BIOCHIMIE, 1979, 61, 913-922.

Preferential incorporation of an exogenous cytokinin, N -benzyladenine, into 18S and 25S ribosomal RNA of tobacco cells

in suspension culture. Bernard TEYSSENDIER DE LA SERVE and J'ean-Pierre JOUANNEAU . (1-2-1979)

Laboratoire de Biochimie Fonetionnelle des Plantes, DOpartement de Biologie Moldcttlaire et Cellalaire, Facult~ des Sciences de Lttminy, 13288 Marseille Cedex 2, France.

Rdsumd.

Summary.

Des cellules de t a b a c exiqeantes eta cytokinine exoq~ne p o u r leur multiplication ont ~t6 raises e n incubation p e n d a n t 10 h e n p r 6 s e n c e d ' u n e cytokinine m a r q u 6 e , la N6-benzyl-[2-3H] Ade, et d e [8-14C]Ado. Apr~s h y d r o l y s e alcaline du RNA total et fractionnement des nucl6otides obtenus, 80 p. cent d e la radioactivit6 SH d u RNA ont 6t6 retrouv6s sous forme du nucl6otide de la /V%benzyl-Ado, li6 de mani~re covalente & des chalnes polynucl6otidiques. Ce nucl~otide n'a p a s 6t6 m a r q u 6 significativement p a r le 14C : la proportion d e ce nucl6otide susceptible de r6sulter d'un transfert du radical b e n z y l e sur des r6sidus a d 6 n i n e pr6sents d a n s les RNA pr6form6s, est inf6rieure & 0,01 p. cent de la quantit6 totale incorpor6e. L'insertion covalente de la N%benzyl-Ade d a n s les RNA int6resse donc la b a s e substitu6e intacte.

Cytokinin-requiring tobacco ceils w e r e incub a t e d for 10 h in the p r e s e n c e of a l a b e l e d cytokinin, N6-benzyl-[2-3H]Ade, a n d of [8-t4C] Ado. After alkaline hydrolysis of total RNA a n d fractionation of the resulting nucleotides, 80 per cent of the 3H radioactivity of RNA w e r e r e c o v e r e d a s the N6-benzyl-Ado nucleotide, c o v a l e n t l y inserted into polynucleotidic chains. The N6-benzy1-Ado nucleotide w a s not significantly l a b e l e d b y 1+C: at most one part of this nucleotide per 10 000 m a y result from a transfer of the b e n z y l m o i e t y to a d e n y l residues in p r e f o r m e d RNA. Thus, the covalent insertion of N6-benzyl-Ade into RNA involves the intact N6-substituted b a s e .

I1 a ~t6 proc6d6 a u fractionnement du RNA total p a r centrifugation en gradient de concentration de s a c c h a r o s e ou p a r 61ectrophorbse en gel de p o l y a c r y l a m i d e . Toutes les esp~ces de RNA identifi~es ont incorpor6 d e l a N6-benzy1Ade. C e p e n d a n t , l a fr6quence d'incorporation, m e s u r 6 e p a r le rapport du h o m b r e d e mol6cules de N~-benzyl-Ade a u n o m b r e total de mol6cules de bases, est 3 & 4 fois plus 61ev6e darts les rRNA 25S et 18S q u e darts les RNA 5S et 4S. Ainsi, la quantit6 de N6-benzyl-Ade incorpor6e d a n s les rRNA 18S et 25S repr6sente 90 p. cent environ d e la quantit6 pr6sente darts le RNA total. L'61ectrophor~se des RNA d6natur6s en pr6sence d e f o r m a m i d e a confirm6 q u e ce sont les mol6cules d e RNA elles-m6mes qui contiennent la N6-benzy1-Ade. To whom all correspondence should be addressed.

Total RNA w a s fractionated either b y sucrose density gradient centrifugation or b y p o l y a c r y l a m i d e gel electrophoresis. All identified RNA species w e r e s h o w n to contain N6-benzyl-Ade. The insertion f r e q u e n c y , m e a s u r e d a s the molecular proportion of N6-benzy1-Ade to the total b a s e content, w a s 3 to 4 times l a r g e r in 25S a n d 18S rRNA t h a n in 5S a n d 4S RNA. The a m o u n t of N6-benzyl-Ade inserted into cytoplasmic ribosomal RNA a c c o u n t e d for a b o u t 90 per cent of the a m o u n t incorporated into total RNA. Electrophoresis of d e n a t u r e d RNA in the pres e n c e of f o r m a m i d e p r o v i d e d additional evid e n c e that N6-benzy1-Ade w a s i n d e e d incorpor a t e d into RNA molecules.

