UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Cell cycle studies in Paramecium : effects of abrupt changes of nutritional state on cell cycle regulation Ching, Ada Sik-Lun 1985

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1985_A6_7 C45.pdf [ 2.65MB ]
Metadata
JSON: 831-1.0096054.json
JSON-LD: 831-1.0096054-ld.json
RDF/XML (Pretty): 831-1.0096054-rdf.xml
RDF/JSON: 831-1.0096054-rdf.json
Turtle: 831-1.0096054-turtle.txt
N-Triples: 831-1.0096054-rdf-ntriples.txt
Original Record: 831-1.0096054-source.json
Full Text
831-1.0096054-fulltext.txt
Citation
831-1.0096054.ris

Full Text

CELL CYCLE STUDIES IN PARAMECIUM: E f t e c t s o-f A b r u p t Changes o f N u t r i t i o n a l S t a t e B . S c , W h i t w o r t h C o l l e g e , Spokane, W a s h i n g t o n . 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Z o o l o g y ) We a c c e p t t h i s t h e s i s a s con-forming t o t h e r e q u i r e d s t a n d a r d on C e l l C y c l e R e g u l a t i o n By ADA SIK-LUN CHING THE UNIVERSITY OF BRITISH November 1985 © Ada S i k - L u n C h i n g , COLUMBIA 1985 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t The U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D epartment of Z o o l o g y The U n i v e r s i t y of B r i t i s h C o l u m b i a V a n c o u v e r , B.C. Canada V6T 2A9 D a t e ABSTRACT The c o n t r o l s o v e r i n i t i a t i o n of DNA s y n t h e s i s , i n i t i a t i o n of c e l l d i v i s i o n , r e g u l a t i o n o f m a c r o n u c l e a r DNA c o n t e n t , and t h e r e l a t i o n s h i p between c e l l mass and growth r a t e were examined i n c e l l s g r o w i n g under n u t r i e n t c o n s t r a i n t , o r i n c e l l s e x p e r i e n c i n g a change i n growth c o n d i t i o n s t h r o u g h n u t r i t i o n a l e n r i c h m e n t < s h i f t - u p ) o r n u t r i t i o n a l s h i f t - d o w n . R e d u c t i o n i n b o t h c e l l mass and DNA c o n t e n t was a c h i e v e d by g r o w i n g Paramecium c e l l s u nder n u t r i t i o n a l l i m i t a t i o n i n t h e c h e m o s t a t . Under t h e e x t r e m e c o n d i t i o n i n t h e c h e m o s t a t , t h e n o r m a l l y b a l a n c e d r e l a t i o n s h i p between DNA c o n t e n t and c e l l mass ( B e r g e r , 1984? K i m b a l l , 1967) i s u n c o u p l e d . The DNA c o n t e n t i n t h e s e c e l l s i s m a i n t a i n e d a t a b o u t 50 u n i t s , b u t c e l l mass can be as l i t t l e a s 24% of n o r m a l . The g e n e r a t i o n t i m e i n t h e s e s l o w g r o w i n g c e l l s was i n c r e a s e d 4 t o 5 t i m e s t h a t o f r a p i d l y g r o w i n g c e l l s ; t h e growth r a t e was a l s o r e d u c e d by a b o u t t h e same p r o p o r t i o n . N u t r i t i o n a l s h i f t - u p was done by t r a n s f e r r i n g t h e c h e m o s t a t c e l l s t o medium o f e x c e s s f o o d . S i m i l a r l y , n u t r i t i o n a l s h i f t - d o w n was p e r f o r m e d by t r a n s f e r r i n g c e l l s e i t h e r t o t h e c h e m o s t a t o r t o e x h a u s t e d medium. The t i m i n g o f DNA s y n t h e s i s i n i t i a t i o n i s l a r g e l y d e t e r m i n e d i n t h e p r e c e d i n g c e l l c y c l e . A l t h o u g h growth r a t e ( p r o t e i n s y n t h e s i s r a t e ) r e s p o n d s q u i c k l y t o t h e new c o n d i t i o n s , t h e t i m i n g o f DNA s y n t h e s i s i n i t i a t i o n i s n o t r e a d j u s t e d i m m e d i a t e l y and r e f l e c t s t h a t o f t h e p a r e n t a l c e l l c y c l e . The r a t e a t w h i c h c e l l s e n t e r S p h a s e however, i s a f f e c t e d by a r e d u c t i o n i n growth r a t e . The c r i t e r i a f o r DNA s y n t h e s i s i n i t i a t i o n a r e not d e t e r m i n e d by c e l l mass per s e . F i r s t , c e l l mass i n c r e a s e s t o ab o u t 180% o f t h e i n i t i a l G l v a l u e a t t h e t i m e o f DNA s y n t h e s i s i n i t i a t i o n f o l l o w i n g a n u t r i t i o n a l s h i f t - u p . T h i s v a l u e i s much g r e a t e r t h a n t h a t o f w e l l - f e d c o n t r o l s ( 1 1 8 % ) . However, t h e i n c r e a s e i n c e l l mass up t o t h e mean t i m e o f DNA s y n t h e s i s i n i t i a t i o n and c e l l d i v i s i o n a r e not s i g n i f i c a n t l y d i f f e r e n t t h a n t h a t o b s e r v e d i n w e l l - f e d c e l l s . T h i s s u g g e s t s a m a s s - r e l a t e d c o n t r o l o v e r i n i t i a t i o n o f DNA s y n t h e s i s . S e c o n d , c e l l s i n i t i a t e DNA s y n t h e s i s even when t h e r e i s a n e t d e c r e a s e i n c e l l mass f o l l o w i n g n u t r i t i o n a l s h i f t - d o w n . Thus, an i n c r e a s e i n c e l l mass per se i s not n e c e s s a r y f o r DNA s y n t h e s i s i n i t i a t i o n . U n l i k e i n i t i a t i o n of DNA s y n t h e s i s , t h e r e g u l a t o r y mechanisms d e t e r m i n i n g t h e m a c r o n u c l e a r DNA s y n t h e s i z e d r e f l e c t s s o l e l y t h e c u r r e n t n u t r i e n t c o n d i t i o n s . C e l l s i n c h e m o s t a t c u l t u r e n o r m a l l y m a i n t a i n a b o u t h a l f t h e normal amount o f DNA (a b o u t 50 u n i t s ) . F o l l o w i n g n u t r i t i o n a l s h i f t - u p c e l l s s y n t h e s i z e lOO u n i t s o f DNA i n s t e a d . S i m i l a r l y c e l l s s y n t h e s i z e o n l y 50 u n i t s of DNA f o l l o w i n g n u t r i t i o n a l s h i f t - d o w n . The amount o f DNA s y n t h e s i z e d , t h e r e f o r e , i s r e l a t e d t o t h e growth r a t e , and as d i s c u s s e d l a t e r , i s a l s o r e l a t e d t o t h e commitment p o i n t t o c e l l d i v i s i o n . T h i s s t u d y a l s o r e v e a l s t h a t t h e p o i n t of i n i t i a t i o n of — i i i — c e l l d i v i s i o n i s not t i m e - d e p e n d e n t . I t d o e s n o t o c c u r a t a f i x e d d u r a t i o n f o l l o w i n g t h e p r e v i o u s f i s s i o n o r t h e i n i t i a t i o n of DNA s y n t h e s i s . The p o i n t o f commitment t o d i v i s i o n o c c u r s a t a b o u t 95 m i n u t e s b e f o r e f i s s i o n r e g a r d l e s s o f growth r a t e . A n a l y s i s o f t h e e f f e c t s o f m a c r o n u c l e a r DNA s y n t h e s i s i n h i b i t i o n i n ccl c e l l s a f t e r t h e t r a n s i t i o n p o i n t f o r d i v i s i o n i n d i c a t e t h a t c e l l s s y n t h e s i z e 50 u n i t s of DNA b e f o r e t h e p o i n t of commitment t o d i v i s i o n . T h i s s u g g e s t s t h a t c e l l s a r e committed t o d i v i d e a f t e r s y n t h e s i z i n g about 50 u n i t s of DNA. F o l l o w i n g t h i s p o i n t , r a p i d l y g r o w i n g c e l l s w i l l p r o d u c e 50 u n i t s o f DNA b e f o r e f i s s i o n ; whereas s l o w g r o w e r s w i l l a c c u m u l a t e an amount p r o p o r t i o n a l t o t h e i r growth r a t e . T h e r e a r e r e a s o n s t o b e l i e v e t h a t t h e t h r e s h o l d v a l u e o f DNA f o r commitment t o c e l l d i v i s i o n may be 41 u n i t s i n s t e a d of 50. - i v-TABLE OF CONTENTS ABSTRACT i i L I S T OF TABLES v i i L I S T OF FIGURES v i i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 MATERIALS AND METHODS 9 S t o c k s and c u l t u r e o f Paramecium 9 E s t a b l i s h m e n t o f c h e m o s t a t c u l t u r e s 9 N u t r i t i o n a l s h i f t s 10 S y n c h r o n i z a t i o n o f c e l l s 11 C y t o l o g i c a l p r o c e d u r e s 11 E s t i m a t i o n o f t h e p r o p o r t i o n o f S pha s e i n t h e c e l l c y c l e 12 S t a t i s t i c a l p r o c e d u r e s 13 RESULTS 14 A. N u t r i t i o n a l l i m i t a t i o n 14 1. E s t a b l i s h m e n t o f c h e m o s t a t c u l t u r e s 14 2. T i m i n g o f i n i t i a t i o n o f DNA s y n t h e s i s i n c h e m o s t a t c u l t u r e s 16 3. T i m i n g o f i n i t i a t i o n o f c e l l d i v i s i o n i n c h e m o s t a t c u l t u r e s 18 4. P r o t e i n s y n t h e s i s a c t i v i t y i n c h e m o s t a t c u l t u r e s 20 -v-B. N u t r i t i o n a l s h i f t - u p 20 1. E f f e c t s o f n u t r i t i o n a l s h i f t - u p on i n i t i a t i o n of DNA s y n t h e s i s 20 2. C e l l mass a t i n i t i a t i o n of DNA s y n t h e s i s 22 3. E f f e c t o f n u t r i t i o n a l s h i f t - u p on m a c r o n u c 1 e a r DNA c o n t e n t and c e l l mass 22 4. E f f e c t of n u t r i t i o n a l s h i f t - u p on p r o t e i n s y n t h e s i s r a t e 23 C. N u t r i t i o n a l s h i f t - d o w n 24 1. E f f e c t of n u t r i t i o n a l s h i f t - d o w n on t i m i n g of DNA s y n t h e s i s 24 2. C e l l mass a t i n i t i a t i o n of DNA s y n t h e s i s 26 3. I n c r e a s e i n m a c r o n u c l e a r DNA c o n t e n t and c e l l mass 26 DISCUSSION 28 C o n t r o l o f i n i t i a t i o n of DNA s y n t h e s i s : 28 T i m i n g o f i n i t i a t i o n o f DNA s y n t h e s i s 28 C e l l mass a t i n i t i a t i o n o f DNA s y n t h e s i s 30 R e g u l a t i o n o f m a c r o n u c l e a r DNA c o n t e n t 33 C o n t r o l o v e r i n i t i a t i o n o f c e l l d i v i s i o n 35 C o m p a r i s o n w i t h o t h e r s y s t e m s 40 BIBLIOGRAPHY 74 -v i -L I S T OF TABLES I. C e l l c y c l e v a r i a b l e s o f c e l l s f o l l o w i n g n u t r i t i o n a l s h i f t - u p . 44 I I . P r o p o r t i o n o f c h e m o s t a t c e l l s i n S phase under e q u i l i b r i u m c o n d i t i o n s . 46 I I I . R a t e o f p r o t e i n s y n t h e s i s i n c h e m o s t a t c u l t u r e u n der e q u i l i b r i u m c o n d i t i o n s . 47 IV. T i m i n g o f i n i t i a t i o n of DNA s y n t h e s i s f o l l o w i n g n u t r i t i o n a l s h i f t - d o w n . 48 V. Mean c e l l mass of c e l l s s y n t h e s i z i n g DNA ('ON') vs. t h o s e t h a t a r e not ('OFF*). 49 V I . C e l l c y c l e v a r i a b l e s f o l l o w i n g n u t r i t i o n a l s h i f t t o e x h a u s t e d medium. 50 V I I . E f f e c t s of change i n growth r a t e on t h e d u r a t i o n o f S p h a s e . 51 V I I I . E f f e c t o f h e a t t r e a t m e n t s t a r t i n g a t 0.75 i n t h e c e l l c y c l e on m a c r o n u c l e a r DNA c o n t e n t a t t h e s u b s e q u e n t f i s s i o n . 52 IX. Mean DNA c o n t e n t s i n c h e m o s t a t c u l t u r e u n d e r e q u i l i b r i u m c o n d i t i o n s . 53 - v i i -L I S T OF FIGURES - v i i i -ACKNOWLEDGEMENTS I would l i k e t o s i n c e r e l y thank Dr. J.D. B e r g e r f o r h i s e n t h u s i a s t i c s u p p o r t , encouragement and g u i d a n c e t h r o u g h o u t t h e c o u r s e of t h i s s t u d y , e s p e c i a l l y i n t h e p r e p a r a t i o n of t h i s m a n u s c r i p t . I would l i k e t o e x p r e s s my a p p r e c i a t i o n t o Dr. T. G r i g l i a t t i and Dr. D. Holm f o r t h e i r h e l p i n p r e p a r i n g t h i s t h e s i s . I am i n d e b t e d t o my f a m i l y f o r t h e c o n s t a n t s u p p o r t and encouragement o v e r t h e y e a r s . F i n a l l y , I w i s h t o thank my husband, S t a n , f o r h i s l o v e and u n d e r s t a n d i n g . R e s e a r c h f u n d s were p r o v i d e d by N a t i o n a l R e s e a r c h C o u n c i l G r a n t # 67-6300 t o Dr. J.D. B e r g e r . INTRODUCTION C e l l s n o r m a l l y m a i n t a i n a r e l a t i v e l y c o n s t a n t c e l l mass and DNA c o n t e n t t h r o u g h t h e c o o r d i n a t i o n o f two f u n d a m e n t a l components o f t h e c e l l c y c l e : t h e growth c y c l e and t h e D N A - d i v i s i o n c y c l e . The c o e f f i c i e n t o f v a r i a t i o n i n c e l l mass i s t y p i c a l l y 1 0 - 1 5 % ( K i m b a l l , 1 9 6 7 ? K i l l a n d e r & Z e t t e r b e r g , 1 9 6 5 ) ; and t h a t o f DNA c o n t e n t i s about 5 % i n mammalian c e l l s ( K i l l a n d e r Z e t t e r b e r g , 1 9 6 5 ) b u t h i g h e r i n c i l i a t e s ( 1 0 - 2 0 % ; C l e f f m a n n , 1 9 6 8 ; K i m b a l l , 1 9 6 7 ; B e r g e r 8< S c h m i d t , 1 9 7 8 ; D o e r d e r , 1 9 7 9 ; D o e r d e r et a/., 1 9 8 1 ) . E v e n t s i n t h e D N A - d i v i s i o n c y c l e i n c l u d e DNA r e p l i c a t i o n and d i v i s i o n ; t h e growth c y c l e i n v o l v e s a c c u m u l a t i o n and s y n t h e s i s of macromo1 e c u 1 e s ( M i t c h i s o n , 1 9 7 0 ) . The c o o r d i n a t i n g mechanisms t h a t c o u p l e t h e growth c y c l e t o t h e D N A - d i v i s i o n c y c l e p r e v e n t c e l l d i v i s i o n f r o m c o n t i n u i n g i n t h e a b s e n c e o f gro w t h , t h e r e b y p r o d u c i n g v e r y s m a l l c e l l s . S i m i l a r l y , growth d o e s n o t c o n t i n u e i n d e f i n i t e l y i n t h e a b s e n c e o f c e l l d i v i s i o n , t h e r e b y p r o d u c i n g v e r y l a r g e c e l l s ( J o h n s t o n et al., 1 9 7 7 ; 1 9 7 9 ) . R e c e n t o b s e r v a t i o n s i n d i c a t e t h a t growth, r a t h e r t h a n p r o g r e s s i o n t h r o u g h t h e D N A - d i v i s i o n c y c l e , i s r a t e - 1 i m i t i n g f o r t h e c e l l c y c l e . T h a t i s , c e l l s n o r m a l l y c o m p l e t e t h e D N A - d i v i s i o n c y c l e f a s t e r t h a n t h e y d o u b l e i n c e l l mass ( H a r t w e l l 8c Unger, 1 9 7 7 J J o h n s t o n et al., 1 9 7 7 ; S i n g e r 8t J o h n s t o n , 1 9 8 3 ) . F o r example, u n u s u a l l y l a r g e c e l l s ( p r o d u c e d e i t h e r by u n e q u a l d i v i s i o n o r by t e m p o r a r y b l o c k a g e o f t h e - 1 -DNA-d i v i s i on c y c l e ) g e n e r a l l y d i v i d e be-fore t h e y d o u b l e t h e i r mass by s h o r t e n i n g G l , t h u s p r o d u c i n g d a u g h t e r c e l l s t h a t a r e w i t h i n t h e normal s i z e r a n g e ( J o h n s t o n et 1 9 7 7 ; Rasmussen & B e r g e r , 1 9 8 2 ; J o h n s t o n 8i S i n g e r , 1 9 8 3 ) . F u r t h e r , p r o l i f e r a t i n g c e l l s grow c o n t i n u o u s l y t h r o u g h o u t G l b e f o r e t h e D N A - d i v i s i o n c y c l e i s i n i t i a t e d . As c e l l s i z e r e m a i n s a p p r o x i m a t e l y c o n s t a n t o v e r t h e c o u r s e o f s e v e r a l g e n e r a t i o n s , g r o w t h d u r i n g 6 1 i n d i c a t e s t h a t t h e D N A - d i v i s i o n c y c l e , w h i c h o c c u p i e s o n l y a p o r t i o n o f t h e c e l l c y c l e , c an t y p i c a l l y be t r a v e r s e d more r a p i d l y t h a n t h e c e l l s c a n d o u b l e t h e i r mass ( J o h n s t o n et al.t 1 9 7 7 ) . The D N A - d i v i s i o n c y c l e becomes t h e o v e r a l l r a t e - 1 i m i t i n g f a c t o r f o r c e l l p r o l i f e r a t i o n o n l y when S ph a s e i s p r o t r a c t e d s i g n i f i c a n t l y . The D N A - d i v i s i o n c y c l e can be d i f f e r e n t i a l l y s l o w e d by t h e p r e s e n c e o f low c o n c e n t r a t i o n s of t h e DNA s y n t h e s i s i n h i b i t o r , h y d r o x y u r e a , o r by g r o w i n g t e m p e r a t u r e - s e n s i t i v e D N A - d i v i s i o n c y c l e m u t a n t s a t s e m i p e r m i s s i v e t e m p e r a t u r e s . Under s u c h c o n d i t i o n s t h e t i m e r e q u i r e d t o c o m p l e t e DNA s y n t h e s i s i s l o n g e r t h a n t h e t i m e needed f o r c e l l s t o d o u b l e i n mass. T h e r e f o r e , t h e p e r f o r m a n c e of t h e D N A - d i v i s i o n c y c l e , n ot growth, becomes r a t e - 1 i m i t i n g f o r t h e c e l l c y c l e ( J o h n s t o n 8c S i n g e r , 1 9 8 5 ; S i n g e r te J o h n s t o n , 1 9 8 5 ; L o r d & Wheals, 1 9 8 3 ) . The c o o r d i n a t i o n between growth and t h e D N A - d i v i s i o n c y c l e c an be r a t i o n a l i z e d by a s s u m i n g t h a t c o m p l e t i o n of a t l e a s t one s t e