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The synthesis of myosin mRNA and myosin in the early development of Xenopus laevis embryos Kreis, Christophe G. 1978

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THE S Y N T H E S I S OF MYOS IN mRNA AND MYOS IN I N THE E A R L Y DEVELOPMENT OF XENOPUS L A E V I S EMBRYOS b y CHR I STOPHE G . K R E I S M . S c . , S a n F r a n c i s c o S t a t e U n i v e r s i t y , 1 9 7 3 A T H E S I S SUBM ITTED I N P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF P H I L O S O P H Y i n THE F A C U L T Y OF GRADUATE STUD I E S ( D e p a r t m e n t o f ZOOLOGY) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMB IA J u l y , 1 9 7 8 © C h r i s t o p h e G. K r e i s , 1 9 7 8 In presenting th is thes is in p a r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without m y v wri t ten permission. Department of ZOOLOGY  The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 18 O c t o b e r , 1978 i i A B S T R A C T A b i o c h e m i c a l a p p r o a c h w a s u s e d t o d e t e c t t h e a p p e a r a n c e o f t h e h e a v y c h a i n o f s k e l e t a l m y o s i n (HCSM) a n d m y o s i n mRNA d u r i n g 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 l a e v i s e m b r y o s . A n a n t i b o d y a g a i n s t t h e HCSM o f a d u l t X> l a e v i s m u s c l e s w a s b i o c h e m i c a l l y c h a r a c t e r i z e d a n d s h o w n t o b e m o n o s p e c i f i c . T h i s a n t i -m y o s i n a n t i b o d y r e a c t e d w i t h e m b r y o n i c p o l y s o m e s s y n t h e s i z i n g m y o s i n a n d w i t h t a d p o l e t a i l m y o s i n . T h i s i n d i c a t e s t h a t t h e m y o s i n s o f a d u l t m u s c l e s , e a r l y e m b r y o n i c m u s c l e s a n d t a d p o l e t a i l s a r e s u f f i c i e n t l y h o m o l o g o u s t o s h a r e s ome a n t i g e n i c d e t e r m i n a n t s . P o l y s o m e s f r o m v a r i o u s s t a g e s o f X . l a e v i s e m b r y o g e n e s i s w e r e r e a c t e d w i t h t h e a n t i - m y o s i n a n t i b o d y . A n a l y s i s o f t h e s e r e a c t i o n s s h o w e d t h a t m y o s i n s y n t h e s i s b e g i n s i n s t a g e 20 e m b r y o s , i n w h i c h a b o u t 7 s o m i t e s h a v e s e g r e g a t e d . T h e RNA f r o m s t a g e 1 2 , s t a g e 1 6 / 1 7 a n d s t a g e 20 e m b r y o s w a s t h e n a n a l y z e d f o r t h e p r e s e n c e o f t h e h e a v y c h a i n m y o s i n mRNA i n o r d e r t o d e t e r m i n e w h e t h e r t h e s y n t h e s i s o f m y o s i n i s u n d e r t r a n s l a t i o n a l o r t r a n s c r i p t i o n a l c o n t r o l . T o t a l RNA p r e p a r a t i o n s f r o m s t a g e d e m b r y o s w e r e f r a c t i o n a t e d o n o l i g o ( d T ) - c e l l u l o s e c o l u m n s a n d f r a c t i o n s t h a t d i d a n d d i d n o t b i n d w e r e t r a n s l a t e d i n a w h e a t g e r m c e l l - f r e e p r o t e i n s y n t h e s i z i n g s y s t e m . T h e t r a n s l a t i o n a l p r o d u c t s w e r e p r e c i p i t a t e d w i t h t h e a n t i - m y o s i n a n t i b o d y a n d c h a r a c t e r i z e d b i o c h e m i c a l l y . M y o s i n mRNA w a s d e t e c t e d b y t h i s m e t h o d i n s t a g e 1 6 / 1 7 e m b r y o s . We c o n c l u d e t h a t s o m i t e s e g r e g a t i o n r e s u l t s i n t h e a p p e a r a n c e o f i i i new myosin mRNA molecules in X. laevis embryos. It seems l i k e l y , by a l l the evidence considered, that a large pool of untranslated myosin mRNA molecules i s not responsible for muscle myosin synthesis. Therefore,: the synthesis of certain proteins in early development i s under transcriptional control. i v T A B L E OF CONTENTS P a g e ABSTRACT i i T A B L E OF CONTENTS i v L I S T OF T A B L E S v L I S T OF F I G U R E S v i ACKNOWLEDGEMENTS v i i i I NTRODUCT ION 1 M A T E R I A L S AND METHODS A 16 RESULTS A 25 C h a r a c t e r i z a t i o n o f a n t i - m y o s i n a n t i b o d y 25 Q u a n t i t a t i v e p r e c i p i t a t i o n o f m y o s i n 27 I s o l a t i o n a n d a n a l y s i s o f e m b r y o n i c p o l y s o m e s 29 B i n d i n g o f a n t i - m y o s i n a n t i b o d y t o e m b r y o n i c p o l y s o m e s 30 P o l y s o m e r e a c t i o n c o n t r o l e x p e r i m e n t s 32 M A T E R I A L S AND METHODS B 64 RESULTS B 73 I s o l a t i o n a n d F r a c t i o n a t i o n o f RNA 73 C e l l - f r e e t r a n s l a t i o n o f X e n o p u s mRNA 75 C e l l - f r e e t r a n s l a t i o n a n a l y s i s o f a p p e a r a n c e o f m y o s i n mRNA d u r i n g X e n o p u s e m b r y o g e n e s i s 76 D I S C U S S I O N 93 CONCLUS IONS 100 L I T E R A T U R E C I T E D 103 V L I S T OF TABLES T a b l e Page 1. •ATPase a c t i v i t y o f Xenopus l a e v i s s k e l e t a l m y o s i n 34 2. Amino a c i d c o m p o s i t i o n o f m y o s i n heavy c h a i n s p r e p a r e d f r o m a d u l t Xenopus l a e v i s s k e l e t a l m u s c l e 35 3. Y i e l d s o f RNA p r e p a r e d f r o m Xenopus embryos by d i f f e r e n t i s o l a t i o n p r o c e d u r e s 82 4. Q u a n t i t i e s o f t o t a l and p o l y s o m a l mRNA i n Xenopus embryos a t s t a g e s 12, 16/17 and 20 83 5. P u r i f i c a t i o n o f p o l y ( A ) + RNA f r o m Xenopus embryos by b i n d i n g t o o l i g o ( d T ) - c e l l u l o s e 84 6. E f f e c t o f magnesium on t o t a l p r o t e i n s y n t h e s i s 85 7. E f f e c t o f p o t a s s i u m a c e t a t e on t o t a l p r o t e i n s y n t h e s i s and m y o s i n s y n t h e s i s 86 8. Dependance o f t o t a l p r o t e i n s y n t h e s i s on added p o l y ( A ) + Xenopus RNA 87 9. Time c o u r s e o f t o t a l p r o t e i n s y n t h e s i s and m y o s i n s y n t h e s i s i n t h e wheat germ p r o t e i n s y n t h e s i z i n g s y s t e m 88 10. I m m u n o p r e c i p i t a t i o n o f t h e c e l l - f r e e r e a c t i o n p r o d u c t w i t h a n t i - m y o s i n a n t i b o d y . 39 11. I m m u n o p r e c i p i t a t i o n o f t h e c e l l - f r e e r e a c t i o n p r o d u c t w i t h a n t i - m y o s i n a n t i b o d y d i r e c t e d by p o l y ( A ) + and p o l y ( A ) RNA f r o m m i x e d s t a g e 12 and s t a g e 20 embryos 90 v i L I S T OF FIGURES F i g u r e Page l a . 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 on a 10% S D S - a c r y l a m i d e g e l o f m y o s i n f r a c t i o n s p u r i f i e d by ammonium s u l f a t e p r e c i p i t a t i o n 36 l b . 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 on a 8% S D S - a c r y l a m i d e g e l o f c o l u m n - p u r i f i e d m y o s i n f r a c t i o n s 38 2. S e r o l o g i c a l i d e n t i t y o f g e l - p u r i f i e d m y o s i n and c r u d e m y o s i n f r o m h i g h s a l t e x t r a c t s o f a d u l t X« l a e v i s m u s c l e 40 3. Phase c o n t r a s t and i n d i r e c t i m m u n o f l u o r e s c e n c e m i c r o g r a p h s o f t h e same f i e l d o f m u s c l e from t h e t h i g h m u s c l e o f a d u l t Xenopus f r o g s . 42 125 4. A u t o r a d i o g r a m o f [ I ] - l a b e l l e d p u r e m y o s i n and 125 [ I ] - l a b e l l e d c r u d e m u s c l e e x t r a c t p r e c i p i t a t e d w i t h a n t i - m y o s i n a n t i b o d y 44 5. A u t o r a d i o y r a m o f i o d i n a t e d t r y p t i c f r a g m e n t s o f e l e c t r o p h o r e t i c a l l y p u r i f i e d m y o s i n and a n t i b o d y -p r e c i p i t a t e d m y o s i n 46 6. P r e c i p i t a t i o n of m y o s i n by a n t i - m y o s i n a n t i b o d y 48 125 7. B i n d i n g o f [ I ] - l a b e l l e d a n t i - m y o s i n a n t i b o d y t o polysomes f r o m v a r i o u s d e v e l o p m e n t a l s t a g e s o f Xenopus l a e v i s embryos 50 125 8. B i n d i n g o f [ I ] - l a b e l l e d a n t i b o d y t o s t a g e 20 polysomes v i i L i s t o f F i g u r e s ( C o n t . ) F i g u r e Page t r e a t e d w i t h RNase p r i o r t o c e n t r i f u g a t i o n 52 9. S e r o l o g i c a l a n a l y s i s o f m y o s i n s f r o m s t a g e 16/17, s t a g e 20 and s t a g e 26 embryos 54 10a. 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 on a 10% S D S - a c r y l a m i d e g e l o f m y o s i n e x t r a c t s o f s t a g e 20 and s t a g e 26 embryos 56 10b. 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 on a 7.5% S D S - a c r y l a m i d e g e l o f e x t r a c t s o f s t a g e 16/17 embryos and t a d p o l e t a i l s 58 125 11. B i n d i n g o f [ I ] - a n t i - m y o s i n t o Xenopus l i v e r polysomes m i x e d 1:1 w i t h t h e 100,000 x g s u p e r n a t a n t o f s t a g e 20 polysomes 60 125 12. B i n d i n g o f [ I ] - a n t i - m y o s i n t o s t a g e 20 polysomes i s o l a t e d i n t h e p r e s e n c e o f m y o s i n heavy c h a i n s 62 13. A n a l y s i s by e l e c t r o p h o r e s i s on a 10% S D S - a c r y l a m i d e g e l o f t r y p t i c f r a g m e n t s o f t r a n s l a t i o n p r o d u c t s p r e c i p i t a t e d w i t h a n t i - m y o s i n a n t i b o d y 91 1 INTRODUCTION A major goal of developmental biology i s to understand the regulatory mechanisms that ensure the synthesis of specific proteins. For the most part, the study of differentiation has become the study of d i f f e r e n t i a l gene expression. Such studies have dealt, because of methodological limitations, with the terminal steps of pathways giving rise to highly specialized tissues, such as skeletal muscle. Efforts to understand the mechanisms that are involved in c e l l differentiation favor two dominant points for control: (1) Transcriptional control and (2) translational control. Transcription includes the synthesis of the i n i t i a l precursor mRNA, the processing of this RNA into mature mRNA and i t s transport to the cytoplasm (Darnell, 1976). Transcriptional control implies that qualitative and/or quantitative changes in the pattern of RNA synthesis lead to alterations in the pool of mRNA sequences available to the protein translating machinery of a c e l l . Thus, the concentration of a particular mRNA in the c e l l determines the rate of synthesis of a particular protein. The term translational control includes situations in which gene expression i s modulated by mechanisms which select or activate particular mRNAs from a pre-existing pool of untranslated mRNA. Thus, a l l cells having the same genes w i l l transcribe a l l those genes into RNA. Differences among cells would result from the a b i l i t y of each c e l l to select which message to translate. ^ It has been shown in unfertilized eggs of sea urchins that 2 v a r i o u s s p e c i e s o f mRNAs a r e s y n t h e s i z e d and some o f them a r e t r a n s l a t e d i n t h e c y t o p l a s m . G a l a u e_t a l . (1976) have d e m o n s t r a t e d t h e e x i s t e n c e o f s o - c a l l e d "complex c l a s s " RNAs ( p r e s e n t i n : 1 - 5 c o p i e s p e r c e l l ) , i n t h e u n f e r t i l i z e d s e a u r c h i n egg, w h i c h p r o b a b l y code f o r m e t a b o l i c enzymes ( h o u s e k e e p i n g enzymes) and s o - c a l l e d " m o r p h o g e n e t i c d e t e r m i n a n t s " ( D a v i d s o n , 1976). I t has a l s o been shown t h a t h i s t o n e and t u b u l i n p r o t e i n s a r e s y n t h e s i z e d p r i o r t o , as w e l l as a f t e r , f e r t i l i z a t i o n i n t h e c y t o p l a s m o f t h e s e a u r c h i n egg ( D a v i d s o n , 1976; Gurdon, 1974). When s e a u r c h i n eggs a r e t r e a t e d a t f e r t i l i z a t i o n w i t h a c t i n o m y c i n D, t h e embryos a r e a b l e t o undergo c l e a v a g e and d e v e l o p up t o t h e g a s t r u l a s t a g e (Terman and G r o s s , 1965). These r e s u l t s i m p l y t h a t p r o t e i n s y n t h e s i s i n e a r l y development o c c u r s on p r e f o r m e d t e m p l a t e s . Hence, i t a p p e a rs t h a t p r o t e i n s s y n t h e s i z e d on complex RNA t e m p l a t e s and h o u s e k e e p i n g RNA t e m p l a t e s ( s u c h as h i s t o n e and t u b u l i n ) a r e under t h e i n f l u e n c e o f a p o s t - t r a n s c r i p t i o n a l d e v e l o p m e n t a l program. S i m i l a r r e s u l t s have been o b t a i n e d when Xenopus ( a m p h i b i a n ) embryos have been t r e a t e d w i t h a c t i n o m y c i n D ( B r a c h e t et^ _ a l . , 1964). A g a i n , c l e a v a g e i n t h e s e embryos does n o t seem t o be a f f e c t e d by t h e d r u g , a l t h o u g h ^ a s t r u l a t i o n and n e u r u l a t i o n a r e b l o c k e d . F u r t h e r m o r e , h i s t o n e s and t u b u l i n a r e s y n t h e s i z e d p r i o r t o and a f t e r f e r t i l i z a t i o n i n Xenopus eggs ( P e s t e l l , 1975; Woodland and Adamson, 1977). I n a d d i t i o n , Darnbrough and F o r d (1976) have shown t h e e x i s t e n c e of v e r y l o n g - l i v e d messages i n t h e c y t o p l a s m o f u n f e r t i l i z e d Xenopus eggs. A n o t h e r c a s e o f documented t r a n s l a t i o n a l c o n t r o l i n Xenopus embryos c o n c e r n s t h e appearance o f enzymes i n v o l v e d i n t h e c o n v e r s i o n o f t y r o s i n e t o m e l a n i n , v i a t h e enzyme t y r o s i n e - D O P A o x i d a s e (Benson and T r i p l e t t , . 1974; D a v i d s o n , 1976). The s y n t h e s i s of t h i s p r o t e i n i s f i r s t d e t e c t a b l e a t t h e n e u r u l a s t a g e , w e l l i n advance o f t h e ap p e a r a n c e o f enzyme a c t i v i t y i n melanphores a t t h e h a t c h i n g s t a g e . Thus, i t a p p e a r s t h a t some genes i n a m p h i b i a n s a r e a c t i v e i n t h e egg and a r e n e c e s s a r y f o r e a r l y d evelopment. I t f o l l o w s f r o m t h i s d i s c u s s i o n t h a t some genes e x p r e s s e d i n e a r l y development a r e c o n t r o l l e d by some f o r m o f t r a n s l a t i o n a l c o n t r o l . However, i t i s p o s s i b l e t h a t genes c o d i n g f o r m o l e c u l e s s u c h as s k e l e t a l m u s c l e m y o s i n may n o t be i n c l u d e d i n t h i s c a t e g o r y s i n c e t h e p r o d u c t s o f t h e s e genes do n o t make t h e i r a p p e a r a n c e u n t i l a f t e r g a s t r u l a t i o n (Gurdon, 1974; D a v i d s o n , 1976). From a l l a v a i l a b l e d a t a , t h e genes c o d i n g f o r s u c h " c e l l t y p e - s p e c i f i c " p r o d u c t s a r e s i n g l e - c o p y sequences whose mRNA p r o d u c t s a r e fo u n d i n h i g h c o n c e n t r a t i o n s i n c e l l s ( G a l a u e t a l . , 1976; P a t e r s o n and B i s h o p , 1977). T h e r e f o r e , t h e i r mRNAs a r e n o t p a r t o f complex c l a s s mRNAs o r o f mRNAs s y n t h e s i z e d f r o m h i g h l y r e p e a t e d genes ( h i s t o n e genes) ( G a l a u e t a l . , 1976; D a v i d s o n , 1976; Adamson and Woodland, 1977). E x p e r i m e n t a l e v i d e n c e a l s o seems t o i n d i c a t e t h a t messenger RNA s y n t h e s i s i n s p e c i a l i z e d c e l l s s u c h as t h e c h i c k o v i d u c t and mus c l e c e l l s i n t i s s u e c u l t u r e i s under t r a n s c r i p t i o n a l c o n t r o l ( P a l m i t e r , 1975; P a t e r s o n and B i s h o p , 1 9 7 7 ) ; t h i s s u g g e s t s t h a t t h i s may a l s o be t r u e o f embryos (Gurdon, 1974). We have examined t h e r e g u l a t i o n o f m y o s i n gene e x p r e s s i o n d u r i n g s o m i t e f o r m a t i o n i n Xenopus embryos. D i f f e r e n t i a t i n g m u s c l e i n Xenopus  l a e v i s embryos undergoes a s e r i e s o f d i s t i n c t m o r p h o l o g i c a l changes. The m o l e c u l a r e v e n t s w h i c h d e t e r m i n e and accompany t h e s e changes i n t h i s e m b r y o n i c s y s t e m , as w e l l as i n o t h e r s , have n o t been f u l l y c h a r a c t e r i z e d . Thus, p r o d u c t i o n o f m y o s i n and o t h e r m u s c l e p r o t e i n s i n X. l a e v i s embryos i s a f a v o u r a b l e s y s t e m f o r s t u d y i n g t h e m o l e c u l a r 4 aspects of myogenesis and sequential gene expression during vertebrate development. The f i r s t somites in Xenopus embryos appear in the head region at the end of neurulation and myosin-like thick filaments are f i r s t v i s i b l e with the light and electron microscope at the early tail-bud stage (Hamilton, 1969; Muntz, 1975; Kullberg et a l . , 1977). Myoblasts in the somites do not appear to fuse u n t i l the onset of metamorphosis (Muntz, 1975). Therefore, the different muscle populations are easily distinguished during the development of Xenopus embryos. Furthermore, pre-fusion myoblasts are well-separated in time from fused myoblasts. Transcriptional versus translational controls in development. Direct evidence for transcriptional and translational controls has come from developmental systems. Intensive studies on erythropoeitic cells and the chick oviduct have now provided some of the most convincing evidence for transcriptional control. Translational control appears to be responsible for the quantitative changes in protein synthesis which occur at f e r t i l i z a t i o n in some species. The f i r s t evidence for message-specific translational components has come from work on translation of chick myosin mRNA in v i t r o . However, there i s also strong evidence that the synthesis of muscle-specific proteins in chick muscle cultures i s mainly controlled at the transcriptional level. The purpose of the present discussion is to examine the evidence for the existence of transcriptional and translational controls during development. Studies i n a few well-defined systems indicate that cells in the terminal compartment of certain lineages synthesize proteins and 5 mRNAs t h a t a r e q u a l i t a t i v e l y d i f f e r e n t f r o m t h o s e f o u n d i n t h e p r o g e n i t o r l i n e a g e c e l l s ( H o l t z e r e_t a l . , 1 9 7 5 ) . E x p e r i m e n t s by A x e l e t a l . (1973) and G r o u d i n e f s t : a l . ( 1 9 7 4 ) , f o r example, p r o v i d e e v i d e n c e t h a t c h i c k e n h e m a t o c y t o b l a s t c e l l s do n o t appear t o s y n t h e s i z e g l o b i n mRNAs a t t h e same r a t e as e r y t h r o b l a s t s . S i m i l a r l y , h y b r i d i z a t i o n e x p e r i m e n t s by t h e s e w o r k e r s , u s i n g a l a b e l l e d cDNA probe complementary t o a d u l t g l o b i n mRNA, show t h a t i f a h e m a t o c y t o b l a s t t r a n s c r i b e s g l o b i n mRNA, i t i s a t a r a t e a t l e a s t 10^ t i m e s l o w e r t h a n t h a t t r a n s c r i b e d by e r y t h r o b l a s t s . I t has a l s o been shown t h a t t h e cDNA h y b r i d i z e d w i t h RNA made by r e t i c u l o c y t e c h r o m a t i n b u t n o t w i t h hRNA made by l i v e r c h r o m a t i n ( A x e l e t a l . , 1973). G r o u d i n e and W e i n t r a u b (1975) a l s o d e m o n s t r a t e d t h a t h e m o g l o b i n mRNA sequences a r e n o t d e t e c t a b l e i n t h e t o t a l RNA o f n o r m a l c h i c k f i b r o b l a s t s . I t i s c l e a r , t h e r e f o r e , t h a t w i t h r e s p e c t t o t h e p r e s e n c e o r absence o f h e m o g l o b i n message, t h e d i f f e r e n c e among r e t i c u l o c y t e , l i v e r and f i b r o b l a s t c e l l s i s t r a n s c r i p t i o n a l i n n a t u r e . However, l o n g - t e r m growth i n t i s s u e c u l t u r e o f c h i c k f i b r o b l a s t s r e s u l t s i n t h e p r o d u c t i o n o f s m a l l amounts o f g l o b i n message as d e t e c t e d by t h e cDNA p r o b e (Humphries e_t a i l . , 1976). The c h i c k o v i d u c t has p r o v e d t o be an e s p e c i a l l y u s e f u l s y s t e m f o r t h e s t u d y o f gene e x p r e s s i o n . A d m i n i s t r a t i o n o f e s t r o g e n t o a newborn c h i c k r e s u l t s i n t h e d i f f e r e n t i a t i o n o f t h r e e d i s t i n c t e p i t h e l i a l c e l l t y p e s f r o m t h e homogeneous p o p u l a t i o n o f m u c o s a l c e l l s ( P a l m i t e r , 1975). One o f t h e s e c e l l t y p e s , t h e t u b u l a r g l a n d c e l l s , s y n t h e s i z e t h e p r o t e i n o v a l b u m i n a f t e r a b out 18 h o u r s . A f t e r h o r m o n a l t r e a t m e n t i s s t o p p e d , o v a l b u m i n s y n t h e s i s d e c l i n e s and becomes u n d e t e c t a b l e . When e s t r o g e n o r p r o g e s t e r o n e ( s e c o n d a r y s t i m u l a t i o n ) i s a d m i n i s t e r e d a seco n d t i m e , o v a l b u m i n i s i n d u c e d a g a i n i n t h e e x i s t i n g t u b u l a r g l a n d 6 c e l l s a f t e r a lag of about 3 hours. This induction precedes a second stage of c e l l u l a r p r o l i f e r a t i o n . Experiments performed on the chick oviduct, which has been secondarily stimulated by s t e r o i d hormones to synthesize ovalbumin, show that the in c r e a s e . i n the rate of ovalbumin J synthesis i s q u a n t i t a t i v e l y c o r r e l a t e d with an increase i n the ovalbumin mRNA content of the oviduct (Palmiter, 1973). T i t r a t i o n of the ovalbumin mRNA sequence, with a cDNA probe complementary to t h i s message, shows that the message i s undetectable i n the absence of s t e r o i d s . A f t e r secondary hormone stimulation, there are 70,000 molecules of ovalbumin mRNA present per tubular gland c e l l (Cox et a l . , 1974; Harris et a l . , 1975) and most of the ovalbumin mRNA (93%) i s present on polysomes. Recently, Thomas and Palmiter (1977) found that ribonucleoprotein (RNP) p a r t i c l e s , i s o l a t e d from n u c l e i of estrogen-stimulated chick oviducts, contain ovalbumin mRNA. Upon hormone withdrawal, the concentration of ovalbumin mRNA i n the RNP p a r t i c l e s r a p i d l y decreases within 24 hours with k i n e t i c s equal to the decrease i n numbers of estrogen receptors i n the nucleus (The numbers of nuclear receptors correlates with the rate of ovalbumin mRNA production). These r e s u l t s imply that ovalbumin mRNA may be stored i n RNP p a r t i c l e s for a short time p r i o r to t r a n s l a t i o n . However, the e s s e n t i a l control f o r ovalbumin synthesis appears to be t r a n s c r i p t i o n a l although secondary controls probably e x i s t at the t r a n s l a t i o n a l and p o s t - t r a n s l a t i o n a l l e v e l s . This hypothesis i s strengthened by the f a c t that the induction of other proteins s p e c i f i c to the oviduct i s mediated by d i f f e r e n t l e v e l s of hormone and has d i f f e r e n t rate constants (Palmiter et a l . , 1972). If regulation of ovalbumin and other o v i d u c t - s p e c i f i c proteins was s t r i c t l y at the t r a n s l a t i o n a l l e v e l , then a l l