Isolation and Chemical Characterization of Nucleolar and Nucleoplasmic Subfraction

7/28/2019 Isolation and Chemical Characterization of Nucleolar and Nucleoplasmic Subfraction

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S T U D I E S O N I S O L AT E D N U C L E I

I I . I so l a t i on and Chemica l Cha rac t e r i za t ion

o f Nuc leo l a r and N uc leop la sm ic Subf rac t ions

R A C H E L E M A G G I O , Ph .D , P H I L I P S I E K E V I T Z , P h .D ,

GEOR GE E . PAL ADE , M.D.

From The Rockefeller Ins titu te

an d

A B S T R A C T

T h i s p a p e r d c s c r i b e s t h c s n b fr a c t io n a t i on o f n u c l e i i s o l a t c d f r o m g u i n e a p i g l i v e r b y t h ep r o c e d u r e p r e s e n t e d i n t h c f ir st r t i c l e o f t h e s e r i es ( 8) . C e n t r i f u g a t i o n i n a d e n s i t y g r a d i c n t

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

s u b f r a c t i o n s : ( a) a n u c l e o l a r s u b f r a c t i o n w h i c h c o n s i s t s m a i n l y o f n u c l c o l i s u r r o u n d c d b y a

v a r i ab l c a m o u n t o f n u c l co l u s -a s s o c ia t e d c h r o m a t i n a n d c o n t a m i n a t e d b y c h r o m a t i n b l o c k s

d e r i v e d p r i m a r i l y f r o m v o n K u p f f c r c e l l n u c l e i ; ( b) a n d ( c) , w o n u c l e o p l a s m i c s u b f ra c t i on s

( I a n d I f ) w h i c h c o n s i s t m a i n l y o f c h r o m a t i n t h r e a ds i n a c oa r s e r ( 1) o r f i n e r ( I I ) d e g r e e o f

f r a g m en t a t i on . T h e p r o t e i n , R N A , a n d D N A c o n t en t o f t h e s e s u b f ra c t i o ns w a s d c t e r m i n e d ,

a n d t h e i r R N A ' s c h a r a c t e r i ze d i n t c r m s o f N a C l -s o l u b i l i t y , n u c l eo t i d e c o m p o si t i o n , a n di n v i v o n u c l e o t i d c t u r n o v e r , u s i n g i n o r g a n i c 3 2 p a s a m a r k c r . T h e r e s u lt s i n d i c a t e t h a t t h e r c

a r e a t I c a s t t h r e c t y p e s o f R N A i n t h e n u c l e u s ( o n c i n t h e n u c l e o l u s a n d t w o i n t h e n u c l e o -

p l a s m o r c h r o m a t i n ) , w h i c h d i f fe r f r o m o n c a n o t h e r i n N a C l - so l u b i l i t y , n u c l eo t i d e c o m -

p o s i t io n , t u r n ov e r , a n d p o s s ib l y s e q u c nc e . P o s s ib l e r e l a ti o n s a m o n g t h e s e R N A ' s a n d t h o s c

o f t h e c y t o p l a s m a r c d i s cu s s e d .

I N T R O D U C T I O N

In contrast with the large literature on nuclearfractions, there ar e rela tively few reports onnuclear subfractions. Most of them deal with theisolation of nucleoli. The problems to be solvedin such a project are: the rupture of the nuclearmembranes, the prevention of gel formation byDNA, 1 a fine dispersion o r solubil izatio n of the

a The abbrevia tions used in this paper are: DNA,deoxyribonucleic acid; RNA, ribonucleic acid;RNP, ribonucleoprotein ; AMP, adenylic acid;

CMP, cytidylic acid; GMP, guanylic acid; UMP,uridylic acid ; ~k UMP, pseudouridylic acid; DOC,deoxycholate; Tris, tris (hydroxymethyl) amino-methane; EDTA, ethylenediaminetetraacetic acid;Pi, inorganic phosphate.

nucleoplasm, and finally, the con comitant preser-vation of the nucleoli. Were these conditions met,theqatte r could be easily separated from the othernuclear materials by differential centrifugation,on account of their large size and grea t density.

A number of different approaches have beenused so far to achieve these conditions. The first,proposed by Litt e t a l . (1) and by Monty e t a l .(2), relied on sonic vibrations to disrupt liver cellnuclei and disperse their nucleoplasm; it yielded anucleolar fraction with a high content of DNA.In the second approach (3, 4), the "solubilization"of the nucleoplasm was obtained through Ca 2+remova l effected by suspending the nucle i in acitrate-c ontaining mediu m; the nuclea r envelope

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w a s s u b s e q u e n tl y r u p t u r e d a n d t h e n u c l e o p l a s md i s p e r s e d b y s t i r r i n g t h e s u s p e n s i o n i n a m i x e r.I n a t h i r d a p p r o a c h ( 5 ) , i s o l a t e d n u c l e i w e r eh o m o g e n i z e d i n a h i g h d e n s i t y m e d i u m a n d t h el i b e r a t e d n u c l e o l i s e p a r a t e d b y d i f f e r e n t i a l c e n t r i -f u g a t i o n u p o n d i l u t i o n o f t h is h o m o g e n a t e . F i n a l l y,

Vi n c e n t ( 6 ) a n d s u b s e q u e n t l y B a h u s ( 7 ) b r o k ec e l ls a n d n u c l e i s i m u l t a n e o u s l y, b y f o r c i n g s t a r -f i sh o o c y t e s t h r o u g h a s m a l l a p e r t u r e . T h e f r e e dn u c l e o l i w e r e s u b s e q u e n t l y i s o l a t e d b y d i f f e r e n t i a lc e n t r i t h g a t i o n .

T h e p r e s e n t p a p e r d e a l s w i t h a p r o c e d u r e f o ro b t a i n i n g n u c l e o l a r a n d n u c l e o p l a s m i c s u b -f r a c t i o n s f r o m n u c l e i i s o l a t e d f r o m g u i n e a p i gl i v e r a s p r e v i o u s l y d e s c r i b e d ( 8 ) ; i t p r e se n t s , i na d d i t io n , d a t a o n t h e R N A ' s o f t h e se n u c l e a rs u b f r a c t i o n s .

E X P E R I M E N T A L

G e n e r a l P r o c e d u r e s

1. CEL L FRACTIONATiON

a . N U C L E A R A ND S U B N U C L E A R F R A C -T I O N S : N u c l e a r f r a c t i o n s w e r e i s o l a te d e i t h e rb y o u r m e t h o d ( 8) o r b y t h a t o f C h a u v e a uet al.( 9 ). I n o u r p r o c e d u r e , t h e t i s s ue is h o m o g e n i z e di n 0 . 8 8 M s u c r o s e - l . 5 m M C aC 1 2 a n d t h e n u c l e ii s o l a t e d b y d i f f e r e n t i a l c e n t r i f u g a t i o n i n a d i s -c o n t i n u o u s d e n s i ty g r a d i e n t ( h o m o g e n a t e o v e r 2 . 2M s u c r o s e - 0 . 5 m M Ca C 12 ). I n t h e p r o c e d u r e o fC h a u v e a u et al. ( 9 ) , b o t h t i s s u e h o m o g e n i z a t i o na n d n u c l e a r i s o l a t i o n a r e c a r r i e d o u t i n 2 . 2 Ms u c r o s e w i t h o u t C a 2 +.

T h e i s o la t i on o f n u c l e o la r a n d n u c l e o p l a s m i cs u b f r a c t i o n s w i l l b e d e s c r i b e d i n d e t a i l u n d e rRESULTS.

b. CYTOPLASMIC FRACTIONS: M i c r o s o m a l ,p o s t m i c r o s o m a l , a n d f i n a l s u p e r n a t e f r a c t i o n s w e r e

i s o l a t e d f r o m g u i n e a p i g l i v e r f o l l o w i n g t h e p r o -c e d u r e s g i v e n in ( 1 0 ) . G u i n e a p i g h e p a t ic a n dp a n c r e a t i c r i b o s o m e s ( R N P p a r t i c l e s ) w e r ep r e p a r e d f r o m D O C - t r e a t e d m i c r o s o m a l f ra c t io n sas in (10) and (11) .

S a m p l e s o fEscherichia colir i b o s o m a l R N A w e r e

o b t a i n e d f r o m D r. D . N a t h a n s . S a m p l e s o f s o l ub l eR N A w e r e k in d l y s u p pl i e d b y D r. J . F. K i r s c h( g u i n e a p i g l i ve r ) a n d D r. G . Z u b a y(E. coli).

~. CHEMICAL PROCEDURES

T h e m e t h o d s u s e d f o r t h e d e t e r m i n a t i o n o fR N A , D N A , t o t a l N , a n d p r o t e i n a r c g i v e n i n( 8 ) .

3. EXTRACTION PROCEDURES

P h o s p h a t e e x t r a c t i o n r e m o v e s li t tl e R N A f r o mn u c l e i i s o l a te d b y o u r p r o c e d u r e ( 8 ) ; h e n c e i t w a so m i t t e d f r o m t h e e x t r a c t io n s c h e d u le o f t h e n u c l e a rs u b f r a c t io n s . T h e 1 u N a C 1 e x t r a c t i o n w a s e x -

t e n d e d t o ~ 1 5 h o u r s b e c a u s e a ft e r sh o r t e r p e ri o d sl a rg e a m o u n t s o f D N A r e m a i n e d u n e x t r a c te d .I n e a c h e x p e r i m e n t n u c l e a r s u b f r a c t i o n s i s o l a t e df r o m 9 t o 1 8 g m f r e s h l i v e r w e r e u s e d , a n d e a c hs u b f r a c t i o n w a s e x t r a c t e d t w i c e w i t h 3 0 t o 4 0 m lo f 1 M N a C l , t h e r e s t o f t h e p r o c e d u r e b e i n g t h es a m e a s i n t h e c a s e o f n u c l e a r f r a c t i o n s ( 8 ) .

4 . RADIOACTIVITY MEASUREMENTS

32 p w a s a d m i n i s t e r e d i n t r a v e n o u s l y a s 1 m li n o rg a n i c p h o s p h a t e s o l u t i o n ( c o n t a i n i n g 1 0 0# M P a n d 3 5 0 ~ c ) t o g u i n e a p i g s f a s t e d f o r 1 d a y.T h e l iv e r s w e r e r e m o v e d u n d e r e t h e r a n e s t he s i aa t s e l e c t e d i n t e r v a l s a f t e r t h e i n j e c t i o n o f th el a b el . T h e R N A ' s o f t h e n u c l e a r a n d s u b n u c l e a rf r a c t i o n s w e r e i s o l a t e d , d i g e s t e d , a n d t h e i r m o n o -n u c l e o t id e s c h r o m a t o g r a p h i c a l l y s e p a r a t e d a s in( 8 ) .

FIGURE 1 Nucleolar subfraction(middle layer ) . At th is leve l, and throughout the upper2/~ of the pellet , the pre paration main ly consists of recognizable nucleoli (nl to ns). M ostof them conta in var iab le amounts of chromat in e i ther in the i r lacunae (n l ,n2 ) , o r as amore or less continuous peripheral shell (n3 to n~) that probably corresponds to the "nu-cleolus-associated chromatin" describedin situ. The nucleolus marked n6 apparent lybroke away wi th the whole layer of chromat in tha t separa ted i tin si tu f rom the sur faceof the nucleus.

