i n g v i a l s .  that  and  p r e c i p i t a t e was washed t w i c e w i t h 0.5 m l o f  t o be n e c e s s a r y t o wash o u t t r a c e s  an  and t h e DNA  The p r e c i p i t a t e  t o M i l l i p o r e membrane f i l t e r s ,  The DNA  preventing discolouration  20% TCA.  was  I t was f o u n d  d i d n o t a g r e e and t h e r e was no l i n e a r r e l a t i o n s h i p o r  between DNA  c o n c e n t r a t i o n and c o u n t s o b t a i n e d .  o f t h e p a p e r seemed t o have v e r y l i t t l e  The geometry  e f f e c t on t h e c o u n t i n g  rate.  S i n c e t h e m i l l i p o r e membrane f i l t r a t i o n t e c h n i q u e was n o t s a t i s factory  a n o t h e r t e c h n i q u e was t r i e d b a s e d on c e n t r i f u g a t i o n .  s o l u t i o n was p r e p a r e d i n M g C l 2 - T r i s b u f f e r led material.  Duplicate aliquots  A DNA  using a double ( H  3  stock  and C^)  label-  o f 0.1, 0.2 and 0.5 m l o f t h i s s o l u t i o n  p l a c e d i n 12 m l S e r v a l l c e n t r i f u g e  tubes.  The t u b e s were k e p t i n an  were  ice-bath.  The t o t a l volume was made up t o 0.5 m l by a d d i n g , where n e c e s s a r y , more o f the  buffer.  t h e DNA  Ten m i c r o l i t e r s o f 1% serum a l b u m i n were added t o each t u b e and  was p r e c i p i t a t e d  w i t h 0.5 m l o f c o l d 20% TCA.  The TCA s o l u t i o n  was  added d r o p w i s e w i t h c o n s t a n t s h a k i n g , and t h e t u b e was t h e n shaken i n a V o r t e x J r . mixer.  The s u s p e n s i o n s w e r e . k e p t i n t h e i c e - b a t h  complete p r e c i p i t a t i o n . refrigerated  centrifuge  The c e n t r i f u g e and c e n t r i f u g e d  f o r 20 m i n . t o a s s u r e  t u b e s were t h e n p l a c e d i n a S e r v a l l f o r 20 m i n u t e s a t 30.000xg.  s u p e r n a t a n t s were c a r e f u l l y and q u a n t i t a t i v e l y removed w i t h a P a s t e u r and  placed i n clean dry t e s t tubes.  The p r e c i p i t a t e s  The pipette  were washed t w i c e by  s u s p e n s i o n i n 0.5 m l o f 1% TCA f o l l o w e d by c e n t r i f u g a t i o n .  The  supernatants  71 were c o m b i n e d and t h e r e s u l t i n g s o l u t i o n was t r e a t e d a s t h e a c i d s o l u b l e fraction.  The p r e c i p i t a t e s were washed w i t h 2 m l o f e t h a n o l : e t h e r  m i x t u r e and w i t h 2 m l o f a n h y d r o u s e t h e r .  The r e s i d u e s were d r i e d in_ v a c u o  o v e r n i g h t and t h e n s u s p e n d e d i n 0.7 m l o f w a t e r . deoxyribonuclease  (50:50)  To t h e s u s p e n s i o n  s o l u t i o n was a d d e d , c o r r e s p o n d i n g  t o 20/^^ DNase.  0.1 m l o f The  tubes  Q were i n c u b a t e d i n t h e s h a k i n g w a t e r b a t h f o r 7 h o u r s a t 37 C. the a c i d - i n s o l u b l e ^material completely  By t h i s  d i s s o l v e d , and a 0.5 m l a l i q u o t o f t h e  s o l u t i o n was t a k e n f o r c o u n t i n g i n t h e s y s t e m d e s c r i b e d on page r e s u l t s o f t h i s experiment c o u n t s o f t h e DNA  The  a r e p r e s e n t e d i n T a b l e V I . where t h e r e c o v e r y o f  a f t e r p r e c i p i t a t i o n w i t h TCA and p u r i f i c a t i o n i s compared  w i t h t h e u n t r e a t e d DNA  solution.  of d u p l i c a t e experiments  The r e s u l t s shown i n T a b l e V I . a r e t h e a v e r a g e  and i n d i c a t e t h a t t h e p r e c i p i t a t i o n and p u r i f i c a t i o n  t e c h n i q u e r e s u l t s i n no l o s s o f r a d i o a c t i v i t y .  The p r o c e d u r e  i s q u a n t i t a t i v e and  t h e r e i s a good c o r r e l a t i o n between t h e c o a c e n t r a t i o n o f l a b e l l e d DNA ing  rates.  periments 6.  time,  and  count-  T h i s t e c h n i q u e was u s e d t o c o u n t a c i d s o l u b l e m a t e r i a l i n t h e e x on e n z y m i c d e g r a d a t i o n o f DNA. 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 a c i d - s o l u b l e f r a c t i o n  during the enzymatic  d e g r a d a t i o n o f DNA  The s u p e r n a t a n t  obtained  solutions.  s o l u t i o n s obtained a f t e r the p r e c i p i t a t i o n of the  a c i d - i n s o l u b l e m a t e r i a l w i t h TCA, were e x t r a c t e d t h r e e t i m e s w i t h 5 m l o f a n h y d r o u s e t h e r , t o remove TCA f r o m t h e s o l u t i o n . moved by b u b b l i n g N2 t h r o u g h were c o u n t e d  0.01m  with C ^  o f e t h e r were r e -  t h e s o l u t i o n , and 0.5 m l a l i q u o t s o f t h e s o l u t i o n  i n the toluene s c i n t i l l a t o r  Previous experiments  Traces  or  s y s t e m as d e s c r i b e d on page &b .  i n d i c a t e d no q u e n c h i n g i n t h e p r e s e n c e  MgCl2, 0.04M T r i s b u f f e r pH 8.9.  of  72 TABLE V I .  R e c o v e r y o f C o u n t s i n DNA a f t e r TCA P r e c i p i t a i o n and P u r i f i c a t i o n  DNA  O r i g i n a l DNA solution  samples  0.1 m l  TCA  0.2 m l  0.5 m l  precipitated DNA  0.1 m l  0.2 m l  0.5 m l  Cpm-Bg on red s c a l e r  313  635  1599  320  632  1598  Cpm-Bg on Qreen s c a l e r  441  909  2227  460  917  2384  73 RESULTS AND  I.  Experiments with T r i t i u m L a b e l l e d  1. labelled  DI5CU5SIDN  DNA  D e s i g n o f t h e _Ln v i v o e x p e r i m e n t s and  p r e p a r a t i o n of the  tissue.  In t h e e x p e r i m e n t s d e s c r i b e d t h e r e i n t h e a n i m a l s two  tritium  main g r o u p s .  The  f i r s t group s e r v e d  were d i v i d e d i n t o  as a t y p e o f a c o n t r o l .  These a n i -  m a l s were i n j e c t e d w i t h t h e r a d i o a c t i v e p r e c u r s o r 24 h o u r s b e f o r e I t was DNA  hoped t h a t i f t h e r e were m e t a b o l i c a l l y d i f f e r e n t f r a c t i o n s amongst t h e  m o l e c u l e s o f t h e s m a l l i n t e s t i n a l e p i t h e l i u m , t h e p r e c u r s o r w o u l d be  e q u i l i b r a t e d w i t h them d u r i n g t h e 24 hour p e r i o d . val  sacrifice.  p r o v e d t o be v e r y c o n v e n i e n t  h a n d l e d a t t h e same t i m e o f day, By t h i s t e c h n i q u e ,  animals  namely;  i n the  t h e s i s i n v i v o c o u l d be c i r c u m v e n t e d  between 9.30  (1B8).  The  would a l s o support  the hypothesis,  h o u r s an a l m o s t c o m p l e t e e q u i l i b r a t i o n s h o u l d be The hours.  animals  (189) activi-  reception days f o r t h e  t h a t i n 24  obtained.  were s t a r v e d d u r i n g t h e p o s t - i n j e c t i o n p e r i o d o f  They were t h e n k i l l e d and  t h e DNA  was  ad-  maxima i n i n c o r p o r a t i o n , one  r e p o r t e d t u r n o v e r t i m e o f 1.5  e x t r a c t e d from the  s c r a p i n g s o f i n t e s t i n a l mucosa as d e s c r i b e d p r e v i o u s l y .  be  morning.  the s p e c i f i c  t h e o t h e r a f t e r 15 t o 24 h o u r s o f t h e  of the r a d i o a c t i v e m a t e r i a l . i n t e s t i n a l e p i t h e l i u m (188)  could  biosyn-  I t has a l s o been shown thymidine,  inter-  samples i s o l a t e d a t d i f f e r e n t t i m e s a f t e r t h e  m i n i s t r a t i o n Df t h e p r e c u r s o r , i n d i c a t e d two o c c u r i n g a f t e r 3 h o u r s and  - 11.30  d i u r n a l v a r i a t i o n s i n t h e DNA  i n j e c t i o n of t r i t i a t e d  t i e s o f t h e i n t e s t i n a l DNA  t h i s time  s i n c e a l l of the experimental  t h e e f f e c t s o f any  that a f t e r intravenous  Moreover,  well  pooled  24  74 In t h e s e c o n d group o f e x p e r i m e n t s  t h e a n i m a l s were e x p o s e d t o t h e  r a d i o a c t i v e p r e c u r s o r o n l y f o r s h o r t p e r i o d s o f t i m e , i . e . 5, ID o r 20 m i n utes p r i o r t o s a c r i f i c e .  I n t h e s e c a s e s t h e r a t s were s t a r v e d f o r 24 h o u r s  before receiving the r a d i o a c t i v e m a t e r i a l . o r 3 male W i s t a r r a t s  I n each e x p e r i m e n t ,  (180 - 200g e a c h ) were u s e d .  groups o f 2  Each a n i m a l w s a  injected  w i t h 0.5 m l o f a s o l u t i o n c o n t a i n i n g 0.0B3mc o f t r i t i a t e d - t h y m i d i n e and 8.2/^.mof t h y m i d i n e c a r r i e r i n p h y s i o l o g i c a l s a l i n e . dose i s b a s e d on t h e p r e v i o u s e x p e r i e n c e  The r e a s o n f o r t h i s  i n t h i s l a b o r a t o r y (177) w h i c h  showed s a t i s f a c t o r y i n c o r p o r a t i o n o f t h e p r e c u r s o r i n t o t h e d i f f e r e n t o f r a t w i t h l o w e r o r s i m i l a r amounts o f r a d i o a c t i v i t y .  organs  The r o u t e o f i n j e c -  t i o n was i n t r a p e r i t o n e a l , s u b c u t a n e o u s i n t h e d o r s a l r e g i o n , o r i n t r a v e n o u s through  the t a i l  vein.  I n two experiments, when t h e i n t r a v e n o u s  injection  t e c h n i q u e was u s e d , 0.83mc o f t r i t i a t e d - t h y m i d i n e was i n j e c t e d i n t o each animal. on  The r a d i o a c t i v e DNA was p r e p a r e d  f r o m t h e mucosa and f r a c t i o n a t e d  ECTEOLA-cellulose.  2.  F r a c t i o n a t i o n on  ECTEOLA-cellulose  P r e v i o u s r e p o r t s (101,122-124,160) c o n c e r n i n g t h e f r a c t i o n a t i o n o f DNA on E C T E O L A - c e l l u l o s e  exchangers,  showed t h a t v e r y complex  p r o f i l e s were o b t a i n e d f o r t h e d i f f e r e n t p r e p a r a t i o n s s t u d i e d . t i a l l y confirmed chromatographic  i n the present i n v e s t i g a t i o n .  chromatographic T h i s was  essen-  F i g u r e 16 r e p r e s e n t s t h e  p r o f i l e o f a DNA p r e p a r a t i o n o b t a i n e d f r o m r a t i n t e s t i n a l  mucosa by t h e method o f Medawar and Zubay ( 1 5 9 ) .  On i n s p e c t i o n o f t h e chroma-  t o g r a m , s e v e r a l p e a k s c a n be n o t i c e d , some o f w h i c h were e l u t e d by sodium c h l o r i d e s o l u t i o n s o f i n c r e a s i n g c o n c e n t r a t i o n s ( F r a c t i o n s I I and I I I . ) B e n d i c h e t a l . (101) have f o u n d t h a t f r a c t i o n s o b t a i n e d w i t h N a C l c o n c e n t r a t i o n s o f 0.2M  0.8  o to  CM  0.6-\  +J  Ul  rt  < U o  c rt  0.4  Jl  O XI  <  0.2  F r a c t i o n No Tube No. : Figure 16.  B  | i "j  " I  "»  Fractionation mucosa of r a t .  I  iv | v I vi  | vii  |viu( |v |  [  50 on E C T E O L A - c e l l u l o s e of  X  T  xi  I  100 tritiated  xii  | vm|  xiv  150 DNA from the i n t e s t i n a l  76 or lower, represent  o l i g o n u c l e o t i d e s not l a r g e r than o c t a n u c l e o t i d e s .  r e l a t i v e l y l a r g e peak I I I e l u t e d w i t h 0.6M N a C l a l s o r e p r e s e n t s low m o l e c u l a r  The  relatively  w e i g h t m a t e r i a l a c c o r d i n g t o B e n d i c h e_t a l . ( 1 0 1 ) .  Occasion-  a l l y , w i t h o t h e r DNA p r e p a r a t i o n s , a c o n s i d e r a b l e amount o f 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 was o b t a i n e d  w i t h t h e column e q u i l i b r a n t , 0.001M p h o s p h a t e  b u f f e r pH7, a f t e r t h e column had a d s o r b e d t h e a p p l i e d DNA s o l u t i o n , had been washed f r e e o f n o n - a d s o r b e d u l t r a - v i o l e t a b s o r b i n g and  then l e f t o v e r n i g h t  a t 4°C.  m a t e r i a l with the b u f f e r ,  Tftree e x p l a n a t i o n s c a n be o f f e r e d f o r t h i s  phenomenon: (a)  t h e column r e l e a s e d some o f t h e a d s o r b e d m a t e r i a l due t o t h e d e crease  (b)  i n temperature  the o r i g i n a l preparation contained  some l o w m o l e c u l a r  w e i g h t mat-  e r i a l , w h i c h c o p r e c i p i t a t e d w i t h t h e DNA d u r i n g t h e e t h a n o l  preci-  pitation tests (c)  depolymerization  o f t h e l a r g e r DNA p o l y n u c l e o t i d e s may have  occur-  r e d a f t e r t h e m a t e r i a l was a d s o r b e d on t h e r e s i n . The f i r s t  p o s s i b i l i t y , would c o n t r a d i c t Bendich's o r i g i n a l r e p o r t t h a t tempera-  t u r e c h a n g e s have l i t t l e e f f e c t on t h e c a p a c i t y o f t h e E C T E O L A - c e l l u l o s e and  t h a t m a t e r i a l a d s o r b e d a t 24°C i s n o t r e l e a s e d f r o m t h e r e s i n on c h i l l i n g o  t o 4 C.  The s e c o n d a l t e r n a t i v e was e l i m i n a t e d by an e x p e r i m e n t i n w h i c h  t h e DNA p r e p a r a t i o n p r e c i p i t a t e d t h r e e t i m e s w i t h e t h a n o l and e x h a u s t i v e l y d i a l y z e d was f r a c t i o n a t e d on t h e e x c h a n g e r .  Even a f t e r t h i s t r e a t m e n t  c o n s i d e r a b l e amount o f 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 was o b t a i n e d  E.  a  i n Fraction  The t h i r d e x p l a n a t i o n i s p r e f e r r e d , s i n c e e x p e r i m e n t s d e s c r i b e d and d i s -  cussed l a t e r i n d i c a t e a p o s s i b l e degradation l o s e exchanger  itself.  o f t h e DNA by t h e E C T E O L A - c e l l u -  77 The  e l u t i o n p r o f i l e i n F i g u r e 16 a l s o i n d i c a t e s t h a t most o f  u l t r a v i o l e t absorbing VII,  m a t e r i a l was  V I I I , X, X I , X I I and  XIV).  eluted with a l k a l i n e solvents,  According  (Fractions  t o K i t (122,123) t h e more m a t e r i a l  e l u t e d by a l k a l i n e s o l u t i o n s , t h e l e s s d e g r a d a t i v e t h e e x t r a c t i o n o f DNA.  the  i s the procedure used f o r  This statement i s v a l i d o n l y , i f the comparison i s  made on t h e same e x c h a n g e r , w i t h t h e same e x c h a n g i n g c a p a c i t y and S i n c e , i n t h e p r e s e n t i n v e s t i g a t i o n , t h e same e l u t i o n s c h e d u l e and c e l l u l o s e with properties s i m i l a r to that reported r e s u l t s are comparable w i t h h i s .  The  N  content. ECTEOLA-  by K i t were u s e d ,  r e l a t i v e l y low amount o f  the  material  e l u t e d by n e u t r a l sodium c h l o r i d e s o l u t i o n s , w o u l d i n d i c a t e t h a t t h e  DNA  preparation  mole-  was  i n h i g h l y p o l y m e r i z e d f o r m , and  had  a r e l a t i v e l y high  c u l a r weight. E a r l i e r i n v e s t i g a t i o n s (101,121-124) i n d i c a t e d t h a t t h e graphic and  p r o f i l e s o f t h e DNA  preparations  were r e p r o d u c i b l 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 were s t r i c t l y i d e n t i c a l .  i n t h e p r e s e n t work i t was  i f the  obtained  and  same s p e c i e s  ( r a t ) were n o t i d e n t i c a l a l t h o u g h t h e method and  f r o m t h e same s o u r c e o f t i s s u e ( i n t e s t i n a l mucosa)  were s t r i c t l y d e f i n e d  and  conditions  identical.  s u s p e c t e d t h a t a p o s s i b l e r e a s o n f o r t h e d i f f e r e n c e s may  been d i f f e r e n t d e g r e e s o f s o n i c a t i o n . DNA  this,  found t h a t the e l u t i o n p a t t e r n s of the d i f f e r e n t  preparations  I t was  methods  In c o n t r a s t t o  DNA  of preparation  chromato-  Doty et al.(190)  s o l u t i o n s t o s o n i c waves o f 9 k c y c l e s / s e c  reported,  t h a t by  f o r warying t i m e s , the  have exposing  molecules  7B were d e g r a d e d t h r o u g h d o u b l e - c h a i n  s c i s s i o n , producing  base p a i r e d h e l i c a l s t r u c t u r e i n t a c t . were p r o d u c e d t h a t c o v e r e d  fragments having the  I n t h i s way homolagous s a m p l e s o f DNA  a 25 f o l d r a n g e i n m o l e c u l a r  weight.  Experiments, i n  w h i c h t h e homogenate o f t h e i n t e s t i n a l mucosa o f r a t was s o n i c a t e d f o r a l o n g e r p e r i o d o f t i m e (1.25  min.) seemed t o c o n f i r m t h i s f i n d i n g .  p r o f i l e o f t h i s DNA p r e p a r a t i o n i s shown i n F i g u r e 1 7 . of the u l t r a v i o l e t absorbing  The c h r o m a t o g r a p h i c  I t c a n be seen t h a t most  m a t e r i a l was e l u t e d by n e u t r a l sodium c h l o r i d e  s o l u t i o n s , i n d i c a t i n g e x t e n s i v e damage,', o r p o s s i b l e breakdown o f t h e DNA molecules. To e x p l o r e t h i s p o s s i b i l i t y , a f u r t h e r e x p e r i m e n t was c a r r i e d o u t i n w h i c h t h e i n t e s t i n a l mucosa o f f o u r r a t s was p o o l e d made up i n p h y s i o l o g i c a l s a l i n e - V e r s e n e two  parts.  