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UBC Theses and Dissertations

Studies on the valine transfer RNAs and their genes in Drosophila melanogaster Addison, William Robert 1982

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STUDIES ON THE VALINE TRANSFER RNAS AND THEIR GENES IN DROSOPHILA MELANOGASTER by WILLIAM ROBERT ADDISON B . S c , U n i v e r s i t y of Regina, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n 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 a u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA February, 1982 (c) W i l l i a m Robert Addison, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of B i o c h e m i s t r y The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date M a r c h 5, 1932 DE-6 (3/81) 1 1 . Abstract The coding p r o p e r t i e s of the 3 major v a l i n e tRNA isoacc e p t o r s of V a l Drosophila melanogaster, . the n u c l e o t i d e sequences of tRNA^ and Val tRNA^ and the nucleotide sequences of genes f o r these two tRNAs have V a l been determined. V a l y l - t R N A ^ binds s t r o n g l y to ribosomes i n r e s -ponse to the t r i n u c l e o t i d e GUA and to a l e s s e r extent w i t h GUU and GUG. V a l V a l y l - t R N A ^ binds s t r o n g l y i n the presence of GUG and very weakly V a l w i t h the other 3 t r i p l e t s whereas valyl - t R N A ^ binds s t r o n g l y i n the presence of GUU, GUC, and GUA and weakly w i t h GUG. V a l V a l The n u c l e o t i d e sequences of tRNA^ b and tRNA^ were d e t e r -mined by a combination of techniques. For both tRNAs most of the sequence was determined by the method of Stanley and V a s s i l e n k o . The sequences at the 5' and 3'-ends of the molecules were determined by wandering-spot a n a l y s i s . Regions of the molecules that could not be sequenced by these two techniques were determined by the g e l read-off method. The use of tRNA modified w i t h chloroacetaldehyde t o overcome problems i n sequencing RNA by the g e l read-off method caused by secondary s t r u c t u r e i n the RNA i s described. The n u c l e o t i d e sequence of tRNAy a l i s : GUUU CCGUm1GGUG^AGCGGDU 4 m (acp 3 U)AUCACA1'CUGCCmUIACAms CGCAGAAGm7 GCCCCCGG'H'C Gm1 AUCCCGGGCGGAAACACCA. About 50% of the U residues a t p o s i t i o n 20 are modified to acp 3U. One of the C residues a t p o s i t i o n 48 or 49 i s probably modified to m5C. The n u c l e o t i d e sequence of tRNA^f 1 i s : GUUUCCGUAGUGS1 AGCGGDacp3 UAUCACG*GUGCUUC ACACGCACAAGm7-J b GDCCCCGGTfCGm1 AACCC GGGCGGGAACACCA. The C residue at p o s i t i o n 48 i s probably modified to m5C. The observed codon responses of the two tRNAs are d i s -cussed i n r e l a t i o n to the anticodons found. V a l The two tRNA^ genes of the recombinant plasmid pDt55 were sequenced by the Maxam and G i l b e r t method. This plasmid h y b r i d i z e s to the V a l 70BC s i t e on the polytene chromosomes, a major s i t e of tRNA^ h y b r i d i -z a t i o n . The two genes are of opposite p o l a r i t y and are separated by 525 bp of DNA. The genes have i d e n t i c a l sequences, which correspond to that V a l expected from the sequence of tRNA^ V a l The n u c l e o t i d e sequence of the t R N A ^ gene of recombinant plasmid pDt78R was a l s o determined. This plasmid h y b r i d i z e s to the 84D V a l s i t e , a major s i t e of tRNA^ h y b r i d i z a t i o n . The sequence of the gene V a l corresponds to that expected from the sequence of t R N A ^ • Comparison of the v a l i n e tRNA genes sequenced i n t h i s study and those determined by other workers shows that tRNA genes from major s i t e s of Va l V a l tRNA^ or tRNA^ h y b r i d i z a t i o n to polytene chromosomes c o r r e s -V a l V a l pond e x a c t l y to the tRNA sequences w h i l e tRNA genes from minor V a l s i t e s of tRNA h y b r i d i z a t i o n d i f f e r a t 4 p o s i t i o n s from the sequences expected on the b a s i s of the tRNA sequences. The p o s s i b l e s i g n i f i c a n c e of t h i s f i n d i n g i s d i s c u s s e d . 1 i v . TABLE OF CONTENTS Page Abstract i i Table of Contents i v L i s t of Tables v i i i L i s t of Figures i x Acknowledgments x i D e d i c a t i o n x i i Abbreviations x i i i I n t r o d u c t i o n 1 I . S t r u c t u r e of the tRNA Molecule 1 A. Primary S t r u c t u r e - RNA Sequencing 1 B. Secondary S t r u c t u r e 5 C. T e r t i a r y S t r u c t u r e 7 D. M i t o c h o n d r i a l tRNAs 11 E. M o d i f i e d Nucleotides Found i n tRNAs 12 I I . Functions of Transfer RNA 14 A. P r o t e i n Synthesis 14 B. Regulation of T r a n s c r i p t i o n 16 C. Aminoacyl Group Transfer 17 D. tRNAs and tRNA-Like S t r u c t u r e s Associated w i t h V i r u s e s .. 19 I I I . D r o s o p h i l a melanogaster tRNA 20 IV. Transfer RNA Genes 21 A. E. c o l i 21 B. Organelles 23 C. Yeast 24 D. Xenopus 26 V. Page V. Drosophila melanogaster tRNA Genes • 27 A. Gene Number 27 B. Gene L o c a t i o n 28 C. O r g a n i z a t i o n of Cloned Drosophila tRNA Genes 30 V I . T r a n s c r i p t i o n of Eukaryotic tRNA Genes 34 V I I . Processing of tRNA T r a n s c r i p t s 37 V I I I . The Present I n v e s t i g a t i o n 39 M a t e r i a l s and Methods • •• 43 I . General 43 A. Thin-Layer Chromatography Solvents 43 B. S c i n t i l l a t i o n Counting 43 C. Polyacrylamide Gel E l e c t r o p h o r e s i s 43 D. Agarose Gel E l e c t r o p h o r e s i s 43 E. Autoradiography 44 F. P u r i f i c a t i o n of tRNA^f,1 and tRNA$[al. 44 G. Synthesis of [5'- 3 2P]pCp 44 H. I s o l a t i o n and C h a r a c t e r i z a t i o n of acp 3U 45 I I . T r i n u c l e o t i d e - S t i m u l a t e d Binding of Valyl-tRNA to Ribosomes 48 A. Synthesis of V a l i n e Codons 48 B. I s o l a t i o n of [ 3 H ] V a l y l - t R N A V a l Isoacceptors 49 C. Determination of Codon T r i p l e t Stimulated Binding of V a l y l - t R N A V a l to Ribosomes 50 I I I . Synthesis of I 2 5 I - C T P 51 IV. I s o l a t i o n of tRNA N u c l e o t i d y l Transferase from Yeast 54 A. Enzyme I s o l a t i o n 54 B. tRNA N u c l e o t i d y l Transferase Assay 55 v i . Page V. End-Labelling of tRNA 56 A. 3' End-La b e l l i n g . 56 B. 5' End-Labelling 57 VI . RNA Sequence A n a l y s i s 58 A. Stanley and Vassilenko Method 58 B. Wandering-Spot A n a l y s i s 60 C. Gel Read-Off Method 61 D. M o d i f i c a t i o n of tRNA w i t h Chloroacetaldehyde 62 V I I . Recombinant Plasmid DNA I s o l a t i o n 62 V I I I . R e s t r i c t i o n Mapping of Recombinant Plasmids 64 A. R e s t r i c t i o n Endonuclease Cleavage of DNA 64 B. R e s t r i c t i o n Mapping 64 IX. DNA Sequence A n a l y s i s 64 R e s u l t s and D i s c u s s i o n 66 I . The Coding P r o p e r t i e s of the Drosophila V a l i n e tRNAs 66 I I . The Nucleotide Sequence of p_. melanogaster tRNA^3-'- 74 A. The Stanley and Vassilenko Method 74 B. Wandering-Spot A n a l y s i s 85 C. Sequencing by the Gel Read-Off Method: Chloroacetaldehyde M o d i f i c a t i o n as an Aid to RNA Sequencing 90 D. Sequencing tRNA End-Labelled w i t h l 2 5I-CMP 96 E. Homologies Between tRNA)[ a l and Other V a l i n e tRNAs ........ 96 I I I . The Nucleotide Sequence of D. melanogaster tRNA^f,1 ...... 101 A. Nucleotide Sequence Determination. 101 B. Features of the tRNA^g 1 Sequence...,,..,.,..,.,.,... 116 C. Homologies between tRNA^3,1 and Other V a l i n e tRNAs ....... 117 v i i • Page IV. The Nucleotide Sequence of tRNA^ a l Genes of pDt55 120 A. Strategy Used to Sequence the tRNA)( a l Genes of pDt55 120 B. The Nucleotide Sequence of the tRNA Genes of pDt55 126 C. Other tRNA^3-*- Genes of Drosophila: Comparison to the tRNA^ a l Genes of PDt55 137 V. The Nucleotide Sequences of tRNAYjf,1 Genes 144 A. The Nucleotide Sequence of the tRNAYjf,1 Gene of pDt78R 144 B. Other tRNA^ §-*- Genes: Comparisons w i t h the tRNA^I 1 Gene of pDt78R 151 Bibli o g r a p h y • 157 v i i i . LIST OF TABLES Page Table I . S i t e s of Drosophila melanogaster Genes on the Polytene Chromosomes 30 Table I I . Nucleoside A n a l y s i s of tRNAsVa-*- from Drosophila 40 Table I I I . Recombinant Plasmids Containing Drosophila t R N A V a l Genes 42 Table IV. Homology between tRNA^3-'- and Other Sequenced V a l i n e tRNAs and V a l i n e tRNA Genes 99 Table V. Chromatographic M o b i l i t i e s of Nucleoside-5'-Phosphates on C e l l u l o s e TLC P l a t e s i n Solvent D 112 Table V I . Homology between tRNAVj3,-'- and Other Sequenced V a l i n e tRNAs and V a l i n e tRNA Genes 119 i x . LIST OF FIGURES Page Figure 1. The c l o v e r l e a f s t r u c t u r e of tRNA 6 Figure 2. Two views of the t e r t i a r y s t r u c t u r e of yeast tRNA p' i e 8 Figure 3. T e r t i a r y hydrogen-bonds of yeast tRNA^e 9 Figure 4. Model f o r a t t e n u a t i o n i n the IS. c o l i t r p operon 17 Figure 5. Removal of the i n t e r v e n i n g sequence from yeast tRNA^he precursor 26 Figure 6. tRNA genes at the 42A r e g i o n of the Drosophila chromosome.. 31 Figure 7. Separation of [ 1 " C ] v a l y l - t R N A s V a l of crude Dros o p h i l a tRNA on an RPC-5 column 40 Figure 8. Chromatography of the uridine-5'-phosphate f r a c t i o n recovered from the QAE-Sephadex column on BioRad AG1-X2 46 Figure 9. Autoradiogram of a two-dimensional chromatogram used to separate 1 2 5 I - C T P from u n l a b e l l e d CTP 52 Figure 10. P u r i f i c a t i o n of Drosophila v a l y l - t R N A ^ a l i s o a c c e p t o r s by RCP-5 chromatography 67 Figure 11. The coding p r o p e r t i e s of the Drosophila v a l i n e tRNAs 69 Figure 12 The n u c l e o t i d e sequence of I), melanogaster tRNA^3-*-arranged as a c l o v e r l e a f 75 Figure 13. Stanley and Vassilenko sequencing of t R N A ^ a l : PEI-c e l l u l o s e chromatography of [ 5 ' - 3 2P]pNp's 78 Figure 14. Stanley and Vassilenko sequencing of tRNAYj3-^: I d e n t i f i -c a t i o n of modified n u c l e o t i d e s by t h i n l a y e r chromatography.. 82 Figure 15. Wandering-spot a n a l y s i s of the 5'-terminal n u c l e o t i d e s of tRNA5[ a 1.., 86 Figure 16. Wandering-spot a n a l y s i s of the 3'-terminal n u c l e o t i d e s of tRNA^ 3 1 88 Figure 17. Reaction of chloroacetaldehyde w i t h c y t i d i n e 91 Figure 18. Chloroacetaldehyde m o d i f i c a t i o n r e l i e v e s band compression on sequencing g e l s of the A43-G57 re g i o n of tRNA^3^" 93 X. Page Figure 19. I d e n t i f i c a t i o n of the n u c l e o t i d e present at p o s i t i o n 33 of tRNAV3-1- 97 Figure 20. The n u c l e o t i d e sequence of I), melanogaster tRNA^g-'-arranged as a c l o v e r l e a f 102 Figure 21. Stanley and Vas s i l e n k o sequencing of tRNA^g-'- 104 Figure 22. Wandering-spot a n a l y s i s of the 5'-end of tRNAYj3,! 108 Figure 23. Wandering-spot a n a l y s i s of the 3'-end of tRNA^f,1 110 Figure 24. Gel read-off sequencing of tRNA^ f,-*-: The n u c l e o t i d e sequence of the v a r i a b l e arm of tRNA^f,1 113 Figure 25. The r e s t r i c t i o n map of plasmid pDt55 ................. 122 Figure 26. I d e n t i f i c a t i o n of the Hinf I r e s t r i c t i o n fragment of pDt55 that contains the tRNA^ a l genes , 124 Figure 27. Strand separation of Dde I fragments of pDt55 DNA ......... 127 Figure 28. Maxam and G i l b e r t sequencing: The f i r s t tRNA^3-*- gene of PDt55 ' 129 Figure 29. The s t r a t e g y used to sequence the tRNA^3-*- genes of pDt55 131 Fig u r e 30. The n u c l e o t i d e sequence of a segment of the Drosophila DNA i n s e r t of plasmid pDt55 134 Fig u r e 31. P o s s i b l e h a i r - p i n s i n the 5 ' - f l a n k i n g sequences of the tRNAX a l genes of pDt55 136 Fi g u r e 32. The n u c l e o t i d e sequence of segments of the Drosophila DNA i n s e r t s of plasmids pDt92R, pDtl20R and pDtT3 7777777 138 Fig u r e 33. The r e s t r i c t i o n map of plasmid pDt78R 145 Figure 34. The s t r a t e g y used to sequence the tRNA^-'- gene of pDt78R 147 Fig u r e 35. The n u c l e o t i d e sequences of two segments of Drosophila DNA c o n t a i n i n g tRNA^g 1 149 Figure 36. The n u c l e o t i d e sequences of segments of the Drosophila DNA i n s e r t s of plasmids pDt48 and pDt41R 153 x i . Acknowledgment s I wish to thank Gordon Tener and Ian G i l l a m f o r much encouragement, guidance and good advice during the course of t h i s work. I would a l s o l i k e to thank A l l e n Delaney, Shizu Hayashi, Dave C r i b b s , C a r o l i n e A s t e l l and Rob Dunn f o r many h e l p f u l d i s c u s s i o n s . DEDICATION TO MY PARENTS X1X1. Abbreviations Used A 2 6 0 - absorbance at 260 nm A 2 6 0 u n i t - the amount of m a t e r i a l g i v i n g an absorbance 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 at 260 nm at n e u t r a l pH acp 3U - 3-(3-amino-3-carboxypropyl)uridine bp - base-pair Cm - 2'-0-methylcytidine D - d i h y d r o u r i d i n e i 6 A - N 6-isopentenyladenosine kb - k i l o base-pair m5C - 5-methylcytidine m1 G - 1-methylguanosine m7G - 7-methylguanosine MOPS - morpholinopropane s u l f o n i c a c i d OAc - acetate P E I - c e l l u l o s e - polyethyleneimine impregnated c e l l u l o s e Pu - a purine nucleoside Py - a pyrimidine nucleoside Q - 7-(4,5-cis-dihydroxy-l-cyclopenten-3-ylaminomethyl)-7-deazaguanosine QAE-Sephadex - quaternary aminoethyl Sephadex RNase - ribonuclease RPC-5 - reverse phase chromatography system 5 rT - ri b o s y l t h y m i n e t 6 A - N-[(9-8-D-ribofuranosylpurin-6-yl)carbamoyl]threonine TLC - t h i n l a y e r chromatography Um - 2'-0-methyluridine V a l 3a - tRNA^I 1 tRNA^fj tRNAX a l a-(carboxyamino)-4,9-dihydro-4,6-dimethyl-9-oxo-lH-imidazo[1,2-a]-purine-7-butyric acid dimethyl ester 1. I n t r o d u c t i o n In 1955 C r i c k (Judson, 1979) postu l a t e d the existence of a c l a s s of "adaptor" molecules capable of ch e m i c a l l y combining w i t h an amino a c i d and of hydrogen bonding to s p e c i f i c sequences i n a template n u c l e i c a c i d . Adaptors w i t h such p r o p e r t i e s were proposed as intermediates r e q u i r e d f o r the t r a n s f e r of genetic i n f o r m a t i o n from the n u c l e o t i d e sequences of n u c l e i c a c i d s to the amino a c i d sequences of p r o t e i n s . Three years l a t e r Zamecnik and h i s coworkers, us i n g 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, (Hoagland et a l . , 1958) detected small RNA molecules w i t h some of the p r o p e r t i e s pre-d i c t e d f o r the adaptors. The study of the s t r u c t u r e and f u n c t i o n of t r a n s -f e r RNA, as the adaptors came to be c a l l e d , continues to be an a c t i v e f i e l d of research. In the s e c t i o n s that f o l l o w the s t r u c t u r e and f u n c t i o n of tRNA and tRNA genes are b r i e f l y d e s c r i b e d . I . S t r u c t u r e of the tRNA Molecule A. Primary S t r u c t u r e - RNA Sequencing A p r e r e q u i s i t e f o r understanding how tRNA f u n c t i o n s at the molecular l e v e l i s knowledge of i t s primary s t r u c t u r e . In 1965 Hol l e y published the A l a f i r s t n u c l e o t i d e sequence of any n u c l e i c a c i d , yeast tRNA ( H o l l e y et^ a l . , 1965). U l t r a v i o l e t spectrophotometry was used to i d e n t i f y each mononu-c l e o t i d e produced during sequence a n a l y s i s . The s e n s i t i v i t y of t h i s method of d e t e c t i o n i s low, and l a r g e amounts of p u r i f i e d tRNA (95 mg) were r e -quired to determine the n u c l e o t i d e sequence. High r e s o l u t i o n tRNA p u r i f i c a -t i o n procedures and. the use of tRNA l a b e l l e d irv v i v o or in. v i t r o w i t h the ra d i o i s o t o p e phosphorus-32 have made f e a s i b l e the r a p i d sequencing of very s m a l l amounts of tRNA. A commonly used method of tRNA sequence a n a l y s i s evolved from H o l l e y ' s o r i g i n a l s t r a t e g y (reviewed by S i l b e r k l a n g et_ a l _ . , 1979). F i r s t , the 2. nucleoside composition of the tRNA i s determined. Knowledge of the number and nature of the modified n u c l e o t i d e s present i n the tRNA i s provided by t h i s a n a l y s i s . These data are required f o r a l l methods of tRNA sequence determination. Next, the tRNA i s digested to completion w i t h RNase T^ (cleaves at G r e s i d u e s ) or RNase A (cleaves at C and U residues) and the 5'-hydroxyl groups of the r e s u l t i n g o l i g o n u c l e o t i d e s are phosphorylated w i t h [ 3 2P]phosphate. The r a d i o l a b e l l e d o l i g o n u c l e o t i d e s are separated 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 acetate i n the f i r s t dimension f o l l o w e d by chroma-tography on ion-exchange paper or TLC p l a t e s i n the second dimension. Each p u r i f i e d o l i g o n u c l e o t i d e i s then p a r t i a l l y digested w i t h snake venom phos-phodiesterase and subjected to "wandering spot" a n a l y s i s . In t h i s a n a l y s i s the phosphodiesterase d i g e s t undergoes e l e c t r o p h o r e s i s at pH 3.5 on c e l l u -l o s e acetate s t r i p s which separates the RNA fragments according to t h e i r charges. At t h i s pH the charge of an o l i g o n u c l e o t i d e r e f l e c t s i t s base composition. The RNA fragments are t r a n s f e r r e d from the c e l l u l o s e acetate s t r i p to DEAE-cellulose TLC p l a t e s and f r a c t i o n a t e d according t o s i z e by homochromatography, a process of displacement a n a l y s i s by an u n l a b e l l e d mixture of o l i g o n u c l e o t i d e s . A f t e r autoradiography of the chromatogram, the n u c l e o t i d e sequence of the o r i g i n a l o l i g o n u c l e o t i d e can be deduced from the p a t t e r n of m o b i l i t y s h i f t s d i s p l a y e d by i t s l a b e l l e d degradation products. Removal of an A, G, C or U residue from the 3'-end of an o l i g o n u c l e o t i d e r e s u l t s i n b a s e - s p e c i f i c s h i f t s i n the d i r e c t i o n and d i s t a n c e the remaining o l i g o n u c l e o t i d e migrates during "wandering-spot" a n a l y s i s . S i m i l a r sequenc-i n g of l a r g e o l i g o n u c l e o t i d e s produced by p a r t i a l RNase T^ or RNase A cleavage of the tRNA allows ordering of a l l the sequenced fragments i n t o a unique tRNA sequence. Recently, a new method of RNA sequencing has been developed that i s s i m i l a r , i n p r i n c i p l e , to the Maxam and G i l b e r t DNA sequencing procedure 3. ( D o n i s - K e l l e r et_ a l . , 1977; Simoncsits et_ a l _ . , 1977). Transfer RNA i s l a b e l l e d at the 5' or 3'-end w i t h 3 2 P . In separate r e a c t i o n s the tRNA i s p a r t i a l l y d igested w i t h b a s e - s p e c i f i c ribonucleases such as RNase T^ f o r cleavage at G r e s i d u e s , RNase f o r cleavage at A and G r e s i d u e s , RNase A f o r cleavage at C and U and RNase Phy I f o r cleavage at a l l bases except C. P a r t i a l h y d r o l y s i s w i t h a l k a l i or hot, aqueous formamide produces i n d i s c r i m -i n a t e cleavage of the RNA. The products of each r e a c t i o n are a p p l i e d to a s e r i e s of s l o t s i n a denaturing polyacrylamide g e l and f r a c t i o n a t e d accord-i n g to s i z e by e l e c t r o p h o r e s i s . Autoradiography of the g e l r e v e a l s the p o s i t i o n of each G, A, U and C residue i n the RNA. In p r a c t i c e , tRNA sequences d e r i v e d by the " g e l r e a d - o f f " method are o f t e n ambiguous. The most s e r i o u s d i f f i c u l t y i s poor d i s c r i m i n a t i o n between U and C residues by the nucleases. More s p e c i f i c ribonucleases from chicken l i v e r (Boguski et a l . , 1980), Staphylococcus aureus and Neurospora (Krupp and Gross, 1979) have been demonstrated to overcome t h i s problem i n t h e i r proponent's hands but are not yet widely used. Because the g e l read-off method does not i d e n t i f y the p o s i t i o n s of modified bases i t s u t i l i t y i n tRNA sequence a n a l y s i s i s l i m i t e d . F i n a l l y , the strong secondary s t r u c t u r e c h a r a c t e r i s t i c of tRNAs r e s u l t s i n poor enzymatic cleavage i n p a r t s of the molecule. This makes i n t e r p r e t a t i o n of the g e l s d i f f i c u l t . C o nditions f o r b a s e - s p e c i f i c chemical cleavage of RNA have been d e v e l -oped by P e a t t i e (1979). The d i s c r i m i n a t i o n between U and C residues i s reported to be e x c e l l e n t and secondary s t r u c t u r e does not a f f e c t the reac-t i v i t y of the RNA w i t h the sequencing reagents. In s p i t e of these advan-tages the chemical sequencing method i s not widely used at present. This may r e f l e c t d i f f i c u l t y i n reproducing the cleavage c o n d i t i o n s used by P e a t t i e i n the o r i g i n a l paper. Although the g e l read-off method cannot, by i t s e l f , be used to determine tRNA sequences i t can provide data supporting 4. sequences obtained by other methods. An RNA sequencing procedure developed by Stanley and Vassilenko (1979) has proved p a r t i c u l a r l y u s e f u l i n sequencing tRNAs. This method r e q u i r e s p u r i f i e d RNA that i s homogeneous i n l e n g t h and that has a phosphate group e s t e r i f i e d to i t s 5'-hydroxyl group. The RNA i s subjected to very l i m i t e d , random h y d r o l y s i s . The c o n d i t i o n s of h y d r o l y s i s ensure that the small number of molecules cleaved only once g r e a t l y exceeds the number that undergo m u l t i p l e cleavages. Cleavage of a p o l y n u c l e o t i d e chain at a s i n g l e s i t e r e s u l t s i n two fragments, only one of which has a f r e e 5'-hydroxy 1 group. These hydroxyl groups are l a b e l l e d w i t h [ 3 2P]phosphate and the l a b e l l e d fragments are f r a c t i o n a t e d by s i z e on a denaturing polyacrylamide g e l . The autoradiograph of the g e l r e v e a l s a "ladder" of bands. Each band i s e x c i s e d from the g e l , i t s RNA i s e l u t e d from the g e l fragment and then hydrolysed to mononucleotides. Only the 5' n u c l e o t i d e of each fragment i s r a d i o a c t i v e l y l a b e l l e d . This n u c l e o t i d e i s i d e n t i f i e d by t h i n l a y e r chro-matography i n s e v e r a l solvent systems. By i d e n t i f y i n g the n u c l e o t i d e at the 5'-end of a l l the fragments the sequence of the RNA can be determined. Each n u c l e o t i d e of the sequence can be t r e a t e d i n d i v i d u a l l y , thus modified n u c l e -o t i d e s can be chromatographed i n the solvent systems best s u i t e d f o r t h e i r i d e n t i f i c a t i o n . This f e a t u r e i s p a r t i c u l a r l y u s e f u l i n the sequencing of tRNA. Gupta and Randerath (1979) and Tanaka e_t al. (1980) have developed m o d i f i c a t i o n s of the Stanley and Vassilenko procedure. The ladder of RNA fragments on the polyacrylamide g e l i s p r i n t e d d i r e c t l y onto ion-exchange TLC p l a t e s . The RNA fragments are digested w i t h ribonuclease i_n s i t u and the p l a t e s are then developed i n an appropriate solvent system or the n u c l e o t i d e s are separated by e l e c t r o p h o r e s i s . The TLC p l a t e s are a u t o r a d i o -graphed and the n u c l e o t i d e sequence of the RNA can be determined. The modi-5. f i e d procedures have two advantages over the o r i g i n a l method. F i r s t , the manipulations r e q u i r e d i n e x c i s i n g and e l u t i n g the RNA fragments are e l i m i -nated. This r e s u l t s i n a great saving i n time and e f f o r t . Second, l e s s RNA i s r e q u ired t o determine a n u c l e o t i d e sequence. Much of the f l e x i b i l i t y inherent i n the Stanley and Vassilenko method i s , however, l o s t i n the more r a p i d procedures derived from i t . The advances made i n RNA sequencing methods i n the l a s t few years mean that a complete tRNA sequence can now be obtained using very small amounts of p u r i f i e d tRNA (10-20 ug). As a r e s u l t sequencing tRNAs from a wider v a r i e t y of organisms becomes p r a c t i c a b l e . Comparison of the primary s t r u c -ture of tRNAs from many sources w i l l undoubtedly deepen our understanding of tRNA s t r u c t u r e , f u n c t i o n and e v o l u t i o n . B. Secondary S t r u c t u r e A l a In t h e i r paper presenting the primary s t r u c t u r e of yeast tRNA H o l l e y and h i s coworkers (1965) described three p o s s i b l e secondary s t r u c -t ures f o r the RNA. Since 1965 about 175 tRNAs have been sequenced (Gauss and S p r i n z l , 1981). A l l these tRNAs can be f o l d e d i n t o a form s i m i l a r t o one of those proposed by H o l l e y , the " c l o v e r l e a f " form shown i n Figure 1 ( R i c h and RajBhandary, 1976). The most prominent fe a t u r e s of the s t r u c t u r e are the f o u r base-paired stem regions, three of which are close d by nonbase-paired loops. The acceptor stem contains the 5' and 3'-ends of the tRNA. I t c o n s i s t s of a 7 base-pair stem and 4 unpaired n u c l e o t i d e s . The 5'-hydroxyl group i s phosphorylated w h i l e the 3'-ends of a l l tRNAs have the same -CCA sequence. The d i h y r o u r i d i n e arm ("D-arm") has a 3 or 4 base-pair stem and a loop of v a r i a b l e s i z e , ranging from 7 to 11 n u c l e o t i d e s . Two regions of v a r i a b l e l e n g t h , a and 8, f l a n k the p a i r of G residues always found i n the D-loop. a and 8 c o n t a i n from 1 to 3 n u c l e o t i d e s each. These are u s u a l -6. o -i ?-?" s o -o-c I -?70 D LOOP 'R* -A • / . • is M-<b_o-Y-o' ACCEPTOR STEM T * C LOOP O — O — O - O - f i I , 5 0 t - * /3 I -o I -o I 45 VARIABLE LOOP ANTIC0D0N LOOP / Y I U \ o I H / - o — o 35 A N T I C O D O N Figure 1. The C l o v e r l e a f S t r u c t u r e of tRNA l y p y r i m i d i n e s , a high p r o p o r t i o n of which i s d i h y d r o u r i d i n e . The anticodon arm i s made up of a 5 base-pair stem and a 7 nuc l e o t i d e loop. The t h i r d r e g i o n of v a r y i n g l e n g t h i n tRNAs i s the v a r i a b l e loop. Two c l a s s e s of tRNAs can be d i s t i n g u i s h e d by the s i z e of t h e i r v a r i a b l e loops. Most tRNAs f a l l i n t o C l a s s 1, tRNAs w i t h 4 or 5 unpaired n u c l e o t i d e s i n t h i s r e g i o n . Tyr Leucine and ser i n e accepting tRNAs and p r o k a r y o t i c tRNAs J form Class 2. They have a long v a r i a b l e arm of 13-21 n u c l e o t i d e s which can form a stem and loop s t r u c t u r e . The TVC-arm ("T-arm") c o n s i s t s of a 5 base-pair stem and a 7 n u c l e o t i d e loop. When the c l o v e r l e a f s t r u c t u r e s of sequenced tRNAs are compared, i t becomes apparent that the nu c l e o t i d e s at c e r t a i n p o s i t i o n s i n the s t r u c t u r e are s t r o n g l y conserved. Some p o s i t i o n s are almost i n v a r i a b l y occupied by the same n u c l e o t i d e . These are: U8, A14, G18, G19, A21, U33, T54, Y55, C56, A58, C61, C74, C75 and A76 (Figure 1 ) . Other p o s i t i o n s are almost always occupied by a pyrimidine (Y) and other s i t e s by purines (R)« These are: Y l l , R15, R24, Y32, R37, Y48, R57 and Y60 (Figure 1 ) . Purine 37, 7. adjacent to the 3' end of the anticodon (H i n Figure 1) i s of t e n hyper-modified and probably p l a y s a r o l e i n the codon-anticodon i n t e r a c t i o n . Purine 15 and py r i m i d i n e 48 (R +, Y + i n Figure 1 ) , though widely sepa-rated i n the c l o v e r l e a f s t r u c t u r e , are u s u a l l y complementary. This sug-gested that they might be c l o s e enough to form a base-pair i n the t e r t i a r y Phe s t r u c t u r e of tRNA. X-ray d i f f r a c t i o n s t u d i e s of yeast tRNA have con-firmed t h i s p r e d i c t i o n and shown that 20 of the 23 s t r o n g l y conserved nucle-o t i d e s i n t h i s tRNA are involved i n maintaining the molecule's t e r t i a r y s t r u c t u r e ( R i c h and RajBhandary, 1976). The patterns of primary and secondary s t r u c t u r e described above are g e n e r a l i z a t i o n s drawn from many tRNA sequences, and any p a r t i c u l a r tRNA species may d i f f e r i n some respects from t h i s p a t t e r n . One c l a s s of tRNAs, the i n i t i a t o r methionine tRNAs, are d i s t i n c t l y d i f f e r e n t from other tRNAs. Met P r o k a r y o t i c tRNA^ l a c k s a base-pair at the 5'-end of the acceptor stem and has an A11*U24 base-pair i n the D-stem r a t h e r than the usual Y11*R24 p a i r . In eukaryotic tRNA\ T54¥55 i s replaced by A54U55 Met and Y60 i s replaced by A. In tRNA^ of higher eukaryotes the normally i n v a r i a n t U33 adjacent to the anticodon i s replaced by a C re s i d u e . C. T e r t i a r y S t r u c t u r e In 1968 tRNA was c r y s t a l l i z e d f o r the f i r s t time. This development meant the powerful technique of X-ray d i f f r a c t i o n a n a l y s i s could be used t o determine the three-dimensional s t r u c t u r e of tRNA. I t was s e v e r a l years before the h i g h l y ordered c r y s t a l s required f o r high r e s o l u t i o n X-ray c r y s t a l l o g r a p h y c o u l d be prepared. In 1975 two research groups published Phe the c r y s t a l s t r u c t u r e of yeast tRNA t o 2.5 A r e s o l u t i o n (reviewed by Kim, 1978; R i c h and RajBhandary, 1976). Phe Yeast tRNA i s a f l a t , L-shaped molecule (Figure 2 ) ( R i c h and RajBhandary, 1976). I t i s 20-25 A t h i c k and each arm of the "L" i s about 8. Acceptor Stem 1>C Stem Tij/C Stem Acceptor Stem \ L 3' Acceptor End Phe Figure 2. Two Views of the T e r t i a r y S t r u c t u r e of Yeast tRNA 70 A long. The 4 h e l i c a l stem regions p r e d i c t e d i n the c l o v e r l e a f s t r u c -ture of tRNA are indeed present i n the t e r t i a r y s t r u c t u r e . The acceptor stem i s stacked on the T-stem to form one limb of the "L". The other limb i s made up of the D-stem stacked onto the anticodon stem. In the l a t t e r h e l i x there i s a 25° d e v i a t i o n from l i n e a r i t y between the two component stem Phe regions. The double-stranded regions of tRNA approximate an RNA A h e l i x . In t h i s h e l i x the base-pairs are t i l t e d w i t h respect to the h e l i x a x i s and do not i n t e r s e c t w i t h the a x i s . This r e s u l t s i n a 6 A hole running through the center of the h e l i x . The A h e l i x has a very deep major groove and a very shallow minor groove. There are 11 base-pairs per h e l i c a l t u r n . The T, D and v a r i a b l e loops are a l l c l u s t e r e d together where the two arms of the "L" i n t e r s e c t . There, a complex array of hydrogen bonds p l a y s an important r o l e i n maintaining the molecule's t e r t i a r y s t r u c t u r e . The anticodon loop i s found at one end of the molecule. The conformation of t h i s loop ensures that the anticodon i s r e a d i l y a c c e s s i b l e f o r hydrogen 9. bonding to the codon on the ribosome. At the other end of the molecule the -CCA sequence, the s i t e of aminoacylation, extends out i n t o the solvent i n a co n t i n u a t i o n of the acceptor stem h e l i x . Phe What fo r c e s hold the i n t r i c a t e tRNA s t r u c t u r e together? Of great importance are t e r t i a r y hydrogen-bonds formed between the bases. These bonds are shown i n Figure 3 (Kim, 1978). 5'<nd A OH s'-end 73 C C A pG. • C C • G G voC a a s , e m G ° U As • U U . A T-orm ~\V • A (C)M 6 ® I L A , - ^ G ^ D-arm (Cm) J GmA5A T-arm a a -stem ^ I g l A C A G — AAUUC GCACCAoh iGr< ^ ^ © U G Um'C UUAGGCGp ICL D-arm (j . m»C ac-arm (Cm) " A GmA A ,Phe At l e f t the c l o v e r -Figure 3. T e r t i a r y Hydrogen-Bonds of Yeast tRNA~ Phe l e a f form of tRNA , at r i g h t a drawing that more c l o s e l y resembles the t e r t i a r y s t r u c t u r e of tRNA Phe Stron g l y conserved n u c l e o t i d e s are i n v o l v e d i n almost a l l of these bonds. Phe This suggests that i n t e r a c t i o n s s i m i l a r to those seen i n yeast tRNA are present i n other tRNAs. Hydrogen bonds form a network h o l d i n g the two arms of the tRNA i n the c o r r e c t o r i e n t a t i o n to one another. A l l the base-pairs i n the major groove of the D-stem are i n v o l v e d i n t e r t i a r y hydrogen bonds w i t h the v a r i a b l e loop. The conserved GG doublet i n the D-loop i s bonded to the ¥C sequence i n the T-loop. U r i d i n e 8 and A9, lo c a t e d between the acceptor stem and the D stem, are hydrogen bonded to A14 and A23 r e s p e c t i v e -l y . These bonds may help s t a b i l i z e the sharp bend i n the RNA chain i n the 10. U8-U12 re g i o n . With the exception of the G19-C56 bond none of the t e r t i a r y hydrogen bonds are of the Watson-Crick type. A wide v a r i e t y of bonds are observed i n c l u d i n g reverse Watson-Crick bonds (G15-C48) and reverse Hoogsteen bonds (U8-A14; 1 5 4 - 1 1 1 ^ 5 8 ) . Other important i n t e r a c t i o n s i n v o l v e hydrogen bonds between bases and the 2'-hydroxyl group of r i b o s e or the oxygen of phosphate r e s i d u e s . These bonds probably p l a y an important part i n maintaining tRNA s t r u c t u r e but are d i f f i c u l t to detect w i t h c e r t a i n t y by X-ray c r y s t a l l o g r a p h y . Sharp bends i n the p o l y n u c l e o t i d e c h a i n appear to be s t a b i l i z e d by these types of hydrogen bonds. For example, A21 i s bonded to the 2'-hydroxyl of U8 at the bend i n the chain between the acceptor and D-stems. S i m i l a r l y , the sharp bends i n the T-loop and anticodon loop are probably s t a b i l i z e d by bonds between V55 and phosphate 58 i n the former and between U33 and phosphate 36 i n the l a t t e r . Phe F i v e t i g h t l y bound d i v a l e n t metal ions have been l o c a t e d i n tRNA ( R i c h et a l . , 1980). A l l these ions are o c t a h e d r a l l y coordinated by water molecules and phosphate groups. One of the Mg ions binds at the 5'-end of the tRNA and the others are found at sharp bends i n the RNA where seg-ments of the n e g a t i v e l y charged p o l y n u c l e o t i d e backbone come i n t o c l o s e Phe contact w i t h one another. Highly ordered c r y s t a l s of tRNA can only be formed i f spermine i s present. Two molecules of spermine are ass o c i a t e d w i t h each tRNA i n these c r y s t a l s . One i s found near the v a r i a b l e loop and l i k e l y s t a b i l i z e s the bend i n the U8-U12 re g i o n of the molecule. The other binds to the major groove of the RNA h e l i x at the top of the anticodon stem. A major c o n t r i b u t i o n to the s t a b i l i t y of tRNA s t r u c t u r e i s made by the extensive base-stacking present i n the molecule. A l l but 5 bases i n t R N A P h e are i n v o l v e d i n base-stacking (Holbrook et a l . , 1978). One column of s t a c k i n g i n t e r a c t i o n s i n v o l v e s the acceptor stem, T-stem and T-loop. A 11. second column, roughly perpendicular to the f i r s t , i n v o l v e s the D-loop and stem and the anticodon arm. Phe Since the s t r u c t u r e of yeast tRNA was published, the t e r t i a r y s t r u c t u r e s of s e v e r a l other tRNAs have been determined at varying degrees of Met Asp r e s o l u t i o n (E. c o l i tRNA f , Woo et a l . , 1980; yeast tRNA , Moras et a l . , 1980; yeast tRNA^ e t, Shevitz e_t £l., 1980). The s t r u c t u r e of Phe yeast tRNA appears t o be t y p i c a l of at l e a s t those tRNAs w i t h a small v a r i a b l e loop. This i s not s u r p r i s i n g s i n c e the major determinants of tRNA s t r u c t u r e , the c l o v e r l e a f p a t t e r n of base-pairing and the s t r o n g l y conserved n u c l e o t i d e s , are common to almost a l l tRNAs. The s t r u c t u r e of tRNA i n s o l u t i o n i s very s i m i l a r to i t s s t r u c t u r e i n a c r y s t a l l a t t i c e . A l a r g e body of data gathered by a wide v a r i e t y of t e c h -niques i n c l u d i n g o l i g o n u c l e o t i d e b i n d i n g , t r i t i u m exchange, b a s e - s p e c i f i c chemical m o d i f i c a t i o n and NMR spectroscopy supports t h i s c o n c l u s i o n (reviewed by Kim, 1978). D. M i t o c h o n d r i a l tRNAs M i t o c h o n d r i a l tRNAs (mt tRNA) are exceptions to the general p a t t e r n of secondary s t r u c t u r e seen i n other tRNAs. The m i t o c h o n d r i a l genome contains genes f o r a set of mt tRNAs required f o r p r o t e i n synthesis w i t h i n the orga-n e l l e . In lower eukaryotes the m i t o c h o n d r i a l genome i s r e l a t i v e l y l a r g e (about 80 kb i n y e a s t ) . Most of the tRNAs produced i n these mitochondria are, judging from the sequence of t h e i r genes, s i m i l a r i n s t r u c t u r e to c y t o -plasmic tRNAs (ye a s t , Bonitz and T z a g o l o f f , 1980; A s p e r g i l l u s n i d u l a n s , Kochel et a l . , 1981). A l l these tRNAs can be fo l d e d i n t o a standard c l o v e r -l e a f s t r u c t u r e but some l a c k a few of the i n v a r i a n t n u c l e o t i d e s found i n cytoplasmic tRNAs. The m i t o c h o n d r i a l genomes of higher eukaryotes are about o n e - f i f t h the s i z e of that of yeast (Borst and G r i v e l l , 1981). Genes found i n these small 12. genomes code f o r tRNAs that d i f f e r g r e a t l y from t h e i r cytoplasmic counter-p a r t s (human, Anderson et a l . , 1981; mouse, Van E t t e n et^ a l . , 1980). Most of the d i f f e r e n c e s l i e i n the D and T-loops. The D-loop ranges from 3 to 10 n u c l e o t i d e s i n l e n g t h and the T-loop can be from 3 to 9 n u l c e o t i d e s long. Some or a l l of the normally i n v a r i a n t n u c l e o t i d e s found i n these loops are m i s s i n g . The most extreme example of the d i f f e r e n c e s between m i t o c h o n d r i a l Ser and cytoplasmic tRNAs i s mt tRNA^y (de B r u i j n et^ a l . , 1980; A r c a r i and Brownlee, 1980). This tRNA completely l a c k s a D-arm. In c o n t r a s t , the mammalian mt t R N A L e u gene codes f o r a tRNA w i t h a l l the s t r u c t u r a l f e a -tures common to cytoplasmic tRNAs (Van E t t e n et_ a l . , 1981). Homologous m i t o c h o n d r i a l tRNAs from d i f f e r e n t mammalian species show great sequence Thr divergence. For example, human and bovine mt tRNA are only 74% homo-logous. Homology between equivalent cytoplasmic tRNAs from d i f f e r e n t mam-malian species i s u s u a l l y complete. E v i d e n t l y , mammalian m i t o c h o n d r i a l tRNA genes are e v o l v i n g much more r a p i d l y than t h e i r cytoplasmic counterparts (Borst and G r i v e l l , 1981). E. M o d i f i e d Nucleotides Found i n tRNAs The l a r g e number and wide v a r i e t y of unusual n u c l e o t i d e s found i n tRNAs are a c h a r a c t e r i s t i c f e a t u r e of these n u c l e i c a c i d s (reviewed by Nishimura, 1978). Over 50 d i f f e r e n t modified bases have been i s o l a t e d from tRNA. Most of these r e s u l t from methylation of the base or r i b o s e m o i e t i e s of nucleo-t i d e s or from replacement of oxygen atoms i n the bases by s u l f u r . More extensive a l t e r a t i o n s i n n u c l e o t i d e s t r u c t u r e produce the hypermodifled bases. Most of the modified n u c l e o t i d e s occur only at one or a few charac-t e r i s t i c p o s i t i o n s i n the tRNA s t r u c t u r e . Often the same modified n u c l e o t i d e or i t s d e r i v a t i v e s occupy the same s i t e i n homologous tRNAs from a wide v a r i e t y of organisms. This suggests the modified bases of tRNA play important r o l e s i n tRNA s t r u c t u r e and f u n c t i o n . 