The regulation of actin gene expression in "Xenopus" embryos

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T H E R E G U L A T I O N O F A C T I N G E N E E X P R E S S I O N

IN X E N O P U S E M B R Y O S .

A t h e s i s s u b m i t t e d for t h e d e g r e e o f d o c t o r o f p h i l o s o p h y .

N i c h o l a s D. Lakin.

A n i m a l m o l e c u l a r g e n e t i c s g r o u p D e p a r t m e n t of B i o l o g i c a l S c i e n c e s U n i v e r s i t y o f W a r w i c k .

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T H IS T H ESIS H A S B EEN M IC R O F IL M E D E X A C T L Y A S R E C E IV E D

T H E BR ITISH ( LIBRARY

D O C U M E N T SUPPLY C E N T R E

Boston Spa, Wetherby

W est Yorkshire, LS23 7 B Q

i C O N T E N T S .

T a b l e o f c o n t e n t s . ii

T a b l e o f f i g u r e s . xi

A c k n o w l e d g e m e n t s . xvii

D e c l a r a t i o n . x viii

S u m m a r y . x ix

ii T a b l e o f c o n t e n t s .

Page C h a p t e r 1: I N T R O D U C T I O N .

1.1. T h e e a r l y d e v e l o p m e n t o f X e n o p u s . 1 1.2. T h e a c t i n g e n e f amily. 5 1.3. A c t i n g e n e s a n d the r e g u l a t i o n of t h e i r 8

e x p r e s s i o n .

1 . 3 . 1 . A c t i n g e n e s t r u c t u r e a n d e x p r e s s i o n . 8 1 . 3 . 2 . D N A s e q u e n c e e l e m e n t s i n v o l v e d in the 13

e x p r e s s i o n of a c t i n g e n e s .

1 . 3 . 3 . C i s - a c t i n g s e q u e n c e s a n d t r a n s - a c t i n g 15 f a c t o r s i n v o l v e d in the c o n t r o l o f c a r d i a c

a c t i n g e n e e x p r e s s i o n .

1 . 3 . 4 . C i s - a c t i n g s e q u e n c e s a n d t r a n s - a c t i n g 19 f a c t o r s i n v o l v e d in t he c o n t r o l o f s k e l e t a l

a c t i n g e n e e x p r e s s i o n .

1 . 3 . 5 . T h e r o l e o f the C A r G b o x in t he e x p r e s s i o n 25 o f the p r o t o - o n c o g e n e c - f o s .

1 .4. E l e m e n t s i n v o l v e d in the e x p r e s s i o n of o t h e r 30 m u s c l e - s p e c i f i c genes.

C h a p t e r 2: M A T E R I A L S .

2 . 1 . G e n e r a l m a t e r i a l s . 44 2 . 2 . S t o c k s o l u t i o n s . 45 2 . 3 . B a c t e r i o l o g i c a l m edia. 45 2 . 4 . B a c t e r i a a n d pla s m i d s . 46 2 . 4 . 1 . G e n o t y p e s of E. c o l i s t r a i n s . 46 2 . 4 . 2 . P l a s m i d vec t o r s . 46

C h a p t e r 3: M E T H O D S .

3 . 1 . O o c y t e s e g g s a nd e m b r y o s . 47 3 . 1 . 1 . O o c y t e s . 47 3 . 1 . 2 . E ggs a n d embryos. 47 3 . 2 . M i c r o - i n j e c t i o n of f e r t i l i s e d eggs. 48 3 . 2 . 1 . P r e p a r a t i o n of D N A f or m i c r o - i n j e c t i o n . 48 3 . 2 . 2 . M i c r o - i n j e c t i o n of DNA. 48 3 . 3 . D i s s e c t i o n of e m b r y o s . 49 3 . 4 . N u c l e i c a c i d i s o l a t i o n f r o m em b r y o s . 49 3 . 5 . S t a n d a r d s u b - c l o n i n g t e c h n i q u e s . 50 3 . 5 . 1 . R e s t r i c t i o n e n z y m e d i g e s t s . 50 3 . 5 . 2 . P r e p a r a t i o n of p l a s m i d v e c t o r s f or 50

s u b - c l o n i n g .

3 . 5 . 3 . L i g a t i o n s . 50 3 . 5 . 4 . E. c o l i p l a s m i d t r a n s f o r m a t i o n s . 51 3 . 5 . 5 . P o l y m e r a s e c h a i n r e a c t i o n . 52 3 . 6 . S mall s c a l e p r e p a r a t i o n o f p l a s m i d DNA. 52 3 . 7 . L arge s c a l e p r e p a r a t i o n o f p l a s m i d D N A and 53

p u r i f i c a t i o n b y c a e s i u m c h l o r i d e / e t h i d i u m b r o m i d e c e n t r i f u g a t i o n .

3 . 8 . E l e c t r o p h o r e s i s of n u c l e i c a c i d s . 55 3 . 8 . 1 . N o n - d e n a t u r i n g a g a r o s e g e l s . 55 3 . 8 . 2 . I s o l a t i o n of r e s t r i c t i o n f r a g m e n t s f r o m 55

a g a r o s e gels.

3 . 8 . 3 . N o n - d e n a t u r i n g p o l y a c r y l a m i d e gels. 56 3 . 8 . 4 . D e n a t u r i n g p o l y a c r y l a m i d e g els. 56 3 . 9 . P r e p a r a t i o n of s y n t h e t i c o l i g o n u c l e o t i d e s . 57 3 . 9 . 1 . G el i s o l a t i o n of s y n t h e t i c D N A 57

o l i g o n u c l e o t i d e s .

3 . 9 . 2 . A n n e a l i n g of c o m p l e m e n t a r y s y n t h e t i c 58 o l i g o n u c l e o t i d e s

3 . 1 0 . M e t h o d s o f r a d i o l a b e l l i n g D N A . 59 3 . 1 0 . 1 . N i c k t r a n s l a t i o n . 59 3 . 1 0 . 2 . E n d - l a b e l l i n g D N A w i t h ^ - 3 2 P - A T P and T4 59

p o l y n u c l e o t i d e k i n a s e .

3 . 1 0 . 3 . L a b e l l i n g D NA f r a g m e n t s b y i n - f i l l i n g . 6 0 3 . 1 1 . S e q u e n c i n g of p l a s m i d DNA. 61 3 . 1 2 . S o u t h e r n b l o t t i n g . 6 3 3 . 1 3 . P r i m e r e x t e n s i o n . 65 3 . 1 4 . C h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e a ssays. 66 3 . 1 4 . 1 . P r e p a r a t i o n of c h l o r a m p h e n i c o l 66

iv

3 .14.2. C h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e a s s a y s

66

3.15. P r e p a r a t i o n o f p r o t e i n e x t r a c t s for b a n d 68 s h i f t a s s a y s a n d D N A f o o t p r i n t i n g .

3 . 15.1. X e n o p u s l a e v i s o o c y t e s , e g g s and e m b r y o s . 68 3.15.2. A d u l t X e n o p u s l a e v i s t i s s u e s . 69 3.16. B a n d s h i f t a s s a y s . 69 3.17. A n t i b o d y b a n d s h i f t a s s a y s . 70 3.18. M e t h y l a t i o n i n t e r f e r e n c e a n d m e t h y l a t i o n 71

p r o t e c t i o n f o o t p r i n t i n g .

R E S U L T S A N D D I S C U S S I O N .

C h a p t e r 4: T h e p r e l i m i n a r y l o c a l i s a t i o n o f c i s - a c t i n g r e g u l a t o r y s e q u e n c e s i n the p r o m o t e r o f the X e n o p u s b o r e a l i s s k e l e t a l a c t i n g ene.

I n t r o d u c t i o n . 73

4.1. P r e l i m i n a r y c h a r a c t e r i s a t i o n of the X e n o p u s 76 b o r e a l i s s k e l e t a l a c t i n g e n e p romoter.

4.2. C A r G b o x 3 is e s s e n t i a l f or the full a c t i v i t y of 79 t he X e n o p u s b o r e a l i s s k e l e t a l a c t i n gene.

vi

C h a p t e r 5: T h e p r o m o t e r o f the X e n o p u s b o r e a l i s s k e l e t a l a c t i n g ene b i n d s t r a n s - a c t i n g f a c t o r s in v i t r o .

I n t r o d u c t i o n . 84

5 .1. I d e n t i f i c a t i o n o f a s e q u e n c e - s p e c i f i c t r a n s - 85 a c t i n g f a c t o r ( s ) that i n t e r a c t s w i t h the

X e n o p u s b o r e a l i s s k e l e t a l a c t i n g e n e p r o m o t e r in v i t r o .

5.2. P r e l i m i n a r y l o c a l i s a t i o n o f f a c t o r b i n d i n g s i t e s 87 in t h e p r o m o t e r o f the X e n o p u s b o r e a l i s s k eletal a c t i n g e n e p r o m o t e r .

5.3. D N A f o o t p r i n t i n g of the - 1 6 7 / - 4 2 r e g i o n of the 90 X e n o p u s b o r e a l i s s k e l e t a l a c t i n g ene p r omoter.

5.4. C A r G b o x 3 a n d C A r G b oxl c a n c o m p e t e f or the 93 b i n d i n g o f f a c t o r s to the X e n o p u s b o r e a l i s

s k e l e t a l a c t i n gene.

5.5. D i s c u s s i o n . 95

C h a p t e r 6: C A r G b o x 3 a n d C A r G b o x l b ind s i m i l a r , yet d i s t i n c t p r o t e i n s .

