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Studies on DNA and DNA polymerases from the intestinal mucosa of rat Leung, Fred Ying Toy 1968

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STUDIES ON DNA AND DNA POLYMERASES FROM THE INTESTINAL MUCOSA OF RAT by FRED YING TOY LEUNG B.S.P., University of B r i t i s h Columbia, 1962 M.S.P., University of B r i t i s h Columbia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s thesis as conforming to the required standard for the degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF BRITISH COLUMBIA July, 196 8 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g ree t h a t 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 r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rpo se s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n -t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department n f Biochemistry The U n i v e r s i t y o f B r i t i s h Co l umb i a Vancouve r 8, Canada Date A&frkwAJutos /C j, /9£§ i ABSTRACT PART I The base compositions of deoxyribonucleic acid, i s o l a t e d from whole c e l l s , n u c l e i , and mitochondria of rat i n t e s t i n a l mucosa were compared- DNA from whole c e l l s or nuclei was frac-tionated by column chromatography on methylated albumin k i e s e l -guhr, MAK. The guanine plus cytosine content of these DNA fractions was determined mainly by the method of heat denatura-tio n , although the methods of acid hydrolysis and equilibrium centrifugation i n cesium chloride solutions were also used. The mole % G+C for the DNA fractions from whole c e l l u l a r extracts ranged between 34.39 and 52.92, while the nuclear DNA fractions showed a range between 37.31 and 50.97. Although the main DNA band which was eluted with 0.6 M NaCl solution had separated into two or three peaks, the detection of a major base compo-s i t i o n a l class of DNA was not evident. Unfractionated DNA from whole c e l l s or nuclei has a G+C content of 42.2 which corresponds to a midpoint of thermal denaturation, Tm of 86.6° C. DNA i s o -lated from the mitochondria of i n t e s t i n a l mucosa was observed to have a Tm of approximately 85.0° C and a density of 1.702 g/cm3. This density corresponded to the value determined for unfraction-ated DNA from the whole mucosa c e l l s . PART II In i n i t i a l experiments, DNA polymerase from Escherichia c o l i was is o l a t e d and p a r t i a l l y p u r i f i e d by treatment with strepto-mycin, ammonium su l f a t e , and by chromatography on DEAE-cellulose. After these introductory experiments, DNA polymerases from the small i n t e s t i n a l mucosa of the r a t were studied. Using suitable assay systems with lf*C-2-dTTP or JltC-8-dATP, both a r e p l i c a t i v e and a terminal DNA nucleotidyl-transferase were detected i n extracts of nu c l e i . The r e p l i c a t i v e enzyme incorporated a labeled precursor into a native or heat denatured DNA primer i n the presence of a l l four complementary triphosphates. The terminal enzyme p r e f e r e n t i a l l y incorporated single deoxyri-bonucleoside triphosphates onto the terminal position of heat denatured DNA primers. Treatment of the DNA products formed i n the terminal addition reaction with snake venom phosphodiester-ase indicated that the labeled precursors were added to 3'-hydroxy terminal positions of the chains. A heterogeneous nature of the DNA polymerases from r at i n t e s t i n a l mucosa was indicated by the appearance of three f r a c -tions of enzyme a c t i v i t y following DEAE-cellulose chromatography. A d i s t i n c t peak of terminal-addition enzyme a c t i v i t y was detected by rechromatography on DEAE-cellulose. Gel f i l t r a t i o n through Sephadex G-150 or G-200 and sucrose density gradient centrifuga-t i o n showed that these DNA polymerases varied i n molecular si z e s . The molecular weights of the DNA polymerase fractions were e s t i -mated to be between 2.5 x 10 k and 3 x 10 5 by comparisons with marker proteins. ACKNOWLEDGEMENTS The author would l i k e to express h i s sincere thanks to Dr. S. H. Zbarsky f o r the advice and en-couragement extended to him throughout the course of this research project. The advice and kind co-operation of Dr. M. Smith on the operation of the a n a l y t i c a l ultracentrifuge are greatly appreciated. Thanks are due also to Dr. W. H. Chase of the Department of Pathology, Uni-v e r s i t y of B r i t i s h Columbia for kindly performing the electron micrographs. The many he l p f u l discussions with Dr. I. Hynie on gel f i l t r a t i o n studies are also appreciated. Personal assistance from the University of B r i t i s h Columbia i n the form of Graduate Fellowships is very g r a t e f u l l y acknowledged. i v TABLE OF CONTENTS Page INTRODUCTION 1 PART I Subcellular location of DNA 1 Possible subunit nature of DNA 3 Physical chemical heterogeneity of DNA 6 The metabolic heterogeneity of DNA 9 The present investigation 11 PART II Enzymatic synthesis of DNA 13 The i n t r a c e l l u l a r location of DNA polymerases 15 Replication of DNA 16 Multiple nature of DNA polymerases 19 The present investigation 2 3 EXPERIMENTAL PART I 1. Preparation of r a t i n t e s t i n a l mucosa c e l l s and th e i r subcellular components 25 2. I s o l a t i o n of DNA from whole i n t e s t i n a l c e l l s and from subcellular components 29 3. P a r t i a l p u r i f i c a t i o n of DNA preparations by ribonu-' clease 31 4. Fractionation of DNA on MAK column 32 5. Some physico-chemical characterizations on DNA 35 6. Determination of the base composition of DNA 35 a. Method of acid hydrolysis and paper chromatography . 35 v Page b. Method of thermal denaturation 36 c. Method of CsCl density gradient centrifugation 38 RESULTS AND DISCUSSION PART I 1. Isolation of DNA from the i n t e s t i n a l mucosa 42 2. Fractionation of DNA by MAK column 47 3. The base composition of DNA is o l a t e d from the i n t e s t i -nal mucosa of the r a t 53 EXPERIMENTAL PART II A. DNA polymerase from Escherichia c o l i 1. Assay system 71 2. Growth and harvest of bacteria 73 3. Preparation of DNA polymerase extract 7 4 4. P u r i f i c a t i o n of E. c o l i DNA polymerase ;.. 75 a. Streptomycin p r e c i p i t a t i o n ;.. 75 b. Ammonium sulfate f r a c t i o n a t i o n 75 c. Chromatography on DEAE-cellulose 76 B. DNA polymerase from small i n t e s t i n a l mucosa of the rat 1. Assay systems 77 a. DNA polymerase (Replicative) 77 b. Terminal deoxynucleotidyl transferase 78 2. Preparation of DNA polymerase extracts from rat i n t e s t i n a l mucosa 80 a. Preparation of i n t e s t i n a l e p i t h e l i a l c e l l s 80 b. I s o l a t i o n of the nuclear f r a c t i o n from the e p i t h e l i a l c e l l s 81 v i Page c. Extraction of DNA polymerases from the nuclei 81 3. Preparation of enzyme extracts from i n t e s t i n a l t i s s u e . 82 4. Extraction of DNA polymerase from nuclei and cyto-plasm prepared i n non-aqueous media 83 5. P u r i f i c a t i o n of DNA polymerases from the i n t e s t i n a l mucosa 85 a. Ammmonium sulfate f r a c t i o n a t i o n 85 b. DEAE-cellulose chromatography 86 c. Hydroxylapatite chromatography 91 d. Sephadexi gel f i l t r a t i o n 92 ei. Sucrose density gradient centrifugation 9 4 6. Enzymatic degradation of synthetic DNA products a. Assay for snake venom phosphodiesterase a c t i v i t y .. 96 b. Action of the venom phosphodiesterase on DNA products synthesized enzymatically 9 7 7. Assay for DNase I a c t i v i t y i n DNA polymerase prepara-tions 100 RESULTS AND DISCUSSION PART II A. DNA polymerase from E. c o l i 102 1. Characteristics of the E. c o l i assay system 102 2. I s o l a t i o n and p u r i f i c a t i o n of E. c o l i DNA polymerase .107 B. DNA nucleotidyltransferases from the small i n t e s t i n a l mucosa of their at 109 1. Characteristics of the i n t e s t i n a l assay system 109 2. Extraction of DNA nucleotidyltransferases from the small i n t e s t i n a l mucosa of the r a t 112 3. I n t r a c e l l u l a r location of DNA nucleotidyltransferases from the i n t e s t i n a l mucosa 113 v i i Page 4 . ' Studies on the p u r i f i c a t i o n of DNA nucleotidyltrans-ferases from the small i n t e s t i n a l mucosa of the rat . . 1 1 8 a. Ammonium sulfate f r a c t i o n a t i o n 1 1 9 b. DEAE-cellulose chromatography 1 2 1 c. Hydroxylapatite chromatography 1 3 2 d. Sephadex gel f i l t r a t i o n . . . . 1 3 6 e. Sucrose density gradient centrifugation 1 4 9 5 . Requirements of the r e p l i c a t i v e DNA polymerase from ra t i n t e s t i n a l mucosa 1 5 9 6 . Terminal DNA nucleotidyltransferase i n nuclei of rat i n t e s t i n a l mucosa 1 6 6 Nature of the products from the terminal-addition and r e p l i c a t i v e reactions 1 6 7 7 . DNase I a c t i v i t y i n DNA polymerase extracts from the i n t e s t i n a l mucosa 1 7 3 SUMMARY Part I 1 7 8 Part II 1 7 9 BIBLIOGRAPHY 1 8 3 v i i i TABLES Page I. Relative Rates for Thymidine Incorporation i n DNA from Rat Tissues 12 I I . E f f e c t of Ribonuclease Treatment on the Mitochondrial DNA Extracts from Rat I n t e s t i n a l Mucosa 46 I I I . The Base Composition of DNA from Rat I n t e s t i n a l Mucosa ; 5 7 IV. The Mole per cent Guanine plus Cytosine of Rat Intes-t i n a l Mucosa DNA from i t s Thermal Denaturation Tem-perature 6 1 V. Determination of the Guanine plus Cytosine content of DNA i s o l a t e d from the I n t e s t i n a l Mucosa of the Rat . . 69 VI. Primer requirements for the E. c o l i DNA polymerase Reaction . . 1 0 3 VII. The E f f e c t of Using Various Volumes of E. c o l i DNA Polymerase Extract i n the Assay System 1 0 5 VIII. E f f e c t of Incubation Time on the DNA Polymerase A c t i v i t y of Crude C e l l Extracts from E. c o l i 1 0 6 IX. Ammonium Sulfate Fractionation of E. c o l i Extract . . . 1 0 8 X. P u r i f i c a t i o n of E. c o l i DNA Polymerase I l l XI. DNA Polymerase A c t i v i t y i n Extracts from Rat I n t e s t i -nal Mucosa 1 1 5 XII. DNA Polymerase A c t i v i t y i n Subcellular Extracts from Rat I n t e s t i n a l Mucosa 1 1 7 XIII. DNA Polymerase A c t i v i t i e s i n Nuclear Extracts from Rat I n t e s t i n a l Mucosa following Ammonium Sulfate Fractionation 1 2 0 XIV. Concentration of Protein Solutions by U l t r a f i l t r a t i o n - 1 2 5 XV. Comparison of DNA Polymerase A c t i v i t y from Nuclear Extracts of Rat I n t e s t i n a l Mucosa following Chromato-graphy on DEAE-cellulose and Hydroxylapatite 1 3 5 XVI. S t a b i l i t y of DNA Polymerases from the I n t e s t i n a l Mucosa of the Rat following Chromatography on Hydroxy-l a p a t i t e 1 3 7 ix Page XVII. Comparison of Peak I and II for DNA Nucleotidyl-transferase A c t i v i t y a f t e r Sephadex G-150 Chroma-tography 141 XVIII. Sephadex Gel F i l t r a t i o n s , 148 XIX. Sucrose Density Gradient Centrifugation of DNA Poly-merase Preparations from Rat I n t e s t i n a l Mucosa 155 XX. P u r i f i c a t i o n of Rat I n t e s t i n a l DNA Nucleotidyl-transferase 158 XXI. Requirements of the Replicative DNA Polymerase from Rat I n t e s t i n a l Mucosa 162 XXII. Properties of Terminal DNA Nucleotidyltransferase i n Nuclei of Rat I n t e s t i n a l Mucosa 16 8 XXIII. DNase I A c t i v i t y i n Rat I n t e s t i n a l Mucosa Prepara-tions .175 X FIGURES Page 1. A microdensitometer tracing of the banding pattern of DNA following equilibrium centrifugation i n a CsCl solution 40 2. Electron micrograph of mitochondria from r a t i n t e s t i n a l mucosa . 44 3. Chromatography of DNA from r a t i n t e s t i n a l mucosa on MAK column 48 4. Chromatography of DNA on MAK column ... 50 5. Rechromatography on MAK of the main DNA f r a c t i o n 51 6. Chromatography of 5 mg of DNA from rat i n t e s t i n a l mucosa on MAK column . . 52 7. Chromatography of nuclear DNA from rat i n t e s t i n a l mucosa c e l l s on MAK column 54 8. Chromatography of DNA on MAK column 55 9. Heat denaturation and cooling curves of DNA from r a t i n t e s t i n a l mucosa 59 10. A comparison of the Tm values as a function of the DNA fractions obtained from the main peak of DNA following MAK chromatography 6 2 11. A comparison of the Tm values as a function of the DNA fractions from the .main peak of DNA following MAK chromatography 6 3 12. Heat denaturation p r o f i l e of mitochondrial DNA from r a t i n t e s t i n a l mucosa 65 13. Microdensitometer tracings of DNA samples from CsCl density gradient studies 68 14. C a l i b r a t i o n curve of r e f r a c t i v e index as a function of potassium chloride buffer concentrations 89 15. Rate of release of p-nitrophenol by the action of snake venom phosphodiesterase on p-nitrophenylthymidine-5-phosphate 9 8 x i Page 16. Chromatography of DNA polymerase from E. c o l i B on DEAE-cellulose 110 17. Chromatography on DEAE-cellulose of a protein extract from the nuclei of rat i n t e s t i n a l mucosa 122 18. Chromatography on DEAE-cellulose of an enzyme prepara-ti o n which had been stored 12 4 19. Rechromatography on DEAE-cellulose of the peak I DNA polymerase f r a c t i o n 127 20. Rechromatography on DEAE-cellulose of the peak II DNA polymerase f r a c t i o n 128 21. Rechromatography on DEAE-cellulose of the peak III DNA polymerase f r a c t i o n 129 22. Sephadex G-100 gel f i l t r a t i o n of the DNA polymerase fractions from DEAE-cellulose 131 23. Chromatography on hydroxylapatite of a DNA polymerase f r a c t i o n .134 24. Sephadex G-150 gel f i l t r a t i o n of a DNA polymerase f r a c t i o n from DEAE-cellulose 139 25. Sephadex G-150 gel f i l t r a t i o n of a DNA polymerase extract from rat i n t e s t i n a l mucosa 143 26. Standard curve for the estimation of the molecular weight of protein samples on the basis of t h e i r e l u t i o n volumes from G-150 ........144 27. Sephadex G-200 gel f i l t r a t i o n of a DNA polymerase extract from rat i n t e s t i n a l mucosa 146 28. Sucrose gradient centrifugation of DNA polymerase extract from r a t i n t e s t i n a l mucosa 151 29. Sucrose gradient centrifugation of DNA polymerase f r a c t i o n from the G-200 gel f i l t r a t i o n 152 30. Sucrose gradient centrifugation of DNA polymerase f r a c t i o n , peak I, from the G-200 ..153 31. Sucrose gradient centrifugation of DNA polymerase f r a c t i o n , peak I I , from the G-200 .154 32. Standard curve for the estimation of the molecular weight of protein samples on the basis of t h e i r sedi-mentation c o e f f i c i e n t s 157 x i i Page 33. E f f e c t of pH on the a c t i v i t y of DNA polymerase from rat i n t e s t i n a l mucosa 161 34. Hydrolysis of 1 lfC-8-dAMP-DNA by snake venom phospho-diesterase. Labeled DNA was formed by the terminal-addition reaction 170 35. Hydrolysis of 1^C-S-dAMP-DNA by snake venom phospho-diesterase. Labeled DNA was prepared by the action of r e p l i c a t i v e DNA polymerase ;....172 36. Chromatography on DEAE-cellulose of a DNA polymerase extract. Assay for DNase I a c t i v i t y .....176 x i i i LIST OF ABBREVIATIONS DNA deoxyribonucleic acid RNA ribonucleic acid DNase deoxyribonuclease RNase ribonuclease NAD nicotinamide adenine dinucleotide TRIS tris(hydroxymethyl)aminomethane EDTA ethylenediaminetetraacetate TCA trichloroacetic acid DEAE diethylaminoethyl-MAK methylated albumin kieselguhr column for chromatography Tm midpoint of thermal denaturation cpm counts per minute (radioactivity) -9 n nano (10 ) p pico (10 ^ ) - 1 -INTRODUCTION Considerable advances have been made i n the past decade on the study of the structure and the b i o l o g i c a l function of DNA from bacteria and viruses. Due to the apparent complexity of mammalian systems, s i m i l a r studies on t h i s macromolecule from higher organisms have been limited. Recent advances i n methodo-logy and instrumentation have made i t possible to further i n v e s t i -gate such complex problems as the physical and chemical hetero-geneity of DNA i n mammalian c e l l s , and the enzymatic synthesis of DNA from these systems. By way of an introduction to the present investigation, the following discussion i s limited to the current experimental evidence which i s relevant to a study of a heterogeneous population of DNA and of the biosynthesis of this nucleic acid. PART I Subcellular location of DNA: Deoxyribonucleic acid which i s recognized as the c a r r i e r of genetic information, has "generally been considered to be lo c a l i z e d s o l e l y i n the nucleus. In the past few years, however, evidence has accumulated which showed that a few per cent of the t o t a l c e l l u l a r DNA were present i n extranuclear compartments. E a r l i e r investigators have shown the presence of DNA i n chloro-plasts (1), kinetoplasts from protozoon (2), kappa p a r t i c l e s from paramecia (3) and i n yeast mitochondria (4). Luck and Reich (5) provided the f i r s t convenient biochemical c r i t e r i a for - 2 -distinguishing the mitochondrial DNA from the nuclear DNA. These authors found that by equilibrium centrifugation of DNA i n cesium chloride solutions, the mitochondrial DNA from Neurospora crassa showed a density which was markedly d i f f e r e n t from that of the nuclear DNA. The existence of DNA i n mitochondria from animal species has been confirmed by several detailed biochemical characterizations. Mitochondrial DNAs have been reported by Kalf (6) from lamb heart, by Schneider (7) from rat l i v e r , by Rabino-witz (8) from chick embryo heart and l i v e r , by Corneo et at. (9) from guinea-pig l i v e r , by Kroon et at. (10) from sheep heart, and by DuBuy et at. (11) from mouse brain. Extranuclear DNA has also been detected i n the microsomes (12) and i n the centrioles (13) of the c e l l . In the l a t t e r organelle, a double-stranded DNA was reported by Granick and Gibor (13) to be i n a procen-t r i o l e unit which was concerned with c e n t r i o l e d i f f e r e n t i a t i o n and cytoplasmic microtubule formation. In addition to the extranuclear located DNA, another minor DNA component has been detected i n the nucleus by equilibrium centrifugation i n CsCl density gradients. A s a t e l l i t e band l i g h t e r than the major nuclear band was found after centrifuga-t i o n of a DNA extract from mouse tissue (9). The exclusive nuclear location of this s a t e l l i t e has been confirmed (14), and was distinguished from the mitochondrial DNA which bands at a d i f f e r e n t buoyant density. The nuclear s a t e l l i t e band consisted of approximately 10 per cent of the t o t a l 1 c e l l u l a r DNA as re-ported by Chun and L i t t l e f i e l d (15). In crabs, a l i g h t s a t e l l i t e - 3 -band accounted for 30 per cent of the DNA from Cancer borealis and 11 per cent i n C. ivvovatus (16). The crab s a t e l l i t e band consisted of 93 per cent adenine and thymine, and was found to correspond clo s e l y with the synthetic copolymer, poly dAT (17). Recent investigations by Smith (18) have indicated that the crab dAT i s located i n the c e l l nucleus. Not a l l s a t e l l i t e bands were l i g h t e r than the major nuclear DNA because s a t e l l i t e bands from the nuclei of guinea pig and c a l f thymus contained bands denser than the average DNA (9). Possible subunit nature of DNA: The concept that the chromosome consisted of a single-continu-ous DNA molecule has been established for viruses (19). Such a DNA structure w i l l probably be proven to occur i n bacteria as well, but has not been established for higher organisms. C a v a l i e r i and Rosenberg (20) and Taylor (21) have proposed several models for the structure of DNA i n chromosomes. In t h e i r models, DNA with single strand breaks or with subunits are joined end to end with peptide l i n k s . Bendich et at. (22) observed that the r e s i -dual protein associated with sperm DNA after deproteinization, contained a considerable amount of serine and threonine. Although these amino acids may be of protamine i n o r i g i n , these authors proposed that phosphodiester bonds could have been formed between the DNA and the carboxyl and hydroxyl groups of the amino acids. The degradation of the DNA by hydroxylamine which was expected to cleave the ester linkages of the amino acids produced DNA subunits approximately 5 x 10 5 i n molecular weight. In a second - 4 -study by Welsh (23), DNA with molecular weight 6 x 10 5 was obtained i f p u r i f i e d c a l f thymus nuclei were repeatedly extracted with water or exposed to an anion-exchange res i n before l y s i s . Lesko and Emery (24) have reported a subunit nature for c a l f thymus DNA after hydroxylamine and papain treatment. Since the exact nature of the attachment of these residual proteins to DNA has not been established, the significance of these experiments which proposed peptide links between DNA subunits remains to be determined. Another approach to the question of a subunit structure for DNA was made through physical studies of DNA subfractions after chromatography on a methylated albumin-kieselguhr column (MAK). Sponar et at. (25) have studied c a l f thymus DNA which was resolved on a MAK column into several fract i o n s . Further studies of these fractions were made by using a thermal denaturation method which measures the increase i n hyperchromicity of a heated DNA sample. According to Pivec et at. (26), the use of a wavelength at 235 my allowed the detection of several d i s t i n c t steps i n the tem-perature p r o f i l e s . These authors (26,27) observed seven d i s t i n c t melting transitions and suggested that c a l f thymus DNA might have as many as seven d i f f e r e n t types of subunits with d i f f e r e n t guanine plus cytosine contents. Due to the complexity of mammalian DNA, i t remains to be substantiated i f these proposed subunits are separate physical species or are merely regions of the DNA that contains d i f f e r e n t base compositions. Taylor (2 8) has recently presented evidence on the rate of DNA chain growth of mammalian chromosomes. The rate was followed - 5 -by the use of a density-label, 3H-bromodeoxyuridine which was i n -corporated into the DNA of Chinese hamster c e l l s i n culture. There appeared to be several independently r e p l i c a t i n g units per chromosome which increased i t s length at a rate of 1 to 2 y per minute. Taylor indicated that small subunits of the DNA were probably joined end to end during chromosome r e p l i c a t i o n to form a single molecular unit. Okazaki et at. (29) have studied the mechanism of DNA chain growth i n b a c t e r i a l and v i r a l genomes, and have postulated a 'discontinuous' mechanism for chromosome r e p l i c a t i o n in vivo. As w i l l be subsequently discussed under DNA r e p l i c a t i o n , this proposal would be contrary to the usually accepted mechanisms of a 'continuous' DNA r e p l i c a t i o n . Okazaki et at. (29) observed that when the organism was exposed to a radioactive DNA precursor for a short period of time and the DNA was i s o l a t e d , most of the l a b e l was recovered i n small molecules with sedimentation c o e f f i c i e n t s 10 to 11S. These small units may l a t e r be joined together by a polynucleotide joining enzyme to forma continuous strand i n the chromosome. Caution i s needed i n the interpretation of experimental data which show the presence of DNA with low molecular weight. Davison (30) has shown that during the i s o l a t i o n of DNA, de-gradation occurs under low shear forces. The DNA units observed by various authors, as e a r l i e r described, might have resulted from a single DNA chain which had been degraded at points along the chain which are susceptible to shear. - 6 -Physical and chemical heterogeneity of DNA: A preparation of DNA might be heterogeneous i n molecular weight, conformational state, base composition, and i n i t s se-quence of bases. Several methods of separation and characteriza-tion of DNA samples have been developed on the basis of these natural v a r i a t i o n s . Rosenkranz and Bendich (31) reported the f r a c t i o n a t i o n of native DNA by ion-exchange chromatography on ECTEOLA-cellulose (epichlorohydrintriethanolaminecellulose) according to molecular s i z e . Frankel and Crampton (32) observed that the chromatography of DNA from c a l f thymus on polymethacrylic acid (IRC-50) resulted i n the f r a c t i o n a t i o n of DNA on the basis of i t s base composition and partly on i t s molecular s i z e . Early studies by Mandell and Hershey (33) showed that methylated albumin kieselguhr columns separated DNA from E.- c o l i , bacteriophages T and T into fractions which d i f f e r e d i n molecular size; subse-2 h quently Sueoka and Cheng (34) reported that the MAK column was also able to fractionate DNAs of d i f f e r i n g base composition from E. c o l i and crab tissue. Chromatography of nucleic acid extracts from KB-cells on sphere-condensed agarose was found to separate DNA from ribosomal and transfer RNA on the basis of t h e i r molecu-l a r sizes (35). McCallum and Walker (36) have reported that chromatography of DNA on a column of hydroxylapatite gave DNA subfractions which d i f f e r e d i n base composition, and i n the secondary structure of the molecule. Schildkraut et al. (37) have shown that native and denatured DNA were separated by centrifugation to equilibrium i n a CsCl density gradient, and - 7 -that DNA samples with high and low G+C contents were also separ-able. Fractionation of native DNA from mammalian and b a c t e r i a l sources by counter-current d i s t r i b u t i o n has been found by Kidson and Kirby (38) to depend more on the degree of strand separation than on i t s base composition. Alberts (39) also showed that f r a c t i o n a t i o n of nucleic acids i n a dextran-polyethylene g l y c o l two-phase system depended on t h e i r degree of double-helical nature i n which single-stranded DNA was separated from the native form. After the DNA has been separated into i t s several components, suitable characterization procedures are needed to evaluate these subfractions. Since no single method has been devised to suitably characterize a nucleic acid, several procedures should be used for comparative purposes. In an extensive review by Eigner and Doty (40), the molecular weights of several DNAs have been e s t i -mated by sedimentation c o e f f i c i e n t , i n t r i n s i c v i s c o s i t y and l i g h t scattering measurements. Autoradiography and electron microscopy techniques have also been used for the estimation of the molecular weights of DNA (41). Probably the most widely used method for characterizing d i f f e r e n t types of DNA i s based on measurements of t h e i r content of guanine plus cytosine which includes 5-methyl cytosine (G+C). Direct chemical analysis of DNA hydrolysates from plants and animals has shown that the average base composition for these species i s between 40 to 45 per cent G+C (42). The DNA i s o l a t e d from cert a i n species of animals was found to contain a composi-- 8 -t i o n a l range of up to 20 per cent G+C. In contrast, the mean base composition of DNA from bacteria and viruses varied from 25 to 75 per cent G+C between d i f f e r e n t species (43), but the DNA i s o l a t e d from one species was rather homogeneous with only a few per cent G+C v a r i a t i o n around the mean value. The conventional acid hydrolysis method for determining the base composition of DNA i s suitable for milligram quantities. This procedure i s d i f f i c u l t for measuring the G+C content of several subfractions of DNA i n microgram quantities. Two i n d i r e c t methods which are widely used to determine the G+C content of DNA are based on the thermal denaturation of DNA and on i t s buoyant density i n CsCl solutions. A spectrophotometric method was developed by Marmur and Doty (44) for the thermal denaturation of DNA samples. Double-stranded DNA denatures within a temperature range of 60 to 100° C, and at a given i o n i c strength, the midpoint of the hyperchromic change, Tm, at 260 my i s c h a r a c t e r i s t i c of each DNA. The base composition of DNA, expressed i n terms of percentage of guanine plus cytosine bases i s l i n e a r l y related to the Tm values. The advantages of this method are i t s experimental s i m p l i c i t y , the r e p r o d u c i b i l i t y of the.results, and the small amount of DNA required, from 20 to 50 yg, for the determination. Some of the limi t a t i o n s of t h i s method are i t s restriction'.to the range of base composition for which i t was established, or between 25 to 75 mole per cent G+C, to maintain l i n e a r i t y of Tm and G+C content, and i t s s e n s i t i v i t y to changes i n i o n i c environment - 9 -which produces higher or lower Tm values. The method of equilibrium density gradient centrifugation i n cesium chloride solutions has been widely used for the analy-s i s of DNA nucleotide composition. Sueoka et al. (45) have found that the buoyant density of DNA preparations was a l i n e a r func-ti o n of the base composition, increasing with G+C content. Afte r denaturation, the buoyant density of the DNA increases by 0.016 g/cm3. The usefulness of this method was that only 1 to 3 yg of p a r t i a l l y p u r i f i e d DNA was required for each study. This pro-cedure also provided information as to the presence of single or double-stranded DNA as well as to the heterogeneity i n base compo-s i t i o n with regards to G+C content of the DNA. Progress i n the determination of the sequence of a nucleic acid has been reviewed by Burton (46) and Rajbhandary and Stuart (47) who indicated that a homogeneous preparation of the macro-molecule i s needed for such a study. In view of the preceeding evidence on the heterogeneous nature of DNA i n higher organisms, the elucidation of the base sequence of a gene from such a system has been limited. The metabolic heterogeneity of DNA: The compositional heterogeneity of the DNA preparations studied in v i t r o has also been supported by in vivo investigations of the metabolism of DNA. In one of the e a r l i e r studies, Bendich et al. (50) studied the metabolic heterogeneity