13. Modified n u c l e o t i d e s i n the f i r s t , or "wobble", p o s i t i o n of the a n t i -codon (when w r i t t e n i n the conventional 5' to 3' d i r e c t i o n ) are d i r e c t l y i n v o l v e d i n the codon-anticodon i n t e r a c t i o n . Unmodified A or U residues are almost never found i n the "wobble" p o s i t i o n , adenosine commonly being modi-f i e d to i n o s i n e ( I ) . In ribosome-binding experiments I i n t h i s p o s i t i o n i s capable of p a i r i n g ("wobbling") w i t h A, C or U i n the t h i r d p o s i t i o n (3'-end) of the codon. U r i d i n e i n the "wobble" p o s i t i o n i s o f t e n modified t o 2 - t h i o u r i d i n e or i t s d e r i v a t i v e s . These n u c l e o t i d e s w i l l p a i r only w i t h A i n the t h i r d p o s i t i o n of the codon. In E s c h e r i c h i a c o l i tRNA a U i n the "wobble" p o s i t i o n i s sometimes modified to u r i d i n - 5 - o x y a c e t i c a c i d a l l o w i n g i t to base-pair w i t h A, G or U i n the codon. The hypermodified base Q (derived from G) or i t s g l y c o s y l a t e d d e r i v a t i v e s are found i n the f i r s t p o s i t i o n of the anticodon of some tRNAs. This base w i l l p a i r w i t h e i t h e r U or C but has g r e a t e r a f f i n i t y f o r U. The t h i r d p o s i t i o n (3'-end) of the anticodon base-pairs w i t h the f i r s t p o s i t i o n (5'-end) of the codon during t r a n s l a t i o n . This i n t e r a c t i o n must be very s p e c i f i c i f e r r o r s i n p r o t e i n s y n t h e s i s are to be avoided. I f a tRNA has an A residue i n the t h i r d p o s i t i o n of the anticodon the A i s almost i n v a r i a b l y f l a n k e d on the 3' side by a hydrophobic base such as ] f - i s o -pentenyladenine, Y base, or t h e i r d e r i v a t i v e s . I f a tRNA has a U residue i n the t h i r d p o s i t i o n of the anticodon the h y d r o p h i l i c nucleoside t 6 A or i t s d e r i v a t i v e s are found immediately 3' to the anticodon. The f u n c t i o n of these hypermodified bases may be to s t a b i l i z e the A-U base-pair between the f i r s t p o s i t i o n of the codon and the t h i r d p o s i t i o n of the anticodon. G or C residues at the t h i r d p o s i t i o n of the anticodon are flanked by simple methy-l a t e d purines or by unmethylated A. Other modified bases are found at s p e c i f i c s i t e s i n the tRNA molecule. Some of these are n e a r l y u n i v e r s a l such as rT and V i n the T-loop and 14. d i h y d r o u r i d i n e i n the D-loop of most tRNAs. Some, such as m7G, are found only i n tRNAs s p e c i f i c f o r c e r t a i n amino a c i d s . Yet others are found only i n the tRNAs of some organisms. For example, 4 - t h i o u r i d i n e i s found only i n p r o k a r y o t i c tRNA w h i l e 5-methylcytosine i s found only i n that of eukary-otes. The f u n c t i o n of modified n u c l e o t i d e s such as these i s poorly under-stood. I I . Functions of Transfer RNA A. P r o t e i n Synthesis The most fundamental f u n c t i o n of tRNA i s i t s r o l e i n p r o t e i n synthe-s i s . Aminoacyl-tRNA i s a c r u c i a l intermediate i n the t r a n s f e r of informa-t i o n from the n u c l e o t i d e sequence of a mRNA to the amino a c i d sequence of a p r o t e i n . The formation of aminoacyl-tRNA i s c a t a l y z e d by the aminoacyl-tRNA synthetases (reviewed by I g l o i and Cramer, 1978; Cramer e_t a l . , 1980). These enzymes (ENZ) c a t a l y z e a two step r e a c t i o n . In the f i r s t step the amino a c i d (AA) i s a c t i v a t e d by forming a mixed anhydride w i t h AMP: ENZ + AA + ATP ^ ENZ*AA-AMP + P P i In the second step the amino a c i d i s e s t e r i f i e d to the f r e e 2' or 3'-hydroxyl groups of the cognate tRNA: ENZ* AA-AMP + tRNA AA-tRNA + AMP + ENZ To ensure that p r o t e i n s y n t h e s i s i s accurate an aminoacyl-tRNA synthetase must d i s p l a y high s p e c i f i c i t y f o r i t s s u b s t r a t e s . Because s t r u c t u r a l d i f -ferences among some amino acid s are so s l i g h t substrate binding alone cannot e x p l a i n the accuracy d i s p l a y e d by the synthetases. "Proofreading" of some k i n d i s needed to prevent m i s a c y l a t i o n of tRNA. A number of proof- reading mechanisms have been proposed. In one scheme, p r o f f e r e d by Fersht (1980), a l l amino a c i d s l a r g e r than the c o r r e c t one are simply excluded from the synthetase's aminoacylation s i t e because of t h e i r s i z e . Amino a c i d s smaller or the same s i z e as the c o r r e c t substrate (e.g. v a l i n e i n s t e a d of i s o l e u c i n e ) may be t r a n f e r e d to the tRNA. These misacylated tRNAs are substrates f o r a second, h y d r o l y t i c , s i t e . The c o r r e c t aminoacyl-tRNA i s excluded from the h y d r o l y t i c s i t e because of i t s s i z e or chemical p r o p e r t i e s and i s released from the enzyme. Other mechanisms have been proposed which d i f f e r i n d e t a i l from that of Fersht but share the same two-step d i s c r i m i n a -t i o n process ( I g l o i and Cramer, 1978). The f e a t u r e s of tRNA recognized by the aminoacyl-tRNA synthetases are poorly known. Chemical m o d i f i c a t i o n studies i n d i c a t e the acceptor stem, D-stem, anticodon loop and v a r i a b l e loop are a l l i n v o l v e d i n tRNA r e c o g n i -Phe t i o n by the synthetases. In the c r y s t a l s t r u c t u r e of tRNA most of these regions l i e on the si d e of the tRNA enclosed by the two arms of the L-shaped molecule. The complex process of p r o t e i n s y n t h e s i s i n eukaryotes i s b r i e f l y out-l i n e d below (reviewed by Weissbach and Ochoa, 1976; Revel, 1977; Benne and Hershey, 1978). 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 r e q u i r e s the formation of a Met ternary complex between the charged i n i t i a t o r tRNA, methionyl-tRNA^ , GTP and an i n i t i a t i o n f a c t o r eIF-2. This complex binds to the 40S ribosomal subunit w i t h the p a r t i c i p a t i o n of f a c t o r s eIF-3 and eIF-4C. Other i n i t i a -t i o n f a c t o r s (eIF4A, eIF4B, e l F l ) promote the binding of mRNA to the 40S subunit. ATP h y d r o l y s i s occurs a t t h i s s t e p . The f a c t o r eIF-5 i s required f o r the j o i n i n g of the 60S ribosomal subunit to the 40S subunit. Concomi-t a n t l y GTP i s hydrolyzed and the i n i t i a t i o n f a c t o r s are released from the Met i n i t i a t i o n complex. In t h i s i n i t i a t i o n complex Met- tRNA^ i s i n the ribosome's p e p t i d y l (P) s i t e (Revel, 1977). During e l o n g a t i o n a te r n a r y complex of aminoacyl-tRNA, an e l o n g a t i o n f a c t o r (EF-1) and GTP binds to the ribosome. H y d r o l y s i s of GTP releases an EF-1*GDP complex from the ribosome and leaves the aminoacyl-tRNA s p e c i f i e d by the codon being t r a n s l a t e d at the ribosome's aminoacyl (A) s i t e . The p e p t i d y l t r a n s f e r a s e a c t i v i t y of the ribosome c a t a l y z e s the t r a n s f e r of the p e p t i d y l (or methionyl) group from the tRNA at the P s i t e to the f r e e amino group of aminoacyl tRNA at the A s i t e . In a concerted s e r i e s of r e a c t i o n s deacylated tRNA i s removed from the P s i t e , the newly formed peptidyl-tRNA i s t r a n s -f e r r e d from the A to the P s i t e and the ribosome moves to the next codon. T r a n s l o c a t i o n i s accompanied by GTP h y d r o l y s i s and r e q u i r e s the e l o n g a t i o n f a c t o r EF-2. At t h i s point the next c y c l e of e l o n g a t i o n can begin. When a termi n a t i o n codon occupies the A s i t e a r e l e a s e f a c t o r (RF). binds to the ribosome i n a GTP dependent r e a c t i o n . P e p t i d y l t r a n s f e r a s e then hydrolyses the peptidyl-tRNA bond, r e l e a s i n g the p r o t e i n . GTP h y d r o l y s i s occurs and the RF i s released from the ribosome. D i s s o c i a t i o n of the ribosome i n t o i t s component subunits can then occur, p o s s i b l y c a t a l y z e d by the i n i t i a t i o n f a c t o r eIF-3. B. R e g u l a t i o n of T r a n s c r i p t i o n Aminoacyl-tRNA play s a r o l e i n the r e g u l a t i o n of s e v e r a l b a c t e r i a l operons. A l a r g e body of evidence supports a model f o r the r e g u l a t i o n of these operons proposed by Lee and Yanofsky (1977). As an example, the regu-l a t i o n of the t r p operon of E. c o l i w i l l be b r i e f l y described i n terms of t h i s model (Yanofsky, 1981; P i a t t , 1981). The t r p operon c o n s i s t s of a r e g u l a t o r y sequence and 5 genes coding f o r enzymes i n the pathway from chorismate to tryptophan. T r a n s c r i p t i o n of the operon i s c o n t r o l l e d by an operator-repressor system and by a t t e n u a t i o n . Only the l a t t e r c o n t r o l mechanism w i l l be described here. The leader sequence of t r p operon mRNA contains a ribosome binding s i t e , coding sequence f o r a short peptide con-t a i n i n g two adjacent tryptophan residues and a t r a n s c r i p t i o n t e r m i n a t i o n s i g n a l (the a t t e n u a t o r ) . The terminator c o n s i s t s of a G-C r i c h region of dyad symmetry followed immediately by a t r a c t of u r i d i n e r e s i d u e s . Termina-17. t i o n at t h i s s i t e i s prevented i f the G-C r i c h region cannot form a h a i r -p i n . The leader r e g i o n of the t r a n s c r i p t can form a number of mutually e x c l u s i v e secondary s t r u c t u r e s (Figure 4 ) ( P l a t t , 1981). Region 1 contains i ' No Excess Trp T rp -s lo rved t rans la t ion Te rm ina t ion No te rm ina t i on Terminat ion j Figure 4. Model f o r A t t e n u a t i o n i n the E. c o l i t r p Operon the end of the peptide coding sequence, i n c l u d i n g the two tryptophan codons. Region 2 i s complementary to region 1 but part of i t can base-pair w i t h r e g i o n 3. Regions 3 and 4 can form the e s s e n t i a l stem and loop s t r u c t u r e of the t e r m i n a t i o n s i g n a l . In Yanofsky's model t r a n s l a t i o n of the leader pep-t i d e i s t i g h t l y coupled to t r a n s c r i p t i o n of the operon. I f the tryp t o p h a n y l -tRNA co n c e n t r a t i o n i n the c e l l i s low, the ribosome s t a l l s when i t reaches the tandem tryptophan codons. The ribosome covers region 1 of the t r a n -s c r i p t . Region 2, t h e r e f o r e , base-pairs to region 3 as soon as the l a t t e r i s t r a n s c r i b e d . This prevents formation of the h a i r - p i n required f o r t e r m i -n a t i o n and t r a n s c r i p t i o n of the s t r u c t u r a l genes of the operon can occur. I f , however, tryptophanyl-tRNA i s abundant i n the c e l l the ribosome reads through the tryptophan codons and s t a l l s at the peptide's t e r m i n a t i o n codon. At t h i s s i t e the ribosome covers regions 1 and 2 l e a v i n g region 3 and 4 f r e e to form a h a i r - p i n when they are t r a n s c r i b e d . Termination of t r a n s c r i p t i o n occurs a t the attenuator and the s t r u c t u r a l genes of the operon cannot be expressed. C. Aminoacyl Group Transfer In a d d i t i o n to i t s u n i v e r s a l p a r t i c i p a t i o n i n p r o t e i n synthesis 18. aminoacyl-tRNA serves as a donor of the aminoacyl group i n a few biosy n -t h e t i c r e a c t i o n s . L y s y l p h o s p h a t i d y l g l y c e r o l i s one of the major l i p i d s found i n Staphylococcus aureus. This l i p i d i s synthesized by t r a n s f e r of l y s i n e from l y s y l - t R N A ^ S to the 3'-hydroxyl group of p h o s p h a t i d y l g l y c e r o l . A l a n y l -p h o s p h a t i d y l g l y c e r o l , a l i p i d found i n C l o s t r i d i u m w e l c h i i , i s made i n an analogous manner ( S o f f e r , 1974). A l l eukaryotes assayed t o date c o n t a i n an enzyme that can t r a n s f e r a r g i n i n e from a r g i n y l - t R N A ^ r ^ to the t e r m i n a l amino group of acceptor p r o t e i n s ( S o f f e r , 1980). To serve as an acceptor a p r o t e i n must have an N-terminal a s p a r t a t e , glutamate or c y s t e i n e r e s i d u e . A s i m i l a r enzyme a c t i v i t y i n Gram-negative b a c t e r i a t r a n s f e r s l e u c i n e , phenylalanine or methionine from t h e i r tRNAs to p r o t e i n s w i t h N-terminal a r g i n y l , l y s y l or h i s t i d y l r e s i d u e s . The p h y s i o l o g i c a l f u n c t i o n of these enzymes i s not understood. The r i g i d c e l l w a l l s of b a c t e r i a are made of murein. In t h i s complex substance strands of peptidoglycan are c r o s s l i n k e d by short peptides. The le n g t h and sequence of these l i n k e r peptides v a r i e s from species to species but they are u s u a l l y made by the s e q u e n t i a l t r a n s f e r of amino a c i d s from aminoacyl-tRNA to the N-terminus of the growing l i n k e r ( S o f f e r et a l . , 1974). In Staphylococcus epidermidis the l i n k e r peptide contains s e r i n e and Ser g l y c i n e r e s i d u e s . This organism contains 1 tRNA iso a c c e p t o r and 2 Gly tRNA J i s o a c c e p t o r s that cannot f u n c t i o n i n p r o t e i n s y n t h e s i s . These tRNAs do, however, p a r t i c i p a t e i n c e l l w a l l s y n t h e s i s . Roberts (1974) has determined the n u c l e o t i d e sequence of the g l y c i n e i s o a c c e p t o r s . In the c l o v e r l e a f form these tRNAs have a 6 base-pair anticodon stem while the anticodon loop i s reduced to 5 n u c l e o t i d e s . The GWC sequence found i n almost a l l tRNAs i s replaced by GUGC and the GG sequence u s u a l l y present i n the D-loop i s replaced by UU. A C residue s u b s t i t u t e s f o r the purine a d j a -cent to the anticodon i n other tRNAs. These changes may produce tRNAs w i t h t e r t i a r y s t r u c t u r e s optimized f o r t h e i r f u n c t i o n i n c e l l w a l l s y n t h e s i s and that preclude t h e i r p a r t i c i p a t i o n i n p r o t e i n s y n t h e s i s . D. tRNAs and tRNA-Like St r u c t u r e s Associated w i t h V i r u s e s T r a n s f e r RNA i s i n t i m a t e l y a s s o c i a t e d w i t h animal r e t r o v i r u s r e p l i c a -t i o n . During r e t r o v i r u s i n f e c t i o n a v i r a l reverse t r a n s c r i p t a s e produces a DNA copy of the RNA v i r a l genome. L i k e other DNA polymerases, reverse t r a n s c r i p t a s e r e q u i r e s a primer. In these v i r u s e s a s p e c i f i c tRNA, of c e l l u l a r o r i g i n , serves t h i s f u n c t i o n (reviewed by Dahlberg, 1980). In the avian r e t r o v i r u s e s s t u d i e d , the primer i s a tRNA^ r^ species w h i l e i n the murine v i r u s e s tRNA^ r°, or i n some cases t R N A ^ S , i s used. Only the 3' t e r m i n a l 16-18 n u c l e o t i d e s of the tRNA h y b r i d i z e to the v i r a l RNA at the priming s i t e near the 5'-end of the v i r a l genome. Each r e t r o v i r u s v i r i o n c ontains two copies of the RNA genome, to which the priming tRNAs are alr e a d y h y b r i d i z e d , 80-100 molecules of reverse t r a n s c r i p t a s e , and 80-100 a d d i t i o n a l tRNA molecules. These tRNAs are a subset of t o t a l host c e l l tRNA. They are probably s e l e c t e d f o r i n c l u s i o n i n the v i r i o n by a s s o c i a t i o n w i t h reverse t r a n s c r i p t a s e molecules. The 3'-ends of many pl a n t v i r u s e s c o n t a i n remarkable s t r u c t u r e s that can be aminoacylated in_ v i t r o (reviewed by Haenni and C h a p e v i l l e , 1980). V i r u s e s c l a s s i f i e d i n the same group u s u a l l y accept the same amino a c i d . For example, t u r n i p y e l l o w mosaic v i r u s and other tymoviruses accept v a l i n e w h i l e bromoviruses, t y p i f i e d by brome mosaic v i r u s (BMV), accept t y r o s i n e . In a d d i t i o n to aminoacyl-tRNA synthetases the v i r u s e s are substrates f o r other enzymes, such as RNase P, tRNA n u c l e o t i d y l t r a n s f e r a s e and some nucle-o t i d e modifying enzymes, th a t normally act upon tRNA s u b s t r a t e s . The n u c l e -o t i d e sequences at the 3'-ends of s e v e r a l of these v i r u s e s are known. They 20. show l i t t l e s t r u c t u r a l resemblance to the i s o a c c e p t i n g tRNA of the host c e l l . For example, BMV contains regions homologous to the acceptor stem and anticodon arm of host c e l l t R N A ^ r but i n the v i r a l RNA these homologies are separated by only 5 n u c l e o t i d e s . Further s t u d i e s of the 3'-ends of pl a n t v i r u s e s may y i e l d new i n s i g h t s i n t o features of tRNA s t r u c t u r e recog-n i z e d by aminoacyl-tRNA synthetases and other enzymes. I I I . Drosophila melanogaster tRNA The w e l l developed s t a t e of Drosophila genetics makes t h i s organism a t t r a c t i v e as a system i n which to study tRNA s t r u c t u r e and f u n c t i o n . The n u c l e o t i d e sequences of 5 Drosophila tRNAs have been published, tRNA^ l u, t R N A f 8 , tRNA^ y S, t R N A M e t and t M M f 6 (Gauss and S p r i n z l , 1981). Though the number of known sequences i s small a few conclusions can be drawn at t h i s p r e l i m i n a r y stage. As expected Drosophila tRNAs can be drawn i n the standard c l o v e r l e a f form and c o n t a i n the s t r o n g l y conserved n u c l e o t i d e s present i n most tRNAs. Drosophila Phe tRNA2 , l i k e that of Bombyx mori, i s unusual among eukaryotic tRNAs . I t has ml G adjacent to the anticodon r a t h e r than the hyper-modified Y base. Homology between Drosophila tRNAs and the corresponding tRNAs of ve r t e b r a t e s i s great. This i n d i c a t e s that genes f o r cytoplasmic tRNAs have evolved very s l o w l y i n the higher eukaryotes. Drosophila goes through s e v e r a l d i s t i n c t stages i n i t s l i f e c y c l e : embryo (egg), f i r s t , second and t h i r d i n s t a r l a r v a , pupa and a d u l t . The tRNA species present i n f i r s t and t h i r d i n s t a r l a r v a e and i n a d u l t f l i e s were r e s o l v e d by RPC-5 chromatography. (White et a l . , 1973a). A t o t a l of 99 tRNA species were detected; of these 63 were major i s o a c c e p t o r s • The p a t t e r n of major i s o a c c e p t o r s f o r c y s t e i n e , glutamine, methionine, s e r i n e and threonine was found to change during the course of development. The 21. i s o a c c e p t o r patterns f o r asparaglne, a s p a r t i c a c i d , h i s t i d i n e and t y r o s i n e show changes i n tRNA m o d i f i c a t i o n during development (White et^ a l . , 1973b). The "&" forms of these tRNAs c o n t a i n a d e r i v a t i v e of base Q at the f i r s t p o s i t i o n of the anticodon. The "V" forms c o n t a i n G at t h i s p o s i t i o n . During l a r v a l growth the amount of Q-containing i s o a c c e p t o r s i n a l a r v a decreases. In an a d u l t f l y , however, the p r o p o r t i o n of each tRNA species i n the i> form i s g r e a t l y increased. The p a t t e r n s of tRNA is o a c c e p t o r s present i n young and o l d f l i e s were i n v e s t i g a t e d by Hosbach and K u b l i (1979 a,b). Transfer RNA i s o l a t e d from o l d males (35 days) could not be aminoacylated to the same extent as the tRNA of young males (5 days). Most of the amino a c i d s t e s t e d showed a drop i n charging of 10-25% but l e u c i n e acceptance was reduced by 50%. The a c t i v i t y of the aminoacyl-tRNA synthetases f o r some amino acid s d i d not change w i t h age w h i l e the synthetases f o r a l a n i n e , l e u c i n e , s e r i n e and a r g i n i n e were reduced by 50% i n the o l d f l i e s . The i s o a c c e p t o r p a t t e r n s f o r a number of amino a c i d s were determined i n o l d and young f l i e s . Only tRNAs c o n t a i n i n g Q base show a change i n p a t t e r n w i t h age. Those i s o a c c e p t o r s c o n t a i n i n g Q ($ forms) increase w i t h age w h i l e the ^ forms decrease. IV. Transfer RNA Genes A. E_. c o l i E_. c o l i contains about 60 tRNA genes (Brenner et a l . , 1970). Genetic and biochemical evidence suggested that most tRNA genes were t i g h t l y c l u s -t e r e d at s e v e r a l s i t e s on the chromosome (Smith, 1976; Ikemura and Ozeki, 1977). These f i n d i n g s have been confirmed by DNA sequence a n a l y s i s of tRNA operons. The 7 rRNA operons of _E. c o l i a l l c o n t a i n tRNA genes (Lund et_ a l . , 1976; Morgan et a l . , 1980). W i t h i n these operons rRNA genes are arranged i n 22. the order 16S rRNA-spacer-23S rRNA-spacer-5S rRNA (Young et^ a l _ . , 1979a). Tra n s f e r RNA genes are found i n two regions of the operon, i n the spacer separating the 16S and 23S rRNA genes and d i s t a l to the 5S gene. Three of H e the operons have i d e n t i c a l 16S-23S spacers each c o n t a i n i n g a tRNA and a A l a tRNA^g gene. The spacer i n the other operons i s s h o r t e r and contains a tRNA^"" gene. The two types of spacer share extensive regions of sequence homology which may be important f o r accurate processing of the operons' primary t r a n s c r i p t s . Four rRNA operons c o n t a i n tRNA genes d i s t a l to the 5S gene. Two have a s i n g l e tRNA^ s^ gene i n t h i s r e g i o n , i n Asp Trp another the tRNA^ y i s t i g h t l y l i n k e d t o a tRNA v gene w h i l e i n the Thr f o u r t h a tRNA^ gene i s found i n the spacer between two 5S genes (Young, 1979b; Sekiya et a l . , 1980; Duester and Holmes, 1980). Those tRNA genes not as s o c i a t e d w i t h rRNA operons are found i n c l u s -t e r s , each c l u s t e r forming a s i n g l e t r a n s c r i p t i o n u n i t . Each c l u s t e r may c o n t a i n m u l t i p l e copies of a s i n g l e tRNA species (e.g. Duester et a l . , 1981) or genes f o r s e v e r a l d i f f e r e n t tRNAs (e.g. Nakajima et^ a_l., 1981). W i t h i n a c l u s t e r the spacers between genes vary g r e a t l y i n le n g t h (9-200 bp) but are t y p i c a l l y 20-40 bp l o n g . With few exceptions (Schedl e_t _ a l . , 1974) E_. c o l i tRNA genes code f o r the t e r m i n a l -CCA sequence common to a l l tRNAs. The sequences needed f o r t r a n s c r i p t i o n of tRNA operons are the same as those found i n other E_. c o l i operons. About 35 n u c l e o t i d e s upstream from the t r a n s c r i p t i o n s t a r t s i t e i s a sequence r e l a t e d to TTGACA. This element of the promoter i s thought to i n t e r a c t w i t h the <^  subunit of RNA p o l y -merase. The "Pribnow box", TATPuATPu, i s found 10 n u c l e o t i d e s upstream from the s t a r t s i t e and i s thought to be recognized by RNA polymerase core ( c o n t a i n i n g only o^BB' subunits) (reviewed by Pribnow, 1979; Rosenberg and Court, 1979). In tRNA operons about 40 n u c l e o t i d e s separate the i n i t i a -t i o n s i t e from the f i r s t tRNA gene. At the other end of the operons are 23. the t r a n s c r i p t i o n t e r m i n a t i o n s i t e s . These can be of two types. Both c o n t a i n a G"C-rich region of dyad symmetry. The t r a n s c r i p t of t h i s region can presumably form a h a i r - p i n s t r u c t u r e . In rho-independent terminators the i n v e r t e d repeat i s followed by a s e r i e s of adjacent T residues i n the non-transcribed s t r a n d . Termination occurs i n the corresponding s e r i e s of U residues i n the t r a n s c r i p t . Rho-dependent terminators l a c k the T - r i c h sequence and r e q u i r e the presence of rho f a c t o r f o r e f f i c i e n t t e r m i n a t i o n (Pribnow, 1979; Duester et a l . , 1981). When the co n c e n t r a t i o n of any aminoacyl-tRNA becomes the r a t e - l i m i t i n g f a c t o r i n p r o t e i n s y n t h e s i s , E_. c o l i c e l l s undergo a complex change i n metabolism known as the s t r i n g e n t response. A prominent f e a t u r e of the st r i n g e n t response i s a 10-20 f o l d r e d u c t i o n i n the synthesis of rRNA and tRNA ( G a l l a n t , 1979). Travers (1980) has noted that promoters s e n s i t i v e to the s t r i n g e n t response have a G*C-rich sequence between the Pribnow box and the t r a n s c r i p t i o n s t a r t s i t e . Sequenced tRNA operons f o l l o w t h i s p a t t e r n (Rossi et^ a l . , 1980; Duester et_ a l . , 1981; Nakajima et_ a l _ . , 1981). How, or even i f , these sequences are in v o l v e d i n reducing s t a b l e RNA synthesis during the s t r i n g e n t response i s unknown. B. Organelles Mitochondria and c h l o r o p l a s t s both c o n t a i n s m a l l , c i r c u l a r DNA mole-c u l e s . These genomes code f o r a complement of tRNAs required f o r the t r a n s -l a t i o n of m i t o c h o n d r i a l and c h l o r o p l a s t mRNAs. Human m i t o c h o n d r i a l DNA (mt DNA) i s 16.5 kb long and codes f o r 2 rRNAs, 13 p r o t e i n s , and 22 tRNAs of h i g h l y unusual s t r u c t u r e (Anderson et a l . , 1981). The most notable feature of t h i s genome i s i t s extreme economy of o r g a n i z a t i o n . Both strands of the DNA are completely t r a n s c r i b e d but the heavy (H) strand t r a n s c r i p t i s the precursor of the mi t o c h o n d r i a l rRNAs, most of the mRNAs and most of the tRNAs. U s u a l l y , there are no spacer n u c l e o t i d e s between the gene t r a n -24. s c r i p t s ; i n s t e a d tRNA sequences are i n t e r s p e r s e d among the rRNA and mRNA sequences (Montoya, 1981; O j a l a , 1981). A t t a r d i and h i s coworkers have proposed a "tRNA punctuation" model f o r the processing of the H strand primary t r a n s c r i p t ( O j a l a , 1981). In t h i s model mRNAs and rRNAs are gene-rated by nucleases that p r e c i s e l y cut the t r a n s c r i p t at the 5' and 3'-ends of the tRNA sequences. Yeast mt DNA i s about 5 times l a r g e r than that of higher eukaryotes (Borst and G r i v e l l , 1981). The extreme compactness of o r g a n i z a t i o n charac-t e r i s t i c of smaller m i t o c h o n d r i a l genomes i s absent i n yeast. Most yeast mt tRNA genes are c l u s t e r e d together i n one segment of the m i t o c h o n d r i a l DNA. The genes f o r spinach c h l o r o p l a s t tRNAs are l o c a t e d at many p o s i t i o n s i n the c h l o r o p l a s t DNA. C l u s t e r s of tRNA genes are found at some s i t e s H e A l a (Steinmetz et_ a l . , 1980). Recently, genes f o r tRNA and tRNA were discovered i n the spacer between the 16S and 23S rRNA genes of maize c h l o r o -p l a s t s (Koch et_ a l . , 1981). This arrangement i s reminiscent of the tRNA genes found i n some E_. c o l i rRNA operons. The c h l o r o p l a s t i s o l e u c i n e and a l a n i n e tRNA genes show strong homology to the corresponding p r o k a r y o t i c tRNAs but c o n t a i n enormous i n t e r v e n i n g sequences of 949 and 806 bp respec-t i v e l y . C. Yeast Saccharomyces c e r e v i s i a e contains about 360 tRNA genes per h a p l o i d genome coding f o r between 40 and 60 d i f f e r e n t tRNA species (Feldmann, 1976). By studying the s e n s i t i v i t y of yeast tRNA genes to U.V. l i g h t Feldmann (1977) determined t h a t , u n l i k e those of E. c o l i , most yeast tRNA genes have monocistronic t r a n s c r i p t i o n u n i t s . Subsequently, sequence analy-s i s of tRNA genes from a wide v a r i e t y of eukaryotes i n d i c a t e s that the great m a j o r i t y of eukaryotic tRNA genes are t r a n s c r i b e d m o n o c i s t r o n i c a l l y . Excep-t i o n s to t h i s general r u l e are, however, known. In bakers' yeast a 25. As D AT 2 tRNA F gene i s found only 10 bp downstream from a tRNA gene (Schmidt et a l . , 1980). S i m i l a r l y , i n Schizosaccharomyces pombe 7 bp separates a c P T- Mpt tRNA gene from one coding f o r tRNA^ (Mao et_ a J . , 1980). Tran-s c r i p t i o n of e i t h e r gene p a i r i n a Xenopus oocyte e x t r a c t produces a dimeric tRNA precursor. The arrangement of nuclear yeast tRNA genes on the chromosomes i s not known i n d e t a i l . Genetic studies have shown that none of the 8 yeast Tyr tRNA genes are t i g h t l y l i n k e d and that they are found on 6 d i f f e r e n t chromosomes. EcoR I fragments bearing 7 of these genes have been cloned. Only one of the fragments contains more than a s i n g l e tRNA gene (Olson et a l . , 1979). S i m i l a r c l o n i n g experiments by Beckmann et a l . (1977) support the hypothesis t h a t i n yeast tRNA genes are not c l u s t e r e d but are widely d i s t r i b u t e d on the chromosomes. W i t h i n some yeast tRNA genes are sequences not found i n the mature Tvr tRNA. O r i g i n a l l y discovered i n the tRNA J genes (Goodman et_ a l . , 1977), i n t e r v e n i n g sequences ranging i n s i z e from 14 to 34 bp have been found i n yeast phenylalanine, s e r i n e , l e u c i n e and tryptophan tRNA genes (Valenzuela et a l . , 1978; Olson et^ a l . , 1981; Venegas eX a l . , 1979; Ogden et_ a l . , 1979). The s i t e of the i n t e r v e n i n g sequence i n the gene i s always between the f i r s t and second n u c l e o t i d e s a f t e r the anticodon. I t i s i n t r i g u i n g that the codons t r a n s l a t e d by the products of these genes a l l begin w i t h U. In a f a m i l y of i s o a c c e p t i n g tRNAs, genes f o r some isoacc e p t o r s may c o n t a i n an i n t e r v e n i n g sequence while other, even c l o s e l y r e l a t e d , i s o a c c e p t o r s may not (Olson e_t a _ l . , 1981). The t r a n s c r i p t s of genes w i t h i n t e r v e n i n g sequences can a l l be folded i n t o a secondary s t r u c t u r e w i t h c e r t a i n common f e a t u r e s . In the tRNA precursors the anticodon may base-pair to a complementary r e g i o n i n the i n t e r v e n i n g sequence. There are two loops i n the proposed secondary s t r u c t u r e , the beginning of the f i r s t loop and the end of the second are the 26. s i t e s at which the i n t e r v e n i n g sequence i s excised during processing of the precursor (Figure 5 ) ( S e l k e r and Yanofsky, 1980). 5! / C i A-<?