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Temperature-sensitive mutations affecting DNA synthesis in Paramecium aurelia Peterson, Eric Lane 1974

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TEMPERATURE-SENSITIVE MUTATIONS AFFECTING DNA SYNTHESIS IN PARAMECIUM AURELIA by ERIC LANE PETERSON B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Genetics We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1974 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 m e n t of the r e -quirements f o r an advanced degree a t The U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the 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 study. I f u r t h e r agree 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 copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Depart-me'nt or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t • copying or 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 allowed without my w r i t t e n p e r m i s s i o n . Department of Genetics The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada V6T 1W5 Date ~ 7 y ] ./ ABSTRACT i 198 t e m p e r a t u r e - s e n s i t i v e mutants of Paramecium a u r e l i a were i s o l a t e d f o l l o w i n g n i t r o s o g u a n i d i n e mutagenesis. In some experiments, mutants were recovered w i t h the a i d of a bromouracil (BU) s e l e c t i o n system developed by Baumann (1973). 56 mutants showed c e s s a t i o n of c e l l d i v i s i o n w i t h i n one c e l l c y c l e a t the r e s t r i c t i v e temperature and were d e s i g n a t e d t s - 0 . 14 of the ts-O's showed a g r e a t e r than 90% r e d u c t i o n i n mac-r o n u c l e a r DNA s y n t h e s i s a t the r e s t r i c t i v e temperature. Two ts - 0 , DNA-defective l i n e s continued p r o t e i n s y n t h e s i s a t g r e a t e r than 5 0% the normal r a t e f o l l o w i n g a r r e s t of DNA syn-t h e s i s . Hence, these two mutants may be d i r e c t l y a f f e c t e d i n the r e p l i c a t i o n process i t s e l f . The two mutants are a l -l e l i c and, i n a d d i t i o n , a t h i r d "leaky" a l l e l e was r e c o v e r e d . Comparison of experiments i n which e i t h e r BU s e l e c t i o n or no s e l e c t i o n was employed shows t h a t a g r e a t e r than t e n - f o l d enrichment f o r t s mutants r e s u l t e d form BU s e l e c t i o n . i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES i v LIST OF FIGURES W ACKNOWLEDGEMENT v v i INTRODUCTION . 1 MATERIALS AND METHODS 11 RESULTS . • . IV DISCUSSION 46 BIBLIOGRAPHY 54 i i i LIST OF TABLES Table Page I. V e g e t a t i v e S u r v i v a l d u r i n g S e l e c t i o n 18 I I . V e g e t a t i v e and Exautogamous V i a b i l i t y d u r i n g S e l e c t i o n 23 I I I . V e g e t a t i v e and Exautogamous V i a b i l i t y f o l l o w -i n g Mutagenesis 24 IV. Mutant Y i e l d s of BU S e l e c t i o n Groups 26 V. Mutant Y i e l d s of Non-selected Groups 27 VI. Exautogamous Seg r e g a t i o n of t s Heterozygotes.. 29 3 V I I . H-thymine I n c o r p o r a t i o n of t s Mutants 39 3 V I I I . H-leucine I n c o r p o r a t i o n of t s Mutants 42 IX. Comparison of BU Selectednand Non-selected Group Y i e l d s 47 C'ioup Y l e i d o i v LIST OF FIGURES F i g u r e Page 1. Nuclear R e o r g a n i z a t i o n i n P. a u r e l i a 4 2. O r g a n i z a t i o n of the Nuclear Events i n the C e l l C y c l e i n P. a u r e l i a 6 3. V i a b i l i t y d u r i n g S e l e c t i o n 20 4. Complementation M a t r i x f o r 4X S e l e c t i o n Group 3 2 5. Complementation M a t r i x f o r 4Y S e l e c t i o n Group 34 6. Complementation M a t r i x f o r t s - f a t Mutants.... 