There are a few masses of recognizable chromatiu derived from liver cell nuclei (ch);

the most impor tan t contaminant of the prepara t ion i s represented by f ragments of leucocyt ic nuc le i (ln) and b locks of compact chromat in(ok) der ived f rom von Kupffer ce l lnuclei .

Pelle t f ixed in 0.039 M OsO4 in 0.88 M sucrose ad justed to pH 7.8 with Tris; euIbeddedin methacry la te . Sec t ion s ta ined wi th Pb(OH)2. X 16 ,000.

294 THE JOURNAL OF CEL L BIOLOGY VOLUME8, 1968


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RACHELE MAGG IO, PHILIP SIEKEVITZ, AND GEORGE ]~. PAL~.DE Isolated Nuclei. 11 29 5


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T h e f in a l c h r o m a t o g r a m s w e r e e x a m i n e d u n d e ru l t r a v i o l e t l i g h t t o l o c a l i z e t h e m o n o n u c l e o t i d es p o t s , a n d s u b s e q u e n t l y e x p o s e d f o r 3 d a y s t oK o d a k " n o s c r e e n " x - r a y f il m to o b t a i n t h e i ra u t o r a d i o g r a p h s . F i v e a u t o r a d i o g r a p h i c s p o ts w e r eo b t a i n e d , o f w h i c h f o u r w e r e c o n g r u e n t i n s iz ea n d l o c a t i o n w i t h t h e m o n o n u c l e o t i d e s p o t s

d e t e c t e d i n t h e u l t r a v i o l e t . 2 - h e f i f t h a u t o r a d i o -g r a p h i c s p o t a p p e a r e d w e l l s e p a r a t e d f r o m t h o s eo f t h e m o n o n u c l e o t i d e s , d i d n o t a b s o r b u l t r a v i o l e tl i g h t , a n d w a s i d e n t i f i e d a s i n o rg a n i c p h o s p h a t eb y t h e s p r a y r e a g e n t o f B a n d u r sk i a n d A x e l r o d02) .

A f t e r t h e e s t i m a t i o n o f it s n u c l e o t i d e c o n c e n -t r a t i o n ( 8 ) , th e e n t i r e e x t r a c t o f e a c h s p o t w a sd e p o s i t e d o n m e t a l p l a n c h e t s , a d r o p o f a c e t o n ea d d e d t o g i v e a u n i f o r m d r y i n g , a n d t h e s o l u t i o ne v a p o r a t e d u n d e r i n f r a r e d l i g h t . R a d i o a c t i v i t yw a s d e t e r m i n e d w i t h a N u c l e a r C h i c a g o g a s fl o wc o u n t e r p r o v i d ed w i t h a " m i c r o m i l " w i n d o w. T h er a d i o a c t i v i ty r e g i s t er e d p e r s a m p l e v a r i e d f r o m

5 0 0 t o 3 ,0 0 0 c P M f r o m o n e 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 r a d i o a c t i v i t y w a s o b t a i n e d a s cP Mp e r m o l e o f e a c h n u c l e o t i d e o r a s cp M p e r m gR N A . T h e l a t t e r v a l u e w a s o b t a i n e d b y d i v i d i n gt h e s u m o f al l c o u n t s r e c o r d e d i n t h e f o u r n u c l e o -t i d es b y th e c a l c u l a t e d a m o u n t o f R N A .

F o r t h e d e t e r m i n a t i o n o f th e r e c o v e r y o f n u c l e o -t i d e s a n d r a d i o a c t i v i t y a f t e r t h e c h r o m a t o g r a p h y,t o t a l R N A a n d t o t a l r a d i o a c t i v i ty w e r e m e a s u r e di n s a m p l e s o f t h e o r ig i n a l a l k a l in e R N A h y d r o l -y s a t e ( 8 ) . ( A c o n t r o l o f r a d i o a c t i v i t y l o ss e s i n -c u r r e d a t t h e e x t r a c t i o n s t a g e o f th e c h r o m a t -o g r a m s w a s n o t c a r r ie d o u t . ) T h e r e s ul ts s h o w e dt h a t n u c l e o t i d e r e c o v e r y a m o u n t e d t o 7 0 t o 7 5p e r c e n t , w h e r e a s t h e r a d i o a c t i v i t y r e c o v e r e d i nn u c l e o t i d e s d i d n o t e x c e e d 4 0 t o 5 0 p e r c e n t o f t h et o t a l . S i n c e a w e l l s e p a r a t e d r a d i o a c t i v e s p o t w a si d e n t i f i e d a s i n o rg a n i c p h o s p h a t e , i t i s a s s u m e dt h a t t h e l o w e r r a d i o a c t i v i t y r e c o v e r y i s d u e t oc o n t a m i n a t i o n o f t h e a l k a l in e h y d r o l y s a t e b yn o n - R N A = P. A s r e p e a t e d l y d is c u ss e d i n th e p a s t(cf. r e f e r e n c e 1 3) , c o n t a m i n a t i o n b y n o n - n u c l e i ca c i d 3 ~p i s a n i m p o r t a n t s o u r c e o f e r r o r w h e nd e t e r m i n i n g t h e r a d i o a c t i v it y o f R N A f r a c t i o n si s o la t e d b y th e S c h m i d t - T h a n n h a u s e r p r o -c e d u r e . T h e i s o l a t e d m o n o n u c l e o t i d e s a r e a p -p a r e n t l y f r e e o f s u c h c o n t a m i n a t i o n .

5 . PS E U D O U R I D I N E D E T E R M I N AT I O N S

To f i n d o u t w h e t h e r %b U M P i s p r e s e n t i n t h eR N A ' s o f n u c l e a r a n d s u b n u c l c a r f r a c t i on s , w eu s e d a m o d i f i c a t i o n o f t h e m e t h o d s o f Yu a n d

A l l e n ( 1 4 ) a n d D u n n ( 1 5 ) c a p a b l e o f d e t e c t i n gs m a l l a m o u n t s o f ~b U M P, a s fo r e x a m p l e t h a to b t a in e d f r o m ~ 0 . 4 m g s o l ub le R N A .

I n a f i r s t s t e p , t h e h y d r o l y z e d R N A s a m p l e sw e r e s u b m i t t e d t o e l e c t r o p h o r e s i s i n 0 .0 5 M N ac i t r a t e b u f f e r a t 4 o n W h a t m a n 3 M M f i lt e rp a p e r w h i c h c a u s e d th e U M P a n d ~b U M P t o

m i g r a t e t o g e t h e r a s a s p o t w e l l s e p a r a t e d f r o m t h es p o t s o f t h e o t h e r m o n o n u c l e o t i d e s . A f t e r e l e c t r o -p h o r e s is , t h e p a p e r w a s d r i e d a n d t h e s p o t c o n -t a i n i n g U M P a n d ~b U M P c u t o u t a n d e x t r a c t e dw i t h a l a rg e v o l u m e o f H 2 0 . T h e e x t r a c t w a sc o n c e n t r a t e d in vacuoa n d f i n a l l y d is s o l v e d i n 1 t o2 m l o f 0 . 0 5 M N a a c e t a t e b u f f e r, p H 5 . 3. I n t h es e c o n d s t e p , t h e m o n o n u c l e o t i d e s o f th e c o n c e n -t r a t e d e x t r a c t w e r e d e p h o s p h o r y l a t e d b y i n c u b a -t i o n w i t h p r o s t a t i c p h o s p h o m o n o e s t e r a s e a t 3 7 f o r 8 t o 1 0 h o u r s a c c o r d i n g t o t h e m e t h o d o fS c h m i d t ( 1 6 ) , t h e r e a c t i o n m i x t u r e b e i n g s u b s e -q u e n t l y c o n c e n t r a t e d in vacuo. I n t h e f i n a l s t e pp s e u d o u r i d i n e w a s s e p a r a t e d f r o m u r i d i n e b y t h e

d e s c e n d i n g c h r o m a t o g r a p h y o f t h is c o n c e n t r a t ei n t h e n - b u t a n o l / w a t e r ( 8 6 / 1 4 ; v / v ) s o l v e nt o fM a r k h a m a n d S m i t h ( 1 7 ) . I n t hi s so l v e n t t h e R Io f t h e ~ /, u r i d i n e i s h a l f t h a t o f u r i d i n e ( 1 4 ). F o ra d e q u a t e s e p a r a t i o n , i t w a s f o u n d n e c e s s a r y t or u n t h e c h r o m a t o g r a m f o r ~ 4 d a y s a t r o omt e m p e r a t u r e . ~b u r i d i n e w a s i d e n t i f i e d b y i t su l t r a v i o l e t a b s o r p t i o n s p e c t r a a t p H 1 a n d 1 2 ( 1 8 )a n d b y i t s r e a c t i o n w i t h o r c i n o l ( 1 8 ). F o r i t sq u a n t i t a t i v e d e t e r m i n a t i o n s , a n E 2~ 0 v a l u e o f 8 . 5X 103 in 0 .01 N HC1 was used (18) .

T h e m e t h o d d e s c r i b e d w a s w o r k e d o u t w i t h as a m p l e o f ~b U M P k i n d l y s u p p l ie d b y D r. W . E .C o h n , a n d w a s c h e c k e d b y d e t e r m i n i n g t h ea m o u n t s o f ~b U M P p r e s e n t i n s o lu b l e R N Af r a c t i o n s o b t a i n e d f r o m v a r i o u s s o u r c e s . B yu l t r a v i o l e t s c a n n i n g , w e w e r e a b l e t o d e t e c ta m o u n t s o f ~b u ri d in e ( ~b U M P ) a s s m a l l a s 0 . 0 2 5t o 0 . 0 3 / ~ m o l e . W h e n n o s p o t w a s v i s i b l e , t h er e g i o n o f t h e p a p e r t o w h i c h ~ b u ri d in e w a s e x -p e c t e d t o m i g r a t e w a s c u t o u t a n d e x t r a c t e d( w i t h 0 . 01 N H C 1 ) ; s p e c t r a o f th e s e e x t r a c t s w e r er o u t i n e l y r e c o r d e d .

R E S U L T S

A . N u c l e a r S u b f r a c t i o n a t i o n

1 . P R E L I M I N A R Y E X P E R I M E N T S

a . C A 2+ C H E L AT I O N : T h i s a p p r o a c h w a s t r i e din a few exper iments by suspending i so la ted nucle ii n a 0 . 02 M c it r a t e - 0 .5 m M E D T A - 0 . 8 8 M o r2.2

2 9 6 T H E JO U R N A L OF C E L L BI O LO G Y V O L U M E 1 8 , 1 9 6 3


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FIGURE ~ Nucleolar subfraction (bo t tom l aye r ) . Nuc leo li (n l t o ns ) a r e st i l l t he ma in c omp onen t a t t h i sl e v e l i n t h e p e l l e t , b u t t h e c o n t a m i n a t i o n b y c h r o m a t i n b l o c k s a n d l a rg e r f r a g m e n t s(nf) o f c o m p a c tnuc le i o r ig ina t ing f rom l eucocy tes(ln) and von Kupffe r ce l l s (ck) i s no t i ceab ly inc reased .

Many nuc leo l i show a more o r l e s s con t inuous co rona o f a s soc ia t ed eh romat in (n l t o n4 ) and somec a m e o u t w i t h t h e w h o l e l a y e r o f c h r o m a t i n i n t e r p o s e d b e t w e e n t h e m a n d t h e s u r f ac e o f t h e n u c l e u s ( n s ) .

