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Studies on transfer RNA and transfer RNA genes in Drosophila melanogaster Dunn, Robert James 1977

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STUDIES ON TRANSFER RNA AND TRANSFER RNA GENES IN DROSOPHILA MELANOGASTER by ROBERT JAMES DUNN B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 ^Robert James Dunn, 1977 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t i -of th is thes is for f inanc ia l gain sha l l not be allowed without my wr i t ten permission. i on Department of /^)ioc\.t**\i){'v The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date OcAil/7? A b s t r a c t In the prese n t study D r o s o p h i l a me1anogaster was used t o d e f i n e the o r g a n i z a t i o n and e x p r e s s i o n of tRNA genes. The three major D r o s o p h i l a v a l i n e tRNAs were i s o l a t e d and p u r i -f i e d by standard chromatographic techniques. N u c l e o s i d e V a l a n a l y s i s i n d i c a t e d t h a t of these tRNAs o n l y tRNA^ c o n t a i n e d i n o s i n e . A l l t h r e e tRNAs^"1" c o n t a i n e d r i b o t h y m i d i n e , t h e r e -V a l f o r e they resemble y e a s t tRNA i n t h i s r e g ard but not the V a l mammalian tRNAs which l a c k r i b o t h y m i d i n e . The p u r i f i e d tRNAs were l a b e l l e d w i t h 1 2 5 I and used t o determine the l o c a t i o n o f the genes f o r these tRNAs u t i l i z i n g the technique of i n s i t u h y b r i d i z a t i o n to s a l i v a r y gland V a l chromosomes. tRNA^ h y b r i d i z e d c o n s i s t e n t l y to one s i t e on the r i g h t arm of the second chromosome, 56D, which i s c l o s e V a l to the s i t e of 5S RNA, 56F. tRNA^^ h y b r i d i z e d to two s i t e s , 84D and 92B, both on the r i g h t arm of the t h i r d chromosome. The l a b e l l i n g o f s i t e 84D was approximately twice as heavy as t h a t of 92B. Dr. A. Delaney (unpublished) has shown t h a t V a l approximately 13 genes code f o r tRNA 3 b per h a p l o i d genome. The i n s i t u h y b r i d i z a t i o n data suggests t h a t the 13 genes are d i v i d e d such t h a t approximately 8 genes are a t s i t e 84D and 5 genes are a t s i t e 92B. Evidence t o support t h i s s u p p o s i t i o n i s d e r i v e d from V a l measurements on the amount of tRNA 3 f c ) i n mutant f l i e s d e f i c i e n t or d u p l i c a t e d f o r s i t e 84D on one of t h e i r two homologous V a l t h i r d chromosomes. tRNA-, amounts, measured r e l a t i v e t o the 3 b other tRNA i s o a c c e p t o r s decrease 31% i n the d e f i c i e n c y and i n c r e a s e 30% i n the d u p l i c a t i o n . These r e s u l t s demon-V a l s t r a t e a d i r e c t r e l a t i o n s h i p of the amount of tRNA^^ to gene dosage because the d u p l i c a t i o n has 8 e x t r a genes, which i s a 30% i n c r e a s e and the d e l e t i o n has 8 fewer genes, a 30% decrease. F i n a l l y , i t was shown t h a t the amount of t o t a l V a l tRNA i n c r e a s e d by 17% i n the d u p l i c a t i o n but d i d not de-crease i n the d e l e t i o n . T h i s r e s u l t demonstrates the amount of v a l i n e tRNA i s under a type of c o n t r o l i n which the amount of t o t a l v a l i n e tRNA i s i n c r e a s e d t o compensate f o r the de-f i c i e n c y o f a s i n g l e i s o a c c e p t o r . A l s o the coding proper-Ser t i e s o f f o u r tRNA i s o a c c e p t o r s were determined. i i i . TABLE OF CONTENTS Page A b s t r a c t i Table of Contents i i i L i s t o f Tables vi L i s t o f F i g u r e s . v i i Acknowledgements ix D e d i c a t i o n x A b b r e v i a t i o n s xi I n t r o d u c t i o n 1 I. S t r u c t u r e o f the tRNA Molecule 1 I I . B i o l o g i c a l F u n c t i o n s o f T r a n s f e r RNA . . . . 6 A. P r o t e i n S y n t h e s i s 6 B. C o n t r o l of T r a n s c r i p t i o n 9 C. T r a n s f e r RNA and V i r a l I n f e c t i o n . . . . 12 D. The Aminoacyl T r a n s f e r a s e s 13 I I I . T r a n s f e r RNA P o p u l a t i o n s 15 A. B a c i l l u s s u b t i l u s 15 B. Hormone E f f e c t s on tRNA 17 C. T r a n s f e r RNA i n C o l l a g e n S y n t h e s i z i n g T i s s u e s 19 D. T r a n s f e r RNA i n the S i l k Gland of Bombyx mori' 20 E. T r a n s f e r RNA i n the R e t i c u l o c y t e . . . . 21 F. T r a n s f e r RNA i n Cancer C e l l s 24 IV. D r o s o p h i l a melanogaster tRNA 24 iv. Page M a t e r i a l s 28 Methods 29 I. Growth of D r o s o p h i l a melanogaster 29 I I . P r e p a r a t i o n of Aminoacyl-tRNA Synthetases . . 29 I I I . The Aminoacylation Reactions 3 0 A. Assay of Amino A c i d Acceptance i n S o l u t i o n 30 B. Assay of Amino A c i d Acceptance of Column F r a c t i o n s 31 C. P r e p a r a t i o n of Aminoacyl-tRNA f o r Chromatography 32 IV. P r e p a r a t i o n of T r a n s f e r RNA from D r o s o p h i l a 33 V. F r a c t i o n a t i o n of tRNA 33 A. B i o - G e l A-0.5 M Chromatography 33 B. B D - c e l l u l o s e Chromatography 34 C. Sepharose 6B Chromatography 35 D. RPC-5 Chromatography 35 VI. N u c l e o s i d e A n a l y s i s 36 A. The T r i t i u m L a b e l l i n g Method 36 B. A n a l y s i s of N u c l e o s i d e s Detected by U.V. Absorbance 38 V I I . Ribonuclease T-^  F i n g e r p r i n t A n a l y s i s . . . . 38 V I I I . In S i t u H y b r i d i z a t i o n 41 IX. P r e p a r a t i o n o f S e r i n e Codons 42 A. S y n t h e s i s 42 B. I d e n t i f i c a t i o n 43 X. Determination of T r i n u c l e o t i d e S t i m u l a t e d B i n d i n g of Seryl-tRNA t o Ribosomes 43 V . Page R e s u l t s and D i s c u s s i o n 45 I. P u r i f i c a t i o n and tRNAs 45 A. B D - C e l l u l o s e Chromatography 45 B. Sepharose 6B Chromatography 48 C. P u r i f i c a t i o n of RNA Species on RPC-5 Columns 48 i . 5S RNA 52 i i . Three Valyl-tRNA Species 52 i i i . P u r i f i c a t i o n of Non-Valine tRNA Species 72 I I . N u c l e o s i d e A n a l y s i s of P u r i f i e d tRNAsVa"''. . . 81 I I I . RNase T^ F i n g e r p r i n t A n a l y s i s 93 IV. In S i t u H y b r i d i z a t i o n of tRNAs 99 ' V. A n a l y s i s of tRNA Mutants 104 A. Amounts of V a l i n e Acceptance i n the Mutant S t r a i n s • 107 B. RPC-5 A n a l y s i s of V a l i n e I s o a c c e p t o r s . . 110 VI. Summary of t h e . E f f e c t s of Gene Dosage on the Amounts of tRNA^gl . 123 Co n c l u s i o n 124 Ser A n a l y s i s of the Coding P r o p e r t i e s of tRNA 126 B i b l i o g r a p h y 135 vi. Table 1, Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8, Table 9, LIST OF TABLES Page Amino a c i d acceptance assays of pools A - J from the Sepharose Column 2B 51 Nuc l e o s i d e a n a l y s i s o f tRNA^ 1^ of y e a s t by the t r i t i u m l a b e l l i n g method 84 Va 1 Nuc l e o s i d e a n a l y s i s of tRNAs from D r o s o p h i l a melanogaster 87 Q u a n t i t a t i o n of o l i g o n u c l e o t i d e s prepared by RNase T i d i g e s t i o n o f t R N A s V a l 96 L i s t o f the genotypes of s t r a i n s o f D r o s o p h i l a w i t h d e f i c i e n c i e s o r d u p l i c a -t i o n s a t s i t e 84D 106 V a l i n e acceptance of tRNA prepared D r o s o p h i l a stocks w i t h d e f i c i e n c i e s o r d u p l i c a t i o n s a t s i t e 84D 108 Amounts of t R N A V a l i s o a c c e p t o r s meas-ured by chromatography i n RPC-5 system A . 113 Amounts of tRNA V a"^ i s o a c c e p t o r s measured by chromatography i n RPC-5 system C . . . . 116 V a l Amounts of tRNA i s o a c c e p t o r s measured by combined chromatography i n RPC-5 systems A and C 122 Table 10. C h a r a c t e r i z a t i o n of t r i n u c l e o t i d e s by RNase T 2 and venom phosphodiesterase d i g e s t i o n 128 v i i . LIST OF FIGURES Page F i g u r e 1. B D - c e l l u l o s e chromatography of D r o s o p h i l a tRNA . . 46 F i g u r e 2. Sepharose 6B chromatography . . . . . . . . 4 9 F i g u r e 3. 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 5S RNA . . 53 F i g u r e 4. RPC-5 chromatography of 5S RNA 54 F i g u r e 5. RPC-5 chromatography of D r o s o p h i l a tRNAVal . . . . . . 56 V a l F i g u r e 6. P u r i f i c a t i o n o f t R N A ^ by RPC-5 chromatography 59 F i g u r e 7. A n a l y t i c a l RPC-5 chromatography of tRNA^al 61 V a l F i g u r e .8. RPC-5 chromatography of tRNA 3 a and tRNAY a l 63 V a 1 V a1 F i g u r e 9. P u r i f i c a t i o n o f t R N A ^ and tRNA^ . . . . 65 V a l F i g u r e 10. Chromatography of tRNA^^ 6 8 F i g u r e 11. F r a c t i o n a t i o n of t R N A ^ 1 and t R N A ^ 1 . . . 70 F i g u r e 12. RPC-5 chromatography of methionine .,. a c c e p t i n g tRNA rec o v e r e d from the Sepharose 6B column 73 F i g u r e 13. RPC-5 chromatography of th r e o n i n e a c c e p t i n g tRNA recovered from the Sepharose 6B column . . . . . . . . . . . . 75 F i g u r e 14. RPC-5 chromatography of tRNA c o n t a i n -i n g a s p a r t i c a c i d acceptance a c t i v i t y . . . 77 F i g u r e 15. RPC-5 chromatography of tRNA c o n t a i n -i n g a l a n i n e , glutamic a c i d , and g l y c i n e a c c e p t o r a c t i v i t y 79 F i g u r e 16. N u c l e o s i d e a n a l y s i s of y e a s t t R N A j 1 ^ u s i n g the t r i t i u m l a b e l l i n g technique . . . 82 F i g u r e 17. N u c l e o s i d e a n a l y s i s of t h r e e p u r i f i e d t R N A s V a l u s i n g the t r i t i u m l a b e l l i n g t echnique 86 Page F i g u r e 18. Nucle o s i d e a n a l y s i s o f t h r e e p u r i f i e d tRNAsVal . . . . . 89 V a l F i g u r e 19. Unknown n u c l e o s i d e A* from tRNA.. . . . . 91 Ja F i g u r e 20. RNase T]_ f i n g e r p r i n t a n a l y s i s of t h r e e p u r i f i e d t R N A s V a l . . . 94 F i g u r e 21. In s i t u h y b r i d i z a t i o n of t R N A ^ l and tRNA^3-'- t o D r o s o p h i l a s a l i v a r y gland chromosomes . F i g u r e 22. Schematic r e p r e s e n t a t i o n of t h i r d chromo-somes w i t h a b e r r a t i o n s i n the r e g i o n 84D. . 105 F i g u r e 23. RPC-5 chromatography i n system A of V a l -t R N A V a l from f l i e s w i t h d u p l i c a t i o n s or d e f i c i e n c i e s a t s i t e 84D I l l F i g u r e 24. RPC-5 chromatography i n system C of V a l - t R N A V a l from f l i e s w i t h d u p l i c a -t i o n s o r d e f i c i e n c i e s a t s i t e 84D 114 F i g u r e 25. RPC-5 chromatography of tRNAY a l 118 F i g u r e 26. RPC-5 chromatography i n system A of t R N A V a l from which tRNAYf 1 has been removed by chromatography i n system C . . . 120 F i g u r e 27. Chromatography of S e r - t R N A S e r on RPC-5 . . 129 Ser F i g u r e 28. B i n d i n g of D r o s o p h i l a Ser-tRNA 2 and Ser-tRNA^ e r to E. c o l i ribosomes 131 Ser F i g u r e 29. B i n d i n g of D r o s o p h i l a Ser-tRNA 5 and Ser-tRNA§ j e r to E. c o l i ribosomes 133 ix. ACKNOWLEDGEMENTS I wish t o thank Gordon Tener f o r h i s guidance and support throughout the p r e p a r a t i o n of t h i s work. A s p e c i a l thanks to Ian G i l l a m f o r s h a r i n g c o u n t l e s s ideas and techniques. I am a l s o indebted t o Bradley White, A l l e n Delaney and Tom G r i g l i a t t i f o r h e l p f u l d i s c u s s i o n s , and to Tom G r i g l i a t t i , S h i z u Hayashi and Tom Kaufman f o r c o n t r i b u t i o n s t o the i n s i t u h y b r i d i z a t i o n experiments. I a l s o wish to thank M a r j o r i e G r i e v e f o r prepar-i n g many of the f i g u r e s i n the t h e s i s . TO SUSIE XV . A b b r e v i a t i o n s Used A,C,G,U Ap,Cp,Gp,Up pA,pC,pG,pU A',C fG'»-fU' ADP,CDP,GDP,UDP ATP,CTP,GTP P i DNA RNA mRNA rRNA tRNA tRNA V a l V a l Val-tRNA Ala,Arg,Asp,Glu, Gin, Gly, Lys , Met, T h r , V a l EDTA DE A E - c e l l u l o s e B D - c e l l u l o s e TCA RPC A 2 6 o A 2 6 o u n i t the r i b o n u c l e o s i d e s o f the fou r bases; adenine, c y t o s i n e , guanine and u r a c i l . the 3 1 - r i b o n u c l e o s i d e monophosphates. the 5 ' - r i b o n u c l e o s i d e monophosphates. the 2 1 , 3 ' , 5 1 - n u c l e o s i d e t r i a l c o h o l s . the 5 ' - r i b o n u c l e o s i d e diphosphates. the 5 * - r i b o n u c l e o s i d e t r i p h o s p h a t e s . i n o r g a n i c phosphate d e o x y r i b o n u c l e i c a c i d r i b o n u c l e i c a c i d messenger r i b o n u c l e i c a c i d ribosomal r i b o n u c l e i c a c i d t r a n s f e r r i b o n u c l e i c a c i d n onacylated v a l i n e tRNA aminoacylated v a l i n e tRNA amino a c i d s ; a l a n i n e , a r g i n i n e , a s p a r t i c a c i d , g l utamic a c i d , glutamine, g l y c i n e , l y s i n e , methionine, t h r e o n i n e , v a l i n e . e t h y l e n e d i a m i n e t e t r a a c e t a t e 0 - ( d i e t h y l a m i n o e t h y l ) c e l l u l o s e b e n z o ylated D E A E - c e l l u l o s e t r i c h l o r o a c e t i c a c i d r e v e r s e phase chromatography absorbance a t 2 60 nm the amount of m a t e r i a l g i v i n g an absorb-ance of 1.0 i n 1.0 ml of s o l u t i o n i n a 1 cm l i g h t path a t 260 nm a t n e u t r a l pH. xii. RNase - r i b o n u c l e a s e m1A,-i 6A>-t 6 A f Am, - The minor n u c l e o s i d e s ; 1-methyl adenosine, m5C,Cm,m1G,m7G, N 6 - i s o p e n t e n y l adenosine, N 6 - ( N - t h r e o n y l -I,dU,ip,Um,rT carbomyl) adenosine, 2 '-0-methyl adenosine, 5-methyl c y t i d i n e , 2 1 - 0 - m e t h y l c y t i d i n e , 1-methyl guanosine, N 7-methyl guanosine, i n o s i n e , 5, 6-dihydrouridine, p s e u d o u r i d i n e , 2'-0-methyl u r i d i n e , r i b o t h y m i d i n e , r e -s p e c t i v e l y . KBTn - [ 3H]-potassium borohydride 1. I n t r o d u c t i o n I. S t r u c t u r e of the tRNA Molecule S e v e r a l r e c e n t reviews are a v a i l a b l e on the p r o p e r t i e s of tRNA molecules (1-4). The sequence of about 100 s p e c i e s of tRNA are known and a l l can be drawn wit h the " c l o v e r l e a f " secondary s t r u c t u r e o r i g i n a l l y proposed by H o l l e y e t a l . (5). Phe Yeast tRNA drawn m the c l o v e r l e a f s t r u c t u r e xs shown xn Phe F i g u r e A. X-ray c r y s t a l o g r a p h y o f tRNA from y e a s t has F i g . A. The N u c l e o t i d e Sequence of Yeast P h e n y l a l a n i n e tRNA A C 75 C A pG - C C - G G - C 70 G U 5A - U " " A 60 '5 U G A C A C m A ° G ACUCn?G1(] ^ ' - ' ' I V G 0 T i i i i n'cu GUG ^ C G A G A C C ? L U"''G 55 G " ni •G . Q 20 2C - G A "5 I I I C - G A - U 30G -nffc.K) A - -y-Cm A U -LJ- y GmA A 35 p r o v i d e d a model t e r t i a r y s t r u c t u r e f o r tRNA molecules (6,7). F i g u r e B i s a drawing of the t e r t i a r y s t r u c t u r e proposed by Kim e t a l . (6). The tRNA molecule i s seen as two h e l i c a l arms i n an L shape. The amino a c i d acceptor end i s a t one end of the L, the anticodon loop i s a t the oth e r end of the L. The f a c t t h a t a number of tRNA molecules w i l l c o - c r y s t a l l i z e i n the same l a t t i c e suggests t h a t t h i s s t r u c t u r e of tRNA may be a g e n e r a l one (8) . A l l mature tRNA s p e c i e s c o n t a i n m o d i f i e d n u c l e o s i d e s . The work on t h i s s u b j e c t has been reviewed (9,10,148). These minor bases have been d i v i d e d i n t o t h r e e d i s t i n c t c l a s s e s . One c l a s s occurs a t the f i r s t p o s i t i o n of the antico d o n . A second c l a s s occurs a d j a c e n t to the 3'-OH end of the a n t i -codon. The t h i r d c l a s s i n c l u d e s a l l m o d i f i e d n u c l e o s i d e s not i n c l u d e d i n the f i r s t two c l a s s e s . A b r i e f review of these n u c l e o s i d e s w i l l serve the purposes of t h i s t h e s i s . M o d i f i e d n u c l e o s i d e s which occur i n the f i r s t p o s i t i o n of the anticodon can i n f l u e n c e the codon r e c o g n i t i o n of the tRNA. U r i d i n e - 5 - o x y a c e t i c a c i d o c c u r s i n the f i r s t p o s i t i o n of the anticodon i n E. c o l i tRNA? e r (11) and E. c o l i tRNAY a l (12). U r i d i n e - 5 - o x y a c e t i c a c i d p a i r s w i t h U as w e l l as A V a l and G i n the codon sequence. T h e r e f o r e tRNAi can p a i r 3. w i t h GUU as w e l l as GUA and GUG (13). Inosine i s a m o d i f i e d n u c l e o s i d e found i n the f i r s t p o s i t i o n which can p a i r w i t h U, C, or A. I t i s found i n E. c o l i tRNA^ r g (14), y e a s t tRNA^ l a (15), y e a s t t R N A f e r (16), r a t l i v e r t R N A S e r (17), y e a s t t R N A V a l (18), and y e a s t t R N A 1 l e (19). S e v e r a l 2 - t h i o u r i d i n e d e r i v a t i v e s are found i n the f i r s t G l u p o s i t i o n o f the antico d o n . E. c o l i tRNA2 c o n t a i n s 5 methyl-aminomethyl-2-thiouridine a t the f i r s t p o s i t i o n . Yeast tRNA2"^s has 2 - t h i o - 5 - a c e t i c a c i d methyl e s t e r u r i d i n e a t P l 11 the f i r s t p o s i t i o n (20,21). Yeast tRNA3 has 2 - t h i o - 5 - a c e t i c a c i d methyl e s t e r u r i d i n e i n * the f i r s t p o s i t i o n and has been shown t o p a i r w i t h GAA but not GAG (22). A pos-s i b l e f u n c t i o n f o r the 2 - t h i o u r i d i n e m o d i f i c a t i o n s may be to r e s t r i c t base p a i r i n g . The s t r u c t u r e of a m o d i f i e d guanosine r e s i d u e c a l l e d Q has r e c e n t l y been r e p o r t e d (23) . Q i s pr e s e n t i n tRNAs i n t e r a c t i n g w i t h codons c o n t a i n i n g U. rand C a t the t h i r d p o s i -t i o n o f the codon and A i n the second p o s i t i o n (24) . The tRNA w i t h Q has a g r e a t e r a f f i n i t y f o r the codon ending i n U compared t o the codon ending i n C. A m o d i f i e d form of Q i s p r e s e n t i n D r o s o p h i l a (25) and i n r a t l i v e r (26). The m o d i f i e d Q of r a t l i v e r i s i d e n t i c a l t o the Q of E. c o l i except t h a t an a d d i t i o n a l sugar moiety i s at t a c h e d . In r a t l i v e r the a t t a c h e d sugar can be e i t h e r mannose or g a l a c t o s e . Q and m o d i f i e d Q are the o n l y 7-deaza n u c l e o t i d e s known to occur i n tRNA. The f u n c t i o n o f t h i s m o d i f i e d n u c l e o s i d e i s not understood. 4. The second c l a s s of m o d i f i e d n u c l e o s i d e s are those t h a t occur a d j a c e n t to the 3'-terminal n u c l e o t i d e of the a n t i -codon. The i s o p e n t e n y l d e r i v a t i v e s of adenosine are i n t h i s c l a s s . They occur e x c l u s i v e l y i n tRNAs which r e c o g n i z e codons beginning w i t h U. In E. c o l i the predominant i s o p e n t e n y l d e r i v a t i v e i s 2-methylthio-N 6 - ( A 2 — i s o p e n t e n y l ) a d e n o s i n e (ms 2£ 6A) (27). In y e a s t and r a t l i v e r N 6 - ( A 2 - i s o p e n t e n y l ) adenosine ( i 6 A ) i s found (28,29). An enzyme which removes the i s o p e n t e n y l s i d e c h a i n from i 6 A i n tRNA i s prese n t i n L a c t o b a c i l l u s a c i d o p h i l u s and i n bovine l i v e r (30). N 6 - ( A 2 - i s o p e n t e n y l ) a d e n o s i n e e x h i b i t s c y t o k i n i n a c t i v i t y i n p l a n t s (31). The r e l a t i o n s h i p o f t h i s a c t i v i t y t o tRNA f u n c t i o n s , i f any, i s not known. A second m o d i f i e d n u c l e o s i d e o c c u r r i n g a d j a c e n t t o the Phe anticodon i s the base Y, f i r s t d i s c o v e r e d i n tRNA of baker's y e a s t (32). T h i s base i s now c a l l e d wybutosine. A m o d i f i c a t i o n o f t h i s base c a l l e d wybutoxine or peroxy Y has been demonstrated i n l i v e r (33) . A t h i r d Y n u c l e o s i d e c a l l e d Phe wyosine has been i s o l a t e d from T o r u l o p s i s u t i l i s tRNA (35). Wyosine i s the parent compound of wybutosine and wybutoxine. The Y bases are hydrophobic i n nature. They may f u n c t i o n i n an analogous manner to the i 6 A m o d i f i c a t i o n s . Both types of m o d i f i c a t i o n occur next t o anticodons which r e c o g n i z e Phe codons beg i n n i n g w i t h U. Removal of Y base from tRNA of y e a s t reduces the codon dependent ribosome b i n d i n g of the 5. tRNA but does not a f f e c t a m i n o a c y l a t i o n (130). D r o s o p h i l a Phe tRNA does not c o n t a i n a Y base (36). N [ 9 - ( 3 - D - r i b o f u r a n o s y l ) p u r i n - 6 - y l carbamoyl]threonine ( t 6 A ) and i t s d e r i v a t i v e s are found adjacent to the a n t i -codon. These m o d i f i e d n u c l e o s i d e s are p r e s e n t i n y e a s t , E. c o l i , and mammals (37). t 6 A occurs i n tRNAs which r e c o g -n i z e codons b e g i n n i n g w i t h A. 6-Methyladenosine has been found next to the 3'-OH terminus of the anticodon i n tRNAY a l of E. c o l i (38,39). 2-Methyladenosine i s p r e s e n t a d j a c e n t to the anticodon of E. c o l i t R N A A r g (14), t R N A A S p (40), t R N A ? ^ (41), t R N A 2 l u (20) and t R N A ? 1 S (42) . Yeast t R N A A l a (15). and T. u t i l i s t R N A A l a (43,44,45) have 1-methyl i n o s i n e a d j a c e n t to the anticodon (15). 