solution.  and a s t o c k homogenate was The homogenate was d i v i d e d i n t o  One p a r t was e x p o s e d t o s o n i c a t i o n f o r 0.7 m i n u t e s and t h e o t h e r f o r  1 m i n u t e and t h e DNA was e x t r a c t e d f r o m b o t h p r e p a r a t i o n s s i m u l t a n e o u s l y e x a c t l y t h e same c o n d i t i o n s .  under  The e l u t i o n p a t t e r n s o f t h e two p r e p a r a t i o n s i n  F i g u r e s 18A and B r e v e a l e d c o n s i d e r a b l e d i f f e r e n c e s between t h e two DNA s a m p l e s . On c a l c u l a t i n g t h e p e r c e n t a g e d i s t r i b u t i o n o f t h e u l t r a v i o l e t a b s o r b i n g in the f r a c t i o n s eluted with i n c r e a s i n g  1  NaCl c o n c e n t r a t i o n s , with i n c r e a s i n g  NH^ and NaOH c o n c e n t r a t i o n s , i t was f o u n d t h a t 41.9, u l t r a v i o l e t absorbing  m a t e r i a l was e l u t e d i n t h e s e  t h e DNA p r e p a r a t i o n o b t a i n e d 0.7 m i n .  material  44.4 and 1 3 . 7 % o f t h e t o t a l  fractions respectively, i n  f r o m t h e sample e x p o s e d t o s o n i c o s c i l l a t i o n f o r  I n t h e 1 m i n . sample t h e d i s t r i b u t i o n o f t h e t o t a l a b s o r b a n c e i n t h e  above m e n t i o n e d f r a c t i o n s was 28.6,  27.3 and 4 4 . 1 % .  I t i s d i f f i c u l t t o e x p l a i n why DNA f r o m mucosa e x p o s e d t o s o n i c l a t i o n s f o r 0.7 m i n .  s h o u l d y i e l d more l o w e r m o l e c u l a r  DNA f r o m t i s s u e e x p o s e d f o r 1 m i n u t e . o f t h e two DNA p r e p a r a t i o n s o b t a i n e d  oscil-  weight m a t e r i a l , than  The d i f f e r e n c e between e l u t i o n  patterns  f r o m t h e same t i s s u e and same a n i m a l s ,  may  79  o CO OJ  •p  rt o o rt J-(  o w  <  0.2-  I  IX  |  x  | xi  |  Xu  | XIII  Xiv | x v |  Fraction NoP 50 Tube Number F i g u r e 17.  100  F r a c t i o n a t i o n on ECTEOLA-cellulose o f t r i t i a t e d DNA i s o l a t e d from r a t i n t e s t i n a l mucosa exposed t o s o n i c o s c i l l a t i o n f o r 1.25 min.  80  (1.23) 0.8-1  < i  L  ©  0.6-  0.4-  0.2-  i  rT  o CO  CM •P  a!  ii  I 1  I III  I  v  IV  I VI  I  VII I V i l l i  IX  I  X  | XII I XIII I  I XI ]  XIV  I X V  <P  o c  (1.47)  \ ©  C5 .Q  U  o m  <  0.8n  0.6-  0.4-  I  0.2-  ^ — *  e  I  X  i  1 ii  I  iii  I vv I  vI  VI I v n  I Villi  IX I  X. I X.I I M l I X I U I  xiv  I  *v  F r a c t i o n No. 50  Figure  100 Tube Number 18. F r a c t i o n a t i o n on ECTEOLA-cellulose o f t r i t i a t e d DNA from r a t i n t e s t i n a l mucosa. (A) T i s s u e exposed t o s o n i c o s c i l l a t i o n f o r 0.7 min (B) T i s s u e exposed t o sonic o s c i l l a t i o n f o r 1.0 min.  81 be due  t o some o t h e r d e g r a d a t i v e  step during the i s o l a t i o n or  fractionation  procedure.  D i f f e r e n c e s m i g h t a l s o a r i s e as a r e s u l t o f a g g r e g r a t i o n o r  t i o n o f DNA  d u r i n g s t o r a g e even a t -15°C.  f r o m mucosa s o n i c a t e d f o r 1 m i n . was  I t s h o u l d be p o i n t e d o u t , t h a t t h e  s t o r e d f o r 2 weeks b e f o r e  When t h e s e e x p e r i m e n t s were c a r r i e d o u t t h e r e was marked c h a n g e s d u r i n g The t h e DNA  little  reason  fractionation. to suspect  and B.  The  was  amount o f u l t r a v i o l e t a b s o r b i n g  described similar findings.  material stored.  They r e p o r t e d  that  p r e p a r a t i o n s i s o l a t e d f r o m t h e same s o u r c e , by t h e same method d i d n o t  i d e n t i c a l e l u t i o n p a t t e r n s on ECTEOLA c o l u m n s . enhanced by a g e i n g  t h e DNA  s o l u t i o n f o r a few weeks i n t h e c o l d .  by t h e i r f r a c t i o n a t i o n p a t t e r n s on In p h y s i c o c h e m i c a l m o l e c u l a r w e i g h t and  give  M o r e o v e r , t h e s e v a r i a t i o n s were On t h e b a s i s o f  t h i s f i n d i n g , t h e y q u e s t i o s e d t h e f e a s i b i l i t y o f " f i n g e r p r i n t i n g " DNA  and  solution  o b t a i n e d 2 weeks a f t e r  i n t h e d i f f e r e n t peaks changed c o n s i d e r a b l y i n t h e m a t e r i a l w h i c h was Klouwen and W e i f f e n b a c h (160)  of  ECTEOLA-cellulose  I n t h i s e x p e r i m e n t t h e same s t o c k  u s e d , b u t t h e p r o f i l e shown i n F i g u r e 19B  t h a t i l l u s t r a t e d i n F i g u r e 19A.  DNA  any  r e s u l t s o f e x p e r i m e n t showing t h e p o s s i b l e e f f e c t s o f s t o r a g e  on r e p r o d u c i b i l i t y o f c h r o m a t o g r a p h i c f r a c t i o n a t i o n on  was  DNA  storage.  a r e i l l u s t r a t e d i n F i g u r e s 19A o f DNA  disaggrega-  preparations  ECTEOLA-cellulose.  s t u d i e s on DNA  Hermans (191)  found d i f f e r e n c e s i n  v i s c o s i t y between p r e p a r a t i o n s i s o l a t e d f r o m t h e same  under i d e n t i c a l c o n d i t i o n s , and i t was  f u r t h e r shown (160)  that these  source physico-  o chemical  p r o p e r t i e s change d u r i n g t h e s t o r a g e o f DNA  s o l u t i o n s a t 0-3  f r a c t i o n a t i o n on ECTEOLA i s g o v e r n e d by t h e s i z e and  shape o f t h e DNA  ( 1 0 1 , 121-124) i t i s p o s s i b l e t h a t d u r i n g t h e s t o r a g e t h e s e c h a n g e s m i g h t b r i n g a b o u t a d i f f e r e n c e i n t h e s i z e and r e s u l t i n g i n a change i n t h e e l u t i o n p a t t e r n .  The  .  Since  molecules  physicochemical  shape o f t h e  molecules,  above e x p l a n a t i o n w o u l d  not account f o r the d i f f e r e n c e s of the chromatographic p r o f i l e s of the  DNA  still  82  (2.06)  1.0,  r=* E l  I I w 1 iii I w I V ] vi  vu| v i u  p71  X~  F r a c t i o n No  | xi \ xu | xiii | 100  50  XIV  J  XV  150  Tube Number F i g u r e 19.  (A) F r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e o f t r i t i a t e d DNA o b t a i n e d from the i n t e s t i n a l mucosa of r a t s i n j e c t e d with the p r e c u r s o r 10 m i n . p r i o r to sacrifice. (B) T h i s sample i s an a l i q u o t o f the stock s o l u t i o n o f DNA i l l u s t r a t e d i n A, but f r a c t i o n a t i o n was performed 2 weeks l a t e r .  83 preparations s t u d i e d during the present i n v e s t i g a t i o n . percentage  Table VII i l l u s t r a t e s  d i s t r i b u t i o n of the t o t a l u l t r a v i o l e t absorbing m a t e r i a l i n the  main a r e a s o f t h e ECTEOLA-chromatograms o f t h e d i f f e r e n t DNA  the t i s s u e , i t i s f e l t t h a t the d i f f e r e n c e s observed e i t h e r r e p r e s e n t a d i f f e r e n c e i n t h e s t a t e o f DNA  changer m a t e r i a l i t s e l f .  was  i n the v a r i o u s  T h i s l a t t e r e x p l a n a t i o n was  Since  e x t r a c t e d from  from experiment  a n i m a l s o r t h e y i n d i c a t e damage o r d e g r a d a t i o n o f t h e DNA  three  preparations.  most o f t h e s e s a m p l e s were f r a c t i o n a t e d v e r y soon a f t e r t h e DNA  the  to  experiment  experimental  molecules  by t h e  f u r t h e r supported  by  exthe  r e s u l t s o b t a i n e d on Sephadex c h r o m a t o g r a p h y o f t h e i n d i v i d u a l f r a c t i o n s o f ECTEOLAc h r o m a t o g r a m s , and  3.  a l s o by t h e r e s u l t s o f r a d i o a c t i v i t y measurements.  C h r o m a t o g r a p h y on Sephadex c o l u m n s o f t h e f r a c t i o n s o f DNA  ECTEOLA-cellulose  obtained  by  chromatography.  During the course of l i q u i d s c i n t i l l a t i o n counting of the d i f f e r e n t f r a c t i o n s , i t was NH  •J  suspected t h a t the presence  of high c o n c e n t r a t i o n s of s a l t  would cause a severe quenching of the r a d i o a c t i v i t y .  I t was  Since  t e c h n i q u e o f g e l f i l t r a t i o n has been shown (166-168) t o be u s e f u l i n t h e  t h i s method was  tried first.  an e x p e r i m e n t .  F i g u r e 20A  m a t e r i a l s from high molecular weight F i g u r e s 20A  when a f r a c t i o n e l u t e d by 0.4M G-50  column.  NH^  i n 2M N a C l was  separa-  i s the diagram  such  eluted obtained  f r a c t i o n a t e d on t h e Sephadex  A c o n s i d e r a b l e o v e r l a p p i n g o f 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 and  c h l o r i d e p o s i t i v e substance repeated  and B i l l u s t r a t e t h e r e s u l t s o f  F i g u r e 20B  the  substances,  r e p r e s e n t s an i n d i v i d u a l f r a c t i o n w h i c h was  by 0.05M N a C l f r o m t h e ECTEOLA-column, and  and  f e l t , therefore,  t h a t t h e f r a c t i o n s s h o u l d be c o m p l e t e l y d e s a l t e d p r i o r t o c o u n t i n g .  t i o n o f low m o l e c u l a r w e i g h t  DNA  was  n o t i c e d i n both cases.  The  fractionation  was  s e v e r a l t i m e s w i t h o t h e r f r a c t i o n s o b t a i n e d on ECTEOLA c h r o m a t o g r a p h y ,  b u t t h e method has n o t p r o v e d  t o be p r a c t i c a l f o r c o m p l e t e r e m o v a l o f t h e c h l o r i d e  B4 TABLE V I I .  Comparison o f t h e Percentage o f t h e U l t r a v i o l e t Absorbing M a t e r i a l i n t h e Main F r a c t i o n s O b t a i n e d f r o m ECTEOLAC h r o m a t o g r a p h y o f t h e DNA P r e p a r a t i o n s . o f t h e t o t a l a b s o r b a n c e a t 260  DNA preparation  I A l l fractions eluted with i n c r e a s i n g NaCl concentration  II A l l fractions eluted with i n c r e a s i n g NH. concentration"  III A l l fractions eluted with i n c r e a s i n g NaOH concentration  H -thymidine i n j e c t e d subcutane o u s l y 24 h r . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1.25 min<  65.7  27.7  H -thymidine i n j e c t e d i n t r a p e r i t o n e a l l y 24 h r . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  28.6  27.3  44.1  H -thymidine i n j e c t e d i n t r a p e r i t o n e a l l y 24 h r . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 0.7 min.  41.9  44.4  13.7  H -thymidine i n j e c t e d subcutane o u s l y 20 m i n . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  29il  34.9  36.0  H -thymidine i n j e c t e d subcutane o u s l y 10 m i n . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  28.7  61.4  H -thymidine i n j e c t e d subcutaneously 5 min. b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  17.8  52.7  29.5  H -thymidine i n j e c t e d intraveno u s l y 5 min. before s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  20.0  69.0  11.0  H -thymidine i n j e c t e d i n t r a v e n o u s l y 5 min. before s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  48.3  42.6  6.6  9.9  9.1  85 U l t r a v i o l e t absorption Qualitative Cl" test  0.181  ©  0.12-  0.06•4 ** o CD OJ  •P  a ©.  o c  8  12  16  18  >  Tj a  ci Xi  £0 •rl  u o w  CJ  Xi  0.12  n  r+ + +  0.06 _  8  12  Tube Number Figure 20.  T  18  16  Chromatography on Sephadex of DNA f r a c t i o n s eluted from ECTEOLA-cellulose. (A) DNA f r a c t i o n from ECTEOLA eluted by 0.05M NaCl (B) DNA f r a c t i o n from ECTEOLA eluted by 0.4M NH i n 2M NaCl. 3  86  i o n s from t h e 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 .  Moreover, t h e r a t e o f f l o w o f t h e  e f f l u e n t was v e r y s l o w , due t o t h e h i g h v i s c o s i t y o f most o f t h e DNA p r e p a r a t i o n s . The  b a s i s o f Sephadex f r a c t i o n a t i o n t e c h n i q u e i s c l a i m e d t o be (168) t h a t m o l e -  c u l e s o f low m o l e c u l a r weight  s u c h as s a l t s c a n d i f f u s e r e l a t i v e l y f r e e l y  the network s t r u c t u r e o f t h e g e l g r a i n s . s t r i c t e d i n t h e i r d i f f u s i o n through the network s t r u c t u r e .  Moderately  l a r g e m o l e c u l e s w i l l be r e -  t h e g e l g r a i n s d e p e n d i n g on t h e p o r o s i t y o f  Large molecules a r e c o m p l e t e l y prevented from e n t e r i n g  t h e g e l g r a i n s and w i l l n o t be r e t a r d e d a t a l l by t h e column m a t r i x . molecular weight  through  substances  are eluted f i r s t  from t h e c o l u m n .  Thus, l a r g e  In t h e p r e s e n t i n -  v e s t i g a t i o n some 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 was e l u t e d b e f o r e c h l o r i d e i o n a p p e a r e d and p r e s u m a b l y t h i s s h o u l d be h i g h m o l e c u l a r w e i g h t m a t e r i a l . for  t h e incomplete removal  o f c h l o r i d e f r o m t h i s DNA f r a c t i o n c o u l d be t h e v i s c o s i t y  of  the preparations.  of  t h e sample t h e l e s s c o m p l e t e  I t has been c l a i m e d  (166-168) t h a t t h e h i g h e r t h e v i s c o s i t y  i s the s e p a r a t i o n of the low molecular weight  s t a n c e s from t h e high m o l e c u l a r weight m a t e r i a l i n t h e m i x t u r e . et  The r e a s o n  Recently  Skidmore  a l . (192) a l s o u s e d Sephadex c h r o m a t o g r a p h y f o r d e s a l t i n g DNA f r a c t i o n s  ECTEOLA-cellulose  sub-  after  c h r o m a t o g r a p h y , and c l a i m e d t h a t t h e method was e f f e c t i v e .  C l o s e r i n s p e c t i o n o f t h e i r d a t a i n d i c a t e s , however, c o n s i d e r a b l e o v e r l a p p i n g o f 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 and s a l t - b a s e c o n t e n t . The  d i a g r a m s o f F i g u r e s 20A and B i l l u s t r a t e a n o t h e r i n t e r e s t i n g o b -  s e r v a t i o n , n a m e l y , t h e e l u t i o n o f 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 i n two p e a k s , f r o m t h e 5ephadex c o l u m n .  The a p p e a r a n c e o f t h e second  m a t e r i a l i n t h e same  e f f l u e n t as the c h l o r i d e suggests t h a t i t i s e i t h e r high molecular weight m a t e r i a l r e t a r d e d i n i t s p a s s a g e t h r o u g h t h e c o l u m n , o r more l i k e l y , component.  a low molecular  S i n c e t h e s a m p l e s i n F i g u r e s 2QA and B r e p r e s e n t 5 m l f r a c t i o n s  from i n d i v i d u a l peaks e l u t e d from E C T E O L A - c e l l u l o s e ,  weight taken  i t i s rather surprising to  f i n d h e t e r o g e n e i t y i n s i z e w i t h i n such s m a l l f r a c t i o n s .  Bendich  et_ a l . (101)  87  r e c h r o m a t o g r a p h e d s e v e r a l t i m e s on ECTEOLA an o r i g i n a l peak and f o u n d ,  that  a f t e r each r e c h r o m a t o g r a p h y f u r t h e r f r a c t i o n a t i o n o f t h e o r i g i n a l peak  occurred.  They c o n c l u d e d f r o m t h i s , t h a t t h e o r i g i n a l c h r o m a t o g r a p h y o f t h e DNA caused incomplete.separation  o f t h e i n d i v i d u a l f r a c t i o n s and f u r t h e r chroma-  tography r e s u l t e d i n p u r i f i c a t i o n o f the o r i g i n a l f r a c t i o n .  Another  o f t h i s phenomenon i s a l s o p o s s i b l e , and t h a t i s t h e d e g r a d a t i o n DNA i t s e l f , by t h e e x c h a n g e r m a t e r i a l . by t h e a l k a l i n e e l u e n t  explanation  of the o r i g i n a l  The p o s s i b i l i t y o f d e g r a d a t i o n  effected  i s u n l i k e l y b e c a u s e DNA i s known t o be d e g r a d e d v e r y  by m i l d l y a l k a l i n e c o n d i t i o n s .  Moreover, very  little  e a r l y f r a c t i o n s e l u t e d by d i l u t e  n e u t r a l s a l t s o l u t i o n s a l s o showed t h i s h e t e r o g e n e i t y  4.  sample  i n s i z e (See F i g u r e 2 0 A ) .  Removal o f i n o r g a n i c c o n s t i t u e n t s f r o m t h e DNA f r a c t i o n s by d i a l y s i s .  Since  Sephadex c h r o m a t o g r a p h y d i d n o t g i v e c o m p l e t e d e s a l t i n g o f t h e  DNA p r e p a r a t i o n s ,  a more c l a s s i c a l method, namely d i a l y s i s was t r i e d .  Preliminary  investigations indicated that d i a l y s i s generally increased  the counting  the f r a c t i o n s .  However, l a t e r  This f i n d i n g i s represented  i n Figure  21.  