the respective mRNAs should be synthesized 7 at the same rate upon hormone stimulation. There are other c e l l types which show similar developmental kinetics to the chick oviduct (Gurdon, 1974; Baker and Shapiro, 1977; Reeves, 1977). For example, vitellogenin mRNA is undetectable in livers of male Xenopus laevis (Baker and Shapiro, 1977). For 3 hours after primary estrogen administration, vitellogenin mRNA remains undetectable. However, 4 1/2 hours after hormone stimulation, these mRNA sequences are detectable. Sixty to 65 days after hormone stimulation, the concentration of vitellogenin mRNA molecules drops down to very low levels. The results of these studies imply that the production of vitellogenin i s transcriptionally controlled. The main points to emerge from this review are the following: Gene transcription is controlled in systems whose developmental program is initiated by an external stimulus such as a hormone. However, the hormones can only affect those cells which have, in the course of their differentiation, acquired the appropriate receptors. Thus, these agents enhance a regulatory mechanism which is already present in the cells that respond. Translational control in muscle differentiation. Evidence for translational control mechanisms during muscle differentiation has come from the work of Gros, Heywood, Sarkar and their colleagues. A study of RNA synthesis during muscle development has been carried out by Buckingham et a l . (1974) on primary cultures from fetal calf muscle. Ribonucleic acid extracted from the muscle cells 8 3 a f t e r p u l s e - l a b e l l i n g w i t h [ H ] - u r i d i n e was a n a l y z e d on s u c r o s e g r a d i e n t s , and mRNA was i d e n t i f i e d on t h e b a s i s o f i t s b i n d i n g t o p o l y ( U ) f i l t e r s . I t was o b s e r v e d t h a t a l a b e l l e d peak o f 26S RNA was p r e s e n t i n d i v i d i n g p r e c u r s o r m y o b l a s t s . T h i s peak was presumed t o be m y o s i n mRNA on t h e b a s i s o f i t s s e d i m e n t a t i o n r a t e . E x a m i n a t i o n o f t h e c y t o p l a s m i c l o c a l i z a t i o n o f t h e 26S RNA i n d i c a t e d t h a t i t was p r e d o m i n a n t l y n o n - p o l y s o m a l . The h a l f - l i f e o f t h e 26S RNA was 10 h o u r s b e f o r e f u s i o n o f c e l l s and i n c r e a s e d by a f a c t o r o f 5-6 j u s t b e f o r e f u s i o n . A f t e r f u s i o n , t h e 26S RNA was f o u n d m o s t l y on pol y s o m e s . These d a t a s u g g e s t t h a t p o s t - t r a n s c r i p t i o n a l c o n t r o l s , a c t i n g on messenger s t a b i l i t y and u t i l i z a t i o n , may be i m p o r t a n t i n t h e t e r m i n a l d i f f e r e n t i a t i o n o f m u s c l e . The weakness i n t h i s argument l i e s i n t h e f a i l u r e o f t h e s e w o r k e r s t o c o n v i n c i n g l y i d e n t i f y t h e 26S RNA. The p r e - f u s i o n 26S RNA may code f o r n o n - s k e l e t a l m y o s i n o r f o r a p r o t e i n o t h e r t h a n m y o s i n . Bag and S a r k a r (1976) have i s o l a t e d a m y o s i n heavy c h a i n mRNP p a r t i c l e (MHC mRNP) f r o m t h e n o n - p o l y s o m a l f r a c t i o n o f homogenates o f 14 d a y - o l d c h i c k e m b r y o n i c m u s c l e s . They showed t h a t t h e p a r t i c l e and the p r o t e i n - f r e e RNA d e r i v e d f r o m t h e p a r t i c l e were e f f e c t i v e i n programming t h e s y n t h e s i s o f m y o s i n i n a r e t i c u l o c y t e c e l l - f r e e s y s t e m as j u d g e d by g e l e l e c t r o p h o r e s i s . F u r t h e r m o r e , i t was shown t h a t t h e mRNP p a r t i c l e s e x i s t a s 2 d i s t i n c t t y p e s o f m a c r o m o l e c u l a r complexes i n d i f f e r e n t f r a c t i o n s o f t h e c y t o p l a s m : a r e l a t i v e l y p r o t e i n -d e f i c i e n t p a r t i c l e i n t h e polysomes where t h e mRNAs a r e t r a n s l a t e d and a p r o t e i n - r i c h p a r t i c l e i n t h e n o n - p o l y s o m a l compartment where t h e y a r e no t t r a n s l a t e d . The t i s s u e s t u d i e d by Bag and S a r k a r i s d i f f e r e n t i a t e d and i t i s n o t c l e a r as t o w h e t h e r t h e n o n - p o l y s o m a l mRNA p a r t i c l e s a r e m e r e l y p r e s e n t i n e x c e s s o f t h e t r a n s l a t i o n a l c a p a c i t y o f t h e c e l l (Gurdon, 1974). A n o t h e r p r o p o s a l t o a c c o u n t f o r c o n t r o l o f p r o t e i n s y n t h e s i s , d e r i v e d f r o m e x p e r i m e n t s on c h i c k e n embryo l e g m u s c l e , i s t h a t o f t h e e x i s t e n c e o f m e s s a g e - s p e c i f i c f a c t o r s f o r t h e t r a n s l a t i o n o f s k e l e t a l m y o sin. Rourke and Heywood (1972) f o u n d t h a t o n l y m u s c l e i n i t i a t i o n f a c t o r s ( p r o t e i n s removed f r o m m u s c l e r i b o s o m e s by w a s h i n g w i t h s a l t ) were c a p a b l e o f d i r e c t i n g m y o s i n heavy c h a i n s y n t h e s i s and t h a t r e t i c u l o c y t e f a c t o r s c o u l d n o t s u b s t i t u t e f o r m u s c l e f a c t o r s when s a l t - w a s h e d m u s c l e r i b o s o m e s were us e d . S i n c e t h e i n i t i a t i o n f a c t o r s i s o l a t e d by Heywood and c o l l e a g u e s were n o t shown t o be c o m p l e t e l y p u r e , i t i s p o s s i b l e t h a t o t h e r f a c t o r s p r e s e n t i n t h e r i b o s o m a l s a l t wash m i g h t a c t s e l e c t i v e l y t o a l l o w c o m p l e t i o n o f t h e i n t a c t m y o s i n m o l e c u l e , w h i c h o t h e r w i s e m i g h t be p r o d u c e d o n l y as an u n d e t e c t e d f r a g m e n t . I t w i l l be shown l a t e r i n t h i s t h e s i s t h a t t h e p r o b l e m o f p r e m a t u r e t e r m i n a t i o n o f p o l y p e p t i d e c h a i n s i n c e l l - f r e e systems has t o be c o n s i d e r e d ( A t k i n s e t a l . , 1 975). Heywood and Kennedy (1976) have a l s o s u g g e s t e d t h a t a s u b s t a n c e w i t h some o f t h e p r o p e r t i e s o f RNA (tcRNA) was i s o l a t e d f r o m d i a l y s a t e s o f m u s c l e i n i t i a t i o n f a c t o r s and t h i s tcRNA can i n h i b i t t r a n s l a t i o n o f m y o s i n mRNA i t i c e l l - f r e e s y s t e m s . However, q u e s t i o n s c o n c e r n i n g t h e degree o f s p e c i f i c i t y o f t h i s r e a c t i o n and t h e f u n c t i o n o f t h i s tcRNA i n v i v o r e m a i n t o be answered. I s m y o s i n s y n t h e s i s r e g u l a t e d a t t h e t r a n s l a t i o n a l l e v e l ? The e x p e r i m e n t s p r e s e n t e d i n t h i s s e c t i o n do n o t p r o v i d e an answer t o t h i s q u e s t i o n . T r a n s l a t i o n a l c o n t r o l as a p r i m a r y mechanism f o r t h e r e g u l a t i o n o f my o s i n s y n t h e s i s p r e s u p p o s e s t h a t m y o s i n mRNA e x i s t s i n d i v i d i n g m u s c l e p r e c u r s o r c e l l s i n an u n s t a b l e f o r m o r i n an u n t r a n s l a t a b l e form. When t h e m u s c l e p r e c u r s o r c e l l s s t o p d i v i d i n g , t h e m y o s i n mRNA 10 w o u l d be s t a b i l i z e d o r a c t i v a t e d . Such a mechanism can n o t be unambiguously r u l e d o u t . However, a t r a n s l a t i o n a l mechanism o f t h i s s o r t c a n n o t a c c o u n t f o r t h e r a p i d i n c r e a s e i n m y o s i n mRNA o b s e r v e d d u r i n g d i f f e r e n t i a t i o n i n v i t r o (Strohman e t a l . , 1977). I t has been c l e a r l y e s t a b l i s h e d t h a t Xenopus o o c y t e s and eggs have no mechanism by w h i c h t h e y can e x c l u d e t h e t r a n s l a t i o n o f messages c h a r a c t e r i s t i c o f o t h e r c e l l t y p e s (Gurdon, 1974; L a s k e y e t a l . , 1977). I t seems l i k e l y t h a t t r a n s l a t i o n a l c o n t r o l i s used t o make q u a n t i t a t i v e a d j u s t m e n t s t o a p a t t e r n o f p r o t e i n s y n t h e s i s d e t e r m i n e d p r i m a r i l y by t h e s y n t h e s i s o f new messages (Gurdon, 1974). T r a n s c r i p t i o n a l c o n t r o l i n m u s c l e d i f f e r e n t i a t i o n . The e x p e r i m e n t s d e s c r i b e d i n t h i s s e c t i o n appear t o d e m o n s t r a t e t h a t t h e e s s e n t i a l c o n t r o l f o r m y o s i n s y n t h e s i s i s t r a n s c r i p t i o n a l , a l t h o u g h s e c o n d a r y c o n t r o l s may e x i s t a t t h e t r a n s l a t i o n a l and p o s t -t r a n s l a t i o n a l l e v e l s . H o l t z e r e t a l . (1957) showed, by t h e use of f l u o r e s c e i n - l a b e l l e d a n t i b o d i e s , t h a t s k e l e t a l m y o s i n s y n t h e s i s i s t e m p o r a l l y c o r r e l a t e d w i t h t h e s e g r e g a t i o n o f s o m i t e s i n c h i c k embryos i n v i v o . T h i s i m p l i e s t h a t s k e l e t a l m y o s i n i s n o t s y n t h e s i z e d i n m u s c l e p r e c u r s o r c e l l s . However, t h i s r e s u l t has t o be i n t e r p r e t e d w i t h c a u t i o n s i n c e t h e myosin a n t i b o d y may have n o t been a b l e t o d e t e c t s m a l l q u a n t i t i e s o f m y o s i n . S u b s e q u e n t l y , B i s c h o f f and H o l t z e r (1970) showed t h a t m u s c l e p r e c u r s o r c e l l s c u l t u r e d i n v i t r o s y n t h e s i z e a m y o s i n heavy c h a i n t h a t i s e l e c t r o p h o r e t i c a l l y s i m i l a r t o m y o s i n i s o l a t e d f r o m myotubes. F u r t h e r m o r e , myosins f r o m m u s c l e p r e c u r s o r c e l l s o r s u c h non-myogenic c e l l s as f i b r o b l a s t s , n e r v e c e l l s , smooth m u s c l e c e l l s o r gut e p i t h e l i a l 11 c e l l s do n o t r e a c t w i t h s k e l e t a l m u s c l e a n t i b o d y i n O u c h t e r l o n y d o u b l e - d i f f u s i o n t e s t s ( C h i e t a l . , 1975; H o l t z e r e t a l . , 1 9 7 6 ) . However, m y o s i n s f r o m n o n - d i v i d i n g m y o b l a s t s , myotubes and mature m u s c l e do r e a c t w i t h s k e l e t a l m y o s i n a n t i b o d y . These r e s u l t s s u g g e s t t h a t m u s c l e p r e c u r s o r c e l l s s y n t h e s i z e m y o s i n s t h a t a r e t h e p r o d u c t s o f s t r u c t u r a l genes d i f f e r e n t f r o m t h o s e t h a t t r a n s c r i b e m y o s i n mRNAs i n p o s t - m i t o t i c m y o b l a s t s . A s t u d y o f RNA s y n t h e s i s d u r i n g myogenesis has been c a r r i e d o u t by Strohman e t a l . (1977) on p r i m a r y c u l t u r e s o f 12-day c h i c k embryo b r e a s t m u s c l e . R i b o n u c l e i c a c i d e x t r a c t e d f r o m p r e - f u s i o n and p o s t -f u s i o n c e l l s was t r a n s l a t e d i n a r e t i c u l o c y t e a s s a y s y s t e m and t h e t r a n s l a t i o n a l p r o d u c t s were p r e c i p i t a t e d w i t h an a n t i b o d y a g a i n s t s k e l e t a l m u s c l e m y o s i n . I t was f o u n d t h a t u n f u s e d c u l t u r e s o f c e l l s c o n t a i n e d a s m a l l amount o f my o s i n mRNA, w h i c h c o u l d be s y n t h e s i z e d by t h e s m a l l amount o f p r e c o c i o u s myotubes and m y o b l a s t s p r e s e n t i n u n f u s e d c u l t u r e s . I n f u s e d c u l t u r e s , however, t h e y f o u n d a 3 0 - f o l d i n c r e a s e i n m y o s i n mRNA a c t i v i t y . Thus, t h i s r e s u l t s u g g e s t s t h a t m y o b l a s t f u s i o n , w h i c h i s an e a r l y and e a s i l y r e c o g n i z e d p h e n o t y p i c m a n i f e s t a t i o n o f m u s c l e d i f f e r e n t i a t i o n , i s a s s o c i a t e d w i t h s y n t h e s i s o f m y o s i n mRNA and my o s i n . P a t e r s o n and B i s h o p (1977) a n a l y z e d t h e sequence c o m p l e x i t y , f r e q u e n c y d i s t r i b u t i o n and c o d i n g c a p a c i t y o f the mRNA p o p u l a t i o n s o f p r i m a r y c h i c k embryo m u s c l e c u l t u r e s a t d i f f e r e n t s t a g e s o f myogenesis. P r e - f u s i o n c u l t u r e s and f u s e d m y o f i b r i l l a r c u l t u r e s a l l c o n t a i n e d 17,000 d i f f e r e n t mRNA sequenc e s . The m y o f i b r i l c u l t u r e s a l s o c o n t a i n e d about 2500 sequences i n h i g h e r c o n c e n t r a t i o n and s i x sequences i n e x c e p t i o n a l l y h i g h c o n c e n t r a t i o n , each p r e s e n t i n about 15,000 c o p i e s p e r n u c l e u s . These sequences were shown t o be 10 t i m e s l e s s common i n p r e - m y o g e n i c 12 c u l t u r e s . The c o n c e n t r a t i o n o f t h e s e sequences i n rayofibrillar c u l t u r e s c o r r e l a t e d w e l l w i t h t h e c a p a c i t y o f t h e mRNA t o s t i m u l a t e t h e c e l l - f r e e s y n t h e s i s o f m u s c l e - s p e c i f i c p r o t e i n s s u c h as m y o s i n , as j u d g e d by co m p a r i s o n o f t r y p t i c p e p t i d e s o f c e l l - f r e e s y n t h e s i z e d m y o s i n and a u t h e n t i c m y o s i n . The e v i d e n c e p r e s e n t e d i n t h i s s e c t i o n s t r o n g l y s u p p o r t s t h e c o n c l u s i o n t h a t under b o t h i n v i v o and i n v i t r o c o n d i t i o n s , myogenesis i s p r o b a b l y r e g u l a t e d by t r a n s c r i p t i o n a l r a t h e r t h a n t r a n s l a t i o n a l c o n t r o l mechanisms. The s k e l e t a l m y o s i n s y n t h e s i z e d by p r e - and p o s t -f u s i o n c h i c k embryo m y o b l a s t s has been c h a r a c t e r i z e d by p r e c i p i t a t i o n w i t h s k e l e t a l m y o s i n heavy c h a i n a n t i b o d y and by t r y p t i c p e p t i d e a n a l y s i s . The 26S RNA c o d i n g f o r t h i s e m b r y o n i c s k e l e t a l m y o s i n has been t r a n s l a t e d i n c e l l - f r e e systems and t h e p r o d u c t s c h a r a c t e r i z e d by a n t i b o d y p r e c i p i t a t i o n and by t r y p t i c p e p t i d e f i n g e r p r i n t i n g . T h e r e f o r e , c h i c k m y o s i n mRNA does n o t appear t o be s t o r e d i n p r e c u r s o r m u s c l e c e l l s i n an i n a c t i v e form. However, t h e s e r e s u l t s do n o t r u l e o u t changes i n t h e s t a b i l i t y o f s e l e c t e d mRNA sequences as t h e mechanism r e g u l a t i n g t h e c o n c e n t r a t i o n o f mRNA i n t h e c y t o p l a s m . M y o g e n e s i s i n Xenopus l a e v i s embryos. I n Xenopus embryos, t h e f i r s t i n d i c a t i o n o f s o m i t e f o r m a t i o n i s fo u n d a t s t a g e 17, t h e l a t e n e u r a l f o l d s t a g e (Nieuwkoop and F a b e r , 1967). The s o m i t e s a r e masses o f mesodermal c e l l s w i t h a s m a l l c e n t r a l c a v i t y . The d o r s a l p a r t o f t h e i n n e r w a l l o f t h e s o m i t e i s t h e s o u r c e o f s o m a t i c m u s c l e i n a v e r t e b r a t e ' s body and i s c a l l e d t h e myotome. The c e l l s o f t h e myotome a r e o r i g i n a l l y o r i e n t a t e d w i t h t h e i r l o n g axes v e r t i c a l t o t h e body a x i s and a r e a r r a n g e d a r o u n d a c e n t r a l c a v i t y 13 c a l l e d t h e m y o c o e l i c c a v i t y . The p r o c e s s o f s o m i t e s e g r e g a t i o n p r o g r e s s e s i n a c r a n i o - c a u d a l d i r e c t i o n . F o r example, by s t a g e 20, when t h e n e u r a l f o l d s a r e f u s e d , s i x t o seven a n t e r i o r s o m i t e s have been i n d i v i d u a l i z e d . As t h e s o m i t e s become s e g r e g a t e d f r o m t h e s o m i t i c mesoderm, t h e i r c e l l u l a r d i f f e r e n t i a t i o n a l s o p r o c e e d s i n a c r a n i o - c a u d a l d i r e c t i o n . H a m i l t o n (1969) o b s e r v e d t h a t t h e m y o c o e l i s o b l i t e r a t e d b e f o r e s e g m e n t a t i o n s t a r t s and t h a t t h e f i n a l l o n g i t u d i n a l o r i e n t a t i o n o f m y o b l a s t s i s a c h i e v e d by a r o t a t i o n o f t h e myotome c e l l s t h r o u g h 90°. The g r a d u a l r o t a t i o n o f t h e myotome c e l l s can be seen i n sequence a l o n g t h e a x i s , s i n c e i t s t a r t s i n t h e most c r a n i a l s o m i t e s and f o l l o w s on i n a c r a n i o - c a u d a l sequence t h r o u g h t h e s e r i e s . Between s t a g e 20, when t h e n e u r a l t u b e has c l o s e d and s t a g e 22, when t h e eyes a r e b e g i n n i n g t o p r o t r u d e , t h e r e i s no s i g n o f s t r i a t e d m y o f i b r i l s i n t h e s o m i t e s when v i e w e d w i t h t h e l i g h t m i c r o s c o p e . A l s o , t h e embryo does n o t re s p o n d t o any f o r m o f s t i m u l a t i o n , i m p l y i n g t h a t t h e myotome c e l l s a r e i n c a p a b l e o f c o n t r a c t i o n (Muntz, 1975). By s t a g e s 22 t o 24, t h e embryo c o n t a i n s 12-15 s o m i t e s and a few t h i n m y o f i b r i l s a r e v i s i b l e by l i g h t m i c r o s c o p y i n t h e myotomes. A t t h e s e s t a g e s , t h e embryo res p o n d s t o d i r e c t m e c h a n i c a l and e l e c t r i c a l s t i m u l a t i o n i n t h e n e c k r e g i o n . By t h e t i m e t h e embryo i s fr e e - s w i m m i n g ( s t a g e s 3 2 - 4 6 ) , t h e myotome c e l l s c o n t a i n m y o f i b r i l s b u t r e m a i n u n i n u c l e a t e . I t i s o n l y a t t h e o n s e t o f metamorphosis ( s t a g e s 48-50) t h a t myotome c e l l s become m u l t i n u c l e a t e , p o s s i b l y by f u s i o n w i t h s a t e l l i t e c e l l s a t t h e ends o f t h e f i b r e s (Muntz, 1975). T h i s o c c u r s i n myotome m u s c l e s l o n g a f t e r c o n t r a c t i l i t y and n e r v o u s c o n t r o l have a p p e a r e d . Thus, t h e myotome c e l l s o f Xenopus d e v e l o p t o a f u l l y f u n c t i o n a l s t a t e and l a r g e s i z e 14 w h i l e t h e y r e m a i n u n i n u c l e a t e (Muntz, 1 9 7 5 ) . I n t h e e a r l y s t a g e s o f met a m o r p h o s i s , when t h e myotome m u s c l e c e l l s become m u l t i n u c l e a t e , t h e h i n d l i m b m u s c u l a t u r e a l s o d e v e l o p s , b u t i t s s t r u c t u r e i s q u i t e d i f f e r e n t ; f r o m t h e myotome m u s c l e b e c a u s e i t a p p e a r s t o be m u l t i n u c l e a t e f r o m t h e v e r y o n s e t , when t h e s t r i a t e d m y o f i b r i l s f i r s t a p p e a r (Muntz, 1975). T h e r e f o r e , i n t h e myotome m u s c l e c e l l s t h e e s t a b l i s h m e n t o f c o n t r a c t i l i t y and m u l t i n u c l e a t i o n ( t h a t i s , f u s i o n ) a r e n o t c a u s a l l y r e l a t e d as may be t h e c a s e w i t h h i n d l i m b m u s c l e . The p r e c e d i n g d e s c r i p t i o n o f Xenopus embryo myogenesis r a i s e s s e v e r a l q u e s t i o n s : 1. Do p r e s u m p t i v e m y o b l a s t s , t h a t i s , t h o s e c e l l s p r e s e n t i n t h e s o m i t e s p r i o r t o s e g r e g a t i o n , s y n t h e s i z e a l l t h e m o l e c u l e s p r e s e n t i n a f u n c t i o n a l m y o b l a s t b u t a t l o w e r l e v e l s ? 2. When i n em b r y o n i c development i s i t p o s s i b l e t o d e t e c t s k e l e t a l m y o s i n mRNA and myo s i n p r o t e i n ? 3. S i n c e many non-myogenic c e l l s s y n t h e s i z e a c t i n and m y o s i n , a r e t h e s e p r o t e i n s t h e p r o d u c t o f t h e same g e n e ( s ) as s k e l e t a l m u s c l e myosin? 4. I s t h e m y o s i n s y n t h e s i z e d i n t h e myotome c e l l s o f Xenopus embryos p r i o r t o f u s i o n , t h e p r o d u c t o f t h e same g e n e ( s ) as t h e m y o s i n o f a d u l t s k e l e t a l m u s c l e ? 5. I s t h e t r a n s c r i p t i o n and t r a n s l a t i o n o f s k e l e t a l m y o s i n mRNA c o u p l e d w i t h f u s i o n o f m y o b l a s t s ? The p r e s e n t s t u d y was d e s i g n e d t o be an e x p l o r a t o r y b a s e f o r a n s w e r i n g some o f t h e q u e s t i o n s a s k e d i n t h i s r e v i e w . A m o n o s p e c i f i c a n t i b o d y a g a i n s t t h e heavy c h a i n o f a d u l t Xenopus s k e l e t a l m u s c l e m y o s i n 15 was prepared and characterized. The antibody was then used to demonstrate the developmental appearance of myosin mRNA and myosin protein i n Xenopus embryos. I t appears that myogenesis i n Xenopus somites i s regulated at the l e v e l of t r a n s c r i p t i o n . 16 MATERIALS AND METHODS A C h e m i c a l s . U n l e s s o t h e r w i s e s t a t e d , a l l c h e m i c a l s u s e d were o f h i g h e s t p u r i t y and were o b t a i n e d e i t h e r f r o m Sigma, F i s h e r o r M a l l i n k r o d t . P h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF) was o b t a i n e d f r o m Sigma. Enzyme grade ammonium s u l p h a t e was o b t a i n e d f r o m Schwarz/Mann. D E A E - c e l l u l o s e and Sepharose 4B were o b t a i n e d f r o m P h a r m a c i a . R i b o n u c l e a s e - f r e e s u c r o s e was o b t a i n e d f r o m Schwarz/Mann. D i e t h y l p y r o c a r b o n a t e was o b t a i n e d f r o m Sigma. A c r y l a m i d e and N,N-methylene b i s a c r y l a m i d e came f r o m Eastman O r g a n i c C h e m i c a l s . Sodium d o d e c y l s u l p h a t e (SDS) was 125 o b t a i n e d f r o m BDH. Na I was p u r c h a s e d f r o m New E n g l a n d N u c l e a r . P r e p a r a t i o n o f s k e l e t a l m y o s i n . M y o s i n was p r e p a r e d f r o m t h e back and s u p e r f i c i a l t h i g h m u s c l e s o f X. l a e v i s a d u l t f r o g s by t h e p r o c e d u r e o f W i k m a n - C o f f e l t (1973) w i t h some m o d i f i c a t i o n . The t i s s u e was minced i n 2.5 volumes o f wash b u f f e r (5 mM so d i u m phosphate (pH 7 . 0 ) ) , 1 mM EDTA, 1 mM sodium p y r o p h o s p h a t e , 1 mM PMSF and 5 mM d i t h i o t h r e i t o l (DTT) and homogenized i n a S o r v a l l Omni-mixer a t f u l l s p e e d . The homogenate was c e n t r i f u g e d a t 9,000 x g f o r 5 m i n u t e s and t h e r e s u l t i n g p e l l e t washed t w i c e more i n wash b u f f e r f o l l o w e d by c e n t r i f u g a t i o n a t 9,000 x g f o r 5 m i n u t e s . Most o f t h e s o l u b l e p r o t e i n s were removed by t h i s p r o c e d u r e . The p e l l e t s f r o m t h e above c e n t r i f u g a t i o n s were t h e n e x t r a c t e d w i t h 1 volume o f 50 mM sod i u m p h o s p h a t e b u f f e r (pH 7 . 5 ) , 0.5 M K C l , 10 mM sodium p y r o p h o s p h a t e , 17 5 mM A T P , 5 mM DTT , 0 . 