UBC Theses and Dissertations

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UBC Theses and Dissertations

Use of benzoylated deae-cellulose for the isolation of glycine transfer ribonucleic acids of yeast and… Warrington, Robert Charles 1970

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THE USE OF BENZOYLATED DEAE-CELLULOSE FOR THE ISOLATION OF GLYCINE TRANSFER RIBONUCLEIC ACIDS OF YEAST AND FOR THE DEVELOPMENT OF NEW METHODS FOR SEQUENCE DETERMINATION OF NUCLEIC ACIDS by Robert Char les War r ing ton , B . S c , The U n i v e r s i t y of B r i t i s h Columbia , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n . t h e Department of B iochemis t ry We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study . I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department 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 understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Depa rtment 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 ABSTRACT Par t I of t h i s t h e s i s d e s c r i b e s a method f o r the i s o l a t i o n of a s p e c i f i c f a m i l y of aminoacy l - tRNAs . React ion of the ami noacy 1-tRNAs w i th the N_-hyd roxysucc i n im i de e s t e r of 2-naphthoxyacet i c a c i d produces N^-2-naphthoxyacety 1 am i noacy 1-tRNAs which possess s u f f i c i e n t a f f i n i t y f o r B D - c e l l u l o s e to a l l o w t h e i r s e p a r a t i o n from unmodif ied tRNA by s imple chromatographic s t e p s . The N_-2-naphthoxyacety 1 amino a c i d is removed by m i l d a l k a l i n e h y d r o l y s i s and the i s o - a c c e p t o r tRNAs are separated by chromatography on B D - c e l l u l o s e . t R N A ^ ^ and t R N A ^ ^ have been p u r i f i e d from brewer 's yeast (Saccharomyces  c e r e v i s i ae) by t h i s method and a s i m p l i f i e d procedure fo r the large s c a l e i s o l a t i o n of these tRNAs was developed. The ob jec t of Par t II of t h i s t h e s i s was to develop a new method of n u c l e o t i d e sequence a n a l y s i s based on the f o l l o w i n g s t e p s : ( i ) s p e c i f i c i n t r o d u c t i o n of hydrophobic groups onto the 3 ' - or 5 ' " t e r m i n a l s of p o l y n u c l e o t i d e s , ( i i ) subsequent c leavage of the a p p r o p r i a t e l y d e r i v a t i z e d polymer under c o n d i t i o n s which favor rupture of on ly one bond per molecule of polymer, ( i i i ) s e p a r a t i o n of fragments bear ing the d e r i v a t i v e from those not bear ing the d e r i v a t i v e , ( iv ) r e s o l u t i o n of d e r i v a t i z e d fragments accord ing to cha in l e n g t h , (v) complete degradat ion of the i i i p u r i f i e d fragments from step ( i v ) w i t h the reagent used to generate these fragments fo l l owed by i d e n t i f i c a t i o n of the components so l i b e r a t e d from each f ragment , and f i n a l l y , ( v i ) assembly of the data obta ined from step (v) to order the composite fragments from 5' and 3' ends to d e r i v e an unambiguous pr imary sequence. It was reasoned that N_-2 -naphthoxyacety lg l ycy l - tRNA^^ cou ld be used as a model polymer bear ing an a p p r o p r i a t e 3 1 -d e r i v a t i v e , that BD-cel1u1ose chromatography could e f f e c t step ( i i i ) , that DEAE-cel1ulose-7 M urea column chromatography cou ld e f f e c t step ( iv ) and that e i t h e r D E A E - c e l l u l o s e chromatography or combined c h r o m a t o g r a p h i c - e l e c t r o p h o r e t i c techniques cou ld e f f e c t step (v ) . A method f o r p l a c i n g a hydrophobic d e r i v a t i v e onto the 5 l - t e r m i n u s of p o l y n u c l e o t i d e s needed to be deve loped . F u r t h e r , f t was necessary to d i s c o v e r i f a v a i l a b l e endonuc1 eases could degrade the model polymer in a manner approaching random, s i n g l e - h i t k i n e t i c s . To a s c e r t a i n the f e a s i b i l i t y of the proposed method, two s t u d i e s were under taken . F i r s t , the 3 '~ terminal fragments r e -leased by exhaus t i ve d i g e s t i o n of Nh2 - n a p h t h o x y a c e t y l g l y c y l -tRNA^'^ w i t h RNase were i s o l a t e d by the procedure to v e r i f y the e f f i c i e n c y of a number of the requ i red s t e p s . The fragments (from the r e s u l t s of the n u c l e o s t d e - a n a l y s i s and on the assumption that the 3 '~sequence cy t idy1y1(3 1~5 1 ) cy t idy1y1(3 1S 1 l adenos ine -i s common to a l l f u n c t i o n a l tRNAs) are (Cp,Ap)CpCpA and (Up ,Ap ,Cp ,Cp) CpCpA. The r e l a t i v e y i e l d s of the two fragments suggested they were der i ved from t R N A ^ l y and t R N A ^ ^ , r e s p e c t i v e l y . Second, a study was undertaken of the degradat ion products l i b e r a t e d from crude and p u r i f i e d g l y c y l - t R N A s as we l l as from crude and p u r i f i e d N_-2-naphthoxyacety 1 g 1 ycy 1 -tRNAs by RNase T^  (and, to a l e s s e r e x t e n t , by p a n c r e a t i c RNase). RNase T^  was found to degrade crude g l y c y l - t R N A w i th a .product d i s t r i b u t i o n s u i t a b l e fo r t e s t i n g the f e a s i b i l i t y of the method. It was not p o s s i b l e , however, to l a b e l s p e c i f i c a l l y the g l . y c y l - o l i g o n u c l e o t i d e s w i t h a hydrophobic group in the presence of other degradat ion p roduc ts . Fragmentat ion pat te rns s u i t a b l e f o r execut ion of the proposed procedure were not obta ined w i t h crude or p u r i f i e d N [-2 -naphthoxyacetylglycyl - tRNAs w i t h e i t h e r RNase T^  or w i th p a n c r e a t i c RNase. S tud ies on the pH-dependent a c t i v i t y of RNase T^  degradat ion of tRNA showed, in a d d i t i o n to the expected optimum at pH 7-5, a second pH.optimum at pH 4.5. T h e . l a t t e r a c t i v i t y was d r a m a t i c a l l y augmented by the presence of 7 M u r e a . Stud ies on the s p e c i f i c i t i e s of severa l RNase T^  p repara t ions under va r ious c o n d i t i o n s showed the presence of s u b s t a n t i a l l e v e l s of adenosine as an end-group of tRNA degradat ion p roducts . P r e l i m i n a r y r e s u l t s of a search f o r reagents capable of adding s p e c i f i c a l l y an aromat ic res idue onto the 5 ' " te rminus of p o l y n u c l e o t i d e s demonstrated that r e a c t i n g e i t h e r 2 - n a p h t h y l -phosphoromorphol i date or 2 ,^4-d i n i t rof 1 uorobenzene wi th tRNA r e s u l t e d in increased b i n d i n g between the modi f ied tRNAs and B D - c e l 1 u l o s e . P a 9 e PART ONE: THE USE OF BENZOYLATED DEAE-CELLULOSE FOR THE ISOLATION OF GLYCINE TRANSFER RIBONUCLEIC ACIDS OF YEAST . 1 TABLE OF CONTENTS 2 LIST OF FIGURES h INTRODUCTION 7 EXPERIMENTAL 27 RESULTS AND DISCUSSION 39 REFERENCES 70 PART TWO: THE USE OF BENZOYLATED DEAE-CELLULOSE FOR THE DEVELOPMENT OF NEW METHODS FOR SEQUENCE DETERMINATION OF NUCLEIC ACIDS . . 80 TABLE OF CONTENTS 81 LIST OF FIGURES 83 LIST OF TABLES 84 INTRODUCTION 88 EXPERIMENTAL 105 RESULTS AND DISCUSSION 119 REFERENCES 181 ACKNOWLEDGEMENT I wish to thank Dr. G.M. Tener f o r h i s encouragement, d i r e c t i o n and c o n s t r u c t i v e c r i t i c i s m throughout the course of t h i s work. TO TARISSA for i o r i t y l e s s o n s ABBREVIATIONS tRNA T r a n s f e r R i b o n u c l e i c A c i d t R N A G l y The tRNAs which accept g l y c i n e t R N A ^ 1 y A S p e c i f i c Member of the Group of tRNAs which accept g l y c i n e DNA Deoxy r ibonuc le i c A c i d RNA R i b o n u c l e i c A c i d mRNA Messenger R i b o n u c l e i c A c i d ATP Adenosine Tr iphosphate A Adenosine C C y t i d i n e G Guanosine U U r i d i n e ip Pseudou r i d i ne D, D i h y d r o u r i d i n e T Ribothymidine DEAE- D ie thy laminoethy l B D - c e l l u l o s e Benzoylated DEAE-ce11u1ose UV U l t r a v i o l e t A 2 gg Absorbance at 260 nm ^260 u n ' t ' ^ n e ^260 u n ' t ' s t n a t amount of m a t e r i a l which when d i s s o l v e d in 1 ml of so l ven t w i l l g i ve an absorbance of one in a c e l l w i th a l i g h t path of 1 cm. nm Nanometer 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 Tr i s Tr i s(hydroxymethy1)ami nomethane RNase Ribonuclease Enz i te -RNase P a n c r e a t i c r ibonuc lease bound c o v a l e n t l y to carboxymethy l -ee l 1 u l o s e DEPC D ie thy lpy rocarbonate DTT D i t h i o t h r e i t o l ' PPO 2 , 5 - d i p h e n y l o x a z o l e Dimethyl > - P 0 P 0 P 1 ,k-b\s-2- ( i » - m e t h y l - 5 - p h e n y J o x a z o l y 1 ) benzene DCC D i c y c l o h e x y 1 c a r b o d i i m i d e PART ONE THE USE OF BENZOYLATED DEAE-CELLULOSE FOR ISOLATION OF GLYCINE TRANSFER RIBONUCLEIC ACIDS OF YEAST P a 9 e PART ONE INTRODUCTION 7 EXPERIMENTAL 27 (1) P r e p a r a t i o n of 2 -naphthoxyacety 1 e s t e r of N_-hydroxysucc in imide . 27 (2) Chromatographic Methods 27 (3) S o l u t i o n s I - IV 29 (k) P r e p a r a t i o n of G l y c y l - t R N A Synthetase . . . . . . 29 (5) Assays fo r G lyc ine - tRNA Acceptor A c t i v i t y 31 (6) Determinat ion of Maximum Charging of tRNA v; i th G l y c i n e 33 1 2 (7) Charging of C - g l y c i n e to tRNA in Large Sca le . . . 33 (8) Charging of ^ C - g l y c i n e to tRNA 3*t (9) Terminat ion of the React ion and Phenol E x t r a c t i o n . . 3^ t (10) Naphthoxyacety la t ion of Combined ^ C - and ^ C -G l y c y l - t R N A Prepara t ions 35 (11) Removal of the Nh2-naphthoxyacety1-g1ycy1 -tRNA from the Remainder of the tRNA 36 (12) Removal of the h[-2-naphthoxyacety 1 g 1 y c i n e Group from tRNA 37 (13) Chromatography of P u r i f i e d G l y c i n e tRNAs on B D - c e l l u l o s e 37 Table of Contents (Continued) Page (14) Chromatography of P u r i f i e d G l y c i n e tRNAs on DEAE-Sephadex A-25 37 RESULTS AND DISCUSSION. . . 39 General Procedure fo r the I s o l a t i o n of t R N A ^ y and t R N A G l y hi Stud ies w i th Stage I Enzyme . 51 Stud ies w i t h "Homologous" tRNA and G l y c y l - t R N A Synthetase 55 Further P u r i f i c a t i o n of t R N A ^ l y and t R N A ^ 7 59 Large Sca le I s o l a t i o n of t R N A G l y and t R N A ^ 7 from Crude tRNA 63 SUMMARY 69 REFERENCES 70 LIST OF FIGURES F i g u r e 1. C l o v e r - L e a f Models o f a Number o f tRNAs. . . 16 F i g u r e 2. G e n e r a l i z e d C l o v e r - L e a f S t r u c t u r e f o r tRNA 17 F i g u r e 3- Chromatography o f Brewer's Y e a s t tRNA on BD-cel l u l o s e AO F i g u r e h. P r o c e d u r e f o r the S p e c i f i c I n t r o d u c t i o n o f the 2 - N a p h t h o x y a c e t y l - G r o u p o n t o the 3'-Terminal o f t R N A G 1 Y kk F i g u r e 5- I n f l u e n c e o f Magnesium L e v e l s on the S y n t h e s i s o f G l y c y l - t R N A hi F i g u r e 6. I n f l u e n c e o f P r e - H e a t i n g and o f L e v e l s o f tRNA on the F i n a l Y i e l d o f 1 i*C-G l y c y l - t R N A 48 F i g u r e 7- I s o l a t i o n o f N[-2-naphthoxyacety 1-g l y c y l - t R N A kS F i g u r e 8. Chromatography o f t R N A G 1 Y I s o l a t e d w i t h Stage II Enzyme 52 F i g u r e 9- Chromatography o f Stage I Enzyme on Hydroxy 1 apat i t e 53 F i g u r e 10. Chromatography o f tRNA G 1Y I s o l a t e d w i t h Stage I Enzyme 56 F i g u r e 11. Chromatography o f tRNA G 1Y I s o l a t e d w i t h "Homologous" Stage I Enzyme 58 F i g u r e 12. Chromatography o f t R N A G , Y on DEAE-Sephadex A-25 61 F i g u r e 13. Chromatography o f t R N A G 1 Y on BD-c e l l u l o s e i n the P r e s e n c e o f EDTA a t A c i d pH 62 L i s t of F igures (Continued) F igure 14. Chromatography of t R N A G 1 y on DEAE-Sephadex A-25 64 Figure 15- P u r i f i c a t i o n of N_-2-naphthoxyacety 1 -g l y c y l - t R N A G , Y f' rom 5 g Untreated Crude tRNA 66 Figure 16. Chromatography of t R N A ^ y I so la ted from 5 g Untreated Crude tRNA 68 I wish to thank Mrs. D. Blew f o r her s k i l f u l a s s i s t a n c e in performing some of the assays shown in F igure 9-INTRODUCTION T r a n s f e r r i b o n u c l e i c a c i d (tRNA) has been termed " t h e u l t i m a t e achievement o f m o l e c u l a r e v o l u t i o n " (1). W h i l e t h i s may seem an e x a g g e r a t e d o p i n i o n , i t i s c e r t a i n t h a t tRNA i s a complex m o l e c u l e adapted t o p e r f o r m a c r i t i c a l r o l e i n p r o t e i n b i o s y n t h e s i s . T h i s f a m i l y o f n u c l e i c a c i d s p o s s e s s e s a c o m p l e x i t y o f s t r u c t u r e and a v e r s a t i l i t y o f f u n c t i o n which s u p e r s e d e the term " a d a p t o r " o r i g i n a l l y a p p l i e d t o i t (2) . 0 The e x i s t e n c e o f twenty s m a l l " a d a p t o r " n u c l e i c a c i d s was p o s t u l a t e d by C r i c k (2) t o e x p l a i n the i n f o r m a t i o n f l o w from DNA t o RNA to p r o t e i n s . C r i c k ' s p r o p o s a l s t a t e d t h a t d i s t i n c t enzymes would c o v a l e n t l y b i n d a s p e c i f i c amino a c i d t o a p a r t i c u l a r a d a p t o r and the aminoacy1ated-adaptors would then i n t e r a c t w i t h s p e c i f i c n u c l e o t i d e sequences o f the " t e m p l a t e " n u c l e i c a c i d s . S h o r t l y t h e r e a f t e r , RNA f r a c t i o n s p o s s e s s i n g the p r o p e r t i e s o f C r i c k ' s a d a p t o r s were d e s c r i b e d (3, k) and h i s p r o p o s a l s have s i n c e been l a r g e l y s u b s t a n t i a t e d . A d e t a i l e d d i s c u s s i o n o f a l l the e v e n t s i n v o l v e d i n the c o n v e r s i o n o f g e n e - i n f o r m a t i o n t o gen e - p r o d u c t s i s beyond the scope o f t h i s i n t r o d u c t i o n . Review a r t i c l e s (5, 6) and r e v i e w volumes (7, 8) a r e a v a i l a b l e . The f o l l o w i n g d i s c u s s i o n o u t l i n e s the s a l i e n t a s p e c t s o f tRNA's i n v o l v e m e n t i n the p r o c e s s o f c o n v e r t i n g g e n e - i n f o r m a t i o n to g e n e - p r o d u c t s . The amino a c i d sequence of a p a r t i c u l a r p r o t e i n i s d i c t a t e d by the sequence of n u c l e o t i d e s in that segment of the DNA known as the s t r u c t u r a l gene. The i n f o r m a t i o n of the s t r u c t u r a l gene i s a c c u r a t e l y conver ted i n t o an e q u i v a l e n t sequence of RNA by the a c t i o n of DNA-dependent RNA polymerase . The product of t h i s t r a n s c r i p t i o n s tep i s messenger RNA (mRNA) which then needs to be t r a n s l a t e d . It i s at the s tage of t r a n s l a t i o n that tRNA f u n c t i o n s . Here, the i n f o r m a t i o n in the l i n e a r sequence of n u c l e o t i d e s in the mRNA d i r e c t s the fo rmat ion of a l i n e a r p o l y p e p t i d e w i t h each t r i p l e t of n o n - o v e r l a p p i n g n u c l e o t i d e s d i r e c t i n g the i n s e r t i o n o f one s p e c i f i c amino a c i d i n t o the p o l y p e p t i d e . The t r a n s l a t i o n s tep i n v o l v e s r ibosomes, mRNA, aminoacy l - tRNAs and a number of p r o t e i n f a c t o r s . Aminoacy l - tRNAs are formed by aminoacy l - tRNA synthetases which s e l e c t a s p e c i f i c tRNA out of a l a r g e c o l l e c t i o n o f molecu les o f s i m i l a r s i z e , c o m p o s i t i o n and o v e r a l l confo rmat ion and e s t e r i f y the c o r r e c t amino a c i d to that RNA. The r e a c t i o n may be w r i t t e n as fo11ows: NH: ATP + R1~CH-C02 + Enzyme1 o Ti tRNA v v tRNA, Enzyme1 + t R N A - O - C - C H - ^ + AMP I H II Enzyme, . . . R , - C H-O0P-0CH O 1 1 | 2 0 Ad + OH OH The terminal adenosine of the 3 l - t e r r n i n a l sequence - c y t i d y1 y1(3 1 -5 1 ) c y t i d y l y1(3 1 -5 1 ) a d e n o s i n e which is common to a l l f u n c t i o n a l tRNAs is the s i t e of aminoacy1 a t i o n . The r e a c t i o n must be s t r i n g e n t s i n c e s e l e c t i o n e i t h e r of the wrong amino a c i d or the wrong tRNA w i l l lead to i n s e r t i o n of the amino a c i d at an i n c o r r e c t p o s i t i o n . Such mistakes are very r a r e . The aminoacy1 -tRNAs possess three s p e c i f i c n u c l e o t i d e s (the ant icodon) which b a s e - p a i r s p e c i f i c a l l y w i th three mRNA n u c l e o t i d e s (a codon) . The codon-ant icodon p a i r i n g , which invo lves hydrogen-bonding of both the c l a s s i c a l and w o b b l e - t y p e , is ant i -para11 e l in that the tRNA ant icodon is read 3' to 5' w h i l e the codon t r i p l e t is read 5' to 31• In b a c t e r i a i n i t i a t i o n of t r a n s l a t i o n requ i res the i n t e r a c t i o n fMet of the s p e c i f i c i n i t i a t i n g tRNA-N_-f ormy lmeth iony 1 - tRNA - w i t h both the mRNA and the 30S subunit of the r ibosome. The unique t r i p l e t of the mRNA which a l l o w s t o t a l in -phase t r a n s l a t i o n d i r e c t s the format ion of t h i s i n i t i a t i o n complex. Only a f t e r t h i s step is completed can the other r ibosomal subuni t bind and t r a n s l a t i o n proper b e g i n . The i n i t i a t o r tRNA serves to s e l e c t the beginning codon and, because of i t s formylated a-amino group, fo rces d i r e c t i o n a l s p e c i f i c i t y on s y n t h e s i s . The c o d o n - t r i p i e t ad jacent to the i n i t i a t o r - t r i p i e t next p a i r s w i th a second aminoacy1 -tRNA to set the stage fo r format ion of the f i r s t pept ide bond. The a-amino group of the second aminoacyl - tRNA a t t a c k s the carboxy l group of the methionine res idue borne by the i n i t i a t o r tRNA. The r e a c t i o n re leases the i n i t i a t o r tRNA and converts the other tRNA in to a peptidy1 - tRNA. The mRNA somehow "moves" or t r a n s -locates to accommodate p o s i t i o n i n g of a t h i r d aminoacy l - tRNA. Once the t r a n s l o c a t i o n is completed and the t h i r d aminoacyl - tRNA is p o s i t i o n e d , another pept ide bond is formed. By a r e p e t i t i o n of these s t e p s , the mRNA is t r a n s l a t e d from the 51 to 3' d i r e c t i o n w i th the growing po l ypept ide i n c r e a s i n g in length from the ct-amino te rmina l to the carboxy l t e r m i n a l . P o l y p e p t i d e s y n t h e s i s proceeds u n t i l a s t o p - s i g n a l or t e r m i n a t i o n t r i p l e t is reached. T r a n s l a t i o n then ceases and the completed po l ypept ide is re leased from both the ribosome and that tRNA cor responding to the penul t imate t r i p l e t under the d i r e c t i o n of the r e l e a s e f a c t o r s . In a d d i t i o n to r ibosomes, messenger RNA and tRNA, a la rge number of " p r o t e i n f a c t o r s " are necessary at the three stages of t r a n s l a t i o n - that i s , in i n i t i a t i o n , e l o n g a t i o n and t e r m i n a t i o n . tRNA appears not to be invo lved in the l a t t e r step except in suppress ion of premature t e r m i n a t i o n . The a b s o l u t e requirement fo r tRNA in both the i n i t i a t i o n and e l o n g a t i o n phases of t r a n s l a t i o n is we l l documented. The d i s c u s s i o n above a p p l i e s in d e t a i l to prokaryotes and in p r i n c i p l e to eukaryotes . But tRNA has more r o l e s to p lay than as the coup ler between g e n e - i n f o r m a t i o n and g e n e - p r o d u c t s . T r a n s f e r RNA can be i n v o l v e d i n f o u r e x t r a - t r a n s l a t i o n a l f u n c t i o n s : ( i ) ,|n cases where the c o n n e c t i o n w i t h p r o t e i n s y n t h e s i s i s not i m m e d i a t e l y a p p a r e n t . T h i s r o l e seems t o i n v o l v e a c l a s s of r e a c t i o n s e x p l o i t i n g t h e a c t i v a t e d s t a t e o f amino a c i d s e s t e r i f i e d t o tRNA. For example, c e r t a i n m i c r o - o r g a n i s m s have tRNAs which a r e i n v o l v e d i n p e p t i d o g 1 y e a n . s y n t h e s i s (9). A l s o , s o l u b l e enzymes w h i c h c a t a l y z e t r a n s f e r o f a r g i n i n e , l e u c i n e and p h e n y l -a l a n i n e from t h e i r tRNAs t o pre-formed a c c e p t o r p r o t e i n s have been d e s c r i b e d (10, 11). F u r t h e r , a hormone-dependent appearance o f Ser 0-phosphosery1-tRNA i n r o o s t e r - 1 i v e r e x t r a c t s has been r e p o r t e d (13) • A l a n y l - t R N A ^ a i s r e q u i r e d f o r s y n t h e s i s o f a l a n y l p h o s p h a t i d y l -g l y c e r o l i n C l o s t r i d i u m w e l c h i i and l y s y l - t R N A ^ 5 f o r the s y n t h e s i s of l y s y l p h o s p h a t i d y l g l y c e r o l i n S t a p h y l o c o c c u s a u r e u s . ( 1 4 , 15). In some o f t h e s e c a s e s (9, ., .„ ') , t h e tRNA - s p e c i e s i n v o l v e d may be s p e c i a l i z e d s i n c e they cannot s e r v e i n p r o t e i n b i o s y n t h e s i s . ( i i ) In r e p r e s s i o n o f c o - r e p r e s s i o n o f s p e c i f i c genomes. That c h a r g e d tRNAs may be i n v o l v e d i n c o n t r o l o f de novo s y n t h e s i s o f a number o f amino a c i d s has been d i s c u s s e d ( 1 6 ) . E v i d e n c e t o d a t e i s l a r g e l y c i r c u m s t a n t i a l and d e t a i l s a r e not c l e a r . However, f o r p r o d u c t i o n o f p h e n y l a l a n i n e (17), h i s t i d i n e (18, 19) and b r a n c h e d - c h a i n amino a c i d s (20, 21, 22, 2 3 ) , a p a r t i a l i n v o l v e m e n t of tRNA seems l i k e l y . In a d d i t i o n t o i t s r o l e i n c o n t r o l o f genomes f o r b i o s y n t h e t i c p a t h w a y s , tRNA a p p e a r s t o be i n v o l v e d i n r e g u l a t i o n o f tRNA a n d rRNA p r o d u c t i o n (24). ( i i i ) In c o n t r o l o f t h e r a t e o f t r a n s l a t i o n o f mRNA. The p r o p o s a l by I t a n o (25) p o s t u l a t i n g t h a t t h e r a t e o f h e m o g l o b i n s y n t h e s i s m i g h t be c o n t r o l l e d by s p e c i f i c c o d o n s w h o s e tRNAs w e r e i n s h o r t s u p p l y , was f o r m a l i z e d i n t h e " m o d u l a t i o n h y p o t h e s i s " o f Ames a n d H a r t m a n (26). T h e h y p o t h e s i s s t a t e d t h a t t h e r a t e o f r e a d i n g o f mRNA was d e p e n d e n t on m o d u l a t o r tRNAs w h i c h w e r e e i t h e r p r e s e n t i n s m a l l a m o u n t s o r i n a l t e r e d f o r m s s o t h a t t h e i r a v a i l a b i l i t y was t h e r a t e - 1 i m i t i n g s t e p i n p r o t e i n - b i o s y n t h e s i s . T h e m o d u l a t i o n phenomenon h a s b e e n o b s e r v e d i n v i t r o d u r i ng t r a n s l a t i o n o f b o t h s y n t h e t i c (27) and n a t u r a l (28) m e s s e n g e r RNAs. T h e s e r e s u l t s s u g g e s t t h a t s i m i l a r c o n t r o l i s p o s s i b l e i n v i v o a n d t h e G i n i n t e r e s t i n g p o s s i b i l i t y t h a t t h e a v a i l a b i l i t y o f g1utaminy1 -tRNA i n c e r t a i n m i c r o - o r g a n i s m s may a c t as a c o n t r o l p o i n t h a s b e e n d e s c r i b e d (12). S i n c e l i t t l e i s known a b o u t t h e r e g u l a t i o n o f s y n t h e s i s o f t R N A s , i t i s s p e c u l a t i v e t o c o n s i d e r how v a r y i n g l e v e l s o f a s p e c i f i c i s o -a c c e p t i n g s p e c i e s o f tRNA m i g h t be a c h i e v e d . One p o s s i b i l i t y , h o w e v e r , i s t o c o n t r o l t h e m a t u r a t i o n o f t R N A s . tRNAs w h i c h a r e a l t e r e d by u n d e r - m e t h y l a t i o n (29, 30) o r by i r o n - d e p e n d e n t m o d i f i c a t i o n s (31» 32) p r o v i d e e x a m p l e s o f how i m m a t u r e tRNAs may h a v e a l t e r e d c a p a c i t y f o r e i t h e r a m i n o a c i d a c c e p t a n c e o r p r o t e i n s y n t h e s i s i n b a c t e r i a l s y s t e m s . S i m i l a r c o n t r o l s may be o p e r a t i v e i n e u k a r y o t i c s y s t e m s s i n c e h o r m o n a l c o n t r o l o f tRNA m e t h y l a s e s (33) and o f tRNA a c t i v i t y i t s e l f (3*0 have been r e p o r t e d . ( i v ) In d e t e r m i n i n g what k i n d s o f mRNA s p e c i e s can be t r a n s l a t e d . T h i s r e g u l a t i o n c o u l d be a c h i e v e d by d e s t r o y i n g s p e c i f i c tRNAs w h i c h read codons no l o n g e r needed o r by p r o d u c i n g tRNAs not p r e v i o u s l y p r e s e n t i n the c e l l w h i c h a 11ow t r a n s 1 a t i o n o f new mRNAs. E v i d e n c e f o r t h i s k i n d o f c o n t r o l comes from v i r u s -i n f e c t e d c e l l s . For example, the s p e c i f i c d e s t r u c t i o n o f a p a r t i c u l a r l e u c i n e tRNA f o l l o w i n g phage T^ i n f e c t i o n o f Esc h e r i c h i a c o l i (35) may be the phage's way o f s t o p p i n g h o s t p r o t e i n s y n t h e s i s . Animal v i r u s e s have been shown t o c o n t a i n s p e c i f i c h o s t - c e l l tRNAs (36) and t o d i r e c t s y n t h e s i s o f new tRNAs (37). C e r t a i n b a c t e r i o p h a g e s a l s o d i r e c t s y n t h e s i s o f tRNAs d i f f e r e n t from tRNAs p r e s e n t i n the host p r i o r t o i n f e c t i o n (38, 39)- The f u n c t i o n o f t h e s e s p e c i e s i s n o t - c l e a r but i t seems r e a s o n a b l e t o suppose t h a t they a r e i n v o l v e d i n a c h i e v i n g p r e f e r e n t i a l t r a n s l a t i o n o f v i r a l genomes. For the number o f r o l e s i t p l a y s , t h e tRNA m o l e c u l e i s d e c e p t i v e l y s i m p l e . I t s m o l e c u l a r w e i g h t o f 25,000 t o 30,000 makes i t a p o l y n u c l e o t i d e o f 75 t o 85 n u c l e o t i d e s and the s m a l l e s t o f the common n u c l e i c a c i d s . In a d d i t i o n t o i t s s m a l l n e s s , tRNA i s unique i n i t s . h i g h c o n t e n t o f "odd" bases. A l a r g e number o f th e s e m o d i f i e d bases have been c h a r a c t e r i z e d ( h O ) . T r a n s c r i p t i o n of tRNA genes produces m o l e c u l e s d e v o i d o f m o d i f i e d bases and th e s e p r e c u r s o r tRNA m o l e c u l e s (41) a r e then changed by c o n v e r s i o n oT normal bases t o t h e i r m o d i f i e d c o u n t e r p a r t s (h2, 4 3 ) . The m a t u r a t i o n p r o c e s s appears t o be complex s i n c e t h e r e a r e many m o d i f i e d b a s e s . Knowledge o f the b i o s y n t h e s i s o f tRNA i s i n i t s i n f a n c y . P r e c u r s o r - t R N A has been d e s c r i b e d (hh, h5, k6) but the o r d e r , number and c o n t r o l o f s t e p s . c o n v e r t i n g immature tRNA t o i t s f u n c t i o n a l l y c o m p l e t e c o u n t e r p a r t remain l a r g e l y unknown. The A l a r e c e n t s y n t h e s i s o f the s t r u c t u r a l gene f o r tRNA s h o u l d a l l o w an i n v e s t i g a t i o n o f some o f t h e s e u n c e r t a i n t i e s (47). Trends a p p a r e n t i n the l i t e r a t u r e (h5, h(>, hi, 48, 49) suggest t h a t a c o n c e r t e d e f f o r t t o e l u c i d a t e not o n l y the s t e p s o f m a t u r a t i o n o f tRNA but a l s o i t s . c o n t r o l i s imminent. The m o l e c u l a r c o m p l e x i t y o f tRNA s u g g e s t s t h a t the e v o l u t i o n o f an i m p r e s s i v e amount o f e n z y m a t i c machinery was r e q u i r e d f o r i t s p r o d u c t i o n . What i s known o f the f i n a l p r o d u c t o f t h i s e n z y m a t i c machinery Here, c o n s i d e r a b 1 y more i n f o r m a t i o n i s a v a i l a b l e . The p r i m a r y sequences o f many d i f f e r e n t tRNAs a r e known. T r a n s f e r RNAs from the y e a s t Saccharomyces c e r e v i s i a e which have been sequenced i n c l u d e t R N A A 1 a (50, 51) , t R N A S e r (52) , t R N A P h e (53) , t R N A T y r (54) , t R N A V a l (55) and t R N A A s p (56). For the y e a s t T o r u l o p s i s u t i 1 i s t R N A V a l (57) , t R N A l 1 e u (58) and t R N A T y r (59) have been sequenced. The sequences o f t R N A J y r ( 6 0 ) , tRNA1/'* (60) , s u j , ( t R N A 1 / r (60) , t R N A P h e (61, 6 2 ) , t R N A f M e t ( 6 3 ) , t R N A M e t (6h), t R N A V a 1 (65) , -tRNA L e U (66) and t R N A T r p (67) o f E s c h e r i c h i a c o l i a r e known.tRNA P h e of wheat germ and tRNA (69) of rat l i v e r are a l s o known. P a r t i a l sequences f o r t R N A A l a (E_. co l i) (70) and t R N A M e t (yeast) (71) have been pub 1i shed. A r e p r e s e n t a t i v e sample of sequences is shown in F igure 1. A l l of the pub l i shed pr imary sequences can be w r i t t e n to conform to the c l o v e r - l e a f secondary s t r u c t u r e model which i s shown in a g e n e r a l i z e d form in F igure 2. The c l o v e r - l e a f model was o r i g i n a l l y proposed by Hoi ley (50) and the p r o b a b i l i t y of f i n d i n g new tRNA spec ies which w i l l not conform to i t now appears remote (72) . A d e t a i l e d a n a l y s i s of pr imary sequences and comparisons of secondary sequences w i l l not be attempted here . These fea tu res have been reviewed r e c e n t l y (41, 73)- A number of s i m i l a r i t i e s and g e n e r a l i t i e s can be gleaned from such comparat ive e x e r c i s e s . Some of these are noted in F igure 2 and are o u t l i n e d below. The c l o v e r - l e a f model a l lows tRNA to be d i v i d e d in to f i v e t o p o l o g i c a11 y - d i s t i n e t r e g i o n s : (1) the amino a c i d arm c o n t a i n i n g a seven-membered b a s e - p a i r e d h e l i x or stem ( i ) and terminated w i t h a s i n g l e - s t r a n d e d reg ion of four r e s i d u e s ; the amino a c i d can be e s t e r i f i e d to the 3 l _ h y d r o x y l group of the adenosine res idue which terminates the l a t t e r r e g i o n , (2) the d i h y d r o u r i d i n e arm ( l ) w i th i t s h e l i x of 3 to k pa i red bases ( i i ) and h i g h l y v a r i e d loop which appears to be d i r e c t l y i m p l i c a t e d in a m i n o a c y l -tRNA synthetase r e c o g n i t i o n (74; however, see 75, 76), (3) the ant icodon arm whose h e l i x of f i v e pa i red bases ( i i i ) supports the Clover-leaf Models of a Number of tRNAs CGCGG,, I I I ! AOH C C A p G - C G - C G - U C - G G - C U - U U A G G C C U U A A 0 I C A - C -G U C C G G f U / C GO G^CGCGoinw Cy» * c—GAG C-G C-G C-G U * U I1"" • GC Alanine tRNA yeast G - C G - U A - U U - A O G A U G A C A C c U A . 0 CUCG.A I I I I I G G I I I I1'"' C U G U G T J , C G G A G A G C G 0 _ ^ C ^ ' * C - G A ' G 7 m . A - U G G— Cjm, rA~*A U G A A Y 2*Om« Phenylalanine tRNA yoasl C A P C — G U — A C — G U — G C — G G — C Q Q _ D G A A U C C C G C U C A , „ _° C C G A I I I I I G Gr«r.. I I l N ™ G G G C G - t y C 0 D A C - G A A A — u G A A — U G — C A—ifr C A U A" 0 5 *" ' G ^ A Tyrosine tRNA yeast c u" C G P G — C G—U C-G A—U A—U C - G A U—A U CGUCCUGA JCCGG I I I I I GA II I GCAGGtyC n AGGCGoim* Csmt ° O A A A-U,, G , G A G« A - U " A — U G —C A-<fc <h A II /^•op«nl IGA Serine tRNA yeast i G A j c u u c c°M C A , G - C G - U U - A U - A U - A C - G G — C ^ U - G G G G U C C U A , „ C C L ) G G , M . I I I I I G G G G C A ^ C C A G ^ C D D A U * - A . 5 S * C - G A C U - A G - C C - G Valine tRNA Saccharomyces cerevisiae *G U U u c* C A -C - U - A - A - A C - G Q £ 0 G A J / U GGGUCCUA D C U G G , m . I I I I I G G n GGCA £ C A G T * C C - G A U - A G - C , C - G Valine tRNA Torulopsis utilis •GA AOH C A P G — C G - C U - A G - C G - C G - C Q £ C . . . , , ! ) ' C U U C C U A A C C C U I I I i I p 1 1 1 1 GAAGGtyC C C A A A G G G A G - C C ^ -  U U R C - G C . A A A-y G U ^ G C A Su* G-C A - V C A C u A G' Tyrosine tRNA E. coli - A UC AOM C C A P C — A G - C C - G G — C G — C G - C „ >A U* C G G C C U A A C C G A G G I I I I I A U ' ''JL r G U C G G T ^ C " R A G C U C G C I . G G O U — A / G "A C — G A R , G - C G G — C «•....£-<fc U. A C A U /V-Formylmethionine tRNA E. coli • G A , AOH C A P G - C G - C G - C U - A G - C A - U r-GA U * T B O C U G C C C U A n CUCGA I I I I I G 2 T i l l GGCGGTd,C G ^ A G A G C A C I , , W  G G A C - G ^ U G " ' C - G G G U - A C - G C - G A C A U A 6"" Z A C Valine tRNA 2 E.coli Genera l i zed C l o v e r - L e a f S t r u c t u r e f o r tRNA (adapted from references 138. and 72) I, I I , I I I , IV = loops or unpaired r e g i o n s . ( i ) , ( i i ) , ( i i i ) , ( iv ) and (v) = h e l i c a l or b a s e - p a i r e d r e g i o n s . S o l i d c i r c l e s = bases in he 1ica1 - r e g i o n s pa i red by hydrogen bonds i n c i d a t e d by c e n t r a l dot Empty .c i rc1es = bases u s u a l l y unpaired in l o o p - r e g i o n s 5and ^ = areas of s i g n i f i c a n t v a r i a t i o n in both numbers and k inds of bases ; the numbers in parentheses near these symbols r e f e r to lower and upper l i m i t of number of bases o c c u r r i n g at such r e g i o n s . Pu = pur ine n u c l e o s i d e . Py = p y r i m i d i n e n u c l e o s i d e . P = phosphate ( 5 ' - t e r m i n a l of tRNA). 