1-Methyl guanosine occurs next t o the anticodon i n E. c o l i t R N A L e u (46), y e a s t t R N A L e u (47), and y e a s t t R N A A s p (48) . A f u n c t i o n a l r o l e f o r a m o d i f i e d base adjacent to the anticodon has been demonstrated by G e f t e r and R u s s e l l (49). They showed t h a t t y r o s i n e tRNA which l a c k e d the i s o p e n t e n y l m o d i f i c a t i o n was d e f e c t i v e i n ribosome b i n d i n g and would not support i n v i t r o p r o t e i n s y n t h e s i s . The t h i r d group of m o d i f i e d n u c l e o s i d e s are those not i n c l u d e d i n the two c l a s s e s above. One example of t h i s c l a s s i s p s e u d o u r i d i n e . Pseudouridine i s p r e s e n t i n the T^C loop of most tRNA molecules. One c l a s s of tRNAs which do not have t h i s p s e u d o u r i d i n e m o d i f i c a t i o n are the eucaryote 6. i n i t i a t o r tRNAs (50,51,52,53,54). Yeast, wheat germ, salmon and mammalian i n i t i a t o r tRNAs l a c k the pseudouridine and the r i b o t h y m i d i n e m o d i f i c a t i o n s u s u a l l y found i n the TtyC loop. T h i s change i n m o d i f i c a t i o n may be important f o r the f u n c t i o n o f the i n i t i a t o r tRNAs i n the i n i t i a t i o n o f p r o t e i n s y n t h e s i s . The T-ip m o d i f i c a t i o n s may f a c i l i t a t e the i n t e r -a c t i o n of tRNA wit h 5S RNA a t the ribosome d u r i n g p r o t e i n s y n t h e s i s e l o n g a t i o n steps (55). I I . The B i o l o g i c a l F u n c t i o n s of T r a n s f e r RNA A. P r o t e i n S y n t h e s i s The primary b i o l o g i c a l r o l e f o r tRNA i s to t r a n s l a t e the g e n e t i c i n f o r m a t i o n encoded i n p o l y n u c l e o t i d e s i n t o a s p e c i f i c amino a c i d sequence d u r i n g p r o t e i n s y n t h e s i s . The f i r s t s tep i n t h i s process i s the attachment o f f r e e amino a c i d t o the 2 1 , 3 ' - h y d r o x y l terminus of the tRNA molecule. T h i s r e a c t i o n i s c a t a l y z e d by the aminoacyl-tRNA synthetases. I t r e q u i r e s magnesium and ATP. A s p e c i f i c synthetase i s u t i l i z e d f o r each amino a c i d . The f i r s t step of the r e a c t i o n i s a c t i v a -t i o n o f the amino a c i d i n the form of an aminoacyladenylate. In the second step the amino a c i d i s t r a n s f e r r e d t o the tRNA molecule. , , Mg 1) AA + ATP . E-AA-AMP + PPi 2) E-AA-AMP +. tRNA , AA-tRNA + AMP + E tRNAs t e r m i n a t i n g i n 2'- or 3'-deoxyadenosine (56) or term-i n a t i n g i n 2 1(3')-amino-2'(3')deoxyadenosine (57) have been 7. s y n t h e s i z e d . S t u d i e s u t i l i z i n g these m o d i f i e d tRNAs have shown t h a t the amino a c i d i s a t t a c h e d to the 2'-hydroxyl i n some tRNAs, the 3'-hydroxyl i n other tRNAs, and e i t h e r 2' or 3' i n y e t o t h e r tRNAs. Recently Hecht and C h i n a u l t have compared the p o s i t i o n of a m i n o a c y l a t i o n f o r y e a s t and E. c o l i (58). They f i n d t h a t the p o s i t i o n of a m i n o a c y l a t i o n i s con-served d u r i n g e v o l u t i o n . Only t R N A T r p i s aminoacylated i n d i f f e r e n t p o s i t i o n s i n these two organisms. In eucaryotes separate aminoacyl-tRNA synthetases can e x i s t i n o r g a n e l l e s such as m i t o c h o n d r i a (59). These enzymes are s p e c i f i c f o r m i t o c h o n d r i a l tRNAs. The work of S. Ochoa, W.F. Anderson and t h e i r coworkers u t i l i z i n g i n v i t r o p r o t e i n s y n t h e s i z i n g systems d e r i v e d from mammalian r e t i c u l o c y t e s and Artemia s a l i n a embryos has demon-s t r a t e d t h a t the steps i n e u c a r y o t i c t r a n s l a t i o n are b a s i c a l l y i d e n t i c a l to the p r o c a r y o t i c system (60,61). The t r a n s l a -t i o n of mRNA i n the r a b b i t r e t i c u l o c y t e system i i s o u t l i n e d below. . . . Met . . A s p e c i a l i n i t i a t o r tRNA, tRNA^ i s u t i l i z e d i n the i n i t i a t i o n of p r o t e i n s y n t h e s i s . T h i s tRNA i s f u n c t i o n a l l y Me t Me t analogous to p r o c a r y o t e tRNA^ (62). Although Met-tRNA^ i s not f o r m y l a t e d i n v i v o , i t can be formylated i n v i t r o u t i l i z i n g the b a c t e r i a l t r a n s f o r m y l a s e (63). The process of i n i t i a t i o n begins w i t h the s e l e c t i o n of the i n i t i a t o r tRNA by a p r o t e i n analogous to p r o c a r y o t e IF-2. Adams e t a l . (64) have demonstrated two f a c t o r s i n the 8. r e t i c u l o c y t e system w i t h IF-2 a c t i v i t y , one c a l l e d IF-M1 and the other IF-MP. E i t h e r f a c t o r can c a t a l y z e the forma-t i o n of methionyl-puromycin i n the presence of GTP, A-U-G, 4OS s u b u n i t s , 60S subunits and f a c t o r IF-M2. IF-MP i s b e l i e v e d to f u n c t i o n i n v i v o . I f n a t u r a l m-RNA i s used as template, IF-MP i s more e f f i c i e n t than IF-M1 i n c a t a l y z i n g m e t h i o n y l -puromycin s y n t h e s i s (64). Secondly, antibody t o IF-M1 does not i n h i b i t amino a c i d i n c o r p o r a t i o n i n the Artemia s a l i n a system (64). IF-MP s e l e c t s the i n i t i a t o r tRNA and forms the Met t e r n a r y complex IF-MP-Met-tRNA^ -4OS subunxt-GTP. F a c t o r IF-M3 s e l e c t s the mRNA and c a t a l y z e s the formation of the Met 8OS-mRNA-Met-tRNA. complex. F a c t o r s IF-M2A, IF-M2B„ and IF-M2Bg are a l s o r e q u i r e d to form the 80S i n i t i a t i o n complex i n the r e t i c u l o c y t e system. T h i s complex can now begin the e l o n g a t i o n c y c l e . The f i r s t step i n e l o n g a t i o n i s the b i n d i n g of aminoacyl-tRNA and GTP to a p r o t e i n f a c t o r c a l l e d EF-1. EF-1 i n t e r -a c t s w i t h the ribosome and t r a n s f e r s the tRNA to the A s i t e . H y d r o l y s i s o f GTP a l s o occurs d u r i n g t h i s p r o c e s s . T h i s step r e q u i r e s codon-anticodon r e c o g n i t i o n between mRNA a t the A s i t e and aa-tRNA bound to EF-1. Next, p e p t i d e bond formation i s c a t a l y z e d by p e p t i d y l t r a n s f e r a s e . F a c t o r EF-2 c a t a l y z e s the movement of peptidyl-tRNA from the A s i t e t o the P s i t e w i t h GTP h y d r o l y s i s o c c u r r i n g a t t h i s s t e p . T ermination o f t r a n s l a t i o n occurs when a t e r m i n a t i o n codon (UAA, UAG, UGA) reaches the. A s i t e of the ribosome. Reticulocyte releasing factor (RF) binds to the A s i t e . This binding i s GTP dependent. The nascent peptide and RF are released with the hydrolysis of GTP. B. Control of Transcription tRNA has been implicated i n the control of certa i n bio-synthetic operons i n Salmonella (65,66,67). The h i s t i d i n e operon i s a clu s t e r of nine genes coding for enzymes re-quired for h i s t i d i n e biosynthesis. Six classes of regulatory mutants were selected as bacteria which were r e s i s t a n t to the h i s t i d i n e analogue 1,2,4-triazole-3-alanine. These bacteria w i l l synthesize h i s t i d i n e i n the presence of the analogue/ whereas i n wild-type bacteria, h i s t i d i n e synthesis suppressed by the analogue. His 0 mutations are c l a s s i c a l operator constitutive mutations. The f i v e other classes of His regulatory mutations implicate tRNA i n control of t h i s operon. His R mutations are mutations i n the s t r u c t u r a l His gene for tRNA . His U and His W have reduced l e v e l s of His tRNA and are believed to specify enzymes required for tRNA processing steps. Mutants at His S have altered histidyl-tRNA synthetase enzymes. These mutations increase H i s the l e v e l s of non-acylated tRNA . His T mutations are the most informative mutations of the group. The His T protein i s an enzyme required to convert two uridine r e s i -His dues to pseudouridines in' the anticodon loop of tRNA Bacteria with His T mutations lack pseudouridine i n the a n t i c o d o n loop and the His operon i s derepressed. The pseudouridine i n the G T i j i C loop i s not a f f e c t e d by t h i s mutation. P r o t e i n s y n t h e s i s i s not d i s r u p t e d by H i s T mutations, t h e r e f o r e the His T tRNA can s t i l l f u n c t i o n i n the t r a n s l a t i o n a l apparatus. T h i s o b s e r v a t i o n suggests a H i s s p e c i f i c r o l e f o r the tRNA molecule, i n p a r t i c u l a r the pseudouridines i n the anticodon loop, i n the r e g u l a t i o n of the His operon. Recently A r t z and Broach have proposed a model f o r c o n t r o l of the His operon (68). They propose than an a c t i v a t o r f a c t o r , as y e t unknown, i n t e r a c t s w i t h H i s His-tRNA and RNA polymerase to s t i m u l a t e t r a n s c r i p t i o n of the operon. In t h e i r model the His G enzyme, phospho-r i b o s y l t r a n s f e r a s e , a c t s as a n e g a t i v e e f f e c t o r of the operon. A t h i r d c o n t r o l l i n g element of the operon i s guanosine-5 1-diphosphate-3 1-diphosphate. T h i s f a c t o r , which accumulates i n s t r i n g e n t c e l l s s t a r v e d f o r amino a c i d s , has been shown to s t i m u l a t e t r a n s c r i p t i o n of the operon. tRNA has a l s o been r e p o r t e d to be i n v o l v e d i n the c o n t r o l of b i o s y n t h e t i c operons f o r the branched c h a i n amino a c i d s i n b a c t e r i a . V a l i n e , i s o l e u c i n e and l e u c i n e synthetase mutants are derepressed f o r the b i o s y n t h e t i c enzymes f o r t h e i r r e s p e c t i v e amino a c i d s (69). His U tRNA p r o c e s s i n g mutants a l s o cause d e r e p r e s s i o n of the i l v operons (70) . F u r t h e r evidence f o r the involvement of t R N A L e u and t R N A I l e i n r e g u l a t i o n of the i l v enzymes comes 11. from s t u d i e s on the His T mutants i n Salmonella (71). A l l t R N A L e u and one tRNA1"*"6 s p e c i e s c o n t a i n u r i d i n e r e s i d u e s i n the anticodon loop which are m o d i i f e d t o pse u d o u r i d i n e by the H i s T enzyme. The i s o l e u c i n e , l e u c i n e and v a l i n e b i o -s y n t h e t i c enzymes are not r e p r e s s i b l e by added branched c h a i n amino a c i d s i n H i s T c e l l s . However, growth r a t e of His T c e l l s i s normal. Cortese e t a l . (71) propose t h a t pseudo-u r i d i n e r e s i d u e s i n the anticodon loops of t R N A L e u and t R N A I l e f u n c t i o n i n r e g u l a t i o n o f the operons and not i n the t r a n s -l a t i o n a l r o l e of the tRNA. The tryptophan operon of E. c o l i has two t r a n s c r i p t i o n c o n t r o l s i t e s . The op e r a t o r s i t e binds the t r p r e p r e s s o r — L-tryptophan complex. A second s i t e , c a l l e d the a t t e n u a t o r s i t e , binds tRNA^'r^> and an u n i d e n t i f i e d p r o t e i n . When aminoacylated tRNA T r^ i s bound to the a t t e n u -a t o r s i t e , t r a n s c r i p t i o n o f the t r p operon i s terminated. Mutations i n tryptophanyl-tRNA synthetase (193) or i n t R N A T r p i t s e l f (194) have been shown to lower the e f f i c i e n c y of t r a n s c r i p t i o n t e r m i n a t i o n a t the a t t e n u a t o r t r a n s c r i p t i o n c o n t r o l s i t e . Yeast which have a temperature s e n s i t i v e i s o l e u c y l - t R N A synthetase show d e r e p r e s s i o n of the i s o l e u c i n e b i o s y n t h e t i c enzymes a t the r e s t r i c t i v e temperature (72,73). A s i m i l a r study on a l i n e o f Chinese Hamster ovary c e l l s c o n t a i n i n g a temperature s e n s i t i v e asparaginyl-tRNA synthetase has been Asn r e p o r t e d (74). Reduced l e v e l s of charged tRNA i n t h i s c e l l l i n e cause the l e v e l of asparagine synthetase t o i n -crease 300 to 400 per c e n t . These s t u d i e s i n d i c a t e t h a t the r o l e of tRNA i n the c o n t r o l o f t r a n s c r i p t i o n may not be l i m i t e d to pr o c a r y o t e systems, but i s a more g e n e r a l phenomenon. C. T r a n s f e r RNA and V i r a l I n f e c t i o n T r a n s f e r RNA f u n c t i o n s i n the r e p l i c a t i o n of c e r t a i n RNA v i r u s e s . Rous sarcoma v i r u s and av i a n m y e l o b l a s t o s i s v i r u s are RNA v i r u s e s which u t i l i z e RNA dependent DNA polymerase i n t h e i r r e p l i c a t i o n p r o c e s s e s . Host c e l l t r y p t o -phan tRNA i s used t o prime the r e v e r s e t r a n s c r i p t a s e r e a c -t i o n (75,76). The t R N A T r p binds t o the 3'-terminal 27 n u c l e o t i d e s o f RSV RNA and to the 3' t e r m i n a l 16 n u c l e o t i d e s of AMV RNA (77) . P r o l i n e tRNA i s r e p o r t e d t o be a s s o c i a t e d i n a s i m i l a r manner w i t h AKR nmrine leukemia v i r u s (78). C e r t a i n p l a n t RNA v i r u s e s can be aminoacylated a t the 3' terminus. The RNAs of the t u r n i p y e l l o w mosaic v i r u s group can be aminoacylated w i t h v a l i n e (79). A l l f o u r RNAs of brome mosaic v i r u s (BMV) w i l l accept t y r o s i n e (80). Sequence a n a l y s i s of the 3' t e r m i n a l sequences of each of the f o u r BMV RNAs has been r e p o r t e d (80). RNA 3 and RNA 4 are i d e n t i c a l . RNA 2 d i f f e r s by o n l y one n u c l e o t i d e and RNA 1 by o n l y two n u c l e o t i d e s . Tobacco mosaic v i r u s w i l l accept h i s t i d i n e (81). Although no d e f i n i t e r o l e f o r the tRNA moiety on the 3' terminus of these RNA v i r u s e s i s known, H a l l and Wepprich have proposed an i n t e r e s t i n g hypothesis (82). The amino-13. a c y l a t e d v i r u s e s have been shown to b i n d e u c a r y o t i c elonga-t i o n f a c t o r one (EF-1) (83,84). The RNA phage Q3 u t i l i z e s E. c o l i e l o n g a t i o n f a c t o r EF-Tu-Ts as two s u b u n i t s of the enzyme Q3 r e p l i c a s e . Thus H a l l and Wepprich s p e c u l a t e t h a t EF-1 may f u n c t i o n i n an analogous manner f o r the p l a n t RNA v i r u s r e p l i c a s e s . The aminoacyl-tRNA group c o u l d b i n d the EF-1 a s s o c i a t e d w i t h the r e p l i c a s e and promote r e p l i c a t i o n of the v i r a l RNA. U n f o r t u n a t e l y no s u p p o r t i n g evidence f o r t h i s i n t e r e s t i n g model i s known. D. The Aminoacyl-tRNA T r a n s f e r a s e s Aminoacyl-tRNA t r a n s f e r a s e s are a group of enzymes which t r a n s f e r amino a c i d s from aminoacyl-tRNA t o a c c e p t o r mole-c u l e s without the involvement of ribosomes. In one case a c l e a r b i o l o g i c a l f u n c t i o n f o r these enzymes i s known. The aminoacyl-tRNA-N-acetylmuramylpentapeptide t r a n s f e r a s e s p a r t i c i p a t e i n the b i o s y n t h e s i s of the b a c t e r i a l c e l l w a l l (85) . These enzymes s y n t h e s i z e the i n t e r p e p t i d e b r i d g e s between two st r a n d s of p e p t i d o g l y c a n . The b r i d g e s are s y n t h e s i z e d by stepwise a d d i t i o n of amino a c i d s d e r i v e d from aminoacyl-tRNA by the t r a n s f e r a s e enzyme. Staphylococcus  epidermis c o n t a i n s g l y c i n e and s e r i n e r e s i d u e s i n the i n t e r -p e p t i d e b r i d g e of p e p t i d o g l y c a n . Two s p e c i e s of g l y c i n e tRNA, comprising 40% of the t o t a l g l y c i n e acceptance, are b e l i e v e d to f u n c t i o n as donors of g l y c i n e f o r the c e l l w a l l (86) . These tRNAs do n o t ^ p a r t i c i p a t e i n p r o t e i n s y n t h e s i s . 14. They c o n t a i n GUGC i n p l a c e of the u s u a l GT^C i n loop IV. T h i s a l t e r a t i o n r e s u l t s i n l o s s of ribosome b i n d i n g a c t i v i t y . These tRNAs are examples of tRNA molecules which have a f u n c t i o n not i n v o l v i n g t r a n s l a t i o n of mRNA. The a m i n o a c y l - t R N A - p h o s p h a t i d y l g l y c e r o l t r a n s f e r a s e s t r a n s f e r l y s y l and a l a n y l r e s i d u e s from tRNA to the g l y c e r o l group of p h o s p h a t i d y l g l y c e r o l . A l y s y l - t R N A - p h o s p h a t i d y l -g l y c e r o l t r a n s f e r a s e has been demonstrated i n Staphylococcus  aureus and C l o s t r i d i u m w e l c h i i (87,88). C. w e l c h i i a l s o has an a l a n y l - t R N A - p h o s p h a t i d y l g l y c e r o l t r a n s f e r a s e a c t i v i t y . The f u n c t i o n of these t r a n s f e r a s e s and o f the aminoacyl-p h o s p h a t i d y l g l y c e r o l groups i n the membranes of these c e l l s i s not known. A t h i r d group of t r a n s f e r a s e s are the aminoacyl-tRNA-p r o t e i n t r a n s f e r a s e s . A s o l u b l e enzyme i n E. c o l i t r a n s f e r s l e u c i n e and p h e n y l a l a n i n e from tRNA to the amino t e r m i n a l b a s i c amino a c i d r e s i d u e s of acceptor p r o t e i n s (89,90). One enzyme i s r e s p o n s i b l e f o r both t r a n s f e r a s e a c t i v i t i e s . Recently the enzyme has been r e p o r t e d to t r a n s f e r methionine as w e l l (91). A mutant l a c k i n g t h i s enzyme has abnormal growth c h a r a c t e r i s t i c s due to i n c r e a s e d p r o l i n e oxidase a c t i v i t y (92). The r o l e of the t r a n s f e r a s e i n c o n t r o l l i n g p r o l i n e oxidase a c t i v i t y i s not known. Revertants of the mutant t h a t have r e g a i n e d t r a n s f e r a s e a c t i v i t y have w i l d -type p r o l i n e oxidase a c t i v i t y . S o f f e r has suggested t h a t the 15. t r a n s f e r a s e may modify a r e c e p t o r p r o t e i n which can i n -f l u e n c e the p r o l i n e oxidase a c t i v i t y (93). E u c a r y o t i c organisms have been shown t o c o n t a i n a r g i n y l -tRNA-protein t r a n s f e r a s e a c t i v i t y . T h i s enzyme has been demonstrated i n mammalian t i s s u e s and i n y e a s t (94,95). An i n v i v o acceptor p r o t e i n f o r t h i s enzyme i s not known. In v i t r o experiments show t h a t albumin, t h y r o g l o b u l i n (96), e r y t h r o c y t e membrane p r o t e i n s (94), and non-histone chromatin p r o t e i n s (97) can be a r g i n y l a t e d a t the amino terminus by t h i s enzyme. No evidence f o r a b i o l o g i c a l r o l e of these t r a n s f e r a s e r e a c t i o n s has been demonstrated. I I I . T r a n s f e r RNA P o p u l a t i o n s As o u t l i n e d above, t r a n s f e r RNA can f u n c t i o n i n many b i o l o g i c a l r o l e s . One approach to the study of tRNA c e l l u l a r f u n c t i o n i s to study tRNA p o p u l a t i o n s i n c e l l s which are d e v e l o p i n g or i n c e l l s s p e c i a l i z e d f o r a p a r t i c u l a r f u n c t i o n . Many s t u d i e s of t h i s type have been r e p o r t e d , some of which are d i s c u s s e d below. A. B a c i l l u s s u b t i l i s B. s u b t i l i s has two d i s t i n c t l i f e s t ages, a growing stage and a dormant or spore stage. S e v e r a l r e p o r t s of changes i n v a l i n e , l y s i n e and t y r o s i n e tRNA l e v e l s d u r i n g the s h i f t from one stage to the next have been r e p o r t e d (99,100,101). The o r i g i n a l workers used methylated albumin k i e s e l g u h r (MAK) 16. chromatography to f r a c t i o n a t e tRNA i s o a c c e p t o r s . V o i d has extended these s t u d i e s u s i n g the RPC-5 chromatography system which w i l l r e s o l v e more i s o a c c e p t o r s than the MAK system (102,103). tRNAs s p e c i f i c f o r s e v e r a l amino a c i d s were screened f o r stage s p e c i f i c changes. Growth stage-dependent changes i n the r e l a t i v e amounts of i s o a c c e p t o r s f o r tRNAs charged w i t h g l y c i n e , t y r o s i n e , l e u c i n e , s e r i n e , t h r e o n i n e , asparagine and a r g i n i n e were demonstrated. The e x i s t e n c e of a s p e c i e s found on l y i n one growth stage was found f o r l y s i n e , glutamic a c i d and a r g i n i n e . V o i d has shown t h a t the changes i n t R N A T y r , t R N A L e u , t R N A T r p and t R N A L y s occur a t d i f f e r e n t times d u r i n g the change from v e g e t a t i v e t o dormant c e l l type (103). These changes may r e f l e c t d i f f e r e n t c e l l u l a r p r o c e s s e s . Many of the changes i n tRNA p a t t e r n s are due t o d i f f e r -ences i n m o d i f i c a t i o n . S i n g h a l and V o i d have shown t h a t e x p o n e n t i a l l y growing c e l l s have tRNA which i s undermethyl-ated (104). A d i f f e r e n c e i n m o d i f i c a t i o n has been r e p o r t e d f o r the two t R N A T y r s p e c i e s . tRNAT y r i s predominant i n Tvr . . growing c e l l s . t R N A 2 i s predominant i n s t a t i o n a r y c e l l s and spores. K e i t h e t a l . have shown t h a t these tRNAs are Tvr i ; . i d e n t i c a l except t h a t tRNAi c o n t a i n s ^ A which i s m o d i f i e d to m s 2 i 6 A i n tRNA^ y r (105). These authors suggest t h a t t h i s d i f f e r e n c e i n m o d i f i c a t i o n of a n u c l e o s i d e present i n the anticodon loop may serve a r e g u l a t o r y f u n c t i o n of some k i n d . The o n l y c l e a r demonstration of a r e g u l a t o r y r o l e f o r tRNA m o d i f i c a t i o n , the Salmonella His T mutants, i n v o l v e s pseudo-17. u r i d i n e m o d i f i c a t i o n s i n the anticodon l o o p . No c l e a r regu-l a t o r y r o l e f o r the m o d i f i c a t i o n o f B_. s u b t i l i s t y r o s i n e tRNA has been demonstrated. B. Hormone E f f e c t s on tRNA Many s t u d i e s have been r e p o r t e d on t r a n s f e r RNA popula-t i o n s i n c e l l s s p e c i a l i z e d f o r a p a r t i c u l a r f u n c t i o n . One w e l l s t u d i e d system i s the c h i c k o v i d u c t . Treatment o f c h i c k s w i t h estrogen causes d i f f e r e n t i a t i o n of the o v i d u c t and the s y n t h e s i s of s p e c i f i c p r o t e i n s , ovalbumin, conalbumin and lysozyme. O'Malley e t a l . have shown t h a t tRNA l e v e l s i n c r e a s e f i v e - f o l d r e l a t i v e to othe r s t a b l e RNA s p e c i e s a f t e r estrogen treatment (106). T r a n s f e r RNA methylase a c t i v i t y a l s o i n c r e a s e s (107). Sharma e t a l . (108) have developed a c e l l f r e e p r o t e i n s y n t h e s i z i n g system d e r i v e d from E r l i c h a s c i t e s c e l l s which i s dependent on added t r a n s f e r RNA. Using t h i s system they s t u d i e d the e f f i c i e n c y of tRNA ex-t r a c t e d from o v i d u c t t o t r a n s l a t e ovalbumin messenger RNA. tRNA e x t r a c t e d from estrogen t r e a t e d c h i c k o v i d u c t or from mature hen o v i d u c t c o u l d support ovalbumin s y n t h e s i s a t a r a t e 50-70% h i g h e r