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UBC Theses and Dissertations

Studies on brain nuclear RNA polymerase and chromatin transcription Singh, Vijendra Kumar 1972

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STUDIES ON BRAIN NUCLEAR RNA POLYMERASE AND CHROMATIN TRANSCRIPTION by VIJENDRA KUMAR SINGH B . S c , U n i v e r s i t y of Lucknow, 1964 M.Sc. , U n i v e r s i t y of Lucknow, 1966 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 t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1972 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Co lumbia , I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re ference and s tudy . I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copy ing 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 ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department o f ^\j(r^J^MJ~S^~yy^-The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8. Canada 2-7. m i ABSTRACT In order to elaborate the nature of the mechanisms c o n t r o l l i n g the t r a n s c r i p t i o n of genes i n the c e l l s of higher organisms, studies have been c a r r i e d out on b r a i n nuclear RNA polymerase and the t r a n s c r i p t i o n of b r a i n chromatin. Foremost, s u i t a b l e conditions were developed for the s o l u b i l i -z a tion of RNA polymerase i n high y i e l d s from p u r i f i e d n u c l e i of beef b r a i n . The s o l u b i l i z e d enzyme was p a r t i a l l y p u r i f i e d by ammonium sulphate f r a c t i o n a t i o n followed by DEAE-cellulose chromato-graphy. By t h i s procedure, two DNA-dependent RNA polymerase a c t i v i t i e s , designated as RNA polymerase I and RNA polymerase I I , were resolved. These were p a r t l y characterized on the basis of t h e i r d i f f e r i n g c a t a l y t i c p r o p e r t i e s . I | RNA polymerase II e x h i b i t s a p r e f e r e n t i a l requirement f o r Mn as the d i v a l e n t cation and heat-denatured DNA as the template, i s markedly stimulated by 0.2 M KC1 and s e l e c t i v e l y i n h i b i t e d by the toxin, a-amanitin. On the other hand, polymerase I prefers Mg as the d i v a l e n t cation and native DNA as the template, i s con-siderably i n h i b i t e d by 0.2 M KC1 and i s not a f f e c t e d by a-amanitin. The capacity of RNA synthesis i n v i t r o by RNA polymerase i s o -lated from b r a i n n u c l e i was markedly enhanced by polyamines such as spermidine or spermine. Spermidine exerted a much more pro-nounced e f f e c t on polymerase II than on polymerase I . Evidence i s presented suggesting that RNA polymerase II a c t i v i t y may be - i -- i i -p r e f e r e n t i a l l y stimulated by spermidine. Yeast RNA i n h i b i t e d the a c t i v i t y of polymerase II and spermidine counteracted t h i s i n h i b i t i o n almost completely, i n d i c a t i n g that spermidine may act by circumventing the p r o d u c t - i n h i b i t i o n . The product of the polymerase II r e a c t i o n sedimented at around 18 S i n a sucrose-density gradient and appeared to be a complex of the type Enzyme-DNA-RNA. The template a c t i v i t y of the i s o l a t e d b r a i n chromatin f o r b r a i n nuclear RNA polymerase II and E. c o l i RNA polymerase was l e s s than 25% than that of the pure c a l f thymus DNA. This g r e a t l y repressed template capacity of the chromatin was probably due to the a c i d -soluble chromosomal proteins. The b r a i n polymerase II was 3-4 times more a c t i v e with a c i d - t r e a t e d chromatin than pure DNA as template whereas the E. c o l i enzyme was almost equally a c t i v e with e i t h e r of these two templates. The RNA synthesised on e i t h e r native or acid-treated chromatin as template by b r a i n polymerase II was somewhat smaller i n s i z e than the RNA made on pure DNA as template. It appears that the co n t r o l of t r a n s c r i p t i o n of genes i n mam-malian c e l l s could be mediated by the m u l t i p l i c i t y of the trans-c r i p t i v e enzyme, RNA polymerase, and by the p h y s i o l o g i c a l state of the template as w e l l . - i i i -TABLE OF CONTENTS Page L i s t of Tables v i i L i s t of Figures v i i i L i s t of Abbreviations x i Acknowledgements x i i CHAPTER ONE: INTRODUCTION 1.1. General 1 1.2. Functional considerations of RNA polymerase i n the t r a n s c r i p t i o n of DNA 2 A. S t r u c t u r a l c h a r a c t e r i s t i c s of b a c t e r i a l RNA polymerase 4 B. Functional aspects of the subunit model of b a c t e r i a l RNA polymerase 6 1.3. Concepts of r e g u l a t i o n of genetic t r a n s c r i p t i o n 13 1.4. Present status of RNA polymerase i n mammalian c e l l s 17 1.5. T r a n s c r i p t i o n of mammalian chromatin 23 1.6. Polyamines and the RNA polymerase a c t i v i t y 26 1.7. The problem of t r a n s c r i p t i o n i n b r a i n 28 1.8. B i o l o g i c a l system and strategy f o r the study of RNA polymerase and chromatin template 30 - i v -Page CHAPTER TWO: MATERIALS AND METHODS 2.1. Materials 35 A. Chemicals 35 B. B i o l o g i c a l system 37 2.2. Methods 37 A. Processing of t i s s u e , the cerebral cortex 37 B. I s o l a t i o n of n u c l e i from cerebral cortex 38 C. S o l u b i l i z a t i o n and i n i t i a l f r a c t i o n a t i o n of RNA polymerase 39 D. P a r t i a l p u r i f i c a t i o n of RNA polymerase 40 a. Preparation of DEAE-cellulose b. DEAE-cellulose chromatography of the enzyme E. Measurement of RNA polymerase a c t i v i t y 42 a. RNA polymerase assay b. Measurement of r a d i o a c t i v i t y F. Preparation and other treatments of cerebral chromatin 44 G. Chemical analysis of RNA, DNA and protein 46 H. Miscellaneous methods 47 a. Analysis of contaminating nucleases b. Analysis of RNA product by sucrose-density gradient c e n t r i f u g a t i o n - V -Page CHAPTER THREE: EXPERIMENTAL RESULTS 3.1. S o l u b i l i z a t i o n and p a r t i a l p u r i f i c a t i o n of b r a i n nuclear RNA polymerase 49 A. P u r i t y of i s o l a t e d n u c l e i 49 B. S o l u b i l i z a t i o n and separation of b r a i n nuclear RNA polymerases 52 3.2. C a t a l y t i c properties of br a i n nuclear RNA poly-merases 59 A. General c h a r a c t e r i s t i c s of RNA polymerase re a c t i o n 59 B. E f f e c t of enzyme concentration 62 C. E f f e c t of pH 65 D. E f f e c t of divalent cations 65 E. E f f e c t of KC1 65 F. Template requirements 69 G. Relative incorporation of ribonucleoside triphosphates 73 H. E f f e c t of a-amanitin toxin 74 I. E f f e c t of c e r t a i n a n t i b i o t i c s 76 3.3. Polyamines and the a c t i v i t y of multiple forms of RNA polymerase from b r a i n n u c l e i 78 A. E f f e c t of spermidine on multiple a c t i v i t i e s of br a i n nuclear RNA polymerase 80 - vi -Page B. I n h i b i t i o n of polymerase II a c t i v i t y by c a l f thymus histone and yeast RNA and e f f e c t of spermidine on t h i s i n h i b i t i o n 87 C. The nature of RNA synthesized by polymerase II under the influence of spermidine 91 3.4. T r a n s c r i p t i o n of br a i n chromatin by b r a i n nuclear RNA polymerase II 94 A. Certain c h a r a c t e r i s t i c s of br a i n chromatin 97 B. Template a c t i v i t y of br a i n chromatin f o r polymerase II of b r a i n n u c l e i 101 CHAPTER FOUR: DISCUSSION AND CONCLUSIONS 4.1. M u l t i p l e forms of DNA-dependent RNA polymerase 114 4.2. Modulation of RNA polymerase a c t i v i t y by polyamines 120 4.3. T r a n s c r i p t i o n of br a i n chromatin by mammalian RNA polymerase 124 4.4. Concluding remarks 129 BIBLIOGRAPHY 134 - v i i -LIST OF TABLES Table Page The content of DNA i n the nuclear preparations of beef cerebral cortex 51 I I P a r t i a l p u r i f i c a t i o n of mul t i p l e RNA polymerases from beef b r a i n n u c l e i 57 I I I Requirements of RNA polymerase s o l u b i l i z e d from beef b r a i n n u c l e i 60 IV Treatment of the r e a c t i o n product by RNase, DNase and a l k a l i 61 VI Reaction requirements of b r a i n nuclear RNA polymerases I and II The r e l a t i v e rates of t r a n s c r i p t i o n of native and heat-denatured DNA templates by b r a i n nuclear RNA polymerases VII R e l a t i v e incorporation of nucleotides by b r a i n nuclear RNA polymerases VIII E f f e c t of polyamines on RNA polymerase II of b r a i n c e l l n u c l e i 63 72 75 79 IX The e f f e c t of spermidine on b r a i n nuclear RNA polymerase a c t i v i t y which was i n h i b i t e d by yeast RNA and c a l f thymus histone X Chemical composition of beef b r a i n chromatin XI The e f f e c t of a-amanitin and r i f a m p i c i n on br a i n nuclear RNA polymerase II and E. c o l i RNA poly-merase as d i r e c t e d by CT-DNA, chromatin and dehistonized chromatin templates 89 100 110 - v i i i -LIST OF FIGURES Figure Page 1 The low magnification electron micrographs of nuclear preparations from beef cerebral cortex 50 2 An o u t l i n e f o r the p a r t i a l p u r i f i c a t i o n of br a i n nuclear RNA polymerase 55 3 Chromatographic r e s o l u t i o n of b r a i n nuclear RNA polymerases on a DEAE-cellulose column 56 4 The e f f e c t of increasing amounts of enzyme p r o t e i n on RNA synthesis 64 5 The enzyme a c t i v i t y - pH r e l a t i o n s h i p 66 6 The e f f e c t of di v a l e n t cations on br a i n nuclear RNA polymerases 67 7 The e f f e c t of KC1 on b r a i n nuclear RNA polymerases 68 8 The e f f e c t of KC1 on b r a i n nuclear RNA polymerase I I a c t i v i t y with respect to divalent cations 70 9 The influence of KC1 on the rate of r e a c t i o n of RNA polymerase II of br a i n n u c l e i 71 10 Relat i v e t r a n s c r i p t i o n of heat-denatured and native DNA by b r a i n nuclear RNA polymerase II 74 11 The e f f e c t of a-amanitin on b r a i n nuclear RNA polymerases I and II 77 12 The e f f e c t of c e r t a i n a n t i b i o t i c s on two RNA polymerases of b r a i n n u c l e i 77 13 The influence of spermidine on two RNA polymerases of b r a i n n u c l e i 81 14 The e f f e c t of spermidine on b r a i n nuclear RNA polymerases with regard to dival e n t c a t i o n 82 15 The k i n e t i c s of spermidine stim u l a t i o n of br a i n nuclear RNA polymerase II with regard to template 84 - i x -Figure Page 16 The k i n e t i c s of spermidine sti m u l a t i o n of b r a i n nuclear RNA polymerase II with regard to template 85 17 The k i n e t i c s of spermidine sti m u l a t i o n of b r a i n nuclear RNA polymerase II with regard to template 86 18 The i n h i b i t i o n of b r a i n nuclear polymerase II a c t i v i t y by c a l f thymus histone and yeast RNA 88 19 The influence of spermidine on the histone-i n h i b i t e d RNA polymerase II a c t i v i t y 90 20 The counteracting e f f e c t of spermidine on RNA-i n h i b i t e d RNA polymerase II a c t i v i t y 92 21 Sucrose-density gradient p r o f i l e of RNA synthesized by polymerase II under the ac t i o n of spermidine 93 22 Sucrose-density gradient p r o f i l e of RNA synthesized by b r a i n nuclear RNA polymerase II 95 23 The nature of RNA synthesized by b r a i n nuclear RNA polymerase II under the influence of spermidine 96 24 An o u t l i n e for the preparation of chromatin and dehistonized chromatin from beef cerebral cortex 98 25 The UV-absorption spectra of beef b r a i n chromatin and CT-DNA 99 26 The e f f e c t of KC1 on the chromatin-templated RNA polymerase II a c t i v i t y 102 27 Template a c t i v i t y of b r a i n chromatin 103 28 Template a c t i v i t y of b r a i n dehistonized chromatin for b r a i n nuclear RNA polymerase II 105 29 Template a c t i v i t y of b r a i n dehistonized chromatin f o r E. c o l i RNA polymerase 106 - x -Figure Page 30 The r e l a t i v e rates of t r a n s c r i p t i o n of chromatin, dehistonized chromatin and CT-DNA by br a i n nuclear RNA polymerase II 107 31 The r e l a t i v e rates of t r a n s c r i p t i o n of chromatin, dehistonized chromatin and CT-DNA by E. c o l i RNA polymerase 108 32 Sucrose-density gradient p r o f i l e of RNA transcribed from chromatin, dehistonized chromatin and CT-DNA by b r a i n nuclear RNA polymerase II 112 - x i -LIST OF ABBREVIATIONS ATP Adenosine-5'-triphosphate BSA Bovine serum albumin cpm Counts per min CTP Cytidine-5'-triphosphate CT-DNA Calf thymus DNA DEAE Diethylaminoethyl DNA Deoxyribonucleic acid DNase Deoxyribonuclease DTT D i t h i o t h r e i t o l EDTA Ethylenediaminetetraacetic acid GTP Guanosine-5'-triphosphate RNA Ribonucleic a c i d RNase Ribonuclease rpm Revolutions per min SDS Sodium dodecyl sulphate TCA T r i c h l o r o a c e t i c a c i d TDG 0.05 M T r i s - H C l , pH 8.0, 0.5 mM DTT, 30% (V/V) g l y c e r o l T r i s Trishydroxymethylaminomethane UMP Uridine-5'-monophosphate UTP Uridine-5'-triphosphate - x i i -ACKNOWLEDGEMENTS The author expresses h i s sincere gratitude to Dr. S.C. Sung for h i s ent h u s i a s t i c guidance, valuable suggestions and encouraging discussions throughout t h i s work. He thanks Drs. V.J. O'Donnell and M. Smith f o r t h e i r c r i t i c a l reading of th i s t h e s i s , and Dr. Sh i r l e y Su Gillam for her constructive discussions of some parts of t h i s work. The author wishes to extend s p e c i a l thanks to Drs. P.L. and E.G. McGeer for t h e i r keen i n t e r e s t i n t h i s work. The a i d of Dr. T. H a t t o r i i n the el e c t r o n microscopic c h a r a c t e r i z a t i o n of the nuclear preparations i s g r a t e f u l l y acknowledged. Thanks are also due to Mr. Abe Benjamin f o r moral support. The author i s most pleased to acknowledge the debt he owes to Miss Joanne A l l a n f o r her excellent and s k i l l e d typing and f o r her i n i t i a t i v e i n the preparation of the manuscript of t h i s t h e s i s . This research was supported by a grant to Dr. S.C. Sung from the Medical Research Council of Canada. CHAPTER ONE: INTRODUCTION 1.1. GENERAL Genetic information, c a r r i e d i n DNA nucleotide sequences, i n l i v i n g c e l l s i s expressed by the mechanisms of " t r a n s c r i p t i o n " and " t r a n s l a t i o n " . The transmission of DNA-coded messages into RNA molecules i s known as t r a n s c r i p t i o n , whereas t r a n s l a t i o n r e s u l t s i n the formation of s p e c i f i c c e l l proteins whose proper-t i e s are determined by these molecules, i . e . DNA — > RNA — > Protein. The whole process i s most commonly known as the "Central Dogma" of molecular biology (Crick, 1958). Thus, one of the most impor-tant aspects of DNA function i s the production of RNA. Con-sequently, an understanding of the molecular mechanism of the c o n t r o l of t r a n s c r i p t i o n i s important i n d e f i n i n g c e l l u l a r pro-cesses. In the c e l l s of higher organisms, the nucleus i s the p r i n c i p a l s i t e of genetic information. A large body of evidence has shown that the biosynthesis of the major portion of c e l l u l a r RNA i s r e s t r i c t e d to the c e l l nucleus i n close a s s o c i a t i o n with DNA (Prescott, 1964). Since the protein synthesizing machinery i s l o c a l i z e d i n the cytoplasm (see Zamecnik, 1970), the RNA mole-cules d i r e c t l y involved i n p r o t e i n synthesis have to be trans-ported from the nucleus into the cytoplasm before they can mani-fe s t any function. Thus, the r e g u l a t i o n of expression of genetic - 2 -message i n mammalian c e l l s may occur at more than one l e v e l : (1) at the l e v e l of t r a n s c r i p t i o n of DNA by the s e l e c t i v e repression or a c t i v a t i o n of RNA synthesis, (2) at the l e v e l of intranuc l e a r processing and s e l e c t i v e transport of RNA mole-cules across the nuclear membrane into the cytoplasm, or (3) at the l e v e l of t r a n s l a t i o n of RNA molecules i n t o s p e c i f i c proteins i n the cytoplasm. From the viewpoint of c e l l u l a r d i f f e r e n t i a t i o n , i t i s generally believed that the c o n t r o l of gene expression i n higher organisms i s at the t r a n s c r i p t i o n a l l e v e l , and i t remains to be seen i f t r a n s l a t i o n a l c o n t r o l i s an important feature (see Watson, 1970). This thesis i s devoted to the study of regulation occur-r i n g at the l e v e l of RNA polymerase. 1.2. FUNCTIONAL CONSIDERATIONS OF RNA POLYMERASE IN THE TRANS- CRIPTION OF DNA In the c e l l , the synthesis of RNA of s p e c i f i c base compo-s i t i o n s i s catalyzed by the t r a n s c r i p t i v e enzyme, c a l l e d RNA polymerase (nucleoside triphosphate:RNA n u c l e o t i d y l transferase, EC 2.7.7.6) and proceeds on a DNA template according to the following r e a c t i o n (Scheme 1): - 3 -n. r u n. ATP L + UTP + GTP + CTP RNA Polymerase DNA Template Mn + + or Mg*4' ; ^ AMP UMP GMP n. CMP + ( i ^ + n 2 + n 3 + n 4) PP ± Scheme 1. General Reaction of DNA-Dependent RNA Polymerase RNA polymerase was f i r s t demonstrated independently by Weiss and Gladstone (1959) i n r a t l i v e r n u c l e i and Hurwitz et a l . (1960) and Stevens (1960) i n Esc h e r i c h i a c o l i . Since then i t s wide occurrence among animals, plants and micro-organisms has been shown (see Hurwitz & August, 1963). In view of i t s function, t h i s enzyme would be expected to be present ubiquitously i n nature. In b a c t e r i a , the synthesis of a l l c e l l u l a r RNA (rRNA, mRNA and tRNA) i s mediated by a s i n g l e RNA polymerase. Thus, DNA-dependent - 4 -RNA polymerase u n e q u i v o c a l l y p l a y s a c e n t r a l r o l e i n the process of g e n e t i c t r a n s c r i p t i o n . Since RNA polymerase from b a c t e r i a l sources i s a very com-plex molecule (see Burgess, 1971), i t i s important to d i s c u s s the s t r u c t u r a l and f u n c t i o n a l c h a r a c t e r i s t i c s of t h i s enzyme. A. STRUCTURAL CHARACTERISTICS OF BACTERIAL RNA POLYMERASE The b a c t e r i a l RNA polymerase i s a l a r g e molecule of 360,000-490,000 molecular weight and has a very complicated subunit s t r u c t u r e (Burgess et a l . , 1969; Z i l l i g et a l . , 1970; Burgess & T r a v e r s , 1970; Chamberlin, 1970; Burgess, 1971). The a c t i v e form contains f i v e s u b u n i t s : one beta prime subunit (S')» one beta subunit ( $ ) , one sigma subunit ( a ) , two alpha subunits (a) and one omega subunit (to), designated i n order of i n c r e a s i n g e l e c t r o p h o r e t i c m o b i l i t y . The polymerase molecule can be d i s s o c i a t e d i n t o i t s s u b u n i t s , i n the presence or absence of s u l f h y d r y l reagents l i k e g-mercaptoethanol, by treatment w i t h a v a r i e t y of denaturing agents, such as SDS, 8 M urea or guanidine h y d r o c h l o r i d e (Walter et_ a l _ . , 1968; Burgess, 1969). The subunits can be separated a n a l y t i c a l l y by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and the estimated values f o r t h e i r molecular weights are: 6* » 165,000 ± 15,000, g = 155,000 ± 15,000, a = 95,000 ± 5,000, - 5 -a = 39,000 ± 2,000 and co = 9,000 ± 2,000. The attachment of one p a r t i c u l a r subunit ( i . e . the sigma) to the other subunits i n the complete RNA polymerase molecule ( i . e . the holoenzyme = ft'Bo^oxr) i s not very f i r m as i t i s e a s i l y separated by phosphocellulose chromatography, l e a v i n g behind the minimal enzyme ( i . e . , the core enzyme = Q'^OL^U). This subunit model of RNA polymerase was o r i g i n a l l y d iscovered i n E. c o l i (Burgess e_t a l . , 1969) and now known to e x i s t i n other m i c r o b i a l systems, such as B a c i l l u s s u b t i l i s ( A v i l a jBt a l . , 1970; L o s i c k e_t a l . , 1970) and Azotobacter v i n e - l a n d i i (Krakow & von der Helm, 1970). P r e l i m i n a r y work on the sub-u n i t s t r u c t u r e of e u k a r y o t i c RNA polymerases a l s o r e v e a l s some s i m i l a r i t i e s , but a s i g m a - l i k e a c t i v i t y has not been detected (Chambon et a l . , 1970; Weaver et a l . , 1971) . R e c e n t l y , a very c a r e f u l study has r e s o l v e d some important d i f f e r e n c e s between holoenzyme and core enzyme i n response to i o n i c s t r e n g t h (Berg & Chamberlin,. 1970). These might w e l l e x p l a i n the previous confusion on the p h y s i c a l c h a r a c t e r i s t i c s of b a c t e r i a l RNA polymerase (see Richardson, 1969). I t was found that h o l o -enzyme has a sedimentation c o e f f i c i e n t ( S2Q °^ ^ a t t n e i o n i c s t r e n g t h (u) of higher than 0.1 and i t i n c r e a s e s to 23 S i f u i s l e s s than 0.1. The core enzyme e x h i b i t s an ^ v a l u e of 12.5 S at i o n i c s t r e n g t h of 0.26 and i f the i o n i c s t r e n g t h i s decreased then the S„ n value i n c r e a s e s to 44-48S. However, - 6 -both the holoenzyme and core enzyme possess a sedimentation value of as low as 9 S under c o n d i t i o n s of very high i o n i c s t r e n g t h (u = 3 . 5 ) . The sigma subunit has a S^Q w value of 4 . 5 - 5 . 0 S. The summary of the subunit model of RNA polymerase i s o u t l i n e d i n Scheme 2 . HOLOENZYME ^ SIGMA + CORE ENZYME (Complete enzyme) ( I n i t i a t i o n f a c t o r ) (Minimal enzyme) a 4 9 0 , 0 0 0 MW 9 5 , 0 0 0 MW 4 0 0 , 0 0 0 MW 1 4 . 9 S 4 . 5 S 1 2 . 