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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 C a t h e r i n e Mezei D i p l . Pharm*, U n i v e r s i t y of M e d i c a l S c i e n c e s , Budapest 1954 M.Sc, U n i v e r s i t y o f B r i t i s h Columbia, 1960 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of B i o c h e m i s t r y We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d f o r the 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 Columbia A p r i l 1964 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of • B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission-Department of Biochemistry The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada Date May 7, 1964. The University of 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 D i p l . Pharm. University of Medical Sciences . M.Sc, The University of B r i t i s h Columbia, 1960 THURSDAY, MAY 7, 1964, at 9:30 A.M. IN ROOM 231, MEDICAL SCIENCES BUILDING, BLOCK A \ of CATHERINE MEZEI Budapest, Hungary, 1954 COMMITTEE IN CHARGE Chairman: F.H. Soward J.J.R. Campbell M. Darrach P.H. J e l l i n c k A.W. Matthews Ross Stewart G.M. Tener S.H.' Zbarsky External Examiner: R.O. Hurst Department of Biochemistry Queen's University THE BIOSYNTHESIS OF DEOXYRIBONUCLEIC ACID IN VIVO IN THE INTESTINAL MUCOSA OF RAT ABSTRACT The i n vivo biosynthesis of deoxyribonucleic acid (DNA) from l a b e l l e d thymidine has been investigated i n rat i n t e s t i n a l mucosa. The DNA preparations were fra c -tionated 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 counting methods. In the f i r s t experiments the DNA was i s o l a t e d from the i n t e s t i n a l mucosa of rats which had received H3-thymidine 5, 10 or 20 minutes or 24 hours before sac-r i f i c e . When the macromolecules were fractionated on ECTEOLA-cellulose the re s u l t s obtained were incon-c l u s i v e because no d e f i n i t s pattern of incorporation of r a d i o a c t i v i t y was observed i n the f r a c t i o n s . Chromatography on ECTEOLA-cellulose was considered unsatisfactory, because of the v a r i a t i o n s i n the e l u t i o n patterns of DNA preparations from, experiment to experi-ment and evidence i n d i c a t i n g degradation of DNA during the f r a c t i o n a t i o n procedure. In subsequent experiments f r a c t i o n a t i o n on methylated albumin-kieselguhr (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 injected intravenously with H 3-thymidine and 24 hours l a t e r with C^-thymidine. The rats were k i l l e d 20 or 40 minutes a f t e r the 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 the 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 reprodu-c i b l e e l u t i o n patterns were obtained even a f t e r storage of the DNA solutions. The main DNA peak was always eluted at the same range of sodium chloride concentra-t i o n and 95-97 percent of the r a d i o a c t i v i t y was eluted i n t h i s peak. Each subfraction comprising the.main peak was examined for H 3 and C ^ a c t i v i t y . By studying the H^/cl^ r a t i o s of the fra c t i o n s newly synthesized material could be compared with older, presumably sta-b i l i z e d DNA. When the animals were exposed to the CL^-l a b e l l e d thymidine for 40 minutes the H 3/C 1^ r a t i o s of the subfractions were constant, i n d i c a t i n g no metabolic differences between the newly synthesized DNA ( C ^ -l a b e l l e d and the. "o l d " (H 3-labelled) DNA. However, when the time of exposure to the C ^ - l a b e l l e d -precursor • i h vivo was 20 minutes, the '&?/G^ r a t i o s of subtrac-tions increased as the sodium chloride concentration of the eluant increased. These r e s u l t s indicated some metabolic differences amongst these f r a c t i o n s . Stepwise enzymatic degradation by snake venom pho-sphodiesterase of the double l a b e l l e d DNA preparations, and the main peak obtained a f t e r MAK chromatography, indicated the incorporation of thymidine into newly synthesized and " o l d " DNA occurred well within the chain. GRADUATE STUDIES F i e l d of Study: Biochemistry Endocrinology Enzymol. ogy Intermediary Metabolism Molecular Structure and B i o l o g i c a l Function V.J. O'Donnell W.J. Polglase Staff of the Depart-ment of Biochemistry Staff of the Depart-ment of Biochemistry Related Studies: Advanced Mammalian Physiology Immunochemistry Introduction to Viruses Neurochemistry Staff of the Depart-ment of Physiology D.C.B. Duff J.E. Bismanis P.L. McGeer PUBLICATION A Comparison of Methods for the I s o l a t i o n of Deoxyri-bonucleic Acid from Small Amounts of Rat Tissue. C. Mezei and S. H. Zbarsky. Can. J . Biochem. & Physiol. 40, 1167 (1962). i ABSTRACT The ir> v i v o b i o s y n t h e s i s of 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 t h ymidine 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. Because of the suggested p h y s i c o -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 of DNA o f mammalian o r i g i n , 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 thus o b t a i n e d 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 the f i r s t experiments the DNA was i s o l a t e d from the i n t e s t i n a l mucosa of 3 r a t s which had r e c e i v e d H - t h y m i d i n e 5, 10 o r 20 minutes o r 24 hours b e f o r e s a c r i f i c e . The method of e x t r a c t i o n of the DNA was based on the d i s i n t e g r a t i o n of t i s s u e s by hi g h frequency 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 of n u c l e o p r o t e i n from the 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 of DNA w i t h chloroform-amyl a l c o h o l m i x t u r e s . When the macromolecules were f r a c t i o n a t e d on ECTEOLA-cellulose, 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 , the 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 e p a t t e r n of r a d i o a c t i v i t y was observed i n the f r a c t i o n s . 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 experiment t o experiment. Because of these f i n d i n g s i t was d i f f i c u l t t o draw any c o n c l u s i o n s . Chromatography on ECTEOLA-cellulose 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 of the e l u t i o n p a t t e r n s of DNA p r e p a r a t i o n s were not r e p r o d u c i b l e from experiment t o ex-periment, the chromatographic p r o f i l e s changed on s t o r a g e of the DNA s o l u t i o n s , and because o f evidence i n d i c a t i n g d e g r a d a t i o n of DNA p r e p a r a t i o n s d u r i n g the f r a c t i o n a t i o n procedure. Because of the 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 methylated a l b u m i n - k i e s e l g u h r (MAK) columns was employed i n the subsequent experiments. A l s o , as a more s e n s i t i v e means towards 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 the components i s o l a t e d from the DNA o f i n t e s t i n a l mucosa, 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 3 14 H -thymidine and 24 hours 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 or 40 minutes a f t e r the 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 the i n t e s t i n a l mucosa by the phenol treatment 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 columns 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 ob-t a i n e d even a f t e r s t o r a g e o f the DNA s o l u t i o n s . The main DNA peak was always e l u t e d at the same range 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 found t h a t 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 the main DNA peak. Because of 3 14 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 the main peak was examined f o r H and C a c t i -3 14 v i t y . By s t u d y i n g the H /C r a t i o s o f the i 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 the animals were ex-14 3 14 posed t o the C - l a b e l l e d thymidine f o r 40 minutes the H /C - r a t i o s o f the sub-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 the newly s y n t h e s i z e d DNA ( C 1 4 - l a b e l l e d ) and the " o l d " ( H 3 - l a b e l l e d ) DNA. However, when the time o f exposure t o the C ^ - l a b e l l e d p r e c u r s o r in_ v i v o was 20 minutes, the H^/C^ r a t i o s o f the s u b f 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 o f 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 . The r e l a t i o n o f these f i n d i n g s t o the 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 the i n t e s t i n a l mucosa of r a t has been d i s c u s s e d . Stepwise enzymatic d e g r a d a t i o n by snake venom phosphodiesterase o f 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 chromato-graphy, i n d i c a t e d the i n c o r p o r a t i o n o f 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 . S. H. Zbarsky i i i ACKNOWLEDGEMENTS The author wishes t o express her s i n c e r e thanks and appre-c i a t i o n t o Dr. S. H. Zbarsky f o r h i s c o n t i n u a l a d v i c e and encourage-ment. The many h e l p f u l d i s c u s s i o n s w i t h Dr. G. M. Tener, Dr. P. H. J e l l i n c k and Dr. G. G. J a c o l i are g r e a t l y a p p r e c i a t e d . Thanks are due a l s o t o Dr. M. Smith o f the 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, f o r h i s k i n d c o -o p e r a t i o n i n the i n i t i a l s tages o f the MAK chromatography. Dr. I . A. S r e t e r of the Department o f Anatomy, U n i v e r s i t y o f B r i t i s h Columbia, and Dr. Wm. L. Dunn k i n d l y performed i n t r a v e n o u s i n j e c 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 s a t v a r i o u s t i m e s . Thanks are due a l s o t o Mrs. B. E. Stewart and Miss B. Ford f o r t h e i r 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 the work. I t i s a p l e a s u r e t o acknowledge the 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 the form of S t u d e n t s h i p s . i v 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 B i o s y n t h e s i s 1 The Conversion o f Thymidine t o Thymine N u c l e o t i d e s and DNA 3 The Enzymatic 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 of DNA S y n t h e s i s 9 The R e p l i c a t i o n of DNA 10 The P h y s i c a l and Chemical H e t e r o g e n e i t y o f DNA 17 The Chromatography of DNA on S u b s t i t u t e d C e l l u l o s e -(ECTEOLA) Columns 18 The Chromatography of DNA 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 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 o f DNA 23 The Pr e s e n t I n v e s t i g a t i o n 27 EXPERIMENTAL I . M a t e r i a l s 30 1. E x p e r i m e n t a l animals 30 i 2. R a d i o a c t i v e m a t e r i a l s 30 I I . P r e p a r a t i o n o f DNA from S m a l l I n t e s t i n a l Mucosa of Rat 30 1. Method of Medawar and Zubay (159) 30 2. .Method of C o l t e r e t a l . (147) 32 I I I . F r a c t i o n a t i o n o f the DNA P r e p a r a t i o n s 33 1. F r a c t i o n a t i o n on ECTEOLA-cellulose exchanger 33 V Page 2. F r a c t i o n a t i o n on MAK columns 34 3. D e s a l t i n g o f the DNA f r a c t i o n s a f t e r Chromatography .. 38 G e l f i l t r a t i o n 40 D i a l y s i s 41 IV. Enzymatic Degradation of DNA 42 1. The p u r i f i c a t i o n o f snake venom phosphodiesterase from the crude venom 42 2. Test f o r Deoxyribonuclease a c t i v i t y i n the snake venom phosphodiesterase p r e p a r a t i o n s 44 3. Assay o f the snake venom phosphodiesterase a c t i v i t y . . . 45 4. Stepwise d e g r a d a t i o n o f DNA by the p u r i f i e d snake venom phosphodiesterase 47 v. R a d i o a c t i v e Counting Procedures 49 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 49 2. 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 the double ( C 1 4 and H 3) l a b e l l e d DNA p r e p a r a t i o n s 52 3. The p u r i f i c a t i o n and c o u n t i n g o f the 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 and c o u n t i n g o f the phenol l a y e r s ... 69 5. The p u r i f i c a t i o n and c o u n t i n g of the a c i d - i n s o l u b l e m a t e r i a l o b t a i n e d d u r i n g the enzymatic d e g r a d a t i o n of DNA s o l u t i o n s 69 6. The p u r i f i c a t i o n and c o u n t i n g o f the a c i d - s o l u b l e f r a c t i o n o b t a i n e d d u r i n g the enzymatic d e g r a d a t i o n . of DNA s o l u t i o n s 71 RE5ULT5 AND DISCUSSION I . Experiments w i t h T r i t i u m L a b e l l e d DNA 73 1. Design o f the i n v i v o experiments and p r e p a r a t i o n of the t r i t i u m l a b e l l e d t i s s u e 73 v i Page 2. F r a c t i o n a t i o n on ECTEOLA-cellulose 74 3. Chromatography on Sephadex columns of the f r a c t i o n s o f DNA o b t a i n e d by ECTEOLA-cellulose chromatography 83 4. Removal of i n o r g a n i c c o n s t i t u e n t s from the DNA f r a c t i o n s by d i a l y s i s 87 3 5. Time-course of the i n c o r p o r a t i o n of H -thymidine i n t o the DNA of subcutaneously i n j e c t e d r a t s 89 6. E f f e c t o f the r o u t e of i n j e c t i o n on the s p e c i f i c a c t i v i t i e s o f the DNA p r e p a r a t i o n s 91 7. 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 the DNA f r a c t i o n s o b t a i n e d by ECTEOLA-cellulose chromatography 93 I I . Experiments w i t h DNA L a b e l l e d w i t h Both Carbon-14 and T r i t i u m 98 1. Design of the i n v i v o experiments and p r e p a r a t i o n of the double l a b e l l e d t i s s u e 98 2. The r e p r o d u c i b i l i t y o f the MAK f r a c t i o n a t i o n method 99 3. Comparison of the MAK chromatographic p r o f i l e s o f the DNA p r e p a r a t i o n s o b t a i n e d by the methods of Medawar and Zubay (159) and C o l t e r et a l . (147) from the same e x p e r i m e n t a l animals 106 4. The C"^ and e l u t i o n p r o f i l e s o f 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 comparison o f the H3/C 1 4 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 chromatography 113 I I I . Measurement of R a d i o a c t i v i t y i n the Protein-RNA I n t e r - phases of the T i s s u e E x t r a c t s Obtained by the Methods o f C o l t e r et a l . (147) and Medawar and Zubay (159) 126 IV. Enzymatic Degradation of the Double L a b e l l e d DNA P r e p a r a t i o n s 129 SUMMARY 136 BIBLIOGRAPHY 139 v i i TABLES Page I . E l u t i o n Schedule f o r DNA Chromatography on ECTEQLA-C e l l u l o s e Anion-Exchanger 35 I I . B u f f e r S o l u t i o n s f o r Chromatography on MAK 37 I I I . Composition of Standards C o n t a i n i n g the S i n g l e Isotope H o r C f Used f o r the D e t e r m i n a t i o n of the 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 IV. Composition of Standards C o n t a i n i n g the S i n g l e Isotope H 3 o r C-*-4, Used f o r the D e t e r m i n a t i o n of the 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 V. E f f e c t of 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 the T r i t i u m and Carbon 14 Assays of 4 D i f f e r e n t Standards 64-65 V I . Recovery of Counts i n DNA a f t e r TCA 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 72 V I I . Comparison o f the Percentage of the U l t r a v i o l e t Absor-b i n g M a t e r i a l i n the Main F r a c t i o n s Obtained from ECTEOLA-Chromatography of the DNA p r e p a r a t i o n s 84 3 V I I I . I n c o r p o r a t i o n of 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 92 IX. Comparison o f the Percentage of the R a d i o a c t i v i t y i n the Main F r a c t i o n s Obtained from ECTEOLA-Chromatography DNA P r e p a r a t i o n s 95 X. Comparison of F r a c t i o n a t i o n on MAK Column of A l i q u o t s of DNA P r e p a r a t i o n '.101 X I . Comparison o f the Percentage of the U l t r a v i o l e t Absorbing M a t e r i a l i n the F r a c t i o n s Obtained from MAK-Chromatography of C 1 4 and H 3 L a b e l l e d DNA P r e p a r a t i o n s 108 14 3 X I I . Comparison o f the % D i s t r i b u t i o n of C and H a c t i v i t i e s i n the Main and Minor F r a c t i o n s of DNAs E l u t B d from MAK Columns 119 3 14 X I I I . Comparison of the H /C A c t i v i t i e s i n the DNA F r a c t i o n s o b t a i n e d by MAK Chromatography 122 3 14 XIV. The R a t i o s of H /C i n the Interphase and Phenol Layers Obtained During the P r e p a r a t i o n o f DNA 128 v i i i FIGURES Page 1. P o s t u l a t e d mechanism f o r extending a DNA c h a i n 8 2. R e p l i c a t i o n of DNA, a c c o r d i n g t o the mechanism of Watson and C r i c k 12 3. 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 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 13 4. 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 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 of the h y p o t h e t i c a l s p i n n i n g mechanism 15 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 39 6. The e f f e c t of 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 46 7. Time course of the h y d r o l y s i s of p - n i t r o p h e n y l - t h y m i d i n e - 5 -phosphate by snake venom phosphodiesterase 48 B. 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 Table I I I determined on the red s c a l e r 55 9. 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 2 from Table I I I determined on the red s c a l e r 56 10. 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 5? I l l 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 Table I I I determined on the r e d s c a l e r 58 12. 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 red s c a l e r 59 13. 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 6 from Table IV determined on the r e d s c a l e r 60 14. 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 61 3 14 15. 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 s t a n d a r d samples and the r a t i o o f the counts o b t a i n e d on red t o green s c a l e r 68 16. F r a c t i o n a t i o n on ECTEOLA-cellulose of t r i t i a t e d DNA from the i n t e s t i n a l mucosa of r a t 75 I x 17. F r a c t i o n a t i o n on ECTEOLA-cellulose of 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 79 18. F r a c t i o n a t i o n on ECTEOLA-cellulose of 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 80 19. (A) 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 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 w i t h the p r e c u r s o r 10 min. p r i o r t o s a c r i f i c e 82 (B) Sample of an a l i q u o t o f the 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, but f r a c t i o n a t i o n performed 2 weeks l a t e r 82 20. Chromatography on 5ephadex of DNA f r a c t i o n s e l u t e d from ECTEOLA-cellulose 85 21. 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 chromatographed on ECTEOLA-cellulose column 88 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 3-thymidine 90 23. F r a c t i o n a t i o n o f DNA by chromatography on MAK 102 24. F r a c t i o n a t i o n o f DNA by chromatography on MAK 103 25. Chromatography of DNA on a m o d i f i e d MAK column 105 26. Chromatography of DNA on a m o d i f i e d MAK column 107 27. 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 method of C o l t e r et a l 110 28. Chromatography on MAK column of 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 method of C o l t e r et a l . . I l l 29. Chromatography on MAK column of DNA from i n t e s t i n a l mucosa of r a t prepared by method o f Medawar and Zubay 112 30. Chromatography on MAK column of 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 method of C o l t e r e t . a_l 115 31. Chromatography on MAK of 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 method of C o l t e r e_t a l 116 32. 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 chroma-tographed on MAK column 117 33. 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 chroma-tographed on MAK column 118 X 34. 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 123 35. Enzymatic de g r a d a t i o n of 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 method of C o l t e r e t a l . 132 36. 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 133 x i LIST OF ABBREVIATIONS DNA d e o x y r i b o n u l c e i c a c i d RNA r i b o n u c l e i c a c i d s-RNA s o l u b l e r i b o n u c l e i c a c i d DNase d e o x y r i b o n u c l e a s e TMP the 5'-phosphate of 2 ' - d e o x y r i b o s y l thymine TDP the 5' (pyro-) diphosphate of thymidine TTP the 5' (pyro-) t r i p h o s p h a t e of thymidine dGPPP the 5' (pyro-) t r i p h o s p h a t e of deoxyuanosine dAPPP the 5' (pyro-) t r i p h o s p h a t e of deoxyadenosine dCPPP the 5' (pyro-) t r i p h o s p h a t e of d e o x y c y t i d i n e PP pyrophosphate dAT a copolymer of deoxyadenylate and deoxythymidylate dGdC a polymer c o n s i t i n g of homopolymers of deoxyguanylate and d e o x y c y t i d y l a t e p o l y (AU) a polymer c o n s i s t i n g o f homopolymers of a d e n y l a t e and urid y l d - t e p o l y (dAT) a polymer c o n s i s t i n g of homopolymers of.deoxyadenylate and t h y m i d y l a t e MAK methylated albumin k i e s e l g u h r column f o r chromatography mc m i l l i c u r i e g microgram m micromole m m i l l i m i c r o n Cpm counts per minute Bg background counts per minute TCA t r i c h l o r o - a c e t i c a c i d 1 INTRODUCTION The E a r l y I n v e s t i g a t i o n s of DNA B i o s y n t h e s i s Although 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 t o the c o n c l u s i o n 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 from simple substances, they gave no i n d i c a t i o n of m a t e r i a l s used o r the mechanisms i n v o l v e d . The major imp-etus f o r the development of our present knowledge was p r o v i d e d by the use of i s o t o p e s as t r a c e r s i n b i o l o g i c a l experiments. In 1940 Hevesy (1) demonstrated t h a t i n o r g a n i c 32 phosphate l a b e l l e d w i t h P was i n c o r p o r a t e d r a p i d l y i n t o the DNA when a d m i n i s t e r e d 15 i n v i v o t o a n i m a l s . Using N l a b e l l e d ammonium c i t r a t e , Barnes and Schoenheimer (2) showed t h a t ammonia i s a p r e c u r s o r of n u c l e i c a c i d p u r i n e s and p y r i m i d i n e s i n the r a t and the pigeon. Other i n v e s t i g a t i o n s (3) have demonstrated t h a t many simple substances, as w e l l as n u c l e i c a c i d bases, n u c l e o s i d e s and n u c l e o t i d e s can be u t i l i z e d i n the b i o s y n t h e s i s of 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 of 32 P i n t o the DNA: those such as bone marrow, s p l e e n , thymus, and i n t e s t i n a l mucosa, which may be regarded as p r o l i f e r a t i n g t i s s u e s i n which the uptake of i s o t o p e i s ; h i g h , and the remainder t y p i f i e d by l i v e r , kidney and b r a i n , i n which i s o t o p e content i s s m a l l and c e l l d i v i s i o n minimal i n the normal a n i m a l . I t has been found (4,5) t h a t t h e r e i s a decrease w i t h age i n the r a t e of f o r m a t i o n of l a b e l l e d DNA i n thymus, 32 lymph nodes and s p l e e n , and the uptake of P by the DNA of weanling r a t l i v e r i s more r a p i d than i n the a d u l t animal ( 6 ) . T h e r e f o r e , i t i s g e n e r a l l y accepted t h a t 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 DNA o c c u r s as a r e s u l t of the s y n t h e s i s o f a d d i t i o n a l DNA which accompanies m i t o s i s . N u c l e i c a c i d s y n t h e s i s i n r e g e n e r a t i n g l i v e r was f i r s t s t u d i e d w i t h t r a c e r t e c h n i q u e s by Brues e t a l . (7) who found t h a t r a d i o a c t i v e phosphate was i n c o r p o r a t e d e x t e n s i v e l y i n t o the DNA and t h i s l a b e l was r e t a i n e d f o r a c o n s i d e r a b l e t i m e . 2 T h i s was c o n f i r m e d by s e v e r a l o t h e r workers (8-11) u s i n g d i f f e r e n t l a b e l l e d p r e -c u r s o r s . The b u l k of e x p e r i m e n t a l evidence i n d i c a t e d t h a t DNA i n n o n - d i v i d i n g c e l l s i s m e t a b o l i c a l l y s t a b l e . These o b s e r v a t i o n s s e t DNA a p a r t , s i n c e most c e l -l u l a r c o n s t i t u t e n t s , even RNA and p r o t e i n may i n c o r p o r a t e r e c e n t l y a d m i n i s t e r e d 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 . Attempts to e l u c i d a t e the f a t e of the phosphorus of DNA 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) but r e c e n t l y Ives and Barnum (17,18) demonstrated q u i t e c l e a r l y the m e t a b o l i c s t a b i l i t y o f DNA phosphorus d u r i n g the 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 . Moreover P o t t e r e t a l . (19-23) p o i n t e d out the 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 DNA i n v i v o and in_ v i t r o . As can be seen, the experiments 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 c o n c e r n i n g b i o s y n t h e t i c p r o c e s s e s . One has t o work on the 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 about the p o s s i b l e mechanisms i n v o l v e d i n these e v e n t s . 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 new n u c l e i c a c i d molecules 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 . The o v e r a l l p i c t u r e c o n c e r n i n g the b i o s y n t h e s i s of DNA can be o u t l i n e d as f o l l o w s : (a) the b i o s y n t h e s i s of p u r i n e and p y r i m i d i n e r i b o n u c l e o s i d e monophosphates, (b) the p h o s p h o r y l a t i o n o f these monophosphates to the diphosphate stage and the con-v e r s i o n of these r i b o n u c l e o t i d e s t o the c o r r e s p o n d i n g d e o x y r i b o n u c l e o t i d e s , (c) the p h o s p h o r y l a t i o n of the d e o x y r i b o n u c l e o s i d e diphosphates t o the t r i p h o s p h a t e stage and (d) the p o l y m e r i z a t i o n of the 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 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 the presence of an a p p r o p r i a t e DNA primer. S i n c e 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 b i o s y n t h e s i s a t the polymerase l e v e l , the f i r s t t h r e e s t e p s o u t l i n e d above are not d i s c u s s e d i n any d e t a i l . Because l a b e l l e d thymidine was used as a r a d i o a c t i v e p r e c u r s o r , the r e -l a t i o n s h i p between t h i s compound and DNA i s c o n s i d e r e d b r i e f l y i n the f a l l o w i n g s e c t i o n . 3 The Conversion o f Thymidine 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) the u t i l i z a t i o n o f the preformed b u i l d i n g b l a c k s ( e . g . bases, 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 these compounds are a v a i l a b l e , w h i l e the de novo, pathways w i l l be used o n l y when the bases are not a v a i l a b l e . Higher organisms have both jde novo and "preformed" pathways f o r both p u r i n e and p y r i m i d i n e b i o s y n t h e s i s present i n v a r y -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 extremely v i g o r o u s enzymes f o r b r e a k i n g down the 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 the l i v e r . Thymine i s a poor p r e c u r s o r and no data on i t s i n c o r p o r a t i o n i n t o DNA i n whole animal experiments have been r e p o r t e d , although thymine i s v i g o r o u s l y c o n v e r t e d t o thymidine r i b o s i d e i n the presence of u r i d i n e and deoxyur.idine ( 2 5 ) . The 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 low i n v i v o , w h i l e the 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 . However, the d e o x y n u c l e o s i d e , 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 t h y m i d y l i c a c i d (26-28) w i t h o u t under-going 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 o r o t h e r c e l l c on-s t i t u e n t s . The compound i s r a p i d l y c o n v e r t e d t o thymine, which i s c a t a b o l i z e d by the l i v e r w i t h i n 1 - 2 hours a f t e r i n j s e c t i o n ( 2 4 ) . During t h i s time i t i s a l s o c o n v e r t e d t o TMP by thymidine k i n a s e and thence t o TTP which i s u t i l i z e d f o r DNA s y n t h e s i s . 14 The p a t t e r n of p h o s p h o r y l a t i o n r e a c t i o n s i n . v i t r o o f C - l a b e l l e d t h y -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 ) . These workers used leukemic s p l e e n and lymph nodes of mice. 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 i s not an i n t e r -mediate i n the f o r m a t i o n of TTP, the l a t t e r compound being formed by pyrophosphate a d d i t i o n t o TMP. However, the o b s e r v a t i o n s of Bollum and P o t t e r (23) would be c o m p a t i b l e w i t h the sequence thymidine — » T M P — » T D P >TTP i f the l a s t s t e p o c c u r s so r a p i d l y , t h a t the c o n c e n t r a t i o n of TDP i s a t a l l times i n s u f f i c i e n t t o be d e t e c t e d . R e s u l t s o f f r a c t i o n a t i o n experiments on e x t r a c t s of E h r l i c h a s c i t e s c e l l s (30) have shown t h a t t h r e e enzymes are i n v o l v e d i n the f o r m a t i o n o f TTP 4 from t h y m i d i n e . I t has p r e v i o u s l y been shown t h a t TTP may be farmed from t h y -midine by p r e p a r a t i o n s from r e g e n e r a t i n g r a t l i v e r (23,31,32) which d i f f e r s from normal l i v e r i n p o s s e s s i n g the necessary k i n a s e s (32-34). Weissman e t a l (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 and TMP k i n a s e s appearing b e f o r e TDP k i n a s e . The i n c r e a s e and decrease i n the a c t i v i t y o f k i n a s e s i s c o i n c i d e n t w i t h the i n c r e a s e i n DNA s y n t h e s i s i n the r e g e n e r a t i n g l i v e r . A s i m i l a r f i n d i n g was observed i n c u l t u r e s of L c e l l s . Thus, the a v a i l a b i l i t y o f the d i s t i n c t k i n a s e s f o r conver-s i o n of thymidine t o TTP and the s e q u e n t i a l appearance of these enzymes d u r i n g 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 the p o s t u l a t e d scheme of Bollum and P o t t e r ( 2 3 ) . P o t t e r and Nygaard (35) t r i e d t o e s t a b l i s h whether t h i s r e a c t i o n sequence i s a l s o c o m p a t i b l e w i t h i n v i v o f i n d i n g s . As the r e s u l t of a very r a p i d e q u i l i b r a t i o n of r a d i o a c t i v i t y between TDP and TTP the m e t a b o l i c i n t e r r e l a t i o n -s h i p s of these two compounds c o u l d not be assessed from t h e i r data and, f u r t h e r -more from the p o i n t o f view of 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 e q u a l l y w e l l s a t i s f y the requirements f o r an immediate p r e c u r s o r o f DNA. T h e i r d a t a , t h e r e f o r e , permit no c o n c l u s i o n as t o the p o s s i b l e i d e n t i t y o f the mechanisms of DNA s y n t h e s i s from thymidine in_ v i v o and i n v i t r o . The Enzymatic S y n t h e s i s of DNA Since the p i o n e e r work of Kornberg and h i s c o l l e g u e s (36) the l a s t stage i n DNA b i o s y n t h e s i s , namely the 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 macrambla-c u l e , has a t t r a c t e d the a t t e n t i o n of many s c i e n t i s t s . The enzyme DNA polymerase, p u r i f i e d from E. c o l i (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 of DNA by a mechanism t h a t i n v o l v e s r e p l i c a t i o n of a DNA primer. A c c o r d i n g t o Kornberg (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 t r i p h o s p h a t e s commonly found i n DNA, and the DNA i t s e l f . The o v e r a l l r e a c t i o n can be summar-i z e d as f o l l o w s : 5 n TPPP n dGPPP n dAPPP n dCPPP enzyme TP dGP dAP dCP + DNA DNA + 4(n)PP n 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 the s y n t h e s i s o f a new DNA c h a i n i n response t o d i r e c t i o n s from a DNA t e m p l a t e ; these d i r e c t i o n s are d i c t a -t e d by the hydrogen-bonding r e l a t i o n s h i p o f adenine t o thymine and o f guanine t o c y t o s i n e . There are f i v e major l i n e s o f evidence t h a t support t h i s t h e s i s . o f the DNA produced by the enzyme. In the enzymatic r e a c t i o n 90 - 95% of the DNA sample comes from s u b s t r a t e s used i n the r e a c t i o n . The enzymatic product 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 weight double-stranded DNA i s o l a t e d from n a t u r a l sources ( 3 9 ) . 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 the neighborhood of 25 and reduced v i s -c o s i t y o f 40 d l / g w i t h an average m o l e c u l a r weight of 6 m i l l i o n . When the DNA i s heated, i t s r o d - l i k e shape c o l l a p s e s and the molecule becomes a compact, randomly c o i l e d s t r u c t u r e . The d i g e s t i o n products 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 are ver y s i m i l a r t o those o b t a i n e d when n a t u r a l DNA i s used.