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UBC Theses and Dissertations

Studies on the processing of polyoma virus RNA Petric, Martin 1973

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STUDIES ON THE PROCESSING OF POLYOMA VIRUS RNA by MARTIN PETRIC B.Sc. McMaster U n i v e r s i t y M.Sc. McMaster U n i v e r s i t y A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF FOR THE DEGREE OF PHILOSOPHY In the Department of M i c r o b i o l o g y We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May 1973 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f )V\ / c i<jJ The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date M4<f. /</ / ??Jr ABSTRACT The main o b j e c t i v e of these s t u d i e s was to analyze the processing of polyoma v i r u s RNA t r a n s c r i p t s . An examination was made of the i n t r a c e l l u l a r d i s t r i b u t i o n of polyoma v i r u s RNA molecules synthesized l a t e during productive i n f e c t i o n i n mouse kidney c e l l s . The v i r u s s p e c i f i c RNA from whole c e l l s , and t h e i r nuclear, cytoplasmic and polyribosomal f r a c t i o n s were compared w i t h respect t o sedimentation behaviour (on sucrose g r a d i e n t s ) , e l e c t r o -p h o r e t i c m o b i l i t y (on polyacrylamide g e l s ) and base sequence homology (by competition h y b r i d i z a t i o n t e s t s ) . Sedimentation and e l e c t r o p h o r e t i c a n a l y s i s revealed marked het e r o g e n e i t y i n polyoma RNA from a l l c e l l f r a c t i o n s . This heterogeneity and s i z e d i s t r i b u t i o n were e s s e n t i a l l y the same on d i m e t h y l s u l f o x i d e sucrose g r a d i e n t s . The v i r a l RNA resedimented t r u e to i t s o r i g i n a l d i s t r i b u t i o n on a sucrose gradient. The polyoma RNA from d i f f e r e n t regions of a sucrose g r a d i e n t contained common sequences as rev e a l e d by c r o s s - c o m p e t i t i o n h y b r i d i z a t i o n experiments. However, the p o l y -ribosome a s s o c i a t e d polyoma RNA was devoid of the ^>28s species found i n the other f r a c t i o n s . Thus i t appeared t h a t polyoma RNA, i n c l u d i n g RNA of apparent s i z e i n excess of one genome len g t h , was synthesized i n the nucleus and cleaved t o smaller peices i n a s s o c i a t i o n w i t h the p o l y r ibo some s. i i The accumulation of the polyoma RNA i n the nuclear f r a c t i o n pro-ceded at a r a t e s i m i l a r to t h a t of the c e l l u l a r RNA, wh i l e the c o r r e s -ponding r a t e of accumulation of v i r a l RNA i n the cytoplasmic f r a c t i o n was s i m i l a r t o the c e l l RNA f o r 30 minutes, but d i d not increase, as d i d t h a t of the c e l l RNA, w i t h increased l a b e l l i n g times. The polyoma RNA l a b e l l e d i n the c e l l s during a 15 minute pulse was s i g n i f i c a n t l y l a r g e r (U6$ sedimented f a s t e r than 28s) than t h a t l a b e l l e d f o r 2 hours (25$ sedimented f a s t e r than 28s). Pulse and chase st u d i e s w i t h A c t i n o -mycin D and excess u r i d i n e revealed t h a t the m a j o r i t y (up t o 75$) of the polyoma RNA l a b e l l e d i n a kO minute pulse was subsequently degraded w i t h i n the nucleus w i t h i n one hour of chase, w h i l e the remainder d i s -appeared from the nucleus at a slower r a t e i n the next f i v e hours of chase. I n a d d i t i o n , the l a r g e r (^28s) polyoma RNA i n the nuclear f r a c t i o n was degraded s l i g h t l y f a s t e r than the s m a l l (£l8s) RNA. In the cytoplasmic f r a c t i o n , the q u a n t i t y of l a b e l l e d v i r a l RNA decreased s l o w l y during the chase p e r i o d and a l l s i z e c l a s s e s were degraded at approximately the same r a t e . Polyadenylate sequences were found a s s o c i a t e d w i t h polyoma RNA. The percentage of the v i r a l RNA molecules c o n t a i n i n g polyadenylate sequences increased as the RNA was processed from the n u c l e i through t o the polyribosomes, suggesting t h a t o n l y p o l y (A) c o n t a i n i n g polyoma RNA molecules can be p r o p e r l y processed. The methodology u t i l i z e d i n the i s o l a t i o n and d e t e c t i o n of p o l y (A) sequences was examined i n more d e t a i l . The method of i s o l a t i o n of p o l y (A) sequences, by b i n d i n g to p o l y (u) f i x e d to g l a s s f i b e r f i l t e r s , was found to be h i g h l y dependent on the s a l t c o n c e n t r a t i o n of the b i n d i n g b u f f e r , through i t d i d s e l e c t f o r non-ribosomal heterogeneous c e l l u l a r RNA which had ribonuclease r e s i s t a n t p o l y (A) s t r e t c h e s . The method of e x t r a c t i o n of the RNA was found to have a considerable e f f e c t on the subsequent b i n d i n g p r o p e r t i e s of these molecules. i v Table of Contents Page CAHPTER I: INTRODUCTION 1. H i s t o r y 1 2. Nomenclature 1 3. V i r u s P r o p e r t i e s 2 a. Morphology 2 b. P h y s i c a l and chemical p r o p e r t i e s 2 i . The py v i r i o n 2 i i . Hemagglutinins 3 i i i . Resistance to p h y s i c a l and chemical agents 3 i v . Polyoma DNA k v. P r o t e i n s 5 k. The V i r u s I n f e c t e d C e l l 6 5. The Productive or L y t i c I n f e c t i o n 8 a. Adsorption and p e n e t r a t i o n 8 b. E a r l y RNA synthesis 9 c. Enzyme i n d u c t i o n 9 d. DNA synthesis 10 e. V i r a l RNA synthesis 11 f. V i r a l p r o t e i n synthesis ih 6. Biochemistry of RNA Synthesis i n Mammalian C e l l s . . Ik a. Processing of RNA ih b. Polyadenylate sequences 17 7. Purpose of the Research P r o j e c t 18 V Table of Contents Page CHAPTER I I : A. .MATERIALS 1. C e l l s 19 2. V i r u s 19 3. Growth Medium and Other B i o l o g i c a l Compounds . . . 19 k. Chemicals 21 5. S o l u t i o n s 22 B. METHODS 1. Primary Mouse Kidney C e l l C u l t u r e s . . . . 26 2. Secondary Mouse Embryo C e l l C ultures 27 3. BHK-21 C e l l s and PyH C e l l s 27 h. Growth of Polyoma V i r u s 28 a. The i n v i t r o i n f e c t i o n . . . . 28 b. The i n v i v o i n f e c t i o n 28 c. Further treatment and a n a l y s i s of the py v i r u s p r e p a r a t i o n 29 d. Hemagglutination assay 29 5. P r e p a r a t i o n of Polyoma Type I DNA (py I DNA) . . . 30 6. L a b e l l i n g and Pr e p a r a t i o n of RNA from py I n f e c t e d C e l l s 32 a. L a b e l l i n g . 32 b. Phenol e x t r a c t i o n 33 c. A n a l y s i s and storage of RNA 3^ -.vi Table of Contents Page 7. M o d i f i c a t i o n s of the RNA E x t r a c t i o n Method . . . . 3^ a. Use of pronase 3^+ b. Use of phenol-chloroform 35 c. Use of phenol at pH 9.0 35 8. C e l l F r a c t i o n a t i o n 35 a. Cytoplasmic and nuclear e x t r a c t s 35 b. Polyribosomes 3^ 9- P r e p a r a t i o n of Ribosomal RNA 37 10. F r a c t i o n a t i o n of RNA 38 a. Sucrose gradients 38 b. Polyacrylamide g e l e l e c t r o p h o r e s i s 38 11. H y b r i d i z a t i o n 39 a. Denaturation and f i x a t i o n of DNA 39 b. DNA-RNA h y b r i d i z a t i o n kO 12. P o l y (A) Studies hi a. F i l t r a t i o n through M i l l i p o r e f i l t e r s . . . . kl b. F i l t r a t i o n through p o l y (U)-GF/C f i l t e r s . . . kl CHAPTER I I I : RESULTS-I : C h a r a c t e r i z a t i o n of Py RNA i n I n f e c t e d C e l l s 1. Polyoma V i r u s RNA Synthesis During the Course of V i r u s I n f e c t i o n ^3 2. Comparison of Py RNA Synthesis i n MK, ME and BHK-21 C e l l s k6 v i i Table of Contents Page 3. a. Size of py RNA i n i n f e c t e d c e l l s U9 b. Sedimentation on DMSO sucrose gradients . . . ^9 c. Polyacrylamide g e l e l e c t r o p h o r e s i s U9 k. Resedimentation of Py RNA on Sucrose Gradients . . 58 5. Polyoma RNA from Nuclear and Cytoplasmic F r a c t i o n s of V i r u s I n f e c t e d C e l l s 59 6. Leakage of RNA from N u c l e i During F r a c t i o n a t i o n . . 62 7. Polyribosomal RNA 65 8. Comparison of Nuclear and Cytoplasmic RNA by Competition H y b r i d i z a t i o n 68 9- Comparison of Nuclear and Polyribosomal Py RNA by Competition H y b r i d i z a t i o n 71 10. Comparison of Large and Small Py RNA by Competition H y b r i d i z a t i o n 71 CHAPTER IV: RESULTS-II: Processing of Py RNA i n V i r u s I n f e c t e d C e l l C u l t u r e s 1. K i n e t i c s of L a b e l l i n g of Nuclear and Cytoplasmic Py RNA 76 2. Py RNA i n Pulse L a b e l l e d C e l l s 80 3. Pulse and Chase Studies 83 k. Size D i s t r i b u t i o n of Py RNA i n Pulse and Chase Studies . . 87 5. Involvement of Polyadenylate Sequences w i t h Py RNA . 91 v i i i Table of Contents Page CHAPTER V: RESULTS-III: Studies on the I s o l a t i o n and Enumeration of P o l y (A) Containing RNA Molecules 1. Parameters Involved i n B i n d i n g P o l y (A) Containing RNA t o M i l l i p o r e F i l t e r s 95 2. Ribonuclease Resistance of RNA Bound t o M i l l i p o r e . 9^ 3. Capacity, Washing and Sampling of M i l l i p o r e F i l t e r s w i t h Bound RNA 99 a. Washing 99 b. C a p a c i t y of M i l l i p o r e f i l t e r s f o r b i n d i n g RNA a s s o c i a t e d w i t h p o l y (A) sequences 102 c. Sampling of M i l l i p o r e f i l t e r s 103 k. B i n d i n g of RNA to M i l l i p o r e , N i t r o c e l l u l o s e and t o P o l y - u r i d y l a t e - G l a s s F i b e r F i l t e r s 10k 5. E f f e c t s of S a l t Concentration on the I n t e r a c t i o n Between C e l l u l a r RNA and P o l y (u)-GF/C F i l t e r s . . 107 o 6. B i n d i n g of H-Adenosine L a b e l l e d RNA t o Other P o l y n u c l e o t i d e s 107 7. E l u t i o n of RNA from P o l y (U)-GF/C F i l t e r s . . . . 112 8. B i n d i n g of Ribosomal RNA t o P o l y (U)-GF/C F i l t e r s . 115 9. Sucrose Gradient Sedimentation of RNA which was Bound and E l u t e d from P o l y (U)-GF/C F i l t e r s . . . 118 10. E x t r a c t i o n of RNA from I n f e c t e d Mouse Kidney C e l l s by Three D i f f e r e n t Techniques 121 i x Table of Contents Page CHAPTER VI: DISCUSSION 1. V i r a l RNA Synthesis i n I n f e c t e d C e l l s 125 2. Size D i s t r i b u t i o n of Py RNA i n I n f e c t e d C e l l s . . . 126 3. Py RNA i n Nuclear, Cytoplasmic and Polyribosomal F r a c t i o n s 128 k. Processing of Py RNA i n I n f e c t e d C e l l s 132 5. Polyadenylate Sequences 135 6. P o l y (A) I s o l a t i o n Techniques 136 LITERITURE CITED 1^ 2 L i s t of Tables Table Page 1. Py RNA Synthesis i n MK, ME and BHK-21 C e l l s . kQ 2. E l u t i o n of Py RNA from Polyacrylamide Gels. 53 33 P o l y (A) Content of Py I n f e c t e d C e l l RNA. 93 h. Resistance of M i l l i p o r e Bound and Non-Bound 98 RNA t o P a n c r e a t i c Ribonuclease. 5a + b Comparison of RNA Bind i n g t o M i l l i p o r e N i t r o - 106 c e l l u l o s e and P o l y (U)-GF/C F i l t e r s . 6. B i n d i n g of 3n-adenosine L a b e l l e d RNA t o GF/C 111 F i l t e r s Containing P o l y (A) and P o l y (C). 7. B i n d i n g of RNA E x t r a c t e d by Three D i f f e r e n t 123 Methods t o P o l y (U)-GF/C F i l t e r s . L i s t of Figure s T e n t a t i v e Scheme f o r the L y t i c Cycle of Polyoma V i r u s i n C o n t a c t - I n h i b i t e d Primary Mouse Kidney Tissue C u l t u r e s C e l l s . P r o duction of V i r u s and V i r a l RNA i n I n f e c t e d C e l l s . Sedimentation of Py RNA through (A) Sucrose Gradient: (B) DMSO Sucrose Gradient. Polyacrylamide Gel E l e c t r o p h o r e s i s of Py I n f e c t e d C e l l RNA. Resedimentation of py RNA i n a Sucrose Gradient. Sucrose Gradient Sedimentation of Py RNA from (A) N u c l e i and (B) Cytoplasm, of Py In f e c t e d MK C e l l s . Sucrose Gradient Sedimentation of Py RNA from WPkO Cytoplasm of Py I n f e c t e d MK C e l l s . Sucrose Gradient Sedimentation of (A) Cyto-plasmic E x t r a c t of Mock I n f e c t e d C e l l s : (B) Cytoplasmic E x t r a c t of Py I n f e c t e d C e l l s and (c) Polyribosome A s s o c i a t e d RNA of Py In f e c t e d C e l l s . Competition H y b r i d i z a t i o n Between Nuclear and Cytoplasmic RNA. L i s t of Figures Competition H y b r i d i z a t i o n Between Nuclear and Polyribosomal Py RNA. Competition H y b r i d i z a t i o n Between Large and Small Py RNA. V a r i a b l e Pulse L a b e l l i n g of I n f e c t e d C e l l RNA. Sucrose Gradient Sedimentation of Pulse L a b e l l e d Py RNA. Pulse and Chase Studies on I n f e c t e d C e l l RNA. Sucrose Gradient A n a l y s i s of Py RNA from Pulse Chase Study. E f f e c t s of Washing on M i l l i p o r e Bound RNA. The E f f e c t of S a l t Concentration on RNA Bi n d i n g to P o l y (U)-GF/C F i l t e r s . E l u t i o n of RNA from P o l y (U)-GF/C F i l t e r s . B i n d i n g of Ribosomal RNA t o P o l y (U)-GF/C F i l t e r s . Sucrose Gradient Sedimentation of RNA which was Bound and E l u t e d from P o l y (U)-GF/C F i l t e r s . ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. J.J.R. Campbell and Dr. D.M. McLean, for providing this opportunity for study and research. The advice and supervision of Dr. J.B. Hudson is greatly appreciated. Thanks are also due to my committee members, and fellow graduate students, for helpful discussions, and also to L. McGrath, G. Steele, L. Waterfield, and B. Bryant for technical assistance. I am indebted to R. Morgan for the typing of this thesis. Finally, I wish to extend my gratitude to my wife, Naomi for her cooperation and the many sacrifices she made throughout the course of this investigation. ABBREVIATIONS BHK - baby hamster kidney c e l l s cpm - counts per minute "^C - carbon - ik C i - Curie d a l t o n - atomic mass u n i t DNA - deox y r i b o n u c l e i c a c i d DNAse - deoxyribonuclease d-RNA - DNA-like RNA EDTA - e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d FUdR - d e o x y r i b o f l u o r o u r i d i n e 7 - gamma ( r a d i a t i o n ) GF/C - gl a s s f i b e r f i l t e r s - type C G + C - guanine plus cytosine JH - t r i t i u m Hn RNA - heteronuclear RNA HAU - hemagglutination u n i t s m-RNA - messenger RNA ml-RNA - messenger-like RNA ( i l - m i c r o l i t e r laCi - m i c r o c u r i e Hgm - micrograms urn - micrometer (micron) XV mCi - m i l l i c u r i e ME - mouse embryo ( c e l l ) MK - mouse kidney ( c e l l ) MBB - M i l l i p o r e b i n d i n g b u f f e r MEM - minimum e s s e n t i a l medium moi - m u l t i p l i c i t y of i n f e c t i o n nm - nanometer NP-kO - nonidet P-hO PD - p e t r i d i s h (9 cm) 32p _ phosphorus-32 PBS - phosphate b u f f e r e d s a l i n e py - polyoma py-H - polyoma transformed hamster c e l l s p o l y (A) - polyadenylate p o l y (C) - p o l y c y t i d y l a t e p o l y (G) - pol y g u a n i d y l a t e p o l y (T) - polythymidylate p o l y (U) - p o l y u r i d y l a t e p f u - plaque forming u n i t s p i - post i n f e c t i o n ( a f t e r i n f e c t i o n ) PUBB - p o l y u r i d i n e b i n d i n g b u f f e r RNA - r i b o n u c l e i c a c i d r-RNA - ribosomal RNA RNAse - ribonuclease RNP - r i b o n u c l e o p r o t e i n RSB - r e t i c u l o c y t e standard b u f f e r SDS - sodium dodecyl sulphate SVkO•• - simian v i r u s kO S - sedimentation c o e f f i c i e n t (seconds) s-RNA - s o l u b l e RNA ( l M NaCI) STE - sodium t r i s EDTA b u f f e r SSC - standard s a l i n e c i t r a t e t-RNA - t r a n s f e r RNA T-antigen - tumor antigen t r i s - t r i s hydroxymethylaminomethane ( b u f f e r ) UV - u l t r a v i o l e t ( r a d i a t i o n ) 1 CHAPTER I: INTRODUCTION 1. H i s t o r y The p a t h o l o g i c a l m a n i f e s t a t i o n s of polyoma (py) v i r u s i n f e c t i o n were f i r s t observed i n 1951 as p a r o t i d gland tumors i n mice e x p e r i -m e n t a l l y i n j e c t e d w i t h c e l l f r e e e x t r a c t s from leukemic mice (^ +1). This agent was subsequently shown to be present i n h e a l t h y mice under n a t u r a l c o n d i t i o n s but to l e a d r a r e l y to tumor development (^2). In 1958 the v i r u s was f i r s t c u l t i v a t e d i n v i t r o (30) (29) and i t s p r o p e r t i e s were subsequently determined (98). I n i960 i t was observed th a t some py i n f e c t e d mouse and hamster c e l l s d i s p l a y e d a change i n growth p a t t e r n . They l o s t the property of contact i n h i b i t i o n and hence grew i n dense whorls,, although i n the case of the hamster c e l l s l i t t l e or no detectable i n f e c t i o u s v i r u s was produced (1C4). On t r a n s p l a n t a t i o n i n t o newborn hamsters, these c e l l s caused tumors to develop (21). The above observers c a l l e d these c e l l s v i r u s t r a n s -formed c e l l s . 2. Nomenclature The name polyoma was given to the v i r u s by Eddy and Stewart i n 1958 (30). This name i n d i c a t e s that the v i r u s i s capable of causing tumors at many s i t e s i n the i n f e c t e d animal. Because of i t s 2 similarity to simian virus ho (SVhO) and rabbit papilloma, i t has been included in the group papovavirus (71). 3. Virus Properties a. Morphology Electron microscope studies on negatively stained preparations of py virus have shown i t to be kO-k-5 nm in diameter, and lacking a limiting membrane (57). The virus particle has cubic symmetry comprising of 72 capsomers in an icosehedral arrangement (51) (10). b. Physical and Chemical Properties i . The py virion Polyoma virus can be purified by plaque purification (31), by banding in cesium chloride or rubidium chloride, or by sedimentation in sucrose gradients (16) (n6). The latter three of these methods have also proved useful in characterizing the virus. The virus has a density in cesium chloride of 1.32 gm per ml (16), and a sedi-mentation coefficient on sucrose gradients of 250 s (H6). Initial purification of polyoma in CsCl gradients revealed the presence of two types of particle^ of which the heavier, 'with a 3 buoyant density of 1.32 gm per ml, was the DNA containing particle, while the lighter, with a buoyant density of 1.28 gm per ml, was the empty capsid ( l 6 ) . Later work expanded on this observation and showed that the heavy component consisted of infective virions of a buoyant density of 1.33 gm per ml and non infective pseudovirions of a buoyant density of 1.315 gm per ml which contained partly cellular DNA (70). i i . Hemagglutinins Polyoma virus has the ability to agglutinate guinea pig and hamster erythrocytes and human type 0 erythrocytes at k°C, though not at 37°C (30). A useful assay for the virus titer has been developed using this property of the virus (9I4-). i i i . Resistance to physical and chemical agents. Viral infectivity is resistant to diethyl ether (U3), trypsin (30), RNAse or DNAse (2^-), repeated freezing and thawing ( 8 ), or 3 minutes of sonication (3k). Although the viral infectivity is sensitive to ultraviolet radiation, the transforming ability is up to five times less sensitive for an equivalent dosage (63). For storage purposes lyophilization or freezing at -70°C have proven very effective (k3) (kk)• k i v . Polyoma DNA E a r l y i n polyoma research, i t was found t h a t DNA, e x t r a c t e d from p a r o t i d tumors, was capable, on i n j e c t i o n i n t o newborn mice, of causing tumor formation (64) (48). This e f f e c t was abrogated on d i g e s t i n g the DNA w i t h DNAse p r i o r t o i n j e c t i o n . I t was l a t e r shown t h a t py DNA, e x t r a c t e d from p u r i f i e d v i r i o n s grown i n c e l l c u l t u r e s , possessed t h i s tumorgenic p r o p e r t y and was moreover i n f e c t i o u s i n mouse embryo c e l l s (2k) (107). Polyoma DNA was found to have a buoyant d e n s i t y on CsCl of 1,709 gm per ml and a base composition of 48$ G + C (93) (17). This base composition, along w i t h a unique base sequence and i n f e c t i o u s p r o p e r t i e s , d i s t i n g u i s h e s py from other papova v i r u s e s (117). The v i r a l DNA has a molecular weight of 3 x 10^ (15) (109) and comprises about 10 t o 12$ of the mass of the v i r i o n ( l l 6 ) . Polyoma v i r u s DNA may be separated i n t o three d i s t i n c t types c a l l e d type I , I I and I I I . When py DNA i s sedimented on a n e u t r a l pH sucrose gradient i n 0.2 M NaCI, these three types sediment at 20s, l6s and Iks r e s p e c t i v e l y (113). An equivalent s e p a r a t i o n may be achieved by c e n t r i f u g a t i o n on n e u t r a l pH CsCl at a d e n s i t y of 1. 