p i n t h e D N A - d i v i s i o n c y c l e i s s i z e d e p e n d e n t ( J o h n s t o n et si., 1 9 7 7 ) . A c e l l s i z e r e q u i r e m e n t ( o r some p r o p e r t y r e l a t e d - 2 -t o s i z e ) f o r i n i t i a t i o n of DNA s y n t h e s i s i s o b s e r v e d i n b o t h b u d d i n g and f i s s i o n y e a s t , Paramecium and some mammalian c e l l l i n e s . T h i s s i z e r e q u i r e m e n t may be c r y p t i c a s i n w i l d - t y p e f i s s i o n y e a s t i n w h i c h t h e s i z e c o n t r o l o v e r i n i t i a t i o n o f m i t o s i s i s o p e r a t i v e , t h u s c a u s i n g c e l l s a t d i v i s i o n t o be s u f f i c i e n t l y l a r g e t o a l l o w immediate i n i t i a t i o n o f DNA s y n t h e s i s. The c e l l s i z e r e q u i r e m e n t f o r i n i t i a t i o n of DNA s y n t h e s i s i n b u d d i n g y e a s t c a n be d e m o n s t r a t e d by e x a m i n i n g t h e k i n e t i c s of i n i t i a t i o n o f DNA s y n t h e s i s o f a b n o r m a l l y s m a l l c e l l s . T h e s e c e l l s c a n be o b t a i n e d e i t h e r t h r o u g h n i t r o g e n s t a r v a t i o n , o r a s s m a l l d a u g h t e r c e l l s f r o m normal c u l t u r e s . In b o t h c a s e s , t h e c e l l s have an e x t e n d e d c e l l c y c l e . The i n c r e a s e i n c y c l e l e n g t h c a n be a c c o u n t e d f o r by t h e l o n g e r G l p e r i o d i n s m a l l c e l l s . D u r i n g t h e G l p e r i o d t h e y grow t o a c r i t i c a l s i z e b e f o r e DNA s y n t h e s i s i n i t i a t i o n and bud emergence can o c c u r ( J o h n s t o n et al., 1977; H a r t w e l l & Unger, 1977). C e l l c y c l e l e n g t h i s a l s o i n c r e a s e d w i t h s l o w e r growth r a t e s . The m a j o r e x t e n s i o n o c c u r s i n t h e unbudded p o r t i o n p r i o r t o t h e a l p h a - f a c t o r d e p e n d e n t s t e p , o r ' s t a r t ' . Thus, r e g u l a t i o n o f c e l l s i z e and c o o r d i n a t i o n w i t h t h e D N A - d i v i s i o n c y c l e r e s i d e s i n t h e G l phase o f t h e c y c l e j u s t p r i o r t o bud i n i t i a t i o n i n t h i s o r g a n i s m ( L o r i n c z & C a r t e r , 1979). S t u d i e s on a s m a l l mutant i n f i s s i o n y e a s t , wee-1, has p r o v i d e d c o n s i d e r a b l e i n f o r m a t i o n on t h e c o n t r o l o f growth and d i v i s i o n i n t h i s o r g a n i s m . The wee 1-50 mutant d i v i d e s a t -3-6 about t h e same c e l l s i z e a s w i l d - t y p e c e l l s a t 25 C, b u t a t h a l f t h a t s i z e a t 35*C ( N u r s e , 1975) . On t r a n s f e r f r o m 25*C t o 35 C, t h e c e l l s a r e a c c e l e r a t e d i n t o m i t o s i s . S i m i l a r l y , upon t r a n s f e r f r o m n u t r i e n t r i c h medium t o n u t r i e n t p o o r medium, w i l d - t y p e c e l l s a r e a d v a n c e d i n t o t h e c e l l d i v i s i o n s t a g e a t a s m a l l e r s i z e . In c o n t r a s t , m i t o s i s i s d e l a y e d when wee-mutants a r e s h i f t e d f r o m 35° C t o 25*C, o r when w i l d - t y p e c e l l s a r e t r a n s f e r r e d f r o m n u t r i e n t p o o r t o n u t r i e n t r i c h medium. These f i n d i n g s i n d i c a t e a s i z e c o n t r o l . o v e r m i t o s i s ( F a n t e s & N u r s e , 1977). F u r t h e r a n a l y s i s o f t h e wee m u t a n t s and s m a l l w i l d t y p e c e l l s has r e v e a l e d an a d d i t i o n a l s i z e c o n t r o l o v e r t h e i n i t i a t i o n of DNA s y n t h e s i s . B o t h wee m u t a n t s ( a t 35°C) and s m a l l w i l d t y p e c e l l s have a l o n g e r 61 p e r i o d t h a n n o r m a l , and DNA s y n t h e s i s t a k e s p l a c e i n c e l l s w i t h s i m i l a r s i z e s . Thus, a minimum c e l l s i z e r e q u i r e m e n t f o r DNA s y n t h e s i s must e x i s t . W i l d t y p e c e l l s , w h i c h a r e n o r m a l l y above t h i s minimum s i z e a f t e r f i s s i o n , i n i t i a t e DNA s y n t h e s i s a f t e r a v e r y s h o r t G l . Whereas wee m u t a n t s , which a r e s m a l l e r t h a n t h i s c r i t i c a l s i z e a t t h e s t a r t of t h e c e l l c y c l e , have t o grow t o t h e t h r e s h o l d s i z e d u r i n g t h e l o n g e r G l b e f o r e DNA s y n t h e s i s c a n p r o c e e d . A g a i n , c o o r d i n a t i o n o f t h e growth c y c l e w i t h t h e D N A - d i v i s i o n c y c l e i s a c h i e v e d t h r o u g h s i z e c o n t r o l mechanisms which r e g u l a t e t h e t i m i n g o f DNA s y n t h e s i s o r c e l l d i v i s i o n . One s i z e c o n t r o l m e c h a n i s m i s o p e r a t i v e i n s m a l l c e l l s ( c o n t r o l D v e r t h e i n i t i a t i o n of DNA s y n t h e s i s ) and a n o t h e r i n l a r g e r -4-ones ( i n i t i a t i o n of m i t o s i s ) . C i l i a t e s a r e u s e f u l o r g a n i s m s f o r s t u d y i n g t h e c o o r d i n a t i o n between growth and D N A - d i v i s i o n c y c l e s b e c a u s e b o t h c e l l s i z e and m a c r o n u c l e a r gene dosage can be m a n i p u l a t e d i n d e p e n d e n t l y . F o r example, c e l l mass can be v a r i e d g r e a t l y by t h e u s e o f gene m u t a t i o n s t h a t a l t e r t h e s i z e of t h e c e l l by s p e c i f i c a l l y b l o c k i n g t h e D N A - d i v i s i o n c y c l e w i t h o u t a f f e c t i n g g rowth {e.g. t h e ccl mutant i n Paramecium, P e t e r s o n ic B e r g e r , 1976; Rasmussen 8t B e r g e r , 1984); o r by r e s t r i c t i n g t h e growth of t h e c e l l c o r t e x , t h e r e b y r e d u c i n g t h e c e l l mass {e.g. t h e sm2 mutant i n Paramecium', J o n e s 8c B e r g e r , 1982); o r by p r o d u c i n g d a u g h t e r c e l l s t h a t d i f f e r i n c e l l mass {e.g. t h e conical mutant i n Tetrahymena\ D o e r d e r et al., 1975; S c h a e f e r ic C l e f f m a n n , 1982). S i m i l a r l y , gene d o s a g e can be a l t e r e d by gene m u t a t i o n s w h i c h c a u s e p a r t i a l o r c o m p l e t e m i s s e g r e g a t i o n of t h e m a c r o n u c l e u s t o d a u g h t e r c e l l s {e.g. t h e am o r t h e tarn m u t a t i o n i n Paramecium, S o n n e b o r n , 1954; R u i z et al., 1976; Cohen & B e i s s o n , 1980). Gene c o n c e n t r a t i o n can a l s o be changed by s u r g i c a l l y r e m o v i n g e i t h e r p a r t of t h e c y t o p l a s m o r p a r t o f t h e m a c r o n u c l e u s ( F r a z i e r , 1973). B o t h t h e v a r i a t i o n i n t h e s i z e of t h e m a c r o n u c l e u s and t h e f r e q u e n t u n e q u a l d i s t r i b u t i o n of DNA t o d a u g h t e r m a c r o n u c l e i a r e t o l e r a t e d b e c a u s e t h e m a c r o n u c l e u s i s p o l y g e n o m i c (Reviews: R a i k b v , 1970, 1982); i t c o n t a i n s a l a r g e number o f f u n c t i o n a l l y r e d u n d a n t h a p l o i d s u b u n i t s ( O r i a s ic F l a c k s , 1975; D o e r d e r et al., 1977). S u b s t a n t i a l v a r i a t i o n i n DNA c o n t e n t can t h e r e f o r e -5-o c c u r w i t h o u t i n v i a b i l i t y . The a v a i l a b i l i t y o f b o t h ccl and tarn m u t a n t s g r e a t l y f a c i l i t a t e s t h e s t u d y on c e l l c y c l e r e g u l a t i o n i n Paramecium. In t h i s o r g a n i s m , t h e i n i t i a t i o n of t h e D N A - d i v i s i o n c y c l e , a s i n d i c a t e d by t h e i n i t i a t i o n o f DNA s y n t h e s i s , n o r m a l l y o c c u r s when c e l l mass e q u a l s o r e x c e e d s 118% o f t h e mean G l v a l u e — t h e c e l l mass a t i n i t i a t i o n o f DNA s y n t h e s i s i n normal c e l l s . T h i s o c c u r s w i t h o r w i t h o u t an i n t e r v e n i n g G l p e r i o d (Rasmussen & B e r g e r , 1982; 1984). T i m i n g o f i n i t i a t i o n o f DNA s y n t h e s i s i s a l s o a f f e c t e d by c h a n g e s i n gene d o s a g e (macronuc1 e a r DNA c o n t e n t ) . S i s t e r c e l l s w i t h d i f f e r e n t DNA c o n t e n t i n i t i a t e DNA s y n t h e s i s a t s i m i l a r t i m e s ( B e r g e r , 1982b; Rasmussen & B e r g e r , 1986) s u g g e s t i n g t h a t gene d o s a g e - d e p e n d e n t e v e n t s i n G l p l a y l i t t l e r o l e i n d e t e r m i n i n g t h e t i m i n g of i n i t i a t i o n o f DNA s y n t h e s i s . However, t h e p a r e n t a l DNA c o n t e n t has a s i g n i f i c a n t e f f e c t on t h i s e v e n t . C e l l s w i t h lower p a r e n t a l DNA c o n t e n t i n i t i a t e DNA s y n t h e s i s s l i g h t l y s o o n e r t h a n c e l l s w i t h g r e a t e r p a r e n t a l DNA (Rasmussen, 1984; Rasmussen & B e r g e r , 1986). T h e s e o b s e r v a t i o n s s u g g e s t t h a t t h e t i m i n g o f i n i t i a t i o n o f DNA s y n t h e s i s i s e s t a b l i s h e d d u r i n g t h e p r e c e d i n g c e l l c y c l e . The h y p o t h e s i s t h a t t h e t i m i n g of i n i t i a t i o n o f DNA s y n t h e s i s i s e s t a b l i s h e d p r i o r t o d i v i s i o n d u r i n g t h e p r e v i o u s c e l l c y c l e c a n be f u r t h e r t e s t e d by a l t e r i n g t h e l e n g t h o f t h e c e l l c y c l e . T h i s can be a c c o m p l i s h e d by t r a n s f e r r i n g c e l l s f r o m one s e t o f n u t r i t i v e c o n d i t i o n s t o a n o t h e r , and e x a m i n i n g t h e s u b s e q u e n t t i m i n g of i n i t i a t i o n o f DNA s y n t h e s i s and o t h e r -6-c e l l c y c l e p a r a m e t e r s . 1+ t h e t i m i n g o-f i n i t i a t i o n o+ DNA s y n t h e s i s i s e s t a b l i s h e d p r i o r t o f i s s i o n , t h e n t h e t i m i n g of i n i t i a t i o n o f DNA s y n t h e s i s i n t h e f i r s t c e l l c y c l e f o l l o w i n g t h e n u t r i t i o n a l s h i f t s h o u l d r e f l e c t t h e t i m i n g of DNA s y n t h e s i s i n i t i a t i o n i n t h e p a r e n t a l c e l l c y c l e , and not t h a t f o u n d i n c e l l s u n d e r g o i n g e q u i l i b r i u m g r o w t h under t h e new s e t of c o n d i t i o n s . As growth i s n o r m a l l y t h e r a t e - 1 i m i t i n g p r o c e s s i n t h e c e l l c y c l e , a s i z e r e q u i r e m e n t f o r t h e i n i t i a t i o n of DNA s y n t h e s i s a n d / o r m i t o s i s p l u s , i n some c a s e s , a c o n s t a n t S+G2 p e r i o d would p r e v e n t t h e c e l l s f r o m becoming t o o s m a l l ( K i m b a l l et al., 1971; Nasmyth, 1979; N u r s e T h u r i a u x , 1977; J o h n s t o n et al., 1977; H a r t w e l l k Unger, 1977; J a g a d i s h fit C a r t e r , 1977; R i v i n & Fangman, 1980). In Paramecium, t h e l e n g t h o f t h e c e l l c y c l e i s d e t e r m i n e d by t h e d u r a t i o n o f t h e G l p e r i o d p l u s t h e t i m e needed f o r DNA s y n t h e s i s and m a c r o n u c l e a r d i v i s i o n ( t h e r e i s no G2 p e r i o d ; B e r g e r , 1971; T u c k e r et al., 1980). P r e v i o u s s t u d i e s have i n d i c a t e d t h a t c e l l s s y n t h e s i z e s i m i l a r amounts Df DNA i n e a c h c e l l c y c l e r e g a r d l e s s of t h e i n i t i a l DNA c o n t e n t . The amount s y n t h e s i z e d i s e q u i v a l e n t t o t h e mean p o s t - f i s s i o n amount. Thus, i n d u c e d v a r i a t i o n s i n m a c r o n u c l e a r DNA c o n t e n t d u r i n g f i s s i o n a r e r e d u c e d by a b o u t 50% d u r i n g e a c h c y c l e , and t h e m a c r o n u c l e a r DNA c o n t e n t i s r e g u l a t e d t oward t h e p o p u l a t i o n mean ( B e r g e r ic S c h m i d t , 1978; B e r g e r , 1978, 1979). F u r t h e r s t u d i e s showed t h a t t h e amount o f DNA made i s a f f e c t e d by b o t h c e l l mass and m a c r o n u c l e a r DNA c o n t e n t , and i s p r o p o r t i o n a l t o -7-t h e lower of t h e two v a r i a b l e s . I t can o n l y be i n c r e a s e d when b o t h v a r i a b l e s a r e i n c r e a s e d s i m u l t a n e o u s l y ( B e r g e r , 1982aj Rasmussen ic B e r g e r , 1982). When c e l l s mass i s v a r i e d by t h e a c t i o n o f gene m u t a t i o n s o r by a c h ange i n g r o w t h r a t e , DNA c o n t e n t a l s o v a r i e s c o o r d i n a t e l y ( K i m b a l l , 1967; B e r g e r , 1984). T h e s e r e s u l t s s u g g e s t t h a t t h e mechanism c o n t r o l l i n g t h e amount of DNA s y n t h e s i z e d i s s e n s i t i v e t o t h e growth c o n d i t i o n s . Thus, one would e x p e c t c e l l s t o p r o d u c e an amount of DNA t h a t i s c h a r a c t e r i s t i c o f t h e new growth c o n d i t i o n s when c e l l s a r e t r a n s f e r r e d f r o m one s e t of n u t r i t i v e c o n d i t i o n s t o a n o t h e r . R e l a t i v e l y l e s s i s known ab o u t t h e c o n t r o l o f i n i t i a t i o n o f c e l l d i v i s i o n . C e l l d i v i s i o n and t e r m i n a t i o n o f DNA s y n t h e s i s n o r m a l l y o c c u r when c e l l s have s y n t h e s i z e d a s t a n d a r d amount o f DNA, a l t h o u g h t h e c o n t r o l f o r t h e amount of DNA made i s e x e r t e d w e l l b e f o r e t h e end o f m a c r o n u c l e a r DNA s y n t h e s i s . M o r e o v e r , c e l l s can d i v i d e w i t h o u t c o m p l e t i n g t h e f u l l s c h e d u l e of DNA s y n t h e s i s (Rasmussen et al.t 1985). Thus, t h e l e n g t h o f t h e c e l l c y c l e i s d e p e n d e n t upon growth r a t e , t h e i n i t i a l DNA c o n t e n t o f t h e c e l l , and t h e r a t e of DNA a c c u m u l a t i o n . T h i s s t u d y , t h e r e f o r e , e x a m i n e s t h e r e s p o n s e o f c e l l s t o n u t r i t i o n a l s h i f t s , t o d e t e r m i n e , i n p a r t i c u l a r , t h e t i m i n g o f DNA s y n t h e s i s , t h e amount of DNA p r o d u c e d , and t h e change i n growth r a t e s d u r i n g t h e c e l l c y c l e f o l l o w i n g t h e change i n growth c o n d i t i o n s . -8-MATERIALS AND METHODS Stocks and culture of Paramecium Paramecium tetraurelia ( S o n n e b o r n , 1975) s t r a i n d 4 - i O O i was grown i n p h o s p h a t e - b u f f e r e d C e r o p h y l medium (So n n e b o r n , 1970) w i t h Enterobacter aerogenes as t h e -food s o u r c e . T h i s s t r a i n , d4-1001, d e r i v e d -from t h e w i l d - t y p e s t o c k 51-S, c a r r i e s ccl, a r e c e s s i v e t e m p e r a t u r e - s e n s i t i v e m u t a t i o n t h a t b l o c k s m a c r o n u c l e a r DNA s y n t h e s i s a t 34.4°C ( P e t e r s o n B e r g e r , 1976; Rasmussen k B e r g e r , 1984). C u l t u r e medium was grown o v e r n i g h t a f t e r i n n o c u l a t i o n and t h e pH a d j u s t e d t o 6.8-7.0 p r i o r t o u s e . T h r e e a n t i b i o t i c s were added t o t h e c u l t u r e medium f o r t h e c h e m o s t a t t o k i l l and p r e v e n t growth of b a c t e r i a : G e n t a m i c i n , 10 ug/ml; p e n n i c i l i n , 0.25 ug/ml; s t r e p t o m y c i n , 0.25 ug/ml. To a v o i d k i l l i n g t h e Paramecium c e l l s , t h e s e c o n c e n t r a t i o n s were d i l u t e d a f u r t h e r two f o l d w i t h s t e r i l i z e d C e r o p h y l medium b e f o r e b e i n g pumped i n t o t h e c h e m o s t a t . Heat t r e a t m e n t s were c a r r i e d o u t i n a w a t e r b a t h m a i n t a i n e d a t t e m p e r a t u r e s w i t h i n +/-0.02°C o f t h e s e t t i n g . Establishment of chemostat cultures Chemostat c u l t u r e s were e s t a b l i s h e d i n a o n e - s t a g e c o n t i n u o u s c u l t u r e a p p a r a t u s ( B e l l c o , USA) w i t h a c a p a c i t y o f 0.5 l i t r e . The a p p a r a t u s was a s s e m b l e d a s e p t i c a l l y and i n n o c u l a t e d w i t h a p p r o x i m a t e l y 200 ml. of Paramecium c u l t u r e . T h ese c e l l s were c o l l