of E. c o l i DNA by resolving DNA samples i s o l a t e d from cultures grown i n the presence of thymine or 5-bromouracil. The separation of DNA - 10 -molecules containing 5-bromouracil from those lacking t h i s base by chromatography on ECTEOLA indicated to these authors that the DNA was metabolically heterogeneous. In a more recent study, Sampson et at. (51) have demonstrated that the DNA from a variety of growing plant tissues could be resolved into two d i s t i n c t fractions on a MAK column. A smaller molecular weight f r a c t i o n had a r e l a t i v e l y rapid rate of turnover. By the use of synchro-nized HeLa c e l l cultures and a density-labeling technique, Mueller and Kajiwara (52) demonstrated the existence of an early- and a l a t e - r e p l i c a t i n g form of DNA. The c e l l s were i n i t i a l l y grown i n a medium containing 3H-thymidine. After several generations of random growth, the c e l l s were resynchronized and exposed to 5-bromodeoxyuridine. The 3H l a b e l was detected i n a heavy, bromo-deoxyuridine containing hybrid DNA that r e p l i c a t e d early i n the second synchronized c e l l cycle. The possible existence of two metabolic forms of DNA i n rat i n t e s t i n a l mucosa has been demon-strated i n this laboratory by Mezei and Zbarsky (53). Doubly labeled DNA was i s o l a t e d from the mucosa of rats injected i n t r a -venously with 3H-thymidine and 24 hours l a t e r with 1 hC-thymidine. A comparison of the 3H/1'*C r a t i o of DNA fractionated on a MAK column indicated the metabolic heterogeneity of the i n t e s t i n a l DNA. Different tissues have been shown to incorporate labeled nucleic acid precursors into t h e i r DNA at d i f f e r e n t rates. In general, tissues i n which c e l l d i v i s i o n was infrequent, i . e . adult l i v e r and kidney, incorporated labeled precursors only - 1 1 -s l i g h t l y . However, tissues with a high mitotic rate such as thy-mus, i n t e s t i n a l mucosa, and bone marrow showed a considerable i n -corporation of the precursor ( 5 4 ) . A high incorporation of pre-cursors into the DNA would indicate active DNA synthesis and metabolism. Such a comparative study was c a r r i e d out by Bollum and Potter ( 5 5 ) who determined the r e l a t i v e 3H-thymidine incor-poration into DNA of various rat tissues. Their results are shown i n Table 1 . The present i n v e s t i g a t i o n : Previous investigations i n t h i s laboratory by Mezei and Zbarsky ( 5 3 ) have indicated that the DNA i s o l a t e d from the i n -t e s t i n a l mucosa of the rat was metabolically as well as p h y s i c a l l y heterogeneous. The f i r s t part of this work i s concerned with a comparative study of the physico-chemical c h a r a c t e r i s t i c s of i n t e s t i n a l DNA i s o l a t e d from the whole c e l l and i t s subfractions. The method of Colter et al. ( 5 6 ) was followed for the i s o -l a t i o n of DNA from the rat i n t e s t i n a l mucosa. The i n t e s t i n a l DNA was chromatographed on a MAK column which should fractionate the DNA according to i t s molecular size ( 3 3 ) and i t s base com-posi t i o n ( 3 4 ) . The guanine plus cytosine content of these sub-fractions was determined using several physico-chemical methods. The chemical procedure of acid hydrolysis of the DNA to i t s free base constituents followed by chromatographic separation and spectrophotometric estimation was used according to Wyatt ( 5 7 ) . Since the physical properties of thermal denaturation ( 4 4 ) and buoyant density i n CsCl ( 5 8 ) were l i n e a r l y related to the average Table I Relative Rates for Thymidine Incorporation in DNA from Rat Tissues Tissue Relative rates* Thymus 100 Regenerating l i v e r 70 Intestine 30 Spleen 8 Liver 5 Lung 4 Brain 3 Skeletal muscle 1 * The rates are r e l a t i v e to thymus which i s set at 100. These experimental results were reported by Bollum and Potter (55). - 13 -base composition of DNA, these techniques were also adopted for assessing the intramolecular heterogeneity of i n t e s t i n a l DNA samples. From these studies, an attempt was made to assess the rela t i o n s h i p of the DNA is o l a t e d from the nu c l e i , mitochon-d r i a , and from the whole i n t e s t i n a l mucosa c e l l s on the basis of th e i r G+C contents. PART II Enzymatic synthesis of DNA: As an introduction to the second part of the subsequent i n -vestigation, the present discussion i s concerned with the enzy-matic polymerization of deoxyribonucleoside triphosphates i n re-lationship to DNA r e p l i c a t i o n . The r e p l i c a t i o n of DNA appears to be catalyzed by the enzyme DNA nucleotidyltransferase, more commonly known as DNA polymerase. This enzyme was f i r s t i s o l a t e d from Escherichia c o l i by Kornberg et at. (59). DNA polymerases were subsequently reported by Davidson et at. (60) from E h r l i c h ascites tumor, by Bollum from c a l f thymus gland (61), and by Mantsavinos et at. (62) from re-generating r a t l i v e r . The biosynthesis of DNA in v i t r o required the presence of DNA primer, Mg + + ions, and deoxyribonucleoside 5'-triphosphates, dATP, dGTP, dCTP, and dTTP i n addition to the enzyme. DNA polymerase catalyzed the formation of polydeoxy-ribonucleotides as represented i n the following reaction sequence: - 14 -n dATP dAMP n dGTP + DNA enzyme dGMP DNA + 4n PP. 1 n dCTP Mg ++ dCMP n dTTP dTMP n The r e p l i c a t i o n of a h e l i c a l DNA template by E. c o l i DNA poly-merase produced a macromolecule which showed a molar proportion of bases expected of a complementary copy of the template (6 3). While i t has not been possible to determine the complete sequence of nucleotides i n DNA, as yet, a procedure which i s termed 'near-est neighbour frequency analysis' has been devised by Josse et al. (64). By the use of this procedure, as described by these authors, i t was possible to determine that the base order of the synthetic DNA product was sp e c i f i e d by the base sequence of the primer. The 'nearest neighbour' technique also showed that the two strands of the product had opposite p o l a r i t i e s . Although the DNA poly-merase was able to produce an exact copy of the primer DNA, these enzymes were not s p e c i f i c with regards to the b i o l o g i c a l source of the primer (65). DNA polymerase from various sources as bacteria, phages or mammalian tiss u e , however, showed a difference i n the degree of activ a t i o n by the primer for net DNA synthesis. When enzyme pre-parations from E. c o l i or B. s u b t i l i s were used, net synthesis of polydeoxyribonucleotide greatly exceeded the amount of primer added to the reaction mixture as reported by Richardson et al. (66), and Okazaki et al. (67). In contrast to these observations DNA polymerases from mammalian tissue did not give r i s e to poly-nucleotides i n excess of the amount of primer DNA which was added - 15 -to the reaction mixture (65,68). Recently, Goulian et al. (69) have reported a DNA polymerase induced i n the host, E. c o l i , by i n f e c t i o n with phage T\ that also catalyzed the synthesize of DNA only to the amount of single-stranded primer added. The i n t r a c e l l u l a r location of DNA polymerases: In the b a c t e r i a l system, Ganesan and Lederberg (70) have found that the membrane f r a c t i o n of E. c o l i c e l l s contained a substantial proportion of t h e . t o t a l c e l l DNA polymerase. These authors also observed that when c e l l s were grown i n a medium containing a radioactive DNA precursor, the membrane f r a c t i o n was i n i t i a l l y labeled i n a pulse experiment. This pulse-labeled f r a c t i o n was subsequently chased by growth i n a nonlabeled medium which indicated that DNA biosynthesis took place i n a membrane-bound fraction-. In mammalian c e l l preparations, DNA polymerases were o r i -g i n a l l y detected i n the soluble supernatant fractions a f t e r high-speed centrifugation (61,71). Although not a l l of the super-natant could be regarded as representative of the c e l l cytoplasm, thi s location of the enzyme was unexpected since only low enzyme a c t i v i t y was found i n the corresponding nuclear preparation (72). By the use of an aqueous extraction medium containing 2 mM C a + + ions, Main and Cole (73) demonstrated that DNA polymerase a c t i -v i t y was evenly d i s t r i b u t e d between the nuclear and the cyto-plasmic fractions following high-speed centrifugation of a homo-genate of r a t thymus tis s u e . B i r n i e and Fox (74) found a s i m i l a r d i s t r i b u t i o n of enzyme a c t i v i t y a f t e r treatment of mouse embryo - 16 -c e l l s , but the s p e c i f i c a c t i v i t y was markedly greater i n the nuclear extract. The presence of cytoplasmic DNA polymerase ac-t i v i t y might have resulted from the passage of t h i s enzyme through the nuclear membrane during the homogenization i n an aqueous medium. Smith and Keir (75) have demonstrated, however, that rapidly f r o -zen and l y o p h i l i z e d c a l f thymus tissue s t i l l contained DNA poly-merase a c t i v i t y i n both the nuclei and the cytoplasm following the separation of these constituents under nonaqueous conditions. When nuclei were prepared i n nonaqueous solvents from regenera-ting rat l i v e r or from Novikoff hepatoma, a greater DNA polymerase a c t i v i t y was detected i n this organelle than i n the cytoplasm (76,77). The presence of DNA polymerase i n the nuclei was, therefore, demonstrated and was believed to catalyze the r e p l i c a t i o n of DNA i n t h i s c e l l u l a r constituent. A s i m i l a r functional r e l a t i o n s h i p might be made to explain the existence of DNA polymerase i n the cytoplasm. This l a t t e r enzyme might have existed f r e e l y i n the c e l l sap or might be associated with the DNA i n mitochondria. Parson and Simpson (78) have recently reported the incorporation of 1 ltC-2-TTP into the DNA of i s o l a t e d mitochondria from the rat l i v e r . It appeared that this DNA was undergoing r e p l i c a t i o n which might indicate the presence of a DNA polymerase i n t h i s organelle. Replication of DNA: The view that the genetic information was contained i n the - 17 -sequence of nucleotides i n DNA has led to extensive investiga-tions on the r e p l i c a t i o n of t h i s macromolecule. On the basis of the Watson-Crick model for l i n e a r molecules of DNA, r e p l i c a t i o n proceeds by a semiconservative mechanism i n which only one h a l f of the parental structure i s passed on to i t s daughter molecules. Experimental evidence by Meselson and Stahl (79) who analyzed the banding patter of N 1 5/N 1 4 hybrid E. c o l i DNA aft e r centrifugation i n CsCl density gradients, supported t h i s mechanism of r e p l i c a t i o n . Since the DNA i n several organisms i s c i r c u l a r , another mechanism has been postulated for i t s r e p l i c a t i o n . The autoradio-graphic technique of Cairns (41) showed the f i r s t v i s u a l evidence for the r e p l i c a t i o n of c i r c u l a r DNA. From Cairn's study, i t appeared that r e p l i c a t i o n proceeded i n a c i r c u l a r manner from one fixed point and i n only one d i r e c t i o n . Yoshikawa and Sueoka (80) have also presented i n d i r e c t evidence for a single r e p l i c a t i o n point per chromosome i n B a c i l l u s s u b t i l i s . After r e p l i c a t i o n was complete, the DNA product was separated from the template i n a c i r c u l a r and double-stranded form. Since the two strands of the DNA have been shown to be of opposite p o l a r i t y , the r e p l i c a t i o n of both strands of the DNA from one fixed point i n the chain requires that simultaneous syn-thesis occur i n the 3' to 5' d i r e c t i o n on one chain and i n a 5 1 to 3' d i r e c t i o n on the other. Mitra and Kornberg (81) have shown that DNA polymerase i s only able to r e p l i c a t e a DNA strand, in v i t r o , i n a 5' to 3' d i r e c t i o n . At present no enzyme has been demonstrated which can r e p l i c a t e i n the 3' to 5' d i r e c t i o n . - 1 8 -Recently Okazaki et at. ( 2 9 ) have presented evidence that the E. c o l i chromosome was formed by the i n i t i a l synthesis of small DNA fragments. These authors postulated that i f a 'discontinuous 1 mechanism of chain growth existed in vivo, the DNA polymerase could have formed small chains of DNA at the growing point which were subsequently joined into a continuous chain i n both strands. The hypothesis of a discontinuous chain growth mechanism was encouraged by the discovery of polynucleotide-joining enzymes (ligases) i n normal and Ti+ phage-infected E. c o l i . DNA joining enzyme was discovered by G e l l e r t ( 8 2 ) i n normal E. c o l i . In order to demonstrate polynucleotide-joining a c t i v i t y , the coenzyme nicotinamide adenine dinucleotide (NAD) was required ( 8 3 ) . The Tit phage ligase required the presence of ATP for a c t i v i t y ( 8 4 , 8 5 ) . Both enzymes were able to catalyze the forma-t i o n of a 3 ' - 5 1-phosphodiester bond between the 3'-hydroxyl and the 5'-phosphate termini of DNA. The discovery of DNA ligases was an important factor which led to the in v i t r o synthesis of the f i r s t b i o l o g i c a l l y active DNA. Kornberg and coworkers have reported e a r l i e r the r e p l i c a t i o n of E. c o l i DNA in v i t r o , but the product as observed i n the electron microscope was branched, renatured rea d i l y after heat treatment ( 8 6 ) and was b i o l o g i c a l l y inactive ( 8 7 ) . Such physical anomalies which might have resulted from the f a i l u r e of DNA poly-merase to r e p l i c a t e both strands of a double-stranded template simultaneously from one end were absent when single-stranded c i r c u l a r DNA from phage M 1 3 or (f>X174 was used as the template - 19 -(88). The DNA produced from these phage templates was l i n e a r and s t i l l b i o l o g i c a l l y i n a c t i v e . In a recent report by Goulian et al. (89), a b i o l o g i c a l l y active DNA was synthesized in v i t r o by the use of both DNA polymerase and DNA ligase. The procedure used by these authors depended upon the r e p l i c a t i o n of a si n g l e -stranded <j>X174 DNA by DNA polymerase from E. c o l i . The ends of the newly synthesized chain were joined by the DNA ligase to form a f u l l y covalent duplex c i r c l e . Separation of the c i r c l e s re-quired limited DNase treatment followed by heat denaturation and density gradient sedimentation as described by Goulian et al. (90). The synthetic DNA c i r c l e s were i n f e c t i v e i n E. c o l i spheroplasts, and were serving as templates in vivo. Multiple nature of DNA polymerases: DNA polymerases from b a c t e r i a l and mammalian sources have been shown to polymerize the four deoxyribonucleoside 5'-triphos-phates i n the presence of a primer DNA to form a double-helical DNA of high molecular weight. An enzyme with t h i s function has been termed a 1 r e p l i c a t i v e ' DNA nucleotidyl-transferase (91). It i s characterized by the r e p l i c a t i o n of the 3'-hydroxy-termi-nated strand of a DNA primer. Canellakis et al. (92) have described a p u r i f i e d enzyme fr a c t i o n from B. s u b t i l i s which phosphorylated deoxythymidine-3' diphosphate to deoxythymidine-3 1 triphosphate i n the presence of ATP. The possible existence of a DNA polymerase that s p e c i f i -c a l l y catalyzes the incorporation of th i s deoxythymidine-3 1 triphosphate would permit both strands to be rep l i c a t e d simul-- 20 -taneously from one point i n the chain. Ordered r e p l i c a t i o n would r e s u l t by the polymerization of 5'-triphosphates along one strand with concomitant polymerization of 3 1-triphosphates along the other. The existence of a DNA polymerase which was s p e c i f i c for 3 1-triphosphates has not, as yet, been demonstrated. DNA polymerases have been shown to repair damaged DNA. In an experiment to demonstrate th i s a b i l i t y of the enzyme, Richard-son and Romberg (93) prepared a DNA template which was p a r t i a l l y degraded by a nuclease s p e c i f i c for the 3'-hydroxyl ends of DNA. When a DNA molecule which had a double-stranded central region with single-stranded ends was added to a DNA polymerase reaction, the enzyme catalyzed the restoration of the DNA molecule to i t s o r i g i n a l length. Both b a c t e r i a l (94) and mammalian (95) DNA polymerase pre-parations have been shown to promote the terminal addition of deoxyribonucleotide residues at the 3'-hydroxyl end of primer chains. In view of the possible presence of an enzyme which catalyzes the addition of nucleotides to ends of a DNA primer, t h i s enzyme was termed a 'terminal 1-DNA nucleotidyltransferase (91) to d i f f e r e n t i a t e i t from the ' r e p l i c a t i v e ' enzyme previously mentioned. Krakow and coworkers (96) were the f i r s t to describe a terminal enzyme from c a l f thymus nuclei which displayed a re-quirement for heated DNA primer, Mg + + ions, and cysteine. Kato et at. (97) have described a terminal enzyme from c a l f thymus cytoplasm which had several d i f f e r e n t c h a r a c t e r i s t i c s from the nuclear terminal enzyme of thi s tissue. Some of these differences - 2 1 -included primer s p e c i f i c i t i e s , requirement for sulfhydryl s t a b i -l i z e r s , and t h e . a b i l i t y to form complexes with DNA. At present the b i o l o g i c a l role of terminal DNA transferase has not been determined. Keir ( 9 1 ) has presented the hypothesis that the terminal enzyme was o r i g i n a l l y a c a t a l y t i c subunit of a complex DNA polymerase molecule. The basis for his discussion was that a complex DNA polymerase composed of several subunits might be expected to possess properties which were si m i l a r to the i n d i v i d u a l subunits, but d i f f e r e d i n i t s s p e c i f i c i t y towards substrates and cofactors, i . e . Mg + + ions; DNA primer. Ono and Iwamura ( 9 8 ) have observed that DNA polymerases from rat fetus, regenerating r a t l i v e r and fas t growing hepatomas were separated as large molecular species by gel f i l t r a t i o n , and preferred a denatured DNA primer. DNA polymerases from adult l i v e r and slow growing hepatomas were separated into two molecular sizes on the gel. The larger protein species preferred a denatured DNA primer while the smaller protein showed equal a c t i v i t y with a native or denatured DNA. Although at least two DNA polymerases are i n d i -cated i n th i s l a t t e r study, these authors have not indicated that i f any terminal enzyme a c t i v i t y was present i n these f r a c t i o n s . The molecular weights of several DNA polymerases from bac-teriophage, bacteria, and mammalian sources have recently been reported. A T i * phage induced DNA polymerase ( 6 9 ) had a molecular weight of 1 1 2 , 0 0 0 and contained 15 sulfhydryl residues i n i t s protein composition. In contrast, the DNA polymerase from i t s host, E. c o l i was estimated to be 1 0 9 , 0 0 0 i n molecular weight - 22 -and contained 3 sulfhydryl groups. Baldwin (99) has estimated the molecular size of E. c o l i DNA polymerase by sedimentation studies. It has a sedimentation c o e f f i c i e n t of 5.6S which cor-responds to a molecular weight of 10 5. Even with the use of highly p u r i f i e d enzymes for t h i s study, some physical hetero-geneity were detected. By the use of acrylamide gel e l e c t r o -phoresis of an enzyme preparation from E. c o l i , C a v a l i e r i and Carrol (10 0) have observed an apparent separation of DNA poly-merase into several molecular species. At least three bands of DNA polymerase a c t i v i t y were detected from the gel pattern and corresponded to molecular weights of 110,000, 58,000 and 24,000 as determined by equilibrium sedimentation and electrophoretic mobility. The r a t i o s of these weights are approximately 4:2:1 which suggested to these authors of a possible tetramer-dimer-monomer rel a t i o n s h i p . Zimmerman has estimated the molecular weight of a DNA polymerase from Micrococcus l y s o d e i k t i c u s to be about 80,000 by Sephadex G-100 gel f i l t r a t i o n (101). Yoneda and Bollum (102) have reported the separation of 'terminal'; and ' r e p l i c a t i v e ' DNA nucleotidyltransferases from c a l f thymus gland by G-100 and G-200 gel f i l t r a t i o n . The re-p l i c a t i v e enzyme was estimated to be about 110,000 while the terminal enzyme was approximately 37,000 i n molecular weight. From a comparison of the molecular weights of DNA poly-merases from various organisms, variations i n molecular size occurred between d i f f e r e n t species as well as i n the same species. These experiments have indicated the possible exis-tence of multiple forms of DNA polymerases which might have some - 23 -b i o l o g i c a l r e l a t i o n s h i p to the previously discussed heterogeneous nature of DNA. The present inves t i g a t i o n : Since i n t e s t i n a l tissue has a high mitotic rate as well as a rapid replacement time for DNA (54) , the presence of an active DNA polymerase i n the in t e s t i n e was indicated. The second part of the present work was undertaken to explore t h i s p o s s i b i l i t y i n small i n t e s t i n a l mucosa of the ra t . Extracts from whole i n t e s t i -nal mucosa c e l l s as well as from nuclei were assayed for the presence of th i s enzyme. According to the protocol of Bollum (61), p u r i f i c a t i o n of an active extract of th i s enzyme was attempted using ammonium sulfate f r a c t i o n a t i o n and chromatography on DEAE-cellulose and on hydroxylapatite. The apparent formation of two major and one minor f r a c t i o n of DNA polymerase a c t i v i t y following chromatography on DEAE-cellulose led to further charac-t e r i z a t i o n studies on these f r a c t i o n s . Using suitable assay systems with 1 4 C - l a b e l e d deoxyribonucleoside triphosphates, both a ' r e p l i c a t i v e 1 and a 'terminal' DNA nucleotidyltransferase were detected i n one or more of these subfractions. The DNA products from these enzymatic reactions were characterized by hydrolysis with snake venom phosphodiesterase. Since a high deoxyribonu-clease I a c t i v i t y has been demonstrated to be present i n c e l l free extracts from i n t e s t i n a l mucosa tissue of the rat (103), i t was also of i n t e r e s t to study the possible r e l a t i o n s h i p of th i s nuclease to DNA polymerase a c t i v i t y . The molecular weights of - 24 -several DNA polymerase components from the i n t e s t i n a l mucosa were estimated by G-150 and G-200 gel f i l t r a t i o n , and by sucrose density gradient sedimentation. - 25 -PART I EXPERIMENTAL 1. Preparation of Rat I n t e s t i n a l Mucosa Cel l s and Their Subcellu- l a r Components: Suitable methods for preparing the i n t e s t i n a l mucosa tissue and for i s o l a t i n g the subcellular components were required p r i o r to the extraction of deoxyribonucleic acids from these constituents. When DNA was to be is o l a t e d from whole mucosa c e l l s , i n t e s t i n a l mucosa scrapings which were not further p u r i f i e d were adequeate ' for this purpose. Further p u r i f i c a t i o n of these scrapings was required, however, when nuclei and mitochondria were prepared from these c e l l s . Male Wistar rats weighing approximately 200 g each were ob-tained from the vivarium of the University of B r i t i s h Columbia. These rats were starved about 20 hours to reduce t h e i r i n t e s t i n a l contents before they were used i n the experiments. Each animal was k i l l e d by a blow to the head and decapitated. As quickly as possible, the small i n t e s t i n e was removed and cut into about 10 cm segments which were flushed free of contents with cold neutral 0.15 M sodium chloride-10 mM ethylenediamine t e t r a a c e t i c acid solution. The segments were cut open, applied to a c h i l l e d glass plate, and the mucosal epithelium scraped from the muscularis with the edge of a microscropic s l i d e . These i n t e s t i n a l scrapings were rapidly frozen by placing them i n l i q u i d nitrogen. When DNA was i s o l a t e d from whole c e l l u l a r extracts, as w i l l be sub-- 26 -sequently described, this frozen tissue was used. In i n i t i a l experiments, i t was observed that the nuclei from a homogenate of i n t e s t i n a l mucosal scrapings did not sediment cleanly at low cen t r i f u g a l forces (700 xg), but were suspended i n a j e l l y - l i k e mass. This s i t u a t i o n did not appear to occur i f the i n t e s t i n a l scrapings were f i r s t washed with a dextran solution as described by Clark and Porteous (10 4). When subcellular components from i n t e s t i n a l mucosa c e l l s were required, the mucosal scrapings were d i r e c t l y suspended i n cold 6%-dextran, Krebs-Ringer phos-phate buffer, pH 7.4. The dextran was purchased from General Biochemicals, Chagrin F a l l s , Ohio, i n c l i n i c a l grade form of approximately 186,000 M.W. A 1:10 (w/v) suspension of the mucosa scrapings was homogenized for one minute at low speed with a Potter-Elvehjem type homogenizer. The pooled homogenate was f i l t e r e d through cheesecloth prewetted with 6%-dextran solution. The f i l t r a t e was centrifuged i n a Serval r e f r i g e r a t e d centrifuge at 1700 xg for 5 minutes. A well-packed sediment and a s l i g h t l y turbid supernatant were obtained. The sediment was resuspended i n 20 volumes of Krebs-Ringer phosphate solution, pH 7.4, and again centrifuged at 1700 xg for 5 minutes. The f i n a l sediment contained the washed e p i t h e l i a l c e l l s , and was used for the i s o l a t i o n of the nuclear and mitochondrial f r a c t i o n s . The washed c e l l s were suspended i n 0.3 M sucrose-5 mM ethylenediamine tetraacetate, pH 7.4 to give a 1:10 w/v mixture. This suspension was homogenized i n a Potter-Elvehjem type homo-genizer at a medium speed, about 20 00 rpm, for one minute. The - 27 -r e s u l t i n g homogenate was then fractionated by centrifugation i n the SS-1 fixed angle rotor with 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 -fuge, 4° C. A well packed sediment was obtained by centrifugation at 1,000 x g for 10 min. and was observed by l i g h t microscopy to contain the c e l l n u c l e i . The r e s u l t i n g supernatant was re-moved and recentrifuged at 10,000 xg for 20 min. to y i e l d a mitochondrial p e l l e t . In further experiments i n which only the c e l l nuclei were required, 3 mM CaCl2 was added to the 0.3 M sucrose sol u t i o n . Calcium ions are believed to reduce the f r a g i l i t y of the nuclei by hardening the nuclear membrane and by reducing gel formation (10 5). These ions have been shown to be harmful to mitochondria, however, by causing them to swell and' to uncouple oxidative phosphorylation (106). The nuclei from the i n t e s t i n a l mucosal c e l l s were further p u r i f i e d . A nuclear p e l l e t was suspended i n 9 volumes of 0.25 M sucrose solution, and 20 ml of t h i s suspension were c a r e f u l l y layered over an equal volume of 0.34 M sucrose-3 mM CaCl2 solu-t i o n i n a l u s t e r o i d centrifuge tube of 50 ml capacity. The sample was centrifuged at 1500 xg for 15 min. i n the SS-1 rotor of the S e r v a l l r e f r i g e r a t e d centrifuge, 4° C. The sediment was c o l l e c t e d and resuspended i n 9 volumes of 2.4 M sucrose-1 mM CaCl2 solution according to the method of Widnell and Tata (107). This suspension was centrifuged at 4° C for 1 hour at 21,500 rpm (50,000 xg) i n a Spinco model L ultracentrifuge i n the no. 30 rotor. A whitish p r e c i p i t a t e containing the nuclei was c o l l e c t e d - 28 -at the bottom of the tube while the contaminating whole c e l l s , mitochondria and erythrocytes, as described by Widnell and Tata (10 7), formed a layer near the top of the tube. The supernatant solution including the contaminating material w a's discarded while the sediment was recovered and resuspended i n 1 to 2 ml of 0.25 M sucrose solution. This suspension was centrifuged at 1000 xg for 10 min. to sediment the p u r i f i e d nuclei and the nuclei were stored at -20° C p r i o r to DNA extraction. In order to pur i f y the mitochondrial f r a c t i o n recovered from the i n t e s t i n a l mucosa homogenate, isopycnic density gradient centrifugation was used according to the method of Rabinowitz et al. (8). The mitochondrial p e l l e t from the i n i t i a l c e n t r i -fugation was resuspended i n 9 volumes of 0.25 M sucrose-2 mM EDTA solution. This suspension was centrifuged at 10,000 xg for 15 min. i n the S e r v a l l r e f r i g e r a t e d centrifuge. A further sus-pension and centrifugation were used to give a twice washed mitochondrial p e l l e t . The washed mitochondrial f r a c t i o n was suspended i n 1 to 2 ml volume of 0.25 M sucrose solution and layered over a pre-formed l i n e a r sucrose gradient with a density range between 1.1366, 1.06 M sucrose-1 mM EDTA, and 1.2462, 1.93 M sucrose-1 mM EDTA. The tubes were centrifuged at 24,000 rpm for 2 hours at 4° C i n the Spinco model L ultracentrifuge i n the SW 25.1 rotor. After centrifugation, the narrow protein bands were c a r e f u l l y pipetted off i n successive layers from the top of the gradient. Generally two bands corresponding to l i g h t and heavy mitochondria were obtained and were d i l u t e d with - 29 -four volumes of 0.25 M sucrose-2 mM EDTA solution. The mitochon-d r i a were recovered by centrifugation at 10,000 xg f o r 20 min. and were stored at -20° C p r i o r to DNA extraction. The fractions were i d e n t i f i e d by phase-contrast and by electron microscopy. 