P repa r a t ion fo r e l ec t ron mic rosco py a s fo r F ig . 1 . X 16 ,000 .

suc rose fol lowed by 30 minu te s o f s t i r r ing a t 4 Th es u s p e n s i o ns w e r e s u b s e q u e n t l y f r a c t i o n a t e d b yd i ff e ren t i a l cen t r i fuga t ion wi th o r w i thou t a d i s -c o n t i n u o u s d e n s i t y g r a d i e n t (cf. r e f e r e n c e 8 ) . T h ee n s u i n g f r a c t io n s w e r e m o n i t o r e d b y e l e c t r o n m i c r o s -c o p y a n d b i o c h e m i c a l d e t e r m i n a t i o n s . A f t e r t h i st r e a t m e n t o n l y a f e w n u c l e i r e m a i n e d i n t a c t ; t h en u c l e o l i d i d n o t d i s p e r s e , b u t t h e y d i d n o t s e p a r a t ef r o m t h e n u c l e o p l a s m , w h i c h f o r m e d a g e l . S i m i l a rR N A / D N A r a t io s w e r e f o u n d i n a ll fr a c t io n s . A t -

t e m p t s t o r e d u c e t h e v i sc o s it y o f t h e c o m m o n n u c l e o -p l a s m - n u c l e o l a r f r a c t i o n s b y f u r t h e r d i l u t i o n w i t hc i t r a t e - E D TA o r b y d e o x y r i b o n u c l e a s e d i g e s t i o nfai led.

b . D I S R U P T I O N B Y S O N I C V I B R A T I O N S : N u -

c l e i w e r e r e s u s p e n d e d i n v a r i o u s m e d i a a n d s u b -j ec t e d fo r va r i e d pe r iods to son ic v ib ra t ions in aR a y t h e o n m a g n e t o s t r i c t i o n o s c i ll a t o r o p e r a t i n g a t1 0 , 0 0 0 c y c l e s / s e e , t h e r e s u l t s b e i n g m o n i t o r e d b yp h a s e c o n t r a s t m i c r o s c o p y. S o n i c a t i o n i n u n b u f f c r e d0 . 8 8 M s u c r o s e ( p H ~ 6 . 5 ) d i s r u p t e d a r e l a t i v el y s m a l lp e r c e n t a g e o f n u c l e i ; t h e n u c l e o l i l i b e ra t e d i n t h ep r o c e s s a p p e a r e d i n t a c t a n d f r e e o f c o a r s e n u c l e o -p l a s m t a b s ; m o r e o v e r t h e y r e t a i n e d t h e i r s i z e a n dre f r ac t iv i ty ove r l on g pe r iods o f s to rage a t 4 Fo r

th i s r eason 0 .88 M suc rose was chosen a s a basa lm e d i u m f o r f u r t h e r e x p e r i m e n t s c a r r i e d o u t t o i n -c rease the e ff i c i ency o f nuc l ea r d i s rup t ion .

p H and ionic strength effects: T h e a d d i t i o n o f 0. 1p h o s p h a t e b u f f e r , p H 7 . l , t o t h e b a s a l s u c r o se m e d i u m

RACHELE MAGGIO, PHILIP SIEKEVITZ, AND GEORG E E. PALADEIsolated Nuclei. lI 29 7


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caused a marked decrease in the frequency of un-broken nuclei. The freed nucleoli appeared un-damaged, but their isolation was precluded by ageneral precipitation of the nucleoplasm whichoccurred shortly after sonication. A further rise inpH was tried without concomitant increase in ionicstrength (cf. reference 19) by adjusting the pH of the

sucrose solution with Tris just before use. At pH 7.5to 8.0 only occasional nuclei remained intact; thefreed nucleoll retained their size and refractivity;the nucleoplasm was finely dispersed and did notagglutinate even after prolonged storage. At pH 8.5general solubilization of all nuclear structuresoccurred.

Other media, recommended for maintainingnucleohistones in solution, e.g. saline-EDTA (20),were also tried with or without sucrose, but foundunsatisfactory, for the liberated nucleoli swelled andfinally disintegrated.

Duration o f son ic treatmen t: This variable wasinvestigated by subjecting aliquots of a nuclearsuspension to sonic vibrations for 15, 20, 25, and 27

minutes and by following by phase microscopy thefrequency of intact nuclei in the treated suspensions.Intact nuclei were common in the 15 minute sampleand only occasionally encountered in those treatedfor 20 and 27 minutes. Nuclear disruption was foundto be influenced also by the concentration of nucleiin the original suspensions, which must therefore becontrolled.

C, F R A C T I O N AT I O N O F S O N I C A L LY T R E AT E D

SUSPENSIONS: Afte r trying variou s systems an d

centrifugal fields, we succeeded in separating nucleo-plasm from nucleoli by using a discontinuous densitygradient in which the lower phase was brought upto 2.2 M sucrose. A two (0.88 M/2.2 M sucrose) and athre e (0.88 M/1.5 M/2.2 M sucrose) laye r s ystem we retried and found to be equally effective in the separa-tion of nucleoli from nucleoplasm.

The required centrifugal field was determined bymonitoring for the absence of nucleoli in the bandformed at the interface bet ween 0.88 M/2.2 M sucrosein a two layer system. Nucleoli were only occasion-ally encountered when the field was set at 20,000 gfor 60 minutes. Nucleolar recovery was improvedwhen crowding at this interface was avoided bydilution of the sonicated suspension and by using thelarger interface provided by the SW 25 rotor.

2. FINAL METHOD FOR SUBFRACTIONATING

NUCLEI

The best results obtained in these preliminaryexperiments determined the param eters of thefinal procedure that follows:

Nuclei isolated from 9 gm fresh liver tissue wereresuspended by careful stirring with a glass rodin 0.88 i sucrose adjust ed to pH 7.8 wit h a fewdrops of Tris just before use. The suspension wasmade up to 12 ml and divided into 4 ml aliquots,using plastic tubes for the Spinco 40.3 rotor. Thetubes were capped, introduced into the water-filled chamber of the magnetostriction oscillatorand submitted to vibrations of 10,000 cycles/see.

FIGURE 3 Part of an isolated nucleus in a nuclear fraction prepared from guinea pig liver.The nucleus shows a large nucleolus surrounded and penetrated by chromatin threads(Chl and eh2 respectively). The nucleolar mass (nm) has a distinct fine granular texture.

The nucleolar lacunae (1) appear distended and "empty," presumably extracted, exceptfor those which contain some chromatin threads. The nuclear envelope marked ne bearsattached ribosomes (r) on its outer membrane.

The nucleoplasm contains, in addition to chromatin threads, some large, dense granules(peg) and clusters of fibrillar and/or granular material of finer texture (eft. The formermay correspond to Watson's perichromatin granules (51).

Pell et fixed in 3.3 M HC H0 in 0.1 M phos phat e buffer, pH 7.6, followed by 0.089 MOsO4 in the same buffer; embedded in methacrylate. Section stained with Pb(OH)2.X 47,000.

FIGURE ~ Nucleolar subfraction. A comparison with Fig. 3 shows that isolated nucleoliretain the size and general morphology they have in intact nuclei; the nucleolar massappears, however, more compact and as a result i ts granular t exture is less clearly visible.Most lacunae are "empty" and seem to be limited by a relatively sharp boundary (b).

The chrornatin threads have aggregated into coarser strands (chl, ch2) than in intact

nuclei and appear as pale, relatively homogeneous masses either in the lacunae (ch2) orat the peri phery of the nucleoli (Chl). The nucleolus marked n, just grazed by the secti on,shows no cut open lacuna.

Pell et fixed in 0.039 M OsO4 in 0.88 M sucrose a djus ted t o pH 7.8 wi th Tris; met h-acrylate embedded. Section stained with Pb(OH)2. X 47,000.

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for 5 minutes , fo l lowed by 2 to 3 minutes ofcool ing of the tubes in an ice ba th . This cyc le wasrepea ted 4 t imes to g ive a to ta l of 20 minute sonict r e a t m e n t . I n a f e w e x p e r i m e n t s t h e o p e r a t i o n w a srepea te d un t i l a to ta l of 27 minu te sonica t ion wasreached. Af ter sonica t ion , the suspens ion wasdi lu ted 1 :3 wi th 0 .88 M sucrose , ad jus ted to pH

7.8 wi th Tr is , and 16 to 18 ml a l iquots ther eofwere laye red on 10 ml of 2 .2 M sucrose ad jus ted int h e s a m e w a y t o t h e s a m e p H . T h e t w o l a y e rgradients were subsequent ly cent r i fuged a t 20 ,000g for 60 minutes in an SW 25 ro tor. At the end ofthe cent r i fugat ion the tubes showed: (a ) A t rans-p a r e n t s u p e r n a t e (nucleoplasmic subfraction H) inthe upper layer which was co l lec ted by p ipe t t ing .I t was opt ica l ly empty in the phase cont ras tmicroscope , but upon cent r i fugat ion a t 105 ,000g for 120 minutes ( ro tor No. 40) gave a v is ib lepe l le t and a superna te , he reaf te r re fer red to asfinal supernate. B o t h c o n t a i n e d T C A p r e c i p i t a b l enucleop rote ins or pro te ins . (b) A ban d of whi tem a t e r i a l (nucleoplasmic sub]faction I) a t the 0 .88~/2 . 2 M in ter face , which was co l lec ted by cut t ingthe tube be low i t . In the phase cont ras t micro-scope i t conta ined f i lamentous e lements andoccas ional ly a few nucleol i . Upon d i lu t ion wi thH20 to a f ina l sucrose concent ra t ion of ~- ,0 .88M and cent r i fugat ion a t 60 ,000 g for 60 minutes( ro tor No. 40) i t y ie lded a superna te and a whi tep e l l e t . T h e l a t t e r c o n t a i n e d a l l t h e T C A p r e -c ip i tab le ma ter ia l of the band . (c) A pe l le t a t theb o t t o m (nucleolar subfraction) which in the phasecont ras t microscope appeared to cons is t main lyo f n u c l e o l i w i t h a f e w c o n t a m i n a t i n g f i l a m e n to u ss t ruc tures , p resumably chromat in threads . Theelec t ron microscopy of these nuclear subfrac t ionsis descr ibed in de ta i l in the next sec t ion .

3. ELECTRON MICROSCOPY

a . M E T H O D S P r e l i m i n a r y e x p e r i m e n t s s h o w e dtha t the f ina l appea ranc e of i so la ted nucleol i was

markedly inf luenced by the f ixa t ion procedure .B y c o m p a r i s o n w i t h c o n t r o ls m a i n t a i n e d i n t h esonica t ion medium, nucleol i swel led and los tdens i ty whe n f ixed in 3 .3 M formald ehyde in 0 .88M sucrose wi th ace ta te -Veron al buffer ( f ina lpH ~8 .3) ; or in 0 .039 M OsO4 in 0 .88 M sucrosebuffered wi th ace ta te -Ve ronal ( f ina l pH 7 .8), o r

wi th 0 .1 M phosph ate ( f ina l pH 7 .1) . S imi larresu l t s were obta ined when the i so la ted nucleol iwere f ixed f i r s t in HCHO and then in OsO4followin g proc edu res (b) and (c) descr ibed in (8).The re was no de tec tab le swel l ing even af te r 15-hou r susp ension in 0 .039 M OsO4 in 0.88 M sucrosew h e n t h e c o n c e n t r a t i o n o f th e p h o s p h a t e b u ff erwas reduced to 0 .05 M and the pH lowered to 6 .0or 5 .5 Equal ly sa t i s fac tory resu l t s were obta inedwi th 0 .039 m OsO4 in 0 .88 M unbuffered sucroseor in 0 .88 M sucrose ad jus ted to pH 7 .8 wi th T r is .