e c t e d f r o m e i t h e r an e x p o n e n t i a l l y g r o w i n g -9-c u l t u r e o f w e l l - f e d c e l l s o r a s t a r v e d c u l t u r e w i t h more t h a n 95% o f t h e c e l l s i n autogamy. C u l t u r e medium was t h e n f e d i n t o t h e c h e m o s t a t a t a c o n s t a n t r a t e u s i n g a p e r i s t a l t i c pump ( B u c h l e r " p o l y s t a l t i c " ^ ) ) . The c u l t u r e i n t h e c h e m o s t a t was s t i r r e d c o n t i n u o u s l y by a m a g n e t i c s t i r r e r t o e n s u r e even d i s t r i b u t i o n o f b o t h Paramecium and b a c t e r i a l c e l l s . The c h e m o s t a t c u l t u r e a p p r o a c h e d e q u i l i b r i u m a f t e r t h r e e d a y s . T h i s was i n d i c a t e d by a c o n s t a n t c e l l d e n s i t y ( a p p r o x i m a t e l y 6000-7000 c e l l s / m l a t d i l u t i o n r a t e of 15 m l / h r ) . D i v i d i n g c e l l s were c o l l e c t e d f r o m s a m p l e s t a k e n f r o m t h e c h e m o s t a t . F o r e a c h sample, t h e p e r i o d of c o l l e c t i o n n e v e r e x c e e d e d 15 m i n u t e s t o e n s u r e c o n s t a n t n u t r i t i o n a l c o n d i t i o n s . The growth r a t e a t e q u i l i b r i u m was c a l c u l a t e d f r o m t h e d i l u t i o n r a t e of t h e c h e m o s t a t ( e q u i l i b r i u m g r o w t h r a t e = volume of c h e m o s t a t c u l t u r e / f l o w r a t e ; E a z i n , 1981). Nutritional shifts N u t r i t i o n a l e n r i c h m e n t ( s h i f t - u p ) was done by t r a n s f e r r i n g newly d i v i d e d c e l l s f r o m a s t e a d y s t a t e c h e m o s t a t c u l t u r e t o medium w i t h e x c e s s f o o d . The f i r s t c e l l c y c l e f o l l o w i n g t h e n u t r i t i o n a l s h i f t - u p was d e s i g n a t e d t h e r e c o v e r y c y c l e and t h e s e c o n d , t h e s e c o n d r e c o v e r y c y c l e . N u t r i t i o n a l s h i f t - d o w n was done by t r a n s f e r r i n g w e l l - f e d c e l l s e i t h e r t o e x h a u s t e d medium o r t o t h e c h e m o s t a t . C o n t r o l c e l l s were a l l o w e d t o grow e x p o n e n t i a l l y i n t h e p r e s e n c e o f e x c e s s f o o d . - l O -E x h a u s t e d medium was p r e p a r e d by r e m o v i n g Paramecium c e l l s , g r a s s d e b r i s and b a c t e r i a f r o m t h e c h e m o s t a t e f f l u e n t by f i l t r a t i o n and c e n t r i f u g a t i o n . A l l q u a n t i t a t i v e measurements were n o r m a l i z e d t o t h e p o s t - f i s s i o n c o n t r o l v a l u e s . The number of c e l l s measured i n e a c h g r o u p i s f r o m 35 t o 40. C e l l c y c l e l e n g t h was e x p r e s s e d as a f r a c t i o n o r p e r c e n t a g e of t h e c o n t r o l c y c l e l e n g t h u n l e s s s t a t e d o t h e r w i s e . Synchronization of cells S y n c h r o n o u s c e l l s were o b t a i n e d by hand s e l e c t i o n o f d i v i d i n g c e l l s f r o m e x p o n e n t i a l g r o w t h - p h a s e c u l t u r e s . F o r e a c h sample, 40-50 d i v i d i n g c e l l s were s e l e c t e d w i t h i n a 5 m i n u t e p e r i o d . Cyto logical procedures M i c r o f 1 u o r i m e t r y was used t o e s t i m a t e DNA and p r o t e i n c o n t e n t o f i n d i v i d u a l c e l l s t h a t had been s t a i n e d by t h e A c r i f l a v i n - P r i m u l i n method o f C o r n e l i s s e and Ploem <1976). A c r i f l a v i n s t a i n s DNA and p r i m u l i n s t a i n s p r o t e i n . S p ecimen p r e p a r a t i o n and a u t o r a d i o g r a p h y were c a r r i e d o u t as d e s c r i b e d by B e r g e r <1982a). L a b e l l i n g o f Paramecium c e l l s w i t h t r i t i a t e d - t h y m i d i n e l a b e l l e d b a c t e r i a were c a r r i e d o u t as p r e v i o u s l y d e s c r i b e d ( B e r g e r , 1971). The r e l a t i v e p r o t e i n s y n t h e s i s r a t e i n i n d i v i d u a l c e l l s -11-was d e t e r m i n e d by p u l s e - l a b e l 1 i n g c e l l s w i t h t r i t i a t e d l e u c i n e ( B e r g e r , 1982). I m m e d i a t e l y a f t e r t h e l a b e l l i n g p e r i o d c e l l s were k i l l e d i n 3% e t h a n o l and t h e n f i x e d i n d i v i d u a l l y o n t o a l b u m i n - c o a t e d m i c r o s c o p e s l i d e s . Measurements o f DNA and p r o t e i n c o n t e n t s were made b e f o r e a u t o r a d i o g r a p h y . The a c t i v i t y p e r u n i t mass o f c y t o p l a s m was e s t i m a t e d by t h e o p t i c a l e x t i n c t i o n of t h e e x p o s e d a u t o r a d i o g r a p h o v e r a s t a n d a r d a r e a o f 25um*. The a r e a was c h o s e n ne a r t h e a n t e r i o r end of t h e c e l l t h a t i s c l e a r o f f o o d v a c u o l e s ( B e r g e r , 1982a). T h e s e v a l u e s were t h e n n o r m a l i z e d t o t h e p o s t - f i s s i o n c o n t r o l v a l u e s of w e l l - f e d c e l l s and. r e p r e s e n t t h e s p e c i f i c r a t e o f p r o t e i n s y n t h e s i s . The c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s was e s t i m a t e d by m u l t i p l y i n g t h e s p e c i f i c r a t e of p r o t e i n s y n t h e s i s by t h e r e l a t i v e mass ( p r o t e i n c o n t e n t ) of t h e e e l 1 . Est imat ion of the proportion of S phase in the cell cycle The r e l a t i v e d u r a t i o n o f S p e r i o d i n t h e c e l l c y c l e c an be e s t i m a t e d f r o m t h e f r a c t i o n of c e l l s i n ' t h e p o p u l a t i o n which a r e s y n t h e s i z i n g DNA. As t h e f r e q u e n c y of c e l l s o f any p a r t i c u l a r age i n an a s y n c h r o n o u s c u l t u r e d e c r e a s e s e x p o n e n t i a l l y w i t h age w i t h i n a c e l l c y c l e , t h e p r o p o r t i o n s of l a b e l l e d and n o n - l a b e l l e d c e l l s do not a l l o w d i r e c t e s t i m a t i o n of t h e t i m i n g o f i n i t i a t i o n o f DNA s y n t h e s i s . A c o r r e c t i o n o f d a t a i s p e r f o r m e d a c c o r d i n g t o a f o r m u l a d e s c r i b e d by Nachtwey 8c Cameron ( 1 9 6 8 ) : -12-t<term?fT = InC <N<term*/N>+1 J / In 2 where tftermS = t i m e s p e n t i n t h e t e r m i n a l p h a s e , T = g e n e r a t i o n t i m e , Nfterm? - number o f c e l l s i n t h e t e r m i n a l p h a s e , and N = t o t a l number of e e l I s . As t h e r e i s no G2 p e r i o d i n t h e c e l l c y c l e o f Paramecium, t h e t i m e s p e n t i n t h e t e r m i n a l p h a s e i s e q u i v a l e n t t o t h e t i m e s p e n t i n t h e S pha s e and c e l l d i v i s i o n . As t h e l a t t e r i n t e r v a l i s s h o r t ( l e s s t h a n 30 m i n u t e s ) , tfterm? s h o u l d g i v e a r e l a t i v e l y good e s t i m a t e of t h e d u r a t i o n of S ph a s e i n t h e c e l l eye 1e. Stat ist ical procedures S t a t i s t i c a l p r o c e d u r e s u s e d i n t h e a n a l y s i s o f d a t a a r e d e s c r i b e d by S o k a l & R o h l f ( 1 9 6 9 ) , p r o p a g a t e d e r r o r s have been c a l c u l a t e d a c c o r d i n g t o B e e r s (1953); and mean v a l u e s a r e shown w i t h t h e i r s t a n d a r d e r r o r s . -13-RESULTS A. Nutritional limitation 1. Establishment of chemostat cultures Chemostat c u l t u r e s were s t a r t e d e i t h e r f r o m a w e l l - f e d p o p u l a t i o n o r f r o m a c e l l p o p u l a t i o n w i t h more t h a n 95% of t h e c e l l s i n autogamy. Changes i n c e l l u l a r DNA and p r o t e i n c o n t e n t f o l l o w i n g t h e i n i t i a t i o n of a c h e m o s t a t c u l t u r e were examined a t r e g u l a r i n t e r v a l s f o r a t l e a s t one week. D i v i d i n g c e l l s were s e l e c t e d f r o m t h e c h e m o s t a t a t e a c h sample p o i n t and f i x e d . The c e l l s were t h e n s t a i n e d and t h e i r DNA and p r o t e i n c o n t e n t s were e s t i m a t e d . When a c h e m o s t a t c u l t u r e was s t a r t e d w i t h w e l l - f e d c e l l s , autogamy o c c u r r e d soon a f t e r t h e i n i t i a t i o n o f t h e c u l t u r e as n u t r i e n t a v a i l a b i l i t y d e c r e a s e d . T h i s c o r r e s p o n d s t o t h e i n i t i a l d r o p i n DNA c o n t e n t ( F i g . 1 ) . The change i n DNA c o n t e n t was s i m i l a r t o t h e change i n t h e t o t a l volume o f m a c r o n u c l e i o b s e r v e d a f t e r c o n j u g a t i o n i n s t a r v e d c e l l s ( B e r g e r , 1974). As t h e new m a c r o n u c l e i ( m a c r o n u c l e a r a n l a g e n ) d e v e l o p e d , t h e DNA c o n t e n t e i t h e r i n c r e a s e d t o a v a l u e c o m p a r a b l e t o t h a t o f w e l l - f e d c e l l s ( about 82 u n i t s ; F i g . I B ) , o r became e s t a b l i s h e d a t a lower v a l u e ( F i g . IA) , d e p e n d i n g on t h e s e v e r i t y o f s t a r v a t i o n . C e l l mass a l s o d e c r e a s e d f o l l o w i n g t h e i n i t i a t i o n o f t h e c h e m o s t a t c u l t u r e ( F i g . 1 ) . The i n i t i a l d e c r e a s e i n c e l l mass -14-i s p a r t i a l l y due t o a r e d u c t i o n i n t h e t o t a l number o f f o o d v a c u o l e s . The number o f f o o d v a c u o l e s i n w e l l - f e d c e l l s d e c r e a s e d 80% w i t h i n 45 m i n u t e s f o l l o w i n g a t r a n s f e r o f t h e c e l l s t o e x h a u s t e d medium ( F i g . 2 ) . C e l l mass was a l s o r e d u c e d by 18% w i t h i n 30 m i n u t e s ( F i g . 3 ) . Food v a c u o l e s were l o s t a t a much s l o w e r r a t e d u r i n g t h e 30 t o 40 m i n u t e i n t e r v a l ( F i g . 2 ) , and c e l l mass r e m a i n e d r e l a t i v e l y c o n s t a n t ( F i g . 3 ) . Thus, t h e r a p i d r e d u c t i o n i n c e l l mass c a n n o t be c a u s e d s i m p l y by t h e a u t o l y s i s o f t h e c y t o p l a s m , and t h e d e c r e a s e i n c e l l mass i n w e l l - f e d c e l l s f o l l o w i n g t h e t r a n s f e r t o t h e c h e m o s t a t i s p a r t l y due t o a n e t r e d u c t i o n i n t h e b a c t e r i a l c o n t e n t c o n t a i n e d i n f o o d v a c u o l e s . The p o p u l a t i o n d e n s i t y and p o s t - f i s s i o n DNA and p r o t e i n c o n t e n t o f t h e c e l l s were r e l a t i v e l y c o n s t a n t a s t h e c u l t u r e a p p r o a c h e d e q u i l i b r i u m ( F i g . 1 ) . In p a r t i c u l a r , t h e p o s t - f i s s i o n DNA c o n t e n t of c h e m o s t a t c e l l s was m a i n t a i n e d a t a p p r o x i m a t e l y 50 u n i t s ( F i g . 1A), i n d i c a t i n g t h a t u nder n u t r i t i o n a l l i m i t a t i o n , c e l l s make a p p r o x i m a t e l y 50 u n i t s o f DNA i n s t e a d o f 100 u n i t s a s t h e y do when w e l l - f e d . On t h e o t h e r hand, r e l a t i v e c e l l mass ( p r o t e i n c o n t e n t ) d e c r e a s e d t o as l i t t l e a s 30 u n i t s o r l e s s when t h e growth r a t e was r e d u c e d ( T a b l e 1, P i ) . M a c r o n u c l e a r DNA c o n t e n t s s i g n i f i c a n t l y below 50 were not o b s e r v e d i n t h e s e v e r y s m a l l c e l l s ( T a b l e 1, D i ) , i n d i c a t i n g t h a t t h e t i g h t c o u p l i n g between DNA and p r o t e i n c o n t e n t o b s e r v e d i n w e l l f e d c e l l s ( B e r g e r , 1984) i s not m a i n t a i n e d a t v e r y low growth r a t e s . T h i s r e s u l t f u r t h e r -15-s u g g e s t s e i t h e r t h a t a minimum DNA c o n t e n t o f a p p r o x i m a t e l y 50 u n i t s i s n e c e s s a r y t o m a i n t a i n v i a b i l i t y when c e l l mass and g rowth r a t e a r e d r a s t i c a l l y r e d u c e d , o r t h a t t h e p r o d u c t i o n o f a b o u t 50 u n i t s of DNA i s a n e c e s s a r y p r e c o n d i t i o n f o r f i s s i o n . As d i s c u s s e d below, t h e l a t t e r h y p o t h e s i s i s f a v o r e d . F o l l o w i n g t h e e s t a b l i s h m e n t o f e q u i l i b r i u m i n t h e c h e m o s t a t , a change i n t h e f l o w r a t e would c a u s e b o t h t h e DNA c o n t e n t and c e l l mass t o change i n a c o o r d i n a t e d f a s h i o n <Fig. I B ) . R e d u c t i o n o f f l o w r a t e below 15 m l / h r ( g e n e r a t i o n t i m e = 33 h o u r s ) l e a d s t o a s u b s t a n t i a l d e c r e a s e i n t h e number of d i v i d e r s ; c e l l d e n s i t y d r o p s a s e q u i l i b r i u m i s r e e s t a b l i s h e d a t a lower c e l l d e n s i t y . 2. Timing of initiation of DNA synthesis in chemostat cu Itu res S t u d i e s w i t h o t h e r e u k a r y o t i c s y s t e m s s u c h as y e a s t and mammalian c e l l l i n e s i n d i c a t e t h a t G l i s t h e most v a r i a b l e p o r t i o n of t h e c e l l c y c l e . By c o n t r a s t , t h e t i m e t a k e n f o r a c e l l t o p r o g r e s s f r o m t h e b e g i n n i n g of S p h a s e t h r o u g h m i t o s i s i s r e m a r k a b l y c o n s t a n t , r e g a r d l e s s of t h e g e n e r a t i o n t i m e ( R e v i e w s : P a r d e e et al., 1978; C a r t e r , 1981; Y a n i s h e v s k y Be G r e t c h e n , 1981.) Thus, t h e d u r a t i o n of S p h a s e i n Paramecium i s examined i n c h e m o s t a t c e l l s i n w h i c h t h e l e n g t h of t h e c e l l c y c l e i s g r e a t l y i n c r e a s e d due t o a s l o w g r o wth r a t e . The r e l a t i v e d u r a t i o n of t h e S p e r i o d w i t h i n t h e c e l l c y c l e was e s t i m a t e d f r o m t h e f r a c t i o n o f c e l l s i n t h e -16-p o p u l a t i o n u n d e r g o i n g DNA s y n t h e s i s . F i v e s a m p l e s of c e l l s were c o l l e c t e d f r o m t h e c h e m o s t a t w i t h a d i l u t i o n t i m e o f 28.9 h o u r s . T h e s e a s y n c h r o n o u s c e l l s a m p l e s were l a b e l l e d w i t h l a b e l l e d b a c t e r i a f o r 15-20 m i n u t e s . A u t o r a d i o g r a p h s were p r e p a r e d , and t h e p e r c e n t a g e o f c e l l s w i t h l a b e l l e d m a c r o n u c l e i was s c a r e d . The p r o p o r t i o n of c e l l s i n S ph a s e was e s t i m a t e d t o be 0.65+/-0.05 ( T a b l e 2 ) . The e s t i m a t e d d u r a t i o n D f S phase was a p p r o x i m a t e l y 18.8 h o u r s . Thus, S p h a s e i n c h e m o s t a t c e l l s d i d n ot o c c u p y t h e same d u r a t i o n as i n r a p i d l y g r o w i n g c e l l s (i.e. 3.62 h o u r s ) . T h i s i n d i c a t e s t h a t u nder e q u i l i b r i u m g rowth c o n d i t i o n s i n a c h e m o s t a t c e l l c y c l e s t a g e s a r e p r o p o r t i o n a t e l y e x t e n d e d . DNA s y n t h e s i s i n i t i a t i o n n o r m a l l y o c c u r s a t 0.25 o f t h e c e l l c y c l e i n w e l l - f e d c e l l s , and c o n t i n u e s u n t i l m a c r o n u c l e a r e l o n g a t i o n . N u c l e a r e l o n g a t i o n normal 1y o c c u r s a p p r o x i m a t e l y h a l f w a y i n t o c e l l d i v i s i o n a t a b o u t 0.87 o f a normal c e l l c y c l e ( T u c k e r et al., 1980). S i n c e c e l l d i v i s i o n b e g i n s a t a c o n s t a n t i n t e r v a l b e f o r e f i s s i o n (90-100 m i n u t e s ) r e g a r d l e s s of growth r a t e ( s e e b e l o w ) , t e r m i n a t i o n of DNA s y n t h e s i s would t a k e p l a c e a t ab o u t 0.8 h o u r s p r i o r t o f i s s i o n . T h e r e f o r e , i n i t i a t i o n o f DNA s y n t h e s i s i n t h e c h e m o s t a t c e l l s o c c u r s a p p r o x i m a t e l y 9.3(+/-1.445) h o u r s a f t e r f i s s i o n ( t h e g e n e r a t i o n t i m e minus t h e d u r a t i o n o f S p l u s 0.8 h o u r s ) . T h i s c o r r e s p o n d s t o a c e l l c y c l e p o s i t i o n o f 0.32+/-0.05 o f a c e l l c y c l e when n o r m a l i z e d . T h i s v a l u e i s not s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t i n w e l l - f e d c o n t r o l s (0.25+/-0.03). -17-A d d i t i o n a l i n f o r m a t i o n c o n c e r n i n g t h e t i m i n g o f DNA s y n t h e s i s i n i t i a t i o n i n c h e m o s t a t c u l t u r e s comes f r o m t h e n u t r i t i o n a l - s h i f t e x p e r i m e n t s ( s e e b e l o w ) . C e l l s i n i t i a t e DNA s y n t h e s i s a t a t i m e c o r r e s p o n d i n g t o 0.25 i n t h e p a r e n t a l c e l l c y c l e f o l l o w i n g a n u t r i t i o n a l e n r i c h m e n t ( s e e b e l o w ) . As t h e s e c e l l s were c o l l e c t e d o r i g i n a l l y f r o m t h e c h e m o s t a t , t h i s f i n d i n g s t r o n g l y s u g g e s t t h a t c e l l s i n t h e c h e m o s t a t c u l t u r e i n i t i a t e DNA s y n t h e s i s a t 0.25 o f t h e c e l l c y c l e . 