I n t r o d u c t i o n . 98

6.1. C A r G b o x l a n d C A r G b ox3 s y n t h e t i c 99 o l i g o n u c l e o t i d e s b i n d s e q u e n c e - s p e c i f i c

f a c t o r s jjn v i t r o .

vii

6 . 3 . C A r G b o x l b i n d s a p r o t e i n w h i c h i s a n t i g e n i c a l l y 104 r e l a t e d t o the s e r u m r e s p o n s e f a c t o r .

6 . 4 . C A r G b o x 3 b i n d s a p r o t e i n w h i c h i s a n t i g e n i c a l l y 108 d i s t i n c t f r o m S R F .

6 . 5 . D i s c u s s i o n .

Ill-C h a p t e r 7: T h e s p a t i a l a nd t e m p o r a l d i s t r i b u t i o n of C A r G b o x b i n d i n g f a c t o r s in X e n o p u s .

I n t r o d u c t i o n . 118 7 .1. C A r G b o x b i n d i n g a c t i v i t i e s a r e p r e s e n t 119

t h r o u g h o u t the e mbryo.

7 .2. C A r G b o x 3 b i n d s a f a c t o r ( s ) t h a t is p r e s e n t 121 t h r o u g h o u t e a r l y X e n o p u s d e v e l o p m e n t .

7 .3. D i s c u s s i o n . 123

C h a p t e r 8: S e q u e n c e s in a d d i t i o n to t h e C A r G b ox m o t i f s a re r e q u i r e d f or t h e e x p r e s s i o n o f t h e X e n o p u s b o r e a l i s s k e l e t a l a c t i n g e n e .

I n t r o d u c t i o n . 129 8.1. A 1 56 n u c l e o t i d e r e g i o n o f the X e n o p u s b o r e a l i s 130

v i l i

8 . 2 . C A r G b o x 3 , C A r G b o x l , o r a c o m b i n a t i o n o f b o t h 13A are not s u f f i c i e n t to d r i v e the t i s s u e - s p e c i f i c e x p r e s s i o n o f a h e t e r o l o g o u s p r o m o t e r .

8 .3. A r e g i o n o f t he X e n o p u s b o r e a l i s s k e l e t a l a c t i n 138 g e n e p r o m o t e r u p s t r e a m o f the C A r G b o x 3 m o t i f

is r e q u i r e d f o r the e x p r e s s i o n o f t he g ene w h e n s e q u e n c e s d o w n s t r e a m o f n u c l e o t i d e - A 2 are a b s e n t f r o m t he g ene.

8 . A. D i s c u s s i o n . 1A2

C h a p t e r 9: T h e u p s t r e a m r e g u l a t o r y e l e m e n t of the X e n o p u s b o r e a l i s s k e l e t a l a c t i n gene b i n d s a t r a n s - a c t i n g f a c t o r ( s ) in v i t r o .

I n t r o d u c t i o n . 1A8

9 .1. T h e X e n o p u s b o r e a l i s U R E b i n d s a p r o t e i n ( s ) 1A9 in v i t r o .

9.2. T he U RE, in c o n j u n c t i o n w i t h C A r G b o x l a nd 1 50 C A r G b o x 3 , is i n s u f f i c i e n t to d i r e c t the

t i s s u e - s p e c i f i c e x p r e s s i o n of a h e t e r o l o g o u s p r o m o t e r .

C h a p t e r 10: S e q u e n c e s l o c a t e d b e t w e e n t he C A r G boxl m o t i f a n d the T A T A b ox of t he X e n o p u s b o r e a l i s s k e l e t a l a c t i n g e n e i n t e r a c t w i t h a t r a n s - a c t i n g f a c t o r ( s ) _in v i t r o .

I n t r o d u c t i o n . 157 1 0 . 1 . A t r a n s - a c t i n g f a c t o r ( s ) b i n d s t he - 8 3 / - A 2 158

r e g i o n o f the X e n o p u s b o r e a l i s s k e l e t a l a c t i n g e n e iji v i t r o .

1 0 . 2 . D i s c u s s i o n . 160

C h a p t e r 11: G e n e r a l d i s c u s s i o n . 164

A P P E N D I X .

A p p e n d i x 1: T h e e x p r e s s i o n o f a h u m a n c a r d i a c a c t i n g e n e in d e v e l o p i n g X e n o p u s l a e v i s embryos. I n t r o d u c t i o n . 171 A . 1.1. T he s p a t i a l e x p r e s s i o n of a h u m a n c a r d i a c a c t i n 172

g e n e i n s t a g e 30 X e n o p u s l a e v i s e m b r y o s .

A . 1.2. The t e m p o r a l e x p r e s s i o n of a h u m a n c a r d i a c a c t i n 175 g ene i n d e v e l o p i n g X e n o p u s l a e v i s em b r y o s .

A . 1.3. The a n a l y s i s of s e q u e n c e e l e m e n t s i m p o r t a n t in 178 the e x p r e s s i o n o f a h u m a n c a r d i a c a c t i n g ene in X e n o p u s l a e v i s e m b r y o s .

T A B L E O F FIGURES.

F i g u r e . p r e c e d i n g Page

1 . 1 . E v o l u t i o n a r y c o n s e r v e d C A r G box s e q u e n c e s 17 in m u s c l e - s p e c i f i c g e n e s .

1 . 2 . S t r u c t u r a l c o m p a r i s o n o f the M y o D f a m i l y o f 39 m y o g e n i c r e g u l a t o r y f a c t o r s .

4 . 0 . S t r u c t u r e o f the c o m p l e t e X e n o p u s b o r e a l i s 74 s k e l e t a l a c t i n g ene.

4 . 1 . A n a l y s i s o f t r a n s c r i p t s f r o m e m b r y o s i n j e c t e d 77 w i t h X e n o p u s b o r e a l i s s k e l e t a l a c t i n p r o m o t e r

d e l e t i o n p l a s m i d s .

4 . 2 . A n a l y s i s of t r a n s c r i p t s from e m b r y o s m i c r o - 79 i n j e c t e d w i t h d e l e t i o n c o n s t r u c t s c o n t a i n i n g an a d d i t i o n a l C A r G b o x 3 m o t i f .

4 .3. C A r G b o x s e q u e n c e s o f t he c a r d i a c a n d s k e l e t a l 81 a c t i n g e n e s .

5.1. B and s h i f t a n a l y s i s o f t he X e n o p u s b o r e a l i s 86 s k e l e t a l a c t i n g e n e p r o m o t e r .

5.2. B and s h i f t a n a l y s i s o f X e n o p u s b o r e a l i s s k e l e t a l 88 a c t i n g e n e p r o m o t e r f r a g m e n t s c o n t a i n i n g

p r o g r e s s i v e l y l e s s 5' f l a n k i n g s e q u e n c e s .

5.3A. D MS p r o t e c t i o n f o o t p r i n t i n g of the X e n o p u s 91 b o r e a l i s s k e l e t a l a c t i n gene p r o m o t e r .

5 . 3 B . D M S i n t e r f e r e n c e f o o t p r i n t i n g of the X e n o p u s 91 b o r e a l i s s k e l e t a l a c t i n g e n e prom o t e r .

5 . A. C o m p e t i t i o n o f b i n d i n g to the X e n o p u s b o r e a l i s 93 s k e l e t a l a c t i n g e n e p r o m o t e r b y s y n t h e t i c C A r G

b o x o l i g o n u c l e o t i d e s .

5 . 5 . C o m p a r i s o n o f D N A f o o t p r i n t s g e n e r a t e d o v e r 95 v a r i o u s a c t i n C A r G b o x l m o t i f s .

6 . 1 . C A r G b o x o l i g o n u c l e o t i d e s b i n d s e q u e n c e - s p e c i f i c 99 t r a n s - a c t i n g f a c t o r s iji v i t r o .

6 . 2 . 1 . C A r G b o x o l i g o n u c l e o t i d e s e x h i b i t d i s t i n c t 102 c o m p e t i t i o n p a t t e r n s .

6 . 2 . 2 . C o m p e t i t i o n a n a l y s i s o f C A r G box b i n d i n g 10A p r o t e i n s u s i n g i n c r e a s i n g a m o u n t s of C A r G

b o x c o m p e t i t o r s .

6 . 3 . 1 . C o m p a r i s o n o f C A r G b o x a n d S RE s e q u e n c e s . 105 6 . 3 . 2 . A p r o t e i n w h i c h is a n t i g e n i c a l l y s i m i l a r 106

to S R F b i n d s the X e n o p u s b o r e a l i s C A r G boxl m o tif.

6 . A. A p r o t e i n w h i c h is a n t i g e n i c a l l y d i s t i n g u i s h a b l e 1 08 f r o m S R F b i n d s the X e n o p u s b o r e a l i s s k e l e t a l actin C A r G b o x 3 m o t i f .

7 . 1 . 1 . D i s t r i b u t i o n of C A r G b ox b i n d i n g f a c t o r s 119 t h r o u g h o u t the e m b r y o .

7 .1.2. T i s s u e d i s t r i b u t i o n o f C A r G b o x 3 b i n d i n g 120 p r o t e i n s .

xi 11

7 . 2 . D i s t r i b u t i o n of C A r G b o x 3 b i n d i n g p r o t e i n s 121 t h r o u g h o u t X e n o p u s d e v e l o p m e n t .

8 . 1 . 1 . D i a g r a m m a t i c r e p r e s e n t a t i o n o f the p l a s m i d 132

p A g b A 5 ( + ) .

8.1.2.

Primer extension analysis of embryos injected

133

w i t h

pXbgbAô, pAg b , or p A g b A 5 (

+

).