The las t two mixtures were used to f ixin situpel le t s of nuc le ar subfrac t ions ob ta ined as de-scr ibed , the o ther condi t ions for f ixa t ion , em-bedding , and m icroscopy be ing the same as g ivenin (8) for nuclear pellets.

b. R E S U LT S : NucIeolar subfractions: T h e p e l -l e t w a s m a i n l y c o m p r i s e d o f r e c o g n i z a b l e n u c l e o l iwhich genera l ly re ta ined the s ize and shape theyhad in in tac t l iver ce l ls and in i so la ted nucle i(F ig . 1) . The nucleolar lacunae were , however,f r e q u e n t l y e n l a rg e d a n d t h e i r c o n t e n t d i l u t e d o rext rac ted . C onverse ly the mater ia l sur roundingt h e l a c u n a e a p p e a r e d m o r e c o n d e n s e d t h a n insitu and i t s gran ular tex ture was less ev ide nt(cf.Figs . 3 and 4) . In many p laces the dense nucleolarm a t e r i a l w a s s h a r p l y d e l i n e a t e d t o w a r d s t h eouts ide of the lacuna e , g iv ing the impress ion tha ta def in i te boundary ex is ted a t tha t leve l . In manyother p laces the nucleol i appeared in cont inui tyw i t h r e c o g n i z a b l e c h r o m a t i n m a t e r i a l l o c a t e de i t h e r w i t h i n t h e l a c u n a e o r a t t h e p e r i p h e r y o fthe nucleolar masses (Figs. 5 and 6). This associa-t ion was c lear ly demonst ra ted in sec t ions s ta ined

FIGURES 5 and 6 These micrograp hs show isolated nucleoli surrounded by a shell ofnucleolus-associated chromatin (chl) which appears thin and more or less continuousin Fig. 5, and discontinuous but thicker in Fig. 6. The chromatin located in some nucleolarlacunae is marked ch2. Remnants of nuclear surface are visible in Fig. 5 atns.

The nueleolar mass, which in this preparation appears less dense than chromatin, iscondensed to a point that obscures its granular texture.

Differences in the size of the nucleolar profiles and in the number and size of theirlacunae are mainly due to the position of the sections: medial in Fig.5, lateral in Fig. 6.Pellet fixed in 0.089 M Os04 in 0.88 M sucrose adjusted to pH 7.8 with Tris; em bedde d

in nlethacrylate. Sections stained with u ranyl ace tate followed by Ph(OH)2. Fig. 5,X 78,000; Fig. 6, X 66,000.

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w i t h b o t h u r a n y l a c e t a t e a n d l e a d h y d r o x i d e(cf. r e f e r e n c e 8 ) i n w h i c h t h e d e n s i t y o f c h r o m a t i nw a s m a r k e d l y i n c r e a s e d . T h i s " n u c l e o l u s - a s s o c i -a t e d c h r o m a t i n " (cf. r e f e r e n c e 2 1 ) s e e m e d t or e s i s t s o n i c d i s r u p t i o n m o r e s u c c e s s f u l l y w h e nl o c a t e d i n a r e l a t i v e l y t h i n l a y e r b e t w e e n t h en u c l e a r s u r f a c e a n d t h e n u c l e o l a r m a s s . I n a d d i -t i o n to b e i n g c o n t a m i n a t e d b y t h is t y p e o f c h r o m -a t i n d e r i v e d f r o m l i v e r c e ll n u c l e i , t h e p r e p a r a t i o nc o n t a i n e d b l o c k s o f d e n s e c h r o m a t i n ( F i g s. 1 a n d2 ) w h i c h , o n a c c o u n t o f t h e i r s h a p e , d e n s i t y, a n dt e x t u r e , c o u l d b e t r a c e d b a c k t o t h e n u c l e i o f v o nK u p f f e r c e l ls a n d l e u c o c y te s . A s a l r e a d y m e n -t i o n e d , t h e s e n u c l e i a r e c h a r a c t e r i z e d b y a p e r i p h -e r a l c o n c e n t r a t i o n o f c o n d e n s e d c h r o m a t i n ;a l t h o u g h t h e y a r e i n m i n o r i t y i n o u r n u c l e a rf r a c ti o n s, t h e i r c h r o m a t i n i s f r a g m e n t e d i n t ob l o c k s r a t h e r t h a n b e i n g f i n e l y d i s p e r s e d b y s o n i ct r e a t m e n t , a n d t h e s e b l o c k s a r e l a rg e a n d h e a v ye n o u g h t o b e c o m e t h e m o s t i m p o r t a n t c o n -

t a m i n a n t i n th e n u c l e o l a r p e l le t s. C o u n t s i n ar e p r e s e n t a t i v e p r e p a r a t i o n g a v e 5 2 5 c h r o m a t i nb l o c k s f o r 8 61 n u c l e o l i . O n a v o l u m e b a s i s th i sc o n t a m i n a t i o n i s e s t i m a t e d a t ~ 3 0 p e r c en t .

Nucleoplasmic subfractions:T h e t w o f r a c t i o n sc o n s i s t o f t h e s a m e m a t e r i a l : d e n s e , k i n k y, f i l a -m e n t o u s o r r i b b o n - l i k e s t r u c t u r e s o f v a r i a b l el e n g t h a n d ~ 4 0 t o 50 m / z i n d i a m e t e r ( F ig s . 7 a n d8 ) . T h e s e f i l a m e n t s , w h i c h a r e s h o r t e r i n t h es e c o n d , a n d l o n g e r i n t h e f i r s t f r a c t i o n , d e r i v ef r o m t h e f i n e t e x t u r e d r e g i o n s o f t h e n u c l e o p l a s mv i s i b le i n i n t r a c e l l u l a r a s w e l l a s i n i s o l a t e d n u c l e i ,a n d h e n c e r e p r e s e n t t h e n u c l e a r c h r o m a t i n . T h i s

c h r o m a t i n a p p a r e n t l y u n d e r g o e s a g g r e g a t i o n o rr e o r g a n i z a t i o n i n t o c o a r s e r f i la m e n t s u p o n r u p t u r eo f t h e n u c l e a r e n v e l o p e , a n a s s u m p t i o n s u p p o r t e db y t h e e x i s te n c e o f i n t e r m e d i a t e a p p e a r a n c e s i np a r t i a l ly r u p t u r e d n u c l e i, a n d b y t h e d e m o n -s t r a t i o n o f f i n e r f i b r il s ( 2 .5 t o 5 . 0 m # ) w i t h i n t h ei s o l a t e d f i l a m e n t s ( F i g . 9 ) . T h e b o t t o m l a y e r o ft h e f i r st f r a c t i o n c o n t a i n s f r a g m e n t e d n u c l e i i nw h i c h t h e r e o r g a n i z a t i o n m e n t i o n e d i s e v i d e n t( F i g . 8 ) a n d i n w h i c h t h e c o a r s e n e d c h r o m a t i nt h r e a d s f r e q u e n t l y f o r m e l a b o r a t e n e t w o r k s. T h es a m e l a y e r i s c o n t a m i n a t e d b y a f e w i n ta c tn u c l e o l i . B o t h f r a c t i o n s a r e f r e e o f r e c o g n i z a b l en u c l e o l a r f ra g m e n t s . W e w e r e n o t a b l e t o t r a c et h e f a te o f t h e o t h e r n u c l e a r c o m p o n e n t s ( c o ar s en u c l e o p l a s m i c g r a n u l e s a n d f i l a m e n t s ) d u r i n g t h ef r a c t i o n a t i o n p r o c e d u r e b e c a u s e , i f s t il l p r e s e n t ,t h e y d o n o t d i f f e r e n o u g h i n t h e i r g e n e r a l a p -p e a r a n c e f r o m t h e m a i n c o m p o n e n t o f t h e n u c l e o -p l a s m i c f r a c t i o n s ,i .e. , t h e f r a g m e n t s o f c oa r s e n e d

c h r o m a t i n f i la m e n t s . T h e f a t e o f t h e n u c l e a re n v e l o p e i s a l s o u n k n o w n .

c . C O M M E N TS " I t s h o u l d b e c l e a r t h a t t h en u c l e a r s u b f r a c t io n a t i o n a c h i e v e d r e p r e s e n tso n l y t h e f i r st a p p r o x i m a t i o n t o t h e s o l u t i o n o f ad i f f ic u l t p r o b l e m . T h e n u c l e o l a r s u b f r a c t i o n i sr e c o g n i z a b l y c o n t a m i n a t e d b y c h r o m a t i n f r o mt w o d i f f e r e n t s o u r ce s ( p e r i n u c l e o l a r c h r o m a t i n a n dc h r o m a t i n b l oc k s) . T h e n u c l e o p l a s m i c f r a c ti o n sa r e a p p a r e n t l y m o r e h o m o g e n e o u s , b u t t h i sp r o b a b l y r e f l e c t s t h e i n s u f f i c i e n c y o f o u r m o r-p h o l o g i c a l c r i t e r i a r a t h e r t h a n a t r u l y s u cc e s s f u lf r a c t io n a t i o n . O n t h e o n e h a n d , k n o w l e d g e a b o u t

FIGURE 7 Second nucleoplasmic subfraction.The prepara t ion cons is ts of smal l f rag-ments of chromat in threads(ch) which appear less uni form and genera l ly th icker thanthose seen in isolated nuclei (see Fig. 3). F or this reason, i t c an no t be ascertain edwhether or not the pe lle t conta ins o ther nuc leoplasmic components know n to ex is tinsitu (large, dense granules and clusters of f ine granules or f i laments).

Pellet f ixed in 0.039 M OsO4 in 0.88 M sucrose adjus ted to pH 7.8 with T ris;embedded in me thacry la te . Sec t ion s ta ined wi th Pb(OH)2. X 31 ,000.

FIGURE 8 Firs t nudeoplasmic subfraet ion(bot tom of the pe l le t ) . At th is leve l theprepara t ion cons is ts main ly of f ragments of nuc lear ehromat in , each appear ing asa ne twork of recognizable chromat in threads(n f l to nfa) . Some of the f ragments

comefrom the sur face of the nucleus as indica ted by the curva ture of one of the i rsides (nf l , nf4) .Denser and coarser f ragments(ck) probably represent von Kupffer

ce l l chromat in .Nucleol i (n) and recognizable nuclcolar f ragments a re only occas ionally encountereda t the very bot tom of the pe l le t . The upper ~ of the pe l le t i s s imi la r in composi t ionto the second nucleop lasmic sub fraction (Fig. 7).

Prepara t ion for e lec t ron microscopy as for F ig . 5 . X 31 ,000.

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the organiza t ion of in te rphase nucle i a t th isd imens ional leve l i s s t i l l very l imi ted , and on theo t h e r h a n d w e c a n n o t r e l i a b l y t r a c e t h r o u g h t h ef r a c t i o n a ti o n p r o c e d u r e a n u m b e r o f n u c l e o p la s m i cc o m p o n e n t s r e c o g n i z a b l e in situ. For th is reason ,we prefer to re fer to these f rac t ions as nuc leo-p l a s m i c , a l t h o u g h t h e i r m a i n c o m p o n e n t i s

FIGURE 9 Chromatin strands in the 2nd nucleo-plasmic subJraction.The strands show dense dots (d)and lines (1) on their sectioned surface, presumablyprofiles of sectioned fibrils ~5 to 30 A in diameter.