3. Timing of initiation of cell division in chemostat cut I tut res P r e v i o u s s t u d i e s i n d i c a t e t h a t t h e commitment p o i n t f o r c e l l d i v i s i o n i n b o t h Tetrahymena and Paramecium i s c o i n c i d e n t w i t h i n i t i a t i o n o f o r a l m o r p h o g e n e s i s and m i c r o n u c l e a r m i t o s i s (Review: B e r g e r , 1984). In Tetrahymena, o r a l m o r p h o g e n e s i s and c e l l d i v i s i o n o c c u p y an i n t e r v a l of f i x e d d u r a t i o n a t t h e end of t h e c e l l c y c l e w hich i s i n d e p e n d e n t o f t h e g e n e r a t i o n t i m e ( S u h r - J e s s e n et alt 1977). I f t h i s i s s o i n Paramecium, t h e n c e l l s g r o w i n g i n c h e m o s t a t c u l t u r e s s h o u l d become co m m i t t e d t o d i v i s i o n t h e same number o f m i n u t e s p r i o r t o f i s s i o n as o c c u r s i n r a p i d l y g r o w i n g c e l l s a t t h e same t e m p e r a t u r e , even t h o u g h t h e g e n e r a t i o n t i m e o f c h e m o s t a t c e l l s i s a t l e a s t 4-5 t i m e s l o n g e r t h a n t h a t of w e l l - f e d c e l l s . T h e r e f o r e , t h e r e l a t i v e s t a g e o f t h e p o i n t o f commitment t o c e l l d i v i s i o n w i t h i n t h e c e l l c y c l e i n t h e s e s l o w g r o w i n g c e l l s s h o u l d be l a t e r t h a n -18-t h a t i n w e l 1 - f e d c e l l s . The t e m p e r a t u r e - s e n s i t i v e mutant, ccl, was u s e d t o map t h e r e l a t i v e p o s i t i o n of t h e p o i n t o f commitment t o d i v i s i o n w i t h i n t h e c e l l c y c l e i n b o t h w e l l - f e d and c h e m o s t a t c e l l s . In w e l l - f e d c e l l s , t h e p o i n t of commitment o c c u r s a t 0.73+/-0.02 w i t h i n t h e c e l l c y c l e , Qr 90-100 m i n u t e s p r i o r t o t h e median t i m e of d i v i s i o n ( P e t e r s o n tc B e r g e r , 1976; Rasmussen Be B e r g e r , 1984). To examine t h e k i n e t i c s o f commitment t o c e l l d i v i s i o n i n c h e m o s t a t c e l l s w i t h an e s t i m a t e d g e n e r a t i o n o f 27.8 h o u r s , t h i r t e e n g r o u p s , e a c h c o n t a i n i n g 50 a s y n c h r o n o u s ccl c e l l s , were t r a n s f e r r e d f r o m t h e c h e m o s t a t t o a n u t r i e n t r i c h medium. They were t h e n s h i f t e d i m m e d i a t e l y t o t h e r e s t r i c t i v e t e m p e r a t u r e and m a i n t a i n e d a t t h a t t e m p e r a t u r e f o r a p p r o x i m a t e l y one c e l l c y c l e d u r a t i o n . The c e l l s were t h e n c o u n t e d t o d e t e r m i n e t h e f r a c t i o n a l i n c r e a s e i n c e l l number a f t e r t h e t e m p e r a t u r e s h i f t . The r e l a t i v e p o s i t i o n o f t h e p o i n t o f commitment t o c e l l d i v i s i o n w i t h i n t h e c e l l c y c l e was t h e n e s t i m a t e d by t h e r e s i d u a l c e l l d i v i s i o n method o f H o w e l l et al. ( 1 9 7 5 ) . The p o i n t of commitment t o c e l l d i v i s i o n was e s t i m a t e d t o be a t 0.96+/-0.02 i n t h e c e l l c y c l e . T h i s c o r r e s p o n d s t o a p o i n t 66.7+/-39.7 m i n u t e s p r i o r t o d i v i s i o n i n a 27.8 c e l l c y c l e . T h i s i n t e r v a l i s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e 95 m i n u t e i n t e r v a l o b s e r v e d i n w e l l - f e d c e l l s , b u t d o e s d i f f e r s i g n i f i c a n t l y f r o m t h e r e l a t i v e l o c a t i o n of t h e t r a n s i t i o n p o i n t i n w e l l - f e d c e l l s (0.73+/-0.02). -19-Protein synthesis activity in chemostat cultures D i v i d i n g c e l l s were c o l l e c t e d f r o m t h e c h e m o s t a t and t h e p r o t e i n s y n t h e s i s r a t e was d e t e r m i n e d a s d e s c r i b e d i n M a t e r i a l s and Methods. The s p e c i f i c r a t e o f p r o t e i n s y n t h e s i s ( a c t i v i t y p e r u n i t mass) i n c h e m o s t a t c e l l s d e c r e a s e s t o 70-85% o f t h e l e v e l f o u n d i n w e l l - f e d c e l l s ( T a b l e 3 ) . The c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s shows a much g r e a t e r d e c r e a s e due t o t h e s m a l l s i z e o f t h e c e l l s . The a v e r a g e p r o t e i n s y n t h e s i s r a t e i n e a c h o f t h e f o u r e x p e r i m e n t s was t h e r e f o r e s i m i l a r t o , o r s m a l l e r t h a n t h e c o r r e s p o n d i n g c e l l mass ( p r o t e i n c o n t e n t ; T a b l e 3 ) . As t h e s p e c i f i c r a t e o f p r o t e i n s y n t h e s i s r e f l e c t s t h e e f f i c i e n c y of t h e p r o t e i n s y n t h e s i z i n g s y s t e m , t h e s e f i n d i n g s s u g g e s t t h a t t h e r e l a t i v e l y low r a t e o f p r o t e i n s y n t h e s i s i s due t o b o t h a d e c r e a s e i n c e l l mass and, p r e s u m a b l y , a r e d u c t i o n i n t h e c o n c e n t r a t i o n o f a c t i v e r i b o s o m e s . B. Nutritional shift-up 1. Effects of nutritional shift-up on initiation of DNA synthesis D i v i d i n g c e l l s were s e l e c t e d f r o m a sample t a k e n f r o m t h e c h e m o s t a t and were t r a n s f e r r e d t o medium w i t h e x c e s s f o o d . The -20-s u b s e q u e n t p a t t e r n o-f i n i t i a t i o n o f DNA s y n t h e s i s i n t h e s e c e l l s was examined by l a b e l l i n g w i t h t r i t i a t e d - t h y m i d i n e (from f e e d i n g on l a b e l l e d b a c t e r i a ) and a u t o r a d i o g r a p h y . DNA s y n t h e s i s n o r m a l l y b e g i n s a t 0.25 i n t h e c e l l c y c l e i n w e l l - f e d c e l l s ( B e r g e r , 1971, 1973). F o l l o w i n g t h e t r a n s f e r , s e v e r a l p o s s i b i l i t i e s e x i s t f o r t h e t i m i n g o f DNA s y n t h e s i s i n t h e s e c e l l s : 1) c e l l s may r e s p o n d q u i c k l y t o t h e new growth c o n d i t i o n s and i n i t i a t e DNA s y n t h e s i s a t 0.25 o f t h e r e c o v e r y c e l l c y c l e (e.g. a t 3.25 h r . a f t e r f i s s i o n when t h e r e c o v e r y c e l l c y c l e d u r a t i o n i s 13 h o u r s ) ; o r 2) c e l l s may i n i t i a t e DNA s y n t h e s i s a t t h e t i m e a t w h i c h DNA i n i t i a t i o n would have o c c u r r e d i f t h e n u t r i t i o n a l c o n d i t i o n s had not been changed (e.g. a t 6.5 hr a f t e r f i s s i o n when t h e p a r e n t a l c y c l e l e n g t h i s 26 h o u r s ) ; o r 3) c e l l s may i n i t i a t e a t a t i m e c o r r e s p o n d i n g t o 0.25 of t h e e q u i l i b r i u m c e l l c y c l e l e n g t h o b s e r v e d under t h e new c o n d i t i o n s (1.5 h r ) . Two s e r i e s o f e x p e r i m e n t s were done t o d i s c r i m i n a t e among t h e s e a l t e r n a t i v e s . In one s e r i e s , g r o u p s o f h a n d - s y n c h r o n i z e d c e l l s were l a b e l l e d w i t h l a b e l l e d b a c t e r i a f o r 20 min., a t 15 t o 30 min. i n t e r v a l s f r o m 1 t o 4 h o u r s a f t e r f i s s i o n . They were t h e n washed f r e e of b a c t e r i a and f i x e d . In a s e c o n d s e r i e s o f e x p e r i m e n t s , c e l l s were l a b e l l e d f r o m 5 t o 8 h o u r s a f t e r f i s s i o n . A u t o r a d i o g r a p h s were p r e p a r e d . M a c r o n u c l e i were s c o r e d f o r t h e p r e s e n c e o r a b s e n c e o f DNA s y n t h e s i s a s i n d i c a t e d by a s i g n i f i c a n t c o n c e n t r a t i o n o f g r a i n s over n u c l e i . -21-In two e x p e r i m e n t s , t h e mean t i m e s o f DNA s y n t h e s i s i n i t i a t i o n were 6.6 h o u r s and 7.9 h o u r s a f t e r f i s s i o n r e s p e c t i v e l y ( F i g . 4 ) . In b o t h c a s e s , i n i t i a t i o n of DNA s y n t h e s i s o c c u r r e d a t a t i m e c o r r e s p o n d i n g t o 25% o f t h e p a r e n t a l c e l l c y c l e (26 and 32 h o u r s r e s p e c t i v e l y ) . No DNA s y n t h e s i s o c c u r r e d p r i o r t o 6 h o u r s a f t e r f i s s i o n . T h e s e r e s u l t s d e m o n s t r a t e t h a t t h e t i m i n g o f DNA s y n t h e s i s i s s e t d u r i n g t h e p r e c e d i n g c e l l c y c l e , and i s not a f f e c t e d by t h e i n c r e a s e d r a t e of growth e x p e r i e n c e d a f t e r t h e n u t r i t i o n a l s h i f t - u p a t f i s s i o n ( s e e b e l o w ) . 2. Cell mass at initiation of DNA synthesis In t h e r e c o v e r y c e l l c y c l e f o l l o w i n g t h e n u t r i t i o n a l s h i f t - u p , c e l l mass a t i n i t i a t i o n of DNA s y n t h e s i s was 133% o f t h e i n i t i a l G l v a l u e ; an i n c r e a s e o f 24.5+/-5.4 and 27.2+/-4.34 u n i t s r e s p e c t i v e l y i n t h e two e x p e r i m e n t s when n o r m a l i z e d t o t h e c o n t r o l v a l u e s . T h e s e v a l u e s a r e s l i g h t l y g r e a t e r t h a n t h o s e o f w e l l - f e d c o n t r o l s i n w h i c h i n i t i a t i o n of DNA s y n t h e s i s o c c u r s a t a c e l l mass of 118% o f t h e i n i t i a l l e v e l , an i n c r e a s e of 18+/-2.5 u n i t s when n o r m a l i z e d t o t h e mean (Rasmussen 6e B e r g e r , 1982). 3. Effect of nutritional shift-up on macronuclear DNA content and cell mass -22-Two g r o u p s of a p p r o x i m a t e l y 40 d i v i d i n g c e l l s were h a n d - s e l e c t e d f r o m a c h e m o s t a t c u l t u r e . One g r o u p was f i x e d i m m e d i a t e l y a f t e r f i s s i o n and was used t o d e t e r m i n e t h e i n i t i a l DNA and p r o t e i n c o n t e n t o f t h e c e l l s . The s e c o n d g r o u p was a l l o w e d t o p r o g r e s s t h r o u g h t h e c e l l c y c l e i n t h e p r e s e n c e of e x c e s s f o o d b e f o r e b e i n g f i x e d and s t a i n e d . C e l l s s y n t h e s i z e d an a v e r a g e of 98.6+/-4 u n i t s of DNA and 107.6+/-4.8 u n i t s of p r o t e i n i n t h e r e c o v e r y c e l l c y c l e r e g a r d l e s s of t h e i n i t i a l DNA o r p r o t e i n c o n t e n t ( T a b l e 1, d e l t a D, d e l t a P ) . T h e s e v a l u e s a r e not s i g n i f i c a n t l y d i f f e r e n t f r o m t h e normal v a l u e s f o r w e l l - f e d c e l l s (lOO u n i t s ) . Thus, u n l i k e t i m i n g o f DNA s y n t h e s i s , t h e mechanisms r e g u l a t i n g DNA and p r o t e i n c o n t e n t a r e q u i c k l y r e s e t t o r e f l e c t t h e new n u t r i e n t c o n d i t i o n s . 4. Effect of nutritional shift-up on protein synthesis rate The p r o t e i n s y n t h e s i s r a t e o f c e l l s d u r i n g t h e r e c o v e r y c e l l c y c l e was d e t e r m i n e d i n two s e r i e s of e x p e r i m e n t s . In one s e r i e s , A, t h e s y n c h r o n i z e d c e l l s a m p l e s were l a b e l l e d f r o m 1 t o 4 h o u r s a f t e r f i s s i o n . In t h e o t h e r s e r i e s , B, t h e c e l l s were l a b e l l e d f r o m 4 t o 8 h o u r s a f t e r f i s s i o n . The r a t e of p r o t e i n s y n t h e s i s o f w e l l - f e d c e l l s l a b e l l e d f r o m 0.5 t o 5 h o u r s was u s e d as t h e c o n t r o l . The p r o t e i n s y n t h e s i s r a t e i n c r e a s e d l i n e a r l y t h r o u g h o u t -23-t h e c e l l c y c l e i n b o t h g r o u p s o f c o n t r o l c e l l s , a l t h o u g h t h e r a t e o f i n c r e a s e d i f f e r e d s l i g h t l y between t h e two e x p e r i m e n t s <Fig. 5 a , b ) . Such v a r i a t i o n i n growth r a t e of c e l l s g r o w i n g under s i m i l a r c u l t u r e c o n d i t i o n s i s not uncommon ( K i m b a l l et al. 1959; K i m b a l l , 1967). T h e s e d i f f e r e n c e s have been a t t r i b u t e d t o s l i g h t v a r i a t i o n s i n t h e c u l t u r e c o n d i t i o n s and i n t h e s t a t e o f t h e c e l l s ( K i m b a l l et al., 1959). F o l l o w i n g n u t r i t i o n a l s h i f t - u p , t h e s p e c i f i c r a t e o f p r o t e i n s y n t h e s i s ( a c t i v i t y p e r u n i t mass) i n c r e a s e d q u i c k l y and r e a c h e d 90% o f t h e c o n t r o l l e v e l by 2.5 h o u r s a f t e r n u t r i t i o n a l s h i f t - u p ( F i g . 8 ) . At t h i s t i m e t h e c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s , however, was o n l y a bout 50% o f t h a t o f p o s t - f i s s i o n c o n t r o l s ( F i g . 6) due t o t h e s m a l l s i z e o f t h e e x p e r i m e n t a l c e l l s . By a bout 5 hour a f t e r f i s s i o n , t h e c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s i n e x p e r i m e n t a l c e l l s r e a c h e d t h e normal l e v e l ( F i g . 7 ) . The r a t e o f p r o t e i n s y n t h e s i s c o n t i n u e d t o i n c r e a s e d u r i n g t h e 4 t o 8 hour i n t e r v a l w h i l e t h e s p e c i f i c r a t e r e m a i n e d r e l a t i v e l y c o n s t a n t as d i d t h a t o f t h e c o n t r o l c e l l s ( F i g . 7 <U F i g . 9 r e s p e c t i v e l y ) . C. Nutritional shift-down 1. Effect of nutritional shift-down on timing of DNA synthesis G r o u p s Df a p p r o x i m a t e l y 40 d i v i d i n g c e l l s were -24-h a n d - s e l e c t e d f r o m a w e l l - f e d c u l t u r e and t r a n s f e r r e d t o e x h a u s t e d medium. Between 45 min. and 3.5 h r . a f t e r f i s s i o n g r o u p s o f c e l l s were t r a n s f e r r e d t o medium w i t h l a b e l l e d b a c t e r i a f o r 20 min. They were t h e n washed f r e e o f b a c t e r i a and f i x e d . The p a t t e r n o f i n i t i a t i o n o f DNA s y n t h e s i s was examined by a u t o r a d i o g r a p h y . In t h r e e d i f f e r e n t e x p e r i m e n t s , DNA s y n t h e s i s began a t 1.25 t o 1.50 h o u r s a f t e r f i s s i o n ( F i g . 1 0 ) . T h e s e t i m e s c o r r e s p o n d e d t o 0.25 i n t h e p a r e n t a l c e l l c y c l e . However, t h e r a t e of i n c r e a s e i n t h e f r a c t i o n of c e l l s s y n t h e s i z i n g DNA i n t h e s e e x p e r i m e n t s was lower t h a n n o r m a l , and v a r i e d i n v e r s e l y w i t h t h e s e v e r i t y of s t a r v a t i o n . The l o n g e r t h e l e n g t h o f t h e c e l l c y c l e , t h e lower t h e s l o p e of t h e r e g r e s s i o n l i n e f o r DNA s y n t h e s i s i n i t i a t i o n ( F i g . 