* analogs such as b r o m o u r a c i l and azaguanine can 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 expected t h a t some l a t i t u d e i n the s t r u c t u r e o f the bases can 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 hydrogen bondings. D e o x y u r i d i n e t r i p h o s p h a t e o r 5-bromodeoxyuridine t r i p h o s p h a t e supported DNA s y n t h e s i s when used i n p l a c e of thymidine t r i p h o s p h a t e but not when s u b s t i t u t e d f o r the t r i p h o s p h a t e s o f deoxyadenosine, deoxyguanosine o r d e o x y c y t i d i n e . I t has a l s o been noted (42) t h a t 5-methyl and 5-bromocytosine s p e c i f i c a l l y r e p l a c e d 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. These f i n d i n g are b e s t i n t e r p r e t e d on the b a s i s o f hydrogen bonding o f the adenine-thymine and g u a n i n e - c y t o s i n e t y p e . * Unpublished evidence based 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 behaviour suggests newly s y n t h e s i z e d DNA d i f f e r s from the n a t u r a l m a t e r i a l . The f i r s t l i n e of evidence i s d e r i v e d from s t u d i e s o f the p h y s i c a l nature Second, on the 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 6 The t h i r d l i n e o f evidence comes from a n a l y s i s of the base r a t i o s of the .enzymatically s y n t h e s i z e d DNA. In experiments u s i n g DNA from d i f f e r e n t sources as p r i m e r s , the e n z y m a t i c a l l y s y n t h e s i z e d product r e p l i c a t e d r a t h e r f a i t h f u l l y the base c o m p o s i t i o n of p r i m e r s ( 4 3 ) . The f o u r t h type of evidence i s p r o v i d e d by the experiments of Josse e_t a l ( 4 4 ) . He d e s c r i b e d a technique which e s t a b l i s h e d t h a t DNA from a g i v e n source d i r e c t s the s y n t h e s i s of a product i n which the f o u r bases occur next to one another i n the 16 p o s s i b l e arrangements not at random but i n a p a t t e r n of f r e q u e n c i e s unique f o r t h a t DNA. Moreover, the e n z y m a t i c a l l y s y n t h e z i d e d DNA showed comple-mentary p a i r i n g of adenine t o thymine and guanine t o c y t o s i n e between the s t r a n d s o f o p p o s i t e p o l a r i t y , as proposed by the Watson and C r i c k model (4 5 ) . F i n a l l y , DNA s y n t h e s i s r e q u i r e s the DNA primer and a l l f o u r d e o x y r i b o n u c l e o -s i d e t r i p h o s p h a t e s . Without added DNA t h e r e i s no template f o r hydrogen bonding and w i t h o u t 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 stops e a r l y and a b r u p t l y because o f l a c k of a hydrogen-bonding mate f o r one o f the bases i n the template ( 3 6 ) . As f a r as the nature of the primer DNA i s concerned, i t has been demonstra-t e d t h a t heat denatured DNA can s e r v e as an e x c e l l e n t primer ( 4 6 ) . Sinsheimer has i s o l a t e d from CpX 174 v i r u s a s i n g l e s t r a n d e d DNA (47) and i t i s a l s o an e x c e l l e n t primer f o r the polymerase system ( 4 4 ) . I t s h o u l d be mentioned, t h a t the DNA i n the s i n g l e s t r a n d e d 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 form when the most p u r i f i e d enzyme p r e p a r a t i o n s are used (36,48). With such p r e -p a r a t i o n s the n a t i v e , double str a n d e d DNA i s i n e r t u n l e s s i t i s heated or p r e -t r e a t e d s l i g h t l y w i t h d e o x y r i b o n u c l e a s e . R e c e n t l y Richardson e_t a l (49) d e s c r i b e d the f r a c t i o n a t i o n o f E. c o l i polymerase on h y d r o x y l a p a t i t e . On chromatography 2 peaks were o b t a i n e d , one major peak, which c o n t a i n e d 90% of the added polymerase a c t i v i t y , and a minor peak. T h i s l a t t e r component i n c r e a s e s the priming a c t i v i t y o f n a t i v e DNA by 2 t o 10 f o l d . The new component has been shown t o be a phosphatase which s p l i t s 3'-phosphoryl e s t e r s a t the ends of DNA c h a i n s and thereby i n c r e a s e s the priming a c t i v i t y o f n a t i v e DNA. T h i s enzyme might be a very important com-ponent i n c o n t r o l l i n g DNA b i o s y n t h e s i s i n v i v o and t r a n s f o r m i n g n a t i v e non-primer DNA i n t o the primer s t a t e . 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 of the d e o x y r i b o n u c l e o t i d e s u b s t r a t e s i s l a c k i n g an extremely s m a l l y e t s i g n i f i c a n t q u a n t i t y of n u c l e o t i d e i s l i n k e d t o the DNA p r i m e r . T h i s s y n t h e s i s was c a l l e d the " l i m i t e d r e a c t i o n " , and under these c i r c u m s t a n c e s a few d e o x y r i b o n u c l e o t i d e s are added t o the n u c l e o -s i d e ends of some of the DNA 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 b l o c k e d f o r l a c k of the m i s s i n g n u c l e o t i d e . A p o s t u l a t e d mechanism f o r extending the DNA c h a i n i s shown i n F i g u r e 1. In t h i s mechanism the d e o x y r i b o c l e o s i d e 5 ' - t r i p h o s p h a t e i s a t t a c k e d by the 3'-hydroxyl group at the growing end of a p o l y d e o x y n u c l e o t i d e c h a i n i n o r g a n i c pyrophosphate i s e l i m i n a t e d , and the c h a i n i s lengthened by one u n i t . Krakow _et _al (51-53) r e p o r t e d the p u r i f i c a t i o n of an enzyme f r a c t i o n from c a l f thymus nue 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 of d e o x y r i b o n u c l e o t i d e s and 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 of DNA. The enzyme appears to be 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^ + and DNA, but i n c o n t r a s t t o polymerase, the r e a c t i o n i s i n h i b i t e d by the presence of a f u l l complement of the d e o x y r i b o n u c l e o s i d e t r i p h o s p h a t e s ; o p t i m a l i n c o r p o r a t i o n proceeds when o n l y one d e o x y r i b o n u c l e o s i d e t r i p h o s p h a t e i s present d u r i n g 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 the l i m i t e d r e a c t i o n y mediatedcby polymerase and may a l s o p l a y a r o l e i n the s y n t h e s i s of DNA. I t was suggested (36) t h a t these r e a c t i o n s r e p r e s e n t 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 r e a c t i o n s have been d i s c o v e r e d (54-56) i n which polymer s y n t h e s i s o c c u r s i n the absence of added DNA a f t e r l a g p e r i o d s . In one case the product i s a copolymer, composed e x c l u s i v e l y of deoxyadenylate and deoxythymidylate i n ex-a c t l y a l t e r n a t i n g sequence. In the second case, the product c o n t a i n s o n l y 8 Figure 1. Postulated mechanism for extending a DNA chain. (According to r e f . ( 3 6 ) ) . 9 deoxyguanylate and d e o x y c y t i d y l a t e arranged as hydrogen bonded homopolymers. When e i t h e r polymer, dAT or dGdC i s i s o l a t e d and used as a primer f o r the 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 of primer added, and e x t e n s i v e s y n t h e s i s of an i d e n t i c a l polymer o c c u r s . Thus Kornberg e t a l (54-56) concluded, t h a t the polymers are s y n t h e s i z e d de novo d u r i n g the l a g p e r i o d and t h a t the f i r s t few molecules of polymers serve as a primer f o r r e p l i c a t i o n . A d d i t i o n a l experiments having a b e a r i n g on the b i o l o g i c a l s i g n i f i c a n c e of DNA polymerase have been r e p o r t e d . Litman 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 polymerase i n the presence of d e n s i t y - l a b e l l e d denatured t r a n s f o r m i n g DNA primer c a t a l y z e d the s y n t h e s i s o f p r i m e r - f r e e b i o l o g i c a l l y a c t i v e m o l e c u l e s . The C e l l u l a r L o c a t i o n of DNA S y n t h e s i s . In mammalian t i s s u e s DNA polymerase a c t i v i t y can be r e a d i l y demonstrated i n the supernatant 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 of d i s r u p t e d c e l l suspensions (22,59-61). T h i s f i n d i n g was unexpected, p a r t i c u l a r l y when i t was found t h a t h i g h a c t i v i t i e s o f the enzyme c o u l d not be demonstrated i n the 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 . I t was spec-u l a t e d ( 6 2 ) , t h a t the enzyme was so h i g h l y s o l u b l e , t h a t most of i t was l e a c h e d out i n t o the e x t r a c t i o n medium from the n u c l e i and/or cytoplasm under the e x t r a -c t i o n c o n d i t i o n s used. A c c o r d i n g l y , when n u c l e i of 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 prepared i n nonaqueous medium by m o d i f i c a t i o n s o f the Behrens procedure (63), they c o n t a i n e d an a c t i v e polymerase (62,64-66). The presence of polymerase i n n u c l e i i s o l a t e d from sea u r c h i n embryos was a l s o d e s c r i b e d by Mazia and Hine-gardner ( 6 7 ) . The exact l o c a t i o n o f the s i t e o f DNA b i o s y n t h e s i s i n the c e l l i s u n s e t t l e d at the p r e s e n t . Smith and K e i r (62) tend t o b e l i e v e t h a t t h e r e i s an equal d i s t r i b u t i o n o f the polymerase between nu c l e u s and cytoplasm of the thymus c e l l . The q u e s t i o n then a r i s e s : What i s the f u n c t i o n of the c y t o p l a s m i c p o l y -merase? I f the t o t a l polymerase were c o n c e n t r a t e d i n the v i c i n i t y o f the n u c l e a r 10 membrane, o u t s i d e and 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 would then be a v a i l a b l e f o r the e x i s t i n g knowledge of the l o c a t i o n and s y n t h e s i s of DNA i n v i v o , f o r i t i s not d i f f i c u l t t o imagine a process whereby s u i t a b l y s m a l l and d i f f u s i b l e u n i t s of DNA might be r e p l i c a t e d a t the n u c l e a r membrane by the c o n c e r t e d a c t i o n o f n u c l e a r and c y t o p l a s m i c polymerase. In t h i s c o n n e c t i o n , i t i s of i n t e r e s t t h a t B e r n a r d i and Sadron (68) have d i s c o v e r e d a DNase from c h i c k e n e r y t h r o c y t e s which degrades 5 DNA t o double h e l i c a l s u b - u n i t s of m o l e c u l a r weight 5-6x10 ; f u r t h e r , the c a p a c i t y t o prime the polymerase r e a c t i o n i s c o n f e r r e d on DNA s o l u t i o n s a f t e r exposure t o m i l d l y a l k a l i n e pH v a l u e s . I t appears t h e r e f o r e t h a t t h e r e are mechanisms a v a i l -a b l e f o r the c o n v e r s i o n of DNA 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 enzymic a c t i o n and pH r e g u l a t i o n . The more complex o p e r a t i o n of reassembly of the massive chromosomal apparatus c o u l d then be c o n f i n e d t o the n u c l e u s and thus the con-c e p t u a l problems of passage o f very 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 the n u c l e a r membrane would no l o n g e r e x i s t ( 6 2 ) . The R e p l i c a t i o n of DNA In s p i t e of the advances i n our knowledge i n DNA r e p l i c a t i o n t h e r e i s s t i l l c o n s i d e r a b l e c o n t r o v e r s y . S e v e r a l schemes f o r the r e p l i c a t i o n of DNA molecule have been proposed ( 6 9 ) : (a) c o n s e r v a t i v e , the parent molecule i s p r e -served i n t a c t and one e n t i r e l y newly-formed daughter molecule i s added, (b) semi-c o n s e r v a t i v e , each molecule c o n s i s t s of one o l d and one new h a l f , (c) d i s p e r s i v e , p a r e n t a l and o f f s p r i n g components form p a r t of a l l m o l e c u l e s . The complementary arrangement of p u r i n e and p y r i m i d i n e bases o f the two p o l y n u c l e o t i d e c h a i n s of DNA suggested t o Watson and C r i c k (70) t h a t DNA might r e p l i c a t e i t s e l f d i r e c t l y by having each c h a i n of DNA serve as a template f o r the f o r m a t i o n o f i t s complementary c h a i n . They h y p o t h e s i z e d t h a t the two s t r a n d s of the o r i g i n a l double h e l i x s e p a r a t e , and each p u r i n e and 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 hydrogen bonds a complementary 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 . These f r e e n u c l e o t i d e s i n the form of d e o x y r i b o n u c l e o t i d e - 5 ' - t r i p h o s p h a t e s a l r e a d y possess the necessary f r e e energy f o r p o l y e s t e r i f i c a t i o n and are j o i n e d t o one another by the DNA polymerase enzyme ( 3 6 ) , a f t e r having been h e l d i n p l a c e on the p a r e n t a l template c h a i n , t o form a new 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. Thus, a f t e r growth _de novo o f complementary r e p l i c a s has taken p l a c e along both 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 , two molecules are now present 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 and sequences, and hence i n f o r m a t i o n a l c o n t e n t , w i t h the p a r e n t a l double h e l i x . At t h i s p o i n t , one c y c l e of r e p l i c a t i o n i s complete. These events are r e p r e s e n t e d i n F i g u r e 2 ( 7 1 ) . The Watson-Crick r e p l i c a t i o n mechanism (70) makes an important p r e d i c t i o n 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 molecule over the r e p l i c a d u plexes, namely, t h a t the p a r e n t a l double h e l i x becomes e q u a l l y d i s t r i b u t e d over the two daughter molecules o f the f i r s t r e p l i c a t i o n c y c l e . In subsequent 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 of the o r i g i n a l p a r e n t a l DNA atoms s h o u l d o c c u r , s i n c e the 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 the p a r e n t a l DNA molecule romain i n t a c t . For i n s t a n c e , among the f o u r daughter molecules generated by the second r e p l i c a t i o n c y c l e , t h e r e are present two m olecules which each c o n t a i n one of the 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 molecules which c o n t a i n no p a r e n t a l substance. T h i s mode o f d i s t r i b u t i o n of the 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 , i n c o n t r a d i c t i o n t o a c o n s e r v a t i v e d i s t r i b u t i o n which e n t i r e l y conserves the i n t e g r i t y o f the p a r e n t a l DNA i n the r e p l i c a t i o n p r o c e s s . Under c o n s e r v a t i v e r e p l i c a t i o n , t h e r e i s among the daughter DNA molecules generated by one o r more r e p l i c a t i o n c y c l e s , always one i n d i v i d u a l whose atoms are e n t i r e l y p a r e n t a l w h i l e the s u b s t a n -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 novo. The s e m i - c o n s e r v a t i v e and con-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 d u r i n g two gen-e r a t i o n c y c l e s are r e p r e s e n t e d i n F i g u r e 3 ( 7 1 ) . 12 F i g u r e 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 t h e mechanism o f Watson and C r i c k . ( A c c o r d i n g t o | r e f . ( 7 1 ) ) . 13 Original parvnt moLtcuit First gerura.tt.ort. daughter molectU&s Figure 3. Semiconservative and conservative d i s t r i b u t i o n of the two parental polynucleotide chains as possible 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 of DNA. (According to r e f . ( 7 1 ) ) . 14 The main stu 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 Meselson and 5 t a h l ( 7 2 ) . By the use of t h e i r t e c h n i q u e 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 i u m s e d i m e n t a t i o n these workers proved t h a t the r e p l i c a -t i o n o f the DNA o f E. c o l i does indeed r e s u l t i n the s e m i - c o n s e r v a t i v e d i s t r i b u t i o n p r e d i c t e d by the mechanism of Watson and C r i c k (70); a f t e r E. c o l i which have been l a b e l l e d by growing i n a medium c o n t a i n i n g N^ ""\ are a l l o w e d t o grow f o r 2 more gen-14 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 the normal i s o t o p e N , h a l f o f the b a c t e r i a l DNA molecules are o f a d e n s i t y c o r r e s p o n d i n g t o the i s o t o p e r a t i o 50% N"^ and 50% N ^ w h i l e the o t h e r h a l f o f the molecules have a d e n s i t y c o r r e s p o n d i n g t o 100% N ^ . The M e s e l s o n - S t a h l experiment has been amply c o n f i r m e d u s i n g c e l l u l a r systems o t h e r than E. c o l i (73-7B). R e c e n t l y C a i r n s ( 7 9 ) demonstrated by the use o f a u t o -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 the c i r c u l a r chromosome o f E. c o l i r e p l i -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. He l a b e l l e d b a c t e r i a l DNA by growing the 3 organisms i n the presence o f H - t h y m i d i n e , and e x t r a c t e d the DNA by very g e n t l e methods t h a t a v o i d e d , as f a r as p o s s i b l e , t u r b u l a n c e and shear. E l e c t r o n m i c r o -graphs of 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 the b a c t e r i a l DNA was c i r c u l a r , and had a m o l e c u l a r weight o f about 2000 m i l l i o n , a l e n g t h o f about 1mm l o n g , and these dimensions corresponded t o the e n t i r e b a c t e r i a l chromosome. By t h i s t e c h -nique some of the DNA molecules were c a p t u r e d i n the r e p l i c a t i v e s t a g e , and showed t h a t d u p l i c a t i o n o f the molecule s t a r t e d at one p o i n t i n : t h e - c i r c l e , the 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 , the t e r m i n a l ends of the f o r k being j o i n e d t o g e t h e r . S i n c e the 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 than two d i v i s i o n c y c l e s , most of the p a r e n t a l DNA molecules 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 the r e p l i c a t i o n o f these h y b r i d m o l e c u l e s , t h e main body of the DNA molecule 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 the br a n c h i n g p a i n t ane o f the l i m b s was l a b e l -l e d i n both s t r a n d s , and the 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 . 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 o f a c i r c l e o f DNA i s presented i n F i g u r e 4. Because of o b j e c t i o n s c o n c e r n i n g the f e a s i b i l i t y o f unwinding the very l o n g DNA double h e l i c e s (80-82), a l t e r n a t i v e hypotheses have been suggested. Dounce has 15 Figure 4. A diagrammatic representation of the r e p l i c a t i o n of a c i r c l e of DNA. The black t r i a n g l e marks the p o s i t i o n of the hypothetical spinning mechanism. (According to r e f . ( 7 9 ) ) . 16 proposed a mechanism (83) i n which one s t r a n d o f the DNA double h e l i x i s c o p i e d e x a c t l y w h i l e the o t h e r s t r a n d i s prevented from f u n c t i o n i n g by some o t h e r f a c t o r , which may be h i s t o n e . B u t l e r (84) envisaged the DNA and RNA polymerase as a k i n d o f d i s k w i t h two h o l e s o r s l o t s each of which can accommodate one o f the s t r a n d s o f the primer DNA. The d i s k would f i r s t a t t a c h i t s e l f t o one end o f the DNA f i b r e W i t h the end of one s t r a n d i n each h o l e . The complementary n u c l e o t i d e f o r the g i v e n p o s i t i o n i n the sequence w i l l now take i t s p l a c e , and as these are a t t a c h e d one by one t o the growing c h a i n the d i s k w i l l move forward along the DNA f i b r e , r o t a t i n g as i t goes and t h e r e b y unwinding the double stranded h e l i x . T h i s a v o i d s the d i f f i c u l t i e s which have been suggested o f f i n d i n g a mechanism f o r unwinding the h e l i x . C a v a l i e r i and Rosenberg (85-88) suggested t h a t the conserved u n i t of DNA 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 o f double h e l i x . Each daughter c e l l , t h e r e f o r e , r e c e i v e s one o l d and one newly s y n t h e s i z e d double h e l i x , j o i n e d t o g e t h e r as a p a i r . These are c a l l e d " b i u n i a l m o l e c u l e s " , the bonds o f which are c o n s i d e r e d as a weak type of c o n n e c t i o n . Thus, r e p l i c a t i o n would not i n v o l v e s t r a n d s e p a r a t i o n . The mechanism may w e l l be s i m i l a r t o t h a t o c c u r i n g when messenger RNA i s s y n t h e s i z e d on an undenatured DNA template ( 8 9 ) . The e x p e r i m e n t a l evidence f o r the e x i s t e n c e of these b i u n i a l molecules- was o b t a i n -ed by means of enzyme k i n e t i c s (87) x-ray k i n e t i c s (90) and e l e c t r o n microscopy (9 1 ) . Recent r e s u l t s of C a v a l i e r i ' s group a l s o seem t o support t h i s h y p o t h e s i s . I t has been shown (92), t h a t DNA polymerase can u t i l i z e p o l y (AU) as a template f o r the s y n t h e s i s of homopolymeric p o l y (dAT). Zimmerman and Geiduschek (93) have found t h a t c r o s s l i n k e d DNA can serve as a primer f o r RNA and DNA i n DNA dependent systems. Thus, i t appears p o s s i b l e t h a t the bases of the double 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 " without s t r a n d s e p a r a t i o n . 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 which the c o n f i g u r a t i o n o f the template remains h e l i c a l d u r i n g the r e p l i c a t i o n of the DNA. 17 In o r d e r t o t e s t C a v a l i e r i ' s model, DNA samples of b a c t e r i a l o r i g i n have been s t u d i e d by x-ray s c a t t e r i n g t e c h n i q u e s ( 9 4 ) . The e x p e r i m e n t a l r e s u l t s are i n e x c e l l e n t agreement w i t h the Watson-Crick model (70), and f u r t h e r demonstrated t h a t the molecules are 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 s i d e a s s o c i a t i o n s . The r e s u l t s were i d e n t i c a l f o r both u n i t a r y and b i u n i a l mole-c u l e s and a p p a r e n t l y d i d not support C a v a l i e r i ' s s u g g e s t i o n s . The x-ray 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 experiments, however, on the assumption t h a t the two u n i t a r y molecules were l o o s e l y a s s o c i a t e d i n the b i u n n i a l m o l e c u l e , the average d i s t a n c e between them being l a r g e compared t o the diameter. The ex-periments o f Shooter and Baldwin (95) are a l s o i n apparent c o n t r a d i c t i o n t o the C a v a l i e r i model of DNA r e p l i c a t i o n . In a study of a l k a l i n e d e g r a d a t i o n o f heavy 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, these workers demonstrated t h a t a t the pH where heavy DNA f i r s t showed t h a t increment i n buoyant d e n s i t y c o r r e s p o n d i n g t o complete d e n a t u r a t i o n , the d e n s i t y o f the h y b r i d i n c r e a s e d by l e s s than o n e - f i f t h o f t h i s increment. I f the h y b r i d had been composed o f a b i u n i a l molecule (one heavy u n i t a r y double h e l i x and one l i g h t u n i t a r y double 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 should have appeared a t a pH at which the heavy u n i t a r y molecule denatured. The P h y s i c a l and Chemical H e t e r o g e n e i t y of DNA Whatever the a c t u a l mechanism o f DNA d u p l i c a t i o n may be, t h e r e i s s t i l l a p u z z l i n g problem i n b i o c h e m i c a l s t u d i e s of DNA p r e p a r a t i o n s . Although i t has been shown t h a t i n the 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 molecule o f DNA per i n f e c t i o u s p a r t i c l e (96,97) and the DNA can be e x t r a c t e d as molecules o f uniform l e n g t h and c o m p o s i t i o n , DNA p r e p a r a t i o n s o f animal and b a c t e r i a l o r i g i n c o n t a i n molecules of 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 . The techniques which have been em-p l o y e d t o demonstrate t h i s inhomogeneity are as f o l l o w s : d i f f e r e n t i a l c e n t r i f u g a -t i o n ( 9 8 ) , 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 d u r i n g t h e r m a l d e n a t u r a t i o n ( 9 9 ) , chromatography on v a r i o u s types o f columns, (100-103) 1 8 and through the use of 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 the l a t t e r v e r y m i l d t e c h n i q u e bimodal 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 from mouse (104,105) guinea p i g (104) o r marine c r a b s , such as Cancer b o r e a l i s and Cancer i c t i t a t u s (105). J o s h i et_ a l (106) d e s c r i b e d the presence 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) demonstrated the presence o f s a t e l l i t e bands t o the 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 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 i t 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 anion exchangers. S e v e r a l i n v e s t i g a t o r s (101-103) e x p l o r e d t h i s p o s s i b i l i t y , and the r e s u l t has been the a d a p t a t i o n o f column; chromatographic t e c h n i q u e s such as f r a c t i o n a t i o n on the s u b s t i t u t e d c e l l u l o s e exchanger, ECTEOLA-cellulose 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 (MAK) columns. The g e n e r a l p r o p e r t i e s and the 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 these two t e c h -niques are c o n s i d e r e d i n the f o l l o w i n g s e c t i o n . The Chromatography of DNA on S u b s t i t u t e d Cellulose-(ECTEOLA) Columns. ECTEOLA-cellulose was o r i g i n a l l y prepared by Peterson and Sober (111), who r e a c t e d sodium c e l l u l o s e w i t h a mi x t u r e 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 the 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 the o t h e r con-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 and c a p a c i t i e s can be o b t a i n e d (112-114). Bendich (112) compared the 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 exchangers t o DNA f r a c t i o n a t i o n and demonstrated t h a t ECTEOLA was the most s u i t a b l e f o r t h i s purpose. On the b a s i s o f s e d i m e n t a t i o n s t u d i e s (115,116) and l i g h t s c a t t e r i n g measurements (101) i t was concluded t h a t the f r a c t i o n a t i o n procedure 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 . Mononucleotides were not r e t a i n e d on the 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 hepta and o c t a n u c l e o t i d e s were e l u t e d by low s a l t c o n c e n t r a t i o n s (101). On the b a s i s o f these experiments Bendich concluded 19 (101) t h a t the DNA f r a c t i o n s e l u t e d a t NaCl c o n c e n t r a t i o n s h i g h e r than 0.2M would be l a r g e r than o c t a n u c l e o t i d e s . A c c o r d i n g t o Bendich (101) the f r a c t i o n s o b t a i n e d from 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 base c o m p o s i t i o n . I t i s f e l t however, t h a t these might 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 than f r a c t i o n a t i o n a c c o r d i n g t o the base c o n t e n t o f the DNA. An i n t e r e s t i n g f e a t u r e of the a n a l y t i c a l data i s the f a i l u r e i n many cases t o o b t a i n u n i t y r a t i o s f o r the adenine/thymine and g u a n i n e / c y t o s i n e p a i r s (101), and on the b a s i s of t h i s Bendich q u e s t i o n e d the adequacy of the 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 C r i c k (70) as a complete e x p l a n a t i o n f o r DNA r e d u p l i c a t i o n i n chromosomes. The ECTEOLA f r a c t i o n a t i o n t e chnique was r e p o r t e d t o g i v e r e p r o d u c i b l e chromatographic p r o f i l e s o f DNA 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 sources (101), and DNA o b t a i n e d from the same source (human g r a n u l o c y t e ) by two d i f f e r e n t p r o-cedures gave e s s e n t i a l l y the same p a t t e r n s (101). A c c o r d i n g t o Bendich e t a l (101) the chromatograms of DNA from d i f f e r e n t sources are d i f f e r e n t , and each o f the 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 the p r o f i l e : c a l f thymus, E. c o l i , . Wi c e r e u s , pneumoccus, T6r phage and T 6 r t phage. Large d i f f e r e n c e s were observed i n the p r o f i l e s o f DNAs d e r i v e d from d i f f e r e n t organs of the same s p e c i e s , e.g. r a t k i d ney, b r a i n and i n t e s t i n e . I t should be mentioned t h a t most o f the p r e v i o u s i n v e s t i g a t o r s concluded (117) t h a t DNA was 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 from d i f f e r e n t organs of a g i v e n s p e c i e s were i d e n t i c a l . Bendich c l a i m e d the r e s o l v i n g power of ECTEOLA-cellulose was shown t o be s a t i s f a c t o r y i n experiments w i t h normal E. c o l i and 5-bromouracil l a b e l l e d E. c o l i , and a l t e r a t i o n s i n the DNA from c a l f thymus following treatment w i t h mutagenic agents were a l s o r e f l e c t e d i n the chromatographic p r o f i l e s (101). In I960 d i Mayorca et a l (118) r e p o r t e d t h a t samples of normal and leukemic 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 chromatography. Moreover, the 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 e l u t e d w i t h lower s a l t c o n c e n t r a t i o n s was i n c r e a s e d i n case of DNA samples o b t a i n e d from leukemic p a t i e n t s a f t e r treatment 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 o b t a i n e d from the exchanger at n e u t r a l pH were found t o r e t a i n t h e i r i n t e g r i t y as was 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 and p r e c i p i t a t i o n w i t h e t h a n o l (119). R a i s i n g the pH of the f r a c t i o n s t o pH13 r e s u l t e d i n the p r o d u c t i o n o f the hyperchromic e f f e c t a t 260 m/A, which i s r e c o g -n i z e d as c h a r a c t e r i s t i c of n a t i v e DNA. B i o l o g i c a l a c t i v i t y was r e t a i n e d i n the 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 showed b i o l o g i c a l a c t i v i t i e s comparable o r 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 (101). I t was found (101) 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 put back upon the column, the major p o r t i o n of the m a t e r i a l was again e l u t e d a t p r e c i s e l y t h a t c o n c e n t r a t i o n of s o l u t e which i n i t i a l l y caused the f r a c t i o n t o be removed from the column. In 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 found both a t lower and h i g h e r c o n c e n t r a t i o n s o f s o l u t e . I f the main peak, from the f i r s t rechromatography ex-periment was again p l a c e d back on the column, the s@me be h a v i o r was observed. Bendich i n t e r p r e t e d these 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 the chromatogram was heterogeneous, and subsequent rechromatography o f such peak e f f e c t s f u r t h e r p u r i f i c a t i o n . On the b a s i s of the experiments c a r r i e d out by Bendich and co-workers, i t 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 the e x t e n t and n a t u r e o f h e t e r o g e n e i t y c o u l d p r o v i d e a b a s i s f o r demonstrating g e n e t i c d i f f e r e n c e s between t i s s u e s a t the chem-i c a l l e v e l . However, f u r t h e r i n v e s t i g a t i o n s of 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 . C o n t r a r y t o Bendich et a l , (101) Kondo and Qsawa (115), S. K i t (121-122) and Smith and Kaplan (125) have noted no d i f f e r e n c e s i n the chromato-gr a p h i c p r o f i l e s of DNA p r e p a r a t i o n s from malignant and normal t i s s u e s . The chromatographic e l u t i o n p a t t e r n s o f l u n g , thymus, kidney and l i v e r o f the mouse and kidney of the r a t , have a l s o been s t u d i e d (121-124) and no marked d i f f e r e n c e s s e r e observed. K i t f e l t t h a t these d i s c r e p a n c i e s were probably due t o d i f f e r e n c e s i n the procedures used f o r the p r e p a r a t i o n of DNA 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 the exchangers used by the 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 s c h e d u l e s . 