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The DNA sequence and transcriptional analyses of Drosophila melanogaster transfer RNA valine genes Rajput, Bhanu 1982

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THE DNA SEQUENCE AND TRANSCRIPTIONAL ANALYSES OF DROSOPHILA MELANOGASTER TRANSFER RNA VALINE GENES by BHANU RAJPUT B . S . , W a s h i n g t o n S t a t e U n i v e r s i t y , 1976 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 8 0 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 MICROBIOLOGY UNIVERSITY OF B R I T I S H COLUMBIA We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d . THE UNIVERSITY OF B R I T I S H COLUMBIA A u g u s t , 1982 © B h a n u - . R a j p u t , 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 m\C g.0 fe\OUOfrV The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date g-yX/ Oct. DE-6 (3/81) A b s t r a c t The n u c l e o t i d e s e q u e n c e o f t h e s i n g l e D r o s o p h i 1 a m e i a n o g a s t e r tRNA gene c o n t a i n e d i n t h e r e c o m b i n a n t p l a s m i d , p D t l 2 0 R was d e t e r m i n e d by t h e Maxam and G i l b e r t m e t h o d . T h i s p l a s m i d h y b r i d i z e s t o t h e 90 BC s i t e on t h e V a l D r o s o p h i l a p o l y t e n e c h r o m o s o m e s , a m i n o r s i t e o f t R N A 4 h y b r i d i z a t i o n . The V a l n u c l e o t i d e s e q u e n c e o f t h e tRNA 4 gene p r e s e n t i n p D t l 2 0 R d i f f e r s a t f o u r V a l p o s i t i o n s f r o m t h e s e q u e n c e e x p e c t e d f r o m t h a t o f t R N A 4 . The f o u r d i f f e r e n c e s o c c u r a t n u c l e o t i d e s 1 6 , 2 9 , 41 and 57 i n t h e coding r e g i o n . C o m p a r i s o n o f t h e DNA s e q u e n c e o f p D t l 2 0 R t o t h a t o f t h e p l a s m i d p D t 9 2 R , w h i c h a l s o h y b r i d i z e s t o t h e 90 BC s i t e , i n d i c a t e s t h a t t h e D r o s o p h i l a f r a g m e n t s c o n t a i n e d i n t h e s e t w o p l a s m i d s a r e e i t h e r a l l e l e s o r r e p e a t s . The i m p l i c a t i o n s o f t h e s e f i n d i n g s a r e d i s c u s s e d . An n i v i t r o t r a n s c r i p t i o n s y s t e m was d e v e l o p e d f r o m a D r o s o p h i l a S c h n e i d e r I I c e l l l i n e . T h i s h o m o l o g o u s c e l l - f r e e e x t r a c t s u p p o r t s p e c i f i c a n d a c c u r a t e t r a n s c r i p t i o n o f v a r i o u s D r o s o p h i l a t R N A V a l g e n e s . The m a j o r p r o d u c t o f t r a n s c r i p t i o n i s a tRNA p r e c u r s o r w h i c h i s p r o c e s s e d t o a tRNA s i z e d s p e c i e s . T r a n s f e r RNA v a l i n e g e n e s o r i g i n a t i n g f r o m d i f f e r e n t s i t e s on t h e D r o s o p h i l a c h r o m o s o m e s a r e t r a n s c r i b e d a t d i f f e r e n t r a t e s . C o m p a r i s o n o f t h e s e q u e n c e s i n t h e i n t e r n a l p r o m o t e r r e g i o n s o f t h e v a r i o u s g e n e s i n d i c a t e s t h a t t h e f e w d i f f e r e n c e s w i t h i n t h e c o d i n g r e g i o n s may n o t be r e s p o n s i b l e f o r t h e o b s e r v e d d i f f e r e n c e i n t h e r a t e s o f t r a n s c r i p t i o n . T h i s c o n c l u s i o n i s s u b s t a n t i a t e d by s t u d i e s w i t h h y b r i d g e n e s c o n s t r u c t e d d u r i n g t h e c o u r s e o f t h i s w o r k . P r e l i m i n a r y e v i d e n c e i n d i c a t e s t h a t t h e V a l tRNA gen e w h i c h i s t r a n s c r i b e d a t t h e h i g h e s t r a t e may be p r e c e d e d i n i t s 5 ' - f l a n k i n g r e g i o n by a p o s i t i v e l y m o d u l a t i n g s e q u e n c e . V a l The p r e c u r s o r RNAs d i r e c t e d by v a r i o u s tRNA g e n e s a r e a l s o p r o c e s s e d a t d i f f e r e n t r a t e s . T r a n s c r i p t i o n and p r o c e s s i n g e x p e r i m e n t s w i t h h y b r i d g e n e s s u g g e s t t h a t n u c l e o t i d e c h a n g e s w i t h i n t h e c o d i n g r e g i o n , w h i c h do n o t a f f e c t t h e r a t e o f t r a n s c r i p t i o n , i n f l u e n c e t h e r a t e o f p r o c e s s i n g . Time c o u r s e a n d c o m p e t i t i o n e x p e r i m e n t s d e m o n s t r a t e t h a t a t l e a s t t w o k i n e t i c s t e p s a r e r e q u i r e d f o r t h e f o r m a t i o n o f a s t a b l e t r a n s c r i p t i o n c o m p l e x . S t u d i e s w i t h an j_n v i t r o c o n s t r u c t e d m u t a n t m i s s i n g i n n u c l e o t i d e s 51-61 i n t h e tRNA c o d i n g r e g i o n s u g g e s t s t h a t t h i s d e l e t e d r e g i o n ( w h i c h i s h i g h l y c o n s e r v e d i n e u k a r y o t i c t R N A s ) may be i n v o l v e d i n t h e p r i m a r y i n t e r a c -t i o n r e q u i r e d f o r tRNA g e n e t r a n s c r i p t i o n . i v , TABLE OF CONTENTS P a g e A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s i x L i s t o f F i g u r e s x A c k n o w l e d g e m e n t s x i i i D e d i c a t i o n x i v A b b r e v i a t i o n s xv I n t r o d u c t i o n 1 I . S t r u c t u r e o f t r a n s f e r RNAs 1 A . P r i m a r y and s e c o n d a r y s t r u c t u r e 1 B . T e r t i a r y s t r u c t u r e . 3 I I . T r a n s f e r RNA a n d tRNA g e n e s i n D r o s o p h i 1 a m e l a n o g a s t e r 4 A . T r a n s f e r RNAs i n D r o s o p h i 1 a 4 B . Number o f D r o s o p h i l a tRNA g e n e s 5 C . O r g a n i z a t i o n o f t R N A g e n e s i n D r o s o p h i l a 6 1 . I n s i t u h y b r i d i z a t i o n - l a r g e s c a l e o r g a n i z a t i o n o f tRNA g e n e s 6 2 . A n a l y s i s o f tRNA gene c l u s t e r s - f i n e s t r u c t u r e o f tRNA gene o r g a n i z a t i o n 7 D. S t r u c t u r e o f tRNA g e n e s 8 I I I . T r a n s c r i p t i o n o f RNA p o l y m e r a s e I I I g e n e s 9 A . E u k a r y o t i c RNA p o l y m e r a s e s 9 B . RNA p o l y m e r a s e I I I t r a n s c r i p t i o n s y s t e m s 11 C . T r a n s c r i p t i o n c o n t r o l r e g i o n s f o r p o l y m e r a s e I I I g e n e s 12 D. B i o s y n t h e s i s o f e u k a r y o t i c t R N A 14 1 . tRNA t r a n s c r i p t i o n u n i t . . . . 14 2 . t R N A t r a n s c r i p t i o n c o n t r o l r e g i o n s 15 3 . P r o c e s s i n g o f tRNA t r a n s c r i p t s 17 V , P a g e I V . The p r e s e n t i n v e s t i g a t i o n 19 M a t e r i a l s and M e t h o d s 21 M a t e r i a l s 21 I . R e a g e n t s 21 I I . N u c l e o t i d e s 22 I I I . R a d i o a c t i v e m a t e r i a l s • 22 I V . Enzymes 23 V . B a c t e r i a l and D r o s o p h i l a s t r a i n s 23 M e t h o d s 24 I . G e n e r a l 24 A . P l a s m i d DNA i s o l a t i o n 24 B . D i g e s t i o n o f DNA w i t h r e s t r i c t i o n e n z y m e s 24 C . A g a r o s e g e l e l e c t r o p h o r e s i s 24 D. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s 24 E. A u t o r a d i o g r a p h y 25 F . I s o l a t i o n o f n u c l e i c a c i d f r o m p o l y a c r y l a m i d e g e l s 25 1 . E l e c t r o e l u t i o n 25 2 . S o a k i n g 26 G. I o d i n a t i o n o f t R N A 26 I I . DNA s e q u e n c e a n a l y s i s 27 A . 3 ' e n d - l a b e l l i n g o f r e s t r i c t i o n f r a g m e n t s 27 B . S e c o n d a r y r e s t r i c t i o n enzyme c l e a v a g e 2 8 C . S t r a n d s e p a r a t i o n 28 D. DNA s e q u e n c e a n a l y s i s - Maxam and G i l b e r t m e t h o d 28 I I I . I n v i t r o t r a n s c r i p t i o n a n a l y s i s 29 A . C e l l c u l t u r e 29 B . P r e p a r a t i o n o f D r o s o p h i l a c e l l - f r e e e x t r a c t ( S . 1 0 0 ) 29 v i , P a g e C . I n , v i t r o t r a n s c r i p t i o n a n d e l e c t r o p h o r e t i c a n a l y s i s o f RNA . . 31 D. RNA.DNA h y b r i d i z a t i o n 31 E. f i n g e r p r i n t a n a l y s i s RNA t r a n s c r i p t s 32 I V . I n v i t r o m u t a g e n e s i s 33 V a l A . S e p a r a t i o n o f two tRNA4 g e n e s c o n t a i n e d i n p D t 5 5 33 1 . P r e p a r a t i o n o f r e s t r i c t i o n f r a g m e n t s 33 2 . 5 ' e n d - l a b e l l i n g o f H i n d l l l l i n k e r 39 3 . P o l I t r e a t m e n t t o f i l l up s i n g l e - s t r a n d e d e n d s 39 4 . 3 2 P . H i n d l l l l i n k e r a d d i t i o n t o 'DNA - b l u n t end l i g a t i o n 40 5 . H i n d l l l d i g e s t i o n o f 3 2 P . l i n k e r - DNA 40 6 . P r e p a r a t i v e g e l e l e c t r o p h o r e s i s and e l e c t r o e l u t i o n 40 7 . P r e p a r a t i o n o f p h o s p h a t a s e - t r e a t e d l i n e a r pBR322 4 1 8 . L i g a t i o n o f 3 2 P . l i n k e r - DNA t o p h o s p h a t a s e t r e a t e d , H i n d l l l d i g e s t e d pBR322 41 9 . T r a n s f o r m a t i o n o f E . c o l i ( S F 8 ) 41 1 0 . S e l e c t i o n o f a m p i c i l l i n r e s i s t a n t c o l o n i e s 42 1 1 . T e s t f o r t e t r a c y c l i n e s e n s i t i v i t y ; H o g n e s s p r o c e d u r e 42 1 2 . S c r e e n i n g f o r r e c o m b i n a n t c l o n e s 43 B . C o n s t r u c t i o n o f h y b r i d g e n e s and d e l e t i o n m u t a n t s 43 1 . H y b r i d s b e t w e e n p D t 0 . 3 and- pDt92R DNAs 44 a . p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) h y b r i d 44 b . p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) h y b r i d 44 2 . H y b r i d s b e t w e e n p D t 0 . 3 and pDt78R DNAs 4 5 a . p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) h y b r i d 45 b . p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) h y b r i d 4 5 3 . C o n s t r u c t i o n o f d e l e t i o n m u t a n t s 46 a . I n t e r n a l d e l e t i o n m u t a n t - pDt0.3A 51-61 46 b . D e l e t i o n o f t h e 5 ' - f l a n k i n g s e q u e n c e - p D t 0 . 3 d l 5 ' - 1 8 . . 46 v i i , P a g e R e s u l t s 48 I . DNA s e q u e n c e a n a l y s i s o f p l a s m i d p D t l 2 0 R 48 V a l A . S t r a t e g y u s e d t o s e q u e n c e t h e t R N A 4 gene c o n t a i n e d i n p D t l 2 0 R 48 B . The n u c l e o t i d e s e q u e n c e o f t h e tRNA gene o f p D t l 2 0 R 62 I I . T r a n s c r i p t i o n o f c l o n e d t R N A g e n e s f r o m D r o s o p h i l a m e l a n o g a s t e r i n a h o m o l o g o u s c e l l - f r e e e x t r a c t 69 A . S y n t h e s i s o f d i s t i n c t RNA s p e c i e s d i r e c t e d by p l a s m i d DNA c o n t a i n i n g a D r o s o p h i l a t R N A ^ g 1 g e n e 6 9 B . A u t h e n t i c i t y o f t h e RNA t r a n s c r i p t s , R N A - I a n d R N A - I I , d i r e c t e d by pDt78R 74 C . P r e c u r s o r - p r o d u c t r e l a t i o n s h i p b e t w e e n t h e m a j o r t r a n s c r i p -t i o n p r o d u c t s 76 1 . Time c o u r s e o f RNA s y n t h e s i s 76 2 . RNA-I i s p r o c e s s e d t o R N A - I I 76 3 . T-L f i n g e r p r i n t a n a l y s i s o f R N A - I and R N A - I I 86 D. P r o p e r t i e s o f D r o s o p h i l a ( S c h n e i d e r I I ) c e l l f r e e e x t r a c t . . . . 86 1 . E n h a n c e m e n t o f t r a n s c r i p t i o n i n t h e p r e s e n c e o f a n A T P -g e n e r a t i n g s y s t e m 86 2 . Optimum KC1 c o n c e n t r a t i o n 89 3 . D i v a l e n t m e t a l i o n o p t i m a 8 9 4 . a - a m a n i t i n s e n s i t i v i t y p r o f i l e 98 5 . E f f e c t o f t e m p l a t e c o n c e n t r a t i o n 98 I I I . K i n e t i c s o f t r a n s c r i p t i o n o f c l o n e d tRNA g e n e s 103 A . Time c o u r s e o f RNA s y n t h e s i s 103 B . C o m p a r i s o n o f t h e r a t e s o f t r a n s c r i p t i o n o f c l o n e d t R N A g e n e s 112 C . S t a b i l i t y o f t h e t r a n s c r i p t i o n c o m p l e x 115 I V . I n v i t r o m u t a g e n e s i s 115 A . C o n s t r u c t i o n o f r e c o m b i n a n t p l a s m i d s '. 115 B . C h a r a c t e r i z a t i o n o f t h e i n v i t r o c o n s t r u c t e d p l a s m i d s 118 v i i i , Page C. T r a n s c r i p t i o n of i n v i t r o constructed plasmids 126 1. T r a n s c r i p t i o n of hybrid clones 126 a. T r a n s c r i p t i o n of pDt0.3-78R hybrid plasmids 126 b. T r a n s c r i p t i o n of pDt0.3-92R hybrid plasmids 132 2. T r a n s c r i p t i o n of d e l e t i o n mutants 132 a. T r a n s c r i p t i o n of pDtO.3dl5'-18 132 b. T r a n s c r i p t i o n of pDtO.3 A 51-61 133 D. Complex s t a b i l i t y experiments using pDtO.3 A51-61 DNA 136 Discussion 143 Va X I. Nucleotide sequence of Drosophila tRNA 4 genes 143 A. Nucleotide sequence of pDtl20R: Comparison to the sequence of other tRNA^ gene c o n t a i n i n g plasmids 143 V a l B. Nucleotide sequence of the tRNA4 gene of pDtl20R: Comparison to other t R N A ^ a l genes of Drosophi 1 a 147 V a l C. Are the tRNA 4 - l i k e genes expressed i n vivo? 148 I I . T r a n s c r i p t i o n of Drosophila t R N A V a l genes 152 A. T r a n s c r i p t i o n using Drosophila Schneider II c e l l - f r e e e x t r a c t 152 V a l B. Different' tRNA genes t r a n s c r i b e at d i f f e r e n t rates i n v i t r o 153 C. Sequence dependence of t r a n s c r i p t i o n r a t e 156 1. E f f e c t of gene i n t e r n a l changes on the rate of tRNA gene t r a n s c r i p t i o n 156 2. Influence of f l a n k i n g sequences on the rate of tRNA gene t r a n s c r i p t i o n 161 3. Role of conserved sequence blocks A and B i n t r a n s c r i p t i o n of tRNA genes 163 D. Influence of sequence on maturation of tRNA precursors 170 I I I . K i n e t i c s of tRNA t r a n s c r i p t i o n 173 Bi b l i o g r a p h y 176 i x , L I S T OF TABLES P a g e Val T a b l e I . S i t e s o f D r o s o p h i l a m e l a n o g a s t e r t R N A g e n e s on t h e p o l y t e n e c h r o m o s o m e s 7 T a b l e I I . S p e c i f i c c h e m i c a l d e g r a d a t i o n o f DNA 30 T a b l e I I I . C h a r a c t e r i s t i c s o f t h e p a r e n t DNA p l a s m i d s u s e d i n t h e w o r k d e s c r i b e d i n t h i s t h e s e s 34 T a b l e I V . RNA.DNA h y b r i d i z a t i o n 75 T a b l e V . C o m p a r i s o n o f t h e r a t e s o f t r a n s c r i p t i o n 129 L I S T OF FIGURES P a g e F i g u r e 1 . The c l o v e r l e a f s t r u c t u r e o f tRNA 2 Phe F i g u r e 2 . Two v i e w s o f t h e t e r t i a r y s t r u c t u r e o f y e a s t tRNA . . . . 3 Phe F i g u r e - 3 . T e r t i a r y h y d r o g e n b o n d s o f y e a s t tRNA 4 F i g u r e 4 . The s t r a t e g y u s e d f o r i n v i t r o c o n s t r u c t i o n o f p l a s m i d s . . 3 6 , 3 7 F i g u r e 5 . Scheme f o r c l o n i n g a DNA f r a g m e n t a f t e r l i n k e r l i g a t i o n . . 38 F i g u r e 6 . D e t e r m i n a t i o n o f t h e number o f c l e a v a g e s i t e s f o r t h e R. e n d o n u c l e a s e Xmal i n p D t l 2 0 R 50 V a l F i g u r e 7 . Maxam and G i l b e r t s e q u e n c i n g o f t h e t R N A 4 g e n e c o n t a i n e d i n p D t l 2 0 R 53 F i g u r e 8 . The s t r a t e g y u s e d t o s e q u e n c e a s e g m e n t o f p D t ! 2 0 R DNA " c o n t a i n i n g t h e t R N A ^ a l gene 55 F i g u r e 9 . D i g e s t i o n o f p D t l 2 0 R w i t h E c o R I u n d e r d i f f e r e n t r e s t r i c t i o n c o n d i t i o n s 58 F i g u r e 1 0 . R e s t r i c t i o n a n a l y s i s o f p D t l 2 0 R DNA u s i n g R. e n d o n u c l e a s e , S a u 9 6 I 61 F i g u r e 1 1 . S t r a n d s e p a r a t i o n o f S a u 9 6 I f r a g m e n t 4 o f p D t l 2 0 R 64 F i g u r e 1 2 . C o n f i r m a t i o n o f t h e Xmal r e s t r i c t i o n s i t e i n t h e t R N A g e n e o f p D t l 2 0 R 66 F i g u r e 1 3 . The n u c l e o t i d e s e q u e n c e o f a s e g m e n t o f t h e D r o s o p h i l a DNA i n s e r t o f p l a s m i d p D t l 2 0 R 68 V a l F i g u r e 1 4 . T r a n s c r i p t i o n o f pDt78R DNA c o n t a i n i n g D r o s o p h i l a t R N A 3 b gene 71 F i g u r e 1 5 . T r a n s c r i p t i o n o f d i f f e r e n t p l a s m i d s c a r r y i n g D r o s o p h i l a t R N A V a l g e n e s 73 F i g u r e 1 6 . Time c o u r s e a s s a y 78 F i g u r e 1 7 . P r o c e s s i n g o f RNA-I d i r e c t e d by p D t 0 . 3 t o R N A - I I 80 F i g u r e 1 8 . P r o c e s s i n g o f R N A - I d i r e c t e d by pDt78R t o R N A - I I 82 F i g u r e 1 9 . P r o c e s s i n g o f RNA-I d i r e c t e d by pDt92R t o R N A - I I 84 F i g u r e 2 0 . RNase T-j_ f i n g e r p r i n t s o f t h e m a j o r t r a n s c r i p t i o n p r o d u c t s f r o m pDt78R a n d p D t 5 5 88 F i g u r e 2 1 . E f f e c t o f an A T P - g e n e r a t i n g s y s t e m on s p e c i f i c t r a n s c r i p -t i o n i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t 91 x i » P a g e F i g u r e 2 2 . E f f e c t o f KC1 c o n c e n t r a t i o n on s p e c i f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t 93 +2 F i g u r e 2 3 . E f f e c t o f Mg c o n c e n t r a t i o n on s p e c i f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t 95 + 2 F i g u r e 2 4 . E f f e c t o f Mn c o n c e n t r a t i o n on s p e c i f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t 97 F i g u r e 2 5 . E f f e c t o f a - a m a n i t i n on s p e c i f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t 100 F i g u r e 2 6 . E f f e c t o f p D t 0 . 3 DNA c o n c e n t r a t i o n on t r a n s c r i p t i o n i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t 102 F i g u r e 2 7 . E f f e c t o f i n c r e a s i n g amount o f p D t 0 . 3 DNA on s p e c i f i c t r a n s c r i p t i o n 1 0 5 F i g u r e 2 8 . T r a n s c r i p t i o n w i t h v a r y i n g a m o u n t s o f d i f f e r e n t t e m p l a t e DNAs 107 F i g u r e 2 9 . I n h i b i t i o n o f s p e c i f i c t r a n s c r i p t i o n by i n c r e a s i n g a m o u n t s o f pBR322 DNA 109 F i g u r e 3 0 . T i m e c o u r s e o f RNA s y n t h e s i s w i t h p D t 0 . 3 DNA as t h e t e m p l a t e I l l F i g u r e 3 1 . C o m p a r i s o n o f t h e r a t e s o f t r a n s c r i p t i o n o f d i f f e r e n t r e c o m b i n a n t p l a s m i d s c a r r y i n g t R N A v g e n e s 114 F i g u r e 3 2 . S t a b i l i t y o f t h e t r a n s c r i p t i o n c o m p l e x 117 F i g u r e 3 3 . DNA s e q u e n c e s o f p a r e n t and i n v i t r o c o n s t r u c t e d p l a s m i d s 120 F i g u r e 3 4 . Maxam and G i l b e r t s e q u e n c i n g o f t h e tRNA g e n e c o n t a i n e d i n t h e d e l e t i o n m u t a n t , p D t 0.3A 51-61 123 F i g u r e 3 5 . R e s t r i c t i o n a n a l y s i s o f t h r e e i n v i t r o c o n s t r u c t e d p l a s m i d s 125 F i g u r e 3 6 . T r a n s c r i p t i o n o f p a r e n t a n d i n v i t r o c o n s t r u c t e d p l a s m i d s 128 F i g u r e 3 7 . C o m p a r i s o n o f t r a n s c r i p t i o n and p r o c e s s i n g e f f i c i e n c e s . o f p l a s m i d s p D t 0 . 3 and p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) 131 F i g u r e 3 8 . T r a n s c r i p t i o n o f t h e d e l e t i o n m u t a n t , p D t 0.3A51 - 6 1 135 F i g u r e 3 9 . S t a b i l i t y o f h a l f t R N A m o l e c u l e s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t 138 x i i , P a g e F i g u r e 4 0 . T r a n s c r i p t i o n c o m p l e x s t a b i l i t y e x p e r i m e n t s i n v o l v i n g t h e d e l e t i o n m u t a n t , p D t 0 . 3 A 5 l - 6 1 140 F i g u r e 4 1 . The d e l e t i o n m u t a n t , p D t O . 3A 51-61 c o m p e t e s w i t h t r a n s c r i p t i o n o f i n t a c t tRNA g e n e s 142 F i g u r e 4 2 . N u c l e o t i d e s e q u e n c e s o f s e g m e n t s o f t h e D r o s o p h i l a DNA i n s e r t s o f p D t ! 2 0 R , p D t 9 2 R , p D t l 4 and p D t 5 5 1 4 5 - 1 4 6 F i g u r e 4 3 . The n u c l e o t i d e s e q u e n c e o f D r o s o p h i l a m e l a n o g a s t e r tRNA ^ a l a r r a n g e d a s a c l o v e r l e a f 150 F i g u r e 4 4 . C o m p a r i s o n o f DNA s e q u e n c e s p o s t u l a t e d t o be i m p o r t a n t i n t r a n s c r i p t i o n o f tRNA g e n e s 158 F i g u r e 4 5 . The p o s t u l a t e d t R N A - l i k e s t r u c t u r e o f t h e RNA t r a n s c r i p t d i r e c t e d by p D t 0 .3A 51-61 167 x i i i , ACKNOWLEDGEMENTS I w i s h t o t h a n k D r . G . B . S p i e g e l m a n f o r f r u i t f u l d i s c u s s i o n s and h e l p i n t h e s u c c e s s f u l c o m p l e t i o n o f t h i s w o r k . I w o u l d a l s o l i k e t o t h a n k D r . G . M . T e n e r and D r . R . C . M i l l e r , J r . f o r c o n s t r u c t i v e c r i t i c i s m . My t h a n k s t o D r . C . R . A s t e l l and D r . D . L . C r i b b s f o r t e a c h i n g me DNA and RNA s e q u e n c i n g m e t h o d s . F i n a l l y , I w o u l d l i k e t o t h a n k L o v e r n e D u n c a n f o r t e c h n i c a l a s s i s t a n c e . DEDICATED TO THE DEAR MEMORY OF MY FATHER AND BROTHER NAVIN ABBREVIATIONS USED A 2 6 0 - a b s o r b a n c e a t 260nm bp - b a s e p a i r s kb - k i l o b a s e p a i r s m . w t . - m o l e c u l a r w e i g h t p o l I - p o l y m e r a s e I p o l y m e r a s e I I I g e n e s - g e n e s t r a n s c r i b e d by RNA p o l y m e r a s e I I R. e n d o n u c l e a s e - r e s t r i c t i o n e n d o n u c l e a s e RNase - r i b o n u c l e a s e R P C - 5 - r e v e r s e p h a s e c h r o m a t o g r a p h y s y s t e m 5 5S DNA - DNA c o d i n g f o r 5S RNA tDNA - DNA c o d i n g f o r t R N A TLC - t h i n l a y e r c h r o m o t o g r a p h y V a l 3b - t R N A ^ 1 V a l 4 - V a l t R N A 4 1. I n t r o d u c t i o n A r e m a r k a b l e f e a t u r e i n t h e d i s c o v e r y o f t r a n s f e r RNA ( t R N A ) was i t s p r e d i c t i o n by F . H . C . C r i c k ( 1 ) . In 1 9 5 5 , i n a n o t e e n t i t l e d , "On D e g e n e r a t e T e m p l a t e s and t h e A d a p t o r H y p o t h e s i s , " C r i c k p o s t u l a t e d t h e e x i s t e n c e o f " a d a p t o r " m o l e c u l e s c a p a b l e o f c o m b i n i n g c h e m i c a l l y w i t h an a m i n o a c i d and o f h y d r o g e n b o n d i n g t o s p e c i f i c s e q u e n c e s i n a t e m p l a t e n u c l e i c a c i d . M o l e c u l e s w i t h s u c h p r o p e r t i e s w e r e p r o p o s e d t o p l a y a k e y r o l e i n t r a n s m i s s i o n o f g e n e t i c i n f o r m a t i o n f r o m t h e n u c l e o t i d e s e q u e n c e s o f n u c l e i c a c i d s t o t h e a m i n o a c i d s e q u e n c e s o f p r o t e i n s d u r i n g p r o t e i n s y n t h e s i s . S m a l l RNA m o l e c u l e s w i t h some o f t h e s e p r o p e r t i e s w e r e d i s c o v e r e d by H o a g l a n d e t a\_. ( 2 ) t h r e e y e a r s l a t e r . S i n c e i t s d i s c o v e r y i n 1 9 5 8 , r e s e a r c h i n t h e f i e l d o f tRNA s t r u c t u r e and b i o s y n t h e s i s h a s t a k e n i m p o r t a n t s t r i d e s ( 3 - 6 ) . The work r e p o r t e d h e r e c o n c e r n s t h e s t r u c t u r e and t r a n s c r i p t i o n o f V a l D r o s o p h i l a tRNA g e n e s . B a c k g r o u n d i n f o r m a t i o n r e l e v a n t t o t h i s s u b j e c t i s b r i e f l y d e s c r i b e d i n t h e f o l l o w i n g s e c t i o n s . I . S t r u c t u r e o f t r a n s f e r RNAs A . P r i m a r y and s e c o n d a r y s t r u c t u r e A l a I n 1965 H o l l e y et a l _ . ( 7 ) p u b l i s h e d t h e s e q u e n c e o f y e a s t tRNA , t h e f i r s t n u c l e o t i d e s e q u e n c e o f a n y n u c l e i c a c i d . They d e s c r i b e d t h r e e p o s s i b l e s e c o n d a r y s t r u c t u r e s f o r t h e t R N A . S i n c e 1 9 6 5 s e q u e n c e s o f o v e r t w o h u n d r e d tRNAS h a v e become a v a i l a b l e ( 8 ) . They a r e 7 5 - 9 0 n u c l e o t i d e s l o n g . A l l t h e s e t R N A s c a n be f o l d e d i n t o a f o r m s i m i l a r t o one o f t h o s e p r o p o s e d by H o l l e y , t h e ' c l o v e r l e a f f o r m shown i n F i g . 1 ( 3 ) . The p r o m i n e n t f e a t u r e s o f t h e s t a n d a r d tRNA s t r u c t u r e a r e t h e f o u r b a s e - p a i r e d s t e m r e g i o n s , t h r e e o f w h i c h a r e c l o s e d by n o n b a s e - p a i r e d l o o p s . The a c c e p t o r s t e m c o n s i s t s o f a 7 b a s e p a i r s t e m a n d f o u r u n p a i r e d n u c l e o t i d e s a n d i t a l s o c o n t a i n s t h e 5 ' and 3 ' - e n d s o f t h e t R N A . The 5 ' - e n d i s p h o s p h o r y l a t e d w h i l e t h e 3 ' - e n d s o f a l l t R N A s a r e s i n g l e - s t r a n d e d t a i l s A N T I C O D O N L O O P A N T I C O D O N F i g u r e V . The C l o v e r l e a f S t r u c t u r e o f tRNA w i t h t h e s e q u e n c e NCCA. . The d i h y d r o u r i d i n e arm ( D - a r m ) h a s a 3 o r 4 b a s e s t e m a n d a l o o p o f v a r i a b l e s i z e , r a n g i n g f r o m 7-11 n u c l e o t i d e s . Two r e g i o n s - , o f v a r i a b l e l e n g t h , a a n d f3 » f l a n k t h e p a i r o f G r e s i d u e s a l w a y s f o u n d i n t h e D - l o o p . a and 3 c o n t a i n 1 t o 3 n u c l e o t i d e s e a c h . The a n t i c o d o n arm i s made up o f a 5 b a s e p a i r s t e m and a 7 n u c l e o t i d e l o o p . The t h i r d r e g i o n o f v a r y i n g l e n g t h i n t R N A s i s t h e v a r i a b l e l o o p ( a l s o known as t h e e x t r a - a r m ) . I t s l e n g t h v a r i e s f r o m 4 - 2 1 n u c l e o t i d e s . The T * C - a r m ( T - a r m ) c o n s i s t s o f a 5 b a s e p a i r s t e m a n d a 7 n u c l e o t i d e l o o p . C o m p a r i s o n o f t h e c l o v e r l e a f s t r u c t u r e s o f s e q u e n c e d t R N A s i n d i c a t e s t h a t n u c l e o t i d e s a t c e r t a i n p o s i t i o n s i n t h e s t r u c t u r e a r e w e l l c o n s e r v e d . Some p o s i t i o n s a r e a l m o s t i n v a r i a b l y o c c u p i e d by t h e ' same n u c l e o t i d e . T h e s e a r e : U 8 , A 1 4 , G 1 8 , G 1 9 , A 2 1 , U 3 3 , G 5 3 , T 5 4 , ¥ 5 5 , C 5 6 , C 6 1 , C 7 4 , C 7 5 , and A76 ( F i g . 1 ) . O t h e r p o s i t i o n s a r e a l m o s t a l w a y s o c c u p i e d .by a p y r i m i d i n e (Y) and s t i l l o t h e r s by p u r i n e s ( R ) . T h e s e i n c l u d e Y l l , R 1 5 , R 2 4 , Y 3 2 , R 3 7 , Y 4 8 , R57 a n d Y60 ( F i g . 1 ) . . The n u c l e o t i d e a t p o s i t i o n 37 i s o f t e n h y p e r m o d i f i e d (H i n F i g . 1.) a n d p r o b a b l y p l a y s a r o l e i n c o d o n - a n t i c o d o n i n t e r a c t i o n s . P u r i n e 15 a n d p y r i m i d i n e 4 8 (R , Y i n F i g . 1) a r e u s u a l l y C o m p l e m e n t a r y a n d maybe i n v o l v e d i n t h e f o r m a t i o n o f a t e r t i a r y b a s e p a i r . B . T e r t i a r y s t r u c t u r e I n 1 9 7 5 , t w o r e s e a r c h g r o u p s p u b l i s h e d t h e 3 - d i m e n s i o n a l c r y s t a l Phe Phe s t r u c t u r e o f y e a s t tRNA ( r e v i e w e d i n r e f . 3 , 9 ) . Y e a s t tRNA i s a f l a t , L - s h a p e d m o l e c u l e ( F i g . 2 ; 3 ) . A c o m p l e x a r r a y o f h y d r o g e n b o n d s p l a y s an i m p o r t a n t r o l e i n m a i n t a i n i n g i t s t e r t i a r y s t r u c t u r e . T h e s e b o n d s a r e more c l e a r l y s e e n i n F i g . 3 ( 9 ) . S t r o n g l y c o n s e r v e d n u c l e o t i d e s a r e i n v o l v e d i n a l m o s t a l l o f t h e s e b o n d s . T h i s s u g g e s t s t h a t t e r t i a r y i n t e r a c t i o n s s i m i l a r t o t h o s e s e e n i n y e a s t t R N A P h e a r e p r e s e n t i n o t h e r t R N A s . The c o n s e r v e d GG d o u b l e t i n t h e D - l o o p i s b o n d e d t o t h e y C n u c l e o t i d e s i n t h e T - l o o p . U8 and A9 a r e h y d r o g e n b o n d e d t o A14 and A23 r e s p e c t i v e l y . W i t h t h e e x c e p t i o n o f G 1 9 - C 5 6 b o n d , n o n e o f t h e t e r t i a r y h y d r o g e n b o n d s i s o f t h e W a t s o n - C r i c k t y p e . O Q ' l t t m A OH 3 e n d C - Send pG, . c C • G G T O C G » U As • U Dorm U • A y < " m ] © G A C A © " ^ (G® (AL / t G ) Q Q - i t e m Q0 '•> iC)U C ^ G ^ tC) ^ © A C A G — A A U U C G C A C C A O H ix. o . . . rfe^©U G Um'C U U A G G C G p © D , (AK, , . O R M A ^ i r -trg. tG) • 'Ci IC> • nfG-mjG • C • G -C • G A • U (Cm> A i.\J) Y Gm A A ,»C acotm V • \' • '•• Phe F i g u r e 3 . T e r t i a r y H y d r o g e n - B o n d s o f Y e a s t t R N A . A t : l e f t t h e C l o v e r l e a f f o r m o f t R N A ^ h e , a t r i g h t a d r a w i n g t h a t more c l o s e l y r e s e m b l e s t h e t e r t i a r y s t r u c t u r e o f t R N A P h e . Phe X - r a y ' d i f f r a c t i o n s t u d i e s o f y e a s t tRNA show t h a t 20 o f t h e 23 s t r o n g l y c o n s e r v e d n u c l e o t i d e s i n t h i s tRNA a r e i n v o l v e d i n m a i n t a i n i n g t h e m o l e c u l e ' s t e r t i a r y s t r u c t u r e . I I . T r a n s f e r RNA a n d tRNA g e n e s i n D r o s o p h i l a m e l a n o g a s t e r A . T r a n s f e r RNAS i n D r o s o p h i l a On R P C - 5 c o l u m n s , D r o s o p h i 1 a a m i n o a c y l - t R N A s c a n be r e s o l v e d i n t o . 63 m a j o r and 36 m i n o r p e a k s ( 1 0 ) i n d i c a t i n g a t o t a l o f 99 d i f f e r e n t s p e c i e s o f t R N A . I t h a s b e e n s u g g e s t e d ( 1 1 ) t h a t s e v e r a l c h r o m a t o g r a p h i c a l l y d i s t i n c t f o r m s o f i s o a c c e p t i n g t R N A s h a v e t h e same n u c l e o t i d e s e q u e n c e and a r e p r o b a b l y p r o d u c t s o f t h e same g e n e . T h a t i s . , t h e s e a r e h o m o g e n e i c s p e c i e s r e s u l t i n g f r o m d i f f e r e n t d e g r e e s o f p o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n . E x a m i n a t i o n o f t h e i s o a c c e p t i n g p a t t e r n s f r o m t h r e e d i f f e r e n t s t a g e s o f d e v e l o p m e n t , f i r s t i n s t a r , t h i r d i n s t a r a n d a d u l t f l i e s showed t h a t a p p r o x i m a t e l y o n e - t h i r d o f t h e 99 tRNA p e a k s u n d e r w e n t some q u a n t i t a t i v e c h a n g e ( T O ) . The v a l y l - t f l N A p r o f i l e s r e m a i n e d e s s e n t i a l l y c o n s t a n t t h r o u g h o u t d e v e l o p m e n t . 5. The v a l i n e t R N A s f o r m one o f t h e m o s t e x t e n s i v e l y c h a r a c t e r i z e d i s o a c c e p t o r f a m i l i e s f r o m D r o s o p h i l a . C h r o m a t o g r a p h y on R P C - 5 i n one b u f f e r Val s y s t e m (pH 4 . 0 , 10 mM M g C l 2 ) s e p a r a t e d D r o s o p h i l a m e l a n o g a s t e r v a l y l - t R N A i n t o 7 p e a k s d e s i g n a t e d 1 t o 7 a c c o r d i n g t o t h e o r d e r o f e l u t i o n f r o m t h e c o l u m n ( 1 0 , 1 2 ) . Of t h e 7 p e a k s , 3 and 4 w e r e t h e m a j o r o n e s . U s i n g a s e c o n d r e s o l v i n g b u f f e r (pH 3 . 8 , 1 mM E D T A ) , f r a c t i o n 3 was f u r t h e r r e s o l v e d i n t o 3a a n d 3b ( 1 2 ) . Val Val Val The t h r e e m a j o r v a l i n e i s o a c c e p t i n g s p e c i e s , t R N A ^ , t R N A 3 b and tRNA4 h a v e d i f f e r e n t b a s e c o m p o s i t i o n and c o d i n g p r o p e r t i e s . In r i b o s o m e b i n d i n g Val Val Val s t u d i e s ( 1 2 , 1 3 ) , t R N A 3 a r e s p o n d e d s t r o n g l y t o GUA, t R N A 3 b t o GUG and t R N A 4 t o GUU, GUC and GUA. T h e r e f o r e , t o g e t h e r t h e t h r e e m a j o r v a l i n e s p e c i e s r e c o g n i z e a l l f o u r v a l i n e c o d o n s , GUN. Q u i t e r e c e n t l y , t h e n u c l e o t i d e Val Val s e q u e n c e s o f tRNA3]-) ( 1 3 ) and t R N A 4 ( 1 3 , 14) h a v e b e e n d e t e r m i n e d . T h e s e t R N A s show g r e a t h o m o l o g y t o t h e e q u i v a l e n t v e r t e b r a t e t R N A s ( 1 3 , 14) i n d i c a t i n g s t r o n g c o n s e r v a t i o n o f t h e s e s e q u e n c e s d u r i n g t h e e v o l u t i o n o f t h e e u k a r y o t e s . B . Number o f D r o s o p h i l a tRNA g e n e s 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 by R i t o s s a e t a l _ . ( 1 5 ) i n d i c a t e d t h a t 0 . 0 1 5 % o f t h e t o t a l D r o s o p h i 1 a DNA c o d e d f o r t R N A , e q u i v a l e n t t o 750 tRNA g e n e s p e r h a p l o i d g e n o m e . Weber and B e r g e r ( 1 6 ) e s t i m a t e d t h e t o t a l number o f tRNA g e n e s p e r h a p l o i d genome o f D r o s o p h i l a t o be 5 9 0 . T h i s f i g u r e i s p r o b a b l y more a c c u r a t e t h a n t h e e a r l i e r one b e c a u s e t h e 4S RNA p r o b e u s e d was o f h i g h e r p u r i t y . The k i n e t i c c o m p l e x i t y o b s e r v e d i n d i c a t e d 59 f a m i l i e s o f tRNA g e n e s . T h e s e s t u d i e s t h e r e f o r e s u g g e s t an a v e r a g e o f 1 0 - 1 3 g e n e s f o r e a c h d i f f e r e n t tRNA s e q u e n c e i n D r o s o p h i 1 a . S u c h r e d u n d a n c y o f tRNA g e n e s a l s o a p p e a r s t o be t h e c a s e f o r o t h e r e u k a r y o t e s e . g . y e a s t ( 1 7 , 18) and X e n o p u s ( 6 , 1 9 ) . On t h e o t h e r h a n d t h e number o f d i f f e r e n t s p e c i e s o f tRNA i n E - c o l i a p p e a r s t o be s i m i l a r t o t h a t i n e u k a r y o t e s ( 6 , 1 7 ) , b u t t h e t o t a l number o f tRNA g e n e s 6 . i s o n l y 60 ( 2 0 ) . The number o f g e n e s f o r a p a r t i c u l a r tRNA c a n be e s t i m a t e d by d i g e s t i n g D r o s o p h i l a DNA t o c o m p l e t i o n w i t h r e s t r i c t i o n e n z y m e s and t h e n h y b r i d i z i n g t h e s e p a r a t e d f r a g m e n t s ( o n g e l ) t o l a b e l l e d tRNA o f i n t e r e s t . D u d l e r e t j f L ( 2 1 ) and T e n e r ^ t a]_. ( 2 2 ) u s e d t h i s a p p r o a c h t o e s t i m a t e 1 7 - 1 9 g e n e s f o r t R N A ^ a l Val and 6 - 7 g e n e s f o r t R N A 3 b r e s p e c t i v e l y . S o l u t i o n h y b r i z a t i o n s t u d i e s i n d i c a t e d 5 and 10 g e n e s r e s p e c t i v e l y f o r t R N A ^ a l ( 2 3 ) and t R N A ^ 1 ^ ) . The l a t t e r v a l u e s may n o t be v e r y a c c u r a t e b e c a u s e o f t h e d i f f i c u l t i e s e n c o u n t e r e d d u r i n g t h i s p r o c e d u r e ( 2 2 , 2 3 ) . C . O r g a n i z a t i o n o f tRNA g e n e s i n D r o s o p h i l a 1 . I n s i t u h y b r i d i z a t i o n - l a r g e s c a l e o r g a n i z a t i o n o f tRNA g e n e s The a v a i l a b i l i t y o f e x t e n s i v e g e n e t i c i n f o r m a t i o n and o f many m u t a n t s t o g e t h e r w i t h t h e e x i s t e n c e o f p o l y t e n e s a l i v a r y g l a n d c h r o m o s o m e s makes D r o s o p h i l a an i d e a l s y s t e m f o r s t u d y i n g tRNA g e n e o r g a n i z a t i o n and e x p r e s s i o n . The t e c h n i q u e o f j_n s i t u h y b r i d i z a t i o n ( 2 4 ) o f tRNA t o p o l y t e n e c h r o m o s o m e s h a s g r e a t l y i n c r e a s e d o u r k n o w l e d g e o f tRNA g e n e l o c a t i o n i n D r o s o p h i l a . S t e f f e n s o n and Wimber ( 2 5 ) and E l d e r et ail_. ( 2 6 ) h y b r i d i z e d t o t a l 4S RNA t o p o l y t e n e c h r o m o s o m e s and i d e n t i f i e d 68 and 63 s i t e s o f h y b r i d i z a t i o n r e s p e c t i v e l y , r a n d o m l y s c a t t e r e d o v e r a l l t h e c h r o m o s o m e s e x a m i n e d . T h e s e n u m b e r s a r e much l e s s t h a n t h e 5 9 0 - 7 5 0 g e n e s d e t e c t e d by i n 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 ( 1 5 , 1 6 ) . The _i_n s i t u r e s u l t s t h e r e f o r e i m p l y t h a t many c h r o m o s o m a l s i t e s c o n t a i n s e v e r a l tRNA g e n e s . I n s i t u h y b r i d i z a t i o n w i t h s i n g l e , p u r i f i e d tRNA s p e c i e s h a s a l l o w e d l o c a l i z a t i o n o f g e n e s f o r o v e r 20 tRNA i s o a c c e p t o r s ( r e v i e w e d i n r e f . 2 7 ) . Two c o n c l u s i o n s c a n be r e a c h e d f r o m t h e s e s t u d i e s : i ) tRNA g e n e s a r e s c a t t e r e d o v e r t h e c h r o m o s o m e s . S i t e s o f h y b r i d i z a t i o n w h i c h p r e s u m a b l y c o n t a i n tRNA g e n e s h a v e b e e n f o u n d on a l l t h e c h r o m o s o m e s e x c e p t f o r t h e 7, v e r y s m a l l f o u r t h o n e . i i ) In g e n e r a l , i n d i v i d u a l t R N A s h y b r i d i z e t o more t h a n one s i t e on t h e c h r o m o s o m e s s u g g e s t i n g t h a t g e n e s f o r a p a r t i c u l a r tRNA o c c u r a t s e v e r a l d i s p e r s e d s i t e s . A s i n g l e s i t e may c o n t a i n more t h a n Va 1 o n e g e n e . F o r e x a m p l e , t R N A 3 £ h y b r i d i z e s t o r e g i o n s 8 4 D , 92B and 90BC on t h e r i g h t arm o f c h r o m o s o m e 3 ( 3 R ) . B a s e d on t h e number o f g r a i n s o v e r t h e s e s i t e s , i t was c o n c l u d e d t h a t t h e y , c o n t a i n e d 5 , 4 a n d 1 g e n e s ( o r m u l t i p l e s t h e r e o f ) , r e s p e c t i v e l y ( 2 8 ) . The c h r o m o s o m a l s i t e s f o r t h e t h r e e m a j o r v a l i n e i s o a c c e p t o r s a r e p r e s e n t e d i n T a b l e 1 ( 2 9 ) . T a b l e I , S i t e s o f D r o s o p h i l a m e l a n o g a s t e r t R N A V a l g e n e s on t h e p o l y t e n e c h r o m o s o m e s I s o a c c e p t o r M a j o r s i t e L o c a t i o n M i n o r s i t e * L o c a t i o n V a l 3a 64D 3L - -V a l 3b 8 4 D , 92B 3R 90BC 3R V a l 4 56D 2R 8 9 B , 90BC 3R 70BC 3L * A b o u t 1/5 t h e s i l v e r g r a i n s a r e f o u n d o v e r a m i n o r s i t e a s a r e f o u n d o v e r a m a j o r s i t e o f h y b r i d i z a t i o n . 2 . A n a l y s i s o f tRNA gene c l u s t e r s - f i n e s t r u c t u r e o f tRNA g e n e  o r g a n i z a t i o n The d e v e l o p m e n t o f t e c h n i q u e s s u c h a s gene c l o n i n g , r e s t r i c t i o n m a p p i n g and DNA s e q u e n c e a n a l y s i s h a s made t h e s t u d y o f t h e f i n e s t r u c t u r e o f tRNA g e n e o r g a n i z a t i o n p o s s i b l e . The 42A r e g i o n o n c h r o m o s o m e s 2 o f D r o s o p h i l a c o n t a i n s a m a j o r c l u s t e r o f tRNA g e n e s ( 3 0 , 3 1 ) . D a v i d s o n and h i s c o l l e a g u e s c l o n e d o v e r l a p p i n g f r a g m e n t s o f D r o s o p h i l a DNA t h a t s p a n 94 kb f r o m t h i s r e g i o n ( 3 0 , 3 1 ) . They showed t h a t a r e g i o n o f 46 kb c o n t a i n e d 18 tRNA g e n e s : 8 f o r t R N A ^ s n , 4 f o r tRNA 2 » 5 f o r t R N A 2 y , and a s i n g l e tRNA g e n e . T h e s e g e n e s a r e 8 , i r r e g u l a r l y s p a c e d and a r e n o t a l l t r a n s c r i b e d f r o m t h e same DNA s t r a n d . S e v e r a l c l u s t e r s o f g e n e s o c c u r . T h e s e c l u s t e r s may c o n t a i n g e n e s f o r t h e same tRNA o r f o r s e v e r a l t R N A s . T r a n s f e r RNA g e n e c l u s t e r s l o c a t e d a t o t h e r c h r o m o s o m a l s i t e s h a v e b e e n i s o l a t e d and c h a r a c t e r i z e d ( 3 2 - 3 6 ) . The o r g a n i z a t i o n o f t h e s e c l u s t e r s i s s i m i l a r t o t h a t s e e n a t r e g i o n 4 2 A . The i n s i t u h y b r i d i z a t i o n s t u d i e s and t h e a n a l y s e s o f t h e tRNA gene c l u s t e r s i n d i c a t e t h a t t h e o r g a n i z a t i o n o f t h e t R N A g e n e s i n D r o s o p h i l a f o l l o w s no f i x e d p a t t e r n and t h a t t h e tRNA g e n e s a r e a r r a n g e d i n d i s p e r s e d c l u s t e r s . T h i s a r r a n g e m e n t i s i n t e r m e d i a t e b e t w e e n t h a t s e e n i n X e n o p u s ( 6 , 1 7 , 19) and y e a s t ( 6 , 1 7 , 1 8 ) . I d e n t i c a l tRNA g e n e s a p p e a r t o be t i g h t l y c l u s t e r e d i n X e n o p u s . On t h e o t h e r h a n d , i n y e a s t , t h e t R N A g e n e s a r e n o t s i g n i f i c a n t l y c l u s t e r e d . D. S t r u c t u r e o f tRNA g e n e s E l e v e n o u t o f 18 tRNA g e n e s f o u n d a t t h e 42A l o c u s h a v e b e e n s e q u e n c e d . The DNA s e q u e n c e s o f many o f t h e tRNA g e n e s f o u n d a t t h e o t h e r c h r o m o s o m a l s i t e s a r e a l s o k n o w n . T h e s e g e n e s s h a r e t h e f o l l o w i n g f e a t u r e s : i ) None c o d e s f o r t h e - C C A s e q u e n c e f o u n d a t t h e 3 ' - e n d s o f a l l t R N A s . i i ) The 3 ' - e n d s o f a l l t h e g e n e s a r e f o l l o w e d by r u n s o f T r e s i d u e s i n t h e n o n c o d i n g s t r a n d s . Leii i i i ) E x c e p t f o r t h e two tRNA g e n e s f r o m t h e 50AB s i t e ( 3 4 ) no o t h e r D r o s o p h i l a tRNA g e n e s e q u e n c e d t o d a t e c o n t a i n s an i n t e r v e n i n g s e q u e n c e . In Leu f a c t t h e g e n e s f o r tRNA a r e t h e f i r s t i n D r o s o p h i l a f o u n d t o c o n t a i n s u c h i n s e r t s . S e v e r a l tRNA g e n e s f r o m o t h e r e u k a r y o t e s a l s o c o n t a i n i n t e r v e n i n g s e q u e n c e s ( 6 , 3 7 ) . i v ) The 5 ' ^ f l a n k i n g s e q u e n c e s o f t h e v a r i o u s tRNA g e n e s do n o t show a n y s i m i l a r i t i e s . H o w e v e r , some g e n e s c o d i n g f o r t h e same tRNA e . g . t R N A 2 Y S ( 3 0 , 3 1 , 3 8 ) , t R N A A r g ( 3 1 ) , t R N A I l e a n d t R N A L e u ( 3 4 ) h a v e r e g i o n s o f h o m o l o g y i n t h e i r 5 ' - f l a n k i n g s e q u e n c e s . The s i g n i f i c a n c e o f many o f t h e s e c o n s e r v e d s e q u e n c e s i s n o t k n o w n . 