1146

Specialia

e a c h n u t r i e n t regime. D e t a i l s of t h e e x t r a c t i o n p r o c e d u r e h a v e b e e n g i v e n in a p r e v i o u s c o m m u n i c a t i o n 5. T o t a l c h l o r o p h y l l , c h l o r o p h y l l a a n d c h l o r o p h y l l b were d e t e r m i n e d b y t h e m e t h o d of V e r n o n 6. T o t a l p r o t o c h l o r o p h y l l ( a s s u m i n g no c o n v e r s i o n to chlorophyll), a n d t h e a m o u n t of p r o t o c h l o r o p h y l l t r a n s f o r m e d to c h l o r o p h y l l were estimated by the method of Smith and Benitez7 From t h e v a l u e s t h u s o b t a i n e d , t h e r a t e s of t r a n s f o r m a t i o n of p r o t o c h l o r o p h y l l t o c h l o r o p h y l l were c a l c u l a t e d . Results and discussion. F i g u r e 1 shows t h e t i m e - c o u r s e of f o r m a t i o n of p r o t o c h l o r o p h y l l in seedlings g r o w n Under p o t a s s i u m d e f i c i e n c y a n d full n u t r i e n t c o n d i t i o n s . D u r i n g t h e first 10 d a y s of s t a r v a t i o n , p o t a s s i u m deficiency d i d n o t affect t h e c o n c e n t r a t i o n of p r o t o c h l o r o p h y l l in t h e m a i z e seedlings. B e y o n d t h i s p o i n t t h e deficiency r e s u l t e d in a d r a s t i c d e c r e a s e in t h e c o n c e n t r a t i o n of t o t a l p r o t o c h l o r o p h y l l . A s i m i l a r p a t t e r n of effect was o b s e r v e d for c h l o r o p h y l l s a a n d b. Seedlings m a i n t a i n e d u n d e r p o t a s s i u m deficiency s h o w e d i n i t i a l r a t e s of c o n v e r s i o n of p r o t o c h l o r o p h y l l t o chlorop h y l l t h a t were a p p r o x i m a t e l y e q u a l t o t h e r a t e s r e c o r d e d

EXPERIENTIA 33/9

for c o n t r o l seedlings (figure 2). T h e r a t e s increased u n d e r b o t h n u t r i e n t regimes d u r i n g t h e first 10 d a y s of feeding w i t h n u t r i e n t s o l u t i o n s a n d t h e r e a f t e r decreased. T h i s decrease was m o r e p r o n o u n c e d in t h e p o t a s s i u m - d e f i c i e n t seedlings t h a n in t h e c o n t r o l seedlings. It can thus be concluded that the decrease in chlorophyll f o r m a t i o n d u r i n g p o t a s s i u m deficiency is due, a t l e a s t in p a r t , t o a d e c r e a s e d f o r m a t i o n of p r o t o c h l o r o p h y l l a n d a d e c r e a s e d r a t e of t r a n s f o r m a t i o n o f p r o t o c h l o r o p h y l l t o c h l o r o p h y l l . B o g o r a d s s u g g e s t e d t h a t in p l a n t s chlorosis is g e n e r a l l y d u e t o a b l o c k in p o r p h y r i n f o r m a t i o n a t a v e r y e a r l y p o i n t in t h e b i o s y n t h e t i c c h a i n . P o t a s s i u m d e f i c i e n c y - i n d u c e d chlorosis w o u l d seem t o b e d u e t o a n a d d i t i o n a l b l o c k l a t e r in t h e c h a i n .

5 6 7 8

I.C. Onwueme and A. O. Lawanson, Planta, Berl. 110, 81 (1973). L.P. Vernon, Analyt. Chem. 32, 1144 (1960). J . H . C . Smith and A. Benitez, Plant Physiol. 29, 135 (1954). L. Bogorad, in: Plant Biochemistry, p. 753. Ed. J. Bonner and J. E. Varner. Academic Press, New York and London 1965.