2 mM P M S F a n d 1 mM EDTA . A f t e r 1 0 m i n u t e s o n i c e , t h e s u s p e n s i o n w a s c e n t r i f u g e d a t 2 0 , 0 0 0 x g f o r 2 0 m i n u t e s . T h e p e l l e t w a s r e - e x t r a c t e d a n d t h e s u p e r n a t a n t s w e r e c o m b i n e d . T h o s e s u p e r n a t a n t s a r e c a l l e d t h e " a c t o m y o s i n f r a c t i o n " . T h e s u p e r n a t a n t s w e r e made 38% i n ammon ium s u l f a t e a t 4 ° C a n d t h e f r a c t i o n s p r e c i p i t a t i n g b e t w e e n 38% a n d 4 5 % s a t u r a t i o n w i t h ( N H . ) „ S 0 . w e r e c o l l e c t e d . T h e 4 2 4 p r e c i p i t a t i o n w i t h ammon i um s u l f a t e w a s r e p e a t e d o n c e m o r e a n d t h e p r e c i p i t a t e s w e r e d i s s o l v e d i n t h e n e c e s s a r y b u f f e r s made 5 0 % v / v i n g l y c e r o l a n d s t o r e d a t - 2 0 ° C . A T P a s e a c t i v i t y . F o r m y o s i n A T P a s e d e t e r m i n a t i o n s , m y o s i n i n 5 0 % ( v / v ) g l y c e r o l w a s p r e c i p i t a t e d a t l o w i o n i c s t r e n g t h (9 v o l u m e s o f 1 mM T r i s - H c l ( p H 7 . 0 ) , 1 mM EDTA a n d 0 . 2 mM PMSF ) a n d r e s u s p e n d e d i n 0 . 0 5 M s o d i u m p y r o p h o s p h a t e ( p H 7 . 5 ) , 0 . 5 M N a C l , a n d 1 mM DTT. T h e s o l u t i o n w a s a p p l i e d t o a 1 . 4 cm x 50 cm c o l u m n o f D E A E - c e l l u l o s e ( W h a t m a n D E - 5 2 ) , e q u i l i b r a t e d w i t h t h e s ame b u f f e r . M y o s i n w a s e l u t e d w i t h a l i n e a r g r a d i e n t o f 0 t o 0 . 5 M N a C l i n 2 0 mM N a p y r o p h o s p h a t e , p H 7 . 5 . F r a c t i o n s c o l l e c t e d w e r e s c a n n e d a t n m t ' i e m y ° s i - n p a a k w a s i d e n t i f i e d b y s o d i u m d o d e c y l s u l p h a t e / g e l e l e c t r o p h o r e s i s ( S D S / G E ) . F o r s ome d e t e r m i n a t i o n s , m y o s i n w a s a p p l i e d t o a 1 . 4 cm x 5 0 cm c o l u m n o f S e p h a r o s e 4B ( P h a r m a c i a ) e q u i l i b r a t e d w i t h 0 . 6 M N a C l , 1 0 mM s o d i u m p h o s p h a t e ( pH 7 . 5 ) , 1 mM s o d i u m p y r o p h o s p h a t e , 1 mM s o d i u m a z i d e , 2 mM DTT. E l u t e d f r a c t i o n s w e r e c o l l e c t e d a n d m o n i t o r e d f o r m y o s i n a s p r e v i o u s l y . A T P a s e a c t i v i t y o f t h e c o l u m n - p u r i f i e d m y o s i n w a s m e a s u r e d b y i n c u b a t i n g s a m p l e s w i t h 2 mM A T P , 10 mM H e p e s ( pH 7 . 0 ) , 0 . 6 M K C l a n d e i t h e r 2 mM E D T A , 10 mM C a C l „ o r 5 mM M g C l „ . T h e r a t e o f o r t h o p h o s p h a t e 18 p r o d u c t i o n was measured by t h e method o f Taussky and S c h o r r (1972) and was l i n e a r w i t h t i m e f o r up t o 1 h o u r . C o l u m a - p u r i f i e d m y o s i n had a s p e c i f i c a c t i v i t y (umoles Pi/mg o r p r o t e i n / m i n ) o f 2.0 i n t h e p r e s e n c e 2+ o f EDTA, 0.45 i n t h e p r e s e n c e o f Ca and no a c t i v i t y was d e t e c t a b l e 2+ i n t h e p r e s e n c e o f Mg . P r o t e i n d e t e r m i n a t i o n s were c a r r i e d o u t by th e method o f Lowry a t a l . (1951), u s i n g a p p r o p r i a t e p r o t e i n r e f e r e n c e s t a n d a r d s . B i o l o g i c a l methods. Embryos were o b t a i n e d and h a n d l e d u s i n g p r o c e d u r e s d e s c r i b e d by Gurdon (1967) and s t a g e d a c c o r d i n g t o Nieuwkoop and Faber (1967). Embryos were d e j e l l i e d w i t h 2% c y s t e i n e - H C l (pH 7.8) c o n t a i n i n g 0.001% each o f s o d i u m b e n z y l p e n i c i l l i n and s t r e p t o m y c i n s u l f a t e , washed i n s t e r i l e pond w a t e r and f r o z e n a t -70°C P r e p a r a t i o n and f r a c t i o n a t i o n o f polysomes. Polysomes were p r e p a r e d e s s e n t i a l l y as d e s c r i b e d by Woodland (1 9 7 4 ) . The h o m o g e n i z a t i o n b u f f e r c o n t a i n e d 0.3 M K C l , 10 mM M g C l 2 , 200 mM T r i s (pH 7.4, a t room t e m p e r a t u r e ) , 4 ug/ml o f p o l y v i n y l s u l p h a t e , 0.13% o f d i e t h y l p y r o c a r b o n a t e (DEP) o r 50 ug/ml o f h e p a r i n . A l l s o l u t i o n s were s t e r i l i z e d w i t h 10 u l / 1 0 0 m l o f DEP. E x c e s s DEP was d e s t r o y e d by warming a t 70°C f o r 1 h o u r . Homogenates were p r e p a r e d w i t h embryos w h i c h had been s t o r e d a t -70°C. 500-2000 embryos were homogenized i n 5-15 m l o f h o m o g e n i z i n g b u f f e r u s i n g a l o o s e - f i t t i n g Dounce homogenizer. A f t e r f i v e s t r o k e s w i t h t h e p e s t l e , t h e homogenate was b r o u g h t t o 0.5% i n sodium d e o x y c h o l a t e and NP-40. Three t o f o u r more s t r o k e s w i t h t h e p e s t l e were 19 f o l l o w e d by c e n t r i f u g a t i o n a t 12,000 x g f o r 5 m i n u t e s a t 4°C. The s u p e r n a t a n t f l u i d r e m a i n i n g a f t e r c e n t r i f u g a t i o n was l a y e r e d o n t o 2.0 m l o f h o m o g e n i z a t i o n b u f f e r c o n t a i n i n g 2.5 M s u c r o s e and was c e n t r i f u g e d i n a Beckman SW 27 r o t o r a t 25,000 RPM f o r 4.0 h o u r s . P e l l e t e d p olysomes were r e s u s p e n d e d i n h o m o g e n i z a t i o n b u f f e r and l a y e r e d o v e r a 20-50% s u c r o s e g r a d i e n t ( 1 2 . 0 . m l ) . The g r a d i e n t s were c e n t r i f u g e d a t 20,000 RPM f o r 2 h o u r s i n t h e Beckman SW 27 r o t o r a t 4°C. G r a d i e n t s were m o n i t o r e d f o r a b s o r p t i o n a t 254 nm i n a LKB f l o w c e l l o r f r a c t i o n s were c o l l e c t e d and m a n u a l l y s c a n n e d i n t h e model 2000 G i l f o r d s p e c t r o p h o t o m e t e r . When Xenopus l i v e r o r c h i c k embryo t h i g h m u s c l e homogenates were p r e p a r e d , f r e s h t i s s u e was u s e d and t h e polysomes were i s o l a t e d as d e s c r i b e d above. I m m u n o p r e c i p i t a t i o n o f polysomes. I m m u n o p r e c i p i t a t i o n o f polysomes was p e r f o r m e d as d e s c r i b e d by P a l m i t e r e t a l . (1972) and Rhoads e t a l . ( 1 9 7 3 ) . S u c r o s e g r a d i e n t f r a c t i o n s were suspended i n i m m u n o p r e c i p i t a t i o n b u f f e r (0.6 M N a C l , 125 5 mM MgC± 2, 10 mM T r i s , pH 7.5) and r e a c t e d w i t h 250 pg o f [ I ] - a n t i -m y o s i n a n t i b o d y d i s s o l v e d i n t h e same b u f f e r . A f t e r 12 h o u r s o f i n c u b a t i o n a t 4°C, p u r e a n t i g e n and u n l a b e l l e d a n t i b o d y were added t o t h e r e a c t i o n m i x t u r e and i n c u b a t e d f o r 12 h o u r s . The p r e c i p i t a t e d complex was c e n t r i f u g e d t h r e e t i m e s t h r o u g h a d i s c o n t i n u o u s g r a d i e n t o f 0.5 M and 1.0 M s u c r o s e ( 1 % T r i t o n X-100, 1% s o d i u m d e o x y c h o l a t e , 0.6 M N a C l ) . The p e l l e t s were washed i n i m m u n o p r e c i p i t a t i o n b u f f e r c o n t a i n i n g 1% T r i t o n X-100 and 1% sodium d e o x y c h o l a t e , d i g e s t e d w i t h 0.7 m l o f P r o t o s o l (New E n g l a n d N u c l e a r ) o v e r n i g h t and c o u n t e d i n a s c i n t i l l a t i o n 20 c o u n t e r . I o d i n a t i o n o f p r o t e i n s . I o d i n a t i o n o f myosin and a n t i - m y o s i n a n t i b o d y was p e r f o r m e d as d e s c r i b e d by P a l a c i o s e t a l . ( 1 9 7 2 ) . The s p e c i f i c a c t i v i t y o f m y o s i n ranged f r o m 9 x 10^ cpm/mg o f p r o t e i n t o 9 x 10^ cpm/mg o f p r o t e i n . F o r a n t i - m y o s i n a n t i b o d y t h e s p e c i f i c a c t i v i t y r a n g e d f r o m 4 x 10^ 9 cpm/mg o f p r o t e i n t o 1.0 x 10 cpm/mg o f p r o t e i n ( 4 5 % c o u n t i n g e f f i c i e n c y ) . P r e p a r a t i o n o f a n t i - m y o s i n a n t i b o d y . Ammonium s u l f a t e - f r a c t i o n a t e d m y o s i n o r c o l u m n - p u r i f i e d m y o s i n was d i s s o l v e d i n Laemmli e l e c t r o p h o r e s i s sample b u f f e r ( L a e m m l i , 1970) and 2.5 mg o f t h e m a t e r i a l was a p p l i e d t o a p r e p a r a t i v e 5% SDS-p o l y a c r y l a m i d e s l a b g e l (L a e m m l i , 1970). A f t e r e l e c t r o p h o r e s i s , s i d e s t r i p s were c u t f r o m t h e g e l s and s t a i n e d w i t h Coomassie B l u e t o d e t e r m i n e t h e l o c a t i o n o f t h e m y o s i n bands. A l t e r n a t i v e l y , t h e whole g e l was s t a i n e d w i t h Coomassie B l u e . The r e g i o n s o f t h e g e l c o n t a i n i n g t h e m y osin band were c u t o u t , and t h e p r o t e i n was e l u t e d f r o m t h e g e l by e l e c t r o p h o r e s i s o f t h e g e l s l i c e , w h i c h was pack e d i n t o a d i s p o s a b l e P a s t e u r p i p e t t e c o v e r e d a t t h e anode end w i t h a d i a l y s i s b ag ( R e e v e s , p e r s o n a l c o m m u n i c a t i o n ) . The p r o t e i n p u r i f i e d i n t h i s manner was homogeneous when a n a l y z e d on a se c o n d S D S - p o l y a c r y l a m i d e g e l and m i g r a t e d w i t h t h e same m o b i l i t y (210,000 d a l t o n s ) as s t a n d a r d m y o s i n . No C p r o t e i n s o r M p r o t e i n s a p p e a r e d i n t h e e l u t e d m y o s i n bands. A n t i s e r u m t o t h e e l e c t r o p h o r e t i c a l l y - p u r i f i e d heavy c h a i n s k e l e t a l m y o s i n was p r e p a r e d by i n o c u l a t i n g r a b b i t s by s t a n d a r d 21 p r o c e d u r e s ( C l a u s e n , 1969). P r i m a r y i n j e c t i o n s i n t o t h r e e r a b b i t s were done i n t h e p r e s e n c e o f Freund's c o m p l e t e a d j u v a n t and were p e r f o r m e d i n t r a m u s c u l a r l y . S e c o n d a r y i n j e c t i o n s i n t h e p r e s e n c e o f i n c o m p l e t e a d j u v a n t were g i v e n s u b c u t a n e o u s l y . Serum was t e s t e d f o r a n t i - m y o s i n a c t i v i t y by i m m u n o d i f f u s i o n a g a i n s t c r u d e m u s c l e homogenates and e l e c t r o p h o r e t i c a l l y - p u r i f i e d m y o s i n . Gammaglobulin was p r e p a r e d f r o m t h e r a b b i t serum a c c o r d i n g t o p u b l i s h e d methods ( C l a u s e n , 1969) and, when n e c e s s a r y , f r e e d o f r i b o n u c l e a s e by p a s s a g e o v e r combined CMC-D E A E - c e l l u l o s e c o l u m r B a c c o r d i n g t o S h a p i r o e t a l . ( 1 9 7 4 ) . A f t e r r e p e a t e d ammonium s u l p h a t e p r e c i p i t a t i o n s t h e serum gammaglobulin f r a c t i o n was d i s s o l v e d i n w a t e r , l y o p h i l i z e d and s t o r e d a t -20°C. F o r u s e , p o r t i o n s o f t h e d r y powder were d i s s o l v e d i n a p p r o p r i a t e b u f f e r s a t c o n c e n t r a t i o n s o f 30 t o 50 u n i t s o f p r o t e i n p e r m l . G e l e l e c t r o p h o r e s i s . P o l y a c r y l a m i d e s l a b g e l s were p o l y m e r i z e d and r u n a c c o r d i n g t o t h e method o f Laemmli and F a v r e ( 1 9 7 3 ) . S t a c k i n g and s e p a r a t i n g g e l s were made f r o m s t o c k s o l u t i o n s c o n t a i n i n g one p a r t o f b i s a c r y l a m i d e t o 37.5 p a r t s o f a c r y l a m i d e ( r e c r y s t a l l i z e d ) . S e p a r a t i n g g e l s were u s e d 24 h o u r s a f t e r m a n u f a c t u r e . Samples were d i l u t e d w i t h 63 mM T r i s - H C l (pH 6.8), 3% sodium d o d e c y l s u l p h a t e , 1% 2 - m e r c a p t o e t h a n o l , 10% g l y c e r o l and 0.002% Bromophenol B l u e . The amount o f p r o t e i n i n t h e samples was a l w a y s d e t e r m i n e d t o i n s u r e t h a t t h e SDS t o p r o t e i n r a t i o was i n e x c e s s o f 1.4 g o f SDS t o 1 g o f p r o t e i n . E l e c t r o p h o r e s i s was c a r r i e d o u t a t room t e m p e r a t u r e f o r 30 m i n u t e s a t 50 v o l t s , t h e n a t 100 v o l t s f o r about 5 h o u r s and s t o p p e d when t h e Bromophenol B l u e f r o n t had m i g r a t e d 10 cm i n t o t h e s e p a r a t i n g g e l . 22 When c o n c e n t r a t i o n s o f p r o t e i n s o r m y o s i n were l e s s t h a n 1 mg/ml i n a volume o f l e s s t h a n 30 p l , t h e S D S - a c r y l a m i d e e l e c t r o p h o r e t i c p r o c e d u r e o f P a t e r s o n and Strohman (1972) was a d o p t e d . The use o f a s t a c k i n g g e l was o m i t t e d . The pH o f t h e sample b u f f e r was r a i s e d f r o m 6.8 t o 8.6. The samples were h e a t e d i n a b o i l i n g w a t e r b a t h f o r 4 m i n u t e s i n s t e a d o f 1 m i n u t e . Under t h e s e c o n d i t i o n s ( h i g h e r pH o f t h e sample b u f f e r and h e a t i n g o f t h e sample f o r l o n g e r t h a n 1 m i n u t e ) m y o s i n p r e p a r a t i o n s do n o t p r e c i p i t a t e as much a t t h e o r i g i n o f t h e SDS-a c r y l a m i d e g e l . E l e c t r o p h o r e s i s was c a r r i e d o ut a t 45 v o l t s f o r about 12 h o u r s and s t o p p e d when t h e Bromophenol B l u e f r o n t had m i g r a t e d 10 cm i n t o t h e g e l . G e l s were s t a i n e d f o r 1-4 h o u r s i n 0.25% (w/v) Coomassie B l u e , 35% (w/v) m e t h a n o l , 10% (w/v) a c e t i c a c i d and d e s t a i n e d i n 35% (v / v ) m e t h a n o l , 10% (w/v) a c e t i c a c i d . F o r p r e p a r a t i v e g e l s , i t was i m p o r t a n t t o s t a i n and d e s t a i n t h e g e l s i n t h e minimum amount o f t i m e t o p r e v e n t damage t o p r o t e i n s . G e l s were p l a c e d i n 5% (v/ v ) m e t h a n o l , 7.5% a c e t i c a c i d i n o r d e r t o r e s t o r e t h e o r i g i n a l s i z e . G e l s l a b s were d r i e d f o r a u t o r a d i o g r a p h y . P r o t e i n d e t e r m i n a t i o n . P r o t e i n c o n c e n t r a t i o n s were e s t i m a t e d by t h e method o f Lowry e t a l . ( 1 9 5 1 ) , u s i n g b o v i n e serum a l b u m i n as t h e p r o t e i n s t a n d a r d ( S i g m a ) . M o l e c u l a r w e i g h t d e t e r m i n a t i o n s . The a p p a r e n t m o l e c u l a r w e i g h t o f m y o s i n heavy c h a i n s was d e t e r m i n e d by r e l a t i v e m o b i l i t i e s upon t h e same 5% o r 7.5% a c r y l a m i d e - S D S g e l w i t h t h e f o l l o w i n g p r o t e i n s as m o l e c u l a r w e i g h t s t a n d a r d s : r a b b i t s k e l e t a l m u s c l e m y o s i n ( 2 1 2 , 0 0 0 ) , E. c o l i B - g a l a c t o s i d a s e ( 1 3 0 , 0 0 0 ) , 23 and r a b b i t s k e l e t a l m u s c l e p h o s p h o r y l a s e b (911,000). The r e l a t i v e m o l e c u l a r w e i g h t o f t h e heavy c h a i n o f e m b r y o n i c and a d u l t m y o s i n was d e t e r m i n e d t o be about 210,000. •, Amino a c i d a n a l y s i s . Samples were h y d r o l y z e d i n 6N H C l i n vacuo a t 110°C f o r 18, 24 o r 48 h o u r s . H y d r o l y s a t e s were a n a l y z e d on a Beckman amino a c i d a n a l y z e r . P a r t i a l t r y p t i c d i g e s t i o n o f i m m u n o p r e c i p i t a t e d m y o s i n and p u r i f i e d  m y o sin. The c r u d e a c t o m y o s i n f r a c t i o n ( s ee p r e p a r a t i o n o f s k e l e t a l myosin) was p r e c i p i t a t e d a t low i o n i c s t r e n g t h (9 volumes o f d i s t i l l e d w a t e r c o n t a i n i n g 2 mM EDTA) and p e l l e t e d by c e n t r i f u g i n g a t 40,000 x g f o r 10 m i n u t e s . The p e l l e t was d i s s o l v e d a t a c o n c e n t r a t i o n o f l e s s t h a n 1 mg/ml o f p r o t e i n i n a b u f f e r c o n t a i n i n g 10 mM sodium p h o s p h a t e (pH 7.8), 0.3 M sodium p h o s p h a t e and i o d i n a t e d as d e s c r i b e d above. The r e a c t i o n p r o d u c t s were t h e n d i a l y z e d f o r t w e l v e h o u r s a g a i n s t a b u f f e r c o n t a i n i n g 0.5 M N a C l , 0.015 M T r i s - H C l (pH 7.5) and 0.1 M PMSF. The d i a l y z e d p r o t e i n s were t h e n l y o p h i l i z e d and r e s u s p e n d e d i n l e s s t h a n 1 m l o f t h e above d i a l y s i s b u f f e r . M y o s i n was t h e n p r e c i p i t a t e d w i t h a n t i - m y o s i n a n t i b o d y ( s e e above) and t h e p r e c i p i t a t e was washed by c e n t r i f u g a t i o n t h r o u g h a d i s c o n t i n u o u s s u c r o s e g r a d i e n t ( s e e a b o v e ) . The m y o s i n - a n t i b o d y complex was t h e n e l e c t r o p h o r e s e d on a S D S - a c r y l a m i d e g e l and, a f t e r s t a i n i n g w i t h Coomassie B l u e , t h e S D S - a c r y l a m i d e g e l was exposed t o X - r a y f i l m . A f t e r a u t o r a d i o g r a p h y , t h e p u t a t i v e m y o s i n bands were c u t o u t f r o m t h e g e l s and t h e m y o s i n was e l u t e d f r o m g e l s l i c e s by e l e c t r o p h o r e s i s i n a P a s t e u r p i p e t t e ( s e e s e c t i o n on p r e p a r a t i o n o f 24 a n t i - m y o s i n a n t i b o d y ) . The c o n t e n t s o f t h e d i a l y s i s bag were t h e n p r e c i p i t a t e d w i t h 10% t r i c h l o r o a c e t i c a c i d t o remove most o f t h e SDS (Weber and Osborne, 1975). T h i s p r o c e d u r e was r e p e a t e d t w i c e . F i n a l l y , t h e m y o s i n was d i s s o l v e d a t a c o n c e n t r a t i o n o f 1 mg/ml i n 0.6 M N a C l , •-- 0.015 M T r i s - H C l (pH 7.4), 0.1% B - m e r c a p t o e t h a n o l and mixed w i t h t r y p s i n a t 0.005 t h e w e i g h t o f myosin ( B u r r i d g e and B r a y , 1975). The d i g e s t i o n was a l l o w e d t o p r o c e e d a t 37°C f o r 30 m i n u t e s and was s t o p p e d by b o i l i n g i n SDS g e l sample b u f f e r ( s e e s e c t i o n on e l e c t r o p h o r e s i s ) . N o n - d e n a t u r i n g c o n d i t i o n s were used f o r p r o t e o l y s i s b e c a u s e u n d e r t h e s e c o n d i t i o n s t r y p s i n p r o d u c e s f r a g m e n t s l a r g e enough t o be s e p a r a t e d by e l e c t r o p h o r e s i s waich are d i s t i n c t i v e f o r a l a r g e number o f myosins ( B i r o eit a l . , 1 972). Of c o u r s e , i t i s i m p o s s i b l e t o s p e c i f y w h e t h e r t r y p t i c p e p t i d e s o f myosins a r i s e f r o m p r i m a r y , s e c o n d a r y o r t e r t i a r y s t r u c t u r e s o f my o s i n . E l e c t r o p h o r e s i s was c a r r i e d o ut i n 10% S D S - a c r y l a m i d e . Column- and g e l -p u r i f i e d m y osin was i o d i n a t e d and t r y p s i n i z e d i n t h e same way as i m m u n o p r e c i p i t a t e d myosin and was r u n on t h e same S D S - a c r y l a m i d e g e l s f o r c o m p a r i s o n s . 25 RESULTS A C h a r a c t e r i z a t i o n o f a n t i - m y o s i n a n t i b o d y . A n t i - m y o s i n i m m u n o g l o b u l i n s were p r e p a r e d f r o m serum o f r a b b i t s immunized w i t h e l e c t r o p h o r e t i c a l l y p u r i f i e d m y o s i n . I n t h e s e e x p e r i m e n t s , i t i s i m p o r t a n t t h a t t h e a n t i b o d y m o n o s p e c i f i c a l l y r e c o g n i z e o n l y t h e heavy c h a i n o f m y o s i n b e c a u s e any o t h e r c r o s s - r e a c t i n g m u s c l e p r o t e i n s m i g h t be s y n t h e s i z e d a t d i f f e r e n t d e v e l o p m e n t a l s t a g e s . To a s c e r t a i n t h a t t h e immunogen w h i c h r u n s on S D S - a c r y l a m i d e g e l s w i t h a m o l e c u l a r w e i g h t o f 210,000 d a l t o n s i s i n d e e d m y o s i n , t h e f o l l o w i n g t e s t s were p e r f o r m e d : 1) The ATPase a c t i v i t y o f a c h e m i c a l l y p u r i f i e d m y o s i n p r e p a r a t i o n was measured. 2) The amino a c i d c o m p o s i t i o n o f t h e p u t a t i v e m y o s i n was compared w i t h t h a t o f a n o t h e r v e r t e b r a t e . 3) The r e a c t i o n o f p u r i f i e d m y o s i n and crude m u s c l e e x t r a c t was compared by i m m u n o d i f f u s i o n ( C l a u s e n , 1969). 4) Crude m u s c l e e x t r a c t s were l a b e l l e d 125 i n v i t r o w i t h [ I ] u s i n g l a c t o p e r o x i d a s e ( P a l a c i o s e t a l . , 1972) and p r e c i p i t a t e d w i t h a n t i - m y o s i n a n t i b o d y . The p r e c i p i t a t e d a n t i g e n / a n t i b o d y complex was r u n on S D S - a c r y l a m i d e g e l s and t h e r a d i o a c t i v e p r o t e i n compared t o a u t h e n t i c m y o s i n bands o b t a i n e d on S D S - a c r y l a m i d e g e l s a f t e r i m m u n o p r e c i p i t a t i o n and t h e r e s u l t i n g p e p t i d e s compared t o t h o s e d e r i v e d f r o m p u r i f i e d m y o s i n . The a s s a y f o r ATPase a c t i v i t y i n 0.6 M K C l and v a r i o u s i o n s was used t o f o l l o w t h e p u r i f i c a t i o n o f m y o s i n f r o m a d u l t f r o g s . The r e l a t i v e ATPase a c t i v i t i e s o f a d u l t f r o g s k e l e t a l m y o s i n ( c o l u m n - p u r i f i e d ) and s k e l e t a l m u s c l e m y o s i n s o b t a i n e d f r o m o t h e r s c u r c e s ( l i t e r a t u r e I 26 v a l u e s ) a t 25°C (pH 7.5) i n t h e p r e s e n c e o f v a r i o u s i o n s a r e summarized i n T a b l e 1. I n t h e p r e s e n c e of EDTA, Xenopus m y o s i n ATPase was h i g h e r t h a n t h a t o f r a b b i t r e d m u s c l e b u t l o w e r than, t h a t o f r a b b i t w h i t e 2+ m u s c l e . S i m i l a r r e s u l t s were o b t a i n e d i n t h e p r e s e n c e o f Ca . These r e s u l t s i n d i c a t e t h a n t h e m y o s i n was p r o b a b l y e x t r a c t e d f r o m m u s c l e s w h i c h c o n t a i n e d a m i x t u r e o f r e d and w h i t e m u s c l e f i b r e s . I n t h e p r e s e n c e 2+ o f Mg no ATPase a c t i v i t y was d e t e c t a b l e , i n d i c a t i n g t h a t a c t i n was n o t c o - p u r i f i e d w i t h t h e m y o s i n . I t can be seen i n F i g . 1 t h a t c o l u m n - p u r i f i e d m y o s i n c o n t a i n e d l i t t l e d e t e c t a b l e a c t i n . A t 2 mM ATP t h e Xenopus m u s c l e enzyme had an a c t i v i t y d o u b l e t h a t a t 1 mM ATP. On t h e b a s i s o f t h e enzyme a c t i v i t y s t u d i e s , t h e m y o s i n heavy c h a i n band i d e n t i f i e d on SDS g e l s a p p e a r s t o be s k e l e t a l m u s c l e m y o s i n . T a b l e 2 summarizes t h e r e s u l t s o f two ami no a c i d a n a l y s e s p e r f o r m e d on t h e heavy c h a i n s o f m u s c l e m y o s i n i s o l a t e d f r o m SDS g e l s . The r e s u l t s i n d i c a t e t h a t t h e amino a c i d c o m p o s i t i o n o f s k e l e t a l m y o s i n f r o m Xenopus i s s i m i l a r t o t h e amino a c i d c o m p o s i t i o n f r o m r a b b i t s k e l e t a l m u s c l e . The r e a c t i o n o f t h e a n t i - m y o s i n a n t i b o d y w i t h p u r i f i e d o r w i t h c r u d e m u s c l e e x t r a c t s gave p r e c i p i t a t i o n l i n e s o f i d e n t i t y , and no s p u r l i n e s were o b s e r v e d as shown i n F i g . 