9 . A r e r e d u n d a n t tRNA g e n e s i d e n t i c a l ? - In most c a s e s w h e r e t h e DNA s e q u e n c e s a r e k n o w n , t h i s a p p e a r s t o be t r u e . The r e d u n d a n t g e n e s f o r A s n L v s Ajtrcj ' tRNA , t R N A 2 and t R N A 2 f r o m r e g i o n 42A a r e i d e n t i c a l ( 3 0 , 3 1 ) . A Lys t R N A 2 g e n e i s o l a t e d f r o m a n o t h e r s i t e , 42E i s i d e n t i c a l t o t h o s e f r o m 42A l i e r e g i o n ( 3 2 , 3 8 ) . F i v e g e n e s f o r tRNA f r o m r e g i o n 50AB ( 3 4 ) a r e a l l l i e Leu i d e n t i c a l t o t h e s i n g l e tRNA 1 g e n e f r o m 42A ( 3 0 ) . The t w o tRNA " s p l i t " Leu g e n e s f r o m 50 AB r e g i o n e n c o d e t h e same m a t u r e tRNA , t h o u g h t h e i n t e r -v e n i n g s e q u e n c e s d i f f e r ( 3 4 ) . H o w e v e r , n o t a l l g e n e s c o d i n g f o r a s i n g l e tRNA h a v e i d e n t i c a l s e q u e n c e s . H e t e r o g e n e i t y i n c l o s e l y r e l a t e d tRNA g e n e s e q u e n c e s was f o u n d i n a c l o n e d c l u s t e r o f t R N A G 1 U g e n e s . F o u r o u t o f f i v e t R N A ^ ^ g e n e s a r e i d e n t i c a l b u t one c o n t a i n s a C t o T t r a n s i t i o n a t t h e f o u r t h n u c l e o t i d e o f Met t h e m a t u r e tRNA s e q u e n c e ( 3 3 ) . Genes f o r tRNA w i t h s e q u e n c e s m a t c h i n g a n d d i f f e r i n g f r o m t h e known tRNA s e q u e n c e a r e p r e s e n t a t r e g i o n 61D ( 3 6 ) . Val N o n - i d e n t i c a l s e q u e n c e s i s o l a t e d by h y b r i d i z a t i o n t o tRNA 4 ( 3 9 , p r e s e n t Lys Ser, w o r k ) , tRNAs ( 4 0 ) and tRNA 7 ( 4 1 ) h a v e b e e n f o u n d r e c e n t l y . T h e s e d i f f e r -i n g g e n e s d e r i v e f r o m d i s t i n c t c h r o m o s o m a l s i t e s . A t p r e s e n t , i t i s n o t known w h e t h e r s u c h v a r i a n t g e n e s w h i c h d i f f e r f r o m t h e i s o l a t e d tRNA s p e c i e s a r e t r a n s c r i b e d _i_n v i v o . I I I . T r a n s c r i p t i o n o f RNA p o l y m e r a s e I I I g e n e s A . E u k a r y o t i c RNA p o l y m e r a s e s In p r o k a r y o t e s a l l g e n e s a r e t r a n s c r i b e d by a s i n g l e RNA p o l y m e r a s e ( 4 2 ) . In e u k a r y o t e s , h o w e v e r , t h e r e a r e a t l e a s t t h r e e RNA p o l y m e r a s e s w h i c h d i f f e r i n t h e i r s t r u c t u r e , l o c a l i z a t i o n and f u n c t i o n ( r e v i e w e d i n r e f . 4 3 , 4 4 ) . P o l y m e r a s e I , l o c a l i z e d i n t h e n u c l e o l u s , t r a n s c r i b e s 1 8 S , 28S a n d 5 . 8 S r R N A ; p o l y m e r a s e I I f o u n d i n t h e n u c l e u s t r a n s c r i b e s h e t e r o -g e n e o u s n u c l e a r RNA (mRNA p r e c u r s o r s ) and p o l y m e r a s e I I I , a l s o p r e s e n t i n t h e n u c l e u s , t r a n s c r i b e s a v a r i e t y o f l o w m o l e c u l a r w e i g h t RNAs i n c l u d i n g 10, 5S r R N A , t R N A s , a d e n o v i r u s v i r u s - a s s o c i a t e d (VA) RNA, some s m a l l n u c l e a r RNAs and t h e A l u f a m i l y o f DNA s e q u e n c e s ( 4 5 , 4 6 ) . A l l t h r e e p o l y m e r a s e s a r e o f h i g h m o l e c u l a r w e i g h t and a r e c o m p o s e d o f f r o m 1 0 - 1 5 d i s t i n c t s u b u n i t s . M u l t i p l e f o r m s o f t h e same enzyme may e x i s t due t o s l i g h t v a r i a t i o n s i n t h e s u b u n i t c o n t e n t . The t h r e e m a j o r p o l y m e r a s e s h a v e c h a r a c t e r i s t i c c a t a l y t i c and c h r o m a t o g r a p h i c p r o p e r t i e s a n d t h e y a r e a l s o d i s t i n g u i s h e d by t h e i r u n i q u e s e n s i t i v i t i e s t o t h e f u n g a l t o x i n , a - a m a n i t i n . The c l a s s I a n d I I e n z y m e s h a v e b e e n r e a d i l y i s o l a t e d and i d e n t i f i e d f r o m many d i f f e r e n t s o u r c e s and t h e i r s t r u c t u r e s a r e w e l l i d e n t i f i e d ( r e v i e w e d i n r e f . 4 3 ) . T h e s e s t u d i e s s u g g e s t t h a t t h e m a i n s t r u c t u r a l f e a t u r e s o f e n z y m e s w i t h i n a g i v e n c l a s s a r e s i m i l a r i n d i f f e r e n t o r g a n i s m s . S t r u c t u r a l a n a l y s i s o f t h e c l a s s I I I p o l y m e r a s e s l a g g e d b e h i n d t h e o t h e r c l a s s e s due t o t h e l o w p o l y m e r a s e I I I c o n t e n t o f m o s t c e l l t y p e s . B u t , w i t h more c a r e f u l i s o l a t i o n p r o c e d u r e s , RNA p o l y m e r a s e I I I f r o m two l o w e r e u k a r y o t e s , y e a s t ( 4 7 , 4 8 ) and amoeba ( 4 9 ) and f r o m s e v e r a l h i g h e r e u k a r y o t e s ( 5 0 ) i n c l u d i n g D r o s o p h i l a ( 5 1 - 5 3 ) h a s b e e n i s o l a t e d and c h a r a c t e r i z e d . RNA p o l y m e r a s e I I I f r o m a l l t h e s e s o u r c e s shows a v e r y s i m i l a r s u b u n i t c o m p o s i t i o n d i s t i n c t f r o m t h a t o f c l a s s I and I I e n z y m e s . H o w e v e r , s i g n i f i c a n t d i f f e r e n c e s h a v e b e e n n o t e d b e t w e e n t h e a - a m a n i t i n s e n s i t i v i t i e s o f t h e c l a s s I I I e n z y m e s f r o m y e a s t , i n s e c t s and a n i m a l s ( 4 4 ) . I n g e n e r a l , t h e a n i m a l e n z y m e s a r e m o d e r a t e l y s e n s i t i v e t o a - a m a n i t i n (50% i n h i b i t i o n a t 2 0 - 3 0 / u g / i n l ) , w h i l e t h e y e a s t enzyme i s l e s s s e n s i t i v e (50% i n h i b i t i o n a t 3 0 0 - 5 0 0 / u g / m l ) and t h e i n s e c t enzyme i s more r e s i s t a n t t o t h e t o x i n . RNA p o l y m e r a s e I I I f r o m t h e two i n s e c t s o u r c e s , Bombyx m o r i ( 5 0 ) and D r o s o p h i l a ( 5 1 ) l a c k an a n a l o g o u s p o l y p e p t i d e s u b u n i t p r e s e n t i n t h e a n i m a l e n z y m e s ( 5 0 ) . W h e t h e r t h e a b s e n c e o f t h i s p o l y p e p t i d e i s r e l a t e d t o t h e a l t e r e d a - a m a n i t i n s e n s i t i v i t y o f i n s e c t c l a s s I I I r e m a i n s t o be d e t e r m i n e d . S i n c e t h e r e a r e t h r e e c l a s s e s o f RNA p o l y m e r a s e t r a n s c r i b i n g t h r e e d i f f e r e n t s e t s o f g e n e s , i t i s p r e s u m e d t h a t e a c h c l a s s o f enzyme r e c o g n i z e s s p e c i f i c n u c l e o t i d e s e q u e n c e t h a t i s common o n l y t o t h e g e n e s w h i c h i t t r a n s c r i b e s . T h i s a s s u m p t i o n i s p r o v i n g t o be t r u e . A l t h o u g h i d e n t i f i c a t i o n o f a p r o m o t e r o f RNA p o l y m e r a s e I t r a n s c r i p t i o n i s y e t t o be a c c o m p l i s h e d , p r e l i m i n a r y r e s u l t s i n d i c a t e t h a t f o r X e n o p u s 18S a n d 28S rRNA g e n e s , t h e s e q u e n c e d e f i n e d by n u c l e o t i d e s - 1 2 t o +16 i s e s s e n t i a l f o r t r a n s c r i p t i o n i n i t i a t i o n ( c i t e d i n r e f . 5 4 ) . The d e t e r m i n a n t s f o r RNA p o l y m e r a s e I t e r m i n a t i o n a l s o a p p e a r t o be d i f f e r e n t f r o m t h o s e s p e c i f y i n g t e r m i n a t i o n f o r o t h e r p o l y m e r a s e s ( 5 5 ) . Genes t r a n s c r i b e d by p o l y m e r a s e I I h a v e b e e n c h a r a c t e r i z e d a s h a v i n g two c o n t r o l r e g i o n s u p s t r e a m o f t h e s i t e a t w h i c h t r a n s c r i p t i o n i s i n i t i a t e d ( 5 6 ) . And y e t a n o t h e r a r r a n g m e n t a p p e a r s t o be t h e c a s e f o r g e n e s t r a n s c r i b e d by p o l y m e r a s e I I I ( t o be d e s c r i b e d b e l o w ) . F r a c t i o n a t i o n s t u d i e s a l s o i n d i c a t e t h a t t h e t r a n s c r i p t i o n f a c t o r s r e q u i r e d f o r a c c u r a t e t r a n s c r i p t i o n o f t h e t h r e e c l a s s e s o f g e n e s a r e d i f f e r e n t ( 5 7 - 6 0 ) . B . RNA p o l y m e r a s e I I I t r a n s c r i p t i o n s y s t e m s E a r l y s t u d i e s showed t h a t when p u r e RNA p o l y m e r a s e I I I f r o m X e n o p u s  l a e v i s o o c y t e s was i n c u b a t e d w i t h p u r i f i e d 5S DNA t e m p l a t e , n o n s p e c i f i c t r a n s c r i p t i o n e n s u e d ( 6 1 ) . H o w e v e r , i n t a c t c h r o m a t i n c o n t a i n i n g 5S RNA g e n e s y i e l d e d s p e c i f i c t r a n s c r i p t i o n ( 6 1 - 6 3 ) . S i m i l a r c o n c l u s i o n s w e r e r e a c h e d f r o m a n a l o g o u s s t u d i e s on mouse 5S RNA and tRNA ( 6 4 ) an VA RNA ( 6 5 ) g e n e s . T h e s e s t u d i e s t h e r e f o r e i n d i c a t e d t h a t RNA p o l y m e r a s e I I I , DNA t e m p l a t e and c h r o m a t i n - a s s o c i a t e d c o m p o n e n t s w e r e n e c e s s a r y f o r a c c u r a t e t r a n s c r i p t i o n o f t h e s e g e n e s . I n d e e d , more r e c e n t f r a c t i o n a t i o n and r e c o n s t i t u t i o n e x p e r i m e n t s show t h a t m u l t i p l e f a c t o r s i n a d d i t i o n t o RNA p o l y m e r a s e I I I a r e r e q u i r e d f o r t r a n s c r i p t i o n o f 5S RNA, tRNA and VA RNA g e n e s ( 5 9 , 6 0 ) . In o r d e r t o i d e n t i f y t h e c o m p o n e n t s e s s e n t i a l f o r g e n e e x p r e s s i o n , t r a n s c r i p t i o n s y s t e m s i n w h i c h p u r i f i e d DNA t e m p l a t e s c a n be s e l e c t i v e l y t r a n s c r i b e d h a v e b e e n d e v e l o p e d . A c c u r a t e t r a n s c r i p t i o n h a s b e e n shown t o o c c u r when 5S RNA ( 6 6 , 67) and tRNA g e n e s f r o m many d i f f e r e n t s o u r c e s ( 6 8 - 7 1 ) a r e m i c r o i n j e c t e d i n t o X e n o p u s o o c y t e n u c l e i . P u r i f i e d 5S RNA ( 7 2 ) and tRNA g e n e s f r o m D r o s o p h i l a ( 3 8 , 7 3 , 7 4 ) , y e a s t ( 7 5 ) , Bombyx ( 7 6 , 77) and X e n o p u s ( 7 8 ) a r e a l s o f a i t h f u l l y t r a n s c r i b e d i n e x t r a c t s made f r o m X e n o p u s o o c y t e n u c l e i . A t p r e s e n t , RNA p o l y m e r a s e I I I c e l l - f r e e t r a n s c r i p t i o n s y s t e m s h a v e b e e n d e v e l o p e d f r o m X e n o p u s o o c y t e s ( 7 2 , 7 9 ) , X e n o p u s k i d n e y c e l l s ( 8 0 ) , human KB c e l l s ( 8 0 , 81) mouse p l a s m a c y t o m a c e l l s ( 8 0 ) , H e l a c e l l s ( 8 2 ) , Bombyx m o r i p o s t e r i o r s i l k g l a n d s and o v a r i e s ( 8 3 ) , D r o s o p h i 1 a Kc c e l l s ( 8 4 ) and w h o l e y e a s t c e l l s ( 8 5 ) . T h e s e e x t r a c t s a c c u r a t e l y t r a n s c r i b e 5S RNA ( 7 9 , 8 0 , 8 4 , 8 5 ) , tRNA ( 3 4 , 8 3 - 8 5 ) and VA RNA ( 8 0 - 8 2 , 8 4 ) g e n e s a s w e l l as t h e A l u f a m i l y o f DNA s e q u e n c e s ( 4 5 , 4 6 ) . C o m p a r i s o n o f t r a n s c r i p t i o n i n h o m o l o g o u s and h e t e r o l o g o u s s y s t e m s shows t h a t RNA p o l y m e r s e I I I g e n e s f r o m d i f f e r e n t s o u r c e s a r e t r a n s c r i b e d a c c u r a t e l y i n b o t h t h e s e s y s t e m s . H o w e v e r , some s t u d i e s i n d i c a t e t h e f o l l o w i n g : i ) e x t r a c t s d e r i v e d f r o m v a r i o u s c e l l t y p e s d i f f e r i n t h e i r a b i l i t y t o d i s c r i m i n a t e b e t w e e n d i f f e r e n t g e n e s ( 8 0 ) ; i i ) c e r t a i n g e n e s a r e t r a n s c r i b e d more e f f i c i e n t l y i n a h o m o l o g o u s s y s t e m t h a n i n a h e t e r o l o g o u s o n e ( 8 4 ) ; i i i ) t h e r e may be d i f f e r e n c e s b e t w e e n t h e h e t e r o l o g o u s and h o m o l o g o u s s y s t e m s w i t h r e g a r d t o t h e DNA s e q u e n c e s r e q u i r e d f o r t r a n s c r i p t i o n o f a tRNA g e n e ( 8 3 , 8 6 ) . F o r t h i s r e a s o n , r e s u l t s o b t a i n e d w i t h h e t e r o l o g o u s s y s t e m s s h o u l d be i n t e r p r e t e d w i t h c a u t i o n . C . T r a n s c r i p t i o n c o n t r o l r e g i o n s f o r p o l y m e r a s e I I I g e n e s By a n a l o g y w i t h b a c t e r i a l p r o m o t e r s , i t was e x p e c t e d t h a t i n s p e c t i o n o f t h e s e q u e n c e s a d j a c e n t t o t h e 5 ' - e n d s o f RNA p o l y m e r a s e I I I g e n e s w o u l d r e v e a l a c o n s e r v e d s e q u e n c e a t w h i c h t r a n s c r i p t i o n was i n i t i a t e d . E x a m i n a t i o n 13. o f s e q u e n c e s p r e c e d i n g s e v e r a l e u k a r y o t i c g e n e s t r a n s c r i b e d by RNA p o l y m e r a s e I I I r e v e a l e d a f e w common f e a t u r e s ( 8 7 ) . In o r d e r t o d e t e r m i n e t h e DNA s e q u e n c e s e s s e n t i a l f o r 5S RNA t r a n s c r i p t i o n i n i t i a t i o n , B r o w n and h i s c o l l e a g u e s ( 8 8 , 8 9 ) c o n s t r u c t e d a s e r i e s o f d e l e t i o n m u t a n t s by s y s t e m a t i c a l l y d e l e t i n g a f r a g m e n t o f X e n o p u s b o r e a l i s s o m a t i c 5S DNA f r o m t h e 5 ' and t h e 3 ' s i d e s o f t h e g e n e . T r a n s c r i p t i o n o f t h e s e s e t o f m u t a n t s g a v e a s u r p r i s i n g r e s u l t . The minimum s e q u e n c e r e q u i r e d t o d i r e c t t r a n s c r i p t i o n i n i t i a t i o n o f 5S DNA l a y i n t h e c e n t r e o f t h e g e n e , b e t w e e n n u c l e o t i d e s 50 and 83 o f t h e c o d i n g r e g i o n . The c o n s e r v e d r e g i o n s p r e c e d i n g t h e s e g e n e s ( m e n t i o n e d a b o v e ) a p p a r e n t l y a r e n o t e s s e n t i a l f o r 5S RNA s y n t h e s i s ; h o w e v e r , t h e y c o u l d i n f l u e n c e t h e e x a c t s i t e o f i n i t i a t i o n . The l e n g t h o f t h e t r a n s c r i p t o b t a i n e d f o r e a c h d e l e t i o n m u t a n t w i t h i n t a c t i n t e r n a l c o n t r o l r e g i o n and f o r " m a x i g e n e s " ( w i t h e x t r a n u c l e o t i d e s i n s e r t e d b e t w e e n t h e c o n t r o l r e g i o n and t h e n o r m a l s i t e o f i n i t i a t i o n ) was a p p r o x i m a t e l y t h e s a m e , i n d i c a t i n g t h a t i n i t i a t i o n o c c u r r e d a f i x e d d i s t a n c e u p s t r e a m f r o m t h e c o n t r o l r e g i o n . D e l e t i o n m u t a n t s w i t h 5 ' - f l a n k i n g r e g i o n s h o r t e r t h a n 26 n u c l e o t i d e s w e r e t r a n s c r i b e d w i t h d e c r e a s e d e f f i c i e n c i e s , s u g g e s t i n g t h a t t h e r a t e o f t r a n s c r i p t i o n i n i t i a t i o n was i n f l u e n c e d by s e q u e n c e s a t o r u p s t r e a m f r o m t h e s t a r t s i t e . To s e a r c h f o r o t h e r c o m p o n e n t s w h i c h m i g h t d i r e c t a c c u r a t e i n i t i a t i o n o f 5S DNA, o o c y t e h o m o g e n a t e s h a v e b e e n f r a c t i o n a t e d . In a d d i t i o n t o RNA p o l y m e r a s e I I I , a t l e a s t t w o p r o t e i n f a c t o r s a r e r e q u i r e d ( 9 0 ) one o f w h i c h , d e s i g n a t e d T F I I I A , h a s b e e n p u r i f i e d ( 9 1 ) . When T F I I I A i s m i x e d w i t h 5S DNA, i t b i n d s t o a r e g i o n i n t h e c e n t r e ( n u c l e o t i d e s 4 5 - 9 6 ) o f b o t h o o c y t e and s o m a t i c v a r i a n t s o f 5S RNA g e n e s ( 9 1 ) . T h i s r e g i o n i n c l u d e s t h a t r e q u i r e d f o r i n i t i a t i o n o f t r a n s c r i p t i o n ( n u c l e o t i d e s 5 0 - 8 3 ) . F u r t h e r m o r e , t h e a b i l i t y o f d e l e t e d f r a g m e n t s o f 5S DNA t o b i n d T F I I I A c o r r e l a t e s w i t h t h e i r a b i l i t y t o t r a n s c r i b e 5S RNA i n v i t r o ( 9 2 ) . T h e s e r e s u l t s s u g g e s t t h a t t h e i n f l u e n c e o f t h e c e n t r a l r e g i o n on t r a n s c r i p t i o n i n i t i a t i o n i s m e d i a t e d by T F I I I A . T F I I I A i s n o t r e q u i r e d f o r t h e e x p r e s s i o n o f o t h e r g e n e s t r a n s c r i b e d by RNA p o l y m e r a s e I I I ( 9 1 ) . The j_n v i t r o t r a n s c r i p t i o n e x t r a c t s s u p p o r t 5S RNA s y n t h e s i s f r o m b o t h o o c y t e o r s o m a t i c t y p e 5S RNA g e n e s . T h u s , a l t h o u g h t h e y e f f i c i e n t l y a n d f a i t h f u l l y t r a n s c r i b e 5S DNA, t h e j_n v i t r o s y s t e m s do n o t show a n y d e v e l o p m e n t a l c o n t r o l . R e c e n t s t u d i e s ( 9 3 - 9 5 ) i n d i c a t e t h a t 5S RNA g e n e s a s p a r t o f i n t a c t c h r o m o s o m e s m a i n t a i n t h e i r d e v e l o p m e n t a l l y r e g u l a t e d s t a t e j_n v i t r o . Do o t h e r g e n e s t r a n s c r i b e d b y RNA p o l y m e r a s e I I I a l s o h a v e i n t e r n a l c o n t r o l r e g i o n s ? E x p e r i m e n t s a n a l o g u e s t o t h o s e w i t h X e n o p u s 5S RNA g e n e s show t h a t an i n t r a g e n i c r e g i o n l o c a t e d b e t w e e n p o s i t i o n s +9 and +72 i s e s s e n t i a l f o r t r a n s c r i p t i o n o f a d e n o v i r u s VA RNA gene ( 8 2 ) . T h e r e i s a l s o e v i d e n c e s u p p o r t i n g a n i n t e r n a l c o n t r o l r e g i o n f o r tRNA g e n e s ( b e l o w ) . S e q u e n c e h o m o l o g i e s h a v e b e e n r e p o r t e d b e t w e e n t h e 5S RNA gene c o n t r o l r e g i o n a n d a n a l o g o u s i n t e r n a l r e g i o n s o f t h e VA RNA and t h e tRNA g e n e s ( 8 2 , 8 6 , 8 9 , 9 6 , 9 7 ) . H o w e v e r , t h e r e i s l e s s h o m o l o g y b e t w e e n 5S RNA and tRNA g e n e s t h a n b e t w e e n VA RNA and tRNA g e n e s . T h i s o b s e r v a t i o n i s f u r t h e r s u b s t a n t i a t e d by f r a c t i o n a t i o n s t u d i e s w h i c h i n d i c a t e t h a t common f a c t o r s a r e r e q u i r e d f o r t r a n s c r i p t i o n o f VA RNA and tRNA g e n e s and t h a t an a d d i t i o n a l f a c t o r i s r e q u i r e d f o r t r a n s c r i p t i o n o f 5S RNA g e n e s ( 5 9 , 6 0 , 9 1 ) . T h e r e f o r e , RNA p o l y m e r a s e I I I may u s e v a r i o u s b i n d i n g f a c t o r s s p e c i f i c f o r d i f f e r e n t c o n t r o l s e q u e n c e s w i t h i n tRNA g e n e s c o m p a r e d t o 5S RNA g e n e s . D. B i o s y n t h e s i s o f e u k a r y o t i c tRNA 1 . tRNA t r a n s c r i p t i o n u n i t DNA s e q u e n c e i n f o r m a t i o n o b t a i n e d s o f a r ( s e e S e c t i o n I I . D ) i n d i c a t e s t h a t i n m o s t c a s e s e u k a r y o t i c tRNA g e n e s a r e o r g a n i z e d as s i n g l e t r a n s c r i p t i o n u n i t s . T h i s i s u n l i k e t h e c a s e i n p r o k a r y o t e s , w h e r e m u l t i m e r i c t r a n s c r i p t i o n u n i t s a r e q u i t e common ( 6 ) . E x c e p t i o n s t o t h e r u l e i n e u k a r y o t e s h a v e b e e n r e p o r t e d . In two d i f f e r e n t y e a s t s p e c i e s , S . pombe ( 9 8 ) and S . c e r e v i s i a e ( 9 9 ) , two tRNA c o d i n g r e g i o n s w e r e f o u n d t o be t i g h t l y l i n k e d , s e p a r a t e d by 7 and 10 n u c l e o t i d e s r e s p e c t i v e l y . T h e s e t R N A s w e r e t r a n s c r i b e d a s d i m e r i c p r e c u r s o r s . As was n o t e d e a r l i e r ( S e c t i o n I I I . B ) , tRNA g e n e s f r o m v a r i e d s o u r c e s a r e t r a n s c r i b e d f a i t h f u l l y i n a number o f d i f f e r e n t c e l l - f r e e t r a n s c r i p t i o n s y s t e m s . T h e s e s y s t e m s h a v e made i t p o s s i b l e t o i s o l a t e tRNA p r e c u r s o r s f o r f u r t h e r c h a r a c t e r i z a t i o n . To d a t e , t h e t r a n s c r i p t i o n i n i t i a t i o n and t e r m i n a t i o n s i t e s i n s e v e r a l tRNA g e n e s h a v e b e e n i d e n t i f i e d ( r e v i e w e d i n r e f . 3 7 ) . T h e s e s t u d i e s h a v e shown t h a t t r a n s c r i p t i o n i n i t i a t i o n i n tRNA g e n e s o c c u r s 3 t o 19 n u c l e o t i d e s b e f o r e t h e 5 ' - e n d o f t h e m a t u r e t R N A - c o d i n g r e g i o n . The p r i m a r y t r a n s c r i p t s a n a l y z e d c o n t a i n an u n c a p p e d p u r i n e n u c l e o s i d e t r i p h o s p h a t e a s t h e i n i t a l b a s e , w h i c h i s f o l l o w e d by a p y r i m i d i n e . The t r a n s c r i p t s t e r m i n a t e w i t h a u r i d y l a t e s t r e t c h s e v e r a l n u c l e o t i d e s b e y o n d t h e 3 ' - e n d o f t h e tRNA c o d i n g r e g i o n . S t u d i e s w i t h X e n o p u s 5S DNA i n d i c a t e t h a t t e r m i n a t i o n o f 5S RNA s y n t h e s i s r e q u i r e a c l u s t e r o f f o u r o r more T r e s i d u e s i n t h e n o n c o d i n g s t r a n d o f t h e DNA and i s e n h a n c e d by an a d j a c e n t G+C r i c h r e g i o n ( 8 7 , 1 0 0 ) . M o r e v e r , two y e a s t t R N A T y r m u t a t i o n s w h i c h i n c r e a s e a s t r i n g o f t h r e e c o n s e c u t i v e T r e s i d u e s i n t h e m i d d l e o f t h e g e n e t o f i v e o r s i x T r e s i d u e s c a u s e p r e m a t u r e t e r m i n a t i o n w i t h i n t h e T c l u s t e r ( 9 6 ) . P r o k a r y o t i c t e r m i n a t i o n s e q u e n c e s i n c l u d e T c l u s t e r s , G + C - r i c h r e g i o n s and a l s o d y a d s y m m e t r i e s ( 8 7 , 1 0 1 ) . 2 . tRNA t r a n s c r i p t i o n c o n t r o l r e g i o n s S e v e r a l l i n e s o f e v i d e n c e i n d i c a t e d t h a t t h e tRNA gene t r a n s c r i p t i o n was d i r e c t e d by c o n t r o l r e g i o n ( s ) w i t h i n t h e g e n e . The f i r s t i n d i c a t i o n was t h e o b s e r v a t i o n t h a t t h e 5 ' - f l a n k i n g s e q u e n c e s o f t h e tRNA g e n e s w e r e 1 6 . u s u a l l y n o t c o n s e r v e d ( 3 7 , 9 8 ) . M o r e i m p o r t a n t l y , s t e p w i s e r e m o v a l o f t h e 5 ' - f l a n k i n g s e q u e n c e s l e a v i n g o n l y 15 ( 1 0 2 ) o r 6 bp ( 7 6 ) o r f i n a l l y none o f t h e 5 ' - s e q u e n c e s o u t s i d e t h e t r a n s c r i p t i o n u n i t ( 3 8 ) d i d n o t a b o l i s h t h e t r a n s c r i p t i o n a l a c t i v i t y o f t h e t r u n c a t e d DNA j_n v i v o o r j_n v i t r o . In c o n t r a s t , a f r a g m e n t c o n t a i n i n g t h e e n t i r e 5 ' - f l a n k i n g r e g i o n and t h e f i r s t Met 30 n u c l e o t i d e s o f t h e X e n o p u s t R N A g e n e , b u t l a c k i n g t h e r e s t o f t h e g e n e , c o u l d n o t s u p p o r t tRNA s y n t h e s i s ( 7 8 ) . A s i n g l e b a s e c h a n g e a t p o s i t i o n T y r 56 w i t h i n t h e s t r u c t u r a l g e n e o f t h e y e a s t SUP4 tRNA J g e n e a l s o p r e v e n t e d i n i t i a t i o n o f t r a n s c r i p t i o n ( 9 6 ) . I n v i t r o d e l e t i o n o f c l o n e d tRNA g e n e s , an a p p r o a c h o r i g i n a l l y u s e d by B r o w n ' s g r o u p o n 5S RNA g e n e s ( 8 8 , 8 9 ) h a s now shown t h a t t h e tRNA g e n e s a l s o c o n t a i n i n t r a g e n i c p r o m o t e r s ( 8 6 , 9 7 , 1 0 3 , 1 0 4 ) . B u t u n l i k e t h e 5S DNA c o n t r o l r e g i o n , t h e g e n e - i n t e r n a l tDNA p r o m o t e r i s d i s c o n t i n u o u s ; t h e t w o s p l i t r e g i o n s d e s i g n a t e d t h e A a n d t h e B b l o c k s ( 9 7 ) h a v e t h e a p p r o x i m a t e c o o r d i n a t e s 8 - 1 9 and 5 2 - 6 2 , r e s p e c t i v e l y . Two l i n e s o f e v i d e n c e s u p p o r t t h e n o t i o n o f d i s c o n t i n u o u s o r s p l i t i n t r a g e n i c p r o m o t e r s : c h i m e r i c tRNA g e n e s c o n t a i n i n g t h e 5 ' - h a l f o f o n e g e n e and t h e 3 ' - h a l f o f a n o t h e r c a n be t r a n s c r i b e d w e l l ( 9 7 , 1 0 5 ) ; and t r a n s c r i p t i o n c a n a l s o o c c u r a f t e r t h e r e p l a c e m e n t o f t h e c e n t r a l r e g i o n o f t R N A g e n e s w i t h DNA o f v e r y d i f f e r e n t s e q u e n c e s ( 9 7 , 1 0 3 , 1 0 5 ) . The c e n t r a l r e g i o n s a p p e a r t o h a v e a s p a c i n g f u n c t i o n b e c a u s e t h e e f f i c i e n c y o f t r a n s c r i p t i o n d e p e n d s on t h e l e n g t h o f t h e r e p l a c e d DNA; t h e o p t i m a l d i s t a n c e b e t w e e n t h e A a n d B b l o c k s b e i n g 3 0 - 4 0 bp ( 9 7 , 1 0 3 , 1 0 5 ) . I n n a t u r a l tRNA g e n e s , t h i s d i s t a n c e c a n v a r y , t h e v a r i a b i l i t y b e i n g due t o t h e l e n g t h o f t h e v a r i a b l e arm and t h e p r e s e n c e w i t h i n c e r t a i n tRNA g e n e s o f an i n t e r v e n i n g s e q u e n c e . S t u d i e s by J o h n s o n e t a l _ . ( 1 0 6 ) and W a l l a c e e t a l . ( 1 0 7 ) d e m o n s t r a t e d t h a t a d d i t i o n o r d e l e t i o n o f i n t e r v e n i n g s e q u e n c e ( I V S ) r e g i o n w i t h i n a tRNA g e n e d i d n o t a f f e c t t r a n s c r i p t i o n o f t h e g e n e . 17. The p r e s e n c e o f IVS may i n f l u e n c e t h e e f f i c i e n c y o f t r a n s c r i p t i o n by e x p a n d i n g t h e d i s t a n c e b e t w e e n t h e A a n d t h e B b l o c k s . An i n t r i g u i n g f e a t u r e o f t h e A and B b l o c k s e q u e n c e s i s t h e i r c l o s e c o r r e l a t i o n w i t h t h e m o s t c o n s e r v e d r e g i o n s o f b a c t e r i a l and e u k a r y o t i c t R N A s : t h e i n v a r i a n t n u c l e o t i d e s U 8 , U 1 4 , G18 and G19 a r e e n c o d e d by t h e A b l o c k , w h i l e G 5 3 , T 5 5 , C 5 6 , A58 and C61 a r e f o u n d i n t h e B b l o c k . T h i s o b s e r v a t i o n h a s l e d H a l l e t aj_. ( 1 0 8 ) t o p r o p o s e a t e r t i a r y i n t e r a c t i o n m o d e l i n w h i c h t h e y s u g g e s t t h a t t h e tRNA g e n e s a s s u m e a t R N A - l i k e t e r t i a r y s t r u c t u r e d u r i n g t r a n s c r i p t i o n i n i t i a t i o n . As w i t h 5S RNA g e n e t r a n s c r i p t i o n , t h e d e l e t i o n s t u d i e s i n v o l v i n g tRNA g e n e s i n d i c a t e t h a t i n m o s t c a s e s t h e 5 ' - f l a n k i n g s e q u e n c e s h a v e o n l y a m i n o r e f f e c t on t r a n s c r i p t i o n , m e r e l y s p e c i f y i n g t h e e x a c t p o i n t o f i n i t i a t i o n ( 8 6 , 9 7 , 1 0 3 , 1 0 4 ) . H o w e v e r , o t h e r s t u d i e s show t h a t t h e 5 ' -f l a n k i n g s e q u e n c e s c a n p l a y a much more s i g n i f i c a n t r o l e i n m o d u l a t i n g t h e l e v e l o f tRNA g e n e t r a n s c r i p t i o n . The 5 ' - f l a n k i n g s e q u e n c e s h a v i n g b o t h a n e g a t i v e ( 3 8 , 1 0 9 , 1 1 0 ) and a p o s i t i v e ( 8 3 ) e f f e c t o n tDNA t r a n s c r i p t i o n h a v e b e e n d e s c r i b e d . 3 . P r o c e s s i n g o f tRNA t r a n s c r i p t s The p r i m a r y t r a n s c r i p t s o f tRNA g e n e s i n b o t h p r o k a r y o t e s a n d e u k a r y o t e s c o n t a i n n u c l e o t i d e s e q u e n c e s n o t p r e s e n t i n t h e m a t u r e t R N A s . The p r o c e s s i n g o f t h e s e t r a n s c r i p t s t o p r o d u c e f u n c t i o n a l tRNA m o l e c u l e s i s b r i e f l y d e s c r i b e d b e l o w ( r e v i e w e d i n r e f . 5 ) . The j_n v i vo and j_n v i t r o t r a n s c r i p t i o n s t u d i e s d e s c r i b e d a b o v e i n d i c a t e t h a t t h e X e n o p u s o o c y t e n u c l e i and t h e c e l l - f r e e e x t r a c t s f r o m v a r i o u s s o u r c e s c o n t a i n a l l t h e e n z y m e s n e c e s s a r y f o r a c c u r a t e p r o c e s s i n g o f tRNA p r e c u r s o r s ; t r a n s c r i p t i o n s y s t e m s d e r i v e d f r o m X e n o p u s ( 6 9 , 7 5 , 9 8 ) and Hel a c e l l s ( I I I ) a l s o c o n t a i n e n z y m e s r e q u i r e d f o r t h e r e m o v a l o f t h e i n t e r v e n i n g s e q u e n c e s . E x c e p t f o r t h e s p l i c i n g e n z y m e ( s ) , t h e a c t i v i t i e s 1 8 . i n v o l v e d i n t h e p r o c e s s i n g o f e u k a r y o t i c tRNA p r e c u r s o r s a p p e a r t o be l i k e t h o s e o f p r o k a r y o t e s . I n b a c t e r i a l t R N A b i o s y n t h e s i s , t w o e n d o n u c l e a s e s a r e i m p l i c a t e d : RNase P g e n e r a t e s t h e m a t u r e 5 ' t e r m i n u s and RNase P2 a p p a r e n t l y c l e a v e s b e t w e e n m a t u r e tRNA r e g i o n s o f p o l y c i s t r o n i c p r e c u r s o r s . A 3 ' t o 5 ' e x o n u c l e a s e ( s ) i s a l s o n e e d e d t o t r i m o f f e x t r a 3 ' n u c l e o t i d e s . RNase P - l i k e a c t i v i t y and an e x o n u c l e a s e c a p a b l e o f p r o c e s s i n g t h e 3 ' - e n d s o f tRNA p r e c u r s o r s h a v e b e e n i s o l a t e d f r o m e u k a r y o t i c c e l l s ( 1 1 2 ) . T h e y a r e c a p a b l e o f p r o c e s s i n g b o t h e u k a r y o t i c and p r o k a r y o t i c tRNA p r e c u r s o r s . A n o v e l enzyme w h i c h r e m o v e s l o n g 3 ' t r a i l e r s e q u e n c e s by a s i n g l e e n d o n u c l e o l y t i c c l e a v a g e h a s b e e n r e p o r t e d i n X e n o p u s GV e x t r a c t ( 7 6 , 77) a n d q u i t e r e c e n t l y i n an y e a s t c e l l e x t r a c t ( c i t e d i n . r e f . 1 1 3 ) . A RNase P 2 - l i k e a c t i v i t y must a l s o be p r e s e n t i n t h e GV e x t r a c t s t o m a t u r e t h e d i m e r i c tRNA p r e c u r s o r s ( 9 8 , 9 9 ) . U n l i k e t h o s e o f E - c o l i , e u k a r y o t i c tRNA p r e c u r s o r s do n o t c o n t a i n t h e 3 ' t e r m i n a l -CCA s e q u e n c e . T h i s s e q u e n c e i s a d d e d p o s t - t r a n s c r i p t i o n a l l y p r e s u m a b l y by a 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 ( 1 1 4 ) p r e s e n t i n t h e s e e x t r a c t s . Some e u k a r y o t i c tRNA p r e c u r s o r s c o n t a i n i n t e r v e n i n g s e q u e n c e s . A b e l s o n and h i s c o - w o r k e r s ( 1 1 5 , 116) i n v e s t i g a t e d t h e s p l i c i n g r e a c t i o n u s i n g a s o l u b l e e x t r a c t f r o m y e a s t . They f o u n d t h a t s p l i c i n g o c c u r s i n t w o s t e p s . F i r s t , an e n d o n u c l e a s e a c t i v i t y e x c i s e s t h e i n t e r v e n i n g s e q u e n c e t o p r o d u c e t w o h a l f m o l e c u l e s . The 3 ' - e n d g e n e r a t e d by t h e e x c i s i o n i s p h o s p h o r y l a t e d w h i l e t h e new 5 ' - e n d b e a r s a f r e e h y d r o x y l g r o u p . The s e c o n d s t e p i s t h e ATP d e p e n d e n t l i g a t i o n o f t h e t w o h a l f - m o l e c u l e s . An a c t i v i t y c a p a b l e o f l i g a t i n g 3 ' - p h o s p h o r y l a t e d end t o a 5 ' - h y d r o x y l e n d was d e s c r i b e d h e r e f o r t h e f i r s t t i m e . T r a n s f e r RNA m a t u r a t i o n i s a c o m p l e x p r o c e s s . The i n t r a c e l l u l a r l o c a t i o n o f m a t u r a t i o n o f 5 ' and 3 ' - e n d s ( 7 0 , 71) and s p l i c i n g ( 1 1 7 ) a n d 19 . t h e o r d e r i n w h i c h t h e p r o c e s s i n g ( 6 9 , 76) and m o d i f i c a t i o n ( 1 1 8 ) s t e p s o c c u r h a v e b e e n d e t e r m i n e d . The c o n c l u s i o n s f r o m t h e s e s t u d i e s a r e t h a t t h e p r o c e s s i n g and t h e m o d i f i c a t i o n e v e n t s o c c u r i n a v e r y s e q u e n t i a l f a s h i o n and t h a t a l l t h e p r o c e s s i n g s t e p s a r e l o c a t e d i n t h e n u c l e u s . I V . The p r e s e n t i n v e s t i g a t i o n The v a l i n e t R N A s ( 1 2 - 1 4 ) and r e c o m b i n a n t p l a s m i d s c o n t a i n i n g tRNA g e n e s ( 3 9 , 1 1 9 , 1 2 0 ) f r o m D r o s o p h i l a m e l a n o g a s t e r a r e v e r y w e l l c h a r a c t e r i z e d . The c o d i n g p r o p e r t i e s o f t h e t h r e e m a j o r i s o a c c e p t o r s , t R N A ^ a l , t R N A^j^ a n d t R N A 4 a l Val Val a r e k n o w n . The RNA s e q u e n c e s o f tRNA 3 ^ and tRNA 4 h a v e r e c e n t l y become a v a i l a b l e . Dunn e t aj_. ( 1 1 9 ) i s o l a t e d s e v e n p l a s m i d s c o n t a i n i n g t R N A V a l g e n e s o n d i f f e r e n t l e n g t h D r o s o p h i l a DNA. T h e s e f r a g m e n t s o r i g i n a t e f r o m f o u r d i f f e r e n t s i t e s on t h e D r o s o p h i l a c h r o m o s o m e s . In t h e p r e s e n t i n v e s t i g a t i o n t h e DNA s e q u e n c e o f a tRNA gene c o n t a i n e d i n p l a s m i d p D t l 2 0 R was d e t e r m i n e d . O t h e r members i n t h i s l a b o r a t o r y h a v e s e q u e n c e d s e v e r a l o f t h e o t h e r g e n e s c o d i n g f o r t R N A ^ 1 and t R N A 4 a l ( 1 3 , 3 9 ) . C o m p a r i s o n o f t h e s e q u e n c e s o f d i f f e r e n t g e n e s c o d i n g f o r t h e same tRNA t o t h a t o f t h e c o r r e s p o n d i n g tRNA r e v e a l e d t h a t some g e n e s v a r i e d a t a f e w n u c l e o t i d e s i n t h e c o d i n g r e g i o n . T r a n s f e r RNA p r o d u c t s c o r r e s p o n d i n g t o t h e v a r i a n t g e n e s e q u e n c e s h a v e n o t b e e n d e t e c t e d s o f a r . Val The DNA s e q u e n c e a n a l y s e s o f c l o n e d tRNA g e n e s p r o v i d e d w e l l c h a r a c t e r i z e d t e m p l a t e s f o r s t u d i e s o f gene e x p r e s s i o n . One o f t h e p r i m a r y g o a l s o f t h i s i n v e s t i g a t i o n was t o d e t e r m i n e t h e DNA s e q u e n c e s i n v o l v e d i n t h e Val r e g u l a t i o n o f D r o s o p h i l a tRNA g e n e s . F o r t h i s p u r p o s e t h e d e v e l o p m e n t o f a h o m o l o g o u s t r a n s c r i p t i o n s y s t e m was deemed n e c e s s a r y b e c a u s e o f t h e p r o b l e m s a s s o c i a t e d w i t h t h e u s e o f h e t e r o l o g o u s o n e s ( r e f e r t o s e c t i o n I I I . B ) . The a v a i l a b i l i t y o f s u c h a s y s t e m w o u l d a l l o w one t o a n s w e r q u e s t i o n s s u c h a s : A r e t h e v a r i a n t g e n e s t r a n s c r i b e d i n v i t r o ? Do t h e y t r a n s c r i b e a t t h e same 20 . r a t e as t h e g e n e s f o r w h i c h a tRNA p r o d u c t h a s b e e n d e m o n s t r a t e d ? A r e t h e r e d i f f e r e n c e s i n t h e i n t e r n a l c o n t r o l r e g i o n s o f g e n e s w h i c h t r a n s c r i b e w i t h d i f f e r e n t e f f i c i e n c i e s ? Do t h e 5 ' - f l a n k i n g s e q u e n c e s e x e r c i s e a n y i n f l u e n c e on t r a n s c r i p t i o n o f t h e s e g e n e s ? A r e t h e p r e c u r s o r p r o d u c t s d i r e c t e d by Val v a r i o u s tRNA g e n e s p r o c e s s e d a t t h e same r a t e ? The work p r e s e n t e d h e r e s e e k s t o a n s w e r some o f t h e a b o v e q u e s t i o n s . M a t e r i a l s and M e t h o d s R e a g e n t s  M a t e r i a l A c r y l a m i d e ( U l t r a p u r e ) A g a r o s e powder Ammonium p e r s u l f a t e A m p i c i l l i n B i s ( N , N 1 m e t h y l e n e b i s a c r y l a m i d e ) ( u l t r a p u r e ) B o r i c a c i d B r o m o p h e n o l b l u e C e l l u l o s e a c e t a t e s t r i p s ( 3 x 5 5 c m , N o . 2 5 0 0 ) C e s i u m C h l o r i d e ( t e c h n i c a l g r a d e ) C h i o r a m p h e n i c o l DEAE - c e l l u l o s e DEAE - c e l l u l o s e TLC p l a t e ( P o l y g r a m C e l 3 0 0 DEAE) D i a l y s i s t u b i n g D i a l y s i s c l i p s D i m e t h y l S u l p h a t e D i t h i o t h r e i t o l EDTA E p p e n d o r f t u b e s E t h i d i u m b r o m i d e F o r m a m i d e ( R e a g e n t g r a d e ) G l y c e r o l S o u r c e M i l e s L a b s . , I n c . B i o - R a d L a b o r a t o r i e s MCB M a n u f a c t u r i n g C h e m i s t s , I n c . S i g m a C h e m i c a l C o . M i l e s L a b s , I n c . A m e r i c a n S c i e n t i f i c a n d C h e m i c a l s C o . (Amachem) F i s c h e r S c i e n t i f i c C o . S c h l e i c h e r and S c h u e l l K a w e c k i B e r y l c o I n d u s t r i e s , I n c . S i g m a C h e m i c a l C o . Whatman L t d . M a c h e r y - N a g e l VWR S c i e n t i f i c I n c . S p e c t r u m M e d i c a l I n d u s t r i e s E a s t m a n Kodak C o . S i g m a C h e m i c a l C o . S i g m a C h e m i c a l C o . E v e r g r e e n S c i e n t i f i c , I n c . S i g m a C h e m i c a l C o . F i s c h e r S c i e n t i f i c C o . Amachem M a t e r i a l H y d r a z i n e ( R e a g e n t g r a d e ) I n t e n s i f y i n g s c r e e n s ( D u p o n t C r o n e x L i g h t n i n g P l u s ) 2 - m e r c a p t o e t h a n o l P h o s p h o c r e a t i n e P h o s p h o r u s p e n t o x i d e P i p e r i d i n e P y r i d i ne S e p h a d e x ( G - 2 5 , G - 1 0 0 ) S o d i u m l a u r y l S u l p h a t e (SDS) TEMED T e t r a c y c l i n e T h i a m i n e HCl T h y m i d i n e U r e a ( U l t r a p u r e ) U r i d i n e Whatman (3MM) f i l t e r p a p e r X - r a y f i l m s X y l e n e c y a n o l Y e a s t tRNA I I . N u c l e o t i d e s A T P , C T P , G T P , UTP d A T P , d C T P , d G T P , dTTP I I I . R a d i o a c t i v e m a t e r i a l s 1 2 5 I o d i n e ( Y - 3 2 P ) ATP ( a - 3 2 P ) d C T P , dGTP ( a - 3 2 P ) UTP 22. S o u r c e F i s c h e r S c i e n t i f i c C o . P i c k e r Mai 1 i n c k r o d t S i g m a C h e m i c a l C o . Mai 1 i n c k r o d t F i s c h e r S c i e n t i f i c C o . F i s c h e r S c i e n t i f i c C o . P h a r m a c i a S i g m a C h e m i c a l C o . B i o - R a d L a b o r a t o r i e s S i g m a C h e m i c a l C o . S i g m a C h e m i c a l C o . S i g m a C h e m i c a l C o . S c h w a r z / M a n n , I n c . S i g m a C h e m i c a l C o . Whatman L t d . P i c k e r E a s t m a n Kodak C o . P r o v i d e d by D r . G . S p i e g e l man S i g m a C h e m i c a l C o . New E n g l a n d N u c l e a r a n d Amersham 23, I V . V . a ) b) c ) M a t e r i a l  Enzymes C a l f i n t e s t i n a l a l k a l i n e p h o s p h a t a s e ( C I A P ) C r e a t i n e p h o s p h o k i n a s e E - c o l i DNA p o l y m e r a s e I ( K l e n o w f r a g m e n t ) L y s o z y m e ( e g g w h i t e ) P r o n a s e P r o t e i n a s e K R e s t r i c t i o n e n d o n u c l e a s e s : D d e l , E c o R I , H i n d l l l , H p a l l , S a u 9 6 I , S m a l , Xmal Xmal Fnu4HI R i b o n u c l e a s e ( S a n k y o ) T 4 p o l y n u c l e o t i d e K i n a s e T 4 p o l y n u c l e o t i d e l i g a s e S o u r c e B o e h r i n g e r M a n n h e i m S i g m a C h e m i c a l C o . B o e h r i n g e r M a n n h e i m S i g m a C h e m i c a l C o . B o e h r i n g e r M a n n h e i m B o e h r i n g e r M a n n h e i m New E n g l a n d B i o l a b s (NEB) W o r t h i n g t o n G i f t f r o m D r . C . A s t e l l C a l b i o c h e m New E n g l a n d B i o l a b s New E n g l a n d B i o l a b s , B o e h r i n g e r M a n n h e i m B a c t e r i a l a n d D r o s o p h i l a S t r a i n s E - c o l i S F 8 was o b t a i n e d f r o m M. O l s o n ( 1 2 1 ) . B a c t e r i a l s t r a i n c o n t a i n i n g t h e p l a s m i d , pBR322 ( 1 2 2 ) was p r o v i d e d b y H, B o y e r . D r o s o p h i ! a S c h n e i d e r I I c e l l s w e r e o b t a i n e d f r o m D r . C . L a i r d , U. o f Washi n g t o n . 