The effect of ribonuclease on rat-liver parenchymal cells in suspension

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FIG. 3. - - F r a c t i o n a t i o n by various methods, of RNA f r o m liver cells in suspension i n c u b a t e d w i t h o u t RNase or w i t h 1 j~g of R N a s e / m l for 1 h o u r at 28°C. After i n c u b a t i o n , the cells were w a s h e d a n d RNA isolated b y Method II. B a n d D : RNA from cells i n c u b a t e d w i t h o u t RNase. C a n d E : RNA f r o m cells i n c u b a t e d w i t h RNase. A : a n a l y t i c a l u l t r a c e n t r i f u g e r u n (the picture was t a k e n a f t e r 20 m i n u t e s at 50740 r e v / m i n u t e ; u p p e r picture, + R N a s e ; lower picture, -RNase). B and C : sucrose density g r a d i e n t r u n s (in a 25.1 rotor for 18 h o u r s at 22500 r e v / m i n u t e ) . D a n d E : MAK runs.

BIOCHIMIE, 1973, 55, n ° 6-7.

Effecl of RNase on liver cell s u s p e n s i o n s . RNA left in acid-insoluble form in the ceils after t r e a t m e n t w i t h RNase. A n a l y t i c a l u l t r a c e n t r i f u g e , MAK and density g r a d i e n t runs of this RNA (fig. 3) s h o w e d that it was e x t e n s i v e l y d e p o l y m e rised, w h e n d e r i v e d from cells treated w i t h 1 Itg of R N a s e / m l for 1 hour. Fig. 4 s h o w s that even 0.1 ~tg of R N a s e / m l for 90 m i n u t e s at 28°C w a s as

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It has been r e p o r t e d e a r l i e r (for r e f e r e n c e s , see the i n t r o d u c t i o n ) that c o n t r o l l e d t r e a t m e n t of r i b o s o m e s w i t h RNase in vitro does not affect t h e i r m o r p h o l o g i c a l i n t e g r i t y or t h e i r ability to f u n c t i o n in an in vitro p r o t e i n - s y n t h e s i s i n g system, though t h e i r RNA is largely degraded. It t h e r e f o r e s e e m e d w o r t h w h i l e to investigate if

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FIG. 4. - - Sucrose density gradient runs of RNA from liver cells in suspension incubated with 0.0015 .ug (A), 0.005 !~g (B), 0.025 :~tg (C), and 0.1 Ixg (D) of RNase/ml for 20 minutes at 28°C. After incubation (done, for A, in Krehs-Ringer bicarbonate, and for others, in Krebs-Ringer phosphate buffer), the cells were washed and RNA isolated by Method II. The sucrose density gradient centrifugation was done in a SW 25.1 rotor for 18 hours at 22500 rev/minute.

effective. As the RNase c o n c e n t r a t i o n was l o w e r e d b e l o w 0.1 ~ g / m l , the extent of p a r t i a l d e p o l y m e r i s a t i o n o b s e r v e d d e c r e a s e d ; 0.0015 , g of R N a s e / m l for 20 m i n u t e s h a d a p p a r e n t l y no effect on cellular RNA. E v e n t h o u g h exogenous RNase (1 ~tg/ml) caused e i t h e r d e g r a d a t i o n (to acid-soluble material~ or e x t e n s i v e p a r t i a l d e p o l y m e r i s a t i o n (to acid-insoluble material) of the rRNA of the ceils in 20 minutes, t h e r e w a s a r e d u c t i o n of only about 20 p. cent in the tRNA a c t i v i t y of the cells by RNase u n d e r these c o n d i t i o n s (table II) (tile p o s s i b i l i t y that the exogenous RNase caused cleavage of some of the i n t e r n u c l e o t i d e linkages in tRNA in such a w a y that the r e s u l t i n g fragments could be held t o g e t h e r by h y d r o g e n bonds, c a n n o t be ruled out ; such tRNAs m a y retain t h e i r biological a c t i v i t y ; for r e f e r e n c e s , see Bhargava et al. r24]).