50 gm per ml (119). Under a l k a l i n e c o n d i t i o n s (pH 12.5) py DNA separated i n a d i f f e r e n t manner. I n sucrose gradients at pH 12.5 (52a) or CsCl (103) at a d e n s i t y of 1.50 at pH 12.5, Py DNA separated i n t o three components of which type I sedimented at 53s, while type I I 5 sedimented at l8s and l 6 s , and type I I I at l 6 s . Type I DNA may be separated from types I I and I I I by i s o p y c n i c c e n t r i f u g a t i o n i n n e u t r a l C s C l at a d e n s i t y of 1,600 gm per ml i n the presence of ethidium bromide (83). A s i m i l a r s e p a r a t i o n may a l s o be achieved by the use of hydroxyapatite column chromatography (5). Type I polyoma DNA (py I DNA) has been shown to be a double stranded c i r c u l a r h e l i c a l molecule s u p e r c o i l e d upon i t s e l f . A s i n g l e s t r a n d break l e d t o the conversion of type I i n t o type I I molecules, t h a t i s , the s u p e r c o i l to the open c i r c l e form (113) (103). I t was r e c e n t l y shown that the r e p l i c a t i o n of py DNA i n v o l v e s enzyme mediated st r a n d opening and c l o s i n g (^9). Py DNA I I I was found to have a molecular weight s i m i l a r to types I and I I but a s l i g h t l y lower d e n s i t y on CsCl. This i s b e l i e v e d t o be a r e s u l t of i t being, to a l a r g e p a r t , c e l l u l a r i n o r i g i n . V i r i o n s c o n t a i n i n g t h i s form of c e l l u l a r DNA are c a l l e d pseudovirions and make up v a r y i n g proportions of v i r a l preparations (70). v. P r o t e i n s I n i t i a l s t u d i e s on py p r o t e i n s have revealed t h a t the v i r u s con-t a i n e d one major c a p s i d p r o t e i n or one major p r o t e i n and an i n t e r n a l component (101) (33). More recent data have shown th a t the v i r u s con-t a i n s s i x or seven polypeptides having molecular weights between 86,000 and 15,000. Of these p r o t e i n s , P-2 which i s thought to be a c a p s i d p r o t e i n , accounts f o r 50 t o 70$ of the v i r a l p r o t e i n content. I n a d d i t i o n , there i s evidence t h a t p r o t e i n s P-5 to P-7 are host c e l l h i s t o n e s ( 8 l ) . k. The V i r u s I n f e c t e d C e l l Polyoma v i r u s has been observed to i n t e r a c t w i t h mammalian c e l l s i n three d i f f e r e n t ways. In the a b o r t i v e i n f e c t i o n , which was the predominant i n t e r a c t i o n of py v i r u s w i t h r a t embryo c e l l s , the v i r u s was taken up by the c e l l s , and l e d t o the eventual s t i m u l a t i o n of c e l l u l a r DNA synthesis at 10 t o l 6 hours p i . The v i r u s was e v e n t u a l l y l o s t from most c e l l s and no f u r t h e r e f f e c t s were observed i n the m a j o r i t y of the c e l l s i n the p o p u l a t i o n (89). Hamster c e l l s a l s o took up py v i r u s e f f i c i e n t l y (h). However, only BHK 21 c e l l s showed a s t i m u l a t i o n of DNA synthesis i n the absence of v i r u s r e p l i c a t i o n . I n a d d i t i o n extensive i n t e g r a -t i o n of the v i r a l DNA i n t o the c e l l DNA was observed i n these c e l l s (2) (5^). This s i t u a t i o n a l s o appeared to represent an a b o r t i v e i n f e c t i o n . The second type of i n t e r a c t i o n was transformation. This s i t u -a t i o n occurred i n 0 . 1 to 12$ of the c e l l s i n v i r u s i n f e c t e d hamster or r a t c u l t u r e s (89) (104). The a f f e c t e d c e l l s l o s t t h e i r c a p a c i t y f o r contact i n h i b i t i o n and t h e i r o r i e n t e d growth p a t t e r n , and hence p i l e d up i n heavy whorls. These c e l l s a l s o had a l t e r e d morphology, 7 an increased growth r a t e and a higher c a p a c i t y t o be s u c c e s s f u l l y t r a n s p l a n t e d i n t o animals (101) (22) (99) (67). At l e a s t part of the c h a r a c t e r i s t i c s of the transformed c e l l s i s thought to be due to membrane changes. This phenomenon was r e f l e c t e d by the increased a b i l i t y of the transformed c e l l s to b i n d concanavalin A and be a g g l u t i n a t e d as compared to normal c e l l s ( 9 ) -Transformed c e l l s have been shown to c a r r y on the synthesis of v i r a l DNA and RNA and p o s s i b l y v i r a l p r o t e i n (115) ( 3 ) (36). Only a f r a c t i o n of the v i r a l genome could t r a n s c r i b e i n t o v i r a l RNA (69), the l a t t e r accounting f o r l e s s than 0.025</0 of the t o t a l RNA syn-t h e s i z e d i n the c e l l ( 3 ) (53). A r e l a t i v e l y l a r g e amount of py v i r u s was found to be needed to cause a t r a n s f o r m a t i o n event i n an i n f e c t e d c e l l c u l t u r e . k 5 Depending on the c e l l and v i r u s types, between 10 and 10 plaque forming u n i t s (pfu) of v i r u s were needed f o r such an event (67), although o n l y one v i r u s p a r t i c l e was apparently r e s p o n s i b l e f o r the transformation. Since p u r i f i e d py DNA could cause t r a n s f o r m a t i o n (2^), i t was considered to be the a c t i n g p r i n c i p l e r e s p o n s i b l e f o r t h i s event. Polyoma v i r u s has been s u c c e s s f u l l y rescued from transformed r a t c e l l s (35)- Attempts to rescue the v i r u s from py transformed hamster c e l l s have however met w i t h f a i l u r e (5k). This could be accounted f o r by the f a c t t h a t these c e l l s appeared to harbour an incomplete v i r a l genome (53). 8 Because transformed c e l l s develop i n t o tumors when t r a n s p l a n t e d i n t o newborn mice and hamsters, they are considered t o be an " i n  v i t r o " model f o r tumor formation by py v i r u s i n animals. The t h i r d type of v i r u s c e l l i n t e r a c t i o n , the l y t i c or productive i n f e c t i o n w i l l be d e a l t w i t h i n more d e t a i l below. 5. The Productive or L y t i c I n f e c t i o n . The l y t i c i n f e c t i o n w i t h py v i r u s was found to be r e s t r i c t e d almost e n t i r e l y t o mouse c e l l s , p a r t i c u l a r l y mouse kidney and mouse embryo c e l l s . There have been observations t h a t py v i r u s r e p l i c a t e s i n kidneys of newborn hamsters (46), but hamster c e l l s i n general do not support the r e p l i c a t i o n of py v i r u s " i n v i t r o " (54). I n con-sequence, o n l y the mouse kidney and mouse embryo c u l t u r e systems w i l l be considered. a. A d s o r p t i o n and P e n e t r a t i o n I n a mouse kidney c e l l c u l t u r e i n f e c t e d w i t h py v i r u s at a m u l t i p l i c i t y of i n f e c t i o n (moi) of 100 pfu per c e l l , 60$ or more of the c e l l s have been observed t o produce v i r u s at 30 hours post i n f e c t i o n ( p i ) . I n a mouse embryo c e l l c u l t u r e , i n f e c t e d w i t h py v i r u s , an moi of 1000, 90$ t o 100$ of the c e l l s have been shown to be v i r u s producers (118) (97). These stu d i e s showed th a t i n c o n t r a s t 9 t o the t r a n s f o r m a t i o n phenomenon, the l y t i c or productive i n f e c t i o n a c t i v e l y i n v o l v e d the m a j o r i t y of the c e l l s of the i n f e c t e d c u l t u r e , and hence could be s t u d i e d i n a more q u a n t i t a t i v e f a s h i o n . Studies on adsorption k i n e t i c s of py v i r u s i n d i c a t e d t h a t 50$ of the v i r u s was taken up by mouse embryo c e l l s i n 30 minutes, and n e a r l y 100$ of the v i r u s was taken up i n k hours ( l l 8 ) . Through autoradiography, i t was shown th a t the l a b e l l e d DNA of the i n f e c t i n g v i r u s appeared i n the cytoplasm by three hours and i n the nucleus by s i x hours a f t e r i n f e c t i o n ( 6 l ) . b. E a r l y RNA Synthesis A f t e r the v i r u s was uncoated i n the i n f e c t e d c e l l , at l e a s t p a r t of the v i r a l DNA was t r a n s c r i b e d i n t o e a r l y py RNA (3). The syn t h e s i s of t h i s RNA was e s s e n t i a l f o r the eventual progress of the i n f e c t i o n and i f i n t e r f e r e d w i t h , the i n f e c t i o n c y c l e ceased (37a)- This RNA w i l l be considered i n more d e t a i l below. c. Enzyme Induc t i o n F o l l o w i n g the beginning of e a r l y py RNA sy n t h e s i s , there was an i n d u c t i o n of some enzymes i n v o l v e d i n DNA and py r i m i d i n e b i o s y n -t h e s i s (27) (62) (59)- I t i s not known i f any of these enzymes are coded f o r by v i r a l genes. d. DNA Synthesis I n mouse embryo (ME) c e l l c u l t u r e s , i n f e c t e d w i t h py v i r u s at low m u l t i p l i c i t y , c e l l DNA synthesis was s t i m u l a t e d at 12 t o 15 hours p i . In t h i s case, the commencement of synthesis of v i r a l DNA lagged about 3 hours behind the^ s t i m u l a t i o n of the c e l l u l a r DNA synthesis (12). On the other hand, i t has been reported t h a t under c o n d i t i o n s of h i g h i n f e c t i o u s m u l t i p l i c i t y , ME c e l l c u l t u r e s underwent a depression i n t h e i r DNA synthesis ( 7 )• I n mouse kidney (MK) c e l l s i n f e c t e d w i t h py v i r u s , v i r a l DNA synthe s i s has been observed as e a r l y as 12 hours p i , while c e l l u l a r DNA sy n t h e s i s was s t i m u l a t e d at about Ik hours a f t e r i n f e c t i o n (H2) (UL) (50). V i r a l DNA synth e s i s reached a maximum at about 35 hours p i when i t accounted f o r 11$ of the t o t a l DNA synth e s i s as measured by i n c o r p o r a t i o n of H-thymidine. About 1$ of the synthesized DNA was e v e n t u a l l y encapsidated ( i l l ) (50). Studies on the r e p l i c a t i o n of the py DNA showed t h a t the r e p l i -cative. form e x i s t e d as type I I DNA which contained at l e a s t two s i n g l e stranded regions and t h a t there was r e p l i c a t i o n i n two d i r e c t i o n s along the molecule (6 ). I n t h i s regard, enzymes have been found i n the n u c l e i of i n f e c t e d c e l l s which could induce s i n g l e stranded n i c k s i n t o the s u p e r h e l i c a l DNA molecule. These enzyme molecules could a l s o have been i n v o l v e d i n a s w i v e l mechanism f o r DNA r e p l i c a t i o n ( l l ) . e. V i r a l RNA Synthesis The development o f e f f i c i e n t techniques of DNA-RNA h y b r i d i z a t i o n has made i t p o s s i b l e to study py RNA synth e s i s i n i n f e c t e d c e l l s . The l e v e l of py RNA synth e s i s was found to be d r a m a t i c a l l y increased a f t e r the r e p l i c a t i o n of py DNA. Hence the py RNA synthesized p r i o r to py DNA synthesis i s r e f e r r e d t o as " e a r l y " py RNA while t h a t synthesized a f t e r DNA synth e s i s i s r e f e r r e d t o as " l a t e " py RNA ( 3 )• I n i t i a l s t u d i e s on e a r l y py RNA were performed i n ME c e l l s where i t was shown t h a t t h i s species of py RNA made up t o 0.0Uf0 of the l a b e l l e d ME c e l l RNA and was synthesized up to 16 hours p i (3 )• I n l a t e r s t u d i e s done i n py i n f e c t e d MK c e l l c u l t u r e s e a r l y py RNA was shown t o be synthesized from 6 t o 12 hours p i , when i t made up 0.003 to 0.006$ of the t o t a l p u l s e l a b e l l e d c e l l RNA. Late py RNA synth e s i s was then observed to begin at 12 hours and t o continue up t o 30 hours p i . At t h i s time up to 3 t o 6$ of the pulse l a b e l l e d c e l l RNA was py RNA. I f py DNA synth e s i s was blocked w i t h FUdR i n the i n f e c t e d c e l l o n l y e a r l y py RNA synthesis occurred (55)-A summary of the c h r o n o l o g i c a l order of the metabolic events o u t l i n e d above may be seen i n f i g . 1 ( 110). A comparison by competition h y b r i d i z a t i o n between l a t e and e a r l y py RNA i n terms of base sequence, showed th a t the e a r l y py RNA was at l e a s t p a r t l y homologous to the l a t e py RNA. I n a d d i t i o n , most of the e a r l y RNA species were a l s o found to be t r a n s c r i b e d l a t e i n i n f e c t i o n (55). F i g . 1: L e n g t h of p h a s e i v a n e s , 3 7 ' C 12 - 3 0 h o ' - ' S m i n d i v i d u a l c e l l s ? 7 * C 2'h - 5 d a y s a d s o r p t i o n o f P y v i r u s P y D f J A p e n t - f a t e s i n t o n u c l e u s 1 . . e a r l y e a r l y " P y m e s s e n g e r R N A . i n t e g r a t i o n ' of P y D N A i n t o m o u s e c h r o m o s o m a l D N A „ e a r l y " P y m e s s e n g e r R N A t r a n s c r i b e d f r o m i n t e g r a t e d P y D N A , 1 a p p e a r a n c e o f i n t r a n u c l e a r P y - s p e c i f i c T - a n t i g e n c ' . i ^ a i i o r . o t c e l l u l a r 0 N A -s y n t h e s i i i n g a p p a r a t u s • p s y c h r o s e n s i t i v e e v e n t ( s ) - - - -R £ P > . : A T : C N C F T M E H O S T ( QC! b, mMOS'S ) 1 1 - ricss cfif (actor (s) I o n s e t o f v i r d D N A s y n t h e s i s t r c s c r i p t i o n o* ' p o l y c i s t r o n i c ) „ l a te "Py m e s s e n g e r R N A t r a n s p o r t i n t o c y t o p l a s m s y n t h e s i s o f c a p s i d p r o t e i n I f a n s p o r t i n t o n u c l e u s I a s s e m b l y ot p r o g e n y v i r u s l y s i s c f h o s t c e l t F i o . 1. Tenta t ive scheme fur lIn: lyt ic cycle of polyoma virus in contact- inhibited pr imary mouse kil l i icy t issue culture cells. T i n : mat keil :isyttclirotiy of llie time course of infect ion '.Fiji. '£ and ref. \a-c) is due to tin: vary ing length, in indiv idual • oils, of phase 1. If l 'y- induced synthesis of viral and cellular DNA i.- inhibited with .VHuorodcoNy uridine, little if any cap-id protein'" •'' or chromosomal protein - is sy nt hesized; however, the. events of phase 1 which lead Wi the appearance of T-atilit-'ciM" and to the subsequent act ivat ion of the cellular DNA-synthesizing apparatus 1 " " r l - lake place just as they do in the absence of the inhibitor. ( I l O ^ Preliminary studies on the size of the viral RNA revealed that i t sedimented heterogeneously between ks and k^s in a sucrose gradient (55). The processing of py RNA has been studied to a limited degree (58). Nuclear py RNA was found to be larger than polysomal py RNA. It was also shown that a large portion of the former was degraded within the nucleus without being further processed. Similar studies have been done on SVkO RNA in terms of size and processing. BSC-1 cells infected with SV^ O, contained polysome associated, early viral RNA of 8 x 10^ daltons, while the corres-ponding late RNA appeared to be 7.9 x 10 and 5.6 x 10 daltons (302). In SV^+O infected VERO cells, early viral RNA from polysomes sedimented at 15 to 17s (5.6 x 10 daltons) while the corresponding late RNA 6 "5 sedimented at 28s (1.7 x 10 ) and 17s (6 x 10"J (68). In these same cells, SYhO specific nuclear RNA sedimented heterogeneously, though there was a predominance of RNA sedimenting around 3^ -s, while species sedimenting as fast as 50s were also observed. Preliminary studies have suggested that SVkO specific RNA which sedimented faster than 28s may be cell-viral linear hybrid molecules(56). In this regard, i t is of interest to note that in SVkO transformed cells, virus specific RNA sedimenting faster than 28s had been found (66). This observation along with the evidence that some transformed cell lines harbour less than one f u l l viral genome (53) lends credence to the hypothesis of linear hybrid RNA composed of both cell and viral base sequences. f. Viral Protein Synthesis On a theoretical basis, the py genome of 3 x 10 daltons, of which 50$ is transcribed in the virus infected cell (69), could yield an RNA molecule of 1.5 x 10^ daltons. This in turn could code for about 1.5 x 10 daltons of protein of which not a l l need be incorporated into the virion. The proteins coded for by early py RNA are essential for the progress of the infection, as shown by use of protein specific metabolic inhibitors (37a). The only detectable early virus specific protein identified so far is the T antigen (38), while the late viral proteins are believed to be structural viral proteins.which have been discussed previously. More insight into early viral proteins has been obtained by immunoprecipitation assay with SVkO. In this case, late viral proteins from infected cells revealed five distinct precipitin bands, while with cells treated with Ara C, a DNA synthesis inhibitor, or trans-formed cells, only two of these bands were present (25). 6. Biochemistry of RNA Synthesis in Mammalian Cells a. Processing of RNA RNA synthesis in mammalian cells has been studied extensively owing to the development of efficient labelling procedures, specific inhibitors and the occurrence in the cells of large quantities of distinct RNA species, such as ribosomal RNA (r-RNA) and transfer RNA (t-RNA). Ribosomal RNA has been shown to make up about 80% of the cells' RNA content, while t-RNA and other s-RNA made up about 19$. The remainder is mostly messenger RNA (m-RNA) ( l 8 ) . Actinomycin D, a rapid inhibitor of RNA synthesis, has proven to be a valuable tool in the study of the processing of RNA (39)-Although this drug has been criticized with respect to its side effects (75), most of the information about RNA processing obtained through the use of actinomycin D has been confirmed by alternative approaches (88). A strong argument favouring the use of Actinomycin D in our system is that the drug does not affect the replication of RNA viruses (8U), or in low concentrations of the drug, of polyoma virus (13). It is, nevertheless, a worthwhile policy to confirm findings on RNA processing obtained with the help of Actinomycin D by an alternate method. Through the use of Actinomycin D i t was shown that r-RNA is transcribed as a molecule sedimenting at h^s which is then sequent-ially processed into intermediate forms, sedimenting at his and 36s. The 36s intermediate breaks down into l8s ribosomal RNA and a 32s intermediate which breaks down into 28s ribosomal RNA. It is the l 8 s , 28s and 7s RNA molecules which form the ribosomes (18). These studies reveal that the processing of RNA in mammalian cells involves degradation of a l a r g e molecule t o smaller u n i t s as w e l l as the complete breakdown of a s u b s t a n t i a l p o r t i o n of the o r i g i n a l l y t rans c r i b e d RNA molecule. The model f o r p r o c e s s i n g of r-RNA proved inadequate to e x p l a i n the p r ocessing of m-RNA (95) (92) (73). I n i t i a l s t u d i e s on m-RNA 32 l a b e l l e d w i t h P i n d i c a t e d t h a t a la r g e p r o p o r t i o n of the l a b e l l e d molecules sedimented f a s t e r than ^5s. Though these molecules were not a s s o c i a t e d w i t h polyribosomes, they d i d have a DNA l i k e base composition (95), and d i d not appear t o be a s s o c i a t e d w i t h the molecules as were the r-RNA molecules (105). These two p r o p e r t i e s d i f f e r e n t i a t e t h i s form of RNA from the r-RNA (73) ( l 8 ) . Because t h i s RNA was found mainly i n the nucleus i t has been termed h e t e r o -nuclear RNA (Hn RNA). Since i t resembled m-RNA or DNA i n base composition^ t h i s RNA has a l s o been r e f e r r e d to as messenger-like RNA (ml-RNA) or DNA l i k e RNA (d-RNA) (85) (37). The Cascade Hypothesis i s a model proposed to e x p l a i n the processing of ml RNA to m-RNA (87) (86). The model s t a t e s t h a t 3 t o 10$ of the c e l l DNA i s t r a n s c r i b e d i n t o a heterogeneous a r r a y of ml RNA molecules v a r y i n g from 1 to 15 x 10^ daltons. These molecules, which have a number of re p e a t i n g sequences, become as s o c i a t e d w i t h p r o t e i n immediately a f t e r t r a n s c r i p t i o n to form r i b o n u c l e o p r o t e i n complexes (RNP). The l e s s s t a b l e regions of these molecules are degraded and the more s t a b l e regions of the molecules then sediment between 6 and 35s (0.05 to 2 x 10^ daltons) These RNA molecules associated with protein may then be stored in the nucleus or transported into the cytoplasm. There they may be stored as RNP complexes or processed further into the polysomes for translation. In duck erythroblast cells, i t was found that i t takes about 20 minutes for newly labelled RNA to reach the cytoplasm where the half l i f e of the RNA was 3 to 6 hours. b. Polyadenylate Sequences Additional significant features of the ml RNA described above arertl) the biphasic shape of their decay curves ( suggesting two possible fates of ml RNA molecules); 2) only 20 to 2% of such RNA is transported into the cytoplasm,and 3) the presence of adenosine rich sequences. A study of the m-RNA and Hn-RNA (or ml-RNA) molecules revealed that these species have covalently linked with them a 200 nucleo-tide long polyadenylic acid (poly(A)) region, at the 3' end of the molecule (6o) (82) (91). There is evidence that the poly A sequence was added post-transcriptionally (20) (79) and appeared to function in the processing or stabilization of the Hn-RNA and m-RNA (72). Initial quantitative studies have shown that 10 to 20$ of the nucleoplasmic RNA and k6 to 60$ of the polyribosome associated RNA contained poly(A) sequences (90). However more recent studies have shown t h a t i n mouse L c e l l s , 20$ of the Hn-RNA and 100$ of the m-RNA con t a i n poly(A)sequences (kO). These data along w i t h the p r e v i o u s l y mentioned o b s e r v a t i o n t h a t o n l y 20$ t o 25$ of the Hn-RNA entered the cytoplasm from the nucleus, i n d i c a t e t h a t p o s s i b l y o n l y Hn-RNA molecules possessing p o l y A sequences are processed to m-RNA. In c e l l s i n f e c t e d w i t h nuclear DNA v i r u s e s , the a s s o c i a t i o n of v i r a l m-RNA wi t h p o l y A has a l s o been observed. Simian v i r u s kO, the DNA of which has no p o l y A or p o l y dT regions corresponding i n length to p o l y A described above, nevertheless has p o l y (A)associated w i t h i t s RNA. A q u a n t i t a t i v e study has shown th a t 50 to 70$ of the SVkO m-RNA from polyribosomes possesses poly(A) sequences ( l l 4 ) . A s i m i l a r study w i t h adenovirus type 2 has rev e a l e d t h a t w h i l e the v i r a l DNA contained no long poly(dA) or poly(dT)sequences, the adenovirus m-RNA from polyribosomes of i n f e c t e d c e l l s had up to 65 to 80$ of i t s molecules a s s o c i a t e d w i t h poly(A) (79)-7. Purpose of the Research P r o j e c t . The o r i g i n a l o b j e c t i v e was to use the py RNA as a model system to study the a n a l y s i s and processing of a def i n e d species of RNA which could be e a s i l y and q u a n t i t a t i v e l y detected i n the presence of the c e l l u l a r RNAs. In t h i s context, i t was a l s o of i n t e r e s t t o determine i f py v i r u s genes are t r a n s c r i b e d , processed, and t r a n s l a t e d i n the same manner as the c e l l u l a r genes. CHAPTER I I : MATERIALS AND METHODS A. M a t e r i a l s 1. C e l l s Primary mouse kidney c e l l s (MK) and secondary mouse embryo c e l l s (ME) were prepared from randomly bred, white Swiss mice, purchased from the Connaught M e d i c a l Research L a b o r a t o r i e s , Toronto or from the F a c u l t y of Medicine Animal U n i t , U n i v e r s i t y of B r i t i s h Columbia. A continuous l i n e of baby hamster kidney c e l l s , BHK-21 (clone 13) and a continuous l i n e of polyoma v i r u s transformed hamster c e l l s , PyH, were purchased from M i c r o b i o l o g i c a l A s s o c i a t e s , Bethesda, U.S.A. 2. V i r u s The w i l d type l a r g e plaque forming s t r a i n of polyoma v i r u s used i n these experiments was obtained from Dr. R. W e i l , Swiss I n s t i t u t e f o r Experimental Cancer Research. 