I n c r e a s e d ! R N A _ s y n t h e s i s d u r i n g lpreconjugation and its effect on lpair f o r m a t i o n in ~Tetrahvmena t ~ A. R o n a n d O. H o r o v i t z

The Department o/Anatomy and Embryology, The Hebrew University Hadassah Medical School, P. O. Box 1172, Jerusalem (Israel), 1 March 1977 Summary. I n c r e a s e d R N A synthesis, m a i n l y m R N A , occurs in T e t r a h y m e n a v e r y s h o r t l y before t h e p a i r i n g w h i c h t a k e s place a f t e r c o n j u g a t i o n h a s b e e n i n d u c e d . Specific i n h i b i t i o n of m R N A s y n t h e s i s b y c o r d y c e p i n delays pairing. T h e s e x u a l p h a s e in t h e ciliated p r o t o z o a n T e t r a h y m e n a is a n i n d u c i b l e d e v e l o p m e n t a l process. F o r m a t i o n of a cell p a i r b e t w e e n 2 m a t i n g - t y p e s r e q u i r e s s e v e r a l s t e p s : 1. D e p r i v a t i o n of n u t r i e n t s . 2. A specific cell-cell i n t e r action. 3. F u s i o n of cell surfaces of b o t h m a t i n g - t y p e s . T h e a c t u a l p a i r i n g is p r e c e d e d b y a process w h i c h m a k e s possible t h e cellular r e c o g n i t i o n a n d a t t a c h m e n t . T h i s pro-

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Fig. 1. RNA synthesis during costimulation and pair formation during the process of conjugation. After mixing starved cultures of mating types, 10 ~.Clml 8H-uridine was added at 1-h-intervals for 10 rain. The radioactivity was determined by precipitation with cold 10 % TCA. Solid line: RNA synthesis during costimulation and pair formation of conjugating Tetrahymena. The curves were calculated and drawn relative to the straight control line. Dotted straight line: RNA synthesis in starved controls. Each starved mating type alone served as a control. The dotted line represents the calculated mean of both mating types. Left curve: nH-uridine inqqr~oration into phenol-chloroform extracted RNA. Right curve: 3H-uridine incorporation into entire Tetrahymena cells.

cess i n v o l v e s a t l e a s t 2 m a j o r stages: i n i t i a t i o n a n d cos t i m u l a t i o n 2. I n i t i a t i o n is i n d u c e d b y s t a r v a t i o n in b u f fers s u c h as 10 mlV[ Trig or 50 m M Tricine for 2 or m o r e h ( d e p e n d i n g on t h e m a t i n g t y p e s used a t 28-30~ a n d is i n d e p e n d e n t of t h e p r e s e n c e of t h e c o m p l e m e n t a r y m a t i n g t y p e s 2,3. A f t e r m i x i n g m a t i n g t y p e s w h i c h were p r e v i o u s l y i n i t i a t e d , t h e r e is a lag p e r i o d of 4 5 - 6 0 m i n u n t i l t h e first p a i r s of c o n j u g a t e s are formed. Obviously, there= fore, d u r i n g t h i s lag p e r i o d some f o r m of c o m m u n i c a t i o n is n e e d e d b e t w e e n cells of c o m p l e m e n t a r y m a t i n g t y p e s 3, ,. Since t h e a f o r e m e n t i o n e d processes of i n i t i a t i o n , costim u l a t i o n a n d fusion o c c u r s e q u e n t i a l l y 3 , it is possible t o e x a m i n e e a c h step s e p a r a t e l y . I n o r d e r to u n d e r s t a n d more about the macromolecular events during costimulation, we h a v e i n v e s t i g a t e d t h e p a t t e r n of R N A s y n t h e s i s in r e l a t i o n to t h i s p e r i o d of c o n j u g a t i o n in T e t r a h y m e n a . I n a d d i t i o n , t h e effect of c o r d y c e p i n , a n R N A s y n t h e s i s i n h i b i t o r , was e x a m i n e d on R N A s y n t h e s i s as well as o n p a i r f o r m a t i o n . Materials and methods. Cell cultures. T e t r a h y m e n a p y r i formis m a t i n g t y p e s W H 6 a n d WH52 of s y n g e n I (obt a i n e d f r o m t h e A m e r i c a n T y p e C u l t u r e Collection) were m a i n t a i n e d s e p a r a t e l y in 2 % p r o t e o s e p e p t o n e (Difco) a t 28 ~ Cells used in t h e e x p e r i m e n t s were o b t a i n e d b y ino c u l a t i n g 100 m l of a m e d i u m c o n t a i n i n g 0.5% p r o t e o s e p e p t o n e , 0.5% B a c t o t r y p t o n e , 0 . 0 1 % y e a s t e x t r a c t , 0.1%