24. M e t h o d s I . G e n e r a l A . P l a s m i d DNA i s o l a t i o n P l a s m i d DNA was p r e p a r e d as d e s c r i b e d by Dunn e t a l _ . ( 1 1 9 ) e x c e p t t h a t c e l l s w e r e grown i n M9S medium s u p p l e m e n t e d w i t h 200 /ug/ml u r i d i n e ( 1 2 3 ) , 0 . 5 u g / m l t h i a m i n e a n d 10 j j g / m l t h y m i d i n e , and t h e p l a s m i d DNA was p u r i f i e d by one o r t w o s u c c e s s i v e c e s i u m c h l o r i d e e q u i l i b r i u m c e n t r i f u g a t i o n s t e p s ( 1 2 0 ) . B . D i g e s t i o n o f DNA w i t h r e s t r i c t i o n e n z y m e s R e a c t i o n c o n d i t i o n s f o r r e s t r i c t i o n e n z y m e s w e r e s i m i l a r t o t h o s e recommended by t h e m a n u f a c t u r e r s . R e s t r i c t i o n b u f f e r s w e r e g e n e r a l l y p r e p a r e d a t c o n c e n t r a t i o n s 1 0 - f o l d h i g h e r t h a n t h e f i n a l c o n c e n t r a t i o n i n t h e r e a c t i o n m i x . F o r c o m p l e t e d i g e s t i o n o f DNA, t h e r e a c t i o n m i x t u r e u s u a l l y c o n t a i n e d 1 . 0 u n i t ( a s d e f i n e d by t h e s u p p l i e r ) o f r e s t r i c t i o n enzyme p e r ug o f DNA a n d t h e i n c u b a t i o n t i m e was 2 h o u r s . F o r r e s t r i c t i o n enzyme p r e p a r a t i o n s , known t o be c o n t a m i n a t e d w i t h n u c l e a s e s ( e . g . Xmal f r o m W o r t h i n g t o n ) , o p t i m u m r e s t r i c t i o n c o n d i t i o n s w e r e d e t e r m i n e d . C . A g a r o s e g e l e l e c t r o p h o r e s i s A g a r o s e g e l s ( 0 . 5 - 2%) w e r e p r e p a r e d and r u n i n T r i s - p h o s p h a t e b u f f e r [ 4 0 mM T r i s . H C l (pH 8 . 0 ) , 20 mM N a H 2 P 0 4 , 1 mM E D T A ] . H i n d l l l d i g e s t e d * DNA ( s i z e r a n g e 0 . 4 5 kb - 2 3 k b ; 124) was g e n e r a l l y u s e d a s m . w t . m a r k e r i n a g a r o s e g e l s . D. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s A 45% a c r y l a m i d e s t o c k s o l u t i o n c o n s i s t e d o f a c r y l a m i d e and N , N -m e t h y l e n e - b i s a c r y l a m i d e i n a 3 0 : 1 r a t i o . P o l y a c r y l a m i d e g e l s w e r e p r e p a r e d by p o l y m e r i z a t i o n o f a m i x t u r e c o n t a i n i n g t h e a p p r o p r i a t e amount o f t h e 45% ( 3 0 : 1 ) a c r y l a m i d e s t o c k s o l u t i o n , 90 mM T r i s - b o r a t e b u f f e r (pH 8 . 3 ) , 2 mM EDTA, 0 . 0 5 % ( w / v ) ammonium p e r s u l p h a t e and TEMED. TEMED was a d d e d t o a f i n a l c o n c e n t r a t i o n o f 0 . 1 % ( v / v ) . D e n a t u r i n g g e l s c o n t a i n e d 7M u r e a . A n a l y t i c a l and p r e p a r a t i v e g e l s w e r e u s u a l l y 1 . 5 mm t h i c k w h i l e s e q u e n c i n g g e l s w e r e 0 . 5 mm t h i c k . S a m p l e s w e r e m i x e d w i t h 0 . 2 - 0 . 5 v o l u m e l o a d i n g s o l u t i o n [ 7 2 mM T r i s - b o r a t e (pH 8 . 3 ) , 24 mM EDTA, 0 . 8 % S D S , 40% g l y c e r o l , 0 . 1 % b r o m o p h e n o l b l u e (BPB) and 0 . 1 % x y l e n e c y a n o l ( X C ) ] b e f o r e l o a d i n g n o n - d e n a t u r i n g g e l s o r w i t h 0 . 5 v o l u m e 10M u r e a , 0 . 1 % B P B , 0 . 1 % XC f o r l o a d i n g d e n a t u r i n g g e l s . S a m p l e s t o be l o a d e d on d e n a t u r i n g g e l s w e r e h e a t e d a t 9 5 ° C f o r 2 m i n . j u s t b e f o r e l o a d i n g . F o r b e t t e r r e s o l u t i o n o f s m a l l e r f r a g m e n t s , d e n a t u r i n g g e l s w e r e p r e - r u n f o r a p p r o x i m a t e l y 30 m i n . b e f o r e l o a d i n g t h e s a m p l e s . U n l a b e l l e d DNA f r a g m e n t s w e r e v i s u a l i z e d by s t a i n i n g t h e g e l w i t h 1 j j g / m l e t h i d i u m b r o m i d e f o r 15 m i n . , t h e n i l l u m i n a t i n g w i t h u l t r a v i o l e t l i g h t . R a d i o a c t i v e l y l a b e l l e d DNA (RNA) f r a g m e n t s w e r e v i s u a l i z e d by a u t o r a d i o g r a p h y ( b e l o w ) . H p a l l d i g e s t e d pBR322 ( s i z e r a n g e 9 bp - 612 b p ; ( 1 2 5 ) was g e n e r a l l y u s e d as m . w t . m a r k e r i n p o l y a c r y l a m i d e g e l s . The s i z e s o f unknown DNA f r a g m e n t s w e r e d e t e r m i n e d by l i n e a r r e g r e s s i o n a n a l y s i s . E. A u t o r a d i o g r a p h y P o l y a c r y l a m i d e g e l s w e r e a u t o r a d i o g r a p h e d by f i r s t w r a p p i n g t h e m i n S a r a n W r a p , t h e n o v e r l a y i n g t h e m w i t h a p i e c e o f X - r a y f i l m ( K o d a k X - O m a t , Kodak N S - 5 T ) . The f i l m s w e r e e x p o s e d a t 4 ° C o r - 2 0 ° C a n d d e v e l o p e d a c c o r d i n g t o t h e m a n u f a c t u r e r ' s 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 , t h e f i l m was s a n d w i c h e d b e t w e e n t h e g e l and an i n t e n s i f y i n g s c r e e n ( D u p o n t C r o n e x L i g h t n i n g - P l u s ; 1 2 6 ) , and e x p o s e d a t - 2 0 ° o r - 7 0 ° C . F . I s o l a t i o n o f n u c l e i c a c i d f r o m p o l y a c r y l a m i d e g e l s 1 . E l e c t r o e l u t i o n DNA f r a g m e n t s w e r e l o c a l i z e d by s t a i n i n g o r a u t o r a d i o g r a p h y ( d e s c r i b e d i n s e c t i o n s D and E) and t h e p o r t i o n o f t h e g e l c o n t a i n i n g t h e d e s i r e d DNA f r a g m e n t was e x c i s e d w i t h a s c a l p e l and e l e c t r o e l u t e d w i t h 8 0 mM T r i s - a c e t a t e , pH 7 . 0 b u f f e r . E l e c t r o e l u t i o n was c a r r i e d o u t a t 3 - 4 m A / t u b e o v e r n i g h t a t room t e m p e r a t u r e , b u t 4 h o u r s w e r e s u f f i c i e n t . The D N A - c o n t a i n i n g e l u a t e ( 3 0 0 - 5 0 0 JJI ) was r e m o v e d f r o m t h e d i a l y s i s t u b i n g and c o l l e c t e d i n 1 . 8 ml e p p e n d o r f t u b e s , t h e e l u a t e was e x t r a c t e d o n c e w i t h b u t a n o l ( e q u i l i b r a t e d w i t h w a t e r ) b e f o r e e t h a n o l p r e c i p i t a t i o n . The DNA was p r e c i p i t a t e d by t h e a d d i t i o n o f s o d i u m a c e t a t e t o a c o n c e n t r a t i o n o f 0 . 3 M and 3 - 4 v o l u m e s o f 95% e t h a n o l . W h e n e v e r r e q u i r e d , e i t h e r 5 - 1 0 jjg o f y e a s t tRNA o r 0 . 1 6 mM GTP was u s e d as c a r r i e r . DNA was p r e c i p i t a t e d f o r 1 5 - 3 0 m i n . i n a d r y i c e / e t h a n o l b a t h o r o v e r n i g h t i n t h e - 7 0 ° C f r e e z e r . The DNA 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 i n an E p p e n d o r f ( B r i n k m a n ) c e n t r i f u g e f o r 5 m i n . a t room t e m p e r a t u r e . The s u p e r n a t a n t was d i s c a r d e d , t h e p r e c i p i t a t e was d r i e d b r i e f l y i n a d e s s i c a t o r and was r e s u s p e n d e d i n an a p p r o p r i a t e s o l u t i o n f o r f u r t h e r u s e . R e c o v e r y o f DNA by e l e c t r o e l u t i o n was g e n e r a l l y 7 0 - 9 5 % . 2 . S o a k i n g RNA f r o m p o l y a c r y l a m i d e g e l was u s u a l l y e l u t e d by s o a k i n g t h e g e l p i e c e s i n 0 . 4 ml 0 . 5 M ammonium a c e t a t e , 10 mM m a g n e s i u m a c e t a t e , 1 mM EDTA and 0 . 1 % SDS ( 1 2 7 ) f o r 1 - 2 d a y s a t 4 ° C . R e c o v e r y o f RNA ( a p p r o x i m a t e l y 100 n u c l e o -t i d e s ) f r o m 10% - 7M u r e a g e l a f t e r s o a k i n g f o r 48 h o u r s was 5 0 - 7 0 % . RNA f r o m t h e e l u a t e was r e c o v e r e d by e t h a n o l p r e c i p i t a t i o n . G. I o d i n a t i o n o f t r a n s f e r RNA T r a n s f e r RNA ( 2 - 3 jug) was i o d i n a t e d w i t h J 2 5 I ( 2 . 5 m C i ; New E n g l a n d N u c l e a r ) a c c o r d i n g t o t h e m e t h o d o f C o m m e r f o r d ( 1 2 8 ) as d e s c r i b e d by H a y a s h i e t a l . ( 2 9 ) . The i o d i n a t e d tRNA was p u r i f i e d by a p p l y i n g t h e r e a c t i o n m i x t u r e t o a s m a l l D E A E - c e l 1 u l o s e c o l u m n ( 0 . 8 x 3 c m ; w a s h e d w i t h a b o u t 100 ml 0 . 3 M N a C l , 0 . 0 3 M s o d i u m a c e t a t e , pH 4 . 5 ) and t h e n by e l u t i n g w i t h 1 . 5 M N a C l , 0 . 5 M s o d i u m a c e t a t e , pH 4 . 5 . F r a c t i o n s o f 0 . 4 ml w e r e c o l l e c t e d , and t h e r a d i o -a c t i v i t y i n e a c h f r a c t i o n was d e t e r m i n e d . The peak f r a c t i o n s w e r e p o o l e d and w e r e r u n on a S e p h a d e x G - 2 5 c o l u m n ( 0 . 7 x 4 5 cm) f o r f u r t h e r p u r i f i c a t i o n and t o o b t a i n t h e tRNA i n t h e d e s i r e d b u f f e r (70% f o r m a m i d e , 0 . 5 M K C 1 , 5 mM EDTA, 23 mM KOH, 60 mM K H 2 P 0 4 a n d K 2 H P 0 4 , pH 7 . 0 ) . The peak f r a c t i o n s w e r e p o o l e d and s t o r e d a t - 2 0 ° C . 1 jul o f t h e p o o l e d G - 2 5 f r a c t i o n s c o n t a i n e d 1 . 9 x }0k c p m . I I . DNA s e q u e n c e a n a l y s i s A . 3 ' e n d - l a b e l l i n g o f r e s t r i c t i o n f r a g m e n t s R e s t r i c t i o n f r a g m e n t s w i t h 5 ' - e x t e n d e d s e q u e n c e s w e r e l a b e l l e d a t t h e i r 3 ' - e n d s w i t h E - c o l i DNA p o l y m e r a s e I ( K l e n o w f r a g m e n t ) and [ a - 3 2 P ] l a b e l l e d d e o x y n u c l e o s i d e t r i p h o s p h a t e ( 1 2 9 ) . R e s t r i c t i o n f r a g m e n t s ( a p p r o x . 5 jj.g) w e r e l a b e l l e d by i n c u b a t i n g t h e r e s t r i c t i o n d i g e s t ( 2 0 - 1 0 0 j u l ) w i t h 5 0 - 7 0 j j C i o f t h e a p p r o p r i a t e [ a - 3 2 P ] d N T P , 2 jul o f e a c h o f t h e a p p r o p r i a t e u n l a b e l l e d dNTP ( 0 . 5 mM e a c h ) and 2 u n i t s o f E - c o l i DNA p o l y m e r a s e I ( K l e n o w f r a g m e n t ) a t room t e m p e r a t u r e f o r 15 m i n . The r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 1 p i 1M EDTA; i t was e x t r a c t e d o n c e w i t h p h e n o l ( s a t u r a t e d w i t h w a t e r ) and t w i c e w i t h e t h e r . E x c e s s e t h e r was b l o w n o f f and t h e l a b e l l e d DNA was p u r i f i e d by p u t t i n g i t t h r o u g h a 1 ml S e p h a d e x G - 1 0 0 c o l u m n . The c o l u m n was w a s h e d w i t h a b u f f e r c o n t a i n i n g 2 mM T r i s . H C l , pH 7 . 4 , 0 . 0 5 mM EDTA and 0 . 4 ml f r a c t i o n s w e r e c o l l e c t e d . The l a b e l l e d DNA u s u a l l y e l u t e d i n t h e s e c o n d 0 . 4 ml f r a c t i o n . The l a b e l l e d DNA was e t h a n o l p r e c i p i t a t e d i n p r e s e n c e o f 5 /ug y e a s t t R N A . T h e r e a r e t w o ways o f g e t t i n g a s i n g l e e n d - l a b e l l e d f r a g m e n t f r o m a DNA f r a g m e n t w h i c h i s l a b e l l e d a t b o t h e n d s : i ) c l e a v i n g t h e d o u b l e e n d - l a b e l l e d f r a g m e n t w i t h a s e c o n d r e s t r i c t i o n enzyme w h i c h w o u l d g i v e t w o s i n g l e e n d -l a b e l l e d f r a g m e n t s o r i i ) s e p a r a t i n g t h e two s t r a n d s o f t h e d o u b l e e n d -l a b e l l e d f r a g m e n t w h i c h w o u l d g e n e r a t e t w o s i n g l e s t r a n d s e a c h l a b e l l e d a t o n e e n d . 28 . B . S e c o n d a r y r e s t r i c t i o n enzyme c l e a v a g e A l i q u o t s o f d o u b l e e n d - l a b e l l e d f r a g m e n t w e r e d i g e s t e d w i t h a s e r i e s o f r e s t r i c t i o n e n z y m e s , e l e c t r o p h o r e s e d on an a n a l y t i c a l p o l y a c r y l a m i d e g e l ( 1 . 5 x 2 0 0 x 4 0 0 mm) a n d t h e g e l was a u t o r a d i o g r a p h e d . The r e s t r i c t i o n enzyme w h i c h g a v e t w o f r a g m e n t s w i t h l e n g t h s t h a t c o u l d be s e q u e n c e d e a s i l y and s e p a r a t e d f r o m e a c h o t h e r , was u s e d t o d i g e s t t h e r e s t o f t h e l a b e l l e d f r a g m e n t . The r e s t r i c t e d s a m p l e was e l e c t r o p h o r e s e d on a p r e p a r a t i v e p o l y a c r y l a m i d e g e l (1 . 5 x 200 x 4 0 0 mm) a n d t h e g e l was a u t o r a d i o g r a p h e d . U s i n g t h e a u t o r a d i o g r a m a s t h e t e m p l a t e , t h e l a b e l l e d f r a g m e n t s w e r e e x c i s e d a n d e l e c t r o e l u t e d a s d e s c r i b e d i n s e c t i o n I . F . I . A f t e r e t h a n o l p r e c i p i t a t i o n t h e s a m p l e was r e a d y f o r s e q u e n c i n g . C . S t r a n d s e p a r a t i o n An a l i q u o t o f t h e d o u b l e e n d - l a b e l l e d f r a g m e n t was t e s t e d t o d e t e r m i n e t h e o p t i m u m c o n d i t i o n s f o r s t r a n d s e p a r a t i o n . The r e s t o f t h e s a m p l e t h e n was s t r a n d s e p a r a t e d by a d d i n g an e q u a l v o l u m e o f 2X s t r a n d d e n a t u r i n g s o l u t i o n (20% g l y c e r o l , 0 . 6 M NaOH, 0 . 0 0 2 M EDTA, 0 . 1 % B P B , and 0 . 1 % X C ) . The s a m p l e was a p p l i e d i m m e d i a t e l y t o a n o n d e n a t u r i n g p o l y a c r y l a m i d e g e l ( 1 . 5 x 200 x 4 0 0 mm) and e l e c t r o p h o r e s e d a t 1 5 0 - 2 0 0 V ( 4 - 5 V/cm) t i l l t h e t r a c k i n g d y e s , BPB a n d X C , had moved t o p o s i t i o n s w h i c h i n d i c a t e d a d e q u a t e r e s o l u t i o n o f t h e c o m p l e m e n t a r y DNA s t r a n d s ( p r e d e t e r m i n e d ) . The v o l t a g e n e v e r was a l l o w e d t o e x c e e d 200 V . T h i s p r e v e n t e d o v e r h e a t i n g t h e g e l ; i t i s b e l i e v e d ( D r . C . A s t e l l , p e r s o n a l c o m m u n i c a t i o n ) t h a t t h e s t r a n d s s e p a r a t e b e c a u s e t h e y a c q u i r e u n i q u e c o n f o r m a t i o n and h e a t w o u l d p r e v e n t t h e s t r a n d s f r o m d o i n g s o . The g e l was a u t o r a d i o g r a p h e d and t h e d e s i r e d f r a g m e n t s w e r e made r e a d y f o r s e q u e n c i n g a s d e s c r i b e d a b o v e ( s e c t i o n I I B ) . D. DNA s e q u e n c e a n a l y s i s - Maxam and G i l b e r t m e t h o d S i n g l e e n d - l a b e l l e d DNA f r a g m e n t s ( w h i c h c o n t a i n e d a t l e a s t 3 x 1 0 5 C e r e n k o v cpm) w e r e s e q u e n c e d by t h e c h e m i c a l m e t h o d o f Maxam and G i l b e r t 29, ( 1 2 7 ) . The p r o t o c o l f o l l o w e d was e x a c t l y a s d e s c r i b e d i n r e f e r e n c e 1 2 7 . The summary o f t h e c h e m i c a l r e a c t i o n s u s e d i s g i v e n i n T a b l e 1 1 . The s e q u e n c e r e a c t i o n s w e r e e l e c t r o p h o r e s e d on 8 - 2 5 % p o l y a c r y l a m i d e g e l s ( 0 . 5 x 2 0 0 x 4 0 0 mm) and t h e g e l s w e r e . e x p o s e d t o X - r a y f i l m s w i t h o r w i t h o u t i n t e n s i f y i n g s c r e e n a t - 2 0 ° C . I I I . I n v i t r o t r a n s c r i p t i o n a n a l y s i s A . C e l l C u l t u r e D r o s o p h i l a S c h n e i d e r I I c e l l s w e r e g r o w n i n g l a s s r o l l i n g b o t t l e s a t 22 - 2 5 ° C i n D r o s o p h i 1 a medium s u p p l e m e n t e d w i t h 13% f e t a l b o v i n e s e r u m and 50 / j g / m l g e n t a m y c i n . C e l l s w e r e h a r v e s t e d by c e n t r i f u g a t i o n a t 8 0 0 0 RPM f o r 10 m i n . (GSA) g e n e r a l l y a f t e r 7 d a y s o f g r o w t h when t h e c e l l d e n s i t y was a p p r o x i m a t e l y 0 . 5 - 1 . 0 x 1 0 7 c e l l s / m l . B . P r e p a r a t i o n o f D r o s o p h i l a c e l l - f r e e e x t r a c t ( S . 1 0 0 ) E x t r a c t s f r o m t h e D r o s o p h i 1 a S c h n e i d e r I I c e l l l i n e w e r e p r e p a r e d e s s e n t i a l l y a s d e s c r i b e d by Wu and Z u b a y ( 1 3 0 ) and W e i l eit a K ( 8 0 ) w i t h t h e f o l l o w i n g m o d i f i c a t i o n s . C e l l s w e r e h a r v e s t e d by c e n t r i f u g a t i o n a t 5 , 8 6 0 x g f o r 10 m i n . a n d w a s h e d t h r e e t i m e s i n 3 0 - 4 0 v o l u m e s o f a b u f f e r c o n t a i n i n g 30 mM T r i s . H C l (pH 7 . 9 ) , 120 mM KC1 , 5 mM M g C l 2 and 0 . 5 mM DTT. The p a c k e d c e l l w e i g h t was m e a s u r e d a f t e r c e n t r i f u g a t i o n a t 4 , 3 4 0 x g f o r 10 m i n . a n d t h e c e l l p e l l e t s w e r e r e s u s p e n d e d i n 2 v o l u m e s (1 v o l u m e = 1 gm p a c k e d c e l l w e i g h t ) o f a h y p o t o n i c b u f f e r [ 1 0 mM T r i s . H C l (pH 7 . 9 ) , 10 mM K C 1 , 1 . 5 mM M g C l 2 and 0 . 5 mM D T T ] . The c e l l s w e r e a l l o w e d t o s w e l l f o r 1 h o u r on i c e and t h e n w e r e b r o k e n by 12 s t r o k e s o f a B - t y p e D o u n c e h o m o g e n i z e r . The e x t e n t o f c e l l l y s i s was m o n i t o r e d w i t h a p h a s e c o n t r a s t l i g h t m i c r o s c o p e and was g e n e r a l l y g r e a t e r t h a n 9 5 % . To t h e l y s a t e was a d d e d 0 . 1 2 5 c e l l v o l u m e o f a s o l u t i o n c o n t a i n i n g 0 . 2 3 M T r i s . H C l (pH 7 . 9 ) , 1 . 2 7 M KC1 , 0 . 0 4 M g C l 2 and 0 . 5 mM DTT, and t h e l y s a t e was c e n t r i f u g e d a t 1 0 0 , 0 0 0 x g f o r 60 m i n . The s u p e r n a t a n t was c o l l e c t e d and g l y c e r o l was a d d e d t o a f i n a l c o n c e n t r a t i o n o f 20% ( v / v ) . The 30. T a b l e I I . S p e c i f i c C h e m i c a l D e g r a d a t i o n o f DNA B a s e M o d i c a t i o n B a s e R e m o v a l S t r a n d S c i s s i o n C l e a v a g e S p e c i f i c i t y T a r g e t s i n DNA R e a g e n t R e a g e n t R e a g e n t P u r i n e s DMS 0 . 1 N H C 1 , 0 ° C 0 . 1 N a l k a l i , 9 0 ° C 70% A 30% G P u r i n e s DMS p i p e r i d i n e p i p e r i d i n e 100% G P y r i m i d i n e s H y d r a z i n e p i p e r i d i ne p i p e r i d i n e 50% C 50% T C y t o s i ne H y d r a z i n e p l u s IM NaCl p i p e r i d i ne p i p e r i d i n e 95% C 3 1 . e x t r a c t was s t o r e d i n a l i q u o t s a t - 7 0 ° C . C . I n v i t r o t r a n s c r i p t i o n and e l e c t r o p h o r e t i c a n a l y s i s o f RNA S t a n d a r d t r a n s c r i p t i o n r e a c t i o n s w e r e p e r f o r m e d i n a f i n a l v o l u m e o f 50 / j l . The r e a c t i o n m i x t u r e c o n s i s t e d o f 20 mM T r i s . H C l (pH 7 . 9 ) , 80 mM K C 1 , 5 mM M g C l 2 » 3 mM DTT, 2 . 5 jug/ml a - a m a n i t i n , 6 U/ml c r e a t i n e p h o s p h o k i n a s e , 5 mM c r e a t i n e p h o s p h a t e ( 8 4 ) , 10% g l y c e r o l , 60 juM e a c h o f t h e t h r e e u n l a b e l l e d r i b o n u c l e o s i d e t r i p h o s p h a t e s , 25 uM [ a - 3 2 p ] UTP ( 3 - 5 C i / m m o l e ) , 2 jug p l a s m i d DNA and 25 jul D r o s o p h i l a S . 1 0 0 e x t r a c t . R e a c t i o n s w e r e s t a r t e d by t h e a d d i t i o n o f t h e c e l l - f r e e e x t r a c t and w e r e i n c u b a t e d a t room t e m p e r a t u r e f o r 2 h o u r s . D e p a r t u r e s f r o m s t a n d a r d c o n d i t i o n s - a r e n o t e d i n t h e f i g u r e l e g e n d s . T r a n s c r i p t i o n r e a c t i o n s w e r e s t o p p e d by t h e a d d i t i o n o f 2 . 5 / j l o f 2 mg/ml P r o t e i n a s e K a n d s o d i u m d o d e c y l s u l f a t e (SDS) t o 0 . 5 % . A f t e r 30 m i n . a t room t e m p e r a t u r e , t h e m i x t u r e was p h e n o l e x t r a c t e d and t h e a q u e o u s p h a s e was c o l l e c t e d . The n u c l e i c a c i d s w e r e p r e c i p i t a t e d by t h e a d d d i t i o n o f 5 /jg y e a s t t R N A , s o d i u m a c e t a t e t o a c o n c e n t r a t i o n o f 0 . 3 M , and 3 - 4 v o l u m e s o f 95% e t h a n o l . F o l l o w i n g e t h a n o l p r e c i p i t a t i o n , RNAs w e r e d i s s o l v e d i n 2 . 5 jul w a t e r and 2 . 5 jul o f a s o l u t i o n c o n t a i n i n g 10 M u r e a , 0 . 1 % (w/v) b r o m o p h e n o l b l u e and 0 . 1 % (w/v) x y l e n e c y a n o l . J u s t b e f o r e l o a d i n g t h e g e l s , t h e s a m p l e s w e r e h e a t e d a t 1 0 0 ° C f o r 2 m i n . The RNAs w e r e a n a l y z e d by e l e c t r o p h o r e s i s on 10% p o l y a c r y l a m i d e g e l s (1 x 175 x 220 mm) c o n t a i n i n g 7 M u r e a . The g e l s w e r e e x p o s e d t o Kodak X - r a y f i l m s (RP X-0MAT) w i t h o r w i t h o u t D u p o n t C r o n e x L i g h t n i n g P l u s i n t e n s i f y i n g s c r e e n s . To d e t e r m i n e t h e r a d i o a c t i v i t y i n s p e c i f i c RNA b a n d s , t h e a p p r o p r i a t e g e l b a n d s w e r e e x c i s e d u s i n g t h e a u t o -r a d i o g r a p h s a s t e m p l a t e s and t h e C e r e n k o v r a d i a t i o n was m e a s u r e d . D. RNA.DNA h y b r i d i z a t i o n P l a s m i d D N A s , p B R 3 2 2 , pDt41R a n d pDt78R w e r e b o u n d t o c e l l u l o s e n i t r a t e f i l t e r s ( S c h l e i c h e r and S c h u e l l , BA85) a s d e s c r i b e d by L a r s e n e t _al_. ( 1 3 1 ) 32, w i t h some m o d i f i c a t i o n s . The DNAs w e r e d i l u t e d t o 25 jug/ml i n 1 ml 0 . 1 x SSC ( l x S S C i s 0 . 1 5 M s o d i u m c h l o r i d e and 0 . 0 1 5 M s o d i u m c i t r a t e , pH 7 . 0 ) and d e n a t u r e d by h e a t i n g a t 1 0 0 ° C u n t i l t h e i n c r e a s e d by a b o u t 4 0 % . The s o l u t i o n was c h i l l e d , a n d t h e c o n c e n t r a t i o n o f SSC was i n c r e a s e d t o 6 X ; i t was d r i p p e d t h r o u g h w e t t e d n i t r o c e l l u l o s e f i l t e r s ( 3 6 mm d i a m . ) a t a r a t e o f 10 m l / m i n . F i l t e r s w e r e a i r - d r i e d o v e r n i g h t and c u t i n t o s m a l l f i l t e r s f o r h y b r i d i z a t i o n ( e a c h s m a l l f i l t e r c o n t a i n e d a p p r o x i m a t e l y 2 . 5 /jg D N A ) . R N A . DNA h y b r i d i z a t i o n s w e r e p e r f o r m e d i n 1 ml b u f f e r c o n t a i n i n g 20% f o r m a m i d e , 6 x SSC (pH 7 . 0 ) and 0 . 1 % SDS a t 6 5 ° C f o r 24 h o u r s ( 1 3 1 ) . The h y b r i d i z a t i o n v i a l c o n t a i n e d 2 b l a n k , 2 p B R 3 2 2 , e i t h e r 2 pDt41R o r 2 pDt78R f i l t e r s and [ a - 3 2 P ] UTP l a b e l l e d RNA. The RNA u s e d f o r h y b r i d i z a t i o n was o b t a i n e d f r o m t h e a p p r o p r i a t e e x c i s e d g e l b a n d s by t h e p r o c e d u r e d e s c r i b e d i n s e c t i o n I . F . 2 . A f t e r h y b r i d i z a t i o n , f i l t e r s w e r e w a s h e d o n c e i n 6 x S S C , 0 . 1 % SDS and t w i c e i n 6 x SSC a t 6 5 ° C f o r 10 m i n . e a c h . They w e r e d r i e d a t 6 5 ° C f o r 30 m i n . , and t h e r a d i o a c t i v i t y on t h e i n d i v i d u a l f i l t e r was d e t e r m i n e d by s c i n t i l l a t i o n c o u n t i n g . E . T ^ f i n g e r p r i n t a n a l y s i s o f RNA t r a n s c r i p t s RNA t r a n s c r i p t s l a b e l l e d w i t h [ a - 3 2 P ] UTP w e r e e l u t e d f r o m g e l s l i c e s , e t h a n o l p r e c i p i t a t e d w i t h 0 . 1 6 mM GTP and w e r e d i g e s t e d w i t h T ^ r i b o n u c l e a s e . The 5 u l r e a c t i o n c o n s i s t e d o f 3 . 5 u l l a b e l l e d RNA (minimum 5 0 0 0 C e r e n k o v c p m ) , 0 . 5 jul 10X T x b u f f e r ( 1 0 0 mM T r i s , pH 7 . 5 , 10 mM EDTA) and 5 u n i t s T x r i b o n u c l e a s e . The r e a c t i o n was i n c u b a t e d a t 3 7 ° C f o r 30 m i n . and was s t o p p e d by t h e a d d i t i o n o f 1 jul 100 mM EDTA (pH 4 . 7 ) . The r e a c t i o n m i x was d e s s i c a t e d and was r e s u s p e n d e d i n 2 / j l w a t e r . O l i g o n u c l e o t i d e s w e r e f r a c t i o n a t e d i n t h e f i r s t d i m e n s i o n by e l e c t r o -p h o r e s i s on a S h a n d o n f l a t b e d e l e c t r o p h o r e s i s a p p a r a t u s ( 1 3 2 ) . The s a m p l e (2 j u l ) was a p p l i e d t o a t h o r o u g h l y b l o t t e d s t r i p o f c e l l u l o s e a c e t a t e ( 3 x 55 cm) t h a t had b e e n s o a k e d i n a b u f f e r c o n t a i n i n g 5% a c e t i c a c i d , 7M u r e a and 1 mM 33 . EDTA (pH 3 . 5 ) . A s m a l l v o l u m e o f m a r k e r d y e s o l u t i o n [ 0 . 3 3 % ( w / v ) x y l e n e c y a n o l F F , o r a n g e G and a c i d f u c h s i n ] was s p o t t e d on e i t h e r s i d e o f t h e s a m p l e a t t h e o r i g i n . W h i l e t h e s a m p l e and d y e s s o a k e d i n t o t h e c e l l u l o s e a c e t a t e s t r i p , t h e r e s t o f t h e s t r i p , e x c e p t f o r a b o u t 1 . 5 cm a r o u n d t h e o r i g i n was c o v e r e d w i t h m y l a r s h e e t s , t h e n w i t h g l a s s p l a t e s . A f t e r t h e s a m p l e had s o a k e d i n , t h e o r i g i n was c o v e r e d w i t h m y l a r s h e e t and g l a s s and b o t h e n d s o f t h e s t r i p w e r e c o n n e c t e d by 3MM f i l t e r p a p e r w i c k s ( w e t t e d w i t h r u n n i n g b u f f e r -5% a c e t i c a c i d , 1 mM EDTA, a d j u s t e d t o pH 3 . 5 w i t h p y r i d i n e ) t o t h e e l e c t r o d e c o m p a r t m e n t s . E l e c t r o p h o r e s i s was c a r r i e d o u t a t 3000V ( w i t h w a t e r - c o o l i n g ) f o r 5 0 - 6 0 m i n . when t h e d i s t a n c e b e t w e e n t h e o r a n g e and b l u e d y e s was 1 3 - 1 4 c m . The s e p a r a t e d o l i g o n u c l e o t i d e s w e r e t r a n s f e r r e d f r o m t h e c e l l u l o s e a c e t a t e s t r i p t o a 20 x 20 cm DEAE - c e l l u l o s e TLC p l a t e ( P o l y g r a m C e l 300 D E A E , M a c h e r y - N a g e l ) by t h e p r o c e d u r e o f S o u t h e r n , 1974 ( 1 3 3 ) . The TLC p l a t e was w a s h e d w i t h w a t e r t o r e m o v e u r e a a l o n g t h e t r a n s f e r l i n e , t h e n a i r - d r i e d . The t r a n s f e r r e d o l i g o n u c l e o t i d e s w e r e s e p a r a t e d i n t h e s e c o n d d i m e n s i o n b y h o m o c h r o m a t o g r a p h y e s s e n t i a l l y as d e s c r i b e d by J a y e t ^1_. ( 1 3 4 ) . The o r i g i n on t h e TLC p l a t e was w e t t e d w i t h 3MM f i l t e r p a p e r s t r i p w h i c h had b e e n s o a k e d i n w a t e r and t h e p l a t e was d e v e l o p e d w i t h Homo-mix V ( 1 3 4 ) a t 6 5 ° C t i l l t h e s o l v e n t f r o n t r e a c h e d t h e t o p o f t h e p l a t e . The p l a t e was a i r - d r i e d and a u t o r a d i o g r a p h e d . I V . I n v i t r o m u t a g e n e s i s V a l A . S e p a r a t i o n o f t w o tRNA ^ g e n e s c o n t a i n e d i n p D t 5 5 The c h a r a c t e r i s t i c s o f t h e p a r e n t p l a s m i d s u s e d i n t h e work d e s c r i b e d i n t h i s t h e s i s a r e g i v e n i n T a b l e I I I . V a l The e x p e r i m e n t a l s t r a t e g y u s e d t o s e p a r a t e t h e t w o t R N A 4 g e n e s c o n t a i n e d i n p D t 5 5 i s shown i n F i g . 4A and F i g . 5 r e p r e s e n t s t h e f l o w c h a r t f o r t h e g e n e r a l p r o c e d u r e u s e d . 1 . P r e p a r a t i o n o f t h e r e s t r i c t i o n f r a g m e n t s T a b l e I I I . C h a r a c t e r i s t i c s o f t h e p a r e n t DNA p l a s m i d s u s e d i n t h e work d e s c r i b e d i n t h i s t h e s i s P l a s m i d t R N A gene c o d e d f o r S i z e o f D r o s o p h i l a H i n d i 11 i n s e r t ( k b ) I n s i t u H y b r i d i -z a t i o n s i t e R e f . p D t 5 5 V a l 4 ( 2 ) + 8 . 0 70 BC 3 9 , 119 pDt92R V a l 4 * 0 . 5 90 BC 3 9 , 120 p D t l 2 0 R V a l 4 * 2 . 4 90 BC 3 9 , 120 pDt78R V a l 3b 5 . 2 84 D 1 3 , 119 + p D t 5 5 c o n t a i n s t w o t R N A 4 g e n e s w h i c h h a v e s e q u e n c e s i d e n t i c a l t o t h a t o f t R N A V 1 a l ( 1 4 , 3 9 ) . V a l * P l a s m i d s , pDt92R a n d p D t l 2 0 R c o d e f o r a t R N A 4 g e n e w h i c h d i f f e r s a t . f o u r p o s i t i o n s i n t h e c o d i n g r e g i o n c o m p a r e d t o t h e s e q u e n c e o f t R N A 4 a _ ( 1 4 , 3 9 ) . F i g u r e 4 . The s t r a t e g y u s e d f o r i n v i t r o c o n s t r u c t i o n o f p l a s m i d s . The f i g u r e s a r e shown t o s c a l e ( 1 . 0 cm - 80 n u c l e o t i d e s ) . The s o l i d , o p e n and h a t c h e d b a r s r e p r e s e n t t h e t R N A V a l g e n e c o n t a i n e d w i t h i n t h e p l a s m i d s , p D t 0 . 3 , pDt92R a n d p D t 7 8 R , r e s p e c t i v e l y . 5 ' and 3 ' d e n o t e t h e o r i e n t a t i o n o f t h e n o n c o d i n g DNA s t r a n d . The s o l i d s q u a r e s show t h e p r e s e n c e o f H i n d l l l l i n k e r s . The numbers on t h e l i n e d r a w i n g s i n d i c a t e t h e l e n g t h i n b a s e p a i r s o f s e g m e n t s b e t w e e n s h o r t v e r t i c a l l i n e s . H i n , D, H , S , X , F and Hpa d e s i g n a t e t h e c l e a v a g e s i t e s f o r R. e n d o n u c l e a s e s , H i n f l , D d e l , H i n d l l l , S m a l , X m a l , Fnu4HI and H p a l l , r e s p e c t i v e l y . A. Separation of two fRNA 4 genes of pOt55 Hmf I frnqment nf pDttt i — i * * 3- g 7" • ; 212 •— i £22 : ( i a n , < 62a B. pDK>3-92R hybrids » 3 X H I i i ? pDt92R 1 ^ "7 LJ 2 3 0 • »9 • • 282 • H S X l_ s Ji li I 2M , 23fi_ 8X H C. pDt03-78R hybrids pDtO-3 L_ pntO-3f5')-78R(3') PDf78R(5')-Q-3(3') D. Deletion mutants oDtO-3 pDtO-3A5l-6l pntn-^ri lfS ' - lB X H H J . I DDt78R I _245_ 245 49Q0 F Hpo Hpo H 235 ,11, S3 , 168 , 161 , H 1 Hpo H I 1 1 . 83 J f i L 3QO 4 9 0 0 a -I 0& , 3 8 . F i g u r e 5 . Scheme f o r c l o n i n g a DNA f r a g m e n t a f t e r l i n k e r l i g a t i o n P l a s m i d DNA 1 R e s t r i c t i o n e n d o n u c l e a s e d i g e s t i o n ( s t e p 1) I P o l I t r e a t m e n t t o f i l l up p r o t r u d i n g s i n g l e - s t r a n d t a i l s ( s t e p 3) I H i n d l l l K i n a s e v 3 2 P - H i n d l 1 1 H i n d l l l l i n k e r , l i n k e r * l i n k e r * l i g a t i o n ( b l u n t e n d ) ( s t e p 4) [ y - 3 2 P ] A T P I ( s t e p 2) j H i n d l l l d i g e s t i o n o f 3 2 P . l i n k e r - D N A ( s t e p 5) i P h e n o l i P r e p a r a t i v e g e l e l e c t r o p h o r e s i s ( s t e p 6 ) I El e c t r o e l u t i o n I P r e p a r a t i o n o f p B R 3 2 2 , > L i g a t i o n w i t h p B R 3 2 2 , H i n d l l l , CIAP ( s t e p 8) H i n d l l l , C I A P ( s t e p 7) T r a n s f o r m a t i o n o f E - c o l i , S F 8 ( s t e p 9 ) I Am ( s t e p 11) S e l e c t i o n o f p c o l o n i e s ( s t e p 10) S T e s t f o r T e t R e p l i c a P l a t e , H o g n e s s I 1 S c r e e n i n g f o r r e c o m b i n a n t c l o n e s ( s t e p 12) 3 9 , P l a s m i d , pDt55 ( 1 0 0 jug) was d i g e s t e d w i t h t h e R. e n d o n u c l e a s e , H i n f l and e l e c t r o p h o r e s e d on a 5% p r e p a r a t i v e p o l y a c r y l a m i d e g e l (1 . 5 x 175 x 220 mm). A 1.1 kb H i n f l f r a g m e n t ( w h i c h was p r e v i o u s l y shown t o c o n t a i n t h e t w o g e n e s ; 13) was e x c i s e d , e l e c t r o e l u t e d and d i g e s t e d w i t h a s e c o n d R. e n d o -n u c l e a s e , D d e l . The D d e l d i g e s t was e l e c t r o p h o r e s e d on a 5% p o l y a c r y l a m i d e g e l a n d t w o f r a g m e n t s , 0 . 3 kb and 0 . 6 kb w e r e e x c i s e d , e l e c t r o e l u t e d and e t h a n o l p r e c i p i t a t e d . The 0 . 3 kb f r a g m e n t was f l a n k e d by t w o D d e l s i t e s and t h e 0 . 6 kb f r a g m e n t was b o u n d e d by D d e l and H i n f l s i t e s . 2 . 5 ' e n d - l a b e l l i n g o f H i n d l l l l i n k e r - d(CAAGCTTG) ( 1 3 5 ) H i n d l l l l i n k e r as s u p p l i e d by t h e m a n u f a c t u r e r d i d n o t c o n t a i n a 5 ' p h o s p h a t e , t h e r e f o r e b e f o r e i t c o u l d be u s e d f o r l i g a t i o n , i t h a d t o be p h o s p h o r y l a t e d . I n o r d e r t o f o l l o w t h e i n c o r p o r a t i o n o f t h e l i n k e r i n t o t h e DNA, t h e l i n k e r was p h o s p h o r y l a t e d w i t h 3 2 P . The r e a c t i o n m i x c o n s i s t e d o f 1 . 2 p i H i n d l l l l i n k e r ( a p p r o x . 0 . 5 p g , 100 p m o l e s ) , 50 p C i [ Y - 3 2 P ] ATP ( ~ 2 0 0 0 C i / m m o l e ) , 1 / j l 10 x k i n a s e b u f f e r ( 0 . 7 M T r i s . H C l , pH 7 . 4 , 0 . 1 M M g C l 2 , 50 mM DTT) a n d 1 p i T 4 p o l y n u c l e o t i d e k i n a s e ( 5 U / p l ) . The v o l u m e was made up t o 10 p i w i t h w a t e r and t h e r e a c t i o n was i n c u b a t e d a t 3 7 ° C f o r 30 m i n . Then 1 p i 10 x k i n a s e b u f f e r , 1 p i 10 mM A T P , 1 p i T 4 k i n a s e and 7 p i w a t e r w e r e a d d e d , and t h e r e a c t i o n was i n c u b a t e d f o r an a d d i t i o n a l 30 m i n a t 3 7 ° C . The r e a c t i o n was s t o p p e d by e x t r a c t i n g o n c e w i t h p h e n o l ( w a t e r - s a t u r a t e d ) and t w i c e w i t h e t h e r . E x c e s s e t h e r was b l o w n o f f , and t h e 3 2 P . H i n d l l l l i n k e r was s t o r e d a t - 2 0 ° C . 3 . P o l I t r e a t m e n t t o f i l l up s i n g l e - s t r a n d e d e n d s ( 1 3 5 ) B o t h t h e D d e l a n d H i n f l s i t e s f l a n k i n g t h e 0 . 3 kb and 0 . 6 kb f r a g m e n t s ( s e c t i o n I V . A . 1 ) h a v e 5 ' - e x t e n d e d s e q u e n c e s . B e f o r e l i g a t i o n t o H i n d l l l l i n k e r , t h e s i n g l e - s t r a n d e d t e r m i n i w e r e f i l l e d i n w i t h P o l I . The 45 p i p o l y m e r a s e r e a c t i o n c o n s i s t e d o f t h e f o l l o w i n g : 5 p i r e s t r i c t i o n f r a g m e n t ( 0 . 2 - 0 . 5 p g ) , 4 . 5 p i 10 x P o l . I b u f f e r ( 0 . 6 M T r i s . H C l , pH 7 . 4 , 0 . 0 8 M M g C l 2 ) , 2 40, jul 0 . 2 M 0 - m e r c a p t o e t h a n o l , 4 jul 10 mM A T P , 0 . 8 jul. o f e a c h o f t h e dNTP (10 mM e a c h ) , 4 u n i t s E - c o l i p o l y m e r a s e I ( K l e n o w f r a g m e n t ) and w a t e r t o b r i n g t h e t o t a l v o l u m e t o 4 5 JJ1 . The r e a c t i o n was i n c u b a t e d a t 1 0 ° C f o r 15 m i n . 4 . 3 2 P . H i n d l l l l i n k e r a d d i t i o n t o DNA - b l u n t e n d l i g a t i o n ( 1 3 5 ) . B l u n t e n d l i g a t i o n i s n o t a s e f f i c i e n t a r e a c t i o n a s c o h e s i v e end l i g a t i o n ; t h e r e a c t i o n i s n o t l i n e a r w i t h r e s p e c t t o t h e DNA l i g a s e c o n c e n t r a t i o n and r e q u i r e s much l a r g e r a m o u n t s o f enzyme ( 1 0 - 3 0 f o l d ) t h a n c o h e s i v e - e n d l i g a t i o n ( 1 3 5 ) . To o v e r c o m e t h e i n e f f i c i e n c y o f b l u n t e n d l i g a t i o n r e a c t i o n , a 2 - 5 f o l d e x c e s s enzyme was u s e d and t h e r e a c t i o n was i n c u b a t e d o v e r n i g h t . A l s o t o m a x i m i z e l i g a t i o n o f l i n k e r t o DNA, a 2 0 - 3 0 f o l d m o l a r e x c e s s o f t h e l i n k e r o v e r t h e DNA was u s e d . The b l u n t - e n d l i g a t i o n r e a c t i o n c o n s i s t e d i n a d d i n g 1 . 5 jul 10X P o l . I b u f f e r , 0 . 8 jul 0 . 2 M 3 - m e r c a p t o e t h a n o l , 1 . 5 p i 10 mM A T P , 9 . 2 j j l 3 2 P . H i n d l l l l i n k e r ( a p p r o x i m a t e l y 50 p i c o m o l e s ) and 3 / j l DNA l i g a s e [1 / j l = 1 u n i t ( B R L ) = a p p r o x . 4 0 0 u n i t s ( N E B ) ] t o t h e p o l y m e r a s e r e a t i o n ( s t e p 3 a b o v e ) . The r e a c t i o n was i n c u b a t e d a t 1 4 ° - 1 6 ° C o v e r n i g h t . 5 . H i n d l l l d i g e s t i o n o f 3 2 P . l i n k e r - D N A ( 1 3 5 ) The l i g a t i o n r e a c t i o n f r o m s t e p 4 ( ~ 60 j u l ) was d i l u t e d w i t h 250 jul 7 mM MgCl 2 and 60 mM NaCl and was h e a t e d a t 6 5 ° C f o r 5 m i n , t o i n a c t i v a t e t h e e n z y m e s . 10 jul o f H i n d I I I ( 5 U / j u l ) was a d d e d and t h e r e a c t i o n was i n c u b a t e d a t 3 7 ° C o v e r n i g h t ; s u b s e q u e n t l y i t was e x t r a c t e d o n c e w i t h p h e n o l , o n c e w i t h e t h e r and was e t h a n o l p r e c i p i t a t e d . 6 . P r e p a r a t i v e g e l e l e c t r o p h o r e s i s and e l e c t r o e l u t i o n The H i n d l l l d i g e s t e d 3 2 P.1 i n k e r - D N A and an a l i q u o t o f 3 2 P . 1 i n k e r - D N A b e f o r e H i n d l l l d i g e s t i o n w e r e e l e c t r o p h o r e s e d on a 7% p o l y a c r y l a m i d e g e l , and t h e g e l was a u t o r a d i o g r a p h e d . The s a m p l e p r i o r t o H i n d l l l d i g e s t i o n showed a l a d d e r o f o l i g o m e r s a n d a b a n d a t t h e p o s i t i o n e x p e c t e d f o r t h e i n p u t r e s t r i c t i o n f r a g m e n t ( 0 . 3 o r 0 . 6 k b ) . The s a m p l e a f t e r H i n d l l l d i g e s t i o n showed no l i n k e r p o l y m e r s , s u g g e s t i n g t h a t H i n d l l l d i g e s t i o n was c o m p l e t e . The b a n d ( s ) c o r r e s p o n d i n g t o t h e l e n g t h o f t h e i n p u t DNA ( 0 . 3 o r 0 . 6 k b ) was e x c i s e d , e l e c t r o e l u t e d , e t h a n o l p r e c i p i t a t e d , r e s u s p e n d e d i n 8 /J] 0 . 2 x l i g a s e b u f f e r ( 1 0 x l i g a s e b u f f e r i s 0 . 6 6 M T r i s - , H C 1 , pH 7 . 4 , 66 mM MgCl 2 , 0 . 1 M DTT) and s t o r e d a t - 2 0 ° C . 7 . P r e p a r a t i o n o f p h o s p h a t a s e - t r e a t e d l i n e a r pBR322 ( 1 3 5 ) pBR322 (15 jjg) was d i g e s t e d w i t h 15 u n i t s o f H i n d l l l i n a 50 jul r e a c t i o n . A f t e r t w o h o u r s i n c u b a t i o n a t 3 7 ° C , t h e r e a c t i o n was d i l u t e d w i t h 4 5 0 p i o f w a t e r and was h e a t e d a t 6 5 ° C f o r 10 m i n . t o i n a c t i v a t e t h e r e s t r i c t i o n e n z y m e . F o l l o w i n g t h i s , 5 jul IM T r i s , pH 9 . 0 and 1 . 5 >ul ( 3 u n i t s ) c a l f i n t e s t i n a l a l k a l i n e p h o s p h a t a s e ( C 1 A P ) was a d d e d and t h e r e a c t i o n was i n c u b a t e d a t 6 5 ° C f o r 30 m i n . A f t e r 30 m i n . , a n o t h e r 1 . 5 jul CIAP was a d d e d a n d t h e r e a c t i o n was i n c u b a t e d f o r an a d d i t i o n a l 30 m i n . a t 6 5 ° C . The enzyme C I A P was i n a c t i v a t e d by e x t r a c t i n g t h r e e t i m e s w i t h p h e n o l and t h e l a s t t r a c e s o f p h e n o l w e r e r e m o v e d by e x t r a c t i n g w i t h e t h e r t h r e e t i m e s . The DNA was e t h a n o l p r e c i p i t a t e d , r e s u s p e n d e d i n 0 . 2 ml 10 mM T r i s . H C l (pH 7 . 4 ) , 1 mM EDTA (1 jul = 0 . 7 5 jug) and s t o r e d a t - 2 0 ° C . P h o s p h a t a s e - t r e a t e d , l i n e a r p B R 3 2 2 , was t e s t e d f o r i t s a b i l i t y t o s e r v e a s a s u b s t r a t e f o r t h e l i g a t i o n r e a c t i o n and a l s o f o r r e s i d u a l p h o s p h a t a s e a c t i v i t y . In b o t h i n s t a n c e s t h e r e s u l t was n e g a t i v e a s e x p e c t e d . 8 . L i g a t i o n o f 3 2 P . l i n k e r - D N A t o p h o s p h a t a s e - t r e a t e d , H i n d l H - d i g e s t e d  pBR322 ( 1 3 5 ) To t h e 8 jul o f 3 2 P . l i n k e r - D N A ( s t e p 6) was a d d e d 2 jul ( 1 5 0 ng) H i n d l l l -d i g e s t e d , p h o s p h a t a s e - t r e a t e d pBR322 ( s t e p 7 ) , 1 . 5 /ul l O x l i g a s e b u f f e r , 1 . 5 J JI 10 mM ATP and t h e m i x t u r e was h e a t e d a t 3 7 ° C f o r 2 m i n . 2 jul (2 u n i t s ) T DNA l i g a s e was a d d e d a n d t h e r e a c t i o n was i n c u b a t e d a t 1 4 - 1 6 ° C o v e r n i g h t . 