BIOCHIMIE, 1973, 55, n ° 6-7.

exogenGus RNase e n t e r i n g the cells affected t h e i r r i b o s o m e s similarly. F o r this purpose, labelled cells d e r i v e d from liver tissue labelled in vivo with 32p-phosphate w e r e i n c u b a t e d w i t h o u t and w i t h RNase (1-5 ~g/ml), and t h e i r r i b o s o m e s isolated using unlabelled r i b o s o m e s from l i v e r as a c a r r i e r . R i b o s o m e s from RNase-treated cells, dissociated c o m p l e t e l y (as by EDTA t r e a t m e n t : fig. 5) or p a r t i a l l y (as by d i a l y s i s : fig. 6) b e h a v e d e x a c t l y as r i b o s o m e s f r o m the u n t r e a t e d cells or f r o m the slices. On the o t h e r hand, the RNA f r o m ribosomes isolated from RNase-treated cells w a s found to be e x t e n s i v e l y d e p o l y m e r i s e d , unlike tile RNA from u n t r e a t e d cells (fig. 5). Almost all ( 7 96 p. cent) of the r a d i o a c t i v i t y in RNA of the r i b o s o m e s f r o m RNase-treated cells w a s a c i d - p r e c i p i t a b l e , s h o w i n g thai exogenous RNase led to the cleavage of rRNA only to large, a c i d - p r e c i p i t a b l e products.

B. Vijaya Kumar and P. M. Bhargava.

666

In an e x p e r i m e n t in w h i c h RNase w a s a d d e d to t h e cell s u s p e n s i o n after i n c u b a t i o n at 28°C f o r 20 m i n u t e s , a n d j u s t b e f o r e t h e a d d i t i o n o f b e n t o n i t e a n d p o l y v i n y l sulphate, the RNA of the

R N a s e (fig. 5) is, t h e r e f o r e , u n l i k e l y to b e a n a r t i f a c t of t h e p r e p a r a t i o n o f r i b o s o m e s o r o f t h e i r RNA, a n d w o u l d s e e m to b e a c o n s e q u e n c e o f t h e action on r i b o s o m e s of RNase taken up by the

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FIG. 5. - - Sucrose density gradient runs of ribosomes (A, B) and of their RNA (C, D), derived f r o m liver cells in suspension incubated for 20 m i n u t e s at 28°C w i t h o u t RNase (A, B) or w i t h 1 j~g of R N a s e / m l (B, D). The cell suspension w a s p r e p a r e d f r o m in vivo 32P-labelled liver. "]'he celts w e r e incubated, washed and h o m o g c n i s e d : a homogenate derived f r o m an unlabelled liver was added (as a source of carrier ribosomes) and ribosomes isolated and dissociated with 0.2 M NaC1 and 0.01 M EDTA in 0.01 M tris-HC1 buffer (pH 7.4), as in the t e x t ; RNA w a s isolated from t h e m by Method II. For A and B, the sucrose density g r a d i e a t c e a t r i f u g a t i o n was done for 12 h o u r s at 18000 r e v / m i n u t e , and for C and D, for 23 hours at 18000 r e v / m i n u t e , in a SW 25.1 rotor. • • : radioactivity (due to ribosomes or RNA from the incubated cells). O O : absorbancy (due to carrier ribosomes or their RNA).

r i b o s o m e s w a s f o u n d n o t to b e a f f e c t e d (fig. 7). Further, the unlabelled rRNA from the carrier h o m o g e n a t e w a s n o t a f f e c t e d w h e n it w a s i s o l a t e d f r o m a m i x t u r e of this h o m o g e n a t e w i t h the h o m o gen,ate o f a c e l l s u s p e n s i o n i n c u b a t e d w i t h R N a s e (fig. 5). T h e o b s e r v e d d e p o l y m e r i s a t i o n of t h e cell s u s p e n s i o n rRNA on i n c u b a t i o n of the cells w i t h