Key words : Cytokinins - N6-benzyladenine - rRNA Tobacco cells. 63

914

B. T e y s s e n d i e r de la Serve a n d J.-P. J o u a n n e a u .

Introduction. T h e s t i m u l a t i o n of m i t o s e s of p l a n t cells is o n e of t h e e s s e n t i a l i m p a c t s of t h e s u p p l y of s o m e N 6substituted adenines or their analogues, described as cytokilnins [11. T h e m o l e c u l a r m e c h a n i s m ( s ) o f t h i s s p e c i f i c a c t i v i t y is still u n k n o ~ v n in s p i t e of w i d e s p r e a d i n v e s t i g a t i o n s in t h i s field (see f o r i n s t a n c e [2]). In our laboratory, kinetic studies of the growth r e s p o n s e of t o b a c c o cell s u s p e n s i o n s h a v e p r e v i 0 u s l y s h o w n t h a t t h e s u p p l y of c y t o k i n i n to t h e c u l t u r e m e d i u m w a s r e q u i r e d f o r a b o u t one h a l f g e n e r a t i o n p e r i o d (i.e. 18 h) b e f o r e t h e o p t i m a l sti. m u l a t i o n of m i t o s e s ~vas u l t i m a t e l y o b s e r v e d [3]. F u r t h e r s t u d i e s [4, 5, 6] s u g g e s t e d t h a t a b r o a d i m p a c t of t h e c y t o k i n i n f a c t o r on m a s s b i o s y n t h e t i c o r m e t a b o l i c steps in the cell w a s q u i t e unlikely. T h e c y t o k i n i n r a t h e r s e e m e d to c o n t r o l the e f f i c i e n c y of o n e o r a s m a l l n u m b e r of m o l e c u l a r e v e n t s c o n n e c t e d to t h e m i t o t i c c y c l e [3, 7]. T w o l i n e s of e x p e r i m e n t s r e c e n t l y p r o d u c e d s o m e r e s u l t s of t n t e r e s t a b o u t t h e m o l e c u l a r i m p a c t of c y t o k i n i n s . F i r s t , it h a s b e e n s h o w n t h a t s o m e p l a n t cell p r o t e i n s e x h i b i t a s t r o n g s p e c i f i c a f f i n i t y f o r N 6 - s u b s t i t u t e d a d e n i n e s of t h e c y t o k i n i n g r o u p . N e v e r t h e l e s s , little is k n o w n on the p a t h w a y s in w h i c h s u c h p r o t e i n s m a y be efficient. S e c o n d , it w a s shoavn in t w o l a b o r a t o r i e s [8, 9, 10j t h a t s o m e c y t o k i n i n s , s u p p l i e d as f r e e bases, w e r e in p a r t r e c o v e r e d as c o v a l e n t l y h o u n d nuc l e o t i d e s in R N A c h a i n s of t o b a c c o tissue c u l t u r e s o r cell s u s p e n s i o n s . M o r e o v e r , u s i n g t o b a c c o t i s s u e c u l t u r e s a n d a d o u b l e l a b e l e d p r e c u r s o r , it w a s s h o w n [8, 11] t h a t N ~ - b e n z y l a d e n i n e , a p o t e n t c y t o k i n i n f a c t o r , ~vas i n s e r t e d i n t o R~NA w i t h o u t a n y s i g n i f i c a n t loss o r g a i n of t h e N6-aryl c y c l e . F r o m t h e first e x p e r i m e n t of t h e p r e s e n t w o r k , it w i l l he s e e n h o w w e a r r i v e d at t h e s a m e conclusion. It h a d b e e n k n o w n f o r s o m e y e a r s t h a t d i s t i n c t t R N A s p e c i e s c o n t a i n e d a h y p e r m o d i f i e d b a s e bel o n g i n g to t h e c y t o k i n i n g r o u p , a d j a c e n t to t h e 3' e n d of t h e a n t i c o d o n [12]. I n t h i s case, t h e l a t e r a l c h a i n of t h e h y p e r m o d i f i e d b a s e is t r a n s f e r r e d o n t o t h e f r e e -N,H 2 g r o u p of t h e a d e n i n e n u c l e o tide previously transcribed in the proper position n e x t to t h e a n t i c o d o n [13]. T h e d i r e c t i n s e r t of N 6 - s u b s t i t u t e d a d e n i n e s in t h e B N A c h a i n s of t o b a c c o cells is o b v i o u s l y a d i f f e r e n t p r o c e s s . T h e a i m o f t h e p r e s e n t w o r k w a s to i d e n t i f y t h e R N A s p e c i e s to w h i c h t h e i n t a c t N 6 - s u b s t i t u t e d a d e n i n e s w e r e l i n k e d a n d to e s t i m a t e t h e m o l e c u -