9 . T r a n s f o r m a t i o n o f E - c o l i ( S F 8 ) E - c o l i ( S F 8 ) was t r a n s f o r m e d w i t h t h e l i g a t e d DNA e s s e n t i a l l y a s 42, d e s c r i b e d by Dunn e t a l _ . (119). J u s t b e f o r e t r a n s f o r m a t i o n , 0 . 1 ml o f 0 . 0 3 M C a C l 2 was a d d e d t o t h e l i g a t i o n r e a c t i o n ( s t e p 8 ) . The m i x t u r e was a d d e d t o 0 . 2 ml o f c o m p e t e n t c e l l s (119) and i n c u b a t e d on i c e f o r 60 m i n . A f t e r 60 m i n . o n i c e , t h e c e l l s w e r e h e a t - s h o c k e d by i n c u b a t i n g a t 4 2 ° C f o r 10 m i n . The b a c t e r i a t h e n w e r e d i l u t e d i n t o 10 ml L u r i a b r o t h ( L B ) and g r o w n f o r 3 h o u r s a t 3 0 ° C . R 1 0 . S e l e c t i o n o f a m p i c i l l i n r e s i s t a n t (Amp ) c o l o n i e s The t r a n s f o r m a t i o n m i x ( s e c t i o n 9 ) was p l a t e d on LB a g a r p l a t e s c o n t a i n i n g 50 /jg/ml a m p i c i l l i n (119) t o s e l e c t f o r b a c t e r i a w i t h p l a s m i d DNA. S e v e r a l c o n t r o l s w e r e p e r f o r m e d t o a s s e s s t h e q u a l i t y o f t h e p l a s m i d v e c t o r u s e d i n t h e t r a n s f o r m a t i o n p r o c e s s : a) s u p e r c o i l e d pBR322 g a v e a h i g h 7 R t r a n s f o r m a t i o n f r e q u e n c y ( ~1 x 10 Amp t r a n s f o r m a n t s / / j g o f D N A ) , b) l i n e a r 4 R pBR322 g a v e a 2 0 0 - f o l d l o w e r t r a n s f o r m a t i o n f r e q u e n c y (~5 x 10 Amp c o l o n i e s / p g ) , c ) Hi n d 1 1 1 - C I A P t r e a t e d pBR322 d e c r e a s e d t h e t r a n s f o r m a t i o n f r e q u e n c y a n o t h e r 1 0 - f o l d ( ~ 5 x 10^ A m p R c o l o n i e s / p g ) a n d t h i s f r e q u e n c y d i d n o t i n c r e a s e a f t e r s e l f - 1 i g a t i o n o f t h e H i n d l l I - C I A P t r e a t e d p B R 3 2 2 . The f r e q u e n c y o f t r a n s f o r m a t i o n w i t h t h e l i g a t e d DNA had a w i d e r a n g e q R a n d v a r i e d f r o m 1 - 8 x 10 Amp t r a n s f o r m a n t s / i j g . s 1 1 . T e s t f o r t e t r a c y c l i n e s e n s i t i v i t y ( T e t ) ; H o g n e s s p r o c e d u r e Amp c o l o n i e s w e r e e i t h e r p i c k e d a n d s t a b b e d o r r e p l i c a p l a t e d o n t o LB a g a r p l a t e s c o n t a i n i n g 20 /jg/ml t e t r a c y c l i n e . A b o u t 100% o f t h e Amp c o l o n i e s R o b t a i n e d w i t h e i t h e r s u p e r c o i l e d o r l i n e a r pBR322 w e r e T e t a s e x p e c t e d ; b u t o n l y 8 5 - 9 0 % o f t h e Amp c o l o n i e s o b t a i n e d w i t h H i n d l I I - C I A P t r e a t e d pBR322 w e r e T e t . I t h a s b e e n r e p o r t e d ( 1 3 5 , 1 3 6 ) t h a t d e l e t i o n s a t t h e H i n d l l l s i t e o f pBR322 a r e s o m e t i m e s g e n e r a t e d d u r i n g H i n d l l l d i g e s t i o n and p h o s p h a t a s e t r e a t m e n t ; c o l o n i e s t r a n s f o r m e d w i t h t h e s e m o l e c u l e s w i l l be Amp] T e t b u t w o u l d n o t c o n t a i n r e c o m b i n a n t DNA ( b e l o w ) . R e c o m b i n a n t s , i . e . A m p R t r a n s f o r m a n t s , w h i c h h a v e i n s e r t e d a p i e c e o f R S R DNA a t t h e H i n d l l l s i t e , s h o u l d be Amp T e t . 2 0 - 7 0 % o f t h e Amp c o l o n i e s S R S o b t a i n e d w i t h t h e l i g a t e d DNA w e r e T e t - n o t a l l o f t h e s e Amp T e t c o l o n i e s c o n t a i n e d r e c o m b i n a n t DNA ( d i s c u s s e d a b o v e ) when t e s t e d d i r e c t l y . R c I n o r d e r t o a v o i d t h e p r o b l e m o f p i c k i n g up f a l s e p o s i t i v e Amp T e t " c o l o n i e s , Amp t r a n s f o r m a n t s o b t a i n e d w i t h t h e l i g a t e d DNA w e r e r e p l i c a p l a t e d o n t o n i t r o c e l l u l o s e f i l t e r s and grown on LB a g a r p l a t e s c o n t a i n i n g 50 /jg/ml a m p i c i 1 1 i n ( 1 1 9 ) . The f i l t e r s w e r e t r e a t e d a c c o r d i n g t o t h e p r o c e d u r e o f G r u n s t e i n and H o g n e s s ( 1 3 7 ) as d e s c r i b e d by Dunn e t a l _ . ( 1 1 9 ) a n d h y b r i d i z e d t o [ 1 2 5 I ] t R N A 4 a l ( 1 1 9 ) . 1 2 . S c r e e n i n g f o r r e c o m b i n a n t c l o n e s A p o s i t i v e c o l o n y a f t e r h y b r i d i z a t i o n was i n o c u l a t e d i n t o 10 ml LB and g r o w n o v e r n i g h t a t 3 0 ° C . A 250 ml p l a s m i d p r e p a r a t i o n was made f r o m t h i s o v e r n i g h t c u l t u r e a c c o r d i n g t o t h e m e t h o d d e s c r i b e d e a r l i e r ( s e c t i o n I . A ) e x c e p t t h a t e v e r y t h i n g was r e d u c e d by o n e - f o u r t h . The DNA was a n a l y z e d b e f o r e and a f t e r d i g e s t i o n w i t h a p p r o p r i a t e r e s t r i c t i o n e n z y m e s t o d e t e r m i n e i f t h e i n s e r t e d DNA was c o r r e c t a n d was o f t h e e x p e c t e d m . w t . The c l o n e c o n t a i n i n g t h e 0 . 3 kb i n s e r t was f u r t h e r i d e n t i f i e d by DNA s e q u e n c e a n a l y s i s . The c l o n e s c o n t a i n i n g t h e 0 . 3 kb and t h e 0 . 6 kb DNA i n s e r t s ( d e r i v e d f r o m p D t 5 5 ) w i l l be r e f e r r e d t o a s p D t 0 . 3 and p D t 0 . 6 r e s p e c t i v e l y . B . C o n s t r u c t i o n o f h y b r i d g e n e s and d e l e t i o n m u t a n t s The s t r a t e g y u s e d t o c o n s t r u c t t h e h y b r i d g e n e s and t h e d e l e t i o n m u t a n t s i s shown i n F i g . 4 B , C , D. The g e n e r a l p r o c e d u r e u s e d i s a s o u t l i n e d i n F i g . 5 , e x c e p t f o r m o d i f i c a t i o n s a t two s t a g e s : i ) In t h e p r e p a r a t i o n o f r e s t r i c -t i o n f r a g m e n t s f o r c l o n i n g ( s t e p 1 i n t h e s c h e m e ) . i i ) In t h e a d d i t i o n o f H i n d l l l l i n k e r s t o t h e f r a g m e n t ( s t e p s 3 - 6 i n t h e s c h e m e ) . When t h e h y b r i d o r t h e d e l e t e d f r a g m e n t t o be c l o n e d was b o u n d e d by 2 H i n d l l l s i t e s , s t e p s 3 - 6 w e r e o m i t t e d ; i f t h e f r a g m e n t was f l a n k e d by one o r no H i n d l l l s i t e , s t e p s 3 - 6 w e r e i n c l u d e d . 1 . H y b r i d s b e t w e e n p D t 0 . 3 and pDt92R DNAs ( F i g . 4B) a ) p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) h y b r i d P l a s m i d s p D t 0 . 3 a n d pDt92R ( 1 0 0 /jg e a c h ) w e r e d i g e s t e d w i t h H i n d l l l , t h e d i g e s t s w e r e e l e c t r o p h o r e s e d on a 5% p o l y a c r y l a m i d e g e l , and t h e H i n d l l l i n s e r t DNAs w e r e r e c o v e r e d by e l e c t r o e l u t i o n . B o t h t h e i n s e r t DNAs w e r e d i g e s t e d by a s e c o n d R. e n d o n u c l e a s e , S a u 9 6 1 , t h e d i g e s t s w e r e e l e c t r o -p h o r e s e d on a 12% p o l y a c r y l a m i d e g e l and f r a g m e n t s 99 bp and 230 bp ( f r o m p D t 0 . 3 ; 3 9 ) a n d 2 3 6 bp a n d 252 bp ( f r o m p D t 9 2 R | 3 9 ) l o n g w e r e i s o l a t e d b y e l e c t r o e l u t i o n . E a c h o f t h e 9 9 , 230 and 252 bp f r a g m e n t was f l a n k e d by a H i n d l l l and a Sau961 s i t e ; t h e 236 bp f r a g m e n t was b o u n d e d by two Sau961 s i t e s . The 230 bp f r a g m e n t f r o m p D t 0 . 3 ( c o n t a i n i n g t h e 5 ' - f l a n k i n g s e q u e n c e and V a l t h e f i r s t 44 n u c l e o t i d e s o f t h e t R N A 4 c o d i n g r e g i o n ) was l i g a t e d t o t h e 236 V a l bp f r a g m e n t f r o m pDt92R ( c o n t a i n i n g t h e l a s t 29 n u c l e o t i d e s o f t h e t R N A 4 c o d i n g r e g i o n and t h e 3 ' - f l a n k i n g s e q u e n c e ) . The l i g a t i o n r e a c t i o n c o n t a i n e d 5 JJI 230 bp f r a g m e n t ( ~ 0 . 2 j u g ) , 5 ^1 236 bp f r a g m e n t ( - . 0 . 2 j u g ) , 1 . 5 jul 10 x l i g a s e b u f f e r , 1 . 5 jul 10 mM ATP and 2 jul (2U) T DNA l i g a s e . The r e a c t i o n was i n c u b a t e d a t 1 4 - 1 6 ° C o v e r n i g h t . The h y b r i d f r a g m e n t o f i n t e r e s t ( 4 6 6 b p ) was f l a n k e d by o n e H i n d l l l a n d o n e S a u 9 6 I s i t e . H i n d l l l l i n k e r was p r e f e r e n t i a l l y a d d e d t o t h e S a u 9 6 I end by t h e p r o c e d u r e d e s c r i b e d i n s e c t i o n A ( s t e p s 3 - 6 ) , e x c e p t t h a t t h e p o l y -m e r i z a t i o n s t e p d i d n o t i n c l u d e a n y c o l d d A T P ; dAMP i s t h e f i r s t n u c l e o t i d e r e q u i r e d t o s t a r t f i l l i n g t h e H i n d l l l s i t e b u t i s n o t r e q u i r e d t o f i l l up t h e Sau961 s i t e . The r e s t o f t h e p r o c e d u r e ( s t e p s 7 - 1 2 ) r e m a i n e d t h e s a m e , b ) p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) h y b r i d The 252 bp f r a g m e n t f r o m pDt92R ( c o n t a i n i n g t h e 5 ' - f l a n k i n g s e q u e n c e and V a l t h e f i r s t 44 n u c l e o t i d e s o f t h e t R N A 4 c o d i n g r e g i o n ) was l i g a t e d t o t h e 45. 99 bp f r a g m e n t f r o m p D t 0 . 3 ( c o n t a i n i n g t h e l a s t 29 n u c l e o t i d e s o f t h e t R N A 4 c o d i n g r e g i o n a n d t h e 3 ' - f l a n k i n g s e q u e n c e ) . The r e a c t i o n c o n d i t i o n s w e r e t h e same as t h o s e d e s c r i b e d a b o v e . The h y b r i d f r a g m e n t o f r e l e v a n c e (351 bp) was f l a n k e d by t w o H i n d l l l s i t e s , t h e r e f o r e , s t e p s 3 - 6 w e r e o m i t t e d . The r e s t o f t h e p r o c e d u r e r e m a i n e d t h e s a m e . The two h y b r i d c l o n e s , p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) , and p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) w e r e c h a r a c t e r i z e d by d i g e s t i o n w i t h a p p r o p r i a t e r e s t r i c t i o n e n z y m e s and DNA s e q u e n c e a n a l y s e s . 2 . H y b r i d s b e t w e e n p D t 0 . 3 and pDt78R DNAs ( F i g . 4C) a ) p D t Q . 3 ( 5 ' ) - 7 8 R ( 3 ' ) h y b r i d p D t 0 . 3 and pDt78R ( 1 0 /jg e a c h ) DNAS w e r e s e q u e n c i a l l y d i g e s t e d w i t h Xmal a n d H i n d l l l , t h e d i g e s t s w e r e e l e c t r o p h o r e s e d on a 10% p o l y a c r y l a m i d e g e l and f r a g m e n t s 84 bp and 2 4 5 bp ( f r o m p D t 0 . 3 ; 3 9 ) and 300 bp and 4 . 9 kb ( f r o m p D t 7 8 R ) l o n g w e r e r e c o v e r e d by e l e c t r o e l u t i o n . E a c h o f t h e f o u r f r a g m e n t s was f l a n k e d by a H i n d l l l a n d a Xmal s i t e . The 245 bp f r a g m e n t f r o m p D t 0 . 3 ( c o n t a i n i n g t h e 5 ' - f l a n k i n g s e q u e n c e and t h e f i r s t 59 n u c l e o t i d e s o f t h e t R N A 4 a l c o d i n g r e g i o n ) was l i g a t e d t o t h e 4 . 9 kb f r a g m e n t f r o m pDt78R ( c o n t a i n i n g t h e l a s t 14 n u c l e o t i d e s o f t h e t R N A ^ 1 c o d i n g r e g i o n and t h e 3 ' - f l a n k i n g s e q u e n c e ) . The l i g a t i o n r e a c t i o n c o n d i t i o n s w e r e t h e same a s t h o s e d e s c r i b e d u n d e r s e c t i o n 1 ( a ) a b o v e . The h y b r i d f r a g m e n t o f i n t e r e s t ( - 5 . 2 k b ) was f l a n k e d by two H i n d l l l s i t e s , h e n c e s t e p s 3 - 6 w e r e o m i t t e d . The r e s t o f t h e p r o c e d u r e r e m a i n e d t h e s a m e . The h y b r i d c l o n e was c h a r a c t e r i z e d by a p p r o p r i a t e r e s t r i c t i o n and DNA s e q u e n c e a n a l y s e s . b ) p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) h y b r i d The 300 bp f r a g m e n t f r o m pDt78R ( c o n t a i n i n g t h e 5 ' - f l a n k i n g s e q u e n c e and t h e f i r s t 59 n u c l e o t i d e s o f t h e t R N A ^ a l c o d i n g r e g i o n ) was l i g a t e d t o t h e 84 46. V a l bp f r a g m e n t f r o m p D t 0 . 3 ( c o n t a i n i n g t h e l a s t 14 n u c l e o t i d e s o f t h e t R N A 4 c o d i n g r e g i o n a n d t h e 3 ' - f l a n k i n g s e q u e n c e ) . The r e l e v a n t h y b r i d f r a g m e n t was 384 bp l o n g a n d was f l a n k e d by t w o H i n d l l l s i t e s . The r e s t o f t h e p r o c e d u r e r e m a i n e d t h e same a s d e s c r i b e d f o r p D t O . 3 ( 5 ' ) - 7 8 R ( 3 ' ) h y b r i d . The h y b r i d c l o n e , p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) was i d e n t i f i e d by d i g e s t i o n w i t h a p p r o p r i a t e r e s t r i c t i o n e n z y m e s . 3 . C o n s t r u c t i o n o f d e l e t i o n m u t a n t s ( F i g . 4D) a ) I n t e r n a l d e l e t i o n m u t a n t - p D t 0 . 3A 51 -61 The p l a s m i d p D t 0 . 3 ( - 1 0 0 jug) was d i g e s t e d w i t h H i n d l l l . The H i n d l l l i n s e r t DNA was i s o l a t e d and c l e a v e d w i t h H p a l l . In o r d e r t o s e e a l l t h e f r a g m e n t s g e n e r a t e d by H p a l l c l e a v a g e , o n e - t e n t h o f t h e H p a l l d i g e s t i o n m i x was l a b e l l e d w i t h E - c o l i DNA p o l . I ( K l e n o w f r a g m e n t ) and [ > - 3 2 P ] d C T P . The l a b e l l e d s a m p l e was a d d e d t o t h e r e s t o f t h e H p a l l c l e a v e d DNA and t h e w h o l e m i x was e l e c t r o p h o r e s e d on a 12% p o l y a c r y l a m i d e g e l . A u t o r a d i o g r a p h y showed t h r e e b a n d s o f t h e f o l l o w i n g l e n g t h s : 2 3 5 , 83 and 11 b p . The 235 and 8 3 bp f r a g m e n t s w e r e e x c i s e d , e l e c t r o e l u t e d a n d l i g a t e d a s d e s c r i b e d i n t h e p r e v i o u s s e c t i o n s . The l i g a t e d f r a g m e n t o f i n t e r e s t (318 bp) was f l a n k e d by two H i n d I I I s i t e s . The r e s t o f t h e p r o c e d u r e t o c l o n e t h i s f r a g m e n t ( s t e p s 7 - 1 2 , F i g . 5) was as d e s c r i b e d b e f o r e . O m i t t i n g t h e 11 bp f r a g m e n t i n t h e l i g a t i o n r e a c t i o n w o u l d c r e a t e a 11 bp d e l e t i o n o f n u c l e o t i d e s 51 and 61 ( 3 9 ) i n - t h e c o d i n g r e g i o n . T h i s d e l e t i o n m u t a n t was c h a r a c t e r i z e d by a p p r o p r i a t e r e s t r i c t i o n and DNA s e q u e n c e a n a l y s e s . b ) D e l e t i o n o f t h e 5 ' - f l a n k i n g s e q u e n c e - p D t 0 . 3 d l 5 ' - 1 8 p D t 0 . 3 ( ~ 1 0 0 / u g ) was d i g e s t e d w i t h H i n d l l l , t h e H i n d l l l i n s e r t DNA was i s o l a t e d and was d i g e s t e d w i t h a s e c o n d e n z y m e , F n u 4 H l . The d i g e s t was e l e c t r o p h o r e s e d on a 12% p o l y a c r y l a m i d e g e l and a f r a g m e n t 161 bp l o n g was r e c o v e r e d by e l e c t r o e l u t i o n . The 161 bp f r a g m e n t was f l a n k e d by a Fnu4Hl a n d a H i n d l l l s i t e and c o n t a i n e d o n l y 18 n u c l e o t i d e s 5 ' t o t h e b e g i n n i n g o f 47. t h e c o d i n g r e g i o n ( 3 9 ) . H i n d l l l l i n k e r was a d d e d t o t h e e n d s o f t h e 161 bp f r a g m e n t and t h e f r a g m e n t was c l o n e d i n t o E - c o l i ( S F 8 ) a c c o r d i n g t o s t e p s 3 - 1 2 ( F i g . 5 , s e c t i o n A ) . The d e l e t i o n m u t a n t , p D t O . 3 d l 5 1 - 1 8 was i d e n t i f i e d by d i g e s t i o n w i t h a p p r o p r i a t e r e s t r i c t i o n e n z y m e s . 48 . Results I. DNA sequence a n a l y s i s of plasmid pDt!20R The recombinant plasmid pDtl20R contains 2.4 kb of Drosophila DNA i n s e r t e d i n t o the H i n d l l l s i t e of the plasmid v e c t o r , pBR322 (Table I I I ) . V a l pDtl20R was presumed to contain a tRNA gene on the basis of i t s h y b r i d i z a -r i 2 5 , V a l , 4 t i o n t o [ l ] - t R N A 4 (119). In s i t u h y b r i d i z a t i o n of pDtl20R t o Drosophila polytene chromosomes showed that i t s Drosophila i n s e r t DNA o r i g i n a t e d from the V a l 90 BC s i t e (120, 39), one of the minor s i t e s of tRNA4 h y b r i d i z a t i o n (29). V a l The nucleotide sequence of a segment of pDtl20R c o n t a i n i n g the tRNA 4 gene i s presented i n the f o l l o w i n g s e c t i o n s . V a l A. Strategy used to sequence the tRNA^, gene contained i n pDt!20R Plasmid pDt120R c a r r i e s a f a i r l y l a r g e piece (2.4 kb) of Drosophila DNA. V a l The f i r s t o b j e c t i v e i n sequencing the tRNA 4 gene contained i n t h i s plasmid was to i s o l a t e a smaller fragment of DNA which contained the gene and which was amenable to sequence determination by the Maxam and G i l b e r t method. V a l , P r e l i m i n a r y RNA sequence a n a l y s i s of tRNA 4 (14) showed that i t contained the sequence CCCGGG, the r e c o g n i t i o n sequence of R. endonuclease Xmal. The a d d i t i o n a l f a c t that vector pBR322 d i d not have any r e c o g n i t i o n s i t e f o r Xmal made the presence of t h i s s i t e w i t h i n the gene a very convenient s t a r t i n g V a l point f o r sequencing the tRNA4 gene of pDtl20R. Plasmid pDtl20R was digested with Xmal to determine the number of Xmal V a l s i t e s and hence an estimate of the maximum number of tRNA 4 genes i t contained. The r e s u l t s of t h i s a n a l y s i s are shown i n F i g . 6. Dig e s t i o n of pDtl20R with Xmal (lane 3) generated a s i n g l e fragment 6.6 kb long, which approximated the s i z e of the l i n e a r recombinant plasmid ( 6.8 kb) quite w e l l . The r e s u l t suggested that pDtl20R contained only one Xmal s i t e , hence only V a l one tRNA 4 gene. In order to determine the l o c a t i o n of the Xmal s i t e w i t h i n the i n s e r t 49. F i g u r e 6 . D e t e r m i n a t i o n o f t h e number o f c l e a v a g e s i t e s f o r t h e R. e n d o n u -c l e a s e Xmal i n p D t l 2 0 R The p l a s m i d , p D t l 2 0 R ( l /jg) was d i g e s t e d w i t h t h e R. e n d o n c u l e a s e Xmal ( l a n e 3 ) , H i n d l l l ( l a n e 4) and X m a l , H i n d l l l ( l a n e 5 ) , t h e r e s u l t i n g f r a g m e n t s w e r e 3 ' e n d - l a b e l l e d a s d e s c r i b e d i n M e t h o d s , e l e c t r o p h o r e s e d on a 4% p o l y -a c r y l a m i d e g e l and t h e g e l was a u t o r a d i o g r a p h e d . L a n e 2 shows pBR322 DNA d i g e s t e d w i t h H i n d l l l and L a n e 1 c o n t a i n s H i n d l l l c l e a v e d l a m b d a DNA a s m . w t . m a r k e r . 50. 5 1 , DNA, p D t l 2 0 R was c l e a v e d w i t h Xmal and H i n d l l l and t h e r e s u l t i n g f r a g m e n t s w e r e e n d - l a b e l l e d . L a n e 5 shows t h a t X m a l , H i n d l l l d i g e s t i o n g e n e r a t e d t h r e e f r a g m e n t s a p p r o x i m a t e l y 4 . 4 k b , 2 . 1 kb and 270 bp l o n g . The sum o f t h e l a t t e r t w o a p p r o x i m a t e s t h e 2 . 4 kb s i z e o f t h e D r o s o p h i l a DNA i n p D t l 2 0 R ( l a n e 4 ) , h e n c e t h e s e f r a g m e n t s m u s t o r i g i n a t e f r o m t h e i n s e r t DNA; t h e f o r m e r f r a g m e n t c o r r e s p o n d s t o t h e l e n g t h o f l i n e a r pBR322 ( c o m p a r e w i t h l a n e 2 ) . p D t l 2 0 R ( 5 p g ) was d i g e s t e d w i t h Xmal ( o r H i n d l l l ) , t h e d i g e s t s w e r e l a b e l l e d a p p r o p r i a t e l y w i t h [ a - 3 2 p ] d C T P ( s e e M a t e r i a l s and M e t h o d s ) t o l a b e l t h e Xmal ( o r t h e H i n d l l l ) s i t e and t h e n c l e a v e d w i t h H i n d l l l ( o r X m a l ) t o g e n e r a t e s i n g l e e n d - l a b e l l e d f r a g m e n t s . The f r a g m e n t s w e r e s e p a r a t e d by e l e c t r o p h o r e s i s on p r e p a r a t i v e p o l y a c r y l a m i d e g e l s and f r a g m e n t s 270 bp and 2 . 1 kb w e r e i s o l a t e d and s e q u e n c e d a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . The a u t o r a d i o g r a m o f one s u c h s e q u e n c i n g e x p e r i m e n t i s shown i n F i g . 7. The s e q u e n c e o b t a i n e d f r o m t h e two 270 bp f r a g m e n t s l a b e l l e d e i t h e r a t t h e Xmal o r t h e H i n d l l l s i t e p r o v i d e d t h e c o m p l e t e s e q u e n c e o f t h e 270 bp f r a g m e n t ( F i g . 8 ) . The a d d i t i o n a l s e q u e n c e o b t a i n e d f r o m t h e 2.1 kb f r a g m e n t l a b e l l e d a t t h e Val Xmal s i t e c o m p l e t e d t h e s e q u e n c e o f t h e t R N A 4 g e n e c o n t a i n e d i n p D t l 2 0 R ( F i g s . 7 and 8 ) . The s t r a t e g y u s e d t o c o n f i r m t h e DNA s e q u e n c e o b t a i n e d f r o m t h e e x p e r i m e n t s d e s c r i b e d a b o v e and t o g e t a d d i t i o n a l s e q u e n c e o f t h e f l a n k i n g r e g i o n s , i s s u m m a r i z e d i n F i g . 8 . P r e l i m i n a r y r e s t r i c t i o n m a p p i n g e x p e r i m e n t s had i n d i c a t e d t h e p r e s e n c e o f an E c o R I , r a t h e r an E c o R I * s i t e ( d i s c u s s e d b e l o w ) , 170 bp f r o m t h e Xmal s i t e . E c o R I i s r e p o r t e d t o r e c o g n i z e and c l e a v e t h e s e q u e n c e AATT i n a d d i t i o n t o i t s n o r m a l r e c o g n i t i o n s e q u e n c e , GAATTC, u n d e r c o n d i t i o n s o f l o w i o n i c s t r e n g t h a n d pH 8 . 5 ( 1 3 8 ) . T h i s 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 y o f E c o R I u n d e r t h e s e c o n d i t i o n s i s t e r m e d t h e E c o R I * a c t i v i t y . I t was o b s e r v e d t h a t when p D t l 2 0 R was d i g e s t e d w i t h E c o R I u n d e r s t a n d a r d F i g u r e 7 . Maxam and G i l b e r t s e q u e n c i n g o f t h e t R N A 4 g e n e c o n t a i n e d i n p D t l 2 0 R . p D t l 2 0 R DNA (5 /jg) was c l e a v e d w i t h X m a l , t h e f r a g m e n t was 3 ' e n d -l a b e l l e d a n d t h e n d i g e s t e d w i t h H i n d l l l as d e s c r i b e d i n M e t h o d s . The t w o f r a g m e n t s , 270 bp and 2.1 kb ( s e e F i g . 1) w e r e i s o l a t e d and t h e i r DNA s e q u e n c e was d e t e r m i n e d by t h e Maxam and G i l b e r t m e t h o d . S a m p l e s o f t h e s e q u e n c i n g r e a c t i o n s s p e c i f i c f o r G ( l a n e s 1 a n d 5 ) , A + G ( l a n e s 2 and 6 ) , T + C ( l a n e s 3 and 7) a n d C ( l a n e s 4 and 8) w e r e a p p l i e d t o a d e n a t u r i n g 25% p o l y -a c r y l a m i d e g e l and e l e c t r o p h o r e s e d a t 1 8 0 0 V ( 4 5 V/cm) f o r 4 h o u r s . The 270 bp f r a g m e n t ( l a n e s 1 - 4 ) shows t h e s e q u e n c e o f t h e n o n c o d i n g s t r a n d w h e r e a s t h e 2 . 1 kb f r a g m e n t ( l a n e s 5 - 8 ) t h a t o f t h e c o d i n g s t r a n d . The 5 ' and 3 ' - e n d s o f t h e t R N AY g e n e a r e i n d i c a t e d . 5 3 . F i g u r e 8 . The s t r a t e g y u s e d t o s e q u e n c e a s e g m e n t o f p D t l 2 0 R DNA c o n t a i n i n g t h e t R N A ^ a l g e n e . V a l The s o l i d b a r r e p r e s e n t s t h e t R N A 4 gene o f p D t l 2 0 R . The b r o k e n l i n e s d e n o t e pBR322 s e q u e n c e s . S , E , H , X and E* d e s i g n a t e r e s t r i c t i o n s i t e s f o r R. e n d o n u c l e a s e s , S a u 9 6 I , E c o R I , H i n d l l l , Xmal and E c o R I * , r e s p e c t i v e l y . The n u m b e r s 1 , 2 a n d 3 i n d i c a t e t h e f i r s t , s e c o n d and t h i r d S a u 9 6 I s i t e r e s p e c t i v e l y . E a c h a r r o w r e p r e s e n t s a n u c l e o t i d e s e q u e n c e d e t e r m i n e d by t h e m e t h o d o f Maxam and G i l b e r t (1 cm « 60 n u c l e o t i d e s ) . F o r s e q u e n c i n g , p D t ! 2 0 R DNA was e n d - l a b e l l e d a t t h e c l e a v a g e s i t e s o f R. e n z y m e s l i s t e d i n t h e s i d e c o l u m n . A r r o w s p o i n t i n g t o t h e r i g h t i n d i c a t e t h a t t h e s e q u e n c e o f t h e c o d i n g s t r a n d was d e t e r m i n e d ; t h o s e p o i n t i n g t o t h e l e f t i n d i c a t e t h a t t h e s e q u e n c e o f t h e n o n c o d i n g s t r a n d was d e t e r m i n e d . Restriction enzyme •Xma I Hind III EcoR I Sou96 (i) S E H (2) S X (3) S J L E c o R I r e s t r i c t i o n c o n d i t i o n s ( 1 0 0 mM T r i s . H C l , pH 7 . 4 , 50 mM N a C l , 5 mM M g C l 2 ) i t g a v e t w o m a j o r f r a g m e n t s , a p p r o x i m a t e l y 470 bp and 6 . 2 kb l o n g ( F i g . 9 , l a n e 4 ) . The l a r g e r f r a g m e n t was e x p e c t e d f r o m c l e a v a g e a t t h e s i n g l e E c o R I s i t e i n p B R 3 2 2 ; t h e 470 bp b a n d was a l w a y s v e r y f a i n t and i t m u s t o r i g i n a t e f r o m an E c o R I s i t e w i t h i n t h e i n s e r t DNA. When p D t l 2 0 R DNA was c l e a v e d w i t h E c o R I and H i n d l l l , f o u r b a n d s w e r e s e e n c o n s i s t e n t l y ( l a n e 5 ) ; 4 . 4 kb and 2 . 4 kb f r a g m e n t s c o r r e s p o n d t o l i n e a r pBR322 a n d p D t l 2 0 R i n s e r t DNA r e s p e c t i v e l y ( c o m p a r e l a n e 5 w i t h l a n e s 8 and 9 ) and 2 . 0 kb and 0 . 4 4 kb f r a g m e n t s add up t o t h e l e n g t h o f i n s e r t DNA, i n d i c a t i n g t h e i r o r i g i n i n t h e i n s e r t DNA. The two r e s u l t s t o g e t h e r s u g g e s t e d t h a t t h e E c o R I s i t e w i t h i n t h e i n s e r t DNA was o n l y p a r t i a l l y c l e a v e d by E c o R I . R e s t r i c t i o n u n d e r c o n d i t i o n s more s p e c i f i c f o r E c o R I r e c o g n i t i o n ( 8 0 mM N a C l ) g a v e s i m i l a r r e s u l t s ( l a n e s 1 2 , 1 3 ) . H o w e v e r , when p D t ! 2 0 R was d i g e s t e d w i t h E c o R I ( l a n e 2) o r E c o R I , H i n d l l l ( l a n e 3) u n d e r c o n d i t i o n s o f l o w i o n i c s t r e n g t h and h i g h pH ( 2 0 mM T r i s . H C l , pH 8 . 5 , 2 mM MgCl 2 ) » t h e c l e a v a g e a t t h e E c o R I s i t e w i t h i n t h e i n s e r t DNA was more c o m p l e t e . Many o t h e r i n t e r m e d i a t e l e n g t h b a n d s w e r e o b s e r v e d i n b o t h r e a c t i o n s , p r e s u m a b l y o r i g i n a t i n g f r o m c l e a v a g e a t o t h e r E c o R I * s i t e s i n pBR322 a n d p D t l 2 0 R i n s e r t DNA. I t was t h u s c o n c l u d e d t h a t t h e s i t e w i t h i n t h e i n s e r t DNA, w h i c h g a v e r i s e t o 2 . 0 kb and 0 . 4 4 kb E c o R I , H i n d l l l f r a g m e n t s , was an E c o R I * s i t e ( a n d t h i s was c o n f i r m e d l a t e r by t h e DNA s e q u e n c e a n a l y s i s ) . I t m u s t be n o t e d t h a t , when t h e E c o R I , H i n d l l l f r a g m e n t s w e r e u s e d f o r DNA s e q u e n c e a n a l y s i s , t h e E c o R I d i g e s t i o n was p e r f o r m e d u n d e r s t a n d a r d E c o R I r e s t r i c t i o n c o n d i t i o n s t o p r e v e n t c o n t a m i n a t i o n o f t h e 2 . 0 kb and 0 . 4 4 kb f r a g m e n t s w i t h o t h e r f r a g m e n t s o f s i m i l a r m . w t . , w h i c h a r e g e n e r a t e d u n d e r E c o R I * r e s t r i c t i o n c o n d i t i o n s ( c o m p a r e l a n e s 3 and 5 ) . DNA s e q u e n c e a n a l y s i s o f t h e s e t w o f r a g m e n t s c o n f i r m e d p a r t o f t h e s e q u e n c e o b t a i n e d e a r l i e r and a d d e d a n o t h e r 140 n u c l e o t i d e s o f t h e 3 ' - f l a n k i n g r e g i o n t o t h e e x i s t i n g 57 . F i g u r e 9 . D i g e s t i o n o f p D t l 2 0 R w i t h E c o R I u n d e r d i f f e r e n t r e s t r i c t i o n c o n d i t i o n s . p D t l 2 0 R DNA (1 jug) was d i g e s t e d w i t h E c o R I u n d e r s t a n d a r d E c o R I r e s t r i c t i o n c o n d i t i o n s ( l a n e s 4 - 6 ) , u n d e r c o n d i t i o n s more s p e c i f i c f o r E c o R I r e c o g n i t i o n ( l a n e s 12 and 13) and u n d e r c o n d i t i o n s o f l o w i o n i c s t r e n g t h and h i g h pH ( l a n e s 2 and 3) as d e s c r i b e d i n R e s u l t s . The d i g e s t s i n l a n e s 3 , 5 a n d 12 w e r e f u r t h e r c l e a v e d w i t h H i n d l l l and t h a t i n l a n e 6 w i t h X m a l . L a n e 1 c o n t a i n s u n c u t p D t l 2 0 R DNA, l a n e 7 p D t l 2 0 R d i g e s t e d w i t h S m a l and H i n d l l l , l a n e 8 p D t l 2 0 R c l e a v e d w i t h H i n d l l l and l a n e 9 pBR322 c u t w i t h H i n d l l l . l a n e s 10 and 11 show m . w t . m a r k e r s , H i n d l l l c l e a v e d l a m b d a DNA and H p a l l c l e a v e d pBR322 r e s p e c t i v e l y . The s a m p l e s w e r e e l e c t r o p h o r e s e d on a 5% p o l y a c r y l a m i d e g e l , t h e g e l was s t a i n e d w i t h e t h i d i u m b r o m i d e and was p h o t o g r a p h e d a f t e r i l l u m i n a t i n g w i t h u l t r a v i o l e t l i g h t . 5 8 . s e q u e n c e ( F i g . 8 ) . The DNA s e q u e n c e a v a i l a b l e up t o t h i s p o i n t i n d i c a t e d t h e p r e s e n c e o f r e c o g n i t i o n s e q u e n c e s f o r t h e R. e n d o n u c l e a s e Sau961 a t s e v e r a l c o n v e n i e n t l o c a t i o n s ( F i g . 8 ) . The two f r a g m e n t s g e n e r a t e d by t h e t h r e e Sau961 s i t e s V a l w e r e u s e d t o c o n f i r m t h e s e q u e n c e o f t h e t R N A 4 gene and a l s o t o c o n f i r m t h e p r e s e n c e o f Xmal and E c o R I * s i t e s . P l a s m i d s p D t l 2 0 R and pBR322 w e r e d i g e s t e d w i t h S a u 9 6 1 ; t h e r e s u l t i n g f r a g m e n t s w e r e e n d - l a b e l l e d , a p p l i e d t o a d j a c e n t s l o t s i n a 5% p o l y a c r y l a m i d e g e l , and t h e f r a g m e n t s w e r e s e p a r a t e d by e l e c t r o p h o r e s i s ( F i g . 1 0 ) . When t h e f r a g m e n t s p r e s e n t i n t h e two l a n e s w e r e c o m p a r e d , l a n e 2 c o n t a i n i n g t h e p D t l 2 0 R - Sau961 d i g e s t showed e i g h t e x t r a b a n d s ( l a b e l l e d 1 - 8 i n t h e o r d e r o f d e c r e a s i n g m . w t ) . The a v a i l a b l e DNA s e q u e n c e d a t a p r e d i c t e d t h a t t h e t w o Sau961 f r a g m e n t s o f i n t e r e s t w o u l d be 308 bp ( f l a n k e d by t h e f i r s t two Sau961 s i t e s shown i n F i g . 8) and 236 bp ( f l a n k e d by t h e 2nd and t h e 3 r d Sau961 s i t e s shown i n F i g . 8 ) l o n g ; t h e f o r m e r c o n t a i n s a H i n d l l l s i t e a n d t h e l a t t e r an Xmal s i t e . F r a g m e n t s 4 , 5 and 6 ( F i g . 1 0 ) w h i c h h a v e m . w t s . ( r a n g i n g f r o m 2 2 0 -3 5 0 bp) s i m i l a r t o t h e r e l e v a n t f r a g m e n t s ( 2 3 6 , 308 bp) w e r e e x c i s e d , e l e c t r o e l u t e d and a l i q u o t s o f e a c h DNA w e r e d i g e s t e d e i t h e r w i t h H i n d l l l o r X m a l . F r a g m e n t s 4 a n d 5 w e r e c l e a v e d w i t h H i n d l l l b u t n o t w i t h X m a l , h e n c e t h e s e two f r a g m e n t s m u s t f o r m t h e j u n c t i o n s b e t w e e n t h e v e c t o r pBR322 and t h e D r o s o p h i l a DNA i n p D t l 2 0 R , w i t h f r a g m e n t 4 f o r m i n g t h e j u n c t i o n on t h e l e f t ( i t s m o l e c u l a r w e i g h t b e i n g c l o s e r t o 3 0 8 bp) i n F i g . 8 and f r a g m e n t 5 t h a t o n t h e r i g h t . F r a g m e n t 6 was c l e a v e d w i t h Xmal b u t n o t w i t h H i n d l l l and t h e r e f o r e r e p r e s e n t s t h e f r a g m e n t f l a n k e d by t h e 2nd and t h e 3 r d S a u 9 6 I s i t e s i n F i g . 8 . F r a g m e n t s 4 a n d 6 w e r e u s e d f o r DNA s e q u e n c e a n a l y s i s . T h e r e c o g n i t i o n s e q u e n c e f o r S a u 9 6 I i s GGNCC; t h u s d e p e n d i n g on what N i s , a f r a g m e n t c o u l d be p r e f e r e n t i a l l y l a b e l l e d a t t h e d e s i r e d e n d . Sau 60 . F i g u r e 1 0 . R e s t r i c t i o n a n a l y s i s o f p D t l 2 0 R DNA u s i n g R. e n d o n u c l e a s e , S a u 9 6 I . P l a s m i d s p D t l 2 0 R (2 >ug) and pBR322 (1 jug) w e r e d i g e s t e d w i t h S a u 9 6 I and t h e r e s u l t i n g f r a g m e n t s w e r e 3 ' e n d - l a b e l l e d . The s a m p l e s w e r e e l e c t r o -p h o r e s e d on a 57o p o l y a c r y l a m i d e g e l and t h e g e l was a u t o r a d i o g r a p h e d . The 8 u n i q u e b a n d s i n t h e p D t l 2 0 R / S a u 9 6 I d i g e s t ( l a n e 2) n o t f o u n d i n t h e p B R 3 2 2 / S a u 9 6 I d i g e s t ( l a n e 1) a r e numbered w i t h a r r o w s . 6 1 . — 7 62. 961 d i g e s t s o f p D t l 2 0 R w e r e l a b e l l e d i n t h e . p r e s e n c e o f [ a - 3 2 P ] d C T P and dGTP o r 0 - 3 2 P ] d T T P a n d d G T P . F r a g m e n t 4 ( w h i c h h a s S a u 9 6 I s e q u e n c e , GGCCC a t e i t h e r e n d ) , l a b e l l e d a t b o t h e n d s , was i s o l a t e d f r o m t h e f o r m e r d i g e s t ; s i n g l e l a b e l l e d e n d s w e r e g e n e r a t e d by s t r a n d s e p a r a t i o n ( F i g . 1 1 ) . F r a g m e n t 6 ( w i t h S a u 9 6 I s e q u e n c e , GGCCC a t one end and GGTCC a t t h e o t h e r ) , r e c o v e r e d f r o m e i t h e r d i g e s t , was s i n g l e e n d - l a b e l l e d b u t a t o p p o s i t e e n d s w i t h r e s p e c t t o e a c h o t h e r . The s e q u e n c e o b t a i n e d f r o m t h e s e f o u r V a l f r a g m e n t s c o n f i r m e d t h e s e q u e n c e o f t h e t R N A 4 g e n e d e t e r m i n e d by e a r l i e r e x p e r i m e n t s ( F i g . 8) and a l s o t h e p r e s e n c e o f t h e Xmal ( F i g . 12) and E c o R I * s i t e s . B . The n u c l e o t i d e s e q u e n c e o f t h e tRNA g e n e o f p D t ! 2 0 R The n u c l e o t i d e s e q u e n c e o f a s e g m e n t o f p D t l 2 0 R DNA c o n t a i n i n g t h e t R N A g e n e i s p r e s e n t e d i n F i g . 1 3 . The DNA s e q u e n c e o f t h e f i r s t 500 n u c l e o t i d e s was o b t a i n e d a t l e a s t t w i c e e i t h e r by s e q u e n c i n g b o t h s t r a n d s o r by s e q u e n c i n g t h e same s t r a n d f r o m a d i f f e r e n t r e s t r i c t i o n f r a g m e n t . The l a s t 50 n u c l e o t i d e s w e r e s e q u e n c e d o n l y o n c e ( F i g . 8 ) . The n u c l e o t i d e s e q u e n c e o f t h e tRNA g e n e i s u n d e r l i n e d ; i t i s w r i t t e n i n t h e c o n v e n t i o n a l 5 ' t o 3 ' d i r e c t i o n and o n l y t h e n o n c o d i n g s t r a n d i s shown ( F i g . 1 3 ) . The g e n e i s G C - r i c h ( ~60% GC) c o m p a r e d t o t h e f l a n k i n g r e g i o n s w h i c h a r e f a i r l y A T - r i c h ( 2 0 - 3 0 % G C ) ; t o t a l D r o s o p h i l a DNA i s a b o u t 40% GC. The g e n e d o e s n o t c o d e f o r t h e 3 ' - t e r m i n a l CCA s e q u e n c e f o u n d i n a l l m a t u r e t R N A s ( 8 ) . T h i s f i n d i n g i s i n common w i t h o t h e r e u k a r y o t i c tRNA g e n e s ( 3 7 ) . A s h o r t d i s t a n c e (11 n u c l e o t i d e s ) b e y o n d t h e 3 ' - e n d o f t h e g e n e i s a c l u s t e r o f T r e s i d u e s (T ) w h i c h a r e t h o u g h t t o be i n v o l v e d i n t h e t e r m i n a t i o n o f t r a n s c r i p t i o n ( 8 7 , 1 0 0 ) . The DNA s e q u e n c e a l s o c o n f i r m e d t h e p r e s e n t o f t h e Xmal s i t e (CCCGGG) a t n u c l e o t i d e s 2 7 6 - 2 8 1 a n d t h e E c o R I * s i t e (GAAATTC) a t n u c l e o t i d e s 4 4 6 - 4 5 2 . N o t e t h a t t h e l a t t e r s e q u e n c e h a s an e x t r a A when c o m p a r e d t o t h e n o r m a l r e c o g n i t i o n s e q u e n c e o f F i g u r e 1 1 . S t r a n d s e p a r a t i o n o f S a u 9 6 I f r a g m e n t 4 o f p D t ! 2 0 R . The DNA f r o m f r a g m e n t 4 ( F i g . 10) was r e c o v e r e d by e l e c t r o e l u t i o n a n d e t h a n o l p r e c i p i t a t i o n . The p r e c i p i t a t e was d i s s o l v e d i n s t r a n d d e n a t u r i n g s o l u t i o n ( M e t h o d s ) a n d a p p l i e d i m m e d i a t e l y t o a 5% n o n d e n a t u r i n g p o l y a c r y l a -m i d e g e l . E l e c t r o p h o r e s i s was c a r r i e d o u t a t 200 V ( 5 V/cm) f o r 18 h o u r s and t h e g e l was a u t o r a d i o g r a p h e d . ' 0 ' d e s i g n a t e s t h e o r i g i n on t h e g e l , s s t h e s e p a r a t e d s t r a n d s , a n d d s t h e r e s i d u a l amount o f t h e o r i g i n a l , u n d e n a t u r e d , d o u b l e s t r a n d e d DNA f r a g m e n t . 64. V a l F i g u r e 1 2 . C o n f i r m a t i o n o f t h e Xmal r e s t r i c t i o n s i t e i n t h e tRN/\4 gene o f pDtl2 0 R . The DNA s e q u e n c e o f t h e S a u 9 6 I f r a g m e n t 6 ( F i g . 1 0 ) was d e t e r m i n e d by t h e m e t h o d o f Maxam a n d G i l b e r t . The e l e c t r o p h o r e s i s c o n d i t i o n s w e r e i d e n t i c a l t o t h o s e d e s c r i b e d i n t h e l e g e n d t o F i g . 7 . L a n e s 1 - 4 r e p r e s e n t s e q u e n c i n g r e a c t i o n s s p e c i f i c f o r C , T + C , A + G and G , r e s p e c t i v e l y . The s e q u e n c e shown i s t h a t o f t h e c o d i n g s t r a n d . The r e c o g n i t i o n s e q u e n c e f o r t h e R. e n z y m e , Xmal ( C C C G G G ) , i s i n d i c a t e d . 66 , F i g u r e 1 3 . The n u c l e o t i d e s e q u e n c e o f a s e g m e n t o f t h e D r o s o p h i 1 a DNA i n s e r t o f p l a s m i d p D t l 2 0 R . The s e q u e n c e o f o n l y t h e n o n c o d i n g s t r a n d i s s h o w n . The tRNA gene i s u n d e r l i n e d . The n u c l e o t i d e s o v e r l i n e d a t p o s i t i o n s 2 3 2 , 2 4 5 , 257 and 273 a r e d i f f e r e n t f r o m t h a t e x p e c t e d f r o m t h e t R N A l p 1 s e q u e n c e . The Nucleotide Sequence of pDt120R 10 20 30 40 50 60 70 80 90 100 AAGCTTCGAG GTAGGTATGT AGCTTCACGG CTTGCTGCTT AAGTTGTTAC AATACCATTG GGAGGAGAGT GGG TAAAGG CAAGCCACTA TATAAGCGT 110 120 130 140 150 160 170 180 190 200 CACTTTTTAA ATTATTTCCT ATAATTACAT TTTATAATTA CTTTGTGCTT TTATTATAAC AGATATATTT GCTAACTTAT CTTAAATTGT CTATGAGGAA 2 10 220 230 _ 240 _ 250 _260 270 _ 280 290 300 AACGTTCGTC ATCCGAGTTT CCGTGGTGTA GTGGTTATCA CATCCGCCTA ACACGCGGAA GGCCCCCGGT TCAATCCCGG GCGGAAACAG TTGGAATTTA 310 320 330 340 350 360 370 380 390 400 TTTTTTGCTA AATATTTATT TATCATAATG TTCAGTTGTA AAACACACAT AGCTAATAGT ATTTATAGCT GCATCATGGC CTTAAACTTA TCACGTTGCT 4 10 420 430 440 450 - 460 470 480 490 500 TTTGCTTCAA GGCCTCGTGC TTCTTACGAT CCACATTTTT AAGCAGAAAT TCTTGAAATT TCCTACGCAT ATCTAACGAT AGACCTGTAT TTCGAAGGTC 510 520 530 540 550 CAACCTCTCA AGAACCTTGT TGCAGCTAAT TATCTTCATC AAATGCTTGC CAAAGTC oo 69 , EcoRI (GAATTC). Val The tRNA gene in pDtl20R hybridizes with tRNA4 (119, 120) but i ts sequence differs from that expected from the tRNA^ a l sequence (14) at four positions overlined in figure 13: at residues 232 (C + T),245 (T+C), 257 (A->G) and 273 (G+A). These sites correspond respectively to positions 16 (in the D loop), 29, 41 (which code for a base pair in the anticodon stem) and 57 (in Ty Val C loop) in the tRNA4 sequence (14). It has the expected AAC (nucleotides 250-252) sequence in the anticodon region. The possible effect(s) of these Val nucleotide changes in the tRNA4 gene is discussed below. II. Transcription of cloned tRNA genes from Drosophila melanogaster in a  homologous ce l l - f ree extract A. Synthesis of dist inct RNA species directed by plasmid DNA containing a Val Drosophila tRNA 3 b gene The ce l l - f ree extract (S.100) was prepared from Drosophila Schneider II ce l l s as described in Materials and Methods. It was tested for i ts abi l i ty to in i t ia te specif ic transcription on an exogenous template, pDt78R, containing a Val tRNA 3 b gene (Table- III). F ig . 