Br ves communications

BIOCHIMIE, 1977, 59, 105-109.

Effect of low doses of Actinomycin D on RNA synthesis in chick embryo fibroblasts transformed by Rous sarcoma virus.

(*)

Gis61e CONNAN ~', G. SUSKIND ( ' * ) a n d G. F . RABOTTI. Laboratoire de Mddecine E x p d r i m e n t a l e du Coll~ge de France, U 112 de I'LN.S.E.R.M. 11 Place Marcelin Berthelol - - 75231 Paris, Cedex 05. (8-9-1976). INTRODUCTION. L o w d o s e s of A c t i n o m i c y n D (AD) h a v e a differ e n t i a l i n h i b i t o r y effect o n t h e s y n t h e s i s of all c l a s s e s o f RNA, d e p e n d i n g o n c o n c e n t r a t i o n a n d t i m e of e x p o s u r e [1, 2]. On t h i s b a s i s A D h a s b e e n e m p l o y e d as a specific p r o b e f o r t r a n s c r i p t i o n of b o t h c e l l u l a r a n d v i r a l R N A i n a v a r i e t y of cell s y s t e m s . It h a s b e e n s u g g e s t e d t h a t t h e d i f f e r e n c e s in i n h i b i t i o n of t h e t r a n s c r i p t i o n of s o m e c l a s s e s o f R N A m a y be r e l a t e d to t h e p r o p e r t y o f i n t e r c a l a t i o n of AD a t specific s i t e s o n t h e l e s s e r c u r v a t u r e of t h e DNA h e l i x r e s u l t i n g i n c o m p e t i t i o n w i t h t h e b i n d i n g to p r o t e i n s r e g u l a t i n g g e n e t r a n s c r i p t i o n [3-7] or p o s s i b l y to t h e i n t e r f e r e n c e with the translocation of RNA polymerase along the t e m p l a t e [,3, 8]. W h e r e a s i n v e s t i g a t i o n s of t h e w e l l - k n o w n i n h i b i t o r y effect of AD o n R o u s S a r c o m a V i r u s (RSV) s y n t h e s i s [9, 10, 11] h a v e b e e n t h e o r i g i n a l b a s i s f o r t h e

[12] t h e r e v e r s i b l e i n h i b i t o r y effect o f AD o n v i r i o n f o r m a t i o n a n d t h e s u b s e q u e n t i n c r e a s e i n s y n t h e s i s of R N A in i n f e c t e d cells [13] are not fully explained by the integration of the RSV g e n o m e a s DNA p r o v i r u s , h u t m a y i n v o l v e a l s o r e g u l a t o r y m e c h a n i s m s of c e l l u l a r R N A t r a n s c r i p t i o n . I n p r e v i o u s s t u d i e s it h a s b e e n s h o w n b y q u a n t i t a t i v e a u t o r a d i o g r a p h y in t h e l i g h t a n d i n t h e e l e c t r o n m i c r o s c o p e t h a t R N A s y n t h e s i s i n n u c l e o l i of R S V i n f e c t e d cells i n h i b i t e d b y lo'w d o s e s o f AD is recov e r e d a t a f a s t e r r a t e t h a n in u n i n f e c t e d cells a l t h o u g h t h e i n i t i a l u p t a k e o f AD is s i m i l a r in t r a n s f o r m e d a n d i n c o n t r o l cells [13]. T h e b i o c h e m i c a l c h a r a c t e r i z a t i o n of t h e t r a n s c r i p t i o n p r o d u c t s o f R S V - i n f e c t e d cells d u r i n g t h e r e c o v e r y f r o m i n h i b i t i o n b y lo'w d o s e s of AD is t h e s u b j e c t of the present communication. It w i l l be s h o w n t h a t , a f t e r AD i n h i b i t i o n , t r a n s c r i p t i o n o f h e t e r o g e n o u s RNA ( H n - R N A , > 45S), r - R N A (z~5S), 4S a n d 5S R N A s r e s u m e s in t r a n s f o r m e d cells 60' m i n e a r l i e r a n d at a f a s t e r r a t e t h a n i n u n i n f e c t e d cells. T h e i n c o r p o r a t i o n r a t e of [3H] u r i d i n e of t h i s ~ n - R N A a s xvell a s t h a t of p r e - r i b o s o m a l (r-RNA) a n d o f 4S a n d 5S R N A s is a l s o r e s u m e d a t a f a s t e r r a t e in t r a n s f o r m e d ceils a s c o m p a r e d t o n o r m a l cells. I n t r a n s f o r m e d a s w e l l as i n n o r m a l c e l l s r e c o v e r y of t h e s y n t h e s i s of H n - R N A p r e e e e d s t h a t o f r - R N A , s u g g e s t i n g a r e g u l a t o r y effect of s o m e k i n d of H n - R N A in r - R N A s y n t h e s i s . MATERIAL AND METHODS. Tissue cultures : Primary cultures of chicken embryo fibroblasts from Edimbourgh Leghorn Brown (*) P a r t i e de t h ~ s e q u i s e r a s o u t e n u e p a r Mae C o n nan. (**) V i r u s S t u d i e s Section, V i r a l O n c o l o g y B r a n c h , N a t i o n a l C a n c e r I n s t i t u t e , B e t h e s d a 20014 M a r y l a n d , USA. To w h o m all c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