3. Growth medium and other b i o l o g i c a l compounds Dulbecco modi f i e d minimal e s s e n t i a l medium (MEM) purchased i n powder form from Grand I s l a n d B i o l o g i c a l Company (GIBCO) was used f o r growing c e l l s . The medium was s t e r i l i z e d by f i l t r a t i o n through M i l l i p o r e membrane of pore diameter 0.22 um and made up to 100 u n i t s per ml i n p e n i c i l l i n , 100 ugm per ml i n streptomycin and 0.25 ugm per ml i n fungizone. I n l a t e r p o r t i o n s of the research, the f i r s t two of the above a n t i b i o t i c s were re p l a c e d by gentamicin ( B i o c u l t L a b o r a t o r i at 50 ugm per ml. The medium was a l s o supplemented w i t h 10$ newborn c a l f serum purchased from GIBC0 or f e t a l c a l f serum purchased from Flow L a b o r a t o r i e s , R o c k v i l l e , U.S.A. Guinea p i g blood, f o r hemagglutination s t u d i e s was obtained from a guinea p i g kept o n l y f o r t h i s purpose. Name of Compound Actinomycin D U r i d i n e Cordycepin Polyadenylate P o l y c y t i d y l a t e P o l y u r i d y l a t e Ribonuclease A Deoxyribonuclease T r y p s i n H-uridine (k2 Ci/m mole) H-adenosine (35 Ci/m mole) ^H-uridine (28 Ci/m mole) H-adenosine (12.1 Ci/m mole) "^ C - u r i d i n e (60 mCi/m mole) Dealer Merck, Sharp and Dohme, Montreal ( G i f t ) Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company Sigma Chemical Company: M i l e s L a b o r a t o r i e s Sigma Chemical Company Worthington Biochemical Co. DIFC0 New England Nuclear Co. New England Nuclear Co. Amersham Searle Co. Amersham Searle Co. Amersham Searle Co. h. Chemicals Name Ethidium bromide Cesium c h l o r i d e Nonidet-P-^O (KP.ko) Reagents f o r polyacrylamide g e l e l e c t r o p h o r e s i s S p e c t r a f l u o r Agarose Dealer Calbiochem Schwartz Mann Co. Laboratory of Dr. M. Smith ( o r i g i n a l l y purchased from S h e l l O i l Co.) Eastman Organic Chemicals Ltd. Amersham Searle Co. Bausch and Lomb Co. A l l remaining reagents were purchased from F i s h e r Chemical Co. A l l water used was g l a s s d i s t i l l e d . A l l glassware used i n operations i n v o l v i n g n u c l e i c acids was washed w i t h chromic a c i d . A l l t i s s u e c u l t u r e g l a s s was s t e r i l i z e d by a u t o c l a v i n g at 15 p s i of steam f o r 20 minutes f o l l o w e d by he a t i n g i n an oven at 170°C f o r 6 hours. 5. S o l u t i o n s Phosphate Bu f f e r e d S a l i n e (PBS) (26) NaCl 0.1370 M KC1 0.0027 M Na 2HP0^ 0.0081 M KH 2P0^ 0.0015 M M g C l 2 0.0005 M C a C l 2 0.0009 M Stock S a l i n e NaCl 0.15 M RNA B u f f e r (55) NaCl 0.1 M CT^COONa 0. 01 M M g C l 2 0.001 M adjusted w i t h HCl t o pH 5.2 R e t i c u l o c y t e Standard B u f f e r (RSB°) (80) NaCl 0.01 M Mg C l 2 0.0015 M T r i s 0.01 M adjusted w i t h HCl t o pH 7-5 23 RSB+ NaCI 0.1 M MgCl2 0.015 M Tris 0.01 M adjusted with HCl to pH 7-5 f. Standard Saline Citrate Solution ( l x SSC) (52a) NaCI 0.15 M sodium citrate 0.015 M A stock solution was made up to 2h x SSC and diluted with distilled water to required concentration. Each large batch was treated with a few drops of CHCl^ as a preservative. g. STE Buffer (80) NaCI 0.1 M EDTA 0.001 M Tris 0.01 M adjusted with HCl to pH 7. 5 Scintillation Fluid Spectrafluor k2 ml Toluene 958 ml Polyacrylamide Gel E l e c t r o p h o r e s i s S o l u t i o n s ( ) ( l ) Running b u f f e r T r i s 0 . 0 4 M CH^COONa 0. 02 M EDTA (Wa 2) 0 .001 M .SDS 0 . 2 $ (weight/volume) (w/v) adjusted w i t h HCl t o pH 7 .4 (2) Acrylamide g e l s o l u t i o n s (106) S o l u t i o n A: Acrylamide 90$ (w/ v) N,N-bis methylene acrylamide 2 . 0 $ (w/v) T r i s 0. 48 M adjusted w i t h HCl to pH 8.4 S o l u t i o n B: Ammonium persulphate 0 . 2 $ (w/v) NM'N'tetramethylethylenediamine 0 .1$ (w/v) S o l u t i o n C: Agarose T r i s 0 . 5 $ (w/v) 0.243 M T r i s s o l u t i o n adjusted w i t h HCl to pH p r i o r t o a d d i t i o n of agarose 8 . 4 M i l l i p o r e B i n d i n g B u f f e r (MBB) (65) K C 1 0 . 5 M T r i s 0 . 0 1 M MgClg 0 . 0 0 1 M adjusted w i t h HCl to pH 7-5 25 P o l y u r i d y l a t e B i n d i n g B u f f e r (90) NaCl 0.1 M T r i s 0.01 M adjusted w i t h HCl t o pH 7-5 1. Hank's Balanced S a l t S o l u t i o n (modified) (55) Glucose 0.0055 M NaCl 0.1^70 M KC1 0.0053 M KHgPO^ O.OOkk M Na 2HP0^ 0.0150 M Phenol red 0.0006 M S t e r i l i z e d by a u t o c l a v i n g at 15 l b s steam f o r 20 minutes. m. SDS-EDTA B u f f e r (50 ) SDS 0.6$ (w/v) EDTA 0.01 M adjusted w i t h 1 M NaOH t o pH 7.0 n. A l s e v e r ' s S o l u t i o n Glucose 0.11 M Sodium C i t r a t e 0.027 M NaCl 0.072 M S t e r i l i z e d by a u t o c l a v i n g at 15 l b s steam f o r 20 minutes. 26 B. Methods 1. Primary Mouse Kidney Cell Cultures Baby mice, 7 to 10 days old, were killed and washed in 95$ ethanol. Their kidneys were aseptically removed with sterile forceps through a dorsal incision. This operation was carried out in an ultraviolet light sterilized hood. The kidneys were macerated mechanically by forcing them through a plastic 10 ml syringe whose needle had been replaced by a ho mesh stainless steel screen. The tissue was then incubated in 0.25$ trypsin in Hank's solution for 20 to ho minutes at room temperature, on a magnetic stirrer. The cells were collected by centrifugation in an ambient temperature International CS centrifuge for 10 minutes at 2000 rpm in kO ml centrifuge tubes. The cells were then washed by resuspending them in fresh Hank's solution and again centrifuging them out of solution. This procedure was repeated two more times. Following the final wash, the centrifuged cell pellet-was re-suspended in MEM prewarmed to 37°C and containing 10$ calf serum, at a resuspending ratio of two mouse kidneys per 10 ml of MEM. Ten ml aliquots of the cell suspension were dispensed into 90 mm tissue culture petri dishes (Falcon Plastics) which were then incubated at 37°C in an atmosphere of 5$ C 0 2 and saturating humidity in a Rational Appliance Co. C 0 2 incubator. The cells formed monolayers within 2k hours. The overlaying medium and cell debris were removed from the monolayers by aspiration and 10 ml of fresh MEM containing 10$ calf serum was added to each culture. Following the medium change, the cell monolayers became confluent within the next kQ hours. 2. Secondary Mouse Embryo Cell Cultures Mouse embryos aseptically removed from their mothers in their second week of gestation were treated as were the kidneys, above, to produce primary mouse embryo cell cultures. The confluent cell monolayers were dissociated with Hank's solution containing 0.25$ trypsin, washed with Hank's solution, resuspended at half of the original concentration in MEM+5$ serum and again dispensed into petri dishes. The cells of new monolayer cultures which became confluent within 2k hours, were termed secondary mouse embryo cells. 3. BHK 21 Cells and PyH Cells These cells were grown as monolayer cultures on 90 mm petri dishes. The cells were subcultured by trypsinizing the confluent monolayer cultures, as was done for the mouse embryo cells, and dispensing the cells in MEM+5$ calf serum into new petri dishes at l / 3 of their original concentration. The monolayer cultures became confluent within 2k to 36 hours. k. Growth of Polyoma Virus A l l the py virus used in this study was grown in primary MK cell cultures. These cultures were infected by two methods termed 1 in vitro' and 'in vivo'. a. The 'in vitro' infection: This method was used both in the production of virus stocks and in studies involving virus growth and development. The overlaying medium of the confluent MK cultures was removed by aspiration and 0.5 ml of py virus suspension containing about 2 x 10^ plaque forming units (pfu) added to each petri dish. During the adsorption period of one hour, the inoculated monolayer cultures were kept in a CO^  incubator. Every 20 to 30 minutes they were with-drawn, and briefly rotated at an angle to redistribute the virus inoculum over the entire monolayer surface. Following the adsorption period, 10 ml of MEM lacking serum were added to each petri dish culture,which was further incubated for ky to kQ hours. The overlaying MEM was then aseptically collected and analyzed for virus content by the hemagglutination assay, while the remaining cell monolayer was saved for DNA extraction. b. The 'in vivo' infection: Newborn mice were each injected sub-cutaneously with 0.03 ml of py virus containing at least 10^ pfu. At the age of 12 to ik days the mice were sacrificed and their kidneys used to make MK cultures. After forming a confluent monolayer, these cell cultures were monitored by hemagglutination assay every 12 hours for the appearance of py virus in the medium. By about the fourth day after the confluent monolayer of cells had formed, the level of virus in the medium reached 80,000 hemagglutination units (HAU) per ml. At this point the medium was collected for its virus while the remaining monolayer was used for DNA extraction. c. Further Treatment and Analysis of the py Virus Preparation. The medium containing the virus was chilled to k°C and sonicated with a Bronwill Biosonic III on probe setting-of kO, for 2 minutes. The virus was then dispensed in 5 ml amounts into either sterile glass vials or plastic tubes (Falcon Plastics) and frozen at -70°C in a Revco freezer until needed. d. Hemagglutination Assay The virus was titrated using guinea pig blood kept in an equal volume of Alsever's solution at k°C for a period not exceeding 3 weeks. The blood was washed twice with stock saline and the final pellet re-suspended up to l<f0 (v/v) in stock saline. Twelve serial twofold dilutions of 0.025 ml of the virus were made in a microtiter plate. To each of these were added 0.025 ml of 1PJ0 guinea pig blood, and the plates placed at 1+°C for 1 to 6 hours. The reciprocal of the highest dilution which produced hemagglutination was used to calculate the virus titer. Periodically the reproducibility of this assay was tested by also doing a plaque assay on the same virus preparation. The virus titer calculated by the hemagglutination assay and obtained by the plaque assay were found to be in close agreement throughout this study. 5. Preparation of Polyoma Type 1 DNA (py I DNA) (50) (83) Infected MK cell monolayers whose medium had been harvested for virus as outlined above, were used as a source of py I DNA. The cells were washed five times with PBS containing 0.001 M EDTA, and lyzed by addition of 0.8 ml of SDS-EDTA buffer to each petri dish. The dishes were gently rotated at an angle until the entire monolayer was exposed to the buffer. The lysates were collected into a 30 ml Corex (Corning) centrifuge tube with the aid of a rubber policeman. The contents of the tube were made up to 1 M in NaCI and mixed by gently inverting the tube 20 times. After the preparation had been allowed to stand at k°C for 8 to l 6 hours, i t was centrifuged at 12,500 rpm for 20 minutes in an SS 3^ rotor at h°C in a Sorvall RC2B centrifuge. While the pellet was dis-carded, the supernatant was mixed with an equal volume of phenol s a t u r a t e d w i t h 1 M t r i s b u f f e r at pH 8.0 at ^ °C. The mixture was c e n t r i f u g e d at 10,000 rpm f o r 10 minutes t o separate the phenol from the aqueous phase... The aqueous phase was r e - e x t r a c t e d w i t h phenol as above and again r e - e x t r a c t e d w i t h chloroform c o n t a i n i n g U$ isoamyl-a l c o h o l . The aqueous phase from t h i s l a s t e x t r a c t i o n was mixed w i t h two volumes of ethanol and kept overnight at k°C t o p r e c i p i t a t e the DNA. The ethanol p r e c i p i t a t e , 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 10,000 rpm a t k°C f o r 10 minutes, was r e d i s s o l v e d i n 0.2 x SSC, 0.0001 M EDTA. The e q u i v a l e n t of 10 t o 15 ml of the o r i g i n a l c e l l l y s a t e was d i s s o l v e d inc.a k ml volume. The s o l u t i o n was made up t o a d e n s i t y of 1.60 gm per ml w i t h CsCl and t o a f i n a l c o n c e n t r a t i o n of 100 |agm per ml i n ethidium bromide. A Bausch and Lomb refractometer was used to de t e r -mine the r e f r a c t i v e index of the s o l u t i o n (a reading of 1.390 corresponded t o a CsCl d e n s i t y of 1.6o gm per ml). A l i q u o t s of about 5 ml of t h i s s o l u t i o n were dispensed i n t o 21-i n c h by 5/8 i n c h n i t r o c e l l u l o s e tubes whose inner surfaces had been t r e a t e d w i t h 1$ s i l a n e . The tubes were f i l l e d t o the top w i t h p a r a f f i n o i l , capped and plac e d i n t o a Beckman Type 50 r o t o r and c e n t r i f u g e d f o r 36 hours at 20°C at kO,000 rpm i n a Beckman L2-65 B u l t r a c e n t r i f u g e . F o l l o w i n g c e n t r i f u g a t i o n the tubes were observed against a l i g h t background f o r the presence of deep r e d bands i n the gradient. The l o c a t i o n of these was marked on the tube surface. The gradients were collected into fifteen equal fractions by puncturing the bottom of the tubes and dripping the contents through. The fractions containing the lower band, which was located around the middle of the gradient, were pooled and combined with the corresponding fractions from other parallel gradients. After adjustments of the density of these solutions to 1.60 gm per ml, they were again centrifuged and fractionated under the above conditions. The pooled fractions containing the lower band were then extracted three times with equal volumes of isopropanol (saturated with aqueous CsCl) to remove the ethidium bromide. The CsCl was then removed by dialyzing the preparation against 1000 volumes of 0.01 x SSC and 0.0001 M EDTA for at least 12 hours. The prep-aration was now considered to consist of pure py I DNA. 6. Labelling and Preparation of RNA from py Infected Cells (55) a. Labelling 3 Polyoma virus infected cultures were labelled with H-uridine by replacing the overlaying medium of the monolayers with 2 ml of MEM containing a total of kO uCi of H-uridine. The petri dishes were rotated at an angle every 30 minutes to ensure even distribution of the medium over the cell surface. After 2 hours of incubation the radioactive medium was decanted and the monolayers washed five times at room temperature with PBS and twice with RNA buffer. One ml of RNA buffer containing 1.0$ SDS was added to the petri dishes and distributed over the surface by gentle rotation. The cell lysates were collected into a 15 ml or 30 ml Corex tube with the help of a rubber policeman. b. Phenol Extraction The cell lysate was mixed with an equal volume of phenol (saturated in RNA buffer) by vigorous agitation. The mixture was incubated in a water bath at 6o°C for 3 to k minutes, again agitated and plunged into an ice water bath, for an additional 5 minutes. The preparation was then centrifuged at 10,000 rpm at k°C for 10 minutes in a Sorvall centrifuge. The phenol phase was discarded while the interphase and the aqueous phase were re-extracted two more times with RNA buffer saturated phenol at k°C. In these two extractions the aqueous phase alone was collected. This aqueous phase from the last extraction was made up to 0.2 M in NaCl and mixed with 2 volumes of ethanol and kept at -20°C for at least k hours to precipitate the RNA. The precipitate was collected by centrifugation at 10,000 rpm for 10 minutes at k°C, made up to 2 ml in RNA buffer at h°C and reprecipitated with ethanol under above conditions. This ethanol p r e c i p i t a t e was resuspended i n 2 ml of RNA b u f f e r at pH 7-2, and t r e a t e d w i t h RNAse f r e e DNAse at 20 ugm per ml f o r 20 minutes at 37°C. The r e a c t i o n was stopped by adding SDS t o a c o n c e n t r a t i o n of 0.5$ (w/v) and the p r e p a r a t i o n was e x t r a c t e d w i t h c o l d phenol saturated i n RNA b u f f e r . The aqueous e x t r a c t was p r e c i p i t a t e d two more times w i t h ethanol, and f i n a l l y made up to 0.1 to 0.3 ml 0.001 M EDTA. c. A n a l y s i s and Storage of RNA Samples of t h i s RNA (10 u l ) were analyzed f o r absorbance spectrum i n the u l t r a v i o l e t range on a Beckman DBG spectrophotometer and f o r the r a d i o a c t i v i t y on a Nuclear Chicago Mark I I s c i n t i l l a t i o n counter which had a background of 15 cpm. A sample was a l s o h y b r i d i z e d w i t h denatured py I DNA t o measure the amount of py RNA present. The remainder of the RNA was f r o z e n at -70°C. I t was g e n e r a l l y used between 1 to 4 weeks a f t e r f r e e z i n g . 7. M o d i f i c a t i o n s o f the RNA E x t r a c t i o n Method a. Use of Pronase Pronase ( f u n g a l protease (Sigma)) was used i n con j u n c t i o n w i t h 1$ SDS i n RNA b u f f e r to l y s e the i n f e c t e d c e l l monolayer. The l y s i s was c a r r i e d out f o r 15 minutes f o l l o w e d by phenol e x t r a c t i o n as out-l i n e d above. b. Use of Phenol-Chloroform (77) A preparation of equal parts phenol and chloroform saturated in MA buffer was used in place of phenol. The extraction was done at h°C. c. Use of Phenol at pH 9.0 (65) A preparation of phenol saturated with 0.05 M KC1, 0.05 M tris pH 9-0 and 0.001 M MgClg was used in place of phenol. The extraction was also carried out at 4°C. 8. Cell Fractionation a. Cytoplasmic and Nuclear Extracts (76) (l4) 3 Virus infected H-uridine labelled MK cell monolayer cultures were washed 5 times with RSB+, and treated with 0.4 ml of RSB+ containing 1$ NPUO which was spread over each monolayer by gentle rocking. The cultures were kept at 4°C for 10 minutes and period-ically rocked. The monolayers, which were already showing signs of disintegration, were scraped with a rubber policeman into a 6 ml Dounce homogenizer (Kontes) and further disrupted by two or three strokes. The broken cell preparation was collected into a 15 ml conical centrifuge tube and centrifuged for 10 minutes at 2000 rpm in a swinging bucket rotor in an International Clinical Centrifuge. The supernatant which contained the cytoplasmic extract was removed with a Pasteur pipette while the pellet was resuspended in RSB°for 5 to 10 minutes, and again homogenized with five or six strokes of the Dounce homogenizer. The preparation was again centrifuged as outlined above, the supernatant discarded while the pellet which contained the nuclei was collected and resuspended in RNA buffer. Both the cytoplasmic extract and the nuclei, which were made up to 0.5$ in SDS (w/v), were extracted with phenol saturated with RNA buffer at pH 5'-2. In i n i t i a l work in this study no detergent was used. Instead, the washed cells were swollen in RSIPfor 15 minutes, scraped off the petri dish with a rubber policeman and disrupted with 5 or 6 strokes of a Dounce homogenizer. The remaining procedure was as outlined above. b. Polyribosomes (80) For a polyribosome preparation, the cytoplasmic extract was layered on a preformed 15$ to 30$ (w/v), RSB+ sucrose gradient in a 3I- by 1 inch nitrocellulose tube. The gradient was centrifuged at k°C for 2 hours at 25,000 rpm in an SW27 rotor in a Beckman L 2 -65 B ultracentrifuge. The gradients were fractionated into 1 ml f r a c t i o n s on an Isco ( i n s t r u m e n t a t i o n S p e c i a l t i e s Inc.) f r a c t i o n c o l l e c t o r and u l t r a v i o l e t monitor by downward displacement of the 3 gradient. I f the cytoplasmic e x t r a c t was from an H-uridine l a b e l l e d p r e p a r a t i o n , 25 [ i l samples of each f r a c t i o n were analyzed f o r r a d i o -a c t i v i t y . A ppropriate f r a c t i o n s of the above gradient were pooled, d i l u t e d 1 : 1 w i t h i c e c o l d RSB + and again sedimented on an S W 2 7 . 1 r o t o r at k°C f o r 6 hours at 27,000 rpm to p e l l e t the polyribosomes. These were resuspended i n RNA b u f f e r , made up t o 0.5$ (w/v) w i t h SDS and the RNA e x t r a c t e d w i t h hot phenol as o u t l i n e d above. A l t e r n a t i v e l y pools of f r a c t i o n s were made up to 0.5$ (w/v) i n SDS and mixed w i t h 2 volumes of ethanol. The p r e p a r a t i o n was kept at -20°C overnight t o p r e c i p i t a t e the RNA. The p r e c i p i t a t e was resuspended i n RNA b u f f e r and e x t r a c t e d once w i t h hot phenol. 