8 . 1 . 3 . S e q u e n c e o f the d e l e t e d X e n o p u s l a e v i s p - g l o b i n 13A p r o m o t e r i l l u s t r a t i n g t he two t r a n s c r i p t i o n

s t a r t s i t e s u s e d b y t h e X e n o p u s b o r e a l i s s k e l e t a l a c t i n g e n e p r o m o t e r f ragment.

8 . 2 . 1 . S e q u e n c e s o f o l i g o n u c l e o t i d e s u s e d i n t h e 135

c o n s t r u c t i o n o f p l a s m i d s p A g b B 3 , p A g b B l

p A g b B 3 / B l f . ) and p A g b B 3 / B l ( - ) .

8 . 2 . 2 . D i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e c o n s t r u c t s 136

p A g b B 3 , p A g b B l , p A g b B 3 / B l ( + ) and

p A g b B 3 / B l ( - ) .

8 . 2 . 3 . P r i m e r e x t e n s i o n a n a l y s i s o f n u c l e i c a c i d 136 i s o l a t e d f r o m e m b r y o s i n j e c t e d w i t h p l a s m i d s

pAg b , pXbgbAô, p A g b A 5 (

+ ), pAgbB3, pAgbBl

p A g b B 3 / B l ( + ) and p A g b B 3 / B l ( - ) .

8 . 2 . A. S o u t h e r n b l o t a n a l y s i s o f e m b r y o s i n j e c t e d w i t h 137

p l a s m i d s p A g b , p X b g b A ô , p A g b A 5 ( + ) , p A g b B 3 ,

p A g b B l , PA g b B 3 / B l ( ♦ ) a nd p A g b B 3 / B l ( - ) .

8 . 3 . 1 . D i a g r a m m a t i c r e p r e s e n t a t i o n of the c o n s t r u c t s 1A0

x i v

8 . 3 . 2 . P r i m e r e x t e n s i o n a n a l y s i s o f n u c l e i c a c i d 1 40 i s o l a t e d f r o m e m b r y o s i n j e c t e d w i t h p l a s m i d s

p A g b , p X b g b A ó , p A g b A 5 ( + ) , p A g b A ó ,

p A g b A 8 + , p A g b B 3 , p A g b B l , p A g b B 3 / B l ( + )

and p A g b B 3 / B l ( - ) .

8 . 3 . 3 . S o u t h e r n b l o t a n a l y s i s o f e m b r y o s i n j e c t e d w i t h 141

p l a s m i d s p A g b , p X b g b A ó , p A g b A 5 ( + ) , p A g b A ó ,

p A g b A 8 + , p A g b B 3 , p A g b B l , p A g b B 3 / B l ( + )

and p A g b B 3 / B l (-) .

8 . 4 . C o m p a r i s o n o f U R E s e q u e n c e s w i t h s e q u e n c e s in the 147 - 4 1 / + 2 8 r e g i o n o f the X e n o p u s b o r e a l i s s k e l e t a l a c t i n gene.

9 . 1 . T he X e n o p u s b o r e a l i s s k e l e t a l a c t i n g e n e U R E b i n d s 149 a t r a n s - a c t i n g f a c t o r ( s ) iji_ v i t r o .

9 . 2 . 1 . D i a g r a m m a t i c r e p r e s e n t a t i o n of the p l a s m i d 151 p A g b C B - U R E .

9.2.2.

extension analysis of embryos injected

152

with the plasmids p A g b A 5 ( + ), p A g b A ó ,

p A g b A 8 + , p A g b B 3 / B l ( ♦ ) a n d p A g b C B - U R E .

1 0 . 1 . 1 . D i a g r a m m a t i c r e p r e s e n t a t i o n o f the 159 o l i g o n u c l e o t i d e B l / B a l .

1 0 . 1 . 2 . T he - 8 3 / - 4 2 r e g i o n o f t he X e n o p u s b o r e a l i s 159 s k e l e t a l a c t i n g e n e b i n d s a t r a n s - a c t i n g

f a c t o r ( s ) in v i t r o .

11.1

A.i

A. 1 .

A . I .

A .I .

A . I .

A . I .

A . I .

D i a g r a m m a t i c r e p r e s e n t a t i o n of the c i s - a c t i n g r e g u l a t o r y s e q u e n c e s , a n d the t r a n s - a c t i n g f a c t o r s w i t h w h i c h t h e y i n t e r a c t , t hat a r e i m p o r t a n t for the e x p r e s s i o n o f the X e n o p u s b o r e a l i s s k e l e t a l a c t i n gene.

.1. D i a g r a m m a t i c r e p r e s e n t a t i o n of the p l a s m i d p H C A 4 8 5 .

.2. T he h u m a n c a r d i a c a c t i n gene p r o m o t e r is c a p a b l e o f d r i v i n g the t i s s u e - s p e c i f i c e x p r e s s i o n o f a C A T r e p o r t e r g e n e in the a x i s r e g i o n o f X e n o p u s l a e v i s e m b r y o s . .3. D i a g r a m m a t i c r e p r e s e n t a t i o n o f t he p l a s m i d

p R E485.

.4. A 4 8 5 b p f r a g m e n t o f the h u m a n c a r d i a c a c t i n g e n e p r o m o t e r p r o d u c e s c o r r e c t l y i n i t i a t e d t r a n s c r i p t s in the a x i s r e g i o n o f X e n o p u s l a e v i s e m b r y o s .

.1. T he t e m p o r a l e x p r e s s i o n o f a h u m a n c a r d i a c a c t i n g e n e in X e n o p u s l a e v i s emb r y o s . 1.1. D i a g r a m m a t i c r e p r e s e n t a t i o n of t he h u m a n

c a r d i a c a c t i n p r o m o t e r d e l e t i o n p l a s m i d s P H C A 4 8 5 , p H C A l 77, p H C A 1 5 3 , p H C A 1 3 1 , p H C A 9 6 a nd p H C A 6 4 .

3.2. The p r o m o t e r a c t i v i t i e s o f the p l a s m i d s pH C A 4 8 5 p H C A 1 7 7 a n d p H C A 1 5 3 w h e n i n j e c t e d i nto X e n o p u s l a e v i s e m b r y o s .

xv 1 68 1 72 1 72 174 1 74

1 7 6

179

X V i

A . 1 . 3 . 3 . D i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e h u m a n c a r d i a c a c t i n p r o m o t e r d e l e t i o n p l a s m i d s p R E 4 8 5 , p R E l 77, p R E 1 5 3 , p R E 1 3 1 , p R E 9 6 and pRE64.

A . 1 . 3 . 4 . The p r o m o t e r a c t i v i t i e s of t he p l a s m i d s pRE485, p R E l 7 7

,

181

X V 1 i A c k n o w l e d g e m e n t s .

I acknowledge my supervisor, Hugh Woodland, and thank him for his guidance and help throughout the course of this project. Grateful thanks are also due to both Hugh Woodland and Liz Jones for the many hours they spent injecting and dissecting embryos. I would also like to thank Dr. Mark Boardman for his technical assistance, in addition to his encouragement and support w hen things were going badly. My thanks are also due to Darrin Smith for various materials, in addition to many useful discussions.

I acknowledge Dr. T. Mohun, Bob Old and Darrin Smith for their generous gifts of anti-SRF, anti-THR and anti-Oct-1 antibodies respectively. I also acknowledge Dr. L. Kedes for the Human cardiac actin promoter plasmids. I acknowledge the financial support of the Medical Research Council.

xviii

D e c l a r a t i o n .

A l l t h e r e s u l t s p r e s e n t e d in t h i s t h e s i s w e r e o b t a i n e d b y the a u t h o r , a p a r t f r o m t h o s e s p e c i f i c a l l y i n d i c a t e d in the text. A ll i n j e c t i o n s a n d a l m o s t a ll t h e d i s s e c t i o n s o f e m b r y o s w e r e p e r f o r m e d b y H u g h W o o d l a n d . In a d d i t i o n , the r e s u l t s p r e s e n t e d in C h a p t e r A w e r e o b t a i n e d b y Dr. M a r k B o a r d m a n in the e a r l y p a r t o f ray s t u d i e s h e r e , b ut t h e y p r o v i d e i m p o r t a n t b a c k g r o u n d i n f o r m a t i o n for the r e s t of t he w ork.

x i x

SUMMARY

The main aim of this work was to identify the important cis-acting regulatory sequences, and the trans-acting factors with which they interact, which are required for the tissue-specific expression of the Xenopus borealis skeletal actin gene.

All sequences necessary and sufficient for the correct spatial and temporal expression of the Xenopus borealis skeletal actin gene are located in a 156 bp fragment of the gene that spans from nucleotide residues -197 to -42 in its promoter. This region of the skeletal actin promoter contains three imperfect repeats of a CArG sequence motif that has been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion analysis of the promoter of the Xenopus borealis skeletal actin gene, using Xenopus micro-injection techniques as a transient assay system for promoter activity, have identified that CArG box3 is essential for skeletal actin gene expression.

By using band shift assays I have demonstrated that, under my assay conditions, CArG box2 is unable to bind any proteins in vitro . Conversely, the CArG boxl sequence exhibits two binding activities on band shift analysis. One of these is antigenically related to the transcription factor SRF, whilst the second appears to be distinct from this protein. CArG box3 also interacts with a protein in vitro. Although this sequence exhibits a similar shift to that of the CArG boxl/SRF complex on band shift analysis, my experiments suggest that this protein is distinct from SRF.