1 0 ) . A l t h o u g h t h e f i r s t c e l l s t o i n i t i a t e DNA s y n t h e s i s d i d so a t t h e normal t i m e , t h e median t i m e o f i n i t i a t i o n was l a t e r t h a n normal b e c a u s e o f t h e s l o w r i s e i n t h e f r a c t i o n of c e l l s s y n t h e s i z i n g DNA. The mean t i m e f o r i n i t i a t i o n o f DNA s y n t h e s i s was not r e a c h e d u n t i l 1.8 h o u r s t o 3.5 h o u r s a f t e r f i s s i o n , d e p e n d i n g on t h e n u t r i t i o n a l c o n d i t i o n s ( T a b l e 4 ) . T h e s e r e s u l t s s u g g e s t t h a t t i m i n g of DNA s y n t h e s i s i s g o v e r n e d by two f a c t o r s : t h e t i m e o f f i r s t i n i t i a t i o n o f DNA s y n t h e s i s a p p e a r s t o be s e t a c c o r d i n g t o t h e c o n d i t i o n s o f t h e p r e c e d i n g c e l l c y c l e . However, t h e r a t e a t w h ich c e l l s e n t e r t h a t s u b s e t o f c e l l s s h o w i n g s u f f i c i e n t DNA s y n t h e s i s t o be d e t e c t e d a u t o r a d i o g r a p h i c a l 1 y i s a f f e c t e d by t h e a l t e r e d n u t r i t i v e c o n d i t i o n s . P r e v i o u s work has shown t h a t -25-t h e r a t e of DNA s y n t h e s i s i s s t r o n g l y c o r r e l a t e d w i t h t h e r a t e o f p r o t e i n s y n t h e s i s ( B e r g e r , 1982a). C o n s e q u e n t l y , s e v e r e n u t r i e n t l i m i t a t i o n may s i g n i f i c a n t l y i n c r e a s e t h e t i m e r e q u i r e d f o r some o f t h e c e l l s t o a c c u m u l a t e s u f f i c i e n t l a b e l l e d DNA t o be s c o r e d a s " l a b e l l e d " . 2. Cell mass at initiation of DNA synthesis The mean p r o t e i n c o n t e n t o f c e l l s t h a t were s y n t h e s i z i n g DNA was h i g h e r t h a n t h o s e t h a t were not ( T a b l e 5 ) . Thus, c e l l mass may p l a y an i m p o r t a n t r o l e i n t h e t i m i n g o f DNA s y n t h e s i s . In t h e c e l l c y c l e f o l l o w i n g n u t r i t i o n a l s h i f t - d o w n , c e l l mass a t i n i t i a t i o n of DNA s y n t h e s i s i s lower t h a n n o r m a l , w i t h a d e c r e a s e o f 40% i n one e x p e r i m e n t (G.T. >24 h r s . ) , o r an i n c r e a s e o f o n l y 5.9% (G.T. = 10 h r s . ) and 11% (G.T. =6.33 h r s ) r e s p e c t i v e l y i n t h e o t h e r two e x p e r i m e n t s . T h i s i s s i g n i f i c a n t l y lower t h a n c o n t r o l c e l l s i n which t h e c e l l mass has i n c r e a s e d 18% by t h e t i m e o f i n i t i a t i o n o f DNA s y n t h e s i s . 3. Increase in macronuclear DNA content and cell mass C e l l s made an a v e r a g e o f 59+/-4.9 u n i t s o f DNA and 33.S+/-4.49 u n i t s of p r o t e i n i n t h e c e l l c y c l e f o l l o w i n g -26-n u t r i t i o n a l s h i f t t o e x h a u s t e d medium ( T a b l e 6, d e l t a D), compared t o lOO u n i t s f o r c o n t r o l c e l l s . -27-DISCUSSION Control of initiation of DNA synthesis Timing of initiation of DNA synthesis T h i s s t u d y e x a mines t h e c r i t e r i a f o r i n i t i a t i o n o f DNA s y n t h e s i s u nder v a r i o u s n u t r i t i o n a l c o n d i t i o n s . The t i m i n g o f DNA s y n t h e s i s i s l a r g e l y d e t e r m i n e d i n t h e p r e c e d i n g c e l l c y c l e . As shown by b o t h n u t r i t i o n a l s h i f t - u p and s h i f t - d o w n e x p e r i m e n t s , t h e t i m i n g o f DNA s y n t h e s i s r e f l e c t s t h e c o n d i t i o n s p r i o r t o t h e n u t r i t i o n a l s h i f t , and i s not r e a d j u s t e d i n t h e c e l l c y c l e f o l l o w i n g t h e s h i f t . F o r example, t h e growth r a t e r e s p o n d s q u i c k l y and i n c r e a s e s f o l l o w i n g t h e n u t r i t i o n a l s h i f t - u p . P r o t e i n s y n t h e s i s a c t i v i t y i s normal f o r a t l e a s t 1.5 h o u r s b e f o r e DNA s y n t h e s i s i n i t i a t i o n . T h e r e f o r e , t h e t i m i n g of DNA s y n t h e s i s i n t h e r e c o v e r y c e l l c y c l e i s not a f f e c t e d by t h e i n c r e a s e d r a t e o f growth e x p e r i e n c e d a f t e r t h e n u t r i t i o n a l s h i f t - u p a t f i s s i o n . The i n c r e a s e i n c e l l mass, t h e number of a c t i v e r i b o s o m e s , t h e r a t e s o f rRNA and p r o t e i n s y n t h e s i s may a l l c o n t r i b u t e t o t h e change i n g r o w t h r a t e f o l l o w i n g t h e n u t r i t i o n a l s h i f t . A r a p i d i n c r e a s e i n a l l of t h e t h e s e components has been o b s e r v e d i n Tetrahymena upon r e f e e d i n g of s t a r v e d c e l l s ( D r e i s i g et al., 1984). F o l l o w i n g n u t r i t i o n a l d o w n - s h i f t , t h e t i m i n g o f i n i t i a t i o n o f DNA s y n t h e s i s f o l l o w s t h a t o f t h e p a r e n t a l c e l l c y c l e e x c e p t t h a t t h e r a t e a t w hich c e l l s e n t e r t h e S phase i s a f f e c t e d by -28-t h e a l t e r e d g r o w t h c o n d i t i o n s . The mean t i m e o f DNA s y n t h e s i s i n i t i a t i o n , t h e r e f o r e , i s s l i g h t l y d e l a y e d d e p e n d i n g on t h e s e v e r i t y o f n u t r i e n t l i m i t a t i o n . The p r e s e n t o b s e r v a t i o n s t h a t t h e t i m i n g of DNA s y n t h e s i s i s d e t e r m i n e d p r i o r t o f i s s i o n , and i s not a f f e c t e d by s u b s e q u e n t i n c r e a s e s i n growth r a t e i s i n c o n s i s t e n t w i t h c o n c e n t r a t i o n d e p e n d e n t m o d e l s f o r c o n t r o l o f c e l l c y c l e e v e n t s (Review: F a n t e s et al. , 1975). The o b s e r v a t i o n s t h a t c o n d i t i o n s r e g u l a t i n g i n i t i a t i o n of DNA s y n t h e s i s a r e e s t a b l i s h e d i n t h e p r e v i o u s c e l l c y c l e , and a r e n o t a f f e c t e d by c h a n g e s i n gene c o n c e n t r a t i o n o c c u r r i n g a t f i s s i o n ( B e r g e r , 1982b) a r e c o n s i s t e n t w i t h t h e s t a b l e i n h i b i t o r m o d e l . In t h i s model, t h e i n h i b i t o r i s p u l s e d s y n t h e s i z e d a t some p o i n t a f t e r i n i t i a t i o n o f DNA s y n t h e s i s i n t h e p r e v i o u s c e l l c y c l e . The a b s o l u t e amount p r o d u c e d i s t h e r e f o r e d e p e n d e n t on t h e p a r e n t a l gene d o s a g e . I n i t i a t i o n o f DNA s y n t h e s i s would o c c u r i n t h e c u r r e n t c e l l c y c l e when t h e c o n c e n t r a t i o n o f i n h i b i t o r i s d i l u t e d below a c r i t i c a l l e v e l by g r o w t h . T h e r e f o r e , t h e t i m i n g o f DNA s y n t h e s i s i n i t i a t i o n i s s e t p r i o r t o f i s s i o n , and i s a f f e c t e d o n l y by t h e r a t e o f g r o w t h . However, as shown by b o t h n u t r i t i o n a l s h i f t - u p and s h i f t - d o w n , g r o w t h r a t e r e s p o n d s q u i c k l y t o t h e new c o n d i t i o n s , b u t t h e t i m i n g of DNA s y n t h e s i s i n i t i a t i o n r e m a i n s a t 0.25 o f t h e p a r e n t a l c y c l e l e n g t h . T h i s o b s e r v a t i o n makes t h e s t a b l e i n h i b i t o r model i n v a l i d f o r Paramecium as d o e s t h e f i n d i n g t h a t an i n c r e a s e i n r e l a t i v e c e l l mass d o e s not b r i n g about DNA s y n t h e s i s i n i t i a t i o n ( s e e -29-b e l o w ) . No f o r m a l model i s c o n s i s t e n t w i t h a l l o b s e r v a t i o n s r e g a r d i n g r e g u l a t i o n of DNA s y n t h e s i s i n i t i a t i o n i n Paramecium. Cell mass at initiation of DNA synthesis P r e v i o u s s t u d i e s have a l s o d e m o n s t r a t e d t h a t c e l l mass p l a y s a s i g n i f i c a n t r o l e i n i n i t i a t i o n of DNA s y n t h e s i s i n w e l l - f e d c e l l s . W e l l - f e d c e l l s w i t h a p o s t - f i s s i o n mass o f 118% o f t h e mean v a l u e o r g r e a t e r i n i t i a t e DNA s y n t h e s i s i m m e d i a t e l y a f t e r f i s s i o n w i t h no i n t e r v e n i n g G l p e r i o d . T h i s i s c o n s i s t e n t e i t h e r w i t h a c e l l s i z e o r a low gene c o n c e n t r a t i o n r e q u i r e m e n t f o r i n i t i a t i o n of DNA s y n t h e s i s . The o b s e r v a t i o n t h a t c h a n g e s i n gene d o s a g e a t f i s s i o n have no e f f e c t on t h e t i m i n g o f DNA s y n t h e s i s a r g u e s a g a i n s t a gene c o n c e n t r a t i o n d e p e n d e n t c o n t r o l mechanism f o r t h e i n i t i a t i o n o f DNA s y n t h e s i s a c t i n g d u r i n g t h e G l p e r i o d i n t h i s o r g a n i s m . The p r e s e n t o b s e r v a t i o n s on t h e p a t t e r n of c e l l mass a c c u m u l a t i o n a f t e r n u t r i t i o n a l s h i f t s f u r t h e r s u p p o r t s a s i z e - r e l a t e d c o n t r o l o v e r t h e i n i t i a t i o n of DNA s y n t h e s i s . F o l l o w i n g a n u t r i t i o n a l s h i f t - u p , c e l l mass i n c r e a s e s t o a b o u t 180% o f t h e i n i t i a l G l v a l u e a t t h e t i m e of DNA s y n t h e s i s i n i t i a t i o n . T h i s v a l u e i s r e l a t i v e l y much g r e a t e r t h a n t h a t i n w e l l - f e d c o n t r o l s and i n d i c a t e s t h a t a r e l a t i v e i n c r e a s e i n c e l l mass p a s t t h e normal l e v e l f o r i n i t i a t i o n of DNA s y n t h e s i s (118%) i s not s u f f i c i e n t f o r i n i t i a t i o n of DNA s y n t h e s i s . However, t h e a b s o l u t e amount of p r o t e i n a c c u m u l a t e d i n t h e s e -30-c e l l s a f t e r n u t r i t i o n a l e n r i c h m e n t up t o t h e p o i n t o f i n i t i a t i o n o f DNA s y n t h e s i s i s on a v e r a g e 2 5 . 8 5 ( + / - 6 . 9 ) % o f normal w e l l - f e d p o s t - f i s s i o n v a l u e s , and t h e a v e r a g e amount of p r o t e i n a c c u m u l a t e d i n one c e l l c y c l e i s 108<+/-4.8) u n i t s . T h e s e v a l u e s a r e s l i g h t l y , b u t not s i g n i f i c a n t l y , g r e a t e r t h a n t h o s e o f w e l l - f e d c o n t r o l s (18+/-2.5 and 100+/-4 u n i t s r e s p e c t i v e l y ) . T h e r e f o r e , t h e a b s o l u t e i n c r e a s e i n c e l l mass, but not t h e r e l a t i v e i n c r e a s e , may be i m p o r t a n t i n d e t e r m i n i n g i n i t i a t i o n of DNA s y n t h e s i s . I f t h i s i s t r u e , t h e n i t i s a l s o a p p a r e n t t h a t t h e a b s o l u t e c e l l mass i n c r e a s e r e q u i r e d f o r i n i t i a t i o n of DNA s y n t h e s i s i s r a p i d l y r e s e t f o l l o w i n g n u t r i t i o n a l s h i f t s . On t h e o t h e r hand, t h e a b s o l u t e i n c r e a s e i n c e l l mass o b s e r v e d p r i o r t o i n i t i a t i o n o f DNA s y n t h e s i s a f t e r n u t r i t i o n a l s h i f t - u p may be p u r e l y c o i n c i d e n t a l , and i s n o t d i r e c t l y r e l a t e d t o t h e mechanisms c o n t r o l l i n g i n i t i a t i o n o f DNA s y n t h e s i s . T h i s l a t t e r h y p o t h e s i s i s f a v o r e d . S t u d i e s i n ccl c e l l s a l s o s u g g e s t t h a t an a b s o l u t e i n c r e a s e i n mass i n G l i s not s u f f i c i e n t f o r i n i t i a t i o n o f DNA s y n t h e s i s (Rasmussen & B e r g e r , 1982). When ccl c e l l s were b l o c k e d e a r l y i n G l , DNA s y n t h e s i s d i d not b e g i n i m m e d i a t e l y upon r e t u r n t o p e r m i s s i v e c o n d i t i o n s . Even t h o u g h t h e r e was a s i g n i f i c a n t i n c r e a s e i n c e l l mass, t h e c e l l s had t o p r o g r e s s t h r o u g h t h e r e s t o f G l p h a s e b e f o r e i n i t i a t i o n c o u l d o c c u r (Rasmussen & B e r g e r , 1982, 1984; Rasmussen, 1984). Thus, t h e a t t a i n m e n t o f a c r i t i c a l c e l i s i z e a l o n e i s not s u f f i c i e n t t o b r i n g a bout i n i t i a t i o n o f DNA s y n t h e s i s . O t h e r c e l l u l a r -31-components r e l a t e d t o c e l l mass, p o s s i b l y t h e a c c u m u l a t i o n o-f s t a b l e RNA ( s u c h as r i b o s o m a l RNA), may be i m p o r t a n t f o r i n i t i a t i o n o f DNA s y n t h e s i s ( J a u k e r , 1975, 1983? B e r g e r , 1984). A low R N A / p r o t e i n r a t i o n e a r t h e b e g i n n i n g o f S p h a s e i s o b s e r v e d i n s e v e r a l s t u d i e s on Tetrahymena ( C l e f f m a n n et al., 1979; S e y f e r t , 1980; S c h a e f e r ic C l e f f m a n n , 1982). T h e s e f i n d i n g s s u g g e s t t h a t a low RNA c o n c e n t r a t i o n i s c o r r e l a t e d w i t h i n i t i a t i o n o f DNA r e p l i c a t i o n . I f a low R N A / p r o t e i n r a t i o i s r e l a t e d t o i n i t i a t i o n of DNA s y n t h e s i s , c e l l s t h a t have a lower t h a n normal RNA c o n t e n t s h o u l d i n i t i a t e e a r l y . A s h o r t e n e d G l p e r i o d i s i n d e e d f o u n d i n Tetrahymena c e l l s when t h e r a t e o f RNA s y n t h e s i s i s r e d u c e d by a c t i n o m y c i n t r e a t m e n t d u r i n g t h e l a t t e r p a r t of t h e p r e v i o u s c e l l c y c l e ( J a u k e r , 1975), and when t h e t r a n s c r i p t i o n a l a c t i v i t y i s r e d u c e d i n c e l l s w i t h a low DNA c o n t e n t . A low DNA c o n t e n t i s o b t a i n e d e i t h e r t h r o u g h an i n h i b i t i o n o f DNA s y n t h e s i s , o r t h r o u g h an u n e q u a l p a r t i t i o n i n g o f m a c r o n u c l e u s d u r i n g d i v i s i o n ( S e y f e r t , 1977; W o r t h i n g t o n e t a l . , 1976; D o e r d e r ic D e B a u l t , 1978). O b s e r v a t i o n s on c e l l s f o l l o w i n g a n u t r i t i o n a l step-down i n t h i s s t u d y a l s o i n d i c a t e t h a t t h e a p p a r e n t s i z e r e q u i r e m e n t f o r i n i t i a t i o n o f DNA s y n t h e s i s c a n n o t be a f u n c t i o n o f c e l l mass per se. I n i t i a t i o n of DNA s y n t h e s i s (and p r o g r e s s i o n t h r o u g h t h e c e l l c y c l e ) o c c u r s even when t h e r e i s a n e t d e c r e a s e i n c e l l mass. T h e r e i s e v i d e n c e , t h o u g h , t h a t b i g g e r c e l l s t e n d -32-t o i n i t i a t e s l i g h t l y e a r l i e r t h a n s m a l l e r c e l l s . When c e l l s a r e t r a n s f e r r e d t o n u t r i t i o n a l l y l i m i t i n g c o n d i t i o n s ( c h e m o s t a t ) t h e y c o n t i n u e t o t r a v e r s e t h e c e l l c y c l e b u t t h e c e l l s become p r o g r e s s i v e s m a l l e r . T h e r e f o r e i f t h e r e i s a c e l l r e l a t e d r e q u i r e m e n t f o r i n i t i a t i o n i t i s r e l a t i v e r a t h e r t h a n a b s o l u t e . DNA s y n t h e s i s o c c u r a t 0.25 of a c e l l c y c l e i n t h e s e c h e m o s t a t c e l l s . A s s u m i n g t h a t c e l l mass i n c r e a s e s e x p o n e n t i a l l y ( K i m b a l l , 1967) and d o u b l e s i n e a c h c e l l c y c l e , t h e mass a t t h e p o i n t of i n i t i a t i o n u n der e q u i l i b r i u m c o n d i t i o n s would be 118% o f t h e i n i t i a l v a l u e . The a b s o l u t e i n c r e a s e i n c e l l mass up t o t h e p o i n t o f DNA s y n t h e s i s i n i t i a t i o n would be e q u i v a l e n t t o t h e p o s t - f i s s i o n c e l l mass t i m e s t h e r e l a t i v e i n c r e a s e ( 1 . 