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 experiments f o r the c r i t i c a l e v a l u a t i o n o f the number of f a c t o r s p e r t i n e n t t o the chromato-graphy 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 exchangers (112). Using 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 found t h a t so long as the same chromatographic procedures were v i g o r o u s l y adhered t o , the e l u t i o n p r o f i l e s were r e p r o d u c i b l e . The percent o f DNA e l u t e d by 0.6M sodium c h l o r i d e (which supposedly r e p r e s e n t s low m o l e c u l a r weight DNA s p e c i e s (101) ) depended on the f o l l o w i n g f a c t o r s : (a) The n i t r o g e n c o n t e n t o f the c e l l u l o s e exchanger. The lower the n i t r o g e n con-t e n t , the 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 procedure used i n p r e p a r i n g the DNA. When the DNA was prepared by the Hammarsten method (126) most of the DNA was e l u t e d i n the r e g i o n of 0.6M NaCl, whereas p r e p a r a t i o n s by the 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 (128) r e q u i r e d 2M NaCl 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 ex t e n t o f de o x y r i b o n u c l e a s e a c t i v i t y d u r i n g the p r e p a r a t i o n o f the t i s s u e . When a u t o l y s i s took p l a c e the amount o f DNA e l u t e d i n the r e g i o n o f low 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 which the t i s s u e was homogenized. When the t i s s u e was blended a t high speed the f r a c t i o n e l u t e d by 0.6M NaCl 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 the DNA. Heat denatured p r e p a r a t i o n s were e l u t e d mostly by low s a l t c o n c e n t r a t i o n . The same was found 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 the DNA p r e p a r a t i o n s . The chromato-g r a p h i c p r o f i l e s were not s i g n i f i c a n t l y m o d i f i e d when the DNA p r e p a r a t i o n s were t r e a t e d w i t h c h y m o t r y p s i n , o r when the p r o t e i n c o n t e n t of the p r e p a r a t i o n was i n -c r e a s e d . 22 On the b a s i s of the above f i n d i n g s , K i t suggested t h a t DNA e l u t e d by low s a l t c o n c e n t r a t i o n s from ECTEOLA-cellulose exchangers i s probably an e x p e r i m e n t a l a r t e f a c t . Other 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 the t i s s u e DNA i s p a r t l y degraded 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 very low m o l e c u l a r weight 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 c y c l e a n imal c e l l s c o n t a i n some s i n g l e stranded DNA, c o u l d not be excluded by these ex-periments. The Chromatography of DNA 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. In 1955 Lerman (102) i n t r o d u c e d the use of columns o f me t h y l a t e d albumin absorbed on k i e s e l g u h r f o r the f r a c t i o n a t i o n o f DNA. M a n d e l l and Hershey (129) developed t h i s method t o the p o i n t which p e r m i t t e d s e p a r a t i o n o f DNA and RNA by t h e i r m o l e c u l a r s i z e . These workers have shown (97) t h a t phage T2 DNA can be i s o l a t e d as r a t h e r uniform l a r g e m o l e c u l e s . Furthermore, by a p p l y i n g a c o n t r o l l e d s h e a r i n g t o the DNA they c o u l d i d e n t i f y fragments of h a l f and q u a r t e r the s i z e o f the o r i g i n a l molecules on the column. On the b a s i s of t h i s f i n d i n g , they c o n c l u d -ed t h a t the DNA of phage T2 e x i s t s i n the phage p a r t i c l e i n the form of one o r more molecules of i d e n t i c a l s i z e . The experiments of 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 the column, such as r e c o g n i t i o n o f base c o m p o s i t i o n and o f hydrogen bond c o n t e n t o f DNA. I t was found 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 e l u t e d at low&r s a l t c o n c e n t r a t i o n than i s n a t i v e DNA. Using t h i s method, a n a t u r a l l y e x i s t -i n g "deoxypolyadenine-thymine"in a marine c r a b Cancer b o r e a l i s was i s o l a t e d from the main DNA of the a n i m a l . 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 RNA, and i t was found t h a t i t not 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, but a l s o 16 S and 23 5 r i b o s o m a l RNA. Sueoka and Yamane (131) were a b l e t o r e s o l v e the amino-acyl-RNA o f d i f f e r e n t 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 the 16 amino-23 acyl-s RNAs examined. Using this technique, Hayashi ejt al;(132) could detect in RNA from induced c e l l s the presence of components not detectable in noninduced controls. It was also found by 5ueoka and 5piegelman (133) that, by the use of two isotopic labels to identify RNA synthesized during various periods i n the T2-E. c o l i system, the two RNAs were always easily distinguishable on MAK columns i f they were synthesized at different times and iden t i c a l i f they came from the same period. The molecular fate of DNA in genetic transformation of pneumococcus (139) was also followed by this technique. Fractionation on MAK showed that very soon after, or during i t s introduction into the c e l l , DNA i s converted partly into low-molecular weight fragments and partly into single stranded polynucleotides. A portion of this l a t t e r component was partly incorporated into the host DNA. The same technique was used by Pouwels et_ al-(135) who used chromatography on MAK to investigate the molecular fate of DNA of bacteriophage T4 after infection of E. co l i B . He was able to separate purified phage DNA from the host DNA and RNA, and showed that the host DNA was gradually degraded to oligonucleotides, whereas the phage DNA remained in double stranded form. Others (136,137) have carried out similar experiments. MAK chromatography proved to be in some respects even superior to cesium chloride density gradient centrifugation. For example, Hayashi et al.(13B) pre-pared adequate amount of the replicative form of <J>X 174 virus by employing chromatography on MAK. The DNA of bacteriophage SPB,, which when denatured, yields two components di f f e r i n g in buoyant density in cesium chloride gradients was separ-able into these components on an MAK column (139). The Metabolic Heterogeneity of DNA If the heterogeneity of DNA preparations i s indeed an in vivo phenomenon, one would automatically raise the question - What i s the bi o l o g i c a l significance of this compositional heterogeneity? During the la s t 10-15 years several i n v e s t i -gators-, attempted the demonstration of metabolic heterogeneity of DNA preparations. 24 One of the e a r l i e s t r e p o r t s d e s c r i b i n g a p r e f e r e n t i a l l a b e l l i n g o f the d i f f e r e n t DNA f r a c t i o n s o b t a i n e d from r a t t i s s u e s was t h a t of Bendich e_t a l . (140). These workers f r a c t i o n a t e d DNA of r a t s m a l l i n t e s t i n e , s p l e e n , pancreas, k i d n e y , b r a i n , t e s t i s and r e g e n e r a t i n g l i v e r i n t o two f r a c t i o n s , d i f f e r i n g i n s o l u b i l i t y i n 0.87 percent NaCl. The r a t i o of 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 found t o vary from organ t o organ. When the metabolism o f these DNA f r a c t i o n s was s t u d i e d 14 u s i n g C l a b e l l e d formate as the n u c l e i c a c i d p r e c u r s o r , i t was found t h a t the s p e c i f i c a c t i v i t i e s of the n u c l e i c a c i d bases i n the two f r a c t i o n s were d i s s i m i l a r . Large d i f f e r e n c e s c o u l d be seen i n the case of t e s t i s and k i d n e y , and not so s t r i k i n g d i f f e r e n c e s i n pancreas, spleen and s m a l l i n t e s t i n e . A d d i t i o n a l meta-b o l i c d i f f e r e n c e s between the two DNA f r a c t i o n s were i n d i c a t e d a l s o by the d i s -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 i s o t o p e i n a number o f the base c o n s t i t u e n t s of some of the organs. Bendich e t a l . ( 1 4 1 ) a l s o s t u d i e d the p o s s i b l e m e t a b o l i c h e t e r o g e n e i t y o f E. c o l i DNA. The a p p l i c a t i o n of the ECTEOLA f r a c t i o n a t i o n t e chnique t o DNA samples i s o l a t e d from E_. c o l i grown i n the presence o f 5-bromo-uracil f o r 18-24 hours i n -d i c a t e d t o them t h a t the DNA p o p u l a t i o n was heterogeneous w i t h r e s p e c t t o the i n c o r p o r a t i o n of t h i s p y r i m i d i n e . S i m i l a r r e s u l t s have been o b t a i n e d w i t h t r a n s -forming pneumococcal DNA (142). On 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 p r i n c i p l e , f r a c t i o n s were o b t a i n e d which were d i f f e r e n t i n t r a n s f o r m i n g a c t i v i t i e s . 14 In 1956 F r i e d k i n and Wood (143) i n c u b a t e d C l a b e l l e d thymidine i n the presence of v a r i o u s r a b b i t t i s s u e s and c h i c k e n bone marrow. A f t e r i n c u b a t i o n the DNA was e x t r a c t e d from the t i s s u e s by the detergent procedure o f Kay et a l . ( 1 4 4 ) . Three s u c c e s s i v e 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 p r e p a r a t i o n s . I t was found t h a t the s p e c i f i c a c t i v i t i e s o f these DNAs d i f f e r e d w i d e l y . Furthermore, when these i n d i v i d u a l DNA f r a c t i o n s were f r a c t i o n a t e d on ECTEOLA-cellulose column, 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 d e v e l o p i n g the column a l s o showed h e t e r o -g e n e i t y i n s p e c i f i c a c t i v i t i e s . 25 F r a n k e l and Crampton (145) f r a c t i o n a t e d DNA on columns o f p o l y m e t h a c r y l i c a c i d ( A m b e r l i t e IRC-50) i n the magnesium form. I t was observed t h a t DNA c o u l d be e l u t e d from the 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 base c o m p o s i t i o n . When the n u c l e i c 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 tumor c e l l s t h a t had been exposed t o C"^ l a b e l l e d formate or l a b e l l e d thymidine both in_ v i v o and in_ v i t r o , was chromatographed by 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 i n s p e c i f i c a c t i v i t i e s as w e l l as i n base c o m p o s i t i o n . The a f f i n i t y o f the r e s i n was found t o be g r e a t e r f o r molecules r i c h i n adenine and thymine. These s t u d i e s r e v e a l e d two i n t e r e s t i n g p r o p e r t i e s of DNA s y n t h e s i s i n a c t i v e l y d i v i d i n g c e l l s . F i r s t a f t e r 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 , amounting t o not mare than 3% o f the t o t a l DNA, c o n t a i n e d as much as 11% of the t o t a l r a d i o a c t i v i t y . Second, e x c l u s i v e o f t h i s p o r t i o n , the DNA c o u l d be f r a c t i o n a t e d so as t o e x h i b i t almost a t w o f o l d v a r i a t i o n i n s p e c i f i c a c t i v i t y . K u s a k a r i et a l . ( 1 4 6 ) who i n v e s t i g a t e d the i n t r a c e l l u l a r t r a n s f e r of n u c l e i c 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 l a b e l l e d 4-amino-5-imidozole-14 carboxamide o r C l a b e l l e d o r o t i c a c i d , the DNA i s o l a t e d from the n u c l e i by the phenol e x t r a c t i o n method of C o l t e r e t a l . ( 1 4 7 ) , had a l l of the r a d i o a c t i v i t y i n the f r a c t i o n e l u t e d by 0.06M NaCL from ECTEOLA columns. The o t h e r f r a c t i o n s con-t a i n e d no r a d i o a c t i v i t y . I t has been shown t h a t newly synthesized DNA c o u l d be d i s t i n g u i s h e d from the b u l k of c e l l u l a r DNA by i t s r e l a t i v e r e s i s t a n c e t o s o n i c v i b r a t i o n (148). The DNA of lysozyme l y s a t e s of E_. c o l i c e l l s was found l a r g e l y i n the p a r t i c u l a t e f r a c t i o n s sedimenting at low c e n t r i f u g a l f o r c e s . B r i e f s o n i c treatment s h i f t e d most of t h i s m a t e r i a l i n t o the h i g h speed s u p e r n a t a n t . However, a s m a l l amount of DNA was not s o l u b i l i z e d _ e v e n 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 t h i s r e f r a c t -ory m a t e r i a l was very r i c h i n newly synthesiBed DNA. Ben-Porat et. a l . ( 1 4 9 ) d e s c r i b e d the e x i s t e n c e of a more r a p i d l y l a b e l l e d "nascent" DNA i n p r e p a r a t i o n s of E_. c o l i . T h i s m a t e r i a l seemed t o be somehow 26 a s s o c i a t e d w i t h p r o t e i n , because i t c o u l d be e x t r a c t e d from the p r o t e i n r i c h i n t e r p h a s e 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 of DNA p r e p a r a t i o n s by the Sevag (150) . 3 procedure. Bach (151) 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 H l a b e l l e d t h ymidine i n t o the DNA of a microsomal f r a c t i o n from He La c e l l s . T h i s microsomal DNA became l a b e l l e d p r i o r t o n u c l e a r DNA d u r i n g s h o r t - t i m e exposures, and the microsomes c o n t a i n e d , i n a d d i t i o n t o RNA polymerase, a s i g n i f i c a n t p a r t o f the c e l l u l a r DNA polymerase a c t i v i t y . Changes i n the p h y s i c a l s t a t e o f DNA d u r i n g the 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 by R o l f e (152). T h i s i n v e s t i g a t o r observed a s a t e l l i t e DNA band of i n c r e a s e d buoyant d e n s i t y and one o f decreased buoyant d e n s i t y i n s t u d y i n g n u c l e i c a c i d p r e p a r a t i o n s from e x p o n e n t i a l l y growing b a c t e r i a a t s e d i m e n t a t i o n e q u i l i b r i u m i n C s C l . P u l s e r a d i o a c t i v e l a b e l -l i n g s t u d i e s have shown t h a t DNA molecules i n the e a r l i e s t stages o f r e p l i c a t i o n are found i n a t l e a s t two s p e c i f i c d e n s i t y r e g i o n s . One of these r e g i o n s c o r r e s -ponds to the denser s a t e l l i t e band, another i s c o n t a i n e d w i t h i n the r e g i o n of the p r i n c i p a l DNA b a n d and corresponds to the l i g h t s a t e l l i t e band. F r a n k e l (153) e x t r a c t e d an unusual DNA from T2 phage i n f e c t e d b a c t e r i a i n which the t e r m i n a l s t a g e s o f phage growth were prevented by an i n h i b i t o r of p r o t e i n s y n t h e s i s . T h i s DNA was shown t o possess an u n u s u a l l y h i g h m o l e c u l a r weight, by i t s s t r o n g b i n d i n g on MAK column, r a p i d s e d i m e n t a t i o n , and extreme f r a g i l i t y under hydrodynamic shear. Probably the c l e a r e s t demonstration of a m e t a b o l i c a l l y d i f f e r e n t DNA was r e p o r t e d by 5ampson e_t a l . ( 1 5 4 ) . These i n v e s t i g a t o r s r e s o l v e d DNA from a v a r i e t y of growing p l a n t t i s s u e s on a MAK column i n t o two d i s t i n c t f r a c t i o n s . One of these 5 has an average m o l e c u l a r weight o f 10 and i s c h a r a c t e r i z e d by a r e l a t i v e l y r a p i d r a t e of t u r n o v e r . The base c o m p o s i t i o n of t h i s DNA appears t o d i f f e r from i t s h i g h m o l e c u l a r weight c o u n t e r p a r t . 27 The Present I n v e s t i g a t i o n On the b a s i s of e x p e r i m e n t a l evidence c o n c e r n i n g the p o s s i b l e m e t a b o l i c h e t e r o g e n e i t y of DNA, an i n v e s t i g a t i o n was undertaken t o e x p l o r e t h i s p o s s i b i l i t y i n mammalian systems. The c h o i c e o f t i s s u e was the mucosa o f s m a l l i n t e s t i n e o f r a t , which has been shown t o possess a h i g h m i t o t i c a c t i v i t y and a r a p i d r e p l a c e -ment time f o r DNA (13,155,156). To approach the problem, s u i t a b l e i s o l a t i o n and f r a c t i o n a t i o n t e c h n i q u e s had t o be found. P r e v i o u s i n v e s t i g a t i o n s i n t h i s l a b -o r a t o r y (157,158) i n d i c a t e d t h a t the i s o l a t i o n procedure o f Medawar and Zubay (159) was r e l a t i v e l y m i l d and non-degradative. The y i e l d s o f DNA o b t a i n e d by t h i s procedure were q u i t e h i g h , c o n t a m i n a t i n g RNA was g r e a t l y reduced, and the secondary s t r u c t u r e of DNA was not e f f e c t e d a p p r e c i a b l y by the h i g h frequency s o n i c o s c i l -l a t i o n s . For most o f the present i n v e s t i g a t i o n , t h i s method was t h e r e f o r e adopted, but i n the l a t t e r stages of the experiments, the technique o f C o l t e r et a l . (147) was used because of the r e l a t i v e l y g r e a t e r ease of o p e r a t i o n s . By the use o f phenol e x t r a c t i o n and the avoidance of s o n i c d e g r a d a t i o n , and e t h a n o l p r e c i p i t a -t i o n of the DNA, 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 and d e n a t u r a t i o n was minimized i n C o l t e r ' s t e c h n i q u e . As had been i n d i c a t e d , the s u b s t i t u t e d anion exchanger, ECTEOLA-cellulose has been adapted 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 of DNA. T h i s method seemed to o f f e r promise f o r e x p l o r i n g the p o s s i b l e h e t e r o g e n e i t y of DNA p r e p a r a t i o n s from r a t i n t e s t i n a l mucosa. I t was f e l t f u r t h e r , t h a t a s e n s i -t i v e i n d i c a t o r of a m e t a b o l i c h e t e r o g e n e i t y would be the i n c o r p o r a t i o n o f a r a d i o -a c t i v e p r e c u r s o r . The 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 purpose was t h y m i -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 i n c o r p o r a t e d i n t o the DNA (26-28). The s p e c i a l m e t a b o l i c d i f f e r e n c e s or p r e c u r s o r r e l a t i o n s h i p s of the 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 demonstrated i n experiments, where the animals are exposed t o the s p e c i f i c DNA p r e c u r s o r o n l y f o r a s h o r t p e r i o d of time (5,10,20 minutes b e f o r e s a c r i f i c e ) . The experiments which are d e s c r i b e d 28 t h e r e i n , however, tended 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 . Moreover, f u r t h e r d i f f i c u l t i e s were encountered when i t was found t h a t f r a c t i o n a t i o n o f DNA on ECTEOLA-cellulose d i d not y i e l d r e p r o d u c i b l e chromatographic p r o f i l e s as was c l a i m e d by o t h e r s (101,121-124). During the time of the present i n v e s t i g a t i o n , 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 Weiffenbach (160). In view of the d i f f i c u l t i e s encountered, e s p e c i a l l y w i t h the f r a c t i o n a t i o n o f DNA on ECTEOLA-cellulose, i t was f e l t t o be important and necessary t o l o o k f o r o t h e r f r a c t i o n a t i o n methods. Chromatography on MAK columns seemed t o be p r o m i s i n g , s i n c e the c o n d i t i o n s of use are m i l d , and no l o s s of 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 (129). A c c o r d i n g l y , s e v e r a l experiments were c a r r i e d out w i t h t h i s m a t e r i a l and i t was found t o g i v e 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 . The e l u t i o n p a t t e r n s of the DNA p r e p a r a t i o n s were, however, d i f f e r e n t from those o b t a i n e d on ECTEOLA-cellulose. To assure an 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 f r a c t i o n s , use was made of the double l a b e l l i n g t e c h n i q u e . The animals were f i r s t g i v e n t r i -14 tium l a b e l l e d t h y m i d i n e , and 24 hours l a t e r they were given C - l a b e l l e d t h y m i d i n e . The time of exposure t o t h i s l a t t e r p r e c u r s o r was r e l a t i v e l y s h o r t (20 o r 40 m i n u t e s ) . Thus DNA molecules s y n t h e s i z e d d u r i n g the s h o r t exposure times c o u l d be d i s t i n g u i s h e d from the b u l k of the c e l l u l a r DNA which would c a r r y the t r i t i u m l a b e l . The comparison of the H^/C^ r a t i o s of the d i f f e r e n t f r a c t i o n s of DNA p r e p a r a t i o n s c o u l d then be used as an i n d i c a t o r of m e t a b o l i c d i f f e r e n c e s . The r e s u l t s have i n d i c a t e d , t h a t the d i f f e r e n c e s which were detedted a f t e r 20 minutes were no l o n g e r present a f t e r 40 minutes. As an e s s e n t i a l p a r t of i n v e s t i g a t i o n s of DNA b i o s y n t h e s i s in_ v i v o from l a b e l l e d p r e c u r s o r s , i t was of importance t o know whether 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 thymidine i s a measure of the f o r m a t i o n of new and c h a r a c t e r i s t i c DNA o r r e p r e s e n t s the a d d i t i o n of the d e o x y r i b o n u c l e o s i d e t o the p r e - e x i s i t n g ends o f 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 , the time course o f 29 the enzymatic d e g r a d a t i o n of the DNA was f o l l o w e d by the measurement of the r e -l e a s e o f r a d i o a c t i v i t y and u l t r a v i o l e t absorbing 1 m a t e r i a l i n the a c i d s o l u b l e 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 . For t h i s purpose, a p u r i f i e d snake venom phosphodiesterase was used, which has been shown (161) t o l i b e r a t e s tepwise ,1 i 5'-mononucleotides from the 3 -OH ends of p o l y n u c l e o t i d e s . The e x p e r i m e n t a l r e s u l t s o b t a i n e d 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 p r e c u r s o r o c c u r r e d 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 c h a i n s of DNA. 30 EXPERIMENTAL I M a t e r i a l s 1. E x p e r i m e n t a l a n i m a l s . Male W i s t a r r a t s weighing 180-200g each, from t h e co l o n y o f the U n i v -e r s i t y o f B r i t i s h Columbia were used i n the experiments. 2. R a d i o a c t i v e m a t e r i a l s . T r i t i a t e d t h y midine was o b t a i n e d from Schwartz B i o r e s e a r c h , I n c . The l a b e l was i n the thymine \ moiety a t p o s i t i o n s not d e f i n e d by the s u p p l i e r . Two 14 samples o f 2-C -t h y m i d i n e were used. One was purchased from the Ra d i o c h e m i c a l C e n t r e , Amersham, England, and the second was prepared by Dr. 5. H. Zbarsky a c c o r d i n g t o F r i e d k i n and Roberts (162). 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 from c h a r c o a l columns (163) and by chroma-tography on Whatman No. 40 paper 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 : water: f o r m i c a c i d (66:35:5). T r i t i a t e d water s t a n d a r d was o b t a i n e d from New England 14 N u c l e a r Corp., and C - l a b e l l e d sodium carbonate was s u p p l i e d by the N u c l e a r Chicago Corp. Other reagents are d e s c r i b e d i n the a p p r o p r i a t e s e c t i o n s . I I P r e p a r a t i o n o f DNA from S m a l l I n t e s t i n a l Mucosa o f Rat. 1. Method o f Medawar and Zubay (159) Reagents: 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 : — 8.78g o f sodium c h l o r i d e and 3.72g o f ethy 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 water. The pH o f the 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 the f i n a l volume of t h e s o l u t i o n was made up t o 1000 ml. 3M Sodium c h l o r i d e s o l u t i o n : — 175.35g of NaCl was d i s s o l v e d i n d i s t i l l e d water t o o b t a i n a f i n a l volume of 1000 ml. Chloroform-amyl a l c o h o l m i x t u r e : — 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 amyl 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. Any s o l u t i o n s remaining a f t e r 4 weeks were d i s c a r d e d . 31 Procedure: A f t e r the r e q u i r e d time each e x p e r i m e n t a l animal was k i l l e d by a blow on the head and d e c a p i t a t e d . The s m a l l i n t e s t i n e was removed and c u t i n t o 10 cm. segments which were f l u s h e d f r e e of 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 . The segments were s p l i t open and a p p l i e d t o a c h i l l e d g l a s s p l a t e . The mucosal e p i t h e l i u m was then scraped from the m u s c u l a r i s w i t h the edge of a microscope s l i d e and p l a c e d i n l i q u i d n i t r o g e n . The s c r a p i n g s were t h o r o u g h l y mixed and from the pooled mucosa an a l i q u o t was immediately used f o r the e x t r a c t i o n of the DNA. 3g of i n t e s t i n a l mucosa was homogenized b r i e f l y (10 - 20 s t r o k e s ) w i t h a T e f l o n homogenizer i n a f i n a l volume of about 60 ml 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 was exposed t o 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 (9 K c y c l e s / s e c . ) f o r 0.7-1 min. In o r d e r t o assure complete breakdown of c e l l and n u c l e a r membranes, but prevent d e g r a d a t i o n of the DNA, the s o n i c treatment was c o n t i n u e d up t o the p o i n t , when m i c r o s c o p i c examination of t h e sample s t a i n e d w i t h t o l u i d i n e b l u e (164) showed no i n t a c t c e l l s or n u c l e i i n the homogenate. The n u c l e a r fragments were f r e e d of the o t h e r d e b r i s by washing 6-B times w i t h 60 ml of n e u t r a l s a l i n e -Versene s o l u t i o n . The washing was 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 n u c l e a r m a t e r i a l was c o l l e c t e d each time by c e n t r i f u g i n g f o r 8 minutes a t 1448 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 washed n u c l e a r fragments were resuspended i n 45 ml of 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 of a S e r v a l l Omni Mixer and homogenized f o r 1 min. a t a speed o f 3000 r e v o l u t i o n s per minute a t 3°C. N i n e t y ml of 3M. NaCl s o l u t i o n was a t once added and b l e n d -i n g was c o n t i n u e d f o r another 20 minutes. The o p a l e s c e n t s o l u t i o n was emul-s i f i e d by shaking w i t h 30-40 ml of CHCl^-amyl a l c o h o l m i x t u r e f o r 10 min. on a v i b r a t o r y shaker. The emulsion was 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 the 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 the aqueous and i n t e r p h a s e l a y e r s were r e t a i n e d s e p a r a t e l y . The e m u l s i f i c a t i o n process was repeated w i t h the 32 aqueous l a y e r up t o 9 times 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 was 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 from the aqueous l a y e r by a d d i t i o n o f an equal 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 again i n the minimum amount of 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 and c e n t r i f u g e d i f necessary t o o b t a i n a c r y s t a l c l e a r s o l u t i o n . DNA was p r e -c i p i t a t e d from the supernatant w i t h e t h a n o l as p r e v i o u s l y . The f i b r o u s m a t e r i a l was washed s e v e r a l times w i t h 5-10 ml of 75% e t h a n o l then d r i e d i n vacuo a t room temperature over phosphorus pentoxide f o r 24 hours. The dry p r e p a r a t i o n was weighed, 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 s o l -u t i o n c o n t a i n i n g 0.05M NaCl, Q.01M sodium c i t r a t e i n 0.05M phosphate pH 7. The f i n a l c o n c e n t r a t i o n of these s o l u t i o n s was 1 mg DNA/ml. The s o l u t i o n s were s t o r e d a t -15°C. 2. Method of C o l t e r e t a l . (147) Reagents: Sodium ch 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 NaCl, 2.72g potassium dihydrogen phosphate and 3.72g ethylenediamine 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 d i s t i l l e d water. The pH o f the s o l u t i o n was a d j u s t e d t o 7.3 w i t h IN NaOH, and the volume made up t o 1 l i t r e w i t h d i s t i l l e d water. 10% sodium deoxycholate s o l u t i o n : — The s o l u t i o n c o n t a i n e d : 10g of sodium deo-x y c h o l a t e i n 100 ml. Water s a t u r a t e d phenol: — 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 and 25 ml of d i s t i l l e d water. Procedure: The t i s s u e s were prepared as d e s c r i b e d under method 1. F i v e 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 the b l e n d e r cup of a S e r v a l l Omni Mixer and 45 ml of sodium-chloride-Versene-phosphate b u f f e r s o l u t i o n , 2.5 ml o f 10% sodium deoxycholate and 50 ml o f water s a t u r a t e d phenol 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 bath and the m i x t u r e was homogenized at a 33 low speed c o r r e s p o n d i n g t o a Power-stat s e t t i n g o f 20v. F o l l o w i n g homogeniza-t i o n the emulsion was c e n t r i f u g e d f o r 10 min. 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 the upper aqueous l a y e r c o n t a i n i n g the DNA was c a r e -f u l l y removed from the lower phenol phase and denatured p r o t e i n which c o l l e c t e d a t the i n t e r f a c e . 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 e x t r a c t i o n s w i t h e q ual volumes of water s a t u r a t e d phenol. These were of 5 minutes d u r a t i o n and were c a r r i e d out i n the c o l d on a mechanical shaker. A f t e r each e x t r a c t i o n , the phenol and aqueous phases were separated by a b r i e f c e n t r i f u g a t i o n at 4340xg i n the 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 . Traces o f phenol were removed from the 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^ through the s o l u t i o n . The s o l u t i o n was then s t o r e d o v e r n i g h t at 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 minutes at 4340xg i n the 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 DNA was p r e c i p i t a t e d by the a d d i t i o n o f an e q u a l volume of e t h a n o l . The p r e p a r a t i o n was then t r e a t e d as d e s c r i b e d i n method 1. In some experiments the 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 the c l e a r e x t r a c t i n the 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 NaCl i n 0.05M phosphate b u f f e r , pH 6.3. The d i a l y z e d s o l u t i o n was then kept at -15°C. I l l F r a c t i o n a t i o n o f the 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 ECTEOLA-cellulose exchanger. The ECTEOLA-cellulose used i n t h i s study was o b t a i n e d from 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 i n water, and washed s e v e r a l times t o remove f i n e s , then w i t h 1.0N NaOH u n t i l the supernatant gave zero 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,**. The ECTEOLA was giv e n a f i n a l wash w i t h 4N ammonium car b o n a t e , i n which i t was s t o r e d . A s l u r r y o f 0.5g of ECTEOLA i n 8 ml of 4N ammonium carbonate was poured 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 g l a s s 34 d i s c a t the lower end and was equipped w i t h stopcock t o r e g u l a t e the r a t e o f f l o w o f the e f f l u e n t . P r i o r t o chromatography the column was washed w i t h 0.001M phosphate pH 7, u n t i l the e f f l u e n t was f r e e o f ammonium i o n , as checked by spot t e s t i n g w i t h N e s s l e r ' s r e a g e n t . The e f f l u e n t was a l s o checked f o r u l t r a -v i o l e t a b s o r p t i o n at 260m/*. Approximately 2.5-3.5 mg DNA ( e q u i v a l e n t t o 50-70.000 absorbance u n i t s * a t 260™^) i n 0.05M NaCl 0.001M phosphate b u f f e r pH7 were a p p l i e d on the column. The s o l u t i o n was then a l l o w e d t o p e r c o l a t e by g r a v i t y through the exchanger at a r a t e not exceeding 2 t o 4 ml per hour, and i t was washed i n w i t h 10-15 ml of 0.001M phosphate b u f f e r . The column was al l o w e d t o remain o v e r n i g h t a t 3°C, a f t e r which i t was washed w i t h 30 ml o f 0.001M phosphate b u f f e r . The d i s c o n t i n u o u s e l u t i o n schedule was i d e n t i c a l t o t h a t used by K i t (122-124) and i s d e s c r i b e d i n Table I. F i v e ml f r a c t i o n s were c o l l e c t e d by means of a G i l s o n automatic f r a c t i o n c o l l e c t o r ( c o n s t a n t volume o p e r a t i o n ) over a p e r i o d o f 2 days. The s o l v e n t s passed through the column under the f o r c e o f g r a v i t y a t an average r a t e o f 30 ml/hour. The amount of DNA e l u t e d was measured a t 260nry«by means of a Beckman Dli). spectrophotometer. In some cases the amount o f the DNA was a l s o measured by the d e t e r m i n a t i o n o f the deoxy-r i b o s e c o n t e n t o f the f r a c t i o n u s i n g the Dische (165) diphenylamine r e a c t i o n . 2. F r a c t i o n a t i o n on MAK 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 purchased i n the grade s o l d as " 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 the J o h n s - M a n v i l l e Prod. I n c . The e s t e r i f i e d albumin was a g i f t from Dr. M. Smith o f the 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. * Absorbance u n i t s are d e f i n e d as the absorbance observed w i t h the Beckman DU spectrophotometer a t 260rvV«for a 1 cm l i g h t path x 10 x the volume, i n ml, of the s o l u t i o n a p p l i e d t o the column 35 TABLE I E l u t i o n Schedule f o r DNA Chromatography on ECTEOLA-Cellulose Anion-Exchanger F r a c t i o n T o t a l * Composition Volume i n ml E 30 0.001M sodium phosphate pH7 I 30 0.05M NaCl, 0.001M sodium phosphate pH7 I I 30 0.2M NaCl, 0.001M sodium phosphate pH7 I I I 30 0.6M NaCl, 0.001M sodium phosphate pH7 IV 30 0.8M NaCl, 0.001M sodium phosphate pH7 V 30 2..0M NaCl, 0.01M sodium phosphate pHB VI 30 0.01M NH 3, 2M NaCl V I I 30 V I I I 60 IX 30 X 30 XI 30 X I I 30 X I I I 30 XIV 30 XV 3D 0.1M NH 3, 2M NaCl 0.2M NH 3, 2M NaCl 0.3M NH 3, 2M NaCl 0.4M NH 3, 2M NaCl 0.5M NH 3, 2M NaCl 0.6M m 3 , 2M NaEl 1.0M NH 3, 2M NaCl 0.5M NaOH 1.0M NaOH The amount o f these e l u a n t s were s l i g h t l y v a r i a b l e from experiment t o experiment A l l o f these e l u a n t s (except NaOH 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 -form t o prevent b a c t e r i a l and f u n g a l growth. 