A combination of the CArG boxl and CArG box3 motifs is unable to confer muscle-specific gene expression on a heterologous promoter. Furthermore, I have identified an upstream regulatory element (URE) in the Xenopus borealis skeletal actin gene promoter that spans from nucleotides -197 to -167 that is required for the expression of the gene, at least when sequences between nucleotide -42 and +28 are absent.

The URE of the Xenopus borealis skeletal actin gene is capable of interacting with a trans-acting factor(s) in vitro. In addition to this a further region of the gene which spans from nucleotide residues -83 to -42 is also capable of interacting with a factor(s) in vitro.

XX A b b r e v i a t i o n s .

ATP, dATP.j1 a d e n o s i n e t r i p h o s p h a t e , d e o x y a d e n o s i n e d d A T P

J

BSA bovine s e r u m a l bumin.

C A M P c yclic a d e n o s i n e m o n o - p h o s p h a t e . c D N A c o m p l e m e n t a r y DNA.

Ci Curie.

C I A P c alf i n t e s t i n a l a l k a l i n e p h o s p h a t a s e . C T P , d C T P 'Ï c y t i d i n e t r i p h o s p h a t e , d e o x y c y t i d i n e d d C T P I t r i p h o s p h a t e , d i d e o x y c y t i d i n e t r i p h o s p h a t e . D N A d e o x y r i b o n u c l e i c a cid.

DTT d i t h i o t h r e i t o l .

d T T P , J thy m i d i n e t r i p h o s p h a t e , d e o x y t h y m i d i n e d d T T P

J

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 a t e . F S H fo l l i c l e s t i m u l a t i n g h o r m o n e . GTP, d G T P ,jI g u a n o s i n e t r i p h o s p h a t e , d e o x y g u a n o s i n e d d G T P

j

Kb k i l o b a s e p airs.

1 litre.

M B T m i d - b l a s t u l a t r a n s i t i o n . M CK m u s c l e c r e a t i n e k i n a s e . ml m i l l i l i t r e .

xx i

M ops 3 - ( N - m o r p h o l i n o ) p r o p a n e s u l f o n i c a c i d ng nan o g r a m .

nt n u c l e o t i d e . Pg pico g r a m . R N A r i b o n u c l e i c acid. r pm r e v o l u t i o n s per m i n u t e . r RNA r i b o s o m a l RNA. SDS s o d i u m d o d e c y l s u l f a t e . SRE s e r u m r e s p o n s e e l e m e n t . SRF s erum r e s p o n s e fa c t o r . SV40 s i m i a n v i r u s 40. THR th y r o i d h o r m o n e r e c e p t o r . c T n I c a r d i a c t r o p o n i n I sTn I s k e l e t a l t r o p o n o n I c T N T c a r d i a c t r o p o n i n T. sTNT s k e l e t a l t r o p o n i n T.

Tris T r i s ( h y d r o x y m e t h y l )a m i n o m e t h a n e . tRNA t r a n s f e r RNA.

ug m i c r o g r a m . ul m i c r o l i t r e .

otion

C H A P T E R

1

I n t r o d u c t i o n .

1 . 1 T h e e a r l y d e v e l o p m e n t of X e n o p u s .

The e a r l y s t a g e s o f Xe n o p u s e r a bryogenesis s h a r e m a n y g e n e r a l c h a r a c t e r i s t i c s w i t h o ther v e r t e b r a t e s . T h e s t u d y o f this o r g a n i s m h a s e n j o y e d p a r t i c u l a r a t t e n t i o n b e c a u s e the e m b r y o s are l a r g e a nd e a s i l y m a n i p u l a t e d , as we ’ll as b e i n g r e a d i l y a v a i l a b l e t h r o u g h o u t t he year. I n a d d i t i o n to t his, the e m b r y o s d e v e l o p r a p i d l y a n d i n d e p e n d e n t l y , w i t h t he b o d y p l a n b e i n g e s t a b l i s h e d a n d t i s s u e - s p e c i f i c g ene a c t i v a t i o n o c c u r r i n g w i t h i n 24 h o u r s o f f e r t i l i s a t i o n . T h e f o l l o w i n g s e c t i o n b r i e f l y s u m m a r i s e s the e m b r y o l o g i c a l c h a n g e s m o s t r e l e v a n t to an u n d e r s t a n d i n g of t h e w o r k d e s c r i b e d in t h i s th e s i s .

The u n f e r t i l i s e d X e n o p u s e gg is a l arge a s y m m e t r i c cell. M a n y y o l k p l a t e l e t s a r e c o n c e n t r a t e d at its v e g e t a l pole, w h i l s t at the a n i m a l p o l e t h e s e p l a t e l e t s a r e m u c h l e s s d e n s e a n d the r egion is d a r k l y p i g m e n t e d by m e l a n i n g r a n u l e s l o c a t e d n e a r the cell s u r f a c e . W h e r e a s the a n i m a l - v e g e t a l p o l a r i t y is e s t a b l i s h e d d u r i n g ooge n e s i s , d o r s a l - v e n t r a l p o l a r i t y is e s t a b l i s h e d b y a s e r i e s of e v e n t s d i r e c t l y a f t e r f e r t i l i s a t i o n of the egg. T h e s u c c e s s f u l s p e r m a l w a y s e n t e r s in the a n i m a l h e m i s p h e r e o f t h e e g g . This t r i g g e r s a r o t a t i o n in the e ggs c y t o p l a s m , r e s u l t i n g in the a p p e a r a n c e o f a l i g h t l y p i g m e n t e d b and (the g r e y c r e s c e n t ) on t h e o p p o s i t e s i d e o f the e g g to s p e r m e n t r y . T h e g r e y c r e s c e n t m a r k s t he d o r s a l side o f the

I n t r o d u c t i o n

e m b r y o a n d is c e n t r e d on the p o i n t w h e r e c e l l s w i l l l a t e r s tart i n v a g i n a t i n g to p r o d u c e the i n t e r n a l s t r u c t u r e s o f t he em b r y o .

T he f i r s t few h o u r s of d e v e l o p m e n t ( u n t i l s t a g e 8) i n v o l v e t w e l v e v e r y r a p i d , s y n c h r o n o u s c e l l d i v i s i o n s ( t h e c l e a v a g e st a g e ) , e a c h of w h i c h ( e x c l u d i n g t he f irst c e l l d i v i s i o n ) t akes a b o u t 35 m i n u t e s o n a v e r a g e ( N e w p o r t a n d K i r s c h n e r , 1 9 8 2 ) . O n l y a l i t t l e n e w t r a n s c r i p t i o n is o b s e r v e d o v e r t h i s period. H o w e v e r at s t a g e 8, the raid-bias tula t r a n s i t i o n (MBT), t r a n s c r i p t i o n of m a n y g e n e s is a c t i v a t e d , or a c c e l e r a t e s , and the c e l l d i v i s i o n s b e c o m e a s y n c h r o n o u s .

In t e r m s of d e v e l o p m e n t a l s p e c i f i c a t i o n , the e a r l y b l a s t u l a e m b r y o c o n s i s t s o f t w o c ell t y pes, t hose at the a n i m a l p o l e a nd t hose at the v e g e t a l p o l e . If a n i m a l p ole c e l l s a r e c u l t u r e d in i s o l a t i o n t h e y d e v e l o p to f o r m e p i d e r m i s , w h e r e a s v e g e t a l c e l l s d e v e l o p into p r e d o m i n a n t l y e n d o d e r m a l ti s s u e s w h e n c u l t u r e d in i s o l a t i o n . I ndeed, t h e e q u a t o r i a l r e g i o n of a 64 c e l l s t a g e em b r y o , w h i c h f o r m s the m e s o d e r m a l t i s s u e s in a n o r m a l l y d e v e l o p i n g e m b r y o , d e v e l o p s i n t o e c t o d e r m a l t i s s u e w h e n c u l t u r e d in i s o l a t i o n . H o w e v e r , if t hese c e l l s a r e i s o l a t e d l a t e r t han the 64 c e l l s tage t h e y d e v e l o p i n t o s u b s t a n t i a l a m o u n t s of m e s o d e r m a l t issue, w i t h the a d d i t i o n o f some e c t o d e r m ( N a k a m u r a e t a l . . 1970).

One i n t e r p r e t a t i o n o f this is t hat m e s o d e r m i n d u c t i o n d e p e n d s on an i n t e r a c t i o n b e t w e e n the a n i m a l and v e g e t a l r e g i o n s o f the b l a s t u l a . T h i s t h e o r y was c o n f i r m e d by the e x p e r i m e n t s o f Ogi (1967, 1969) and N i e u w k o o p ( 1 9 6 9 ) s h o w i n g that, a s p r e v i o u s l y known, a n i m a l a n d v e g e t a l p o l e s o f e m b r y o s c u l t u r e d s e p a r a t e l y

I n t r o d u c t i o n

d e v e l o p into e p i d e r m i s a n d e n d o d e r m r e s p e c t i v e l y . H o w e v e r , by c o m b i n i n g a n i m a l a nd v e g e t a l f r a g m e n t s a v a r i e t y o f m e s o d e r m a l t i s s u e s w ere f o rmed. N i e u w k o o p l a t e r d e m o n s t r a t e d t hat the m e s o d e r m was f o r m e d e n t i r e l y f rom the e c t o d e r m a l c o m p o n e n t of the e m b r y o , as a r e s u l t o f i n d u c t i o n by t h e p r o s p e c t i v e e n d o d e r m ( S u d a r w a t i a n d N i e u w k o o p , 1 971; N i e u w k o o p a n d Ub b e l s , 1972).