than tRNA e x t r a c t e d from c h i c k s w i t h -drawn from es t r o g e n treatment. A l l t h r e e tRNAs c o u l d support t r a n s l a t i o n o f endogenous mRNA or added r a b b i t g l o b i n mRNA at equal r a t e s . These r e s u l t s suggest t h a t estrogen induces changes i n the tRNA p o p u l a t i o n of t a r g e t t i s s u e s . These changes r e s u l t i n ,a tRNA p o p u l a t i o n adapted f o r e f f i c i e n t t r a n s l a t i o n of estrogen induced messenger RNA. 18. Estrogen induced changes i n the tRNAs have been s t u d i e d by B e r n f i e l d and h i s coworkers (109,110,111). Estrogen treatment induces the s y n t h e s i s of the s e r i n e r i c h y o l k phosphoprotein p h o s v i t i n i n c h i c k e n l i v e r . tRNA ex-t r a c t e d from the l i v e r s of c h i c k s t r e a t e d w i t h estrogen has decreased l e v e l s of tRNA? e r r e l a t i v e t o t R N A f e r and tRNA^ e r. Ser The tRNA acceptance i s i n c r e a s e d over c o n t r o l l e v e l s and t h i s i n c r e a s e has been shown to be due to decreased degrada-t i o n , not to i n c r e a s e d s y n t h e s i s (109). The decreased Ser amounts of tRNAi may be due t o s e l e c t i v e l o s s o f t h i s i s o -acceptor d u r i n g e x t r a c t i o n of the tRNA. Maenpaa and B e r n f i e l d Ser (111) f i n d t h a t tRNAi i s i n c r e a s e d i n the tRNA p o p u l a t i o n s bound to ribosomes and i n the n u c l e i . They suggest t h a t Ser tRNAi i s u t i l i z e d e x t e n s i v e l y f o r p h o s v i t i n s y n t h e s i s . During e x t r a c t i o n the n u c l e i and ribosomal tRNA p o p u l a t i o n s Ser were l o s t r e s u l t i n g i n an apparent lowering of the tRNAi l e v e l s i n estrogen t r e a t e d l i v e r s . P i t u i t a r y hormones induce spermatogenesis i n salmon. Urquhart has s t u d i e d the tRNA i n salmon t e s t e s a t f o u r succes-s i v e stages of development (112). The tRNA p o p u l a t i o n s are s p e c i a l i z e d f o r p r o d u c t i o n of the b a s i c n u c l e a r p r o t e i n s . Arginine and lysine tRNAs are p r e s e n t i n i n c r e a s e d amounts when compared t o l i v e r tRNA. A r g i n i n e tRNA i n c r e a s e s s t e a d i l y through spermatogenesis w h i l e l y s i n e tRNA i n c r e a s e s i n i t i a l l y , and then decreases a f t e r the second stage. These changes c o r r e l a t e 19. with the synthesis of ly s i n e r i c h histones early i n spermatogenesis and the synthesis of arginine r i c h protamines in the l a t e r spermatogenic stages. C. Transfer RNA i n Collagen Synthesizing Tissues Collagen i s a major protein of connective t i s s u e . Collagen contains 33% glycine, 23% proline and hydroxyproline and 11% alanine. Maenpaa and Ahonen (113), and Lanks and Weinstein (114) have studied tRNA populations i n granula-t i o n tissue which i s producing extensive amounts of collagen. They f i n d that the leve l s of glycine, proline, ly s i n e and arginine tRNAs are increased i n the granulating tissue when compared to l i v e r . Analysis of these tRNAs on BD-cellulose and MAK columns showed quantitative changes but no q u a l i -t a t i v e changes. This s p e c i a l i z a t i o n of tRNA for collagen synthesis was also reported for embryonic chicken bone (115). These workers compared chicken tendon and embryonic bone to brain, heart and l i v e r t issues. They f i n d that l e v e l s of glycine, proline and arginine tRNAs are increased in the collagen producing t i s s u e . One species of glycine tRNA, tRNA^^ G G U i s increased. They predict that the codons GGC and GGU are Val prevalent i n the collagen mRNA. The l e v e l of tRNA i s decreased i n the collagen producing tissue. Collagen i s low i n v a l i n e . The increased l e v e l s of arginine tRNA are 20. not due to s p e c i a l i z a t i o n f o r c o l l a g e n s y n t h e s i s . A r g i n i n e may be r e q u i r e d f o r the s y n t h e s i s of an undefined c e l l u l a r component i n these t i s s u e s . D. T r a n s f e r RNA i n the S i l k Gland of Bombyx mori The s i l k gland of B. mori i s d i v i d e d i n t o three p a r t s . The p o s t e r i o r p a r t i s s p e c i a l i z e d f o r producing a p r o t e i n c a l l e d f i b r o i n . The middle p a r t i s s p e c i a l i z e d f o r produc-in g a p r o t e i n c a l l e d s e r i c i n . Development o f the s i l k gland begins a t the f i r s t day of the f i f t h l a r v a l i n s t a r and con-t i n u e s u n t i l the e i g h t h day. During t h i s development the amount of tRNA i n the middle p a r t i n c r e a s e s f i v e - f o l d w h i l e the amount i n the p o s t e r i o r p a r t i n c r e a s e s t e n - f o l d (116, 117,118). tRNAs s p e c i f i c f o r a l a n i n e , g l y c i n e , s e r i n e , and t y r o s i n e reach a maximum a t day seven i n the p o s t e r i o r p a r t . These tRNAs have i n c r e a s e d t h i r t y - f o l d . The non-f i b r o i n s p e c i f i c tRNAs stop i n c r e a s i n g by day f o u r . At t h i s stage these tRNAs have i n c r e a s e d by 6-7 times the o r i g i n a l amounts. A s i m i l a r i n c r e a s e i n s e r i c i n s p e c i f i c tRNAs occurs i n the middle p a r t . Gage (119), and Majima and Shimura (12 0) have r e p o r t e d t h a t no s e l e c t i v e a m p l i f i c a t i o n of the s i l k gland g l y c i n e tRNA genes occurs d u r i n g develop-ment. F o u r n i e r e t a l . (121) r e p o r t t h a t the turnover of tRNA s p e c i e s l a b e l l e d i n c u l t u r e o c c u r s a t the same r a t e i n f i b r o i n s p e c i f i c and n o n - s p e c i f i c tRNA s p e c i e s . T h e r e f o r e i t appears t h a t the a d a p t a t i o n of s i l k gland tRNA i s due to i n c r e a s e d s y n t h e s i s and p r o c e s s i n g of s p e c i f i c tRNAs. A l a n i n e , g l y c i n e , and s e r i n e c o n s t i t u t e 87% of the amino a c i d s p r e s e n t i n f i b r o i n . The most p r e v a l e n t codons i n the f i b r o i n message are GCU f o r a l a n i n e , GGU and GGA f o r g l y c i n e , and UCA f o r s e r i n e (122). G a r e l (123) proposes t h a t the s i l k gland i s o a c c e p t o r s are q u a n t i t a t i v e l y adapted to p r o v i d e i n c r e a s e d tRNAs s p e c i f i c f o r these codons. Delaney and S i d d i q u i have measured the i n v i v o l e v e l s of aminoacyl-tRNA i n the p o s t e r i o r s i l k gland (118). In the e a r l y growth stages of the s i l k gland f i b r o i n i s not b e i n g produced. A t t h i s stage c e l l u l a r c o n s t i t u e n t s and f i b r o i n mRNA accumulate. Only 40% of the tRNAs are amino-a c y l a t e d a t t h i s stage. In the l a t e r stages when f i b r o i n p r o d u c t i o n i s maximal, 80% of the tRNAs are aminoacylated. These s t u d i e s demonstrate t h a t the s i l k gland tRNA p o p u l a t i o n i s t a i l o r e d to the p r o d u c t i o n of s p e c i f i c s i l k p r o t e i n s . T h i s a d a p t a t i o n i s brought about by c e l l u l a r con-t r o l of the tRNA genes. In t h i s case the process of develop-ment i n c l u d e s s p e c i f i c c o n t r o l of tRNA genes. E. T r a n s f e r RNA i n the R e t i c u l o c y t e R e t i c u l o c y t e s are the anucleate p r e c u r s o r s of e r y t h r o -c y t e s . R e t i c u l o c y t e s c o n t a i n the apparatus necessary f o r the t r a n s l a t i o n of p r e v i o u s l y s y n t h e s i z e d messenger RNA. The p r o t e i n s y n t h e s i z e d i s 85 to 90% hemoglobin. S e v e r a l workers have demonstrated t h a t r e t i c u l o c y t e tRNA i s adapted to the s y n t h e s i s of the a and 3 chains of hemoglobin. Anderson and G i l b e r t u t i l i z e d a tRNA dependent i n v i t r o p r o t e i n s y n t h e s i s system d e r i v e d from r e t i c u l o c y t e s t o study the r o l e of tRNA i n t h i s system (124). T h i s i n v i t r o system t r a n s l a t e d endogenous hemoglobin mRNA when tRNA was added. A d d i t i o n o f r a b b i t r e t i c u l o c y t e tRNA supported the s y n t h e s i s a t a r a t e 40 perc e n t g r e a t e r than r a b b i t l i v e r tRNA. The r e t i c u l o c y t e tRNA ap p a r e n t l y c o n t a i n s more o f a l i m i t i n g tRNA s p e c i e s . Furthermore when r e t i c u l o c y t e tRNA was added i n l i m i t i n g amounts s y n t h e s i s of a ch a i n was reduced to 50 percent t h a t o f 3 c h a i n . With excess r e t i c u l o c y t e tRNA equal amounts o f a and 8 c h a i n were produced. The codons r e q u i r i n g the l i m i t i n g tRNA s p e c i e s may be more abundant i n the a c h a i n message than i n the 8 c h a i n message. Sharma e t a l . r e p o r t e d an analogous study u t i l i z i n g an a s c i t e s c e l l f r e e p r o t e i n s y n t h e s i s system which r e q u i r e d tRNA and added mRNA (125). They found t h a t r e t i c u l o c y t e tRNA would not f a c i l i t a t e t r a n s l a t i o n o f o v i d u c t or encephalomyocarditis v i r u s RNA e f f i c i e n t l y whereas l i v e r tRNA allowed t r a n s l a t i o n of these two RNAs as w e l l as r e t i c u l o c y t e mRNA. The f a i l u r e of the r e t i c u l o c y t e tRNA t o support t r a n s l a t i o n r e s u l t e d i n premature t e r m i n a t i o n of the non-homologous messages. T h i s premature t e r m i n a t i o n may have r e s u l t e d from d e f i c i e n c i e s of s p e c i f i c tRNAs needed t o i n t e r a c t w i t h c e r t a i n cddons presen t i n the non-homologous messages but not presen t i n the r e t i c u l o c y t e mRNA. L i v e r tRNA which i s not adapted t o s y n t h e s i s o f a p a r t i c u l a r p r o t e i n does not c o n t a i n d e f i c -i e n c i e s of c e r t a i n tRNAs. H i l s e and R u d l o f f (126) r e p o r t such a d e f i c i e n c y f o r Gin t R N A C A A . They f i n d t h a t o n l y two percent of r e t i c u l o c y t e t R N A G l n responds t o the codon CAA w h i l e 25% of l i v e r t R N A G l n responds t o CAA. Furthermore they show t h a t r a b b i t Hb mRNA does not c o n t a i n the codon CAA. T h i s r e s u l t demonstrates the a d a p t a t i o n of r e t i c u l o c y t e tRNA and p r o v i d e s a p o s s i b l e e x p l a n a t i o n f o r the r e s u l t s o f Sharma e t a l . (125). L i t t and Kabat have s t u d i e d tRNA a d a p t a t i o n i n sheep r e t i c u l o c y t e s (127). Hemoglobins A and B have no i s o l e u c i n e r e s i d u e s and s i x to e i g h t methionine r e s i d u e s . Hemoglobin C on the oth e r hand has two i s o l e u c i n e r e s i d u e s and o n l y two methionine r e s i d u e s . R e t i c u l o c y t e s which s y n t h e s i z e Hb A and Hb B have two to th r e e times the amount of i s o -l e u c i n e acceptor a c t i v i t y . R e t i c u l o c y t e s which make Hb-C have i n c r e a s e d l e v e l s of one methionine i s o a c c e p t o r . T h i s study demonstrates a d a p t a t i o n o f t r a n s f e r RNA l e v e l s w i t h i n two p o p u l a t i o n s o f the same c e l l type. Smith and McNamara (128) have compared h i s t i d i n e and i s o l e u c i n e tRNA l e v e l s i n r a b b i t r e t i c u l o c y t e s t o those o f r a b b i t l i v e r . Hemoglobin has a h i g h h i s t i d i n e content but a low i s o l e u c i n e content. H i s t i d i n e tRNA l e v e l i s i n c r e a s e d by a f a c t o r of two i n the r e t i c u l o c y t e w h i l e i s o l e u c i n e i s reduced by a f a c t o r o f two. 24. F. T r a n s f e r RNA i n Cancer C e l l s Many s t u d i e s have r e v e a l e d changes i n i s o a c c e p t o r pat-t e r n s i n malignant c e l l s . These s t u d i e s have been reviewed and catalogued (129,130,131). Many of these changes have been shown t o be due to n u c l e o t i d e m o d i f i c a t i o n . For Phe . Phe example, hepatoma tRNA resembles normal l i v e r tRNA Phe except f o r m o d i f i c a t i o n s (132) . tRNA from neuroblastoma c e l l s l a c k s the peroxy Y m o d i f i c a t i o n i n 85% of the molecules. Phe In normal t i s s u e s , o n l y 6-10% of tRNA are m i s s i n g t h i s m o d i f i c a t i o n (133). D e s p i t e the v a s t wealth of i n f o r m a t i o n , on changes i n tRNA i s o a c c e p t o r p a t t e r n s i n n e o p l a s i a i t i s s t i l l not p o s s i b l e to a s s i g n a r o l e f o r these changes i n the b i o l o g y of cancer. IV. D r o s o p h i l a melanogaster tRNA T r a n s f e r RNA i s i n v o l v e d i n many processes w i t h i n the e u c a r y o t i c c e l l . D r o s o p h i l a melanogaster i s an organism whose g e n e t i c s i s w e l l s t u d i e d and many mutants are a v a i l -a b l e . These a t t r i b u t e s w i l l be u s e f u l to molecular b i o l -o g i s t s attempting t o t r a c e the many d i v e r s e f u n c t i o n s o f tRNA molecules. D r o s o p h i l a tRNA has been analyzed i n f l i e s from t h r e e developmental stages (134,135). RPC-5 chromatography was used t o r e s o l v e 63 major and 36 minor tRNA s p e c i e s . There were s e v e r a l q u a n t i t a t i v e changes i n tRNA i s o a c c e p t o r l e v e l s d u r i n g development from f i r s t i n s t a r to a d u l t . Changes were noted i n t R N A M e t , t R N A T h r , and t R N A C y s . The f o u r s p e c i e s o f tRNA which c o n t a i n a Q-type n u c l e o s i d e were a l t e r e d i n a c h a r a c t e r i s t i c manner (136). The Q - l i k e base i n D r o s o p h i l a resembles the n u c l e o s i d e Q from E. c o l i but i s not i d e n t i c a l to E. c o l i Q (137, 25,). The n u c l e o t i d e Q i s s y n t h e s i z e d from G i n a p o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n r e a c t i o n (195). t R N A A s p , t R N A A s n , t R N A H l S , and t R N A T y r c o n t a i n p a i r s o f i s o a c c e p t o r s which d i f f e r o n l y i n the presence of e i t h e r an unmodified G or a Q-type m o d i f i c a t i o n i n the anticod o n . The l e v e l s of the Q - l i k e n u c l e o s i d e c o n t a i n i n g s p e c i e s decrease r e l a t i v e to the G c o n t a i n i n g s p e c i e s d u r i n g development from the egg stage t o the pupal stage. During pupation the amounts of Q c o n t a i n i n g tRNA s p e c i e s i n c r e a s e d r a m a t i c a l l y (25). A mutant of D r o s o p h i l a c a l l e d suppressor of sable has been shown to c o n t a i n more - Q - l i k e n u c l e o -s i d e c o n t a i n i n g i s o a c c e p t o r s than the w i l d - t y p e f l i e s a t a l l stages of development (25,138). The most probable e x p l a n a t i o n of t h i s e f f e c t i s a d e r e p r e s s i o n o f the Q syn-t h e s i z i n g system i n the mutant d u r i n g the l a r v a l stages. The suppressor of sa b l e mutation w i l l suppress the expres-s i o n of an eye c o l o r mutation c a l l e d v e r m i l i o n . The v e r -m i l i o n - f l i e s have reduced tryptophan p y r r o l a s e a c t i v i t y . Jacobson had r e p o r t e d t h a t the t R N A T y r l a c k i n g the Q - l i k e n u c l e o s i d e c o u l d b i n d to tryptophan p y r r o l a s e and i n h i b i t i t s a c t i v i t y w h i l e the tRNA c o n t a i n i n g the Q - l i k e n u c l e o s i d e c o u l d not b i n d (139). The developmental changes i n the tRNAs c o n t a i n i n g the Q - l i k e m o d i f i c a t i o n c o u l d r e f l e c t r o l e s played by these tRNAs i n c o n t r o l l i n g enzymes such as t r y p t o -phan p y r r o l a s e . I f the v e r m i l i o n .mutation a f f e c t e d t r y p t o -phan p y r r o l a s e i n such a way as to make i t s e n s i t i v e t o the non Q c o n t a i n i n g t R N A T y r , the suppressor of s a b l e mutation would rescue the v e r m i l i o n f l i e s by r e d u c i n g the l e v e l s of non Q c o n t a i n i n g t R N A T y r . T h i s i n t e r p r e t a t i o n i s now i n doubt. Jacobson e t a l . (140) have r e p o r t e d t h a t they cannot repeat the tRNA mediated i n h i b i t i o n o f tryptophan pyrrolase.. A study by Mishke e t a l . a l s o f a i l e d i n attempts t o r e p e a t these experiments (141). S e v e r a l D r o s o p h i l a tRNA s p e c i e s have been p u r i f i e d and Phe t h e i r minor base compositionsdetermined. D r o s o p h i l a tRNA i s unusual i n t h a t i t does not c o n t a i n the hypermodified Y base ( 3 6 ) . The tRNAs (143) have a l s o been analyzed. Ser The D r o s o p h i l a tRNAs which r e c o g n i z e codons beginning w i t h U c o n t a i n the i s o p e n t e n y l adenosine m o d i f i c a t i o n . The m o d i f i e d bases prese n t i n l y s i n e , glutamate, and glutamine tRNAs have a l s o been analyzed (141). Atwood (191) has suggested t h a t a c l a s s of mutations i n D r o s o p h i l a c a l l e d Minutes may be mutations of the tRNA genes. There are approximately 60 Minute l o c i known. White et a l . (134) have shown t h a t a t o t a l of 99 tRNA s p e c i e s can be r e s o l v e d on RPC-5 columns. Many of these s p e c i e s may be due to p a r t i a l m o d i f i c a t i o n of c e r t a i n tRNAs, t h e r e f o r e the estimate of 99 tRNA s p e c i e s c o u l d be an o v e r e s t i m a t e . A k i n e t i c a n a l y s i s of RNA--DNA h y b r i d i z a t i o n data has been r e p o r t -ed r e c e n t l y (145). T h i s a n a l y s i s i n d i c a t e d 59 genes f o r D r o s o p h i l a tRNA w i t h an average redundancy of 10 c o p i e s each. The number o f Minute mutations i s i n rough agreement w i t h t h i s b i o c h e m i c a l e s t i m a t i o n of the number of tRNA gene f a m i l i e s . Two other s t u d i e s support the Minute h y p o t h e s i s . F l i e s c a r r y -Y i n g a d e l e t i o n f o r the Minute (3)h_ l o c u s have a reduced Thr T v amount of tRNA3 . A l s o a Minute i s a s s o c i a t e d w i t h the tRNAs genes l o c a l i z e d by the i n s i t u h y b r i d i z a t i o n assay (147). F u r t h e r a n a l y s i s of the tRNA genes i n D r o s o p h i l a i s r e q u i r e d b e f o r e the Minute hy p o t h e s i s can be s u b s t a n t i a t e d . The purpose of t h i s t h e s i s i s to i n v e s t i g a t e the r o l e s of t r a n s f e r RNA and t r a n s f e r RNA genes i n D r o s o p h i l a . V a l i n e V a l tRNAs were chosen because the t h r e e major tRNAs c o u l d be r e a d i l y p u r i f i e d . The o r g a n i z a t i o n of the genes f o r the v a l i n e tRNAs was s t u d i e d u t i l i z i n g the p u r i f i e d tRNAs as probes. Then the f u n c t i o n of these genes was s t u d i e d through the a n a l y s i s of mutants c a r r y i n g d e l e t i o n s and d u p l i c a t i o n s f o r the tRNA genes. In a d d i t i o n the coding p r o p e r t i e s of the Ser Ser tRNAs are r e p o r t e d i n t h i s t h e s i s . tRNAs must t r a n s -l a t e the codons of the group UCX as w e l l as the group AGPy. The coding s t u d i e s were performed to t e s t the h y p o t h e s i s t h a t those s e r i n e i s o a c c e p t o r s which c o n t a i n the m o d i f i e d n u c l e o s i d e i 6 A would respond to the codons UCX and not to the codons AGPy. 28. M a t e r i a l s Whatman D E A E - c e l l u l o s e (DE-22) and chromatography paper were from W. and R. B a l s t o n L t d . Sephadex G-25 and Sepharose 4B from Pharmacia. B i o - G e l A-0.5 M was o b t a i n e d from B i o -rad L a b o r a t o r i e s . The RPC-5 m a t e r i a l s Adogen 464 and Plaskon CTFE-2300 powder were from Ashland Chemical Co. and A l l i e d Chemical Corp. r e s p e c t i v e l y . High p r e s s u r e columns were purchased from Chromatronix Inc. C e l l u l o s e t h i n l a y e r .1 p l a t e s and p o l y e t h y l e n i m i n e t h i n l a y e r p l a t e s were from Eastman Kodak Co. and BrinkmannInstruments L t d . re s p e c -t i v e l y . C e l l u l o s e n i t r a t e f i l t e r s , pore s i z e 0.45 ym, were from M i l l i p o r e Corp. A l l common chemicals, b u f f e r s , amino a c i d s , e t c . were of reagent grade and o b t a i n e d commercially. R a d i o a c t i v e amino a c i d s , [ 3 2P]orthophosphate-HCl and c a r r i e r f r e e Na 1 2 5 I were from New England Nuclear Corp. [ 3H]-Potassium borohydride was from Amersham/Searle. P a n c r e a t i c RNase, p u r i f i e d snake venom phosphodies-t e r a s e , E. c o l i a l k a l i n e phosphatase, and g l y c e r a l d e h y d e phosphate dehydrogenase ( r a b b i t muscle) were from Worthing-ton B i o c h e m i c a l s . RNase T i and RNase T2 were from C a l b i o -chem. T4 p o l y n u c l e o t i d e kinase and M. l y s o d e i k t i c u s p o l y -n u c l e o t i d e phosphorylase were from P-L B i o c h e m i c a l s . Myokinase and 3-phosphoglycerate k i n a s e (yeast) were from Sigma Chemical Co. 29. Methods I. Growth of D r o s o p h i l a melanogaster The Samarkand s t r a i n of D r o s o p h i l a melanogaster was grown at 22°C i n l a r g e p l a s t i c cages. The growth medium was prepared a c c o r d i n g to Lewis (149). A d u l t s were har-v e s t e d w i t h carbon d i o x i d e and s t o r e d a t -70°C. Mutant s t r a i n s of D r o s o p h i l a were grown i n p i n t b o t t l e s a t 22°C on the same growth medium as the Samarkand w i l d - t y p e s t r a i n . I I . P r e p a r a t i o n of Aminoacyl-tRNA Synthetases D r o s o p h i l a aminoacyl-tRNA synthetases were prepared a c c o r d i n g t o White and Tener (150). A l l procedures were per-formed a t 0-4°C. Frozen D r o s o p h i l a a d u l t s (50 gms) were added to 200 mis of 10 mM T r i s HCl (pH 7.5), 10 mM Mg(0Ac) 2, 10 mM 2-mercaptoethanol, and 1 mM p h e n y l m e t h y l s u l f o n y l -f l u o r i d e ( b u f f e r A ) . The f l i e s were homogenized f o r one minute a t top speed i n a V i r t i s homogenizer, l e f t to c o o l f o r two minutes, and