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UBC Theses and Dissertations

Modualtion [sic] of transcription by sequences contained in the 5’-flanking region of a Drosophila melanogaster… Sajjadi, Fereydoun G. 1985

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LA M O D U A L T I O N O F T R A N S C R I P T I O N BY S E Q U E N C E S C O N T A I N E D I N T H E 5 ' - F L A N K I N G R E G I O N O F A 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 ^ 1 G E N E by F E R E Y D O U N G . B . A . , U n i v e r s i t y o f S A J J A D I N o r t h C a r o l i n a , 1 9 8 2 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F M A S T E R O F S C I E N C E i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( T h e G e n e t i c s P r o g r a m m e ) 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 T H E 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 A u g u s t 1 9 8 5 ® F e r e y d o u n G . S a j j a d i , 1 9 8 5 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 The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date ( i i ) A B S T R A C T T r a n s f e r RNA g e n e s r e q u i r e " p o s i t i v e " 5 ' - f l a n k i n g s e q u e n c e s t o d i r e c t e f f i c i e n t t r a n s c r i p t i o n . I n o r d e r t o d e l i m i t t h e m o d u l a t o r y s e q u e n c e s p r e s e n t i n t h e 5 ' - f l a n k o f V a l a D r o s o p h i l a t R N A ^ g e n e , a n e x t e n s i v e s e r i e s o f d e l e t i o n m u t a n t s w a s c o n s t r u c t e d a n d e n d - p o i n t s d e t e r m i n e d b y d i -d e o x y s e q u e n c i n g . T h e m u t a n t s w e r e t r a n s c r i b e d jLn v i t r o i n 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 - f r e e e x t r a c t . T w e n t y n u c l e o t i d e s o f t h e 5 ' - f l a n k i m m e d i a t e l y a d j a c e n t t o t h e m a t u r e t R N A c o d i n g s e q u e n c e w e 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 . N e g a t i v e m o d u l a t o r y s e q u e n c e s w e r e c o n t a i n e d b e t w e e n p o s i t i o n s - 2 0 t o - 3 0 a n d - 4 5 t o - 7 0 r e l a t i v e t o t h e m a t u r e c o d i n g s e q u e n c e . T h e - 4 5 t o - 7 0 s e q u e n c e s h a r e s h o m o l o g y w i t h i n h i b i t o r y s e q u e n c e s p r e v i o u s l y d e s c r i b e d i n t h e 5 ' - f l a n k o f t R N A g e n e s , e x c e p t t h a t t h i s s e q u e n c e w a s s i g n i f i c a n t l y l a r g e r i n l e n g t h . S e q u e n c e s c o n t a i n e d b e -t w e e n p o s i t i o n s - 3 8 a n d - 4 5 a c t a s p o s i t i v e m o d u l a t o r y s e q u e n c e s w h i c h e n h a n c e t h e l e v e l o f t r a n s c r i p t i o n . I n a d d i t i o n , a T r a n s c r i p t i o n M o d u l a t i o n E l e m e n t ( T M E ) w a s i d e n t i f i e d b e t w e e n n u c l e o t i d e s - 3 3 a n d - 3 8 . T h e T M E w a s a l s o f o u n d 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 v a r i o u s o t h e r t R N A g e n e s a n d p r e l i m i n a r y d a t a s u g g e s t s t h a t i t e n h a n c e s t r a n s c r i p t i o n e f f i c i e n c y t h r o u g h i t s p o s i t i o n r e l a t i v e t o t h e D a n d T c o n t r o l r e g i o n s ( i i i ) T A B L E O F C O N T E N T S 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 i i L i s t o f T a b l e s v L i s t o f F i g u r e s v i A c k n o w l e d g e m e n t s v i i i A b b r e v i a t i o n s i x I n t r o d u c t i o n 1 I C l a s s I I I g e n e s 1 I I T r a n s c r i p t i o n In v i t r o 2 I I I 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 5S r R N A a n d t R N A g e n e s 3 I V M o d u l a t o r y s e q u e n c e s c o n t a i n e d 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 t R N A g e n e s 7 , M a t e r i a l s a n d M e t h o d s 11 A . L a r g e - s c a l e i s o l a t i o n o f r e c o m b i n a n t DNA 11 i ) P l a s m i d p D t 5 5 - 0 . 3 ; h o s t E . c o l i S F 8 11 i i ) P l a s m i d p U C 8 ; h o s t E . c o l i J M 8 3 12 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 d o n u c l e a s e s 14 C . A g a r o s e a n 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 14 D . I s o l a t i o n o f DNA f r o m a g a r o s e g e l s 15 E . I s o l a t i o n o f DNA f r o m p o l y a c r y l a m i d e g e l s 16 F . T r e a t m e n t o f DNA w i t h e x o n u c l e a s e B A L - 3 1 16 G . E n d - l a b e l l i n g a n d f i l l - i n r e a c t i o n s o f DNA 17 H . A u t o r a d i o g r a p h y 18 I . L i g a t i o n o f DNA i n t o p U C 8 18 J . T r a n s f o r m a t i o n o f E . c o l i s t r a i n J M 8 3 a n d s e l e c t i o n o f r e c o m b i n a n t s c a r r y i n g c l o n e d f r a g m e n t s 19 K . S m a l l - s c a l e i s o l a t i o n o f p l a s m i d DNA 19 ( i v ) P a g e L . P r e p a r a t i o n o f p l a s m i d DNA f o r s e q u e n c e a n a l y s i s 20 M . DNA s e q u e n c i n g 21 N . L n v i t r o t r a n s c r i p t i o n a n d a n a l y s i s o f R N A p r o d u c t s 22 0 . C a l c u l a t i o n s a n d s t a t i s t i c a l a n a l y s i s 22 R e s u l t s 24 I 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 Ik I I T r a n s c r i p t i o n o f m u t a n t t D N A s i n 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 - f r e e e x t r a c t 51 D i s c u s s i o n 77 R e f e r e n c e s 98 ( v ) L I S T O F T A B L E S P a g e T a b l e I C o m p a r i s o n o f t h e e f f e c t s o f p B R 3 2 2 v s . p U C 8 o n t h e r a t e o f V a l t r a n s c r i p t i o n o f . t D N A ^ - 1 7 9 56 T a b l e I I T r a n s c r i p t i o n o f d e l e t i o n e n d - p o i n t s . . 5 8 ( v i ) L I S T O F F I G U R E S P a g e F i g u r e 1 R e s t r i c t i o n map o f p D t 5 5 - 0 . 3 a n d V a l p D t D N A u 25 F i g u r e 2 C o n s t r u c t i o n o f 5 ' - f l a n k i n g d e l e t i o n s 28 F i g u r e 3 C r e a t i o n o f B A L - 3 1 d e l e t i o n s 3 0 F i g u r e 4 S c r e e n i n g o f d e l e t i o n m u t a n t s 33 F i g u r e 5 T h e DNA s e q u e n c e o f A 5 ' - 3 7 ( A ) a n d 5 * - 3 5 ( B ) 36 F i g u r e 6 D i s t r i b u t i o n o f d e l e t i o n s f r o m A 5 ' - 4 9 a n d A 5 ' - 3 7 39 F i g u r e 7 T h e DNA s e q u e n c e o f A 5 ' - 3 3 ( A ) a n d A 5 ' - 2 6 ( B ) 42 F i g u r e 8 T h e DNA s e q u e n c e o f A 5 ' - 3 0 ( A ) a n d A 5 ' - 2 0 ( B ) 44 F i g u r e 9 D i s t r i b u t i o n o f d e l e t i o n s f r o m A 5 ' - 3 3 47 F i g u r e 10 P o s i t i o n o f d e l e t i o n e n d - p o i n t s 4 9 F i g u r e 11 E f f e c t o f t e m p l a t e DNA c o n c e n t r a t i o n o n t h e r a t e o f t r a n s c r i p t i o n f o r V a l t D N A ^ - 1 7 9 52 F i g u r e 12 D o u b l e r e c i p r o c a l p l o t o f d a t a V a l f r o m t r a n s c r i p t i o n o f t D N A ^ - 1 7 9 , A 5 ' - 4 5 a n d A 5 ' - 3 0 61 F i g u r e 13 A u t o r a d i o g r a m o f p r o d u c t s f r o m t h e V a l t r a n s c r i p t i o n o f t D N A ^ - 1 7 9 a n d A 5 ' - 4 9 63 F i g u r e 14 A u t o r a d i o g r a m o f p r o d u c t s f r o m t h e t r a n s c r i p t i o n o f A 5 ' - 4 5 a n d A 5 ' - 3 8 66 F i g u r e 15 A u t o r a d i o g r a m o f p r o d u c t s f r o m t h e t r a n s c r i p t i o n o f A 5 ' - 3 3 a n d A 5 ' - 2 5 68 F i g u r e 16 A u t o r a d i o g r a m o f p r o d u c t s f r o m t h e t r a n s c r i p t i o n o f A 5 ' - 2 0 R ' a n d A 5 ' - 2 0 R* 71 ( v i i ) P a g e F i g u r e 17 A u t o r a d i o g r a m o f p r o d u c t s f r o m t h e t r a n s c r i p t i o n o f A 5 ' - 1 7 a n d A 5 ' - 1 0 73 F i g u r e 18 C o m p a r i s o n o f i n h i b i t o r y - l i k e s e q u e n c e s . 7 9 F i g u r e 19 T N N C T a T M E ? 84 F i g u r e 20 A - R e s t o r a t i o n o f t h e T M E i n t h e 5 ' -V a l f l a n k o f t D N A . d e l e t i o n m u t a n t s 4 A 5 ' - 2 0 a n d A 5 ' - 1 7 87 B - R e s t o r a t i o n o f a T M E - l i k e s e q u e n c e i n t h e 5 ' - f l a n k o f t D N A , d e l e t i o n 4 m u t a n t s A 5 ' - 3 7 a n d A 5 ' - 3 5 " . . 8 7 ( v i i i ) A C K N O W L E D G E M E N T S I t h a n k t h e m e m b e r s o f my c o m m i t t e e : G e o r g e B . S p i e g e l m a n , R o b e r t C . M i l l e r J r . , G o r d o n M . T e n e r a n d T o m A . G r i g l i a t t i . I am g r a t e f u l t o R o b e r t C . M i l l e r J r , f o r h i s c o n t i n u e d e n c o u r a g e m e n t a n d i n d e b t e d t o G e o r g e B, S p i e g e l m a n f o r h i s h e l p , e n t h u s i a s m a n d n o n - t i r i n g p a t i e n c e . I a l s o t h a n k t h e m e m b e r s o f my l a b , p a s t a n d p r e s e n t f o r t h e i r c o l l a b o r a t i o n a n d c o m r a d e r y t h r o u g h o u t my r e s e a r c h . I f i n a l l y t h a n k N a n c y C . B a t e s f o r h e r '. t i m e s p e n t t y p i n g t h i s m a n u s c r i p t . ( i x ) A B B R E V I A T I O N S BME 2 - m e r c a p t o e t h a n o l b p b a s e p a i r s DNA d e o x y r i b o n u c l e i c a c i d D T T d i t h i o t h r e i t o l E D T A e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d E G T A e t h y l e n e g l y c o l - b i s - ( 3 - a m i n o e t h y l e n e e t h e r ) N , N ' -t e t r a a c e t i c a c i d h r s h o u r s K d K i l o d a l t o n m i n m i n u t e s m o l e c u l a r w e i g h t N a O A c s o d i u m a c e t a t e RNA r i b o n u c l e i c a c i d S D S d o d e c y l s o d i u m s u l f a t e t R N A t r a n s f e r r i b o n u c l e i c a c i d X - g a l 5 - b r o m o - 4 - c h l o r o - 3 - i n d o n y 1 B - D - g a l a c t o p y r a n o s i d e 1 I N T R O D U C T I O N I C l a s s I I I g e n e s RNA p o l y m e r a s e I I I ( P o l I I I ) i s a n e n z y m e ( ~ 7 0 0 K d ) r e s p o n s i b l e f o r t h e t r a n s c r i p t i o n o f a c l a s s o f R N A s w h i c h h a v e b e c o m e d e f i n e d a s c l a s s I I I g e n e s . T h e s t r u c t u r e a n d c h a r a c t e r i s t i c s o f P o l I I I h a v e b e e n r e v i e w e d b y R o e d e r ( 1 9 7 6 ) a n d S p i n d l e r ( 1 9 7 8 ) . O n e c l a s s o f g e n e s t r a n s c r i b e d b y P o l I I I a r e m i d d l e -r e p e t i t i v e s e q u e n c e s f r o m t h e h u m a n A l u f a m i l y . I t h a s b e e n s u g g e s t e d t h a t t h e s e s e q u e n c e s f u n c t i o n a s r e g i o n s o f DNA r e p l i c a t i o n ( H a y n e s a n d J e l i n e c k , 1 9 8 1 ) . R e c e n t l y a r e p e a t e d " b r a i n - s p e c i f i c " s m a l l RNA h a s b e e n s h o w n t o b e t r a n s c r i b e d b y P o l I I I . T h e 82 n u c l e o t i d e s e q u e n c e h a s b e c o m e t e r m e d a n I D s e q u e n c e a n d i s b e l i e v e d t o b e r e s p o n -s i b l e f o r r e g u l a t i o n o f P o l I I I t r a n s c r i p t i o n i n t h e b r a i n ( S u t c l i f f e e t a l . , 1 9 8 4 a ; b ) . O t h e r g e n e s t r a n s c r i b e d b y P o l I I I i n c l u d e t h e a d e n o v i r u s - a s s o c i a t e d ( V A ) R N A , 5S RNA a n d t R N A g e n e s . G e n e s t r a n s c r i b e d b y P o l I I I a l l s h a r e c e r t a i n c h a r a c -t e r i s t i c s . O n e o f t h e f e a t u r e s o f c l a s s I I I g e n e s i s t h e p r e s e n c e o f a c l u s t e r 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 o f t h e i r 3 ' - f l a n k i n g s e q u e n c e s . RNA p o l y m e r a s e I I I w a s s h o w n t o t e r m i n a t e t r a n s c r i p t i o n i n 5S RNA a n d t R N A g e n e s a t f o u r o r m o r e T r e s i u e s w i t h o u t t h e a d d i t i o n o f p r o t e i n f a c t o r s o t h e r t h a n t h o s 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 i n i t i a t i o n . T h e a b s e n c e o f t h e t e r m i n a t i o n s i g n a l r e s u l t s i n t h e p r o d u c t i o n o f r u n - o n t r a n s c r i p t s ( B o g e n h a g e n a n d B r o w n , 1 9 8 1 ; C o z z a r e l l i e t a l . , 1 9 8 3 ; W a t s o n e t a l . , 1 9 8 4 ) . T h e c o d i n g s e q u e n c e o f P o l I I I g e n e s i s c a p a b l e o f p r o -m o t i n g t r a n s c r i p t i o n . E u k a r y o t i c t R N A g e n e s a l l c o n t a i n t w o h i g h l y c o n s e r v e d i n t e r n a l p r o m o t e r s e q u e n c e s r e f e r r e d t o a s B o x A a n d B o x B ( T r a b o n i e t a l . , 1 9 8 2 ) . A l u - f a m i l y a n d VA RNA g e n e s a l s o c o n t a i n B o x A a n d B - l i k e s e q u e n c e s ( F o w l k e s a n d S h e n k , 1 9 8 0 ; C i l i b e r t o e t a l . , 1 9 8 3 ) . T h e 5S RNA c o d i n g s e q u e n c e s h a r e s h o m o l o g y a n d i s f u n c t i o n a l l y e q u i v a l e n t t o o n l y t h e B o x A o f t R N A g e n e s ( C i l i b e r t o e t a l . , 1 9 8 3 ) . T h e c o n s e r v e d s e q u e n c e s h a v e b e e n s h o w n t o b i n d f a c t o r s ' r e q u i r e d f o r t h e p r o m o t i o n o f t r a n s c r i p t i o n b y P o l I I I ( T a y l o r a n d S e g a l l , 1 9 8 5 ; J o h n s o n - B u r k e a n d S o i l , 1 9 8 5 ) . T r a n s c r i p t i o n i n v i t r o E a r l y t r a n s c r i p t i o n s t u d i e s s h o w e d t h a t p u r i f i e d P o l I I I i s o l a t e d f r o m X e n o p u s l a e v i s c o u l d n o t s u p p o r t s e l e c t i v e t r a n s c r i p t i o n o f c l o n e d 5S RNA g e n e s , e v e n t h o u g h P o l I I I m a i n t a i n e d p o l y m e r i z i n g a c t i v i t y o n 5S RNA o o c y t e c h r o m a t i n t e m p l a t e s ( P a r k e r a n d R o e d e r , 1 9 7 7 ) . T h i s s u g -g e s t e d t h e p o s s i b l e r e q u i r e m e n t o f a d d i t i o n a l t r a n s c r i p t i o n f a c t o r s . S o l u b l e c e l l - f r e e e x t r a c t s t o e n a b l e f a i t h f u l P o l I I I t r a n s c r i p t i o n JLJI v i t r o w e r e f i r s t d e v e l o p e d f o r V A RNA u s i n g h u m a n KB c e l l s ( H a r r i s a n d R o e d e r , 1 9 7 8 ; W e i l e t a l . , 1 9 7 9 ; W u , 1 9 8 0 ) a n d H e L a c e l l s ( G u i l f o y l e a n d W e i n m a n n , 1 9 8 1 ) . T h e s e c e l l - f r e e e x t r a c t s w e r e f o u n d t o a l s o t r a n s -c r i b e c e r t a i n t r a n s c r i p t i o n a l u n i t s o f a b o u t 3 0 0 b p w h i c h b e c a m e d e f i n e d a s t h e A l u - f a m i l y o f s e q u e n c e s ( D u n c a n e t 3 a l . , 1979; JeXinek et a l . , 1980). Subsequently other c e l l - f r e e t r a n s c r i p t i o n systems from S-100 e x t r a c t s have been used to f a i t h f u l l y t r a n s c r i b e VA, 5S RNA and tRNA genes. A l s o , i t has been shown that heterologous e x t r a c t s have decreased s t r i n g e n c y i n d i r e c t -ing tRNA t r a n s c r i p t i o n ±n_ v i t r o . The s t r i n g e n c y v a r i e s between d i f f e r e n t organisms, s i n c e c e r t a i n e x t r a c t s such as HeLa and Xenopus are l e s s s e l e c t i v e i n d i r e c t i n g t r a n s c r i p -t i o n of D r o s o p h i l a heterologous tRNA genes than are yeast e x t r a c t s (Schaack et a l . , 1984; Schaack and So'll, 1985). Most t r a n s c r i p t i o n systems s t u d i e d have shown m u l t i p l e rounds of t r a n s c r i p t i o n i n i t i a t i o n . I l l T r a n s c r i p t i o n c o n t r o l regions f o r 5S rRNA and tRNA genes A promoter i s d e f i n e d as the sequence of the DNA r e q u i r e d to d i r e c t b i n d i n g of polymerase to i n i t i a t e t r a n s -c r i p t i o n . T e l f o r d et a l . (1979) were able to d e l i m i t the Met promoter r e g i o n f o r a tRNA ^ 'gene to w i t h i n 22 bp of the 5 ' - f l a n k by i n j e c t i o n i n t o Xenopus o o c yjte s . Xenopus oocyte was used to t r a n s c r i b e a s e r i e s of 5' and 3' d e l e -t i o n s of a 120 n u c l e o t i d e Xenopus b o r e a l i s 5S rRNA gene. The aim was to d e l i m i t the c o n t r o l r e g i o n s f o r i n v i t r o RNA s y n t h e s i s (Sakonju et a l . , 1980; Bogenhagen et a l . , 1980). I t was found that the 5 ' - f l a n k i n g sequences and up to 50 n u c l e o t i d e s of the gene could be d e l e t e d before a f f e c t i n g t r a n s c r i p t i o n . The 3' boundary f o r the c o n t r o l r e g i o n of the 5S rRNA gene was found to be at approximately +83 r e l a t i v e to the i n i t i a t i o n point of the gene (+1). 