1 2 3 4

Supported by Stiftung Volkswagenwerk, research grant No. 112273. P . J . Bruns and T. B. Brussared, J. exp. Zool. 188, 337 (1974). J. Wolfe, Dev. Biol. 35, 221 (1973). J . W . McCoy, J. exp. Zool. 780, 271 (1972).

15. 9. 1977

Speeialia

s o d i u m a c e t a t e a n d 0.1% K 2 H P O 4 a t p H 7.2. A f t e r 2 d a y s of g r o w t h a t 28~ t h e ceils of e a c h m a t i n g t y p e were removed and utilized during their exponential growth phase. S h i f t d o w n . Cells were c o n c e n t r a t e d for s h i f t - d o w n b y c e n t r i f u g a t i o n w i t h a GLC-2 (Sorvall) c e n t r i f u g e a t 1000 r p m for 1-2 min. E a c h m a t i n g t y p e was w a s h e d sep a r a t e l y 3 t i m e s in 0.05 M t r i c i n e b u f f e r (pH 7.2) a n d res u s p e n d e d to a c o n c e n t r a t i o n of 4 - 5 • 105 cells/ml, a n d m a i n t a i n e d for a b o u t 24 h u n d e r s t a r v a t i o n . Conjugation. The mating reaction was induced by mixing 1 m l of a n e q u a l c o n c e n t r a t i o n of e a c h m a t i n g t y p e in 15m l E r l e n m e y e r flasks w h i c h were t h e n i n c u b a t e d a t 30 ~ in a w a t e r b a t h . C o n j u g a t i n g o r g a n i s m s were fixed b y a d d i n g g l u t a r a l d e h y d e t o t h e m (final c o n c e n t r a t i o n : 1.5%). S a m p l e s were c o u n t e d a t 1 - h - i n t e r v a l s w i t h a Neu-

200 I

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Fig. 2. Incorporation of 3H-uridine into RNA during eostimulation and pair formation of conjugating Tetrahymena. After phenolchloroform extraction, mRNA was isolated on poly-U-sepharose columns, rRNA represents phenol-chloroform extracted RNA which could not be isolated on poly-U-sepharose.

1.^1 Control 100 uu . . . o - . . o . . - . - o . - - - - ~ . - - - - . o - - - - - = - - - - - o • 0/0 r +Cordycepin

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Fig. 3. The effect of cordycepin on the rate of pair formation and RNA synthesis in conjugating Tetrahymena. Cordycepin was added at the initial mixing time of the mating types. The 2 upper lines represent the inhibitory effects of cordycepin on RNA synthesis as compared to a control without eordyeepin. The 2 lower lines represent the inhibitory effects of cordycepin on pair formation, as compared to a control to which no cordycepin was added. The rate of pair formation during conjugation was determined analyzing a minimum of 400 cells per count.