BIOCHIMIE, 1979, 61, n ° 8.

l a r p r o p o r t i o n of s u c h c y t o k i n i n s r e c o v e r e d f r o m d i f f e r e n t R N A species. T a k i n g i n t o a c c o u n t t h e already mentioned kinetic studies, the cytokinin w a s a d d e d to t h e i n c u b a t i o n m e d i a o n l y d u r i n g h a l f t h e t i m e s p a n r e q u i r e d for an o p t i m a l m i t o t i c efficiency. A p r e l i m i n a r y a c c o u n t of t h i s w o r k h a s a l r e a d y b e e n p r e s e n t e d [14].

Materials and Methods. CELL CULTURES AND INCUBATIO~ CONDITIONS. A cytokinin-requiring clonal cell line (II 19), isolated from a permanent strain of tobacco cells (Nicotiana tabaeum, c.v. Wisconsin 38), was used [15]. Cell suspensions were maintained in shaken liquid medium as previously described [7]. Early stationary phase cells were washed in eytokinin-free culture medium. The cells were further incubated in the same medium at a cell density of 1.0 to 1.2 g (fresh weight) in 25 ml (ca. 200 000 eells/ml). After 17 h at 25°C, cell suspensions were supplemented w i t h tritiated N6benzyl-adenine (N6-benzyl-[2-3H]Ade, 17.6 Ci/mmole), [8-~4C]Ado and 10 ~M each nucleoside Guo, Urd and Cyd. N6-benzyl-Ade and nucleoside solutions were sterilized by aseptic filtration. Concentrations and radioaetivities of labeled precursors will be specified for each experiment. Cells were incubated in flasks shaken for 10 h at 25°C under white light (approx. 1000 lux). RADIOACTIVE PBECUnSORS,

N6-henzyl-[2-3H]Ade ~eas synthesized from [2-3H] Ade (specific radioactivity 23 Ci/mmole) obtained from The Radiochemical Centre (Amersham, England). The chemical purity of the radioactive eytokinin was controlled as previously described [10]. [8-14C]Ado (46.5 m C i / m m o l e ) was purchased from C.E.A. (Saelay, France). l ~ N A EXTRACTION FROM TOBACCO CELLS.

Reagent volumes are given for 1 g of starting blotted cells. After incubation, ceils were collected on a 50 Ix nylon filter and washed five times at 4°C ~,vith 10 ml of 10 mM KC1, 5 mM Na._,-EDTA, 50 IxM N6-benzyl-Ade, pH adjusted to 5.7 w i t h KOH. Cells 'were then frozen by liquid nitrogen and stored at --20°C. The frozen cells 'were lha'wed in the RNA extraction mixture : 3 ml buffer A, 2 ml phenolic solution. Buffer A [16] : 20 mM Tris-HCl, pH 7.2; 10 mM NaC1 ; 10 mM Na~_-EDTA ; 50 ixg/ml sodium polyvinyls u l p h a t e ; 2 m g / m l (National Lead Co, Houston, Texas). Fifty ~M N6-benzyl-Ade and 50 IxM N6-benzyl-Ado were added to bring about the isotopic dilution of tritiated precursors possibly adsorbed on RNA [10]. Phenolic solution [17] : I00 ml p h e n o l ; 14 ml m - c r e s o l ; 100 mg 8-hydroxyquinoline ; 0,5 g sodium dodecylsulphate (SDS). This solution was saturated with buffer A. The thawed ceils were disrupted at 0°C w i t h an 3 800

> 56 000

from N6-benzyl-Ado-3'-p

>

>

4 200

62 000

Values measured : A) in nucleotide fractions obtained by Dowex 1 chromatography of a n a l k a l i n e hydrolysate of total RNA. B) in N6-benzyl-Ado, obtained by phosphatase hydrolysis of the relevant nucleotides collected in experiment A. TABLE II.