1 8 ) . Thus, t h e c e l l mass r e q u i r e d f o r i n i t i a t i o n of DNA s y n t h e s i s becomes p r o p o r t i o n a l t o t h e growth r a t e o f t h e c e l l . C e l l s w i t h a maximum growth r a t e w i l l i n i t i a t e DNA s y n t h e s i s when t h e a b s o l u t e i n c r e a s e i n c e l l mass i s 18% o f t h e p o s t - f i s s i o n v a l u e ; whereas s l o w g r o w e r s w i l l i n i t i a t e a t a f r a c t i o n of t h a t v a l u e (18%) t h a t i s p r o p o r t i o n a l t o t h e i r s i z e . Regulation of macronuclear DNA content U n l i k e t h e t i m i n g of i n i t i a t i o n of DNA s y n t h e s i s , t h e amount o f DNA made d u r i n g t h e c e l l c y c l e r e f l e c t s s o l e l y t h e c u r r e n t n u t r i t i o n a l c o n d i t i o n s . C e l l s a t e q u i l i b r i u m i n t h e c h e m o s t a t m a i n t a i n a p o s t - f i s s i o n DNA c o n t e n t of ab o u t 50 u n i t s , s u g g e s t i n g t h a t t h e y o n l y s y n t h e s i z e a b o u t h a l f t h e -33-normal amount p e r c e l l c y c l e u nder n u t r i e n t c o n s t r a i n t . When w e l l - f e d c e l l s a r e t r a n s f e r r e d t o e x h a u s t e d medium, t h e amount of DNA p r o d u c e d i s a l s o r e d u c e d t o s l i g h t l y more t h a n 50 u n i t s ( s e e a l s o n e x t s e c t i o n ) . But when c e l l s a r e t r a n s f e r r e d f r o m t h e c h e m o s t a t t o a n u t r i e n t r i c h medium, t h e y p r o d u c e lOO u n i t s o f DNA i n t h e c e l l c y c l e i m m e d i a t e l y f o l l o w i n g t h e n u t r i t i o n a l s h i f t - u p as do w e l l - f e d c e l l s . T h e s e f i n d i n g s i n d i c a t e t h a t t h e mechanism r e g u l a t i n g DNA c o n t e n t i s s e n s i t i v e t o t h e growth r a t e o f t h e c e l l . T h i s s h o u l d n o t be s u r p r i s i n g s i n c e c e l l s need t o a d j u s t gene d o s a g e t o m a i n t a i n a c h a r a c t e r i s t i c gene c o n c e n t r a t i o n (gene d o s a g e / c e l l m a s s ) . As t h e c e l l mass i n c r e a s e s (or d e c r e a s e s ) r a p i d l y due t o a change i n growth r a t e , gene c o n c e n t r a t i o n i s a l s o d r a s t i c a l l y r e d u c e d (or i n c r e a s e d ) . The c e l l ' s r e g u l a t o r y mechanisms r e s p o n d by m aking a h i g h e r ( o r lower) amount of DNA. The b a s i s of t h i s r e g u l a t o r y mechanism has been r e v e a l e d by a n a l y s i s o f t h e e f f e c t s of c h a n g e s i n gene d o s a g e and c e l l mass on t h e r a t e of p r o t e i n s y n t h e s i s . At any p o i n t i n t h e c e l l c y c l e t h e r a t e o f p r o t e i n s y n t h e s i s i s l i m i t e d by t h e lower o f t h e r e l a t i v e v a l u e s o f t h e two v a r i a b l e s . B o t h DNA s y n t h e s i s and c e l l g r owth seem t o be l i m i t e d by t h e c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s , and t h e r a t e s of DNA s y n t h e s i s and c e l l g r o w t h i n c r e a s e above t h e normal l e v e l o n l y when b o t h gene d o s a g e and c e l l mass a r e i n c r e a s e d ( B e r g e r , 1982a? Rasmussen & B e r g e r , 1982). The s i t u a t i o n f o l l o w i n g n u t r i t i o n a l s h i f t - u p i s somewhat -34-a n a l o g o u s t o one where t h e i n i t i a l c e l l mass i s normal b u t t h e i n i t i a l DNA c o n t e n t i s r e d u c e d , g i v i n g a lower gene c o n c e n t r a t i o n . T h e s e c e l l s more t h a n d o u b l e t h e i r DNA c o n t e n t by s y n t h e s i z i n g 100 u n i t s o f DNA b e f o r e f i s s i o n ( B e r g e r , 1979). T h i s l e a d s t o r a p i d r e e s t a b 1 i s h m e n t o f t h e a p p r o p r i a t e gene c o n c e n t r a t i o n . The g r o w t h r a t e - s e n s i t i v e r e g u l a t o r y mechanism f o r DNA c o n t e n t w i l l be d i s c u s s e d f u r t h e r i n t h e n e x t s e c t i on. Control over initiation of cell division Two major a s p e c t s o f t h e c o n t r o l o v e r c e l l d i v i s i o n a r e r e v e a l e d i n t h i s s t u d y . F i r s t , t h e mechanism f o r t h e i n i t i a t i o n of c e l l d i v i s i o n i s n o t t i m e - d e p e n d e n t . N e i t h e r t h e a b s o l u t e d u r a t i o n nor t h e r e l a t i v e p r o p o r t i o n o f S p h a s e i n t h e c e l l c y c l e i s s i m i l a r among c e l l s w i t h d i f f e r e n t growth r a t e s ( T a b l e 7 ) . T h e r e f o r e , i n i t i a t i o n of c e l l d i v i s i o n i s not d e t e r m i n e d by a ' t i m e r ' w h i c h r u n s f r o m t h e i n i t i a t i o n o f DNA r e p l i c a t i o n t o f i s s i o n . P r e v i o u s s t u d i e s a l s o i n d i c a t e t h a t t h e i n i t i a t i o n o f c e l l d i v i s i o n d o e s n o t o c c u r a t a f i x e d i n t e r v a l f o l l o w i n g t h e p r e v i o u s f i s s i o n . C e l l s w i t h h i g h l y r e d u c e d DNA c o n t e n t r e q u i r e t h r e e t o f i v e t i m e s t h e normal c e l l c y c l e d u r a t i o n t o r e a c h f i s s i o n ( B e r g e r , 1979), e v e n though t h e t i m i n g o f DNA s y n t h e s i s i n i t i a t i o n i s normal ( B e r g e r , 1982b). S e c o n d , commitment t o d i v i s i o n o c c u r s a b o u t 95 m i n u t e s b e f o r e f i s s i o n r e g a r d l e s s of g r o w t h r a t e . A n a l y s i s o f t h e e f f e c t s Df b l o c k a g e o f m a c r o n u c l e a r DNA s y n t h e s i s i n c c l c e l l s -35-i m m e d i a t e l y a f t e r t h e p o i n t of commitment t o c e l l d i v i s i o n shows t h a t normal c e l l s have a c c u m u l a t e d a b o u t 50 u n i t s o f new DNA a t t h e p o i n t o f commitment t o d i v i s i o n ( T a b l e 8 ) . S i n c e t h e r a t e of DNA s y n t h e s i s and t h e growth r a t e a r e b o t h p r o p o r t i o n a l t o t h e c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s ( B e r g e r , 1982a? Rasmussen k B e r g e r , 1982); and t h e t i m e i n t e r v a l between commitment t o d i v i s i o n and f i s s i o n i s c o n s t a n t , s l o w l y g r o w i n g c e l l s would be e x p e c t e d t o have lower t h a n normal DNA c o n t e n t a t t h e end o f m a c r o n u c l e a r S p e r i o d , even though t h e i n c r e a s e i n DNA c o n t e n t o c c u r i n g between f i s s i o n and t h e p o i n t o f commitment t o d i v i s i o n i s n o r m a l . The amount s y n t h e s i z e d f o l l o w i n g t h e p o i n t of commitment t o d i v i s i o n would be p r o p o r t i o n a l t o t h e r a t e of g r o w t h . T h i s h y p o t h e s i s e x p l a i n s t h e n u t r i e n t s e n s i t i v i t y o f t h e mechanisms r e g u l a t i n g DNA c o n t e n t , and a l s o why t h e l o w e s t DNA c o n t e n t o b s e r v e d i n newly d i v i d e d c e l l s sampled f r o m t h e c h e m o s t a t i s not s i g n i f i c a n t l y d i f f e r e n t f r o m 50 u n i t s r e g a r d l e s s o f growth r a t e . C e l l s t h a t have s y n t h e s i z e d l e s s t h a n 50 u n i t s of DNA do not d i v i d e . The DNA i n c r e a s e r e q u i r e d f o r c e l l d i v i s i o n may be s l i g h t l y l e s s t h a n t h e 50 u n i t s s u g g e s t e d above f o r two r e a s o n s . F i r s t , t h e i n c r e a s e i n DNA c o n t e n t t o 0.75 i n t h e c e l l c y c l e i s a v e r a g e d f r o m f o u r d i f f e r e n t e x p e r i m e n t s . A l t h o u g h c a r e was t a k e n t o h e a t - t r e a t c e l l s a t t h e same p o i n t i n t h e c e l l c y c l e , c e l l s may have a l r e a d y p a s s e d t h e p o i n t o f commitment t o c e l l d i v i s i o n when s h i f t e d t o t h e r e s t r i c t i v e c o n d i t i o n a t 0.75 o f t h e c o n t r o l c y c l e l e n g t h . As c c l c e l l s do -36-not d i v i d e i f t h e y a r e h e a t - t r e a t e d b e f o r e t h e t r a n s i t i o n p o i n t f o r c e l l d i v i s i o n , t h e l o w e s t i n c r e a s e i n DNA c o n t e n t , 42 u n i t s , may be a c l o s e r e s t i m a t e o f t h e c r i t i c a l amount f o r commitment t o c e l l d i v i s i o n ( T a b l e 8 ) . S e c o n d l y , DNA c o n t e n t between 43 and 50 u n i t s a r e f o u n d i n c h e m o s t a t s a m p l e s ( T a b l e 1, D i ) . T h i s i n d i c a t e s t h a t c e l l s do d i v i d e a f t e r making s l i g h t l y l e s s t h a n 50 u n i t s o f DNA. As d i s c u s s e d above, c e l l s a r e c o m m i t t e d t o d i v i s i o n a f t e r s y n t h e s i z i n g a t h r e s h o l d amount of DNA, a f t e r w hich d i v i s i o n w i l l p r o c e e d a t t h e s c h e d u l e d t i m e . The amount of DNA made d u r i n g t h e l a t t e r i n t e r v a l s h o u l d be p r o p o r t i o n a l t o t h e growth r a t e . Based on t h i s a s s u m p t i o n , t h e t h e amount o f DNA p r o d u c e d d u r i n g a c e l l c y c l e c an be c a l c u l a t e d a s f o l l o w s : Di = Dt + <100-Dt> <r> (1) where Di i s t h e i n c r e a s e i n DNA c o n t e n t d u r i n g t h e c e l l c y c l e , Dt i s t h e t h r e s h o l d DNA c o n t e n t , and r i s t h e r e l a t i v e g r o w t h r a t e . In r a p i d l y g r o w i n g c e l l s r i s l . O . I f e q u a t i o n (1) i s s o l v e d f o r t h e t h r e s h o l d DNA c o n t e n t , Dt, we o b t a i n : Dt = (Di - 100r>/<l-r> (2) When t h e t h r e s h o l d DNA i n c r e m e n t (Dt) i s c a l c u l a t e d f r o m e q u a t i o n (2) u s i n g d a t a f r o m t h e c h e m o s t a t c u l t u r e s , a v a l u e of 41 u n i t s i s o b t a i n e d a s t h e e s t i m a t e of t h e t h r e s h o l d DNA i n c r e m e n t . T h i s v a l u e i s s i m i l a r t o b o t h t h e l o w e s t p o s t - f i s s i o n DNA c o n t e n t of c h e m o s t a t c e l l s o b s e r v e d , and t h e minimum s i z e o f t h e DNA i n c r e m e n t o b t a i n e d f r o m s t u d i e s on ccl -37-c e l l s . T h i s f i n d i n g , t h e r e f o r e , s t r o n g l y s u g g e s t s t h a t t h e t h r e s h o l d i n c r e m e n t o f DNA c o n t e n t f o r commitment t o c e l l d i v i s i o n i s n e a r e r t o 40 t h a n t o 50 u n i t s . S i n c e c e l l s m a i n t a i n e d a minimum DNA c o n t e n t of a t l e a s t 40 u n i t s when t h e g r o w th r a t e was d e c r e a s e d , t h i s i m p l i e s t h a t t h e c o n t r o l o v e r i n i t i a t i o n o f c e l l d i v i s i o n d o e s not change w i t h growth c o n d i t i o n s . I t i s g o v e r n e d by t h e amount o f DNA made, o r by o t h e r c e l l u l a r components r e g u l a t e d by t h e gene d o s a g e , s u c h as t h e t o t a l amount o f s t a b l e RNA o r RNA s y n t h e s i s r a t e i n t h e c e l l . A s i m i l a r g r o w t h r e l a t e d c o n t r o l o v e r i n i t i a t i o n o f c e l l d i v i s i o n has been o b s e r v e d i n t h e Tetrahymena mutant, conical. D a u g h t e r c e l l s d i f f e r i n t h e amount o f c y t o p l a s m b u t have m a c r o n u c l e i w i t h s i m i l a r DNA c o n t e n t ( D o e r d e r et al., 1975). The two s i s t e r c e l l s , d e s p i t e t h e i r d i f f e r e n c e i n c e l l mass, a c c u m u l a t e s i m i l a r amounts of RNA and p r o t e i n d u r i n g t h e c o u r s e of t h e c e l l c y c l e ( S c h a e f e r &: C l e f f m a n n , 1982). N e l s o n et al. (1981) have a l s o shown t h a t Tetrahymena c e l l s have a s t a n d a r d s i z e a t i n i t i a t i o n o f o r a l m o r p h o g e n e s i s , a p o i n t w hich c o r r e s p o n d s c l o s e l y t o t h e p o i n t o f commitment t o c e l l d i v i s i o n . T h i s c r i t i c a l s i z e i s about 1.8 t i m e s t h e i n i t i a l p o s t - f i s s i o n s i z e and i s i n d e p e n d e n t of g e n e r a t i o n t i m e and g r o w th r a t e . J a u k e r (1975, 1983) a l s o a r g u e s t h a t t h e i n i t i a t i o n of c e l l d i v i s i o n i s c o r r e l a t e d w i t h t h e a c c u m u l a t i o n of a c r i t i c a l mass o f s t a b l e RNA (rRNA). A s i z e - r e l a t e d c o n t r o l o v e r i n i t i a t i o n of c e l l d i v i s i o n -38-may be p a r t i c u l a r l y i m p o r t a n t t o p r e v e n t c e l l s i n s t a t i o n a r y p h a s e f r o m b e c o m i n g t o o s m a l l . A s t a t i o n a r y p o p u l a t i o n o f Tetrahymena c o n t a i n s c e l l s i n a l l s t a g e s o f t h e c e l l c y c l e ( R e u t e r et al., 1980). Thus Tetrahymena, and p o s s i b l y Paramecium, a r e c a p a b l e of p r o g r e s s i n g t h r o u g h t h e c e l l c y c l e when s t a r v e d . They a r e not a r r e s t e d i n G l , a s i n y e a s t (Review: C a r t e r , 1981); n e i t h e r do t h e y become q u i e s c e n t and e n t e r i n t o a GO s t a t e , a s i n mammalian c e l l s ( R e v i e w s : S t i l e s et al, 1981; Y a n i s h e v s i y 8c S t e i n , 1981). Such s l o w l y ' c y c l i n g ' s t a r v e d c u l t u r e s c an be k e p t v i a b l e f o r an e x t e n d e d p e r i o d of t i m e ( s e v e r a l weeks i n Paramecium). Thus t h e r e g u l a t o r y mechanisms must s t i l l o p e r a t e u n d e r s u c h . e x t r e m e c o n d i t i o n s , e ven though t h e c e l l s became v e r y s m a l l . I t s h o u l d be n o t e d , however, t h a t u nder t h e e x t r e m e c o n d i t i o n s e i t h e r i n a c h e m o s t a t o r i n s t a t i o n a r y c u l t u r e s , t h e n o r m a l l y b a l a n c e d r e l a t i o n s h i p between DNA c o n t e n t and c e l l mass i n Paramecium ( B e r g e r , 1984; K i m b a l l , 1967) i s u n c o u p l e d . W h i l e DNA c o n t e n t i s m a i n t a i n e d a t 50 u n i t s , t h e c e l l s t o l e r a t e a much s m a l l e r c e l l mass , as l i t t l e a s 24%. T h i s o b s e r v a t i o n i s f u r t h e r e v i d e n c e t h a t t h e D N A - d i v i s i o n c y c l e c a n be t r a v e r s e d much f a s t e r t h a n t h e growth c y c l e , a s s u g g e s t e d by J o h n s t o n et al. (1977) and many o t h e r s . -39-Comparison with other systems T h e r e i s i n s u f f i c i e n t e v i d e n c e t o i n d i c a t e t h a t t h e t i m i n g o f DNA s y n t h e s i s i n i t i a t i o n i s p r e s e t i n t h e p r e v i o u s c e l l c y c l e i n Tetrahymena. When Tetrahymena c e l l s a r e t r e a t e d w i t h h y d r o x y u r e a t o b l o c k DNA s y n t h e s i s l a t e i n t h e c e l l c y c l e , c e l l s a r e ad v a n c e d i n t o DNA s y n t h e s i s ( W o r t h i n g t o n et al., 1976). B l o c k a g e o f RNA s y n t h e s i s l a t e i n t h e c e l l c y c l e a l s o r e d u c e s t h e l e n g t h o f G l p e r i o d i n t h e nex t c e l l c y c l e ( J a u k e r , 1975). B o t h o f t h e s e o b s e r v a t i o n s a r e a l s o c o n s i s t e n t w i t h a gene c o n c e n t r a t i o n model i n wh i c h i n i t i a t i o n o f DNA s y n t h e s i s i s i n d u c e d by a low gene c o n c e n t r a t i o n o r by a low RNA t o p r o t e i n r a t i o . When c e l l s i z e i s r e d u c e d by s t a r v a t i o n i n Paramecium, c e l l s b e g i n DNA s y n t h e s i s a t t h e p r e d e t e r m i n e d t i m e . On t h e o t h e r hand, when c e l l s i z e i s r e d u c e d by u n e q u a l d i v i s i o n o f c y t o p l a s m i n c e l l s w i t h normal s i z e m a c r o n u c l e i i n Tetrahymena, i n i t i a t i o n o f DNA s y n t h e s i s i s d e l a y e d <Schaefer and C l e f f m a n n , 1982). T h e s e and o t h e r o b s e r v a t i o n s (Review: B e r g e r , 1984) wh i c h d e m o n s t r a t e s t h e e f f e c t of a r e d u c e d G l gene c o n c e n t r a t i o n on t h e t i m i n g of S, s t r o n g l y f a v o r t h e n o t i o n t h a t c o n d i t i o n s f o r i n i t i a t i o n o f DNA s y n t h e s i s i n Tetrahymena a r e not s e t i n t h e p r e v i o u s c e l l c y c l e . As i n Tetrahymena, c o n d i t i o n s f o r i n i t i a t i o n of DNA s y n t h e s i s a r e not d e t e r m i n e d b e f o r e t h e p r e s e n t G l p e r i o d i n y e a s t . When y e a s t c e l l s a r e s h i f t e d f r o m one growth c o n d i t i o n -40-t o a n o t h e r , c e l l s i n i t i a t e DNA s y n t h e s i s (as i n d i c a t e d by bud i n i t i a t i o n ) a t a s i z e c h a r a c t e r i s t i c o f t h e new growth r a t e ( J o h n s t o n et al., 1977, 1979; F a n t e s 8c N u r s e , 1977) u n l e s s t h e y have r e a c h e d t h e commitment p o i n t f o r bud i n i t i a t i o n . T h e r e f o r e , i f c e l l s a r e t r a n s f e r r e d f r o m medium s u p p o r t i n g a s l o w e r growth r a t e t o one t h a t s u p p o r t s a h i g h e r growth r a t e , t h e G l p hase i s e x t e n d e d and i n i t i a t i o n of DNA s y n t h e s i s i s d e l a y e d ; i f c e l l s a r e t r a n s f e r r e d f r o m r i c h medium t o p o o r medium t h e y w i l l be a d v a n c e d i n t o DNA s y n t h e s i s f o l l o w i n g a s h o r t e r G l . Thus i n y e a s t , t h e t i m i n g o f i n i t i a t i o n o f DNA s