rate of evalua-  t i o n o f t h e p e r c e n t a g e r e c o v e r i e s o f t h e r a d i o a c t i v i t y a f t e r ECTEOLA f r a c t i o n a t i o n , revealed  i n some c a s e s l o s s e s o f a s much a s 3 0 % o f t h e t o t a l r a d i o a c t i v i t y  to the column.  applied  I t was s u s p e c t e d t h a t t h i s was due t o some h i g h l y r a d i o a c t i v e l o w  m o l e c u l a r w e i g h t s u b s t a n c e i n t h e f r a c t i o n s w h i c h m i g h t have been l o s t d u r i n g t h e d i a l y s i s procedure. yzed against  Therefore,  i n an e x p e r i m e n t t h e u n f r a c t i o n a t e d  DNA was  d i l u t e s a l t s o l u t i o n s i n t h e c o l d , and p r e c i p i t a t e d s e v e r a l t i m e s ,  reasonably constant  s p e c i f i c a c t i v i t y o f t h e sample was o b t a i n e d .  i t y c o u l d be d e t e c t e d  i n t h e d i a l y z a t e o f t h e u n f r a c t i o n a t e d DNA.  d i s t i l l e d water.  The DNA  The d i a l y z a t e s were e v a p o r a t e d i n a " f l a s h "  o r a t o r a t 25-30°C and t h e r e s i d u e s w a t e r and c o u n t e d .  until  No r a d i o a c t i v -  was t h e n f r a c t i o n a t e d on an E C T E O L A - c e l l u l o s e column and t h e f r a c t i o n s were yzed against  dial-  sample dialevap-  were t a k e n up i n a known volume o f d i s t i l l e d  S i g n i f i c a n t r a d i o a c t i v i t y was f o u n d i n t h e d i a l y z a t e o f t h e  88  Radioactivity after dialysis R a d i o a c t i v i t y before d i a l y s i s  120n  100  80-  S a  20  40  60  80  100  Tube Number F i g u r e 21.  E l u t i o n of r a d i o a c t i v i t y of t r i t i a t e d DNA chromatographed on ECTEOLA-cellulose column. The r a d i o a c t i v i t y was determined i n the e l u a t e s shown i n F i g u r e 17 b e f o r e (shaded areas) and a f t e r ( s o l i d l i n e ) d i a l y s i s o f the i n d i v i d u a l f r a c t i o n s . :  89  first  f r a c t i o n o f t h e DNA.  260 myt*.  one  c h r o m a t o g r a p h e d i n an  i - p r o p y l a l c o h o l : NH^  u l t r a v i o l e t absorbing  i n 0.1N  HC1.  The  curve,  s p o t was  observed.  absorbance a t 40  c o n e : H^O  i n s p e c t i n g t h e chromatograms u n d e r u l t r a v i o l e t lamp two  d e f i n i t e absorption  and  a l s o a very high  A l i q u o t s o f t h i s s o l u t i o n were a p p l i e d t o Whatman No.  p a p e r and On  T h i s d i a l y z a t e had  a c i d washed (7:1:2) s y s t e m .  faint fluorescent  O n l y t h e u l t r a v i o l e t s p o t had  same s o l u t i o n a t pH  11.2  had  a maximum a b s o r p t i o n  p r o b a b l y i d e n t i c a l w i t h an unknown u l t r a v i o l e t a b s o r b i n g  w h i c h can  e x t r a c t e d from the d i a l y s i s t u b i n g w i t h d i s t i l l e d water. d i r e c t l i q u i d s c i n t i l l a t i o n counting  t h e r a d i o a c t i v i t y was paper.  The  /  not  of the  It  t h a t i t i s not  sample has  a r e a s on  n o t been e l u c i d a t e d , b u t  p r e s e n t o r i g i n a l l y i n t h e u n f r a c t i o n a t e d DNA,  exhaustively  d i a l y z e d p r i o r t o f r a c t i o n a t i o n . The  is felt,  s i n c e the  c u l a r w e i g h t , d i a l y z a b l e r a d i o a c t i v e m a t e r i a l c o u l d be o b t a i n e d  the  dialyzate  sample  f i n d i n g , t h a t low  ECTEOLA f r a c t i o n a t i o n , seemed t o i n d i c a t e some d e g r a d a t i o n  was  p a p e r chromatogram  a s s o c i a t e d w i t h the u l t r a v i o l e t absorbing  f r a c t i o n o f t h e DNA  absorb-  substance,  c h e m i c a l n a t u r e o f t h e h i g h l y r a d i o a c t i v e compound i n t h e  of the f i r s t  was  277..5m «  at  i n g s p o t was  f o u n d , t h a t an  a  w i t h a maximum a t 275r»v«and a minimum a t 262.5~»/«  a minimum a t 264.5wi/4.Later e x p e r i m e n t s showed t h a t t h i s u l t r a v i o l e t  be  and  f r o m DNA  o f t h e DNA  by  moleafter the  exchanger.  3 5.  Time-course of the  i n c o r p o r a t i o n o f H - t h y m i d i n e i n t o t h e DNA  of  subcutaneously i n j e c t e d r a t s . In some p r e l i m i n a r y e x p e r i m e n t s , g r o u p s o f two cutaneously  i n t h e b a c k s and  t r i t i a t e d thymidine precursor obtained 22.  f o r the  V e r y low  specific  r a t s were i n j e c t e d s u b -  t h e r a t s were s a c r i f i c e d a f t e r b e i n g f o r 5, 10 o r 20 m i n u t e s o r 24  a c t i v i t i e s o f t h e s e DNA  hours.  preparations  i n c o r p o r a t i o n of the r a d i o a c t i v e precursor  was  exposed t o The  time curve  i s shown i n  achieved  the  during  Figure the  90  Figure 22.  S p e c i f i c a c t i v i t i e s o f DNA o b t a i n e d from r a t s i n j e c t e d subcutaneously w i t h H -thymidine.  91 first  10 m i n u t e s .  40 o r 60 m i n u t e s ,  U n f o r t u n a t e l y , no d a t a a r e a v a i l a b l e f o r s p e c i f i c a c t i v i t i e s a t b u t by c o m p a r i n g t h e v a l u e s o b t a i n e d f o r 20 m i n u t e s and 24 h o u r s ,  one c a n assume t h a t , b y 60 m i n u t e s a p l a t e a u on t h e c u r v e w o u l d be r e a c h e d .  This  a s s u m p t i o n i s s u p p o r t e d by t h e d a t a o f P o t t e r and N y g a a r d (35) and S c h w a r t z e t a l . (193) who a l s o d e m o n s t r a t e d , t h a t w i t h i n 60 m i n u t e s t h e i n c o r p o r a t i o n o f r a d i o a c t i v e thymidine reached  i n t o t h e DNA o f s p l e e n , thymus, bone marrow and s m a l l i n t e s t i n e  a maximum p l a t e a u .  I t i s i n t e r e s t i n g t o note, that the i n i t i a l  portion  o f t h e s p e c i f i c a c t i v i t y - t i m e c u r v e i n F i g u r e 2 2 , i s v e r y s i m i l a r i n shape t o t h a t r e p o r t e d by P o t t e r and N y g a a r d f o r r a t s p l e e n DNA  6.  E f f e c t o f t h e r o u t e o f i n j e c t i o n on t h e s p e c i f i c a c t i v i t i e s o f t h e DNA  preparations.  S i n c e v e r y l o w amounts o f r a d i o a c t i v i t y were i n c o r p o r a t e d d u r i n g t h e short-time experiments,  u s i n g t h e s u b c u t a n e o u s i n j e c t i o n t e c h n i q u e , some o t h e r  modes o f i n j e c t i o n were t r i e d .  T a b l e V I I I shows t h e p e r c e n t a g e  incorporation of  t h e i n j e c t e d r a d i o a c t i v i t y p e r mg o f DNA, f o r t h e d i f f e r e n t r o u t e s o f a d m i n i s t r a tion.  H i g h e s t i n c o r p o r a t i o n was o b s e r v e d  i n t r a p e r i t o n e a l route of i n j e c t i o n . is  i n t h e 24 hour e x p e r i m e n t ,  Objection to the intraperitoneal  t h a t t h e t i s s u e b e i n g s t u d i e d , t h e i n t e s t i n e , becomes s u r r o u n d e d  cursor.  I t was f e l t ,  condition.  using the  by t h e p r e -  t h a t t h i s does n o t c o n s t i t u t e a p h y s i o l o g i c a l l y  For t h a t reason  injection  normal  t h e i n t r a v e n o u s r o u t e o f i n j e c t i o n was s t u d i e d .  The amount o f r a d i o a c t i v i t y was h i g h e r i n t h e DNA i s o l a t e d f r o m t h e 3 i n t e s t i n a l mucosa o f r a t s s a c r i f i c e d 5 m i n u t e s a f t e r r e c e i v i n g H - t h y m i d i n e by intravenous i n j e c t i o n through  the t a i l vein.  T h i s was e x p e c t e d ,  since the radio-  a c t i v e p r e c u r s o r i s t r a n s p o r t e d d i r e c t l y t o t h e c e l l s by t h e b l o o d s t r e a m , w h e r e as by t h e s u b c u t a n e o u s t e c h n i q u e  t h e p r e c u r s o r has t o p a s s t h r o u g h  compartments b e f o r e e n t e r i n g t h e b l o o d .  various  A d m i n i s t r a t i o n o f t h e p r e c u r s o r by  cell  92 TABLE V I I I  I n c o r p o r a t i o n o f H -Thymidine i n t o the D i f f e r e n t DNA Samples I s o l a t e d  Route o f i n j e c t i o n of precursor  Time o f exposure t o the precursor  Injected dose o f H thymidine i n mc  % Incorporation of H^-thymidine p e r mg o f DNA isolated  subcutaneous  24 h r .  0.083  0.03  intraperitoneal  24 h r .  0.083  0.07  intraperitoneal  24 h r .  0.083  0.08  subcutaneous  20 min.  0.083  0.006  subcutaneous  10 min.  0.083  0.001  subcutaneous  5 min.  0.083  0.0009  intravenous  5 min.  0.083  0.003  intravenous  5 min.  0.83  0.006  93 i n t r a v e n o u s i n j e c t i o n was  7.  employed f o r l a t e r  experiments.  S p e c i f i c a c t i v i t y - t i m e i n t e r r e l a t i o n s h i p s i n t h e DNA  by E C T E O L A - c e l l u l o s e  fractions obtained  chromatography.  On t h e b a s i s o f t h e c o n c l u s i o n s p o s t u l a t e d by B e n d i c h e_t a l _ . (101)  that  t h e f r a c t i o n s o b t a i n e d on E C T E O L A - c e l l u l o s e r e p r e s e n t e d d i f f e r e n t p o l y m e r i c s p e c i e s o f DNA,  i t was  a l s o expected t h a t d i f f e r e n c e s i n i n c o r p o r a t i o n of l a b e l l i n g  e r i a l m i g h t be o b s e r v e d i n t h e f r a c t i o n s .  I t m i g h t be e x p e c t e d , t h a t t h e  a c t i v i t i e s would be d i f f e r e n t i n t h e s u p p o s e d l y l o w e r m o l e c u l a r w e i g h t i f these f r a c t i o n s e x i s t indeed i n v i v o .  p r e c u r s o r r e l a t i o n s h i p s o f t h e d i f f e r e n t DNA  Any  specific  fractions,  Such d i f f e r e n c e s m i g h t i n d i c a t e  precursor r e l a t i o n s h i p s to the higher molecular weight f r a c t i o n s .  mat-  certain  special  f r a c t i o n s o u g h t t o be more d i s t i n c t l y  demonstrated  i n t h e " s h o r t t i m e " e x p e r i m e n t s , where t h e a n i m a l s a r e e x p o s e d  s p e c i f i c DNA  precursor, t r i t i a t e d thymidine, only f o r a short period of time.  I t m i g h t be assumed, t h a t as t h e t i m e o f e x p o s u r e t o t h e t r i t i a t e d i n c r e a s e d , the e x t e n t o f i n c o r p o r a t i o n would  to the  thymidine  s h i f t f r o m one f r a c t i o n t o t h e  o t h e r , thus r e v e a l i n g p o s s i b l e metabolic d i f f e r e n c e s or p r e c u r s o r i n t e r r e l a t i o n s h i p s amongst t h e s e d i f f e r e n t  fractions.  The f u n d a m e n t a l e x p e r i m e n t a l r e q u i r e m e n t f o r a p p r o a c h i n g t h i s  problem,  i s t o have f r a c t i o n a t i o n and i s o l a t i o n p r o c e d u r e s w h i c h a r e r e p r o d u c i b l e f r o m e x p e r i m e n t t o e x p e r i m e n t , and w h i c h w o u l d exists i n vivo.  r e p r e s e n t t h e s t a t e o f t h e DNA  A t p r e s e n t , even t h e m i l d e s t i s o l a t i o n p r o c u e d u r e s  DNAs, do n o t seem t o s a t i s f y t h i s c o n d i t i o n c o m p l e t e l y .  as i t  f o r mammalian  As f a r as t h e ECTEOLA-  c e l l u l o s e f r a c t i o n a t i o n p r o c e d u r e i s c o n c e r n e d , i t has been shown d u r i n g t h i s i n v e s t i g a t i o n t h a t t h e method i s n o t as r e p r o d u c i b l e as i t was (101,121-124).  c l a i m e d by o t h e r s  F u r t h e r , because the q u a n t i t y of u l t r a v i o l e t absorbing m a t e r i a l  e l u t e d i n t h e d i f f e r e n t f r a c t i o n s was  d i f f e r e n t from experiment t o  experiment,  94 i t was  f e l t t o be more p r a c t i c a l t o d i v i d e t h e ECTEOLA-chromatograms i n t o  main a r e a s , namely; f r a c t i o n  I r e p r e s e n t i n g a l l t h e peaks e l u t e d w i t h i n c r e a s i n g  c o n c e n t r a t i o n s o f n e u t r a l sodium c h l o r i d e s o l u t i o n , and the lower molecular absorbing fraction  weight p o l y n u c l e o t i d e s ;  fraction  m a t e r i a l e l u t e d w i t h i n c r e a s i n g NH^  o f t h e chromatograms o f each DNA  concentrations and  ultraviolet  i n 2M N a C l ,  and  1.0N  The  NaOH.  T a b l e IX summarizes t h e  f o r t h e t r i t i u m - l a b e l l e d DNA  results prepara-  I t can be s e e n , t h a t t h e d i s t r i b u t i o n o f t h e r a d i o a c t i v e m a t e r i a l  d i f f e r e n t from experiment t o experiment.  No  d e f i n i t e or c h a r a c t e r i s t i c  c a n be o b s e r v e d i n t h e " s h o r t e x p o s u r e t i m e e x p e r i m e n t s " obtained those  representing  c a l c u l a t e d f o r t h e s e t h r e e main a r e a s  preparation:..  o f t h e r a d i o a c t i v e measurements o b t a i n e d  probably  I I a l l the  I I I r e p r e s e n t i n g t h e m a t e r i a l o b t a i n e d w i t h 0.5  d i s t r i b u t i o n o f t h e t o t a l r a d i o a c t i v i t y was  tions.  three  f r o m t h e 24 h o u r i n j e c t e d a n i m a l s .  obtained  I f one  or i n the  was  pattern  preparations  compares thesB r e s u l t s  with  i n T a b l e V I I , a s i m i l a r i t y can be n o t i c e d between t h e p a t t e r n  of  fl t h e p e r c e n t a g e d i s t r i b u t i o n o f t h e r a d i o a c t i v i t y and e r i a l i n t h e t h r e e f r a c t i o n s o f most o f t h e DNA however, t h e most s t r i k i n g b e i n g  t h e DNA  r a d i o a c t i v i t y was  samples.  obtained  p r e c u r s o r 20 m i n u t e s p r i o r t o s a c r i f i c e .  u l t r a v i o l e t absorbing There are  exceptions,  from r a t s i n j e c t e d w i t h  In t h i s p r e p a r a t i o n , 54.5%  m a t e r i a l was  contained  the  of the  e l u t e d w i t h n e u t r a l s a l t s o l u t i o n s , whereas o n l y 2 9 . 1 %  t o t a l u l t r a v i o l e t absorbing  mat-  in this fraction.  total  of  the  This i n -  d i c a t e s a d e f i n i t e l y h i g h e r s p e c i f i c a c t i v i t y i n F r a c t i o n I compared t o F r a c t i o n s I I and  III.  S i m i l a r f i n d i n g s can be o b s e r v e d i n two  5 minute experiments.  t h e o t h e r 5 m i n u t e e x p e r i m e n t , however, a d i f f e r e n t p a t t e r n was  obtained.  t h e r a d i o a c t i v i t y seemed t o be u n i f o r m l y d i s t r i b u t e d i n a l l o f t h e On  c a l c u l a t i n g the percentage recovery  a t i o n i n t h i s p r e p a r a t i o n ^ i t was  c o u n t s were l o s t d u r i n g t h e f r a c t i o n a t i o n .  85% o f the  Since observations  v i o u s l y i n d i c a t e d t h a t l o s s e s of r a d i o a c t i v i t y occurred mostly  Here  fractions.  of the t o t a l r a d i o a c t i v i t y a f t e r  found, that approximately  In  fractiontotal  described from the  pre-  first  95 TABLE I X .  Comparison o f the Percentage o f t h e R a d i o a c t i v i t y i n t h e Main F r a c t i o n s O b t a i n e d f r o m ECTEOLA-Chromatography DNA P r e p a r a t i o n s % of the t o t a l  DNA preparation  I All fractions eluted with i n c r e a s i n g NaCl concentration  radioactivity  II A l l fractions eluted with i n c r e a s i n g NH^ concentration  III A l lfractions eluted with i n c r e a s i n g NaOH concentration  H -thymidine i n j e c t e d subcutane o u s l y 24 hr.' b e f o r e s a c r i f i c e T i s s u e was s o n i c a t e d f o r i . 2 5 m i n .  71.7  26.8  1.5  3 H -thymidine i n j e c t e d i n t r a p e r i t o n e a l l y 24 h r . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  37.1  24.B  3B.1  45.4  40.9  13.7  54.5  35.3  10.2  25.5  64.7  9.8  30.3  45.5  24.2  17.4  62.0  19.6  61.9  31.0  7.1  ,,3 H -thymidine i n j e c t e d i n t r a p e r i t o n e a l l y 24 h r . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 0.7 m i n . 3 H -thymidine i n j e c t e d subcutane o u s l y 20 m i n . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n . 3 H -thymidine i n j e c t e d subcutane o u s l y 10 m i n . b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n . 3 H -thymidine i n j e c t e d subcutane o u s l y 5 min. b e f o r e s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 min 3 H -thymidine i n j e c t e d i n t r a v e n o u s l y 5 min. before s a c r i f i c e . T i s s u e Bias s o n i c a t e d f o r 1 m i n . 3 H -thymidine i n j e c t e d i n t r a v e n o u s l y 5 min. before s a c r i f i c e . T i s s u e was s o n i c a t e d f o r 1 m i n .  