the homogenization step was repeated. N u c l e i , m i t o c h o n d r i a , and c e l l d e b r i s were removed by cen-t r i f u g a t i o n a t 40,000 g_ f o r 15 minutes. The supernatant was c e n t r i f u g e d a t 105,000 g f o r two hours i n a Beckman Model L u l t r a c e n t r i f u g e t o p e l l e t the ribosomes. The super-natant was made 0.3 M i n NaCl and washed through a DE-22 DE A E - c e l l u l o s e column (1.2 cm x 15 cm) e q u i l i b r a t e d i n b u f f e r A c o n t a i n i n g 0.3 M NaCl. T h i s step removes tRNA from the synthetase p r e p a r a t i o n . The A 2 8 o absorbing m a t e r i a l 30. washed from the column wi t h 0.3 M NaCl was pooled and d i a l y z e d a g a i n s t two l i t e r s of b u f f e r A c o n t a i n i n g 50 per-cent g l y c e r o l . The con c e n t r a t e d p r o t e i n s o l u t i o n was s t o r e d i n a l i q u o t s (1 ml) a t -70°C. Before the aminoacyla-t i o n r e a c t i o n each a l i q u o t was thawed a t 4°C and passed through a smal l Sephadex G-25 column (1.2 cm x 20 cm) e q u i l i b r a t e d i n b u f f e r A. T h i s g l y c e r o l - f r e e p r e p a r a t i o n w i l l be r e f e r r e d t o as crude synthetase p r e p a r a t i o n . I l l . The Aminoacylation Reactions The a m i n o a c y l a t i o n r e a c t i o n s were performed a c c o r d i n g to White and Tener (150) . A. Assay o f Amino A c i d Acceptance i n S o l u t i o n T r a n s f e r RNA was aminoacylated a t room temperature f o r t h i r t y minutes i n a volume of 0.1 to 0.2 ml. The concen-t r a t i o n s of r e a c t i o n components f o r most of the r e a c t i o n s were 50 mM T r i s HCI (pH 7.5), 10 mM MgCla, 4 mM ATP, 0.5 mM CTP, 30 mM KC1, 125 yM [lkC] amino a c i d and 100 yM of each of the ni n e t e e n common [ 1 2C] amino a c i d s e x c l u d i n g the l a b e l l e d amino a c i d . Optimal c h a r g i n g of c e r t a i n amino a c i d s r e q u i r e d s p e c i a l c o n d i t i o n s : the a s p a r t i c a c i d r e a c t i o n c o n t a i n e d 250 yM [ l l fC] a s p a r t i c a c i d , 20 mM MgCl 2, 2 mM ATP; f o r glutamic a c i d and glutamine the mixtures were m o d i f i e d to c o n t a i n 200 yM [lhC] amino a c i d and 2.5 mM MgCl2; the p r o l i n e assay mixture c o n t a i n e d 250 yM [11*C] p r o l i n e ; and the methionine assay c o n t a i n e d 250 yM [1'*C] methionine and 5 mM MgCl.2 • A d d i t i o n of crude synthetase p r e p a r a t i o n s t a r t e d the r e a c t i o n s . A l i q u o t s (SO ]Ul) removed a t 10 minutes, 20 minutes and minutes were adsorbed into f i l t e r d i s k s (Whatman 2.4 cm 3 MM). The d i s k s were immediately washed i n the f o l l o w i n g i c e c o l d s o l u t i o n s f o r 20 minutes each: 10% TCA, 66% e t h a n o l con-t a i n i n g 0.5 M NaCl, 10% TCA, twice i n 5% TCA and t h r e e times i n a s o l u t i o n of e t h a n o l ether (3:1). R a d i o a c t i v i t y was determined by immersion of the d i s k i n 5 ml of toluene s c i n t i l l a t i o n f l u i d (4 gm Omnifluor, New England Nuclear, i n 1 l i t e r o f t o l u e n e ) . Reactions c o n t a i n i n g h i g h l y p u r i -f i e d t r a n s f e r RNAs co n t a i n e d one A 2 6 0 u n i t of D r o s o p h i l a 5S RNA as c a r r i e r . A l l a m i n o a c y l a t i o n s r e p o r t e d i n t h i s t h e s i s were shown to reach a p l a t e a u w i t h i n 20 minutes by the f i l t e r d i s k assay. B. Assay f o r Amino A c i d Acceptance of Column F r a c t i o n s Column f r a c t i o n s were assayed by the procedure of Cherayil e t a l . (151) as m o d i f i e d by White and Tener (150). A l i q u o t s (0.1 ml) of each f r a c t i o n were a p p l i e d to f i l t e r paper d i s k s (Whatman 2.4 cm 3 MM). The d i s k s were d r i e d and s a l t removed by washing i n 75% e t h a n o l c o n t a i n i n g 30 mM KC1. The d i s k s were then incubated w i t h 0.1 ml of r e a c t i o n mixture c o n t a i n i n g r a d i o a c t i v e amino a c i d s , r e a c t i o n components, and crude aminoacyl synthetase p r e p a r a t i o n as d e t a i l e d i n S e c t i o n I I I - A . Immersion of the d i s k s i n i c e c o l d 10% TCA terminated the r e a c t i o n s . The d i s k s were washed and the r a d i o a c t i v i t y determined e x a c t l y as des-c r i b e d i n s e c t i o n TTI-A. In most cases these r e a c t i o n s and washings were c a r r i e d out on f i l t e r d i s k s s t a p l e d to a 8 x 125 cm s t r i p o f r i b b e d p l a s t i c (Handi-Mat). T h i s pro-cedure reduces the mani p u l a t i o n s of i n d i v i d u a l d i s k s . C. P r e p a r a t i o n o f Aminoacyl-tRNA f o r Chromatography tRNA was aminoacylated i n 0.2 ml r e a c t i o n mixtures as d e s c r i b e d i n s e c t i o n III-A. F i v e m i c r o l i t e r a l i q u o t s were removed and a p p l i e d t o f i l t e r d i s k s a t 10 minutes, 20 min-utes and 30 minutes. These d i s k s were washed and counted as d e s c r i b e d i n s e c t i o n III-A. Only r e a c t i o n s which had reached p l a t e a u c h a r g i n g w i t h i n 20 minutes were used f o r chromatography. The remaining volume of am i n o a c y l a t i o n mixture was d i l u t e d t o 2.0 ml w i t h i c e c o l d 0.1 M NaOAc (pH 4.0) and loaded onto a s m a l l D E A E - c e l l u l o s e column (0.7 cm x 4 cm) e q u i l i b r a t e d i n 10 mM NaOAc (pH 4.5), 10 mM MgCl 2, 1 mM 2-mercaptoethanol and 0.25 M NaCl ( b u f f e r B). The column was then washed w i t h 20 ml of b u f f e r B. The tRNA was e l u t e d w i t h 10 ml of b u f f e r B c o n t a i n i n g 0.75 M NaCl. T h i s aminoacyl-tRNA was d i l u t e d t o the a p p r o p r i a t e s a l t c o n c e n t r a t i o n b e f o r e l o a d i n g on the RPC-5 column. When p r e p a r i n g aminoacyl-tRNA f o r RPC-5 chromatography i n System C d e s c r i b e d below, b u f f e r C was s u b s t i t u t e d f o r b u f f e r B i n the procedure. B u f f e r C contained 10 mM Na formate (pH 3.8), 1 mM EDTA, 1 mM 2-mercaptoethanol and 0.25 NaCl. IV. P r e p a r a t i o n of T r a n s f e r RNA from D r o s o p h i l a T h i s procedure i s a m o d i f i c a t i o n of the phenol method of K i r b y (152) t h a t was developed by White and Tener (150). A l l procedures were c a r r i e d out a t 0-4°C. A d u l t f l i e s were homogenized i n the homogenization mixture (10 ml mixture per gm of f l i e s ) w i t h a P o l y t r o n homogenizer (Kinematica GMBH, S w i t z e r l a n d ) . The homogenization mixture c o n t a i n e d equal volumes of 88% phenol and b u f f e r D[10 mM T r i s (pH 7.5), 10 mM Mg(OAc) 2, 1 mM 2-mercaptoethanol, and 0.1 M NaCl]'. A f t e r c e n t r i f u g a t i o n and removal of the aqueous l a y e r , the phenol l a y e r was washed w i t h b u f f e r D (5 ml/gm of f l i e s ) . The a d d i t i o n of 2.5 volumes of 95% e t h a n o l t o the combined aqueous l a y e r s p r e c i p i t a t e d RNA, DNA, and contaminating p o l y -s a c c h a r i d e s . A f t e r s t a n d i n g o v e r n i g h t a t -20°C the p r e c i p -i t a t e 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 , d i s s o l v e d i n b u f f e r D, and loaded onto a D E A E - c e l l u l o s e column e q u i l i b r a t e d i n the same b u f f e r . The column was washed w i t h ten column volumes of b u f f e r D c o n t a i n i n g 0.3 M NaCl to remove p o l y -s a c c h a r i d e s . B u f f e r D c o n t a i n i n g 1.0 M NaCl was used to e l u t e the tRNA. F i n a l l y the RNA was p r e c i p i t a t e d w i t h e t h a n o l (2.5 volumes), c o l l e c t e d by c e n t r i f u g a t i o n , d i s -s o l v e d i n water, d i a l y z e d a g a i n s t d i s t i l l e d water, and then l y o p h i l i z e d . V. F r a c t i o n a t i o n of tRNA A. B i o - G e l A-0.5 M Chromatography In some cases crude tRNA p r e p a r a t i o n s were chromatographed 34. on agarose (Bio-Gel A-0.5 M, Biorad) columns to remove hi g h m o l e c u l a r weight RNA. The l y o p h i l i z e d tRNA p r e p a r a t i o n was f i r s t d i s s o l v e d i n 2.0 ml d i s t i l l e d water. T h i s s o l u t i o n was heated to 70°C f o r 5 minutes and then loaded onto the A-0.5 M column (1.2 cm x 42 cm). The RNA was e l u t e d w i t h d i s t i l l e d water a t a flow r a t e of 1.5 ml per minute. RNA was d e t e c t e d as absorbance a t 260 nm. B. Benzoylated D E A E - c e l l u l o s e Chromatography B D - c e l l u l o s e was prepared a c c o r d i n g to G i l l a m e t a l . (153) and passed through a 50 mesh s i e v e (0.3 mm opening). The column (1.8 cm x 110 cm) was packed w i t h the i o n ex-changer suspended i n 2 M NaCl and washed w i t h t h i s s o l u t i o n u n t i l the absorbance ( A 2 6 0 ) of the e l u a t e was l e s s then 0.05. The column was loaded and e l u t e d a c c o r d i n g to White and Tener (150). A l l s o l u t i o n s c o n t a i n e d 10 mM MgCl 2 and the a p p r o p r i a t e amounts o f NaCl. The temperature was 22°C and the flow r a t e was 30 ml per hour. Approximately 9300 A 2 6 0 u n i t s of crude tRNA were d i s s o l v e d i n 0.1 M NaCl and loaded onto the column which had been e q u i l i b r a t e d i n 0.3 M NaCl. A f t e r 500 ml of 0.35 M NaCl was washed through the column a l i n e a r g r a d i e n t c o n t a i n i n g 3 l i t e r s each of 0.35 M NaCl and 1.2 M NaCl was a p p l i e d . F i f t e e n ml f r a c t i o n s were c o l l e c t e d and the RNA d e t e c t e d as absorbance a t 2 60 nm. C. Sepharose 6B Chromatography Chromatography of tRNA on Sepharose 6B was c a r r i e d out by the procedures of Holmes e t a l . (154). A column (1.6 cm x 95 cm) was f i l l e d w i t h Sepharose 6B beads suspended i n 1.5 M ( N H O 2 S C K . tRNA d i s s o l v e d i n 1.5 M (NHO 2SCH was a p p l i e d t o the top of the column and e l u t e d w i t h a l i n e a r g r a d i e n t o f d e c r e a s i n g c o n c e n t r a t i o n of ammonium s u l f a t e d e r i v e d from 500 gm of 1.5 M (NHO 2SCK and 500 gm o f d i s -t i l l e d water. The flow r a t e was 17 ml per hour a t 22°C. Peak f r a c t i o n s ( A 2 6 0 ) were pooled, d i a l y z e d t o remove the s a l t , and l y o p h i l i z e d . D. RPC-5 Chromatography The RPC-5 chromatography system d e s c r i b e d by Pearson e t a l . (155) was used except the RPC-5 was prepared by a new method. Four ml of Adogen 464 was added t o 500 ml of 10 mM NaOAc (pH 4.5), 10 mM M g C l 2 i n a Waring blendor. A f t e r b r i e f mixing to suspend the Adogen, 75 gm of Plaskon CTEF-2300 powder was s l o w l y added as the blendor s t i r r e d a t low speed. The mixture was then s t i r r e d a t h i g h speed f o r 10 minutes. The r e s u l t i n g RPC-5 suspension was degassed and passed through a 200 mesh s i e v e . The RPC-5 was twice suspended i n 500 ml of f r e s h b u f f e r and l e f t to s e t t l e b e f o r e b e i n g loaded i n t o the column. Chromatography on the RPC-5 mat r i x was c a r r i e d out i n thr e e d i f f e r e n t b u f f e r systems u s i n g l i n e a r g r a d i e n t s of increasing NaCl concentrations. System A contained 10 mM NaOAc (pH 4.0 or pH 4.5), 10 mM M g C l 2 , and 1 mM 2-mercap-toethanol. System B had 10 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 and 1 mM 2-mereaptoethanol. System C contained 10 mM Na formate (pH 3.8), 1 mM ElDTA, and 1 mM 2-mercaptoethanol. Chromatography was ca r r i e d out at temperatures varying from 25°C to 45°C i n jacketed columns (Chromatronix) u t i -l i z i n g a Haake water bath to control the temperature. A Milton Roy pump was used to force the eluent through the column. When radioactive aminoacyl-tRNA was being f r a c -tionated aliquots of each f r a c t i o n were dissolved i n s c i n -t i l l a t i o n c o c k t a i l (66 gm Omnifluor, 8.1 I Xylene, 3.1 I Triton N-101) and the r a d i o a c t i v i t y determined i n a s c i n -t i l l a t i o n counter (Nuclear Chicago, Isocap 300). The count-ing e f f i c i e n c y was determined by counting aliquots of [ l l fC] and [3H] l a b e l l e d amino acids dissolved i n the s c i n t i l l a t i o n c o c k t a i l . VI. Nucleoside Analysis A. The Tritium Labelling Method The base composition of p u r i f i e d RNA samples was deter-mined using the procedures described by Randerath et a l . (156). Approximately 0.1 A 2eo unit of p u r i f i e d tRNA was digested overnight at 37°C i n a volume of 50 y l containing 1.5 umole Bicine pH 8.0, 0.5 ymole MgCl 2/ 5 yg snake-venom phosphodiesterase, 10 yg RNase A and 8.4 yg E. c o l i phosphomonoesterase. Then the d i g e s t was t r e a t e d a t room temperature w i t h sodium p e r i o d a t e a t a c o n c e n t r a t i o n of 19.2 yg i n 60 y l f o r 2 hours i n the dark to o x i d i z e the c i s d i o l s t o d i a l d e h y d e s . A f t e r t h i s step the r e a c t i o n mix was made 17 mM i n K2HPO1+ (pH 6.8) and 0.438 ymoles KBT^ were added ( s p e c i f i c a c t i v i t y 3.8 Ci/mmole). A f t e r r e a c t i o n a t room temperature i n the dark f o r 2 hours the remaining KBT-t was h y d r o l y s e d by i n c u b a t i o n f o r t h i r t y minutes a f t e r the a d d i t i o n of one volume of 1 N a c e t i c a c i d . The r e a c -t i o n was blown dry and taken up i n 15 y l o f 0.1 N formic a c i d An a l i q u o t c o n t a i n i n g approximately 10 7 cpm was s p o t t e d onto a p l a s t i c backed c e l l u l o s e t h i n l a y e r p l a t e (Eastman 6064) and s e p a r a t i o n achieved by two dimensional chromato-graphy. Development i n the f i r s t dimension was i n a c e t o -n i t r i l e , e t h y l a c e t a t e , n-butanol, i s o p r o p a n o l , 6 N aqueous ammonia (7-2-1-1-2.7, v/v) to the 17 cm mark. The second dimen-s i o n was i n t-amyl a l c o h o l , methyl e t h y l ketone, a c e t o n i t r i l e e t h y l a c e t a t e , H 2 0 , formate (4-2-1.5-2-1.5-0.18, v/v) to 5 cm on a paper wick (Whatman No. 1 paper) used to extend the sheet. The d r i e d chromatograms were coated w i t h a 7% s o l u t i o n of PPO i n e t h e r . The r a d i o a c t i v e areas were l o c a l i z e d by f l u o r o g r a p h y . a t -70°C f o r 3 days u s i n g Kodak X-ray f i l m (RP-Royal X-Omat). The n u c l e o s i d e t r i -a l c o h o l s from each spot were e l u t e d w i t h 1 ml of 2 N N H i t O H . The r a d i o a c t i v i t y i n each spot was determined by d i s s o l v i n g an a l i q u o t i n s c i n t i l l a t i o n c o c k t a i l and c o u n t i n g on the ISOCAP 300 (Nuclear-Chicago). 38. B. A n a l y s i s of N u c l e o s i d e s Detected by U.V. Absorbance P u r i f i e d tRNA s p e c i e s were d i g e s t e d to n u c l e o s i d e s and f r a c t i o n a t e d a c c o r d i n g t o Rogg e t a l . (157). Approximately 4 A 26o u n i t s o f RNA were incubated a t 37°C o v e r n i g h t i n 0.1 ml of 20 mM ammonium formate (pH 7.6), 0.5 mM MgCl 2 c o n t a i n i n g 15 yg p a n c r e a t i c RNase (Worthington), 15 yg snake venom phosphodiesterase (Worthington), 22 yg E. c o l i a l k a l i n e phosphatase (Worthington), and one drop c h l o r o f o r m to i n h i b i t microbes. A f t e r 12 hours 2.5 yg of RNase T i (Calbiochem) was added and the r e a c t i o n continued f o r 2 hours. The h y d r o l y s a t e was s p o t t e d i n a 1.2 cm l i n e on a c e l l u l o s e t h i n l a y e r sheet (Eastman 6064 20 cm x 20 cm). The f i r s t dimension was developed i n 1 - b u t a n o l - i s o b u t y r i c a c i d - cone. NEUOH - water (75:37.5:2.5:25 by volume) to 5 cm on a paper wick e x t e n s i o n (Whatman 31). The second dimension, developed f o r 20 cm, was s a t u r a t e d (NEU) 2SO11., 0.1 M NaOAc pH 6, i s o p r o p a n o l (79:19:2 by volume). The n u c l e o s i d e s were d e t e c t e d under UV l i g h t . The u v absorbing m a t e r i a l was e l u t e d w i t h d i s t i l l e d water and i t s spectrum of U.V. absorbance measured on a Cary 11 r e c o r d i n g spectrometer. V I I . Ribonuclease T i F i n g e r p r i n t A n a l y s i s P u r i f i e d tRNA samples were d i g e s t e d w i t h RNase T i and l a b e l l e d w i t h [ 3 2P] a c c o r d i n g t o Szekely and Sanger (158). ATP l a b e l l e d i n the y p o s i t i o n w i t h [ 3 2P] was prepared a c c o r d i n g t o the method of Schendel and Wells (159) . No attempt was made to determine the s p e c i f i c a c t i v i t y o f the ATP. Schendel and Wells r e p o r t a s p e c i f i c a c t i v i t y o f approximately 1000 Ci/mmole (159). The l a b e l l e d o l i g o -n u c l e o t i d e s were f r a c t i o n a t e d a c c o r d i n g to Sanger e t a l . (160). The second dimension was chromatography on p o l y -e t h y l e n i m i n e t h i n l a y e r p l a t e s i n s t e a d of i o n o p h o r e s i s on DEAE paper. The PEI t h i n l a y e r method was developed by Southern and M i t c h e l l (161) and G r i f f i n (162). P u r i f i e d tRNA samples (20 yg) were d i g e s t e d f o r 3 hours a t 45°C i n T r i s HCI (pH 7.4), 1 mM EDTA w i t h 2.5 yg of RNase T i (Calbiochem) i n a volume of 15 y l . The d i g e s t s were f r o z e n a t -20°C u n t i l r e q u i r e d . The l a b e l l i n g r e a c t i o n c o n t a i n e d 2 yg of the o l i g o -n u c l e o t i d e d i g e s t , 50 y C i of [y- 3 2P]ATP, one nmole ATP and 3 u n i t s of T4 p o l y n u c l e o t i d e k i n a s e (P.L. Biochemicals) i n 8-10 y l of 10 mM T r i s - H C l (pH 7.4), 5 mM MgCl 2 and 5 mM 2-mercaptoethanol. Incubation was a t 37°C f o r 30 minutes. The r e s i d u a l [ Y - 3 2 P ] A T P was h y d r o l y z e d by the a d d i t i o n of myosin, a Ca a c t i v a t e d ATPase. Myosin was prepared a c c o r d i n g t o Wikman e t a l . (163). The crude myosin was f u r t h e r p u r i f i e d by g e l e x c l u s i o n chromatography on Sephar-ose 4B. The ATPase a c t i v i t y from t h i s column was concen-t r a t e d i n a membrane cone ( D i a f l o PM-10) and d i a l y z e d a g a i n s t 50 mM T r i s (pH 7.5), 10 mM 2-mercaptoethanol, 0.5 M KC1 and 1 mM EDTA. T h i s s o l u t i o n had a c o n c e n t r a t i o n of 2 mg p r o t e i n per ml and was s t o r e d a t -70°C u n t i l r e q u i r e d . The l a b e l l e d o l i g o n u c l e o t i d e d i g e s t was made 10 mM i n C a C l 2 and then t r e a t e d w i t h 8 yg of myosin f o r 15 minutes a t 37°C. S i n c e the [ y - 3 2 P ] A T P a l s o c o n t a i n e d s i g n i f i c a n t l a b e l a t the 6 p o s i t i o n , i t s d egradation product ADP would produce a dark spot on the chromatogram. The ADP spot c o u l d be s i g n i f i c a n t l y reduced by the a d d i t i o n of myokinase (Sigma) to the enzyme d i g e s t 7.5 minutes a f t e r the myosin had been added. A f t e r the ATPase step 0.5 ymoles of EDTA (pH 7.0) were added to c h e l a t e the C a + + and M g + + p r e s e n t i n the mixture. F i n a l volume was 17-20 y l . F i v e y l of l a b e l l e d d i g e s t was a p p l i e d to a c e l l u l o s e a c e t a t e s t r i p soaked i n 5% a c e t i c a c i d - p y r i d i n e (pH 3.5), 7 M urea and s u b j e c t e d to e l e c t r o p h o r e s i s f o r 1.5 hr a t 600 v o l t s a t a temperature of 30°C on a f l a t p l a t e e l e c t r o -p h o r e s i s apparatus (Savant Instrument I n c . ) . The o l i g o -n u c l e o t i d e s were t r a n s f e r r e d to prewashed (10% NaCl, 2 M p y r i d i n e formate pH 2.2, H 20) PET p l a t e s . The p l a t e s were developed to the 10 cm mark i n 1.6 M p y r i d i n i u m formate (pH 3.5) and then to the top w i t h 2.3 M p y r i d i n i u m formate (pH 3.5). The r a d i o a c t i v i t y was determined by autoradiography f o r t h r e e hours u s i n g Kodak (RP-Royal X-Omat) f i l m . Areas of r a d i o a c t i v i t y were cu t out and p l a c e d i n 1.0 ml of 0.1 M HCl. A l i q u o t s of these s o l u t i o n s were counted i n the Cerenkov ;channel on the Isocap 300 s c i n t i l l a t i o n counter. I I . In S i t u H y b r i d i z a t i o n The work r e p o r t e d u s i n g t h i s technique was performed i n c o l l a b o r a t i o n w i t h Dr. T.A. G r i g l i a t t i , Dr. T. Kaufman and Dr. S. Hayashi. The p u r i f i e d tRNA was i o d i n a t e d w i t h [ 1 2 5 I ] by the procedure of Commerford (164). Two mCi of c a r r i e r f r e e [ 1 2 5 I ] p l u s 3 u l of 0.2 M a c e t i c a c i d were t r e a t e d w i t h sodium s u l f i t e (6 nmoles) i n a volume of 10 y l f o r 15 minutes a t room temperature. Sodium a c e t a t e (3.55 ymoles, pH 4.0), 5 yg tRNA and 60 nmoles t h a l l i u m t r i c h l o r -i d e were added g i v i n g a f i n a l volume of 25 y l . Incubation was f o r 20 minutes a t 60°C. The r e a c t i o n mixture was then loaded onto a column of h y d r o x y l a p a t i t e packed i n t o a Pasteur p i p e t t e . The column was e q u i l i b r a t e d i n 50 mM NaH2POit (pH 6.8). The [ 1 2 5 I ] was washed from the column w i t h 50 mM NaHaPOit (pH 6.8). The RNA was e l u t e d with 0.2 M NaH 2P0 4 (pH 6.8). Unstable [ 1 2 5 I ] was removed from the RNA by a d d i t i o n of 1/10 volume of 0.1 M sodium s u l f i t e and h e a t i n g t o 70°C f o r 30 minutes. The h y d r o x y l a p a t i t e step was r e -peated and the RNA d i a l y z e d t h r e e times a g a i n s t 500 ml of 2 x SSC. Modinated tRNA prepared i n t h i s manner had a s p e c i f i c a c t i v i t y o f approximately 10 8 dpm/yg. The i o d i n a t e d tRNA was h y b r i d i z e d t o D r o s o p h i l a s a l i v a r y gland chromosomes a c c o r d i n g to the method of G a l l and Pardue (165). The s a l i v a r y glands were prepared from the s t r a i n of D r o s o p h i l a c a r r y i n g the mutation c a l l e d g i a n t (166,167). S a l vary glands i n t h i s mutant c o n t a i n chromosomes w i t h roughly 42. twice the degree of polyteny p r e s e n t i n w i l d type f l i e s . Autoradiography was f o r two weeks u s i n g I l f o r d K.5 l i q u i d emulsion. IX. P r e p a r a t i o n of S e r i n e Codons A. S y n t h e s i s T r i n u c l e o t i d e s r e p r e s e n t i n g the s i x s e r i n e codons were s y n t h e s i z e d by means of p o l y n u c l e o t i d e phosphorylase from Micrococcus l y s o d e i k t i c u s by the procedure of Thach and Doty (168). The r e a c t i o n t o prepare XpYpN con t a i n e d 0.4 M NaCl, 0.2 M g l y c i n e (pH 9.3), 10 mM MgCl 2, 1 mM NDP, 6 mM XpY and 0.25 mg enzyme. • The r e a c t i o n mixture was heated to 60°C f o r 5 min b e f o r e adding the enzyme (112). A f t e r i n c u b a t i o n f o r 4 hr a t 34°C the r e a c t i o n was p l a c e d i n a b o i l i n g water bath f o r 4 minutes. Excess NDP was dephos-p h o r y l a t e d by the a d d i t i o n of 0.17 mg a l k a l i n e phosphatase (Worthington) f o r 1 hour a t 37°C. The mixture was d i l u t e d t o 60 ml w i t h H 2 0 and loaded onto a D E A E - c e l l u l o s e column (0.7 x 14 cm) which had been prewashed i n 0.5 M NH - tHC0 3 and then H 2 0 . The column was e l u t e d with a l i n e a r g r a d i e n t of 150 ml H 2 0 and 150 ml 0.2 M N H 4 H C O 3 a t 0.75 ml per minute. The t r i n u c l e o t i d e s e l u t e d as a peak of A 2 6 o a f t e r the d i n u c l e o s i d e monophosphate peak. The product was d e s a l t e d by repeated e v a p o r a t i o n i n ethanol/water a t 35°C under reduced p r e s s u r e . B. I d e n t i f i c a t i o n Approximately 1 A 2 6 o u n i t of each t r i n u c l e o t i d e was d i g e s t e d with snake venom phosphodiesterase and r i b o -nuclease T 2 . The products were separated by descending paper chromatography i n s o l v e n t s A, B, or by h i g h v o l t a g e e l e c t r o p h o r e s i s on Whatman #40 paper i n 50 mM NaOAc (pH 4.5). The products were i d e n t i f i e d by m o b i l i t y and UV s p e c t r a a t pH 2 and pH 11. The y i e l d of each product was c a l c u l a t e d from i t s absorbance a t wavelength of maximum absorbance a t pH 5. S o l v e n t s f o r Paper Chromatography A. I s o b u t y r i c a c i d - cone. Ammonium hydroxide - H 20 (66/1/33;: v/v) B. 95% e t h a n o l - 1 M NH^OAc pH 7.2 (7/3\. v/v) X. Determination of T r i n u c l e o t i d e S t i m u l a t e d B i n d i n g of  Seryl-tRNA to Ribosomes T h i s procedure i s e s s e n t i a l l y t h a t of Leder and Nirenberg (169). Ribosomes prepared from E. c o l i MRE 600 were a g i f t from Dr. Nadine Urquhart. Each assay contained 100 ymoles T r i s - H C l (pH 7.2), 20 ymoles Mg(OAc) 2 50 ymoles KC1, 24 A 2 e 0 u n i t s of ribosomes per ml i n a volume of 50 y l . The amount of [ 1 4 C ] s e r y l - t R N A i n each 50 y l r e a c t i o n v a r i e d from 14.4 to 2.5.8 pmoles. The [ 1 4 C ] s e r y l - t R N A had p r e v i o u s l y been f r a c t i o n a t e d on RPC-5 i n system A (pH 4.5) as d e s c r i b e d above. Peaks of r a d i o a c t i v i t y c orresponding to seryl-tRNA i s o a c c e p t o r s were pooled, p r e c i p i t a t e d w i t h 2.5 volumes e t h a n o l and s t o r e d a t -2 0°C u n t i l r e q u i r e d . The t r i n u c l e o -t i d e c o n c e n t r a t i o n s v a r i e d from 0 to 4.14 nmoles per assay. The r e a c t i o n s were incubated f o r 20 minutes a t 25°C. The assay was d i l u t e d 5 times w i t h i c e c o l d wash b u f f e r (0.1 M T r i s - H C l pH 7.2, 50 mM KC1, 20 mM Mg(OAc). 