4 Thus the c o n t r o l r e g 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 was defined by p o s i t i o n s +50 and +83. A l s o , the p o s i t i o n of the c o n t r o l r e g i o n w i t h i n the 5S RNA gene along with the immediate 5 ' - f l a n k i n g sequences were found to s p e c i f y the s i t e of i n i t i a t i o n , some 50 bp upstream of the i n t e r n a l c o n t r o l r egion (Sakonju et a l . , 1980; Bogenhagen et a l . , 1980). Though the t r a n s c r i p t i o n of tRNA genes was found to have a g r e a t e r requirement f o r the 5 ' - f l a n k i n g sequences than f o r 5S RNA genes, two i n t e r n a l conserved sequences (Box A and Box B) were i d e n t i f i e d w i t h i n tRNA genes ( C i l i -berto et a l . , 1983; Stewart et a l . , 1985; Murphy and Bar-a l l e , 1984). The c o n t r o l regions had been p r e v i o u s l y shown to be r e s p o n s i b l e f o r d i r e c t i n g t r a n s c r i p t i o n of a Met Xenopus l a e v i s tRNA ^ gene ( H o f s t e t t e r et a l . , 1981). Since i t was observed that the 5 ' - f l a n k i n g sequences of s e v e r a l c l a s s I I I genes rev e a l e d very l i t t l e or no ap-parent sequence homology, i t seemed l i k e l y that these sequences played a major r o l e i n promoter r e c o g n i t i o n . Therefore the n o t i o n that c l a s s I I I promoters were i n t e r n a l became strengthened ( H o f s t e t t e r et a l . , 1981). C i l i b e r t o et a l . (1983) have shown that 5S RNA and tRNA genes f a l l i n t o two d i f f e r e n t c l a s s e s . F i r s t they showed that the 34 bp i n t e r n a l c o n t r o l r e g i o n (ICR) of the somatic 5S RNA gene from Xenopus b o r e a l i s c o n s i s t s of two separate components and can be s p l i t by i n s e r t i o n . o f nucleo-t i d e s to produce a t r a n s c r i p t i o n a l l y a c t i v e maxigene. Second, the f i r s t 11 bp of the ICR were shown to be homo-5 Pro logous and f u n c t i o n a l l y e q u i v a l e n t to the box A of a tRNA gene, s i n c e h y b r i d genes c o n s t r u c t e d from the 5S and tRNA genes were e f f i c i e n t l y t r a n s c r i b e d i n Xenopus l a e v i s oocytes However, no Box B consensus sequence i s found i n 5S RNA genes. In a d d i t i o n tRNA and 5S RNA genes i n i t i a t e t r a n s -c r i p t i o n at very d i f f e r e n t d i s t a n c e s from t h e i r mature cod-ing sequences. F i n a l l y , 5S RNA genes bind a s p e c i f i c t r a n -s c r ipitL on f a c t o r that does not i n t e r a c t with tRNA genes ( S a -konju et a l . , 1981). The l i m i t s of the i n t e r n a l c o n t r o l regions i n tRNA genes have been more c l e a r l y d e f i n e d f o r a D r o s o p h i l a A r 2 tRNA £ gene (pArg) (Sharp et a l . , 1981). One promoter element was d e f i n e d by p o s i t i o n s 8-25 and was r e f e r r e d to as the D - c o n t r o l or Box A (Sharp et a l . , 1982). P o s i t i o n s 50-58 encode the T - c o n t r o l or Box B i n t e r n a l promoter. In a d d i t i o n , the 5 ' - h a l f of the tRNA^ r^ genes c o n t a i n i n g the D - c o n t r o l r e g i o n alone was found to d i r e c t RNA s y n t h e s i s i n Kc c e l l and Xenopus oocyte e x t r a c t s . D e l e t i o n of the 5 ' - f l a n k i n g sequences and D - c o n t r o l r e g i o n a b o l i s h t r a n s -c r i p t i o n i n Kc c e l l e x t r a c t , but not i n Xenopus oocyte e x t r a c t (Sharp et a l . , 1983). The d i s t a n c e s e p a r a t i n g the D and T c o n t r o l regions i n pArg could be i n c r e a s e d between 12 and 77 n u c l e o t i d e s without decreasing the e f f i -c i ency of t r a n s c r i p t i o n or the a b i l i t y to compete i n the b i n d i n g of t r a n s c r i p t i o n f a c t o r s i n D r o s o p h i l a Kc c e l l ex-t r a c t (Dingermann et a l . , 1983). The d i s t a n c e between the D and T c o n t r o l regions i s v a r i a b l e among n a t u r a l l y occur-r i n g tRNA genes. Some tRNAs c o n t a i n i n t e r v e n i n g sequences and i t i s not unusual to f i n d c o n s i d e r a b l e v a r i a t i o n i n the lengths of t h e i r e x t r a arms (S t a n d r i n g , et a l . , 1981; B a l d i , et a l . , 1983) . An experiment i n c r e a s i n g the d i s t a n c e between the two Pro c o n t r o l r e g ions of a C. elegans tRNA gene found that a d i s t a n c e of 40 to 50 n u c l e o t i d e s allows f o r optimal t r a n s -c r i p t i o n . In a d d i t i o n , a h y b r i d tRNA gene c o n t a i n i n g L G u Pro c o n t r o l r e g ions from a tRNA and tRNA genes was found to t r a n s c r i b e e f f i c i e n t l y i n Xenopus oocytes, showing that at l e a s t i n t h i s case the c o n t r o l regions are independent t r a n s c r i p t i o n u n i t s ( C i l i b e r t o , et a l . , 1982). A number of s t u d i e s have focused on the e f f e c t s of changes i n the conserved i n t e r n a l c o n t r o l regions of tRNA genes. Using s i t e - d i r e c t e d ±n_ v i t r o mutagenesis, s i n g l e and double poin t mutations were created i n the i n -v a r i a n t n u c l e o t i d e s of c e r t a i n tRNA genes which r e s u l t e d i n a decrease i n t r a n s c r i p t i o n . Such e f f e c t s f u r t h e r strengthened the n o t i o n that promoters of tRNA genes were i n t e r n a l . Although these r e s i d u e s are i m p l i c a t e d i n f a c t o r b i n d i n g , there have been i n c o n s i s t e n c i e s i n the e f f e c t s of n u c l e o t i d e changes between d i f f e r e n t tRNA genes. It would appear then that s u b s t i t u t e d bases i n the i n t e r n a l c o n t r o l r e g i o n a f f e c t t r a n s c r i p t i o n d i f f e r e n t l y i n d i f -f e r e n t t r a n s c r i p t i o n systems (Stewart et a l . , 1985). The r o l e of Pol I I I t r a n s c r i p t i o n complexes f o r 5S RNA arid tRNA genes has been reviewed by Brown (1984) , Lassar, et 6a1 a l . (1985) and s t u d i e d i n d e t a i l by Andrews et a l . (1984) and Lassar et a l . (1985). Some of the f a c t o r s that are r e q u i r e d f o r Pol I I I t r a n s c r i p t i o n have been analysed by f r a c t i o n a t i o n of crude c e l l u l a r e x t r a c t s ( S e g a l l et a l . , 1980v';; Shastry et a l . , 1982; Klekamp and Weil, 1982; John-son-Burke, 1983). The f i r s t t r a n s c r i p t i o n f a c t o r p u r i f i e d and shown to bind to the i n t e r n a l c o n t r o l r e g i o n of 5S RNA genes was a p o l y p e p t i d e of approximately 40 Kd (TF IIIA) (Engelke et a l . , 1980) . T r a n s c r i p t i o n - c o m p e t i t i o n assays were developed i n order to measure the a b i l i t y of i n c r e a s i n g amounts of one gene to i n h i b i t t r a n s c r i p t i o n of a second gene. Worming-ton et a l . (1981) found that the 40 Kd p r o t e i n which i s a p o s i t i v e t r a n s c r i p t i o n . f a c t o r a c t s as a l i m i t i n g component i n the competition assay. Furthermore, d e l e t i o n of the 5 ' - f l a n k i n g r e g i o n reduced the competitive s t r e n g t h of somatic 5S DNA, but did not a f f e c t oocyte 5S DNA i n oocyte n u c l e a r e x t r a c t . Dirigermann et a l . (1983) proposed a model i n which two t r a n s c r i p t i o n f a c t o r s , 6 and x i n t e r a c t with the D and T c o n t r o l r e g i ons r e s p e c t i v e l y and r e s u l t i n template a c t i -v a t i o n by c o o p e r a t i v e mechanism. In t h i s model, the 6 f a c t o r binds to the D - c o n t r o l and the T f a c t o r binds to the T - c o n t r o l to b r i n g about s t a b l e complex formation. Sepa-r a t i o n of the two c o n t r o l regions i n t e r f e r e s with cooper-a t i v e s t a b l e complex formation as measured by competition experiments. T r a n s c r i p t i o n competition assays using 5' and 3' d e l e t i o n d e r i v a t i v e s of a tRNA"^^ gene (pArg) showed 7 that the presence of both the D and T c o n t r o l regions i s necessary f o r maximum com p e t i t i v e s t r e n g t h and that both the 5' and 3 ' - f l a n k i n g sequences c o n t r i b u t e to the competi-t i v e a b i l i t y of the D and T c o n t r o l regions r e s p e c t i v e l y (Sharp et a l . , 1983; Schaack et a l . , 1983). Two t r a n s c r i p t i o n f a c t o r s designated TF IIIB or Fac-tor B ( 6 ) , M r 260 Kd, and TF IIIC or Factor C ( T ) , Mr 300 Kd ; are r e q u i r e d f o r the t r a n s c r i p t i o n of tRNA genes ( S t i l l -man et a l . , 1984;Ruetet a l . , 1984; Johnson-Burke and So'll, 1985). However, i n a d d i t i o n to TF IIIB and TF I I I C , 5S RNA genes r e q u i r e a t h i r d t r a n s c r i p t i o n f a c t o r TF IIIA ( G o t t e s f e l d and Bloomer, 1982; Hanas et a l . , 1984; Smith et a l . , 1984; Bogenhagen et a l . , 1985). To promote the formation of the most s t a b l e complex p r i o r to t r a n s c r i p t i o n by P o l I I I , the bindi n g order of t r a n s c r i p t i o n f a c t o r s i n 5S RNA genes i s b e l i e v e d to be TF IIIA, TF IIIC and TF IIIB (Setzer and Brown, 1985). The order of bindi n g of f a c t o r s i n tRNA genes i s b e l i e v e d to be TF IIIC f o l l o w e d by TF IIIB, I n t e r e s t i n g l y , s t a b l e complex formation has been shown to be i n f l u e n c e d by the 5 ' - f l a n k i n g sequences i n both tRNA and 5S RNA genes (Johnson-Burke and S o l i , 1985; T a y l o r and Se-g a l l , 1985). IV Modulatory sequences contained i n the 5 ' - f l a n k i n g sequences  of tRNA genes I t i s w e l l e s t a b l i s h e d that 5 ' - f l a n k i n g sequences of tRNA genes modulate t r a n s c r i p t i o n . The study of tRNA gene t r a n s c r i p t i o n r e v e a l e d a gr e a t e r requirement f o r the 5'-f l a n k i n g s e q u e n c e s t h a n f o r 5S r R N A g e n e s ( D e F r a n c o e t a l . , A l a 1 9 8 0 ) . A c l o n e d B o m b y x m o r i t R N A ^ g e n e l a c k i n g a l l b u t 11 n u c l e o t i d e s o f i t s 5 ' - f l a n k w a s u n a b l e t o d i r e c t e f f i -c i e n t t r a n s c r i p t i o n i n B o m b y x e x t r a c t , b u t w a s t r a n s c r i b e d i n X e n o p u s e x t r a c t ( S p r a g u e e t a l . , 1 9 8 0 ) . I t w a s l a t e r A l a s h o w 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 t R N A g e n e b e t w e e n p o s i t i o n s - 3 7 a n d - 1 6 r e l a t i v e t o t h e m a t u r e c o d i n g s e q u e n c e w e r e r e q u i r e d f o r e f f i c i e n t t r a n s c r i p t i o n i n a h o m o l o g o u s e x t r a c t ( L a r s o n e t a l . , 1 9 8 3 ) . T h e 5 ' - f l a n k i n g s e q u e n c e r e q u i r e m e n t s h a v e a l s o b e e n P r o s h o w n f o r a C . e l e g a n s t R N A g e n e ( C i l i b e r t o e t a l . , 1 9 8 2 a ) T v r a n d a y e a s t t R N A J g e n e ( S h a w a n d O l s o n , 1 9 8 4 ) . I n f a c t , i t h a s b e e n s h o w n t h a t t h e 5 ' - f l a n k i n g r e g i o n o*f a y e a s t L G u t R N A ^ g e n e i s s u f f f i c i e n t t o c o n s t i t u t e a P o l I I I p r o m o t e r w i t h e i t h e r t h e A o r B B o x s e q u e n c e s i n 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 ( J o h n s o n a n d R a y m o n d , 1 9 8 4 ) . A d e l e t i o n o f L 6 TJ. t h e 5 ' - f l a n k i n g s e q u e n c e o f t h i s y e a s t t R N A ^ g e n e t o p o s i -t i o n - 2 w a s s t i l l a c t i v e i n X e n o p u s e x t r a c t s , b u t n e a r l y i n e r t i n a h o m o l o g o u s e x t r a c t ( R a y m o n d a n d J o h n s o n , 1 9 8 3 ) . A t e r m i n a t i o n s e q u e n c e , . a s p a r t o f a n u n d e c a n u c l e o t i d e w a s f o u n d t o b e r e s p o n s i b l e : £ ~ o ; r r e d u c e d t e m p l a t e a c t i v i t y i n D r o s o p h i l a t R N A ^ s g e n e s w h e n p r e s e n t i n t h e 5 ' - f l a n k i n g r e g i o n . T h e i n h i b i t o r y e f f e c t s o f t h i s s e q u e n c e w e r e r e d u c e d b y i t s p o s i t i o n i n g a w a y f r o m t h e m a t u r e c o d i n g s e -q u e n c e a n d i n h i b i t i o n r e m o v e d b y i t s d e l e t i o n f r o m t h e 5 ' -f l a n k ( D e F r a n c o e t a l . , 1 9 8 1 ) . S i m i l a r s e q u e n c e s h a v e b e e n A r g f o u n d i n t h e 5 ' - f l a n k i n g r e g i o n o f a D r o s p h i l a t R N A 6 g e n e V a l ( D i n g e r m a n n e t a l . , 1 9 8 2 ) a n d a D r o s o p h i l a t R N A ^ g e n e reported i n t h i s study. The only other i n h i b i t o r y sequence found has been a 12 bp 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 sequence Met i n the 5 ' - f l a n k i n g r e g i o n of two tRNA genes from Xenopus  l a e v i s ( Hipskind and C l a r k s o n , 1983). The most ex t e n s i v e d e l e t i o n study of the 5' and 3' f l a n k i n g sequences of' a Pol I I I gene have been f o r a Droso-p h i l a tRNA A^ 8 gene (pArg) by Schaack et a l . (1984). Dele-t i o n of the 5 ' - f l a n k i n g sequences to p o s i t i o n -36 r e s u l t e d i n a higher l e v e l of t r a n s c r i p t i o n than f o r pArg. The i n -creased e f f i c i e n c y of t r a n s c r i p t i o n was a b o l i s h e d by d e l e -t i o n to p o s i t i o n -33 and t r a n s c r i p t i o n e f f i c i e n c y was only 12% of pArg f o r d e l e t i o n -32 (Schaack et a l . , 1985). In a d d i t i o n , the K value f o r d e l e t i o n s between p o s i t i o n s -41 m and -33 was found to be i n c r e a s e d r e l a t i v e to pArg. Fur-ther d e l e t i o n of the 5'-flank to p o s i t i o n -17 r e s u l t e d i n a gradual l o s s 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 , with d e l e t i o n -11 t r a n s c r i b i n g at l e s s than 1% of pArg. As a r e s u l t , the 5':- f l a n kL :ng modulatory.sequences of pArg were d e l i m i t e d by p o s i t i o n s -60, -33 and -11 (Schaack et a l . , 1984). Although negative modulatory sequences have been i d e n t i f i e d i n the 5 ' - f l a n k s of tRNA genes, no conserved or unique p o s i t i v e 5 ' - f l a n k i n g sequence has so f a r been i d e n t -i f i e d . The purpose of t h i s study was to f u r t h e r i n v e s t i g a t e the e f f e c t s of 5 ' - f l a n k i n g sequences on t r a n s c r i p t i o n modu-l a t i o n and to i d e n t i f y any conserved sequences that may be Val present i n an a c t i v e D r o s o p h i l a tRNA ^ gene. As a r e s u l t of t h i s work, I have d e l i m i t e d an e x t e n s i v e i n h i b i t o r y sequence 45 n u c l e o t i d e s away from the mature coding sequence 10 as w e l l as a sequence TNNCT (N r e f e r r i n g to any n u c l e o t i d e ) l o c a t e d between p o s i t i o n s -30 and -40 of t h i s and other tRNA genes. The p e n t a n u c l e o t i d e appears to f u n c t i o n as a p o s i t i v e modulatory element depending upon i t s p o s i t i o n r e l a t i v e to the D and T c o n t r o l r e g i o n s . 