5 S Scheme 2 . Subunit Model of E. c o l i RNA Polymerase B. FUNCTIONAL ASPECTS OF THE SUBUNIT MODEL OF BACTERIAL RNA  POLYMERASE Although the subunit s t r u c t u r e of RNA polymerase appears to be defin e d now, at present very l i t t l e i s known about the f u n c t i o n of i n d i v i d u a l s u b u n i t s . Undoubtedly, the sigma component i s e s s e n t i a l f o r the i n i t i a t i o n of s p e c i f i c RNA molecules. The beta prime and beta subunits may be i n v o l v e d i n the primary b i n d i n g of the enzyme to DNA - 7 -while nothing i s known about the function of alpha and omega com-ponents . Burgess e_t a l . (1969) i n i t i a l l y reported the r e v e r s i b l e separation of two f u n c t i o n a l components of E. c o l i RNA polymerase and c a l l e d them "core" enzyme and "sigma" f a c t o r . They found that the core enzyme i s v i r t u a l l y i n a c t i v e on phage T^ DNA template, but the ad d i t i o n of sigma f a c t o r stimulates t h i s a c t i v i t y . The degree of stim u l a t i o n was dependent upon the q u a l i t y of DNA template, being much higher with double-stranded T^ DNA (50-75-fold) than the c a l f thymus or denatured DNA (1.5 - 5-f o l d ) . This f i n d i n g was in t e r p r e t e d to mean that perhaps the core enzyme has l o s t the s p e c i f i c i t y while sigma f a c t o r restores i t . Various l i n e s of evidence have now established that the core enzyme transcribes DNA u n s p e c i f i c a l l y while the sigma f a c t o r i s required f o r the s p e c i f i c i n i t i a t i o n of RNA chains (Bautz et a l . , 1969; Summers & S i e g e l , 1969; Sugiura ejt al. , 1970). I t has also been shown that a f t e r chain i n i t i a t i o n sigma i s released from the DNA-enzyme complexes (Travers & Burgess, 1969) and thus may be re-used i n the s o - c a l l e d "sigma c y c l e " , as i l l u s t r a t e d i n Scheme 3. - 8 -Scheme 3. The Sigma Cycle At present, i t i s not c l e a r whether the s p e c i f i c i t y of tr a n -s c r i p t i o n i s determined by sigma f a c t o r or core enzyme or both i n a co-operative manner. Though the mode of a c t i o n of sigma subunit remains obscure, i t may be implicated i n the opening of double-stranded DNA at the promotor regions (Hinkle & Chamberlin, 1970; Ishihama et a l . , 1971). - 9 -Both beta prime and beta subunits bind to polyanionic compounds l i k e DNA, RNA, phosphocellulose and heparin i n d i c a t i n g that these p r o t e i n subunits may have template binding s i t e s . Recently, the 3' subunit has been designated as the DNA-binding component. This reasoning was based on the f i n d i n g that the separated 8 ' subunit, but not the other subunits, e f f i c i e n t l y binds to DNA and i s retained on membrane f i l t e r s under non-denaturing conditions (Sethi et a l . , 1970). On the other hand, beta appears to be the c a t a l y t i c subunit as suggested by the data on a n t i b i o t i c - r e s i s t a n c e . The a n t i b i o t i c rifamycin i n h i b i t s the i n i t i a t i o n step of RNA synthesis by binding to RNA polymerase rather than the template (Sippel & Hartmann, 1968; Wehrli et a l . , 1968; L i l l ejt a_l. , 1970). Using r a d i o a c t i v e r i f a -14 mycin ( C - l a b e l l e d ) , the s i t e of action of t h i s a n t i b i o t i c was shown to be core enzyme and not the sigma component (di Mauro ejt a l . , 1969; Wehrli & Staehelin, 19 70). Z i l l i g and h i s co-workers, based on t h e i r recent work on the r e c o n s t i t u t i o n of active enzyme from subunits i s o l a t e d from r i f a m y c i n - s e n s i t i v e and rifamycin-r e s i s t a n t mutant RNA polymerases, have c l e a r l y demonstrated that beta subunit i s the s i t e of action of rifamycin a n t i b i o t i c (Rabussay & Z i l l i g , 1969; H e i l & Z i l l i g , 1970). S i m i l a r l y , another a n t i b i o t i c , s t r e p t o l y d i g i n , which i n t e r a c t s with core RNA polymerase and prevents the elongation of RNA chains (Cassani e_t _al., 1970), has been shown to bind S subunit ( H e i l & Z i l l i g , 1970). The mechanism of action of B' and g subunits of RNA polymerase i s unknown. - 10 -Besides sigma f a c t o r , several other p r o t e i n factors have been described which influence the t r a n s c r i p t i o n r e a c t i o n markedly. These are M-, p s i - and rho-factors. M-Factor was i s o l a t e d from a high s a l t -wash of E. c o l i ribosomes (Davison et a l . , 1969). I t has a sedimen-t a t i o n value of approximately 5 S and i n crude preparations i t stimu-l a t e s the a c t i v i t y of core enzyme 30-50-fold on T^ and X phage DNA. It i s d i f f e r e n t from sigma f a c t o r and may influence a step immediately a f t e r i n i t i a t i o n of RNA chains. The l a t t e r remains to be e s t a b l i s h e d . Travers et a l . (1970) have i s o l a t e d a p r o t e i n f a c t o r from ribosome-free supernatant of E. c o l i c e l l s and c a l l e d i t p s i - f a c t o r (^) . This f a c t o r sediments around 3 S and stimulates the synthesis of ribosomal RNA s e v e r a l hundred f o l d . The involvement of p s i - f a c t o r f o r ribosomal RNA synthesis has now been questioned on the b a s i s of the f i n d i n g that an ordered and p r e f e r e n t i a l synthesis of ribosomal RNA i n v i t r o occurs without added p s i - f a c t o r (Pettijohn, 1972). In a d d i t i o n , Roberts (1969) has i s o l a t e d a p r o t e i n f a c t o r from E. c o l i c e l l s which causes the termination of RNA synthesis i n v i t r o at d i s t i n c t s i t e s on DNA template producing RNA molecules that are probably i d e n t i c a l to messenger RNA. This was c a l l e d rho-factor ( p ) . Recently, a p r o t e i n has been i s o l a t e d from phage T^-infected E. c o l i c e l l s , which i n t e r -feres with the i n i t i a t i o n of RNA chains, behaves l i k e the r i f a m y c i n a n t i b i o t i c does and appears to antagonize the a c t i o n of sigma f a c t o r (Mahadik et^ al., 1972). Although cytoplasmic factors a f f e c t i n g the a c t i v i t y of mammalian RNA polymerase have been reported, t h e i r nature, function and mode of a c t i o n remains to be elucidated (Stein & Hausen, 1970; S e i f a r t , 1970). - 11 -In summary, the enzymatic machinery of DNA t r a n s c r i p t i o n involves the following steps (Scheme 4) : Sigma Factor Core RNA Polymerase (E) Holoenzyme DNA Primary Binding of Holoenzyme to DNA Recognition of I n i t i a t i o n S i t e by Sigma Factor Nucleoside Triphosphates RNA Chain I n i t i a t i o n Sigma Factor Released and Recycled .RNA Rho Factor RNA Chain Termination Binding of Rho Factor DNA RNA RNA Chain Growth Nucleoside Triphosphates Scheme 4. The Summary of T r a n s c r i p t i o n a l Events - 12 -(1) Binding of RNA polymerase to DNA template: Free enzyme r e -v e r s i b l y binds to double-stranded DNA i n a n o n - s p e c i f i c manner. Beta prime and beta subunits are involved i n t h i s function. Certain i n h i b i t o r s , such as tRNA, heparin, high s a l t concentration and p r o f l a v i n s u l f a t e , prevent t h i s binding. This step i s s e n s i -t i v e to rifamycin. (2) I n i t i a t i o n of RNA chains: The enzyme locates a s p e c i f i c binding s i t e , perhaps a pyrimidine c l u s t e r . A highly stable com-plex i s formed at t h i s s p e c i f i c s i t e at temperatures between 15°C -37°C i n the presence of low i o n i c strength. The sigma subunit plays a key r o l e i n the r e c o g n i t i o n of the i n i t i a t i o n s i g n a l and the t r a n s c r i p t i o n begins with the incorporation of f i r s t 5'-ribo-nucleoside triphosphate, almost always a purine nucleotide. (3) Elongation of RNA chains: The f i r s t phosphodiester bond i s formed by the incorporation of the second ribonucleoside t r i p h o s -phate. The sigma f a c t o r i s released and u t i l i z e d again i n a c y c l i c manner. The RNA chains grow i n the 5' to 3' d i r e c t i o n only. (4) Termination of RNA chains: The binding of rho-factor to the DNA-enzyme complex, which i s already busy i n making RNA, terminates the growth of RNA chains. As a consequence of t h i s , free RNA molecules are released i n v i t r o , which are probably i d e n t i c a l to n a t u r a l messenger RNA molecules. - 13 -1.3. CONCEPTS OF REGULATION OF GENETIC TRANSCRIPTION Jacob and Monod (1961) put forward an elegantly simple model fo r the genetic regulation of p r o t e i n synthesis, now most popularly known as the "operon" model. In essence, the operon model contains the concept of separate and s p e c i f i c genes ( i . e . the regulatory genes) involved i n the c o n t r o l of other genes ( i . e . the s t r u c t u r a l genes). I t also states that the product of the regulatory genes i s a repressor molecule which w i l l repress the gene function by binding to a s p e c i f i c s i t e on DNA (operator) and thereby preventing the t r a n s c r i p t i o n of genes by RNA polymerase. Thus the repressor i s an e s s e n t i a l element of t h i s c o n t r o l mechanism. In f a c t , the s a l i e n t features, i . e . , the s t r u c t u r e , expression and r e g u l a t i o n of a s i n g l e operon have now been inve s t i g a t e d (see Beckwith & Zipser, 1970), and the operon model appears to be proven at l e a s t i n b a c t e r i a . In a d d i t i o n to t h i s negative c o n t r o l , the elements of a p o s i t i v e c o n t r o l system acting at the l e v e l of i n i t i a t i o n of gene t r a n s c r i p t i o n by RNA polymerase have recently been characterized (see Burgess, 1971). By considering the involvement of formally s i m i l a r mechanisms, B r i t t e n and Davidson (1969) have recently advanced a theory f o r the r e g u l a t i o n of gene a c t i v i t y i n the higher eukaryotic organisms. The key point of t h e i r proposal i s that the change i n gene a c t i v i t y during diverse states i s a consequence of concerted a c t i v a t i o n of one or more sets of "producer genes" ( s i m i l a r to s t r u c t u r a l genes). - 14 -The function of producer genes i s regulated by " a c t i v a t o r RNA" molecules which are synthesized by " i n t e g r a t o r gene" or genes ( s i m i l a r to regulatory genes). The function of i n t e g r a t o r gene(s) l i n k e d to "sensory gene" (another sequence on DNA) i s to induce the t r a n s c r i p t i o n of many producer genes i n response to a s i n g l e molecular stimulus, e.g., hormones. Since most s t i m u l i (or inducing agents ) w i l l not bind to sensory gene DNA i n a sequence-s p e c i f i c manner, an intermediary s t r u c t u r e , such as a p r o t e i n , w i l l be required. Thus, i n t h i s model, the gene r e g u l a t i o n i s accomplished by sequence-specific binding of an a c t i v a t o r RNA and not of chromosomal proteins. Although there i s no proof f o r the occurrence of t h i s model i n higher organisms, i t i s subject to experimental a n a l y s i s . These considerations suggest that there are two major ways i n which the c o n t r o l of synthesis of c e l l u l a r RNA on DNA templates can be accomplished: (1) the regulation could be mediated at the l e v e l of RNA polymerase v i a changes i n the a c t i v i t y or l e v e l of enzyme i t s e l f , or (2) the r e g u l a t i o n could occur at the l e v e l of DNA template v i a modulations i n the a v a i l a b i l i t y or s t r u c t u r e of s p e c i f i c genes (DNA). Recently,observations i n m i c r o b i a l systems have, i n f a c t , explored the p o s s i b i l i t y of r e g u l a t i o n of gene t r a n s c r i p t i o n operative at the l e v e l of RNA polymerase. The subunit s t r u c t u r e of a basic molecule of E. c o l i RNA polymerase was defined (Burgess - 15 -ejt a l . , 1969), and consequently, i t was demonstrated that the presence of s p e c i f i c p r otein factors (regulatory subunits) i s required f o r the t r a n s c r i p t i o n of s p e c i f i c genes during b a c t e r i o -phage i n f e c t i o n (Travers, 1971; Bautz et a l . , 1970; Hager et a l . , 1970). The synthesis of a completely new bacteriophage T ? -s p e c i f i c RNA polymerase has been described as another means of gene c o n t r o l i n phage ^ - i n f e c t e d E. c o l i c e l l s (Chamberlin et a l . , 1970). Phosphorylation (Martelo et a l . , 1970) and the interconversion bet-ween adenylated and non-adenylated forms (Chelala e_t al_. , 1971) of E. c o l i RNA polymerase has been reported to represent a d d i t i o n a l c o n t r o l mechanisms of DNA-dependent RNA synthesis. Although there has been no evidence for the existence of s i m i l a r regulatory mechanisms i n higher organisms, some important questions can now be r a i s e d : (1) how RNA polymerase catalyzes the synthesis of a l l types of c e l l u l a r RNA ( i . e . rRNA, mRNA and tRNA)? (2) do many stimulators of RNA synthesis (e.g. hormones) modulate the ac-t i v i t y or l e v e l of RNA polymerase? (3) i s there any c o n t r o l of l e v e l or a c t i v i t y of RNA polymerase during c e l l u l a r d i f f e r e n t i a t i o n or v i r u s i n f e c t i o n ? and f i n a l l y (4), how p r e c i s e l y the c o n t r o l of t r a n s c r i p t i o n could be mediated through RNA polymerase? Unlike b a c t e r i a and other micro-organisms, the RNA polymerase i n higher organisms i s l o c a l i s e d e x c l u s i v e l y , with the exception of mitochondria (Saccone ej: a l . , 1967; Kuntzel & Schafer, 1970; T s a i ejt al_., 1971), i n the c e l l nucleus i n very close a s s o c i a t i o n with the genetic m a t e r i a l . The chromosome of eukaryotes, as opposed to - 16 -prokaryotes, i s a highly complex structure (see Hearst & Botchan, 1970) i n which the nuclear genes (DNA) e x i s t tightly-bound to an' array of both basic as w e l l as a c i d i c proteins and RNA molecules (Bonner et a l . , 1968b). The s t r u c t u r e of chromosomal DNA i n higher organisms appears to be f u r t h e r complicated according to a recent model (Crick, 1971). One of the most prominent features of Crick's proposal (1971) i s that the s i t e s i n eukaryotic DNA which are recognized by regulator molecules are not double-stranded but com-p r i s e single-stranded regions of unwound DNA. At the present time the nature of regulatory molecules i s unknown, but i f they are RNA as has been suggested ( B r i t t e n & Davidson, 1969) then i t would be very s i g n i f i c a n t f o r the r e c o g n i t i o n s i t e s to be s i n g l e -stranded regions of DNA in. order that complementary base-pairing can take place. However, the model lacks experimental support. In view of c e l l u l a r d i f f e r e n t i a t i o n , the complex st r u c t u r e of eukaryotic genome, i t s much l a r g e r s i z e and the occurrence of "repeated" sequences i n chromosomal DNA ( B r i t t e n & Kohne, 1968), the r e g u l a t i o n of gene t r a n s c r i p t i o n i n higher organisms would appear to be a more complicated process than i s known today i n the prokaryotes. The most popular hypothesis which i s frequently used to explain the s e l e c t i v e gene t r a n s c r i p t i o n i n animal c e l l s i s the one which proposes that the synthesis of s p e c i f i c RNA molecules i s regulated by the macromolecules associated with the DNA i n the - 17 -nucleo-protein matrix (Bonner et^ a l . , 1968b; Paul et a l . , 1970). According to t h i s view, the r e g u l a t i o n i s accomplished by a general repression of RNA synthesis by histone p r o t e i n s , but the s p e c i f i c i t y of the repression i s determined by other chromosomal components, e i t h e r the a c i d i c proteins (Paul & Gilmour, 1968; Gilmour & Paul, 1970; Spelsberg & H n i l i c a , 1971) or chromosomal RNA molecules (Bekhor et a l . , 1969; Huang & Huang, 1969) inde-pendently or both i n co-operation. From the foregoing d e s c r i p t i o n i t would appear that the present knowledge about the r e g u l a t i o n of DNA t r a n s c r i p t i o n i n higher organisms i s very inadequate and, i n f a c t , does not provide any clue to the mechanisms of r e g u l a t i o n . Nevertheless, there i s a general b e l i e f among various workers i n the f i e l d that the c o n t r o l of gene t r a n s c r i p t i o n i n higher organisms i s accomplished by mechanisms which are b a s i c a l l y analogous, i f not i d e n t i c a l , to those found i n b a c t e r i a . From t h i s point of view, t h i s t h e s i s w i l l be concerned p r i m a r i l y with studies of how the a c t i v i t y of RNA poly-merase could be c o n t r o l l e d i n mammalian c e l l s . 1.4. PRESENT STATUS OF RNA POLYMERASE IN MAMMALIAN CELLS U n t i l r e c e n t l y , most studies on DNA-dependent RNA polymerase i n higher organisms have employed i s o l a t e d n u c l e i or "aggregate-enzyme" preparations. This a c t i v i t y has now been reported i n mito-chondria (Saccone et a l . , 1967; Kuntzel & Schafer, 1970; T s a i et^ - 18 -a l . , 1971). In nuclear studies, the RNA polymerase a c t i v i t y i s most frequently assayed i n the absence of exogenously added DNA template because the endogenous nucleo-protein complex of the i n t a c t n u c l e i i t s e l f serves the function of template. Goldberg (1961) reported that the RNA polymerase a c t i v i t y of "aggregate-enzyme" preparations i s stimulated i f the i o n i c strength of the r e a c t i o n mixture i s r a i s e d by ammonium sulphate. Subsequently, Windell and Tata (1964) found that the stimulatory e f f e c t of ammonium sulphate on RNA polymerase a c t i v i t y of i n t a c t I | n u c l e i i s d i f f e r e n t i a l with respect to the dival e n t cation (Mn I j or Mg ) present i n the assay mixture. They showed that ammonium sulphate stimulated RNA polymerase a c t i v i t y of i s o l a t e d r a t l i v e r I | | | n u c l e i i n the presence of Mn , but not Mg . Moreover, they found I | that the RNA synthesized i n the presence of Mn (plus ammonium sulphate) resembled DNA-like RNA (AU-rich) and that synthesized i n the presence of Mg (minus ammonium sulphate) resembled ribosomal RNA (GC-rich) i n terms of base composition and nearest-neighbour frequency (Windell & Tata, 1966). In the l i g h t of evidence that ribosomal RNA i s synthesized i n the nucleolus (Perry, 1962; Brown & Gurdon, 1964; McConkey & Hopkins, 1964; Liau et a l . , 1965) and that nucleolar preparations contain a DNA-dependent RNA polymerase (Ro ejt a_l. , 1964; Tsukada & Lieberman, 1964; Liau ejt a l . , 1965; Jacob eijt a_l. , 1968) and based on t h e i r own observations, - 19 -Windell and Tata (1966) proposed that the n u c l e i of mammalian c e l l s contain two DNA-dependent RNA polymerase a c t i v i t i e s , namely, the I | j | Mn /ammonium sulphate-stimulated a c t i v i t y and the Mg -stimulated a c t i v i t y . In support of t h i s proposal, the hi g h - r e s o l u t i o n auto-radiographic studies on the int r a n u c l e a r l o c a l i z a t i o n of these two enzymic a c t i v i t i e s demonstrated that the Mn /ammonium sulphate-stimulated r e a c t i o n which makes a more DNA-like RNA occurs p r i m a r i l y I | i n the extranucleolar region whereas the Mg -stimulated r e a c t i o n which synthesizes ribosomal type RNA takes place p r i m a r i l y i n the nucleolus (Pogo et a l . , 1967; Maul & Hamilton, 1967). In a d d i t i o n , the evidence i n favour of the above notion that the n u c l e i of animal c e l l s would contain two types of DNA-dependent RNA polymerases comes from the studies of c e r t a i n hormones which stimulate nuclear RNA synthesis, and c e r t a i n i n h i b i t o r s which ++ s e l e c t i v e l y suppress the synthesis of nuclear RNA. The Mg -stimulated RNA polymerase a c t i v i t y i s found to increase at an e a r l i e r time than does the Mn /ammonium sulphate-stimulated a c t i v i t y , as assayed i n v i t r o using rat l i v e r n u c l e i i s o l a t e d a f t e r i n vivo administration of tr i i o d o t h y r o n i n e or testosterone (Tata, 1966; Liao et_ a_l. , 1965). During a primary a c t i o n , an increase i n Mg -stimulated RNA polymerase a c t i v i t y only has been observed i n response to various other hormones, e.g., c o r t i c o s t e r o i d s (Sereni & Barnabei, 1967; Lukacs & Sekeris, 1967; Yu & Feigelson, - 20 -1971), growth hormone (Pegg & Korner, 1965; Tata, 1966; Janne & Raina, 1969), o e s t r a d i o l (Hamilton e_t a l . , 1968) and hydrocortisone (Jacob et a l . , 1969; Sajdel & Jacob, 1971). Among the i n h i b i t o r s , extremely low concentrations of a tox i n , a-amanitin, which i s a small b i c y c l i c octapeptide (M.W. 1,000) from poisonous green mush-room, Amanita phalloides (see Wieland, 1968), dramatically i n h i b i t s I | the Mn /ammonium sulphate-stimulated a c t i v i t y of i n t a c t n u c l e i I | while the Mg -stimulated a c t i v i t y i s af f e c t e d very l i t t l e or not at a l l (Stirpe & Fiume, 1967). Although ammonium sulphate may d i f f e r e n t i a l l y a f f e c t the putative nucleolar and nucleoplasmic (extranucleolar) RNA poly-merase a c t i v i t i e s of the i n t a c t n u c l e i , i t i s also l i k e l y that ammonium sulphate simply enhances the a v a i l a b i l i t y of DNA for a common enzyme by the d i s s o c i a t i o n of proteins from the endogenous nucleo-protein template. The l a t t e r p o s s i b i l i t y appears to be more l i k e l y since the e f f e c t of higher concentrations of s a l t on the Increased template a c t i v i t y of chromatin preparations have, i n f a c t , been reported (Marushige & Bonner, 1966; Breuer & F l o r i n i , 1966; Georgiev et_ a l . , 1966). Thus, i t i s impossible to r u l e out the p o s s i b i l i t y of d i f f e r e n t i a l template e f f i c i e n c y i n the presence of ammonium sulphate. - 21 -U n t i l recently, the question of whether more than one f u n c t i o n a l RNA polymerase e x i s t s i n the n u c l e i of mammalian c e l l s has remained unresolved, p r i m a r i l y due to lack of methods of s o l u b i l i z a t i o n of t h i s enzyme i n good y i e l d s . Although some workers were able to extract nuclear RNA polymerase using b u f f e r s of low i o n i c strength (Chambon et^ a l . , 1965; Furth & Ho, 1965; B a l l a r d & Williams-Ashman, 1966; Ishihama, 1967; Cunningham et a l . , 1968; Frederick et a l . , 1969; Goldberg et a l . , 1969) or high i o n i c strength ( S e i f a r t & Sekeris, 1969; Spelsberg £t a l . , 1969), the y i e l d s are r e l a t i v e l y low, and i n each case only a s i n g l e enzyme a c t i v i t y i s reported which resembles, i n c a t a l y t i c p r o p e r t i e s , Mn /ammonium sulphate-stimulated a c t i v i t y of i s o l a t e d n u c l e i . I f more than one form of RNA polymerase did e x i s t , as o r i g i n a l l y observed i n i n t a c t n u c l e i by Windell and Tata (1964), then the f a i l u r e to resolve them a f t e r s o l u b i l i z a t i o n could be due to one of the following reasons: (1) s e l e c t i v e e x t r a c t i o n methods or conditions may be required; (2) lack of s u i t a b l e a n a l y t i c a l procedures f o r