s t r a i n (ELB) s u p p l i e d b y Dr. L a c o u r , V i l l e j u i f , F r a n c e , w e r e p r e p a r e d b y s t a n d a r d t e c h n i q u e s f r o m 9/10 d a y s old e m b r y o s . T h e e m b r y o s u t i l i z e d in t h i s ~ ' o r k w e r e of p h e n o t y p e C / E , c h f +, gs +. C u l t u r e m e d i u m c o n s i s t e d of m i n i m u m e s s e n t i a l m e d i u m (MEM) s u p p l e m e n t e d w i t h 10 p e r c e n t t r y p t o s e p h o s p h a t e b r o t h a n d 10 p e r c e n t i n a c t i v e c a l f s e r u m a n d a n t i b i o t i c s . T h e cells w e r e c u l t i v a t e d in 75 c m 2 ( F a l c o n ) p l a s t i c flasks. Virus infection and RNA labelling : the non defective S e h m i d t - B u p p i n s t r a i n (Sr-RSV-2) b e l o n g i n g to a n t i g e n i c s u b - g r o u p B [14] w a s u s e d a n d c u l t u r e s h a n d l e d as p r e v i o u s l y d e s c r i b e d [13]. Cells w e r e i n f e c t e d at a m u l t i p l i c i t y of i n f e c t i o n (MOI) of 0,5-1. At t h e t i m e of AD t r e a t m e n t t h e p r o p o r t i o n o f t r a n s f o r m e d cells r e a c h e d 90-95 p e r c e n t o f t h e t o t a l cell p o p u l a t i o n i n t h e i n f e c t e d c u l t u r e s . U n i n f e c t e d cells w e r e used when subcontinent. U n i n f e c t e d a n d t r a n s f o r m e d cells w e r e w a s h e d in p h o s p h a t e - b u f f e r e d s a l i n e (PBS) a n d t r e a t e d w i t h AD at d i f f e r e n t c o n c e n t r a t i o n s a n d t i m e - l e n g t h s of e x p o s u r e . AD (gift f r o m Dr. A l p e r t M e r c k - S h a r p e a n d D o h m e C ° , "West P o i n t , PA) w a s d i s s o l v e d a n d s t o r e d as a n a q u e o u s s o l u t i o n o f 100 ~xg/ml a c c o r d i n g to t h e m e t h o d of Sa'wicki a n d G o d m a n [15]. C e l l u l a r R N A w a s l a b e l l e d f o r 60 m i n u t e s w i t h 50 t x C i / m l of [3HI u r i d i n e (sp. act. 20 C i / m m M o l e ) s u p p l i e d b y t h e C o m m i s sariat h l'Energie Atomique, Saelay, France). This l a b e l l i n g t i m e is n e e d e d f o r t h e d e t e c t i o n o f t h e m a t u ration products of pre-ribosomal RNA and for efficient m a r k i n g of t h e l o w m o l e c u l a r w e i g h t RNA. R N A extraction and p o l y - a c r y l a m i d e - g e l electrophorests (PAGE). R N A w a s e x t r a c t e d f r o m w h o l e cells to m i n i m i z e t h e l o s s o f r e g u l a t o r y f a c t o r s [16]. T h e p r o c e d u r e o f S c h e r r e r a n d D a r n e l l [17] as m o d i f i e d b y T i o l l a i s [18] was employed. Briefly this requires two extractions with phenol-solution (distilled phenol, acetate buffer 0.01 M, p H 5, c r i s t a l l i z e d 1 p e r c e n t SDS, 8 - h y d r o x y q u i n o l e i n e 0.1 p e r cent) a t 50 ° f o r 30 r a i n f o l l o w e d by two extractions with chloroform-iso-amyl alcohol (5/1). R N A w a s r e c o v e r e d b y p r e c i p i t a t i o n w i t h t w o v o l u m e s of e t h a n o l a f t e r a d d i t i o n of 1/10 v o l u m e o f a 2 M s o l u t i o n of NaC1 a t - - 2 0 ° C . A f t e r c e n t r i f u g a t i o n t h e p e l l e t is d i s s o l v e d i n 100 m l o f w a t e r c o n t a i n i n g 0,1 p e r c e n t SDS. T h e t e c h n i q u e o f a n a l y t i c a l P A G E a s d e s c r i b e d b y T i o l l a i s [19] u s i n g r e c r i s t a l l i z e d a c r y l anaide a n d b i s - a c r y l a m i d e , W a s e m p l o y e d [20]. F o r t h e a n a l y s i s of h i g h m o l e c u l a r w e i g h t R N A t h e gel c o n t a i n e d 1.7 p e r c e n t a c r y l a m i d e , 0.5 p e r c e n t a g a r o s e ( B e h r i n g - W e r k ) , a n d w a s r u n at 200 V f o r 5 h o u r s . F o r t h e lo'w m o l e c u l a r w e i g h t R N A t h e gel c o n t a i n e d 8 p e r c e n t a c r y l a m i d e a n d r u n a t 130 V f o r 16 h o u r s . U n d e r t h e s e c o n d i t i o n s 4S a n d 5S R N A s a r e s e p a r a t e d . Gels w e r e c a s t i n 7 r a m . OD p l a s t i c t u b e s 25 c m i n l e n g t h .