11 MATERIALS AND METHODS A. L a r g e - s c a l e i s o l a t i o n of recombinant DNA i ) Plasmid pDt55-0.3; host E. C o l i SF8 A loop of g l y c e r o l c u l t u r e was used to i n n o c u l a t e 2.5 mis of L u r i a broth supplemented with 50 yg/ml ampi-c i l l i n . The c u l t u r e was grown overnight at 37° C with vigorous shaking. The overnight c u l t u r e was used to i n -noculate 250 mis of M9 s a l t s media ( C l e w e l l and H e l i n s k i , 1972) supplemented with 0.002% u r i d i n e , 0.005% thymidine and 0.001% thiamine i n a 1L f l a s k . The c u l t u r e was shaken v i g o r o u s l y at 30° C u n t i l the OD 6 0 0 of the c u l t u r e was approximately 0.6, at which poin t chloramphenicol (80 mg/ml i n 95% ethanol) was added to a f i n a l c o n c e n t r a t i o n of 200 yg/ml. The i n c u b a t i o n of c u l t u r e continued f o r an a d d i t i o n a l 14-18 hrs i n order to amplify plasmid copy num-ber. B a c t e r i a were harvested by c e n t r i f u g a t i o n at 10,000x g f o r 12 min. at 4° C i n a So r v a l GSA r o t o r . The b a c t e r i a l p e l l e t was resuspended i n 1.25 mis of i c e - c o l d 25% sucrose, 0.05 M T r i s - C l [pH 8.0] and the c e l l suspension t r a n s f e r r e d to 10 ml polycarbonate tubes. Next, '2.5 mis of 0.5 M EDTA were added and a f t e r a 15 min. i n c u b a t i o n on i c e , 0.25 mis of a f r e s h l y prepared s o l u t i o n of egg white lysozyme at 5 mg/ml i n 25% sucrose, 0.05 M T r i s - C l [pH 8.0] (Sigma) were added. The suspension was mixed by i n v e r t i n g the tube s e v e r a l times and incubated on i c e f o r 25 min.. Bac-t e r i a l c e l l s were l y s e d by the r a p i d a d d i t i o n of 1.8 mis of 2% T r i t o n X-100 and f u r t h e r i n c u b a t i o n on i c e f o r 10 12 min. The l y s a t e was c e n t r i f u g e d i n a Beckman 70.1 T i r o t o r at 65,000 x g f o r 1 hr. at 4° C and the supernatant was t r a n s f e r r e d to a 10 ml graduated c y l i n d e r . For every ml of l y s a t e , 1.13 gms of cesium c h l o r i d e were added and the s a l t d i s s o l v e d by ge n t l e a g i t a t i o n . Ethidium bromide (2 mg/ ml'.- i n dH 20) was then added at 0.5 ml f o r every 7 mis of cesium c h l o r i d e s o l u t i o n and the plasmid DNA c e n t r i f u g e d to e q u i l i b r i u m i n a Beckman 70.1 T i r o t o r at 109,000xg, at 4° C f o r 40 hrs. The s u p e r c o i l e d plasmid DNA band was removed, e x t r a c t e d 5 times with two volumes of dH 20-satur-ated n-butanol and d i a l y z e d overnight :against 4L of 10 mM T r i s - C l [pH 7.4], 1 mM EDTA and 10 mM NaCl, at 4° C. F o l -lowing the dete r m i n a t i o n of plasmid c o n c e n t r a t i o n at 0 D 2 6 0 (1 OD 2 6 0=50 yg/ml of DNA), 1 y'g of DNA was run on a 0.7% agarose g e l to assess the degree of s u p e r h e l i c i t y . i i ) Plasmid pUC 8; host E. c o l i JM83 A l o o p f u l of g l y c e r o l c u l t u r e was used to i n n o c u l a t e 5 mis of 2YT medium ( M a n i a t i s et a l . , 1982) supplemented with 50 yg/ml a m p i c i l l i n (2YT/amp) and the c u l t u r e was grown overnight as p r e v i o u s l y d e s c r i b e d ; 0.4 mis of the overnight c u l t u r e was used to i n n o c u l a t e 25 mis of 2YT/amp i n a 100 ml f l a s k and shaken at 37° C u n t i l the c u l t u r e reached l a t e l og phase ( 0 D 6 0 0 ~ 0 . 6 ) ; a l l of t h i s c u l t u r e was i n turn used to i n n o c u l a t e 500 mis of 2YT/amp i n a 2L f l a s k which was incubated at 37° C with vigorous shaking u n t i l the 0D 6oo of the c u l t u r e was ~0.4, at which poin t 2.5 mis of chloramphenicol (34 mg/ml i n ethanol) was added to a f i n a l c o n c e n t r a t i o n of 170 yg/ml. -Incubation was continued with vigorous shaking f o r a f u r t h e r 14-18 h r s . The a l k a l i l y s i s procedure d e s c r i b e d by M a n i a t i s et a l . (1982) was used to i s o l a t e DNA, with the 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 were harvested i n a S o r v a l GSA r o t o r at 10,000 x g, 4° C, f o r 12 min. Treatment with the l y s o -zyme and subsequent l y s i s were c a r r r i e d out as d e s c r i b e d , except that the c e l l s were maintained i n 250 ml p l a s t i c b o t t l e s . The l y s a t e was c e n t r i f u g e d i n a S o r v a l GSA r o t o r at 4 ° C , 13,000xg f o r 20 min. and the c l e a r e d l y s a t e t r a n s f e r r e d to two 50 ml polycarbonate tubes. F o l l o w i n g p r e c i p i t a t i o n with i s o p r o p a n o l , the DNA was r e -covered by c e n t r i f u g a t i o n at 12,000 x = g i n a S o r v a l SS34 r o t o r f o r 30 min. at 23° C. The n u c l e i c a c i d p e l l e t was then washed with 5 mis of 70% ethanol and f o l l o w i n g b r i e f d e s s i c a t i o n was d i s s o l v e d i n 5.5 mis of TE (10 mM T r i s - C l , 1 mM EDTA) [pH 8.0]. Cesium c h l o r i d e and ethidium bromide were added as p r e v i o u s l y d e s c r i b e d i n S e c t i o n A - i ) and the contaminating r e s i d u a l p r o t e i n removed by a 5 min. c e n t r i f u g a t i o n i n the S o r v a l SS34 r o t o r at 6,000xg. The s o l u t i o n of DNA and cesium c h l o r i d e was t r a n s f e r r e d to 13.5 ml p o l y a l l o m e r tubes and c e n t r i f u g e d i n a Beckman 70.1 T i r o t o r as d e s c r i b e d i n S e c t i o n A - i ) . 14 D i g e s t i o n of DNA with r e s t r i c t i o n endonucleases R e s t r i c t i o n endonucleases were obtained from New England B i o l a b s and P.L. Biochemicals and were used i n accordance with the manufacturer's s p e c i f i c a t i o n s . In ge n e r a l , 10 u n i t s of r e s t r i c t i o n enzyme per yg of DNA were used and i n c u b a t i o n was f o r 2-jhrs. at 37° C (1 u n i t of enzyme a c t i v i t y i s the amount of enzyme r e q u i r e d to completely d i g e s t 1 yg of DNA i n 60 min. i n a t o t a l volume of 50 u l at 37° G). Agarose and polyacrylamide g e l e l e c t r o p h o r e s i s A l l n a t i v e g e l s contained TBE b u f f e r (0.089 M T r i s -Base; 0.089 M B o r i c A c i d ; 0.002 M EDTA); the sample run-ning dye contained 2X TBE, 50% g l y c e r o l , 0.25% xylene c y l a n o l and 0.25% bromophenol blue. E i t h e r 0.7%, 0.8% or 1% agarose g e l s (Biorad) were used to analyze s u p e r c o i l e d or r e s t r i c t e d plasmid DNA r e s p e c t i v e l y . M i n i - g e l s were cast on 5cm x 7.5cm g l a s s s l i d e s and contained 0.001% ethidium bromide ( E t B r ) . The g e l s were e l e c t r o p h o r e s e d at 60 m amp., constant c u r r e n t . Native polyacrylamide g e l s (17cm x 23cm), e i t h e r 6% or 8%, were prepared using a stock s o l u t i o n of 45% a c r y l -amide (43.5% acrylamide: 1.5% N,N'-methylene b i s a c r y l a m i d e ) TBE b u f f e r (as above), 0.05% ammonium p e r s u l f a t e (w/v) and 0.1% (v/v) TEMED (N,N*,N'-tetramethylene diamine), DNA samples were run at 200 v o l t s and s t a i n e d i n 0.1% ethidium bromide f o r 15 min. p r i o r to viewing with a UV t r a n s i l l u m -i n a t o r . 15 Denaturing polyacrylamide gels (8% polyacrylaraide, 7 M urea) were used f o r the a n a l y s i s of t r a n s c r i p t i o n products. C o n d i t i o n s f o r g e l p r e p a r a t i o n were as d e s c r i b e d above except that the TBE b u f f e r contained 0.05 M T r i s -Base, 0.05 M B o r i c Acid and 0.001 M EDTA. Samples were d i s s o l v e d i n 5 y l of dH 20 and 10 y l of running dye (8 M urea, 20 mM T r i s - C l [pH 7.5], 1 mM EDTA, 0.05% xylene c y l a n o l and bromophenol blue) were added p r i o r to heat-d e n a t u r a t i o n ot 90° C f o r 2 min.. Products were run at 600 v o l t s f o r 1.5 hrs. and tRNA bands detected by auto-radiography at -70° C. For DNA sequencing gels (20cm x 36cm), between 2 y l and 3 y l of denatured samples were loaded onto the w e l l s of e t i t h e r 8% or 10% g e l s c o n t a i n i n g 7 M urea. C o n d i t i o n s f o r the p r e p a r a t i o n of gels were as d e s c r i b e d f o r t r a n s -c r i p t i o n g e l s , except that the running dye contained 90% formamide (BRL), 25 mM EDTA and 0.05% xylene c y l a n o l and bromophenol blue; g e l s were run at 1700 v o l t s . Native p o l y a c r y l a mide, denaturing polyacrylamide and DNA sequencing gels were 1.5mm, 0.75 mm and 0.35 mm i n t h i c k n e s s , r e s p e c t i v e l y . D. I s o l a t i o n of DNA from agarose gels R e s t r i c t i o n fragments were recovered from 0%8%gagar.ose m i n i - g e l s by e l e c t r o p h o r e s i s i n t o a w e l l , which had been cut out of the g e l i n f r o n t of the l e a d i n g edge of the band and contained TBE and 40% g l y c e r o l . The DNA (200 y l -300 y l ) was e x t r a c t e d three times with dH 20-saturated 16 n - b u t a n o l , once w i t h p h e n o l / c h l o r o f o r m (24:24:1, p h e n o l , c h l o r o f o r m , i s o a m y l a l c o h o l ) and t w i c e w i t h d H 2 O - s a t u r a t e d e t h e r . A f t e r t h e a d d i t i o n of sodium a c e t a t e [pH 8.0] t o 0.25 M, t h e DNA was p r e c i p i t a t e d t w i c e w i t h two volumes of 95% e t h a n o l . I s o l a t i o n of DNA from p o l y a c r y l a m i d e g e l s F o l l o w i n g d i g e s t i o n and e l e c t r o p h o r e s i s of DNA f r a g -ments, p o l y a c r y l a m i d e g e l s were s t a i n e d by etitridium bromide and g e l s l i c e s 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 were e x c i s e d from t h e g e l . G e l s l i c e s were c u t up and p l a c e d i n t o 1.5 ml E p p e n d o r f t u b e s and c o v e r e d w i t h TE b u f f e r (10 mM T r i s - C l [pH 7 . 2 ] , 1 mM EDTA) c o n t a i n i n g 0.2 M N a C l . The t u b e s were t h e n p l a c e d a t 50° C f o r 16 h r s . and t h e e l u a t e 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 removed and p u r i f i e d on NACS PREPAC (BRL) i o n exchange m i n i - c o l u m n s . P r o c e d u r e s f o r b i n d i n g , e l u t i o n and p r e c i p -i t a t i o n of DNA 'were as d e s c r i b e d i n t h e NACS A p p l i c a t i o n s Manual ( B R L ) . T r e a t m e n t of DNA w i t h e x o n u c l e a s e BAL-31 A l l r e a c t i o n s were c a r r i e d out i n a t o t a l volume of 50 y l , a t 30° C. N u c l e a s e BAL-31 (New E n g l a n d B i o l a b s ) was used a t 1 u n i t p e r y g o f l i n e a r i z e d p l a s m i d DNA (3-5 y g o f DNA per r e a c t i o n ) under b u f f e r c o n d i t i o n s recommended by t h e m a n u f a c t u r e r . I n c u b a t i o n t i m e s were v a r i e d between 1 and 4 min. and t h e r e a c t i o n s were t e r -m i n a t e d by t h e a d d i t i o n of c o l d EGTA to a f i n a l c o n c e n -17 t r a t i o n of 0.02 M ( t o t a l volume= 25 y 1 ) . Samples were d i l u t e d three times with dH 20 and p r e c i p i t a t e d with two volumes of 95% e t h a n o l . In order to d>etermine the extent of the d e l e t i o n s , ~0.5 yg of exonucleased DNA were d i g e s t e d with Hind I I I and K l e n o w - l a b e l l e d at the 3'-end with a- 3 2PdCTP (see sec-t i o n H). The l a b e l l e d DNA was loaded d i r e c t l y onto a 6% n a t i v e p o lyacrylamide g e l with the a p p r o p r i a t e DNA s i z e c o n t r o l ( i . e . the same fragment d i g e s t e d with Hind I I I and l a b e l l e d , but not t r e a t e d with exonuclease). F o l -lowing autoradiography (~16 h r s . at 4° C), the sample c o n t a i n i n g the a p p r o p r i a t e - s i z e d DNA fragments was t r e a t e d with Klenow i n the presence of a l l four u n l a b e l l e d dNTP's. Blunt-ended fragments were e x t r a c t e d once with an equal volume (100 y l ) of phenol/chloroform, twice with dH 2 0-s a t u r a t e d ether and p r e c i p i t a t e d twice with two volumes of 95% ethanol as before, except that 5 y l of 0.04 M ATP were added to the second p r e c i p t i t a t i o n as c a r r i e r . The DNA was washed with 1 ml of 80% e t h a n o l , d r i e d and sus-pended i n an a p p r o p r i a t e volume of TE [pH 8.0], p r i o r to c l o n i n g . G. E n d - l a b e l l i n g and f i l l - i n r e a c t i o n s of DNA The Klenow fragment of E. c o l i ",DNA polymerase was used to l a b e l . o r f i l l - i n 5' extended ends generated by d i g e s t i o n of r e s t r i c t i o n endonucleases or exonuclease BAL-31. Reactions were c a r r i e d out i n a t o t a l volume of 30 y l at 25° C i n Hin b u f f e r (6.6 mM MgCl , 6.6 mM T r i s - C l [pH 7.4], 18 50 mM NaCl and 6.6 mM BME) and contained 1 y1 of each dNTP (2 mM stock) f o r u n l a b e l l e d r e a c t i o n s . E n d l a b e l -l i n g r e a c t i o n s were c a r r i e d out with 1 y l (10 yCi) of a - 3 2 P dCTP (3.16 x 10 3Ci/mmole) as w e l l as 3 u n i t s of Klenow (BRL or P.L. Bio c h e m i c a l s ) enzyme and no u n l a b e l l e d dCTP. H. Autoradiography Gels c o n t a i n i n g r a d i o a c t i v e l y l a b e l l e d DNA or RNA were covered with a l a y e r of cellophane (Saran Wrap) and autoradiographed using 3M H i L i t e X-Ray f i l m . Native and denaturing g e l s were placed at 4° C and -70° C r e s p e c t i v e l y . DNA sequencing g e l s were d r i e d onto Whatmann 3MM f i l -t e r paper using a Biorad Slab Gel D r i e r Model 1125B p r i o r to autoradiography at room temperature. Exposure time was f o r 16-20 h r s . and the X-Ray f i l m was developed a c c o r d i n g to the manufacturer's s p e c i f i -c a t i o n s I. L i g a t i o n of DNA i n t o pUC 8 The ve c t o r pUC 8 ( V i e i r a and Messing, 1982) (10 yg i n 20 y l ) was di g e s t e d with 20 u n i t s of Sma I f o r 3 hrs. at 37° C. A 1 yg a l i q u o t of DNA was e l e c t r o p h o r e s e d on a 0.7% agarose m i n i - g e l to ensure l i n e a r i z a t i o n . The DNA was suspended i n TE [pH 8.0] to a f i n a l c o n c e n t r a t i o n of 0.1 yg/y1 a f t e r the i n a c t i v a t i o n of the enzyme at 6 5 0 C f o r 5 min. L i n e a r i z e d pUC 8 DNA was stored at -20° C. L i g a t i o n r e a c t i o n s were c a r r i e d out i n a t o t a l volume of 13 y l c o n t a i n i n g 50 mM T r i s - C l [pH/7.8], 10 mM MgCl^, 15 mM d i t h i o t h r e i t o l , 1 mM spermidine, 0.5 mM ATP and 50 yg/ml BSA. The t o t a l amount of DNA i n the r e a c t i o n was 0.5 yg at a 1:1 molar r a t i o of i n s e r t to v e c t o r . Four u n i t s of T h D N A l i g a s e (New England B i o l a b s or P-L Biochemicals) and 0.2 u n i t s of T i+ RNA l i g a s e (P-L B i o -chemicals) were added to each l i g a t i o n r e a c t i o n and reac-t i o n s were incubated at 12°C-16°C ov e r n i g h t . Transformation of E . c o l i s t r a i n JM83 and s e l e c t i o n of  recombinants c a r r y i n g cloned fragments The procedure d e s c r i b e d by M a n i a t i s et a l . (1982) was used, except that c e l l s were grown i n 2YT medium and only h a l f the volume of c e l l s (50 mis) was t r e a t e d with C a C l 2 / T r i s - C l [pH 8.0]. A l l subsequent volumes were adjusted to account f o r the decreased number of c e l l s . Heat-shocked -c e l l s were incubated at 37° C f o r 1 hr. to allow e x p r e s s i o n of a m p i c i l l i n r e s i s t a n c e before p l a t i n g . F o l l o w i n g i n c u b a t i o n , 90 Ul of the transformant c e l l suspension was p l a t e d on 2YT/amp agar p l a t e s with 50 Ul of 2% X-gal (Sigma) and incubated overnight at 30° C. White c o l o n i e s c a r r y i n g recombinant plasmids were picked and grown i n 2 mis of 2YT/amp at 3 7 0 C with vigorous a e r a t i o n f o r plasmid s c r e e n i n g . Recombinants were store d at - 7 0 0 C i n v i a l s c o n t a i n i n g 4 mis of 2YT/amp, 20% g l y c e r o l ( v / v ) . S m a l l - s c a l e i s o l a t i o n of plasmid DNA The a l k a l i n e l y s i s method de s c r i b e d by M a n i a t i s et a l . (1982) was used to i s o l a t e , recombinant plasmid DNA from 2 ml 20 c u l t u r e s . F o l l o w i n g the f i n a l p r e c i p i t a t i o n with 95% eth a n o l , the dry p e l l e t was suspended i n the a p p r o p r i a t e r e s t r i c t i o n b u f f e r and di g e s t e d f o r 2.5 hrs i n a . t o t a l volume of 30 y l c o n t a i n i n g 20 yg of DNase-free RNase A (Sigma). DNase was removed from RNase by heating to 90° C fo r 10 min. L. P r e p a r a t i o n of plasmid DNA f o r sequence a n a l y s i s For s c r e e n i n g recombinants, plasmid DNA from 2 ml c u l t u r e s of c e l l s was prepared as de s c r i b e d i n the pre---ceeding s e c t i o n . However, the c l e a r e d l y s a t e (400 y l ) was p r e c i p i t a t e d with two volumes of 95% ethanol and loaded onto a NACS mini-column (BRL) i n s t e a d of e x t r a c t i o n with phenol/ chloroform (see S e c t i o n E ) . P u r i f i e d plasmid DNA that had been e l u t e d from a NACS mini-column and p r e c i p i t a t e d with ethanol was sus-pended i n 25 y l of TE [pH 8.0] and allowed to s i t at room temperature f o r 10 min. f o l l o w i n g a d d i t i o n of 5 y l of 5 N NaOH. To the denatured DNA were added 210 y l dH 20, 10 y l 2 mM T r i s - C l [pH 8.0], 50 y l M HC1, 30 y l 3 M NaOAc and 700 y l of 95% e t h a n o l . The ethanol p r e c i p i t a t e was washed with 700 y l of 80% etha n o l , d r i e d i n vacuo and suspended i n 18 y l of ~TE - [ pH 8.0]. In a few cases DNA from CsCl was used f o r sequencing, but t h i s DNA r e q u i r e d f u r t h e r p u r i f i c a t i o n on a NACS m i n i -column p r i o r to sequence a n a l y s i s . 