36 if" E s t e r i f i e d albumin s o l u t i o n : — A 1% s o l u t i o n of t h i s was prepared by d i s -s o l v i n g l g i n 100 ml of d i s t i l l e d water. The c o m p o s i t i o n o f b u f f e r s o l u t i o n s f o r washing the columns and e l u t i n g the DNA are shown i n Table I I . P r o t e i n c o a t e d C e l i t e : — the p r e p a r a t i o n of t h i s m a t e r i a l was performed as d e s c r i b e d by Mandel and Hershey (129). In t h i s procedure 20g of C e l i t e i n 100 ml o f b u f f e r 2 was b o i l e d t o e x p e l a i r and then c o o l e d t o room temperature. F i v e ml of e s t e r i f i e d albumin was added, w i t h s t i r r i n g and then 20 ml of add-i t i o n a l b u f f e r 2 was poured i n t o the s l u r r y . 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 2 x 30 cm chromatographic column, which had a pad of powdered c e l l u l o s e on the s i n t e r e d g l a s s d i s k . The column was formed by p l a c i n g 5-10 ml p o r t i o n s o f the suspension i n the tube and f o r c i n g the excess s a l i n e under an a i r p r e s -2 s u r e ^ o f 3 l b / i n . The f i n i s h e d column which o c c u p i e s about 50 ml, was washed w i t h 200-300 ml of b u f f e r 4 at the p r e s s u r e s t a t e d . The e n t i r e c o n t e n t s of the column were suspended i n 125 ml of b u f f e r 3, s a t u r a t e d w i t h CHCl^ and used as r e q u i r e d . The unused suspension was 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 r e -q u i r e d . P r e p a r a t i o n of the column f o r chromatography The p r e p a r a t i o n o f the column was e s s e n t i a l l y as d e s c r i b e d by Mandel and Hershey (129). The column was composed of t h r e e l a y e r s i n a 2 x 30 cm chromatographic tube. I t was prepared as f o l l o w s : Suspensions o f C e l i t e were b o i l e d i n t h r e e beakers: (1) 8g i n 40 ml b u f f e r 2,(2) 6g i n 40 ml of b u f f e r 3, (3) l g i n 10 ml of b u f f e r 3. Two ml o f 1% e s t e r i f i e d albumin was added t o the f i r s t beaker w i t h s t i r r i n g , and then 15 ml of a d d i t i o n a l b u f f e r 2. The f i r s t l a y e r o f the column was formed w i t h t h i s suspension an c e l l u l o s e powder as d e s c r i b e d above, and the excess p r o t e i n was washed down i n t o the column w i t h b u f f e r 1 t o 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 . Then 10 ml of p r o t e i n -c oated c e l i t e was added t o the suspension i n beaker two. A c c o r d i n g t o Mandel and Hershey, t h i s m i x t u r e should not be prepared i n advance because 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 on MAK B u f f e r number Composition o f B u f f e r *  1. D.05M sodium c h l o r i d e i n 0.Q5M phosphate 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 phosphate 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 phosphate 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 phosphate 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 phosphate b u f f e r pH 6.7 * A l l o f these 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 prevent b a c t e r i a l and f u n g a l growth. 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 the uncoated C e l i t e . The second l a y e r o f the column was b u i l t up w i t h t h i s suspension i n the same manner as the f i r s t and was covered w i t h the c o n t e n t s o f the t h i r d beaker, which s e r -ved as a mechanical b a r r i e r t o the working p o r t i o n s o f the column. The column was washed promptly w i t h b u f f e r 1 u n t i l the o p t i c a l d e n s i t y o f the e f f l u e n t was z e r o , and the r e f r a c t i v e index o f the e f f l u e n t was the same as t h a t o f the e l u a n t . When more than 20.000 absorbance u n i t s o f DNA were a p p l i e d on the column, the amounts o f m a t e r i a l s i n the second l a y e r were doubled. The washed column was loaded 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 c o n t a i n i n g 15 -40.000 absorbance u n i t s i n 50 ml of b u f f e r 1. A l i n e a r sodium c h l o r i d e g r a -d i e n t was e s t a b l i s h e d u s i n g 400 ml o f b u f f e r 1 i n the m i x i n g chamber and 400 ml o f b u f f e r 4 i n the r e s e r v o i r . The g r a d i e n t e l u t i o n had t o be mai n t a i n e d under an a i r pressure o f 3 l b / i n . t o o b t a i n a f l o w r a t e o f about 25 - 30 m l / hour. In l a t e r experiments a B u c h l e r Micro-pump was s u b s t i t u t e d f o r the a i r pr e s s u r e system. 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 assayed f o r DNA c o n t e n t as d e s c r i b e d on page 3^-. The g r a d i e n t s were monitored by r e f r a c t i v e index 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 . These were determined on an Abbe 60 r e f r a c t o m e t e r (Bellingham and S t a n l e y I n c . ) . The inst r u m e n t was c a l i b r a -t e d by measuring the 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 NaCl s o l u t i o n s o f known c o n c e n t r a t i o n i n 0.05M phosphate b u f f e r . The c a l i b r a t i o n curve o b t a i n e d i s shown i n F i g u r e 5 and the c o m p o s i t i o n o f an unknown s o l u t i o n was determined by r e f e r e n c e t o t h i s c u r v e . 3. D e s a l t i n g o f the DNA f r a c t i o n s a f t e r chromatography. In experiments where the r a d i o a c t i v i t y o f the DNA f r a c t i o n s was t o be determined i t was necessary t o remove the 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 found t h a t s a l t s such as sodium c h l o r i d e and phosphate markedly reduced the 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 below were 39 Figure 5. C a l i b r a t i o n curve for measurement of NaCl concentration by refractometry. 40 S t u d i e d t o determine t h e i r u s e f u l n e s s and convenience f o r d e s a l t i n g the f r a c t i o n s . G e l f i l t r a t i o n G e l 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 the s e p a r a t i o n o f substances w i t h d i f f e r e n t m o l e c u l a r dimensions (166) and i s c l a i m e d t o be p a r t i c u l a r l y u s e f u l f o r d e s a l t i n g high m o l e c u l a r weight substance such as p r o t e i n (167). The use of p a r t i c u l a r d e x t r a n g e l s f o r s e p a r a t i o n a c c o r d i n g t o s i z e was i n t r o -duced by Porath and F l o d i n (168). The range o f s e p a r a t i o n i s i n c r e a s e d w i t h d e c r e a s i n g 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 . The p r e p a r a t i o n of the Column: In these experiments Sephadex G-50 was used and o b t a i n e d from Pharmacia, U p s a l a , Sweden. The dry Sephadex powder was suspended i n tap-water and s t i r -r e d f o r a few minutes t o a l l o w i t t o s w e l l . I t was then washed w i t h d i s t i l l e d water s e v e r a l times t o remove f i n e s . The volume o f water was chosen so t h a t the r a t i o supernatant t o sediment was 10:1. B e f o r e b e i n g packed, the column (2 x 30 cm) was mounted v e r t i c a l l y and f i l l e d w i t h t a p water a t room tempera-t u r e . A f u n n e l l a r g e enough t o h o l d one l i t r e o f s o l u t i o n was mounted on the top of the column through a rubber s t o p p e r . The system was then f i l l e d w i t h water up t o the f u n n e l . Care was taken t h a t no a i r bubbles were present i n the column. The suspension of 5ephadex was added t o the f u n n e l and s t i r r e d i n the f u n n e l w i t h a motor-driven s t i r r e r d u r i n g the packing procedure. The bottom o u t l e t from the column was opened t o a l l o w the suspension t o f l o w a t a r a t e o f 5 t o 20 ml per minute. A r i s i n g h o r i z o n t a l boundary o f the packed m a t e r i a l was c o n s i d e r e d evidence o f good p a c k i n g . A f t e r a l l of the m a t e r i a l had s e t t l e d , the f u n n e l and c o n n e c t i n g tube were removed and a f i l t e r paper w i t h a diameter s l i g h l y s m a l l e r than the bore o f the column tube was p l a c e d on the h o r i z o n t a l even s u r f a c e o f the bed. To l e t the bed s t a b i l i z e , i t was p e r c o l a t e d f o r s e v e r a l hours, p r e f e r a b l y o v e r n i g h t , w i t h g l a s s d i s t i l l e d water. 41 A DNA f r a c t i o n (5 ml volume) o b t a i n e d from ECTEOLA-chromatography was then a p p l i e d on the column, and the m a t e r i a l was e l u t e d w i t h d i s t i l l e d water. The 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 assayed 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 at 260 nrj^and f o r c h l o r i d e i o n by the AgNQ^ p r e c i p i t a t i o n t e s t . D i a l y s i s D i a l y s i s i s one o f the most common methods f o r d e s a l t i n g l a r g e mole-c u l a r weight substances. For the experiments d e s c r i b e d h e r e i n , seamless c e l l u l o s e t u b i n g , 3/4" diameter was used. P u r i f i c a t i o n of the d i a l y s i s t u b i n g . I t was found t h a t the t u b i n g c o n t a i n e d l a r g e amounts 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 s which had a peak a b s o r p t i o n at 2 7 4 I T 1 / * . I t was necessary t h e r e f o r e t o t r e a t t h i s t u b i n g t o remove 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 s . The f o l l o w i n g p u r i f i c a t i o n procedure suggested by Dr. G. G. J a c o l i (169) was used t o e l i m i n -a t e t h i s blank a b s o r p t i o n : the d i a l y s i s t u b i n g was c u t i n t o segements about 50 cm l o n g and f l u s h e d through w i t h d i s t i l l e d water. The p i e c e s o f t u b i n g were then p l a c e d i n t o a beaker, covered w i t h d i s t i l l e d water and the c o n t e n t s of the beaker were b o i l e d . The water was d i s c a r d e d , the t u b i n g was covered w i t h 0.5N NaOH and the b o i l i n g was r e p e a t e d . The brownish supernatant was a g a i n d i s c a r d e d and washing w i t h b o i l i n g d i s t i l l e d water was repeated u n t i l the supernatant was n e u t r a l and had n e g l i g i b l e a b s o r p t i o n a t 274m /««. The washed t u b i n g was kept i n the c o l d u n t i l used. The DNA f r a c t i o n s o b t a i n e d by ECTEOLA o r MAK chromatography were p l a c e d i n s m a l l bags prepared from t h i s t u b i n g . The d i a l y s i s was c a r r i e d out i n the c o l d room a t 4°C, a g a i n s t r u n n i n g t a p 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. In the l a t t e r case the bags were a g i t a t e d by use o f a magnetic s t i r r e r . In those c a s e s , when the 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 a f t e r chromatography had very 42 l i t t l e u l t r a v i o l e t a bsorbing m a t e r i a l , the f r a c t i o n s composing one s m a l l peak on the chromatogram were combined, d i a l y z e d and evaporated t o almost dryness i n a f l a s h e v a p o r a t o r . The r e s i d u e was made up t o a s m a l l known volume, and 0.5 ml a l i q u o t s o f t h i s s o l u t i o n were counted. IV Enzymatic Degradation of DNA 1. The p u r i f i c a t i o n o f snake venom phosphodiesterase from the crude venom; M a t e r i a l s : L y o p h i l i z e d C r o t a l u s adamanteus venom was purchased from Ross A l l e n R e p t i l e I n s t . C a r b o x y m e t h y l - c e l l u l o s e wps a product o f the 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 water, the pH o f the 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 the volume was made up t o 1000 ml 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 ml 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 g l a c i a l a c e t i c a c i d i n 1000 ml h^O, pH a d j u s -t e d 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 ml H^O. Procedure: (a) The p r e p a r a t i o n o f venom phosphodiesterase was performed e s s e n t i a l l y a c c o r d i n g t o the procedure o f Koerner and Sinsheimer (170). F i v e hundred mg of crude venom was d i s s o l v e d i n 30.0 ml o f d i s t i l l e d water, and the s o l u t i o n was l e f t a t room temperature f o r an hour. The s o l u t i o n was then c e n t r i f u g e d f o r 15 minutes a t 3030xg on the 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 . To 29.5 ml of the c l e a r supernatant s o l u t i o n 20.0 ml of 0.5M A c e t a t e b u f f e r pH4 was 43 added, and the 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 the c o l d room (4°C) where a l l the subsequent procedures 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 tubes and 36.25 ml of acetone a t - 2 0 ^ p i p e t t e d r a p i d l y on t o the b u f f e r e d venom. The tubes were covered t i g h t l y w i t h aluminium f o i l and s w i r l e d r a p i d l y t o mix the r e a g e n t s . A f t e r i t sto o d f o r 30 minutes, the mix-t u r e was c e n t r i f u g e d and the supernatant l i q u i d was decanted i n t o another c e n t r i f u g e tube. A s l i g h t l y t u r b i d supernatant l i q u i d was observed a t t h i s s t a g e . Seven ml o f acetone were added, and the s o l u t i o n was s t o r e d f o r 12 hours i n stoppered t u b e s . I t was then c e n t r i f u g e d and the c l e a r supernatant s o l u t i o n decanted i n t o another c e n t r i f u g e tube. S i x ml of acetone 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 stand f o r 30 minutes. The 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 ml of d i s t i l l e d water. (b) Chromatography on C a r b o x y m e t h y l - c e l l u l o s e was performed a c c o r d i n g t o F e l i x e_t a l . ( 1 7 1 ) . The acetone p r e c i p i t a t e i n 5.0 ml of d i s t i l l e d water 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 of carboxy-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 the same b u f f e r . The p r o t e i n was e l u t e d stepwise u s i n g 90 ml of D.05M a c e t a t e b u f f e r pH6, 90 ml o f 0.2M a c e t a t e b u f f e r pH6 t and f i n a l l y 90 ml of 0.5M a c e t a t e b u f f e r pH6 i n the o r d e r s t a t e d . F r a c t i o n s o f 3 mis were c o l l e c t e d at a f l o w r a t e o f 6 ml/hour io and a t a temperature o f 3 C. The f r a c t i o n s were assayed 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»vv*«, and f o r phosphodiesterase a c t i v i t y . The f o l l o w -i n g procedure was developed f o r q u a l i t a t i v e assay o f phosphodiesterase a c t i v i t y i n the f r a c t i o n s . One t e n t h ml o f the s u b s t r a t e s o l u t i o n ( c o m p o s i t i o n o f 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 tube and i n c u b a t e d i n a shaking water-bath a t 37°C. One drop of s o l u t i o n from a given f r a c t i o n was added i n t o the t e s t tube and the appearance o f y e l l o w c o l o u r , due t o the 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 minute was accepted as a p o s i t i v e r e a c t i o n f o r phosphodiesterase a c t i v i t y . 44 2. Te 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 the snake venom phosphodies-t e r a s e p r e p a r a t i o n s . 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 ml of b u f f e r c o n s i s t i n g of 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 — 200/tjof DNase I (Worthington Co.) was d i s s o l v e d i n 1.0 ml of 0.1M a c e t a t e , 0.01M M g C l 2 b u f f e r pH7. For the ste p w i s e d e g r a d a t i o n o f DNA by snake venom ph 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 from c o n t a m i n a t i n g endonucleases. I t i s known (172-174) t h a t a c t i o n o f an endonuclease on DNA s o l u t i o n s i s accompanied by a marked decrease i n v i s c o s i t y of these 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 assay f o r the d e t e r m i n a t i o n o f DNase a c t i v i t i e s . W i l l i a m s et al_. (175) showed t h a t when the i n t r i n s i c v i s c o s i t i e s 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 the number o f d i a s t e r bond c l e a v a g e s per DNA m o l e c u l e , the 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 drop i n the i n -t r i n s i c v i s c o s i t y a t the i n i t i a l phase of the r e a c t i o n , whereas venom phospho-d i e s t e r a s e caused a decrease 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 the 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 expected, t h a t d u r i n g the f i r s t 30 - 200 minutes o f i n c u b a t i o n a snake venom phosphodiesterase 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 from DNase a c t i v i t y , would not e f f e c t a p p r e c i a b l y the v i s c o s i t y o f a DNA s o l u t i o n . To t e s t t h i s , the f o l l o w i n g assay method was developed: 5.0 ml of the 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 Ostwald v i s c o s i m e t e r , which had an average f l o w time o f 0.77 min. f o r the b u f f e r a l o n e . The v i s c o s i m e t e r was immersed i n a water-bath a t 37°C. The DNase o r venom phosphodiesterase s o l -u t i o n s were a l s o i n c u b a t e d a t the same temperature. The time o f f l o w o f the 45 DNA s o l u t i o n was determined by u s i n g a stop-watch. At zero time. 0*1 ml of 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 the DNA s o l u t i o n , the m i x t u r e was t h o r o u g h l y mixed and the f l o w - t i m e was measured. During the f o l l o w i n g 30 minutes these were repeated a t 5 min. i n t e r v a l s . F i g u r e 6 shows the 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 -t e d 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 the 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 -ose f r a c t i o n a t i o n o f the acetone p r e c i p i t a t e d venom ph o s p h o d i e s t e r a s e , the acetone 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 the 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 phospho-d i e s t e r a s e . From the e f f e c t on the f l o w time shown i n F i g u r e 6, i t was concluded t h a t the phosphodiesterase p r e p a r a t i o n which was f r a c t i o n a t e d on carboxymethyl-c e l l u l o s e had e s s e n t i a l l y no endonuclease a c t i v i t y and t h a t the endogeneous DNase a c t i v i t y of the crude m a t e r i a l was e l u t e d probably i n the f i r s t peak. The r e s u l t c o n f i r m s the f i n d i n g s o f F e l i x et_ a l . (171) who o r i g i n a l l y d e s c r i b e d the f r a c t i o n a -t i o n o f snake venom phosphodiesterase from Bothrops a t r o x venom. 3. Assay o f the snake venom phosphodiesterase a c t i v i t y . Reagents: 0.1M T r i s - 0.Q02M Magnesium a c e t a t e b u f f e r pH8.9-12.llg o f T r i s (hydroxy-methyl 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 the pH o f the 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 the s o l -u t i o n was made up t o 1 l i t r e . 5 u b s t r a t e s o l u t i o n — 13.6 mg of p-ni 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 ml o f the b u f f e r s o l u t i o n . Snake venom phosphodiesterase s o l u t i o n — One mg of Worthington 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 ml of b u f f e r s o l u t i o n . 46 O DNase A Acetone pr e c i p i t a t e d snake venom diesterase O F i r s t peak from carboxymethyl c e l l u l o s e chromatography ^ Snake venom diesterase a f t e r carboxymethyl c e l l u l o s e chromatography I . I O T 'CO © -t-> 3 C • H s c E o 1.00 -E 0.90-0.80 1 1 1 r-10 15 20 25 Reaction Time i n Minutes 30 Figure 6, The e f f e c t of enzymatic degradation on the v i s c o s i t y of DNA solutions. 47 Procedure: The assay f o r d i e s t e r a s e a c t i v i t y was performed e s s e n t i a l l y as d e s c r i b e d by R a z z e l and Khorana (176). One and one h a l f mis of s u b s t r a t e s o l u t i o n and 1.4 ml o f the 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 c u r v e t t e (1 cm l i g h t p a t h ) . At zero time 0.1 ml o f enzyme s o l u t i o n was added and the s o l u t i o n was mixed t h o r o u g h l y . The 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 t o the l i b e r a t i o n of p a r a n i t r o p h e n y l a t e was measured a t 400*ry*with a Cary r e c o r d i n g spectrophoto-meter equipped w i t h a t h e r m o s t a t a b l e c e l l compartment maintained a t 37°C. 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 of 1.2 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 of Q . l / t m o f s u b s t r a t e . F i g u r e 7 shows t h a t , when the 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 p l o t t e d a g a i n s t the 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 o b t a i n e d . The s l o p e of the l i n e o b t a i n e d f o r the Worthington 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 of 0.016/min. whereas t h a t of the c a r b o x y m e t h y l - c e l l u l o s e p u r i f i e d p h o s p h o d i s t e r a s e i s 0.014/min. These v a l u e s correspond t o the h y d r o l y s i s of 0.078/am and 0 . 0 6 6 / t m o f s u b s t r a t e per hour by these enzyme p r e p a r a t i o n s . I t was found t h a t the p u r i f i e d snake venom phosphodiesterase s o l u t i o n h y d r o l y z e d about 6.6/*»viof s u b s t r a t e /hour / O . l ml of the o r i g i n a l enzyme s o l u t i o n . T h i s v a l u e corresponded t o about 17/t^ of the Worthington venom phosphodiesterase per 0.1 ml of the s o l u t i o n . 4. Stepwise d e g r a d a t i o n of ONA by the p u r i f i e d snake venom phosphodies-t e r a s e . 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 of M g C l ^ h ^ O and 4.B5g of 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 water, the pH o f the 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 HC1 and the volume was made up t o 1000 ml w i t h d i s t i l l e d water. 1% serum albumin s o l u t i o n : — c o n t a i n s l g albumin/100 ml of d i s t i l l e d water 48 O Worthington enzyme 0.4 0.3 " o o +•> a o v G a u o < 0.2 -0.1 -^ Carboxymethyl c e l l u l o s e p u r i f i e d enzyme Figure 7. 10 15 20 Reaction Time i n Minutes Time course of the hydrolysis of p-nitrophenyl-thymidine-5-phosphate by snake venom phospho-diesterase. 49 20% TCA — c o n t a i n s 20g TCA/100 ml o f d i s t i l l e d water. 2% TCA — c o n t a i n s 2g TCA/100 ml of d i s t i l l e d water. Procedure: Radioactiv/e DNA o r the 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 from the main DNA peak a f t e r MAK chromatography was d i s s o l v e d i n 0.01M M g C l 2 - 0.04M T r i s b u f f e r pH 8.9, 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 per 5 ml. Four and one h a l f ml o f the r e s u l t i n g DNA s o l u t i o n were i n c u b a t e d w i t h 0.5 ml o f t h e p u r i f i e d snake venom phosphodiesterase s o l u t i o n at 37°C. The amount o f the p u r i f i e d enzyme used corresponded i n a c t i v i t y t o 80 or 160/^j of Worthington p h o s p h o d i e s t e r a s e . The m i x t u r e was 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 time i n t e r v a l s a l i q u o t s were withdrawn and p l a c e d i n 12 ml S e r v a l l c e n t r i f u g e t u b e s . The r e a c t i o n was stopped by the dropwise a d d i t i o n of 0.5 ml of c o l d 20% TCA, and the 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 sepa r a t e d by c e n t r i f u g a -t i o n . The r a d i o a c t i v i t y o f both f r a c t i o n s were determined by a procedure d e s c r i b e d i n a subsequent s e c t i o n . The absorbance of the a c i d - s o l u b l e f r a c t i o n was measured i n the u l t r a v i o l e t r e g i o n . Due t o the h i g h amount of TCA p r e s e n t i n the a c i d - s o l u b l e p a r t , the 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 o f these s o l u t i o n s were r a t h e r d i s t o r t e d . The 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 determined a f t e r e x t r a c t i o n t h r e e times w i t h e t h e r . As "zero time c o n t r o l s " 0.5 ml a l i q u o t s o f the o r i g i n a l DNA s o l u t i o n w ithout added enzyme were used. V. R a d i o a c t i v e Counting 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 of 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 DNA p r e p a r a t i o n s was determined i n an automatic T r i - C a r b l i q u i d s c i n t i l l a t i o n s pectrometer (Packard Instrument Co., 314A). The samples were c h i l l e d a t -3°C 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 temperature d u r i n g c o u n t i n g . The 50 c o u n t i n g r a t e s on the r e d channel were recorded 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 v o l t s . 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 tap s e t t i n g , was s e l e c t e d which gave maximum c o u n t i n g e f f i c i e n c y . M a t e r i a l s : 2y5 - d i p h e n y l o x a z o l e (PPO) and 2,2'~paraphenylene-5-phenyloxazole (POPQP) were o b t a i n e d from the Packard I n s t . Co. Toluene was purchased from Baker and Adamson Co. Hyamine 10-X ( D i - i s o b u t y l c r e s o x y ethoxy e t h y l d i m e t h y l b e n z y l ammonium c h l o r i d e monohydrate) was o b t a i n e d from Rohm and Haas, P h i l a -d e l p h i a . S o l v e n t s and S o l u t i o n s : Hyamine Hydroxide s o l u t i o n — Hyamine 10-X was r e c r y s t a l l i z e d from t o l u e n e a s ; . f o l l o w s : ; 1.4 kg hyamine 10-X was d i s s o l v e d i n 2.51 t o l u e n e w i t h h e a t i n g , and the s o l u t i o n was f i l t e r e d w h i l e hot. The s o l u t i o n was r e f r i g e r a t e d a t 4TJ o v e r n i g h t . R e c r y s t a l l i z a t i o n was repeated u n t i l the mother l i q u i d was c o l o u r l e s s . 96g of the r e c r y s t a l l i z e d hyamine 10-X were d i s s o l v e d i n 100 ml g l a s s d i s t i l l e d methanol and 1.8 ml d i s t i l l e d water was added. The s o l u t i o n was a l l o w e d t o reach room temperature, then 25.5 g Ag^O were added, w i t h v i g o r -ous s h a k i n g . The suspension was s t i r r e d f o r 10 minutes w i t h a magnetic s t i r r e r and q u i c k l y c e n t r i f u g e d f o r about 10 minutes a t 810 x g a t room temperature. The supernatant was decanted i n t o a c l e a r g l a s s stoppered b o t t l e , and was 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 . The s o l u t i o n was t i t r a -t e d w i t h IN HC1 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 . The c o n c e n t r a t i o n ranged between 0.8 and 1.0N. S c i n t i l l a t o r s o l u t i o n — 6.3 g of PPO and 0.262 g o f PQP0P were d i s s o l v e d i n 1000 ml of r e d i s t i l l e d t o l u e n e . P r e p a r a t i o n of the 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 from 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 c o u n t i n g 51 system to be the 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 thymine: 0.4 ml of a s o l u t i o n o f thymine i n 0.1 NHC1 1.0 ml of hyamine hydroxide 3.0 ml o f a b s o l u t e e t h a n o l 5.0 ml of t o l u e n e c o n t a i n i n g 0.63% PPO and 0.0262% P0P0P The samples were made up i n 20 ml g l a s s v i a l s . S i n c e d u r i n g the f r a c t i o n a t i o n procedures 5.0 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 thought t o be more rea s o n a b l e t o assay 0.5 ml a l i q u o t s of these f r a c t i o n s . Moreover, s i n c e the a c t i v i t y o f the DNA samples was low i n most c a s e s , i t was f e l t d e s i r a b l e t o i n -c r e a s e the volume o f the sample t o be assayed. Under these c o n d i t i o n s , however, the volume of 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 ml t o a v o i d phase s e p a r a t i o n when the samples were c h i l l e d . Each sample was counted f o r 30 t o 50 minutes. In e a r l y experiments 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 the s i d e s of the v i a l s . I t 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 quenching e f f e c t on 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 above. D u p l i c a t e s of each sample were counted. Under these c o n d i t i o n s t h e r e was a l i n e a r r e l a t i o n s h i p between the amount o f DNA present and the r a d i o a c t i v i t y d e t e c t e d . The r e l a t i v e c o u n t i n g e f f i c i e n c y of t h i s system i s 3.0% u s i n g an approximate s t a n d a r d of t r i t i a t e d t h y m i d i n e . In 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 e f f i c i e n c y o f the i n s t r u m e n t and f o r v a r i a t i o n s i n the c o n c e n t r a t i o n o f the hyamine hydrox-3 yde, d u p l i c a t e 0.5 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 s o l u t i o n i n d i s t i l l e d water were counted w i t h each s e r i e s of experiments and the necessary c o r r e c t i o n s were made. 52 14 3 2. 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 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 . The e f f e c t i v e n e s s o f l i q u i d s c i n t i l l a t o r s 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 the d i f f e r e n c e i n the e n e r g i e s o f the b e t a p a r t i c l e s o f 14 t r i t i u m and carbon-C make i t p o s s i b l e t o assay f o r both i s o t o p e s i n a double-l a b e l l e d compound. The maximum beta energy f o r i s 0.018Mev and f o r C"*"^  0.155M ev and the two i s o t o p e s may be counted i n one o p e r a t i o n i n the Packard T r i - C a r b l i q u i d s c i n t i l l a t i o n spectrometer, by proper s e l e c t i o n o f high v o l -tage and d i s c r i m i n a t o r - s e t t i n g s . The two channels f o r p u l s e - h e i g h t d i s c r i m i n a -t i o n are o b t a i n e d by means of 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. The output o f each channel i s f e d i n t o i t s r e s p e c t i v e s c a l e r . A l l p u l s e s o f energy be-tween AA' t o B are recorded on the red s c a l e r and p u l s e s from B t o C are r e -g i s t e r e d by the green s c a l e r . To o b t a i n optimum s e t t i n g s , the 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 the hig h v o l t a g e tap s e t t i n g s f o r both 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 determined on the 14 graph a t which C and t r i t i u m c o u l d be counted i n the r e d s c a l e r w i t h h i g h 14 e f f i c i e n c i e s , w h i l e the C c o u l d be counted i n the green s c a l e r w i t h few o r no t r i t i u m p u l s e s coming i n t o t h a t c h a n n e l . 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 tap 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 making up a s e r i e s o f sta n d a r d s o l u t i o n s c o n t a i n i n g both o f the 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 tap s e t t i n g s . The c o u n t i n g o f the double l a b e l l e d compounds was performed on a Packard T r i - C a r b l i q u i d s c i n -t i l l a t i o n s p e c t r o m e t e r , model 314 AX. 