T h i s r e l a t i v e l y s i m p l e m o d e l of m e s o d e r m i n d u c t i o n w as c o m p l i c a t e d by the f i n d i n g s that v e n t r a l v e g e t a l b l a s t o m e r e s i n d u c e d l ittle or no m u s c l e f rom a n i m a l p ole c e l l s , d e s p i t e the fact that m u s c l e of t h e e m b r y o is f o r m e d from b l a s t o m e r e s of the v e n t r a l p art of t h e e mbryo. T h i s i n c o n s i s t e n c y o f c ell f ates h as l e a d to t h e 'three s i g n a l ' m o d e l o f m e s o d e r m i n d u c t i o n as p r o p o s e d b y S l a c k a nd c o - w o r k e r s ( S m i t h a n d Slack, 1983; S l a c k £ t a l . . 1 9 0 4 ; S m i t h e_t a l . , 1985; D a l e a n d Slack, 1987). The f irst two s i g n a l s in this m o d e l o r i g i n a t e f rom the ve g e t a l h e m i s p h e r e of t h e em b r y o . O ne o n the d o r s a l s i d e of the e m b r y o i nduces p r e d o m i n a n t l y n o t o c h o r d , w h i l s t o n the ve n t r a l side a s econd s i gnal i n d u c e s ve n t r a l m e s o d e r m s u c h as b lood, m e s e n c h y m e a nd m e s o t h e l i u m . The t h i r d signal o f this m odel o r i g i n a t e s f rom the n e w l y f o r m e d d o r s a l m e s o d e r m . T h i s s ignal a c t s w i t h i n the m e s o d e r m g e r m l i n e to ' d o r s a l i z e ' a d j a c e n t v e n t r a l mes o d e r m .

In r ecent y e a r s w o r k h as s u g g e s t e d that the fa c t o r s r e s p o n s i b l e for cell f a t e s d u r i n g m e s o d e r m i n d u c t i o n ( m e s o d e r m i n d u c i n g factors, or M I F s ) are r e l a t e d to p e p t i d e g r o w t h fa c t o r s (PGFs) (for r e v i e w see Smith, 1989).

I n t r o d u c t i o n

F o l l o w i n g the o n s e t o f m e s o d e r m a l i n d u c t i o n g a s t r u l a t i o n o c c u r s w h e n c e l l s i n v a g i n a t e t h r o u g h t he b l a s t o p o r e , s t a r t i n g at a p o s i t i o n in t he e q u a t o r i a l r e g i o n o p p o s i t e t he s p e r m e n t r y p oint, k n o w n as the d o r s a l lip of t he b l a s t o p o r e . E n d o d e r m a l a n d m e s o d e r m a l c e l l s g r a d u a l l y m o v e i n s i d e t h e e m b r y o to l o c a t i o n s w h e r e t h e y w i l l s u b s e q u e n t l y f o r m the m a j o r i t y o f the i n t e r n a l o r g a n s . It i s at the e nd o f this p r o c e s s ( stage 12^) that s ome m e s o d e r m a l c e l l s , w h i c h l a t e r f o r m m y o t o m e s , b e g i n to e x p r e s s the c a r d i a c a n d s k e l e t a l a c t i n g e n e s ( G u r d o n e_t a l . , 1985, W i l s o n et a h , 1 9 8 6 ) .

O v e r the f o l l o w i n g h o u r s n e u r u l a t i o n t a k e s p l a c e , l e a d i n g to the e v e n t u a l c r e a t i o n o f the n e u r a l t u b e a n d p r i m i t i v e n e r v o u s s ystem. The f i r s t s o m i t e is r e c o g n i s e d at s t a g e 17, a f t e r the i n i t i a l d e t e c t i o n o f t h e £?C-actin p r o t e i n s ( S t u r g e s s e_t a l . , 1980). I ndeed, the a c t i v a t i o n of t h e 0 ( - * c t i n g e n e s d u r i n g e a r l y X e n o p u s d e v e l o p m e n t o c c u r s a bout 8 h o u r s b e f o r e m o r p h o l o g i c a l d i f f e r e n t i a t i o n of m u s c l e tissue t a k e s p lace. T h i s m a k e s the s t u d y of s a r c o m e r i c a c t i n gene e x p r e s s i o n o f p a r t i c u l a r in t e r e s t in d e v e l o p m e n t f or two r e a s o n s . F i r s t l y it a c t s as a e a r l y m a r k e r for m u s c l e c o m m i t m e n t a nd s e c o n d , it m a y be r e g u l a t e d b y g e n e a c t i v a t o r s w h i c h o p e r a t e at t h e v e r y e a r l i e s t s t a g e s of m u s c l e d i f f e r e n t i a t i o n .

A l t h o u g h p r o g r e s s is b e i n g made it is s t i l l u n c l e a r as to h o w t he f a c t o r s r e s p o n s i b l e f or m e s o d e r m i n d u c t i o n d i c t a t e the fate of p r e s u m p t i v e m u s c l e c ells a n d a c t i v a t e the m y o g e n i c p r o g r a m m e . To this e n d m u c h work h a s b e e n p e r f o r m e d on s t u d y i n g the m e c h a n i s m s by w h i c h m u s c l e - s p e c i f i c g e n e s a r e a c t i v a t e d ,

I n t r o d u c t i o n

w i t h the a i m of w o r k i n g b a c k w a r d s to t h e p o i n t w h e n a n i m a l c e l l s f i r s t r e c e i v e a s i g n a l f r o m v e g e t a l c ells. T h e r e m a i n d e r o f t h i s c h a p t e r is c o n c e r n e d w i t h r e v i e w i n g t his l ine of r e s e a r c h in b o t h a m p h i b i a a n d o t h e r v e r t e b r a t e s .

1.2 T h e a c t i n g e n e family.

A c t i n is a s t r u c t u r a l p r o t e i n f o u n d in a l l e u k a r y o t i c c e lls. It e x i s t s in a s m a l l n u m b e r o f c l o s e l y r e l a t e d i s o f o r m s w h i c h h ave d i v e r s e f u n c t i o n s in v a r i o u s d i f f e r e n t c ell types. All t h e s e i s o f o r m s c a n be m a i n t a i n e d as m o n o m e r s ( G - a c t i n ) , or t hey c an p o l y m e r i s e to f o r m f i l a m e n t s ( F - a c t i n ) .

In s a r c o m e r i c m u s c l e ( s k e l e t a l a n d h e a r t m u s c l e ) F - a c t i n e x i s t s as t h i n f i l a m e n t s w h i c h i n t e r d i g i t a t e w i t h m y o s i n . M y o s i n is a t t a c h e d to the a c t i n via a g l o b u l a r d o m a i n on the m y o s i n m o l e c u l e w h i c h is h i n g e d to the r e s t of the c o m p l e x . It is b e l i e v e d that a c y c l i c p r o c e s s of b i n d i n g , r o t a t i o n a r o u n d the h i n g e a nd d i s s o c i a t i o n d r i v e s a s l i d i n g a c t i o n o f m y o s i n f i l a m e n t s o v e r a c t i n f i l a m e n t s , t h u s f a c i l i t a t i n g m u s c l e c o n t r a c t i o n . T h i s p r o c e s s r e q u i r e s t he h y d r o l y s i s of ATP, a n d is i n h i b i t e d by d e c r e a s i n g i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s .

In a d d i t i o n to t he role o f a c t i n in m u s c l e c o n t r a c t i o n o t h e r a c t i n i s o f o r m s f o r m a v i t a l c o m p o n e n t o f the c y t o s k e l e t o n in b o t h m u s c l e a n d n o n - m u s c l e cells. T h e y are i n v o l v e d in d i f f e r e n t a s p e c t s o f c ell m o t i l i t y , i n c l u d i n g c e l l m o v e m e n t , c y t o k i n e s i s , c y t o p l a s m i c t r a n s p o r t , s e c r e t i o n and p h a g o c y t o s i s (for r e v i e w s ee C l a r k e a nd S p u d i c h , 1977).

I n t r o d u c t i o n

T h e m u l t i p l e i s o f o r m s o f act in d e s c r i b e d i n t he p r e c e d i n g p a r a g r a p h s w e r e f irst i d e n t i f i e d b y t h e v a r i a t i o n i n m o b i l i t y of a c t i n p r o t e i n s on i s o - e l e c t r i c f o c u s s i n g g e l s ( G a r r e l s a nd G i b s o n , 1976; W h a l e n e_t_ a l . . 1 9 7 6 ) . U s i n g t his t e c h n i q u e at l e a s t t hree a c t i n isoforins, the Q( , ^2, a n d $ a c t i n s , w e r e i d e n t i f i e d . H o w e v e r it was n o t u n t i l V a n d e k e r c k h o v e a n d W e b e r ( 1978a, 1978b, 1 978c a n d 1 9 7 9 ) s e q u e n c e d d i f f e r e n t i s o f o r m s f r o m a n u m b e r o f d i f f e r e n t t i s s u e s t h a t at l e a s t s i x d i f f e r e n t a c t i n t ypes w e r e i d e n t i f i e d . T w o o f t h e s e , t he a n d ft a c t i n i s o f o r m s , are c o - e x p r e s s e d in a l l m a m m a l i a n n o n - m u s c l e c e l l s so far s t u d i e d ( V a n d e k e r c k h o v e a n d W e b e r 1 979) a n d a r e t h e a c t i n t y p e s that are t he c o m p o n e n t s o f t he c y t o s k e l e t o n . T h e (X a c t i n g e n e f a m i l y c l a s s i c a l l y c o n s i s t s of a n O i - s k e l e t a l a c t i n w h i c h is o b s e r v e d s o l e l y in the s k e l e t a l m u s c l e , a n d an 0^ - c a r d i a c a ctin, w h i c h p r e d o m i n a t e s in t h e h e a r t mu s c l e . S m o o t h m u s c l e c o n t a i n s two a c t i n i s o f o r m s , a n © < - t y p e w h i c h p r e d o m i n a t e s in the a o r t a for e x a m p l e , a n d a | £ -type t h a t is at h i g h e r r e l a t i v e l e v e l s in the s t omach.