BIOCHIMIE, 1973, 55, n ° 6-7.

cel]s. T h i s v i e w is f u r t h e r s u p p o r t e d b y t h e o b s e r v a t i o n that i n c u b a t i o n of liver slices w i t h RNase h a d no effect w h a t s e v e r on the intrac e l l u l a r r i b o s o m e s o r o n t h e i r R N A (fig. 8). S i n c e r i b o s o m e s i s o l a t e d f r o m cells i n c u b a t e d with RNase, although apparently morphologieally

E f f e c t of R N a s e on liver celt suspensions. intact, contained only partially depolymerised RNA, it s e e m e d l i k e l y t h a t t h e p e r c e n t a g e of ribosomes conlaining undepolymerized native RNA w i t h i n these cells w a s relatively small,

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if a n y s u c h r i b o s o m e s w e r e p r e s e n t at all. It w a s , t h e r e f o r e , a r g u e d t h a t if t h e s t r u c t u r a l i n t e g r i t y of RNA w a s e s s e n t i a l f o r t h e b i o l o g i c a l a c t i v i t y of t h e r i b o s o m e s , t h e r i b o s o m e s f r o m R N a s e - t r e a t e d cells s h o u l d not w o r k in a p r o t e i n s y n t h e s i s i n g s y s t e m in vitro as e f f i c i e n t l y as r i b o s o m e s f r o m u n t r e a t e d cells. T a b l e III s h o w s t h a t t h e r i b o s o m e s f r o m R N a s e - t r e a t e d cells

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Fro. 6. Sucrose density gradient r u n s of ribosomes from liver ceils in suspension incubated for 20 m i n u t e s at 28°C w i t h o u t RNase (A) or with 5 ttg of RNase/m! (B). In vivo-labelled liver cells were incubated, and ribo. somes isolated following addition of a source of carrier ribosomes, as in Fig. 6. They were partially dissociated by dialysis against 0.01 M tris-HC1 buffer (pH 7.4). The sucrose density gradient centrifugation was done for 12 h o u r s at 18000 r e v / m i n u t e in a SW 25.1 rotor.

30

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FI6. 7. - - Sucrose density gradient centrifugation of RNA f r o m ribosomes of cells incubated for 0 m i n u t e at 0°C w i t h 1 ~ug of RNase/ml. T h e ribosomes were isolated f r o m the in vivolabelled cells immediately after the addition of RNase, using carrier ribosomes, as in Fig. 6. RNA was isolated f r o m the ribosomes by Method II. The sucrose density gradient c e n t r i f u g a t i o n was done for 23 hours at 18000 r e v / m i n u t e in a SW 25.1 rotor.

c a r r i e d out p o l y U - d i r e c t e d p o l y p h e n y l a l a n i n e s y n t h e s i s n e a r l y as e f f i c i e n t l y as r i b o s o m e s f r o m tlle u n t r e a t e d c e l l s d i d . T h i s s h o w s t h a t t h e s t r u c t u r a l i n t e g r i t y of r R N A w i t h i n t h e r i b o s o m e s ill a w h o l e cell is n o t e s s e n t i a l f o r t h e i r b i o l o g i c a l f u n c t i o n in situ.

TABLE III (*).

Doly U-directed polyphenylalanine synlhesising ability of ribosomes from liver cells in suspension incubated withonl RNase or with 1 ~Lg of RNase/ml f o r PO minutes at 28°C.

Source of ribosomes

Unincubated ceils . . . . . . . . . . . . . . . . . . . . . . . . . . . Cells incubated for 20 minutes without R N a s e . . . Cells incubated for 20 minutes with 1 ~tg of RNase/ml . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Radioactivity in protein (counts/minute/rng ribosomes) Expt. t

Expt. 2

Expt. 3

1731 1611

2778 3245

1455 1701

1642

2278

1585

(*) The incubation with 14C-phenylalanine was carried out for 40 m i n u l e s ; 0-minute values have been subtracted.