G. Connan, G. S u s k i n d and G. F. Rabolti.

106

A l i q u o t s of 30 p,g RNA 'were a n a l y s e d in each r u n . The buffer used for electrophoresis contained Tris 0.097 M, b o r i c acid 0.097 M EDTA 0.02 M, SDS 0.2 p e r cent p H 8.4 Gels 'were f r a c t i o n e d i n t o 2 m m slides a n d t h e f r a c t i o n s e l u t e d in 0.8 m l NCS ( A m e r s h a m Searle, Corp.) at 50 ° f o r 2 h o u r s a n d c o u n t e d in a l i q u i d s c i n t i l l a t o r a f t e r a d d i t i o n of 6 m l NE 233 ( N u c l e a r E n t r e p r i s e L i m i t e d , Scotland). RESULTS. P r e l i m i n a r y e x p e r i m e n t s ~were p e r f o r m e d u s i n g d i f f e r e n t c o n c e n t r a t i o n s of AD a n d different t i m e s of e x p o s u r e (see t a b l e I). The c o n c e n t r a t i o n of 0,04 ~ g / m l

TABLE I. Time ol incubation with AD {minutes)

Time of labelling with 3H-U (minutes)

Delay between withdrawal ol AD and beginning of extra, orion (hours)

0,2

180

10

0 10 24

0,2

90

60

0 24

AD ~tg/ml

5 6

10 24 0,02

90

60

C o m p a r i s o n of figure 1A a n d 1B s h o w s r e c o v e r y of Hn-RNA s y n t h e s i s t a k e s place 1 h o u r a h e a d of t h a t of r - R N A in b o t h t y p e s of cells.