s e l e c t i v e r e s o l u t i o n ; or (3) greater i n s t a b i l i t y and thus s e l e c t i v e d e t e r i o r a t i o n of one or more enzymic a c t i v i t i e s during extraction and c h a r a c t e r i s a t i o n . Taking a l l these parameters i n t o consideration, Roeder and Rutter (1969) have now described the existence of m u l t i p l e forms of DNA-dependent RNA polymerase i s o l a t e d from the n u c l e i of two eukaryotes, r a t l i v e r and sea urchin. A f t e r s o l u b i l i z a t i o n of nuclear enzyme a c t i v i t y , two major RNA polymerase a c t i v i t i e s were - 22 -resolved on a DEAE-sephadex column and were characterized based on cer-t a i n c a t a l y t i c p r o p e r t i e s : (1) RNA polymerase I - t h i s enzyme a c t i v i t y i s shown to be activ a t e d more by Mg than Mn (the Mn /Mg a c t i v i -t i e s r a t i o at t h e i r optimum concentrations i s about one), s l i g h t l y stimu-l a t e d by ammonium sulphate at low concentrations only (around 0.05 M) and prefers native DNA rather than heat-denatured DNA as template. (2) RNA polymerase II - t h i s enzyme a c t i v i t y i s a c t i v a t e d by Mn i n pre-I | | | [ | ference to Mg (the Mn /Mg a c t i v i t i e s r a t i o at t h e i r optimum con-centration i s about 3-5), g r e a t l y stimulated by ammonium sulphate at r e -l a t i v e l y higher concentrations only (around 0.15 M) and pr e f e r s heat-denatured DNA to native DNA as template. These workers substantiated t h e i r findings by showing that RNA polymerase I i s l o c a l i z e d i n the nuc-l e o l a r preparations whereas the RNA polymerase II i s of nucleoplasmic o r i g i n (Roeder & Rutter, 1970). Also, i t was found that very low con-centrations of a-amanitin toxin i n h i b i t s RNA polymerase II almost com-p l e t e l y whereas i t has no e f f e c t on RNA polymerase I ( L i n d e l l et^ a l . , 1970; Kedinger e_t a l . , 1970). In a d d i t i o n to these two RNA polymerases, a minor component c a l l e d RNA polymerase I I I has been reported i n rat l i v e r and sea urchin (Roeder & Rutter, 1969), but i t s c h a r a c t e r i z a t i o n and f u n c t i o n a l s i g n i f i c a n c e remains to be learned. S i m i l a r l y , the n u c l e i of c a l f thymus (Chambon et a l . , 1970), bovine thymus (Goldberg & Moon, 1970), Xenopus l a e v i s (Roeder et a l . , 1970; T o c c h i n i - V a l e n t i n i & Crippa, 1970), yeast (Ponta et_ a l . , 1971) and maize (St r a i n et_ a l . , 1971) have now been shown to contain more than one form of DNA-dependent RNA polymerase. - 23 -1.5. TRANSCRIPTION OF MAMMALIAN CHROMATIN In the interphase nucleus, the chromosomes appear i n t h e i r extended form, and are of great i n t e r e s t from the point of view of c e l l u l a r d i f f e r e n t i a t i o n because during t h i s period they carry out both DNA r e p l i c a t i o n and RNA synthesis. Such chromo-somes, i n morphological terms are c o l l e c t i v e l y described as "chromatin". The chromatin consists of a net-work of DNA, RNA and proteins, and thus contains the genetic material i n the nucleus of a l l eukaryotic c e l l s (Bonner et a l . , 1968b). The d i f f e r e n t i a t e d c e l l s of an organism appear to contain the same genetic information (see Gurdon, 1970), but the popu-l a t i o n s of RNA transcribed vary from one t i s s u e to another (Marushige & Bonner, 1966; Paul & Gilmour, 1966; Smith et a l . , 1969). These observations are compelling evidence of s e l e c t i v e gene t r a n s c r i p t i o n r e s u l t i n g from the o r g a n - s p e c i f i c "masking" of genes i n mammalian chromosomes (see Paul e_t a_l. , 1970). Stedman and Stedman (1950) proposed that the basic proteins of c e l l n u c l e i are gene reg u l a t o r s . This view stands as a land-mark i n the advancement of the concept of d i f f e r e n t i a l gene a c t i v i t y and the regulatory r o l e of chromosomal DNA-bound proteins. Huang and Bonner (1962) f i r s t reported that RNA synthesis i n v i t r o on chromatin template i s much lower than on an equivalent amount - 24 -of pure, p r o t e i n - f r e e DNA. They concluded that much of the DNA i n chromatin i s masked and not a v a i l a b l e for RNA synthesis. S i m i l a r observations were made by others ( A l l f r e y et a l . , 1963; Paul & Gilmour, 1966). Later on, i t was found that although the a b i l i t y of chromatin to support RNA synthesis i s g r e a t l y reduced, the f u l l template capacity of chromatin can be r e s -tored i f histone proteins are removed (Marushige & Bonner, 1966; Paul & Gilmour, 1966; Georgiev ejr a l . , 1966). Since then, a great deal of work has been done on the chemistry and biology of histone proteins (Stellwagen & Cole, 1969; DeLange & Smith, 1971; P h i l l i p s , 1971), yet t h e i r function as gene regulators i s not f u l l y understood. Present opinion, however, i s that histones might provide a mechanism of general repression, but by themselves they do not determine the s p e c i f i c i t y of genome masking (see Georgiev, 1969; A l l f r e y , 1971; E l g i n et a l . , 1971). In an attempt to provide a mechanism of s p e c i f i c i t y of gene t r a n s c r i p t i o n , s e v e r a l proposals have been made: (1) The enzymatic modifications of histone s t r u c t u r e i n -v o l v i n g group s u b s t i t u t i o n s , such as a c e t y l a t i o n and methylation ( A l l f r e y e_t a l . , 1964; Pogo et a l . , 1966; A l l f r e y , 1970) and phosphorylation (Kleinsmith e_t al_. , 1966; Langan, 1970) have been suggested to impose s e l e c t i v i t y i n the t r a n s c r i p t i o n of chromatin DNA. In view of the lack of s p e c i f i c i t y of recog-- 25 -n i t i o n of DNA regions having t h e i r histones modified, the physio-l o g i c a l r o l e of e i t h e r of these mechanisms remains to be established. (2) Nuclear polyanions, such as nuclear RNA, might f u n c t i o n as de-repressors of RNA synthesis i n a s e l e c t i v e manner (Frenster, 1965). Due to lack of experimental evidence, t h i s mechanism i s a matter of speculation only. (3) In recent years, much a t t e n t i o n has been paid to a s o - c a l l e d "non-histone f r a c t i o n " of chromatin. This f r a c t i o n contains both a c i d i c proteins and chromosomal RNA and evidently appears to mediate the t i s s u e - s p e c i f i c r e s t r i c t i o n of chromatin t r a n s c r i p t i o n , as d i s -cussed below. Based on the experiments on r e c o n s t i t u t i o n of chromatin, which has been pre-treated to d i s s o c i a t e one or the other component s e l e c t i v e l y , two opinions have emerged: ( i ) The regulatory molecules of t i s s u e - s p e c i f i c i t y of gene t r a n s c r i p t i o n might be a c i d i c proteins (Paul & Gilmour, 1968; Spelsberg & H n i l i c a , 1971). These workers have, found that the non-histone f r a c t i o n i s e s s e n t i a l f o r the re-c o n s t i t u t i o n of chromatin, with the maintenance of a high degree of f i d e l i t y of t r a n s c r i p t i o n as measured by h y b r i d i z a t i o n com-p e t i t i o n assay. ( i i ) The regulatory molecules of t i s s u e - s p e c i f i c gene t r a n s c r i p t i o n might be chromosomal RNA molecules (Bekhor et_ a l . , 1969; Huang & Huang, 1969). These researchers have found that the chromosomal RNA of the non-histone f r a c t i o n i s necessary f o r the f a i t h f u l r e c o n s t i t u t i o n of chromatin having absolute - 26 -s p e c i f i c i t y of t r a n s c r i p t i o n as determined by h y b r i d i z a t i o n com-p e t i t i o n assay. Some a d d i t i o n a l support i n favour of each of these two opinions may be obtained by the f a c t that chromosomal a c i d i c proteins (Platz et a l . , 1970; M a c G i l l i v r a y e_t a l . , 1971; Teng et a l . , 1971; Wang, 1971) as w e l l as chromosomal RNA (Mayfield & Bonner, 1971) e x h i b i t t i s s u e - s p e c i f i c i t y . In a d d i t i o n , nuclear a c i d i c proteins have been shown to stimulate the tran-s c r i p t i o n of chromatin by b a c t e r i a l RNA polymerase (Kamiyama & Wang, 1971; Teng et a l . , 1971). 1.6. POLYAMINES AND THE RNA POLYMERASE ACTIVITY In recent years, much a t t e n t i o n has been paid to i n v e s t i g a t i n g the r o l e of polyamines because they manifest numerous e f f e c t s i n a v a r i e t y of b i o l o g i c a l systems (see Bachrach, 1970; Cohen, 1971). The polyamines l i k e spermidine and spermine are non-protein n i t r o -genous bases and occur ubiquitously i n nature (Tabor & Tabor, 1964). In mammals, they are found i n the highest concentrations i n a c t i v e l y d i f f e r e n t i a t i n g t i s s u e s , such as prostate gland (Rodes & Williams-Ashman, 1964), regenerating l i v e r (Dykstra & Herbst, 1965; Raina et a l . , 1966), l a c t a t i n g mammary gland (Neish & Key, 1968), developing chick embryo (Caldarera et a l . , 1965) and developing b r a i n (Shimizu et a l . , 1965a; Pearce & Schanberg, 1969). I n t r a -c e l l u l a r l y , polyamines are d i s t r i b u t e d p r e f e r e n t i a l l y i n the nucleus, but considerable q u a n t i t i e s are also found i n the cytoplasm (Shimizu et a l . , 1965b; Stevens, 1966; Raina & Telaranta, 1967). - 27 -In a d d i t i o n to the above information, there i s increasing evidence to permit speculation that polyamines may play some r o l e i n growth processes or c e l l p r o l i f e r a t i o n by r e g u l a t i n g RNA metabolism (see Bachrach, 1970). In t h i s regard, i t i s i n t e r e s t i n g to note that there i s a very good c o r r e l a t i o n i n the changes i n RNA and polyamine l e v e l s i n many cases, such as developing chick embryo (Moruzzi et a l . , 1968), Drosophila melanogaster (Dion & Herbst, 1967), developing b r a i n (Caldarera et a l . , 1969) and developing eggs of Xenopus l a e v i s ( R u s s e l l , 1970). In a d d i t i o n , polyamines have been shown to stimulate DNA-dependent RNA poly-merase a c t i v i t y from m i c r o b i a l systems (Krakow, 1963; Abraham, 1968; Peterson et^ a l . , 1968). S i m i l a r l y , the RNA synthesizing capacity of i s o l a t e d n u c l e i of mammalian c e l l s i s also enhanced i n the presence of spermidine or spermine (MacGregor & Mahler, 1967; Caldarera ^ t a l . , 1968; B a r b i r o l i et a l . , 1971a). By v i r t u e of t h e i r h i g h l y c a t i o n i c nature, polyamines bind strongly to n u c l e i c acids (Tabor & Tabor, 1964) and thereby might influence RNA polymerase a c t i v i t y . However, t h i s remains to be established. The foregoing r e s u l t s are i n d i c a t i v e of the f a c t that polyamines could play some r o l e i n the r e g u l a t i o n of RNA syn-t h e s i s , yet t h e i r p h y s i o l o g i c a l r o l e i s unknown. In the l i g h t of the knowledge that they are present i n the mammalian c e l l nucleus, that they bind to n u c l e i c acids and that they stimulate - 28 -DNA-dependent RNA polymerase i n v i t r o , i t i s l i k e l y that they could mediate some co n t r o l i n the functioning of RNA polymerase. How t h i s i s accomplished i s not understood. Investigations on t h i s l i n e of research are also described i n t h i s t h e s i s . 1.7. THE PROBLEM OF TRANSCRIPTION IN BRAIN P h y s i o l o g i c a l l y , the b r a i n i s a unique organ and i n morpho-l o g i c a l terms i t i s a very complex and heterogeneous t i s s u e . Attempts to i n v e s t i g a t e a molecular basis of learning and memory phenomena, highly s p e c i f i c of b r a i n f u n c t i o n , have l e d to the proposal that RNA may be the engram f o r memory (Hyden, 1960 & 1967). Two l i n e s of biochemical evidence are i n favour of p o s s i b l e involvement of macromolecules i n b r a i n f u n c t i o n : (1) an increased synthesis of RNA and p r o t e i n takes place i n some regions of b r a i n during learning, and (2) the c o n s o l i d a t i o n of "long-term" memory i s impaired by i n h i b i t o r s of synthesis of RNA and p r o t e i n . However, at the present time, the c o r r e l a t i o n between behavioural phenomena and the synthesis of s p e c i f i c macromolecules i s rather feeble (see Glassman, 1969; Gaito, 1969; Becker, 1971). Since the c e l l u l a r s p e c i f i c i t y i n higher organisms i s thought to derive from t r a n s c r i p t i v e properties of nuclear chromatin (Bonner et a l . , 1968b; Paul et a l . , 1970), i t i s quite l i k e l y that the unique s t r u c t u r a l and f u n c t i o n a l c h a r a c t e r i s t i c s of the brain are r e l a t e d to the s p e c i f i c properties of nuclear - 29 -chromatin, at l e a s t , i n terms of d i s t i n c t i v e patterns of RNA syn-th e s i s . Thus, the r a d i c a l approach to sol v i n g the problem of neuroscience as r e l a t e d to molecules and behaviour w i l l depend upon the study of t r a n s c r i p t i o n a l phenomena and the r e g u l a t i o n of gene function i n nerve c e l l s . The understanding of the mechanism of gene t r a n s c r i p t i o n and i t s c o n t r o l i n b r a i n i s very i n f a n t i l e (see Mandel, 1969) and the problem s t i l l remains unexplored, possibly due to the lack of good i n v i t r o systems. U n t i l now, the i n v e s t i g a t i o n s on t h i s t o p i c i n b r a i n have employed RNA polymerase, being assayed i n i n t a c t n u c l e i (Bondy & Waelsch, 1965; Dutton & Mahler, 1968) or i n "aggregate-enzyme" preparation (Barondes, 1964). None of these RNA poly-merase preparations i s dependent on exogenous DNA because the nucleo-pr o t e i n complex by i t s e l f serves the function of template f o r RNA synthesis. Obviously, these preparations are not s u i t a b l e f o r the purpose of studying the mechanism of RNA t r a n s c r i p t i o n from DNA and i t s c o n t r o l . Therefore, the e l u c i d a t i o n of t r a n s c r i p t i o n a l events i n the b r a i n i s of the utmost importance before any corre-l a t i o n between molecules and behaviour i s a c t u a l l y conceived. This thesis i s p r i m a r i l y devoted to such an i n v e s t i g a t i o n . - 30 -1.8. BIOLOGICAL SYSTEM AND STRATEGY FOR THE STUDY OF RNA  POLYMERASE AND CHROMATIN TEMPLATE For the purpose of studying the RNA polymerase and the pos s i b l e involvement of t h i s enzyme and the chromatin template i n the c o n t r o l of RNA synthesis, beef b r a i n has been employed as the b i o l o g i c a l system. In the l i g h t of previous knowledge on mammalian RNA polymerase (Windell & Tata, 1966) , i t was con-sidered that nerve c e l l s might contain more than one f u n c t i o n a l species of RNA polymerase. In ad d i t i o n , i t was considered that the template could mediate a fundamental regulatory r o l e i n the t r a n s c r i p t i o n of genes by RNA polymerase, mainly, because the i n vivo template i n mammalian c e l l s i s a network of nuclear genes (DNA), proteins and RNA molecules rather than a naked DNA template (see Hearst & Botchan, 1970). The primary goal of t h i s work was two-fold. At f i r s t , the i s o l a t i o n of a RNA polymerase from b r a i n which w i l l be free of endogenous template so that the exogenously added DNA template can be used to study RNA synthesis i n v i t r o . Secondly, the es-tablishment of the existence of more than one RNA polymerase i n b r a i n , not i n the i n t a c t n u c l e i but i n the s o l u b i l i z e d form. The experimental approach to t h i s analysis was to obtain the complete s o l u b i l i z a t i o n of nuclear RNA polymerase a c t i v i t y , f o l -lowed by the subsequent r e s o l u t i o n of putative m u l t i p l e RNA poly-- 31 -merases by chromatographic methods. Although mitochondria also contain RNA polymerase a c t i v i t y (Saccone et a l . , 1967; Kuntzel & Schafer, 1970; T s a i et a l . , 1971), the studies to be described i n t h i s thesis have been r e s t r i c t e d to the nuclear RNA polymerase, l a r g e l y because t h i s enzyme i s l o c a l i z e d p r i m a r i l y i n the nucleus of the c e l l . While t h i s work using b r a i n t i s s u e was s t i l l i n progress, the m u l t i p l e RNA polymerases have re c e n t l y been shown i n some eukaryotic systems, such as, r a t l i v e r and sea urchin embryos (Roeder & Rutter, 1969) and thymus (Kedinger et a l . , 1970; Goldberg & Moon, 1970), but to date there i s no such report on b r a i n t i s s u e . Thus, a major part of t h i s thesis has been concerned with the a n a l y s i s of RNA polymerase i n b r a i n . The strategy f o r the study of chromatin t r a n s c r i p t i o n by a mammalian RNA polymerase, which i s also an e s s e n t i a l part of t h i s t h e s i s , i s based on sev e r a l grounds. I n v e s t i g a t i o n s , thus f a r , on the determination of t r a n s c r i p t i o n a l properties of chromatin have employed b a c t e r i a l RNA polymerases and i t has been repeatedly observed that only 5-20% of the t o t a l DNA i n chromatin i s a v a i l a b l e for t r a n s c r i p t i o n by b a c t e r i a l RNA polymerase (Marushige & Bonner, 1966; Paul & Gilmour, 1966; Tan & Miyagi, 1970). These obser-vations have implied that the remainder of the eukaryotic genome i s masked by DNA-bound macromolecules (Bonner et a l . , 1968b; Paul - 32 -et a l . , 1970). For several reasons, the data obtained so f a r from these studies are not open to d e f i n i t i v e i n t e r p r e t a t i o n . One of the most serious drawbacks of these studies i s the use of b a c t e r i a l RNA polymerase for determining the tr a n -s c r i p t i o n a l s p e c i f i c i t y of mammalian chromatin. By v i r t u e of s p e c i f i c i t y and heterogeneity i n eukaryotes, not only at c e l -l u l a r l e v e l but also at genomic l e v e l , i t i s po s s i b l e that b a c t e r i a l and mammalian RNA polymerases may e x h i b i t d i f f e r e n t s p e c i f i c i t i e s , perhaps with respect to the i n i t i a t i o n of RNA chains to be transcribed. I t i s also l i k e l y that the binding s i t e s on the DNA f o r RNA polymerases of e n t i r e l y d i f f e r e n t o r i g i n may be d i f f e r e n t or, at the same time, the s t r u c t u r a l components of chromatin (chromosomal p r o t e i n or RNA molecules) may invoke mechanisms of absolute s p e c i f i c i t y with respect to eukaryotic rather than prokaryotic RNA polymerase. There are c e r t a i n findings which might favour these considerations: ( i ) Rifamycin a n t i b i o t i c i s known to prevent b a c t e r i a l t r a n s c r i p t i o n by binding to b a c t e r i a l RNA polymerase p r o t e i n rather than DNA template, whereas i t has no e f f e c t on animal RNA polymerase (Hartmann et a l . , 1967; Wehrli et a l . , 1968). On the other hand, a-amanitin toxin i s known to i n h i b i t t r a n -s c r i p t i o n by binding to mammalian RNA polymerase rather than DNA template, but i t i s without any influence on the b a c t e r i a l - 33 -RNA polymerase a c t i v i t y (Jacob e_t a_l. , 1970). D i f f e r e n t i a l r e s -ponses of rifamycin and a-amanitin towards b a c t e r i a l and mammalian RNA polymerases could, i n f a c t , r e f l e c t some r a d i c a l d i f f e r e n c e between these two enzymic proteins. ( i i ) The DNA-RNA h y b r i d i z a t i o n assays, which were used to characterize the number and kinds of RNA molecules transcribed i n  v i t r o by b a c t e r i a l RNA polymerase from chromatin DNA, might not r e f l e c t locus s p e c i f i c i t y , because probably they are the measure of more redundant sequences present i n eukaryotic DNA ( B r i t t e n & Kohne, 1968; M e l l i & Bishop, 1969; McCarthy & Church, 1970; Kennell, 1971). Also, the p h y s i c a l c h a r a c t e r i s t i c s which govern the s p e c i f i c i t y of h y b r i d i z a t i o n assay are not c l e a r l y understood at the present time. ( i i i ) Newer data on the structure of chromatin suggest that as much as 35-50% of the DNA i n chromatin may not be masked by chromosomal proteins (Clark & F e l s e n f e l d , 1971; I t z h a k i , 1971). (iv) Just r e c e n t l y , some evidence has been presented that the rat l i v e r and m i c r o b i a l RNA polymerases bind to and t r a n s c r i b e d i f f e r e n t s i t e s on the chromatin DNA (Butterworth et a l . , 1971). (v) Some workers have now begun to cast serious doubts on the b i o l o g i c a l v a l i d i t y of those data on chromatin t r a n s c r i p t i o n which were previously obtained using b a c t e r i a l RNA polymerase (Clark & F e l s e n f e l d , 1971; Butterworth et a l . , 1971). - 34 -The foregoing r e s u l t s suggest the p o s s i b i l i t y that c e r t a i n d i s t i n c t i v e c h a r a c t e r i s t i c s of chromatin t r a n s c r i p t i o n may emerge i f mammalian RNA polymerase, instead of b a c t e r i a l enzyme, i s used. This thesis w i l l also deal with such a study. The experimental approach to t h i s type of study was to prepare chromatin from beef b r a i n , t r e a t i t to remove chromosomal proteins s e l e c t i v e l y and then compare the template capacity f o r RNA synthesis by b r a i n RNA polymerase (a homologous system) and b a c t e r i a l RNA polymerase (a heterologous system). - 35 -CHAPTER TWO: MATERIALS AND METHODS 2.1. MATERIALS A. CHEMICALS 1. Ribonucleoside Triphosphates (ATP, GTP, CTP and UTP), disodium s a l t s were purchased from Sigma or Calbiochem. Co. Stock so l u t i o n s were prepared i n d i s t i l l e d water and stored at -20°C. 3 3 2. [H]-Ribonucleoside 5 1-Triphosphates ( H-ATP, Spec. Act. 3 3 24.5 Ci/mmole; H-GTP, Spec. Act. 1 Ci/mmole; H-CTP, Spec. Act. 3 20.1 Ci/mmole; H-UTP, Spec. Act. 18-25 Ci/mmole), t e t r a l i t h i u m s a l t s i n ethanol were purchased from Schwarz BioResearch Corp., New York. These were stored at -20°C a f t e r desired d i l u t i o n s with d i s t i l l e d water. 3. Calf thymus DNA was obtained from Worthington Biochemical Corporation. Stock so l u t i o n s (1-2 mg/ml) i n 0.01 M NaCl were pre-pared by gentle s t i r r i n g at 4°C with the help for a small magnetic bar and were stored at -20°C. 4. Bovine serum albumin, c r y s t a l l i n e , was obtained from Calbiochem. 