21 M. DNA sequencing A l l dideoxy and de o x y 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 were pH adjusted (to pH 7.0) with T r i s - C l and t h e i r c o n c e n t r a t i o n s determined by spectrophotometry at ap p r o p r i a t e wavelengths as d e s c r i b e d by M a n i a t i s et a l . (1982). A l l dideoxy mixes were made as de s c r i b e d by Messing et a l . (1981). Stocks of d i d e o x y n u c l e o t i d e s (5 mM) and deoxynucleotides ;( 10 ..mM);were a l i q u o t e d , maintained at -20° C and thawed only once. Denatured DNA (18 y l ) was used f o r dideoxy sequencing as d e s c r i b e d by Sanger et a l . (1977) using both the forward and r e v e r s e primers s u p p l i e d i n s o l u t i o n (P-L B i o c h e m i c a l s ) . Reactions contained 9 y l of DNA i n T.E. b u f f e r [pH 8.0], 1.2 y l of lOx Hin b u f f e r and 2 y l of the a p p r o p r i a t e primer s o l u t i o n . The mixtures were incubated at 55° C f o r 5 min. and an a d d i t i o n a l 5 min. at room temperature, at which time 1 y l of 0.1 M DTT, 1 y l 0.015 mM dATP, 1 y l (10 yCi) of a - 3 2 P dATP (NEN, 3.16 x 10 3 Ci/mmole) and 1 y l (3 u n i t s ) Klenow (BRL) were added. The mixture was a l i q u o t e d i n t o four tubes c o n t a i n i n g 2 y l of the a p p r o p r i a t e dideoxy-n u c l e o s i d e t r i p h o s p h a t e / deoxynucleoside t r i pho sphat e i i m i x (P-L B i o c h e m i c a l s ) . The f i n a l volume was 6 y l and r e a c t i o n s were c a r r i e d out at 30° C. A f t e r i n c u b a t i o n f o r 15 min., 1 y l of c o l d chase n u c l e o t i d e mix c o n t a i n i n g 0.3 mM dATP, dTTP, dCTP, dGTP and 0.3 u n i t s of Klenow were added to each of the four r e a c t i o n tubes and i n c u b a t i o n continued f o r an a d d i t i o n a l 15 min.. Reactions were stopped by the a d d i t i o n of .10 y l of formamide mix (see S e c t i o n C) and 22 b o i l e d f o r 3 min.. In v i t r o t r a n s c r i p t i o n and a n a l y s i s of RNA products T r a n s c r i p t i o n r e a c t i o n s (50 y l ) contained 19 mM T r i s - C l [pH 7.9]; 110 mM KC1, 7 mM MgCl 2 , 3 mM DTT, 2.5 yg/ml a - a m a n i t i n (Boerhinger Mannheim), 6.5 u n i t s / m l c r e a t i n e phosphokinase, 5 mM phosphocreatine, 6.0 mM of ATP, GTP, CTP, 25 M a - 3 2 P UTP (3.5 Ci/mmole), 5 to 50 ng of template DNA, up to 1 yg of pUC 8 DNA and 25 y l of D r o s o p h i l a Schneider II S100 c e l l e x t r a c t , as d e s c r i b e d by Rajput et a l . (1982). Reactions were c a r r i e d out at 24° C f o r 90 min. and terminated by the a d d i t i o n of 60 y l Stop mix (170 yg/ml sRNA [P-L B i o c h e m i c a l s ] , 0.5% SDS, 50 mM NaOAc) and 90 y l dH 20. The mixture was e x t r a c t e d with 200 y l of phenol and the aqueous phase p r e c i p i t a t e d with two volumes of 95% ethanol and 10 y l of 0.09 M ATP. The p e l l e t was d r i e d i n vacuo and suspended i n 5 y l of dH 20 and e l e c t r o p h o r e s e d on an 8% p o l y a c r y l a m i d e g e l c o n t a i n i n g 7 M urea. F o l l o w i n g autoradiography, g e l s l i c e s c o n t a i n i n g r a d i o a c t i v e tRNA bands were excised from the g e l and counted f o r Cerenkov r a d i a t i o n i n order to q u a n t i t a t e the amount of product. C a l c u l a t i o n s and s t a t i s t i c a l a n a l y s i s Val A l l t r a n s c r i p t i o n s f o r pDt ^ and mutant : tDNAs were c a r r i e d out using 5,7,10,18,25 and 50 ng of template DNA and pUC 8 DNA to a f i n a l c o n c e n t r a t i o n of 1 y g . Tran-s c r i p t i o n s were the r e s u l t of two d i f f e r e n t S100 e x t r a c t s . 2 3 Val Val C o n t r o l t r a n s c r i p t i o n s for.pDt ^ -179 (tDNA ^ ) were i n c l u d e d i n every experimental run. Values f o r V J max and K apparent (K app) were d e r i v e d from the c a l c u l a t i o n m m and p l o t of a l i n e a r . r e g r e s s e d l i n e of a Lineweaver-Burke p l o t (1/V vs 1/S) and expressed r e l a t i v e to V m a x f o r Val tDNA ^ -179 from that experiment. F i n a l l y , the values de r i v e d from the separate . e x t r a c t s were • averaged. 24 RESULTS I C o n s t r u c t i o n of recombinant plasmids Plasmid pDt 55-0.3 (see F i g u r e 1-A) c o n t a i n s a 317 bp Val D r o s o p h i l a i n s e r t coding f o r tRNA ^ and had p r e v i o u s l y been cloned i n t o the Hind I I I s i t e of pBR322 i n both o r i e n -t a t i o n s (Rajput et a l . , 1982). This plasmid i s a d e r i v a t i v e Val of pDt 55, c o n t a i n i n g two i d e n t i c a l tRNA ^ genes (from 70 BC on the l e f t arm of chromosome 3) 525 bp apart and i n o p posite p o l a r i t y (Dunn et a l . , 1979; Addison et a l . , 1982). In t h i s study, the EcoRl/BamHl fragment of pDt 55-0.3 Val c o n t a i n i n g the tRNA ^ gene was i s o l a t e d and f o l l o w i n g d e l e t i o n mutagenesis, i n s e r t e d i n t o the v e c t o r pUC 8. Plasmid pUC 8 c o n t a i n s nine unique r e s t r i c t i o n enzyme s i t e s i n a p o l y l i n k e r sequence i n s e r t e d i n t o the coding r e g i o n f o r the amino p o r t i o n of the fragment of the 3 g a l a c t o s i d a s e gene ( V i e i r a and Messing, 1982). In a d d i t i o n , i t c o n t a i n s the o r i g i n of r e p l i c a t i o n and the 3 lactamase gene of pBR322. This c o n s t r u c t thus allows the p o s i t i v e s e l e c t i o n of inserted DNA fragments on the b a s i s of a m p i c i l l i n r e s i s t a n c e and the l a c k of c o l o r p r o d u c t i o n on i n d i c a t o r p l a t e s . In a d d i t i o n , both forward and reverse sequencing primers (complementary to e i t h e r strand) may be used f o r r a p i d dideoxy sequencing of cloned fragments. C o n s t r u c t i o n of the i n i t i a l set of 5 ' - f l a n k i n g d e l e t i o n mutants e n t a i l e d e x c i s i o n and i s o l a t i o n of the 692 bp EcoRl/BamHl fragment of pDt 55-0.3, followed by v a r i o u s lengths of i n c u b a t i o n time with nuclease BAL-31 (Gray et a l . , 1975). Due to the 3' exonuclease a c t i v i t y of BAL-31, the 25 F i g u r e 1 R e s t r i c t i o n map of pDt55-0.3 and Val pDtDNA * A- Plasmid pDt55-0.3 c o n t a i n i n g a 317 bp fragment i n s e r t e d i n t o the Hind I I I s i t e of vector pBR322 i n the 5' to 3' o r i e n t a t i o n Val and encoding tRNA ^ . The EcoRl/BamHl f r a g -ment was used to c o n s t r u c t BAL-31 d e l e t i o n s which were recloned i n t o the Sma I s i t e of v e c t o r pUC8. _ p B R sequences D r o s o p h i l a sequences M M gene B- F i g u r e showing the c o n s t r u c t of a t y p i c a l V a l A5' subclone of tDNA ,. . The 5' to 3' o r i e n -4 t a t i o n i s shown here, but blunt-ended f r a g -ments were cloned i n both o r i e n t a t i o n s . Res-t r i c t i o n s i t e s B, E, H, P and S r e f e r to BamHl, EcoRl, Hind I I I , Pst I and Sal I r e s p e c t i v e l y . Val The tRNA ^ gene i s boxed and P r e f e r s to the d i r e c t i o n of Pol I I I t r a n s c r i p t i o n . pUC 8 sequences _____ D r o s o p h i l a sequences pBR sequences a___ gene 27 Val 3 ' - f l a n k i n g r e g i o n of the tRNA ^ gene r e q u i r e d p r o t e c t i o n and t h i s was achieved by maintenance of the 346 bp Hind I I I / BamHl fragment of pBR322 from pDt 55-0.3 (see scheme out-' l i n e d i n Fi g u r e 2). Approximately 60 yg of pDt 55-0.3 were d i g e s t e d with EcoRl and BamHl and the fragment separated on an agarose g e l . The 692 bp fragment was p u r i f i e d as d e s c r i b e d i n M a t e r i a l s and Methods, a l i q u o t e d i n t o 6 tubes each c o n t a i n -ing 1.2 yg of i n s e r t DNA and t r e a t e d with 1 u n i t of BAL-31 f o r v a r i o u s times to o b t a i n d i f f e r e n t ranges of d e l e t i o n s (see F i g u r e 3). F o l l o w i n g d i g e s t i o n , the extent of the d e l e t i o n s was determined by the removal of small a l i q u o t s ( c o n t a i n i n g ~0.4 yg of BAL-31 DNA) from each time p o i n t . The DNA i n a l i q u o t s was d i g e s t e d with Hind III, 3' end-l a b e l l e d with c t - 3 2 P dCTP and electrophoresed on an 8% poly a c r y l a m i d e g e l .as d e s c r i b e d i n M a t e r i a l s and Methods. S i z e markers (0.5 yg of l a b e l l e d Msp I d i g e s t of pBR322) were a l s o e l e c t r o p h o r e s e d , though gels i n other BAL-31 experiments contained undeleted gene DNA as s i z e marker. Fig u r e 3 shows the autoradiogram of e n d - l a b e l l e d DNA from the BAL-31 experiment. A l l time p o i n t s contained one u n i t of nuclease BAL-31, except lane 3, which contained twice as much enzyme. With i n c r e a s i n g time, of with constant time and i n c r e a s i n g enzyme a p r o g r e s s i v e decrease i n the average s i z e of bands was seen. The i n t e n s e broad band at the top of the g e l corresponded to the Hind III/.BamHl fragment from pBR322. I t can be seen that d i g e s t i o n gave r i s e to the predominance of c e r t a i n d i s c r e t e bands at each time p o i n t . 28 F i g u r e 2 C o n s t r u c t i o n of 5 ' - f l a n k i n g d e l e t i o n s The scheme used f o r the c r e a t i o n of 5 ' - f l a n k -Va 1 ing sequences of tDNA * . The 692 bp EcoRl/BamHl fragment of pDt55-0.3 was i s o l a t e d and subjected to treatment with exonuclease BAL-31 f o r v a r i o u s time p o i n t s . Approximately 0.5 yg of exonu-cl e a s e d DNA from each time poin t was d i g e s t e d with Hind I I I and 3' e n d - l a b e l l e d with a - 3 2 P dCTP using the Klenow fragment of DNA Pol I. The extent of the d e l e t i o n s was assessed on an 8% p o l y a c r y l a m i d e gel f o l l o w i n g autoradiography. DNA from the a p p r o p r i a t e time point was then blunt-ended with Klenow, p u r i f i e d and cloned i n t o the Sma I s i t e of vector pUC 8. pDt55 0.3 EcoRl/BamHl; i s o l a t e fragment 29bp 317bp 346bp BAL-31 various time p o i n t s Various s i z e d e l e t e d fragments Remove ~0.5 yg from each d e l e t i o n time point Hind I I I Klenow end-label with a - 3 2 P dCTP i i i Run on an 8% p o l y a c r y l . , / gel i i i i i i i I Autoradiogram One or more d e l e t i o n time p o i n t s i s s e l e c t e d S Klenow blunt-end with a l l four dNTPs ! j P u r i f y i fragments i T Clone i n t o Sma I s i t e of pUC 8 ! i i i i Pick white c o l o n i e s i i bcreen 30 Figu r e 3 C r e a t i o n of BAL-31 d e l e t i o n s An autoradiogram of a t y p i c a l 8% polyacrylamide ., gel showing the extent of BAL-31 d e l e t i o n s . Reactions contained ~1.2 yg of DNA fragments and 1 u n i t of BAL-31 (except i n lane 3 where 2 u n i t s were used) i n a t o t a l volume of 10 y l . Incubation times (at 30° C) were v a r i e d between 1 and 6 min. as i n d i c a t e d above lanes 2 through 6. Lane 1 c o n t a i n s Msp I fragments of pBR322. 31 32 This was due to the "pausing" of BAL-31 at c e r t a i n nucleo-t i d e s and was a common occurrence. In the experiment shown i n F i g u r e 3, DNA corresponding to d e l e t i o n s i n lanes 3 and 6 was presumed to c o n t a i n the a p p r o p r i a t e s i z e ranges of fragments corresponding to the Msp I s i z e markers. The remainder of DNA from d e l e t i o n s shown i n lanes 3 and 6 was pooled, t r e a t e d with Klenow, the l a r g e fragment of DNA Pol I (BRL), to ensure blunt ends and f o l l o w i n g p u r i f i c a t i o n , cloned i n t o the Sma I s i t e of pUC 8 as d e s c r i b e d i n M a t e r i a l s and Methods (see a l s o F i g u r e 1-B). White transformants (50-100) were picked randomly from p l a t e s c o n t a i n i n g ^200 transformants each and i n n o c u l a t e d i n t o 2 mis of 2YT/amp. Mini-prep plasmid DNA prepared from these c u l t u r e s as d e s c r i b e d i n M a t e r i a l s and Methods was d i g e s t e d with EcoRl and Hind I I I and e l e c t r o p h o r e s e d on 6% polyacrylamide g e l s to determine the presence of the c o r r e c t s i z e ranges of i n s e r t s . P o s i t i v e clones r e s u l t e d i n the l i b e r a t i o n of two small fragments, corresponding to the tRNA gene and the f l a n k i n g pBR322 fragment, from the vector pUC 8. About 80 clones were screened and an example of a t y p i c a l g e l showing scr e e n i n g of 12 clones i s shown i n F i g u r e 4. Lane 9 r e s u l t e d from the p a r t i a l d i g e s t i o n of DNA and lanes 10 and 11 were considered n e g a t i v e . The r e -mainder of the clones were p o s i t i v e and were screened by d i -deoxy-nucleotide sequencing. U s u a l l y ~ 6 5 % of c o l o n i e s on each p l a t e were blue and 70-80% of the white c o l o n i e s contained an i n s e r t from the cloned fragment. F o l l o w i n g p r e l i m i n a r y s c r e e n i n g of transformants, DNAs 33 Figure A Screening of d e l e t i o n mutants A 6% polyacrylamide g e l c o n t a i n i n g 12 cloned d e l e t i o n fragments i s shown. Fo l l o w i n g m i n i -prep l y s i s from 2 ml c u l t u r e s of white c o l o n i e s and p u r i f i c a t i o n of plasmid DNA, the clones were d i g e s t e d with EcoRl and Hind I I I . The DNA was screened by e l e c t r o p h o r e s i s on po l y -acrylatnide g e l s as d e s c r i b e d i n M a t e r i a l s and Methods. 