2. I s o l a t i o n of DNA from Whole I n t e s t i n a l C e l l s and from  Subcellular Components: The i s o l a t i o n of DNA from r a t i n t e s t i n a l mucosa tissue was conducted according to the procedure of Colter et at. (56). Five g of frozen tissue were transferred to the large blender cup of a S e r v a l l omni-mixer. The following solutions of 45 ml of 1.0 M NaCl-10 mM EDTA-20 mM K phosphate, pH 7.3, 2.5 ml of 10% sodium deoxycholate solution, and 50 ml of 75% w/w phenol solution were added to the cup. After the suspended tissue had defrosted, the mixture was homogenized at 0° C using a low blender speed corresponding to about 250 0 rpm for a 30 min. period. After this time, the emulsion was centrifuged for 10 min. at 27,000 xg i n the S e r v a l l r e f r i g e r a t e d centrifuge. The upper aqueous layer containing the DNA was c a r e f u l l y removed from the lower phenol phase. The aqueous solution was subjected to two further extractions with equal volumes of water-saturated phenol. These extractions were carried out for 5 min. on a mechanical shaker. Each extraction was followed by centrifuga-t i o n at 4300 xg i n the S e r v a l l centrifuge for a 5 min. period. Traces of phenol were removed from the DNA solution by 4 or 5 b r i e f ether extractions, and residual ether was eliminated by - 30 -bubbling N 2 through the solution. This solution was stored at 4° C overnight and was c l a r i f i e d by centrifugation, i f necessary. The addition of an equal volume of absolute e.thanol, at 0° C, to the aqueous solution p r e c i p i t a t e d the DNA fibers which were wound onto a glass rod. A further volume of absolute ethanol was added to the solution to remove any remaining DNA. The t o t a l y i e l d of DNA was approximately 25 to 30 mg from 5 g of tissue. The DNA was washed with 75% ethanol, dried in vacuo, and stored at -20° C p r i o r to further characterization studies. The method used for the i s o l a t i o n of DNA from the nuclei of i n t e s t i n a l mucosa c e l l s was about the same as that described for DNA i s o l a t i o n from whole c e l l s . A 2.5 g sample of frozen nuclei was thawed i n a solution which contained 2 3 ml of sodium chloride-EDTA-phosphate buffer and 1.3 ml of 10% sodium deoxycholate. This mixture was s t i r r e d with a magnetic s t i r r e r for 30 min., at 4° C. Afte r this time, 25 ml of 75% phenol solution were added to the flask and the contents gently shaken for an additional 30 min. at 4° C. The mixture was then centrifuged at 27,000 xg for 10 min. and the upper aqueous phase was c a r e f u l l y removed and treated as before. A y i e l d of approximately 20 mg of nuclear DNA was obtained. DNA was i s o l a t e d from the mitochondria of i n t e s t i n a l mucosa c e l l s using a modified phenol extraction medium as previously described. The frozen mitochondria were suspended inT3 volumes of 0.15 M NaCl-10 mM EDTA-0.1 M T r i s buffer, pH 8.1, containing 1% sodium deoxycholate. The suspension was s t i r r e d for 30 min. - 31 -with a magnetic s t i r r e r at 4° C. An equal volume of 75% phenol-0.1 M Tris-HCl buffer, pH 8.1, was added to the mixture and shaken for an additional 30 min. on a mechanical shaker. The emulsion was centrifuged at 10,000 xg for 10 min., and the upper aqueous phase was removed. After two further washings with 75% phenol-T r i s solution, a protein free interface was observed between the water-phenol layers following centrifugation at 4300 xg for 5 min. Residual phenol was removed from the aqueous solution by extraction with ether as before. DNA was preci p i t a t e d from the soluti o n by adding two volumes of cold absolute ethanol and was recovered by centrifugation at 12,000 xg for 15 min. The DNA was dissolved i n 0.15 M NaCl-0.015 M sodium c i t r a t e solution, pH 7.0 (SSC). 3. P a r t i a l P u r i f i c a t i o n of DNA Preparations by Ribonuclease: DNA i s o l a t e d from r at i n t e s t i n a l tissue was treated with ribonuclease to remove any contaminating RNA. A stock solution of RNase was prepared by dissolving 5.4 mg bovine pancreas RNase (Worthington) i n 1 ml of standard saline c i t r a t e solution, pH 7. This solution was heated at 90° C for 10 min. to degrade any deoxyribonuclease which may have been present. The enzyme solu-t i o n was cooled and s u f f i c i e n t RNase solution was added to a solution of DNA to give a f i n a l concentration of 10 0.ug enzyme per ml. After incubation at 37° C for 1 hour, this solution was poured into c e l l u l o s e tubing and dialyzed against SSC solution for 2 hours, at 4° C. The SSC solution was renewed and the - 32 -d i a l y s i s was continued overnight. The DNA solution was removed from the d i a l y s i s tubing and the DNA was pre c i p i t a t e d from solu-t i o n by the addition of two volumes of cold absolute ethanol. The DNA was removed with a glass rod or by centrifugation and dissolved i n about 3 ml of SSC solution. 4. Fractionation of DNA on Methylated Albumin Column: Methylated albumin was prepared according to the procedure of Mandell and Hershey (33). Five g of bovine albumin powder, f r a c t i o n V from Armour Pharmaceuticals, were suspended i n 500 ml of absolute methanol and 4.2 ml of 12N hydrochloric acid were added. The protein dissolved and eventually formed a p r e c i p i t a t e . After allowing the mixture to stand i n the dark for 3 to 4 days with occasional shaking, tfe:^precipitate was c o l l e c t e d by c e n t r i -fugation. The p r e c i p i t a t e was washed twice with methanol and twice with anhydrous ether. The l a s t ether wash was removed and the remaining ether evaporated i n a i r . The dried methylated albumin was reduced to a powder and stored over KOH. Methylated albumin-coated kieselguhr (MAK) was prepared following the protocol of Mandell and Hershey (33). Kieselguhr was purchased from Johns-Manville Prod. Inc. 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 . A suspension of 20 g. kieselguhr i n 100 ml of 0.1 M NaCl-0.05 M Na phosphate buffer, pH 6.7, was boiled to expel a i r , and then cooled to room temperature. Five ml of 1% methylated albumin was added, with s t i r r i n g , and then 20 ml of additional 0.1 M buffered saline was added. The solution was - 33 -transferred into a chromatographic column (2 x 30 cm) over a pad of c e l l u l o s e powder, standard grade (Whatman). Five to ten ml portions of the suspension were placed i n the column and excess saline solution was forced out under an a i r pressure of 3 l b s / i n 2 . The MAK i n the column was washed with about 300 ml of 0.4 M NaCl-0.0 5 M Na phosphate buffer, pH 6.7, at the same pressure. The contents of the column were suspended i n 125 ml of 0.4 M buffered saline solution. This suspension was stored at 4° C i n the presence of a few drops of chloroform, as preservative, and was stable for several weeks. The column used for the fractionation of DNA was composed of three layers packed i n a 2 x 30 cm chromatographic column. The column contents were b u i l t up as follows: Layer 1: 8 g of kieselguhr were boiled i n 40 ml of 0.1 M NaCl-0.05 M sodium phosphate buffer, pH 6.7. After cooling, 2 ml of 1% methylated albumin solution were added, and an addi-t i o n a l 15 ml of 0.1 M buffered saline were added. This s l u r r y was used to form the f i r s t layer i n the column on a pad of c e l l u -lose powder. Excess protein was washed free with 0.1 M saline buffer. Layer 2: 6 g of kieselguhr were bo i l e d and cooled i n 40 ml of 0.4 M NaCl-0.05 M sodium phosphate buffer, pH 6.7. Ten ml of MAK were added to the suspension, and the t o t a l suspension was used to form the second layer i n the column. Layer 3: One g of kieselguhr was b o i l e d i n 10 ml of 0.4 M buffered s a l i n e , and was used to form the t h i r d layer i n the - 34 -column. This f i n a l layer acted as a mechanical b a r r i e r to the working part of the column. An a i r pressure of 3 l b s / i n 2 was applied to form each layer of the column. The fi n i s h e d column was washed with 0.05 M NaCl-0.05 M sodium phosphate buffer, pH 6.7, u n t i l the e f f l u e n t had the same r e f r a c t i v e index as the eluant. In certain experiments, the amount of MAK i n the second layer was doubled to allow the a p p l i -cation of larger DNA samples, of up to 100 absorbancy units at 260 my. Generally, 20 absorbancy units i s equivalent to 1 mg of DNA (121). The washed MAK column was loaded with a DNA preparation containing 0.5 to 2 mg of the sample i n 50 ml of 0.05 M sodium chloride buffer. Elution of the column was achieved by the passage of a phosphate buffer with increasing s a l t concentration through the MAK. Such a system was obtained by using 400 ml of 0.05 M saline buffer pH 6.7 i n the mixing chamber and 400 ml of 1.5 M saline buffer pH 6.7 i n the reservoir which enabled the formation of a l i n e a r concentration gradient of NaCl through the column. A buchler micro-pump was used to obtain a column flow rate of about 25-30 ml/hr. Five ml e f f l u e n t fractions were colle c t e d i n an automatic f r a c t i o n c o l l e c t o r and the s a l t con-tents of these fractions were monitored by r e f r a c t i v e index measurements using a c a l i b r a t i o n curve previously prepared. Following chromatography, the DNA fractions were desalted by d i a l y s i s i n c e l l u l o s e tubing. Seamless c e l l u l o s e tubing, 3/4 inch d i a . , was thoroughly washed with b o i l i n g 10% NaOH - 35 -solution and rinsed repeatedly with d i s t i l l e d water u n t i l the rinsings were neutral. The washed tubing i n approximately 20 cm segments was suitable for the d i a l y s i s of 5 ml samples. The DNA fraction s from the MAK column were dialyzed against standard saline c i t r a t e solution, pH 7, at 4° C. During the d i a l y s i s , the solution of SSC was gently s t i r r e d with a magnetic s t i r r e r . 5. Some Physicochemical Characteristics of DNA: The nitrogen content of i n t e s t i n a l mucosa DNA was deter-mined by a micro-Kjeldahl d i s t i l l a t i o n method (108). The phos-phorus content of the DNA was determined according to the pro-cedure of B a r t l e t t (109). The l a t t e r procedure employed ammon-ium molybdate solution and Fiske-Subba Row reagent for the colorimetric determination. From the o p t i c a l density of a DNA solution at maximum ab-sorption, usually at 257.5 my, and i t s determined phosphorus content, the atomic extinction c o e f f i c i e n t with respect to phosphorus can be calculated according to the rel a t i o n s h i p by Chargaff (110). This value, designated as e(P) i s equal to O.D. at max. X x atomic wt. P/conc. P g . l x 1 cm l i g h t path. An s(P) value for DNA samples has been used as a guide to the native form of the preparation. According to Chargaff (110), DNA with values higher than 7200 was considered to be denatured. 6. Determination of the Base Composition of DNA: a. Method of acid hydrolysis and paper chromatography: The DNA from the rat i n t e s t i n a l mucosa was hydrolyzed with - 36 -98% formic acid according to the method of J e r v e l l , et al. (111). The solution was sealed i n a glass tube and heated for 2 hours i n an oven at 165° C. After t h i s time, the sample was cooled to room temperature and then to -20° C. The tube was opened cautious-ly and the contents were thawed; poured into a 10 ml beaker. The excess formic acid was removed in vacuo and the residue taken up i n a few drops of 0.1 N HC1. The purine and pyrimidine bases of the hydrolyzate were separated by paper chromatography by a descending technique on Whatman no. 1 paper using Wyatt's solvent (112). After the bases were located on the paper by examination under u l t r a v i o l e t l i g h t , they were eluted from the paper with 0.1 N HC1 using the technique of cutting the paper into f i n e pieces and packing them into drawn out glass tubing resembling a Pasteur pipette (113) . The proportion of each base was deter-mined qua n t i t a t i v e l y by u l t r a v i o l e t spectrophotometry using a Cary-15 recording spectrophotometer. b. Method of thermal denaturation: The procedure of Marmur and Doty (44) was followed for the determination of the guanine plus cytosine content of rat i n -t e s t i n a l mucosa DNA by heat denaturation. Materials and Reagents: (i) DNA is o l a t e d from the small i n t e s t i n a l mucosa of the rat was used. Studies were made on the crude extract or on the MAK fractionated DNA samples. ( i i ) 0.15 M NaCl-0.015 M sodium c i t r a t e solution, pH 7.0 is designated as standard saline c i t r a t e (SSC) solution. - 37 -Procedure: The DNA was dissolved i n SSC to a concentration of approxi-mately 20 to 25 yg/ml and 3 ml of th i s solution was placed i n a quartz cuvette having a 1 cm l i g h t path. Absorbance was measured at 260 my i n a G i l f o r d model 2000 multiple sample absorbance recorder which has a cuvette chamber equipped for heating the DNA samples. The temperature i n the chamber was raised by c i r c u l a t i n g ethylene g l y c o l (bp. 198-200° C) which was heated i n a thermostat regulated c i r c u l a t o r . After f i r s t measuring the absorbance of the DNA solution at 25° C, the temperature of the chamber was raised quickly to about 5° C below the estimated onset of the melting region. The temperature was then raised about 1° C per 5 min. period u n t i l the sample showed no further increase i n absorbance. The r e l a t i v e absorbance of the DNA sample at each temperature was determined by di v i d i n g the absorbance at this temperature by the value at 25° C. The temperature correspond-ing to half the maximum increase i n the r e l a t i v e absorbance i s designated as the midpoint of thermal denaturation, Tm, according to Marmur and Doty (4 4). According to the experimental obser-vations of these authors, the Tm value for a number of DNA samples has a l i n e a r r e l a t i o n s h i p to i t s guanine plus cytosine content at a p a r t i c u l a r i o n i c strength. This rel a t i o n s h i p i n standard saline c i t r a t e i s represented by the equation Tm =• 69.3 + 0.41 (G+C). A l i n e s t a r t i n g from a Tm value of 69.3° C has a slope of 0.41° C per 1% r i s e i n the G+C content. - 38 -c. Method of CsCl density gradient centrifugation: The base composition of rat i n t e s t i n a l mucosa DNA was determined also by cesium chloride density gradient analysis following the procedure of Schildkraut et al. (58). Materials and Reagents: (i) Sample DNA: Small i n t e s t i n a l mucosa DNA from the rat was i s o l a t e d as e a r l i e r described. ( i i ) Reference DNA: Pseudomonas fluorescens DNA which _3 has a density of 1.721 g cm and G+C mole % of 63 was a g i f t from Dr. M. Smith. ( i i i ) Cesium chloride: Optical grade CsCl was obtained from Gallard-Schlesinger Chem. Manuf. Corp. New York. A 7 M CsCl stock solution was prepared by dissol v i n g 130 g of CsCl i n 70 ml of 0.01 M Tris-HCl buffer, pH 8.5. Procedures: A t y p i c a l composition used for the analysis of a DNA sample by CsCl density gradient centrifugation i s as follows: 1) 0.93 ml of CsCl stock solution 2) 0.001 ml reference DNA solution, 0.5 mg/ml 3) 0.0 4 ml sample DNA of unknown density, 50 ug/ml 4) 0.2 ml of 0.01 M T r i s buffer, pH 8.5 The density of the f i n a l solution was adjusted to approximately -3 1.710 g cm with small amounts of s o l i d CsCl or by varying the volume of 0.01 M Tis buffer. This density was adjusted to give a DNA which would band near the center of the gradient i n the c e l l . This density was estimated by a r e f r a c t i v e index method - 39 -(114) to be approximately D2o 1.4010. Suitable concentrations for the DNA sample to be measured were 1 to 2 yg with 0.5 to 1 yg for the reference DNA. Approximately 0.75 ml of the f i n a l CsCl solution was placed i n the centrifuge c e l l using a 1 ml syringe and a no. 22 needle without a beveled t i p . A f l a t tipped needle reduced the danger of scratching the Kel-F center-piece . The c e l l containing the sample to be examined was placed i n an An-D rotor with an appropriate counterbalance (7 g). Centric fugation was c a r r i e d out i n the Spinco model E a n a l y t i c a l u l t r a -centrifuge at 44,770 rpm at 25° C. After 16 to 20 hours of centrifugation, the samples i n the c e l l were at equilibrium. At t h i s point, u l t r a v i o l e t absorption photographs were taken on the DNA samples during the centrifugation with Kodak commercial f i l m . (The ultracentrifuge used was equipped with an u l t r a v i o l e t o p t i c a l system.) After developing the f i l m , the DNA bands corresponding to the sample and the reference material were traced with a Joyce-Loebel double beam recording microdensito-meter . A t y p i c a l microdensitometer tracing of an u l t r a v i o l e t absorption photograph of approximately 1 yg of i n t e s t i n a l DNA of unknown density and 0.5 yg of Pseudomonas fluoresoens DNA i s shown i n Figure 1. The distances measured on the tracing are as follows: a to d = distance from top to bottom of the c e l l (15.1 cm), a to c = distance from peak of reference DNA band to the top of the c e l l (11.1 cm); a to b = distance from 40 -1 u U o w < Density Figure 1. A microdensitometer tracing of the banding pattern of DNA following equilibrium centrifugation i n a CsCl solution. The reference DNA i s from Pseudomonas fluovesoens. - 41 -peak of unknown DNA to the top of the c e l l (9.8 cm). The center-piece cavity i s known to be 1.6 cm i n length. Therefore, the magnification of the tracing i s . 1 5 . 1 / 1 . 6 . M u l t i p l i c a t i o n by the r e c i p r o c a l of t h i s r a t i o converted each magnified distance to the actual distance from the c e n t r i p e t a l end of the c e l l . The actual distance of each band from the center of r o t a t i o n i s 5 .7 cm greater when the An-D two-cell rotor i s used. In this example, the peak of the reference DNA band i s 5 .7 + 1.176 = 6.876 cm (r 0) from the center of rotation, and the unknown peak i s 5 .7 + 1.038 = 6.738 cm (r) from the center of r o t a t i o n . The buoyant density of the DNA at a distance r from the center of rotation was calculated according to the following equation (58) : — 10 — 3 p = p 0 + 4 . 2 w 2 ( r 2 - r 0 2 ) x 10 g cm where p 0 = density of the standard DNA; co = speed of rotation i n radians sec. 1 ; r 0 = distance of the standard DNA from the center of r o t a t i o n . At 44,770 rpm, the above equation i s s i m p l i f i e d to the following — 3 r e l a t i o n s h i p : p = p 0 + 0.0092 ( r 2 - r o 2 ) g cm . By the use of this equation, the density of the unknown DNA i n the present - 3 example was calculated to be 1.70 4 g cm The corresponding G+C content of the DNA was obtained by using the following l i n e a r r e l a t i o n s h i p of Schildkraut et al. (58) : (G+C) = p - 1.660 g cm"1/0.098 where (G+C) = mole f r a c t i o n guanine plus cytosine i n native DNA. For the example c i t e d above, the G+C content was 0.449 or contained 44.9 per cent guanine plus cytosine. PART I RESULTS and DISCUSSION 1. Iso l a t i o n of DNA from the i n t e s t i n a l mucosa: Crude rat i n t e s t i n a l mucosa scrapings were suitable for the i s o l a t i o n of DNA when whole c e l l extracts were used for DNA studies. The combination of a high s a l t concentration, detergent, and phenol as described by Colter et at. (56) was found to e f f e c t i v e l y deproteinize the DNA from the i n t e s t i n a l mucosa as well as to give an adequate y i e l d of DNA for the present study, 25 to 30 mg DNA from 5 g tis s u e . This DNA was found to have an average nitrogen and phosphorus content of 14.5 and 9.0 per cent respectively. The r a t i o of the N/P i s 1.61 which i s close to the t h e o r e t i c a l ^alue of 1.65 according to Watson and Crick (122), and agrees with values previously determined for rat i n t e s t i n a l DNA by Mezei and Zbarsky (113) using another DNA i s o l a t i o n pro-cedure. On the basis of the atomic extinction c o e f f i c i e n t value with respect to phosphorus, e(P), the i n t e s t i n a l DNA i s o l a t e d with the present procedure showed an average value of 6700, at 260 my. Generally native DNA from mammalian sources exhibits an e(P) value close to 6600, at 260 my, regardless of the base composition as described by Chargaff (110). As w i l l be subse-quently discussed under the heat denaturation of DNA, these i n t e s t i n a l DNA samples showed the c h a r a c t e r i s t i c melting p r o f i l e for a native double-stranded DNA structure. A p a r t i a l p u r i f i c a t i o n of i n t e s t i n a l mucosa scrapings was - 43 -necessary when the subcellular organelles, nuclei and mitochondria, were required for the extraction of DNA. The presence of a heavy mucus coating over the i n t e s t i n a l c e l l s caused the formation of a j e l l y - l i k e p e l l e t upon centrifugation of the crude c e l l homogenate. An i n i t i a l washing of the i n t e s t i n a l tissue before c e l l disruption was necessary. A 6 per cent dextran Krebs-Ringer phosphate solu-t i o n was used to remove this mucin material according to the pro-cedure of Clark and Porteous (104). Following homogenization of dextran washed c e l l s , a better separation of the subcellular com-ponents by centrifugation was obtained. In order to make comparative studies of DNA from the nuclei and mitochondria, these subcellular components required further p u r i f i c a t i o n . The method of separating nuclei by centrifugation through a dense sucrose medium as o r i g i n a l l y developed by Chauveau (115) who used a 2.2 M sucrose solution and modified by Widnell and Tata (107) who used 2.4 M sucrose was found to give high purity nuclei from the intestinal mucosa c e l l s , but the y i e l d of nuclei was only 15 to 20 per cent by weight of the o r i g i n a l nu-clear sample used. The p u r i f i c a t i o n of mitochondria by sucrose density gradient centrifugation also gave a f a i r l y pure mitochon-d r i a l preparation as shown i n the electron micrograph of Figure 2. The y i e l d of mitochondria was very low, i . e . less than 0.05 g from 5 g of mucosal tissue. The i s o l a t i o n of DNA from the nuclei or mitochondria of i n t e s t i n a l mucosa c e l l s was achieved by a detergent and phenol treatment as before except that the homogenization i n a S e r v a l l - 44 -Figure 2. Electron micrograph of mitochondria from ra t i n t e s t i n a l mucosa. The mitochondria were prepared by-sucrose density gradient centrifugation. Magnification 20,000 times. - 45 -omni-mixer step was omitted. Although the blender speed used, about 2500 rpm, was well below the c r i t i c a l speed considered to degrade DNA according to Hershey and Burgi (116), as good or even a s l i g h t l y better y i e l d of nuclear DNA, of about 22 mg form 2 g nucl e i , was obtained using a gentle s t i r r i n g and shaking of the nuclei i n the phenol extraction medium. A similar gentle phenol extraction procedure was used for the i s o l a t i o n of DNA from the mitochondria. The y i e l d of DNA from this l a t t e r organelle was d i f f i c u l t to estimate because of the high content of RNA i n the nucleic acid extract. The removal of RNA from the mitochondrial extracts would simplify the characterization of mitochondrial DNA which accounts for less than 1% of the t o t a l c e l l u l a r DNA (117). In preliminary experiments on the DNA extracts from mitochondria of i n t e s t i n a l mucosa c e l l s , a posi t i v e o r c i n o l test (118) and an i n d e f i n i t e diphenylamine test (119) were obtained. Acid hydrolysis and paper chromatography of this extract indicated the presence of a l l four RNA bases, A, G, C and U but without detectable thymine which i s usually c h a r a c t e r i s t i c of DNA. In subsequent experi-ments , the crude mitochondrial DNA extracts were treated with ribonuclease to remove residual RNA which contaminated these preparations. The l i g h t and heavy mitochondrial fractions re-covered from sucrose denstiy gradient centrifugation were separ-ately extracted for DNA, and these extracts were treated with RNase. Table II shows two experiments i n which d i f f e r e n t volumes of DNA extraction medium were used, 1:10 w/v and 1:2 w/v for - 46 -Table II Ef f e c t of Ribonuclease Treatment on the Mitochondrial DNA Extracts from Rat I n t e s t i n a l Mucosa Mitochondrial Fraction from sucrose gradient RNase a treatment Volume*3 ml. O.D. at 260 my I. Light mitochondria before 12 0.38 after I I 0.17 Heavy mitochondria before 10 0.68 after I I 0.27 II. Light mitochondria before 2 3.86 after i i 2.82 Heavy mitochondria before 2 3.65 after I I 1.43 Bovine pancreas RNase was added to give a f i n a l con-centration of 100 yg enzyme per ml solution. The solutions were dialyzed against SSC solution, pH 7. Volume of DNA extracts of the mitochondria. - 47 -experiments 1 and 2 respectively. As indicated, both, l i g h t and heavy mitochondrial extracts contained a substantial amount of material which was sensitive to RNase treatment. After d i a l y s i s , the material which was retained i n the c e l l u l o s e tubing gave a pos i t i v e diphenylanine t e s t , but might s t i l l contain RNA which was not completely degraded by the RNase. Nass et at. (120) have reported such a RNase r e s i s t a n t ribose-containing component i n DNA extracts from r a t l i v e r mitochondria. 2. Fractionation of DNA by MAK column: Several adsorbents such as hydroxylapatite (McCallum and Walker, 36), methacrylic carboxylic acid (IRC-50, Frankel and Crampton, 32), and ECTEOLA c e l l u l o s e (Rosenkranz and Bendich, 31) have been used for the column chromatography of DNA, but the methylated albumin-kieselguhr column has been the most widely used. The early studies by Mandell and Hershey (33) showed that MAK could separate DNA into fractions d i f f e r i n g i n molecular s i z e . Subsequently, Sueoka and Cheng (34) reported that these columns could also fractionate DNA according to i t s base compo-s i t i o n i n which fractions with higher G+C content were eluted from the column f i r s t . As described under the experimental procedure, a standard MAK column was used for the chromatography of r a t i n t e s t i n a l mucosa DNA. A t y p i c a l e l u t i o n pattern for the chromatography of a 2 mg sample of i n t e s t i n a l DNA which was i s o l a t e d from a whole c e l l homogenate i s shown i n Figure 3. The u l t r a v i o l e t ab-sorption p r o f i l e at 260 my shows a major peak was eluted at Figure 3. Chromatography of DNA from rat i n t e s t i n a l mucosa on MAK column. Elution was carried out with a gradient of NaCl i n 0.0 5 M phosphate, pH 6.7 - 49" -approximately 0.65 M NaCl. This peak would correspond to a double h e l i c a l DNA f r a c t i o n which was shown by Sueoka and Cheng (121) to be eluted from a s i m i l a r l y prepared MAK column by a NaCl concentration of 0.6 to 0.75 M. A forepeak as well as several smaller U.V. absorbing bands were usually found adjacent to the main DNA peak. If the DNA extracts were f i r s t treated with ribonuclease and the ribonucleotides were removed by d i a l y s i s before chromatography, several of the smaller peaks were absent following MAK chromatography of this preparation as shown i n Figure 4. The small forepeak of U.V. material was s t i l l present and was found by o r c i n o l and diphenylamine tests to be mostly ribose containing material with a small amount of deoxyribose material. When the main DNA f r a c t i o n at 0.65 M NaCl was con-centrated to a small volume on a fl a s h evaporator, and dialyzed against 0.1 M NaCl, rechromatography of this DNA f r a c t i o n gave only one peak eluted by 0.6 M NaCl as shown i n Figure 5. A small forepeak of material was not present i n this chromatography. The material which formed this, forepeak was, therefore, present i n the o r i g i n a l DNA solution, and was not formed i n the column during the fr a c t i o n a t i o n . When up to 100 O.D. units of DNA, 5 mg, were used for the fra c t i o n a t i o n , the amount of material used to form the second layer of the column was doubled. The second layer of such a column would contain 12 g kieselguhr with 20 ml of MAK suspension. As shown i n Figure 6, the eluti o n pattern for about 5 mg of DNA from a t o t a l c e l l extract shows the separation of the main peak into two closely related f r a c t i o n s . Figure 4. Chromatography of DNA on MAK column. The DNA sample used was the same as that i n Figure 3, but has been treated with RNase before chromatography. - 51 -Figure 5. Rechromatography on MAK of the main DNA f r a c t i o n as shown i n Figure 4. - 52 -Figure 6. Chromatography of 5 mg of DNA from rat i n -t e s t i n a l mucosa on MAK column. The second layer of the column was doubled. - 53 -In a s i m i l a r experiment, the fr a c t i o n a t i o n of DNA from the nuclei also showed the double-peaked DNA band as shown i n Figure 7. As w i l l be subsequently discussed, the base composition of these fractions from the t o t a l and the nuclear DNAs were compared. 