5. Yeast RNA, highly polymerized, was obtained from Worthington Biochemical Corporation. A 1 mg/ml s o l u t i o n i n d i s t i l l e d water was prepared f r e s h . - 36 -6. Calf thymus t o t a l histone f r a c t i o n was obtained from Worthington Biochemical Corporation and a f r e s h l y prepared s o l u t i o n was used. 7. 8-Mercaptoethanol was obtained from Eastman Kodak Co., New York. 8. D i t h i o t h r e i t o l was obtained from Calbiochem. 9. Spermine tetrahydrochloride and spermidine t r i h y d r o c h l o r i d e were dissol v e d i n d i s t i l l e d water j u s t before the enzyme assay. 10. Ammonium sulphate, c r y s t a l l i n e , enzyme grade was obtained from Mann Research Laboratories. 11. Sucrose, density-gradient grade, c r y s t a l l i n e , u l t r a p u r e was obtained from Mann Research Laboratories. This was s p e c i f i c a l l y used i n the analysis of RNA product by sucrose-density gradient c e n t r i f u g a t i o n . 12. DEAE-Cellulose (Cellex D) was obtained from BioRad Laboratories. 13. Ribosomal RNA was kindl y provided by Dr. K. Marushige, then at the U n i v e r s i t y of B r i t i s h Columbia, Vancouver. TM 14. Soluene -100 was obtained from Packard Instrument Co. 15. Whatman GF/C glass f i l t e r s , 2.4 cm i n diameter, were purchased from Reeve-Angel Co., New Jersey. - 37 -16. S c i n t i l l a t i o n f l u i d , a mixture of 240 g of naphthalene, 15 g of 2,5-diphenyloxazole (PPO) and 150 mg of 1,4-bis-(5-phenyl-oxazolyl-2)-benzene (POPOP) dissol v e d i n one l i t e r each of toluene, 1,4-dioxane and 95% ethanol. 17. Actinomycin D was a g i f t from Merck, Sharp & Dohme. 18. Rifamycin was purchased from Mann Research Laboratories. 19. a-amanitin was a generous g i f t from Dr. T. Wieland of the Max-Planck I n s t i t u t e , Heidelberg, Germany. 20. Pancreatic ribonuclease and deoxyribonuclease were purchased from N u t r i t i o n a l Biochemicals Co. 21. Esch e r i c h i a c o l i DNA-dependent RNA polymerase (a DEAE-c e l l u l o s e f r a c t i o n ) was kind l y supplied by Dr. S h i r l e y Su Gillam of the U n i v e r s i t y of B r i t i s h Columbia, Vancouver. 22. A l l other chemicals were reagent grade. B. BIOLOGICAL SYSTEM Beef b r a i n . This was generously supplied by the I n t e r c o n t i n e n t a l Packers Ltd., Vancouver, through the courtesy of Mr. M. Knight. 2.2. METHODS A. PROCESSING OF TISSUE, THE CEREBRAL CORTEX Beef brains were obtained fresh, packed i n i c e , from the slaughterhouse. The ce r e b r a l hemispheres were cut-off from the remaining b r a i n . These were cleaned free of dural membranes and - 38 -blood c l o t s , as f a r as p o s s i b l e , with the help of a forcep. The cerebral cortex was then r i n s e d into a large volume of i c e - c o l d 0.25 M sucrose and b l o t t e d on Whatman paper before use. B. ISOLATION OF NUCLEI FROM CEREBRAL CORTEX The i s o l a t i o n of p u r i f i e d n u c l e i from cerebral cortex was c a r r i e d out based on the method of Chauveau et a l . (1956), as modified i n the following manner. A l l experimental manipulations were done at 0-4°C. 15 g of cerebral t i s s u e was cut into small pieces with a pair of s c i s s o r s and homogenized i n 3 volumes of i c e - c o l d Medium I (2.2 M sucrose containing 1 mM MgC^) i n a glass Potter-Elvehjem homogenizer f i t t e d with a Teflon pestle (Clearance 0.006 - 0.009 inch, s i z e C, Arthur H. Thomas Co.), employing 5 to 6 up and down motor strokes, driven at powersetting #35. The homogenate was immediately mixed with 2 more volumes of the Medium I. 25 ml a l i q u o t s of t h i s homogenate were layered on top of 5 ml of Medium I contained i n a c e l l u l o s e n i t r a t e tube for the Spinco rotor SW 25.1. A f t e r 60 min c e n t r i f u g a t i o n at 22,500 rpm i n a Beckman Model L U l t r a c e n t r i f u g e , the n u c l e i sedimented at the bottom of the tube as a gelatinous mass (white to l i g h t pink i n c o l o u r ) . The n u c l e i were separated from c e l l debris and cytoplasmic components (which remain as a p e l l i c l e on the top) by decantation with the help of a spatula. The tubes were kept i n an inverted p o s i t i o n f o r - 39 -about 2 min to drain the adhering sucrose s o l u t i o n and the i n s i d e of the tubes was then wiped with c e l l u l o s e wipe-paper. The i s o l a t e d n u c l e i were e i t h e r used f r e s h or stored at 0°C fo r periods of 12-20 h. C. SOLUBILIZATION AND INITIAL FRACTIONATION OF RNA POLYMERASE The p u r i f i e d n u c l e i , which were i s o l a t e d by the method out-l i n e d above, were used for the s o l u b i l i z a t i o n of RNA polymerase. The n u c l e i were gently suspended using a Potter-Elvehjem homogenizer i n the s o l u b i l i z a t i o n medium which contained 0.05 M T r i s - H C l b u f f e r , pH 7.9, 0.01 M DTT, 0.2 M KC1 and 30% (V/V) g l y c e r o l . A f t e r about 5 min, the viscous nuclear suspension was sonicated i n 20-25 ml portions f o r a period of 90 sec at 0°C using a Branson S o n i f i e r (9 bursts of 10 sec each at 4.2 amp voltage with an i n t e r v a l of 20 sec a f t e r each burst f o r c o o l i n g ) . The nuclear sonicate was centrifuged at 127,000 x g/h i n a Spinco Rotor #50 ( i n routine work at 37,000 x g/1.5 h i n a S o r v a l l Centrifuge, Model RC-2). The p e l -l e t was discarded while the c l e a r supernatant (Fraction I) was h a l f -saturated with respect to ammonium sulphate by the drop-wise a d d i t i o n of saturated ammonium sulphate s o l u t i o n which was pre-adjusted to pH 7.9 with l i q u i d ammonia. While adding the ammonium sulphate, the s o l u t i o n was s t i r r e d slowly but constantly using a magnetic bar at 0°C. A f t e r 45 min of a d d i t i o n a l s t i r r i n g , the p r e c i p i t a t e formed was c o l l e c t e d by c e n t r i f u g a t i o n at 27,000 x g/h and sus-- 40 -pended i n TDG-buffer containing 0.05 M NH^Cl. A f t e r d i a l y s i s against the same b u f f e r f o r 3-5 h at 4°C, the non-dialyzate was c l a r i f i e d by ce n t r i f u g i n g at 27,000 x g/30 min to remove any r e s i d u a l m a t e r i a l . The d i a l y z e d supernatant (Fraction II) was e i t h e r immediately sub-jected to DEAE-cellulose chromatographic analysis or stored at -65°C f o r periods no longer than a day. Throughout t h i s procedure, e s p e c i a l l y at the s o l u b i l i z a t i o n and ammonium sulphate f r a c t i o n a t i o n steps, extra care was taken to avoid any f r o t h i n g which otherwise develops and renders lower r e -coveries of the enzyme a c t i v i t y . D. PARTIAL PURIFICATION OF RNA POLYMERASE (a) Preparation of DEAE-cellulose: Commercially obtained DEAE-cellulose (Cellex D), exchange capacity 0.84 meq/g, was thoroughly washed before use according to the i n s t r u c t i o n s of Peterson and Sober (1962). The absorbent was repeatedly used a f t e r washing with 0.5 N NaOH. Washed DEAE-cellulose was suspended i n 0.05 M T r i s - H C l , pH 8.0, decanted o f f and was re-suspended i n the e q u i l i b r a t i n g buffer (TDG-buffer containing 0.05 M NH.Cl). - 41 -(b) DEAE-cellulose chromatography of the enzyme: Columns of approximately 1 cm diameter x 20 cm dimensions were used. DEAE-cellulose s l u r r y i n the e q u i l i b r a t i n g b u f f e r was allowed to pack by g r a v i t a t i o n a l force. For f i n a l e q u i l i b r a t i o n of the column, 6-8 bed volumes of the e q u i l i b r a t i n g b u f f e r were allowed to percolate through j u s t before use. Preparations of s o l u b i l i z e d enzyme (Fraction II containing 6-10 mg protein/ml) were loaded on the column. A f t e r the enzyme s o l u t i o n had been absorbed on to the column bed, the column was washed with approximately 3 bed volumes of the e q u i l i b r a t i n g buf-f e r , followed by the e l u t i o n of enzyme r o u t i n e l y by stepwise i n -crease i n the concentration of NH^Cl i n TDG-buffer (at f i r s t , from 0.05 M to 0.15 M, then to 0.3 M, and f i n a l l y to higher than 0.3 M). In some a n a l y t i c a l work, the e l u t i o n was c a r r i e d out by a l i n e a r - g r a d i e n t of NH^Cl i n TDG-buffer, produced by mixing 100 ml each of 0.05 M NH^Cl and 0.6 M NH^Cl contained i n separate beakers (250 ml capacity) connected by a tygon p l a s t i c tube. 3 ml f r a c t i o n s were usually c o l l e c t e d at a flow rate of 30-40 ml/h. Storage of a l l enzyme f r a c t i o n s was conducted at -65°C i n the presence of higher concentrations of g l y c e r o l , 30-50% (V/V), f o r periods of no longer than 2 weeks. They were thawed at 0°C j u s t p r i o r to the enzyme preparation or enzyme assay and were u t i l i z e d usually within a week. - 42 -E. MEASUREMENT OF RNA POLYMERASE ACTIVITY The determination of RNA polymerase a c t i v i t y was based upon the o r i g i n a l r e a c t i o n of Weiss (1960), which measures the i n i t i a l r ate of incorporation of r a d i o a c t i v i t y from a l a b e l l e d ribonucleo-side triphosphate into TCA-insoluble material (RNA). (a) RNA Polymerase Assay: For the standard assay, the re a c t i o n mixture contained 30 mM Tr i s - H C l b u f f e r , pH 8.0, 3 mM MnCl 2, 6 mM MgCl 2, 10 mM B-mercapto-ethanol, 0.6 mM each of ATP, CTP and GTP, 1 uCi of [5- 3H]-UTP (Spec. Act., 18-25 Ci/mmole), 100 yg of CT-DNA and the enzyme pre-paration i n a f i n a l volume of 0.5 ml. Incubation was c a r r i e d out at 37°C f o r 20 min. The r e a c t i o n was terminated by quick quenching of the assay tubes into an ice-bath, followed by the addit i o n of 2 ml of i c e -cold 10% (W/V) TCA and thorough mixing on a Vortex mixer. E i t h e r 0.3 ml of a 2% (W/V) BSA s o l u t i o n or 0.2 ml of a 1% (W/V) yeast RNA s o l u t i o n were then added. A f t e r standing f o r 10 min on i c e , the TCA-precipitated samples were processed as described below fo r the measurement of r a d i o a c t i v i t y . (b) Measurement of R a d i o a c t i v i t y : This was c a r r i e d out eit h e r by c e n t r i f u g a t i o n or by f i l t r a t i o n methods as described below: - 43 -(i ) Centrifugation Method: TCA-precipitated samples con-t a i n i n g 0.6 mg BSA were analyzed by t h i s method. The samples were centrifuged for 4 min i n a c l i n i c a l model centrifuge. The p r e c i -p i t a t e thus c o l l e c t e d was washed 3 times with 5 ml portions of i c e -cold 5% (W/V) TCA. TCA was drained o f f by leaving the tubes i n an inverted p o s i t i o n f o r about 3 min. F i n a l l y , the p r e c i p i t a t e TM was dissol v e d i n 0.2 ml of Soluene • -100 and with the help of a Pasteur pipet i t was tr a n s f e r r e d to the counting v i a l s containing 10 ml of the s c i n t i l l a t i o n f l u i d . ( i i ) F i l t r a t i o n Method: TCA-precipitated samples containing 0.2 mg of yeast RNA were analyzed by t h i s method. The samples were f i l t e r e d through the g l a s s - f i b e r f i l t e r s (Whatman GF/C). Each assay tube was rinsed onto the f i l t e r with 5 ml of i c e - c o l d 5% (W/V) TCA. Every f i l t e r was then washed 5 times with 5 ml portions of cold 5% (W/V) TCA, a i r - d r i e d and transferred to the counting v i a l s . F i l t e r s were dispersed i n 10 ml of s c i n t i l l a t i o n TM f l u i d i n the presence of 0.3 ml Soluene -100. Both these methods of r a d i o a c t i v i t y measurement were used because p r a c t i c a l l y there was no d i f f e r e n c e ( q u a l i t a t i v e or quanti-t a t i v e ) i n the data obtained. The v i a l s were cooled overnight at 4°C and the r a d i o a c t i v i t y of the samples was counted i n a Nuclear-Chicago S c i n t i l l a t i o n Spectrometer, Model 6848. The counting e f f i c i e n c y was us u a l l y about 32%. - 44 -Unless otherwise defined, standard incubation and assay conditions were used f o r the determination of enzyme a c t i v i t y . The additions into or deletions from the standard assay system of any component i n i n d i v i d u a l experiments are given i n the legend f o r the appropriate f i g u r e or ta b l e . In order to main-t a i n the s e n s i t i v i t y of the enzyme assay, the determinations 3 were done at non-saturating concentrations of UTP, the H-l a b e l l e d precursor which was used to monitor the synthesis of RNA i n v i t r o . One u n i t of enzyme a c t i v i t y corresponds to the inc o r p o r a t i o n of one picomole of UMP i n t o RNA i n 20 min at 37°C under the stan-dard conditions f o r enzyme assay. The RNA polymerase a c t i v i t y i s expressed as the s p e c i f i c a c t i v i t y of UMP (picomoles/mg of protein) or the s p e c i f i c r a d i o -a c t i v i t y (counts per min/mg of protein) incorporated i n t o RNA. Whenever required, native CT-DNA (2 mg/ml i n 0.01 M NaCl) was heat-denatured, j u s t p r i o r to the enzyme assay, i n b o i l i n g water f o r 10 min and was quickly quenched i n i c e . F. PREPARATION AND OTHER TREATMENTS OF CEREBRAL CHROMATIN Chromatin was prepared at 0 -4°C by a method modified from that of Clark and F e l s e n f e l d (1971). The p u r i f i e d n u c l e i , i s o l a t e d from cerebral cortex, were gently homogenized i n EDTA-buffer (0.02 M EDTA-Na 0.08 M NaCl and 0.005 M T r i s - H C l , f i n a l pH 5 . 4 ) . The - 45 -nuclear homogenate was centrifuged at 9,750 x g/10 min and the p e l l e t so c o l l e c t e d was washed twice with EDTA-buffer, followed by two a d d i t i o n a l washings with 0.02 M T r i s - H C l , pH 8.0. The f i n a l gelatinous p e l l e t was suspended i n 0.02 M T r i s - H C l , pH 8.0 and was sheared i n a Branson S o n i f i e r f o r 1.5 min at 0°C (9 bursts of 10 sec each at 3 amp voltage with an i n t e r v a l of 20 sec a f t e r every burst f o r c o o l i n g ) . The sonicated s o l u t i o n was centrifuged at 12,100 x g/30 min and the r e s u l t i n g supernate constituted the f i n a l chromatin product. The chromatin, stored at 0°C, was used to study the template a c t i v i t y w i t h i n 24 h a f t e r preparation. Removal of histones from cerebral chromatin was c a r r i e d out at 0°C by adding 0.25 ml of i c e - c o l d 2 N H^O^/ml of chromatin s o l u t i o n ( f i n a l concentration of H^SO^ D e i n g 0*4 N ) • The s o l u t i o n was thoroughly mixed on a Vortex mixer, allowed to stand at 0°C for 30 min with occasional mixing and then centrifuged at 27,000 x g/20 min. The p e l l e t thus obtained was washed with cold d i s -t i l l e d water using a glass rod and was suspended by gentle homo-genization i n 0.02 M T r i s - H C l , pH 8.0. This m a t e r i a l , prepared fresh was used as the dehistonized chromatin template. The d e - p r o t e i n i z a t i o n of cerebral chromatin was based on the method of Marushige and Dixon (1969). - 46 -G. CHEMICAL ANALYSIS OF RNA, DNA AND PROTEIN The separation of RNA, DNA and protein into i n d i v i d u a l f r a c t i o n s was c a r r i e d out according to the method of Schneider (1957). An ali q u o t of the test sample was p r e c i p i t a t e d at 0°C with TCA ( f i n a l concentration 5% W/V), washed twice with 5% (W/V) TCA and once with 95% ethanol (5 ml portions each time). Insoluble p r e c i p i t a t e was diss o l v e d i n 2 ml of 0.2 N KOH and l e f t overnight at 37°C. 0.05 ml of 70% p e r c h l o r i c acid was added while the sample was pre-cooled to 0°C. The supernatant obtained by c e n t r i -fugation i n a c l i n i c a l model centrifuge was used f o r RNA deter-mination by o r c i n o l r e a c t i o n (Schneider, 1957). The residue was washed once with 5% (W/V) TCA and extracted i n 5% (W/V) TCA at 90°C f o r 15 min. DNA i n the extract was determined by diphenyl-amine r e a c t i o n (Schneider, 1957). The f i n a l p e l l e t was washed once with 95% ethanol, diss o l v e d i n d i l u t e a l k a l i and was used for p r o t e i n determination by the method of Lowry et a l . (1951). Yeast RNA, CT-DNA ( i n 5% TCA) and bovine serum albumin were employed as the standards for the determination of RNA, DNA and p r o t e i n , r e s p e c t i v e l y . For the measurement of small q u a n t i t i e s of RNA, DNA and protein i n the samples, a l i q u o t s were pipetted out without going through the above e x t r a c t i o n procedure. - 47 -The determination of basic.proteins (histones) was c a r r i e d out i n the following manner: An a l i q u o t of chromatin f r a c t i o n was separately treated with H^SO^ (0.4 N) and the acid-soluble f r a c t i o n was mixed with 3 volumes of 95% ethanol and l e f t overnight at -20°C. The p r e c i p i t a t e obtained by c e n t r i f u g a t i o n at 27,000 x g/20 min was d i s s o l v e d i n d i l u t e a l k a l i and the content of histone proteins was determined by the method of Lowry e_t a l . (1951) . The absorbance at 260 nm f o r n u c l e i c acids and 280 nm for proteins was followed f o r the purpose of q u a l i t a t i v e a n a l y s i s . H. MISCELLANEOUS METHODS (a) Analysis of Contaminating Nucleases: The a c t i v i t i e s of ribonuclease and deoxyribonuclease enzymes were assayed by measuring the release of acid-soluble material i n 0.7 N p e r c h l o r i c acid according to Rosenbluth and Sung (1969). (b) Analysis of RNA Product by Sucrose-Density Gradient  C e n t r i f u g a t i o n A l l enzyme assays f o r the purpose of RNA product a n a l y s i s by sucrose-density gradient c e n t r i f u g a t i o n were c a r r i e d out f o r 60 min i n the presence of 3-fold higher r a d i o a c t i v e concentration of 3 H-UTP ( i . e . , 3 yCi/assay instead of 1 uCi/assay i n the standard r e a c t i o n mixture). - 48 -At the end of the incubation, the assay tubes were im-mediately cooled i n i c e and 0.1 ml of c a r r i e r UTP (20 mM s o l u t i o n ) was added. Whenever treated with SDS, 0.1 ml of a 1% (W/V) SDS s o l u t i o n was added, followed by incubation at 37°C for 5 min. A 0.4 ml a l i q u o t of t h i s assay mixture was c a r e f u l l y layered on top of a l i n e a r gradient of sucrose, pre-cooled to 4°C. The sucrose-gradients were prepared (using a Density-Gradient s e d i -mentation system of Buchler Instruments Inc., New Jersey) by mixing 2.3 ml each of 5% (W/V) and 20% (W/V) sucrose s o l u t i o n s i n 0.01 M T r i s - H C l , pH 8.0 containing 0.1 M NH^Cl. A f t e r c e n t r i -fugation at 39,000 rpm/5 h i n a Spinco Rotor #SW 39, f r a c t i o n s of 15 drops were c o l l e c t e d by p i e r c i n g with a hypodermic needle from the bottom of the c e l l u l o s e n i t r a t e tube. Fractions were made TCA-insoluble i n the presence of 0.2 mg of yeast RNA as the c a r r i e r and were processed f o r the measurement of r a d i o a c t i v i t y according to the f i l t r a t i o n method (see Chapter 2.2 E, b). Unlabelled r a t l i v e r RNA was used as a reference. A f t e r the centrifuge run, f r a c t i o n s of 15 drops were d i l u t e d with 3 ml portions of d i s t i l l e d water and the absorbance at 260 nm was read i n a Beckman Model DU Spectrophotometer. - 49 -CHAPTER THREE: EXPERIMENTAL RESULTS 3.1. SOLUBILIZATION AND PARTIAL PURIFICATION OF BRAIN NUCLEAR RNA  POLYMERASE A. PURITY OF ISOLATED NUCLEI The nuclear p e l l e t , i s o l a t e d according to the method o u t l i n e d i n Chapter 2.2B, contained highly p u r i f i e d and i n t a c t n u c l e i from both neuronal and g l i a l c e l l populations of cerebral t i s s u e ( F i g . IA). The c r i t e r i a used to define the types of n u c l e i from beef cerebral cortex are those described by others (Sporn et_ a l . , 1962; Lovtrup-Rein & McEwen, 1966; Kato & Kurokawa, 1967; Rappoport et a l . , 1969). The neuronal n u c l e i are considerably l a r g e r i n s i z e than the g l i a l n u c l e i , and display a l i g h t background of the nucleoplasmic chromatin with a prominent nucleolus. The n u c l e i from g l i a l c e l l s have denser nucleoplasm without apparent n u c l e o l i . Some of the important mor-phological features of nuclear s t r u c t u r e , such as the double-layered nuclear envelope, nucleoplasmic d i f f u s e d chromatin and a prominent nucleolus, were v i s u a l i z e d c l e a r l y i n a t y p i c a l neuronal nucleus (F i g . IB). As judged by low magnification e l e c t r o n microscopy, there was no contamination of whole c e l l or any other cytoplasmic organelles, f o r example, the mitochondria. As shown i n Table I, the y i e l d s of nuclear DNA and RNA, on the average, were 402 ug/g wet tissue and 98 ug/g wet t i s s u e , r e s p e c t i v e l y . The RNA to DNA - 50 -FIG. 1. THE LOW MAGNIFICATION ELECTRON MICROGRAPHS OF NUCLEAR PREPARATIONS FROM BEEF CEREBRAL CORTEX (A) Showing n u c l e i from two d i f f e r e n t c e l l p o p u l a t i o n s o f b r a i n , namely, t h e s m a l l e r g l i a l - c e l l n u c l e i (GN) and the l a r g e r n e u r o n a l - c e l l n u c l e i (NN). x 9,710. (B) Showing t h e s a l i e n t f e a t u r e s o f n u c l e a r s t r u c t u r e i n a t y p i c a l n e u r o n a l n u c l e u s , i . e . t h e d o u b l e -l a y e r e d n u c l e a r e n v e l o p e (NE), n u c l e o p l a s m i c c h r o -m a t i n (Np) and n u c l e o l u s ( N c ) . x 15,048. F r e s h l y p r e p a r e d n u c l e i were embedded i n 2% a g a r , s l i c e d and f i x e d i n 4% g l u t a r a l d e h y d e i n 0.1 M p h o s p h a t e b u f f e r , pH 7.5, and were p o s t - f i x e d i n 1% OsO, i n t h e same b u f f e r . The s e c t i o n s o f 60-90 mp 4 t h i c k n e s s were s t a i n e d w i t h u r a n y l magnesium a c e t a t e and l e a d c i t r a t e . The p h o t o g r a p h s were t a k e n u s i n g a P h i l i p s 300 e l e c t r o n m i c r o s c o p e . - 51 -TABLE I . THE CONTENT OF DNA AND RNA IN THE NUCLEAR PREPARATIONS OF BEEF CEREBRAL CORTEX RNA (ug/g wet t i s s u e ) DNA (ug/g wet t i s s u e ) Mass Ra t i o s ^ D N A 82.4 380 0.217 104.0 376 0.276 98.0 370 0.265 100.0 448 0.223 110.0 470 0.234 96.0 370 0.259 * Mean: 98.4 402.3 0.245 S i x independent determinations were conducted i n d u p l i c a t e as described i n Methods (Chapter 2.2E, a) - 52 -mass r a t i o of approximately 0.245 i s of the same order as that described by others f o r the p u r i f i e d nuclear f r a c t i o n s from the br a i n of an adult animal (Sporn et a l . , 1962; Lovtrup-Rein & McEwen, 1966; Balazs & Cocks, 1967). I t should be noted that a lower mass r a t i o of RNA to DNA i s biochemical evidence f o r f r e e -dom of i s o l a t e d n u c l e i from contaminating cytoplasmic RNA (see . Wang, 1967; Busch, 1967). B. SOLUBILIZATION AND SEPARATION OF BRAIN NUCLEAR RNA POLYMERASES RNA polymerase from various b a c t e r i a l sources i s s o l u b i l i z e d simply by mechanical d i s r u p t i o n of c e l l s i n the presence of low i o n i c strength b u f f e r (Chamberlin & Berg, 1962). The s a t i s f a c t o r y and consistent methods of p u r i f i c a t i o n of b a c t e r i a l RNA polymerase, which y i e l d s i g n i f i c a n t q u a n t i t i e s of the highly p u r i f i e d enzyme ( i . e . the y i e l d s of 100-400 mg of enzyme per kg of wet c e l l s and p u r i t i e s above 95%), have been described ( Z i l l i g ej: a l . , 1966; Burgess, 1969; Berg et a l . , 1971). Similar means of s o l u b i l i z i n g the nuclear RNA polymerase from mammalian sources have not been s u c c e s s f u l , p o s s i b l y because nuclear enzyme i s firmly-bound to the in s o l u b l e nucleoprotein matrix (Weiss, 1960). This i s one of the major reasons that the progress i n the f i e l d of mammalian RNA polymerase has been very slow. In recent years, various attempts to s o l u b i l i z e the nuclear RNA polymerase have included gentle homogenization, l y s i s or s o n i -cation e i t h e r i n i s o t o n i c buffer s o l u t i o n s around neutral pH or - 53 -i n b uffers of high i o n i c strength i n s l i g h t l y a l k a l i n e pH range. The necessity f o r a v a r i e t y of conditions required to extract nuclear enzyme might be due to the d i f f e r e n c e i n mammalian sources used. We f a i l e d to extract RNA polymerase from beef b r a i n n u c l e i by gentle homogenization or l y s i s i n i s o t o n i c b u f f e r s o l u t i o n s . Preliminary success i n s o l u b i l i z i n g b r a i n nuclear RNA polymerase was obtained by s o n i c a t i o n i n a b u f f e r of low i o n i c strength containing higher concentrations of g l y c e r o l (Singh & Sung, 1970, 1971a). Subsequently, i t was found that more enzyme can be extracted by homogenizing the r e s i d u a l m a t e r i a l i n Tris-OH, pH 10.4 (Singh & Sung, 1971b) or more e f f e c t i v e l y i n 0.2 M KC1 contained i n the s o l u b i l i z a t i o n medium. The replacement of KC1 by (NH^.