34 L a n e 1 2 3 4 5 6 7 8 9 10 11 12 35 from p o s i t i v e i n s e r t s judged to be i n the d e s i r e d s i z e range were sequenced. Plasmid DNA from 2 mis of c u l t u r e was prepared f o r forward and reverse sequencing as d e s c r i b e d i n M a t e r i a l s and Methods and 2-3 y l a l i q u o t s of the dideoxy sequencing r e a c t i o n were electr.ophoresed on 8% sequencing g e l s . An example of a t y p i c a l sequencing g e l showing the forward (F') r e a c t i o n i s seen i n Figure 5. Screening df." the d e l e t i o n of the 692 bp fragment r e s u l t e d i n recovery of mutants extending to p o s i t i o n s -179, -138, -136, -129, -75, -48, -38, -8 and +19. These end-points were obtained a f t e r sequencing of 32 c l o n e s . Clones were r o u t i n e l y sequenced i n forward and r e v e r s e d i r e c t i o n s as part of the s c r e e n i n g procedure i n s t e a d of f i r s t determining the o r i e n t a t i o n of the i n s e r t . In order to o b t a i n an e x t e n s i v e array of d e l e t i o n end-p o i n t s (to allow the determination of the e f f e c t of s i n g l e n u c l e o t i d e changes) some of the cloned d e l e t i o n s were r e -d e l e t e d f o r short p e r i o d s and sub-cloned. The procedure i n v o l v e d the i s o l a t i o n of the mutant EcoRl/BamHl fragment from .the v e c t o r pUC 8 (see F i g u r e 1-B), p u r i f i c a t i o n and short exonuclease treatment with BAL-31 (up to 4 min.). A l l f u r t h e r procedures were as p r e v i o u s l y d e s c r i b e d for. the pDt55-0.3 -fragment. D e l e t i o n s beginning with A5'-75 r e s u l t e d i n mutants ending at n u c l e o t i d e p o s i t i o n s -49, -47 and -20. Mutants d e r i v e d as a r e s u l t of three separate experiments i n v o l v i n g d e l e t i o n of A 5 ' - 4 9 extended to p o s i t i o n s -45, -43, -42, -41, -37, -33, -29, -27 and -17. The s i z e d i s t r i b u t i o n of 36 Fi g u r e 5 "The DNA sequence of A5'-37 (A) and A5'-35 (B) The DNA sequence of these mutants was determined as d e s c r i b e d i n M a t e r i a l s and Methods. Lanes.1-4 represent the forward sequencing r e a c t i o n s s p e c i f i c f o r C, T, A and G r e s p e c t i v e l y . The DNA sequence isshown on the s i d e of the auto-radiograph. Note that i n A, A5'-36 becomes A5'-37 and i n B, A5'-34 becomes A5'-35 due to the adjacent., sequence of vector DNA. The j u n c t i o n between the vecto r and d e l e t e d DNA i s marked by an arrow on the sequence. 38 mutants a r i s i n g from A5'-49 i n one e x p e r i m e n t a r e seen i n F i g u r e 6-A. D e l e t i o n s p r e f e r e n t i a l l y , t e r m i n a t e d a t c e r t a i n s p e c i f i c n u c l e o t i d e p o s i t i o n s ( e g . -49, -43 and -37) and t e r m i n a t i o n a t o t h e r s i t e s s u c h as -31, -32, -23, -22 e t c . d i d . n o t o c c u r ( o r too k p l a c e v e r y i n f r e q u e n t l y and t h e r e -f o r e were n e v e r d e t e c t e d ) . The s e q u e n c e s of A5'-33 and A5'-26 (-33 b e i n g a p r e f e r e n t i a l s t o p s i t e ) a r e shown i n F i g u r e 7. The r e m a i n i n g d e l e t i o n s , A5'-35, -30, -28, -26, -25, -20, -10, -9, -8, -3 and -2 were d e r i v e d f r o m d e l e t i o n s o f A5'-37 i n t h r e e s e p a r a t e e x p e r i m e n t s ( s e e F i g u r e 6-B). Seq u e n c e s f o r A5'-30 and A5'-20 a r e shown i n F i g u r e 8. The r e p e t i t i o n of t h e s e e x p e r i m e n t s was n e c e s s a r y , s i n c e t h e n u c l e a s e BAL-31 t e r m i n a t e d p r e f e r e n t i a l l y a t c e r t a i n "pause - s i t e s " on t h e 5 ' - f l a n k i n g s e q u e n c e s , t h e r e -by g i v i n g r i s e t o d e l e t i o n s r e p e a t e d l y e n d i n g a t t h e same p o s i t i o n s . As one r e s u l t , s e v e r a l i d e n t i c a l d e l e t i o n s were r e c o v e r e d c l o n e d i n o p p o s i t e o r i e n t a t i o n s so t h a t i t was p o s s i b l e t o compare t h e e f f e c t s of v e c t o r s e q u e n c e s on t h e e x p r e s s i o n of t h e s e mutant tDNAs. D e l e t i o n s t e r m i n a t i n g a t n u c l e o t i d e p o s i t i o n s -31 or -32 were n e v e r o b t a i n e d , p r e s u m a b l y due t o the n a t u r e o f th e two A r e s i d u e s a t t h e s e p o s i t i o n s . I t was t h e r e f o r e a t t e m p t e d t o o b t a i n A5'-31 and A5'-32 u s i n g t h e combined a c t i o n s o f T_. DNA p o l y m e r a s e and n u c l e a s e SI on A5'-33. The r a t i o n a l e was t o l i n e a r i z e t h e p l a s m i d c a r r y i n g A5'-33.with BamHl and a l l o w t h e T^ p o l y m e r a s e t o "chew b a c k " t o p o s i t i o n -30 on t h e c o d i n g s t r a n d u s i n g i t s 3' to 5' e x o n u c l e a s e a c t i v i t y and a t t h e same time i n c o r p o r a t e 39 Fi g u r e 6 D i s t r i b u t i o n of d e l e t i o n s from A5'-49 and A5'-37 A- Bar-graph showing the extent of d e l e t i o n s a r i s i n g from a s i n g l e BAL-31 experiment using A5'-49 as the s t a r t i n g DNA. The d e l e t i o n end-points are i n d i c a t e d on the X-axis and t h e i r frequency of occurrence from sc r e e n i n g of a t o t a l of 18 mutants i s i n d i c a t e d on the Y-a x i s . B- The s i z e d i s t r i b u t i o n of mutants derived from one experiment i n v o l v i n g f u r t h e r d e l e t i o n of A5'-37. Data i s from the sequence of 17 mutant clones . A 4-> o c CD __ C J CD L i . -49 -45 -43-42-41 -37 .33 .29 _27 .23 A End_point 7 B 6-5-1 • _37 _35 _33 _30 _28_27_26_25 -20 A Enci_point 42 Figu r e 7 The DNA sequence of A5'-33 (A) and A5'-26 (B) The sequence of mutant DNAs was determined as des c r i b e d i n M a t e r i a l s and Methods. Lanes 1-4 represent the reverse sequencing r e a c t i o n s s p e c i f i c f o r C, T, A and G r e s p e c t i v e l y . The DNA sequence i s shown on the s i d e of the auto-radiograph and the j u n c t i o n between the vecto r and d e l e t e d DNA i s marked by an arrow on the sequence. 43 44 Figu r e 8 The DNA sequence of A5'-30 (A) and A5'-20 (B) The sequence of mutant DNAs was determined as de s c r i b e d i n M a t e r i a l s and Methods. Lanes 1-4 represent the re v e r s e (A) and forward (B) se-quencing r e a c t i o n s s p e c i f i c f o r C, T, A and G r e s p e c t i v e l y . The DNA sequence i s shown on the s i d e of the autoradiograph and the j u n c t i o n between the vecto r and d e l e t e d DNA i s marked by an arrow on the sequence. 46 dTTP at p o s i t i o n s -31 and -32 by i t s 5' to 3' polymerase a c t i v i t y . The overhang r e s u l t i n g from the c o m p e t i t i v e exonuclease and polymerase a c t i v i t i e s was e x c i s e d by nuclease SI and the plasmid was r e c i r c u l a r i z e d by T^ DNA l i g a s e . However, these experiments gave r i s e to mutant end-p o i n t s t e r m i n a t i n g beyond the -31 s i t e . F i g u r e 9-A shows the d i s t r i b u t i o n - o f the r e s u l t a n t mutants screened i n t h i s experiment. The experiment was then repeated with Mung-Bean nuclease s u b s t i t u t e d f o r SI nuclease. Mung-Bean nuclease i s r e p o r t e d to not have as strong an exonuclease a c t i v i t y as nuclease SI at double stranded t e r m i n i (Laskowski, 1980). In t h i s experiment a l l d e l e t i o n s screen terminated at e i t h e r -33 or -30 (see'".Figure 9-B) . The attempts to d e r i v e -31 or -32 were t h e r e f o r e abandoned. A summary of a l l d e l e t i o n end-points reported i n t h i s study i s presented i n F i g u r e 10. 47 Figu r e 9 D i s t r i b u t i o n of d e l e t i o n s from A5'-33 A- D i s t r i b u t i o n of end-points from an experiment to o b t a i n A5'-31 or A5'-32. Reactions were c a r r i e d out by d i g e s t i o n of 2 Ug of A5'-33 DNA with BamHl and treatment with 6 u n i t s of T\ DNA polymerase i n the presence of dTTP ( f i n a l c o n c e n t r a t i o n of 0.5 mM) at 37° C f o r 4 min. i n a t o t a l volume of 12 - y l . DNA was the t r e a t e d wtih 70 u n i t s of nuclease SI at 37° C f o r 30 min. ( v o l : 40 y l ) . DNA was then l i g a t e d and transformed as d e s c r i b e d i n M a t e r i a l s and Methods. B- As i n Fig u r e 9-A, except that 30 u n i t s of Mung-Bean nuclease was s u b s t i t u t e d f o r nuclease SI. The r e a c t i o n was continued f o r 30 min. at 30° C i n a t o t a l volume of 50 y l . D e l e t i o n end-points are i n d i c a t e d on the X-axis and t h e i r frequency of occurrence on the Y-a x i s . A 5-4-1 -— // — -30 _27 _10_9 _3_2 A End_point B _33 _30 49 Figu r e 10 P o s i t i o n of d e l e t i o n end-points Sequence of the 5 ' - f l a n k i n g r e g i o n (non-coding strand) of pDt55-0.3 to p o s i t i o n -70 ( r e l a t i v e to the mature coding sequence) i s shown. Arrows i n d i c a t e d e l e t i o n end-points analyzed i n t h i s t h e s i s . _ 4 4 4 4- -4-4 4 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 4 4 4 4 _tn + + 4-^4- * 4 _ t n 4 4 G G T C G C T G A A G T T G G 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 O 51 T r a n s c r i p t i o n of mutant tDNAs i n a D r o s o p h i l a (Schneider  I I ) c e l l - f r e e e x t r a c t D e l e t i o n mutants of pDt 55-0.3 DNA were t r a n s c r i b e d i n v i t r o i n a homologous Schneider II S100 c e l l - f r e e ex-t r a c t as d e s c r i b e d i n M a t e r i a l s and Methods. The i n i t i a l experiment to determine the optimum range of DNA concen-t r a t i o n s i n the t r a n s c r i p t i o n r e a c t i o n contained from 0.05 u g to 0.3 u g of t D N A ^ 1 -179 DNA. The tRNA products were run on g e l s , autoradiographed, the gel s l i c e s c o r -responding to the bands excised and counted f o r Cerenkov r a d i a t i o n . F i g u r e 11-A shows that a l l 1 values obtained were approximately the same, presumably because the t r a n -s c r i p t i o n r e a c t i o n had been sat u r a t e d with template DNA even at the lowest DNA i n p u t . Since saturation took place at the lowest l e v e l of DNA i n p u t , a seven point input DNA range of between 0.0035 yg Val to 0.05 yg was t e s t e d with tDNA ^ -179 to see i f a DNA curve e f f e c t could be obtained. Between 0.005 yg and 0.05 yg of template DNA input there was a steady i n c r e a s e i n the r a t e of t r a n s c r i p t i o n (see F i g u r e 11-B). A l l sub-sequent t r a n s c r i p t i o n curves we re c a r r i e d out with input DNAs ranging between 0.005 yg and 0.05 yg as d e s c r i b e d i n M a t e r i a l s and Methods. In a l l t r a n s c r i p t i o n s pUC 8 DNA was added to a t o t a l of 1 yg per r e a c t i o n to counter an i n h i b i t o r reported to be present (S£. Louis and Spiegelman, 1985). In previous t r a n s c r i p t i o n r e a c t i o n s pBR322 had been used :to counter the e f f e c t . o f an i n h i b i t o r i n the S100 e x t r a c t s (Fowlkes 52 y . F i g u r e 11 E f f e c t of template DNA c o n c e n t r a t i o n on Val the r a t e of t r a n s c r i p t i o n f o r tDNA ^ -179 The v e l o c i t y of r e a c t i o n f o r tDNA^"*" -179 (cpm/hr x 10~3) i s p l o t t e d as a f u n c t i o n of template c o n c e n t r a t i o n . The t o t a l amount of DNA i n each r e a c t i o n was 1 yg (adjusted by adding pUC 8 DNA) and r e a c t i o n s were c a r r i e d out f o r 90 min. at 24° C and analyzed as d e s c r i b e d i n M a t e r i a l s and Methods. Val A- tDNA ^ -179 template input was v a r i e d between 50 ng •. and 300 ng. Val B- tDNA ^ -179 template input was v a r i e d between 3.3 ng and 50 ng. A T e m p l a t e DNA (ng) " B 55 and Shenk, 1980; Sharp et a l . , 1983; St. Louis and Spiegelman, 1985), but i t was found that the a d d i t i o n of pUC 8 DNA r e s u l t e d i n i n c r e a s e d t r a n s c r i p t i o n e f f i c i e n c y r e l a t i v e to the a d d i t i o n of pBR322 (see Table I ) . In Val an experiment using between 0.01 yg and 0.03 yg of tDNA ^ -179 template DNA, t r a n s c r i p t i o n r e a c t i o n s were c a r r i e d out with e i t h e r pBR322;„ or pUC 8 s e r v i n g as non-template DNA. The maximal amount of t r a n s c r i p t i o n with pUC 8 se r v -ing as non-template DNA, was reached with 0.05 yg of tem-p l a t e input DNA. When pBR322 was used as non-template DNA, Val the maximal amount of t r a n s c r i p t i o n f o r tDNA ^.-179 r e s u l t e d only a f t e r the a d d i t i o n of at l e a s t 0.3 yg of •'" template DNA. Furthermore, 1 at 0.05 yg of template DNA in p u t , there was 1.5 times more t r a n s c r i p t i o n product with pUC 8 as non-template DNA than with pBR322. Since pUC 8 lead to a gre a t e r amount of t r a n s c r i p t i o n product (i,e. higher e f f i c i e n c y ) at lower l e v e l s of template DNA i n p u t , pBR322 was r e p l a c e d with pUC 8 as non-template DNA i n subsequent t r a n s c r i p t i o n s . To analyze t r a n s c r i p t i o n , the amount of product con-ta i n e d i n polyacrylamide g e l s f o l l o w i n g 90 min. r e a c t i o n times, was determined by e x c i s i o n of the g e l s l i c e s con-t a i n i n g the t r a n s c r i p t i o n products and Cerenkov counting. Values corresponding to the v e l o c i t y of t r a n s c r i p t i o n (v = cpm of product per 90 min'. r e a c t i o n time= r a t e of r e a c t i o n ) were c o r r e c t e d to cpm per hour and analyzed by the method of St. Louis and Spiegelman (1985). The method assumes that the k i n e t i c s of t r a n s c r i p t i o n conform to those of a 56 Table I Comparison of the e f f e c t s of pBR322 vs. pUC 8 on the r a t e of t r a n s c r i p t i o n of Val t'DNA , -179 4 T r a n s c r i p t i o n of -179 was c a r r i e d out with 0.01 yg, 0.05 yg and 0.3 yg of input DNA as d e s c r i b e d i n M a t e r i a l s and Methods. Non-template DNAs were added to a f i n a l t o t a l c o n c e n t r a t i o n of 1 yg. Non-template DNA added VpBR 322 VpUC 8 Template DNA ( u g ) (cpm/hr. x l O ~ 3 ) (cpm/hr x 10~ 3 ) 0.01 5.2 6.3 0.05 10.8 16.0 0.30 16.4 15.9 57 c l a s s i c one s u b s t r a t e enzyme r e a c t i o n and can be analyzed by the Lineweaver-Burke method. The r e c i p r o c a l of the values f o r v e l o c i t y of the r e a c t i o n and s u b s t r a t e input (S= jjg of template DNA) were p l o t t e d and ^ m a x a n d ^ m ap-parent (K app) were de r i v e d ff.em a l i n e a r -.regressed l i n e of 1/V vs 1/S. To counter day to day v a r i a t i o n s , each set of t r a n s c r i p t i o n s of d e l e t i o n mutant tDNAs was accompanied Val by a s i x - p o i n t input of tDNA ^ -179 s e r v i n g as a c o n t r o l . Due to the r e p e t i t i o n of -179 t r a n s c r i p t i o n , a number of independent measurements f o r app were obtained which were then used to d e r i v e the standard d e v i a t i o n f o r i t s K app m r as an estimate of the accuracy of the data. The values f o r V were expressed as percent i n c r e a s e max r r Val or decrease of the value of V f o r tDNA , -179 t r a n s -max 4 c r i b e d on the same day. Each d e l e t i o n end-point was t r a n s -c r i b e d with two d i f f e r e n t a c t i v e S100 c e l l - f r e e e x t r a c t s due to the v a r i a b i l i t y 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 between d i f f e r e n t S100 preps. The v a r i a b i l i t y was approximately 20% i n the f i n a l r a t e of t r a n s c r i p t i o n . However the per-cent i n c r e a s e and decrease i n V f o r a l l d e l e t i o n end-max p o i n t s was n e a r l y the same i n both e x t r a c t s . For some of the d e l e t i o n s t r a n s c r i p t i o n was repeated more than twice i n order to confirm data. The percent i n c r e a s e s and decreases i n V and values f o r K app f o r each d e l e t i o n mutant were max m r r c a l c u l a t e d and the average of the two d i f f e r e n t values from the two d i f f e r e n t e x t r a c t s was reported (see Table I I ) . An example of the d o u b l e - r e c i p r o c a l p l o t obtained with 58 Table II T r a n s c r i p t i o n of d e l e t i o n end-points Summary of r e s u l t s obtained from s i x - p o i n t t r a n s -c r i p t i o n s of mutant tDNAs with two d i f f e r e n t S100 e x t r a c t s . The V and K app were averaged and max m r r & are presented f o r each d e l e t i o n end-point. The Val V r e l a t i v e to tDNA . -179 i s expressed as % i n -max 4 crease or % decrease. The c o n c e n t r a t i o n f o r K app m ^ r (nM) were c a l c u l a t e d by e s t i m a t i n g the s i z e of Val plasmid clones to be 3 Kb; except f o r tDNA ^ -179, where 3.4 Kb was used as the s i z e of plasmid c l o n e . 