1, 2, 3 = bases of the a n t i c o d o n . W = wobble p o s i t i o n . D = d i h y d r o u r i d i n e . T = r i bothym i d i ne. A , C , U, G = normal a b b r e v i a t i o n s of the four major pur ine and p y r i m i d i n e n u c l e o s i d e s . D Arm A t C i c P9-9 OH Amino A c i d Arm ( i ) ( 1 - 2 ) o l d ' y A C IV Pu T^C Arm 9 ® . • (,-3)cAO G G g A (1 -2 ) ^ - • • J b ° , o ( . - 3 ) ••• 9-9 99. ( i i i ) Ex t ra Arm o ( 2 - 1 3 ) Ant icodon Arm U N A " 0 - © - © Ant icodon w -O-O-CK Codon seven-membered 1 oop (| l) wh i ch c o n t a i n s the a n t i c o d o n ; the three c i r c l e d bases in t h i s loop make up the a n t i c o d o n ; the wobble-p o s i t i o n is the f i r s t of these bases (77), (^) the Ti(jC-arm whose f i v e base pa i red stem ( iv ) bears a loop (IV) of seven n u c l e o t i d e s which may be invo lved in r ibosomal b ind ing (78), f i n a l l y , (5) the e x t r a arm or lump ( I I I ) which l i e s between the T^C-arm and the ant icodon arm; i t shows marked v a r i a t i o n from tRNA to tRNA. S i g n i f i c a n t homologies appear when tRNAs are compared t h i s way but i r r e g u l a r i t i e s e x i s t . For example, the d i h y d r o u r i d i n e arm shows such v a r i a t i o n that tRNA"*"yr of E_. co l i lacks d i hyd rour id i ne complete ly (60) . The c l o v e r - l e a f model revea ls that odd bases tend to occupy loops rather than b a s e - p a i r e d stems in most tRNAs. The odd bases probably serve in r e c o g n i t i o n phenomena and in maintenance of tRNA s t r u c t u r e . The p h y s i c a l bas i s f o r the secondary s t r u c t u r e - d e t e r m i n i n g r o l e of the mod i f ied bases is not w e l l understood. For example, 2 2 N -dimethy1 guanosine (m^G) occurs as the n u c l e o s i d e at the "bend" between the stem of the d i h y d r o u r i d i n e arm and the stem of the Ser ant icodon arm of a number of yeast tRNA spec ies and ra t l i v e r tRNA but not in any E_. co l i tRNA of known sequence (see F igure 1). An 2 examinat ion of model compounds c o n t a i n i n g m G^ showed that w h i l e normal hydrogen bonding is prevented there is no need f o r bends 2 (due to an i n a b i l i t y of the m G^ to s tack w i th ad jacent bases) to 2 occur at p o s i t i o n s occupied by m G in tRNA (79)- The r e l a t i o n s h i p of content and p o s i t i o n of mod i f ied bases to s t r u c t u r e and f u n c t i o n of tRNA c l e a r l y needs f u r t h e r e l u c i d a t i o n . Advantages other than a convenient means of d e f i n i n g s p e c i f i c regions are provided by the c l o v e r - l e a f model . For example, i t has been reported that pheny la lan ine - tRNA synthetase of yeast i s ab le to aminoacy late E_. co l i tRNA^ a^ as w e l l as yeast 80) Phe and wheat germ tRNA (7^). These tRNA spec ies show l i t t l e apparent s i m i l a r i t y i f compared as pr imary sequences; however, i f arranged in c l o v e r - l e a f fo rm, remarkable homology is ev ident in the d i h y d r o u r i d i n e arms. Th is o b s e r v a t i o n serves to emphasize the a c t u a l and p o t e n t i a l va lue of the model . Recogn i t ion of f u n c t i o n a l and t o p o l o g i c a l s i m i l a r i t i e s in tRNAs is of prime importance in f o r m u l a t i o n of d e t a i l e d molecu lar models of tRNA (72, 81 , 82) and in d e l i n e a t i n g var ious enzyme-tRNA r e c o g n i t i o n s i t e s (7^ » 80, 82). Progress in t h i s area w i l l s u r e l y prov ide an e x c i t i n g . g l i m p s e of the e v o l u t i o n a r y aspects of molecu lar b i o l o g y . At the present time much e f f o r t is being expended to determine the t e r t i a r y s t r u c t u r e of tRNA and to r e l a t e the s t r u c t u r e to f u n c t i o n . D e t a i l e d molecu lar models have been proposed (72, 81, 82). D e f i n i t e in fo rmat ion regarding s t r u c t u r e - f u n c t i o n r e l a t i o n -sh ips is l i m i t e d but the number of researchers and range of approaches used holds promise fo r imminent r e s u l t s . Even a summary of approaches and t e n t a t i v e c o n c l u s i o n s cannot be attempted here . The f o l l o w i n g s h o u l d , however, demonstrate the i n t e r e s t these p r o b l e m s a r e r e c e i v i n g . A p p r o a c h e s s u c h a s t h e s u s c e p t i b i l i t y o f t R N A t o a t t a c k b y v a r i o u s n u c l e a s e s (84) a n d a n u m b e r o f c h e m i c a l r e a g e n t s (81, 85) u n d e r a v a r i e t y o f a m b i e n t c o n d i t i o n s h a v e b e e n u s e d t o s t u d y t e r t i a r y s t r u c t u r e a n d e n z y m e r e c o g n i t i o n s i t e s . P h y s i c a l t e c h n i q u e s s u c h a s n u c l e a r m a g n e t i c r e s o n a n c e (86, 87), e l e c t r o n s p i n r e s o n a n c e (88, 89), o p t i c a l r o t a r y d i s p e r s i o n (90, 91), c i r c u l a r d i c h r o i s m (91, 92), f 1 u o r e s c e n c e ( 9 3 , 94), c o m b i n e d p r o b e - f l u o r e s c e n c e t e c h n i q u e s (95), p h o t o c h e m i c a l p r o b e s (96) a n d X - r a y a n a l y s i s o f t R N A i n s o l u t i o n (97) a n d i n c r y s t a l f o r m (98, 99) h a v e b e e n u s e d t o s t u d y t h e c o n f o r m a t i o n o f t R N A . A p p r o a c h e s s u c h a s n u c l e a s e d i g e s t i o n o f t R N A - a m i n o a c y 1 -t R N A - s y n t h e t a s e c o m p l e x e s (100) a n d u s e o f b o t h h o m o l o g o u s a n d h e t e r o l o g o u s m e t h y l a s e s t o m o d i f y t R N A s (101) h a v e b e e n u s e d t o t r y t o e s t a b l i s h w h a t a r e a s o f t h e t R N A a r e a v a i l a b l e f o r e n z y m e -i n t e r a c t i o n . T h e f r a g m e n t - r e c o n s t i t u t i o n m e t h o d w h i c h a t t e m p t s t o d e l i n e a t e f u n c t i o n a l r e g i o n s r e l a t e d t o a m i n o a c y l - t R N A s y n t h e t a s e r e c o g n i t i o n (76, 102) h a s b e e n e x t e n d e d t o s t u d i e s o f r i b o s o m a l -b i n d i n g r e q u i r e m e n t s (103) a n d t h e a n t i c o d o n - c o d o n b i n d i n g r e q u i r e m e n t s (10*0 • T h e t e c h n i q u e o f u s i n g h e t e r o l o g o u s s y s t e m s t o a n a l y z e t h e r e c o g n i t i o n . o f . t R N A b y t h e a m i n o a c y l - t R N A s y n t h e t a s e s (105) w i l l p r o b a b l y b e e x t e n d e d t o e x a m i n e b o t h s t r u c t u r a l (7k, 80) a n d e v o l u t i o n a r y . p r o b 1 e m s . P e r h a p s o n e o f t h e m o s t p r o m i s i n g a p p r o a c h e s p r e s e n t l y a v a i l a b l e f o r t h e a n a l y s i s o f s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s i n tRNA is the technique developed by J . D . Smith and co -workers (106a, b) . The procedure is based on g e n e t i c s e l e c t i o n and a n a l y s i s of the a b i l i t y of E_. co l i s u s | | | tRNA^^"" mutants to f u n c t i o n in s u p p r e s s i o n . Mutat ions which r e s u l t in e i t h e r r e v e r s i o n from s u p p r e s s o r - p o s i t i v e to s u p p r e s s o r - n e g a t i v e p r o p e r t i e s or convers ion to t e m p e r a t u r e - s e n s i t i v e suppress ion (106b) can be used to probe numerous aspects of tRNA s t r u c t u r e and f u n c t i o n . Cons iderab le success has a l ready been obta ined w i t h mutants r e s u l t i n g from s i n g l e - b a s e changes at var ious p o s i t i o n s in the tRNA. It is l i k e l y that t h i s procedure can f a c i l i t a t e examinat ion of the f o l l o w i n g aspects of t r a n s f e r RNA: ( i ) c o n t r o l of the t r a n s c r i p t i o n of the s t r u c t u r a l genes f o r tRNAs, ( i i ) d e l i n e a t i o n of maturat ion-enzyme r e c o g n i t i o n s i t e s , ( i i i ) c o n t r i b u t i o n of modi f ied bases to f i n a l f u n c t i o n and s t r u c t u r e , ( iv ) d e l i n e a t i o n of a l t e r a t i o n s lead ing to conformat iona l i n f l u e n c e , (v) d e f i n i t i o n of a l t e r a t i o n s in a l l types of r e c o g n i t i o n s i t e s as w e l l as (v i ) f u n c t i o n a l aspects such as changes in the three ant icodon p o s i t i o n s . U l t i m a t e l y , X - r a y a n a l y s i s of both tRNA and tRNA-enzyme complexes w i l l p rov ide a complete d e s c r i p t i o n of t e r t i a r y s t r u c t u r e and c e r t a i n s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s . P r e r e q u i s i t e to the understanding of many aspects of tRNA s t r u c t u r e and f u n c t i o n is the a v a i l a b i l i t y of methods f o r i s o l a t i o n of homogeneous tRNAs and f o r chemical a n a l y s i s of those s p e c i e s . T h i s t h e s i s i s concerned w i t h both o f t h e s e problems. P a r t I r e l a t e s t he a u t h o r ' s c o n t r i b u t i o n s t o the development o f a g e n e r a l p r o c e d u r e f o r the i s o l a t i o n o f s p e c i f i c t r a n s f e r RNA s p e c i e s ( 1 0 7 ) . I t s h o u l d be emphasized t h a t the a u t h o r ' s r o l e was one o f development r a t h e r than i n v e n t i o n . P a r t II d e s c r i b e s a t t e m p t s t o d e v e l o p a novel s e q u e n c i n g t e c h n i q u e f o r p o l y -nuc1eot i des. The need f o r i s o l a t i o n o f homogeneous tRNA s p e c i e s has r e s u l t e d i n development o f a number o f methods. These methods can be d i v i d e d i n t o two g e n e r a l types - one r e l y i n g on d i f f e r e n c e s i n p h y s i c a l p r o p e r t i e s o f tRNAs and the second r e l y i n g on the d i f f e r e n c e i n c h e m i c a l r e a c t i v i t y o f aminoacy1ated-tRNAs and tRNAs which a r e not aminoacy1ated. Methods which attempt t o r e s o l v e tRNA s p e c i e s on the b a s i s o f p h y s i c a l p r o p e r t y d i f f e r e n c e s i n c l u d e c o u n t e r c u r r e n t d i s t r i b u t i o n (108, 109, 1 10) , 1 i q u i d - 1 i q u i d p a r t i t i o n ( 1 1 1 ) , p a r t i t i o n column chromatography ( 1 1 2 ) , reversed-.phase chromatography (113, l i * * , 115), gel f i l t r a t i o n (116), chromatography on m e t h y l a t e d a l b u m i n - k i e s e l g u h r ( 1 1 7 ) , m e t h y l a t e d a l b u m i n s i l i c i c a c i d ( 1 1 8 ) , h y d r o x y l a p a t i t e (119), D E A E - c e l l u l o s e (120, 121, 122), DEAE-Sephadex (122, 123, 124), c a t i o n i c exchange s t a r c h (125) and b e n z o y l a t e d DEAE-cellulose?(126). In p r i n c i p l e , t h e s e methods a r e d e s i g n e d f o r f r a c t i o n a t i o n o f tRNA r a t h e r than p u r i f i c a t i o n o f homogeneous s p e c i e s . In p r a c t i c e , however, p r o c e d u r e s used t o p u r i f y tRNAs f o r sequence s t u d i e s have g e n e r a l l y invo lved a p p l i c a t i o n of one or combinat ion of these p h y s i c a l methods. The second general c l a s s of p u r i f i c a t i o n methods f o r tRNA takes advantage of the d i f f e r e n c e in chemical r e a c t i v i t y between the e s t e r i f i e d and n o n - e s t e r i f i e d tRNAs as a means of s e p a r a t i n g the two. The s t r i c t s p e c i f i c i t y of the aminoacyl - tRNAs synthetases a l lows s e l e c t i v e e s t e r i f i c a t i o n of a s p e c i f i c tRNA f a m i l y w i thout erroneous e s t e r i f i c a t i o n . Those tRNAs which are not e s t e r i f i e d are s u s c e p t i b l e to pe r iodate o x i d a t i o n . The o x i d i z e d spec ies w i l l couple w i th hydraz ine d e r i v a t i v e s w h i l e e s t e r i f i e d spec ies w i l l no t . Such c o u p l i n g a l lows s e p a r a t i o n of p rotected from o x i d i z e d tRNA (127). A l t e r n a t i v e l y , the aminoacyl - tRNAs may be reacted w i t h reagents to produce i n s o l u b l e - d e r i v a t i v e s which are r e a d i l y separab le from f r e e tRNAs (128, 129, 130). Whi le the p r i n c i p l e of these methods makes them s u i t e d to i s o l a t i o n of s p e c i f i c tRNA f a m i l i e s , they are not in popular use because of p r a c t i c a l d i f f i c u l t i e s encountered. U n t i l r e c e n t l y , methods in popular use f o r p u r i f i c a t i o n of tRNA r e l i e d e x c l u s i v e l y on p h y s i c a l property d i f f e r e n c e s and inc luded counte rcur rent d i s t r i b u t i o n , chromatography on DEAE-Sephadex, reversed -phase p a r t i t i o n chromatography and B D - c e l l u l o s e chromatography. The s i m p l i c i t y , a b i l i t y to handle la rge amounts of tRNA and success in a c h i e v i n g the d e s i r e d r e s o l u t i o n have made these the favored methods. Chromatographic methods, however, are becoming more popular than counte rcur rent d i s t r i b u t ion methods s i n c e they are f a s t e r , t e c h n i c a l l y s i m p l e r , not as s e n s i t i v e to temperature changes and can be performed more r e a d i l y on large or small s c a l e than the counte rcu r ren t d i s t r i b u t i o n approach. Chromatographic methods on m a t e r i a l s such as DEAE-Sephadex are much used s i n c e c o n d i t i o n s such as pH, temperature , i o n i c s t reng th and content of o r g a n i c so l vents can be v a r i e d so as to ach ieve the des i red r e s o l u t i o n . The reversed-phase p a r t i t i o n systems ach ieve remarkable r e s o l u t i o n of i s o - a c c e p t o r tRNAs and the a v a i l a b i l i t y of four reversed-phase p a r t i t i o n systems (113, 11**, 115) has g r e a t l y expanded the u t i l i t y of t h i s procedure. Methylated albumin on s i l i c i c a c i d a l l o w s f r a c t i o n a t i o n of tRNA in g rea te r amount and w i t h h igher r e s o l u t i o n than i t s p recursor methylated albumin on k i e s e l g u h r (117, 118). The p r e p a r a t i o n of benzoylated-DEAE-cel1u1ose (BD-ce l1ulose) prov ided a m a t e r i a l which gave good r e s o l u t i o n of tRNAs and a l lowed development of a novel method f o r p u r i f i c a t i o n of s p e c i f i c tRNAs (107)- BD-ce l1ulose a l l o w s c o n s i d e r a b l e f r a c t i o n -a t i o n r o f tRNA spec ies from a v a r i e t y of sources (131, 132, 133, 13^, 135, 136, 137). The r e s o l u t i o n achieved appears , in p a r t , to be r e l a t e d to the r e l a t i v e amounts of hydroph-Mic and hydro -phobic c h a r a c t e r i s t i c s of the va r ious tRNA spec ies s i n c e t h e i r r e l a t i v e a f f i n i t i e s fo r B D - c e l l u l o s e r e f l e c t pat terns observed from coun t e r cu r r en t d i s t r i b u t i o n ( c . c . d . ) s t u d i e s (138). T rans fe r RNA s p e c i e s o f l o w h y d r o p h o b i c c h a r a c t e r move w i t h t h e a q u e o u s p h a s e i n c . c . d . a n d e l u t e e a r l y f r o m B D - c e l l u l o s e w h i l e t h o s e o f h i g h e r h y d r o p h o b i c c h a r a c t e r move w i t h t h e o r g a n i c p h a s e i n c . c . d . a n d A l a e l u t e l a t e f r o m B D - c e l l u l o s e (138). E x a m p l e s a r e tRNA ( y e a s t ) and tRNA ( y e a s t ) r e s p e c t i v e l y . T h e h i g h a f f i n i t y B D - c e l l u l o s e h a s f o r s p e c i f i c t R N A s a l l o w e d Phe tRNA o f y e a s t , w h i c h c o n t a i n s an u n u s u a l l y h y d r o p h o b i c b a s e Y, t o be h i g h l y e n r i c h e d by s i m p l e p a s s a g e o f b u l k tRNA o f y e a s t t h r o u g h a B D - c e l l u l o s e c o l u m n (139)- I t was r e a s o n e d o n t h e b a s i s o f t h i s o b s e r v a t i o n t h a t t h e h y d r o p h o b i c n u c l e o t i d e Y o f Phe tRNA was r e s p o n s i b l e f o r t h e s t r o n g b i n d i n g o f t h a t s p e c i e s o f tRNA t o B D - c e l l u l o s e (159). T h i s p r o p o s a l was r e c e n t l y c o n f i r m e d Phe s i n c e tRNA p r e v i o u s l y t r e a t e d w i t h m i l d a c i d t o r e m o v e Y b a s e w i t h o u t c h a i n s c i s s i o n was o b s e r v e d t o e l u t e f r o m B D - c e l l u l o s e e a r l y i n t h e s a l t g r a d i e n t (151). t R N A P h e , t R N A T y r , t R N A T r p h a v e b e e n e x t e n s i v e l y p u r i f i e d by e x p l o i t i n g t h e i n c r e a s e d a f f i n i t y o f t h e s e tRNA s p e c i e s f o r BD-c e l l u l o s e f o l l o w i n g e s t e r i f i c a t i o n w i t h t h e i r r e s p e c t i v e a r o m a t i c a m i n o a c i d s ( 1 4 0 , 139, 1^1). E s t e r i f i c a t i o n s u f f i c i e n t l y a l t e r s t h e i r a f f i n i t y f o r B D - c e l l u l o s e t h a t h i g h p u r i f i c a t i o n i s a c h i e v e d a f t e r s i m p l e c h r o m a t o g r a p h i c s t e p s . By a n a n a l o g o u s p r o c e d u r e , a new g e n e r a l l y a p p l i c a b l e m e t h o d f o r i s o l a t i o n o f h i g h l y p u r i f i e d tRNA s p e c i e s h as b e e n d e v e l o p e d . I t i n v o l v e s s e l e c t i v e i n t r o d u c t i o n o f a n a r o m a t i c r e s i d u e o n t o t h e aminoacy1ated-tRNA and hence a 1 lows . p u r i f i c a t i o n of a s p e c i f i c f a m i l y of tRNAs (107, 142, 143)• The method prov ides a means of s e l e c t i n g a c t i v e from i n a c t i v e tRNA spec ies (75, 8 3 , 144). The method is a combinat ion of the chemical and p h y s i c a l approaches to tRNA p u r i f i c a t i o n . It is the r e s u l t of a c o l l a b o r a t i v e e f f o r t d i r e c t e d by Dr. G.M. Tener and involved Dr. Ian G i l l a m , Dr. E. Wimmer, Dr. I. Maxwe l l , Dr. S. M i l l w a r d , M. von T ige rs t rom and D. Blew. Par t I of t h i s t h e s i s r e l a t e s the a u t h o r ' s part in expanding the u t i l i t y of the method and in extending s t u d i e s on g l y c i n e tRNAs from yeast i n i t i a t e d by Dr. S. M i l l w a r d . EXPERIMENTAL Large S c a l e I s o l a t i o n of G 1 y c i n e - S p e c i f i c tRNA from B o e h r i n g e r and Soehne Brewer's Y e a s t . (1) P r e p a r a t i o n o f 2-naphthoxyacety1 e s t e r o f N ^ h y d r o x y s u c c i n i m i d e . The method i s e s s e n t i a l l y t h a t d e s c r i b e d i n r e f e r e n c e 145. To 2 - n a p h t h o x y a c e t i c a c i d (4.04 g, 20 mmoles) and N - h y d r o x y s u c c i n i m i d e (2.3 g, 20 mmoles) d i s s o l v e d i n d i o x a n e (40 m l , m a i n t a i n e d o v e r sodium m e t a l ) was added d i c y c l o h e x y l c a r b o d i i m i d e (4.2 g, 20 mmoles). The s o l u t i o n was s h a k e n to d i s s o l v e the c a r b o d i i m i d e and a f t e r 2 hours a t 23° the i n s o l u b l e d i c y c l o h e x y 1 u r e a was f i l t e r e d and washed w i t h d i o x a n e . The combined f i l t r a t e s and washings were c o n c e n t r a t e d under reduced p r e s s u r e t o a s y r u p . The s y r u p was d i s s o l v e d i n i s o p r o p a n o l f o r r e c r y s t a 1 1 i z a t i o n . The p r o d u c t from the f i r s t r e c r y s t a 1 1 i z a t i o n (3-9 g) gave a m e l t i n g p o i n t o f 145-147°. (2) C h r o m a t o g r a p h i c Methods. B D - c e l l u l o s e ( f u l l y b e n z o y l a t e d ) , p r e p a r e d by r e a c t i o n o f DEAE-cel1u1ose w i t h b e n z o y l c h l o r i d e as d e s c r i b e d i n r e f e r e n c e 126, was ground and s i e v e d i n the wet s t a t e through e i t h e r a 30 mesh (0.6 - mm opening) or a 50 mesh (0.3 ~ mm opening) s c r e e n and f r e e d o f f i n e p a r t i c l e s by r e p e a t e d s e t t l i n g and d e c a n t a t i o n . D u r i n g t h e s i e v i n g a n d t h e r e m o v a l o f t h e f i n e s , t h e B D - c e l l u l o s e w a s m a i n t a i n e d i n s o l u t i o n s c o n t a i n i n g d i l u t e (0.1 - 0.5 M) s o d i u m c h l o r i d e t o p r e v e n t e x c e s s i v e g e n e r a t i o n o f f i n e s . T h e B D - c e l l u l o s e w a s p a c k e d i n t o c o l u m n s b y a d d i n g a s l u r r y o f t h e ' r e s i n in2 'M s o d i u m c h l o r i d e ( w h i c h h a d b e e n f r e e d o f t r a p p e d a i r b y b r i e f b o i l i n g a t r o o m t e m p e r a t u r e u n d e r r e d u c e d p r e s s u r e ) t o a c o l u m n h a l f f i l l e d w i t h 2 M s o d i u m c h l o r i d e . T h e s l u r r y w a s a l l o w e d t o s e t t l e u n t i l a p p r o x i m a t e l y 2-3 c m o f B D -c e l l u l o s e h a d p a c k e d . T h e c o l u m n s t o p - c o c k w a s t h e n o p e n e d b u t t h e l i q u i d l e v e l w a s a l w a y s k e p t a b o v e t h e p a c k e d s u r f a c e o f t h e e x c h a n g e r . T h e s l u r r y w a s a d d e d u n t i l t h e d e s i r e d d e p t h o f b e d w a s o b t a i n e d . F o r c h r o m a t o g r a p h y w i t h f i n e (50 - m e s h ) B D -c e l l u l o s e , c o l u m n p r e p a r a t i o n w a s p e r f o r m e d b y c o n n e c t i n g a r e s e r v o i r t o t h e t o p o f t h e c o l u m n a n d m a i n t a i n i n g a p p r o x i m a t e l y 5 p o u n d s p r e s s u r e p e r s q u a r e i n c h . d u r i n g p a c k i n g . P r e s s u r e w a s m a i n t a i n e d w i t h t h e a i d . o f a n a i r p r e s s u r e r e g u l a t o r . T h e p a c k e d c o l u m n w a s w a s h e d w i t h 2 M s o d i u m c h l o r i d e u n t i l t h e e l u a t e h a d a c c e p t a b l y l o w a b s o r b a n c e 0.025). F o l l o w i n g t h e s e w a s h i n g s t e p s , t h e c o l u m n w a s t h e n w a s h e d w i t h t h e s o l u t i o n u s e d t o s t a r t t h e e l u t i o n a n d t h e t R N A ( d i s s o l v e d i n t h e l a t t e r s o l u t i o n ) w a s a p p l i e d t o t h e c o l u m n a n d r i n s e d i n . A f t e r a b r i e f w a s h i n g w i t h t h e s o l u t i o n u s e d t o e q u i l i b r a t e t h e c o l u m n , p o s i t i v e l i n e a r g r a d i e n t s o f s o d i u m c h l o r i d e w e r e a p p l i e d i n t h e u s u a l m a n n e r . F o r s t e p - w i s e c h r o m a t o g r a p h y o n B D - c e l 1 u 1 o s e , t h e w a s h i n g p r o c e d u r e was performed w i t h 1 M o r 2 M sodium c h l o r i d e c o n t a i n i n g 19% (v/v) e t h a n o l u n t i l the e l u a t e showed a c c e p t a b l y low a b s o r b a n c e . The column was then unpacked and the washed BD-c e l l u l o s e made i n t o a s l u r r y w i t h 2 M sodium c h l o r i d e c o n t a i n i n g no e t h a n o l and repacked as above. The repacked columns were then t r e a t e d as d e s c r i b e d above. S t e p - w i s e chromatography on BD-cel l u l o s e (30 - mesh s i z e ) i s d e s c r i b e d i n Step I I . (3) S o l u t i o n s l - I V . Each s o l u t i o n c o n t a i n e d 10 mM magnesium c h l o r i d e and 10 mM a c e t i c a c i d (pH 4.5 by a d d i t i o n o f NaOH). In a d d i t i o n , s o l u t i o n s I -1V had the f o l l o w i n g a d d i t i v e s : s o l u t i o n I, 0.3 M sodium c h l o r i d e ; s o l u t i o n I I , 1.0 M sodium c h l o r i d e ; s o l u t i o n I I I , 1.0 M sodium c h l o r i d e , 4.1% e t h a n o l ; s o l u t i o n IV, 1.5 M sodium c h l o r i d e , 19% e t h a n o l . (4) P r e p a r a t i o n o f G l y c y l - tRNA S y n t h e t a s e . T h i s p r o c e d u r e i s . a m o d i f i c a t i o n o f the method o f L a z a r u s e_t aj_. (146) used by M i l l w a r d ( 1 4 7 ) . Two pounds o f F l e i s c h m a n ' s y e a s t cake ( S t a n d a r d Brands L t d . ) were crumpled i n t o two l i t e r s o f t o l u e n e c o n t a i n i n g e x c e s s dry i c e ( d u r i n g the a d d i t i o n o f y e a s t and f o r one hour a f t e r the a d d i t i o n o f y e a s t an e x c e s s o f d r y i c e was m a i n t a i n e d i n the t o l u e n e ) . A f t e r one hour, the t o l u e n e was d e c a n t e d and the r e s i d u e a l l o w e d t o s t a n d f o r one hour a t room temperature, t o remove e x c e s s d r y i c e . The p r e p a r a t i o n was s t o r e d a t 3"5° f o r 16 hours a f t e r which e x c e s s t o l u e n e was d e c a n t e d . To the r e s i d u e 200 ml o f 1 M T r i s - H C l , pH 8.0, b u f f e r ( c h i l l e d t o 3~5°) was added. A f t e r an a d d i t i o n a l 23 hours a t 3"5° the pH o f the p a s t e was brought t o 7-5 by a d d i t i o n o f kO ml o f 1 M T r i s - H C l , pH 8.0, and the p a s t e was c e n t r i f u g e d (800 x g_ f o r 40 minutes a t 3-5°) • For a l l o p e r a t i o n s d e s c r i b e d below, the enzyme p r e p a r a t i o n was m a i n t a i n e d i n i c e - b a t h s u n l e s s noted o t h e r w i s e . The t o l u e n e -foam l a y e r o v e r the c l e a r aqueous l a y e r was removed by a s p i r a t i o n . To the combined aqueous l a y e r s (312 ml t o t a l ) was added 2 g s t r e p t o m y c i n s u l f a t e per 100 ml o f s o l u t i o n . A d d i t i o n was over a 20-minute p e r i o d w i t h f r e q u e n t s t i r r i n g . The p r e c i p i t a t e w h i c h formed a f t e r 30 minutes was c o l l e c t e d by c e n t r i f u g a t i o n as above and d i s c a r d e d . To each 100 ml o f s u p e r n a t a n t s o l u t i o n was added 25.4 g o f ammonium s u l f a t e . F o l l o w i n g a d d i t i o n o f ammonium s u l f a t e (30 m i n ) , t he p r e c i p i t a t e which formed a f t e r kS minutes was c o l l e c t e d by c e n t r i f u g a t i o n and d i s c a r d e d . To each 100 ml o f s u p e r n a t a n t s o l u t i o n a f u r t h e r 17-0 g ammonium s u l f a t e was added e x a c t l y as d e s c r i b e d above. The p r e c i p i t a t e formed was c o l l e c t e d and s t o r e d as a p e l l e t a t -20° u n t i l needed. Immediately b e f o r e u s e , a p o r t i o n o f t h i s m a t e r i a l was d i s s o l v e d i n b u f f e r I (10 mM p o t a s s i u m phosphate (pH-7-5), 1 mM m e r c a p t o e t h a n o l , and 0.1 mM EDTA c o n t a i n i n g k0% g l y c e r o l by volume) and d e s a l t e d by chromatography on a Sephadex G-25 column e q u i l i b r a t e d i n the b u f f e r I. T h i s i s r e f e r r e d t o as s t a g e I p u r i f i c a t i o n . 4a. Chromatography o f G l y c y l - t R N A S y n t h e t a s e P r e p a r a t i o n s on H y d r o x y l a p a t i t e . In some e x p e r i m e n t s , c r u d e s y n t h e t a s e p r e p a r a t i o n s were chromatographed on h y d r o x y l a p a t i t e . The m a t e r i a l from the Sephadex G-25 column was loaded o n t o a column (26 x 5 cm) o f h y d r o x y l a p a t i t e ( B i o - G e l HTP) e q u i l i b r a t e d w i t h b u f f e r I. A g r a d i e n t o f p o t a s s i u m phosphate (1.5 l i t e r b u f f e r I t o 1.5 l i t e r o f b u f f e r l l ) ( b u f f e r II c o n t a i n e d a l l components o f b u f f e r I, e x c e p t p o t a s s i u m phosphate was 0.3 M) was a p p l i e d a t 3 -4° and f r a c t i o n s c o l l e c t e d and assayed f o r v a r i o u s aminoacyl-tRNA s y n t h e t a s e a c t i v i t i e s . F r a c t i o n s c o n t a i n i n g g l y c y l - t R N A s y n t h e t a s e a c t i v i t y were po o l e d and the combined f r a c t i o n s a r e r e f e r r e d t o as enzyme p u r i f i e d t o s t a g e I I . (5) Assays f o r G l y c i n e - t R N A A c c e p t o r A c t i v i t y . Two p r o c e d u r e s were used. Method I was a m o d i f i c a t i o n of the p r o c e d u r e g i v e n i n r e f e r e n c e 126. Column e l u a t e (one volume) was added t o a s o l u t i o n (one volume) made by c o m b i n i n g equal volumes of w a t e r , s t a g e 'I e n z y m e ( c o n t a i n i n g g l y c e r o l ) and r a d i o a c t i v e g l y c i n e mix. The r a d i o a c t i v e mix c o n t a i n e d per ml: 66 ymoles magnesium c h l o r i d e , 140 ymoles p o t a s s i u m c h l o r i d e , 500 ymoles sodium c a c o d y l a t e (pH 7-3), 2 ymoles EDTA, 6.6 ymoles 14 / \ Na -ATP and 1 ymole C - g l y c i n e (10 y C i ) . F o l l o w i n g i n c u b a t i o n ' (at room t e m p e r a t u r e , u n l e s s s t a t e d o t h e r w i s e ) a sample (usua11y 50 y 1) was w ithdrawn and a p p l i e d t o a f i 1 t e r paper d i s k (Whatman 1, 2.4 cm d i a m e t e r ) and washed a t 0° f o r a minimum o f 10 m i nutes w i t h o c c a s i o n a l s t i r r i n g i n each o f the f o l l o w i n g b a t h s : two c o n s e c u t i v e baths o f 10% (w/v) o f t r i c h l o r o a c e t i c a c i d , one of.3 5 % (v/v) e t h a n o l and one o f d i e t h y l e t h e r . Each bath c o n t a i n e d a t l e a s t 10 ml s o l v e n t per paper d i s k e x c e p t f o r the d i e t h y l e t h e r bath which c o n t a i n e d a p p r o x i m a t e l y 5 ml s o l v e n t per d i s k . The d i s k s were f i n a l l y s p r e a d on g l a s s p l a t e s and d r i e d w i t h the a i d o f a heat lamp. The r a d i o a c t i v i t y was d e t e r m i n e d i n a s c i n t i l l a t i o n s p e c t r o m e t e r by s u s p e n d i n g t h e d i s k s i n 5 ml o f s c i n t i l l a t i o n f l u i d c o n t a i n i n g 3 g o f PPO and 0.3 g o f d i m e t h y l P0P0P per l i t e r o f t o l u e n e . Method 2 was based on the p r o c e d u r e d e s c r i b e d i n r e f e r e n c e 148. The amino a c i d a c c e p t o r a s s a y s were performed on Whatman 3 mm, 2.4 cm d i a m e t e r paper d i s k s . A l i q u o t s (100 y l ) from column f r a c t i o n s were added t o . i n d i v i d u a 1 d i s k s . The d i s k s were a l l o w e d t o d r y u n t i l the wet sheen had d i s a p p e a r e d and were then t r a n s f e r r e d i n t o a v e s s e l c o n t a i n i n g c o l d 75% (v/v) e t h a n o l and 30 mM p o t a s s i u m c h l o r i d e . The d i s k s were washed f o r about 10 m i nutes w i t h o c c a s i o n a l s t i r r i n g . The s o l v e n t was d i s c a r d e d and t h e d i s k s were washed once more w i t h c o l d 75% (v/v) e t h a n o l and 30 mM p o t a s s i u m c h l o r i d e . Excess s o l v e n t was then removed and the d i s k s were d r i e d ( s l o w l y ) under a heat lamp. The d r y d i s k s were supported on s t a i n l e s s s t e e l p ins anchored in a styrofoam b l o c k . A 100 u l a l i q u o t of a s o l u t i o n made by combining equal volumes of stage l : enzyme, water and r a d i o a c t i v e g l y c i n e mix (which conta ined the same c o n c e n t r a t i o n s of components as the r a d i o -a c t i v e g l y c i n e mix desc r ibed f o r Method l) was added to the suspended papers so that the enzyme-amino a c i d mix was un i fo rmly d i s t r i b u t e d . The styrofoam b lock was then placed in a g l a s s box l i n e d wi th moist paper towe ls . The g l a s s box was sealed to mainta in a humid atmosphere dur ing the incubat ion per iod of 30 min . The d i s k s were then removed, washed w i th TCA, ethanol and d i e t h y l e t h e r , d r i e d and the T C A - i n s o l u b l e r a d i o a c t i v i t y determined e x a c t l y as d e t a i l e d f o r Method I. (6) Determinat ion of Maximum Charging of tRNA w i th Glyc i ne. The k i n e t i c s of charg ing of g l y c i n e to smal l sample of crude tRNA by enzyme prepared as in Step h was i n v e s t i g a t e d . Leve ls of enzyme, amino a c i d , magnesium ion and ATP a l l o w i n g maximum charg ing of tRNA in 10 min at 30° were determined. Detai of a t y p i c a l experiment are prov ided in the legend to F igure k. 1 2 (7) Charging of C - g l y c i n e to tRNA on Large S c a l e . 1 2 Concent rat ions of c o - f a c t o r s and C-amino a c i d ( sca led up d i r e c t l y from c o n d i t i o n s found in s e c t i o n 6) f o r o p t i m a ] c h a r g i n g o f 5 g of c r u d e tRNA i n 150 ml t o t a l volume were: M g C l 2 , 20 mM; KC1, 40 mM; sodium c a c o d y l a t e , 170 mM, pH 7.4; EDTA, 0.3 mM; m e r c a p t o e t h a n o l , 0.4 mM; ATP, 4.4 mM; g l y c i n e , 3.3 mM; f i n a l pH 7-35. F i v e grams tRNA was added t o t h i s s o l u t i o n and the m i x t u r e a l l o w e d t o warm up t o 30°. Stage I enzyme ( d e s a l t e d i m m e d i a t e l y b e f o r e use) was added and the m i x t u r e s t i r r e d w h i l e i n c u b a t i n g a t 30° f o r 10 min. The amount o f enzyme added was d e t e r m i n e d by s c a l i n g up the number o f u n i t s r e q u i r e d from Step 6. (8) C h a r g i n g o f ^ C - g l y c i n e t o tRNA. To a s o l u t i o n (200 y l ) c o n t a i n i n g the same c o n c e n t r a t i o n s o f components as s e c t i o n 7 ( w i t h o m i s s i o n o f tRNA and g l y c i n e ) was added 200 y l of u n i f o r m l y l a b e l l e d ^ C - g l y c i n e (0.5 mCi and 0.324 mg i n 10 mM H C l ) , 200 y l 10 mM NaOH and 200 y l H 20 c o n t a i n i n g 20 mg tRNA. An a p p r o p r i a t e l e v e l o f enzyme was added and the m i x t u r e was i n c u b a t e d 10 min a t 30°. (9) T e r m i n a t i o n of the R e a c t i o n and Phenol E x t r a c t i o n . The r e a c t i o n s i n s t e p s 7 and 8 were stopped by a d d i t i o n o f i c e c o l d 4.0 mM sodium f o r m a t e b u f f e r (pH 4.0) u n t i l t he pH 1 2 was 4.5 (35 ml was r e q u i r e d f o r C-sample, 400 y l was used f o r ^ C - s a m p l e ) . Phenol (180 ml and 1 ml f o r the ^ C - and p r e p a r a t i o n s , r e s p e c t i v e l y ) was added w i t h v i g o r o u s s t i r r i n g a n d t h e m i x t u r e s l e f t a t r o o m t e m p e r a t u r e f o r 30 m i n . T h e m i x t u r e s w e r e c e n t r i f u g e d , t h e a q u e o u s l a y e r s w e r e r e m o v e d a n d t h e p h e n o l l a y e r s w e r e r e - e x t r a c t e d w i t h s o l u t i o n I (50% o f o r i g i n a l v o l u m e 1 2 o f a q u e o u s l a y e r ) . T h e a q u e o u s l a y e r s f r o m b o t h C - g 1 y c y 1 - t R N A 1 4 a n d C - g 1 y c y 1 - t R N A p r e p a r a t i o n s w e r e c o m b i n e d a n d p r e c i p i t a t e d w i t h 2 . 5 v o l u m e s o f 95% e t h a n o l . T h e p r e c i p i t a t e w h i c h f o r m e d a f t e r t h e s o l u t i o n w a s s t o r e d o v e r n i g h t a t -20° w a s c o l l e c t e d b y c e n t r i f u g a t i o n a n d w a s h e d w e l l w i t h 1 l i t r e o f c o l d w a s h I (270 m l 1 M N a C l , 30 m l 0.1 M s o d i u m a c e t a t e , p H 4 . 5 , a n d 600 m l o f 95% e t h a n o l ) f o l l o w e d b y 0 . 5 l i t r e o f w a s h II (5 m l 1 M M g C l , 5 m l 0.1 M s o d i u m a c e t a t e , p H 4 . 5 , a n d 490 m l 95% e t h a n o l ) . T h e t R N A w a s c o l l e c t e d b y c e n t r i f u g a t i o n a n d w e l l d r a i n e d a t e a c h s t e p t o r e m o v e a s m u c h e t h a n o l a s p o s s i b l e . (10) N a p h t h o x y a c e t y 1 a t i o n o f C o m b i n e d ' ^ C - a n d " * * C -G l y c y l - t R N A P r e p a r a t i o n s . T h e p r o d u c t f r o m s t e p . 