Incorporation o[ N6-benzyl-[2-~H]Ade and o[ [8-1~C]Ado into total RNA and major RNA species. RNA fractious

Total

25S

I8S

Relative amount : per cent of total RNA 3H/t~C ratio

100 0.93

52.3 0.87

27.4 0.98

Specific radioactivity of adenyllc m~cleotides: t4C dpm/nmole

461

I n c o r p o r a t e d N6-benzyl-Ade • - - Frequency: pmoles/zxmole nucleotide - - Amount : per cent of the amount i n c o r p o r a t e d into total RNA

3.16 100

439 3.48 57.5

416 3.52 30.5

45+5S 20.3 0.45 349 0.89 5.8

Incubation conditions : 0.09 lxM N6-benzyl-[2-aH]Ade ; 50 p.M [8-14C]Ado (1.1 mCi/mmole). The amount of each RNA species was measured by planimetry of each peak area, after the A~o absorbanee scanning of an eleetrophoregram of total RNA on polyaerylamide-agarose gel. On the e l e c t r o p h o r e g r a m of figure 2, chloroplastic or m i t o c h o n d r i a l rRNA did not appear. These RNA w e r e e i t h e r u n r e s o l v e d by the e l e c t r o p h o r e sis p r o c e d u r e or w e r e p r e s e n t in t h e total RNA in a m o u n t u n d e r t h e detectable limit. These possibilities w e r e e x a m i n e d in c o n t r o l e x p e r i m e n t s . T h e total RNA w a s p r e p a r e d f r o m late s t a t i o n a r y phase cells w h i c h are k n o w n to c o n t a i n a n u m b e r of d i f f e r e n t i a t e d c h l o r o p l a s t s [23]. The RNA w a s ann_ lyzed on p o l y a c r y l a m i d e - a g a r o s e gel : t w o small UV a b s o r b i n g peaks 'were r e s p e c t i v e l y s e p a r a t e d f r o m 25S and 18S rR,NA ( e l e c t r o p h o r e g r a m not shown) ; each of these peaks a m o u n t e d to about 5 p e r cent of its c y t o p l a s m i c counterpa~'t. T w o

BIOCHIMIE, 1979, 61, n ° 8.

w e r e estimated at 1.0 × 106 and 0.58 × 106 daltons by a c o m p a r i s o n w i t h 25S and 18S rRNA, suggesting these peaks to be due to c h l o r o p l a s t i c rRNA. These results suggested that the amount of o r g a n e l l e RNA was too small to be detected in total RNA of y o u n g e r cells i n c u b a t e d as in the exp e r i m e n t of figure 2 a n d t h a t the organelle RNA could not a c c o u n t for the ~H or 14C r a d i o a c t i v i t i e s m e a s u r e d in the total RNA of these cells. The 3H/14C values m e a s u r e d in the above exper i m e n t s suggested that the m a j o r species of cytop l a s m i c RNA i n c o r p o r a t e d N"-benzyl-[2-ZH]Ade and that the i n c o r p o r a t i o n rate w a s g r e a t e r in the

9'20

B. Tegssendier de la Serve and J.-P. Jouanneau.

18S or the 25S rl~,NA t h a n in the 4S + 5S RNA fraction. Nevertheless, it was necessary to distinguish b e t w e e n a~H r a d i o a c t i v i t y as accounted for by [2-3H]Ade or N6-benzyl-[2-~H]Ade i n each of the isolated RNA species.