^SO^,. which has been u t i l i z e d f o r the purpose of s o l u b i l i z a t i o n of r a t l i v e r or sea urchin enzyme (Roeder & Rutter, 1969) or c a l f thymus enzyme (Kedinger et a l . , 1970), di d not s o l u b i l i z e the RNA poly-merase a c t i v i t y from b r a i n n u c l e i . Approximately, 80% of the nuclear RNA polymerase was s o l u b i l i z e d by s o n i c a t i o n i n the presence of 0.2 M KC1 dissolved i n the s o l u b i l i z a t i o n medium as given i n Chapter 2.2. From the point of view of c a l c u l a t i n g the recoveries of s o l u b i l i z e d enzyme, i t should be noted that the extracted enzyme i s almost completely dependent on the a d d i t i o n of exogenous DNA whereas the a c t i v i t y of nuclear suspension i s not, because the endogenous nucleo-protein complex serves the function of template. The d i f f e r e n c e i n template function may influence the - 54 -y i e l d of soluble enzyme markedly. Therefore, the expression of recovery of RNA polymerase a c t i v i t y s o l u b i l i z e d from i s o l a t e d b r a i n n u c l e i i s i n r e l a t i v e terms only. The s o l u b i l i z e d RNA polymerase was p a r t i a l l y p u r i f i e d by ammonium sulphate f r a c t i o n a t i o n and DEAE-cellulose chromatography, as ou t l i n e d i n Figure 2. Two peaks of RNA polymerase a c t i v i t y were resolved on a DEAE-cellulose column. The f i r s t peak of en-zymic a c t i v i t y was eluted i n the flow-through (at 0.05 M NH^Cl concentration) at the end of pro t e i n peak followed by a second peak of enzymic a c t i v i t y eluted at 0.3 M NH^Cl concentration ( F i g . 3). These peaks of polymerase a c t i v i t y , which were characterized by c e r t a i n c a t a l y t i c properties (to be des-cribed i n the next s e c t i o n of t h i s Chapter), were termed RNA polymerase I and polymerase I I , r e s p e c t i v e l y , i n conformation of the terminology developed for mammalian RNA polymerases (Roeder & Rutter, 1969). The data obtained from a t y p i c a l experiment on the p a r t i a l p u r i f i c a t i o n of b r a i n nuclear RNA polymerases are summarized i n Table I I . Although there was no s i g n i f i c a n t i n -crease i n the s p e c i f i c a c t i v i t y of sonicated extract on ammonium sulphate f r a c t i o n a t i o n , t h i s step has the merit of concentrating the r e l a t i v e l y large volumes of soluble enzyme. The chromato-graphy on a DEAE-cellulose column r e s u l t e d i n an approximately 12-fold increase i n the s p e c i f i c a c t i v i t y of RNA polymerase II while the s p e c i f i c a c t i v i t y of RNA polymerase I remained without - 55 -FIG. 2. AN OUTLINE FOR THE PARTIAL PURIFICATION OF BRAIN NUCLEAR RNA POLYMERASE P u r i f i e d N u c l e i S o n i c a t i o n i n s o l u b i l i z a t i o n medium at 0°C/90 sec 125,000 x g/60 min OR 37,000 x g/90 min Sonicated E x t r a c t P e l l e t ( d i s c a r d ) (NH^) 2SO^ F r a c t i o n a t i o n (0-50% s a t u r a t i o n ) 27,000 x g/60 min P r e c i p i t a t e Supernatant ( d i s c a r d ) D i a l y s i s a g a i n s t 0.05 M NH^Cl i n TDG-buffer DEAE-Cellulose Chromatography RNA Polymerase I and I I (The d e t a i l s of each step are given i n Chapter 2.2) - 56 -FIG. 3. CHROMATOGRAPHIC RESOLUTION OF BRAIN NUCLEAR RNA POLYMERASES ON A DEAE-CELLULOSE COLUMN About 56 mg p r o t e i n of the ammonium sulphate f r a c t i o n (0-50% s a t u r a t i o n ) , which has been dialyzed against the e q u i l i b r a t i n g b u f f e r , was loaded on a DEAE-cellulose column (1.2 x 18 cm). A f t e r washing with one column volume of e q u i l i b r a t i n g buffer, the e l u t i o n was accom-pl i s h e d by a l i n e a r gradient of NH^Cl (0.05 - 0.6 M) dissol v e d i n TDG-buffer, the t o t a l volume being 200 ml. Fractions of 3.5 ml each were c o l l e c t e d . Aliquots of 0.1 ml were assayed for RNA polymerase a c t i v i t y with native CT-DNA as template under the conditions given i n Methods (Chapter 2.2E, a). (o o) Absorbance at 280 nm; (• •) RNA polymerase a c t i v i t y expressed as the r a d i o a c t i v i t y 3 (cpm) of H-UMP incorporated; ( ) NH^Cl concen-t r a t i o n gradient. TABLE I I . PARTIAL PURIFICATION OF MULTIPLE RNA POLYMERASES FROM BEEF BRAIN NUCLEI Fractions T o t a l Protein T o t a l A c t i v i t y A c t i v i t ^ S p e c i f i c A c t i v i t y Fold (mg) (Units) C ^ y (Units/mg) P u r i f i c a t i o n Sonicated Extract* Ammonium Sulphate 141.75 56.78 124.34 99.45 100 80 0.875 1.75 1 2 DEAE-Cellulose** Chromatography (a) Polymerase I (b) Polymerase II 6.07 3.40 11.08 36.06 8.9 29 1.82 10.59 2.08 12.1 * The high-speed supernatant, obtained from the n u c l e i i s o l a t e d from 120 g wet b r a i n t i s s u e , constituted the sonicated extract. ** Most active fractions from a DEAE-cellulose column (1.2 x 19 cm) are included i n t h i s Table. - 58 -any s i g n i f i c a n t p u r i f i c a t i o n . I t should be considered that the s p e c i f i c a c t i v i t y of sonicated extract i s predominantly due to polymerase II and hence the DEAE-cellulose chromatography does not b r i n g about any s i g n i f i c a n t increase i n the s p e c i f i c a c t i v i t y of polymerase I. The d e t e r i o r a t i o n of polymerase I a c t i v i t y during the p u r i f i c a t i o n procedure may also a f f e c t the s p e c i f i c a c t i v i t y of this enzyme. Usually, the y i e l d of t o t a l enzyme a c t i v i t y recovered from the DEAE-cellulose column was about 90% of that of the f r a c t i o n charged on to i t . The RNA polymerase a c t i v i t y of sonicated extract as w e l l as that of chromatographed f r a c t i o n s was very unstable and thus could not be preserved f o r longer periods. The addition of substrates ( r i b o -nucleoside triphosphates) did not prevent loss of enzyme a c t i v i t y . Native c a l f thymus DNA (0.1 mg/ml) and bovine serum albumin (0.1 mg/ ml) were somewhat e f f e c t i v e i n the s t a b i l i z a t i o n of the enzyme a c t i v i t y . The d e t e r i o r a t i o n of the enzyme a c t i v i t y was prevented most e f f e c t i v e l y by high concentrations of g l y c e r o l (higher than 25%) i f the enzyme f r a c t i o n s are stored at -65°C. Under these con-d i t i o n s , the sonicated extract could be stored f o r about 2 weeks without any s i g n i f i c a n t loss of enzyme a c t i v i t y , about 65-75% of RNA polymerase II a c t i v i t y could be preserved f o r about 10 days and only 50% of RNA polymerase I was detectable a f t e r about 2 days of storage. The r a t i o of absorbance at 280 nm to 260 nm i n the enzy-ma t i c a l l y most act i v e f r a c t i o n s was usually around 1.0, i n d i c a t i n g the presence of some n u c l e i c acid material as the contamination. - 59 -On the other hand, there was no detectable contaminating a c t i v i t y of ribonuclease or deoxyribonuclease as determined at pH 8.0, the pH value at which the br a i n nuclear RNA polymerase a c t i v i t y i s maximally assayed. 3.2. CATALYTIC PROPERTIES OF BRAIN NUCLEAR RNA POLYMERASES A. GENERAL CHARACTERISTICS OF RNA POLYMERASE REACTION The r e s u l t s summarized i n Tables I II and IV e s t a b l i s h that the assay f o r RNA polymerase a c t i v i t y , which has been r o u t i n e l y used i n t h i s work, measures the synthesis of RNA as d i r e c t e d by DNA template. This conclusion i s supported by the f a c t that the s o l u b i l i z e d RNA polymerase from beef b r a i n n u c l e i requires the I | | | presence of bu f f e r , a divalent c a t i o n (Mn or Mg ), a s u l f h y d r y l reagent (8-mercaptoethanol), a l l four ribonucleoside triphosphates (ATP, UTP, CTP and GTP) and DNA. The omission of any of these components from the complete r e a c t i o n mixture s i g n i f i c a n t l y de-3 creased the incorporation of H-UTP int o TCA-insoluble p r e c i p i t a t e . Moreover, t h i s i ncorporation was considerably i n h i b i t e d (up to the extent of 65%) by the presence of added pancreatic DNase and actinomycin D (Table I I I ) . The i n h i b i t i o n by deoxyribonuclease i s r e l a t e d with the h y d r o l y s i s of the DNA template whereas a c t i n o -mycin D has been shown to bind to c e r t a i n regions on the DNA tem-pla t e and thereby prevent the RNA synthesis (see Goldberg & Friedman, 1971). These data c l e a r l y e s t a b l i s h that the RNA polymerase a c t i v i t y - 60 -TABLE I I I . REQUIREMENTS OF RNA POLYMERASE SOLUBILIZED FROM BEEF BRAIN NUCLEI Reaction mixture UMP i n c o r p o r a t e d i n t o RNA pmoles/mg p r o t e i n % of c o n t r o l Complete* 1.560 100 - Mn^, Mg"" 0.575 37 - ATP 0.448 28 - ATP, CTP, GTP 0.264 17 - DNA 0.268 17 - enzyme 0.043 <3 - g-mercaptoethanol 1.220 78 + DNase (25 ug/ml) 0.645 41 + Actinomycin D (2.5 ug/ml) 0.545 35 * The complete r e a c t i o n mixture was the same as des c r i b e d i n Methods (Chapter 2.2E, a ) . Incubation was c a r r i e d out at 37°C f o r 20 min. - 61 -TABLE IV. TREATMENT OF THE REACTION PRODUCT BY RNase, DNase AND ALKALI Ac i d - i n s o l u b l e % of c o n t r o l t i o n s * cpm Control 2042 100 Control + None 2758 136 Control + RNase (25 yg/ml) 1381 68 Control + DNase (25 yg/ml) 2071 101 Control + RNase (25 yg/ml) + DNase (25 yg/ml) 1057 51 Control + KOH (0.2 N, overnight at 37°C) 132 6 * Additions were made at the end of 20 min incubation at 37°C (Control) and the rea c t i o n mixture was f u r t h e r incubated f o r 30 min at 37°C. - 62 -of brain nuclear sonicated extract i s DNA-dependent. The RNA nature of the re a c t i o n product was demonstrated by i t s s e n s i t i v i t y e i t h e r to ribonuclease or to a l k a l i n e d i g e s t i o n , while DNase was without any e f f e c t (Table IV). These experiments were conducted using a crude soluble preparation of br a i n nuclear RNA polymerase. Subsequent work on the p u r i f i c a t i o n of t h i s enzyme led to the r e s o l u t i o n of two RNA polymerase a c t i v i t i e s on a DEAE-cellulose column (as discussed e a r l i e r i n t h i s Chapter), and, therefore, i t was e s s e n t i a l to re-examine the requirements f o r RNA synthesis by the separated enzymic f r a c t i o n s . As shown i n Table V, the a c t i v i t y of both RNA polymerases I and II were dependent on DNA and required the presence of a divalent cation, and s u l f h y d r y l reagent and a l l four ribonucleoside triphosphates. B. EFFECT OF ENZYME CONCENTRATION The incorporation of l a b e l l e d UTP into a c i d - i n s o l u b l e m a t e r i a l , as catalyzed by RNA polymerases I and II from beef b r a i n n u c l e i , was found to be d i r e c t l y p r o p o r t i o n a l to the amount of enzyme i n the range of 0-200 ug ( F i g . 4). In a l l the experiments reported i n this t h e s i s , the amount of enzyme used was within t h i s range. From Figure 4 i t i s cl e a r that polymerase I e l i c i t e d almost h a l f the a c t i v i t y of polymerase II at around s i m i l a r concentrations of enzymic proteins. - 63 -TABLE V. REACTION REQUIREMENTS OF BRAIN NUCLEAR RNA POLYMERASES I AND I I Reaction Mixture R a d i o a c t i v i t y of UTP incorporated RNA Polymerase I RNA Polymerase II cpm/assay % of Control cpm/assay % of Control * Complete (Control) 560 100 1850 100 - Mn"1"1", Mg""" 32 5.9 23 1.2 - 8-mercaptoethanol 356 63.5 1130 61 - ATP 122 21.8 172 9.3 - ATP, CTP, GTP 135 24 178 9.6 - DNA 28 5 55 2.9 - Enzyme 18 3.2 23 1.2 * The complete reaction mixture was the same as described i n Methods (Chapter 2.2E, a) containing 156 ug and 110 yg prot e i n of the most a c t i v e f r a c t i o n s of polymerases I and I I from DEAE-cellulose column, r e s p e c t i v e l y . Incubation was fo r 20 min at 37°C. - 64 -/ ENZYME PROTEIN ( pg ) FIG. 4. THE EFFECT OF INCREASING AMOUNTS OF ENZYME PROTEIN ON RNA SYNTHESIS The RNA s y n t h e s i s was c a r r i e d out at 37°C f o r 20 min using v a r i o u s amounts of polymerase I (o o) or polymerase I I ( • - - - • ) contained i n the complete r e a c t i o n mixture as given i n Chapter 2.2E, a. - 65 -C. EFFECT OF pH The enzyme a c t i v i t y - p H r e l a t i o n s h i p of two RNA polymerases i s o l a t e d from beef b r a i n n u c l e i i s depicted i n Figure 5. As seen i n t h i s Figure, the a c t i v i t y of both the polymerases was maximally assayed near pH 8.0. D. EFFECT OF DIVALENT CATIONS I | | | The requirements f o r divalent cations (Mn or Mg ) of b r a i n nuclear RNA polymerases I and II are shown i n Figure 6. The ++ ++ a c t i v i t y of RNA polymerase I was stimulated more by Mg than Mn I | | | reaching an optimum around 8 mM f o r Mg and about 3 mM f o r Mn I | | | ( F i g . 6A). The r a t i o of Mn /Mg - a c t i v i t i e s f o r t h i s enzyme (at t h e i r optimum concentrations) was lower than one. On the con-I | | j t r a r y , RNA polymerase II preferred Mn to Mg with optimum concentrations of about 3 mM and 6-8 mM, r e s p e c t i v e l y ( F i g . 6B). I | | | At these optimum concentrations, the r a t i o of Mn /Mg - a c t i v i t i e s f o r polymerase II was approximately 3-4. Moreover, both the en-I j | | zymes exhibited highest a c t i v i t y i n the presence of Mn and Mg together. This e f f e c t was not a d d i t i v e because the enzyme ++ -In-a c t i v i t y observed i n the presence of Mn plus Mg was never equal I | | | to the sum of the enzymic a c t i v i t i e s measured with Mn or Mg alone. E. EFFECT OF KC1 Figure 7 depicts the e f f e c t of various concentrations of KC1 on the a c t i v i t y of polymerase I and polymerase II with native DNA - 66 -7 8 9 10 pH - range FIG. 5. THE ENZYME ACTIVITY-pH RELATIONSHIP The a c t i v i t y of polymerase I (o o) and polymerase I I ( • - - - - • ) was assayed i n the presence of 0.03 M T r i s -b u f f e r s of d i f f e r e n t pH v a l u e s . Other assay c o n d i t i o n s were the same as described i n Chapter 2.2E, a. - 67 -FIG. 6. THE EFFECT OF DIVALENT CATIONS ON BRAIN NUCLEAR RNA POLYMERASES The a c t i v i t i e s of RNA polymerase I (6A) and II (6B) as determined i n the presence of various concen-++ 4+ t r a t i o n s of Mn (o o) , Mg —- —•) and I | j | Mn plus Mg (o o). The amount of enzymic pr o t e i n per assay was 146 yg and 99 yg for poly-merases I and I I , r e s p e c t i v e l y . Incubation was c a r r i e d out at 37°C f o r 20 min. - 68 -A d d e d KCI ( x 1 0 M ) FIG. 7. THE EFFECT OF KCI ON BRAIN NUCLEAR RNA POLYMERASES Polymerase I (o o) and Polymerase I I (•— — —•) were assayed i n the presence of v a r i o u s amounts of KCI under the standard c o n d i t i o n s (see Chapter 2.2E, a ) . - 69 -as the template. The two enzymes responded to KC1 p r o f i l e s d i f -f e r e n t i a l l y , i . e . polymerase I e x h i b i t e d maximal a c t i v i t y around 0.05 M s a l t w h i l e polymerase I I e x h i b i t e d a r a t h e r sharp optimum at about 0.2 M KC1. Conceivably,at 0.2 M c o n c e n t r a t i o n of KC1, polymerase I I was approximately 50% greater than the c o n t r o l (without KC1) whereas polymerase I was i n h i b i t e d by about 51%. In a d d i t i o n , the s t i m u l a t i o n of polymerase I I by KC1 (near 0.2 M) I | was found to be much more pronounced i n the presence of Mn or I | | | | | Mn p l u s Mg than i n the presence of Mg alone ( F i g . 8). Furthermore, the a d d i t i o n of KCl at 0.16 M, but not at 0.04 M, r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n the r a t e of RNA s y n t h e s i s by polymerase I I ( F i g . 9 ) . Thus the s t i m u l a t o r y e f f e c t of K C l appears to be s e l e c t i v e f o r polymerase I I o n l y . F. TEMPLATE REQUIREMENTS E a r l i e r i t was shown th a t RNA polymerase I and polymerase I I , i s o l a t e d from the n u c l e i of beef c e r e b r a l c o r t e x , r e q u i r e d the presence of exogenously added n a t i v e DNA as the template (Table V). In t h i s r egard, i t was thought that these two RNA polymerases might d i s p l a y some preference towards n a t i v e or heat-denatured DNA. K i n e t i c data summarized i n Table VI show th a t polymerase I i s more a c t i v e w i t h n a t i v e DNA as template than w i t h denatured DNA (the r a t i o of denatured/native DNA a c t i v i t y being l e s s than one). - 70 -CO I o >-< co CO < a. o 3 -£ 2 - 1 , Added KCI ( x10 M ) FIG. 8. THE EFFECT OF KCI ON BRAIN NUCLEAR RNA POLYMERASE I I ACTIVITY WITH RESPECT TO DIVALENT CATIONS RNA polymerase I I (81 ug p r o t e i n ) was assayed i n the ++ presence of d i f f e r e n t c o n c e n t r a t i o n s of KCI w i t h Mn j j _|__| alone (•— — — • ) , Mg alone (x x) , and Mn I | plus Mg ( o — — — — o ) . Other assay c o n d i t i o n s were the same as those given i n Chapter 2.2E, a. 2 0 4 0 6 0 8 0 T IME ( min ) FIG. 9. THE INFLUENCE OF KCl ON THE RATE OF REACTION OF RNA POLYMERASE I I OF BRAIN NUCLEI RNA polymerase I I (81 yg p r o t e i n ) was assayed using standard r e a c t i o n mixture f o r d i f f e r e n t periods of i n c u b a t i o n i n the absence of KCl (o o) or i n the presence of 0.04 M KCl (x x) or 0.16 M KCl (• . ) . TABLE VI. THE RELATIVE RATES OF TRANSCRIPTION OF NATIVE AND HEAT-DENATURED DNA TEMPLATES BY BRAIN NUCLEAR RNA POLYMERASES DNA Template Time of Ratio of Enzyme* Incubation Native Heat-denatured Heat-denatured/Native (min) cpm/assay cpm/assay Activity Polymerase I 15 215 136 0.63 30 383 141 0.37 45 456 141 0.31 Polymerase II 10 508 1620 3.19 20 815 2591 3.18 40 1204 4263 3.54 60 1702 6087 3.51 * The most active fractions from a DEAE-Cellulose column of RNA polymerase I (135 yg protein) and RNA polymerase II (73 yg protein) were assayed under standard reaction conditions (see Chapter 2.2E, a). - 73 -Conversely, the a c t i v i t y of polymerase I I was about 3 - f o l d g r e a t e r w i t h denatured DNA than w i t h n a t i v e DNA as template. Moreover, the s a t u r a t i o n of polymerase I I a c t i v i t y occurs at much lower q u a n t i t i e s of denatured DNA than n a t i v e DNA ( F i g . 