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 (179). The d e t e r m i n a t i o n o f optimum hig h v o l t a g e tap 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 necessary t o know the 14 3 quenching e f f e c t o f hyamine and DNA on the C and H 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 samples were prepared. Each sample c o n t a i n e d 0.5ml 14 o f the s i n g l e i s o t o p e ( e i t h e r 0.5 ml of C - l a b e l l e d Na^CO^ stand a r d c o n t a i n -i n g 44,400 dpm/min or 0.5 ml of 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), the amounts of e t h a n o l and s c i n t i l l a t o r s o l u t i o n as d e s c r i b e d p r e -v i o u s l y and v a r i a b l e amounts of DNA and Hyamine Hydroxide as shown i n Table I I I . The compounds were added t o the v i a l s i n the 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 , hyamine, e t h a n o l and s c i n t i l l a t o r s o l u t i o n . P r e v i o u s o b s e r v a -t i o n s from another l a b o r a t o r y (180) i n d i c a t e d t h a t a naphthalene/dioxane system (c o m p o s i t i o n of naphthalene/dioxane s o l v e n t : 1% PPO, 0.05% P0P0P, 5% napthalene i n dioxane: e t h y l e n e g l y c o l monoethyl e t h e r 5 ; l ) gave approx-i m a t e l y 10% 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 samples were counted. I t was o f i n t e r e s t , t h e r e f o r e , t o compare t h i s system w i t h the t o l u e n e s c i n t i l l a t o r . T able IV shows the c o m p o s i t i o n of the samples prepared i n the n a p t h a l e n e / dioxane system. The 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 the r e d channel of sam-p l e s 1 - 6 are 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 of c u r v e s o b t a i n e d by p l o t t i n g the c o u n t i n g r a t e s on the green s c a l e r a g a i n s t the h i g h v o l t a g e tap s e t t i n g s at 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 , these diagrams r e s u l t e d i n the 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 -i n g the volume of Hyamine Hydroxide t o 1.0 ml has a d e f i n i t e quenching e f f e c t 14 3 on both C and H c o u n t i n g r a t e s . The c o u n t i n g r a t e s are g e n e r a l l y lower i n samples 2, 4 and 6, and the peaks o f maximum e f f i c i e n c i e s are s h i f t e d towards t h e r i g h t ( 179), (b) t h e presence o f added DNA has no a p p r e c i a b l e e f f e c t on the 14 c o u n t i n g r a t e s , (see F i g u r e 10, sample 3 ) , (c) the e f f i c i e n c i e s o f both C and 3 H l a b e l l e d samples were h i g h e r i n the napthalene/dioxane system (Sample 5, F i g u r e 1 2 ) . T h i s system, however, had the b i g disadvantage t h a t a f t e r 2 - 3 days of s t o r a g e , the s o l u t i o n s became d i s c o l o u r e d . For t h a t reason, the t o l u e n e s c i n t i l l a t o r system was used f o r f u r t h e r s t u d i e s . Diagrams 8 - 1 4 a l s o 54 TABLE I I I Composition o f Standards C o n t a i n i n g the S i n g l e Isotope H 3 o r C 1 4 , Used f o r the D e t e r m i n a t i o n o f the 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 the Toluene S c i n t i l l a t o r System Sample DNA i n hyamine hydroxide number n_g i n ml  1 - 0.5 2 - 1.0 3 0.5 0.5 4 0.5 1.0 TABLE IV t 3 14 Composition o f Standards C o n t a i n i n g the S i n g l e Isotope H o r C , Used f o r the D e t e r m i n a t i o n of the 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 the Naphthalene/ Dioxane S c i n t i l l a t o r System. Sample DNA i n ftyamine naphthalene/ number mg hydr o x i d e i n ml 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 I T 24-, 18 to X s o 12 A 6 i Figure 8, T~ 2 T T i 1 r 3 4 5 6 7 8 High Volatage Tap Setting r 10:100 «V10:70 '^10:50 /-10:40 «-10:30 ^10:20 Integral discriminator bias curves for sample 1 from Table III determined on the red scaler For d e t a i l s see text. 56 High Voltage Tap Setting Figure 9. Integral discriminator bias curves for sample 2 from Table III determined on the red scaler. For d e t a i l s see text. 57 High Voltage Tap Setting Figure 10. Integral discriminator bias curves for sample 3 from Table III determined on the red scaler. For d e t a i l s see text. 58 14 24-r r f to o s ft 18-12-Figure 11, 1 2 3 4 5 6 7 8 High Volatge Tap Setting Integral discriminator bias curves for sample 4 from Table III determined on the red scaler. For d e t a i l s see text. 59 ,14 24 T FT 18 to s o 12 -6 -2 T 3 ^10:100 r10:70 <>V10:50 t<_/10:40 18118 4 5 i 6 8 Figure 12, High Voltage Tap Setting Integral discriminator bias curves for sample 5 from Table IV determined on the red scaler. For d e t a i l s see text. 60 1 I i I 1 1 1 1 r 1 2 3 4 5 6 7 8 9 H i g h V o l t a g e Tap S e t t i n g F i g u r e 1 3 . I n t e g r a l d i s c r i m i n a t o r b i a s c u r v e s f o r s a m p l e 6 f r o m T a b l e I V d e t e r m i n e d o n t h e 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 Voltage Tap Setting Figure 14. Integral discriminator bias curves for sample 1 from Table III determined on the green s c a l e r . The curve represents the average of the determination of 6 d i f f e r e n t window settings. For d e t a i l s see text. 62 i n d i c a t e d t h a t optimum h i g h v o l t a g e tap s e t t i n g s f o r double l a b e l l e d compounds 14 sho u l d be e i t h e r 4 or 5. At these h i g h v o l t a g e s e t t i n g s , both t r i t i u m and C c o n t a i n i n g samples were counted 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 the r e d 14 s c a l e r , whereas C was counted w i t h a r e a s o n a b l y good e f f i c i e n c y on the green s c a l e r w i t h o u t too much i n t e r f e r e n c e by counts a r i s i n g from t r i t i u m p u l s e s . To o b t a i n optimum d i s c r i m i n a t o r s e t t i n g s , 4 sta n d a r d samples c o n t a i n i n g both _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 high v o l t a g e 14 tap s e t t i n g s , and the percentage r e c o v e r i e s o f both C and t r i t i u m were c a l -c u l a t e d a c c o r d i n g t o the d i s c r i m i n a t o r - r a t i o method i n r e f . (181,182). T h i s method uses the counts/min. on the green s c a l e r t o those of the red s c a l e r , which were o b t a i n e d from s t a n d a r d samples of each of the i s o t o p e s . O k i t a _ t a l . (181) d e r i v e d the f o l l o w i n g e q u a t i o n f o r the c a l c u l a t i o n o f and C'''4 dpm i n a sample c o n t a i n i n g both i s o t o p e s : .3 H dpm = b N l - H2 b - a re d s c a l e r H e f f i c i e n c y f a c t o r and b ( N 2 - a l ^ ) L dpm = j j green s c a l e r C e f f i c i e n c y f a c t o r where = net cpm on r e d s c a l e r - " " green s c a l e r 3 a _ net cpm of H on green s c a l e r net cpm of H 3 on red s c a l e r h net cpm of C"*~^  on green s c a l e r • _ c j^_= a net cpm of C on r e d s c a l e r The c o m p o s i t i o n of the f o u r double (H^ and C"^) l a b e l l e d s t a n d a r d s o l u t i o n s was as f o l l o w s : 2 j_4 0.5 ml of stan d a r d ( c o n t a i n i n g both H and C i s o t o p e s ) 0.5 ml of hyamine hydroxide 4.0 ml of a b s o l u t e e t h a n o l 5.0 ml of s c i n t i l l a t o r s o l u t i o n 63 Table V shows the 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 the t r i t i u m 14 and C assays of 4 d i f f e r e n t s t a n d a r d s . 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 the data f o r standard 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 s e t t i n g 10:20:100 Background counts on red s c a l e r = 17.5 cpm Background counts on green s c a l e r = 33.8 cpm T o t a l counts on r e d s c a l e r = 1047.3 cpm T o t a l counts on green s c a l e r = 749.6 cpm l\l = T o t a l counts on red s c a l e r - bg = 1047.3 - 17.5 1029.8 cpm N = T o t a l counts on green s c a l e r - bg = 749.6 -33.8 715.8 cpm a = 0 (a and b were determined from F i g u r e s 8,14 r e s p e c t i v e l y ) b = 0.87 3 r e d s c a l e r H e f f i c i e n c y f a c t o r = 0.01685 I* green 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 determined from F i g u r e s 8,14 r e s p e c t i v e l y ) B N - N 2 (0.87x1029.B-K715.B 3 - i — — ± — 0.87-0 = 12,300 0.01685 H dpm = b - a re d s c a l e r e f f i c i e n c y f a c t o r b(N - al\l ) 0.87(715.8-0x1029.8) - 1 4 . z 0.87 - 0 = 9107 C dpm . b_=_e 0 > 0 7 a 6 green s c a l e r e f f i c i e n c y f a c t o r From i n s p e c t i o n o f the dat a i n Table V- i t was concluded t h a t standards 1 - 3 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 s e t t i n g s 10:30:100 and hig h v o l -tage t a p s e t t i n g 5. Standard 4 was an e x c e p t i o n and a l s o the widest v a r i a t i o n s from the t h e o r e t i c a l counts were observed on a s s a y i n g this,^sample. 64 Table V. E f f e c t o f Vol 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 the T r i t i u m and Carbon 14 Assays o f 4 D i f f e r e n t Standards. 3 14 Standard 1 C o n t a i n s T h e o r e t i c a l l y 15•680 H dpm and 8880i.C dpm. D i s c r i m i n a t o r Cpm-bg Cpm-bg ,14 % d e v i a t i o n % d e v i a t i o n &. h i g h v o l t a g e on r e d on green H dpm C dpm from theo- from theo-tap s e t t i n g s s c a l e r s c a l e r r e c o v e r e d r e c o v e r e d r e t i c a l H 3 r e t i c a l C^-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 1029.8 715.8 12,300 9107 737.0 2064.7 17,130 8543 1863.6 677.4 13,742 8889 1318.2 2123.6 15,260 8885 2570.9 707.1 12,590 8332 1842.9 2102.1 16,450 8785 3153.3 69B.2 12,160 8794 2331.6 2135.5 15,406 8855 21.6 9.2 12.4 2.7 19.7 4.9 22.5 1.8 2.5 3.8 0.1 0.1 6.2 1.1 1.0 0.3 Standard 2 C o n t a i n s t h e o r e t i c a l l y 15,680 dpm and 4440 C ^ dpm. D i s c r i m i n a t o r Cpm-bg Cpm-bg ^ &. h i g h v o l t a g e on r e d on green H dpm r 1 4 A C dpm % d e v i a t i o n % d e v i a t i o n from theo:? from t h e o - , 3 14 tap s e t t i n g s s c a l e r s c a l e r r e c o v e r e d r e c o v e r e d r e t i c a l H r e t i c a l C 10:.20sl00 t a p 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 5 659.3 344.7 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 4.7 0.6 1477.1 1071.1 16,540 4345 5.5 2.1 65 Table V. c o n t i n u e d Standard 3 Co n t a i n s t h e o r e c t i c a l l y 31,360 dpm and 8S80 dpm C ^ D i s c r i m i n a t o r Cpm-bg Cpm-bg 3 C"^ dpm % d e v i a t i o n % d e v i a &. h i g h v o l t a g e on r e d on green H dpm from theo- from thi tap s e t t i n q s s c a l e r s c a l e r r e c overed r e c o v e r e d r e t i c a l r e t i c a l 10:20:100 t a p 4 1330.3 677.0 32,740 9043 4.4 1.8 10:20:100 tap 5 1008.5 2099.9 33,780 8600 7.7 3.2 10:30:100 tap 4 2333.9 640.0 34,950 8398 11.4 5.4 10:30:100 tap 5 1746.4 2171.8 31,308 8931 0.2 0.6 10:40:100 tap 4 3116.7 691.3--. 30,870 8729 1.6 1.7 10:40:100 tap 5 2432.3 2083.9 34,866 8644 11.2 2.7 10:50:100 tap 4 3787.6 679.2 32,890 8658 4.9 2.5 10:50:100 tap 5 3018.8 2103.0 35,016 8572 11.6 3.5 -14 Standard 4 Contain s T h e o r e c t i c a l l y 7840 dpm H 3 and 8880 dpm C ^ D i s c r i m i n a t o r L h i g h v o l t a g e Cpm-bg on r e d Cpm-bg on green dpm C dpm % d e v i a t i o n from t h e o ^ % devia-from thi tap s e t t i n q s s c a l e r s c a l e r r e c overed recovered r e t i c a l H r e t i c a l 10:20:100 tap 4 890.4 699.4 5136 8899 34.5 0.2 10:20:100 tap 5 601.8 2120.2 7835 8828 0.1 0.6 10:30:100 tap 4 1672.9 664.0 ,7569 8722 3.5 1.8 10:30:100 tap 5 1099.8 2096.4 7112 8739 9.3 1.6 10:40:100 tap 4 2315.0 701.3 5245 8855 33.1 0.3 10:40:100 tap 5 1577.9 2070.4 8355 8678 6.6 2.3 10:50:100 tap 4 2900.1 696.2 4969 8888 36.6 0.1 10:50:100 tap 5 2015.7 2098.8 7584 8692 3.3 2.1 66 T h i s was expected, 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 assays H"^  dpm/C^ dpm should be between 1-15. T h i s r a t i o , , was l e s s than one f o r Standard 4. 'On'the b a s i s o f the data o f graphs and Table V i t was decided t h a t , f o r the r o u t i n e assays o f double l a b e l l e d compounds the f o l l o w i n g system would be adopted: 3 14 0.5 ml of a s o l u t i o n o f H and C l a b e l l e d m a t e r i a l i n d i s t i l l e d water 0.5 ml of hyamine hydroxide 4.0 ml o f a b s o l u t e e t h a n o l 5.0 ml o f s c i n t i l l a t o r s o l u t i o n The samples were counted f o r 10 t o 100 minutes (depending on the 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 high v o l t a g e t a p s e t t i n g o f 5 w i t h the " 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 from time t o time a c c o r d i n g t o changes i n e f f i c i e n c y o f the apparatus, u s u a l l y observed a f t e r s e r v i c i n g . S i n c e i n double i s o t o p e c o u n t i n g one i s not c o u n t i n g a t the "balance p o i n t " o r peak of the 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 cause a s i g n i f i c a n t change i n the c o u n t i n g r a t e . T h e r e f o r e , a number of standards c o n t a i n i n g known but v a r i a b l e r a t i o s o f H"^  dpm, dpm were counted w i t h each s e r i e s o f e x p e r i -ments and the necessary c o r r e c t i o n s f o r in 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 Prockop and Eb e r t (183) have r e p o r t e d a s i m p l e method which 3 , 14 per m i t s the 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 r a t i o s i n p a r t i a l l y 3 14 p u r i f i e d b i o l o g i c a l m a t e r i a l s . In t h i s method the known r a t i o s o f H dprn/C dpm of a s e r i e s o f standards were p l o t t e d a g a i n s t the 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. T h e i r r e s u l t s i n -d i c a t e d a l i n e a r r e l a t i o n s h i p . I t was a l s o found t h a t a change i n the s l o p e o f the stan d a r d curve r e f l e c t s changes i n the s t a t e o f the i n s t r u m e n t . 67 In the present i n v e s t i g a t i o n , t h e r e f o r e , t h i s method was adopted f o r c h e c k i n g the i n s t r u m e n t from experiment to experiment. The t e chnique i s a l s o 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 of H"Vc^ r a t i o s i n unknown samples. F i g u r e 15 shows the s t a n d a r d c u r v e s o b t a i n e d , when the cpm o b t a i n e d on the r e d scaler/cpm on the green s c a l e r were p l o t t e d a g a i n s t the known H"Vc^ dpm o f the v a r i o u s standard s o l u t i o n s . Knowing the counts on the two s c a l e r s 3. 14 the H /C r a t i o s of an unknown sample can be determined from the graph. In F i g u r e 15 i s i l l u s t r a t e d the change which occured at one o c c a s i o n when the i n -strument was s e r v i c e d . The 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 a g r e a t e r s i o p e , i n d i t i n g . n i c h e , e f f i c i e n c y f o r H 3 c o u n t i „ 9 . 3. The p u r i f i c a t i o n and c o u n t i n g of the 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 . A f t e r c a r e f u l l y removing the DNA c o n t a i n i n g aqueous l a y e r the i n t e r -phase l a y e r s o b t a i n e d d u r i n g the d e p r o t e i n i z a t i o n s t e p s i n the i s o l a t i o n of t h e DNA (147,159) were p u r i f i e d and prepared f o r c o u n t i n g of the r a d i o a c t i v i t y as f o l l o w s : The i n t e r p h a s e l a y e r was l i f t e d w i t h a s p a t u l a from the f r o z e n "phenol l a y e r , o r from the chloroform-amyl a l c o h o l l a y e r . The suspension was e x t r a c t e d w i t h 5 ml of i c e c o l d 20% TCA u s i n g a Vortex J r . mixer, then i t was t r e a t e d two more times w i t h 5 ml o f 2% TCA. The suspension which formed each time was c e n t r i f u g e d 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 . The p r e c i p i t a t e was then e x t r a c t e d f o u r times w i t h 20-30 ml e t h e r , f o l l o w e d by c e n t r i f u g a t i o n each time at 926xg a t room temperature. The r e s i d u e was d r i e d i r _ vacuo a t o room temperature and was f u r t h e r d r i e d at 100 C i n the oven f o r 5-8 hours. The c o u n t i n g of the d r i e d and p u r i f i e d powder was performed a c c o r d i n g t o Brown and Badman (184). To 10 mg of dry powdered i n t e r p h a s e 0.5 ml of IN KOH was added and the m i x t u r e was b o i l e d i n a water-bath f o r 10 min. A f t e r t h a t 0.5 ml of hyamine hyd r o x i d e was added t o the c l e a r a l k a l i n e s o l u t i o n s l o w l y , 68 1.8 n October 1963 December 1963 Figure 15. 4 6 8 10 Theoretical H 3/C 1 4 Linear 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 of the counts obtained on red to green scaler. 69 w i t h s t i r r i n g . The s o l u t i o n was then n e u t r a l i z e d w i t h 0.5 ml o f 2Ni HC1.and c e n t r i f u g e d a t 2500xg. 0.5 ml a l i q u o t s of the c l e a r supernatant l a y e r were used f o r c o u n t i n g i n the 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 of the phenol l a y e r s (147) The phenol 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 of the t i s s u e s a c c o r d i n g t o the method of C o l t e r et_ a l (147) were combined and 1/4 o f the volume of water was added. The m i x t u r e was shaken f o r 5 minutes and c e n t r i f u g e d a t 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 . A second aqueous e x t r a c t i o n of the phenol l a y e r s was a l s o performed and the combined aqueous e x t r a c t s were shaken 4 times w i t h e q ual volumes of e t h e r . The s o l u t i o n was evaporated almost t o dryness i n a F l a s h - e v a p o r a t o r at room temperature, the c o n c e n t r a t e taken up i n 2.0 ml of water and 0.5 ml a l i q u o t s o f t h i s s o l u t i o n were counted 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 the a c i d - i n s o l u b l e m a t e r i a l o b t a i n -ed d u r i n g the enzymatic d e g r a d a t i o n of DNA s o l u t i o n s . To count the a c i d - i n s o l u b l e m a t e r i a l o b t a i n e d i n the experiments i n which the double l a b e l l e d DNA was exposed t o snake venom phosphodiesterase i t was important t o p u r i f y the m a t e r i a l . Presence of any quenching m a t e r i a l , such as s a l t s , i n the r a d i o a c t i v e sample would i n f l u e n c e the r a t i o s o f the counts o b t a i n e d i n the two c h a n n e l s . For t h a t reason v a r i o u s t e c h n i q u e s were s t u d i e d 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 m a t e r i a l . S e v e r a l authors 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 counted on M i l l i p o r e membranes (185-187). T h i s technique was t r i e d f i r s t . A s t o c k s o l u t i o n was prepared from t r i t i u m l a b e l l e d DNA ( c o n c e n t r a t i o n 1 mg/ml) and d u p l i c a t e a l i q u o t s of t h i s s o l u t i o n r a n g i n g from 0.1 t o 0.5 ml 70 were p l a c e d i n m i c r o - t e s t t u b e s . Ten m i c r o l i t e r s o f 1% albumin s o l u t i o n was added t o each t u b e , the 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 and the DNA and 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 ml of c o l d 20% TCA. The p r e c i p i t a t e was q u a n t i t a t i v e l y t r a n s f e r r e d t o M i l l i p o r e membrane f i l t e r s , (HA 0.45 whit e p l a i n 25 mm d i a m e t e r ) . The DNA p r e c i p i t a t e was washed t w i c e w i t h 0.5 ml of i c e c o l d 2% TCA and then w i t h 5.0 ml t o l u e n e . The t o l u e n e treatment was found t o be necessary t o wash out t r a c e s o f occluded TCA from the membrane, thu9 p r e v e n t i n g d i s c o l o u r a t i o n o f the f i l t e r . The membranes were a i r d r i e d f o r an hour and p l a c e d h o r i z o n t a l l y i n t o the c o u n t i n g v i a l s . F i v e ml of 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 the samples were counted. I t was found t h a t the d u p l i c a t e s d i d not agree 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 p r o p o r t i o n a l i t y between DNA c o n c e n t r a t i o n and counts o b t a i n e d . The geometry o f the paper seemed t o have very l i t t l e e f f e c t on the c o u n t i n g r a t e . S i n c e the m i l l i p o r e membrane f i l t r a t i o n t e c hnique was not s a t i s -f a c t o r y another technique was t r i e d based on c e n t r i f u g a t i o n . A DNA s t o c k s o l u t i o n was prepared i n MgCl2-Tris b u f f e r u s i n g a double ( H 3 and C^) l a b e l -l e d m a t e r i a l . D u p l i c a t e a l i q u o t s o f 0.1, 0.2 and 0.5 ml o f t h i s s o l u t i o n were p l a c e d i n 12 ml S e r v a l l c e n t r i f u g e t u b e s . The tubes were kept i n an i c e - b a t h . The t o t a l volume was made up to 0.5 ml by adding, where n e c e s s a r y , more of the b u f f e r . Ten m i c r o l i t e r s o f 1% serum albumin were added t o each tube and the DNA was p r e c i p i t a t e d w i t h 0.5 ml o f c o l d 20% TCA. The TCA s o l u t i o n was added dropwise w i t h c o n s t a n t s h a k i n g , and the tube was then shaken i n a Vortex J r . m i x er. The suspensions were.kept i n the i c e - b a t h f o r 20 min. t o assure complete p r e c i p i t a t i o n . The c e n t r i f u g e tubes were then p l a c e d 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 c e n t r i f u g e d f o r 20 minutes at 30.000xg. The supernatants 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 Pasteur p i p e t t e and p l a c e d i n c l e a n dry t e s t tubes. The p r e c i p i t a t e s were washed t w i c e by suspension i n 0.5 ml of 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 combined and the 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 as the a c i d s o l u b l e f r a c t i o n . The p r e c i p i t a t e s were washed w i t h 2 ml of e t h a n o l : e t h e r (50:50) m i x t u r e and with 2 ml of anhydrous e t h e r . The r e s i d u e s were d r i e d in_ vacuo o v e r n i g h t and then suspended i n 0.7 ml of water. To the suspension 0.1 ml o f de o x y r i b o n u c l e a s e s o l u t i o n was added, c o r r e s p o n d i n g t o 20/^^ DNase. The tubes Q were i n c u b a t e d i n the shaking water bath f o r 7 hours a t 37 C. By t h i s t i m e , the a c i d - i n s o l u b l e ^ m a t e r i a l c o m p l e t e l y d i s s o l v e d , and a 0.5 ml a l i q u o t o f the s o l u t i o n was taken f o r c o u n t i n g i n the system d e s c r i b e d on page The r e s u l t s o f t h i s experiment are presented i n Table V I . where the r e c o v e r y o f counts o f the DNA 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 the u n t r e a t e d DNA s o l u t i o n . The r e s u l t s shown i n Table V I . are the average o f d u p l i c a t e experiments and i n d i c a t e t h a t the 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 procedure 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 the c o a c e n t r a t i o n of l a b e l l e d DNA and count-i n g r a t e s . T h i s technique was used t o count a c i d s o l u b l e m a t e r i a l i n the ex-periments on enzymic d e g r a d a t i o n o f DNA. 6. P u r i f i c a t i o n and c o u n t i n g o f the a c i d - s o l u b l e f r a c t i o n o b t a i n e d d u r i n g the enzymatic d e g r a d a t i o n o f DNA s o l u t i o n s . The supernatant s o l u t i o n s o b t a i n e d a f t e r the p r e c i p i t a t i o n o f 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 times w i t h 5 ml o f anhydrous e t h e r , t o remove TCA from the s o l u t i o n . Traces o f e t h e r were r e -moved by b u b b l i n g N2 through the s o l u t i o n , and 0.5 ml a l i q u o t s o f the s o l u t i o n were counted i n the t o l u e n e s c i n t i l l a t o r system as d e s c r i b e d on page &b . P r e v i o u s experiments w i t h C ^ o r i n d i c a t e d no quenching i n the presence o f 0.01m MgCl2, 0.04M T r i s b u f f e r pH 8.9. 72 TABLE V I. Recovery o f Counts 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 samples O r i g i n a l DNA s o l u t i o n TCA p r e c i p i t a t e d DNA 0.1 ml 0.2 ml 0.5 ml 0.1 ml 0.2 ml 0.5 ml Cpm-Bg on red s c a l e r Cpm-Bg on Qreen s c a l e r 313 635 1599 441 909 2227 320 632 1598 460 917 2384 73 RESULTS AND DI5CU5SIDN I . Experiments w i t h T r i t i u m L a b e l l e d DNA 1. Design of the _Ln v i v o experiments and p r e p a r a t i o n of the t r i t i u m l a b e l l e d t i s s u e . In the experiments d e s c r i b e d t h e r e i n the animals were d i v i d e d i n t o two main groups. The f i r s t group served as a type of a c o n t r o l . These a n i -mals were i n j e c t e d w i t h the r a d i o a c t i v e p r e c u r s o r 24 hours b e f o r e s a c r i f i c e . I t was 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 the DNA molecules of the 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 , the p r e c u r s o r would be w e l l e q u i l i b r a t e d w i t h them d u r i n g the 24 hour p e r i o d . Moreover, t h i s time i n t e r -v a l proved t o be very convenient s i n c e a l l of the e x p e r i m e n t a l animals c o u l d be handled a t the same time of day, namely; between 9.30 - 11.30 i n the morning. By t h i s t e c h n i q u e , the e f f e c t s o f any d i u r n a l v a r i a t i o n s i n the DNA b i o s y n -t h e s i s i n v i v o c o u l d be circumvented (1B8). I t has a l s o been shown (189) t h a t a f t e r i n t r a v e n o u s i n j e c t i o n o f t r i t i a t e d t h y m i d i n e , the s p e c i f i c a c t i v i -t i e s o f the i n t e s t i n a l DNA samples i s o l a t e d at d i f f e r e n t times a f t e r the ad-m i n i s t r a t i o n Df the p r e c u r s o r , i n d i c a t e d two maxima i n i n c o r p o r a t i o n , one o c c u r i n g a f t e r 3 hours and the o t h e r a f t e r 15 t o 24 hours of the r e c e p t i o n of the r a d i o a c t i v e m a t e r i a l . The r e p o r t e d t u r n o v e r time of 1.5 days f o r the i n t e s t i n a l e p i t h e l i u m (188) would a l s o support the h y p o t h e s i s , t h a t i n 24 hours an almost complete e q u i l i b r a t i o n should be o b t a i n e d . The animals 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 of 24 hours. They were then k i l l e d and the DNA was e x t r a c t e d from the pooled s c r a p i n g s of 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 . 74 In the second group of experiments the animals were exposed t o the 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 ti m e , i . e . 5, ID or 20 min-u t e s p r i o r t o s a c r i f i c e . In these cases the r a t s were s t a r v e d f o r 24 hours b e f o r e r e c e i v i n g the r a d i o a c t i v e m a t e r i a l . In each experiment, groups o f 2 o r 3 male W i s t a r r a t s (180 - 200g each) were used. Each animal w as i n j e c t e d w i t h 0.5 ml of 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 thymidine 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 reason f o r t h i s dose i s based on the p r e v i o u s e x p e r i e n c e i n t h i s l a b o r a t o r y (177) which 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 the p r e c u r s o r i n t o the d i f f e r e n t organs o f r a t w i t h lower o r s i m i l a r amounts of r a d i o a c t i v i t y . 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 , subcutaneous i n the 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 v e i n . In two experiments, when the i n t r a v e n o u s i n j e c t i o n t e c hnique was used, 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 a n i m a l . The r a d i o a c t i v e DNA was prepared from the mucosa and f r a c t i o n a t e d on 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 the f r a c t i o n a t i o n o f DNA on ECTEOLA-cellulose exchangers, showed t h a t very complex chromatographic p r o f i l e s were o b t a i n e d f o r the 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 h i s was essen-t i a l l y c o n f i r m e d i n the present i n v e s t i g a t i o n . F i g u r e 16 r e p r e s e n t s the chromatographic 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 from r a t i n t e s t i n a l mucosa by the method of Medawar and Zubay (159). On i n s p e c t i o n o f the chroma-togram, s e v e r a l peaks can be n o t i c e d , some of which 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 . ) Bendich et a l . (101) have found 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 NaCl c o n c e n t r a t i o n s of 0.2M 0.8 o to CM +J rt <U o c rt O XI < 0.6-\ 0.4 0.2 Jl F r a c t i o n No B | i "j " I "» I iv | v I v i | vii |viu( |v | [ X T xi I xii | vm| xiv Tube No. : 50 100 150 Figure 16. F r a c t i o n a t i o n on ECTEOLA-cel lulose of t r i t i a t e d DNA from the i n t e s t i n a l mucosa of r a t . Ul 76 o r lower, r e p r e s e n t 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 . The 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 NaCl a l s o r e p r e s e n t s r e l a t i v e l y low m o l e c u l a r weight m a t e r i a l a c c o r d i n g t o Bendich e_t a l . (101). O c c a s i o n -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 of u l t r a v i o l e t ab-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 the column e q u i l i b r a n t , 0.001M phosphate b u f f e r pH7, a f t e r the column had adsorbed the 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 non-adsorbed 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 w i t h the b u f f e r , and then l e f t o v e r n i g h t at 4°C. Tftree e x p l a n a t i o n s can be o f f e r e d f o r t h i s phenomenon: (a) the column r e l e a s e d some o f the adsorbed m a t e r i a l due t o the de-cre a s e i n temperature (b) the o r i g i n a l p r e p a r a t i o n c o n t a i n e d some low m o l e c u l a r weight mat-e r i a l , which c o p r e c i p i t a t e d w i t h the DNA d u r i n g the e t h a n o l p r e c i -p i t a t i o n t e s t s (c) d e p o l y m e r i z a t i o n o f the l a r g e r DNA p o l y n u c l e o t i d e s may have o c c u r -red a f t e r the m a t e r i a l was adsorbed on the 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 changes have l i t t l e e f f e c t on the c a p a c i t y o f the ECTEOLA-cellulose and t h a t m a t e r i a l adsorbed a t 24°C i s not r e l e a s e d from the r e s i n on c h i l l i n g o t o 4 C. The second a l t e r n a t i v e was e l i m i n a t e d by an experiment i n which the 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 times 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 the exchanger. Even a f t e r t h i s treatment a c o n s i d e r a b l e amount of 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 i n F r a c t i o n E. 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 experiments 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 d e g r a d a t i o n o f the DNA by the ECTEOLA-cellu-l o s e exchanger i t s e l f . 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 the 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 w i t h a l k a l i n e s o l v e n t s , ( F r a c t i o n s V I I , V I I I , X, X I , X I I and X I V ) . A c c o r d i n g t o K i t (122,123) the 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 , the l e s s d e g r a d a t i v e i s the procedure used f o r the e x t r a c t i o n o f DNA. T h i s statement i s v a l i d o n l y , i f the comparison i s made on the same exchanger, w i t h the same exchanging c a p a c i t y and N c o n t e n t . S i n c e , i n the present i n v e s t i g a t i o n , the same e l u t i o n schedule and ECTEOLA-c e l l u l o s e w i t h p r o p e r t i e s s i m i l a r t o t h a t r e p o r t e d by K i t were used, the r e s u l t s are comparable w i t h h i s . The r e l a t i v e l y low amount of m a t e r i a l 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 , would i n d i c a t e t h a t the DNA p r e p a r a t i o n was i n h i g h l y p o l y m e r i z e d form, and had a r e l a t i v e l y h i g h mole-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 the chromato-g r a p h i c p r o f i l e s o f the DNA p r e p a r a t i o n s were r e p r o d u c i b l e i f the methods and 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 . In c o n t r a s t t o t h i s , i n the present work i t was 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 DNA p r e p a r a t i o n s o b t a i n e d from the same source of t i s s u e ( i n t e s t i n a l mucosa) and same s p e c i e s ( r a t ) were not i d e n t i c a l although the method and c o n d i t i o n s of p r e p a r a t i o n were s t r i c t l y d e f i n e d and i d e n t i c a l . I t was suspected t h a t a p o s s i b l e reason f o r the d i f f e r e n c e s may have been d i f f e r e n t degrees of s o n i c a t i o n . Doty e t a l . ( 1 9 0 ) r e p o r t e d , t h a t by exposing DNA s o l u t i o n s t o s o n i c waves of 9 k c y c l e s / s e c f o r warying t i m e s , the molecules 7B were degraded through doub l e - c h a i n s c i s s i o n , producing fragments having the 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 . In t h i s way homolagous samples o f DNA were produced t h a t covered a 25 f o l d range i n m o l e c u l a r weight. Experiments, i n which the homogenate o f the i n t e s t i n a l mucosa of 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 time (1.25 min.) seemed t o c o n f i r m t h i s f i n d i n g . The chromatographic 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 17. I t can be seen t h a t most o f the u l t r a v i o l e t a bsorbing 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,', or p o s s i b l e breakdown of the DNA mo l e c u l e s . 