59 End-point ^ i n c r e a s e (+) or decrease (4.) K m app K app m . i n V r e l a t i v e to -1/9 (Ug> (nM) Val tDNA 4 -179 0 0.038* 0.338 A5'-70 0 0.021 0.212 A5'-49 44.31 0.046 0.464 A5'-48 43. 5t 0.052 0.524 A5'-47 18.Ot 0.043 0.434 A5'-45 66. 5 + 0.029 0. 292 A5'-43 20. 7 + 0.038 0.384 A5'-42 20.Ot 0.039 0.394 A5'-41 16.2t 0.042 0.424 A5'-38 19.2t 0.036 0.364 A5'-37 43 . 44 0.017 0.172 A5'-35 30.24 0.011 0.112 A5'-33 88.24 0.025 0 . 252 A5'-30 91 .44 0 . 0 0 5 * * 0.051** A5 '-29 100.04 - -A5'-28 98.04 0 . 0 0 6 * * 0.060** A5'-27 93.64 0.014 0. 142 A5 '-26 79.44 0.024 0 . 242 A5'-25 90. 24 0.022 0 . 222 A5'-20F' 43.74 0.020 0. 202 A5'-20R' 90.04 0.036 0.364 A5'-17 49.44 . 0.012 0.122 A5'-10 97 .04 0.006** 0.060** A5'-8 95.84 0.007** 0.070** A5 ' -2 100.04 _ _ * i n d i c a t e s standard deviation. ± 0.007 * i n d i c a t e s i n a c c u r a t e numbers as a r e s u l t of very low l e v e l s of t r a n s c r i p t i o n 60 V a l d a t a from t h e t r a n s c r i p t i o n s o f tDNA ^ -170, A5'-45 and V a l A5'-30 i s p r e s e n t e d i n F i g u r e 12. Most p o i n t s f o r tDNA ^ -179 and A5,;|"45 f a l l on t h e l i n e . The V and K app c o u l d max m r r be e s t i m a t e d g r a p h i c a l l y from t h e i n t e r c e p t s , however, t h e y were c a l c u l a t e d n u m e r i c a l l y f r o m t h e i n t e r c e p t s of l e a s t -s q u a r e s l i n e s . The e f f e c t of t h e d e l e t i o n t o p o s i t i o n -45 was t o r a i s e t h e V and lower t h e K app r e l a t i v e t o -179. max m v v D e l e t i o n A~5 '- 3 0 r e s u l t e d i n a s t e e p s l o p e , c o r r e s p o n d i n g to v e r y s m a l l v a l u e s f o r V and K app. The c o r r e l a t i o n max m r r Val c o e f f i c i e n t s f o r tDNA * -179, A5'-45 and A 5 '-30 were c a l -c u l a t e d t o be 0.995, 0.997 and 0.98 r e s p e c t i v e l y . V a l u e s d e r i v e d f o r V 's and K app's from t h e t r a n s c r i p t i o n o f max m r r r a l l d e l e t i o n e n d - p o i n t s i s t a b u l a t e d i n T a b l e I I . Note t h a t a t low t r a n s c r i p t i o n l e v e l s t h e r e was i n c r e a s e d v a r i a b i l i t y i n t h e v a l u e s f o r K app '(tJfeese v a l u e s a r e m marked w i t h a*.* i n T a b l e I I ) . A number of d e l e t i o n s a p p e a r e d t o i d e n t i f y 'sequences which m o d u l a t e t r a n s c r i p t i o n t h e s e a r e d e s c r i b e d i n more d e t a i l i n t h e D i s c u s s i o n . D e l e t i o n o f t h e 5 ' - f l a n k to p o s i t i o n -70 d i d not a f f e c t t h e V , but d i d l o w e r t h e K app by 45%. The -70 max m ^ 3 d e l e t i o n removed s e v e r a l p y r i m i d i n e t r a c t s l o c a t e d between p o s i t i o n s -179 and -70. D e l e t i o n o f t h e 5 ' - f l a n k t o r e s i -due -49 r e s u l t e d i n a 44% i n c r e a s e i n V r e l a t i v e t o t h e max w i l d - t y p e -179 f l a n k , w i t h a s l i g h t i n c r e a s e i n K m app. F i g u r e 13 shows t h e a u t o r a d i o g r a m of a g e l c o n t a i n i n g t r a n s -Val c r i p t i o n p r o d u c t s from tDNA * -179 (A) and A5'-49 ( B ) . T r a n s c r i p t i o n s were c a r r i e d out w i t h 0.005, 0.007, 0.01, 0.018, 0.025 and 0.05 y g of t e m p l a t e DNA i n p u t as d e s c r i b e d 61 Fi g u r e 12 Double r e c i p r o c a l p l o t of data from t r a n s c r i p t i o n of tDNA^^-179, A5'-45 and A5'-30 Gel . s l i c e s corresponding to t r a n s c r i p t i o n products were cut out and the amount of product determined by Cerenkov r a d i a t i o n . The data was p l o t t e d as 1/V (V=cpm/hr) versus 1/S (S=mass of template i n yg) Using steady s t a t e k i n e t i c a n a l y s i s of St. Louis and Spiegelman (1985), the 1/K app and 1/V were ° m V i f max c a l c u l a t e d from the x and y i n t e r c e p t s on the graph. • = -179 A = -45 • = -30 62 63 Figu r e 13 Autoradiogram of products from the Val t r a n s c r i p t i o n s of tDNA ^ -179 and A5'-49 T r a n s c r i p t i o n s were c a r r i e d out using a t o t a l DNA c o n c e n t r a t i o n of 1.0 yg per r e a c t i o n and t r a n s -c r i p t i o n products were analyzed on polyacrylamide g e l s as d e s c r i b e d i n M a t e r i a l s and Methods. An autoradiograph r e p r e s e n t i n g the e l e c t r o p h o r e t i c Val s e p a r a t i o n of l a b e l l e d tRNAs from tDNA ^ -179 and A5'-49 i s shown i n F i g u r e s 13-A and 13-B r e s p e c t i v e l y . Lanes 1-6 show the t r a n s c r i p t i o n products of 0.005 Val to 0.05 Mg of tDNA * -179. Lanes 7-12 show the t r a n s c r i p t i o n products of 0.005 to 0.05 yg of A5'-49. 65 M a t e r i a l s and Methods. As with d e l e t i o n s to -70, d e l e t i o n s extending to p o s i t i o n s -49 r e s u l t e d i n the removal of an a d d i t i o n a l set of p y r i m i d i n e r e s i d u e s and t e r m i n a t i o n -l i k e sequences found between -70 and -49 (see F i g u r e 10),. D e l e t i o n s between A5'-'59 and A 5'-45 d i s r u p t e d the sequence GGCAG and were accompanied bya.66.5% i n c r e a s e i n V r e l a t i v e to -179 and a 74% decrease i n K app. An max m autoradiogram of a g e l c o n t a i n i n g t r a n s c r i p t i o n products from A5'-49 i s seen i n F i g u r e 14-A. T r a n s c r i p t i o n s were c a r r i e d out as p r e v i o u s l y d e s c r i b e d i n M a t e r i a l s and Methods and the amount of product determined by Cerenkov r a d i a t i o n following e l e c t r o p h o r e s i s of t r a n s c r i p t i o n products on an 8% denaturing p o l y a c r y l a m i d e g e l . For d e l e t i o n s ending at p o s i t i o n s -43, -42, -41 and -38 there was l i t t l e change i n app r e l a t i v e to -179. The V m a x f o r these clones remained high with approximately a 20% i n c r e a s e over the -179 c o n t r o l DNA (Table I I ) . The f i r s t drop i n V occurredwhen the n u c l e o t i d e at -38 was max d e l e t e d . The -3.7 d e l e t i o n was accompanied by a sharp decrease (.55%) i n K app. An autoradiogram showing the £.tfxaanscription products d i r e c t e d by A5'-38 i s shown i n F i g u r e 14-B. D e l e t i o n -37 r e s u l t e d i n a V that was 43% ° max lower that that obtained with -179 and 62.6% lower than the V f o r A5'-38. The V 's f o r A5'-35 and A5'-33 were max max reduced by 30% and 88% r e s p e c t i v e l y r e l a t i v e to -179 and the K m app's remained low (71% down r e l a t i v e to -179 f o r A 5'-35). An autoradiogram of products from the t r a n s -c r i p t i o n of A5'-35 i s shown i n F i g u r e 15-A. The t r a n -66 F i g u r e 14 Autoradiogram of products from the t r a n s c r i p t i o n of A5'-45 and A5'-38 T r a n s c r i p t i o n s were c a r r i e d out using a t o t a l DNA c o n c e n t r a t i o n of 1.0 yg per r e a c t i o n and t r a n s -c r i p t i o n products were analyzed on pol y a c r y l a m i d e gels as d e s c r i b e d i n M a t e r i a l s and Methods. An autoradiogram r e p r e s e n t i n g the e l e c t r o p h o r e t i c s e p a r a t i o n of l a b e l l e d tRNAs from A5'-45 and A5'-38 i s shown i n F i g u r e s 14-A and 14-B r e s p e c t i v e l y . Lanes 1-6 show the t r a n s c r i p t i o n products of 0.005 to 0.05 yg of A5'-45. Lanes 7-12 show the t r a n s -c r i p t i o n products of 0.005 to 0.05 yg of A5'-38. il A 5 _ 4 5 t\ 5'_ 3 8 68 F i g u r e 15 A u t o r a d i o g r a m of p r o d u c t s from t h e t r a n s c r i p t i o n of A5'-33 and A5'-25 T r a n s c r i p t i o n s were c a r r i e d out u s i n g a t o t a l DNA c o n c e n t r a t i o n of 1.0 pg per r e a c t i o n and t r a n s -c r i p t i o n p r o d u c t s were 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 as d e s c r i b e d i n M a t e r i a l s and Methods. An a u t o r a d i o g r a m r e p r e s e n t i n g t h e e l e c t r o p h o r e t i c s e p a r a t i o n of l a b e l l e d tRNAs from A5'-33 and A5'-25 i s shown i n F i g u r e s 15-A and 15-B r e s p e c t i v e l y . L a nes 1-6 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 of 0.005 to 0.05 pg o f A5'-33. La n e s 7-12 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 of 0.005 t o 0.05 Ug o f A5'-25. 61 A 5 _ 3 3 1 2 3 4 5 6 A 5 _ 2 5 7 8 9 10 11 12 A B 70 s i t i o n between an-increased V f o r A5'-38 ( F i g u r e 14-B) max ° to an 88% decrease 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 f o r A5'-33 (see F i g u r e 15-A) was the r e s u l t of d i s r u p t i o n of the se-quence TCGCT (see Fig u r e 10). Further d e l e t i o n to r e s i d u e -30 maintains t r a n s c r i p t i o n e f f i c i e n c y at a g r e a t l y reduced l e v e l . No t r a n s c r i p t i o n was detected f o r A5'-29 and a d d i -t i o n a l d e l e t i o n s of the 5'-flank to p o s i t i o n -20 (R') main-t a i n e d t r a n s c r i p t i o n at approximately 90% lowered e f f i c i e n c y with a s l i g h t i n c r e a s e f o r A5'-26 (Table I I ) . The t r a n s -c r i p t i o n products of A5'-25 are seen i n Fig u r e 15-B. D e l e t i o n s extending to r e s i d u e -20 were obtained i n two o r i e n t a t i o n s i n the vecto r (designated by R' and F' c o r r e s -ponding to the 5' to 3' and the 3' to 5' o r i e n t a t i o n s r e l a t i v e to the l a c Z gene i n the vecto r pUC 8, r e s p e c t i v e l y ) and the t r a n s c r i p t i o n e f f i c i e n c y of both clones was compared. Dele-' t i o n to A5'-20 R' (see F i g u r e 20-A f o r sequence) i n c r e a s e d the K app from the 42% decrease obtained with the A5'-25 mutant m r r to the l e v e l f o r -179. T r a n s c r i p t i o n of the -20 d e l e t i o n i n the opposite o r i e n t a t i o n (A5'-20 F') r e s u l t e d i n a higher V and a lower K app than found with A5'-20 R' (Table II max m r r and F i g u r e 16-A and 16-B). D e l e t i o n -20 F' t r a n s c r i b e d at Val 44% decreased e f f i c i e n c y r e l a t i v e to tDNA ^ -179 which was s u r p r i s i n g given the extent of the d e l e t i o n . The t r a n s -c r i p t i o n e f f i c i e n c y f o r A5'-17 ( o r i e n t a t i o n 5' to 3' i . e . R 1) was a l s o found to be unexpectedly high at approximately 49% decreased e f f i c i e n c y r e l a t i v e to -179 (Table II and Figu r e 17-A). Both A5'-20:F* and A5'-17 R* have lowered K apparents (47% and 68%, r e s p e c t i v e l y ) compared to that m 71 F i g u r e 16 Autoradiogram of products from the t r a n s c r i p t i o n of A5'-20 R' and A5'-20 F' T r a n s c r i p t i o n s were c a r r i e d out using a t o t a l DNA c o n c e n t r a t i o n of 1.0 yg per r e a c t i o n and t r a n s -c r i p t i o n products were analyzed on polyacrylamide gels as des c r i b e d i n M a t e r i a l s and Methods. An autoradiogram r e p r e s e n t i n g the e l e c t r o p h o r e t i c s e p a r a t i o n of l a b e l l e d tRNAs from A5'-20 R' and A5'-20 F' i s shown i n F i g u r e s 16-A and 16-B, r e s p e c t i v e l y . Lanes 1-6 show the t r a n s c r i p t i o n of products of 0.005 to 0.05 yg of A5'-20 R'. Lanes 7-show the t r a n s c r i p t i o n products of 0.005 to 0.05 yg of A5'-20-F'. 72 A 73 F i g u r e 17 Autoradiogram of products from the t r a n s c r i p t i o n of A5'-17 and A 5 ' - 1 0 T r a n s c r i p t i o n s were c a r r i e d out using a t o t a l DNA c o n c e n t r a t i o n of 1.0 yg per r e a c t i o n and t r a n s -c r i p t i o n products were analyzed on polyacrylamide gels as des c r i b e d i n M a t e r i a l s and Methods. An autoradiogram r e p r e s e n t i n g the e l e c t r o p h o r e t i c s e p a r a t i o n of l a b e l l e d tRNAs from A5'-17 and A5'-10 i s shown i n Fi g u r e s 17-A and 17-B r e s p e c i v e l y . Lanes 1-6 show the t r a n s c r i p t i o n products of 0.005 to 0.05 yg of A5'-17. Lanes 7-12 show the t r a n s c r i p t i o n products of. 0.005 to 0.05 yg of A5'-10. 74-__ 5_ 10 7 8 9 10 11 12 75 for -179. : !.,'=,'. D e l e t i o n s to p o s i t i o n s -10 ( F i g u r e 17-B) and -8 d i s -played very low l e v e l s of t r a n s c r i p t i o n , approximately 97% lower than -179 and no t r a n s c r i p t i o n was detected f o r A5'-2 (Table I I ) . F i n a l l y , i t i s i n t e r e s t i n g to note that i n i t i a l data from an experiment comparing the e f f e c t s of pUC 8 versus pBR322 as non-template DNAs, suggested t h a t . t r a n s c r i p t i o n s using pUC 8 DNA would probably r e s u l t i n a lower app; s i n c e a g r e a t e r amount of t r a n s c r i p t i o n product was ob-t a i n e d at lower DNA input l e v e l s of pUC 8. However, .the Val opposite was observed when app f o r tDNA ^ (Table II) was compared to that of pDt55-0.3 where pBR322 had been used as non-template DNA ( S t . Louis and Spiegelman, 1985). In an experiment comparing the e f f e c t s of pUC 8 on pDt55-0.3 Val and tDNA * -179 with 0.0033 to 0.05 yg of template DNA in p u t , i t was found that the r a t e of t r a n s c r i p t i o n was Val g r e a t e r f o r pDt55-0.3 than f o r tDNA * -179 between 0.01 and 0.25 yg of template DNA i n p u t . The f a s t e r r a t e of r e a c t i o n between the two DNA i n p u t s t h e r e f o r e l e d to the lower values f o r K- app f o r pDt55-0.3, even though the r a t e of t r a n s c r i p t i o n was higher and the t r a n s c r i p t i o n r e a c t i o n Val reached s a t u r a t i o n at lower inputs of tDNA ^ -179. An experiment to t e s t v t h e e f f e c t s of pBR322 versus pUC 8 s e r v i n g as non-template DNAs using 0.3 yg of pDt55-0.3 as template DNA showed that when 0.7 yg of pUC 8 DNA was added to the r e a c t i o n , the amount of t r a n s c r i p t i o n product was twice the amount obtained with 0.7 yg of pBR322 s e r v i n g as non-template DNA. T h i s r e s u l t was c o n s i s t e n t i n experiments with three d i f f e r e n t S-100 e x t r a c t s . 