3. The base composition of DNA i s o l a t e d from the i n t e s t i n a l  mucosa of the r a t : Three methods for the determination of the base composition of DNA i s o l a t e d from the i n t e s t i n a l mucosa of the rat were com-pared. Direct chemical analysis, thermal denaturation and buoyant density centrifugation i n CsCl were used to assess the intermolecular heterogeneity of DNA from the small i n t e s t i n e . In the e a r l i e r experiments, a l l three methods were used to study the t o t a l c e l l u l a r DNA extracts. Subsequently, the thermal denaturation method was found to be suitable for determining the G+C content, and was used for comparing the nuclear and the mitochondrial fractions of DNA to the t o t a l c e l l u l a r DNA samples. The d i r e c t chemical analysis method was performed by an acid hydrolysis of the i n t e s t i n a l DNA followed by the separation of the nucleic-acid bases on paper chromatograms. For th i s study, a whole c e l l extract of DNA was fractionated on the MAK column. The e l u t i o n p r o f i l e for the chromatography of 100 O.D. units of i n t e s t i n a l DNA i s shown i n Figure 8. A wide elut i o n band for the main DNA p r o f i l e was obtained which appeared to have segregated into three d i s t i n c t peaks which were grouped as fractions I (tubes no. 56-65), II (tubes no. 67-74) and - 54 -I I • ' • i i i i u i i It 1 1 0 20 h0 60 80 100 120 Tube Number Figure 7. Chromatography of nuclear DNA from rat i n -t e s t i n a l mucosa c e l l s on MAK column. A 5 mg DNA sample was used. The second layer of the column was doubled. - 55 -J _ J • ' I I I L 0 20 1+0 60 80 100 Tube Number Figure 8. Chromatography of DNA on MAK column. A 5 mg sample of i n t e s t i n a l mucosa DNA from a whole c e l l extract was used. The second layer of the MAK column was doubled. - 56 -III (tubes no. 75-82) for chemical analysis. The DNA was re-covered from these fractions by reducing the volume of the solu-t i o n on a f l a s h evaporator, and then p r e c i p i t a t i n g the DNA with-a' few drops of cold 1 N perchloric acid. After acid hydrolysis of the DNA and recovery of the free bases as described under the experimental procedure, the base composition of the DNA samples were calculated as shown i n Table I I I . These values represent the average composition of the pooled fractions and could be compared to the mole per cent guanine and cytosine determined for the t o t a l c e l l u l a r DNA of 43.6. The group I DNA f r a c t i o n contained the peak eluted f i r s t from the MAK column and showed a G+C content.of 46.53. The group III f r a c t i o n which was eluted with a higher s a l t concentration gave a lower G+C content of 39.97. The group II f r a c t i o n showed a base composition of 43.12 mole % G+C which corresponds to the mean value of the t o t a l unfractionated i n t e s t i n a l DNA. Chemical analysis, thus, i n d i -cated that the rat i n t e s t i n a l DNA was heterogeneous i n base composition and that the molecular species with a higher G+C con-tent was eluted from the MAK column f i r s t as previously reported by Sueoka and Cheng (34). Thermal denaturation has proven to be a simple and rapid procedure for the determination of the base composition of DNA. Marmur and Doty (44) have shown that the temperature at the midpoint of the hyperchromic change at 260 my, designated as the Tm, i s c h a r a c t e r i s t i c of each DNA sample, and i s l i n e a r l y related Table III The Base Composition of DNA from Rat I n t e s t i n a l Mucosa DNA source Base Proportions, mole % A+T mole G+C G A C T G + C Whole c e l l a 23.3 28.6 20.3 27.8 1.29 43.6 MAK fractions I 23.56 26.95 22.97 26.51 1.15 46.53 II 22.30 27.63 20.82 29.24 1.32 43.12 III 20.36 29.19 19.61 30.84 1.59 39.97 Data according to C.Y. Lee and S.H. Zbarsky (103). ^The DNA sample used was extracted from the whole mucosa c e l l s and was fractionated on a MAK column. - 58 -to i t s average guanine and cytosine content. According to these authors, the rapid hyperchromic change of up to 40% has i t s o r i g i n i n the sequential unwinding of the base pairs i n the native h e l i x DNA to form single stranded chains which were o r i -g i n a l l y s t a b i l i z e d by the tiree hydrogen bonds between G+C and two between A+T. Typical hyperchromic p r o f i l e s f o r samples of rat i n t e s t i n a l DNAs which were measured at 260 my i n a standard saline c i t r a t e solution are shown i n Figure 9. The central l i n e with an average Tm of 86.6° C i s c h a r a c t e r i s t i c of t o t a l i n t e s t i n a l DNA i s o l a t e d from mucosal tissue. Two other Tm p r o f i l e s are shown which represent subfractions of the DNA following MAK chromatography. A f r a c t i o n eluted near the front of the main peak would have a higher Tm and G+C content. After heat denaturation, the i n t e s t i -nal DNA from the total or nuclear components of the c e l l did not show the a b i l i t y to renature as indicated by the gradual decrease i n the hyperchromicity over the whole temperature range during slow cooling. Heterogeneous DNA from higher organisms are generally unable to show s p e c i f i c reunion of i t s complementary single strands once the two strands have separated. Geiduschek (123) has previously found that i f a heated DNA sample was rapid l y cooled from the elevated temperature, those molecules i n which the two strands were s t i l l united at one or more points due to r e s i s t a n t G+C pairs would rapidly re-form v i r t u a l l y a l l of the inter-chain base pairs to give the o r i g i n a l native conformation. - 59 -1.5 r 1 A 1.3 1.2 1.1 —o— -~ 50 60 70 80 Temperature (*C) 90 100 Figure 9. Heat denaturation and cooling curves of DNA from r a t i n t e s t i n a l mucosa. The samples were studied i n standard saline c i t r a t e solution, pH 7. Total i n t e s t i n a l DNA - o o - Tm = 86.6; cooling curve. A leading f r a c t i o n of DNA from chromatography on MAK Tm = 8 8.5 —•—•— ; a rear f r a c t i o n , Tm = 8 4.5° C -*—*• . - 60 -A comparison of the DNA i s o l a t e d from the whole c e l l and from the subcellular components was made on the basis of guanine and cytosine contents as determined by the technique of thermal de-naturation. DNA from rat i n t e s t i n a l mucosa was extracted from the whole c e l l or the ruclei, and was fractionated on MAK as pre-viously described i n Figures 6 and 7. These fractions were separately dialyzed i n SSC and then heat denatured to determine t h e i r Tm values. On the basis of the r e l a t i o n s h i p of Tm and mean G+C content as described by Marmur and Doty (44), the mole per cent G+C was calculated for the DNA f r a c t i o n s . The results from such a study are shown i n Table IV. When the t o t a l c e l l u l a r DNA fract i o n s were compared as a function of t h e i r Tm values as shown i n Figure 10, a gradual decrease i n Tm value from early to l a t e r fractions was observed. There appeared to be several fractions with Tm values near 86.6° C which i s the mean value of the t o t a l unfractionated DNA from r a t i n t e s t i n a l mucosa. A similar decrease i n the Tm values was obtained from the nuclear DNA fractions as shown i n Figure 11. A few fractions with a Tm value of 86.6° C were also found. After f r a c t i o n number 86, the Tm values showed a gradual increase with increasing fractions eluted from the MAK. This change i n the Tm pattern may r e f l e c t the a b i l i t y of the MAK to fractionate DNA on the basis of i t s molecular size as well as on i t s G+C content according to Sueoka and Cheng (34). Due to the small apparent v a r i a t i o n between the Tm and the corresponding G+C content of the DNA subtractions - 61 -Table IV The Mole per cent Guanine plus Cytosine of Rat I n t e s t i n a l Mucosa DNA from i t s Thermal Denaturation Temperature. DNA Fraction Tm, °C Mole % G+C* Total c e l l sample 86.6 42.19 Whole c e l l extract, fractionated on a MAK column. #73 91.0 52.92 74 90.6 51.95 75 89.4 49.02 76 87.4 44.14 77 87.1 43.41 78 86.6 42.19 79 86.5 41.95 80 86.4 41.70 81 85.6 39.75 82 84.1 36.09 83 84.0 35.85 84 83.4 34.39 Nuclear DNA extract, fractionated on a MAK column. #71 90.2 50.97 72 89.2 48.53 73 88.5 46.82 74 88.5 46.58 75 87.7 44.87 76 87.4 44.14 77 86.6 42.19 78 86.6 42.19 79 86.5 41.95 80 86.3 41.46 81 85.9 40.48 82 85.8 40.24 83 85.3 39.02 84 85.1 38.53 85 85.0 38.29 86 84.6 37.31 87 84.8 37.80 88 85.3 39.02 89 85.4 39.27 90 85.6 39.75 91 85.7 40.00 92 86.2 41.20 93 86.8 42.70 94 87.0 43.17 * G+C = Tm-69.3/0.41 according to Marmur and Doty (44). ' • 1 • • 1 1 • • • • • • 73 75 77 79 81 83 85 Fraction Number Fraction 10. A comparison of the Tm values as a function of the DNA fractions obtained from the main peak of DNA following MAK chromatography. The DNA was iso l a t e d from a whole c e l l extract of rat i n t e s t i n a l mucosa. . 91 o • • • i i i i i i i i i 66 71 76 81 86 91 96 Fraction Number Figure 11. A comparison of the Tm values as a function of the DNA fractions obtained from the main peak of DNA following MAK chromatography. The DNA was is o l a t e d from nuclei of rat i n t e s t i n a l mucosa c e l l s . - 64 -from both the whole c e l l and nuclear extracts, i t was not possible to i d e n t i f y the existence of one or more major types of DNA with a c h a r a c t e r i s t i c physical constant. A nucleic acid extract of mitochondria from rat i n t e s t i n a l mucosa was heat denatured and the increase i n hyperchromicity was followed i n a spectrophotometer as before. Both the samples i s o l a t e d from the l i g h t and the heavy mitochondria gave s i m i l a r bimodal p r o f i l e s as shown i n Figure 12. Two Tm values were observed at approximately 60° and 85° C. Although these samples had been treated with ribonuclease to remove RNA, the lower temperature p r o f i l e may not be due to DNA but to the nondia-lyzable RNA core f r a c t i o n . As the quantity of nucleic acid material that was of DNA nature was very low, probably less than 10 yg, attempted p u r i f i c a t i o n s of these samples on a MAK column of 1/10 the standard size were unsuccessful. The higher Tm value at about 85° C was c h a r a c t e r i s t i c of a double-stranded DNA as indicated by i t s melting p r o f i l e . This value i s lower than the average Tm value of 86.6° C found for the t o t a l c e l l u l a r or the nuclear DNAs. A Tm value for mitochondrial DNA of 85° C would correspond to a mole % of 3 8.29 G+C, but was not unique since a DNA f r a c t i o n from the nuclei has a s i m i l a r G+C content. Such an observation may not exclude the p o s s i b i l i t y that t h i s i s indeed a mitochondrial DNA because Corneo et at. (9) have found that mitochondrial DNA from r a t l i v e r , beef heart, beef l i v e r , and mouse l i v e r had i d e n t i c a l densities and G+C contents - 65 -1 • • i i i i • • 20 4-0 60 80 100 Temperature C'C.) Figure 12. Heat denaturation p r o f i l e of mitochondrial DNA from r a t i n t e s t i n a l mucosa. Two Tm values are apparent at approximately 60° and 85° C. - 66 -with the nuclear DNA from the same tissue. Since contamina-ti o n by fragments of DNA from disintegrated nuclei was d i f f i -c u l t to eliminate, the question of the purity of the mito-chondrial DNA preparations was always present. Attempts to remove th i s contamination by repeated washing of the i s o l a t e d mitochondria or by treatment of the i n t a c t mitochondria with DNase p r i o r to DNA i s o l a t i o n have generally been used by previous investigators (6,8). In the present study, the method of repeated washings of the mitochondria from the int e s t i n e was used because e a r l i e r studies by Kalf (6), and by Nass, et al. (120) have indicated that DNA added to a mitochondrial suspension was not adsorbed to the surface of the mitochondria, and that after repeated washings, no release of material which absorbs at 260 my was noted even after DNase treatment. The G+C content of i n t e s t i n a l mucosa DNA was also de-termined by density gradient sedimentation i n a solution of CsCl since Schildkraut et al. (58) have demonstrated that the buoyant density of DNA was d i r e c t l y proportional to i t s guanine plus cytosine content. Equilibrium centrifugation was ca r r i e d out on selected samples of rat i n t e s t i n a l mucosa DNA which had been fractionated on a MAK column. By a comparison of these fractions to a reference DNA, Pseudomonas f-tuar§scens, with a density of 1.721 g/cm3; mole % G+C = 62, the density of the unknown samples were determined. The t o t a l unfractionated DNA from whole i n t e s t i n a l mucosa c e l l s - 6 7 -had a density of 1 . 7 0 2 which corresponds to a 4 2 . 9 mole % G+C. In comparison, f r a c t i o n number 8 1 which was eluted from a MAK column as shown i n Figure 6 , had a density of 1 . 7 0 4 g/cm3; mole % G+C = 4 4 . 9 while f r a c t i o n number 88 showed a density of 1 . 6 9 9 g/cm3; mole % G+C = 3 9 . 8 . These results are shown by the microdensitometer tracings i n Figure 1 3 . A heterogeneity i n the G+C content and hence the base composition of DNA i s o l a t e d from the i n t e s t i n a l mucosa of the r a t was observed following fr a c t i o n a t i o n on MAK. Although the main band of DNA eluted with 0 . 6 5 M NaCl had separated into two or three d i s t i n c t peaks i n several of the f r a c t i o n -ations, each peak was not composed of one type of DNA with unique physical chemical c h a r a c t e r i s t i c s , but appeared to consist of several subfractions of varying base composition. Generally, the DNA sample eluted i n the leading f r a c t i o n con-tained the highest G+C content and progressively decreased to the l a s t f r a c t i o n with the lowest G+C content. Such re-sults were observed by comparing the three methods used for determining the G+C content of DNA fractions from MAK chroma-tography as shown i n Table V. Studies on DNA i s o l a t e d from the whole mucosa c e l l showed e s s e n t i a l l y the same r e s u l t s from a l l three methods. The DNA i s o l a t e d from the nuclei showed a si m i l a r pattern of heterogeneity i n i t s base compo-s i t i o n . DNA i s o l a t e d from the mitochondria appeared to have a si m i l a r Tm value as one of the subfractions of nuclear DNA. Attempts to measure the density of the mitochondrial - 68 -Buoyant Density of Rat I n t e s t i n a l Mucosa DNA i n CsCl t o t a l DNA 1 . 7 0 2 reference DNA 1 . 7 2 1 c I f r a c t i o n n o . 8 8 DNA 1 . 6 9 9 reference DNA 1 . 7 2 1 Figure 13. Microdensitometer tracings of DNA samples equilibrated i n CsCl density gradients by centrifugation at 44,770 rpm. The reference DNA i s from Ps eudomonas fluoresceins; the i n t e s t i n a l DNA fractions were obtained by MAK chromatography. - 69 -Table V Determination of the Guanine plus Cytosine content of DNA i s o l a t e d from the I n t e s t i n a l Mucosa of the Rat Source of MAK Tm Density Mole % DNA Fractions °C g/cm3 G+C Acid hydrolysis whole c e l l I II III Thermal denaturation whole c e l l 86.6 - 42. 19 75 89.4 - 49. 02 77 87.1 - 43. 41 81 85.6 - 39. 75 nuclei 72 89 .2 - 48. 53 77 86.6 - 42. 19 86 84.6 - 37. 31 mitochondria 85.0 - 38. 29 CsCl density gradient centrifugation whole c e l l - 1 .702 42. 9 81 - 1 .704 44. 9 88 - 1 .699 39. 8 mitochondria - 1.70 2 (approx.) The MAK fractions were taken from the main peak of DNA eluted with 0.6 M NaCl. For a more complete comparison of the heat d e n a t u r a t i o n f r a c t i o n s , re-fer to Table IV. 43.6 46.53 43.12 39.97 - 70 -DNA samples by CsCl density gradient centrifugation have re-sulted i n the appearance of a diffuse band on the photographs which corresponded to the density of the whole c e l l DNA. These results appear to agree with the studies by Corneo et al. (9) who showed that the densities of mitochondrial and nuclear DNA from the rat l i v e r were the same. The present characterizations on the mitochondrial DNA from rat i n t e s t i -nal mucosa are, nevertheless, inconclusive. PART II EXPERIMENTAL Before attempting to i s o l a t e and study the more complex mammalian DNA nucleotidyltransferases, i t was of p r a c t i c a l value to f i r s t characterize t h i s enzyme from the simpler b a c t e r i a l systems. As the DNA polymerase from Escherichia c o l i has been extensively characterized by Kornberg and co-workers (63), t h e i r procedures were used for the i s o l a t i o n and p u r i f i c a t i o n of this enzyme from E. c o l i B c e l l s . A. DNA Polymerase from Escherichia c o l i 1. Assay System: The p u r i f i c a t i o n of DNA polymerase from E. c o l i was followed by measuring the conversion of radioac t i v e l y l a b e l l e d deoxyribonucleoside triphosphate into an acid-insoluble product (63). Reagents: The reaction mixture i n a t o t a l volume of 0.3 ml con-• tained: 20 ymoles glycine buffer, pH 9.2 (20 y l of 1 M) 2 ymoles MgCl 2 (20 y l of 0.1 M) 0.3 ymoles 2-mercaptoethanol (30 y l of 0.01 M) 10 yg rat i n t e s t i n a l DNA (20 y l of 0.5 mg/ml) 10 nmoles dATP (10 y l of 1 ymole/ml) 10 nmoles dGTP (10 y l of 1 ymole/ml) - 72 -10 nmoles dCTP (10 y l of 1 ymole/ml) 10 nmoles dTTP (10 y l of 1 ymole/ml) 1.65 nmoles llfC-2-dTTP (5 y l of 4 x 10 7 c.p.m./ymole) 0.1 ml enzyme solution (25-100 yg protein) Procedure: I n t e s t i n a l mucosa DNA from the rat was used i n i t s native or denatured form as a template for the enzyme. When a denatured template was required for the study, the DNA solution was placed i n a 100° C water bath for 10 minutes, and then rapidly cooled to 0° C. In conducting the assay, the reaction mixture was i n -cubated for 15 to 30 min at 37° C i n a water bath. The reac-t i o n was stopped by c h i l l i n g i n an i c e bath, and by the addi-t i o n of 0.5 ml of cold 20% t r i c h l o r o a c e t i c acid (TCA) solu-t i o n . After 5 min at 0° C, another 2 ml of cold 5% TCA solu-t i o n were added. A glass f i l t e r disk (Whatman GF/C, 2.4 cm dia.) was placed on a M i l l i p o r e f i l t e r assembly, and the mixture was f i l t e r e d through the membrane with the aid of weak suction. A M i l l i p o r e f i l t e r disk (0.45 y pore s i z e , 2.4 cm dia.) was also found to be suitable for retaining the acid-insoluble material, but solvents such as ethanol which caused the membrane to swell and prevented further f i l t r a t i o n , must be avoided. The f i l t e r disk and i t s re-tained contents were washed with 20 to 25 ml of cold 5% TCA solution, and allowed to dry i n the a i r . The disk was then placed into a standard v i a l used for counting r a d i o a c t i v i t y , - 73 -and 10 ml o f a s c i n t i l l a t i o n s o l u t i o n c o n s i s t i n g o f 5 g o f 2 , 5 - d i p h e n y l o x a z o l e (PPO) and 300 mg o f 1 , 4 - b i s - 2 - ( 4 - m e t h y l -5 - p h e n y l o x a z o l y l ) - b e n z e n e ( a l s o known as d i m e t h y l POPOP) i n one l i t r e o f t o l u e n e were added. The samples were counted i n a ' 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 (314AX, P a c k a r d ) . A c o n t r o l r e a c t i o n m i x t u r e w i t h o u t added enzyme s o l u -t i o n or DNA template was i n c u b a t e d and t r e a t e d as f o r the assay of an enzyme s a m p l e . 2. Growth and H a r v e s t o f B a c t e r i a : M a t e r i a l s and R e a g e n t s : An Escherichia coli s t r a i n B c u l t u r e w h i c h was used i n the p r e s e n t i n v e s t i g a t i o n was a g i f t f rom D r . W. J . P o l g l a s e . E. coli B was grown i n a medium c o n t a i n i n g 1.1 p e r c e n t K 2 HE0 i t , 0.85 per cent K H 2 P 0 i t , 0.6 p e r c e n t D i f c o y e a s t e x t r a c t , and 1 per c e n t g l u c o s e . The medium was s t e r i l i z e d by h e a t i n g i n an a u t o c l a v e f o r 15 min a t 20 0 ° F under 15 l b s / i n 2 steam p r e s s u r e . P r o c e d u r e : A 100 ml f r a c t i o n o f growth medium was a s e p t i c a l l y t r a n s f e r r e d t o a s t e r i l e 500 ml E r l e n m e y e r f l a s k . T h i s medium was i n o c u l a t e d by b a c t e r i a f rom a c u l t u r e s l o p e of E. coli B . The f l a s k was i n c u b a t e d a t 3 7 ° C f o r 18 h o u r s . A f t e r t h i s t i m e , the heavy growth c u l t u r e was a s e p t i c a l l y t r a n s f e r r e d t o a 4 l i t r e E r l e n m e y e r f l a s k w h i c h c o n t a i n e d - 74 -19 00 ml of growth medium. The culture i n the 4 1 flask was incubated at 37° C for 18 hours on a shaking water bath. The 30 1 of growth medium i n the Biogen tank was pre-heated to 37° C. Using vigorous aeration and ag i t a t i o n , the 2 1 inoculum of E. c o l i B c e l l s were added to the growth medium. A small sample was taken at the s t a r t of the incu-bation and at one hour i n t e r v a l s , thereafter. These samples were measured at 600 my i n a spectrophotometer to determine the phase of exponential growth. The cultures were harvested about 2 hours a f t e r the end of exponential growth. The c e l l s were co l l e c t e d i n a Sharpies supercentrifuge at 44,000 r.p.m. and were washed by suspension i n 0.5 per cent NaCl-0.5 per cent KC1 (1:3 w/v). After centrifugation of the washed c e l l s , a y i e l d of about 6 g per l i t r e of culture was obtained. These c e l l s were used d i r e c t l y or stored at -20° C u n t i l required for extraction. 3. Prepration of DNA Polymerase Extract: The method of Lehman et al. (63) was e s s e n t i a l l y used for the preparation of a DNA polymerase extract from E. c o l i . An 80 g batch of packed frozen c e l l s was thawed i n 320 ml of 0.05 M gly c y l g l y c i n e buffer, pH 7.0 using slow mechanical s t i r r i n g . Approximately 80 ml fractions of t h i s suspension were treated for 15 min i n t e r v a l s i n a Bronwill 20-KC sonic o s c i l l a t o r . The pooled suspension of disrupted c e l l s were centrifuged for 15 min at 12,0 00 x g i n a S e r v a l l r e f r i g e r a t e d centrifuge and gave a s l i g h t l y turbid supernatant. The pro-t e i n content of the supernatant solution (Fraction I) was - 75 -determined by the method of Lowry et al. (12 4), and was found to be 17.6 mg per ml. 4. P u r i f i c a t i o n of E. c o l i DNA Polymerase (a) Streptomycin p r e c i p i t a t i o n : A 5 per cent streptomycin sulphate solution (27 ml) was added over a 10 min period with constant s t i r r i n g to the Fraction I extract (350 ml). After 10 min, the suspension was centrifuged at 10,000 xg for 15 min i n the S e r v a l l re-frig e r a t e d centrifuge. The supernatant f l u i d was discarded and the thick, s t i c k y p r e c i p i t a t e was transferred to a 125 ml Erlenmeyer f l a s k . The p r e c i p i t a t e was suspended by slow mechanical s t i r r i n g for about 21 hours i n 45 ml of potassium phosphate buffer, 0.05 M, pH 7.4. This suspension, at 0° C was homogenized i n a S e r v a l l omnimixer for 30 min at low speed. The supernatant f l u i d (Fraction II) was co l l e c t e d by centrifugation at 12,000 xg for one hour, and was found to have a protein content of 4.2 mg/ml. (b) Ammonium sulfate f r a c t i o n a t i o n . A 56 ml portion of Fraction II was brought to 90 ml by the addition of potassium phosphate buffer, 0.02 M, pH 7.4. A further 9 ml of potassium phosphate buffer, 1 M, pH 6.5 and 0.9 ml of a 0.1 M 2-mercaptoethanol solution were added. With s t i r r i n g , 2 4.7 g of ammonium sulfate were added to the solution to make i t 40 per cent saturated. After 10 min s t i r r i n g at 4° C, the pr e c i p i t a t e which formed was removed by centrifugation at 12,000 xg for 10 min. To - 76 -the supernatant f l u i d , an additional 9 . 6 g of ammonium s u l -fate were slowly added with constant s t i r r i n g . The 55 per cent saturated solution was s t i r r e d i n the cold for iChmin, and the p r e c i p i t a t e that formed was coll e c t e d by centrifuga-t i o n at 1 2 , 0 0 0 xg for 10 min. This f i n a l p r e c i p i t a t e was dissolved i n 9 ml of potassium phosphate buffer, 0 . 0 2 M, pH 7 . 2 . This solution (Fraction I I I ) was stored at - 2 0 ° C before further use. (c) Chromatography on DEAE-cellulose Materials and Reagents: i) Diethylaminoethyl c e l l u l o s e was purchased from Brown Company and had a capacity of 0 . 8 9 m i l l i e q u i v a l e n t s per g. i i ) Potassium phosphate buffers, pH 6 . 5 , containing 0 . 0 1 M 2-mercaptoethanol were prepared as follows: a s u f f i -cient volume of a stock 1 M potassium phosphate solution was di l u t e d with d i s t i l l e d water to 0 . 0 5 , 0 . 1 , 0 . 2 , and 0 . 5 M. i n phosphate concentrations. After adjusting the 2-mercap-toethanol concentration to give a f i n a l value of 0 . 0 1 M and the pH to 6 . 5 , the solutions were each made up to 10 ml. Procedure: A column of DEAE-cellulose ( 1 x 1 1 cm) was prepared according to the procedure of Lehman et at. ( 6 3 ) , and washed with approximately 5 0 0 ml of 0 . 0 2 M K 2 H P O i ( - 0 . 0 1 M 2-mercap-toethanol solution. The column was equilibr a t e d with, another 5 0 0 ml of the same solution. Three ml of Fraction I I I which - 77 -contained 3.8 mg/ml protein was di l u t e d to 8 ml with the equi-l i b r a t i n g solution, and passed through the column at a rate of 12 ml per hour. Fractions of 1.5 ml were co l l e c t e d i n a small f r a c t i o n c o l l e c t o r (Metaloglass) at 4° C. The column was washed with 3 ml of the s t a r t i n g solution, and then eluted with the potassium phosphate buffers (pH 6.5)-0.01 M 2-mercaptoethanol as follows: 8 ml of 0.05 M, 10 ml of 0.1 M, 7 ml of 0.2 M, and 10 ml of 0.5 M. The protein content of the fractions was determined by measuring i t s absorbance i n a spectrophotometer at 280 my. Each f r a c t i o n was assayed for DNA polymerase a c t i v i t y as previously described. The majority of the enzyme a c t i v i t y was eluted with 0.2 M buffer (Fraction IV). B. DNA Polymerases from Small I n t e s t i n a l Mucosa of the Rat. 1. Assay Systems: (a) DNA polymerase (Replicative deoxynucleotidyl trans- ferase) . The a c t i v i t y of DNA polymerase was measured by the incor-poration of a given labeled nucleotidyl precursor into an acid insoluble product as described above for the E. c o l i system. The following assay system was similar to that described by Bollum (125) for the assay of DNA polymerase a c t i v i t y from c a l f thymus gland. Reagents: The reaction mixture ( t o t a l volume 0.25 ml) contained: - 78 -20 ymoles Tris-HCl buffer, pH 8.1 2 ymoles MgCl 2 0.3 ymoles 2-mercaptoethanol 16 nmoles dATP 16 nmoles dGTP 16 nmoles dCTP 16 nmoles dTTP 1.65 nmoles 1"c-2-dTTP (5 y l of 4 x 10 7 c.p.m./ymole) 15 yg r a t i n t e s t i n a l DNA (30 y l of a solution containing 0.5 mg DNA/ml) 0.1 ml Enzyme solution (25-75 yg protein) (b) Terminal deoxynucleotidyl transferase. The assay procedure used was similar to that reported by Krakow et at. (95). This method measured the c a t a l y t i c a c t i v i t y of an enzyme which p r e f e r e n t i a l l y incorporated single deoxyribo-nucleoside triphosphates onto the terminal position of s i n g l e -stranded primers. Reagents: The reaction mixture ( t o t a l volume 0.25 ml) contained: 20 ymoles Tris-HCl buffer, pH 7.4 2 ymoles MgCl 2 2.5 ymoles cysteine 0.3 ymoles 2-mercaptoethanol 16 nmoles dTTP 1.65 nmoles 1 "*C-2-dTTP (4 x 10 7 c.p.m./ymole) 15 yg heat denatured i n t e s t i n a l mucosa or E. c o l i DNA - 79 -0.1 ml enzyme solution (25-50 yg protein). The DNA was denatured by heating at 100° C for 10 min followed by rapid cooling to 0° C. Variations i n the compon-ents of these assay systems were made from time to time to s u i t the experiment under study. For example, comparisons were made between the effectiveness of potassium phosphate buffer, pH 7.4 and the Tris-HCl buffer, and between the e f f i c i e n c y of 1 I fC-2-dTTP and llfC-8-dATP as precursors. Procedure: An incubation time of up to 60 min has been used for certain experiments, but for the general screening of a number of f r a c t i o n s , a 15 min incubation time was found to be adequate. At t h i s time, the incorporation of labeled precursors was at a lin e a r rate. A f t e r the incubation period i n a water bath held at 37° C, the reaction mixture was rapidl y cooled to 0° C, and the macromolecules, i . e . DNA and protein, were precipitated with 0.5 ml cold 20% TCA. After 5 min of c h i l l i n g , a further 2 ml of cold 5% TCA were added, and the pr e c i p i t a t e was co l l e c t e d on 2.4 cm glass f i l t e r disks (Whatman GF/C). The disks were held on a M i l l i p o r e f i l t e r assembly and washed with 20-25 ml of cold 5% TCA to remove residual labeled precursors (126). The f i l t e r disks and i t s retained contents were allowed to dry i n the a i r or were f i r s t washed with 95% ethanol and e'ther before drying. A s i m i l a r recovery of r a d i o a c t i v i t y from either drying method was obtained as measured by a l i q u i d s c i n t i l l a t i o n system des-cribed e a r l i e r for the>assay of E. c o l i DNA polymerase. - 80 -2. Preparation of DNA Polymerase Extracts from Rat I n t e s t i n a l  mucosa The method of Clark and Porteous (10 4) was followed for the preparation of mucin-free i n t e s t i n a l e p i t h e l i a l c e l l s , and for the i s o l a t i o n of the c e l l n u c l e i . (a) Preparation of i n t e s t i n a l e p i t h e l i a l c e l l s : Materials and reagents: i) Male rats of the Wistar s t r a i n were obtained from the vivarium of the University of B.C. These animals were not starved before use, and had an average weight of 200 g each. i i ) 6% dextran-Krebs Ringer phosphate solution was pre-pared according to the method of Clark and Porteous (10 4). Dextran was purchased from General Biochemicals, Chagrin F a l l s , Ohio i n c l i n i c a l grade form of approximately 186,000 MW. Procedure: Generally 4 to 5 rats were used per experiment and each animal was k i l l e d by a blow to the head and decapitated. The small intestines were rapidiy removed and t h e i r contents flushed out with cold Krebs-Ringer phosphate buffer, pH 7.4. Mucosal scrapings were obtained by stroking the opened in t e s t i n e with a glass s l i d e , and were suspended i n 6% dextran-Krebs Ringer phosphate solution, 0° C. The suspension of mucosal tissue, 1:10 w/v, was homogenized i n a glass homogenizer of the Potter-Elveh jem type with a Teflon-tipped pestle. Homogenization was car r i e d out for one minute under a low pestle speed of about - 81 -500 rpm. The viscous homogenate was f i l t e r e d through four layers of cheese cl o t h , and the c e l l s c o l l e c t e d by centrifuging the f i l t r a t e i n the S e r v a l l refrigerated centrifuge at 1700 xg for 5 min. This p e l l e t was washed by resuspension i n 20 volumes of Krebs-Ringer phosphate buffer, and recentrifuged at 1700 xg for 5 min. These washed e p i t h e l i a l c e l l s were suitable for enzyme extraction when whole c e l l studies were used. (b) Isolation of the nuclear f r a c t i o n from the i n t e s t i n a l  e p i t h e l i a l c e l l s . Mucin-free e p i t h e l i a l c e l l s were obtained as described above. These washed c e l l s were suspended i n 10 volumes of 0.3 M sucrose-5 mM EDTA solution, pH 7.4, and homogenized for one min i n a glass homogenizer under a medium pestle speed of about 2000 rpm. The r e s u l t i n g homogenate was centrifuged i n the S e r v a l l r e f r i g e r -ated centrifuge. A nuclear p e l l e t which was obtained by c e n t r i -fugation at 1000 xg for 10 min was used for the preparation of a crude enzyme extract. (c) Extraction of DNA polymerases from the nuclei A l l operations were carried out between 0° and 4° C. The nuclear p e l l e t i s o l a t e d from the i n t e s t i n a l mucosa was suspended i n 5 volumes of an enzyme extraction solution which consisted of 50 mM Tris-HCl buffer, pH 7.5, 5 mM MgCl 2 and 5 mM 2-mercapto-ethanol. This suspension was homogenized for 3 min at a low speed of about 250 0 rpm i n a S e r v a l l omni-mixer (Ivan S o r v a l l , Norwalk, Conn.). The homogenate was transferred to a f l a s k and s t i r r e d with a magnetic s t i r r e r for 10 min. After t h i s time, the nuclear extract was centrifuged i n the preparative u l t r a c e n t r i -- 82 -fuge (Beckman, Model L) at 1 0 5 , 0 0 0 xg for 2 hours using a no. 30 rotor. The high speed supernatant contained the soluble proteins from the nuclei i n a concentration of 5 to 8 mg per ml. 3. Preparation of Enzyme Extracts from I n t e s t i n a l Tissue: Leblond and Walker ( 1 2 7 ) have observed that the renewal of the e p i t h e l i a l c e l l s i n the intestine occurred as a continuous physiological phenomenon. By following the incorporation of a 32P-DNA precursor into the i n t e s t i n e , these authors found a higher mitotic rate i n c e l l s from the lower crypt portion of the e p i t h e l i a l l i n i n g . After a 2 3 hour period, the 3 2 P - l a b e l e d DNA was found i n c e l l s near the ti p s of the v i l l i and these c e l l s were f i n a l l y ejected into the lumen. The following comparative study was made i n an attempt to determine i f the lower crypt area of the i n t e s t i n a l mucosa contained a higher DNA polymerase a c t i v i t y than the ti p s of the v i l l i . Reagents: i) A wash solution which consisted of 6% dextran, 0 . 