• A-I C-A-I A -2 W 0 - A ^ ©u C-A-C u u I % C-G A-0 G-C C A U G S. cerevisiae Phe Figure 5. Removal of the Intervening Sequence from Yeast tRNA Precursor Intervening sequences are not unique to yeast tRNA genes. S i m i l a r i n t e r v e n i n g sequences have been found i n tRNA genes of D r o s o p h i l a , Xenopus, and Neurospora (Robinson and Davidson, 1981; M u l l e r and Clarkson, 1980; Selker and Yanofsky, 1980). The f u n c t i o n of i n t e r v e n i n g sequences i n tRNA genes i s unknown. D. Xenopus In c o n t r a s t t o the dispersed arrangement of tRNA genes i n y e a s t , the tRNA genes of Xenopus l a e v i s are organized i n tandemly repeated a r r a y s . H y b r i d i z a t i o n data i n d i c a t e there are 8000 tRNA genes i n Xenopus coding f o r about 43 types of tRNA (Clarkson et a l . , 1973a). The number of genes f o r d i f f e r e n t tRNA species i s q u i t e v a r i a b l e . For example, there are 310 genes f o r tRNA^ e t but only 170 genes f o r tRNA^ e t (Clarkson et a l . , 1973a). Clarkson e_t a l . (1973b) sheared Xenopus DNA and c e n t r i f u g e d i t t o e q u i l i b r i u m on a CsCl d e n s i t y g r a d i e n t . Even when the DNA was of high aver-Met Met V a l age molecular weight the genes f o r tRNA^ , tRNA^ and tRNA were segregated from one another on DNA fragments of d i f f e r e n t buoyant 27. Met d e n s i t y ( " c r y p t i c " s a t e l l i t e DNAs). DNA c o n t a i n i n g tRNA^ genes was p a r t i a l l y p u r i f i e d by repeated d e n s i t y gradient c e n t r i f u g a t i o n . R e s t r i c t i o n Met a n a l y s i s of t h i s DNA showed that the tRNA^ genes were l o c a t e d on a tandemly repeated 3.1 kb fragment of DNA (Clarkson and Kurer, 1976). The Met repeat u n i t was cloned and contained 2 tRNA^ genes and s i n g l e copies of 6 other tRNA genes (Clarkson et_ a l _ . , 1978; H o f s t e t t e r et_ a l . , 1981). In t h i s repeat u n i t the genes are i r r e g u l a r l y spaced and are t r a n s c r i b e d from both strands of DNA. I t i s l i k e l y the other " s a t e l l i t e " bands c o n t a i n tandemly repeated f a m i l i e s of tRNA genes. The arrangement of tRNA genes i n Xenopus p a r a l l e l s that of the h i g h l y redundant 5S RNA, rRNA, and histone genes i n t h i s organism. The arrangement of these genes may be adapted to the requirements of oogenesis when l a r g e amounts of s t a b l e RNAs are accumulated. V. Drosophila melanogaster tRNA Genes A. Gene number One of the most elementary questions that can be asked about Drosophila tRNA genes i s : how many tRNA genes are there i n the Drosophila genome? R i t o s s a et a l . (1966) attempted to answer t h i s question by q u a n t i t a t i n g the h y b r i d i z a t i o n of a tRNA probe ( t o t a l mixed tRNAs) to Drosophila DNA. They found that 0.015% of the t o t a l D rosophila DNA coded f o r tRNA, equivalent to 750 tRNA genes per h a p l o i d genome. In a more recent study Weber and Berger (1976) followed the k i n e t i c s of h y b r i d i z a t i o n of Drosophila 4S RNA to Drosophila DNA. By measuring the r a t e at which the probe h y b r i d i z e d to the DNA the k i n e t i c complexity of the tRNA genes could be determined ( B r i t t e n et a l . , 1974). The observed complexity i n d i c a t e d there are 59 f a m i l i e s of tRNA genes i n Dros o p h i l a. The t o t a l number of tRNA genes was estimated to be about 590 per h a p l o i d genome. This f i g u r e i s probably more accurate than 28. the e a r l i e r estimate because the 4S probe used i n these experiments was of higher p u r i t y . The 56 gene f a m i l i e s detected by h y b r i d i z a t i o n are s i g n i f i -c a n t l y fewer than the approximately 90 species of tRNA found i n Drosophila by White e_t a l . (1973a). The h y b r i d i z a t i o n technique would probably not detect s l i g h t heterogeneity among tRNA genes, t h i s would lead to an under-estimate of sequence complexity. The d i f f i c u l t i e s i n determining tRNA gene number by h y b r i d i z a t i o n were demonstrated by the s t u d i e s of Delaney (Tener et a l . , 1980). P u r i f i e d tRNA is o a c c e p t o r s were used as probes t o determine the number of genes f o r i n d i v i d u a l tRNA sp e c i e s . The " s a t u r a t i o n " l e v e l of h y b r i d i z a t i o n was found to vary g r e a t l y w i t h the RNA c o n c e n t r a t i o n , though i n a l l cases there was an excess of RNA present. For example, the number of Lv s tRNA^ genes ranged from 4 to 18 depending on the RNA:DNA r a t i o . The hy b r i d s that d i d form were of two c l a s s e s , one about 10 times more s t a b l e Lvs than the other. Seven of 18 tRNA^ genes formed l a b i l e hybrids w i t h the probe, the remainder were much more s t a b l e . The number of genes f o r a p a r t i c u l a r tRNA can be estimated by d i g e s t i n g Drosophila DNA to completion w i t h r e s t r i c t i o n enzymes and determining the number of DNA fragments (separable by g e l e l e c t r o p h o r e s i s ) that c o n t a i n genes f o r the tRNA of i n t e r e s t . This approach was used to estimate the V a l number of genes f o r two v a l i n e i s o a c c e p t o r s : 17-19 tRNA^ genes V a l (Dudler, 1981) and 6-7 tRNA^ b genes were detected (Tener et_ a l . , 1980). B. Gene L o c a t i o n The f i r s t i n f o r m a t i o n about the l o c a t i o n of tRNA genes i n the Drosophila genome came from the e a r l y h y b r i d i z a t i o n s t u d i e s of R i t o s s a et a l . ( 1 9 6 6 ) . They were able to show that few, i f any, tRNA genes were a s s o c i -ated w i t h rRNA genes i n Drosoph i l a . Since then the technique of in_ s i t u h y b r i d i z a t i o n of tRNA to polytene chromosomes has g r e a t l y increased our 29. knowledge of tRNA gene l o c a t i o n i n Drosophila. Polytene chromosomes are found i n a number of Drosophila t i s s u e s , notably the l a r v a l s a l i v a r y gland. They are the r e s u l t of repeated rounds of DNA r e p l i c a t i o n i n the absence of c e l l d i v i s i o n . A l l the chromatids r e s u l t i n g from the r e p l i c a t i o n of each chromosomal arm are e x a c t l y a l i g n e d w i t h one another (Beermann, 1972; Rudkin, 1972). Polytene chromosomes are much t h i c k e r and longer than m i t o t i c chromosomes. Th e i r most s t r i k i n g f e a -t u r e i s a p a t t e r n of bands seen on a l l the chromosomal arms. The p a t t e r n of bands i s constant f o r a given stock of f l i e s and they serve as u s e f u l chro-mosomal markers. A system of numbers and l e t t e r s i s used to describe the l o c a t i o n of s i t e s on the polytene chromosomes (L e f e v r e , 1976). In s i t u h y b r i d i z a t i o n ( G a l l and Pardue, 1969) i s widely used to l o c a t e genes on Drosophila polytene chromosomes. In t h i s technique a 3H or 1 2 5 1 l a b e l l e d probe i s h y b r i d i z e d to i t s genes i n a squashed pre p a r a t i o n of the polytene chromosomes from l a r v a l s a l i v a r y glands. Subsequent auto-radiography r e v e a l s the s i t e s of h y b r i d i z a t i o n as s i l v e r g r a i n s c l u s t e r e d above s p e c i f i c l o c i on the chromosomes. In s i t u h y b r i d i z a t i o n of t o t a l 4S RNA to polytene chromosomes re v e a l s 54 s i t e s of h y b r i d formation, 26 strong s i t e s and 28 weak s i t e s ( Steffensen and Wimber, 1971; E l d e r et^ a l . , 1980). The s i t e s are randomly s c a t t e r e d over the chromosomal arms w i t h the exception of the X chromosome, which has p r o p o r t i o n a l l y fewer s i t e s than the oth e r s . No tRNA genes were detected on the small f o u r t h chromosome. Any tRNA genes i n the u n d e r - r e p l i c a t e d and h i g h l y condensed heterochromatin may have escaped d e t e c t i o n . The number of tRNA s i t e s i d e n t i f i e d on the chromosomes i s much l e s s than the 600-750 genes detected by jLn v i t r o h y b r i d i z a t i o n s t u d i e s . This i m p l i e s that many chromo-somal s i t e s c o n t a i n s e v e r a l tRNA genes. Atwood has hypothesized t h a t the c l a s s of about 55 dominant mutations 30. known as Minutes i s caused by d e l e t i o n of tRNA genes ( R i t o s s a et_ a l . , 1966). The Minute phenotype (delayed development, small b r i s t l e s , and r e c e s s i v e l e t h a l i t y ) was a t t r i b u t e d to reduced p r o t e i n s y n t h e s i s due to suboptimal l e v e l s of tRNA. The l o c a t i o n of Minute mutations does not c o i n -c i d e , on more than a chance b a s i s , w i t h the l o c a t i o n of tRNA genes ( E l d e r et a l . , 1980). Genetic evidence gathered by Huang and Baker (1976) supports the c o n c l u s i o n that the Minute phenotype i s not due to d e f i c i e n c i e s f o r tRNA genes. Genes coding f o r 25 p u r i f i e d tRNA is o a c c e p t o r s have been l o c a t e d by i n s i t u h y b r i d i z a t i o n (reviewed by Hayashi et_ a l . , 1981a). In g e n e r a l , genes f o r a p a r t i c u l a r tRNA species are found at more than one s i t e . These s i t e s may or may not be on the same chromosomal arm and a s i n g l e s i t e can c o n t a i n genes f o r s e v e r a l d i f f e r e n t tRNAs. The chromosomal s i t e s f o r the 3 major v a l i n e i s o a c c e p t o r s are presented i n Table I (Hayashi et a l . , 1980, 1981a). Table I . S i t e s of Drosophila melanogaster tRNA Genes on the Polytene Chromosomes Isoacceptor Major S i t e L o c a t i o n Minor S i t e 3 L o c a t i o n V a l 3a 64D 1_ 2 3L - -V a l 3b 84D3_4 92B 1_ 9 3R 3R 90BC 3R V a l 4 56D 3_ 7 70BC 2R 3L 90BC 89B 3R 3R a - About 1/5 the s i l v e r g r a i n s are found over a minor s i t e as are found over a major s i t e of h y b r i d i z a t i o n . C. Organization of Cloned Drosophila tRNA Genes The technique of r n s i t u h y b r i d i z a t i o n of tRNAs to polytene chromosomes 31. provides much i n f o r m a t i o n about the l a r g e s c a l e o r g a n i z a t i o n of tRNA genes. The development of techniques such as gene c l o n i n g , r e s t r i c t i o n mapping and DNA sequence a n a l y s i s has made study of the f i n e s t r u c t u r e of tRNA genes p o s s i b l e . No uniform p a t t e r n of tRNA gene o r g a n i z a t i o n has emerged from these s t u d i e s . Rather, Drosophila tRNA genes are found to be organized i n a v a r i e t y of ways. Davidson and h i s colleagues cloned overlapping fragments of Drosophila DNA that span 94 kb from the 42A region of chromosome 2 (Hovemann et a l . , A S H 1980; Yen and Davidson, 1980). A t o t a l of 18 tRNA genes: 8 tRNA , 4 t R N A ^ 8 , 5 t R N A ^ and 1 t R N A I l e , have been l o c a t e d i n t h i s l a r g e segment of DNA. The o r g a n i z a t i o n of these genes i s i l l u s t r a t e d i n Figure 6 (Yen and Davidson, 1980). Si 5 Figure 6. tRNA Genes at the 42A Region of the Drosophila Chromosome. Arrows denote p o l a r i t y of the genes, arrowheads are at the 3'-ends of the genes. They are widely dispersed and i r r e g u l a r l y spaced w i t h i n the c e n t r a l 46 kb of the DNA segment. Several c l u s t e r s of genes occur. These c l u s t e r s may c o n t a i n i d e n t i c a l genes or genes f o r s e v e r a l tRNAs. I t i s noteworthy that wherever i d e n t i c a l genes are c l u s t e r e d two of the genes are of opposite Lv s p o l a r i t y . The arrangement of some tRJM^ genes as c l o s e l y l i n k e d i n v e r t e d repeats may e x p l a i n the p u z z l i n g r e s u l t s of h y b r i d i z a t i o n of tRNA^ y s to Drosophila DNA (Tener ejt a l . , 1980) described i n a previous s e c t i o n (V.A). Under h y b r i d i z a t i o n c o n d i t i o n s two such genes could form h a i r - p i n s t r u c t u r e s w i t h each other r a t h e r than an RNA:DNA h y b r i d . Eleven of the 18 genes have been sequenced. None contains an i n t e r -vening sequence or codes f o r the -CCA end of the tRNA. A l l the genes f o r a given tRNA species are i d e n t i c a l . A l l the sequenced genes are followed by runs of T residues i n the non-coding st r a n d . These are thought to be t e r m i -n a t i o n s i g n a l s f o r RNA polymerase I I I (Valenzuela e_t a l . , 1977; Silverman et a l . , 1979). Some genes coding f o r the same tRNA have regions of homology i n Lvs t h e i r 5' - f l a n k i n g sequences. A l l but one of the cloned tRNA^ genes i n the 42A r e g i o n have a sequence c l o s e l y r e l a t e d to GGCAGTTTTTA about 25 bp Lvs upstream from the gene. A s i m i l a r sequence i s found 5' to a tRNA^ gene from a d i f f e r e n t , but not p r e c i s e l y known, chromosomal l o c a t i o n (De Franco et a l . , 1980). S i m i l a r l y , a l l the sequenced tRNA^ r^ genes from the 42A r e g i o n have a conserved n u c l e o t i d e sequence (TCTTNACA or TGTTACA) about 20 n u c l e o t i d e s 5' to the genes (Yen and Davidson, 1980). A cloned fragment of DNA that h y b r i d i z e s to the 50AB s i t e on chromosome 2 codes f o r a c l u s t e r of 5 t R N A I l e genes and 2 t R N A L e u genes (Robinson and Davidson, 1981). H e A l l the tRNA genes are preceded by a region s t r o n g l y homologous to GCNTTTTG. At a s i m i l a r p o s i t i o n , about 25 bp before the coding sequence, GANTTTGG precedes the t R N A L e u genes. The s i g n i f i c a n c e of these conserved sequences i s not yet known. A number of other plasmids c o n t a i n i n g Drosophila tRNA genes have been i s o l a t e d and c h a r a c t e r i z e d . Some of these d i s p l a y f e a t u r e s of gene o r g a n i -z a t i o n not found at the 42A s i t e . The genes f o r tRNA mentioned above are the f i r s t i n Drosophila found to c o n t a i n i n t e r v e n i n g sequences (Robinson and Davidson, 1981). The two i n t e r v e n i n g sequences d i f f e r from one another 33. i n s i z e but d i s p l a y regions of homology w i t h the i n t e r v e n i n g sequence found i n the yeast tRNA^ e u genes. Hershey and Davidson (1980) cloned a group of overlapping fragments of Drosophila DNA spanning 22 kb of sequence. W i t h i n t h i s DNA segment they found a 1.1-2 kb fragment to be d u p l i c a t e d . Each repeat u n i t contains a s i n g l e tRNA^"^ gene. In s i t u h y b r i d i z a t i o n s t u d i e s showed that t h i s repeat u n i t occurs only at the 56F s i t e . The arrangement of these tRNA genes p a r a l l e l s the tandem arrangement of repeat u n i t s c o n t a i n i n g 5S RNA genes, a l s o found at 56F (Prensky et a l . , 1973). I t w i l l be i n t e r e s t i n g to see how other tRNA genes, known to be present a t 56EF (Hayashi, et^ a l . , 1981b), are arranged. Heterogeneity i n c l o s e l y r e l a t e d tRNA gene sequences was found i n a cloned c l u s t e r of tRNA G^ u genes (Hosbach et_ a l . , 1980). Four of 5 t R N A ^ u genes are i d e n t i c a l but one contains a C to T t r a n s i t i o n at p o s i -t i o n 4 of the non-transcribed s t r a n d . Homologies among the f l a n k i n g sequences of these genes i n d i c a t e that the present gene c l u s t e r arose from a s i n g l e a n c e s t r a l gene by a combination of gene d u p l i c a t i o n and unequal c r o s s i n g over. Although few of the hundreds of tRNA genes i n the Drosophila genome have been thoroughly studied some g e n e r a l i z a t i o n s about the o r g a n i z a t i o n of these genes can be made. Many tRNA genes occur i n c l u s t e r s of a few genes. The c l u s t e r s o f t e n c o n t a i n genes f o r more than one species of tRNA. W i t h i n a c l u s t e r tRNA genes are i r r e g u l a r l y spaced and may be t r a n s c r i b e d from both strands of DNA. Frequently, c l o s e l y spaced i d e n t i c a l tRNA genes are arranged as i n v e r t e d repeats. Often genes f o r a p a r t i c u l a r tRNA species are preceded by a short sequence common to other genes f o r the same tRNA. A s i n g l e chromosomal tRNA h y b r i d i z a t i o n s i t e may c o n t a i n s e v e r a l gene c l u s t e r s as w e l l as i s o l a t e d tRNA genes. 34. VI. T r a n s c r i p t i o n of Eukaryotic tRNA Genes Eukaryotes c o n t a i n 3 types of RNA polymerase. Each type t r a n s c r i b e s a unique c l a s s of genes. XRNA polymerase I t r a n s c r i b e s rRNA genes, RNA p o l y -merase I I synthesizes the precursors to mRNAs and RNA polymerase I I I t r a n -s c r i b e s genes f o r a number of small RNAs i n c l u d i n g tRNA and 5S RNA. A l l three polymerases are of hi g h molecular weight and are composed of from 10 to 15 subunits (Paule, 1981). The n u c l e o t i d e sequences of many euk a r y o t i c genes t r a n s c r i b e d by RNA polymerase I I I have been determined. By analogy w i t h b a c t e r i a l promoters i t might be expected that i n s p e c t i o n of the sequences adjacent t o the 5'-ends of these genes would r e v e a l a conserved sequence, the RNA polymerase I I I promoter. This expectation has not been f u l f i l l e d . The 5 ' - f l a n k i n g sequences of 5S and tRNA genes show no s t r o n g l y conserved sequences. \ A number of approaches have been used to determine the f e a t u r e s of a tRNA gene e s s e n t i a l f o r e f f i c i e n t t r a n s c r i p t i o n . Kurjan et_ a l . (1980) Tyr s e l e c t e d spontaneous mutants of the yeast SUP4 tRNA locus that could no longer produce f u n c t i o n a l suppressor tRNA molecules. S i x t y - n i n e mutants i n the tRNA gene were mapped to 10 t i g h t l y l i n k e d s i t e s . Several mutant genes from each of these c l u s t e r s were cloned and the n u c l e o t i d e sequence of each mutant gene was determined. None of the mutants had changes i n the se-quences f l a n k i n g the gene. This suggests the gene's promoter does not l i e i n these f l a n k i n g regions. T r a n s c r i p t i o n of 29 of the sequenced mutant genes was t e s t e d i n a t r a n s c r i p t i o n system derived from Xenopus kidney c e l l s (Koski et^ a l . , 1980). Most of the mutant genes were t r a n s c r i b e d although changes i n the l e n g t h or q u a n t i t y of the t r a n s c r i p t were noted i n some cases. Two of the mutant genes were not t r a n s c r i b e d i n t h i s system. Both are a l t e r e d at the same s i t e w i t h i n the gene. In one mutant, the C residue corresponding to the i n v a r i a n t C56 of the T^C sequence i n the tRNA i s 35. changed to a G. In the other, i t i s changed to a U. Thus, i t i s l i k e l y t h a t the C-G base-pair a t t h i s s i t e i n the gene i s an important part of the RNA polymerase I I I promoter. The elements of the eukaryotic tRNA gene promoter have been more pre-c i s e l y defined by the s t u d i e s of B i r n s t i e l and h i s coworkers (Kressmann et a l . , 1979; H o f s t e t t e r e_t £l. , 1981). From a poi n t i n the 5 ' - f l a n k i n g Met sequence of a cloned Xenopus tRNA^ gene a s e r i e s of d e l e t i o n mutants were c r e a t e d , each w i t h p r o g r e s s i v e l y more of the 5 ' - f l a n k i n g sequence or gene sequence removed. The a b i l i t y of each mutant gene to support s p e c i f i c t r a n s c r i p t i o n i n v i t r o or a f t e r i n j e c t i o n i n t o Xenopus oocytes was t e s t e d . A l l of the 5 * - f l a n k i n g sequence and up to 9 bp of coding sequence could be d e l e t e d without impairment of t r a n s c r i p t i o n . These changes i n the 5'-f l a n k i n g sequence d i d a l t e r the exact s i t e of t r a n s c r i p t i n i t i a t i o n . Tran-s c r i p t i o n d i d not occur i f more than the f i r s t 10 n u c l e o t i d e s of the gene were d e l e t e d . Studies of a s i m i l a r s e r i e s of d e l e t i o n s from the 3'rend of the gene showed th a t d e l e t i o n of the 3 ' - f l a n k i n g sequence d i d not prevent t r a n s c r i p t i o n of the gene. A mutant gene l a c k i n g the 3'-end of the coding sequence (from n u c l e o t i d e 55 on) as w e l l as the 3 ' - f l a n k i n g sequence could not be t r a n s c r i b e d . Two d e l e t i o n mutants were created by removing sequences of DNA from w i t h i n the gene. These mutants lacked DNA between p o s i t i o n s 12 and 28 or between p o s i t i o n s 33 and 46. Neither gene could be t r a n s c r i b e d i n  v i t r o . T r a n s c r i p t i o n of the l a t t e r mutant gene, but not the former, could be r e s t o r e d by r e p l a c i n g the deleted s e c t i o n w i t h DNA of unrelated se-quence. This i n d i c a t e s t h a t the p a r t i c u l a r n u c l e o t i d e sequence between p o s i t i o n 12 and 28 i s required f o r t r a n s c r i p t i o n w h i l e the sequence of the DNA between p o s i t i o n s 33 and 46 does not matter so long as i t i s at l e a s t 13 base-pairs i n l e n g t h . I n s e r t i o n mutants were made by i n s e r t i n g short pieces Met of DNA at s e v e r a l s i t e s i n the tRNA. gene. A l l the i n s e r t i o n mutants could be t r a n s c r i b e d . The t r a n s c r i p t s of these mutants had the same i n i t i a -t i o n p o i n t as the t r a n s c r i p t of the w i l d - t y p e gene but were longer by the number of n u c l e o t i d e s introduced i n t o the tRNA gene when the mutants were con s t r u c t e d . Met These experiments i n d i c a t e that the tRNA^ gene contains an i n t e r n a l promoter sequence. A model f o r t h i s promoter has been proposed by H o f s t e t t e r et a l . (1981). The promoter has two e s s e n t i a l elements, one lo c a t e d between n u c l e o t i d e s 8 and 30 and the other between p o s i t i o n s 51 and 72. These regions c o i n c i d e w i t h two h i g h l y conserved sequences i n eukary-o t i c tRNA genes, GTPuGCGPyAGTNGG (acceptor stem, D-arm) and GGTTCGA(A/T)-PyCC(T-arm) r e s p e c t i v e l y . The middle p o r t i o n of the gene maintains the two elements of the promoter at a c r i t i c a l d i s t a n c e from one another. This d i s t a n c e may be increased but any s i g n i f i c a n t decrease destroys promoter a c t i v i t y . Other genes t r a n s c r i b e d by RNA polymerase I I I , such as the 5S RNA genes of Xenopus and the adenovirus VAI gene, c o n t a i n i n t e r n a l promoters Met s i m i l a r to that found i n the tRNA^ gene (Sakonju e_t_ a l . , 1980; Bogenhagen e t _ a l . , 1980; Fowlkes and Shenk, 1980). Met In t h e i r study of the Xenopus tRNA^ gene H o f s t e t t e r et_ a l . (1981) found that the gene's 5' - f l a n k i n g sequence had only a minor e f f e c t on t r a n s c r i p t i o n , merely a l t e r i n g s l i g h t l y the exact p o i n t of i n i t i a t i o n . S everal other s t u d i e s i n d i c a t e that the 5 ' - f l a n k i n g sequence can play a much more s i g n i f i c a n t r o l e i n the c o n t r o l of tRNA gene t r a n s c r i p t i o n . De Franco Lvs et a l . (1980) t r a n s c r i b e d two cloned Drosophila tRNA£ genes i n the Xenopus germinal v e s i c l e system. One gene was t r a n s c r i b e d about 10 times more e f f i c i e n t l y than the other. The 5 ' - f l a n k i n g sequence of the poor l y t r a n s c r i b e d gene was shown by these workers to be r e s p o n s i b l e f o r i t s t r a n -s c r i p t i o n a l i n a c t i v i t y . Both genes are downstream from a conserved sequence found near many Drosophila . t R N A ^ 8 genes ( S e c t i o n V.C). 37. A l a Sprague et_ a l . (1980) t r a n s c r i b e d a cloned Bombyx mori tRNA2 gene i n two systems, a Xenopus germinal v e s i c l e e x t r a c t and a system derived from Bombyx t i s s u e s . The i n t a c t gene was w e l l t r a n s c r i b e d i n both systems. I f a l l but 11 bp of the gene's 5 ' - f l a n k i n g sequence was removed the gene was s t i l l t r a n s c r i b e d i n the heterologous Xenopus system but not i n the homolo-gous Bombyx system. In c o n t r a s t to one of the Drosophila t R N A ^ 8 genes described above, the 5'-flanking sequence of the gene s t i m u l a t e s r a t h e r than i n h i b i t s t r a n s c r i p t i o n . Thus, i t i s concluded that sequences i n the 5 ' -f l a n k i n g region seem to play a r o l e i n modulating the a c t i v i t y of adjacent tRNA genes. V I I . Processing of tRNA T r a n s c r i p t s The primary t r a n s c r i p t s of tRNA genes, i n both prokaryotes and eukary-otes, c o n t a i n n u c l e o t i d e sequences not present i n the mature tRNAs. The processing of these t r a n s c r i p t s to produce f u n c t i o n a l tRNA molecules i s b r i e f l y described below (reviewed by Mazzara and McClain, 1980). The events, and p a r t i c u l a r l y the enzymology, of tRNA processing are best understood i n E_. c o l i . In prokaryotes the primary t r a n s c r i p t u s u a l l y contains s e v e r a l tRNA sequences separated by spacers. Enzymatic cuts are made w i t h i n the spacers l i b e r a t i n g monomeric tRNA precursors that are then trimmed to mature s i z e . RNase P2 i s an endonuclease that can cut tRNA precursors i n the spacer r e g i o n . Another endonuclease, RNase P, cuts mono-meric pre-tRNAs, p r e c i s e l y at the 5'-end of the tRNA sequence. While endo-n u c l e o l y t i c cleavage by RNase P generates the mature 5'-end of the tRNA, the 3'-end i s produced by the a c t i o n of an exonuclease. A great d e a l of confu-s i o n e x i s t s as t o which nuclease i s r e s p o n s i b l e f o r t h i s trimming in v i v o . RNase D removes n u c l e o t i d e s from the 3'-end of a precursor but stops when i t reaches the t e r m i n a l -CCA sequence (Ghosh and Deutscher, 1980). RNase D may 38. be the same enzyme as the p r e v i o u s l y described RNases P3 and Q. Other nu-c l e a s e s that have been i m p l i c a t e d i n E. c o l i tRNA processing are BN exonu-clease and RNase I I I (Mazzara and McClain, 1980). The primary t r a n s c r i p t s of e u k a r y o t i c tRNA genes are u s u a l l y mono-meric. An RNase P - l i k e a c t i v i t y and an exonuclease capable of processing the 3'-ends of tRNA precursors have been detected i n a wide v a r i e t y of e u k a r y o t i c c e l l s . These enzymes have been p a r t i a l l y p u r i f i e d from Bombyx  mori (Garber and Altman, 1979). They are capable of a c c u r a t e l y processing both e u k a r y o t i c and p r o k a r y o t i c tRNA precursors. U n l i k e those of E_. c o l i , e u k a r y o t i c tRNA precursors do not c o n t a i n the 3'-terminal -CCA sequence. This sequence i s added p o s t - t r a n s c r i p t i o n a l l y by the enzyme tRNA n u c l e o t i d y l t r a n s f e r a s e . Some eu k a r y o t i c tRNA precursors c o n t a i n i n t e r v e n i n g sequences. Abelson and h i s coworkers have used a s o l u b l e e x t r a c t from yeast t h a t e x c i s e s these i n t e r v e n i n g sequences to i n v e s t i g a t e the s p l i c i n g r e a c -t i o n (Peebles et_ a l . , 1979; Knapp et_ a l . , 1979). S p l i c i n g occurs i n 2 s t e p s . F i r s t an endonuclease a c t i v i t y e x c i s e s the i n t e r v e n i n g sequence to produce two h a l f - m o l e c u l e s . The 3'-end generated by the e x c i s i o n i s phos-phorylated w h i l e the new 5'-end bears a f r e e hydroxyl group. The second step i s the ATP dependent l i g a t i o n of the two h a l f - m o l e c u l e s . T r a n s f e r RNA maturation i s a complex process. The i n t r a c e l l u l a r l o c a -t i o n of processing and the order i n which the processing steps occur was Tyr i n v e s t i g a t e d by Melton et_ a l . (1980). Plasmids c a r r y i n g a yeast tRNA gene were i n j e c t e d i n t o Xenopus oocytes. At s e v e r a l times a f t e r i n j e c t i o n the nucleus and cytoplasm of some of the oocytes were separated, the RNA of each f r a c t i o n was i s o l a t e d and the processing intermediates were f r a c t i o n -ated by polyacrylamide g e l e l e c t r o p h o r e s i s . The primary t r a n s c r i p t was 108 n u c l e o t i d e s long and was confined t o the nucleus. Trimming of n u c l e o t i d e s from the 5'-end of t h i s t r a n s c r i p t generated d i s c r e t e processing i n t e r -39. mediates 104 and 97 n u c l e o t i d e s long. Base m o d i f i c a t i o n s at s p e c i f i c s i t e s i n the tRNA were already present i n the l a r g e r intermediate. Next a l l the e x t r a n u c l e o t i d e s were removed from the 5' and 3'-ends of the tRNA, the -CCA sequence was added, and base m o d i f i c a t i o n occurred at other s i t e s i n the tRNA. The product was an intermediate 92 n u c l e o t i d e s l o n g . The f i n a l step was the e x c i s i o n of the i n t e r v e n i n g sequence. A l l these processing steps occurred w i t h i n the nucleus. V I I I . The Present I n v e s t i g a t i o n The v a l i n e tRNAs of D_. melanogaster are an a t t r a c t i v e system i n which to study the s t r u c t u r e of tRNA is o a c c e p t o r s and the genes that code f o r them. The i n v e s t i g a t i o n of Drosophila v a l i n e tRNAs reported i n t h i s t h e s i s can be d i v i d e d i n t o two p a r t s . In the f i r s t p a r t , the r e l a t i o n s h i p between the coding p r o p e r t i e s and the n u c l e o t i d e sequences of Drosophila v a l i n e tRNA iso a c c e p t o r s was i n v e s t i g a t e d . These s t u d i e s were prompted by r e p o r t s of V a l the p e c u l i a r coding behavior of r a b b i t l i v e r tRNA^^,^ as revealed by the ribosome-binding assay (Jank et a l . , 1977a). This tRNA, w i t h an i n o s i n e residue i n the f i r s t p o s i t i o n of the anticodon, binds to ribosomes i n the presence of any v a l i n e codon (GUA, GUC, GUU or GUG) but most s t r o n g l y i n the i V a l presence of GUG. The wobble hypothesis p r e d i c t s that tRNA^j^,^ should bind to ribosomes i n the presence of any v a l i n e codon except GUG V a l ( C r i c k , 1966). The n u c l e o t i d e sequence of r a b b i t l i v e r t R N ^ ( X A C ) ^ a S s e v e r a l unusual features which may be r e s p o n s i b l e f o r i t s anomalous coding V a l p r o p e r t i e s (Jank et_ a l . , 1977). Seven Drosophila tRNA i s o a c c e p t o r s V a l have been resolved by RPC-5 chromatography, 3 major species tRNA^ a , V a l V a l V a l t R NA^ and tRNA^ , and the minor species tRNA^ , tRNA^ a : L,tRNA^ a l and tRNA^ a l ( F i g u r e 7) (Dunn et al., 1978). In t h i s study the coding p r o p e r t i e s of the 3 major v a l i n e i s o a c c e p t o r s were E a 3r-4& 5 ' " 1 ' W Val-tRNA^' D -6 I 3a A i i i i V/ 1 V 40. 6 0 8 0 100 120 140 160 4 0 6 0 8 0 . 100 120 140 160 180 Fraction Number F i g u r e 7. (a) Separation of [- ^ C ] v a l y l - t R N A s V a ^ of crude D r o s o p h i l a tRNA on an RPC-5column (0.9 x 21 cm) using e l u t i o n system A, a 100 ml l i n e a r g r a d i e n t of sodium c h l o r i d e from 0.5 to 0.7 M i n 10 mM MgCl2, 1 mM 2-mercaptoethanol, and 10 mM sodium a c e t a t e pH 4.0. Flow r a t e 0.25 ml/min; f r a c t i o n s i z e , 0.5 ml. (b) Separation of [ ^ C ] v a l y l -t R N A ^ a l as i n (a) but w i t h e l u t i o n system C, a l i n e a r gradientoof sodium c h l o r i d e from 0.5 to 0.7 M, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 10 mM sodium formate pH 3.8. Table I I . . Nucleoside A n a l y s i s of t R N A s V a l from Drosophila Number of residues per 75-nucleotide tRNA molecule tRNA),," tRNA ji'1 tRNA*"1 Nuclco-side I" H* 1 I I I 11 A 14.6 15.1 14.6 15.2 13.7 14.7 U X.7 8.7 8.8 7.8 8.1 6.3 O 16.2 17.X 18.7 23.9 16.9 21.0 c 20.5 18.3 22.8 18.0 22.3 19.0 <!' 5.7 4.6 3.7 4.0 4.0 4.0 hU -> i Nt'' 2.3 Nt • 1.6 Nt rT 1 .0 1.1 1.0 1.2 1.0 1.0 m'A 1 .0 0.80. 0.83 0.73 1.0 0.90 I I V G 0.S4 1 . 1 0.80 1 .1 0.72 0.92 m'C 0.X0 0.73 0.88 0.96 2.0 1.7 m ' C i i --- — _.. — 0.81 1.0 i -i i t i n Nt 0.75'' Nt Nt i . 1.3 Urn Nt 0.75'1 Nl — Nt . 0.9 Am Nl 0.41 Nt — Nt — X 1.7 — A* 0.80'' — — ... N 0.57'' — -- — "Columns labelled I ate \;ilues obtained by ihc iriiium-labellini; technique ( Mulch.lis ami Methods). 'Corumns labelled II are values obtained from the ultraviolet spectra ol" separated nucleosides (Materials and Methods). ' Nl, not tested. ''I'stiiuatcd from two poorly resolved nucleoside spots. 'Assuiniui: a molar extinction of 14 (>00 at 2(»0 rim. 'Assuming a molar extinction of WOO at 2<»0 mil. \ 41. determined by the ribosome-blndlng assay of Nlrenberg and Leder (1964). The V a l V a l nuc l e o t i d e sequences of tRNA^ and tRNA^^ were determined by a combination of modern RNA sequencing methods. The base composition of the Drosophila v a l i n e tRNAs (Table I I ) , p r e v i o u s l y determined by Dunn (Dunn, et a l . , 1978), provided i n f o r m a t i o n e s s e n t i a l to the sequence determinations. In the second part of t h i s i n v e s t i g a t i o n the n u c l e o t i d e sequences of Drosophila v a l i n e tRNA genes were determined. Dunn et_ a l . (1979) i s o l a t e d V a l and c h a r a c t e r i z e d recombinant plasmids c a r r y i n g tRNA genes (Table V a l I I I ) . In the present i n v e s t i g a t i o n the two tRNA^ genes of plasmid V a l pDt55 and the s i n g l e tRNA^ gene of plasmid pDt78R were sequenced. Other i n v e s t i g a t o r s i n t h i s l a b o r a t o r y have sequenced s e v e r a l of the other genes coding f o r these two tRNAs. As a r e s u l t d i f f e r e n t genes f o r the same tRNA can be compared to each other and t o the n u c l e o t i d e sequence of the tRNA. Such comparisons may help answer some important questions about Drosophila tRNA gene s t r u c t u r e and expression. Are r e g u l a t o r y sequences present i n the DNA f l a n k i n g Drosophila tRNA genes? Are the gene sequences the same as the sequences of the tRNAs that h y b r i d i z e to them? Are genes f o r the same tRNA from d i f f e r e n t chromosomal l o c a t i o n s i d e n t i c a l ? How i s the sequence homogeneity of wid e l y s c a t t e r e d copies of a tRNA gene main-tained? 42. Table I I I . Recombinant Plasmids Containing Drosophila t R N A V a l Genes Each of the recombinant plasmids listed here was cleaved by Hindlll, electrophoresed in agarose gels and hybridized with complementary 1 5 5I- labeled tRNA. All of the inserted Hindlll fragments from the original isolates are listed; the 4.4 kb pBR322 parental fragmei is omitted. In order to distinguish/different plasmids containing similar sized Hindlll inserts, the plasmids were cleaved with other endonucleases and analyzed by the procedure of Southern (1975). Frequently all of the fragments are listed in this Table; these include fragments liberated from the parental pBR322 DNA as well as those from the Drosophila insert DNA. Sometimes only the fragments which anneal with the [ 1 I 5 I]tRNA are listed. tRNA Plasmid Prep. Fragments  No. No. Hindlll Other nucleases pDt21 2 5.2*,0.6 3.4 a c* pDt41R 3 2.0*,5.2 pDt48 3 2.4* pDt78R 4 5.2*, 2.2 3.4 a c* pDt85 4 5.2*,4.2 3 4ac* pDtl4 2 12.0* 0.4b* pDt23 2 12.0* 0.4b* pDt55 4 8.0* 4.7,3.3a c;0.8*,0.6b c* pDt62 4 8.0*,3.1 4.7,3.3a c pDt70 4 8.0*,3.2,1.1 4.7,3.3a c pDt92 4 1.7,0.5* 0.3b*;1.3e* pDtl09 5 8.0* 4.7*, 3.3 a c pDtllO 5 2.0*,5.4 2.0*,2.1,2.7ac pDtl l2 5 8.0* 9.0*,3.3a pDtll3' 5 8.0* 9.0*,3.3a pDtl l4 5 8.0*,2.5,1.9 ' 4.7*, 3.7,3.3,2.6,1.7,1.4; p D t l l 5 5 8.0* 9.0*,3.3a pDtl l7 5 8.0* 9.0*,3.3a;4.7*,3.3ac pDtl l8 5 8.0* 9.0*,3.3a p D t l l 9 5 8.0* 9.0*,3.3a;0.8*,0.6*bc pDtl20 5 2.0*,5.4 3.2a*;1.9e* a Fragment generated by digestion with EcoRI. b Fragment generated by digestion with Haelll. c Fragment generated by digestion with Hindlll. d Fragment generated by digestion with Psll. e Fragment generated by digestion with Hhal. * Insert to which [ l 2 5 I ] tRNA hybridizes as determined by the procedures of Southern (1975). R signifies that the parent plasmid has been recloned to give a new plasmid containing only the Hindlll fragment bearing the tRNA gene. 43, M a t e r i a l s and Methods I . General A. T h i n - l a y e r Chromatography Solvents Solvent A: 0.8 M ( N H 4 ) 2 S 0 4 , 10 mM Na2EDTA Solvent B: i s o b u t y r i c a c i d : 30% NH^OH: 1 mm EDTA 66:1:33 Solvent C: 0.1 M sodium phosphate b u f f e r pH 6.8: (NH^SO^: 1-propanol 100:60:2 v/w/v Solvent D: 2-propanol: 12 N HC1: H 20 70:15:15 B. S c i n t i l l a t i o n Counting Aqueous samples c o n t a i n i n g 3H, 1 *C or 1 2 5 1 were prepared f o r counting by mixing them w i t h 7 volumes of a s c i n t i l l a t i o n c o c k t a i l con-t a i n i n g 66 g Omnifluor, 8.1 1 xylene and 3.1 1 T r i t o n N-101. Rad i o a c t i v e p r e c i p i t a t e s deposited on paper, g l a s s f i b r e or n i t r o c e l l u l o s e f i l t e r s were counted i n a c o c k t a i l c o n t a i n i n g 12 g PPO, 0.3 g dimethyl POPOP and 8 1 toluene. The r a d i o a c t i v i t y of samples c o n t a i n i n g 3 2 P was measured i n water. The r a d i o a c t i v i t y of samples was measured i n a Nuclear Chicago Isocap 300 s c i n t i l l a t i o n counter. C. Polyacrylamide Gel E l e c t r o p h o r e s i s Acrylamide stock s o l u t i o n (45%) was deionize d by s t i r r i n g i t succes-s i v e l y w i t h Dowex-1 (OH ) and Dowex-50 (H +) r e s i n . Polyacrylamide g e l s of the re q u i r e d p o r o s i t y were prepared by p o l y m e r i z a t i o n of a mixture c o n t a i n i n g the appr o p r i a t e c o n c e n t r a t i o n of acrylamide, N,N-methylene-bisacrylamide (1/20 con c e n t r a t i o n of acrylamide, Eastman), 45 mM T r i s - b o r a t e b u f f e r pH 8.3, 1 mM EDTA, 0.08% t h i o u r e a and 0.15% v/v hydrogen peroxide (30%). Denaturing g e l s c o n t a i n e d , 7M urea. A n a l y t i c a l and sequencing g e l s were u s u a l l y 0.5 mm t h i c k w h i le p r e p a r a t i v e g e l s were 1.5 mm t h i c k . D. Agarose Gel E l e c t r o p h o r e s i s Agarose g e l s (0.5-2% agarose) were prepared by g e l l i n g an agarose 44. (Biorad) s o l u t i o n c o n t a i n i n g 45 mM T r i s - b o r a t e pH 8.3, 1 mM EDTA, 1 ug/ml ethidium bromide i n a Stud i e r - t y p e g e l apparatus (McDonell et a l . , 1977). The sample w e l l s were f i l l e d w i t h e l e c t r o p h o r e s i s b u f f e r and the DNA samples (30 M l ) , c o n t a i n i n g 10% sucrose and 0.1% bromphenol blu e , were c a r e f u l l y added. The w e l l s were sealed by covering them w i t h warm agarose s o l u t i o n . E l e c t r o p h o r e s i s was done at 100-250 V on a Savant f l a t - b e d e l e c t r o p h o r e s i s apparatus cooled w i t h running tap water. Gels were exposed to u l t r a v i o l e t l i g h t and photographed through an orange f i l t e r u sing P o l a r o i d Type 57 or 667 f i l m . E. Autoradiography TCL p l a t e s or polyacrylamide g e l s were autoradiographed by f i r s t wrap-ping them i n Saran Wrap then p r e s s i n g them i n t o i n t i m a t e contact w i t h X-ray f i l m (Kodak X-OMAT R, Kodak NS-5T, Agfa-Gevaert C u r i x RP-1). The f i l m s were exposed at -20°C and developed according to the manufacturers' i n s t r u c -t i o n s . When e x t r a s e n s i t i v i t y was r e q u i r e d , the f i l m was pre f l a s h e d (Laskey and M i l l s , 1977), sandwiched between the g e l or TLC p l a t e and an i n t e n s i -f y i n g screen (Dupont Cronex L i g h t n i n g - P l u s ) , and exposed at -70°C. V a l V a l F. P u r i f i c a t i o n of tRNA,? and tRNA, Jb 4 V a l V a l tRNA^ and tRNA^ were p u r i f i e d by Dr. I.C. G i l l a m as p r e v i -o u s l y described (Dunn et a l . , 1978). G. Synthesis of f5'- 3 2P]pCp [5'- 3 zP]pCp was prepared by i n c u b a t i n g a 10 u l r e a c t i o n mixture c o n t a i n i n g 1.2 mM Cp(2' and 3*), 20 mM T r i s HC1 pH 8.3, 10 mM MgCl 2, 10 mM d i t h i o t h r e i t o l , 35 uM [ $ - 3 2 P ] A T P (>300 Ci/mmol, Amersham) and 2 u n i t s of T4 p o l y n u c l e o t i d e kinase (P-L Biochemicals) f o r 60 min at 37°C. The r e a c t i o n was stopped by heating the mixture to 100°C f o r 1 min. The [5'- 3 2P]pCp was used without f u r t h e r p u r i f i c a t i o n . 