0 24

Cells w e r e e x p o s e d to different d o s e s of AD f o r var i o u s t i m e s l a b e l l e d w i t h [3H] u r i d i n e a n d c h a s e d f o r different l e n g t h of t i m e , as sho'wn. Cells g r o w n in AD free m e d i u m 'were r u n in p a r a l l e l . RNA s y n t h e s i s 'was m e a s u r e d by PAGE.

z rr

.=

F16. 1.

Incorporation of [3H] uridine in RNA a f t e r AD t r e a t m e n t (0,04 ~ g / m l f o r 90 m i n ) a n d chase (see text). A : Hn-RNA, B : rRNA, C : 5S RNA a n d D : 4S RNA. 0--0--0 : U n i n f e c t e d cells, 0 - - 0 - - 0 : T r a n s f o r m e d cells.

D u r i n g c h a s e p o s t AD t r e a t m e n t (fig. 1 A) a p r o gressive r e d u c t i o n of Hn-RNA t r a n s c r i p t i o n is o b s e r ved in b o t h series of c u l t u r e s w h i c h r e a c h e s a m i n i m u m at the 3rd h o u r . The rate of H n - R N A i n h i b i t i o n is m o r e r a p i d in t r a n s f o r m e d cells t h a n in u n i n f e c t e d cells.

P r e - r i b o s o m a l RNA r e m a i n s t o t a l l y i n h i b i t e d u n d e r t h o s e c o n d i t i o n s d u r i n g t h e first 3 h o u r s of c h a s e (fig. 1B) in b o t h series of c u l t u r e s . Af t h e 4th h o u r r-RNA s y n t h e s i s is r e s u m e d a n d r e a c h e s a 100 p e r cent r a t e a f t e r 6 h o u r s , in t r a n s f o r m e d cells. I n t h e u n i n f e c ted cells s y n t h e s i s is r e s u m e d at t h e 5 t h h o u r a n d r e a c h e s 60 p e r cent r a t e a f t e r 10 h o u r s .

4

60

90

RSV t r a n s f o r m e d a n d u n i n f e c t e d c u l t u r e s w e r e w a s h e d 'with f r e s h m e d i u m a n d o v e r l a i d w i t h m e d i u m c o n t a i n i n g AD. A f t e r e x p o s u r e to t h e d r u g t h e cells w e r e c a r e f u l l y w a s h e d ~¢ith f r e s h m e d i u m a n d l a b e l l e d w i t h [3H] u r i d i n e i m m e d i a t l y (time 0) or a f t e r c h a sing f o r v a r i o u s t i m e s (table I). Each e x p e r i m e n t w a s r u n in p a r a l l e l w i t h t r a n s f o r m e d a n d u n i n f e c t e d cult u r e s t r e a t e d i d e n t i c a l l y in AD-free m e d i u m . The recov e r y r a t e of the different c l a s s e s of RNA in c u l t u r e s t r e a t e d w i t h AD, e x p r e s s e d as p e r c e n t of [3HI u r i d i n e i n c o r p o r a t i o n of t h a t of c u l t u r e s in AD-free m e d i u m , is p r e s e n t e d in figure 1. T h o s e d a t a w e r e o b t a i n e d b y p l o t t i n g t h e s u r f a c e r a t i o s of pies r e s o l v e d b y PAGE analysis.

S y n t h e s i s of H n - R N A is r e s u m e d 60 m i n e a r l i e r a n d at f a s t e r r a t e in t r a n s f o r m e d cells as c o m p a r e d to cont r o i s a n d t h e m a x i m u m r a t e is r e a c h e d a f t e r 5 h o u r s of chase in t r a n s f o r m e d a n d n o r m a l cells.

0 3 0,04

a n d e x p o s u r e t i m e of 00 m i n w e r e c h o s e n b e c a u s e f u l l r e c o v e r y a f t e r i n h i b i t i o n of RNA s y n t h e s i s w a s o b s e r v e d 24 h o u r s p o s t t r e a t m e n t .

o ~ ~ o o ".~ ~ ~ ~ --

A 80 p e r cent i n h i b i t i o n of t r a n s c r i p t i o n of lo~v m o l e c u l a r 'weight RNA is o b s e r v e d a f t e r AD t r e a t m e n t . As w i t h H n - R N A a n d p r e - r i b o s o m a l RNA, s y n t h e s i s is r e s u m e d at a f a s t e r r a t e in t r a n s f o r m e d cells as c o m p a r e d to n o r m a l cells (fig. 1 C a n d D). O u r d a t a

lOO 80 60 40

\

,'7 //

,

o

\

20

log

,,o~ o...