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 experiment was c a r r i e d out i n which the i n t e s t i n a l mucosa of f o u r r a t s was pooled and a sto c k homogenate was 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 s o l u t i o n . The homogenate was d i v i d e d i n t o two p a r t s . One p a r t was exposed t o s o n i c a t i o n f o r 0.7 minutes and the o t h e r f o r 1 minute and the DNA was e x t r a c t e d from both 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 under e x a c t l y the same c o n d i t i o n s . The e l u t i o n p a t t e r n s o f the 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 the two DNA samples. On c a l c u l a t i n g the percentage d i s t r i b u t i o n 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 the f r a c t i o n s e l u t e d w i t h 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 , w i t h 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 found t h a t 41.9, 44.4 and 13.7% of the 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 m a t e r i a l was e l u t e d i n these f r a c t i o n s r e s p e c t i v e l y , i n the DNA p r e p a r a t i o n o b t a i n e d from the sample 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. In the 1 min. sample the d i s t r i b u t i o n o f the t o t a l absorbance i n the above mentioned f r a c t i o n s was 28.6, 27.3 and 44.1%. I t i s d i f f i c u l t t o e x p l a i n why DNA from mucosa exposed t o s o n i c o s c i l -l a t i o n s f o r 0.7 min. should y i e l d more lower m o l e c u l a r weight m a t e r i a l , than DNA from t i s s u e exposed f o r 1 minute. The d i f f e r e n c e between e l u t i o n p a t t e r n s of the two DNA p r e p a r a t i o n s o b t a i n e d from the same t i s s u e and same a n i m a l s , may 79 o CO O J •p rt o o rt J-( o w < 0.2-Fraction NoP I IX | x | xi | Xu | XIII Xiv | x v | 50 Tube Number 100 Figure 17. Fractionation on ECTEOLA-cellulose of 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 to sonic o s c i l l a t i o n for 1.25 min. 80 o C O CM •P a! <P o c C5 .Q U o m < 0.8-1 0.6-0.4-0.2-0.8n 0.6-0.4-0.2-I (1.23) < i L rT © i I 1 ii I III I I V v I V I I V I I I V i l l i IX I X I XI ] | XII I XIII I X I V I X V (1.47) \ © X ^ — * e I i 1 i i I i i i I vv I v I Fraction No. VI I v n I V i l l i I X I X. I X.I I M l IXIUI xiv I *v 50 100 Tube Number Figure 18. Fractionation on ECTEOLA-cellulose of t r i t i a t e d DNA from rat i n t e s t i n a l mucosa. (A) Tissue exposed to sonic o s c i l l a t i o n for 0.7 min (B) Tissue exposed to sonic o s c i l l a t i o n for 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 s t e p d u r i n g the i s o l a t i o n or f r a c t i o n a t i o n procedure. D i f f e r e n c e s might a l s o a r i s e as a r e s u l t of a g g r e g r a t i o n or d i s a g g r e g a -t i o n of DNA d u r i n g s t o r a g e even a t -15°C. I t s h o u l d be p o i n t e d o u t , t h a t the DNA from mucosa s o n i c a t e d f o r 1 min. was s t o r e d f o r 2 weeks b e f o r e f r a c t i o n a t i o n . When these experiments were c a r r i e d out t h e r e was l i t t l e reason t o suspect any marked changes d u r i n g s t o r a g e . The r e s u l t s o f experiment showing the p o s s i b l e e f f e c t s o f s t o r a g e of the DNA on r e p r o d u c i b i l i t y o f chromatographic f r a c t i o n a t i o n on ECTEOLA-cellulose are i l l u s t r a t e d i n F i g u r e s 19A and B. In t h i s experiment the same sto c k s o l u t i o n o f DNA was used, but the p r o f i l e shown i n F i g u r e 19B was o b t a i n e d 2 weeks a f t e r t h a t i l l u s t r a t e d i n F i g u r e 19A. The 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 i n the 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 the m a t e r i a l which was s t o r e d . Klouwen and Weiffenbach (160) d e s c r i b e d s i m i l a r f i n d i n g s . They r e p o r t e d t h a t DNA p r e p a r a t i o n s i s o l a t e d from the same source, by the same method d i d not g i v e 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 columns. Moreover, these v a r i a t i o n s were enhanced by ageing the DNA s o l u t i o n f o r a few weeks i n the c o l d . On the b a s i s of t h i s f i n d i n g , they q u e s t i o s e d the f e a s i b i l i t y of " f i n g e r p r i n t i n g " DNA p r e p a r a t i o n s 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 ECTEOLA-cellulose. In p h y s i c o c h e m i c a l 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 m o l e c u l a r weight and 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 from the same source and 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) t h a t these p h y s i c o -o c h e m i c a l p r o p e r t i e s change d u r i n g the s t o r a g e of DNA s o l u t i o n s a t 0-3 . Since f r a c t i o n a t i o n on ECTEOLA i s governed by the s i z e and shape o f the DNA molecules (101, 121-124) i t i s p o s s i b l e t h a t d u r i n g the s t o r a g e these p h y s i c o c h e m i c a l changes might b r i n g about a d i f f e r e n c e i n the s i z e and shape o f the m o l e c u l e s , r e s u l t i n g i n a change i n the e l u t i o n p a t t e r n . The above e x p l a n a t i o n would s t i l l 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 82 1.0, (2.06) r=* E l I I w 1 iii I w I V ] vi vu| viu p71 X~ F r a c t i o n No 50 Tube Number | xi \ xu | xiii | 100 XIV J XV 150 Figure 19. (A) F r a c t i o n a t i o n on ECTEOLA-cel lulose of t r i t i a t e d DNA obtained from the i n t e s t i n a l mucosa of r a t s in jec ted with the precursor 10 min. p r i o r to s a c r i f i c e . (B) Th i s sample i s an a l iquot of the stock s o l u t i o n of 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 p r e p a r a t i o n s s t u d i e d d u r i n g the present i n v e s t i g a t i o n . Table V I I i l l u s t r a t e s the percentage 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 a b s o r b i n g m a t e r i a l i n t h e t h r e e main areas of the ECTEOLA-chromatograms of the d i f f e r e n t DNA p r e p a r a t i o n s . Since most of these samples were f r a c t i o n a t e d very soon a f t e r the DNA was e x t r a c t e d from 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 from experiment t o experiment 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 the s t a t e of DNA i n the v a r i o u s e x p e r i m e n t a l animals or they i n d i c a t e damage o r d e g r a d a t i o n of the DNA molecules by the ex-changer m a t e r i a l i t s e l f . T h i s l a t t e r e x p l a n a t i o n was f u r t h e r supported by the r e s u l t s o b t a i n e d on Sephadex chromatography of the i n d i v i d u a l f r a c t i o n s of ECTEOLA-chromatograms, and a l s o by the r e s u l t s of r a d i o a c t i v i t y measurements. 3. Chromatography on Sephadex columns of the f r a c t i o n s o f DNA o b t a i n e d by ECTEOLA-cellulose chromatography. During the course of 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 the d i f f e r e n t DNA f r a c t i o n s , i t was suspected t h a t the presence of h i g h c o n c e n t r a t i o n s of s a l t and NH would cause a severe quenching of the r a d i o a c t i v i t y . I t was f e l t , t h e r e f o r e , •J t h a t the f r a c t i o n s should 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 . S i n c e the technique of 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 the s e p a r a -t i o n of low m o l e c u l a r weight m a t e r i a l s from h i g h m o l e c u l a r weight substances, t h i s method was t r i e d f i r s t . F i g u r e s 20A and B i l l u s t r a t e the r e s u l t s o f such an experiment. F i g u r e 20A 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 which was e l u t e d by 0.05M NaCl from the ECTEOLA-column, and F i g u r e 20B i s the diagram o b t a i n e d when a f r a c t i o n e l u t e d by 0.4M NH^ i n 2M NaCl was f r a c t i o n a t e d on the Sephadex G-50 column. A c o n s i d e r a b l e o v e r l a p p i n g of 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 was n o t i c e d i n both c a s e s . The f r a c t i o n a t i o n was repeated s e v e r a l times 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 chromatography, but the method has not proved t o be p r a c t i c a l f o r complete removal of the c h l o r i d e B4 TABLE V I I . Comparison of the Percentage o f the U l t r a v i o l e t Absorbing M a t e r i a l i n the Main F r a c t i o n s Obtained from ECTEOLA-Chromatography of the DNA P r e p a r a t i o n s . DNA p r e p a r a t i o n of the t o t a l absorbance a t 260 I I I A l l f r a c t i o n s A l l f r a c t i o n s e l u t e d w i t h e l u t e d w i t h i n c r e a s i n g NaCl i n c r e a s i n g NH. c o n c e n t r a t i o n c o n c e n t r a t i o n " I I I A l l f r a c t i o n s e l u t e d w i t h i n c r e a s i n g NaOH c o n c e n t r a t i o n H -thym i d i n e i n j e c t e d subcutan-e 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 6.6 H -thymi d i n e 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 min. 28.6 27.3 44.1 H - t h y m i d i n e 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 -thym i d i n e i n j e c t e d subcutan-e o u s l y 20 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. 2 9 i l 34.9 36.0 H -thym i d i n e i n j e c t e d subcutan-e o u s l y 10 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. 28.7 61.4 9.9 H -thymi d i n e i n j e c t e d subcutan-e 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. 17.8 52.7 29.5 H -thym i d i n e i n j e c t e d i n t r a v e n -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. 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. 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. 48.3 42.6 9.1 85 0.181 0.12-0.06-o CD O J •P a © . o c ci Xi u o w Xi U l t r a v i o l e t absorption Q u a l i t a t i v e Cl" test 8 12 16 0.12 n 0.06 _ 8 12 Tube Number T 16 © •4 ** > 18 Tj a • r l CJ £0 r+ + + 18 Figure 20. Chromatography on Sephadex of DNA frac 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 NH3 i n 2M NaCl. 86 i o n s from 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 . Moreover, the r a t e o f f l o w o f the e f f l u e n t was very slow, due t o the high v i s c o s i t y o f most of the 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 chnique i s c l a i m e d t o be (168) t h a t mole-c u l e s of low m o l e c u l a r weight such as s a l t s can d i f f u s e r e l a t i v e l y f r e e l y through the network s t r u c t u r e o f the g e l g r a i n s . Moderately l a r g e molecules w i l l be r e -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 g e l g r a i n s depending on the p o r o s i t y o f the network s t r u c t u r e . Large molecules are c o m p l e t e l y prevented from e n t e r i n g the g e l g r a i n s and w i l l not be r e t a r d e d a t a l l by the column m a t r i x . Thus, l a r g e m o l e c u l a r weight substances are e l u t e d f i r s t from the column. In the present 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 appeared and presumably t h i s s h o u l d be high m o l e c u l a r weight m a t e r i a l . The reason f o r the inc o m p l e t e removal o f c h l o r i d e from t h i s DNA f r a c t i o n c o u l d be the v i s c o s i t y of the p r e p a r a t i o n s . I t has been c l a i m e d (166-168) t h a t the h i g h e r the v i s c o s i t y of the sample the l e s s complete i s the s e p a r a t i o n of the low m o l e c u l a r weight sub-s t a n c e s from the high m o l e c u l a r weight m a t e r i a l i n the m i x t u r e . R e c e n t l y Skidmore et a l . (192) a l s o used Sephadex chromatography f o r d e s a l t i n g DNA f r a c t i o n s a f t e r ECTEOLA-cellulose chromatography, and c l a i m e d t h a t the 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 data 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 of 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 diagrams of F i g u r e s 20A and B i l l u s t r a t e another i n t e r e s t i n g ob-s e r v a t i o n , namely, the e l u t i o n o f u l t r a v i o l e t a bsorbing m a t e r i a l i n two peaks, from the 5ephadex column. The appearance of the second m a t e r i a l i n the 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 h i g h m o l e c u l a r weight m a t e r i a l r e t a r d e d i n i t s passage through the column, or more l i k e l y , a low m o l e c u l a r weight component. Since the samples i n F i g u r e s 2QA and B r e p r e s e n t 5 ml f r a c t i o n s taken from i n d i v i d u a l peaks e l u t e d from ECTEOLA-cellulose, i t i s r a t h e r s u r p r i s i n g t o 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 rechromatographed s e v e r a l times on ECTEOLA an o r i g i n a l peak and found, t h a t a f t e r each rechromatography f u r t h e r f r a c t i o n a t i o n o f the o r i g i n a l peak o c c u r r e d . They concluded from t h i s , t h a t the o r i g i n a l chromatography of the DNA sample caused i n c o m p l e t e . s e p a r a t i o n o f the 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 e x p l a n a t i o n 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 the de g r a d a t i o n o f the o r i g i n a l DNA i t s e l f , by the exchanger m a t e r i 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 e f f e c t e d by the a l k a l i n e e l u e n t i s u n l i k e l y because DNA i s known t o be degraded ver y l i t t l e 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 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 i n s i z e (See F i g u r e 20A). 4. Removal of i n o r g a n i c c o n s t i t u e n t s from the DNA f r a c t i o n s by d i a l y s i s . S i n c e Sephadex chromatography d i d not g i v e complete d e s a l t i n g o f the 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 . P r e l i m i n a r y i n v e s t i g a t i o n s i n d i c a t e d t h a t d i a l y s i s g e n e r a l l y i n c r e a s e d the c o u n t i n g r a t e o f the f r a c t i o n s . T h i s f i n d i n g i s r e p r e s e n t e d i n F i g u r e 21. However, l a t e r e v a l u a -t i o n of the percentage r e c o v e r i e s o f the 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 , r e v e a l e d i n some cases l o s s e s o f as much as 30% of the t o t a l r a d i o a c t i v i t y a p p l i e d t o the column. I t was suspected 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 low m o l e c u l a r weight substance i n the f r a c t i o n s which might have been l o s t d u r i n g the d i a l y s i s procedure. T h e r e f o r e , i n an experiment the u n f r a c t i o n a t e d DNA was d i a l -yzed a g a i n s t d i l u t e s a l t s o l u t i o n s i n the 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 , u n t i l r e a s o n a b l y c o n s t a n t s p e c i f i c a c t i v i t y o f the sample was o b t a i n e d . No r a d i o a c t i v -i t y c o u l d be d e t e c t e d i n the d i a l y z a t e o f the u n f r a c t i o n a t e d DNA. The DNA sample was then f r a c t i o n a t e d on an ECTEOLA-cellulose column and the f r a c t i o n s were d i a l -yzed a g a i n s t d i s t i l l e d water. The d i a l y z a t e s were evaporated i n a " f l a s h " evap-o r a t o r at 25-30°C and the r e s i d u e s were taken up i n a known volume of d i s t i l l e d water and counted. 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 found i n the d i a l y z a t e o f the 88 Radioactivity a f t e r d i a l y s i s 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 Tube Number 80 100 Figure 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 eluates shown i n Figure : 17 before (shaded areas) and a f t e r ( s o l i d line) d i a l y s i s of the i n d i v i d u a l f r a c t i o n s . 8 9 f i r s t f r a c t i o n of the DNA. T h i s d i a l y z a t e had a l s o a very h i g h absorbance a t 260myt*. A l i q u o t s of t h i s s o l u t i o n were a p p l i e d t o Whatman No. 40 a c i d washed paper and chromatographed i n an i - p r o p y l a l c o h o l : NH^ c o n e : H^O (7:1:2) system. On i n s p e c t i n g the chromatograms under u l t r a v i o l e t lamp two f a i n t f l u o r e s c e n t and one u l t r a v i o l e t absorbing spot was observed. Only the u l t r a v i o l e t spot had a d e f i n i t e a b s o r p t i o n c u r v e , w i t h a maximum a t 275r»v«and a minimum a t 262.5~»/« i n 0.1N HC1. The same s o l u t i o n at pH 11.2 had a maximum a b s o r p t i o n a t 277 . . 5m / « and a minimum at 264.5wi/4.Later experiments showed t h a t t h i s u l t r a v i o l e t absorb-i n g spot was probably 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 substance, which can be 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. I t was found, t h a t an 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 c o u n t i n g of the paper chromatogram the r a d i o a c t i v i t y was not 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 a b s o r b i n g areas on the paper. The c h e m i c a l nature of the h i g h l y r a d i o a c t i v e compound i n the d i a l y z a t e o f the f i r s t f r a c t i o n of the DNA sample has not been e l u c i d a t e d , but i s f e l t , t h a t i t i s not present o r i g i n a l l y i n the u n f r a c t i o n a t e d DNA, s i n c e the sample was e x h a u s t i v e l y 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 f i n d i n g , t h a t low mole-c u l a r weight, 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 from DNA a f t e r 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 deg r a d a t i o n of the DNA by the exchanger. 3 5. Time-course of the i n c o r p o r a t i o n of H -thymidine i n t o the 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 experiments, groups of two r a t s were i n j e c t e d sub-cu t a n e o u s l y i n the backs and the r a t s were s a c r i f i c e d a f t e r being exposed t o the t r i t i a t e d t hymidine p r e c u r s o r f o r 5, 10 or 20 minutes o r 24 hours. The time curve o b t a i n e d f o r the s p e c i f i c a c t i v i t i e s o f these DNA p r e p a r a t i o n s i s shown i n F i g u r e 22. Very low 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 p r e c u r s o r was achieved d u r i n g the 90 Figure 2 2 . S p e c i f i c a c t i v i t i e s of DNA obtained from rats injected subcutaneously with H -thymidine. 91 f i r s t 10 minutes. U n f o r t u n a t e l y , no data are 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 40 or 60 minutes, but by comparing the v a l u e s o b t a i n e d f o r 20 minutes and 24 hours, one can assume t h a t , by 60 minutes a p l a t e a u on the curve would be reached. T h i s assumption i s supported by the data o f P o t t e r and Nygaard (35) and Schwartz e t a l . (193) who a l s o demonstrated, t h a t w i t h i n 60 minutes the 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 i n t o the DNA of s p l e e n , thymus, bone marrow and s m a l l i n t e s t i n e reached 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, t h a t the i n i t i a l p o r t i o n of the s p e c i f i c a c t i v i t y - t i m e curve i n F i g u r e 22, i s very 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 Nygaard f o r r a t sple e n DNA 6. E f f e c t o f the r o u t e o f i n j e c t i o n on the s p e c i f i c a c t i v i t i e s o f the DNA p r e p a r a t i o n s . S i n c e very low amounts of 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 the s h o r t - t i m e experiments, u s i n g the subcutaneous 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 of i n j e c t i o n were t r i e d . Table V I I I shows the percentage i n c o r p o r a t i o n o f the i n j e c t e d r a d i o a c t i v i t y per mg of DNA, f o r the 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 -t i o n . H i g h e s t i n c o r p o r a t i o n was observed i n the 24 hour experiment, u s i n g the i n t r a p e r i t o n e a l r o u t e o f i n j e c t i o n . O b j e c t i o n t o the i n t r a p e r i t o n e a l i n j e c t i o n i s t h a t the t i s s u e being s t u d i e d , the i n t e s t i n e , becomes surrounded by the p r e -c u r s o r . I t was f e l t , t h a t t h i s does not 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 normal c o n d i t i o n . For t h a t reason the 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 of r a d i o a c t i v i t y was h i g h e r i n the DNA i s o l a t e d from the 3 i n t e s t i n a l mucosa of r a t s s a c r i f i c e d 5 minutes a f t e r r e c e i v i n g H -thymidine by i n t r a v e n o u s i n j e c t i o n through the t a i l v e i n . T h i s was expected, s i n c e the r a d i o -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 the c e l l s by the b l o o d stream, where-as by the subcutaneous technique the p r e c u r s o r has t o pass through v a r i o u s c e l l compartments b e f o r e e n t e r i n g the b l o o d . A d m i n i s t r a t i o n o f the p r e c u r s o r by 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 o f p r e c u r s o r Time of exposure t o the p r e c u r s o r I n j e c t e d dose of H -thymidine i n mc % I n c o r p o r a t i o n of H^-thymidine per mg o f DNA i s o l a t e d subcutaneous 24 h r . 0.083 0.03 i n t r a p e r i t o n e a l i n t r a p e r i t o n e a l 24 h r . 24 h r . 0.083 0.083 0.07 0.08 subcutaneous 20 min. 0.083 0.006 subcutaneous 10 min. 0.083 0.001 subcutaneous 5 min. 0.083 0.0009 i n t r a v e n o u s 5 min. 0.083 0.003 i n t r a v e n o u s 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 employed f o r l a t e r experiments. 7. 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 the DNA f r a c t i o n s o b t a i n e d by ECTEOLA-cellulose chromatography. On the b a s i s of the c o n c l u s i o n s p o s t u l a t e d by Bendich e_t a l _ . (101) t h a t the f r a c t i o n s o b t a i n e d on ECTEOLA-cellulose 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 mat-e r i a l might be observed i n the f r a c t i o n s . I t might be expected, t h a t the s p e c i f i c a c t i v i t i e s would be d i f f e r e n t i n the supposedly lower m o l e c u l a r weight f r a c t i o n s , i f t hese f r a c t i o n s e x i s t indeed i n v i v o . Such d i f f e r e n c e s might i n d i c a t e c e r t a i n p r e c u r s o r r e l a t i o n s h i p s t o the h i g h e r m o l e c u l a r weight f r a c t i o n s . Any s p e c i a l p r e c u r s o r r e l a t i o n s h i p s of the d i f f e r e n t DNA f r a c t i o n s ought to be more d i s t i n c t l y demonstrated i n the " s h o r t time" experiments, where the animals are exposed t o the s p e c i f i c DNA p r e c u r s o r , t r i t i a t e d t h y m i d i n e , o n l y f o r a s h o r t p e r i o d of t i m e . I t might be assumed, t h a t as the time of exposure t o the t r i t i a t e d t hymidine i n c r e a s e d , the e x t e n t of i n c o r p o r a t i o n would s h i f t from one f r a c t i o n t o the o t h e r , thus r e v e a l 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 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 these d i f f e r e n t f r a c t i o n s . The fundamental e x p e r i m e n t a l requirement f o r approaching 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 procedures which are r e p r o d u c i b l e from experiment t o experiment, and which would r e p r e s e n t the s t a t e of the DNA as i t e x i s t s i n v i v o . At p r e s e n t , even the m i l d e s t i s o l a t i o n procuedures f o r mammalian DNAs, do not 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 f a r as the ECTEOLA-c e l l u l o s e f r a c t i o n a t i o n procedure i s concerned, 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 the method i s not as r e p r o d u c i b l e as i t was c l a i m e d by o t h e r s (101,121-124). 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 a bsorbing m a t e r i a l e l u t e d i n the 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 the ECTEOLA-chromatograms i n t o t h r e e 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 the 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 of 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 probably r e p r e s e n t i n g the lower m o l e c u l a r weight p o l y n u c l e o t i d e s ; f r a c t i o n I I a l l 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 e l u t e d w i t h i n c r e a s i n g NH^ c o n c e n t r a t i o n s i n 2M NaCl, and f r a c t i o n I I I r e p r e s e n t i n g the m a t e r i a l o b t a i n e d w i t h 0.5 and 1.0N NaOH. The d i s t r i b u t i o n of the t o t a l r a d i o a c t i v i t y was c a l c u l a t e d f o r these t h r e e main areas of the chromatograms of each DNA preparation:.. Table IX summarizes the r e s u l t s o f the r a d i o a c t i v e measurements o b t a i n e d f o r the t r i t i u m - l a b e l l e d DNA p r e p a r a -t i o n s . I t can be seen, t h a t the d i s t r i b u t i o n of the r a d i o a c t i v e m a t e r i a l was d i f f e r e n t from experiment t o experiment. No d e f i n i t e o r c h a r a c t e r i s t i c p a t t e r n can be observed i n the " s h o r t exposure time experiments" or i n the p r e p a r a t i o n s o b t a i n e d from the 24 hour i n j e c t e d a n i m a l s . I f one compares thesB r e s u l t s w i t h those o b t a i n e d i n Table V I I , a s i m i l a r i t y can be n o t i c e d between the p a t t e r n of fl the percentage 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 and u l t r a v i o l e t a b s o r b i n g mat-e r i a l i n the t h r e e f r a c t i o n s of most of the DNA samples. There are e x c e p t i o n s , however, the most s t r i k i n g b e i n g the DNA o b t a i n e d from r a t s i n j e c t e d w i t h the p r e c u r s o r 20 minutes p r i o r t o s a c r i f i c e . In t h i s p r e p a r a t i o n , 54.5% of the t o t a l r a d i o a c t i v i t y was 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 29.1% o f the 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 m a t e r i a l was c o n t a i n e d i n t h i s f r a c t i o n . T h i s 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 I I I . S i m i l a r f i n d i n g s can be observed i n two 5 minute experiments. In the o t h e r 5 minute experiment, however, a d i f f e r e n t p a t t e r n was o b t a i n e d . Here the r a d i o a c t i v i t y seemed to 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 of the f r a c t i o n s . On c a l c u l a t i n g the percentage r e c o v e r y 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 f r a c t i o n -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 found, t h a t a p p r o x i m a t e l y 85% o f the t o t a l counts were l o s t d u r i n g the f r a c t i o n a t i o n . S i n c e o b s e r v a t i o n s d e s c r i b e d p r e -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 o f r a d i o a c t i v i t y occurred mostly from the f i r s t 95 TABLE IX. Comparison of the Percentage of the R a d i o a c t i v i t y i n the Main F r a c t i o n s Obtained from ECTEOLA-Chromatography DNA P r e p a r a t i o n s % of the t o t a l r a d i o a c t i v i t y I I I I I I A l l f r a c t i o n s A l l f r a c t i o n s A l l f r a c t i o n s DNA e l u t e d w i t h e l u t e d w i t h e l u t e d w i t h p r e p a r a t i o n i n c r e a s i n g NaCl i n c r e a s i n g NH^ i n c r e a s i n g NaOH c o n c e n t r a t i o n c o n c e n t r a t i o n c o n c e n t r a t i o n H -thymidine i n j e c t e d subcutan-e 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 min. 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 min. ,,3 H -t h y m i d i n e 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. 3 H -thym i d i n e i n j e c t e d subcutan-e o u s l y 20 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 -thym i d i n e i n j e c t e d subcutan-e o u s l y 10 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 -thym i d i n e i n j e c t e d subcutan-e 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 -thym i d i n e i n j e c t e d i n t r a v e n -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 Bias s o n i c a t e d f o r 1 min. 3 H -thym i d i n e i n j e c t e d i n t r a v e n -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. 71.7 26.8 1.5 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 96 f r a c t i o n s , t h i s l o s s c o u l d have obscured 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 lower s p e c i f i c a c t i v i t y i n the f i r s t f r a c t i o n s . DNA samples from animals i n j e c t e d 24 hours 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 s p e c i f i c 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 . Even i n these samples, however, the percentage 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 e l u t e d by a l k a l i n e solventsseems t o be s l i g h t l y lower than of those e l u t e d by NaCl. There are no e x t e n s i v e i n v e s t i g a t i o n s of ECTEOLA-cellulose f r a c t i o n a t i o n o f DNA u s i n g r a d i o i s o t o p e t e c h n i q u e s . The experiments of Bendich e t a l . (141) 14 w i t h 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. are probably comparable w i t h the 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 the DNA p r e p a r a t i o n s o f the 24 hour "exposure t i m e " experiments. These i n v e s t i g a t o r s have grown JE. c o l i i n the presence of 14 C - 5 - b r o m o u r a c i l and the DNA was e x t r a c t e d from the organisms 24 hours l a t e r . Chromatographic s e p a r a t i o n of the 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 a b s o r b i n g peaks. The peak e l u t e d w i t h 0.5N NaOH had v e r y low s p e c i f i c a c t i v i t y , and they 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 the e s s e n t i a l g e n e t i c DNA, whereas the o t h e r f r a c t i o n s might 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 r e s u l t s d e s c r i b e d i n the present 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 minutes 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 of r a d i o a c t i v i t y was i n c o r p o r a t e d i n t o the 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 . The e x p e r i m e n t a l r e s u l t s of F r i e d k i n and Wood (143) are a l s o i n apparent c o n t r a d i c t i o n t o Bendich's f i n d i n g , a l t h o u g h the two 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 used mammalian t i s s u e s and in_ 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 the r a d i o -14 a c t i v e p r e c u r s o r (C - t h y m i d i n e ) . 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 DNA was 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 procedure of Kay et a l . (144). I t was found, t h a t the DNA f r a c t i o n most d i f f i c u l t t o ex-t r a c t from the t i s s u e by the d e t e r g e n t had the h i g h e s t s p e c i f i c a c t i v i t y . More-ove r , when the DNA f r a c t i o n i s o l a t e d by the detergent procedure was f r a c t i o n a t e d 97 on ECTEOLA-column, the 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 the h i g h e s t 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) assumed t h a t DNA molecules most d i f f i c u l t t o e x t r a c t w i t h the d e t e r g e n t , and t o e l u t e from the columns, r e p r e s e n t the s p e c i e s which 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 the b i o s y n t h e s i s of DNA. The d e f i n i t e s p e c i f i c a c t i v i t y p a t t e r n r e p o r t e d by F r i e d k i n and Wood (143) 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 f r a -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 not obaserved i n any o f the DNA p r e p a r a t i o n s s t u d i e d d u r i n g the present i n v e s t i g a t i o n . The r e p o r t o f Qsawa and S a k a k i (194) seems t o c o n t r a d i c t a l l of the p u b l i c a t i o n s c o n c e r n i n g 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 d i f f e r e n t 32 DNA f r a c t i o n s . 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 the f r a c t i o n s of r a b b i t appendix 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 not 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 and a l s o , t o a l e s s e r e x t e n t , the 24 hour i n c o r p o r a -t i o n experiments 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 a b s o r b i n g peaks. The r e s u l t s o b t a i n e d w i t h the ECTEOLA-cellulose f r a c t i o n a t i o n procedure are very d i f f i c u l t t o e v a l u a t e , due t o the n o n - r e p r o d u c i b i l i t y o f chromatographic p r o f i l e s , and t o 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 a f t e r d i a l y s i s . Moreover, i n most of the s h o r t exposure time experiments, the average l e v e l o f r a d i o a c t i v i t y was very low i n the samples, making the e v a l u a t i o n of the counts very d i f f i c u l t and i n a c c u r a t e . I f the e x p e r i m e n t a l r e s u l t s o b t a i n e d from the 20 min. and the two 5 min. i n j e c t e d animals are not e x p e r i m e n t a l a r t e -f a c t s , one c o u l d envisage some me t a b o l i c d i f f e r e n c e s between the law m o l e c u l a r weight and h i g h e r m o l e c u l a r weight f r a c t i o n s . These m e t a b o l i c d i f f e r e n c e s i f they e x i s t , c o u l d not be very s t r i k i n g , s i n c e a f t e r exposure t o the p r e c u r s o r f o r very 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 d e t e c t e d i n - a l l o f the f r a c t i o n s . 98 I I . Experiments w i t h DNA L a b e l l e d w i t h Both Carbon-14 and T r i t i u m 1. Design of the i n . v i v o experiments and p r e p a r a t i o n of the double l a b e l l e d t i s s u e . Because of the 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 on ECTEOLA-cellulose, a t t e n t i o n was c e n t e r e d on the use o f MAK columns, because 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 o f f e r e d advantages which would be s u i t a b l e f o r the present purposes. To f u r t h e r i n c r e a s e the s e n s i t i v i t y f o r 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 the d i f f e r e n t DNA f r a c t i o n s , 14 use was made of the double l a b e l l i n g t e c h n i q u e . 