Frequency of N~-benzgl-[2-~H]Ade in major RNA species. To measure the actual a m o u n t of N6-benzyl-Ade i n c o r p o r a t e d into each RNA species, the total RNA was f r a c t i o n a t e d by sucrose g r a d i e n t centrifugation. 25S, 18S a n d 4S + 5S RNA w e r e separately recovered from the relevant fractions by ethanol p r e c i p i t a t i o n a n d s u b m i t t e d to an alkaline h y d r o lysis. The resulting nucleotides were chromatographed o n Dowex I as already described. Since N6benzyl-[2-aH]Ade has been s h o w n to be i n c o r p o rated w i t h o u t isotopic dilution, the m e a s u r e m e n t of the ~H r a d i o a c t i v i t y of N6-benzyl-Ado nucleotides allowed to calculate the n u m b e r of molecules i n c o r p o r a t e d as a fraction of the total n u m b e r of c h r o m a t o g r a p h e d nucleotides. As seen i n table II, the i n c o r p o r a t i o n frequencies of N6-benzyl-[2-aH]Ade i n the m a j o r R~NA species were in agreement w i t h t h e i r ~H/I*C ratios : the f r e q u e n c y was 3.5 to 4 times larger i n 25S or 18S rRNA t h a n i n the 4S ~- 5S RNA mixture. Moreover, the specific 1~C radioactivities of the Ado nucleotides recovered from every R NA species w e r e similar. If the label of RNA b y [8-1~C]Ado is taken as a reliable m e a s u r e m e n t of the rate of sy.n_ thesis, the h i g h e r Ne-benzyl-Ade f r e q u e n c y in rRNA c o m p a r e d to the 4S ~- 5S RNA m i x t u r e was not d u e to different rates of synthesis of these RNA species. This result implies that the exogenous c y t o k i n i n is p r e f e r e n t i a l l y i n c o r p o r a t e d into rl~.'NA. T a k i n g into a c c o u n t the p r o p o r t i o n of each RNA species in the total RNA, it was calculated (table II) that 88 p e r cent of the N6-benzylAde i n total RNA was i n c o r p o r a t e d into rRNA.

Electrophoresis of RNA under denaturing conditions. It was not yet excluded that at least a part of the i n c o r p o r a t e d N6-henzyl-Ade m i g h t be i n s e r t e d i n small oligonucleotide c h a i n s associated by noncovalent b i n d i n g to some of the m a j o r R,NA species. On the other h a n d , in the previous analysis of the total RNA, 5.S R.NA w a s not resolved from 4S RNA ; nor coul,d 5.8S RNA be observed since its dissociation from 25.S RNA requires d e n a t u r a tion [24!. C o n s i d e r i n g this, the total RNA was den a t u r e d a n d f r a c t i o n a t e d u n d e r c o n d i t i o n s allow i n g a p r o p e r separation of low m ~ l e c u l a r weight RNA.

BIOCHIMIE, 1979, 61, n ° 8.

P o l y a c r y l a n f i d e gel electrophoresis in formamide resolved five B.NA species (fig. 5A). Compared to control E. colt RNA, t h e i r different molecular weights were estimated at 1.1 × 106, 0.7 × l06, 52 000, 40 000 a n d 25 000 daltons ; they were referred to as, 25S, 18S, 5.~S, 5S and 4.S RNA respectively. As a l r e a d y observed u n d e r n o n denatur i n g conditions, all I%NA species were both aH and 14C labeled. The aH/14C ratios of d e n a t u r e d 25S and 18S RNA were in agreement w i t h the relevant ratios of native molecules. To obtain accurate m e a s u r e m e n t s of the radioactivities of the low molecular weight RNA fractions, a large q u a n t i t y of d e n a t u r e d RNA was f r a c t i o n a t e d on a composite f o r m a m i d e gel (fig. 5;B). It was noticed that the aH/14C ratio of the 5.8S RNA was almost equal to the ratio of the 25S RNA w h e r e a s the 5S RNA and the 4S R,NA ratios were close together but different from the formers. These results were evidence that N~-benzyl[2-~H]Ade was really i n s e r t e d into the p o l y n u cleotide c h a i n of the m a j o r RNA species. They also confirmed the p r e f e r e n t i a l i n c o r p o r a t i o n into 25S a n d 18S rRNA.