36 7. T r a n s i t i o n P o i n t s of t s - f a t Mutants 44 v ACKNOWLEDGEMENT I wish to thank Dr. James D. Berger for his advice, assistance and encouragement during the course of t h i s work. I also thank my associates, Don Jones, Wayne Kale. Glenn Morton and Phyl Riseborough. Special thanks go to Adele Hunter for her help with experiment 6 and to my relentless collegue Paul Baumann who developed the BU selec-tion system. v i 1 INTRODUCTION The i s o l a t i o n and a n a l y s i s of c o n d i t i o n a l temperature-s e n s i t i v e (ts_) mutants i s a powerful approach toward the understanding of the o r g a n i z a t i o n of the c e l l c y c l e . P a r t i -c u l a r l y s i g n i f i c a n t are c e l l c y c l e mutants, those mutants which are c o n d i t i o n a l l y d e f e c t i v e i n gene f u n c t i o n s r e q u i r e d a t s p e c i f i c stages of the c e l l c y c l e . H a r t w e l l (1974) and c6-workers, u s i n g t s c e l l c y c l e mutants, have g e n e t i c a l l y dissected the c e l l c y c l e of the y e a s t Saccharomyces c e r e v i s i a e , i d e n t i f y i n g 32 n u c l e a r genes r e s p o n s i b l e f o r s t a g e - s p e c i f i c f u n c t i o n s . S i m i l a r s t r a t e g i e s have been employed, though as y e t w i t h l e s s success, i n such d i v e r s e eukaryotes as U s t i l a g o maydis (Unrau and H o l l i d a y , 197 0) , Tetrahymena p y r i -formis ( F r a n k e l , u n p u b l i s h e d ) , Physarum polycephalum (Haugli and Dove, 1972) and mammalian c e l l s i n c u l t u r e ( L i ^ k a y , 1974; Roscoe, e t a l , , 1973a; 1973b; Wang, 1974). In a d d i t i o n , ^ e x t e n s i v e s t u d i e s have been conducted w i t h comparable pro-k a r y o t i c mutants (reviewed, S l a t e r and Schaechter, 1974) . An obvious s t a g e - s p e c i f i c process i n most c e l l c y c l e s i s the p e r i o d of n u c l e a r DNA r e p l i c a t i o n . Temperature-s e n s i t i v e mutants a f f e c t e d i n DNA r e p l i c a t i o n have been r e -covered i n y e a s t ( H a r t w e l l , 1971), U s t i l a g o (Unrau and H o l l i -day, 1970; Jeggo, e t al_. , 1973) mammalian c e l l s ( L i s k a y , 1974; Smith and Wigglesworth, 1973; 1974) and v a r i o u s b a c t e r i a l 2 s p e c i e s (Gross, e t a_l. , 1968; S p r a t t and Rowbury, 1971; Gross, 1972) . In a l l systems examined c r i t i c a l l y i t has been found p o s s i b l e to uncouple c e l l growth from DNA r e p l i c a t i o n . That i s , i n h i b i t i o n of DNA r e p l i c a t i o n , e i t h e r by way of a muta-t i o n or an exogenous chemical i n h i b i t o r , need not r e s u l t i n the immediate c e s s a t i o n of o t h e r processes r e l a t e d to c e l l growth ( M i t c h i s o n , 1971). Agents causing such s p e c i f i c i n -h i b i t i o n probably have as t a r g e t s , f u n c t i o n s i n t i m a t e l y i n -v o l v e d w i t h the process of DNA r e p l i c a t i o n i t s e l f . H a r t w e l l (1974) has shown t h a t i n yeast the maintenance of c e l l growth i s a g e n e r a l f e a t u r e of c e l l s which are a r r e s -ted at any stage of the c e l l d i v i s i o n c y c l e . Thus, mainte-nance of p r o t e i n and RNA s y n t h e s i s f o l l o w i n g m u t a t i o n a l a r r e s t i s an i n d i c a t i o n t h a t a mutation does i n f a c t d i r e c t l y a f f e c t a c e l l c y c l e s t e p . Furthermore, a c h a r a c t e r i s t i c common to the t e r m i n a l phenotypes of c e l l s a r r e s t e d by c e l l c y c l e b l o c k s i s abnormally l a r g e s i z e (Unrau and H o l l i d a y , 1970; M i t c h i s o n , 1971; H a r t w e l l , 1974). The broad aim of the p r e s e n t study has been to i s o l a t e and c h a r a c t e r i z e t s c e l l c y c l e mutants of the h o l o t r i c h o u s c i l i a t e Paramecium a u r e l i a with p a r t i c u l a r emphasis on those mutants s p e c i f i c a l l y a f f e c t e d i n DNA s y n t h e s i s . A v e g e t a t i v e P. a u r e l i a c e l l c o n t a i n s two types of nuc-l e i , two d i p l o i d m i c r o n u c l e i and a macronucleus which con-t a i n s 800-900 h a p l o i d e q u i v a l e n t s of DNA (Woodard, e_t a l . , 1961; A l l e n and Gibson, 1972; Morton, 1974), the o r g a n i z a -3 t i o n of which i s u n c e r t a i n . The macronucleus i s the "somatic" nucleus of the c e l l i n t h a t i t , r a t h e r than the m icronucleus, determines the phenotype of the c e l l . Moreover, d u r i n g each n u c l e a r r e o r g a n i z a t i o n (conjugation or autogamy) the macro-nucleus i s a u t o l y z e d and r e p l a c e d by a new macronucleus of m i c r o n u c l e a r o r i g i n . During n u c l e a r r e o r g a n i z a t i o n ( F i g . 