1147

b a u e r H e m a c y t o m e t e r a n d t h e p e r c e n t a g e of c o n j u g a t i n g ceils was c a l c u l a t e d . A m i n i m u m of 400 cells were c o u n t e d in e a c h sample. Cell labelling. Cells were labelled w i t h 10 ~zC/ml of 3Hu r i d i n e (specific a c t i v i t y 25 Ci/mmole, A m e r s h a m , E n g land). R N A s y n t h e s i s was m e a s u r e d b y t h e i n c o r p o r a t i o n of ~H - u r i d i n e i n t o whole cells, following p r e c i p i t a t i o n w i t h cold 10% TCA, or b y t h e e x t r a c t i o n of R N A w i t h p h e n o l c h l o r o f o r m as d e s c r i b e d elsewhere 5. F r a c t i o n a t i o n of R N A o n p o l y - ( U ) - s e p h a r o s e . S a m p l e s of p h e n o c h l o r o f o r m e x t r a c t e d R N A were p u t o n poly-(U)s e p h a r o s e c o l u m n s a n d f r a c t i o n a t e d as d e s c r i b e d elsew h e r e 5. T h e r a d i o a c t i v i t y of e a c h f r a c t i o n was d e t e r m i n e d in t h e T C A p r e c i p i t a n t . C o r d y c e p i n (3' d e o x y adenosine) ( S i g m a C h e m i c a l Co.) was m a d e into a s t o c k Solution of 500 ~zg/ml in buffer. I n p r e l i m i n a r y dose t o l e r a n c e tests, a dose 0I c o r d y c e p i n as h i g h as 100 ~zg/ml h a d no effect of t h e s u r v i v a l or m o r p h o l o g y of t h e o r g a n i s m s e v e n a f t e r 48 h of t r e a t m e n t . Since c o n c e n t r a t i o n s of 50 txg/ml a n d 100 lxg/ml yielded t h e s a m e effect, we chose to use t h e lower dose. E a c h of t h e s t a r v e d m a t i n g t y p e cultures, t e s t e d s e p a r a t e l y , s e r v e d as a control. Results. R N A s y n t h e s i s d u r i n g t h e i n d u c t i o n of c o n j u g a t i o n in T e t r a h y m e n a . W h e n c o m p l e m e n t a r y m a t i n g t y p e s of s t a r v e d T e t r a h y m e n a were m i x e d for t h e ind u c t i o n of c o n j u g a t i o n , u n d e r o u r e x p e r i m e n t a l conditions, t h e r e w a s a lag p e r i o d of a b o u t 1.5 h u n t i l t h e first c o n j u g a t i n g p a i r s were recorded. W h e n R N A s y n t h e s i s w a s m e a s u r e d f r o m t h e i n i t i a l m i x i n g t i m e of t h e m a t i n g t y p e s for a p e r i o d of 4 - 5 h t h e r e a f t e r , a n increase in t h e r a t e of R N A s y n t h e s i s was o b s e r v e d . T h e m a x i m a l R N A synthesis stimulation was measured after 1-2 h from the i n i t i a l m i x i n g t i m e (figures 1 a n d 2). A t t h e e n d of t h i s 2-h-period, t h e first c o n j u g a t i n g p a i r s could be o b s e r v e d . 4 - 5 h a f t e r t h e i n i t i a l m i x i n g of t h e m a t i n g types, t h e r a t e of c o n j u g a t i o n r e a c h e d 5 0 - 6 0 % of t h e t o t a l p o p u l a t i o n a n d R N A s y n t h e s i s d e c r e a s e d to t h e level of t h e c o n t r o l c u l t u r e s (figure 1). T h e t o t a l a m o u n t of R N A , s y n t h e s i z e d d u r i n g t h e first 6 h a f t e r t h e i n i t i a l m i x i n g , was e x t r a c t e d a n d f r a e t i o n a t e d on p o l y - ( U ) - s e p h a r o s e c o l u m n s . T h e results s h o w e d t h a t t h e s y n t h e s e s of m R N A a n d r R N A were b o t h s t i m u l a t e d , p a r t i c u l a r l y t h e former. T h e r e s u l t of t h e s e e x p e r i m e n t s are p l o t t e d in figure 2. T h e i n h i b i t i o n of R N A s y n t h e s i s b y c o r d y c e p i n . T h e a b o v e r e s u l t s suggest t h a t R N A s y n t h e s i s m i g h t b e ess e n t i a l for t h e process of c o n j u g a t i o n a n d especially during t h e i n i t i a t i o n phase. Therefore, t h e i n h i b i t i o n of R N A s y n t h e s i s d u r i n g t h e first 2 h f r o m t h e i n i t i a l m i x i n g t i m e of t h e m a t i n g - t y p e s m i g h t also i n h i b i t or d e l a y c o n j u g a tion. I n o r d e r to i n v e s t i g a t e t h i s p o i n t , a series of exp e r i m e n t s was p e r f o r m e d to d e t e r m i n e t h e effect of cordycepin, a RNA synthesis inhibitor, on conjugation. F i g u r e 3 d e m o n s t r a t e s t h e effect of 50 txg/ml c o r d y c e p i n on t h e c o n j u g a t i o n a n d R N A s y n t h e s i s of T e t r a h y m e n a c u l t u r e s w h e n t h e i n h i b i t o r was a d d e d t o t h e e x p e r i m e n t a l c u l t u r e s a t t h e m i x i n g t i m e of t h e m a t i n g types. W h e n t h e r a t e of c o n j u g a t i o n was d e t e r m i n e d , t h e r e was a 1 h d e l a y in t h e p a i r i n g process of c o n j u g a t i o n following t h e c o r d y c e p i n t r e a t m e n t . 4 h a f t e r a d d i n g t h e drug, t h e r e was n o s i g n i f i c a n t difference b e t w e e n c o r d y c e p i n - t r e a t e d a n d n o n - t r e a t e d c u l t u r e s in t h e a m o u n t of R N A synthesis. R N A s y n t h e s i s m e a s u r e m e n t s following c o r d y c e p i n t r e a t m e n t r e v e a l e d a decrease of 3 5 - 4 0 % in 3 H - u r i d i n e i n c o r p o r a t i o n a f t e r t h e first h o u r f r o m t h e initial m i x i n g t i m e (figure 3). O u r i m p r e s s i o n was t h a t a f t e r 3-4 h t h e o r g a n i s m s r e c o v e r e d f r o m t h e effect of t h e drug, a n d step-