9. P r e p a r a t i o n of Ribosomal RNA (80) Ik Monolayers of BHK or PyH c e l l s were l a b e l l e d w i t h C-uridine at 0.2 uCi/ml i n 5 ml of MEM f o r l 6 hours. The l a b e l l i n g medium was removed and the c e l l s incubated an a d d i t i o n a l 8 hours w i t h non-l a b e l l e d MEM. The cytoplasmic e x t r a c t of these c e l l s was made up to 0.5$ (w/v) i n SDS and e x t r a c t e d twice w i t h hot phenol satura t e d i n RNA b u f f e r . RNA i n 25 M-1 amounts was pl a c e d i n t o v i a l s and f r o z e n at -70°C. 38 1 0 . F r a c t i o n a t i o n of RNA a. Sucrose Gradients ( 8 0 ) F i v e ml sucrose gradients of 5 to 2 0 $ sucrose i n STE-SDS b u f f e r were formed i n a Buchler two chamber gradient maker. On these were l a y e r e d 25 to 200 ugm of RNA i n a volume not exceeding 2 0 0 u l . The gr a d i e n t s were c e n t r i f u g e d at 4 2 , 0 0 0 rpm i n a Beckman L2-65 B u l t r a -c e n t r i f u g e f o r 2-g- hours at 15°C, then f r a c t i o n a t e d by downward d i s -placement i n t o 20 equal f r a c t i o n s . Samples of 10 u l were withdrawn from each f r a c t i o n and assayed f o r r a d i o a c t i v i t y . P e r i o d i c a l l y the f r a c t i o n s of the gradient were analyzed f o r ^260 o n a B e c ^ m a n DBG spectrophotometer. b. Polyacrylamide G e l E l e c t r o p h o r e s i s Qo5) Polyacrylamide g e l s were made up by mixing one p a r t s o l u t i o n A, one p a r t s o l u t i o n B and two p a r t s of molten s o l u t i o n C at room temperature, and then pouring the mixture i n t o 150 mm by l6 mm g l a s s tubes sealed at the bottom by p a r a f i l m stoppers. The s o l u t i o n was allowed 15 minutes to harden, whereupon the tubes were blocked a t the other end w i t h a d i a l y s i s membrane, and turned upside down i n a Buchler e l e c t r o p h o r e s i s stand. The gels were allowed to polymerize f o r two hours, the p a r a f i l m plugs removed and chambers f i l l e d w i t h running b u f f e r . The RNA was made up t o 50 \±L i n 10 M EDTA and 30$ (w/v) i n sucrose and 0.0001$ bromophenol blue. The mixture was a p p l i e d t o the s u rface of the g e l w i t h a syringe. The anode was connected to the bottom of the g e l and a current of 5 v per cm of g e l a p p l i e d . The e l e c t r o p h o r e s i s was continued f o r 2g- hours i n which time the dye marker had migrated to w i t h i n 1 cm of the bottom of the g e l . The g e l s were removed from the tubes under tap water pressure and s l i c e d w i t h a r a z o r blade i n t o 75 equal pieces. These were deposited d i r e c t l y i n t o s c i n t i l l a t i o n v i a l s , which contained 2 ml of methanol to dehydrate the s l i c e s . E i g h t ml of s c i n t i l l a t i o n f l u i d were added t o each v i a l , and these were counted f o r r a d i o -a c t i v i t y . 11. H y b r i d i z a t i o n (52) (52a) a. Denaturation and F i x a t i o n of DNA A known mass of Py I DNA i n 0.01 X SSC was denatured by h e a t i n g i n a b o i l i n g waterbath f o r 20 to 25 minutes, then by r a p i d l y c h i l l -i n g i n an icewater bath t o prevent r e n a t u r a t i o n . This DNA was made up t o 50 ml i n 6 x SSC and passed by g r a v i t y f l o w through a h.5 cm M i l l i p o r e f i l t e r presoaked i n 6 x SSC. The f i l t e r was then washed w i t h two 20 ml volumes of 6 x SSC under s l i g h t vacuum a s s i s t a n c e . The f i l t e r was p a r t i a l l y d r i e d and cut i n t o at l e a s t 20 equal s i z e 1+0 d i s c s O.65 cm i n diameter. Care was taken t h a t o n l y the surface i n contact w i t h the DNA was used. By t h i s procedure, o n l y about two t h i r d s of the f i l t e r area exposed to the DNA was recovered. These f i l t e r s contained approximately equal p r e c a l c u l a t e d amounts of denatured DNA. The f i l t e r s were a i r d r i e d , heated i n an oven at 78°C f o r 2 to 1+ hours. They were then s t o r e d at room temperature f o r periods up to 6 months. Blank f i l t e r s were prepared i n an i d e n t i c a l f a s h i o n , but without DNA. b. DNA-RNA H y b r i d i z a t i o n The RNA p r e p a r a t i o n was made up i n a s c i n t i l l a t i o n v i a l to a t o t a l volume of 1 ml i n 6 X SSC, 0.0001 M EDTA and 0.1$ (w/v) SDS. To t h i s were added d u p l i c a t e f i l t e r s c o n t a i n i n g DNA and d u p l i c a t e blank f i l t e r s . The v i a l s were t i g h t l y sealed and incubated at 65°C f o r 20 to 2k hours i n a water bath. The f i l t e r s were marked w i t h a p e n c i l f o r i d e n t i f i c a t i o n . The f i l t e r s were removed, washed i n batch w i t h 50 ml of 2 x SSC, and incubated f o r 30 minutes at 37°C i n 50 ml of 2 x SSC c o n t a i n i n g 20 ugm per ml of RNAse A. They were again washed w i t h 50 ml of 2 x SSC and counted f o r r a d i o a c t i v i t y . The r a d i o a c t i v i t y of the blank f i l t e r s was subtracted from r a d i o a c t i v i t y of the DNA c o n t a i n -i n g f i l t e r s i n a l l c a l c u l a t i o n s . in 12. P o l y (A) Studies a. F i l t r a t i o n through M i l l i p o r e F i l t e r s (65) A sample of l a b e l l e d RNA was made up t o 1.0 ml i n M i l l i p o r e b i n d i n g b u f f e r (MBB) and c h i l l e d t o h°C. The s o l u t i o n was passed by g r a v i t y f l o w through a M i l l i p o r e f i l t e r , presoaked i n MBB. The f i l t e r was washed i n MBB and the f i l t r a t e and washings c o l l e c t e d , and t h e i r r a d i o a c t i v e content determined. The amount o f py s p e c i f i c RNA on the M i l l i p o r e f i l t e r was determined by i n c u b a t i n g the f i l t e r , cut i n t o f o u r equal s e c t i o n s , i n a s c i n t i l l a t i o n v i a l c o n t a i n i n g 1 ml of 0.5$ SDS (w/v), 6 x SSC, 0.001 M EDTA and two M i l l i p o r e f i l t e r s each bearing 0.25 M-gm of py I DNA. The o r i g i n a l cut up M i l l i p o r e f i l t e r s served t o de t e r -mine the background. b. F i l t r a t i o n through P o l y (U)-GF/C F i l t e r s (90) A s o l u t i o n of p o l y u r i d y l a t e ( p o l y U) at 1.0 mg per ml was made up i n d i s t i l l e d water. Of t h i s 0.150 ml were dispensed onto g l a s s f i b r e f i l t e r s (Whatman GF/C). The f i l t e r s were allowed 2 hours at 37°C t o dry. The dry f i l t e r s were i r r a d i a t e d w i t h a 15 watt germi-c i d a l u l t r a v i o l e t lamp at 22 cm from the f i l t e r f o r 10 minutes each s i d e . About 70$ of the poly(u)was r e t a i n e d by the f i l t e r s a f t e r they were washed w i t h 20 ml of d i s t i l l e d water. A volume of 0.5 ml of RNA made up at room temperature i n p o l y -u r i d y l a t e b i n d i n g buffer(PUBB) was placed on one of the above f i l t e r s ( a l s o presoaked i n PUBB) and passed through under g r a v i t y flow. The f i l t e r was then washed w i t h k.5 ml of PUBB and d r i e d under a low vacuum. The f i l t r a t e and washings were c o l l e c t e d and t h e i r r a d i o a c t i v i t y determined by p r e c i p i t a t i o n w i t h 6$ t r i c h l o r o a c e t i c a c i d (TCA). The RNA bound t o the f i l t e r was removed by e l u t i n g the f i l t e r w i t h 1.5 ml of PUBB which was 0.001 M i n NaCI, 0.001 M i n t r i s pH 7.5. The e l u t e d RNA was made up to 0.2 M i n NaCI and p r e c i p i t a t e d w i t h 2 volumes of ethanol. CHAPTER I I I : RESULTS-I C h a r a c t e r i z a t i o n of Py RNA i n I n f e c t e d C e l l s 1. Polyoma V i r u s RNA Synthesis During the Course of V i r u s I n f e c t i o n A p r e l i m i n a r y step i n the study of py v i r u s RNA was t o determine the p a t t e r n of RNA synthesis during the course of i n f e c t i o n i n our p a r t i c u l a r system. As o u t l i n e d i n f i g . 2 , i n f e c t e d MK c e l l c u l t u r e s 3 were l a b e l l e d w i t h H-uridine, at v a r i o u s times a f t e r i n f e c t i o n , and t h e i r RNA e x t r a c t e d and p u r i f i e d . Samples of each RNA p r e p a r a t i o n were h y b r i d i z e d w i t h py I DNA to determine the r e l a t i v e amount of py RNA synthesized. The appearance of t h i s RNA i n the i n f e c t e d c u l t u r e w i t h respect to time a f t e r i n f e c t i o n , i s represented by the s o l i d l i n e i n f i g . 2 which revealed t h a t there was a very low l e v e l of v i r u s RNA i n the c e l l c u l t u r e up to l 6 hours p i , whereupon i t i n -creased l i n e a r l y up to kO hours p i , a f t e r which i t remained constant. The o v e r l a y i n g medium of each c u l t u r e was saved, and was ana-l y z e d f o r py v i r u s t i t e r by hemagglutination. The p a t t e r n of v i r u s r e l e a s e i s shown by the broken l i n e i n f i g . 2 . V i r u s was f i r s t detected at 3 2 to kO hours a f t e r i n f e c t i o n and was then r e l e a s e d i n i n c r e a s i n g amounts w i t h time. These r e s u l t s not o n l y confirmed p r e v i o u s l y reported i n f o r m a t i o n of v i r a l RNA s y n t h e s i s (55), but they were a l s o important from a Fig. 2: Production of Virus and Viral RNA in Infected Cells Twenty-one confluent primary MK monolayer cultures were infected with py virus at an moi of 50 pfu/cell. At specified times pi, the overlaying medium was removed from a group of 3 dishes, pooled and frozen at -70°C. The cell cultures were then incubated with -uridine at kO uCi/2 ml of MEM/dish for 2 hours. The RNA was extracted from the cells and fractions comprising one eighth of each preparation were analyzed for py RNA content. A total of about 300 |_igm of RNA with a specific activity of about 3,500 cpm/|_igm were obtained from each of the above samples. The virus titer of the overlaying medium was determined by hemagglutination. F i g . 2: t e c h n i c a l p o i n t of view i n t h a t they demonstrated t h a t i n the system used i n t h i s l a b o r a t o r y v i r a l RNA may be l a b e l l e d from 2h to 36 hours p i . Other r e l e v a n t i n f o r m a t i o n to come out of t h i s experiment was th a t at kO hours p i about 1.5$ of the l a b e l l e d RNA was v i r u s s p e c i f i c and at 16 hours p i o n l y 0.02$ was v i r u s s p e c i f i c w h i le no net decrease i n the t o t a l c e l l RNA synthesis was observed throughout the i n f e c t i o n (data not shown). These values are a l s o i n agreement w i t h previous r e p o r t s (55). 2. Comparison of Py RNA Synthesis i n MK, ME and BHK-21 C e l l s Although py v i r u s r e p l i c a t e s mainly i n mouse c e l l s , there have been r e p o r t s t h a t i t r e p l i c a t e s i n hamster t i s s u e (46). An attempt was t h e r e f o r e made t o compare v i r a l RNA synth e s i s i n py i n f e c t e d MK, ME and BHK-21 c e l l s at 8 hours and ho hours a f t e r they were i n f e c t e d w i t h py v i r u s . As shown i n t a b l e 1, BHK-21 c e l l s e x h i b i t e d no s i g n i f i c a n t s y n t h e s i s of py RNA at any time a f t e r i n f e c t i o n , w h i l e ME c e l l s and MK c e l l s e x h i b i t e d an increase of py RNA t o 0.005$ (twice the background l e v e l ) at 8 hours and 0.2$ at kO hours a f t e r i n f e c t i o n . I t i s of i n t e r e s t t o note t h a t w h i l e py v i r u s was found to i n f e c t BHK-21 c e l l s and i n t e g r a t e i t s DNA i n t o the c e l l u l a r DNA.. (2), there was no detectable t r a n s c r i p t i o n of the v i r a l RNA. Table I: Py RNA Synthesis in MK, ME and BHK-21 Cells. Six petri dish cultures of secondary ME cells and of BHK-21 cells were treated as follows: Two cultures of each cell type were labelled with "H-uridine for 2 hours at 200 pCi/2 ml of MEM/dish, and their RNA extracted and purified. The remaining cultures were infected with py virus at an moi of 50 pfu/cell. At 6 hours pi and 38 hours pi, two petri dishes of each cell type were labelled with -uridine as outlined above, and their RNA extracted and purified. Samples (6o (igm) of each RNA preparation were analyzed for radio-activity and hybridized with 1 |_igm of py I DNA. Specific activity of ME RNA was about 50,000 cpm/ugm and that of BHK-21 RNA was about 100,000 cpm/Vgm. The above experiment was repeated with BHK-21 and primary MK cell cultures. Specific activity of MK cell RNA was 3,000 cpm/figm. Values of RNA hybridized at k-0 hours were 850, 60 and 7,000 for MK, BHK-21 and ME RNAs. Table I: Py RNA synthesis in MK, ME and BHK-21 Cells $ cpm hybridized with Py DNA Time pi. Mouse Kidney Mouse Embryo BHK-21 0 hours 8 hours hO hours 0 0.008$ 0. 23$ 0.003$ 0.005$ 0. 20$ 0.0007$ 0.0004$ 0.0003$ 49 3. a. Size of Py RNA i n I n f e c t e d C e l l s P r e l i m i n a r y s t u d i e s on py RNA have shown t h a t i t sedimented heterogeneously i n sucrose gradients and d i d not appear to co n t a i n any p o p u l a t i o n of d i s c r e t e s i z e molecules (55). These r e s u l t s were confirmed and extended i n the present study. Thus as shown i n f i g . 3(a) the py RNA was found to be a heterogeneous p o p u l a t i o n of molecules, sedimenting from 4s t o beyond 453?,. b. Sedimentation on DMSO Sucrose Gradients In order t o r u l e out the p o s s i b i l i t y t h a t the heterogeneous behaviour of py RNA during sedimentation was due t o c o n f i g u r a t i o n a l changes between molecules of s i m i l a r s i z e , RNA from the same pr e p a r a t i o n as used i n f i g . 3(a) was c e n t r i f u g e d on a sucrose gradient made up i n dimethyl s u l f o x i d e (DMSO). Sedimentation i n DMSO tends t o minimize the secondary s t r u c t u r e i n RNA molecules (lOO). As seen i n f i g . 3(b), the v i r a l RNA s t i l l sedimented h e t e r o -geneously throughout the gradient as i n f i g . 3(a). c. Polyacrylamide g e l E l e c t r o p h o r e s i s Polyacrylamide g e l e l e c t r o p h o r e s i s was a l s o used as a method f o r a n a l y s i s of py RNA s i z e d i s t r i b u t i o n , s i n c e the method r e s o l v e s Fig. 3: Sedimentation of Py RNA through (A) Sucrose Gradient, (B) DMSO Sucrose Gradient. The RNA was obtained from py infected MK cell cultures infected o at an input multiplicity of 50 pfu/cell, incubated with H-uridine between 29 and 31 hours pi at kO p.Ci/2 ml/dish, and harvested for RNA extraction at 31 hours pi. Specific activity of the RNA was 3,500 cpm/ngm. About 50 |agm of this RNA were sedimented through a sucrose gradient or DMSO sucrose gradient. After removal of 10 p.1 of each fraction for total radioactivity measurement on glass fibre filters, the fractions were pooled in pairs and used for the hybridization reaction with filters bearing 0\ 5 M-gm of py DNA. Prior to hybridiza-tion, the DMSO fractions were dialyzed against 1,000 volumes of 0.001 M EDTA to remove the DMSO. Direction of sedimentation was to the left. Table 2:. E l u t i o n of Py RNA from Polyacrylamide Gels. Four 5 0 ugm q u a n t i t i e s of nuclear RNA ( 7 , 0 0 0 cpm/ugm) from py i n f e c t e d MK c e l l s were r e s p e c t i v e l y placed i n t o 13 x 1 5 0 mm t e s t tubes. To each of 2 t e s t tubes were added 0 . 5 ml of unpoly-merized agarose-polyacrylamide s o l u t i o n ; t o one t e s t tube o n l y polyacrylamide s o l u t i o n , and to one t e s t tube d i s t i l l e d water. The tubes were kept at room temperature f o r 2 hours and the contents of the tubes placed i n a h y b r i d i z a t i o n mixture w i t h 1 . 0 ugm of py 1 DNA. One agarose-polyacrylamide g e l was macerated w i t h a pasteur p i p e t t e p r i o r to h y b r i d i z a t i o n . Table 2: Elution of Py RNA from Polyacrylamide Gels Parameter CPM hybridized RNA in polya crylami de agarose RNA in polya crylamide agarose (macerated) RNA in polyacrylamide RNA in solution io Py CPM recovered 132i+ 7k 1^30 80 1560 88 1791 100 F i g , k: Polyacrylamide Gel E l e c t r o p h o r e s i s of Py I n f e c t e d C e l l RNA. Polyacrylamide g e l s , prepared as o u t l i n e d i n the Methods chapter, were overlayed w i t h 100 ug of the t o t a l c e l l RNA p r e p a r a t i o n used Ik i n f i g . 2. I n a d d i t i o n , about 1,000 cpm of C-ribosomal RNA prepared from py H c e l l s were a l s o l a y e r e d on the g e l . The RNA was e l e c t r o -phoresed at 7 v/cm of g e l f o r 3 hours, and s l i c e d manually. The s l i c e s were i n d i v i d u a l l y dehydrated i n s c i n t i l l a t i o n v i a l s w i t h 2 ml of absolute methanol, and counted by a d d i t i o n of 8 ml of s c i n t i l l a t i o n f l u i d t o the v i a l s . A f t e r counting f o r r a d i o a c t i v i t y , the s l i c e s were removed from the toluene, washed twice w i t h 95$ ethanol, and twice w i t h 6 x SSC. They were then s e q u e n t i a l l y pooled i n groups of 5 s l i c e s , and h y b r i d i z e d w i t h 0.5 ug of py DNA. D i r e c t i o n of m i g r a t i o n i s t o the r i g h t . Fig. 5: Resedimentation of py RNA in a Sucrose Gradient. Purified, radioactive (10,000 cpm/ugm) RNA was prepared from two MK cell cultures which had been infected with py virus at moi of 100 and labelled with ^H-uridine at kO \iC±/2 ml of MEM/dish. The RNA (100 ngm) was layered on each of 2 sucrose gradients, centrifuged and fractionated. The fractions of one gradient, pooled in threes, were hybridized with py I DNA, while the fractions of the second gradient were monitored for radioactivity, pooled as indicated in fig. 5(a) and the pools were precipitated with ethanol. The ethanol precipitates were resuspended in 0.001 M EDTA and again sedimented on individual sucrose gradients. The fractions of the gradients were monitored for radioactivity and fractions, pooled in groups of 3, hybridized with py I DNA. 57 F i g . 5: 6 12 18 6 12 18 FRACTION NUMBER RNA species according t o t h e i r molecular weights (106). A p r e l i m i n a r y study on the e l u t i o n o f py RNA from the g e l s l i c e s was done as out-l i n e d i n t a b l e 2. This study i n d i c a t e d t h a t i n c u b a t i o n of the RNA trapped i n the polyacrylamide agarose g e l i n a h y b r i d i z a t i o n mixture l e d t o the e l u t i o n of at l e a s t 75$ of the py RNA. This method was t h e r e f o r e considered adequate f o r monitoring py RNA d i s t r i b u t i o n on g e l s a f t e r e l e c t r o p h o r e s i s of i n f e c t e d c e l l RNA. The r e s u l t s of such a study are shown i n f i g . k. The v i r a l RNA appeared t o migrate heterogeneously w i t h a s i z e range s i m i l a r to t h a t found i n sucrose g r a d i e n t s . k. Resedimentation of Py RNA on Sucrose Gradients To f u r t h e r t e s t t h a t the sedimentation c h a r a c t e r i s t i c s of py RNA were not due t o an a r t e f a c t , the RNA which sedimented to d i f f e r e n t regions of a sucrose g r a d i e n t , was r e c e n t r i f u g e d on another sucrose gradient. As shown i n f i g . 5, v i r u s i n f e c t e d c e l l RNA was f r a c t i o n a t e d on a sucrose g r a d i e n t (5a), and RNAs i n f r a c t i o n s corresponding t o regions of 28s + (5b), 28s to l8s (5c) and l8s t o Us (5d) were r e c e n t r i f u g e d . The v i r a l RNA resedimented t r u e to i t s o r i g i n a l sedimentation value, w i t h a f r a c t i o n s e d i -menting as s l i g h t l y smaller u n i t s . These may have been due t o breakdown during h a n d l i n g or incomplete r e s o l u t i o n on the o r i g i n a l g r adient. 5. Polyoma RNA from Nuclear and Cytoplasmic F r a c t i o n s of V i r u s I n f e c t e d C e l l s . The p r e p a r a t i o n of nuclear and cytoplasmic e x t r a c t s from MK c e l l s proved d i f f i c u l t , u s i n g conventional techniques (76), since the c e l l s would not r e a d i l y detach from the monolayer. A c c o r d i n g l y , the procedure was modifi e d so th a t washed c e l l monolayers were e i t h e r swollen w i t h RSB or t r e a t e d w i t h RSB + c o n t a i n i n g 1$ NPUO, f o r 15 minutes. The swollen or detergent t r e a t e d c e l l s were then removed from the c u l t u r e d i s h w i t h a rubber policeman. Although about h a l f of the c e l l s were broken by t h i s method, as seen by phase c o n t r a s t microscopy, the resuspended c e l l s had to be f u r t h e r t r e a t e d i n a Dounce homogenizer t o give near complete breakage of the c e l l s . The i s o l a t e d nuclear f r a c t i o n of the above p r e p a r a t i o n was then resuspended i n RSB and f u r t h e r d i s r u p t e d w i t h a Dounce homogenizer. No whole c e l l s could be detected by phase co n t r a s t microscopy i n the nuclear f r a c t i o n so t r e a t e d . When the supernatant of the r e -homogenized nuclear f r a c t i o n was f u r t h e r examined, i t was found to co n t a i n about 10$ of the r a d i o a c t i v i t y and py RNA content of the cytoplasmic e x t r a c t . This RNA a l s o sedimented on a sucrose gradient i n a manner i n d i s t i n g u i s h a b l e from t h a t of the cytoplasmic e x t r a c t . C e l l breakage was monitored by phase microscopy which revealed whole, cytoplasm f r e e n u c l e i . • I n f e c t e d c e l l s were al s o l a b e l l e d w i t h H-thymidine f o r l 6 hours, f u r t h e r incubated i n non-radioactive F i g . 6: Sucrose Gradient Sedimentation of Py RNA from (A) N u c l e i and (B) Cytoplasm, of Py I n f e c t e d MK C e l l s . Conditions of i n f e c t i o n , l a b e l l i n g , sedimentation and h y b r i d -i z a t i o n were the same as i n legend to f i g . 