9 w a s d i s s o l v e d i n 400 m l o f c o l d (3"4°) 0 .10 M t r i e t h a n o l a m i n e - h y d r o c h l o r i d e c o n t a i n i n g 10 mM m a g n e s i u m c h l o r i d e ( p H 4 .5) a n d t h e s o l u t i o n w a s m a i n t a i n e d a t 0 ° . T o 40 m l o f t e t r a h y d r o f u r a n ( d r i e d b y p a s s a g e t h r o u g h a c o l u m n o f d r y a l u m i n a i m m e d i a t e l y b e f o r e u s e ) w a s a d d e d 1.8 g o f N _ - h y d r o x y -s u c c i n i m i d e e s t e r o f 2 - n a p h t h o x y a c e t i c a c i d . F i f t e e n m l o f t h e t e t r a h y d r o f u r a n s o l u t i o n w a s a d d e d t o t h e t R N A s o l u t i o n w i t h v i g o r o u s s t i r r i n g , t h e p H o f t h e s o l u t i o n w a s b r o u g h t t o 8 .0 b y d r o p w i s e a d d i t i o n o f 1 M s o d i u m h y d r o x i d e , a n d t h e r e m a i n d e r of the te t rahyd ro fu ran s o l u t i o n was added dropwise over 5 minutes . The r e a c t i o n was a l lowed to proceed f o r another 1 0 minutes (the pH was mainta ined at 8 .0 throughout t h i s per iod) when the pH was brought q u i c k l y to 4.5 by a d d i t i o n of g l a c i a l a c e t i c a c i d . Two volumes of 95% ethanol were.added. The p r e c i p i t a t e which formed a f t e r the s o l u t i o n was s tored overn ight at - 2 0 ° was c o l l e c t e d by c e n t r i f u g a t i o n , washed w i t h a l i t e r of wash I I and f i n a l l y d i s s o l v e d in 7 0 0 ml of s o l u t i o n I . ( 1 1 ) Removal of N H 2 - n a p h t h o x y a c e t y 1 g l y c y 1 - t R N A from the Remainder of tRNA. The tRNA s o l u t i o n above was loaded onto a column ( 1 1 0 x 3 cm) of B D - c e l l u l o s e (30 mesh) e q u i l i b r a t e d in s o l u t i o n I . When load ing was c o m p l e t e d , . t h e column was washed w i t h s o l u t i o n I . When the A ^ ^ Q of the e l u a t e approached z e r o , washing w i t h s o l u t i o n I was stopped and s o l u t i o n I I was a p p l i e d . As b e f o r e , s o l u t i o n I I washing was d i s c o n t i n u e d when the A ^ Q of the e l u a t e approached zero and s o l u t i o n I I I was a p p l i e d . F i n a l l y , s o l u t i o n I V was a p p l i e d as b e f o r e . Flow rates were approx imate ly 7 ml per minute and 2 0 ml f r a c t i o n s were c o l l e c t e d and examined f o r A ^ ^ Q and r a d i o a c t i v i t y . For de te rminat ion of r a d i o a c t i v i t y , a l i q u o t s were added to B r a y ' s s o l u t i o n fo r s c i n t i l l a t i o n count ing ( 1 4 9 ) . (12) Removal o f the N^-2-naphthoxyacety 1 g 1 yc i ne Group from tRNA. tRNA (7900 A 2 £ Q u n i t s ) e l u t e d from B D - c e l l u l o s e w i t h s o l u t i o n IV was c o l l e c t e d by p r e c i p i t a t i o n and c e n t r i f u g a t i o n and d i s s o l v e d i n 120 ml M T r i s - H C l (pH 8.5)- The s o l u t i o n was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 2.5 ho u r s . G l a c i a l a c e t i c a c i d was used t o b r i n g t h e pH t o 4.5, the a c i d i f i e d s o l u t i o n was made 10 mM i n magnesium c h l o r i d e and 2.5 volumes o f 35% e t h a n o l were added. The tRNA was c o l l e c t e d and washed as d e s c r i b e d above. (13) Chromatography o f P u r i f i e d G l y c i n e tRNAs on B D - C e l l u l o s e . The p r o c e d u r e f o l l o w e d was as d e s c r i b e d i n the c h r o m a t o g r a p h i c methods s e c t i o n above (Step 2 ) . D e t a i l s f o r i n d i v i d u a l e x p e r i m e n t s a r e p r o v i d e d i n the legends t o the a p p r o p r i a t e f i g u r e s . (14) Chromatography o f P u r i f i e d G l y c i n e tRNAs on DEAE-Sephadex A-25. The p r o c e d u r e o f M i y a z a k i et_ aj_. (150) was f o l l o w e d . DEAE-Sephadex A-25 was a l l o w e d t o s w e l l i n water (1-2 da y s , room t e m p e r a t u r e ) and e q u i l i b r a t e d w i t h 2 mM ammonium s u l f a t e , 20 mM p o t a s s i u m a c e t a t e (pH 5-3) c o n t a i n i n g 5% (v/v) d i m e t h y l f o r m a m i d e . E q u i l i b r a t i o n was achieved by e x t e n s i v e washing of the exchanger w i th the l a t t e r s o l u t i o n on a Buchner f u n n e l . Columns were packed as desc r ibed f o r the p r e p a r a t i o n of B D - c e l l u l o s e (50 mesh) columns. tRNA was d i s s o l v e d in the s t a r t i n g e luent at a c o n c e n t r a t i o n of approx imate ly 2 mg/ml. The s o l u t i o n was loaded onto the columns which were p r e v i o u s l y e q u i l i b r a t e d w i th the s t a r t i n g e l u e n t . Flow rates were maintained at 0.3 ml/min per 2 cm column c r o s s - s e c t i o n a l a r e a . G e n e r a l l y , the t o t a l g rad ien t volume employed was 20 column volumes. D e t a i l s fo r i n d i v i d u a l experiments are supp l ied in the legends to the cor responding f i gures . RESULTS AND DISCUSSION A p r e p a r a t i o n of tRNA from a g iven source may c o n t a i n as many as f i f t y or more components. At l e a s t one tRNA e x i s t s f o r each of the amino a c i d s involved in p r o t e i n s y n t h e s i s . More g e n e r a l l y , a g iven amino a c i d possesses a tRNA f a m i l y of two or more members. The la rge number of tRNA spec ies and the apparent l y s u b t l e d i f f e r e n c e s between them make, t h e i r s e p a r a t i o n d i f f i c u l t . A v a i l a b l e techniques which ach ieve r e s o l u t i o n of tRNA p repara t ions in to la rge numbers of i soaccepto rs f r e q u e n t l y employ a n i o n -exchangers w i th l i p o p h i l i c c h a r a c t e r i s t i c s . Methy lated albumin on k i e s e l g u h r , reverse phase chromatography, and B D - c e l l u l o s e are members of t h i s c l a s s of m a t e r i a l s . One member in p a r t i c u l a r - B D - c e l l u l o s e - achieves s i g n i f i c a n t r e s o l u t i o n of tRNAs from a number of sources . The e l u t i o n p r o f i l e of tRNA from Brewer 's yeast chromatographed on B D - c e l l u l o s e is shown in F igure 3, upper frame. (This f i g u r e is taken from re ference (126) and is not work done by the au thor . ) A g rad ien t of sodium c h l o r i d e (0.4 M to 1.0 M in the presence of 0.01 M magnesium c h l o r i d e ) e l u t e s most of the a p p l i e d m a t e r i a l . Ex tens ive r e s o l u t i o n of va r ious tRNA f a m i l i e s is seen by an examinat ion of the lower frames of F igure 3. These show r e s u l t s FIGURE 3 (from reference 126) Chromatography of Brewer's Yeast tRNA on B D - c e l l u l o s e F r a c t i o n a t i o n of 5 g (70 x 10 A 2 ^ Q u n i t s ) of tRNA from Brewer 's yeast (Boehringer) was performed on a column (3-2 x 110 cm) of BD-ce l l u l o s e . The sample was a p p l i e d in 500 ml of 0.45 M sodium c h l o r i d e and 0.01 M magnesium s u l f a t e and e l u t e d w i t h the i n d i c a t e d (dashed l i n e ) g r a d i e n t of sodium c h l o r i d e (a t o t a l of 10 1.), c o n t a i n i n g 10 mM magnesium s u l f a t e . F r a c t i o n s were 20 ml/15 min . At the end of the g rad ien t (tube 497) e l u t i o n was cont inued w i th 1.0 M sodium chl o r i de - 10 mM mag nesi um s u l f a t e c o n t a i n i n g 10% (v/v) 2-me thoxye th ano 1. o f a s s a y s f o r amino a c i d a c c e p t o r a c t i v i t i e s o f t h e v a r i o u s f r a c t i o n s . The b a s i s f o r t h i s r e s o l u t i o n has been d e s c r i b e d i n some d e t a i 1 (126, 147). Not a l l tRNA a p p l i e d t o B D - c e l l u l o s e i s e l u t e d by s a l t g r a d i e n t . 1 T r a n s f e r RNA p o s s e s s i n g a c c e p t o r a c t i v i t y f o r p h e n y l a l a n i n e e l u t e s o n l y i f o r g a n i c compounds l i k e u r e a , d i m e t h y l formamide o r a l i p h a t i c a l c o h o l s a r e added t o the s a l t Phe s o l u t i o n . The b e h a v i o u r o b s e r v e d f o r tRNA ( y e a s t ) appears t o be due t o the p r e s e n c e o f the u n u s u a l l y h y d r o p h o b i c base Y whi c h i s l o c a t e d next t o t h e a n t i c o d o n o f t h i s tRNA (151). A l t e r e d a f f i n i t y f o r B D - c e l l u l o s e i s ob s e r v e d i f c e r t a i n tRNAs a r e chromatographed on t h i s exchanger i n t h e i r a m i n o a c y l a t e d s t a t e . For example, t y r o s y 1 - t R N A ^ y r and tryptophanyl-tRNA^'"' 3 i n t e r a c t s u f f i c i e n t l y s t r o n g l y w i t h B D - c e l l u l o s e t h a t development o f a f a c i l e p r o c e d u r e f o r t h e i r i s o l a t i o n was p o s s i b l e (139, 141). Phe S i m i l a r l y , a l t e r e d a f f i n i t y between non-charged and charged tRNA a l l o w e d L i t t t o o b t a i n t h i s s p e c i e s i n v e r y pure form ( 1 4 0 ) . The p r e v i o u s d i s c u s s i o n i n d i c a t e s t h a t the p r e s e n c e o f a h y d r o p h o b i c m o i e t y a t a s u i t a b 1e p o s i t i o n i n a tRNA m o l e c u l e has a d i s p r o p o r t i o n a t e l y l a r g e i n f l u e n c e on the b e h a v i o u r o f t h a t s p e c i e s d u r i n g chromatography on B D - c e l l u l o s e . The r e m a i n d e r . o f t h i s t h e s i s r e l a t e s , i n two p a r t s , a t t e m p t s t o x T h a t f r a c t i o n o f a tRNA p r e p a r a t i o n w h i c h i s e l u t e d from B D - c e l l u l o s e w i t h 1 M sodium c h l o r i d e c o n t a i n i n g k.7% e t h a n o l , but not w i t h 1 M sodium c h l o r i d e a l o n e i s r e f e r r e d t o as the e t h a n o l - f r a c t i o n . U n l e s s s t a t e d o t h e r w i s e , a l l tRNA p r e p a r a t i o n s used i n t h i s s t u d y were f r e e d o f e t h a n o l - f r a c t i o n p r i o r t o use. e x p l o i t t h i s o b s e r v a t i o n . In p r i n c i p l e , the e x p l o i t a t i o n r e l i e s on the s p e c i f i c i n t r o d u c t i o n o f a h y d r o p h o b i c group o n t o a p a r t i c u l a r p o l y -n u c l e o t i d e which c o n f e r s a s u f f i c i e n t l y t e n a c i o u s i n t e r a c t i o n between the m o d i f i e d polymer and B D - c e l l u l o s e t o a l l o w p h y s i c a l s e p a r a t i o n of the m o d i f i e d from u n m o d i f i e d polymers w i t h s i m p l e c h r o m a t o g r a p h i c s t e p s . The f i r s t p a r t r e l a t e s the a u t h o r ' s p a r t i n e n l a r g i n g on a g e n e r a l method f o r i s o l a t i o n o f s p e c i f i c tRNAs d e v e l o p e d i n t h i s l a b o r a t o r y . The o t h e r , p r e s e n t e d i n t h e second p a r t o f t h i s t h e s i s , d e s c r i b e s p r o g r e s s i n development o f a novel s e q u e n c i n g procedu r e . G e n e r a l P r o c e d u r e f o r t h e I s o l a t i o n o f t R N A G 1 y a n d tRNA^1Y_ The p r o c e d u r e f o r the i s o l a t i o n o f s p e c i f i c tRNAs i s shown d i a g r a m m a t i c a l l y i n F i g u r e h. In t h i s c a s e , t h e o b j e c t i s t o i s o l a t e the i s o a c c e p t i n g s p e c i e s o f tRNA f o r g l y c i n e . G l y c y l - t R N A s y n t h e t a s e i s used t o e s t e r i f y s p e c i f i c a 1 1 y i t s c o g n a t e tRNAs. T h i s f i r s t s t e p , termed the c h a r g i n g - s t e p , e x p l o i t s the r i g o r o u s s e l e c t i v i t y o f the aminoacyl-tRNA s y n t h e t a s e s . Those tRNAs l a c k i n g i n t a c t 3'"hydroxy 1 t e r m i n a l s w i l l not be charged u n l e s s r e p a i r enzyme i s p r e s e n t ( 1 5 2 ) ; dimers o f tRNA may be charged (142 ) . E s t e r i f i c a t ion i s a c h i e v e d w i t h o u t c o n c o m i t a n t e s t e r i f i c a t i o n o f o t h e r amino a c i d s t o t h e i r a c c e p t o r s by s u p p l y i n g pure amino Procedure for the Specific Introduction of the 2-Naphthoxyacety1• Group Onto the 3'-Terminal of tRNA G l y. tRNA + glycine ATP, glycyl-tRNA synthetase tRNA + tRNA-0-C-CH2-NH2 O C, pH8.0, 10 minutes O I H ? tRNA + tRNA-0-C-CH2-N-C-CH2-O-a c i d a n d p a r t i a l l y p u r i f i e d e n z y m e . A m i n o a c i d s p o s s e s s a n a - a m i n o g r o u p w h e n e s t e r i f i e d t o t h e i r t R N A s a n d i t i s t h e a - a m i n o g r o u p w h i c h a l l o w s t h e s p e c i f i c i n t r o d u c t i o n o f a h y d r o p h o b i c m o i e t y i n t o t R N A s b y r e a c t i o n w i t h t h e N _ - h y d r o x y -s u c c i n i m i d e e s t e r o f 2 - n a p h t h o x y a c e t i c a c i d . T h i s r e a c t i o n i s r e f e r r e d t o a s t h e d e r i v a t i z a t i o n s t e p . T h e p r o d u c t , a - _ N -n a p h t h o x y a c e t y 1 g l y c y 1 - t R N A ^ ^ , i n t h i s c a s e , b i n d s s t r o n g l y e n o u g h t o B D - c e l l u l o s e t o a l l o w p h y s i c a l s e p a r a t i o n o f t h i s m a t e r i a l f r o m i t s u n m o d i f i e d c o u n t e r p a r t s i n s i m p l e c h r o m a t o g r a p h i c s t e p s . T h e d e r i v a t i z a t i o n r e a c t i o n p r o c e e d s p o o r l y f o r s e r y l - t R N A (152) a n d t h r e o n y 1 - t R N A (154). T h e p r e d i c t i o n t h a t l y s y l - t R N A m a y r e a c t a t b o t h a - a m i n o a n d e - a m i n o g r o u p s h a s b e e n c o n f i r m e d (143). I n a l l o t h e r c a s e s t e s t e d , t h e r e a c t i o n p r o c e e d s a s d e s c r i b e d . T h e d e r i v a t i z e d a m i n o a c i d i s r e a d i l y c l e a v e d f r o m t h e p u r i f i e d t R N A b y b r i e f t r e a t m e n t w i t h 1 M T r i s - h y d r o c h 1 o r i c a c i d b u f f e r ( p H 8.5) a t r o o m t e m p e r a t u r e o n c e s e p a r a t i o n h a s b e e n a c h i e v e d . T h e e n r i c h e d t R N A f r a c t i o n m a y t h e n b e c h r o m a t o g r a p h e d o n B D - c e l l u l o s e w i t h g r a d i e n t - e l u t i o n . T h e f i n a l s t e p c o n v e n i e n t l y s e p a r a t e s i s o a c c e p t i n g t R N A s p e c i e s a n d p r o v i d e s f u r t h e r p u r i f i c a t i o n . A m o r e d e t a i l e d d i s c u s s i o n o f t h e p u r i f i c a t i o n p r o c e d u r e f o l l o w s . T h e c h a r g i n g s t e p o f t h e p r o c e d u r e i s p e r f o r m e d u n d e r c o n d i t i o n s o p t i m i z e d f o r y i e l d o f g l y c y l - t R N A . F a c t o r s s u c h as pH, ATP, monovalent ions and magnesium ion c o n c e n t r a t i o n s are op t imized fo r a p a r t i c u l a r l eve l of s u b s t r a t e and enzyme. The c r i t i c a l concentrat ion -dependence that g l y c i n e - t R N A synthetase has on magnesium l e v e l s is shown in F igure 5-That the crude tRNA used in these s t u d i e s does not possess components present in a denatured form (155) is shown in F igure 6. P r e - h e a t i n g of tRNA p r i o r to a d d i t i o n of enzyme d id not inc rease the f i n a l y i e l d of g l y c y l - t R N A . (This treatment reduced the y i e l d fo r the high leve l of tRNA samples , p o s s i b l y due to h y d r o l y s i s of ATP dur ing the heat ing s t e p . ) For the unheated samples the t w o - f o l d h igher tRNA c o n c e n t r a t i o n sample gave e s s e n t i a l l y double the y i e l d of g l y c y l - t R N A compared to the lower tRNA c o n c e n t r a t i o n sample. Th is a l l o w s the c o n c l u s i o n that complete charg ing was being obta ined at the high tRNA l e v e l and was used r o u t i n e l y as a t e s t f o r o p t i m i z a t i o n . On the bas i s of a study l i k e that shown in F igure 6, 1-5 g of tRNA was charged, p h e n o l - e x t r a c t e d and d e r i v a t i z e d and f i n a l l y separated by s tepwise chromatography on B D - c e l l u l o s e . The procedures used were e s s e n t i a l l y as desc r ibed in Steps 7 to 11 of the Experimental S e c t i o n . F igure 7 shows a t y p i c a l s e p a r a t i o n of j^-2 -naphthoxyacety1g1ycy1 -tRNAs from unmodif ied tRNA. The product from the charg ing and d e r i v a t i z i n g steps was loaded onto a B D - c e l l u l o s e column which was developed as descr ibed in the Exper imental S e c t i o n , Step 11. E l u t i o n w i th molar sodium c h l o r i d e FIGURE 5 I n f l u e n c e o f Magnesium L e v e l s on the S y n t h e s i s o f G l y c y l - t R N A . The i n c o r p o r a t i o n o f C - g l y c i n e i n t o tRNA was measured as 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 a s s a y e d by Method I a f t e r 10, 25 and 45 m i n u t e s i n c u b a t i o n a t 22°. Enzyme p u r i f i e d t o s t a g e II as d e s c r i b e d . i n P a r t I, E x p e r i m e n t a l S e c t i o n , Step 4 was added t o 14 a s o l u t i o n w i t h 2.5 yg tRNA, 25 y l C - g l y c i n e m i x t u r e d e s c r i b e d i n P a r t I, E x p e r i m e n t a l S e c t i o n , Step 7 and v a r i o u s l e v e l s o f magnesium c h l o r i d e . For the two p o i n t s t o t h e l e f t o f z e r o magnesium c h l o r i d e , 1 ymole and 2 ymoles ( r e s p e c t i v e l y ) o f EDTA were added per mg tRNA. FIGURE 6 I n f l u e n c e o f P r e - h e a t i n g and o f L e v e l s o f tRNA on F i n a l Y i e l d o f 1 i | C - G l y c y l - t R N A . D u p l i c a t e 5 mg and 10 mg samples of c r u d e tRNA d i s s o l v e d i n 25 o r 50 ul o f H20 ( r e s p e c t i v e l y ) were added t o 50 ] i l of the m i x t u r e d e s c r i b e d i n P a r t I, E x p e r i m e n t a l S e c t i o n , Step 7 ( o m i t t i n g g l y c i n e ) , 50 u l o f ^ - g l y c i n e (20 uC i and 10.09 ymoles per ml) and H20 (when n e c e s s a r y ) t o a f i n a l volume o f 150 u]. Two s o l u t i o n s (a s i n g l e 5 mg and a s i n g l e 10 mg t R N A - c o n t a i n i n g s o l u t i o n ) were heated t o 60° f o r 10 minutes and a l l o w e d t o c o o l t o 30°. Then, 100 y l o f Stage I enzyme was added to each tube. A l i q u o t s (25 u l ) were removed a t the t i m e s shown and the a c i d -p r e c i p i t a b l e ^ C - g l y c i n e d e t e r m i n e d by Method I (® and 5 mg tRNA unheated a n d . h e a t e d , r e s p e c t i v e l y , p r i o r t o a s s a y , 0 and A , 10 mg tRNA unheated and heated r e s p e c t i v e l y , p r i o r t o a s s a y ) . I s o l a t i o n o f N-2-naphthoxyacety1g1ycy1 -tRNA. One g o f tRNA minus e t h a n o l - f r a c t i o n was e s t e r i f i e d w i t h 12 14 C- and C - g l y c i n e by Stage I enzyme and s u b s e q u e n t l y d e r i v a t i z e d to y i e l d N_-2-naphthoxyacety 1 g 1 ycy 1 - tRNA e s s e n t i a l l y as d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n , Steps 7—11. The m i x t u r e was loaded o n t o a column (2.5 x 35 cm) o f B D - c e l l u l o s e i n s o l u t i o n I and t h e column was washed w i t h s o l u t i o n I. At the p o i n t s i n d i c a t e d , s o l u t i o n s I I , I I I and IV were a p p l i e d . S o l i d l i n e : A ^ ^ QJ d o t t e d l i n e : r a d i o a c t i v i t y from * ^ C - g 1 y c i n e . F r a c t i o n s were 20 ml/5 min. FRACTION NUMBER removed the bulk of the a p p l i e d tRNA but l i t t l e r a d i o a c t i v i t y . More unmodif ied tRNA bound n o n - s p e c i f i c a11y was removed by e l u t i o n w i t h 1.0 molar sodium c h l o r i d e c o n t a i n i n g k.1% e t h a n o l . To t h i s stage on ly a t r a c e of r a d i o a c t i v i t y - rep resent ing n o n - d e r i v a t i z e d g l y c y l - t R N A - was e l u t e d . F i n a l l y , that tRNA which was both charged and d e r i v a t i z e d was re leased from the column by e l u t i o n w i t h 1.5 M sodium c h l o r i d e c o n t a i n i n g 19% e t h a n o l . This step e l u t e d approx imate ly 8% of the o r i g i n a l tRNA and g reater than %% of the ' ^ C - g l y c i n e l a b e l . 1 k An examinat ion of the d i s t r i b u t i o n of C - l a b e l in the va r ious f r a c t i o n s in t h i s f i g u r e shows that the d e r i v a t i z a t i o n r e a c t i o n was v i r t u a l l y q u a n t i t a t i v e . That f r a c t i o n e l u t e d by so l ven t IV ( f r a c t i o n IV) c o n t a i n s a l l spec ies of tRNA ab le to accept g l y c i n e as w e l l as some i m p u r i t i e s . The N_-2-naphthoxyacety 1 g 1 yc i ne group was removed and the tRNA recovered as descr ibed in s tep 12 of the Experimental S e c t i o n . The acceptor a c t i v i t y f o r a g iven amino a c i d g e n e r a l l y res ides in more than a s i n g l e spec ies of tRNA. Thus, sub-f r a c t i o n a t i o n of f r a c t i o n IV was necessary . For t h i s purpose, g r a d i e n t chromatography on B D - c e l l u l o s e was used. In crude yeast tRNA, g l y c i n e acceptor a c t i v i t y was found to be the h ighes t f o r any amino a c i d and to be present in two major peaks a f t e r BD-cel1ulose.chromatography (126). F r a c t i o n IV s h o u l d , t h e r e f o r e , c o n t a i n two major components capable of a c c e p t i n g g l y c i n e when chromatographed under the c o n d i t i o n s o f F i g u r e 3- As a n t i c i p a t e d , two g 1 y c i n e - a c c e p t i n g components were found ( F i g u r e 8) f o l l o w i n g c h r o m a t o g r a p h i c r e s o l u t i o n o f f r a c t i o n IV under t h e s e c o n d i t i o n s . The P r°f'' e °f F i g u r e 8 s u g g e s t s t h a t v e r y l i t t l e m a t e r i a l o t h e r than g 1 y c i n e - a c c e p t o r a c t i v i t y was p r e s e n t . The f i r s t peak, termed t R N A ^ y , had a h i g h s p e c i f i c a c c e p t o r a c t i v i t y - 2.1 mmoles g l y c i n e a c c e p t e d per A ^ Q u n i t - w h i l e the second peak, t R N A ^ y , showed somewhat lower s p e c i f i c a c t i v i t y . Thus, t R N A ^ y peak i s p r o b a b l y pure a t t h i s s t a g e w h i l e some c o n t a m i n a t i o n o f t R N A ^ y i s a p p a r e n t . S t u d i e s w i t h Stage I Enzyme. The tRNAs shown i n F i g u r e 8 were i s o l a t e d w i t h s t a g e II enzyme wh i c h i s p a r t i a l l y - p u r i f i e d g l y c y l - t R N A s y n t h e t a s e . F i g u r e 9 shows the A^gQ p r o f i l e and some aminoacyl-tRNA s y n t h e t a s e s p r e s e n t i n a t y p i c a l r e s o l u t i o n o f s t a g e I' enzyme on hyd r o x y -l a p a t i t e . A t t e m p t s t o o b t a i n f u r t h e r p u r i f i c a t i o n o f t h e g l y c y l -tRNA s y n t h e t a s e were hampered by the l a b i l i t y o f the enzyme. The l a b i l i t y o f g l y c y l - t R N A s y n t h e t a s e o f y e a s t has been r e p o r t e d by B l a c k and Hazel (156). I t i s now a p p a r e n t t h a t g l y c y l - t R N A s y n t h e t a s e i s c o l d - s e n s i t i v e and t h a t the p r e s e n c e o f mercapto-e t h a n o l may speed t h e l o s s o f a c t i v i t y on c o l d s t o r a g e (157)-The u n e x p l a i n e d l a b i l i t y o f the enzyme r e q u i r e d f o r the c h a r g i n g -s t e p r e p r e s e n t e d an o b s t a c l e t o development o f l a r g e s c a l e methods FIGURE 8 G1 y Chromatography of tRNA I so la ted w i t h Stage II Enzyme. T rans fe r RNA (1,025 A 2 6 q u n i t s ) obta ined by charg ing 1 g of tRNA minus e t h a n o l - f r a c t i o n w i th Stage II enzyme and subsequent ly d e r i v a t i z i n g and p u r i f y i n g t R N A ^ ' y e s s e n t i a l l y accord ing to Steps 7~12 of the Experimental S e c t i o n was chromatographed on a column (1.2 x 98 cm) of B D - c e l l u l o s e e lu ted w i t h the i n d i c a t e d (dashed l i n e ) g rad ien t of sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e ( t o t a l of 1 1.). Flow r a t e , 1 ml/1 m i n ; 18 min f r a c t i o n s . At the i n d i c a t e d p o i n t , e l u t i o n was cont inued w i t h 1.5 M sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e in ]k% (v/v) e t h a n o l . S o l i d l i n e : A _ / n ; dot ted l i n e : acceptor a c t i v i t y f o r g l y c i n e . Chromatography of Stage I Enzyme on H y d r o x y l a p a t i t e Stage I enzyme was prepared , d e s a l t e d and f i n a l l y chromatographed on a column (26 x 5 cm) of h y d r o x y l a p a t i t e (B io -Ge l HTP) as descr ibed in Exper imental S e c t i o n , Steps 4 and 4a. E l u t i o n was achieved wi th a g rad ient of potassium phosphate (3 1. t o t a l ) from 10 mM to 0.3 M ( c o n t a i n i n g the o ther i n g r e d i e n t s desc r ibed in Step 4a). L i g h t s o l i d l i n e : c o n d u c t i v i t y r e a d i n g ; heavy s o l i d l i n e : ^280'' d o t t e d , dashed and dot -dashed l i n e s : a c t i v i t i e s fo r the aminoacyl - tRNA synthetases noted . F r a c t i o n s were 20 ml/20 min . CO LA FRACTION NUMBER f o r p u r i f i c a t i o n of tRNA^ y and tRNA^ . However, the enzyme was more s t a b l e in the crude s t a t e than in the p a r t i a l l y p u r i f i e d s t a t e . It was reasoned that i f the enzyme p u r i f i e d on ly to stage I would g i ve s u f f i c i e n t l y pure products then an i n v e s t i g a t i o n of methods to s t a b i l i z e the p a r t i a l l y p u r i f i e d enzyme would be unnecessary . T h e r e f o r e , g l y c i n e - t R N A synthetase p u r i f i e d to stage I was used to charge crude tRNA (1 g) w i th g l y c i n e . Stage I enzyme is a comparat i ve l y impure p r e p a r a t i o n . It was necessary to take p recaut ions to avoid e s t e r i f i c a t i o n of tRNAs other than g l y c i n e - t R N A w i t h amino ac ids which might a r i s e from p r o t e o l y s i s in the crude enzyme p r e p a r a t i o n . A smal l p o r t i o n of stage I enzyme was d e s a l t e d and used to determine opt imal c o n d i t i o n s fo r e s t e r i f i c a t ion of g l y c i n e to tRNA. The r e a c t i o n was opt imized to a l l o w maximum charg ing w i t h i n 10 minutes . For la rge s c a l e c h a r g i n g , the remainder of the same g l y c y l - t R N A synthetase p r e p a r a t i o n was d e s a l t e d and the amount of enzyme needed was c a l c u l a t e d by comparing A^gQ u n i t s from the s m a l l - s c a l e s tudy . In t h i s way, the enzyme was used in less than 1 hour of complet ion of the Sephadex G-25 chromatography s t e p . In the shor t i n t e r v a l between d e s a l t i n g and use , the enzyme was s tored at -20° to i n h i b i t p r o t e o l y s i s . Th is procedure proved s a t i s f a c t o r y and has been used r o u t i n e l y . The charged tRNA was d e r i v a t i z e d , p u r i f i e d , hydro lyzed and s u b f r a c t i o n a t e d as b e f o r e . The r e s u l t s are shown ,in F igure 1 0 and a re s i m i l a r to those obta ined us ing Stage II enzyme wi th one e x c e p t i o n . The A ^ ^ Q p r o f i l e revea ls a t h i r d peak w i t h no acceptor a c t i v i t y f o r g l y c i n e . However, the s p e c i f i c acceptor a c t i v i t y fo r the two major peaks e s s e n t i a l l y mimics va lues seen in F igure 8 . The s p e c i f i c acceptor a c t i v i t y found fo r the g l y c i n e - t R N A s prepared w i t h enzyme p u r i f i e d to Stage I and Stage II were s i m i l a r . Crude enzyme p repara t ions have been found, however, to r a p i d l y dest roy c e r t a i n amino a c i d s such as a r g i n i n e , p r o l i n e and g l y c i n e . To avoid i n c l u s i o n of such a c t i v i t i e s the range of ammonium s u l f a t e f r a c t i o n a t i o n used to p rov ide Stage I enzyme was reduced from the 0 to S0% s a t u r a t i o n range (which is r o u t i n e l y used to o b t a i n crude aminoacyl - tRNA synthetases) to a 2 5 to 60% s a t u r a t i o n range. Th is g r e a t l y reduced p r o t e i n y i e l d and removed the g l y c i n e - d e s t r o y i n g a c t i v i t y w i thout i n f l u e n c i n g the l e v e l of g l y c i n e e s t e r i f i c a t i o n to crude tRNA. S tud ies w i th "Homologous" tRNA and G l ycy l - tRNA Synthetase . A hetero logous system may have been employed in t h i s work. A b a k e r ' s yeast enzyme p r e p a r a t i o n is used to s e l e c t brewer 's yeast t R N A s . 2 Both yeasts are commercial p repara t ions and l i t t l e i s known of the genet i c p u r i t y o f . y e a s t (or yeasts ) i n v o l v e d . It was, t h e r e f o r e , d e s i r a b l e to i s o l a t e tRNA us ing enzyme prepared from a "homologous" source - that is to i s o l a t e brewer 's yeast ' 2 B a k e r ' s yeast t R N A ^ e r and German brewer 's yeast t R N A ^ e r seem to be i d e n t i c a l ( 4 1 , 1 5 8 ) and both yeasts are Saccharomyces c e r e v i s i a e . The b a k e r ' s yeast used in t h i s labora to ry and the brewer 's yeast used by Boehr inger and Soehne to prepare the tRNA used in t h i s study a l s o appear to be Saccharomyces c e r e v i s i a e ; ( 1 5 9 ) • FIGURE 10 Chromatography of t R N A ^ y I so la ted w i t h Stage I Enzyme. The tRNA (1,400 A^^Q u n i t s ) obta ined by t r e a t i n g f r a c t i o n s 178-210 of F igure 7 accord ing to Step 12 of the Experimental S e c t i o n was chromatographed on a column (1.2 x 98 cm) of BD-c e l l u l o s e e l u t e d w i t h the i n d i c a t e d (dashed l i n e ) g rad ien t of sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e ( t o t a l of 2 1.). Flow r a t e , 1 ml/1 m i n ; 20 ml per f r a c t i o n . At the i n d i c a t e d p o i n t , e l u t i o n was cont inued w i t h 1.5 M sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e in 1k% (v/v) e t h a n o l . S o l i d l i n e : ^260' dotted l i n e : acceptor a c t i v i t y f o r g l y c i n e . ro b OJ b b 01 b 1.5 M NaCl +14% ETHANOL o o JL o p o o p ^ Ol 0) ->i o° MOLARITY OF SODIUM CHLORIDE ro ^ cn co g l y c i n e i soaccepto r tRNAs us ing a brewer 's yeast enzyme preparat i o n . From a sample of the yeast used by Boehringer and Soehne to prepare tRNA, a crude aminoacyl - tRNA synthetase p r e p a r a t i o n was prepared as descr ibed in the Experimental S e c t i o n . In t h i s c a s e , however, an ammonium s u l f a t e f r a c t i o n from a 0 to 80% s a t u r a t i o n range was used. G l y c i n e i soacceptor tRNA spec ies were i s o l a t e d and s u b - f r a c t i o n a t e d as b e f o r e . F igure 11 shows that r e s u l t s are c l e a r l y s i m i l a r to r e s u l t s of F igure 10. The t h i r d peak, l a c k i n g g l y c i n e acceptor a c t i v i t y , is more s u b s t a n t i a l in t h i s c a s e . In an attempt to c h a r a c t e r i z e the m a t e r i a l present in f r a c t i o n s 60 to 80 ( i n c l u s i v e ) of F igure 11, the f r a c t i o n s were pooled and the m a t e r i a l present was p r e c i p i t a t e d w i th ethanol and c o l l e c t e d by c e n t r i f u g a t i o n . The product was then d i s s o l v e d in a s o l u t i o n c o n t a i n i n g the components requ i red to e s t e r i f y 1 h amino a c i d s to t h e i r r e s p e c t i v e tRNAs. A mixed C-amino a c i d mix ture (un i formly l a b e l l e d a l g a l - p r o t e i n hydro l ysate ) and crude enzyme was then added to charge tRNAs p resent . A f t e r an a p p r o p r i a t e incubat ion t ime , the mix ture was e x t r a c t e d w i th phenol and the presumed tRNAs and aminoacy1 - tRNAs were repeated ly p r e c i p i t a t e d and r e d i s s o l v e d to remove u n e s t e r i f i e d amino a c i d s . F i n a l l y , the products were d i g e s t e d w i t h a l k a l i to remove the . e s t e r i f i e d amino a c i d s . The a l k a l i n e h y d r o l y s a t e was subjected FIGURE 11 Chromatography of t R N A ^ ' y I so la ted w i th "Homologous" Stage I Enzyme The tRNA (395 ^2(>0 u n ' t S / obta ined by charg ing hOO mg of crude tRNA minus e t h a n o l - f r a c t i o n w i th "homologous" stage I enzyme (see tex t f o r exp lanat ion ) and subsequent ly d e r i v a t i z i n g and p u r i f y i n g Gly tRNA 7 accord ing to steps 7~12 of the Exper imental S e c t i o n was chromatographed on a column (97 x 1.5 cm) of B D - c e l l u l ose e l u t e d w i t h the i n d i c a t e d (dashed l i n e ) g r a d i e n t of sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e ( t o t a l of 2 1.). Flow r a t e , 1 ml/min ; 18 m l / f r a c t i o n . At the i n d i c a t e d po in t , e l u t i o n was cont inued w i t h 1.5 M sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e in ]k% (v/v)ethanol . S o l i d l i n e : ^260'' dot ted l ' n e : acceptor a c t i v i t y fo r g l y c i n e . FRACTION NUMBER t o p a p e r c h r o m a t o g r a p h y ( d e s c e n d i n g ; s o l v e n t , n - b u t a n o l : g 1 a c i a 1 a c e t i c a c i d : w a t e r : : 6 0 : 1 5 : 2 5 b y v o l u m e ) a n d t h e d e v e l o p e d c h r o m a t o g r a m w a s s c a n n e d w i t h a s t r i p - c o u n t e r t o l o c a t e r a d i o -a c t i v i t y . T h e o n l y s i g n i f i c a n t l a b e l c a r r i e d t h r o u g h t h e s e s t e p s w a s t e n t a t i v e l y i d e n t i f i e d a s l y s i n e b y c o m p a r i s o n o f R f v a l u e s o f a u t h e n t i c l y s i n e a n d r a d i o a c t i v i t y r e l e a s e d b y a l k a l i n e h y d r o l y s i s o f t h e e s t e r i f i e d t R N A . T h i s s u g g e s t s t h e p r e s e n c e o f t R N A ' ~ y s i n f r a c t i o n s 6 0 t o 80 o f F i g u r e 1 1 . N e i t h e r t h e n a t u r e n o r t h e o r i g i n o f t h e p e a k i n q u e s t i o n i s k n o w n b e y o n d t h i s i n c o m p l e t e s t u d y . F u r t h e r P u r i f i c a t i o n o f t R N A G 1 y a n d t R N A ^ 7 . t R N A G ^ y o b t a i n e d a t S t e p 13 o f t h e i s o l a t i o n p r o c e d u r e o u t l i n e d i n t h e E x p e r i m e n t a l - S e c t i o n g e n e r a l l y p o s s e s s e d a s p e c i f i c a c c e p t o r a c t i v i t y w h i c h i s h i g h e r t h a n u s u a l ( v a l u e s o f 2 . 1 n m o l e g l y c i n e a c c e p t e d p e r A ^ ^ Q u n i t h a v e b e e n f o u n d ) . H o w e v e r , v a l u e s c l o s e t o 2 . 0 h a v e b e e n r e p o r t e d ( 1 4 0 ) . T h e v a l u e o f t h e s p e c i f i c a c c e p t o r a c t i v i t y o f a p u r i f i e d t R N A i s d e p e n d e n t o n t h e c h a i n l e n g t h a n d b a s e c o m p o s i t i o n o f t h e t R N A . F u r t h e r , i t d e p e n d s o n t h e c o n d i t i o n s u s e d t o m e a s u r e t h e A ^ ^ Q v a l u e a n d a r e a s o n a b l e c r i t e r i o n f o r p u r i t y s h o u l d i n c l u d e a m e a s u r e o f a m o u n t o f a m i n o a c i d a c c e p t e d p e r p h o s p h a t e r e s i d u e : ; i n a d d i t i o n t o a v a l u e o f a m i n o a c i d a c c e p t e d p e r A ^ ^ Q u n i t . T h e d e g r e e o f p u r i t y i s e s t a b l i s h e d c o n c l u s i v e l y a f t e r d e t a i l e d c h e m i c a l ana 1ys i s . To f u r t h e r t e s t the p u r i t y of t R N A ^ y , the method of Takemura and co -workers f o r f r a c t i o n a t i o n of tRNAs was used . The i r procedure employs DEAE-Sephadex A-25-. chromatography and s e l e c t i v e e l u t i o n . o f tRNAs is achieved w i t h ammonium s u l f a t e g r a d i e n t s which c o n t a i n dimethy1formamide. Minor contaminants present in tRNA^" 7 7 p repara t ions should be detected a f t e r chromatography on the l a t t e r system s i n c e the p r o b a b i l i t y of t h e i r behaving the same on B D - c e l l u l o s e and DEAE-Sephadex A-25 should be s m a l l . The t R N A ^ y gave a s i n g l e , symmetrical A ^ Q p r o f i l e (F igure 12) a f t e r DEAE-Sephadex A-25 chromatography. No change in the s p e c i f i c acceptor a c t i v i t y was observed and i t was t h e r e f o r e concluded that t R N A ^ y was e s s e n t i a l l y pure p r i o r to t h i s chromatographic s t e p . t R N A G ^ y is obta ined in a less pure s t a t e than i t s i s o -a c c e p t i n g p a r t n e r . Grad ient chromatography on B D - c e l l u l o s e in the absence of magnesium and at a c i d pH reso lves components which tend to e l u t e together under neut ra l c o n d i t i o n s in the presence of magnesium (147)- F igure 13 shows t R N A G ' y reso lved on B D - c e l l u l o s e by use of an EDTA-contain ing sodium c h l o r i d e g rad ien t which was buf fe red at pH 3-5- Three peaks absorbing l i g h t at 260 nm are seen. The second of two minor peaks accepts g l y c i n e and probably represents contaminat ion of tRNA G ^ y w i t h t R N A ^ y . The major peak, , t R N A G ' y now possesses a s p e c i f i c acceptor a c t i v i t y near that FIGURE 12 G1 v Chromatography o f tRNA^ y on DEAE-Sephadex A-25. The t R N A ^ y (545 ^260 u n ' t s ) o b t a i n e d by p o o l i n g f r a c t i o n s 41-55 ( i n c l u s i v e ) o f F i g u r e 6 was chromatographed on a column (52.5 x 1.4 cm) o f DEAE-Sephadex A-25 e l u t e d w i t h the i n d i c a t e d (dashed l i n e ) g r a d i e n t o f ammonium s u l f a t e c o n t a i n i n g 20 mM p o t a s s i u m a c e t a t e , pH 5-3 and \% (v/v) r e d i s t i l l e d d i m e t h y l f o r m a m i d e ( t o t a l o f 2 1.). Flow r a t e 0.55 ml/min; 32 min f r a c t i o n s . A f t e r f r a c t i o n 105 was c o l l e c t e d , e l u t i o n was c o n t i n u e d w i t h 2M ammonium s u l f a t e , 20 mM p o t a s s i u m a c e t a t e and \% (v/v) dimethy1formamide . 14 S o l i d l i n e : . ^260' d o t t e d l i n e : i n c o r p o r a t i o n o f C - g l y c i n e d e t e r m i n e d by Method 2. ABSORBANCE AT 260 NM FIGURE 13 Chromatography, of t R N A ^ y on B D - C e l l u l o s e in the Presence of EDTA at Ac id pH t R N A ^ ^ (pooled from f r a c t i o n s 56-72 i n c l u s i v e of F igure 8, f r a c t i o n s £1-72 of F igure 10 and f r a c t i o n s 48-57 o f F igure 11, 398 f^2S0 U N ' T S T O T A 0 w a s chromatographed on a column (92 x 1.5 cm) of B D - c e l l u l o s e e l u t e d w i th the i n d i c a t e d (dashed l i n e ) g r a d i e n t of sodium c h l o r i d e c o n t a i n i n g 1 mM EDTA and 5 mM sodium formate , pH 3-5 ( t o t a l of 2 1.). Flow rate 1 ml/min; 20 min f r a c t i o n s . 