BIOCHIMIE, 1973, 55, n ° 6-7.

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B. Vijaya K u m a r and P. M. Bhargava.

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E[fect of exogenous RNase o17 endogenous degradation of RNA at 37°C.

m a t e r i a l i n t h e p r e s e n c e of R N a s e (1 I~g/ml) w a s f o u n d to b e p a r t i a l l y d e p o l y m e r i s e d i n all t h e fractionation systems used ; such RNA from ceils i n c u b a t e d a t 37°C i n t h e a b s e n c e of R N a s e w a s e a r l i e r f o u n d to g i v e n o r m a l f r a c t i o n a t i o n p a t t e r n s o n MAK a n d S e p h a d e x c o l u m n s El91.

It h a s b e e n r e p o r t e d e a r l i e r El9] t h a t i n c u b a t i o n of r a t - l i v e r p a r e n c h y m a l c e i l s at 37°C r e s u l t s i n t h e a c t i v a t i o n of a n i n t r a c e l l u l a r n u c l e a s e . T h e question that we now asked was whether this degradation was already operating at the maximal l e v e l i n t h e a b s e n c e of e x o g e n o u s R N a s e , o r c o u l d it b e a c c e l e r a t e d b y t h e e x o g e n o u s e n z y m e ?

DISCUSSION.

Uptake o[ RNase.

T a b l e IV s h o w s t h a t R N a s e (1 ~ g / m l ) e n h a n c e d the endogen.ous degradation. The total degradat i o n i n c r e a s e d w i t h t h e c o n c e n t r a t i o n of R N a s e u p t o t h e l i m i t of a b o u t 70 p. c e n t of t h e t o t a l c e l l u l a r RNA. R N A left u n d e g r a d e d to a c i d - s o l u b l e

When liver cells in suspension are incubated w i t h R N a s e , a p r o g r e s s i v e l y i n c r e a s i n g a m o u n t of t h e e n z y m e is f o u n d to b e a s s o c i a t e d w i t h t h e cells. T h i s a s s o c i a t i o n c a n n o t r e p r e s e n t a d s o r p -

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FIG. 8 . F r a e t i o n a t i o n by various m e t h o d s of RNA f r o m liver slices i n c u b a t e d w i t h 1 ug of R N a s e / m l for 1 h o u r at 37°C. After i n c u b a t i o n , the slices were w a s h e d a n d RNA extracted by Method II. The f r a c t i o n a t i o n p a t t e r n s given here were s u p e r i m p o s a b l e on the corresponding p a t t e r n for RNA o b t a i n e d f r o m the slices i n c u b a t e d ill the absence of RNase. A : a n a l y t i c a l u l t r a c e n t r i f u g e r u n (picture was t a k e n a f t e r 21 r~inutes at 50740 r e v / m i n u t e ) . B : sucrose density g r a d i e n t r u n (in a 25.1 r o t o r for 16 h o u r s at 24000 r e v / m i n u t e ) . C : MAK run.

BIOCHIMIE, 1973, 55, u ° 6-7.

E f f e c t of R N a s e on liver cell s u s p e n s i o n s . TABLE

669

IV (*).

Percentage degradation of RNA to acid-sohlble material on incubation of liver cells in suspension without RNase or with varying concentration of RNase at 37°C. RNA

degraded (p. cent)

Concentration o[ RNase (rtg/ml) (*') t5 minutes

0 ............................. 1 ............................. 10 .............................

100 . . . . . . . . . . . .

-................