y n t h e s i s r e s p o n d s t o t h e a l t e r e d e n v i r o n m e n t i m m e d i a t e l y a f t e r a medium s h i f t , whereas i n Paramecium, t h e r e s p o n s e t o t h e new g rowth c o n d i t i o n i s not shown u n t i l t h e s e c o n d c e l l c y c l e f o l l o w i n g t h e s h i f t . D e s p i t e t h e d i f f e r e n c e s d e s c r i b e d above, c e l l c y c l e r e g u l a t i o n w i t h r e f e r e n c e t o p o i n t s of c o o r d i n a t i o n o f D N A - d i v i s i o n and g r o w t h c y c l e s i n y e a s t s , Paramecium and Tetrahymena i s r e m a r k a b l y s i m i l a r . T h e s e i n c l u d e t h e c e l l s i z e r e l a t e d c o n t r o l a t i n i t i a t i o n of DNA s y n t h e s i s , and t h e growth r e l a t e d c o n t r o l o v e r i n i t i a t i o n o f c e l l d i v i s i o n (i.e. a c e l l s i z e r e q u i r e m e n t i n f i s s i o n y e a s t , t h e amount of DNA, o r p o s s i b l y RNA, made i n Paramecium, and t h e amount o f RNA o r p r o t e i n made i n Tet raft ymena) . In b o t h c a s e s , t h e c o n t r o l mechanism i s s e n s i t i v e t o t h e growth c o n d i t i o n s . A c o n s t a n t , minimum s i z e - r e l a t e d r e q u i r e m e n t e x i s t s when t h e growth r a t e i s -41-below a c e r t a i n l e v e l . Above t h i s growth r a t e , a c r i t i c a l s i z e r e q u i r e m e n t may be n e c e s s a r y f o r t h e c o m p l e t i o n of t h e c e l l c y c l e e v e n t s , b u t t h e t h r e s h o l d v a l u e i t s e l f i s d e p e n d e n t on t h e growth r a t e o f t h e c e l l . O b s e r v a t i o n s on t h e c o n t r o l o v e r i n i t i a t i o n o f c e l l d i v i s i o n s u g g e s t t h a t t h e t h r e s h o l d v a l u e o b s e r v e d f o r growth r a t e h i g h e r t h a n t h e minimum i s j u s t a r e s u l t o f n e t i n c r e a s e i n s i z e p r o p o r t i o n a l t o t h e growth r a t e a f t e r t h e r e q u i r e m e n t i s r e a c h e d . In c o n c l u s i o n , t h i s s t u d y has r e v e a l e d f i v e m a j o r p o i n t s c o n c e r n i n g c e l l c y c l e r e g u l a t i o n i n Paramecium. F i r s t , i n i t i a t i o n o f DNA s y n t h e s i s i s d e t e r m i n e d i n t h e p r e c e d i n g c e l l c y c l e , and i s not a f f e c t e d by t h e r a t e o f growth e x p e r i e n c e d f o l l o w i n g a n u t r i t i o n a l s h i f t a t f i s s i o n . S e c ond, t h e r e i s a m a s s - r e l a t e d c o n t r o l o v e r DNA s y n t h e s i s i n i t i a t i o n , b u t i n c r e a s e i n c e l l mass per se i s not s u f f i c i e n t f o r i n i t i a t i o n of DNA r e p l i c a t i o n . T h i r d , t h e amount o f DNA made r e f l e c t s t h e c u r r e n t n u t r i e n t c o n d i t i o n s , and i s r e l a t e d t o t h e growth r a t e of t h e c e l l . F o u r t h , t h e mechanism f o r i n i t i a t i o n of c e l l d i v i s i o n i s not t i m e - d e p e n d e n t . C e l l d i v i s i o n d o e s not o c c u r a t a f i x e d i n t e r v a l f o l l o w i n g t h e p r e v i o u s f i s s i o n o r t h e i n i t i a t i o n o f DNA s y n t h e s i s . F i f t h , commitment t o c e l l d i v i s i o n o c c u r s a f t e r c e l l s have s y n t h e s i z e d a s t a n d a r d amount (about 41 u n i t s ) of DNA, a f t e r w h i c h d i v i s i o n w i l l p r o c e e d a t t h e s c h e d u l e d t i m e . T h i s p o i n t l i e s 90-100 m i n u t e s b e f o r e -42-f i s s i o n r e g a r d l e s s o f t h e t o t a l c y c l e l e n g t h . T h e r e a r e many q u e s t i o n s w hich r e m a i n unanswered. T h e s e i n c l u d e t h e f o l l o w i n g : What i s ( a r e ) t h e mechanisms w h i c h c o n t r o l i n i t i a t i o n o f DNA s y n t h e s i s and c e l l d i v i s i o n ? What r o l e d o e s t h e c o n c e n t r a t i o n o r t h e a b s o l u t e amount of RNA p l a y i n t h e c o n t r o l of c e l l c y c l e e v e n t s ? Do c e l l s i n s t a t i o n a r y p h a s e c u l t u r e s c o n t i n u e p r o g r e s s i o n t h r o u g h t h e c e l l c y c l e ? I f so, what i s t h e i r minimum DNA c o n t e n t ? How do c e l l s behave a t o t h e r growth r a t e s ? T h e s e a r e q u e s t i o n s t h a t need t o be r e s o l v e d i n o r d e r t o g a i n b e t t e r p e r s p e c t i v e on t h e c o n t r o l p r o c e s s e s i n v o l v e d i n t h e r e g u l a t i o n o f t h e c e l l c y c l e i n Paramecium a s w e l l as i n o t h e r o r g a n i s m s . -43-T a b l e 1. Cell cycle variables of cells foI lowing nutrition a I shift-up. •# Di -- i n i t i a l DNA c o n t e n t P i -- i n i t i a l c e l l mass ( p r o t e i n c o n t e n t ) Df -- f i n a l DNA c o n t e n t Pf -- f i n a l p r o t e i n c o n t e n t d e l t a D — DNA i n c r e m e n t d e l t a P -- p r o t e i n i n c r e m e n t G.T. (exp) -- c e l l c y c l e l e n g t h of e x p e r i m e n t a l c e l l s ( h r ) G.T. — g e n e r a t i o n t i m e (hr) * A l l v a l u e s e x c e p t g e n e r a t i o n t i m e s a r e e x p r e s s e d as p e r c e n t a g e s o f c o n t r o l v a l u e s -44-Table 1. Cell cycle variables of cells /O/ZOK/BJ nutritional shift-up expt. 1 Di Pi Df Pf delta D delta P G.T.(exp) 6.T. (control) 83-37a 66.411.7 28.010.8 165.211.0 146.612.1 98.8*2.0 110.6*2.7 N.D. N.D. 83-37b 56.2*1.6 41.7*1.0 139.8*0.9 152.6*1.4 83.6*184 110.9*1.7 N.D. N.D. 83-41 44.9i3.0 N.D. 126.4*3.2 N.D. 81.4*4.3 N.D. 12.9 N.D. 83-42 72.4*4.2 32.910.9 191.1*3.7 137.8*0.7 117.0*5.6 106.0*1.1 13.3 N.D. 83-43 N.D. 34.3*1.8 N.D. 134.6*2.4 N.D. 100.3*3.0 12.3 6.0 83-44 58.6*3.2 44.6*1.6 144.9*2.5 143.0*2.0 86.3*4.0 98.512.6 12.4 5.0 83-45 56.415.6 40.710.8 162.514.2 106.3410.8 106.117.0 84.311.2 11.4 5.0 83-48 66.413.4 29.711.3 157.213.6 154.813.2 90.814.9 125.H3.5 14.5 5.8 83-49 54.2*1.6 24.6*1.5 140.5*3.0 62.3*2.5 86.313.5 100.012.5 N.D. N.D. 84-3 47.311.7 N.D. 164.414.8 N.D. 117. U5.1 N.D. 15.3 7.0 84-5 48.012.5 32.012.2 132.213.0 116.412.0 84.313.9 83.612.9 13.9 6.5 84-7 65.111.7 N.D. 194.812.1 N.D. 129.7*2.7 N.D. 13.7 N.D. 84-11 43.513.5 N.D. 142.417.8 N.D. 98.9*8.6 N.D. 13.5 N.D. 84-15 55.3H.7 43.5*0.8 N.D. 171.8*0.8 N.D. 128.3*1.1 13.8 6.3 84-16 76.913.4 67.9H.5 196.513.3 198.2*0.9 119.6*4.7 139.3*1.8 11.0 6.3 84-18 88.214.7 52.8*1.3 190.0*3.4 N.D. 101.8*5.8 N.D. 15.9 5.8 84-19 52.6H.6 32.3H.0 133.4*2.9 N.D. 80.8*3.3 N.D. 15.1 6.3 84-25 49.7H.1 39.6*0.8 144.6*2.6 144.2*1.2 94.9*2.7 104.6*1.5 15.0 6.1 NeantSE N 98.614.0 16 107.614.8 12 -45-TABLE 2 Proportion of chemostat cells in S phase under equilibrium conditions. Group # ON tt OFF F r a c t i o n P r o p o r t i o n a l i n S p h a s e D u r a t i o n o-f S p h a s e 1 27 16 2 39 35 3 33 13 4 28 38 5 35 28 O. 63 0. 53 0. 72 0. 42 O. 56 O. 70 0.61 O. 78 O. 51 0. 64 Sample mean 0.57±0.05 O . 6 5 ± 0 . 0 5 # ON number o+ c e l l s l a b e l l e d # OFF number o+ c e l l s n ot l a b e l l e d -46-TABLE 3 /fate of protein synthesis in chemostat culture under equiIibrium conditions. E x p t . P i EXTN P - a c t 83-43 24. 6 ± 1 . 8 8 4 . 8 ± 0 . 02 2 0 . 9 ± 2 . O 83-45 40. 7 ± 0 . 8 8 1 . 5 ± 0 . 03 2 3 . 5 ± 1 . 4 83-48 29. 7 ± 1 . 3 7 2 . 6 ± 0 . 02 17.4 ± 0 . 9 83-49 24. 6±i . 8 8 4 . 8 ± 0 . 02 2 0 . 9 ± 2 . 0 P i — c e l l mass ( p r o t e i n c o n t e n t ) as % o f c o n t r o l v a l u e s P - a c t — c e l l u l a r r a t e o-f p r o t e i n s y n t h e s i s as % o-f c o n t r o l v a l u e s EXTN — s p e c i f i c r a t e o f p r o t e i n s y n t h e s i s as % o f c o n t r o l v a l u e s -47-TABLE 4 Timing of initiation of DNA synthesis following nutritional sh i f t-down. E x p t . ** 84- 34 84-31 Mean t i m e D - f i n i t i a t i o n o-f DNA s y n t h e s i s (Hr) 1. 8 3.5 E x p e r i m e n t a l G.T. (Hr) 6. 3 10. O C o n t r o l G.T. (Hr) 5. 7 6.2 -48-TABLE 5 Mean cell mass of cells synthesizing DNA CON'} v s . those that are not < 'OFF'> . t i m e (Hr) mean p r o t e i n c o n t e n t * F r a c t i o n of c o n t r o l O 140±i.6 1. 00 2 ON 150±2.0 1.07 2 OFF 143±2.O 1. 02 2.5 ON 144±2.4 1. 03 2.5 OFF 136±3.O 0.97 3 ON 153±4.0 1. 09 3 OFF 144±2.0 1 . 03 3.5 ON 148 ± 2 . 7 1. 06 3.5 OFF 146±2.6 1. 04 • P r o t e i n c o n t e n t s a r e e x p r e s s e d i n a r b i t r a r y u n i t s . -49-TABLE 6 Cell cycle x/arishies following nutritional shift to exhausted mediurn. Di 1 0 0 . 4 ± 3 . 8 P i 1 0 0 . 4 ± 4 . 2 D-f 1 5 9 . 1 ± 3 . 2 Pf 1 3 3 . 8 ± 1 . 6 ^D 5 9 . 1 ± 5 . 0 A P 3 3 . 8 ± 1 . 5 G.T. (Hr) 6.3 Cont.ro 1 G.T. (Hr) 5.7 Di and P i a r e i n i t i a l DNA c o n t e n t and c e l l mass r e s p e c t i v e l y ; D-f and P-f a r e - f i n a l DNA c o n t e n t and c e l l mass r e s p e c t i v e l y ; A d and ^ P a r e DNA and P r o t e i n i n c r e m e n t i n one c e l l c y c l e r e s p e c t i v e l y ; and G.T. d e n o t e s g e n e r a t i o n t i m e i n h o u r s . A l l v a l u e s a r e a v e r a g e d f r o m a sample of 35 t o 40 c e l l s . -50-TABLE 7 Effects of change in growth rate on the duration of S phase. E x p t . D u r a t i o n of S* P r o p o r t i o n o f S G e n e r a t i o n t i m e * Cont roI 1 3.62 0.62 6.0 Chemostat culture 2 18.8 0.65 28.9 Nutritional shift-up 3 4.78 0.37 13.O 4 4.33 0.31 13.9 Nutritional shift-down 5 3.46 0.55 6.33 6 6.88 0.69 10.O • G e n e r a t i o n t i m e s and d u r a t i o n o f S a r e e x p r e s s e d i n h o u r s . -51-TABLE 8 Effect of heat t reatment starting at 0. 75 in the cell cycle on macranuclear DNA content at the subsequent fission.* C o n t r o l c e l i s E x p e r i m e n t a l e e l I s E x p e r i -m e n t a l a s % o f c o n t r o l E x p t . Mean DNA N Mean DNA N & D 1 6 8 . 4 ± 1 . 4 9 40 4 8 . 4 ± 1 . 8 3 22 71 42 2 5 6 . 7 ± 1 . 9 2 25 4 3 . 1 ± 1 . 8 3 34 76 52 3 15 7 . 7 ± 1 . 1 3 31 1 1 4 . 8 ± 2 . 7 5 52 73 46 4 12 6 . 6 ± 2 . 9 6 39 9 8 . 3 ± 3 . 2 8 36 78 56 Samp 1e mean 49±3.1 •X-DNA c o n t e n t s a r e e x p r e s s e d i n a r b i t r a r y u n i t s , d e l t a D -- i n c r e a s e i n DNA c o n t e n t . -52-TABLE 9 Mean DNA contents in chemostat culture under equilibrium cond i tions. E x p t . Mean DNA Flow r a t e (rol/hr) G e n e r a t i o n t i m e (Hr) 84-3 47.3*1.67 15.O 33.3 84-5 47.9*2.5 16.0 31.3 84-7 65.1*1.68 15.0 33.3 84-11 43.5*3.54 18.5 27.0 84-15 55.3*1.66 17.0 29.4 84-19 52.6*1.55 18.O 27.8 84-25 49.7*1.08 17.O 29.4 Sample mean 51.6*2.7 30.2*1.0 Minimum G e n e r a t i o n Time ( c o n t r o l ) 5.5 Hr. Mean growth r a t e a s a p e r c e n t a g e o-f c o n t r o l 18% -53-F i g u r e 1 (A & B ) . Changes i n c e l l u l a r DNA and p r o t e i n c o n t e n t w i t h t i m e i n a c h e m o s t a t c u l t u r e . < (^) ) = p r o t e i n c o n t e n t ; < ) = DNA c o n t e n t . (O ) = Flow r a t e i n m l / h r . DNA and p r o t e i n c o n t e n t a r e e x p r e s s e d as a p e r c e n t a g e o-f t h e i r i n i t i a l v a l u e s . - 5 4 -Percentage of initial level [%) cn o o o J U ~ o o O o o o * m> t o mi a o >-^ M T| O "0 3 = O H«M D O MtM TT O O ro Flow rate(mUhr) DD Percentage of initial level (%) o h ° 3 N) O O o o o CJI O _ 1 _ p tot O o O O o o o n b a t - k n D i ha* o m n •n • u f 5 I -I 3 O ro O O Flow rate(ml/hr) F i g u r e 2. K i n e t i c s of -food v a c u o l e s l o s t ( e x p r e s s e d as p e r c e n t a g e of t h e i n i t i a l v a l u e ) w i t h t i m e i n c e l l s t r a n s f e r r e d t o e x h a u s t e d medium. -56-Time (min.) - s i -F i g u r e 3. Changes i n p r o t e i n c o n t e n t ( e x p r e s s e d as p e r c e n t a g e of t h e i n i t i a l v a l u e ) w i t h t i m e i n c e l l s t r a n s f e r r e d t o e x h a u s t e d medium. -58-C 0) c o o c CD -•—• o 100 H c o 0J E 80 —1 1 20 Time (min.) 40 F i g u r e 4. K i n e t i c s o f i n i t i a t i o n o f DNA s y n t h e s i s f o l l o w i n g n u t r i t i o n a l s h i f t - u p . A r r o w s i n d i c a t e t h e mean t i m e o f DNA s y n t h e s i s f o r e a c h of t h e two e x p e r i m e n t a l g r o u p s ( O & • ). <D> = C o n t r o l c e l l s ; d a t a o b t a i n e d f r o m Rasmussen ic B e r g e r , 1982. -60-cells with labelled macronuclei (%) F i g u r e 5. Changes i n c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s w e l l - f e d c e l l s . P r o t e i n s y n t h e s i s a c t i v i t y i s e x p r e s s e d as p e r c e n t a g e of t h e p o s t - f i s s i o n v a l u e . -62-F i g u r e 6. Changes i n c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s 0-4 h o u r s f o l l o w i n g n u t r i t i o n a l s h i f t - u p . P r o t e i n s y n t h e s i s a c t i v i t y i s e x p r e s s e d a s a p e r c e n t a g e o f p o s t - f i s s i o n c o n t r o l v a l u e s . -64-F i g u r e 7. Changes i n c e l l u l a r r a t e o f p r o t e i n s y n t h e s i s 4-8 h o u r s f o l l o w i n g n u t r i t i o n a l s h i f t - u p . P r o t e i n s y n t h e s i s a c t i v i t y i s e x p r e s s e d as a p e r c e n t a g e o f p o s t - f i s s i o n c o n t r o l v a l u e s . -66-Protein synthesis activity (%) i .ON 3 CO CD oo H a a i d 101 a F i g u r e 8. Changes i n s p e c i f i c r a t e o f p r o t e i n s y n t h e s i s 0-3 h o u r s f o l l o w i n g n u t r i t i o n a l s h i f t - u p . (• ) = c o n t r o l c e l l s ; <0 ) = e x p e r i m e n t a l c e l l s . S p e c i f i c p r o t e i n s y n t h e s i s r a t e i s e x p r e s s e d as a p e r c e n t a g e o f p o s t - f i s s i o n c o n t r o l v a l u e s . -68-100 H CD o CD -•—» c c cu "o I— C L O CD CL 80 -60 -i 0 I 1 ' ' ' i o experimental • control cells Time (Hr.) F i g u r e 9. Changes i n s p e c i f i c p r o t e i n s y n t h e s i s r a t e ( e x p r e s s e d as p e r c e n t a g e o f p o s t - f i s s i o n c o n t r o l v a l u e s ) 4-8 h o u r s f o l l o w i n g n u t r i t i o n a l s h i f t - u p . -70-4^ i H 3 CD 0 0 Specific protein synthesis rate (%) OD o o o w lot 101 a KM KM 101 Id lot F i g u r e 10. K i n e t i c s o f i n i t i a t i o n o f DNA s y n t h e s i s f o l l o w i n g t r a n s f e r t o e x h a u s t e d medium. A r r o w s i n d i c a t e t h e t i m e DNA s y n t h e s i s f i r s t b e g i n s i n e a c h o f t h e e x p e r i m e n t . <Q) = c o n t r o l c e l l s ; d a t a o b t a i n e d f r o m Rasmussen & B e r g e r , 1982. -72-cells with labelled macronuclei (%) BIBLIOGRAPHY, B a z i n , M.J. 1981. T h e o r y o f c o n t i n u o u s c u l t u r e . In C o n t i n o u j c u l t u r e o f c e l l s . P.H. C a l c o t t . ed. CRC P r e s s . F l o r i d a . USA. V o l . 1 , 27-62. B e e r s , Y. 1953. I n t r o d u c t i o n t o t h e t h e o r y o f e r r o r . A d dison-Wes1ey. Cambridge, Mass. B e r g e r , J.D. 1971. K i n e t i c s o f i n c o r p o r a t i o n of DNA p r e c u r s o r s f r o m i n g e s t e d b a c t e r i a i n t o m a c r o n u c l e a r DNA o f Paramecium sure/ia. J . P r o t o z o o l . 18, 419-429. B e r g e r , J.D. 1973. N u c l e a r d i f f e r e n t i a t i o n and n u c l e i c a c i d s y n t h e s i s i n w e l l - f e d e x c o n j u g a n t s of Paramecium aurelia. Chromosoma. 42, 247-68. B e r g e r , J.D. 1974. S e l e c t i v e a u t o l y s i s o f n u c l e i a s a SQurc* of DNA p r e c u r s o r s i n Paramecium aurelia e x c o n j u g a n t s . J . P r o t o z o o l . 21, 145-152. B e r g e r , J.D. 1978. Downward r e g u l a t i o n o f m a c r o n u c l e a r DNA c o n t e n t i n Paramecium tetraurelia: The e f f e c t s of e x c e s s m a c r o n u c l e a r DNA c o n t e n t on s u b s e q u e n t DNA and p r o t e i n c o n t e n t and t h e c e l l c y c l e . Exp. C e l l Res. 114, 253-261. B e r g e r , J.D. 1979. R e g u l a t i o n o f m a c r o n u c l e a r DNA c o n t e n t i n Paramecium tetraurelia. J . P r o t o z o o l . 