Preparation for electron microscopy as for Fig. 7except that the section was doubly stained in uranylaceta te followed by Pb(OH)~. X ~50,000.

def in i te ly chrom at in . I t should a lso be noted tha ta m o n g o u r n u c l e a r s u b f r a c t io n s t h e r e i s n o e q u i v a -len t of the " nuc lea r sap " o f the cy to logis t s.

B . C h e m i s t r y

1. GRoss CHEMICAL COMPOSITION

Like the parenta l nuc lear f rac t ion (8) , thenuclear subfrac t ions (Table I ) showed con-

s i d er a b l e v a r i a ti o n s i n t h e a m o u n t s o f R N A ,D N A a n d p r o t e i n r e c o v e r e d p e r g r a m f re s h ti ss u e.The re la t ive concent ra t ions expressed as per-centages or ra t ios were not iceably more s tab le .

The nucleolar f rac t ion represents 6 to I1 percent of the or ig ina l nuc lear mass and cons is t s of~ 4 p e r c e n t R N A , 9 t o 1 0 p e r c e n t D N A , a n d 8 6to 87 per cent pro te in (Table I ) , the cor respondingf igures for the nuc leoplasmic f rac t ions be ing ~2per cent , 10 to 14 per cent , and 84 to 88 per cent ,respec t ive ly. (For these ca lcu la t ions the amounto f R N A + D N A + p r o t e i n w a s e q u a t e d t o 1 00 .)The nucleolar subfrac t ions accounted for 11 to 17p e r c e n t o f t h e R N A , 5 t o 7 p e r c e n t o f t h e D N Aand 6 to 10 per cent of the pro te in o f the nucle arf r a c ti o n ( Ta b l e I I ) . I n t e r m s o f D N A a n d p r o t e in ,R N A w a s 2 t i m e s m o r e c o n c e n t r a t e d i n t h enucleolar than in the nucleoplasmic f rac t ions .S ince the nucleolar f rac t ion i s heavi ly contamin-a t e d b y p e r i n u c le a r c h r o m a t i n a n d p e r i p h e r a lc h r o m a t i n b l oc k s, a n a t t e m p t w a s m a d e t o re d u c et h is c o n t a m i n a t i o n b y m o r e e x t e n d e d s o n ict rea tment . The resu l t s obta ine d af te r 27 minutesof sonic nuclear d is rup t ion d id not show, however,a n y s t r i k i n g i m p r o v e m e n t o v e r t h e u s u a l p r e p a r a -t ions (Tables I and I I ) .

S i n c e b y m o r p h o l o g i c a l c r i t e r i a a v a i l a b l e a tpresent the nucleoplasmic f rac t ions appearh o m o g e n e o u s , i t m a y b e t e n t a t i v e ly c o n c l u d e dt h a t t h e i r R N A r e p r e s e n t s c h r o m a t i n o r c h r o m o -some-assoc ia ted RNA in situ. The conclus ion i sbased on the assumpt ion tha t sonic t rea tmentdoes not resu l t in ex tens ive nucleolar d is rupt iona n d c o n t a m i n a t i o n o f t h e n u c l e o p l a s m f r a c ti o n s

b y u n r e c o g n i z a b l e n u c l e o l a r f r a g m e n ts . T h eRN A of the nuc leolar f rac t ion , however, i s c lear lya m i x t u r e o f n u c l e o l a r a n d c h r o m o s o m a l R N A .If a l l the nucleolar DNA is cons idered to representc h r o m a t i n c o n t a m i n a t i o n , t h e n ~ 4 0 p e r c e n t o fthe nucleola r RN A is a l so of chro mat in or ig in .

2. NACL EXTRACTIONS

RESULTS: Tab le I I I g ives the resul t s of twoe x p e r i m e n t s i n w h i c h t h e R N A , D N A , a n d p r o t e i nc o n t e n t s w e r e d e t e r m i n e d i n n u c l e o p r o t e i nf rac t ions obta ined by NaC1 ext rac t ion f romnucleolar and nuclcoplasmic subfrac t ions . In thecase of nuc leola r subfrac t ions, f igures a re g iven

only for the sa l t residue , for the ex t rac t ion remov esl i t tl e mater ia l and not more than 5 to l0 p er cento f t h e t o t a l n u c l e o l a r R N A . S i n c e t h e n u c l e o l a rs u b f ra c t io n i s k n o w n t o b e c o n t a m i n a t e d b y

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TA B L E I

Chemical Composition o f Nuclea r Sub fractio ns

N u c l e o p l a s m i c s u b f r a c t i o n I i s t h e m a t e r i a l a t t h e 0 . 88 M / 2. 2 M s u c r o s e i n t e r fa c e ; n u c l e o p l a s m i cs u b f r a c t i o n I I i s t h e e n t i r e m a t e r i a l i n t h e 0 .8 8 M s u c r o s e s u p e r n a t e .

R N A , D N A , a n d p r o t e i n w e r e d e t e r m i n e d o r c a l c u l a t e d a s i n (8 ).

No. of ex- RNA/ RNA/

periments Subfraction RNA DNA Protein Protein DNA

3

3

mg/gm* per centll mg/ gm* per centl] mg/gm* per cenO[

N u c l e o l a r ~ 0 . 0 0 9 4 . 0 0 . 0 2 2 1 0 . 0 0 . 1 9 8 6 . 0 0 . 0 4 7 0 . 6 4N u c l e o p l a s m i c I 0 . 0 1 4 2 . 0 0 . 1 0 0 1 3 .4 0 . 6 3 8 6 . 6 0 . 02 4 0 . 2 6N u c l e o p l a s m i c I I 0 . 0 2 9 2 . 2 0 . 1 7 3 1 3 . 5 1 . 0 9 8 4 . 3 0 . 0 2 6 0 . 2 8

N u c l e o l a r 0 . 0 0 9 4 . 1 0 . 0 2 0 8 . 9 0 . 1 9 8 7 . 0 0 . 0 4 7 0 . 4 5N u c l e o p l a s m i c I 0 . 0 3 2 2 . 2 0 . 1 8 6 1 2 . 0 1 . 2 3 8 5 . 0 0 . 0 2 6 0 . 1 7N u c l e o p l a s m i c I ! 0 . 0 3 9 2 . 0 0 . 2 0 0 1 0 . 7 1 . 6 4 8 7 . 3 0 . 0 2 4 0 . 1 9

N u c l e o l a r $ 0 . 0 11N u c l e o p l a s m i c I 0 . 0 1 8N u c l e o p l a s m i c I I 0 . 0 3 2

* p e r g r a m o r i g i n a l t i ss u e .s u b f r a c t i 0 n a t i o n a f t e r 2 0 - m i n u t e so n i c t r e a t m e n t .

s u b f r a c t i o n a t i o n a f t e r 2 7 - m i n u t e s o n i c t r e a t m e n t .II t h e s u m o f R N A -+- D N A --k p r o t e i n i n e a c h s u b f r a c t i o n b e i n g c o n s i d e r e d 1 00 t h e v a l u e s g i v e n a r e t h e a v e r a g e s o f t h e t h r e e e x p e r i m e n t s . T h e v a l u e s i n t h e 2 7 - m i n u t e s o n i c a t i o ne x p e r i m e n t s w e r e w i t h i n a f ew p e r c e n t o f t h e a v e r a g e , w h i l e t h o s e i n t h e 2 0 - m i n u t e s o n i c a t i o n e x p e r i -m e n t s w e r e w i t h i n 2 0 p e r c e n t o f t h e a v e r a g e .

TA B L E I I

Distr ibution of RNA , DN A and Protein in Hepatic Nuclear Subfraetions(Guinea Pig)

T h e d a t a i n Ta b l e I w e r e r e c a l c u l a t e d t o g iv e t h e p e r c e n t a g e d i s t r i b u t i o n o fR N A , D N A a n d p r o t e i n a m o n g n u c l e a r s u b f r ac t i o n s.

Distributionl]No. of Per cent of total

experiments Subfractions nuclear ma~ RNA DN A Protein

1"

32

N u c l e o l a r 9 . 8 1 7 . 2 7 . 5 1 0 . 0N u c l e o p l a s m i c I 3 3 . 2 2 8 . 8 3 4 . 0 3 3 . 0N u c l e o p l a s m i c I I 5 7 . 0 5 4 . 0 5 8 . 5 5 7 . 0N u c l e o l a r 6 . 3 11 .5 4 . 9 6 . 3N u c l e o p l a s m i c I 4 0 . 7 4 0 . 0 4 6 . 0 5 0 . 0N u c l e o p l a s m i c I I 5 3 . 0 4 8 . 5 4 9 . 1 5 3 . 0

* S u b f r a c t i o n a t i o n a f t e r 2 0 - m i n u t e s o n ic t r e a t m e n t .S u b f r a c t i o n a t i o n a f t e r 2 7 - m i n u t e s o n i c t r e a t m e n t .

C a l c u l a t e d a s p e r c e n t a g e o f t h e s u m o f R N A , D N A , a n d p r o t e i n o f w h o l e n u c l e i .11 C a l c u l a t e d a s p e r c e n t a g e o f t h e t o t a l R N A , o r D N A , o r p r o t e i n i n e a c h o f t h et h r e e s u b f r a c t i o n s .

c h r o m a t i n , a n d s i nc e t he n u c l e o p l a s m i c s u b -f r a c t i o n s c o n t a i n a s i z a b l e a m o u n t o f s a l t e x -

t r a c t a b l e R N A ( s ee b e l o w ), i t is c o n c e i v a b l e t h a tt h e s m a ll a m o u n t o f R N A e x t r a c t e d f r o m t h e

n u c l e o l a r s u b f r a c t i o n i s e n t i r e l y d u e t o n u c l e o -p l a s m i c c o n t a m i n a t i o n . U s i n g t h e s a l t e x t r a c t i o nd a t a i n Ta b l e I I I a n d a s s u m i n g t h a t t r u ly

n u c l e o l a r R N A i s n o t e x t r a c t a b l e b y s a lt , it c a n

RACHELE MAaaIo, PHILIP SIEKEVITZ, AND GEORGE E. PALADEIsolated Nuclei . I I 30 5


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b e c a l c u l a te d t h a t 2 5 t o 4 0 p e r c e n t o f th e R N Ai n t h e n u c l e o l a r s u b f r a c t i o n i s o f n u c l e o p l a s m i co r i g i n . T h i s f i g u r e a g r e e s w i t h o t h e r e s t i m a t e s o fn u c l e o p l a s m i c c o n t a m i n a t i o n : ~ 3 0 p e r c e n t o nt h e b a s i s o f m i c r o s c o p i c o b s e r v a t i o n s , a n d ~ 4 0p e r c e n t o n t h e b a s i s o f D N A d i s t r ib u t i o n .

T h e s a l t e x t r a c t o f th e p e l l e t o b t a i n e d b yc e n t r i f u g i n g ( 1 0 5 , 0 0 0 g f o r 6 0 m i n u t e s ) t h ec o m b i n e d n u c l o p l a s m i c f r a c t io n s I a n d I Ia c c o u n t e d f o r 1 5 t o 2 0 p e r c e n t o f t h e R N A , 6 0t o 8 0 p e r c e n t o f t h e D N A , a n d a b o u t 3 5 p e r c e n t

f r a c t i o n ( r a t ) a r e s i d u e i n w h i c h t h e y r e c o g n i z e d ,b y l i g h t m i c r o s c o p y, i n t a c t n u c l e o l i i n a d d i t i o n t or e s i d u a l c h r o m o s o m e s .