96 f r a c t i o n s , t h i s l o s s c o u l d have o b s c u r e d  the c o u n t i n g r e s u l t s o b t a i n e d , showing  an a p p a r e n t l y l o w e r s p e c i f i c a c t i v i t y i n t h e f i r s t f r a c t i o n s . animals  DNA  samples from  i n j e c t e d 24 h o u r s b e f o r e s a c r i f i c e , showed a s h i f t o f h i g h e r  a c t i v i t i e s towards the l a t e r f r a c t i o n s . percentage  Even i n t h e s e s a m p l e s , however, t h e  d i s t r i b u t i o n o f r a d i o a c t i v i t y i n t h e f r a c t i o n s e l u t e d by  solventsseems  specific  t o be s l i g h t l y l o w e r t h a n o f t h o s e e l u t e d by  alkaline  NaCl.  T h e r e a r e no e x t e n s i v e i n v e s t i g a t i o n s o f E C T E O L A - c e l l u l o s e o f DNA  using radioisotope techniques.  The  experiments  of Bendich  fractionation  e t a l . (141)  14 with C  - 5 - b r o m o u r a c i l - l a b e l l e d _. c o l i DNA.  f r a c t i o n a t i o n r e s u l t s o b t a i n e d f o r t h e DNA time" experiments.  are probably comparable with  the  p r e p a r a t i o n s o f t h e 24 h o u r " e x p o s u r e  These i n v e s t i g a t o r s have grown JE. c o l i i n t h e p r e s e n c e  of  14 C  -5-bromouracil  and t h e DNA  was  e x t r a c t e d f r o m t h e o r g a n i s m s 24 h o u r s  C h r o m a t o g r a p h i c s e p a r a t i o n o f t h e d i f f e r e n t DNA  f r a c t i o n s r e v e a l e d some d i f f e r -  ences i n the s p e c i f i c a c t i v i t i e s o f the u l t r a v i o l e t absorbing e l u t e d w i t h 0.5N  later.  peaks.  The  peak  NaOH had v e r y low s p e c i f i c a c t i v i t y , and t h e y i n t e r p r e t e d t h i s  f i n d i n g t o mean t h a t t h i s peak c o u l d r e p r e s e n t t h e e s s e n t i a l g e n e t i c DNA, t h e o t h e r f r a c t i o n s m i g h t have some o t h e r " b i o l o g i c a l f u n c t i o n " .  The  whereas  results  d e s c r i b e d i n t h e p r e s e n t i n v e s t i g a t i o n seem t o c o n t r a d i c t t h i s f i n d i n g s i n c e even d u r i n g 5 m i n u t e s i n c o r p o r a t i o n t i m e s c o n s i d e r a b l e amount o f  radioactivity  was  The  i n c o r p o r a t e d i n t o t h e f r a c t i o n s e l u t e d by sodium h y d r o x i d e .  r e s u l t s o f F r i e d k i n and Wood (143) f i n d i n g , a l t h o u g h t h e two  are a l s o i n apparent  experimental  c o n t r a d i c t i o n to  Bendich's  systems are not s t r i c t l y comparable s i n c e F r i e d k i n  and  Wood u s e d mammalian t i s s u e s and i n _ v i t r o i n c o r p o r a t i o n c o n d i t i o n s f o r t h e r a d i o 14 a c t i v e p r e c u r s o r (C DNA  was  o f Kay  -thymidine).  A f t e r two  hours o f i n c u b a t i o n the r a d i o a c t i v e  e x t r a c t e d from the t i s s u e s i n t h r e e p o r t i o n s w i t h the d e t e r g e n t et a l . (144).  I t was  f o u n d , t h a t t h e DNA  t r a c t f r o m t h e t i s s u e by t h e d e t e r g e n t had o v e r , when t h e DNA  f r a c t i o n most d i f f i c u l t  the highest s p e c i f i c a c t i v i t y .  f r a c t i o n i s o l a t e d by t h e d e t e r g e n t  procedure  was  procedure to  exMore-  fractionated  97 on ECTEOLA-column, t h e f r a c t i o n s o b t a i n e d w i t h h i g h l y a l k a l i n e s o l v e n t s had highest s p e c i f i c a c t i v i t i e s .  F r i e d k i n and Wood (143)  most d i f f i c u l t t o e x t r a c t w i t h t h e d e t e r g e n t , and  assumed t h a t DNA  molecules  t o e l u t e from the columns,  r e p r e s e n t t h e s p e c i e s w h i c h i s more c l o s e l y a s s o c i a t e d w i t h p r o t e i n d u r i n g b i o s y n t h e s i s o f DNA.  The  F r i e d k i n and Wood (143)  d e f i n i t e s p e c i f i c a c t i v i t y pattern reported  p r e p a r a t i o n s s t u d i e d during the present The  r e p o r t o f Qsawa and  not obaserved  the  by  i n d i c a t i n g i n c r e a s i n g s p e c i f i c a c t i v i t i e s i n gNA  c t i o n s e l u t e d by more a l k a l i n e s o l u t i o n s was  the  fra-  i n any o f t h e  DNA  investigation.  S a k a k i (194)  seems t o c o n t r a d i c t a l l o f  p u b l i c a t i o n s concerning a d i f f e r e n t i a l uptake of the p r e c u r s o r i n t o the  the  different 32  DNA  fractions.  T h e i r experiment  i n d i c a t e s a homogeneous d i s t r i b u t i o n o f P  l a b e l l i n g i n t o t h e f r a c t i o n s o f r a b b i t a p p e n d i x DNA.  The  r e s u l t s of the  present  i n v e s t i g a t i o n do n o t seem t o be i n agreement w i t h t h i s f i n d i n g , s i n c e s h o r t exposure time experiments t i o n experiments  and a l s o , t o a l e s s e r e x t e n t , t h e 24 h o u r i n c o r p o r a -  i n d i c a t e d a r a t h e r non  uniform d i s t r i b u t i o n of the r a d i o -  a c t i v i t y i n the d i f f e r e n t u l t r a v i o l e t absorbing The  r e s u l t s obtained with the ECTEOLA-cellulose  a r e v e r y d i f f i c u l t t o e v a l u a t e , due p r o f i l e s , and dialysis.  fractionation  to the n o n - r e p r o d u c i b i l i t y of  to the l o s s of r a d i o a c t i v e m a t e r i a l from the f r a c t i o n s  M o r e o v e r , i n most o f t h e s h o r t e x p o s u r e t i m e e x p e r i m e n t s ,  l e v e l o f r a d i o a c t i v i t y was  f r o m t h e 20 m i n .  and t h e two  f a c t s , one c o u l d e n v i s a g e  procedure  chromatographic after the average  v e r y low i n t h e s a m p l e s , making t h e e v a l u a t i o n o f  t h e c o u n t s v e r y d i f f i c u l t and i n a c c u r a t e .  weight  peaks.  5 min.  I f the experimental r e s u l t s  i n j e c t e d animals are not e x p e r i m e n t a l  some m e t a b o l i c d i f f e r e n c e s between t h e l a w  and h i g h e r m o l e c u l a r w e i g h t  obtained  fractions.  molecular  These m e t a b o l i c d i f f e r e n c e s i f  t h e y e x i s t , c o u l d n o t be v e r y s t r i k i n g , s i n c e a f t e r e x p o s u r e t o t h e f o r v e r y s h o r t p e r i o d s , r a d i o a c t i v i t y was  arte-  detected i n - a l l of the  precursor  fractions.  98 II.  Experiments  1. labelled  Design  w i t h DNA  L a b e l l e d w i t h B o t h Carbon-14 and T r i t i u m  of the i n . v i v o experiments  and  preparation of the  double  tissue. B e c a u s e o f t h e i n c o n c l u s i v e r e s u l t s o b t a i n e d by f r a c t i o n a t i o n  ECTEOLA-cellulose,  a t t e n t i o n was  c e n t e r e d on t h e use o f MAK  o t h e r i n v e s t i g a t o r s c l a i m e d t h a t f r a c t i o n a t i o n on MAK w o u l d be s u i t a b l e f o r t h e p r e s e n t p u r p o s e s . for  on  columns, because  o f f e r e d advantages which  To f u r t h e r i n c r e a s e t h e  sensitivity  d e t e c t i n g p o s s i b l e m e t a b o l i c d i f f e r e n c e s amongst t h e d i f f e r e n t DNA  fractions, 14  use was  made o f t h e d o u b l e  l a b e l l i n g technique.  The  simultaneous  use o f C  3 and H - l a b e l l e d p r e c u r s o r s u n d e r i d e n t i c a l e x p e r i m e n t a l c o n d i t i o n s i s v e r y f u l i n experiments  i n w h i c h t h e p o s s i b l e t u r n o v e r o f a p r o d u c t and  t i o n of the p r e c u r s o r i n t o the product mental animals.  use-  the i n c o r p o r a -  has t o be measured i n t h e same e x p e r i -  Since previous observations i n the present i n v e s t i g a t i o n i n -  d i c a t e d t h a t d u r i n g t h e 24 h o u r e x p o s u r e t i m e , t h e r a d i o a c t i v e p r e c u r s o r w e l l i n c o r p o r a t e d i n t o e v e r y f r a c t i o n o f t h e DNA,  i t was  o f t i m e w o u l d be s u i t a b l e t o l a b e l t h e b u l k o f t h e DNA  was  f e l t that t h i s length  i n t h e i n t e s t i n a l mucosa  of r a t . Each o f a g r o u p o f 5 male W i s t a r r a t s was  through  t h e t a i l v e i n w i t h 0.5  injected intravenously 3  ml o f a s o l u t i o n c o n t a i n i n g 0.1  of H  -thymidine  and  8,2yt<.mof t h y m i d i n e c a r r i e r i n p h y s i o l o g i c a l s a l i n e .  for  24 h o u r s f o l l o w i n g t h e i n j e c t i o n o f t h e r a d i o a c t i v e m a t e r i a l , and each 14  r e c e i v e d i n t r a v e n o u s l y 0.5 and  The  mc  a n i m a l s were s t a r v e d  ml o f a s o l u t i o n c o n t a i n i n g 0.033 mc  8.2/<.*nof t h y m i d i n e , c a r r i e r i n p h y s i o l o g i c a l s a l i n e .  The  C  then  -thymidine  a n i m a l s were  killed  20 o r 40 m i n u t e s a f t e r t h e s e c o n d i n j e c t i o n . I t has been shown (181) t h a t , 3 14 3 f o r a c c u r a t e a s s a y s o f H and C i n t h e same s a m p l e , t h e i s o t o p e r a t i o n s , H : C^ , s h o u l d be between 1 t o 15. I t was hoped, t h a t by c h o o s i n g t h e s e t i m e 3 14 i n t e r v a l s and i s o t o p e r a t i o s f o r i n j e c t i o n , t h e d e s i r e d r a t i o o f H :C 4  99 i n v i v o w o u l d be indicated  obtained.  that the  2.  The  desired  mammalian o r i g i n .  and  centration 0.705M. was  two  Philipson  La c e l l s on  When t h e DNA  was  c o l u m n s were r e p o r t e d by  p r o f i l e s o f DNAs o b t a i n e d by  DNA  species,  (130)  the  be  demonstrated t h a t  eluted  be  such d i f f e r e n c e s d e t e c t e d by  MAK  (137)  columns.  He  f o r the  preliminary the  Res.)  fractions.  The  e l u t i o n of the  total soluble  average con-  peak o f DNA  was  at the  same sodium c h l o r i d e  rather  concentration preparations  surprising considering  the  other f r a c t i o n a t i o n techniques.  fractionation.  on  s i n g l e DNA  The  possibility,  peak r e p r e s e n t  different  S i n c e Sueoka and  hydrogen bond c o n t e n t , i t was  and  multiple-peak  column d i f f e r e n t i a t e s between n u c l e i c  amongst t h e DNA  as  (129,134-135) u s i n g b a c t e r i a l  e x c l u d e d by t h e s e i n v e s t i g a t i o n s .  the  peak  acid  Cheng  preparations  hoped t h a t , i f t h e r e  m o l e c u l e s o f r a t i n t e s t i n a l mucosa, t h e s e w o u l d M o r e o v e r , i t has  been shown (129) eluted  from the  that  low  column by  lower  concentrations. To  by  chroma-  suceeded i n s e p a r a t i n g  m o l e c u l a r weight or shear-degraded p r e p a r a t i o n s are salt  MAK  r e p o r t e d the chromatography of  i n d i c a t i n g a s i n g l e peak f o r DNA  o f d i f f e r e n t b a s e c o m p o s i t i o n and are  of the  u s e d t h i s method f o r f r a c t i o n a t i n g DNAs o f  i n d i v i d u a l f r a c t i o n s i n the  c o u l d not  method.  d e v e l o p m e n t (129)  other investigators  T h i s f i n d i n g was  however, t h a t  preparations  rechromatographed i n a narrower g r a d i e n t , a s i n g l e  Similar findings,  phage DNAs.  fractionation  and  calculated  o b t a i n e d a g a i n , w h i c h was  MAK  MAK  d i f f e r e n t h i g h m o l e c u l a r w e i g h t RNA  o f sodium c h l o r i d e  originally.  MAK  d o u b l e l a b e l l e d DNA  obtained.  (102)  investigators  In 1961  a c i d s f r o m He  DNA  r a t i o was  introduction  t o g r a p h y r e l a t i v e l y few  RNA,  measurements o f t h e  r e p r o d u c i b i l i t y of the  5ince the  nucleic  The  t e s t the  r e p r o d u c i b i l i t y of the  e x p e r i m e n t s were c a r r i e d out  method o f Medawar and were f r a c t i o n a t e d .  Zubay (159)  MAK  f r a c t i o n a t i o n t e c h n i q u e , some  i n which d u p l i c a t e  s a m p l e s o f DNA  and  (Cal.Corp. Biochem.  A sample o f s o l u b l e  c o m m e r c i a l DNA RNA  was  also  chromatographed.  prepared  100 T a b l e s X A and  B summarize t h e d i s t r i b u t i o n o f t h e u l t r a v i o l e t a b s o r b i n g  material  amongst t h e main f r a c t i o n s e l u t e d . I t can awar and was  be n o t e d , t h a t i n t h e DNA  Zubay (159)  sample o b t a i n e d  by t h e method o f  a p p r o x i m a t e l y 50% o f t h e t o t a l u l t r a v i o l e t a b s o r b i n g  e l u t e d w i t h a sodium c h l o r i d e s o l u t i o n c o r r e s p o n d i n g t o an i n i t i a l  t i o n o f 0.66M i n t h e e l u a t e . m a t e r i a l was  In t h e c a s e o f t h e c o m m e r c i a l DNA  e l u t e d by l o w e r s a l t c o n c e n t r a t i o n s  perhaps a higher  amount o f d e g r a d a t i o n  or i m p u r i t i e s .  t h o u g h t h e s e c o n d sample o f each t y p e o f DNA the . f i r s t ,  was  t o 0.21M  material  concentra-  s a m p l e , more NaCl) i n d i c a t i n g  I t i s noteworthy t h a t a l -  c h r o m a t o g r a p h e d 2 weeks a f t e r  t h e r e were no marked c h a n g e s o b s e r v e d i n t h e e l u t i o n p a t t e r n .  w o u l d t e n d t o s u g g e s t , t h a t no period of  (0.09  s i g n i f i c a n t c h a n g e s had  occured during  This  t h e 2 week  storage. When s-RNA was  absorbing  m a t e r i a l was  a p p l i e d on t h e c o l u m n , 92.7%  of the t o t a l  e l u t e d a t an i n i t i a l N a C l c o n c e n t r a t i o n  ultraviolet  o f 0.35M.  The  r e s u l t s i n d i c a t e a q u i t e reasonable r e p r o d u c i b i l i t y of the e l u t i o n p a t t e r n t h e MAK  Med-  f r a c t i o n a t i o n technique.  (130,137) t h a t DNA An  can  The  previous  observation  be s e p a r a t e d f r o m s-RNA has  i n t e r e s t i n g p o i n t may  of s e v e r a l workers  been c o n f i r m e d .  be m e n t i o n e d i n c a s e o f t h e DNA  e x t r a c t e d by t h e method o f Medawar and  with  Zubay ( 1 5 9 ) .  preparation  This preparation  had  been  3 obtained  from r a t s allowed  sacrifice.  to incorporate  A l i q u o t s o f t h i s sample have been p r e v i o u s l y f r a c t i o n a t e d i n d u p l i c a t e  e x p e r i m e n t s on E C T E O L A - c e l l u l o s e , and obtained tions.  H - t h y m i d i n e f o r 10 m i n u t e s p r i o r t o  a v e r y complex e l u t i o n p r o f i l e has  c o n s i s t i n g o f s e v e r a l p e a k s e l u t e d by d i f f e r e n t N a C l and These f i n d i n g s were r e p r e s e n t e d  on MAK  f r a c t i o n a t i o n o n l y one  23 and  24.  The  continuously.  i n Figures  main peak was  obtained,  r e s t of the u l t r a v i o l e t absorbing The  19 A and  B.  On  NH^  concentra-  the other  as i t i s shown i n  m a t e r i a l was  been  hand,  Figures  e l u t e d more o r  less  d i f f e r e n c e s a r e r a t h e r s t r i k i n g between t h e e l u t i o n p r o f i l e s  101 TABLE X .  A.  C o m p a r i s o n o f F r a c t i o n a t i o n on MAK Column o f A l i q u o t s o f DNA  DNA sample  Percent of t o t a l  P r e p a r e d by t h e method o f Medawar and Zubay (159(  B.  absorbance In f r a c t i o n s e l u t e d by 0.66-0.73M N a C l  1st  sample  16.1  49.1  2nd  sample*  17.6  48.6  In f r a c t i o n s e l u t e d by 0.09-0.21M N a C l  C o m m e r c i a l DNA  .*  In f r a c t i o n s e l u t e d by 0.09-0.13M N a C l  Preparation  In f r a c t i o n s e l u t e d by 0.42-0.86M N a C l  1st  sample  42.1  32.4  2nd  sample*  36.9  29.5  The 2nd s a m p l e s were c h r o m a t o g r a p h e d 2 weeks l a t e r  F r a c t i o n a t i o n R e s u l t s o f s-RNA o f Y e a s t  Percent of t o t a l In f r a c t i o n s e l u t e d by 0.1 t o 0.27M N a C l  4.9  absorbance In f r a c t i o n s e l u t e d b y 0.35-0.57M N a C l  92.7  102  A NaCl  gradient  0.3  1.2  0.2  o CO OJ  +>  a  o o c a  Xi  +> c rt  Xi  w  u o w  <  0.8  G  •H  0.1 rt «H  0 >> +>  rt A  A  -I  20  40  I  60  ,  80  j  100  120  140  Tube Number F i g u r e 23.  F r a c t i o n a t i o n o f DNA by chromatography on MAK. The DNA was prepared by the method of Medawar and Zubay (159).  0.4  103  A  NaCl  gradient  fl  0.3  1.2  0.2  o  co  OJ  -p rt <D O C  0.8  rf J3  c rt  U  o  iH  w fl  CO .Q  0.1  o  rt «w 0 >> +>  •H A  & A  rt  A  11 20 F i g u r e 24.  40  60 80 100 120 140 Tube Number F r a c t i o n a t i o n of DNA by chromatography on MAK. The DNA used was a sample of the same p r e p a r a t i o n as F i g u r e 23, but which had been s t o r e d 2 weeks i n the c o l d .  0.4  104 obtained  u s i n g E C T E O L A - c e l l u l o s e and MAK, w i t h t h e same DNA p r e p a r a t i o n .  