2 and washed onto M i l l i p o r e f i l t e r s (type HA pore s i z e 0.45 micron). The f i l t e r s were washed w i t h 10 ml of wash b u f f e r , d r i e d under an i n f r a - r e d lamp and the r a d i o a c t i v i t y adsorbed t o the f i l t e r determined by l i q u i d s c i n t i l l a t i o n c o u n t i n g . 45. R e s u l t s and D i s c u s s i o n I. P u r i f i c a t i o n o f tRNAs As a f i r s t s tep toward the a n a l y s i s of tRNAs and the genes f o r tRNAs a procedure was developed t o p u r i f y i n d i -v i d u a l tRNA s p e c i e s . T h i s procedure u t i l i z e s B D - c e l l u l o s e chromatography as d e s c r i b e d by White and Tener (150) and a new chromatographic system d e s c r i b e d by Holmes e t a l . (154). T h i s l a t t e r method f r a c t i o n a t e s tRNA on the b a s i s of the d i f f e r e n t a f f i n i t i e s which i n d i v i d u a l tRNA s p e c i e s have f o r agarose i n the presence of h i g h c o n c e n t r a t i o n s of ammonium s u l f a t e . The order of e l u t i o n o f tRNA s p e c i e s i s thought t o depend on the r e l a t i v e hydrophobic nature o f the tRNA molecules (154). On the oth e r hand B D - c e l l u l o s e and RPC-5 chromatography u t i l i z e i o n exchange as w e l l as hydro-phobic i n t e r a c t i o n s t o f r a c t i o n a t e tRNAs. T r a n s f e r RNAs g e n e r a l l y e l u t e from these ion-exchange columns i n the same order> B D - c e l l u l o s e having a h i g h e r c a p a c i t y but l e s s r e -s o l v i n g power then RPC-5. The p u r i f i c a t i o n method d e s c r i b e d below takes advantage of the h i g h c a p a c i t y of the B D - c e l l u l o s e method, the new f r a c t i o n a t i o n on Sepharose 6B and the hig h r e s o l u t i o n o f the RPC-5 method. A. B D - C e l l u l o s e Chromatography Crude tRNA was f r a c t i o n a t e d on a B D - c e l l u l o s e column a c c o r d i n g t o White and Tener (150). F r a c t i o n s were assayed f o r the acceptance of v a l i n e , l y s i n e , t h r e o n i n e and a r g i n i n e ( F i g . 1) . The p o o l c o n t a i n i n g v a l i n e acceptance was se-l e c t e d f o r f u r t h e r a n a l y s i s . F i g u r e 1. B D - c e l l u l o s e chromatography of D r o s o p h i l a tRNA. Crude D r o s o p h i l a tRNA (9300 A 2 6 o u n i t s ) was chromato-graphed on t h i s B D - c e l l u l o s e column (1.8 x 110 cm). F r a c -t i o n s (15 ml) were e l u t e d w i t h a 6 l i t e r l i n e a r g r a d i e n t of NaCl (0.35 M t o 1.2 M) at a flow r a t e of 30 ml per hour. Samples o f f r a c t i o n s (0.1 ml) were-assayed f o r a r g i n i n e , l y s i n e , t h r e o n i n e and v a l i n e a c c e p t o r a c t i v i t y u s i n g the procedure d e s c r i b e d i n Methods. Absorbance of each f r a c -t i o n a t 260 nm ( s o l i d l i n e ) ; counts per minute [ 1 1 +C] amino a c i d accepted per 0.1 ml (dotted l i n e s ) . 48... B. Sepharose 6B Chromatography The v a l i n e a c c e p t i n g f r a c t i o n s were d i v i d e d i n t o e a r l y and l a t e p o o l s , l a b e l l e d A and B r e s p e c t i v e l y ( F i g . 1). These pools were f r a c t i o n a t e d on the Sepharose 6B column u s i n g a r e v e r s e s a l t g r a d i e n t ( F i g . 2). The peaks of A 2 6 0 absorbance were pooled, d i a l y z e d a g a i n s t water and l y o p h i -l i z e d . The d e s a l t e d RNA p o o l s were assayed f o r acceptance of aminoacids known t o e l u t e i n the e a r l y f r a c t i o n s from the B D - c e l l u l o s e column (150). The r e s u l t s of t h i s assay f o r the 'experiment shown i n f i g u r e 2B are g i v e n i n Table 1. I t should be noted t h a t these r e s u l t s are not q u a n t i t a t i v e . The assays were performed under c o n d i t i o n s of suboptimum amino a c i d c o n c e n t r a t i o n w i t h the purpose of determining which tRNA s p e c i e s were pr e s e n t i n each peak. Two c o n c l u s i o n s can be drawn. F i r s t , the Sepharose column g i v e s good r e s o -l u t i o n f o r t R N A M e t , t R N A A s p , t R N A V a l , t R N A T h r , t R N A G l u and t R N A G l n . Secondly, i t seems t h a t tRNA 6"^ i s not r e s o l v e d as a sharp peak i n t h i s system. P u r i f i c a t i o n of tRNAG"*"y might be b e t t e r achieved by another type of column. As demonstrated below peak J c o n t a i n s 5S RNA. C. P u r i f i c a t i o n of tRNA Species on RPC-5 columns The B D - c e l l u l o s e column and the Sepharose 6B column pro-v i d e d s e v e r a l p o o l s of RNA from which i n d i v i d u a l tRNA s p e c i e s and 5S RNA were p u r i f i e d by RPC-5 chromatography. F i g u r e 2. Sepharose 6B chromatography. Pooled f r a c t i o n s l a b e l l e d A and B i n f i g u r e 1 were chromatographed on t h i s Sepharose 6B column (1.6 x 95 cm). F r a c t i o n s (3.4 ml) were e l u t e d w i t h a l i n e a r r e v e r s e s a l t g r a d i e n t (500 gm of 1.5 M ammonium s u l f a t e t o 500 gm of d i s t i l l e d water) c o n t a i n i n g 10 mM M g C l 2 a t a flow r a t e o f 20 ml per hour. F i g u r e A was obt a i n e d when 1672 A 2 6 o u n i t s of P o o l A ( f i g . 1) were chromatographed, f i g u r e B was obta i n e d when 1854 A 2 6 0 u n i t s o f Pool B ( f i g . 1) were chromatographed. Absorbance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) . Table 1. Amino A c i d Acceptance Assays of Pools A - J from Sepharose Column 2B Amino A c i d Acceptance (pmoles / A 2 6 o u n i t ) F r a c t i o n V a l A l a Gly Thr Met Glu Gin Asp A 15 0 0 398 49 15 8 , a nt B 91 7 34 2 0: 399 10 10 nt C 163 0 147 4 189 8 205 nt D 592 27 180 4 108 nt nt n t E 174 27 243 4 13 29 20 n t F 14 67 222 3 12 179 68 nt G 45 103 286 4 7 195 21 nt H 448 26 17 0 2 11 19 n t I nt nt nt nt n t nt n t 650 J 1 0 2 0 0 nt nt 2 a n t = not t e s t e d (i) 5S RNA The l a r g e s t f r a c t i o n c o l l e c t e d from the Sepharose c o l -umn i s p o o l J ( F i g . 2). Poo l J does not accept any of the amino a c i d s t e s t e d and i s presumed to be 5S RNA. Three ob-s e r v a t i o n s support t h i s s u p p o s i t i o n . F i r s t , p o o l J RNA g i v e s a s t r o n g band o f s t a i n e d m a t e r i a l a t the p o s i t i o n of 5S RNA on 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 . Only t r a c e s o f 4S m a t e r i a l are p r e s e n t ( F i g . 3). Secondly, p o o l J g i v e s two RNA peaks a f t e r chromatography on RPC-5 i n system A, pH 4.5 ( F i g . 4). These two peaks have been demonstrated f o r a u t h e n t i c 5S RNA by G r i g l i a t t i e t a l . (147). These two peaks probably r e s u l t from p a r t i a l h y d r o l y s i s o f the t r i -phosphate at the ;5'terminus o f ' t h e 5S RNA s i n c e treatment of the 5S RNA w i t h PME changes i t s e l u t i o n p a t t e r n , g i v i n g a s i n g l e peak (171). T h i r d l y i n s i t u h y b r i d i z a t i o n of Sepharose p o o l J RNA, l a b e l l e d w i t h 1 2 5 I , to s a l i v a r y gland chromosomes l a b e l s the c y t o l o g i c a l area 56 F e x c l u s i v e l y (172). T h i s area i s known to be the l o c a t i o n o f the 5S genes i n D r o s o p h i l a (173). These r e s u l t s demonstrate the u s e f u l n e s s of Sepharose 6B chromatography i n the p r e p a r a -t i o n o f D r o s o p h i l a 5S RNA. ( i i ) Three Valyl-tRNA Species On RPC-5 chromatography i n system A (10 mM NaOAc, pH V a l 4.0; 10 mM MgCl 2; 1 mM 2 mercaptoethanol) tRNA separates i n t o s e v e r a l minor and two major peaks ( F i g . 5). The l e a d -V a l i n g major peak e a r l i e r d e s i g n a t e d tRNA. (134) was 1 2 3 4 5 6 Figure 3. Polyacrylamide g e l e l e c t r o p h o r e s i s of 5S RNA. RNA recovered from pool J i n Figure 2 was f r a c t i o n -ated on a 12% polyacrylamide g e l i n 1% a c e t i c a c i d ad-just e d to pH 3.5 w i t h NaOH, 1 mM EDTA and 8 M urea. E l e c t r o p h o r e s i s was f o r 18 hours at 600 v o l t s . Lane 1, p u r i f i e d yeast f R N A p h e (10 yg); lane 2, pool J ( f i g . 2) RNA (10 yg); lane 3-6, crude 4S RNA i n amounts of 8 yg, 12 yg, 20 yg and 40 yg r e s p e c t i v e l y . The g e l was s t a i n e d w i t h ethidium bromide (1 yg/ml) f o r 2 hr and photographed under UV l i g h t . 54. F i g u r e 4. RPC-5 chromatography of 5S RNA. Approximately 69 A 2 6 o u n i t s o f po o l J RNA ( f i g . 2) were chromatographed on t h i s RPC-5 column (0.9 x 60 cm). F r a c t i o n s (3.0 ml) were e l u t e d w i t h a 600 ml l i n e a r grad-i e n t o f NaCl (0.50 M to 0.75 M) i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.5; 10 mM MgCl 2; 1 mM 2-mercaptoethanol). The flow r a t e was 30 ml/hr and the temperature was 37°C. 2.0 1.8 1.6 1.4 -1.2 -1.0 8 6 R P C - 5 C h r o m a t o g h o f 5 S R N A 0 F i g u r e 5. RPC-5 chromatography o f D r o s o p h i l a t R N A V a l . D r o s o p h i l a 4S RNA (5 A 2 6 0 u n i t s ) was aminoacylated w i t h [ 1 ^ C ] - V a l i n e as d e s c r i b e d i n Methods. T h i s prep-a r a t i o n o f [ 1 4 C ] - V a l - t R N A V a l (40,000 cpm) was chromato-graphed on an RPC-5 column (0.9 x 21 cm) a t 37°C w i t h a flow r a t e of 15 ml per hour. F r a c t i o n s (0.5 ml) were e l u t e d w i t h a l i n e a r g r a d i e n t of NaCl (0.50 M t o 0.65 M) i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.5; 10 mM M g C l 2 j 1 mM 2-mercaptoethanol). Approximately 96% o f the r a d i o a c t i v i t y was recovered i n the g r a d i e n t . Counts per minute i n each f r a c t i o n ( s o l i d l i n e ) . 57. Fraction Number subsequently shown to c o n t a i n two s p e c i e s now desi g n a t e d tRNAY a l and tRNAY a l. 3a 3b V a l tRNA2^ was p u r i f i e d from p o o l H o b t a i n e d from the Sepharose column i n f i g u r e 2A. Pool H RNA was chromato-graphed on the RPC-5 system A a t pH 4.0 and assayed f o r acceptance of v a l i n e . The r e s u l t s of t h i s experiment are V a l shown i n f i g u r e 6A. The f r a c t i o n s c o n t a i n i n g t R N A ^ were pooled and rechromatographed on RPC-5 system B (10 mM T r i s HCl, pH 8.0; 10 mM MgCl 2; 1 mM 2-mercaptoethanol). The tRNA^a"'' o b t a i n e d from t h i s column, shown i n f i g u r e 6B was judged t o be of h i g h p u r i t y by two c r i t e r i a : (a) i t accepted 1760 pmoles of v a l i n e per A 2 6 o u n i t , and (b) i t ran as a s i n g l e peak when chromatographed on the t h i r d RPC-5 system, system C (10 mM Na formate,pH 3.8; 1 mM EDTA 1 mM 2-mercaptoethanol), as shown i n f i g u r e 7. V e i l V e i l tRNA 3 a and tRNA^ were o b t a i n e d from p o o l D of the Sepharose column i l l u s t r a t e d i n f i g u r e 2B. They were p u r i f i e d by chromatography i n RPC-5 system A and then chromato graphy i n RPC-5 system C. These columns are i l l u s t r a t e d i n f i g u r e s 8, 9A, and 9B. The acceptance of p u r i f i e d tRNA^"*" was 1647 pmoles per A 2 6 o u n i t and t h a t o f tRNA^a"'" was 1705 pmoles per A 2 6 o u n i t . In the s t u d i e s d e s c r i b e d i n a l a t e r s e c t i o n i t w i l l V a l be important t o show the e l u t i o n p o s i t i o n o f t R N A ^ i n TT-a "I TT-s "I the RPC-5 system r e l a t i v e t o t h a t o f t R N A ^ and tRNA^. . To determine t h i s p o s i t i o n [ 1 ^ C ] V a l - t R N A V a l (crude tRNA F i g u r e 6. P u r i f i c a t i o n of tRNA^k by RPC-5 chromatography. 6A. Pool H RNA (170 A 2 6 o u n i t s ) from the columns i l l u s -t r a t e d i n f i g u r e s 2A and 2B was chromatographed on t h i s RPC-5 column (2.4 x 42 cm) a t 37°C. F r a c t i o n s (9.4 ml) were e l u t e d w i t h a l i n e a r g r a d i e n t of NaCl (0.475 M t o 0.625 M) i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.0; 10 mM MgCl2,* 1 mM 2 mercaptoethanol) a t a flow r a t e o f 160 ml per hour. Samples of f r a c t i o n s (0.1 ml) were assayed f o r v a l i n e a cceptor a c t i v i t y u s i n g the procedure d e s c r i b e d i n Methods. Absorbance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) ; counts per minute [ 1 ^ C ] - V a l i n e accepted per 0.1 ml (dotted l i n e ) . V a l 6B. The f r a c t i o n s c o n t a i n i n g tRNA3j-) (80 A 2 6 o u n i t s ) i n f i g u r e 6A were pooled and rechromatographed on t h i s RPC-5 column (2.4 x 42 cm) a t 37°C. F r a c t i o n s (10.0 ml) were e l u t e d w i t h a l i n e a r g r a d i e n t o f NaCl (0.50 M to 0.70 M) i n RPC-5 b u f f e r system B (10 mM T r i s - H C l , pH 8.0; 10 mM MgCl 2; 1 mM 2-mercaptoethanbl) a t a flow r a t e o f 160 ml per hour. The po o l o f tRNAYg-1- co n t a i n e d 48 A 2 6 o u n i t s of tRNA. 61. F i g u r e 7. A n a l y t i c a l RPC-5 chromatography of t R N A ^ 1 . Va 1 P u r x f i e d t R N A ^ (0.22 A 2 eo u n i t s ) was chromato-graphed on t h i s RPC-5 column (0.9 x 20 cm) i n RPC-5 b u f f e r system C (10 mM Na formate, pH 3.8; 1 mM EDTA) a t 37°C w i t h a flow r a t e o f 2 0 ml per hour. The tRNA was e l u t e d w i t h a l i n e a r g r a d i e n t (100 ml) o f NaCl (0.50 M to 0.80 M) and the absorbance of the e l u a n t a t 254 nm recorded. F i g u r e 8. RPC-5 chromatography of tRlSTA^f1 and tRNA^ Pooled RNA f r a c t i o n s (76 A 2 6 0 u n i t s ) l a b e l l e d D i n f i g u r e 2B were chromatographed on t h i s RPC-5 column (2.5 x 42 cm) i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.0; 10 mM MgCl 2; 1 mM 2-mercaptoethanol) a t 37°C. F r a c t i o n s (9.9 ml) were e l u t e d w i t h a 2 l i t e r g r a d i e n t o f NaCl (0.525 M t o 0.650 M) a t a flow r a t e o f 160 ml per hour. Samples of f r a c t i o n s (0.1 ml) were assayed f o r v a l i n e acceptor a c t i v i t y u s i n g the procedure d e s c r i b e d i n Methods. Absorbance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) ; counts per minute [ 1 h C ] - v a l i n e accepted per 0.1 ml (dotted l i n e ) . 6 4 . Fraction Number 65. F i g u r e 9. P u r i f i c a t i o n of t R N A ^ and tKNAj . 9A. The p o o l of tRNAY a l ( f i g . 8, 26 A 2 6 o u n i t s ) was chrom-atographed on t h i s RPC-5 column (0.9 x 62 cm) i n RPC-5 b u f f e r system C (10 mM Na formate, pH 3.8; 1 mM EDTA; 1 mM 2-mercaptoethanol) a t 37°C. F r a c t i o n s (3.0 ml) were e l u t e d w i t h a 600 ml l i n e a r g r a d i e n t of NaCl (0.550 M to 0.725 M) a t a flow r a t e of 30 ml per hour. Samples of f r a c t i o n s (50 y l ) were assayed f o r v a l i n e acceptor a c t i v -i t y u s i n g the procedure d e s c r i b e d i n Methods. 9B. A p o r t i o n of the p o o l of t R N A Y a l ( f i g . 8, 20 A 2 6 o u n i t s ) was chromatographed e x a c t l y as d e s c r i b e d above except t h a t the g r a d i e n t of NaCl was a l t e r e d (0.550 M to 0.675 M) and the samples of f r a c t i o n s assayed f o r acceptance o f v a l i n e were i n c r e a s e d t o a volume of 0.1 ml. Absorbance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) ; counts per minute [ l l fC] - V a l i n e accepted per 50 y l i n A or per 0.1 ml i n B (dotted l i n e s ) . Fraction Number l a b e l l e d w i t h [ 1 **C] v a l i n e ) was f r a c t i o n a t e d on the Sepharose 6B column, as d e s c r i b e d i n f i g u r e 10A. The t R N A ^ 1 peak was pooled and rechromatographed on the RPC-5 system A a t V a l pH 4.0. T o t a l tRNA l a b e l l e d w i t h t r i t i u m was cochrom-atographed w i t h the [ 1 ^ C] Val'-tKNA^ 1 and the r e s u l t s shown V a l i n F i g u r e 10B. T h i s r e s u l t shows t h a t the tRNA^^ e l u t e s i n the e a r l y e l u t i n g major t R N A V a l peak ( o r i g i n a l l y c a l l e d V a l tRNA-j ) on the RPC-5 system A. In subsequent s t u d i e s (Results and D i s c u s s i o n , s e c t i o n V) mutants i n v o l v i n g the genes f o r t R N A ^ 1 , but not the oth e r t R N A V a l genes, changed the amounts o f the tRNA^ 5 1 1 peak r e l a t i v e to the other t R N A V a l when d e t e c t e d by u s i n g the RPC-5 system A. V a l F u r t h e r experiments have demonstrated t h a t the tRNA^ peak c o n t a i n s two t R N A V a l s p e c i e s (tRNA^ 3 1 and tRNA^ a l) . F i g u r e 11A i l l u s t r a t e s the po o l o f tRNA^ 3 1 from RPC-5 system A which was rechromatographed on the Sepharose 6B column V a l shown i n f i g u r e 11B. T h i s diagram i l l u s t r a t e s t h a t tRNA-o 3. and t R N A ^ 1 are d e r i v e d from the t R N A ^ 1 peak. tRNA^ 3 1 V a l and tRNA 3 b c o n s t i t u t e 27% and 73% r e s p e c t i v e l y o f the tRNA^^" peak. T h i s e x p l a i n s why two major tRNAVa"*" s p e c i e s c o u l d be p u r i f i e d from p o o l C of the Sepharose column ( f i g . 2A) as d e s c r i b e d i n f i g u r e 8 and a t h i r d major tRNA^ a^ c o u l d be p u r i f i e d from p o o l H as d e s c r i b e d i n f i g u r e 7. V a l Three major tRNAs are re c o v e r e d although o n l y two major s p e c i e s can be d e t e c t e d by e i t h e r the RPC-5 system A ( f i g . 5) or the Sepharose system ( f i g . 10A). The presence of 68. J F i g u r e 10. Chromatography of tRNA^gl. 10A. D r o s o p h i l a 4S RNA (5 A 2 6 o u n i t s ) was aminoacylated w i t h [ J l t C ] - V a l i n e as d e s c r i b e d i n Methods. T h i s prep-a r a t i o n (40,000 cpm) was chromatographed on a Sepharose 6B column (1 x 40 cm) i n 10 mM NaOAc (pH 4.5), 10 mM MgCl 2, and 1 mM 2-mercaptoethanol. F r a c t i o n s (1.2 ml) were e l u t e d w i t h a 240 ml l i n e a r r e v e r s e ammonium s u l f a t e g r a d i e n t (1.4 M t o 0 M) a t a flow r a t e of 7.5 ml per hour. Samples (0.3 ml) were d i s s o l v e d i n s c i n t i l l a t i o n c o c k t a i l and the r a d i o a c t i v i t y determined by l i q u i d s c i n -t i l l a t i o n c o u n t i n g . The recover y o f r a d i o a c t i v i t y was approximately 70%. The p o o l o f tRNAY^l was washed onto a D E A E - c e l l u l o s e column (1.0 x 6 cm), washed w i t h 10 mM NaCl i n RPC-5 b u f f e r A (10 mM NaOAc, pH 4.0; 10 mM MgCl 2; and 1 mM 2-mercaptoethanol), and e l u t e d w i t h 0.75 M NaCl i n RPC-5 b u f f e r A. 10B. D r o s o p h i l a 4S RNA (5 A 2 6 o u n i t s ) was aminoacylated w i t h [ 3 H ] - V a l i n e as d e s c r i b e d i n Methods. T h i s prepara-t i o n o f [ 3 H ] - V a l - t R N A V a l (650,000 cpm) was mixed w i t h the p r e p a r a t i o n o f [ 1^C]-Val-tRNAYg 1 (6900 cpm) d e s c r i b e d above and chromatographed on the RPC-5 column (0.9 x 22 cm) i n RPC-5 system A. F r a c t i o n s (0.5 ml) were e l u t e d w i t h a 100 ml l i n e a r g r a d i e n t o f NaCl (0.50 M t o 0.65 M) at 37°C a t a flow r a t e of 15 ml per hour. Approximately 87% of the [ l l fC] and 95% of the [ 3H] r a d i o a c t i v i t y was recovered i n the g r a d i e n t . 