0,/.~p~.j

//-

80 60 4(~ 20

/ ,

1

2

3 4

5

6":10h

"!

2

3 4

5

hours of chase post A D treatement

BIOCHIMIE, 1977, 59, n ° 1.

h,

6"10h

R N A s t j n t h e s i s in R S V - i n f e c t e d cells t r e a t e d w i t h A D .

107

Ai

I

455 3~S

,,

1!

45S

!i

j!

/

,.

$

,A("

Z'": .'. v ~ / \ r-.-.. "W~ z

Z8s Bii

All

45s

1!

8$

0 x

'°

'i

FIG. 2.

A

32

A

,d!

PAGE analysis o/Hn-RNA and r-RNA alter inhibition by AD (0.04 ~ g / m l f o r 90 m i n ) of uninfected and RSV-lransformed cells after 3 h (AI a n d B~)., after 5h (A~ a n d B~I) a n d 6 h (A,11 a n d Bin) c h a s e . Left

side : u n i n f e c t e d ceils, r i g h t t r a n s f o r m e d cells.

X--X--X

0

cells t r e a t e d w i t h AD.

• c o n t r o l cells n o t t r e a t e d w i t h AD r u n in p a r a l l e l in e a c h e x p e r i m e n t .

g ,,,

side :

A c r 3 q a m i d e 1.7 %, a g a r o s e 0.5 % 180 V r u n n i n g t i m e 5 h.

n

h

i

A

21Is

III

|Ill

45S

11

"i//

45s 28s

1Is

10 32

".,'..j

L

12

16

Cm

12

do n o t a l l o w to d e t e r m i n e t h e b e g i n n i n g of t h e s y n t h e s i s f o r 5S a n d 4S R N A s . P A G E a n a l y s i s d i a g r a m s o f t r a n s c r i p t i o n a t select e d t i m e s o f c h a s e p o s t - A D t r e a t m e n t is s h o w n i n figure 2. A f t e r 3 h o u r s p o s t AD t r e a t m e n t figure 2 A1 a n d B1 sho'w t h a t h i g h m o l e c u l a r w e i g h t I~NA is c o m p l e t e l y i n h i b i t e d i n b o t h t y p e s o f cells. A f t e r 5 h o u r s o f c h a s e Hn-RN'A a n d r - R N A a r e s y n t h e t i z e d a t a d i f f e r e n t r a t e i n n o r m a l a n d t r a n s f o r m e d cells. I n t h e l a t t e r , t h e r a t e s of s y n t h e s i s a r e 70 p e r c e n t of

BIOCHIMIE, 1977, 59, n ° 1.

16

¢m

t h o s e o f c o n t r o l cells f o r b o t h R N A species, ~vhereas in n o r m a l cells t h e s e v a l u e s a r e 16 p e r c e n t a n d 40 p e r c e n t f o r r - R N A a n d H n - R N A r e s p e c t i v e l y (fig. 2, A 11 a n d B 11). A n o r m a l r a t e of s y n t h e s i s is r e s u m e d i n u n i n f e c t e d cells o n l y 6 h o u r s a f t e r r e m o v a l o f AD (fig. 2, A 111 a n d B 111). T h e profiles o f P A G E a n a l y s i s i n figure 2 a l s o s h o w t h a t c l e a v a g e of p r e - r i b o s o m a l R N A is n o t affected b y AD t r e a t m e n t i n b o t h t y p e s o f cells.

G. C o n n a n ,

108

G. S u s k i n d

and

G. F . R a b o t t i .

A 4,5

G ©

FIG. 3.

PAGE analysis of low molecular weight RNA. S a m e c o n d i t i o n s as i n

4.5

15

x

figure 2 a f t e r 3 h 30 m i n o f c h a s e .

(:1_

A : u n i n f e c t e d cells. B : f o r m e d cells.