explanations are possible: graphy a r e e x p e r i m e n t a l  e i t h e r t h e m u l t i p l e peak p r o f i l e s o f ECTEOLA  a r t e f a c t s p r o d u c e d by t h e e x c h a n g e r , o r MAK  previously that observations  chromato-  chromatography  r e s u l t s i n an i n c o m p l e t e r e s o l u t i o n o f t h e h e t e r o g e n e o u s DNA p o p u l a t i o n . f e l t t h a t b o t h e x p l a n a t i o n s m i g h t be t r u e t o some e x t e n t .  Two  I t h a s been  It i s  discussed  regarding the n o n - r e p r o d u c i b i l i t y o f chromatographic  p a t t e r n s , l o s s o f r a d i o a c t i v i t y d u r i n g d i a l y s i s f r o m t h e DNA f r a c t i o n s a f t e r ECTEOLA c h r o m a t o g r a p h y , i n d i c a t e some changes  and p e r h a p s t h e Sephadex c h r o m a t o g r a p h i c e x p e r i m e n t s m i g h t  i n t h e DNA p r e p a r a t i o n s b r o u g h t a b o u t by t h e ECTEOLA-exchanger  itself. An i n d i c a t i o n t h a t t h e main peak o b t a i n e d by MAK c h r o m a t o g r a p h y  under  c o n d i t i o n s d e s c r i b e d c o u l d be r e s o l v e d f u r t h e r , was s u g g e s t e d i n an e x p e r i m e n t i n w h i c h DNA was c h r o m a t o g r a p h e d was d o u b l e d .  on a c o l u m n , i n w h i c h t h e s i z e o f t h e s e c o n d l a y e r  T h i s i s t h e l a y e r which c o n t a i n s t h e p r o t e i n a t e d C e l i t e  e l u t i o n p a t t e r n i s shown i n F i g u r e 25 w h i c h was o b t a i n e d  f r o m a DNA  (129).  The  preparation  e x t r a c t e d by t h e method o f C o l t e r _e_t a l . (147) f r o m a n i m a l s a l l o w e d t o i n c o r p o r a t e 14 the C  - l a b e l l e d p r e c u r s o r 40 m i n u t e s p r i o r s a c r i f i c e .  I t c a n be seen t h a t , a  s p l i t has o c c u r r e d i n t h e main peak, i n d i c a t i n g p e r h a p s an i n h o m o g e n e i t y o f t h e DNA s p e c i e s w i t h i n t h e main peak. periments.  T h i s f i n d i n g was c o n f i r m e d i n s u b s e q u e n t e x -  I t has t o be m e n t i o n e d , t h a t i n F i g u r e 25 o n l y one s m a l l peak p r e c e d e d  i m m e d i a t e l y t h e main DNA peak i n s t e a d o f t h e two u s u a l l y o b s e r v e d i n l a t e r e x p e r i m e n t s u s i n g DNA p r e p a r e d by t h e method o f C o l t e r ( 1 4 7 ) .  In t h i s  particular  e x p e r i m e n t , t h e i n c o m p l e t e s e p a r a t i o n o f t h e peaks m i g h t have been due t o some s l i g h t v a r i a t i o n i n the rate of flow of the eluant, or l o c a l v a r i a t i o n i n the sodium c h l o r i d e c o n c e n t r a t i o n  gradient.  Of f u r t h e r i n t e r e s t i s t h e e x p e r i m e n t i n w h i c h t h e sample o f t h e DNA m e n t i o n e d a b o v e , was c h r o m a t o g r a p h e d  on a MAK c o l u m n , i n w h i c h o n l y t h e C e l i t e  component o f t h e s e c o n d l a y e r o f t h e column  was d o u b l e d .  The e l u t i o n p a t t e r n i s  105  Absorbance at 260 m/t, Absorbance at 595 m//t (Dische NaCl  test)  gradient  1.2-  -1.2  0.8-  -0.8 0)  v a a  Xi  u  o w  Xi  < 0.4.  -0.4  120 100 60 80 Tube Number Chromatography of DNA on a m o d i f i e d MAK column. The amount of m a t e r i a l s i n the second l a y e r was d o u b l e d . The DNA was prepared by the method of C o l t e r et a l . ( l 4 7 ) from animals allowed to i n c o r p o r a t e TTRe C - l a b e l l e d p r e c u r s o r 40 min. p r i o r to sacrifice. 40  Figure  25.  1  106 shown i n F i g u r e 26.  Under t h e s e c o n d i t i o n s t h e r e was no s p l i t  i n t h e main peak.  However, a much c l e a r e r s e p a r a t i o n o f t h e u l t r a v i o l e t a b s o r b i n g peaks o c c u r r e d similar ing  t o t h e f i n d i n g s shown i n F i g u r e 25.  In b o t h c a s e s , t h e b a c k g r o u n d  absorb-  u l t r a v i o l e t m a t e r i a l almost completely disappeared. Further supporting the concept of the heterogeneity of the  fractions  o f t h e main DNA  individual  peak a r e t h e e x p e r i m e n t s o f Cheng and Sueoka (103)  w h i c h w i l l be d i s c u s s e d l a t e r . To summarize t h e e x p e r i m e n t a l f i n d i n g s c o n c e r n i n g t h e of  MAK c h r o m a t o g r a p h i c t e c h n i q u e , T a b l e X I compares t h e d i s t r i b u t i o n  u l t r a v i o l e t absorbing material i n the d i f f e r e n t 14 of  reproducibility  the v a r i o u s C  ibility  a r e a s o f t h e MAK  chromatograms  3 and H  labelled  DNA  preparations.  A r e a s o n a b l y good r e p r o d u c -  was f o u n d f o r t h e c h r o m a t o g r a p h i c p r o f i l e s o f t h e DNA  ed f r o m e x p e r i m e n t t o e x p e r i m e n t .  This i s especially  v i o l e t a b s o r b i n g a r e a s o b t a i n e d by an i n i t i a l  preparations obtain-  t r u e f o r t h e main u l t r a -  N a C l c o n c e n t r a t i o n o f 0.66M, and  f o r t h e s m a l l peaks i m m e d i a t e l y p r e c e d i n g t h e main DNA initial  of the  peak, and e l u t e d by an  N a C l c o n c e n t r a t i o n o f 0.45M ( F r a c t i o n I I , T a b l e X I ) .  I t was a l s o  found,  t h a t t h e r e c o v e r i e s o f t h e 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 f r o m t h e column was v e r y good. 3.  Comparison  o f t h e MAK  c h r o m a t o g r a p h i c p r o f i l e s o f t h e DNA p r e p a r a t i o n s  o b t a i n e d by t h e methods o f Medawar and Zubay (159) and C o l t e r e t a l . (147) f r o m the  same e x p e r i m e n t a l a n i m a l s . P r e v i o u s e x p e r i m e n t s showed, t h a t c h r o m a t o g r a p h y  cellulose  gave e l u t i o n  p r o f i l e s which d i f f e r e d  In t h e s e e x p e r i m e n t s t h e DNA  w i t h t h e DNA  on ECTEOLA-  from experiment t o e x p e r i m e n t .  was p r e p a r e d by t h e method o f Medawar and Z u b a y .  I t was f e l t t o be o f i n t e r e s t , c o l u m n s o f DNA  o f DNA  t h e r e f o r e , t o study the e l u t i o n  p a t t e r n on  p r e p a r e d by t h i s method and t o compare t h i s w i t h r e s u l t s  MAK  obtained  e x t r a c t e d by t h e p r o c e d u r e o f C o l t e r e t a l . ( 1 4 7 ) f r o m t h e same  107  1.6  Absorbance at 260 m/c Absorbance at 595 m.//. (Dische t e s t ) NaCl g r a d i e n t  1.2-  0.8  -  o o a a  1.2  - 0.8  u  o  -  0.4-  Figure  26.  0.4  60 80 100 Tube Number Chromatography of DNA on a modified MAK column. The C e l i t e component of the second l a y e r was d o u b l e d . The DNA sample was taken from the same stock s o l u t i o n as i n F i g u r e 25.  108 TABLE X I . Comparison o f t h e Percentage o f t h e U l t r a v i o l e t Absorbing M a t e r i a l i n t h e F r a c t i o n s O b t a i n e d f r o m MAK-Chromatography of C and H L a b e l l e d DNA P r e p a r a t i o n s 1 4  3  DNA* preparation  14 C - t h y m i d i n e i n j e c t e d 40 m i n . p r i o r t o s a c r i f i c e . DNA p r e p a r e d by t h e method o f C o l t e r e t a l . (147) 14 C - t h y m i d i n e i n j e c t e d 40 m i n . p r i o r t o s a c r i f i c e . DNA p r e p a r e d by t h e method o f C o l t e r e_t a l . (147) and p u r i ' f i e d by e t h a n o l p r e c i p i t a t i o n 14 C - t h y m i d i n e i n j e c t e d 40 m i n . p r i o r t o s a c r i f i c e . DNA p r e p a r e d by t h e method o f Medawar and Zubay (159) 14 C - t h y m i d i n e i n j e c t e d 40 m i n . p r i o r t o s a c r i f i c e . DNA p r e p a r e d by t h e method o f C o l t e r e t a l . (147) 14 C - t h y m i d i n e ' i n j e c t e d 20 m i n . p r i o r t o s a c r i f i c e . DNA p r e p a r e d by t h e method o f C o l t e r e t a l . (147) 14 C - t h y m i d i n e i n j e c t e d 20 m i n . p r i o r t o s a c r i f i c e . DNA p r e p a r e d by t h e method o f C o l t e r e t a l . (147)  % o f t o t a l absorbance fractions  i n the  I II III N a C l M= N a C l M= N a C l M= 0.09-0-.44 0.45-0.65 0.66-0.73  IV N a C l M= 0.74.0.90  Total absorbance a t 260 recovered  20.4  15.6  53.0  11.0  101.7  19.9  13.1  55.5  11.5  96.1  70.1  1.5  111.2  28.4  27.7  10.1  60.2  2.0  108.0  27.4  9.9  59.9  2.8  104.4  vl8.3  13.3  60.7  7.4  92.4  * A l l a n i m a l s r e c e i v e d H - l a b e l l e d t h y m i d i n e 24 h r . b e f o r e t h e C p r e c u r s o r was a d m i n i s t e r e d .  -labelled  109 pooled mucosal t i s s u e . t e r n s o f DNA represents  Figures  27,28 i l l u s t r a t e t h e c h r o m a t o g r a p h i c e l u t i o n  s a m p l e s i s o l a t e d by t h e a DNA  preparation  p h e n o l e x t r a c t i o n method ( 1 4 7 ) .  w h i c h was  f u r t h e r p u r i f i e d by two  pat-  Figure  ethanol  28  precipi-  t a t i o n s t e p s , whereas F i g u r e 27 i s t h e chromatogram o f t h e d i a l y z e d e x t r a c t t h e t i s s u e f r o m w h i c h t h e DNA p r o f i l e s are very on MAK  s i m i l a r , and  was  not  purified further.  Figure  f r o m t h e e x t r a c t ; by, e t h a n o l . -  29 shows t h e e l u t i o n p a t t e r n o f DNA  s o n i c a t i o n method ( 1 5 9 ) . NaCl concentration  These c h r o m a t o g r a p h i c  suggest t h a t f o r purposes of r o u t i n e chromatography  c o l u m n , i t i s n o t n e c e s s a r y t o i s o l a t e DNA  precipitation.  W i t h t h i s DNA  t h e main peak was  p r e p a r e d by  A higher  peak.  The  two  m a t e r i a l was  missing  e x t r a c t e d by in  s i z e and  ments and  absorbing  i n Figure  e l u t e d at the very  n i n g o f t h e f r a c t i o n a t i o n , i n d i c a t i n g p e r h a p s some d e g r a d a t i o n i s , however, n o t v e r y e x t e n s i v e ,  However,  s m a l l peaks which precede  phenol e x t r a c t e d samples are c o m p l e t e l y  amount o f u l t r a v i o l e t a b s o r b i n g  This degradation  same  as t h e sample e x t r a c t e d by t h e p h e n o l method ( 1 4 7 ) .  e l u t e d i n t h e main DNA  main peak i n t h e  the  e l u t e d at the  i n s p e c t i o n o f t h e d a t a i n T a b l e XI i n d i c a t e s t h a t more u l t r a v i o l e t m a t e r i a l was  of  29. begin-  o f t h e DNA  s i n c e o t h e r DNA  the  sample.  preparations  t h e p h e n o l method showed o c c a s i o n a l l y t h e a p p e a r a n c e o f p e a k s s i m i l a r a t t h e same p l a c e on t h e c h r o m a t o g r a m .  diphenyhamine t e s t s i n d i c a t e d t h a t the  concentration  o f 0.09M c o n t a i n e d  of deoxyribose-positive peaks r e p r e s e n t degradation  material.  little  M o r e o v e r , r a d i o a c t i v e measure-  peaks e l u t e d by an i n i t i a l  o r no r a d i o a c t i v i t y and  very  s m a l l amount  These f i n d i n g s seemed t o i n d i c a t e , t h a t  contaminating u l t r a v i o l e t absorbing  salt  m a t e r i a l s , r a t h e r than  the DNA  products. From t h e r e s u l t s o f t h e c o m p a r i s o n i t can  p r e p a r e d by t h e method o f Medawar and t h a n does DNA  p r e p a r e d by  t h a t t h e methods u s i n g  Zubay (159)  be c o n c l u d e d t h a t t h e  u n d e r g o e s no more  t h e method o f C o l t e r e t a l .  phenol e x t r a c t i o n introduce  degradation  It i s generally  little  DNA  damage t o t h e  accepted primary  110  A  NaCl g r a d i e n t Jl  0.6  - 1.0 o CO  OJ  0.4  •p rt  0.8  o o fl  4-> C  rt  rt Xi  u w o  fl •rl  Xi  -0.6  <  0.4  0.2-  •0.2  -i— 20 Figure  27.  —i—  40  -i—  60 80 Tube Number  100  120  Chromatography on MAK column of double l a b e l l e d DNA prepared from i n t e s t i n a l mucosa of r a t by the method of C o l t e r et _ a l . (147). The DNA was not p u r i f i e d by e t h a n o l p r e c i p i t a t i o n . The animals were exposed to C - t h y m i d i n e 40 m i n . before s a c r i f i c e .  Ill  A  0.6  NaCl g r a d i e n t  Jl  1.2  4J o  0.4  CO OJ  0.8  A  •p  A  a  +-> •C  o o a  cS  a  rH  J2  U  O  tfi  < 0.4  0.2  20 F i g u r e 28.,  40  60 Tube Number  Chromatography on MAK column o f double l a b e l l e d DNA o b t a i n e d from the i n t e s t i n a l mucosa of r a t by the method of C o l t e r e t j o l . (147). The animals were exposed t o C - l a b e l l e d thymidine 40 min. b e f o r e s a c r a f i c e . The DNA was p u r i f i e d by p r e c i p i t a t i o n with ethanol..  112  0.8 -]  A  NaCl g r a d i e n t  -1.2  0.6  O  CD OJ  0.4  4-> ci  0.8  0  O fl  fl  ci  jo  ci  u  3  o CO  <  c •r-l  0.2_  o  A  ci 55  o  —i  20 Figure  29.  i  1—  120 100 60 80 Tube Number Chromatography on MAK column of DNA from i n t e s t i n a l mucosa of r a t prepared by the method of Medawar and Zubay (159). 40  -0.4  113 and s e c o n d a r y s t r u c t u r e o f DNA.  Because o f t h e g r e a t e r ease o f o p e r a t i o n , t h e  p h e n o l e x t r a c t i o n t e c h n i q u e was a d o p t e d f o r f u r t h e r e x p e r i m e n t a t i o n .  4.  The C  14  and H  3  e l u t i o n p r o f i l e s o f t h e d o u b l e l a b e l l e d DNA 3  and t h e c o m p a r i s o n o f t h e H /C MAK  preparations,  14 r a t i o s i n the d i f f e r e n t f r a c t i o n s o b t a i n e d a f t e r  chromatography. To i n c r e a s e t h e s e n s i t i v i t y o f t h e MAK  investigators  fractionation technique, several  (102,134,135) i n t r o d u c e d r a d i o a c t i v e l a b e l l i n g t o d e t e c t m i n o r p h y s i c o -  c h e m i c a l d i f f e r e n c e s between d i f f e r e n t n u c l e i c a c i d p r e p a r a t i o n s . 32 l a b e l l i n g experiments P  For pulse  l a b e l l e d i n o r g a n i c phosphate i s used e x t e n s i v e l y .  main d i s a d v a n t a g e o f t h i s method i s however, t h a t t h e i s o t o p e i s n o t f o r DNA.  One  specific  5ampson e t a l . (154) u s e d t h i s t e c h n i q u e t o d e m o n s t r a t e t h e e x i s t e n c e o f  a m e t a b o l i c a l l y l a b i l e DNA  i n growing p l a n t t i s s u e s .  By e x p o s i n g wheat s e e d s t o  32 P - i n o r g a n i c p h o s p h a t e , two t y p e s o f l a b e l l e d DNAs were s e p a r a t e d on MAK c o l u m n s . The t i m e c o u r s e o f t h e a p p e a r a n c e and d i s a p p e a r a n c e o f t h e r a d i o a c t i v e p h o s p h a t e 31 was f o l l o w e d i n t h e s e DNAs, a f t e r " c h a s i n g " w i t h P p h o s p h a t e . The h i g h m o l e c u l a r 32 w e i g h t DNA behaved i n t h e e x p e c t e d f a s h i o n ; t h e s p e c i f i c a c t i v i t y o f P -DNA decreased o n l y s l i g h t l y f o l l o w i n g exposure o f r o o t s t o P t h e s p e c i f i c a c t i v i t y o f l o w m o l e c u l a r w e i g h t DNA  32  phosphate.  By  contrast,  decreased to the vanishing  point  31 f o l l o w i n g t h e same e x p o s u r e t o P  .  T h i s i n d i c a t e d t o them t h e l a t t e r DNA  species  was more l a b i l e t h a n t h e f o r m e r . To i n s u r e even more s e n s i t i v e d e t e c t i o n o f c h r o m a t o g r a p h i c t h e d o u b l e l a b e l l i n g t e c h n i q u e has been e x t e n s i v e l y used ( 1 8 1 ) . Sueoka and S p i e g e l m a n 3 H  and C  example,  (133) were a b l e t o d e t e c t c h r o m a t o g r a p h i c d i f f e r e n c e s between  14 - l a b e l l e d RNA  p r e p a r a t i o n s o b t a i n e d f r o m t h e T2 - E_. c o l i s y s t e m . 32 3  t h e s i m u l t a n e o u s use o f P f o r m o f t h e cbx  For  differences,  and H  By  l a b e l l i n g t h e r e p l i c a t i v e and s i n g l e s t r a n d e d  174 v i r u s were e a s i l y i d e n t i f i e d on MAK  columns  (138).  114 I t was e x p e c t e d ,  t h e r e f o r e , t h a t i f d u r i n g t h e b i o s y n t h e s i s o f DNA,  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 i s i n somewhat d i f f e r e n t p h y s i c a l o r f o r m , s u c h a DNA s p e c i e s m i g h t be d e t e c t e d by MAK c h r o m a t o g r a p h y .  chemical To i n s u r e  a more s e n s i t i v e means o f d e t e c t i o n o f c h r o m a t o g r a p h i c d i f f e r e n c e s , a d o u b l e l a b e l was  i n t r o d u c e d t o t h e DNA.  To compare DNAs l a b e l l e d i n . v i v o f o r s h o r t e r  o r l o n g e r p e r i o d s i t i s d e s i r a b l e t o have t h e two One  avoids thereby  c o m p l i c a t i o n s o f v a r i e d p o o l s i z e s and 14  entry.  