Discussion. It has been p r e v i o u s l y s h o w n that w h e n tobacco cells were s u p p l e m e n t e d w i t h N6-benzyl-Ade or N6-furfuryl-Ade, the N6-substituted a d e n i n e was i n part recovered i n RNA [8, 9, 10]. We first endeavoured to ascertain w h e t h e r N6-benzyl-Ade was covalently i n s e r t e d in RNA chains or w h e t h e r N6-benzyl-Ade-containing metabolites or the free base itself were tightly b o u n d to purified RNA. Alk a l i n e h y d r o l y s i s of R~NA y i e l d e d N6-benzyl-Ado2'-P and N6-benzyl-Ado-3'-P. The structures of these nucleotides w e r e conclusive evidence of the covalent linkage of N6-benzyl-Ado n u c l e o t i d e into p o l y r i b o n u c l e o t i d e chains. Nevertheless, it could not be p r e c l u d e d that these chains might be oligonucleotides, h y d r o g e n - b o u n d to the RNA molecules. The d e n a t u r a t i o n of RNA has s h o w n that the N~-benzyl-Ade-bearing molecules w e r e R,NA themselves, as identified by t h e i r electrophoretic mobilities u n d e r d e n a t u r i n g conditions. This result b r o u g h t forth the p r o b l e m of the identification of the m e c h a n i s m b y w h i c h N6-benzyI-Ade w a s inserted into R~A. Except 5S RNA [25], m a t u r e RNA of ettkaryotic ceils c o n t a i n modified bases [26]. Among others, N6-(he-isopentenyl)-Ade is adjacent to the 3' end of some tRNA a n t i c o d o n s w h i c h recognize codons s t a r t i n g with letter U ; such bases are synthesized by m o l e c u l a r modification of t r a n s c r i p t e d a d e n i n e [12, 13]. E1liott a n d Murray [27] suggested that the a d e n i n e

B e n z y l a d e n i n e in R N A . of tRNA might be t r a n s b e n z y l a t e d by a process of this type. Our e x p e r i m e n t s are not i n agreement w i t h this hypothesis : w e showed that the f r a c t i o n of N6-benzyl-Ade i n s e r t e d into RNA c h a i n s b y t r a n s b e n z y l a t i o n c a n n o t be larger t h a n 1 molecule per 10 000 inserted into RNA. Our results agree w i t h the reports of other authors [8, 11] w h i c h p r o v i d e d evidence that N6-[~tH]benzyl-[a4C]Ade was i n c o r p o r a t e d into the RNA of tobacco tissues w i t h o u t change in the aH/I~C ratio. This body of evidence proves that the i n s e r t i o n of intact cytok i n i n molecules into R~A takes place by a mechanism different from the formation of h y p e r m o d i fled bases p r e s e n t next to the a n t i c o d o n of some tRNA. The question is w h e t h e r the i n s e r t i o n of intact N6-substituted a d e n i n e s is a t r a n s c r i p t i o n a l e r r o r or alternatively a p o s t - t r a n s c r i p t i o n a l p h e n o m e n o n w h i c h might be relevant to the c y t o k i n i n activity. The i n c o r p o r a t i o n frequencies of N6-benzyl-Ade in the various RNA species are low. According to our estimations, they are lower t h a n the e r r o r rate of E. colt RNA polymerase, as m e a s u r e d in vitro [281. However, the estimations of table II refer to the total q u a n t i t y of the relevant RNA species. These values are ¢ by default >> since the n u m b e r of target molecules of N6-substituted Ade is only an u n k n o w n fraction of the total RNA. D u r i n g a 10 h i n c u b a t i o n , the net weight increase i n RNA is n e a r 30 per cent ( u n p u b l i s h e d ) . On the other h a n d , the N6-benzyl-Ade c o n c e n t r a t i o n in the m e d i u m was 0.05 to 0.1 ~M, well u n d e r s a t u r a t i o n c o n d i tions [29]. It shoul,d also be observed that endogenous N 6substituted Ade molecules are likely t~ be present, even in c y t o k i n i n - r e q u i r i n g cells. It is expected that these endogenous bases w i l l compete w i t h the exogenous tracer for the i n c o r p o r a t i o n into RNA. This suggestion is further s u p p o r t e d by the evidence that exogenous N6-furfuryl-Ade a n d N6-(A2isopentenyl)-Ade are i n d e e d competitive i n h i b i tors of the N6-benzyl-Ade i n c o r p o r a t i o n , w h e r e a s exogenous free a d e n i n e is a very poor competitor [29]. F o r these reasons, the i n c o r p o r a t i o n frequencies of N6-benzyl-[2-ZH]Ade, as m e a s u r e d in table II, are u n d e r e s t i m a t e s of the s a t u r a t i n g load of 1RNA by c y t o k i n i n s . The i n c o r p o r a t i o n of N6-benzyl-Ade in the 4S I~NA fraction raised other questions. It is not u n likely that such fractions c o n t a i n e d fragments of high u m l e c u l a r weight RNA species, mixed w i t h u n c h a r a c t e r i z e d i n d i v i d u a l tRNA species. MoreoBIOC,HIMIE,

1 9 7 9 , 61, n ° 8.