77 DISCUSSION An extensive s e r i e s of d e l e t i o n s extending i n t o the Val 5 '-flank of the D r o s o p h i l a tRNA ^ gene were ..made using nuclease BAL-31. Deletions -.' were subsequently screened and t h e i r ends sequenced. Mutants were then t r a n s c r i b e d i n an i n v i t r o homologous t r a n s c r i p t i o n system to d e l i m i t modulatory sequences present i n the 5 ' - f l a n k i n g r e g i o n . The data obtained as a r e s u l t of these t r a n s c r i p t i o n s was used to d e r i v e values f o r V and K app f o r each d e l e t i o n max m r r end-point as p r e v i o u s l y d e s c r i b e d . The t r a n s c r i p t i o n data was c o n s i s t e n t with previous o b s e r v a t i o n s which found that the a d d i t i o n of c e r t a i n non-template DNAs con v e r t e d : the t r a n s c r i p t i o n k i n e t i c s to a h y p e r b o l i c f u n c t i o n of DNA i n p u t , which could be analyzed by steady s t a t e k i n e t i c s (Sharp et a l . , 1983; St. Louis and Spiegelman, 1985). Using the same r a t i o n a l e , the t r a n s c r i p t i o n f o r each d e l e t i o n end-point was analyzed using values f o r i t s K m app (the apparent a f f i n i t y of the t r a n s c r i p t i o n complex or some component for. the template DNA) and V m a x (the maximum v e l o c i t y of - the r e a c t i o n ; template a c t i v i t y or e f f i c i e n c y ) The p a r e n t a l sub-clone from pDt55-0.3 had 179 bp of 5 ' - f l a n k i n g sequence and d e l e t i o n of the f l a n k i n g sequence to p o s i t i o n -70 did not a f f e c t the V , but lowered the max K m app s l i g h t l y (Table I I ) . T h i s may have been due to the removal of a p y r i m i d i n e t r a c t betweeen p o s i t i o n s -85 and -80. The s t r e t c h of thymidines i s s i m i l a r to t r a n s c r i p t i o n t e r m i n a t i o n s i g n a l s found at the 3' end of tRNA genes (Bogenhagen and Brown, 1981 ; C o z z a r e l 1 i et a l . , 1983; Watson 78 e t a l . , 1 9 8 4 ) . T h e p r e s e n c e o f t h e s e t e r m i n a t i o n - l i k e s e q u e n c e s i n t h e 5 ' - f l a n k i n g r e g i o n s o f t R N A g e n e s h a s p r e v i o u s l y r e p o r t e d t o b e i n h i b i t o r y t o t r a n s c r i p t i o n , i n c o L v s a s s h o w n f o r a t R N A ^ ( D e F r a n c o e t a l . , 1 9 8 1 ; D i n g e r -m a n n e t a l . , 1 9 8 2 ) . T h e t R N A ^ g e n e c o n t a i n s a n u n d e c a -n u c l e o t i d e i n t h e 5 ' - f l a n k i n g s e q u e n c e c o n s i s t i n g o f a p e n t a n u c l e o t i d e t h y m i d i n e t r a c t , f l a n k e d b y p u r i n e r e s i d u e s o n e i t h e r s i d e ( F i g u r e 1 8 ) . T h e s e q u e n c e i s l o c a t e d b e t w e e n p o s i t i o n s - 2 4 a n d - 1 3 a n d i t s r e m o v a l r e m o v e d t r a n s c r i p t i o n a l r e p r e s s i o n . I n a d d i t i o n , p o s i t i o n i n g o f : t h e u n d e c a n u c l e o t i . d e f u r t h e r u p s t r e a m f r o m t h e m a t u r e c o d i n g r e g i o n i n c r e a s e d t h e l e v e l o f t r a n s c r i p t i o n ( D e F r a n c o e t a l . , 1 9 8 1 ) . V a l D e l e t i o n o f t D N A . t o r e s i d u e - 4 9 r e s u l t e d i n a 44% 4 i n c r e a s e i n V r e l a t i v e t o t h e u n d e l e t e d - 1 7 9 f l a n k max a c c o m p a n i e d b y a s l i g h t i n c r e a s e i n a p p . I t i s v e r y l i k e l y t h a t t h e i n c r e a s e i n V w h i c h r e s u l t e d f r o m t h e : J max r e m o v a l o f 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 - 7 0 a n d - 4 9 i s c o n s i s t e n t w i t h t h e e f f e c t s o f r e m o v i n g t h e t D N A ^ y s i n -h i b i t o r y s e q u e n c e . I t c a n b e s e e n f r o m F i g u r e 18 t h a t t h e d e l e t e d s e q u e n c e s b e t w e e n - 7 0 a n d - 5 6 s h a r e a l a r g e d e g r e e o f h o m o l o g y t o t h e t D N A ^ y s i n h i b i t o r y s e q u e n c e . F i g u r e 18 s h o w s t h a t t h e s e q u e n c e r e s p o n s i b l e f o r i n h i b i t i o n i n V a l t D N A ^ - i s m u c h m o r e e x t e n s i v e i n l e n g t h t h a n t h a t f o u n d i n t h e 5 ' - f l a n k o f t R N A L ^ S g e n e s ( D e F r a n c o e t a l.;1980, V a l 1 9 8 1 ) . T h e i n h i b i t o r y - l i k e s e q u e n c e f r o m t D N A ^ i s v e r y s i m i l a r t o t h e t D N A ^ 1 ^ 3 s e q u e n c e b e t w e e n p o s i t i o n - 7 0 a n d - 6 1 , b u t i s a l s o f o l l o w e d b y a t r a c t o f f i v e t h y m i d i n e s 79 F i g u r e 18 Comparison of i n h i b i t o r y - l i k e sequences Represented are the i n h i b i t o r y sequences from: tDNA A r g17D, tDNA L^ S, n u c l e o t i d e s -71 to -45, -49 to -45 and -26 to -18 from tDNA Vf 1. 4 80 I n h i b i t o r y sequence from pl7DArg GGATTTTTG I n h i b i t o r y sequence from tDNA L^ S •25 -14 GGCAGTTTTTG Nu c l e o t i d e s --45 from tDNA 71 to Val 4 -70 -56 -49 -45 ACCAGTTTTATTTTTGAYYYYYGGCAG Nuc l e o t i d e s --45 from tDNA 49 to Val 4 -49 -45 GGCAG. Nu c l e o t i d e s -26 to -18 from t D N A ^ 1 -26 -18 RYYYYYYYYR .= a n u c l e o t i d e mismatch with the t D N A ^ 2 S s e c l u e n c e Y= p y r i m i d i n e R= purine 81 f l a n k e d by two purine r e s i d u e s ( F i g u r e 18). The r e s u l t i s a repeated s e r i e s of t e r m i n a t i o n - l i k e sequences. The L v s tDNA ^ i n h i b i t o r y sequence alone r e s u l t e d i n poor t r a n -s c r i p t i o n a c t i v i t y when placed c l o s e to the gene. Val The g r e a t e s t i n c r e a s e i n V f o r tDNA , r e s u l t e d b max 4 from the d e l e t i o n of sequences between -49 and -45. A 66.5% i n c r e a s e i n V and a 76% drop i n K app r e s u l t e d max r m when the sequence GGCAG was d i s r u p t e d ( F i g u r e s 10 and 18). I n t e r e s t i n g l y , the sequence e x a c t l y matches the f i r s t L v s f i v e nucleotides of the tDNA ^ i n h i b i t o r y sequence (De Franco et a l . , 1981). I t i s t h e r e f o r e apparent that the i n h i b i t o r y sequence contained w i t h i n the 5'-flank of Val tDNA ^ -179 i s 25 n u c l e o t i d e s i n l e n g t h , l o c a t e d between p o s i t i o n s -45 and -70 i n the 5'-flank and i n f l u e n c e s the l e v e l of t r a n s c r i p t i o n 45 nucleotides away from the mature coding sequence. The extent of i n h i b i t i o n caused by t h i s sequence appears to be gr e a t e r than the i n h i b i t i o n found with the tDNA^2 S sequence c o n s i d e r i n g the l a r g e d i s t a n c e Val from the tDNA ^ gene. When the former was moved f u r t h e r upstream, away from the gene the l e v e l of t r a n s c r i p t i o n was enhanced (De Franco et a l . , 1981). I t would be i n t e r e s t i n g to t e s t the e f f e c t s of t h i s e xtensive sequence Val from tDNA ^ on the t r a n s c r i p t i o n of a tRNA gene i f i t were to be placed c l o s e r to the mature coding sequence. The dramatic i n c r e a s e i n V f o r A5'-45 did not max p e r s i s t with f u r t h e r d e l e t i o n of the 5' - f l a n k . The d e l e t i o n of only two n u c l e o t i d e s to p o s i t i o n -43 decreased the e f f i c i e n c y of t r a n s c r i p t i o n to the l e v e l of A5'-47. The 82 Val K app a l s o i n c r e a s e d to the l e v e l of tDNA . -179. In m r r 4 f a c t d e l e t i o n s to A5'-38 a l l r e s u l t e d i n an approximately 20% i n c r e a s e d V r e l a t i v e to -179 with no apparent max change i n K app. The f i r s t drop i n V from the l e v e l found f o r e max Val tDNA ^ -179, occured at n u c l e o t i d e -37, accompanied by a drop i n K m app. The d e l e t i o n of a s i n g l e T r e s i d u e at -38 gave r i s e to a 60% d i f f e r e n c e i n V and a 43% drop ° max r e l a t i v e to -179. D e l e t i o n to p o s i t i o n -33 had an even Val greater e f f e c t , lowering V 88% r e l a t i v e to tDNA , -179 b ° max 4 It would appear that sequences between -33 and -38 are r e q u i r e d f o r e f f i c i e n t t r a n s c r i p t i o n . N u c l e o t i d e s betweeen -33 and -45 could t h e r e f o r e be regarded as p o s i t i v e modulatory sequences. To date no conserved or unique p o s i t i v e modulatory sequence has been i d e n t i f i e d f o r any c l a s s I I I gene. Some sequence homology does e x i s t i n the 5'-flank of c e r t a i n D r o s o p h i l a tRNA genes, but the f u n c t i o n of such sequences i s unknown (Indik and T a r t o f , 1982). Yet the requirement f o r a " p o s i t i v e " 5 ' - f l a n k i n g sequence f o r e f f i c i e n t t r a n s c r i p t i o n has been shown i n the silkworm 5S RNA (Morton and Sprague, 1982, 1984) and tRNA genes (Sprague et a l . , 1980; Larson et a l . , 1983). Modulation of t r a n s c r i p t i o n by 5 ' - f l a n k i n g sequences a l s o occurs i n the tRNA genes of yeast, but to a l e s s e r extent (Shaw and Olson, 1984; Raymond and Johnson, 1983; Johnson and Raymond, 1984). The i n f l u e n c e s of the 5 ' - f l a n k i n g sequences upon 83 t r a n s c r i p t i o n are best observed i n homologous t r a n s - : c r i p t i o n systems (Schaack et a l . , 1984). T r a n s c r i p t i o n of A r 2 d e l e t i o n mutants d e r i v e d from a tRNA ^ gene (pArg) showed l e s s 5'-flank sequence dependence i n HeLa c e l l e x t r a c t than when t r a n s c r i b e d i n homologous Kc c e l l ex- : t r a c t . The e f f e c t of sequences i n the 5'-flank was a l s o found to be even l e s s dependent upon the source of e x t r a c t when the pArg d e l e t i o n mutants were t r a n s c r i b e d i n Xenopus oocyte and yeast e x t r a c t s (Schaack and S6*11 , 1985). These s t u d i e s would suggest that the dependence on the 5 ' - f l a n k i n g sequences i s a general phenomenon that v a r i e s i n magnitude between d i f f e r e n t organisms. The dependence on the e f f e c t of 5 ' - f l a n k i n g sequences upon the source of e x t r a c t i s supported by an experiment i n which pl7D Arg c o n t a i n i n g an i n h i b i t o r y sequence i n i t s 5'-f l a n k and was thereby i n a c t i v e i n homologous Kc c e l l ex-t r a c t , was t r a n s c r i b e d i n HeLa c e l l e x t r a c t only so long as no Kc c e l l e x t r a c t was added to the r e a c t i o n (Dinger-mann et a l . , 1982). The e f f e c t s of d e l e t i o n s from Val tDNA ^ -179 on t r a n s c r i p t i o n have not been t e s t e d i n l e s s s t r i n g e n t heterologous e x t r a c t s . The only e x t e n s i v e study of the tRNA 5'-flank p r i o r Arg to t h i s i n v e s t i g a t i o n was of a D r o s o p h i l a tRNA ^~ gene (Schaack et a l . , 1984). Though there i s l i t t l e sequence Val homology between pArg and tDNA ^ , both genes c o n t a i n a p e n t a n u c l e o t i d e TNNCT between p o s i t i o n s -38 and -33 (see Val F i g u r e 19). D e l e t i o n tDNA ^ A5'-38 showed an i n c r e a s e d l e v e l of t r a n s c r i p t i o n r e l a t i v e to -179. D e l e t i o n s i n 84 Fi g u r e 19 TNNCT a TME? Various tRNA genes, the p o s i t i o n s of t h e i r conserved p e n t a n u c l e o t i d e and r e l a t i v e t r a n s c r i p t i o n e f f i c i e n c i e s are t a b u l a t e d . The symbols f o r t r a n s c r i p t i o n e f f i c i e n c i e s a re: += e f f i c i e n t +(-)= i n e f f i c i e n t -(+)= <1% of w.t. - = no t r a n s c r i p t i o n Footnotes 1 Rajput et a l . , 1982 2 MacKay and Spiegelman, per s o n a l communication 3 Leung, per s o n a l communication 4 C r i b b s , 1982; St. Louis and Spiegelman, 1985 5 Schaack et a l . , 1984 6 DeFranco et a l . , 1982 7 Dingermann et a l . , 1982 8 Cooley et a l . , 1984 9 St. Louis and Spiegelman, 1985 Gene tDNA V a l p D t s s - o . e ^ 1 P Y H 4 8 - p A r g p67R-Arg p E l . 7 - A r g pE3.8-Arg V a l pDt92R * V a l pDt78R pAva4-Arg Sequence T r a n s c r i p t i o n •38 -34 -23 -19 E f f i c i e n c y TCGCT & TCTCT •39 - 3 5 1 TGGCT •38 - 3 4 s TTTCT -36 - 3 2 3 TGCCT -40 r 3 6 3 TATC,J -27 - 2 2 3 TAGCT -39 -35 -29 - 2 5 2 TGTCT & TGTCT -48 -44 -44 -40 TTGCT & TAACT No TME 2 No TME 3 + + ( - . ) : pl7D A r g No TME' p35D Arg p l l F Arg pHi s ; ( p 4 8 F H i s ) p y H i s P D t 3 9 R - L ^ pDt59R Ly s " 5 Ly s P D t l 6 " 4 - ? 7 4 S G 3T p D t l 6 - 4 - 7 7 7 P D t 5 - Ser No TME 7 . •'. ( t o -40) No TME 7 -46 - 4 2 8 TTGCT -22 - 1 8 8 TTGCT -36 - 3 2 6 TACCT -38 -34 6 TTGCT -39 -351* TAGCT No TME'" No TME ""• - ( + ) 7 - ( + ) 7 + 8 - ( + ) 8 + (-) 9 + (-) 9 + 86 pArg extending to p o s i t i o n -33 r e s u l t e d i n a 35% lower t r a n s c r i p t i o n e f f i c i e n c y than obtained with pArg and t r a n s -c r i p t i o n e f f i c i e n c y was only 12% of pArg with d e l e t i o n s extending to p o s i t i o n -32 (Schaack and So'll, 1985). The data i s c o n s i s t e n t with the r e s u l t s found i n t h i s study which showed a dramatic decrease 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 with the e l i m i n a t i o n of the p e n t a n u c l e o t i d e (Table I I ) , Val though the e f f e c t s are more pronounced f o r tDNA ^ . The data suggests that the p e n t a n u c l e o t i d e f u n c t i o n s as an element which a c t s to enhance t r a n s c r i p t i o n . Therefore f o r d i s c u s s i o n purposes the sequence w i l l be r e f e r r e d to as a T r a n s c r i p t i o n Modulation Element (TME). Val The 5 ' - f l a n k i n g r e g i o n of tDNA ^ con t a i n s two copies of the TME ( F i g u r e 10). However, the TME l o c a t e d between p o s i t i o n s -19 and -23 would appear to l a c k any p o s i t i v e i n f l u e n c e on t r a n s c r i p t i o n . The r e l a t i o n s h i p between the p o s i t i o n of the TME and i t s e f f e c t on t r a n s c r i p t i o n i s a l s o i l l u s t r a t e d by the t r a n s c r i p t i o n of the -20 d e l e t i o n s . D e l e t i o n -20 F' has a s i n g l e TME l o c a t e d between p o s i t i o n s -39 and -43, due to sequences found i n the v e c t o r . The TME i n A5'—20 R' i s between p o s i t i o n s -19 and. -23 and was crea t e d through a combination of vecto r and 5 ' - f l a n k i n g D r o s o p h i l a sequences. A5'-20 R' was a very i n e f f i c i e n t template as compared to i t s counterpart A5'-20 F', which was t r a n s c r i b e d at 50% higher e f f i c i e n c y (Table I I ) . I t would th e r e -f o r e appear that the p o s i t i o n of the TME r e l a t i v e to 87 Fi g u r e 20-A R e s t o r a t i o n of the TME i n the Val 5'-flank of tDNA . d e l e t i o n mutants 4 A5'-20 and A5'-17 The n u c l e o t i d e sequences f o r A5*-20 F', A5'-20 R' and A5'-17 and t h e i r a d j o i n i n g vector sequences are presented. A5'-20 F' has the TME t r a n s l o c a t e d to p o s i t i o n -38. A5'-20 R* has the TME t r a n s -l o c a t e d to p o s i t i o n -18 and con t a i n s a TME-like sequence at p o s i t i o n -41. A5'-17 a l s o c o n t a i n s a TME-like sequence, but l o c a t e d at p o s i t i o n -39. -B R e s t o r a t i o n of a TME_like sequence i n the 5'-flank of Val tDNA , d e l e t i o n mutants A5'-37 4 and A5'-35 The n u c l e o t i d e sequences f o r A5'-38, -37, -35 and -33 and t h e i r a d j o i n i n g vector sequences are presented. 88 A + -AO -30 -20 A5'-20 F* AGCTTGG CTGCAGGTCG ACGGATCCCC CTGCCGCTT 4-A5'-20 R' ACAGCTA TGACCATGAT TACGAATTCC CTGCCGCTT 4-A5'-17 R' AAACAGC TATGACCATG ATTACGAATT CCCCCGCTT B + -30 A5'-38 F' GGATCCCCTCGCT GAA 4-A5'-37 R' CGAATTCCCCGCT GAA 4-A5'-35 F* GGATCCCCT GAA 4-A5'-33 F' GGATCCCC GAA Sequences from pUC 8 are u n d e r l i n e d , - i d e n t i f i e s the TME or TME-like sequences, 4- i n d i c a t e s d e l e t i o n end-point 89 the mature coding sequence e f f e c t s the l e v e l of t r a n s -c r i p t i o n . The 5 ' - f l a n k i n g sequences of a number of other D r o s o p h i l a tRNA genes which had been t r a n s c r i b e d ±ri v i t r o was examined. Figure 19 c o n t a i n s a l i s t of v a r i o u s tRNA genes and the p o s i t i o n s of t h e i r p e n t a n u c l e o t i d e s i n the 5'- f l a n k . Other v a l i n e and a r g i n i n e tRNA genes which c o n t a i n the TME between -30 and -40 are e f f i c i e n t i n t r a n s c r i p t i o n (as i n d i c a t e d by a + i n Fi g u r e 19). In the case of A5'-20R', the phenomenon r e s u l t i n g i n the g r e a t l y reduced r a t e ; of t r a n s c r i p t i o n would seem s i m i l a r to that found f o r pE3.8-Arg which has a TME t r a n s l o c a t e d c l o s e r to the mature coding sequence, r e s u l t i n g i n an i n -e f f i c i e n t l e v e l of t r a n s c r i p t i o n ( i n d i c a t e d by a +(-) i n Val F i g u r e 19). A tRNA ^ gene contained on the plasmid pDt92-R which has 4 copies of the TME does not d i r e c t t r a n s -c r i p t i o n and a p s e u d o - h i s t i d i n e tRNA gene (p | His) with a TME d i s p l a c e d c l o s e r to the mature coding sequence (-18 to -22) t r a n s c r i b e s at l e s s than 1% e f f i c i e n c y when His compared to i t s counte r p a r t pHis. The tRNA gene (pHis) c o n t a i n s a TME between p o s i t i o n s -42 and -46 i n the 5'-flank and i s e f f i c i e n t i n d i r e c t i n g t r a n s c r i p t i o n (Cooley et a l . , 1984). Therefore the positioning of the TME 5' to -30 does not seem to lower t r a n s c r i p t i o n e f -f i c i e n c y . Val D e l e t i o n end-point tDNA ^ A5'-17 a l s o t r a n s c r i b e d at an unusually high l e v e l ; 41% higher than A5/-20 R\.