3(a). Seventy-five ngm of nuclear RNA and 150 pgm of cytoplasmic RNA w i t h r e s p e c t i v e s p e c i f i c a c t i v i t i e s of 7,200 cpm/|agm, and 1,700 cpm/pgm, were used. medium f o r 8 hours, and nuclear and cytoplasmic f r a c t i o n s separated as above. Less than 3$ of the TCA p r e c i p i t a b l e r a d i o a c t i v i t y was found i n the cytoplasmic f r a c t i o n , i n d i c a t i n g t h a t the n u c l e i were not s i g n i f i c a n t l y d i s r u p t e d (ik). A q u a n t i t a t i v e study on a number of preparations of nuclear and cytoplasmic f r a c t i o n s showed th a t about 75$ of the mass of RNA i s e x t r a c t e d from these preparations as compared to d i r e c t e x t r a c t i o n from the whole c e l l c u l t u r e s . I n general, the nuclear RNA mass was about 2 0 $ t h a t of the c y t o -plasmic RNA, w h i l e the t o t a l u r i d i n e r a d i o a c t i v i t y of the RNA from c u l t u r e s l a b e l l e d f o r 2 hours, was about equal i n the nuclear and cytoplasmic e x t r a c t s . When RNA p r e p a r a t i o n s , p u r i f i e d from nuclear and cytoplasmic f r a c t i o n s of v i r u s i n f e c t e d c e l l s , were c e n t r i f u g e d on sucrose g r a d i e n t s , as shown i n f i g . 6, the py RNA was found t o sediment as d i d the py RNA from whole c e l l s . 6. Leakage of RNA from N u c l e i During F r a c t i o n a t i o n The above r e s u l t s were f u r t h e r analyzed f o r cross contamination between nuclear and cytoplasmic RNAs. The d i s t r i b u t i o n of t o t a l r a d i o a c t i v i t y r e v e a l e d t h a t i n the nuclear RNA there was a b a r e l y detectable peak at l8s though no peak at ks, which i n d i c a t e d a n e g l i g i b l e amount of contamination w i t h cytoplasmic RNA. The c y t o -F i g . 7: Sucrose Gradient Sedimentation of Py RNA from NPl+O Cytoplasm of Py I n f e c t e d MK C e l l s . Conditions o f i n f e c t i o n and l a b e l l i n g were described i n the legend t o f i g . 2. S p e c i f i c a c t i v i t y of the NPUO cytoplasmic MA was 860 cpm/ pgm. Seventy-five (igm of t h i s RNA were analyzed by the u s u a l sucrose gradient method. I n t h i s case, f r a c t i o n s were pooled i n groups of three f o r the h y b r i d i z a t i o n t e s t w i t h f i l t e r s b e aring 0.5 Mgm of py DNA. 6k 65 plasmic RNA gradient d i d c o n t a i n r a d i o a c t i v i t y sedimenting beyond 28s, though there was no d i s t i n c t 45s peak present, thus demonstrating t h a t the cytoplasmic RNA was r e l a t i v e l y f r e e of nuclear RNA (76). I n order t o f u r t h e r s u b s t a n t i a t e t h i s c o n c l u s i o n , a d i f f e r e n t method of RNA e x t r a c t i o n was used which i n v o l v e d the use of NPUO and no mechanical breakage of the c e l l s . The cytoplasmic e x t r a c t obtained under these c o n d i t i o n s was e x t r a c t e d f o r RNA. This RNA sedimented on a sucrose gradient as shown i n f i g . 7- The py RNA was detected throughout the gradient as i n f i g . 6(b), though no s i g n i f i c a n t amount of c e l l u l a r RNA sedimenting beyond 28s was detected. 7. Polyribosomal RNA In order to c h a r a c t e r i z e the f u n c t i o n a l py s p e c i f i c m-RNA, i t was necessary t o examine the polysomes of the i n f e c t e d c e l l s . As shown i n f i g . 8(a) and 8 ( b ) , the l e v e l of polyribosomes i n the i n -f e c t e d c e l l c u l t u r e was twice the l e v e l i n the contact i n h i b i t e d u n i n f e c t e d c e l l c u l t u r e s which i n d i c a t e d an increase i n the synthesis of f u n c t i o n a l m-RNA i n the i n f e c t e d c e l l s . The s i z e d i s t r i b u t i o n of py RNA from the polyribosomes was co n s i d e r a b l y d i f f e r e n t from t h a t of the nuclear and cytoplasmic f r a c t i o n s . No v i r u s s p e c i f i c RNA sedimenting f a s t e r than 28s was detected, implying that the l a r g e py RNA i n the cytoplasm was cleaved upon e n t e r i n g the polyribosomes or simply d i d not i n c o r p o r a t e i n t o the polyribosomes. The i s o l a t e d Fig. 8: 'Sucrose Gradient Sedimentation of (A) Cytoplasmic Extract of Mock Infected Cells: . (B) Cytoplasmic Extract of Py Infected Cells and (C) Polyribosome Associated RNA of Py Infected Cells. Cultures of mock-infected and py infected (50 pfu/cell) MK cells were incubated with H-uridine at kO uCi/2 ml/dish between 30 to 32 hours pi and harvested at 32 hours pi for cell fractionation. The cytoplasmic fractions obtained by Dounce homogenization were analyzed on 15 to 30$ RSB+ sucrose gradients (A, B). Infected polyribosome fractions sedimenting faster than 80s were pooled, and extracted for RNA, 150 ugm of which were analyzed on a 5 "bo 20$ sucrose gradient (C) as outlined in legend to fig. 2. Specific activity of the poly-ribosomal RNA was 600 cpm/ugm. polyribosomes were predominantly hexamers which i n d i c a t e d t h a t no s i g n i f i c a n t breakdown of the m-RNA had occurred during t h e i r i s o l a -t i o n . I t should be p o i n t e d out however, t h a t no species of py RNA sedimenting f a s t e r than 28s could be found i n the subribosomal r e g i o n of the g r a d i e n t shown i n f i g . 8(b), although c e l l u l a r RNA exceeding 28s i n sedimentation was found here. 8. Comparison of Nuclear and Cytoplasmic RNA by Competition H y b r i d i z a t i o n A study of the base sequence homology between the nuclear and cytoplasmic py RNAs was performed i n order t o f u r t h e r compare these 1 molecules. M i l l i g r a m q u a n t i t i e s of non r a d i o a c t i v e and r a d i o a c t i v e RNA were prepared both from the nuclear and cytoplasmic f r a c t i o n s of i n f e c t e d c e l l s . As depicted i n f i g . 9, i n c r e a s i n g amounts of non l a b e l l e d RNA were h y b r i d i z e d w i t h a set amount of py I DNA bound to f i l t e r s . The f i l t e r s were then washed and incubated w i t h the r a d i o a c t i v e RNA. The f r a c t i o n of the l a b e l l e d py RNA t h a t h y b r i d -i z e d w i t h the f i l t e r as a f u n c t i o n of the c o n c e n t r a t i o n of the non l a b e l l e d py RNA i s shown. Both the nuclear and cytoplasmic py RNA competed e f f i c i e n t l y w i t h themselves and each other, which i n d i c a t e d t h a t i n terms of base sequence homology, the two py RNA preparations resembled each other c l o s e l y . F i g . 9: Competition H y b r i d i z a t i o n Between Nuclear and Cytoplasmic RNA. Nuclear and cytoplasmic RNA were obtained as o u t l i n e d i n the M a t e r i a l s and Methods, u s i n g the Dounce homogenization procedure. The s p e c i f i c r a d i o a c t i v i t y o f nuclear RNA was 11,200 cpm/ugm and of the cytoplasmic RNA was 4,525 cpm/ugm. The h y b r i d i z a t i o n mixture of the f i r s t h y b r i d i z a t i o n step contained the i n d i c a t e d amounts of u n l a b e l l e d RNA, and 0.1 ugm of py I DNA. In.the second step, the h y b r i d c o n t a i n i n g f i l t e r s were incubated w i t h 20 ugm of the l a b e l l e d RNA. Both h y b r i d i z a t i o n s were c a r r i e d on f o r 2k hours. The 100$ value of the graph denotes 300 cpm f o r nuclear RNA and 200 cpm f o r cytoplasmic RNA. The upper l i n e denotes a c o n t r o l h y b r i d i z a t i o n w i t h n o n - l a b e l l e d yeast RNA and l a b e l l e d nuclear RNA. The lower curve the f o l l o w i n g : *> l a b e l l e d nuclear RNA, u n l a b e l l e d nuclear RNA. • l a b e l l e d nuclear RNA, u n l a b e l l e d cytoplasmic RNA. A l a b e l l e d cytoplasmic RNA, u n l a b e l l e d cytoplasmic RNA. o l a b e l l e d cytoplasmic RNA, u n l a b e l l e d nuclear RNA. 9. Comparison of Nuclear and Polyribosomal py RNA by Competition H y b r i d i z a t i o n . A s i m i l a r study was done, comparing polyribosomal py RNA w i t h nuclear py RNA from i n f e c t e d c e l l c u l t u r e s . As shown i n f i g . 10, the nuclear py RNA competed e f f i c i e n t l y w i t h polyribosomal py RNA i n d i c a t i n g t h a t a l l sequences i n polyribosomal py RNA were present i n the nuclear py RNA. However the polyribosomal py RNA d i d not compete e f f i c i e n t l y w i t h nuclear py RNA, nor w i t h i t s e l f . I n t h i s regard, i t was d i f f i c u l t t o say w i t h c e r t a i n t y t h a t the polyoma sequences present i n the nuclear RNA were conserved as the py RNA was processed to the polysomes. I t i s b e l i e v e d t h a t the low amounts of polysomal py RNA a v a i l a b l e accounted f o r t h i s low e f f i c i e n c y o f h y b r i d i z a t i o n . 10. Comparison of Large and Small py RNA by Competition H y b r i d i z a t i o n . Polyoma s p e c i f i c cytoplasmic RNA populations sedimenting beyond 20s and below 18s on a sucrose gradient were compared by competition h y b r i d i z a t i o n , as shown i n f i g . 11. Both RNA species competed e f f i c i e n t l y w i t h themselves and w i t h each other, i n d i c a t -i n g t h a t the l a r g e py RNA had a very s i m i l a r base sequence to the s m a l l py RNA. Fig. 10: Competition Hybridization Between Nuclear and Polyribosomal Py RNA. Nuclear and polyribosomal py RNA were, obtained as outlined in the Methods. Since a number of batches of infected cells were necessary, care was taken that both the nuclei and polyribosomes from each batch were used in equal amounts in each pool. Polyribosomal RNA was con-sidered to be a l l RNA from ribosomes sedimenting in excess of 80s. In addition, one third of each batch preparation was always labelled with H-uridine at 60 uCi/2 ml/dish in order to obtain the radioactive RNA. The specific radioactivity of the nuclear RNA was 13,000 cpm/ugm and of the polyribosomal RNA was about 2,000 cpm/ugm. The hybridization mixture of the fi r s t step involved indicated amounts of unlabelled RNA with 0.1 ugm of py I DNA. In the second step 20 ugm of nuclear RNA or 60 ugm of polysomal radioactive RNA were used. Both hybridizations were carried out for 2k hours. The 100$ value of the graph denotes 1,200 cpm for the nuclear RNA and 200 cpm for the polysomal RNA. The upper line denotes a control hybridization with non-labelled yeast RNA and labelled nuclear RNA. The lower curve the following: o labelled polyribosomal RNA, non-labelled nuclear RNA A labelled polyribosomal RNA, non-labelled polyribosomal RNA • labelled nuclear RNA, non-labelled polyribosomal RNA g\ labelled nuclear RNA, non-labelled nuclear RNA Fig. 11: Competition Hybridization Between Large and Small Py RNA. Approximately 1 mg of labelled (specific activity 4,525 cpm/ugm) and 2 mg of non-labelled cytoplasmic RNA were centrifuged through 5 to 2 0 $ STE SDS sucrose gradients in an SW40 rotor for k. 5 hours at 15°C. The gradients were fractionated, monitored for a n d r a < ^ - ° ~ activity. The ^> l8s and < l8s fractions were separately pooled for each gradient. The adjacent fractions of the pools were omitted. The RNA was recovered by ethanol precipitation. The indicated amounts of unlabelled RNA were used in each hybridization mixture along with 0 . 1 ugm of py I DNA. In the second step, 50 ugm of small RNA or 25 ugm of large RNA were used in each appropriate mixture. The values of the hybrids were 150 cpm in the case of the large RNA and 75 cpm in the case of the small RNA. The upper line denotes a control hybrid-ization with non-labelled yeast RNA and labelled large RNA. The lower curve the following: A labelled large RNA, 0 labelled large RNA, A labelled small RNA, m labelled small RNA, unlabelled large RNA unlabelled small RNA unlabelled small RNA unlabelled large RNA 76 CHAPTER IV: RESULTS II Processing of Py RNA in Virus Infected Cell Cultures 1. Kinetics of Labelling of Nuclear and Cytoplasmic Py RNA In the previous chapter, the py RNA from different compartments of the infected cell was characterized with respect to base sequence homology and size distribution. It therefore became possible to study the actual processing of py RNA from its transcription time onward. A fi r s t step in this direction was to study the kinetics of labelling of py RNA in the nuclear and cytoplasmic fractions of the infected cells. In the experiment, the results of which are depicted in fig. 12, py infected cells were exposed to H-uridine for periods of 15 minutes to k hours. The RNA was extracted from these cells, purified and analyzed for absorbancy, radioactivity and also hybridized with py I DNA to determine the virus specific content. The incorporation of radioactive label into the total nuclear RNA started immediately, and increased at a constant rate. On the other hand, the incorporation of label into the total cytoplasmic RNA was very slow for about one hour, whereupon i t began to increase at a rate parallel to that of the nuclear RNA. This lag in the incorporation of the label was considered to reflect the time F i g . 12: V a r i a b l e Pulse L a b e l l i n g of I n f e c t e d C e l l RNA F i f t e e n MK c e l l c u l t u r e s , i n f e c t e d w i t h py v i r u s at an input o m u l t i p l i c i t y of 100 p f u / c e l l , were l a b e l l e d at 30 hours p i w i t h H-u r i d i n e at 100 uCi/ 2 ml/dish. At 15, 30, 60, 120 and 2^0 minutes a f t e r l a b e l l i n g , RNA was e x t r a c t e d from 3 of the above c u l t u r e s , p u r i f i e d and monitored f o r and r a d i o a c t i v i t y . One eighth of each RNA sample (50 ugm of cytoplasmic RNA and 15 ugm of nuclear RNA) was incubated w i t h 0.5 Mgm of py I DNA f o r h y b r i d i z a t i o n . A l l values were normalized w i t h respect t o the A^g^ °^ ^ e n u c-'- e a r a n t^ cytoplasmic RNA preparations. nuclear RNA - dark symbols cytoplasmic RNA - l i g h t symbols F i g . 12; 1 2 3 4 H O U R S taken f o r the l a b e l l e d RNA t o be t r a n s p o r t e d from the nucleus to the cytoplasm i n the i n f e c t e d MK c e l l s . The i n c o r p o r a t i o n of l a b e l i n t o the py RNA i n the nuclear f r a c t i o n i n creased at a constant r a t e , s i m i l a r to t h a t of the c e l l u l a r RNA, up to two hours, a f t e r which i t slowed down. On the other hand, the cytoplasmic py RNA was observed to become l a b e l l e d at a constant r a t e , which was p a r a l l e l t o the cytoplasmic c e l l RNA f o r the f i r s t hour, but d i d not undergo a sharp increase as d i d the l a t t e r over a longer l a b e l l i n g p e r i o d . A comparison of the v i r a l and c e l l u l a r RNA i n the above study showed t h a t the n u c l e i contained about 10 times as much of the l a b e l l e d py RNA as d i d the cytoplasm. Of the t o t a l l a b e l l e d RNA i n the nuclear f r a c t i o n , o n l y about 1% was v i r u s s p e c i f i c , r e g a r d l e s s of the l a b e l l i n g p e r i o d . This i m p l i e d t h a t during t r a n s c r i p t i o n a constant p o r t i o n of the t r a n s c r i b e d RNA was v i r a l RNA which was then processed i n the n u c l e i at a r a t e s i m i l a r t o the c e l l RNA. On the other hand, the percentage of the l a b e l present as py RNA i n the cytoplasmic RNA decreased a f t e r 30 minutes of l a b e l l i n g . This most l i k e l y r e f l e c t e d the accumulation o f long l i v e d r-RNA i n the c y t o -plasm. The py RNA was synthesized and processed i n the nuclear f r a c t i o n at a r a t e comparable to the t o t a l c e l l RNA. I t s appearance i n the cytoplasmic f r a c t i o n was s i m i l a r to th a t of the t o t a l c e l l RNA f o r the f i r s t 30 minutes, a f t e r which i t d i d not appear t o accumulate at the rate of the cell RNA, but increased at a rate which was about 10$ of its rate of increase in the nuclear fraction. In similar experi-ments the cytoplasmic RNA made up to 20$ of the nuclear py RNA, but this fraction also remained constant regardless of the labelling period. The implication of these data was that regardless of the time of pulse, only 10 to 20$ of the viral RNA transcribed in the nuclear fraction was processed on to the cytoplasmic fraction in py infected MK cell cultures. 2. Py RNA in Pulse Labelled Cells. The size distribution of py RNA pulse labelled for 15 minutes was examined on a sucrose gradient as shown in fig. 13. Based on the results of the above experiment (fig. 12) the labelled viral RNA was s t i l l mainly in the nuclear fraction and hence would be subject to a smaller degree of processing than py RNA labelled for a 2 hour period (shown in fig. 3)• As shown in fig. 13, the 15 minute pulse labelled RNA sedimented in a heterogeneous fashion, predominantly faster than l8s with up to 46$ sedimenting faster than 28s. Since no distinct labelled r-RNA peaks were evident in the gradient, the RNA was considered to have been subject to a minimal amount of processing. Although shorter pulse labelling studies were not performed (since an insufficient amount of viral RNA would be labelled), this study alone suggested Fig. 13: Sucrose Gradient Sedimentation of Pulse Labelled Py RNA. Py infected (moi 100 pfu/cell) MK cells were labelled at 31 hours pi with H-uridine at 200 uCi/2 ml/dish for 15 minutes. RNA was extracted and purified from these cells, and 75 ugm of this RNA (specific activity 1,l6o cpm/ugm) were analyzed on a sucrose gradient as outlined in the Methods. The gradient fractions were monitored for A^ g- , 10 ul sampled for radioactivity, and the remainder pooled sequentially in pairs and hybridized with 0.5 ugm of py I DNA. Direction of sedimentation was to the left. 82 Fig. 13: 5 1 0 1 5 2 0 F R A C T I O N N U M B E R that the py RNA species were transcribed as a population of large . molecules, predominantly greater than 28s. A similar size distribution of py RNA labelled for 20 minutes was recently observed by other workers (1). 3. Pulse and Chase Studies. Pulse and chase studies, which involved pulse labelling of cells for a brief period of time, followed by the addition of Actinomycin D to stop incorporation of the label, have proven useful in studying the synthesis and processing of r-RNA. A similar type of approach to study the processing of py RNA in infected cells was attempted by the use of not only Actinomycin D, but also excess non labelled uridine, and cordycepin. The behaviour of the cellular RNA and viral RNA in the nuclear and cytoplasmic -fractions, under the above conditions of pulse and chase, was examined. The result of such an experiment is shown in fig. 14(a) and (b). In Actinomycin D treated cells, the radioactivity of the total nuclear RNA showed a rapid rate of decay in the first hour., followed by a slower rate for the remaining 5 hours of chase. A total decrease of 82$ was observed throughout the entire chase period. The radio-activity of the py nuclear RNA behaved in a similar fashion, with the radioactivity decreasing by 90$ during the six hour chase period. In the same cell cultures, the total cytoplasmic RNA radioactivity Fig. Ik: Pulse and Chase Studies on Infected Cell RNA. Twenty-four py infected (moi 100 pfu/cell) MK cell cultures were pulse labelled with H-uridine, at 100 uCi/2 ml/dish for kO minutes. The RNA was immediately extracted from two cultures, while the remainder were washed with MEM and one half incubated with MEM containing 5 ugm/ml of Actinomycin D, and the other half ' -k with MEM containing 2 x 10 M uridine. RNA was extracted from these cultures at 1, 2 and 6 hours after the chase began. The purified RNA preparations were monitored for A^g^, radioactivity, and one eighth of each sample (12 ugm of nuclear RNA, and kO ugm of cytoplasmic RNA) hybridized with 0.5 M-gm of py I DNA to deter-mine viral RNA content. The data expressed in this figure were a l l normalized to a constant mass of respective nuclear or cyto-plasmic RNA. A. Total cell RNA B. Viral RNA C. % cpm hybridized Actinomycin D chase, nuclear RNA urindine chase, nuclear RNA Actinomycin D chase, cytoplasmic RNA uridine chase, cytoplasmic RNA. Fig. Ik: 2 4 6 H O U R S O F C H A S E 86 i n c r e a s e d about three f o l d , w h i l e the corresponding py s p e c i f i c r a d i o -a c t i v i t y decreased about three f o l d during the chase w i t h Actinomycin D. A comparison of the above values ( f i g . 14(c)) showed t h a t i n the nucleus the py RNA makes up a constant f r a c t i o n of about 3$ of the c e l l RNA. On the other hand i n the cytoplasm, the py RNA f r a c t i o n of the c e l l RNA decreased during the chase p e r i o d from 2$ t o 0.1$. This was b e l i e v e d to be due t o the d i f f e r e n t r a t e s of processing of the py RNA and the longer l i v e d species of c e l l u l a r RNA such as r-RNA. The shapes of the decay curves of the py RNA i n the nuclear and cytoplasmic f r a c t i o n s throughout the chase w i t h Actinomycin D, i n d i c a t e d t h a t the p r o c e s s i n g of py RNA was b i p h a s i c i n nature. In the f i r s t hour over 75$ of the v i r a l RNA was degraded i n the nucleus, without being t r a n s p o r t e d t o the cytoplasm. I n the next f i v e hours, the r a t e s of l o s s of py RNA from the nucleus and cytoplasm were n e a r l y p a r a l l e l , which was c o n s i s t e n t w i t h processing from the nucleus t o the cytoplasm and degradation i n the cytoplasm. -k I n a p a r a l l e l chase experiment, 10 M u r i d i n e was used i n place of Actinomycin D. As shown i n f i g . l U , the c e l l u l a r nuclear r a d i o -a c t i v i t y appeared t o remain constant, r e f l e c t i n g the f a c t t h a t i n c o r -3 p o r a t i o n of H-uridine continued, t o some extent, i n the presence of excess u n l a b e l l e d u r i d i n e . This was thought t o be due t o the l a r g e i n t r a c e l l u l a r p o o l of u r i d i n e compounds i n mammalian c e l l s (18). The nuclear py RNA r a d i o a c t i v i t y a l s o showed a more gradual decrease com-pared w i t h the Actinomycin D t r e a t e d c e l l s . The u r i d i n e chased c e l l RNA r a d i o a c t i v i t y from the cytoplasmic f r a c t i o n appeared t o be r e l a t i v e l y u n a f f e c t e d by the a d d i t i o n of u r i d i n e up to s i x hours of chase. The l a b e l l e d RNA continued to accumulate i n t h i s f r a c t i o n , i n contrast t o the s i t u a t i o n i n the Actinomycin D chase. The v i r u s s p e c i f i c r a d i o a c t i v i t y remained at a constant l e v e l showing no decrease. These r e s u l t s were i n agreement w i t h those of the Actinomycin D chase, on the assumption t h a t the excess u r i d i n e was an i n e f f i c i e n t chasing system. I n a s i m i l a r experiment, cordycepin at 50 ugm/ml was used to produce a chase. The e f f e c t on the t o t a l and v i r a l nuclear and c y t o -plasmic RNA was intermediate between t h a t of Actinomycin D and u r i d i n e . Although t h i s drug i n h i b i t s the processing of m-RNA (76a), i t d i d not a f f e c t the e n t r y of l a b e l l e d py RNA i n t o the cytoplasm. Since the process was not i n v e s t i g a t e d f u r t h e r , i t i s p o s s i b l e that i n s u f f i c i e n t q u a n t i t i e s of cordycepin were used t o completely b l o c k the processing of v i r a l RNA. k. Size D i s t r i b u t i o n of Py RNA i n Pulse and Chase Studies Pulse l a b e l l e d and chased RNA from nuclear and cytoplasmic f r a c t i o n s of i n f e c t e d c e l l s were analyzed on sucrose g r a d i e n t s . As shown i n f i g . 