40 60 80 100 60 80 100 120 Fraction Number CY 70. F i g u r e 11. F r a c t i o n a t i o n o f tRNA V a l l3a and tRNA V a l 3b IIA. D r o s o p h i l a 4S RNA (5 A 2 e o u n i t s ) was aminoacylated w i t h [ 3 H ] V a l i n e as d e s c r i b e d i n Methods. T h i s p r e p a r a -t i o n (700,000 cpm) was chromatographed on an RPC-5 column (0.9 x 21 cm) i n RPC-5 system A (10 mM NaOAc, pH 4.0; 10 mM MgCl 2; and 1 mM 2-mercaptoethanol) a t 37°C a t a flow r a t e of 15 ml per hour. F r a c t i o n s (0.5 ml) were e l u t e d w i t h a 100 ml l i n e a r g r a d i e n t o f NaCl (0.50 M to 0.65 M). Samples of f r a c t i o n s (0.2 ml) were d i s s o l v e d i n s c i n t i l l a t i o n c o c k t a i l and the r a d i o a c t i v i t y d e t e r -mined. The r a d i o a c t i v i t y e l u t i n g under the peak l a b e l l e d 3 was p r e c i p i t a t e d w i t h i c e - c o l d e t h a n o l (2.5 volumes). Counts per minute ."[-H]Valine per 0.2 ml ( s o l i d l i n e ) , r a d i o a c t i v i t y pooled and p r e c i p i t a t e d i s designated by the shaded area. IIB. The 13H] V a l - t R N A 3 ^ 3 b (100 , 000 cpm) recovered from the RPC-5 column d e s c r i b e d i n A was d i s s o l v e d i n 10 mM NaOAc (pH 4.5), 10 mM MgCl 2, 1 mM 2-mercaptoethanol and 1.4 M ammonium s u l f a t e b e f o r e being loaded onto a Seph-arose 6B column (0.8 x 110 cm) e q u i l i b r a t e d i n the same b u f f e r . F r a c t i o n s -(1.5= ml) were e l u t e d w i t h a 300 ml r e v e r s e ammonium s u l f a t e g r a d i e n t (1.4 M to 0.0 M) a t a flow r a t e of 7.5 ml per hour. Approximately 81% of the r a d i o a c t i v i t y was e l u t e d by the g r a d i e n t [ 3 H ] V a l per 1.0 ml ( s o l i d l i n e ) . 72. t R N A ^ 1 w i l l complicate the q u a n t i t a t i v e a n a l y s i s o f t R N A ^ 1 d e s c r i b e d l a t e r i n t h i s t h e s i s ( s e c t i o n V ) . ( i i i ) P u r i f i c a t i o n o f Non-Valine tRNA Species C e r t a i n tRNA s p e c i e s which do not accept v a l i n e are a l s o p r e s e n t i n the f r a c t i o n s d e r i v e d from the Sepharose 6B columns. S e v e r a l o f these tRNAs can be p u r i f i e d u s i n g procedures analogous to those d e s c r i b e d f o r the th r e e v a l y l -tRNA s p e c i e s . F i g u r e 13 i l l u s t r a t e s the p r o f i l e o b t ained from the chromatography of p o o l A ( f i g . 2) from the Sepharose columns on RPC-5 i n system B a t pH 8.0. Two major peaks of A 2 6 0 absorbance are due to threonine-tRNA s p e c i e s . F i g u r e 14 shows the r e s u l t s of the f r a c t i o n o f t R N A A s p from Sepharose p o o l I ( f i g . 2) on RPC-5 a t pH 8.0. The major peak of A 2 6 o absorbance from t h i s column was pooled and found t o accept 900 pmoles a s p a r t i c a c i d per A 2 6 0 u n i t . T h i s tRNA i s not pure, but f u r t h e r chromatography on RPC-5 system A and RPC-5 system C should r e s u l t i n a p u r i f i e d t R N A A s p . F i g u r e 12 i l l u s t r a t e s the f r a c t i o n a t i o n of the methio-n i n e a c c e p t i n g Sepharose p o o l B ( f i g . 2) on RPC-5 i n system A. Pool A from t h i s column d i d not c o n t a i n s i g n i f i -c a n t methionine a c c e p t o r a c t i v i t y . P o o l C was f u r t h e r Met f r a c t i o n a t e d a t pH 8.0 i n RPC-5 system B. The f i n a l tRNA from t h i s p o o l accepted methionine t o a l e v e l o f 1660 pmoles per A 2 6 o u n i t . F i g u r e 12. RPC-5 chromatography o f methionine a c c e p t i n g tRNA recovered from the Sepharose 6B column. Pooled f r a c t i o n s (280 A 2 6 0 u n i t s ) l a b e l l e d B i n f i g -ure 2A were chromatographed on an RPC-5 column (2.4 x 42 cm at 37°C i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.0; 10 mM MgCl 2; and 1 mM 2-mercaptoethanol). F r a c t i o n s (10 ml) were e l u t e d w i t h a 2 l i t e r l i n e a r g r a d i e n t o f NaCl (0.475 M t o 0.650 M) a t a flow r a t e o f 160 ml per hour. Absorbance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) . F i g u r e 13. RPC-5 chromatography of t h r e o n i n e a c c e p t i n g tRNA recovered from the Sepharose 6B column. Pooled f r a c t i o n s (107 A 2 6 0 u n i t s ) l a b e l l e d A i n f i g u r e s 2A and 2B were chromatographed on an ; RPC-5 column (2.4 x 42 em) a t 37°C i n RPC-5 b u f f e r system B (10 mM T r i s - H C l , pH 8.0; 10 mM M g C l 2 ; 1 mM 2-mercaptoethanol). F r a c t i o n s (9.7 ml) were e l u t e d w i t h a 2 l i t e r l i n e a r g r a d i e n t of NaCl (0.50 M to 0.75 M) a t a flow r a t e of 160 ml per hour. Samples (0.1 ml) of f r a c t i o n s were assayed' f o r v a l i n e acceptor a c t i v i t y u s i n g the procedure d e s c r i b e d i n Methods. Absorbance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) ; counts per minute [ 1^C]Threonine accepted per 0.1 ml (dotted l i n e s ) . Fract ion Number F i g u r e 14. RPC-5 chromatography of tRNA c o n t a i n i n g as-p a r t i c a c i d acceptance a c t i v i t y . Pooled f r a c t i o n s (210 A 2 6 0 u n i t s ) c o n t a i n i n g as-p a r t i c a c i d a c c e p t o r a c t i v i t y ( l a b e l l e d H i n f i g u r e s 2A and 2B) were chromatographed on an RPC-5 column (2.4 x 42 cm) i n RPC-5 b u f f e r system B (10 mM T r i s , pH 8.0; 10 mM MgCl 2; and 1 mM 2-mercaptoethanol). F r a c t i o n s (9.9 ml) were e l u t e d w i t h a 2 l i t e r l i n e a r NaCl g r a d i e n t (0.50 M to 0.70 M) a t a flow r a t e of 160 ml per hour. Approximately 66% of the tRNA was recovered w i t h the grad-i e n t , 25% was e l u t e d w i t h a 1.5 M NaCl wash. Absorb-ance of each f r a c t i o n a t 260 nm ( s o l i d l i n e ) . 78. 79. F i g u r e 15. RPC-5 chromatography o f tRNA c o n t a i n i n g a l a -n i n e , glutamic a c i d , and g l y c i n e acceptor a c t i v i t y . Pooled f r a c t i o n s (177 A 2 6 0 u n i t s ) taken from the f r a c t i o n s l a b e l l e d E, F and G i n f i g u r e 2B were chrom-atographed on an RPC-5 column (0.9 x 60 cm) a t 37°C i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.0; 10 mM MgCl 2; 1 mM 2-mercaptoethanol). F r a c t i o n s (3.0 ml) were e l u t e d w i t h a 600 ml l i n e a r g r a d i e n t o f NaCl (0.50 M to 0.65 M) a t a flow r a t e o f 3 0 ml per hour. Samples (0.1 ml) of f r a c -t i o n s were assayed f o r acceptance o f [ 1 ^ C ] l a b e l l e d a l a n i n e , g l u t a m i c a c i d and g l y c i n e as d e s c r i b e d i n Methods. Approximately 89% of the absorbance a t 2 60 nm was e l u t e d by the g r a d i e n t . Absorbance o f each f r a c t i o n a t 260 nm ( s o l i d l i n e ) , counts per minute [ 1 h C ] A l a n i n e (open c i r c l e s ) , [ 1 4 C ] G l u t a m i c a c i d ( c r o s s e s ) , and [ 1 4 C ] -G l y c i n e ( c l o s e d c i r c l e s ) accepted per 0.1 ml. Absorbance 260 nm 81. F i n a l l y f r a c t i o n s E, F, and G ( f i g . 2) were combined and f r a c t i o n a t e d on the RPC-5 system A. The r e s u l t s o f t h i s column are i l l u s t r a t e d i n f i g u r e 15. S e l e c t e d areas of t h i s column were assayed f o r the acceptance o f glutamate, g l y c i n e and a l a n i n e . The r e s u l t i n g p r o f i l e s i n d i c a t e t h a t t h i s procedure i s a promising route to the p u r i f i c a t i o n o f these tRNAs. V a l I I . N u c l e o s i d e A n a l y s i s of P u r i f i e d tRNAs The n u c l e o s i d e composition of t R N A ^ 1 , t R N A ^ 1 and tRNA^ a l was analyzed by the s e n s i t i v e t r i t i u m d e r i v a t i v e method of Randerath e t a l . (156) . The i d e n t i t i e s of the n u c l e o s i d e s were i n f e r r e d from t h e i r p o s i t i o n on the two-dimensional t h i n l a y e r p l a t e s by r e f e r e n c e t o p u b l i s h e d m o b i l i t i e s o f n u c l e o -s i d e d e r i v a t i v e s i n the same system (156). The r e l i a b i l i t y of t h i s method was t e s t e d by a p p l y i n g i t to p u r i f i e d tRNA^ l y from y e a s t . The sequence of t h i s tRNA has been r e p o r t e d (170). The t r i t i u m d e r i v a t i v e map of the n u c l e o s i d e t r i a l c o h o l s d e r i v e d from t h i s tRNA i s i l l u s t r a t e d i n F i g u r e 16. Table 2 c o n t a i n s the y i e l d o f each of the n u c l e o s i d e d e r i v a t i v e s . S e v e r a l background spots are a l s o d e t e c t e d . The spots l a b e l l e d 1, 2 and 3 r e s u l t from p a r t i a l r e d u c t i o n of p e r i o -date o x i d i z e d guanosine, p s e u d o u r i d i n e , and adenosine r e -s p e c t i v e l y to the monoaldehydes (174). The f o u r other back-ground spots are of unknown o r i g i n . A l l of the expected minor n u c l e o s i d e s were d e t e c t e d . The 5-methyl c y t o s i n e t r i -a l c o h o l was d e t e c t e d a t twice the expected amount, p o s s i b l y 82. F i g u r e 16. N u c l e o s i d e a n a l y s i s of y e a s t tRNAj_ y u s i n g the t r i t i u m l a b e l l i n g technique. Approximately 0.1 A 2 6 0 u n i t of y e a s t t R N A f l y was d i g e s t e d and l a b e l l e d w i t h t r i t i u m as d e s c r i b e d i n Methods. Two m i c r o l i t e r s of the l a b e l l e d d i g e s t c o n t a i n -i n g 6 x 10 6 cpm were a p p l i e d to each of two c e l l u l o s e t h i n l a y e r p l a t e s (20 x 20 cm). Chromatography and sub-sequent f l u o r o g r a p h y are d e s c r i b e d i n Methods. Chrom-at o g r a p h i c s o l v e n t s were as f o l l o w s ; f i r s t dimension was a c e t o n i t r i l e , e t h y l a c e t a t e , n-butanol, i s o p r o p a n o l , 6 N aqueous ammonia (7-2-1-1-2.7 by volume), second dimension was t-amyl a l c o h o l , methyl e t h y l ketone, a c e t o n i t r i l e , e t h y l a c e t a t e , H 20, formic a c i d (4-2-1.5-2-1.5-0.18 by v o l -ume) . Fluorography was f o r 72 hours a t -70°C. The n u c l e o -s i d e t r i a l c o h o l s are denoted by the a b b r e v i a t i o n s as des-c r i b e d i n the t a b l e of a b b r e v i a t i o n s on page xi. 83. 84 . Table 2. N u c l e o s i d e A n a l y s i s o f t R N A G l y of Yeast by the  T r i t i u m L a b e l l i n g Method Nucle o s i d e T r i a l c o h o l Experimental P r e d i c t e d A' 12.5 13 U' 11.7 11 G' 17.8 21 C' 20.0 18 au' 2.3 2 3.7 4 m 5c' 1.9 1 i ^ G * 0.74 1 T 1 1 1 P r e d i c t e d v a l u e s c a l c u l a t e d from the sequence g i v e n i n r e f e r e n c e 17 0. 85. due to contamination from the h e a v i l y l a b e l l e d c y t o s i n e t r i a l c o h o l spot. ' The t r i t i u m d e r i v a t i v e method as demonstrated on y e a s t Gly . . . tRNA^ J p r o v i d e s r e l i a b l e i n f o r m a t i o n on the types and amounts of m o d i f i e d n u c l e o s i d e s i n tRNA samples. However, the p e r c e n t composition o f the major bases determined by t h i s method was i n e r r o r by as much as 15% f o r the G' and C' v a l u e s c a l c u l a t e d f o r y e a s t tRNA^ l v i n Table 2. The e r r o r s i n the q u a n t i t a t i v e e s t i m a t i o n s c o u l d be due to two p o s s i b l e sources. The sample of tRNA^ l v may be l e s s than pure or the chemical d e r i v a t i z a t i o n technique may p r e f e r -e n t i a l l y l a b e l C because the r e d u c t i o n of the other major n u c l e o s i d e s i s not complete. With the r e s e r v a t i o n t h a t the major base composition r e s u l t s may be u n r e l i a b l e , the t r i t i u m d e r i v a t i v e technique was a p p l i e d t o the th r e e p u r i f i e d V a l D r o s o p h i l a tRNAs ( F i g . 17). The r e l a t i v e amounts of r a d i o a c t i v i t y p r e s e n t i n each n u c l e o s i d e t r i a l c o h o l spot i s presented i n Table 3 along w i t h the r e s u l t s of the second n u c l e o s i d e a n a l y s i s presented below. These r e s u l t s are d i s -cussed i n the next s e c t i o n . A s e r i o u s drawback to the t r i t i u m d e r i v a t i v e n u c l e o s i d e a n a l y s i s i s the i n a b i l i t y t o d e t e c t 2-0-methylated n u c l e o -s i d e s . To c o r r e c t t h i s d e f i c i e n c y and t o c o n f i r m the n u c l e o s i d e composition d e r i v e d from the d e r i v a t i v e technique, V a l 3 t o 4 A 2 6 o u n i t s of each tRNA were h y d r o l y z e d t o n u c l e o -s i d e s , f r a c t i o n a t e d , and q u a n t i t a t e d by UV a b s o r p t i o n as t R N A V ^ t R N A ^ tRNA^ ' ® 0 , n r hU g|y 0 ° 0T' B O Origin B x'O h^ s l y 0' Or B.~. ^ B O © O"' O V Origin o ' Origin V a l F i g u r e 17. N u c l e o s i d e a n a l y s i s of t h r e e p u r i f i e d tRNAs u s i n g the t r i t i u m l a b e l l i n g technique. V a l Approximately 0.1 A 2 6 o of each tRNA was d i g e s t e d and l a b e l l e d w i t h t r i t i u m as d e s c r i b e d i n Methods. Approx-imately 5 x 10 5 cpm of each l a b e l l e d d i g e s t was a p p l i e d to each of two c e l l u l o s e t h i n l a y e r p l a t e s and chromato-graphy c a r r i e d out as d e s c r i b e d i n Methods and i n f i g u r e 16. Fluorography was f o r 72 hours at -7 0°C. 87. Table 3. Nucle o s i d e A n a l y s i s of tRNAs from D r o s o p h i l a  melanogaster N u c l e o s i d e tRNAY a l 3 a t R N A ya l 4 A B A B Number of residues/75 n u c l e o t i d e tRNA : molecule A 14.6 15.2 14.6 15.1 13.7 14.7 U 8.8 7.8 8.7 8.7 8.1 6.3 G 18.7 23.9 16.2 17.8 16.9 21.0 C 22.8 18.0 20.5 18.3 22.3 19.0 i> 3.7 4.0 5.7 4.6 4.0 4.0 dU 2.3 n t c 2.2 nt 1.6 nt rT 1.0 1.2 1.0 1.1 1.0 1.0 n^A 0.83 0.73 1.0 0.80 1.0 0.90 m7G 0.80 1.1 0.84 1.1 0.72 0.92 m 5C 0.88 0.96 0.80 0.73 2.0 1.7 m:G - - - - 0.81 1.0 I - - - - - 1.3 Cm nt - nt 0.75<* nt 1.3 Um nt - nt 0.75 d nt 0.9 Am nt - nt 0.41 nt -X - 1.7 - - -A* - - - 0.80 - -Columns l a b e l l e d A are v a l u e s o b t a i n e d by the t r i t i u m l a b e l l i n g technique (Methods). Columns l a b e l l e d B are v a l u e s o b t a i n e d from the U.V. s p e c t r a of separated n u c l e o s i d e s (Methods). n t = not t e s t e d Estimated from two p o o r l y r e s o l v e d n u c l e o s i d e s p o t s . 88. d e s c r i b e d i n Methods s e c t i o n VI-B. The r e s u l t s o f t h i s study are shown i n F i g u r e 18 and Table 3. The n u c l e o s i d e s were i n i t -i a l l y i d e n t i f i e d by r e f e r e n c e to a standard n u c l e o s i d e map i n Rogg e t al. (157). They were p o s i t i v e l y i d e n t i f i e d by r e c o r d i n g UV s p e c t r a a t pH 2 and pH 11. V a l t R N A ^ i s the l e a s t m o d i f i e d tRNA i n the v a l i n e s e r i e s (Table 3 ) . I t c o n t a i n s one r e s i d u e o f rT,m 7G,m 1A and m5C; two r e s i d u e s of dU and f o u r r e s i d u e s o f ty per molecule o f tRNA. No 2-0-methyl d e r i v a t i v e s were d e t e c t e d i n t h i s molecule. The major disagreement noted between the two n u c l e o s i d e a n a l y s i s methods are the v a l u e s c a l c u l a t e d f o r G and C. An analogous disagreement was noted f o r the model experiment on tRNA^"^ u s i n g the f r a c t i o n d e r i v a t i v e method (Table 2 ) . Th e r e f o r e the n o n - d e r i v a t i z e d n u c l e o s i d e a n a l y s i s v a l u e s f o r the major base compositions are c o n s i d e r e d more r e l i a b l e . V a l N u c l e o s i d e s d e r i v e d from tRNA., d i d not r e s o l v e w e l l 3a i n the n o n - d e r i v a t i z e d n u c l e o s i d e a n a l y s i s . U n f o r t u n a t e l y V a l the supply of p u r i f i e d tRNA, was too low to permit a r e -<5 a V a l peat experiment. Two problems w i t h the tRNA, experiment should be noted. 2'-0-Methyl c y t i d i n e and 2'-0-methyl u r i d i n e d i d not r e s o l v e adequately and q u a n t i t a t i o n o f these n u c l e o s i d e s was est i m a t e d from the combined absorbance o f two p o o r l y r e s o l v e d s p o t s . A l s o an u l t r a v i o l e t absorbing spot l a b e l l e d X was d e t e c t e d . The spectrum of t h i s spot c o n t a i n e d a broad absorbance maximum from 260 to 270 nm. V a l F i g u r e 18. N u c l e o s i d e a n a l y s i s o f t h r e e p u r i f i e d tRNAs Approximately 4 A 2 6 o u n i t s of each p u r i f i e d tRNA were d i g e s t e d as d e s c r i b e d i n Methods. Each d i g e s t , i n a volume of 0.1 ml, was spotted onto a c e l l u l o s e t h i n l a y e r p l a t e (20 x 20 cm) and developed f i r s t w i t h 1-b u t a n o l , i s o b u t y r i c a c i d , cone ammonia, water (7 5-37.5-2.5-25 by volume) and i n the second dimension w i t h s a t -u r a t e d ammonium s u l f a t e , 0.1 M Na a c e t a t e (pH 6), i s o -propanol (79-19-2 by volume). The n u c l e o s i d e s were v i s -u a l i z e d under an u l t r a v i o l e t lamp and c h a r a c t e r i z e d as d e s c r i b e d i n Methods. 90. T h i s m a t e r i a l i s p o s s i b l y a d i n u c l e o t i d e c o n t a i n i n g 2'-0-methyladenosine. I t s presence may e x p l a i n the low y i e l d of 2'-O-methyladenosine. With these r e s e r v a t i o n s taken i n t o account, the a n a l y s i s of tRNAY3''" shows t h a t i t c o n t a i n s more cl m o d i f i e d n u c l e o s i d e s than tRNAY a l. tRNAYf 1 c o n t a i n s an 3b 3b e x t r a ¥ r e s i d u e and three 2'-0-methyl m o d i f i c a t i o n s . An unknown n u c l e o s i d e with absorbance s p e c t r a resembling adeno-s i n e was d e t e c t e d . T h i s n u c l e o s i d e i s l a b e l l e d A* i n f i g -ure 18B. The UV spectrum of t h i s n u c l e o s i d e i s shown i n f i g u r e 19. The r e l a t i o n s h i p of A* to the spot X 1 d e t e c t e d by the t r i t i u m l a b e l l i n g method ( f i g . 17B) i s not c l e a r . The spot X' co n t a i n e d enough r a d i o a c t i v i t y to account f o r two n u c l e o s i d e r e s i d u e s . tRNA^a''" c o n t a i n s ir^G and I which are not present i n the Va l V a l other two tRNAs . A l s o tRNA^ c o n t a i n s an e x t r a m5C and two 2'-0-methyl d e r i v a t i v e s . T h i s n u c l e o s i d e a n a l y s i s data V a l i n d i c a t e s t h a t tRNA^^ has a lower degree o f m o d i f i c a t i o n than tRNA^a"*" and tRNA^3'"'". The major base composition of Va l V a l tRNAs are very s i m i l a r except t h a t t R NA^ appears t o have a reduced amount of guanosine. The presence o f i n o s i n e V a 1 i n tRNA* suggests t h a t i t w i l l code f o r the codons GUA, GUC and GUU (10). V a l The tRNAs from a number of sources have been sequenced. Those from baker's y e a s t (175) and T o r u l o p s i s u t i l i s (176) con-t a i n i n o s i n e i n the f i r s t p o s i t i o n o f the anticodon. They a l s o c o n t a i n rT,m1G,m5C,m1A, dU and s e v e r a l ^ ' s . The ye a s t 91. 220 240 260 280 300 320 Wavelength nm F i g u r e 19. Unknown n u c l e o s i d e A* from tRNA^ a x. o a U l t r a v i o l e t a b s o r p t i o n s p e c t r a of n u c l e o s i d e A* r e -covered from the t h i n l a y e r chromatogram of tRNAY.f-'-n u c l e o s i d e d i g e s t d e s c r i b e d i n f i g u r e 18B. Absorbance measured a t pH 2.0 ( s o l i d l i n e ) , absorbance measured a t pH 11.0 (dotted l i n e ) . t R N A j a l c o n t a i n s mJG wh i l e T. u t i l i s t R N A V a l does not. On the o t h e r hand, the major v a l i n e tRNA from mouse myeloma c e l l s (177) resembles D r o s o p h i l a tRNA^ a l i n t h a t i t a l s o c o n t a i n s i n o s i n e . The mouse tRNA i s i d e n t i c a l t o tRNA^ a i V a l from r a b b i t (178). These mammalian tRNAs c o n t a i n ^,dU,m1A,m5C and Cm which are l i k e w i s e found i n the Dros-V a l o p h i l a tRNA^ . A d i s t i n c t i v e f e a t u r e of the mammalian t R N A s V a l i s the absence of rT u s u a l l y found w i t h i n the T^C V a l loop. D r o s o p h i l a tRNAs c o n t a i n rT and t h e r e f o r e probably resemble y e a s t t R N A V a i i n the TTJJC l o o p . A second d i s t i n c -V a l t i v e f e a t u r e o f the mammalian tRNAs i s t h a t they recog-n i z e a l l f o u r v a l i n e codons, i m p l y i n g t h a t the i n o s i n e can "wobble" p a i r w i t h GUG as w e l l as GUA, GUU and GUC (178). V a l Yeast tRNA^ which c o n t a i n s i n o s i n e i n the anticodon does V a l not r e c o g n i z e GUG (179). The f a c t t h a t tRNA 3 b does not c o n t a i n i n o s i n e suggests t h a t i t may be r e q u i r e d to t r a n s -V a l l a t e GUG and t h a t D r o s o p h i l a tRNA^ may resemble y e a s t tRNA^ a X more than i t resembles mammalian t R N A s V a x . V a l t RNA 3 a i s more e x t e n s i v e l y m o d i f i e d than the othe r V a l tRNAs . T h i s may i n d i c a t e a d a p t a t i o n o f t h i s tRNA mole-His c u l e to a s p e c i a l c e l l u l a r f u n c t i o n . The tRNA molecules of Salmonella c o n t a i n pseudouridine m o d i f i c a t i o n s which appear t o f u n c t i o n i n c e l l u l a r r e g u l a t i o n but are not e s s e n t i a l to p r o t e i n s y n t h e s i s (67) . The e x t e n s i v e l y modi-V a l f i e d tRNA 3 a may a l s o f u n c t i o n i n a non p r o t e i n s y n t h e t i c r o l e . t R N A ^ 1 and tRNA^ 3 1, which are the most abundant V a l tRNAs , would be s u f f i c i e n t to s u s t a i n p r o t e i n s y n t h e s i s i f t R N A ^ 1 codes f o r GUG. I f t h i s were t r u e , then t R N A Y a l 3b 3a would be f r e e to evolve i n t o a tRNA s p e c i e s which c o u l d serve c e l l u l a r f u n c t i o n s other than mRNA t r a n s l a t i o n . T h i s suppos-i t i o n can be t e s t e d i n D r o s o p h i l a . I f i t were t r u e , d e l e -V a l t i o n s of the tRNA- genes should have d i f f e r e n t phenotypes •5 a V a l than d e l e t i o n s of the tRNA genes i n v o l v e d i n p r o t e i n syn-t h e s i s . I f these experiments gave p o s i t i v e r e s u l t s , t h i s system might p r o v i d e i n s i g h t i n t o the o b s e r v a t i o n t h a t f o r many amino a c i d s more i s o a c c e p t i n g tRNAs are pr e s e n t than are r e q u i r e d t o t r a n s l a t e mRNAs. I I I . RNase T i F i n g e r p r i n t A n a l y s i s V a l The n u c l e o s i d e analyses of the three D r o s o p h i l a tRNAs showed d i f f e r e n c e s i n composition w i t h i n t h i s group of i s o -a c c e p t o r s . An attempt was made to determine i f any p a i r of these i s o a c c e p t o r s are the product of a s i n g l e gene, t h a t i s i f they are homogene'ic. Two i s o a c c e p t o r s c o u l d have the same primary sequence w i t h d i f f e r e n c e s i n m o d i f i c a t i o n t h a t r e s u l t i n d i f f e r e n c e s i n chromatographic p r o p e r t i e s . V a l RNase T ti d i g e s t s of the three tRNAs were l a b e l l e d w i t h [ 3 2P] u s i n g [ y - 3 2 P ] A T P and T4 p o l y n u c l e o t i d e k i n a s e and the product f i n g e r p r i n t e d as d e s c r i b e d i n Methods. The auto-radiograms o b t a i n e d are shown i n F i g . 