45. H. I s o l a t i o n and C h a r a c t e r i z a t i o n of acrJ_U 3-(3-Amino-3-carboxypropyl) uridine-5'-phosphate was i s o l a t e d from E_. c o l i tRNA by the f o l l o w i n g method. Crude E. c o l i B tRNA (0.5 g) (Schwarz-Mann) was d i s s o l v e d i n 5 ml 50 mM NH^OAc b u f f e r pH 5-3 c o n t a i n i n g 2.5 mM EDTA, heated to 100°C f o r 2 min, cooled and incubated w i t h 0.4 mg of nuclease P^ (Calbiochem) f o r 16 h at 37°C. At the end of t h i s time a f u r t h e r 0.2 mg of nuclease P^ was added and i n c u b a t i o n was continued f o r 7 more hours. 82% of the RNA was rendered a c i d s o l u b l e by the nuclease t r e a t -ment. The enzyme d i g e s t was d i l u t e d w i t h 25 ml of 50 mM Na^O^ pH 10.5, 50 mM NaCl and a p p l i e d to a QAE-Sephadex column (1.2 x 39 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h the d i l u t i o n b u f f e r . Nucleoside-5'-phosphates were e l u t e d from the column w i t h a 600 ml l i n e a r gradient of NaCl (0.05 M to 0.8 M) i n 50 mM Na 2C0 3 pH 10.5. The u l t r a v i o l e t a b s o r p t i o n of the e l u t e d f r a c -t i o n s was monitored at 260 nm and the major n u c l e o t i d e i n each peak of absorbance was i d e n t i f i e d by i t s u l t r a v i o l e t a bsorption spectrum. The f r a c t i o n s making up the t h i r d peak, c o n t a i n i n g mostly uridine-5'-phosphate, were pooled and a c i d i f i e d w i t h 12N HC1 to pH 3. The n u c l e o t i d e s were de s a l t e d by a d s o r p t i o n on c h a r c o a l as described by Thomson (1960). The desalted n u c l e o t i d e s (500 u n i t s ) were d i s s o l v e d i n 10 ml d i s t i l l e d H 20 and a p p l i e d t o a 0.6 x 8 cm column of BioRad AG1-X2 r e s i n (200-400 mesh, formate form). The n u c l e o t i d e s were e l u t e d w i t h a 100 ml l i n e a r gradient of formic a c i d (0 to 3 M). The e l u t i o n p r o f i l e of the column i s presented i n Figure 8. Peaks 4 and 5 contained n u c l e o t i d e s w i t h u l t r a v i o l e t a b sorption spectra (at pH 5 and 12) s i m i l a r to those of acp 3U (Ohashi et a l . , 1974). A l i q u o t s (0.2-0.5 ^260 u n i t s ) from each peak were a p p l i e d to a c e l l u l o s e TLC p l a t e (Eastman) and developed i n Solvent B. The p l a t e was thoroughly d r i e d , the spots of UV absorbing m a t e r i a l were lo c a t e d and the chromatogram was sprayed w i t h 0.1% n i n h y d r i n i n water-saturated 1-butanol. 46. Figure 8. Chromatography of the uridine-5'-phosphate f r a c t i o n recovered from the QAE-Sephadex column on BioRad AG1-X2. F r a c t i o n s from the QAE-Sephadex column c o n t a i n i n g predominantly u r i d i n e -5'-phosphate (503 &260 u n i t s ) were pooled, d e s a l t e d , d i s s o l v e d i n d i s t i l -l e d water and a p p l i e d to a column (0.6 x 8 cm) of BioRad AG1-X2 (200-400 mesh, formate form) that had been thoroughly washed w i t h d i s t i l l e d water. Adsorbed n u c l e o t i d e s were e l u t e d w i t h a 100 ml l i n e a r gradient of formic a c i d (0 to 3 M) at a f l o w - r a t e of 20 ml/hr. Eluted n u c l e o t i d e s were detected by t h e i r UV absorbance at 260 nm. F r a c t i o n s corresponding to peaks of UV absorbance (1-5) (marked by s o l i d l i n e s i n the Figure) were pooled and reduced to dryness over KOH at reduced pressure (10 mm Hg). The residue from each pool was d i s s o l v e d i n 1 ml d i s t i l l e d water and c h a r a c t e r i z e d as described i n the t e x t . Peak 5 was found to co n t a i n acp 3U-5 1-phosphate (7.8 A£60 u n i t s ) . FRACTION NUMBER 48. The p l a t e was heated to 80°C f o r 5 min. Only m a t e r i a l from peak 5 gave the p o s i t i v e n i n h y d r i n r e a c t i o n expected of acp 3U (Ohashi et a l . , 1974). The p o s i t i o n on the TLC p l a t e of t h i s n i n h y d r i n p o s i t i v e m a t e r i a l c o i n c i d e d w i t h the p o s i t i o n of the UV absorbing m a t e r i a l i n peak 5. Some of the n u c l e o t i d e i n peak 5 was dephosphorylated by s e a l i n g 8 u l of 0.1 M HCl c o n t a i n i n g 2 A^^Q u n i t s of the n u c l e o t i d e i n a c a p i l l a r y tube and heating the tube i n the autoclave to 126°C f o r 75 min. The contents of the tube were a p p l i e d to a c e l l u l o s e TCL p l a t e and developed i n solvent D. Two spots of UV absorbing m a t e r i a l were seen on the chromatogram p o s s i b l y because dephosphorylation was not complete. Both spots were n i n h y d r i n p o s i t i v e and one had an very s i m i l a r to that reported f o r acp 3U (Ohashi et a l . . , 1974). I I • T r i n u c l e o t i d e - s t i m u l a t e d Binding of Valyl-tRNA t o Ribosomes A. Synthesis of V a l i n e Codons The f o u r v a l i n e codons, GUA, GUG, GUC and GUU were made by the p o l y -n u c l e o t i d e phosphorylase c a t a l y s e d a d d i t i o n of the appropriate n u c l e o t i d e to a GpU primer (Thach and Doty, 1965). Each 0.4 ml r e a c t i o n mixture t y p i c a l l y contained 0.2 M g l y c i n e pH 9.3, 0.4 M NaCl, 10 mM Mg(0Ac) 2 > 0.1 mM CuS0 4, 0.8 mM NDP, 5 mM GpU (Serva Feinbiochemica) and 1 u n i t primer-dependent p o l y n u c l e o t i d e phosphorylase (Micrococcus l y s o d e i k t i c u s , . P-L Bi o c h e m i c a l s ) . The r e a c t i o n mixture was heated to 70°C f o r 5 min then cooled before adding the enzyme. A f t e r i n c u b a t i o n a t 37°C f o r 12-36 h the r e a c t i o n was stopped by heating the mixture t o 100°C f o r 2 min. Unreacted NDP was dephosphory-l a t e d by i n c u b a t i n g the mixture w i t h 0.2 mg b a c t e r i a l a l k a l i n e phosphatase (Worthington) f o r 1 h at 37°C. The r e a c t i o n mixture was d i l u t e d to 8 ml wi t h d i s t i l l e d 1^0 and a p p l i e d to a column of DEAE-cellulose (^0^ form, 0.7 x 50 cm). The n u c l e o t i d e s were e l u t e d w i t h a l i n e a r g radient (300 ml) of (NH 4)HC0 3 from 0 t o 0.275 M. The t r i n u c l e o t i d e diphosphate product was detected as a peak of absorbance at 260 nm that emerged from the column a f t e r the major peak of unreacted d i n u c l e o s i d e monophosphate s t a r t i n g m a t e r i a l . The t r i n u c l e o s i d e diphosphate s o l u t i o n was desalted by repeated evaporation w i t h ethanol/water under reduced pressure. The y i e l d s of t r i n u -c l e o s i d e diphosphate ranged from 48% f o r GpUpA to 10% f o r GpUpG. The t r i n u c l e o s i d e diphosphates were c h a r a c t e r i z e d by h y d r o l y s i n g a small amount (0.5 u n ^ t ) °f each i n 10% p i p e r i d i n e at 100°C f o r 2 h or w i t h RNase A (4 u n i t s i n l O i u l 10 mM T r i s - H C l pH 7.5) at room temperature f o r 2 h. The hydro l y s a t e s were a p p l i e d t o c e l l u l o s e TLC p l a t e s along w i t h the appropriate n u c l e o t i d e and nucleoside standards and the p l a t e was d e v e l -oped i n Solvent B. A l l the t r i n u c l e o s i d e diphosphates gave the expected h y d r o l y s i s products. V a l B. I s o l a t i o n of [j_H]Valy 1-1RNA Isoacceptors Crude Dros o p h i l a tRNA was i s o l a t e d from a d u l t Oregon R f l i e s by the method of Roe (1975). Crude tRNA (1.3 mg) was aminoacylated w i t h 0.1 mCi L - [ 2 , 3 , 4 - 3 H ] v a l i n e (10 Ci/mmol, New England Nuclear) using the c o n d i t i o n s of White and Tener (1973c) f o r the r e a c t i o n . A f t e r aminoacylation the reac-t i o n mixture (1.25 ml) was d i l u t e d w i t h 3.5 ml of 50 mM NaOAc pH 4.5, 0.3 M NaCl, 10 mM Mg(0Ac) 2 and a p p l i e d to a sm a l l DEAE-cellulose column (1.1 x 5 cm) e equilibrated w i t h the same b u f f e r . The column was washed w i t h 60 ml of the above d i l u t i o n b u f f e r then the tRNA was e l u t e d by i n c r e a s i n g the NaCl c o n c e n t r a t i o n to 1.1 M. 1 ml f r a c t i o n s were c o l l e c t e d manually and those f r a c t i o n s c o n t a i n i n g [ 3H]valyl-tRNA were pooled. The n u c l e i c a c i d was p r e c i p i t a t e d w i t h 2 volumes of ethanol at -20°C and the p r e c i p i t a t e c o l -l e c t e d on a M i l l i p o r e f i l t e r (Type HA), washed w i t h ethanol and d r i e d . V a l The 3 major [ 3H]valyl-tRNA i s o a c c e p t o r s were separated by RPC-5 chromatography using the b u f f e r systems of Dunn et a l . (1978). The p r e c i p i -t a t e d tRNA (above) was d i s s o l v e d i n 3 ml of 0.45 M NaCl i n Bu f f e r A (10 mM •50. NaOAc pH 4.0, 10 mM MgCl 2, 1 mM 2-mercaptoethanol) and a p p l i e d to an RPC-5 column (0.9 x 60 cm) e q u i l i b r a t e d w i t h the same b u f f e r . The tRNA was el u t e d from the column w i t h a l i n e a r gradient (600 ml) of B u f f e r A c o n t a i n i n g 0.5 to 0.65 M NaCl. The column temperature was 37°C and the flow r a t e was 15 ml/h. F r a c t i o n s c o n t a i n i n g the two major peaks of r a d i o a c t i v i t y , c o r r e s -ponding to [ 3 H ] v a l y l - t R N A ^ 3 b and [ 3H] v a l y - t R N A ^ a l ( F i g . 10A), were each pooled and p r e c i p i t a t e d w i t h ethanol as described above. The two v a l i n e tRNA f r a c t i o n s were f u r t h e r p u r i f i e d by RPC-5 chromatography i n a second b u f f e r system. Each sample was d i s s o l v e d i n 3 ml of Bu f f e r C (10 mM sodium formate pH 3.8, 1 mM Na 2 EDTA, 1 mM 2-mercaptoethanol) con-t a i n i n g 0.45 M NaCl and a p p l i e d to an RPC-5 column (0.9 x 60 cm) that had been e q u i l i b r a t e d w i t h the second b u f f e r . The column was e l u t e d w i t h a l i n e a r gradient (600 ml) of Bu f f e r B c o n t a i n i n g 0.55 to 0.75 M NaCl, the column temperature and flo w r a t e were the same as f o r the previous column. The 3 p u r i f i e d [ 3H]valyl-tRNA isoacceptors ( F i g . 10B, F i g . 10C) were each p r e c i p i t a t e d w i t h ethanol and the p r e c i p i t a t e s were c o l l e c t e d on M i l l i p o r e f i l t e r s f o r storage at -70°C. V a l C. Determination of Codon T r i p l e t - Stimulated Binding of Valyl-tRNA to  Ribosomes The codons recognized by each of the v a l i n e tRNA is o a c c e p t o r s were determined using the ribosome binding assay of Nirenberg and Leder (1964). Each ribosome-binding r e a c t i o n (50 u l ) contained 0.1 M Tris-OAc pH 7.2, 20 V a l mM Mg(0Ac) 2, 50 mM KC1, 2 A 2 6 Q u n i t s of ribosomes, 4 pmol [ 3H]valyl-tRNA i s o a c c e p t o r and from 0 to 5 nmoles of one of the v a l i n e codon t r i p l e t s . The re a c t i o n s were incubated at 24°C f o r 30 min then stopped by the a d d i t i o n of 5 volumes of i c e - c o l d wash b u f f e r (0.1 M Tris-OAc pH 7.2, 20 mM Mg(0Ac) 2 > 50 mM KC1). The ribosomes were c o l l e c t e d on M i l l i p o r e f i l t e r s (Type HA) and washed w i t h 120 ml of c o l d wash b u f f e r . The f i l t e r s were d r i e d and the 51. bound r a d i o a c t i v i t y measured by s c i n t i l l a t i o n counting. I I I . Synthesis of 1 2 5 I - C T P 1 2 5 1 - C T P ( 1 2 5 I - 5 - i o d o c y t i d i n e - 5 ' - t r i p h o s p h a t e ) of high s p e c i f i c a c t i v i t y was made by a m o d i f i c a t i o n of the procedure of Scherberg and R e f e t o f f (1974). Two Ml of c a r r i e r - f r e e N a 1 2 5 I (Amersham, 16 mCi/Mg of i o d i n e , approximately 550 mCi/ml) was added to 18 Ml of c o l d (0°C) 44 mM CTP, 0.17 M NaOAc pH 4.0, 1.1 mM T l C l ^ i n a capped 1.5 ml c o n i c a l polypropylene tube and heated to 70°C f o r 20 min. The mixture was c h i l l e d on i c e and 2 M1 of 2-mercaptoethanol was added. To p u r i f y the 1 2 5 I - C T P , the r e a c t i o n mix-ture was a p p l i e d as a 1 cm streak near one corner of a 20 x 20 cm PEI-c e l l u l o s e TLC p l a t e (HC0~ form)(Polygram Cel 300 PEI, Machery-Nagel). The p l a t e was developed w i t h 0.2 M NH^HCO^ 10 mM EDTA i n the f i r s t dimension, d r i e d thoroughly, and developed w i t h 0.1 M NaH^PO^, pH 6.8 (NaOH)/(NH 4) 2S0 4/0.25 M EDTA/l-propanol (100 ml:60 g:4 ml:2 ml) i n the second dimension. This procedure separates 1 2 5 I - C T P from unreacted CTP and from iodin a t e d s i d e products ( 1 2 5I-CDP, 1 2 5I-CMP, e t c . ) . The 1 2 5 I - C T P , which was l o c a t e d on the TLC p l a t e by autoradiography (Figure 9 ) , was scraped from the p l a t e and e l u t e d from the scrapings w i t h 6 x 0.5 ml p o r t i o n s of 2.5 M NaCl. The 1 2 5 I-CTP was desalted by applying i t to a column (0.7 x 1.0 cm) of acid-washed c h a r c o a l . The column was washed w i t h 15 ml d i s t i l l e d water and the 1 2 5 I-CTP was e l u t e d w i t h 2 ml of e t h a n o l / water/30% ammonium hydroxide (20:20:5). The e l u a t e was reduced to dryness over ?2®5 * n a v a c u u m d e s s i c a t o r at 10 mm Hg and the residue taken up i n 5 0 M 1 ethanol:water (1:1) f o r storage at -20°C. Twenty-five percent of the input 1 2 5 1 could be incorporated i n t o 1 2 5 I - C T P by t h i s method, g i v i n g a product c o n t a i n i n g e s s e n t i a l l y u n d i l u t e d 1 2 5 I - C T P . 52. Fi g u r e 9. Autoradiogram of a two-dimensional chromatogram used t o separate 1 2 5 I - C T P from u n l a b e l l e d CTP. . Shaded areas represent UV-absorbing m a t e r i a l s . The P E I - c e l l u l o s e TLC p l a t e was developed i n 0.2 M ( N H ^ I K ^ , 10 mM EDTA i n the f i r s t dimen-s i o n (1) and i n 0.1 M NaH 2P0 4, pH 6.8 (NaOH)/(NH 4) 2SO 4/0.25 M EDTA/l-propanol (100 ml:60 g:4 ml:2 ml) i n the second dimension ( 2 ) . 54. IV. I s o l a t i o n of tRNA N u c l e o t i d y l Transferase from Yeast A. Enzyme I s o l a t i o n tRNA n u c l e o t i d y l t r a n s f e r a s e was i s o l a t e d from bakers' yeast by m o d i f i -c a t i o n s to the procedure of Mo r r i s and Herbert (1970). Compressed bakers' yeast (125 g, Fleishmann) was crumbled i n t o f i n e pieces and added to a Dewar f l a s k c o n t a i n i n g 300 ml of toluene at -70°C. The temperature was maintained at -70°C f o r 3 h by the a d d i t i o n of dry i c e as needed. The fro z e n yeast was c o l l e c t e d on a Buchner funnel (no paper), t r a n s f e r r e d to a 250 ml beaker and 40 ml of 1.25 M MOPS b u f f e r pH 8 (NaOH) c o n t a i n i n g 10 mM EDTA and 10 mM 2-mercaptoethanol were added. The r e s t of the enzyme p u r i f i c a t i o n was c a r r i e d out at 4°C. The c e l l s were thawed on i c e f o r 8 h, excess toluene was removed and the c e l l s were allowed to autolyse f o r 24 h. During a u t o l y s i s i t was important to prevent the pH of the au t o l y s a t e from f a l l i n g below pH 7.5. The pH was adjusted by a d d i t i o n of 30% NH^OH as needed. C e l l u l a r d e b r i s was removed from the au t o l y s a t e by c e n t r i f u g a t i o n at 12,000 x g f o r 15 min. The supernatant was brought to 58% s a t u r a t i o n w i t h (NH 4) 2S0^ by a d d i t i o n of saturated ( N H ^ S O ^ (saturated at 4°C) i n 50 mM T r i s - H C l pH 7.5, 25 mM EDTA and s t i r r e d f o r 2 h. The p r e c i p i t a t e d p r o t e i n s were c o l l e c t e d by c e n t r i f u g a t i o n at 13,200 x g f o r 20 min and sus-pended i n 40% saturated ( N H 4 ) 2 S 0 4 , 20 mM T r i s - H C l pH 7.5, 10 mM EDTA, 15 mM MgCl 2, 10 mM 2-mercaptoethanol. A f t e r s t i r r i n g f o r 2 h the s o l u t i o n was c l a r i f i e d by c e n t r i f u g a t i o n at 16,000 x g f o r 20 min. One volume of saturated ( N H ^ S O ^ i n 25 mM T r i s - H C l pH 7.5, 10 mM 2-mercaptoethanol was added to the supernatant and the mixture was s t i r r e d f o r 30 min at 4°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 at 16,000 x g f o r 20 min, drained w e l l and d i s s o l v e d i n 10 ml of 20% g l y c e r o l , 5 mM T r i s - H C l pH 7-5, 10 mM 2-mercaptoethanol ( g l y c e r o l - b u f f e r ) . The enzyme s o l u t i o n was desa l t e d on a column (2 x 47 cm) of Sephadex G-25 (coarse) e q u i l i b r a t e d w i t h 55. g l y c e r o l - b u f f e r . The peak of m a t e r i a l absorbing at 280 nm from the Sephadex column was d i l u t e d to < 1 &280 un^t/m-'- w i t h g l y c e r o l - b u f f e r and a p p l i e d t o a wide, short column (5.5 x 2 cm) of DEAE-cellulose. The m a t e r i a l not adsorbed on the DEAE-cellulose column was a p p l i e d to a column (1.2 x 10 cm) of phosphocellulose (Whatman P 11) that had been e q u i l i b r a t e d w i t h 20% g l y c e r o l , 25 mM T r i s - H C l pH 7.5, 1 mM EDTA and 10 mM 2-mercaptoethanol. The enzyme was e l u t e d from the column w i t h a l i n e a r gradient (100 ml t o t a l volume) of 0 to 0.8 M NaCl i n column b u f f e r . The column f r a c t i o n s (1 ml) were assayed f o r tRNA n u c l e o t i d y l t r a n s f e r a s e a c t i v i t y and the enzyme was seen to emerge as a broad peak centered about 0.5 M NaCl. The enzyme was concentrated by d i l u t i n g the pooled a c t i v e f r a c t i o n s from the phosphocellu-l o s e column w i t h 3 volumes of 40% g l y c e r o l , 0.25 M T r i s - H C l pH 7.5, 1 mM EDTA, 10 mM 2-mercaptoethanol and applying the s o l u t i o n to a s m a l l column of phosphocellulose (1.2 x 2 cm) e q u i l i b r a t e d w i t h the d i l u t i o n b u f f e r . The enzyme was e l u t e d w i t h d i l u t i o n b u f f e r c o n t a i n i n g 0.6 M NaCl. The a c t i v e f r a c t i o n s from the column were pooled, d i v i d e d i n t o 0.5 ml a l i q u o t s and stored at -70°C. A t o t a l of 4700 u n i t s (defined below) of a c t i v i t y were recovered and the enzyme co n c e n t r a t i o n was 0.65 u n i t s / p l . B. tRNA N u c l e o t i d y l Transferase Assay Substrate f o r the tRNA n u c l e o t i d y l t r a n s f e r a s e assay, was prepared by periodate o x i d a t i o n of the 3'-terminal r i b o s e residue of tRNA followed by the l y s i n e c a t a l y s e d 3 - e l i m i n a t i o n of the o x i d i z e d n u c l e o s i d e . The 3'-phosphate of the shortened RNA was removed w i t h a l k a l i n e phosphatase. The 3'-terminal n u c l e o t i d e was removed from 50 mg of crude yeast tRNA by the method of Khym and U z i e l (1968). The product tRNA ("tRNA-CC") was obtained as i t s cetyltrimethylammonium s a l t . This p r e c i p i t a t e was converted to the ammonium s a l t by e x t r a c t i n g i t 3 times w i t h 0.1 M NH^OAc i n 70% et h a n o l . The enzyme assay mixture (0.125 ml) contained 50 mM g l y c i n e pH 9.5 (NaOH), 10 mM MgS0 4 > 0.15 mM CTP, 6 A 2 6 Q u n i t s tRNA-CC, 2 mM [ 3H]ATP (4 mCi/mmol) and 10 p i of sample s o l u t i o n . The mixture was incubated f o r 40 min at 37°C then 2 ml c o l d (0°C) 5% TCA was added. The p r e c i p i t a t e d RNA was c o l l e c t e d on a g l a s s f i b r e f i l t e r (Reeve Angel) and the f i l t e r s were washed s u c c e s s i v e l y w i t h two 2 ml p o r t i o n s of 5% TCA, 5 ml 95% ethanol and 5 ml ether, d r i e d and counted i n a s c i n t i l l a t i o n counter. One u n i t of tRNA n u c l e o t i d y l t r a n s f e r a s e i s the amount re q u i r e d to i n c o r p o r a t e 1 nmol of ATP i n t o substrate tRNA i n 1 min at 37°C under the above assay c o n d i t i o n s . V. End-Labelling of tRNA A. 3' End-Labelling R a d i o l a b e l l e d n u c l e o t i d e s were incorp o r a t e d i n t o the 3'-end of tRNA molecules by e i t h e r tRNA n u c l e o t i d y l t r a n s f e r a s e or T4 RNA l i g a s e . In pr e p a r a t i o n f o r l a b e l l i n g w i t h n u c l e o t i d y l t r a n s f e r a s e , the 3'-CCA end of the p u r i f i e d tRNA was removed by l i m i t e d d i g e s t i o n of the tRNA w i t h snake venom phosphodiesterase ( S p r i n z l je_t a l . , 1972). A 10 p i r e a c t i o n mixture c o n t a i n i n g 10 Pg tRNA, 0.6 u n i t s ( R a z z e l l , 1963) of snake venom phospho-d i e s t e r a s e (Worthington), 50 mM T r i s , pH 8.0 and 10 mM MgCl 2 was incubated at 25°C f o r 30 min. The r e a c t i o n was stopped by heating the mixture to 100°C f o r 1 min. An [a- 3 2P]AMP residue c o u l d be incorporated i n t o the 3'-end of tRNA by i n c u b a t i n g 10 pg of snake venom phosphodiesterase t r e a t e d tRNA i n 10 p i of 100 mM T r i s - H C l b u f f e r pH 9.0, c o n t a i n i n g 100 mM KC1, 10 mM magnesium a c e t a t e , 2 mM d i t h i o t h r e i t o l , 1 mM EDTA, 0.15 mM CTP, 60 pCi [a- 3 2P]ATP (12 Ci/mmol, Amersham) and 1.2 u n i t s of tRNA nucleo-t i d y l t r a n s f e r a s e f o r 20 min at 32°C. The r e a c t i o n was stopped by adding 10 p l 7 M urea c o n t a i n i n g 0.5% xylene cyanol and bromphenol blue. The mixure was a p p l i e d to the sample s l o t i n a 16 x 17 x 0.15 cm 20% polyacrylamide g e l c o n t a i n i n g 7 M urea. A f t e r e l e c t r o p h o r e s i s the t e r m i n a l l y l a b e l l e d tRNA was 57-... lo c a t e d i n the g e l by autoradiography and e l u t e d from the g e l s l i c e by soak-i n g i n 0.5 ml 0.1 M sodium a c e t a t e , 0.1% SDS f o r 1-2 days at 4°C. The l a b e l l e d tRNA and 10 ug of added c a r r i e r tRNA was p r e c i p i t a t e d from the e l u a t e w i t h 3 volumes of ethanol and was used f o r sequence a n a l y s i s . tRNA n u c l e o t i d y l t r a n s f e r a s e was a l s o used to i n c o r p o r a t e 1 2 SI-CMP residues i n t o the 3'-ends of tRNA molecules. L a b e l l i n g was c a r r i e d out i n a 10 u l volume c o n t a i n i n g 2 ug phosphodiesterase-treated tRNA, 6-9 x 10 7 dpm 1 2 5 I - C T P , 50 mM g l y c i n e b u f f e r , pH 9.5, 10 mM MgCl 2, 0.1 mM ATP, 5 mM 2-mercaptoethanol and 1.2 u n i t s of tRNA n u c l e o t i d y l t r a n s f e r a s e . A f t e r i n c u b a t i o n at 32°C f o r 45 min 10 u l of a saturated s o l u t i o n of urea con-t a i n i n g 0.5% xylene cyanol and 0.5% bromphenol blue was added to stop the r e a c t i o n . The r e a c t i o n mixture was a p p l i e d to the sample s l o t i n a 16 x 17 x 0.15 cm 20% polyacrylamide g e l c o n t a i n i n g 7 M urea. A f t e r e l e c t r o p h o r e s i s the t e r m i n a l l y l a b e l l e d tRNA was l o c a t e d on the g e l by autoradiography, e l u t e d from the g e l s l i c e by soaking i n 0.4 ml 0.1 M NaOAc, pH 6.0, 0.1% SDS, 1 mM EDTA f o r 1 to 2 days at 4°C, and then p r e c i p i t a t e d from the e l u a t e w i t h ethanol i n the presence of 20 ug c a r r i e r E_. c o l i tRNA. The p r e c i p i -t a t e was r i n s e d w i t h c o l d (-20°C) 70% e t h a n o l , d r i e d i n vacuo and d i s s o l v e d i n water. 60 to 80% of the input 1 2 5 I - C T P was recovered i n the tRNA. tRNA was a l s o l a b e l l e d at the 3'-end w i t h [5'- 3 2P]pCp using phage T4 RNA l i g a s e (P-L Biochemicals) as described by England and Uhlenbeck (1978). B. 5' End-Labelling L a b e l l i n g the 5'-end of the v a l i n e tRNAs using [#- 3 2P]ATP and polynu-c l e o t i d e kinase was i n e f f i c i e n t unless the tRNA was f i r s t cut i n the a n t i -codon loop by l i m i t e d d i g e s t i o n w i t h RNase U 2 to give h a l f molecules. The V a l 10 u l r e a c t i o n mixture c o n t a i n i n g 2.5 ug tRNA , 10 mM ammonium acetate b u f f e r , pH 4.5 and 0.01 u n i t of RNase U"2 was incubated at 4°C f o r 1 h, heated to 100°C f o r 1 min, then c h i l l e d on i c e . 1 u l of 1 M T r i s - H C l 58.-pH 7.5 and 1 u l 100 mM MgCl2 were added t o the r e a c t i o n mixture and the tRNA fragments were dephosphorylated by adding 1 u n i t of c a l f i n t e s t i n e a l k a l i n e phosphatase (Boehringer Mannheim) and in c u b a t i n g the mixture a t 45°C f o r 30 min. The r e a c t i o n was stopped by adding 2 u l 50 mM n i t r i l o -t r i a c e t i c a c i d , 1 P i 2-mercaptoethanol, 1 U l 1% SDS and heating i t to 100°C f o r 2 min. The RNA was p r e c i p i t a t e d w i t h 3 volumes of ethanol a t -70°C, the p r e c i p i t a t e was r i n s e d w i t h 95% ethanol and d r i e d i n vacuo. The RNA fragments were e n d - l a b e l l e d w i t h [ 3 2P]phosphate by i n c u b a t i n g them i n a 10 u l r e a c t i o n mixture c o n t a i n i n g 50 mM T r i s - H C l pH 8.0, 50 mM NaCl, 20 mM KC1, 10 mM MgCl 2, 2 mM spermine h y d r o c h l o r i d e , 2 mM d i t h i o t h r e i t o l , 20-30 uCi ['i ,- 3 2P]ATP (2000-3000 Ci/mmol, Amersham) and 1.5 u n i t phage T4 polynucleotide kinase (New England B i o l a b s ) at 37°C f o r 30 min. The kinase r e a c t i o n was stopped by adding 10 u l of 7 M urea c o n t a i n i n g 0.5% xylene cyanol and bromphenol blue and was a p p l i e d to the sample s l o t i n a 35 x 15 x 0.05 cm denaturing 20% polyacrylamide g e l . A f t e r e l e c t r o p h o r e s i s l a b e l l e d tRNA fragments were l o c a t e d on the g e l by autoradiography. Fragments appro-ximately 35 n u c l e o t i d e s long were el u t e d from g e l s l i c e s and used f o r sequence a n a l y s i s . V I . RNA Sequence A n a l y s i s A. Stanley and V a s s i l e n k o Method V a l V a l The sequencing of tRNA^^ and tRNA^ was done by a combina-t i o n of methods. Most of the n u c l e o t i d e sequence was obtained using the method of Stanley and V a s s i l e n k o (1978). P u r i f i e d tRNA was prepared f o r sequencing by in c u b a t i n g i t i n a 10 u l r e a c t i o n mixture c o n t a i n i n g 45 ug tRNA, 50 mM g l y c i n e pH 9.5 (NaOH), 10 mM MgS0 4, 2 mM ATP, 0.15 mM CTP, 2 mM d i t h i o t h r e i t o l , 1.2 u n i t s tRNA n u c l e o t i d y l t r a n s f e r a s e and 2 u n i t s T4 p o l y n u c l e o t i d e kinase (New England B i o l a b s ) f o r 40 min at 37°C. 20 u l of 59. saturated urea s o l u t i o n c o n t a i n i n g 0.1% xylene cyanol and bromphenol blue was added to the r e a c t i o n mixture before i t was a p p l i e d to 3 wide (2.5 cm) sample s l o t s i n a denaturing 20% polyacrylamide g e l (35 x 15 x 0.15 cm). E l e c t r o p h o r e s i s was c a r r i e d out u n t i l the xylene cyanol marker dye had reached the bottom of the g e l . The g e l was stained w i t h ethidium bromide (1 Ug/ml), the topmost f l u o r e s c e n t band was excis e d and the RNA e l u t e d by soaking the g e l fragment overnight i n 1.5 ml of 0-1 M NaOAc pH 6.0, 0.1% SDS, 1 mM EDTA at room temperature. The e l u t e d RNA was p r e c i p i t a t e d w i t h 3 volumes of ethanol and the p r e c i p i t a t e was d r i e d i n vacuo. The Stanley and Vassilenko (1978) sequencing procedure was modified i n the f o l l o w i n g ways. G e l - p u r i f i e d tRNA (2 ug) was sealed i n a gl a s s c a p i l -l a r y tube w i t h 10 U l of e i t h e r 5 mM MOPS b u f f e r pH 7.2, 0.1 mM EDTA or 10 mM NH^OAc pH 4.5, 1 mM EDTA and heated to 100°C f o r 3 min. The acetate b u f f e r produced l i m i t e d , random h y d r o l y s i s of the tRNA more c o n s i s t e n t l y than d i d the MOPS b u f f e r . A l k a l i n e h y d r o l y s i s of the l a b e l l e d RNA fragments was replaced by d i g e s t i o n w i t h 0.05 u n i t RNase T 2 i n 10 u l of 10 mM NH^OAc b u f f e r pH 4.6 c o n t a i n i n g 2 mM EDTA f o r 16 h at 37°C. The l a b e l l e d nucleoside-5' , 3'-bisphosphates (pNp's) were i d e n t i f i e d by comparing t h e i r m o b i l i t i e s on P E I - c e l l u l o s e TLC p l a t e s (Polygram Cel 300 PEI, Machery-Nagel) i n Solvent A and on c e l l u l o s e TLC p l a t e s (E. Merck or Eastman Kodak) i n Solvent B w i t h published values ( S i l b e r k l a n g et_ al., 1979; Cr i b b s , 1979). Modified pNp's were incubated w i t h 1 ug nuclease P^ (Calbiochem) f o r 16 h at 25°C i n 5 u l 10 mM NH40Ac b u f f e r pH 4.6, 2 mM EDTA and the r e s u l -t i n g nucleoside-5'-phosphates were i d e n t i f i e d by chromatography on c e l l u l o s e TLC p l a t e s i n Solvent B and Solvent C or D. In some cases the l a b e l l e d RNA fragments were hydrolysed d i r e c t l y to nucleoside-5-phosphates w i t h nuclease 6 0 . B. Wandering-spot A n a l y s i s The sequences near the 5' and 3'-ends of the tRNAs were determined by wandering spot a n a l y s i s . The procedure followed was a m o d i f i c a t i o n of that of S i l b e r k l a n g eit a l . (1979). E n d - l a b e l l e d RNA and 5 pg of c a r r i e r RNA were d i s s o l v e d i n 4 y l of deionized 98% formamide and p a r t i a l l y hydrolysed by heating the s o l u t i o n f o r 1 h at 100°C. 1 y l of a dye mix c o n t a i n i n g 0.33% of xylene cyanol FF, orange G and a c i d f u c h s i n was added to the hydro-l y s a t e and the mixture was a p p l i e d to a thoroughly b l o t t e d s t r i p of c e l l u -l o s e acetate membrane (3 x 55 cm, S c h l e i c h e r and S c h u e l l No. 2500) th a t had been soaked i n a b u f f e r c o n t a i n i n g 5% HOAc, 7 M urea and 5 mM EDTA (pH 3.5). While the drop of RNA hydrolys a t e soaked i n t o the c e l l u l o s e acetate s t r i p the sample was covered w i t h an i n v e r t e d 1 ml beaker to r e t a r d evaporation. The r e s t of the s t r i p , except f o r about 1.5 cm around the o r i g i n , was covered w i t h a l a y e r of Saran Wrap. A f t e r the sample had soaked i n , the s t r i p was covered w i t h s i n g l e l a y e r of Saran Wrap and connected by 3 MM paper wicks to the e l e c t r o d e compartments of a Shandon high voltage e l e c t r o -p h o r e s i s apparatus. The e l e c t r o p h o r e s i s b u f f e r contained 5% HOAc, pH 3.5 ( p y r i d i n e ) and 5 mM EDTA. E l e c t r o p h o r e s i s was done at 500 V t i l l the dyes had separated, then the v o l t a g e was increased t o 3000 V. E l e c t r o p h o r e s i s was stopped when the xylene cyanol dye had migrated 6-8 cm. The separated 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 from the c e l l u l o s e acetate membrane to a 20 x 40 cm DEAE-cellulose TLC p l a t e (Polygram C e l 300 DEAE, Machery-Nagel) by the method of Southern (1974). The TLC p l a t e was washed i n d i s t i l l e d ^ 0 , d r i e d , and taped to a g l a s s p l a t e (20 x 38 cm). The TLC p l a t e was developed w i t h 1 mM EDTA at 65°C t i l l the solvent f r o n t was 10 cm beyond the o r i g i n , then the p l a t e was t r a n s f e r r e d to a second tank where development was continued w i t h Homo-mix V (Jay et a l . , 1974) at 65°C t i l l the solvent f r o n t reached the top of the p l a t e . The p l a t e was d r i e d and a u t o r a d i o -61. graphed. C. Gel Read-Off Method RNA sequencing by p a r t i a l , base-specific,enzymatic cleavage of end-l a b e l l e d RNA ( g e l read-off method) was used to f i l l i n gaps i n the sequence and con f i r m r e s u l t s obtained by other methods. The procedures described are m o d i f i c a t i o n s of those of D o n i s - K e l l e r et^ a l . (1977), Simoncsits et_ a l . (1977) and Gupta and Randerath (1977). Each RNase A (Worthington) or RNase T^ (Calbiochem) r e a c t i o n was done i n 10 u l of a b u f f e r c o n t a i n i n g 6 mM sodium c i t r a t e pH 5.0, 6 mM EDTA, 4.2 M urea and 0.5 ug of tRNA (end-l a b e l l e d tRNA plus c a r r i e r tRNA). The 1.9 ml p l a s t i c c e n t r i f u g e tubes (Evergreen) c o n t a i n i n g the r e a c t i o n s were heated to 100°C f o r 20 s then c h i l l e d on i c e before the enzymes were added. U s u a l l y s e v e r a l r e a c t i o n s , each c o n t a i n i n g a d i f f e r e n t amount of enzyme, were done t o ensure that a good d i s t r i b u t i o n of p a r t i a l d i g e s t i o n products was obtained. From 0.01-0.005 u n i t of RNase A and 0.1-0.01 u n i t of RNase were used. The rea c -t i o n s were incubated f o r 15 min at 50°C. RNase U 2 (Calbiochem, 0.01-0.001 u n i t ) and RNase Phy I (Enzo Biochem, 1-0.1 u n i t ) were incubated f o r 15 min a t room temperature i n 10 u l r e a c t i o n s c o n t a i n i n g 10 mM NH^OAc pH 4.5, 1 mM EDTA and 0.5 ug RNA. A l l the enzymatic r e a c t i o n s were stopped by adding 1 u l of 0.5% SDS, 50% 2-mercaptoethanol to each r e a c t i o n and heating them to 100°C f o r 30 s. The samples were reduced to dryness i n a vacuum d e s i c c a t o r . Those samples c o n t a i n i n g urea were d i s s o l v e d i n 5 u l d i s t i l l e d 1^0 c o n t a i n i n g 0.1% xylene cyanol and bromphenol blu e , the remaining samples were d i s s o l v e d i n 7 M urea c o n t a i n i n g the same dyes. The samples were heated to 100°C f o r 30 s and a p p l i e d to the s l o t s of a p o l y -acrylamide sequencing g e l . A "ladder" of fragments derived from breaks at every phosphodiester bond i n the tRNA was produced by p a r t i a l h y d r o l y s i s of the RNA i n aqueous 62. formamide. Formamide was deioniz e d by s t i r r i n g i t c o n s e c u t i v e l y w i t h Dowex-1 (OH ) and Dowex-50 (H +) r e s i n and was stored at 4°C. End-l a b e l l e d RNA (1 ug) and 12 u l of 66% formamide were sealed i n a g l a s s c a p i l l a r y tube and heated at 100°C f o r 20 min. 1 u l of 0.1 M NH^OAc pH 4.5, 10 mM EDTA was added to the contents of the tube before the hydrolysate was a p p l i e d t o a s l o t i n a sequencing g e l . D. M o d i f i c a t i o n of tRNA w i t h Chloroacetaldehyde The occurrence of f i v e consecutive Cp residues (C^-C,.