15(a) and (b), a l l s i z e c l a s s e s of nuclear RNA decreased i n the chase w i t h Actinomycin D. The g r e a t e s t decrease was observed by the l a r g e py RNA, and the smallest decrease w i t h the s m a l l py RNA. Fig. 15: Sucrose Gradient Analysis of Py RNA from the Pulse-Chase Study. RNA preparations from pulse and chase studies such as outlined in fig. Ik were analyzed on sucrose gradients. About 200 ugm of cytoplasmic RNA (specific activities from k-9 to 730 cpm/ugm) were analyzed on the gradients which are shown in sections B and F. Similar analyses of about 60 ugm of nuclear RNA (specific activities from 8,000 to 1,5^0 cpm/ugm) are shown in sections A and E . The fractionated sucrose gradients were analyzed for virus specific radioactivity. Fractions 1-8, 9-lk and 15-20 from each preparation were respectively summed for large, medium and small py RNA, and these sums appear opposite to their corresponding gradients. Sections A to D correspond to Actinomycin D chased RNA, while sections E to H are for uridine chased RNA. A l l values were normalized to a constant mass of nuclear or cytoplasmic RNA. For Sections A, B, E , F: control —Q-2 hours chase — 6 hours chase -Ar-For Sections C, D , G, H: 'large RNA' (region of fractions 1-8) - 0 - - O -'medium RNA' (region of fractions 9-lh) -• - - •• 'small RNA' (region of fractions 15-20) - A - - A -8 9 90 These data i n d i c a t e t h a t there was a p r e f e r e n t i a l breakdown of the l a r g e nuclear py RNA t o smaller molecules during the chase period. A s i m i l a r a n a l y s i s of the cytoplasmic py RNA from a pulse and chase study ( f i g . 15(b), ( d ) ) , revealed t h a t a l l s i z e c l a s s e s were degraded at a v e r y s i m i l a r r a t e . Though here again the s m a l l py RNA appeared most s t a b l e . The r a t e s of decay of the v a r i o u s s i z e c l a s s e s of py RNA pulse l a b e l l e d and chased w i t h excess u r i d i n e , were not as pronounced as w i t h Actinomycin D. These r e s u l t s , shown i n f i g . 1 5 ( e ) , ( f ) , ( g ) , ( h ) , d i d none the l e s s , agree w i t h those obtained w i t h the Actinomycin D chase i n t h a t the decay curves f o l l o w e d a s i m i l a r p a t t e r n . 5. Involvement of Polyadenylate Sequences w i t h Py RNA. Polyadenylate ( p o l y (A)) sequences, attached t o the 3' terminus of the m-RNA, have been shown to be i n v o l v e d i n the processing of these molecules (65). The r o l e of p o l y (A) i n regard to the pro-cessing of py RNA was t h e r e f o r e examined i n the py i n f e c t e d MK c e l l system. P u r i f i e d RNA from py i n f e c t e d c e l l s as w e l l as from t h e i r nuclear cytoplasmic and polyribosomal components was analyzed f o r the presence of p o l y (A) sequences, by i t s a b i l i t y t o b i n d t o M i l l i -pore f i l t e r s i n the presence of 0.5 M MBB. As shown i n Table 3, the degree of b i n d i n g to M i l l i p o r e increases as the py RNA i s Table 3: Poly (A) Content of Py Infected Cell RNA These are average values derived from three separate experiments measuring poly (A) content from various components of the virus infected cell. Less than 30 ugm of each RNA was used. One sixth of the surface area of each f i l t e r was assayed for radioactivity while the rest was used for hybridization. Table 3: P o l y (A) Content of Py I n f e c t e d C e l l RNA. R M $ of t o t a l RNA $ of Py RNA c o n t a i n i n g p o l y (A) c o n t a i n i n g p o l y (A) T o t a l c e l l Nuclear Cytoplasmic Polysomal 8.3 - 0.2 10.9 - 2.3 lk.6 + 2 . 8 31$ 16.3 t h 6.1 i 1.3 30.2 1 1.1 58$ processed from the n u c l e i through to the polyribosomes. Hence the percentage of p o l y (A) c o n t a i n i n g molecules i s lowest i n the nuclear f r a c t i o n and highest i n the polyribosomes. This suggests t h a t o n l y polyadenylate c o n t a i n i n g py RNA molecules can be p r o p e r l y processed and be i n c o r p o r a t e d i n t o polyribosomes f o r t r a n s l a t i o n . CHAPTER V: RESULTS III Studies on the Isolation and Enumeration of Poly (A)  Containing MA Molecules 1. Parameters Involved in Binding Poly (A) Containing RNA to Millipore Filters (72) In the process of measuring the relationship between poly (A) sequences of py RNA and the degree of processing, a number of parameters of poly (A) binding to Millipore were examined. The f i r s t such parameter examined was the efficiency of poly (A) binding to Millipore in the presence of MBB containing sodium (Na+MBB) compared with MBB containing potassium (K+MBB). In each case approxi-mately 5$ of the total cell RNA radioactivity bound to the Millipore f i l t e r , the values being 5.3$ for Na+MBB and 5.7$ for K+MBB. The potassium salt was therefore used in the ensuing studies to accord with other workers. The second parameter examined was the elution of bound RNA from the Millipore f i l t e r . One of the RNA containing filters was placed in a 1 ml solution of 0.1 M Tris (pH 9.0) and 0.5$ SDS and incubated at k°C for 30 minutes. This solution and f i l t e r were sampled for radio-activity and after the former was brought to pH 7-0 with HCl, were assayed for py RNA by hybridization to py I DNA. Another Millipore f i l t e r c o n t a i n i n g an equal amount of RNA was plac e d d i r e c t l y i n t o a h y b r i d i z a t i o n mixture (0.5$ SDS, 6 x SSC and 0.001 M EDTA) co n t a i n i n g 0.5 M-gm of py I DNA. I n the former case, 0.1+5$, and i n the l a t t e r case, 0.85$ of the cpm bound t o the f i l t e r subsequently h y b r i d i z e d . Thus py RNA e l u t e d from M i l l i p o r e more e f f i c i e n t l y i f the f i l t e r was pla c e d d i r e c t l y i n t o the h y b r i d i z a t i o n mixture; r a t h e r than w i t h the e l u t i n g b u f f e r , f o l l o w e d by h y b r i d i z a t i o n . I n a d d i t i o n , i t was observed t h a t the o r i g i n a l M i l l i p o r e f i l t e r s , which were e l u t e d by p l a c i n g them d i r e c t l y i n t o the h y b r i d i z a t i o n mixture, r e t a i n e d v i r t u a l l y no r a d i o a c t i v i t y . This process of e l u t i o n was, t h e r e f o r e , used i n a l l subsequent experiments. 2. Ribonuclease Resistance of RNA Bound to M i l l i p o r e . Polyoma i n f e c t e d c e l l RNA pre p a r a t i o n s , l a b e l l e d w i t h H-3 u r i d i n e or H-adenosine, were passed through M i l l i p o r e f i l t e r s i n MBB, sampled f o r t o t a l r a d i o a c t i v i t y and s t o r e d f r o z e n i n 0.5 x SSC. The RNA, p r e c i p i t a t e d out of the f i l t r a t e s by the a d d i t i o n of 2 volumes of ethanol, was r e d i s s o l v e d i n 0.5 x SSC, and sampled f o r r a d i o a c t i v i t y . Both the f i l t e r s and the f i l t r a t e s were incubated w i t h p a n c r e a t i c RNAse, at 37°C, r a p i d l y c h i l l e d t o 0°C, and made up to 6$ i n TCA. The TCA i n s o l u b l e r a d i o a c t i v i t y on both f i l t e r s and f i l t r a t e s was determined. As shown i n t a b l e k, the H-adenosine l a b e l l e d RNA bound t o M i l l i p o r e f i l t e r s was more RNAse r e s i s t a n t 97 Table k: Resistance of M i l l i p o r e Bound and Non-Bound RNA to Pa n c r e a t i c Ribonuclease. •a Thirty-one ugm of H-adenosine l a b e l l e d RNA ( s p e c i f i c a c t i v i t y 1,600 cpm/p-gm) and k-3 ugm of ^H-uridine l a b e l l e d RNA ( s p e c i f i c a c t i v i t y 3,100 cpm/ugm) were s e p a r a t e l y made up to 0.5 ml i n MBB, passed through M i l l i p o r e f i l t e r s at k°C and washed w i t h k.5 ml of i c e c o l d MBB. The f i l t e r s , of which one s i x t h of the exposed area was sampled f o r r a d i o -a c t i v i t y , were f r o z e n i n 1.0 ml of 0 . 5 x SSC at -70°C wh i l e the RNA was recovered from the pooled f i l t r a t e s and washes by p r e c i p i t a t i o n w i t h 2 volumes of ethanol. The RNA from the ethanol p r e c i p i t a t e s were a l s o made up t o 1 ml of 0 . 5 x SSC and along w i t h the f i l t e r s were i n d i v i d u a l l y mixed w i t h 10 ugm of p a n c r e a t i c RNAse and incubated at 37°C f o r kO minutes. The preparations were then p r e c i p i t a t e d w i t h 6$ TCA at 0°C and the p r e c i p i t a t e s , c o l l e c t e d on g l a s s f i b e r f i l t e r s , were counted f o r r a d i o a c t i v i t y . Table k: Resistance of Millipore Bound and Non-Bound RNA to Pancreatic Ribonuclease. of •RlVTAcp Isotope No RNAse RNAse /0 .1~~ resistant 3 cpm on f i l t e r H-adenosine 3,091 cpm 217 cpm 7.05 (bound) ^H-uridine k,96o cpm Ilk cpm 2.27 cpm in filtrate ^-adenosine 5^,981 cpm 587 cpm 1.03 (non-bound) "TI-uridine 1^3,3U8 cpm l,k00 cpm 1.01 than the corresponding ~TI-uridine l a b e l l e d RNA, while t h i s d i f f e r e n c e was not evident w i t h RNA from the f i l t r a t e s from these two species. These data provided evidence t h a t the RNA bound t o the M i l l i p o r e f i l t e r s i n MBB had RNAse r e s i s t a n t "^H-adenosine l a b e l l e d regions which c o u l d w e l l have been p o l y (A) sequences, w h i l e the RNA from the same pr e p a r a t i o n , which d i d not b i n d to M i l l i p o r e , l a c k e d t h i s property. 3. Capacity, Washing and Sampling of M i l l i p o r e F i l t e r s w i t h Bound RNA. (a) Washing An a n a l y s i s was made of the degree of washing needed to remove 5 non s p e c i f i c a l l y bound RNA from a M i l l i p o r e f i l t e r . As depicted i n f i g . l6, two s i m i l a r q u a n t i t i e s of H-adenosine l a b e l l e d RNA and 3 H-uridine l a b e l l e d RNA, were s e p a r a t e l y passed through M i l l i p o r e f i l t e r s . The f i l t e r s were washed w i t h successive volumes of MBB, and the RNA was c o l l e c t e d from the washes and f i l t r a t i o n by ethanol p r e c i p i t a t i o n . F i g u r e 16 shows th a t i f the f i l t e r s , through which RNA has been f i l t e r e d , were washed w i t h 0.5 ml of MBB, about 75$ of the now bound RNA was removed. I f they were washed w i t h an a d d i t i o n a l 1.0 ml, up t o of the non bound RNA was removed. Washing w i t h F i g . l 6 : E f f e c t s of Washing on M i l l i p o r e Bound RNA. Two 50 ugm q u a n t i t i e s of H-adenosine l a b e l l e d RNA ( l , 6 0 0 cpm/ ugm) and ^H-uridine l a b e l l e d RNA ( 3 , 0 0 0 cpm/ugm) from v i r u s i n f e c t e d c e l l s , were f i l t e r e d i n d i v i d u a l l y through M i l l i p o r e . The f i l t e r s were washed w i t h i n c r e a s i n g volumes of MBB and the washes c o l l e c t e d s e p arately. The r a d i o a c t i v i t y of the f i l t e r s was determined d i r e c t l y and that of the washes was obtained from the TCA p r e c i p i t a t e of each wash. I n a p a r a l l e l experiment, o n l y one t h i r d of each wash was TCA p r e c i p i t a t e d w h i l e the remainder was mixed w i t h 2 volumes of ethanol. The RNA p r e c i p i t a t e d by the ethanol was resuspended i n 0 . 5 x SSC, incubated f o r 3 0 minutes w i t h p a n c r e a t i c RNAse at 10 ugm/ml, and p r e c i p i t a t e d w i t h i c e c o l d 6 $ TCA. The TCA i n s o l u b l e m a t e r i a l was counted. •3 H-adenosine l a b e l l e d RNA -£0-") 3 H - u r i d i n e l a b e l l e d RNA - (-0^) F i g . l6 : VOLUME OF MBB <ml> g r e a t e r volumes (2.5 ml to 8.5 ml) removed a l l of the non bound RNA. From these data, the p r o t o c o l of applying the RNA t o the f i l t e r i n 0. 5 ml of MBB and e l u t i n g w i t h 5.k ml of MBB was developed. 3 An examination of the RNAse r e s i s t a n c e of H-adenosine and - u r i d i n e l a b e l l e d RNA from such washes re v e a l e d t h a t the f i l t r a t e and 0.5 ml wash of the former species had 2.88$ RNAse r e s i s t a n t TCA i n s o l u b l e r a d i o a c t i v i t y , w h i le the corresponding value f o r the l a t t e r RNA was 1.61+$. These values d i d not change i f the f i l t e r s were washed w i t h a t o t a l of 2.5 ml of MBB. Further studies showed tha t i f the above p r o t o c o l , of washing w i t h k.5 ml of MBB, was 3 employed, the H-adenosine l a b e l l e d RNA i n the pooled f i l t r a t e s and washes was about 1.0$ RNAse r e s i s t a n t . These data i n d i c a t e t h a t washing the RNA bound t o the M i l l i p o r e f i l t e r w i t h MBB removes o n l y the non P o l y (A) c o n t a i n i n g species of RNA. (b) Cap a c i t y of M i l l i p o r e F i l t e r s f o r Binding RNA A s s o c i a t e d w i t h P o l y (A) Sequences. 3 Four a l i q u o t s c o n t a i n i n g 5, 10, 15, and 30 ugm of H-adenosine l a b e l l e d py i n f e c t e d c e l l RNA ( s p e c i f i c a c t i v i t y 1,600 cpm/ugm) were a p p l i e d t o M i l l i p o r e f i l t e r s , which were washed w i t h 1+. 5 ml of MBB. For each r e s p e c t i v e RNA sample, 7.5$, 8.1$, 5.7$ and 5.8$ of the counts were found t o b i n d to the M i l l i p o r e f i l t e r . Thus i n t h i s and other s i m i l a r experiments, the extent of b i n d i n g was pro-p o r t i o n a l to the amount of RNA f i l t e r e d . (c) Sampling of M i l l i p o r e F i l t e r s . . Since i n c u b a t i n g the M i l l i p o r e f i l t e r , c o n t a i n i n g the bound RNA i n a h y b r i d i z a t i o n mixture, was found to be the best way of e l u t i n g the RNA, a sample of the uneluted f i l t e r was necessary t o determine the t o t a l r a d i o a c t i v i t y bound. The method chosen was t o punch out a c i r c l e which was one s i x t h of the f i l t e r area exposed to the RNA. I n order to i n v e s t i g a t e the r e l i a b i l i t y of t h i s technique, three M i l l i p o r e f i l t e r s , through which were f i l t e r e d equal amounts of l a b e l l e d i n f e c t e d c e l l RNA, were sampled by t h i s technique from three d i f f e r e n t s i t e s on the exposed f i l t e r areas. The t o t a l r a d i o a c t i v i t y per f i l t e r , obtained from the sum of the three samples of the f i l t e r , was i n c l o s e agreement f o r a l l three f i l t e r s . However, the r a d i o a c t i v i t i e s of s i n g l e samples from the same f i l t e r d i f f e r e d by kO t o 60$. I t was concluded from these data t h a t the RNA bound t o M i l l i p o r e f i l t e r s t o a r e -p r o d u c i b l e extent, although i t d i d not b i n d homogeneously across the f i l t e r surface. Hence t h i s method of sampling M i l l i p o r e f i l t e r s was not accurate, though the use of m u l t i p l e f i l t e r s f o r each determination would be adequate. h. B i n d i n g of RNA t o M i l l i p o r e , N i t r o c e l l u l o s e and to P o l y -u r i d y l a t e - G l a s s F i b e r F i l t e r s . I n order t o g a i n some i n s i g h t i n t o the nature of the b i n d i n g process, a comparison was made between M i l l i p o r e f i l t e r s (composed of mixed c e l l u l o s e e s t e r s ) , and Scheicher and S c h u e l l n i t r o c e l l u l o s e f i l t e r s i n terms of t h e i r a b i l i t y to b i n d RNA from i n f e c t e d c e l l s . As shown i n t a b l e 5(a), the n i t r o c e l l u l o s e f i l t e r bound much more RNA than d i d the M i l l i p o r e f i l t e r . I n a d d i t i o n a smaller f r a c t i o n of the RNA which bound t o the n i t r o c e l l u l o s e f i l t e r was RNAse r e s i s t a n t , compared w i t h RNA which bound t o M i l l i p o r e f i l t e r s . These data, though l i m i t e d i n scope, d i d nevertheless show a profound d i f f e r e n c e i n the b i n d i n g p r o p e r t i e s of the n i t r o c e l l u l o s e and M i l l i p o r e f i l t e r s . Taken by themselves, these observations i m p l i e d t h a t the i n t e r a c t i o n between p o l y (A) and M i l l i p o r e was more complex than a n t i c i p a t e d . On the other hand, a comparison of the RNA b i n d i n g between M i l l i p o r e and polyuridylate-GF/C ( p o l y (U)-GF/C) f i l t e r s , as shown i n t a b l e 5'(D)> r e v e a l e d t h a t the l a t t e r system was r e l a t i v e l y i n -e f f i c i e n t i n b i n d i n g c e l l RNA or RNAse r e s i s t a n t ^H-adenosine l a b e l l e d RNA. Since the system was considered t o work on a p r i n -c i p l e d i f f e r e n t from M i l l i p o r e b i n d i n g , the f a c t t h a t the degree of p o l y (u)-GF/C b i n d i n g of RNA was not s i m i l a r to the M i l l i p o r e b i n d i n g makes an unambiguous i n t e r p r e t a t i o n of the b i n d i n g r e s u l t s Table 5(a): Two 20 ugm preparations of H-adenosine l a b e l l e d py i n f e c t e d c e l l RNA ( s p e c i f i c a c t i v i t y 2,l6o cpm/ugm) were made up t o 0.5 ml i n MBB and f i l t e r e d through M i l l i p o r e and N i t r o c e l l u l o s e ( S c h l i e c h e r and S c h u e l l ) f i l t e r s . One s i x t h of each f i l t e r was sampled f o r r a d i o a c t i v i t y and the f i l t r a t e s and washes were mixed w i t h ethanol to p r e c i p i t a t e the RNA. The RNA p r e c i p i t a t e was resuspended i n 0.5 x SSC of which one quarter was sampled f o r r a d i o a c t i v i t y and the r e s t , together w i t h the f i l t e r s , incubated w i t h p a n c r e a t i c RNAse at 10 ugm/ml f o r kO minutes at 37°C. The dig e s t e d RNA was p r e c i p i -t a t e d w i t h 6</0 TCA f o r r a d i o a c t i v e determination. Table 5(b): The operations performed above were repeated w i t h M i l l i p o r e f i l t e r s and GF/C f i l t e r s on which was bound p o l y (U). The b i n d i n g of p o l y (u) and GF/C was described i n the Methods. The b i n d i n g b u f f e r f o r a p p l i c a t i o n and washing of the RNA on the f i l t e r was 0.12 M NaCl, 0.01 M T r i s pH 7-0. Table 5(a) and 5(b): Comparison of RNA b i n d i n g t o M i l l i p o r e , N i t r o c e l l u l o s e and P o l y (u)-GF/C F i l t e r s 5(a) 7(b) F i l t e r P a r a m e t e r ^ - ^ ^ 8 M i l l i p o r e N i t r o c e l l u l o s e cpm bound t o f i l t e r 1,130 10, 4oo cpm i n f i l t r a t e 34,000 2k, 300 "jo cpm bound to f i l t e r 3.2 29.1 cpm of bound RNA which was RNAse r e s i s t a n t 80 332 F i l t e r P a r a m e t e r ^ ^ ^ 6 M i l l i p o r e P o l y (U)-GF/C cpm bound t o f i l t e r 3,993 851 cpm i n f i l t r a t e 72,288 60,920 $ cpm bound to f i l t e r 5.25 1.38 cpm of bound 228 124 RNA t h a t was RNAse r e s i s t a n t d i f f i c u l t . I t thus became important to examine the above RNA b i n d i n g systems f u r t h e r , i n order t o e x p l a i n the d i s c r e p a n c i e s . 5. E f f e c t s of S a l t Concentration on the I n t e r a c t i o n Between C e l l u l a r RNA and P o l y (u)-GF/C F i l t e r s . The e f f e c t s of the s a l t c o n c e n t r a t i o n of the p o l y (U) b i n d i n g b u f f e r (PUBB) on the RNA b i n d i n g by the p o l y (U)-GF/C f i l t e r s i s depicted i n f i g . 17. E v i d e n t l y , the extent of the RNA b i n d i n g was s t r o n g l y i n f l u e n c e d by the s a l t concentration. Although the RNA b i n d i n g t o the p o l y (U)-GF/C f i l t e r s d i d not appear to have reached a plateau, i t d i d show a d i s t i n c t r e p r o d u c i b l e b i p h a s i c e f f e c t above 0.5 M NaCl. On the other hand, i n c r e a s i n g the s a l t c o n c e n t r a t i o n had no detectable e f f e c t on the b i n d i n g of t h i s same RNA onto i r r a d i a t e d GF/C f i l t e r s l a c k i n g p o l y (U). 6. Binding of H-Adenosine L a b e l l e d RNA t o Other P o l y n u c l e o t i d e s . I n an e f f e c t t o d i s t i n g u i s h between the s p e c i f i c b i n d i n g of the long p o l y (A) t r a c t of RNA t o p o l y (u) on the f i l t e r and any l e s s s p e c i f i c b i n d i n g t h a t might occur due t o "A r i c h " regions of these molecules, the RNA preparations used f o r f i g . 1 7 were exposed to GF/C f i l t e r s c o n t a i n i n g f i x e d polyadenylate or p o l y c y t i d y l a t e . I n t h i s case i t was assumed t h a t the p o l y (A) and p o l y (C) would F i g . 