En consecuencia, se le aplica la medida so-

LO CNI

5sRNA +

+

tRNA +

+

F ig .3. Treatm ent o f 428 p a r tic le s w ith 0.5M NaCl, u n its o f 428 rib o n u c le o p ro te in p a r tic le s is o la te d from stage 1 oocytes were d ia ly s e d a g a in s t b u ffe r c o n ta in in g 0.5M N a d f o r I6 h r a t 4 ° C. (A) sucrose g ra d ie n t showing d is s o c ia tio n p ro d u cts as a s in g le peak sedim enting about 78. The p o s itio n o f th e o r ig in a l 428 peak i s shown (broken l i n e ) . (B) C sd d e n s ity g ra d ie n t a n a ly s is o f ribonuce o p ro te in complexes co nta in ed in the 78 peak. R e la tiv e amounts o f RNA are in d ic a te d by r a d io a c tiv it y (th e ovary was o r ig in a lly la b e lle d w ith * H -u rid in e as d e scrib e d i n 8 e c tio n 1 :4 ). The th e o r e tic a l p o s tio n o f complexes form ed between 3,2 and 1 m olecules o f tRNA and a s in g le p ro te in and between 1 m olecule o f 58 RNA and P33 are in d ic a te d . (# ) RNA r a d io a c t iv it y ; (A) d e n s ity o f CsCl.

— Sedimentation s eg 5SRNA/P33 l2tRNA 1.6 E ycn 1.4% 3tRNAi URNA ► 15 1 .2 ^ 10 5 Gradient fractions

la b e l is recovered as fr e e RNA a t the bottom o f the tube (1.65g/cm^ , F ig .S B ); over 95JS o f the la b e l i s removed from the m id d le o f the g ra d ie n t in th e form o f s ta b le rib o n u c le o p ro te in complexes. These complexes a re d e te cte d as fo u r peaks o f r a d io a c t iv it y which correspond i n d e n s ity to those expected from s in g le p ro te in m olecules i n com b ina tio n w ith 1, 2 and 3 m olecules o f tRNA, and w ith 1 m olecule o f 58 RNA. Due to the f ix a t io n procedure, the p ro te in components o f in d iv id u a l peaks cannot be id e n t if ie d . N e ve rthe le ss, th e peak a t the d e n s ity p o s itio n o f 1.58g/cm? corresponds to th e value expected from 58 RNA i n co m b ina tio n w ith the P33 cleavage p ro du ct o f P48 (th e co n te n t r a t io o f P33:P48 in th e 428 p re p a ra tio n used f o r t h is p a r tic u la r experim ent was about 1 :1 ). Although the peak w ith lo w e st d e n s ity (1.47g/cm * ) c o u ld c o n ta in e ith e r P48 o r P43 in co m b ina tio n w ith a s in g le tRNA m o le cu le , th e re m a in in g two peaks a t 1.55g/cm^ and 1.62g/cm* are more l i k e l y to c o n ta in P48 in co m b ina tio n w ith 2 and 3 m olecules o f tRNA, re s p e c tiv e ly , since o nly t h is p ro te in was found to be capable o f m u ltip le b in d in g o f tRNA i n i n v i t r o re co m b in a tio n s tu d ie s ( F ig . 1 ).

1:21 H e te ro g e n e ity in C om position o f 428 P a r tic le s

The s t a t ic p ic tu r e o f 428 p a r tic le s i s one o f a W etrameric complex o f the b a sic monomeric u n it co m p risin g 2 m olecules o f P48, 1 m olecule o f P43, 1 m olecule o f 58 RNA and 3 m olecules o f tRNA. T h is p ic tu r e does n o t in c o rp o ra te any view o f the p a r tic le being a dynamic p a r tic le p o s s ib ly in flu e n c in g t r a n s c r ip tio n o f 58 RNA o r tRNA and th e processes o f rRNA t r a n s c r ip tio n and ribosom e assem bly. However, some h e te ro g e n e ity in th e 428 p a r tic le s i s observed when fr a c tio n s are

take n across the 428 re g io n o f sucrose g ra d ie n ts and i n o v a rie s take n a t d if fe r e n t stages o f developm ent.