14 shows the autoradiogram of the poly-acrylamide gel electrophoresis of the RNAs synthesized by the extract. Incubation with pDt78R (lanes 2 and 5) resulted in the synthesis of two major products (designated RNA-I and RNA-II) which were not present in a minus DNA control (lane 4) or when pBR322 DNA was added (lane 3). Lane 6 shows that the products in lane 5 were RNase sensit ive. These results demonstrate that RNA-I V a 1 and RNA-II are specif ic to the Drosophila DNA containing the tRNA* gene. 3 b The larger RNA, RNA-I, was the predominant product in al l transcription reactions and its size varied with the DNA template (Fig. 15). The size of RNA-I directed by pDt78R (lane 6) and that directed by pDt55, pDt92R and pDtl20R (Table III; lanes 1-3) respectively, was estimated to be 95 and 100 nucleotides long by comparison to denatured Hpall fragments of pBR322 DNA 70 . F i g u r e 1 4 . T r a n s c r i p t i o n o f pDt78R DNA c o n t a i n i n g D r o s o p h i l a t R N A ^ g e n e . T r a n s c r i p t i o n r e a c t i o n s w e r e c a r r i e d o u t and a n a l y z e d as d e s c r i b e d i n M e t h o d s . ( 0 ) r e p r e s e n t s t h e o r i g i n o f e l e c t r o p h o r e s i s and ( I ) and ( I I ) d e n o t e t h e p o s i t i o n s o f R N A - I and R N A - I I . L a n e s 1 and 7 c o n t a i n P 2 5 I ] - t R N A Y a l m a r k e r . R e a c t i o n s 2 and 5 c o n t a i n 2 p g pDt78R DNA, r e a c t i o n 3 c o n t a i n s 2 yjg pBR322 DNA and r e a c t i o n 4 c o n t a i n s no a d d e d DNA. R e a c t i o n 6 a l s o c o n t a i n s 2 jug pDt78R DNA, b u t f o l l o w i n g 2 h o u r s i n c u b a t i o n , i t was t r e a t e d w i t h 20 /jg/ml RNase A a t 3 7 ° C f o r 30 m i n . L a n e s 1 - 4 and l a n e s 5 - 7 w e r e t a k e n f r o m s e p a r a t e g e l s . 71. 1 2 3 4 ., 5 6 7 t F i g u r e 1 5 . T r a n s c r i p t i o n o f d i f f e r e n t p l a s m i d s c a r r y i n g D r o s o p h i 1 a tRNA g e n e s . T r a n s c r i p t i o n r e a c t i o n s w i t h e q u i m o l a r amount o f e a c h g e n e o f t h e i n d i c a t e d p l a s m i d w e r e p e r f o r m e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s . L a n e s 1 - 3 a n d 6 show t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h p l a s m i d s , p D t 5 5 , p D t 9 2 R , p D t l 2 0 R a n d p D t 7 8 R , r e s p e c t i v e l y . L a n e s 4 and 5 c o n t a i n [ 1 2 T ] - t R N A ^ a l and H p a l l c l e a v e d pBR322 DNA r e s p e c t i v e l y . 4 73. 74. ( l a n e 5 ) . The s i z e o f R N A - I I , t h e s m a l l e s t o f t h e s p e c i f i c RNA p r o d u c t s , was V a l c o n s t a n t f o r a l l DNA t e m p l a t e s ( F i g . 1 5 ) ; i t was s i m i l a r i n s i z e t o t h e tRNA4 m a r k e r ( l a n e 4 ; b o t h t R N A ^ ^ a n d t R N A a r e 76 n u c l e o t i d e s l o n g ; 1 3 , 1 4 ) a n d c o u l d r e p r e s e n t t h e m a t u r e tRNA p r o d u c t . B . A u t h e n t i c i t y o f t h e RNA t r a n s c r i p t s , R N A - I and R N A - I I , d i r e c t e d by  pDt78R To d e m o n s t r a t e t h a t RNA-I and R N A - I I w e r e s p e c i f i c tRNA t r a n s c r i p t s , l a b e l l e d j_n v i t r o p r o d u c t s d i r e c t e d by pDt78R w e r e e l u t e d f r o m b a n d s on p o l y a c r y l a m i d e g e l s and h y b r i d i z e d t o n i t r o c e l l u l o s e b o u n d pDt78R and pDt41R D N A s , e a c h o f w h i c h c o n t a i n s a D r o s o p h i l a t R N A ^ ^ g e n e ( 1 1 9 , 1 3 ) . A n a l y s i s o f h e t e r o d u p l e x f o r m e d b e t w e e n pDt78R and pDt41R DNAs shows s i g n i f i c a n t h o m o l o g y V a l o n l y i n t h e t R N A 3 b c o d i n g r e g i o n ( 1 3 9 ) . T h u s , i f RNA-I a n d R N A - I I w e r e a u t h e n t i c tRNA t r a n s c r i p t s , t h e y s h o u l d h y b r i d i z e t o b o t h pDt78R and pDt41R D N A s . The r e s u l t o f o n e s u c h h y b r i d i z a t i o n a n a l y s i s i s shown i n T a b l e I V . R N A - I h y b r i d i z e d t o pDt78R a n d pDt41R DNAs 34 a n d 39 t i m e s m o r e t h a n t o t h e i r r e s p e c t i v e pBR322 c o n t r o l s . T h e r e f o r e , m o s t o f t h e RNA c o n t a i n e d i n t h e RNA-I V a l b a n d h y b r i d i z e d s p e c i f i c a l l y t o D r o s o p h i l a DNA c o n t a i n i n g t R N A 3 b g e n e . The e f f i c i e n c y o f h y b r i d i z a t i o n o f R N A - I I t o pDt78R and pDt41R DNAs was a b o u t 5 - 8 f o l d l o w e r t h a n t h a t o f R N A - I . The r e a s o n f o r t h i s i s n o t c l e a r ; i t i s p o s s i b l e t h a t R N A - I I h a s a g r e a t e r s e c o n d a r y s t r u c t u r e w h i c h makes i t l e s s a c c e s s i b l e f o r h y b r i d i z a t i o n . A l t h o u g h t h e h y b r i d i z a t i o n e f f i c i e n c y was l o w , R N A - I I a l s o h y b r i d i z e d t o b o t h pDt78R a n d pDt41R D N A s , i n d i c a t i n g t h a t i t was a l s o an a u t h e n t i c t r a n s c r i p t i o n p r o d u c t . H y b r i d i z a t i o n o f RNA-I a n d R N A - I I t o pDt41R was a b o u t 80% and 50% o f t h a t t o pDt78R r e s p e c t i v e l y . T h i s r e s u l t was n o t t o t a l l y u n e x p e c t e d b e c a u s e d e t a i l e d h y b r i d i z a t i o n s t u d i e s i n v o l v i n g t h e s e r i 2 5 T ^ ---Val t w o p l a s m i d DNAs a n d [ 5 I ] - t R N A 3 b showed t h a t t h e e f f i c i e n c y o f h y b r i d i z a t i o n r i p c -, V a l ' o f [ T ] - t R N A 3 b t o pDt41R DNA was 3 0 - 5 0 % l o w e r t h a n t h a t t o pDt78R DNA ( 1 4 0 ) . DNA s e q u e n c e a n a l y s e s a l s o showed t h a t pDt78R and pDt41R d i f f e r a t f o u r T a b l e I V . RNA.DNA h y b r i d i z a t i o n [ a - 3 2 P ] UTP l a b e l l e d pDt78R t r a n s c r i p t i o n p r o d u c t s , RNA-I and R N A - I I w e r e h y b r i d i z e d t o p B R 3 2 2 , pDt41R and pDt*78R DNAs b o u n d t o n i t r o c e l l u l o s e f i l t e r s a s d e s c r i b e d i n M a t e r i a l s and M e t h o d s . The t o t a l amount o f r a d i o a c t i v i t y i n e a c h v i a l was a p p r o x i m a t e l y 3 6 , 0 0 0 c p m . The cpm v a l u e s shown i n t h e t a b l e w e r e o b t a i n e d a f t e r s u b t r a c t i n g t h e b l a n k c p m . pDt78R t r a n s c r i p t i o n cpm h y b r i d i z e d % i n p u t cpm p r o d u c t V i a l 1 V i a l 2 h y b r i d i z e d pBR322 pDt78R pBR322 pDt41R pDt78R pDt41R R N A - I 220 7517 147 5794 2 0 . 0 % 1 5 . 5 % R N A - I I 159 1408 141 6 8 0 3 . 7 % 1 . 8 % p o s i t i o n s i n t h e c o d i n g r e g i o n ( 1 3 , L e u n g and T e n e r , p e r s o n a l c o m m u n i c a t i o n ) . T h e s e 4 d i f f e r e n c e s may c a u s e l o w e r e f f i c i e n c y o f h y b r i d i z a t i o n o f pDt78R t r a n s c r i p t i o n p r o d u c t s t o pDt41R t h a n t o pDt78R DNA. C . P r e c u r s o r - p r o d u c t r e l a t i o n s h i p b e t w e e n t h e m a j o r t r a n s c r i p t i o n p r o d u c t s T r a n s f e r RNA i s s y n t h e s i z e d a s a p r e c u r s o r w h i c h i s p r o c e s s e d a f t e r -w a r d s t o g i v e t h e m a t u r e tRNA p r o d u c t ( r e v i e w e d i n r e f . 6 ) . A n o t h e r way t o d e m o n s t r a t e t h e a u t h e n t i c i t y o f t h e RNA t r a n s c r i p t s made i n t h e S . 1 0 0 e x t r a c t i s t o show t h a t t h e t r a n s c r i p t s a r e r e l a t e d a s p r e c u r s o r and p r o d u c t . The p r e c u r s o r - p r o d u c t r e l a t i o n s h i p b e t w e e n RNA-I and R N A - I I i s d e m o n s t r a t e d by t h e d a t a i n F i g s . 1 6 - 1 9 . 1 . T i m e c o u r s e o f RNA s y n t h e s i s F i g . 16 shows a d e t a i l e d t i m e c o u r s e o f s y n t h e s i s o f s p e c i f i c t r a n s c r i p t s f r o m p l a s m i d , p D t 0 . 3 , w h i c h c a r r i e s a t R N A J a l g e n e ( M a t e r i a l s and 0 M e t h o d s , s e c t i o n I V . A ) . B a n d s l a b e l l e d a ( R N A - I ) , b and c ( R N A - I I ; F i g . 16A) w e r e e x c i s e d , t h e r a d i o a c t i v i t y i n t h e i n d i v i d u a l b a n d s was m e a s u r e d and p l o t t e d a g a i n s t t h e t i m e o f i n c u b a t i o n w i t h t h e S . 1 0 0 e x t r a c t ( F i g . 1 6 B ) . The d a t a i n F i g . 16 show t h a t t h e a c c u m u l a t i o n o f R N A - I I ( a n d RNA d e s i g n a t e d b) l a g g e d b e h i n d t h e s y n t h e s i s o f R N A - I , s u g g e s t i n g t h a t t h e p r o d u c t i o n o f t h e s e p r o d u c t s d e p e n d e d on p r i o r s y n t h e s i s o f R N A - I . 2 . R N A - I i s p r o c e s s e d t o R N A - I I To d e m o n s t r a t e t h a t RNA-I c o u l d be c o n v e r t e d t o R N A - I I , RNA-I was e l u t e d f r o m p o l y a c r y l a m i d e g e l s l i c e s , r e i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t and t h e n a n a l y z e d on p o l y a c r y l a m i d e g e l s ( F i g s . 1 7 A , 1 8 A , 1 9 A ) . R a d i o a c t i v i t y i n t h e i n d i v i d u a l b a n d s was d e t e r m i n e d and p l o t t e d a s t h e p e r c e n t a g e o f t h e i n i t i a l amount o f RNA-I a g a i n s t t h e t i m e o f i n c u b a t i o n w i t h t h e S . 1 0 0 e x t r a c t ( F i g s . 1 7 B , 1 8 B , 1 9 B ) . The DNA t e m p l a t e s u s e d f o r t h e e x p e r i m e n t s d e s c r i b e d i n F i g s . 1 7 - 1 9 w e r e p D t 0 . 3 , pDt78R a n d pDt92R r e s p e c t i v e l y ( T a b l e I I I ) . 77, F i g u r e 1 6 . Time c o u r s e a s s a y . A . T r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g 1 jug p D t 0 . 3 DNA w e r e p e r f o r m e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s , e x c e p t t h a t t h e r e a c t i o n s w e r e s t o p p e d a t t h e i n d i c a t e d t i m e s . L a n e 1 c o n t a i n s [ 1 2 5 I j - t R N A^p 1 m a r k e r . R e a c t i o n s i n l a n e s 2 - 1 4 w e r e s t o p p e d a f t e r 1 0 , 2 0 , 3 0 , 4 5 , 6 0 , 7 5 , 9 0 , 1 0 5 , 1 2 0 , 1 5 0 , 1 8 0 , 210 and 240 m i n . o f i n c u b a t i o n , r e s p e c t i v e l y . A r r o w s a , b and c d e s i g n a t e t h e p o s i t i o n s o f RNA-I ( p r e c u r s o r p r o d u c t ) , i n t e r m e d i a t e RNA s p e c i e s a n d R N A - I I ( m a t u r e tRNA l e n g t h s p e c i e s ) , r e s p e c t i v e l y . B . B a n d s l a b e l l e d a ( s o l i d c i r c l e s ) , b ( o p e n c i r c l e s ) and c ( s o l i d s q u a r e s ) w e r e e x c i s e d f r o m t h e g e l , t h e r a d i o a c t i v i t y i n t h e i n d i v i d u a l b a n d s was d e t e r m i n e d by c o u n t i n g C e r e n k o v r a d i a t i o n ( e f f i c i e n c y o f c o u n t i n g a p p r o x . 20%) a n d t h e dpm v a l u e s w e r e p l o t t e d a g a i n s t t h e t i m e o f i n c u b a t i o n w i t h t h e S . 1 0 0 e x t r a c t . 78. F i g u r e 1 7 . P r o c e s s i n g o f RNA-I d i r e c t e d by p D t 0 . 3 t o R N A - I I . A . A s e r i e s o f i d e n t i c a l t r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g 1 jug p D t 0 . 3 DNA w e r e p e r f o r m e d a n d a n a l y z e d a s d e s c r i b e d i n M e t h o d s . B a n d s c o r r e s p o n d i n g t o RNA-I w e r e e x c i s e d , and t h e RNA was r e c o v e r e d f r o m t h e g e l . A l i q u o t s o f RNA-I ( - 1 9 x l O 4 dpm) w e r e r e - i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r 1 5 , 3 0 , 4 5 , 6 0 , 7 5 , 9 0 , 105 and 120 m i n . ( l a n e s 4-11 r e s p e c t i v e l y ) . The p r o d u c t s o f p r o c e s s i n g w e r e a n a l y z e d on a 10% - 7M u r e a p o l y a c r y l a m i d e g e l . B a n d s l a b e l l e d a , b a n d c c o r r e s p o n d t o R N A - I , t h e i n t e r m e d i a t e RNA s p e c i e s and R N A - I I , r e s p e c t i v e l y . L a n e 1 c o n t a i n s [ 1 2 5 I ] - t R N AY A L m a r k e r , l a n e 2 s h o w s a c o n t r o l t r a n s c r i p t i o n r e a c t i o n c o n t a i n i n g 1 jjg p D t 0 . 3 DNA and l a n e 3 i s a R N A - I c o n t r o l w h i c h was n o t i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t . B . The b a n d s a ( c i r l c e s ) , b ( s q u a r e s ) and c ( t r i a n g l e s ) w e r e e x c i s e d f r o m t h e g e l , t h e r a d i o a c t i v i t y i n t h e i n d i v i d u a l b a n d s was d e t e r m i n e d and p l o t t e d a s p e r c e n t a g e o f r a d i o a c t i v i t y i n t h e i n i t i a l amount o f R N A - I (19 x 1 0 ^ dpm) a g a i n s t t h e t i m e o f i n c u b a t i o n w i t h t h e S . 1 0 0 e x t r a c t . '08 8.1. F i g u r e 1 8 . P r o c e s s i n g o f RNA-I d i r e c t e d by pDt78R t o R N A - I I . A . T h i s e x p e r i m e n t was p e r f o r m e d a s d e s c r i b e d i n t h e l e g e n d t o F i g . 17 e x c e p t t h a t a l i q u o t s o f R N A - I d i r e c t e d by pDt78R w e r e i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r 1 5 , 3 0 , 4 5 , 6 0 , 90 and 120 m i n . ( l a n e s 3 - 8 r e s p e c t i v e l y ) . L a n e 1 shows a t r a n s c r i p t i o n c o n t r o l c o n t a i n i n g 1 ug pDt78R DNA and l a n e 2 i s a RNA-I c o n t r o l . B . B a n d s a ( c i r c l e s ) , b ( s q u a r e s ) and c ( t r i a n g l e s ) w e r e e x c i s e d , t h e r a d i o a c t i v i t y i n t h e i n d i v i d u a l b a n d s d e t e r m i n e d and p l o t t e d a s p e r c e n t a g e o f r a d i o a c t i v i t y i n t h e i n i t i a l amount o f RNA-I (6 x 1 0 4 dpm) a g a i n s t t h e t i m e o f i n c u b a t i o n w i t h t h e S . 1 0 0 e x t r a c t . percent dpm O O " 03 CO cn CD ' Z8 8 3 . F i g u r e 1 9 . P r o c e s s i n g o f RNA-I d i r e c t e d by pDt92R t o R N A - I I . A . RNA-I d i r e c t e d by pDt92R was i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r 3 0 , 6 0 , 9 0 a n d 1 2 0 m i n . ( l a n e s 3 - 6 r e s p e c t i v e l y ) . The b a n d s l a b e l l e d a , b , c a n d d c o r r e s p o n d t o R N A - I , t h e two i n t e r m e d i a t e RNA s p e c i e s and R N A - I I , r e s p e c t i v e l y . L a n e 1 shows a t r a n s c r i p t i o n c o n t r o l c o n t a i n i n g 1 p g pDt92R DNA a n d l a n e 2 i s a RNA-I c o n t r o l . B . B a n d s a ( s o l i d c i r c l e s ) , b ( o p e n c i r c l e s ) , c ( s q u a r e s ) a n d d ( t r i a n g l e s ) w e r e e x c i s e d , t h e r a d i o a c t i v i t y i n t h e i n d i v i d u a l b a n d s was d e t e r m i n e d and p l o t t e d a s p e r c e n t a g e o f r a d i o a c t i v i t y i n t h e i n i t i a l amount o f RNA-I (4 x 1 0 4 dpm) a g a i n s t t h e t i m e o f i n c u b a t i o n w i t h t h e S . 1 0 0 e x t r a c t . 85. The d a t a i n F i g s . 1 7 - 1 9 d e m o n s t r a t e t h e f o l l o w i n g : I ) R e g a r d l e s s o f t h e DNA t e m p l a t e u s e d , t h e r e was a p r o g r e s s i v e c o n v e r s i o n o f RNA-I i n t o s m a l l e r RNA s p e c i e s , w i t h R N A - I I b e i n g t h e s m a l l e s t RNA p r o d u c t . In a d d i t i o n , t h e sum o f t h e r a d i o a c t i v i t y i n t h e p r e c u r s o r and p r o d u c t s d i d n o t d r o p s i g n i f i c a n t l y , s u g g e s t i n g m i n i m a l n o n s p e c i f i c d e g r a d a t i o n o f R N A s . Thus RNA-I c o u l d s e r v e a s a p r e c u r s o r f o r p r o c e s s i n g t o R N A - I I by t h e S . 1 0 0 e x t r a c t , i i ) The p a t t e r n o f c o n v e r s i o n o f RNA-I t o R N A - I I i n t h e p r o c e s s i n g e x p e r i m e n t s v a r i e d w i t h d i f f e r e n t DNA t e m p l a t e s . F o r e x a m p l e t h e r e w e r e two i n t e r m e d i a t e s p e c i e s ( b , c ) b e t w e e n R N A - I and R N A - I I w i t h pDt92R a s t e m p l a t e ( F i g . 19A) b u t o n l y one ( b ; t h o u g h i t may be o f s l i g h t l y d i f f e r e n t m . w t . ) w i t h pDt78R ( F i g . 18A) o r p D t 0 . 3 ( F i g . 17A) a s t e m p l a t e s , i i i ) The e f f i c i e n c y o f c o n v e r s i o n o f R N A - I t o R N A - I I i n t h e p r o c e s s i n g r e a c t i o n s v a r i e d w i t h d i f f e r e n t t e m p l a t e s . RNA-I d i r e c t e d by p D t 7 8 R , was c o n v e r t e d t o R N A - I I a t t h e h i g h e s t r a t e . T h i s c a n be s e e n f r o m t h e l e v e l o f t h e i n t e r m e d i a t e p r o d u c t ; t h i s s p e c i e s f r o m pDt78R ( F i g . 18B) was on t h e d e c l i n e a f t e r 30 m i n . , b u t t h e l e v e l o f t h e c o r r e s p o n d -i n g s p e c i e s f r o m p D t 0 . 3 ( F i g . 17B) was s t i l l on t h e r i s e t i l l 120 m i n . A l s o t h e r i s e i n t h e l e v e l o f R N A - I I f r o m pDt78R was much s h a r p e r t h a n t h a t f r o m p D t 0 . 3 o r pDt92R ( F i g . 1 9 B ) . A t 120 m i n . , R N A - I I f r o m pDt78R r e p r e s e n t e d a b o u t 38% o f t h e i n u t RNA, w h e r e a s R N A - I I f r o m p D t 0 . 3 r e p r e s e n t e d o n l y a b o u t 8% s i g n i f y i n g a 5 - f o l d d i f f e r e n c e i n t h e e f f i c i e n c y o f c o n v e r s i o n o f R N A - I , d i r e c t e d by t h e s e t w o t e m p l a t e s , t o R N A - I I . The r a t e o f c o n v e r s i o n o f RNA-I d i r e c t e d by p D t 0 . 3 t o t h e i n t e r m e d i a t e s p e c i e s ( b ) was much h i g h e r t h a n t h e r a t e o f p r o c e s s i n g o f t h e i n t e r m e d i a t e s p e c i e s t o R N A - I I . S i m i l a r r e s u l t s w e r e o b s e r v e d w i t h RNA-I d i r e c t e d by p D t 9 2 R ; t h e r a t e o f c o n v e r s i o n o f R N A - I t o t h e f i r s t i n t e r m e d i a t e s p e c i e s ( b ) was q u i t e h i g h b u t t h e a p p e a r a n c e o f t h e s e c o n d i n t e r m e d i a t e s p e c i e s ( c ) and R N A - I I ( d ) l a g g e d f a r b e h i n d . 86 , The r e s u l t s o b t a i n e d i n t h e p r o c e s s i n g e x p e r i m e n t s i n v o l v i n g t h r e e d i f f e r e n t DNA t e m p l a t e s s u g g e s t s e q u e n c e d e p e n d e n c e i n t h e c o n v e r s i o n o f R N A - I t o R N A - I I ( s e e s e c t i o n I V . C l ) . 3 . T_1 f i n g e r p r i n t a n a l y s i s o f R N A - I a n d R N A - I I The t r a n s c r i p t i o n p r o d u c t s , RNA-I and R N A - I I , d i r e c t e d by pDt78R ( p a n e l s a and b) and p D t 0 . 3 ( p a n e l s c and d) w e r e f u r t h e r c h a r a c t e r i z e d by f i n g e r p r i n t a n a l y s e s o f t h e i r RNase T j d i g e s t s ( F i g . 2 0 ) . C o m p a r i s o n o f t h e f i n g e r p r i n t s o f RNA-I and R N A - I I ( f o r b o t h DNA t e m p l a t e s ) showed t h a t t h e p a t t e r n s w e r e v e r y s i m i l a r ; some d i f f e r e n c e s b e t w e e n t h e f i n g e r p r i n t s o f RNA-I a n d R N A - I I w e r e e x p e c t e d b e c a u s e o f t h e d i f f e r e n c e i n t h e i r s i z e s . T h u s , t h e T-j^  f i n g e r p r i n t r e s u l t s a l s o s u p p o r t t h e f i n d i n g t h a t RNA-I and R N A - I I a r e r e l a t e d RNA p r o d u c t s . D. P r o p e r t i e s o f D r o s o p h i l a ( S c h n e i d e r I I ) c e l l - f r e e e x t r a c t To e s t a b l i s h o p t i m a l c o n d i t i o n s f o r s p e c i f i c g e n e t r a n s c r i p t i o n , v a r i o u s c h a r a c t e r i s t i c s o f t h e t r a n s c r i p t i o n s y s t e m w e r e e x a m i n e d w i t h r e s p e c t t o s p e c i f i c RNA s y n t h e s i s . T h e s e p a r a m e t e r s w e r e t e s t e d g e n e r a l l y w i t h t w o e x t r a c t s made a t d i f f e r e n t t i m e s and g e n e r a l l y w i t h t w o o r more d i f f e r e n t DNA t e m p l a t e s . To d e t e r m i n e o p t i m a , r e a c t i o n c o n d i t i o n s w e r e v a r i e d and t h e p r o d u c t s w e r e e l e c t r o p h o r e s e d on p o l y a c r y l a m i d e g e l s . The i n c o r p o r a t i o n i n t o t h e RNA was d e t e r m i n e d by e x c i s i n g t h e g e l a r e a s and m e a s u r i n g C e r e n k o v r a d i a t i o n . The d a t a shown i n F i g s . 2 1 - 2 5 r e p r e s e n t one t y p i c a l e x p e r i m e n t and r a d i o a c t i v i t y i n c o r p o r a t e d i n o n l y RNA-I i s r e p o r t e d . R e s u l t s w i t h R N A - I I f o l l o w e d t h e same t r e n d as R N A - I . 1 . E n h a n c e m e n t o f t r a n s c r i p t i o n i n t h e p r e s e n c e o f an A T P - g e n e r a t i n g s y s t e m D i n g e r m a n n et a]_. ( 8 4 ) o b s e r v e d s t i m u l a t i o n o f t r a n s c r i p t i o n o f a AJTQ" D r o s o p h i l a t R N A 2 g e n e i n t h e p r e s e n c e o f an A T P - g e n e r a t i n g s y s t e m c o n s i s t i n g o f c r e a t i n e p h o s p h o k i n a s e and c r e a t i n e p h o s p h a t e . To t e s t w h e t h e r t h i s 87 . F i g u r e 2 0 . RNase T, f i n g e r p r i n t s o f t h e m a j o r t r a n s c r i p t i o n p r o d u c t s f r o m pDt78R i n d p D t 5 5 . The RNAs l a b e l l e d w i t h [ a - 3 2 P ] U T P w e r e d i g e s t e d w i t h T , ( M e t h o d s ) and t h e f r a g m e n t s w e r e s e p a r a t e d by e l e c t r o p h o r e s i s a t pH 3.5~ on c e l l u l o s e -a c e t a t e s t r i p s i n t h e f i r s t d i m e n s i o n and by h o m o c h r o m a t o g r a p h y o n D E A E - c e l 1 u l o s e p l a t e s i n t h e s e c o n d d i m e n s i o n . The RNAs a n a l y z e d i n p a n e l s ( a ) and ( b ) c o r r e s p o n d r e s p e c t i v e l y t o pDt78R t r a n s c r i p t i o n p r o d u c t s , RNA-I a n d R N A - I I . P a n e l s ( c ) and ( d ) r e p r e s e n t f i n g e r p r i n t s o f p D t 5 5 t r a n s c r i p t i o n p r o d u c t s , RNA-I and R N A - I I , r e s p e c t i v e l y . B , P and Y mark t h e p o s i t i o n s o f b l u e , p i n k a n d y e l l o w t r a c k i n g d y e s . 88. 8 9 , o b s e r v a t i o n was t r u e f o r t h e t r a n s c r i p t i o n s y s t e m d e s c r i b e d h e r e , pDt78R DNA was t r a n s c r i b e d i n t h e p r e s e n c e ( F i g . 21 s o l i d c i r c l e s ) and t h e a b s e n c e ( o p e n c i r c l e s ) o f c r e a t i n e p h o s p h o k i n a s e and c r e a t i n e p h o s p h a t e . T h e r e was a c o n s i d e r a b l e s t i m u l a t i o n o f t r a n s c r i p t i o n a t a l l t i m e p o i n t s e x a m i n e d i n t h e p r e s e n c e o f t h e A T P - g e n e r a t i n g s y s t e m . The r e a s o n f o r t h e s t i m u l a t i o n i s n o t known b u t a s was s p e c u l a t e d by D i n g e r m a n n e t ^1_. ( 8 4 ) , a c o m p o n e n t i n t h e t r a n s c r i p t i o n s y s t e m may be e n e r g y r e q u i r i n g o r i n h i b i t e d by t h e ADP l e v e l s p r e s e n t i n t h e e x t r a c t . 2 . Optimum KC1 c o n c e n t r a t i o n The d a t a i n F i g . 22 shows t h e e f f e c t o f v a r i a b l e KC1 c o n c e n t r a t i o n s on t h e t r a n s c r i p t i o n o f pDt78R ( t r i a n g l e s ) and p D t l 2 0 R ( c i r c l e s ) D N A s . The l o w e s t KC1 c o n c e n t r a t i o n t e s t e d was 60 mM, w h i c h was t h e amount o f KC1 p r e s e n t i n t h e S . 1 0 0 e x t r a c t . The KC1 o p t i m u m f o r t r a n s c r i p t i o n was r a t h e r s h a r p and was 70 mM f o r pDt78R and 80 mM f o r p D t l 2 0 R . 3 . D i v a l e n t m e t a l i o n o p t i m a +2 The e f f e c t o f v a r i a b l e Mg c o n c e n t r a t i o n s on t h e t r a n s c r i p t i o n o f pDt78R ( c i r c l e s ) and p D t l 2 0 R ( t r i a n g l e s ) i s shown i n F i g . 2 3 . The l o w e s t +2 c o n c e n t r a t i o n t e s t e d was 2 . 4 mM w h i c h was t h e c o n c e n t r a t i o n o f Mg c o n t r i b u t e d +2 by t h e S . 1 0 0 e x t r a c t . The o p t i m u m r a n g e f o r Mg f o r pDt78R t r a n s c r i p t i o n was r a t h e r n a r r o w a n d c e n t e r e d a r o u n d 5 mM; f o r p D t l 2 0 R t r a n s c r i p t i o n t h e o p t i m u n +2 Mg c o n c e n t r a t i o n r a n g e d f r o m 3 . 5 - 5 mM. The e f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f M n + 2 on t r a n s c r i p t i o n was d e t e r m i n e d o n l y w i t h pDt78R DNA a s t e m p l a t e ( F i g . 2 4 ) . A l l t h e r e a c t i o n s + 2 + 2 c o n t a i n e d 2 . 4 mM Mg , t h e c o n c e n t r a t i o n o f Mg c o n t r i b u t e d by t h e S . 1 0 0 e x t r a c t . F o r t h i s r e a s o n i t was n o t p o s s i b l e t o d e t e r m i n e t h e a b s o l u t e +2 +2 o p t i m a l Mn c o n c e n t r a t i o n . The Mn o p t i m u m u n d e r t h e s e c o n d i t i o n s was 0 . 2 mM and h i g h e r c o n c e n t r a t i o n s w e r e d r a m a t i c a l l y i n h i b i t o r y ; 2 mM M n C l 2 c o m p l e t e l y i n h i b i t e d t r a n s c r i p t i o n . S t i m u l a t i o n o f t r a n s c r i p t i o n o f pDt78R 90. F i g u r e 2 1 . E f f e c t o f an A T P - g e n e r a t i n g s y s t e m on s p e c i f i c t r a n s c r i p t i o n i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . pDt78R DNA (2 u g ) was t r a n s c r i b e d i n t h e p r e s e n c e ( s o l i d c i r c l e s ) and t h e a b s e n c e ( o p e n c i r c l e s ) o f 6 U/ml c r e a t i n e p h o s p h o k i n a s e and 10 mM c r e a t i n e p h o s p h a t e and t h e r e a c t i o n s w e r e i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r v a r y i n g l e n g t h s o f t i m e . The r a d i o a c t i v i t y i n c o r p o r a t e d i n RNA-I was d e t e r m i n e d as d e s c r i b e d i n M e t h o d s . I I 1 I I 3 0 6 0 9 0 120 180 Time (min) 92, 0 F i g u r e 2 2 . E f f e c t o f KC1 c o n c e n t r a t i o n on s p e c i f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . T r a n s c r i p t i o n r e a c t i o n s w e r e p e r f o r m e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s , e x c e p t t h a t v a r y i n g a m o u n t s o f KC1 w e r e u s e d . S t a n d a r d r e a c t i o n s c o n t a i n e d e i t h e r pDt78R ( t r i a n g l e s ) o r p D t l 2 0 R ( c i r c l e s ) DNA and t h e i n d i c a t e d c o n c e n t r a t i o n o f K C 1 . The i n s e t shows t h e t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h pDt78R DNA and 6 0 , 7 0 , 8 0 , 9 0 , 100 and 100 mM K C 1 , i n l a n e s 1 - 6 r e s p e c t i v e l y . L a n e 7 c o n t a i n s [ 1 2 5 I ] - t R N A V a l m a r k e r . J I I I I I 4 0 8 0 120 KCI (mM) F i g u r e 2 3 . E f f e c t o f Mg c o n c e n t r a t i o n on s p e c f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . T r a n s c r i p t i o n r e a c t i o n s w e r e c o n d u c t e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s , e x c e p t t h a t v a r y i n g a m o u n t s o f M g + 2 w e r e u s e d . S t a n d a r d r e a c t i o n s c o n t a i n e d e i t h e r pDt78R ( c i r c l e s ) o r p D t l 2 0 R ( t r i a n g l e s ) DNA and t h e i n d i c a t e d c o n c e n t r a t i o n o f M g + 2 . The i n s e t shows t h e t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h pDt78R DNA and 2 . 4 , 2 . 9 , 3 . 4 , 3 . 9 , 4 . 9 and 5 . 9 mM M g + 2 i n l a n e s 1 - 6 r e s p e c t i v e l y . L a n e 7 c o n t a i n s [ 1 2 5 I ] - t R N A Y a l m a r k e r . F i g u r e 2 4 . E f f e c t o f Mn c o n c e n t r a t i o n on s p e c i f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . T r a n s c r i p t i o n r e a c t i o n s w e r e c a r r i e d o u t and a n a l y z e d as d e s c r i b e d i n M e t h o d s , e x c e p t t h a t v a r y i n g a m o u n t s o f M n + 2 w e r e u s e d i n p l a c e o f Mg + 2 . The r e a c t i o n s c o n t a i n e d pDt78R DNA and t h e i n d i c a t e d c o n c e n t r a t i o n o f M n + 2 . The i n s e t shows t h e t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h pDt78R DNA a n d 0 . 0 , 0 . 1 , 0 . 2 , 0 . 5 , l.Q, 2 . 0 and 3 . 0 mM M n + 2 i n l a n e s 2 - 8 r e s p e c t i v e l y . L a n e 1 c o n t a i n s [ 1 2 5 I ] - t R N A 4 m a r k e r . 98, + 2 was a b o u t 4 - f o l d h i g h e r w i t h MgCl 2 t h a n w i t h M n C l 2 . The p r e s e n c e o f Mn i n t h e t r a n s c r i p t i o n r e a c t i o n s s t i m u l a t e d t h e s y n t h e s i s o f an RNA s l i g h t l y l a r g e r t h a n R N A - I . The n a t u r e o f t h i s RNA was n o t i n v e s t i g a t e d f u r t h e r . 4 . a-amanitin s e n s i t i v i t y p r o f i l e S e n s i t i v i t y t o t h e i n h i b i t o r a - a m a n i t i n h a s b e e n u s e d t o c l a s s i f y e u k a r y o t i c RNA p o l y m e r a s e s i n t o t h r e e c l a s s e s , n a m e l y I , I I a n d I I I ( 4 3 , 4 4 ) . A s e r i e s o f r e a c t i o n s w i t h v a r y i n g c o n c e n t r a t i o n s o f a - a m a n i t i n w e r e p e r f o r m e d ( F i g . 2 5 ) t o d e t e r m i n e t h e RNA p o l y m e r a s e r e s p o n s i b l e f o r t h e s p e c i f i c t r a n s c r i p t i o n o b s e r v e d h e r e . Low c o n c e n t r a t i o n s o f a - a m a n i t i n ( l a n e s 2 - 5 ) had v e r y l i t t l e e f f e c t on s p e c i f i c t r a n s c r i p t i o n o f pDt78R DNA i n t h e D r o s o p h i l a e x t r a c t . C o n c e n t r a t i o n s as h i g h a s 250 jug/ml and 1 mg/ml w e r e f o u n d t o i n h i b i t RNA s y n t h e s i s 60% a n d 70% r e s p e c t i v e l y . H i g h t o l e r a n c e o f a - a m a n i t i n h a s b e e n r e p o r t e d a s a common f e a t u r e o f i n s e c t RNA p o l y m e r a s e I I I ( 4 4 , 5 1 , 8 3 , 1 4 1 , 1 4 2 ) . I n s e n s i t i v i t y t o l o w c o n c e n t r a t i o n s and i n h i b i t i o n by c o n c e n t r a t i o n b e l o w 1 mg/ml o f a - a m a n i t i n r u l e o u t RNA p o l y m e r a s e I I a n d I r e s p e c t i v e l y as b e i n g i n v o l v e d i n t r a n s c r i p t i o n o b s e r v e d h e r e . Thus t h e d a t a i n F i g . 25 p o i n t s t o RNA p o l y m e r a s e I I I a s t h e p o l y m e r a s e r e s p o n s i b l e f o r s p e c i f i c t r a n s c r i p t i o n o b s e r v e d w i t h t h e D r o s o p h i l a e x t r a c t . 5 . E f f e c t o f t e m p l a t e c o n c e n t r a t i o n The e f f e c t o f v a r y i n g p D t 0 . 3 DNA c o n c e n t r a t i o n on s p e c i f i c RNA s y n t h e s i s i s shown i n F i g s . 26 and 2 7 . The a u t o r a d i o g r a m i n F i g . 26 shows t h a t as t h e c o n c e n t r a t i o n o f p D t 0 . 3 DNA was i n c r e a s e d , t h e r e was a c o n c o m i t a n t i n c r e a s e i n t h e s y n t h e s i s o f a l a r g e r p r o d u c t ( s ) w h i c h d i d n o t e n t e r t h e g e l . S u c h a h i g h m . w t . p r o d u c t was a l s o n o t e d by W e i l e t ^1_. ( 8 0 ) . T h i s p r o d u c t was n o t a n a l y z e d f u r t h e r . T h e r e was a l s o an i n c r e a s e i n n o n s p e c i f i c t r a n s c r i p t i o n ( s e e n by t h e i n c r e a s e i n b a c k g r o u n d ) w i t h i n c r e a s i n g DNA c o n c e n t r a t i o n p o s s i b l y c a u s e d by n o n s p e c i f i c b i n d i n g o f RNA p o l y m e r a s e a n d / o r " f a c t o r s " . F i g u r e 2 5 . E f f e c t o f a - a m a n i t i n on s p e c i f i c RNA s y n t h e s i s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . T r a n s c r i p t i o n r e a c t i o n s w e r e p e r f o r m e d and a n a l y z e d as d e s c r i b e d i n M e t h o d s , e x c e p t t h a t v a r y i n g a m o u n t s o f a - a m a n i t i n w e r e u s e d . S t a n d a r d r e a c t i o n s c o n t a i n e d pDt78R DNA and t h e i n d i c a t e d c o n c e n t r a t i o n o f a - a m a n i t i n . The i n s e t shows t h e t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h pDt78R DNA and 0 . 0 , 0 . 1 , 0 . 5 , 2 . 5 , 5 0 , 1 0 0 , 250 and 1 0 0 0 jug/ml a - a m a n i t i n , i n l a n e s 2 - 9 r e s p e c t i v e l y . L a n e s 1 and 10 c o n t a i n [ 1 2 5 I ] - t R N A y a l m a r k e r . ioo 1 0 1 . F i g u r e 2 6 . E f f e c t o f p D t 0 . 3 DNA c o n c e n t r a t i o n on t r a n s c r i p t i o n i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . T r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g v a r y i n g amounts o f p D t 0 . 3 DNA w e r e p e r f o r m e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s e x c e p t t h a t t h e r e a c t i o n s w e r e i n c u b a t e d f o r 90 m i n . L a n e s 1 - 1 4 show t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h 0 . 1 , 0 . 2 , 0 . 4 , 0 . 6 , 0.8, 1 . 0 , 1 . 5 , 2 . 0 , 2 . 5 , 3 . 0 , 3 . 5 , 4 . 0 , 4 . 5 and 5 . 0 jug p D t 0 . 3 DNA, r e s p e c t i v e l y . I and I I d e n o t e t h e p o s i t i o n s o f RNA-I and R N A - I I . 1 0 3 . The g r a p h i n F i g . 27 shows t h a t a t l o w c o n c e n t r a t i o n s o f p D t 0 . 3 DNA, t h e r e was a l m o s t a l i n e a r i n c r e a s e i n t h e s y n t h e s i s o f t h e s p e c i f i c RNA p r o d u c t s , w i t h maximum t r a n s c r i p t i o n a c h i e v e d b e t w e e n 0 . 6 - 1 . 0 p g o f p D t 0 . 3 DNA; f u r t h e r i n c r e a s e i n DNA c o n c e n t r a t i o n c a u s e d a s i g n i f i c a n t i n h i b i t i o n o f s p e c i f i c t r a n s c r i p t i o n . S i m i l a r r e s u l t s w e r e o b t a i n e d when p D t 7 8 R , pDt92R a n d p D t l 2 0 R ( T a b l e I I I ) w e r e u s e d a s DNA t e m p l a t e s ( F i g . 2 8 ) . The o p t i m u m DNA c o n c e n t r a t i o n v a r i e d s l i g h t l y f r o m t h a t o f p D t 0 . 3 , w i t h pDt92R and p D t l 2 0 R s h o w i n g an o p t i m u m r a n g e b e t w e e n 0 . 6 and 1 . 5 ug and pDt78R b e t w e e n 0 . 8 and 1 . 5 f i g . I n h i b i t i o n o f s p e c i f i c t r a n s c r i p t i o n was a l s o o b s e r v e d w i t h t h e s e t e m p l a t e s when t h e i r r e s p e c t i v e o p t i m u m DNA c o n c e n t r a t i o n was e x c e e d e d , b u t t h e d e c l i n e was more g r a d u a l t h a n t h a t s e e n w i t h p D t 0 . 3 DNA. The d a t a i n F i g . 29 show t h a t when v a r y i n g a m o u n t s o f pBR322 DNA w e r e a d d e d t o r e a c t i o n s c o n t a i n i n g 0 . 4 p g p D t 0 . 3 DNA, t h e r e was a d r a m a t i c i n h i b i t i o n o f s p e c i f i c t r a n s c r i p t i o n a f t e r t h e t o t a l amount o f DNA i n t h e r e a c t i o n e x c e e d e d 0 . 8 p g , t h e DNA o p t i m u m f o r p D t 0 . 3 t r a n s c r i p t i o n ( R e f e r t o F i g . 2 7 ) . T h i s r e s u l t s u g g e s t s t h a t t h e m e c h a n i s m o f i n h i b i t i o n o f s p e c i f i c t r a n s c r i p t i o n when t h e DNA o p t i m u m i s e x c e e d e d i s o f a n o n - s p e c i f i c n a t u r e . I I I . K i n e t i c s o f t r a n s c r i p t i o n o f c l o n e d tRNA g e n e s A . T i m e c o u r s e o f RNA s y n t h e s i s P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t d i f f e r e n t p l a s m i d s c o n t a i n i n g Val tRNA g e n e s t r a n s c r i b e d a t d i f f e r e n t e f f i c i e n c i e s ( F i g s . 2 7 , 2 8 ) . To i n v e s t i g a t e t h e r a t e s o f t r a n s c r i p t i o n d i r e c t e d by d i f f e r e n t t e m p l a t e s , i t was n e c e s s a r y t o l o o k a t t h e t i m e c o u r s e o f RNA s y n t h e s i s f i r s t . A t y p i c a l t i m e c o u r s e o f RNA s y n t h e s i s d i r e c t e d by p D t 0 . 3 i s shown i n F i g . 3 0 . A d i s t i n c t l a g was o b s e r v e d i n t h e r a t e o f s y n t h e s i s l a s t i n g a p p r o x i -m a t e l y 30 m i n . F o l l o w i n g t h e l a g i n t h e r e a c t i o n , t h e r a t e o f s y n t h e s i s was l i n e a r f o r up t o two h o u r s . A l l p l a s m i d s t e s t e d showed t h i s b i p h a s i c t i m e 1 0 4 . F i g u r e 2 7 . E f f e c t o f i n c r e a s i n g amount o f p D t 0 . 3 DNA on s p e c i f i c t r a n s c r i p -t i o n . The sum o f t h e r a d i o a c t i v i t y p r e s e n t i n t h e m a j o r t r a n s c r i p t i o n p r o d u c t s f r o m i n d i v i d u a l r e a c t i o n s shown i n F i g . 26 was d e t e r m i n e d and p l o t t e d a g a i n s t t h e c o n c e n t r a t i o n o f DNA. 1 0 5 . To to 50 to to DNA (ug) 106 . F i g u r e 2 8 . T r a n s c r i p t i o n w i t h v a r y i n g a m o u n t s o f d i f f e r e n t t e m p l a t e DNAs. T r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g v a r y i n g a m o u n t s o f pDt78R ( s o l i d c i r c l e s ) , p D t l 2 0 R ( o p e n c i r c l e s ) a n d pDt92R ( s q u a r e s ) DNA w e r e p e r f o r m e d a n d a n a l y z e d a s d e s c r i b e d i n M e t h o d s . The sum o f t h e r a d i o a c t i v i t y p r e s e n t i n t h e m a j o r t r a n s c r i p t i o n p r o d u c t s was d e t e r m i n e d and p l o t t e d a g a i n s t c o n c e n t r a t i o n o f DNA. a s to it zo z5 to DNA (gg) 1 0 8 , F i g u r e 2 9 . I n h i b i t i o n o f s p e c i f i c t r a n s c r i p t i o n by i n c r e a s i n g a m o u n t s o f pBR322 DNA. T r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g 0 . 4 jug p D t 0 . 3 DNA and v a r y i n g a m o u n t s o f pBR322 DNA w e r e p e r f o r m e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s . The sum o f t h e r a d i o a c t i v i t y p r e s e n t i n t h e m a j o r t r a n s c r i p t i o n p r o d u c t s d i r e c t e d by p D t 0 . 3 was d e t e r m i n e d and p l o t t e d a g a i n s t t h e c o n c e n t r a t i o n o f pBR322 DNA i n t h e r e a c t i o n s . 1 0 9 , i pBR322 (pg) 1 1 0 , F i g u r e 3 0 . Time c o u r s e o f RNA s y n t h e s i s w i t h p D t 0 . 3 DNA a s t h e t e m p l a t e . The RNA b a n d s shown i n t h e g e l i n F i g . 16 w e r e e x c i s e d and t h e sum o f t h e r a d i o a c t i v i t y p r e s e n t i n b a n d s a , b and c was d e t e r m i n e d and p l o t t e d a g a i n s t t h e t i m e o f i n c u b a t i o n w i t h t h e S . 1 0 0 e x t r a c t . I l l , 2 0 4 0 6 0 8 0 100 120 Time of incubation (min) i i i 112. c o u r s e o f s y n t h e s i s . The l e n g t h o f t h e l a g p e r i o d was e f f e c t i v e l y t h e same r e g a r d l e s s o f t h e s l o p e o f t h e l i n e a r p o r t i o n o f t h e s y n t h e s i s c u r v e . The l i n e a r s l o p e o f t h e t i m e c o u r s e e x t r a p o l a t e d t o 20 m i n . on t h e a b s c i s s a , i n d i c a t i n g t h a t an a s s e m b l y s t e p r e q u i r i n g a p p r o x i m a t e l y 20 m i n . i s n e c e s s a r y f o r t h e l i n e a r r a t e o f t r a n s c r i p t i o n b e t w e e n 30 and 120 m i n . V a l B . C o m p a r i s o n o f r a t e s o f t r a n s c r i p t i o n o f c l o n e d tRNA g e n e s The DNA d e p e n d e n c e o f t h e t r a n s c r i p t i o n r e a c t i o n was i n v e s t i g a t e d u s i n g a 90 m i n . r e a c t i o n t i m e s i n c e t h i s was w e l l w i t h i n t h e l i n e a r p o r t i o n o f t h e t i m e c o u r s e . As t h e l a g t i m e f o r d i f f e r e n t p l a s m i d s was e s s e n t i a l l y i d e n t i c a l , t h e DNA d e p e n d e n c e c u r v e s m e a s u r e d t h e r a t e o f r e a c t i o n . T r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g v a r y i n g a m o u n t s o f DNA w e r e p e r f o r m e d V a l f o r v a r i o u s t R N A p l a s m i d s . The sum o f r a d i o a c t i v i t y p r e s e n t i n t h e m a j o r t r a n s c r i p t i o n p r o d u c t s was d e t e r m i n e d and p l o t t e d a g a i n s t t h e amount o f DNA i n t h e r e a c t i o n m i x ( F i g s . 2 7 , 2 8 ) . T h e s e r e s u l t s showed t h a t f o r s e v e r a l p l a s m i d s , t h e c o n c e n t r a t i o n o f DNA y i e l d i n g maximum tRNA t r a n s c r i p t i o n was a r a t h e r b r o a d r a n g e . H e n c e , i t was d i f f i c u l t t o d e t e r m i n e t h e e x a c t DNA c o n c e n t r a t i o n t h a t y i e l d e d maximum tRNA t r a n s c r i p t i o n . F o r t h i s r e a s o n , t h e DNA d e p e n d e n c e o f t h e r a t e o f t r a n s c r i p t i o n was d e t e r m i n e d f r o m t h e i n i t i a l s l o p e o f t h e DNA c u r v e ( F i g . 3 1 ) . S i n c e t h e p l a s m i d s v a r i e d i n s i z e , t h e DNA c o n c e n t r a t i o n s w e r e e x p r e s s e d a s g e n e e q u i v a l e n t s a f t e r n o r m a l i z i n g t h e m . w t s . o f t h e v a r i o u s p l a s m i d s t o t h a t o f p D t 0 . 3 . A l s o , b e c a u s e t h e t r a n s c r i p t s w e r e l a b e l l e d w i t h [ a - 3 2 P ] U T P , t h e a p p r o x i m a t e U - c o n t e n t o f t h e t r a n s c r i p t s was t a k e n i n t o a c c o u n t i n d e t e r m i n i n g t h e r a t e s o f t r a n s c r i p t i o n . The p l a s m i d , p D t 0 . 3 , d e r i v e d f r o m p D t 5 5 t r a n s c r i b e d a t t h e h i g h e s t r a t e ( 2 x l O ^ d p m / u g / m i n ) . P l a s m i d s pDt92R and p D t l 2 0 R , c a r r y i n g v a r i a n t g e n e s f o r t R N A ^ a l ( T a b l e I I I ) t r a n s c r i b e d r e s p e c t i v e l y a t r a t e s 8 and 6 f o l d l o w e r t h a n p D t 0 . 