^) i n the V a l v a r i a b l e loop and T-stem of tRNA^ could not be s a t i s f a c t o r i l y demon-s t r a t e d by e i t h e r the Stanley and V a s s i l e n k o or the g e l read-off methods, presumably because of strong secondary s t r u c t u r e i n t h i s r e g i o n . M o d i f i c a -t i o n of the RNA w i t h chloroacetaldehyde allowed the sequence t o be res o l v e d . A 50% s o l u t i o n (v/v) of chloroacetaldehyde was prepared by heating 25 u l of 97% chloroacetaldehyde dimethyl a c e t a l ( A l d r i c h ) w i t h an equal volume of 0.2 M HC1 i n a sealed g l a s s tube at 100°C f o r 30 min. End-l a b e l l e d tRNA plus c a r r i e r tRNA (2-5 ug) was d i s s o l v e d i n 15 u l of 1 M sodium acetate b u f f e r pH 4.0, 1 mM EDTA c o n t a i n i n g 2 u l of the c h l o r o -acetaldehyde s o l u t i o n . The s o l u t i o n was sealed i n a g l a s s c a p i l l a r y tube and heated to 100°C f o r 1 min, then c h i l l e d on i c e . The contents of the tube were e x p e l l e d i n t o 100 u l of 0.1 M sodium acetate b u f f e r pH 4.0 con-t a i n i n g 0.1 mM EDTA and the s o l u t i o n was incubated at 80°C f o r 20 min. The RNA was p r e c i p i t a t e d w i t h ethanol and i t s sequence was determined by the g e l read-off method. V I I . Recombinant Plasmid DNA I s o l a t i o n B a c t e r i a c o n t a i n i n g recombinant plasmids were grown i n M9S medium (Champe and Benzer, 1962) c o n t a i n i n g 0.4% glucose, 0.25% casamino a c i d s , 0.025% MgSO, , 0.02% u r i d i n e and 0.001% thiamine. Cultures were incubated 63. at 30°C w i t h vigorous a g i t a t i o n (300 rpm) i n an a i r shaker bath (New Brunswick S c i e n t i f i c ) u n t i l the k,cr, of the c u l t u r e was about 0.6. Ch l o r -650 amphenicol (80 mg/ml i n 95% ethanol) was then added to a f i n a l concentra-t i o n of 200 Mg/ml and in c u b a t i o n was continued f o r 12-16 h. The c e l l s i n a 1 l i t e r c u l t u r e were c o l l e c t e d by c e n t r i f u g a t i o n and suspended i n 10 ml of co l d b u f f e r (0°C) c o n t a i n i n g 12.5% sucrose, 25 mM T r i s - H C l pH 8 and 0.25 M EDTA (pH 8 ) . Egg white lysozyme (2.5 ml, Sigma 3x c r y s t a l l i z e d , 5 mg/ml) was added and the mixture was incubated f o r 15 min at 0°C. The c e l l s were l y s e d by r a p i d l y i n j e c t i n g 7 ml of 2% T r i t o n X-100 i n t o the c e l l suspension w i t h a syringe (50 ml, 18 gauge needle). The l y s a t e was c e n t r i f u g e d at 25,000 rpm f o r 1 h i n a Beckman 30 r o t o r . The supernatant was ex t r a c t e d w i t h 20 ml phenol:CHCl 3 (1:1, e q u i l i b r a t e d w i t h 50 mM T r i s - H C l pH 8 ) . The upper phase was r e e x t r a c t e d as above, then twice w i t h 20 ml p o r t i o n s of CHCl^iisoamyl a l c o h o l (24:1). The d e p r o t e i n i z e d c l e a r e d l y s a t e was i n c u -bated w i t h 1 ml of RNase A (1 mg/ml, Sigma, heated 100°C/5 min) f o r 10 min at 37°C then i t was ext r a c t e d once w i t h phenol:CHC1 3 and once w i t h CHCl^iisoamyl a l c o h o l as above. N u c l e i c a c i d was p r e c i p i t a t e d w i t h 3 volumes of ethanol at -20°C f o r 16 h. The p r e c i p i t a t e was d i s s o l v e d i n 10 ml 10 mM T r i s - H C l pH 8, 10 mM NaCl, 1 mM EDTA then 3.75 mg of ethidium bromide and 10 g of CsCl (Koweki Berylco) were d i s s o l v e d i n the DNA s o l u -t i o n . The s o l u t i o n was c e n t r i f u g e d to e q u i l i b r i u m at 35,000 rpm i n a Beckman Ti50 r o t o r . Plasmid DNA was c o l l e c t e d from the CsCl g r a d i e n t . The DNA s o l u t i o n was ex t r a c t e d 3 times w i t h water-saturated 1-butanol and twice w i t h ether then d i a l y z e d against 1 l i t e r of 50 mM NaCl, 10 mM T r i s - H C l pH 8, 1 mM EDTA. The b u f f e r was changed once during the course of d i a l y s i s . The plasmid DNA was p r e c i p i t a t e d w i t h 3 volumes of ethanol and d i s s o l v e d i n 1 ml of 5 mM T r i s - H C l pH 8, 5 mM NaCl, 0.5 mM EDTA. In some cases the DNA was p u r i f i e d f u r t h e r on a second CsCl g r a d i e n t , i n others CsCl gradients were : 64 . replaced by chromatography on a Bio-Gel A-5m column. V I I I . R e s t r i c t i o n Mapping of Recombinant Plasmids A. R e s t r i c t i o n Endonuclease Cleavage of DNA R e s t r i c t i o n b u f f e r s A, B and C each contained 6 mM T r i s - H C l pH 7.5, 6 mM MgCl 2 and 6 mM 2-mercaptoethanol. The NaCl co n c e n t r a t i o n of the three b u f f e r s d i f f e r e d , being 6 mM, 50 mM and 150 mM r e s p e c t i v e l y . B u f f e r D con-tained 100 mM T r i s - H C l pH 7.5, 10 mM MgCl 2 and 6 mM 2-mercaptoethanol. Decreased cleavage s i t e s p e c i f i c i t y (EcoR I * , Dde I * a c t i v i t i e s ) was d i s -played i f DNA was cut w i t h EcoR I or Dde I i n a low s a l t b u f f e r c o n t a i n i n g 20 mM T r i s HC1 pH 8.5, 2 mM MgCl 2, 5 mM 2-mercaptoethanol ( P o l i s k y et a l . , 1975). For complete d i g e s t i o n of plasmid DNA the r e a c t i o n mixture contained 0.5 u n i t of r e s t r i c t i o n enzyme per ug of DNA and the i n c u b a t i o n time was 2 h. A u n i t of enzyme i s defined as the amount required to completely d i g e s t 1 ug of phage X DNA i n 15 min at 37°C. R e s t r i c t i o n endonuclease Xma I was prepared by the method of Endow and Roberts (1977) or was the g i f t of Dr. C. A s t e l l . R e s t r i c t i o n enzymes Fnu4H I and FnuE I were the g i f t of Dr. M. Smith. B. R e s t r i c t i o n Mapping R e s t r i c t i o n maps of plasmids pDt55 and pDt78R were constructed by the m u l t i p l e enzyme d i g e s t method described by Danna (1980). Hind I I I cut phage X DNA provided s i z e standards (23.1, 9.9, 6.6, 4.4, 2.46, 2.15 and 0.49 kb) u s e f u l i n determining the s i z e of r e s t r i c t i o n fragments. The method of Southern (1979) was used to estimate the l e n g t h of DNA fragments from t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s . IX. DNA Sequence A n a l y s i s The n u c l e o t i d e sequence of plasmid DNA r e s t r i c t i o n fragments was d e t e r -mined using the Maxam and G i l b e r t (1980) procedure as modified by Dr. A. Delaney. 65. R e s t r i c t i o n fragments were l a b e l l e d f o r sequencing by i n c u b a t i n g the DNA (5-20 pg) i n 20 p i of r e s t r i c t i o n b u f f e r B c o n t a i n i n g 30-80 uCi of the appropriate [a- 3 2P]deoxynucleoside triphosphate (2000-3000 Ci/mmol, Amersham) and 1 u n i t of the Klenow fragment of E_. c o l i DNA polymerase I (Boehringer Mannheim) f o r 15 min at room temperature. The s o l u t i o n was mixed w i t h an equal volume of 15% sucrose c o n t a i n i n g ^ 0.1% xylene cyanol and bromphenol blue and a p p l i e d to the sample s l o t of a t h i n , non-denaturing polyacrylamide g e l (35 x 15 x 0.05 cm) f o r f r a c t i o n a t i o n of the l a b e l l e d DNA fragments by e l e c t r o p h o r e s i s . I f the l a b e l l e d fragments were to be strand-separated, s o l i d urea ( t o a c o n c e n t r a t i o n of 50% w/v) and the dyes were added to the l a b e l l i n g r e a c t i o n mixture. The s o l u t i o n was then heated t o 100°C f o r 3-5 min and q u i c k l y a p p l i e d to the sample s l o t of a non-denaturing polyacrylamide g e l . The sample was subjected to e l e c t r o p h o r e s i s at 400 V f o r 10 min then at 100-250 V t i l l the d e s i r e d degree of s e p a r a t i o n had been achieved. Some fragments that could not be strand-separated at room temperature separated c l e a n l y i f the e l e c t r o p h o r e s i s was performed at 4°C. Sequence data were s t o r e d , e d i t e d and analyzed using the SEQNCE computer program of Dr. A. Delaney. 66. Results and D i s c u s s i o n I . The Coding P r o p e r t i e s of the Drosophila V a l i n e tRNAs The ribosome-binding assay developed by Nirenberg and Leder (1964) i s used f o r determining the coding p r o p e r t i e s of tRNA i s o a c c e p t o r s . In the assay E. c o l i ribosomes are "programmed" w i t h a t r i n u c l e o s i d e diphosphate ( " t r i p l e t " ) of known sequence. These ribosomes w i l l bind aminoacyl-tRNAs w i t h anticodons complementary to the t r i p l e t . Ribosome-bound aminoacyl-tRNA can be separated from unbound m a t e r i a l by f i l t e r i n g the r e a c t i o n mixture through a n i t r o c e l l u l o s e membrane. Only ribosome-bound aminoacyl-tRNA i s r e t a i n e d on the f i l t e r . In determining the codon r e c o g n i t i o n p r o p e r t i e s of i n d i v i d u a l tRNA iso a c c e p t o r s i t i s important that the is o a c c e p t o r s are completely separated from one another. In t h i s study the v a l i n e tRNA is o a c c e p t o r s i n a crude mixture of Drosophila tRNAs were aminoacylated w i t h L - [ 3 H ] v a l i n e and V a l separated by chromatography on two successive RPC-5 columns. tRNA^ V a l and tRNA^ were resolve d by chromatography i n a b u f f e r c o n t a i n i n g Mg"^ ions (Figure 10A). [ 3 H ] v a l y l - t R N A V a ^ from each of these peaks was chromatographed on a second column i n a b u f f e r c o n t a i n i n g EDTA (no f r e e Mg"*"*"). This chromatographic system separated tRNA^3''" i n t o two sub-Vo 1 Vfl1 Va 1 s p e c i e s , t R N A 3 a a n d t R N A 3 b ( F i S u r e 1 0 B ) a n d freed tRNA^ V a l V a l of contaminating tRNA„ and tRNA„, (Figure IOC). Ja Jb The response of the 3 major v a l i n e tRNAs to each of the four v a l i n e V a l codon t r i p l e t s (GUG, GUA, GUU and GUC) i s shown i n Figure 11. tRNA" V a l responded s t r o n g l y to GUA and weakly to GUU and GUG. tRNA^^ respond-V a l ed only to GUG. tRNA^ responded s t r o n g l y to GUU, GUC and GUA and weakly to GUG. On the b a s i s of ribosome-binding experiments, tRNA n u c l e o t i d e sequences, 67. Figure 10. P u r i f i c a t i o n of Drosophila v a l y l - t R N A V a l i s o a c c e p t o r s by RPC-5 chromatography A. Crude Drosophila tRNA (54 A26O u n i t s ) was aminoacylated w i t h [ 3 H ] v a l i n e and chromatographed on an RPC-5 column i n b u f f e r A (10 mM NaOAc, pH 4 . 0 ; 10 mM Mg(0Ac)2 5 and 1 mM 2-mercaptoethanol) as described i n M a t e r i a l s and Methods. The numbers denote the e l u t i o n p o s i t i o n s of the 6 v a l i n e tRNA i s o a c c e p t o r s . Those f r a c t i o n s c o n t a i n i n g v a l y l - t R N A ^ 3 1 or v a l y l - t R N A ^ 3 1 that were pooled f o r f u r t h e r p u r i f i c a t i o n are i n d i c a t e d by the s o l i d b l ack l i n e s . B. V a l y l - t R N A ^ 3 1 was f r a c t i o n a t e d i n t o two subspecies, v a l y l -t R N A ^ (3a) and v a l y l - t R N A ^ 3 1 (3b), by RPC-5 chromatography i n b u f f e r B (50 mM sodium formate, pH 3.8; 1 mM EDTA; 1 mM 2-mercaptoethanol) as described i n M a t e r i a l s and Methods. The v a l y l - t R N A V a l of f r a c t i o n s marked by s o l i d b l a c k l i n e s was used f o r the ribosome-binding experiments. C. V a l y l - t R N A ^ 3 1 was p u r i f i e d by RPC-5 chromatography i n b u f f e r B as described i n M a t e r i a l s and Methods. V a l y l - t R N A V a l of those f r a c t i o n s marked by the s o l i d b l a c k l i n e was used f o r the ribosome-binding experiments. 69. Figure 11. The coding p r o p e r t i e s of the Drosophila v a l i n e tRNAs. Binding of Drosophila [ 3H]-tRNA to E_. c o l i ribosomes i n the presence of t r i p l e t s : GUU (•), GUC (•), GUA (•), GUG ( o ) , and UCG ( A ) . Each 50 y l r e a c t i o n mixture contained 20 mM magnesium ace t a t e , 50 mM KC1, 0.1 M T r i s - a c e t a t e pH 7.2, 2 A260 u n i t salt-washed ribosomes (1 &260 u n i t i n the case of V a l 3a) and [ 3 H] valyl-tRNA, i . e . , V a l 3a, 0.77 pmol; V a l 3b, 2.2 pmol; and V a l 4, 1.4 pmol. A f t e r 20 min at 25°C, the ribosome complexes were c o l l e c t e d on M i l l i p o r e f i l t e r s , washed, d r i e d , and the r a d i o a c t i v i t y determined i n a l i q u i d s c i n t i l l a t i o n counter. Val 3a — a G U A ^ - • • G U U '''....o... - o G U G — . • G U C - i i_ 1 2 3 4 5 ' 6 5 4 3 2 1 NA 7 Val 3b •oGUG / G U U . — • G U C ••••°GUA 1 2 3 4 5 N O M O L E S O F T R I P L E T Va l 4 / °-G U C GUA • t * • o G U G • * 1 2 3 4 5 71. the o r g a n i z a t i o n of the genetic code, and s t r u c t u r a l c o n s i d e r a t i o n s , C r i c k (1966) proposed the "wobble" hypothesis. C r i c k p o s t u l a t e d that there i s a c e r t a i n amount of "play" or wobble p o s s i b l e i n the b a s e - p a i r i n g between the f i r s t ("wobble") n u c l e o t i d e of the anticodon and the t h i r d n u c l e o t i d e of the codon. This wobble allows some n u c l e o t i d e s i n the f i r s t p o s i t i o n of the anticodon to form unorthodox base-pairs w i t h the codon. The wobble r u l e s , as formulated by C r i c k , a l l o w the f o l l o w i n g base-pairs: 1st p o s i t i o n of 3rd p o s i t i o n of anticodon codon A p a i r s w i t h U C G G " " C, U U " " A, G I " " A, C, U The wobble r u l e s a l l o w p r e d i c t i o n s to be made about the anticodon V a l sequences of the Drosophila v a l i n e tRNAs. Thus t R N A ^ and Val tRNA^ would be p r e d i c t e d to have the anticodons CAC and IAC respec-t i v e l y . The n u c l e o t i d e sequences of these tRNAs, presented l a t e r i n t h i s study (Figure 12, Figure 20) confirm these p r e d i c t i o n s . The marked p r e f e r -V a l ence of tRNA^ f o r the GUA t r i p l e t over the GUG t r i p l e t suggests that the wobble p o s i t i o n of t h i s tRNA contains a 2 - t h i o u r i d i n e residue (or d e r i -V a l v a t i v e ) ( N i s h i m u r a , 1979). However, tRNA. binds weakly to ribosomes i n response to the GUU t r i p l e t , behavior not c o n s i s t e n t w i t h the presence of a t h i o u r i d i n e residue i n the f i r s t p o s i t i o n of the anticodon (Nishimura, 1979). The u n i d e n t i f i e d nucleoside N found i n the nucleoside a n a l y s i s of V a l tRNA^^ (Table I I ) may be r e s p o n s i b l e f o r i t s somewhat unusual coding p r o p e r t i e s . V a l The coding p r o p e r t i e s and n u c l e o t i d e sequences of tRNA^^,^ from yeast, r a b b i t l i v e r , r a t l i v e r and Drosophila are now known. Yeast V a l tRNA^^,^ has the coding p r o p e r t i e s p r e d i c t e d by the wobble hypothe-V a l s i s (Mirzabekov et^ a l . , 1968). In c o n t r a s t , r a b b i t l i v e r tRNA^ I A C^ binds most s t r o n g l y to ribosomes i n the presence of GUG, somewhat l e s s strongly i n the presence of GUU and weakly i n the presence of the other two v a l i n e t r i p l e t s (Jank et_ a l . , 1977a). Such base-pairing between an IAC anticodon and a GUG codon v i o l a t e s the wobble bas e - p a i r i n g r u l e s . I t could V a l be argued that some unique s t r u c t u r a l f e a t u r e of r a b b i t l i v e r t R N A ^ ^ ^ V a l i s r e s p o n s i b l e f o r i t s unusual coding p r o p e r t i e s . However, t R N A ^ ^ ^ of r a t a s c i t e s hepatoma c e l l s , w i t h a n u c l e o t i d e sequence i d e n t i c a l to V a l tRNA of r a b b i t l i v e r , binds to ribosomes s o l e l y i n response to (IAC ) GUU (Shindo-Okada et a l . , 1981). The ribosome-binding assay c o n d i t i o n s were V a l very s i m i l a r f o r the two mammalian tRNA^^,^ s p e c i e s . C l e a r l y f u r t h e r s t u d i e s of these two tRNAs are required to r e c o n c i l e these c o n t r a -V a l d i c t o r y f i n d i n g s . Drosophila tRNA^ i s q u i t e s i m i l a r to yeast V a l tRNA-£j.^ ,j i n i t s coding p r o p e r t i e s but does show some ribosome-b i n d i n g i n the presence of the GUG t r i p l e t s . How c l o s e l y do ribosome-binding s t u d i e s of the codon-anticodon i n t e r a c -t i o n r e f l e c t the i n t e r a c t i o n s that occur i n vivo? M i t r a et^ a l . (1977) attempted to answer t h i s question by t r a n s l a t i n g phage MS2 RNA i n an E_. c o l i p r o t e i n s y n t h e s i z i n g system i n which p r o t e i n synthesis was completely depen-V a l dent on the a d d i t i o n of p u r i f i e d valyl-tRNA i s o a c c e p t o r s . The c a p a c i t y of each i s o a c c e p t o r to t r a n s l a t e the v a l i n e codons of MS2 coat p r o t e i n mRNA V a l was determined. In t h i s system a l l v a l i n e tRNAs (E_. c o l i tRNA^^,^, t R N A ^ c ^ yeast t R N A ^ 3 ^ , T o r u l o p s i s u t i l i s t R N A ^ ^ ) t r a n s l a t e d a l l v a l i n e codons. The authors concluded t h a t , i n t h e i r system, the codon-anticodon i n t e r a c t i o n was much l e s s r e s t r i c t i v e than ribosome-bindi n g s t u d i e s suggested. Holmes e^ t a l . (1978) showed that the e r r o r r a t e i n a p r o t e i n s y n t h e s i z i n g system s i m i l a r to that described above was very h i g h w i t h misreading o c c u r r i n g i n 15-30% of the p r o t e i n synthesized. Further experiments under more p h y s i o l o g i c a l c o n d i t i o n s i n d i c a t e d that codon anticodon b a s e - p a i r i n g was l e s s r e s t r i c t i v e than p r e d i c t e d by the wobble hypothesis but much l e s s i n d i s c r i m i n a t e than o r i g i n a l l y suggested by M i t r a e t a l . ( M i t r a e_t a l . , 1979; Goldman et a l . , 1979: L u s t i g et a l . , 1981). Genetic evidence suggests t h a t , in_ v i v o , codon-anticodon i n t e r a c t i o n s are at l e a s t as r e s t r i c t i v e as those p r e d i c t e d by the wobble hypothesis. E. c o l i contains 4 species of g l y c i n e tRNA. Ribosome-binding experiments Gly showed that only t R N A ^ ^ ^ t r a n s l a t e s the GGA g l y c i n e codon. There Gly i s only 1 copy of the t R N A ^ ^ ^ gene i n the _E. c o l i genome. There-f o r e , e l i m i n a t i o n of t h i s gene should be l e t h a l to the c e l l . A mutation i n Gly which t R N A ^ ^ ^ has been changed i n t o an AGA suppressor tRNA i s indeed l e t h a l i n the h a p l o i d s t a t e (Murgola and Pagel, 1980). In Ser Schizosaccharomyces pombe there are two genes f o r a t R N A ^ ^ ^ that t r a n s l a t e s the UCA codon. Munz et^ a l . (1981) i n a c t i v a t e d one of these genes i n one s t r a i n of S. pombe and the other gene i n another s t r a i n . When the two s t r a i n s were crossed spores c o n t a i n i n g both of the i n a c t i v a t e d genes Ser were not v i a b l e . The c e l l s d i d c o n t a i n a t R N A ^ g ^ is o a c c e p t o r that should have been a b l e , according to the wobble hypothesis, to t r a n s l a t e the Ser UCA codons i n the absence of the t R N A ^ U ( ^ species. This suggests that i n o s i n e may not wobble base-pair w i t h adenosine in_ v i v o . Perhaps the r e c o g n i t i o n of NNA codons by INN anticodons, commonly observed i n the ribosome-binding assay, i s a r t i f a c t u a l . The Mg c o n c e n t r a t i o n (20 mM) i n the ribosome-binding assay mixture i s much higher than the p h y s i o l o g i c a l c o n c e n t r a t i o n (4 mM i n b a c t e r i a , Lusk et_ a l . , 1968; 1 mM i n r a t c e l l s , Veloso ejb a_l., 1973). High Mg c o n c e n t r a t i o n i s known to reduce the s p e c i f i c i t y of some a n t i c o d o n - t r i p l e t i n t e r a c t i o n s i n the ribosome-binding assay (Rudloff and H i l s e , 1975). The suggestion that i n o s i n e cannot wobble V a l base-pair w i t h adenosine in_ v i v o i s a t t r a c t i v e . Drosophila tRNA^ 74. would then t r a n s l a t e only GUU and GUC codons, GUA codons would be t r a n s l a t e d V a l V a l s o l e l y by t R N A 3 a a n d t R N A 3 b w o u l d t r a n s l a t e GUG codons. The Va l amounts of the three major tRNA species i n Drosophila (Figure 7) would a l s o roughly p a r a l l e l the frequency of v a l i n e codons i n the mRNAs of animals (30 GUC + GUU:5 GUA:33 GUG, Grantham et a l . , 1981). Va l I I . The Nucleotide Sequence of D. melanogaster tRNA7| V a l The n u c l e o t i d e sequence of tRNA^ , determined i n t h i s study i s shown i n c l o v e r l e a f form i n Figure 12. A combination of 3 d i f f e r e n t RNA sequencing techniques was used to determine the sequence. The r e s u l t s obtained by each of these techniques are described and discussed below. A. The Stanley and Vassilenko Method Most of the tRNA sequence was obtained by the Stanley and Vassilenko method (n u c l e o t i d e s 1, 2, 8-32, 34-46 and 51-71). The [ 5 1 - 3 2 P ] n u c l e o s i d e - 5 ' , 3'-bisphosphates (pNp's) produced by t h i s technique were separated by TLC on P E I - c e l l u l o s e ion-exchange p l a t e s and c e l l u l o s e p l a t e s i n Solvents A and B r e s p e c t i v e l y . The pNp's were i d e n t i f i e d by comparing t h e i r chromatographic m o b i l i t i e s to published values ( S i l b e r k l a n g et_ a l . , 1979; Cri b b s , 1979). The sequence data obtained by the Stanley and Vassilenko method was, f o r almost a l l p o s i t i o n s i n the tRNA, unambiguous (Figure 13). Experience has shown that the most important f a c t o r i n o b t a i n i n g such r e s u l t s i s high tRNA p u r i t y . Heterogeneity i n the length of the tRNA, presumably due to hy d r o l y -s i s during storage, manifests i t s e l f as a "messy" ladder of 5' en d - l a b e l l e d fragments. In such a ladder there are a great many bands, the bands are fuzzy and they vary g r e a t l y i n i n t e n s i t y . To avoid these problems tRNA to be sequenced by the Stanley and Vassilenko method was r o u t i n e l y p u r i f i e d by g e l e l e c t r o p h o r e s i s j u s t before use (see M a t e r i a l s and Methods). 75. Figure 12. The n u c l e o t i d e sequence of V_. melanogaster tRNA^3-'- arranged as a c l o v e r l e a f . The u r i d i n e residue at p o s i t i o n 20 i s p a r t i a l l y modified t o acp 3U. One of the c y t i d i n e r esidues between p o s i t i o n s 47 and 50 i s probably modi-f i e d to m5C. T r a n s c r i p t i o n of the t R N A ^ a l - l i k e genes of plasmids pDt92R, pDtl20R and pDtl4 would produce a tRNA i d e n t i c a l to tRNA^ 3 1 except f o r the replacement of C16, U29, A41 and G57 by U,C,G and A respec-t i v e l y . Val D.m. tRNA * 4 AQH 7 6 c A P G C P U A U A 70 Urn A 5 C G 60 C ' ^^C G U ^ A u C G G G C C f V r n V G C C C G G G C A C C D . . U 25 m 7 G U A A G ocp^U V o " g . A C U • A 6 30 G • C 40 C G Cm m5C u a i A c 35 Stanley and Vassilenko sequencing i s uniquely s u i t e d to determining the i d e n t i t y and p o s i t i o n of modified n u c l e o t i d e s i n tRNAs. Those pNp's t h a t , because of t h e i r chromatographic behavior or p o s i t i o n i n the p u t a t i v e tRNA sequence, were suspected of being modified were c h a r a c t e r i z e d more f u l l y by f i r s t converting them to [5'- 3 2P]nucleoside-5'-phosphates (pN's) using the 3'-phosphatase a c t i v i t y of nuclease P^ then i d e n t i f y i n g them by t h e i r m o b i l i t y on c e l l u l o s e TLC p l a t e s developed i n s o l v e n t s B (Figure 14) and C. The i d e n t i f i c a t i o n of s e v e r a l modified n u c l e o t i d e s deserves s p e c i a l comment. The chromatographic m o b i l i t y of the pNp from p o s i t i o n 9 of tRNAY a l (Solvent A, R „ = 0.83; Solvent B, R . = 0.71) d i d not 4 ' pUp ' pAp match any of the published m o b i l i t y values f o r modified pNps. The sequence V a l o f [ 5 ' - 3 2 P ] e n d l a b e l l e d tRNA^ obtained by the g e l read-off method showed that the RNA was s l o w l y cleaved at n u c l e o t i d e 9 by RNases T-^  and This suggested that n u c l e o t i d e 9 was a modified G r e s i d u e . On the b a s i s of the nucleoside a n a l y s i s (Table I I ) 1-methylguanosine (m lG) was assigned to p o s i t i o n 9. This assignment i s c o n s i s t e n t w i t h the observation that mlG i s found only at p o s i t i o n 9 i n p r e v i o u s l y sequenced eukaryotic tRNAs (Dirheimer e_t al., 1979). About 50% of the u r i d i n e residues at p o s i t i o n 20 ( F i g u r e 13) are modi-f i e d to 3-(3-amino-3-carboxypropyl) u r i d i n e (acp 3U). Authentic acp 3U-5'-phosphate, i s o l a t e d from a nuclease P^ d i g e s t of crude 15. c o l i tRNA and c h a r a c t e r i z e d by i t s UV a b s o r p t i o n spectrum, p o s i t i v e n i n h y d r i n r e a c t i o n and the chromatographic p r o p e r t i e s of the nucleoside (Ohashi et a l . , 1974), was used as a standard f o r i d e n t i f i c a t i o n of the modified n u c l e o t i d e . The V a l presence of acp 3U i n tRNA^ i s c o n s i s t e n t w i t h the work of White V a l (1980) who has shown that Drosophila tRNA rea c t s w i t h cyanogen bromide and the N-hydroxysuccinimide e s t e r of naphthoxyacetic a c i d , reagents thought to react w i t h the amino group of acp 3U i n tRNAs. An acp 3U 7 8 . F i g u r e 1 3 . S t a n l e y a n d V a s s i l e n k o s e q u e n c i n g o f t R N A ^ 3 - ' - : P E l - c e l l u l o s e c h r o m a t o g r a p h y o f [ 5 ' - 3 z P ] p N p ' s . t R N A ^ 3 ^ w a s s e q u e n c e d b y t h e S t a n l e y a n d V a s s i l e n k o m e t h o d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e r e s u l t i n g [ 5 ' - 3 2 P ] p N p ' s w e r e i d e n t i f i e d b y c h r o m a t o g r a p h y o n P E l - c e l l u l o s e T L C p l a t e s i n S o l v e n t A . T h e n u c l e o t i d e s a t p o s i t i o n s 5 7 - 5 1 , 4 6 - 3 4 a n d 3 1 - 8 o f t R N A ^ a l w e r e i d e n t i -f i e d b y t h i s m e t h o d . P o s i t i o n s a t w h i c h t h e s e q u e n c e o f t R N A ^ 3 - * -d i f f e r s f r o m a h y p o t h e t i c a l t r a n s c r i p t o f t h e t R N A j J 3 - 1 — l i k e g e n e s o f p l a s m i d s p D t 9 2 R , p D t l 2 0 R a n d p D t l 4 a r e m a r k e d ( A ) . T h e A a n d C r e s i d u e s s e e n b e t w e e n m s C 3 8 a n d A 3 7 a r e a r e p e t i t i o n o f A 3 7 a n d C 3 6 . T h e o r d e r o f G a n d C r e s i d u e s s e e n b e t w e e n C 5 1 a n d m 7 G 4 6 i s n o t k n o w n b e c a u s e o f b a n d c o m p r e s s i o n i n t h e l a d d e r o f 5 ' e n d - l a b e l l e d f r a g m e n t s g e n e r a t e d d u r i n g s e q u e n c i n g b y t h e S t a n l e y a n d V a s s i l e n k o m e t h o d . 79. I m # G C V T G G C 55 G G A A G A 45 A C G m C 40 A C A 35 I CmU C G U C ^ A C A 30 A 25 81. 1 c u A(acp3)U D 20 G G C • G 15 A V G U G 10 G U 82. Figure 14. Stanely and Vassilenko sequencing of tRNA^al: I d e n t i f i c a -t i o n of modified n u c l e o t i d e s by t h i n - l a y e r chromatography. The [5'- 3 2P]3',5'-bisphosphates of modified n u c l e o t i d e s produced by the Stanley and V a s s i l e n k o method were dephosphorylated to the corresponding [ 3 2P]nucleoside-5'-phosphate by d i g e s t i o n w i t h nuclease P]^ as described i n M a t e r i a l s and Methods. These n u c l e o t i d e s were i d e n t i f i e d by chromato-graphy on c e l l u l o s e TLC p l a t e s developed i n Solvent C. I f commercially a v a i l a b l e , the 5'-phosphates of the appropriate modified nucleosides were chromatographed on the same TLC p l a t e s as standards. The p o s i t i o n s of these standards a f t e r chromatography were detected by t h e i r UV absorbance and are c i r c l e d i n the autoradiograms shown i n the F i g u r e . 83. 84. r e s i d u e , a l s o designated nucleoside X, was detected i n the nucleoside analy-V a l s i s of tRNA^^ but not i n those of the other two major v a l i n e i s o a c -ceptors (Table I I ) . The reason why acp 3U escaped d e t e c t i o n i s not appar-ent. I t may have simply run o f f the end of the TLC p l a t e used to separate V a l the nucleosides during the a n a l y s i s . A l t e r n a t i v e l y , the tRNA^ and Va l tRNA^ used f o r nucleoside a n a l y s i s may have contained s i g n i f i c a n t l y l e s s acp 3U than the sequenced tRNAs. An acp 3U residue i s found at p o s i t i o n 20 i n the D-loop of both Veil Asp tRNA^ and r a t l i v e r t R N A ^ y ^ , the only other sequenced euk a r y o t i c tRNA that contains t h i s n u c l e o t i d e (Chen and' Roe, 1978). tRNAs f o r the same amino a c i d s c o n t a i n acp 3U i n both Drosophila and r a t (Asp, H e , Thr, Tyr and V a l tRNAs; White, 1980). This d i s t r i b u t i o n i s qui t e d i f f e r e n t from that found among E_. c o l i tRNAs (Lys, Phe, V a l , H e , Arg and Met tRNAs; Friedman, 1973). The p o s i t i o n of acp 3U w i t h i n E_. c o l i tRNAs i s a l s o d i f f e r e n t from i t s p o s i t i o n i n the tRNAs of higher eukaryotes. In prokaryotes and plan t c h l o r o p l a s t s acp 3U i s found e x c l u s i v e l y 3' to the 7-methylguanosine (m7G) residue i n the v a r i a b l e arm of those tRNAs that c o n t a i n m7G (Gauss and S p r i n z l , 1981). acp 3U has not been detected i n yeast tRNA (White, 1980). In RNA the phosphodiester bond between a 2'-0-methylated n u c l e o t i d e and i t s 3' neighbouring n u c l e o t i d e i s r e s i s t a n t to h y d r o l y s i s by a l k a l i and most V a l r i b o n u c l e a s e s . This had two consequences f o r the sequencing of tRNA^ by the Stanley and Vassilenko method. F i r s t , since the bond between Cm32 and U33 was not cleaved during p a r t i a l h y d r o l y s i s of the tRNA the nuc l e o t i d e at p o s i t i o n 33 was not l a b e l l e d and could not, t h e r e f o r e , be i d e n t i f i e d by t h i s sequencing method. Second, RNase T 2 d i g e s t i o n of that l a b e l l e d f r a g -ment w i t h Cm33 as i t s 5'-terminal n u c l e o t i d e d i d not produce a pNp but a di n u c l e o s i d e t r i p h o s p h a t e , [5'- 3 2P]pCmpUp. D i g e s t i o n of t h i s n u c l e o t i d e 85. w i t h nuclease P^, however, l i b e r a t e d [5'- 3 2P]pCm which could be e a s i l y i d e n t i f i e d (Figure 14). V a l Stanley and Vassilenko sequencing showed that Drosophila tRNA^ has a t y p i c a l T-loop w i t h rT at p o s i t i o n 54 and a C residue at p o s i t i o n 60. V a l In c o n t r a s t , the T-loops of mammalian tRNA^^,^ are unique among sequenced cytoplasmic tRNAs i n having a U residue at p o s i t i o n 54 and an A at p o s i t i o n 60 ( P i p e r and C l a r k , 1974; P i p e r , 1975; Chen and Roe, 1977; Jank et_ a l . , 1977a; Shindo-Okada et_ a l . , 1981). These two n u c l e o t i d e s could base-p a i r to produce a tRNA w i t h a 6 base-pair T-stem and a 5 n u c l e o t i d e T-loop. Gross and h i s coworkers have produced some evidence favouring such a s t r u c -ture (Jank et j a l . , 1977b). As p r e v i o u s l y described i n t h i s D i s c u s s i o n V a l ( S e c t i o n I ) mammalian tRNA. . has unusual coding p r o p e r t i e s while C -LAC ) V a l D r o s o p h i l a tRNA^ has coding p r o p e r t i e s c o n s i s t e n t w i t h the wobble hypothesis. Comparison of the two tRNA s t r u c t u r e s suggests that the anoma-V a l lous coding p r o p e r t i e s of the mammalian tRNA^^,^ may be r e l a t e d to i t s unusual T-loop s t r u c t u r e . B. Wandering-Spot A n a l y s i s V a l Wandering-spot a n a l y s i s of e n d - l a b e l l e d tRNA^ was used to d e t e r -mine the sequences at the 5' and 3'-ends of the molecule ( n u c l e o t i d e s 1-16, V a l and 62-76). The wandering-spot a n a l y s i s of the 5'-end of tRNA^ i s presented i n Figure 15. The 5'-terminal n u c l e o t i d e was shown to be pG by the Stanley and V a s s i l e n k o method. The residue at p o s i t i o n 4 i s r e s i s t a n t to the formamide-induced h y d r o l y s i s used i n the wandering-spot procedure. This would be expected i f n u c l e o t i d e 4 was 2'-0-methylated. Of the two ribose-methylated nucleosides detected i n the nucleoside a n a l y s i s (Table I I ) 2' -O-methylcytidine had been detected at p o s i t i o n 32 by the Stanley and Vassilenko method. Therefore, the 2'-O-methyluridine (Urn) residue was assigned to p o s i t i o n 4. A Um residue at t h i s p o s i t i o n could hydrogen bond 86. Figure 15. Wandering-spot a n a l y s i s of the 5'-terminal n u c l e o t i d e s of t R N A f a l . was cleaved i n the anticodon loop w i t h RNase U2 and 5' e n d - l a b e l l e d w i t h [ 3 2P]phosphate as described i n M a t e r i a l s and Methods. The l a b e l l e d RNA fragment c o n t a i n i n g the 5'-end of tRNA)[ a l (2.5 x 10 s cpm Cerenkov) was d i s s o l v e d i n 5 p i of 98% formamide and heated to 100°C f o r 50 min. The p a r t i a l l y hydrolysed RNA was subjected to wandering-spot a n a l y s i s as described i n M a t e r i a l s and Methods. In the f i r s t dimension ( 1 ) , e l e c t r o p h o r e s i s at pH 3.5, the xylene cyanol marker dye was allowed to migrate 6 cm. A "10 mM KOH" homomix prepared according to the procedure of S i l b e r k l a n g e_t al. (1979) was used f o r homochromatography i n the second dimension ( 2 ) . R and Y are the p o s i t i o n s of the red and y e l l o w marker dyes. The i d e n t i f i c a t i o n of the 2'-O-methyluridine residue at p o s i t i o n 4 i s discussed i n the t e x t . 88. Figure 16. Wandering-spot a n a l y s i s of the 3'-terminal n u c l e o t i d e s of tRNA^ a l. tRNAj[ a l was l a b e l l e d at the 3'-end w i t h [5'- 3 2P]pCp as described i n M a t e r i a l s and Methods. The e n d - l a b e l l e d RNA (4.4 x lO^cpm Cerenkov) was d i s s o l v e d i n 5 u l 98% formamide and p a r t i a l l y hydrolysed by heating the s o l u t i o n to 100°C f o r 1 h. The p a r t i a l hydrolysate was subjected to wandering-spot a n a l y s i s as described i n M a t e r i a l s and Methods. The xylene cyanol marker dye (B) was allowed to migrate 8 cm during e l e c t r o p h o r e s i s i n the f i r s t dimension ( 1 ) . "10 mM KOH" homomix ( S i l b e r k l a n g et a l . , 1979) was used f o r homochromatography i n the second dimension ( 2 ) . R shows the p o s i -t i o n of the red marker dye. 0 2 90. to the complementary A residue at p o s i t i o n 69. Ribose-methylated nucleo-t i d e s are present at p o s i t i o n 4 of a l l sequenced g l y c i n e and p r o l i n e tRNAs Ser (Gauss and S p r i n z l , 1981) and tRNA^ of D_. melanogaster ( C r i b b s , 1979). With these exceptions n u c l e o t i d e s w i t h methylated base or sugar residues are very rare i n the aminoacyl stem of tRNAs (Gauss and S p r i n z l , 1981). V a l Figure 16 shows the wandering-spot a n a l y s i s of the 3'-end of tRNA^ The i n t e r p r e t a t i o n of the m o b i l i t y s h i f t s was s t r a i g h t f o r w a r d except f o r the presence of a f a i n t p a t t e r n of spots " p a r a l l e l i n g " the major one ( i n d i c a t e d by the arrow i n Figure 16). The n u c l e o t i d e sequence i n f e r r e d from e i t h e r p a t t e r n i s the same. The weaker spots d i s p l a y much greater m o b i l i t y i n the f i r s t dimension ( e l e c t r o p h o r e s i s at pH 3.5) than the corresponding major spots but e x a c t l y the same m o b i l i t y i n the second dimension (homochromato-graphy). For wandering spot a n a l y s i s the tRNA was e n d - l a b e l l e d w i t h [5'- 3 2P]pCp using phage T4 RNA l i g a s e . The s e r i e s of minor spots are probably the r e s u l t of a small amount of the RNA being l a b e l l e d w i t h a [5'- 3P]pUp contaminant i n the [5'- 3 2P]pCp used to l a b e l the tRNA. Replacement of 3'-terminal pCp by pUp would increase the negative charge at pH 3.5 of otherwise i d e n t i c a l o l i g o n u c l e o t i d e s and hence increase t h e i r e l e c t r o p h o r e t i c m o b i l i t y . For example, at pH 3.5 ApCp i s c a l c u l a t e d to have a charge of -0.52 w h i l e ApUp would have a charge of -1.36 (Brown, 1979). C. Sequencing by the Gel Read-Off Method: Chloroacetaldehyde M o d i f i c a t i o n as an A i d to RNA Sequencing The g e l read-off method was used t o determine the n u c l e o t i d e sequence of regions of the tRNA that could not be sequenced by the other two methods ( n u c l e o t i d e s 33, 46-52). Chloroacetaldehyde m o d i f i c a t i o n of the tRNA f a c i l i t a t e d sequencing by t h i s method. 91. At pH 4.0 chloroacetaldehyde r e a c t s w i t h nonbase-paired c y t i d i n e and adenosine residues i n n u c l e i c a c i d s to form f l u o r e s c e n t etheno d e r i v a t i v e s . These d e r i v a t i v e s cannot form Watson-Crick base-pairs w i t h other n u c l e o t i d e s (Kochetkov et_ a l . , 1971; B a r r i o £t a l . , 1972; Kimura et_ a l . , 1977; Biernat et a l . , 1978). The c u r r e n t l y accepted mechanism f o r the r e a c t i o n of chloracetaldehyde w i t h c y t i d i n e residues i s presented i n Figure 17 (Krzyzosiak et a l . , 1979). Figure 17. Reaction of Chloroacetaldehyde w i t h C y t i d i n e - Compounds 1, 2, 3 and 4 are r e s p e c t i v e l y chloroacetaldehyde, c y t i d i n e , hydroxyethno-c y t i d i n e and et h e n o c y t i d i n e . The r a t e - l i m i t i n g dehydration step i s a c i d c a t a l y s e d . Adenosine r e a c t s w i t h chloroacetaldehyde i n an analogous manner. Thus i f a tRNA were denatured and then modified w i t h chloroacetaldehyde i t should l o s e much of i t s secondary s t r u c t u r e . In the technique developed i n t h i s study e n d - l a b e l l e d tRNA i s heated to 100°C at pH 4.0 w i t h a 6.6% chloroacetaldehyde s o l u t i o n (v/v) f o r 1 min. The r e a c t i o n mixture i s g r e a t l y d i l u t e d w i t h b u f f e r (pH 4.0) and incubated at 80°C f o r 20 min. The modified tRNA i s then sequenced by the g e l read-off method. I f the 80°C post-treatment i s omitted very fuzzy bands are seen on autoradiographs of the sequencing g e l s . K r z y z o s i a k ej: a l . (1981) have r e c e n t l y shown that such a "maturation" step i s required f o r the dehydration of the s t a b l e hydroxyetheno r e a c t i o n intermediate t o form the etheno d e r i v a t i v e . The s e r i e s of f i v e c y t i d i n e residues at p o s i t i o n s 47-51 ( v a r i a b l e loop, T-stem regions) could not be determined by e i t h e r the g e l read-off or Stanley and Vassilenko methods. E l e c t r o p h o r e s i s of p a r t i a l enzymatic or formamide V a l d i g e s t s of tRNA^ on denaturing polyacrylamide g e l s e x h i b i t e d both strong band compression and incomplete enzymatic cleavage i n the v a r i a b l e loop Phe and T-stem r e g i o n s . In yeast tRNA these regions are i n v o l v e d i n strong secondary and t e r t i a r y i n t e r a c t i o n s w i t h other parts of the molecule ( R i c h and V a l RajBhandary, 1976). S i m i l a r i n t e r a c t i o n s i n tRNA^ may be p a r t i c u l a r l y strong because of the high G-C content of these regions. The observed band compression i n d i c a t e s that the products of such RNase cleavage as does occur f o l d back on themselves and migrate anomalously during polyacrylamide g e l e l e c t r o p h o r e s i s . V a l Figure 18 compares the r e s u l t s obtained when unmodified tRNA^ and V a l chloroacetaldehyde modified tRNA^ are sequenced by the g e l read-off method. The severe band compression and inadequate enzymatic cleavage V a l observed when i n t a c t tRNA^ was the substrate (Figure 18A) was r e l i e v e d when the RNA was modified i n t h i s way before sequencing (Figure 18B). The ribonucleases used f o r sequencing d i s p l a y the same substrate s p e c i f i c i t y towards modified RNA as to the unmodified form. This i s s u r p r i s i n g c o n s i d e r -ing the s t r i c t requirement of RNase A f o r an unsubs t i t u t e d n i t r o g e n at p o s i -t i o n 3 of pyrimi d i n e s i n i t s substrate (Richards and Wyckoff, 1971). Thus, the ethenocytidine residues produced by m o d i f i c a t i o n w i t h chloroacetaldehyde would not be expected to be s i t e s f o r RNase A cleavage. Tolman et a l . (1974) modified d i n u c l e o s i d e monophosphates c o n t a i n i n g c y t i d i n e w i t h c h l o r o a c e t a l d e -93. Figure 18. Chloroacetaldehyde m o d i f i c a t i o n r e l i e v e s band compression on sequencing gels of the A43-G57 region of tRNA^ a l. A. tRNAY 3 1 was l a b e l l e d at the 3'-end w i t h [ct- 3 2P]ATP and tRNA n u c l e o t i d y l t r a n s f e r a s e ( M a t e r i a l s and Methods). The e n d - l a b e l l e d tRNA was sequenced by the g e l read-off method ( M a t e r i a l s and Methods). 