1 k S o l i d l i n e : A , n ; dotted l i n e : i n c o r p o r a t i o n of C - g l y c i n e , e x p e c t e d f o r a homogeneous tRNA. However, t h e r e i s e v i d e n c e f o r f u r t h e r c o n t a m i n a t i o n by t h e l a c k o f c o i n c i d e n c e o f A ^ Q p r o f i l e and a c c e p t o r a c t i v i t y . t R N A G ^ y from t h i s column was chromatographed on DEAE-Sephadex A -25v Some minor c o n t a m i n a n t s a r e a p p a r e n t ( F i g u r e 1 4 ) ; however, the peak o f the ab s o r b a n c e p r o f i l e i s e s s e n t i a l l y c o i n c i d e n t w i t h the peak o f a c c e p t o r a c t i v i t y . The e n r i c h e d f r a c t i o n s a s s a y t o t h e same l e v e l o f p u r i t y as a c h i e v e d f o r t R N A G , y and thus both t R N A G l y and t R N A G ^ y have been e n r i c h e d t o a p p a r e n t homogeneity. I t had been o b s e r v e d e a r l i e r (147) t h a t t R N A G , y c o u l d be r e s o l v e d i n t o two components f o l l o w i n g chromatography unde-c o n d i t i o n s used i n F i g u r e 13- In t h i s c a s e , however, the tRNA was o b t a i n e d from g r a d i e n t chromatography o f t o t a l c r u d e tRNA and not by the c h a r g i n g - d e r i v a t i z a t i o n p r o c e d u r e . A l t h o u g h t R N A G ^ y i s o b t a i n e d by the l a t t e r t e c h n i q u e i n lower r e l a t i v e y i e l d than by the f o r m e r , i t c o n t a i n s o n l y a s i n g l e g l y c i n e a c c e p t o r a c t i v i t y . T h i s f i n d i n g has been o b s e r v e d r e p e a t e d l y and w i t h a v a r i e t y o f enzyme p r e p a r a t i o n s . L a r g e S c a l e I s o l a t i o n o f t R N A G ' y and t R N A G 1 y from Crude  tRNA. The s t u d i e s d e s c r i b e d here were d e s i g n e d t o - p r o v i d e a s i m p l e , l a r g e - s c a l e method f o r i s o l a t i o n o f . h i g h l y p u r i f i e d tRNA. A number o f s i m p l i f i c a t i o n s o v e r the o r i g i n a l p r o c e d u r e .(107) FIGURE 14 Chromatography o f t R N A ^ 1 y on DEAE-Sephadex A-25 t R N A G l y ( f r a c t i o n s 75 t o 95 o f F i g u r e 13; 442 u n i t s ) was chromatographed on a column (52.5 x 1.4 cm) of DEAE-Sephadex A - 2 5 e l u t e d w i t h the i n d i c a t e d (dashed l i n e ) g r a d i e n t o f ammonium s u l f a t e c o n t a i n i n g 2 mM p o t a s s i u m a c e t a t e and \% (v/v) r e d i s t i l l e d d i m e t h y l f o r m a m i d e ( t o t a l o f 1800 m l ) . Flow r a t e 0 .55 ml/min; 30 min f r a c t i o n s . S o l i d l i n e : ^260' d o t t e d l i n e : i n c o r p o r a t i o n 14 o f C - g l y c i n e d e t e r m i n e d by method 2. ABSORBANCE AT 260 NM O o CPM PER MILLILITER XIO • * * * O " O o bo ro b MOLARITY OF ( N H 4 ) 2 S 0 4 O ho ON 4=-have been d e v e l o p e d . Crude g l y c i n e - t R N A s y n t h e t a s e gave f i n a l p r o d u c t s o f h i g h p u r i t y so t h a t the use o f p a r t i a l l y p u r i f i e d enzyme was u n n e c e s s a r y . The i n t e r a c t i o n o f the N^-2-naphthoxy-a c e t y l g l y c y l - t R N A and B D - c e l l u l o s e was s u f f i c i e n t l y t e n a c i o u s Phe t h a t the e t h a n o l - f r a c t i o n o f c r u d e tRNA which c o n t a i n s tRNA X107) d i d not need t o be removed b e f o r e the tRNA was used i n the pur i f i c a t i o n o f t R N A ^ y and t R N A ^ y . F i n a l l y , sham-acy 1 a t i o n 3 was not c a r r i e d out s i n c e t R N A ^ y and t R N A ^ y p r e p a r e d from tRNA samples which had been o r had not been sham-acy1ated were o f s i m i l a r p u r i t y . These o b s e r v a t i o n s s i m p l i f i e d the p u r i f i c a t i o n p r o c e d u r e . The d e t a i l s i n the E x p e r i m e n t a l S e c t i o n a r e a c u l m i n a t i o n o f t h e s e s t u d i e s . The r e s u l t s o f the i s o l a t i o n o f Nh2-naphthoxy-a c e t y l g l y c y l - t R N A from 5 g o f c r u d e , u n t r e a t e d tRNA a r e shown i n F i g u r e 15- The d e t a i l s o f p r e p a r a t i o n a r e p r o v i d e d i n the l e g e n d . 1 k V i r t u a l l y a l l o f the C i s e l u t e d i n the f i n a l peak o f the A^^Q p r o f i l e . The m a t e r i a l e l u t i n g ahead o f the l a t t e r peak i s t h e Phe e t h a n o l - f r a c t i o n w h i c h c o n t a i n e d tRNA as judged by the p r e s e n c e o f Y-base i n t h i s m a t e r i a l (159)• The f i n a l peak, s u b - f r a c t i o n a t e d 3 S h a m - a c y l a t i o h i n v o l v e s r e a c t i n g c r u d e tRNA w i t h the N_-hyd r o x y s u c c i n im ide e s t e r o f 2 - n a p h t h o x y a c e t i c a c i d under d e r i v a t i z a t i o n c o n d i t i o n s . That p o r t i o n o f the c r u d e tRNA wh i c h r e a c t s i s removed from the u n m o d i f i e d tRNA by s t e p w i s e chromatography as d e s c r i b e d i n Step 12 o f t h e E x p e r i m e n t a l S e c t i o n . The RNA e l u t e d by t h e 1 M sodium c h l o r i d e wash i s then used as a s o u r c e o f tRNA f o r the p u r i f i c a t i o n p r o c e d u r e . FIGURE 15 P u r i f i c a t i o n o f N^-2-Naphthoxyacety 1 g1ycy1-tRNA G^^ from 5 g o f U n t r e a t e d Crude tRNA. The t R N A G ^ y p r e s e n t in 5 9 o f c r u d e u n t r e a t e d tRNA was e s t e r i f i e d w i t h g l y c i n e u s i n g s t a g e II enzyme, d e r i v a t i z e d and chromatographed on B D - c e l l u l o s e e x a c t l y as d e s c r i b e d i n Steps 7 to- 11 o f the E x p e r i m e n t a l S e c t i o n . Column s i z e 110 x 3 cm; f l o w r a t e k ml/min; 20 ml f r a c t i o n s . S o l i d l i n e : ^260' d o t t e d l i n e : r a d i o a c t i v i t y from " ^ C - g l y c i n e . on BD-cel l u l o s e a f t e r removal o f the N_-2-naph t h o x y a c e t y 1 g 1 yc i ne r e s i d u e , g i v e s two components a c c e p t i n g g l y c i n e and a t h i r d , minor peak w i t h no g l y c i n e a c t i v i t y . These r e s u l t s a r e shown i n F i g u r e 16. The t R N A G 1 y i s o f h i g h p u r i t y ; t R N A G l y i s o f lower p u r i t y . However, as shown e a r l i e r h i g h l y p u r i f i e d tRNA*j' y i s a v a i l a b l e f o l l o w i n g f u r t h e r c h r o m a t o g r a p h i c r e s o l u t i on. FIGURE 16 Chromatography of t R N A ^ ' y I so la ted from 5 g of Crude, Untreated tRNA F r a c t i o n s kOO to 450 o f F igure 15 were pooled and t r e a t e d e x a c t l y as desc r ibed in Step 12 of the Exper imental S e c t i o n . The r e s u l t a n t tRNA (5,900 A^^Q u n i t s ) was chromatographed on a column (115 x 3 cm) of B D - c e l l u l o s e e l u t e d w i th the i n d i c a t e d (dashed l i n e ) g rad ient of sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e ( t o t a l of 10 1.). Flow rate 1 ml/min; 20 ml f r a c t i o n s . At the i n d i c a t e d p o i n t , e l u t i o n was cont inued w i th 1.5 M sodium c h l o r i d e c o n t a i n i n g 10 mM magnesium c h l o r i d e and \k% (v/v) e t h a n o l . S o l i d l i n e : A , • dotted l i n e : acceptor a c t i v i t y f o r g l y c i n e . SUMMARY A method f o r the i s o l a t i o n o f a f a m i l y o f aminoacyl-tRNAs has been d e s c r i b e d . The p r o c e d u r e i n v o l v e s r e a c t i o n o f the aminoacyl-tRNAs w i t h the IN-hydroxysucc i n im i de e s t e r o f 2-naphthoxy-a c e t i c a c i d . The N - 2-naphthoxyacety1 aminoacyl-tRNAs w h i c h r e s u l t p o s s e s s s u f f i c i e n t a f f i n i t y f o r B D - c e l l u l o s e t o a l l o w t h e i r p h y s i c a l s e p a r a t i o n from u n m o d i f i e d c o u n t e r p a r t s by s i m p l e c h r o m a t o g r a p h i c s t e p s . 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PART TWO THE USE OF BENZOYLATED DEAE-CELLULOSE FOR THE DEVELOPMENT OF NEW METHODS FOR SEQUENCE DETERMINATION OF NUCLEIC ACIDS PART TWO INTRODUCTION 88 EXPERIMENTAL 105 1 ^  (1) P r e p a r a t i o n of C - g l y c y l tRNA (crude) and N_-2 -naphthoxyacetyl - 1 / *C-glycyl tRNA (crude) . . . 105 (2) Large Sca le I s o l a t i o n of N-2-Naphthoxyacety1-g l y c y l - t R N A 2 1 ^ . 106 (3) Chelex 100 Chromatography 106 (4) Magnesium Determinat ion 107 (5) Assays f o r RNase A c t i v i t y 107 (6) Assay of the S p e c i f i c i t y of R ibonuclease T^  . 107 (7) Column Chromatography 109 (8) D e s a l t i n g of O l i g o n u c l e o t i d e s . 110 (9) Method fo r I s o l a t i o n and P u r i f i c a t i o n of R ibonuclease T , - R e l e a s e d 3 l - t e r m i n a l Fragments of N_-2-N a p h t h o x y a c e t y l g l y c y l - t R N A G 1 y 111 (10) Nuc leos ide A n a l y s i s of Peaks I and II O l i g o -nuc leot i des 11 k (11) Synthes is of 2 -Naphthy1phosphoromorphol idate. . . 115 (12) Formation of Cety1trimethylammonium S a l t of tRNA . 116 (13) React ion of Naphthy1phosphoromorpholidate w i t h Cety1trimethy1ammonium S a l t of tRNA . . . . 117 T a b l e o f C o n t e n t s ( C o n t i n u e d ) (14) R e a c t i o n o f tRNA w i t h 1 - F l u o r o - 2 , 4 - d i n i t r o -benzene 118 RESULTS AND DISCUSSION 119 I s o l a t i o n and P a r t i a l C h a r a c t e r i z a t i o n o f the R i b o n u c l e a s e T ^ -Released 3 ' ~ t e r m i n a l Fragments o f N _ - 2 - N a p h t h o x y a c e t y l g l y c y l - t R N A G 1 y 119 S t u d i e s on P a r t i a l D e g r a d a t i o n o f N.-2-N a p h t h o x y a c e t y 1 g l y c y 1 -tRNA w i t h R i b o n u c l e a s e T^ 134 S t u d i e s on t h e S t a b i l i t y , pH-Dependent A c t i v i t y and S p e c i f i c i t y o f R i b o n u c l e a s e T^ 141 S t u d i e s on D i g e s t i o n o f G l y c y l - t R N A by R i b o n u c l e a s e T^ 154 S t u d i e s on P a r t i a l D e g r a d a t i o n o f N^-2-naphthoxy-ace t y 1 g 1 y c y 1 -tRNA^' y w i t h P a n c r e a t i c R i b o n u c l e a s e . . 164 A l t e r n a t e Methods f o r L a b e l l i n g the 3'~ t e r m i n a l Group o f P o l y n u c l e o t i d e s 168 S t u d i e s on Methods f o r D e r i v a t i z i n g the 5 ' - t e r m i n a l o f tRNA 172 SUMMARY 180 REFERENCES 181 LIST OF TABLES Table 1. In f luence of Var ious So lvents on the Absorbance of Crude tRNA 137 Table 2. S p e c i f i c i t y of Var ious Ribonuclease T P r e p a r a t i o n s 148 LIST OF FIGURES PART TWO F igure 1. Schematic Diagram of the Proposed Sequence Method 96 Figure 2. Chromatography of R ibonuclease T j -d i gested j^-2 -naphthoxyacety 1 g 1 ycy 1 -tRNA G 1 ^ on B D - c e l l u l o s e . . . . . . 123 F igure 3. D e s a l t i n g of N^2 -naphthoxyacety1-g1ycy1 -o l igonuc1eot ides on N a p h t h o y l - c e l 1 u lose 125 F igure k. Chromatography of N^-2-naphthoxy-a c e t y1g l y c y1 - o l i g o n u c l e o t i d e s on DEAE-cel l u l o s e . 127 F igure 55. Chromatography of Peak I O l i g o -n u c l e o t i d e on DEAE-cel 1 u lose 128 F igure 6. Nuc leos ide A n a l y s i s of Peak I O l i g o n u c l e o t i d e 129 F igure 7. Chromatography of Peak II O l i g o n u c l e o t i d e on DEAE-ce11u1ose . . . 131 F igure 8. Nuc leos ide A n a l y s i s of Peak II O l i g o n u c l e o t i d e 132 F igure 9- Magnesium Determinat ion 136 F igure 10. DEAE-cel1ulose Chromatography of a P a r t i a l R ibonuclease T^  D i g e s t i o n of Nh2 - n a p h t h o x y a c e t y l g l y c y l - t R N A . . . . . UO F igure 11. pH A c t i v i t y P r o f i l e f o r R ibonuclease T^  Degrading tRNA in the Presence and Absence of 7 M urea Ikk F igure 12. E f f e c t of D ie thy lpy rocarbonate on Ribonuclease T-| A c t i v i t y 1^9 L i s t o f F i g u r e s ( C o n t i n u e d ) F i g u r e 13. E f f e c t o f D i t h i o t h r e i t o ! on R i b o n u c l e a s e T-| A c t i v i t y 151 F i g u r e 14. Chromatography o f R i b o n u c l e a s e on BD-cel l u l o s e . . . 153 F i g u r e 15- Chromatography o f RNase T - j - d i g e s t e d , P h e n o l - E x t r a c t e d G l y c y l - t R N A on D E A E - c e l l u l o s e 156 F i g u r e 16. Chromatography o f RNase T ^ - d i g e s t e d , P h e n o l - E x t r a c t e d , Naphthoxyacety1 a t e d G l y c y l - t R N A on DEAE-cel1u1ose . . . . 157 F i g u r e 17. D E A E - c e l l u l o s e Chromatography o f the P r o d u c t s O b t a i n e d from B D - c e l l u l o s e Chromatography of G l y c y l - t R N A w h i c h had P r e v i o u s l y Been D i g e s t e d w i t h R i b o n u c l e a s e , P h e n o l - E x t r a c t e d and N a p h t h o x y a c e t y l a t e d 1 58 F i g u r e 18. E f f e c t o f N a p h t h o x y a c e t y l a t i n g In-d i g e s t e d tRNA on B D - c e l l u l o s e Chromatography . . 161 F i g u r e 19- B D - c e l l u l o s e Chromatography o f G l y c y l - t R N A D i g e s t e d w i t h R i b o n u c l e a s e T-| , P h e n o l - E x t r a c t e d and Naphthoxy-a c e t y l a t e d i n the P r e s e n c e o f G l y c e r o l F i g u r e 20. DEAE-cel1u1ose Chromatography o f NH2-n a p h t h o x y a c e t y l g l y c y l - t R N A Y ^ D i g e s t e d w i t h E n z i t e - R N a s e . . . ' 165 F i g u r e 21. Reagents w h i c h C o u p l e w i t h t h e 3'-terminus o f P e r i o d a t e - 0 x i d i z e d RNAs 169 F i g u r e 22. Presumed R e a c t i o n of 2 - N a p h t h y l -p h o s p h o m o r p h o l i d a t e w i t h tRNA . . . . 176 F i g u r e 23- P o s s i b l e R e a c t i o n s o f DNFB w i t h tRNA 178 I wish to thank Mr. W. Bo lzer f o r h i s s k i l f u l a s s i s t a n c e in determin ing the n u c l e o s i d e a n a l y s i s of Peak I and Peak It o l i g o n u c l e o t i d e s . Solvent I n_-butanol (60 m l ) - g l a c i a l a c e t i c a c i d (15 ml) - water (25 ml) Solvent I I e thy l a l c o h o l (70 ml) - 1 M ammonium a c e t a t e , pH 7-5 (30 ml) So lvent I I I i so -propanol (70 ml) - concent rated ammonia (10 ml) - water (20 ml) INTRODUCTION P a r t II o f t h i s t h e s i s d e s c r i b e s a t t e m p t s t o d e v e l o p a novel p r o c e d u r e f o r sequence d e t e r m i n a t i o n o f n u c l e i c a c i d s . A b r i e f d i s c u s s i o n o f sequence methods used t o d a t e w i l l s e r v e t o i n d i c a t e t h e p o t e n t i a l u s e f u l n e s s and s i g n i f i c a n c e o f the proposed method. Reviews o f n u c l e i c a c i d s e q u e n c i n g p r o c e d u r e s a r e a v a i l a b l e (1, 2, 3, 4) . The methodology o f n u c l e i c a c i d r e s e a r c h has o f t e n p a r a l l e l e d , i n p r i n c i p l e , the m e thodology.developed by p r o t e i n c h e m i s t s . T h i s i s no l e s s t r u e f o r sequence s t u d i e s . Two main methods e x i s t f o r d e t e r m i n a t i o n o f n u c l e o t i d e sequences and both a r e p r e - d a t e d by o p e r a t i o n a l l y e q u i v a l e n t p r o c e d u r e s d e v e l o p e d f o r p r o t e i n c h e m i s t r y . The f i r s t p r o c e d u r e mimics the Edman d e g r a d a t i o n f o r p r o t e i n s e q u e n c i n g (5) and i s known i n n u c l e i c a c i d v e r n a c u l a r as the W h i t f e l d : ' d e g r a d a t i o n (6, 7). O p e r a t i o n a l l y , the Edman and W h i t f e l d d e g r a d a t i o n s i n v o l v e s t e p w i s e d e g r a d a t i o n o f t h e polymer from a s p e c i f i c end; each c y c l e o f d e g r a d a t i o n l i b e r a t e s the t e r m i n a l u n i t w h i c h i s s u b s e q u e n t l y i d e n t i f i e d . R e p e t i t i o n o f t h e s e s t e p s would o b v i o u s l y y i e l d the t o t a l p r i m a r y sequence of any polymer. The l i m i t a t i o n o f t h i s approach i s t h a t each s t e p i s not c o m p l e t e l y q u a n t i t a t i v e and hence, a f t e r a number o f s t e p s t h e i d e n t i f i c a t i o n o f the n e w l y - r e l e a s e d u n i t i s e q u i v o c a l . The W h i t f e l d d e g r a d a t i o n i n v o l v e s t h r e e s t e p s . The c i s - d i o l group o f the 3 1~termina1 i s o x i d i z e d w i t h p e r i o d a t e t o a d i a l d e h y d e . Second, the t e r m i n a l p h o s p h o d i e s t e r bond i s c l e a v e d by p r i m a r y amine-cata1yzed B - e l i m i n a t i o n which r e l e a s e s the t e r m i n a l base and i t s degraded r i b o s e m o i e t y . F i n a l l y , t he polymer i s made ready f o r a second c y c l e o f d e g r a d a t i o n once the 3'"phosphate group i s removed by the a c t i o n o f phospho-monoesterase. T h i s approach has seen marked improvement s i n c e i t s o r i g i n a l f o r m u l a t i o n (8, 9) and has a l l o w e d d e t e r m i n a t i o n o f the sequences o f o l i g o n u c l e o t i d e s o f c h a i n l e n g t h near twenty (10, 11). More g e n e r a l use o f t h i s approach w i l l probab1y r e s u 1 t from t h e r e c e n t development o f a machine which a u t o m a t i c a l l y performs the s t e p s o f the W h i t f e l d - d e g r a d a t i o n (12). The second method, w h i c h has a l l o w e d d e t e r m i n a t i o n o f a number o f p r i m a r y sequences o f s m a l l n u c l e i c a c i d s , d e r i v e s from the S a n g e r - o v e r l a p method used t o a n a l y z e the s t r u c t u r e o f i n s u l i n (13). Here, polymers a r e degraded c o m p l e t e l y i n a t l e a s t two d i f f e r e n t ways t o y i e l d o l i g o m e r s . The sequence o f each o l i g o m e r i s d e t e r m i n e d and the d a t a assembled i s used t o c o n s t r u c t r e g i o n s o f unambiguous o v e r l a p . S i n c e many s i m i l a r but not i d e n t i c a l f r a gments may be pr o d u c e d , the r e q u i r e m e n t s f o r unambiguous assignment can make sequence d e t e r m i n a t i o n o f l a r g e polymers a d i f f i c u l t t a s k . For both p r o t e i n and tRNA s e q u e n c e - s t u d i e s , the d i f f i c u l t y o f o r d e r i n g the v a r i o u s fragments i s reduced by the p r e s e n c e o f a number o f c h e m i c a l l y d i s t i n c t monomeric u n i t s . T h i s advantage does not e x i s t i n o t h e r RNA s p e c i e s s i n c e , f o r the most p a r t , t h e r e a r e but f o u r d i s t i n c t monomeric u n i t s . The approach d e v e l o p e d by R.W. Hoi l e y t o d e t e r m i n e the n u c l e o t i d e sequence o f t R N A ^ 3 (14) p a r a l l e l s , i n p r i n c i p l e , t he Sanger-method used t o sequence i n s u l i n . In the now c l a s s i c a l "Hoi l e y - a p p r o a c h " t o tRNA s e q u e n c i n g , the polymer i s f i r s t degraded c o m p l e t e l y w i t h r i b o n u c l e a s e T^ (which h y d r o l y z e s the RNA next t o G and s e v e r a l G d e r i v a t i v e s ) and s i m i l a r l y w i t h p a n c r e a t i c r i b o n u c l e a s e (which c l e a v e s the polymer a t both p y r i m i d i n e n u c l e o t i d e s and most o f the m o d i f i e d bases d e r i v e d from the l a t t e r n u c l e o t i d e s ) . The fragments l i b e r a t e d by the e x h a u s t i v e e n z y m a t i c d e g r a d a t i o n a r e then s e p a r a t e d and c h a r a c t e r i z e d . C h a r a c t e r i z a t i o n g e n e r a l l y i n v o l v e s d e t e r m i n a t i o n o f the n u c l e o t i d e c o m p o s i t i o n as w e l l as f u r t h e r e n z y m a t i c d e g r a d a t i o n e i t h e r w i t h the c o m p l i m e n t a r y e n d o n u c l e a s e o r w i t h the e x o n u c l e a s e s s p l e e n p h o s p h o d i e s t e r a s e o r snake-venom p h o s p h o d i e s t e r a s e . The d a t a assemb1ed are used t o d e t e r m i n e the o r d e r o f n u c l e o t i d e s i n each fragment. Once the l a t t e r s t e p i s c o m p l e t e , i t i s p o s s i b l e t o r e c o n s t r u c t p o r t i o n s o f the o r i g i n a l tRNA m o l e c u l e s by a c o m p a r i s o n o f the f r a g m e n t s . The t o t a l unambiguous a s s i g n m e n t o f f r a g m e n t - o r d e r i s p o s s i b l e o n l y a f t e r l a r g e fragments o f the tRNA a r e o b t a i n e d by p a r t i a l e n d o n u c l e a s e d i g e s t i o n . N o r m a l l y , the l a t t e r step is performed under c o n d i t i o n s which s t a b i l i z e the t e r t i a r y - s t r u c t u r e of the tRNA so that the s u s c e p t i b i l i t y of the s u b s t r a t e to nuclease a t t a c k is l i m i t e d . I s o l a t i o n of the la rge fragments fo l lowed by exhaust i ve d i g e s t i o n wi th a nuc lease prov ides in fo rmat ion on which fragments are adjacent to one another in the i n t a c t mo lecu le . Once a number of la rge fragments have been a n a l y z e d , i t is p o s s i b l e to d e r i v e an unambiguous order f o r a l l f ragments . The methodology of tRNA sequence d e t e r m i n a t i o n has progressed to a po in t where the t o t a l sequence a n a l y s i s of any tRNA i s a f a i r l y r o u t i n e procedure. GiI ham 1 s recent review (4), which d e s c r i b e s and eva luates recent advances in techniques of RNA sequence a n a l y s i s , obv ia tes the need f o r d e t a i l e d d i s c u s s i o n h e r e , as do the reviews of Hoi ley (3). Zachau (15) and von Ehrens te in (16). The l a t t e r a r t i c l e s d i s c u s s a n a l y s i s of tRNA. The t e c h n i c a l advances made in tRNA sequencing are r e l a t e d p r i m a r i l y to the speed and ease of o b t a i n i n g , p u r i f y i n g and sequencing o l i g o m e r i c d e r i v a t i v e s of tRNA spec ies rather than any change in the p r i n c i p l e s of a n a l y s i s . Th is is t rue as w e l l f o r s t u d i e s of l a r g e r molecules such as v i r a l and r ibosomal RNA. Some of these advances are mentioned here . For example, methods which r e s t r i c t the s p e c i f i c i t y of commonly used nucleases T^  and p a n c r e a t i c RNase (17, 18), as w e l l as newly desc r ibed nucleases such as r i b o n u c l e a s e U (19) have provided s u c c e s s f u l means of o b t a i n i n g s e l e c t i v e fragments u s e f u l f o r o v e r l a p p i n g . F u r t h e r , E n t e r o b a c t e r sp. c o n t a i n a r i b o n u c l e a s e which c l e a v e s o n l y a t c y t i d y l i c a c i d r e s i d u e s (20) and t h i s enzyme may prove t o be o f c o n s i d e r a b l e u t i l i t y f o r sequence s t u d i e s . Development o f p u r i f i c a t i o n p r o c e d u r e s such as homochromatography on paper (21) and t h i n - l a y e r (22) as w e l l as e 1 e c t r o p h o r e t i c s e p a r a t i o n o f l a r g e fragments o f h i g h m o l e c u l a r w e i g h t RNA have proven to be e x t r e m e l y v a l u a b l e (23, 2k). P a r t i c u l a r l y u s e f u l has been the development o f a mapping-procedure i n which r a d i o a c t i v e l y 1 abel1ed nuc1e i c ac i ds (^P) a r e d i g e s t e d and f r a c t i o n a t e d on a t w o - d i m e n s i o n a l system (25). Subsequent r a d i o a u t o g r a p h y y i e l d s a f i n g e r - p r i n t o f components whose p o s i t i o n r e v e a l s the base-c o m p o s i t i o n and o f t e n the p r i m a r y - s e q u e n c e o f o l i g o n u c l e o t i d e s d i r e c t l y . The t e c h n i q u e ' s r e q u i r e m e n t f o r s m a l l amounts o f s t a r t i n g m a t e r i a l , i t s s i m p l i c i t y , speed and d i r e c t a p p l i c a b i l i t y t o homochromatographic and e l e c t r o p h o r e t i c r e s o l u t i o n o f l a r g e fragments may be c o n t r a s t e d w i t h d i f f i cu^l t i es o f p r e p a r i n g (and p r o b a b l y i n w o r k i n g w i t h ) the h i g h l e v e l s o f r a d i o a c t i v i t y r e q u i r e d However, the s u c c e s s o f the method, which has become known as the " S a n g e r - t e c h n i q u e " , speaks f o r i t s e l f . A method f o r o b t a i n i n g f i n g e r - p r i n t s . b y t h i s same p r o c e d u r e from n o n - r a d i o a c t i v e n u c l e i c a c i d s has been d e s c r i b e d (26). I f RNA m o l e c u l e s can be s y n t h e s i z e d i n v i t r o o t h e r methods o f a n a l y s i s a r e p o s s i b l e . For example, the synchronous RNA p r o d u c t i o n w i t h QJB-repl i c a s e d i r e c t e d by phage 0J3RNA minus s t r a n d a l l o w e d C. Weissmann and co-workers t o d e t e r m i n e a sequence o f 175 n u c l e o t i d e s b e g i n n i n g from the 5'~terminus o f Qj3~plus s t r a n d (27). S p e c i f i c s t r e t c h e s o f RNA which ranged from 20 t o 160 n u c l e o t i d e s i n l e n g t h were p r e p a r e d by s y n c h r o n i z e d 32 enzyme s y n t h e s i s . By s u p p l y i n g n u c l e o s i d e a - P t r i p h o s p h a t e s s e p a r a t e l y o r a l l f o u r s i m u l t a n e o u s l y , the s t r e t c h e s were sequenced e i t h e r by t h e Sanger-mapping p r o c e d u r e o r n e a r e s t - n e i g h b o u r a n a l y s i s . By removing a l i q u o t s a t p r o g r e s s i v e l y l a t e r s t a g e s o f s y n t h e s i s , i t was p o s s i b l e t o o b s e r v e the time a t which d i f f e r e n t s e c t i o n s o f the p r o d u c t were s y n t h e s i z e d . T h i s t ime-element has s i g n i f i c a n t p o t e n t i a l s i n c e i t p r o v i d e s a means o f o r d e r i n g f r a g m e n t s . The Weissmann-approach w i l l a l m o s t c e r t a i n l y be a p p l i e d t o se q u e n c i n g n u c l e i c a c i d s f r o m . s o u r c e s o t h e r than b a c t e r i o p h a g e s . For example, c e r t a i n animal v i r i o n s possess t h e i r own polymerases (28, 29) and th e s e may be used t o produce mRNAs i n v i t r o ( 3 0 ) . Thus, mRNAs o p e r a t i v e i n animal c e l l s may be s t u d i e d by the We issmann-approach. The r e c e n t r e p o r t s d e s c r i b i n g the p u r i f i c a t i o n o f t he 1ac-operon (31a) and i n d i c a t i n g t h a t a s i m i l a r p r o c e d u r e may be a p p l i c a b l e t o the i s o l a t i o n o f any b a c t e r i a l genome (31b) both s u g g e s t t h a t t he Weissmann-method c o u l d be a p p l i e d t o n o n - v i r a l RNAs. In t h i s c a s e , DNA-dependent RNA-polymerase c o u l d be used t o produce l a b e l l e d RNAs from the genes i n v i t r o . F u r t h e r , a procedure us ing the " r e p a i r - p r o p e r t i e s " of DNA-polymerase has a l lowed p a r t i a l sequences of the cohes ive ends of bacter iophage A and 186 DNA to be determined (32) . The l a t t e r procedure , which should prove to be of c o n s i d e r a b l e u t i l i t y f o r d e t e r m i n a t i o n of DNA sequences, mimics the Weissmann-procedure in both execut ion and advantage. The s e q u e n c e - a n a l y s i s of la rge RNA spec ies is t e c h n i c a l l y f e a s i b l e - indeed such s t u d i e s are a c t i v e l y under way (27, 3 3 , 3 4 ) . One problem may w e l l a r i s e w i t h attempts to sequence l a r g e r p o l y n u c l e o t i d e s . Th is is the need to o b t a i n and order la rge fragments which a l l o w c o n s t r u c t i o n of unambiguous o v e r l a p s . For example, t h i s problem prevented d e t e r m i n a t i o n of the primary A l a sequence of tRNA u n t i l Hoi ley and Penswick succeeded in o b t a i n i n g h a l f - m o l e c u l e s by c o n t r o l l e d enzymatic d i g e s t i o n (35) . The need f o r o b t a i n i n g s e l e c t i v e f ragmentat ions in l a r g e r RNA spec ies becomes apparent i f one c o n s i d e r s the s i z e of such spec ies and lack of odd bases which serve as i n t e r n a l re ference p o i n t s . Many la rge RNAs have both d e f i n i t e secondary s t r u c t u r e and a number of s i t e s where s e l e c t i v e c leavage is p o s s i b l e (36, 37 3 8 ) . Other methods of r e s t r i c t i n g nuc lease a c t i v i t y to s p e c i f i c l o c i have been desc r ibed (39, 40 , 4 1 , 4 2 ) . New methods f o r o b t a i n i n g and o r d e r i n g la rge fragments w i l l be necessary i f p r e s e n t l y a v a i l a b l e sequence methods a re to be extended to d e t e r m i n a t i o n of pr imary sequences of la rge p o l y n u c l e o t i d e s . S e l e c t i v e c l e a v a g e s a c h i e v e d by r e s t r i c t i n g n u c l e a s e a c c e s s i b i l i t y and s e l e c t i v e s e g m e n t - l a b e l l i n g by the Weissmann-procedure w i l l u n d o u b t e d l y f e a t u r e i n t h e s e s t u d i e s . The o b j e c t o f the f o l l o w i n g s t u d y was t o d e v e l o p a method o f RNA sequence a n a l y s i s which employs a novel approach t o the o r d e r i n g o f f r a g m e n t s . A s i m i l a r approach has been c o n s i d e r e d from a t h e o r e t i c a l p o i n t o f v i e w (43). The proposed r o u t e i n v o l v e s l a b e l l i n g e i t h e r the 3'"terminus o r 5'"terminus o f a n u c l e i c a c i d f o l l o w e d by p a r t i a l h y d r o l y s i s o f the polymer. The l a b e l a t the t e r m i n a l c o u l d s e r v e as a marker f o r p o s i t i o n i n g each n u c l e o t i d e a c c o r d i n g t o i t s d i s t a n c e from the m o d i f i e d t e r m i n a l and, most i m p o r t a n t , as a means of s e p a r a t i n g fragments b e a r i n g the l a b e l from t h o s e l a c k i n g i t . By a p p r o p r i a t e m a n i p u l a t i o n s , such a method c o u l d be used t o o r d e r o l i g o m e r i c u n i t s w i t h o u t r e l y i n g on o v e r l a p s . A t h e o r e t i c a l a p p l i c a t i o n o f the.method t o a h y p o t h e t i c a l polymer w i l l s e r v e t o c l a r i f y the s t e p s , advantages and p o t e n t i a l problems such a method might i n v o l v e . T h i s i s shown diagram-a t i c a l l y i n F i g u r e 1. C o n s i d e r a p o l y n u c l e o t i d e w i t h t h r e e s i t e s s u s c e p t i b l e t o c l e a v a g e by a p a r t i c u l a r e n d o n u c l e a s e . E x h a u s t i v e d i g e s t i o n w i t h t h i s n u c l e a s e y i e l d s f o u r fragments A, B, C and D, whose sequences a r e abc, d e f , ghi and j k l , r e s p e c t i v e l y , as d e t e r m i n e d by s t a n d a r d methods. The method proposed here would be a 11ow'ordering o f t h e s e f o u r fragments by Schematic Diagram of the Proposed Sequence Method. (1) Polynucleot ide Exhaustive Nuclease Digest ion A + B + C + D where A = abc, B = def , C = g h i , D = j k l the polynucleotide = (A,B,C,D) (2) Polynucleot ide + L-X ( . -Polynucleotide i Co l lec t a l1 L-ended Fragments L-A + BCD L-AB + CD L-ABC + D Resolve a l l L -ended According to Size i ; * L-A • L-AB L-ABC ' • I ' Digest Each Fragment Exhaustively with Nuclease I and Identify the Components A A + B A + B + C I I Lef t member is A B is next to A C is next to B This analys is leaves only fragment D unaccounted f o r ; the primary sequence is A-B-C-D or abcdefgh i jk l . the f o l l o w i n g s t e p s . F i r s t , the polymer i s m o d i f i e d by the s p e c i f i c i n t r o d u c t i o n of a group L a t a p a r t i c u l a r t e r m i n a l - the 5 ' " t e r m i n a 1 , f o r example. Then, the n u c l e a s e i s a l l o w e d t o degrade the m o d i f i e d polymer under c o n d i t i o n s which f a v o r c l e a v a g e o f no more than one bond per m o l e c u l e . In t h i s way, a l l p o s s i b l e fragments w i l l be r e p r e s e n t e d i n the d i g e s t i o n m i x t u r e i n r o u g h l y e q u i v a l e n t amounts. The L-ended fragments are then s e p a r a t e d from t h e o t h e r f r a g m e n t s . Next, the i s o l a t e d L-ended fragments a r e r e s o l v e d on the b a s i s o f s i z e and each p u r i f i e d fragment i s e x h a u s t i v e l y d i g e s t e d w i t h the n u c l e a s e . The p r o d u c t s a r e r e s o l v e d and i d e n t i f i e d . As shown i n F i g u r e ! , t h e i n f o r m a t i o n o b t a i n e d by t h e s e s t e p s would a l l o w u n e q u i v o c a l assignment o f the p r i m a r y sequence as 5 1 - L - a b c d e f g h i j k l . A s i m i l a r a n a l y s i s c o u l d be performed from the 3 ' - t e r m i n a l and f o r long p o l y m e r s , v a r i o u s c o m b i n a t i o n s c o u l d be used. I t was reasoned t h a t t h e methodology needed f o r t h i s a pproach was e i t h e r a v a i 1 a b l e o r c o u l d be d e v e l o p e d . P u r i f i e d N _ - 2 - n a p h t h o x y a c e t y l g l y c y l - t R N A G 1 y c o u l d s e r v e as a model polymer b e a r i n g an a p p r o p r i a t e 3 ' - d e r i v a t i v e . F o l l o w i n g p a r t i a l d e g r a d a t i o n o f the model polymer, the fragments b e a r i n g the lThe n o t a t i o n N _ - 2 - n a p h t h o x y a c e t y l g l y c y l - t R N A 1 y r e f e r s t o a m i x t u r e o f the c o r r e s p o n d i n g d e r i v a t i v e s of t R N A G 1 Y ' and t R N A G 1 Y . N^-2-naphthoxyacety 1 g 1 ycy 1 - res i due c o u l d be s e p a r a t e d from fragments not b e a r i n g the group by s t e p w i s e chromatography on B D - c e l l u l o s e . D E A E - c e l 1 u l o s e c o u l d be used t o s e p a r a t e fragments a c c o r d i n g t o s i z e . F o l l o w i n g e x h a u s t i v e - d i g e s t i o n o f the f r a g m e n t s , the c o m p o s i t e p r o d u c t s c o u l d be r e s o l v e d by e i t h e r c h r o m a t o g r a p h i c p r o c e d u r e s o r combined e l e c t r o p h o r e t i c - c h r o m a -t o g r a p h i c p r o c e d u r e s . The r e m a i n i n g s t e p s would be t o f i n d r e a g e n t s w h i c h would s p e c i f i c a l l y i n t r o d u c e a r o m a t i c groups o n t o t h e 5 ' ~ t e r m i n a l and, most i m p o r t a n t , t o e s t a b l i s h c o n d i t i o n s under which a v a i l a b l e n u c l e a s e s c o u l d degrade the model polymer i n a manner a p p r o a c h i n g random, s i n g l e - h i t k i n e t i c s . A more d e t a i l e d d i s c u s s i o n o f v a r i o u s a s p e c t s o f the proposed sequence method f o l l o w s . The i d e a l s i t u a t i o n f o r a c h i e v i n g the a n a l y s i s proposed here i s t o degrade each polymer once and o n l y once i n a c o m p l e t e l y random f a s h i o n . In t h i s way, a l l p o s s i b l e f ragments w i l l be p r e s e n t i n e q u i v a l e n t amounts. W h i l e i t i s . n o t p o s s i b l e t o o b t a i n t r u l y s i n g l e - h i t k i n e t i c s , t h i s c o n d i t i o n may be a p p r o x i m a t e d by:employing low l e v e l s o f e n d o n u c l e a s e and by a l l o w i n g t h e d e g r a d a t i o n t o proceed o n l y a s h o r t t i m e . 2 2 A r i g o r o u s m a t h e m a t i c a l a n a l y s i s o f t h e c o u r s e o f d e g r a d a t i o n o f a p o l y n u c l e o t i d e by an e n d o n u c l e a s e might p'rovide a u s e f u l b a s i s f o r e x p e r i m e n t a t i o n . However, even i f the problem i s f o r m u l a t e d w i t h g r e a t l y s i m p l i f i e d a s s u m p t i o n s , i t i s u n l i k e l y t h a t s o l u t i o n s can be o b t a i n e d (44a) f o r f e a t u r e s o t h e r than the e x p e c t e d s u r v i v a l o f o r i g i n a l polymers as the r e a c t i o n proceeds (44b, 44c). The use of low enzyme to s u b s t r a t e r a t i o should encourage s i n g l e - h i t k i n e t i c s . F u r t h e r , the short r e a c t i o n time and the use of c o n d i t i o n s f a v o r i n g s i n g l e - h i t degradat ion should d iscourage excess i ve format ion of low molecu lar weight f ragments. While i t is d e s i r a b l e to o b t a i n a l l products in e q u i v a l e n t amounts, the r e l a t i v e y i e l d s are not c r i t i c a l provided enough of each fragment is present to a l l o w i t s components to be i d e n t i f i e d . An important f a c t o r to cons ider is the a c t u a l number of p o t e n t i a l c leavage s i t e s possessed by the polymer. As the number of c leavage s i t e s becomes l a r g e , the number of d i s t i n c t fragments w i l l become too la rge f o r p r a c t i c a l execut ion of the method . 