5S

©

×--×--× - - - -

5S

'

RSV-trans-

cells t r e a t e d w i t h AD.

n o r m a l cells n o t t r e a t e d w i t h AD r u n in p a r a l l e l in e a c h experiment,

A c r y l a m i d e 8 % 130 V r u n n i n g t i m e 16 h. ,N,"6 .4¢4"..~,.*.. %.r~," ~'~,

9

12

cm

9

F i g u r e 3 (A a n d B) sho'ws t h e r e s u l t s f o r t h e l o w m o l e c u l a r w e i g h t R N A s a f t e r 3 h 30 m i n o f c h a s e . A g a i n o n e c a n see t h a t 5S a n d 4S R N A a r e s y n t h e t i z e d at a f a s t e r r a t e i n t r a n s f o r m e d as c o m p a r e d to u n i n f e c t e d cells. DISCUSSION.

12

cm

I n c o n c l u s i o n it it s h o w n h e r e t h a t t r a n s c r i p t i o n o f s o m e c l a s s e s of c e l l u l a r R N A e s c a p e m o r e r a p i d l y f r o m t h e i n h i b i t o r y a c t i o n of AD i n R S V - t r a n s f o r m e d cells t h a n i n u n i n f e c t e d cells. T h e p o s s i b i l i t y h a s b e e n r a i s e d [16, 23] t h a t AD c o u l d act u p o n r - R N A s y n t h e s i s t h r o u g h t h e H n - R N A o r t h r o u g h a r e g u l a t o r y p r o t e i n . S o m e of o u r r e s u l t s c o u l d h e e x p l a i n e d i n t h i s 'way.

T h e m o r e r a p i d r e c o v e r y of R N A s y n t h e s i s f r o m AD i n h i b i t i o n o b s e r v e d i n R S V t r a n s f o r m e d cells c o m p a r e d to t h a t of u n i n f e c t e d cells [1.3, 21] h a s b e e n s t u d i e d here by PAGE analysis of the transcription products. By s u c h a n a l y s i s it is s h o w n t h a t t h i s e a r l i e r r e c o v e r y i n t r a n s f o r m e d cells i n v o l v e s a l l c l a s s e s of R N A w h i l e previous studies based on high resolution autoradiography had suggested that ribosomal RNA was the o n l y f r a c t i o n i n v o l v e d in t h i s p h e n o m e n o n [13].

T h i s 'work w a s a i d e d b y a s u b v e n t i o n f r o m la F o n d a t i o n p o u r la R e c h e r c h e M6dicale F r a n q a i s e w h i c h we gratefully ackno'wledge here.

The new results presented here show that after t r e a t m e n t w i t h 0.04 i ~ g / m l o f AD f o r 90 r a i n t h e t r a n s c r i p t i o n of H n - R N A a n d r - R N A is r e s u m e d in t r a n s f o r m e d cells 60 m i n a h e a d of t h a t o b s e r v e d i n u n i n f e c t e d cells. T h e s e d a t a w e r e o b t a i n e d in s p i t e of t h e f a c t t h a t t h e p e n e t r a t i o n o f AD i n t r a n s f o r m e d cells w a s n o t a l t e r e d c o m p a r e d to t h a t i n u n i n f e c t e d cells. T h i s ~was s h o w n u s i n g AD a n d h i g h r e s o l u t i o n a u t o r a d i o g r a p h y w h e r e no d i f f e r e n c e c o u l d be d e m o n s t r a t e d i n t h e u p t a k e of t h e d r u g b e t w e e n R S V t r a n s f o r m e d cells a n d u n i n f e c t e d c o n t r o l cells [13]. O t h e r authors have reported similar results, still other w o r k e r s h a d o b s e r v e d d i f f e r e n c e s i n p e r m e a b i l i t y to AD a t t h e cell m e m b r a n e b e t w e e n n o r m a l a n d t r a n s f o r m e d cells in a n o t h e r s y s t e m : B H K cells i n f e c t e d b y v a r i o u s v i r u s e s [22]. T h e d i s c r e p a n c y h o w e v e r m a y he e x p l a i n e d b y t h e d i f f e r e n t t e e h n i q u e s u s e d . F u r t h e r m o r e t h e d a t a p r e s e n t e d i n figure 1 s h o w i n g t h a t t h e AD i n h i b i t i o n of t h e r a t e of s y n t h e s i s of all c l a s s e s o f R N A is h i g h e r i n t r a n s f o r m e d as c o m p a r e d to n o r real eells d u r i n g t h e first 3 h o u r s , is i n e o m p a t i h l e w i t h a d e f e c t i n d r u g p e n e t r a t i o n i n t r a n s f o r m e d cells c o m p a r e d to t h e u n i n f e c t e d cells. It is u n l i k e l y t h e n t h a t o u r f i n d i n g s m i g h t be t h e r e s u l t of d i f f e r e n t p e r m e a b i l i t y to t h e d r u g in t h e t w o c e l l - s y s t e m s .