5

A. Ron, O. Horovitz and I. Sarov, J. molec. Evol. 8, 137 (1976).

1148

Specialia

wise r e c o v e r e d f r o m its i n h i b i t o r y effect o n R N A synthesis. I n o r d e r t o s t u d y t h e ' r e c o v e r y ' p e r i o d m o r e s y s t e m a t i c a l l y , we p e r f o r m e d a d d i t i o n a l e x p e r i m e n t s i n which cordycepin was added at different intervals after t h e i n i t i a l m i x i n g t i m e of t h e m a t i n g - t y p e s . R e s u l t s s h o w e d t h a t t h e t i m e of a p p l i c a t i o n of c o r d y c e p i n d i d n o t effect t h e l e n g t h of t h e lag period, w h i c h r e m a i n e d 1-1.5 h, b u t d i d effect t h e r a t e of c o n j u g a t i o n . W h e n c o r d y c e p i n was a d d e d i m m e d i a t e l y a f t e r t h e i n i t i a l m i x i n g t i m e of t h e m a t i n g - t y p e s , or w i t h i n 1 h t h e r e a f t e r , t h e r e was a s i g n i f i c a n t d e l a y in t h e r a t e of p a i r f o r m a t i o n . T h e a d d i t i o n of t h e d r u g l a t e r t h a n 2 h f r o m t h e i n i t i a l m i x i n g p o i n t of t h e m a t i n g - t y p e s h a d little effect o n t h e r a t e of p a i r f o r m a t i o n (figure 4). R N A s y n t h e s i s in cells t r e a t e d w i t h c o r d y c e p i n a t t h e i n i t i a l m i x i n g t i m e of t h e m a t i n g - t y p e s d e c r e a s e d b y 6 0 - 8 0 % a f t e r t h e first h. A p p l i c a t i o n of c o r d y c e p i n a f t e r 2 - 3 h f r o m t h e initial m i x i n g p o i n t of t h e m a t i n g - t y p e s d e c r e a s e d t h e level of R N A

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