17: The E f f e c t of S a l t Concentration on RNA Bindi n g t o P o l y (u)-GF/C F i l t e r s . V i r u s i n f e c t e d c e l l RNA l a b e l l e d w i t h ^H-uridine ( s p e c i f i c a c t i v i t y 3,370 cpm/ugm) was made up i n q u a n t i t i e s of 9-0 ugm i n the PUBB concentrations i n d i c a t e d on the graph, passed through p o l y (U) -G-F/C f i l t e r s and washed w i t h 8.0 ml of the same PUBB concentration. The RNA was p r e c i p i t a t e d out of the f i l t r a t e s w i t h 6% TCA and the f i l t e r s and TCA i n s o l u b l e RNA i n the f i l t r a t e s were counted f o r r a d i o a c t i v i t y . Blank f i l t e r s were t r e a t e d i d e n t i c a l l y except t h a t they d i d not co n t a i n p o l y (U). ( • ) Blank GF/C f i l t e r ( O ) P o l y (U)-GF/C f i l t e r F i g . 17: N a C l C O N C E N T R A T I O N I N P D B B Table 6': B i n d i n g of ^H-adenosine L a b e l l e d RNA t o GF/C F i l t e r s C o ntaining P o l y (A) and P o l y (C). The RNA p r e p a r a t i o n used i n f i g . 17 was made up i n equal q u a n t i t i e s of 0.5 ugm i n the PUBB concentrations i n d i c a t e d and passed through the GF/C f i l t e r s c o n t a i n i n g e i t h e r p o l y (A) or p o l y (C) as o u t l i n e d i n the legend to f i g . 17. The p o l y (A) and p o l y (C) were attached t o the f i l t e r by UV i r r a d i a t i o n as was o u t l i n e d i n the Methods. Table 6,.- B i n d i n g of -Adenosine L a b e l l e d RNA t o GF/C F i l t e r s C ontaining P o l y (A) and P o l y (C). NaCl c o n c e n t r a t i o n % cpm bound to $ cpm bound to i n PuBB P o l y (A)-GF/C F i l t e r P o l y (C)-GF/C F i l t e r 0.01 M 1.15 0.82 0.1 M 1.73 2.kk 0.3 M U.90 N.A. 0.5 M 8.00 19.26 0.9 M k5.k N.A. 1.5 M 56.3 23.6 f i x to GF/C f i l t e r s by U.V. l i g h t to the same degree as p o l y (U) d i d . The r e s u l t s d i s p l a y e d i n t a b l e 6 i n d i c a t e d t h a t there e x i s t e d "U r i c h " and "G r i c h " regions i n the RNA which caused i t t o b i n d t o such a degree t o p o l y (A) and p o l y (C) f i l t e r s . A l t e r n a t i v e l y some non s p e c i f i c b i n d i n g to p o l y (A) and p o l y (C) may occur at the h igher s a l t c o ncentration. Since a h i g h degree of b i n d i n g to the above p o l y n u c l e o t i d e s occurred at moderate s a l t concentrations (1.3 M - 0.5 M) these data could not be used to d i s t i n g u i s h between the s a l t concentrations which would l e a d to s p e c i f i c b i n d i n g of p o l y (A) t r a c t s and t h a t which promoted the b i n d i n g of "A r i c h " regions. 7. E l u t i o n of RNA from P o l y (U)-GF/C F i l t e r s . Another approach was attempted to r e s o l v e which s a l t concen-t r a t i o n would produce s p e c i f i c b i n d i n g of p o l y (A) t o the p o l y (u), and which would l e a d t o non s p e c i f i c b i n d i n g . This i n v o l v e d examining the d i s s o c i a t i o n of the p o l y (A) c o n t a i n i n g RNA from the p o l y (U). As shown i n f i g . 18, p o l y (u)-GF/C f i l t e r s onto which RNA had been bound i n 1.5 M PUBB were e l u t e d w i t h lower concentrations of s a l t . I t was thought t h a t authentic p o l y ( A ) -p o l y (u) h y b r i d s would d i s s o c i a t e at a d i f f e r e n t s a l t c o n c e n t r a t i o n from the non s p e c i f i c a l l y bound RNA. The s a l t dependent e l u t i o n F i g . 18: E l u t i o n of RNA from P o l y (U)-GF/C F i l t e r s . Equal q u a n t i t i e s ( l 8 ugm) of H-adenosine l a b e l l e d RNA ( s p e c i f i c a c t i v i t y 3,370 cpm/ugm) were a p p l i e d t o each of seven p o l y (u)-G-F/c f i l t e r s . Through each f i l t e r was then passed 10 ml of PUBB whole s a l t c o n c e n t r a t i o n v a r i e d from 1.5 M to 0. 01 M as shown i n the graph. The RNA e l u t e d by t h i s treatment was p r e -c i p i t a t e d w i t h 6% TCA and counted f o r r a d i o a c t i v i t y along w i t h the p o l y (u)-GF/c f i l t e r s . The percentage of cpm e l u t e d was the cpm e l u t e d as a f r a c t i o n of the t o t a l cpm which was bound to the f i l t e r p r i o r to e l u t i o n . observed was s i m i l a r to the reverse of the b i n d i n g curve. The p o i n t of i n f l e c t i o n of the curve occurred between 0.3 and 0 .5 M PUBB, though t h i s was not a sharp change. Although these r e s u l t s d i d not d i s t i n g u i s h between p o l y (A)-poly (U) hy b r i d s , and RNA non s p e c i f i c a l l y bound to p o l y (U), they d i d prove t h a t i t was f e a s i b l e to e l u t e RNA from the poly (U) GF/C f i l t e r s by usi n g 0.001 M NaCI. 8. Binding of Ribosomal RNA t o P o l y (U) -GF/C F i l t e r s A p o s s i b l e e l u c i d a t i o n of the problem of n o n - s p e c i f i c b i n d i n g of RNA to p o l y (U)-GF/C f i l t e r s could be obtained by studying the b i n d i n g of l a b e l l e d r-RNA to. these f i l t e r s , e s p e c i a l l y as a 14 f u n c t i o n of the s a l t c o n c e n t r a t i o n of the PUBB. To do t h i s , C-u r i d i n e l a b e l l e d r-RNA was passed through p o l y (U)-GF/C f i l t e r s at d i f f e r e n t PUBB concentrations. As i l l u s t r a t e d i n f i g . 19, there was a low l e v e l of b i n d i n g of RNA t o the f i l t e r s which i n t h i s case, though not i n others, increased w i t h the PUBB concentration. However, the maximum of 2.5$ achieved w i t h the highest PuBB con-c e n t r a t i o n was i n marked c o n t r a s t w i t h the 12$ of the l a b e l which bound when i n f e c t e d c e l l RNA was so assayed. These data give strong evidence t h a t the non ribosomal RNA was r e s p o n s i b l e f o r t t h e b i n d i n g to the p o l y (u)-GF/C f i l t e r s . F i g . 19: B i n d i n g of Ribosomal RNA to P o l y (u)-GF/C F i l t e r s . S i x equal 7- 5 M-gm samples of C l a b e l l e d ribosomal RNA ( s p e c i f i c a c t i v i t y 2,175 cpm/ugm) from BHK-21 c e l l s were made up to 1.0 ml w i t h the v a r i o u s PUBB concentrations shown i n the graph. These were f i l t e r e d through p o l y (u)-GF/C f i l t e r s and the RNA i n the f i l t r a t e s p r e c i p i t a t e d w i t h TCA. The f i l t e r s and the TCA i n s o l u b l e RNA i n the f i l t r a t e s were counted f o r r a d i o a c t i v i t y . Fig. 19: ft P 0 - 5 M 1.0 M 1.5 NaCl CONCENTRATION IN PUBB 9. Sucrose Gradient Sedimentation of RNA which was Bound and E l u t e d from P o l y (U)-GF/C F i l t e r s . A.more d e t a i l e d a n a l y s i s of the H-adenosine l a b e l l e d RNA th a t was bound to, and e l u t e d from, p o l y (U)-GF/C f i l t e r s was made by c e n t r i f u g i n g t h i s RNA, and i t s RNAse digest, on sucrose gradients as shown i n f i g . 20. This RNA was very heterogeneous i n s i z e sedimenting from the 4s to greater than 45s regions of the sucrose gradient. I n a d d i t i o n , i t was found t o possess no detectable l a b e l l e d ribosomal RNA species such as were seen i n a s i m i l a r but u n f i l t e r e d RNA p r e p a r a t i o n ( f i g . 3). This i n f o r m a t i o n along w i t h the obs e r v a t i o n t h a t r-RNA d i d not b i n d to p o l y (U)-GF/C were s t r o n g l y i n d i c a t i v e t h a t the RNA which binds to poly (u)-GF/C f i l t e r s was n e i t h e r r-RNA nor t-RNA, but could have been Hn-RNA and/or m-RNA. I t was al s o observed t h a t the RNA which bound t o the p o l y (U)-GF/C f i l t e r s i n 0.3 M NaCl and subsequently e l u t e d sedimented i n a s i m i l a r f a s h i o n to th a t shown i n f i g . 20. The RNAse digested RNA d i d not appear t o sediment f a s t e r than 5s. This was i n t e r p r e t e d to mean that e i t h e r the RNA exam-ined d i d not c o n t a i n p o l y (A) sequences sedimenting from 5 to 7s as observed i n other systems (19), or that the RNAse d i g e s t i o n was too severe and r e s u l t e d i n a very l i m i t e d breakdown of some of the p o l y (A) sequences. These RNAse r e s i s t a n t TCA i n s o l u b l e H-adenosine l a b e l l e d counts comprised up to 4$ of the undigested F i g . 20: Sucrose Gradient Sedimentation of RNA Which Was Bound and E l u t e d from P o l y (u)-GF/C F i l t e r s . Twenty-five ugm of ^H-adenosine l a b e l l e d i n f e c t e d c e l l RNA ( s p e c i f i c a c t i v i t y 4,500 cpm/ugm) were f i l t e r e d through each of two p o l y (U)-GF/C f i l t e r s , e l u t e d from them w i t h 0.001 M PUBB and p r e c i p i t a t e d w i t h ethanol. The p r e c i p i t a t e , resuspended i n 0.1 x SSC, was d i v i d e d i n t o three equal p a r t s , of which one p a r t was l a y e r e d on a sucrose g r a d i e n t , w h i l s t the other two p a r t s were together incubated w i t h p a n c r e a t i c RNAse at 10 ugm/ml. The r e a c t i o n was stopped by the a d d i t i o n of SDS to a f i n a l concen-t r a t i o n of 0.5$, and p r e p a r a t i o n l a y e r e d on a sucrose gradient. C e n t r i f u g a t i o n and f r a c t i o n a t i o n were done, as o u t l i n e d i n F i g . 3, except t h a t c e n t r i f u g a t i o n was at 42,000 rpm f o r 3 hours and o n l y the TCA p r e c i p i t a b l e RNA of each f r a c t i o n was counted. ( O ) l a b e l l e d RNA C • ) RNAse t r e a t e d l a b e l l e d RNA arrows i n d i c a t e 28s, l8s and 4s markers. Fig. 20: RNA. I n c o n t r a s t , RNAse di g e s t e d - u r i d i n e l a b e l l e d RNA gave no detec t a b l e TCA p r e c i p i t a b l e r a d i o a c t i v i t y on the top of the gradient or elsewhere. 10. E x t r a c t i o n of RNA from I n f e c t e d Mouse Kidney C e l l s by Three D i f f e r e n t Techniques. The r e s u l t s i n the above s e c t i o n show th a t very l i t t l e 5 t o 7s RNAse r e s i s t a n t H-adenosine l a b e l l e d RNA may be detected i n RNAse di g e s t s of RNA e l u t e d from p o l y (u)-GF/C f i l t e r s . This prompted an i n v e s t i g a t i o n i n t o the nature of the RNA, and p a r t i c u l a r l y i t s p o l y (A) r e g i o n t h a t i s bound to the p o l y (u)-GF/C f i l t e r . A method of i n v e s t i g a t i n g t h i s would be t o prepare the c e l l RNA by d i f f e r e n t methods, one of which has been shown t o y i e l d complete recovery of p o l y (A) sequences (77). A c c o r d i n g l y , RNA was prepared from three p a i r s of i n f e c t e d c e l l monolayer c u l t u r e s l a b e l l e d w i t h H-adenosine, by the standard pH 5.2 phenol e x t r a c t i o n at 6o°C; by the phenol-chloroform^ e x t r a c t i o n ; and by phenol e x t r a c t i o n at pH f h e l a t t e r two techniques y i e l d e d some DNA i n the e x t r a c t s but t h i s was removed by the DNAse treatment. A f r a c t i o n of the RNA i s o l a t e d and p u r i -f i e d by each of the three techniques was passed through a p o l y (U)-GF/C f i l t e r i n 0 . 3 M NaCl and 0.1 M NaCl. As shown i n t a b l e 7, the b i n d i n g of the RNA e x t r a c t e d by the standard pH 5.2 phenol Table 7?: Binding of RNA E x t r a c t e d by Three D i f f e r e n t Methods to P o l y (U)-GF/C F i l t e r s . 3 Three preparations o f H-adenosine l a b e l l e d RNA were obtained by e x t r a c t i n g i n f e c t e d MK c e l l c u l t u r e s w i t h phenol pH 5 .2 at 6o°C; w i t h phenol-chloroform at room temperature, and wi t h phenol pH 9.0. They had r e s p e c t i v e s p e c i f i c a c t i v i t i e s of 6,250, 7,500 and 8,000 cpm/ugm. One p a i r of ik ugm samples from each of these preparations was passed through i n d i v i d u a l p o l y (U)-GF/C f i l t e r s i n 0.1 or 0.3 M PUBB. The f i l t e r s were counted as was the TCA i n s o l u b l e RNA i n the f i l t r a t e s . Table 7: B i n d i n g of RNA E x t r a c t e d by Three D i f f e r e n t Methods 'to P o l y (U)-GF/C F i l t e r s . cpm on cpm i n $ cpm bound f i l t e r f i l t r a t e t o f i l t e r phenol 60°C pH 5.2 1,121 84,670 1.30 0.1 M PUBB phenol CHClo pH 5.2 7,235 98,026 6.86 0.1 M PUBB phenol pH 9.0 2,1+13 70,467 3.30 0.1 M PUBB phenol 6o°C pH 5-2 3,598 90,987 3.82 0.3 M PUBB phenol CHClo pH 5.2 12,032 98,942 10.80 0.3 M PUBB phenol pH 9.0 4,539 68,767 6.20 0.3 M PUBB at 60 method bound the l e a s t e f f i c i e n t l y , w h i l e the phenol-chloroform e x t r a c t e d RNA bound most e f f i c i e n t l y . The phenol (pH 9-0) e x t r a c t e d RNA was intermediate i n b i n d i n g to p o l y (U)-GF/C f i l t e r s . F u r t h e r t e s t s were done on these three RNA p r e p a r a t i o n s . Samples of RNA were sedimented on sucrose gradients to check i f there was some unique component present which would account f o r increased b i n d i n g . None was detected, though the phenol-chloroform e x t r a c t e d RNA. d i d not c o n t a i n as much r a p i d l y sedimenting m a t e r i a l . The o v e r a l l y i e l d of RNA, the r a d i o a c t i v i t y , s p e c i f i c a c t i v i t y and py RNA content were s i m i l a r f o r a l l three p r e p a r a t i o n s . Samples of these RNA p r e p a r a t i o n s were then bound t o p o l y (u)-GF/C f i l t e r s , e l u t e d , p a r t of the sample d i g e s t e d w i t h RNAse, and the digested and undigested RNA sedimented on i n d i v i d u a l sucrose g r a d i e n t s . A c o n s i d e r a b l y greater amount of the phenol chloroform e x t r a c t e d RNA was RNAse r e s i s t a n t as compared t o the phenol pH 5.2 e x t r a c t e d RNA. The non d i g e s t e d RNA sedimented heterogeneously i n a sucrose gradient, not u n l i k e t h a t of f i g . 19, though i t appeared t o be composed of s l i g h t l y smaller s i z e c l a s s e s . The RNAse r e s i s t a n t RNA d i d not however have a prominant 5 to 7s peak. This l a s t experiment r a i s e s a number of questions about the RNA p r e p a r a t i o n techniques and the b a s i s f o r b i n d i n g of RNA t o p o l y (U)-GF/C f i l t e r s , as w e l l as p r o v i d i n g a p o s s i b l e e x p l a n a t i o n f o r the r e s u l t s so f a r obtained i n t h i s chapter. These issues w i l l be discussed i n the next chapter. CHAPTER VI: DISCUSSION The purpose of t h i s study as s t a t e d i n the I n t r o d u c t i o n , was to examine the processing of py RNA i n v i r u s i n f e c t e d c e l l s . The l a s t three chapters have d i r e c t l y or i n d i r e c t l y c o n t r i b u t e d to t h i s study. 1. V i r a l RNA Synthesis i n I n f e c t e d C e l l s . I n order to c o r r e l a t e t h i s work w i t h other s t u d i e s on polyoma v i r u s , the time course of v i r a l RNA synth e s i s was measured. This time course, under the c o n d i t i o n s used i n the experiments performed i n t h i s t h e s i s , was found t o be comparable to th a t of other workers (3 ) ( H 2 ) . I t was a l s o of i n t e r e s t t o note, although r e s u l t s were not shown, t h a t a r e l a t i v e l y constant amount of c e l l RNA was l a b e l l e d throughout the i n f e c t i o n . This confirmed previous observ-a t i o n s (112) t h a t the i n i t i a l stages of v i r a l r e p l i c a t i o n d i d not have much e f f e c t on the c e l l u l a r RNA metabolism. The o b s e r v a t i o n t h a t py i n f e c t e d BHK-21 c e l l s d i d not produce de t e c t a b l e amounts of. v i r a l RNA, w h i l e under i d e n t i c a l c o n d i t i o n s MK and ME c e l l s produced v i r a l RNA, and v i r a l DNA was shown to i n t e g r a t e i n t o the BHK-21 c e l l s ( 2 ), was of i n t e r e s t . Hamster t i s s u e has moreover been shown t o be capable of supporting the r e p l i c a t i o n of py v i r u s (46), and the presence of v i r a l RNA has a l s o been demonstrated i n transformed hamster c e l l s (53)• The above o b s e r v a t i o n denotes t h a t e i t h e r a very s m a l l percentage of v i r u s i n f e c t e d BHK-21 c e l l s produce v i r a l RNA, or t h a t the e n t i r e c e l l p o p u l a t i o n produces py RNA but i n l e s s than one te n t h the q u a n t i t y produced i n p r o d u c t i v e l y , i n f e c t e d ME c e l l s e a r l y i n i n f e c t i o n . I t i s p o s s i b l e t h a t o n l y the i n f e c t e d BHK-21 c e l l s , which are destined t o be transformed, synthesize v i r a l RNA at l e v e l s s i m i l a r to ME c e l l s e a r l y i n i n f e c t i o n . Under such circumstances, the d e t e c t i o n of t h i s v i r a l RNA would have been beyond the scope of the technique used. 2. Size D i s t r i b u t i o n of Py RNA i n I n f e c t e d C e l l s . The o b s e r v a t i o n t h a t py RNA from i n f e c t e d MK c e l l s had a heterogeneous s i z e d i s t r i b u t i o n from 4s to ^  45s when analyzed i n a sucrose g r a d i e n t , was i n agreement w i t h other workers (55) ( l ) (58). This phenomenon has been more r i g o r o u s l y examined by sedimentation i n DMSO sucrose g r a d i e n t s , by polyacrylamide g e l e l e c t r o p h o r e s i s , and by resedimentation of RNA from d i f f e r e n t regions of the sucrose g r a d i e n t . A l l of these s t u d i e s showed t h a t py RNA co n s i s t e d of a heterogeneous p o p u l a t i o n of molecules. Since py DNA i s a s m a l l molecule (mw 3 x 10^ (lOS)) i t would not be expected t o code f o r a la r g e v a r i e t y of monocistr.onic m-RNA molecules, nor f o r a p o l y c i s t -r o n i c m-RNA r e p r e s e n t i n g a complete DNA t r a n s c r i p t which was great e r than 1.5 x 10^ ( 28s). Although the hydrogen bond mediated form-a t i o n of aggregates, to account f o r py M A sedimenting f a s t e r than 28s, has been r u l e d out by use of DMSO sucrose gra d i e n t s , i t was s t i l l p o s s i b l e t h a t the smaller py MA molecules could have been due to RNAse mediated breakdown. This p o s s i b i l i t y was u n l i k e l y , s i n c e i n cases where A^g^ measurements of the gradient f r a c t i o n s were performed, the 28s r-RNA f r a c t i o n was a d i s t i n c t peak c o n t a i n i n g about twice the absorbance of the l8s r-MA, as would be expected f o r undegraded mammalian c e l l RNA. Likewise the MA preparations were a l s o always scanned f o r absorbance at 260 nm and 280 nm and A26o the 2^8~0 ra"ki° w a s always 2.0, which i n d i c a t e d t h a t the RNA was f r e e of s u f f i c i e n t p r o t e i n contamination to i n f l u e n c e i t s sediment-a t i o n p r o p e r t i e s . A number of hypotheses could e x p l a i n the heterogeneous d i s t r i -b u t i o n of py RNA. I t i s p o s s i b l e t h a t the py MA undergoes a very r a p i d turnover, as was observed f o r ^ x 174 DNA (47), hence a number of d i f f e r e n t s i z e s of p a r t i a l l y s ynthesized and broken down MA c o u l d account f o r the heterogeneity. I n t h i s case the 28s py RNA would have been t r a n s c r i b e d from the c o n t i n u a t i o n of t r a n s -c r i p t i o n of a s i n g l e c i r c u l a r genome. The f a c t t h a t py DNA i s a c i r c u l a r molecule supports t h i s hypothesis. A l t e r n a t i v e l y , py RNA may be t r a n s c r i b e d from the py DNA which was i n t e g r a t e d i n t o the c e l l DNA (2). I n t h i s case, the l a r g e number of d i f f e r e n t s i z e molecules could be due t o t h i s i n t e g r a t e d v i r a l DNA t h a t had under-gone f u r t h e r recombinations w i t h the c e l l DNA. As w e l l s e v e r a l 128 copies of v i r a l DNA may i n t e g r a t e i n tandem and thus t r a n s c r i b e d would l e a d to the formation of py RNA 28s. F i n a l l y , i t i s p o s s i b l e t h a t v i r a l DNA may be t r a n s c r i b e d from i n t e g r a t e d py DNA i n such a way t h a t i t i s a l i n e a r h y b r i d of v i r a l and c e l l RNA. I f the v i r a l DNA has many i n t e g r a t i o n p o i n t s , a number of d i f f e r e n t s i z e h y b r i d molecules could a r i s e which would be greater than 28s.. This l a s t hypothesis i s supported by the o b s e r v a t i o n t h a t i n py transformed c e l l s , the v i r a l RNA has a s i m i l a r s i z e d i s t r i b u t i o n (66). In these c e l l s the v i r a l DNA was not o n l y i n t e g r a t e d i n t o the c e l l DNA but was l i k e l y smaller than one complete genome i n s i z e . S i m i l a r observations have been made w i t h SVkO transformed c e l l s (68). 3. Py RNA i n Nuclear, Cytoplasmic and Polyribosomal F r a c t i o n s . The py RNA i n the nuclear and cytoplasmic f r a c t i o n s of i n f e c t e d c e l l s was s i m i l a r i n both s i z e and base composition. I n co n t r a s t to these observations, most of the v i r a l RNA i n the n u c l e i of SVkO i n f e c t e d c e l l s was l a r g e r than i n the cytoplasmic e x t r a c t s (68) (102). I n order t o r u l e out the p o s s i b i l i t y t h a t anomalies i n c e l l f r a c t i o n a t i o n techniques were r e s p o n s i b l e f o r the s i m i l a r i t y between nuclear and cytoplasmic py RNA, two methods of f r a c t i o n -a t i n g the i n f e c t e d c e l l s were used, namely, Dounce homogenization (76) and NPUO treatment (lh). I n a l l cases, phase c o n t r a s t microscopy r e v e a l e d almost q u a n t i t a t i v e breakage of c e l l s and v i r t u a l absence of cytoplasmic "tabs" on the n u c l e i . According t o the c r i t e r i a of Penman ( 76 ) these c e l l f r a c t i o n s were r e l a t i v e l y " c l e a n " judging from the s m a l l amounts of heterogeneous-nuclear RNA i n the cytoplasm and of l8s RNA i n the n u c l e i . Although i t i s probable t h a t some cross contam-i n a t i o n occurred ( i e . leakage of nuclear RNA i n t o the cytoplasm and b i n d i n g of ribosomes to nuclear membranes) such l e v e l s could not account f o r the amount of ^  28s v i r a l RNA i n the cytoplasm. I f i t i s assumed t h a t the presence of ">28s RNA i n the c y t o -plasm was due t o "leakage" from the n u c l e i during c e l l f r a c t i o n a t i o n then a comparison of the amount of r a d i o a c t i v e RNA i n t h i s r e g i o n of a sucrose gradient between nuclear and cytoplasmic f r a c t i o n s would i n d i c a t e the maximum extent of t h i s "leakage". These values were t h e r e f o r e c a l c u l a t e d f o r three separate experiments which comprised a complete a n a l y s i s of nuclear and cytoplasmic v i r a l RNA (using the Dounce homogenization technique) on sucrose gradients The values f o r t o t a l >28s r a d i o a c t i v e RNA ranged from 8.5$ t o 2 1 . 