9 % NaCl and 2 mM EDTA was prepared. The pH of thi s solution was adjusted to 7 . 4 with 0 . 1 N NaOH before the f i n a l volume was made up to 1 l i t r e . i i ) A homogenization-extraction medium which contained 10 mM T r i s , 5 mM MgCl2, and 1 mM 2-mercaptoethanol was prepared. The pH of thi s solution was adjusted to 7 . 4 with 0 . 1 M KH 2POif and the solution made up to volume with water. Procedure: Four male Wistar rats were s a c r i f i c e d and the upper two-- 83 -thirds of t h e i r small intestines removed. The i n t e s t i n a l con-tents and mucus material were removed as completely as possible by flushing the i n t e s t i n e several times with the cold 6% dextran wash solution. Using a l i g h t stroke with a microscopic s l i d e , the upper layer of mucosa which should contain the t i p s of the v i l l i were removed from the opened i n t e s t i n e , and suspended i n the homogenization medium at 0° C. By the use of a second heavier scraping with the glass s l i d e , the c e l l s near the base of the v i l l i should be obtained. The t o t a l y i e l d s of the upper and the lower c e l l u l a r scrapings were about 5.8 g and 4.6 g respectively. These fractions were separately homogenized i n four volumes of 0.01 M Tris-phosphate homogenization medium with the glass homogenizer f i t t e d with a Teflon pestle. The homogenate was centrifuged at 10,000 xg for 20 min i n the S e r v a l l r e f r i g e r a t e d centrifuge. The supernatant which con-tained the soluble proteins from the whole c e l l was c e n t r i -fuged at 105,000 xg for one hour. This f i n a l supernatant rep-resented the cytoplasmic and f i r s t nuclear extract of the c e l l s . The p e l l e t from the 10,000 xg centrifugation which would contain the nuclear residue was rehomogenized i n one-half the o r i g i n a l volume of buffer, and centrifuged at 10 5,000 xg for one hour. The supernatant fractions from the high-speed centrifugations were assayed for DNA polymerase a c t i v i t y as previously described. 4. Extraction of DNA polymerase from Nuclei and Cytoplasm  Prepared i n Non-Aqueous Media; In order to investigate the subcellular location of DNA - 84 -polymerases from the small i n t e s t i n a l mucosa of the ra t , the procedure of Kay et al. (12 8) was used to i s o l a t e nuclei and cytoplasm. This method involved the use of non-aqueous s o l -vents on rapidly frozen and l y o p h i l i z e d tissue to minimize the possible transfer of protein components from the nuclei to the cytoplasm during the i s o l a t i o n procedure. A l l operations were performed at 0-4° C. Small i n t e s t i n a l mucosa scrapings were obtained from three male ra t s , Wistar s t r a i n of about 200 g each, and were immediately frozen i n l i q u i d N 2. A t o t a l of 8.4 g of frozen tissue was broken into small pieces i n a cold mortar and pestle. L y o p h i l i -zation of th i s tissue was carr i e d out for 48 to 52 hours i n a bench top freeze dryer (New Brunswick S c i e n t i f i c , N.J.). A y i e l d of 1.6 g of thoroughly dried mucosa tissue was homogenized i n 20 ml of l i g h t l i q u i d petroleum. After homogenization i n a Potter-Elvehjem apparatus, the mixture was f i l t e r e d through four layers of cheese cloth. The f i l t r a t e was centrifuged at 1500 xg for 5 min i n the S e r v a l l refrigerated centrifuge. The super-natant f l u i d was removed and recentrifuged at 29,000 rpm for one hour i n the no. 30 rotor with the Spinco model L ultra c e n t r i f u g e . A cytoplasmic p e l l e t was obtained and retained for further t r e a t -ment . The 1500 xg sediment from the above procedure was washed by two resuspensions and centrifugations at 1500 xg for 5 min. After the second wash, the sediment was suspended i n a mixture of n-hexane-CClit (1.25:1, v/v, and densities of 0.6603 and 1.595 - 85 -respectively) and centrifuged at 1500 xg for 5 min. This pro-cedure was repeated twice with the supernatant f l u i d being discarded i n each case. The f i n a l sediment was suspended i n 25 ml of a n-hexane-CClij (1:2, v/v) and was centrifuged at 3,000 xg for 30 min. After discarding the supernatant f l u i d , the sedi-ment was homogenized with n-hexane-CCli, (1:1, v/v) mixture and centrifuged at 1500 xg for 5 min. The p e l l e t was recovered, washed with l i g h t l i q u i d petroleum at 0° C, and centrifuged at 1500 xg for 5 min to give the f i n a l washed nuclear f r a c t i o n . The cytoplasmic and nuclear p e l l e t s were a i r - d r i e d and stored i n a desiccator at -20° C. Yields of 0.9 g and 0.3 g were obtained for the dried nuclear and cytoplasmic powders respec-t i v e l y . These desiccated powders were separately extracted with an aqueous extraction medium i n a 1:5 w/v suspension. This enzyme extraction medium enzyme extraction medium contained 0.01 M potassium phosphate buffer, pH 7.5, 5 mM MgCl2, and 1 mM 2-mercaptoethanol. Homogenization was carr i e d out i n the Potter apparatus, and the homogenates were centrifuged i n the no. 30 rotor at 29,000 rpm for one hour. The high-speed supernatants from the extracts of the nuclei and the cytoplasm were assayed for DNA polymerase a c t i v i t y . 5. P u r i f i c a t i o n of DNA Polymerases from the I n t e s t i n a l  Mucosa. (a) Ammonium sulfate f r a c t i o n a t i o n : To a 45 ml volume of nuclear extract at 4° C, 10.9 4 g of s o l i d ammonium sulfate were slowly added to give a 40% saturated - 86 -solution. This solution was continuously s t i r r e d with a magnetic s t i r r e r during the addition of the s a l t and was s t i r r e d for an additional 30 min, thereafter. The pre c i p i t a t e which formed was co l l e c t e d by centrifugation at 12,000 xg for 20 min i n the Ser v a l l r e f r i g e r a t e d centrifuge. This i n i t i a l sediment was termed the 40% ammonium sulfate f r a c t i o n . The supernatant solution from the f i r s t centrifugation was removed and treated with an additional 6.6 g of s o l i d ammonium sulfate as i n the f i r s t f r a c t i o n a t i o n . After s t i r r i n g for 30 min at 4° C, the mixture was centrifuged at 12,000 xg for 20 min. The second p r e c i p i t a t e was termed the 60% ammonium s u l -fate f r a c t i o n . The 40% and the 60% fractions were each dissolved i n 2 ml of 0.05 M Tris-HCl buffer, pH 8.1, 5 mM MgCl 2-5 mM 2-mercaptoethanol solution. These solutions were dialyzed against one l i t r e of the same buffer solution, and were assayed for DNA polymerase a c t i v i t y . (b) DEAE-cellulose chromatography: Materials and Reagents: i) DEAE-cellulose (N,N 1-diethylaminoethyl c e l l u l o s e , Cellex-D, Bio-Rad Laboratory) with an exchange capacity of 0.7 m i l l i -equivalents per g was further washed to remove yellow u l t r a v i o l e t absorbing material as described by Moore, et al. (129). Approxi-mately 20 g of the ce l l u l o s e were washed with 1 l i t r e of 1 N NaOH, and then rinsed with water to ne u t r a l i t y . The ce l l u l o s e was further washed with ethanol and ether, and was dried at room temperature. It was then suspended i n 6 mM Tris-phosphate - 87 -buffer, pH 8.0. i i ) Preparation of the---column: A chromatographic column 1 x 45 cm was packed with the p u r i f i e d DEAE ce l l u l o s e to a depth of 30 cm, under an a i r pressure of 10 p . s . i . as described by Peterson and Sober (130). The column was then washed with about 500 ml of 5 mM Tris-phosphate buffer, pH 8.0 before use. Procedure: A 20 ml solution of r a t i n t e s t i n a l extract containing 5 mg protein per ml was applied to the equilibrated column and allowed to soak i n by gravity. Five ml fractions were co l l e c t e d i n a ref r i g e r a t e d f r a c t i o n c o l l e c t o r from the s t a r t of the sample a p p l i -cation. The column was maintained at 3-4° C throughout the f r a c -tionation by c i r c u l a t i n g a cooled re f r i g e r a n t (25% ethylene glycol/water solution) through the jacket of the column. After the protein solution had completely entered the adsorbant, 20 0 ml of 5 mM Tris-phosphate, pH 8, were washed through the column to remove unadsorbed and loosely adsorbed material. Absorbed material was eluted by a parabolic chloride gradient as described by Moore et at. (129). Three open 250 ml Erlenmeyer flasks were bridged i n series by means of U-tubes. The f i r s t flask which was connected to the column, and the second each contained 20 0 ml of the s t a r t i n g buffer (5 mM Tris-phosphate, pH 8.0). Both solu-tions were s t i r r e d with magnetic s t i r r e r s . Flask number three contained 200 ml of the l i m i t buffer (1.0 M potassium chloride, 0.05 M dibasic potassium phosphate, and 0.01 M 2-mercaptoethanol, pH 6.5). The flow rate of this column was between 12 and 15 ml - 88 -per hour. Fractions recovered from the column were measured for u l t r a -v i o l e t absorbancy at 260 and 280 my on the G i l f o r d 2000 spectro-photometer. The protein content per tube was estimated either by i t s o p t i c a l density at 280 my (131) or by the method of Lowry et al. (124). By measuring the r e f r a c t i v e indices of the fractions and comparing these valuestto a c a l i b r a t i o n curve of r e f r a c t i v e index against potassium chloride buffer concentrations, as shown in Figure 14, the s a l t gradient for the column was deter-mined. Replicative and terminal DNA nucleotidyl transferase a c t i v i t i e s were measured on selected fractions as described under assay systems. i) Concentration of protein solutions: A membrane f i l t e r which i s available from the Amicon Corp. (Cambridge, Mass.) was found to be suitable for concentrating d i l u t e protein solutions. The membrane (UM-1) which has an a r b i t r a r i l y fixed retentive capacity for p a r t i c l e s of 10,000 molecular weight or larger was used for the u l t r a f i l t r a t i o n of DNA polymerase solutions. Solutions such as those recovered from a DEAE-cellulose column were concentrated i n the model-50 'Diaflow' apparatus under a nitrogen pressure of 45 p . s . i . After concentration, the 3 to 5 ml volume retained i n the appar-atus was suitable for further study. Although the s a l t concen-trations i n these solutions were not increased by the f i l t r a t i o n method, these concentrated solutions were further dialyzed i n - 89 -1.3M+0r 1.33001 1 . , . . 1 . • • , 0 0.2 O A 0.6 0.8 1.0 Molarity of KCl Figure 14. C a l i b r a t i o n curve of r e f r a c t i v e index as a function of potassium chloride buffer concentrations. The buffer contained 0.0 5 M dibasic potassium phosphate and 0.01 M 2-mercaptoethanol, pH 6.5 - 90 -ce l l u l o s e membranes against 5 mM Tris-phosphate buffer, pH 8.0 to reduce the i o n i c strength of the eluting buffer used i n column chromatography. i i ) Chromatography on DEAE-cellulose Column I I . The concentrated protein fractions from the o r i g i n a l DEAE-ce l l u l o s e chromatography were further chromatographed on a small DEAE-cellulose column of 1 x 11 cm. Fractionation on t h i s second column was achieved by using step-wise e l u t i o n as e a r l i e r describ-ed for the p u r i f i c a t i o n of E. c o l i DNA polymerase. In t h i s step of the p u r i f i c a t i o n of i n t e s t i n a l DNA polymerases, a separation of the 1 r e p l i c a t i v e 1 from the 'terminal-addition' DNA nucleotidyl-transferase a c t i v i t i e s appeared to have occurred. Procedure: A column (1 x 11 cm) of DEAE-cellulose (washed Cellex-D) was prepared i n a glass tube which was f i t t e d with a sintered glass disk. The adsorbant was packed under an a i r pressure of 3 p . s . i . and washed with one l i t r e of 0.02 M K2YiVOh, 0.01 M 2-mercaptoethanol solution before use. An enzyme solution con-taining 3 to 4 mg protein per ml was loaded onto the column under g r a v i t a t i o n a l force. Fractions of 1.5 ml were coll e c t e d i n the Metaloglass compact f r a c t i o n c o l l e c t o r at 4° C. A further 3 ml of the potassium phosphate solution was passed through the column, and then the adsorbed proteins were eluted with the following concentrations of potassium phosphate buffers (pH 6.5) -0.01 M 2-mercaptoethanol: 8 ml of 0.05 M, 10 ml of 0.1 M, 7 ml of 0.2 M, and 10 ml of 0.5 M. The o p t i c a l densities - 91 -of the fractions were measured at 260 and 280 my and the s a l t concentration of the eluted fractions were measured by the r e f r a c t i v e index method. (c) Hydroxylapatite chromatography: Procedures: i) Preparation of the column: Hydroxylapatite powder (Bio-Gel HTP, Bio-Rad Laboratories) was suspended i n 0.02 M potassium phosphate buffer, pH 6.8 con-taining 0.01 M 2-mercaptoethanol. A column (1 cm x 10 cm) was prepared by pouring the material as a s l u r r y and applying an a i r pressure of 3 p . s . i . The column was washed with about 350 ml of 0.0 2 M potassium phosphate buffer -0.01 M 2-mercaptoethanol, pH 6.8 before use. i i ) E l u t i on of the column: An enzyme solution containing from 3 to 5 mg protein per ml was applied to the hydroxylapatite column under an a i r pressure of 3 p . s . i . The column was washed with 30 ml of 0.05 M potassium phosphate buffer, pH 6.8 and the adsorbed material was eluted with a li n e a r gradient of 0.05 to 0.5 M i n potassium phosphate concentration. Buffer solutions which contained 0.01 M 2-mercaptoethanol i n a t o t a l volume of 300 ml were used. The column's flow rate was adjusted to about 12 ml per hour. Five ml fractions were coll e c t e d and these fractions were measured for s a l t concentration by the r e f r a c t i v e index method. The protein content of each f r a c t i o n was measured by a spectrophotometer at 280 mu. Thfemajor peak of DNA polymerase a c t i v i t y was eluted with approximately 0.2 M potassium phosphate buffer. - 92 -(d) Sephadex gel f i l t r a t i o n : The method of molecular-serve chromatography on Sephadex gels was used for the estimation of the molecular sizes of DNA polymerases as well as for the p u r i f i c a t i o n of these enzymes. The procedures used for preparing the gels and for packing the columns were as described i n the 'Technical Data Sheets' which. were supplied by Pharmacia (132). Materials and Reagents: i) Sephadex G-150 and Sephadex G-20 0 were obtained from Pharmacia, Uppsala, Sweden. Both- gels had a p a r t i c l e size of 40-120 y diameter. The G-150 had a water regain of 15 ± 1.5 and a bed volume of 20-30 ml/g dry bead. The G-200 had a water r e -gain of 20 ± 2.0 with a bed volume of 30-40 ml/g dry Sephadex. i i ) A 2.5 x 100 cm jacketed Sephadex column was packed with G-150, and a 2.5 x 45 cm jacketed Sephadex column was used for the G-200 beads. i i i ) 0.1 M potassium phosphate buffer, pH 7.2 - 1 mM 2-mercaptoethanol. iv) Blue dextran 2000 (Pharmacia, Uppsala, Sweden) with an average molecular weight of 2 x 10 6 was used for determining the void volume of the columns. The concentration of t h i s marker was measured at an o p t i c a l density of 280 or 625 my. v) Protein markers with known molecular parameters-1. Chymotrypsinogen-A (MW 25,000, Mann Research Labora-tories) ; - 93 -2. Y~ Gl°kulin (human, MW 160,000, Mann Research Labora-o torxes), Stokes radius = 52 A (133); 3. Catalase (MW 250,000, Calbiochem., no. 2190), Stokes radius = 52 A (133). Procedure: The Sephadex gels were suspended i n 0.1 M potassium phos-phate buffer, pH 7.2 with 1 mM 2-mercaptoethanol, and allowed to swell 3 to 4 days at 4° C (or at room temperature) before use. Columns were packed by allowing the swollen gels, pre-viously deaerated under reduced pressure, to enter the column as a s l u r r y . The column was packed i n a single continuous manner to avoid trapping a i r bubbles. A l l procedures were per-formed at 4° C. The column was equilibrated by washing with about 2 1 of 0.1 M phosphate buffer. Protein samples and suitable markers i n a one to two ml volume were applied through a syringe and flow adaptor connected to the bottom of the column. Upward flow e l u t i o n which should produce a smaller hydrostatic pressure on the gels was found to give an adequate flow rate. Fractions of about 5 ml were c o l -lected i n a re f r i g e r a t e d f r a c t i o n c o l l e c t o r . Protein markers such as chymotrypsinogen-A and y-globulin were measured at 280 my. A standard chromatography run was re-quired to determine the e l u t i o n volume of these markers. A catalase marker was included i n the sample to be examined, and this marker was measured for a c t i v i t y according to the method of Chance and Maehly (134). - 94 -A 2 ml volume of the substrate which was prepared by mixing 0.2 ml of 30% H 20 2 i n 50 ml of 0.05 M potassium phosphate buffer, pH 7.0 was mixed with 1 ml of the sample to be examined. The de-crease i n absorbance at 240 my was graphically recorded, and the fr a c t i o n which showed the greatest rate of decrease was taken as the point of maximum catalase a c t i v i t y . The void volume of the column was estimated by measuring the quantity of buffer required to elute a marker such as blue dextran which was excluded from the G-150 or G-200 beads. The t o t a l volume of the column was measured by determining the volume of buffer required to elute a small molecule as L i C l from the gel . A conductivity meter (Radiometer, Copenhagen, Denmark) was used to determine the f r a c t i o n of maximum s a l t concentration, (e) Sucrose density gradient centrifugation: Materials and Reagents: i) 50 mM Tris-HCl buffer, pH 7.5. This solution was pre-pared by dissolving 6.05 g T r i s (Sigma 121) i n d i s t i l l e d water. The pH was adjusted to 7.5 with d i l u t e HC1 and the volume was made up to 1 l i t r e with d i s t i l l e d water. i i ) Sucrose solutions: 1. 7.5% sucrose i n 50 mM Tris-HCl buffer, pH 7.5. 2. 25% sucrose i n 50 mM Tris-HCl buffer, pH 7.5. i i i ) Protein markers: 1. Chymotrypsinogen-A (MW 25,000, Mann) one mg of dry powder was dissolved i n 0.5 ml 50 mM T r i s buffer. 2. Catalase (MW 250,000, Calbiochem., 50 mg/2.5 ml). - 95 -A 0.025 ml f r a c t i o n of catalase solution was diluted to 0.5 ml with 50 mM T r i s buffer, iv) Enzyme extracts from the rat i n t e s t i n a l mucosa were ob-tained from the high-speed supernatant of the crude homogenate or from the Sephadex G-200 eluants which were concentrated by u l t r a -f i l t r a t i o n through Diaflow membranes. Procedure: A l i n e a r sucrose gradient from 7.5 to 25 per cent concentra-tion was prepared i n % x 2 inch c e l l u l o s e n i t r a t e tubes (Beckman) according to the procedure of Martin and Ames (135). Enzyme solu-tions and protein markers i n 0.1-0.15 ml were c a r e f u l l y layered over the preformed gradients. These prelayered tubes were loaded into the model SW-50L swinging bucket rotor, and were centrifuged i n the model L Spinco ultracentrifuge (Beckman) at a rotor speed of 45,000 r.p.m. for 20 hours, at 4° C. The time of centrifugation was taken as the period from the s t a r t of acceleration of the rotor to the s t a r t of deceleration. After t h i s time, the centrifuge tubes were c a r e f u l l y removed from the rotor and a hole punched i n the bottom of each tube with a needle apparatus which was similar to the one described by Martin and Ames (135). F i f t e e n drop fractions were collected i n small test tubes, and each f r a c t i o n was d i l u t e d with 0.5 ml of 50 mM T r i s -HC1 buffer, pH 7.5, before assay. The procedure used for measuring the protein markers was the same as that described for the Sephadex gel fr a c t i o n s . DNA polymerase a c t i v i t i e s were detectable using the standard assay system. - 96 -The l i n e a r sucrose gradients were reproducible i n d i f f e r e n t tubes when they were prepared under the same conditions. There-fore under similar centrifugational conditions, the r a t i o of the distance traveled by any two substances from the meniscus would be constant. When a protein of known sedimentation c o e f f i c i e n t was used, i t was possible to estimate the 'S* value for an un-known substance. The r a t i o 'r' could be determined aft e r any time of centrifugation as: _ distance traveled from meniscus by unknown " d i s t a n c e traveled from meniscus by standard Proteins of the same p a r t i a l volume would give: S„„ of unknown = S_„ of standard x r 20,w 20,w Generally most globular proteins have a p a r t i a l s p e c i f i c volume of 0.725 cm3 per g (135). 6. Enzymatic Degradation of Synthetic DNA Products. (a) Assay for snake venom phosphodiesterase a c t i v i t y (136): Materials and Reagents: i) Lyophilized Crotalus adamanteus venom was purchased from the Ross A l l e n Reptile I n s t i t u t e , S i l v e r Springs, F l o r i d a . The venom phosphodiesterase was prepared according to the procedure of Koerner and Sinsheimer (137), and further p u r i f i e d by chroma-tography on carboxymethylcellulose as described by F e l i x et at. (138). An enzyme solution of 1 mg/ml was prepared i n 0.1 M T r i s -HC1 buffer, pH 8.9. i i ) 0.1 M Tris-0.00 2 M Magnesium acetate buffer, pH 8.9. This solution required 12.1 g T r i s (Sigma 121) and 0.43 g - 97 -magnesium acetate i n d i s t i l l e d water. The pH of the solution was adjusted to 8.9 with acetic acid and the solution was d i l u t e d to 1 l i t r e with d i s t i l l e d water. i i i ) Substrate (1 uM/ml) 13.6 mg of p-nitrophenylthymidine-5'-phosphate was dissolved i n 25 ml of the T r i s buffer solution. Procedure: One and one-half ml of a solution containing p-nitrophenyl-thymidine-5'-phosphate and 1.4 ml of 0.1 M Tris-HCl buffer were pipetted into a cuvette maintained at 37° C i n the cuvette chamber of the G i l f o r d 2000 automatic recording spectrophotometer. At zero time, 0.1 ml of the snake venom phosphodiesterase solu-tion was added to the cuvette and the contents were thoroughly mixed. The increase i n absorbance at 400 my due to the l i b e r a -tion of yellow p-nitrophenylate was recorded. The l i b e r a t i o n of 0.1 ymole of p-nitrophenol i n a t o t a l volume of 3 ml corresponds to an increase i n absorbance of 0.4 units (Figure 15). (b) Action of the venom phosphodiesterase on DNA products  synthesized enzymatically. The DNA products synthesized by the in v i t r o action of the terminal addition enzyme and the r e p l i c a t i v e enzyme from the nuclei of the i n t e s t i n a l mucosa were treated with the venom phosphodiesterase. Reagents: i) 0.1 M Tris-HCl buffer (pH 8.9) -0.01 M MgCl 2 solution: 12.11 g T r i s (Sigma 121) and 2.03 g MgCl 2 (MW 203.3) were d i s -solved i n d i s t i l l e d water; the pH of the solution was adjusted - 98 -Figure 15. Rate of release of p-nitrophenol by the action of snake venom phosphodiesterase on p-nitrophenyl thymidine-5' phosphate. - 99 -to 8.9 with d i l u t e HC1 and the volume was made up to 1 l i t r e . Procedure: The DNA products were synthesized using the in v i t r o assay system for DNA nucleotidyltransferases as e a r l i e r described, but the quantities were scaled up four f o l d . These products were labeled by the incorporation of 1 4C-dATP or 1 4C-dTTP and were recovered from the incubation mixture by p r e c i p i t a t i o n with 20% TCA. After centrifugation at 2000 r.p.m. for 10 min, the sedi-ment was dissolved i n ,3 ml of 0.1 M Tris-HCl buffer system. This solution was incubated at 37° C with a 0.5 ml solution of p u r i f i e d snake venom phosphodiesterase. After various time i n t e r v a l s , 0.5 ml aliquots were withdrawn and placed into a 12 ml conical centrifuge tube. The reaction was stopped as quickly as possible by the addition of 0.5 ml of cold 20% TCA solution. One drop of 1% bovine serum albumin solution was added as a p r e c i p i t a t i o n medium. The acid-soluble mononucleo-tides were recovered by centrifugation at 2000 r.p.m. for 10 min. The absorbance of the acid-soluble supernatant f r a c t i o n was measured at 260 my. The r a d i o a c t i v i t y released into the acid soluble f r a c t i o n was measured. Counting of Radioactivity: Counting was carried out on a Packard model 314A T r i -Carb l i q u i d s c i n t i l l a t i o n spectrometer. The sample v i a l s were prepared to contain 0.5 ml of the aqueous radioactive solution, 0.5 ml of 1 M Hyamine hydroxide i n methanol and 5 ml of a Polyether-611 solvent system. This- l a t t e r solvent CP-6111 was - 100 -developed by Davidson and Feigelson (139) and consisted of the following: 0.05% l,4-bis-2 (5-phenyloxazolyl)-benzene, (POPOP) 0.96 g 1.2% 2,5-diphenyloxazole, (PPO) 23 g Anisole and 1,2-dimethoxyethane, 200 ml of each p-dioxane 1200 ml 7. Assay for Deoxyribonuclease I A c t i v i t y i n Small I n t e s t i - nal Mucosa Preparations. In order to compare the effects of DNase I on the DNA poly-merase enzyme, selected samples with polymerase a c t i v i t y were also tested for DNase I a c t i v i t y . (a) Assay for DNase I A c t i v i t y : Materials and Reagents: i) 0.1 M Ammonium acetate buffer-0.01 M MgCl2, pH 6.5: 7.7 g NHUC2H3O2 and 2.03 g MgCl2 were dissolved i n d i s t i l l e d water; the pH was adjusted to 6.5 with d i l u t e acetic acid. Suf-f i c i e n t d i s t i l l e d water was added to make one l i t r e . i i ) DNA Substrate - 50 yg/ml of native E. c o l i DNA (Worth-ington) i n 0.1 M ammonium acetate - 0.01 M MgCl 2 buffer, pH 6.5. Procedure: Deoxyribonuclease-I (E.C. 3.1.4.5) a c t i v i t y was determined by the method of Kunitz (131) which depends on the degradation of DNA to acid-soluble fragments. This change i n the DNA struc-ture can be measured spectropfiotometrically at 2 60 my. Three ml of the DNA substrate solution were pipetted into a cuvette maintained at 37° C i n the cuvette chamber of the G i l f o r d 2000 - 101 -automatic recording spectrophotometer. At zero time, 0.1 ml of an enzyme extract was added to the substrate solution and mixed thoroughly. Increase i n the absorbance at 260 my was followed for a 10 min period. The increase i n o p t i c a l density was found to be l i n e a r for the f i r s t 5-10-min of the reaction, and t h i s rate was proportional to the concentration of enzyme present. One unit of enzyme was defined by Kunitz (131) as an increase i n absorbance of 0.001 per min under the above assay conditions. The assay system was standardized for the rat i n t e s t i n a l mucosa extracts with deoxyribonuclease-I prepared from this tissue according to the method of Lee et at. (103). - 102 -PART II RESULTS and DISCUSSIONS In the present investigation, DNA polymerase from E. c o l i was i s o l a t e d and characterized p r i o r to the study of a sim i l a r enzyme from the small i n t e s t i n a l mucosa of the ra t . It was of p r a c t i c a l value to review the c h a r a c t e r i s t i c s of an established enzyme before attempting to study such an enzyme from the mamma-l i a n system. 1. Characteristics of the E. c o l i Assay System. (a) E f f e c t of pH and divalent metals: In the standard assay system, the use of glycine buffer pH 9.2 produces a pH of approximately 8.7 i n the t o t a l mixture. This i s the optimal pH value i n glycine buffer while a maximal a c t i v i t y i s obtained at pH 7.4 i n potassium phosphate buffer as described by Richardson et at. (66). The presence of MgCl2 i s e s s e n t i a l for detectable enzyme a c t i v i t y . At pH 7.4 i n potassium phosphate buffer under the conditions of the standard assay, the optimal Mg + + concentration i s 7 x 10~ 3 M (66). (b) Requirement for DNA primer: As reported by Romberg and coworkers (66), DNA polymerase from E. c o l i u t i l i z e s heat-denatured and native DNA at approxi-mately equal rates. With the present enzyme preparation from E. c o l i , a similar primer a c t i v i t y was observed as shown i n Table VI. At reduced temperatures, 20° C, native DNA i s a less e f f i c i e n t primer than a heat-denatured or p a r t i a l l y s ingle-stranded DNA (140). Synthetic deoxyribo-oligonucleotides of - 103 -Table VI Primer Requirements for the DNA Polymerase from E. c o l i Incubation DNA polymerase a c t i v i t y from E. c o l i 1 ltC-2-dTMP incorporation mixture i n DNA primer per mg protein Counts/minute pmoles Enzyme + native DNA 842 21 Enzyme + denatured DNA 79 8 20 Enzyme omit DNA 37 1 The composition of the assay mixture i s as described i n the experimental for the standard incubation mixture. 