10), p o i n t i n g to i t s preference f o r denatured DNA as template. G. RELATIVE INCORPORATION OF RIBONUCLEOSIDE TRIPHOSPHATES As shown i n Table V I I , polymerase I as w e l l as polymerase I I c a t a l y z e s the i n c o r p o r a t i o n of a l l f o u r bases i n t o TCA-insoluble products (RNA). The r a t i o s of A + U/G + C i n c o r p o r a t e d under the d i r e c t i o n of n a t i v e CT-DNA by polymerase I and I I were 0.65 and 0.96 r e s p e c t i v e l y . Although these values might i n d i c a t e that polymerase I sy n t h e s i z e s "GC-rich" RNA and polymerase I I produces "AU-rich" RNA, more s o p h i s t i c a t e d data such as those obtained from h y b r i d i z a t i o n - c o m p e t i t i o n assays w i l l be r e q u i r e d to j u s t i f y the v a l i d i t y of t h i s statement. I t should be p o i n t e d out that no attempt was made to exclude the formation of homo-polymers, i f any, i n these experiments and thus these base r a t i o s might not represent the true f u n c t i o n of two enzymic a c t i v i t i e s . H. EFFECT OF a-AMANITIN TOXIN a-Amanitin i s a h i g h l y t o x i c s m a l l c y c l i c peptide and was I | i n i t i a l l y shown to i n h i b i t d r a s t i c a l l y the Mn /ammonium su l p h a t e -s t i m u l a t e d RNA polymerase a c t i v i t y of r a t l i v e r i n t a c t n u c l e i - 74 -~ 2 CO I < CO CO < Q. ( J 0 / / / 0 50 100 DNA ( jug ) 150 FIG. 10. RELATIVE TRANSCRIPTION OF HEAT-DENATURED AND NATIVE DNA BY BRAIN NUCLEAR RNA POLYMERASE II RNA polymerase (92 pg protein) was assayed in the presence of various amounts of heat-denatured CT-DNA ( A — A) or native CT-DNA (• •) . Other enzyme assay conditions were the same as described elsewhere (see Chapter 2.2E, a). - 75 -TABLE V I I . RELATIVE INCORPORATION OF NUCLEOTIDES BY BRAIN NUCLEAR RNA POLYMERASES Nucleoside Triphosphate* ( 3H-Labelled) A + U G + C R a d i o a c t i v i t y Incorporated (cpm) Polymerase I Polymerase I I ATP 1780 1757 UTP 900 903 CTP 3042 1389 GTP 1404 1373 0.65 0.96 Separate i n c u b a t i o n s were c a r r i e d out as des c r i b e d i n Methods (Chapter 2.2E, a) w i t h n a t i v e CT-DNA as template. The amounts of enzymic p r o t e i n were 147 yg and 73 yg f o r polymerase I and p o l y -merase I I r e s p e c t i v e l y . * The f i n a l c o n c e n t r a t i o n of each l a b e l l e d n u c l e o s i d e t r i p h o s p h a t e (1 yCi/assay) during enzyme assays was the same (0.1 mM). - 76 -(Stirpe & Fiume, 1967). The high s p e c i f i c i t y of a c t i o n of a-amanitin makes t h i s t o x i n an invaluable t o o l f o r the study of RNA polymerase i n animal c e l l s (Novello & S t i r p e , 1970; Sekeris e_t a l . , 1970; Shaaya & Sekeris, 1970). In view of the high s e l e c -t i v i t y of a-amanitin a c t i o n , i t s e f f e c t on b r a i n nuclear RNA polymerases was tested. Figure 11 i l l u s t r a t e s that a-amanitin i n h i b i t e d polymerase II almost completely while the a c t i v i t y of polymerase I i s not influenced by t h i s toxin. Although the mechanism of d i f f e r e n t i a l response of two polymerases for a-amanitin i s unknown, i t might i n d i c a t e the existence of some s t r u c t u r a l d i f f e r e n c e i n d i f f e r e n t polymerases. I t i s important to note that a-amanitin a c t i o n i s apparently r e l a t e d to poly-merase protein rather than DNA template (Kedinger ej: al. , 1970; Meihlac e_t aJL. , 1970; Jacob et a l . , 1970; L i n d e l l e_t a l . , 1970). I. EFFECT OF CERTAIN ANTIBIOTICS The e f f e c t of two a n t i b i o t i c s , namely, actinomycin D and r i f a m p i c i n was in v e s t i g a t e d with b r a i n nuclear RNA polymerases I and I I . Figure 12 shows that various concentrations of a c t i n o -mycin D i n h i b i t e d both the enzymic a c t i v i t i e s up to the extent of 70%. The a n t i b i o t i c rifamycin i s a very potent i n h i b i t o r of E. c o l i RNA polymerase. I t has been used as a very important t o o l f o r the e l u c i d a t i o n of subunit function of b a c t e r i a l RNA polymerase because r i f a m p i c i n i n t e r a c t s with the polymerase rather than DNA template - 77 -FIG. 11. THE EFFECT OF a-AMANITIN ON BRAIN NUCLEAR RNA POLYMERASES I AND I I RNA polymerase I (•— — —•) and RNA polymerase I I (o o) were s e p a r a t e l y assayed i n the presence of v a r i o u s amounts of a-amanitin. The enzyme assay c o n d i t i o n s were the same as described i n Methods, Chapter Two. FIG. 12. THE EFFECT OF CERTAIN ANTIBIOTICS ON TWO RNA POLY-MERASES OF BRAIN NUCLEI Polymerase I was assayed i n the presence of v a r i o u s q u a l i t i e s of actinomycin D ( o — — — o ) and r i f a m p i c i n (• • ) . Other assay c o n d i t i o n s were the same as mentioned i n Chapter Two. Under s i m i l a r c o n d i t i o n s , polymerase I I was s e p a r a t e l y assayed i n the presence of actinomycin D (o o) and r i f a m p i c i n (x -x) . 100 _J o cc \-z o o o T> 2.5 5 7.5 alpha-AMANITIN ( pg ) 2.5 5 7.5 ANTIBIOTIC (/ig ) FIG .11 FIG.12 - 78 -(see L i l l et a l . , 1970; Wehrli & S t a e h e l i n , 1970; Z i l l i g et a l . , 1970). I t was th e r e f o r e i n t e r e s t i n g to t e s t the e f f e c t of r i f a m -p i c i n on b r a i n n u c l e a r RNA polymerases. The data a l s o d e p i c t e d i n Fig u r e 12 demonstrate that r i f a m p i c i n i s without any de t e c t a b l e i n f l u e n c e on e i t h e r of the two polymerases from b r a i n n u c l e i . T h i s f i n d i n g i s i n accordance w i t h that of others who have observed that b a c t e r i a l RNA polymerase but not mammalian RNA polymerase i s i n h i b i t e d by the r i f a m y c i n s (Hartmann et a l . , 1967; We h r l i et a l . , 1968). 3.3. POLYAMINES AND THE ACTIVITY OF MULTIPLE FORMS OF RNA POLYMERASE  FROM BRAIN NUCLEI As d i s c u s s e d i n the i n t r o d u c t o r y chapter, polyamines i n f l u e n c e the s y n t h e s i s of RNA i n a v a r i e t y of c e l l s and at t h e i r low concen-t r a t i o n s they s t i m u l a t e DNA-dependent RNA polymerase a c t i v i t y assayed i n i n t a c t n u c l e i of mammalian c e l l s . The f a c t that p o l y -amines have been shown to preserve the nucl e a r morphology (MacGregor & Anderson, 1960) and that b r a i n c e l l n u c l e i c o n t a i n , at l e a s t , two RNA polymerase a c t i v i t i e s (as demonstrated i n previous s e c t i o n s of t h i s c h a p t e r ) , i t was very i n t e r e s t i n g to i n v e s t i g a t e the e f f e c t of polyamines on the 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 RNA polymerases. The data presented i n Table V I I I r e v e a l that the a c t i v i t y of RNA polymerase I I was inc r e a s e d up to 186% of the c o n t r o l (100%) by spermidine and up to 140% of c o n t r o l (100%) by spermine w i t h t h e i r - 79 -TABLE VI I I . EFFECT OF POLYAMINES ON RNA POLYMERASE II OF BRAIN CELL NUCLEI Enzyme A c t i v i t y Enzyme A c t i v i t y Spermidine , % of Spermine , % of / w \ cpm/assay .. , u> cpm/assay . (mM) y J c o n t r o l (mM) r c o n t r o l 0 1451 100 0 1451 100 1 1738 120 1 1490 103 2 1940 134 2 1746 120 3 2229 154 3 1955 135 4 2602 179 4 2028 140 6 2700 186 6 1545 107 10 2008 138 10 1281 89 RNA polymerase II (125 yg protein) was assayed under standard assay conditions (see Chapter 2.2E, a) i n the presence of various concentrations of spermidine or spermine. - 80 -optimum concentrations around 4-6 mM and 3-4 mM, r e s p e c t i v e l y . Since spermidine e l i c i t e d a much more pronounced sti m u l a t i o n than did spermine, the former polyamine was chosen for further study. I t should be mentioned that spermidine could not be substituted I | | | f o r Mn or Mg as the cation i n the re a c t i o n mixture. A. EFFECT OF SPERMIDINE ON MULTIPLE ACTIVITIES OF BRAIN NUCLEAR  RNA POLYMERASE The e f f e c t of spermidine on the a c t i v i t y of polymerase I and polymerase I I i s shown i n Figure 13. This Figure i l l u s t r a t e s that the a d d i t i o n of spermidine (5-6 mM) r e s u l t e d i n an increase of 65-80% over the c o n t r o l (without spermidine) i n polymerase II a c t i v i t y while the a c t i v i t y of polymerase I was only 30-50% stimulated. This observation might i n d i c a t e that the two enzymes responded to spermidine somewhat d i f f e r e n t l y . In order to delineate t h i s e f f e c t f urther, the a c t i v i t i e s of polymerases I and II were determined i n the presence of t h e i r preferred divalent cation. The r e s u l t s of these experiments are depicted i n Figure 14, which c l e a r l y demonstrates that the magnitude of stimulation by sper-midine of polymerase II a c t i v i t y was much higher (about 250% of I | | | the control) i n the presence of Mn than i n the presence of Mg I | alone (Mn being the preferred d i v a l e n t cation for polymerase II as described before i n Figure 6B). However, polymerase I was only I | 40-50% stimulated by spermidine i n the presence of Mg only and - 81 -200 175 O DC I-Z o O 150 L L O O S 125 1 0 0 ^ -• • 2 3 4 SPERMIDINE ( mM ) FIG. 13. THE INFLUENCE OF SPERMIDINE ON TWO RNA POLYMERASES OF BRAIN NUCLEI Independent assays were c a r r i e d out i n the presence of various concentrations of spermidine f o r RNA polymerase I (A A) and RNA polymerase II (• — — — • ) . Other assay conditions were those described i n Chapter 2.2E, a. - 82 -FIG. 14. THE EFFECT OF SPERMIDINE ON BRAIN NUCLEAR RNA POLYMERASES WITH REGARD TO DIVALENT CATION I | Polymerase I was assayed i n the presence of Mn I | (3 mM) only (A. •) or Mg (6 mM) only (• •) . The a c t i v i t y of polymerase I I was ++ determined i n the presence of Mn ( o — — — o) I | or Mg ( A A ) . Other assay c o n d i t i o n s were the same as given i n Chapter 2.2E, a. The concentrations of spermidine present during the enzyme assays were those as denoted i n the f i g u r e . 2 3 4 SPERMIDINE ( mM ) - 83 -t h i s stimulatory e f f e c t i s suppressed i f Mn i s the divalent I | cation i n the assay mixture (Mg being the preferred d i v a l e n t cation f o r polymerase I as shown previously i n Figure 6A). If spermidine stimulated polymerase II p r e f e r e n t i a l l y , as revealed by the above st u d i e s , then there should be some s p e c i f i c i t y of spermidine s t i m u l a t i o n with respect to native and denatured DNA as template because t h i s enzyme prefers denatured DNA (as shown e a r l i e r i n Table V I ) . This was investigated by following the k i n e t i c s and the data are summarized i n Figures 15, 16, and 17. I | | | The k i n e t i c data obtained i n the presence of both Mn and Mg showed that the stimulatory e f f e c t of spermidine (4 mM) required the presence of native DNA as template for polymerase II a c t i v i t y ( F i g . 15). Under these conditions, spermidine had l i t t l e or no e f f e c t i n enhancing the enzyme a c t i v i t y which was primed with heat-denatured DNA as template. This e f f e c t , however, v a r i e d depending upon the diva l e n t c a t i o n present i n the r e a c t i o n mixture. As depicted i n Figure 16, the a c t i v i t y of polymerase II assayed In I j the presence of Mn only was found to be stimulated by spermidine (4 mM) with either native or heat-denatured DNA as template. In the presence of Mg alone, spermidine (4 mM) was somewhat stimu-l a t o r y with native DNA template only but i t was r e l a t i v e l y i n h i b i -tory with heat-denatured DNA template ( F i g . 17). Thus spermidine stimulated the a c t i v i t y of polymerase II with both native as w e l l - 84 -T I M E , m j n FIG. 15. THE KINETICS OF SPERMIDINE STIMULATION OF BRAIN NUCLEAR RNA POLYMERASE I I WITH REGARD TO TEMPLATE I [ The enzyme was assayed i n the presence of Mn (3 mM) I | and Mg (6 mM) w i t h n a t i v e DNA template (without spermidine, o o, and w i t h 4 mM spermidine • — • ) , and w i t h heat-denatured DNA template (without spermidine, A A, w i t h 4 mM spermidine, A A ) . - 85 -T I M E , min FIG. 16. THE KINETICS OF SPERMIDINE STIMULATION OF BRAIN NUCLEAR RNA POLYMERASE I I WITH REGARD TO TEMPLATE I j The enzyme was assayed i n the presence of Mn (3 mM) only w i t h n a t i v e DNA template (without spermidine, o o, and w i t h 4 mM spermidine • — — — • ) , and with.heat-denatured DNA template (without spermidine, A A, w i t h 4 mM spermidine, A - A ) . - 86 -FIG. 17. THE KINETICS OF SPERMIDINE STIMULATION OF BRAIN NUCLEAR RNA POLYMERASE I I WITH REGARD TO TEMPLATE j | The enzyme was assayed i n the presence of Mg (6 mM) only w i t h n a t i v e DNA template (without spermidine, o o, and w i t h 4 mM spermidine • — • ), and w i t h heat-denatured DNA template (without sper-midine, A A, w i t h 4 mM spermidine, A— A ) . - 87 -as denatured DNA as template i n the presence of Mn o n l y . With I | denatured DNA, t h i s e f f e c t was suppressed i f Mg was supplemented I | to the Mn - c o n t a i n i n g assay system, and spermidine was i n h i b i t o r y I | r a t h e r than s t i m u l a t o r y i f Mg was the only d i v a l e n t c a t i o n . I | These observations tend to suggest that the Mn -primed RNA p o l y -merase a c t i v i t y may be p r e f e r e n t i a l l y s t i m u l a t e d by spermidine. B. INHIBITION OF POLYMERASE I I ACTIVITY BY CALF THYMUS HISTONE  AND YEAST RNA AND EFFECT OF SPERMIDINE ON THIS INHIBITION Histones and RNA are found i n mammalian c e l l nucleus and that the molecules of a s i m i l a r nature, i f added to the assay m i x t u r e , s i g n i f i c a n t l y i n h i b i t e d the a c t i v i t y of RNA polymerase I I from beef b r a i n n u c l e i ( F i g . 18), i t was tempting to see i f polyamines could e i t h e r prevent or overcome t h i s i n h i b i t i o n . In order to perform such a study, r e c o n s t i t u t e d systems were employed and the r e s u l t s of these experiments are given i n Table IX. The a d d i t i o n of 100 ug yeast RNA or 200 ug c a l f thymus h i s t o n e f r a c t i o n i n t o the r e a c t i o n mixture r e s u l t e d i n a c o n s i d e r a b l e degree of i n h i b i t i o n ( greater than 60%) of polymerase I I a c t i v i t y . Spermidine (5 mM) added at the end of the t w e n t i e t h minute of i n c u b a t i o n s t i m u l a t e d the yeast RNA i n h i b i t e d enzyme a c t i v i t y (over 2 f o l d i n c r e a s e ) , but i t has almost no e f f e c t on the enzyme a c t i v i t y which was i n h i b i t e d by c a l f thymus h i s t o n e . F i g u r e 19 i l l u s t r a t e s that the i n a b i l i t y of - 88 ->«g / A S S A Y FIG. 18. THE INHIBITION OF BRAIN NUCLEAR RNA POLYMERASE II ACTIVITY BY CALF THYMUS HISTONE AND YEAST RNA The enzyme a c t i v i t y was measured i n the presence of various amounts of c a l f thymus histone ( o — — — o ) and yeast RNA (• • ) . - 89 -TABLE IX. THE EFFECT OF SPERMIDINE ON BRAIN NUCLEAR RNA POLYMERASE I I ACTIVITY WHICH WAS INHIBITED BY YEAST RNA AND CALF THYMUS HISTONE Assay Mixture Time o f i n c u b a t i o n 20 min 60 min S p e c i f i c a c t i v i t y (pmoles/mg p r o t e i n ) S p e c i f i c a c t i v i t y (pmoles/mg p r o t e i n ) Increase by spermidinet (%) EXPERIMENT 1 Complete Complete + spermidine* Complete + yeast RNA Complete + yeast RNA + spermidine* 3.10 3.10 1.14 1.14 4.06 5.65 1.54 3.20 40 108 EXPERIMENT 2 Complete Complete + spermidine* Complete + c a l f thymus h i s t o n e Complete + c a l f thymus h i s t o n e + spermidine* 3.99 3.99 0.695 0.695 6.45 8.00 1.34 1.42 24 The enzyme a c t i v i t y was assayed i n the absence o r i n the presence of yeast RNA (0.1 mg/assay) or c a l f thymus h i s t o n e (0.2 mg/assay) using complete r e -a c t i o n mixture as given elsewhere (Chapter 2.2E, a ) . * Spermidine (5 mM) was added at the end of the twentieth minute of i n -cubation p e r i o d and the r e a c t i o n was continued u n t i l 60 min at 37°C. t The i n c r e a s e i n enzyme a c t i v i t y by spermidine was c a l c u l a t e d based on the c o n t r o l v a l u e (the enzyme a c t i v i t y without spermidine). - 90 -2000 < c/> 1000 < O. O o> o o _L 0.5 1 1.5 H i s t o n e D N A FIG. 19. THE INFLUENCE OF SPERMIDINE ON THE HISTONE-INHIBITED RNA POLYMERASE I I ACTIVITY The enzyme a c t i v i t y was determined i n the presence of v a r i o u s amounts of c a l f thymus h i s t o n e (without spermidine, o — — — — o , and w i t h 5 mM spermidine, • — — — •) using standard c o n d i t i o n s of enzyme assay. - 91 -spermidine to overcome the h i s t o n e - i n h i b i t i o n of polymerase I I a c t i v i t y i s not dependent upon the amount of h i s t o n e present during the assay. Moreover, the co u n t e r a c t i n g e f f e c t of spermidine (5 mM) of yeast R N A - i n h i b i t i o n was a l s o found to be independent of quan-t i t i e s o f yeast RNA (50-250 yg) present i n the assay system ( F i g . 20). C. THE NATURE OF RNA SYNTHESIZED BY POLYMERASE I I UNDER THE  INFLUENCE OF SPERMIDINE The data presented i n the foregoing s e c t i o n s c l e a r l y demon-s t r a t e d that the a c t i v i t y of RNA polymerase I I i s profoundly s t i m u l a t e d by spermidine. Therefore, i t was very a l l u r i n g to i n v e s t i g a t e the nature of RNA produced by polymerase I I under the a c t i o n of spermidine. The experiments to conduct t h i s type of study i n c l u d e d the suc r o s e - d e n s i t y g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s of RNA sy n t h e s i z e d i n v i t r o by polymerase I I i n the absence or presence of spermidine. Figure 21 shows that the RNA sy n t h e s i z e d i n 60 min w i t h or without spermidine sediments as a s i n g l e peak. 3 Since the r a d i o a c t i v i t y of H-UTP i n c o r p o r a t e d i n t o RNA under the i n f l u e n c e of spermidine i s much higher than i n the presence of spermidine, i n d i c a t i n g that the t o t a l amount of RNA sy n t h e s i z e d i n v i t r o i s in c r e a s e d by spermidine. This would suggest that perhaps polymerase I I under the a c t i o n of spermidine t r a n s c r i b e s the same segments of DNA because the nature of newly-synthesized RNA was i n d i s t i n g u i s h a b l e by sucrose-density g r a d i e n t c e n t r i f u g a t i o n . - 92 -FIG. 20. THE COUNTERACTING EFFECT OF SPERMIDINE ON RNA-INHIBITED RNA POLYMERASE I I ACTIVITY The enzyme a c t i v i t y was measured i n the presence of v a r i o u s q u a n t i t i e s of yeast RNA (without sper-midine, • and w i t h 5 mM spermidine, o o) using standard r e a c t i o n mixture. I n -cubation was c a r r i e d out at 37°C f o r 20 min. 2 0 % F R A C T I O N NUMBER 5 % Sucrose S u c r o s e FIG. 21. SUCROSE-DENSITY GRADIENT PROFILE OF RNA SYNTHESIZED BY POLYMERASE I I UNDER THE ACTION OF SPERMIDINE The enzyme was assayed under the c o n d i t i o n s des-c r i b e d i n Chapter Two w i t h spermidine (5 mM) or without spermidine. At the end of 60 min i n c u b a t i o n , the r e a c t i o n product was separated by sucrose-density g r a d i e n t c e n t r i f u g a t i o n according to the procedure o u t l i n e d i n Methods (see Chapter 2.2H, b ) . The 3 r a d i o a c t i v i t y of H-UMP inco r p o r a t e d i n t o RNA, which was synth e s i z e d w i t h spermidine (• •) or without spermidine (A A) , i s p l o t t e d . - 94 -Subsequently, i t was found that the product of polymerase II a c t i v i t y may be a complex of the type Enzyme-DNA-RNA rather than free RNA. This was substantiated by the f i n d i n g that the r a d i o -a c t i v i t y peak of RNA s h i f t e d towards a l i g h t e r region i f the rea c t i o n mixture had been treated with SDS p r i o r to i t s analysis by sucrose-density gradient c e n t r i f u g a t i o n (Fig. 22). Treatment of r e a c t i o n mixture with SDS denatures the enzyme pr o t e i n and di s s o c i a t e s i t from the Enzyme-DNA-RNA complex, and thus r e s u l t e d i n a s h i f t of r a d i o a c t i v i t y peak of RNA towards a l i g h t e r region of the sucrose-density gradient. This f i n d i n g i s compatible with the f a c t that the product of RNA polymerase r e a c t i o n i s an Enzyme-DNA-RNA complex (Bremer & Konrad, 1964). I t should be pointed out that the pattern of RNA synthesized i n the presence or absence of spermidine was found to be s i m i l a r no matter whether the r e a c t i o n mixture was treated or not with SDS before analysis by sucrose-density gradient c e n t r i f u g a t i o n ( F i g . 23). This Figure also i l l u s t r a t e s the f a c t that the product of the rea c t i o n i s somewhat smaller i n s i z e than 18 S ribosomal RNA of the r a t l i v e r used as a reference. 3.4. TRANSCRIPTION OF BRAIN CHROMATIN BY BRAIN NUCLEAR RNA  POLYMERASE II In experiments, reported i n e a r l i e r sections of th i s Chapter, a commercial grade of p u r i f i e d c a l f thymus native DNA has been - 95 -0 5 10 15 2 0 2 5 20% F R A C T I O N N U M B E R 5% S u c r o s e S u c r o s e FIG. 22. SUCROSE-DENSITY GRADIENT PROFILE OF RNA SYNTHESIZED BY BRAIN NUCLEAR RNA POLYMERASE I I The enzyme assay c o n d i t i o n s were the same as those given i n Chapter Two. A f t e r 60 min i n c u b a t i o n , the r e a c t i o n product was t r e a t e d w i t h SDS f o r 5 min at 37°C p r i o r to the a n a l y s i s by sucrose-density g r a d i e n t c e n t r i f u g a t i o n (see Chapter 2.2H, b ) . The r a d i o -a c t i v i t y of RNA i n the untreated (• •) and SDS-treat e d ( o — — — o) r e a c t i o n p r o d u c t , i s diagrammed. - 96 -FIG. 23. THE NATURE OF RNA SYNTHESIZED BY BRAIN NUCLEAR RNA POLYMERASE I I UNDER THE INFLUENCE OF SPERMIDINE The enzyme assay and the s e p a r a t i o n of RNA were c a r r i e d out according to the procedure o u t l i n e d i n the legend to Fi g u r e 21, except that the r e a c t i o n product was t r e a t e d w i t h SDS p r i o r to i t s a n a l y s i s by sucrose-density g r a d i e n t c e n t r i f u g a t i o n . The r a d i o a c t i v i t y of RNA, which was syn t h e s i z e d i n the presence of 5 mM spermidine (• — •) or i n the absence of spermidine (A A) t i s p l o t t e d . Absorbance at 260 nm (o o) represent the r a t l i v e r ribosomal RNA which was simultaneously run as a r e f e r e n c e . 