3 . N U C L E O T I D E C O M P O S I T IO N O F V A R I O U S

N U C L E A R R N A ' s

R E S U LT S: Ta b l e I V g i v e s t h e n u c l e o t i d ec o m p o s i t i o n o f t h e R N A ' s o f t h e n u c l e o l a r a n dn u c l e o p l a s m i c f r a c t i o n s a n d o f t h e s a l t in s o l u b l ea n d s a l t e x t r a c t a b l e R N A ' s o b t a i n e d f r o m s o m eo f t h e s e f r a c t i o n s .

T A B L E I I I

The R NA , D N A , and Protein Content of Na Cl Extracts and Residues ofNuclear Subfractions

E x t r a c t i o n w i t h 1 M N a C I w a s c a r r i e d o u t o v e r n i g h t i n t h e c o l d w i t h s t i r r in g

N u c l e a r s u b f r a c t io n s R N A D N A P r o t e i n

N u c l e o l a rN a C 1 r e s i d u eN u c l e o p l a s m i c

P e l l e tN a C 1 r e s i d u eN a C I e x t r a c t

F i n a l s u p e r n a t e

mg per cent~ mg per cent~ mg per cent~

0 . 0 6 6 1 8 . 1 " 0 . 0 6 5 4 . 2 1 . 5 0 9 . 5

0 . 1 8 7 5 1 . 8 0 . 1 7 5 11 . 3 6 . 7 3 4 2 . 80 .077 21 . 1 1 .200 78 .0 5 .62 35 .90 . 0 3 2 9 . 0 0 . 1 0 0 6 . 5 1 . 8 5 11 . 8

N u c l e o l a rN a C 1 r e s i d u e 0 . 1 0 8 1 6 . 3 " 0 . 2 4 5 8 . 0 1 . 7 2 7 . 5

N u c l e o p l a s m i cP e l l e t

N a C 1 r e s i d u e 0 . 4 0 9 6 1 . 5 0 . 4 8 0 1 5 . 8 8 . 8 4 3 8 . 5N a C 1 e x t r a c t 0 . 0 9 7 1 4 . 6 1 . 9 5 0 6 4 . 0 7 . 8 9 3 4 . 5

F i n a l s u p e r n a t e 0 . 05 1 7 . 6 0 . 3 7 5 1 2 .2 4 . 4 9 1 9 . 5

* I n o t h e r e x p e r i m e n t s i t w a s f o u n d t h a t o n l y 5 t o 1 0 p e r c e n t o f t h e t o t a l n u c l e o l a rR N A (i.e. 1 t o 2 p e r c e n t o f t o t a l n u c l e a r R N A ) c o u l d b e e x t r a c t e d w i t h 1 M N a C 1 .

P e r c e n t a g e o f R N A , o r D N A , o r p r o t e i n i n t h e t o t a l r e c o v e r a b l e m a t e r i a l o f t h es u b f r a c t i o n s .

o f t h e p r o t e i n o f th e n u c l e i ( m o r e p r e c i s e l y o f t h et o t a l R N A , D N A , a n d p r o t e i n r e c o v e re d i n a l ln u c l e a r s u b f r a c t i o n s ) . T h e f i n a l s u p e r n a t e o f th ec o m m o n p e l l e t c o n t a i n e d ~ 1 0 p e r c e n t o f t h en u c l e a r R N A a n d D N A t o g e t h e r w i t h 1 0 t o 20p e r c e n t o f i t s p r o t e i n . T h e f i g u re s g i v e n c o n c e r nt h e w h o l e s u p e r n a t e , f o r i n t h i s c a s e t h e s a l ti n s o l u b l e f r a c t i o n a p p e a r s t o b e n e g l i g ib l e .

C O M M E N TS : O u r f a i l u r e t o e x t r a c t a s i g -n i f i ca n t a m o u n t o f n u c l e o l a r R N A b y s a lt c a n b ec o r r e l a t ed w i t h t h e r e s ul t s p u b l is h e d b y Z b a r s k i ia n d G e o r g i e v ( 2 2 ) : u s i n g a l o w e r s a l t c o n c e n -t r a t io n , t h e y o b t a i n e d f r o m a h e p a t i c n u c l e a r

T h e s a l i e n t f i n d i n g i s t h e p e c u l i a r b a s e c o m -p o s i ti o n o f t h e R N A o f t h e n u c l e o l a r f r a c t i o n : i ti s c h a r a c t e r i z e d b y a h i g h G M P, a n d a l o w A M Pa n d C M P c o n t e n t , a n d a s su c h i t is d i f f e re n t f r o mt h e R N A ' s o f a ll t h e o t h e r c e ll f r a c ti o n s e x a m i n e d ,n u c l e a r a s w e l l a s c y t o p l a s m i c(cf. Ta b l e I V i nr e f e re n c e 8) . It s A M P / C M P r a t io , fo r i n s t a n c e ,i s c l os e t o 1 , w h e r e a s t h e c o r r e s p o n d i n g v a l u e f o rn u c l e o p l a s m i c R N A ' s i s n e a r l y d o u b l e. T h e s a l te x t r a c t o f th e n u c l e o l a r R N A r e t a i n s t h is p e c u l i a rc o m p o s i t i o n. T h e t w o n u c l e o p l a s m i c s u b f r a c t io n s ,a l r e a d y k n o w n t o b e s i m i l a r i n t h e i r g r o s s c h e m i s -t r y, a r e a l s o s i m i l a r i n t h e n u c l e o t i d e c o m p o s i t i o n

3 0 6 THE JOURNAL OF CELL BtOLOG Y VOLUME 18, 1963


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of the i r RN A's . This f inding re inforces ourcontent ion tha t the two subfrac t ions do notr e p r e s e n t d i s t i n c t n u c l e a r c o m p o n e n t s , b u tref lec t d i ffe rences in the d egree of f ragm enta t io no f c h r o m a t i n . I n o t h e r e x p e r i m e n t s t h e t w on u c l e o p l a s m i c f r a c t i o n s w e r e c o m b i n e d , a n dcent r i fuged to y ie ld a superna te and a pe l le t whichwere subjec ted to sa l t ex t rac t ion (Table IV) . Th ed a t a o n t h e n u c l e o t i d e c o m p o s i t i o n o f th e N a C 1i n s o l u b l e a n d N a C 1 e x t r a c t a b l e R N A ' s a r e h a r dt o i n t e r p r e t b e c a u s e t h e y c o n c e r n o n l y t w o e x p e r i-ments and show re la t ive ly smal l d i ffe rences inbase composi t ion . Tenta t ive ly they sugges t tha tt h e t w o R N A ' s a r e d i f f e r e n t . O n e c o u l d p o i n t o u tin th is respec t tha t in both exper iments theU M P / G M P r a t io w a s c l os e t o 1 i n t h e R N A o f

c u r r e n c e o f r i b o n u c l e o p r o t e in s i n c h r o m o s o m e sa n d c h r o m a t i n , c o r r e s p o n d i n g t o o u r n u c l e o -p lasmic f rac t ions , has a l ready been s tudied andd i s c u s s e d b y K a u f m a n n et al. (23) , Mirsky andR i s ( 2 4 ) , a n d Ta y l o r a n d M c M a s t e r ( 2 5 ) . Vi n c e n t( 7 ) h a s m e n t i o n e d t h e h i g h G M P c o n t e n t o fn u c l e o l a r R N A a s c o m p a r e d t o c y t o p l a s m i cRNA in s ta r f i sh oocytes . Eds t r6m et al. (26) alsof o u n d t h a t n u c l e o l a r R N A h a s a h i g h e r G M P a n da lo w e r C M P c o n t e n t t h a n c y t o p l a s m i c R N A ,again in Asterias rubens oocytes . To th is ex tentwe conf i rm the i r resu l t s , bu t the agreement i snot genera l : they (26) d id not f ind a low AM Pc o n t e n t i n t h e n u c l e o l a r R N A a s w e d i d , a n dc o n v e r s el y w e d i d n o t d e t e c t a h ig h A M P c o n t e n tin the nucleoplasm, as they d id (26) . From

TA B L E I V

Nucleotide Composition of the RNA's Extracted from Nuclear Subfractions

Moles/100 moles of total nucleotidesNuclear subfractions UMP GMP CMP AMP A-~-C/G-}-U

Nucleo lar* 22 .4 50 .8 14 .5 12 .3N u c l e o p l a s m i c

I (midd le interfac e)* 28.7 33.8 14.9 22.6I I (upper layer )* 26 .4 34 .9 14 .8 23 .9

N u c l e o p l a s m i c p e l l e tNaG1 residue~ 32.0 33.5 12.0 22.5NaC1 extrae t~ 25.2 35.2 15.6 24.0

Fina l superna te 26 .0 33 .0 14 .0 27 .0

0 .37

0.600.63

0.530.660.69

* Ave rage of three ex per iments .Average of two exper iments .

the res idue and cons ide rably smal le r than 1 intha t of the ex t rac t . Ot he r d i ffe rences be twee nthese two RNA's wi l l be d iscussed in the nextsection.

T h e n u c l e o t i d e c o m p o s i t io n o f th e R N A o f t h ef ina l superna te i s qui te s im i lar to tha t of the R NAof the sa l t ex t rac tab le n ucleopla smic f rac t ion .S i n c e th e f i n a l s u p e r n a t a n t R N A i s a l m o s t c o m -ple te ly sa l t ex t rac tab le , the forme r f inding sugges tst h a t t h e n o n - s e d i m e n t a b l e n u c l e o p r o t e i n r e p r e -sents tha t f ine ly d ispersed ch rom at in of then u c l e o p l a s m i n w h i c h , s a lt e x t r a c t a b l e R N Ap r e d o m i n a t e s .

COMMENTS: Som e of ~he_ fea tures of theR N A ' s o f o u r n u c l e a r s u b f r a c ti o n s a r e s i m i l a r t othose descr ibed in the l i te ra ture for comparablemater ia l s i so la ted f rom other sources . The oc-

autoradiographic s tudies i t was a l so infer redt h a t t he A M P / C M P r a ti o o f n u c l eo l a r R N A i sc o n s i d e r a b ly l o w e r t h a n t h a t o f t h e R N A o f t h eres t of the n ucleus (27) (cf. Ta b l e I V ) . T h e s a l tinso luble RN A of whole nuclea r f rac t ions iso la tedf rom l iver and o ther t i ssues (28) has a h igh G M P/U M P r a t io , w h i c h m i g h t r e f le c t t h e h i g h G M Pc o n t e n t o f th e N a C l - i n s o l u b le n u c l e o l a r R N A .