. 60 minntes

t20 minutes I 180 minutes

13.3 -4- 9.0

2 1 .6 -+- 9.0 34.9 - - -~- lO.O 39.0 ~- 11.0 58.0 1 63 .84 66.8 12.0 -3 7 . 7 -457.9 -'~ 9 O 166. 2 -F- 9.0 71.1 ____~8.0

(*) Acid-insoluble RNA was estimated in the total incubation mixture by Method I. Percentages are on the value for 0 hour. ('*) Values for 0 and 100 ~tg/ml are averages of 4-10 experiments and are followed by tile standard deviation. tion on the cell surface alone as at least a p a r t of the RNase taken up was f o u n d in the cell supernatant f r a c t i o n in an u n c h a n g e d form, and as in the p r e s e n c e of the e n z y m e (but not in its absence) the rRNA within the cells was p r o g r e s sively h y d r o l y s e d . On the c o n t r a r y , w h e n l i v e r slices w e r e i n c u b a t e d w i t h RNase, m u c h s m a l l e r a m o u n t s of the enzyme, if any, w e r e f o u n d to be associated w i t h the slices, and no detectable effect on the RNA of the slices w a s observed. These o b s e r v a t i o n s s h o w that l i v e r cell suspensions can take up RNase, a n d that the uptake in the slices, if any, is m u c h s m a l l e r w h e n c m n p a r e d to the cells in suspension. T h e uptake in the cell suspensions is u n l i k e l y to be a e o n s e q u e n c e of m e t a b o l i c death as l i v e r cell suspensions p r e p a r e d by the m e t h o d used here, r e s p i r e , o x i d i s e m a n y of the c o m m o n l y oxidisable snbstrates, s y n t h e s i s e RNA and p r o t e i n ( i n c l u d i n g s e r u m albumin), take up and metabolise lipids, are viable by the criteria of s t a i n a b i l i t y by t r y p a n blue and a b i l i t y to t r a n s p o r t n o n - m e t a b o l i s a b l e a m i n o a c i d s , and e x h i b i t m e t a b o l i c a c t i v i t y for p r o l o n g e d p e r i o d s u n d e r suitable c o n d i t i o n s (for r e f e r e n c e s , see B h a r g a v a [25, 26] ; D i c k s o n [27]). T h e a c q u i s i t i o n by l i v e r p a r e n c h y m a l cells of the ability to take up RNase m a y t h e r e f o r e r e p r e s e n t a specific change in t h e i r p r o p e r t i e s c o n s e q u e n t to an alter a t i o n in t h e i r level of organisation. This c h a n g e c o u l d be a result of the r e m o v a l of influences such as those of cells o t h e r than the p a r e n c h y m a t cells in liver, a n d / o r of the e x t r a c e l l u l a r material. This v i e w is s u p p o r t e d by the o b s e r v a t i o n that r e a g g r e g a t i o n of l i v e r p a r e n c h y m a l cells in the i u t r a p e r i t o n e a l c a v i t y results in a loss of the a b i l i t y to take up RNase [28]. O t h e r specific perm e a b i l i t y changes o c c u r r i n g in the p a r e n c h y m a l

BIOCH1MIE, 1973, 55, n ° 6-7.

cells on d i s p e r s i o n of l i v e r to a single cell suspension h a v e been d e s c r i b e d [25, 26].