l a b e l s on t h e same p r e c u r s o r .  In the present  investigation C  d i f f e r e n t paths o f  3 - and  H - l a b e l l e d thymidine  were u s e d  as s p e c i f i c DNA p r e c u r s o r s . 14 I t can be e x p e c t e d , t h a t i f t h e C - l a b e l l e d DNA s y n t h e s i z e d i n t h e d u r i n g t h e 40 o r 20 m i n u t e s e x p o s u r e t i m e t o t h e p r e c u r s o r was d i f f e r e n t  animals  3 f r o m t h e o l d o r p a r e n t a l H - l a b e l l e d DNA s y n t h e s i z e d d u r i n g t h e 24 h o u r s o f e x 3 p o s u r e t i m e , t h e e l u t i o n p r o f i l e s o f H and i f t h e two  and  14  c h e m i c a l l y , t h e e l u t i o n p r o f i l e s o f H and  C  s h o u l d be i d e n t i c a l , o r t h e  ratio  14  o f H :C  c o u n t s s h o u l d be c o n s t a n t . The  e l u t i o n p a t t e r n s o f t h e d o u b l e l a b e l l e d DNA p r e p a r a t i o n s a r e shown  i n F i g u r e s 25,27,30,31. absorbing  These F i g u r e s i l l u s t r a t e t h e e l u t i o n o f t h e m a t e r i a l  a t 260rry«. as t h e f u n c t i o n o f N a C l c o n c e n t r a t i o n .  the r e s u l t s o f q u a n t i t a t i v e Dische t e s t s f o r deoxyribose The  Similarly,  l a b e l l e d DNAs a r e t h e same o r v e r y s i m i l a r p h y s i c a l l y and 3  3  14 C s h o u l d be d i f f e r e n t .  radiochromatograms are represented  InFigures  25,30,31  a r e also;,shown)(165).  i n F i g u r e s 32,33.  I f one compares t h e 14 3 e l u t i o n p a t t e r n s o f 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 and t h e C and H a c t i v i t i e s i n t h e d i f f e r e n t f r a c t i o n s o f t h e d i f f e r e n t DNA p r e p a r a t i o n s t h e f o l l o w i n g c o n 14 3  e l u s i o n s can be made:  Most o f t h e r a d i o a c t i v i t y r e p r e s e n t i n g b o t h C  a c t i v i t i e s i s i n c o r p o r a t e d i n t o t h e main peak e l u t e d by 0.66M N a C l . s e r v a t i o n s are f u r t h e r supported  and H These  b y t h e d a t a i n T a b l e XIT, w h i c h show t h a t  obbe-  tween 94-97% o f t h e t o t a l r a d i o a c t i v i t y e l u t e d f r o m t h e column i s i n c o r p o r a t e d 14 3 i n t o t h e main DNA peak. The C :H e l u t i o n p r o f i l e s a r e a l m o s t i d e n t i c a l i n  115  Absorbance a t 260 m/<Absorbance a t 595 m/^ (Dische t e s t ) NaCl g r a d i e n t  1.6 -  1.2 -  o c ci U  0.8  o w  -0.8  0.4-  -0.4  A  40 F i g u r e 30,  A  60 Tube Number  80  100  120  Chromatography on MAK column o f double l a b e l l e d DNA o b t a i n e d from i n t e s t i n a l mucosa of r a t by the method of C o l t e r e t a l . ( 1 4 7 ) . The animals were exposed t o C -thymidine 20 min. before sacrifice. 1  116  Absorbance a t 260 m./*Absorbance a t 595 myd(Dische t e s t ) NaCl g r a d i e n t  Tube Number F i g u r e 31.  Chromatography on MAK o f double l a b e l l e d DNA o b t a i n e d from i n t e s t i n a l mucosa o f r a t by the method o f CoJ^er e t _al.(147). The animals were exposed t o C -thymidine 20 min. b e f o r e s a c r i f i c e .  117  C ^ activity  ©  1  H  activity  2CQ  0) •H  4-> •H > •H  •P  O  ©  to  1.  F i g u r e 32,  i •  1  120 80 100 60 Tube Number E l u t i o n o f r a d i o a c t i v i t y o f double l a b e l l e d DNA chromatographed on MAK column. The r a d i o a c t i v i t y i n F i g u r e A was determined i n the e l u a t e s shown i n F i g u r e 25, and the r a d i o a c t i v i t y o f t h a t of F i g u r e B corresponded t o F i g u r e 27. 20  40  118  0  5n  4-  C  1 4  Activity  H° A c t i v i t y 3-  icn cu  •rl •P •H > •rl -t-> O <  ©  o (3  to  2-  120 60 80 100 Tube Number E l u t i o n o f r a d i o a c t i v i t y o f double l a b e l l e d DNA chromatographed on MAK column. The r a d i o a c t i v i t y i n F i g u r e A was determined i n the e l u a t e s shown i n F i g u r e 30, and t h a t o f F i g u r e B corresponded t o F i g u r e 31. 20  F i g u r e 33,  40  119 TABLE X I I . 14 3 Comparison o f t h e % D i s t r i b u t i o n o f C and H A c t i v i t i e s i n t h e Main and M i n o r F r a c t i o n s o f DNAs E l u t e d f r o m MAK Columns.  % of total DNA* sample  14 C - t h y m i d i n e i n j e c t e d 40 min. p r i o r t o s a c r i f i c e . DNA e x t r a c t e d by p h e n o l method 14 C - t h y m i d i n e i n j e c t e d 40 min. p r i o r t o s a c r i f i c e . DNA was e x t r a c t e d by p h e n o l method and p r e c i p i t a t e d by ethanol 14 C - t h y m i d i n e i n j e c t e d 40 min. p r i o r t o s a c r i f i c e . DNA was p r e p a r e d by t h e s o n i c a t i o n method (159) 14 C - t h y m i d i n e i n j e c t e d 40 min. p r i o r t o s a c r i f i c e . P h e n o l method (147) 14 C - t h y m i d i n e i n j e c t e d 20 min. p r i o r t o s a c r i f i c e . P h e n o l method (147) 14 C - t h y m i d i n e i n j e c t e d 20 min. p r i o r t o s a c r i f i c e . P h e n o l method (147)  *  peak e l u t e d with 0.66M NaCl  H  3  activity  % of total  Combined minor peaks  peak e l u t e d with 0.66M NaCl  C  1 4  activity combined minor peaks  97.5  2.5  97.3  2.7  96.4  3.6  97.1  2.9  96.1  3.9  98.0  2.0  97.9  2.1  95.9  4.1  96.6  3.4  94.0  4.0  95.3  4.7  94.8  5.2  A l l a n i m a l s r e c e i v e d H - l a b e l l e d t h y m i d i n e 24 h o u r s b e f o r e t h e C p r e c u r s o r was a d m i n i s t e r e d .  -labelled  120 each c a s e . low  (5-10  V a r i a t i o n s can cpm  inaccurate sidered  above b a c k g r o u n d ) .  and  T h i s made c a l c u l a t i o n o f t h e H^/C^  very  r a t i o s very  t h e r e f o r e , v a r i a t i o n s i n t h e s e a r e a s on t h e d i a g r a m s a r e n o t  con-  significant. On  c a l c u l a t i n g the percentage r e c o v e r i e s of r a d i o a c t i v i t y a f t e r  c h r o m a t o g r a p h y i t was  f o u n d t h a t i n a l l c a s e s 95 t o 100%  a p p l i e d on t h e column was o b s e r v e d f r o m any Since  recovered.  of the t o t a l  MAk  radioactivity  M o r e o v e r , no l o s s o f r a d i o a c t i v i t y  was  fraction after dialysis.  the t h r e e  c o n s i s t e n t l y obtained p r o c e d u r e , i t was  s m a l l e r peaks p r e c e d i n g  f o r a l l DNA  preparations  t h e main DNA  obtained  f r a c t i o n were  by t h e p h e n o l e x t r a c t i o n  of i n t e r e s t t o i n v e s t i g a t e f u r t h e r whether these areas  m a t e r i a l c o n t a i n i n g DNA. DNA  be n o t i c e d i n t h e a r e a s where t h e a c t i v i t i e s a r e  The  two  s m a l l e r peaks i m m e d i a t e l y p r e c e d i n g  represent  the  main  peak were o f s p e c i a l i n t e r e s t , s i n c e t h e s e were e l u t e d w i t h a p p r o x i m a t e l y  same N a C l c o n c e n t r a t i o n et a l . (154).  The  as the  u l t r a v i o l e t absorbing  longer  o n l y 20 min.  possible explanations.  m a t e r i a l w h i c h i s n o t DNA.  F i r s t , the peaks  by  Sampson  even i n t h e DNA  before  sacrifice.  obtained I t was  represent  Second, t h e f r a c t i o n s c o u l d  s p e c i e s of such a high r a t e of t u r n o v e r ,  detectable  reported  f a c t t h a t i n none o f t h e e x p e r i m e n t s were t h e s e p e a k s f o u n d t o  r a d i o a c t i v e , s u g g e s t s two  i n d i c a t e a DNA  " m e t a b o l i c a l l y l a b i l e " DNA  the  t h a t r a d i o a c t i v i t y i s no  from animals r e c e i v i n g the  precursor  f o u n d , however, t h a t t h e p h e n o l l a y e r  the t i s s u e e x t r a c t , which presumably c o n t a i n s  acid-soluble materials,  of  contained  14 considerable  amounts o f C  r a d i o a c t i v e precursor  may  - a c t i v i t y even a f t e r 20 m i n u t e s p o s t i n j e c t i o n . t h e r e f o r e be  a v a i l a b l e f o r t h e s y n t h e s i s o f DNA.  measurement o f t h e d e o x y r i b o s e c o n t e n t o f t h e d i f f e r e n t f r a c t i o n s o f t h e c h r o m a t o g r a m s , however, i n d i c a t e d t h a t t h e concentration  o f 0.45M c o n t a i n e d  also represented  peaks e l u t e d w i t h an i n i t i a l  n e g l i g i b l e amounts o f DNA.  as t h e shaded a r e a s i n F i g u r e s  r i b o s e p o s i t i v e m a t e r i a l was  The  25,26,30,31.  e l u t e d i n t h e main DNA  The MAK  NaCl  These f i n d i n g s Most o f t h e  are  deoxy-  peak, i n f u r t h e r agreement  be  121 3 with the r a d i o a c t i v i t y determinations. Since H be r e g a r d e d as s p e c i f i c p r e c u r s o r s o f t h e DNA  14 and C  - l a b e l l e d thymidine can  (26-28) and t h e d i p h e n y l a m i n e  test  gave p o s i t i v e r e a c t i o n s o n l y f o r t h o s e a r e a s w h i c h c o n t a i n e d r a d i o a c t i v i t y , i t seems t h a t t h e o t h e r s m a l l peaks on t h e chromatograms r e p r e s e n t m a t e r i a l  which  i s n o t DNA, b u t w h i c h i s c l o s e l y a s s o c i a t e d w i t h t h e DNA and i s n o t removed  even  after extensive d i a l y s i s . A l t h o u g h f u r t h e r i d e n t i f i c a t i o n o f t h e two s m a l l peaks i m m e d i a t e l y p r e c e d i n g i t h e main DNA peak has n o t been a t t e m p t e d , i t i s s u s p e c t e d t h a t t h e y may r e p r e s e n t s o l u b l e RNA.  The b a s i s o f t h i s a s s u m p t i o n i s t h a t when y e a s t s-RNA  was f r a c t i o n a t e d on t h e MAK c o l u m n , t h e m a t e r i a l was e l u t e d a t a p p r o x i m a t e l y t h e same N a C l c o n c e n t r a t i o n .  Moreover,  t h e f r a c t i o n a t i o n e x p e r i m e n t o f L a c k s (134)  w i t h t o t a l n u c l e i c a c i d e x t r a c t s f r o m pneumococcal  c e l l s a l s o i n d i c a t e d t h a t two  s m a l l peaks i m m e d i a t e l y p r e c e d i n g t h e DNA f r a c t i o n were s-RNA. 14 3 Although the e l u t i o n patterns of C and H a c t i v i t i e s o f t h e 20 o r 40 m i n u t e d o u b l e l a b e l l i n g e x p e r i m e n t s d i d n o t r e v e a l any s t r i k i n g d i f f e r e n c e s , t h e 3 . 14 c a l c u l a t i o n o f t h e H /C r a t i o s o f t h e i n d i v i d u a l f r a c t i o n s o f t h e main DNA peaks i n d i c a t e d t h a t some v a r i a t i o n s e x i s t e d . 3 o f t h e H /C  T a b l e X I I I summarizes t h e r a t i o s  14 a c t i v i t i e s o f t h e f r a c t i o n s o f DNA samples o b t a i n e d a f t e r  chromatography.  An i n t e r e s t i n g d i f f e r e n c e became a p p a r e n t , when each  making up t h e main DNA peak was a n a l y z e d f o r r a d i o a c t i v i t y . a r e r e p r e s e n t e d i n F i g u r e 34.  MAK  fraction  The d a t a o b t a i n e d  The d i a g r a m i n d i c a t e s a r a t h e r c o n s t a n t r a t i o  o f H^/C"^ t h r o u g h o u t t h e main DNA peak i n t h e 40 m i n u t e e x p o s u r e t i m e e x p e r i m e n t s . 4  In t h e c a s e o f 20 m i n u t e e x p e r i m e n t s t h e r a t i o s o f H^/C^  are higher i n the l a t e r 14  f r a c t i o n s , i n d i c a t i n g a d e f i n i t e s h i f t towards lower r e l a t i v e C these f r a c t i o n s .  content i n  Due t o t h e v a r i a t i o n s e n c o u n t e r e d i n i n v i v o work, t h e a v e r a g e  l e v e l o f r a d i o a c t i v i t y i n t h e DNA p r e p a r a t i o n s o f t h e d u p l i c a t e e x p e r i m e n t s a r e n o t t h e same, b u t t h e main f e a t u r e s ( e i t h e r a h o r i z o n t a l l i n e o r an upward c u r v e ) a r e r e m a r k a b l y s i m i l a r i n each s e t o f e x p e r i m e n t s .  I t s h o u l d be p o i n t e d o u t t h a t  122 TABLE X I I I C o m p a r i s o n o f t h e H^/ C a c t i v i t i e s i n t h e DNA F r a c t i o n s o b t a i n e d by MAK C h r o m a t o g r a p h y . 1 4  The r a t i o s o f H /C  activities  i n the fractions  F r a c t i o n number and m o l a r i t y o f N a C l i n DNA preparation*  eluant**  Ill 0.66 - 0.73  I .0.090.44  IV 0.730.77  V 0.78 0.90  1  2  3  4  5  6  7  4.6  5.3  5.4  5.3  5.6  5.5  5.0  6.7  2.8  9.7  -  6.6  6.5  6.4  5.6  5.7  5.7  6.8  7.2  3.4  C -thymidine i n j e c t e d 40 m i n . b e f o r e s a c r i fice. DNA p r e p a r e d by method o f C o l t e r e t a l . (147)  -  7.8  7.5  7.8  7.3  7.4  7.5  7.7  10.9  C"*" -thymidine i n j e c t e d 20 m i n . b e f o r e s a c r i fice. DNA p r e p a r e d by method o f C o l t e r e t a l . (147)  3.3  8.9 10.5 10.8 11.4 11.5 11.8 10.9  14.3  14 C -thymidine i n j e c t e d 40 m i n . b e f o r e s a c r i fice. DNA p r e p a r e d by method o f C o l t e r e t a l . (147). 14 C -thymidine i n j e c t e d 40 m i n . b e f o r e s a c r i fice. DNA p r e p a r e d by method o f Medawar and Zubay ( 1 5 9 ) .  -  4  C -thymidine injected 20 m i n . b e f o r e s a c r i fice. DNA p r e p a r e d by method o f C o l t e r e t a l . (147)  14.4  1 4  -  4.1  5.3  6.8  7.5  7.5  7.B  8.8  8.1  1  3 14 * A l l a n i m a l s r e c e i v e d H - l a b e l l e d t h y m i d i n e 24 h r . b e f o r e t h e C l a b e l l e d p r e c u r s o r was a d m i n i s t e r e d . ** F r a c t i o n activity  I I e l u t e d by 0.45-0.65M N a C l d i d n o t c o n t a i n  any r a d i o -  10.5  123  40 Min. C  14  i n c o r p o r a t i o n , exp. 1  40  "  "  20  "  •*  20  "  "  12.0  " 2  —^ l  " 2  A  © O  n  10.0  8.01  6.0  4.0'  Figure  34.  F r a c t i o n Number 14 H /C r a t i o s o f the f r a c t i o n s o f the DNA peak o b t a i n e d by MAK chromatography. 3  124 r e c e n t l y Cheng and Sueoka (103) d e m o n s t r a t e d t h a t c h r o m a t o g r a p h y on MAK c o l u m n s o f DNAs o f a n i m a l o r i g i n y i e l d e d on e l u t i o n by N a C l g r a d i e n t i n which t h e i n d i v i d u a l f r a c t i o n s d i f f e r e d i n d e n s i t y . higher  NaCl c o n c e n t r a t i o n s  one main DNA peak,  The f r a c t i o n s e l u t e d by  had l o w e r d e n s i t i e s i n a C s C l d e n s i t y g r a d i e n t .  This  f i n d i n g i n d i c a t e s t h a t t h e l a t e r f r a c t i o n s i n t h e main DNA peak had l o w e r g u a n i n e cytosine content. tograms. there  The c o n t r a r y  was t r u e f o r t h e e a r l i e r f r a c t i o n s o f MAK c h r o m a -  The r e s u l t s o f Cheng and Sueoka a r e b e s t i n t e r p r e t e d by a s s u m i n g  i s a heterogeneity  that  i n b a s e c o m p o s i t i o n o f t h e f r a c t i o n s o f t h e main DNA  v  peak.  Recently  Sponar e t a _ . (195) a l s o r e p o r t e d  t h a t c a l f thymus DNA on chroma-  t o g r a p h y on MAK c o l u m n s y i e l d e d f r a c t i o n s d i f f e r i n g i n g u a n i n e - c y t o s i n e and  content  i n molecular weight. Since'inthe  present i n v e s t i g a t i o n , d u p l i c a t e experiments i n d i c a t e d a def-  i n i t e s h i f t towards higher  concentration precursor,  r a t i o s i n t h e f r a c t i o n s e l u t e d by h i g h e r 14  t h e s h o r t e r 20 m i n u t e s e x p o s u r e t i m e t o t h e C  i t i s c o n c l u d e d t h a t t h e r e m i g h t be m e t a b o l i c  fractions. species  during  H"Vc"^  These f i n d i n g s c o u l d  i n d i c a t e a higher  i n f r i c t i o n s 1 - 4 , i n Figure 14  t i m e s r e l a t i v e l y more C f r a c t i o n s 5-6.  34,  since during  - t h y m i d i n e was i n c o r p o r a t e d  -labelled  heterogeneity  turnover  salt  i n these  r a t e o f t h e DNA  t h e 20 m i n u t e s e x p o s u r e  i n t o them, t h a n i n t o  A f t e r 40 m i n u t e s i n c o r p o r a t i o n t h e s e d i f f e r e n c e s d i s a p p e a r e d and  a r e l a t i v e l y uniform incorporation of C ^ - l a b e l l e d precursor each o f t h e s e f r a c t i o n s . DNA f r a c t i o n s a r e n o t v e r y  The s u g g e s t e d m e t a b o l i c  was o b t a i n e d  into  differences i n the d i f f e r e n t  s t r i k i n g , however, s i n c e t h e e l u t i o n p a t t e r n o f  carbon-14 f o l l o w e d t h a t o f t h e t r i t i u m .  