When the m a te ria l th a t sedim ents in th e 428 re g io n o f a sucrose g ra d ie n t i s se p la t e d in t o th re e fr a c tio n s co rre sp on d ing to the heavy s id e , m iddle and t r a i l i n g sid e o f the 428 peak ( F ig .4 ) , co m p o sitio n a l v a r ia tio n i s observed. P olyacrylam id e g el e le c tro p h o re s is shows th a t P43 i s underrepresented i n th e f r a c t io n ta ke n a t th e heavy side of the 428 peak. The g e l w hich i s s ta in e d w ith s ilv e r s a lts , a ls o i^ows th e RNA components w hich are re co gn ize d as n e g a tiv e ly s ta in in g bands. I t can be seen th a t the p ro p o rtio n s o f 58 RNA and tRNA components i s s im ila r i n a l l th re e fr a c tio n s . These o b s e rv a tio n s w ould suggest t h a t th e re i s n o t a sim ple r e la tio n s h ip w ith in th e p a r tic le s o f one type o f RNA w ith one p ro te in and th e o th e r type o f RNA w ith the o th e r p ro te in .

F u rth e r h e te ro g e n e ity is observed when 428 p a r tic le s are is o la te d from o v a rie s a t d if fe r e n t stages o f developm ent. T h is h e te ro g e n e ity can be seen w ith re sp e ct to both p ro te in and RNA, F ig .5 shows the r e la t iv e abundance o f p ro te in s i n 428 p a r tic le s is o la te d frcm ovary th a t c o n ta in e d : (1) o n ly stage 1 (c le a r ) o ocyte s, (2 ) m a in ly stage 2 (w h ite ) o ocyte s, and (3) m a in ly p o s t-s ta g e 2 (y o lk y ) o o cyte s. A lthough the c o n tr ib u tio n o f 428 m a te ria l is m a in ly from stages 1-2 (6 5 ,9 3 ), i t can be seen th a t p ro te in co m p o sitio n changes from being m o stly P48 in e a r ly ovary to b eing m o stly P43 i n more m ature ovary. In a d d itio n , RNA from th e th re e p re p a ra tio n s was measured by scanning g e ls c o n ta in in g e le o tro p h o re tic a lly -s e p a ra te d RNA sp ecies. T h is a n a ly s is showed th e r a t io i n A^^^ o f 58 RNA to tRNA changed from 1.51 to 0.23 on g oing

F ig .4 . A n a ly s is o f fr a c tio n s across th e 428 rib o n u c le o p ro te in peak is o la te d frcm stage 1 oocytes. (A) sucrose g ra d ie n t p r o f ile showing fr a c tio n s colleeJsa^ from the le a d in g side (1 ), M id d le (2 ) and t r a i l i n g s id e (3) o f the 428 peak. In m a te ria l from t h is e a r ly oogenic stage, 428 m a te ria l exceeds th a t o f ribos<xnes(808) and sm a ll rib o n u c le o p ro te in com plexes(7S). (B) equal volumes o f the 428 fr a c tio n s were precipîfeo,V;«A w ith e th a n o l and prepared f o r S D 8-polyacrylam ide g e l e le c tro p h o re s is . 8 ta in in g w ith s ilv e r s a lts r e s u lts in dark p r o te in bands and n e g a tiv e ly -s ta in e d RNA bands. (Pm) in d ic a te s p ro te in s a s s o cia te d w ith mRNA w hich cosedim ent w ith 428 p a r tic le s as mRNP p a r t ic le s (2 0 3 ). P33 i s a cleavage p ro du ct o f P48 (see t e x t ) .

— Sedimentation

5sRNA-