3 It is necessary to r e c o g n i z e , however, that only a l i m i t e d number of the t o t a l popu la t ion of fragments w i l l be requ i red to perform the a n a l y s i s . For the case of 20 c leavage s i t e s , fo r example, there e x i s t s the p o s s i b i l i t y of generat ing 230 d i s t i n c t fragments of which but 20 w i l l be d i s t i n c t fragments bear ing the o r i g i n a l 3 ' " terminus and an equal number w i l l bear the o r i g i n a l 5 1 - t e r m i n u s . A n a l y s i s of the s m a l l e s t ten d i s t i n c t 3 F o r a polymer of n c leavage s i t e s i t is p o s s i b l e to generate (n+1 (n+2) .j d i s t i n c t products by random cleavage provided 2 the composi t ions of fragments between c leavage po in ts are d i s t i n c t (45). For t h i s same polymer, on ly n of the t o t a l p o p u l a t i o n of fragments w i l l possess the o r i g i n a l l e f t -te rminus . S i m i l a r l y , n of the t o t a l popu la t ion of fragments w i l l possess the o r i g i n a l r i g h t - t e r m i n u s . 3 1 - f r a g m e n t s a n d t h e s m a l l e s t t e n 5 ' ~ e n d e d f r a g m e n t s c a n p r o v i d e t h e t o t a l s e q u e n c e . T h e p r e s e n c e o f s e c o n d a r y s t r u c t u r e i n c e r t a i n R N A s a n d t h e p o t e n t i a l o f e x p l o i t i n g i t t o r e s t r i c t n u c 1 e a s e - a c c e s s i b i 1 i t y h a s b e e n d i s c u s s e d a b o v e . I t m i g h t b e p o s s i b l e t o v a r y t h e n u m b e r o f p o t e n t i a l c l e a v a g e s i t e s f r o m a m a x i m u m v a l u e t o a l o w v a l u e m e r e l y b y m a n i p u l a t i n g t h e a m o u n t o f s t r u c t u r e a n R N A p o s s e s s e s . T h e o r e t i c a l l y , i t w o u l d b e p o s s i b l e t o a c h i e v e t h e n u m b e r o f c l e a v a g e p o i n t s w h i c h g e n e r a t e s a n i d e a l n u m b e r o f f r a g m e n t s t o p e r f o r m t h e a n a l y s i s . I t i s w o r t h w h i l e t o d i s c u s s , i n a n a p r i o r i m a n n e r , t h e s i t u a t i o n s w h i c h m i g h t b e m e t i n a t t e m p t i n g t h e p r o p o s e d a n a l y s i s , f i r s t o n a t R N A u s i n g r i b o n u c l e a s e T ^ d i g e s t i o n a n d s e c o n d , o n a l a r g e m o l e c u l a r w e i g h t R N A u n d e r c o n d i t i o n s i n w h i c h t h e n u m b e r o f c l e a v a g e s i t e s i s s m a l l . T h i s w i l l i n d i c a t e a n u m b e r o f f e a t u r e s o f t h i s p r o p o s e d m e t h o d n o t y e t d i s c u s s e d o r s u g g e s t e d . T o b e g i n , c o n s i d e r a p p l i c a t i o n o f t h e p r o p o s e d s e q u e n c e m e t h o d t o a t R N A . I f t h e p a r t i a l e n d o n u c l e a s e - d e g r a d a t i o n s t e p i s p e r f o r m e d w i t h r i b o n u c l e a s e T ^ , a l l g u a n y l i c a c i d r e s i d u e s a r e p o t e n t i a l c l e a v a g e s i t e s . T h e s e r e s i d u e s o c c u r i n t h e p r i m a r y s e q u e n c e a c c o r d i n g t o . n e a r - r a n d o m d i s t r i b u t i o n a n d h e n c e g u a n y l i c a c i d r e s i d u e s m a y o c c u r s i n g l y o r c l u s t e r e d . I t i s p r o b a b l e t h a t p a r t i a l R N a s e T ^ d i g e s t i o n c o u l d r e s u l t i n f o r m a t i o n o f a n u m b e r o f o l i g o m e r s d i f f e r i n g o n l y b y a s i n g l e g u a n y l i c a c i d r e s i d u e . These fragments could be d i f f i c u l t to separate and ana lyze i f the parent fragments were of c o n s i d e r a b l e l e n g t h . However, i f i n fo rmat ion were a v a i l a b l e o n ' t h e d i g e s t i o n products l i b e r a t e d by exhaust i ve d i g e s t i o n wi th p a n c r e a t i c RNase, such c l u s t e r s of g u a n y l i c a c i d res idues could be read i l y i d e n t i f i e d . Thus, the t o t a l degradat ion of the tRNA by p a n c r e a t i c RNase and RNase and t o t a l a n a l y s i s and c h a r a c t e r i z a t i o n of the products p r i o r to a p p l i c a t i o n of the proposed method would be u s e f u l in two ways. F i r s t , c e r t a i n over laps and hence, the order of c e r t a i n fragments would be deduced from the p r e l i m i n a r y s t u d i e s . Second, those regions r i c h in pur ines and p a r t i c u l a r l y those r i c h in g u a n y l i c a c i d res idues would be complete ly br idged and a n t i c i p a t e d from the p a n c r e a t i c RNase s t u d i e s . T h e r e f o r e , the a c t u a l number of fragments requ i red to deduce the t o t a l pr imary sequence could be s i g n i f i c a n t l y reduced. Another f e a t u r e of t h i s method deserves emphasis. If the complete RNase T^  d i g e s t i o n products are known and c h a r a c t e r -i z e d , then once t e r m i n a l - f r a g m e n t s have been separated from non - te rmina l fragments and from each o t h e r , the products l i b e r a t e d by exhaus t i ve d i g e s t i o n of the p u r i f i e d fragments need o n l y - b e i d e n t i f i e d to perform the a n a l y s i s . The i d e n t i f i c a t i o n step could be performed w i t h smal l amounts of m a t e r i a l . For example, the composi t ion of short fragments could be determined by chromatography on D E A E - c e l l u l o s e columns under c o n d i t i o n s known to ach ieve high r e s o l u t i o n of o l i g o n u c l e o t i d e mixtures (46). As fragments increase in s i z e , two-d imensional systems such as combined e l e c t r o -p h o r e t i c - c h r o m a t o g r a p h i c procedures (47) could be of v a l u e . Mapping procedures prov ide f a s t e r r e s o l u t i o n w i t h lower l e v e l s of m a t e r i a l t h a n . i s p o s s i b l e w i t h column procedures . That they do not ach ieve the q u a n t i t a t i v e r e s u l t s obta ined w i t h column procedures i s i r r e l e v a n t s i n c e mere i d e n t i f i c a t i o n is the o b j e c t i v e . Thus, the f a c t that smal l amounts of each component a re requ i red to perform the proposed a n a l y s i s counters a p o t e n t i a d isadvantage of the procedure. Th is d isadvantage is that in order to generate s u f f i c i e n t l e v e l s of u s e f u l f ragments , a c o n s i d e r a b l e amount of the o r i g i n a l m a t e r i a l may be r e q u i r e d . D i r e c t a p p l i c a t i o n of the proposed method as d e t a i l e d f o r s t u d i e s of tRNA sequences is not expected to be f e a s i b l e f o r s t u d i e s of the sequences of high molecu lar weight RNAs. In the l a t t e r c a s e , the la rge number of fragments which could be generated as w e l l as the d i f f i c u l t y in s e p a r a t i n g la rge o l i g o n u c l e o t i d e s which d i f f e r on ly by a few res idues would make a p p l i c a t i o n of the method i m p r a c t i c a l . With some m o d i f i c a t i o n s in approach, however, the method proposed might be of c o n s i d e r a b l e v a l u e . For example, i t could be used to extend sequence i n f o r m a t i o n i n from t h e two ends o f l a r g e RNAs. C o n s i d e r a b l y more v a l u e c o u l d be g a i n e d i f the p r o c e d u r e were a p p l i e d t o l a r g e RNAs i n which n u c l e a s e - a c c e s s i b i 1 i t y was mark e d l y reduced by s e c o n d a r y - s t r u c t u r e r e s t r i c t i o n as d i s c u s s e d beiow. Thach and B o e d t k e r (38) have r e p o r t e d t h a t l i m i t e d d i g e s t i o n o f R17 RNA w i t h p a n c r e a t i c RNase forms a p p r o x i m a t e l y ten fragments r e v e a l e d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f p r o d u c t s . Of t h e s e f r a g m e n t s , a t l e a s t f o u r were s t i l l i n p o s s e s s i o n o f the o r i g i n a l 3 l - t e r m i n u s . T h e i r r e s u l t s p r o v i d e an example of how the method proposed i n t h i s t h e s i s c o u l d be o f v a l u e f o r a n a l y s i s o f h i g h molecu 1 a r ' w e i g h t RNAs. The RNA would f i r s t be r e a c t e d w i t h r e a g e n t s c a p a b l e o f l a b e l l i n g a s p e c i f i c t e r m i n u s . N ext, p a r t i a l d e g r a d a t i o n o f the m o d i f i e d polymers under r e s t r i c t i v e c o n d i t i o n s as d e s c r i b e d by Thach and B o e d t k e r (38) , r e s o l u t i o n o f t e r m i n a l from n o n - t e r m i n a l l a b e l l e d f r a g m e n t s by B D - c e l l u l o s e chromatography, ** f o l l o w e d by r e s o l u t i o n on p o l y a c r y l a m i d e g e l s would p r o v i d e two f a m i l i e s ^ C e r t a i n s i n g l e - s t r a n d e d RNAs b i n d t e n a c i o u s l y t o B D - c e l l u l o s e (48) . If B D - c e l l u l o s e were a b l e t o d i m i n i s h the s e c o n d a r y - s t r u c t u r e o f such s p e c i e s o r the f r a g m e n t a t i o n p r o c e s s caused e x t e n s i v e s i n g l e - s t r a n d e d r e g i o n s t o form, then the i n c r e a s e d a f f i n i t y f o r B D - c e l l u l o s e r e s u l t i n g from t h e i n t r o d u c t i o n o f h y d r o p h o b i c groups might be d i m i n i s h e d t o a p o i n t where t h e s e p a r a t i o n o f fragments b e a r i n g d e r i v a t i v e s from t h o s e not p o s s e s s i n g d e r i v a t i v e s would be i m p o s s i b l e . Should t h i s p rove t o be the c a s e , a l t e r n a t e r o u t e s a c h i e v i n g the r e s o l u t i o n c o u l d be a t t e m p t e d . The p r i n c i p l e o f the method, however, would remain the same. o f fragments d e r i v e d from s p e c i f i c ends o f the m o l e c u l e . The v a r i o u s fragments c o u l d be used t o s t u d y r e q u i r e m e n t s f o r the i n i t i a t i o n o f t r a n s l a t i o n , t o s t u d y t h e i n f l u e n c e o f mRNA m a t e r i a l on the t r a n s l a t i o n o f more d i s t a l genes and t o d e t e r m i n e the o r d e r o f genes w i t h i n the o r i g i n a l polymer by i n v i t ro t r a n s l a t i o n . ( E x p e r i m e n t s l i k e t h i s have been performed on fragments o f R17 RNA (37.) F u r t h e r , the proposed p r o c e d u r e would g e n e r a t e and o r d e r l a r g e segments o f the RNA amenable to e x t e n d i n g the " S a n g e r - a n a 1 y s i s " w h i c h has a l r e a d y been performed on segments o f t h i s RNA (33)• P r o g r e s s made i n development o f the proposed sequence method w i l l now be p r e s e n t e d . EXPERIMENTAL (1) P r e p a r a t i o n o f * V-glycyl t R N A ^ r u d e and Naphthoxy-a c e t y l - ^ C - g l y c y l t R N A C r u d e . To 500 y l o f a s o l u t i o n c o n t a i n i n g the same components and the same c o n c e n t r a t i o n o f components as l i s t e d i n Step 7 of E x p e r i m e n t a l S e c t i o n o f P a r t I ( o m i t t i n g tRNA and g l y c i n e ) was 1 4 added 50-100 y l o f C - g 1 y c i n e ( u n i f o r m l y l a b e l l e d , 1 mCi and 9 ymoles per m i l l i l i t e r o f 10 mM h y d r o c h l o r i c a c i d ) and an equal volume o f 10 mM sodium h y d r o x i d e , 50 mg o f B o e h r i n g e r and Soehne s o l u b l e r i b o n u c l e i c a c i d , 200-400 y l o f H^ O and 1-2 ml o f a c r u d e tRNA s y n t h e t a s e p r e p a r a t i o n p r e p a r e d e x a c t l y as d e s c r i b e d i n Step 4 of E x p e r i m e n t a l S e c t i o n o f P a r t I. The combined components were i n c u b a t e d 10-20 minutes a t room t e m p e r a t u r e (22°) f o l l o w i n g a d d i t i o n o f enzyme. Then 1 ml o f i c e - c o l d 4 M sodium f o r m a t e (pH 4.0) and 2.5 volumes o f c o l d 95% e t h a n o l were added. The p r e c i p i t a t e w h i c h formed was c o l l e c t e d by c e n t r i f u g a t i o n , washed w i t h an e t h a n o l - a c e t a t e b u f f e r m i x t u r e (5 ml 1 M magnesium c h l o r i d e , 5 ml 1 M sodium a c e t a t e (pH 4.5), 490 ml 95% e t h a n o l ) t o remove r a d i o a c t i v i t y and p h e n o l - e x t r a c t e d e s s e n t i a l l y as d e s c r i b e d i n Step o f E x p e r i m e n t a l S e c t i o n o f P a r t I t o remove p r o t e i n . The aqueous-phases were combined and the tRNA was p r e c i p i t a t e d w i t h e t h a n o l as above. The p r e c i p i t a t e was r o u t i n e l y d i s s o l v e d i n 10 mM sodium a c e t a t e c o n t a i n i n g 2 mM EDTA (pH 5.4) i f 1^C-g1ycy1 -tRNA was d e s i r e d . I f N_-2-naphthoxyacety ] - C-g 1 ycy 1 - tRNA was d e s i r e d , the p r e c i p i t a t e was d i s s o l v e d i n c o l d 0.1 M t r i e t h a n o l a m i n e -h y d r o c h l o r i c a c i d (pH 4.4) and r e a c t e d w i t h 20 mg o f N.-hydroxy-s u c c i n i m i d e e s t e r o f 2 - n a p h t h o x y a c e t i c a c i d as d e s c r i b e d i n E x p e r i m e n t a l S e c t i o n o f P a r t I, Step 10. The p r e c i p i t a t e o b t a i n e d by a d d i t i o n o f e t h a n o l t o the r e a c t i o n m i x t u r e (whose pH was made 4.5) was r i n s e d w i t h t h e e t h a n o l - s o d i u m a c e t a t e b u f f e r m i x t u r e and f i n a l l y d i s s o l v e d i n 10 mM sodium a c e t a t e c o n t a i n i n g 2 mM EDTA (pH 5-4). Both 1^C-g1ycy1 -tRNA and i t s n a p h o x y a c e t y l a t e d c o u n t e r p a r t were s t o r e d i n the acetate-EDTA b u f f e r s a t -20°. (2) Large S c a l e I s o l a t i o n o f N_-2-naphthoxyacety 1 g 1 ycy 1 - tRNA^ ' y . The i n i t i a l s t a g e s o f the p r e p a r a t i o n o f N[-2-naphthoxy-a c e t y l g l y c y l - t R N A ^ ^ were e x a c t l y as d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n o f P a r t I. Steps 4 t o 11 were c a r r i e d o ut as d e s c r i b e d . The m a t e r i a l from the B D - c e l l u l o s e column e l u t e d by s o l u t i o n IV was r o u t i n e l y re-chromatographed a t pH 4-;;5 i n 1 M sodium c h l o r i d e on B D - c e l l u l o s e columns w i t h e l u t i o n a c h i e v e d by e t h a n o l g r a d i e n t s (0 t o 30% volume bas i s ) . (3) C h e l e x 100 Chromatography. N [ - 2 - N a p h t h o x y a c e t y l g l y c y l - t R N A ^ ' y from I tern 2 above was p r e c i p i t a t e d by a d d i t i o n o f 2.5 volumes o f 95% e t h a n o l , the p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n , r i n s e d w e l l w i t h e t h a n o l - a c e t a t e b u f f e r (490 ml 95% e t h a n o l , 5 ml 10 mM sodium a c e t a t e (pH 4.5), 5 ml 1 M magnesium c h l o r i d e ) and f i n a l l y d i s s o l v e d i n a s m a l l volume of 10 mM sodium a c e t a t e (pH 4.5). The r e s u l t a n t s o l u t i o n was loaded o n t o a column (1.2 x 1 4 cm) o f C h e l e x 100 e q u i l i b r a t e d w i t h 10 mM sodium a c e t a t e (pH 4.5). The column was d e v e l o p e d w i t h the l a t t e r b u f f e r and f r a c t i o n s w i t h h i g h A ^ ^ Q and r a d i o a c t i v i t y were poo l e d and s t o r e d a t -20° u n t i l needed. ( 4 ) Magnesium D e t e r m i n a t i o n . M i c r o e s t i m a t i o n of magnesium was performed as d e s c r i b e d by Orange and R h e i n ( 4 9 ) . D e t a i l s a r e g i v e n i n the legend t o F i g u r e 9-(5) Assays f o r RNase A c t i v i t y . A s s a y s f o r r i b o n u c l e a s e T^ a c t i v i t y were performed e s s e n t i a l l y as d e s c r i b e d by U c h i d a and Egami (51), e x c e p t c r u d e s o l u b l e RNA was used t h r o u g h o u t . D e t a i l s a r e g i v e n i n t h e 1egend t o F i g u r e 11. (6) Assay o f the S p e c i f i c i t y o f R i b o n u c l e a s e Ty The method d e v e l o p e d i n t h i s l a b o r a t o r y (52) was used to t e s t the s p e c i f i c i t y o f r i b o n u c l e a s e . To 15 mg o f c r u d e tRNA d i s s o l v e d i n 2 ml o f a p p r o p r i a t e b u f f e r was added r i b o n u c l e a s e T^ (8 x 10 W o r t h i n g t o n u n i t s o r 100 Sankyo u n i t s ) , a few drops o f c h l o r o f o r m and t h e m i x t u r e was i n c u b a t e d a t 22° o v e r n i g h t . To t h i s s o l u t i o n 9-0 ml o f 0.1 M T r i s - h y d r o c h l o r i c a c i d (pH 9.0), 0.25 ml o f 1 M MgCl^ and 15 mg c r u d e l y o p h i l i z e d snake venom were added and the m i x t u r e was i n c u b a t e d a t 37°. A f t e r one h o u r , an a d d i t i o n a l 0.25 ml 1 M magnesium c h l o r i d e and 15 mg c r u d e snake venom was added. The pH o f the i n c u b a t i o n m i x t u r e was m a i n t a i n e d between 8.8 and 9.0 t h r o u g h o u t the d i g e s t i o n p e r i o d of 18 h o u r s . The d i g e s t i o n m i x t u r e was then a p p l i e d t o a column (1.2 x 40 cm) of DEAE-cel1u1ose ( c a r b o n a t e form) e q u i l i b r a t e d w i t h H^O. E l u t i o n w i t h H^O was c o n t i n u e d a f t e r l o a d i n g u n t i l the e l u a t e had no a b s o r b a n c e a t 260 nm and then 0.4 M ammonium c a r b o n a t e was a p p l i e d . M o l a r ammonium c a r b o n a t e was a p p l i e d t o the column when the 0.4 M wash was c o m p l e t e . That p o r t i o n o f a p p l i e d m a t e r i a l e l u t e d by 0.4 M ammonium c a r b o n a t e was p o o l e d , taken r e p e a t e d l y t o d r y n e s s ( w i t h o c c a s i o n a l a d d i t i o n o f c o n c e n t r a t e d ammonia) by e v a p o r a t i o n under reduced p r e s s u r e a t 50°. The r e s i d u e was d i s s o l v e d i n 10 ml o f 0.1 M T r i s - a c e t a t e (pH 9-0) c o n t a i n i n g 25 mM magnesium c h l o r i d e . Crude snake venom (15 mg) and b a c t e r i a l phosphomonoesterase (25 .Ul o f a 34 u n i t , 10 mg per ml s o l u t i o n ) and a few drops o f c h l o r o f o r m were added and the s o l u t i o n was i n c u b a t e d a t 37° f o r 18 h o u r s . The d i g e s t i o n p r o d u c t s were loaded o n t o a column (1.8 x 120 cm) o f DEAE-Sephadex A-25 e q u i l i b r a t e d w i t h 40 mM ammonium c a r b o n a t e . The column was e l u t e d w i t h the l a t t e r s o l u t i o n . The f l o w r a t e o f 30 ml per hour was m a i n t a i n e d w i t h the a i d o f a pump and 20 m i n u t e (10 ml) f r a c t i o n s were c o l l e c t e d . N u c l e o s i d e s were then i d e n t i f i e d by U V - s p e c t r a l ana l y s i s . (7) Column Chromatography. DEAE-cel1u1ose (Whatman F i b r o u s DE22, 1.0 m-equiva1ent/g d r y w e i g h t ) was s t i r r e d i n t o 0.5 M HC1 (15 ml o f HC1 per g d r y D E A E - c e l 1 u l o s e ) , l e f t f o r 30 m i n u t e s , f i l t e r e d and washed on a Buchner f u n n e l u n t i l t he e f f l u e n t pH was 4. The exchanger was then s t i r r e d i n t o 0.5 M NaOH (15 ml NaOH per g d r y DEAE-c e l l u l o s e ) , l e f t 30 minutes and f i l t e r e d as above u n t i l t he e f f l u e n t pH was 8. The exchanger was s t i r r e d i n t o 0.5 M HC1 (e x c e s s ) and degassed under reduced p r e s s u r e . The DEAE-c e l l u l o s e was then added t o 2 M sodium c h l o r i d e and the f i n e s were removed by r e p e a t e d s e t t l i n g and d e c a n t a t i o n . When the a c e t a t e form o f the D E A E - c e l l u l o s e was r e q u i r e d , t he exchanger was washed w i t h 2 M sodium a c e t a t e (pH 4.5) on a Buchner f u n n e l . The exchanger was c o n v e r t e d t o the c a r b o n a t e form by the l a t t e r p r o c e d u r e e x c e p t 2 M ammonium c a r b o n a t e was used. Columns were packed u s i n g t he same t e c h n i q u e s as employed f o r t he p r e p a r a t i o n o f B D - c e l l u l o s e columns d e s c r i b e d i n P a r t I, E x p e r i m e n t a l S e c t i o n , C h r o m a t o g r a p h i c Methods. Flow r a t e s were m a i n t a i n e d w i t h t he a i d o f a pump and were r o u t i n e l y s e t a t 2 one ml per minute per cm column c r o s s - s e c t i o n a l a r e a . E l u t i o n was m o n i t o r e d w i t h the a i d o f an Isco UV-monitor r e c o r d i n g a b s o r b a n c e a t 254 nm. For d e t e r m i n a t i o n o f r a d i o a c t i v i t y , a l i q u o t s were added t o Bray's s o l u t i o n f o r s c i n t i l l a t i o n c o u n t i n g (53)• B D - c e l l u l o s e columns were p r e p a r e d and run as d e s c r i b e d i n P a r t I, E x p e r i m e n t a l S e c t i o n , Step 2. B i o - G e l P-2 was s w o l l e n i n H^O o v e r n i g h t , packed and e q u i l i b r a t e d w i t h a p p r o p r i a t e b u f f e r b e f o r e use. N a p h t h o y l - c e l 1 u l o s e was pre-washed w i t h 95% e t h a n o l , then w i t h 47-5% e t h a n o l , 10 mM sodium a c e t a t e (pH 4.5) and f i n a l l y e q u i l i b r a t e d w i t h 10 mM sodium a c e t a t e (pH 4.5) b e f o r e use. , Chromatography on D E A E - c e l l u l o s e w i t h l i n e a r g r a d i e n t s o f ammonium c a r b o n a t e (pH 8.6) was performed w i t h the a i d o f a g r a d i e n t - g e n e r a t o r e s s e n t i a l l y as d e s c r i b e d i n r e f e r e n c e (54). The g r a d i e n t s o l u t i o n s were c o n t a i n e d i n two Erlenmeyer f l a s k s o f e qual c r o s s - s e c t i o n a t equal h e i g h t so t h a t the g r a d i e n t would be l i n e a r . The v e s s e l s were c o n n e c t e d by a s i p h o n and s t o p p e r e d but each had an a i r b l e e d c o n s i s t i n g o f 2 meters o f t h i n - b o r e (0.06 i n c h e s ) p o l y e t h y l e n e t u b i n g . The a i r b l e e d s a l l o w e d a i r t o e n t e r the system as t h e l i q u i d was removed by pumping from t h e v e s s e l c o n t a i n i n g t h e l e s s c o n c e n t r a t e d s o l u t i o n , ( c o n s t a n t s t i r r i n g was m a i n t a i n e d i n t h i s v e s s e l w i t h the a i d o f a m a g n e tic s t i r r e r ) . The a i r b l e e d s a l s o s e r v e d as a k i n e t i c b a r r i e r t o d i f f u s i o n o f the v o l a t i l e components of the b u f f e r system. (8) D e s a l t i n g o f O l i g o n u c l e o t i d e s . O l i g o n u c l e o t i d e s were d e s a l t e d on D E A E - c e l l u l o s e w i t h t r i ethy1ammon i um b i ca rbonate ( 5 5 ) • O l i g o n u c l e o t i d e s b e a r i n g the j^-2-naphthoxyacety1 - C-g l y c y l s u b s t i t u e n t were d e s a l t e d on naphthoy1 e e l 1u1ose as d e s c r i b e d i n S e c t i o n 9 o r on B i o - G e l P-2 columns e q u i l i b r a t e d w i t h 10 mM sodium a c e t a t e (pH 5-8) c o n t a i n i n g 19% (v/v) e t h a n o l . (9) Method f o r I s o l a t i o n and P u r i f i c a t i o n o f R i b o n u c l e a s e T ^ - R e l e a s e d 3 ' - t e r m i n a l Fragments o f N[-2-Naphthoxyacety 1 g l y c y l - t R N A ^ . ( i ) D i g e s t i o n w i t h R i b o n u c l e a s e and Chromatography on B D - c e l l u l o s e . G l y To p u r i f i e d N^-2-naphthoxyacety 1 g 1 ycy 1 -tRNA^ £ (788 u n i t s and 91 x 10^ cpm) i n 10 ml 10 mM sodium a c e t a t e (pH 4.5) was added r i b o n u c l e a s e T^ (9 x 10 u n i t s o f W o r t h i n g t o n RT^) and c h l o r o f o r m (2 d r o p s ) . The m i x t u r e was i n c u b a t e d 18 hours a t room t e m p e r a t u r e (26°). The i n c r e a s e i n a b s o r b a n c e a t 260 nm was.28%. The d i g e s t was d i l u t e d t o 25 ml w i t h s o l u t i o n I ( t h e s e a r e e x a c t l y as d e s c r i b e d i n P a r t I, E x p e r i m e n t a l S e c t i o n Step 3) and loaded o n t o a column 1.2 x 12 cm o f B D - c e l l u l o s e e q u i l i b r a t e d i n s o l u t i o n I. (The c o m p o s i t i o n o f s o l u t i o n s I, I I , I I I and IV a r e d e s c r i b e d i n P a r t I, E x p e r i m e n t a l S e c t i o n , Step 3.) The column was d e v e l o p e d as d e s c r i b e d i n P a r t I, E x p e r i m e n t a l S e c t i o n , Step 11. The f i n a l s o l u t i o n a p p l i e d t o the column was s o l u t i o n IV e x c e p t i t c o n t a i n e d 47.5% (y/v) e t h a n o l . ( i i ) Removal o f E t h a n o l and N a C l . The m a t e r i a l e l u t e d by the l a t t e r s o l u t i o n (97-5 ^260 u n ' t s a n c ' 9^ x 10^ cpm) was p o o l e d , the e t h a n o l was removed under reduced p r e s s u r e u s i n g a r o t a r y e v a p o r a t o r and the e t h a n o l -f r e e s o l u t i o n was loaded (90 A^^Q u n i t s and 73 x 10** cpm) o n t o a column (10 x 2.8 cm) o f naphthoy1-eel 1ulose. The column was e l u t e d w i t h 10 mM sodium a c e t a t e (pH 4.5) and then w i t h 20 mM sodium a c e t a t e (pH 4.5) c o n t a i n i n g 47-5% (v/v) e t h a n o l . ( i i i ) Chromatography on DEAE-ce11u1ose i n the P r e s e n c e o f 7 M u r e a . M a t e r i a l e l u t e d by the l a t t e r s o l u t i o n (52.5 A, 260 u n i t s and 68 x 10** cpm) was p o o l e d , the e t h a n o l removed as b e f o r e , and the remainder made 7 M w i t h r e s p e c t t o u r e a . The pH was a d j u s t e d t o 5-05 and the r e s u l t a n t s o l u t i o n was loaded o n t o a column (80 x 0.6 cm) o f DEAE-cel1u1ose ( a c e t a t e form) e q u i l i b r a t e d w i t h 10 mM sodium a c e t a t e (pH 5-05) c o n t a i n i n g 7 M u r e a . The column was d e v e l o p e d w i t h a g r a d i e n t o f 200 ml e q u i l i b r a t i n g b u f f e r t o 200 ml 0.7 M sodium a c e t a t e c o n t a i n i n g 7 M urea (pH 5-05). When the g r a d i e n t had e x p i r e d , a wash o f 1.4 M sodium a c e t a t e c o n t a i n i n g 7 M urea (pH 5-05) was a p p l i e d . Flow r a t e s were 20 ml per hour and 20 m i n u t e (6.6 ml) f r a c t i o n s were c o l l e c t e d . The major peak, peak I , had 26.5 A, 260 4 u n i t s and 43 x 10 cpm, w h i l e a minor peak, peak I I , had 11.5 4 / ^260 u n ' t s a n (^ ^ x ^ c P m ' a n e a r ' y peak w i t h l i t t l e A^^ n p o s s e s s e d a p p r o x i m a t e l y 9 x 10 cpm p r o b a b l y r e p r e s e n t e d N_-2-n a p h t h o x y a c e t y 1 - g l y c i n e . 5 ( i v ) F u r t h e r C h r o m a t o g r a p h i c C h a r a c t e r i z a t i o n o f Peak I 01igonuc1eot i d e . The p o o l e d f r a c t i o n s of peak I were d i l u t e d t o 200 ml by t h e a d d i t i o n o f H^O and the s o l u t i o n was loaded o n t o a column (1.2 x 10 cm) of DEAE-cel1u1ose ( c a r b o n a t e form) e q u i l i b r a t e d w i t h w a t e r . The column was washed w i t h h^O t o remove urea and f i n a l l y w i t h 1 M ammonium c a r b o n a t e (pH 8.6). The e l u t e d m a t e r i a l (23-7 ^60 u n i t s and hi x 10 cpm) was l e f t a t room t e m p e r a t u r e f o r 3 h o u r s , d i l u t e d t w e n t y - f o l d and loaded o n t o a column (0.9 x 52 cm) o f DEAE-cel1ulose e q u i l i b r a t e d i n 10 mM ammonium c a r b o n a t e . A gradient(10 mM t o 1 M ammonium c a r b o n a t e (pH 8.6), 300 ml each) c o n t a i n e d i n a "low d i f f u s i o n " g r a d i e n t system ( d e s c r i b e d above under c h r o m a t o g r a p h i c s e c t i o n ) was a p p l i e d . (v) F u r t h e r R i b o n u c l e a s e T^ Treatment and Ch r o m a t o g r a p h i c C h a r a c t e r i z a t i o n o f Peak II O l i g o n u c l e o t i d e . The pH o f the p o o l e d peak II m a t e r i a l was a d j u s t e d 5The e l u t i o n p o s i t i o n o f J ^ - 2 - n a p h t h o x y a c e t y l g l y c i n e under s i m i l a r c o n d i t i o n s was found by t r e a t i n g c r u d e tRNA c o n t a i n i n g N_-2-naphthoxyacety1 - C - g l y c y l - t R N A w i t h d i l u t e ammonium h y d r o x i d e and c h r o m a t o g r a p h i n g the n e u t r a 1 i z e d s o l u t i o n on DEAE-cel1u1ose under s i m i l a r c o n d i t i o n s . The N_-2~naph t h o x y a c e t y 1 g 1 yc i ne group was l o c a t e d by a s s a y i n g column f r a c t i o n s f o r r a d i o a c t i v i t y . t o pH 8.6 by a d d i t i o n o f T r i s - H C l b u f f e r ( f i n a l c o n c e n t r a t i o n o f T r i s 1M), t h e s o l u t i o n was l e f t 3 hours a t room t e m p e r a t u r e and then d i l u t e d f i f t y - f o l d . The r e s u l t a n t s o l u t i o n was d e s a l t e d on a DEAE-ce11u1ose column w i t h triethy1ammonium b i c a r b o n a t e . A l l o f the r a d i o a c t i v i t y e l u t e d w i t h the f i r s t s a l t (20 mM) wash. The f i n a l s a l t wash (1 M) e l u t e d 10.8 A„,„ 260 u n i t s o f m a t e r i a l which was then d r i e d by e v a p o r a t i o n under reduced p r e s s u r e w i t h the a i d o f a r o t a r y e v a p o r a t o r . The r e s i d u e was d i s s o l v e d i n 2 ml 0.1 M ammonium b i c a r b o n a t e , pH 8.0. To t h i s s o l u t i o n was ..added 600 u n i t s (Worth i ngton) o f r i b o n u c l e a s e T^ and a few drops o f CHC1^ • A f t e r 12 h o u r s , the d i g e s t was made t o 25 ml by a d d i t i o n o f wa t e r and a n a l y z e d on a DEAE-ce11u1ose column (0.6 x 52 cm) de v e l o p e d w i t h ammonium c a r b o n a t e as d e s c r i b e d f o r . p e a k ( . o l i g o n u c l e o t i d e . (10) N u c l e o s i d e A n a l y s i s o f Peaks I and II 0 1 i g o n u c l e o t i d e s . The o l i g o n u c l e o t i d e s o f Peak I and Peak II c a r r i e d t o s t e p s ( i v ) and (v) above ( r e s p e c t i v e l y ) were i n d e p e n d e n t l y d e s a l t e d w i t h triethy1ammonium b i c a r b o n a t e . To 5-5 ^2S0 un'ts o f Peak I o l i g o n u c l e o t i d e (18.2 A£go u n ' t s t o t a l ) d i s s o l v e d i n 300 y l of 0.1 M ammonium b i c a r b o n a t e (pH 8.0) was added 50 y l o f a s o l u t i o n o f 35 u n i t s o f phosphomonoesterase and 10 mg o f cr u d e snake venom per ml. D i g e s t i o n was f o r 5 hours a t room t e m p e r a t u r e . The d i g e s t i o n m i x t u r e was d r i e d , d i s s o l v e d i n 75 y l o f 0.4 M ammonium f o r m a t e , pH 4.7, and a n a l y z e d by chromatography on a column (21 x 0.8 cm) o f Bio-Rad A-6 as d e s c r i b e d i n r e f e r e n c e (56.). The o l i g o n u c l e o t i d e o f Peak II (6.2 ^2S0 u n ' t s t o t a l ) w a s t r e a t e d as above, e x c e p t t h a t 75 y l of- t h e phosphomonoesterase-c r u d e snake venom p r e p a r a t i o n was added. (The d i g e s t i o n s to n u c l e o s i d e l e v e l and the subsequent c h r o m a t o g r a p h i c r e s o l u t i o n o f components were k i n d l y performed by Mr. W. B o l z e r . ) (11) S y n t h e s i s o f 2 - N a p h t h y l p h o s p h o r o m o r p h o l i d a t e . The p r o c e d u r e used f o l l o w e d r e f e r e n c e (57) • E i g h t mEqj(600 mg) o f 2-naphthy 1 phosphate, d i s o d i u m s a l t d i s s o l v e d i n 5 ml H^ O was loaded o n t o a 1.2 x 10 cm Dowex 50 column (Bio-Rad AG50W x 12, 200 t o 400 mesh; hydrogen f o r m ) . As l o a d i n g proceeded a p r e c i p i t a t e appeared. The w a t e r wash showed s t r o n g a b s o r b a n c e a t 325 nm so r e t e n t i o n was not c o m p l e t e . A f t e r the w a t e r wash, e l u t i o n was c o n t i n u e d w i t h a w a t e r p l u s 19% (v/v) e t h a n o l wash u n t i l no f u r t h e r a b s o r p t i o n a t 325 nm appeared. The combined w a t e r and w a t e r - e t h a n o l washes were c o n c e n t r a t e d a t reduced p r e s s u r e t o remove e t h a n o l , the pH was brought t o 7 by a d d i t i o n o f m o r p h o l i n e and the s o l u t i o n was c o n c e n t r a t e d t o a s y r u p . To t h e l a t t e r was added 10 ml t - b u t a n o l , 8 ml o f H^O, 1 ml (12.5 mmoles) o f m o r p h o l i n e and the r e s u l t a n t s o l u t i o n heated t o r e f l u x . To the r e f l u x i n g s o l u t i o n was added a^  s o l u t i o n o f 2.58 g (12.5 mmoles) d i c y c l o h e x y l c a r b o d i i m i d e i n 17 ml o f t - b u t a n o l . A d d i t i o n was d r o p w i s e over a 4 hour p e r i o d ; r e f l u x i n g was c o n t i n u e d t h r o u g h o u t the a d d i t i o n and f o r an a d d i t i o n a l 4 hours a f t e r a d d i t i o n was c o m p l e t e . The p r o d u c t was l e f t o v e r n i g h t , r o t a r y - e v a p o r a t e d t o d r y n e s s , w a t e r (15 ml) was added and the s o l u t i o n was l e f t a f u r t h e r 16 hours t o be s u r e a l l DCC had decomposed. The d i e y e 1ohexy1urea was removed by f i l t r a t i o n and the f i l t e r was r i n s e d l i b e r a l l y w i t h w a t e r . The combined f i l t r a t e s (250 ml) were e x t r a c t e d w i t h e t h e r ( f o u r times w i t h 100 ml p o r t i o n s ) ; the combined e t h e r l a y e r s were e x t r a c t e d w i t h w a t e r (2 x 30 ml) and the combined aqueous l a y e r s were c o n c e n t r a t e d to d r y n e s s . P y r i d i n e was added and the s o l u t i o n was e v a p o r a t e d t o a s y r u p ; t h i s was repeated s e v e r a l t i m e s . P y r i d i n e (5 ml) and 1 N sodium h y d r o x i d e (10 ml) were added and the m i x t u r e was e x t r a c t e d w i t h d i e t h y l e t h e r ( s e v e r a l times w i t h an equal volume). F i n a l l y , t h e aqueous phase was d i l u t e d t o 0.2 N sodium h y d r o x i d e and the s o l u t i o n was s t o r e d at 4°. For r e a c t i o n w i t h 5'~guanosine monophosphate or w i t h . tRNA, the nap h t h y l p h o s p h o r o m o r p h o 1 i d a t e was c o n v e r t e d t o i t s t r i e t h y l ammonium s a l t by passage t h r o u g h a Dowex 50 column (triethy1ammonium f o r m ) . (12) F o r m a t i o n o f Cety1trimethylammonium S a l t o f tRNA. The p r o c e d u r e d e s c r i b e d i n r e f e r e n c e (57) was f o l l o w e d . F i f t y mg cety1trimethy1ammonium bromide i n 10 ml H^O was added to 50 mg o f c r u d e tRNA d i s s o l v e d i n 10 ml 10 mM T r i s - H C l (pH 7-5) b u f f e r . The s o l u t i o n s were mixed, l e f t o v e r n i g h t a t k , c e n t r i f u g e d , and the p r e c i p i t a t e was washed f i v e times w i t h 10 ml H^O. The p r e c i p i t a t e was d i s s o l v e d i n p y r i d i n e and e v a p o r a t e d t o d r y n e s s . T h i s l a t t e r s t e p was r e p e a t e d s e v e r a l t imes. (13) R e a c t i o n o f Naphthy1phosphoromorpholidate w i t h C e t y l t r i m e t h y l a m m o n i u m S a l t o f tRNA. One hundred umoles of triethy1ammonium form o f 2-n a p h t h y l p h o s p h o r o m o r p h o l i d a t e i n H^ O and 100 umoles t r i - N -h e x y l a m i n e i n 5 ml o f p y r i d i n e were combined and r e p e a t e d l y e v a p o r a t e d t o d r y n e s s w i t h a d d i t i o n o f anhydrous p y r i d i n e between e v a p o r a t i o n s . Cety1trimethy1ammonium s a l t o f tRNA (2 umoles) i n p y r i d i n e was added t o t h e above s o l u t i o n o f the m o r p h o l i d a t e and t r i - N - h e x y 1 amine i n p y r i d i n e . The combined s o l u t i o n s were e v a p o r a t e d to d r y n e s s r e p e a t e d l y w i t h a d d i t i o n of anhydrous p y r i d i n e . A i r passed through d r y i n g tubes was a l l o w e d to e n t e r the e v a c u a t e d f l a s k s a t f i n a l s t a g e s . The f i n a l d r i e d m i x t u r e was t a k e n up i n 5 ml o f anhydrous dimethy1su1 f o x i d e . The r e a c t i o n was l e f t f o r 1 week a t room t e m p e r a t u r e . A f t e r t h i s t i m e , two volumes o f 1 M sodium c h l o r i d e was added (heat was g e n e r a t e d ) and seven volumes o f e t h a n o l (95%) was added. The p r e c i p i t a t e w h i c h formed a f t e r 1 hour a t -20° was c o l l e c t e d by c e n t r i f u g a t i o n , r e d i s s o l v e d i n 1 M sodium c h l o r i d e and c o l l e c t e d as b e f o r e . The s t e p s were r e p e a t e d once more and t h e p r e c i p i t a t e was f i n a l l y d i s s o l v e d i n s o l u t i o n I, and loaded o n t o a B D - c e l l u l o s e column (1.2 x 10 cm). The B D - c e l l u l o s e column was d e v e l o p e d as d e s c r i b e d under I tern 9 o f t h i s E x p e r i m e n t a l S e c t i o n . (14) R e a c t i o n o f tRNA w i t h 2 , 4 - D i n i t r o f l u o r o b e n z e n e (DNFB). The r e a c t i o n c o n d i t i o n s used were adapted from r e f e r e n c e (58) . S i n c e t h e i r e x p e r i m e n t a l d e t a i l s a r e g i v e n o n l y i n C h i n e s e , a d e t a i l e d m o d i f i c a t i o n o f t h e i r p r o c e d u r e i s d e s c r i b e d h e r e : t o 100 mg o f tRNA ( w i t h e t h a n o l - f r a c t i o n removed (59) ) d i s s o l v e d i n 8 ml o f 4% sodium b i c a r b o n a t e was added 0.36 mg DNFB d i s s o l v e d i n 10 ml o f 95% e t h a n o l . A d d i t i o n was over an 80 m i n u t e p e r i o d . D u r i n g the a d d i t i o n and t h r o u g h o u t the a d d i t i o n a l 100 minute r e a c t i o n p e r i o d , v i g o r o u s s t i r r i n g was m a i n t a i n e d w i t h the a i d o f a m a g n e tic s t i r r e r . The r e a c t i o n was t e r m i n a t e d by a d d i t i o n o f 5 ml 2 M sodium a c e t a t e (pH 4.3), 5 ml 1 M sodium c h l o r i d e , 1.5 ml 1 M magnesium c h l o r i d e and 150 ml o f c o l d 95% e t h a n o l . The p r e c i p i t a t e . w h i c h formed a f t e r s t o r a g e a t -20° f o r 2 hours was c o l l e c t e d and washed w i t h a n o t h e r 200 ml o f c o l d e t h a n o l . F i n a l l y , the p r e c i p i t a t e was d i s s o l v e d i n 20 ml s o l u t i o n I and e x t r a c t e d t h r e e times w i t h equal volume o f d i e t h y l -e t h e r . The aqueous s o l u t i o n was then loaded o n t o a column (1.2 x 10 cm) o f B D - c e l l u l o s e and d e v e l o p e d as d e s c r i b e d i n I tern 9 above. RESULTS AND DISCUSSION The o b j e c t o f t h i s s t u d y was t o d e v e l o p a novel, method o f n u c l e o t i d e sequence a n a l y s i s based on the f o l l o w i n g s t e p s : ( i ) s p e c i f i c , independent i n t r o d u c t i o n o f h y d r o p h o b i c r e a g e n t s o n t o the 3 I _ and 5 l _ t e r m i n a l s o f p o l y n u c l e o t i d e s , ( i i ) subsequent c l e a v a g e of the a p p r o p r i a t e l y d e r i f v a t i z e d polymer under c o n d i t i o n s w h i c h f a v o r r u p t u r e o f o n l y one bond per polymer, ( i i i ) s e p a r a t i o n o f fragments b e a r i n g the d e r i v a t i v e from t h o s e not b e a r i n g t h e d e r i v a t i v e , ( i v ) r e s o l u t i o n o f d e r i v a t i z e d f r a g m e n t s a c c o r d i n g t o c h a i n l e n g t h , (v) complete d e g r a d a t i o n o f t h e p u r i f i e d fragments from s t e p ( i v ) w i t h the reagent used t o g e n e r a t e t h e s e fragments f o l l o w e d by i d e n t i f i c a t i o n o f the components so l i b e r a t e d from each f r a g m e n t , and f i n a l l y , ( v i ) assembly of the d a t a o b t a i n e d from s t e p (v) t o o r d e r the c o m p o s i t e fragments from 5' and 3' ends to d e r i v e an unambiguous p r i m a r y sequence. A s c h e m a t i c d i a g r a m i n d i c a t i n g the p r i n c i p l e o f the method.has been p r e s e n t e d i n the i n t r o d u c t i o n . I s o l a t i o n and P a r t i a l C h a r a c t e r i z a t i o n o f t h e R i b o n u c l e a s e T.