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 under 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 very use-f u l i n experiments i n which the p o s s i b l e t u r n o v e r of a product and 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 i n t o the product has t o be measured i n the same e x p e r i -mental a n i m a l s . S i n c e p r e v i o u s o b s e r v a t i o n s 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 the 24 hour exposure t i m e , the r a d i o a c t i v e p r e c u r s o r was w e l l i n c o r p o r a t e d i n t o every f r a c t i o n of the DNA, i t was f e l t t h a t t h i s l e n g t h o f time would be s u i t a b l e t o l a b e l the b u l k of the DNA i n the i n t e s t i n a l mucosa o f r a t . Each of a group of 5 male W i s t a r r a t s was i n j e c t e d i n t r a v e n o u s l y 3 through the t a i l v e i n w i t h 0.5 ml of a s o l u t i o n c o n t a i n i n g 0.1 mc of H - t h y m i d i n e and 8,2yt<.mof thymidine 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 animals were s t a r v e d f o r 24 hours f o l l o w i n g the i n j e c t i o n of the r a d i o a c t i v e m a t e r i a l , and each then 14 r e c e i v e d i n t r a v e n o u s l y 0.5 ml of a s o l u t i o n c o n t a i n i n g 0.033 mc C - t h y m i d i n e and 8.2/<.*nof thymidine, 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 animals were k i l l e d 20 or 40 minutes a f t e r the second 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 assays of H and C i n the same sample, the i s o t o p e r a t i o n s , H : C^ 4, should be between 1 to 15. I t was hoped, t h a t by choosing these time 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 , the d e s i r e d r a t i o o f H :C 99 i n v i v o would be o b t a i n e d . The measurements o f the double l a b e l l e d DNA p r e p a r a t i o n s i n d i c a t e d t h a t the d e s i r e d r a t i o was o b t a i n e d . 2. The r e p r o d u c i b i l i t y of the MAK f r a c t i o n a t i o n method. 5in c e the i n t r o d u c t i o n (102) and development (129) o f the MAK chroma-tography r e l a t i v e l y few i n v e s t i g a t o r s used t h i s method f o r f r a c t i o n a t i n g DNAs o f mammalian o r i g i n . In 1961 P h i l i p s o n (137) r e p o r t e d the chromatography of t o t a l n u c l e i c a c i d s from He La c e l l s on MAK columns. He suceeded i n s e p a r a t i n g s o l u b l e RNA, DNA and two d i f f e r e n t h i g h m o l e c u l a r weight RNA f r a c t i o n s . The average con-c e n t r a t i o n o f sodium c h l o r i d e c a l c u l a t e d f o r the e l u t i o n o f the peak of DNA was 0.705M. When the DNA was rechromatographed i n a narrower g r a d i e n t , a s i n g l e peak was o b t a i n e d a g a i n , which was e l u t e d at the same sodium c h l o r i d e c o n c e n t r a t i o n as o r i g i n a l l y . S i m i l a r f i n d i n g s , i n d i c a t i n g a s i n g l e peak f o r DNA p r e p a r a t i o n s on MAK columns were r e p o r t e d by o t h e r i n v e s t i g a t o r s (129,134-135) u s i n g b a c t e r i a l and phage DNAs. T h i s f i n d i n g was r a t h e r s u r p r i s i n g c o n s i d e r i n g the m u l t i p l e - p e a k p r o f i l e s of DNAs o b t a i n e d by o t h e r f r a c t i o n a t i o n t e c h n i q u e s . The p o s s i b i l i t y , however, t h a t the i n d i v i d u a l f r a c t i o n s i n the s i n g l e DNA peak r e p r e s e n t d i f f e r e n t DNA s p e c i e s , c o u l d not be excluded by these i n v e s t i g a t i o n s . S i n c e Sueoka and Cheng (130) demonstrated t h a t the column d i f f e r e n t i a t e s between n u c l e i c a c i d p r e p a r a t i o n s o f d i f f e r e n t base c o m p o s i t i o n and hydrogen bond c o n t e n t , i t was hoped t h a t , i f t h e r e are such d i f f e r e n c e s amongst the DNA molecules of r a t i n t e s t i n a l mucosa, these would be d e t e c t e d by MAK f r a c t i o n a t i o n . Moreover, i t has been shown (129) t h a t low m o l e c u l a r weight o r shear-degraded p r e p a r a t i o n s are e l u t e d from the column by lower s a l t c o n c e n t r a t i o n s . To t e s t the r e p r o d u c i b i l i t y of the MAK f r a c t i o n a t i o n t e c h n i q u e , some p r e l i m i n a r y experiments were c a r r i e d out i n which d u p l i c a t e samples of DNA prepared by the method of Medawar and Zubay (159) and commercial DNA ( C a l . C o r p . Biochem. Res.) were f r a c t i o n a t e d . A sample of s o l u b l e RNA was a l s o chromatographed. 100 T a b l e s X A and B summarize the d i s t r i b u t i o n of 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 amongst the main f r a c t i o n s e l u t e d . I t can be noted, t h a t i n the DNA sample o b t a i n e d by the method o f Med-awar and Zubay (159) a p p r o x i m a t e l y 50% of the 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 m a t e r i a l was 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 c o n c e n t r a -t i o n of 0.66M i n the e l u a t e . In the case of the commercial DNA sample, more m a t e r i a l was e l u t e d by lower s a l t c o n c e n t r a t i o n s (0.09 t o 0.21M NaCl) i n d i c a t i n g perhaps a h i g h e r amount of d e g r a d a t i o n o r i m p u r i t i e s . I t i s noteworthy t h a t a l -though the second sample of each type of DNA was chromatographed 2 weeks a f t e r the . f i r s t , t h e r e were no marked changes observed i n the e l u t i o n p a t t e r n . T h i s would tend t o suggest, t h a t no s i g n i f i c a n t changes had occured d u r i n g the 2 week p e r i o d of s t o r a g e . When s-RNA was a p p l i e d on the column, 92.7% o f the 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 m a t e r i a l was e l u t e d a t an i n i t i a l NaCl c o n c e n t r a t i o n of 0.35M. The r e s u l t s i n d i c a t e a q u i t e r e a s o n a b l e r e p r o d u c i b i l i t y o f the e l u t i o n p a t t e r n w i t h the MAK f r a c t i o n a t i o n t e c h n i q u e . The p r e v i o u s o b s e r v a t i o n of s e v e r a l workers (130,137) t h a t DNA can be s e p a r a t e d from s-RNA has been c o n f i r m e d . An i n t e r e s t i n g p o i n t may be mentioned i n case of the DNA p r e p a r a t i o n e x t r a c t e d by the method o f Medawar and Zubay (159). T h i s p r e p a r a t i o n had been 3 o b t a i n e d from r a t s a l l o w e d t o i n c o r p o r a t e H - t h y m i d i n e f o r 10 minutes p r i o r t o s a c r i f i c e . A l i q u o t s of 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 experiments on ECTEOLA-cellulose, and a very complex e l u t i o n p r o f i l e has been o b t a i n e d c o n s i s t i n g of s e v e r a l peaks e l u t e d by d i f f e r e n t NaCl and NH^ c o n c e n t r a -t i o n s . These f i n d i n g s were r e p r e s e n t e d i n F i g u r e s 19 A and B. On the o t h e r hand, on MAK f r a c t i o n a t i o n o n l y one main peak was o b t a i n e d , as i t i s shown i n F i g u r e s 23 and 24. The r e s t of 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 was e l u t e d more o r l e s s c o n t i n u o u s l y . The d i f f e r e n c e s are r a t h e r s t r i k i n g between the e l u t i o n p r o f i l e s 101 TABLE X . A. Comparison o f F r a c t i o n a t i o n on MAK Column of A l i q u o t s o f DNA P r e p a r a t i o n DNA sample Percent o f t o t a l absorbance Prepared by the method of Medawar and Zubay (159( In f r a c t i o n s e l u t e d by 0.09-0.13M NaCl In f r a c t i o n s e l u t e d by 0.66-0.73M NaCl 1 s t sample 16.1 49.1 2nd sample* 17.6 48.6 Commercial DNA In f r a c t i o n s e l u t e d by 0.09-0.21M NaCl In f r a c t i o n s e l u t e d by 0.42-0.86M NaCl 1 s t sample 42.1 32.4 2nd sample* 36.9 29.5 . * The 2nd samples were chromatographed 2 weeks l a t e r B. F r a c t i o n a t i o n R e s u l t s o f s-RNA of Yeast Percent o f t o t a l absorbance In f r a c t i o n s e l u t e d by 0.1 t o 0.27M NaCl In f r a c t i o n s e l u t e d by 0.35-0.57M NaCl 4.9 92.7 102 0.3 0.2 0.1 o CO O J +> a o o c a Xi u o w Xi < A A 20 A NaCl gradient 1.2 0.8 +> c rt w G •H rt «H 0 >> +> rt 0.4 -I I , j 40 60 80 100 Tube Number 120 140 Figure 23. F r a c t i o n a t i o n of DNA by chromatography on MAK. The DNA was prepared by the method of Medawar and Zubay (159). 103 0.3 0.2 0.1 o co OJ -p rt <D O C rf J3 U o CO .Q & A A A NaCl gradient fl A 20 c rt iH w fl o rt «w 0 >> +> •H rt 11 1.2 0.8 0.4 40 60 80 Tube Number 100 120 140 Figure 24. 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 preparat ion as Figure 23, but which had been stored 2 weeks i n the c o l d . 104 o b t a i n e d u s i n g ECTEOLA-cellulose and MAK, w i t h the same DNA p r e p a r a t i o n . Two e x p l a n a t i o n s are p o s s i b l e : e i t h e r the m u l t i p l e peak p r o f i l e s o f ECTEOLA chromato-graphy are e x p e r i m e n t a l a r t e f a c t s produced by the exchanger, or MAK chromatography r e s u l t s i n an incomplete r e s o l u t i o n o f the heterogeneous DNA p o p u l a t i o n . I t i s f e l t t h a t both e x p l a n a t i o n s might be t r u e t o some e x t e n t . I t has been d i s c u s s e d p r e v i o u s l y t h a t o b s e r v a t i o n s r e g a r d i n g 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 from the DNA f r a c t i o n s a f t e r ECTEOLA chromatography, and perhaps the Sephadex chromatographic experiments might i n d i c a t e some changes i n the DNA p r e p a r a t i o n s brought about by the ECTEOLA-exchanger i t s e l f . An i n d i c a t i o n t h a t the main peak o b t a i n e d by MAK chromatography 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 suggested i n an experiment i n which DNA was chromatographed on a column, i n which the s i z e o f the second l a y e r was doubled. T h i s i s the l a y e r which c o n t a i n s the p r o t e i n a t e d C e l i t e (129). The 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 which was o b t a i n e d from a DNA p r e p a r a t i o n e x t r a c t e d by the method of C o l t e r _e_t a l . (147) from animals 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 minutes p r i o r s a c r i f i c e . I t can be seen t h a t , a s p l i t has occurred i n the main peak, i n d i c a t i n g perhaps an inhomogeneity o f the DNA s p e c i e s w i t h i n the main peak. T h i s f i n d i n g was conf i r m e d i n subsequent ex-periments. I t has t o be mentioned, t h a t i n F i g u r e 25 o n l y one s m a l l peak preceded immediately the main DNA peak i n s t e a d o f the two u s u a l l y observed i n l a t e r ex-periments u s i n g DNA prepared by the method of C o l t e r (147). In t h i s p a r t i c u l a r experiment, the incomplete s e p a r a t i o n o f the peaks might have been due t o some s l i g h t v a r i a t i o n i n the r a t e o f f l o w o f the e l u a n t , o r 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 g r a d i e n t . Of f u r t h e r i n t e r e s t i s the experiment i n which the sample o f the DNA mentioned above, was chromatographed on a MAK column, i n which o n l y the C e l i t e component of the second l a y e r o f the column was doubled. 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 test) NaCl gradient 1.2-0.8-0.4. 0) v a a Xi u o w Xi < Figure 25. 40 100 -1.2 -0.8 -0.4 120 60 80 Tube Number Chromatography of DNA on a modified MAK column. The amount of mater ia ls i n the second layer was doubled. The DNA was prepared by the method of C o l t e r et a l . ( l 4 7 ) from animals allowed to incorporate TTRe C 1 - l a b e l l e d precursor 40 min. p r i o r to s a c r i f i c e . 106 shown i n F i g u r e 26. Under these c o n d i t i o n s t h e r e was no s p l i t i n the main peak. However, a much c l e a r e r s e p a r a t i o n of the 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 s i m i l a r t o the f i n d i n g s shown i n F i g u r e 25. In both c a s e s , the background absorb-i n g u l t r a v i o l e t m a t e r i a l almost c o m p l e t e l y d i s a p p e a r e d . F u r t h e r s u p p o r t i n g the concept of the h e t e r o g e n e i t y of the i n d i v i d u a l f r a c t i o n s o f the main DNA peak are the experiments of Cheng and Sueoka (103) which w i l l be d i s c u s s e d l a t e r . To summarize the 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 the r e p r o d u c i b i l i t y o f MAK chromatographic t e c h n i q u e , Table XI compares the d i s t r i b u t i o n 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 the d i f f e r e n t areas of the MAK chromatograms 14 3 o f the v a r i o u s C and H 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 s o n a b l y good reproduc-i b i l i t y was found f o r the chromatographic p r o f i l e s o f the DNA p r e p a r a t i o n s o b t a i n -ed from experiment t o experiment. T h i s i s e s p e c i a l l y t r u e f o r the main u l t r a -v i o l e t a b s o r b i n g areas o b t a i n e d by an i n i t i a l NaCl c o n c e n t r a t i o n of 0.66M, and f o r the s m a l l peaks immediately p r e c e d i n g the main DNA peak, and e l u t e d by an i n i t i a l NaCl c o n c e n t r a t i o n of 0.45M ( F r a c t i o n I I , Table X I ) . I t was a l s o found, t h a t the r e c o v e r i e s 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 from the column was ver y good. 3. Comparison of the MAK chromatographic p r o f i l e s o f the DNA p r e p a r a t i o n s o b t a i n e d by the methods of Medawar and Zubay (159) and C o l t e r et a l . (147) from 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 experiments showed, t h a t chromatography of DNA on ECTEOLA-c e l l u l o s e 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 from experiment t o experiment. In these experiments the DNA was prepared by the method of Medawar and Zubay. I t was f e l t t o be of i n t e r e s t , 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 MAK columns of DNA prepared 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 o b t a i n e d w i t h the DNA e x t r a c t e d by the procedure o f C o l t e r et a l . ( 1 4 7 ) from the same 107 1.6 1.2-0.8 0 .4-o o a a u o Absorbance at 260 m/c Absorbance at 595 m.//. (Dische test ) NaCl gradient - 1.2 - 0.8 - 0.4 Figure 26. 60 80 100 Tube Number Chromatography of DNA on a modified MAK column. The C e l i t e component of the second layer was doubled. The DNA sample was taken from the same stock so lu t ion as i n Figure 25. 108 TABLE X I . Comparison of the Percentage of the U l t r a v i o l e t A b sorbing M a t e r i a l i n the F r a c t i o n s Obtained from MAK-Chromatography of C 1 4 and H 3 L a b e l l e d DNA P r e p a r a t i o n s DNA* p r e p a r a t i o n % of t o t a l absorbance i n the f r a c t i o n s I I I I I I IV NaCl M= NaCl M= NaCl M= NaCl M= 0.09-0-.44 0.45-0.65 0.66-0.73 0.74.0.90 T o t a l absorbance a t 260 r e c o v e r e d 14 C -thym 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 prepared by the method of C o l t e r et a l . (147) 14 C -thymidine 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 prepared by the 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 -thym 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 prepared by the method of Medawar and Zubay (159) 14 C -thym 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 prepared by the method of C o l t e r et a l . (147) 14 C -thymidine' 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 . DNA prepared by the method of C o l t e r e t a l . (147) 14 C -thym 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 . DNA prepared by the method o f C o l t e r e t a l . (147) 20.4 15.6 53.0 11.0 19.9 13.1 55.5 11.5 28.4 70.1 27.7 10.1 60.2 27.4 9.9 59.9 vl8.3 13.3 60.7 1.5 2.0 2.8 7.4 101.7 96.1 111.2 108.0 104.4 92.4 * A l l animals r e c e i v e d H - l a b e l l e d thymidine 24 h r . b e f o r e the 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 . 109 pooled mucosal t i s s u e . F i g u r e s 27,28 i l l u s t r a t e the chromatographic e l u t i o n p a t -t e r n s o f DNA samples i s o l a t e d by the phenol e x t r a c t i o n method (147). F i g u r e 28 r e p r e s e n t s a DNA p r e p a r a t i o n which was f u r t h e r p u r i f i e d by two e t h a n o l p r e c i p i -t a t i o n s t e p s , whereas F i g u r e 27 i s the chromatogram of the d i a l y z e d e x t r a c t o f the t i s s u e from which the DNA was not p u r i f i e d f u r t h e r . These chromatographic p r o f i l e s are very s i m i l a r , and suggest t h a t f o r purposes of r o u t i n e chromatography on MAK column, i t i s not necessary t o i s o l a t e DNA from the e x t r a c t ; by, ethanol.-p r e c i p i t a t i o n . F i g u r e 29 shows the e l u t i o n p a t t e r n of DNA prepared by the s o n i c a t i o n method (159). With t h i s DNA the main peak was e l u t e d at the same NaCl c o n c e n t r a t i o n as the sample e x t r a c t e d by the phenol method (147). However, i n s p e c t i o n of the data i n Table 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 a b s o r b i n g m a t e r i a l was e l u t e d i n the main DNA peak. The two s m a l l peaks which precede the main peak i n the phenol e x t r a c t e d samples are c o m p l e t e l y m i s s i n g i n F i g u r e 29. A h i g h e r amount of 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 a t the very b e g i n -n i n g of the f r a c t i o n a t i o n , i n d i c a t i n g perhaps some de g r a d a t i o n of the DNA sample. T h i s d e g r a d a t i o n i s , however, not v e r y e x t e n s i v e , s i n c e o t h e r DNA p r e p a r a t i o n s e x t r a c t e d by the phenol method showed o c c a s i o n a l l y the appearance o f peaks s i m i l a r i n s i z e and a t the same p l a c e on the chromatogram. Moreover, r a d i o a c t i v e measure-ments and diphenyhamine t e s t s i n d i c a t e d t h a t the peaks e l u t e d by an i n i t i a l s a l t c o n c e n t r a t i o n o f 0.09M c o n t a i n e d l i t t l e o r no r a d i o a c t i v i t y and very s m a l l amount o f d e o x y r i b o s e - p o s i t i v e m a t e r i a l . These f i n d i n g s seemed t o i n d i c a t e , t h a t the peaks r e p r e s e n t c o n t a m i n a t i n g 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 s , r a t h e r than DNA d e g r a d a t i o n p r o d u c t s . From the r e s u l t s of the comparison i t can be concluded t h a t the DNA prepared by the method of Medawar and Zubay (159) undergoes no more de g r a d a t i o n than does DNA prepared by the method o f C o l t e r e t a l . I t i s g e n e r a l l y accepted t h a t the methods u s i n g phenol e x t r a c t i o n i n t r o d u c e l i t t l e damage t o the primary 110 0.6 0.4 o C O OJ •p rt o o fl rt Xi u o w Xi < 0.2-A NaCl gradient Jl - i — 20 — i — 40 60 Tube Number - i — 80 - 1.0 0.8 4-> C rt fl •rl -0 .6 0.4 •0.2 100 120 Figure 27. 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 ra t by the method of C o l t e r et _al. (147). The DNA was not p u r i f i e d by ethanol p r e c i p i t a t i o n . The animals were exposed to C -thymidine 40 min. before s a c r i f i c e . I l l 0.6 0.4 0.2 A NaCl gradient Jl 4J o CO O J •p a o o a a J2 U O tfi < A A +-> • C cS rH 20 40 60 Tube Number 1.2 0.8 0.4 Figure 28., Chromatography on MAK column of double l a b e l l e d DNA obtained from the i n t e s t i n a l mucosa of rat by the method of Colter et jol. (147). The animals were exposed to C - l a b e l l e d thymidine 40 min. before 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 gradient 0.6 -1.2 0.4 O CD OJ 4-> ci 0 O fl ci jo u o CO < 0.2_ A 20 — i i 1— 40 60 80 Tube Number 100 fl ci 3 c •r-l o ci 55 o 120 Figure 29. 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). 0.8 -0.4 113 and secondary s t r u c t u r e of DNA. Because of the g r e a t e r ease o f o p e r a t i o n , the phenol e x t r a c t i o n technique was adopted 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 . 14 3 4. The C and H e l u t i o n p r o f i l e s o f the double l a b e l l e d DNA p r e p a r a t i o n s , 3 14 and the comparison of the H /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 chromatography. To i n c r e a s e the s e n s i t i v i t y o f the MAK f r a c t i o n a t i o n t e c h n i q u e , s e v e r a l i n v e s t i g a t o r s (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 minor physico-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 . For p u l s e 32 l a b e l l i n g experiments P 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 . One main disadvantage of t h i s method i s however, t h a t the i s o t o p e i s not s p e c i f i c f o r DNA. 5ampson et a l . (154) used t h i s t e c h n i q u e t o demonstrate the e x i s t e n c e of 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 exposing wheat seeds t o 32 P - i n o r g a n i c phosphate, two types of l a b e l l e d DNAs were sepa r a t e d on MAK columns. The time course of the appearance and disappearance o f the r a d i o a c t i v e phosphate 31 was f o l l o w e d i n these DNAs, a f t e r " c h a s i n g " w i t h P phosphate. The h i g h m o l e c u l a r 32 weight DNA behaved i n the expected f a s h i o n ; the s p e c i f i c a c t i v i t y o f P -DNA de-32 c r e a s e d 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 phosphate. By c o n t r a s t , the s p e c i f i c a c t i v i t y o f low m o l e c u l a r weight DNA decreased t o the v a n i s h i n g p o i n t 31 f o l l o w i n g the same exposure t o P . T h i s i n d i c a t e d t o them the l a t t e r DNA s p e c i e s was more l a b i l e than the former. 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 of chromatographic d i f f e r e n c e s , the double l a b e l l i n g t e c hnique has been e x t e n s i v e l y used (181). For example, Sueoka and Spiegelman (133) were a b l e t o d e t e c t chromatographic d i f f e r e n c e s between 3 14 H and C - 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 from the T2 - E_. c o l i system. By 32 3 the simultaneous use of P and H l a b e l l i n g the 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 form of the cbx 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 expected, t h e r e f o r e , t h a t i f d u r i n g the b i o s y n t h e s i s of DNA, the newly s y n t h e s i z e d molecule i s i n somewhat d i f f e r e n t p h y s i c a l or c h e m i c a l form, such a DNA s p e c i e s might be det e c t e d by MAK chromatography. 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 chromatographic d i f f e r e n c e s , a double l a b e l was i n t r o d u c e d t o the 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 the two l a b e l s on the same p r e c u r s o r . 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 d i f f e r e n t paths o f 14 3 e n t r y . In the present i n v e s t i g a t i o n C - and H - l a b e l l e d thymidine were used 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 expected, t h a t i f the C - l a b e l l e d DNA s y n t h e s i z e d i n the animals d u r i n g the 40 or 20 minutes exposure time to the p r e c u r s o r was d i f f e r e n t 3 from the 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 the 24 hours o f ex-3 14 posure t i m e , the e l u t i o n p r o f i l e s o f H and C should be d i f f e r e n t . S i m i l a r l y , i f the two and l a b e l l e d DNAs are the same o r very s i m i l a r p h y s i c a l l y and 3 14 c h e m i c a l l y , the e l u t i o n p r o f i l e s of H and C should be i d e n t i c a l , o r the r a t i o 3 14 o f H :C counts should 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 the double l a b e l l e d DNA p r e p a r a t i o n s are shown i n F i g u r e s 25,27,30,31. These F i g u r e s i l l u s t r a t e the e l u t i o n of the m a t e r i a l a b s o r b i n g a t 260rry«. as the f u n c t i o n of NaCl c o n c e n t r a t i o n . In F i g u r e s 25,30,31 the r e s u l t s o f q u a n t i t a t i v e D i s c h e t e s t s f o r d e o x y r i b o s e are also;,shown)(165). The radiochromatograms are r e p r e s e n t e d i n F i g u r e s 32,33. I f one compares the 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 the C and H a c t i v i t i e s i n the d i f f e r e n t f r a c t i o n s o f the d i f f e r e n t DNA p r e p a r a t i o n s the f o l l o w i n g con-14 3 e l u s i o n s can be made: Most of the 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 both C and H 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 the main peak e l u t e d by 0.66M NaCl. These ob-s e r v a t i o n s are f u r t h e r supported by the data i n Table XIT, which show t h a t be-tween 94-97% o f the t o t a l r a d i o a c t i v i t y e l u t e d from the column i s i n c o r p o r a t e d 14 3 i n t o the main DNA peak. The C :H e l u t i o n p r o f i l e s are almost i d e n t i c a l i n 115 1.6 -1.2 -0.8 0.4-o c ci U o w Absorbance at 260 m/<-Absorbance at 595 m/^  (Dische test) NaCl gradient A A 40 60 80 Tube Number 100 -0.8 -0.4 120 Figure 30, Chromatography on MAK column of double l a b e l l e d DNA obtained from i n t e s t i n a l mucosa of rat by the method of Colter et al.(147). The animals were exposed to C 1 -thymidine 20 min. before s a c r i f i c e . 116 Absorbance at 260 m./*-Absorbance at 595 myd-(Dische test) NaCl gradient Tube Number Figure 31. Chromatography on MAK of double l a b e l l e d DNA obtained from i n t e s t i n a l mucosa of rat by the method of CoJ^er et _al.(147). The animals were exposed to C -thymidine 20 min. before s a c r i f i c e . 117 C Q 0) • H 4-> • H > • H •P O to 2 -1 . C 1^ a c t i v i t y H a c t i v i t y © © 20 i • 1 40 60 Tube Number 80 100 120 Figure 32, El u t i o n of r a d i o a c t i v i t y of 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 Figure A was determined i n the eluates shown i n Figure 25, and the r a d i o a c t i v i t y of that of Figure B corresponded to Figure 27. 1 1 8 cn cu •rl •P •H > • r l -t-> O < o (3 to 5n 4-3-C 1 4 A c t i v i t y H° A c t i v i t y 0 i -2-© 20 40 60 80 Tube Number 100 120 Figure 33, Elution of r a d i o a c t i v i t y of double l a b e l l e d DNA chromatographed on MAK column. The radio-a c t i v i t y i n Figure A was determined i n the eluates shown i n Figure 30, and that of Figure B corresponded to Figure 31. 119 TABLE X I I . 14 3 Comparison o f the % D i s t r i b u t i o n of C and H A c t i v i t i e s i n the Main and Minor F r a c t i o n s o f DNAs E l u t e d from MAK Columns. % of t o t a l H 3 a c t i v i t y % o f t o t a l C 1 4 a c t i v i t y DNA* sample peak e l u t e d w i t h Combined minor peak e l u t e d w i t h combined minor 0.66M peaks 0.66M peaks NaCl NaCl 14 C -thymidine 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 phenol method 97.5 2.5 97.3 2.7 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 phenol method and p r e c i p i t a t e d by e t h a n o l 96.4 3.6 97.1 2.9 14 C -thym 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 prepared by the s o n i c a t i o n method (159) 96.1 3.9 98.0 2.0 14 C -thymidine 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 . Phenol method (147) 97.9 2.1 95.9 4.1 14 C -thymidine 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 . Phenol method (147) 96.6 3.4 94.0 4.0 14 C -thym 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 . Phenol method (147) 95.3 4.7 94.8 5.2 * A l l animals r e c e i v e d H - l a b e l l e d thymidine 24 hours b e f o r e the 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 . 120 each case. V a r i a t i o n s can be n o t i c e d i n the areas where the a c t i v i t i e s are very low (5-10 cpm above background). T h i s made c a l c u l a t i o n of the H^/C^ r a t i o s v e r y i n a c c u r a t e and t h e r e f o r e , v a r i a t i o n s i n these areas on the diagrams are not con-s i d e r e d s i g n i f i c a n t . 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 MAk chromatography i t was found t h a t i n a l l cases 95 t o 100% of the t o t a l r a d i o a c t i v i t y a p p l i e d on the column was r e c o v e r e d . Moreover, no l o s s of r a d i o a c t i v i t y was observed from any f r a c t i o n a f t e r d i a l y s i s . S i n c e the t h r e e s m a l l e r peaks preceding the main DNA f r a c t i o n were c o n s i s t e n t l y o b t a i n e d f o r a l l DNA p r e p a r a t i o n s o b t a i n e d by the phenol e x t r a c t i o n procedure, i t was 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 r e p r e s e n t m a t e r i a l c o n t a i n i n g DNA. The two s m a l l e r peaks immediately pr e c e d i n g the main DNA peak were of s p e c i a l i n t e r e s t , s i n c e these were e l u t e d w i t h a p p r o x i m a t e l y the same NaCl c o n c e n t r a t i o n as the " m e t a b o l i c a l l y l a b i l e " DNA r e p o r t e d by Sampson et a l . (154). The f a c t t h a t i n none o f the experiments were these peaks found t o be r a d i o a c t i v e , suggests two p o s s i b l e e x p l a n a t i o n s . F i r s t , the peaks r e p r e s e n t u l t r a v i o l e t a b s o r b i n g m a t e r i a l which i s not DNA. Second, the f r a c t i o n s c o u l d i n d i c a t e a DNA s p e c i e s of such a h i g h r a t e of t u r n o v e r , t h a t r a d i o a c t i v i t y i s no l o n g e r d e t e c t a b l e even i n the DNA o b t a i n e d from animals r e c e i v i n g the p r e c u r s o r o n l y 20 min. b e f o r e s a c r i f i c e . I t was found, however, t h a t the phenol l a y e r of the t i s s u e e x t r a c t , which presumably 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 , c o n t a i n e d 14 c o n s i d e r a b l e amounts of C - a c t i v i t y even a f t e r 20 minutes post i n j e c t i o n . The r a d i o a c t i v e p r e c u r s o r may t h e r e f o r e be a v a i l a b l e f o r the s y n t h e s i s of DNA. The measurement of the d eoxyribose content of the d i f f e r e n t f r a c t i o n s of the MAK chromatograms, however, i n d i c a t e d t h a t the peaks e l u t e d w i t h an i n i t i a l NaCl c o n c e n t r a t i o n of 0.45M c o n t a i n e d n e g l i g i b l e amounts of DNA. These f i n d i n g s are a l s o r e p r e s e n t e d as the shaded areas i n F i g u r e s 25,26,30,31. Most of the deoxy-r i b o s e p o s i t i v e m a t e r i a l was e l u t e d i n the main DNA peak, i n f u r t h e r agreement 121 3 14 w i t h the r a d i o a c t i v i t y d e t e r m i n a t i o n s . Since H and C - l a b e l l e d t h ymidine can be regarded as s p e c i f i c p r e c u r s o r s o f the DNA (26-28) and the diphenylamine t e s t 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 those areas which 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 the o t h e r s m a l l peaks on the chromatograms r e p r e s e n t m a t e r i a l which i s not DNA, but which i s c l o s e l y a s s o c i a t e d w i t h the DNA and i s not removed even a f t e r e x t e n s i v e d i a l y s i s . A lthough f u r t h e r i d e n t i f i c a t i o n o f the two s m a l l peaks immediately p r e -c e d i n g ithe main DNA peak has not been attempted, i t i s suspected t h a t they may r e p r e s e n t s o l u b l e RNA. The b a s i s of t h i s assumption 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 the MAK column, the m a t e r i a l was e l u t e d a t ap p r o x i m a t e l y t h e same NaCl c o n c e n t r a t i o n . Moreover, the f r a c t i o n a t i o n experiment o f Lacks (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 from 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 immediately p r e c e d i n g the DNA f r a c t i o n were s-RNA. 14 3 Although the e l u t i o n p a t t e r n s o f C and H a c t i v i t i e s o f the 20 or 40 minute double l a b e l l i n g experiments d i d not 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 , the 3 . 14 c a l c u l a t i o n o f the H /C r a t i o s of the i n d i v i d u a l f r a c t i o n s o f the 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 . Table X I I I summarizes the r a t i o s 3 14 of the H /C a c t i v i t i e s o f the 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 MAK chromatography. An i n t e r e s t i n g d i f f e r e n c e became apparent, when each f r a c t i o n making up the main DNA peak was analyzed f o r r a d i o a c t i v i t y . The dat a o b t a i n e d are r e p r e s e n t e d i n F i g u r e 34. The diagram 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"^4 throughout the main DNA peak i n the 40 minute exposure time experiments. In the case o f 20 minute experiments the r a t i o s o f H^/C^ are h i g h e r 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 c o n t e n t i n these f r a c t i o n s . Due t o the v a r i a t i o n s encountered i n i n v i v o work, the average l e v e l o f r a d i o a c t i v i t y i n the DNA p r e p a r a t i o n s of the d u p l i c a t e experiments are not the same, but the 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 curve) are remarkably s i m i l a r i n each s e t of experiments. I t sho u l d be p o i n t e d out t h a t 122 TABLE X I I I Comparison of the H^/ C 1 4 a c t i v i t i e s i n the DNA F r a c t i o n s o b t a i n e d by MAK Chromatography. The r a t i o s o f H /C a c t i v i t i e s i n the f r a c t i o n s F r a c t i o n number and m o l a r i t y o f NaCl i n e l u a n t * * DNA p r e p a r a t i o n * I .0.09-0.44 I l l 0.66 - 0.73 IV 0.73-0.77 V 0.78 0.90 1 2 3 4 5 6 7 14 C -thym i d i n e i n j e c t e d 40 min. b e f o r e s a c r i -f i c e . DNA prepared by method of C o l t e r e t a l . (147). 