T he six c l a s s i c a l a c t i n i s o f o r m s a r e m e m b e r s o f a h i g h l y c o n s e r v e d f a m i l y of p r o t e i n s . F or e x a m p l e o n l y 4 o u t of 375 a m i n o a c i d s d i f f e r b e t w e e n the c a r d i a c a n d s k e l e t a l 0(-a c t i n s . I n d e e d , in the m ost w i d e l y d i v e r g e n t e x a m p l e o n l y 2 4 a n d 25 a m i n o a c i d s d i f f e r b e t w e e n the c y t o s k e l e t a l , a nd c a r d i a c a n d s k e l e t a l a c t i n s r e s p e c t i v e l y ( V a n d e k e r c k h o v e a n d W e b e r , 1979). I n t e r e s t i n g l y , the N - t e r m i n u s is t h e m o s t d i v e r g e n t r e g i o n of the a c t i n m o l e c u l e . I ndeed, the f i r s t t hree or f our a m i n o a c i d s a re c h a r a c t e r i s t i c o f e a c h a c t i n i s o f o r m . T h e y a r e a l w a y s

I n t r o d u c t ion

a c i d i c r e s i d u e s ( e i t h e r g l u t a m a t e or a s p a r t a t e ) , b ut t heir e xact s e q u e n c e d i f f e r s b e t w e e n i s o f o r m s . V a n d e k e r c k h o v e a nd W e b e r (1 9 8 1 a ) e x p l o i t e d t his f a c t a n d d e v e l o p e d a s i m p l e a s s a y s y s t e m to i d e n t i f y o t h e r a c t i n i s o f o r m s in o t h e r o r g a n i s m s b y a p r o t e i n - c h e m i c a l a n a l y s i s o f t he N - t e r m i n a l p e p t i d e s o f a c t i n p r o t e i n s .

Ho w e v e r , the c l a s s i c a l m o d e l , d e v e l o p e d u s i n g m a m m a l s , of there b e i n g o n l y s ix s t r i c t l y c o n s e r v e d a c t i n i s o f o r m s , w i t h the s k e l e t a l , c a r d i a c a n d s m o o t h m u s c l e a c t i n s b e i n g e x p r e s s e d t i s s u e - s p e c i f i c a l l y , r e p r e s e n t s a o v e r s i m p l i f i c a t i o n o f the s i t u a t i o n in o t h e r v e r t e b r a t e s . F or e x a m p l e , t he s t u d y of c y t o s k e l e t a l a c t i n s in a m p h i b i a b y V a n d e k e r c k h o v e a n d W e b e r (1981b) led to the p r o p o s a l t h a t a n y o r d e r o f a c i d i c a m i n o a c i d s at the N - t e r m i n u s of t h e s e p r o t e i n s m i g h t be f u n c t i o n a l . Eight d i f f e r e n t a r r a n g e m e n t s a r e t h e r e f o r e p o s s i b l e ( t y p e s 1- 8), a nd at l e a s t s i x o f t h e s e h a v e b e e n i d e n t i f i e d _in v ivo ( V a n d e k e r c k h o v e and W e b e r , 1 9 8 1 b ; B e r g s m a £ £ a l . , 1985). De s p i t e the a d v a n c e s p r e v i o u s l y d e s c r i b e d , the a c c u r a t e s t u d y of the d i s t r i b u t i o n o f v a r i o u s d i f f e r e n t a c t i n t y p e s w as li m i t e d b y the i n a b i l i t y o f the a s s a y s y s t e m u s e d to d i s t i n g u i s h b e t w e e n c l o s e l y r e l a t e d i s o f o r m s , s u c h as s k e l e t a l a nd c a r d i a c ( X - a c t i n s . It w a s n o t u n t i l t he a c t i n s w e r e s t u d i e d at the n u c l e i c a c i d l e v e l that a m o r e d e t a i l e d a c c o u n t of a c t i n is o t y p e e x p r e s s i o n in d i f f e r e n t c e l l s w a s a c c o m p l i s h e d .

I n t r o d u c t i o n

1 . 3 A c t i n g e n e s a n d t he r e g u l a t i o n o f t h e i r e x p r e s s i o n .

1 .3.1 A c t i n g e n e s t r u c t u r e and e x p r e s s i o n .

T he h igh a m i n o a c i d c o n s e r v a t i o n b e t w e e n a c t i n i s o f o r m s m e a n s that the c o d i n g s e q u e n c e s of t h e i r g e n e s a r e v e r y s i m i l a r . T his has e n a b l e d w o r k e r s to u s e o n e D N A p r o b e t o s creen f o r m a n y a c t i n g e n e i s o f o r m s . A c t i n c D N A s f r o m o r g a n i s m s as d i s t a n t l y r elated as D i c t y o s t e l i u m a nd D r o s o p h i l a c a n act as s u i t a b l e p robes f or the v e r t e b r a t e g e n o m e ( see C r o s s , 1984; E n g e l et a l . , 1981). C o n s e q u e n t l y , by h y b r i d i s i n g a c D N A p r o b e to a g enomic S o u t h e r n b l o t a n d w a s h i n g at l o w s t r i n g e n c y an e s t i m a t e o f the a c t i n g e n e n u m b e r c an be o b t a i n e d b y c o u n t i n g the n u m b e r of h y b r i d i s i n g b a n d s . T his, a n d o t h e r e x p e r i m e n t a l a p p r o a c h e s h a v e b e e n u s e d to e s t i m a t e t he n u m b e r o f a c t i n r e l a t e d seq u e n c e s in b o t h the h u m a n a n d m o u s e g e n o m e s (Engel e_t a l . , 1981; H u m p h r i e s e_t al. , 1981; M i n t y e_t a l . . 1983). In c o n t r a s t to i n i t i a l s u s p i c i o n that t h e r e w e r e s i x a c t i n r e l a t e d seq u e n c e s in the g e n o m e (i.e. o n e f or e a c h a ctin i s o f o r m ) , there t u r n o ut to be a p p r o x i m a t e l y 2 0 - 3 0 a c t i n r e l a t e d seq u e n c e s in t he h u m a n g e n o m e ( E n g e l et a l . , 1981; H u m p h r i e s et a l ., 1981) a n d a m i n i m u m of 2 0 in the m o u s e ( M inty e_t a l . , 1983). In o t h e r o r g a n i s m s a c t i n g e n e n u m b e r v a r i e s f r o m one in yeast (Ng a n d A b e l s o n , 1980; G a l l w i t z a n d S e i d e l , 1980), to six in D r o s o p h i l a ( T o b i n e_t a l . . 1980; F y r b e r g e_t a l . , 1981), s even to e l e v e n in c h i c k e n s ( C l e v e l a n d £ t a l . , 1980; S c h w a r t z and Rothblum, 1980) a n d 11 to 20 in s ea u r c h i n s ( Durica e_t a l . . 1980; S c h e l l a r et a l . , 1981). H o w e v e r the f a c t that the h a m s t e r

I n t r o d u c t i o n

h a s o n l y 5 a c t i n l i k e s e q u e n c e s in i t s g e n o m e (Doderaont e_t a l . , 1 9 8 2 ) su g g e s t s t h a t the l a r g e n u m b e r o f a c t i n s e q u e n c e s i n the h u m a n a nd m o u s e m a y not a ll be n e c e s s a r y . I n m an, at l e a s t , m a n y c opies a p p e a r to be d i s p e r s e d , p r o c e s s e d - a c t i n p s e u d o g e n e s (Ng et a l . , 1985).

A c t i n - l i k e s e q u e n c e s p r e s e n t in the g e n o m e a p p e a r to be u n c l u s t e r e d a nd _in s i t u h y b r i d i s a t i o n h a s r e v e a l e d that m a n y of t h e s e s e q u e n c e s a r e s c a t t e r e d o n d i f f e r e n t c h r o m o s o m e s . I n deed, it h a s been s h o w n that a c t i n g e n e s as c l o s e l y r e l a t e d as the s k e l e t a l a n d c a r d i a c O ^ - a c t i n s a r e l o c a t e d o n d i f f e r e n t c h r o m o s o m e s ( C z o s n e k e_t a l . , 1 9 8 3 ) . T h i s w o u l d a r g u e a g a i n s t v a r i o u s a c t i n i s o f o r m s b e i n g d e r i v e d f r o m the s ame t r a n s c r i p t i o n a l u n i t by d i f f e r e n t i a l p r o c e s s i n g of the R N A , as h a s b e e n d e m o n s t r a t e d w i t h o t h e r m u s c l e s p e c i f i c g e n e s s u c h as t h e m y o s i n l i g h t c h a i n s 1 a n d 3 g e n e l o c u s (e.g. N a b e s h i m a et a l . . 1984).