-R e l e a s e d 3'-Terminal Fragments o f N - 2 - n a p h t h o x y a c e t y l g l y c y l -t R N A G 1 y . The f e a s i b i l i t y o f the proposed method was a s s e s s e d by a t t e m p t i n g t o i s o l a t e and c h a r a c t e r i z e the 3'-terminal fragments r e l e a s e d by e x h a u s t i v e r i b o n u c l e a s e T d i g e s t i o n o f N - 2-naphthoxy-a c e t y l g l y c y l - t R N A y u s i n g the p r o c e d u r e s s u g g e s t e d i n the I n t r o d u c t i o n t o P a r t I I . T h i s s t u d y c o n s t i t u t e s a t e s t f o r a l l o f the n e c e s s a r y s t e p s e x c e p t t h a t o f o b t a i n i n g p a r t i a 1 - d i g e s t i o n o f the polymer which approaches random, s i n g l e - h i t d e g r a d a t i o n . F i r s t , t he a b i l i t y o f B D - c e l l u l o s e t o pe r f o r m the s e p a r a t i o n of u n m o d i f i e d o l i g o n u c l e o t i d e s o f v a r y i n g s i z e s from m o d i f i e d o l i g o n u c l e o t i d e s w i l l be a s c e r t a i n e d . Problems such as a c h i e v i n g q u a n t i t a t i v e r e c o v e r y from B D - c e l l u l o s e , o f o b t a i n i n g a s a t i s f a c t o r y method o f ' d e s a l t i n g o l i g o n u c l e o t i d e s b e a r i n g an a r o m a t i c r e s i d u e , o f t e s t i n g the s t r i n g e n c y o f the B D - c e l l u l o s e s e l e c t i o n s t e p by the subsequent s i z e - r e s o l u t i o n and c o m p o s i t e p r o d u c t - c h a r a c t e r i z a t i o n p r o c e d u r e s w i l l a l s o be examined. N _ - 2 - n a p h t h o x y a c e t y l g l y c y l - t R N A ^ y w a s i s o l a t e d and p u r i f i e d as d e s c r i b e d i n P a r t i o f t h i s t h e s i s . G e n e r a l l y , f o r l a r g e s c a l e i s o l a t i o n , t he p r o c e d u r e s f o l l o w e d were tho s e d e s c r i b e d i n Steps 1-11 o f the E x p e r i m e n t a l S e c t i o n o f P a r t I. G r a d i e n t chromatography of m a t e r i a l o b t a i n e d by c a r r y i n g out t h e s e p r o c e d u r e s t o s t e p 13 r e v e a l e d s u b s t a n t i a l l e v e l s o f c o n t a m i n a n t s which d i d not s i g n i f i c a n t l y i n t e r f e r e w i t h the u l t i m a t e p u r i t y o b t a i n e d f o r t R N A ^ l y o r t R N A ^ 7 (see F i g u r e 16 o f P a r t I, f o r example). S e v e r a l methods f o r f u r t h e r p u r i f y i n g the N_-2-n a p h t h o x y a c e t y l g l y c y l - t R N A ^ y p r e p a r a t i o n have been at t e m p t e d w i t h l i m i t e d s u c c e s s . A l l the components o b t a i n e d by e l u t i o n o f the B D - c e l l u l o s e column w i t h s o l u t i o n IV i n Step 1 1 , P a r t I p o s s e s s a h i g h a f f i n i t y f o r t h i s e xchanger. T h e r e f o r e , f u r t h e r f r a c t i o n a t i o n a t t e m p t s employed B D - c e l l u l o s e chroma-to g r a p h y w i t h g r a d i e n t e l u t i o n a c h i e v e d by r e a g e n t s known t o d i s r u p t h y d r o p h o b i c b o n d i n g . G r a d i e n t s o f m e t h a n o l , d i m e t h y l f o r m a m i d e and e t h a n o l were used. L e v e l s o f 30% (v/v) methanol i n 1 . 5 M sodium c h l o r i d e o r 5% (v/v) dimethy 1formamide i n 2 M sodium c h l o r i d e d i d not e l u t e N k 2-naphthoxyacety 1 g 1 ycy 1 -tRNA*?1 y from BD-c e l l u l o s e nor d i d t h e s e t r e a t m e n t s r e s u l t i n any s i g n i f i c a n t p u r i f i c a t i o n . The d e r i v a t i z e d tRNAs were q u a n t i t a t i v e l y r e c o v e r e d from the columns by e l u t i o n w i t h 1 . 5 M sodium c h l o r i d e c o n t a i n i n g 13% (v/v) of e t h a n o l o r o v e r the range 1 5 _ 2 0 % e t h a n o l (v/v) i f g r a d i e n t s from 0 t o 28.5% (v/v) e t h a n o l i n p r e s e n c e o f h i g h s a l t . w e r e used. However, l i t t l e p u r i f i c a t i o n r e s u l t e d . 6 N - 2 - n a p h t h o x y a c e t y l g l y c y l - t R N A G 1 y was e x h a u s t i v e l y d i g e s t e d w i t h r i b o n u c l e a s e T^ and the p r o d u c t s were chromatographed on BD-6 A p r o c e d u r e w h i c h c o u l d p r o v i d e pure and homogeneous N - 2-naphthoxy-a c e t y l g 1 y c y 1 - t R N A G l y or the c o r r e s p o n d i n g d e r i v a t i v e o f i t s i s o -a c c e p t i n g p a r t n e r would be t o . f o l l o w the c o m p l e t e p u r i f i c a t i o n p r o c e d u r e d e s c r i b e d i n P a r t I , c h a r g e and f i n a l l y d e r i v a t i z e the a p p r o p r i a t e l y p u r i f i e d and c h a r a c t e r i z e d t R N A G l y and tRNA^W s p e c i e s . However, t h i s l e n g t h y p r o c e d u r e w a s c o n s i d e r e d unwarranted f o r the r e q u i r e m e n t s h e r e . c e l l u l o s e . The r e s u l t s a r e shown i n F i g u r e 2. A l l o f the r a d i o a c t i v i t y o r i g i n a l l y p r e s e n t i n the g l y c i n e m o i e t y o f N_-2-naphthoxyacety1 - ^ C - g 1 y c y 1 - t R N A ^ y i s r e t a i n e d by B D - c e l l u l o s e u n t i l h i g h l e v e l s o f e t h a n o l a r e i n c o r p o r a t e d i n t o the e l u t i o n system. C o n v e r s e l y , the b u l k o f the d i g e s t i o n p r o d u c t s a r e e l u t e d by s a l t - s o l u t i o n s a l o n e . These r e s u l t s s u ggest t h a t o n l y m a t e r i a l s t i l l b e a r i n g the o r i g i n a l 3 ' - t e r m i n u s b i n d s t e n a c i o u s l y t o B D - c e l l u l o s e . The next s t e p was t o f r e e the m o d i f i e d o l i g o n u c l e o t i d e s from the h i g h l e v e l s o f e t h a n o l and o f s a l t needed f o r t h e i r e l u t i o n from B D - c e l l u l o s e . E t h a n o l was removed by e v a p o r a t i o n under reduced p r e s s u r e . The d e s a l t i n g o f o l i g o n u c l e o t i d e s b e a r i n g h y d r o p h o b i c a d d i t i v e s p r e s e n t e d a t e c h n i c a l d i f f i c u l t y u n t i l n a p h t h o y 1 - e e l 1u1ose was employed f o r t h i s purpose. O r i g i n a l l y , B i o - G e l P-2 columns ( w h i c h a r e r o u t i n e l y employed f o r d e s a l t i n g o f o l i g o n u c l e o t i d e s (60)) were used. However, d i f f i c u l t i e s were e n c o u n t e r e d when the m o d i f i e d o l i g o n u c l e o t i d e s were of s h o r t c h a i n l e n g t h s i n c e m a t e r i a l b e a r i n g the Nh2-naphthoxy-1 k a c e t y 1 g 1 y c i n e group (as i n d i c a t e d by C) i n t e r a c t e d w i t h t h e 7 E t h a n o l r a t h e r than some o t h e r r e a g e n t such as urea was employed to r e l e a s e the . 3 1 ~ t e r m i n a 1 fragments from B D - c e l l u l o s e . T h i s c h o i c e was governed by the f a c t t h a t e t h a n o l can be r e a d i l y removed by e v a p o r a t i o n under reduced p r e s s u r e . For i s o l a t i o n o f l a r g e r m o d i f i e d o 1 i g o n u c 1 e o t i d e s the use o f 8 M urea may be a d v i s a b l e as p r e c i p i t a t i o n o f p r o d u c t s c o u l d o c c u r w i t h the l e v e l s o f s a l t and e t h a n o l employed i n F i g u r e 2. Chromatography o f R i b o n u c l e a s e T ^ - d i g e s t e d N^-2-naphthoxyacety 1-g l y c y l - t R N A G l y on B D - c e l l u l o s e . N [ - 2 - N a p h t h o x y a c e t y l g l y c y l - t R N A G l y (788 u n i t s and 91 x 10^ cpm) d i s s o l v e d i n 10 ml 10 mM sodium a c e t a t e (pH 4.5) c o n t a i n i n g r i b o n u c l e a s e T (9 x 10^ u n i t s o f W o r t h i n g t o n R T ^ was i n c u b a t e d 18 hr a t 26°. The d i g e s t was chromatographed i n a s t e p w i s e manner w i t h e l u t i o n a c h i e v e d by the i n d i c a t e d s o l u t i o n s . S o l i d l i n e : ^260' d o t t e d l i n e : r a d i o a c t i v i t y from 1 \ - g 1 y c i n e . F r a c t i o n s were 10 ml/5 min. CN o CD CM I-< LU O < m i r o CO CD < 16 12 8 0.3M NaCl I.OM NaCL » l • , i ft. 0 I.OM NaCL + I.5M NaCL-h 4 . 7 % ETHANOL 4 7 . 5 % ETHANOL 1 I ru T 5 « i 4 O x 3 _ E 2 \ E • o 20 FRACTION NUMBER 1 I 3 0 ............... T 0 B i o - G e l P-2 such t h a t p a r t o f the r a d i o a c t i v i t y e l u t e d l a t e r from the column than d i d the s a l t peak. N a p h t h o y 1 - e e l l u l o s e ( o r i g i n a l l y p r e p a r e d by Dr. I.C. G i l l am i n t h i s l a b o r a t o r y ) was found t o be u s e f u l f o r d e s a l t i n g o l i g o -n u c l e o t i d e s b e a r i n g h y d r o p h o b i c r e s i d u e s . The m o d i f i e d o l i g o -n u c l e o t i d e s a r e s e l e c t i v e l y absorbed by the naphthoy1-eel 1ulose and c o n t a m i n a t i n g s a l t s can be removed by washing the column w i t h d i l u t e b u f f e r . The m o d i f i e d o l i g o n u c l e o t i d e s a r e s u b s e q u e n t l y r e c o v e r e d by e l u t i o n w i t h d i l u t e b u f f e r c o n t a i n i n g e t h a n o l . F i g u r e 3 i n d i c a t e s t h a t s a l t i s e f f i c i e n t l y removed from m o d i f i e d o l i g o n u c l e o t i d e s by t h i s p r o c e d u r e . The r e c o v e r y o f r a d i o a c t i v i t y i s e s s e n t i a 11y q u a n t i t a t i v e whereas a c o n s i d e r a b l e l o s s o f m a t e r i a l a b s o r b i n g l i g h t a t 260 nm i s o b s e r v e d . However, much of t h i s m a t e r i a l i s u n d o u b t e d l y non-nuc1eotide i n n a t u r e s i n c e e l u t i o n o f B D - c e l l u l o s e w i t h the l e v e l s o f e t h a n o l employed i n F i g u r e 2 i n v a r i a b l y l i b e r a t e s a s u b s t a n t i a l amount o f non-n u c l e o t i d e m a t e r i a l w h i c h a b s o r b s a t 260 nm. The p r o d u c t s from the n a p h t h o y l - e e l l u l o s e column were f r e e d o f e t h a n o l as b e f o r e and chromatographed on DEAE-cel1ulose i n the p r e s e n c e o f 7 M u r e a . T h i s s t e p was performed a t pH 5-05 t o p r o t e c t t h e a 1ka1i- 1abi1e bond between the h y d r o p h o b i c group and the o l i g o n u c l e o t i d e s . Chromatography under t h e s e c o n d i t i o n s s h o u l d s e p a r a t e o l i g o n u c l e o t i d e s a c c o r d i n g t o net c h a r g e and D e s a l t i n g o f N ^ 2 - n a p h t h o x y a c e t y 1 g 1 y c y 1 - o l i g o n u c 1 e o t i d e s on N a p h t h o y 1 - C e l l u l o s e . F r a c t i o n s 27 t o 31 of F i g u r e 2 (97-5 A 2 6 q u n i t s and 89 x 10 cpm) were p o o l e d , t h e e t h a n o l was removed and the p r o d u c t s were chromatographed (90 A^^Q u n i t s and 73 x 10 cpm) on a column (10 x 2.8 cm) o f n a p h t h o y 1 - e e l l u l o s e i n a s t e p w i s e manner w i t h e l u t i o n a c h i e v e d by the i n d i c a t e d s o l u t i o n s . S o l i d l i n e : ^ 2So'' dashed l i n e : c o n d u c t i v i t y , i n mi 11iMhos; d o t t e d l i n e : r a d i o -1 k a c t i v i t y from C - g l y c i n e . F r a c t i o n s were 20 ml/10 min. A B S O R B A N C E A T 2 S O N M ro O J J > cpm / mix I0~ 4 i i i i ° o C O O o N O C O N D U C T I V I T Y : hence e s s e n t i a l l y a c c o r d i n g t o s i z e (61). F i g u r e 4 shows t h a t two major peaks ( l a b e l l e d Peak I and Peak I I ) were p r e s e n t i n the p r o d u c t s l i b e r a t e d from the n a p h t h o y l -c e l l u l o s e column. A number o f minor peaks o c c u r as do minor 14 l e v e l s o f r a d i o a c t i v i t y i n a d d i t i o n t o a C-peak c e n t e r e d a t f r a c t i o n 20. T h i s l a t t e r peak i s p r o b a b l y Nh2-naphthoxyacety1 -g l y c i n e " * . The c o n t a m i n a t i o n known t o be p r e s e n t i n the H_~2-n a p h t h o x y a c e t y l g l y c y l - t R N A G 1 y p r e p a r a t i o n does not appear t o make a s i z e a b l e c o n t r i b u t i o n t o the p a t t e r n o b s e r v e d i n F i g u r e 4 . That m a t e r i a l p r e s e n t i n Peak I was r e c o v e r e d , i n c u b a t e d a t pH 8.6 t o remove the M^-2-naphthoxyacety 1 g 1 ycy 1-group, d e s a l t e d and f i n a l l y chromatographed as d e s c r i b e d i n the legend t o F i g u r e 5- T h i s c h r o m a t o g r a p h i c system was chosen f o r two r e a s o n s . F i r s t , i t i n v o l v e s e l u t i o n a t a d i f f e r e n t pH v a l u e than the c h r o m a t o g r a p h i c s t e p p r e c e e d i n g i t . Thus, c o n t a m i n a n t s which e l u t e w i t h the major peak from the s i z i n g - c o l u m n s h o u l d not e l u t e a t the same p o s i t i o n f o l l o w i n g t h i s s h i f t i n pH from 5-05 t o 8.6. F u r t h e r , t h i s c h r o m a t o g r a p h i c system has been shown t o be a p a r t i c u l a r l y p o w e r f u l means o f r e s o l v i n g o l i g o n u c l e o t i d e s (46, 5 4 ) . As i n d i c a t e d by F i g u r e 5 , Peak I m a t e r i a l c o n s i s t e d o f a s i n g l e component. The n u c l e o s i d e a n a l y s i s r e v e a l e d a s t o i c h i o m e t r y o f a d e n o s i n e and c y t i d i n e o f 2 t o 3, r e s p e c t i v e l y ( F i g u r e 6). T h i s a n a l y s i s agrees w i t h the r e s u l t s o f H e r b e r t and Smith (62, 63) who o b s e r v e d a sequence CpApCpCpA-OH f o r the 3 l - t e r m i n a l FIGURE 4 Chromatography o f N ^ 2 - n a p h t h o x y a c e t y l g l y c y l - o l i g o n u c l e o t i d e s on DEAE-cel1u1ose. M a t e r i a l i n f r a c t i o n s 16-20 o f F i g u r e 3 (52.5 ^260 u n ' t s a n c ' 68 x 10** cpm) was p o o l e d , t h e e t h a n o l was removed, the s o l u t i o n was made 7 M ur e a and pH 5-05 and t h e p r o d u c t s were chromatographed on a column (80 x 0.6 cm) o f DEAE-cel 1ulose ( a c e t a t e form) w i t h e l u t i o n a c h i e v e d by t h e i n d i c a t e d (dashed l i n e ) g r a d i e n t o f sodium a c e t a t e (pH 5.05) c o n t a i n i n g 7 M urea ( t o t a l volume 400 m l ) . Flow r a t e 20 ml/min; 20 min f r a c t i o n s . S o l i d l i n e : ^60' d o t t e d l i n e : 14 r a d i o a c t i v i t y from C - g l y c i n e . F r a c t i o n s 39 t o 47: Peak I; f r a c t i o n s 53 t o 59: Peak I I . ABSOREIANCE AT 260 NM O — K) CM £ bi o o o o o o p p o o — ro 01 A bi ij) ->i bo MOLARITY OF SODIUM ACETATE i 1 1 i i i i N * °> 0 0 O r o £ cpm /ml x I 0 " 3 * r ro M M Q> 00 o (VJ ^ Chromatography o f Peak I O l i g o n u c l e o t i d e on DEAE-cel1ulose. The m a t e r i a l i n Peak I o f F i g u r e h (26.5 A 260 u n i t s a n d ^ x 10^ cpm) was d e s a l t e d and i n c u b a t e d a t pH 8.6 f o r 3 h r s as d e s c r i b e d i n the E x p e r i m e n t a 1 . S e c t i o n , I tern S, and chromatographed on a column (52 x 0.9 cm) o f D E A E - c e l l u l o s e ( c a r b o n a t e form) w i t h e l u t i o n a c h i e v e d by the i n d i c a t e d (dashed l i n e ) g r a d i e n t o f ammonium c a r b o n a t e pH 8.6 (600 ml t o t a l v o l ume). Flow r a t e 55 m l / h r ; 15 min f r a c t i o n s . ( F r a c t i o n s 15 and 16 s h o u l d be p l o t t e d as 0.56 and O.83 A , r e s p e c t i v e l y ) . > o CD m 3 3 ABSORBANCE AT 260 NM O o o o o o o ro OJ CT) O o o o o o o-l J l r o O " OJ o 4 V O i 1 1 1 1 r \ \ \ \ \ V \ i i i i >i o p o o p — ro l p . c n co o M O L A R I T Y O F A M M O N I U M C A R B O N A T E N u c l e o s i d e A n a l y s i s o f Peak I O l i g o n u c l e o t i d e . F r a c t i o n s 14-17 of F i g u r e 5 were combined, d e s a l t e d , d r i e d and d i s s o l v e d i n 0.1 M ammonium b i c a r b o n a t e , pH 8.0 (18.6 A 0 /-„ v 260 u n i t s t o t a l ) . To 5-46 u n i t s was added 50 y l o f a s o l u t i o n c o n t a i n i n g 35 u n i t s o f phosphomonoesterase and 10 mg o f cr u d e snake venom per ml. A f t e r d i g e s t i o n (5 h r s at room te m p e r a t u r e ) the p r o d u c t s were a n a l y z e d on a column (21 x 0.8 cm) o f Bio-Rad A-6; e l u t i o n was a c h i e v e d by 0.4 M ammonium f o r m a t e , pH 4.7 a t 50°. F r a c t i o n s were 1 ml/1 min. FRACTION NUMBER o f y e a s t g l y c i n e tRNA. ( I t s h o u l d be mentioned a t t h i s p o i n t 14 t h a t a second component which p o s s e s s e d l a b e l from C - g l y c i n e f o l l o w i n g RNase - h y d r o l y s i s o f "^C-g1ycy1-tRNA^ 1 y was ob s e r v e d but not i s o l a t e d o r c h a r a c t e r i z e d by t h e s e w o r k e r s . F u r t h e r , B e r g q u i s t , i n a s t u d y o f f o u r g l y c i n e tRNAs o f y e a s t ( 6 4 ) , proposed a 3'"terminus of CpApCpCpCpA-OH f o r t h e s e i s o a c c e p t o r s . ) M a t e r i a l i n Peak II o f F i g u r e 4 was r e c o v e r e d , f r e e d o f urea and s a l t , i n c u b a t e d a t pH 8.6 t o remove N_-2-naphthoxyacety 1-g l y c i n e group and r e d i g e s t e d w i t h r i b o n u c l e a s e . The l a t t e r s t e p was performed t o be c e r t a i n t h a t Peak II m a t e r i a l was not an i n c o m p l e t e l y d i g e s t e d fragment w h i c h c o u l d ' b e c o n v e r t e d t o Peak I o l i g o n u c l e o t i d e on f u r t h e r i n c u b a t i o n w i t h r i b o -n u c l e a s e Jy F o l l o w i n g t h e s e s t e p s , the p r o d u c t s were chromato-graphed on DEAE-ce11u1ose e x a c t l y as performed f o r a n a l y s i s o f Peak I o l i g o n u c l e o t i d e . F i g u r e 7 i n d i c a t e s t h a t s l i g h t c o n t a m i n a t i o n was p r e s e n t . The s i n g l e , major component e l u t e d l a t e r i n the g r a d i e n t than d i d the Peak I component. These r e s u l t s s u g gest t h a t the Peak I t component i s not an i n c o m p l e t e l y degraded p r e c u r s o r o f Peak I component but r a t h e r i t i s d i s t i n c t and 1 a r g e r . The r e s u l t s of the n u c l e o s i d e a n a l y s i s o f Peak t l o l i g o -n u c l e o t i d e f u r t h e r i n d i c a t e t h e u n i q u e n e s s o f t h i s f r a g m e n t . F t g u r e 8 shows t h a t no g u a n o s i n e r e s i d u e s appeared a f t e r snake Chromatography o f Peak II O l i g o n u c l e o t i d e on D E A E - C e l l u l o s e . M a t e r i a l p r e s e n t i n Peak II o f F i g u r e k (11.5 ^260 u n ' t s a n c ' 11 x 10 cpm) was i n c u b a t e d 3 hrs a t pH 8.6, d e s a l t e d and f u r t h e t r e a t e d w i t h r i b o n u c l e a s e T^ f o r 12 hrs ' :as d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n , I tern 9- The m a t e r i a l (11 A^^Q u n i t s ) was chromatographed on a column (52 x 0.6 cm) o f DEAE-cel1ulose ( c a r b o n a t e form) w i t h e l u t i o n a c h i e v e d by the i n d i c a t e d (dashed l i n e ) g r a d i e n t o f ammonium c a r b o n a t e , pH 8.6 ( t o t a l volume 600 m Flow r a t e 55 ml/min; 15 ml f r a c t i o n s . A B S O R B A N C E AT 2 6 0 N M n > o H O 00 m 33 o o o o o — ro J> cn oo ° MOLARITY OF AMMONIUM CARBONATE FIGURE 8 N u c l e o s i d e A n a l y s i s o f Peak II O l i g o n u c l e o t i d e . F r a c t i o n s 22 and 23 of F i g u r e 7 were combined, d e s a l t e d , d r i e d and d i s s o l v e d i n 500 y l o f 0.1 H ammonium b i c a r b o n a t e , pH 8 .0 (6.2 &2()0 u n ' t s t o t a l ) , c o n t a i n i n g 75 y l o f a s o l u t i o n c o n t a i n i n g 35 u n i t s o f phosphomonoesterase and 10 mg o f c r u d e snake venom per m l. A f t e r d i g e s t i o n (5 h r s a t room t e m p e r a t u r e ) the p r o d u c t s were a n a l y z e d on a column (21 x 0 .8 cm) o f Bio-Rad A -6 w i t h e l u t i o n a c h i e v e d by 0 .4 M ammonium f o r m a t e , pH 4 . 7 , a t 50°. F r a c t i o n s were 1 ml/1 min. CN venom and phosphomonoesterase d e g r a d a t i o n , p r o v i n g i t was indeed a l i m i t - d i g e s t i o n p r o d u c t . F u r t h e r , the n u c l e o s i d e a n a l y s i s r e v e a l e d a s t o i c h i o m e t r y o f 1 u r i d i n e t o 2 a d e n o s i n e t o k c y t i d i n e r e s i d u e s . Peak II o l i g o n u c l e o t i d e , t h e n , i s p r o b a b l y a h e p t a n u c l e o t i d e . The r a t i o o f r a d i o a c t i v e l a b e l i n Peaks I and II as w e l l as the r a t i o o f o l i g o n u c l e o t i d e m a t e r i a l i n t h e s e peaks seen i n F i g u r e k s u g g e s t t h a t they may be d e r i v e d from t R N A ^ y and t R N A ^ y , r e s p e c t i v e l y . On the b a s i s o f t h i s a s s u m p t i o n i t can t e n t a t i v e l y be c o n c l u d e d t h a t d i f f e r e n c e s i n the p r i m a r y sequences o f tRNA^' y and t R N A ^ ^ have been f o u n d . The 3 l _ t e r m i n a l sequences a r e p r o b a b l y GpCpApCpCpA-OH (63) and Gp(Cp,Cp,Up,Ap) CpCpA-OH, r e s p e c t i v e l y . F u r t h e r c h a r a c t e r i z a t i o n o f b o t h fragments i s r e q u i r e d to f i n a l i z e t he s t r u c t u r e s . The l i m i t - c o n d i t i o n s t u d y p r e s e n t e d above d e m o n s t r a t e d t h e e f f e c t i v e n e s s o f B D - c e l l u l o s e i n p e r f o r m i n g the s o r t i n g - s t e p , i n t r o d u c e d the u t i l i t y o f a new d e s a l t i n g method and e s t a b l i s h e d the e f f e c t i v e n e s s o f the s i z i n g - p r o c e d u r e and the h i g h - r e s o l u t i o n column. A f t e r t h e s e s t u d i e s were i n i t i a t e d , a paper by E. Wimmer and M.E. Reichmann appeared w h i c h d e s c r i b e d the u t i l i t y o f BD-c e l l u l o s e in i s o l a t i n g the 3 l _ t e r m i n u s o f s a t e l l i t e t o b a c c o n e c r o s i s v i r u s RNA t o which had been added an a r o m a t i c r e s i d u e ( 6 5 ) . T h e i r p r o c e d u r e i s t h e r e f o r e o p e r a t i o n a l l y s i m i l a r t o what has j u s t been d e s c r i b e d , e x c e p t t h a t t h e y have used t h i s as an end-group method and not as t h e b a s i s o f a p r o c e d u r e f o r i s o l a t i n g a f a m i l y o f fragments as i s the goal h e r e . S i m i l a r l y , F i e r s and c o - w o r k e r s have d e s c r i b e d (66) an o p e r a t i o n a l l y s i m i l a r method f o r the i s o l a t i o n o f the 5 I _ t e r m i n u s o f RNA but a g a i n . t h e p r o c e d u r e has been used as an end-group method. In t h i s c a s e , the 5 , - t e r m i n u s o f MS2 RNA 1 2c was r e a c t e d w i t h p h e n y l d i a z o m e t h a n e (or p- I-phenyIdiazomethane) to form a y b e n z y l - (or p- I-benzy 1 -) 5 1 _ t r i phosphory 1 guanos i ne ended m o l e c u l e . Subsequent c l e a v a g e and chromatography o f p r o d u c t s on B D - c e l l u l o s e a l l o w e d a f a c i l e method o f i s o l a t i n g the b e n z y l a t e d - 5 ' - t e r m i n a l fragment. S t u d i e s on P a r t i a l D e g r a d a t i o n o f N-2-naphthoxyacety1-g l y c y l - t R N A w i t h R i b o n u c l e a s e . 1 k A s t u d y o f n u c l e a s e a c t i o n on N^-2-naphthoxyacety 1 - C-g l y c y l - t R N A was next i n i t i a t e d t o a t t e m p t t o f i n d c o n d i t i o n s a p p r o x i m a t i n g random, s i n g l e - h i t d e g r a d a t i o n . The f i r s t s t e p was t o e s t a b l i s h c o n d i t i o n s i n w h i c h tRNA would be l e a s t l i k e l y t o p o s s ess s i g n i f i c a n t s e c o n d a r y and t e r t i a r y s t r u c t u r e . T h i s c o n d i t i o n might be met when tRNA was d i s s o l v e d i n s o l u t i o n s d e v o i d o f magnesium,1ow i n i o n i c s t r e n g t h , and c o n t a i n i n g h i g h c o n c e n t r a t i o n s o f c h a o t r o p i c r e a g e n t s . tRNA p r e p a r a t i o n s were f r e e d o f magnesium by passage t h r o u g h -columns o f Bio-Rad Chelex-100 as d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n . T h i s r e s i n i s composed o f a s t y r e n e l a t t i c e b e a r i n g i m i n o d i a c e t i c a c i d exchange groups and F i g u r e 9 shows t h a t i t e f f e c t i v e l y removes magnesium. tRNA p r e p a r a t i o n s p r e c i p i t a t e d i n the p r e s e n c e o f magnesium t o wh i c h EDTA had been added t o c h e l a t e r e s i d u a l magnesium were found ( F i g u r e 9) t o c o n t a i n h i g h l e v e l s o f r e s i d u a l magnesium. The a b i l i t y o f tRNA t o compete w i t h EDTA f o r magnesium i o n i s not known but i t has been r e p o r t e d t h a t a s m a l l number o f d i v a l e n t ions per tRNA m o l e c u l e p l a y a major r o l e i n m a i n t a i n i n g s t r u c t u r e . Cohn et_ aj_. (67) o b s e r v e d t h a t t h e s e d i v a l e n t i o n s , a p p r o x i m a t e l y 6 per tRNA m o l e c u l e , r e s i d e a t s t r o n g , c o o p e r a t i v e b i n d i n g s i t e s i n the tRNA m o l e c u l e . The as s a y f o r magnesium i o n used i n F i g u r e 9 s h o u l d d e t e c t t h e s e l e v e l s o f magnesium i n the C h e l e x - t r e a t e d s a m p l e s . 8 D i l u t e sodium a c e t a t e was r o u t i n e l y used t o b u f f e r the N_-2-naphthoxyacety1g1ycy1 -tRNA p r e p a r a t i o n s and a d d i t i o n o f urea to a f i n a l c o n c e n t r a t i o n o f 7 molar was used t o p r o v i d e a hydrogen- and hydro p h o b i c - b o n d d i s r u p t i n g a g e n t . T a b l e I shows r e a d i n g s f o r a b s o r p t i o n o f 260 nm l i g h t o b s e r v e d f o r equal w e i g h t s o f c r u d e tRNA d i s s o l v e d i n v a r i o u s s o l u t i o n s . On the 8 T h i s s t a t e m e n t w i l l not be t r u e i f the s t a b i l i t y o f the C l a y t o n Yellow-magnesium complex i s l e s s than the s t a b i l i t y o f the tRNA-magnesium complex. That the r e a c t i o n i s performed i n 7'5% sodium h y d r o x i d e s u g g e s t s t h i s i s u n 1 i k e l y however. Magnesium D e t e r m i n a t i o n . S o l u t i o n s c o n t a i n e d 250 y l o f C l a y t o n Y e l l o w (7-5 mg per 100 ml H 20), 250 y l p o l y v i n y l a l c o h o l (0.1% s o l u t i o n , 99% h y d r o l y z e d , v i s c o s i t y 28-32 cps a t 23°), 250 y l o f H 20 c o n t a i n i n g e i t h e r magnesium s u l f a t e o r tRNA samples and 500 y l 7.5% sodium h y d r o x i d e . The a d s o r p t i o n o f the a l k a l i n e s o l u t i o n s was measured a t 560 nm. The s t a n d a r d c u r v e was p r e p a r e d o v e r the range o f 0.5 t o 5.0 yg magnesium s u l f a t e per 250 y l . C o n t r o l samples were ^-2-naphthoxy-a c e t y l g l y c y l - t R N A p r e p a r e d as d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n , I tern I; C h e l e x - t r e a t e d samples were e q u i v a l e n t l e v e l s o f s i m i l a r tRNA p r e p a r a t i o n s chromatographed on Chelex-100 as d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n p r i o r t o a s s a y . S t a n d a r d C a l i b r a t i o n Curve f o r Magnesium. o < 0.3 o L A 4J 0.2 ro <D o c ro 0.1 1 1 .0 2.0 3-0 yGram Magnes i um. o LTV O c ro < 0.3 0.2 0.1 Magnesium D e t e r m i n a t i o n o f C o n t r o l and C h e l e x - T r e a t e d tRNA P r e p a r a t i o n s , Cont r o l C h e l e x - T r e a t e d 0.125 0.25 0.375 mGram tRNA. 0.50 0.625 T a b l e i . I n f l u e n c e o f V a r i o u s S o l v e n t s on the Absorbance o f Crude tRNA. c , . . " Observed Absorbance S o l u t i o n a t 260 nm H 20 1.03 7 M u r e a , 20 mM sodium a c e t a t e , 2 mM EDTA, pH 5-4 1.00 7 M u r e a , 5 mM EDTA, pH 5-4 1.07 0.3 M sodium c h l o r i d e 0.83 A l l s o l u t i o n s (10 ml t o t a l volume) r e c e i v e d 10 y l o f a s o l u t i o n o f 50 mg c r u d e tRNA d i s s o l v e d i n 1 ml H2O. Absorbance a t 260 nm was read a g a i n s t the a p p r o p r i a t e s o l u t i o n w i t h no tRNA. b a s i s o f t h i s s i m p l e h y p e r c h r o m i c i t y s t u d y , tRNAs d i s s o l v e d i n d i l u t e a c e t a t e o r EDTA and 7 M urea p o s sess l i t t l e s t r u c t u r e . Agents c a p a b l e o f d i s r u p t i n g s t r u c t u r e i n p o l y n u c l e o t i d e s a r e a l s o c a p a b l e o f d i s r u p t i n g the s t r u c t u r e o f p o l y p e p t i d e s . Both r i b o n u c l e a s e and p a n c r e a t i c RNase were found t o r e t a i n t h e i r a c t i v i t y i n 7 M u r e a . I t was next n e c e s s a r y t o d e c i d e what n u c l e a s e t o use. Of the two commonly used endonuc1 eases r i b o n u c l e a s e T, c l e a v e s a t fewer s i t e s i n the model polymer than p a n c r e a t i c RNase and i t was s e l e c t e d f o r t h i s r e a s o n . ( F o r r i b o n u c l e a s e the number o f c l e a v a g e s i t e s assumes a v a l u e o f 17~25 f o r most tRNAs whereas f o r p a n c r e a t i c RNase the v a l u e may range from 30-40.) The proposed r o u t e , t h e n , was t o p e r f o r m a p a r t i a l d i g e s t i o n w i t h r i b o n u c l e a s e T^ , remove the enzyme from t h e degraded IN-2-naphthoxyacety1g1ycine-tRNA, and a n a l y z e the d e g r a d a t i o n p r o d u c t s by chromatography on DEAE-cel1u1ose i n the p r e s e n c e o f 7 M u r e a . The c o n d i t i o n s sought were the f o l l o w i n g : ( i ) A^£Q p r o f i l e s showing components r a n g i n g from s m a l l s i z e t o i n t a c t tRNA s i z e w i t h a d i s t r i b u t i o n ( i n d i c a t e d by the ^260 P r°f'^ e) t e n d i n g toward l a r g e r m o l e c u l e s , and ( i i ) a d i s t r i b u t i o n o f "^C (which r e v e a l s the p o p u l a t i o n o f o l i g o n u c l e o t i d e s s t i l l b e a r i n g the o r i g i n a l 3 l - e n d o f the polymer) i n d i c a t i n g the f o r m a t i o n of a l a r g e number o f fragments r a n g i n g i n s i z e from the l i m i t RNase T . - r e l e a s e d fragments t o D i g e s t i o n s o f c r u d e tRNA c o n t a i n i n g N_-2-naphthoxyacety 1 -14 C-glycy1-tRNA w i t h r i b o n u c l e a s e were performed w i t h v a r y i n g l e v e l s o f enzyme a t v a r i o u s t e m p e r a t u r e s f o r v a r i o u s t i m e s . R e a c t i o n s were performed a t pH 5-4 and a t the t e r m i n a t i o n o f the r e a c t i o n , the pH was r a p i d l y lowered t o 4.5 o r 4.0 and h i g h l e v e l s o f m e r c a p t o e t h a n o l were added t o i n h i b i t the r i b o n u c l e a s e (69). The d i g e s t i o n p r o d u c t s were loaded i m m e d i a t e l y o n t o D E A E - c e l l u l o s e columns f o r a n a l y s i s . A l a r g e number o f e x p e r i m e n t s were pe r f o r m e d . F i g u r e 10 shows the r e s u l t s o f a t y p i c a l e x p e r i m e n t . 14 A l t h o u g h a number o f C-peaks r a n g i n g i n s i z e from the l i m i t fragment ( e l u t i n g a t f r a c t i o n 41-45) t o a p p a r e n t l y i n t a c t m o l e c u l e s a r e a p p a r e n t , t h e r e i s e v i d e n c e o f a marked tendency f o r f o r m a t i o n o f the l i m i t - d i g e s t i o n fragment. (The peak o f r a d i o a c t i v i t y c e n t r e d a t f r a c t i o n 26 i s p r o b a b l y N_-2-naphthoxyacety 1 - ^ V-g l y c i n e 5 . . ) I n i t i a l l y , i t was reasoned t h a t the l a t t e r f e a t u r e might be e l i m i n a t e d by r e d u c i n g the e x t e n t o f d e g r a d a t i o n . However, i t was l a t e r o b s e r v e d t h a t the nuc1 e a s e - a c t i v i t y was not b e i n g c o n t r o l l e d a f t e r the o r i g i n a l d i g e s t i o n p e r i o d . M e r c a p t o -e t h a n o l was found t o be i n e f f e c t i v e i n c o n t r o l l i n g r i b o n u c l e a s e T^ as has been r e p o r t e d by o t h e r s (70, 71). 