9'21

ver, it is not k n o w n w h e t h e r Na-benzyl-Ade is inserted into each tRNA species w i t h the same freq u e n c y or w h e t h e r some species electively incorporate such molecules. The essential p o i n t emphasized by our experiments is that the f r e q u e n c y of the N6-benzyl-Ade i n s e r t i o n into the 18S a n d 25S rRNA is 3.5 to 4 times larger t h a n into 4S or 5S RNA. This preferential i n c o r p o r a t i o n of N6-substituted bases was ind e p e n d e n t l y r e p o r t e d by other authors [8, 9, 30], although their e x p e r i m e n t a l a p p r o a c h was quite different from ours. It should be u n d e r l i n e d that these authors used i n c u b a t i o n s of the tissues w i t h the tracer c y t o k i n i n d u r i n g several weeks. I n our experiments, the i n c u b a t i o n s p a n was restricted to 10 h, the time necessary to later observe the onset of mitoses i n control cultures. These converging findings strongly suggest that the differences b e t w e e n the i n s e r t i o n frequencies of N6-substituted Ade into rtENA on the one h a n d , 4S a n d 5S on the other, are significant a n d detect specific m o l e c u l a r processes. The hypothesis of a t r a n s c r i p t i o n a l e r r o r is not d i s c a r d e d b y this evidence : for instance, the error rate of type HI RNA p o l y m e r a s e t r a n s c r i b i n g 4S a n d 5S RNA species might be s m a l l e r t h a n the e r r o r rate of type I RNA p o l y m e r a s e t r a n s c r i b i n g rRNA. I n agreement w i t h the results of a n a l r e a d y m e n t i o n e d report [30], a small difference might exist b e t w e e n the i n c o r p o r a t i o n rates into 18S a n d 25S rRNA, the significance of w h i c h is not clear. On the other h a n d , if the linkage of N¢-benzyl-Ade were a p o s t - t r a n s c r i p t i o n a l process, the f r e q u e n c y of N6-benzyI-Ade i n rRNA p r e c u r s o r s should be tower t h a n i n m a t u r e rRNA : such a difference ~nay exist ,Mthough our results are still p r e l i m i n a r y . At last, it was f o u n d that d e n a t u r e d fragments of the large rBNA s u b u n i t exhibited sim i l a r ~H/14C ratios. This result does not p r o v i d e a d d i t i o n a l i n f o r m a t i o n on the i n s e r t i o n process. It should be m e n t i o n e d that Mural et al. [30] inc o r p o r a t e d labeled N6-furfuryl-Ade into tobacco rRNA a n d s t u d i e d their complete h y d r o l y s i s products b y RNases A a n d T I : it t u r n e d out that the r a d i o a c t i v i t y per nucleotide was larger in di - or t r i - n u c l e o t i d e s e n d i n g by C or U than i n the oligonucleotides t e r m i n a t e d by G. It should be recalled that a d e n i n e acts as a very poor c o m p e t i t o r of the Ng-benzyl-Ade i n s e r t i o n into RNA of tobacco cells w h e r e a s some cytokin i n s are active competitors [29]. This fact might p r o v i d e some evidence in favor of a specific insertion of Ng-substituted Ade. The above results are the first steps towards an e x p l a n a t i o n of the

922

B. Teyssendier de la Serve and J.-P. Jouanneau.

i n c o r p o r a t i o n of i n t a c t c y t o k i n i n b a s e s i n t o RNA, w h e t h e r a t r a n s c r i p t i o n a l e r r o r or a p o s t - t r a n s criptional insertion.

Acknowledgements. This ~mor/¢ was supported by the > (Contrat n ° 73-7-1855). The s kilful and competent technical assistance of M. C. Durand is gratefully acknowledged. We are indebted to Dr. C. Pdaud-Leno~l for useful suggestions and his constant interest in this work.