2 0 % Sucrose F R A C T I O N N UMBER 5 % Sucrose - 97 -u t i l i z e d as template f o r b r a i n nuclear RNA polymerases. However, the genetic material i n the d i f f e r e n t i a t e d c e l l s i s represented by chromatin, a matrix of DNA, RNA and p r o t e i n molecules. Moreover, the RNA polymerase a c t i v i t y i n mammalian c e l l s i s confined p r i m a r i l y i n the nucleus, being f i r m l y associated with the chromatin matrix. Therefore, i t was of i n t e r e s t to determine the a b i l i t y of i s o l a t e d enzymes to tra n s c r i b e RNA from chromatin, a template of more physio-l o g i c a l nature. In order to do so, chromatin was i s o l a t e d from cerebral cortex and RNA polymerase II from the same tiss u e was used to t r a n s c r i b e RNA o f f the DNA i n cerebral chromatin ( r e c o n s t i t u t i n g a homologous system). The observations made on t h i s type of study are described i n t h i s Section. I t should be pointed out that be-cause of the greater i n s t a b i l i t y of polymerase I, polymerase II was u t i l i z e d to i n v e s t i g a t e the t r a n s c r i p t i v e properties of the i s o l a t e d chromatin. A. CERTAIN CHARACTERISTICS OF BRAIN CHROMATIN The soluble chromatin prepared by a method, as ou t l i n e d i n Figure 24, exhibited a sharp peak of absorption-maximum at 260 nm, which a l -so coincided with the peak of native c a l f thymus DNA (Fig. 25). The r a t i o of 280 nm/260 nm absorbance i n chromatin preparations was usually around 0.6. The absorbance at 320 nm was p r a c t i c a l l y n e g l i g i b l e i n -d i c a t i n g the absence of t u r b i d i t y i n the soluble chromatin, which was found to contain DNA, RNA and proteins i n the mass r a t i o s of 1:0.09: 1.92 (Table X). A l l these features of soluble b r a i n chromatin are compatible with those which have been described for the chromatin preparations from.various other tissues (see Bonner et a l . , 1968a; see Hearst & Botchan, 1970). - 98 -FIG. 24. AN OUTLINE FOR THE PREPARATION OF CHROMATIN AND DEHISTONIZED CHROMATIN FROM BEEF CEREBRAL CORTEX P u r i f i e d N u c l e i Homogenized i n EDTA-buffer and c e n t r i f u g e d at 9,750 x g/10 min Washed twice w i t h EDTA-buffer and twice w i t h 0.02 M T r i s - H C l b u f f e r , pH 8.0. Suspended i n the same b u f f e r and sheared by s o n i c a t i o n f o r 1.5 min/0°C 12,100 x g/30 min  P e l l e t Supernate (d i s c a r d ) Supernate (Soluble Chromatin) P e l l e t ( d i s c a r d ) Treatment w i t h 0.4 N H„SO./0°C 27,000 x g/20 min P e l l e t Suspended i n 0.02 M T r i s - H C l b u f f e r , pH 8.0 (Dehistonized Chromatin) Supernate (Histone F r a c t i o n ) (The d e t a i l s of each step are given i n Chapter 2.2) - 99 -FIG. 25. THE UV-ABSORPTION SPECTRA OF BEEF BRAIN CHROMATIN AND CT-DNA The absorbance spectrum of a d i l u t e d sample of b r a i n chromatin ( - - - - ) or c a l f thymus DNA ( ) was recorded using a S p e c t r o n i c , Model 505 (Bausch & Lomb In c . , N.Y.). - 100 -TABLE X. CHEMICAL COMPOSITION OF BEEF BRAIN CHROMATIN Component Mass R a t i o * DNA 1.0 RNA 0.09 Histone p r o t e i n 1.02 Non-histone p r o t e i n 0.9 * Average values of three d e t e r m i n a t i o n s . The determinations were c a r r i e d out i n d u p l i c a t e a f t e r e x t r a c t i o n of each component as d e s c r i b e d i n Methods (Chapter 2.2E, a ) . - 101 -B. TEMPLATE ACTIVITY OF BRAIN CHROMATIN FOR POLYMERASE I I OF  BRAIN NUCLEI In order to manipulate the c o r r e c t values f o r the t r a n s c r i p t i o n of b r a i n chromatin by b r a i n n u c l e a r polymerase I I , i t was e s s e n t i a l to check the endogenous RNA polymerase a c t i v i t y i n the s o l u b l e chro-matin p r e p a r a t i o n s . F i g u r e 26 d e p i c t s that such chromatin p r e p a r a t i o n s contained almost n e g l i g i b l e amounts of endogenous RNA polymerase a c t i v i t y , assayed even i n the presence of as high as 0.4 M KCI. I t should be pointed out t h a t the c o n d i t i o n s of preparing chromatin from p u r i f i e d n u c l e i are those which are very d e t r i m e n t a l to the n u c l e a r RNA polymerase a c t i v i t y and perhaps do not s o l u b i l i z e i t as some polymerase a c t i v i t y i s d e t e c t a b l e i n the discarded chromatin p e l l e t . This f i g u r e a l s o shows that s o l u b l e chromatin served as a template f o r added enzyme (polymerase I I ) , whose a c t i v i t y i s f u r t h e r s t i m u l a t e d by the presence of KCI, reaching an optimum around 0.25 M. When compared w i t h c a l f thymus n a t i v e DNA as template, the i s o l a t e d chromatin from beef b r a i n supported RNA s y n t h e s i s i n a homologous system by b r a i n nuclear RNA polymerase I I up to l e s s than 25% ( F i g . 27). Moreover, the s y n t h e s i s of RNA by polymerase I I on d e p r o t e i n i z e d chromatin ( b r a i n DNA) was of an equal magnitude to that obtained w i t h c a l f thymus DNA, i n d i c a t i n g that the source of DNA used as a reference f o r comparison does not matter. These r e -- 102 -CM I o 20 15 >-5 10 < -Q. o / / / s \ 2 i l KCl (x10 M ) FIG. 26. THE EFFECT OF KCl ON THE CHROMATIN-TEMPLATED RNA POLYMERASE I I ACTIVITY B r a i n n u c l e a r RNA polymerase I I (76 yg p r o t e i n ) was assayed i n the presence of v a r i o u s concen-t r a t i o n s of KCl using s o l u b l e chromatin (con-t a i n i n g 60 yg DNA) from beef b r a i n as the tem-p l a t e ( o — — — o ) . Other assay c o n d i t i o n s were the same as des c r i b e d i n Methods. The en-dogenous RNA polymerase a c t i v i t y of the s o l u b l e chromatin (without added enzyme) was s i m u l -taneously determined (• — • ) . 4 - 103 -DNA (^ig) 150 FIG. 27. TEMPLATE ACTIVITY OF BRAIN CHROMATIN B r a i n nuclear RNA polymerase I I (0.051 mg p r o t e i n ) was assayed i n the presence of v a r i o u s amounts of c a l f thymus DNA, obtained from Worthington B i o -chemical Corporation (•— — —•) , d e p r o t e i n i z e d chromatin (• •) and equivalent amounts of DNA i n n a t i v e chromatin (o o ) . - 104 -s u i t s suggest that most of the genomic a c t i v i t y i n d i f f e r e n t i a t e d b r a i n c e l l s i s somehow repressed. Figure 28 demonstrates t h a t i f d e h i s t o n i z e d chromatin from b r a i n (the chromatin which had been t r e a t e d w i t h 0.4 N ^SO^ to remove h i s t o n e p r o t e i n s , see Figure 24) was u t i l i z e d as the tem-p l a t e f o r polymerase I I , the r e s u l t i n g enzyme a c t i v i t y was found to be about 3 - f o l d g r e a t e r than that obtained w i t h pure DNA. However, s i m i l a r l y prepared d e h i s t o n i z e d chromatin from c e r e b r a l t i s s u e was as a c t i v e as pure DNA as a template f o r E. c o l i RNA polymerase ( F i g . 29). A l s o the r a t e of a c t i v i t y of polymerase I I ( F i g . 30), but not E. c o l i enzyme ( F i g . 31), was about three times g r e a t e r w i t h d e h i s t o n i z e d chromatin than pure DNA as template (both the enzymes being assayed under s i m i l a r c o n d i t i o n s i n the presence of equ i v a l e n t amounts o f DNA i n d e h i s t o n i z e d chromatin). These observations imply that an a c t i v a t i n g mechanism of DNA t r a n s c r i p t i o n e x i s t s i n the genome of b r a i n c e l l s which i s o n l y d e t e c t a b l e by using an homologus enzyme ( i . e . b r a i n nuclear RNA polymerase I I ) but not by using an heterologous enzyme ( i . e . E. c o l i RNA polymerase). RNA polymerase I I of b r a i n n u c l e i and E. c o l i enzyme ex-h i b i t e d a s e l e c t i v e response towards a-amanitin t o x i n and the a n t i b i o t i c r i f a m p i c i n , i . e . polymerase I I of b r a i n n u c l e i primed w i t h CT-DNA, chromatin and d e h i s t o n i z e d chromatin i s almost com-- 105 -D N A ( / j g ) FIG. 28. TEMPLATE ACTIVITY OF BRAIN DEHISTONIZED CHROMATIN FOR BRAIN NUCLEAR RNA POLYMERASE I I Br a i n nuclear RNA polymerase I I (0.051 mg p r o t e i n ) was assayed i n the presence of c a l f thymus DNA (•— — — •) and equ i v a l e n t amounts of DNA i n de-h i s t o n i z e d chromatin ( • — — • ) and n a t i v e chromatin (o o) . - 106 -0 20 4 0 6 0 D N A ( pg ) FIG. 29. TEMPLATE ACTIVITY OF BRAIN DEHISTONIZED CHROMATIN FOR E. COLI RNA POLYMERASE E. c o l i RNA polymerase (0.0225 mg p r o t e i n ) was assayed i n the presence of c a l f thymus DNA (•— — — • ) and e q u i v a l e n t amounts of DNA i n d e h i s t o n i z e d chromatin (• •) and n a t i v e chromatin (o o) . - 107 -FIG. 30. THE RELATIVE RATES OF TRANSCRIPTION OF CHROMATIN, DEHISTONIZED CHROMATIN AND CT-DNA BY BRAIN NUCLEAR RNA POLYMERASE I I The enzyme (76 yg p r o t e i n ) was assayed i n the presence of chromatin (60 yg DNA, o o) or d e h i s t o n i z e d chromatin (60 yg DNA, • •) or CT-DNA (60 yg, • — • ) as template. Other c o n d i t i o n s of enzyme assay were the same as given i n Chapter 2.2E, a. 5 r T I M E (min ) - 108 -FIG. 31. THE RELATIVE RATES OF TRANSCRIPTION OF CHROMATIN, DEHISTONIZED CHROMATIN AND CT-DNA BY E. COLI RNA POLYMERASE The enzyme (22.5 yg p r o t e i n ) was assayed i n the presence of chromatin (60 yg DNA, o o) or d e h i s t o n i z e d chromatin (60 yg DNA,B H) or CT-DNA (60 yg, • •) as template. Other c o n d i t i o n s of enzyme assay were the same as f o r b r a i n n u c l e a r polymerase I I (see F i g . 30), except that c a r r i e r DTP (0.1 mM) was included i n the r e -a c t i o n mixture. T I M E ( min ) - 109 -pletely inhibited by a-amanitin but not by rifampicin whereas E. c o l i enzyme primed with the same templates is inhibited by rifampicin but not by a-amanitin (Table XI). Since the pattern of sensitivity in each case was unaltered by the presence of three different types of templates, indicating that some portion (which is required for the interaction with a-amanitin or rifam-picin in respective cases) may be structurally different in the molecule of RNA polymerases obtained from two organisms of widely distinct evolutionary origin. In this regard, i t is important to note that the mechanism of action of a-amanitin and rifampicin i s related to the polymerase protein and not to the DNA template (for discussion see Chapter 1 and also Goldberg & Friedman, 1971). As described in the foregoing section, the removal of histones from cerebral chromatin accentuated RNA synthesis in vitro by brain nuclear RNA polymerase II to levels even higher than those obtained with pure DNA, i t was very enticing to look for the nature of RNA transcribed from the DNA i n chromatin and in dehistonized chromatin. The nature of RNA synthesized by polymerase II with pure CT-DNA, chromatin and dehistonized chromatin as templates was analyzed by sucrose-density gradient centrifugation. Figure 32 shows that RNA transcribed on chromatin and dehistonized chromatin as templates sediments as a single peak in slightly lighter regions than the - 110 -TABLE X I . THE EFFECT OF a-AMANITIN AND RIFAMPICIN ON BRAIN NUCLEAR RNA POLYMERASE I I AND E. COLI RNA POLYMERASE AS DIRECTED BY CT-DNA, CHROMATIN AND DEHISTONIZED CHROMATIN TEMPLATES Enzyme Template a-Amanitin R i f a m p i c i n (% of (ug/assay) c o n t r o l ) (% of (ug/assay) c o n t r o l B r a i n Nuclear Polymerase I I CT-DNA 0 2.5 5.0 100 9.5 8.5 0 2.5 5.0 100 98 97 Chromatin De h i s t o n i z e d chromatin 0 2.5 5.0 0 2.5 5.0 100 7.4 8.0 100 5.5 5.6 0 2.5 5.0 0 2.5 5.0 100 96 97 100 95.8 96 E. c o l i Polymerase CT-DNA 0 2.5 5.0 100 98.5 99.6 0 2.5 5.0 100 3.5 3.4 /Continued. - I l l -TABLE X I . THE EFFECT OF a-AMANITIN AND RIFAMPICIN ON BRAIN NUCLEAR RNA POLYMERASE I I AND E. COLI RNA POLYMERASE AS DIRECTED BY CT-DNA, CHROMATIN AND DEHISTONIZED CHROMATIN TEMPLATES Continued Enzyme Template a-Amanitin R i f a m p i c i n of (% of (yg/assay) c o n t r o l ) (yg/assay) c o n t r o l ) E. c o l i Polymerase Chromatin 0 2.5 5.0 100 101 99 0 2.5 5.0 100 4.2 3.6 Deh i s t o n i z e d chromatin 0 2.5 5.0 100 100 99.5 0 2.5 5.0 100 3.2 3.4 B r a i n n u c l e a r polymerase I I (56 yg protein/assay) and E. c o l i polymerase (22.5 yg protein/assay) were assayed i n the absence ( c o n t r o l ) o r presence of v a r i o u s amounts of e i t h e r a-amanitin or r i f a m p i c i n . The amount of DNA template" i n each case was 60 yg/assay. Other c o n d i t i o n s of enzyme assay were the same as des c r i b e d i n Methods (Chapter 2.2E, a ) . - 112 -FIG. 32. SUCROSE-DENSITY GRADIENT PROFILE OF RNA TRANSCRIBED FROM CHROMATIN, DEHISTONIZED CHROMATIN AND CT-DNA BY BRAIN NUCLEAR RNA POLYMERASE I I Polymerase I I (112 yg p r o t e i n ) was s e p a r a t e l y assayed using 60 yg each of chromatin DNA, d e h i s t o n i z e d chromatin DNA and CT-DNA. A f t e r 60 min i n c u b a t i o n , the r e a c t i o n mixtures were t r e a t e d w i t h SDS and the RNA product was analyzed by sucrose d e n s i t y g r a d i e n t 3 c e n t r i f u g a t i o n . The r a d i o a c t i v i t y of H-UMP i n -corporated i n t o RNA, which was t r a n s c r i b e d from chromatin (o o) or d e h i s t o n i z e d chromatin (• •) or CT-DNA (• •) , i s diagrammed. - 113 -RNA which i s made on CT-DNA. This f i n d i n g i l l u s t r a t e s two points: (1) RNA chains transcribed from the naked DNA (free of chromosomal pr o t e i n and RNA molecules) are longer i n length than those syn-thesized with chromatin or dehistonized chromatin, and (2) no unique species of RNA i s made on chromatin from which most of the histone proteins have been s e l e c t i v e l y removed. Although a de-f i n i t i v e i n t e r p r e t a t i o n w i l l require an extensive analysis by more s p e c i f i c assays such as hybridization-competition measurements, nevertheless, i t appears as i f the as s o c i a t i o n of chromosomal macromolecules with the genome brings about reduction i n s i z e of RNA transcribed i n v i t r o . • - 114 -CHAPTER FOUR. DISCUSSION AND CONCLUSIONS 4.1. MULTIPLE FORMS OF DNA-DEPENDENT RNA POLYMERASE Although DNA-dependent RNA polymerase enzyme, which catalyzes the i n corporation of ribonucleotides i n t o RNA as i n s t r u c t e d by DNA, was f i r s t i d e n t i f i e d i n nuclear preparations from r a t l i v e r (Weiss & Gladstone, 1959), the study of RNA polymerase from various eukary-o t i c organisms has proceeded very slowly. This slow progress could be a t t r i b u t e d to the f a c t that the i n t r i n s i c enzyme a c t i v i t y of i n t a c t n u c l e i i s low and that many d i f f i c u l t i e s are encountered i n obtaining a soluble enzyme f r a c t i o n which w i l l depend on exogenous DNA f o r i t s c a t a l y t i c function. Recent studies on RNA polymerase from n u c l e i of eukaryotic c e l l s have t e s t i f i e d to a l l these d i f f i -c u l t i e s (see Chapter One). Despite numerous i n i t i a l d i f f i c u l t i e s , various t r i a l e x p eri-ments i n v o l v i n g a v a r i e t y of conditions eventually l e d to the s o l u -b i l i z a t i o n of RNA polymerase i n high y i e l d s from beef b r a i n n u c l e i . The experimental data, as presented i n the preceding Chapter of t h i s thesis,demonstrate the existence of at le a s t two fu n c t i o n a l species of DNA-dependent RNA polymerase i n the n u c l e i of br a i n c e l l s which are designated as RNA polymerase I and polymerase I I . These enzymes are r e a d i l y separated from each other by chromatographic procedures and appear to be d i s t i n c t e n t i t i e s . There i s no evidence to ru l e out the p o s s i b i l i t y of the interconversion of one form to the other. - 115 -In ad d i t i o n to chromatographic r e s o l u t i o n , the separated RNA polymerase species are d i s t i n g u i s h a b l e based on some of t h e i r d i f f e r i n g c a t a l y t i c p roperties; f o r example, the p r o f i l e s of manganese, magnesium and potassium chl o r i d e , the r e l a t i v e t r a n s c r i p t i v e a c t i v i t y with native and denatured DNA templates and the s e n s i t i v i t y to a-I | | | amanitin toxin are very d i f f e r e n t f o r each polymerase. The Mn /Mg a c t i v i t y r a t i o i s about 3-4 times greater f o r b r a i n nuclear poly-merase II than for polymerase I, whose a c t i v i t i e s are maximal at high and low i o n i c strengths, r e s p e c t i v e l y . Moreover, polymerase I prefers native DNA to denatured DNA as template whereas polymerase II i s about 3-4 times more a c t i v e with denatured DNA than with native DNA as the template. At very low concentrations, a-amanitin s e l e c -t i v e l y i n h i b i t s RNA polymerase II almost completely while RNA polymerase I i s r e s i s t a n t to a-amanitin. These properties of b r a i n nuclear polymerase I and polymerase II are s i m i l a r to those which have been reported recently f o r two species of nuclear DNA-dependent RNA polymerase of other eukaryotes (Roeder & Rutter, 1969; Chambon et a l . , 1970; Goldberg & Moon, 1970; Roeder et a l . , 1970; T o c c h i n i - V a l e n t i n i & Crippa, 1970; Ponta et a l . , 1971; S t r a i n et a l . , 1971). An a d d i t i o n a l species of nuclear RNA polymerase ( c a l l e d polymerase III) has been described i n sea urchin (Roeder £ Rutter, 1969) and i n yeast ( B l a t t i et a l . , 1970; Adman & H a l l , 1971; Ponta et ^1., 1971). This enzyme i s not detected i n the n u c l e i of b r a i n c e l l s . However, i t should be mentioned that polymerase I I I i n the - 116 -aquatic fungus, B l a s t o c l a d i e l l a emersonii, has been shown to be derived from the contaminating mitochondria (Horgen & G r i f f i n , 1971) and thus t h i s enzyme a c t i v i t y may be of mitochondrial o r i g i n instead of being a nuclear component. As discussed i n the Introductory Chapter, the concept of mu l t i p l e RNA polymerases i s not new. The existence of, at l e a s t , two RNA polymerase a c t i v i t i e s was i n i t i a l l y detected by Windell and Tata (1964) i n i s o l a t e d n u c l e i of r a t l i v e r . Subsequent work led these workers to propose that the n u c l e i of eukaryotic c e l l s contain two a c t i v i t i e s of DNA-dependent RNA polymerase, namely, the [ | | | Mg -stimulated a c t i v i t y and the Mn /ammonium sulphate-stimulated a c t i v i t y (Windell & Tata, 1966). This view was further strengthened by the h i g h - r e s o l u t i o n autoradiographic studies on the i n t r a n u c l e a r l o c a l i z a t i o n of these two RNA polymerase a c t i v i t i e s (Pogo et^ a l . , 1967; Maul & Hamilton, 1967). The i s o l a t i o n and separation of two RNA polymerases from the nuclear extracts of beef b r a i n (as reported i n t h i s thesis) and of other eukaryotic tissues (Roeder & Rutter, 1969; Chambon et_ a l . , 1970; Goldberg & Moon, 1970; Roeder et_aL, 1970; T o c c h i n i - V a l e n t i n i & Crippa, 1970; Ponta et a l . , 1971; S t r a i n et a l . , 1971) constitutes one of the most convincing pieces of evidence to confirm the e a r l i e r proposal of Windell and I | Tata (1966). Moreover, the Mg -stimulated a c t i v i t y (designated I | RNA polymerase I) and the Mn /ammonium sulphate-stimulated a c t i v i t y (designated RNA polymerase II) has been l o c a l i z e d i n the n u c l e o l a r - 117 -and nucleoplasmic regions r e s p e c t i v e l y (Roeder & Rutter, 1970). From the viewpoint of r e g u l a t i o n of RNA synthesis i n eukaryotic organisms, the occurrence of s e l e c t i v e l y l o c a l i z e d DNA-dependent RNA polymerases merits important a t t e n t i o n . The synthesis of ribosomal RNA takes place i n the nucleolus (Brown & Gurdon, 1964; Penman et a l . , 1966; Perry, 1967; Reeder & Brown, 1971) whereas DNA-like RNA synthesis occurs predominantly i n the nucleoplasm (Perry et a l . , 1964; Georgiev, 1967), the extranucleolar region of the nucleus which contains the bulk of the nuclear DNA. Since polymerase I i s of the nucleolar o r i g i n while polymerase II i s l o c a l i z e d i n the nucleoplasm (Roeder & Rutter, 1970), these enzymes may be implicated i n the s p e c i f i c t r a n s c r i p t i o n of nucleolar genes and extranucleolar genes, r e s p e c t i v e l y . Rutter and h i s c o l -laborators (1970) have substantiated t h i s hypothesis based on t h e i r f i n d i n g that the RNA synthesized by i s o l a t e d n u c l e i i n the presence of a-amanitin i s predominantly ribosomal RNA while i n the absence of t h i s t o x i n i t i s non-ribosomal nuclear RNA, as determined by hybridization-competition assays. Thus i t i s l i k e l y that poly-merase I functions i n the t r a n s c r i p t i o n of nucleolar DNA (tran-s c r i p t s being p r i m a r i l y ribosomal RNA) whereas polymerase II i s i n -volved i n the t r a n s c r i p t i o n of a greater range of nucleoplasmic genes (producing more of DNA-like RNA or messenger RNA). In a d d i t i o n , - 118 -i f polymerase I I I i s a nuclear component then i t may have a unique t r a n s c r i p t i v e r o l e i n the extranucleolar regions (perhaps t r a n s f e r RNA?) ( B l a t t i et a l . , 1970). In e a r l i e r work with i s o l a t e d n u c l e i , the Mg -primed and I | Mn /ammonium sulphate-stimulated RNA polymerase a c t i v i t i e s were found to make GC-rich RNA (ribosomal type) and AU-rich (DNA-like), r e s p e c t i v e l y (Windell & Tata, 1966). In these studies i t was also shown that the e f f e c t of ammonium sulphate on Mn -primed polymerase a c t i v i t y i s perhaps r e l a t e d to the dis s o c a t i o n of c e r t a i n chromo-somal proteins with the r e s u l t a n t a c t i v a t e d t r a n s c r i p t i o n of those genes which code f o r DNA-like RNA. L i a u et al. (1965) have demon-stra t e d that p a r t i a l removal of acid-soluble proteins from the i s o l a t e d n u c l e o l i s h i f t s the base composition of the RNA synthesized i n v i t r o (from GC-rich to AU-rich). These observations imply that the chromosomal proteins associated with the endogenous templates (nucleolar and extranucleolar DNA) may r e s t r i c t , i n part, the i n -volvement of separate RNA polymerases i n the s e l e c t i v e synthesis of ribosomal or messenger RNA even though these enzymes are found i n a s s o c i a t i o n with t h e i r respective templates. Thus pr o t e i n f a c t o r s , perhaps s i m i l a r to those necessary f o r s p e c i f i c t r a n s c r i p t i o n during bacteriophage i n f e c t i o n (see Travers, 1971; Bautz et a l . , 1970; Summers & S i e g e l , 1970; Hager et a l . , 1970), provide a good means of reg u l a t i n g the t r a n s c r i p t i o n of s p e c i f i c genes by separate RNA - 119 -polymerases. In t h i s regard, i t i s important to point out that c e r t a i n protein factors have been described very r e c e n t l y which are capable of modulating the a c t i v i t y of RNA polymerases from the n u c l e i of green coconuts, Cocus n u c i f e r a (Mondal et a l . , 1972). Furthermore, many hormones have been shown to a c t i v a t e gene t r a n s c r i p t i o n (see Tata, 1966). Although the mechanism of I | a c t i o n i s not yet clear i t appears as i f the Mg -primed RNA polymerase a c t i v i t y i s s e l e c t i v e l y modulated i n response to s e v e r a l hormones (Pegg & Korner, 1965; Tata, 1966; Sereni & Barnabei, 1967; Lukacs & Sekeris, 1967; Hamilton e_t aj.. , 1968; Jacob et a l . , 1969; Yu & Feigelson, 1971). Recently hydrocortisone was shown to i n -crease the a c t i v i t y of RNA polymerase I i s o l a t e d from r a t l i v e r n u c l e i a f t e r the i n vivo administration of the hormone (Sajdel e_t a l . , 1971). These workers have presented evidence to show that hydrocortisone stimulates the polymerase I a c t i v i t y by an a l i o s t e r i c mechanism. Smuckler and Tata (1971) have observed s i g n i f i c a n t i n -creases i n the l e v e l s of polymerase I i s o l a t e d from the hepatic n u c l e i of ra t s treated with growth hormone or t r i i o d o t h y r o n i n e . These findings do suggest that the t r a n s c r i p t i v e r o l e of RNA i s profoundly under the c o n t r o l of c e r t a i n hormones. However, the p r e c i s i o n of t h i s c o n t r o l mechanism remains to be e s t a b l i s h e d . The present knowledge about the eukaryotic RNA polymerase I and II i s inadequate to permit a d e f i n i t e i n t e r p r e t a t i o n - 120 -of t h e i r f u n c t i o n i n g i n the s e l e c t i v e s y n t h e s i s of ribosomal and messenger RNA, r e s p e c t i v e l y . N evertheless, the exi s t e n c e o f m u l t i p l e forms of RNA polymerase could reasonably be i m p l i c a t e d i n the s y n t h e s i s of d i s t i n c t species of c e l l u l a r RNA i n h i g h e r organisms. Although there i s great p o t e n t i a l i n mediating a r e g u l a t i o n of gene t r a n s c r i p t i o n by d i f f e r e n t RNA polymerases, the mechanism of such a c o n t r o l system i s f a r from being under-stood. This i s p o s s i b l y due to the f a c t that many a d d i t i o n a l c o n t r o l mechanisms, such as those i n v o l v i n g chromosomal macro-molecules and hormones, operate i n eukaryotes and thus complicate any fundamental system of r e g u l a t i o n . Therefore, the t r a n s c r i p t i v e s p e c i f i c i t y does not appear to be s o l e l y due to d i s t i n c t s p e c i e s of RNA polymerase l o c a l i z e d s p e c i f i c a l l y w i t h i n the v a r i o u s nuclear s t r u c t u r e s (e.g. n u c l e o l u s and nucleoplasm), but the t r a n s c r i p t i o n a l t r a n s i t i o n s could s t i l l be brought about by other f a c t o r s . 