4.6 5.3 5.4 5.3 5.6 5.5 5.0 6.7 2.8 9.7 14 C -thymidine i n j e c t e d 40 min. b e f o r e s a c r i -f i c e . DNA prepared by method of Medawar and Zubay (159). - 6.6 6.5 6.4 5.6 5.7 5.7 6.8 7.2 3.4 C -thym i d i n e i n j e c t e d 40 min. b e f o r e s a c r i -f i c e . DNA prepared by method of 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"*" 4-thymidine i n j e c t e d 20 min. b e f o r e s a c r i -f i c e . DNA prepared by method of 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 14.4 C 1 4 - t h y m i d i n e i n j e c t e d 20 min. b e f o r e s a c r i -f i c e . DNA prepared by method of C o l t e r e t a l . (147) - 4.1 5.3 6.8 7.5 7.5 7.B 8.8 8.1 1 10.5 3 14 * A l l animals r e c e i v e d H - l a b e l l e d thymidine 24 h r . b e f o r e the 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 I I e l u t e d by 0.45-0.65M NaCl d i d not c o n t a i n any r a d i o -a c t i v i t y 123 40 Min. C 40 " 20 " 20 " 14 incorporation, exp. 1 A " " 2 —^ •* l © " " 2 O 12.0 n 10.0 8.01 6.0 4.0' Figure 34. Fraction Number 3 14 H /C r a t i o s of the f r a c t i o n s of the DNA peak obtained by MAK chromatography. 124 r e c e n t l y Cheng and Sueoka (103) demonstrated t h a t chromatography on MAK columns of DNAs of animal o r i g i n y i e l d e d on e l u t i o n by NaCl g r a d i e n t one main DNA peak, i n which the 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 . The f r a c t i o n s e l u t e d by h i g h e r NaCl c o n c e n t r a t i o n s had lower 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 . T h i s f i n d i n g i n d i c a t e s t h a t the l a t e r f r a c t i o n s i n the main DNA peak had lower guanine-c y t o s i n e c o n t e n t . The c o n t r a r y was t r u e f o r the e a r l i e r f r a c t i o n s o f MAK chroma-tograms. The r e s u l t s of Cheng and Sueoka are best i n t e r p r e t e d by assuming t h a t t h e r e i s a h e t e r o g e n e i t y i n base c o m p o s i t i o n o f the f r a c t i o n s o f the main DNA v peak. R e c e n t l y 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-tography on MAK columns 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 c o n t e n t and i n m o l e c u l a r weight. S i n c e ' i n t h e 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 h i g h e r H"Vc"^  r a t i o s i n the f r a c t i o n s e l u t e d by h i g h e r s a l t 14 c o n c e n t r a t i o n d u r i n g the s h o r t e r 20 minutes exposure time t o the C - l a b e l l e d p r e c u r s o r , i t i s concluded t h a t t h e r e might be me t a b o l i c h e t e r o g e n e i t y i n these f r a c t i o n s . These f i n d i n g s c o u l d i n d i c a t e a h i g h e r t u r n o v e r r a t e o f the DNA s p e c i e s i n f r i c t i o n s 1 - 4 , i n F i g u r e 34, s i n c e d u r i n g t he 20 minutes exposure 14 times r e l a t i v e l y more C -thymi d i n e was i n c o r p o r a t e d i n t o them, than i n t o f r a c t i o n s 5-6. A f t e r 40 minutes i n c o r p o r a t i o n these 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 u n i f orm i n c o r p o r a t i o n o f C ^ - l a b e l l e d p r e c u r s o r was o b t a i n e d i n t o each of these f r a c t i o n s . The suggested m e t a b o l i c d i f f e r e n c e s i n the d i f f e r e n t DNA f r a c t i o n s are not very s t r i k i n g , however, s i n c e the 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 the t r i t i u m . Perhaps these d i f f e r e n c e s would be more d i s t i n c t l y demonstrated i n experiments o f even s h o r t e r exposure time i f the pro-blems o f a s s u r i n g a high enough i n c o r p o r a t i o n of the 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 i s o t o p e r a t i o d i f f e r e n c e s o b t a i n e d i n the present i n v e s t i g a t i o n s h o u l d 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, the 125 d i f f e r e n c e s between 20 and 40 minute exposure time experiments might i n d i c a t e 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 the d i f f e r e n t f r a c t i o n s o f DNA of i n t e s t i n a l mucosa of r a t . However, s i n c e i t i s not known, whether these f r a c t i o n s do i n -deed e x i s t i n . v i v o , the r e s u l t s o b t a i n e d cannot y e t be taken t o i n d i c a t e meta-b o l i c i n t e r r e l a t i o n s h i p s amongst these f r a c t i o n s . Furthermore, i n t e s t i n a l mucosa r e p r e s e n t s a somewhat heterogeneous p o p u l a t i o n of c e l l s , i n which the d i f f e r e n t types of 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 of t u r n o v e r . Since the newly s y n t h e s i z e d DNA 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 , the f r a c t i o n s o f the main DNA peak c o u l d r e p r e s e n t merely v a r i o u s r a t e s of c e l l renewals i n a heterogeneous p o p u l a t i o n . The concept of m e t a b o l i c h e t e r o g e n e i t y of DNA i s d i f f i c u l t t o r e c o n c i l e w i t h the accepted i d e a s c o n c e r n i n g DNA r e p l i c a t i o n . A c c o r d i n g t o these t h e o r i e s once a substance i s i n c o r p o r a t e d i n t o the 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 sense t h a t i t i s passed on from g e n e r a t i o n t o g e n e r a t i o n . The i d e a of DNA r e p l i c a t i o n i s a l s o envisaged as an " a l l o r none" process i n which once the macramolecular d u p l i c a t i o n has s t a r t e d , the newly s y n t h e s i z e d molecules appear as i n t a c t whole e n t i t i e s . There i s some evidence at present (79,97) t h a t the DNA of 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 , but no s i m i l a r r e p o r t s appeared c o n c e r n i n g the DNA of h i g h e r organisms. The t o t a l amount of t h i s macramolecule i n c e l l s from h i g h e r organisms i s much l a r g e r than 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 the average about 2 cm of DNA. Although 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 to d i s p l a y such 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 the DNA of the chromosomes o f the h i g h e r organisms as b e i n g one molecule and i n a c i r c u l a r form. Two r a t h e r tenuous reasons are c i t e d by t h i s i n v e s t i g a t o r t o support t h i s t h e s i s . The f i r s t one i s the experiments o f T a y l o r and Woods (77,78) on the d u p l i c a t i o n of chromosomes i n p l a n t c e l l s . I f a marker, such 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 time d u r i n g the p e r i o d of DNA s y n t h e s i s i n the p l a n t c e l l , both the daughter chromatids are found t o be 126 l a b e l l e d a t the time of c e l l d i v i s i o n . When they become v i s i b l e , however, a f t e r a f u r t h e r round of d u p l i c a t i o n , these two chromosome are each found t o have produced one 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 would be expected i f each of these chromosomes c o n t a i n e d a s i n g l e molecule; but the r e s u l t s are not c o m p e l l i n g evidence f o r t h i s h y p o t h e s i s , s i n c e they t e l l one o n l y about the s e g r e g a t i o n of the m a t e r i a l present and not about the number of s t r a n d s p r e s e n t . The second argument i s based on the c a l c u l a t i o n of the d u p l i c a -t i o n r a t e of the DNA of 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. There i s a case 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 and t h i s i n t u r n , g i v e s some support f o r the i d e a of one molecule f o r each chromosome. •n the o t h e r hand, s e v e r a l authors (196,197) v i s u a l i z e DNA as c o n s i s t i n g o f s u b u n i t s of a r e l a t i v e l y low m o l e c u l a r weight. 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 note t h a t very r e c e n t l y Sponar e t a l . (195) h y p o t h e s i z e d a mech-anism of f o r m a t i o n of DNA molecules by p o l y m e r i z a t i o n of two k i n d s of s u b u n i t s . Furthermore, the m e t a b o l i c a l l y l a b i l e DNA, i f i t e x i s t s i n v i v o , c o u l d have some r o l e o t h e r than g e n e t i c . A p h y s i o l o g i c a l r o l e f o r DNAs of c h l o r a p l a s t s (107) and the 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 suggested (154). I I I . Measurement of R a d i o a c t i v i t y i n the Protein-RNA I n t e r p h a s e s o f the T i s s u e  E x t r a c t s Obtained by the Methods o f C o l t e r et a l . (147) and Medawar and  Zubay (159) Most of the " m i l d t e c h n i q u e s " employed f o r the e x t r a c t i o n of DN^ from 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 disadvantage t h a t the y i e l d o f the p r e p a r a -t i o n s are u s u a l l y very low. 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 of the DNA by d e t e r g e n t s (144), h i g h c o n c e n t r a t i o n of s a l t s (138), are not s u f f i c i e n t t o break the l i n k a g e s between p r o t e i n and DNA m o l e c u l e s . The method of b r e a k i n g down the 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 importance. I t has been found, d u r i n g the present i n v e s t i g a t i o n s , f o r example, t h a t DNA of the i n t e s t i n a l mucosa of the r a t c o u l d not 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 of s a l t s o l u t i o n s , 127 w i t h o u t the a i d of s o n i c d e g r a d a t i o n of the n u c l e a r and c e l l u l a r membranes. Whatever g e n t l e method has been employed t o e x t r a c t the n u c l e i c a c i d s from t h i s t i s s u e , i t has been demonstrated (157), t h a t no more than 40 t o 50% of the t o t a l DNA of the c e l l s c o u l d be i s o l a t e d . S i n ce the aim of the present i n v e s t i g a t i o n was t o demonstrate 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 i n the d i f f e r e n t DNAs of 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 of the macromolecules d u r i n g the e x t r a c t i o n procedure, c o u l d e f f e c t s e r i o u s l y the outcome of the experiments. The f i n d i n g of G o l d s t e i n and Brown (148) 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 , suggested t o them t h a t the chromosomal mat-e 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 . The s t a b l e s t a t e of the newly s y n t h e s i z e d DNA i s probably due t o i t s i n t e r a c t i o n o r c l o s e a s s o c i a -t i o n w i t h p r o t e i n . The experiments of Ben-Porat e t a l . (149) seemed t o c o n f i r m t h i s assumption. These workers demonstrated t h a t the newly formed DNA i n r a b b i t -kidney c e l l s was r e s i s t a n t t o chloroform-amyl a l c o h o l e x t r a c t i o n , and remained i n the protein-RNA r i c h i n t e r p h a s e . Furthermore, when the c e l l s were i n c u b a t e d w i t h r a d i o a c t i v e t h y m i d i n e , and then the r a d i o a c t i v e m a t e r i a l was "chased" w i t h " c o l d " c a r r i e r , the s p e c i f i c a c t i v i t y o f the i n t e r p h a s e DNA which was 13 times h i g h e r than the b u l k of the c e l l u l a r DNA at the time of the a d d i t i o n of non-r a d i o a c t i v e t h y m i d i n e , decreased p r o g r e s s i v e l y w i t h time of i n c u b a t i o n . The s p e c i f i c a c t i v i t y o f the t o t a l DNA i n c r e a s e d c o n c o m i t a n t l y ! ' , S e v e r a l o t h e r s • workers (151,198,199) v i s u a l i z e DNA s y n t h e s i s as p a r t l y b e i ng performed i n the c y t o p l a s m o r microsomes, which 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 and 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 t o 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 complex, but the 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 of t h i s o b s e r v a t i o n i s not c e r t a i n a t p r e s e n t . On the b a s i s of the above f i n d i n g s , i t was of i n t e r e s t t o count the protein-RNA 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 both e x t r a c t i o n procedures 3 14 (147,159) d u r i n g the present i n v e s t i g a t i o n . Table XIV summarizes the H /C r a t i o s of the i n t e r p h a s e l a y e r s o b t a i n e d from the v a r i o u s t i s s u e e x t r a c t s , and 128 TABLE XIV 3 14 The R a t i o s of H /C i n the Interphase and Phenol Layers Obtained During the 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 * H 3/C 1 4 Interphase from the Medawar-Zubay method. 40 min. C - ^ - i n c o r p o r a t i o n experiment Interphase from the method. 40 min. C14. experiment Interphase from the method. 40 min. C14. experiment Interphase from the method. 20 min. C^-4-experiment phenol e x t r a c t i o n • i n c o r p o r a t i o n phenol e x t r a c t i o n • i n c o r p o r a t i o n phenol e x t r a c t i o n - i n c o r p o r a t i o n Interphase from the phenol e x t r a c t i o n method. 20 min. [ ^ - i n c o r p o r a t i o n experiment Phenol l a y e r 40 min. [ > 4 - i n c o r p o r a t i o n experiment Phenol l a y e r 40 min. C - ^ - i n c o r p o r a t i o n experiment Phenol l a y e r 20 min. [ ^ - i n c o r p o r a t i o n experiment Phenol l a y e r 20 min. C - ^ - i n c o r p o r a t i o n experiment 7.1 10.2 17.7 17.0 18.0 a l l C a c t i v i t y 0.3 0.4 0.2 * A l l animals r e c e i v e d H - l a b e l l e d thymidine 24 h r . the 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 b e f o r e • 129 3 14 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 the cases the H /C r a t i o s of the 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 than those o b t a i n e d from the d i f f e r e n t f r a c t i o n s of the c o r r e s p o n d i n g DNA. T h i s might i n d i c a t e 3 h i g h e r amount of the " o l d e r " H - l a b e l l e d m a t e r i a l i n the i n t e r p h a s e l a y e r . At the same time i t should be p o i n t e d out t h a t the same r e s u l t s c o u l d a r i s e from quenching e f f e c t s i n these samples. The e l i m i n a t i o n of quenching i n t h e s e samples i s d i f f i c u l t because of the high amounts of KOH, HC1, and hyamine bydrox-yde used i n the p r e p a r a t i o n of the m a t e r i a l f o r c o u n t i n g (184). O b v i o u s l y b e t t e r methods are r e q u i r e d f o r the simultaneous c o u n t i n g of two i s o t o p e s i n t h i s type o f m a t e r i a l . No f u r t h e r attempt was made to e x p l o r e the f u r t h e r p u r i f i c a t i o n and 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 , 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 counts 14 o b t a i n e d i n the phenol l a y e r s were almost e x c l u s i v e l y due t o C . T h i s f i n d i n g might i n d i c a t e the presence of a c i d - s o l u b l e low m o l e c u l a r weight p r e c u r s o r s i n the phenol l a y e r . I f i t i s assumed t h a t the phenol 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 of the t i s s u e , i t can be concluded, t h a t 24 hours a f t e r i n j e c t i o n , 3 most of the H - l a b e l l e d t h ymidine 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 whereas 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 c o n c e n t r a t i o n even a f t e r 40 or 20 minutes post i n j e c t i o n . IV. Enzymatic Degradation of the Double L a b e l l e d DNA P r e p a r a t i o n s . 3 . 14 Although the d e t e r m i n a t i o n of the H /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 of the DNA p r e p a r a t i o n s might i n d i c a t e the 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 , they would not g i v e an answer t o problems c o n c e r n i n g the n a t u r e and s i t e of 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 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 types of 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 p r e c u r s o r can occur d u r i n g the b i o -s y n t h e s i s of the DNA molecule. The f i r s t type would 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 the c h a i n , whereas the second k i n d would be a d d i t i o n of the 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 the ends of the 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 c h a i n . To i n v e s t i g a t e which one of the two p o s s i b i l i t i e s o c c u r r e d d u r i n g the i n v i v o b i o s y n t h e s i s o f DNA i n the i n t e s t i n a l mucosa of r a t , use was made o f te c h n i q u e s o f step w i s e d e g r a d a t i o n o f DNA molecules from one end. In such an experiment one f o l l o w s the time course o f the r e l e a s e o f the a c i d - s o l u b l e m a t e r i a l ( 5 0 ) . I t i s expected, t h a t i f the i n c o r p o r a t i o n o c c u r r e d by end add-i t i o n o f the p r e c u r s o r , the r e l e a s e o f the r a d i o a c t i v i t y i n the a c i d - s o l u b l e f r a c t i o n would be much h i g h e r than the r e l e a s e 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 the e a r l i e r stages o f the r e a c t i o n . C o n v e r s e l y , i f the i n c o r p o r a t i o n was 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 c h a i n , the r a t e o f r e l e a s e o f r a d i o a c t i v i t y s h o u l d p a r a l l e l the r a t e of the r e l e a s e 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 . S i m i l a r types o f experiments have been d e s c r i b e d by s e v e r a l i n v e s t i g a t o r s , (50-53) f o r t he stepwise d e g r a d a t i o n of DNA and RNA. One of the e a r l i e s t r e p o r t s , des-c r i b i n g t h i s t e c hnique f o r the d e t e r m i n a t i o n o f the l o c 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 i n 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 was t h a t o f A d l e r e t a l . ( 5 0 ) . These workers used snake venom phosphodiesterase f o r the s e q u e n t i a l l i b e 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 from the d e o x y r i b o n u c l e o s i d e ends of DNA c h a i n s . I t has 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 as an exonuclease, l i b e r a t i n g mononucleotides st e p w i s e from the ends of o l i g o n u c l e o t i d e s w i t h 3'-h y d r o x y l end groups. 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 p u r i f i e d snake venom d i e s t e r a s e f o r the step w i s e enzymatic d e g r a d a t i o n o f the double l a b e l l e d DNA p r e p a r a t i o n s , some i n f o r m a t i o n would be o b t a i n e d r e g a r d i n g the mode of i n c o r p o r a t i o n of the 14 . C - l a b e l l e d thymidine d u r i n g the s h o r t i n c o r p o r a t i o n times i n v i v o . To increase'. the s e n s i t i v i t y o f the method, the r e l e a s e o f the r a d i o a c t i v i t y was measured both i n the a c i d - s o l u b l e and 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 o f the i n c u b a t i o n m i x t u r e d u r i n g the course o f the r e a c t i o n . F i g u r e s 35A and B re p r e s e n t the r e s u l t s ob-t a i n e d when a DNA p r e p a r a t i o n ( F i g u r e 35A) from r a t s i n j e c t e d w i t h the C 1 4 - l a b e l l e d p r e c u r s o r 40 min. be f o r e s a c r i f i c e and the main DNA peak of the same DNA 131 p r e p a r a t i o n ( F i g u r e 35B) from MAK chromatography were s u b j e c t e d t o the s t e p w i s e a c t i o n of the enzyme. F i g u r e 36 i l l u s t r a t e s the r e s u l t s o b t a i n e d , when the main 14 DNA peak of a DNA p r e p a r a t i o n from the 20 min. C exposure time experiment was s u b j e c t e d t o the same tre a t m e n t . The 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 the 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 the r e l e a s e o f u l t r a -3 14 v i o l e t a b s o r b i n g m a t e r i a l . Furthermore, the r a t i o s of H :C remained c o n s t a n t i n the a c i d - i n s o l u b l e f r a c t i o n . These are the r e s u l t s t h a t would be expected i f the i n c o r p o r a t i o n o f the p r e c u r s o r o c c u r r e d w e l l w i t h i n the c h a i n . I f b l o c k s o f 14 3 C - l a b e l l e d thymidine were added to the ends of the p a r e n t a l mainly 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"*'4 i n the a c i d - i n s o l u b l e f r a c t i o n should have been 14 i n c r e a s e d d u r i n g the course of the s t e p w i s e d e g r a d a t i o n , and more C a c t i v i t y would be expected 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 s tages of the r e a c t i o n . There are o n l y a few r e p o r t s c o n c e r n i n g the stepwise enzymatic degrada-t i o n of DNA (50-53). One of the e a r l i e s t i s t h a t of A d l e r e t al.who demonstrated 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 p r e -e x i s t i n g DNA c h a i n i n v i t r o . 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 from c a l f thymus n u c l e i , which 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 , namely, the i n c o r p o r a t i o n of r i b o and 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 o f DNA. The enzyme, however, appears t o be d i s t i n c t from polymerase, and the i n c o r p o r a 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 i s i n h i b i t e d i n the presence of the o t h e r t h r e e 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 b i o c h e m i c a l s i g n i f i c a n c e of these r e a c t i o n s are not known, but s e v e r a l p o s s i b i l i t i e s were suggested ( 5 0 ) . The r e a c t i o n between DNA and a s i n g l e d e o x y r i b o n u c l e o s i d e t r i -phosphate may a c t u a l l y be the f i r s t step i n the o v e r - a l l process of enzymatic r e p l i c a t i o n of the DNA primer; then the t e r m i n a l r e p l i c a t i n g c h a i n , formed when f o u r d e o x y r i b o n u c l e o s i d e 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 t o 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 produced by the 132 A A c i d - i n s o l u b l e f r a c t i o n o Absorbance a t 260 m/^  60 120 180 240 300 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. Enzymatic d e g r a d a t i o n o f double l a b e l l e d DNA o b t a i n e d from t h e i n t e s t i n a l mucosa o f r a t by the method o f C o l t e r et a l . ( 1 4 7 ) . The a n i m a l s were exposed t o the C** - l a b e l l e d p r e c u r s o r f o r 40 min.(A) DNA b e f o r e chromatography on MAK. (B) The main peak o b t a i n e d from MAK chromatography. 133 A A c i d - i n s o l u b l e f r a c t i o n 0 Absorbance at 260 m/c ° R a d i o a c t i v i t y 60 120 180 240 Reaction Time i n Minutes Figure 36. Enzymatic degradation 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 precursor for 20 minutes. 134 r o t a t i o n o f ver y few d e o x y r i b o n u c l e o t i d e 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 time by nuclease a c t i o n . Another p o s s i b i l i t y i s t h a t the r e a c t i o n between DNA and a s i n g l e d e o x y r i b o n u c l e o s i d e t r i p h o s p h a t e i s merely a p a r t of the e x t e n s i o n o f the s h o r t e r o f two u n e q u a l l y l o n g c h a i n s o f a DNA double h e l i x and i s not d i r e c t l y r e l a t e d t o the 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 the t e r m i n a l p o s i t i o n o f DNA might i n d i c a t e the p a r t i c i -p a t i o n o f such a complex i n RNA s y n t h e s i s by a c t i n g as the s t a r t i n g • p o i n t 1 fdr - r . the p o l y m e r i z a t i o n of r i b o n u c l e o t i d e s on a DNA template by RNA polymerase. I n v e s t i g a t i o n s , c o n c e r n i n g 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 the i n v i v o b i o s y n t h e s i s o f DNA, are even more l i m i t e d . Zbarsky (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 - l a b e l l e d thymidine i n t o E h r l i c h a s c i t e s tumour both i n . v i v o and v i t r o . 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 s u b j e c t e d t o the stepwise a c t i o n o f snake venom phosphodiesterase at the e a r l y stages o f the i n c u b a t i o n t i m e , more r a d i o a c t i v i t y was rendered a c i d - s o l u b l e than i t would be expected from a random d i s t r i b u t i o n o f the thymine i n the DNA c h a i n . T h i s f i n d i n g i n d i c a t e d t h a t the r a d i o a c t i v e t hymidine was added t o the ends of the DNA c h a i n s d u r i n g the 24 hours i n c o r p o r a t i o n i n . v i v o . These data are r a t h e r s u r p r i s i n g , s i n c e one would expect t h a t , even i f the 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 l o n g i n c o r p o r a t i o n t i m e s , the newly s y n t h -e s i z e d molecules would be l a b e l l e d a l l along the DNA c h a i n s , and appear as new i n d i v i d u a l e n t i t i e s . The e x p e r i m e n t a l r e s u l t s i n the present i n v e s t i g a t i o n a r e r a t h e r i n agreement w i t h the i n v i t r o f i n d i n g s o f Zbarsky (201). He demonstrated t h a t the r a d i o a c t i v i t y was r e l e a s e d very s l o w l y from the i n 1 v i t r o m a t e r i a l , and even i n the 20-hour h y d r a l y z a t e the r a d i o a c t i v i t y p r e s e n t as thymidine-5'-phosphate corresponded t o o n l y 2% of the t o t a l . The e x p e r i m e n t a l r e s u l t s w i t h the double l a b e l l e d DNA p r e p a r a t i o n s are a l s o i n apparent agreement w i t h the 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 fragments of h y b r i d C -N -DNA, R o l f e has excluded the 135 p o s s i b i l i t y t h a t the l i n e a r s u b u n i t s 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 -s i d e . I t appears, from the present i n v e s t i g a t i o n , t h a t even i n experiments where 14 t h e r e was a s h o r t exposure t o the C - l a b e l l e d t h y m i d i n e , the newly s y n t h e s i z e d molecules d i d not become a t t a c h e d end t o end t o the o l d p a r e n t a l n o n - l a b e l l e d or 3 3 14 H - l a b e l l e d m o l e c u l e s . A p a r a l l e l r e l e a s e o f both H and C a c t i v i t y i n the a c i d s o l u b l e p o r t i o n would be expected a l s o , i f the newly s y n t h e s i z e d molecules were 14 of a h y b r i d c o m p o s i t i o n , e.g. one c h a i n c o n t a i n i n g o n l y C l a b e l and the com-3 plementary c h a i n 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 t h y m i d i n e . The r e s u l t s do not exclude the 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 might be found i n experiments where exposure t o the p r e c u r s o r in_ v i v o was of extremely s h o r t d u r a t i o n . 136 SUMMARY The i _ v i v o b i o s y n t h e s i s of DNA from 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 the i n t e s t i n a l mucosa of r a t . Because of the p u b l i s h e d evidence c o n c e r n i n g the p h y s i c o - 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 of DNA p r e p a r a t i o n s o f mammalian o r i g i n , two methods of s e p a r a t i n g the components of the DNA p r e p a r a t i o n s were s t u d i e d . I n i t i a l l y the DNA was f r a c t i o n a t e d by chromatography on ECTEOLA-cellulose c o l -umns, u s i n g the d i s c o n t i n u o u s e l u t i o n schedule o f K i t . T h i s procedure 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 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 the DNA p r e p a r a t i o n s were not r e p r o d u c i b l e from experiment t o experiment. (b) The e l u t i o n p a t t e r n s changed on s t o r a g e o f the DNA s o l u t i o n at -15°C. (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 p r e p a r a t i o n s were degraded d u r i n g f r a c t i o n a t i o n on ECTEOLA-cellulose. 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 appeared i n the f r a c t i o n s e l u t e d by the column e q u i l i b r a n t . R a d i o a c t i v i t y was l o s t from the e l u t e d f r a c t i o n s on d i a l y s i s . 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 heterogeneous 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 experiments the DNA was i s o l a t e d from the i n t e s t i n a l mucosa of r a t s 3 which had r e c e i v e d H -thym i d i n e 5, 10 o r 20 minutes o r 24 hours. When DNA was f r a c t i o n a t e d on ECTEOLA-cellulose, 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 , the 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 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 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 the 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 p a t -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 not 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 of the s h o r t exposure time experiment^ and the l o n g e r 24 hours i n c o r p o r a t i o n experiments. 137 The second method of 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 from r a t i n t e s t i n a l mucosa employed methylated a l b u m i n - k i e s e l g u h r (MAK) columns. With 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 s t o r -age o f the 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 columns than were o b t a i n e d u s i n g ECTEOLA-cellulose. The main DNA peak was always e l u t e d at the same range o f sodium c h l o r i d e c o n c e n t r a t i o n . DNA samples ob-t a i n e d by two methods of p r e p a r a t i o n (147,159) gave very s i m i l a r chromatogra-p h i c p r o f i l e s on MAK. H e t e r o g e n e i t y o f the 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 suggested i n some experiments, i n which the components of the second l a y e r o f the column were i n c r e a s e d . Chromatography o f DNA on these m o d i f i e d columns i n d i c a t e d t h a t f u r t h e r f r a c -t i o n a t i o n o f the main DNA component might be accomplished. As a more s e n s i t i v e means towards 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 the components i s o l a t e d from the DNA of 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 from newly formed m a t e r i a l , double l a b e l l i n g experiments were c a r r i e d 3 out. Rats were f i r s t g i v e n H -thymidine and 24 hours l a t e r were i n j e c t e d w i t h 14 C - t h y m i d i n e . Twenty or f o r t y minutes l a t e r the animals were k i l l e d and the double l a b e l l e d DNA was i s o l a t e d from the i n t e s t i n a l mucosa. By s t u d y i n g the 3 . 14 H /C r a t i o s o f the f r a c t i o n s o b t a i n e d from MAK, 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. For t h i s purpose i t was necessary t o adapt 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 the determina-t i o n o f and H ^ - a c t i v i t i e s i n the double l a b e l l e d DNA samples. I t was found t h a t 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 the main DNA peak. Because 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 the main peak was ex-14 amined. In the experiments, when the animals were exposed t o the C - l a b e l l e d 3 , 14 thymidine f o r 40 minutes, the H /C r a t i o s were c o n s t a n t . However, when the 14 time of exposure t o the C - 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 minutes, the H^/C^-4 r a t i o s o f the s u b f 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 138 o f 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 me 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 . The r e l a t i o n o f these f i n d i n g s t o the 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 the i n t e s t i n a l mucosa of r a t has been d i s c u s s e d . 8. A n a l y s i s of the products of the ste p w i s e enzymatic d e g r a d a t i o n of the double l a b e l l e d DNA by p u r i f i e d snake venom d i e s t e r a s e showed t h a t the r e l e a s e o f r a d i o a c t i v i t y i n t o the a c i d - s o l u b l e m a t e r i a l f o l l o w e d t h a t o f the u l t r a v i o l e t 3 14 absor b i n g substance, whereas the 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 . I t was concluded, t h e r e f o r e , t h a t i n c o r p o r a t i o n o f the 14 C - l a b e l l e d thymidine o c c u r r e d w i t h i n the p o l y n u c l e o t i d e c h a i n , even d u r i n g the s h o r t exposure of 20 minutes. 139 BIBLIOGRAPHY 1. Hevesy, G., R a d i o a c t i v e I n d i c a t o r s , I n t e r s c i e n c e Pub. New York, (1948) 2. Barnes, F. W., J r . , and Schoenheimer, R., J . B i o l . Chem., 151 123, (1943) 3. 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