Autoradiographic evidence for a difference in 3H-thymidine incorporation between intact and cultured roots of pisum

455 AUTORADIOGRAPHIC 3H-THYMIDINE AND

EVIDENCE

FOR

INCORPORATION CULTURED

ROOTS

A DIFFERENCE

BETWEEN

IN

INTACT

OF PISUM 1

J. V A N ' T H O F 2 Biology Department, Brookhaven National Laboratory, Upton, New York 11973, U.S.A. Received August 5, 1965a T H E p r e s e n t s t u d y is p a r t of a series in w h i c h cell d i v i s i o n a n d p a r t i c u l a r l y t h e e v e n t s of t h e m i t o t i c cycle are i n v e s t i g a t e d in e x c i s e d c u l t u r e d roots. One of t h e s t a r t i n g p o i n t s for t h e s e studies was a c o m p a r i s o n of results o b t a i n e d f r o m c u l t u r e d a n d i n t a c t roots. Since D N A s y n t h e s i s is an i n t e g r a l a n d i n d i s p e n s i b l e p a r t of t h e m i t o t i c cycle as d e f i n e d b y H o w a r d a n d P e l c [2], it is r e a s o n a b l e to b e g i n at this point. E x p e r i m e n t s d e s i g n e d to m e a s u r e t h e m i t o t i c cycle d u r a t i o n , t h e p r e s y n t h e t i c p e r i o d (G1), t h e d u r a t i o n of D N A s y n t h e s i s (S period), t h e p o s t s y n t h e t i e p e r i o d (65) a n d m i t o s i s h a v e s h o w n t h a t e x c e p t for t h e G1 period, c u l t u r e d r o o t s h a v e a m i t o t i c cycle v e r y similar to t h a t of i n t a c t root m e r i s t e m cells [3]. D u r i n g t h e s e a n d o t h e r studies, h o w e v e r , it b e c a m e i n c r e a s i n g l y e v i d e n t t h a t 3 H - t h y m i d i n e ( 3 H - T ) i n c o r p o r a t i o n i n t o D N A of i n t a c t a n d c u l t u r e d r o o t m e r i s t e m cells was n o t t h e s a m e q u a n t i t a t i v e l y . T h e p r e s e n t s t u d y was d e s i g n e d to d e t e r m i n e if this s u s p e c t e d difference was i n d e e d true. Materials and Methods. I n t a c t seedlings of P i s u m s a t i v u m (var. A l a s k a ) w i t h p r i m a r y r o o t s of 2.5 to 3.0 cm in l e n g t h w e r e s u s p e n d e d in c o n t i n u o u s l y a e r a t e d H o a g l a n d ' s n u t r i e n t s o l u t i o n a n d a l l o w e d to a c c l i m a t e o v e r n i g h t . T h e n e x t d a y t h e r o o t s w e r e t r a n s f e r r e d to 250 m l b e a k e r s t h a t c o n t a i n e d n u t r i e n t s o l u t i o n a n d 1 /,C/ml 3 H - T (spec. act. 6.7 C / r a M : N e w E n g l a n d N u c l e a r Corp.). T h e s e r o o t s w e r e s a m p l e d for a p e r i o d of t i m e up to 24 hr. T w o g r o u p s of sterile excised r o o t s w e r e used to m e a s u r e 3H--T i n c o r p o r a t i o n . S t e r i l i z a t i o n was p e r f o r m e d before g e r m i n a t i o n b y a m e t h o d a l r e a d y d e s c r i b e d [3]. A f t e r g e r m i n a t i o n , one g r o u p of 1 em r o o t tips was e x c i s e d a n d i m m e d i a t e l y p l a c e d in W h i t e ' s c u l t u r e m e d i u m [4] c o n t a i n i n g 2 p e r c e n t sucrose a n d 1 /~C/ml 3H T. 125 m l E r l e n m e y e r flasks w e r e u s e d as c u l t u r e vessels a n d e a c h c o n t a i n e d 50 ml of r a d i o a c t i v e c u l t u r e m e d i u m a n d 10 r o o t tips. A f t e r 2 h r in c u l t u r e at least 3 r o o t tips w e r e r e m o v e d for s a m p l i n g p u r p o s e s a t v a r i o u s t i m e i n t e r v a l s up to 22 hr. T h e second g r o u p of 1 em e x c i s e d r o o t tips was s u s p e n d e d in W h i t e ' s c u l t u r e m e d i u m w h i c h c o n t a i n e d n e i t h e r sucrose n o r ~H T. 125 m l E r l e n m e y e r flasks w e r e u s e d as c u l t u r e vessels, 50 m l of m e d i u m , 10 r o o t tips p e r flask w e r e also used for t h i s s e c o n d g r o u p of roots. A f t e r 24 hr in a m e d i u m w i t h o u t sucrose, t h e s e c o n d g r o u p of r o o t s w e r e t r a n s f e r r e d to m e d i u m w h i c h c o n t a i n e d sucrose a n d ~ H - T (1 /~C/ml). Maint a i n i n g t h e r o o t s in a m e d i u m w i t h o u t an e x o g e n o u s s u g a r d e p l e t e s t h e c a r b o h y d r a t e 1 Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission. 2 Present address: Department of Botany, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A. Revised version received December 6, 1965. Experimenlal Cell Research 41