9 9 R e d u c t i o n o f d i s u l f i d e groups i n p r o t e i n s by t h i o l r e a g e n t s g e n e r a l l y proceeds more r a p i d l y a t pH v a l u e s more a l k a l i n e than were used here (105). T h i s may a c c o u n t f o r the f a i l u r e o f the t h i o l s used i n t h i s t h e s i s t o i n a c t i v a t e RNase T.. DEAE-Cel 1 u l o s e Chromatography o f a P a r t i a l R i b o n u c l e a s e T'j D i g e s t i o n of N - 2 - n a p h t h o x y a c e t y l g l y c y l - t R N A . • N-2-naphthoxyacety1glycyl^tRNA (5-0 mg crude tRNA' charged w i t h 1 4 C - g l y c i n e and d e r i v a t i z e d as d e s c r i b e d i n P a r t I I , E x p e r i m e n t a l S e c t i o n , I tern l ) d i s s o l v e d i n 0.5 ml o f 5 mM EDTA (pH 5-4) c o n t a i n i n g 7 M urea was d i g e s t e d w i t h 6 yg (3 x 10 W o r t h i n g t o n u n i t s ) o f W o r t h i n g t o n RT1 r i b o n u c l e a s e T^ f o r 4.5 min a t 37° when 1 ml o f 7 M u r e a , 10 mM sodium a c e t a t e (pH 4.5) c o n t a i n i n g 15 y l o f m e r c a p t o e t h a n o l was added. The m i x t u r e was chromatographed on a column (102 x 0.7 cm) o f DEAE-ce11u1ose ( c h l o r i d e form) e l u t e d w i t h the i n d i c a t e d (dashed l i n e ) g r a d i e n t o f sodium c h l o r i d e c o n t a i n i n g 10 mM sodium a c e t a t e , pH 4.5 ( t o t a l volume 600 m l ) . Flow r a t e , 22 m l / h r ; 30 min f r a c t i o n s . Heavy s o l i d l i n e : absorbance a t 254 nm; s o l i d l i n e : r a d i o a c t i v i t y from ' ^ C - g l y c i n e . FRACTION NO S t u d i e s on the S t a b i l i t y , pH-Dependent A c t i v i t y and  S p e c i f i c i t y o f R i b o n u c l e a s e . The need t o c o n t r o l o r remove r i b o n u c l e a s e T^ a f t e r the i n i t i a l d i g e s t i o n s t e p became a p p a r e n t a f t e r numerous a t t e m p t s t o o b t a i n p r e d i c t a b l e . k i n e t i c s o f d e g r a d a t i o n i n s t u d i e s l i k e t h a t d e s c r i b e d i n F i g u r e 10 f a i l e d . P h e n o l , w h i c h i s o f t e n employed t o remove t h i s n u c l e a s e from d i g e s t i o n p r o d u c t s , c o u l d not be used. A n a l y s i s o f r a d i o a c t i v i t y i n the o r g a n i c and aqueous phases f o l l o w i n g phenol 1 4 e x t r a c t i o n o f r i b o n u c l e a s e T^ d i g e s t e d N_-2-naphthoxyacety 1 - C-g l y c y l - t R N A i n d i c a t e d t h a t much o f the r a d i o a c t i v i t y d i s s o l v e d i n the o r g a n i c phase. The l a b e l w hich d i s s o l v e d i n the o r g a n i c phase was not h y d r o l y z e d N_-2-naph t h o x y a c e t y 1 g 1 yc i ne as i n d i c a t e d by t h e f o l l o w i n g s t u d y . Samples o f c r u d e N_-2-naphthoxyacety 1 -14 C-g1ycy1 -tRNA were i n c u b a t e d w i t h d i l u t e ammonium h y d r o x i d e , r i b o n u c l e a s e T o r no a d d i t i v e . F o l l o w i n g i n c u b a t i o n , ammonium h y d r o x i d e was n e u t r a 1 i z e d . a n d . a 1iquots from the t h r e e d i g e s t s were a p p l i e d i n d e p e n d e n t l y t o Whatman 3 mm paper. A f t e r d e s c e n d i n g chromatography had been performed w i t h S o l v e n t I, the r a d i o a c t i v i t y was l o c a t e d by s t r i p - c o u n t i n g . R a d i o a c t i v i t y from the u n t r e a t e d sample was l o c a t e d e x c l u s i v e l y a t the o r i g i n ; r a d i o a c t i v i t y from a 1 k a 1 i - t r e a t e d sample was found a t p o s i t i o n s of Rf 0.79 and 0.83 (presumably N^-2-naphthoxyacety 1-^C-g 1 yc i ne and the c o r r e s p o n d i n g amide, r e s p e c t i v e l y ) and r a d i o a c t i v i t y from the r i b o n u c l e a s e t r e a t e d sample was found a t and near the o r i g i n . No r a d i o a c t i v i t y was found a t p o s i t i o n s c o r r e s p o n d i n g t o Rf 0.7 t o 0.9- These r e s u l t s i n d i c a t e t h a t p h e n o l - s o l u b l e r a d i o a c t i v i t y i s not N_-2-naphthoxyacety 1 g 1 yc i ne but N^-2-naphthoxyacety 1 g 1 ycy 1-ol i g o n u c l e o t ides . (The pH o f s o l u t i o n s d u r i n g p h e n o l - e x t r a c t i o n was c o n t r o l l e d so t h a t h y d r o l y s i s a t t h i s s t a g e was e l i m i n a t e d . ) H e r b e r t and Smith r e p o r t e d p h e n o l - s o l u b i 1 i t y o f t y r o s y 1 - o l i g o n u c 1 e o t i d e s r e l e a s e d 14 by r i b o n u c l e a s e T^ d i g e s t i o n o f C - l a b e l l e d t y r o s y l - t R N A (70) and Zachau has r e p o r t e d t h a t the Y - c o n t a i n i n g h e x a n u c l e o t i d e o b t a i n e d Phe from p a n c r e a t i c RNase d i g e s t i o n o f tRNA ( y e a s t ) was somewhat p h e n o l - s o l u b l e (72). P r e c e d e n t , t h e r e f o r e , e x i s t s f o r the o b s e r v a t i o n t h a t N [ - 2 - n a p h t h o x y a c e t y l g l y c y l - o l i g o n u c l e o t i d e s a r e p h e n o l - s o l u b 1 e . C h l o r o f o r m , w h i c h has been used t o d e n a t u r e n u c l e a s e s (73), was found not t o e x t r a c t t h e s e p r o d u c t s from the aqueous phase, but was i n e f f i c i e n t a t d e n a t u r i n g r i b o n u c l e a s e A l t e r n a t e r o u t e s which would a l l o w removal o f r i b o n u c l e a s e T^ from the p r o d u c t s o r d e s t r u c t i o n o f the enzyme w h i l e s p a r i n g the i n t e g r i t y o f the p r o d u c t s were sought. For example, metal ions such as z i n c were r e p o r t e d t o c o m p l e t e l y i n h i b i t r i b o n u c l e a s e (69), but i n h i b i t i o n c o u l d not be o b s e r v e d . T reatments such.as h e a t - i n a c t i v a t i o n , z i n c - h e a t t r e a t m e n t s and z i n c - h e a t - m e r c a p t o e t h a n o 1 c o m b i n a t i o n s f a i l e d t o a c h i e v e i n a c t i v a t i o n . C o n t r o l was not a c h i e v e d by l o w e r i n g the pH t o 3 and a d d i n g l a r g e amounts o f p e p s i n t o d e s t r o y the n u c l e a s e . An o u t s t a n d i n g d i f f i c u l t y i n t h e s e i n i t i a l s t u d i e s was t h a t a l e n g t h y a s s a y f o r c l e a v a g e c o n d i t i o n s was b e i n g employed. For each e x p e r i m e n t , chromatography on D E A E - c e l l u l o s e f o l l o w e d by a n a l y s i s o f the ^2So P r ° f ' l e s a r , d r a d i o a c t i v i t y d i s t r i b u t i o n s were r e q u i r e d . In o r d e r t o i n v e s t i g a t e the p r o p e r t i e s o f r i b o n u c l e a s e more e f f e c t i v e l y , a m o d i f i c a t i o n o f the assay d e s c r i b e d by U c h i d a and Egami (51) was employed. As d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n , c r u d e tRNA was employed as the s u b s t r a t e . The a b i l i t y o f r i b o n u c l e a s e t o degrade c r u d e tRNA i n the p r e s e n c e and absence o f urea at v a r i o u s pHs was d e t e r m i n e d . F i g u r e 11, which shows the r e s u l t s o f . o n e such s t u d y , r e v e a l s t h a t the pH optimum i n u r e a i s a t l e a s t 3 u n i t s lower than the v a l u e a n t i c i p a t e d f r o m . r e f e r e n c e (69). The phenomenon appears t o be s u b s t r a t e r d e p e n d e n t and p r o b a b l y r e f l e c t s p r o t o n a t i o n o f c y t i d i n e and a d e n o s i n e r e s i d u e s w i t h a c o n c o m i t a n t d e s t a b i 1 i z a t i o n o f d o u b l e - s t r a n d e d r e g i o n s . Urea appears t o f a c i l i t a t e t h i s proposed s t r u c t u r e - r e d u c t i o n , s i n c e the e f f e c t seen i n the aqueous system i s much s m a l l e r . That the optimum f a l l s o f f r a p i d l y a t lower pH v a l u e s may be due t o the r e q u i r e m e n t s f o r c a r b o x y l groups i n RNase t o be i o n i z e d f o r a c t i v i t y (74). pH A c t i v i t y P r o f i l e f o r R i b o n u c l e a s e Tt Degrading tRNA i n the Pr e s e n c e and Absence o f 7 M Urea. S o l u t i o n s c o n t a i n i n g 3 mg crude tRNA d i s s o l v e d i n 250 y l o f H 20 or 7 M u r e a , 250 y l o f 0.2 M o f a p p r o p r i a t e b u f f e r , 100 y l o f 20 mM EDTA (pH 5-4), 300 y l o f HO o r 7 M urea and 25 y l o f W o r t h i n g t o n RT1 r i b o n u c l e a s e T- (300 u n i t s , added a t z e r o time) were i n c u b a t e d 5 min ( f o r u r e a - c o n t a i n i n g s o l u t i o n s ) o r 10 min ( f o r aqueous s o l u t i o n s ) when 0.25 ml o f 0.75% u r a n y l a c e t a t e i n 25% aqueous p e r c h l o r i c a c i d was added. The s o l u t i o n s were i m m e d i a t e l y mixed, c e n t r i f u g e d and 100 y l o f the s u p e r n a t a n t d i l u t e d t o a t o t a l o f 5 ml w i t h H^O. The A 2gg was determ i n e d a g a i n s t c o n t r o l s l a c k i n g r i b o n u c l e a s e Tij c a r r i e d t h r o u g h the same p r o c e d u r e . A c i d - s o l u b l e absorbance a t 260 nm i s p l o t t e d f o r the f o l l o w i n g s o l u t i o n s : o and • , 0.2 M g l y c i n e - H C l b u f f e r (0.2 M g l y c i n e , pH a d j u s t e d by a d d i t i o n o f h y d r o c h l o r i c a c i d ) w i t h and w i t h o u t 7 M u r e a , r e s p e c t i v e l y a t pH 2.2 and 3.0; - • and • , 0.2 M sodium a c e t a t e b u f f e r (0.2 M a c e t i c a c i d , pH a d j u s t e d by a d d i t i o n o f sodium h y d r o x i d e ) w i t h and w i t h o u t 7 M u r e a , r e s p e c t i v e l y a t pH 4.5, 5.0 and 5.4; A and • , 0.2 M T r i s - h y d r o c h 1 o r i c a c i d b u f f e r (0.2 M T r i s , pH a d j u s t e d by a d d i t i o n o f h y d r o c h l o r i c a c i d ) w i t h and w i t h o u t 7 M u r e a , r e s p e c t i v e l y a t pH 7-5 and 8.5. A C I D - S O L U B L E A B S O R B A N C E A T 2 6 0 N M O p O O o o — ro OJ J> CJI I 1 1 1 1 —1 The p o s s i b i l i t y t h a t t h e r e s u l t s seen i n F i g u r e 11 were due t o a c o n t a m i n a n t n u c l e a s e was examined i n s e v e r a l ways. A l l r i b o n u c l e a s e T^ p r e p a r a t i o n s used i n t h e s e s t u d i e s were s u b j e c t e d t o p h e n o l - t r e a t m e n t p r i o r t o use i n o r d e r t o remove p o s s i b l e p hosphatase (75) o r r i b o n u c l e a s e T^ c o n t a m i n a t i o n (68). The l a t t e r two enzymes a r e d e n a t u r e d o r i n s o l u b l e i n p h e n o l , whereas r i b o n u c l e a s e T^ i s r e a d i l y s o l u b l e i n t h i s r e a g e n t . Treatment o f the phenol-phase w i t h e t h e r a l l o w s r e c o v e r y of p u r i f i e d r i b o n u c l e a s e T^ i n q u a n t i t a t i v e y i e l d (68). A v a r i e t y o f r i b o n u c l e a s e T^ p r e p a r a t i o n s were f u r t h e r p u r i f i e d and c h a r a c t e r i z e d as t o t h e i r p H - a c t i v i t y p r o f i l e s . W o r t h i n g t o n RT.^  enzyme was chromatographed on.Sephadex G-75 and on DEAE-c e l l u l o s e w i t h g r a d i e n t e l u t i o n as d e s c r i b e d i n r e f e r e n c e 69-In both c a s e s , a s i n g l e p r o t e i n peak (measured by a b s o r b a n c e a t 220 nm s i n e e neg1igib1e absorbance a t 280 nm was o b s e rved) and a c o r r e s p o n d i n g n u c l e a s e - a c t i v i t y peak was o b s e r v e d . R i b o -n u c l e a s e T^ was a l s o i s o l a t e d from C a l b i o c h e m CB RNase (a c r u d e RNase p r e p a r a t i o n c o n t a i n i n g both r i b o n u c l e a s e T^ and T ) by chromatography on D E A E - c e l 1 u l o s e under c o n d i t i o n s known to s e p a r a t e t h e s e two n u c l e a s e s (68, 69)- The r e g i o n of the e l u t i o n p r o f i l e a t w h i c h r i b o n u c l e a s e T^ was e x p e c t e d t o appear p o s s e s s e d n u c l e a s e a c t i v i t y . Enzyme i s o l a t e d on the l a t t e r column as wel1 as samples of Sankyo r i b o n u c l e a s e T^ and W o r t h i n g t o n RT. enzyme p u r i f i e d both by Sephadex G-75 and D E A E - c e l l u l o s e chromatography a l l showed p H - a c t i v i t y p r o f i l e s as seen i n F i g u r e 11. To f u r t h e r i n v e s t i g a t e the p o s s i b i l i t y t h a t c o n t a m i n a t i n g enzymes were c a u s a l agents f o r the p H - a c t i v i t y p r o f i l e , a s t u d y o f the s p e c i f i c i t y o f t h e s e n u c l e a s e p r e p a r a t i o n s under v a r i o u s c o n d i t i o n s and pH v a l u e s was u n d e r t a k e n . The s p e c i f i c i t y was a s c e r t a i n e d u s i n g an end-group method d e v e l o p e d i n t h i s l a b o r a t o r y (52). The method r e l i e s on the i n a b i l i t y o f c r u d e snake venom enzymes to remove the 5'-phosphate group o f a n u c l e o t i d e w h i c h a l s o b e a r s a 3'-phosphate. ( T h i s c o n d i t i o n i s o b s e r v e d o n l y i f t h e pH of the d i g e s t i o n m i x t u r e i s m a i n t a i n e d above pH 8.8.) Thus, tRNA d i g e s t e d w i t h r i b o n u c l e a s e T^ y i e l d s a m i x t u r e o f n u c l e o t i d e s and o l i g o n u c l e o t i d e s whose 3 ' " t e r m i n a l m o i e t i e s a r e m a i n l y e i t h e r g u a n o s i n e ~ 3 1 - p h o s p h a t e or a g u a n o s i n e -2 ' , 3 ' - c y c l i c phosphate. A d d i t i o n o f c r u d e snake venom t o t h i s m i x t u r e w i l l (at pH 8.8 - 9) degrade a l l r e s i d u e s e x c e p t t h o s e b e a r i n g a 3 l - p h o s p h a t e t o n u c l e o s i d e l e v e l ; t h o s e r e s i d u e s p o s s e s s i n g a'. 3 l - p h o s p h a t e group w i l l be c o n v e r t e d t o n u c l e o t i d e -3 1 , 5 1 " d i p h o s p h a t e s . The l a t t e r p r o d u c t s a r e r e a d i l y s e p a r a t e d from n u c l e o s i d e s and, f o l l o w i n g removal o f t h e phosphate groups w i t h phosphomonoesterase and c r u d e snake venom, th e c o m p o s i t i o n o f the o r i g i n a l n u c l e o s i d e ~ 3 1 , 5 ' ~ d i p h o s p h a t e s can be d e t e r m i n e d as n u c l e o s i d e s . On the b a s i s o f r i b o n u c l e a s e T^ s p e c i f i c i t y , onjly g u a n o s i n e , m e t h y l a t e d g u a n o s i n e d e r i v a t i v e s ( e x c e p t 2'-O-methy] g u a n o s i n e ) , and i n o s i n e a r e a n t i c i p a t e d as b e i n g d e r i v e d from t h e n u c l e o s i d e - 3 1 , 5 1 ~ d i p h o s p h a t e s . T a b l e 2- shows the r e s u l t s o f t h e end-group a n a l y s i s o f a number o f r i b o n u c l e a s e T^ p r e p a r a t i o n s under v a r i o u s c o n d i t i o n s . C l e a v a g e was not as s p e c i f i c as a n t i c i p a t e d , s i n c e s u b s t a n t i a l c l e a v a g e a t a d e n y l i c a c i d r e s i d u e s was a p p a r e n t i n a l l c a s e s . The l a c k o f s p e c i f i c i t y f o r r i b o n u c l e a s e T^ has been r e p o r t e d (76, 77) and d i s c u s s e d (78). As found by C a n t o n i et_ aj_. (77), the use o f h i g h enzyme l e v e l s and long i n c u b a t i o n p e r i o d s may be r e s p o n s i b l e f o r the l o s s o f s p e c i f i c i t y . No e v i d e n c e f o r changed s p e c i f i c i t y i s found i n d i g e s t s performed a t pH k - 5 i n the p r e s e n c e o f 7 M u r e a . The a b i l i t y o f a number o f r e a g e n t s t o i n a c t i v a t e r i b o -n u c l e a s e T.| was s t u d i e d u s i n g t h e m o d i f i e d nuc 1 e a s e - a s s a y o f U c h i d a and Egami (51). R e c e n t l y , d i e t h y l p y r o c a r b o n a t e (DEPC) has been used i n p r o c e d u r e s f o r e x t r a c t i n g n u c l e i c a c i d s from p l a n t (79) and b a c t e r i a l s o u r c e s (80) because o f i t s a b i l i t y t o i n a c t i v a t e ribonuc1 e a s e s . The mechanism by which i t i n a c t i v a t e s p a n c r e a t i c RNase (81) s u g g e s t s t h a t the reagent could r e a c t w i t h n u c l e i c a c i d s as w e l l . T h i s has r e c e n t l y been v e r i f i e d (82, 83a,83b). DEPC was found t o i n a c t i v a t e r i b o n u c l e a s e T^ ( F i g u r e 12), b u t , i n c o n t r a s t t o i t s i r r e v e r s i b l e i n a c t i v a t i o n o f p a n c r e a t i c RNase, the i n a c t i v a t i o n o f r i b o n u c l e a s e T^ was t r a n s i e n t . I f DEPC and r i b o n u c l e a s e T^ were p r e i n c u b a t e d , T a b l e 2. S p e c i f i c i t y o f V a r i o u s R i b o n u c l e a s e T^ P r e p a r a t i o n s , (see t e x t f o r e x p l a n a t i o n s ; a s s a y s were performed as d e s c r i b e d i n the E x p e r i m e n t a l S e c t i o n , I tern 6 ) . r- • J n • A o / „ u n i t s f o r : n ^. _ Enzyme and D i g e s t i o n 260 R a t i o G C o n d i t i o n s c" U A G A W o r t h i n g t o n RT,; pH 7-5, no u r e a N.D. N.D. 11 .7 51 .8 4.4 W o r t h i n g t o n RT,, p h e n o l -e x t r a c t e d ; pH 5-0, 7 M urea 2.4 4.1 11.3 57.8 5.1 W o r t h i n g t o n RT,, phenol -e x t r a c t e d , chromatographed on D E A E - c e l l u l o s e ; pH 4.5; 7 M u r e a 1.8 3-2 4.1 22.8 5-5 Sankyo T,; pH 4.5, 7 M urea 8.4 4.9 10.1 57-5 5-7 N o n - n u c l e o s i d e m a t e r i a l e l u t e s a t the same p o s i t i o n ; d e t e r m i n a t i o n s a r e v a r i a b l e and not e x c l u s i v e l y a measure o f c y t i d i n e p r e s e n t . N.D. = not d e t e r m i n e d . E f f e c t o f D i e t h y l p y r o c a r b o n a t e on R i b o n u c l e a s e T j A c t i v i t y . S o l u t i o n s o f 30 A^Q u n i t s o f p u r i f i e d N_-2-naphthoxyacety 1 -g l y c y 1 - t R N A ^ y i n 1 ml o f 20 mM sodium a c e t a t e c o n t a i n i n g 7 M urea (pH 4 . 5 ) , 200 y l d i e t h y l p y r o c a r b o n a t e o r no a d d i t i v e , 5 u n i t s (Sankyo) o f Sankyo r i b o n u c l e a s e Tj o r no enzyme, were i n c u b a t e d a t 22°. At the time i n d i c a t e d , 100 y l a l i q u o t s were withdrawn and added t o 0.25 ml o f 0.75% u r a n y l a c e t a t e i n 25% aqueous p e r c h l o r i c a c i d . The m i x t u r e was c e n t r i f u g e d , 100 y l o f the s u p e r n a t a n t was added t o 2 ml o f H^O and the absorbance a t 260 nm was d e t e r m i n e d . The A^ Q^ v a l u e s a r e p l o t t e d as a p e r c e n t a g e o f the maximum A^ Q^ v a l u e o b s e r v e d f o r samples which l a c k e d DEPC but c o n t a i n e d r i b o n u c l e a s e Tt. n -, 5 u n i t s o f r i b o n u c l e a s e T ^ # , 5 u n i t s o f r i b o n u c l e a s e T^ and 200 y l o f DEPC a t z e r o t i m e ; O , minus r i b o n u c l e a s e T'^  c o n t r o l and samples i n which 200 y l DEPC was added t o r i b o n u c l e a s e T1| (5 u n i t s ) and p r e - i n c u b a t e d 1 o r 2 hr p r i o r t o a d d i t i o n o f s u b s t r a t e (which was added a t z e r o t i m e ) . Iks R e a c t i o n Time (minutes) DEPC removed by e t h e r e x t r a c t i o n , i n t a c t N.-2-naphthoxyacety 1 -14 C - g l y c y l - t R N A added and the m i x t u r e chromatographed on DEAE-c e l l u l o s e i n the p r e s e n c e o f 7 M u r e a , n e a r l y c o m p l e t e d i g e s t i o n o c c u r r e d d u r i n g chromatography. Chromatography i n the p r e s e n c e o f DEPC was i m p r a c t i c a l s i n c e the reagent decomposed w i t h e v o l u t i o n o f a gas w h i c h p r e c l u d e d p r o p e r a n a l y t i c a l chromatography The a b i l i t y o f a d i t h i o t h r e i t o l - a m m o n i a m i x t u r e t o i n a c t i v a t e r i b o n u c l e a s e T^ has been r e p o r t e d r e c e n t l y (71). The need f o r ammonia (or the need f o r a pH h i g h e r than 4 t o 5) t o o b t a i n t h i s i n a c t i v a t i o n i s . s u g g e s t e d by the r e s u l t s shown i n F i g u r e 13-Even i n the p r e s e n c e o f h i g h l e v e l s o f d i t h i o t h r e i t o 1 (0.1 M) r i b o n u c l e a s e T^ r e t a i n e d c o n s i d e r a b l e a c t i v i t y compared to u n t r e a t e d c o n t r o l s . N - b r o m o s u c c i n i m i d e (NBS) i s a p o t e n t and i r r e v e r s i b l e i n h i b i t o r o f r i b o n u c l e a s e T^ (84) . However^ NBS r a p i d l y degrades n u c l e i c a c i d r e s i d u e s i n tRNA under m i l d c o n d i t i o n s and s h o r t r e a c t i o n times (85) and hence, has not been used i n t h e s e e x p e r i m e n t s . R i b o n u c l e a s e T^ i s a l s o i n h i b i t e d by r e a c t i o n w i t h i o d o a c e t i c a c i d a t pH 5-5 (86). However, t h i s r e a c t i o n i s s l o w and marked p r o t e c t i o n i s p r o v i d e d by p r e s e n c e o f s u b s t r a t e s . P h o t o - o x i d a t i o n . ( 8 7 a , 87b) and r e a c t i o n w i t h 2 - h y d r o x y - 5 ~ n i t r o b e n z y l bromide (98) (a reagent s p e c i f i c f o r t r y p t o p h a n r e s i d u e s ) i n a c t i v a t e the enzyme as does i n c u b a t i o n ©f the enzyme above pH 9.0 (69). These l a t t e r p r o c e d u r e s were E f f e c t of D i t h i o t h r e i t o l on R i b o n u c l e a s e Tlj A c t i v i t y . S o l u t i o n s c o n t a i n e d 3 mg o f crude tRNA d i s s o l v e d i n 1 ml o f 20 mM sodium acetate-, 7 M urea (pH 4.5); 0 o r 8 mg o f DTT added 1 hr p r i o r t o a d d i t i o n o f s u b s t r a t e ; 6000 W o r t h i n g t o n u n i t s o f r i b o n u c l e a s e Tj o r no a d d i t i v e . At the times n o t e d , 500 y l o f 0.75% u r a n y l a c e t a t e i n 25% aqueous p e r c h l o r i c a c i d was added, the m i x t u r e was c e n t r i f u g e d , and 100 y l o f the s u p e r n a t a n t was w i t h d r a w n and d i l u t e d to 5 ml w i t h h^O. The absorbance a t 260 nm was d e t e r m i n e d and p l o t t e d as p e r c e n t a g e o f the maximum v a l u e o b s e r v e d f o r the samples w h i c h c o n t a i n e d r i b o n u c l e a s e T'-j but no DTT. • , r i b o n u c l e a s e T^ a l o n e ; A , 8 mg o f DTT added t o r i b o n u c l e a s e T j i n 1 ml b u f f e r f o r 1 hour p r i o r t o a d d i t i o n o f s u b s t r a t e j - O , no add i t i o n . R e a c t i o n Time ( m i n u t e s ) . c o n s i d e r e d i m p r a c t i c a l f o r . t h e purposes here because they would d e s t r o y the i n t e g r i t y o f not o n l y the n u c l e a s e but a l s o the n u c l e i c a c i d s o r n u c l e i c a c i d d e r i v a t i v e s . A t t e m p t s to b i n d r i b o n u c l e a s e c o v a l e n t l y t o i n s o l u b l e polymers were made s i n c e s e p a r a t i o n o f t h e i n s o l u b l e - p o l y m e r l i n k e d n u c l e a s e from d i g e s t i o n p r o d u c t s c o u l d be a c h i e v e d by f i l t r a t i o n o r c e n t r i f u g a t i o n . A t t e m p t s t o c o u p l e t h e c a r b o x y l groups of the n u c l e a s e t o the h y d r o x y l groups on beads o f Sephadex G-25 w i t h the a i d o f 1 - e y e l o h e x y 1 ~ 3 _ ( 2 - m o r p h o l i n y 1 -4 - e t h y 1 ) - c a r b o d i i m i d e m e t h o - p - t o l u e n e s u l f o n a t e and t o c o u p l e the amino groups o f the enzyme w i t h n i t r o u s - a c i d t r e a t e d A c r y l o z y m e HY were u n d e r t a k e n . ( A c r y l o z y m e HY i s a commercial p o l y -a c r y l a m i d e p r o d u c t c o n t a i n i n g a c i d h y d r a z i d e r e s i d u e s . ) No n u c l e a s e a c t i v i t y was o b s e r v e d a f t e r r e a c t i o n and e x t e n s i v e w a s hing r e q u i r e d t o remove non-cova1ent1y bound n u c l e a s e from t h e i n s o l u b l e p o l y m e r s . F i n a l a t t e m p t s t o remove r i b o n u c l e a s e T, from d i g e s t i o n p r o d u c t s employed chromatography o f the enzyme on B D - c e l l u l o s e and on D E A E - c e l l u l o s e . F i g u r e 14 shows the r e s u l t s o f c h r o m a t o g r a p h i n g r i b o n u c l e a s e on B D - c e l l u l o s e . The s t e p w i s e e l u t i o n s shown r e v e a l e d no n u c l e a s e a c t i v i t y u n t i l h i g h l e v e l s o f e t h a n o l were i n c o r p o r a t e d . T h i s r e m a r k a b l e a f f i n i t y o f the enzyme f o r B D - c e l l u l o s e may be r e l a t e d t o t h e p h e n o l - s o 1 u b i 1 i t y o f t h i s n u c l e a s e . T h i s Chromatography o f R i b o n u c l e a s e Tij on BD-Cel 1 u l o s e . W o r t h i n g t o n r i b o n u c l e a s e T j (3 x 10^ u n i t s ) d i s s o l v e d i n 0.5 ml 20 mM sodium a c e t a t e c o n t a i n i n g 7 M u r e a (pH 4.5) was loaded onto a column (8 x 0.5 cm) o f B D - c e l l u l o s e and d e v e l o p e d by s t e p w i s e e l u t i o n w i t h the i n d i c a t e d s o l u t i o n s ; I, 0.3 M sodium c h l o r i d e ; I I , 1.0 M sodium c h l o r i d e ; I I I , 1.0 M sodium c h l o r i d e , 4.7% (v/v) e t h a n o l ; IVa, 1.5 M sodium c h l o r i d e , 47.5% (v/v) e t h a n o l ; a l l s o l u t i o n s c o n t a i n e d 20 mM sodium a c e t a t e , pH 4.5 and 10 mM magnesium c h l o r i d e . 100 u l a l i q u o t s o f each f r a c t i o n were added to 3 mg of c rude tRNA d i s s o l v e d i n 1 ml o f 20 mM sodium a c e t a t e , c o n t a i n i n g 7M u r e a , pH 4.5- F o l l o w i n g 15 min i n c u b a t i o n , the a c i d - s o l u b l e absorbance a t 260 nm was d e t e r m i n e d as d e s c r i b e d i n the legend o f F i g u r e 13-F r a c t i o n Number. o b s e r v a t i o n s u g g e s t s t h a t B D - c e l l u l o s e c o u l d be used as a means o f removing RNase from n u c l e i c a c i d d e g r a d a t i o n p r o d u c t s p r o v i d e d the l a t t e r do not bear a h y d r o p h o b i c m o i e t y . To t e s t the p o s s i b i l i t y o f e m p l o y i n g DEAE-cel1u1ose t o remove RNase T^ from t h e d i g e s t i o n p r o d u c t s , a combined sample o f N h Z - n a p h t h o x y a c e t y l - ' ^ C - g l y c y l - t R N A ^ ^ (which had been e x h a u s t i v e l y d i g e s t e d w i t h RNase T^) and o f r i b o n u c l e a s e was a p p l i e d t o a column o f DEAE-cel1u1ose and the column was d e v e l o p e d e s s e n t i a l l y as d e s c r i b e d i n F i g u r e 10. An a s s a y o f 14 column f r a c t i o n s f o r both n u c l e a s e a c t i v i t y and C de m o n s t r a t e d t h a t the r i b o n u c l e a s e T^ e l u t e d ahead o f the l i m i t - f r a g m e n t ( i n d i c a t e d by '^C) produced by t h e n u c l e a s e . Thus, a DEAE-c e l l u l o s e c h r o m a t o g r a p h i c s t e p might be employed t o f r e e d i g e s t i o n p r o d u c t s from the enzyme; however, t h i s o b s e r v a t i o n was made l a t e i n t h e s e s t u d i e s and the e x p e r i m e n t has not been p e r f o r m e d . S t u d i e s on D i g e s t i o n o f G l y c y l - t R N A by R i b o n u c l e a s e T^. A f t e r numerous f r u i t l e s s a t t e m p t s t o i n a c t i v a t e r i b o n u c l e a s e 14 T^, t h e f o l l o w i n g q u e s t i o n was a s k e d . Could C-g1ycy1 -tRNA be d i g e s t e d w i t h r i b o n u c l e a s e T^, be p h e n o l - e x t r a c t e d t o remove the enzyme, and then th o s e o l i g o n u c l e o t i d e s s t i l l b e a r i n g the g l y c y l - r e s i d u e be r e a c t e d w i t h the N_-hydroxysucci n i m i d e e s t e r o f 2 - n a p h t h o x y a c e t i c a c i d ? T h i s approach wouId a 11ow f a c i l e c o n t r o l o f d i g e s t i o n and, by r e a c t i n g the r i b o n u c 1 e a s e - f r e e d i g e s t i o n m i x t u r e i n d e p e n d e n t l y w i t h 3'" o r 5 1"termina1= d e r i v a t i z i n g g r o u p s , a s i n g l e d i g e s t i o n c o u l d s u p p l y f a m i l i e s o f fragments s t i l l b e a r i n g the o r i g i n a l 3'~ and 5 1 ~ t e r m i n a 1 s . F i g u r e s 15 and 16 show t h a t r i b o n u c l e a s e can degrade 14 C-g1ycy1 -tRNA i n a manner p r o v i d i n g a n e a r l y i d e a l p r o d u c t d i s t r i b u t i o n . The e x p e r i m e n t s shown i n F i g u r e s 15 and 16 were 14 performed w i t h c r u d e tRNA charged w i t h C - g l y c i n e . D i g e s t i o n s were done a t pH 5.4 ( i n the p r e s e n c e o f 7 M urea and EDTA) and p h e n o l - e x t r a c t e d p r i o r t o chromatography ( F i g u r e 15) or t o n aphthoxyacety1 a t i o n ( F i g u r e 16). The l a s t two e x p e r i m e n t s demonstrated t h a t r i b o n u c l e a s e T^ was c a p a b l e o f g e n e r a t i n g a number o f d i s t i n c t f r a g m e n t s b e a r i n g t h e o r i g i n a l 3 ' ~ t e r m i n a l . F u r t h e r , once the n u c l e a s e was e f f e c t i v e l y removed, c o n d i t i o n s were found w h i c h d i d not r e s u l t i n a w a s t e f u l tendency t o f o r m a t i o n o f the l i m i t - d i g e s t i o n f r agment. W h i l e the a c t u a l number o f d i s t i n c t 3 l - t e r m i n a l b e a r i n g f r a g m e n t s g e n e r a t e d i s not known w i t h c e r t a i n t y , t h e p a t t e r n s o b t a i n e d were s a t i s f a c t o r y f o r the purposes o f a s s e s s i n g t h e f e a s i b i l i t y of t h e proposed method. I t was next n e c e s s a r y t o d i s c o v e r i f t h e n a p h t h o x y a c e t y l a t i s t e p was p r o c e e d i n g w i t h o u t c o n c o m i t a n t r e a c t i o n a t s i t e s o t h e r than t h e a-amino group o f the g 1 y c y 1 - o l i g o n u c 1 e o t i d e s . Crude 14 tRNA c o n t a i n i n g C-g1ycy1 -tRNA was d i g e s t e d , p h e n o l - e x t r a c t e d t o remove the n u c l e a s e , n a p h t h o x y a c e t y 1 a t e d and chromatographed on B D - c e l l u l o s e as d e s c r i b e d i n the legend t o F i g u r e 17-Chromatography o f RNase T ^ - d i g e s t e d , P h e n o l - E x t r a c t e d G l y c y l - t R N A on D E A E - C e l l u l o s e . 14 G l y c y l - t R N A (2.5 mg o f crude tRNA charged w i t h C - g l y c i n e p r e p a r e d as d e s c r i b e d i n P a r t I I , E x p e r i m e n t a l S e c t i o n , . I tern 1, d i s s o l v e d i n 0.25 ml o f 10 mM EDTA (pH 5.4) c o n t a i n i n g 7 M urea was d i g e s t e d w i t h W o r t h i n g t o n r i b o n u c l e a s e T .j ( a p p r o x i m a t e l y 2 x 10^ u n i t s ) f o r 4 min a t 38° when 2.25 ml o f 0.1 M sodium a c e t a t e (pH 5-0) and 1 ml o f phenol (88% l i q u i f i e d , p r e v i o u s l y s a t u r a t e d w i t h 1 M sodium a c e t a t e , pH 5-0) were added. The phases were mixed v i g o r o u s l y , the s o l u t i o n was c e n t r i f u g e d and the p h e n o l -l a y e r was removed. Two a d d i t i o n a l phenol e x t r a c t i o n s were pe r f o r m e d . The combined phenol l a y e r s were e x t r a c t e d w i t h 2 ml 0.1 M sodium a c e t a t e (pH 5-0). The combined aqueous l a y e r s were e x t r a c t e d w i t h d i e t h y l e t h e r , d i l u t e d t o 10 mM a c e t a t e , made 7 M i n ur e a and chromatographed as d e s c r i b e d i n the legend t o F i g u r e 10. Heavy 14 s o l i d l i n e : ^60' s o l i d l i n e : r a d i o a c t i v i t y from C - g l y c i n e . F r a c t i o n s were 15 ml/6.<5 min. cpm x 10 Chromatography o f RNase T . - d i g e s t e d , P h e n o l - E x t r a c t e d , Naphthoxy-a c e t y l a t e d G l y c y l - t R N A on DEAE-Cel1u1ose. G l y c y l u t R N A (2.5 mg crude tRNA charged w i t h " ^ C - g l y c i n e p r e p a r e d as d e s c r i b e d i n P a r t I I , E x p e r i m e n t a l S e c t i o n , I tern 1) d i s s o l v e d i n 0.25 ml o f 10 mM EDTA (pH 5.4) c o n t a i n i n g 7 M urea was d i g e s t e d w i t h W o r t h i n g t o n r i bonucl ease T ( a p p r o x i m a t e l y 4 x 1o"* u n i t s ) f o r 10 min a t 38° and p h e n o l - e x t r a c t e d as d e s c r i b e d i n the legend t o F i g u r e 15- The aqueous phase was n a p h t h o x y a c e t y l a t e d (as d e s c r i b e d i n P a r t I I , E x p e r i m e n t a l S e c t i o n , I tern 1) and the p r o d u c t s were a n a l y z e d on D E A E - c e l l u l o s e as d e s c r i b e d i n the legend t o F i g u r e 10. Heavy s o l i d l i n e : ^260' 14 s o l i d l i n e : r a d i o a c t i v i t y from C - g l y c i n e . F r a c t i o n s were 15 ml/6.5 min. LA D E A E - C e l l u l o s e Chromatography o f the P r o d u c t s O b t a i n e d from BD-C e l l u l o s e Chromatography o f G l y c y l - t R N A which had P r e v i o u s l y Been D i g e s t e d w i t h R i b o n u c l e a s e T^, P h e n o l - E x t r a c t e d and Naphthoxyacety1 a t e d . 