0 $ w h i l e the corresponding percentages f o r the v i r u s s p e c i f i c ">28s RNA (cytoplasm as percent of nucleus) ranged from 27$ to 43$. Therefore unless there was a p r e f e r e n t i a l leakage of v i r a l s p e c i f i c > 28s RNA, as opposed t o c e l l u l a r > 28s RNA, i t had to be concluded t h a t v i r u s s p e c i f i c RNA of great e r than s i n g l e genome s i z e could be t r a n s p o r t e d across the nuclear membrane. This c o n c l u s i o n was supported by the more l i m i t e d s t u d i e s u s i n g the N P 4 0 technique and by examining the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the c e l l 3 f r a c t i o n s of H-thymidine l a b e l l e d c e l l s . I n the l a t t e r case, l e s s than 3% of the l a b e l was found i n the cytoplasmic e x t r a c t , which i n d i c a t e d a minimum of nuclear fragmentation (ih). These s t u d i e s have shown t h a t py RNA i s t r a n s c r i b e d i n the i n f e c t e d c e l l nucleus as a heterogeneous c o l l e c t i o n of RNA molecules of d i f f e r e n t s i z e s , i n c l u d i n g RNA l a r g e r than one genome i n length. A s i m i l a r s i z e d i s t r i b u t i o n of v i r a l RNA was recovered from the cytoplasm, and competition h y b r i d i z a t i o n t e s t s r e v e a l e d t h a t a l l nuclear py RNA sequences were represented i n the cytoplasmic py RNA. V i r u s s p e c i f i c RNA e x t r a c t e d from the polyribosomes was devoid of the "> 28s species, implying t h a t py RNA i n excess of one genome i n l e n g t h was cleaved as p a r t of i t s processing. This cleavage d i d not appear t o be an a r t i f a c t of p u r i f y i n g and c h a r a c t e r i z i n g the RNA, s i n c e the sedimentation behaviour of the polyribosomes i n d i c a t e d t h a t no breakdown had occurred up to t h i s p o i n t i n the procedure. Since the polyribosomes were then immediately d i s r u p t e d w i t h SDS and the RNA e x t r a c t e d w i t h hot phenol, i t i s u n l i k e l y t h a t any enzymatic breakdown of the v i r a l RNA occurred a f t e r t h i s p o i n t . I n a d d i t i o n the ribosomal RNA markers d i d not appear t o have been degraded by t h i s e x t r a c t i o n process. Studies w i t h SVkO i n f e c t e d c e l l s a l s o showed th a t v i r a l RNA i n polyribosomes was smaller than i n the n u c l e i (68). The py RNA found i n the polyribosomes sedimented at or below 18s, corresponding to a maximum molecular weight of about 0.5 x 10 . This could w e l l represent a monocistronic m-RNA coding f o r the c a p s i d p r o t e i n which has a molecular weight of k.5 x 10^ ( 8 l ) . Since t h i s p r o t e i n comprises up to 70$ of the v i r i o n , i t i s p o s s i b l e t h a t the bulk of the py RNA codes f o r t h i s p r o t e i n . I t i s not d e f i n i t e i f a l l py RNA species e v e n t u a l l y become a s s o c i a t e d w i t h the polyribosomes. Because of t e c h n i c a l l i m i t a t i o n s unequivocal r e s u l t s from competition h y b r i d i z a t i o n experiments w i t h py RNA from polyribosomes could not be obtained. I t was s i g n i f i c a n t however, th a t i n py RNA from the cytoplasmic e x t r a c t the small species ( < l 8 s ) could e f f i c i e n t l y compete w i t h l a r g e r py RNA i n a competition h y b r i d i z a t i o n study. I f the s m a l l species represented predominantly those present i n the polyribosomes, then t h i s observ-a t i o n would imply t h a t a l l species of py RNA become e v e n t u a l l y a s s o c i a t e d w i t h the polyribosomes. A comparison of these r e s u l t s w i t h those of other workers, showed that there was agreement with the work of Cheevers (13), who noted t h a t the l e v e l of polyribosomes was s t i m u l a t e d i n i n f e c t e d c e l l s , as was a l s o shown above. On the other hand, a study of the py RNA from the polyribosomes (58) r e v e a l e d t h a t i n a sucrose g r a d i e n t , the m a j o r i t y of the py RNA sedimented below l 8 s , but there a l s o appeared a d i s t i n c t peak of v i r a l RNA at 28s. No evidence f o r t h i s peak was ever observed i n the s t u d i e s described i n t h i s t h e s i s . k. P r o c e s s i n g of py RNA i n I n f e c t e d C e l l s . The study of the k i n e t i c s of l a b e l l i n g of nuclear and cytoplasmic RNA of i n f e c t e d c e l l s l a t e i n i n f e c t i o n r e v e a l e d three important f a c t s : (a) the l e v e l of l a b e l l e d py RNA i n the nucleus was about 10 times as hig h as the l e v e l i n the cytoplasm; (b) i n the nucleus and l a b e l l i n g o f py RNA progressed at the same r a t e as the l a b e l l i n g of the c e l l RNA (c) l a b e l l e d py RNA accumulated i n the cytoplasm at the same r a t e as c e l l RNA f o r 30 minutes of l a b e l l i n g , a f t e r which the l a t t e r accumu-l a t e d f a s t e r . These f a c t s w i l l now be discussed f u r t h e r . The c o n s i s t e n t l y higher l e v e l of py RNA i n the nuclear f r a c t i o n over the cytoplasmic f r a c t i o n i m p l i e d t h a t most of the RNA i n the nucleus may not be processed to the cytoplasm. This i m p l i c a t i o n was confirmed by the pulse and chase s t u d i e s which w i l l be discussed l a t e r That t h i s o b s e r v a t i o n was made at t h i s stage i s v e r y s i g n i f i c a n t since the p o s s i b i l i t y of i t being a p o t e n t i a l a r t e f a c t caused by the use of Actinomycin D i s el i m i n a t e d . The observation?.that i n the nucleus, the v i r a l RNA forms a constant f r a c t i o n of the c e l l u l a r RNA (^1.0$) r e g a r d l e s s of the l a b e l -l i n g time, was a l s o important since i t i m p l i e s that the same mechanism i s i n v o l v e d i n the t r a n s c r i p t i o n of both RNA species. I f t h i s were indeed the case, the v i r a l RNA could then be considered as a t y p i c a l m-RNA and a study of i t s processing would thus be a more meaningful model f o r the c e l l u l a r RNA. In t h i s regard the accumulation of l a b e l l e d v i r a l and c e l l u l a r RNA i n the cytoplasm appeared t o be compatible w i t h the concept of v i r a l RNA being a t y p i c a l m-RNA molecule. For the f i r s t 30 minutes the v i r a l RNA formed a constant f r a c t i o n of the c e l l u l a r RNA, a f t e r which the r a t e of accumulation f o r the c e l l RNA i n the cytoplasm increased, w h ile the r a t e f o r the v i r a l RNA was constant. This o b s e r v a t i o n may be expla i n e d by p o s t u l a t i n g t h a t the RNA processed i n t o the cytoplasm f o r the f i r s t 30 minutes of l a b e l l i n g was mostly m-RNA, a f t e r which the r-RNA and t-RNA, which have longer h a l f l i v e s began to appear. The constant r a t e of accumulation of py RNA may thus be taken as an i n d i c a t i o n of the accumulation of m-RNA i n the cytoplasm. A more d e t a i l e d extension of the k i n e t i c s of l a b e l l i n g was done i n a pulse and chase study which examined the subsequent f a t e of py RNA l a b e l l e d f o r bo minutes, by stopping f u r t h e r l a b e l l i n g of RNA wi t h Actinomycin D or excess u r i d i n e . The most s i g n i f i c a n t o b s e r v a t i o n was t h a t about 75$ of "the l a b e l l e d v i r a l RNA i n the nuclear f r a c t i o n decayed w i t h i n the f i r s t hour of chase without being t r a n s p o r t e d to the cytoplasm. A f t e r the f i r s t hour of chase the r a t e s of decay of the nuclear py RNA and cytoplasmic py RNA were roughly p a r a l l e l . These observations, which were al s o r e p o r t e d by K a j i o k a (58), imply t h a t there are two types of v i r a l RNA l a b e l l e d i n the nuclear f r a c t i o n . The more abundant type i s ver y s i m i l a r to the Hn-RNA i n terms of i t s r a p i d l a b e l l i n g and e q u a l l y r a p i d decay p r o p e r t i e s , while the second type i s a much more s t a b l e form which may have been de r i v e d from the f i r s t type. This o b s e r v a t i o n could a l s o be explained by p o s t u l a t i n g t h a t the two types of RNA are t r a n s c r i b e d from i n t e g r a t e d and non-integrated DNA r e s p e c t i v e l y . A more d e t a i l e d examination of the above RNA on sucrose g r a d i e n t s revealed t h a t the r a p i d l y decaying nuclear RNA was very s i m i l a r i n s i z e d i s t r i b u t i o n t o the more s t a b l e form. Further exam-i n a t i o n of the sucrose gradients a l s o i n d i c a t e d t h a t during the chase, the la r g e (>28s) py RNA was broken down at a s l i g h t l y f a s t e r r a t e than the s m a l l (<l8s) py RNA. Since such a tr e n d was a l s o observed i n the cytoplasm, t h i s i m p l i e s t h a t the l a r g e py RNA i n the course of processing i s broken down t o 'intermediate' and f i n a l l y i n t o 'small s i z e 1 species p r i o r to t r a n s l a t i o n . This argument i s supported by the obs e r v a t i o n t h a t i n py RNA pulse l a b e l l e d f o r 15 minutes, 46$ sedimented f a s t e r than 28s, but i n py RNA l a b e l l e d f o r 2 hours o n l y 25$ sedimented i n t h i s region. Further support comes from the work of Acheson et a l ( 1 ) . This s i m i l a r i t y i n processing of v i r a l and c e l l u l a r RNA of the nuclear f r a c t i o n was a l s o observed i n the pulse and chase study w i t h Actinomycin D. Both species appeared t o decay w i t h s i m i l a r r a t e s w i t h py RNA comprising about 3$ of the c e l l RNA. I t should be po i n t e d out t h a t the above conclusions are based on pulse l a b e l l i n g s t u d i e s as w e l l as pulse and chase st u d i e s u s i n g 135 an i n h i b i t o r of RNA s y n t h e s i s . I t i s s i g n i f i c a n t t h a t the r e s u l t s obtained by a l l these s t u d i e s are at l e a s t q u a l i t a t i v e l y i n agreement. 5. Polyadenylate Sequences. The f r a c t i o n of py RNA molecules t h a t were a s s o c i a t e d w i t h p o l y (A) sequences in c r e a s e d as the py RNA was processed from the nuclear through to the polyribosomal f r a c t i o n of the i n f e c t e d c e l l . I n t h i s regard, py RNA behaved as a t y p i c a l c e l l u l a r species of m-RNA s t u d i e d i n other c e l l l i n e s (65) (19)• The increased a s s o c i a t i o n of the py RNA from the cytoplasmic and p o l y r i b o s o m a l f r a c t i o n s w i t h p o l y (A) may i n d i c a t e t h a t o n l y py RNA which was so m o d i f i e d was capable of being p r o p e r l y processed, w h i l e the r e s t was degraded e i t h e r i n the nuclear or cytoplasmic f r a c t i o n s . This hypothesis would account f o r the r a p i d degradation of py RNA i n the nuclear f r a c t i o n and i t s r a t h e r i n e f f i c i e n t t r a n s f e r to the cytoplasmic and p o l y r i b o s o m a l fractions'. I n a d d i t i o n , when the r e l a t i o n s h i p of p o l y (A) sequences to py RNA i n a pulse and chase experiment was examined (data not shown), the f r a c t i o n of py RNA i n the nuclear f r a c t i o n a s s o c i a t e d w i t h p o l y (A) was seen to d i m i n i s h throughout the chase p e r i o d . This r e s u l t was a l s o i n agreement w i t h the above hypothesis. The method of assaying f o r p o l y (A) sequences was taken, w i t h v e r y few m o d i f i c a t i o n s , d i r e c t l y from Brawerman (65). A considerable v a r i a t i o n was observed i n the values of the degree of p o l y (A) a s s o c i a t i o n w i t h the RNA, hence these r e s u l t s were considered o n l y from a q u a l i t a t i v e p o i n t of view. 6. P o l y (A) I s o l a t i o n Techniques. I n the i n i t i a l s t u d i e s , the r e s u l t s obtained by Brawerman (65) were confirmed and expanded. M i l l i p o r e f i l t e r s were shown to s p e c i f i c a l l y b i n d RNAse r e s i s t a n t ^ H-adenosine l a b e l l e d RNA. The f i l t e r s were not sa t u r a t e d by the amounts of RNA used, nor d i d yeast RNA compete w i t h the b i n d i n g of l a b e l l e d c e l l or v i r a l RNA. A p r o t o -c o l was a l s o e s t a b l i s h e d i n which RNA was a p p l i e d to the f i l t e r i n 0.5 ml and the f i l t e r washed w i t h k.5 ml of b u f f e r . None of the p o l y (A) c o n t a i n i n g RNA was removed by the washing. These studies were e s s e n t i a l from a t e c h n i c a l p o i n t of view. Further s t u d i e s showed t h a t there were u n c e r t a i n t i e s a s s o c i a t e d w i t h t h i s technique. I n c o n t r a s t to the homogeneous d i s t r i b u t i o n of s i n g l e stranded DNA on M i l l i p o r e f i l t e r s (52)', the RNA was not d i s t r i b u t e d homogeneously across the f i l t e r surface, hence methods of sampling the f i l t e r were u n s a t i s f a c t o r y . A considerable d i s -crepancy was a l s o seen between the amount of RNA bound to M i l l i p o r e , n i t r o c e l l u l o s e and p o l y (U)-GF/C f i l t e r s . These observations p l a c e d the a c t u a l mechanism of the b i n d i n g of the RNA t o M i l l i p o r e i n question. Some stu d i e s on the mechanism of b i n d i n g of p o l y (A) sequences t o d i f f e r e n t c e l l u l o s e d e r i v a t i v e s have i m p l i e d t h a t i t was the l i g n i n i m p u r i t i e s i n the c e l l u l o s e which were r e s p o n s i b l e f o r the b i n d i n g p r o p e r t i e s (23). Further a n a l y s i s of the M i l l i p o r e and n i t r o c e l l u l o s e f i l t e r s could p o s s i b l y c l a r i f y t h i s problem. The f a i l u r e of the p o l y (U)-GF/C f i l t e r s , which represent a b e t t e r defined system, to b i n d s i g n i f i c a n t q u a n t i t i e s of v i r a l RNA i n 0.12 M NaCl l e d to f u r t h e r i n v e s t i g a t i o n of t h i s system. The u n c e r t a i n t i e s inherent i n t h i s method were: (a) the o p t i m a l s a l t c o n c e n t r a t i o n to be used, and (b) the amount of 'non-poly (A)' b i n d i n g t h a t would occur at d i f f e r e n t s a l t concentrations. The degree of b i n d i n g as a f u n c t i o n of s a l t c o n c e n t r a t i o n f o l l o w e d a b i p h a s i c p a t t e r n from which 0.9 M NaCl was taken to be the o p t i m a l s a l t c o ncentration. This o b s e r v a t i o n was i n marked con t r a s t w i t h s t u d i e s performed by Kates and co-workers (90) who showed t h a t the op t i m a l s a l t c o n c e n t r a t i o n f o r b i n d i n g p o l y (A) t o the p o l y (u)-GF/C f i l t e r s was 0.12 M NaCl. However, t h i s study was performed w i t h pure l a b e l l e d p o l y (A), hence t h i s r e s u l t may not r e a d i l y apply to an RNA molecule of which o n l y a s m a l l t e r m i n a l p o r t i o n i s p o l y (A). A number of st u d i e s were performed t o i n v e s t i g a t e the degree of n o n - s p e c i f i c b i n d i n g of 'A r i c h ' regions to the p o l y (u) on the f i l t e r as a f u n c t i o n of the s a l t concentration. Ribosomal RNA, which comprises the b u l k of the c e l l RNA, was shown to b i n d to a very s m a l l degree and t h i s was o n l y m a r g i n a l l y increased by increasing the salt concentration. A study of the elution pattern of the RNA, that had been bound to the f i l t e r in 1.5 M NaCI, as a function of the salt concentration, revealed that the RNA eluted in a biphasic manner which reflected a reverse of the above binding pattern. In this study, as well as in a similar study by other workers (90), only 80$ of the RNA bound to the filters was eluted. The nature of the non-eluted RNA was not examined. Neither of these studies led to a differentiation between the poly (A) binding and the non-specific binding by 'A rich' regions. A comparison of the effects of increasing salt concentration on the binding of RNA to poly (A), poly (U) and poly (C)-containing glass fibre filters was made in a further effort to resolve the question of non-specific binding. The high degree of binding to poly (A) and poly (C) filters at even relatively low salt concentr-ation indicated that there must be regions in mammalian cell RNA rich in U and G, an observation also made recently by other workers (74). Moreover, the high degree of binding (60$) to poly (A) filters at high salt concentration indicated that this U rich region may well be associated with the ribosomal RNA, which is the only species present in such large amounts. The question of non-specific binding was partially resolved by sucrose gradient sedimentation of the RNA bound to and eluted from poly (u)-GF/C filters at various salt concentrations (0.1, 0.3 and 0.9 M). The sedimentation pattern of a l l these preparations r e v e a l e d t h a t the RNA was heterogeneously d i s t r i b u t e d throughout the gradi e n t and l a c k i n g any d i s t i n c t r-RNA or t-RNA markers. This o b s e r v a t i o n i n d i c a t e d t h a t o n l y messenger-like RNA or m-RNA would b i n d t o p o l y (u)-GF/C f i l t e r s . No change i n t h i s p a t t e r n was observed f o r the v a r i o u s s a l t concentration's i n which the RNA was bound to the f i l t e r s , i n d i c a t i n g t h a t at l e a s t i n s i z e , the RNA which binds to the f i l t e r s i s the same re g a r d l e s s of the s a l t c o n c e n t r a t i o n i n which i t i s bound. The RNA which bound to and was e l u t e d from the p o l y (U)-GF/C f i l t e r s d i d not appear t o have been degraded since, i f i t represented messenger-like RNA, i t sedimented i n a s i m i l a r manner t o pulse l a b e l l e d c e l l RNA, which was predominantly messenger-like RNA ( f i g . 13). When the above %-adenosine l a b e l l e d RNA,eluted from p o l y (u)-GF/C f i l t e r s , was f i r s t t r e a t e d w i t h RNAse and then analyzed on a sucrose g r a d i e n t , i t s a c i d p r e c i p i t a b l e r a d i o a c t i v i t y sedimented at ks, w h i l e t h a t of s i m i l a r l y t r e a t e d H-uridine l a b e l l e d RNA was h a r d l y detectable even on the top of the gradient. This was taken as f i r m evidence t h a t there are p o l y (A) sequences a s s o c i a t e d w i t h RNA trapped by the p o l y (u)-GF/C f i l t e r s , although these sequences were s h o r t e r than those r e p o r t e d by other workers (19) (32). I n view of t h i s l a s t f i n d i n g , an attempt was made t o determine i f the l e n g t h of the p o l y (A) sequences i s r e s p o n s i b l e f o r the l a c k of b i n d i n g to p o l y (U) i n the presence of the lower s a l t concentra-t i o n s . RNA preparations were e x t r a c t e d by three d i f f e r e n t methods, of which at l e a s t one (phenol-CHCl^) was shown t o give undamaged p o l y (A) sequences (77)- From t h i s i t was found t h a t the method of e x t r a c t i o n of the RNA has a profound e f f e c t upon i t s a b i l i t y to b i n d to p o l y (u). An a n a l y s i s of the s i z e d i s t r i b u t i o n of the (CHCT^-phenol extracted) RNA b i n d i n g t o p o l y (if) before and a f t e r RNAse treatment r e v e a l e d t h a t although the ^H-adenosine l a b e l l e d , RNAse r e s i s t a n t sequences s t i l l sediment around 4s, they appear somewhat l a r g e r i n s i z e and g r e a t e r i n c o n c e n t r a t i o n . ( f i g u r e not shown). These f a c t s p o i n t t o an e x p l a n a t i o n f o r the p o l y (A) b i n d i n g c h a r a c t e r i s t i c s observed above. I f one assumes th a t e x t r a c t i o n w i t h hot phenol, f o l l o w e d by c o l d phenol as done f o r the most p a r t i n t h i s study, i s r e l a t i v e l y i n e f f i c i e n t i n e x t r a c t i n g whole un-fragmented p o l y (A) sequences (a f a c t a l s o shown r e c e n t l y by Perry) (7^), then the p o l y (A) sequences attached t o t h i s RNA could be s h o r t e r . These short sequences, w h i l e s t i l l b i n d i n g to p o l y (U), may not form as s t a b l e a h y b r i d i n low s a l t as i n higher s a l t c oncentrations. When the e x t r a c t i o n was done w i t h phenol c h l o r o -form, shown by the above authors to be e f f i c i e n t i n e x t r a c t i n g whole p o l y (A) sequences, then the longer sequences form s t a b l e hybrids under lower s a l t concentrations. I t was however p u z z l i n g t h a t the p o l y (A) sequences from these RNA preparations s t i l l sedimented at 4s. 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