456

J. Van 't Hal

supply of t h e cells to t h e e x t e n t t h a t cell division stops [5] and p r e s u m a b l y would also reduce the size of th e D N A precursor pool if such a pool did exist. Following t h e transfer from m e d i u m with no sucrose to one which contained 2 per cent sucrose and 3H T, at least three roots were r e m o v e d at various time intervals up to 22 hr. All ex p er i m en t s were p e r f o r m e d at room t e m p e r a t u r e (22-24~ and t h e r o o t cultures were shaken gently at a b o u t 1 cycle per second. A f t e r sampling, the roots were fixed in 3 p a r t s ethanol and 1 p a r t glacial acetic acid, h y d r o l y z e d for 15 min at 60~ with I N HC1, and stained by the Feulgen m e t h o d . E a c h 2 m m root tip was squashed and p r e p a r e d for a u t o r a d i o g r a p h y b y t h e m e t h o d of Conger and Fairchild [1]. The slides were coated with K o d a k N T B liquid emulsion and exposed for 2 days at 4~ Results and discussion.--Autoradiographs show t h a t the a m o u n t of a H - T incorpor a t e d into nuclear D N A during a 22 24 hr period was greater in t h e cells of c u l t u r e d roots t h a n the a m o u n t i n c o r p o r a t e d into the cells of i n t a c t roots (Fig. 1). The n u m ber of silver grains a b o v e the cells of cultured roots was b e y o n d accurate c o u n t i n g (upper portion of Fig. 1) while only a few grains were observed ab o v e i n t a c t root cells (lower portion of Fig. 1). The low n u m b e r of grains observed ab o v e cells from i n t a c t roots was t h e result of fewer 3 H - T molecules in these cells and n o t t h e failure to i nc o r p o r at e the isotope, since a 5 to 12 day increase in exposure of t h e emulsion to 3 H - T p r o d u c e d m a n y more silver grains per nucleus as well as an increase in t h e n u m b e r of labeled cells as a function of t i m e in t h e isotope solution. Comparison of t h e increase in the per cent labeled interphase cells as a f u n c t i o n of t i m e in t h e 3 H - T solution i n d ic a t e d t h a t the root tips which were excised and i m m e d i a t e l y placed in t h e m e d i u m containing sucrose and a H - T h ad a p p r o x i m a t e l y 20 pe r cent of t h e cells in t h e S period (Fig. 2; c u r v e B) while those t h a t u n d e r w e n t c a r b o h y d r a t e s t a r v a t i o n h a d fewer t h a n 4 per cent of t h e cells in S (Fig. 2; cu r v e C). In each case, however, t h e per cent D N A synthesizing cells increased to ab o u t 75 at 22 hr. A different result was observed with t h e i n t a c t roots. E v e n t h o u g h t h e y rem a i n e d in t h e r a d i o a c t i v e solution for 24 hr, t h e per cent labeled cells n e v e r exceeded 15 and was generally a r o u n d 10 (Fig. 2; c u r v e A). The initial difference which exists b e t w e e n c a r b o h y d r a t e s t a r v e d and n o n s t a r v e d roots m a y be due to at least t w o factors. C a r b o h y d r a t e s t a r v a t i o n m a y either reduce t h e n u m b e r of S period cells or it m a y lower t h e m e t a b o l i s m of t h e S period cells to a level insufficient to s u p p o r t D N A m e t a b o l i s m i m m e d i a t e l y upon p r e s e n t a t i o n of sucrose. E x p e r i m e n t s are pre se n t l y being c o n d u c t e d to ascertain which e x p l a n a t i o n is correct. The difference between c u l t u r e d and i n t a c t roots, on t h e o t h e r hand, has at least t h r e e possible explanations. First, it is plausible t h a t the open w o u n d p r o d u c e d b y excision p r o v i d e d a r o u t e to th e proliferating cells b y which 3 H - T t r a v e l e d with little or no control. Once available to these cells, a H - T was readily i n c o r p o r a t e d into t he nuclear D N A . This e x p l a n a t i o n implies t h a t t h er e is some control exercised b y t h e cells of t h e i n t a c t seedling over either a H - T u p t a k e a n d / o r incorporation. E v i d e n c e in s u p p o r t of this i m p l ic a t io n is lacking, however. A second e x p l a n a -