20A-D. The l e v e l of r a d i a c t i v i t y p r e s e n t i n each o l i g o n u c l e o t i d e i s g i v e n i n Table 4. 94. A Val 3b 3 I 7 0 - 8 ,2*9^ 9 9 "6 ADP 017 021 1°9 I8 f 0 ° 2 2 <!>ATP B Val 3a l 0 2 .04 io 0 5 0 11 14 7«»08 1 0 p . 9 o , 3 J ^ ° A D % 8 7 «23 1 6 19 020 »22 2° <3>ATP C Val 4 S * ° 8 A 0 1 ^-Npi A D P O 17 22.'JS?21 23 2 0 > ^ A T P D oPi ADPO A T P C 5 F i g u r e 20. RNase T-]_ f i n g e r p r i n t a n a l y s i s of three p u r i -f i e d tRNAs V a ]-. P u r i f i e d tRNA (20 yg) was d i g e s t e d w i t h RNase and l a b e l l e d with [ 3 2P] u t i l i z i n g T 4 p o l y n u c l e o t i d e k i n a s e as d e s c r i b e d i n Methods. Approximately 4 yg of l a b e l l e d o l i g o n u c l e o t i d e s were f r a c t i o n a t e d i n the f i r s t dimen-s i o n by e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e i n 5% a c e t i c a c i d - p y r i d i n e (pH 3.5), 7 M urea and 1 mM EDTA a t 600 v o l t s f o r 1.5 hours. The o l i g o n u c l e o t i d e s were then t r a n s f e r r e d t o PEI c e l l u l o s e t h i n l a y e r p l a t e s (20 x 20 cm) and chromatographed w i t h 1.6 M p y r i d i n i u m formate (pH 3.5). When the s o l v e n t f r o n t had r i s e n 10 cm the chrom-atograms were t r a n s f e r r e d t o 2.3 M p y r i d i n i u m formate t o complete the chromatography. Autoradiography was f o r 3 hours. The r e s u l t s f o r tRNA^^ 1 are shown i n A, tRNAYf 1 i n B, tRNAY a l i n C, and a c o n t r o l experiment i n D which con-t a i n e d the r e a c t i o n components except f o r the RNase T]_ d i g e s t . Each o l i g o n u c l e o t i d e spot .is numbered and these numbers shown above. 95. A • • B • c • • ' v . > -D • Table 4. Q u a n t i t a t i o n of O l i g o n u c l e o t i d e s Prepared by RNase T i D i g e s t i o n of tRNAs^3"'' Percentage of T o t a l R a d i o a c t i v i t y 3 O l i g o n u c l e o t i d e tRNAY a l tRNAY a l t R N A V a l 3D 3a 4 F i g u r e 2OA F i g u r e 2OB F i g u r e 20C A. a l l t h r e e t R N A s V a l : 1 1.1 1.4 0.58 3 27.1 16.6 27.7 4 3.4 6.0 1.8 7 3.7 1.8 1.0 8 1.7 6.8 7.2 10 2.5 3.0 0.8 11 2.1 2.9 2.9 13 1.6 2.0 1.1 14 18.3 13.2 11.1 17 7.8 6.9 6.8 18 1.7 1.3 2.1 21 3.1 5.2 4.7 B. O l i g o n u c l e o t i d e s w i t h d i f f e r e n t m o b i l i t i e s when comparing: i . tRNA ^ f 1 and tRNAY a l 3b 3a 2 3.5 3.1 6 2.0 2.6 9 1.1 1.6 12 2.6 2.2 15 7.4 6.2 19 1.5 1.7 20 3.6 2.7 22 1.1 3.0 23 - 0.9 cont'd 97. Table 4 (cont'd) i i . t R N A ^ 1 and tRNA^ a l 2 3.5 3.5 5 3.3 4.8 6 2.0 6.1 9 1.1 1.6 12 2.6 1.0 15 7.4 3.6 16 1.6 6.2 20 3.6 1.8 22 1.1 1.7 23 - 0.7 i i i . t R N A ^ 1 and tRNA^ a l 2 3.1 3.5 5 6.7 4.8 6 2.6 6.1 12 2.2 1.0 15 6.2 3.6 16 1.5 6.2 19 1.7 0.8 20 2.7 1.8 22 3.0 1*7 23 0.9 0.7 These v a l u e s r e f e r t o the percentage of r a d i o a c t i v i t y r e -covered from the f i n g e r p r i n t s d e s c r i b e d i n f i g u r e 20. The r a d i o a c t i v i t y p r e s e n t i n the ATP, ADP, and P i spots i s not i n c l u d e d i n t h i s t a b l e . 98. S e v e r a l p o i n t s must be noted when attempting t o i n t e r -p r e t these f i n g e r p r i n t s . F i r s t the spots f o r ATP, ADP and P i shown i n the c o n t r o l ( f i g . 20D) are presen t i n a l l the f i n g e r p r i n t s . Secondly, a l a r g e spot ( l a b e l l e d spot 3) occurs i n the f i n g e r p r i n t s . T h i s spot c o n t a i n s from 16 t o 27% of the r a d i o a c t i v i t y i n c o r p o r a t e d i n t o o l i g o n u c l e o t i d e s . I t may be an a r t i f a c t a r i s i n g from a contaminant present i n the RNase T i p r e p a r a t i o n . Also" the i n c o r p o r a t i o n of r a d i o -a c t i v e phosphate i n t o the o l i g o n u c l e o t i d e s i s not s t o i c h i o -m e t r i c (Table 4 ) . T h i s makes i t i m p o s s i b l e t o q u a n t i t a t e the y i e l d of each o l i g o n u c l e o t i d e and thus makes i t d i f f i -c u l t t o compare the sequence r e l a t e d n e s s of two RNAs. With these r e s e r v a t i o n s a comparison of the m o b i l i t i e s of the de t e c t e d o l i g o n u c l e o t i d e s was made. As shown i n Table 4 each of the RNase T i f i n g e r p r i n t s c o n t a i n s nine or ten o l i g o -n u c l e o t i d e s w i t h m o b i l i t i e s d i f f e r e n t from corresponding o l i g o n u c l e o t i d e s i n the other two f i n g e r p r i n t s . These d i f f e r e n c e s are due to changes i n the m o b i l i t y of o l i g o n u c l e o t i d e s or t o the a d d i t i o n or removal of RNase T i cleavage s i t e s . N u c l e o s i d e m o d i f i c a t i o n c o u l d account f o r many of the changes. Ino s i n e i s d e r i v e d from adenosine. RNase T i c l e a v e s a f t e r i n o s i n e . T h e r e f o r e the i n o s i n e mod-i f i c a t i o n adds a new RNase T i cleavage s i t e t o the molecule. The m1G m o d i f i c a t i o n w i l l r e s t r i c t RNase T i cleavage t o a s u b s t a n t i a l e x t e n t (180). M o d i f i c a t i o n s such as i p , , m5C and A* w i l l a f f e c t the m o b i l i t y of o l i g o n u c l e o t i d e s i n the f i n g e r -p r i n t i n g system. To determine the sequence r e l a t e d n e s s of these tRNAs i t w i l l be important to determine the n u c l e o t i d e sequence of each o l i g o n u c l e o t i d e v i s u a l i z e d i n the f i n g e r p r i n t s i n f i g u r e 20A-C. Proper q u a n t i t a t i o n of each o l i g o n u c l e o t i d e i s a l s o important. Due t o the l a r g e number o f m o d i f i c a t i o n s i n tRNA^fJ 1 and tRNA^ a l as compared to t R N A ^ 1 , i t i s not p o s s i b l e t o prove from these f i n g e r p r i n t s i f any two V a l tRNAs have the same primary sequence. However, the l a r g e number o f changes i n m o b i l i t y shown i n f i g u r e 2 0A-C suggest t h a t the primary sequences are probably d i f f e r e n t f o r a l l three molecules. Experiments d e s c r i b e d below u s i n g V a l the technique o f i n s i t u h y b r i d i z a t i o n of the tRNA i s o -acceptors t o s a l i v a r y gland chromosomes support the hypothesis Y ? 1 and tRNAY' 3b 4 V a l t h a t at l e a s t tRNA^ a l NAY a l have sequence d i f f e r e n c e s . IV. In S i t u H y b r i d i z a t i o n of tRNAs The technique o f i n s i t u h y b r i d i z a t i o n t o p o l y t e n e chromo-somes was p i o n e e r e d by G a l l and Pardue (181). The genes f o r 5S RNA (173) and f o r tRNA^ y s (147) have been l o c a l i z e d t o s p e c i f i c r e g i o n s of the D r o s o p h i l a genome by t h i s t echnique. In c o l l a b o r a t i o n w i t h Dr. T.A. G r i g l i a t t i , Dr. S. Hayashi and T. Kaufman, t h i s method was a p p l i e d u s i n g the t h r e e i s o a c c e p t i n g tRNAsVa''" as h y b r i d i z a t i o n probes. 100. F i g u r e 21: In s i t u h y b r i d i z a t i o n of t R N A ^ and tRNA* to D r o s o p h i l a s a l i v a r y gland cnromosomes. V a l V a l P u r i f i e d t R N A 3 b and t R N A 4 (5 yg each) were l a b e l -l e d w i t h 1 2 5 i t o a s p e c i f i c a c t i v i t y o f approximately 10 8 dpm per yg and then h y b r i d i z e d t o D r o s o p h i l a s a l i v a r y gland chromosomes as d e s c r i b e d i n Methods. A u t o r a d i o -graphy was f o r 16 days. F i g u r e A i l l u s t r a t e s the h y b r i d -i z a t i o n o f [ 1 2 5 I ] tRNAYg- 1- to the t h i r d chromosome a t s i t e s 84D and 92B. F i g u r e B i l l u s t r a t e s the h y b r i d i z a t i o n o f [ 1 2 5 1 ] t R N A V a l t o the second chromosome a t s i t e 56D. Ma g n i f i c a t i o n i s 4400 X. 101 102. The D r o s o p h i l a p o l y t e n e chromosomes are d i v i d e d i n t o 102 segments each c o n t a i n i n g an average of 50 c y t o l o g i c a l l y d i s t i n c t bands. Each of the 102 segments i s s u b d i v i d e d i n t o subsegments which are denoted by a c a p i t a l l e t t e r (182). Genes f o r 5S RNA, f o r example, are l o c a t e d on the r i g h t arm of the second chromosome a t a s i t e l a b e l l e d 56F (173). The t h r e e i s o a c c e p t i n g t RNAs V a l were i o d i n a t e d w i t h [ 1 2 5 I ] to a s p e c i f i c a c t i v i t y of approximately 10 8 dpm per yg as d e s c r i b e d i n Methods. F i g u r e s 21A and 21B are examples of V a l V a l the r e s u l t s o b t a i n e d w i t h t R N A ^ and tRNA^ r e s p e c t i v e l y . V a l Attempts to l o c a l i z e the genes f o r tRNA^ a have not y i e l d e d p o s i t i v e r e s u l t s . T h i s may not be s u r p r i s i n g because hy-b r i d i z a t i o n experiments i n s o l u t i o n have i n d i c a t e d t h a t Va1 - Va1 t RNA 3 a has o n l y 1-3 genes per h a p l o i d genome wh i l e tRNA-^ has 13 genes and t R N A ^ 1 has 8 genes (183) . I f t R N A 3 a l h a S o n l y two genes, the s e n s i t i v i t y of the i n s i t u h y b r i d i z a -t i o n assay may not a l l o w d e t e c t i o n of these genes. V a l t RNA 3 b h y b r i d i z e s to two l o c a t i o n s , area 84D and area 92B, on the r i g h t arm of the t h i r d chromosome. t R N A ^ 1 h y b r i d i z e s to one site,56D, j u s t to the l e f t of the 5S gene l o c a t i o n on the r i g h t arm of the second chromosome. V a l The f a c t t h a t the tRNAs h y b r i d i z e t o d i f f e r e n t r e g i o n s of the D r o s o p h i l a genome suggests t h a t the sequence of these tRNAs are a l l d i f f e r e n t . S i g n i f i c a n t c r o s s -h y b r i d i z a t i o n between the i s o a c c e p t o r s i s not observed. Since t R N A ^ 1 h y b r i d i z a t i o n was not d e t e c t e d these data do not rule out 103. the p o s s i b i l i t y t h a t i t i s a m o d i f i e d form of one of the Va 1 other- tRNAs . The appearance of two bands when h y b r i d i z a t i o n i s per-V a l formed u s i n g the h i g h l y p u r i f i e d tRNA^^ as a probe suggests t h a t , u n l i k e the 5S genes, the gene c o p i e s of i n d i v i d u a l tRNA i s o a c c e p t o r s are not n e c e s s a r i l y c l u s t e r e d i n one l o c a t i o n . Two gene c l u s t e r s appear to e x i s t f o r a s i n g l e tRNA molecule. I f t h i s i s t r u e , i t w i l l c omplicate the model of unequal c r o s s i n g over proposed by G.P. Smith (181) as a mechanism to m a i n t a i n the homogeneity of these genes. Evidence f o r nonadjacent d u p l i c a t e s of tRNA genes has been uncovered i n s t u d i e s on y e a s t . Only one s p e c i e s of t y r o s i n e tRNA has been d e t e c t e d i n y e a s t (185) . However, e i g h t separated l o c i are known t h a t can mutate t o t y r o s i n e i n s e r t i n g amber and ochre suppressors (186) . The t y r o s i n e tRNA of one of these suppressors has been sequenced and the mutation i n the anticodon demonstrated (189). T h i s work proves t h a t one of the suppressor l o c i i s the s t r u c t u r a l gene f o r a t y r o s i n e tRNA. The other seven suppressors have Tvr v ery s i m i l a r phenotypes to the demonstrated tRNA J suppres-sor and t h e r e f o r e are a l s o probably t y r o s i n e tRNA genes (186). In support of t h i s i d e a , H a l l and coworkers have shown t h a t e i g h t separate r e s t r i c t i o n fragments of y e a s t DNA w i l l h y b r i d i z e the t y r o s i n e tRNA (188). I In D r o s o p h i l a the genes f o r tRNA^ a l can be l o c a l i z e d to two separate gene c l u s t e r s . I t i s a l s o p o s s i b l e t h a t other V a l s m a l l e r gene c l u s t e r s f o r t R N A ^ e x i s t but are not d e t e c t e d by the i n s i t u h y b r i d i z a t i o n assay. The e s t i m a t i o n of two V a l gene c l u s t e r s f o r t R N A ^ must be c o n s i d e r e d a minimal es-timate a t t h i s time. These r e s u l t s do p r e d i c t t h a t r e s t r i c -t i o n fragment a n a l y s i s of the D r o s o p h i l a genome should pro-V a l duce a t l e a s t two separate fragments which h y b r i d i z e tRNA3^ . V. A n a l y s i s of tRNA Mutants In s i t u h y b r i d i z a t i o n experiments i n d i c a t e t h a t two gene c l u s t e r s code f o r one tRNA s p e c i e s , namely t R N A ^ 1 . An V a l a n a l y s i s of the l e v e l s of tRNA3^ i n s t r a i n s of D r o s o p h i l a which have e i t h e r a d u p l i c a t i o n or a d e l e t i o n o f the gene c l u s t e r a t area 84D was undertaken. The genotype of the s t r a i n s of f l i e s u t i l i z e d i n these s t u d i e s i s presented i n T a b l e 5 and f i g u r e 22. These s t r a i n s were p r o v i d e d by Dr. T. Kaufman. A r e p o r t on the s e l e c t i o n of some of these s t r a i n s has been p u b l i s h e d (189). A l l f l i e s c a r r i e d w i l d type Oregon R second chromosomes, f o u r t h chromosomes and sex chromosomes. A l l s t r a i n s c a r r y one copy of the t h i r d chromo-some c a l l e d TM-3 (190) which c a r r i e s m u l t i p l e i n v e r s i o n s and suppresses c r o s s i n g over. The c o n t r o l f l i e s c a r r y a normal third;chromosome marked w i t h s e v e r a l mutations d e s c r i b e d i n T able 5. The mutant f l i e s c a r r y t h i r d chromosomes w i t h added or d e l e t e d segments o u t l i n e d i n f i g u r e 22. The bound-a r i e s of the d e l e t i o n s and d u p l i c a t i o n s were determined by t R N A V ^ A j B | C | 0 | 79 1 . , ! - ; » / r ; ; . ' . , ' , 1 : C ' . D i E i F A | B ! 82 H1PI 1 ffiH R3 A | B I* I BS D u p l i c a t i o n J-De f i c i e n c y 1 D e f i c i e n c y 2 >_ F i g u r e 22. Schematic r e p r e s e n t a t i o n o f t h i r d chromosomes w i t h a b e r r a t i o n s i n the r e g i o n 84D. The d u p l i c a t i o n chromosome had an e x t r a segment which i n c l u d e s the area w i t h i n the bar. The d e f i c i e n c y chromo-somes were d e f i c i e n t f o r the r e g i o n s i n c l u d e d w i t h i n the b a r s . H O Ln 106. Table 5. L i s t o f the Genotypes of S t r a i n s o f D r o s o p h i l a w i t h  D e f i c i e n c i e s o r D u p l i c a t i o n s a t s i t e 84-D S t r a i n Genotype of T h i r d Chromosome C o n t r o l K i dsx bx s r e s/TM3 D u p l i c a t i o n K i dsx p P Dp (84-B-84-D)/TM3 D e f i c i e n c y 1 Def. (84-D-84-E)/TM3 D e f i c i e n c y 2 Def. (84-B-84-D)/TM3 A l l o f these s t o c k s c a r r y i d e n t i c a l Oregon-R backgrounds, t h a t i s the second, f o u r t h , and sex chromosomes are w i l d type Oregon R chromosomes. D e s c r i p t i o n o f Marker M u t a t i o n s 3 Genetic Map C y t o l o g i c a l on T h i r d Chromosome P o s i t i o n Map P o s i t i o n bx - b i t h o r a x ; h a l t e r e s en-l a r g e d , metathorax meso-t h o r a c i c 58.8 89E dsx - double sex; females and males transformed i n t o c i n t e r s e x e s 48 84F e - ebony; b l a c k body 70.7 93B-•F K i - Kinked; b r i s t l e s & h a i r s s h o r t & t w i s t e d 47.6 11A- •F P P - pink peach; eyes d u l l ruby 48.0 85A s r - s t r i p e ; dark median s t r i p e on thorax 62.0 90D-•F TM3 - Balancer t h i r d chromosome w i t h m u l t i p l e i n v e r s i o n s . - Suppresses c r o s s i n g over These d e s c r i p t i o n s from r e f e r e n c e 190. 107. examination of s a l i v a r y chromosome squashes from each s t r a i n (189). Atwood (191) has suggested t h a t the c l a s s of muta-t i o n s c a l l e d Minutes may be due t o d e l e t i o n s of tRNA genes. The Def. 2 d e l e t i o n i n f i g u r e 22 does not have a minute pheno-type. I t w i l l be i n t e r e s t i n g t o determine i f the double d e l e t i o n of 84D and 92B w i l l have a minute phenotype. The l e f t m o s t boundary o f the Def. 1 d e l e t i o n l i e s very c l o s e to the tRNA gene c l u s t e r . I t i s not p o s s i b l e to determine c y t o -l o g i c a l l y i f t h i s d e l e t i o n i n c l u d e s the tRNA gene c l u s t e r but b i o c h e m i c a l evidence o u t l i n e d below i n d i c a t e s t h a t the Def. 1 d e l e t i o n i n c l u d e s o n l y p a r t of the tRNA gene c l u s t e r . (A) L e v e l s of V a l i n e Acceptance i n the Mutant S t r a i n s T r a n s f e r RNA was e x t r a c t e d from the f o u r s t r a i n s of f l i e s d e s c r i b e d i n Table 5. The crude tRNA was passed through a B i o - G e l A-0.5 m column to remove hig h molecular weight RNA. The h i g h m o l e c u l a r weight RNA e l u t e s from t h i s column i n the v o i d volume. The tRNA p r e p a r a t i o n s were assayed f o r acceptance of v a l i n e , l y s i n e and a l a n i n e . The r e s u l t s f o r v a l i n e accep-tance were expressed as r a t i o s to the l y s i n e and a l a n i n e v a l u e s i n Table 6. L y s i n e was chosen as a standard because the genes f o r both of the major l y s i n e tRNAs are b e l i e v e d to be l o c a t e d on the second chromosome (192). A l a n i n e was chosen because the a m i n o a c y l a t i o n r e a c t i o n w i t h a l a n i n e as s u b s t r a t e g i v e s a r a p i d and s t a b l e l e v e l of acceptance. 108. Table 6. V a l i n e Acceptance o f tRNA Prepared from D r o s o p h i l a Stocks w i t h D e f i c i e n c i e s or D u p l i c a t i o n s a t S i t e 84-D Amino A c i d Acceptance Stock V a l i n e ( p m o l e s / A 2 6 0 u n i t ) / % of Lysi n e ( p m o l e s / A 2 6 o u n i t ) a C o n t r o l C o n t r o l 0.96 100 D e f i c i e n c y 1 0.92 96 D e f i c i e n c y 2 1.01 105 D u p l i c a t i o n 1.14 118 Va l i n e ( p m o l e s / A 2 e o u n i t ) / A lanine(pmoles/A 2 6 o u n i t ) * 3 C o n t r o l 0.89 100 D e f i c i e n c y 1 0.92 103 D e f i c i e n c y 2 0.92 103 D u p l i c a t i o n 1.02 115 cl These v a l u e s are the average of two experiments each. k These v a l u e s are the average of f o u r experiments each. 109; The r e s u l t s i n Table 6 i n d i c a t e t h a t the d e f i c i e n c y s t o c k s c o n t a i n w i l d type l e v e l s o f t R N A V a l w h i l e the d u p l i c a -V a l t i o n stock has an i n c r e a s e of 17%. tRNA-^ i s r e s p o n s i b l e V a l f o r approximately 35% of the t o t a l tRNA acceptance i n the c o n t r o l f l i e s . A d u p l i c a t i o n o f the e n t i r e t R N A ^ 1 gene complement on one of the t h i r d chromosomes i n a d i -V a l p l o i d f l y would i n c r e a s e the l e v e l of tRNA by 17.5% i f the l e v e l o f tRNA i s d i r e c t l y p r o p o r t i o n a l to the gene dosage, In s i t u h y b r i d i z a t i o n experiments suggest t h a t approximately t w o - t h i r d s o f the t R N A ^ 1 genes are a t s i t e 84D. T h i s sug-g e s t i o n i s made because the number of g r a i n s a t 84D i s roughly twice the number of g r a i n s a t 92D. I f the d u p l i c a -t i o n mutant has d u p l i c a t e d o n l y t w o - t h i r d s of the tRNA3a"'" genes p r e s e n t on one of i t s t h i r d chromosomes, then an i n -V a l crease o f 12% i n t o t a l tRNA acceptance would be p r e d i c t e d i f the d i r e c t r e l a t i o n s h i p of gene dosage t o tRNA l e v e l s i s assumed. The observed average i n c r e a s e o f 17% i s c l o s e to the 17.5% i n c r e a s e p r e d i c t e d f o r a d u p l i c a t i o n o f the V a l whole tRNA^k gene c l u s t e r and more than the 12% i n c r e a s e V a l p r e d i c t e d i f two- t h i r d s of the tRNA^k genes are d u p l i c a t e d . However, the e r r o r i n the acceptance assays i s c o n s i d e r a b l e due t o f a c t o r s such as nucl e a s e s pres e n t i n the aminoacyla-t i o n enzymes p r e p a r a t i o n . Whereas the tRNA from the d u p l i c a -t i o n stock shows an i n c r e a s e i n t o t a l v a l i n e acceptance, the d e l e t i o n s t o c k s do not show s i g n i f i c a n t d i f f e r e n c e s i n t o t a l v a l i n e acceptance. T h i s would imply t h a t the f l i e s can compensate f o r the l o s s o f t R N A ^ 1 genes. A b e t t e r understanding of the e f f e c t o f these mutations on the tRNA V a^" system was achieved by examining the amounts of i n -V a l d i v i d u a l tRNA i s o a c c e p t o r s w i t h i n each s t r a i n . (B) RPC-5 A n a l y s i s o f V a l i n e I s o a c c e p t o r s tRNA e x t r a c t e d from the f o u r s t r a i n s o f f l i e s des-c r i b e d above was charged w i t h [ 1 ! tC] v a l i n e and f r a c t i o n a t e d on the RPC-5 system A a t pH 4.0. The r e s u l t s of t h i s study are i l l u s t r a t e d i n f i g u r e 23. The RPC-5 columns were r e -peated three times each except those f r a c t i o n a t i n g tRNA from the d u p l i c a t i o n mutant which was repeated seven times. The areas under the t R N A ^ ^ ^ peak and under the tRNA^ .3'"'" peak were es t i m a t e d and the averages c a l c u l a t e d i n Table 7. As demonstrated i n F i g u r e s 10A and 10B, 27% of the combined V a l tRNA, peak i n the c o n t r o l f l i e s i s a c t u a l l y due to V a l tRNA* 3a The t ^ C ] v a l i n e tRNA p r e p a r a t i o n s from the f o u r s t r a i n s were chromatographed on RPC-5 system C as i l l u s t r a t e d i n f i g u r e 24 and Table 8. In t h i s system tRNAY a l runs as a -j a V a l peak which e l u t e s b e f o r e the peak c o n t a i n i n g tRNA^ and tRNA^ a l ( f i g . 