2. By t h i s c r i t e r i o n , t h e a n t i - m y o s i n a n t i b o d y i s m o n o s p e c i f i c f o r t h e heavy m y o s i n c h a i n . F u r t h e r m o r e , f l u o r e s c e i n - l a b e l l e d a n t i - m y o s i n a n t i b o d y r e a c t s s p e c i f i c a l l y w i t h g l y c e r i n a t e d m u s c l e s l i c e s , w h i l e non-immune serum shows no s u c h r e a c t i o n ( F i g . 3 ) . T h i s i n d i c a t e s t h a t t h e a a t i - m y o s i n a n t i b o d y r e c o g n i z e s a n t i g e n i c d e t e r m i n a n t s p r e s e n t on t h e g e l - p u r i f i e d m y o s i n , on t h e h i g h s a l t - e x t r a c t e d m y o s i n and on f i x e d t i s s u e s l i c e s . I n a n o t h e r t e s t f o r m o n o s p e c i f i c ! t y , a d u l t f r o g m u s c l e p r o t e i n was e x t r a c t e d i n 0.6 M N a C l and l a b e l l e d i n v i t r o w i t h [ I ] u s i n g t h e t h e p r o c e d u r e o f P a l a c i o s e t a l . ( 1 9 7 2 ) , and t h e n was i n c u b a t e d w i t h non-immune, o r a n t i - m y o s i n a n t i b o d y . A f t e r p e l l e t i n g of t h e m y o s i n -a n t i - m y o s i n c o m p l e x e s , t h e p r e c i p i t a t e s were a n a l y z e d by S D S - a c r y l a m i d e e l e c t r o p h o r e s i s , f o l l o w e d by X - r a y a u t o r a d i o g r a p h y as can be s e e n i n F i g . 4. P r e c i p i t a t i o n o f t h e m u s c l e p r o t e i n s w i t h a n t i - m y o s i n a n t i b o d y r e s u l t e d i n t h e a p p e a r a n c e o f r a d i o a c t i v e m y o s i n on t h e SDS g e l s and no o t h e r p r o t e i n bands e x c e p t o c c a s i o n a l l y low m o l e c u l a r w e i g h t s p e c i e s ( w h i c h may r e p r e s e n t degraded p r o t e i n s ) m i g r a t i n g w i t h t h e Bromophenol B l u e f r o n t were o b s e r v e d on t h e g e l s . When i n c r e a s i n g amounts of t h e m u s c l e e x t r a c t were added t o t h e a n t i b o d y r e a c t i o n m i x t u r e , t h e r e s u l t i n g i m m u n o p r e c i p i t a t e s were n o t c o n t a m i n a t e d w i t h new p r o t e i n s . No l a b e l l e d p r o t e i n s a p p e a r e d on g e l s when non-immune s e r a were used. F i g . 5 shows an X - r a y a u t o r a d i o g r a p h o f a o n e - d i m e n s i o n a l e l e c t r o p h o r e t i c t r y p t i c p e p t i d e map o f p u r e m y o s i n and a n t i b o d y - p r e c i p i t a t e d m y o s i n l a b e l l e d 125 w i t h [ I ] . I t a p p e a r s t h a t t h e l a b e l l e d t r y p t i c f r a g m e n t s o f a n t i b o d y -p r e c i p i t a t e d m y o s i n c o - e l e c t r o p h o r e s e w i t h t h e l a b e l l e d t r y p t i c f r a g m e n t s d e r i v e d f r o m p u r e m y o s i n . I t i s n o t p o s s i b l e t o o b t a i n c o m p l e t e l y i d e n t i c a l p e p t i d e , maps s i n c e f r o m one e x p e r i m e n t t o a n o t h e r t h e s p e c i f i c a c t i v i t y o f t r y p s i n may v a r y and t h e r e may be s u b t l e changes i n i n c u b a t i o n t e m p e r a t u r e . Q u a n t i t a t i v e p r e c i p i t a t i o n o f m y o s i n . A p r e c i p i t i n c u r v e was u s e d t o confix-m t h e r e s u l t s o f t h e O u c h t e r l o n y i m m u n o d i f f u s i o n t e s t s and t h e a n a l y s i s o f i m m u n o p r e c i p i t a t e s i n S D S - a c r y l a m i d e g e l s . 125 The p r e c i p i t i n c u r v e o b t a i n e d w i t h g e l - p u r i f i e d [ I ] - m y o s i n 28 and a n t i - m y o s i n i s shown i n F i g . 6. The c u r v e shows a c l a s s i c a l p r e c i p i t i n c u r v e w i t h one peak, i n d i c a t i n g t h a t o n l y one a n t i g e n i c s p e c i e s i s p r e s e n t ( C l a u s e n , 1969). A d d i t i o n o f i n c r e a s i n g amounts o f r a d i o a c t i v e m y o s i n t o a c o n s t a n t amount o f a n t i - m y o s i n a n t i b o d y (1 mg) r e s u l t s i n t h e f o r m a t i o n o f an a n t i b o d y e x c e s s r e g i o n (below 75 fig), an e q u i v a l e n c e zone a t 75 ug, and f i n a l l y a r e g i o n o f a n t i g e n e x c e s s i n w h i c h t h e i m m u n o p r e c i p i t a t e becomes p r o g r e s s i v e l y s o l u b l e . As e x p l a i n e d i n C l a u s e n ( 1 9 6 9 ) , r a b b i t a n t i b o d i e s when p r e s e n t i n e x c e s s w i l l q u a n t i t a t i v e l y p r e c i p i t a t e an a n t i g e n ( e s p e c i a l l y a l a r g e one s u c h as m y o s i n ) . I n t h e e q u i v a l e n c e zone, a l l f r e e a n t i b o d y and a n t i g e n f o r m a complex, w h i c h i s r e a d i l y p r e c i p i t a b l e . As more a n t i g e n i s added, t h e complex becomes e v e n t u a l l y s o l u b l e b e c a u s e e x c e s s a n t i g e n forms s m a l l e r a g g r e g a t e s w h i c h a r e n o t r e a d i l y p r e c i p i t a t e d . A c c o r d i n g t o F i g . 6, q u a n t i t i e s o f m y o s i n l e s s t h a n 75 ug were q u a n t i t a t i v e l y p r e c i p i t a t e d when added t o a c o n s t a n t amount o f a n t i - m y o s i n a n t i b o d y . F o r example, when th e amount o f r a d i o a c t i v i t y p r e c i p i t a t e d by a n t i - m y o s i n a n t i b o d y was compared t o t h e amount p r e c i p i t a t e d by i c e - c o l d 20% t r i c h l o r o a c e t i c a c i d ( TCA), t h e r e s u l t s a g r e e d w i t h i n 10% f o r amounts o f m y o s i n i n the. nanogram r a n g e . I t was p o s s i b l e , u s i n g t h i s r a d i o i m m u n o a s s a y , t o r e l i a b l y d e t e c t q u a n t i t i e s o f m y o s i n e q u a l t o 0.05-0.1 ng. S i n c e t h e a n t i g e n - a n t i b o d y r e a c t i o n i s a f f e c t e d by v a r i o u s p h y s i c o - c h e m i c a l e v e n t s , s e v e r a l a d d i t i o n a l c o n t r o l s were p e r f o r m e d on 125 t h e m y o s i n a n t i - m y o s i n r e a c t i o n . When [ I ] - m y o s i n was m i x e d w i t h a h i g h s a l t e x t r a c t o f Xenopus l i v e r c e l l s under t h e same c o n d i t i o n s as d e s c r i b e d i n t h e l e g e n d t o F i g . 6, an i m m u n o p r e c i p i t a t i o n c u r v e i d e n t i c a l t o t h e one i n F i g . 6 was o b t a i n e d . Whei?. a h i g h s a l t e x t r a c t 29 o f t a d p o l e t a i l s was i o d i n a t e d and r e a c t e d w i t h a n t i - m y o s i n a n t i b o d y , a g a i n a c u r v e i d e n t i c a l t o t h e one i n F i g . 6 was o b t a i n e d . T h i s r e s u l t i n d i c a t e s t h a t e m b r y o n i c m u s c l e and a d u l t m u s c l e m y o s i n s s h a r e s i m i l a r a n t i g e n i c d e t e r m i n a n t s . The e f f e c t o f r e a c t i o n volume on t h e p r e c i p i t a t i o n o f m y o s i n 125 was d e t e r m i n e d by d i l u t i n g 1 mg of a n t i b o d y and 75 ug o f [ I ] - m y o s i n w i t h v a r i o u s amounts o f r e a c t i o n b u f f e r . Reduced p r e c i p i t a t i o n o c c u r r e d when t h e r e a c t i o n volume was i n c r e a s e d beyond 1.0 m l . The p r e s e n c e o f 0.75 M s u c r o s e , a compound used i n t h e i s o l a t i o n o f p o l y s o m e s , i n t h e 125 r e a c t i o n m i x t u r e r e d u c e d t h e amount o f [ I ] - m y o s i n p r e c i p i t a t e d a t t h e e q u i v a l e n c e p o i n t t o 88% o f t h e v a l u e i n i t s absence. The a n t i - m y o s i n a n t i b o d y t h u s a p p e a r s t o m o n o s p e c i f i c a l l y r e a c t w i t h t h e heavy c h a i n o f s k e l e t a l m u s c l e m y o s i n and i s a b l e t o p r e c i p i t a t e m y o s i n f r o m c r u d e m u s c l e e x t r a c t s . By t h e s e c r i t e r i a t h e a n t i - m y o s i n a n t i b o d y can be used t p p r e c i p i t a t e m y o s i n - s y n t h e s i z i n g polysomes and t o d e t e c t low l e v e l s o f s k e l e t a l m y o s i n p r o t e i n . I s o l a t i o n and a n a l y s i s o f e m b r y o n i c polysomes. T y p i c a l polysome p r o f i l e s f r o m s t a g e s 12, 16/17 and s t a g e 20 X. l a e v i s embryos a r e shown i n F i g s . 7 a , b and c. The polysomes were i s o l a t e d i n t h e p r e s e n c e o f sodium d e o x y c h o l a t e , NP-40, 0.3 M K C l and 0.2M T r i s - H C l . The s a l t and t h e d e t e r g e n t s i n c r e a s e t h e y i e l d o f e m b r y o n i c polysomes (Woodland, 1974). The h i g h s a l t c o n c e n t r a t i o n (0.3 M K C l ) i n t h e h o m o g e n i z a t i o n b u f f e r has two e f f e c t s . F i r s t , i t a p p e a r s t h a t h i g h s a l t d i s s o c i a t e s X- l a e v i s e m b r y o n i c monosomes w h i c h a r e n o t a s s o c i a t e d w i t h mRNA (Woodland, 1974). I n d e e d , F i g . 7 shows t h a t t h e polysome p a t t e r n o f s t a g e s 12 and 16/17 embryos i s dominated by s u b u n i t s . 30 S e c o n d l y , s i n c e myosin, i s s o l u b l e a t h i g h i o n i c s t r e n g t h , t h e use o f a h i g h s a l t c o n c e n t r a t i o n s h o u l d i n c r e a s e t h e y i e l d o f m y o s i n polysomes ( S a r k a r , 1 9 7 6 ) . The 60S s u b u n i t and t h e 80S monosome a r e n o t w e l l s e p a r a t e d i n t h e s e p r o f i l e s b e c a u s e t h e g r a d i e n t s a r e o v e r l o a d e d i n o r d e r t o i n c r e a s e t h e y i e l d o f polysomes ( t h e a r r o w i n each p a n e l o f F i g . 7 i n d i c a t e s t h e p o s i t i o n o f t h e monosome p e a k ) . Whether o v e r l o a d i n g o f g r a d i e n t s causes polysome a g g r e g a t i o n and s e d i m e n t a t i o n t o t h e b o t t o m of c e n t r i f u g e t u b e s w i l l be d i s c u s s e d when d a t a on t h e i m m u n o p r e c i p i t a t i o n o f polysomes i s p r e s e n t e d below. When t h e polysome h o m o g e n i z i n g b u f f e r was made 0.13% v / v i n d i e t h y l p y r o c a r b o n a t e (DEP), i d e n t i c a l p r o f i l e s were o b t a i n e d , s u g g e s t i n g t h a t d e g r a d a t i o n o f polysomes d i d n o t o c c u r . The DEP was added t o t h e h o m o g e n i z i n g b u f f e r t o i n a c t i v a t e n u c l e a s e s t h a t m i g h t cause polysome d e g r a d a t i o n . When s t a g e 20 homogenates were t r e a t e d w i t h RNase, t h e s e d i m e n t a t i o n p a t t e r n s h i f t e d t o t h a t o f s u b u n i t s ( F i g . 8 ) . Treatment o f s t a g e 12 and s t a g e 16/17 homogenates w i t h RNase p r o d u c e d no change i n t h e s e d i m e n t a t i o n p a t t e r n . The e v i d e n c e p r e s e n t e d h e r e i n d i c a t e s t h a t t h e a r e a o f t h e g r a d i e n t s p r e s e n t i n F i g . 7 a , b and c between f r a c t i o n s 6 and 1.2 r e p r e s e n t s polysomes. F u r t h e r m o r e , i s o l a t e d f r a c t i o n s 3 o f t h e s e r e g i o n s o f t h e p o l y s o m a l g r a d i e n t s i n c o r p o r a t e d [ H ] - l e u c i n e when added t o t h e 100,000 x g s u p e r n a t a n t f r o m a wheat germ c e l l - f r e e p r o t e i n s y n t h e s i z i n g s y s t e m , a g a i n s u g g e s t i n g t h e p o l y s o m a l n a t u r e o f t h i s r e g i o n . B i n d i n g o f a n t i - m y o s i n a n t i b o d y t o em b r y o n i c p o l y s o m e s . The a n t i - m y o s i n a n t i b o d y d e s c r i b e d i n t h e p r e c e d i n g s e c t i o n was 31 used t o d e t e r m i n e a t w h i c h s t a g e o f e m b r y o n i c development s k e l e t a l - l i k e m y o s i n i s f i r s t s y n t h e s i z e d on polysomes. Polysomes were i s o l a t e d f r o m d i f f e r e n t e m b r y o n i c s t a g e s and s e p a r a t e d on s u c r o s e d e n s i t y g r a d i e n t s by c e n t r i f u g a t i o n as d e s c r i b e d i n M a t e r i a l s and Methods. A f t e r 125 c e n t r i f u g a t i o n , each polysome f r a c t i o n was r e a c t e d w i t h [ I ] - l a b e l l e d a n t i b o d y and t h e p r e c i p i t a t e s were washed by c e n t r i f u g a t i o n t h r o u g h a d i s c o n t i n u o u s s u c r o s e g r a d i e n t ( s e e M a t e r i a l s and M e t h o d s ) . The r e s u l t s o f b i n d i n g o f l a b e l l e d a n t i b o d y t o polysomes f r o m d i f f e r e n t s t a g e s o f e m b r y o n i c development a r e shown i n F i g . 7 a , b and c. A t s t a g e s 12 ( F i g . 7a) and 16/17 ( F i g . 7b) r a d i o a c t i v i t y i s p r e s e n t a t a low l e v e l t h r o u g h o u t t h e g r a d i e n t s and r e p r e s e n t s n o n - s p e c i f i c b i n d i n g . F i g . 7c shows t h a t a t s t a g e 20 t h e m y o s i n a n t i b o d y i s b i n d i n g w i t h r a p i d l y s e d i m e n t i n g polysomes. The w i d t h o f t h e p u t a t i v e m y o s i n peak o b s e r v e d i n F i g . 7c may be a t t r i b u t e d t o v a r i a b l e r i b o s o m e s p a c i n g , i n c o m p l e t e l o a d i n g o n t o t h e mRNA and m i n o r a g g r e g a t i o n (Latham and D a r n e l l , 1 9 6 5 ) . The f a c t t h a t t h e peak s p r e a d s i n t o t h e a r e a o f t h e polysome p r o f i l e w h i c h has polysome s i z e s o f l e s s t h a n 30S may be due t o d e g r a d a t i o n . To d e m o n s t r a t e t h a t t h i s a n t i b o d y b i n d i n g i s s t a g e -s p e c i f i c , two f u r t h e r t e s t s were used t o a s s e s s t h e p r e s e n c e o r absence o f m y o s i n i n embryos. I n t h e f i r s t t e s t , t h e r e a c t i o n o f h i g h s a l t e x t r a c t s o f s t a g e 12, s t a g e 16/17, s t a g e 20 and s t a g e 26 embryos w i t h a n t i - m y o s i n were compared by i m m u n o d i f f u s i o n ( F i g . 9 ) . O n l y e x t r a c t s o f s t a g e 20 and s t a g e 26 embryos gave p r e c i p i t i n l i n e s . I n t h e s e c o n d t e s t , c r u d e e x t r a c t s o f t h e above s t a g e s were a n a l y z e d by S D S - g e l e l e c t r o p h o r e s i s ( F i g . 1 0 ) . A g a i n , t h e s e g e l p a t t e r n s i n d i c a t e d t h a t d e t e c t a b l e m y o s i n a p p e a r e d o n l y i n e x t r a c t s o f s t a g e 20 and s t a g e 26 embryos. I n a few c a s e s a band w i t h a m o l e c u l a r veight o f 210,000 32 appeared on SDS-acrylamide gels from extracts of stages 1-5 and stages 9-13 embryos (Brock, unpublished observations). Since these bands could not be immunoprecipitated, we assume that they represent non-muscle myosin or some other protein. Polysome reaction control experiments. A number of control experiments were performed to test the specificity of the anti-myosin ahtibody-polysome binding reaction. For example, when polysome preparations from stage 20 or 26 embryos were f i r s t reacted with cold anti-myosin antibody, this resulted in the 125 abolishment of the [ I]-labelled anti-myosin binding. This indicates that the sites for binding anti-myosin antibody on the myosin molecule can be saturated. Other controls also indicated that sera from non-immunized rabbits or anti-ovalbumin antibody from rabbits do not inhibit the binding of labelled anti-myosin antibody to the polysomes. Furthermore, labelled anti-ovalbumin antibody does not bind to stage 20 polysomes. Labelled anti-myosin antibody does not bind to Xenopus li v e r polysomes when they are mixed 1 :1 with the 100,000 x g supernatant from stage 20 embryos (Fig. 11). Furthermore, another control, shown in Fig. 12, indicates that exogenously supplied myosin does not appear to contaminate polysomes when i t i s added to the embryo supernatant prior to sucrose gradient fractionation. In this situation, the free myosin remains i n the supernatant near the top of the gradient and reacts with the anti-myosin antibody. And f i n a l l y , treatment of stage 20 embryo homogenates with RNase prior to sedimentation results in the polysomal profile shown in Fig. 8. As would be expected, labelled anti-myosin 33 binds in this case to the monosome and supernatant regions of the gradient. These results indicate that myosin-synthesizing polysomes are f i r s t observed around stage 20 of Xenopus embryonic development. This implies that myosin i s synthesized at stage 20. A l l controls performed indicate that the anti-myosin antibody i s monospecific for skeletal myosin, that i t binds specifically to polysomes synthesizing myosin, and that i t does not bind to non-skeletal muscle myosin polysomes. 34 TABLE 1 a ATPase a c t i v i t y of Xenopus l a e v i s s k e l e t a l myosin. Assay Myosin-specific ATPase a c t i v i t y b X. l a e v i s Rabbit WM RM CM 1. 1 mM EDTA 0.5M KCl 1 mM ATP 2. 1 mM EDTA 0.5M KCl 2 mM ATP 3. 10 mM Ca 0.5M KCl 2 mM ATP 2+ 4. 5 mM Mg 2 mM ATP 2+ 2.0 3.9 0.45 3.2 1.6 1.1 1.0 0.25 0.35 ^ A l l r eaction mixtures contained 0.2 mg of protein/ml. Reactions were were i n i t i a t e d by the addition of ATP and incubated for 15 min at 25°C. Phosphate hyd r o l y s i s was measured as described i n Materials and Methods A and s p e c i f i c a c t i v i t i e s are reported as pmoles Pi/mg of protein/min. The r e s u l t s are average values of two experiments. 3Data taken from Sreter et a l . (1966). White muscle (WM) , red muscle (RM) , cardiac muscle (CM). 35 TABLE 2 Amino a c i d c o m p o s i t i o n o f m y o s i n heavy c h a i n s p r e p a r e d f r o m a d u l t Xenopus l a e v i s s k e l e t a l m u s c l e . Amino a c i d Xenopus a d u l t R a b b i t R a b b i t m y o s i n s k e l e t a l ^ s k e l e t a l 0 l y s i n e 90 92 93 h i s t i d i n e 17 16 15 a r g i n i n e 46 43 45 a s p a r t a t e . 81 85 84 t h r e o n i n e 48 44 41 s e r i n e 40 39 38 g l u t a m a t e 155 157 163 p r o l i n e 22 22 21 g l y c i n e 42 40 40 a l a n i n e 78 78 75 1/2 c y s t e i n e 9 8.8 9 v a l i n e 45 43 42 m e t h i o n i n e ^ 24 23 21 i s o l e u c i n e 45 42 40 l e u c i n e 84 81 80 t y r o s i n e 21 20 17 p h e n y l a l a n i n e 29 29 28 Expressed as moles per 10~* g of m y o s i n . D a t a are a v e r a g e s of amino a c i d h y d r o l y s e s at 1, 24, 48 and 72 h o u r s . ^ D a t a t a k e n f r o m a l i s t c o m p i l e d by Tonomura ( 1 9 7 3 ) . D a t a t a k e n f r o m H u s z a r and E l z i n g e r ( 1 9 7 2 ) . ^The v a l u e for m e t h i o n i n e was not c o r r e c t e d for l o s s e s d u r i n g h y d r o l y s i s . 36 Figure l a . Electrophoretic separation on a 10% SDS-acrylamide gel of myosin fractions purified by ammonium sulfate precipitation. This gel shows considerable contamination of myosin with actin from various muscle sources. Slots 1, 2 and 3i myosin from thigh muscles of adult Xenopus laevis after one, two and three ammonium sulfate precipitations (30 ug of protein in each slot). Slots 4 and 5: myosin from back muscles of adult X. laevis after one and two ammonium sulfate precipitations (about 15 ug of protein in each sl o t ) . Slot 6: myosin obtained from the t a i l s of pre-metamorphosed X. laevis tadpoles and precipitated twice with ammonium sulfate (about 10 ug of protein in the s l o t ) . It appears from this gel that the myosin heavy chains have similar molecular weights. 37 1 2 3 4 5 6 fx1"* Myosin Actin 38 F i g u r e l b . 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 on an 8% S D S - a c r y l a m i d e g e l o f c o l u m n - p u r i f i e d m yosin f r a c t i o n s . T h i s g e l shows t h a t c o l u m n - p u r i f i e d m y osin c o n t a i n s l i t t l e a c t i n c o n t a m i n a t i o n . A r r o w d e n o t e s p o s i t i o n o f m y o s i n . The n e g a t i v e o f t h e g e l was p r i n t e d on h i g h c o n t r a s t p a p e r t o e l i m i n a t e b a c k g r o u n d s t a i n i n g . S l o t 1: 60 ug o f c o l u m n - : p u r i f i e d ( c . p . ) m y o s i n f r o m t h e t h i g h and b a c k m u s c l e s o f a d u l t X. l a e v i s . S l o t 2: 25 ug o f myosin as i n S l o t 1. S l o t 3: 15 pg o f m y o s i n as i n S l o t 1. S l o t 4: myosin f r o m t a i l s o f pre-metamorphosed t a d p o l e s . S l o t 5: m i x t u r e o f c.p. m y o s i n and t a d p o l e m y o s i n . Myosin Actin 40 F i g u r e 2. S e r o l o g i c a l i d e n t i t y o f g e l - p u r i f i e d m y o s i n and c r u d e m y o s i n f r o m h i g h s a l t e x t r a c t s o f a d u l t X. l a e v i s m u s c l e . P h o t o g r a p h o f an O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n p l a t e s t a i n e d w i t h Amido B l a c k 10B. The g e l i s made o f 1% a g a r o s e p r e p a r e d i n 0 .4 M K C l , 0.03 M PO^ b u f f e r (pH 7.3). The p l a t e was d e v e l o p e d f o r 48 h o u r s i n t h e c o l d (4°c), t h e n washed o v e r n i g h t i n 0 .4 M K C l , 0.03 M PO^ b u f f e r (pH 7.3) a t room t e m p e r a t u r e b e f o r e s t a i n i n g . The c e n t r e w e l l c o n t a i n e d 15 p i o f a n t i - m y o s i n a n t i b o d y ( a b o u t 15 ug o f p r o t e i n ) . W e l l 1 c o n t a i n e d about 20 ug o f a h i g h s a l t e x t r a c t o f a d u l t X. l a e v i s l i v e r . W e l l s 2, 3 and 4 c o n t a i n e d e l e c t r o p h o r e t i c a l l y - p u r i f i e d m y o s i n ( p r o t e i n c o n c e n t r a t i o n about 0.3 u g , 0.6 ug, 1.2 ug r e s p e c t i v e l y ) . W e l l s 5 and 6 c o n t a i n e d c r u d e m y o s i n ( t o t a l p r o t e i n c o n c e n t r a t i o n about 2.5 ug and 15.0 ug r e s p e c t i v e l y ) . I n s e p a r a t e d i f f u s i o n p l a t e s , serum f r o m non-immune r a b b i t s d i d n o t f o r m a p r e c i p i t i n l i n e w i t h g e l -p u r i f i e d m y o s i n or c r u d e m y o s i n . 42 F i g u r e 3. Phase c o n t r a s t and i n d i r e c t i m m u n o f l u o r e s c e n c e m i c r o g r a p h s o f t h e same f i e l d o f m u s c l e f r o m t h e t h i g h m u s c l e o f a d u l t Xenopus f r o g s . G l y c e r i n a t e d m u s c l e was p r e p a r e d as d e s c r i b e d by Pepe ( 1 9 6 7 ) . The l a b e l l i n g o f a n t i - m y o s i n a n t i b o d y w i t h f l u o r e s c e i n i s o t h i o c y a n a t e was p e r f o r m e d as d e s c r i b e d by Pepe ( 1 9 6 6 ) . A R e i c h e r t Z e t o p a n m i c r o s c o p e w i t h an u l t r a v i o l e t s o u r c e and a p p r o p r i a t e f i l t e r s was used f o r f l u o r e s c e n t m i c r o s c o p y . Non-immune serum d i d n o t g i v e a r e a c t i o n w i t h a n o t h e r p i e c e o f m u s c l e . X 400. U3 44 F i g u r e 4. A u t o r a d i o g r a m o f [ J " ' " ' l ] - l a b e l l e d p u r e m y o s i n and [J~*"'l]-l a b e l l e d c r u d e m u s c l e e x t r a c t p r e c i p i t a t e d w i t h a n t i - m y o s i n a n t i b o d y . S l o t 1 c o n t a i n s m y o s i n p r e c i p i t a t e d by a n t i b o d y . S l o t 2 c o n t a i n s i o d i n a t e d m y o s i n e l e c t r o p h o r e t i c a l l y p u r i f i e d . The p r o t e i n s were a n a l y z e d on a S D S - p o l y a c r y l a m i d e s l a b g e l (5% monomer). 