C l o n e d a c t i n g e n e s e q u e n c e s r e v e a l that t h e c o d i n g r e g i o n s of d i f f e r e n t a c t i n isoforras s h o w c o n s i d e r a b l e s i m i l a r i t i e s . T h i s w o u l d be e x p e c t e d f rom the h i g h d e g r e e of a m i n o a c i d c o n s e r v a t i o n b e t w e e n the d i f f e r e n t a c t i n p r o t e i n s . G e n e r a l l y no c o n s e r v a t i o n w i t h i n i n t r o n s e q u e n c e s h a s b e e n o b s e r v e d in v e r t e b r a t e a c t i n genes, a l t h o u g h N g et a l . ( 1 9 8 5 ) a n d N a k a j i m a - I i j i m a et a l . ( 1 9 8 5 ) h ave d i s c o v e r e d s e q u e n c e h o m o l o g i e s in the i n t r o n s of the h u m a n and r at c y t o s k e l e t a l - a c t i n g enes. The p o s i t i o n s of i n t r o n s are e n t i r e l y c o n s e r v e d b e t w e e n s p e c i e s in g e n e s e n c o d i n g a s i n g l e v e r t e b r a t e i s o f o r m ( s e e B u c k i n g h a m a nd M i n t y , 1983), b u t the p a t t e r n is a l t e r e d b e t w e e n d i f f e r e n t

I n t r o d u c t i o n

i s o f o r m s . The p r e s e n c e o f c o n s e r v e d e n h a n c e r s e q u e n c e s w i t h i n t he a c t i n g ene i n t r o n s , s u c h as t h o s e f o u n d i n the m y o s i n g ene, a n d t h e i r role in the c o - o r d i n a t e d e x p r e s s i o n o f t h e s e g e n e s r e m a i n s d o u b t f u l (see s e c t i o n 1 . 3 . 3 a n d 1 . 3 . 4 ) .

E x a m i n a t i o n o f the r e m a i n d e r of t he t r a n s c r i p t i o n a l u n i t r e v e a l s s e q u e n c e h o m o l o g i e s i n the 5' a n d 3' u n t r a n s l a t e d r e g i o n s (5' a n d 3' U TR) o f a c t i n g e n e s . A l t h o u g h the 5' U T R r e v e a l s some c o n s e r v e d s e q u e n c e s , i ts l e n g t h is n o r m a l l y too s h o r t a nd v a r i a b l e to m a k e a n y d i a g n o s t i c c o m p a r i s o n s . C o n v e r s e l y , t he 3' U T R is c o n s i d e r a b l y l o n g e r t h a n the 5' U TR. S e q u e n c e c o m p a r i s o n s of the 3' U T R h a v e s h o w n that the r e g i o n is h i g h l y c o n s e r v e d b e t w e e n a c t i n i s o t y p e s ( Y a f f e e_t a l . . 1 9 8 5 ) . For e x a m p l e a l i g n m e n t o f s e q u e n c e s o f t he r at a n d h u m a n O ^ - a c t i n g e n e s r e v e a l s l o n g s t r e t c h e s w i t h 9 0 - 1 0 0 % i d e n t i t y . H o w e v e r , this h i g h d e g r e e of s e q u e n c e h o m o l o g y o f the 3' U T R is n o t s h a r e d b e t w e e n d i f f e r e n t a c t i n i s o f o r m s . T h i s has b e e n e x p l o i t e d to d i f f e r e n t i a t e s p e c i f i c a c t i n i s o f o r m s at the m R N A l e v e l a n d to s t u d y the s p a t i a l a n d t e m p o r a l e x p r e s s i o n o f s p e c i f i c a c t i n g enes.

T h e c l a s s i c a l m o d e l o f a c t i n g e n e e x p r e s s i o n , as d e t e r m i n e d b y

e x p r e s s e d in a l l c ell t y p e s so far e x a m i n e d , w h i l s t two s m o o t h m u s c l e a c t i n i s o t y p e s a re e x p r e s s e d in s m o o t h m u s c l e c e l l t y p e s . In a d d i t i o n to t hese t h e r e a l s o e x i s t the s k e l e t a l a n d c a r d i a c 0 ^ - a c t i n s w h i c h a r e e x p r e s s e d in the s k e l e t a l a n d c a r d i a c m u s c l e r e s p e c t i v e l y .

H o w e v e r , this m o d e l o f a c t i n g e n e e x p r e s s i o n w a s c o m p l i c a t e d p r o t e i n s e q u e n c i n g , is that the p a n d i s o f o r m s are

-I n t r o d u c t i o n

b y the f i n d i n g s o f M i n t y e_t a l . (1982), w h o c h a r a c t e r i s e d a m o u s e f o e t a l s k e l e t a l m u s c l e c D N A and s h o w e d it to be h o m o l o g o u s to a c a r d i a c a c t i n m R N A e x p r e s s e d i n b o t h f oetal a n d a d u l t h e a r t t i ssue. F u r t h e r m o r e , G u n n i n g e_t a l . (1983) w e r e a b l e to i s o l a t e c a r d i a c a c t i n c D N A s f r o m a h u m a n s k e l e t a l m u s c l e c D N A l i b r a r y . T h e s e o b s e r v a t i o n s s u g g e s t e d that the c a r d i a c a c t i n g e n e is n o t l i m i t e d e x c l u s i v e l y to c a r d i a c t i s s u e . I ndeed, in m o u s e f o e t a l s k e l e t a l m u s c l e c a r d i a c a c t i n t r a n s c r i p t s a re a m a j o r c o m p o n e n t o f the R N A ( 3 0 - 4 0 % of the t o t a l s k e l e t a l m u s c l e a c t i n m e s s a g e in 1 7 - 2 0 d a y o l d foet u s e s ) . M o r e o v e r , in c h i c k e n s k e l e t a l m u s c l e , t h e y a c c o u n t for m o r e t h a n 9 0 % of a c t i n m R N A d u r i n g e a r l y d e v e l o p m e n t ( P a t e r s o n a n d E l d r i d g e , 1984).

It w o u l d a p p e a r that the p a i r e d e x p r e s s i o n o f t h e c a r d i a c a n d s k e l e t a l a c t i n g e n e s a l s o o c c u r s in c a r d i a c m u s c l e , j u d g i n g by the f a c t that c D N A s c o m p l e m e n t a r y to s k e l e t a l a c t i n m e s s a g e are p r e s e n t in c D N A l i b r a r i e s p r e p a r e d f r o m c a r d i a c a c t i n t e m p l a t e ( M a y e r e_t_ a l . , 1 9 84). In a d d i t i o n to this it h a s b e e n r e p o r t e d t hat s k e l e t a l m u s c l e a c t i n m R N A s e q u e n c e s i n the h eart a r e a b o u t 1 0 % of t h e i r a b u n d a n c e in the l e g m u s c l e o f n e w b o r n rats. H o w e v e r a f t e r 80 d a y s of d e v e l o p m e n t their a m o u n t in the h e a r t d e c r e a s e s by a f a c t o r o f 12. F u r t h e r m o r e , l e v e l s of s k e l e t a l a c t i n m R N A in rat a n d m o u s e h e a r t s has b e e n s h o w n to be 2% of t hat o f the c a r d i a c a c t i n m R N A ( S h a n i e t a l . . 1981; M i n t y et a l . . 1 9 8 2 ) .

As a g e n e r a l r ule it w o u l d a p p e a r that b o t h t h e sk e l e t a l a n d c a r d i a c a c t i n i s o f o r m s a re e x p r e s s e d in b o t h s k e l e t a l a nd h e a r t

-I n t r o d u c t i o n

m u s c l e tis s u e s . H o w e v e r , as d e v e l o p m e n t p r o g r e s s e s t h e i r e x p r e s s i o n b e c o m e s i n c r e a s i n g l y d i f f e r e n t i a l l y l o c a l i s e d , w i t h t h e s k e l e t a l m u s c l e a c t i n b e i n g the m a j o r a c t i n m e s s a g e in s k e l e t a l m u s c l e , a n d the c a r d i a c a c t i n m e s s a g e p r e d o m i n a t i n g in the h e a r t . T h i s m o d e l is s u p p o r t e d b y o b s e r v a t i o n s that the s k e l e t a l m u s c l e c e l l line C 2 C 1 2 e x p r e s s e s m o s t l y c a r d i a c a c t i n o n f u s i o n o f m y o b l a s t s to f o r m m y o t o m e s . H o w e v e r t h i s level o f c a r d i a c a c t i n t r a n s c r i p t s l ater d r o p s a n d g i v e s w ay to p r e d o m i n a n t l y s k e l e t a l a c t i n t r a n s c r i p t s ( B a i n s e_t a l . , 1984).

In X e n o p u s m u c h the same p a t t e r n of e x p r e s s i o n e x i s t s . B o t h c a r d i a c a n d s k e l e t a l a c t i n t r a n s c r i p t s a r e f i r s t o b s e r v e d at s t a g e 1 2 ^ - 1 3 o f d e v e l o p m e n t a n d a re r e s t r i c t e d to t h e r e g i o n of the e m b r y o w h i c h w i l l d e v e l o p i nto m u s c l e t i s s u e ( M o h u n et a l . . 1984; W i l s o n e_t a l . , 1986). At l ater s t a g e s o f d e v e l o p m e n t it c a n be s e e n that b o t h t hese a c t i n t r a n s c r i p t s a r e l o c a l i s e d e x c l u s i v e l y to the m u s c u l a r t issue of the e m b r y o s o m i t e s . T h e s e t wo i s o f o r m s m a y c o - e x i s t in e m b r y o n i c m u s c l e u n t i l r e l a t i v e l y l a t e in d e v e l o p m e n t ( s tage 42). H o w e v e r i n a d u l t s k e l e t a l m u s c l e it is p r e d o m i n a n t l y the s k e l e t a l a c t i n m e s s a g e that is d e t e c t e d , w i t h the c a r d i a c a c t i n t r a n s c r i p t b e i n g pr e s e n t at v e r y l o w levels. L i k e w i s e c a r d i a c a c t i n m e s s a g e is t he p r e d o m i n a n t $ “a c t *n m e s s a g e d e t e c t a b l e in a d u l t h e a r t t issue ( M o h u n £t_ a l . , 1984).