Fig. 1.--Photomicrographs taken at two focal planes of meristematic cells from the root tip of a cultured root (upper portion A and B) and an intact root (lower portion C and D). Each root had remained for 22-24 hr in a medium containing ~H-thymidine.

Experimental Cell Research 41

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t i o n is t h a t t h e t h y m i d i n e p o o l size i n t h e cells of i n t a c t s e e d l i n g r o o t s is m u c h g r e a t e r t h a n t h a t of t h e c u l t u r e d r o o t s . S u c h a d i f f e r e n c e w o u l d p r o d u c e a r e l a t i v e i n c r e a s e i n t h e u p t a k e a n d i n c o r p o r a t i o n of 3H T b y t h e e u l t u r e d r o o t s . A t h i r d p o s s i b i l i t y t h a t w o u l d p r o d u c e t h i s d i f f e r e n c e is t h a t t h e i n t a c t r o o t m e r i s t e m cells a r e s u p p l i e d t h y m i d i n e or t h y m i d i n e p r e c u r s o r s f r o m t h e c o t y l e d o n s . E x c i s i o n l i t e r a l l y c u t s o f f 90

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Fig. 2.--The increase in 3H-thymidine labeled interphase cells as a function of time in the radioactive medium. Each point represents the mean per cent of at least three meristems; the standard error of the mean is indicated. Curve A, intact root meristems; curve B, cultured roots placed immediately after excision in culture medium with 2 per cent sucrose and 3H-thymidine; curve C cultured roots subjected to 24 hr of sucrose starvation before being transferred to medium with 2 per cent sucrose and 8H-thymidine.

this source and hence the cultured roots incorporate more 3H-T. In the final analysis, i t is p o s s i b l e t h a t a c o m b i n a t i o n of all t h r e e f a c t o r s m a y b e i n v o l v e d . Two interesting observations which emerge from these studies are that the state of D N A s y n t h e s i s c a n b e r e g u l a t e d i n c u l t u r e d r o o t s b y a p e r i o d of c a r b o h y d r a t e starvation followed by carbohydrate supplementation and that 3H-T incorporation i n t o n u c l e a r D N A o c c u r s m o r e r a p i d l y in c u l t u r e d r o o t s t h a n i n cells of i n t a c t r o o t s . T h e f i r s t o b s e r v a t i o n offers a n e x p e r i m e n t a l s y s t e m w h i c h is v e r y u s e f u l i n s t u d i e s w h e r e t h e a b s e n c e or p r e s e n c e of D N A s y n t h e s i z i n g cells is r e q u i r e d w h i l e t h e s e c o n d o b s e r v a t i o n r e v e a l s still a n o t h e r a r e a w h e r e s t u d i e s o n t h e r e g u l a t i o n of D N A s y n t h e s i s in p l a n t cells m a y b e c o n d u c t e d . Summarg.--Autoradiographic s t u d i e s of i n t a c t a n d c u l t u r e d r o o t m e r i s t e m cells continuously immersed in a medium eontaining 3H-thymidine indicated that inc o r p o r a t i o n i n t o n u c l e a r D N A w a s m u c h g r e a t e r i n t h e c u l t u r e d r o o t cells. T h i s difference persisted even when the cultured roots were carbohydrate starved for 24 h r b e f o r e b e i n g t r a n s f e r r e d t o a m e d i u m t h a t c o n t a i n e d 2 p e r c e n t s u e r o s e a n d 3H-thymidine.