1 Myosin 46 F i g u r e 5. A u t o r a d i o g r a m o f i o d i n a t e d t r y p t i c f r a g m e n t s o f e l e c t r o p h o r e t i c a l l y p u r i f i e d m y o s i n and a n t i b o d y p r e c i p i t a t e d myos S l o t 1 c o n t a i n s i o d i n a t e d m y o s i n d i g e s t e d w i t h t r y p s i n . S l o t 2 125 c o n t a i n s m y o s i n p r e c i p i t a t e d by a n t i b o d y , l a b e l l e d w i t h [ I ] and d i g e s t e d w i t h t r y p s i n . The p e p t i d e f r a g m e n t s were a n a l y z e d on a S D S - a c r y l a m i d e s l a b g e l ( 1 0 % monomer). molecular weight x 10 3 4 8 F i g u r e 6. P r e c i p i t a t i o n o f m y o s i n b y a n t i - m y o s i n a n t i b o d y . [ X'" JI]-m y o s i n h e a v y c h a i n ( 9 x 1 0 ^ cpm p e r m g ) , a n t i - m y o s i n a n t i b o d y ( c o n t a i n i n g a b o u t 40 A „ o n u n i t s p e r m l ) a n d a n t i b o d y f r o m n o n - i m m u n i z e d z oU r a b b i t s ( c o n t r o l ) ( a b o u t 2 0 A „ o n u n i t s p e r m l ) w e r e p r e p a r e d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s A . E a c h r e a c t i o n t u b e c o n t a i n e d 1 2 5 a b o u t 1 mg o f a n t i - m y o s i n a n t i b o d y , [ I ] - m y o s i n i n a t o t a l v o l u m e o f 2 0 0 p l . T h e t u b e s w e r e i n c u b a t e d f o r 12 h o u r s a t 4 ° C a n d p r o c e s s e d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s A . E a c h p o i n t o n t h e g r a p h r e p r e s e n t s a n a v e r a g e o f 4 d i f f e r e n t e x p e r i m e n t s w h i c h a g r e e d w i t h i n 1 0 % . T h e c o n t r o l a n t i b o d y o b t a i n e d f r o m a n o n - i m m u n i z e d r a b b i t 1 2 5 p r e c i p i t a t e d b e t w e e n 0 . 2 t o 0 . 3 % o f t h e [ I ] - m y o s i n . L i n e c o d e s : ( o - © ) , p r e c i p i t a t i o n w i t h a n t i - m y o s i n a n t i b o d y ; (• • ) , p r e c i p i t a t i o n w i t h n o n - i m m u n e s e r u m ( c o n t r o l ) . 49 I Myos in Added (jug) 5 0 F i g u r e 7 . B i n d i n g o f [ X / , : , I ] - l a b e l l e d a n t i - m y o s i n a n t i b o d y t o p o l y s o m e s f r o m v a r i o u s d e v e l o p m e n t a l s t a g e s o f X e n o p u s l a e v i s e m b r y o s . T h e g r a d i e n t s w e r e 2 0 - 5 0 % s u c r o s e a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . -- ( 7 a ) P o l y s o m e s f r o m s t a g e 12 e m b r y o s ( m e d i u m y o l k p l u g s t a g e ) . ( 7 b ) P o l y s o m e s f r o m s t a g e 1 6 / 1 7 e m b r y o s ( n e u r a l f o l d s t a g e ) . ( 7 c ) P o l y s o m e s f r o m s t a g e 20 e m b r y o s ( f u s e d n e u r a l f o l d s s t a g e ) . L i n e 1 2 5 c o d e s : ( ) , o p t i c a l d e n s i t y a t 2 5 4 n m ; ( o — — — o ) , [ I ] - l a b e l l e d a n t i b o d y b o u n d t o p o l y s o m e s . T h e p o s i t i o n o f t h e r i b o s o m e mono some i s i n d i c a t e d b y t h e a r r o w s a n d w a s d e t e r m i n e d b y c o m p a r i s o n t o X e n o p u s l i v e r p o l y s o m e s c e n t r i f u g e d u n d e r t h e s ame c o n d i t i o n s ( s e e M a t e r i a l s a n d M e t h o d s ) . 51 52 125 F i g u r e 8 . B i n d i n g o f [ I ] - l a b e l l e d a n t i b o d y t o s t a g e 2 0 p o l y s o m e s t r e a t e d w i t h R N a s e p r i o r t o c e n t r i f u g a t i o n . P r i o r t o s u c r o s e g r a d i e n t s e d i m e n t a t i o n s t a g e 2 0 h o m o g e n a t e s w e r e t r e a t e d w i t h R N a s e . L i n e 1 2 5 c o d e s : ( ) , o p t i c a l d e n s i t y a t 2 5 4 n m ; ( o o ) , [ I ] - l a b e l l e d a n t i b o d y b o u n d t o p o l y s o m e s . 54 F i g u r e 9. S e r o l o g i c a l a n a l y s i s o f m y o s i n s f r o m s t a g e 16/17, s t a g e 20 and s t a g e 26 embryos. T h i s i s a p h o t o g r a p h o f an O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n p l a t e s t a i n e d w i t h Amido B l a c k . 10B. F o r d e t a i l s o f how t h e p l a t e was p r o c e s s e d s e e F i g . 2. C e n t r e w e l l c o n t a i n e d a b out 15 pg o f a n t i - m y o s i n a n t i b o d y . W e l l 1 c o n t a i n e d a h i g h s a l t e x t r a c t o f X. l a e v i s l i v e r . W e l l 2 c o n t a i n e d about 30 pg o f p r o t e i n f r o m a h i g h s a l t e x t r a c t o f s t a g e 16/17 embryos. W e l l s 3 and 4 c o n t a i n e d a h i g h s a l t e x t r a c t f r o m s t a g e 20 embryos ( a b o u t 75 pg and 15 pg o f p r o t e i n r e s p e c t i v e l y ) . W e l l 5 c o n t a i n e d about 20 pg o f a h i g h s a l t e x t r a c t o f s t a g e 26 embryos. W e l l 6 c o n t a i n e d a b out 60 pg o f c r u d e m y o s i n f r o m a h i g h s a l t e x t r a c t o f a d u l t X. l a e v i s m u s c l e . I n a s e p a r a t e e x p e r i m e n t , low s a l t e x t r a c t s o f s t a g e 12 and 16/17 embryos d i d n o t f o r m a p r e c i p i t i n l i n e when r e a c t e d w i t h a n t i - m y o s i n a n t i b o d y . 56 F i g u r e 10a. 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 on a 10% S D S - a c r y l a m i d e g e l o f myo s i n e x t r a c t s o f s t a g e 20/22 and s t a g e 26/27 embryos. About 3000 embryos o f each s t a g e were p r o c e s s e d f o r myosin p r e p a r a t i o n as d e s c r i b e d i n M a t e r i a l s and Methods A up t o and i n c l u d i n g an (NH^)SO^ p r e c i p i t a t i o n ( 3 8 % - 4 5 % ) . S l o t 1: h i g h s a l t e x t r a c t o f a d u l t Xenopus  l a e v i s m y o s i n . S l o t 2: low s a l t h o m o g e n i z a t i o n e x t r a c t o f s t a g e 20 embryos. S l o t 3: h i g h s a l t i n s o l u b l e p r o t e i n s f r o m s t a g e 20 embryos. S l o t 4: ammonium s u l f a t e - p r e c i p i t a t e d f r a c t i o n o f s t a g e 20 embryos (abo u t 20 ug o f p r o t e i n ) . S l o t 4: ammonium s u l f a t e - p r e c i p i t a t e d f r a c t i o n o f s t a g e 26 embryos (about 20 ug o f p r o t e i n ) . 57 2 3 4 5 Myosin Actin 58 F i g u r e 10b. 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 on a 7% S D S - a c r y l a m i d e g e l o f h i g h s a l t e x t r a c t s o f s t a g e 16/17 embryos and p r e - metamorphosis t a d p o l e t a i l s . About 5000 s t a g e 16/17 embryos were p r o c e s s e d f o r m y o s i n i s o l a t i o n ( s e e M a t e r i a l s and Methods A and F i g . 1 0 a ) . S e v e r a l t a d p o l e t a i l s were p r o c e s s e d i n a s i m i l a r way. S l o t 1: low s a l t e x t r a c t f r o m s t a g e 16/17 embryos (ab o u t 40 ug o f p r o t e i n ) . S l o t 2: ammonium s u l f a t e f r a c t i o n (38-48%) o f t a d p o l e t a i l e x t r a c t s . S l o t 3: h i g h s a l t e x t r a c t f r o m s t a g e 16/17 embryos. 60 F i g u r e 1 1 . B i n d i n g o f [±*"JI]-anti-myosin t o X e n o p u s l i v e r p o l y s o m e s m i x e d 1 :1 w i t h t h e 1 0 0 , 0 0 0 x g s u p e r n a t a n t o f s t a g e 20 p o l y s o m e s . P o l y s o m e s f r o m a d u l t X e n o p u s l i v e r w e r e m i x e d w i t h t h e 1 0 0 , 0 0 0 x g s u p e r n a t a n t f r o m s t a g e 20 e m b r y o s p r i o r t o s u c r o s e g r a d i e n t s e d i m e n t a t i o n . A f t e r c e n t r i f u g a t i o n , f r a c t i o n s w e r e c o l l e c t e d a n d 125 r e a c t e d w i t h [ I ] - a n t i - m y o s i n a s i n M a t e r i a l s a n d M e t h o d s s e c t i o n s . 1 2 5 L i n e c o d e s : ( ) , a b s o r b a n c e p r o f i l e a t 2 5 4 n m ; ( o — — o ) , [ I ] -l a b e l l e d a n t i b o d y b o u n d t o p o l y s o m e s . T h e a r r o w s i n d i c a t e t h e p o s i t i o n o f t h e mono some a n d t h e 3 0 - s o m e p e a k s . Fraction Number 62 F i g u r e 1 2 . B i n d i n g o f ['L*"J1]-anti-myosin t o s t a g e 2 0 p o l y s o m e s i s o l a t e d i n t h e p r e s e n c e o f m y o s i n h e a v y c h a i n s . P u r i f i e d m y o s i n h e a v y c h a i n s w e r e a d d e d t o s t a g e 2 0 h o m o g e n a t e s p r i o r t o s u c r o s e g r a d i e n t s e d i m e n t a t i o n . L i n e c o d e s : ( ) , a b s o r b a n c e p r o f i l e a t 125 2 5 4 n m ; ( o — o ) , [ I ] - l a b e l l e d a n t i b o d y b o u n d t o p o l y s o m e s . 12 11 1 0 9 8 7 6 5 4 3 2 1 B o t t o m T o p Fract ion Number 64 MATERIALS AND METHODS B E m b r y o n i c s t a g i n g . The embryos used f o r i s o l a t i o n o f polysomes and RNA were s t a g e d a c c o r d i n g t o t h e Normal T a b l e o f Xenopus l a e v i s (Daudin) by Nieuwkoop and Faber ( 1 9 6 7 ) . Embryos were s t a g e d a f t e r r e m o v i n g t h e j e l l y c o a t and v i t e l l i n e membrane. As s o o n as 50-100 embryos were s t a g e d , t h e y were washed i n s t e r i l e H o l t f r e t e r ' s medium and f r o z e n i n p l a s t i c v i a l s i n a m i x t u r e o f d r y i c e and e t h a n o l . I t i s n o t p o s s i b l e t o e s t i m a t e how s y n c h r o n o u s t h e embryos were a t each s t a g e . However, i t i s p o s s i b l e t o e l i m i n a t e c e r t a i n s t a g e s f r o m p a r t i c u l a r b a t c h e s . S t a g e 12 embryos m o r p h o l o g i c a l l y d i s p l a y a medium y o l k p l u g and a r e about 13 1/4 h o u r s o l d . T h i s b a t c h i n a l l l i k e l i h o o d c o n s i s t e d o f s t a g e 11 1/2 embryos (12 1/2 h o u r s o l d ) , s t a g e 12 embryos and s t a g e 12 1/2 embryos (14 1/2 h o u r s o l d ) . E a r l i e r s t a g e s ( s t a g e 11) and l a t e r s t a g e s ( s t a g e 13) w o u l d n o t c o n t a m i n a t e t h i s b a t c h b ecause t h e y do n o t d i s p l a y a y o l k p l u g , w h i c h i s e a s i l y seen under t h e l i g h t m i c r o s c o p e . S t a g e 16/17 embryos a r e c h a r a c t e r i z e d by a r e a d i l y d i s t i n g u i s h a b l e n e u r a l f o l d and a r e 18 1/4 t o 18 3/4 h o u r s o l d . I t i s easy t o s e p a r a t e s t a g e 15 embryos f r o m s t a g e 16 embryos be c a u s e t h e n e u r a l f o l d s a r e d i f f e r e n t l y shaped. However, s i n c e i t i s n o t p o s s i b l e t o c o n t r o l ambient t e m p e r a t u r e and p h y s i o l o g i c a l d i f f e r e n c e s d u r i n g s t a g i n g , some s t a g e 15 embryos (17 1/2 h o u r s o l d ) m i g h t be i n c l u d e d i n s t a g e 16 b a t c h e s . S t a g e 17 and s t a g e 18 embryos can e a s i l y be d i s t i n g u i s h e d b e c a u s e t h e n e u r a l f o l d s a r e w e l l s e p a r a t e d p o s t e r i o r l y a t s t a g e 17 b u t a r e v e r y 65 c l o s e t o e a c h o t h e r a t s t a g e 1 8 . H o w e v e r , s t a g e 17 e m b r y o s may c o n t a i n s t a g e 18 e m b r y o s b e c a u s e up t o a n h o u r c a n e l a p s e d u r i n g t h e s t a g i n g o f e v e n s m a l l q u a n t i t i e s o f e m b r y o s . E a r l i e r s t a g e s ( s t a g e 14 ) o r l a t e r s t a g e s ( s t a g e 19 ) w o u l d n o t c o n t a m i n a t e t h e s e b a t c h e s b e c a u s e t h e y c a n b e e a s i l y s p o t t e d . T h e r e f o r e , b a t c h e s o f s t a g e 1 6 / 1 7 e m b r y o s may s p a n a t i m e f r o m 17 1/2 h o u r s ( s t a g e 1 5 ) t o 19 3/4 h o u r s ( s t a g e 1 8 ) . H o w e v e r , m o s t o f t h e e m b r y o s s h o u l d b e a t s t a g e 1 7 . A t s t a g e 20 ( 2 1 3/4 h o u r s o l d ) t h e n e u r a l f o l d s a r e f u s e d , t h e s u t u r e i s s t i l l p r e s e n t , t h e f u t u r e s u c k e r i s c h a r a c t e r i s t i c a n d t h e t w o e y e a n l a g e n a r e b e c o m i n g d u m b - b e l l s h a p e d . T h e r e f o r e , t h i s b a t c h s h o u l d n o t c o n t a i n a n y e a r l i e r s t a g e s . P r e s e n c e o f s t a g e 2 1 e m b r y o s ( 2 2 1/2 h o u r s o l d ) c a n n o t b e r u l e d o u t b a c a u s e o f t i m e f a c t o r s i n v o l v e d i n s t a g i n g . H o w e v e r , s i n c e s t a g e 2 1 e m b r y o s a r e e a s i l y d i s t i n g u i s h e d f r o m s t a g e 2 0 e m b r y o s , c o n t a m i n a t i o n s h o u l d b e m i n i m a l . T h e r e f o r e , i t a p p e a r s t h a t s t a g e 2 0 e m b r y o s w e r e t h e m o s t a c c u r a t e l y s t a g e d . B a t c h e s o f s t a g e 12 e m b r y o s m o s t l i k e l y s p a n 2 h o u r s o f d e v e l o p m e n t , a p e r i o d m a i n l y i n v o l v e d w i t h t h e f o r m a t i o n o f e c t o - , meso - a n d e n d o d e r m a l l a y e r s . B a t c h e s o f s t a g e 1 6 / 1 7 e m b r y o s s p a n n e d t h e l o n g e s t d e v e l o p m e n t a l t i m e p e r i o d ( 2 1/4 h o u r s ) , a t i m e p e r i o d c h a r a c t e r i z e d b y n e u r a l p l a t e f o r m a t i o n a n d t h e s e g r e g a t i o n o f t h r e e t o f o u r s o m i t e s o n e a c h o f t h e e m b r y o s . T h e s e s t a g i n g p r o b l e m s do n o t i n v a l i d a t e t h e f i n d i n g s r e p o r t e d i n t h i s s t u d y . RNA e x t r a c t i o n . a ) M a g n e s i u m p r e c i p i t a t i o n o f p o l y s o m e s . P o l y s o m e s a n d r i b o n u c l e o p r o t e i n p a r t i c l e s f r o m e m b r y o h o m o g e n a t e s w e r e p r e c i p i t a t e d w i t h m a g n e s i u m e s s e n t i a l l y a s d e s c r i b e d b y P a l m i t e r ( 1 9 7 4 ) . A l l b u f f e r s 66 were s t e r i l i z e d w i t h DEP. A l l g l a s s w a r e was h e a t - s t e r i l i z e d and t r e a t e d w i t h a s o l u t i o n o f d i c h l o r o d i m e t h y l s i l a n e . A l l p r o c e d u r e s were p e r f o r m e d a t 4°C. To e a c h b a t c h o f embryos ( 3 0 0 0 ) , w h i c h had been s t o r e d a t -70°C, B u f f e r A (0.2 M Hepes, pH 7.5; 25 mM N a C l , 10 mM M g C l 2 , 1.0% T r i t o n X-100, 0.5% sodium d e o x y c h o l a t e ) was added. The embryos were homogenized i n a Dounce homogenizer ( K o n t e s G l a s s Co.) w i t h 10-20 s t r o k e s o f a l o o s e p e s t l e . The homogenate was c e n t r i f u g e d f o r 5 m i n u t e s a t 10,000 x g; t h e s u p e r n a t a n t was d e c a n t e d i n t o a b e a k e r and an e q u a l volume o f S o l u t i o n B was added (200 mM M g C l 2 , 2.0% T r i t o n X-100, 1 mg/ml h e p a r i n ) . The p e l l e t f r o m t h e p r e v i o u s c e n t r i f u g a t i o n was r e - e x t r a c t e d w i t h B u f f e r A, c e n t r i f u g e d and t h e s e c o n d s u p e r n a t a n t combined w i t h t h e f i r s t . The m i x t u r e was k e p t on i c e f o r 1 h o u r . S u b s e q u e n t l y , t h e m i x t u r e was l a y e r e d s l o w l y o v e r o n e - t h i r d volume o f B u f f e r C (25 mM N a C l , 5 mM M g C l 2 , 20 mM Hepes, pH 7.5, and 1.0 M s u c r o s e ) and spun f o r 10 m i n u t e s a t 27,000 x 8 m a x « The s u p e r n a t a n t s were d e c a n t e d r a p i d l y f r o m t h e t u b e s and any l i p i d o r s u c r o s e a d h e r i n g t o t h e w a l l s was w i p e d w i t h t i s s u e p a p e r . The p e l l e t was t a k e n up i n 20 mM Hepes (pH 7.6), t h e n e x t r a c t e d w i t h p h e n o l and c h l o r o f o r m as d e s c r i b e d by P a l m i t e r ( 1 9 7 4 ) . P h e n o l ( F i s h e r r e a g e n t grade) was r o u t i n e l y made 0.1% i n 8 - h y d r o x y q u i n o l i n e . C h l o r o f o r m was r o u t i n e l y r e d i s t i l l e d . A f t e r t h e o r g a n i c e x t r a c t i o n s , t h e RNA was p r e c i p i t a t e d w i t h 2 volumes o f e t h a n o l a t -20°C. The RNA p e l l e t was t h e n washed t w i c e w i t h 2 m l o f 3 M sodium a c e t a t e (pH 6.0). The RNA was t h e n d i s s o l v e d i n 0.1 M p o t a s s i u m a c e t a t e (pH 7 . 0 ) , p r e c i p i t a t e d w i t h e t h a n o l , and c o l l e c t e d by c e n t r i f u g a t i o n ; t h e p e l l e t was d r i e d and d i s s o l v e d i n 20 mM Hepes (pH 7.4) a t a c o n c e n t r a t i o n o f 1.0 mg/ml. 67 b ) G u a n i d i n i u m h y d r o c h l o r i d e e x t r a c t i o n o f RNA. T h i s e x t r a c t i o n p r o c e d u r e w a s d e v e l o p e d b y W. R u t t e r ( U n i v e r s i t y o f C a l i f o r n i a , S a n F r a n c i s c o ) u s i n g a m o d i f i c a t i o n o f t h e m e t h o d d e s c r i b e d b y C o x ( 1 9 6 8 ) . E m b r y o s w e r e h o m o g e n i z e d a s i n t h e p r e v i o u s s e c t i o n i n B u f f e r A a n d t h e n c e n t r i f u g e d . T h e s u p e r n a t a n t a n d t h e p e l l e t w e r e m a d e 4 M i n g u a n i d i n e - H C l ( S i g m a ) b y t h e a d d i t i o n o f 6 M G u C l ; 2 M p o t a s s i u m a c e t a t e w a s a d d e d t o a c h i e v e a p H o f 5 . 0 . T h e t w o m i x t u r e s w e r e h o m o g e n i z e d 2 0 t i m e s i n t h e D o u n c e h o m o g e n i z e r w i t h a l o o s e p e s t l e a t - 2 0 ° C ( s a l t / i c e ) . T h e h o m o g e n i z a t i o n m i x t u r e c o n t a i n i n g t h e p e l l e t w a s c e n t r i f u g e d a t 5 , 0 0 0 x g f o r 5 m i n u t e s . T h e n , 1/2 v o l u m e o f - 2 0 ° C 9 5 % e t h a n o l w a s a d d e d t o t h e s e p a r a t e s u p e r n a t a n t s a n d t h e m i x t u r e s w e r e p r e c i p i t a t e d a t - 2 0 ° C o v e r n i g h t . T h e i n d i v i d u a l p r e c i p i t a t e s w e r e c e n t r i f u g e d a t 1 0 , 0 0 0 x g f o r 5 m i n u t e s a n d t h e p r e c i p i t a t e s w e r e made 6 M i n G u C l a n d 0 . 1 M i n p o t a s s i u m a c e t a t e ( p H 5 . 0 ) . T h e m i x t u r e s w e r e t h e n p r e c i p i t a t e d w i t h 1/2 v o l u m e o f - 2 0 ° C 9 5 % e t h a n o l a n d p r e c i p i t a t e d f o r 12 h o u r s a t - 2 0 ° C . T h e p r e c i p i t a t e s w e r e c e n t r i f u g e d a t 5 , 0 0 0 x g f o r 1 0 m i n u t e s . T h e p r e c i p i t a t e s w e r e c o m b i n e d a n d s u b s e q u e n t l y d i s s o l v e d i n 2 0 mM H e p e s ( p H 7 . 4 ) a n d e x t r a c t e d w i t h o r g a n i c s o l v e n t s a c c o r d i n g t o P a l m i t e r ( 1 9 7 4 ) . A l l RNA p r e p a r a t i o n s h a d ^dO^lHQ r a t i o s i n t * i e 2 . 1 0 r a n g e . T h e y i e l d s o f RNA e x t r a c t e d b y b o t h m e t h o d s v a r i e d f o r e a c h b a t c h o f 2+ e m b r y o s . G e n e r a l l y , w i t h t h e Mg m e t h o d , t h e y i e l d w a s 1 1 . 2 mg o f R N A / 3 0 0 0 e m b r y o s ; f o r t h e G u C l m e t h o d t h e y i e l d w a s 1 0 . 6 mg o f R N A / 3 0 0 0 e m b r y o s . F o r c o m p a r i s o n , w h e n RNA w a s i s o l a t e d b y t h e h o t p h e n o l m e t h o d o f M c C a r t h y a n d H o y e r ( 1 9 6 4 ) , t h e y i e l d s w e r e a s h i g h a s t h o s e o b t a i n e d 2+ b y t h e Mg p r e c i p i t a t i o n m e t h o d . H o w e v e r , t h e RNA e x t r a c t e d b y h o t p h e n o l w a s n o t a s a c t i v e i n d i r e c t i n g p r o t e i n s y n t h e s i s i n t h e w h e a t g e r m c e l l - f r e e s y s t e m . RNA c o n c e n t r a t i o n s w e r e e s t i m a t e d b y a s s u m i n g 68 2+ t h a t one ^fto u n ^ t °^ i n t h e absence o f Mg was e q u a l t o 50 jag o f RNA. F o r some e x p e r i m e n t s , RNA c o n c e n t r a t i o n s were d e t e r m i n e d by t h e o r c i n o l r e a c t i o n (Merchant e t a l . , 1969). O l i g o ( d T ) - c e l l u l o s e b i n d i n g o f mRNAs. RNA e x t r a c t e d f r o m embryos was s e p a r a t e d f r o m r i b o s o m a l and o t h e r RNAs n o t c o n t a i n i n g p o l y ( A ) by o l i g o ( d T ) - c e l l u l o s e c h r omatography. The o l i g o ( d T ) - c e l l u l o s e was p u r c h a s e d f r o m C o l l a b o r a t i v e R e s e a r c h (Type T-3, l o t 534-35). The samples were p r o c e s s e d b a t c h w i s e ( H a i n e s and P a l m i t e r , 1974; Strohman e t a l . , 1977) p e r m i t t i n g e l u t i o n o f s m a l l amounts o f message i n v e r y s m a l l volumes. The o l i g o ( d T ) - c e l l u l o s e was s t e r i l i z e d w i t h 0.05% DEP. " F i n e s " were removed by e x t e n s i v e w a s h i n g w i t h 0.5 M K C l , 20 mM Hepes (pH 7.5). The o l i g o ( d T ) - c e l l u l o s e was t h e n washed w i t h b i n d i n g b u f f e r (0.5 M K C l , 20 mM Hepes (pH 7 . 5 ) , 1.0% SDS) u n t i l an ^(,0 o f 0 was o b t a i n e d . B i n d i n g o f c o n t r o l p r e p a r a t i o n s o f p o l y ( A ) i s ;s» 95% co m p l e t e under t h e s e c o n d i t i o n s (R. Reeves, p e r s o n a l c o m m u n i c a t i o n ) . To p r e v e n t a g g r e g a t i o n o f RNA, t h e RNA - e t h a n o l p r e c i p i t a t e s were d i s s o l v e d i n d o u b l e - d i s t i l l e d w a t e r and h e a t e d t o 65°C f o r 10 m i n u t e s (Strohman e t a l . , 1977). The RNA m i x t u r e was d i l u t e d w i t h an e q u a l volume o f 2X b i n d i n g b u f f e r b e f o i e m i x i n g w i t h o l i g o ( d T ) -c e l l u l o s e . However, t h e same amount o f b i n d i n g was o b t a i n e d i f a c o n c e n t r a t i o n n o t h i g h e r t h a n 2-3 mg/ml o f RNA/l'.OO mg o l i g o ( d T ) - c e l l u l o s e was used. RNA i n 0.2 m l o f b i n d i n g b u f f e r was m i x e d w i t h 0.2 m l o f o l i g o ( d T ) - c e l l u l o s e and i n c u b a t e d a t room t e m p e r a t u r e f o r 30 m i n u t e s . The m i x t u r e was c e n t r i f u g e d and t h e c e l l u l o s e was washed w i t h b i n d i n g b u f f e r u n t i l no U V - a b s o r b i n g m a t e r i a l was e l u t e d . The bound m a t e r i a l was e l u t e d w i t h s e v e r a l washes o f 20 mM Hepes b u f f e r (pH 7.5). The 69 c e l l u l o s e was t h e n r e g e n e r a t e d by w a s h i n g w i t h 0.1 N KOH and s u b s e q u e n t l y w i t h b i n d i n g b u f f e r . The bound and f l o w - t h r o u g h RNA f r a c t i o n s were t h e n a d j u s t e d t o 0.1 M w i t h p o t a s s i u m a c e t a t e (pH 7 . 0 ) , p r e c i p i t a t e d o v e r n i g h t w i t h e t h a n o l and c o l l e c t e d by c e n t r i f u g a t i o n ; t h e RNA p e l l e t s were d r i e d and d i s s o l v e d i n 20 mM Hepes (pH 7.5). Under t h e s e c o n d i t i o n s , 3000 s t a g e 12 embryos y i e l d e d 54.6 pg o f p o l y ( A ) + RNA ( b o u n d ) ; 3000 s t a g e 16/17 embryos y i e l d e d 61.0 pg o f p o l y ( A ) + RNA and 3000 embryos a t s t a g e 20 y i e l d e d 63.4 pg o f p o l y ( A ) + RNA. P r e p a r a t i o n o f wheat germ l y s a t e s . A c r u d e wheat germ e x t r a c t was p r e p a r e d a c c o r d i n g t o t h e method o f R o b e r t s and P a t e r s o n (1973) w i t h m i n o r m o d i f i c a t i o n s . The e x t r a c t i o n b u f f e r c o n s i s t e d o f 20 mM Hepes (pH 7.6; a d j u s t e d w i t h KOH), 100 mM KOAc, 2 mM C a C l 2 . 