3 . P l a s m i d , p D t 7 8 R , c a r r y i n g a g e n e f o r t R N A ^ a l ( T a b l e I I I ) d i r e c t e d t r a n s c r i p t i o n a t a r a t e 3 - 4 f o l d l o w e r t h a n p D t 0 . 3 . 113. F i g u r e 3 1 . C o m p a r i s o n o f t h e r a t e s o f t r a n s c r i p t i o n o f d i f f e r e n t r e c o m b i n a n t p l a s m i d s c a r r y i n g t R N A V a l g e n e s . The d a t a shown on t h i s f i g u r e i s d e r i v e d f r o m t h o s e shown i n F i g s . 27 a n d 28 e x c e p t t h a t o n l y t h e p o i n t s f o r m i n g t h e i n i t i a l s l o p e a r e shown and t h a t t h e DNA c o n c e n t r a t i o n i s e x p r e s s e d as gene e q u i v a l e n t s . The gene e q u i v a l e n t v a l u e s w e r e d e r i v e d by a s s u m i n g t h a t 0.1 p g o f p D t 0 . 3 DNA c o n t a i n e d 1 g e n e e q u i v a l e n t . Gene e q u i v a l e n t s f o r t h e o t h e r DNAs w e r e o b a i n e d by n o r m a l i z i n g t h e i r m . w t s . t o t h e m . w t . o f p D t 0 . 3 DNA. T r a n s c r i p t i o n d i r e c t e d by p D t 0 . 3 , p D t 7 8 R , p D t l 2 0 R and pDt92R i s r e p r e s e n t e d by s o l i d c i r c l e s , s o l i d s q u a r e s , o p e n s q u a r e s a n d o p e n c i r c l e s , r e s p e c t i v e l y . 1 1 I 1 1 I 1 T G e n e E q u i v a l e n t s 1 1 5 , C . S t a b i l i t y o f t h e t r a n s c r i p t i o n c o m p l e x One p o s s i b i l i t y f o r t h e d i f f e r e n c e i n t r a n s c r i p t i o n r a t e s f o r t h e p l a s m i d s was t h a t a c o m p l e x r e q u i r e d f o r i n i t i a t i o n o f t h e l i n e a r r a t e o f s y n t h e s i s was more s t a b l e f o r some p l a s m i d s t h a n o t h e r s . T h u s , f o r t e m p l a t e s d i r e c t i n g l o w t r a n s c r i p t i o n r a t e s t h e c o m p l e x w o u l d d i s s o c i a t e f r e q u e n t l y r e d u c i n g t h e o v e r a l l r a t e o f t r a n s c r i p t i o n . To e x a m i n e t h i s q u e s t i o n , a c o m p l e x s t a b i l i t y e x p e r i m e n t was d e s i g n e d u s i n g pDt78R and p D t 0 . 3 as t e m p l a t e s . T h e s e p l a s m i d s c a r r y g e n e s whose s e q u e n c e i s i d e n t i c a l t o t h e V a l V a l i s o a c c e p t o r s tRNA 4 and tRNA 3 b r e s p e c t i v e l y ( 1 3 , 1 4 , 3 9 ) . H o w e v e r , t h e p l a s m i d s a r e t r a n s c r i b e d a t d i f f e r e n t r a t e s and t h e p r i m a r y t r a n s c r i p t i o n p r o d u c t f r o m e a c h o f t h e DNAs c a n be s e p a r a t e d o n p o l y a c r y l a m i d e g e l s . To t e s t c o m p l e x s t a b i l i t y , o n e o f t h e t w o t e m p l a t e s was a d d e d a t a c o n c e n t r a t i o n j u s t b e l o w t h e o p t i m u m c o n c e n t r a t i o n . A t i n c r e a s i n g i n t e r v a l s t h e s e c o n d t e m p l a t e was a d d e d , a n d e a c h r e a c t i o n was i n c u b a t e d f o r an a d d i t i o n a l 90 m i n . The t r a n s c r i p t i o n p r o d u c t s ( R N A - I ) w e r e a n a l y z e d on p o l y a c r y l a m i d e g e l s ( F i g s . 32A and 3 2 B ) . When pDt78R was a d d e d t o t h e r e a c t i o n f i r s t , a d d i t i o n o f p D t 0 . 3 b e f o r e . 15 m i n . t o t a l l y i n h i b i t e d s y n t h e s i s o f t h e pDt78R p r i m a r y p r o d u c t ( F i g . 3 2 A ) . B e t w e e n 15 m i n . and 20 m i n . t h e r e was a d r a m a t i c c h a n g e i n t h e r a t i o o f t r a n s c r i p t i o n , s o t h a t a f t e r 20 m i n . , a d d i t i o n o f p D t 0 . 3 DNA had no e f f e c t on pDt78R t r a n s c r i p t i o n . When p D t 0 . 3 was a d d e d t o t h e r e a c t i o n f i r s t , no s y n t h e s i s o f pDt78R was d e t e c t e d , r e g a r d l e s s o f when i t was a d d e d t o t h e r e a c t i o n ( F i g . 3 2 B ) . I V . I n v i t r o m u t a g e n e s i s A . C o n s t r u c t i o n o f r e c o m b i n a n t p l a s m i d s The e x p e r i m e n t a l s t r a t e g y u s e d t o s e p a r a t e t h e two t R N A ^ a l g e n e s c o n t a i n e d i n p D t 5 5 ( T a b l e I I I ) and t o c o n s t r u c t t h e h y b r i d p l a s m i d s and t h e 116 , F i g u r e 3 2 . S t a b i l i t y o f t h e t r a n s c r i p t i o n c o m p l e x . A . 0 . 8 ug o f pDt78R DNA was i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r 5 , 1 0 , 1 5 , 2 0 , 2 5 , 3 0 , 3 5 , 4 5 , 55 and 65 m i n . ( l a n e s 1 - 1 0 r e s p e c t i v e l y ) b e f o r e 0 . 8 jug o f p D t 0 . 3 DNA was a d d e d . A l l t h e r e a c t i o n s w e r e i n c u b a t e d f o r an a d d i t i o n a l 90 m i n . The t r a n s c r i p t i o n p r o d u c t s w e r e a n a l y z e d on a 15% - 7M u r e a p o l y a c r y l a -m i d e g e l t o d i s t i n g u i s h b e t w e e n RNA-I d i r e c t e d by p D t 0 . 3 ( a ) and pDt78R ( b ) . The r e a c t i o n i n l a n e 11 i s a t r a n s c r i p t i o n c o n t r o l c o n t a i n i n g 0 . 8 ug o f pDt78R DNA. L a n e 12 c o n t a i n s [ 1 2 5 I ] - t R N A 4 m a r k e r . B . T h i s e x p e r i m e n t was p e r f o r m e d a s d e s c r i b e d a b o v e , e x c e p t t h a t p D t 0 . 3 ( 0 . 6 u g ) was u s e d a s t h e f i r s t DNA and pDt78R ( 0 . 6 ug) a s t h e s e c o n d DNA ( l a n e s 2-11 r e s p e c t i v e l y ) , a i n t h e f i g u r e d e s i g n a t e s t h e p o s i t i o n o f RNA-I t r a n s c r i b e d by p D t 0 . 3 DNA. L a n e 12 shows a t r a n s c r i p t i o n c o n t r o l c o n t a i n i n g 0 . 6 u g o f p D t 0 . 3 DNA. L a n e 1 h a s L 1 2 5 I ] - t R N A ^ a l m a r k e r . 1 1 7 . 1 1 8 . d e l e t i o n m u t a n t s i s shown i n F i g . 4 ( M a t e r i a l s a n d M e t h o d s , s e c t i o n I V ) . B . C h a r a c t e r i z a t i o n o f t h e i n v i t r o c o n s t r u c t e d p l a s m i d s The new p l a s m i d s g e n e r a t e d i n v i t r o w e r e c h a r a c t e r i z e d by a p p r o p r i a t e r e s t r i c t i o n a n d / o r DNA s e q u e n c e a n a l y s i s . The DNA s e q u e n c e s o f t h e p a r e n t p l a s m i d s and t h e i n v i t r o c o n s t r u c t e d p l a s m i d s a r e shown i n F i g . 3 3 . The s e q u e n c e o f t h e h y b r i d p l a s m i d , p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) was d e d u c e d f r o m t h e DNA s e q u e n c e s o f pDt78R a n d p D t 0 . 3 . The a u t h e n t i c i t y o f t h i s h y b r i d p l a s m i d was d e t e r m i n e d b y r e s t r i c t i o n a n a l y s i s ( s e e b e l o w ) . The DNA s e q u e n c e o f p D t 0 . 3 d e t e r m i n e d h e r e a g r e e d c o m p l e t e l y w i t h t h e c o r r e s p o n d i n g s e q u e n c e p r e s e n t i n p D t 5 5 ( 1 3 , 3 9 ) . The p l a s m i d s , pDt92R and pDt78R w e r e s e q u e n c e d by A d d i s o n e t a]_. ( 1 3 , 3 9 ) . The c o d i n g r e g i o n o f t h e h y b r i d p l a s m i d , p D t 0 . 3 ( 5 ' ) - 9 2 ( 3 ' ) , i s i d e n t i c a l t o t h a t o f p D t 0 . 3 e x c e p t t h a t i t h a s an A i n s t e a d o f a G a t p o s i t i o n 57 ( u n d e r l i n e d ) ; t h e 5 ' and 3 ' - f l a n k i n g s e q u e n c e s c o n t a i n e d i n t h i s p l a s m i d d e r i v e f r o m p D t 0 . 3 a n d pDt92R r e s p e c t i v e l y ( F i g s . 4 , 3 3 ) . On t h e o t h e r h a n d , t h e 5 ' - f l a n k i n g a n d t h e c o d i n g r e g i o n o f t h e h y b r i d p l a s m i d p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) a r e i d e n t i c a l t o t h o s e o f pDt92R e x c e p t f o r t h e p r e s e n c e o f a G i n s t e a d o f an A a t p o s i t i o n 57 ( u n d e r l i n e d ) ; t h e 3 ' - f l a n k i n g s e q u e n c e i s t h a t o f p D t 0 . 3 ( F i g s . 4 , 3 3 ) . The h y b r i d p l a s m i d , p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) , h a s a c o d i n g r e g i o n i d e n t i c a l t o t h a t o f p D t 0 . 3 , e x c e p t t h a t i t h a s a G i n s t e a d o f an A a t p o s i t i o n 6 9 ( o v e r l i n e d ) ; t h e 5 ' and 3 ' - f l a n k i n g s e q u e n c e s o r i g i n a t e f r o m p D t 0 . 3 and pDt92R r e s p e c t i v e l y ( F i g s . 4 , 3 3 ) . The c o d i n g r e g i o n o f p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) i s i d e n t i c a l t o t h a t o f pDt78R e x c e p t f o r an A i n s t e a d o f a G a t p o s i t i o n 69 ( o v e r l i n e d ) ; i t s 5 ' and 3 ' - f l a n k i n g s e q u e n c e s a r e s i m i l a r t o t h o s e o f pDt78R a n d p D t 0 . 3 r e s p e c t i v e l y ( F i g s . 4 , 3 3 ) . The d e l e t i o n m u t a n t , p D t O . 3 A 5 1 - 6 1 , i s s i m i l a r i n a l l r e s p e c t s t o pDt 0 . 3 e x c e p t f o r 11 n u c l e o t i d e s a t p o s i t i o n s 51-61 i n t h e c o d i n g r e g i o n ( F i g . 1 1 9 . F i g u r e 3 3 . DNA s e q u e n c e s o f p a r e n t and i n v i t r o c o n s t r u c t e d p l a s m i d s . The s e q u e n c e s o f t h e v a r i o u s p l a s m i d s shown i n t h i s f i g u r e w e r e d e t e r m i n e d by t h e m e t h o d o f Maxam and G i l b e r t . The s e q u e n c e o f o n l y t h e n o n c o d i n g s t r a n d i s s h o w n . The 5 ' and 3 ' - e n d s o f t h e tRNA g e n e s a r e i n d i c a t e d . The d i f f e r e n c e s b e t w e e n p D t 0 . 3 , p D t 9 2 R , and h y b r i d p l a s m i d s , p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) , p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) a t p o s i t i o n 57 a r e u n d e r l i n e d . S i m i l a r l y , t h e d i f f e r e n c e s b e t w e e n p D t 0 . 3 , p D t 7 8 R , p D t O . 3 ( 5 ' ) - 7 8 R ( 3 ' ) and p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) a t p o s i t i o n 69 a r e o v e r l i n e d . The d a s h e d r e g i o n i n d i c a t e s t h a t t h e n u c l e o t i d e s b e t w e e n p o s i t i o n s 51 and 61 a r e m i s s i n g i n t h e d e l e t i o n m u t a n t , p D t O . 3 A 5 1 - 6 1 . DNA s e q u e n c e s o f p a r e n t and i n v i t r o c o n s t r u c t e d p l a s m i d s i 1 p D t 0 . 3 G T T T C pDt92R G T T T C p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) G T T T C p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) G T T T C pDt78R G T T T C p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) G T T T C p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) G T T T C p D t 0 . 3 ^ 51-61 G T T T C p D t 0 . 3 A G A A G pDt92R G G A A G p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) G G A A G p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) A G A A G pDt78R A C A A G p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) A C A A G p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) A G A A G p D t 0 . 3 A 51-61 A G A A G 10 20 c G T G G T G T A G C G G T T A T c G T G G T G T A G T G G T T A T c G T G G T G T A G T G G T T A T c G T G G T G T A G C G G T T A T c G T A G T G T A G C G G T T A T c G T A G T G T A G C G G T T A T c G T G G T G T A G C G G T T A T c G T G G T G T A G C G G T T A T 50 60 G C C C C C G G T T C G A T C C C G C C C C C G G T T C A A T C C C G C C C C C G G T T C G A T C C c G C C C C C G G T T C A A T C C c G T C C C C G G T T C G A A C C c G T C C C C G G T T C G A A C C c G C C C C C G G T T C G A T C C c G C C C C c 30 40 C A C A T C T G C C T A A C A C G C C A C A T C C G C C T A A C A C G C C A C A T C C G C C T A A C A C G C C A C A T C T G C C T A A C A C G C C A C G T G T G C T T C A C A C G C C A C G T G T G C T T C A C A C G C C A C A T C T G C C T A A C A C G C C A C A T C T G C C T A A C A C G C 3 ' _ 70 i 80 G G G C G G A A A C A G G T G A T A G G G C G G A A A C A G T T G G A A G G G C G G A A A C A G G T G A T A G G G C G A A A A C A G T T G G A A G G G C G G G A A C A T G C G A T C G G G C G G A A A C A G G T G A T A G G G C G G G A A C A T G C G A T C G G G C G G A A A C A G G T G A T A 90 100 p D t 0 . 3 A A C T T T T T T T T T A G T T T T T A pDt92R T T T A T T T T T T G C T A A A T A T T p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) A A C T T T T T T T T T A G T T T T T A p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) T T T A T T T T T T G C T A A A T A T T pDt78R C T T T T T G A A T T A A T T T A T C A p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) A A C T T T T T T T T T A G T T T T T A p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) C T T T T T G A A T T A A T T T A T C A p D t 0 . 3 A 51-61 A A C T T T T T T T T T A T T T A T C A I—1 to o 3 3 ) ; t h e a u t o r a d i o g r a m o f a s e q u e n c e g e l ( F i g . 34) shows t h e r e l e v a n t p o r t i o n m i s s i n g . I t s h o u l d be n o t e d t h a t when a l l t h e r e c o n s t r u c t e d p l a s m i d s d i s c u s s e d a b o v e w e r e d i g e s t e d w i t h H i n d l l l , t h e y g e n e r a t e d H i n d l l l i n s e r t s o f l e n g t h s e x p e c t e d f r o m t h e f r a g m e n t s w h i c h w e r e u s e d f o r t h e o r i g i n a l l i g a t i o n ( r e f e r t o F i g . 4 ) . I n v i t r o c o n s t r u c t e d p l a s m i d s , w h i c h w e r e n o t s e q u e n c e d , w e r e c h a r a c -t e r i z e d by r e s t r i c t i o n a n a l y s i s ( F i g . 3 5 ) . The p l a s m i d , p D t 0 . 6 , when d i g e s t e d w i t h H i n d l l l ( l a n e 1) o r S m a l , H i n d l l l ( l a n e 2 ; Smal i s an i s o s c h i z o m e r o f X m a l ) r e l e a s e d f r a g m e n t s o f l e n g t h s e x p e c t e d f r o m t h e c o r r e s p o n d i n g s e q u e n c e p r e s e n t i n p D t 5 5 ( 1 3 , 3 9 ) ; a 0 . 6 3 kb f r a g m e n t w i t h H i n d l l l d i g e s t i o n a n d a p p r o x i m a t e l y 0 . 5 kb and 160 bp f r a g m e n t s w i t h S m a l , H i n d l l l d o u b l e d i g e s t i o n . The h i g h m . w t . b a n d s e e n w i t h b o t h d i g e s t i o n s r e p r e s e n t s t h e 4 . 4 kb pBR322 f r a g m e n t . The d e l e t i o n m u t a n t , p D t O . 3 d l 5 ' - 1 8 , h a s a H i n d l l l i n s e r t a p p r o x i m a t e l y 170 bp l o n g ( l a n e 3 ) ; when t h i s DNA was c l e a v e d w i t h S m a l , H i n d l l l , i t g e n e r a t e d t w o f r a g m e n t s o f a l m o s t i d e n t i c a l l e n g t h , a b o u t 90 bp l o n g ( l a n e 4 ) . A g a i n , t h e h i g h m . w t . b a n d i n l a n e s 3 and 4 c o r r e s p o n d s t o t h e l i n e a r pBR322 f r a g m e n t . The r e s t r i c t i o n f r a g m e n t s o b t a i n e d w i t h t h e two d i g e s t s w e r e a s p r e d i c t e d f r o m t h e DNA s e q u e n c e ( 1 3 , 3 9 ) . The h y b r i d p l a s m i d , p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) , i s c l e a v e d w i t h H i n d l l l t o r e l e a s e a D r o s o p h i l a i n s e r t a p p r o x i m a t e l y 400 bp l o n g ( l a n e 8 ) ; t h i s s i z e i s e x p e c t e d f r o m t h e l e n g t h s o f t h e t w o f r a g m e n t s ( 3 0 0 b p , 8 4 b p ) l i g a t e d t o g e n e r a t e t h i s h y b r i d i n s e r t ( F i g . 4 ) . D o u b l e d i g e s t i o n o f t h e h y b r i d DNA w i t h S m a l and H i n d l l l ( l a n e 9 ) r e l e a s e d t h e o r i g i n a l 300 bp a n d 84 bp f r a g m e n t s ( c o m p a r e t h i s t o S m a l , H i n d l l l d i g e s t s o f pDt78R a n d p D t 0 . 3 i n l a n e s 7 a n d 6 r e s p e c t i v e l y ) . 122. F i g u r e 3 4 . Maxam and G i l b e r t s e q u e n c i n g o f t h e tRNA g e n e c o n t a i n e d i n t h e d e l e t i o n m u t a n t , p D t O . 3A 5 1 - 6 1 . A 140 bp H i n d l l l , Fnu4HI f r a g m e n t ( l a b e l l e d a t t h e H i n d l l l s i t e ) i s o l a t e d f r o m p D t 0 . 3 A51-61 was s e q u e n c e d by t h e m e t h o d o f Maxam and G i l b e r t . S a m p l e s o f t h e s e q u e n c i n g r e a c t i o n s s p e c i f i c f o r G ( l a n e 1 ) , A + G ( l a n e 2 ) , T + C ( l a n e 3) and C ( l a n e 4) w e r e e l e c t r o p h o r e s e d on a 12% d e n a t u r i n g p o l y -a c r y l a m i d e g e l a t 1 5 0 0 V ( 3 7 . 5 V/cm) f o r 3 . 5 h o u r s . A p o r t i o n o f t h e DNA s e q u e n c e ( n o h c o d i n g s t r a n d ) o f t h e tRNA g e n e c o n t a i n e d i n p D t 0 . 3 A 51-61 i s s h o w n . The a r r o w s e p a r a t i n g t h e t w o C s d e s i g n a t e s t h e p o s i t i o n a t w h i c h 11 n u c l e o t i d e s a r e m i s s i n g f r o m t h i s d e l e t i o n m u t a n t ( r e f e r t o F i g . 3 3 ) . The 3 ' - e n d o f t h e g e n e i s i n d i c a t e d . 123 . 1 2 3 4 124, F i g u r e 3 5 . R e s t r i c t i o n a n a l y s i s o f t h r e e i n v i t r o c o n s t r u c t e d p l a s m i d s . P l a s m i d s p D t 0 . 6 , p D t O . 3 d l 5 1 - 1 8 and p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) w e r e d i g e s t e d w i t h H i n d l l l ( l a n e s 1 , 3 , 8 r e s p e c t i v e l y ) a n d S m a l , H i n d l l l ( l a n e s 2 , 4 , 9 r e s p e c t i v e l y ) and e l e c t r o p h o r e s e d on a 10% p o l y a c r y l a m i d e g e l . L a n e 5 h a s H p a l l c l e a v e d pBR322 a s t h e m . w t . m a r k e r and l a n e s 6 and 7 c o n t a i n S m a l , H i n d l l l d i g e s t s o f p D t 0 . 3 a n d pDt78R r e s p e c t i v e l y . 125 4 5 6 7 8 9 4 * * £ t 3   1 2 6 . C . T r a n s c r i p t i o n o f i n v i t r o c o n s t r u c t e d p l a s m i d s 1 . T r a n s c r i p t i o n o f h y b r i d c l o n e s The t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h h y b r i d and p a r e n t p l a s m i d s a r e shown i n F i g . 36 and t h e e f f i c i e n c i e s o f t r a n s c r i p t i o n o f t h e v a r i o u s p l a s m i d s a r e c o m p a r e d i n T a b l e V . a . T r a n s c r i p t i o n o f p D t 0 . 3 - 7 8 R h y b r i d p l a s m i d s The h y b r i d p l a s m i d , p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) ( l a n e 6 ) showed a t r a n s c r i p t i o n p a t t e r n and an e f f i c i e n c y s i m i l a r t o t h e p a r e n t p l a s m i d , p D t 0 . 3 , ( l a n e 2 , T a b l e V) b u t a p r o c e s s i n g ( o f * R N A - I t o R N A - I I ) e f f i c i e n c y s i m i l a r t o t h a t o f t h e o t h e r p a r e n t p l a s m i d , pDt78R ( l a n e 4 ) . The r a t i o o f t h e r a d i o a c t i v i t y i n c o r p o r a t e d i n R N A - I : R N A - I I f o r e q u i m o l a r a m o u n t s o f p D t 0 . 3 , pDt78R and p D t 0 . 3 ( 5')-78R ( 3 ' ) was 1 2 . 8 , 2 . 5 and 2 . 0 r e s p e c t i v e l y , i n d i c a t i n g t h a t R N A - I d i r e c t e d by pDt78R and p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) was c o n v e r t e d t o R N A - I I 5 t o 6 t i m e s more e f f i c i e n t l y t h a n t h e RNA-I f r o m p D t 0 . 3 . T h e s e r e s u l t s a r e v e r y s i m i l a r t o t h o s e o b t a i n e d f r o m t h e p r o c e s s i n g e x p e r i m e n t s i n s e c t i o n I I . C . 2 . The h i g h t r a n s c r i p t i o n and p r o c e s s i n g e f f i c i e n c y o f p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 1 ) i s c l e a r l y s e e n f r o m t h e d a t a i n F i g . 3 7 . The h y b r i d p l a s m i d s y n t h e s i z e d R N A - I a t a s h i g h a r a t e as p D t 0 . 3 (~2 x 10 d p m / m i n . ) ; h o w e v e r , t h e r a t e o f a p p e a r a n c e o f R N A - I I d i r e c t e d by i t ( 0 . 5 x 10 dpm/min.) was 5 t i m e s h i g h e r t h a n t h a t f o r p D t 0 . 3 ( 0 . 1 x 10 3 d p m / m i n . ) . The t r a n s c r i p t i o n and p r o c e s s i n g r e s u l t s o b t a i n e d w i t h t h e h y b r i d p l a s m i d , p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) , ( l a n e 5) w e r e j u s t t h e r e v e r s e o f t h o s e s e e n w i t h p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) , i . e . , i t s p a t t e r n and e f f i c i e n c y o f t r a n s c r i p t i o n w e r e s i m i l a r t o t h o s e o f pDt78R ( l a n e 4 , T a b l e V) b u t i t s r a t i o o f R N A - I : R N A - I I was c o m p a r a b l e t o t h a t o b t a i n e d w i t h p D t 0 . 3 . The 5 ' - f l a n k i n g s e q u e n c e s and t h e c o d i n g r e g i o n s o f t h e h y b r i d p l a s m i d s , p D t 0 . 3 ( 5')-78R ( 3 ' ) and p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) , a r e i d e n t i c a l t o t h o s e o f p D t 0 . 3 a n d pDt78R r e s p e c t i v e l y , e x c e p t f o r t h e o n e n u c l e o t i d e c h a n g e a t p o s i t i o n 69 i n 127 . F i g u r e 3 6 . T r a n s c r i p t i o n o f p a r e n t and i n v i t r o c o n s t r u c t e d p l a s m i d s . T r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g 0 . 5 jug o f e a c h p l a s m i d DNA w e r e p e r f o r m e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s e x c e p t t h a t t h e r e a c t i o n s w e r e i n c u b a t e d f o r 90 m i n . L a n e s 1 - 9 show t h e t r a n s c r i p t i o n p r o d u c t s o b t a i n e d w i t h p D t 0 . 6 , p D t 0 . 3 , p D t 0 . 3 d l 5 ' - 1 8 , p D t 7 8 R , p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) , p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) , p D t 9 2 R , p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) a n d p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) , r e s p e c t i v e l y . 1 2 8 . 129. T a b l e V , C o m p a r i s o n o f t h e r a t e s o f t r a n s c r i p t i o n The v a l u e s shown i n t h e m i d d l e c o l u m n w e r e o b t a i n e d f r o m t h e i n i t i a l s l o p e o f t h e r e s p e c t i v e DNA c u r v e as d e s c r i b e d u n d e r R e s u l t s s e c t i o n 11 I B . The r e s u l t s shown i n t h e l a s t c o l u m n w e r e an a v e r a g e o f s e v e r a l s i n g l e p o i n t t r a n s c r i p t i o n e x p e r i m e n t s . A l l t h e r a t e s w e r e e x p r e s s e d r e l a t i v e t o t h a t o f p l a s m i d p D t 0 . 3 w h i c h was a s s u m e d t o be 1 . P I a s m i d R e l a t i v e r a t e s o f t r a n s c r i p t i o n R a t e d e t e r m i n e d f r o m t h e i n i t i a l s l o p e o f t h e DNA c u r v e A v e r a g e o f 3 - 5 s i n g l e p o i n t e x p e r i m e n t s p D t 0 . 6 0 . 9 0 0 . 9 0 p D t 0 . 3 1 . 0 0 1 . 0 0 pDt78R 0 . 3 0 0 . 2 5 pDt92R 0 . 1 3 0 . 1 2 p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) 1 . 0 4 1 . 0 0 p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) 0 . 2 3 0 . 2 5 p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) 1 . 0 0 1 . 0 0 p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) 0 . 1 1 0 . 1 1 p D t O . 3 d l 5 ' - 1 8 0 . 5 0 0 . 5 0 p D t l 2 0 R 0 . 2 2 0 . 2 1 130v Figure 37. Comparison of transcription and processing eff ic iencies of plasmids pDt0.3 and pDt0.3(5')-78R(3'). Transcription reactions containing varying amounts of pDt0.3 (circles) and pDt0.3(5')-78R(3') (squares) were carried out and analyzed as described in Methods. The radioactivity incorporated in bands corresponding to RNA-I (solid c i rc les and squares) and RNA-II (open c i rc les and squares) was determined separately and plotted against the DNA concentration expressed as gene equivalents. 160 120 80 40 Gene Equivalents 132. t h e c o d i n g r e g i o n s ( F i g . 3 3 ) ; t h e i r 3 ' - f l a n k i n g s e q u e n c e s w e r e d e r i v e d f r o m pDt78R and p D t 0 . 3 , r e s p e c t i v e l y ( F i g . 4 ) . The r e s u l t s o b t a i n e d w i t h t h e s e two p l a s m i d s i n d i c a t e d t h a t t h e h i g h r a t e o f t r a n s r i p t i o n was a s s o c i a t e d w i t h t h e 5 ' - f l a n k i n g and t h e c o d i n g r e g i o n o f p D t 0 . 3 ; t h e A + G c h a n g e a t p o s i t i o n 69 d i d n o t a f f e c t t h e r a t e o f t r a n s c r i p t i o n . H o w e v e r , t h e i n f o r m a t i o n w h i c h d i c t a t e d e f f i c i e n t p r o c e s s i n g o f t h e e n s u i n g RNA-I a p p e a r e d t o r e s i d e i n t h e 3 ' f r a g m e n t f r o m p D t 7 8 R ; t h e p r e s e n c e o f G a t p o s i t i o n 69 i n t h e c o d i n g r e g i o n a n d / o r t h e 3 ' - f l a n k i n g s e q u e n c e may be s i g n i f i c a n t , b . T r a n s c r i p t i o n o f p D t 0 . 3 - 9 2 R h y b r i d p l a s m i d s The h y b r i d p l a s m i d , p D t O . 3 ( 5 ')-92R(3' ) , ( l a n e 9 ) t r a n s c r i b e d a s e f f i c i e n t l y a s p D t 0 . 3 ( l a n e 2 , T a b l e V ) , b u t t h e RNA-I d i r e c t e d by i t was p r o c e s s e d t o R N A - I I v e r y i n e f f i c i e n t l y , u n l i k e t h a t f r o m p D t 0 . 3 . C o m p l e m e n t a r y r e s u l t s w e r e o b t a i n e d w i t h t h e o t h e r h y b r i d p l a s m i d , pDt92R ( 5 ' ) - 0 . 3 ( 3 ' ) , ( l a n e 8 ) ; i t s t r a n s c r i p t i o n e f f i c i e n c y was s i m i l a r t o pDt92R ( l a n e 7 , T a b l e V ) , h o w e v e r t h e c o n v e r s i o n o f i t s RNA-I t o R N A - I I was a b o u t t w i c e a s e f f i c i e n t a s t h a t o f p D t 9 2 R . T h e s e r e s u l t s e m p h a s i z e d a g a i n t h a t t h e h i g h t r a n s c r i p t i o n e f f i c i e n c y was a s s o c i a t e d w i t h t h e 5 ' ^ f l a n k i n g and t h e c o d i n g r e g i o n o f p D t 0 . 3 , t h e G ^ A c h a n g e a t p o s i t i o n 57 i n t h e c o d i n g r e g i o n d i d n o t a f f e c t t h e r a t e o f t r a n s c r i p t i o n . The p r o c e s s i n g d a t a i n d i c a t e d t h e i n f l u e n c e o f 3 ' - t e r m i n a l s e q u e n c e s on t h e e f f i c i e n c y o f c o n v e r s i o n o f RNA-I t o R N A - I I . The 3 ' - t e r m i n a l s e q u e n c e s o f p D t 0 . 3 d i r e c t e d a s l i g h t l y h i g h e r r a t e o f c o n v e r s i o n o f RNA-I t o R N A - I I c o m p a r e d t o t h o s e f r o m p D t 9 2 R . 2 . T r a n s c r i p t i o n o f d e l e t i o n m u t a n t s a . T r a n s c r i p t i o n o f p D t 0 . 3 d ! 5 ' - 1 8 The d e l e t i o n m u t a n t , p D t 0 . 3 d l 5 ' - 1 8 , i s i d e n t i c a l t o p D t 0 . 3 , e x c e p t t h a t i t h a s o n l y 18 n u c l e o t i d e s o f D r o s o p h i l a DNA 5 ' t o t h e s t a r t o f t h e c o d i n g r e g i o n ( F i g . 4 ) . I t s t r a n s c r i p t i o n p a t t e r n ( l a n e 3) was s i m i l a r t o t h a t o f 133. pDt0.3 (lane 2), but i t transcribed at a rate 2-fold lower than pDt0.3 (Table V). b. Transcription of pDt0.3A 51-61 The plasmid, pDt0.3 A 51-61, is identical to pDt0.3 in al l respects except that i t is missing 11 nucleotides at positions 51-61 in the coding region (Figs. 4, 33, 34). The transcription of this deletion mutant is shown .in F ig . 38. The results in F ig . 38 show that as the concentration of pDt0.3A 51-61 DNA in the reaction mix was increased, there was a concomitant increase in the intensity of a band intermediate in length between RNA-I (-100 nucleotides) and RNA-II (~76 nucleotides) directed by pDt0.3 (lane 8) , and another fuzzy band s l ight ly longer than RNA-I. These new bands were not present in the endogenous (lane 1) or the pBR322 (lane 2) controls, suggesting that they were specif ic to Drosophila DNA contained in pDt0.3 A51-61. Because of the paucity of material, these bands could not be analyzed further. The shorter of the two transcripts directed by pDt0.3 A51 -61 is approximately 85 nucleotides long and could represent the shortened derivative (short by about 11 nucleotides, the length of the deletion) of RNA-I directed by pDt0.3; the higher m.wt. transcript is about 100 nucleotides long and could be due to in i t ia t ion at an ear l ier s i te and/or termination at a later s i te (compared to the in i t ia t ion and termination sites used by pDt0.3). Such uncharacteristic in i t ia t ion and/or termination may be caused by the internal deletion. The eff iciency of transcription of pDt0.3 A51-61 was approximately 2% of that of pDt0.3. In order to ascertain that the low level of transcription observed with pDt0.3 A 51-61 was not due to the instabi l i ty of i ts transcrip-t ion product(s), half tRNA molecules were isolated, incubated with the S.100 extract for different lengths of time and analyzed on polyacrylamide gel (Fig. F i g u r e 3 8 . T r a n s c r i p t i o n o f t h e d e l e t i o n m u t a n t , p D t 0.3A 5 1 - 6 1 . T r a n s c r i p t i o n r e a c t i o n s c o n t a i n i n g 0 . 2 , 0 . 4 , 0 . 8 , 1 . 0 , 1 . 5 jug o f p D t 0 . 3 A 51-61 DNA ( l a n e s 3 - 7 r e s p e c t i v e l y ) w e r e p e r f o r m e d and a n a l y z e d a s d e s c r i b e d i n M e t h o d s e x c e p t t h a t t h e r e a c t i o n s w e r e i n c u b a t e d f o r 90 m i n . R e a c t i o n 1 c o n t a i n s no a d d e d DNA and r e a c t i o n 2 c o n t a i n s 1 jug pBR322 DNA. I a n d I I d e s i g n a t e d t h e p o s i t i o n s o f R N A - I and R N A - I I d i r e c t e d by p D t 0 . 3 ( l a n e 8 ) . 135. 2 3 4 5 6 X 8 . 1 3 6 . 3 9 ) as d e s c r i b e d b e l o w . 1 2 5 Val [ I ] - t R N A 4 was p a r t i a l l y d i g e s t e d w i t h T-j_ r i b o n u c l e a s e and e l e c t r o -p h o r e s e d on a 15% p o l y a c r y l a m i d e g e l ( F i g . 3 9 A ) . T r a n s f e r RNA f r a g m e n t s a b o u t Val 34 n u c l e o t i d e s l o n g ( a r r o w , l a n e 3 ; t R N A 4 i s 76 n u c l e o t i d e s l o n g ; 14) w e r e i s o l a t e d and i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r v a r y i n g p e r i o d s o f t i m e ( F i g . 3 9 B ) . I t c a n be s e e n t h a t t h e 34 n u c l e o t i d e tRNA f r a g m e n t was s t a b l e i n t h e S . 1 0 0 e x t r a c t o v e r two h o u r s . D. C o m p l e x s t a b i l i t y e x p e r i m e n t s u s i n g p D t 0 . 3 A 51-61 DNA E x p e r i m e n t s s i m i l a r t o t h o s e d e s c r i b e d i n s e c t i o n 111C w e r e p e r f o r m e d w i t h p D t 0 . 3 A 51-61 as t h e f i r s t DNA and e i t h e r p D t 0 . 3 o r pDt78R a s t h e 2nd DNA ( F i g s . 4 0 , 4 1 ) . The r e s u l t s i n F i g s . 40A and 41 show t h a t as p D t 0 . 3 A 51-61 i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r more t i m e b e f o r e p D t 0 . 3 was a d d e d , t h e r e was a g r a d u a l i n h i b i t i o n o f t r a n s c r i p t i o n o f p D t 0 . 3 . F o r t h e f i r s t 15 m i n . , o n l y p D t 0 . 3 a p p e a r e d t o be t r a n s c r i b e d , b u t a f t e r 30 m i n . , f a i n t b a n d s a b o u t t h e l e n g t h o f p D t 0.3A 51-61 s h o r t t r a n s c r i p t , ( - 8 5 n u c l e o t i d e s ) w e r e d i s c e r n i b l e . A t no t i m e t e s t e d , was t h e t r a n s c r i p t i o n o f p D t 0 . 3 c o m p l e t e l y s h u t o f f . S i m i l a r r e s u l t s w e r e o b t a i n e d when pDt78R was u s e d a s t h e s e c o n d DNA ( F i g s . 40B and 41) e x c e p t t h a t t h e i n h i b i t i o n o f pDt78R t r a n s c r i p t i o n was s l i g h t l y more g r a d u a l t h a n t h a t s e e n w i t h p D t 0 . 3 DNA. The s h o r t t r a n s c r i p t d i r e c t e d by p D t O . 3 A 51-61 DNA was v i s i b l e b e f o r e t h e f i r s t 15 m i n . 137 . F i g u r e 3 9 . S t a b i l i t y o f h a l f tRNA m o l e c u l e s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . A . [ 1 2 5 I ] - t R N A 4 a l ( 0 . 5 / j g ) was d i g e s t e d w i t h ^ r i b o n u c l e a s e ( 0 . 0 1 U ) i n a 1 0 / u l r e a c t i o n c o n t a i n i n g 10 mM T r i s , pH 7 . 5 and 1 mM EDTA. The r e a c t i o n was i n c u b a t e d a t 3 7 ° C f o r 2 . 5 m i n . and s t o p p e d by t h e a d d i t i o n o f 10 /ul o f 10M u r e a , 0 . 1 % B P B , 0 . 1 % X C . The s a m p l e ( l a n e 3) was e l e c t r o p h o r e s e d on a 15%-7M u r e a p o l y a c r y l a m i d e g e l . L a n e 1 c o n t a i n s H p a l l c l e a v e d pBR322 a s t h e m . w t . m a r k e r a n d l a n e 2 c o n t a i n s u n d i g e s t e d L125! D - t R N A ^ 3 1 . B . T r a n s f e r RNA f r a g m e n t s a p p r o x i m a t e l y 34 n u c l e o t i d e s l o n g ( a r r o w ) w e r e e x c i s e d a n d t h e RNA r e c o v e r e d f r o m t h e g e l . A l i q u o t s o f t h e 34 n u c l e o t i d e l o n g t R N A Y a l f r a g m e n t w e r e i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r 1 5 , 3 0 , 4 5 , 6 0 , 90 a n a 120 m i n . ( l a n e s 2 - 7 r e s p e c t i v e l y ) . L a n e 1 shows a RNA c o n t r o l w h i c h was n o t i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t . 138. 1 2 3 7 6 5 4 3 2 1 B . 139, F i g u r e 4 0 . T r a n s c r i p t i o n c o m p l e x s t a b i l i t y e x p e r i m e n t s i n v o l v i n g t h e d e l e t i o n m u t a n t , p D t 0 . 3 A5 1 - 6 1 • A . p D t O . 3A 51-61 (2 / i g ) was i n c u b a t e d w i t h t h e S . 1 0 0 e x t r a c t f o r 0 , 1 5 , 3 0 , 45 a n d 60 m i n . ( l a n e s 2 - 6 r e s p e c t i v e l y ) b e f o r e p D t 0 . 3 DNA ( 0 . 6 p g ) was a d d e d . A l l t h e r e a c t i o n s w e r e i n c u b a t e d f o r an a d d i t i o n a l 90 m i n . R e a c t i o n s i n l a n e s 1 a n d 7 a r e c o n t r o l s c o n t a i n i n g 2 /jg pDt0.3A 51-61 and 0 . 6 /ug p D t 0 . 3 DNA r e s p e c t i v e l y . B . T h i s e x p e r i m e n t was p e r f o r m e d a s d e s c r i b e d a b o v e e x c e p t t h a t pDt78R ( 0 . 8 jug) was u s e d as t h e s e c o n d DNA ( l a n e s 2 - 6 r e s p e c t i v e l y ) . L a n e s 1 and 7 show c o n t r o l r e a c t i o n s c o n t a i n i n g 2 jug p D t 0 . 3 A 51-61 and 0 . 8 jug pDt78R DNA r e s p e c t i v e l y . The d a t a shown i n F i g s . 40A and B w e r e t a k e n f r o m s e p a r a t e g e l s . 140 . F i g u r e 4 1 . The d e l e t i o n m u t a n t , p D t 0 . 3 A 51-61 c o m p e t e s w i t h t r a n s c r i p t i o n o f i n t a c t t R N A g e n e s . The sum o f t h e r a d i o a c t i v i t y p r e s e n t i n t h e m a j o r t r a n s c r i p t i o n p r o d u c t s d i r e c t e d by p D t 0 . 3 ( c i r c l e s ) a n d pDt78R ( s q u a r e s ) i n F i g . 40 ( l a n e s 2 - 6 ) was d e t e r m i n e d a n d p l o t t e d a s p e r c e n t a g e o f r a d i o a c t i v i t y a t z e r o t i m e ( l a n e s 2) a g a i n s t t h e t i m e a t w h i c h t h e s e c o n d DNA was a d d e d . percent dpm X D i s c u s s i o n V a l I . N u c l e o t i d e s e q u e n c e o f D r o s o p h i l a t R N A d g e n e s A . 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 : C o m p a r i s o n t o t h e DNA s e q u e n c e o f o t h e r V a l t R N A4 g e n e c o n t a i n i n g p l a s m i d s V a l R e c o m b i n a n t p l a s m i d s c o n t a i n i n g D r o s o p h i l a t R N A 4 g e n e s w e r e i d e n t i f i e d V a l by t h e i r a b i l i t y t o h y b r i d i z e w i t h t R N A 4 ( 1 1 9 ) . F o u r d i f f e r e n t s i z e H i n d l l l f r a g m e n t s o f D r o s o p h i l a DNA ( 1 2 . 0 k b , 8 . 0 k b , 2 . 4 kb and 0 . 5 k b ; 119) c o n t a i n -V a l i n g t R N A 4 g e n e s w e r e i s o l a t e d i n t h e p l a s m i d s p D t l 4 , p D t 5 5 , p D t l 2 0 R and pDt92R r e s p e c t i v e l y ( 1 1 9 , 1 2 0 ) . The n u c l e o t i d e s e q u e n c e o f a s e g m e n t o f V a l p D t l 2 0 R DNA c o n t a i n i n g t h e t R N A 4 g e n e was d e t e r m i n e d i n t h e c o u r s e o f t h i s work and i s p r e s e n t e d e a r l i e r ( F i g . 1 3 ) . The n u c l e o t i d e s e q u e n c e s o f t h e V a l t R N A 4 g e n e s c o n t a i n e d i n t h e o t h e r t h r e e p l a s m i d s w e r e d e t e r m i n e d by o t h e r w o r k e r s ( 3 9 ) and a r e p r e s e n t e d i n F i g . 4 2 . P l a s m i d s pDt92R and p D t l 2 0 R b o t h h y b r i d i z e t o t h e 90 BC s i t e on t h e V a l D r o s o p h i ! a p o l y t e n e c h r o m o s o m e s ( 1 2 0 , 3 9 ) , a m i n o r s i t e o f t R N A 4 h y b r i d -i z a t i o n ( 2 9 ) . The s e q u e n c e s o f pDt92R and p D t l 2 0 R a r e shown i n p a n e l A i n F i g . 4 2 , and t h e d i f f e r e n c e s i n pDt92R a r e n o t e d by l e t t e r s u n d e r t h e s e q u e n c e o f p D t l 2 0 R . T h e s e t w o p l a s m i d s d i f f e r a t o n l y e i g h t s i t e s i n t h e 506 bp b e t w e e n t h e H i n d l l l s i t e a t t h e l e f t e n d o f e a c h i n s e r t and a H i n d l l l s i t e o n t h e r i g h t e n d o f t h e pDt92R i n s e r t . p D t l 2 0 R l a c k s t h i s s e c o n d H i n d l l l s i t e , a n d i t s i n s e r t i s a b o u t 1 5 0 0 bp l o n g e r ; s t a r t i n g a t p o s i t i o n 5 1 3 , i t h a s t h e s e q u e n c e AACCTT, w h i c h i n pDT92R a p p a r e n t l y i s AAGCTT, t h e H i n d l l l r e c o g n i t i o n s e q u e n c e . The e i g h t d i f f e r e n c e s i n c l u d e t h r e e t r a n s i t i o n s , f o u r t r a n s v e r s i o n s and one d e l e t i o n ( o r i n s e r t i o n ) . I t i s p o s s i b l e t h a t t h e n o n - i s o g e n i c s t o c k o f D r o s o p h i l a m e l a n o g a s t e r ( O r e g o n R ) , u s e d f o r c l o n i n g t h e tRNA g e n e s , g a v e r i s e t o t h e two p l a s m i d s , pDt92R a n d p D t l 2 0 R . The v a r i a t i o n s i n n u c l e o t i d e s e q u e n c e o u t s i d e t h e c o d i n g r e g i o n may r e p r e s e n t d i f f e r e n c e s i n t h e DNA o f h o m o l o g o u s c h r o m o s o m e s ( a l l e l i c 144. F i g u r e 4 2 . N u c l e o t i d e s e q u e n c e s o f s e g m e n t s o f t h e D r o s o p h i l a DNA i n s e r t s o f p D t l 2 0 R , p D t 9 2 R , p D t l 4 and p D t 5 5 . The DNA s e q u e n c e s o f o n l y t h e n o n c o d i n g s t r a n d s a r e shown e x c e p t f o r t h e s e c o n d g e n e i n p D t 5 5 w h e r e t h e c o d i n g s t r a n d i s p o r t r a y e d . t R N A g e n e s a r e u n d e r l i n e d . A t s i t e s w h e r e t h e s e q u e n c e s o f p D t l 2 0 R and pDt92R d i f f e r , t h e n u c l e o t i d e s f o u n d i n pDt92R a r e shown d i r e c t l y u n d e r t h e c o r r e s p o n d i n g n u c l e o t i d e s i n p D t l 2 0 R . D a s h e s b e n e a t h t h e p D t l 2 0 R s e q u e n c e i n d i c a t e r e g i o n s w h i c h w e r e n o t s e q u e n c e d i n p D t 9 2 R . The G i n p a r e n t h e s e s a t p o s i t i o n 515 i s p o s t u l a t e d t o o c c u r i n t h e c h r o m o s o m e t o a c c o u n t f o r t h e H i n d l l l r e c o g n i t i o n s e q u e n c e f o u n d a t t h i s s i t e . The n u c l e o t i d e s o v e r l i n e d a r e d i f f e r e n t f r o m t h o s e i n t h e g e n e s i n p D t 5 5 and t h o s e e x p e c t e d on t h e b a s i s o f t h e t R N A ^ a l s e q u e n c e . The f i r s t g e n e i n p D t l 4 c o d e s f o r t R N A ? 