Classification of cellular RNA on the basis of susceptibility to RNase. In the absence of RNase, at 28°C, v e r y little RNA of the l i v e r p a r e n c h y m a l cells w a s d e g r a d e d to acid-soluble m a t e r i a l ([19] ; Table I). At this t e m p e r a t u r e , in the p r e s e n c e of the enzyme, a m a x i m u m of about 40 p. cent of the RNA was d e g r a d e d (Table I). At 37°C, even in the absence of the enzyme, a m a x i m u m of about 40 p. cent of the cellular RNA w a s d e g r a d e d by an i n t r a c e l l n l a r nuclease ( [ 1 9 ] ; Table IV). H o w e v e r , the RNA d e g r a d e d by the exogenous e n z y m e at 28°C and the RNA d e g r a d e d by the e n d o g e n o u s e n z y m e at 37°C are likely to be different as the m a x i m a l degradation w a s a d d i t i v e w h e n exogenous e n z y m e was used at 37°C (table,IV). This ~vould suggest that RNA of the cells could be classified into t h r e e types : (a) RNA (ca 40 p. cent of the total RNA) w h i c h m a y be d e g r a d e d to acid-soluble m a t e r i a l only by the e n d o g e n o u s nuclease a c t i v a t e d on disp e r s i o n of l i v e r and i n c u b a t i o n of the cells at 37°C; (b) RNA (ca 40 p. cent of the total RNA) w h i c h m a y be d e g r a d e d to acid-soluble m a t e r i a l only by e x o g e n o u s l y a d d e d n u c l e a s e ; and (c) RNA (ca 20 p. cent of the total RNA) w h i c h is not d e g r a d e d to acid-soIuble m a t e r i a l by e i t h e r e n d o g e n o u s or exogenous nuclease. W h a t the nature of these RNAs m i g h t be is not clear f r o m this investigation. D e g r a d a t i o n of the RNA of the first type c o u l d be p i c k e d up by c e n t r i f u g a t i o n on a sucrose d e n s i t y g r a d i e n t or in an a n a l y t i c a l u l t r a c e n t r i fuge, but not by c h r o m a t o g r a p h y on a MAK or S e p h a d e x c o l u m n [19] ; that of the s e c o n d type could be p i c k e d up also on the MAK and S e p h a d e x columns. 44

B. Vijaya Kumar and P. M. Bhargaoa.

670

E[fect of RNase taken up on the ribosomes. The r i b o s o m e s isolated f r o m cells i n c u b a t e d with RNase exhibited a normal sedimentation profile but contained partially depolymerised RNA. T h e s e r i b o s o m e s w e r e f o u n d to b e n e a r l y as a c t i v e as n a t i v e r i b o s o m e s i n a p o l y U - s t i m u l a t e d polyphenylalanine-synthesising system. The above e f f e c t o f R N a s e o n r i b o s o m e s w i t h i n t h e c e l l s is s i m i l a r to t h a t o b s e r v e d in vitro u s i n g i s o l a t e d r i b o s o m e s (for r e f e r e n c e s , see the i n t r o d u c t i o n ) . T h e s e o b s e r v a t i o n s a r e also i n c o n f o r m i t y w i t h the view now generally held about the structure o f r i b o s o m e s i n w h i c h p a r t of t h e r R N A is e n v i s a g e d to e x i s t i n a h y d r o g e n b o n d e d f o r m i n a c c e s s i b l e o r r e s i s t a n t to e x o g e n o u s n u e l e a s e [14].

l~suM~. 1) On a montr~ que les eellules en suspension du p a r e n e h y m e h~patique du Rat i n e o r p o r e n t la ribonuel~ase 131I. 2) La nucl~ase ineorpor~e d6grade j u s q u ' h 40 p. cent de I'ARN celluIaire en e o n s t i t u a n t s acido-solubles. Cet ARN semble diff6rent de celui qui est d6grad6 par la nucl6ase endog6ne activ6e, apr~s homog~n6isation du tissu ~t l'6tat de suspension cellulaire et incubation h 37°C. 3) La ribonuel~ase incorpor~e h 28°C d~polymdrise p a r t i e l l e m e n t I'ARNr cellulaire de telle sorte que l ' e n s e m b l e de l'int~grit~ structurale ou l'activit~ biologique des ribosomes n ' e s t pas atteinte, n o n plus qne l'activit~ cellulaire de YARN t.

A eknolwledgmenls. The analytical u l t r a c e n t r i f u g e used in t h i s s t u d y was a gift f r o m The Welleome Trust, London. Assistance of Mr. P r a m o d in the ultracentrifuge r u n s is g r a t e f u l l y acknowledged. B.V.K. received a Senior Research F e l l o w s h i p of the Council of Scientific & I n d u s t r i a l Research, New Delhi. REFERENCES. 1. Braehet, J. (1955) Biochim. Biophys. Acta, 18, 247268.

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