2 H 2 0 , 1 mM M g ( 0 A c ) 2 and 1 mM DTT. The wheat germ was a g i f t o f G e n e r a l M i l l s , V a l l e j o , C a l i f o r n i a . The column e l u t i o n b u f f e r c o n t a i n e d 20 mM Hepes (pH 7 . 6 ) , 120 mM KOAc, 5 mM M g ( 0 A c ) 2 and 1 mM DTT. A f t e r d e s a l t i n g on a Sephadex G-25 column, t h e wheat germ homogenate ( t h e S-30 p r e p a r a t i o n ) was c e n t r i f u g e d a t 0°C a t 30,000 x g f o r 10 m i n u t e s . T h i s s t e p r e d u c e s t h e p r o t e i n c o n c e n t r a t i o n o f t h e S-30 w i t h o u t a f f e c t i n g i t s t r a n s l a t i o n a l a c t i v i t y . The S-30 e x t r a c t was s t o r e d i n p o l y p r o p y l e n e m i c r o f u g e t u b e s ( B r i n k m a n , I n c . ) a t -70°C u n t i l needed. A l l embryo RNA t r a n s l a t i o n s r e p o r t e d h e r e were p e r f o r m e d w i t h t h e same S-30 wheat germ p r e p a r a t i o n . C e l l - f r e e p r o t e i n s y n t h e s i s . 14 [ C ] - l e u c i n e (270 p C i / m o l e ) was f r o m New E n g l a n d N u c l e a r . 70 ATP, GTP, c r e a t i n e p h o s p h a t e and c r e a t i n e p h o s p h o k i n a s e were f r o m Sigma, and were p r e p a r e d and s t o r e d as a m a s t e r m i x i n s m a l l a l i q u o t s a t -70°C. The u n l a b e l l e d amino a c i d s were f r o m Sigma a n d were n e u t r a l i z e d b e f o r e a d d i n g them t o t h e m a s t e r mix. The c o m p l e t e c e l l - f r e e s y s t e m i n a f i n a l volume o f 50 u l c o n t a i n e d : 15-20 u l o f wheat germ e x t r a c t , 20 mM Hepes (pH 7.4; a d j u s t e d w i t h KOH), 2 mM DTT, 8 mM c r e a t i n e p h o s p h a t e , 50 u n i t s / m l o f c r e a t i n e p h o s p h o k i n a s e , 25 pM GTP, 150 mM KOAc, 3.0 mM M g ( 0 A c ) 2 , 25 pM o f a l l 19 u n l a b e l l e d amino a c i d s and 0.25 u C i o f [ 1 4 C ] -l e u c i n e (270 u C i / u m o l e ) . A s s a y s were i n c u b a t e d f o r 60 t o 90 m i n u t e s a t 30°C. The r e a c t i o n s were s t o p p e d by a d d i t i o n o f an e q u a l volume of 0.1 N KOH and were t h e n i n c u b a t e d f o r 2 m i n u t e s a t 37°C t o d e a c y l a t e t h e tRNAs. The m i x t u r e s were t h e n p r e c i p i t a t e d w i t h 20% i c e - c o l d t r i c h l o r o a c e t i c a c i d (made 1 mM i n " c o l d " l e u c i n e ) and c o l l e c t e d onto Whatman GF/A f i l t e r s ( p r e v i o u s l y m o i s t e n e d w i t h c o l d 5% TCA). A s s a y t u b e s were r i n s e d two t i m e s w i t h 20% TCA and t h e f i l t e r s were r i n s e d one more t i m e w i t h i c e - c o l d 20% TCA. F i l t e r s were t h e n washed w i t h 95% e t h a n o l and e t h y l e t h e r and c o u n t e d i n A q u a s o l i n a N u c l e a r C h i c a g o I s o c a p s c i n t i l l a t i o n c o u n t e r . A n t i b o d y p r e c i p i t a t i o n o f c e l l - f r e e p r o d u c t s . F o r a n t i b o d y p r e p a r a t i o n , 50 p l wheat germ l y s a t e a s s a y s were t e r m i n a t e d by a d d i t i o n o f a c o l d s o l u t i o n o f 10% T r i t o n X-100 and s o d i u m d e o x y c h o l a t e i n 5 M K C l . S u b s e q u e n t l y , 100 pg of u n l a b e l l e d g e l - p u r i f i e d m y o s i n heavy c h a i n was added, f o l l o w e d by 5 p l o f 0.1 M " c o l d " l e u c i n e c o n t a i n i n g 0.2 mM PMSF. S i n c e t h e s e m i x t u r e s a l w a y s c o n t a i n e d a p r e c i p i t a t e , t h e y were c e n t r i f u g e d i n Beckman m i c r o f u g e t u b e s a t f u l l speed f o r 15 m i n u t e s . The p e l l e t s and t h e s u p e r n a t a n t s 71 were t h e n p r o c e s s e d f o r a n t i b o d y p r e c i p i t a t i o n . The p e l l e t s were r e s u s p e n d e d i n a low i o n i c s t r e n g t h b u f f e r (20 mM Hepes (pH 7 . 5 ) , 1 mM M g C l 2 and 2% T r i t o n X-100 and sodium d e o x y c h o l a t e ) . The m i x t u r e was r e a c t e d o v e r n i g h t w i t h 30-60 ug o f a n t i - m y o s i n o r non-immune a n t i b o d y . The r e s u l t i n g p r e c i p i t a t e s were f r e e d o f n o n - s p e c i f i c a l l y a d s o r b e d m a t e r i a l by s e d i m e n t a t i o n t h r o u g h a d i s c o n t i n u o u s , d e t e r g e n t - c o n t a i n i n g s u c r o s e g r a d i e n t i n m i c r o f u g e t u b e s as p r e v i o u s l y d e s c r i b e d . The t u b e s were t h e n f r o z e n and t h e t i p s were c u t o f f and t h e p r e c i p i t a t e d r a d i o a c t i v i t y c o u n t e d as p r e v i o u s l y . No r a d i o a c t i v i t y above b a c k g r o u n d l e v e l s o f n o n - s p e c i f i c p r e c i p i t a t i o n was d e t e c t e d i n t h e p e l l e t s . The s u p e r n a t a n t s f r o m t h e wheat germ i n c u b a t i o n l y s a t e s ( s e e above) were d i l u t e d w i t h 9 volumes o f a " c o l d " b u f f e r s o l u t i o n (1 mM Hepes (pH 7 . 5 ) , 1 mM EDTA) and l e f t s t a n d i n g f o r 12 h o u r s a t 0°C. The p r e c i p i t a t e d m y o s i n was c o l l e c t e d by c e n t r i f u g a t i o n a t 12,000 x g f o r 15 m i n u t e s and d i s s o l v e d i n 20 mM Hepes (pH 7 . 5 ) , 1 mM M g C ^ , 1% T r i t o n X-100 and s o d i u m d e o x y c h o l a t e . F i n a l l y , 30-60 pg o f a n t i - m y o s i n a n t i b o d y o r non-immune serum were added. The m i x t u r e s were i n c u b a t e d o v e r n i g h t a t 0°C and t h e p r e c i p i t a t e s were p r o c e s s e d as above. T r y p t i c d i g e s t i o n o f c e l l - f r e e t r a n s l a t i o n p r o d u c t s . The a n t i g e n - a n t i b o d y p r e c i p i t a t e s r e s u l t i n g f r o m t h e r e a c t i o n o f a n t i - m y o s i n a n t i b o d y and t h e c e l l - f r e e wheat germ i n c u b a t e s ( s e e p r e v i o u s s e c t i o n ) were e l e c t r o p h o r e s e d on S D S - a c r y l a m i d e g e l s (when p r e c i p i t a t e s r e s u l t i n g f r o m t h e r e a c t i o n o f non-immune r a b b i t s e r a w i t h wheat germ i n c u b a t e s were e l e c t r o p h o r e s e d , no r a d i o a c t i v e m y o s i n band was d e t e c t e d on t h e g e l s ) . The bands m i g r a t i n g i r . t h e 210,000 d a l t o n 72 range were c u t o u t and p r o c e s s e d as d e s c r i b e d i n R e s u l t s A under m y o s i n p u r i f i c a t i o n . The p r o d u c t s were p o o l e d and r e s u s p e n d e d i n 0.6 M N a C l , 0.015 M T r i s - H C l (pH 7 . 4 ) , 0.1% B - m e r c a p t o e t h a n o l ( s e e R e s u l t s A under t h e h e a d i n g T r y p t i c d i g e s t i o n ) and mixed w i t h 2 pg o f t r y p s i n . T h i s m i x t u r e was d i g e s t e d f o r 60 m i n u t e s a t 37°C and t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 4 pg o f t r y p s i n i n h i b i t o r and 20 p l o f Laemmli's SDS sample b u f f e r f o l l o w e d by b o i l i n g f o r 4 m i n u t e s . The m i x t u r e was t h e n c e n t r i f u g e d i n a Beckman m i c r o f u g e f o r 1 m i n u t e and d i a l y z e d f o r a few h o u r s a g a i n s t L a e m m l i 1 s SDS sample b u f f e r ( s e e R e s u l t s A ) . About 70 p l o f t h i s m i x t u r e c o n t a i n i n g about 1397 cpm was e l e c t r o p h o r e s e d ( s e e R e s u l t s A) on a 10% S D S - a c r y l a m i d e g e l and s t a i n e d w i t h Coomassie B l u e . The p o s i t i o n o f t h e bands was d e t e r m i n e d by m e a s u r i n g w i t h a r u l e r and t h e n t h e g e l was f r o z e n and s l i c e d i n t o 2 mm s l i c e s . E a c h s l i c e was p l a c e d i n a c o u n t i n g v i a l and d i g e s t e d w i t h 100 p l o f 30% ^ 0 2 o v e r n i g h t a t 50°C. A f t e r a d d i n g 300 p l o f P r o t o s o l ( N u c l e a r C h i c a g o ) s o l u b i l i z e r and 4 m l o f A q u a s o l s c i n t i l l a t i o n f l u i d , t h e r a d i o a c t i v i t y was d e t e r m i n e d i n a s c i n t i l l a t i o n c o u n t e r . About 87% o f a p p l i e d r a d i o a c t i v i t y was r e c o v e r e d . 73 RESULTS B I s o l a t i o n and f r a c t i o n a t i o n o f RNA. RNA was i s o l a t e d f r o m s t a g e 12, s t a g e 16/17 and s t a g e 20 Xenopus embryos. B a t c h e s o f Xenopus embryos were t r e a t e d i n two d i f f e r e n t ways as a f i r s t s t e p towards i s o l a t i o n o f RNA. I n t h e magnesium p r e c i p i t a t i o n t e c h n i q u e ( P a l m i t e r , 1 9 7 4 ) , p o l y s o m e s , mRNP p a r t i c l e s and h i g h m o l e c u l a r w e i g h t RNAs were p r e c i p i t a t e d 2+ by 0.1 M Mg f r o m t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t . I n t h e g u a n i d i n i u m h y d r o c h l o r i d e t e c h n i q u e , t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t was made 4 M i n g u a n i d i n i u m c h l o r i d e and n u c l e i c a c i d s were p r e c i p i t a t e d by t h e a d d i t i o n o f 1/2 volume o f 95% e t h a n o l . I n b o t h t y p e s o f e x p e r i m e n t , RNA was e x t r a c t e d f r o m t h e p r e c i p i t a t e s by t h e p h e n o l : c h l o r o f o r m p r o c e d u r e as d e s c r i b e d by P a l m i t e r ( 1 9 7 4 ) . T h i s c o m p a r a t i v e a p p r o a c h , u s i n g two d i f f e r e n t RNA e x t r a c t i o n p r o c e d u r e s , s h o u l d m i n i m i z e t h e p o s s i b i l i t y t h a t a n e g a t i v e r a s u l t i n p r o t e i n t r a n s l a t i o n i s due t o RNA d e g r a d a t i o n o r l o s s d u r i n g i s o l a t i o n . T a b l e 3 shows t h a t s i m i l a r amounts o f RNA a r e o b t a i n e d a f t e r 2+ e x t r a c t i o n o f n u c l e i c a c i d f r o m Mg p r e c i p i t a t e s o r from g u a n i d i n i u m -e t h a n o l p r e c i p i t a t e s . R e s u l t s f r o m c e l l - f r e e t r a n s l a t i o n a s s a y s , t o be d e s c r i b e d l a t e r , show t h a t b o t h p r o c e d u r e s y i e l d mRNAs w i t h s i m i l a r t r a n s l a t i o n c h a r a c t e r i s t i c s . As shown i n T a b l e 4, t h e a p p r o x i m a t e amount o f t o t a l RNA and p o l y s o m a l RNA i n Xenopus embryos has been e s t i m a t e d f r o m measurements o f RNA c o n t e n t (Brown and L i t t n a , 1964) and o f polysome c o n t e n t (Woodland, 1974). A c c o r d i n g t o t h i s d a t a , t h e t o t a l 74 RNA c o n t e n t o f Xenopus embryos i s about 12-13 mg/3000 embryos. T h e r e f o r e , t h e y i e l d s shown i n T a b l e 3 i n d i c a t e t h a t t h e magnesium p r e c i p i t a t i o n and g u a n i d i n i u m c h l o r i d e methods gave good y i e l d s o f RNA. T o t a l RNA o b t a i n e d by e i t h e r method was f u r t h e r f r a c t i o n a t e d by o l i g o ( d T ) - c e l l u l o s e chromatography. T a b l e 5 shows t h a t most of t h e t o t a l RNA d i d n o t b i n d t o t h e o l i g o ( d T ) - c e l l u l o s e . The c o n c e n t r a t i o n o f t h e RNA t h a t d i d b i n d t o t h e c e l l u l o s e was i n t h e same r a n g e as t h e amount f o u n d i n polysomes ( T a b l e 3; and Woodland, 1974). As a t e s t o f 3 t h e s p e c i f i c i t y o f b i n d i n g t o o l i g o ( d T ) - c e l l u l o s e , [ H ] - u r i d i n e - l a b e l l e d rRNA f r o m Xenopus o o c y t e s ( i s o l a t e d by Dr. R. Reeves) was mix e d w i t h t h e c e l l u l o s e . The l a b e l l e d rRNA was n o t r e t a i n e d on t h e c e l l u l o s e a f t e r two p a s s a g e s . To t e s t f o r a g g r e g a t i o n , RNA f r o m s t a g e 20 embryos was h e a t e d t o 65°C f o r 10 m i n u t e s , r a p i d l y c o o l e d and s u b j e c t e d t o o l i g o ( d T ) f r a c t i o n a t i o n . The y i e l d s o b t a i n e d were t h e same as t h o s e shown i n T a b l e 5. I t t h e r e f o r e a p p e a r s t h a t , u n d e r t h e c o n d i t i o n s u s e d , p o l y ( A ) + mRNA does n o t f o r m a g g r e g a t e s w i t h rRNA. I t i s assumed, b u t n o t d i r e c t l y p r o v e n , t h a t p o l y ( A ) + and p o l y ( A ) RNAs do n o t f o r m a g g r e g a t e s e i t h e r . A l s o , i t was f o u n d t h a t i t was u n n e c e s s a r y t o add tRNA t o t h e b i n d i n g b u f f e r i n o r d e r t o p r e v e n t n o n - s p e c i f i c a b s o r p t i o n o f RNA t o t h e o l i g o ( d T ) - c e l l u l o s e s i n c e a c o n t r o l e x p e r i m e n t i n d i c a t e d t h a t t h e y i e l d s o f p o l y ( A ) + RNA f r o m s t a g e 20 embryos were i d e n t i c a l i n t h e p r e s e n c e and absence o f tRNA. We t h e r e f o r e c o n c l u d e f r o m t h e s e r e s u l t s t h a t b o t h t h e magnesium p r e c i p i t a t i o n and g u a n i d i n i u m c h l o r i d e methods a r e q u a n t i t a t i v e means o f o b t a i n i n g t o t a l RNAs f r o m Xenopus embryos. The o l i g o ( d T ) -chromatography o f t o t a l c e l l u l a r RNA r e s u l t s i n a p o l y ( A ) + RNA f r a c t i o n w h i c h i s p r o b a b l y n o t a p p r e c i a b l y c o n t a m i n a t e d w i t h rRNA. T h i s 75 c e l l u l o s e - b o u n d f r a c t i o n i s p r o b a b l y composed o f p o l y ( A ) + mRNA d e r i v e d f r o m p o l y s o m a l and mRNP p a r t i c l e s . We cannot r u l e o u t t h e p o s s i b i l i t y t h a t some p o l y ( A ) RNA c o n t a m i n a t e s t h e p o l y ( A ) f r a c t i o n o r t h a t some mRNAs w i t h s h o r t p o l y ( A ) - t a i l s (Rosbash e t a l . , 1977) were n o t bound t o t h e o l i g o ( d T ) - c e l l u l o s e column. C e l l - f r e e t r a n s l a t i o n o f Xenopus mRNA. Messenger RNA f r a c t i o n s were t r a n s l a t e d i n a wheat germ 14 c e l l - f r e e p r o t e i n - s y n t h e s i z i n g s y s t e m u s i n g [ C ] - l e u c i n e as t h e r a d i o a c t i v e p r o t e i n p r e c u r s o r . I n c o r p o r a t i o n o f t h i s amino a c i d i n t o p r o t e i n s was measured by p r e c i p i t a t i o n o f t h e c e l l - f r e e e x t r a c t s w i t h 20% c o l d TCA o r w i t h a n t i b o d y a g a i n s t m y o s i n as d e s c r i b e d i n M a t e r i a l s and Methods. A f i n a l c o n c e n t r a t i o n o f about 3.5 mM magnesium i s optimum f o r t h e c e l l - f r e e t r a n s l a t i o n o f a v a r i e t y o f mRNAs ( E f r o n and M a r c u s , 1973; D a v i e s and K a e s b e r g , 1974; R o b e r t s and P a t e r s o n , 1973). T a b l e 6 shows 2+ t h a t i n t h e p r e s e n c e o f 100 mM KOAc, t h e Mg optimum f o r s t a g e 20 + 2+ p o l y ( A ) RNA i s 3.0 mM. T a b l e 7 i n d i c a t e s t h a t a t 3.0 mM Mg , h i g h c o u n t s i n i m m u n o p r e c i p i t a t e s a r e o b t a i n e d when t h e K + c o n c e n t r a t i o n i s 156 mM. A l t h o u g h a t t h i s K c o n c e n t r a t i o n t h e t o t a l c e l l - f r e e i n c o r p o r a t i o n was f o u n d t o be l e s s t h a t t h a t a t optimum K + c o n c e n t r a t i o n , f o r t h e wheat germ s y s t e m more r a d i o a c t i v e c o u n t s were i m m u n o p r e c i p i t a t e d by a n t i b o d y a t 156 mM KOAc. T h i s r e s u l t i m p l i e s t h a t e i t h e r 156 mM K i s optimum f o r m y o s i n mRNA o r t h a t t h e f i d e l i t y o f t r a n s l a t i o n o f m y o s i n mRNA i s i m p r o v e d a t t h i s c o n c e n t r a t i o n o f p o t a s s i u m . T a b l e 8 shows t h e dependence o f p r o t e i n s y n t h e s i s on t h e amount o f mRNA p r e s e n t . P r o t e i n s y n t h e s i s i s s e e n t o be l i n e a r l y dependent on 76 t h e amount o f added mRNA up t o 10.0 pg p e r i n c u b a t e . Above t h i s mRNA 14 c o n c e n t r a t i o n , i n c o r p o r a t i o n o f t h e [ C ] - l a b e l r e a c h e s a p l a t e a u . T a b l e 9 shows a t i m e - c o u r s e s t u d y o f t h e c e l l - f r e e r e a c t i o n w i t h p o l y ( A ) + mRNA. I t ap p e a r s t h a t a f t e r 90 m i n u t e s o f i n c o r p o r a t i o n t h e s y n t h e t i c r e a c t i o n r e a c h e s a p l a t e a u . F o r some RNAs, a d d i t i o n o f s p e r m i n e t o t h e wheat germ c e l l - f r e e s y s t e m i n c r e a s e s t h e amount o f i s o t o p e i n c o r p o r a t i o n ( A t k i n s e t a l . , 1 9 7 5 ; R o b e r t s e t a l . , 1975; H u n t e r elt a l . , 1 9 7 7 ) . I n t h i s s t u d y , a d d i t i o n o f 40 mM s p e r m i n e had an i n h i b i t o r y e f f e c t on t h e t r a n s l a t i o n o f p o l y ( A ) + and p o l y ( A ) RNA f r o m s t a g e 20 embryos ( i n c o r p o r a t i o n was r e d u c e d about t w o - f o l d ) . A s i m i l a r e f f e c t has been r e p o r t e d f o r s i l k moth c h o r i o n mRNA t r a n s l a t i o n i n t h e wheat germ s y s t e m ( E f s t r a t i a d i s and K a f a t o s , 1976). T h i s r e s u l t may be e x p l a i n e d i n s e v e r a l ways: 1) C e r t a i n e u k a r y o t i c mRNAs may be e f f i c i e n t l y t r a n s l a t e d w i t h o u t p o l y a m i n e s ; 2) There may be a s u f f i c i e n t amount o f p o l y a m i n e s i n t h e wheat germ used i n t h i s s t u d y w h i c h were n o t removed by g e l f i l t r a t i o n (by v i r t u e o f a t t a c h m e n t o f r i b o s o m e s o r some o t h e r c e l l u l a r f a c t o r s ) ; 3) The h i g h magnesium and p o t a s s i u m c o n c e n t r a t i o n used i n t h i s s t u d y may r e p l a c e t h e n e c e s s i t y f o r t h e use o f p o l y a m i n e s ; and 4) The p o l y a m i n e u s e d i n t h i s s t u d y may have been degraded d u r i n g s t o r a g e . C e l l - f r e e t r a n s l a t i o n a n a l y s i s o f appearance o f my o s i n mRNA d u r i n g  Xenopus e m b r y o g e n e s i s . Messenger RNA i s o l a t e d f r o m v a r i o u s s t a g e s o f Xenopus em b r y o g e n e s i s was t r a n s l a t e d and a s s a y e d f o r m y o s i n s y n t h e s i s by i m m u n o p r e c i p i t a t i o n 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 d e n t i f i c a t i o n o f the. heavy c h a i n o f s k e l e t a l m y o s i n as one o f t h e 77 p r o d u c t s s y n t h e s i z e d i n v i t r o under t h e i n f l u e n c e o f added mRNA was a c h i e v e d by i m m u n o p r e c i p i t a t i o n w i t h a n t i - m y o s i n a n t i b o d y shown t o be s p e c i f i c i n s e c t i o n A o f t h e R e s u l t s . F u r t h e r m o r e , t h e p r o t e i n s y n t h e s i z e d i n v i t r o was d i g e s t e d w i t h t r y p s i n and t h e l a b e l l e d p e p t i d e s were shown t o c o - m i g r a t e w i t h heavy c h a i n m y o s i n p e p t i d e s . U s i n g t h e a n t i - m y o s i n a n t i b o d y we c o u l d show ( T a b l e 10) t h a t m y o s i n mRNA a p p e a r s a t t h e l a t e n e u r a l - f o l d s t a g e ( s t a g e s 16/17) o f Xenopus development. T h i s i s a p p r o x i m a t e l y 3 h o u r s b e f o r e t h e appe a r a n c e o f n a s c e n t m y o s i n on polysomes a t s t a g e 20, as shown i n s e c t i o n A o f t h e R e s u l t s . T a b l e 10 shows t h a t between 0.3-2.8% o f t h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o p o l y p e p t i d e s i n t h e c e l l - f r e e s y s t e m i n v i t r o under t h e i n f l u e n c e o f s t a g e 16/17 RNA i s f o u n d i n i m m u n o p r e c i p i t a t e s . The t o t a l i n c o r p o r a t i o n i n t o s y n t h e s i z e d p r o t e i n v a r i e d f r o m one RNA p r e p a r a t i o n t o a n o t h e r as can be see n i n T a b l e 10 ( s t a g e 16/17). T h i s r e s u l t may be due t o t h e f a c t t h a t i n c o r p o r a t i o n i s n o t s t r i c t l y q u a n t i t a t i v e i n t h e wheat germ s y s t e m o r t h a t t h e mRNA p r e p a r a t i o n s d i f f e r f r o m b a t c h t o b a t c h o f embryos. T h i s s e c o n d p o s s i b i l i t y may r e s u l t f r o m a number o f f a c t o r s : 1) D i f f e r e n t b a t c h e s o f embryos a r e n o t o f e x a c t l y t h e same s i z e ( l i g h t m i c r o s c o p y o b s e r v a t i o n s ) ; 2) There was m i n o r i n a c c u r a c y i n embryo s t a g i n g ( t o be d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n ) ; and 3) P o s s i b l y b i o c h e m i c a l changes do n o t n e c e s s a r i l y c o r r e l a t e e x a c t l y w i t h m o r p h o l o g i c a l changes ( M a c k l i n and W o j t k o w s k i , 1973). However, i t can be see n i n T a b l e 10 t h a t a t s t a g e 16/17, t h e number o f c o u n t s i n t h e i m m u n o p r e c i p i t a t e s i s a b out t h e same f o r a l l o f t h e d i f f e r e n t mRNA p r e p a r a t i o n s . T h e r e f o r e , i t seems more l i k e l y t h a t s u b t l e d i f f e r e n c e s i n t h e wheat germ i n c u b a t i o n m i x t u r e s may be r e s p o n s i b l e f o r d i f f e r e n c e s i n t o t a l i n c o r p o r a t i o n . 