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 i n 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 1 0 0 A A G C T T C G A G GTAGGTATGT AGCTTCACGG CTTGCTGCTT AAGTTGTTAC A A T A C C A T T G GGAGGAGAGT GGG TAAAGG C A A G C C A C T A TATAAGCGT G A G A 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 C A C T T T T T A A A T T A T T T C C T A T A A T T A C A T TTTATAATTA CTTTGTGCTT T T A T T A T A A C AGATATATTT G C T A A C T T A T C T T A A A T T G T CTATGAGGAA G 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 A C G T T C G T C ATCCGAGTTT CCGTGGTGTA GTGGTTATCA CATCCGCCTA ACACGCGGAA GGCCCCCGGT T C A A T C C C G G GCGGAAACAG TTGGAATTTA 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 TTCAGTTGTA A A A C A C A C A T AGCTAATAGT ATTTATAGCT GCATCATGGC C T T A A A C T T A TCACGTTGCT 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 GGCCTCGTGC TTCTTACG AT CCACATTTTT AAGCAGAAAT TCTTGAAATT T C C T A C G C A T A T C T A A C G A T AGACCTGTAT TTCGAAGGTC 5 1 0 5 2 0 5 3 0 5 4 0 5 5 0 C A A C C T C T C A AGAACCTTGT T G C A G C T A A T T A T C T T C A T C AAATGCTTGC CAAAGTC (G) 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 TAATGCGCTT T A A A A A A A A G GATGTACTGA AAAATAATGT CTGTACTTTA AACAGGGAAG G T T C A A A T A T TTTGGCAAGT ACGGCAAATT 1 10 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 C A C A A C T T T G ATGTGAAGAA TCCCGTGGCC GAAATAGCTC AGTTGGGAGA GCGTTAGACT GAAGATCTAA AGGTCCCCGG T T C A A T C C C G GGTTTCGGCA 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 TTGCACAAAT TAGGCAGAAC TCGCATAAAA A A A T A A T A T A AATTTTGGAA 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 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 A C A C T T T T T A A T A T T T G A A C GATCTGATAG TTCAATAAGA CCGTTATCCA AGTCTTATTT 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 GA A A AGCTGTGAAT 4 1 0 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 C C A C C C C A T C ACGTGTTTCC GTGGTGTAGT GGTTATCACA TCCGCCTAAC ACGCGGAAGG CCCCCGGTTC AATCCCGGGC 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 T T T T A A T G C A T T T C C C A A A T TATTTTGCCT GTATAACTTA AATATATATA TTTTGTAATG TGATTTATGT G T C A C T T T T G TCGGTCACCT GAAGTGCTTA 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 A A T T G T A T A G TAAGTTTGAG G T C T C C A C T G GCAAACCTCC CCTAGATCAG CGGCATGCCA GAATCTTCGT C C T G G A C T C G C A C C T A C C T C AACTGGAAGC 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 AAGTGCGTAT ACTTAATGAT GGTGGTGAAG TTCAGCCAGA GGT N u c l e o t i d e S i e q u e n c e o f p D t 5 5 9 0 1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 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 1 0 1 2 0 1 3 0 1 4 0 1 5 0 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 1 0 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 G 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 C 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 A C C G G G G G C C 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 d i f f e r e n c e s ) o r d i f f e r e n c e s b e t w e e n r e p e a t e d s e q u e n c e s i n t h e DNA o f t h e same c h r o m o s o m e . A t p r e s e n t , i t i s n o t known w h i c h o f t h e t w o a l t e r n a t i v e s i s c o r r e c t . P l a s m i d p D t 1 4 h y b r i d i z e s t o t h e 89B s i t e on t h e D r o s o p h i 1 a p o l y t e n e V a l c h r o m o s o m e s ( 1 1 9 ) , t h e o t h e r m i n o r s i t e o f tRNA 4 h y b r i d i z a t i o n ( 2 9 ) . The s e q u e n c e o f a 763 bp s e g m e n t o f t h i s p l a s m i d i s shown i n p a n e l B, i n F i g . 4 2 . V a l T h i s r e g i o n c o n t a i n s t w o tRNA g e n e s , a t R N A 4 , and 214 bp u p s t r e a m , a Phe , v t R N A 2 g e n e ; b o t h g e n e s h a v e t h e same p o l a r i t y ( 3 9 ) . A d d i t i o n a l f e a t u r e s c o n t a i n e d i n t h i s s e q u e n c e a r e d e t a i l e d i n r e f e r e n c e 3 9 . V a l P l a s m i d p D t 5 5 h y b r i d i z e s t o one o f t h e m a j o r s i t e s o f t R N A 4 h y b r i d -i z a t i o n , n a m e l y , 70 BC ( 1 1 9 , 2 9 ) . The DNA s e q u e n c e o f a b o u t a 1 0 0 0 bp s e g m e n t o f t h i s p l a s m i d i s shown i n p a n e l C i n F i g . 4 2 . I t c o n t a i n s t w o i d e n t i c a l V a l t R N A 4 g e n e s i n o p p o s i t e p o l a r i t y , 525 bp a p a r t . The t w o p l a s m i d s , p D t 0 . 3 ( c o n t a i n i n g a DNA f r a g m e n t e n c o m p a s s e d by n u c l e o t i d e s 54 and 371) and p D t 0 . 6 ( c o n t a i n i n g a 629 bp f r a g m e n t b e t w e e n n u c l e o t i d e s 371 and a p p r o x i m a t e l y 1 0 0 0 ) , c o n s t r u c t e d d u r i n g t h e c o u r s e o f t h i s work w e r e d e r i v e d f r o m p D t 5 5 . R e f e r -e n c e s 13 a n d 39 a n a l y z e i n d e t a i l a d d i t i o n a l f e a t u r e s p r e s e n t i n t h e s e q u e n c e s V a l f l a n k i n g t h e - t w o t R N A 4 g e n e s . V a l , I n s i t u h y b r i d i z a t i o n s t u d i e s w i t h t R N A 4 ( T a b l e 1 ; 29) i n d i c a t e s f o u r s i t e s o f h y b r i d i z a t i o n t o D r o s o p h i l a c h r o m o s o m e s : 70 BC ( m a j o r ) , 89 B and 90 BC ( m i n o r ) m e n t i o n e d a b o v e and a l s o 56 D ( m a j o r ) . In s e v e r a l i n d e p e n d e n t ' s h o t g u n ' r e c o m b i n a n t DNA e x p e r i m e n t s u n d e r t a k e n by Dunn e t _al_. ( 1 1 9 ) , no p l a s m i d h y d r i d i z i n g t o t h i s l a t t e r s i t e was i s o l a t e d . V a l B . N u c l e o t i d e s e q u e n c e o f t h e tRNA gene o f p D t ! 2 0 R : C o m p a r i s o n t o o t h e r V a l t R N A 4 g e n e s o f D r o s o p h i l a V a l The tRNA g e n e s c o n t a i n e d i n p l a s m i d s p D t l 2 0 R , pDt92R a n d p D t l 4 a r e V a l i d e n t i c a l and d i f f e r f r o m t h e t R N A 4 g e n e s o f p D t 5 5 a t f o u r s i t e s ( o v e r l i n e d i n F i g . 4 2 ) ; t h e n u c l e o t i d e s e q u e n c e o f t h e t w o g e n e s i n p D t 5 5 c o r r e s p o n d s t o 1 4 8 , V a l V a l t h a t f o u n d i n t R N A 4 ( 3 9 , 1 4 ) . The f o u r d i f f e r e n c e s i n c o n t e x t o f t R N A 4 V a l s e q u e n c e a r e shown i n F i g . 4 3 . T r a n s c r i p t i o n o f t h e t R N A 4 - l i k e g e n e s w o u l d p r o d u c e a tRNA w i t h a U i n s t e a d o f a C a t p o s i t i o n 16 ( i n t h e D - l o o p ) , a C-G b a s e p a i r i n s t e a d o f a U29-A41 b a s e p a i r i n t h e a n t i c o d o n s t e m and an A i n s t e a d o f a G a t p o s i t i o n 57 ( i n t h e T * C l o o p ) . V a l C . A r e t h e t R N A ^ - l i k e g e n e s e x p r e s s e d i n v i v o ? A t p r e s e n t a d e f i n i t e a n s w e r t o t h i s q u e s t i o n i s n o t p o s s i b l e . A r g u m e n t s i n f a v o u r o f a n d a g a i n s t t h e s e g e n e s b e i n g e x p r e s s e d i n v i v o c a n be m a d e . V a l i n e i s o a c c e p t i n g s p e c i e s c a n be r e s o l v e d i n t o a t l e a s t s e v e n p e a k s on , % V a l " V a l V a l R P C - 5 c o l u m n s ( 1 2 ) ; t h r e e m a j o r p e a k s l a b e l l e d t R N A 3 a , t R N A 3 b and t R N A 4 V a l V a l V a l V a l a n d f o u r m i n o r s p e c i e s t R N A j , t R N A 2 , tRNA 5 and t R N A g . The t h r e e V a l , x m a j o r t R N A s h a v e d i f f e r e n t n u c l e o t i d e s e q u e n c e s ( 1 3 ) and s e q u e n c e a n a l y s i s V a l V a l V a l o f t R N A 3 a a n d tRNA 3 b showed t h a t t h e tRNA 4 - l i k e g e n e s do n o t c o d e f o r V a l t h e s e t R N A s ( 1 3 ) . In d e t e r m i n i n g t h e s e q u e n c e f o r t R N A 4 , t h e r e was no e v i d e n c e f o r h e t e r o g e n e i t y ( 1 4 ) w h i c h c o u l d be a t t r i b u t e d t o c o n t a m i n a t i o n V a l w i t h t R N A 4 - l i k e t r a n s c r i p t s . The t h r e e m a j o r v a l i n e i s o a c c e p t o r s a l s o h a v e d i f f e r e n t c o d o n p r e f e r e n c e s and t o g e t h e r t h e y r e c o g n i z e a l l f o u r v a l i n e c o d o n s V a l ( 1 2 ) . T h e r e f o r e , t h e s e t h r e e t R N A s s h o u l d be s u f f i c i e n t f o r g r o w t h and d e v e l o p m e n t . V a l I t i s p o s s i b l e t h a t t h e t R N A 4 - l i k e g e n e s c o d e f o r one o f t h e f o u r V a l m i n o r v a l i n e i s o a c c e p t o r s . The m i n o r tRNA s p e c i e s , w h i c h may r e p r e s e n t s m a l l c h a n g e s i n m o d i f i c a t i o n o f t h e b a s e s , h a v e n o t b e e n c h a r a c t e r i z e d b e c a u s e o f t h e p a u c i t y o f m a t e r i a l f o r s u c h i n v e s t i g a t i o n s . I t c a n be a r g u e d t h a t t h e s e g e n e s a r e e x p r e s s e d a t v e r y l o w l e v e l s and c o n s e q u e n t l y a r e d i f f i c u l t t o d e t e c t by R P C - 5 c h r o m a t o g r a p h y . On t h e o t h e r h a n d , t h e s e g e n e s V a l c o u l d be e x p r e s s e d a t h i g h e r l e v e l s b u t t h e t R N A 4 - l i k e p r o d u c t f o r m e d c o u l d be d e f e c t i v e i n tRNA c h a r g i n g a n d t h u s be u n d e r e s t i m a t e d on t h e R P C - 5 F i g u r e 4 3 . The n u c l e o t i d e s e q u e n c e o f D r o s o p h i l a m e l a n o g a s t e r t R N A 4 a r r a n g e d a s a c l o v e r l e a f . V a l T r a n s c r i p t i o n o f t h e t R N A 4 - l i k e g e n e s o f p l a s m i d s p D t l 2 0 R , pDt92R and p D t l 4 w o u l d p r o d u c e a tRNA i d e n t i c a l t o t R N A ^ a l e x c e p t f o r t h e r e p l a c e m e n t o f C l 6 , U 2 9 , A41 and G57 by U , C , G and A r e s p e c t i v e l y . D.m. tRNA 4 A Q H 7 6 C C A pG • C P U • A U • A 70 Um- A 5 C • G U " f ' C G G G C C C C Vr , 1 .V 1 , G C C C G G G G U G c 5 0 j G C A C C 60 D „ U 25 A m a c p U 20 C ' G C U ' A G 30 G • C40 C • G Cm m5C u A i A c 35 c o l i i m n s . Val A number o f f a c t s s u g g e s t t h a t t h e t R N A 4 - l i k e g e n e s may be e x p r e s s e d i n v i v o : i ) The g e n e s o f p D t l 4 , p D t 9 2 R / 1 2 0 R a r e w e l l s e p a r a t e d on t h e c h r o m o s o m e s y e t t h e y h a v e e x a c t l y t h e same s e q u e n c e . T h i s s u g g e s t s t h a t t h e s e q u e n c e h o m o g e n e i t y o f t h e s e g e n e s h a s b e e n a c t i v e l y m a i n t a i n e d , p r e s u m a b l y t o p r e s e r v e t h e i r f u n c t i o n i n t h e c e l l , i i ) t w o o f t h e d i f f e r e n c e s b e t w e e n t h e Val s t r u c t u r e o f t h e t R N A 4 and t h e s e g e n e s i n v o l v e t h e s u b s t i t u t i o n o f a c o m p l e m e n t a r y b a s e p a i r ( C - G f o r U-A) i n t h e a n t i c o d o n s t e m o f t h e p o t e n t i a l Val t r a n s c r i p t o f t h e t R N A 4 - l i k e g e n e ; s u c h c o u p l e d s u b s t i t u t i o n s u g g e s t s a Val f u n c t i o n a l g e n e , i i i ) Any t r a n s c r i p t o f t h e t R N A 4 - l i k e g e n e w o u l d h a v e a l l t h e 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 o f e u k a r y o t i c v a l i n e t R N A s ( c i t e d i n r e f . 13) e x c e p t f o r an A i n s t e a d o f a G r e s i d u e a t p o s i t i o n 57 ( 8 ) . W h i l e a G a t t h i s s i t e i s m o s t common, many t R N A s h a v e b e e n s e q u e n c e d t h a t c o n t a i n an A a t t h a t Val p o s i t i o n , i v ) The p o t e n t i a l t r a n s c r i p t f r o m t h e s e g e n e s w o u l d , l i k e t R N A 4 , h a v e an IAC a n t i c o d o n . E x a m p l e s o f o r g a n i s m s c o n t a i n i n g s l i g h t l y d i f f e r e n t t R N A s e a c h w i t h t h e same a n t i c o d o n ( i s o c o d i n g t R N A ) h a v e b e e n c i t e d ( 1 4 3 - 1 4 7 ) . I s o c o d i n g s p e c i e s show s l i g h t v a r i a t i o n s i n t h e tRNA s e q u e n c e , u s u a l l y s i n g l e bp c h a n g e s a t f e w s i t e s ( 1 4 3 - 1 4 7 ) . One o f t h e m o s t f r e q u e n t s i t e s o f d i f f e r e n c e b e t w e e n i s o c o d i n g t R N A s i s n u c l e o t i d e 57 ( 1 4 3 - 1 4 6 ) ; t h e c h a n g e a t t h i s p o s i t i o n g e n e r a l l y i n v o l v e s a G/A s u b s t i t u t i o n . I n s e v e r a l o r g a n i s m s , new i s o c o d i n g s p e c i e s o f tRNA a r e p r o d u c e d d u r i n g Val c e l l d i f f e r e n t i a t i o n ( 1 4 6 , 1 4 7 ) . I t i s p o s s i b l e t h a t t h e tRNA^ - l i k e g e n e s o f D r o s o p h i l a a r e e x p r e s s e d o n l y i n c e r t a i n t i s s u e s o r o n l y a t s p e c i f i c p e r i o d s d u r i n g d e v e l o p m e n t . The p a t t e r n o f v a l i n e i s o a c c e p t o r s i n D r o s o p h i l a r e v e a l e d by R P C - 5 c h r o m o a t o g r a p h y i s t h e same i n t h e f i r s t and t h e 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 ( 1 0 ) . H o w e v e r , t h e p a t t e r n o f i s o a c c e p t o r s p r e s e n t i n t h e e m b r y o n i c ( e g g ) and p u p a l s t a g e s i s n o t k n o w n . T h e r e a r e o t h e r e x a m p l e s i n t h e l i t e r a t u r e o f t h e s e q u e n c e o f p o s s i b l e tRNA c o d i n g r e g i o n b e i n g d i f f e r e n t f r o m t h e s e q u e n c e o f t h e i s o l a t e d t R N A . T h e s e i n c l u d e D r o s o p h i l a g e n e s f o r t R N A ^ e r ( 4 1 ) , t R N A ^ ^ O ) , t R N / P ^ ( 3 6 ) , t R N A G l L } 3 3 ) , and t R N A H l S ( 1 4 8 ) , human t R N A M e t ( 1 4 9 ) and an S . pombe t R N A G l u g e n e ( 1 5 0 ) . W h e t h e r t h e s e g e n e s a r e e x p r e s s e d i n v i v o i s a l s o n o t k n o w n . V a l I I . T r a n s c r i p t i o n o f D r o s o p h i l a tRNA g e n e s A . T r a n s c r i p t i o n u s i n g D r o s o p h i l a S c h n e i d e r I I c e l l - f r e e e x t r a c t The c e l l - f r e e e x t r a c t s e m p l o y e d f o r t h e t r a n s c r i p t o n s t u d i e s w e r e p o s t - n u c l e a r e x t r a c t s d e r i v e d by h i g h s p e e d c e n t r i f u g a t i o n ( 1 0 0 , 0 0 0 x g) o f c y t o p l a s m i c f r a c t i o n s p r e p a r e d f r o m D r o s o p h i l a S c h n e i d e r I I c e l l s . S i m i l a r e x t r a c t s h a v e b e e n p r e p a r e d f r o m many o t h e r s o u r c e s ( 7 9 - 8 4 ) . W h e r e v e r e s t i m a t e s h a v e b e e n m a d e , s u c h e x t r a c t s h a v e b e e n shown t o c o n t a i n 6 5 - 9 0 % o f t h e c e l l u l a r RNA p o l y m e r a s e I I I ( 8 0 , 6 4 , 6 5 , 151 ) . RNA p o l y m e r a s e I I I i s l o c a l i z e d i n t h e n u c l e u s ( 4 3 , 4 4 ) ; s i n c e m i n i m a l n u c l e a r l y s i s i s o b s e r v e d d u r i n g t h e p r e p a r a t i o n o f t h e s e e x t r a c t s ( o b s e r v e d h e r e , 8 0 , 8 4 ) , RNA p o l y m e r a s e I I I and o t h e r c o m p o n e n t s a s w e l l must l e a k o u t f r o m t h e n u c l e u s d u r i n g c e l l u l a r f r a c t i o n a t i o n t o a c c o u n t f o r t h e i r p r e s e n c e i n t h e S . 1 0 0 e x t r a c t s . I n c u b a t i o n o f DNA t e m p l a t e s c o n t a i n i n g D r o s o p h i l a tRNA g e n e s w i t h t h e S . 1 0 0 e x t r a c t s d e r i v e d f r o m D r o s o p h i l a S c h n e i d e r I I c e l l s r e s u l t e d i n t h e s y n t h e s i s o f t w o m a j o r p r o d u c t s , d e s i g n a t e d RNA-I ( l a r g e r o f t h e two RNA s p e c i e s ) a n d R N A - I I . The c o m b i n e d s i z e ( e l e c t r o p h o r e t i c ) and h y b r i d i z a t i o n a n a l y s e s o f t h e m a j o r t r a n s c r i p t s s u g g e s t e d t h a t t h e c e l l - f r e e e x t r a c t s s u p p o r t e d s e l e c t i v e and a c c u r a t e t r a n s c r i p t i o n o f t h e tRNA g e n e s . I n s e n -s i t i v i t y o f s p e c i f i c t r a n s c r i p t i o n t o l o w c o n c e n t r a t i o n s o f a - a m a n i t i n ( 0 . 1 / j g / m l ) and i n h i b i t i o n by h i g h a - a m a n i t i n c o n c e n t r a t i o n s ( b e l o w 1 m g / m l ) i n d i c a t e d RNA p o l y m e r a s e I I I ( 4 4 , 5 1 , 8 3 , 1 4 1 , 1 4 2 ) as t h e p o l y m e r a s e r e s p o n s i b l e f o r s p e c i f i c t r a n s c r i p t i o n o f tRNA g e n e s o b s e r v e d w i t h t h e D r o s o p h i l a e x t r a c t s . 1 5 3 , R N A - I was t h e p r e d o m i n a n t t r a n s c r i p t i o n p r o d u c t d i r e c t e d by d i f f e r e n t Val tRNA g e n e s and i t s s i z e v a r i e d ( 9 5 - 1 0 0 n u c l e o t i d e s ) w i t h t h e DNA t e m p l a t e e m p l o y e d . The e x a c t t r a n s c r i p t i o n i n i t i a t i o n and t e r m i n a t i o n s i t e s i n t h e Val v a r i o u s tRNA g e n e s w e r e n o t i d e n t i f i e d . The DNA s e q u e n c e s o f a l l t h e Val D r o s o p h i l a tRNA g e n e s ( F i g s . 3 3 , 4 2 ) u s e d f o r t r a n s c r i p t i o n show t h e p r e s e n c e o f a c l u s t e r ( b e t w e e n 5 and 9 ) o f T r e s i d u e s on t h e n o n c o d i n g s t r a n d a s h o r t d i s t a n c e ( 9 - 1 2 n u c l e o t i d e s ) b e y o n d t h e 3 ' ^ e n d o f t h e c o d i n g r e g i o n . The s t r e t c h o f T r e s i d u e s h a v e b e e n i m p l i c a t e d i n t h e t e r m i n a t i o n o f t r a n s c r i p t i o n by RNA p o l y m e r a s e I I I ( 8 7 , 1 0 0 ) . From t h e s i z e o f RNA-I and t h e l o c a t i o n o f t h e T - c l u s t e r a known d i s t a n c e f r o m t h e 3 ' - e n d o f t h e tRNA g e n e , Val i t c a n be e s t i m a t e d t h a t t r a n s c r i p t i o n i n i t i a t i o n i n t h e tRNA g e n e s o c c u r r e d a b o u t 8 - 1 5 n u c l e o t i d e s b e f o r e t h e 5 ' - e n d o f t h e m a t u r e tRNA c o d i n g r e g i o n . T h i s e s t i m a t e a g r e e s w i t h o t h e r s t u d i e s ( r e v i e w e d i n r e f . 37) w h i c h show t h a t t r a n s c r i p t i o n i n i t i a t i o n i n tRNA g e n e s o c c u r s 3 - 1 9 n u c l e o t i d e s 5 ' t o t h e b e g i n n i n g o f t h e c o d i n g r e g i o n . Val R N A - I I , t h e o t h e r m a j o r t r a n s c r i p t i o n p r o d u c t d i r e c t e d by t h e tRNA g e n e s , was c o n s t a n t i n s i z e f o r a l l DNA t e m p l a t e s a n d i t a p p r o x i m a t e d t h e s i z e Val o f p u r i f i e d tRNA 3 b , 4 « The p r o c e s s i n g e x p e r i m e n t s i n d i c a t e d t h a t R N A - I I was d e r i v e d f r o m R N A - I and d e p e n d e d on p r i o r s y n t h e s i s o f R N A - I . None o f t h e Val t R N A g e n e s c o d e s f o r t h e 3 ' - t e r m i n a l CCA s e q u e n c e f o u n d i n a l l m a t u r e t R N A s ( 8 ) . T h e r e f o r e , t h e CCA s e q u e n c e i s p r e s u m a b l y a d d e d p o s t - t r a n s c r i p -t i o n a l l y by a 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 ( 1 1 4 ) i n t h e S . 1 0 0 e x t r a c t . T h i s h a s n o t b e e n d e t e r m i n e d w i t h c e r t a i n t y . T h u s , t h e D r o s o p h i l a S c h n e i d e r I I c e l l - f r e e e x t r a c t a p p e a r s t o s u p p o r t a c c u r a t e t r a n s c r i p t i o n and m a t u r a t i o n o f D r o s o p h i 1 a t R N A V a l g e n e s . B . D i f f e r e n t t R N A V a l g e n e s t r a n s c r i b e a t d i f f e r e n t r a t e s i n v i t r o The r e s u l t s p r e s e n t e d i n F i g . 31 show t h a t t h e D r o s o p h i l a t R N A V a l g e n e s p r e s e n t i n p l a s m i d s p D t 0 . 3 , p D t 7 8 R , pDt92R and p D t l 2 0 R d i r e c t e d V a l t r a n s c r i p t i o n a t d i f f e r e n t r a t e s . p D t 0 . 3 , c a r r y i n g a gene f o r t R N A 4 was t r a n s c r i b e d 3 - 4 t i m e s more e f f i c i e n t l y t h a n pDt78R w h i c h c o n t a i n s a t R N A ^ 1 V a l g e n e . The DNA s e q u e n c e s o f t h e tRNA g e n e s i n p D t 0 . 3 and pDt78R c o r r e s p o n d V a l t o t h e s e q u e n c e s o f t h e tRNA i s o a c c e p t o r s t o w h i c h t h e y h a v e b e e n a s s i g n e d ( 1 3 , 1 4 , 3 9 ) . The p l a s m i d s p D t 0 . 3 and pDt78R a l s o h y b r i d i z e t o t h e c o r r e s -p o n d i n g m a j o r s i t e s ( 7 0 BC a n d 84 D r e s p e c t i v e l y ) on t h e D r o s o p h i 1 a c h r o m o s o m e s ( 1 1 9 , 2 9 ) . T h e s e a r e , t h e r e f o r e , b o n a f i d e tRNA g e n e s . Dunn e t a l . ( 2 8 ) and r e c e n t l y L a r s e n e t ^1_. ( 1 3 1 ) h a v e d e m o n s t r a t e d t h a t a d e l e t i o n o f V a l t h e 84D l o c u s i n D r o s o p h i ! a r e d u c e d t h e l e v e l o f t R N A 3 b i n h e t e r o z y g o u s f l i e s ; t h e f o r m e r s t u d y a l s o showed t h a t a d u p l i c a t i o n o f t h e 84D r e g i o n V a l c a u s e d a p r o p o r t i o n a t e i n c r e a s e i n t h e l e v e l o f t R N A 3 b . I t i s t h u s h i g h l y V a l l i k e l y t h a t t h e t R N A 3 b gene i n pDt78R i s e x p r e s s e d i n v i v o ; t h e p o s s i b i l i t y V a l o f o t h e r t r a n s c r i p t i o n a l l y a c t i v e t R N A 3 b g e n e s i n t h e d e l e t i o n o r i n t h e d u p l i c a t i o n c a n n o t be c o m p l e t e l y r u l e d o u t . V a l V a l The r a t i o o f t R N A 4 : t R N A 3 b i s s l i g h t l y g r e a t e r t h a n one in a d u l t V a l V a l f l i e s ( 1 2 , 1 3 ) . The e s t i m a t e o f t h e t o t a l number o f t R N A 4 and tRNA 3 b g e n e s i n D r o s o p h i l a v a r i e s f r o m 5 - 1 9 ( 2 1 - 2 3 ) and 6 - 1 0 ( 2 2 ) r e s p e c t i v e l y . How t h e s e g e n e s a r e d i s t r i b u t e d a t t h e v a r i o u s s i t e s o f h y b r i d i z a t i o n on t h e D r o s o p h i l a c h r o m o s o m e s ( 2 9 ) and w h e t h e r a l l t h e g e n e s a r e a c t i v e i s n o t k n o w n . I t i s t h u s d i f f i c u l t t o c o r r e l a t e t h e j_n v i t r o a c t i v i t i e s o f t h e V a l t R N A g e n e s i n p D t 0 . 3 and pDt78R t o t h e a c t i v i t i e s o f t h e c o r r e s p o n d i n g g e n e s j_n v i v o . P l a s m i d s , pDt92R a n d p D t l 2 0 R , w e r e t r a n s c r i b e d 8 and 6 f o l d l e s s V a l e f f i c i e n t l y t h a n p D t 0 . 3 r e s p e c t i v e l y . The s e q u e n c e o f t h e tRNA g e n e f o u n d V a l i n pDt92R and p D t l 2 0 R d i f f e r e d a t f o u r p o s i t i o n s c o m p a r e d t o t h e t R N A 4 g e n e V a l c o n t a i n e d i n p D t 0 . 3 ( F i g s . 4 2 , 4 3 ) . The a r g u m e n t s f o r and a g a i n s t t h e t R N A 4 - l i k e g e n e s b e i n g e x p r e s s e d i n v i v o w e r e p r e s e n t e d i n s e c t i o n IC a b o v e . I t V a l s h o u l d be n o t e d t h a t p l a s m i d s , pDt41R and p D t 4 8 ( 1 1 9 ) , w h i c h c o d e f o r t R N A Val g e n e s w h o s e s e q u e n c e s d i f f e r f r o m t h a t o f tRNA 3 t ) ( a n d t h e g e n e p r e s e n t i n p D t 7 8 R ) a t f o u r p o s i t i o n s : n u c l e o t i d e s 5 , 1 6 , 68 and 69 ( 1 3 , L e u n g , D e l a n e y and T e n e r , p e r s o n a l c o m m u n i c a t i o n ) , w e r e a l s o t r a n s c r i b e d p o o r l y i n p r e l i m i n a r y e x p e r i m e n t s . W h e t h e r t h e s e t R N A - l i k e g e n e s a r e e x p r e s s e d i n v i v o i s n o t k n o w n . Val I t i s i n t e r e s t i n g t o n o t e t h a t t h e t R N A ^ - 1 i k e g e n e s o r i g i n a t e f r o m t h e 90 BC ( 3 9 , 1 1 9 , 1 2 0 ) , a m i n o r s i t e o f h y b r i d i z a t i o n on t h e D r o s o p h i l a c h r o m o s o m e s f o r b o t h t h e s e i s o a c c e p t o r s ( 2 9 ) . I t i s p o s s i b l e t h a t h y b r i d i z a t i o n was p o o r a t t h e s e m i n o r s i t e s b e c a u s e o f t h e d i f f e r e n c e s b e t w e e n t h e tRNA p r o b e s u s e d f o r h y b r i d i z a t i o n and t h e g e n e s e q u e n c e s p r e s e n t a t t h e s e s i t e s a n d n o t b e c a u s e m i n o r s i t e s c o n t a i n e d f e w e r g e n e s c o m p a r e d t o t h e i r r e s p e c t i v e m a j o r s i t e s . I f t h i s i s t r u e , t h e n t h e s e t R N A - l i k e g e n e s a r e u n d e r r e p r e s e n t e d by s u c h a n a l y s e s . I f t h e s e g e n e s a r e i n d e e d s o n u m e r o u s , why a r e t h e i r p r o d u c t s n o t o b s e r v e d Jjn v i v o ? I n c o m p l i a n c e w i t h t h e vn v i t r o d a t a , t h e s e g e n e s may be e x p r e s s e d a t l e v e l s w h i c h may n o t be d e t e c t a b l e o r w h i c h may be r e p r e s e n t e d a s m i n o r s p e c i e s . Met C l a r k s o n j i t a]_. ( 1 1 0 ) r e p o r t t h a t t w o X e n o p u s l a e v i s tRNA g e n e s , w h i c h d i f f e r a t a s i n g l e p o s i t i o n i n t r a g e n i c a l l y , a r e t r a n s c r i b e d w i t h v e r y d i f f e r e n t e f f i c i e n c i e s i n a h o m o l o g o u s c e l l - f r e e s y s t e m . The t R N A M e t gene w h i c h i s t r a n s c r i b e d l e s s e f f i c i e n t l y h a s a C i n s t e a d o f a T u s u a l l y f o u n d a t Met p o s i t i o n 64 i n X . l a e v i s tRNA . T h e s e r e s u l t s a g r e e w i t h t h o s e r e p o r t e d Met h e r e . H o w e v e r , s t u d i e s by S o l i et a]_. ( 3 6 ) i n d i c a t e t h a t t w o D r o s o p h i l a t R N A g e n e s , w h i c h d i f f e r by a s i n g l e n u c l e o t i d e a t p o s i t i o n 3 0 , a r e t r a n s c r i b e d w i t h e q u a l e f f i c i e n c i e s . T h e r e f o r e , n o t a l l t R N A - l i k e g e n e s , whose s e q u e n c e s d i f f e r by f e w n u c l e o t i d e s f r o m t h e s e q u e n c e s o f t h e i s o l a t e d t R N A , a r e t r a n s c r i b e d a t l o w l e v e l s J_n v i t r o . T h a t o t h e r f a c t o r s l i k e n u c l e o s o m e f o r m a t i o n may be r e q u i r e d t o m i m i c i n v i v o p r o m o t e r a c t i v i t i e s i s s u g g e s t e d by r e c e n t e x p e r i m e n t s w i t h 5S RNA g e n e s ( 9 4 , 9 5 ) and tRNA g e n e s ( 1 5 2 ) . 156 . C . S e q u e n c e d e p e n d e n c e o f t r a n s c r i p t i o n r a t e 1 . E f f e c t o f g e n e i n t e r n a l c h a n g e s on t h e r a t e o f tRNA gene t r a n s c r i p t i o n R e c e n t s t u d i e s h a v e d e f i n e d t w o r e g i o n s w i t h i n t h e tRNA g e n e a s e s s e n t i a l f o r i n i t i a t i o n o f t r a n s c r i p t i o n . The t w o r e g i o n s h a v e b e e n d e s i g n a t e d a s t h e i n t e r n a l , s p l i t - p r o m o t e r and h a v e b e e n l o c a l i z e d by f o u r g r o u p s t o l i e w i t h i n n u c l e o t i d e s 8 t o 18 ( r e g i o n A) and 51 t o 62 ( r e g i o n B) o f t h e m a t u r e tRNA s e q u e n c e ( 8 6 , 9 7 , 1 0 3 , 1 0 4 ) . T r a n s c r i p t i o n r e s u l t s p r e s e n t e d e a r l i e r i n d i c a t e d t h a t d i f f e r e n t p l a s m i d s c o n t a i n i n g t R N A V a l g e n e s w i t h n e a r l y i d e n t i c a l c o d i n g r e g i o n s w e r e t r a n s c r i b e d a t v e r y d i f f e r e n t r a t e s . Time c o u r s e s t u d i e s w i t h p D t 0 . 3 , pDt78R a n d pDt92R showed t h a t a l l t h e s e p l a s m i d s s u p p o r t e d l i n e a r r a t e s o f t r a n s c r i p t i o n i n t h e D r o s o p h i 1 a S . 1 0 0 e x t r a c t s f o r a t l e a s t two h o u r s . Hence t h e l e s s e f f i c i e n t t r a n s c r i p t i o n o f some o f t h e s e p l a s m i d s i s n o t due t o t h e i n s t a b i l i t y o f t h e i r t r a n s c r i p t s . T h e r e f o r e , t h e d i f f e r e n t r a t e s must r e f l e c t t h e d i f f e r e n c e s i n t h e c o d i n g r e g i o n s a n d / o r t h e d i f f e r e n t s e q u e n c e s w h i c h f l a n k t h e s e g e n e s . The f i r s t f i v e l i n e s i n F i g . 44 show s e q u e n c e s i n r e g i o n s A a n d B o f t h o s e e u k a r y o t i c tRNA g e n e s f o r w h i c h t h e r e i s d i r e c t e v i d e n c e f o r t h e f u n c t i o n o f t h e s e r e g i o n s i n t r a n s c r i p t i o n ( 8 6 , 9 7 , 1 0 3 - 1 0 5 ) . The s e q u e n c e shown i n l i n e 6 i s a g e n e r a l i z e d s e q u e n c e d e r i v e d f r o m t h e f i v e known s e q u e n c e s shown a b o v e i t . L i n e 7 r e p r e s e n t s a g e n e r a l i z e d s e q u e n c e o b t a i n e d b y c o m p a r i s o n o f h i g h l y c o n s e r v e d s e q u e n c e s i n h o m o l o g o u s r e g i o n s o f a l l known e u k a r y o t i c t R N A s e q u e n c e s ( 8 , 1 0 5 ) . The c o r r e s p o n d i n g s e q u e n c e s i n t h e t R N A ^ a g e n e s s t u d i e d h e r e a r e shown i n l i n e s 8 - 1 0 . The l a s t l i n e shows t h e s e q u e n c e s i n h o m o l o g o u s p o s i t i o n s i n t h e d e l e t i o n m u t a n t p D t 0 . 3 A 5 1 - 6 1 . Among t h e t R N A V a l g e n e s , t h e r e a r e f o u r d i f f e r e n c e s i n r e g i o n s A a n d B : a t n u c l e o t i d e s 9 and 59 ( b e t w e e n p D t 0 . 3 and p D t 7 8 R ) , and a t 16 and 57 ( b e t w e e n p D t 0 . 3 a n d p D t 9 2 R ) . The c h a n g e s a t n u c l e o t i d e s 9 , 16 and 57 a r e b a s e 157 . F i g u r e 4 4 . C o m p a r i s o n o f DNA s e q u e n c e s p o s t u l a t e d t o be i m p o r t a n t i n t r a n s c r i p t i o n o f t R N A g e n e s . S e c t i o n A c o m p a r e s t h e s e q u e n c e s f o u n d i n t h e c o n s e r v e d r e g i o n s A ( b e t w e e n n u c l e o t i d e s 8 t o 18) and B ( b e t w e e n n u c l e o t i d e s 5 1 - 6 0 ) and t h e e x t r a arm r e g i o n ( b e t w e e n n u c l e o t i d e s 44 and 4 8 ) o f t h e v a r i o u s t R N A g e n e s . G e n e r a l i z e d s e q u e n c e s ( s e e t e x t f o r d e t a i l s ) f o r r e g i o n s A and B a r e m a r k e d o f f by a r e c t a n g l e . R e f e r e n c e s f r o m w h i c h t h e DNA s e q u e n c e s w e r e o b t a i n e d a r e shown i n b r a c k e t s . S e q u e n c e s f o u n d i n t h e h o m o l o g o u s p o s i t i o n s o f t h e v a r i o u s t R N A V a l g e n e s s t u d i e d h e r e a r e shown b e l o w t h e r e c t a n g l e . R, Y and N s t a n d f o r p u r i n e , p y r i m i d i n e and a n y n u c l e o t i d e , r e s p e c t i v e l y . S e c t i o n B c o m p a r e s t h e 5 ' - f l a n k i n g r e g i o n s o f t h e t R N A V a l g e n e s . +1 d e n o t e s t h e f i r s t n u c l e o t i d e o f t h e tRNA c o d i n g r e g i o n a n d - 5 0 i n d i c a t e s 50 n u c l e o t i d e s 5 ' t o t h e c o d i n g r e g i o n . The 50 n u c l e o t i d e s f l a n k i n g t h e 5 ' - e n d s o f t h e tRNA g e n e s i n p D t l 2 0 R a n d pDt92R a r e i d e n t i c a l e x c e p t f o r t h e d i f f e r e n c e a t p o s i t i o n - 3 8 . tDNA*'9 Drosophila (86) Met tDNA X. laevis (103) tDNA L , u X. laevis (97) tDNA L e u C. elegans (105) tDNA*0 C. elegans (105,155) Rftginn A + 8 +18 TGGCGCAATGG TGGCGCAGCGG TGGCCGAGCGG TGGCCGAGCGG TGGTCTAGTGG Fxtrn arm +44 +48 AGATT AGGTC AGGTC AGGTC Region B + 51 +60 AGGTTCGACT TGGATCGAAA GGGTTCGAAT GGGTTCGAAT GGGTTCAATC Generalized sequence TGGY §NARYGG NGGJTCRANN Conserved sequence motifs (105) TRGYNNARYGG NNGTTCRANN pDt03 Drosophila TGGTGTAGCGG AGGCC CGGTTCGATC pDt78R Drosophila TAGTGTAGCGG AGGTC CGGTTCGAAC pDt92R/l20R Drosophila TGGTGTAGTGG AGGCC CGGTTCAATC pDtO-3A5l-6l TGGTGTAGCGG CGGGCGGAAA B Comparison of 5' flanking sequences -50 - 4 0 -30 -20 -tO +1 pDtQ3 TGGCAGTTGAGGTCGCTGAAGTTGACCTCTCTGCCGCTTAAGTTTCAACTG pDtO-6 AAAGCCTCCTGTGGCTCACAAGAGGCTCTTCGGACGGACTATATTCAACTG pDt78R TTTATACTAGACTTTATAATATAGGTCTTGTGATGTCAGCACCGCCACAAG pDtl20R ATTTGCTAACTTATCTTAAATTGTCTATGA GGAAAACGTTCGTCATCCGAG 92R G 1 5 9 , t r a n s i t i o n s w h e r e a s t h e a l t e r n a t i o n a t p o s i t i o n 59 i s a b a s e t r a n s v e r s i o n . The l a t t e r t h r e e c h a n g e s o c c u r a t p o s i t i o n s w h e r e b o t h t h e known and t h e p r e d i c t e d g e n e r a l i z e d s e q u e n c e s i n d i c a t e a p y r i m i d i n e ( Y ) , p u r i n e ( R ) , o r any n u c l e o t i d e ( N ) , r e s p e c t i v e l y . The n u c l e o t i d e a t p o s i t i o n 9 i s shown t o be a G by t h e g e n e r a l i z e d s e q u e n c e d e r i v e d f r o m f i v e known s e q u e n c e s b u t t h e g e n e r a l i z e d s e q u e n c e o b t a i n e d by t h e c o m p a r i s o n o f more t h a n 80 known e u k a r y o t i c tRNA s e q u e n c e s i n d i c a t e s t h i s p o s i t i o n t o be o c c u p i e d by e i t h e r G , " V a l o r A ( R ) . T h i s c o m p a r i s o n t h e r e f o r e shows t h a t t h e tRNA g e n e s w h i c h s u p p o r t v e r y d i f f e r e n t r a t e s o f t r a n s c r i p t i o n a r e v i r t u a l l y i d e n t i c a l i n t h e i n t e r n a l c o n t r o l s e q u e n c e s . The d i f f e r e n c e s t h a t e x i s t i n t h e s p l i t - p r o m o t e r V a l r e g i o n s o f t h e tRNA g e n e s a r e f o u n d i n p o s i t i o n s w h i c h we p r e s u m e h a v e m i n i m a l e f f e c t on p r o m o t e r f u n c t i o n and a c t i v i t y . T h i s o b s e r v a t i o n i s f u r t h e r s u b s t a n t i a t e d by t h e f a c t t h a t a G57 + A 5 7 [ h y b r i d p l a s m i d , p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) ] s u b s t i t u t i o n had l i t t l e e f f e c t on t h e e f f i c i e n c y o f t r a n s c r i p t i o n o f p D t 0 . 3 . T h e r e i s a p r e c e d e n c e f o r a c h a n g e a t t h i s s e m i - i n v a r i a n t p o s i t i o n ( G 5 7 ^ T57) i n l i t e r a t u r e , w h e r e i t d i d n o t h a v e any e f f e c t on t h e t r a n s c r i p t i o n Met e f f i c i e n c y o f a human t R N A ± g e n e i n v i v o ( 1 5 3 ) o r i n v i t r o ( 1 5 4 ) . K o s k i e t a]_. ( 9 6 ) s t u d i e d t r a n s c r i p t i o n e f f e c t s o f 28 d i f f e r e n t p o i n t tyr m u t a t i o n s w i t h i n y e a s t S u p 4 - 0 tRNA g e n e . They f o u n d t h a t s i n g l e bp a l t e r a t i o n s i n some i n v a r i a n t and s e m i - i n v a r i a n t p o s i t i o n s i n t h e D - l o o p (A r e g i o n ) and t h e T ^ C l o o p (B r e g i o n ) a f f e c t e d t r a n s c r i p t i o n . The m o s t d r a m a t i c c h a n g e was a t t h e i n v a r i a n t p o s i t i o n 5 6 , when a C + G m u t a t i o n g a v e no v i s i b l e t r a n s c r i p t i o n and a C + T c h a n g e c a u s e d a 1 0 - f o l d d e c r e a s e i n t r a n s c r i p t i o n e f f i c i e n c y . M u t a t i o n s a t o t h e r i n v a r i a n t p o s i t i o n s , n u c l e o t i d e 8 (T - * G ) , g a v e no t r a n s c r i p t i o n ( 8 6 ) and a t n u c l e o t i d e 53 (G + T) r e d u c e d t r a n s c r i p t i o n t o 45% o f t h e w i l d t y p e l e v e l ( 1 5 5 ) . On t h e o t h e r h a n d , e x a m p l e s w h e r e m u t a t i o n s had o c c u r r e d o u t s i d e o f t h e two c o n s e r v e d s e q u e n c e b l o c k s - i n t h e a m i n o a c i d - a c c e p t o r s t e m ( A 6 9 G69 c h a n g e i n t h e h y b r i d 160 , p l a s m i d , p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) , r e p o r t e d h e r e ; 9 6 ) , i n t h e a n t i c o d o n s t e m and l o o p ( 9 6 ) and i n t h e e x t r a arm ( 9 6 ) - showed v e r y l i t t l e e f f e c t on t r a n s c r i p t i o n e f f i c i e n c y . I t s h o u l d be n o t e d t h a t t h e r e a r e a t o t a l o f f o u r d i f f e r e n c e s i n t h e c o d i n g r e g i o n s b e t w e e n p D t 0 . 3 and pDt92R ( F i g s . 4 2 , 4 3 ) : two i n t h e D - l o o p and t h e T ^ C l o o p ( d i s c u s s e d a b o v e ) and two c o m p l i m e n t a r y bp c h a n g e s i n t h e a n t i - c o d o n s t e m . From t h e a n a l y s i s p r e s e n t e d a b o v e , t h e l a t t e r t w o d i f f e r e n c e s s h o u l d h a v e a m i n i m a l e f f e c t on t h e e f f i c i e n c y o f t r a n s c r i p t i o n . V a r i a t i o n i n t h e e x t r a - a r m r e g i o n o f tRNA was i n v e s t i g a t e d by C i a m p i e t P r o a l . ( 1 5 5 ) who showed t h a t f o r a tRNA g e n e f r o m C a e n o r h a b d i t i s e l e g a n s , a n y c h a n g e f r o m t h e s e q u e n c e AGGTC r e s u l t e d i n a d e c r e a s e i n t r a n s c r i p t i o n V a l a c t i v i t y . The s e q u e n c e s i n t h e e x t r a - a r m r e g i o n s o f tRNA g e n e s and f o u r o t h e r tRNA g e n e s a r e c o m p a r e d i n F i g . 4 4 . An a n a l y s i s o f t h e e x t r a arm s e q u e n c e s o f n e a r l y a l l known e u k a r y o t i c t R N A s r e v e a l e d t h a t AGGUC was t h e m o s t common s e q u e n c e i n t h i s r e g i o n ( t R N A s w i t h l o n g e x t r a arms w e r e n o t c o n s i d e r e d ; 1 5 5 ) . B u t D r o s o p h i l a t R N A 2 g e n e , w h i c h d i f f e r s f r o m t h i s s e q u e n c e a t t w o p o s i t i o n s ( F i g . 4 4 ) , i s a p p a r e n t l y t r a n s c r i b e d q u i t e e f f i c i e n t l y ( 7 4 , 8 6 ) . A l s o p l a s m i d s , p D t 0 . 3 a n d p D t 9 2 R , a r e i d e n t i c a l i n t h i s r e g i o n and d i f f e r f r o m t h e a b o v e s e q u e n c e a t o n e p o s i t i o n ; pDt78R w h i c h h a s P r o s e q u e n c e i d e n t i c a l t o t h a t o f C . e l e g a n s tRNA g e n e i n t h i s e x t r a arm r e g i o n i s t r a n s c r i b e d a t a r a t e i n t e r m e d i a t e b e t w e e n t h a t o f p D t 0 . 3 and p D t 9 2 R . T h e r e f o r e , t h e s e n u c l e o t i d e s may n o t r e g u l a t e D r o s o p h i l a t R N A ^ a l and tRNA ^ r g P r o g e n e t r a n s c r i p t i o n r a t e s a s t h e y do t h a t o f t h e C . e l e g a n s tRNA g e n e . On t h e o t h e r h a n d , c h a n g e s a t o t h e r p o s i t i o n s i n t h e s e q u e n c e s o f p D t 0 . 3 , pDt92R a n d t R N A 2 g e n e c o u l d c o m p e n s a t e f o r d e v i a t i o n s f r o m t h e AGGTC s e q u e n c e i n t h e e x t r a arm r e g i o n . A n a l y s i s o f t h e d i f f e r e n c e s i n t h e p r o p o s e d i n t e r n a l c o n t r o l r e g i o n s and V a l t h e e x t r a arm r e g i o n o f t h e tRNA g e n e s i n d i c a t e s t h a t t h e s e d i f f e r e n c e s do 161. n o t a c c o u n t f o r t h e d i f f e r e n t t r a n s c r i p t i o n r a t e s d i r e c t e d by t h e s e g e n e s . Hence i t c a n be c o n c l u d e d t h a t t h e d i f f e r e n c e i n t h e r a t e s o f t r a n s c r i p t i o n V a l b e t w e e n t h e d i f f e r e n t D r o s o p h i l a tRNA g e n e s i s p o s s i b l y t h e r e s u l t o f V a l d i f f e r i n g s e q u e n c e s f l a n k i n g t h e v a r i o u s tRNA c o d i n g r e g i o n s . 2 . I n f l u e n c e o f f l a n k i n g s e q u e n c e s on t h e r a t e o f tRNA g e n e t r a n s c r i p t i o n F l a n k i n g s e q u e n c e s h a v e b e e n shown t o m o d u l a t e t h e r a t e o f tRNA t r a n s c r i p t i o n by a number o f p r e v i o u s s t u d i e s . D e l e t i o n s and s u b s t i t u t i o n s i n t h e 5 ' - f l a n k i n g s e q u e n c e s o f tRNA g e n e s c a n r e d u c e t h e i r t r a n s c r i p t i o n a p p r e c i a b l y ( 8 3 , 8 6 , 9 7 ) , a n d may a l s o c h a n g e t h e f r e q u e n c y o f u s e o f a p a r t i c u l a r i n i t i a t i n g n u c l e o t i d e ( 1 0 3 ) . An e x t r e m e e x a m p l e i s t h e t r a n s -A l a c r i p t i o n o f a s i l k w o r m tRNA g e n e w h i c h h a s an a b s o l u t e r e q u i r e m e n t f o r a t l e a s t 11 n u c l e o t i d e s o f i t s n o r m a l 5 ' - f l a n k i n g s e q u e n c e f o r e x p r e s s i o n i n a h o m o l o g o u s c e l l - f r e e e x t r a c t ( 8 3 ) . T h i s i s t h e o n l y e x a m p l e known w h e r e t h e 5 ' - f l a n k i n g s e q u e n c e s e x e r c i s e a p o s i t i v e i n f l u e n c e . In some c a s e s , t h i s c o n t r o l i s e x e r t e d i n a n e g a t i v e w a y . D e F r a n c o e t _al_. ( 3 8 , 1 0 9 ) f o u n d a G + T - r i c h u n d e c a n u c l e o t i d e , GGCAGTTTTTG, t h a t p r e c e d e s s e v e r a l D r o s o p h i 1 a Lys t R N A 2 g e n e s t o be i n h i b i t o r y f o r t r a n s c r i p t i o n when l o c a t e d 13 n u c l e o t i d e s i n f r o n t o f t h e m a t u r e t R N A - c o d i n g r e g i o n . C o m p l e t e r e m o v a l o f t h i s s e q u e n c e e n h a n c e s t r a n s c r i p t i o n by a b o u t 1 0 - f o l d . On t h e o t h e r h a n d , p l a c i n g t h i s s e q u e n c e a p p r o x i m a t e l y 13 n u c l e o t i d e s i n f r o n t o f a n o t h e r g e n e d e p r e s s e s i t s t r a n s c r i p t i o n t o t h e same e x t e n t . P o t e n t i a l i n h i b i t o r y s e q u e n c e s o f a Met d i f f e r e n t k i n d a r e f o u n d i n f r o n t o f a v a r i a n t tRNA g e n e o f X . l a e v i s ( 1 1 0 ) . Met The v a r i a n t g e n e w h i c h d i f f e r e d f r o m t h e b o n a f i d e tRNA g e n e a t a s i n g l e p o s i t i o n i n t r a g e n i c a l l y and a t many p o s i t i o n s i n t h e f l a n k i n g r e g i o n s was t r a n s c r i b e d a t a much l o w e r r a t e . S w i t c h i n g and d e l e t i o n e x p e r i m e n t s r e v e a l e d t h a t t h e r e l a t i v e i n a c t i v i t y o f t h e v a r i a n t t R N A ^ g e n e was due t o t h e p r e s e n c e o f i n h i b i t o r y s e q u e n c e ( s ) i n i t s 5 ' - - f l a n k i n g r e g i o n . Two r e g i o n s w h i c h p r e c e d e t h e v a r i a n t gene a r e o f p o t e n t i a l s i g n i f i c a n c e : o n e i s a s t r e t c h 1 6 2 . o f 1 3 - 1 5 C r e s i d u e s l o c a t e d 4 8 bp u p s t r e a m o f t h e m a t u r e 5 ' - e n d w h i c h c o u l d hamper t h e u n w i n d i n g o f t h e DNA p r i o r t o t r a n s c r i p t i o n . The s e c o n d i s a 9 bp G + C - r i c h a l t e r n a t i n g p u r i n e - p y r i m i d i n e t r a c t 11 n u c l e o t i d e s f r o m t h e 5 ' - e n d o f t h e c o d i n g r e g i o n . S u c h a f e a t u r e h a s a p o t e n t i a l f o r f o r m i n g l e f t h a n d e d DNA h e l i x ( 1 5 6 ) w h i c h h a s b e e n s p e c u l a t e d t o p l a y a r o l e i n r e g u l a t i n g g e n e e x p r e s s i o n ( 1 5 7 ) . Removal o f t h e 3 ' - f l a n k i n g s e q u e n c e s d o e s n o t a p p e a r t o a f f e c t t h e e f f i c i e n c y o f t r a n s c r i p t i o n a l t h o u g h i t may v a r y t h e a c t u a l t e r m i n a t i o n s i t e ( 8 6 , 9 7 , 1 0 3 , 1 0 4 ) . S e a r c h e s o f t h e n u c l e o t i d e s u p s t r e a m f r o m t h e v a l i n e tRNA g e n e s h a v e r e v e a l e d no common s e q u e n c e s a h e a d o f e i t h e r e f f i c i e n t l y o r p o o r l y t r a n s c r i b e d g e n e s ( F i g s . 