10 u l reac-t i o n volumes contained: T T _ - 0.1 u n i t RNase Ti_; A - l - 0.01 u n i t RNase A; A-2 - 0.005 u n i t RNase A; L - 66% formamide, 100°C f o r 60 min. The sequence between p o s i t i o n s 45-52 could not be read because of band compression. B. tRNAVal w a s l a b e l l e d at the 3'-end as above. The e n d - l a b e l l e d tRNA was modified w i t h chloroacetaldehyde ( M a t e r i a l s and Methods) and sequenced by the g e l read-off method. 10 u l r e a c t i o n volumes contained: T]_ -0.01 u n i t RNase Tlt U - l - 0.01 u n i t RNase U 2; U-2 - 0.001 u n i t RNase U 2; L - 66% formamide, 100°C f o r 45 min; A - l - 0.05 u n i t RNase A; A-2 -0.01 u n i t RNase A; P]_ - 1 u n i t RNase Phy I . The n u c l e o t i d e sequence between p o s i t i o n s 45 and 52 could be read when chloroacetaldehyde-modified t R N A V a l was used as the substrate f o r the sequencing r e a c t i o n s . hyde and examined the s e n s i t i v i t y of the etheno d e r i v a t i v e s to h y d r o l y s i s by RNase A. They found e t h e n o c y t i d y l y l u r i d i n e (eCpU) and eCpeA to be completely r e s i s t a n t to h y d r o l y s i s w h i l e eCpG and ECpeC showed s l i g h t h y d r o l y s i s a f t e r prolonged RNase A treatment. I t i s p o s s i b l e , t h e r e f o r e , that RNase A recognizes the modified c y t i d i n e residues but i t i s more l i k e l y that m o d i f i c a t i o n was not complete and that unmodified c y t i d i n e residues were the s i t e s of RNase A cleavage. V a l The nucleoside a n a l y s i s (Table I I ) suggests that tRNA^ cont a i n s two 5-methylcytidine (m 5C) residues per molecule. One of these was l o c a t e d at p o s i t i o n 38; the other i s assigned to a probable p o s i t i o n at residue 48 or 49 since m5C has been found i n t h i s region of other eukaryotic v a l i n e tRNAs (Gauss and S p r i n z l , 1981). The bands corresponding to i n Figure 18B move anomalously f a s t compared to the surrounding c y t i d i n e bands. This suggests m o d i f i c a t i o n of C^g. Ribonuclease Phy I i s o f t e n assumed to hydrolyse a l l phosphodiester bonds i n RNA except those 3' to a C re s i d u e . Figure 18B shows that RNase Phy I cleaves the C-G bond between residues 51 and 52 and residues 56 and 57. The work of P i l l y et^ al_. (1978) showed that the r a t e at which RNase Phy I cleaves a phosphodiester bond depends on the nu c l e o t i d e s both 5' and 3' to the bond. For the 5' - n u c l e o t i d e the r a t e of cleavage decreases i n the order U > G = A > C. For the 3'-nucleotide the order of preference i s A > G = C > U. Thus UpA should be most s u s c e p t i b l e to cleavage by RNase Phy I and CpU most r e s i s t a n t . Other phosphodiester bonds should be of intermediate s e n s i t i v i t y . Of the four CpN phosphodiester bonds, only CpG was cleaved under the p a r t i a l d i g e s t i o n c o n d i t i o n s used f o r sequencing by the g e l read-off method. This made d i s t i n g u i s h i n g UpG from CpG d i f f i c u l t . Bonds 3' to modified u r i d i n e residues such as rT and ¥ were poorly cleaved by RNase Phy I and these residues c o u l d , t h e r e f o r e , be mistaken f o r C re s i d u e s . V a l Nucleotide 33 of tRNA^ (Figure 12) i s flanked on the 5'-side by a 2'-O-methylcytidine r e s i d u e . The phosphodiester bond between these two nucle-o t i d e s i s r e s i s t a n t to h y d r o l y s i s , hence n u c l e o t i d e 33 could not be i d e n t i f i e d V a l by the Stanley and Vassilenko method. tRNA^ was modified w i t h c h l o r o -acetaldehyde, l a b e l l e d at the 5'-end w i t h [ 3 2P]phosphate and sequenced by the g e l read-off method. Figure 19 shows that p o s i t i o n 33 i s occupied by a U Met re s i d u e . With the exception of the i n i t i a t o r tRNA i of higher eukary-otes, a l l sequenced tRNAs have a U at t h i s p o s i t i o n (Gauss and S p r i n z l , 1981). The tRNA was modified w i t h chloroacetaldehyde not because t h i s f a c i l i t a t e d i n t e r p r e t a t i o n of the sequencing g e l but because m o d i f i c a t i o n made e f f i c i e n t l a b e l l i n g of the 5-end of the molecule p o s s i b l e . D. Sequencing tRNA End-Labelled w i t h 1 2 5I-CMP V a l tRNA 4 , l a b e l l e d at the 3'-end w i t h l 2 5I-CMP using the enzyme tRNA n u c l e o t i d y l t r a n s f e r a s e (see M a t e r i a l s and Methods), was sequenced by the g e l read-off method. The r e s u l t s were i n f e r i o r to those obtained w i t h tRNA e n d - l a b e l l e d i n a s i m i l a r manner w i t h [a- 3 2P]AMP residues because of the wide, fuzzy bands produced on autoradiograms of sequencing g e l s when an ! 2 5 i l a b e l was used. V a l E. Homologies Between tRNA7| and Other V a l i n e tRNAs V o l V a l V a l The homology between tRNA* and 18 published tRNA or tRNA gene sequences i s presented i n Table IV. I t should be remembered t h a t , because of the i n v a r i a n t and h i g h l y conserved n u c l e o t i d e s present i n a l l V a l tRNAs, unrelated tRNAs d i s p l a y about 40% homology to each other. tRNA^ d i s p l a y s the great e s t homology (85%) to the mammalian v a l i n e tRNAs. In t h i s V a l tRNA^ i s unexceptional, homologies between Drosophila tRNAs and the Ly s corresponding tRNAs of mammals are high, ranging from 100% f o r tRNA 2 of Drosophila and r a b b i t l i v e r to 92% f o r tRNA^ e t of mammals (Gauss and V a l S p r i n z l , 1981). tRNA^ shows an i n t e r e s t i n g p a t t e r n of homology w i t h Figure 19. I d e n t i f i c a t i o n of the nucleotide present at p o s i t i o n 33 of tRNAX a l• tRNA^3-1 (7 ug) was modified w i t h chloroacetaldehyde as described i n M a t e r i a l s and Methods. The modified tRNA was dephosphorylated then 5' en d - l a b e l l e d w i t h [ 3 2P]phosphate by the procedures of S i l b e r k l a n g et_ a l . (1979). Chloroacetaldehyde-modified tRNA was l a b e l l e d about 5 times more e f f i c i e n t l y then unmodified tRNA by t h i s procedure. The 5' e n d - l a b e l l e d tRNA)[ a l was sequenced by the g e l read-off method as described i n M a t e r i a l s and Methods. 10 u l r e a c t i o n volumes contained: U-l - 0.005 u n i t RNase U*2; U-2 - 0.001 u n i t RNase U2; T - l - 0.01 u n i t RNase Ti_; T-2 - 0.001 u n i t RNase T^; L - 66% formamide, 100°C f o r 45 min; A - l - 0.01 u n i t RNase A; A-2 - 0.001 u n i t RNase A; P - l - 1 u n i t RNase Phy I ; P-2 - 0.1 u n i t RNase Phy I . Nucleotide 33 i s a U re s i d u e . The cause of the band present i n the A - l s l o t between p o s i t i o n s 34 and 35 i s not known. 98. U-l U-2 T-l T-2 L A- l A-2 P-l P - 2 V a l Table IV. Homology Between tRNA^ and Other Sequenced V a l i n e tRNAs and V a l i n e tRNA Genes Source V a l i n e Percent Reference Anticodon Homology D. melanogaster IAC 100 -mammalian IAC 85 1 human p l a c e n t a CAC 85 1 yeast IAC 81 1 yeast CAC 70 1 yeast UAC 61 1 T. u t i l i s IAC 79 1 B. stearothermophilus GAC 60 1 B. s u b t i l i s UAC 57 1 E. c o l i UAC 57 1 E. c o l i 2a* GAC 56 1 E. c o l i 2b GAC 49 1 s p i n i c h c h l o r o p l a s t UAC 48 2 maize c h l o r o p l a s t UAC 41 3 N. cras s a mitochondria UAC 44 1 yeast mitochondria UAC 44 4 mouse mitochondria UAC 42 5 A. n i d u l a n s mitochondria UAC 36 6 human mitochondria UAC 37 7 * E_. c o l i c o n t a i n s two v a l i n e tRNAs, tRNA^^ and t R N A ^ t w i t h the same GAC anticodon. References 1. Gauss and S p r i n z l , 1981 2. Sprouse et a l . , 1981 3. Schwarz et a l . , 1981 4. L i and T z a g o l o f f , 1979 5. Van E t t e n et a l . , 1980 6. Kochel et a l . , 1981 7. Anderson et a l . , 1981 100. V a l the three yeast tRNA i s o a c c e p t o r s . There i s strong homology (81%) to the V a l yeast i s o a c c e p t o r w i t h the same IAC anticodon as tRNA^ . Homology i s V a l V a l a s i g n i f i c a n t l y weaker between tRNA^ and yeast tRNA^^,^ (70%). V a l V a l Yeast tRNA^ U A C^ d i s p l a y s only 61% homology to tRNA^ , about the V a l same l e v e l of homology as i s found between tRNA^ and most b a c t e r i a l V a l V a l V a l tRNA s p e c i e s . tRNA^ d i s p l a y s the l e a s t homology to tRNA of mitochondria and c h l o r o p l a s t s (36-48%). Examination of the s i t e s of homo-logy among the sequenced v a l i n e tRNAs shows a nonrandom d i s t r i b u t i o n of these s i t e s . There i s co n s i d e r a b l y more homology among the loop regions of these tRNAs than i n the stem regions. Eighteen v a l i n e tRNA species or t h e i r genes have been sequenced to date. Inspection of these sequences re v e a l s a number of s i t e s occupied by the same nu c l e o t i d e s i n a l l v a l i n e tRNAs or among the members of a p a r t i c u l a r subset of these sequences ( i n a d d i t i o n , of course, to the i n v a r i a n t n u c l e o t i d e s found i n a l l tRNAs). Some of these s i m i l a r i t i e s have been p r e v i o u s l y noted by Sprouse et a l . (1981). A l l sequenced v a l i n e tRNAs c o n t a i n an A73 residue and a l l , V a l w i t h the exception of maize c h l o r o p l a s t tRNA , conta i n a G10-C25 base-V a l p a i r . A l l the non-organelle tRNA c o n t a i n G1-C72 and C31-G39 bas e - p a i r s . The conserved Y11-R24 base-pair (Figure 1) i s occupied by a U-A p a i r i n a l l V a l sequenced e u k a r y o t i c tRNAs and by a C-G p a i r i n the p r o k a r y o t i c and orga-n e l l e tRNAs. S i m i l a r l y , a l l eukaryotic v a l i n e tRNAs have a G12-C23 base-pair w h i l e the p r o k a r y o t i c and o r g a n e l l e tRNAs have a U-A base-pair at these p o s i -t i o n s . The l a s t n u c l e o t i d e i n the anticodon loop i s C38 i n a l l e u k a r y o t i c , V a l and w i t h the exception of maize c h l o r o p l a s t tRNA , a l l o r g a n e l l e tRNAs. V a l No sequenced p r o k a r y o t i c tRNA has a C residue at t h i s p o s i t i o n . A l l V a l sequenced eu k a r y o t i c tRNA c o n t a i n a U3-A70 and Y27-A43 base-pairs and V a l an A residue at p o s i t i o n 44. Eukaryotic tRNAs a l s o c o n t a i n C49-G65 and C 50-G64 base p a i r s . In b a c t e r i a , w i t h the exception of B. stearothermophilus 101. Va l tRNA , these p o s i t i o n s are occupied by G49-C65 and G50-C64 p a i r s . The conserved n u c l e o t i d e s described above probably play important r o l e s i n the s t r u c t u r e , f u n c t i o n and r e c o g n i t i o n of v a l i n e tRNAs. The Drosophila v a l i n e tRNAs can be e f f i c i e n t l y aminoacylated by E. c o l i valyl-tRNA synthetase (Dr. I.C. G i l l a m , personal communication). Therefore, those conserved n u c l e o t i d e s found e x c l u s i v e l y among eukaryotic v a l i n e tRNAs are probably not i n v o l v e d i n V a l the r e c o g n i t i o n of tRNA by i t s synthetase. V a l I I I . The Nucleotide.Sequence of D. melanogaster tRNA^^ A. Nucleotide Sequence Determination l3b V a l The n u c l e o t i d e sequence of Drosophila tRNA-, i s presented i n Va l Figure 20. The s t r a t e g y used to sequence tRNA^ was a p p l i e d success-V a l f u l l y to the sequence determination of t R N A ^ . Again the Stanley and Vassilenko method provided most of the sequence i n f o r m a t i o n ( n u c l e o t i d e s 9-45, 51-69). No s i n g l e Stanley and Vassilenko experiment allowed the nu c l e o t i d e s at a l l these s i t e s to be i d e n t i f i e d . The r e s u l t s obtained by t h i s method and di s p l a y e d i n Figure 21 a re, t h e r e f o r e , drawn from s e v e r a l d i f f e r e n t e x p e r i -V a l ments. In sequencing tRNA^ a s l i g h t m o d i f i c a t i o n was made i n the Stanley and Va s s i l e n k o procedure. Solvent D (2-propanol:12 N HClirL^O, 70:15:15) was used f o r the separation of l a b e l l e d nucleoside-5'-phosphates on c e l l u l o s e TLC p l a t e s . This solvent proved p a r t i c u l a r l y u s e f u l f o r the i d e n t i -f i c a t i o n of modified pyrimidine n u c l e o t i d e s . Table V l i s t s the r e l a t i v e m o b i l i t y value of s e v e r a l n u c l e o t i d e s i n t h i s solvent system. Va l The 5' and 3'-ends of tRNA^ were sequenced by wandering spot a n a l y s i s . The r e s u l t s of these experiments are shown i n Figures 22 and 23. V a l The 3'-terminal n u c l e o t i d e of tRNA^ was not determined. However, i t i s undoubtedly an A residue s i n c e , without exception, a l l sequenced tRNAs have a 3'-terminal -CCA sequence (Gauss and S p r i n z l , 1981). 102. Figure 20. The n u c l e o t i d e sequence of Jn melanogaster tRNA^f,-L arranged as a c l o v e r l e a f . The C residue a t p o s i t i o n 48 i s probably modified to m5C. T r a n s c r i p -t i o n of the t R N A ^ f ^ - l i k e genes of plasmids pDt48 and pDt41R would produce a tRNA i d e n t i c a l to t R N A ^ l except f o r the replacement of C5, C16, G68 and G69 by U, U, A and A r e s p e c t i v e l y . m. t R N A ^ | 3 b AOH 76 c U - A U G A U 5 C - G A X 65 ~ A G A ^ U G G U G J § ) C G G T v C G C A C D 5 5 D U 25 m G a c P3 U A G A G 4 5 20 X " £ G " C U " A 30 G " C 4 0 C - G U C U A C A C 35 104. Figure 21. Stanley and Vassilenko sequencing of tRNA^f, 1. tRNAYjl 1 (2 ug) was d i s s o l v e d i n 10 u l 10 mM NH 40Ac pH 4.5, 1 mM EDTA and the s o l u t i o n was heated to 100°C f o r 3 min. The RNA fragments produced by t h i s l i m i t e d h y d r o l y s i s were 5' e n d - l a b e l l e d and f r a c t i o n a t e d according to s i z e by e l e c t r o p h o r e s i s on a 20% polyacrylamide g e l as described i n M a t e r i a l s and Methods. A f t e r autoradiography of the g e l , bands c o n t a i n i n g l a b e l l e d RNA fragments were excis e d from the g e l and the RNA was separately e l u t e d from each band and p r e c i p i t a t e d w i t h ethanol ( M a t e r i a l s and Methods). The e l u t e d RNAs were each d i s s o l v e d i n 5 u l 10 mM N H 4 O A C pH 4.5 c o n t a i n i n g 1 ug of nuclease Pj_. A f t e r i n c u b a t i o n at 37°C f o r 14 h the r e s u l t i n g [ 3 2P]nucleoside-5'-phosphates (pN's) were i d e n t i f i e d by t h i n l a y e r chromatography. In the F i g u r e , p o s i t i o n s at which the sequence of tRNA^f 1 d i f f e r s from a h y p o t h e t i c a l t r a n s c r i p t of plasmids pDt48 and pDt41R are marked ( A ) . The o r i g i n s of the chromatograms are a l s o marked ( 0 ) . A. Chromatography of pN's from p o s i t i o n s 35-37 and 58-69 on c e l l u l o s e TLC p l a t e s developed i n Solvent B ( M a t e r i a l s and Methods). The p o s i t i o n s of pG, pA and pi) standards on the t h i n l a y e r p l a t e were detected by t h e i r UV absor-bance and are c i r c l e d on the autoradiogram. B. Chromatography of pN's from p o s i t i o n s 13, 18, 19, 27 and 51-56 on c e l l u -l o s e TLC p l a t e s developed i n Solvent D. The p o s i t i o n of n u c l e o t i d e s t a n -dards on the TLC p l a t e are c i r c l e d on the p l a t e s ' s autoradiogram. C. The r e s u l t s presented i n panel C are from a d i f f e r e n t experiment from those shown i n panels A and B. In t h i s experiment the 5', 3 1 -bisphosphates (pNp's) of nu c l e o t i d e s 37-45 and 47 were i d e n t i f i e d by chromatography on P E I c e l l u l o s e TLC p l a t e s developed i n Solvent A. D. Chromatography of the pN's from p o s i t i o n s 16, 17, 21-26 and 29-34 of tRNA^f 1 on c e l l u l o s e TLC p l a t e s d eveloped i n Solvent B. E. Chromatography of pN's from p o s i t i o n s 9-12 and 14, 15 of t R N A ^ 1 on c e l l u l o s e TLC p l a t e s developed .in Solvent B. F. I d e n t i f i c a t i o n of the acp 3U residue at p o s i t i o n 20 of tRNA^f, 1. pNp's r e s u l t i n g from the Stanley and Vassilenko sequencing of the D-loop o f tRNA^g 1 were chromatographed on P E I - c e l l u l o s e TLC p l a t e s i n Solvent A. 105. 3 4 3 3 3 2 31 3 0 2 9 2 8 2 6 2 5 2 4 2 3 2 2 21 17 16 108. Figure 22. Wandering-spot a n a l y s i s of the 5'-end of tRNA^f,1. In an attempt to cleave tRNA^f,1 at i t s m7G residue ( S i l b e r k l a n g e t a l . , 1979), tRNA^f,1 (2 ug) was incubated at room temperature w i t h 50 mM NaOH, 0.25 mM EDTA (40 u l ) . A f t e r 15 min, 3 ul of 1 M a c e t i c a c i d and 40 ul of a n i l i n e - H C l pH 4.5 were added to the r e a c t i o n mixture and in c u b a t i o n was continued at 37°C f o r 4 h. The cleaved RNA was concentrated by p r e c i p i t a t i o n w i t h e t h a n o l , dephosphorylated and then 5' e n d - l a b e l l e d as described i n M a t e r i a l s and Methods. The l a b e l l e d RNA fragments were sepa-rated from each other by polyacrylamide g e l e l e c t r o p h o r e s i s . Autoradio-graphy of the g e l revealed a l a r g e number of RNA fragments. Several of the most prominent of these bands were exci s e d from the g e l . RNA was e l u t e d from each of the bands. The 5'-terminal n u c l e o t i d e of each fragment was i d e n t i f i e d by i n c u b a t i n g an a l i q u o t of RNA from each band i n 5 ul 10 mM N R A O A C pH 4.5, 1 mM EDTA c o n t a i n i n g 0.1 u n i t of RNase T 2 f o r 3 h at 37 C. The [5*- 3 2P]pNp's produced by the RNase T 2 d i g e s t i o n were i d e n t i -f i e d by TLC on P E I - c e l l u l o s e p l a t e s developed i n Solvent A. One of the fragments had a 5'-terminal G r e s i d u e . The sequence of t h i s fragment was determined by the wandering-spot procedure ( M a t e r i a l s and Methods). The xylene cyanol marker dye was allowed to migrate 6 cm during e l e c t r o p h o r e s i s i n the f i r s t dimension ( 1 ) . Homo-mix V of Jay et a l . (1974) was used f o r homochromatography i n the second dimension ( 2 ) . R i n d i c a t e s the p o s i t i o n of the red marker dye. The mark ( A ) at C5 i n d i c a t e s that a t r a n s c r i p t of the tRNA^f.-'—like genes of plasmids pDt48 and pDt41R would c o n t a i n a U resi d u e at th a t p o s i t i o n . 109. 110. Figure 23. Wandering-spot a n a l y s i s of the 3'-end of tRNA^f, 1. tRNA^f,1 was l a b e l l e d at the 3'-end w i t h [5'- 3 2P]pCp as d e s c r i b -ed i n M a t e r i a l s and Methods. The e n d - l a b e l l e d RNA was heated to 100°C f o r 40 min i n 4 y l of 98% formamide. The p a r t i a l l y hydrolysed RNA was sub-j e c t e d to wandering-spot a n a l y s i s as described i n M a t e r i a l s and Methods. The xylene cyanol marker dye was allowed to migrate 8 cm during e l e c t r o -phoresis i n the f i r s t dimens i o n (1)« Homo—mix V of Jay et al» (1974) was used f o r homochromatography i n the second dimension ( 2 ) . 112. Table V. Chromatographic M o b i l i t i e s of Nucleoside-5'-Phosphates on C e l l u l o s e TLC P l a t e s 3 i n Solvent D Nucleotide R^U pU 1.00 pC 0.59 pA 0.40 pG 0.36 pm7G 0.47 pf 0.72 prT 1.10 pD 0.90 a - c e l l u l o s e on g l a s s TLC p l a t e s (E. Merck) b - m o b i l i t y r e l a t i v e to pU V a l V a l L i k e tRNA^ , the v a r i a b l e loop region of tRNA^ could not be sequenced by the Stanley and V a s s i l e n k o method presumably because of strong secondary s t r u c t u r e of RNA fragments produced by cleavage i n t h i s V a l r e g i o n . Instead tRNA^ was cleaved i n the anticodon by l i m i t e d d i g e s t i o n w i t h RNase to produce h a l f - m o l e c u l e s . The half-molecules c o u l d be e f f i c i e n t l y 5' e n d - l a b e l l e d and were then sequenced by the g e l read-off method. Figure 24 shows an autoradiogram of part of such a sequencing g e l . The n u c l e o t i d e sequence of the v a r i a b l e loop region of V a l tRNA^ can be read from t h i s autoradiogram. The strong band compres-s i o n evident i n the sequencing g e l s of the corresponding r e g i o n i n 3'-end V a l l a b e l l e d tRNA^ (Figure 18A) i s absent i n t h i s autoradiogram. When present i n the same molecule, the p y r i m i d i n e - r i c h v a r i a b l e loop of ^Val „™ AVal 3b tRNA„fx or tRNA^ """" can presumably hydrogen bond to p u r i n e - r i c h regions near the 3'-ends of these tRNAs. Such i n t r a m o l e c u l a r i n t e r a c t i o n s may r e s u l t i n the anomalous e l e c t r o p h o r e t i c behavior observed when 3' end l a b e l l e d tRNA i s sequenced by the g e l read-off method. These i n t e r a c t i o n s would a l s o e x p l a i n the poor 5' e n d - l a b e l l i n g of tRNA fragments produced by V a l V a l cleavage of tRNA^ and tRNA^ i n the v a r i a b l e loop during 113. Figure 24. Gel read-off sequencing of tRNA^f,-^: The n u c l e o t i d e sequence of the v a r i a b l e arm o f tRNA^I 1 . tRNA^f,! w a s p a r t i a l l y hydrolysed w i t h RNase U2 and the r e s u l t -i n g fragments were 5' e n d - l a b e l l e d as described i n M a t e r i a l s and Methods. One of these fragments was sequenced by the g e l read-off method ( M a t e r i a l s and Methods). 10 u l r e a c t i o n volumes contained: NE - no enzyme; U-l -0.01 u n i t RNase U 2; U-2 - 0.001 u n i t RNase U 2; T - 0.02 u n i t RNase T j ; L - 66% formamide, 100°C f o r 20 min; A - l - 0.02 u n i t RNase A; A-2 - 0.01 u n i t RNase A; A-3 - 0.02 u n i t RNase A, 2 u l a l i q u o t s removed and the d i g e s t i o n stopped a f t e r 1, 2, 4, 8 and 16 min of i n c u b a t i o n at 50°C; P - 0.1 u n i t of RNase Phy I . In the autoradiogram of the sequencing g e l a very f a i n t band i s v i s i b l e i n the A - l s l o t at p o s i t i o n 47, the probable s i t e of a D resi d u e . C48 may be modified to m5C. 114. G45 115. Stanley and Vassilenko sequencing of these tRNAs. When 5' e n d - l a b e l l e d tRNA i s sequenced by the g e l read-off method cleavages i n the v a r i a b l e loop by b a s e - s p e c i f i c ribonucleases separate the two i n t e r a c t i n g regions of the tRNA. The e n d - l a b e l l e d products of such cleavages migrate normally on the sequen-c i n g g e l . V a l The nucleoside a n a l y s i s of tRNA^^ i n d i c a t e s that each tRNA mole-cule contains a s i n g l e N 7-methylguanosine (m7G) residue (Table I I ) . This n u c l e o t i d e i s found at only one p o s i t i o n i n sequenced tRNAs, i n the middle of the v a r i a b l e loop ( n u c l e o t i d e 46) (Dirheimer et_ a l . , 1979). Figure 24 shows that none of the sequencing ribonucleases cleave t RNA^ a l at p o s i t i o n 46 but that h y d r o l y s i s i n formamide cleaves par-3b t i c u l a r l y e f f i c i e n t l y there g i v i n g a band i n the formamide ladder at t h i s p o s i t i o n more intense than the other bands. This i s c h a r a c t e r i s t i c of an m7G residue ( f o r example see Figure 18B). Several l i n e s of evidence suggest that there i s a d i h y d r o u r i d i n e (D) V a l residue at p o s i t i o n 47 of tRNA^ . The tRNA i s poorly cleaved by RNase A but appr e c i a b l y cleaved by RNase Phy I at p o s i t i o n 47 (Figure 24). This suggests a modified u r i d i n e residue may be present at t h i s s i t e . Stanley V a l and Vassilenko sequencing of tRNA^^ i n d i c a t e d that a D residue was present i n the v a r i a b l e loop region of the tRNA (Figure 21) although the p o s i t i o n of the n u c l e o t i d e could not be e s t a b l i s h e d by t h i s method. On the b a s i s of these data one of the two D residues detected i n the nucleoside V a l l y s i s of tRNA^k (Table I I ) was assigned to p o s i t i o n 47. This Va] 3b ana V a l assignment i s c o n s i s t e n t w i t h the p a r t i a l sequence of tRNA_, p u b l i s h -ed by Altwegg (1980). V a l t RNA^ i s cleaved at p o s i t i o n 48 by RNase A but not by RNase Phy I . This i n d i c a t e d that a C residue i s present at that p o s i t i o n . Examina-t i o n of Figure 24 shows that the l a b e l l e d RNA fragment produced by RNase A 116. cleavage at p o s i t i o n 48 migrates more slowly during e l e t r o p h o r e s i s than the corresponding fragment produced by h y d r o l y s i s i n hot, aqueous formamide. The reason f o r t h i s anomalous behavior i s not c l e a r . I t i s tempting to speculate that n u c l e o t i d e 48 i s the 5-methylcytidine (m sC) residue V a l detected i n the nucleoside a n a l y s i s of tRNA^^ (Table I I ) . Cribbs (1979) has p r e v i o u s l y noted that cleavage at an m5C residue i n a tRNA by RNase A produced a double band on a sequencing g e l . He a t t r i b u t e d t h i s behavior to p a r t i c u l a r l y e f f i c i e n t RNase A-catalysed ring-opening of c y c l i c phosphates on 3'-terminal m5 C re s i d u e s . I t should be mentioned t h a t , under the c o n d i t i o n used f o r g e l read-off sequencing, the products of forma-mide h y d r o l y s i s and p a r t i a l ribonuclease d i g e s t i o n are thought to bear 2 ' , 3 ' - c y c l i c phosphate moie t i e s at t h e i r 3'-termini (Simoncsits jst a l . , V a l 1977). Thus formamide h y d r o l y s i s at p o s i t i o n 48 of t R N A ^ would y i e l d a product w i t h a c y c l i c phosphate at the newly generated 3'-end. This fragment would migrate normally on the sequencing g e l . Assuming that n u c l e o t i d e 48 i s an m5C re s i d u e , cleavage at t h i s p o s i t i o n w i t h RNase A would produce, according to Crib b s ' p r o p o s a l , s u b s t a n t i a l amounts of RNA wi t h a n o n - c y c l i c 3'-phosphate re s i d u e . This m a t e r i a l may migrate anoma-l o u s l y during e l e c t r o p h o r e s i s . m5C residues are found at p o s i t i o n 48 of a l l sequenced v a l i n e tRNAs of higher eukaryotes (Gauss and S p r i n z l , 1981). V a l B. Features of the tRNA_, Sequence 3D V a l Although the n u c l e o t i d e sequence of t R N A ^ i s very s i m i l a r to V a l t h a t of Drosophila tRNA^ , i t does c o n t a i n a number of unique f e a -V a l t u r e s . Some of these f e a t u r e s are b r i e f l y discussed below. t R N A ^ has a mismatched U4-G69 base-pair i n i t s acceptor stem (Figure 20). Such mismatches are q u i t e common i n the stem regions of tRNAs. C l a r k (1978) has hypothesized that the i r r e g u l a r i t y introduced i n t o double h e l i c a l regions of tRNAs by these mismatched base-pairs are important s i t e s of enzyme rec o g n i -117. V a l t i o n . I t i s i n t e r e s t i n g that i n tRNA^ the same base-pair i s of the Watson-Crick type but that the U4 residue i s modified to 2'-O-methyluridine. Phe Examination of a s p a c e - f i l l i n g model of yeast tRNA , presumed to be sim-V a l i l a r i n s t r u c t u r e to the s t r u c t u r e of tRNA^ , shows that a 2'-0-methyl group on nu c l e o t i d e 4 would l i e i n the shallow groove of the h e l i c a l stem. Some a l t e r a t i o n i n the h e l i x backbone would probably be required to accomo-.Val , _„.Val dat e the bulky methyl group. Thus i n both tRNA^^ and tRNA^ the base-pair between nucl e o t i d e s 4 and 69 i s d i s t i n c t i v e . V a l U n l i k e the case i n tRNA^ , the u r i d i n e residue at p o s i t i o n 20 of t R N A ^ 1 i s completely modified to acp 3 U (Figure 20). As w i t h t R N A j a \ acp 3 U (nucleoside X) was not detected i n the nucleoside a n a l y s i s of t R N A ^ 1 (Table I I ) . V a l tRNA^ has a CAC anticodon. According to the wobble hypothesis (see Results and D i s c u s s i o n , S e c t i o n I ) t h i s tRNA should, t h e r e f o r e , decode only the GUG codon. The tRNA's behaviour during ribosome-binding s t u d i e s was c o n s i s t e n t w i t h t h i s p r e d i c t i o n (Figure 11). The anticodon loop of V a l tRNA^ ends w i t h an unmodified C residue at p o s i t i o n 38. In tRNA^"*" t h i s p o s i t i o n i s occupied by an m5 C r e s i d u e . This d i f f e r e n c e V a l i n methylation of C38 among isoacceptors i s unusual. Human tRNA^^,^ V a l V a l and tRNA. . and the 3 yeast tRNA isoacceptors a l l have unmeth-V a l y l a t e d C at p o s i t i o n 38. In tRNA^^,^ of mouse myeloma c e l l s and r a b b i t l i v e r an m5C residue i s found at t h i s p o s i t i o n . V a l The v a r i a b l e loop of tRNA^ contains a d i h y d r o u r i d i n e residue at V a l p o s i t i o n 47 (Figure 20). In t h i s respect t R N A ^ resembles a l l se-V a l quenced eukaryotic v a l i n e tRNAs except Drosophila tRNA^ which con-t a i n s a C residue at t h i s s i t e . V a l C. Homologies Between tRNA^ and Other V a l i n e tRNAs Va 1 Drosophila tRNA^ contains a l l the i n v a r i a n t and s t r o n g l y con-118. served n u c l e o t i d e s present i n a l l cytoplasmic tRNAs. In a d d i t i o n , i t contains a l l the n u c l e o t i d e s common to e u k a r y o t i c v a l i n e tRNAs noted i n Se c t i o n II.D of the Results and D i s c u s s i o n . One d i f f e r e n c e between "Val tRNA.^ and a l l other sequenced v a l i n e tRNAs i s the G28-C42 base-pair; i n the other tRNAs a pyrimidine 28-purine 42 base-pair i s found at t h i s s i t e . V a l V a l t R N A ^ d i s p l a y s 88% homology to Drosophila tRNA^ . This degree of homology i s much stronger than the 61% homology found between the „»..Val , „„.Val . e corresponding tRNA^^,^ and t R N A ^ j ^ ^ i s o a c c e p t o r s of yeast. There are s e v e r a l p o s s i b l e explanations f o r t h i s . The two Drosophila tRNAs may have diverged from a common a n c e s t r a l sequence more r e c e n t l y than the two species from yeast. A l t e r n a t i v e l y , stronger s e l e c t i v e pressures may have prevented the two Drosophila species from d i v e r g i n g as r a p i d l y a those of yeast. I t i s a l s o p o s s i b l e that the two Drosophila tRNAs are the products of convergent e v o l u t i o n from a n c e s t r a l sequences that were l e s s s i m i l a r to one another. Of course, any combination of these processes i s a l s o p o s s i b l e . Deciding which, i f any, of these a l t e r n a t i v e s i s c o r r e c t i s beyond the scope of t h i s t h e s i s . A recent review by Cedergren et a l . (1981) describes the d i f f i c u l t i e s a s s o c i a t e d w i t h the determination of phylogenetic V a l V a l r e l a t i o n s h i p s among tRNA sequences. While tRNA^^ and tRNA^ of Drosophila are more s i m i l a r than the corresponding tRNAs of yeast they are V a l V a l much l e s s s i m i l a r than the sequenced tRNA^^,^ and t R N A ^ ^ ^ of human pla c e n t a . These two tRNAs d i f f e r by a s i n g l e n u c l e o t i d e change i n the f i r s t p o s i t i o n of the anticodon (Chen and Roe, 1977). I t remains to be V a l seen i f the sequenced human tRNA^^,^ i s the only v a l i n e tRNA i n human placenta w i t h the CAC anticodon. V a l The degree of homology between Drosophila tRNA^^ and the other V a l sequenced v a l i n e tRNAs i s presented i n Table V I . tRNA^^ shows no V a l more homology to Drosophila tRNA^ than i t does to the mammalian V a l Table VI. Homology Between tRNA^ and Other Sequenced V a l i n e tRNAs and V a l i n e tRNA Genes Source V a l i n e Percent Reference Anticodon Homology D. melanogaster CAC 100 -D. melanogaster IAC 88 -mammalian IAC 88 1 human placenta CAC 89 1 yeast IAC 74 1 yeast CAC 77 1 yeast UAC 66 1 T. u t i l i s IAC 70 1 B. stearothermophilus GAC 63 1 B. s u b t i l i s UAC 53 1 E. c o l i UAC 51 1 E. c o l i 2a* GAC 55 1 E. c o l i 2b GAC 49 1 s p i n i c h c h l o r o p l a s t UAC 48 2 maize c h l o r o p l a s t UAC 42 3 N. c r a s s a mitochondria UAC .. 