FtEFEFtENCES. 1. S~oog, F..& Armstrong, D. J. (1970) Annu. Rev. Plant Fhysiol., 21, 359-384. 2. Burrows, W. J. (1975) Current Adv. Plant Set., 7, 837-847. 3. J o u a n n e a u , J. P. & Tandeau de Marsac, N. (1973) Exp. Cell Res., 77, 167-174. 4. Jouanneau, J. P. (1970) Physiol. Plant., 23, 232-244. 5. Fosket, D. E. & Short, K. C. (1973) Physiol. Plant., 28, 14-23. 6. Fosket, D. E., Voll~, M. J. & Goldsmith, M. R. (1977) Plant Physiol., 60, 554-562. 7. Jouanneau, J; P. (1971) Exp. Cell Res., 67, 329-337. 8. Armstrong, D. J., Murat, N., Taller, B. J. ~ Sl~oog, F. (1976) Plant Physiol., 57, 15-22. 9. Murat, N., Taller, B. J., Armstrong, D. J., Skoog, F., Mi~ke, M. A; .& Sehnoes, H. K. (1977) Plant P h y siol., 60, 197-202. 10. Jouanneau, J. P., Gandar, J. C. & P6aud-Leno~l, C. (1977) Plant Sci. Lett., 9, 77-87. 11. Wal~er, G. C., Leonard, N. J., Armstrong, D. J., Murat, N. & Skoog, F. (1974) Plant Physiol., 54, 737743.

BIOCHIMIE, 1979, 61, n ° 8.

12. Nishimura, S. (1972) in ¢ Frog. Nucleic Acid Res. Mol. Biol. >> (Davidson, J. N. & Cohn, W. E. ed.), Vol. 12, pp. 49-85, Academic Press, New York and London. 13. Hall, R. H. (1970) in ~ Frog. Nucleic Acid Res. Mol. Biol.>> (Davidson, J. N. ~, Cohn, W. E. ed.), Vol. 10, pp. 57-86, Academic Press, New York and London. I4. J o u a n n e a u , J. P., Gandar, J. C., Teyssendier de la Serve, B. & P6aud-Leno~l, C. (1978) F e d e r a t i o n of European Societies of P l a n t Physiology. Inaugural Meeting, Edinburgh, July 10-13 1978. Abstracts of invited and poster papers, pp. 276-277. 15. Tandeau de Marsac, N. & J o u a n n e a u , J. P. (1972) Physiol. Vdg., 19, 369-380. 16. Ceechini, J. P. ~ Miassod, Ft. (1976) Biochim. Biophys. Acta, 418, 104-116. 17. Miassod, R., Penon, P., Teissere, M., .qicard, J. & Ceechini, J. P. (1970) Biochim. Biophys. Acta, 224, 423-440. 18. Loening, U. E. (1967) Biochem. J., 102, 251-257. 19. Tiollais, P., Galibert, F., Lepetit, A. ~ Auger, M. A. (1972) Bioehimie, 54, 339-354. 20. Pinder, J. C., Staynov, D. Z. & Gratzer, W. B. (1974) Biochemistry, 13, 5373-5377. 21. Gilbert, W. (1963) J. Mol. Biol., 6, 389-403. 22. Hurlbert, R. B. (1957) in ¢, Methods in Enzymology>> (Colowick, S. P. & Kaplan, N. O. ed.), Vol. III, pp. 785-805, Academic Press, New York and London. 23. Seyer, P., Marty, D., Lescure, A. M. & P~aud-Leno~l, C. (1975) Cell Differ., 4, 187-197. 24. Payne, P. I. & Dyer, T. A. (1972) Nature New Biol., 23.5, I45-147. 25. E r d m a n n , V. A. (1978) Nucl. Acid Res., 5, r l - r l 3 . 26. Perry, R. P. (1976) Annu. Rev. Biochem., 45, 605629. 27. Elliott, D. C. & Murray, A. W. (1972) Biochem. J., 130, 1157-1160. 28. Springgate, C. F. & Loeb, L. A. (1975) J. Mol. Biol., 97, 577-591. 29. J o u a n n e a u , J. P. ~ Gandar, J. C. (1979) C. R. Acad. Set., Paris, 288, 611-614. 30. Murat, N., Armstrong, D. J., Taller, B. J. & Skoog, F. (1978) Plant Physiol., 61, 318-322.

Preferential incorporation of an exogenous cytokinin, N6-benzyladenine, into 18S and 25S ribosomal RNA of tobacco cells in suspension culture.

BIOCHIMIE, 1979, 61, 913-922. Preferential incorporation of an exogenous cytokinin, N -benzyladenine, into 18S and 25S ribosomal RNA of tobacco cells...
802KB Sizes 0 Downloads 0 Views