T h i s p r e c i s e a nd i n t r i c a t e p a t t e r n of e x p r e s s i o n of t he c a r d i a c a n d s k e l e t a l a c t i n g e n e s r a i s e s c h a l l e n g i n g q u e s t i o n s as to the m o l e c u l a r m e c h a n i s m s of a c t i n g e n e e x p r e s s i o n . F u r t h e r m o r e , the s t u d y of the t i s s u e - s p e c i f i c e x p r e s s i o n of

-I n t r o d u c t i o n

a c t i n g e n e s p r o v i d e s a u s e f u l m o d e l to a t t e m p t to g a i n some u n d e r s t a n d i n g o f t he g e n e r a l m e c h a n i s m s o f c e l l - t y p e sp e c i f i c g e n e a c t i v a t i o n . T o a t t e m p t to a n s w e r s o m e o f t h e s e ques t i o n , a n d t o a t t e m p t to g a i n some k n o w l e d g e o f the a c t i v a t i o n o f the m y o g e n i c p r o g r a m as a whole, t he m o l e c u l a r a c t i v a t i o n of both a c t i n a n d o t h e r m u s c l e - s p e c i f i c g e n e s h a s b e e n t he s u b j e c t of m u c h r e s e a r c h o v e r r e c e n t y e a r s . T he f o l l o w i n g s e c t i o n s o u t l i n e some o f the p r o g r e s s m a d e in u n d e r s t a n d i n g t h i s c o m p l e x s e q u e n c e o f ev e n t s .

1 . 3 . 2 D N A s e q u e n c e e l e m e n t s i n v o l v e d in the e x p r e s s i o n of a c t i n g enes.

T h e e m e r g e n c e o f 0( - a c t i n t r a n s c r i p t s d u r i n g m y o b l a s t f u s i o n in c e l l c u l t u r e , or p r i o r to t he f o r m a t i o n o f m u s c l e in Xe n o p u s e m b r y o s , s u g g e s t s that at l e a s t o ne m e c h a n i s m o f c o n t r o l l i n g a c t i n g e n e e x p r e s s i o n is at the t r a n s c r i p t i o n a l level. F u r t h e r m o r e , h e t e r o k a r y o n s t u d i e s d e m o n s t r a t e d t h a t f u s i o n of m u s c l e c e l l s w i t h n o n - m u s c l e t i s s u e s ¿ri v i t r o i n d u c e d the e x p r e s s i o n of m u s c l e - s p e c i f i c g e n e s in an e n v i r o n m e n t in w h i c h t h e y w o u l d o t h e r w i s e be s i l e n t ( B l a u e £ a l . , 1 9 8 5 ) . T h i s w o u l d s u g g e s t that the e m e r g e n c e o f m u s c l e - s p e c i f i c m R N A s p e c i e s on the f u s i o n of m y o b l a s t s to f o r m m u l t i - n u c l e a t e d m y o t u b e s is not c a u s e d b y a n a l t e r a t i o n in the s t a b i l i t y of t h e m e s s a g e in the c y t o p l a s m , or i n d e e d due to the p a c k a g i n g o f t h e R NA in an u n t r a n s l a t e a b l e f orm. E q u a l l y as i m p o r t a n t h o w e v e r , these s t u d i e s d e m o n s t r a t e that t he a c t i v a t i o n o f m u s c l e - s p e c i f i c

-I n t r o d u c t i o n

g e n e s is m e d i a t e d b y d i f f u s i b l e t r a n s - a c t i n g m o l e c u l e s w h i c h a r e t r a n s p o r t e d to the n u c l e i t h r o u g h the c y t o p l a s m . D i r e c t e v i d e n c e t h a t the r e g u l a t i o n o f a c t i n g e n e e x p r e s s i o n is at the l e v e l o f t r a n s c r i p t i o n h a s c o m e f r o m p r o b i n g the t r a n s c r i p t i o n a l s t a t u s o f t h e s e g e n e s in v a r i o u s ti s s u e s by D N a s e l s e n s i t i v i t y t e c h n i q u e s ( C a r m o n e_t a l . , 1 9 8 2 ) . T h e s e s t u d i e s d e m o n s t r a t e d that the s k e l e t a l m u s c l e t ( - a c t i n g ene is D N a s e l s e n s i t i v e , a n d t h e r e f o r e t r a n s c r i p t i o n a l l y a c t i v e , o n l y in n u c l e i i s o l a t e d f r o m d i f f e r e n t i a t e d m u s c l e c e l l s . In n uclei i s o l a t e d f r o m m o n o - n u c l e a t e d m y o b l a s t s a n d b r a i n t i s s u e s , t his s e q u e n c e is r e l a t i v e l y p r o t e c t e d from D N a s e d i g e s t i o n , s u g g e s t i n g that the c h a n g e in h i g h e r o r d e r c h r o m a t i n s t r u c t u r e w h i c h e n a b l e s t r a n s c r i p t i o n to t ake p l a c e , o n l y o c c u r s i m m e d i a t e l y b e f o r e or d u r i n g c e l l f usion. F u r t h e r m o r e , it w a s a l s o d e m o n s t r a t e d that t his s e n s i t i v i t y to D N a s e l does n o t e x t e n d b e y o n d 0 . 7 K b 5' to the t r a n s c r i b e d r e g i o n o f the C ( - a c t i n g e n e .

S i g n i f i c a n t a d v a n c e s in c h a r a c t e r i s i n g t h e c i s - a c t i n g r e g u l a t o r y s e q u e n c e s o f the a c t i n g e n e s , a nd t he i d e n t i f i c a t i o n of the t r a n s - a c t i n g f a c t o r s w i t h w h i c h t hey i n t e r a c t , has b e e n a c h i e v e d i n r e c e n t y e a r s . T h e d e m o n s t r a t i o n t h a t c l o n e d a c t i n g e n e s i n t r o d u c e d i n t o m y o g e n i c c e l l l i n e s a r e e x p r e s s e d in a s i m i l a r m a n n e r to t hat of t h e i r e n d o g e n o u s c o u n t e r p a r t s has p r o v i d e d o n e a s s a y s y s t e m f or t he d e l i n e a t i o n o f i m p o r t a n t c i s - a c t i n g s e q u e n c e s i n v o l v e d in a c t i n g e n e e x p r e s s i o n ( f o r e x a m p l e G r i c h n i c k e_t a l . , 1986; M i n t y a n d K e d e s , 1 9 8 6 ) . H o w e v e r , t his a s s a y s y s t e m w o r k s w i t h a v a r y i n g d e g r e e o f e f f i c i e n c y ,

-I n t r o d u c t i o n

d e p e n d i n g o n the c e l l l i n e s u s e d a nd the g e n e b e i n g s t u d i e d ( see M i n t y et a l . , 1986).

In X e n o p u s . W i l s o n et a l . (1986) f o l l o w e d t h e e x p r e s s i o n o f a m i c r o - i n j e c t e d X e n o p u s b o r e a l i s ca r d i a c a c t i n gene and d e m o n s t r a t e d that t his g e n e was e x p r e s s e d i n b o t h a tem p o r a l a n d s p a t i a l m anner a l o n g w i t h the e n d o g e n o u s a c t i n gene. M i c r o ­ i n j e c t i o n o f c l oned a c t i n g e n e s i n t o d e v e l o p i n g X e n o p u s e m b r y o s t h u s p r o v i d e s an a l t e r n a t i v e a s s a y s y s t e m f or t h e s t u d y of t h e i r e x p r e s s i o n to t he i n t r o d u c t i o n of g e n e s i n t o m y o g e n i c c e l l l i n e s . Indeed, this a s s a y s y s t e m h a s b e e n e x p l o i t e d to i d e n t i f y s e q u e n c e s i n v o l v e d in the e x p r e s s i o n o f the X e n o p u s l a e v i s c a r d i a c a c t i n g e n e ( M o h u n ^ t a l . . 1 986; M o h u n e_t a l . , 1 9 89a; T a y l o r et

a h

In a d d i t i o n to t h e s e t wo a s s a y s y s t e m s t r a n s g e n i c a n i m a l s h a v e a l s o b e e n e x p l o i t e d to s t u d y the e x p r e s s i o n o f a c t i n genes. The r e m a i n d e r of this s e c t i o n o u t l i n e s the p r o g r e s s m a d e by the use o f t h e s e v a r i o u s t e c h n i q u e s in i d e n t i f y i n g c i s - a c t i n g s e q u e n c e s a n d t r a n s - a c t i n g f a c t o r s i m p o r t a n t in the e x p r e s s i o n of the

0*\-a c t i n g e n e s .

1 . 3 . 3 . C i s - a c t i n g s e q u e n c e s and t r a n s - a c t i n g f a c t o r s i n v o l v e d i n the c o n t r o l o f c a r d i a c a ctin g e n e e x p r e s s i o n . P r e l i m i n a r y e x p e r i m e n t s by M i n t y and K e d e s ( 1 9 8 6 ) d e m o n s t r a t e d t hat t h e 5' fla n k i n g r e gion, the first e x o n a n d 2 8 n u c l e o t i d e s o f t h e first i n t r o n of the h u m a n c a r d i a c a c t i n g ene is s u f f i c i e n t to d r i v e the t i s s u e - s p e c i f i c e x p r e s s i o n of a c h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e (CAT) r e p o r t e r g ene w h e n