P e r h a p s t h e s e d i f f e r e n c e s w o u l d be more  d i s t i n c t l y d e m o n s t r a t e d i n e x p e r i m e n t s o f even s h o r t e r e x p o s u r e t i m e i f t h e p r o blems o f a s s u r i n g  a h i g h enough i n c o r p o r a t i o n o f t h e i s o t o p e and o f t i s s u e man-  i p u l a t i o n c o u l d be overcome. The should  isotope r a t i o differences obtained  i n the present i n v e s t i g a t i o n  be i n t e r p r e t e d w i t h some c a u t i o n , however.  As i n d i c a t e d above, t h e  125 d i f f e r e n c e s between 20 and 40 m i n u t e e x p o s u r e t i m e e x p e r i m e n t s m i g h t some 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 d i f f e r e n t f r a c t i o n s o f DNA mucosa o f r a t .  However, s i n c e i t i s n o t known, w h e t h e r  of  indicate  intestinal  t h e s e f r a c t i o n s do i n -  deed e x i s t i n . v i v o , t h e r e s u l t s o b t a i n e d c a n n o t y e t be t a k e n t o i n d i c a t e metab o l i c i n t e r r e l a t i o n s h i p s amongst t h e s e f r a c t i o n s . mucosa r e p r e s e n t s a somewhat h e t e r o g e n e o u s  Furthermore,  intestinal  p o p u l a t i o n of c e l l s , i n which  d i f f e r e n t t y p e s o f c e l l s c o u l d have d i f f e r e n t r a t e s o f t u r n o v e r . newly  s y n t h e s i z e d DNA  the  Since the  r e p r e s e n t s newly s y n t h e s i z e d c e l l s _Ln v i v o , t h e f r a c t i o n s  o f t h e main DNA  peak c o u l d r e p r e s e n t m e r e l y v a r i o u s r a t e s o f c e l l r e n e w a l s i n  a heterogeneous  population.  The c o n c e p t o f m e t a b o l i c h e t e r o g e n e i t y o f DNA w i t h t h e a c c e p t e d i d e a s c o n c e r n i n g DNA  replication.  i s d i f f i c u l t to reconcile  According to these t h e o r i e s  once a s u b s t a n c e i s i n c o r p o r a t e d i n t o t h e g e n e t i c m a t e r i a l i t assumes " i m m o r t a l i t y " i n a s e n s e t h a t i t i s p a s s e d on f r o m g e n e r a t i o n t o g e n e r a t i o n . DNA  The i d e a o f  r e p l i c a t i o n i s a l s o e n v i s a g e d as an " a l l o r none" p r o c e s s i n w h i c h once t h e  m a c r a m o l e c u l a r d u p l i c a t i o n has s t a r t e d , t h e newly s y n t h e s i z e d m o l e c u l e s a s i n t a c t whole e n t i t i e s . DNA  appear  T h e r e i s some e v i d e n c e a t p r e s e n t (79,97) t h a t t h e  o f a s i n g l e phage u n i t o r b a c t e r i a l c e l l i s a s i n g l e m o l e c u l e , b u t no  r e p o r t s a p p e a r e d c o n c e r n i n g t h e DNA t h i s macramolecule  of higher organisms.  similar  The t o t a l amount o f  i n c e l l s f r o m h i g h e r o r g a n i s m s i s much l a r g e r t h a n i n b a c t e r i a .  Each human chromosome, f o r example,  c o n t a i n s on t h e a v e r a g e a b o u t 2 cm o f  DNA.  A l t h o u g h c o n s i d e r a b l e t e c h n i c a l s k i l l w i l l be needed t o d i s p l a y s u c h m o l e c u l e s , C a i r n s (79) e n v i s a g e s t h e DNA  o f t h e chromosomes o f t h e h i g h e r o r g a n i s m s  b e i n g one m o l e c u l e and i n a c i r c u l a r f o r m . by t h i s i n v e s t i g a t o r t o s u p p o r t t h i s t h e s i s .  Two  as  r a t h e r tenuous reasons are c i t e d  The f i r s t one i s t h e e x p e r i m e n t s  o f T a y l o r and Woods (77,78) on t h e d u p l i c a t i o n o f chromosomes i n p l a n t c e l l s .  If  a m a r k e r , s u c h as t r i t i a t e d t h y m i d i n e , i s f e d f o r a s h o r t t i m e d u r i n g t h e p e r i o d o f DNA  s y n t h e s i s i n t h e p l a n t c e l l , b o t h t h e d a u g h t e r c h r o m a t i d s a r e f o u n d t o be  126 l a b e l l e d at the time of c e l l d i v i s i o n .  When t h e y become v i s i b l e ,  however,  a f t e r a f u r t h e r r o u n d o f d u p l i c a t i o n , t h e s e two chromosome a r e each f o u n d have p r o d u c e d one be e x p e c t e d  l a b e l l e d and  one u n l a b e l l e d c h r o m a t i d .  T h i s i s what w o u l d  i f each o f t h e s e chromosomes c o n t a i n e d a s i n g l e m o l e c u l e ;  r e s u l t s are not c o m p e l l i n g evidence  to  but  the  f o r t h i s hypothesis, s i n c e they t e l l  one  o n l y a b o u t t h e s e g r e g a t i o n o f t h e m a t e r i a l p r e s e n t and n o t a b o u t t h e number o f strands present.  The  t i o n r a t e o f t h e DNA  s e c o n d argument i s b a s e d on t h e c a l c u l a t i o n o f t h e d u p l i c a o f b a c t e r i o p h a g e s , b a c t e r i a and  human chromosomes.  i s a c a s e f o r b e l i e v i n g i n a u n i v e r s a l d u p l i c a t i o n r a t e f o r DNA g i v e s some s u p p o r t  f o r t h e i d e a o f one m o l e c u l e  •n t h e o t h e r hand, s e v e r a l a u t h o r s  (196,197) v i s u a l i z e DNA  Furthermore,  r o l e o t h e r than g e n e t i c . and  III.  consisting  h y p o t h e s i z e d a mech-  by p o l y m e r i z a t i o n o f two  t h e m e t a b o l i c a l l y l a b i l e DNA,  as  In t h i s c o n n e c t i o n i t i s  i n t e r e s t i n g t o n o t e t h a t v e r y r e c e n t l y S p o n a r e t a l . (195) molecules  and t h i s i n t u r n ,  f o r each chromosome.  o f s u b u n i t s o f a r e l a t i v e l y low m o l e c u l a r weight.  a n i s m o f f o r m a t i o n o f DNA  There  kinds of subunits.  i f i t e x i s t s i n v i v o , c o u l d have some  A p h y s i o l o g i c a l r o l e f o r DNAs o f c h l o r a p l a s t s (107)  t h e m e t a b o l i c a l l y l a b i l e DNA  p l a n t embryos have been s u g g e s t e d  (154).  Measurement o f R a d i o a c t i v i t y i n t h e P r o t e i n - R N A I n t e r p h a s e s o f t h e T i s s u e E x t r a c t s O b t a i n e d by t h e Methods o f C o l t e r e t a l . (147) and Medawar and Zubay (159)  Most o f t h e " m i l d t e c h n i q u e s " employed f o r t h e e x t r a c t i o n o f DN^ v a r i o u s t i s s u e s have t h e d e f i n i t e d i s a d v a n t a g e t i o n s are u s u a l l y very low. t h e DNA  by d e t e r g e n t s  t h a t the y i e l d of the  from  prepara-  Sometimes g e n t l e c o n d i t i o n s such as e x t r a c t i o n  (144), high c o n c e n t r a t i o n of s a l t s  t o b r e a k t h e l i n k a g e s between p r o t e i n and DNA  molecules.  (138), are not The  down t h e c e l l u l a r and n u c l e a r membranes i s a l s o o f i m p o r t a n c e . d u r i n g t h e p r e s e n t i n v e s t i g a t i o n s , f o r e x a m p l e , t h a t DNA  method o f  of  sufficient breaking  I t has been  found,  o f t h e i n t e s t i n a l mucosa  o f t h e r a t c o u l d n o t be e x t r a c t e d w i t h h i g h c o n c e n t r a t i o n o f s a l t  solutions,  127 without  the a i d of sonic degradation  o f t h e n u c l e a r and c e l l u l a r membranes.  W h a t e v e r g e n t l e method has been employed t o e x t r a c t t h e n u c l e i c a c i d s f r o m  this  t i s s u e , i t has been d e m o n s t r a t e d ( 1 5 7 ) , t h a t no more t h a n 40 t o 50% o f t h e  total  DNA  o f t h e c e l l s c o u l d be i s o l a t e d .  S i n c e t h e aim o f t h e p r e s e n t i n v e s t i g a t i o n  was  to demonstrate p o s s i b l e metabolic  d i f f e r e n c e s i n t h e d i f f e r e n t DNAs o f i n -  t e s t i n a l mucosa, e x t e n s i v e l o s s e s o f t h e m a c r o m o l e c u l e s d u r i n g t h e e x t r a c t i o n p r o c e d u r e , c o u l d e f f e c t s e r i o u s l y t h e outcome o f t h e The  f i n d i n g o f G o l d s t e i n and Brown (148)  experiments.  t h a t newly s y n t h e s i z e d  DNA  i s r e s i s t a n t t o s o n i c o s c i l l a t i o n , s u g g e s t e d t o them t h a t t h e chromosomal mate r i a l assumes a more s t a b l e p h y s i c a l s t a t e d u r i n g r e p l i c a t i o n . o f t h e n e w l y s y n t h e s i z e d DNA t i o n with protein. t h i s assumption. k i d n e y c e l l s was  The  i s probably  due  seemed t o  These w o r k e r s d e m o n s t r a t e d t h a t t h e n e w l y f o r m e d DNA r e s i s t a n t t o c h l o r o f o r m - a m y l a l c o h o l e x t r a c t i o n , and  with r a d i o a c t i v e thymidine,  and  F u r t h e r m o r e , when t h e c e l l s were  t h e n t h e r a d i o a c t i v e m a t e r i a l was  " c o l d " c a r r i e r , t h e s p e c i f i c a c t i v i t y o f t h e i n t e r p h a s e DNA h i g h e r t h a n t h e b u l k o f t h e c e l l u l a r DNA r a d i o a c t i v e thymidine,  confirm in rabbit-  remained incubated  "chased" w i t h  w h i c h was  13  times  at the t i m e o f the a d d i t i o n o f non-  decreased p r o g r e s s i v e l y with time of i n c u b a t i o n .  s p e c i f i c a c t i v i t y o f t h e t o t a l DNA w o r k e r s (151,198,199) v i s u a l i z e DNA  and  stable state  to i t s i n t e r a c t i o n or c l o s e a s s o c i a -  e x p e r i m e n t s o f B e n - P o r a t e t a l . (149)  i n the protein-RNA r i c h i n t e r p h a s e .  cytoplasm  The  The  increased concomitantly!' ,Several others • s y n t h e s i s as p a r t l y b e i n g  performed i n the  o r m i c r o s o m e s , w h i c h a l s o c o u l d mean a c l o s e r a s s o c i a t i o n o f p r o t e i n  newly s y n t h e s i z e d DNA.  I t i s of i n t e r e s t to note, t h a t B i l l e n  (200)  succeed-  ed i n i s o l a t i n g a DNA-polymerase c o m p l e x , b u t t h e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h i s observation On  i s not c e r t a i n a t  present.  t h e b a s i s o f t h e above f i n d i n g s , i t was  of i n t e r e s t to count  the  p r o t e i n - R N A c o n t a i n i n g i n t e r p h a s e s o b t a i n e d by b o t h e x t r a c t i o n p r o c e d u r e s 3 (147,159) d u r i n g t h e p r e s e n t  i n v e s t i g a t i o n . T a b l e XIV  summarizes t h e H  14 /C  r a t i o s o f t h e i n t e r p h a s e l a y e r s o b t a i n e d f r o m t h e v a r i o u s t i s s u e e x t r a c t s , and  128 TABLE X I V 3 14 The R a t i o s o f H /C i n t h e I n t e r p h a s e and P h e n o l L a y e r s O b t a i n e d D u r i n g t h e P r e p a r a t i o n o f DMA  DNA p r e p a r a t i o n * I n t e r p h a s e f r o m t h e Medawar-Zubay method. 40 m i n . C - ^ - i n c o r p o r a t i o n experiment I n t e r p h a s e from t h e phenol e x t r a c t i o n •incorporation method. 40 m i n . 14. experiment Interphase from the phenol e x t r a c t i o n •incorporation method. 40 m i n . 14. experiment Interphase from the phenol e x t r a c t i o n method. 20 m i n . C^ -- i n c o r p o r a t i o n C  C  H /C 3  1 4  7.1  10.2  17.7  -4  experiment Interphase from the phenol e x t r a c t i o n method. 20 m i n . [ ^ - i n c o r p o r a t i o n experiment Phenol  layer  40 m i n . [ > - i n c o r p o r a t i o n e x p e r i m e n t 4  Phenol  layer  40 m i n . C - ^ - i n c o r p o r a t i o n Phenol  17.0  18.0 all C activity 0.3  experiment 0.4  layer  20 m i n . [ ^ - i n c o r p o r a t i o n  experiment  Phenol l a y e r 20 m i n . C - ^ - i n c o r p o r a t i o n  experiment  0.2  * A l l a n i m a l s r e c e i v e d H - l a b e l l e d t h y m i d i n e 24 h r . b e f o r e t h e C - ^ - l a b e l l e d p r e c u r s o r was a d m i n i s t e r e d  •  129  a l s o the v a l u e s o b t a i n e d f o r the phenol l a y e r s .  In most o f t h e c a s e s t h e H  r a t i o s o f t h e p r o t e i n r i c h i n t e r p h a s e s were somewhat h i g h e r t h a n t h o s e f r o m t h e d i f f e r e n t f r a c t i o n s o f t h e c o r r e s p o n d i n g DNA.  T h i s might  3  /C  14  obtained  indicate  3 h i g h e r amount o f t h e " o l d e r " H - l a b e l l e d m a t e r i a l i n t h e i n t e r p h a s e l a y e r . t h e same t i m e i t s h o u l d be p o i n t e d o u t t h a t t h e same r e s u l t s c o u l d a r i s e quenching e f f e c t s i n these samples.  The  HC1,  yde  (184).  used i n the p r e p a r a t i o n o f the m a t e r i a l f o r c o u n t i n g  methods a r e r e q u i r e d f o r t h e s i m u l t a n e o u s No f u r t h e r a t t e m p t  was  from  e l i m i n a t i o n of quenching i n these  s a m p l e s i s d i f f i c u l t b e c a u s e o f t h e h i g h amounts o f KOH,  material.  At  c o u n t i n g o f two  and hyamine  bydrox-  Obviously b e t t e r  i s o t o p e s i n t h i s type  made t o e x p l o r e t h e f u r t h e r p u r i f i c a t i o n  c o u n t i n g of the i n t e r p h a s e m a t e r i a l , but i t i s f e l t ,  of  and  t h a t i f newly s y n t h e s i z e d  14 C  - r i c h DNA  was  p r e s e n t , i t was  so o n l y i n v e r y s m a l l amounts. The 14  o b t a i n e d i n t h e p h e n o l l a y e r s were a l m o s t might i n d i c a t e the presence the phenol l a y e r .  e x c l u s i v e l y due  to C  .  o f a c i d - s o l u b l e low m o l e c u l a r w e i g h t  counts  This  finding  precursors i n  I f i t i s assumed t h a t t h e p h e n o l l a y e r c o n t a i n s a c i d - s o l u b l e  m a t e r i a l s o f t h e t i s s u e , i t can be c o n c l u d e d ,  t h a t 24 h o u r s a f t e r  injection,  3 most o f t h e H - l a b e l l e d t h y m i d i n e was  no l o n g e r a v a i l a b l e f o r DNA b i o s y n t h e s i s ,  14 w h e r e a s some C  - l a b e l l e d p r e c u r s o r was  present i n s i g n i f i c a n t concentration  even I V . aE nf zt ye mra t40 i c o rD e g20r a dmaitniuotne s o fp o st th e iDnojuebcltei o Ln a. b e l l e d DNA  Preparations.  3 . 14 Although i o n s o f t h e DNA  t h e d e t e r m i n a t i o n o f t h e H /C  r a t i o s i n the d i f f e r e n t  fract-  p r e p a r a t i o n s m i g h t i n d i c a t e t h e m e t a b o l i c d i f f e r e n c e s amongst  t h e s e f r a c t i o n s , t h e y w o u l d n o t g i v e an answer t o p r o b l e m s c o n c e r n i n g t h e  nature  and  s i t e of the i n c o r p o r a t i o n of the precursor i n t o the p o l y n u c l e o t i d e c h a i n s .  Two  t y p e s o f i n c o r p o r a t i o n o f the r a d i o a c t i v e p r e c u r s o r can o c c u r d u r i n g the  s y n t h e s i s o f t h e DNA  molecule.  The  first  bio-  t y p e w o u l d be i n c o r p o r a t i o n s . w e l l w i t h i n  t h e c h a i n , w h e r e a s t h e s e c o n d k i n d w o u l d be a d d i t i o n o f t h e d e o x y r i b o n u c l e o s i d e a f t e r c o n v e r s i o n t o t r i p h o s p h a t e , t o t h e ends o f t h e p r e - e x i s t i n g p o l y n u c l e o t i d e  130 chain.  To i n v e s t i g a t e w h i c h one o f t h e two p o s s i b i l i t i e s o c c u r r e d  during the  i n v i v o b i o s y n t h e s i s o f DNA i n t h e i n t e s t i n a l mucosa o f r a t , use was made o f techniques o f stepwise degradation  o f DNA m o l e c u l e s f r o m one e n d .  I n s u c h an  e x p e r i m e n t one f o l l o w s t h e t i m e c o u r s e o f t h e r e l e a s e o f t h e a c i d - s o l u b l e material  (50).  I t i s expected, that i f the i n c o r p o r a t i o n occurred  i t i o n of the precursor,  the release of the r a d i o a c t i v i t y i n the acid-soluble  f r a c t i o n w o u l d be much h i g h e r  than t h e r e l e a s e o f the u l t r a v i o l e t  m a t e r i a l i n the e a r l i e r stages of the r e a c t i o n . was w e l l w i t h i n t h e p o l y n u c l e o t i d e should  Conversely, i f the incorporation  p a r a l l e l the rate of the release o f u l t r a v i o l e t absorbing  the stepwise degradation  o f DNA and RNA.  i n the polydeoxyribonucleotide  material.  by s e v e r a l i n v e s t i g a t o r s , (50-53)  One o f t h e e a r l i e s t r e p o r t s ,  c r i b i n g t h i s technique f o r the determination precursor  absorbing  chain, the rate of release of r a d i o a c t i v i t y  S i m i l a r t y p e s o f e x p e r i m e n t s have been d e s c r i b e d for  by end a d d -  of the l o c a t i o n of the radioactive  c h a i n was t h a t o f A d l e r e t a l . ( 5 0 ) .  T h e s e w o r k e r s used snake venom p h o s p h o d i e s t e r a s e f o r t h e s e q u e n t i a l of deoxyribonucleotides has  from t h e d e o x y r i b o n u c l e o s i d e  liberation  ends o f DNA c h a i n s .  