The synthesis of myosin mRNA and myosin in the early development of Xenopus laevis embryos

Kreis, Christophe G.

doi:10.14288/1.0094638

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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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Title	The synthesis of myosin mRNA and myosin in the early development of Xenopus laevis embryos
Creator	Kreis, Christophe G.
Date Issued	1978
Description	A biochemical approach was used to detect the appearance of the heavy chain of skeletal myosin (HCSM) and myosin mRNA during the early development of Xenopus laevis embryos. An antibody against the HCSM of adult X. laevis muscles was biochemically characterized and shown to be monospecific. This anti-myosin antibody reacted with embryonic polysomes synthesizing myosin and with tadpole tail myosin. This indicates that the myosins of adult muscles, early embryonic muscles and tadpole tails are sufficiently homologous to share some antigenic determinants. Polysomes from various stages of X. laevis embryogenesis were reacted with the anti-myosin antibody. Analysis of these reactions showed that myosin synthesis begins in stage 20 embryos, in which about 7 somites have segregated. The RNA from stage 12, stage 16/17 and stage 20 embryos was then analyzed for the presence of the heavy chain myosin mRNA in order to determine whether the synthesis of myosin is under translational or transcriptional control. Total RNA preparations from staged embryos were fractionated on oligo(dT)-cellulose columns and fractions that did and did not bind were translated in a wheat germ cell-free protein synthesizing system. The translational products were precipitated with the anti-myosin antibody and characterized biochemically. Myosin mRNA was detected by this method in stage 16/17 embryos. We conclude that somite segregation results in the appearance of new myosin mRNA molecules in X. laevis embryos. It seems likely, by all the evidence considered, that a large pool of untranslated myosin mRNA molecules is not responsible for muscle myosin synthesis. Therefore, the synthesis of certain proteins in early development is under transcriptional control.
Subject	RNA Myosins Sea urchins
Genre	Thesis/Dissertation
Type	Text
Language	eng
Date Available	2010-03-05
Provider	Vancouver : University of British Columbia Library
Rights	For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
DOI	10.14288/1.0094638
URI	http://hdl.handle.net/2429/21552
Degree	Doctor of Philosophy - PhD
Program	Zoology
Affiliation	Science, Faculty of Zoology, Department of
Degree Grantor	University of British Columbia
Campus	UBCV
Scholarly Level	Unknown
AggregatedSourceRepository	DSpace

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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. Enhanced d i f f e r e n t i a l s y n t h e s i s o f p r o t e i n s i n a mammalian c e l l -f r e e s y s t e m by a d d i t i o n s o f p o l y a m i n e s . J . B i o l . Chem. 250: 5688. A x e l , R., Cedar, H. and F e l s e n f e l d , G. 1973. S y n t h e s i s o f g l o b i n RNA f r o m duck r e t i c u l o c y t e c h r o m a t i n i n v i t r o . P r o c . N a t . Acad. S c i . USA 70: 2029. Bag, J . and S a r k a r , S. 1976. S t u d i e s on a n o n p o l y s o m a l r i b o n u c l e o -p r o t e i n c o d i n g f o r m yosin heavy c h a i n s f r o m c h i c k e m b r y o n i c m u s c l e s . J . B i o l . Chem. 251: 7600. B a k e r , H.J. and S h a p i r o , D.J. 1977. K i n e t i c s o f e s t r o g e n i n d u c t i o n o f X. l a e v i s v i t e l l o g e n i n mRNA as measured by h y b r i d i z a t i o n t o cDNA. . J . B i o l . Chem. 252: 8428. B a l i n s k y , B . I . 1975. "An I n t r o d u c t i o n t o Emb r y o l o g y . " W.B. 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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UBC Theses and Dissertations

UBC Theses and Dissertations

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

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UBC Theses and Dissertations

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

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