1 4 G l y c y l - t R N A (5.0 mg crude tRNA charged w i t h C - g l y c i n e as d e s c r i b e d i n P a r t I I , E x p e r i m e n t a l S e c t i o n , I tern 2) d i s s o l v e d i n 1 ml 10 mM EDTA (pH 5-4) c o n t a i n i n g 7 M urea was d i g e s t e d w i t h W o r t h i n g t o n r i b o n u c l e a s e T^ (6 x 10 u n i t s ) f o r 10 min at 37°, p h e n o l - e x t r a c t e d and n a p h t h o x y a c e t y 1 a t e d as d e s c r i b e d i n the legend t o F i g u r e 16. The p r o d u c t s were chromatographed on a column (10 x 1.2 cm) o f B D - c e l l u l o s e e x a c t l y as d e s c r i b e d i n the legend t o F i g u r e 2. The m a t e r i a l e l u t e d by the 1.5 M sodium c h l o r i d e 47-5% (v/v) e t h a n o l s o l u t i o n was f r e e d o f e t h a n o l and chromatographed on a column (43 x 3-4 cm) o f B i o - G e l P-2 (50-100 mesh; e l u t i o n was a c h i e v e d w i t h 20 mM sodium a c e t a t e (pH 5 - 8 ) c o n t a i n i n g 19% ( v / v ) e t h a n o l ) . The m a t e r i a l which p o s s e s s e d r a d i o -a c t i v i t y and e l u t e d ahead o f the s a l t peak o f the B i o - G e l P^2 column was combined, the e t h a n o l was removed, the r e s u l t a n t s o l u t i o n was made pH 4.5 and 7 M i n u r e a . The s o l u t i o n was f i n a l l y l o a d e d o n t o a column (0.6 x 4 8 cm) o f DEAE-cel1u1ose. The column was dev e l o p e d as d e s c r i b e d i n the legend t o F i g u r e 10^ ex c e p t t h e : t o t a l g r a d i e n t was 300 ml and f r a c t i o n s were 4 ml/15 min. A 2 S 4 N M S t e p w i s e e l u t i o n ( c a r r i e d out as d e s c r i b e d i n the legend t o F i g u r e 2) r e l e a s e d a p p r o x i m a t e l y 75% o f the t o t a l r a d i o -a c t i v i t y i n the f i n a l h i g h s a l t - e t h a n o l wash. ( i t was a p p a r e n t t h a t the a c y l a t i o n r e a c t i o n had not proceeded t o c o m p l e t i o n s i n c e r a d i o a c t i v i t y was e v i d e n t i n a l l e l u t i o n s o l u t i o n s . O v e r l o a d i n g was not r e s p o n s i b l e s i n c e re-chromatography on a l a r g e r B D - c e l l u l o s e column gave i d e n t i c a l r e s u l t s . ) That f r a c t i o n e l u t e d by 1.5 M sodium c h l o r i d e and kj.5% e t h a n o l was p o o l e d , e t h a n o l was removed and the p r o d u c t s were d e s a l t e d on B i o - G e l P-2 as d e s c r i b e d i n I tern 9 o f the E x p e r i m e n t a l S e c t i o n of P a r t I I . That m a t e r i a l w h i c h e l u t e d . a h e a d o f the s a l t peak on the B i o - G e l P-2 column was f i n a l l y chromatographed on DEAE-c e l l u l o s e . The d e t a i l s and r e s u l t s o f the f i n a l s t e p a r e d e s c r i b e d and shown i n F i g u r e 17- A number o f f e a t u r e s o b s e r v e d i n t h i s f i g u r e w a r r a n t comment. F i r s t , the p o s i t i o n a t which N^-2-naphthoxyacety 1 g 1 yc i ne i s e x p e c t e d to appear p o s s e s s e s no r a d i o a c t i v i t y . N_-2-naphthoxyacety 1 g 1 yc i ne appears t o i n t e r a c t s t r o n g l y w i t h B i o - G e l P-2 and e l u t e s a f t e r the s a l t peak. T h i s e x p l a i n s i t s absence from the r a d i o a c t i v i t y p r o f i l e i n F i g u r e 17- A number o f peaks o f r a d i o a c t i v i t y a r e o b s e r v e d , but t h e r e i s not a c o i n c i d e n c e o f r a d i o a c t i v i t y and m a t e r i a l a b s o r b i n g l i g h t a t 260 nm. T h i s i n d i c a t e s e i t h e r t h a t naphthoxyacety1 a t i o n o f components i n a d d i t i o n to the g l y c y l - e n d e d o l i g o n u c l e o t i d e s was o c c u r r i n g or t h a t the B D - c e l 1 u l o s e . r e s o l u t i o n s t e p was not s e l e c t i n g o n l y n a p h t h o x y a c e t y 1 a t e d components. To d i s t i n g u i s h between t h e s e two p o s s i b i l i t i e s ( o r t o a s c e r t a i n i f both were o c c u r r i n g ) the f o l l o w i n g was done. A sample of c r u d e tRNA was d i g e s t e d w i t h r i b o n u c l e a s e and the d i g e s t i o n m i x t u r e d i v i d e d i n t o two p o r t i o n s . The f i r s t o f t h e s e was chromatographed on B D - c e l l u l o s e as shown i n the lower frame o f F i g u r e 18. The second p o r t i o n was r e a c t e d w i t h the N_-hydroxysucc i n im i de e s t e r o f 2-naphthoxyacet i c a c i d and then chromatographed on B D - c e l l u l o s e e x a c t l y as t h e u n t r e a t e d sample had been. F i g u r e 18 shows t h a t the "sham-a c y l a t i o n " s t e p caused a p p r o x i m a t e l y t h r e e times the amount o f t h e d i g e s t i o n p r o d u c t s t o be f i r m l y bound t o the B D - c e l l u l o s e column than was o b s e r v e d f o r the u n a c y l a t e d c o n t r o l . T h i s s u g g e s t e d t h a t a number o f n a p h t h o x y a c e t y l a b l e s i t e s a r e c r e a t e d by n u c l e a s e d i g e s t i o n . The most.reasonab1e s i t e would be 5 ' - h y d r o x y l groups formed by r i b o n u c l e a s e T^ a c t i o n . To t e s t 14 / \ t h i s p o s s i b i l i t y , C-g1ycy1 -tRNA (c r u d e ) was d i g e s t e d w i t h r i b o n u c l e a s e T^, p h e n o l - e x t r a c t e d and f i n a l l y a c y l a t e d i n a t y p i c a l a c y l a t i o n s o l u t i o n ( P a r t I, Step 10) which a l s o c o n t a i n e d 20% (v/v) g l y c e r o l . The l a t t e r r eagent was added t o d e t e r m i n e i f the presumed 0 - a c y l a t i o n c o u l d be overcome by c o m p e t i t i o n . F i g u r e 19 shows t h a t n a p h t h o x y a c e t y l a t i o n i n the p r e s e n c e o f g l y c e r o l reduced t h e l e v e l o f m a t e r i a l e l u t e d by s o l u t i o n IVa to a v a l u e s i m i l a r to t h a t seen f o r the u n a c y l a t e d c o n t r o l o f F i g u r e 18. However, the r e a c t i o n was i n c o m p l e t e as i n d i c a t e d The E f f e c t o f N a p h t h o x y a c e t y l a t i n g R i b o n u c l e a s e T : - D i g e s t e d tRNA on B D - C e l l u l o s e Chromatography. Crude tRNA (15 mg) was d i g e s t e d w i t h r i b o n u c l e a s e T^ (12 x 10^ W o r t h i n g t o n u n i t s ) f o r 10 min a t 37°, the m i x t u r e was p h e n o l - e x t r a c t e d as d e s c r i b e d i n the legend t o F i g u r e 15- O n e - h a l f o f the s o l u t i o n was n a p h t h o x y a c e t y 1 a t e d as d e s c r i b e d i n the legend to F i g u r e 16. E q u i v a l e n t amounts o f n a p h t h o x y a c e t y 1 a t e d and u n t r e a t e d samples were chromatographed i n d e p e n d e n t l y on B D - c e l l u l o s e (9 x 1.2 cm) w i t h e l u t i o n a c h i e v e d by the i n d i c a t e d s o l u t i o n s (see legend t o F i g u r e 14). F r a c t i o n s i z e , 18 ml (by siiphon) . S o l i d l i n e : ^260 ' Upper frame: n a p h t h o x y a c e t y 1 a t e d sample, Lower frame: c o n t r o l sample. III IVa | Naphthoxyacety 1ated Sample . O v e r a l l r e c o v e r y 9 1 - 5 % ; I 26% e l u t e d by IVa. | C o n t r o l Sample O v e r a l l r e c o v e r y 92%; I 12% e l u t e d by IVa. I 1 T T r 20 F r a c t i o n Number. B D - C e l l u l o s e Chromatography o f G l y c y l - t R N A D i g e s t e d w i t h R i b o n u c l e a s e T, , P h e n o l - E x t r a c t e d and N a p h t h o x y a c e t y 1 a t e d i n the P r e s e n c e o f G l y c e r o l . G l y c y l - t R N A was d i g e s t e d , p h e n o l - e x t r a c t e d , and n a p h t h o x y a c e t y l a t e d e x a c t l y as d e s c r i b e d i n the legend t o F i g u r e 17 ( l e v e l s o f g l y c y l -tRNA and enzyme and e x t e n t o f d i g e s t i o n were i d e n t i c a l ) e x c e p t t h a t the n a p h t h o x y a c e t y l a t i o n was performed i n the p r e s e n c e o f 20% (v/v) o f g l y c e r o l . The p r o d u c t s were chromatographed on a column (9 x 1.2 cm) o f B D - c e l l u l o s e w i t h the i n d i c a t e d s o l u t i o n s (see legend t o F i g u r e 1 4 ) . 11% o f the a p p l i e d ^2(>0 w a s r e l e a s e d from the column when s o l u t i o n IVa was a p p l i e d . S o l i d l i n e : ^260' 14 dashed l i n e : r a d i o a c t i v i t y from C - g l y c i n e . F r a c t i o n s were 10 ml/5 min. F r a c t i o n Number by the p r e s e n c e o f r a d i o a c t i v i t y t h r o u g h o u t t h e e l u t i o n s t e p s and more m a t e r i a l was p r e s e n t i n the f i n a l peak than was a n t i c i p a t e d on the b a s i s o f s e l e c t i n g o n l y g l y c y l - e n d e d o l i g o n u c l e o t i d e s . These r e s u l t s were c o n s i d e r e d s u f f i c i e n t t o d i s c o u r a g e f u r t h e r s t u d i e s . In r e t r o s p e c t , however, the l e v e l o f here was s i m i l a r t o t h a t o b s e r v e d i n r i b o n u c l e a s e T d i g e s t e d , n o n - a c y l a t e d c o n t r o l s . F u r t h e r , a c o n s i d e r a b l e p o r t i o n o f t h i s a p p a r e n t c o n t a m i n a n t may, i n f a c t , be due t o t h e l a r g e amounts o f n o n - n u c l e o t i d e m a t e r i a l l i b e r a t e d from B D - c e l l u l o s e when i t i s t r e a t e d w i t h v e r y h i g h l e v e l s o f e t h a n o l (see F i g u r e 2 f o r e x a m p l e ) . T h i s i n f o r m a t i o n was not a v a i l a b l e when the s t u d i e s d e s c r i b e d i n F i g u r e 19 were b e i n g p e r f o r m e d . Thus, a r e - e x a m i n a t i o n o f t h i s r o u t e i n v o l v i n g d i g e s t i o n o f p u r i f i e d ^ C - g l y c y l tRNA^' y w i t h r i b o n u c l e a s e T^, p h e n o l - e x t r a c t i o n , a c y l a t i o n i n p r e s e n c e o f h i g h l e v e l s o f g l y c e r o l , r e s o l u t i o n o f a c y l a t e d from n o n - a c y l a t e d f r a g m e n t s , d e s a l t i n g and a DEAE-c e l 1ulose c h r o m a t o g r a p h i c r e s o l u t i o n i s w a r r a n t e d . The a b i l i t y o f r i b o n u c l e a s e T^ t o degrade tRNA i n a manner c l o s e t o t h a t r e q u i r e d f o r the method proposed h e r e has been d e m o n s t r a t e d . . I t has not been p o s s i b l e , however, t o a c h i e v e the f r a g m e n t a t i o n p a t t e r n s shown i n F i g u r e s 15 and 16 w i t h e i t h e r c r u d e o r p u r i f i e d N_-2-naphthoxyacety 1 - C - g l y c y l - t R N A . A major e f f o r t t o remove o r d e s t r o y r i b o n u c l e a s e T 1 i n the p r e s e n c e o f o l i g o n u c l e o t i d e s b e a r i n g an a 1 k a 1 i - 1abi1e hydro-p h o b i c m o i e t y p r o v i d e d a p o s s i b l e method ( D E A E - c e l l u l o s e chromatography) o n l y a t the f i n a l s t a g e s o f t h e s e i n v e s t i g a t i o n s . Thus, i t i s s t i l l not known i f r i b o n u c l e a s e T^ can produce f a m i l i e s o f t e r m i n a 1 - b e a r i n g fragments by d e g r a d a t i o n o f c r u d e o r p u r i f i e d d e r i v a t i z e d - t R N A as was o b s e r v e d f o r the c a s e o f crude g l y c y l - t R N A . S t u d i e s on P a r t i a l D e g r a d a t i o n o f N-2-naphthoxyacety1- g l y c y l - t R N A ^ 1 ^ w i t h P a n c r e a t i c R i b o n u c l e a s e . A number o f s t u d i e s w i t h p a n c r e a t i c RNase were u n d e r t a k e n i n an a t t e m p t t o d e m o n s t r a t e the f e a s i b i l i t y o f the proposed method. The commercial p r o d u c t E n z i t e - R N a s e ( p a n c r e a t i c RNase l i n k e d c o v a l e n t l y t o carboxymethy1-ce11u1ose) i s an i n s o l u b l e n u c l e a s e w h i c h can be removed from the r e a c t i o n m i x t u r e by f i l t r a t i o n o r c e n t r i f u g a t i o n . The r e s u l t s o f d i g e s t i n g pur i f i ed j^-2-naphthoxyacety 1 V-gl ycyl-tRNA*? 1 y w i t h E n z i t e -RNase such t h a t 7-6% of the maximum h y p e r c h r o m i c i t y was r e a l i z e d a r e shown i n F i g u r e 20. T h i s d i g e s t i o n was performed i n 10 mM sodium a c e t a t e . (pH 5-5) c o n t a i n i n g 7 M urea a t 45°. The A^^Q p r o f i l e i n d i c a t e s f o r m a t i o n o f a l a r g e number 14 o f p r o d u c t s . However, the C - p r o f i l e i n d i c a t e s t h a t few fragments b e a r i n g the o r i g i n a l 3 l _ t e r m i n u s a r e found. F u r t h e r , a p r e f e r e n t i a l h y d r o l y s i s a t the f i r s t c y t i d y l i c a c i d r e s i d u e 1 k from t h e 3 ' - t e r m i n u s i s i n d i c a t e d by the l a r g e C peak a t FIGURE 20 DEAE-Cel l u l o s e Chromatography o f ^-2-naphthoxyacety 1 g 1 ycy 1 - t R N A ^ y D i g e s t e d w i t h E n z i t e - R N a s e . To N [ - 2 - n a p h t h o x y a c e t y l g l y c y l - t R N A ^ l y (76' A 2 6 q u n i t s and 91 x 10 cpm) i n 1 ml 20 mM sodium a c e t a t e (pH 4.5) was added 420 mg u r e a , 1 ml 20 mM sodium a c e t a t e , 7 M urea (pH 5.65). The m i x t u r e was heated t o 45° and added t o 10 mg E n z i t e - R N a s e ( S e r a v a c ; a c t i v i t y : 54 x 10 u n i t s per mg; 1 u n i t : umoles 21 ,3 1-eye 1ic-CMP h y d r o l y z e d t o 3'~CMP per min per mg o f E n z i t e - R N a s e ; p r i o r to use 10 mg o f E n z i t e - R N a s e was d i s s o l v e d i n 7 M u r e a , 10 mM sodium a c e t a t e , pH 5-6 and r e p e a t e d l y s t i r r e d and c e n t r i f u g e d w i t h f r e s h s o l u t i o n added a f t e r each c y c l e ) w i t h v i g o r o u s s t i r r i n g ( a i d e d by magnetic s t i r r e r ) f o r 1 min.Then the s o l u t i o n was c e n t r i f u g e d . The o b s e r v e d i n c r e a s e i n A ^ Q was 1.6%. The p r o d u c t s were f r e e d from the i n s o l u b l e n u c l e a s e by d e c a n t a t i o n and chromatographed on a column (66 x 0.6 cm) o f DEAE-cel1u1ose e l u t e d w i t h the i n d i c a t e d (dashed l i n e ) g r a d i e n t o f sodium c h l o r i d e c o n t a i n i n g 10 mM sodium a c e t a t e , 7 M u r e a , pH 5-05 ( t o t a l volume 360 m l ) . Flow r a t e 15 ml per h r ; f r a c t i o n s i z e 5 ml. Heavy 14 s o l i d l i n e : ^260' s o ' l ' n e : r a d i o a c t i v i t y from C - g l y c i n e . FRACTION NO f r a c t i o n 4 which i s p r o b a b l y IN_-2-naphthoxyacety 1 - C - g l y c y l -a d e n o s i n e . S i m i l a r p a t t e r n s were o b t a i n e d when p u r i f i e d N.-2-n a p h t h o x y a c e t y 1 - ' \ - g 1 ycy 1 - tRNA"My was d i g e s t e d t o the same e x t e n t a t room t e m p e r a t u r e (22° i n 7 H u r e a , pH 5«5) w i t h both E n z i t e - R N a s e and w i t h p a n c r e a t i c RNase. T h e . r e s u l t s o b s e r v e d i n F i g u r e 20 s u g g e s t t h a t even i n d i l u t e b u f f e r s c o n t a i n i n g 7 M urea a t 45°, t h e ( a p p a r e n t l y ) magnesium-free Nh2-naphthoxyacety1g1ycy1 - t R N A G 1 y must r e t a i n i , z s t r u c t u r a l p r o t e c t i o n a g a i n s t d i g e s t i o n . T h i s s u p p o s i t i o n i s i n d i c a t e d by t h e h i g h y i e l d o f the presumed .N_-2-naphthoxyacety 1 -g l y c y 1 - a d e n o s i n e and by t h e l a c k o f r a d i o a c t i v i t y - l a b e l l e d f ragments between f r a c t i o n 20 and 40. How the r e s u l t s o b t a i n e d w i t h p a n c r e a t i c RNase r e l a t e t o f r a g m e n t a t i o n p a t t e r n s o b t a i n e d w i t h r i b o n u c l e a s e seen i n F i g u r e s 15 and 16 i s not c l e a r . The a p p a r e n t l y s u p e r i o r 14 d i s t r i b u t i o n o f C - c o n t a i n i n g fragments i n the l a t t e r c a s e s may, i n p a r t , be due t o homochromotbgraphic e f f e c t s (21) a c h i e v e d by the l a r g e number o f d e g r a d a t i o n p r o d u c t s which a r e p r e s e n t . For chromatography o f t h e d e g r a d a t i v e p r o d u c t s from the p u r i f i e d tRNAs, the homochromatographic e f f e c t w i l l be much l e s s . N e v e r t h e l e s s , t h e number o f d i s t i n c t fragments g e n e r a t e d by r i b o n u c l e a s e T^ as seen i n F i g u r e s 15 and 16 appears t o be f a r g r e a t e r than t h a t o b served when p a n c r e a t i c RNAse i s used. T h i s i s o b v i o u s l y not e x p e c t e d . F u r t h e r s t u d i e s w h i c h might p r o v i d e some i n s i g h t i n t o t h i s problem can be s u g g e s t e d . P u r i f i e d V^-g 1 ycy 1 " t R N A ^ y c o u l d be d i g e s t e d w i t h r i b o n u c l e a s e , p h e n o l - e x t r a c t e d and the p a t t e r n o f o l i g o n u c l e o t i d e s a n a l y z e d on DEAE-cel1u1ose. D i g e s t i o n s c o u l d be performed on both C h e l e x 100 t r e a t e d samples and on samples which were p r e c i p i t a t e d i n the p r e s e n c e o f magnesium but which were d i s s o l v e d i n E D T A - c o n t a i n i n g s o l u t i o n s . (Note t h a t t h e tRNA used i n F i g u r e s 15 and 16 was not C h e l e x 100 t r e a t e d . ) These s t u d i e s would show the e f f e c t o f C h e l e x t r e a t m e n t v e r s u s EDTA t r e a t m e n t and a l s o the e f f e c t o f u s i n g p u r i f i e d v e r s u s crude tRNA p r e p a r a t i o n s . The p o s s i b l e r o l e o f the IN-2-naph t h o x y a c e t y 1 g 1 yc i ne group on the d i g e s t i o n p a t t e r n s s h o u l d not be i g n o r e d . T h i s r e s i d u e i s s t r o n g l y h y d r o p h o b i c and i t might t h e r e f o r e be e x p e c t e d t o a s s o c i a t e w i t h h y d r o p h o b i c r e g i o n s on o r w i t h i n t h e n u c l e a s e . Such a s s o c i a t i o n can be imagined t o markedly i n f l u e n c e the tendency o f the n u c l e a s e t o a t t a c k a t s i t e s near the h y d r o p h o b i c r e s i d u e . 14 T h i s p o s s i b i l i t y c o u l d be checked by d i g e s t i n g p u r i f i e d C - g l y c y l -Gl V tRNA^ 7 w i t h E n z i t e - R N a s e and comparing .the p a t t e r n s from t h i s e x p e r i m e n t w i t h the r e s u l t s o f F i g u r e 20. I f such i n t e r a c t i o n i s indeed o c c u r r i n g , a l t e r n a t e r o u t e s t o l a b e l l i n g the 3'"terminus w i t h a h y d r o p h o b i c group f o l l o w i n g d i g e s t i o n and enzyme removal c o u l d be i n v e s t i g a t e d . A l t e r n a t e Methods f o r L a b e l l i n g the 3 ' ~ t e r m i n a l Group  o f P o l y n u c l e o t i d e s . E x p e r i m e n t s t o t h i s p o i n t have r e l i e d on the p r e s e n c e o f a f r e e amino group a t the 3'~terminus t o a c h i e v e d e r i v a t i z a t i o n w i t h a h y d r o p h o b i c group. A l t h o u g h one e x c e p t i o n e x i s t s (89) o n l y tRNAs can be e x p e c t e d t o be amenable t o t h i s p r o c e d u r e . T h e r e f o r e , a more g e n e r a l method o f d e r i v a t i z i n g the 3'"terminus o f RNAs w i l l be needed i f the proposed a n a l y s i s i s t o be u t i l i z e d on o t h e r p o l y r i b o n u c l e o t i d e s . Methods o f more g e n e r a l a p p l i c a b -i l i t y a r e i n e x i s t e n c e and most a r e based on the r e a c t i o n o f the p e r i o d a t e - o x i d i z e d 3 l - t e r m i n a l w i t h r e a g e n t s such as i s o n i a z i d (92) ( s e e . F i g u r e 21). The p r o c e d u r e d e v e l o p e d by Hunt (90) w h i c h i n v o l v e s c o u p l i n g o f p e r i o d a t e - o x i d i z e d RNAs w i t h i s o n i a z i d i s an example. T h i s reagent was not s p e c i f i c f o r the o x i d i z e d 3'"terminus however, and r e c e n t l y t h i s reagent has been found t o r e a c t s p e c i f i c a l l y w i t h c y t o s i n e r e s i d u e s i n tRNA under m i l d c o n d i t i o n s (93). S i n c e a s i m i l a r r e a c t i o n o c c u r s w i t h s e m i c a r b a z i d e , the p r o c e d u r e s o f Hunt (90) and Mandeles (91) may be l i m i t e d by t h i s l a c k o f s p e c i f i c i t y . A method w h i c h may be more s p e c i f i c i s the r e a c t i o n o f p e r i o d a t e -o x i d i z e d f ragments w i t h p h e n y l e t h y l a m i n e f o l l o w e d by r e d u c t i o n t o form a s tab 1 e _N_- (pheny 1 e t h y 1 )morphol i ne d e r i v a t i v e (65). T h i s d e r i v a t i v e has been used t o i s o l a t e t h e 3'"terminus o f s a t e l l i t e t o b a c c o n e c r o s i s v i r u s RNA as d e s c r i b e d e a r l i e r . Reagents w h i c h C o u p l e w i t h t h e 3 1 "Terminus o f P e r i o d a t e - O x i d i z e d RNAs. a) I son i a z i d b) S e m i c a r b a z i d e 0 C-NH-NH, 0 H„N-C-NH-NH 2 2 R e c e n t l y , the use o f dimedone i n a method f o r t e r m i n a l l a b e l l i n g o f p e r i o d a t e - o x i d i z e d RNA has been d e s c r i b e d (92). S i n c e t h i s r eagent ( u n l i k e hydrazines', s e m i c a r b a z i d e s and p r i m a r y amines) r e a c t s w i t h a l d e h y d e s which a r e s a t u r a t e d a t the a l d e h e d i c c a r b o n atom, i t s d e r i v a t i v e s a r e not v e r y s u s c e p t i b l e t o B ~ e l i m i n a t i o n . The d e r i v a t i v e i s r e p o r t e d t o be s t a b l e , s p e c i f i c f o r the o x i d i z e d 3'"terminus and t o c a use no c l e a v a g e o f the d e r i v a t i z e d polymer. Thus, t h i s reagent a p p ears t o be s u p e r i o r t o most o f the p r e s e n t l y a v a i l a b l e t e c h n i q u e s ( i n c l u d i n g t h e p e r i o d a t i o n - H-NaBH^ r e d u c t i o n p r o c e d u r e (9^)) f o r l a b e l l i n g the 3 , - t e r m i n u s o f h i g h m o l e c u l a r w e i g h t RNAs. 5~benzy1-5 -methy1 - 1 , 3 _ c y c1 o h e x a n e d i o n e (or the c o r r e s p o n d i n g n a p h t h y 1 - d e r i v a t i v e ) might t h e r e f o r e be r e a g e n t s o f c o n s i d e r a b l e u t i l i t y i n o b t a i n i n g a s p e c i f i c , s t a b l e and h y d r o -p h o b i c d e r i v a t i v e a t the 3'"terminus o f any p o l y r i b o n u c l e o t i d e . I t s h o u l d be p o i n t e d out t h a t a method a l l o w i n g d i r e c t i s o l a t i o n . o f 3 ' ~ t e r m i n a l p o l y n u c l e o t i d e s has been d e v e l o p e d (95, 96). The p r o c e d u r e r e l i e s on the c o u p l i n g . o f the p e r i o d a t e -o x i d i z e d polymer t o a m i n o e t h y l - e e l l u l o s e . Once n o n - t e r m i n a l fragments have been removed, the c o v a l e n t l y - b o n d e d 3 l _ e n d e d fragments a r e l i b e r a t e d from the c e l l u l o s e by t r e a t m e n t w i t h a p r i m a r y amine. T h i s method has the p o t e n t i a l t o p e r f o r m the s e q u e n c e - a n a l y s i s p r o c e d u r e on 3 ' ~ t e r m i n a l fragments as i s b e i n g proposed i n t h i s t h e s i s . R e c e n t l y , a p r o c e d u r e p o s s e s s i n g t h e p o t e n t i a l f o r d i r e c t i s o l a t i o n o f o l i g o r i b o n u c l e o t i d e s p o s s e s s i n g f r e e 2' ,3 ' - c i s d i o l g roups was announced (107). Two c e l l u l o s e d e r i v a t i v e s which p o s s e s s t h e d i h y d r o x y b o r y l group have been prep a r e d (106) and the a b i l i t y o f the l a t t e r group t o form a complex w i t h c i s - g l y c o l m o i e t i e s o f p o l y o l s and n u c l e o s i d e s has been used t o d e v e l o p a t e c h n i q u e f o r s t u d y i n g o l i g o - and p o l y n u c l e o t i d e s (107). For t h e l a t t e r t e c h n i q u e i t was r e p o r t e d (106) t h a t o l i g o - o r p o l y n u c l e o t i d e s c o u l d be s e l e c t i v e l y r e t a i n e d . o n the columns. The r e t e n t i o n was dependent on a number o f f a c t o r s such as the p r e s e n c e o f an u n s u b s t i t u t e d 2',31-d io1 group a t the 3 1 -t e r m i n u s , the n a t u r e o f the bases w i t h i n the v i c i n i t y o f the d i o l , t h e c h a i n l e n g t h and net char g e o f t h e p o l y n u c l e o t i d e and the c o n d i t i o n s used f o r e l u t i o n . The p o s s i b i l i t y o f e x t e n d i n g t h i s p r o c e d u r e t o p o l y n u c l e o t i d e s b e a r i n g t e r m i n a l phosphate (su g g e s t e d i n r e f e r e n c e 106) by i n c o r p o r a t i n g a s o r b i t o l group o n t o the l a t t e r group s u g g e s t s t h a t the p r o c e d u r e c o u l d be extended t o a n a l y s i s o f fr a g m e n t s d e r i v e d from both 3 I _ and 5 l - t e r m i n a l s o f s i n g l e - s t r a n d e d DNAs . I f s e l e c t i v e r e t e n t i o n o f fragments b e a r i n g the m o d i f i e d phosphate was found f o r c o n s i d e r a b l e l e n g t h s o f o l i g o - o r p o l y n u c l e o t i d e s , t h i s t e c h n i q u e would a l s o a l l o w development o f a sequence p r o c e d u r e as proposed i n t h i s t h e s i s . S t u d i e s on Methods f o r D e r i v a t i z i n g t h e 5'-terminal o f tRNA. The f i n a l s e c t i o n o f t h i s d i s c u s s i o n w i l l p r e s e n t the r e s u l t s o f e x p e r i m e n t s which a t t e m p t e d t o l a b e l t h e 5 ' ~ t e r m i n u s o f p o l y n u c l e o t i d e s w i t h a h y d r o p h o b i c d e r i v a t i v e . These s t u d i e s were i n i t i a t e d f o r s e v e r a l r e a s o n s . U n t i l r e c e n t l y , no d i r e c t method f o r t h e i s o l a t i o n o f the 5 ' - t e r m i n a l o f an RNA was a v a i l a b l e ( 6 6 ) . Thus, a method s i m i l a r t o t h a t a l r e a d y p r e s e n t e d f o r t he d i r e c t i so l a t i on o f the 3 ' ~ t e r m i n a l s o f t R N A ^ y and t R N A 2 ' y c o u l d be d e v e l o p e d . F u r t h e r , w i t h t h e a v a i l a b i l i t y o f h y d r o p h o b i c d e r i v a t i v e s a t t h e 5 ' - t e r m i n u s o f p o l y n u c l e o t i d e s , the proposed sequence-method c o u l d be a p p l i e d from both the 5 '~ and 3 1 ~ t e r m i n a 1 s . S i n c e a reagent c a p a b l e o f r e a c t i n g s p e c i -f i c a l l y a t the 5 ' ~ t e r m i n u s o f RNAs (whether a monophosphate or t r i p h o s p h a t e group was p r e s e n t a t the t e r m i n u s ) w i l l a l s o r e a c t w i t h phosphate r e s i d u e s a t both the 5 I _ and 3 1 " t e r m i n a 1 s of p o l y d e o x y r i b o n u c l e o t i d e s , t h e a v a i l a b i l i t y o f such a re a g e n t would a l s o a 1 l o w - s i n g 1 e - s t r a n d e d DNAs t o be s t u d i e d by the proposed sequence p r o c e d u r e . Four main methods f o r l a b e l l i n g o f t h e 5 ' " t e r m i n u s a r e i n 1 O IT e x i s t e n c e . The u t i l i t y o f . pheny1diazomethane (or i t s p- I-l a b e l l e d c o u n t e r p a r t ) i n both r e a c t i n g a t the 5 ' ~ t e r m i n u s o f RNA and a l l o w i n g d i r e c t i s o l a t i o n o f t h e t e r m i n a l fragment has a l r e a d y been d i s c u s s e d . T h i s method appears t o be t h e o n l y - p r e s e n t l y a v a i l a b l e method a l l o w i n g d i r e c t i s o l a t i o n o f the 5'-termina1s o f p o l y r i b o n u c l e o t i d e s . (A method r e l y i n g on d i f f e r e n c e s i n ch a r g e s f o l l o w i n g phosphatase t r e a t m e n t (97) has been used but i t i n v o l v e s s e v e r a l o p e r a t i o n s f o r i t s e x e c u t i o n . ) The method u s i n g p o l y n u c l e o t i d e k i n a s e t o l a b e l t h e 5 l - t e r m i n a l o f n u c l e i c a c i d s has a l s o been d i s c u s s e d (26). A t h i r d method i s t h a t r e p o r t e d by Ralph e t aj_. (98), i n wh i c h the 5 l _ t e r m i n a l phosphate group o f tRNA i s c o n v e r t e d t o the c o r r e s p o n d i n g p h o s p h o r o a n i 1 i d a t e by r e a c t i n g t h e p o l y n u c l e o t i d e w i t h d i i s o p r o p y 1 c a r b o d i i m i d e i n p r e s e n c e o f an e x c e s s o f a n i l i n e i n a w a t e r , dimethy1formamide and t e r t i a r y - b u t a n o l m i x t u r e . S h o r t l y t h e r e a f t e r , a method f o r l a b e l l i n g o f end groups i n p o l y n u c l e o t i d e c h a i n s by r e a c t i n g the cety11rimethy1 -14 ammonium s a l t o f tRNA w i t h C - m e t h y l p h o s p h o r o m o r p h o l i d a t e was r e p o r t e d by Raj Bhandary et_ a_l_. (57). T h i s method was c o n s i d e r e d t o be s u p e r i o r t o the p h o s p h o r o a n i 1 i d a t e r o u t e (98). The c o u p l i n g 14 o f C - m e t h y l p h o s p h o r o m o r p h o l i d a t e w i t h tRNA r e q u i r e d the use o f non-aqueous s o l v e n t s and r a t h e r e x t e n s i v e r e a c t i o n t i m e s . However, the method had the f e a t u r e o f g e n e r a l u t i l i t y i n l a b e l l i n g 5'-phosphomonoester groups o f RNAs and 5 1 - and 3'-phosphomonoester groups p f DNAs. A t t e m p t s to d e v e l o p a method f o r l a b e l l i n g t he 5 1 - t e r m i n u s o f p o l y n u c l e o t i d e s w i t h a h y d r o p h o b i c r e a g e n t were guid e d by the f o l l o w i n g c o n s i d e r a t i o n s : ( i ) t he re a g e n t s h o u l d s p e c i f i c a l l y form a s t a b l e adduct w i t h a poly.nuc 1 eot ide whether i t t e r m i n a t e s w i t h a 5'""rnono-, d i - o r t r i p h o s p h a t e g r o u p , ( i i ) t he reagent s h o u l d r e a c t e x c l u s i v e l y a t the t e r m i n a l pos i t i on , ( i i i ) t he r e a c t i o n s h o u l d proceed i n aqueous medium a t low t e m p e r a t u r e s , near n e u t r a l i t y and i n s h o r t t i m e p e r i o d s , ( i v ) t he reagent s h o u l d be r e a d i l y a v a i l a b l e i n both r a d i o a c t i v e and n o n - r a d i o a c t i v e f o r m s , and f i n a l l y , (v) t he r e s u l t a n t d e r i v a t i v e s h o u l d p o s s e s s s u f f i c i e n t h y d r o p h o b i c i t y t o b i n d the d e r i v a t i z e d polymer f i r m l y t o B D - c e l l u l o s e . I n i t i a l a t t e m p t s were based on the a b i l i t y o f w a t e r -s o l u b l e c a r b o d i i m i d e t o form p h o s p h o d i e s t e r o r pyr o p h o s p h a t e bonds i n aqueous s o l u t i o n (99)- Model systems u s i n g g u a n o s i n e -5 1-monophosphate, 1 - c y c l o h e x y 1 - 3 - ( 2 - m o r p h o l i n y 1 - 4 - e t h y 1 ) -c a r b o d i i m i d e m e t h o - p - t o l u e n e s u l f o n a t e i n sodium 2-(N-morpholine) e t h a n e s u 1 f o n a t e b u f f e r (pH 6.0) were used t o whi c h were added ( i n d e p e n d e n t l y ) 2 - n a p h t h y l - p h o s p h a t e , p - n i t r o p h e n o l , b e n z y l a l c o h o l o r p h e n e t h y l a l c o h o l ( v a r i o u s l e v e l s o f t h e s e r e a g e n t s and c a r b o d i i m i d e s were u s e d ) . A l i q u o t s were removed a t v a r i o u s times and chromatographed on paper w i t h S o l v e n t II o r S o l v e n t II Fo r m a t i o n o f a 5 I -GMP d e r i v a t i v e w i t h an R^ . g r e a t e r than t h a t o f 5 I _GMP was used as t h e b a s i s o f r e a c t i o n . L i t t l e , i f any, c o u p l i n g was o b s e r v e d . S y n t h e s i s o f the e t h y l e s t e r o f a d e n o s i n e - 5 ' - p h o s p h a t e under s i m i l a r c o n d i t i o n s was o b t a i n e d , however. F u r t h e r i n v e s t i g a t ions on the i n f l u e n c e o f v a r y i n g the m olar r a t i o s o f r e a g e n t s and on the pH-.opt ima f o r r e a c t i o n s w h i c h might g i v e the d e s i r e d c o u p l i n g have not been u n d e r t a k e n . The next group o f e x p e r i m e n t s were performed w i t h the o b j e c t o f c o u p l i n g t h e 5 ' " t e r m i n a l monophosphate group o f tRNA w i t h 2 - n a p h t h y l p h o s p h o m o r p h o l i d a t e t o form a p y r o p h o s p h a t e 1inkage between tRNA and 2 - n a p h t h y l p h o s p h a t e (see F i g u r e 2 2 ) . The s y n t h e s i s o f 2 - n a p h t h y l p h o s p h o r o m o r p h o l i d a t e and i t s r e a c t i o n w i t h g u a n o s i n e 5~monophosphate (hydrogen form) and c e t y l t r i -methylammonium s a l t o f tRNA a r e d e s c r i b e d i n . t h e E x p e r i m e n t a l S e c t i o n . The methods a re e s s e n t i a 1 l y t h o s e d e s c r i b e d i n r e f e r e n c e 57. C o u p l i n g of the 2 - n a p h t h y l p h o s p h a t e d e r i v a t i v e t o tRNA was a s s a y e d by s t e p w i s e chromatography on B D - c e l l u l o s e . The amount of tRNA which remained bound t o the column a f t e r e x t e n s i v e 1 M sodium c h l o r i d e wash, .but w h i c h e l u t e d w i t h 1 M sodium c h l o r i d e c o n t a i n i n g 47-5% (v/v) e t h a n o l was assumed to be a measure o f the e x t e n t o f c o u p l i n g . I n i t i a l e x p e r i m e n t s i n d i c a t e d a p p r o x i m a t e l y 20% o f - t h e o r i g i n a l tRNA was r e a c t e d . A UV-spectrum o f the presumed p r o d u c t i n d i c a t e d a minor a b s o r p t i o n peak a t 325 nm w h i l e the remainder o f t h e spectrum was a t y p i c a l s pectrum f o r tRNA. (The a b s o r p t i o n a t 325 nm i n d i c a t e s t h e p r e s e n c e o f the n a p h t h y l p h o s p h a t e d e r i v a t i v e . ) S e v e r a l e x p e r i m e n t s a t t e m p t i n g t o . i n c r e a s e t h e y i e l d gave Presumed R e a c t i o n o f 2 - N a p h t h y l p h o s p h o r y I m o r p h o l i d a t e w s i m i l a r o r lower y i e l d s . The need f o r r i g o r o u s l y anhydrous c o n d i t i o n s t o o b t a i n h i g h y i e l d s i n r e a c t i o n s e m p l o y i n g mor-p h o l i d a t e s may be i m p l i c a t e d i n the d i f f i c u l t i e s o f r a i s i n g y i e l d s . The d i f f i c u l t y o f o b t a i n i n g r e a s o n a b l e y i e l d s and the r e q u i r e m e n t f o r c o u p l i n g i n non-aqueous s o l u t i o n s were c o n s i d e r e d s u f f i c i e n t . t o d i s c o u r a g e . f u r t h e r i n v e s t i g a t i o n s . However, t h e s e s t u d i e s i n d i c a t e d t h a t a h y d r o p h o b i c m o i e t y a t the 5'"terminus o f tRNA c o u l d a c h i e v e the r e q u i r e d b i n d i n g t o B D - c e l l u l o s e . The r e p o r t (58) t h a t 2 , k - d i n i t r o f 1 u o r o b e n z e n e (DNFB) r e a c t s s p e c i f i c a l l y w i t h the t e r m i n a l n u c l e o s i d e - 5 1 - p h o s p h a t e of tRNA s u g g e s t e d t h a t t h i s r e a g e n t might p r o v i d e a means of d e r i v a t i z i n g the 5'~terminus w i t h a h y d r o p h o b i c group. I t i s a l s o known t h a t the r e a g e n t c o n v e r t s r i b o n u c 1 e o s i d e - 2 1 -3 1 - p h o s p h a t e s i n t o r i b o n u c 1 e o s i d e - 2 1 , 3 1 - c y c l i c phosphates and 1 2 n u c l e o s i d e ~ 5 1 - p h o s p h a t e s t o p ,p - d i n u c 1 e o s i d e - 5 1 - p y r o p h o s p h a t e s i n p y r i d i n e and t o n u c l e o s i d e - 5 1 - p h o s p h o r o f l u o r i d a t e s i n o t h e r s o l v e n t s (100, 101, 102, 103). The p o s s i b i l i t y o f f o r m i n g both the d i n i t r o p h e n y 1 - d e r i v a t i v e and p h o s p h o r o f l u o r o - d e r i v a t i v e o f tRNA by r e a c t i o n o f tRNA w i t h DNFB under c o n d i t i o n s d e s c r i b e d i n r e f e r e n c e 58 was s u f f i c i e n t t o d i s c o u r a g e i n i t i a t i o n o f s t u d i e s w i t h t h i s r eagent (see F i g u r e 2 3 ) . However, i t was r e c e n t l y o b s e r v e d (104) t h a t p h o s p h o r o f 1 u o r i d a t e f o r m a t i o n P o s s i b l e R e a c t i o n s o f DNFB w i t h tRNA. o c c u r r e d i n r e a c t i o n o f DNFB w i t h d eoxynuc1eoside~5 1-phosphates i n aqueous s o l u t i o n o n l y i f t r i a 1ky1 amine was p r e s e n t . In the absence o f the l a t t e r s p e c i e s , the r e a c t i o n proceeded to form o n l y the d i n i t r o p h e n y 1 e s t e r o f the d e o x y n u c l e o s i d e ~ 5 1 -phosphate. T h i s o b s e r v a t i o n , p l u s the r e p o r t t h a t t h e DNFB i p r o c e d u r e a l l o w e d a method f o r c h a r a c t e r i z i n g the 5 ' " t e r m i n a l groups of E_. c o l i tRNA ( 108 ) , prompted a s t u d y o f t h e r e a c t i o n of DNFB w i t h tRNA. The c o u p l i n g o f tRNA and DNFB was o u t l i n e d i n the E x p e r i m e n t a l S e c t i o n , I tern 14. The h i g h e s t y i e l d o b t a i n e d was a p p r o x i m a t e l y 60%. The e x t e n t o f r e a c t i o n was ju d g e d by d e t e r m i n i n g t h e amount o f o r i g i n a l tRNA ( p r e v i o u s l y s t r i p p e d o f e t h a n o l - f r a c t i o n ) which remains bound t o B D - c e l l u l o s e a f t e r e x t e n s i v e 1.0 M sodium c h l o r i d e e l u t i o n but wh i c h i s r e l e a s e d by e l u t i o n w i t h 1.0 M sodium c h l o r i d e c o n t a i n i n g k7-5% (v/v) e t h a n o l f o l l o w i n g r e a c t i o n w i t h DNFB. Ex p e r i m e n t s d e s i g n e d t o i n c r e a s e t h i s y i e l d have g i v e n s i m i l a r o r lower l e v e l s o f a p p a r e n t r e a c t i o n . E x t e n s i v e c h a r a c t e r i z a t i o n o f the p r o d u c t has not been performed and a l t h o u g h the r e a c t i o n has been r e p o r t e d t o be s p e c i f i c f o r the 5 l - t e r m i n u s o f tRNA ( 108 ) , u r a c i l and thymine have been r e p o r t e d t o form 2 , 4 - d i n i t r o p h e n y 1 d e r i v a t i v e s (109 ) . In the l a t t e r c a s e , the d i n i t r o p h e n y 1 -group i s a t t a c h e d t o t h e N* o f t h e r i n g . Thus, t h e DNFB method may not be s u f f i c i e n t l y s p e c i f i c f o r the d e r i v a t i z a t i o n o f te r m i n a 1 - p h o s p h a t e b e a r i n g p o l y n u c l e o t i d e s . SUMMARY In summary, P a r t II o f t h i s t h e s i s has o u t l i n e d the p r i n c i p l o f a new method f o r s e q u e n c e - d e t e r m i n a t i o n o f n u c l e i c a c i d s . The p o t e n t i a l advantages and e x p e c t e d l i m i t a t i o n s o f t h e proposed method have been d i s c u s s e d a t both t h e o r e t i c a l and p r a c t i c a l l e v e l s . Two groups o f s t u d i e s u s i n g a model polymer were u n d e r t a k e n t o a s c e r t a i n the f e a s i b i l i t y o f the proposed method. F i r s t , the e f f e c t i v e n e s s o f the method as an end-group p r o c e d u r e has been v e r i f i e d by i s o l a t i n g and p a r t i a l l y c h a r a c t e r i z i n g the 3 l _ t e r m i n a l groups o f t R N A ^ y and tRNA^' y o f y e a s t . 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