24A). The [ 1 V a l - t R N A ^ J 1 peak o b t a i n e d from the RPC-5 column run i n system C was rechromatographed on the RPC-5 i n system A wi t h [ 3H] Val-tRNA^3''". T h i s experiment, shown i n f i g u r e 25, demonstrates t h a t more than 90% o f the V a l t RNA 3 a peak from the RPC-5 column run i n system C i s due 111. F i g u r e 23. RPC-5 chromatography i n system A o f Val-tRNA from f l i e s w i t h d u p l i c a t i o n s or d e f i c i e n c i e s a t s i t e 84D. tRNA (4-5 A 2 6 o u n i t s ) from the f o u r s t o c k s d e s c r i b e d i n t a b l e 5 and f i g u r e 22 was aminoacylated w i t h [ 1 k C ] - V a l i n e (17 3 mCi/mmole) as d e s c r i b e d i n methods. Each prep- -a r a t i o n of [ 1 " c ] - V a l - t R N A V a l (between 40,000 and 70,000 cpm) was chromatographed on an RPC-5 column (0.9 x 21 cm) a t 37°C i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.0; 10 mM MgCl 2; 1 mM 2-mercaptoethanol). F r a c t i o n s (0.5 ml) were e l u t e d w i t h a 100 ml l i n e a r g r a d i e n t o f NaCl (0.50 M to 0.65 M) a t a flow r a t e of 15 ml per hour. Approximately 90-100% o f the r a d i o a c t i v i t y was e l u t e d by the g r a d i e n t . The r a t i o o f peak 3 to peak 4 was c a l c u l a t e d from the measured areas under these peaks. Counts per minute per 0.5 ml ( s o l i d l i n e ) , average v a l u e s o f peak 3 expressed as a percentage of peak 4 (average of 3 experiments f o r A, C and D, and 7 experiments -for B as d e s c r i b e d i n t a b l e 7) i s shown above peak 3 on each f i g u r e . 112. F R A C T I O N N U M B E R Table 7. L e v e l s of tRNA Isoacceptors Measured by Chromatography i n the RPC-5 System A a b d D r o s o p h i l a tRNA j^jJ . /tRNA^ a l Average ± 9 5 % tRNA^ a l/tRNAY a l tRNAYg 1 as . S t r a i n Ja * j D 4 confidence l i m i t s 0 J 4 of c o n t r o l C o n t r o l 1.12,1.16,1.14 1.14 ± .03 0.83 100 D e f i c i e n c y 1 0.96,1.07,1.07 1.04 ± .09 0.73 88 D e f i c i e n c y 2 0.90,0.89,0.86 0.88 ± .03 0.57 69 D u p l i c a t i o n 1.39,1.31,1.46 1.39 ± .05 1.08 130 1.28,1.33,1.41 1.39 The chromatography i s d e s c r i b e d i n F i g u r e 23 . These val u e s are the r a t i o of the areas under the two major peaks shown i n F i g u r e 23. Confidence l i m i t s c a l c u l a t e d from t h i s formula, where Sd i s the standard d e v i a t i o n and 95% l i m i t s = ±(1.96)Sd n i s the number of valu e s These val u e s were obtained by s u b t r a c t i n g 0.31 from the v a l u e s f o r tRNA^a^b/tRNA^ 1. T h i s c o r r e c t i o n can be made because the l e v e l of tRNA^fl i s 31% t h a t o f tRNAY a x i n a l l s t o c k s , as shown i n the t e x t . i—1 L O 114. F i g u r e 24. RPC-5 chromatography i n system C of V a l - t R N A v a from f l i e s w i t h d u p l i c a t i o n s or d e f i c i e n c i e s a t s i t e 84D. tRNA (4-5 A 2 6 o u n i t s ) from the f o u r stocks d e s c r i b e d i n t a b l e 5 and f i g u r e 22 was aminoacylated w i t h [ 3 H ] v a l i n e (824 mCi/mmole) ; as d e s c r i b e d i n Methods. Each p r e p a r a -t i o n of [ 3 H ] V a l - t R N A V a l (between 47,000 and 69,000 cpm) was chromatographed on an RPC-5 column (0.9 x 21 cm) a t 37°C i n RPC-5 system C (10 mM Na formate, pH 3.8; 1 mM EDTA; 1 mM 2-mercaptoethanol). F r a c t i o n s 0O.5 ml) were e l u t e d w i t h a 100 ml l i n e a r g r a d i e n t of NaCl (0.55 M to 0.75 M) a t a flow r a t e of 15 ml per hour. The areas under the t hree major peaks were determined and the r a t i o s of tRNA^ I 1 and tRNA^g 1 to tRNA ^ J determined ( t a b l e 8). The f i g u r e s are normalized such -that the maximum va l u e of the tRNA^ a^ peak i s a t 100% i n each f i g u r e . Counts per min-ute per 0.5 ml expressed as a percentage of the h i g h e s t value i n counts per minute found i n the tRNA^3^; peak ( s o l i d l i n e ) . ' 115. RPC-5 EDTA pH 3.8 Fraction Number 116. Table 8. Amounts of tRNA I s o a c c e p t o r s Measured by Chromato-graphy i n RPC-5 System C a D r o s o p h i l a S t r a i n t R N A ^ V t R N A ^ f t R N A l g 1 as % of C o n t r o l C o n t r o l 0.29 0.73 100 D e f i c i e n c y 1 0.27 0.68 93 D e f i c i e n c y 2 0.29 0.52 71 D u p l i c a t i o n 0.28 0.90 123 Chromatography d e s c r i b e d i n F i g u r e 24. These v a l u e s are the r a t i o o f the areas under the approp-r i a t e peaks i n F i g u r e 24. t o tRNA^ 3 1. The amount of t h i s t R N A 3 a l peak does not change V a l r e l a t i v e t o t R N A 4 & 5 peak i n the f o u r tRNA samples as shown i n f i g u r e 24 and Table 8. These r e s u l t s demonstrate t h a t the d e l e t i o n and d u p l i c a t i o n f o r the r e g i o n 84D d i d not a l t e r the r a t i o of t R N A 3 a l to t R N A ^ 1 . t R N A 3 a l i s 28% o f the amount of tRNAY a^ or 31% of the amount of tRNAY a l i t s e l f . 4'&'D- 4 The tRNA^k"*" peak e l u t e s very c l o s e t o the tRNA^ a^ s p e c i e s i n the columns i l l u s t r a t e d i n f i g u r e 24. Therefore the V a l v a l u e s reported^ f o r the amount of tRNA,^ may be i n f l u e n c e d by the extent of r e s o l u t i o n of tRNAg. a l from tRNA^^ • F o r t h i s reason these columns are not a r e l i a b l e i n d i c a t i o n of V a l tRNA^k amounts. However, they are u s e f u l f o r demonstrating V a l t h a t the amounts of t R N A o can be c a l c u l a t e d r e l i a b l y . *^ a And because t R N A 3 a l and t R N A 3 a l cochromatograph on the RPC-5 V a l system A ( F i g . 24)f the amounts of tRNA 3 a can be s u b t r a c t e d V a l from the combined t R N A 3 a & 3 r ) P e a k t o g i v e a r e l i a b l e v a l u e V a l f o r the q u a n t i t y of tRNA-^ . These c a l c u l a t i o n s are shown i n Table'7. The minor i s o a c c e p t o r tRNA^a"^ does not r e s o l v e from t R N A ^ 1 i n the RPC-5 system C. Th e r e f o r e the va l u e of 28% f o r the amount of tRNA^a''" r e l a t i v e t o tRNA^ c j i s an under-estimate o f the -amount of tRNA^a"'" r e l a t i v e t o tRNA^a"^. A more r e a l i s t i c estimate i s ob t a i n e d from the r e s u l t s of the Sepharose column shown i n f i g u r e 11. T h i s column dem-V a l o n s t r a t e s t h a t f o r the c o n t r o l f l i e s 27% of the t R N A 3 a & 3 k peak i s due to t R N A 3 a l . Since the t R N A 3 a & 3 b P e a k i s 1 1 4 % 118. F i g u r e 25. RPC-5 chromatography of tRNA^ a x. - • — — — j a D r o s o p h i l a tRNA (4 A 2 6 o u n i t s ) was aminoacylated w i t h [ 3 H ] V a l i n e (9.75 Ci/mmole) as d e s c r i b e d i n Methods. The [ 3 H ] V a l - t R N A V a l (720,000 cpm) was chromatographed on a RPC-5 column (0.9 x 21 cm) e x a c t l y as d e s c r i b e d i n f i g u r e 24. Samples of each f r a c t i o n (50 y l ) were d i s s o l v e d i n s c i n t i l l a t i o n c o c k t a i l and the r a d i o a c t i v i t y determined. The r a d i o a c t i v i t y which e l u t e d as the e a r l y peak (Peak 3a i n f i g u r e 24A) was pooled, p r e c i p i t a t e d w i t h i c e - c o l d e t h a n o l (2.5 volumes), and d i s s o l v e d i n a 0.45 M NaCl s o l u t i o n o f RPC-5 b u f f e r A (10 mM NaOAc, pH 4.0; 10 mM Mg C l 2 ; 1 mM 2 - m e r c a p t o e t h a n o l ) D r o s o p h i l a tRNA (4 A 2 6 0 u n i t s ) was aminoacylated w i t h C1 ^ -C]Valine (234 mCi/mmole) as d e s c r i b e d i n .Methods. T h i s [ 1 "*C] V a l - t R N A V a l p r e p a r a -t i o n (16,700 cpm) was mixed w i t h the [ 3H]Val-tRNAYf x p r e p a r a t i o n (60,600 cpm) d e s c r i b e d above and chromato-' graphed on the RPC-5 column (0.9 x 21 cm) a t 37°C i n RPC-5 system A. The f r a c t i o n s (0.5 ml) were e l u t e d w i t h a 100 ml l i n e a r g r a d i e n t of NaCl (0.50 M t o 0.65 M) a t 15 ml per hour. Counts per minute per 0.5 ml [ 3 H ] V a l i n e (dotted l i n e ) , counts per minute per 0.5 ml [ 1'*C]Valine ( s o l i d l i n e ) . 120. F i g u r e 26. RPC-5 chromatography i n system A o f tRNA V d' L from which tRNA^g has been removed by chrom-atography i n system C. tRNA (4-5 A 2 6 o u n i t s ) from the f o u r s t o c k s d e s c r i b e d i n t a b l e 5 and i n f i g u r e 22 was aminoacylated w i t h [lkC]-V a l i n e (234 mCi/mmole) as d e s c r i b e d i n Methods. Each t 1 ^ C ] V a l - t R N A V a l (between 34,000 and 54,000 cpm) was chromatographed i n RPC-5 system C (10 mM Na formate,pH 3.8; 1 mM EDTA; 1 mM 2-mercaptoethanol) on a column (0.9 x 21 cm) a t 37°C w i t h a flow r a t e of 15 ml per hour. F r a c t i o n s (0.5 ml) were e l u t e d w i t h a 100 ml l i n e a r grad-i e n t of NaCl (0.55 M to 0.75 M). The r a d i o a c t i v i t y which e l u t e d a f t e r the tRNAY a x peak, which corresponds to a l l of the t R N A s V a l except tRNAY a l and tRNAYl; 1/ was p r e c i p i -t a t e d w i t h i c e - c o l d e t h a n o l (2.5 volumes) and d i s s o l v e d i n a 0.45 M NaCl s o l u t i o n of RPC-5 b u f f e r A (10 mM NaOAc, pH 4.0; 10 mM M g C l 2 , * 1 mM 2-mercaptoethanol). Each of the f o u r [ 1 4 C ] V a l - t R N A V a l p r e p a r a t i o n s (between 24,500 and 41,000 cpm) o b t a i n e d i n t h i s way was chromatographed on the RPC-5 column (0.9 x 21 cm) i n RPC-5 system A a t 37°C. F r a c t i o n s (0.5 ml) were e l u t e d w i t h a 100 ml l i n e a r g r a d i e n t of NaCl (0.50 M to 0.65 M) a t a flow r a t e of 15 ml per hour. The percentage of r a d i o a c t i v i t y e l u t e d by the g r a d i e n t was 93%, 98%, 96% and 91% i n columns A, B, C and D r e s p e c t i v e l y . Counts per minute t ^ C ] V a l i n e per 0.5 ml ( s o l i d l i n e ) . 121. Fraction Number Table 9. Amounts of tRNA I s o a c c e p t o r s Measured by Com-bined Chromatography i n RPC-5 Systems A and C a D r o s o p h i l a S t r a i n t R N A ^ a l / t R N A j a ± b tRNA3k as % of C o n t r o l C o n t r o l 0.76 100 D e f i c i e n c y 1 0.73 96 D e f i c i e n c y 2 0.59 78 D u p l i c a t i o n 0.92 121 Chromatography d e s c r i b e d i n F i g u r e 26. R a t i o s c a l c u l a t e d from the areas under the two major peaks of r a d i o a c t i v i t y shown i n F i g u r e 26. 123. r e l a t i v e to tRNA^ a l i n the control f l i e s , the amount of tRNA^ 1 can be estimated to be 27% of the t R N A 3 a & 3 b P e a k o r Val 31% r e l a t i v e to tRNA4 . The values given i n Table 7 for Val the amounts of tRNA3^ are obtained by correcting the values of the tRNA3 a^ 3 b peak for tRNA^fJ 1. A t h i r d set of experiments was performed to estimate the amounts of tRNA^ a ± i n these st r a i n s . In these experiments [:^C]Val-tRNA was fractionated on the RPC-5 system C. The V a l r a d i o a c t i v i t y e l u t i n g a f t e r the tRNA 3 a peak had eluted was pooled, p r e c i p i t a t e d with cold ethanol, and rerun on the RPC-5 column i n system A. The net e f f e c t of t h i s procedure i s to chromatographically remove tRNA^ 1 from the combined tRNA^ a^3^ peak. The column p r o f i l e s obtained are presented Val i n figure 26. The calculated amounts of tRNA2j_) r e l a t i v e to tRNA^a^" are presented i n Table 9. Val Summary of the Eff e c t s of Gene Dosage on the Amounts of tRNA-^ The most r e l i a b l e estimate of tRNA^ a l amounts are those presented i n Table 7. These experiments were repeated several times and the re s u l t s corrected for the amounts of Val tRNA 3 a . In spite of some question concerning the r e l i -a b i l i t y of the procedures i n Table 8 and Table 9, a l l three Val procedures gave • similar r e s u l t s . The amount of tRNA3k was decreased by 30% i n the large deficiency and increased by 29% i n the duplication when estimated i n columns run i n RPC-5 system A (Table 7). The r e s u l t s shown i n Tables 8 and 124. 9 show changes which are q u a l i t a t i v e l y i d e n t i c a l t o those i n Table 7 but are of lower magnitude. The amount of tRNA^^ i n the s m a l l d e f i c i e n c y (Def. 2) i s decreased o n l y s l i g h t l y , a 12% decrease i n the most r e l i a b l e e s t i m a t i o n g i v e n i n Table 7. Co n c l u s i o n V a l Three D r o s o p h i l a tRNAs have been p u r i f i e d and char-V a l a c t e r i z e d . tRNA^ c o n t a i n s i n o s i n e and probably f u n c t i o n s as the tRNA r e s p o n s i b l e f o r the t r a n s l a t i o n o f the code-words GUA, GUC and GUU. One or both o f t R N A ^ 1 and t R N A ^ 1 w i l l l i k e l y f u n c t i o n to t r a n s l a t e the codeword GUG. V a l The p u r i f i e d tRNAs were used t o probe the o r g a n i z a -t i o n of the tRNA genes by i n s i t u h y b r i d i z a t i o n . Only one V a l s i t e was found c o n s i s t e n t l y f o r the tRNA^. genes. However, V a l two s i t e s were found f o r t R N A ^ genes. T h i s l a t t e r observa-V a l t i o n suggests t h a t the tRNA 3 b gene c l u s t e r may be d i v i d e d i n t o two separate u n i t s . I t i s p o s s i b l e t h a t h e t e r o g e n e i t y V a l e x i s t s w i t h i n the p u r i f i e d tRNA 3k p r e p a r a t i o n . One or two base changes which do not a f f e c t chromatographic behaviour may e x i s t between two very c l o s e l y r e l a t e d tRNA s p e c i e s which V a l cochromatograph as tRNA3^ . T h i s p o s s i b i l i t y can be i n v e s -V a l t i g a t e d by sequencing the tRNA3j 3 p r e p a r a t i o n o r sequencing the DNA coding f o r tRNA^3"*" a t the two gene s i t e s . The two s i t e s l a b e l l e d i n the i n s i t u h y b r i d i z a t i o n experiment a r e not u n i f o r m l y l a b e l l e d . S i t e 84D i s l a b e l l e d approximately twice as h e a v i l y as s i t e 92B . H y b r i d i z a t i o n experiments i n s o l u t i o n u t i l i z i n g DNA ob t a i n e d from w i l d type f l i e s have shown t h a t 13 genes f o r t E N A ^ 1 a r e p r e s e n t per h a p l o i d genome (183)* The i n s i t u h y b r i d i z a t i o n experiment p r e d i c t e d t h a t the genes would be d i s t r i b u t e d approximately 8 genes a t 84D • and 5 genes a t 92B • because of the d i s t r i b u -t i o n o f l a b e l l i n g over the two s i t e s . H y b r i d i z a t i o n experiments i n s o l u t i o n u t i l i z i n g DNA o b t a i n e d from f l i e s w ith a d e f i c i e n c y a t s i t e 84D. ; (Def. 2) V a l show a r e d u c t i o n of 30% i n tRNA 3 b genes (183) . T h i s r e s u l t i s i n t e r p r e t e d i n the f o l l o w i n g manner. The c o n t r o l f l i e s have 26 tRNA^^ genes per d i p l o i d c e l l (13 on each o f two -t h i r d chromosomes per d i p l o i d c e l l ) . The d e f i c i e n c y f l i e s V a l c a r r y o n l y 18 tRNA-j^ genes per d i p l o i d c e l l (13 on one t h i r d chromosome and 5 on the mutant t h i r d chromosome). T h e r e f o r e , the d e f i c i e n c y stock has 8 fewer genes or 31% lower number of tRNA genes than the w i l d type per d i p l o i d genome. These data from the h y b r i d i z a t i o n i n s o l u t i o n , t h e r e f o r e support the c o n c l u s i o n t h a t o n l y a p o r t i o n of the V a l tRNA 3j 3 genes are p r e s e n t a t s i t e 8 4D. A d i r e c t r e l a t i o n s h i p of gene dosage to the amount of gene product has been demonstrated f o r the enzyme xanthine dehydrogenase i n D r o s o p h i l a (192). T h i s r e l a t i o n s h i p was V a l a l s o observed f o r the tRNA-^ genes. An i n c r e a s e or V a l decrease i n the number of tRNA^^ genes by 30% r e s u l t e d i n an i n c r e a s e o r decrease of 30% i n tRNA amounts. These V a l changes are c a l c u l a t e d r e l a t i v e to the o t h e r tRNA s p e c i e s . However, since i n the deficiency mutants the amount of valine acceptance was e s s e n t i a l l y that of wild type, a form of control must e x i s t to increase the t o t a l valine tRNA popu-l a t i o n i n f l i e s carrying a deletion. This control i s not s p e c i f i c for a single isoacceptor but instead affects a l l the isoacceptors of valine tRNA. I t should also be noted that t h i s control did not function i n the f l i e s carrying the duplication for tRNA^"*" genes since tRNA from these f l i e s did have an increased amount of valine acceptance. Ser Analysis of the Coding Properties of tRNA The genetic code i s redundant. Most amino acids are coded by more than one t r i p l e t codon. Serine i s coded for by six t r i p l e t s , AGC, AGU, UCA, UCC, UCG, UCU. One of the best studied minor nucleosides i n tRNA i s isopentenyl adeno-sine ( i 6 A ) . This hypermodified adenosine occurs adjacent to the 3 1-terminal nucleotide of the anticodon i n some tRNAs which respond to codons beginning i n U (10). A second hyper-modified adenosine, N- [ 9 - ( B-D-ribofuranosyl)purin - 6-yl carbamoyl]threonine (t 6A) occurs only i n tRNAs with codons beginning i n A (10). Dr. B. White i n our laboratory had shown that c e r t a i n Drosophila serine isoacceptors contained either i 6 A or a nucleoside resembling modified t 6 A c a l l e d N- [ 9 - ( 8-D-ribofuranosyl)purin - 6-yl-N-methyl carbamoyl]adeno-sine (mt 6A). In v i t r o t r i p l e t stimulated ribosome binding experiments were performed to determine i f the re l a t i o n s h i p of i 6 A and m£ 6A to codon s p e c i f i c i t y was maintained i n the Ser Drosophila tRNAs 127. The t r i p l e t s were s y n t h e s i z e d as d e s c r i b e d i n Methods. To c o n f i r m t h e i r i d e n t i t y each t r i p l e t was d i g e s t e d by snake venom phosphodiesterase and by RNase T 2 . The c h a r a c t e r i z a -t i o n o f the d i g e s t i o n products i s presented i n Table 10. The i d e n t i t y o f each n u c l e o s i d e was confirmed by uv spec-t r o s c o p y . [ 1 ^ C ] S e r y l - t R N A S e r was f r a c t i o n a t e d on RPC-5 i n system A a t pH 4.5 as shown i n f i g u r e 27. The [ 1 ! * C ] S e r y l -Ser tRNA peaks were pooled, p r e c i p i t a t e d w i t h c o l d e t h a n o l and used f o r the ribosome b i n d i n g assay. The r e s u l t s of these assays are presented i n f i g u r e s 28 and 29. These r e s u l t s c o n f i r m the r e l a t i o n s h i p of i 6 A and mt 6A t o codon Ser Ser R s t r u c t u r e . tRNA;, and tRNA,- c o n t a i n the mt A - l i k e r e s i -Ser due. These two s p e c i e s respond t o AGC and AGU. tRNA,. gave s i g n i f i c a n t response t o UCG which i s due to contamina-Ser Ser R t i o n o f the tRNA 4 by tRNA g . The i A c o n t a i n i n g s p e c i e s Ser Ser tRNA^. and tRNA^ respond t o UCG and UCU r e s p e c t i v e l y . Ser tRNA^ a l s o responds weakly t o UCC and UCA. T h i s response Ser Ser i s p o s s i b l y due to the t a i l i n g o f tRNAg. i n t o the tRNA^ Ser K peak. tRNAg a l s o c o n t a i n s i A. The r e s u l t s o f t h i s study were p u b l i s h e d (143). Table 10. C h a r a c t e r i z a t i o n of T r i n u c l e o t i d e s by RNase T  and Venom Phosphodiesterase D i g e s t i o n RNase T 2 Amount of Products (nmoles) Compound Se p a r a t i o n A Ap C Cp G Gp U Up UCU 1 _ mmm 30 35 28 UCC 2 a - - 33 28 - - - 40 UCA 2 a 34 - - 15 - - - 35 UCG 1 - - - 17 31 - - 26 AGU 2 b - 34 - - - 31 27 -AGC 2 b — 37 30 — - 26 - -Venom Phosphodiesterase A pA C PC G pG U pU UCU 2 a — — — 37 — — 48 38 UCC 2 a - - - 104 - - 41 -UCA 2 b 18 - - 22 - - 33 -UCG 2a - - - 31 - 29 32 -AGU 2 b 44 - - - - 34 - 37 AGC 2 a 47 — 32 — 42 — Chromatography and E l e c t r o p h o r e s i s 1) Paper e l e c t r o p h o r e s i s , 60 v o l t s per cm, 50 mM Na a c e t a t e (pH 4.5) f o r one hour. 2) Descending chromatography on Whatman 40 chromatgraphy paper. Chromatographic s o l v e n t s a I s o b u t y r i c a c i d - cone, ammonia - H 20 (66/1/33, v/v) . b 95% e t h a n o l - 1 M ammonium a c e t a t e pH 7.2 (7/3, v/v) . 129. F i g u r e 27. Chromatography of S e r - t R N A s e r on RPC-5. D r o s o p h i l a tRNA (82 A 2 6 0 u n i t s ) was aminoacylated w i t h [lkC]Serine (154 mCi/mmole) v a s d e s c r i b e d i n Methods. The [ 1 "*C] S e r - t R N A S e r was chromatographed on ahRPC-5 column (0.9 x 62 cm) a t 37°C i n RPC-5 b u f f e r system A (10 mM NaOAc, pH 4.5; 10 mM MgCl 2; 1 mM 2-mercaptoethanol). F r a c -t i o n s (3.0 ml) were e l u t e d w i t h a 600 ml l i n e a r g r a d i e n t of NaCl (0.55 M to 0.70 M) a t a flow r a t e o f 1.0 ml per minute. Samples of f r a c t i o n s (0.2 ml) were d i s s o l v e d i n s c i n t i l l a t i o n c o c k t a i l and the amount of r a d i o a c t i v i t y d e t e r -mined. The r a d i o a c t i v i t y c o r r e s p o n d i n g t o tRNA^ e r, . tRNA| e r, t R N A § e r and tRNASer (shown as bars on the f i g u r e ) was p r e c i p i t a t e d w i t h i c e - c o l d e t h a n o l (2.5 volumes) and s t o r e d a t -20°C u n t i l r e q u i r e d . Counts per minute [ l l f C ] -S e r i n e per 0.2 ml ( s o l i d l i n e ) . { 130. 1 3 1 . F i g u r e 2 8 . B i n d i n g o f D r o s o p h i l a Ser-tRNA2 and Ser-tRNA4 to E. c o l i ribosomes. The t r i p l e t s t i m u l a t e d ribosome b i n d i n g e x p e r i m e n t s g e r were c a r r i e d out as d e s c r i b e d i n Methods. [ 1^C]Ser-tRNA2 ( 1 4 . 4 pmoles) or [ l l fC]Ser tRNA| e r ( 2 3 . 0 pmoles) were bound to E. c o l i ribosomes ( 1 . 2 A 2 6 o u n i t s ) i n 5 0 y l of 2 0 mM magnesium a c e t a t e , 5 0 mM.KCl, and 0 . 1 M T r i s - H C l , pH 7.2 f o r 1 5 minutes. The amount of t r i p l e t was v a r i e d from 0 to 4 . 1 4 nmoles per assay. A l l r e a c t i o n s were c a r r i e d out i n d u p l i c a t e . T r i p l e t s added, UCC ( • ) , UCU ( A ) , UCA (•), UCG ( A ) , AGC ( • ) , and AGU (o); pmoles [ 1^C]Ser-tRNA per f i l t e r ( s o l i d l i n e s ) . 133. Ser S F i g u r e 29. 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