45 i yeast mitochondria UAC 45 4 mouse mitochondria UAC • 40 5 A. n i d u l a n s mitochondria UAC 42 6 human mitochondria UAC 34 7 * c o l i c o n t a i n s two v a l i n e tRNAs, tRNA 2 a and tRNA^^, w i t h the same GAC anticodon. References 1. Gauss and S p r i n z l , 1981 2. Sprouse et a l . , 1981 3. Schwarz et a l . , 1981 4. L i and T z a g o l o f f , 1979 5. Van Etten et a l . , 1980 ' 6. Kochel et a l . , 1981 7. Anderson et a l . , 1981 120,. v a l i n e tRNAs. This suggests that the two Drosophila tRNAs diverged from a common a n c e s t r a l sequence about the time the l i n e s l e a d i n g to arthropods and chordates diverged from each other. tRNA sequence data from a wider v a r i e t y of organisms and more s o p h i s t i c a t e d a n a l y s i s of the data are required to Va l prove or disprove t h i s hypothesis. Drosophila t R N A ^ d i s p l a y s about V a l V a l equal homology to yeast tRNA^^,^ and tRNA^^,^ (about 75%) V a l but d i s t i n c t l y lower homology to yeast t RNA^ U A C^ (66%). B a c i l l u s Va l s tearothermophilus tRNA^g^,^ shows considerably greater homology to Va l V a l tRNA 3£ (63%) (and to a l e s s e r extent tRNA* ) than do other b a c t e r i a l v a l i n e tRNAs (about 52%). Most of the increased homology can be a t t r i b u t e d to the great s i m i l a r i t y i n the acceptor stems of the two tRNAs. V a l V a l tRNA^ > l i k e tRNA^ , d i s p l a y s the l e a s t homology to the v a l i n e tRNAs of s u b c e l l u l a r o r g a n e l l e s (34-48%). V a l IV. The Nucleotide Sequence of tRNA^ Genes The recombinant plasmid pDt55 c o n s i s t s of an 8 kb segment of Drosophila DNA cloned i n t o the Hind I I I r e s t r i c t i o n s i t e of the plasmid v e c t o r pBR322 Va l (Table I I I ) . The plasmid can form an RNA:DNA hyb r i d w i t h tRNA^ and was, t h e r e f o r e , presumed to c o n t a i n a gene f o r t h i s tRNA (Dunn et a l . , 1979). In s i t u h y b r i d i z a t i o n of pDt55 t o Drosophila polytene chromosomes showed that the Drosophila DNA of the plasmid came from the 70BC r e g i o n , one of the V a l major s i t e s of tRNA^ h y b r i d i z a t i o n (Table I ) . In the f o l l o w i n g sec-Va l t i o n s the n u c l e o t i d e sequence of the two tRNA^ genes of pDt55 w i l l be presented and t h e i r sequences w i l l be compared to those of other Drosophila m 7 i V a l tRNA^ genes. V a l A. Strategy Used to Sequence the tRNA^ Genes of pDt55 Plasmid pDt55 c a r r i e s a l a r g e segment of Drosophila DNA. The f i r s t .Val o b j e c t i v e i n sequencing the tRNA^ genes of t h i s plasmid was to iden-121. t i f y s maller fragments of DNA, amenable to sequence determination by the Maxam and G i l b e r t method, that contained the genes of i n t e r e s t . To t h i s end a r e s t r i c t i o n endonuclease cleavage map of the plasmid was constructed. This map i s shown i n Figure 25. The two s i t e s of cleavage by r e s t r i c t i o n V a l endonuclease Xma I were of s p e c i a l s i g n i f i c a n c e . tRNA^ contains the sequence CCCGGG (n u c l e o t i d e s 60-65, Figure 12), the r e c o g n i t i o n sequence of Xma I . Thus the genes f o r t h i s tRNA should be cleaved by t h i s r e s t r i c t i o n enzyme• V a l R e s t r i c t i o n fragments of pDt55 c o n t a i n i n g the tRNA^ genes were i d e n t i f i e d by t h e i r s e n s i t i v i t y to cleavage w i t h Xma I . pDt55 DNA was cleaved w i t h a r e s t r i c t i o n nuclease and the r e s u l t i n g fragments were end-l a b e l l e d . An a l i q u o t of t h i s mixture of l a b e l l e d r e s t r i c t i o n fragments was digested w i t h Xma I . The o r i g i n a l r e s t r i c t i o n enzyme d i g e s t and the DNA that had been cleaved w i t h both the o r i g i n a l enzyme and Xma I were a p p l i e d to adjacent s l o t s i n a polyacrylamide g e l and the r e s t r i c t i o n fragments were separated by e l e c t r o p h o r e s i s . Autoradiography revealed the p o s i t i o n of l a b e l l e d DNA fragments on the g e l . Any fragments present i n DNA cut only w i t h the f i r s t r e s t r i c t i o n enzyme but absent i n the DNA cut w i t h both the f i r s t enzyme and Xma I must c o n t a i n an Xma I r e s t r i c t i o n s i t e and hence Va l perhaps a tRNA^ gene. Figure 26 shows the r e s u l t s of such an e x p e r i -ment. D i g e s t i o n of pDt55 DNA w i t h r e s t r i c t i o n enzyme Hinf I produces a s i n g l e l a r g e fragment (about 1 kb long) s e n s i t i v e to cleavage by Xma I . This fragment was used as the source of DNA f o r most of the sequencing experiments done on plasmid pDt55. In a t y p i c a l sequencing experiment plasmid DNA (20 pg) was cleaved w i t h Hinf I and the fragments were e n d - l a b e l l e d to a low s p e c i f i c a c t i v i t y w i t h 3 2 P (see M a t e r i a l s and Methods). The fragments were separated by el e c t r o p h o r e s i s on a 5% polyacrylamide g e l and the l a b e l l e d fragments were 122. Figure 25. The r e s t r i c t i o n map of plasmid pDt55. Recombinant plasmid pDt55 c o n s i s t s of a 8.1 kb Hind I I I fragment of Drosophila DNA cloned i n t o the s i n g l e Hind I I I s i t e of the plasmid v e c t o r pBR322. A r e s t r i c t i o n endonuclease cleavage map of pDt55 was constructed by the m u l t i p l e enzyme d i g e s t method described by Danna (1980). The two Xma I s i t e s of pDt55 mark the s i t e s of the plasmid's two tRNA^3-1- genes. EcoR I, Hind III pDt55 124. Figure 26. I d e n t i f i c a t i o n of the H i n f I r e s t r i c t i o n fragment of pDt55 that contains the tRNA^ a l genes. pDt55 DNA (20 pg) was d i s s o l v e d i n 400 p i of r e s t r i c t i o n enzyme b u f f e r B ( M a t e r i a l s and Methods) c o n t a i n i n g 22 u n i t s of r e s t r i c t i o n endonu-clease Hinf I (New England B i o l a b s ) and incubated f o r 3.5 h at 37°C. The cleaved DNA was p r e c i p i t a t e d w i t h ethanol and r e d i s s o l v e d i n 50 p i of b u f f e r B c o n t a i n i n g 80 pCi of [a- 3 2p]dATP (Amersham, 2000-3000 Ci/mmol) and 1 u n i t of Klenow DNA polymerase I (Boehringer-Mannheim). An a l i q u o t (10 p i ) of the r e a c t i o n mixture was withdrawn and i t s DNA was p r e c i p i t a t e d w i t h ethanol i n p r e p a r a t i o n f o r cleavage w i t h Xma I . EDTA (5 p l , 0.25 M) and sucrose (15 p i , 40% w/v) were added to the remainder of the r e a c t i o n mixture which was then c h i l l e d on i c e u n t i l i t was loaded onto the g e l . The a l i q u o t of Hinf Icleaved DNA was d i s s o l v e d i n 10 p l of r e s t r i c t i o n b u f f e r A ( M a t e r i a l s and Methods) that contained 2 u n i t s of Xma I . A f t e r i n c u b a t i o n at 37°C f o r 2 h, 3 p l of 40% sucrose c o n t a i n i n g 0.1% xylene cyanol was added and i t , together w i t h the H i n f I-cleaved DNA, was loaded onto a non-denaturing 5% polyacrylamide g e l (15 x 40 x 0.05 cm) and run f o r 16 h at 100 V (2.8 V/cm). The autoradiogram of the g e l showed that a l a r g e Hinf I fragment of pDt55 (marked by the arrow) contained at l e a s t one Xma I cleavage s i t e . Cleavage of t h i s Hinf I fragment w i t h Xma I generated a new, much smaller fragment (*). "0" i s the o r i g i n of the g e l , "XC" marks the p o s i t i o n of xylene cyanol. 126. l o c a t e d by autoradiography. The second l a r g e s t band (Figure 26) on the g e l was ex c i s e d and the DNA was e l u t e d from i t . This DNA was cleaved w i t h any one of s e v e r a l r e s t r i c t i o n enzymes and the fragments were end-labelled to high s p e c i f i c a c t i v i t y . These l a b e l l e d fragments were strand separated as described i n M a t e r i a l s and Methods. Figure 27 shows the autoradiograph of such a strand separation. Bands c o n t a i n i n g s i n g l e - s t r a n d e d fragments of end - l a b e l l e d DNA were excised from the g e l and the DNA was e l u t e d from the g e l s l i c e s . The n u c l e o t i d e sequence of the e l u t e d fragments was determined by the method of Maxam and G i l b e r t (1980). The autoradiogram of one se-quencing experiment i s shown i n Figure 28. The strat e g y used to sequence the two tRNA genes of pDt55 i s summarized i n Figure 29. For 50% of the Hinf I fragment depicted i n Figure 29 both strands of the DNA were sequenced. The r e s t of the DNA, except f o r 70 bp that was sequenced only once, was sequenced at l e a s t twice i n the same d i r e c t i o n . The DNA between the two tRNA genes i s very A-T r i c h (73% A-T) and contains few r e s t r i c t i o n enzyme r e c o g n i t i o n sequences. The l a c k of s u i t a b l e r e s t r i c t i o n enzyme s i t e s made t h i s region d i f f i c u l t to sequence. At pH 8.5 under c o n d i t i o n s of low i o n i c s t r e n g t h r e s t r i c t i o n enzyme EcoR I i s reported to recognize and cleave the sequence AATT i n a d d i t i o n to i t s normal r e c o g n i t i o n sequence, GAATTC ( P o l i s k y et a l . , 1975). The l e s s s p e c i f i c r e c o g n i t i o n p r o p e r t i e s of EcoR I under these c o n d i t i o n s are termed the EcoR I * a c t i v i t y . The use of EcoR I * per-V a l mitted the region between the two tRNA^ genes to be sequenced. The major s i t e s of cleavage by EcoR I * were: AAATTC, GAATTT and GAGTTC• A l l these s i t e s are r e l a t e d t o the normal r e c o g n i t i o n sequence of EcoR I by a s i n g l e base change and a l l r e t a i n at l e a s t 1 G-C base-pair i n the r e c o g n i -t i o n sequence. B. The Nucleotide Sequence of the tRNA Genes of pDt55 V a l The n u c l e o t i d e sequence of the tRNA A genes of plasmid pDt55 i s 127. Figure 27. Strand s e p a r a t i o n of Dde I fragments of pDt55 DNA. pDt55 DNA (20 ug) was cleaved w i t h Hinf I and e n d - l a b e l l e d by i n c o r -p o r a t i o n of [a- 3 2P]dATP i n t o the r e s t r i c t i o n enzyme cleavage s i t e s as described i n F i g . 26 except t h a t only 2 uCi of [a- 3 2P]dATP (2000-3000 Ci/mmol) was used f o r the l a b e l l i n g . The Hinf I fragments were separated by polyacrylamide g e l e l e c t r o p h o r e s i s . The DNA fragments were lo c a t e d on the g e l by autoradiography and the second l a r g e s t Hinf I fragment was e l u t e d from the g e l . This DNA was p r e c i p i t a t e d w i t h ethanol and r e d i s s o l v e d i n 25 p l of b u f f e r c o n t a i n i n g 100 mM T r i s - H C l pH 7.5, 100 mM NaCl, 5 mM MgCl 2, 5 mM 2-mercaptoethanol and 1.25 u n i t of Dde I . A f t e r i n c u b a t i o n at 37°C f o r 2 h, 30 uCi of [a- 3 2P]TTP (Amersham, 2000-3000 Ci/mmol) and 1 u n i t of Klenow DNA polymerase I (Boehringer-Mannheim) was added and i n c u b a t i o n was continued at room temperature f o r 15 min. TTP (1 p l , 0.5 mM was added t o the mixture and 30 s l a t e r EDTA (2 P l , 0.25 M) was added to stop the po l y -merase r e a c t i o n . 3 p l of the r e a c t i o n mixture was withdrawn and mixed w i t h 3 p l of a s o l u t i o n c o n t a i n i n g 20% sucrose w/v, 0.1% xylene cyanol and 0.1% bromphenol blue. This sample was used as a c o n t r o l f o r the strand s e p a r a t i o n . U l t r a p u r e urea (25 mg) was added to the remainder of the reac-t i o n mixture. The s o l u t i o n was heated to 100°C f o r 3 min, then q u i c k l y a p p l i e d to a wide sample s l o t (8 cm) i n a 15 x 15 x 0.05 cm non-denaturing 5% polyacrylamide g e l . The c o n t r o l sample was a p p l i e d to an adjacent narrow sample s l o t ( C ) . The samples were subjected to e l e c t r o p h o r e s i s a t 500 V f o r 45 min. E l e c t r o p h o r e s i s was continued i n the c o l d room (4°C) at 500 V (14.2 V/cm) f o r 4 h. Those bands (numbered 1-7) i n the strand s e p a r a t i o n s l o t that had e l e c t r o p h o r e t i c m o b i l i t i e s d i f f e r e n t from bands i n the c o n t r o l s l o t were presumed to c o n t a i n s i n g l e - s t r a n d e d DNA. DNA from these bands was elu t e d from the g e l and subjected t o Maxam and G i l b e r t sequence a n a l y s i s . "XC" and "BPB" mark the p o s i t i o n s of xylene cyanol and bromphenol blue marker dyes. 129. Figure 28. Maxam and G i l b e r t sequencing: The f i r s t tRNA^3-1 gene of pDt55. The sequence of DNA fragment 4 of Figure 27 was determined by the Maxam and G i l b e r t method as described i n M a t e r i a l s and Methods. Samples of the sequencing r e a c t i o n s were a p p l i e d to a denaturing 12% polyacrylamide g e l and subjected to e l e c t r o p h o r e s i s f o r 5.5 h (4 lanes at l e f t ) or 3 h (4 lanes at r i g h t ) at 1800 V (51 V/cm). Fragment 4 contains the DNA sequence of the untranscribed strand of the f i r s t tRNA^ a l gene of pDt55 ( F i g . 30). The 5' and 3'-ends of the tRNA^3-1 gene are i n d i c a t e d i n the F i g u r e . 130. 131. Figure 29. The s t r a t e g y used to sequence the tRNA^ a l genes of pDt55. Each arrow represents a n u c l e o t i d e sequence determined by the method of Maxam and G i l b e r t (1.5 cm = 100 n u c l e o t i d e s ) . For the sequencing, pDt55 DNA was e n d - l a b e l l e d at cleavage s i t e s f o r the r e s t r i c t i o n endonucleases l i s t e d i n the F i g u r e . Arrows p o i n t i n g t o the l e f t i n d i c a t e t h a t the sequence of the strand shown i n the top panel of the Figure was determined. Arrows p o i n t i n g to the r i g h t i n d i c a t e the sequence of the complementary strand was determined. RESTRICT ION V a l 4 V a l 4 E N Z Y M E 5' 3' 3' 5' X m a 1 _ 4 H i n f 1 ; • 4 D d e 1 4 P--E c o R 1* • 4 •4 • • F n u 4 H 1 • • 4 S a u 9 6 1 -S a u 3 A • •4 • 133. presented i n Figure 30. The sequenced segment of Drosophila DNA contains 2 V a l i d e n t i c a l tRNA^ genes of opposite p o l a r i t y 525 bp a p a r t . With respect to the pDt55 r e s t r i c t i o n map (Figure 25) the f i r s t gene shown i n Figure 30 i s nearest the pBR322-Drosophila DNA boundary. The sequence of both genes V a l corresponds to the sequence of tRNA^ (Figure 12). The sequence homo-logy between the two genes extends 7 bp beyond the 5'-ends and 5 bp beyond the 3'-ends of the s t r u c t u r a l genes. In f a c t , w i t h loop-outs the homology extends f o r 24 bp beyond the 3'-end. These regions of homology may be important f o r the f u n c t i o n i n g of the genes or they may be the r e s u l t of a d u p l i c a t i o n by which a s i n g l e a n c e s t r a l gene gave r i s e to the present gene p a i r . S i m i l a r homologies are noted i n the f l a n k i n g sequences of a group of 5 Drosophila tRNA^"11 genes thought to have a r i s e n by a combination of gene d u p l i c a t i o n and unequal c r o s s i n g over (Hosbach et_ a l . , 1980). A short V a l d i stance beyond the 3'-ends of the tRNA^ genes of pDt55 are c l u s t e r s of T residues i n the non-transcribed strand (Tg f o r the f i r s t gene, T^ f o r the second). These sequences are probably t e r m i n a t i o n s i g n a l s f o r euk a r y o t i c RNA polymerase I I I (Valenzuela et_ a l . , 1977; Silverman et_ a l . , 1979). V a l The 5 ' - f l a n k i n g sequences of the two tRNA^ genes conta i n a number of i n t e r e s t i n g f e a t u r e s . For both genes there i s the p o s s i b i l i t y of forming h a i r - p i n s t r u c t u r e s i n the 5 ' - f l a n k i n g sequences. These h a i r - p i n s , s t a r t i n g 15 bp upstream from the f i r s t gene and 14 bp upstream from the second are shown i n Figure 31. I f loop-outs and G-T base-pairs are allowed, the h a i r - p i n adjacent to the f i r s t gene would have a 13 bp stem and a 7 n u c l e o t i d e loop w h i l e the h a i r - p i n upstream from the second gene would have a 17 bp stem and a 3 n u c l e o t i d e loop. 134. Figure 30. The n u c l e o t i d e sequence of a segment of the Drosophila DNA i n s e r t of plasmid pDt55. The strand shown i s the non-transcribed strand w i t h respect to the f i r s t tRNA^ a l gene and the t r a n s c r i b e d strand of the second tRNA^ a l gene. The two tRNA^3-^ genes are un d e r l i n e d . N u c l e o t i d e S l e q u e n c e o f p D t 5 5 100 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 C T C A G C A G C C A C C T T A A A A T A A T T C T A T T A T C A G T T G T G C T C T T T C C C C T T C A C T G A G C T G A A T A C C A T T A A C A A A G A C A A A C T G C C C A A T C A T T G G G T C 1 10 120 130 140 150 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 T C C T T G A A A C A T T T C C C A T A A A A A T C A C T C A A A T A G A T A C A A T A T A C G A T T T T A T T C A A G C A A C C A G T T T T A T T T T T G A C C C T T G G C A G T T G A G G T C G C T 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 . 3 0 0 G A A G T T G A C C T C T C T G C C G C T T A A G T T T C A A C T G T T T C C G T G G T G T A G C G G T T A T C A C A T C T G C C T A A C A C G C A G A A G G C C C C C G G T T C G A T C C C G G G C G 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 G A A A C A G G T G A T A A A C T T T T T T T T T A G T T T T T A T A C A A T T C G T A T T T T A A G A A A C C A C C A G A C T A A A T G G C T G A G T T C T C C T C T A A C G A T A T T T A G G T A T 4 10 4 2 0 4 3 0 4 4 0 4 5 0 4 6 0 4 7 0 4 8 0 4 9 0 5 0 0 A A A G T A T T T G A G T A T T A A T T G A A A T T T A T A G A T A T G T G C A T A A A T A T T T C A C T T T T T T T T G C T A G T T C T T G A T T G C T C G C T T T A T G T G T A A C T T A A A C G T 5 1 0 5 2 0 5 3 0 5 4 0 5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 6 0 0 T T T A A G C C A T T A G T A T A T A C T T G C A A T A A A G T A T T T A A G C A T T A A T T A A A T T A T C T A T C A A T C C T A G C T T G T T A T T T A G T G T A C G C A T C A T G A G T T A C T T 6 1 0 6 2 0 6 3 0 6 4 0 6 5 0 6 6 0 6 7 0 6 8 0 6 9 0 7 0 0 T G A A G C T T A C A A T T A T T G T A T T A A A A A A A C G T A C A T T A C A T T T T C C T A T T G G A A T T T A T C A T A G A A A A T A T A T G A C C A A A T G A A A C C C T T T T A C T C A A A T 7 1 0 7 2 0 7 3 0 7 4 0 7 5 0 7 6 0 7 7 0 7 8 0 7 9 0 8 0 0 A T G T C A A C T A A A T A C A T C T A C T C C A T A C A G G C T G A C T T A A A A T A T C G C A T A G C A A C T A C A G T T T C A T T G A T A A A A A T T C A A A C A T C T T T T A C A T C A A T C C 8 1 0 8 2 0 8 3 0 8 4 0 8 5 0 8 6 0 8 7 0 8 8 0 8 9 0 9 0 0 G A A T A A C A A A A A A T T A A A A A A T T T T T T C A C C T G T T T C C G C C C G G G A T C G A ACCGGGGGCC T T C T G C G T G T T A G G C A G A T G T G A T A A C C G C T A C A C C A C G G 9 1 0 9 2 0 9 3 0 9 4 0 9 5 0 9 6 0 9 7 0 A A A C A G T T G A A T A T A G T C C G T C C G A A G A G C C T C T T G T G A G C C A C A G G A G G C T T T C G G T T G G G C A A A G T G C C A G T A I 136. B .G A T *G C T G - f C-G T-A G-C G-C A-T G-C. T"G-C A-T C-G G-C - 50 ^G-C„-15 CCT T " G CTT G'C"T G-C T-A G-C c J A t - G ' T-A C-G C-G G-C A-T-_ A - T - c £f C-G C-G A A A C T G C C ACT Figure 31. P o s s i b l e H a i r - p i n s i n the 5'-Flanking Sequences of the  t R N A V a l Genes of pDt55 - A. A p o t e n t i a l h a i r - p i n s t r u c t u r e 15 bp upstream from the f i r s t tRNA^ a l gene of pDt55. The strand shown i s the non-transcribed s t r a n d . B. A p o t e n t i a l h a i r - p i n s t r u c t u r e 14 bp upstream from the second tRNA^ 3 1 gene of pDt55. The strand shown i s the non-t r a n s c r i b e d strand of t h i s gene; i n the complementary strand the stem would be 3 bp longer because G-T base-pairs are co n s i d e r -a b l y more s t a b l e than A-C p a i r s . In the " I n t r o d u c t i o n " ( S e c t i o n V.C) i t was noted that many sequenced Droso p h i l a t R N A ^ 3 genes have a sequence c l o s e l y r e l a t e d to GGCAGTTTTTA about 25 bp upstream from the genes. Examination of the 5'- f l a n k i n g V a l sequences of the two tRNA^ genes shows that the f i r s t gene has a s i m i l a r sequence, GGCAGTT, 49 bp upstream from the gene. Sixty-seven base-p a i r s upstream from the second gene (on the non-transcribed strand) i s a r e l a t e d GGCACTT sequence. At present, the f u n c t i o n of these sequences, i f any, i s unknown. In t h e i r study of human i n i t i a t o r methionine tRNA genes Santos and Met Z a s l o f f (1981) noted that short segments of the tRNA^ gene sequence 137. were repeated i n the 5' - f l a n k i n g sequences of these genes. They reviewed published tRNA gene sequence data and found other examples of d u p l i c a t e d Met sequences s i m i l a r to those a s s o c i a t e d w i t h human tRNA^, genes. In se v e r a l genes f o r which the t r a n s c r i p t i o n s t a r t s i t e s are known they f a l l w i t h i n a 6 or 7 bp sequence that i s a l s o found between p o s i t i o n s 15 and 40 of the s t r u c t u r a l gene. This suggests that the d u p l i c a t e d sequence may play a r o l e i n determining the t r a n s c r i p t i o n i n i t i a t i o n s i t e . The f i r s t V a l tRNA^ gene of pDt55 contains the sequence TCTGCC between p o s i t i o n s 27 and 32 ( n u c l e o t i d e s 260-265, Figure 30). This sequence i s a l s o present 21 nuc l e o t i d e s upstream from the gene ( n u c l e o t i d e s 213-218, Figure 30). I t w i l l be i n t e r e s t i n g to see i f the t r a n s c r i p t of t h i s gene i s i n i t i a t e d V a l w i t h i n the repeated sequence. Segments of the second tRNA^ gene of pDt55 are not repeated i n the sequenced p o r t i o n of the gene's 5 ' - f l a n k i n g sequence. C. Other tRNA^ a l Genes of Drosophila: Comparison to the tRNA^ a l  Genes of pDt55 V a l Recombinant plasmids c o n t a i n i n g tRNA^ genes were i d e n t i f i e d by V a l the c a p a c i t y of these plasmids to h y b r i d i z e w i t h tRNA^ (Dunn et_ a l . , 1979). Four d i f f e r e n t s i z e Hind I I I fragments of Drosophila DNA c o n t a i n i n g V a l tRNA^ genes were i s o l a t e d i n these plasmids (12 kb, 8 kb, 2 kb and V a l 0.5 kb; Table I I I ) . The sequences of the tRNA^ genes of the 8 kb Hind I I I fragment of Drosophila DNA cloned i n pDt55 were presented above. Va l Other workers have sequenced the tRNA genes present i n recombinant plasmids c o n t a i n i n g each of the three other Hind I I I fragments. These sequences are shown i n Figure 32. Plasmids pDt92R and pDtl20R (sequenced by Dr. C. A s t e l l and B. Ra j p u t ) , r e p r e s e n t a t i v e of plasmids c o n t a i n i n g the 0.5 kb and 2 kb Hind I I I fragments r e s p e c t i v e l y , both h y b r i d i z e to the 90C s i t e V a l on the Drosophila polytene chromosomes, a minor s i t e of tRNA^ h y b r i -138. Figure 32. The n u c l e o t i d e sequence of segments of the Drosophila DNA i n s e r t s of plasmids pDt92R, pDtl20R and pDtl4. Only the non-transcribed strands are shown. tRNA genes are underlin e d . At s i t e s where the n u c l e o t i d e sequences of pDtl20R and pDt92 d i f f e r the n u c l e o t i d e s found i n pDt92R are shown d i r e c t l y under the corresponding n u c l e o t i d e s i n pDtl20R. Dashes beneath the pDtl20R sequence i n d i c a t e regions which have not been sequenced i n pDt92R. The G i n parentheses at p o s i t i o n 515 of pDt92R i s postulated to occur i n the chromosome t o account f o r a Hind I I I r e c o g n i t i o n sequence found at t h i s s i t e . The s i t e s a t which the t R N A ^ a l - l i k e genes of these plasmids d i f f e r from the tRNA^ a l genes of pDt55 are o v e r l i n e d i n the F i g u r e . The f i r s t gene i n pDtl4 codes f o r tRNA| h e. T h e N u c l e o t i d e S e q u e n c e o f p D t 1 2 0 R w i t h t h e d i f f e r e n c e s 1n p D t 9 2 R u n d e r t h e s e q u e n c e 10 2 0 3 0 , 4 0 5 0 ' 6 0 7 0 8 0 9 0 100 AAGCTTCGAG G T A G G T A T G T A G C T T C A C G G C T T G C T G C T T A A G T T G T T A C A A T A C C A T T G GGAGGAGAGT GGG T A A A G G C A A G C C A C T A T A T A A G C G T G A G A 1 1 0 120 1 3 0 140 150 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 CACTTTTTAA A T T A T T T C C T A T A A T T A C A T T T T A T A A T T A C T T T G T G C T T T T A T T A T A A C A G A T A T A T T T G C T A A C T T A T C T T A A A T T G T C T A T G A G G A A G 2 10 2 2 0 2 3 0 _ 2 4 0 _ 2 5 0 _ 2 6 0 2 7 0 _ 2 8 0 2 9 0 3 0 0 AACGTTCGTC A T C C G A G T T T C C G T G G T G T A G T G G T T A T C A C A T C C G C C T A A C A C G C G G A A G G C C C C C G G T T C A A T C C C G G G C G G A A A C A G T T G G A A T T T A 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 T T T T T T G C T A A A T A T T T A T T T A T C A T A A T G T T C A G T T G T A A A A C A C A C A T A G C T A A T A G T A T T T A T A G C T G C A T C A T G G C C T T A A A C T T A T C A C G T T G C T A T " C 4 10 4 2 0 4 3 0 4 4 0 4 5 0 4 6 0 4 7 0 4 8 0 4 9 0 5 0 0 T T T G C T T C A A G G C C T C G T G C T T C T T A C G A T C C A C A T T T T T A A G C A G A A A T T C T T G A A A T T T C C T A C G C A T A T C T A A C G A T A G A C C T G T A T T T C G A A G G T C 5 1 0 5 2 0 5 3 0 5 4 0 5 5 0 CAACCTCTCA A G A A C C T T G T T G C A G C T A A T T A T C T T C A T C A A A T G C T T G C C A A A G T C ( G )  T h e N u c l e o t i d e S e q u e n c e o f p D t 1 4 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 T G T A T G C T C A T A A T G C G C T T T A A A A A A A A G G A T G T A C T G A A A A A T A A T G T C T G T A C T T T A A A C A G G G A A G G T T C A A A T A T T T T G G C A A G T A C G G C A A A T T 1 1 0 120 130 140 150 1 6 0 170 180 1 9 0 2 0 0 GACAACTTTG A T G T G A A G A A T C C C G T G G C C G A A A T A G C T C A G T T G G G A G A G C G T T A G A C T G A A G A T C T A A A G G T C C C C G G T T C A A T C C C G G G T T T C G G C A 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0 A T A A T A A T T T T T G C A C A A A T T A G G C A G A A C T C G C A T A A A A A A A T A A T A T A A A T T T T G G A A T A A T T T T A A G G C A T A A T A C A A C A C A T A C C G T A A A C A C T G A 3 10 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 ACACTTTTTA A T A T T T G A A C G A T C T G A T A G TTCAATAAGA C C G T T A T C C A A G T C T T A T T T A A A A T T A T T T A A T C A C C C T C A A A A C C G A A A A G C T G T G A A T 4 10 4 2 0 4 3 0 4 4 0 _ 4 5 0 _ 4 6 0 4 7 0 _ 4 8 0 4 9 0 5 0 0 CCACCCCATC A C G T G T T T C C G T G G T G T A G T G G T T A T C A C A T C C G C C T A A C A C G C G G A A G G C C C C C G G T T C A A T C C C G G G C G G A A A C A T T G G A A A T A T T T A 5 1 0 5 2 0 5 3 0 5 4 0 5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 6 0 0 TTTTAATGCA T T T C C C A A A T T A T T T T G C C T G T A T A A C T T A A A T A T A T A T A T T T T G T A A T G T G A T T T A T G T G T C A C T T T T G T C G G T C A C C T G A A G T G C T T A 6 1 0 6 2 0 6 3 0 6 4 0 6 5 0 6 6 0 6 7 0 6 8 0 6 9 0 7 0 0 AATTGTATAG TA A G T T T G A G G T C T C C A C T G G C A A A C C T C C C C T A G A T C A G C G G C A T G C C A G A A T C T T C G T C C T G G A C T C G C A C C T A C C T C A A C T G G A A G C 7 10 7 2 0 7 3 0 7 4 0 7 5 0 7 6 0 GGGTGCTGTT C C T C T C G C T G A A G T G C G T A T A C T T A A T G A T G G T G G T G A A G T T C A G C C A G A GGT 140. d i z a t i o n (Table I ) . These two plasmids d i f f e r at only 8 s i t e s i n the 506 bp between the Hind I I I s i t e at the l e f t end of each Drosophila DNA i n s e r t and a Hind I I I s i t e at the r i g h t end of the pDt92R i n s e r t . pDtl20R l a c k s t h i s second Hind I I I s i t e and i t s i n s e r t i s about 1500 bp longer. The d i f f e r -ences i n pDt92R are noted i n Figure 32 by l e t t e r s under the sequence of pDtl20R. These two plasmids may have been generated because the DNA used f o r c l o n i n g the tRNA genes was i s o l a t e d from a non-isogenic stock of JJ. melanogaster (Oregon R). The v a r i a t i o n s i n n u c l e o t i d e sequence outside the s t r u c t u r a l genes f o r the tRNAs may represent e i t h e r d i f f e r e n c e s i n the DNA of homologous chromosomes, making the regions f l a n k i n g the tRNA genes " a l l e l i c " , or d i f f e r e n c e s between repeated sequences i n the DNA of the same chromosome. At present, we do not know which a l t e r n a t i v e i s c o r r e c t . V a l Plasmid pDtl4 i s r e p r e s e n t a t i v e of the tRNA^ plasmids c o n t a i n i n g the l a r g e 12 kb fragment of Drosophila DNA. This plasmid h y b r i d i z e s to the Va l 89B s i t e on the polytene chromosomes, the other minor s i t e of tRNA^ h y b r i d i z a t i o n . A 763 bp segment of t h i s plasmid was sequenced by Dr. A Delaney. The sequence i s shown i n Figure 32. This r e g i o n contains two tRNA Va l genes of the same p o l a r i t y . The second i s a tRNA gene l o c a t e d 214 bp Phe downstream from the f i r s t , a tRNA 2 gene. Va 1 The tRNA genes of plasmids pDt92R, pDtl20R and pDtl4 are i d e n t i c a l V a l and d i f f e r from the tRNA^ genes of pDt55 at 4 s i t e s . T r a n s c r i p t i o n V a l of the tRNA^ - l i k e genes would produce a tRNA w i t h a U i n s t e a d of a C at p o s i t i o n 16 ( i n the D-loop), a C-G base-pair i n s t e a d of a U29-A41 base-p a i r i n the anticodon stem and an A57 in s t e a d of the G57 residue present i n Va l V a l the T-loop of tRNA^ (Figure 12). Are these tRNA^ - l i k e genes expressed i n vivo? At present a d e f i n i t e answer to t h i s question cannot be Va l V a l given. Sequence a n a l y s i s of tRNA^ a and tRNA^^ shows that the Va l tRNA^ - l i k e genes do not code f o r these tRNAs. There i s no hetero-141. V a l geneity i n the sequence of tRNA^ that could be a t t r i b u t e d to the V a l presence of t r a n s c r i p t s of the tRNA^ - l i k e genes i n the sample of V a l tRNA^ used f o r sequence determination (Figure 13). I t i s p o s s i b l e V a l that the. tRNA^ - l i k e genes code f o r one of the 4 minor v a l i n e tRNAs of Drosophila (Figure 7). None of these tRNAs have been c h a r a c t e r i z e d because of the p a u c i t y of m a t e r i a l a v a i l a b l e f o r such i n v e s t i g a t i o n s . . V a l A number of f a c t s h i n t that the tRNA^ - l i k e genes may be express-V a l ed and are not i n a c t i v e pseudo-genes. F i r s t , the tRNA^ - l i k e genes of pDtl4 and pDt92R/120R are w e l l separated on the chromosomes yet have e x a c t l y the same sequence. This suggests that the sequence homogeneity of these genes has been a c t i v e l y maintained, presumably to preserve t h e i r a b i l i t y to f u n c t i o n i n the c e l l . I t c o u l d , however, be argued that whatever mechanism maintains the sequence homology of dispersed copies of f u n c t i o n a l tRNA genes a l s o a c t s on pseudo-genes. Second, two of the d i f f e r e n c e s between the V a l V a l s t r u c t u r e of tRNA^ and the tRNA^ - l i k e genes are the s u b s t i t u -t i o n of a C-G base-pair f o r a U-A base-pair i n the anticodon stem of the V a l p o t e n t i a l t r a n s c r i p t of the tRNA^ - l i k e genes (Figure 12). Such V a l coupled s u b s t i t u t i o n s would be very u n l i k e l y i f the tRNA^ - l i k e genes were the product of mutations i n a no n - f u n c t i o n a l gene. T h i r d , a l l the nu-c l e o t i d e s c h a r a c t e r i s t i c of eu k a r y o t i c v a l i n e tRNAs (Res u l t s and D i s c u s s i o n , V a l S e c t i o n II.D) would be present i n any t r a n s c r i p t of the tRNA^ - l i k e gene. Such a t r a n s c r i p t would, however, be the f i r s t v a l i n e tRNA w i t h an A57 r a t h e r than a G57 residue (Gauss and S p r i n z l , 1981). While a G at p o s i -t i o n 57 i s most common, many tRNAs have been sequenced that c o n t a i n an A at that p o s i t i o n . V a l I f the tRNA^ - l i k e genes were expressed the tRNA produced would V a l have, l i k e tRNA^ , an IAC anticodon. There are s e v e r a l examples i n the l i t e r a t u r e of organisms that c o n t a i n s e v e r a l s l i g h t l y d i f f r e n t tRNAs,142. each w i t h the same anticodon ("isocoding tRNA"). For example, yeast Ser Ser tRNA^ and tRNA2 both have an IAG anticodon but d i f f e r from each other a t 3 other s i t e s , one tRNA has a G at p o s i t i o n 57 the other having an A residue at that p o s i t i o n (Zachau et^ a l . , 1966). Bovine T T T I tRNA i s a mixture of up t o 3 subspecies d i f f e r i n g i n the extent of some base m o d i f i c a t i o n s and i n the nu c l e o t i d e s present at 3 p o s i t i o n s ( F o u r n i e r e t a l . , 1978). Two of the s i t e s of v a r i a b i l i t y (C/U 16, G/A 57) are theV a l same as those between tRNA^ and the h y p o t h e t i c a l isocoding V a l Phe tRNA . K e i t h and Dirheimer (1980) reported that tRNA of Bombyx mori p o s t e r i o r s i l k gland i s a mixture of two species; 20% of the tRNA has an A at p o s i t i o n 57 the r e s t contains a G residue at that p o s i t i o n . In s e v e r a l organisms new isoc o d i n g species of tRNA are produced during c e l l d i f f e r e n t i a t i o n . In bovine lens the e p i t h e l i a l c e l l s c o n t a i n a s i n g l e Phe tRNA^ i s o a c c e p t o r w i t h a sequence i d e n t i c a l to that of beef l i v e r Phe tRNA . Lens e p i t h e l i a l c e l l s d i f f e r e n t i a t e to produce lens f i b r e c e l l s . Phe Phe The d i f f e r e n t i a t e d c e l l s c o n t a i n tRNA^ as w e l l as tRNA^ • The Phe two tRNAs d i f f e r by a s i n g l e n u c l e o t i d e . tRNA,, contains G57 wh i l e Phe tRNA^ contains an A residue at t h i s p o s i t i o n ( L i n e_t a l . , 1980). I t i s noteworthy how f r e q u e n t l y i s o c o d i n g tRNAs d i f f e r i n the n u c l e o t i d e present at p o s i t i o n 57. During development of the Bombyx p o s t e r i o r s i l k A l a A l a gland a new species of tRNA i s produced (tRNA^ ) i n a d d i t i o n to A l a the tRNA^, found i n a l l Bombyx t i s s u e s . The two species d i f f e r by a A l a s i n g l e n u c l e o t i d e . The C residue present at p o s i t i o n 40 of tRNA 2 i s A l a replaced by a U or * residue i n tRNA 2 • This introduces a mis-matched base-pair i n t o the anticodon stem of the l a t t e r species (Sprague et a l . , 1977). What i s the f u n c t i o n of the is o c o d i n g tRNAs? Bovine lens f i b r e c e l l s and Bombyx p o s t e r i o r s i l k gland c e l l s make l a r g e amounts of s p e c i f i c p r o t e i n s , lens c r y s t a l l i n s and s i l k f i b r o i n r e s p e c t i v e l y . Lens c r y s t a l l i n s 143. are r e l a t i v e l y r i c h i n phenylalanine while f i b r o i n contains 29% a l a n i n e . Both L i n et a l . (1980) and Sprague et a l . (1977) speculate that the new tRNA isoacce p t o r s produced during the d i f f e r e n t i a t i o n of lens f i b r e and p o s t e r i o r s i l k gland c e l l s are r e q u i r e d f o r the most e f f i c i e n t s y n t h e s i s of the major pr o t e i n s produced by these s p e c i a l i z e d c e l l s . Perhaps the mRNAs f o r these p r o t e i n s c o n t a i n codons that r e q u i r e the new i s o c o d i n g tRNAs f o r t h e i r e f f i c i e n t t r a n s l a t i o n . Evidence f o r such context e f f e c t s has been found i n V a l E. c o l i ( B o s s i and Roth, 1980). I t i s p o s s i b l e that the tRNA^ - l i k e genes of Drosophila are expressed only i n c e r t a i n t i s s u e s or only a t s p e c i f i c periods during development. The p a t t e r n of v a l i n e i s o a c c e p t o r s i n Drosophila revealed by RPC-5 chromatography i s the same i n f i r s t and t h i r d i n s t a r l a r v a e and i n a d u l t f l i e s (White j2t a l . , 1973a). However, the p a t t e r n of i s o a c c e p t o r s present i n the embryonic (egg) and pupal stages remains unknown. V a l V a l In summary, tRNA^ or tRNA^ - l i k e genes have been i s o l a t e d V a l and sequenced from 3 of the 4 s i t e s of tRNA^ h y b r i d i z a t i o n to Drosophila polytene chromosomes. The genes of plasmid pDt55 o r i g i n a t e from V a l V a l a major s i t e of tRNA^ h y b r i d i z a t i o n (70BC). The two tRNA^ V a l genes cloned i n t h i s plasmid correspond to the sequence of tRNA^ . No V a l tRNA^ genes have been cloned from the other major s i t e of V a l V a l tRNA^ h y b r i d i z a t i o n , 56D. The tRNA^ - l i k e genes of plasmids V a l pDt92R, pDtl20R and pDtl4 o r i g i n a t e at the minor s i t e s of tRNA^ h y b r i d i z a t i o n to polytene chromosomes (90BC and 89BC). T r a n s c r i p t i o n of these genes would produce a tRNA that d i f f e r s at 4 s i t e s from the n u c l e o t i d e sequence of tRNA^3^". 