4. PSEUDOURIDYLIC ACID CONTENT

RESULTS: On ly 0 .020 #m oles of ~ /ur id inew e r e f o u n d i n 4 . 4 6 m g t o t a l R N A e x t r a c t e d f r o mo u r n u c l e a r f r ac t i o n. T h e a m o u n t i s e q u i v a l e n tto 1 .25 moles of ~b UM P per 100 moles of UM P.No t races of ~burid ine were found in the RN A'sp r e p a r e d f r o m n u c l e o l a r a n d n u c l e o p l a s m i cf r ac t io n s , t h e s t a r ti n g a m o u n t s o f R N A b e i n g

RACHELE MAGGIO, PHILIP SIEKEVITZ, AND GEORGE ]~. ~)ALADE Isolated Nuclei. I I 30 7


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0.63 mg and 2 .39 mg, respec t ive ly. These negat iveresult s can not be ascr ibed to techn ica l l imi ta t ions ,s ince the procedu re used was capab le of de tec t ing u r i d i n e i n t h e e x p e c t e d a m o u n t s i n R N A ' sknow n to conta in ~b UM P. For examp le , suchresul t s, expressed as percenta ge ~ U M P of UM P,were 12 per cent and 18 per cent for so luble

R N A ' s f r o mE. coli

and guinea p ig l iver, respec t -ive ly. The cor responding da ta in the l i te ra ture a re14 per cent for the so luble RNA ofE. coli (29),25 per cent for the so luble RN A of ra t l iver (15) ,a n d 2 2 p e r c e n t f o r t h e " p H 5 e n z y m e " f r o m t h esame source (30). Fo r the RN A of both hep at ica n d p a n c r e a t i c r i b o s o m e s w e h a v e o b t a i n e d 4p e r c e n t ~b U M P o f U M P. T h e r e a r e n o d i r e c tl ycomparable da ta in the l i te ra ture , bu t a c loseapp roxim at ion i s represented by the f igures of7 .5 per cent (29) and 2 .7 per cent (30) publ i shedf o r t o t a l m i c ro s o m a l R N A . F o r R N A i s o la t e d f r o mE. coli r i b o s o m e s w e o b t a i n e d o n l y 0 . 8 p e r c e n t~b U M P o f U M P a s c o m p a r e d t o 1 . 7 p e r c e n t

found by Osawa (30) .COMMENTS: It is possible that the l imited

a m o u n t o f ~b U M P f o u n d i n t h e t o t a l R N A o f o u rnuclear f rac t ion represents a cy toplasmic con-tamina t ion . I f we assume tha t th i s i s no t the case ,t h a t t h e n u c l e i c o n t a i n s o lu b l e R N A o f t h e i r o w n ,a n d t h a t t h i s R N A c o n t a i n s ~b U M P i n t h e s a m econc ent ra t io n as i t s cy toplasmic counte rpar t , ourd a t a i m p l y t h a t o n l y 5 p e r c e n t o f t h e t o t a l n u c l e a rR N A c o u l d b e s o l u b l e R N A . T h i s i n t e r p r e t a t i o nis in agreement wi th the conclus ion tha t l i t t l e orno so luble RNA occurs in neut ra l ex t rac ts ofthym us nucle i prepara t ions (31) .

I f we take in to ac coun t the l imi t of sens i tiv i tyof our p rocedu re an d the necessar i ly smal l amountso f R N A a v a i l a b l e a s s t a rt i n g m a t e r i a l , a n d a g a i nassume an eq ual ~/, U M P conc ent ra t ion in cy to-p l a s m i c a n d n u c l e a r s o l u b l e R N A , o u r d a t afur ther ind ica te tha t the concen t ra t ion of t ransferR N A m u s t b e b e l o w 1 0 p e r c e n t o f t h e t o t a lR N A o f t h e n u c l e o p l a sm i c s u b f r a c ti o n a n d b e l o w20 per cent of tha t of the nuc leolar subfrac t ion toescape de tec t ion . Accordingly, our resu l t s do notexc lude the presence of t ransfer RN A in l ivernucle i , bu t a re compat ib le only wi th the occur-rence of smal l amounts there in . Meta bol ic ev i -dence for the ex istence of t ransfer RN A in at h y m i c n u c l e i p r e p a r a t i o n h a s b e e n r e p o r t e d ( 32 ).

5. TURNOVER OF RNA's

RESULTS : Pre l im inary exper ime nts wi th shortt ime in vivo labe ling (5, 10, and 30 minu tes)

s h o w e d t h a t t h e h y d r o l y sa t e s o f t h e R N A ' s o fnuclear subfrac t ions a re contaminated , to asurpr i s ing ex tent , by non-nucle ic ac id phosphoruscomp ounds . Al tho ugh the incorpora t ion of a~Pwas very ac t ive , on ly 6 to 12 per cen t of the cou ntsw a s r e c o v e r e d i n t h e i s o l a te d m o n o n u c l e o t i d e s ,the res t be ing present as inorganic phosphate . At

la te r t imes a f te r ~P in jec t ion (1 to 4 hours) , thec o n t a m i n a t i o n , a l t h o u g h n o t i c e a b l y re d u c e d ,r e m a i n e d l a rg e : o n ly ~ 5 0 p e r c e n t o f t h e c o u n tswere recovered in mononucleot ides . As a resu l tof these exper iments , the la te r t ime po in ts werec h o s e n f o r f u r t h e r w o r k o n R N A m e t a b o l i s m ;t h e c o r r e s p o n d i n g d a t a a r e m o r e r e l i a b l e t h a n a te a r l i e r t i m e p o i n t s , b u t t h e a p p r o a c h a d m i t t e d l yc a n n o t d e t e c t r a p i d l y t u r n i n g - o v e r n u c l e a rRN A's i f such are present .

At the 1 hou r poin t (Table V ) , the spec if icradioac t iv i ty of the R NA was s imi lar in thenucleolar and nucleoplasmic subfrac t ions ; i tincreased in para l le l in a l l subfrac t ions be tween

1 and 2 hours , and leve led off in the nucleo plasmicsubfrac t ions be tween 2 and 4 hours . The spec if icac t iv i t ies of the 4 mono nucleo t ides va r ied to ac e r t a i n e x t e n t: t h o se o f A M P a n d U M P w e r es imi lar throughout the nuclear subfrac t ions ; tha to f t h e C M P w a s g e n e ra l ly a b o v e t h e A M P - U M Pl e v e l , e x p e c i a l l y i n t h e R N A o f t h e n u c l e o l a rsubfrac t ion; and f ina lly, tha t of the GM P wasbelow the same leve l in the nucleoplasmic sub-fractions.

S ince no d i ffe rences in the metabol ic ac t iv i t ieso f t he t o t a l R N A ' s o f t h e n u c l e a r s u b f ra c t io n sw e r e d e t e c t e d , t h e i n q u i r y w a s e x t e n d e d t o t h esa l t ex t rac ta b le and sa l t inso luble RN A's obta ine dfrom the same subfrac t ions . The t ime poin tse lec ted for these exper iments was 2 hours , s incep r e vi o u s w o r k h a d r e v e a l e d m a x i m u m R N Alabel ing af te r th i s in te rva l . The resu l t s showed(Table V) tha t the spec if ic rad ioa c t iv i ty of thes a lt e x t ra c t a b l e R N A o f t h e c o m b i n e d n u c l e o -p lasmic sub frac t ions was only ha l f tha t of the sa l ti n s o lu b l e R N A ' s o f t h e s a m e p r e p a r a t i o n . I t w a sa lso ha l f the spec if ic rad ioac t iv i ty of the to ta lR N A o f t h e n u c l e o l a r s u b f ra c t io n . F u r t h e r d i ff e r -e n c e s b e c a m e a p p a r e n t w h e n t h e s p e ci fi c r a d i o -a c t i v i t y o f t h e v a r i o u s m o n o n u c l e o t i d e s w a scons idered . The lowes t va lue was found for theC M P o f t h e s a lt e x t ra c t a b l e R N A o f t h e n u c l e o -p lasmic f rac t ions , and the h ighes t for the CMP ofthe sa l t inso luble RN A o f the same f rac t ions . TheC M P o f t h e t o t a l R N A o f t h e n u c l e o l a r f ra c t i o nshowed an even h igh er spec i f ic rad ioac t iv i ty.

308 THE JOURNALOF CELL BIOLOGY VOLUME18, 1963


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Since the phosphate of the CMP, as isolatedfrom an alkaline hydrolysate, belongs to its nextneighbor in the RNA chain, and since the saltinsoluble RNA of the nucleoplasmic fraction andespecially the RNA of the nucleolar fraction arecharacterized by a high GMP content, our resultssuggest a high frequency of CMP-GMP (rather

than CMP-AMP, or CMP-UMP) sequences in

COMMENTS: The m etab olic propert ies of theRNA of whole nuclear fractions have been re-peatedl y studied in the past. Th e results general lyindicate that it has a greater turnover rate thanthe RNA of other cellular fractions (33-35). As aresult of numerous autoradiographic studies ithas also been inferred that nucleolar RNA is

metabolically more active than any other cellular

TABLE V

Specific Radioactivity of Mono nucleotides Obtained from the RN A' s o f Various Nuclear Subfractionsafter Intravenous Injection of a2p Inorganic Phosphate

The nuclear subfractions and extracts were obtained as described in the text; the R NA was extracted,digested, and the nucleotides isolated by paper chromatography and counted according to themethods described in the text.

Time Per cent ofafter total RNA

injection Nuclear subfractions in fraction

Radioactivi ty (cPM)

RNA* UM P GMP CMP AMP

hrs.

1

mg I~t I~M I~M la~a

Nucleol ar$ 11 70,000 21,900 24,400 30,000 21,300Nucleoplasmic

I (middle interface) 41.5 64,000 21,800 1 9 ,3 0 0 27,000 23,000II (upper layer) 47.5 64,000 20,600 19 , 5 0 0 33,000 20,900

Nucleolar$ 12 .1 1 00 ,0 00 30,000 33,000 53,000 32,000Nucleoplasmie

I (middle inte rface) 39.3 93,000 32,800 27,700 36,500 33,700II (upper layer) 48.6 1 0 0 ,0 0 0 35,500 31,300 45,000 37,000

NucleoplasmicI (middle interface) 39.7 1 0 0 , 0 0 0 36,200 28,500 45,000 36,500

II (upper laye r) 49.1 90,000 32,900 25,000 37,000 33,000

Nuc leo lar 17.2 99,000 35,000 25,600 61,600 34,800Nucleoplasmic pellet

NaC1 res idu e 56.7 96,800 32,740 26,100 47,900 36,800NaC1 extr act 17.8 48,800 20,200 13 ,800 9,900 13 ,300

Fin al supe rnate 8.3 99,500 35,700 28,000 44,000 35,500

* These values were obtained by dividing the sum of the counts in each of the four mononucleotides bythe sum of the amounts of each of these nucleotides after converting ~M nucleotide to mg RNA.

In these experiments, the nucleolar fractions were obtained after 27 minutes of sonication of the nuclearfraction; in the other experiments sonication lasted for 20 minutes. Average of two experiments

the corresponding RNA chains. Conversely thelow specific radioactivity of the CMP obtainedfrom the salt extractable RNA of the nucleo-plasmic fractions may reflect the high frequencyof CMP-GMP and CMP-AMP sequences in thisparticular RNA. This interpretation assumes auniform labeling of all triphosphate precursors ofthe mononucleotides incorporated in the RNAchains.

RNA (36-39), but the possible misleading effectof high RNA concentration in the nucleolus hasbeen discussed by Harr is (27).

Our results indicate clearly the me:abolicdiversity of nuclear RNA and confirm the con-comitant incorporation of asp in chromosomaland nucleolar RNA's, but do not support theview that there is between these two RNA's aprecursor-to-product relationship.