10 yg of primer DNA and 50 yg of protein from the high-speed centrifugation f r a c t i o n were used. Incubations were for 15 min at 37° C. - 104 -from 6 to 14 alternating deoxyadenylate and deoxythymidylate residues (dAT copolymer) can also prime the synthesis (141). After a lag period of several hours, DNA polymerase has also been shown by Schachman, et 'at. (141) to c a t a l i z e de novo dAT synthesis i n the absence of a dAT primer. (c) Requirement f o r deoxyribonucleoside triphosphates: A l l four deoxyr'ibonucleoside-5 '-triphosphates ( i . e . dATP , dCTP, dGTP, dTTP) are required for maximal a c t i v i t y . In the absence of one to three of these substrates, only a small or basal l e v e l of incorporation i s observed. This phenomenon of 'limited-end reaction' i s due to the addition of one or a few molecules of the deoxyribonucleotide at the 3'-hydroxyl terminus of the DNA primer (94). (d) Enzyme extract from E. coli'. In the crude sonicated extract from E. c o l i , the presence of DNA polymerase was detected using the standard assay system. A decrease i n the incorporation of radioactive precursor was observed when a larger volume of the crude extract was used per assay as shown i n Table VII. D i s t i n c t nucleases which degrade the primer or synthetic products are known to be present i n c e l l free extracts, i . e . E. c o l i exonuclease I, I I , I I I ; deoxyribo-nuclease I, I I . A possible competition by such degrading enzymes might have caused the observed decrease i n the recovery of labeled products aft e r 30 mins of incubation as shown i n Table VIII. P u r i f i c a t i o n of the crude enzyme preparation was required i n an attempt to remove such i n t e r f e r i n g substances. - 105 -Table VII The E f f e c t of Using Various Volumes of E. c o l i DNA Polymerase Extract i n the Assay System Volume of enzyme DNA polymerase a c t i v i t y 11*C-2-dTMP incorporation i n DNA/mg protein Counts/minute pmoles 0.02 410 11 0.04 778 19 0.08 525 14 The protein content of the enzyme extract from E. c o l i i s 17.6 mg per ml. The incubation mixture was the same as that i n Table VI with the exception of the volume of enzyme extract used. - 106 -Table VIII E f f e c t of Incubation Time on the DNA Polymerase A c t i v i t y from Crude C e l l Extracts of E. c o l i Time minutes DNA polymerase a c t i v i t y 1 "*C-2-dTMP incorporated*/mg protein Counts/minute pmoles 5 778 19 15 1580 40 30 1235 31 60 1394 35 The quantities of the reactants used i n the i n -cubation mixture were as described under the assay system for E. c o l i DNA polymerase. The DNA used was i n i t s native form. * The labeled precursor was incorporated into an acid-insoluble product. - 107 -2. Iso l a t i o n and P u r i f i c a t i o n of E. c o l i DNA polymerase. The results obtained from the growth and i s o l a t i o n of the E. c o l i c e l l s were e s s e n t i a l l y the same as those described by Lehman, et al. (63) . In an i n i t i a l experiment, sonication for 5 min i n the Bronwill 20 KC sonic o s c i l l a t o r produced an enzyme extract containing 7.5 mg of protein per ml. In a second experi-ment, a sonication time of 15 min was found to give an extract with a protein content of 17.6 mg per ml. In the present investigation, the p u r i f i c a t i o n of DNA poly-merase from E. c o l i B involved the stages of streptomycin sulfate p r e c i p i t a t i o n , ammonium sulfate f r a c t i o n a t i o n , and chromatography on DEAE-cellulose as described by Lehman et al. (63). Fraction-ation of the E. c o l i enzyme extract with streptomycin sulfate gave a f r a c t i o n of DNA polymerase which was s l i g h t l y more active than the crude preparation. Following streptomycin sulfate treatment the enzyme solution was brought to a 40% saturation l e v e l with ammonium sul f a t e . The sediment from the centrifuga-t i o n of this solution contained a minimal amount of DNA polymer-ase a c t i v i t y as shown i n Table IX. At a 55 per cent saturation, the p r e c i p i t a t e contained the major DNA polymerase a c t i v i t y as shown by the incorporation of 1^C-dTTP into an acid insoluble product. According to Lehman et al. (63), the bulk of the deoxyribonucleases was found i n the f i r s t 40% f r a c t i o n while the second f r a c t i o n at 55% saturation contained the DNA polymerases. Following ammonium sulfate f r a c t i o n a t i o n , the active enzyme extract was chromatographed on a DEAE ce l l u l o s e column which - 108 -Table IX Ammonium Sulfate Fractionation Salt E. c o l i DNA polymerase a c t i v i t y Concentration 1 ltC-2-dTMP incorp. i n DNA/mg protein Counts/minute pmoles 40% 27 1 55% 2766 52 The standard DNA polymerase assay system was used with an incubation period of 15 minutes at 37° C. 75 yg of protein from the enzyme fractions were used. - 109 -gave a substantial p u r i f i c a t i o n of the E. c o l i DNA polymerase. An elu t i o n p r o f i l e from the chromatography of th i s enzyme i s shown i n Figure 16. The f r a c t i o n which contained the major portion of the DNA polymerase a c t i v i t y was eluted with a 0.2 M potassium phosphate buffer, pH 6.5. This band of enzyme a c t i -v i t y was low i n protein and nucleic acid content as estimated from the solutions absorbance at 280 and 260 my respectively. The r e s u l t s obtained for the various stages of p u r i f i c a -tion of the E. c o l i enzyme extract are summarized i n Table X. Chromatography on DEAE-cellulose was observed to be the single most e f f e c t i v e stage i n the p u r i f i c a t i o n of the E. c o l i DNA polymerase up to this l e v e l . DNA polymerase which was obtained from the DEAE-cellulose fractions was sa t i s f a c t o r y for use as a standard. The o v e r a l l s p e c i f i c a c t i v i t i e s from the crude sonicated extract to the DEAE-cellulose stage indicate a p u r i -f i c a t i o n of approximately 100 f o l d . DNA Nucleotidyltransferases from the Small I n t e s t i n a l Mucosa of  the Rat. 1. Characteristics of the I n t e s t i n a l Assay System. As observed with the E. c o l i system, i t was d i f f i c u l t to determine the optimal conditions for an enzymatic reaction i n crude c e l l extracts. In order to demonstrate some of the basic requirements for the i n t e s t i n a l DNA nucleotidyl transferases, a p a r t i a l p u r i f i c a t i o n of the enzyme preparations was necessary. Some of these c h a r a c t e r i s t i c s for the transferase reaction w i l l - 110 -1 . 2 1 . 0 0 . 8 0 . 6 0 . 1 ; 0 . 2 Enzyme a c t i v i t y 8 7 . 5 7 5 . 0 6 2 . 5 5 o . o 3 7 . 5 2 5 . 0 1 2 . 5 1 0 2 0 3 0 Fraction Number ll-O - P > 4J U cu tn tc (U I, o < IS Q m o tn +J •H C !=> 0 . 0 2 0 . 0 5 0 . 1 0 . 2 0 . 5 M Potassium Phosphate Buffer, pH 6.5 Figure 16. Chromatography of DNA polymerase from E.. c o l i B on DEAE-cellulose. One enzyme unit incorporates 1 ,pmole 1 ^ C - l a b e l l e d deoxyribonucleotide into an acid-insoluble product i n 15 minutes at 37° C. - I l l -Table X P u r i f i c a t i o n of E. c o l i DNA polymerase Step Volume of enzyme solution ml Protein mg/ml DNA polymerase a c t i v i t y units per mg protein I Sonicated extract 400 II Streptomycin 154 III Ammonium sulfate 10 IV DEAE-cellulose 3 17.6 4.2 6.0 0.25 18.5 23.0 52 .0 1900.0 One unit of DNA polymerase a c t i v i t y i s equal to the incor-poration of 1 picomole of 1 ltC-2-TMP i n an acid-insoluble product under standard assay conditions. The reaction was carried out for 15 min at 37° C. The value shown for DEAE-cel l u l o s e represents the peak f r a c t i o n . - 112 -be presented after the discussion of the p u r i f i c a t i o n steps used. E a r l i e r investigators (125,142) have shown that the basic requirements for the mammalian DNA polymerases were e s s e n t i a l l y the same as those previously described for the b a c t e r i a l system. In the mammalian ' r e p l i c a t i v e ' system, Bollum (125) has shown that the enzyme required the deoxyribonucleoside triphosphates of adenine, guanine, cytosine and thymine, a DNA template, and Mg + + for a c t i v i t y . The 'terminal' deoxynucleotidyltransferase showed a requirement for a single triphosphate precursor, a de-natured DNA primer, and a sulfhydryl compound such as cysteine or 2-mercaptoethanol (143). 2. Extraction of DNA Nucleotidyltransferases from the Small  I n t e s t i n a l Mucosa of the Rat. Very l i t t l e DNA polymerase a c t i v i t y was observed i n the extracts from the small i n t e s t i n a l mucosa of the r a t when the i n t e s t i n a l scrapings were homogenized i n a buffered sucrose medium (0.25 M sucrose-0.15 M KC1-0.2 M Tris-HCl, pH 8). A s l i g h t l y more active enzyme preparation was obtained when the crude homogenate was centrifuged at 105,000 xg for 2 hours and the supernatant solution was used as shown i n Table XI. If the i n t e s t i n a l scrapings were f i r s t washed with a 6 per cent dextran-Krebs Ringer phosphate solution as described by Clark and Porteous (104), an increase i n the DNA polymerase a c t i v i t y was observed. This increase i n a c t i v i t y was probably due to the removal of the high content of mucin normally found to be present i n the rat i n t e s t i n a l mucosa. During homogenization of the tissue scrapings, - 113 -the mucin causes the formation of a gelatinous mass which re-tards c e l l u l a r disruption as well as clean separation of sub-c e l l u l a r components at low c e n t r i f u g a l forces. In unwashed c e l l s the high-speed supernatant would contain less s o l u b i l i z e d enzymes due to this mucin trap. If the i n t e s t i n a l e p i t h e l i a l c e l l s were f i r s t washed with the dextran solution, homogenization followed by subcellular f r a c t i o n a t i o n gave a nuclear p e l l e t which was e a s i l y sedimented at low c e n t r i f u g a l force (600 xg for 10 min) and was less contaminated by subcellular p a r t i c l e s such as mitochondria (104). 3. I n t r a c e l l u l a r Location of DNA Nucleotidyltransferases from  the I n t e s t i n a l Mucosa of the Rat. DNA nucleotidyltransferases from mammalian tissues were o r i g i n a l l y obtained from the high-speed supernatant f r a c t i o n of c e l l s homogenized i n an aqueous medium (55,71,142). Although the supernatant cannot be considered to be exclusively the c e l l cytoplasm, i t contained a high a c t i v i t y of the enzyme while the corresponding sediment which contained the c e l l nuclei showed l i t t l e transferase a c t i v i t y (60). Canellakis and coworkers (96) have i s o l a t e d c a l f thymus nuclei by d i f f e r e n t i a l centrifugation i n 0.25 M sucrose-3 mM CaCl2 which contained an active DNA nucleo-tid y l t r a n s f e r a s e a c t i v i t y . It i s believed that the presence of the C a + + ions allowed the i s o l a t i o n of more nuclei with i n t a c t nuclear membranes (144). The effectiveness of C a + + ions for the s t a b i l i z a t i o n of the nuclei i s o l a t e d from r a t i n t e s t i n a l mucosa was determined - 114 -by comparing the amount of DNA polymerase a c t i v i t y obtained from nuclei i s o l a t e d i n the presence or absence of th i s divalent metal. Although 6% dextran-Krebs Ringer phosphate wasa solution contained 0.11 M CaCl2, i t was discarded with the supernatant during the recovery of the whole e p i t h e l i a l c e l l f r a c t i o n . A solution was prepared for washing the c e l l s , without added C a + + ions, and contained 6% dextran i n 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethane s u l f o n i c acid, "TES" solution. A homogeni-zation medium which contained 0.3 M sucrose -5 mM EDTA, pH 7.4 was used for the i s o l a t i o n of the nu c l e i . In a si m i l a r experi-ment, the nuclei were i s o l a t e d from a homogenate which contained 3 mM CaCl2 i n the sucrose-EDTA medium. Nuclei prepared under these two conditions were extracted for DNA polymerase with a medium containing 50 mM Tris-HCl, 5 mM MgCl2 and 5 mM 2-mercap-toethanol, pH 7.5. The nuclei i s o l a t e d i n the presence of C a + + ions retained a larger proportion of i t s protein and correspond-ing DNA polymerase a c t i v i t y than nuclei i s o l a t e d i n a medium without C a + + as shown i n Table XI. In support of these observa-tions, Main and Cole (73) have reported that the presence of 2 mM C a + + ions i n a medium used for the homogenization of rat thymus tissue reduced the loss of DNA polymerase from the n u c l e i . In the present investigation, the addition of 3 mM CaCl2 i n the homogenization medium used for the i s o l a t i o n of the nuclei did not appear to i n t e r f e r e with the DNA polymerase a c t i v i t y from these nuclear extracts. Furlong (145) observed that the DNA polymerase from Walker 256 tumor was not active when C a + + ions Table XI DNA Polymerase A c t i v i t y i n Extracts from Rat I n t e s t i n a l Mucosa DNA polymerase a c t i v i t y Enzyme Fraction Protein 11*c-2-dTMP incorp. i n DNA/mg protein Fraction Volume mg/ml ml Counts/minute pmoles Whole c e l l homogenate t o t a l 190 19.5 11 0.1 105,000 xg supernatant 175 6.0 84 1.4 Cel l s washed with dextran solution before homogenization 105,000 xg supernatant 157 6.9 768 19.8 Subcellular fractionation 6% dextran-Krebs Ringer phosphate washed c e l l s ; homogenized with 3 mM Ca Nuclei 90 4.8 2565 64 Cytoplasm 125 12.0 795 20 6% dextran 'TES1 buffer washed c e l l s : homogenized without added C a + + Nuclei 90 1.8 1100 28 Cytoplasm 125 13.7 987 25 The reaction mixture contained 25 to 75 yg protein from the enzyme fr a c t i o n s , 15 yg native i n t e s t i n a l DNA, and 1.65 nmoles 1 lfC-2-dTTP i n the presence of 16 nmoles of the four deoxyribonucleoside triphosphates. 2 ymoles of MgCl2 and 0.3 ymoles of 2-mercaptoethanol were included i n a t o t a l volume of 0.25 ml, buffered to pH 8.1. The tubes were incubated for 15 min at 37° C. - 116 -were used as the divalent metal i n place of Mg ions, but that the presence of 4 mM CaCl 2 does not i n h i b i t the polymerase a c t i -v i t y i n the presence of •thex -usual concentration of Mg + + ions. A comparison of the DNA polymerase a c t i v i t y from i n t e s t i n a l e p i t h e l i a l c e l l s i s o l a t e d from the tips of the v i l l i and from the lower crypt area was made as shown i n Table XII. After homogeni-zation of the c e l l s i n Tris-phosphate buffer, both the upper, v i l l i t i p s , and lower, crypt area, supernatant fractions from high-speed centrifugations showed s i g n i f i c a n t amounts of poly-merase a c t i v i t y . The upper cytoplasmic extract appeared to con-ta i n a larger proportion of enzyme a c t i v i t y . The nuclear frac-t i o n represented the re-extraction of the sediment from the f i r s t centrifugation at 10,000 xg for 20 min. A s l i g h t l y greater t o t a l a c t i v i t y for DNA polymerase was found from an extract of nuclei from the lower crypt area than from the corresponding v i l l i t i p s . The question of the i n t r a c e l l u l a r d i s t r i b u t i o n of the DNA polymerases cannot be answered with the present aqueous experi-mental system because these enzymes appear to be highly soluble and can be leached out into the extraction medium from the nuclei and/or cytoplasm. In order to minimize the opportunities for the possible d i f f u s i o n of i n t r a c e l l u l a r components across the nuclear membrane p r i o r to the preparation of the enzyme extracts, the nuclei and the cytoplasm were i s o l a t e d i n non-aqeuous solvents from rapid l y frozen and l y o p h i l i z e d tissue according to the pro-cedure of Kay et al. (128). The nuclear and cytoplasmic f r a c -tions were subseqently extracted with an aqueous medium and were Table XII DNA polymerase a c t i v i t i e s i n subcellular extracts from r at i n t e s t i n a l mucosa Fraction Protein . , . . , _ _ ; . T T , DNA polymerase a c t i v i t y Enzyme Fraction Volume ^ 2 J ml mg/ml Total enzyme units i n f r a c t i o n C e l l s from upper mucosa, v i l l i t ips Supernatant, including cytoplasm 12 11.4 9Q29 Nuclear 5 7.1 2201 C e l l s from lower crypt area Supernatant, including cytoplasm 12 13.5 3402 Nuclear 5 6.4 2376 Subcellular fractions i s o l a t e d by non-aqueous solvents Nuclei 8 6.4 292 Cytoplasm 3 12.6 72 One unit of DNA polymerase incorporated 1 pmole llfC-2-dTMP i n DNA using a simi l a r reaction mixture as described i n Table XI. Incubations were con-ducted for 15 min at 37° C. - 118 -shown to contain DNA polymerase a c t i v i t y as indicated i n Table XII. Although enzyme a c t i v i t y was detected i n both f r a c t i o n s , the nuclear f r a c t i o n was observed to contain a higher t o t a l a c t i v i t y than the cytoplasmic extract. Keir et al. (75,77) have prepared nuclei and cytoplasm from regenerating rat l i v e r , rabbit thymus, and from c a l f thymus using the non-aqeuous s o l -vent technique. When these fractions were extracted for DNA nu-cleotidyltransferase a c t i v i t y , substantial a c t i v i t y was found i n the nuclear f r a c t i o n while a lower but appreciable enzyme a c t i v i t y was present i n the cytoplasm. Evidence from the rat i n t e s t i n a l mucosa supports these observations which indicate that mammalian DNA nucleotidyltransferases are present i n both the nucleus and the cytoplasm of the c e l l . 4, Studies on the .Purification of DNA Nucleotidyltransferases  from the Small I n t e s t i n a l Mucosa of the Rat. The p u r i f i c a t i o n of mammalian DNA polymerases has been con-cerned mainly with the removal of known i n t e r f e r i n g factors as nucleic acids, endogeneous mononucleotides, DNases, phosphodi-esterases, triphosphatases, and other enzymes which may block the biosynthesis of DNA or degrade'-the products. P u r i f i c a t i o n of DNA polymerases from mammalian tissues has been generally found to be more d i f f i c u l t than from b a c t e r i a l systems (61). Mamma-l i a n enzymes appear to be more delicate and losses of enzymatic a c t i v i t y have been observed (146) with the use of conventional fr a c t i o n a t i o n techniques such as ammonium sulfate p r e c i p i t a t i o n . The choice of the p u r i f i c a t i o n procedures to be used as well as - 119 -the sequence i n which they w i l l be applied w i l l depend upon factors such as the source and the amount of tissue available, the s t a b i l i t y of the enzyme during and after the p u r i f i c a t i o n technique, and the degree of p u r i f i c a t i o n desired with the aim of obtaining a native-active enzyme. (a) Ammonium sulfate f r a c t i o n a t i o n : The phenomenon of salting-out proteins from solutions using various concentrations of ammonium sulfate has been routinely used to fractionate DNA polymerase from tissue ex-tracts (61,145,146). In the present work, a solution of soluble proteins extracted from the nuclei of rat i n t e s t i n a l c e l l was brought to 40 per cent saturation by the addition of s o l i d ammonium s u l f a t e . A p r e c i p i t a t e was recovered after c e n t r i f u -gation and was found to contain only a low DNA polymerase a c t i -v i t y . Previous studies by Lee and Zbarsky (10 3) have shown that a high DNase a c t i v i t y was recovered from an i n t e s t i n a l mucosa extract by p r e c i p i t a t i o n with ammonium sulfate at a 20% satura-t i o n l e v e l . In the present study, the 40% saturated solution would give a p r e c i p i t a t e with a high DNase content. After the supernatant solution from the f i r s t f r a c t i o n a t i o n was brought to the 60 per cent saturation l e v e l with ammonium sul f a t e , the recovered p r e c i p i t a t e was found to contain more DNA polymerase a c t i v i t y than observed i n the 40 per cent f r a c t i o n as shown i n Table XIII. Although the DNA polymerase a c t i v i t y was recovered i n the 60% ammonium sulfate f r a c t i o n , the a c t i v i t y of the enzyme following t h i s treatment appeared to be lower than the - 120 -Table XIII DNA polymerase a c t i v i t i e s i n nuclear extracts from r at i n t e s t i -nal mucosa following ammonium sulfate f r a c t i o n a t i o n . Enzyme Reaction C-2-dTMP incorporated i n DNA Time Fraction . , Counts/minute minutes ' I. Whole nuclear 5 159 15 98 30 61 II. Ammonium sulfate 40% saturation 15 9 60% saturation 5 54 15 86 30 49 The quantities of enzyme used i n the above assays were the same, 75 pg/assay, on the basis of the protein concentrations. The reaction mixture contained the remaining materials as described in Table XI. The tubes were incubated for 15 min at 37° C. - 121 -o r i g i n a l a c t i v i t y from the whole nuclear extract. In view of the present results and the possible detrimental effects of using a high s a l t concentration on the s t r u c t u r a l conformation of the mammalian DNA polymerases as described by Keir (91), ammonium sulf a t e f r a c t i o n a t i o n was not included i n the usual scheme for the p u r i f i c a t i o n of the i n t e s t i n a l DNA polymerases, (b) DEAE-cellulose chromatography: A high-speed supernatant f r a c t i o n which contained the solu-ble proteins extracted from the nuclei of rat i n t e s t i n a l mucosa was chromatographed on a column of diethylaminoethyl-cellulose (DEAE-cellulose). The appearance of three separate bands of DNA polymerase a c t i v i t y from a column (1 x 45 cm) of t h i s anion-exchange r e s i n indicated a possible complex nature for this enzyme. A t y p i c a l elution p r o f i l e for the DNA polymerase a c t i -v i t i e s i s shown i n Figure 17. The f i r s t a c t i v i t y detected from this column was associated with the leading peak of unadsorbed proteins and nucleic acid material. Moore and Lee (129) who have used a s i m i l a r sized column of DEAE-cellulose for the chroma-tography of r a t l i v e r soluble proteins also found a fore-peak of high u l t r a v i o l e t absorbing material. In the present study, the elu t i o n p r o f i l e s from six separate experiments using from 12 to 160 mg of rat i n t e s t i n a l proteins, a l l showed the presence of a forepeak of rapidly eluted material. As w i l l be subsequently discussed, rechromatography of this foreband indicated the presence of both a r e p l i c a t i v e and a terminal DNA nucleotidyl-transferase . Units of DNA polymerase a c t i v i t y 3 3 3 d H- o h-1 rt h-1 *l Ul fD H-0 H-iQ 3 rh d H- O H 3 CD Hi fD d 3 ft N H H (0 <^ oi B rt • fD d H-3 O Oi 3 rt 3-fD O fD rj H 0 01 O H rt 3 01 ^ H- £D rt 0 3 rt Oi hi OJ O 3 Oi MIQ Oi rt H P> fD 3 01 t-i DJ d ^ Qi O 3* M O <^ Jl) 01 0) <T3 0) O cn 3 • 3 Oi O O > M o > o 3 rt o 3 01 -PO OJ O h-H- i d i-3 01 fD H-3 rt O M I O fD 01 3 O O rh rh (—1 0) 0) o H' a- o l 3 H H O ui rt 3 01 O H & M fD •-i 0 O 01 3 d CD O rt fD H-3 fD X rt H Oi O rt rh n o rt 3" fD to Ul -J o to M Ul O Ul o 1 Ul 1 o Ul 1 o Absorbance at 280 my r{ 0) O rt H-o 3 d fD 00 Molarity of KC1 - Z3T -- 123 -A gradual e l u t i o n of the proteins from the ion exchange r e s i n was achieved by the use of a parabolic chloride gradient. The second peak of DNA polymerase a c t i v i t y which was eluted near the front of the protein band, at 0 .1 M CI , was the major transferase f r a c t i o n when fresh extracts of the mucosa were used. Enzyme extracts which had been stored at 0 - 5 ° C f o r two or more weeks before chromatography gave a p r o f i l e with a smaller amount of t h i s second band of a c t i v i t y , and an apparent greater proportion of a t h i r d peak of DNA polymerase a c t i v i t y , at 0 . 16 -0 .2 M CI . This e l u t i o n p r o f i l e i s shown i n Figure 18. Rechromatography of the active bands of DNA nucleotidyltransfer- ase . Three separate groups of DNA nucleotidyltransferase a c t i v i t y which corresponded to the three major peaks of a c t i v i t y from the o r i g i n a l DEAE-cellulose column, 1 x 45 cm, were prepared for chromatography on a small DEAE-cellulose column, 1 x 11 cm. Those fractions containing the active DNA polymerases were pooled and concentrated by u l t r a f i l t r a t i o n . The results obtained from the use of this concentration procedure are shown i n Table XIV. As an example, the volume of the f i r s t sample was reduced to one-f i f t h of i t s o r i g i a n l quantity but the amount of absorbance at 2 80 my was only about twice as high after concentration. The loss of u l t r a v i o l e t absorbing material was evident, but the amount of DNA polymerase a c t i v i t y appeared to be mostly retained as measured by the incorporation of radioactive precursors. >1 +J •H > -H +J O rO (D (d QJ E >i H o ft < Q m o w +J •H ID 15.0 12.5 10.0 7.5 5.0 V 2.5 o 00 CN •P (0 CD O C (0 X! U o w < 1.2 1.0 • 0.8 • 0.6 0.4 0.2 • DNA polymerase a c t i v i t y -a- -o-KC1 gradient • • * • -- II • • I i n r i n « 1 .• . .. 1 1 R • j \ ' 20 40 60 80 100 Fraction Number 120 - 1.0 0.8 0.6 0.4 0.2 140 u m o >i +J •H H (C iH O s Figure 18. Chromatography on DEAE-cellulose of a similar enzyme preparation as shown i n Figure 17, but the sample was stored at 4° C for two weeks before chromatography. One unit of enzyme a c t i v i t y i s the same as that previously described. to Table XIV Concentration of protein solutions by u l t r a f i l t r a t i o n Enzyme Fraction Fraction Molarity Absorbance Volume KC1 i n ml Fraction 260 my 280 my 1 k DNA polymerase a c t i v i t y C-2-TMP incorp. i n DNA Counts/minute I. Before concentration 30 After concentration 6 II. Before concentration 57 After concentration 6 II I . Before concentration 6 0 After concentration 3 DEAE-cellulose (tubes 5-15) 0.05 1.561 0.766 0.05 2.166 1.462 DEAE-cellulose (tubes 95-114) 0.07 0.150. 0.457 0.05 0.348 0.617 0.17 0.309 0.331 0.18 1.825 1.561 55 250 5 37 21 51 *Counts/minute represents the r a d i o a c t i v i t y detected per assay by the stan-dard assay procedure. Solutions were concentrated i n the model 50 Diaflow apparatus with UM-1 membranes (Amicon). - 126 -Samples II and III showed e s s e n t i a l l y the same pattern of re-s u l t s . The concentrated enzyme fractions were loaded onto DEAE-cel l u l o s e columns, 1 x 11 cm, and eluted with a stepwise s a l t gradient as described under the p u r i f i c a t i o n procedure for the E. c o l i DNA polymerase. Protein groups I, I I , III were chromato-graphed on DEAE-cellulose columns S - l , S-2, S-3 respectively as shown i n Figures 19, 20 and 21. From an examination of the enzyme a c t i v i t y p r o f i l e from the S-l column, two main bands of 1 r e p l i c a t i v e ' DNA nucleotidyltransferase were observed. The f i r s t enzyme a c t i v i t y was recovered i n the forepeak while the second was recovered i n fractions eluted with 0.1 to 0.2 M potassium phosphate buffer, pH 7.4. A d i s t i n c t peak of 'terminal' DNA nucleotidyltransferase was eluted with 0.0 5 M phosphate buffer. These results were reproducible i n further experiments on the chromatography of s i m i l a r l y prepared enzyme samples on DEAE-cellulose (1 x 11 cm). The method of rechromatography on DEAE-cellulose using step-wise elu t i o n appeared to produce a separation of the ' r e p l i c a t i v e ' and the 'terminal' DNA nucleo-tidyltransferases which were extracted from the nuclei of the rat small i n t e s t i n a l mucosa. The e l u t i o n p r o f i l e for the group II proteins as obtained from the DEAE-cellulose column (S-2) indicated the presence of two r e p l i c a t i v e transferase a c t i v i t i e s . One peak was eluted as an unadsorbed f r a c t i o n while the second occurred i n the 0.05-0.1 M phosphate f r a c t i o n s . No terminal addition enzyme was evident - 127 -3. e o 00 CM - P nJ CD u . Q W O CO «< o.5 0.1). 0 . 3 0.2 0 DNA N u c l e o t i d y l t r a n s f e r a s e -o- - o - R e p l i c a t i v e a c t i v i t y • A • T e r m i n a l a c t i v i t y 0 . 1 • 10 20 Fraction Number _ 1 I 1 L 6 . 2 5 0.02 0.05 0.1 0.2 0 . 5 M Potassium Phosphate Buffer, pH 6.5 -p •H > •H 4-> O (0 <u g >1 N C <u m o CO •P •'H c Figure, 19. Rechromatography on DEAE-cellulose (S-l). of the peak I DNA polymerase f r a c t i o n as shown i n Figure 17. The size of the column was 1 x 11 cm. One unit of r e p l i -cative or terminal enzyme a c t i v i t y incorporated 1 pmole of ll*C-2-dTTP under the assay conditions as previously des-cribed . - 128 -o 00 CM -P ttf Q) U C (0 X! H-o 0 . 8 0 . 6 O.lj. 0.2 DNA N u c l e o t i d y l t r a n s f e r a s e -o- -o- R e p l i c a t i v e a c t i v i t y • A . . A . T e r m i n a l a c t i v i t y n 1 0 f l i n • A . - • • A 1 . in I I . <>--«. 7 . 5 5 . 0 2.5 0 1 0 2 0 Fraction Number 3 0 -p •H ^ •H 4-> O (0 Q) g> >i N c <u o 0) -P •H C 0.02 0.05 0.1 0.2 0.5 M Potassium Phosphate Buffer, pH 6.5 Figure 20. Rechromatography on DEAE-cellulose (S-2) of the peak II DNA polymerase f r a c t i o n as shown i n F i g . 17. The size of the column was 1 x 11 cm. One unit of r e p l i c a t i v e or terminal enzyme a c t i v i t y incorporated 1 pmole of 1'tC-2-dTTP under the assay conditions as previously described. - 129 -e o 00 CM - P (0 0) o c m o 0) 2 . 0 DNA Nucleotidyltransferase -o- -^-Replicative a c t i v i t y • 'Terminal a c t i v i t y 1.5 ' 1.0 • o.5 • 0 10 20 Fraction Number - P - H > •H - P u CD (D m o w - P ID 0.02 0.05 0.1 0.2 0 . 