4.2. MODULATION OF RNA POLYMERASE ACTIVITY BY POLYAMINES As discussed i n Chapter One, polyamines have been p o s t u l a t e d to play an important r o l e i n the c o n t r o l of RNA metabolism, p o s s i b l y by modulating some phase i n RNA s y n t h e s i s . In a d d i t i o n , polyamines have been shown to enhance the RNA s y n t h e s i z i n g c a p a c i t y of i s o l a t e d n u c l e i (MacGregor & Mahler, 1967; Dutton & Mahler, 1968; Calda r e r a et a l . , 1968; B a r b i r o l i et a l . , 1971a). The data reported i n t h i s - 121 -thesis demonstrate that the RNA polymerase i s o l a t e d from beef b r a i n n u c l e i i s s i g n i f i c a n t l y stimulated by spermine or spermidine. The e f f e c t of spermidine i s much more pronounced than spermine, and i t stimulates both the RNA polymerases separated from b r a i n n u c l e i . The magnitude of stimu l a t i o n by spermidine of b r a i n nuclear poly-merase II i s much greater than that of polymerase I, when these enzymes are assayed i n the presence of t h e i r preferred d i v a l e n t c a t i o n . Moreover, the influence of spermidine on the polymerase II a c t i v i t y i s found to be s e l e c t i v e with respect to i t s preferred DNA template. Spermidine stimulated Mn -primed enzyme a c t i v i t y ( i . e . polymerase II) with both native and heat-denatured DNA as the templates. However, the stimulatory e f f e c t of spermidine with heat-denatured DNA was eit h e r l o s t or suppressed i f Mg was added j | to the Mn -containing assay system, and spermidine was i n h i b i t o r y I | rather than stimulatory i f Mg was the only d i v a l e n t cation. These observations are inte r p r e t e d to mean that nucleoplasmic RNA polymerase (or polymerase II) and consequently, the synthesis of DNA-like RNA may be p r e f e r e n t i a l l y stimulated by polyamines such as spermidine. In a preliminary report, R u s s e l l et a_l. (1971) have shown that polyamines elevate RNA polymerase a c t i v i t y of rat l i v e r n u c l e o l i I | above that l e v e l found with other stimulating cations (e.g. Mg , I | j Mn , NH. , e t c . ) . The same workers also observed that anucleolate - 122 -mutants of Xenopus l a e v i s , which lack the capacity to synthesize ribosomal RNA, do not synthesize normal amounts of putrescine and spermidine (Russell, 1971). They inte r p r e t e d t h e i r data to i n d i c a t e an a c t i v a t i o n of ribosomal RNA synthesis by polyamines. T h i s , i n f a c t , contrasts with the observations of polyamine e f f e c t s on b r a i n nuclear RNA polymerase T and polymerase I I , as recorded i n t h i s t h e s i s . On the other hand, the data presented here on the spermidine stim u l a t i o n of separated enzymes are s i m i l a r to those obtained by others (Stirpe & Novello, 1970; B a r b i r o l i et a l . , 1971a). These I | i n v e s t i g a t o r s have observed a greater s t i m u l a t i o n of Mn /ammonium I | sulphate-primed a c t i v i t y than the Mg -primed a c t i v i t y by polyamines. Moreover, based upon the analysis on methylated albumin kieselguhr columns of RNA synthesized i n spermine-treated and untreated chick embryos, i t has been resolved that the magnitude of increase by spermine i n the synthesis of DNA-like RNA i s much greater than that of ribosomal RNA ( B a r b i r o l i et a l . , 1971b). The mechanism of stimu l a t i o n of DNA-dependent RNA polymerase a c t i v i t y by spermidine i s very f a r from being understood. A possible ac t i o n of spermidine i n vivo might be displacement of histones from the DNA template (endogenous template i n interphase nucleus being chromatin) with r e s u l t a n t a c t i v a t i o n of the genetic t r a n s c r i p t i o n . This does not appear to be the case, at l e a s t , as resolved by r e -c o n s t i t u t i o n experiments which indi c a t e d that spermidine could not - 123 -overcome the h i s t o n e - i n h i b i t i o n . However, such a p o s s i b i l i t y f o r spermidine a c t i o n cannot be excluded based on these experiments. Using E. c o l i RNA polymerase i t has been shown that more RNA chains are i n i t i a t e d i n the presence of spermidine (Peterson et^ a l . , 1968). The r e s u l t s reported i n t h i s t h e s i s show that under the i n -f l u e n c e of spermidine more RNA chains are s y n t h e s i z e d by b r a i n n u c l e a r RNA polymerase I I w i t h c a l f thymus DNA as template. I t , t h e r e f o r e , may be reasonable to assume t h a t , at l e a s t i n v i t r o , spermidine may act by causing the r e l e a s e of the RNA product from the Enzyme-DNA-RNA complex which i s formed during the course of the r e a c t i o n . This view gains some support from the f i n d i n g that sper-midine could counteract the i n h i b i t o r y e f f e c t of yeast RNA on b r a i n n u c l e a r RNA polymerase I I . Moreover, the a v a i l a b l e evidence suggests that the polyanions such as tRNA i n h i b i t E. c o l i RNA polymerase by preventing the b i n d i n g of the enzyme to DNA template, and thus i n h i b i t the i n i t i a t i o n of RNA chains (Richardson, 1966). Since spermidine could circumvent the i n h i b i t i o n of RNA polymerase I I a c t i v i t y by yeast RNA, i t i s q u i t e p o s s i b l e that spermidine stimu-l a t e s the i n i t i a t i o n of RNA c h a i n s , not n e c e s s a r i l y from d i f f e r e n t segments of DNA. In doing so, spermidine could act e i t h e r by a c c e l e r a t i n g the b i n d i n g of the enzyme to DNA template (perhaps s p e c i f i c a l l y at the i n i t i a t i o n s i g n a l s ) or by s t a b i l i z i n g the Enzyme-DNA complex. - 124 -4.3. TRANSCRIPTION OF BRAIN CHROMATIN BY MAMMALIAN RNA POLYMERASE Based on the strategy, as discussed i n the Introductory Chapter, the template a c t i v i t y of b r a i n chromatin was studied using a homolo-gous or a heterologous RNA polymerase. The data presented i n t h i s t hesis demonstrate that the capacity of the i s o l a t e d chromatin from beef b r a i n to function as a template for RNA synthesis i n v i t r o by b r a i n nuclear RNA polymerase II i s markedly repressed. In accord with e a r l i e r reports (Huang & Bonner, 1962; A l l f r e y e_t a l . , 1963; Paul & Gilmour, 1966; Georgiev et a l . , 1966) only 20-25% of the normal t r a n s c r i p t i o n , such as that occurring on pure DNA, i s observed by b r a i n polymerase II and also by E. c o l i enzyme. These observations suggest that most of the genome i n nerve c e l l s i s somehow repressed, r e i n f o r c i n g the support for the "masking" hypothesis of gene tra n -s c r i p t i o n i n higher organisms (see Bonner et_ a_l., 1968b; Paul et_ a l . , 1970). The data, such as those r e f e r r e d to above, on the template a c t i v i t y of the i s o l a t e d chromatin preparations by RNA polymerase are d i f f i c u l t to r e c o n c i l e with the recent observations made on the structure of mammalian chromatin (Clark & F e l s e n f e l d , 1971; I t z a h k i , 1971). These workers, based on t h e i r physico-chemical studies i n -volving the t i t r a t i o n of chromatin with the polymers of b a s i c amino acids and the di g e s t i o n by nucleases, have suggested that nearly 35-50% of the DNA i n chromatin i s "naked" and not covered with - 125 -p r o t e i n s . However, i t i s noteworthy that the measurement of RNA s y n t h e s i s on chromatin template by exogenously added RNA polymerase i s a f u n c t i o n of b i o l o g i c a l a c t i v i t y which, i n t u r n , i s dependent on a v a r i e t y of parameters. Thus, the s m a l l discrepancy i n these r e s u l t s i s most l i k e l y r e f l e c t e d by the d i f f e r e n t nature of the experiments. A group of DNA-bound b a s i c p r o t e i n s , the h i s t o n e s , have long been suggested as the repressors of gene expression (Stedman & Stedman, 1950). The r e p r e s s o r f u n c t i o n of histones i s i n d i c a t e d by the f a c t that the s e l e c t i v e removal of h i s t o n e s by a c i d - or detergent-treatment r a i s e s the template c a p a c i t y of the i s o l a t e d chromatin to the same l e v e l as that obtained w i t h pure DNA as template f o r b a c t e r i a l RNA polymerase (Marushige & Bonner, 1966; Paul & Gilmour, 1966; Georgiev et, a l . , 1966; Smart & Bonner, 1971). Moreover, the a c t i v i t y of DNA-dependent RNA polymerase i s g r e a t l y suppressed by histones i n v i t r o (see H n i l i c a , 1967; Georgiev, 1969; Spelsberg et a l . , 1969; and F i g u r e 18 of t h i s t h e s i s ) . A s i m i l a r p a t t e r n of the template a c t i v i t y of d e h i s t o n i z e d chromatin from b r a i n chromatin (the chromatin which had been t r e a t e d w i t h 0.4 N H^SO^ to remove h i s t o n e s ) i s observed using E. c o l i RNA polymerase. However, t h i s p a t t e r n v a r i e s w i t h b r a i n nuclear RNA polymerase II which, i n f a c t , i s about 3-4 times more a c t i v e w i t h d e h i s t o n i z e d chromatin than w i t h pure DNA as the template. This o b s e r v a t i o n i m p l i e s that an a c t i v a t i n g mechanism of the DNA t r a n s c r i p t i o n e x i s t s - 126 -i n the g e n e t i c m a t e r i a l (chromatin) of b r a i n c e l l s which i s only d e t e c t a b l e i n a r e c o n s t i t u t e d homologous system but not i n a heterologous system. Since RNA polymerase I I from b r a i n n u c l e i p r e f e r s heat-denatured DNA r a t h e r than n a t i v e DNA (see Table VI of t h i s t h e s i s ) , i t may be argued that the treatment of n a t i v e chromatin w i t h a c i d might have caused the den a t u r a t i o n of chromatin DNA and t h a t i s why d e h i s t o n i z e d chromatin i s the p r e f e r r e d template to pure DNA f o r t h i s enzyme. However, i t has been documented that the t r e a t -ment of chromatin w i t h 0.4 N H^SO^ at 0°C (the c o n d i t i o n s u t i l i z e d i n the experiments d e s c r i b e d i n t h i s t h e s i s ) does not b r i n g about the de n a t u r a t i o n of chromatin DNA ( f o r d i s c u s s i o n see Bonner e_t a l . , 1968a; H n i l i c a , 1 9 6 7 ; Murray, 1969). The above mentioned argument may a l s o be excluded based on the f i n d i n g that E. c o l i RNA polymerase u t i l i z e s d e h i s t o n i z e d chromatin as a template as good as pure DNA. Such a r e s u l t would not be expected i f the DNA i n d e h i s t o n i z e d chromatin had undergone de n a t u r a t i o n because w i t h de-natured DNA as the template E. c o l i enzyme i s very p o o r l y a c t i v e (Chamberlin & Berg, 1962; Hurwitz et al., 1962; Furth & Loh, 1964; Stevens & Henry, 1964). Furthermore, i t has been shown that ' n i c k s ' i n DNA template (produced by l i m i t e d d i g e s t i o n w i t h DNase) s t i m u l a t e t r a n s c r i p t i o n - 127 -by b a c t e r i a l RNA polymerase (Vogt, 1969). Therefore, i t may be questioned whether the a c t i v a t e d t r a n s c r i p t i o n of b r a i n d e h i s t o n i z e d chromatin by b r a i n RNA polymerase I I i s a consequence of some nu-cl e a s e a c t i o n . In view of the ob s e r v a t i o n that the a c t i v a t e d t r a n -s c r i p t i o n of b r a i n d e h i s t o n i z e d chromatin i s c a t a l y z e d by b r a i n enzyme only and not by E. c o l i RNA polymerase, the p o s s i b i l i t y of nuclease e f f e c t appears very u n l i k e l y . Although the mechanism of a c t i v a t e d t r a n s c r i p t i o n , such as observed h e r e i n , i s not c l e a r , chromosomally-bound macromolecules (e.g. chromosomal RNA or a c i d i c p r o t e i n s ) may be i m p l i c a t e d i n t h i s phenomenon. There i s no evidence f o r d e c i d i n g i f chromosomal RNA or a c i d i c p r o t e i n s are s o l e l y r e s p o n s i b l e f o r t h i s e f f e c t because d e h i s t o n i z e d chromatin contains both of them. In t h i s connection, i t i s of p a r t i c u l a r importance that non-histone p r o t e i n s have been shown to a c t i v a t e the chromatin-templated b a c t e r i a l RNA polymerase (Teng & Hamilton, 1969; Kamiyama & Wang, 1971; Teng et a l . , 1971; Kostraba & Wang, 1972) . Moreover, i t has been reported that non-hi s t o n e p r o t e i n s which s t i m u l a t e chromatin-templated t r a n s c r i p t i o n have p r o t e i n kinase a c t i v i t y (Kamiyama & Dastugue, 1971). I t i s p o s s i b l e that non-histone p r o t e i n s can a c t i v a t e the chromatin t r a n s c r i p t i o n e i t h e r by n e u t r a l i z i n g the i o n i c - c h a r g e e f f e c t s or by cou n t e r a c t i n g the h i s t o n e - r e p r e s s i o n . Therefore, the p o s s i b i l i t y of involvement of chromosomal RNA as a gene a c t i v a t o r , as advanced by Bonner and h i s c o l l a b o r a t o r s (Bekhor et a l . , 1969; M a y f i e l d & Bonner, 1971) cannot be excluded. - 128 -If the non-histone proteins or the chromosomal RNA function as gene a c t i v a t o r s i n the process of genetic t r a n s c r i p t i o n , as discussed above, then RNA synthesis on dehistonized chromatin should be greater than when pure DNA i s used as the template. But this i s not so because b a c t e r i a l RNA polymerase transcribes dehistonized chromatin or pure DNA without any preference (Maru-shige & Bonner, 1966; Paul & Gilmour, 1966; Smart & Bonner, 1971; also t h i s t h e s i s ) . This a n t i c i p a t e d function i s , i n f a c t , resolved by u t i l i z i n g a homologous enzyme system. Thus, i n the former case, the f a i l u r e to detect the a n t i c i p a t e d template a c t i v i t y of the dehistonized chromatin might be due to the use of a heterologous enzyme. At present, i t i s d i f f i c u l t to v i s u a l i z e a d e f i n i t e explanation f o r t h i s kind of discrepancy but two comments are worth making: ( i ) polymerases of d i f f e r e n t o r i g i n s might be highly s e l e c t i v e i n t h e i r a c t i o n , perhaps i n the step of chain-i n i t i a t i o n , and ( i i ) the molecular o r i e n t a t i o n of macromolecules i n the dehistonized chromatin may enforce the s p e c i f i c i t y with homologous RNA polymerase but not with heterologous enzyme. There-fo r e , i t i s considered very l i k e l y that the t r a n s c r i p t i o n a l c o n t r o l mechanisms which operate at the l e v e l of RNA polymerase are highly s p e c i f i c i n mammalian c e l l s . - 129 -4.4. CONCLUDING REMARKS The observations recorded i n t h i s t h e s i s on the t r a n s c r i p t i o n of genes by RNA polymerase i n b r a i n c e l l s , are summarized as f o l l o w s : (1) The c o n d i t i o n s are developed which s o l u b i l i z e d i n h i g h y i e l d s the enzyme RNA polymerase from the n u c l e i of b r a i n c e l l s . By chromatographic a n a l y s i s , the s o l u b i l i z e d enzyme i s separated i n t o two peaks of RNA polymerase a c t i v i t y , designated as RNA polymerase I and polymerase I I . (2) These two polymerases are completely dependent upon exogenously added DNA template. RNA polymerase I p r e f e r s n a t i v e DNA to heat-denatured DNA as template w h i l e RNA polymerase I I u t i l i z e s heat-denatured DNA much b e t t e r than n a t i v e DNA as template. (3) Both the polymerases e x h i b i t an optimum pH around pH 8.0. Th e i r a c t i v i t i e s are g r e a t l y s e n s i t i z e d by actinomycin D. [ | | | (4) The polymerase I r e q u i r e s Mg i n preference to Mn as the d i v a l e n t c a t i o n whereas polymerase I I i s about four times as I | | | a c t i v e w i t h Mn as w i t h Mg . Both the enzymes e x h i b i t maximal I | | | a c t i v i t y i n the presence of Mn plus Mg (at t h e i r optimum con-c e n t r a t i o n s ) . (5) Two RNA polymerases from b r a i n c e l l n u c l e i respond to d i f f e r e n t KCl p r o f i l e s . The a c t i v i t y of polymerase I i s somewhat enhanced by 0.05 M K C l , but i t i s s i g n i f i c a n t l y i n h i b i t e d as the - 130 -concentration of KCI i s r a i s e d to 0.2 M and above. KCI markedly stimulates the polymerase II a c t i v i t y with an optimum concentration around 0.2 M. The stimu l a t i o n by KCI of polymerase II i s much more I | | | | | pronounced i n the presence of Mn or Mn plus Mg than i n the I | presence of Mg alone. (6) RNA polymerase II i s almost completely i n h i b i t e d by the toxin a-amanitin while the a c t i v i t y of polymerase I i s not af f e c t e d by t h i s t o x i n . (7) The polyamines, such as spermidine or spermine, sharply enhanced the a c t i v i t y of both the polymerases. Spermidine exerts a much more pronounced stimulatory e f f e c t on polymerase I I than polymerase I, p a r t i c u l a r l y , when assayed i n the presence of t h e i r preferred divalent cation. The enhancement of polymerase I I a c t i v i t y by spermidine i s also c h a r a c t e r i s t i c of the preferred template f o r t h i s enzyme. (8) The ad d i t i o n of yeast RNA and c a l f thymus histone r e s u l t s i n a considerable degree of i n h i b i t i o n of polymerase II a c t i v i t y . Spermidine appears to counteract the i n h i b i t i o n due to yeast RNA, but not that due to histone. (9) The product of the r e a c t i o n of RNA polymerase I I s e d i -ments around 18 S i n a sucrose-density gradient and apparently i s a complex of the kind Enzyme-DNA-RNA. - 131 -(10) The c a p a c i t y of the i s o l a t e d c e r e b r a l chromatin to serve as a template f o r RNA s y n t h e s i s by a homologous or a heterologous RNA polymerase i s very much lower than that of the pure DNA. Most probably, the a c i d - s o l u b l e chromosomal p r o t e i n s are r e s p o n s i b l e f o r t h i s g r e a t l y reduced template a c t i v i t y of chromatin. (11) The b r a i n n u c l e a r RNA polymerase I I i s 3-4 times more a c t i v e w i t h a c i d - t r e a t e d chromatin than pure DNA as the template, but E. c o l i RNA polymerase uses these two templates without any preference. (12) The RNA s y n t h e s i z e d on chromatin templates ( n a t i v e or acid-treated) by b r a i n polymerase I I i s somewhat s m a l l e r i n s i z e than that made on pure DNA. In the l i g h t o f accumulating evidence that mammalian RNA polymerase d i s p l a y s vast m u l t i p l i c i t y and that the template a c t i v i t y of a more p h y s i o l o g i c a l template can be modulated i n -v a r i a b l y , a dual nature of the t r a n s c r i p t i o n a l c o n t r o l i s a n t i c i -pated. The m u l t i p l e nature of RNA polymerase may p r i m a r i l y be i m p l i c a t e d i n the s y n t h e s i s of d i f f e r e n t types of c e l l u l a r RNA. On the other hand, the template c h a r a c t e r i s t i c s are l i k e l y to be of the utmost importance i n e n u n c i a t i n g the s e c r e t s of c e l -l u l a r d i f f e r e n t i a t i o n . The b r a i n c e l l s are probably no exception to the above statement. - 132 -In s p i t e of the recent advances that have been made i n the f i e l d of mammalian RNA polymerase, a great deal s t i l l remains to be understood, e.g. how m u l t i p l e RNA polymerases recognize the s i g n a l s f o r RNA chain i n i t i a t i o n and termination? What i s the s t r u c t u r e - f u n c t i o n a l r e l a t i o n s h i p i n d i f f e r e n t RNA polymerases? I t i s hoped that f u t u r e r e s e a r c h , p a r t i c u l a r l y i n these areas, w i l l l e a d to major break-throughs i n e l u c i d a t i n g the mechanism of c o n t r o l of RNA s y n t h e s i s i n mammalian c e l l s . The enhancement of the RNA polymerase a c t i v i t y by polyamines i n v i t r o occurs i n those concentrations of polyamines which are w i t h i n the p h y s i o l o g i c a l range. Although such an e f f e c t i s w e l l documented, yet the p h y s i o l o g i c a l involvement of polyamines i n the process of gene t r a n s c r i p t i o n remains to be e s t a b l i s h e d . The p r e f e r e n t i a l s t i m u l a t i o n of polymerase I I by spermidine may sug-gest that the s y n t h e s i s of DNA-like RNA ( i . e . messenger RNA) i s markedly i n f l u e n c e d by polyamines. The data presented here on the t r a n s c r i p t i o n of chromatin DNA by a homologous RNA polymerase r e v e a l c e r t a i n important c h a r a c t e r i s t i c s which are q u a n t i t a t i v e l y d i s t i n c t from those observed using a heterologous enzyme. 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