It  been shawm (161,176) t h a t t h i s enzyme a c t s p r i m a r i l y a s an e x o n u c l e a s e ,  l i b e r a t i n g m o n o n u c l e o t i d e s s t e p w i s e f r o m t h e ends o f o l i g o n u c l e o t i d e s w i t h hydroxyl  the stepwise enzymatic degradation  some i n f o r m a t i o n C  14  3'-  end g r o u p s . I t was f e l t t h e r e f o r e , t h a t by u s i n g  for  des-  w o u l d be o b t a i n e d  - l a b e l l e d thymidine during  p u r i f i e d s n a k e venom d i e s t e r a s e  o f t h e d o u b l e l a b e l l e d DNA  regarding  preparations,  t h e mode o f i n c o r p o r a t i o n o f t h e  . the short incorporation times i n v i v o .  To increase'.  t h e s e n s i t i v i t y o f t h e method, t h e r e l e a s e o f t h e r a d i o a c t i v i t y was measured b o t h in  t h e a c i d - s o l u b l e and i n t h e a c i d i n s o l u b l e f r a c t i o n s o f t h e i n c u b a t i o n  during  the course of the r e a c t i o n .  t a i n e d when a DNA p r e p a r a t i o n precursor  40 m i n . b e f o r e  Figures  35A and B r e p r e s e n t  mixture  t h e r e s u l t s ob-  ( F i g u r e 35A) f r o m r a t s i n j e c t e d w i t h t h e C  s a c r i f i c e and t h e main DNA peak o f t h e same DNA  1 4  -labelled  131 preparation  ( F i g u r e 35B)  a c t i o n o f t h e enzyme.  f r o m MAK  c h r o m a t o g r a p h y were s u b j e c t e d t o t h e  stepwise  F i g u r e 36 i l l u s t r a t e s t h e r e s u l t s o b t a i n e d , when t h e main 14  DNA  peak o f a DNA  p r e p a r a t i o n f r o m t h e 20 m i n .  s u b j e c t e d t o t h e same t r e a t m e n t .  The  C  exposure time experiment  r e s u l t s i n a l l cases are e s s e n t i a l l y  the  same, i n d i c a t i n g t h a t t h e r e l e a s e o f r a d i o a c t i v i t y p a r a l l e l s t h e r e l e a s e o f 3 14 v i o l e t absorbing  material.  F u r t h e r m o r e , t h e r a t i o s o f H :C  i n the a c i d - i n s o l u b l e f r a c t i o n .  remained  was  ultra-  constant  These a r e t h e r e s u l t s t h a t w o u l d be e x p e c t e d i f  the i n c o r p o r a t i o n of the p r e c u r s o r occurred w e l l w i t h i n the c h a i n . I f blocks of 14 3 C - l a b e l l e d t h y m i d i n e were added t o t h e ends o f t h e p a r e n t a l m a i n l y H - l a b e l l e d DNA  c h a i n s , the r a t i o s of  H'Vc"*' i n 4  i n c r e a s e d during the course w o u l d be e x p e c t e d  t h e a c i d - i n s o l u b l e f r a c t i o n s h o u l d have been 14  of the stepwise  degradation,  and more C  i n the a c i d s o l u b l e f r a c t i o n at the i n i t i a l  activity  stages of  the  enzymatic  degrada-  reaction. T h e r e a r e o n l y a few r e p o r t s c o n c e r n i n g t i o n o f DNA  (50-53).  One  the stepwise  o f t h e e a r l i e s t i s t h a t o f A d l e r e t al.who d e m o n s t r a t e d  the l i m i t e d a d d i t i o n of a s i n g l e d e o x y r i b o n u c l e o t i d e t r i p h o s p h a t e t o the e x i s t i n g DNA  chain i n v i t r o .  pre-  C a n e l l a k i s e_t a l . (51-53) d e s c r i b e d an enzyme  p r e p a r a t i o n f r o m c a l f thymus n u c l e i , w h i c h c a t a l y s e d a s i m i l a r r e a c t i o n , n a m e l y , t h e i n c o r p o r a t i o n o f r i b o and o f DNA.  The  d e o x y r i b o n u c l e o t i d e s i n t o the t e r m i n a l p o s i t i o n s  enzyme, however, a p p e a r s t o be d i s t i n c t f r o m p o l y m e r a s e , and  the  i n c o r p o r a t i o n of a s i n g l e deoxyribonucleotide t r i p h o s p h a t e i s i n h i b i t e d i n the presence of the other three d e o x y r i b o n u c l e o t i d e s .  The  b i o l o g i c a l and  biochemical  s i g n i f i c a n c e o f t h e s e r e a c t i o n s a r e n o t known, b u t s e v e r a l p o s s i b i l i t i e s were suggested  (50).  p h o s p h a t e may  The  r e a c t i o n between DNA  and  a single deoxyribonucleoside  a c t u a l l y be t h e f i r s t s t e p i n t h e o v e r - a l l p r o c e s s  r e p l i c a t i o n o f t h e DNA  of  tri-  enzymatic  p r i m e r ; t h e n t h e t e r m i n a l r e p l i c a t i n g c h a i n , f o r m e d when  four deoxyribonucleoside  t r i p h o s p h a t e s are p r e s e n t , would l o o p back, i n o r d e r  o r i e n t i t s e l f along the primer c h a i n .  Such l o o p s c o u l d be p r o d u c e d by  the  to  132  A  Acid-insoluble  fraction  o  A b s o r b a n c e a t 260  m/^  60 120 180 240 R e a c t i o n Time i n M i n u t e s F i g u r e 35.  300  E n z y m a t i c d e g r a d a t i o n o f d o u b l e l a b e l l e d DNA o b t a i n e d f r o m t h e i n t e s t i n a l mucosa o f r a t by t h e m e t h o d o f C o l t e r et a l . ( 1 4 7 ) . The a n i m a l s w e r e e x p o s e d t o t h e C** - l a b e l l e d p r e c u r s o r f o r 40 m i n . ( A ) DNA b e f o r e c h r o m a t o g r a p h y o n MAK. (B) The m a i n peak o b t a i n e d f r o m MAK chromatography.  133  A  Acid-insoluble  0  Absorbance at 260 m/c  °  Radioactivity  60 F i g u r e 36.  fraction  120 180 240 R e a c t i o n Time i n Minutes  Enzymatic d e g r a d a t i o n of the main DNA peak from MAK chromatography of double l a b e l l e d DNA from the i n t e s t i n a l mucosa of r a t s . The animals were exposed to the C - l a b e l l e d p r e c u r s o r f o r 20 minutes.  134 r o t a t i o n o f v e r y few d e o x y r i b o n u c l e o t i d e t i m e by n u c l e a s e a c t i o n . and  u n i t s and c o u l d be d i s s i p a t e d a t a l a t e r  A n o t h e r p o s s i b i l i t y i s t h a t t h e r e a c t i o n between DNA  a s i n g l e deoxyribonucleoside  o f t h e s h o r t e r o f two u n e q u a l l y  triphosphate long chains  i s merely a part of the extension  o f a DNA d o u b l e h e l i x and i s n o t  d i r e c t l y r e l a t e d t o t h e r e p l i c a t i o n o f i n t a c t DNA m o l e c u l e s .  The i n c o r p o r a t i o n  o f r i b o n u c l e o t i d e s i n t o t h e t e r m i n a l p o s i t i o n o f DNA m i g h t i n d i c a t e t h e p a r t i c i p a t i o n o f s u c h a c o m p l e x i n RNA s y n t h e s i s by a c t i n g a s t h e s t a r t i n g • p o i n t the polymerization  o f r i b o n u c l e o t i d e s on a DNA t e m p l a t e by RNA  1  fdr-r.  polymerase.  Investigations, concerning the p a r t i c i p a t i o n of the " l i m i t e d r e a c t i o n " i n t h e i n v i v o b i o s y n t h e s i s o f DNA, a r e even more l i m i t e d .  Z b a r s k y (201) i n v e s t i -  14 gated the i n c o r p o r a t i o n o f C b o t h i n . v i v o and v i t r o . subjected  - l a b e l l e d t h y m i d i n e i n t o E h r l i c h a s c i t e s tumour  When i n v i v o l a b e l l e d E h r l i c h a s c i t e s tumour DNA  was  t o t h e s t e p w i s e a c t i o n o f s n a k e venom p h o s p h o d i e s t e r a s e a t t h e  e a r l y stages of the incubation  t i m e , more r a d i o a c t i v i t y was r e n d e r e d  acid-soluble  t h a n i t w o u l d be e x p e c t e d f r o m a random d i s t r i b u t i o n o f t h e t h y m i n e i n t h e DNA chain.  T h i s f i n d i n g i n d i c a t e d t h a t t h e r a d i o a c t i v e t h y m i d i n e was added t o t h e  ends o f t h e DNA c h a i n s  during  t h e 24 h o u r s i n c o r p o r a t i o n i n . v i v o .  These d a t a  a r e r a t h e r s u r p r i s i n g , s i n c e one w o u l d e x p e c t t h a t , even i f t h e end a d d i t i o n p l a y s a r o l e i n DNA r e p l i c a t i o n d u r i n g  long incorporation times,  e s i z e d m o l e c u l e s w o u l d be l a b e l l e d a l l a l o n g individual entities.  The e x p e r i m e n t a l  t h e DNA c h a i n s ,  even i n t h e 2 0 - h o u r h y d r a l y z a t e  synth-  and a p p e a r a s new  r e s u l t s i n the present i n v e s t i g a t i o n are  r a t h e r i n agreement w i t h t h e i n v i t r o f i n d i n g s o f Z b a r s k y ( 2 0 1 ) . t h a t t h e r a d i o a c t i v i t y was r e l e a s e d  t h e newly  He d e m o n s t r a t e d  v e r y s l o w l y f r o m t h e i n v i t r o m a t e r i a l , and 1  the r a d i o a c t i v i t y present as thymidine-5'-phosphate  c o r r e s p o n d e d t o o n l y 2% o f t h e t o t a l . The  experimental  r e s u l t s w i t h t h e d o u b l e l a b e l l e d DNA p r e p a r a t i o n s a r e  a l s o i n a p p a r e n t agreement w i t h t h e r e p o r t o f R o l f e (202) i n a remote way. By 13 15 i n v e s t i g a t i n g s o n i c f r a g m e n t s o f h y b r i d C -N -DNA, R o l f e has e x c l u d e d t h e  135 p o s s i b i l i t y that the l i n e a r subunits side.  I t appears, from t h e present  t h e r e was a s h o r t e x p o s u r e t o t h e C m o l e c u l e s d i d n o t become a t t a c h e d 3 H - l a b e l l e d molecules.  are a s s o c i a t e d end-to-end i n s t e a d o f s i d e - t o i n v e s t i g a t i o n , t h a t even i n e x p e r i m e n t s where  14  - l a b e l l e d thymidine,  t h e newly  end t o end t o t h e o l d p a r e n t a l n o n - l a b e l l e d o r  A p a r a l l e l r e l e a s e of both H  3  and C  14  a c t i v i t y i n the acid  s o l u b l e p o r t i o n w o u l d be e x p e c t e d a l s o , i f t h e newly s y n t h e s i z e d of a hybrid composition,  synthesized  e.g. one c h a i n c o n t a i n i n g o n l y C  14  m o l e c u l e s were  l a b e l and t h e com-  3 plementary chain c o n t a i n i n g e n t i r e l y H - l a b e l l e d thymidine.  The r e s u l t s do n o t  e x c l u d e t h e p o s s i b i l i t y t h a t end t o end a d d i t i o n m i g h t be f o u n d i n e x p e r i m e n t s where e x p o s u r e t o t h e p r e c u r s o r  in_ v i v o was o f e x t r e m e l y s h o r t  duration.  136 SUMMARY The i _  v i v o b i o s y n t h e s i s o f DNA f r o m l a b e l l e d p r e c u r s o r s has been i n v e s t i g a t e d  i n t h e i n t e s t i n a l mucosa o f r a t . Because o f t h e p u b l i s h e d evidence h e t e r o g e n e i t y o f DNA  t h e DNA  the physico-chemical  and  metabolic  p r e p a r a t i o n s o f mammalian o r i g i n , two methods o f s e p a r a t i n g  t h e components o f t h e DNA Initially  concerning  p r e p a r a t i o n s were s t u d i e d .  was f r a c t i o n a t e d by c h r o m a t o g r a p h y on E C T E O L A - c e l l u l o s e  umns, u s i n g t h e d i s c o n t i n u o u s e l u t i o n s c h e d u l e  of K i t .  T h i s procedure  col-  was  considered u n s a t i s f a c t o r y f o r the f o l l o w i n g reasons: (a)  The e l u t i o n p a t t e r n s o f t h e DNA experiment t o  p r e p a r a t i o n s were n o t r e p r o d u c i b l e f r o m  experiment.  (b)  The e l u t i o n p a t t e r n s c h a n g e d on s t o r a g e o f t h e DNA  (c)  Evidence  was o b t a i n e d i n d i c a t i n g t h a t DNA  d u r i n g f r a c t i o n a t i o n on E C T E O L A - c e l l u l o s e .  s o l u t i o n a t -15°C.  p r e p a r a t i o n s were d e g r a d e d U l t r a v i o l e t absorbing  a p p e a r e d i n t h e f r a c t i o n s e l u t e d by t h e column e q u i l i b r a n t . was l o s t f r o m t h e e l u t e d f r a c t i o n s on d i a l y s i s .  material  Radioactivity  Furthermore, the i n d i v i -  d u a l s m a l l f r a c t i o n s were h e t e r o g e n e o u s a c c o r d i n g t o r e s u l t s o b t a i n e d by g e l f i l t r a t i o n on Sephadex. In some e x p e r i m e n t s t h e DNA was i s o l a t e d f r o m t h e i n t e s t i n a l mucosa o f r a t s 3  w h i c h had r e c e i v e d H - t h y m i d i n e  5, 10 o r 20 m i n u t e s o r 24 h o u r s .  was f r a c t i o n a t e d on E C T E O L A - c e l l u l o s e ,  When DNA  and t h e f r a c t i o n s were a s s a y e d f o r  r a d i o a c t i v i t y , t h e r e s u l t s o b t a i n e d were i n c o n c l u s i v e b e c a u s e t h e d i s t r i b u t i o n of r a d i o a c t i v i t y i n the f r a c t i o n s v a r i e d from experiment t o experiment.  No  d e f i n i t e p a t t e r n o f r a d i o a c t i v i t y was o b t a i n e d i n t h e d i f f e r e n t f r a c t i o n s , i n d i c a t i n g an i n c r e a s i n g o r d e c r e a s i n g s p e c i f i c a c t i v i t y .  Furthermore, the pat-  t e r n o f d i s t r i b u t i o n o f r a d i o a c t i v i t y was n o t s i g n i f i c a n t l y d i f f e r e n t i n between most o f t h e s h o r t e x p o s u r e t i m e e x p e r i m e n t ^ experiments.  and t h e l o n g e r 24 h o u r s i n c o r p o r a t i o n  137 The s e c o n d method o f f r a c t i o n a t i o n o f DNA p r e p a r a t i o n s f r o m r a t i n t e s t i n a l mucosa employed  m e t h y l a t e d a l b u m i n - k i e s e l g u h r (MAK) c o l u m n s .  W i t h t h i s method  o f f r a c t i o n a t i o n , r e p r o d u c i b l e e l u t i o n p a t t e r n s were o b t a i n e d even a f t e r age o f t h e DNA s o l u t i o n s .  Fewer f r a c t i o n s were o b t a i n e d u s i n g MAK  t h a n were o b t a i n e d u s i n g E C T E O L A - c e l l u l o s e .  p h i c p r o f i l e s on MAK.  columns  The main DNA peak was a l w a y s  e l u t e d a t t h e same r a n g 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 . t a i n e d by two methods o f p r e p a r a t i o n  stor-  (147,159)  DNA samples o b -  gave v e r y s i m i l a r  chromatogra-  H e t e r o g e n e i t y o f t h e main DNA peak e l u t e d by an i n i t i a l  sodium c h l o r i d e c o n c e n t r a t i o n o f 0.66Mwas s u g g e s t e d i n some e x p e r i m e n t s , i n w h i c h t h e components o f t h e s e c o n d l a y e r o f t h e column were i n c r e a s e d . Chromatography  o f DNA on t h e s e m o d i f i e d c o l u m n s i n d i c a t e d t h a t f u r t h e r  frac-  t i o n a t i o n o f t h e main DNA component m i g h t be a c c o m p l i s h e d . As a more s e n s i t i v e means t o w a r d s d e t e c t i n g m e t a b o l i c d i f f e r e n c e s amongst t h e components i s o l a t e d f r o m t h e DNA o f i n t e s t i n a l mucosa, and t o d i s t i n g u i s h s t a b l e DNA f r o m n e w l y formed m a t e r i a l , d o u b l e l a b e l l i n g e x p e r i m e n t s were c a r r i e d 3  out.  R a t s were f i r s t g i v e n H - t h y m i d i n e and 24 h o u r s l a t e r were i n j e c t e d  with  14 C  -thymidine.  Twenty o r f o r t y m i n u t e s l a t e r t h e a n i m a l s were k i l l e d  d o u b l e l a b e l l e d DNA was i s o l a t e d f r o m t h e i n t e s t i n a l mucosa.  and t h e  By s t u d y i n g t h e  3 . 14 H /C  r a t i o s o f t h e f r a c t i o n s o b t a i n e d f r o m MAK, newly s y n t h e s i z e d  c o u l d be compared w i t h o l d e r , p r e s u m a b l y s t a b i l i z e d DNA.  material  F o r t h i s purpose i t  was n e c e s s a r y t o a d a p t l i q u i d s c i n t i l l a t i o n c o u n t i n g methods f o r t h e d e t e r m i n a tion of  and H ^ - a c t i v i t i e s i n t h e d o u b l e l a b e l l e d DNA  samples.  I t was f o u n d t h a t 95 - 97 p e r c e n t o f t h e r a d i o a c t i v i t y was e l u t e d i n t h e main DNA peak.  B e c a u s e o f t h i s , each s u b f r a c t i o n c o m p r i s i n g t h e main peak was e x 14 amined. I n t h e e x p e r i m e n t s , when t h e a n i m a l s were e x p o s e d t o t h e C -labelled 3 , 14 t h y m i d i n e f o r 40 m i n u t e s , t h e H /C r a t i o s were c o n s t a n t . However, when t h e 14 time o f exposure t o t h e C H^/C^  -4  - l a b e l l e d p r e c u r s o r i n v i v o was 20 m i n u t e s , t h e  r a t i o s o f t h e s u b f r a c t i o n s i n c r e a s e d a s t h e sodium c h l o r i d e  concentration  138 of the eluant  increased.  amongst t h e s e f r a c t i o n s . and 8.  r e p l i c a t i o n o f DNA  These r e s u l t s i n d i c a t e d some m e t a b o l i c  The r e l a t i o n o f t h e s e f i n d i n g s t o t h e b i o s y n t h e s i s  i n t h e i n t e s t i n a l mucosa o f r a t has been  A n a l y s i s of the products of the stepwise enzymatic degradation l a b e l l e d DNA  differences  discussed. of the double  by p u r i f i e d s n a k e venom d i e s t e r a s e showed t h a t t h e r e l e a s e o f  r a d i o a c t i v i t y into the acid-soluble material followed that of the u l t r a v i o l e t absorbing  3 14 s u b s t a n c e , whereas t h e H /C r a t i o i n the a c i d - i n s o l u b l e f r a c t i o n s  remained c o n s t a n t .  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