78 I t m i g h t b e a r g u e d t h a t m y o s i n mRNA f r o m s t a g e 12 e m b r y o s i s i n a c t i v a t e d b y s ome f a c t o r ( s ) w h i c h c a n n o t b e r e m o v e d b y t h e f r a c t i o n a t i n g p r o c e d u r e s u s e d i n t h i s s t u d y . T o t e s t i f t h i s f a c t o r ( s ) i s c a p a b l e o f i n h i b i t i n g s t a g e 2 0 m y o s i n mRNA t h e f o l l o w i n g m i x i n g e x p e r i m e n t w a s p e r f o r m e d . A b a t c h o f 1 5 0 0 s t a g e 12 e m b r y o s a n d 1 5 0 0 s t a g e 2 0 e m b r y o s w e r e m i x e d a n d h o m o g e n i z e d . P o l y ( A ) - t e r m i n a t e d RNA w a s i s o l a t e d a s p r e v i o u s l y d e s c r i b e d . T a b l e 11 s h o w s t h a t p o l y ( A ) + mRNA o b t a i n e d f r o m t h e s e m i x e d s t a g e 12 a n d s t a g e 2 0 e m b r y o s i s t r a n s l a t e d i n t o i m m u n o p r e c i p i t a b l e m y o s i n . T h e r e f o r e , s t a g e 12 c e l l h o m o g e n a t e s c a n n o t d e g r a d e s t a g e 2 0 mRNA a n d d o n o t c o n t a i n a n e x c e s s o f t h e p u t a t i v e i n h i b i t o r s , w h i c h c o u l d c o m p l e t e l y p r e v e n t t h e t r a n s l a t i o n o f s t a g e 2 0 m y o s i n mRNA i n t h e w h e a t g e r m c e l l - f r e e s y s t e m . I f i t i s a s s u m e d t h a t i n t h e m i x i n g e x p e r i m e n t t h e r e i s a p p r o x i m a t e l y 5 p g o f p o l y ( A ) + f r o m s t a g e 2 0 e m b r y o s i n e a c h w h e a t g e r m i n c u b a t i o n m i x t u r e , t h e n t h e s y n t h e s i s o f m y o s i n r e p r e s e n t s a b o u t 7% o f t o t a l i n c o r p o r a t i o n ( s e e l e g e n d t o T a b l e 1 1 ) . T h i s c a l c u l a t i o n s u g g e s t s t h a t t h e r e i s n o i n h i b i t i o n o f t r a n s l a t i o n o f s t a g e 20 m y o s i n mRNA a n d a l s o , n o a c t i v a t i o n o f u n t r a n s l a t a b l e s t a g e 12 m y o s i n mRNA b y s ome f o r m o f a c t i v a t o r f r o m s t a g e 2 0 c e l l s . I f t h e r e i s a n y u n t r a n s l a t a b l e m y o s i n mRNA i n s t a g e 12 e m b r y o s , i t c a n n o t b e d e t e c t e d b y o u r m e t h o d s b e c a u s e o f t o o l o w c o n c e n t r a t i o n s . O t h e r m o d e l s w h i c h m i g h t e x p l a i n t h e s e r e s u l t s w i l l b e p r e s e n t e d i n t h e d i s c u s s i o n . T h e i m m u n o p r e c i p i t a t e s o f t h e p r o d u c t s s y n t h e s i z e d i n v i t r o u n d e r t h e i n f l u e n c e o f s t a g e 2 0 mRNA w e r e m i x e d w i t h c a r r i e r a u t h e n t i c m y o s i n a n d d i s p l a y e d b y S D S - a c r y l a m i d e e l e c t r o p h o r e s i s ( g e l s n o t s h o w n ) . T h e C o o m a s s i e B l u e b a n d s c o - m i g r a t i n g w i t h a u t h e n t i c h e a v y c h a i n o f s k e l e t a l m u s c l e m y o s i n w e r e c u t o u t a n d t h e p r o t e i n s e l u t e d a n d c o u n t e d 79 14 f o r r a d i o a c t i v i t y . B e t w e e n 90-95% o f [ C ] - l e u c i n e r a d i o a c t i v i t y w a s f o u n d i n t h e " c a r r i e r " m y o s i n b a n d s t h a t w e r e c u t o u t f r o m t h e g e l s . T h e r a d i o a c t i v i t y t h a t d i d n o t m i g r a t e w i t h t h e h e a v y c h a i n o f m y o s i n may b e d u e t o i n c o m p l e t e c h a i n s o f m y o s i n . A s d i s c u s s e d i n R e s u l t s A , n a s c e n t p e p t i d e c h a i n s o n p o l y s o m e s a r e e f f i c i e n t l y i s o l a t e d b y i m m u n o p r e c i p i t a t i o n . T h e f a c t t h a t a b o u t 90-95% o f t h e a n t i b o d y - p r e c i p i t a t e d c e l l - f r e e p r o d u c t c o n s i s t e d o f c o m p l e t e d m y o s i n c h a i n s c a n b e e x p l a i n e d i n s e v e r a l w a y s . H i g h c o n c e n t r a t i o n s o f m a g n e s i u m (0.1 M) , g u a n i d i n i u m c h l o r i d e (4M) a n d h e p a r i n (1 m g / m l ) a r e k n o w n t o i n h i b i t r i b o n u c l e a s e s ( C o x , 1967; R h o a d s e t a l . , 1973; P a l m i t e r , 1973). T h e r e f o r e , i t i s l i k e l y t h a t m y o s i n mRNA w a s n o t d e g r a d e d s i g n i f i c a n t l y d u r i n g i s o l a t i o n d u e t o t h e p r e s e n c e o f t h e s e r e a g e n t s . F o r t h i s r e a s o n t h e a m o u n t o f l o w m o l e c u l a r w e i g h t p e p t i d e s d u e t o t h e t r a n s l a t i o n o f d e g r a d e d m y o s i n mRNA w o u l d b e g r e a t l y r e d u c e d . A l t h o u g h t h e w h e a t g e r m s y s t e m c o n t a i n s e n d o g e n o u s R N a s e s , t h e h i g h p o t a s s i u m a c e t a t e c o n c e n t r a t i o n (156 mM) u s e d i n t h e t r a n s l a t i o n s t a b i l i z e s p o l y s o m e s a n d i t i s p o s s i b l e t h a t t h e r a t e o f n u c l e o l y t i c a t t a c k o n p o l y s o m e - a s s o c i a t e d mRNA i s s l o w e r t h a n o n u n b o u n d mRNA ( H u n t e r e t a l . , 1977). A l t h o u g h t h e r e a s o n s o u t l i n e d a b o v e d o n o t p r e c l u d e t h e p o s s i b i l i t y t h a t t h e m y o s i n a n t i b o d y c a n p r e c i p i t a t e p r e m a t u r e l y t e r m i n a t e d p o l y p e p t i d e s o r i n c o m p l e t e p o l y p e p t i d e c h a i n s s t i l l a t t a c h e d t o p o l y s o m e s ( D a r n b r o u g h a n d F o r d , 1 9 7 6 ) , t h i s i s u n l i k e l y b e c a u s e t h e w h e a t g e r m s y s t e m i n t h i s s t u d y w a s o p t i m i z e d f o r t h e s y n t h e s i s o f h i g h m o l e c u l a r w e i g h t p r o t e i n s ( s p e c i f i c a l l y m y o s i n ) . A s d e s c r i b e d i n a p r e v i o u s s e c t i o n , t h e o p t i m a l c o n d i t i o n s f o r t h e s y n t h e s i s o f m y o s i n w e r e n o t c o i n c i d e n t w i t h t h e o p t i m a l c o n d i t i o n s f o r f o r m a t i o n o f 80 a c i d - p r e c i p i t a b l e m a t e r i a l . A f t e r t h i s s t u d y was c o m p l e t e d , P a t r i n o u - G e o r g o u l a s and John (1977) compared m y o s i n mRNA t r a n s l a t i o n a t 2+ 96 mM K C l and 160 mM K C l (2.0 mM Mg ) i n t h e wheat germ s y s t e m . They fo u n d t h a t a p p r o x i m a t e l y 12 t i m e s more myosin was s y n t h e s i z e d i n t h e p r e s e n c e o f 160 mM K C l t h a n i n t h e p r e s e n c e o f 96 mM K C l . On t h e o t h e r hand, t h e y f o u n d t h a t i n c o r p o r a t i o n i n t o t o t a l p r o t e i n , d e t e r m i n e d as a c i d - i n s o l u b l e r a d i o a c t i v i t y , was g r e a t l y r e d u c e d i n t h e p r e s e n c e o f 160 mM K C l . F u r t h e r m o r e , t h e s e w o r k e r s removed q u a n t i t a t i v e l y t h e low m o l e c u l a r w e i g h t r a d i o a c t i v e p e p t i d e s , s y n t h e s i z e d i n t h e p r e s e n c e o f 160 mM K C l , by p r e c i p i t a t i n g m y o s i n a t low i o n i c s t r e n g t h . The low m o l e c u l a r w e i g h t p e p t i d e s were p r o b a b l y m o s t l y composed of. non-myosin p r o t e i n s s i n c e t h e y a p p e a r e d t o r u n on S D S - a c r y l a m i d e g e l s w i t h a m o l e c u l a r w e i g h t o f 30,000 o r l e s s . I f t h e r e were any i n c o m p l e t e m y o s i n c h a i n s p r e s e n t , t h e y w o u l d most c e r t a i n l y c o n s i s t o f heavy meromyosin c h a i n s (HMM) (MW -110,000 o r l e s s ) w h i c h a r e s y n t h e s i z e d f r o m t h e 5' end o f th e mRNA m o l e c u l e and a r e s o l u b l e a t low i o n i c s t r e n g t h ( S a r k a r , 1976). I n t h e p r e s e n t s t u d y , m y o s i n was p r e c i p i t a t e d f r o m wheat germ l y s a t e s a t low i o n i c s t r e n g t h (see M a t e r i a l s and Methods; S a r k a r , 1976). T h e r e f o r e , t h e f a c t t h a t i n t h i s s t u d y t h e m y o s i n a n t i b o d y p r e c i p i t a t e d p r o t e i n w i t h a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 210,000 d a l t o n s i s due t o t h e f a c t t h a t s t r i n g e n t measures were t a k e n t o e l i m i n a t e n a s c e n t m y o s i n c h a i n s and p r o t e o l y t i c c l e a v a g e o f co m p l e t e m y o s i n m o l e c u l e s . The wheat germ s y s t e m was o p t i m i z e d f o r magnesium and p o t a s s i u m c o n c e n t r a t i o n s o p t i m a l f o r m y o s i n mRNA t r a n s l a t i o n . T h i s p r o b a b l y r e s u l t e d i n a b e t t e r f i d e l i t y o f t r a n s l a t i o n o f m y o s i n mRNA. P r e c a u t i o n s were t a k e n t o r e d u c e d e g r a d a t i o n o f m y o s i n mRNA by i n c l u d i n g i n h i b i t o r s 81 o f r i b o n u c l e a s e s i n t h e i s o l a t i o n p r o c e d u r e . T h e i n v i t r o p r o d u c t w a s p r e c i p i t a t e d a t l o w i o n i c s t r e n g t h p r i o r t o a n t i b o d y p r e c i p i t a t i o n . T h i s r e s u l t e d i n e n r i c h m e n t o f t h e p r e p a r a t i o n w i t h c o m p l e t e d m y o s i n m o l e c u l e s . A l s o , f o r a n t i b o d y p r e c i p i t a t i o n ( s e e p . 7 0 ) a n a t t e m p t w a s made t o i n h i b i t p r o t e o l y s i s o f m y o s i n s y n t h e s i z e d i n t h e c e l l - f r e e s y s t e m b y t r e a t i n g t h e w h e a t g e r m l y s a t e s w i t h T r i t o n X - 1 0 0 , P M S F a n d s o d i u m s u l p h i t e . T h e n e u t r a l l y c h a r g e d d e t e r g e n t T r i t o n X - 1 0 0 p a r t i a l l y d e n a t u r e s s ome p r o t e i n s a n d c o n s e q u e n t l y , may i n h i b i t p r o t e o l y t i c a c t i v i t y . I t i s c o m m o n l y u s e d i n a n t i g e n - a n t i b o d y r e a c t i o n s ( P a l a c i o s e t a l . , 1 9 7 2 ; S t r o h m a n e t a l . , 1 9 7 7 ) . T h e p r o t e a s e i n h i b i t o r PMSF s p e c i f i c a l l y i n h i b i t s s e r i n e p r o t e a s e s ( G l a z e r , 1 9 7 5 ) . I t i s n o t k n o w n i f s e r i n e - l i k e p r o t e a s e s a r e p r e s e n t i n t h e w h e a t g e r m c e l l - f r e e s y s t e m . I n s ome p r e p a r a t i o n s , s o d i u m s u l f i t e ( 2 . 5 mM) w a s a l s o u s e d a n d d i d n o t a f f e c t t h e a m o u n t s o f m y o s i n p r e c i p i t a t e d b y a n t i b o d y . S o d i u m s u l f i t e s u l f o n a t e s t h e d i s u l f i d e b o n d s o f s ome p r o t e i n s a n d i n h i b i t s t h e i r e n z y m a t i c a c t i v i t y ( G l a z e r , 1 9 7 5 ) . No e x p e r i m e n t w a s c a r r i e d o u t t o d e t e r m i n e i f t h e p r e s e n c e o f PMSF a n d / o r s o d i u m s u l f i t e i n t h e w h e a t g e r m s y s t e m d u r i n g i n c u b a t i o n w o u l d i n c r e a s e t h e y i e l d o f m y o s i n s y n t h e s i s . F i n a l l y , s i n c e t h e i n v i t r o r e a c t i o n w a s i n c u b a t e d f o r a b o u t 90 m i n u t e s , i t i s p o s s i b l e t h a t a l a r g e p e r c e n t a g e o f n a s c e n t p e p t i d e c h a i n s w e r e a l l o w e d t o p r o c e e d t o c o m p l e t i o n . A n a l y s i s o f t r y p t i c d i g e s t i o n f r a g m e n t s o f r a d i o a c t i v e m y o s i n , p r e c i p i t a t e d b y a n t i b o d y , i n t h e p r e s e n c e o f c o l d c a r r i e r m y o s i n , w a s c o n s i s t e n t w i t h t h e p r e s e n c e o f r a d i o a c t i v e m y o s i n h e a v y c h a i n ( F i g . 1 3 ) . A p p r o x i m a t e l y 9 p r o m i n e n t r a d i o a c t i v e p e a k s c o i n c i d e w i t h v i s i b l e f r a g m e n t s d e r i v e d f r o m t h e c a r r i e r m y o s i n h e a v y c h a i n . I f t h e a n t i b o d y - p r e c i p i t a t e d p r o t e i n h a d c o n t a i n e d p e p t i d e s o t h e r t h a n s k e l e t a l h e a v y c h a i n m y o s i n , i t w o u l d b e e x p e c t e d t h a t s ome r a d i o a c t i v e f r a g m e n t s 81a would electrophorese independently of the v i s i b l e fragments of the p u r i f i e d c a r r i e r myosin. The r e s u l t s reported here support the conclusion that tra n s l a t a b l e myosin mRNA f i r s t makes i t s appearance during Xenopus development at the l a t e neurula stage (stage 16/17). 82 T A B L E 3 Y i e l d s o f RNA p r e p a r e d f r o m X e n o p u s e m b r y o s b y d i f f e r e n t i s o l a t i o n p r o c e d u r e s . * 1 S t a g e b I s o l a t i o n p r o c e d u r e N u m b e r o f e m b r y o s M g 2 + (mg) G u C l (mg) 12 1 0 . 8 1 0 . 4 3 0 0 0 1 1 . 4 1 0 . 3 3 0 0 0 1 0 . 3 1 0 . 7 3 0 0 0 1 6 / 1 7 1 1 . 2 1 0 . 8 3 0 0 0 1 1 . 0 1 0 . 3 3 0 0 0 1 0 . 7 1 0 . 5 3 0 0 0 2 0 1 0 . 9 1 1 . 2 3 0 0 0 1 1 . 1 1 0 . 3 3 0 0 0 1 0 . 4 1 0 . 5 3 0 0 0 A s s u m p t i o n u s e d i n c a l c u l a t i o n s : 2 0 ^2e0 u n i t s o f R N A i n a b s e n c e o f Mg = 1 m g . b N i e u w k o o p a n d F a b e r ( 1 9 6 7 ) ; s t a g e s o f d e v e l o p m e n t . c 2+ RNA f r o m X e n o p u s e m b r y o s w a s p r e p a r e d u s i n g t h e Mg o r g u a n i d i n i u m c h l o r i d e p r o c e d u r e a n d e x t r a c t e d w i t h p h e n o l : c h l o r o f o r m . 83 T A B L E 4 Q u a n t i t i e s o f t o t a l a n d p o l y s o m a l mRNA i n X e n o p u s e m b r y o s a t s t a g e s 1 2 , 1 6 / 1 7 a n d 2 0 . a S t a g e s DNA c o n t e n g p e r e m b r y o ( n g ) N o . o f c e l l s T o t a l R N A d p e r e m b r y o ( n g ) P o l y s o m a l mRNA p e r e m b r y o ( n g ) 12 3 0 0 4 . 8 x 1 0 4 4 0 0 0 26 1 6 / 1 7 4 0 0 6 . 4 x 1 0 4 4 0 0 0 17 2 0 5 0 0 7 . 9 x 1 0 4 4 0 0 0 25 " T h e s e v a l u e s a r e a p p r o x i m a t e . ^ V a l u e s f o r DNA c o n t e n t w e r e t a k e n f r o m D a v i d ( 1 9 6 5 ) . V a l u e s f o r N o . o f c e l l s w e r e c a l c u l a t e d o n t h e b a s i s o f 6 . 3 p g o f DNA p e r d i p l o i d c e l l ( D a v i d , 1 9 6 5 ) . ^ T a k e n f r o m B r o w n a n d L i t t n a ( 1 9 6 4 ) . C a l c u l a t e d o n t h e b a s i s t h a t 9 0% o f t h e t o t a l RNA i s r i b o s o m a l ( G a l a u , 1 9 7 4 ) , 1 6 . 2 % o f t h e r i b o s o m e s a r e i n p o l y s o m e s a t s t a g e 1 2 , 1 0 . 7 % o f t h e r i b o s o m e s a r e i n p o l y s o m e s a t s t a g e 1 6 / 1 7 a n d 1 5 . 6 % o f t h e r i b o s o m e s a r e i n p o l y s o m e s a t s t a g e 2 0 ( W o o d l a n d , 1 9 7 4 ) . F i n a l l y , 4 . 5 % o f p o l y s o m a l RNA i s mRNA ( G a l a u , 1 9 7 4 ) . 84 TABLE 5 Purification of poly(A) RNA from Xenopus embryos by binding to oligo(dT)-cellulose. Stage Purification step Total RNA applied to oligo(dT)-cellulose Amount (pg) 12 Experiment I: 2 n oligo(dT)-cellulose Flow-through Bound 10,800 9,234 56.6. Exgeriment II: 2 oligo(dT)-cellulose Flow-through Bound 11,400 10,032 57.4 16/17 Exgeriment I: 2 oligo(dT)-cellulose Flow-through Bound 11,200 9,576 54.4 Exgeriment II: 2 oligo(dT)-cellulose Flow-through Bound 10,700 9,341 56.0 20 Exgeriment I: . 2 oligo(dT)-cellulose Flow-through Bound 10,900 8,829 58.4 Exgeriment II: 2 oligo(dT)-cellulose Flow-through Bound 11,100 8,891 58.8 85 TABLE 6 E f f e c t o f magnesium on t o t a l p r o t e i n s y n t h e s i s . 1 Magnesium c o n c e n t r a t i o n (mM) b T o t a l cpm 2.0 600 + 48 2.5 23,528 + 1,011 .3.0 53,939 + 3,236 3.5 49,018 + 1,715 4.0 35,222 + 2,465 4.5 30,000 + 1,200 °A11 i n c u b a t i o n s ^ e r e c a r r i e d o u t f o r 90 min a t 30°C w i t h 10 pg s t a g e 20 p o l y ( A ) RNA p e r 50 p i of a s s a y . b A t a K + c o n c e n t r a t i o n o f 100 mM w i t h a l l o t h e r components as d e s c r i b e d i n M a t e r i a l s and Methods. Background r a d i o a c t i v i t y (no RNA c o n t r o l s ) was about 370 cpm. T h i s v a l u e has been s u b t r a c t e d . 86 TABLE 7 E f f e c t o f p o t a s s i u m a c e t a t e on t o t a l p r o t e i n s y n t h e s i s and m y o s i n s y n t h e s i s . 3 P o t a s s i u m c o n c e n t r a t i o n (mM) b T o t a l cpm° Cpm i n i m m u n o p r e c i p i t a t e s 50 23,528 + 1,411 0 80 47,056 + 2,729 843 + 93 100 58,821 + 3,529 900 + 90 120 48,000 156 27,647 + 1,990 2,109 + 147 160 20,587 + 905 1,600 + 133 170 14,705 + 1,029 767 + 75 " A l l i n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n T a b l e 6. b 2"t A t a Mg c o n c e n t r a t i o n o f 3.0 mM. Ba c k g r o u n d r a d i o a c t i v i t y has been s u b t r a c t e d ( s e e T a b l e 6 ) . ^ I m m u n o p r e c i p i t a t e s were p r e p a r 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. The b a c k g r o u n d r a d i o a c t i v i t y (non-immune serum added t o wheat germ i n c u b a t e s ) amounted t o about 65 cpm and was s u b t r a c t e d . 87 TABLE 8 Dependence o f t o t a l p r o t e i n s y n t h e s i s on added s t a g e 20 + a p o l y ( A ) Xenopus RNA. RNA T o t a l cpm^ (ug) 1.3 4,300 + 430 2.5 9,462 + 870 5.0 17,844 + 1,070 7.5 25,200 + 1,260 10.0 26,910 + 1,345 A l l i n c u b a t i o n s were c a r r i e d o u t f o r 90 min a t 30°C i n a t o t a l volume o f 50 u l . A l l components were a t t h e c o n c e n t r a t i o n s d e s c r i b e d i n M a t e r i a l s and Methods. Background r a d i o a c t i v i t y was s u b t r a c t e d . 88 T A B L E 9 T i m e c o u r s e o f t o t a l p r o t e i n s y n t h e s i s ancL m y o s i n s y n t h e s i s i n t h e w h e a t g e r m p r o t e i n - s y n t h e s i z i n g s y s t e m . 3 T i m e T o t a l cpm' Cpm i n ( m i n ) i m m u n o p r e c i p i t a t e s 0 0 0 5 1 , 9 4 2 + 1 6 1 1 0 3 , 9 5 0 + 3 5 5 0 1 5 6 . 2 0 0 + 5 5 8 0 2 0 7 , 7 6 8 + 5 6 7 3 0 1 2 , 7 6 0 1 , 0 6 0 + 1 1 7 4 0 1 5 , 4 7 5 + 1 , 2 3 8 5 0 1 8 , 0 6 1 + 9 5 7 60 2 5 , 8 0 0 + 1 , 1 3 5 2 , 4 2 5 + 1 9 4 7 0 2 6 , 2 1 0 + 1 , 3 1 0 80 2 7 , 0 0 0 + 1 , 6 2 0 9 0 2 9 , 4 1 1 + 1 , 7 6 5 2 , 6 4 6 + 2 1 8 3 O A l l i n c u b a t i o n s w e r e c a r r i e d o u t a t 30 £ w i t h 1 0 p g of s t a g e 2 0 p o l y _ ( A ) RNA p e r 5 0 p l o f a s s a y . T h e K c o n c e n t r a t i o n w a s 1 5 6 mM, t h e Mg c o n c e n t r a t i o n w a s 3 . 0 mM. A l l o t h e r c o m p o n e n t s a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . ^ R a d i o a c t i v i t y i n b a c k g r o u n d s a m p l e s ( n o RNA) w a s s u b t r a c t e d . Q R a d i o a c t i v i t y i n s a m p l e s p r e c i p i t a t e d w i t h n o n - i m m u n e s e r u m w a s s u b t r a c t e d . . 89 TABLE 10 I m m u n o p r e c i p i t a t i o n o f t h e c e l l - f r e e r e a c t i o n p r o d u c t w i t h a n t i - m y o s i n a n t i b o d y . 3 mRNA 14 I n c o r p o r a t i o n o f [ C ] - l e u c i n e i n t o 100 B/A s o u r c e T o t a l c e l l - f r e e p r o d u c t M y o s i n A B S t a g e 12b 1 9,183 0 0 2 10,664 0 0 3 10,207 0 0 S t a g e 16/17 1 148,404 510 0.3 2 157,277 503 0.3 3 24,607 670 2.7 4 23,661 661 2.8 S t a g e 20C 1 58,294 3,363 6 2 27,893 2,769 10 3 26,470 2,382 9 %heat germ c e l l - f r e e p r o t e i n s y n t h e s i s was c a r r i e d o ut as d e s c r i b e d i n M a t e r i a l s and Methods w i t h 10 pg p o l y ( A ) RNA f o r e a c h e x p e r i m e n t . B l a n k v a l u e s (no RNA added t o t h e wheat germ system) as w e l l as v a l u e s o b t a i n e d w i t h c o n t r o l serum were s u b t r a c t e d . "Each e x p e r i m e n t r e p r e s e n t s mRNA i s o l a t e d f r o m a d i f f e r e n t b a t c h o f embryos. Three o t h e r e x p e r i m e n t s were p e r f o r m e d u s i n g o n e - h a l f as much RNA (5 pg) as i n t h e p r e v i o u s e x p e r i m e n t s . T o t a l i n c o r p o r a t i o n was 6089 cpm and m y o s i n s y n t h e s i s was u n d e t e c t a b l e . °Each e x p e r i m e n t r e s p r e s e n t s mRNA i s o l a t e d f r o m a different b a t c h o f embryos. F o r o t h e r e x p e r i m e n t s u s i n g s t a g e 20 p o l y ( A ) RNA see T a b l e s 7, 8 and 9. 90 T A B L E 11 I m m u n o p r e c i p i t a t i o n o f t h e c e l l - f r e e r e a c t i o n p r o d u c t w i t h a n t i - m y o s i n a n t i b o d y d i r e c t e d b y p o l y ( A ) + a n d p o l y ( A ) RNA f r o m m i x e d s t a g e 12 a n d s t a g e 20 e m b r y o s . mRNA 14 I n c o r p o r a t i o n o f [ C ] - l e u c i n e i n t o 100 B/A s o u r c e T o t a l c e l l - f r e e p r o d u c t M y o s i n A B S t a g e 12 £ s t a g e 2Jj) p o l y ( A ) 1 21,601 1,166 5.4 2 20,000 1,120 5.6 S t a g e 12 + s t a g e 20 p o l y ( A ) 1 14,000 0 0 1500 s t a g e 20 a n d 1500 s t a g e 12 e m b r y o s w e r e + m i x e d a n d h o m o g e n i z e d a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . P o l y ( A ) a n d p o l y ( A ) RNA w e r e i s o l a t e d a n d t r a n s l a t e d i n t h e w h e a t g e r m s y s t e m . c + ' E a c h e x p e r i m e n t c o n t a i n e d 10 p g o f p o l y ( A ) RNA. 9 1 F i g u r e 1 3 . A n a l y s i s b y e l e c t r o p h o r e s i s o n a 1 0% S D S - a c r y l a m i d e g e l o f t r y p t i c f r a g m e n t s o f t r a n s l a t i o n p r o d u c t s p r e c i p i t a t e d w i t h a n t i - m y o s i n a n t i b o d y . R a d i o a c t i v e m y o s i n h e a v y c h a i n s i n t h e p r e s e n c e o f c o l d c a r r i e r - p u r i f i e d m y o s i n h e a v y c h a i n s w e r e o b t a i n e d b y i m m u n o -p r e c i p i t a t i o n a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h e m y o s i n h e a v y c h a i n s w e r e d i g e s t e d w i t h t r y p s i n a n d e l e c t r o p h o r e s e d o n 1 0 % S D S - a c r y l a m i d e g e l s . P h o t o g r a p h r e p r e s e n t s C o o m a s s i e B l u e s t a i n e d g e l . Top p r o f i l e r e p r e s e n t s d e n s i t o m e t r i c s c a n o f t h e p h o t o g r a p h o f t h e g e l . B o t t o m p r o f i l e r e p r e s e n t s r a d i o a c t i v e c o u n t s i n 2 mm s l i c e s o f t h e o r i g i n a l g e l . 