Experimental Cell Research 41

X-ttl~ivctlent isopycnosis

459

T h e a u t h o r is m o s t a p p r e c i a t i v e for tire t e c h n i c a l a s s i s t a n c e of Miss C a r o l A. W o l f a n d t h e p h o t o m i c r o g r a p h s t a k e n b y R o b e r t F. S m i t h .

REFERENCES

CONGER, A. D. and FAIRCHILD, L. M., Slain Teehnol. 28, 281 (1953). HOWARD, A. and PELC, S. R., Heredity Suppl. 6, 261 (1953). VAN 'T HoF, ,I., .1. Cell Biol. 27, 179 (1965). \VHITE, P. 1R., A Handbook of Plant Tissne Culture. Cattell and Co., Inc., Lancaster, Pennsylvania, 1943. 5. WILSON, G. B., ~IoRRISON, J. H. and KNOBLOCH, N., J. Biolihgs. Biochem. Cglol. 5, 411 (1959).

1. 2. 3. 4.

ISOPYCNOTIC

BEHAVIOR IN

THE

X0

OF MOUSE

THE

X-UNIVALENT

OVUM

G. J A G I E L L O 1 . 2 and S. O H N O

Department of Medicine, University of Illinois, Chicago, Ill., and Department of Biology, City of Hope Medical Center, Duarte, Calif., U.S.A. Received October 25, 1965 IN t h e m o u s e , as in m o s t o t h e r m a m m a l s i n c l u d i n g m a n , b o t h tile X a n d t h e Y during male meiosis manifest positive heteropycnosis along their entire length and a s s o c i a t e o n l y e n d - t o - e n d [7], w h i l e d u r i n g f e m a l e meiosis, b o t h X - c h r o m o s o m e s b e h a v e in a n i s o p y c n o t i c m a n n e r a n d t h e free e x c h a n g e of c h i a s m a t a b e t w e e n t h e t w o c a n b e s e e n [8]. M u l d a l [4] a n d o t h e r s h a v e s u g g e s t e d t h a t p o s i t i v e h e t e r o p y c n o s i s of t h e sex c h r o m o s o m e s d u r i n g m a l e m e i o s i s is d u e t o t h e l a c k of h o m o l o g o u s p a i r i n g b e t w e e n t h e t w o . As t h e X a n d t h e Y of m o s t m a m m a l s n o l o n g e r s h a r e a homologous segment, they have to be held together by a nonspecific stickiness, w h i c h is i n d u c e d b y t h e p o s i t i v e l y h e t e r o p y c n o t i c s t a t e . A n o p p o r t u n i t y t o see w h e t h e r t h e m o n o s o m i c s t a t e of t h e s e x e l e m e n t d u r i n g m e i o s i s does i n d e e d d i c t a t e its p o s i t i v e l y h e t e r o p y c n o t i c b e h a v i o r is o f f e r e d b y t h e X 0 f e m a l e m o u s e , 3ltts muscuhls, w h i c h is f e r t i l e [9], u n l i k e t h e h u m a n X 0 f e m a l e . P r e v i o u s l y , o n e X 0 f e t u s in t h e 1 9 t b d a y of g e s t a t i o n w a s o b t a i n e d f r o m a p r e g nant X0 mouse. Ovarian squash preparations yielded numerous pachytene figures, none containing a positively heteropycnotic element. However, the possibility that t h i s X 0 f e t u s w a s a g o n o s o m i e m o s a i c in t h e s a m e m a n n e r as t h e X 0 f e m a l e of ~iicrolus oregoni [6] c o u l d n o t b e r u l e d o u t . T h e s e p a e h y t e n e n u c l e i m a y h a v e b e e n e n d o w e d with the XX bivalent. M o r e r e c e n t l y , d i a k i n e s i s a n d f i r s t m e i o t i c f i g u r e s of f o l l i c u l a r o v a w e r e o b t a i n e d f r o m 12 s e x u a l l y - m a t u r e X 0 m i c e k i n d l y s u p p l i e d b y Drs. B r u c e C a t t a n a c h a n d J a c k I s a a c s o n of t h e I n s t i t u t e of A n i m a l G e n e t i c s , U n i v e r s i t y of E d i n b u r g h . T h e s e 1 Supported by National Foundation Grant no. CRMS-206. 2 Career Development Award no. PHS 1 K3-HD-8443.

Experimental Cell Research 41