1. P e n m a n , S., Vesco, C..& P e n m a n , M. (1968) J. Mol. Biol., 34, 49-69. 2. P e r r y , R. P. ,& K e l l y , D. E. (1970) J. Cell Physiol., 76, 127-139. 3. R e i c h , E. & G o l d b e r g , I. H. (1964) Pro 9. Nucleic Acid Res. MoL Biol., 3, 183-234. 4. D a r z y n k i c w i c z , Z., B o l u n d , L _ - R i n g e r t z , N. R. (1969) Exp. Cell Res., 55, 120. ,a 5. B o l u n d , L. (1970) Exptl. Cell Res., 63, 171-188. 6. B e n e d e t t o , A., Delfini, C., C a r l o n i , G. z~ D j a l z e n k o , W . (1975) J. Cell Biol., 67, 538-550. 7. P e d e r s o n , T. ,& R o b b i n s , E. (1972) J. Cell Biol., 5 5 , 322-327. 8. Sa'wicky, G. S. & G o d m a n , G. C. (1972) J. Cell Biol., 55, 299-309. 9. T e m i n , H. M. (1963) Virology, 20, 577-582 10. B a d e r , J. P. (1964) Virology, 22, 462-468. 11. Vigier, P. ~ Gold6, A. (1964) Virology, 23, 511-519. 12. T e m i n , H. M. (1971) J. Natl. Cancer Inst., 46, IIIVII 13. S u s k i n d , R. G., M i e h e l s o n - F i s k e , S.. H a g u e n a u , Fr. .& R a b o t t i , G. F. (1975) J. Natl. Cancer Inst., 54, 319-360.

BIOCHIMIE, 1977, 59, n ° 1.

W h e t h e r t h e o b s e r v e d d i f f e r e n c e s i n s e n s i t i v i t y to AD i n h i b i t i o n of t r a n s c r i p t i o n a r e r e l a t e d to t h e s t a t e of t r a n s f o r m a t i o n , to v i r a l r e p l i c a t i o n o r to b o t h r e m a i n s to be clarified.

REFERENCES.

RNA

s y n t h e s i s in R S V - i n f e c t e d

14. Ishizaki, R. ~ Vogt, P. (1966) Virology, 30, 375-387. 15. Sa~ceicki, S. G. ~ Godman, G. C. (1971) J. Cell Biol., 50, 746-761. 16. Hadjiolov and Col. (1974) Biochem. J., 138, 321-334. 17. Scherrer, K. ,~ Darnell, J. E. (I962) Biochem. Riophys. Res. Commun., 7, 486-490. 18. Tiollais, P., Galibert, F..¢ Boiron, M. (1971) Ellr. J. Biochem., 18, 35-45.

BIOCHIM1E, 1977, 59, n ° 1.

cells t r e a t e d w i t h A D .

109

19. Tiollais, P., Galibert, F., Lepetit, A. & Auger, M. A. (1972) Biochimie, 3, 339-354. 20. Loening, U. E. (1967) Biochem. J., 102, 251-257. 21. Suskind, R. G., Pry, W. ~ Rabotti, G. F. (1969) Cancer Res., ~9, 1598-1605. 22. Williams, J. G. a MaePherson, J. (1973) J. Cell Biol., 57, 148-158. 23. Lindell, T. J. (1976) Nature, 263, 347-349.

9