5 M Potassium Phosphate Buffer, pH 6.5 Figure 21. Rechromatography on DEAE-cellulose (S-3) of the peak III DNA polymerase f r a c t i o n as shown i n Fig, 17. The s i z e of the column was 1 x 11 cm. One unit of r e p l i c a t i v e or terminal enzyme a c t i v i t y incorporated 1 pmole of 1'*C-2-dTTP under the assay conditions as previously described. - 130 -i n this f r a c t i o n a t i o n . In order to determine i f a difference i n the molecular size caused -the formation of the two r e p l i c a t i v e bands, a Sephadex G-100 column was used for gel f i l t r a t i o n of these two f r a c t i o n s . The results from the G-100 column which was prepared according to the procedure of Whitaker (147) are shown i n Figure 22. Both peaks of r e p l i c a t i v e a c t i v i t y were eluted with the same buffer volume using separate f i l t r a t i o n studies. These re s u l t s indicated that a s i m i l a r i t y i n the molecular r a d i i existed for the two r e p l i c a t i v e f r a c t i o n s , and that the forepeak e a r l i e r mentioned was probably due to unadsorbed DNA polymerase of the same molecular size as the second band. When the group III proteins were rechromatographed on the DEAE-cellulose column (S-3), one major band of protein was re-covered. The eluted fractions contained a band of r e p l i c a t i v e a c t i v i t y at 0.0 5-0.1 M phosphate concentration, but were also associated with an apparent terminal addition a c t i v i t y at several of the f r a c t i o n s . Although the i n i t i a l DEAE-cellulose chromatography of the supernatant proteins from an extract of small i n t e s t i n a l mucosa nuclei indicated that there may be three d i s t i n c t proteins with the same DNA nucleotidyltransferase a c t i v i t y , rechromatography of these fractions on DEAE-cellulose allowed the detection of further sub-fractions. The peak I proteins were shown to contain a separate terminal transferase enzyme from the r e p l i c a t i v e DNA polymerase. Peak II proteins which appear to contain only re-p l i c a t i v e a c t i v i t y may represent the main DNA polymerase struc-- 131 -3 e o 00 CN +J fd <u u c rd X! S-i O tn < 25 r .20 .15 .10 . 0 5 Enzyme a c t i v i t y Forepeak from DEAE-cellulose Middle peak from 11 f 0 6 .25 5.oo 3.75 2.5o 1.25 5 10 15 Fraction Number 20 Figure 22. Sephadex G-100 gel f i l t r a t i o n of the DNA polymerase fractions from DEAE-cellulose chromatography (S-2) as shown i n Fig..20. The eluti o n p r o f i l e s from two separate gel f i l t r a t i o n s are superimposed. Elution was carri e d out with 0.1 M potassium phosphate buffer, pH 7.2. >i 4-> > •V4 4-> U fd <D tn rd U O ft < S3 Q <M O tn 4-> 'A c D - 132 -ture i n a multimeric form as described by Keir (91). The peak III f r a c t i o n of a c t i v i t y appears to contain a mixture of the r e p l i c a t i v e and the-terminal addition enzymes. These results may be explained by the hypothesis of Keir (91) who suggested that the degradation of a multimeric DNA nucleotidyltransferase to a monomeric subunit may r e s u l t i n the formation of the t e r -minal addition enzyme. Hayes et al. (14 8) found that increasing the time of storage of frozen c a l f thymus glands led to increased terminal and decreased r e p l i c a t i v e transferase y i e l d s . A s i m i l a r transformation may have occurred with the rat i n t e s t i n a l trans-ferases. When fresh extracts of the mucosal nuclear f r a c t i o n were chromatographed one DEAE-cellulose as discussed e a r l i e r , peak II contained the major DNA polymerase a c t i v i t y with a smaller peak III a c t i v i t y . After storage at 0-5° C (or a f t e r freezing at -20° C) for two weeks, the peak II a c t i v i t y de-creased and a larger peak III a c t i v i t y resulted. The presence of terminal addition enzyme i n peak III and not i n peak II appeared to support such a hypothesis. (c) Hydroxylapatite chromatography; Chromatography on hydroxylapatite has been shown by Shepherd and Keir (146) and by Okazaki and Kornberg (67) to remove DNase I from DNA polymerase preparations. Bollum et al. (149) have also used this adsorbant for the separation of the r e p l i c a t i v e DNA polymerase from the terminal-addition enzyme of c a l f thymus gland. In the present work, a DNA nucleotidyltransferase preparation from ra t i n t e s t i n a l mucosa which had been p a r t i a l l y p u r i f i e d on DEAE-- 133 -c e l l u l o s e was chromatographed on a hydroxylapatite column. From an examination of tfe.ielution p r o f i l e i n Figure 23, the major r e p l i c a t i v e DNA polymerase peak was eluted with 0.2 M phosphate buffer,- pH 6.8. A shoulder of terminal-addition enzyme was detected at tube number 20. This e l u t i o n p r o f i l e was s i m i l a r to one reported by Yoneda and Bollum (102) who showed that a small shoulder of terminal-addition enzyme a c t i v i t y preceeded the main peak of r e p l i c a t i v e a c t i v i t y following chromatography of a c a l f thymus preparation on hydroxylapatite. An enzyme extract from the nuclei of the i n t e s t i n a l mucosa was p a r t i a l l y p u r i f i e d by chromatography on DEAE-cellulose. The peak f r a c t i o n was found to incorporate 866 pmoles of 1 "*C-2-TMP per mg protein as shown i n Table XV. Following chromatography of the main DNA polymerase band from th i s column on a hydroxy-l a p a t i t e column, the peak f r a c t i o n incorporated 46 5 pmoles of labeled precursor into an acid-insoluble product. From t h i s study, i t appeared that a loss i n enzyme a c t i v i t y had occurred following hydroxylapatite chromatography. As observed by Richardson et al. (150), the E. c o l i DNA polymerase was less active with either a native or denatured c a l f thymus DNA follow-ing chromatography on hydroxylapatite. These authors postulated that the removal of nucleases such as endonuclease I (152), and exonuclease III (151) which were believed to increase the priming capacity of the DNA were removed by the hydroxylapatite treatment. In the present work, the loss of enzyme a c t i v i t y following hydroxylapatite chromatography of the i n t e s t i n a l DNA - 134 -0.12 DNA N u c l e o t i d y l t r a n s f e r a s e R e p l i c a t i v e a c t i v i t y T e r m i n a l a c t i v i t y 0.10 0.07 -0.05 & 0.02 . CD E >i N C CD O 4-> -H CO > 4-) -H - H 4-> C O ID rd 5 .oo-3.75-2.50-1 .:25-A " . A . i -O -O 0.5 0.14. 0.3 0.2 0.1 0 10 20 30 Fraction Number >i0 0 Figure 23. Chromatography on hydroxylapatite of a DNA polymerase f r a c t i o n , peaks II and I I I , from DEAE-cel l u l o s e as shown i n F i g . 17. Elution was carried out with a lin e a r gradient of potassium phosphate -0.01 M 2-mercaptoethanol buffer, pH 6.5. Table XV Comparison of DNA polymerase a c t i v i t y from nuclear extracts of rat i n t e s t i n a l mucosa following chromatography on DEAE-cellulose and hydroxy-l a p a t i t e * Enzyme Fraction Fraction Volume ml Protein mg/ml DNA polymerase a c t i v i t y 1 **C-2-TMP incorp.*/mg protein Counts/minute pmoles DEAE-cellulose I I . Hydroxylapatite 0.2 0.05 33,100 18,100 866 465 The enzyme f r a c t i o n represented the peak tube of DNA polymerase a c t i v i t y , i n c o r p o r a t e d into DNA. - 136 -polymerase might be accounted for by the decrease i n the a b i l i t y of the added DNA to prime the reaction. Yoneda and Bollum (10 2) have shown that both deoxyribonuclease and phosphodiesterase were removed from a c a l f thymus DNA preparation following hydroxy-l a p a t i t e treatment. Hydroxylapatite chromatography has also been found to a f f e c t the s t a b i l i t y of the DNA polymerases. As shown i n Table XVI, both the r e p l i c a t i v e and the terminal-addition enzyme a c t i v i t i e s were decreased following storage of these hydroxylapatite f r a c -tions. Zimmerman (101) has found that Micrococcus l y s o d e i k t i c u s DNA polymerase was also unstable aft e r chromatography on hydroxy-l a p a t i t e . Gottesman and Canellakis (143) have observed that terminal DNA nucleotidyltransferase from c a l f thymus gland was unstable i n the assay systems, at 37° C, and during d i a l y s i s , at 4° C, aft e r hydroxylapatite treatment. In view of the above considerations, and the poor separation of the terminal from the r e p l i c a t i v e enzymes by the hydroxylapatite column as shown i n Figure 23, th i s step was not used for the further p u r i f i c a t i o n of i n t e s t i n a l enzyme preparations, (d) Sephadex gel f i l t r a t i o n : Molecular-sieve chromatography, more commonly termed 'gel-f i l t r a t i o n 1 , has been used for the separation of c a l f thymus DNA polymerase from tfte'-terminal-addition enzyme, and for the e s t i -mation of t h e i r molecular weights (102). Through the use of both a Sephadex G-100 and G-200 column, the molecular sizes of the c a l f thymus enzymes were estimated by Bollum et at. (149) - 137 -Table XVI S t a b i l i t y of DNA polymerases from the i n t e s t i n a l mucosa of the rat following chromatography on hydroxylapatite. Hydroxylapatite DNA nucleotidyltransferase a c t i v i t y 1 lfC-2-dTMP incorporated, counts/minute Fraction Replicative Terminal-addition Before- After-storage Before- After-storage 19 36 4 26 12 20 31 5 46 23 25 123 33 35 15 43 29 11 4 0 Enzyme a c t i v i t y was measured before and after storage at -20° C for one week. The reaction mixture for r e p l i c a t i v e enzyme ac-t i v i t y was the same as that described i n Table XI. Terminal-addition enzyme a c t i v i t y was measured i n a system which con-tained, per 0.25 ml, 20 yM Tris-HCl buffer (pH 7.4), 2yM MgCl 2, 2.5 yM cysteine, 0.3 yM 2-mercaptoethanol, 16 nM dTTP, 1.6 5 nM llfC-dTTP, 15 yg heat-treated DNA, and 50 yg protein from the enzyme f r a c t i o n s . Incubations were for 15 min at 37°. - 138 -to be 110,000 for the r e p l i c a t i v e and 37,000 for the terminal DNA nucleotidyltransferase. A Sephadex G-200 column was also found to p a r t i a l l y separate DNase I from a DNA-nucleotidyl-transferase from Landschutz ascites tumor c e l l s (91). Besides these known applications, gel f i l t r a t i o n was chosen for further studies on i n t e s t i n a l mucosa DNA nucleotidyltransferase because i t i s a mild f r a c t i o n a t i o n method which does not expose the enzyme to great changes i n pH and i o n i c strength. Sephadex G-150 was used for the f i l t r a t i o n of a rat i n t e s t i -nal DNA nucleotidyltransferase preparation which had been pre-viously fractionated on a DEAE-cellulose column and then concen-trated by u l t r a f i l t r a t i o n . Approximately 0.5 g of th i s protein together with non-enzymatic markers, human y-globulin, beef pan-creas chymotrypsinogen-A, and blue dextran (in 1-2 ml) were loaded onto the column. The elut i o n pattern of the non-enzymatic markers was f i r s t determined from a separate f i l t r a t i o n on the same column to act as a standard. From an examination of the r e p l i c a t i v e DNA transferase p r o f i l e as shown i n Figure 24, two peaks of a c t i v i t y were evident. Peak I was eluted ahead of y-globulin (MW 160,000; Stokes radius 52 A) while peak II was eluted approximately with the chymotrypsinogen-A f r a c t i o n (MW 25,000; Stokes radius 20). Comparisons of the presence of r e p l i c a t i v e or terminal a c t i -v i t y and i t s primer requirements were made on the two peaks from the G-150 column. Under the standard r e p l i c a t i v e DNA polymerase assay, peak I appeared to be less active than peak I I . The peak - 139 -3 -g O CO CM -P nJ 00 u (d X! u O Ul .25 .20 .15 .10 .05 DNA polymerase a c t i v i t y B l u e d e x t r a n A f \ f > I I l P ,25 ,20 15 25 35 lj-5 55 15 • .10 - .05 •H GO •P 0 H 04 e \ GO •P n3 •U o Oi o o c ft S EH I U GO rH o e Fraction Number Figure 24. Sephadex G-150 gel f i l t r a t i o n of a DNA polymerase f r a c t i o n , peak II, from DEAE-cellulose chromatography as shown i n F i g . 17. Elution was carr i e d out with 0.1 M potassium phosphate buffer, pH 7.2-1 mM 2-mercaptoethanol. - 140 -I fractions showed nearly equal a c t i v i t i e s i n the presence of heat denatured or native DNA as primers as shown i n Table XVII. The peak II fractions were less active with a denatured primer. The enzyme preparation from both peaks showed a small amount of terminal addition a c t i v i t y . As the amount of labeled precursor incorporated into the acid-insoluble products during the ter-minal addition enzyme assay was less than the amount incor-porated under a r e p l i c a t i v e assay system, these enzyme prepara-tions were not c h a r a c t e r i s t i c of a terminal enzyme as described by Canellakis (143). The a b i l i t y of r e p l i c a t i v e DNA polymerases to catalyze a 'limited' terminal-addition reaction has been des-cribed by previous investigators (review by Keir, 91). In a recent report by Ono and Iwamura (9 8), DNA polymerase from adult r a t l i v e r was separated into two peaks on Sephadex gel f i l t r a t i o n . The larger peak I enzyme preferred denatured DNA as a primer while the smaller molecular species was only active with a native DNA primer. These authors also reported that f e t a l l i v e r showed only the peak I enzyme while regenerat-ing r a t l i v e r showed a major peak I a c t i v i t y but also the peak II enzymes. These experiments on r a t l i v e r tissue suggest the m u l t i p l i c i t y of the mammalian DNA polymerases, and that these enzymes were related to the metabolic state of the tissue. In the present work, the i n t e s t i n a l DNA polymerase extracts were obtained from adult rats i n a l l the experiments which might explain the presence of peaks I and II obtained by gel f i l t r a t i o n . - 141 -Table XVII Comparison of Peak I and II fractions from Sephadex G-150 gel f i l t r a t i o n for DNA nucleotidyltransferase a c t i v i t y . T , .. DNA polymerase a c t i v i t y Incubation _, f / . . J Counts/minute per assay system Peak I Fractions Peak II Fractions 26 27 51 52 Replicative assay Native DNA 191 145 199 187 Denatured DNA 17 8 14 8 52 54 Terminal-addition assay 73 60 51 48 Standard assay procedures were used for the incorporation of 11*C-2-TMP i n DNA. The composition of the assay mix-tures for r e p l i c a t i v e enzyme a c t i v i t y was the same as that described i n Table XI, and for terminal-addition enzyme a c t i v i t y was the same as that described i n Table XVI. - 142 -As the rat i n t e s t i n a l enzymes did not show an absolute prefer-ence for a native or denatured DNA i n peaks I or I I , i t was not possible to d i s t i n g u i s h the two on the bases of the rat l i v e r studies. In a further experiment, an extract of whole i n t e s t i n a l mucosa c e l l s containing about 17 mg protein was f i l t e r e d on Sephadex G-150 as described for the previous run. The sample was eluted with 0.1 M potassium phosphate buffer -ImM 2-mercaptoethanol, pH 7.2. The elution pattern for the DNA poly-merase a c t i v i t y i s shown i n Figure 25, and indicates at least two main enzyme fractions are present. The f i r s t band was excluded by the gel while the second was eluted near the chymo-trypsinogen-A marker. A t h i r d band of DNA polymerase which was not detected before was eluted between the two main fractions of a c t i v i t y . This central band was associated with the main protein peak and contained a very low a c t i v i t y . An estimate of the molecular size of this small f r a c t i o n was made by com-paring the r a t i o of the e l u t i o n volume to the void volume, Ve/Vo, to a standard curve. This standard curve as shown i n Figure 26, was obtained by r e l a t i n g the r a t i o , Ve/Vo, as a function of the logarithm of known molecular weight markers as previously described. The size of the central enzyme was approxi-mately 5.4 x 101* while the enzyme eluted near chymotrypsinogen-A was about 2.6 x 101*. These studies indicate the existence of heterogeneous DNA polymerases i n c e l l free extracts from the small i n t e s t i n a l mucosa. 143 -3. e o 00 CM -P (« CD O c M o tn X> < 0.6 0 .5 o.lj. 0.3 0.2 0.1 DNA polymerase a c t i v i t y 10 I Blue dextran l a 1 20 30 14-0 5o Fraction Number 60 .06 .05 .0i|. .03 .02 .01 70 G 'H CD •P 0 U CU tn g \ 13 CD -P (C H O & M o O c •H ft £ EH I U w CD rH O 6 G Figure 25. Sephadex G-150 gel f i l t r a t i o n of a DNA polymerase extract from r a t i n t e s t i n a l mucosa. Elution was car r i e d out with 0.1 M potassium phosphate buffer, pH 7.2, 1 mM 2-mercaptoethanol. - 144 -o CD > CD 6 iH O > o -P w CD > •H -P fd rH CD 2.8 2.0 1.6 1.2 0.8 0.1+ Chymotryps inogen-A (MW 25,000) Bovine Albumin (MW 6 7,000) Globulin (MW 160,000) li..2 1+.1+ i+,6 1+.8 5.0 Logarithm of Molecular Weight 5.2 Figure 26. Standard curve for the estimation of the molecular weights of protein samples on the basis of t h e i r e l u t i o n volumes from a Sephadex G-150 gel f i l a t r a t i o n . The e f f e c t i v e range for the sample i s between 25,000 and 160,000 i n molecular weight. - 145 -In a recent report by C a v a l i e r i and C a r r o l l (100), three active molecular species of DNA polymerase were is o l a t e d from E. c o l i B c e l l s . Their i s o l a t i o n procedure involved high speed centrifugation of the crude extract followed by acrylamide gel electrophoresis. This procedure separated DNA polymerases into fractions which corresponded to a molecular size of 110,000, 58,000 and 24,000 i n the approximate r a t i o of 4:2:1. Their suggestions of a tetramer, dimer to monomer relat i o n s h i p has not been confirmed as yet, but they postulate that the smallest p a r t i c l e may be a subunit of the native molecule or a l t e r n a t i v e l y , that the small unit i s the native polymerase and that the larger molecules are aggregates of these units. In order to further establish the molecular heterogeneity of the i n t e s t i n a l DNA polymerases, and to study the active f r a c -tion that was excluded by the G-150 beads, a Sephadex G-200 column was used because of i t s wider protein f i l t r a t i o n range from 5,000 to 800,000. An i n t e s t i n a l enzyme extract (8 mg pro-tein) and the markers, blue-dextran 2000, chymotrypsinogen-A, and catalase were loaded onto the G-200 column. The elu t i o n of the sample with 0.1 M potassium phosphate buffer, pH 7.4, gave the p r o f i l e as shown i n Figure 27. Two major fractions of DNA polymerase a c t i v i t y as well as a small f r a c t i o n of excluded enzyme a c t i v i t y were detected. The f i r s t main peak was eluted with 1.86 column volumes while the second was eluted at 3.37 column volumes. The f i r s t f r a c t i o n appeared to be larger than catalase which was eluted at 1.97 column volumes, while the - 146 -3. g o 00 CN -P rrj CD O C rrj Si O CO < 0.5 0.3 0.2 0.1 -0 B l u e d e x t r a n 1 1.0 I b I fi ,DNA p o l l e r a s e a c t i v i t y II A \ _ JO 0.8 0.6 - 0.1+ 0.2 10 20 30 Fraction Number 1+0 50 •H CD -P o u ft g \ T3 CD •P (0 O ft U O o c CU S E H 1 U j -CQ CD cH O g Figure 27. Sephadex G-200 gel f i l t r a t i o n of a DNA polymerase extract from r at i n t e s t i n a l mucosa. Elution was carr i e d out with 0.1 M potassium phosphate buffer, pH 7.2, 1 mM 2-mercaptoethanol. - 147 -second was eluted close to chymotrpsinogen-A which required 3.35 volumes for elu t i o n . The e l u t i o n c h a r a c t e r i s t i c s and the estimated molecular weights of the rat i n t e s t i n a l DNA nucleotidyltransferases are shown i n Table XVIII. The following parameters were used: a. Relative e l u t i o n volume = Ve/Vo, where Vo i s the void volume or the elut i o n volume for a substance that i s com-pl e t e l y excluded from the gel (ie. blue dextran-2000), and Ve i s the elu t i o n volume corresponding to the peak concentration of a solute. The Ve/Vo i s dependent on the packing of the column. At a high elut i o n pressure an increase i n th i s value due to a small Vo can occur. b. The average d i f f u s i o n c o e f f i c i e n t between the l i q u i d phase and the gel phase can be related with the following equation: Kav. = Ve-Vo/Vt-Vo where Vt i s the t o t a l volume of the gel bed (Pharmacia, 132). c. Ackers (133) has proposed a mathematical c o r r e l a t i o n of the d i f f u s i o n c o e f f i c i e n t and the Stokes radius, 'a 1, of a macromolecule based on the Renkin equation: Kd = [ l - j r ] 2 [ l -2.10 4(1") + 2.0 9 (p) 3 - 0.9 5 (p) 5 ] where r i s the e f f e c t i v e pore radius of the gel matrix. A c o r r e l a t i o n of the observed Kd values of samples of known Stokes r a d i i to a/r values has been tabulated by Ackers for the c a l i b r a t i o n of gel columns. After the columns had been calibrated with the macromolecular markers of known Stokes r a d i i , the Stokes r a d i i for the unknown rat i n t e s t i n a l DNA polymerases were determined (Table XVIII). At - 148 -Table XVIII Sephadex Gel F i l t r a t i o n s _ Ve Ve-Vo Fraction ^— —^7— Vo V^ _-Vo Stokes 0 radius A Molecular weight G-150 Column, Vo = 117 ml; V = 390 ml In t e s t i n a l mucosa DNA polymerase Peak I 1.24 0.103 Peak II 2.38 0.593 57 21 >1.6 x 10 5 2.6 x 10 ** Y-Globulin 1.31 0.135 52 160,000a Chymotrypsinogen-A 2.42 0.6 08 20 25,000a G-150 Column, Vo = 115 ml; Vfc = 383 ml I n t e s t i n a l mucosa DNA polymerase Peak I excluded at front Peak l a 1 .55 0 .24 32 5. .4 X 10' ** Peak II 2 .35 0 .59 21 2. .6 X 10' ** G-200 Column, Vo = 43 : ml; V t = 195 ml In t e s t i n a l mucosa DNA polyme ra se Peak I 1 .86 0 .248 56 Peak II 3 .37 0 .684 20 2. .5 X 10' ** Catalase 1 .97 0 .281 52 2. .5 X 10! 5b Chymotrypsinogen- A 3 .35 0 . 677 20 2. .5 X 10' Mann Research Laboratories, New York G.K. Ackers, Biochem. 33 723 (1964). *appropriate values - 149 -least two species of i n t e s t i n a l transferases which had molecular r a d i i of approximately 56 and 20 A were determined from the gel f i l t r a t i o n . Evidence has been presented (154) which strongly indicates that the elu t i o n position of a protein upon Sephadex G-200 chromatography i s correlated with the Stokes radius rather than with the molecular weight of the protein. Previous investigators (147,155) have however, obtained excellent c o r r e l a t i o n between eluti o n volume of a series of globular proteins f i l t e r e d on G-75, G-100, and G-200, and the logarithm of t h e i r molecular weights. A better estimate of the molecular weight of a macro-molecule i n impure form can be made by cor r e l a t i n g the Stokes radius measured by gel f i l t r a t i o n with the sedimentation co-e f f i c i e n t determined by density gradient centrifugation. One such rel a t i o n s h i p i s as follows: M = 6ITr|Nas/(1-vp) where M = molecular weight, a = Stokes radius, S = sedimentation c o e f f i -cient, ~ = p a r t i a l s p e c i f i c volume,n = v i s c o s i t y of medium, p = density of medium, and N = Avogadro's number. (e) Sucrose density gradient centrifugation: Sedimentation patterns were obtained for DNA n u c l e o t i d y l -transferase a c t i v i t i e s by sucrose density gradient centrifugation of the high-speed supernatant from a whole c e l l extract of the rat i n t e s t i n a l mucosa, and the concentrated enzyme a c t i v i t y peaks from the Sephadex G-200 column. G-200 pooled fractions (a. 8-12, b. 13-19, c. 22-31) were concentrated by the u l t r a -f i l t r a t i o n technique on UM-1 membranes. - 150 -The sedimentation p r o f i l e for the enzymes from the whole c e l l extract as shown i n Figure 2 8 indicates the presence of three l i g h t e r fractions above the heavier sedimenting f r a c t i o n . Peaks I and II are heavier and l i g h t e r than the catalase marker, respectively while peak III appears near the chymotrypsinogen-A band. The sedimentation patterns of the Sephadex G-200 fractions are shown i n Figures 29,30,31. Group-'a' proteins representing the larger molecules from G-200 appear to show the major DNA nucleotidyl-transferase a c t i v i t y near the bottom of the tube. Some disaggregation of the enzyme might have caused the appear-ance of scattered low a c t i v i t y throughout the tube. The group-'b' proteins representing the peak I enzymes from G-200 showed a wide band of transferase a c t i v i t y near the center of the sucrose gradient. There i s also detectable enzyme a c t i v i t y near the bottom of the tube (fraction 3) and near the meniscus (frac-t i o n 22). The group- 1c' proteins from peak II which were the small molecular species remained near the meniscus after c e n t r i -fugation, but a f r a c t i o n of high a c t i v i t y was also observed near the bottom of the tube. This l a t t e r a c t i v i t y may be due to the aggregation of the enzyme to i t s e l f or to some unspecified material. Table XIX summarizes the results obtained from the density gradient sedimentation studies. The DNA polymerase bands be-tween the various tubes could be correlated i n reference to catalase which was constant i n a l l the tubes. The distance the enzyme fractions t r a v e l l e d from the meniscus was compared to - 151 -Fraction Number Figure 28. Sucrose gradient centrifugation of DNA polymerase extract from r at i n t e s t i n a l mucosa. Cen t r i -fugation was carried out for 20 hours at 45,000 rpm through a l i n e a r gradient from 7.5 to 25% sucrose i n 50 mM Tris-HCl buffer, pH 7.5. One unit of DNA polymerase incorporated 1 pmole 1 uC-dATP into an acid-insoluble pro-duct i n 15 min, under standard assay conditions. One unit of catalase produced a decrease i n absorbance of 0.01 i n 30 sec. under the assay conditions. - 152 -p. e o CM -P cn -P -H G CD cn CCS rH to -p (0 U ,70 60 50 30 20 10 0 .catalase to 0 u cn •rH G CD S - 2.50 3.75 - 1.25 5 10 15 20 25 30 Fraction Number Figure 29. Sucrose gradient centrifugation of DNA polymerase f r a c t i o n , group a, from the G-200 gel f i l -t r a t i o n as shown i n F i g . 27. The units of catalase and DNA polymerase a c t i v i t y are the same as those pre-viously described i n F i g . 28. •P -H > •H -P O PS CD cn H CD e >1 rH O OJ < Q m o cn •p •H G - 153 -I o CM •P rrj C O •p c QJ tn rrj rH rrj -P rrj U i 10 - 7-5 - 5.0 2.5 o 5 10 15 20 Fraction number 25 30 Figure 30. Sucrose gradient centrifugation of DNA polymerase f r a c t i o n , group b ( I ) , from the G-200 gel f i l t r a t i o n as shown i n F i g . 27. The units of catalase and DNA polymerase a c t i v i t y are the same as those des-cribed i n F i g . 28. - 154 -80 e o C N •P <d cn -P -H C O CD cn rd H fd -P (d u 60 i|0 20 catalase cn P" u cn c CD I l l °N 1 0 7 .5 5.o 2 .5 10 20 Fraction Number 30 Figure 31. Sucrose gradient centrifugation of DNA polymerase f r a c t i o n , group c (II), from the G-200 gel f i l t r a t i o n as shown i n F i g . 27. The units of catalase and DNA polymerase a c t i v i t i e s are as described i n F i g . 28, - 155 -Table XIX Sucrose density gradient centrifugation of DNA polymerase preparations from rat i n t e s t i n a l mucosa Fraction R a t i o a s b 20,w Molecular weight I. DNA polymerase Peak I 1.15 13.0 Peak II 0.77 8.7 Peak III 0.22 2.5 2.5 x 10k Catalase 1.00 11.35 2.5 x 1 0 5 C Chymotryps inogen 0.22 2.5 2.5 x 1 0 ^ II . DNA polymerase Peak I 1.19 13.5 Peak III 0.21 2.4 II I . DNA polymerase Peak I 1.19 13.5 3.0 x 10 5 Peak II 0.76 8.6 1.8 x 10 5 Peak III 0.21 2.4 2.4 x 101* IV. DNA polymerase Peak III 0.21 2.4 Ratio refers to the distance the sample i s from the meniscus i n reference to catalase which i s set at 1. S»„ i s the sedimentation c o e f f i c i e n t corrected to the 20 ,w reference temperature and to water. According to Ackers (133). (3. Mann Research Laboratories, New York. - 156 -that t r a v e l l e d by catalase. The sedimentation c o e f f i c i e n t s for three bands of i n t e s t i n a l DNA nucleotidyltransferase were e s t i -mated as 2.4, 8.6, and 13.5 S as previously described i n the experimental section. The corresponding molecular weights were obtained from a standard curve known Sor. versus molecular 20 ,w weights as given i n Figure 32. These enzyme species would be 2.5 x 10 \ 1.8 x 10 5, and 3 x 10 5 i n molecular weights as e s t i -mated from this c a l i b r a t i o n curve. The smallest p a r t i c l e at 2.5 x 10 agrees with the value obtained from the Sephadex gel studies. A molecular species which had a Stokes radius of 56 might corres-pond to the molecular weight of 1.8 x 10 s which i s larger than y-globulin of 52 Stokes radium and a molecular weight of 1.6 x 10 5. The 3 x 10 s component which i s larger than catalase of 2.5 x 10 5 i n molecular weight might be an aggregate of the smaller enzyme species. Although these molecular parameters have not been d e f i n i t e l y established for the DNA nucleotidyltransferases from the i n t e s t i -nal mucosa of the r a t , there appear to be several species of these enzymes when assayed in v i t r o . As studies on the DNA nucleotidyltransferases from various sources progress, more e v i -dence i s obtained which points to the existence of one or more forms of these enzymes i n E. c o l i (100). Calf thymus gland (10 2), r a t l i v e r (98) , and Landschutz ascites tumour c e l l s (146). The results from the p u r i f i c a t i o n of i n t e s t i n a l mucosa DNA nucleotidyltransferase from the rat are summarized i n Table XX. After subcellular f r a c t i o n a t i o n , the enzyme extracts were obtained - 157 -I i i i i _ 0 5 10 1£ 20 S x 10 sec. Figure 32. Standard curve for the estimation of the molecular weight of protein samples on the basis of th e i r sedimentation c o e f f i c i e n t s determined from 'sucrose density gradient centrifugations. These values were obtained from the l i t e r a t u r e (135,154). Table XX P u r i f i c a t i o n of rat i n t e s t i n a l DNA nucleotidyltransferase Step Method Fraction „ .. Volume Fraction ml Protein mg/ml DNA transferase a c t i v i t y 14C-2-dTMP incorporated*/mg protein Counts/minute pmoles Replicative enzyme I Crude homogenate t o t a l 157 6.9 768 19. 8 II Nuclear extract 90 4.8 . 