RACHELE MAGGIO, PHILIP SIEKEVITZ, AND GEORGE E. PALADE Isolated Nuclei. II 3 0 9


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A d e t a i l e d a n a l y si s o f n u c l e a r R N A t y p es a n dm e t a b o l i s m h a s b e e n c a r r i e d o u t b y Z b a r s k i i a n dh i s c o l l a b o r a t o r s( c f .r e f e r e n c e 4 0 ) , w h o f o u n d ( 4 1)t h a t a f t e r 2 0 m i n u t e s ' l a b e l i n gi n v i v owith 32Pi ,t h e f r a c t i o n w i t h t h e h i g h e s t s p e c i f ic r a d i o a c t i v i t yw a s t h e r e s i d u e o f th e e x t r a c t i o n w i t h 0 . 1 4 MN a C 1 , w h i c h t h e y c a ll e d t h e " n u c l e o l a r - c h r o m o -

s o m a l a p p a r a t u s . " T h e a c t i v i ty o f t h e " h i g hp o l y m e r ic R N A " o f t h e " n u c l e a r s a p " w a s h a lf ash i g h , b u t s t i ll f a r a b o v e t h a t o f v a r i o u s c y t o p l a s m i cR N A ' s ( e . g . ,s o lu b l e a n d r i b o s o m a l R N A ) . T h e r ea r e e v i d e n t s i m i l a r i t i e s b e t w e e n t h e s e f i n d i n g sa n d o u r s , a l t h o u g h a c l e a r a p p r e c i a t i o n o f t h ee x t e n t o f a g r e e m e n t i s h a m p e r e d b y t h e f ac t t h a td i f f e r e n t is o l a t i o n a n d e x t r a c t i o n p r o c e d u r e s w e r eu s e d i n t h e t w o c a s e s . A t p r e s e n t , t h e f u n c t i o n a lr o l e o f t h e v a r i o u s n u c l e a r R N A ' s i s s t il l p o o r l yu n d e r s t o o d ( c f .r e v i e w b y S i r l i n , 4 2) , b e c a u s e o f t h ed i v e r s i t y o f th e s e R N A ' s , t h e d i f f i c u l t y o f is o l a t i n gn u c l e a r s u b f r a c t i o n s a n d o f i n t e r p r e t i n g k i n e t i cd a t a .

D I S C U S S I O N

O u r g e n e r a l r e s u l t s i n d i c a t e t h a t t h e n u c l e a r c o m -p o n e n t s c o n s i d e r e d , i . e . t h e n u c l e o l i a n d t h en u c l e o p l a s m , c o n t a i n d i f f e r e n t ty p e s o f R N Ac h a r a c t e r i z e d b y d i f f e r e n t s o l u b i l i t y in 1 M N a C l ,d i f f e r e n t n u c l e o t i d e c o m p o s i t i o n , a n d d i f f e r e n tm e t a b o l i c p r o p e r t i e s. A t p r e s e n t w e d o n o t k n o wt o w h a t e x t e n t o u r r e s u l t s a r e a f f e c t e d b y t h e p r o -t r a c t e d p r o c e d u r e a n d e x t e n d e d s o n i c a t io n u se df o r t h e i s o l a t i o n o f o u r n u c l e a r s u b f r a c t i o n s .P r e f e r e n t i a l d a m a g e o f s o m e R N A ' s e i t h e r b ys h e a r o r R N a s e a c t i v i t y i s n o t e x c l u d e d , e s p e c i a l l yd u r i n g n u c l e a r s u b f r a c t i o n a ti o n . P a r t i a l a n s w e r st o s u c h q u e s t i o n s m a y c o m e f r o m a c o m p a r i s o nn o w u n d e r w a y b e t w e e n t h e p h y s ic o c h e m i c a lc h a r a c t e ri s t ic s o f R N A ' s e x t r a c t e d f r o m w h o l en u c l e i a n d t h o s e o f R N A ' s o b t a i n e d f r o m n u c l e a rs u b f r a c t i o n s .

T h e R N A o f t h e n u c l e o l a r s u b f r ac t i o n i s c h a r a c -te r iz ed b y i t s inso lub i l i ty in sa l t so lu t ions , i t s h ighG M P a n d l o w A M P c o n t e n t , a n d i ts h i g h tu r n -o v e r r a t e . T h e d i f f e r e n t t u r n o v e r r a t e s o f it s i n d i -v i d u a l m o n o n u c l e o t i d e s s u g g e s t a h i g h f r e q u e n c yo f C M P - G M P s e q ue n ce s i n t he c o r r e s po n d i ngR N A c h a in s . T h e r e a r e r e a s o n s to b e l ie v e t h a ts o m e o f th e s e p e c u l i a r i t i e s , e s p e c i a l l y t h e n u c l e o -t i d e c o m p o s i t i o n , w o u l d b e c o m e m o r e p r o n o u n c e di f i t w o u l d b e p o s s i b l e to r e d u c e t h e n u c l e o p l a s m i cc o n t a m i n a t i o n o f t he f r a c t io n .

T h e R N A o f th e n u c l e o p l a s m i c s u b f ra c t i on sa p p e a r s t o b e a m i x t u r e o f a t le a s t t w o R N A

s p e c i e s , o n e c h a r a c t e r i z e d b y h i g h s o l u b i l i t y i n1 M N a C I a n d r e l a t iv e l y l o w t u r n o v e r r a t e , t h eo t h e r h a v i n g c o n v e r s e f ea t u re s . T h e d i f f e re n c e i nn u c l e o t i d e c o m p o s i t i o n i s n o t p a r t i c u l a r l y p r o -n o u n c e d b u t d i f f e r e n c e s i n t h e t u r n o v e r r a t e o fi n d i v i d u a l m o n o n u c l e o t i d e s i n d i c a t e r a t h e r d i ff e r-e n t s e q u e n c e s i n t h e c o r r e s p o n d i n g R N A c h a i n s .

O n t h e a s s u m p t i o n t h a t t h e s u b n u c l e a r R N A ' ss t u d i e d a r e h o m o g e n e o u s , t h e c o n s i d e r a b l e d i f f e r -e n c e s i n n u c l e o t i d e c o m p o s i t i o n r e v e a l e d b y o u rf i n d i n g s s t r o n g l y s u g g e s t t h a t a r e l a t i o n p r e -c u r s o r - - . p r o d u c t c a n n o t e x is t b e t w e e n t h e v a r i o u sn u c l e o l a r a n d n u c l e o p l a s m i c R N A ' s o n t h e o n eh a n d a n d t h e v a r i o u s c y t o p l a s m i c R N A ' s o n t h eo t h e r. M o r e o v e r , i f t h e n u c l e o t i d e c o m p o s i t i o n o fl i v e r D N A ( 4 3 ) is c o n s i d e r e d , t h e r e i s a g a i n n oe x t e n s i v e s i m i l a r i t y b e t w e e n i t a n d t h a t o f t h eR N A o f a n y n u c l e a r s u bf r a c ti o n . E v i d e n t l y c o m -p l e m e n t a r i t y a s w e l l as p r e c u r s o r - - . p r o d u c t r e l a -t i o n s a r e s t il l p o s s i b le i f t h e v a r i o u s s u b n u c l e a rR N A ' s a r e m i x t u r e s o f t w o o r m o r e d i s t i n ct R N A

s p e c i e s . R e c e n t l y p u b l i s h e d w o r k s u g g e s t s i n d e e dt h a t s u c h m a y b e t h e c as e w i t h t h e n u c l e o la r R N A .G e o r g i e v e t a l . ( 4 1, 4 4 - 4 7 ) h a v e f r a c t i o n a t e dn u c l e a r R N A ' s b y a c o m b i n a t i o n o f s a lt a n dp h e n o l e x t r a c t i o n p r o c e d u r e s a n d h a v e a r r i v e d a tt h e c o n c l u s i o n t h a t t h e s a l t i n s o l u b l e R N A o f t h e" n u c l e o l o - c h r o m o s o m a l a p p a r a t u s " c o n t a i n s af r a c t i o n w i t h a p a r t i c u l a r l y h i g h t u r n o v e r r a t ew h i c h i s t h e p r e c u r s o r o f s o m e c y t o p la s m i c R N A .T h e i n t e r p r e t a t i o n o f t h e i r r e su l ts i s h a m p e r e db y t h e f a c t t h a t r a d i o a c t i v i t y w a s m e a s u r e d i nR N A f r a c t io n s r a t h e r t h a n i n s e p a r a t e d m o n o -n u c l e o t i d e s , a c i r c u m s t a n c e w h i c h i n t r o d u c e s ac e r t a i n d e g r e e o f u n c e r t a i n t y e v e n w h e n p h e n o l -

e x t r a c t e d R N A f r a c t i o n s a r e c o n s i d e r e d( c f .r e f e r e n c e 4 8 ) . A l s o r e c e n t l y, S i b a t a n ie t a l . (49)a n d I t i a t t ( 5 0 ) h a v e r e p o r t e d t h e i s o l a t i o n o f a nR N A f r a c t i o n f r o m t h y m i c ( 4 9) a n d h e p a t i c ( 5 0)n u c l e a r f r a c t i o n s b y a m o d i f i e d p h e n o l e x t r a c t i o np r o c e d u r e . T h i s f r a c t i o n h a s a h i g h e r t u r n o v e rr a t e t h a n a n y o t h e r n u c le a r R N A f r a c t io n . M o r e -o v e r i n b o t h c a s es i t w a s d e m o n s t r a t e d t h a t t h er a d i o a c t i v i t y w a s i n R N A a n d i t w as p o s t u l a te dt h a t t h e n e w R N A f r a c t i o n , su p p o s e d ly t h e " m e s -s e n g e r " R N A , i s p a r t o f t h e n u c l e o l a r R N A .

A l t h o u g h o u r e x p e r i m e n t s h a v e n o t y e t s u c -c e e d e d i n r e s o l v i n g t h e m i x t u r e s o f n u c l e a rR N A ' s i n t o f u n c t i o n a l l y m e a n i n g f u l t y p e s , w eb e l i e v e t h a t t h e y r e p r e s e n t a s t e p i n t h e r i g h td i r e c t i o n b e c a u s e t h e y p u t n u c l e a r f r a c t i o n a t i o no n a s t r u c t u r a l b a s i s , a n d i n t r o d u c e m o r e s t r i n g e n tc r i t e r ia o f m o r p h o l o g i c a l c o n t r o l. W i t h f u r t h e r

3 1 0 T H E J O U R N A L O F C E L L B IO L O G Y V O L U M E 1 8 , 1 9 6 3


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re f inements , based on such premises , the presentn u c l e a r R N A f r a c t i o n s m a y b e r e s o l v e d i n t oh o m o g e n e o u s c o m p o n e n t s o f d e f in e d f u n c t i o n a lsignificance.

Part of this work was ma de possible by a gran t(A-1635) to Dr. Siekevitz from the Natio nal Insti tute

of Arthritis and M etab olic Diseases, Natio nal Insti-tutes of Health, Unite d States Public Health Service.

R E F E R E N C E S

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Dr. M aggio is a Fellow of the Popu lation Council ,Rockefeller Foundation . He r present address is theDepar tmen t of Compara t ive Anatomy, Univers i tyof Palermo, P alermo, I taly.

A preliminary re port of these findings was made atthe meeting of the American Society for Cell Biologyin San Francisco, November, 1962.

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312 Th E JOURNAL OF CELL BIOLOGY VOLUME 18, 1963

Documents

Isolation and Chemical Characterization of Nucleolar and Nucleoplasmic Subfraction