1) both micronuc-l e i undergo m e i o s i s y i e l d i n g e i g h t h a p l o i d products. Seven of these are a u t o l y z e d w h i l e the e i g h t h undergoes a m i t o t i c d i v i s i o n r e s u l t i n g i n two i s o g e n i c h a p l o i d n u c l e i . In the case of c o n j u g a t i o n the p a i r e d c e l l s r e c i p r o c a l l y exchange one of these n u c l e i . The two n u c l e i w i t h i n a g i v e n c e l l then fuse to y i e l d a d i p l o i d nucleus, the synkaryon. Dur-i n g autogamy, which occurs w i t h i n an unpaired c e l l , the two i s o g e n i c h a p l o i d n u c l e i simply fuse to y i e l d a complete-l y homozygous synkaryon. In both r e o r g a n i z a t i o n processes the synkaryon then undergoes two m i t o t i c d i v i s i o n s produc-i n g two macronuclear anlagen which segregate a t the f i r s t f i s s i o n and two presumptive m i c r o n u c l e i which d i v i d e a t the f i r s t f i s s i o n producing the v e g e t a t i v e n u c l e a r c o n s t i t u t i o n . The d u r a t i o n of a P. a u r e l i a v e g e t a t i v e c e l l c y c l e un-der the c o n d i t i o n s s t a n d a r d l y used i n g e n e t i c work (27 C; Cerophyl medium) i s about 5.0 h r . The o r g a n i z a t i o n of the major n u c l e a r events of the c e l l c y c l e i s shown i n F i g u r e d . P. a u r e l i a i s a p a r t i c u l a r l y f a v o r a b l e organism f o r a mutagenic study of t h i s nature owing to the e x i s t a n c e of 4 F i g u r e 1. Nuclear r e o r g a n i z a t i o n i n P. a u r e l i a . 5 MACRONUCLEUS MICRONUCLEI 2 d i p l o i d m i c r o n u c l e i 8 h a p l o i d n u c l e i 2 i s o g e n i c h a p l o i d n u c l e i 2 h a p l o i d n u c l e i d i p l o i d synkaryon 4 d i p l o i d n u c l e i m e i o s i s random s u r v i v a l m i t o s i s AUTOGAMY CONJUGATION 0 0 0 © (exchange) f u s i o n mitoses 6 6 d i f f e r e n -t i a t i o n PRESUMPTIVE MICRONUCLEI fragmentation; a u t o l y s i s MACRONUCLEAR. ANLAGEN 6 F i g u r e 2. O r g a n i z a t i o n of the n u c l e a r events i n the c e l l c y c l e i n P. a u r e l i a . C e l l s e p a r a t i o n i s a t t=0 hr and t=5 hr. Data of Pasternak (1967) and Berger (1971) Woodard, e t a_l. , (1961) r e p o r t a macronuclear phase of 2.5 h r . 7 MACRO S MACRO DIV MICRO S MICRO DIV 0 1 2 3 4 5 TIME IN HOURS AT 27 C 8 autogamy. That i s , mutations induced i n the m i c r o n u c l e i can be rendered homozygous and brought i n t o e x p r e s s i o n i n the macronucleus i n a s i n g l e step by i n d u c i n g autogamy. There are two b a s i c s t r a t e g i e s f o r o b t a i n i n g from a l a r g e mutagen-treated p o p u l a t i o n of c e l l s , mutants s p e c i -f i c a l l y a f f e c t e d i n DNA s y n t h e s i s . On the one hand, one can screen f o r l i n e s t h a t are c o n d i t i o n a l l y n o n - v i a b l e and then t e s t such l i n e s f o r d e f e c t s i n DNA s y n t h e s i s . T h i s approach I w i l l term " i s o l a t i o n without s e l e c t i o n " . On the other hand, one can t r e a t the mass p o p u l a t i o n with an agent which causes the s e l e c t i v e s u r v i v a l of the c l a s s of t s mutant sought, then screen the r e s u l t i n g " e n r i c h e d " p o p u l a t i o n f o r t s mutants. T h i s second, p o t e n t i a l l y more powerful approach would be " i s o l a t i o n w i t h s e l e c t i o n " . S e l e c t i o n has, i n g e n e r a l , not been employed i n the mutagenesis experiments t h a t have y i e l d e d s p e c i f i c DNA mutants. T h i s has mainly been due to the ease w i t h which l a r g e numbers of c e l l l i n e s c o u l d be screened f o r tem-pe r a t u r e s e n s i t i v i t y by r e p l i c a p l a t i n g i n the systems i n q u e s t i o n ( b a c t e r i a , y e a s t and U s t i l a g o ) . However, s e l e c t i o n systems have been used i n some cases, the most e f f e c t i v e system being a procedure based on the syner-g i s t i c e f f e c t of "near UV l i g h t " (the 313 nm UV component of standard f l u o r e s c e n t tubes) and 5-bromouracil (BU) s u b s t i t u t i o n on the i n a c t i v a t i o n of c e l l u l a r DNA (reviewed, 9 Hutchinson, 1973). The r a t i o n a l e of the procedure i s as f o l l o w s : Incubation of mass c u l t u r e s at the r e s t r i c t i v e temperature i n medium c o n t a i n i n g BU i n place of thymine, followed by i r r a d i a t i o n w i t h near UV r e s u l t s i n the s e l e c -t i v e death of c e l l s which continue DNA sy n t h e s i s at t h a t temperature. The p o p u l a t i o n s u r v i v i n g such treatment i s thus, i n theory, enriched f o r c e l l s having temperature-s e n s i t i v e defects i n DNA s y n t h e s i s . Some p r o k a r y o t i c DNA mutants (Pauling and Hamm, 1968; C a r l , 1970) have been ob-t a i n e d w i t h the a i d of t h i s system and a s i m i l a r procedure has been used i n mammalian c e l l c u l t u r e to i n d i r e c t l y se-l e c t n u t r i t i o n a l mutants (Puck and Kao, 1967). In the present study, t s mutants were i s o l a t e d both without s e l e c t i o n and w i t h the a i d of a BU s e l e c t i o n system developed by Baumann (1973). The f a c t t h a t P. a u r e l i a were i n a l l cases c u l t u r e d monaxenically i n the presence of a b a c t e r i a l food organism n e c e s s i t a t e d a m o d i f i c a t i o n of the standard design of the BU procedure. I t has been shown t h a t i n monaxenic c u l t u r e P. a u r e l i a DNA precursors are p r e f e r e n t i a l l y drawn from b a c t e r i a l DNA r a t h e r than from e x t r a c e l l u l a r nucleoside pools (Berger and K i m b a l l , 1964; Berger, 1971). Consider-in g t h i s and a l s o the demonstrated preference of c e l l u l a r enzymes of other organisms f o r thymine as opposed to BU (Boyce and Setlow, 1963) i t was f e l t - t h a t i n order to a-chieve s i g n i f i c a n t replacement of thymine by BU, i t would 10 be necessary to use b a c t e r i a c o n t a i n i n g BU-substituted DNA as the means of i n c o r p o r a t i n g BU i n t o Paramecium DNA (Baumann, 1973). This i n d i r e c t technique f o r o b t a i n i n g BU i n c o r p o r a -t i o n i n non-axenic c u l t u r e was a l s o employed by Haugli and Dove (1972) i n Physarum. The p o l i c y pursued w i t h regard to p u t a t i v e mutants recovered e i t h e r w i t h the a i d of the BU s e l e c t i o n system or without s e l e c t i o n was to r e t a i n f o r i n t e n s i v e study only those which manifested d i v i s i o n a r r e s t w i t h i n the f i r s t c e l l c y c l e f o l l o w i n g a s h i f t to the r e s t r i c t i v e temperature. Such mu-tants were t e s t e d f o r maintenance of DNA and p r o t e i n s y n t h e s i s and those showing f a i l u r e of DNA synthesis and/or mainte-nance of p r o t e i n s y n t h e s i s were f u r t h e r c h a r a c t e r i z e d gene-t i c a l l y and p h y s i o l o g i c a l l y . 