2565 64 III DEAE-cellulose (1x45) a. 25 0.6 10,000 258 b. 50 0.2 33,100 866 c. 45 0.1 29,800 774 IV DEAE-cellulose ( l x l l ) a. 3 0.2 7,500 193 b. 7.5 0.07 36,000 928 V Sephadex G-200 a. 15 0.07 29,667 816 b. 20 0.06 22,979 632 VI Sucrose density gradient a. 1.5 0.009 220,000 4150 b. 2.0 0.008 175,000 3290 c. 1.5 0.009 160,000 3020 Terminal-addition enzyme IV DEAE-cellulose ( l x l l ) 5 0.06 58,000 1490 *Labeled precursor incorporated into DNA. - 159 -from the nuclear f r a c t i o n , and were shown to contain both the r e p l i c a t i v e and terminal-addition enzymes. The enzyme prepara-t i o n used i n step V for Sephadex gel f i l t r a t i o n was obtained from the step III DEAE-cellulose f r a c t i o n s . After rechromato-graphy of the step III enzymes with the small DEAE-cellulose column (1 x 11 cm, 'S'), the recovery of the enzymes was too low for further column chromatography on the Sephadex. The several fractions which are l i s t e d under the method used i n steps III to VI as a, b, c represent the peak tubes of each band. For example, DEAE-cellulose (1 x 45) column chromatogra-phy showed three bands of DNA polymerase a c t i v i t y . The peak f r a c t i o n from each band would correspond to a, b, and c. The volume represents the t o t a l volume of each band or component which contained the DNA polymerase a c t i v i t y . Due to the presence of a low protein content, the sucrose gradient step shows a substantial increase i n the s p e c i f i c a c t i v i t y . In the p u r i f i -cation of the r e p l i c a t i v e enzyme, DEAE-cellulose chromatography was the single most e f f e c t i v e procedure i n increasing the speci-f i c a c t i v i t y of the transferase. The o v e r a l l p u r i f i c a t i o n from the crude high-speed supernatant extract to the sucrose gradient centrifugation step was about 150. Rechromatography on DEAE-ce l l u l o s e appeared to give an increase i n s p e c i f i c a c t i v i t y of the terminal-addition enzyme up to 13 times. 5. Requirements of the Replicative DNA Polymerase i n the  I n t e s t i n a l Mucosa of the Rat. (a) pH Dependence: - 160 -Figure 33 i l l u s t r a t e s the incorporation of labeled t r i -phosphates i n DNA at various pH values of Tris-HCl buffer systems. An optimal a c t i v i t y of the i n t e s t i n a l DNA polymerase was observed at pH 8.0 when either a native or heat denatured DNA was used as the primer i n this buffer system. The use of rat i n t e s t i n a l mucosa or E. c o l i DNA as the primer gave the same results of an optimum a c t i v i t y at pH 8.0. A Tris-HCl system at pH 8.0 was used for the standard buffer system i n the assay of the r e p l i c a -t i v e DNA polymerase a c t i v i t y . When a potassium phosphate buffer medium was used i n the assay system, enzyme a c t i v i t y was maximal i n a pH range of 7.3 to 7.5. These pH optima observed for the i n t e s t i n a l DNA polymerase a c t i v i t i e s at pH 7.4 i n phosphate buffer, and at pH 8.0 i n Tris-HCl solution are i n agreement with pH optimal values obtained from studies by other workers with b a c t e r i a l (101) and mammalian tissues (156) under similar condi-tions. (b) Primer Requirements: The rat i n t e s t i n a l DNA nucleotidyltransferase reaction w i l l not proceed i n the absence of DNA as the template. A heat de-natured (single-stranded) or a native (double helix) DNA serves as a primer for the i n t e s t i n a l enzyme. As l i s t e d i n Table XXI, the peak I enzyme f r a c t i o n from a G-150 gel f i l t r a t i o n showed a s l i g h t preference for the heat denatured DNA is o l a t e d from the rat i n t e s t i n a l mucosa. The peak II enzymes from t h i s same gel column indicated a preference for the native i n t e s t i n a l DNA. When an E. c o l i DNA was used as the primer, a sim i l a r v a r i a t i o n - 161 -5.00 • 3.75 2.50 1.25 / \ / \ i \ i \ i \ / v 8 pH \ Figure 33. Effecto of pH on the a c t i v i t y of DNA poly-merase from rat i n t e s t i n a l mucoss. A Tris-HCl buffer system was used. One unit of DNA polymerase incorporated 1 pmole 11*C-2-dTTP into an acid insoluble product i n 15 min as described i n the assay system used. - 16 2 -Table XXI Requirements of the r e p l i c a t i v e DNA polymerase from r at i n t e s t i n a l mucosa Requirement Enzyme Fraction DNA polymerase a c t i v i t y Enzyme units/mg protein* I. DNA primer native r a t i n t e s t i n a l G-150 I II 580 750 denatured rat i n t e s t i n a l I II 660 470 native r a t i n t e s t i n a l DEAE-cellulose c 760 native E. c o l i c 790 denatured E. c o l i c 890 Omit DNA c 5 II. MgCl 2 b 866 Omit MgCl2 b 3 Add 0.2 mM EDTA b 20 III . A l l four dXTP with 1 "*C-2-dTTP with llfC-8-dATP b b 866 838 Omit three dXTP with 11*C-2-dTTP b 31 IV. 2-mercaptoethanol b 866 Omit 2-mercaptoethanol b 9 V. Glycerol 15% v/v c 794 Omit g l y c e r o l c 739 *One unit of enzyme incorporated 1 pmole 1^C-labeled t r i -phosphate into an acid-insoluble product under standard assay conditions. DEAE-cellulose fractions from a 1 x 45 cm column. - 163 -for native or denatured DNA occurs depending upon the enzymic f r a c t i o n tested. In one enzyme fr a c t i o n c, as shown i n Table XXI, the native DNA from the rat inte s t i n e or from E. c o l i was s l i g h t l y more active. A native rat i n t e s t i n a l DNA was generally used i n the standard assay procedure. In several DNA polymerase catalyzed systems (91,125)', a denatured DNA i s more active i n supporting synthesis than i s native DNA. Mantsavinos et al. (157) have investigated the primer requirements of p a r t i a l l y p u r i f i e d regenerating r at l i v e r DNA polymerase, and found that t h i s enzyme u t i l i z e s native (double-stranded) DNA much more e f f e c t i v e l y than either s i n g l e -stranded or denatured DNA as i t s primer. This greater incorpora-tion with native DNA did not depend on the source of the primer since v i r a l , b a c t e r i a l , and mammalian DNA a l l gave the same re-su l t s . In contrast, p u r i f i e d c a l f thymus DNA polymerase has an absolute requirement for single-stranded or denatured DNA primers (61,102). Calf thymus DNA polymerase cannot use poly-dAT or native DNA as primers unless traces of bovine pancreatic DNase are added to the system (65). The DNA polymerase from Landschutz ascites-tumour c e l l s (146) also preferred a thermally denatured (single-stranded) DNA as i t s primer. The reason for these d i f -ferent primer requirements for the various mammalian DNA poly-merases remains to be elucidated, (c) Ionic environment: As found i n previous DNA biosynthetic systems (61,142), a divalent metal cation i s an absolute requirement for the rat - 164 -i n t e s t i n a l DNA polymerase. A Mg concentration of 5 to 8 mM which i s i n the optimal range for several mammalian systems (61,146) was used for the i n t e s t i n a l enzymes. In the absence of added Mg + + ions, l i t t l e i f any incorporation of the lab e l l e d precursors occurred. Mg + + ions appear to give the highest re-sponse for the DNA polymerases, but Mn + + ions at a concentration of 0.8 mM showed a low a c t i v i t y i n the Walker 256 carcinosarcoma tissue of the rat (145). The DNA polymerase from regenerating ++ ++ rat l i v e r (156) was not active i n the presence of Mn or Ca ions. Furlong (145) and Shepherd and Keir (146) have previously found that the inc l u s i o n of 0.2 to 0.5 mM EDTA i n the assay medium produced an increase i n the DNA polymerase a c t i v i t y . These authors suggested that the EDTA might have removed small quantities of heavy metal cations that i n h i b i t the reaction. When the concentration of EDTA was increased to 1.2 mM, less than 10 per cent of the maximum incorporation remained. These investigators suggested that at a higher EDTA concentration, the Mg + + ions which were required by the DNA polymerase were also chelated. In the present investigation, the addition of 0.2 mM EDTA into the standard assay medium for rat i n t e s t i n a l DNA poly-merase appeared to have caused a lower incorporation of labeled precursors into an acid-insoluble product than was obtained with the control sample as shown i n Table XXI. (d) Requirement for deoxyribonucleoside 5'-triphosphates: Previous studies by Lehman et at. (63) and by Bollum (61) - 165 -have shown that a l l four deoxyribonucleoside 5.'-triphosphates (adenine, guanine, cytosine, and thymine) were required for r e p l i c a t i v e DNA synthesis in v i t r o . The mono or diphosphate forms of these nucleosides were not e f f e c t i v e for the incorpora-t i o n into DNA unless s p e c i f i c kinases were used to convert them into the triphosphate form (158). In the assay system for rat i n t e s t i n a l DNA polymerase, the four triphosphates were used i n equal amounts of about 10-16 nM each. As shown i n Table XXI, either labeled precursor, 11JC-8-dATP or 1 ltC-2-dTTP i n 1.5-2 nM was incorporated into the primer at about the same rate. As observed with other mammalian polymerases (61,156), the omission of one or more of these triphosphates resulted i n a reduction of r e p l i c a t i v e DNA polymerase a c t i v i t y . A basal l e v e l of incor-poration remains i n the presence of only one triphosphate and may be due to the a b i l i t y of the r e p l i c a t i v e enzyme to catalyze a limited end-addition reaction. The r e p l i c a t i v e enzyme from r a t i n t e s t i n a l mucosa appears to catalyze this l a t t e r a c t i v i t y when assayed i n the presence of only one nucleoside triphosphate, (e) S t a b i l i t y of the nucleotidyltransferase: Early experiments indicated that the rat i n t e s t i n a l DNA polymerase was unstable during storage at 0° C and during the incubation period of the assay system. As shown i n Table XXI, the omission of 2-mercaptoethanol i n the assay system led to a substantial loss of a c t i v i t y . These considerations led to the incl u s i o n of 2-mercaptoethanol i n the enzyme extraction medium as well as i n the assay system. In a recent study by Calvin - 166 -et al. (159), i t was shown that the addition of glycero l to the assay medium for rat t e s t i s DNA polymerase enhanced dATP incor-poration into either heat-denatured or native DNA. This e f f e c t was found to be maximal at a concentration of 15-20 per cent (v/v) g l y c e r o l . In the present work, the addition of 15 per cent glycero l into the assay medium for the rat i n t e s t i n a l DNA poly-merase gave a small increase i n the incorporation of 1 4C-dATP into a native DNA primer. 6. Terminal DNA Nucleotidyltransferase i n Nuclei of Rat  In t e s t i n a l Mucosa. Since the f i r s t description by Krakow et al. (95) of an enzyme from c a l f thymus nuclei which incorporates mononucleo-t i d y l residues onto the terminal positions of DNA, several i n -vestigators have confirmed the existence of th i s enzyme (102, 160). Considerable data have accumulated that indicate the existence of a separate nuclear and cytoplasmic (soluble) t e r -minal DNA nucleotidyltransferase from c a l f thymus gland. The soluble enzyme uses oligodeoxynucleotides as i n i t i a t o r s of polymerization (161) while the nuclear enzyme cannot u t i l i z e such primers but functions with a single-stranded DNA (143). Krakow et al. (9 5) have stated a probable association of the nuclear terminal enzyme with DNA. The cytoplasmic enzyme i s not bound to DNA and reconstitution experiments with DNA and the enzyme f a i l to form complexes (102). An absolute requirement for s u l f -hydryl compounds such as cysteine or 2-mercaptoethanol has been shown for the nuclear enzyme (95). This requirement for SH - 167 -s t a b i l a t i o n i s not observed for the soluble enzyme even though i t i s sensitive to sulfhydryl i n h i b i t o r s (97). The cytoplasmic enzyme i s b a s i c a l l y a dATP polymerase and shows a greater rate of incorporation of th i s substrate than the complementary nucleo-tides . The nuclear enzyme incorporates dATP at a rate equal to the other precursors (143). The terminal addition enzyme i n nuclei i s o l a t e d from rat i n t e s t i n a l mucosa possesses c h a r a c t e r i s t i c s which are sim i l a r to the nuclear terminal transferase of c a l f thymus gland. A charac-t e r i s t i c of the i n t e s t i n a l enzyme which was o r i g i n a l l y observed for the c a l f thymus nuclear enzyme i s that the in c l u s i o n of the complementary triphosphate i n the incubation medium causes a 50 per cent or greater i n h i b i t i o n i n the incorporation of the labeled triphosphate as shown i n Table XXII. The i n t e s t i n a l enzyme u t i l i z e s a heat denatured (single-stranded) DNA, but not a native DNA as primer. This terminal enzyme i s activated by the addition of cysteine into the incubation mixture and shows equal incorporation with 1 4C-2-dTTP or 14C-8-dATP as the single species of deoxynucleoside triphosphate. I t seems quite probable that the terminal addition enzyme from nuclei of rat i n t e s t i n a l mucosa i s functionally the same as that described by Canellakis from c a l f thymus nu c l e i . The functional nature of such an enzyme in the c e l l , however, remains to be determined. Nature of the products from the terminal addition reaction: In order to establish the location of the labeled deoxy-ribonucleotide incorporated i n the terminal addition reaction, - 168 -Table XXII Properties of terminal DNA nucleotidyltransferase i n nuclei of r a t i n t e s t i n a l mucosa Ef f e c t on the „ . £ . , , .. Enzyme DNA transferase a c t i v i t y Incubation t-, i • t-. • , , . . i. s t Fraction Enzyme units/mg protein* Deoxyribonucleoside triphosphates: a*Only aitC-2-dTTP DEAE-cellulose Add a l l 4 dXTP (1x45 cm), 10 b"Only llfC-2-dTTP DEAE-cellulose ( l x l l cm), 11 Add a l l 4 dXTP 55 7 108 35 C*0nly ^C-S-dATP DEAE-cellulose ( l x l l cm), 11 Add a l l 4 dXTP 115 34 II. DNA primer: Heat denatured Native DEAE-cellulose ( l x l l cm), 11 106 5 II I . Sulfhydryl s t a b i l i z e r s : Cysteine and 2-mercaptoethanol DEAE-cellulose ( l x l l cm), 11 Omit cysteine " Omit cysteine and 2-mercaptoethanol " 108 36 0 *0ne enzyme unit incorporates 1 pmole 1 h C - l a b e l l e d triphosphate into an acid-insoluble product under standard terminal addi-ti o n enzyme assay conditions. The enzyme fractions used were taken from DEAE-cellulose columns with the dimensions shown i n brackets and the f r a c t i o n number i s shown after the dimensions. - 169 -the DNA products were is o l a t e d and subsequently digested with snake venom phosphodiesterase as described by Adler et al. (94). This venom exonuclease s p e c i f i c a l l y cleaves nucleoside-5 1-phos-phates from the 3 1-hydroxy ends of nucleic acid polymers. The product-DNA was formed by the incorporation of single 1 "*C-dATP precursor into a heat denatured E. c o l i primer with the rat i n -t e s t i n a l terminal transferase. After i s o l a t i n g the labeled pro-ducts, they were hydrolyzed with the phosphodiesterase, and the amount of radioactive mononucleotides released as acid-soluble material was measured by a l i q u i d s c i n t i l l a t i o n procedure. As shown i n Figure 34, over 75 per cent of the 1^C-dAMP o r i g i n a l l y incorporated was released after 90 min of hydrolysis. In th i s time, only up to 10 per cent of the t o t a l DNA was rendered acid soluble as indicated by an increase i n the absorbance at 260 my. In a further experiment with the i n t e s t i n a l terminal trans-ferase, a native E. c o l i DNA was used as the primer for the i n -corporation of the precursor, 1 4C-dATP. Phosphodiesterase hy-dr o l y s i s on the recovered DNA-product showed a steady increase i n absorbance units with the time of hydrolysis. This indicated a cleavage of the DNA, but no release of r a d i o a c t i v i t y was detected i n the acid soluble fractions when counted i n the s c i n t i l l a t i o n system. These results would indicate that the i n t e s t i n a l terminal transferase was not able to use the native DNA as a primer. In consideration of the previous r e s u l t s , i t was of in t e r e s t to prepare a labeled DNA using a native primer and the r e l i c a t i v e 100 R a d i o a c t i v i t y 0 30 60 90 120 150 180 Time, Minutes Figure 34. Hydrolysis of 24C-8-dAMP-DNA by snake venom phosphodiesterase. Labeled DNA was prepared by the nuclear terminal DNA-nucleotidyltransferase a c t i v i t y on denatured DNA i n the presence of labeled dATP. Radioactivity and absorbance were measured on the acid soluble f r a c t i o n s . - 171 -DNA polymerase from rat i n t e s t i n a l mucosa. The standard incuba-t i o n system was used for the incorporation of 1 "^ C-dATP and the four unlabeled nucleoside triphosphates onto a native E. c o l i DNA template. After incubation for 30 min at 37° C, the acid insoluble DNA-product was recovered by p r e c i p i t a t i o n with cold 20 per cent TCA solution. This product was hydrolyzed with venom phsophodiesterase, and the acid soluble f r a c t i o n was measured for r a d i o a c t i v i t y . Figure 35 shows that both the ab-sorbance and the r a d i o a c t i v i t y curves increased s t e a d i l y , but less than 25 per cent of the o r i g i n a l labeled material was released after 150 min of hydrolysis. These observations indicate an i n t e r n a l d i s t r i b u t i o n of the labeled precursor into the DNA» rather than a condensation of the isotope at the 3'-hydroxy terminus. Through the use of the venom phosphodiesterase, i t was possible to show the presence of a d i s t i n c t terminal DNA nucleo-ti d y l t r a n s f e r a s e which i s d i f f e r e n t i n i t s mode of action from the r e p l i c a t i v e enzyme. This c r i t e r i o n can be used to d i f f e r e n -t i a t e the two transferases from the n u c l e i . These two enzymes can also be d i f f e r e n t i a t e d on the basis of t h e i r optimum pH values. In Tris-HCl buffer, the terminal addition enzyme from the r a t i n t e s t i n e shows a pH optimum near 7 while the i n t e s t i n a l r e p l i c a t i v e enzyme has a pH optimum at 8 i n t h i s system. As discussed i n the p u r i f i c a t i o n procedure with DEAE-cellu-lose, enzyme extracts of the i n t e s t i n a l nuclei which had been stored showed a larger proportion of the terminal addition enzyme 100 rH •P 75 -EH m o -P c 0) o 50 -M 0) CM Reaction Time, Minutes Figure 3 5 . Hydrolysis of 1^C-S-dAMP-DNA by snake venom phosphodiesterase. Labeled DNA was prepared by the;action of r e p l i c a t i v e DNA polymerase on a native DNA primer i n the presence of a l l four deoxyribonucleoside triphosphates in which the dATP was labeled t - 173 -upon chromatography. In a previous report by Keir (91), d i f f e r -ent levels of terminal nucleotidyltransferase a c t i v i t y were re-covered from various batches of c a l f thymus gland. Hayes et at. (148) have also shown that storage of c a l f thymus tissue resulted i n a greater recovery of terminal addition enzyme. Sephadex gel f i l t r a t i o n of the terminal enzyme from c a l f thymus gland shows i t i s a smaller molecule than the r e p l i c a t i v e enzyme (102). In the present work, the separation of the rat i n t e s t i n a l DNA poly-merase into at least two major peaks was observed after gel f i l -t r a t i o n on G-150 or G-200 as e a r l i e r described. The peak cor-responding to a 25,000 molecular weight protein on G-150 showed predominantly r e p l i c a t i v e a c t i v i t y with a low terminal a c t i v i t y . After G-200 f i l t r a t i o n , a s i m i l a r sized p a r t i c l e showed s l i g h t l y higher terminal than r e p l i c a t i v e a c t i v i t y . Since these observa-tions were inconsistent, i t was not possible to assign a terminal enzyme function to th i s small molecular weight f r a c t i o n . 7. Deoxyribonuclease I A c t i v i t y i n DNA Polymerase Extracts  from the I n t e s t i n a l Mucosa. Deoxyribonuclease a c t i v i t y i s generally high i n tissues which possess a vigorous growth rate and a rapid DNA synthetic mechanism. Small i n t e s t i n a l mucosa of the r a t i s one such tissue and has also been shown to contain a high DNase I content (103). The follow-ing experiments were performed to determine i f the presence of DNase I i n the DNA polymerase extracts from r a t i n t e s t i n a l mucosa was a f f e c t i n g the biosynthetic reaction. The amounts of DNase I a c t i v i t y i n the crude extract, the nuclear soluble proteins, and - 174 -the high-speed supernatant f r a c t i o n following centrifugation of a whole c e l l extract were measured. As shown i n Table XXIII, a high DNase I a c t i v i t y i s evident i n the crude homogenate prepara-ti o n . After a 10 min incubation period at 37° C, a 57 per cent increase i n absorbancy units was observed. After the mucosa c e l l s were washed i n 6% dextran, homogenized, and centrifuged at high-speed, the supernatant f r a c t i o n showed an absorbancy increase of about 8% when assayed for DNase I a c t i v i t y . An extract of the nuclei had a DNase I a c t i v i t y which corresponded to a 6% increase i n absorbancy at 260 my. The nuclear and cytoplasmic fractions which were prepared i n non-aqueous media contained DNase I a c t i v i t y corresponding to a 3 and a 11% increase i n absorbance, respectively. As previously reported, a l l these i n t e s t i n a l ex-tracts contained DNA polymerase a c t i v i t y . I t was observed, how-ever, that those fractions such as the crude homogenate which contained a high DNase I a c t i v i t y showed a low DNA polymerase a c t i v i t y . The nuclear extracts generally showed the highest DNA polymerase a c t i v i t y and probably contained the lowest amount of DNase I a c t i v i t y . These studies indicate the possible harmful ef f e c t s of a high concentration of DNase I i n crude enzyme pre-parations which were used for the biosynthesis of DNA. After DEAE-cellulose chromatography as shown i n Figure 36, the DNase I a c t i v i t y which was associated with the nuclear ex-t r a c t was eluted with the forepeak of th i s column. Further assays for DNase I a c t i v i t y on subsequent fractions showed l i t t l e i f any detectable a c t i v i t y . The usual DNA polymerase bands of - 175 -Table XXIII Deoxyribonuclease I a c t i v i t y i n rat i n t e s t i n a l mucosa preparations. Fraction Absorbancy change at 260 my % Increase i n Absorbance 1. Crude homogenate 0.375 High-speed supernatant 0.053 Soluble nuclear proteins 0.045 57.0 7.9 6.2 2. Non-aqueous extract Nuclear Cytoplasm 0.021 0 .075 3.0 11.2 3. DEAE-cellulose fractions Tubes 12 to 16 0.013 Tubes 20 to 120 0 2.0 A l l samples were incubated at 37° C for a 10 minute period. DNase I a c t i v i t y was measured according to Kunitz method (131). - 176 -50 r 6.25 DNA polymerase a c t i v i t y I4.O 30 20 10 — O — — -O— — — - o — 5.00 3.75 2.50 1.25 0 20 ho 60 80 100 Fraction Number Figure 36. Chromatography on DEAE-cellulose of a DNA polymerase extract from the nuclei of r a t i n t e s t i n a l mucosa on a similar sized column as described i n F i g . 17. The fractions were assayed for DNase I a c t i v i t y as well as for DNA polymerase a c t i v i t y . One unit of DNase I pro-duced an increase i n absorbancy of 0.001 per 0.1 ml of solution tested i n the reaction conditions as described e a r l i e r . One unit of DNA polymerase incorporated 1 pmole labeled precursor as previously described. / - 177 -a c t i v i t y were present and did not appear to be related to the nuclease a c t i v i t y . In a recent review by Lehman (16 2), arguments are given for and against the possible function of endonucleases i n relationship to DNA synthesis, however, no d i r e c t evidence has so far been given which show DNA polymerases have a d i r e c t requirement for DNases during DNA r e p l i c a t i o n . There was also no apparent re-quirement for nucleases during the action of the transferase from B a c i l l u s s u b t i l i s in v i t r o . I t was possible to remove a l l the nuclease from this DNA polymerase without impairment of the syn-t h e t i c reaction (67). - 178 -SUMMARY PART I Previous investigations i n t h i s laboratory indicated that the DNA i s o l a t e d from r a t i n t e s t i n a l tissue was metabolically as well as physically heterogeneous. Further comparative studies were made on DNA from whole c e l l s and t h e i r subfractions with regards to base compositional rela t i o n s h i p s . Nuclei and mitochondria which were used for the i s o l a t i o n of DNA were obtained from i n t e s t i n a l mucosa c e l l s which had been washed with a dextran solution according to the method of Clark and Porteous (104)'. The chromatography of DNA from whole c e l l s or nuclei on MAK was found to give one major band which was eluted with 0.6 M NaCl solution. Several smaller bands which also showed u l t r a v i o l e t absorbance at 260 my were eluted adja-cent to this main f r a c t i o n , but were not present following chromatography of a s i m i l a r extract which had been treated with ribonuclease. Although the main DNA band had separated into two or three d i s t i n c t peaks i n several of the MAK fractionations, each peak was not composed of DNA with one c h a r a c t e r i s t i c base composition but appeared to consist of several subfractions with varying guanine plus cytosine content. Generally, the DNA sample eluted i n the leading f r a c t i o n contained the highest G+C content, and t h i s decreased progresively to the l a s t DNA f r a c t i o n . This pattern of high to low G+C content was observed for the whole c e l l DNA fractions using the methods of acid hydrolysis, heat denaturation and CsCl density gradient centrifugation. Following - 179 -fra c t i o n a t i o n on MAK, the main f r a c t i o n of nuclear DNA showed a similar decrease i n G+C content from the leading to the central f r a c t i o n s , but the rear fractions of the DNA peak showed a gradual increase i n G+C composition as determined from t h e i r Tm values. The base composition of DNA fractions from whole c e l l s showed a mole G+C per cent range between 52.92 and 34.39. The G+C content of nuclear DNA fractions ranged between 50.97 and 37.31. Several of these fractions as well as the unfractionated DNA from whole c e l l s or nuclei have a G+C content of 42.2 which corresponds to a thermal denaturation midpoint of 86.6° C. An unfractionated DNA preparation from mitochondria of r a t i n t e s t i -nal mucosa showed a Tm value of approximately 85° C. This mito-chondrial DNA showed a Tm value which was similar to one of the values found for a subfraction of nuclear DNA. Further character-i z a t i o n of the mitochondrial DNA by equilibrium centrifugation i n a CsCl solution resulted i n the appearance of a d i f f u s e u l t r a -v i o l e t absorbing band which corresponded i n density to the whole c e l l u l a r DNA of 1.70 2 g/cm3. These observations might be charac-t e r i s t i c of mitochondrial DNA from r a t i n t e s t i n e , but the present results are inconclusive. PART II In i n i t i a l experiments, DNA polymerase from E. c o l i was i s o l a t e d and p a r t i a l l y p u r i f i e d . After chromatography on DEAE-c e l l u l o s e , the a c t i v i t y of t h i s enzyme was increased approxi-mately 100 times i n comparison to the s t a r t i n g preparation. - 180 -DNA polymerases from the small i n t e s t i n a l mucosa of the ra t were studied in v i t r o . Mucosa c e l l s were homogenized i n either aqueous or nonaqueous solvents, and the nuclei were separated from the cytoplasm by centrifugation. DNA polymerase a c t i v i t y was detected i n the nuclear extract and the cytoplasm using either solvent system. A greater enzyme a c t i v i t y was obtained from nuclei of i n t e s t i n a l mucosa c e l l s which have been washed with a dextran-Krebs Ringer phosphate solution before homogenization. Using suitable assay systems with 1 "*C-labeled deoxyribo-nucleoside triphosphates, a ' r e p l i c a t i v e ' and a •'terminal' DNA nucleotidyltransferase were detected i n extracts of the n u c l e i . Following centrifugation of a nuclear homogenate, a high-speed supernatant extract was found to incorporate. xl*C-2-dTTP into a native or heat denatured DNA primer i n the presence of dATP, dGTP, and dCTP. The;.-optimum pH for t h i s enzymatic reaction was 8.0 i n Tris-HCl buffer. The terminal enzyme p r e f e r e n t i a l l y incorporated single deoxyribonucleoside triphosphates onto the terminal p o s i t i o n of heat denatured, single-stranded, DNA primers. Chromatography of the nuclear extracts on DEAE-cellulose resulted i n the detection of three peaks of DNA polymerase a c t i v i t y . The f i r s t a c t i v i t y (I) was found to be associated with a forepeak of unadsorbed protein and nucleic acid material. The two further peaks were eluted with approximately 0.1 (II) and 0.2 M (III) KC1 solutions. Following storage of an enzyme extract, a greater amount of peak III a c t i v i t y appeared to have - 181 -formed. Rechromatography of a l l three peaks from the o r i g i n a l DEAE-cellulose f r a c t i o n a t i o n indicated that the peak II f r a c t i o n was composed of only ' r e p l i c a t i v e ' DNA polymerase a c t i v i t y , while the peak III f r a c t i o n appeared to also contain 'terminal-addition' enzyme a c t i v i t y . A d i s t i n c t peak of 'terminal' enzyme a c t i v i t y was detected by the rechromatography on DEAE-cellulose of the. peak I f r a c t i o n . The terminal enzyme was s t a b i l i z e d by the addition of cysteine to the incubation mixture and was most active at a pH about 7.0. Treatment of the DNA products formed i n the terminal addition reaction with snake venom phosphodi-esterase indicated that the labeled precursors were added to 3'-hydroxy terminal positions of the chains. Several methods which were used for the p u r i f i c a t i o n of DNA polymerases from nuclear extracts of i n t e s t i n a l mucosa appeared to have resulted i n less active enzyme preparations. These methods included ammonium sulfate f r a c t i o n a t i o n and hy-droxy l a p a t i t e chromatography. An investigation of the presence of DNase I a c t i v i t y i n several of the DNA polymerase preparations did not appear to indicate any functional r e l a t i o n s h i p of these two enzymes i n the in v i t r o reactions. A heterogeneous nature of the DNA polymerases from r a t i n t e s t i n a l mucosa was indicated by the appearance of three f r a c -tions of enzyme a c t i v i t y following DEAE-cellulose chromatography. Gel f i l t r a t i o n on G-150 and G-200 of nuclear extracts also showed that these polymerases varied i n molecular sizes. 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