< An examination of upstream v e c t o r sequences re v e a l e d an 90 a l t e r e d TME sequence between p o s i t i o n s -40 and -44. The f i r s t T i n the p e n t a n u c l e o t i d e sequence i s r e p l a c e d with a C, r e s u l t i n g i n the sequence CNNCT ( F i g u r e 20-A). I t may be that the p y r i m i d i n e s u b s t i t u t i o n of the TME r e s u l t s i n the 6% lower t r a n s c r i p t i o n e f f i c i e n c y than f o r A5'-20 R'. I t should be noted that e f f i c i e n t t r a n s c r i p t i o n does not r e q u i r e the presence of two copies of the TME as i l l u s t r a t e d f o r pArg and other tRNA genes l i s t e d i n F i g u r e 19. However, tRNA v a l i n e and a r g i n i n e genes with no -TME f a i l .to t r a n s -c r i b e or are t r a n s c r i b e d very i n e f f i c i e n t l y (Spiegelman and Mackay, unpublished r e s u l t s ) . The requirement f o r a TME i n a r g i n i n e and v a l i n e genes holds true i n a l l cases s t u d i e d , with the p o s s i b l e excep-t i o n f o r pE1.7-Arg which probably c o n t a i n s a TME, though the sequence data i s p r e l i m i n a r y (Spiegelman, personal com m u n i c a t i o n ) . In a d d i t i o n , given that every 10 bp cons-t i t u t e s one turn i n the DNA h e l i x , i t would appear that TMEs which enhance the l e v e l of t r a n s c r i p t i o n (between -30 and -46) f a l l on both s i d e s of the h e l i x . T h e r e f o r e , i t i s u n l i k e l y that the p o s i t i o n of the TME on the sur-face of the h e l i x a l t e r s i t s e f f e c t on t r a n s c r i p t i o n . The i n f l u e n c e of the TME on t r a n s c r i p t i o n probably'occurs due to i t s p o s i t i o n i n the 5'-flank i . e . d i s t a n c e r e l a t i v e to the D and T c o n t r o l r e g i o n s . The TME i s a l s o found i n the 5 ' - f l a n k i n g regions of v a r i o u s other tRNA genes such as 91 L v s tRNA £ which d i r e c t s e f f i c i e n t t r a n s c r i p t i o n (DeFranco et a l . , 1983) and a l s o i n the 5'-flank of t R N A L e U and t R N A I l e U genes, though t h e i r t r a n s c r i p t i o n s have not been st u d i e d or reported (Robinson and Davidson, '1981). While t r a n s c r i p t i o n e f f i c i e n c y i s c o r r e l a t e d with the presence of the p e n t a n u c l e o t i d e TNNCT f o r v a l i n e and a r g i - • nine tRNA genes, the s e r i n e tRNA genes seem to f a l l i n an-other category, s i n c e two s e r i n e genes d i r e c t t r a n s c r i p t i o n i n the absence of a TME ( F i g u r e 19 and C r i b b s , 1982). In * S 6 I * f a c t , the l e v e l of t r a n s c r i p t i o n f o r the tRNA ^ gene from Val pDt5 i s s i m i l a r to that found with tDNA ^ . I n t e r e s t i n g l y , S G r the d e l e t i o n of a tRNA Zj a l l ogene (pDtl6-4) to p o s i t i o n -36 which d i s r u p t s a TME, r e s u l t e d i n a 4 - f o l d decrease i n i t s r a t e of t r a n s c r i p t i o n ( S t . Louis and Spiegelman, 1985). The p o s s i b l e r o l e ( s ) of the TME i s s t i l l open to s p e c u l a t i o n at t h i s p o i n t i n time. Given that the TME sequence (TNNCT) .-.contains two v a r i a b l e n u c l e o t i d e s , the r o l e of the p e n t a n u c l e o t i d e may be s p e c i f i c f o r a p a r t i c u l a r , gene, or more g e n e r a l , depending on which n u c l e o t i d e s are s u b s t i t u t e d . I t may be that the TME overcomes the negative or i n h i b i t o r y e f f e c t s of sequences l o c a t e d i n the 5'-f l a n k i n g r e g i o n s of tRNA genes. By extension i t may be that 5 ' - f l a n k i n g sequences of tRNA genes which do not c o n t a i n a TME sequence^ and are yet e f f i c i e n t l y t r a n s c r i b e d , do not co n t a i n i n h i b i t o r y or negative modulatory sequences which would r e q u i r e the presence of a .TME f o r e f f i c i e n t t r a n s - . c r i p t i o n . The TME could be one of a c l a s s of p o s i t i v e 92 modulatory elements, most of which have not yet ibeen i d e n -t i f i e d . As p r e v i o u s l y d i s c u s s e d , the sequence TNNCT i s not Vv'eatyy s p e c i f i c s i n c e any combination of n u c l e o t i d e s could be s u b s t i t u t e d i n place of NN ( r e f e r to F i g u r e 19); a l s o , a study i n v o l v i n g the exchange of p o s i t i v e 5 ' - f l a n k s of pArg and pHis i n which both genes contained a TME, showed them to be incompatible (Cooley et a l . , 1984). I t i s t h e r e -f o r e h i g h l y l i k e l y that 5 ' - f l a n k i n g sequences are not a r b i -t r a r y and are s p e c i f i c f o r t h e i r own p a r t i c u l a r tRNA genes. Val D e l e t i o n s of the tDNA . 5'-flank to -30 r e s u l t e d :.in. a 4 91% drop i n V r e l a t i v e to -179 and d e l e t i o n of an a d d i -r max t i o n a l n u c l e o t i d e to p o s i t i o n -29 a b o l i s h e d t r a n s c r i p t i o n completely. D e l e t i o n of pArg A5'-32 r e s u l t s i n t r a n s -c r i p t i o n at only 12% of pArg e f f i c i e n c y (Schaack and So'll, Val 1985). Furt h e r d e l e t i o n s of the tDNA ^ 5'-flank to p o s i -t i o n -20 g r a d u a l l y i n c r e a s e the app to the l e v e l of -179 and allow t r a n s c r i p t i o n at 90% decreased e f f i c i e n c y , with a s l i g h t i n c r e a s e at -26. S i m i l a r r e s u l t s were ob-t a i n e d with d e l e t i o n s f o r pArg but with s l i g h t i n c r e a s e s 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 at -25 and -30. I t would t h e r e -f o r e seem that n u c l e o t i d e s between -30 and -20 of the Val tDNA ^ 5'-flank must be i n h i b i t o r y s i n c e t h e i r removal r e s t o r e s t r a n s c r i p t i o n . There s t i l l remain minor d e v i a t i o n s to be e x p l a i n e d . D e l e t i o n s A 5'-47 and A 5'-26 had l e v e l s of t r a n s c r i p t i o n that d e v i a t e d from the apparent tre n d . S i m i l a r obser-v a t i o n s were made with A 5,'-30 and A5'-25 of pArg (Schaack et a l . , 1984). These d e v i a t i o n s ; may be the r e s u l t of r e -93 p l a c i n g and r e p o s i t i o n i n g of 5 ' - f l a n k i n g sequences with sequences contained i n the v e c t o r . D e l e t i o n s extending to p o s i t i o n -10 and beyond the i n i t i a t i o n point s e v e r e l y a f f e c t e d t r a n s c r i p t i o n e f -f i c i e n c y , with no d e t e c t a b l e t r a n s c r i p t i o n f o r A5'-2 (<1% of -179). In pArg, A 5 * — 11 r e s u l t e d i n a t r a n s c r i p -t i o n e f f i c i e n c y of l e s s than 1%. I t i s t h e r e f o r e c l e a r that 5 ' - f l a n k i n g sequences are r e q u i r e d f o r tRNA t r a n s -c r i p t i o n s i n c e the replacement of 5 ' - f l a n k i n g sequences with u n r e l a t e d tRNA 5 ' - f l a n k i n g sequences or v e c t o r sequences does not r e s t o r e t r a n s c r i p t i o n e f f i c i e n c y . These data lead to the c o n c l u s i o n that sequences contained i n the 5 ' - f l a n k i n g regions of tRNA genes are to a high degree r e s p o n s i b l e f o r both p o s i t i v e and negative modu-l a t i o n of t r a n s c r i p t i o n . The modulatory sequences f o r pArg had p r e v i o u s l y been de f i n e d by p o s i t i o n s -60, -33 and -11. In t h i s study we can d e l i m i t the p o s i t i o n s of Val modulatory sequences i n the 5'-flank of tDNA ^ by -70, -45, -38, -33, -20, -10 and -2. Sequences l o c a t e d between -70 and -45 as w e l l as -30 and -20 may be regarded as negative modulatory sequences. Sequences between the mature coding sequence and the i n i t i a t i o n point ( G at p o s i t i o n -9; Spiegelman, unpublished r e s u l t s ) along with sequences contained between -10 and -20 account f o r 5% and 10% of t r a n s c r i p t i o n r e l a t i v e to -179, r e s p e c t i v e l y . Sequences contained between p o s i t i o n s -38 and -45 are p o s i t i v e modulatory sequences that enhance the l e v e l of t r a n s c r i p t i o n , with the TME sequence r e q u i r i n g s t i l l f u r t h e r c h a r a c t e r i z a t i o n . The data i n Table II shows that there i s a substan-t i a l l e v e l of t r a n s c r i p t i o n s t i l l remaining (30% decrease r e l a t i v e to -179) f o r A5'-35, even though the TME i s d i s r u p t e d . An examination of the border sequences ( F i g u r e 20-B) rev e a l e d the r e s t o r a t i o n of TME-like se-quences f o r A5'-37 and -35 as a r e s u l t of sequences con-t a i n e d i n the vect o r ., a case s i m i l a r to that found i n A5'-17 R' ( F i g u r e 20-A). I t would t h e r e f o r e appear that the 5'-flank of D r o s o p h i l a tRNA genes c o n s t i t u t e s a s e r i e s of p o s i t i v e and negative modulatory sequences r e s p o n s i b l e f o r i n -c r e a s i n g or dec r e a s i n g K app-and V v , which together ° G m r r max b determine the o v e r a l l e f f i c i e n c y of that gene. This i s i n accordance with previous f i n d i n g s (Schaack et a l . , 1984; Sharp et a l . , 1983). However, we do not know how w e l l the values f o r K app and V r e f l e c t the true m r r max l e v e l s of t r a n s c r i p t i o n i_n vivo. A model f o r t r a n s c r i p t i o n has been d e r i v e d from the a n a l y s i s of values f o r K app and V from a s e r i e s of m max s e r i n e tRNA genes ( S t . Louis and Spiegelman, 1985). The model p o s t u l a t e d that a K m app r e f e r r e d to the a f f i n i t y of some component(s) i n the r e a c t i o n f o r the DNA template and that the 5 ' - f l a n k i n g sequences of these tRNA^ e r genes would not a f f e c t the app. However, the data presented i n t h i s study show that d e l e t i o n i n the 5'-flank do cause S 6 r* changes i n K m app. Assuming that the tRNA genes a l s o c o n t a i n modualtory sequences i n t h e i r 5 ' - f l a n k s , s i m i l a r 95 e f f e c t s might have been n o t i c e d i n t h e i r s t u d i e s had they c a r r i e d out analogous experiments. The data from Val t h i s study with tDNA ^ are c o n s i s t e n t with those d e r i v e d f o r pArg i n that the 5 ' - f l a n k i n g sequences do c o n t a i n modulatory sequences shown to c o n t r i b u t e to the s t a b i l i t y : of t r a n s c r i p t i o n complexes ( S c h a a c k e t a l . , 1984; Johnson-Burke and S o l i , 1985). Dependence of t r a n s c r i p t i o n on the 5 ' - f l a n k i n g se-quence i s a general phenomenon found f o r tRNA (Sharp et a l . , 1981) and 5S RNA genes (Morton and Sprague, 1984). T r a n s c r i p t i o n of tRNA genes i s b e l i e v e d to be d i r e c t e d by two promoter regions which bind t r a n s c r i p t i o n f a c t o r s ( C i l i b e r t o et a l . , 1982; Sharp et a l . , 1983; Dingermann et a l . , 1983; Johnson et a l . , 1984; Johnson-Burke, 1985). The r o l e of D and T c o n t r o l regions i n binding of f a c t o r s and d i r e c t i n g t r a n s c r i p t i o n has been s t u d i e d i n great d e t a i l (Johnson-Burke, 1983, 1985; S t i l l m a n et a l . , 1984; Ruet et a l . , 1984). Studi e s using point mutations w i t h i n the i n t e r n a l promoters of tRNA genes (Ryan et a l . , 1979; Ciampi et a l . , 1982; Murphy and B a r a l l e , 1983, 1984; Traboni et a l . , 1982, 1984; Stewart et a l . , 1985) found that n u c l e o t i d e changes at c e r t a i n conserved n u c l e o t i d e p o s i t i o n s a f f e c t e d the l e v e l of t r a n s c r i p t i o n , however, these changes were not c o n s i s t e n t between the d i f f e r e n t tRNA genes t e s t e d . For example, a n u c l e o t i d e s u b s t i t u t i o n f o r C19 created a t e n - f o l d decrease i n the _in v i t r o t r a n s -c r i p t i o n of a yeast tRNA^ e U .gene (Newman et a l . , 1983), but s u b s t i t u t i o n s i n G18 and G19 did not cause a dramatic 96 A r s decrease i n t r a n s c r i p t i o n of a D r o s o p h i l a tRNA 6 gene (Stewart e t ; a l . , 1985). A l s o , an A19 change i n Xenopus Met tRNA gene causes a 30% drop i n the l e v e l of t r a n s -c r i p t i o n (Folk arid H o f s t e t t e r , 1983), yet the same n u c l e o t i d e s u b s t i t u t i o n does not a f f e c t the l e v e l of T v r t r a n s c r i p t i o n i n a yeast tRNA y gene ( A l l i s o n et a l . , 1983). Current data suggests that the occurrence of i n -v a r i a n t n u c l e o t i d e s i n tRNAs are a r e s u l t of t h e i r r e -quirement i n tRNA s t r u c t u r e and f u n c t i o n and not an o b l i g a t o r y promoter f u n c t i o n (Stewart et a l . , 1985). Therefore there i s a dual r o l e f o r conserved sequences of c o n t r o l regions i n tRNA genes; conserved r e s i d u e s c o n t r i b u t e to the binding of Pol I I I t r a n s c r i p t i o n f a c -t o r s and a l s o c o n t r i b u t e to the f u n c t i o n of tRNA s t r u c -ture depending on the degree of homology to a c e r t a i n consensus sequence. I t i s w e l l e s t a b l i s h e d that the bound f a c t o r s allow or serve as a s i g n a l to Pol I I I to recogn i z e the u n i t as a tRNA gene with an assigned p o i n t of t r a n s c r i p t i o n i n i t i a t i o n ( H a l l et a l . , 1982; Lassar et a l . , 1983; Sharp et a l . , 1983). T r a n s c r i p t i o n e f f i c i e n c y and the command to i n i t i a t e would then be d i r e c t e d by sequences contained i n the 5'-flank of the tRNA gene. In f a c t Schaack et a l . (1984) have suggested that the i n i t i a l i n t e r a c t i o n of RNA Pol I I I with the gene may occur i n the 5'- f l a n k . I t remains to determine whether t r a n s c r i p t i o n modu-l a t i o n i n v o l v e s the i n t e r a c t i o n of the 5 ' - f l a n k i n g sequence with P ol I I I d i r e c t l y , through a d d i t i o n a l t r a n s -97 c r i p t i o n f a c t o r s that may bind to say, the TME, or a mechanism i n v o l v i n g both phenomena. A number of e x p e r i -ments may be c a r r i e d out to support the n o t i o n that the TME has a f u n c t i o n . E x i s t i n g TME sequences may be a l t -ered by s i t e - s p e c i f i c mutagenesis or placement of a TME sequence between p o s i t i o n s -30 and -40 of a poorly t r a n s -Val c r i b i n g tRNA gene, such asa-tRNA ^ gene from pDt78R. The t r a n s c r i p t i o n of such a l l o g e n e s would f u r t h e r support the requirement of a TME sequence. In a d d i t i o n , compe-t i t i o n experiments could be c a r r i e d out with i s o l a t e d 5 ' - f l a n k i n g sequences that e i t h e r c o n t a i n or l a c k TME sequences. Such s t u d i e s using i s o l a t e d p u r i f i e d complexes would shed more l i g h t on the mechanism of i n t e r a c t i o n i n -v o l v i n g s p e c i f i c f a c t o r s and/or Pol I I I . 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