3 3 , 4 4 B ; 1 3 , 3 9 ) . P l a s m i d s , p D t 0 . 3 and p D t 0 . 6 , w h i c h a r e b o t h t r a n s c r i b e d a t v e r y h i g h r a t e s , show v e r y l i t t l e h o m o l o g y b e y o n d p o s i t i o n - 7 , b u t t h e y do s h a r e some common f e a t u r e s ( 1 3 , 3 9 ) . The 5 ' - f l a n k i n g s e q u e n c e s i n f r o n t o f b o t h t h e s e g e n e s h a v e t h e p o s s i b i l i t y o f f o r m i n g h a i r p i n s t r u c t u r e s , a n d a t - 5 2 p o s i t i o n i n f r o n t o f t h e g e n e i n p D t 0 . 3 i s a s e q u e n c e GGCAGTT, a v a r i a t i o n o f w h i c h (GGCACTT) i s a l s o f o u n d a t - 6 7 n u c l e o t i d e 5 ' o f t h e gene i n p D t 0 . 6 . The f i r s t 7 n u c l e o t i d e s o f t h e G + T - r i c h s e q u e n c e w h i c h p r e c e d e s t h e D r o s o p h i l a t R N A ^ 3 g e n e s ( 3 8 , 1 0 9 ) a r e i d e n t i c a l t o t h e s e q u e n c e a b o v e . A t p r e s e n t , t h e f u n c t i o n o f t h e s e s e q u e n c e s i s n o t k n o w n . H o w e v e r , t h e d e l e t i o n m u t a n t , p D t O . 3 d l 5 ' - 1 8 , w h i c h h a s o n l y 18 n u c l e o t i d e s o f D r o s o p h i l a DNA f l a n k i n g 5 ' o f i t s g e n e was t r a n s c r i b e d a t a 50% l o w e r e f f i c i e n c y t h a n p D t 0 . 3 f r o m w h i c h i t was d e r i v e d . T h i s e f f e c t c o u l d n o t be a t t r i b u t e d t o t h e p r e s e n c e o f pBR322 s e q u e n c e s b e y o n d n u c l e o t i d e - 1 8 , b e c a u s e a d e l e t i o n m u t a n t w i t h t h e D r o s o p h i l a DNA i n s e r t e d i n t h e o p p o s i t e o r i e n t a t i o n g a v e s i m i l a r r e s u l t s . T h e s e r e s u l t s t h e r e f o r e s u g g e s t t h a t some s e q u e n c e ( s ) w i t h a p o s i t i v e mode o f c o n t r o l a r e p r e s e n t b e y o n d p o s i t i o n - 1 8 i n t h e p l a s m i d p D t 0 . 3 . E x p e r i m e n t s i n v o l v i n g s e q u e n t i a l d e l e t i o n o f t h e 5 ' ^ - f l a n k i n g 163, s e q u e n c e s w o u l d r e s o l v e w h e t h e r any o f t h e f e a t u r e s d i s c u s s e d a b o v e i n f l u e n c e s t r a n s c r i p t i o n . S i m i l a r e x p e r i m e n t s r e p e a t e d on a p o o r l y t r a n s c r i b e d t e m p l a t e , Val e . g . p D t 9 2 R , w o u l d d e t e r m i n e w h e t h e r i t s tRNA g e n e i s p r e c e d e d by i n h i b i t o r y s e q u e n c e s . T h e s e e x p e r i m e n t s f o l l o w e d by f l a n k i n g s e q u e n c e s w i t c h i n g s t u d i e s w o u l d e s t a b l i s h w i t h c e r t a i n t y t h e r o l e o f 5 ' - f l a n k i n g Val s e q u e n c e s i n m o d u l a t i n g t r a n s c r i p t i o n o f D r o s o p h i l a tRNA g e n e s . 3 . R o l e o f c o n s e r v e d s e q u e n c e b l o c k s A and B i n t r a n s c r i p t i o n o f tRNA g e n e s D e l e t i o n and i n s e r t i o n a n a l y s e s h a v e shown t h a t tRNA g e n e s c o n t a i n i n t r a g e n i c s p l i t - p r o m o t e r s ( D i s c u s s i o n ) . T h e s e s e q u e n c e s , t e r m e d t h e A and B b l o c k s ( 9 7 ) , h a v e t h e a p p r o x i m a t e c o o r d i n a t e s 8 - 1 9 and 5 1 - 6 2 ( 8 6 , 9 7 , 1 0 3 , 1 0 4 ) r e s p e c t i v e l y by t h e s t a n d a r d tRNA n u m b e r i n g s y s t e m . The o p t i m u m d i s t a n c e b e t w e e n t h e A and t h e B b l o c k s i s 3 0 - 4 0 b p ; t h e e f f i c i e n c y o f t r a n s c r i p t i o n f a l l s i f t h e t w o r e g i o n s a r e c l o s e r t o g e t h e r o r f a r t h e r a p a r t ( 9 7 , 1 0 3 , 1 0 5 ) . The r e g i o n B i s h i g h l y c o n s e r v e d i n a l l tRNA g e n e s ( 8 ) and i t i s s p e c u l a t e d ( 1 0 4 ) t h a t i t i n t e r a c t s w i t h a common e l e m e n t o f t h e t r a n s c r i p -t i o n a l m a c h i n e r y . On t h e o t h e r h a n d , r e g i o n A , w h i c h i s more v a r i a b l e i n s i z e a n d i n s e q u e n c e ( 8 ) , may be r e s p o n s i b l e f o r a more s p e c i f i c c o n t r o l o f t r a n s c r i p t i o n ( 1 0 4 ) . A s t u d y by K r e s s m a n e t jal_. ( 7 8 ) i n d i c a t e d t h a t w h i l e t h e Met 5 ' and 3 ' - h a l v e s o f an X . l a e v i s t R N A i gene a r e u n a b l e t o d i r e c t t r a n s c r i p t i o n i n v i t r o , o n l y t h e 3 ' - h a l f o f e a c h g e n e c a n i n h i b i t t r a n s c r i p -t i o n o f t h e i n t a c t g e n e . T h e r e f o r e , i t h a s b e e n p r o p o s e d ( 1 0 8 ) t h a t t h e p r i m a r y i n t e r a c t i o n r e q u i r e d f o r t r a n s c r i p t i o n o f tRNA g e n e s i s t h e b i n d i n g o f a t r a n s c r i p t i o n f a c t o r t o t h e B b l o c k s e q u e n c e s . T h i s i n i t i a l i n t e r a c t i o n t h e n p r o m o t e s b i n d i n g t o t h e A b l o c k s e q u e n c e s whose f u n c t i o n i s t o p o s i t i o n RNA p o l y m e r a s e I I I f a r t h e r u p s t r e a m , w h e r e i t t h e n i n i t i a t e s t r a n s c r i p t i o n . 1 6 4 . T h e r e a r e r e a s o n s t o r e l e g a t e A b l o c k s e q u e n c e s t o a p o s i t i o n i n g r o l e : i n n a t u r a l tRNA g e n e s , t h e r e i s a g r e a t l a t i t u d e i n t h e d i s t a n c e b e t w e e n t h e A and B b l o c k s f r o m 29 t o > 74 bp ( 9 7 , 1 0 8 ) , b u t t h e d i s t a n c e b e t w e e n t h e tRNA t r a n s c r i p t i o n s t a r t s i t e s and t h e A b l o c k s e q u e n c e s i s l e s s v a r i a b l e , a b o u t 1 0 - 2 7 bp ( 3 7 , 1 0 8 ) . In a d d i t i o n , m u t a n t s w i t h d e l e t i o n s o r i n s e r t i o n s b e t w e e n t h e A and B r e g i o n s i n i t i a t e t r a n s c r i p t i o n a t a p p r o x i m a t e l y t h e same d i s t a n c e f r o m t h e A r e g i o n as d o e s t h e w i l d t y p e tRNA gene ( 9 7 , 1 0 3 - 1 0 5 ) p r o d u c i n g a p p r o p r i a t e l y s h o r t e r o r l o n g e r t r a n s c r i p t s . The t e r t i a r y i n t e r a c t i o n model o f H a l l e t a l _ . ( 1 0 8 ) p r e d i c t s t h a t a n y a l t e r a t i o n w h i c h w e a k e n s t h e t e r t i a r y i n t e r a c t i o n b e t w e e n t h e A ( D - l o o p ) and t h e B ( T * C l o o p ) b l o c k s w o u l d d i m i n i s h t r a n s c r i p t i o n . A more b a s i c p r e m i s e o f t h e i r model i s t h a t t h e e l i m i n a t i o n o f t h e e n t i r e B b l o c k w o u l d a b o l i s h t r a n s c r i p t i o n . Two r e p o r t s i n l i t e r a t u r e do n o t s u p p o r t t h i s a s s u m p t i o n . . . A r g S h a r p e t a]_. ( 8 6 ) , w o r k i n g w i t h a D r o s o p h i l a t R N A 2 g e n e , f o u n d t h a t t r a n s c r i p t i o n was m e r e l y r e d u c e d by 50% a f t e r r e m o v a l o f t h e B b l o c k s e q u e n c e s , and t h a t i t was o n l y a b o l i s h e d when d e l e t i o n s r e a c h e d t h e 3 ' - e n d o f t h e D s t e m . C a r r a r a e t ^1_. ( 1 5 8 ) i n s e r t e d 3 0 0 bp o f mouse DNA i n t o t h e L e u i n t e r v e n i n g s e q u e n c e o f a y e a s t t R N A 3 g e n e . T h i s e x p a n d e d g e n e was e f f i c i e n t l y t r a n s c r i b e d j_n v i t r o , e v e n a f t e r r e m o v a l o f p a r t o f t h e mouse i n s e r t t o g e t h e r w i t h t h e 3 ' - h a l f o f t h e g e n e . H a l l e t aj_. ( 1 0 8 ) e x p l a i n t h e s e d i s c r e p a n c i e s by p r o p o s i n g t h a t t h e s e q u e n c e s u s e d t o r e p l a c e t h e , 3 ' - . - h a l v e s o f A r g L e u t h e D r o s o p h i l a t R N A 2 a n d y e a s t t R N A 3 g e n e s a r e a b l e t o f o r m s u b s t i t u t e B b l o c k s w h i c h f o r m t h e n e c e s s a r y s t r u c t u r e f o r f a c t o r b i n d i n g and f o r t e r t i a r y i n t e r a c t i o n s w i t h t h e n o r m a l A b l o c k s e q u e n c e s . The d e l e t i o n m u t a n t , p D t 0 . 3 A 5 1 - 6 1 , c o n s t r u c t e d d u r i n g t h e c o u r s e o f t h i s w o r k , l a c k s n u c l e o t i d e s 51 t o 61 i n t h e B b l o c k ; t h e r e s t o f t h e s e q u e n c e i s i d e n t i c a l t o t h a t f o u n d i n t h e p l a s m i d , p D t 0 . 3 . No s u c h d e l e t i o n m u t a n t h a s b e e n d e s c r i b e d t o d a t e . The s e q u e n c e p r e s e n t i n t h e h o m o l o g o u s p o s i t i o n ( n u c l e o t i d e s 5 1 - 6 1 ) i n t h e d e l e t e d m u t a n t i s shown i n t h e l a s t l i n e i n F i g . 4 4 A . C o m p a r i s o n t o t h e g e n e r a l i z e d B b l o c k s e q u e n c e shows t h a t i t d i f f e r s o n l y a t n u c l e o t i d e p o s i t i o n s 5 4 , 55 and 5 6 . The RNA t r a n s c r i p t d i r e c t e d by p D t O . 3 A 51-61 c o u l d be d r a w n t o r e p r e s e n t t h e s t r u c t u r e i n F i g . 4 5 . I t h a s a g e n e r a l a p p e a r a n c e o f a tRNA m o l e c u l e w i t h a r e g u l a r a c c e p t o r s t e m a n d D and a n t i c o d o n a r m s . The d i f f e r e n c e s a r e a s f o l l o w s : i ) t h e v a r i a b l e arm h a s o n l y V a l t h r e e n u c l e o t i d e s i n s t e a d o f f i v e a s i n D r o s o p h i l a t R N A 4 . The m o s t r e c e n t c o m p i l a t i o n o f tRNA s e q u e n c e s by S p r i n z l and G a u s s ( 8 ) shows t h a t two y e a s t t R N A s , t R N A V a l ( 1 5 9 ) and tRNA G l y ( 1 6 0 ) , h a v e o n l y t h r e e n u c l e o t i d e s i n t h e e x t r a - a r m r e g i o n . Thus a minimum o f t h r e e n u c l e o t i d e s i n t h i s r e g i o n must p r o v i d e e n o u g h f l e x i b i l i t y t o f o r m a p r o p e r t e r t i a r y s t r u c t u r e . W i t h t h e e x c e p t i o n o f s t u d i e s . b y C i 1 i b e r t o e t ( 1 0 4 ) and C i a m p i et aj_. ( 1 5 5 ) , t h e s e q u e n c e i n t h e v a r i a b l e o r t h e e x t r a arm h a s b e e n shown t o be d i s p e n s a b l e f o r t R N A t r a n s c r i p t i o n ( 8 6 , 9 7 , 1 0 3 ) ; h o w e v e r , i t d o e s a p p e a r t o s e r v e a s p a c e r f u n c t i o n f o r maximum t r a n s c r i p t i o n e f f i c i e n c y ( 9 7 , 1 0 3 - 1 0 5 ) . i i ) The T - s t e m i s v e r y G + C - r i c h l i k e r e g u l a r T - s t e m s , t h o u g h i t h a s o n l y t h r e e bp i n s t e a d o f t h e n o r m a l f i v e . E x c e p t f o r t h e two C r e s i d u e s , t h e T ^ C l o o p i s c o m p l e t e l y m i s s i n g . T r a n s c r i p t i o n s t u d i e s w i t h t h e d e l e t i o n m u t a n t , p D t 0 . 3 A 5 1 - 6 1 shows t h a t i t t r a n s c r i b e s a l t h o u g h a t o n l y a b o u t 2% o f t h e w i l d t y p e g e n e e f f i c i e n c y . T h a t t h i s l o w l e v e l o f t r a n s c r i p t i o n i s n o t due t o t h e i n s t a b i l i t y o f t h e t r a n s c r i p t i s shown by t h e s t a b i l i t y o f h a l f tRNA m o l e c u l e s i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . The s p e c i f i c t r a n s c r i p t i s a p p r o p r i a t e l y s h o r t e n e d a s i f t h e RNA i n i t i a t e d and t e r m i n a t e d a t t h e same s i t e s a s t h e w i l d t y p e RNA t r a n s c r i p t . T h e s e d a t a c a n be i n t e r p r e t e d i n s e v e r a l w a y s : i ) The B b l o c k V a l s e q u e n c e i s e s s e n t i a l f o r t r a n s c r i p t i o n o f t h e D r o s o p h i l a t R N A 4 g e n e ; a l t e r a t i o n o f t h i s r e g i o n i n t h e d e l e t i o n m u t a n t a l l o w s o n l y a v e r y l o w l e v e l o f t r a n s c r i p t i o n , i i ) The l o w e r e d e f f i c i e n c y o f t r a n s c r i p t i o n i s s o l e l y due t o 166 . F i g u r e 4 5 . The p o s t u l a t e d t R N A - l i k e s t r u c t u r e o f t h e RNA t r a n s c r i p t d i r e c t e d by p D t 0 . 3 A 5 1 - 6 1 . A tRNA t r a n s c r i p t d i r e c t e d by t h e p l a s m i d , p D t 0 . 3 A 51-61 c o u l d be a r r a n g e d t o r e p r e s e n t t h e c l o v e r l e a f - 1 i k e s t r u c t u r e shown i n t h i s f i g u r e . 1 6 7 . G G U G A U U A U G U U U C C G U G U G C A C U C U G C A C A A A G G C G G G C . . . I c c c c G G A A G A C G U A A A C 1 6 8 . V a l t h e s h o r t e n i n g o f t h e t R N A 4 g e n e , i i i ) The B b l o c k s e q u e n c e i s r e q u i r e d f o r t h e p r i m a r y e v e n t o f t r a n s c r i p t i o n i n i t i a t i o n . B o t h t h e a l t e r a t i o n o f t h e B b l o c k s t r u c t u r e and t h e s h o r t e n i n g o f t h e d i s t a n c e b e t w e e n t h e A and B b l o c k s a r e r e s p o n s i b l e f o r t h e l o w e f f i c i e n c y o f t r a n s c r i p t i o n . E v i d e n c e and a r g u m e n t s w i l l be p r e s e n t e d b e l o w w h i c h f a v o u r t h e l a s t i n t e r p r e t a t i o n . A c c o r d i n g t o t h e model o f H a l l e t j i l _ . ( 1 0 8 ) , a m u t a n t m i s s i n g t h e B b l o c k s e q u e n c e s h o u l d c o m p l e t e l y e l i m i n a t e t r a n s c r i p t i o n . U n l i k e t h e two o t h e r e x c e p t i o n s d i s c u s s e d a b o v e ( 8 6 , 1 5 8 ) , t h e s e q u e n c e s now o c c u p y i n g t h e d e l e t e d r e g i o n i n p D t 0 . 3 A 51-61 a r e n o t a b l e t o f o r m a s u b s t i t u t e B b l o c k ( w i t h a c o m p l e t e s t e m and l o o p s t r u c t u r e ) . H o w e v e r , i t i s p o s s i b l e t h a t t h i s d e l e t i o n m u t a n t i s a b l e t o d i r e c t a l o w d e g r e e o f t r a n s c r i p t i o n by b i n d i n g a t r a n s c r i p t i o n f a c t o r t o t h e s t i l l e x i s t e n t and p o t e n t i a l l y s t a b l e T - s t e m a t a r e s i d u a l l e v e l . To c a r r y on w i t h t h e a n a l o g y o f H a l l e t a j _ . , s u c h a b i n d i n g t h e n c o u l d p r o m o t e some l e v e l o f t e r t i a r y i n t e r a c t i o n s w i t h t h e c o m p l e t e l y i n t a c t A b l o c k s e q u e n c e s w h i c h t h e n c o u l d d i r e c t i n i t i a t i o n o f t r a n s c r i p t i o n f a r t h e r u p s t r e a m . The p r i m a r y i m p o r t a n c e o f t h e T - s t e m i n tRNA • t r a n s c r i p t i o n i s i n d i c a t e d by t h e s t u d i e s on t h e d e l e t i o n m u t a n t c o n s t r u c t e d by C i l i b e r t o e t a l _ . ( 1 0 4 ) . T h i s d e l e t i o n , w h i c h e l i m i n a t e d t h e T s t e m b u t s t i l l c o n t a i n e d t h e i n v a r i a n t G53-C61 ( w h i c h f o r m t h e T T C l o o p ) n u c l e o t i d e s , r e d u c e d t r a n s c r i p t i o n by 9 9 % . Though t h e d e l e t i o n m u t a n t , p D t 0 . 3 A 5 1 - 6 1 , l a c k s t h e i n v a r i a n t TC r e s i d u e s a t p o s i t i o n s 55 and 56 r e s p e c t i v e l y ( w h i c h f o r m t e r t i a r y b a s e p a i r s w i t h G18 and G19 r e s p e c t i v e l y i n t h e D - l o o p ) , i t i s c o n c e i v a b l e t h a t t h e two u n b a s e - p a i r e d C r e s i d u e s i n e q u i v a l e n t p o s i t i o n s ( F i g . 4 5 ) c o u l d f o r m t e r t i a r y b a s e p a i r s w i t h t h e same G r e s i d u e s i n t h e D - l o o p (A b l o c k ) . In f a c t , t h e model o f H a l l e t _al_. p r e d i c t s t h a t a T ^ C t r a n s i t i o n a t p o s i t i o n 55 s h o u l d e n h a n c e t r a n s c r i p t i o n . A n o t h e r f a c t o r w h i c h c o u l d c o n t r i b u t e t o t h e l o w e f f i c i e n c y o f t r a n s c r i p t i o n o b s e r v e d w i t h t h e d e l e t i o n m u t a n t , p D t 0 . 3 A 5 1 - 6 1 , i s t h e 169 . s h o r t e n i n g o f t h e d i s t a n c e b e t w e e n t h e A and B b l o c k s . The d i s t a n c e A - B i n Val t h e t R N A 4 g e n e i n p D t 0 . 3 i s 30 b p , ( F i g . 4 3 ) , t h a t i n t h e d e l e t e d tRNA g e n e i s 28 bp ( F i g . 4 5 ) . In n a t u r a l tRNA g e n e s , t h i s d i s t a n c e c a n v a r y f r o m 29 t o >74 bp ( 9 7 , 1 0 8 ) . S e v e r a l s t u d i e s i n d i c a t e t h a t s h o r t e n i n g t h e d i s t a n c e b e t w e e n t h e A and t h e B b l o c k s d e c r e a s e s t h e e f f i c i e n c y o f t r a n s c r i p t i o n b u t i t d o e s n o t a p p e a r t o h a v e a n y e f f e c t on t h e p r i m a r y e v e n t o f t r a n s c r i p t i o n i n i t i a t i o n ( 9 7 , 1 0 3 , 1 0 5 ) . The d a t a o f C i l i b e r t o e t a l . ( 1 0 5 ) s u g g e s t t h a t a s h o r t e n i n g by 10 bp w o u l d d e c r e a s e t r a n s c r i p t i o n t o 60% and s h o r t e n i n g by 2 bp w o u l d h a v e m i n i m a l e f f e c t . H o f s t e t t e r et _a_L ( 1 0 3 ) c o n s t r u c t e d X e n o p u s t R N A ^ * g e n e m u t a n t s i n w h i c h t h e y s u b s t i t u t e d t h e s e q u e n c e b e t w e e n +33 t o +46 i n t h e c o d i n g r e g i o n w i t h l i n k e r DNA. A d e l e t i o n m u t a n t i n w h i c h t h e gene was s h o r t e n e d by 2 bp ( c o m p a r e d t o t h e w i l d t y p e g e n e ) g a v e no t r a n s c r i p t i o n . T h e r e f o r e , t h e m a g n i t u d e o f t h e e f f e c t on t r a n s c r i p t i o n due t o t h i s s h o r t e n i n g o f t h e d i s t a n c e b e t w e e n t h e A and t h e B b l o c k s a p p e a r s t o be q u i t e v a r i a b l e a n d d e p e n d s on t h e i n d i v i d u a l tRNA g e n e , on t h e r e g i o n i n w h i c h t h e d e l e t i o n i s c r e a t e d and on t h e s e q u e n c e w h i c h r e p l a c e s t h e d e l e t e d r e g i o n . The e x t e n t t o w h i c h t h e s h o r t e n i n g o f t h e d i s t a n c e b e t w e e n t h e A a n d B r e g i o n s i n t h e g e n e i n p D t O . 3 A 5 1 - 6 1 c o n t r i b u t e d t o t h e l o w e r e d e f f i c i e n c y o f t r a n s c r i p t i o n o f t h i s g e n e i s n o t c l e a r . A l l t h e e u k a r y o t i c tRNA g e n e s s t u d i e d s o f a r h a v e shown t h e r e q u i r e m e n t f o r t h e B b l o c k s e q u e n c e s o r s t r u c t u r e s ( t o g e t h e r w i t h A b l o c k ) f o r t r a n s c r i p -t i o n i n i t i a t i o n ( 8 6 , 9 7 , 1 0 3 , 1 0 4 , 1 5 8 ) . T h e r e f o r e , u n l e s s t h e d e l e t e d Val D r o s o p h i l a t R N A 4 g e n e i s an e x c e p t i o n t o t h i s r u l e , t h e t r a n s c r i p t i o n r e s u l t s o b t a i n e d w i t h t h e d e l e t i o n m u t a n t , p D t O . 3 A 5 1 - 6 1 , i n d i c a t e t h a t t h e a l t e r e d B b l o c k s t r u c t u r e p r e s e n t i n t h i s m u t a n t i s c a p a b l e o f d i r e c t i n g a l o w l e v e l o f t r a n s c r i p t i o n . P r e s u m a b l y , i n t a c t T^ C l o o p and t h e o p t i m u m d i s t a n c e b e t w e e n t h e A and B b l o c k s a r e i m p o r t a n t f e a t u r e s f o r o b t a i n i n g maximum t r a n s c r i p t i o n e f f i c i e n c y . 170. D. I n f l u e n c e o f s e q u e n c e on m a t u r a t i o n o f tRNA p r e c u r s o r s U n l i k e t h e tRNA t r a n s c r i p t i o n i n i t i a t i o n s t u d i e s , a s y s t e m a t i c s t u d y l o o k i n g a t t h e e f f e c t s o f s e q u e n c e c h a n g e s on t h e m a t u r a t i o n s t e p s o f tRNA b i o s y n t h e s i s i s n o t a v a i l a b l e y e t . T h i s i s p a r t l y b e c a u s e o f t h e c o m p l e x n a t u r e o f t h e tRNA b i o s y n t h e t i c p r o c e s s and p a r t l y d u e t o t h e u n a v a i l a b i l i t y o f t h e tRNA p r e c u r s o r s i n a m o u n t s s u f f i c i e n t f o r b i o c h e m i c a l s t u d i e s . W i t h t h e a v a i l a b i l i t y o f i n v i t r o t r a n s c r i p t i o n s y s t e m s w h e r e t h e tRNA p r e c u r s o r s a c c u m u l a t e i n s u f f i c i e n t q u a n t i t i e s , s u c h s t u d i e s s h o u l d become f e a s i b l e . The f o r m a t i o n o f an e u k a r y o t i c tRNA i s a c o m p l e x m u l t i s t e p e n z y m a t i c p r o c e s s w h i c h f i r s t i n v o l v e s t h e s y n t h e s i s o f a p r i m a r y t r a n s c r i p t c o n t a i n i n g a d d i t i o n a l 5 ' and 3 ' - n u c l e o t i d e s a n d , i n some c a s e s , i n t e r v e n i n g s e q u e n c e s . S u b s e q u e n t l y , t h e s e e x t r a n u c l e o t i d e s a r e r e m o v e d , a 3 ' - C C A t e r m i n u s i s a d d e d , a n d c e r t a i n r i b o n u c l e o t i d e m o d i f i c a t i o n s a r e i n t r o d u c e d . T r a n s c r i p t i o n , p r o c e s s i n g and m o d i f i c a t i o n r e a c t i o n s a p p e a r t o be l o c a l i z e d i n t h e n u c l e u s a n d a l l t h e s e s t e p s t a k e p l a c e i n a s e q u e n t i a l f a s h i o n ( s e e I n t r o d u c t i o n ) . A l l t h e e u k a r y o t i c tRNA p r e c u r s o r s s h a r e t h e f o l l o w i n g common f e a t u r e s : t h e y i n i t i a t e w i t h p u r i n e t r i p h o s p h a t e s , t h e i r 5 ' - l e a d e r s e q u e n c e s a r e o n l y a f e w n u c l e o t i d e s l o n g , and t h e i r 3 ' - t r a i l e r s e q u e n c e s t e r m i n a t e i n a s e r i e s o f U r e s i d u e s . S i n c e t h e 5 ' and 3 ' - f l a n k i n g r e g i o n s f r o m v a r i o u s tRNA p r e c u r s o r s v a r y i n s e q u e n c e , i t i s a s s u m e d t h a t t h e l i m i t e d number o f p r o c e s s i n g e n z y m e s known t o be i n v o l v e d i n p r o c e s s i n g o f d i f f e r e n t tRNA p r e c u r s o r s r e c o g n i z e s e c o n d a r y and t e r t i a r y s t r u c t u r e s common t o a l l tRNA p r e c u r s o r s . N i s h i k u r a e t a l . ( m a n u s c r i p t i n p r e p a r a t i o n ) a n a l y z e d t h e e f f e c t o f 18 d i f f e r e n t s i n g l e Tyr n u c l e o t i d e c h a n g e s i n y e a s t tRNA gene on tRNA p r o c e s s i n g by X . l a e v i s o o c y t e s . T h e i r m a i n c o n c l u s i o n was ( c i t e d i n r e f . 117) t h a t t h e m a t u r a t i o n o f t h e 5 ' and 3 ' ^ e n d s was t h e s t e p m o s t s e n s i t i v e t o m u t a t i o n s . A s t u d y by Met H o f s t e t t e r e t ^1_. ( 1 0 3 ) w i t h X . l a e v i s tRNA g e n e i n d i c a t e d t h a t i n f r o g o o c y t e s t h e p r e c u r s o r s m a t u r e d a t a h i g h r a t e f o r a l l t h o s e m u t a n t s i n w h i c h 1 7 1 . s e q u e n c e a l t e r a t i o n s w e r e l o c a t e d e x c l u s i v e l y i n t h e 5 ' o r 3 ' - f l a n k i n g r e g i o n s o r w i t h i n t h e g e n e b u t c l o s e t o t h e a n t i c o d o n l o o p . A l l o t h e r m u t a t i o n s t h a t a l t e r e d t h e s e q u e n c e make up o f g e n e r e g i o n s 1 - 2 8 o r 3 6 - 7 2 p r e v e n t e d e f f i c i e n t p r o c e s s i n g . A s i n g l e n u c l e o t i d e d e l e t i o n i n t h e a c c e p t o r s t e m ( C 3 ) o f a Lys D r o s o p h i l a tRNA g e n e d e c r e a s e d p r o c e s s i n g o f t h e a l t e r e d p r e c u r s o r ( 3 8 ) . A l l t h e s e r e s u l t s s u g g e s t t h a t t h e t e r t i a r y s t r u c t u r e o f t h e p r e c u r s o r s may be c r u c i a l f o r t h e i r p r o p e r r e c o g n i t i o n by t h e v a r i o u s p r o c e s s i n g e n z y m e s . Val P r o c e s s i n g e x p e r i m e n t s w i t h D r o s o p h i l a tRNA g e n e s d e s c r i b e d h e r e Val s u p p o r t t h e a b o v e c o n c l u s i o n . The p l a s m i d , p D t 7 8 R , c o n t a i n i n g a t R N A 3 b g e n e was t r a n s c r i b e d a t a b o u t 3 - 4 f o l d l o w e r r a t e t h a n p D t 0 . 3 c o n t a i n i n g a t R N A ^ 3 1 g e n e . B u t t h e p r e c u r s o r RNA d i r e c t e d by pDt78R was p r o c e s s e d a b o u t 5 - t i m e s m o r e e f f i c i e n t l y t h a n t h a t f r o m p D t 0 . 3 i n t h e D r o s o p h i l a S . 1 0 0 e x t r a c t . T h i s d i f f e r e n c e i n t h e l e v e l o f p r o c e s s i n g was n o t due t o l i m i t e d p r o c e s s i n g a c t i v i t y i n t h e S . 1 0 0 e x t r a c t . The h y b r i d p l a s m i d , p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) was t r a n s c r i b e d a s e f f i c i e n t l y a s p D t 0 . 3 a n d i t d i r e c t e d a p r e c u r s o r RNA w h i c h was p r o c e s s e d a t a r a t e s i m i l a r t o t h a t f o r p D t 7 8 R . The o n l y d i f f e r e n c e b e t w e e n t h e c o d i n g r e g i o n s o f p D t 0 . 3 a n d t h e h y b r i d p l a s m i d i s t h e p r e s e n c e a t p o s i t i o n 69 ( i n t h e a c c e p t o r s t e m ) o f an A i n t h e f o r m e r and a G i n t h e l a t t e r . The A69~*"G69 t r a n s i t i o n w o u l d i n t r o d u c e a non W a t s o n - C r i c k b a s e p a i r a t t h e f o u r t h p o s i t i o n ( U 4 - G 6 9 ) i n t h e a c c e p t o r s t e m o f t h e h y b r i d RNA p r o d u c t . S u c h a U.G b a s e p a i r i s n o r m a l l y f o u n d a t t h i s p o s i t i o n i n Val D r o s o p h i l a t R N A 3 b ( 1 3 ) . U.G i s t h e m o s t common non W a t s o n - C r i c k b a s e p a i r f o u n d i n tRNA ( 1 6 1 ) and C l a r k ( 1 6 1 ) h a s h y p o t h e s i z e d t h a t t h e i r r e g u l a r i t i e s i n t r o d u c e d i n t o t h e d o u b l e h e l i c a l r e g i o n s o f t R N A s by t h e s e m i s - m a t c h e d b a s e p a i r s may be s p e c i a l p o i n t s f o r enzyme r e c o g n i t i o n . The h y b r i d p l a s m i d , p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) , g a v e c o m p l e m e n t a r y r e s u l t s , i . e . , i t t r a n s c r i b e d a t a r a t e s i m i l a r t o pDt78R and i t s p r e c u r s o r RNA was p r o c e s s e d w i t h a l o w e f f i c i e n c y c o m p a r a b l e t o t h a t o f p D t 0 . 3 . T h i s h y b r i d 1 7 2 . p l a s m i d c a r r i e s a g e n e w h i c h h a s t h e c o d i n g s e q u e n c e i d e n t i c a l t o t h a t i n p D t 7 8 R , e x c e p t t h a t i t s p r i m a r y p r o d u c t w o u l d h a v e a n o r m a l W a t s o n - C r i c k b a s e p a i r , U 4 - A 6 9 , i n t h e a c c e p t o r r e g i o n . I t s h o u l d be p o i n t e d o u t t h a t o f t h e 7 - 8 d i f f e r e n t D r o s o p h i l a tRNA g e n e s u s e d f o r t r a n s c r i p t i o n , o n l y t h e g e n e i n pDt78R c o n t a i n s a m i s - m a t c h e d b a s e p a i r i n t h e a c c e p t o r r e g i o n and t h a t t h e p r e c u r s o r RNA d i r e c t e d by i t ( a n d t h e m a n i p u l a t e d DNA t e m p l a t e , p D t 0 . 3 ( 5 ' ) -7 8 R ( 3 ' ) , w h e r e s u c h a c h a n g e was i n t r o d u c e d ) was p r o c e s s e d t o m a t u r e tRNA p r o d u c t w i t h t h e h i g h e s t e f f i c i e n c y . The h y b r i d p l a s m i d s , p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) and p D t 7 8 R ( 5 ' ) - 0 . 3 ( 3 ' ) , d e r i v e d t h e i r 5 ' - f l a n k i n g s e q u e n c e s f r o m p D t 0 . 3 and p D t 7 8 R , a n d t h e i r 3 ' - f l a n k i n g s e q u e n c e s f r o m pDt78R and p D t 0 . 3 r e s p e c t i v e l y . C o m p a r i s o n o f t h e e f f i c i e n c i e s o f p r o c e s s i n g o f t h e p r e c u r s o r RNAs d i r e c t e d by t h e f o u r p l a s m i d s i n d i c a t e s t h a t t h e 5 ' - f l a n k i n g s e q u e n c e s h a v e l i t t l e e f f e c t on p r o c e s s i n g . H o w e v e r , i t c a n be a r g u e d t h a t e i t h e r t h e 3 ' - ^ f l a n k i n g s e q u e n c e f r o m pDt78R a n d / o r t h e i r r e g u l a r b a s e p a i r i n t h e a c c e p t o r r e g i o n i s r e s p o n s i b l e f o r t h e h i g h e f f i c i e n c y o f p r o c e s s i n g s e e n w i t h pDt78R a n d p D t 0 . 3 ( 5 ' ) - 7 8 R ( 3 ' ) . B u t t h e s t u d y by H o f s t e t t e r e t a l _ . ( 1 0 3 ) c i t e d a b o v e and t h e o b s e r v a t i o n i n g e n e r a l t h a t t h e 3 ' - f l a n k i n g r e g i o n s o f t h e v a r i o u s tRNA g e n e s i n c l u d i n g D r o s o p h i l a V a l tRNA g e n e s ( F i g . 3 3 ) v a r y i n s e q u e n c e make t h e i n v o l v e m e n t o f t h e 3 ' f l a n k i n g s e q u e n c e s i n p r o c e s s i n g i m p r o b a b l e . S i m i l a r s t u d i e s a s d e s c r i b e d a b o v e w i t h p l a s m i d s p D t 0 . 3 , pDt92R a n d t h e h y b r i d p l a s m i d s , p D t 0 . 3 ( 5 ' ) - 9 2 R ( 3 ' ) and p D t 9 2 R ( 5 ' ) - 0 . 3 ( 3 ' ) , i n d i c a t e t h a t a G a t p o s i t i o n 57 i n t h e T ¥ C l o o p (B b l o c k ) r e g i o n a l l o w s more e f f i c i e n t p r o c e s s i n g o f p r e c u r s o r tRNA t h a n when i t i s c h a n g e d t o an A . Z a s l o f f e t a l . a l s o r e p o r t e d t h a t a G57 +T57 t r a n s v e r s i o n ( a n U d o e s n o t o c c u r a t p o s i t i o n 57 i n any o f t h e .tRNA s p e c i e s i n e i t h e r p r o k a r y o t e s o r e u k a r y o t e s ; 8 ) d i m i n i s h e d Met p r o c e s s i n g o f t h e p r e c u r s o r RNA d i r e c t e d by human tRNA g e n e _i_n v i v o ( 1 5 3 ) a n d j_n v i t r o ( 1 5 4 ) . The vn v i t r o s t u d y showed t h a t , w h i l e t h e p r i m a r y 173 , t r a n s c r i p t o f t h e n o r m a l g e n e ( w i t h G57) u n d e r w e n t s t e p w i s e e x c i s i o n o f i t s 5 ' a n d 3 ' - t e r m i n a l s e q u e n c e s ( 5 * p r e c e d i n g 3 ' ) , o n l y t h e 5 ' - l e a d e r s e q u e n c e o f Met t h e p r i m a r y t r a n s c r i p t o f t h e v a r i a n t tRNA g e n e was e x c i s e d , r e s u l t i n g i n t h e a c c u m u l a t i o n o f an i n t e r m e d i a t e c o n t a i n i n g t h e u n p r o c e s s e d 3 ' ^ t r a i l e r s e q u e n c e . The e x a c t m e c h a n i s m by w h i c h t h e G57 ^ A 5 7 c h a n g e ( r e p o r t e d h e r e ) a f f e c t s p r o c e s s i n g i s n o t c l e a r . H o w e v e r , p r o c e s s i n g e x p e r i m e n t s w i t h t h e Val p l a s m i d s , pDt92R ( w h i c h c o n t a i n s a tRNA g e n e w i t h an A a t p o s i t i o n 57) i n d i c a t e d t h a t t h e s t e p w h i c h c o n v e r t e d t h e p r e c u r s o r RNA t o t h e f i r s t i n t e r m e d i a t e s p e c i e s was more e f f i c i e n t t h a n t h e s u b s e q u e n t s t e p ( s ) w h i c h p r o c e s s e d t h e i n t e r m e d i a t e p r o d u c t t o t h e m a t u r e tRNA s p e c i e s . T h e r e f o r e , t h e G 5 7 + A 5 7 c h a n g e , l i k e t h e G57 + T 5 7 a l t e r a t i o n , a p p e a r s t o b l o c k o r r e d u c e one o f t h e s t e p s i n v o l v e d i n t h e m a t u r a t i o n o f t h e tRNA p r e c u r s o r . T h e s e s t u d i e s s u g g e s t t h a t t h e s e q u e n c e r e q u i r e m e n t s f o r t h e p r o c e s s i n g o f p r e c u r s o r RNA t o m a t u r e tRNA a r e much more s t r i n g e n t t h a n t h o s e f o r t r a n s c r i p t i o n i n i t i a t i o n . N u c l e o t i d e c h a n g e s , e . g . G 5 7 + A 5 7 , G 5 7 + T 5 7 , A69 + G69 w h i c h do n o t a f f e c t t h e r a t e o f t r a n s c r i p t i o n ( b u t w h i c h may c a u s e s l i g h t a l t e r a t i o n s i n t h e t e r t i a r y s t r u c t u r e ) i n f l u e n c e t h e e f f i c i e n c y o f p r o c e s s i n g . T h e r e f o r e , c e r t a i n e u k a r y o t i c tRNA m u t a t i o n s may n o t a p p e a r i n m a t u r e tRNA s p e c i e s due t o t h e e f f e c t s o f t h e m u t a t i o n on p r e c u r s o r p r o c e s s i n g . T h e s e r e s u l t s s u g g e s t t h a t p o s t - t r a n s c r i p t i o n a l p r o c e s s i n g may p l a y a " s c r e e n i n g " f u n c t i o n i n t h e e x p r e s s i o n o f e u k a r y o t i c t R N A s . I I I . K i n e t i c s o f tRNA t r a n s c r i p t i o n E a r l y s t u d i e s u s i n g p u r i f i e d RNA p o l y m e r a s e I I I and n a k e d 5S RNA and t R N A g e n e s i n d i c a t e d t h a t c o m p o n e n t s o r f a c t o r s o t h e r t h a n RNA p o l y m e r a s e I I I a n d DNA t e m p l a t e w e r e e s s e n t i a l f o r s p e c i f i c t r a n s c r i p t i o n o f t h e s e g e n e s ( I n t r o d u c t i o n ) . S i n c e t h e n f r a c t i o n a t i o n o f c e l l - f r e e e x t r a c t s a n d r e c o n s t i t u t i o n e x p e r i m e n t s ( 5 9 , 6 0 ) h a v e l e n t d i r e c t e v i d e n c e f o r i n v o l v e m e n t o f m u l t i p l e c o m p o n e n t s i n 5S RNA a n d tRNA g e n e t r a n s c r i p t i o n . T h e s e s t u d i e s 174 . i n d i c a t e t h a t t h e r e a r e a t l e a s t t w o f a c t o r s o t h e r t h a n RNA p o l y m e r a s e I I I r e q u i r e d f o r t R N A g e n e t r a n s c r i p t i o n . The t i m e c o u r s e and t h e t r a n s c r i p t i o n c o m p l e x s t a b i l i t y e x p e r i m e n t s d e s c r i b e d h e r e g i v e k i n e t i c e v i d e n c e f o r t h e i n v o l v e m e n t o f m u l t i p l e c o m p o n e n t s i n t h e f o r m a t i o n o f t h e t r a n s c r i p t i o n c o m p l e x . Two c o n c l u s i o n s c a n be d r a w n f r o m t h e d a t a i n F i g s . 32A and 3 2 B . F i r s t , a s t a b l e t r a n s c r i p t i o n c o m p l e x a p p e a r e d t o be f o r m e d w i t h pDt78R s i n c e s y n t h e s i s f r o m p D t 0 . 3 d i d n o t o c c u r i f i t was a d d e d a f t e r a c r i t i c a l t i m e o f 1 5 - 2 0 m i n . T h i s c r i t i c a l t i m e f o r p D t 0 . 3 t e m p l a t e e x c l u s i o n c o r r e s p o n d e d c l o s e l y t o t h e t i m e when t h e o v e r a l l r e a c t i o n a c h i e v e d a l i n e a r r a t e o f s y n t h e s i s . I t s h o u l d be n o t e d t h a t i n s i n g l e t e m p l a t e a s s a y s , p D t 0 . 3 was t r a n s c r i b e d 3 - 4 f o l d more e f f i c i e n t l y t h a n p D t 7 8 R . The s e c o n d c o n c l u s i o n i s t h a t p D t 0 . 3 was much more e f f i c i e n t a t a s s u m i n g an e x c l u s i o n a r y c o m p l e x i n t h e e x t r a c t t h a n p D t 7 8 R . T h u s , pDt78R DNA c o u l d n o t c o m p e t e f o r t r a n s c r i p t i o n u n l e s s i t was a d d e d a c o n s i d e r a b l e t i m e b e f o r e p D t 0 . 3 . S i n c e t h e o v e r a l l l a g i n t h e t i m e c o u r s e f o r RNA s y n t h e s i s i n s i n g l e t e m p l a t e a s s a y s was e f f e c t i v e l y t h e same f o r p D t 0 . 3 a n d p D t 7 8 R , t h e s e r e s u l t s i n d i c a t e a t l e a s t two k i n e t i c s t e p s i n t h e f o r m a t i o n o f t h e t r a n s c r i p -t i o n c o m p l e x . F i r s t , t h e f o r m a t i o n o f an e x c l u s i o n a r y c o m p l e x w h i c h may i n v o l v e b i n d i n g o f a common f a c t o r t h a t may be p r e s e n t i n a l i m i t e d a m o u n t . T h i s s t e p was more r a p i d w i t h p D t 0 . 3 t h a n w i t h pDt78R w h i c h may i m p l y d i f f e r e n t a f f i n i t i e s o f t h e common f a c t o r f o r t h e t w o t e m p l a t e s . S e c o n d , t h e s l o w e r s t e p ( r e q u i r i n g a p p r o x i m a t e l y 2 0 - 3 0 m i n . ) l e a d s t o a c o m p l e t e t r a n s c r i p t i o n c o m p l e x n e e d e d t o d i r e c t t h e l i n e a r r a t e o f t r a n s c r i p t i o n . T h u s , i t a p p e a r s t h a t b e f o r e RNA p o l y m e r a s e b e g i n s t o a c c u m u l a t e t r a n s c r i p t i o n p r o d u c t s , a t l e a s t two e v e n t s must t a k e p l a c e . T r a n s c r i p t i o n f a c t o r s i m p l i c a t e d i n tRNA s y n t h e s i s ( 5 9 , 6 0 ) may be i n v o l v e d i n t h e s e s t e p s . K i n e t i c s and c o m p e t i t i o n s t u d i e s w i t h 5S DNA a l s o i n d i c a t e a t l e a s t two s t e p s i n t h e f o r m a t i o n o f t h e a c t i v e t r a n s c r i p t i o n c o m p l e x ( 9 4 , 9 5 , 1 6 2 , 1 6 3 ) . 1 7 5 . One o f t h e f a c t o r s , T F I I I A , i n v o l v e d i n t h e s t a b l e c o m p l e x f o r m a t i o n h a s b e e n i d e n t i f i e d ( 9 1 , 9 2 , 9 4 , 1 6 4 , 9 0 - 9 2 ) . A t l e a s t two o t h e r c o m p o n e n t s a r e n e c e s s a r y f o r a c c u r a t e t r a n s c r i p t i o n o f 5S DNA ( 5 9 , ' 6 0 ) . E x p e r i m e n t s on c o m p l e x s t a b i l i t y i n v o l v i n g t h e d e l e t i o n m u t a n t , p D t 0 . 3 A 51-61 ( F i g s . 4 0 A , B and 41) i n d i c a t e t h a t i t i s n o t a b l e t o f o r m a s t a b l e , e x c l u s i o n a r y c o m p l e x , a s do p l a s m i d s , p D t 0 . 3 and p D t 7 8 R . I t i s s t i l l a b l e t o c o m p e t e w i t h t r a n s c r i p t i o n o f i n t a c t tRNA g e n e s , t h o u g h p o o r l y . T h e s e r e s u l t s i m p l y a r o l e f o r t h e d e l e t e d r e g i o n (B b l o c k ) i n t h e f o r m a t i o n o f t h e s t a b l e c o m p l e x . The a r g u m e n t u s e d t o e x p l a i n t h e l o w l e v e l o f t r a n s c r i p t i o n d i r e c t e d by t h i s m u t a n t gene ( s e e s e c t i o n I I . C . 3 ) c a n be e m p l o y e d h e r e t o e x p l a i n t h e a b o v e o b s e r v a t i o n s . The d e l e t i o n m u t a n t i s a b l e t o b i n d common t r a n s c r i p t i o n f a c t o r ( s ) a t t h e d e l e t e d s i t e a t a l o w l e v e l . T h i s e x p l a i n s t h e l o w l e v e l o f c o m p e t i t i o n o f f e r e d by t h e m u t a n t g e n e . T h i s b i n d i n g i s n o t s t a b l e e n o u g h t o c o m p l e t e l y e x c l u d e t h e t r a n s c r i p t i o n o f i n t a c t g e n e s . I f t h e f o r m a t i o n o f t h e e x c l u s i o n a r y c o m p l e x i s t h e p r i m a r y i n t e r a c t i o n r e q u i r e d f o r t r a n s c r i p t i o n [ a s i s s u g g e s t e d by t h e e x p e r i m e n t s u s i n g p D t 0 . 3 a s t h e f i r s t DNA ( F i g . 3 2 B ) ] , t h e n t h e s e e x p e r i m e n t s i n d i c a t e t h e b i n d i n g o f a t r a n s c r i p t i o n f a c t o r t o t h e B b l o c k s e q u e n c e s a s t h e p r i m a r y e v e n t w h i c h p r o m o t e s o t h e r s t e p s o f t r a n s c r i p t i o n i n i t i a t i o n . T h i s o b s e r v a t i o n a g r e e s w i t h t h e p r o p o s a l made by H a l l e t a]_. ( 1 0 8 ) . S t a b l e t r a n s c r i p t i o n c o m p l e x e s h a v e b e e n i m p l i c a t e d i n a m e c h a n i s m f o r r e g u l a t i o n o f t r a n s c r i p t i o n o f o o c y t e and s o m a t i c v a r i a n t s o f 5S RNA g e n e s d u r i n g d i f f e r e n t i a t i o n ( 9 3 - 9 5 ) . I t i s n o t known i f c o m p l e x s t a b i l i t y p l a y s a s i m i l a r r o l e i n r e g u l a t i n g t r a n s c r i p t i o n o f v a r i a n t tRNA g e n e s . 1 7 6 , B i b i i o g r a p h y 1 . C r i c k , F . H . C . ( 1 9 5 7 ) B i o c h e m . S o c . Symp. j _ 4 , 2 5 . 2 . H o a g l a n d , M . B . , S t e p h e n s o n , M . L . , S c o t t , J . F . , H e c h t , L . and Z a m e c n i k , P . C . ( 1 9 5 8 ) J . B i o l . Chem. 2 3 1 , 2 4 1 - 2 5 7 . 3 . R i c h , A . and R a j B h a n d a r y , U . L . ( 1 9 7 6 ) A n n . R e v . B i o c h e m . 4 5 , 8 0 5 - 8 6 0 . 4 . A l t m a n , S . e d . ( 1 9 7 8 ) T r a n s f e r RNA, MIT P r e s s , C a m b r i d g e , M a s s . , and L o n d o n . 5 . M a z z a r a , G . P . and M c C l a i n , W . H . ( 1 9 8 0 ) i n T r a n s f e r RNA: B i o l o g i c a l A s p e c t s ( S o i l , D . , A b e l s o n , J . N . and S c h i m m e l , P . 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