144. V a l V. The Nucleotide Sequence of tRNA^ Genes V a l A. The Nucleotide Sequence of the tRNAJj^ Gene of pDt78R The recombinant plasmid pDt78R contains a 5.2 kb fragment of Drosophila V a l DNA that can h y b r i d i z e to tRNA^ (Table I I I ) . The Drosophila DNA of t h i s plasmid was shown by i n s i t u h y b r i d i z a t i o n to have o r i g i n a t e d from the 84D region of the polytene chromosomes, one of the two major s i t e s of V a l tRNA^k h y b r i d i z a t i o n (Table I ) . Thus pDt78R was p r e d i c t e d to co n t a i n V a l a gene f o r tRNA^ . The r e s t r i c t i o n map of pDt78R i s presented i n Figure 33 (the s i t e s of cleavage by Hha I were determined by D. S t . L o u i s ) . P a r t i c u l a r l y noteworthy i s the presence of a s i n g l e Xma I s i t e i n the Va] S b l plasmid. The n u c l e o t i d e sequence of tRNA.,, i n d i c a t e s that V a l t RNA^ genes should c o n t a i n the r e s t r i c t i o n s i t e f o r t h i s enzyme. V a l The stra t e g y developed to sequence the tRNA^ genes of pDt55 was suc-V a l c e s s f u l l y a p p l i e d to the sequence a n a l y s i s of the tRNA^^ gene of pDt78R. The sequencing of pDt78R i s summarized i n Figure 34. Cleavage of pDt78R w i t h the r e s t r i c t i o n endonuclease Hha I produces a 640 bp fragment of DNA (the t h i r d l a r g e s t fragment present i n the d i g e s t ) that contains the V a l tRNA^ gene. This fragment provided a convenient source of DNA f o r many of the sequencing experiments. V a l The n u c l e o t i d e sequence of the t R N A ^ gene of pDt78R i s present-ed i n Figure 35. The sequence of the gene corresponds e x a c t l y to that of V a l t R N A^ • The 3 ' - f l a n k i n g sequence of the gene contains a s e r i e s of 5 consecutive T residues ( i n the non-transcribed s t r a n d ) . L i k e s i m i l a r sequences i n other eukaryotic tRNA genes, t h i s sequence probably serves as a t r a n s c r i p t i o n t e r m i n a t i o n s i g n a l . The f i v e n u c l e o t i d e s immediately adjacent to the 5'-end of the gene together w i t h the f i r s t n u c l e o t i d e of the gene form the sequence CACAAG. This sequence recurs w i t h i n the gene i t s e l f beginning at p o s i t i o n 40 (n u c l e o t i d e 98 i n Figure 35). As p r e v i o u s l y 145. Figure 33. The r e s t r i c t i o n map of plasmid pDt78R. Recombinant plasmid pDt78R c o n s i s t s of a 5.2 kb fragment of Drosophila DNA cloned i n t o the s i n g l e Hind I I I s i t e of the plasmid v e c t o r pBR322. A r e s t r i c t i o n endonuclease cleavage map of pDt78R was constructed by the m u l t i p l e enzyme d i g e s t method described by Danna (1980). The Xma I s i t e marks the l o c a t i o n of the plasmid's s i n g l e tRNA^f,-1 gene. The Hha I r e s t r i c t i o n s i t e s i n the pBR322 DNA of the plasmid are not shown. Hind 111 Pvull pDt78R 147. Figure 34. The s t r a t e g y used to sequence the tRNA^f,1 gene of pDt78R. Each arrow represents a n u c l e o t i d e sequence determined by the method of Maxam and G i l b e r t (1 cm = 20 n u c l e o t i d e s ) . For the sequencing, pDt78R DNA was e n d - l a b e l l e d at cleavage s i t e s f o r the r e s t r i c t i o n endonucleases l i s t e d i n the F i g u r e . Arrows p o i n t i n g to the l e f t i n d i c a t e that the sequence of the non-transcribed strand (shown i n the top panel of the Figure) was d e t e r -mined. Arrows p o i n t i n g to the r i g h t i n d i c a t e the sequence of the comple-mentary strand was determined. RESTR ICT ION E N Z Y M E X m a I. EcoR I* Msp I Taq I FnuE I 149. Figure 35. The n u c l e o t i d e sequences of two segments of Drosophila DNA c o n t a i n i n g tRNA^a)-L genes. The nucleotide sequences of the non-transcribed strands of two t R N A ^ 1 genes, one found i n pDt78R the other sequenced by S i l b e r k l a n g , are shown. The tRNAYjf,-1 genes are u n d e r l i n e d . T h e N u c l e o t i d e S e q u e n c e o f p D t 7 8 R 10 2 0 3 0 4 0 5 0 T C C G T G A A T T T A T A C T A G A C T T T A T A A T A T A G G T C T T G T G A T G T C A G C A C 110 120 130 140 150 A A G G T C C C C G G T T C G A A C C C GGGCGGGAAC A T G C G A T C C T T T T T G A A T T A 2 10 2 2 0 2 3 0 2 4 0 2 5 0 A A G G A C T G T T C A G C C A G A A A C G A A G T T T T T C C G T T C A G A T G T G T T G T T G G 6 0 7 0 8 0 9 0 1 0 0 C G C C A C A A G T T T C C G T A G T G T A G C G G T T A T C A C G T G T G C T T C A C A C G C A C 160 1 7 0 180 1 9 0 2 0 0 A T T T A T C A A T A A T T A T T T T G T A T T A T T T T A C G T T T T T A G T A T G T G G G A A A 2 6 0 G A T G C A T A T G TGAAGGGA V a l T H E N U C L E O T I D E S E Q U E N C E OF THE t R N A 3 b GENE D E T E R M I N E D BY S I L B E R K L A N G 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 C G G C C G T C T C T T A A G A G T T T C C G T A G T G T A G C G G T T A T C A C G T G T G C T T C A C A C G C A C A A G G T C C C C G G T T C G A A C C C G G G C G G G A A C A G T C G A A A T A G T 110 120 130 140 T T T T G T C T T T T T T T A T T T C A T T A C T C A C T A G T T A T T T T G C G A T A A T A T T 151. described ( S e c t i o n IV.B, Results and D i s c u s s i o n ) , d u p l i c a t i o n s of tRNA gene sequences i n the 5 ' - f l a n k i n g sequences of the genes are found f o r one of the Val tRNA^ genes of pDt55 and have been noted i n the tRNA genes of a number of organisms by Santos and Z a s l o f f (1981). These sequences may i n f l u e n c e the s i t e at which t r a n s c r i p t i o n of these tRNAs genes i s i n i t i -a t e d. An i n v e r t e d complement of the CACAAG hexanucleotide occurs 24 bp V a l upstream from , the tRNA^ gene of pDt78R (nu c l e o t i d e s 35-40, Figure 35). H y b r i d i z a t i o n of t h i s complementary sequence to e i t h e r of the two CACAAG sequences would produce h a i r - p i n s t r u c t u r e s i n the DNA. With loop-outs, two longer i n v e r t e d complementary r e g i o n s , one c o n t a i n i n g the i n t r a g e n i c CACAAG sequence and the other i t s i n v e r t e d complement, can be recognized. The re g i o n w i t h i n the gene extends from n u c l e o t i d e 86 to nuc l e o t i d e 103 of Figure 35. An imperfect i n v e r t e d complementary sequence to t h i s r e g i o n i s found between n u c l e o t i d e s 35 and 50 of Figure 35. The s i g n i f i c a n c e , i f any, of these regions i s unknown. The CACAAG sequence i s a l s o found i n the 5 ' - f l a n k i n g sequence of another Drosophila tRNA gene. The sequence occurs 34 bp upstream from the V a l 5'-end ( i n the non-transcribed strand) of the second tRNA^ gene of plasmid pDt55 ( n u c l e o t i d e 933-938, Figure 30). The a s s o c i a t i o n of the V a l CACAAG sequence w i t h two d i f f e r e n t tRNA genes from widely separated chromosomal l o c a t i o n s suggests that the sequence plays some r o l e i n tRNA gene expression. However, the sequence i s not found i n the 5 ' - f l a n k i n g sequences of the other Drosophila tRNA genes discussed i n t h i s t h e s i s . V a l V a l B. Other tRNA^ Genes: Comparisons w i t h the tRNA^ Gene of pDt78R V a l In a d d i t i o n to the t R N A 3 t 8 e n e o f pDt78R three other Va 1 tRNA^ genes have been sequenced. One of these genes i s loc a t e d at V a l the 92B s i t e on the polytene chromosome, a major s i t e of tRNA^^ 152 . h y b r i d i z a t i o n . This gene was sequenced by Dr. M. S i l b e r k l a n g (personal communication). The other two genes were cloned and sequenced i n t h i s labo-r a t o r y . One gene was cloned i n the recombinant plasmid pDt48, the other i n plasmid pDt41R (Table I I I ) . Both of these genes o r i g i n a t e from the minor V a l V a l s i t e of t R N A 3 b h y b r i d i z a t i o n , 90BC (Table I ) . Thus t R N A 3 b genes from each of t h i s tRNA's chromosomal l o c i have been sequenced. V a l The n u c l e o t i d e sequence of a tRNA^^ gene from the 92B s i t e was determined by Dr. M. S i l b e r k l a n g and i s shown i n Figure 35. The t r a n s c r i p t V a l of t h i s gene would have the same sequence as tRNA^^ . Thus the V a l sequence of t h i s gene i s i d e n t i c a l to that of the tRNA^^ gene of pDt78R. The f l a n k i n g sequences of the genes from the two plasmids, however, show no s i g n i f i c a n t homology other than the 5 adjacent T residues that are probably t e r m i n a t i o n s i g n a l s f o r RNA polymerase I I I . Recombinant plasmids pDt48 and pDt41R c o n t a i n segments of Drosophila DNA (2.4 kb and 2.0 kb long r e s p e c t i v e l y ) that can h y b r i d i z e w i t h V a l V a l t R N A ^ • The tRNA^ gene of pDt48 was sequenced by Dr. A. Delaney, t h a t of pDt4lR was sequenced by J . Leung. Their r e s u l t s are shown i n Figure 36. The sequenced p o r t i o n of pDt48 contains two tRNA genes, one V a l Pro f o r a tRNA s i m i l a r to t R N A ^ » the other f o r a tRNA^ U G G^. A V a l t r a n s c r i p t of the tRNA^ - l i k e gene of pDt48 would d i f f e r from tRNAYf 1 at 4 s i t e s . Nucleotides C5, C16, G68 and G69 of t R N A ^ 1 3b 3b would be replaced by U5, U16, A68 and A69 i n a t r a n s c r i p t of the t R N A ^ a l - l i k e gene ( F i g u r e 20). Except f o r the C16 to Ul6 t r a n s i t i o n V a l these d i f f e r e n c e s are not the same as those between tRNA^ and hypo-V a l t h e t i c a l t r a n s c r i p t s of the tRNA^ - l i k e genes of plasmids pDtl20R, V a l pDt92R. or pDtl4. I t i s perhaps s i g n i f i c a n t t h a t , u n l i k e tRNA^^ , a V a l t r a n s c r i p t of the tRNA^ - l i k e gene would have a p e r f e c t l y base-paired aminoacyl stem. With the p o s s i b l e exception of C16, the sequence of 153. Figure 36. The n u c l e o t i d e sequences of segments of the Drosophila DNA i n s e r t s of plasmids pDt48 and pDt41R. The non-transcribed strand of the tRNA^ 3, 1-like genes i s shown. The second gene, coding f o r t R N A P r o , i s of opposite p o l a r i t y to the f i r s t . Homologies between the sequences from pDt48 and pDt41R are marked by a s t e r i s k s . Dashes were i n s e r t e d i n t o the sequences wherever necessary to ensure maximum homology. A 14 bp sequence from p o s i t i o n 325 to 338 of pDt48 i s absent i n pDt41R. A l a r g e segment of pDt4lR, between p o s i t i o n s 409 and 483, has not yet been sequenced. tRNA genes are o v e r l i n e d . D i f f e r e n c e s between the tRNA^-'—like genes of pDt48 and the t R N A ^ 1 gene of pDt78R are u n d e r l i n e d . T h e N u c l e o t i d e S e q u e n c e s o f p D t 4 8 a n d p D t 4 1 R - H o m o l o g i e s b e t w e e n t h e t w o s e q u e n c e s a r e m a r k e d b y a s t e r i s k s . 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 100 4 8 A A A A T A A A T C T A A G T A T G C A A C T T T G G C A A G A T C A G A A G A A T A A G T T A A A C G G C C A T T G A A A A T G T G T T T C T C C A A T T G T T C T A A A A A A A A T G T A A T A A A 1 1 0 1 2 0 1 3 0 140 150 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 4 8 A T T T T A A A A T A A G C A A A T A G T T C C A C A G G A A A C T A G A G T C A T G C A G G T T A G T C C T T T T T G T G T T G T G T G A A C A C A A T A C G C T A T A C T G T T A G T T T A A A C T *************************** ***** ******** 4 1R . T G T G T T G T G T G A A C A C A A T A C G C T A T A G T G T T A T T T T A A A C T 2 1 0 . 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 . 2 8 0 2 9 0 3 0 0 4 8 A A A T G T T T C A T G A T G T T T T C G T A G T G T A G T G G T T A T C A C G T G T G C T T C A C A C G C A C A A G G T C C C C G G T T C G A A C C C G G G C G A A A A C A G A T T G A T T T T T T T ***** ******* ************************************************************************* ** ** ****** 41R A A A T G A T T C A T G A C G T T T T C G T A G T G T A G T G G T T A T C A C G T G T G C T T C A C A C G C A C A A G G T C C C C G G T T C G A A C C C G G G C G A A A A C A A A T C G A C T T T T T T 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 4 8 T T A A T T T C T T T T T A C A T T T T C G A T G A A T C T T A G G G T T G A A A A C G G T A A C A C A A A T A A A A T A T T T T A A T A C C C T T A A G G A A T A A T T G A A A A A A G A C - - C G A * **************,**** * ************************** **** ********* * ********* * *** 41R T A A A T T T C T T T T T A C A T T T T T T A C D e l e t t o n A A A A C G G T A A C A C A A A T A A A A T A T T T G A A T A T C C T T A A G G A T A A T T T G A A A A A A T A A A A C G A 4 10 4 2 0 4 3 0 4 4 0 4 5 0 4 6 0 4 7 0 4 8 0 4 9 0 5 0 0 4 8 T G C T A T A G A A A C G T A C C A A T A T T T G A A T A A G C C A A T G G G G T T G A A A T C C A T A C A T A T T G T T T A C G G G T C A A A C C A T T T A C T T T C T A T A G T T T A A A T A T T T ********* ***************** 4 1R T G C T A T A G A N o t Y e t S e q u e n c e d - - - C T A T A G T T T A A A T A T T T 5 1 0 5 2 0 5 3 0 5 4 0 5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 6 0 0 4 8 T C T T T A A T T T T C A G A A A A A T T A G C A A A G A A A A A A T T T G T A C G T G C G G T T G A - - G T T G A G C A A T A A A A A C A G T A C A G C T G G G C T C A A C C G G G A T T T G A A C C ************** ****** **************** ****** ***** ***** *** **** * **************************** 41R T C T T T A A T T T T C A G G A A A A T T T G C A A A G A A A A A A T T T G A A C G T G C A G T T G A C C G T T G A A C A A C A A A A - T A A C A C A G C T G G G C T C A A C C G G G A T T T G A A C C 6 1 0 6 2 0 6 3 0 6 4 0 6 5 0 6 6 0 6 7 0 6 8 0 6 9 0 7 0 0 4 8 C G G G A C C T C T C G C A C C C A A A G C G A G A A T C A T A C C C C T A G A C C A T T G A G C C T C A T A A C G A G T A T G C A G C T G T T C G C C A G T T T C C A A C T A G G C A G A G C A A G G 41R C G G G A C C T C T C G C A C C C A A A G C G A G 7 1 0 7 2 0 7 3 0 7 4 0 7 5 0 7 6 0 7 7 0 7 8 0 7 9 0 8 0 0 4 8 C A T T T T C T T G A G T G A G C C T C T A A A G A C A A A C A A A A A T C A T T T C T T C G A T G T A A C A A T C T A A A A T A T A T A T T T A G T A A A C A T A G A A C A A T A T C T C T C A G C G 4 8 T C G C T A A G T G 155. V a l tRNA^k shows no heterogeneity at the s i t e s at which i t d i f f e r s from V a l the tRNA^k - l i k e genes (Figure 21, 22). I t i s concluded, t h e r e f o r e , Va1 V a l that tRNA„, does not c o n t a i n t r a n s c r i p t s of the tRNA., - l i k e genes. P r e l i m i n a r y sequencing data i n d i c a t e these genes do not code f o r Va1 Va1 tRNA^ a • The t r a n s c r i p t of the tRNA^ - l i k e genes would c o n t a i n a l l the n u c l e o t i d e s c h a r a c t e r i s t i c of eukaryotic v a l i n e tRNAs (Results and D i s c u s s i o n , S e c t i o n I I . E ) and these genes may w e l l be expressed as one of Va l the minor tRNA species (Figure 7 ) . There i s no s i g n i f i c a n t homology Va l between the DNA sequences f l a n k i n g the tRNA genes of pDt78R and pDt48. The p r o l i n e tRNA gene of pDt48 i s l o c a t e d 287 bp downstream from the Va l t RNA^ - l i k e gene and i s of opposite p o l a r i t y to the l a t t e r . Neither Pro of the two major tRNA species of Drosophila (White e_t a l . , 1973a) has been sequenced so a comparison between the gene sequence and the p r o l i n e Pro tRNA sequence cannnot yet be made. However, the t R N A ^ g ^ gene (non-t r a n s c r i b e d strand) has a sequence very s i m i l a r to that of the corresponding tRNA of mouse and chicken (95% homology)(Gauss and S p r i n z l , 1981). The sequenced p o r t i o n of pDt41R i s very s i m i l a r , but not i d e n t i c a l , to V a l corresponding sequences i n pDt48 (Figure 36). The tRNA^^ - l i k e genes Pro Pro and most of the tRNA genes (the sequencing of the tRNA gene of pDt41R i s not yet completed) of the two plasmids are i d e n t i c a l . Outside the genes, the two plasmids are very s i m i l a r (about 86% homology) but are more V a l divergent than the homologous regions around the tRNA^ - l i k e genes of plasmids pDt92R and pDtl20R. Most of the d i f f e r e n c e s between the sequenced po r t i o n s of pDt48 and pDt41R are due t o s c a t t e r e d base changes or the i n s e r -t i o n or d e l e t i o n of one or two base-pair segments of DNA. There i s , however, a 14 bp sequence present between n u c l e o t i d e s 325 and 339 of pDt48 (Figure 36) that i s not found i n pDt41R. I t seems l i k e l y that both sequences are present i n the Drosophila genome. The Drosophila DNA of pDt41R i s a Hind 156. I l l fragment 2.0 kb long . A fragment of t h i s s i z e was detected by h y b r i -V a l d i z i n g 1 2 5 I - l a b e l l e d t R N A 3 b to a Southern b l o t of Drosophila DNA fragments generated by cleavage of genomic DNA w i t h Hind I I I (Tener et a l . , 1980). pDt48 contains a 2.4 kb Hind I I I fragment of Drosophila DNA. No V a l fragment t h i s s i z e c a r r y i n g t R N A ^ genes was detected i n Hind I I I -cleaved Drosophila DNA (Tener e_t _ a l . , 1980). The 2.0 kb fragment of DNA cloned i n pDt41R i s , t h e r e f o r e , l i k e l y an " a l l e l e " of the fragment cloned i n pDt48. This " a l l e l e " may not have been detected i n Hind I l l - c l e a v e d Drosophila DNA because i t i s present i n low frequency i n the population of f l i e s from which the DNA was i s o l a t e d . I f the 2.0 and 2.4 kb Hind I I I fragments were derived from d i f f e r e n t repeat u n i t s of a DNA segment d u p l i -cated i n the Drosophila genome, they should always be present i n a 1:1 r a t i o i n the Drosophila DNA. In t h i s case both the 2.4 and 2.0 kb fragments should have been detected i n the Hind I I I cleaved genomic DNA. V a l In summary, the tRNA^ genes present at the major s i t e s of V a l t RNA^ h y b r i d i z a t i o n to Drosophila polytene chromosomes (pDt78R from 84D, S i l b e r k l a n g ' s gene sequence from 92B) correspond to the sequence of the V a l t R NA^ • The genes present at the minor h y b r i d i z a t i o n s i t e (pDt48 and V a l pDt41R from 90BC) are s i m i l a r , but not i d e n t i c a l , to the tRNA^^ se-V a l quence. These r e s u l t s p a r a l l e l those obtained f o r the cloned tRNA^ Val V a l genes. Perhaps the genes f o r tRNA^ and tRNA^^ present at the major h y b r i d i z a t i o n s i t e s are i s o l a t e d from the c l o s e l y r e l a t e d V a l V a l tRNA^ - l i k e and tRNA^ - l i k e genes present at the minor s i t e s . Val Val The weaker h y b r i d i z a t i o n of tRNA^ and t R N A ^ to the minor s i t e s f o r these tRNAs would then be due to the decreased homology between the genes at these s i t e s and the tRNA probes used to l o c a t e them. I f t h i s Va1 Va1 hypothesis i s true the genes f o r tRNA^ and t R N A ^ would not be so widely dispersed on the chromosome as p r e v i o u s l y thought. 157. B i b l i o g r a p h y Altwegg, M. (1980) Inaugural D i s s e r t a t i o n , Philosophischen F a k u l t a t I I Der U n i v e r s i t a t Z u r i c h . Anderson, S., Bankier, A.T., B a r r e l l , B.G., de B r u i j n , M.H.L., Coulson, A.R., Drouin, J . , Eperon, I.C., N i e r l i c h , D.P., Roe, B.A., Sanger, F., S c h r e i e r , P.H., Smith, A.J.H., Staden, R. and Young, I.G. (1981) Nature 290, 457-465. A r c a r i , P. and Brownlee, G.G. (1980) N u c l . Acids Res. 8^ , 5207-5212. B a r r i o , J.R., S e c r i s t I I I , J.A. and Leonard N.J. (1972) Biochem. Biophys. Res. Commun. 46_, 597-604. Beckmann, J.S., Johnson, P.F. and Abelson, J . (1977) Science 196, 205-208. Beermann, W. (1972) i n Developmental Studies on Giant Chromosomes (Beermann, W., ed.), R e s u l t s and Problems i n C e l l D i f f e r e n t i a t i o n V o l . 4, pp 1-31, Spr i n g e r - V e r l a g , New York. Benne, R. and Hershey, J.W.B. (1978) J . B i o l . Chem. 253, 3078-3087. B i e r n a t , J . , C i e s i o l k a , J . , G o r n i c k i , P., Adamiak, R.W., Krz y z o s i a k , W.J. and Wiewiorowski, M. (1978) N u c l . Acids Res. _5_, 789-804. Bogenhagen, D.F. , Sakonju, S. and Brown, D.D. (1980) C e l l 1_9, 27-35. Boguski, M.S., H i e t e r , P.A. and Levy, C.C. (1980) J . B i o l . Chem. 255, 2160-2163. B o n i t z , S.G. and T z a g o l o f f , A. (1980) J . B i o l . Chem. 255, 9075-9081. B o r s t , P. and G r i v e l l , L.A. (1981) Nature 290, 443-444. B o s s i , L. and Roth, J . J . (1980) Nature 286, 123-127. Brenner, D.J., F o u r n i e r , M.J. and Doctor, B.P. (1970) Nature 227, 448-451. B r i t t e n , R.J., Graham, D.E., and Neufeld, B.R. (1974) Methods i n Enzymology 29_, 363-418. Brown, N.L. (1979) i n Companion to Biochemistry ( B u l l , A.T., Legnado, J.R., Thomas, J.0. and Ti p t o n , K.F., eds.) V o l . 2, pp 1-48, Longman, London and New York. Cedergren, R.J., Sankoff, D., LaRue, B. and Grosjean, H. (1981) C r i t . Rev. Biochem. 11_, 35-104. Champe, S.P. and Benzer, S. (1962) Proc. N a t l . Acad. S c i . USA ^ 8, 532-546. Chen, E.Y. and Roe, B.A. (1977) Biochem. Biophys. Res. Commun. 7_8, 631-640. Chen, E.Y. and Roe, B.A. (1978) Biochem. Biophys. Res. Commun. 82, 235-245. 158. C l a r k , B.F.C. (1978) i n Transfer RNA (Altman, S., ed.) pp 14-47, MIT Press Cambridge, Mass. and London. Cla r k s o n , S.G., B i r n s t e i l , M.L. and S e r r a , V. (1973a) J . Mol. B i o l . 79, 391-410. Clarkson, S.G., B i r n s t e i l , M.L. and Prudom, I.F. (1973b) J . Mol. B i o l . 79, 411-429. Clarkson, S.G. and Kurer, V. (1976) C e l l 8, 183-195. Clarkson, S.G., Kurer, V. and Smith, H.O. (1978) C e l l 14, 713-724. Cramer, F., von der Haar, F. and I g l o i , G.L. (1980) i n Transfer RNA:  S t r u c t u r e , P r o p e r t i e s and Recognition (Schimmel, P.R., S o i l , D., and Abelson, J.N., eds.) pp 267-279, Cold Spring Harbor Laboratory. C r i b b s , D.L. (1979) M.Sc. Th e s i s , U n i v e r s i t y of B r i t i s h Columbia. C r i c k , F.H.C. (1966) J . Mol. B i o l . 19, 548-555. Dahlberg, J.E. (1980) i n Transfer RNA: B i o l o g i c a l Aspects ( S o i l , D., Abelson, J.N. and Schimmel, P.R., eds.) pp 507-516, Cold Spring Harbor Laboratory. Danna, K.J. (1980) Methods i n Enzymology 65, 449-467. de B r u i j n , M.H.L., S c h r e i e r , P.H., Eperon, I.C., B a r r e l l , B.G., Chen, E.Y., Armstrong, P.W., Wong, J.F.H. and Roe, B.A. (1980) N u c l . A c i d s Res. 8, 5213-5222. DeFranco, D., Schmidt, 0., and S o i l , D. (1980) Proc. N a t l . Acad. S c i . U.S.A. 7_7_, 3365-3368. Dirheimer, G., K e i t h , G., S i l b e r , A.-P. and M a r t i n , R.P. (1979) Transfer RNA:  S t r u c t u r e , P r o p e r t i e s and Recognition (Schimmel, P., S o i l , D. and Abelson, J.N. , eds.) pp 19-41, Cold Spring Harbor Laboratory. D o n i s - K e l l e r , H., Maxam, A.M. and G i l b e r t , W. (1977) Nuc l . Acids Res. 4_, 2527-2538. Dudler, R., Egg, A.H., K u b l i , E., Artavanis-Tsakonas, S., Gehring, W.J., Steward, R. and Schedl, P. (1980) Nuc l . Acids Res. 13_, 2921-2937. Dudler, R.K. (1981) Inaugural D i s s e r t a t i o n , Zoologisches I n s t i t u t der U n i v e r s i t a t Z u r i c h . Duester, G.L. and Holmes, W.M. (1980) Nuc l . Acids Res. 8, 3793-3807. Duester, G., Campen, R.K. and Holmes, W.M. (1981) Nuc l . Acids Res. 2121-2139. Dunn, R., Addison, W.R., G i l l a m , I.C. and Tener, G.M. (1978) Can. J . Biochem. 56, 618-623. 159. Dunn, R., Delaney, A.D., G l l l a m , I.C., Hayashl, S., Tener, G.M., G r l g l l a t t l , T., M i s r a , V., Spurr, M.G., T a y l o r , D.M. and M i l l e r , R.C. J r . (1979) Gene 7, 197-215. E l d e r , R.T., Uhlenbeck, O.C. and Szabo, P. (1980) i n Transfer RNA: B i o l o g i c a l  Aspects (S b ' l l , D., Abeleson, J.N. and Schimmel, P.R., eds.) pp 317-323, Cold Spring Harbor Laboratory. Endow, S.A. and Roberts R.J. (1977) J . Mol. B i o l . 112, 521-529. England, T.E. and Uhlenbeck, O.C. (1978) Nature 275, 560-561. Feldmann, H. (1976) Nucl. Acids Res. 3_, 2379-2386. Feldmann, H. (1977) Nuc l . Acids Res. _4, 2831-2841. F e r s h t , A.R. (1980) i n Transfer RNA: S t r u c t u r e , P r o p e r t i e s and Recognition (Schimmel, P.R., S o i l , D. and Abelson, J.N., eds.) pp 247-254. F o u r n i e r , M., Labouesse, J . , Dirheimer, G., F i x , C. and K e i t h , G. (1978) Biochim. Biophys. Acta 521, 198-208. Fowlkes, D.M. and Shenk, T. (1980) C e l l 22_, 405-413. Friedman, S. (1973) Nature New B i o l . 244, 18-20. G a l l , J.G. and Pardue, M.L. (1969) Proc. N a t l . Acad. S c i . USA 63, 378-383. G a l l a n t , J.A. (1979) Ann. Rev. Genet. L3, 393-415. Garber, R.L. and Altman, S. (1979) C e l l 17_, 389-397. Gauss, D.H. and S p r i n z l , M. (1981) Nuc l . Acids Res. 9_, r l - r 2 3 . Ghosh, R.K. and Deutscher, M.P. (1980) i n Transfer RNA: B i o l o g i c a l Aspects (SOU, D., Abelson, J.N. and Schimmel, P.R., eds.) pp 59-69, Cold Spring Harbor Laboratory. Goldman, E., Holmes, W.M. and H a t f i e l d , G.M. (1979) J . Mol. B i o l . 129, 567-585. Goodman, H.M., Olson, M. and H a l l , B.D. (1977) Proc. N a t l . Acad. S c i USA 74, 5453-5457. Grantham, R., G a u t i e r , C , Gouy, M. Jacobzone, M. and M e r c i e r , R. (1981) Nucl. Acids Res. 9_, r43-r74. Gupta, R.C. and Randerath, K. (1977) Nuc l . Acids Res. 4_, 3441-3454. Gupta, R.C. and Randerath, K. (1979) Nucl. Acids Res. 6, 3443-3458. Haenni, A.-L. and C h a p e v i l l e , F. (1980) i n Transfer RNA: B i o l o g i c a l Aspects (SS11, D., Abelson, J.N. and Schimmel, P.R., eds.) pp 539-556, Cold Spring Harbor Laboratory. 160. Hayashi, S., G i l l a m , I.C., Delaney, A.D., Dunn, R.D., Tener, G.M., G r i g l i a t t i , T.A. and Suzuki, D.T. (1980) Chromosoma _76, 65-84. Hayashi, S., G i l l a m , I.C. and Tener, G.M. (1981a), Manuscript i n p r e p a r a t i o n . Hayashi, S., Addison, W.R., G i l l a m , I . C , G r i g l i a t t i , T.A. and Tener, G.M. (1981b) Chromosoma 82, 385-397. Hershey, N.D. and Davidson, N. (1980) Nucl. Acids Res. 8, 4899-4910. Hoagland, M.B., Stephenson, M.L., S c o t t , J.F., Hecht, L. and Zamecnik, P.C (1958) J . B i o l . Chem. 231, 241-257. H o f s t e t t e r , H., Kressmann, A. and B i r n s t i e l , M.L. (1981) C e l l 24, 573-585. Holbrook, S.R., Sussman, J.L., Warrant, R.W. and Kim, S.-H. (1978) J . Mol. B i o l . 123, 631-660. H o l l e y , R.W., Apgar, J . E v e r e t t , G.A., Madison, J.T., Marquisee, M., M e r r i l l , S.H., Penswick, J.R. and Zamir, A. (1965) Science 147, 1462-1465. Holmes, W.M., H a t f i e l d , G.W. and Goldman, E. (1978) J . B i o l . Chem. 253, 3482-3486. Hosbach, H.A. and K u b l i , E. (1979a) Mech. Ageing Dev. 1£, 131-140. Hosbach, H.A. and K u b l i , E. (1979b) Mech. Ageing Dev. 10, 141-149. Hosbach, H.A., S i l b e r k l a n g , M. and McCarthy, B.J. (1980) C e l l 21, 169-178. Hovemann, B., Sharp, S., Yamada, H. and S o i l , D. (1980) C e l l 19, 889-895. Huang, S.L. and Baker, B.S. (1976) Mut. Res. 34.. 407-414. I g l o i , G.L. and Cramer, F. (1978) i n Transfer RNA (Altman, S., ed.) pp 2 94-349 MIT Pr e s s , Cambridge, Mass. and London. Ikemura, T. and Ozeki, H. (1977) J . Mol. B i o l . 117, 419-446. Jank, P., Shindo-Okada, N., Nishimura, S. and Gross, H.J. (1977a) Nuc l . Acids Res. 4, 1999-2008. Jank, P., Riesn e r , D. and Gross, H.J. (1977b) Nuc l . Acids Res. _4_, 2009-2020. Jay, E., Bambara, R., Padmanabhan, R. and Wu, R. (1974) Nuc l . Acids Res. 1^ , 331-353. Judson, H.F. (1979) The E i g h t h Day of C r e a t i o n , pp 285-293, Simon and Schuster, New York. K e i t h , G. and Dirheimer, G. (1980) Biochem. Biophys. Res. Commun. 92, 109-115. Khym, J.X. and U z i e l , M. (1968) Biochemistry 7, 422-426. 161. Kim, S.-H. (1978) i n Transfer RNA (Altman, S., ed.) pp 248-293 MIT Pr e s s , Cambridge, Mass. and London. Kimura, K., Nak a n i s h i , M., Yamamoto, T. and Tsuboi, M. (1977) J . Biochem. 81, 1699-1703. Knapp, G., Ogden, R.C., Peebles, C.L. and Abelson, J . (1979) C e l l 18^ , 37-45. Koch, W., Edwards, K. and K o s s e l , H. (1981) C e l l 2_5, 203-213. Kochel, H.G., Lazarus, CM., Basak, N. and K u n t z e l , H. (1981) C e l l 23, 625-633. Kochetkov, N.K., Shibaev, V.N. and Kost, A.A. (1971) Tetrahedron L e t t . 22, 1993-1996. K o s k i , R.A., Clarkson, S.G., Kurjan, J . , H a l l , B.D. and Smith, M. (1980) C e l l 22_, 415-425. Kressmann, A., H o f s t e t t e r , H., DiCapua, E., Grosschedl, R. and B i r n s t i e l , M.L. (1979) N u c l . Acids Res. 7_, 1749-1763. Krupp, G. and Gross, H.J. (1979) Nuc l . Acids Res. 3481-3490. Kr z y z o s i a k , W.J., B i e r n a t , J . , C i e s i o l k a , J . , G o r n i c k i , P. and Wiewiorowski, M. (1979) P o l i s h J . Chem. _5_3, 243-252. Krz y z o s i a k , W.J., B i e r n a t , J . , C i e s i o l k a , J . , Gulewicz, K. and Wiewiorowski, M. (1981) Nuc l . Acids Res. 9_, 2841-2851. Kurjan, J . , H a l l , B.D. , G i l l a m , S. and Smith, M. (1980) C e l l 20_, 701-709. Laskey, R.A. and M i l l s , A.D. (1977) FEBS L e t t . J32, 314-316. Lee, F. and Yanofsky, C. (1977) Proc. N a t l . Acad. S c i . USA 74_, 4365-4369. L e f e v r e , G. J r . (1976) i n The Genetics and Biology of Drosophila (Ashburner, M. and N o v i t s k i , E., eds. ) V o l . l a , pp 31-66, Academic Press, London, New York, San F r a n c i s c o . L i , M. and T z a g o l o f f , A. (1979) C e l l 18^ , 47-53. L i n , F.-K., F u r r , T.D., Chang, S.H., Horwitz, J . , A g r i s , P.F. and Ortwerth, B.J. (1980) J . B i o l . Chem. 255, 6020-6023. Lund, E., Dahlberg, J.E., L i n d a h l , L., Jaskunas, S.R., Dennis, P.P. and Nomura, M. (1976) C e l l _7, 165-177. Lusk, J.E., W i l l i a m s , R.J.P. and Kennedy, E.P. (1968) J . B i o l . Chem. 243, 2618-2624. L u s t i g , F., E l i a s , P., Axberg, T., Samuelsson, T., T i t t a w e l l a , I . and L a g e r k v i s t , U. (1981) J . B i o l . Chem. 256, 2635-2643. Mao, J . , Schmidt, 0. and S o i l , D. (1980) C e l l 21, 509-516. 162. Maxam, A.M. and G i l b e r t , W. (1980) Methods i n Enzymology 65_, 499-560. Mazzara, G.P. and McClain, W.H. (1980) i n Transfer RNA: B i o l o g i c a l Aspects ( S o i l , D., Abelson, J.N. and Schimmel, P.R., eds.) pp 3-27, Cold Spring Harbor Laboratory. McDonell, M.W., Simon, M.N. and S t u d i e r , F.W. (1977) J . Mol. B i o l . 110, 119-146. Melton, D.A., De R o b e r t i s , E.M. and Cortese, R. (1980) Nature 284, 143-148. Mirzabekov, A.D., Grunberger, D., K r u t i l i n a , A.I., Holy, A., Bayev, A.A. and Sorm, F. (1968) Biochim. Biophys. Acta 166, 75-81. M i t r a , S.K., L u s t i g , F., Akesson, B., L a g e r k v i s t , U. and S t r i d , L. (1977) J . B i o l . Chem. 252, 471-478. M i t r a , S.K., L u s t i g , F., Akesson, B., Axberg, T., E l i a s , P. and L a g e r k v i s t , U. (1979) J . B i o l . Chem. 254, 6397-6401. Montoya, J . , O j a l a , D. and A t t a r d i , G. (1981) Nature 290, 465-470. Moras, D., Comarmond, M.B., F i s c h e r , J . , Weiss, R., T h i e r r y , J.C., E b e l , J.P. and Giege, R. (1980) Nature 288, 669-674. Morgan, E.A., Ikemura, T., Post, L.E. and Nomura, M. (1980) i n Transfer RNA:  B i o l o g i c a l Aspects ( S ' d l l , D., Abelson, J . , and Schimmel, P.R., eds.) pp 259-266, Cold Spring, Harbor Laboratory. M o r r i s , R.W. and Herbert, E. (1970) Biochem. j ) , 4819-4827. M u l l e r , F. and Clarkson, S.G. (1980) C e l l 19, 345-353. Munz, P., Leupold, U., A g r i s , P. and K o h l i , J . (1981) Nature 294, 187-188. Murgola, E.J. and Pagel, F.T. (1980) J . Mol. B i o l . 138, 833-844. Nakajima, N., Ozeki, H. and Shimura, Y. (1981) C e l l 23_, 239-249. Nirenberg, M. and Leder, P. (1964) Science 145, 1399-1407. Nishimura, S. (1978) i n Transfer RNA (Altman, S., ed.) pp 168-195, MIT Press, Cambridge, Mass. and London. Nishimura, S. (1979) i n Transfer RNA: S t r u c t u r e , P r o p e r t i e s and Recognition (Schimmel, P.R., S o i l , D. and Abelson, J.N., eds.) pp 59-79, Cold Spring Harbor Laboratory. Ogden, R.C., Beckmann, J.S., Abelson, J . , Kang, H.S., S o i l , D. and Schmidt, 0. (1979) C e l l 17, 399-406. Ohashi, Z., Maeda, M., McCloskey, J.A. and Nishimura, S. (1974) Biochem. 13, 2620-2625. O j a l a , D., Montoya, J . and A t t a r d i , G. (1981) Nature 290, 470-474. Olson, M.V., H a l l , B.D., Cameron, J.R. and Davis R.W. (1979) J . Mol. B i o l . 127, 285-295. Olson, M.V., Page, G.S., Sentenac, A., P i p e r , P.W., Worthington, M., Weiss, R.B., and H a l l , B.D. (1981) Nature 291, 464-469. Paule, M.R. (1981) Trends Biochem. S c i . 6_, 128-131. P e a t t i e , D. (1979) Proc. N a t l . Acad. S c i . USA 76, 1760-1764. Peebles, C.L., Ogden, R.C., Knapp, G. and Abelson, J . (1979) C e l l 18^ , 27-35. P i l l y , D., Niemeyer, A., Schmidt, M. and B a r g e t z i , J.P. (1978) J . B i o l . Chem. 253, 437-445. P i p e r , P.W. (1975) Eur. J . Biochem. 51. 295-304. P i p e r , P.W. and C l a r k , B.F.C. (1974) FEBS L e t t . 47, 56-59. P i a t t , T. (1981) C e l l 24^ , 10-23 P o l i s k y , B., Greene, P. G a r f i n , D.E., McCarthy, B.J., Goodman, H.M. and Boyer, H.W. (1975) Proc. N a t l . Acad. S c i . USA 7_2, 3310-3314. Prensky, W., Steffensen, D.M. and Hughes, W.L. (1973) Proc. Nat. Acad. S c i . USA 10, 1860-1864. Pribnow, D. (1979) i n B i o l o g i c a l Regulation and Development (Goldberger, R.F., ed.) V o l . I , pp 219-277, Plenum Press, New York and London. R a z z e l l , W.E. (1963) Methods i n Enzymology 6_, 237-238. Revel, M. (1977) i n Molecular Mechanisms of P r o t e i n B i o s y n t h e s i s (Weissbach, H. and Pestka, S., eds.) pp 245-321, Academic Press, New York, San Francisco and London. R i c h , A. and RajBhandary, U.L. (1976) Annu. Rev. Biochem. 4_5, 805-860. R i c h , A., Quigley, G.J., Teeter, M.M., Ducruix, A. and Woo, N. (1980) i n T r a n s f e r RNA: S t r u c t u r e , P r o p e r t i e s and Recognition (Schimmel, P.R., S o i l , D. and Abelson, J.N., eds.) pp 101-113, Cold Spring Harbor Laboratory. Richards, F.M. and Wyckoff, H.W. (1971) i n The Enzymes 3rd ed. (Boyer, P.D., ed.) V o l . IV, pp 647-806, Academic Press, New York and London. R i t o s s a , F.M., Atwood, K.C., and Spiegelman, S. (1966) Genetics 54, 663-676. Roberts, R.J. (1974) J . B i o l . Chem. 249, 4787-4796. Robinson, R.R. and Davidson, N. (1981) C e l l 12, 251-259. Roe, B.A. (1975) Nuc l . Acids Res. 2, 21-42. Rosenberg, M. and Court, D. (1979) Ann. Rev. Genet. 13, 319-353. 164. R o s s i , J . , Egan, J . , Beraan, M.L. and Landy, A. (1980) i n Transfer RNA: B i o l o g i c a l Aspects ( S o i l , D., Abelson, J.N. and Schimmel, P.R., eds.) pp 221-244, Cold Spring Harbor Laboratory. Rudkin, G.T. (1972) i n Developmental Studies on Giant Chromosomes (Beermann, W., ed.) Re s u l t s and Problems i n C e l l D i f f e r e n t i a t i o n V o l . 4, pp 58-80, S p r i n g e r - V e r l a g , New York. R u d l o f f , E. and H i l s e , K. (1975) Hoppe-Seyler's Z. P h y s i o l . Chem. 356, 1359-1367. Sakonju, S., Brown, D.D., Engelke, D., Ng, S.-Y., Shastry, B.S. and Roeder, R.G. (1981) C e l l 23_, 665-669. Santos, T. and Z a s l o f f , M. (1981) C e l l 23_, 699-709. Schedl, P., Primakoff, P. and Roberts, J . (1974) Brookhaven Symp. B i o l . 26, 53-76. Scherberg, N.H. and R e f e t o f f , S. (1974) Biochim. Biophys. Acta 340, 446-451. S c h e v i t z , R.W., Podjarny, A.D., Krishnamachari, N., Hughes, J . J . and S i g l e r , P.B. (1980) i n Transfer RNA: S t r u c t u r e , P r o p e r t i e s , and Recognition (Schimmel, P.R., S o i l , D. and Abelson, J . , eds.) pp 133-143. Schmidt, 0., Mao, J . , Ogden, R., Beckmann, J . , Sakano, H., Abelson, J . and S o i l , D. (1980) Nature 287, 750-752. Schwarz, Z., K o s s e l , H., Schwarz, E. and Bogorad, L. (1981) Proc. N a t l . Acad. S c i . USA 78_, 4748-4752. Sekiya, T., M o r i , M., Takahashi, N. and Nishimura, S. (1980) Nuc l . Acids Res. 8^ , 3809-3827. S e l k e r , E. and Yanofsky, C. (1980) Nuc l . Acids Res. 8, 1033-1042. Shindo-Okada, N., Kuchino, Y., Harada, F., Okada, N., and Nishimura, S. (1981) J . Biochem. jK), 535-544. S i l b e r k l a n g , M., G i l l u m , A.M. and RajBhandary, U.L. (1979) Methods i n Enzymology 59, 58-109. Silverman, S., Schmidt, 0., S o i l , D. and Hovemann, B. (1979) J . B i o l . Chem. 254, 10290-10294. Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and G u i l l e y , H. (1977) Nature 269, 833-836. Smith, J.D. (1976) Prog. N u c l e i c A c i d Res. Mol. B i o l . 3.6, 25-73. S o f f e r , R.L. (1974) Adv. Enzymol. 40_, 91-139. S o f f e r , R.L. (1980) i n Transfer RNA: B i o l o g i c a l Aspects ( S o i l , D., Abelson, J.N. and Schimmel, P.R., eds.) pp 493-505, Cold Spring Harbor Laboratory. 165. Southern, E.M. (1974) An a l . Biochem. 62, 317-318. Southern, E.M. (1979) A n a l . Biochem. 100, 319-323. Sprague, K.U., Hagenbuchle, 0. and Zuniga, M.C. (1977) C e l l 11_, 561-570. Sprague, K.U., Larson, D. and Morton, D. (1980) C e l l 22, 171-178. S p r i n z l , M., von der Haar, F. and Cramer, F. (1972) Europ. J . Biochem. 25, 262-266. Sprouse, H.M., Kashdan, M., O t i s , L. and Dudock, B. (1981) Nuc l . Acids Res. 9_, 2543-2547. Sta n l e y , J . and V a s s i l e n k o , S. (1978) Nature 274, 87-89. Steffensen, D.M. and Wimber, D.E. (1971) Genetics 69_, 163-178. Steinmetz, A., Mubumbila, M., K e l l e r , M., Burkard, G., W e i l , J.H., D r i e s e l , A.J., Crouse, E.J., Gordon, K., Bohnert, H.-J. and Herrmann, R.G. (1980) i n Transfer RNA: B i o l o g i c a l Aspects ( S o i l , D., Abelson, J.N. and Schimmel, P.R. eds.) pp 281-286, Cold Spring Harbor Laboratory. Tanaka, Y., Dyer, T.A. and Brownlee, G.G. (1980) Nuc l . Acids Res. 8^ , 1259-1272. Tener, G.M., Hayashi, S., Dunn, R., Delaney, A., G i l l a m , I.C., G r i g l i a t t i , T.A., Kaufman, T.C. and Suzuki, D.T. (1980) i n Transfer RNA: B i o l o g i c a l  Aspects ( S o i l , D., Abelson, J.N. and Schimmel, P.R., eds.) pp 295-307, Cold Spring Harbor Laboratory. Thach, R.E. and Doty, P. (1965) Science 148, 632-634. Thomson, R.Y. (1960) i n Chromatographic and E l e c t r o p h o r e t i c Techniques (Smith, I . , ed.) V o l . I , pp 242-243, Wm. Heinemann P u b l i s h e r s , London. Tolman, G.L., B a r r i o , J.R. and Leonard, N.J. (1974) Biochem. 13_, 4869-4878. Travers, A.A. (1980) J . B a c t e r i o l . 141, 973-976. Valenzuela, P., B e l l , G.I., Venegas, A., Sewell, E.T., Masiarz, F.R. , DeGennaro, L . J . , Weinberg, F. and R u t t e r , W.J. (1977) J . B i o l . Chem. 252, 8126-8135. Valenzuela, P., Venegas, A., Weinberg, F., Bishop, R. and Ru t t e r , W.J. (1978) Proc. N a t l . Acad. S c i . USA 75, 190-194. Van E t t e n R.A., Walberg, M.W. and Clayton, D.A. (1980) C e l l 22., 157-170. Veloso, D. , Guynn, R.W., Oskarsson, M., and Veech, R.L. (1973) J . B i o l . Chem. 248, 4811-4819. Venegas, A., Quiroga, M. Z a l d i v a r , J . , R u t t e r , W.J. and Valenzuela, P. (1979) J . B i o l . Chem. 254, 12306-12309. Weber, L. and Berger, E. (1976) Biochem. 15_, 5511-5519. Weissbach, H. and Ochoa, S. (1976) Ann. Rev. Biochem. ^ 5 , 191-216. Wbite, B.N., Tener, G.M., Holden, J . and Suzuki, D.T. (1973a) Develop. B i o l . _33, 185-195. White, B.N., Tener, G.M., Holden, J . and Suzuki, D.T. (1973b) J . Mol. B i o l . 74_, 635-651. White, B.N. and Tener, G.M. (1973c) Can. J . Biochem. 51, 896-902. White, B.N. (1980) Comp. Biochem. P h y s i o l . 67B, 103-107. Woo, N.H., Roe, B.A. and R i c h , A. (1980) Nature 286, 346-351. Yanofsky, C. (1981) Nature 289, 751-758. Yen, P.H. and Davidson, N. (1980) C e l l _22, 137-148. Young, R.A., M a c k l i s , R., and S t e i t z , J.A. (1979a) J . B i o l . Chem. 254, 3264-3271. Young, R.A. (1979b) J . B i o l . Chem. 254, 12725-12731. Zachau, H.G., D u t t i n g , D. and Feldmann, H. (1966) Hoppe-Seyler 1s Z. P h y s i o l . Chem. 347, 212-235. 

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