26, 18-28. B e r g e r , J.D. 1982a. E f f e c t s o f gene d o s a g e on p r o t e i n s y n t h e s i s r a t e i n Paramecium tetraurelia: I m p l i c a t i o n s f o r r e g u l a t i o n of c e l l mass, DNA c o n t e n t and t h e c e l l c y c l e Exp. C e l l Res. 142, 261-275. B e r g e r , J.D. 1982b. I n i t i a t i o n and t e r m i n a t i o n o f m a c r o n u c l e a r DNA s y n t h e s i s i n Paramecium: E f f e c t o f v a r i a t i o n i n m a c r o n u c l e a r DNA c o n t e n t . Can. J . Z o o l . 60, 2501-2506. B e r g e r , J.D. 1984. The c i l i a t e c e l l c y c l e . In: The M i c r o b i a l c e l l c y c l e . P. N u r s e and E. S t r e i b l o v a e d s . CRC P r e s s . B o c a R a t o n , F l a . pp. 191-208. B e r g e r , J.D. and H.J. S c h m i d t . 1978. R e g u l a t i o n o f m a c r o n u c l e a r DNA c o n t e n t i n Paramecium tetraureIia. J . C e l l B i o l . 76, 116-126. C a r t e r , B.L.A. 1981. The c o n t r o l o f c e l l d i v i s i o n i n -74-Saccharomyces cerevisiae. In The c e l l c y c l e . P.C.L. J o h n . ed. Cambridge U n i v e r s i t y P r e s s n , New York, p.99-118. C l e f f m a n n , G. 1968. R e g u l i e r u n g d e r DNS-Menge im M a k r o n u k l e u s von Tetrahymena. E x p t . C e l l Res. 50, 193-207. C l e f f m a n n , G. , W.-0. R e u t h e r and H.-M. S e y f e r t . 1979. I n c r e a s e i n macromo1ecu1ar amounts d u r i n g t h e c e l l c y c l e o f Tetrahymena: A c o n t r i b u t i o n t o c e l l c y c l e c o n t r o l . J . C e l l S c i . 37, 117-124. Cohen, J . and J . B e i s s o n . 1980. G e n e t i c a n a l y s i s of t h e r e l a t i o n s h i p s between t h e c e l l s u r f a c e and t h e n u c l e i i n Paramecium tetraureIia. G e n e t i c s . 95, 797-818. C o r n e l i s s e , C . J . and J . S . Ploem. 1976. A new t y p e o f t w o - c o l o r f l u o r e s c e n c e s t a i n i n g f o r c y t o l o g y s p e c i m e n s . J . H i s t o c h e m . Cytochem. 24, 72-81. D o e r d e r , F.P. 1979. R e g u l a t i o n o f m a c r o n u c l e a r DNA c o n t e n t i n Tetrahymena thermophila. 3. P r o t o z o o l . 26, 28-35. D o e r d e r , F.P. and L . E . D e B a u l t . 1975. C y t o f 1 u o r i m e t r i c a n a l y s i s o f n u c l e a r DNA d u r i n g m e i o s i s , f e r t i l i z a t i o n and m a c r o n u c l e a r d e v e l o p m e n t i n t h e c i l i a t e Tetrahymena pyriformis, s y n g e n 1. J . C e l l S c i . 17, 471. D o e r d e r , F.P. and L . E . D e B a u l t . 1978. L i f e c y c l e v a r i a t i o n and r e g u l a t i o n o f m a c r o n u c l e a r DNA c o n t e n t i n Tetrahymena thermophila. Chromosoma. 69, 1-19. D o e r d e r , F.P., J . D i t a r a n t o and L . E . D e B a u l t . 1981. E v o l u t i o n a r y c o n s t r a i n t s on q u a n t i t a t i v e v a r i a t i o n and r e g u l a t i o n of m a c r o n u c l e a r DNA c o n t e n t i n t h e genus Tetrahymena. 3. C e l l S c i . 49, 177-193. D o e r d e r , F.P., J . F r a n k e l , L.M. J e n k i n s and L . E . D e B a u l t . 1975. Form and p a t t e r n i n c i l i a t e d p r o t o z o a : A n a l y s i s o f a g e n i e mutant w i t h a l t e r e d c e l l s h a p e i n Tetrahymena pyriformis, s y n g e n 1. J . Exp. Z o o l . 192, 237-258. D o e r d e r , F.P., J.H. L i e f and L . E . D e B a u l t . 1977. M a c r o n u c l e a r s u b u n i t s o f Tetrahymena thermophila a r e f u n c t i o n a l l y h a p l o i d . S c i e n c e . 198, 946-948. D r e i s i g , H., A n d r e a s e n , " P . H . and K. K r i s t i a n s e n . 1984. R e g u l a t i o n o f r i b o s o m e s y n t h e s i s i n Tetrahymena pyriformis. 2. C o o r d i n a t i o n of s y n t h e s i s of r i b o s o m a l p r o t e i n s and r i b o s o m a l RNA d u r i n g n u t r i t i o n a l s h i f t up. E u r . J . Biochem. 140, 477-483. -75-F a n t e s , P., and P. N u r s e . 1977. C o n t r o l o f c e l l s i z e a t d i v i s i o n i n f i s s i o n - y e a s t by a g r o w t h - m o d u l a t e d c o n t r o l o v e r n u c l e a r d i v i s i o n . Exp. C e l l Res. 107,377-380. F a n t e s , P.A., W.D. G r a n t , R.H. P r i t c h a r d , P.E. S u d b e r y and A.E. Wheals. 1975. The r e g u l a t i o n of c e l l s i z e and t h e c o n t r o l o f m i t o s i s . J . T h e o r . B i o l . 50, 213-244. F r a z i e r , E . A . J . 1973. DNA s y n t h e s i s f o l l o w i n g g r o s s a l t e r a t i o n s o f t h e n u c 1 e o c y t o p 1asmic r a t i o i n t h e c i l i a t e Stentor coeruleus. Dev. B i o l . 34, 77-92. H a r t w e l l , L.H. and M.W. Unger. 1977. U n e q u a l d i v i s i o n i n Saccharomyces cerex/isiae and i t s i m p l i c a t i o n s f o r t h e c o n t r o l o f c e l l d i v i s i o n . J . C e l l . B i o l . 75, 422-435. H o w e l l , S.H., W.J. B l a s h k o and C M . Drew. 1975. I n h i b i t o r e f f e c t s d u r i n g t h e c e l l c y c l e i n Chlamydomonas r-einhardt i i. J . C e l l B i o l . 67, 126-135. J a g a d i s h , M.N. and B.L.A. C a r t e r . 1977. G e n e t i c c o n t r o l o f c e l l d i v i s i o n i n y e a s t c u l t u r e s a t d i f f e r e n t g r o w t h r a t e s . N a t u r e <Lond.) 269, 145-147. J a u k e r , F. 1975. A f e e d b a c k c o n t r o l o f c e l l c y c l e p a r a m e t e r s i n Tetrahymena. J . C e l l B i o l . 67, 901-904. J a u k e r , F. 1983. A popu1 a t i o n - k i n e t i c a l a p p r o a c h t o RNA f o r m a t i o n and d e g r a d a t i o n i n g r o w i n g and r e s t i n g c e l l s . E x p t . C e l l Res. 143, 163-174. J o h n s t o n , G . C and R.A. S i n g e r . 1983. Growth and t h e c e l l c y c l e of t h e y e a s t Saccharomyces cerevisiae. I. S l o w i n g S p h a s e o r n u c l e a r d i v i s i o n d e c r e a s e s t h e G l c e l l c y c l e p e r i o d . Exp. C e l l Res. 149, 1-14. J o h n s t o n , G.C. and R.A. S i n g e r . 1985. N o v e l c e l l c y c l e r e g u l a t i o n i n t h e y e a s t Sch i zosaccharomyces pomJbei The D N A - d i v i s i o n s e q u e n c e m o d u l a t e s mass a c c u m u l a t i o n . E x p t . C e l l Res. 158, 544-553 J o h n s t o n , G . C , C.W. E r h a r d t , A. L o r i n c z and B.L.A. C a r t e r . 1979. R e g u l a t i o n o f c e l l s i z e i n t h e yeast Saccharomyces cerevisiae. J . B a c t e r i o l . 137, 1-5. J o h n s t o n , G . C , J.R. P r i n g l e and L.H. H a r t w e l l . 1977. C o o r d i n a t i o n o f g r o w t h w i t h c e l l d i v i s i o n i n t h e y e a s t Saccharomyces cerevisiae. Exp. C e l l Res. 105, 79-98. J o n e s , D. and J.D. B e r g e r . 1982. T e m p e r a t u r e - s e n s i t i v e -76-t m u t a t i o n s a f f e c t i n g c o r t i c a l m o r p h o g e n e s i s and c e l l d i v i s i o n i n Paramecium tetraurelia. Can. J . Z o o l . 60, 2296-2308. K i l l a n d e r , D. and A. Z e t t e r b e r g . 1965. Q u a n t i t a t i v e c y t o c h e m i c a l s t u d i e s on i n t e r p h a s e g r o w t h . I. D e t e r m i n a t i o n of DNA, RNA and mass c o n t e n t o f age d e t e r m i n e d mouse f i b r o b l a s t s in vitro and o f i n t e r c e l l u l a r v a r i a t i o n i n g e n e r a t i o n t i m e . E x p t . C e l l Res. 38, 272-284. K i m b a l l , R.F. 1967. P e r s i s t e n t i n t r a c l o n a l v a r i a t i o n i n c e l l d r y mass and DNA c o n t e n t i n Paramecium aurelia. Exp. C e l l Res. 48, 378-394. K i m b a l l , R.F., T.O. C a s p e r s s o n , G. S v e n s s o n and L. C a r l s o n . 1959. Q u a n t i t a t i v e c y t o c h e m i c a l s t u d i e s on Parmecium aurelia I. Growth i n t o t a l d r y w e i g h t measured by s c a n n i n g i n t e r f e r e n c e m i c r o s c o p e and X - r a y a b s o r p t i o n methods. Exp. C e l l Res. 17, 160-172. K i m b a l l , R.F., S.W. P e r d u e , E.H. Chu and J.R. O r t i z . 1971. M i c r o p h o t o m e t r i c and a u t o r a d i o g r a p h i c s t u d i e s on t h e c e l l c y c l e and c e l l s i z e d u r i n g growth and d e c l i n e of C h i n e s e hamster c u l t u r e s . E x p t . C e l l Res. 66, 17-32. L o r i n c z , A. and B.L.A. C a r t e r . 1979. C o n t r o l o f c e l l s i z e a t bud i n i t i a t i o n i n Saccharomyces cerevisiae. 3. Gen. M i c r o b i o l . 113, 287-295. L o r d , P.G. and A.E. Wheals. 1983. R a t e o f c e l l c y c l e i n i t i a t i o n of y e a s t c e l l s when c e l l s i z e i s n o t a r a t e - d e t e r m i n i n g f a c t o r . 3. C e l l S c i . 59, 183-201. M i t c h i s o n , J.M. 1970. The B i o l o g y of t h e C e l l C y c l e . C a m bridge U n i v e r s i t y P r e s s . C ambridge. 313 pp. Nachtwey, D.S. and I . L . Cameron, 1968. C e l l c y c l e A n a l y s i s . In Methods i n c e l l p h y s i o l o g y . D.M. P r e s c o t t , ed. A cademic P r e s s , N.Y. V o l . 3 . 213-259. Nasmyth, K. 1979. A c o n t r o l a c t i n g o v e r i n i t i a t i o n o f DNA r e p l i c a t i o n i n t h e y e a s t Sch i xosaccharomyces pomt>e. 3. C e l l S c i . 36, 155-168. N e l s e n , E.M., J . F r a n k e l and E. M a r t e l . 1981. Development of t h e C i l i a t u r e o f Tetrahymena thermophila I. Temporal c o o r d i n a t i o n w i t h o r a l d e v e l o p m e n t . D e v e l o p . B i o l . 88, 27- 38. N u r s e , P. 1975. G e n e t i c c o n t r o l of c e l l s i z e a t c e l l d i v i s i o n -77-i n y e a s t . N a t u r e (Lond.) 256, 547-551. N u r s e , P. and P. T h u r i a u x . 1977. C o n t r o l s o v e r t h e t i m i n g of DNA r e p l i c a t i o n d u r i n g t h e c e l l c y c l e o f - f i s s i o n y e a s t . E x p t . C e l l Res. 107, 365-375. O r i a s , E. and M. F l a c k s . 1975. M a c r o n u c l e a r g e n e t i c s o f Tetrahymena I. Random d i s t r i b u t i o n o f m a c r o n u c l e a r gene c o p i e s i n Tetrahymena pyriformis, s y n g e n 1. G e n e t i c s . 79, 187-206. P a r d e e , A.B., R. Dubrow, J . L . H a m l i n and R.F. K l e t z i e n . 1978. A n i m a l c e l l c y c l e . Annu. Rev. Biochem. 47, 715-750. P e t e r s o n , E.L. and J.D. B e r g e r . 1976. M u t a t i o n a l b l o c k a g e of DNA s y n t h e s i s i n Paramecium tetraure1ia. Can. J . Z o o l . 54, 2089-2097. R a i k o v , I.B. 1970. The m a c r o n u c l e u s o f c i l i a t e s . In: R e s e a r c h i n P r o t o z o o l o g y I I I . T.T. Chen ed. Pergamon P r e s s . New Y o r k . pp. 1-128. R a i k o v , I.B. 1982. The p r o t o z o a n n u c l e u s . 2nd e d i t i o n . S p r i n g e r V e r l a g . Rasmussen, C D . 1984. S t u d i e s on t h e c e l l c y c l e i n Paramecium tetraurelia. M.Sc. t h e s i s . U n i v e r s i t y of B r i t i s h C o l u m b i a . Rasmussen, C D . and J.D. B e r g e r . 1982. Downward r e g u l a t i o n o f c e l l s i z e i n Paramecium tetraurelia: E f f e c t s o f i n c r e a s e d c e l l s i z e , w i t h o r w i t h o u t i n c r e a s e d DNA c o n e t n t , on t h e c e l l c y c l e . J . C e l l S c i . 57,315-329. Rasmussen, C D . and J.D. B e r g e r . 1984. A gene f u n c t i o n r e q u i r e d f o r c e l l c y c l e p r o g r e s s i o n d u r i n g t h e G l p o r t i o n of t h e c e l l c y c l e and f o r m a i n t e n a n c e o f m a c r o n u c l e a r DNA s y n t h e s i s i n Paramecium tet raureIi a. E x p t . C e l l Res. 155, 593-597. Rasmussen, C D . and J.D. B e r g e r . 1986. E f f e c t s o f i n c r e a s e d c e l l mass and a l t e r e d gene dosage on t h e t i m i n g of m a c r o n u c l e a r DNA s y n t h e s i s i n Paramecium tetraureIia: I m p l i c a t i o n s f o r c e l l c y c l e r e g u l a t i o n . i n p r e s s . Rasmussen, C D . , A.S.L. C h i n g and J.D. B e r g e r . 1985. The f u l l s c h e d u l e o f m a c r o n u c l e a r DNA s y n t h e s i s i s not r e q u i r e d f o r c e l l d i v i s i o n i n Paramecium tetraurelia. J . P r o t o z o o l . 32, 366-368. -78-R i v i n C . J . and W.L. Fangman. 1980. C e l l c y c l e p h a s e e x p a n s i o n i n n i t r o g e n - 1 i m i t e d c u l t u r e s o f Saccharomyces cerevisiae. J . C e l l B i o l . 85, 96-107. R e u t h e r , W.-0., G. C l e f f m a n n and F . J . J a u k e r . 1980. R e a c t i v a t i o n of s t a t i o n a r y Tetrahymena'. A c o n t r i b u t i o n t o t h e q u e s t i o n o f GO s t a t e . Exp. C e l l Res. 126, 343-349. R u i z , F., A. A d o u t t e , M. R o s s i g n o l , J . B e i s s o n . 1976. G e n e t i c a n a l y s i s of m o r p h o g e n e t i c p r o c e s s e s i n Paramecium. I. A m u t a t i o n a f f e c t i n g t r i c h o c y s t f o r m a t i o n and n u c l e a r d i v i s i o n . G e n et. Res. 27, 109-122. S c h a e f e r , E. and G. C l e f f m a n n . 1982. D i v i s i o n and g r o w t h k i n e t i c s o f t h e d i v i s i o n mutant conical o f Tetrahymena. Exp. C e l l Res. 137, 277-286. S e y f e r t , H.-M. 1977. A s h o r t G l p e r i o d i s c o r r e l a t e d w i t h low m a c r o n u c l e a r DNA c o n t e n t i n Tetrahymena. Exp. C e l l Res. 108, 456-459. S e y f e r t , H-M. 1980. Changes of DNA, RNA and p r o t e i n c o n t e n t s of Tetrahymena m a c r o n u c l e i t h r o u g h o u t t h e c e l l c y c l e : A c y t o p h o t o m e t r i c i n v e s t i g a t i o n . M i c r o s c o p i c a A c t a ( s u p p l 4 ) , 26-30. S i n g e r , R.A. and G.C. J o h n s t o n . 1983. Growth and t h e c e l l c y c l e of t h e y e a s t Saccharomyces cerevisiae I I . R e l i e f o f c e l l c y c l e c o n s t r a i n t s a l l o w s a c c e l e r a t e d c e l l d i v i s i o n . Exp. C e l l Res. 149, 15-26. S i n g e r , R.A. and G.C. J o h n s t o n . 1985. I n d i r e c t s u p p r e s s i o n of t h e weel mutant p h e n o t y p e i n Schizosaccharomyces pombe. 158, 533-543. S o k a l , R.R. and F . J . R o h l f . 1969. B i o m e t r y . Freeman, San F r a n c i s c o . S o n n e b o r n , T.M. 1940. The r e l a t i o n o f m a c r o n u c l e a r r e g e n e r a t i o n t o m a c r o n u c l e a r s t r u c t u r e , a m i t o s i s and g e n e t i c d e t e r m i n a t i o n . A n a t . Rec. 78 ( s u p p ) , 53-54. S o n n e b o r n , T.M. 1954. Gene c o n t r o l l e d a b e r r a n t n u c l e a r b e h a v i o u r i n Paramecium aurelia. M i c r o . G e net. B u l l . 11, 24-25. S o n n e b o r n , T.M. 1963. Does p r e f o r m e d c e l l s t r u c t u r e p l a y an e s s e n t i a l r o l e i n c e l l h e r e d i t y ? . In :The N a t u r e o f B i o l o g i c a l D i v e r s i t y . J.M. A l l e n ed. M c G r a w - H i l l Co. New Y o r k . pp. 165-211. -79-S o n n e b o r n , T.M. 1970. Methods i n Paramecium R e s e a r c h . JwrMethods i n C e l l P h y s i o l o g y . D.M. P r e s c o t t ed. 4, 241-339. Academic P r e s s . New Y o r k . S o n n e b o r n , T.M. 1975. The Paramecium aurelia complex o-f -fourteen s i b l i n g s p e c i e s . T r a n s Amer. m i c r o s c . S o c . 94, 155-178. S t i l e s , C D . , B.H. C o c h r a n and C D . S c h e r . 1981. R e g u l a t i o n o-f mammalian c e l l c y c l e by hormones. In The c e l l c y c l e . P . C L . J o h n . ed. Cambridge U n i v e r s i t y P r e s s . N.Y. P.119-138. S u h r - J e s s e n , P.B., J.M. S t e w a r t and L. Rasmussen. 1977. T i m i n g and r e g u l a t i o n o-f n u c l e a r and c o r t i c a l e v e n t s i n t h e c e l l c y c l e o f Tetrahymena pyriformis. J . P r o t o z o o l . 24, 299-303. T u c k e r , J.B., J . B e i s s o n and D . L . J . Roche. 1980. M i c r o t u b u l e s and c o n t r o l o-f m a c r o n u c l e a r ' a m i t o s i s ' i n Paramecium. J . C e l l S c i . 44, 135-151. Worthington„ D.H., M. Salamone and D.S. Nachtwey. 1976. Nuc 1 e o c y t o p 1 asmi c r a t i o r e q u i r e m e n t s -for t h e i n i t i a t i o n o-f DNA r e p l i c a t i o n and f i s s i o n i n Tetrahymena. C e l l T i s s u e K i n e t . 9, 119-130. Y a n i s h e w s k y , R.M. and G.H. S t e i n . 1981. R e g u l a t i o n o f t h e c e l l c y c l e i n e u k a r y o t i c c e l l s . I n t . Review. C y t o l . 69, 223-258. -80-

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096054/manifest

Comment

Related Items