2 5 0 H Distance Migrated (mm) 93 D I S C U S S I O N O u r e x p e r i m e n t s d e m o n s t r a t e t h a t m y o s i n mRNA i s s y n t h e s i z e d i n a t r a n s l a t a b l e f o r m a t a b o u t t h e t i m e t h e f i r s t s o m i t e s a r e s e g r e g a t e d i n X e n o p u s e m b r y o s . M y o s i n i s f i r s t s y n t h e s i z e d w h e n a b o u t 6 - 7 s o m i t e s h a v e s e g r e g a t e d i n t h e e m b r y o s . T h e t i m e l a g b e t w e e n m y o s i n mRNA s y n t h e s i s a n d m y o s i n a p p e a r a n c e i s a b o u t 1 1 / 2 h o u r s . T h e m y o s i n s y n t h e s i z e d i n X e n o p u s e m b r y o s c r o s s - r e a c t s w i t h a d u l t m y o s i n s . T h i s may m e a n t h a t e m b r y o n i c m y o s i n s h a r e s s i m i l a r a n t i g e n s w i t h a d u l t m y o s i n s o r t h a t b o t h e m b r y o n i c a n d a d u l t m y o s i n s a r e b e i n g s y n t h e s i z e d i n e a r l y d e v e l o p m e n t . We h a v e a t t e m p t e d i n t h i s s t u d y t o a n s w e r t h e q u e s t i o n o f w h e n m y o s i n mRNA i s f i r s t p r o d u c e d i n d e v e l o p m e n t . A n a n s w e r t o t h i s q u e s t i o n t h e n r a i s e s t h e m o s t i m p o r t a n t i s s u e : n a m e l y , w h a t e v e n t s p r o d u c e t h e g e n e t i c m a c h i n e r y t h a t a l l o w s o n l y a p a r t i c u l a r c e l l t o p r o d u c e m y o s i n mRNA a n d m y o s i n p r o t e i n ( H o l t z e r a n d S a n g e r , 1 9 7 2 ) ? I n w h a t f o l l o w s we d i s c u s s t h e r e l i a b i l i t y o f t h e t e c h n i q u e s we u s e d t o a n s w e r t h e f i r s t q u e s t i o n . A n t i g e n a n d a n t i b o d y c h a r a c t e r i z a t i o n s . A n a n t i b o d y a g a i n s t t h e h e a v y c h a i n o f s k e l e t a l m u s c l e m y o s i n w a s p r e p a r e d f r o m a d u l t X e n o p u s m u s c l e s t o p r o b e t h e d e v e l o p m e n t a l a p p e a r a n c e o f m y o s i n mRNA a n d m y o s i n i t s e l f . T h e m u s c l e , t o b e u s e d a s i m m u n o g e n , w a s d i s s e c t e d f r o m t h e b a c k a n d s u p e r f i c i a l t h i g h m u s c l e s . T h e m u s c l e s o f t h e b a c k i n a m p h i b i a n s d e v e l o p f r o m t h e m y o t o m e s 94 ( B a l i n s k y , 1 9 7 5 ) . The l i m b m u s c u l a t u r e o f a m p h i b i a n s p r o b a b l y d e v e l o p s f r o m mesodermal c e l l s t h a t o r i g i n a t e i n t h e myotomes ( B a l i n s k y , 1975). T h e r e f o r e , t h e m y o s i n m o l e c u l e i s o l a t e d f r o m a d u l t t i s s u e shoud have some a n t i g e n i c d e t e r m i n a n t s t h a t a r e s i m i l a r t o e m b r y o n i c m y o s i n . F u r t h e r m o r e , t h e immunogen used i n t h i s s t u d y was i s o l a t e d f r o m d e n a t u r i n g S D S - a c r y l a m i d e g e l s and c o n s e q u e n t l y most i f n o t a l l o f i t s a n t i g e n i c d e t e r m i n a n t s s h o u l d have been exposed. We have shown t h a t t h e a n t i b o d y g e n e r a t e d under t h e s e c o n d i t i o n s r e a c t s w i t h a d u l t and s t a g e 20 e m b r y o n i c m y o s i n . T h i s does n o t i m p l y t h a t a d u l t and e m b r y o n i c m y o s i n a r e i d e n t i c a l b u t t h a t t h e y s h a r e a t l e a s t a p a r t i a l s e t o f a n t i g e n i c d e t e r m i n a n t s . S r e t e r et_ a l . (1972) showed t h a t m y o s i n i s o l a t e d f r o m young c h i c k myotubes has t h e same ATPase a c t i v i t y as a d u l t f a s t m u s c l e m y o s i n (FM). These f a c t s and o t h e r s i m p l i e d t h a t e m b r y o n i c m y o s i n (EM) i s s i m i l a r t o a d u l t FM. T r a y e r and P e r r y (1966) on t h e o t h e r hand, found t h a t e m b r y o n i c a v i a n and mammalian m y o s i n r e s e m b l e s l o w a d u l t m u s c l e m y o s i n (SM) as j u d g e d by i m m u n o l o g i c a l t e s t s , ATPase a c t i v i t y and t h e l a c k o f 3 - m e t h y l h i s t i d i n e . S u b s e q u e n t l y , S r e t e r et_ a l . (1975) found t h a t t h e APTase a c t i v i t y o f m y o s i n p r e p a r e d f r o m m u s c l e s o f 3-4 week r a b b i t embryos i s s i m i l a r t o t h e a c t i v i t y o f FM. They a l s o f o u n d some s i m i l a r i t y between t h e t r y p t i c f r a g m e n t s g e n e r a t e d f r o m EM, SM and FM. However, c o n t r a c t i l e p r o t e i n s have been shown t o e x i s t i n non-myogenic c e l l s i n c l u d i n g c l e a v i n g s e a u r c h i n embryos ( S c h r o e d e r , 1973; f o r a r e v i e w see P o l l a r d and W e i h i n g , 1974). I t t h u s appears t h a t Xenopus non-muscle m y o s i n and m u s c l e m y o s i n a r e t h e p r o d u c t s o f d i f f e r e n t genes and do n o t s h a r e many common a n t i g e n i c d e t e r m i n a n t s . I n agreement w i t h t h i s c o n c l u s i o n , H o l t z e r and c o - w o r k e r s ( C h i e t a l . , 1975) have 95 u s e d i m m u n o d i f f u s i o n t o p r e c i p i t a t e m y o s i n e x t r a c t e d f r o m p r e s u m p t i v e c h i c k m y o b l a s t s , f i b r o b l a s t s , n e r v e c e l l s , s m o o t h m u s c l e c e l l s a n d g u t e p i t h e l i a l c e l l s . T h e s e w o r k e r s a l s o f o u n d t h a t o n l y m y o s i n f r o m 5 m y o b l a s t s , m y o t u b e s a n d m u s c l e c e l l s w e r e p r e c i p i t a t e d b y a n t i b o d y A a g a i n s t s k e l e t a l m y o s i n . S i m i l a r l y , P o l l a r d e t a l . ( 1 9 7 6 ) h a v e d e m o n s t r a t e d t h a t a n t i - h u m a n s k e l e t a l m y o s i n a n t i b o d i e s d o n o t p r e c i p i t a t e m y o s i n s f r o m h u m a n u t e r i o r p l a t e l e t s . B u r r i d g e a n d B r a y ( 1 9 7 5 ) h a v e a n a l y z e d n o n - m u s c l e m y o s i n s a n d m u s c l e m y o s i n s f r o m v a r i o u s c h i c k e n t i s s u e s b y c y a n y l a t i o n a n d p r o t e o l y t i c c l e a v a g e . I f t h e c l e a v a g e p a t t e r n s r e f l e c t d i f f e r e n c e s i n p r i m a r y s e q u e n c e s , t h e n s i x d i f f e r e n t m y o s i n s e x i s t i n t h e c h i c k e n , o n e o f t h e m b e i n g s k e l e t a l m u s c l e m y o s i n . I n t e r e s t i n g l y , c h i c k e m b r y o n i c m u s c l e m y o s i n g a v e a p a t t e r n s i m i l a r t o a d u l t l e g . T h u s , i t a p p e a r s t h a t m y o s i n i s s y n t h e s i z e d i n many c e l l t y p e s b u t t h a t o n l y m y o b l a s t s s y n t h e s i z e s k e l e t a l - l i k e m y o s i n , w h i c h i t s e l f may b e h e t e r o g e n e o u s . F u r t h e r m o r e , s k e l e t a l - t y p e m y o s i n c a n b e d e t e c t e d i n v i v o i n c h i c k e m b r y o s u s i n g a n t i b o d i e s a g a i n s t m y o s i n , a t s t a g e 1 3 - 1 5 ( 5 0 - 5 5 h o u r s a f t e r f e r t i l i z a t i o n ) f o r t h e m y o b l a s t s o f t h e b r a c h i a l m y o t o m e s ( H o l t z e r et: a l . , 1 9 5 7 ; M a s a k i a n d Y o s h i z a k i , 1 9 7 4 ) . T h e s e s t u d i e s i m p l y t h a t m y o s i n s y n t h e s i s i s l i n k e d t o s o m i t e s e g r e g a t i o n i n t h e c h i c k . T h e s e r e s u l t s a l l s u g g e s t t h a t , a l t h o u g h d i f f e r e n t m y o s i n g e n e s may b e t u r n e d o n d u r i n g d e v e l o p m e n t , a l a r g e d e g r e e o f s i m i l a r i t y e x i s t s a t t h e l e v e l o f t h e p r i m a r y s e q u e n c e a n d , p e r h a p s , e v e n a t h i g h e r f o l d i n g l e v e l s o f d i f f e r e n t m y o s i n s . T h e r e f o r e , s i n c e t h e a n t i b o d y u s e d i n t h i s s t u d y w a s p r o b a b l y g e n e r a t e d b y a m i c r o h e t e r o g e n e o u s i m m u n o g e n c o m p o s e d o f d i f f e r e n t m y o s i n h e a v y c h a i n s , we f e e l t h a t i t s c r o s s -r e a c t i v i t y w i t h e m b r y o n i c m y o s i n i s n o t a n a r t i f a c t o r e v e n s u r p r i s i n g . 96 A n o t h e r p o i n t n e e d s t o b e s t r e s s e d c o n c e r n i n g t h e p r e p a r a t i o n o f t h e a n t i g e n f o r i m m u n o l o g i c a l w o r k . T h e m y o s i n u s e d i n t h i s s t u d y w a s i s o l a t e d f r o m S D S - a c r y l a m i d e g e l s . C a r e w a s t a k e n n o t t o i n c l u d e i n t h e g e l s l i c e s p r o t e i n s t h a t m i g r a t e w i t h s l i g h t l y h i g h e r o r l o w e r m o b i l i t i e s t h a n t h e m y o s i n h e a v y c h a i n . O f f e r ( 1 9 7 6 ) h a s s h o w n t h a t C - p r o t e i n , w h i c h i s a c o m p o n e n t o f m y o f i b r i l s a n d f r e q u e n t l y c o n t a m i n a t e s s o - c a l l e d p u r e m y o s i n p r e p a r a t i o n s , i s a p o t e n t i m m u n o g e n w h i c h i s c a p a b l e o f e l i c i t i n g a l a r g e a m o u n t o f a n t i b o d y . I t i s t h e r e f o r e i m p o r t a n t t o r e m o v e a l l t r a c e s o f s o - c a l l e d " c o n t a m i n a t i n g p r o t e i n s " d u r i n g t h e p r e p a r a t i o n o f a n a n t i g e n s i n c e i t i s k n o w n t h a t t h e a p p e a r a n c e o f t h e s e p r o t e i n s d u r i n g d e v e l o p m e n t i s c l o s e l y l i n k e d t o t h e a p p e a r a n c e o f m y o s i n ( F i s c h m a n , 1 9 7 0 ) . We c a n n o t r u l e o u t t h e p o s s i b i l i t y t h a t t h e SDS g e l b a n d s u s e d a s a n t i g e n s c o n t a i n p r o t e i n s o t h e r t h a n m y o s i n h e a v y c h a i n s . H o w e v e r , a n y s i g n i f i c a n t c o n t a m i n a t i o n o f t h e m y o s i n p r e p a r a t i o n w i t h m i n o r p r o t e i n s i s u n l i k e l y b e c a u s e t h e p r o t e i n s w o u l d h a v e t o d i s p l a y s i m i l a r c h e m i c a l a n d e l e c t r o p h o r e t i c p r o p e r t i e s t o m y o s i n , i n o r d e r t o h a v e b e e n i n c l u d e d i n t h e a n t i g e n p r e p a r a t i o n . I s o l a t i o n o f m y o s i n mRNA. T h e s t u d i e s r e p o r t e d h e r e o n t h e a p p e a r a n c e o f m y o s i n mRNA s u g g e s t t h a t t h e m e s s a g e i s s y n t h e s i z e d a t l o w l e v e l s d u r i n g t h e l a t e n e u r u l a s t a g e ( 1 6 / 1 7 ) . T h e mRNA i s . n o t d e t e c t a b l e a t t h e m e d i u m y o l k p l u g s t a g e ( s t a g e 1 2 ) u s i n g t h e w h e a t g e r m c e l l - f r e e s y s t e m a n d i m m u n o p r e c i p i t a t i o n . T h e p o s s i b i l i t y t h a t X e n o p u s e m b r y o n i c RNA a n d s p e c i f i c a l l y m y o s i n mRNA w a s d e g r a d e d d u r i n g RNA i s o l a t i o n c a n n o t b e r u l e d o u t c o m p l e t e l y b u t i s u n l i k e l y . A s d i s c u s s e d i n R e s u l t s B , RNA w a s i s o l a t e d 97 i n t h e p r e s e n c e o f r e a g e n t s w h i c h i n a c t i v a t e r i b o n u c l e a s e s . F u r t h e r m o r e , RNA i s o l a t e d f r o m s t a g e 12 and s t a g e 20 embryos mixed t o g e t h e r was c a p a b l e o f c h a l l e n g i n g t h e wheat germ c e l l - f r e e s y s t e m t o s y n t h e s i z e m y o s i n as d e t e r m i n e d by i m m u n o p r e c i p i t a t i o n . T h i s e x p e r i m e n t showed t h a t m y o s i n mRNA i s n o t i n a c t i v a t e d by some s u b s t a n c e p r e s e n t i n s t a g e 12 embryos and t h a t m y o s i n mRNA i s n o t p r e f e r e n t i a l l y degraded i n homogenates o f s t a g e 12 embryos. However, i t i s p o s s i b l e t h a t an i n h i b i t o r y f a c t o r i n p r e - n e u r u l a embryos b i n d s o n l y t o n u c l e o t i d e r e g i o n s p r e s e n t on n o n - p o l y s o m a l RNA m o l e c u l e s . I n t h i s c a s e , s t a g e 20 m y o s i n mRNA w o u l d n o t be i n h i b i t e d s i n c e i t most l i k e l y i s p r e s e n t on polysomes. Such a model has been p r o p o s e d f o r m y o s i n mRNA s t a b i l i z a t i o n i n c h i c k myogenesis (Heywood and Kennedy, 1976). However, c o n c l u s i v e e v i d e n c e i s l a c k i n g f o r t h i s model and t h e p u r i t y o f t h e p u t a t i v e i n h i b i t o r has n o t been e s t a b l i s h e d . Weber e t a l . (1977) have f o u n d t h a t a t h i g h p o t a s s i u m c o n c e n t r a t i o n s , u n m e t h y l a t e d V a c c i n i a mRNAs a r e t r a n s l a t e d a t 15-20% o f th e e f f i c i e n c y o f m e t h y l a t e d RNAs i n r e t i c u l o c y t e and wheat germ c e l l - f r e e systems,. I t i s p o s s i b l e t h a t t h e l a c k o f t h i s m o d i f i e d 5' t e r m i n u s may p r e v t n t t r a n s l a t i o n o f s t a g e 12 m y o s i n mRNA under o u r c o n d i t i o n s ( s e e a l s o F u r u i c h i e t a l . , 1976). However, e v i d e n c e t o d a t e i n d i c a t e s t h a t l a c k o f a m e t h y l a t e d 5' t e r m i n u s on a message r e d u c e s t h e e f f i c i e n c y o f b i n d i n g o f an mRNA t o t h e 40S r i b o s o m a l s u b u n i t b u t does n o t i n h i b i t i t s t r a n s l a t i o n ( K a e s b e r g , 1976). I f m y o s i n mRNA i s s y n t h e s i z e d a t s t a g e 12 o r e a r l i e r i n Xenopus embryos, i t p r o b a b l y can be t r a n s l a t e d b u t cannot be d e t e c t e d by o u r methods because o f low c o n c e n t r a t i o n s . I t c o u l d be argue d t h e n t h a t p r i o r t o s t a g e 16/17 i n Xenopus 98 embryos, m y o s i n mRNA i s p r e s e n t a t s u c h a low c o n c e n t r a t i o n t h a t , i n t h e absence o f a s p e c i f i c Xenopus m y o s i n mRNA r e g u l a t o r y f a c t o r , i t i s n o t t r a n s l a t e d e f f i c i e n t l y enough t o be d e t e c t e d i n a wheat germ c e l l - f r e e s y s t e m . Heywood and co- w o r k e r s (Rourke and Heywood, 1972; <. Heywood and Kennedy,, 1974; Heywood e t a l . , 1974) have r e p o r t e d t h a t c h i c k m y o s i n mRNA i s more e f f i c i e n t l y t r a n s l a t e d i n a r e t i c u l o c y t e c e l l - f r e e s y s t e m i n t h e p r e s e n c e o f m u s c l e i n i t i a t i o n f a c t o r s ( I F - 3 ) , w h i c h make a s p e c i f i c a ppearance i n c h i c k m y o g e n e s i s . However, many mRNAs have been t r a n s l a t e d i n h e t e r o l o g o u s c e l l - f r e e systems and no a b s o l u t e r e q u i r e m e n t f o r s p e c i f i c i n i t i a t i o n f a c t o r s has been d e m o n s t r a t e d ( B l o e m e n d a l , 1972). F o r example, m y o s i n mRNA has been t r a n s l a t e d i n h e t e r o l o g o u s r a b b i t r e t i c u l o c y t e and wheat germ systems w i t h o u t s p e c i f i c r e q u i r e m e n t s (Mondal e t a l . , 1974; Strohman e t . a l . , 1977; P a t e r s o n and B i s h o p , 1977). No doubt b o t h i n v i v o and i n v i t r o many f a c t o r s ( s u c h as i n i t i a t i o n f a c t o r s ) and many c o n d i t i o n s ( s u c h as rib o s o m e c o n c e n t r a t i o n and mRNA p u r i t y ) a r e r e s p o n s i b l e f o r t r a n s l a t i o n a l e f f i c i e n c y . However, so f a r , v a r i o u s s t u d i e s show t h a t m o d i f i c a t i o n s o f e u k a r y o t i c mRNAs and t h e p r e s e n c e o f v a r i o u s t r a n s l a t i o n f a c t o r s p l a y a f a c i l i t a t e r y r a t h e r t h a n an o b l i g a t o r y r o l e i n i n v i t r o t r a n s l a t i o n s . One a r e a r e l a t e d t o t h e t r a n s l a t i o n o f mRNA, w h i c h r e c e n t s t r u c t u r a l s t u d i e s have l e f t u n r e s o l v e d , b u t w h i c h i s an a r e a o f i m p o r t a n c e t o d i f f e r e n t i a t i o n , i s t h e q u e s t i o n o f mRNA t u r n o v e r . Buckingham e t a l . (1974; 1976) have d e m o n s t r a t e d t h a t d i v i d i n g p r e s u m p t i v e m u s c l e c e l l s c o n t a i n a 26S RNA t h a t has a h a l f - l i f e o f 10 h o u r s . As t h e p r e s u m p t i v e m u s c l e c e l l s undergo d i f f e r e n t i a t i o n , t h e h a l f - l i f e o f t h i s 26S RNA i n c r e a s e s . A f t e r f u s i o n o f m u s c l e c e l l s , t h i s 99 26S RNA i s f o u n d on polysomes. However, t o d a t e , t h e p r o t e i n p r o d u c t o f t h i s 26S RNA i n p r e - f u s i o n and p o s t - f u s i o n has n o t been i d e n t i f i e d . The 26S RNA i n p r e - f u s i o n c e l l s may code f o r some non-muscle myosins o r o t h e r m u s c l e c e l l p r o t e i n s . I t i s p o s s i b l e t h a t p r i o r t o n e u r u l a t i o n i n Xenopus embryos, t h e p r e c u r s o r o f m y o s i n mRNA has a s h o r t h a l f - l i f e and c a n n o t be i s o l a t e d undegraded o r i n l a r g e amounts. F u r t h e r m o r e , because o f i t s i n s t a b i l i t y i t may n o t be t r a n s l a t e d . T h i s p o s s i b i l i t y c a nnot be e l i m i n a t e d . However, i f f a i l u r e t o d e t e c t m y o s i n mRNA was e x c l u s i v e l y due t o r a p i d i n t r a n u c l e a r breakdown o f n e w l y - s y n t h e s i z e d m y o s i n mRNA, t h e h a l f - l i f e o f t h e n e w l y - s y n t h e s i z e d message w o u l d p r o b a b l y have t o be much l e s s t h a n 10 h o u r s i n o r d e r n o t t o be d e t e c t e d i n a c e l l - f r e e s y s t e m . The r e s u l t s o f Buckingham e t a l . (1974; 1976) do n o t ag r e e w i t h t h e r e s u l t s o f Strohman and co - w o r k e r s (Hughes e t a l . , 1 9 7 7 ) . I n t h i s s t u d y t h e r a t e s o f s y n t h e s i s o f whole c e l l RNA and m y o s i n 26S RNA f r o m c h i c k e m b r y o n i c b r e a s t m u s c l e c e l l s were measured by a n a l y z i n g b o t h t h e s p e c i f i c a c t i v i t y o f c e l l u l a r p r e c u r s o r UTP p o o l s and t h e r a t e s o f 3 i n c o r p o r a t i o n o f [ H ] - u r i d i n e i n t o t h e s e RNAs. I t was found t h a t t h e o v e r a l l r a t e o f s y n t h e s i s o f whole c e l l RNA remained r e l a t i v e l y c o n s t a n t t h r o u g h o u t myotube development i n c u l t u r e , b u t t h e r a t e o f 26S mRNA s y n t h e s i s was 2 . 5 - f o l d h i g h e r a f t e r c e l l f u s i o n . T h i s e v i d e n c e s t r o n g l y s u p p o r t s t h e c o n c l u s i o n t h a t t h e i n c r e a s e d s y n t h e s i s o f m u s c l e m y o s i n d u r i n g myogenesis i s r e g u l a t e d by t h e c o n c e n t r a t i o n o f mRNA i n t h e c y t o p l a s m r a t h e r t h a n by a c t i v a t i o n o f a p r e - e x i s t i n g mRNA p o p u l a t i o n — t h a t i s , m yogenesis i s p r o b a b l y r e g u l a t e d by t r a n s c r i p t i o n a l r a t h e r t h a n by t r a n s l a t i o n a l c o n t r o l mechanisms ( P a t e r s o n and B i s h o p , 1 9 7 7 ) . F i n a l l y , t o p r o v e t h a t t h e absence o f my o s i n mRNA i n s t a g e 12 100 embryos does n o t r e p r e s e n t t h e l i m i t o f s e n s i t i v i t y o f th e wheat germ a s s a y s y s t e m s , two more c o n t r o l s s h o u l d be r u n i n t h e f u t u r e . F i r s t , i t s h o u l d be a s c e r t a i n e d what t h e s m a l l e s t amount o f m y o s i n mRNA m o l e c u l e s t h a t can be d e t e c t e d i n t h e wheat germ s y s t e m u s i n g a n t i - m y o s i n a n t i b o d y a c t u a l l y i s . S e c o n d l y , when, s m a l l amounts o f m y o s i n mRNA a r e t r a n s l a t e d i n t h e wheat germ s y s t e m , i t s h o u l d be d e t e r m i n e d w h e t h e r t h e r e i s a s y s t e m a t i c l o s s o f m y o s i n mRNA o r n e w l y - s y n t h e s i z e d m y o s i n ( d e g r a d a t i o n o f a c o n s t a n t number o f m o l e c u l e s ) w h i c h w o u l d c r e a t e an a r t i f i c i a l absence o f my o s i n o r my o s i n mRNA upon a n a l y s i s ( P a l m i t e r , 1 9 7 4 ) . C o n c l u s i o n s . The d a t a p r e s e n t e d i n t h i s s t u d y a r e among t h e f i r s t i n f o r m a t i o n , i n X. l a e v i s embryos, on t h e s y n t h e s i s o f a s t r u c t u r a l p r o t e i n r e l a t e d t o a s p e c i f i c p h e n o t y p e . R e c e n t l y , Reeves (1977) showed t h a t Xenopus t a d p o l e s can be i n d u c e d t o s y n t h e s i z e k e r a t i n mRNA by exogenous t h y r o i d hormone t r e a t m e n t . R i b o n u c l e i c a c i d i s o l a t e d f r o m u n i n d u c e d t a d p o l e s d i d n o t s y n t h e s i z e k e r a t i n . These r e s u l t s i m p l y t h a t l i t t l e k e r a t i n mRNA i s p r e s e n t i n u n i n d u c e d t a d p o l e s . T h e r e f o r e , i t a p p ears t h a t t h e s y n t h e s i s o f m y o s i n i n Xenopus s o m i t e s i s c o n t r o l l e d by t h e same k i n d o f mechanisms r e g u l a t i n g t h e s y n t h e s i s o f h o r m o n a l l y - i n d u c e d p r o t e i n s ( k e r a t i n i n X. l a e v i s embryos, v i t e l l o g e n i n i n X. l a e v i s l i v e r s , o v a l b u m i n i n t h e c h i c k o v i d u c t ) . O t h e r p r o t e i n s t h a t have been s t u d i e d i n X. l a e v i s embryos a r e h i s t o n e s , t u b u l i n s and a c t i n s . These p r o t e i n s make t h e i r a p pearance a t v a r i o u s t i m e s b e f o r e and a f t e r f e r t i l i z a t i o n o f t h e embryos ( S c h r o e d e r , 1973; P e s t e l l , 1975; Adamson and Woodland, 1976; B r o c k and Reeves, i n p r e s s ; Woodland, u n p u b l i s h e d r e s u l t s ) . S i n c e a l l o f t h e s e 101 p r o t e i n s a r e needed by c e l l s a t a l l t i m e s , i t i s n o t s u r p r i s i n g t h a t t h e i r mRNAs a r e s y n t h e s i z e d b e f o r e and a f t e r f e r t i l i z a t i o n . I t c o n t r a s t t o o u r s t u d i e s , P e r l m a n e t a l . (1977) have r e p o r t e d t h e p r e s e n c e o f t a d p o l e and a d u l t h e m o g l o b i n mRNA i n Xenopus o o c y t e s . The s y n t h e s i s o f t h i s message may r e p r e s e n t " l e a k a g e " o f g l o b i n gene t r a n s c r i p t i o n ( i m p l y i n g t h a t no b i o l o g i c a l s y s t e m may be t u r n e d o f f c o m p l e t e l y ) , s i n c e t h e amounts d e t e c t e d a r e r e l a t i v e l y low i f one t a k e s i n t o a c c o u n t t h e l a r g e s i z e o f Xenopus o o c y t e s ( P e r l m a n e t a l . , 1977). On t h e o t h e r hand, i t i s p o s s i b l e t h a t i n t h e case o f c e l l - s p e c i f i c p r o t e i n s ( s u c h as m y o s i n , h e m o g l o b i n , e t c . ) , t h e r e s p e c t i v e messages may be s y n t h e s i z e d a t l o w l e v e l s p r i o r t o f e r t i l i z a t i o n . The r a t e s o f s y n t h e s i s o f t h e s e messages may a l s o be c o n t r o l l e d by m o r p h o g e n e t i c d e t e r m i n a n t s s y n t h e s i z e d e a r l y i n o o g e n e s i s . Assuming s u c h a s i t u a t i o n , one can p o s t u l a t e t h a t as development p r o c e e d s , m y o s i n mRNA i s s y n t h e s i z e d a t low r a t e s (and p o s s i b l y even m y o s i n i t s e l f ) and t h e n s e q u e s t e r e d i n t h o s e c e l l s t h a t w i l l g i v e r i s e t o s o m i t e s . The f u n c t i o n o f s u c h a h y p o t h e t i c a l l y s t o r e d message (and/or i t s p r o t e i n ) w o u l d be t o t i d e t h e p r e s u m p t i v e m u s c l e c e l l s o v e r u n t i l t h e y b e g i n new t r a n s c r i p t i o n o f my o s i n mRNAs and t o p l a y an i m p o r t a n t r o l e i n t h i s t r a n s c r i p t i o n . However, u n t i l some d i r e c t e x p e r i m e n t a l e v i d e n c e i s advanced t o s u p p o r t s u c h a h y p o t h e t i c a l s i t u a t i o n f o r m u s c l e d i f f e r e n t i a t i o n , t h e r e s u l t s o f t h e work r e p o r t e d h e r e and f r o m o t h e r l a b o r a t o r i e s ( H o l t z e r e t a l . , 1975; Strohman e t a l . , 1977; P a t e r s o n and B i s h o p , 1977) i n d i c a t e t h a t s u c h a s i t u a t i o n i s n o t t h e g e n e r a l c a s e f o r m u s c l e d i f f e r e n t i a t i o n i n v e r t e b r a t e s . R a t h e r , i t seems t h a t t h e appearance o f s k e l e t a l m y o s i n d u r i n g m u s c l e d i f f e r e n t i a t i o n i s under some fo r m o f t r a n s c r i p t i o n a l c o n t r o l . The q u e s t i o n t h a t r e m a i n s i s 102 what causes a c e l l to synthesize myosin mRNA. 103 LITERATURE CITED Adamson, E.D. and Woodland, H.R. 1974. H i s t o n e s y n t h e s i s i n e a r l y a m p h i b i a n development: h i s t o n e and DNA s y n t h e s e s a r e n o t c o o r d i n a t e d . J . M o l . B i o l . 88: 263. Adamson, E.D. and Woodland, H.R. 1977. Changes i n t h e r a t e o f h i s t o n e s y n t h e s i s d u r i n g o o c y t e m a t u r a t i o n and v e r y e a r l y development o f Xenopus l a e v i s . Dev. B i o l . _5_7: 135. A t k i n s , J . F . , L e w i s , J.B., A n d e r s o n , C.W. and G e s t e l a n d , R.F. 1975. 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Saunders Co., T o r o n t o . Benson, S.C. and T r i p l e t t , E.L. 1974. The s y n t h e s i s and a c t i v i t y o f t y r o s i n a s e d u r i n g development p f t h e f r o g Rana p i p i e n s . Dev. B i o l . 40: 270. B i s c h o f f , R. and H o l t z e r , H. 1970. I n h i b i t i o n o f m y o b l a s t f u s i o n a f t e r one r o u n d o f DNA s y n t h e s i s i n 5 - b r o m o d e o x y u r i d i n e . J . C e l l B i o l . 44: 134. B l o e m e n d a l , H. 1972. Mammalian mRNA. I n "The mechanism o f p r o t e i n s y n t h e s i s and i t s r e g u l a t i o n . " p. 487. ( B o s c h , L., ed.) N o r t h -H o l l a n d , Amsterdam. B r a c h e t , J . , D e n i s , H. and d e V i t r y , F. 1964. The e f f e c t s o f a c t i n o m y c i n D and p u r o m y c i n on morphogenesis i n a m p h i b i a n eggs. Dev. B i o l . 9_: 938. B r o c k , H.W. and Reev e s , R. 1978. 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