11 MATERIALS AND METHODS C u l t u r e of Paramecia General c u l t u r i n g procedures f o r P. a u r e l i a can be found i n Sonneborn (1970) . P. a u r e l i a syngen I4T, stock 51 was grown on Cerophyl rye grass medium w i t h K l e b s i e l l a  aerogenes as the food organism (Sonneborn, 1970) u n l e s s otherwise s p e c i f i e d . A l l c u l t u r e s were maintained a t pH 6.8. P r e p a r a t i o n of B a c t e r i a E s c h e r i c h i a c o l i K-12, s t r a i n 557 (thymine, a r g i n i n e , methionine, tryptophane d e f i c i e n t ) was grown on m o d i f i e d M-9 medium ( P i e r u c c i , 1969) supplemented w i t h tryptophane (25 ug/ml), cassamino a c i d s (Difco) (2.5 mg/ml), thiamine (2.5 ug/ml) and e i t h e r 5 ug/ml thymine ("thymine M-9") or 25 ug/ml 5-bromouracil ("BU-M-9"). B a c t e r i a l c u l t u r e s were grown a t 37 C i n an a g i t a t i n g i n c u b a t o r . (i) "BU E. c o l i " and "thymine E. c o l i " To prepare BU E. c o l i , E . c o l i 557 was grown on thymine M-9 to O.D. 0.20 then washed three times by c e n t r i f u g a t i o n i n thymine-free medium and resuspended a t 0. D. 0.10 i n BU M-9. The c u l t u r e was then incubated i n the dark u n t i l i t reached s t a t i o n a r y phase (0. D. 0.40-0.45) and s t o r e d f o r up to two days i n the dark a t 4 C. 12 Thymine E. c o l i were prepared by growing E. c o l i 557 c u l t u r e s to s t a t i o n a r y phase on thymine M-9. Immediately p r i o r to use the b a c t e r i a were harvested by c e n t r i f u g a t i o n , washed twice and resuspended i n a s m a l l q u a n t i t y of s t e r i l e Cerophyl medium (pH 6.8). 3 ( i i ) H-thymine l a b e l l e d E. c o l i E. c o l i 557 was grown to s t a t i o n a r y phase i n 2 ml thymine 3 M-9 c o n t a i n i n g 25uCi/ml H-thymine (Schwarz-Mann), washed three times and resuspended i n 10 ml s t e r i l e Cerophyl medium. Mutagenesis Procedure and S e l e c t i o n Group Indexing of Mutants Four s u c c e s s f u l mutagenesis experiments were conducted numbered 2,3,4 and 6 (1 and 5 being those of Baumann (1973)). 4 C e l l s were c o n c e n t r a t e d by c e n t r i f u g a t i o n to about 2xx 10 per ml and t r e a t e d i n D r y l ' s s o l u t i o n c o n t a i n i n g N-methyl-N 1-n i t r o - N - n i t r o s o g u a n i d i n e (MNNG) (Sigma) a t 7 5 ug/ml f o r 6 0 min a t room temperature (Kung, 1971). A f t e r t hree washes through D r y l ' s s o l u t i o n the c e l l s were resuspended i n s u f -f i c i e n t b a c t e r i z e d Cerophyl to p r o v i d e f o r three d o u b l i n g s p r i o r to s t a r v a t i o n . The c u l t u r e was then d i v i d e d i n t o sub-groups l e t t e r e d A,B,C, These subgroups, termed " s e l e c -t i o n groups" were kept separate d u r i n g a l l subsequent s e l e c -t i o n and s c r e e n i n g s t e p s . A f t e r autogamy was induced i n a s e l e c t i o n group by s t a r -v a t i o n , 7/8 of the c u l t u r e was d i s c a r d e d and the remaining 1/8 d i l u t e d e i g h t f o l d w i t h new b a c t e r i z e d Cerophyl to pro-13 v i d e , i n theory, f o r three doublings of the remaining c u l -t u r e . T h i s procedure of d i s c a r d and d i l u t i o n was repeated three times a t two day i n t e r v a l s u n t i l the remaining c e l l s had c e r t a i n l y grown, on the average, i n excess of 12 f i s s i o n s , the maximum observed phenotypic l a g i n P. a u r e l i a (Berger, 1969). S e l e c t i o n groups a t t h i s stage were then screened f o r t s mutants e i t h e r w i t h or without p r i o r BU s e l e c t i o n . A mutant i s o l a t e d was indexed a c c o r d i n g to i t s s e l e c t i o n t h group of o r i g i n . For example, the 17 p u t a t i v e mutant d e r i v e d from mutagenesis 4, s e l e c t i o n group Y was named 4Y17. BU S e l e c t i o n Procedure The procedure employed was t h a t developed by Baumann (1973) wi t h m o d i f i c a t i o n s . Samples of l a t e l o g phase c e l l s (1500-2000/ml) were s h i f t e d to the r e s t r i c t i v e temperature (34.5 C) i n a water bath and e q u i l i b r a t e d f o r 30 min. S u f f i c i e n t BU E. c o l i (or thymine E. c o l i i n c o n t r o l e x p e r i -ments) was then added to produce a very t u r b i d s o l u t i o n (0. D. g r e a t e r than 0.45). The c e l l s were then incubated i n the dark f o r 6 h r . a t 3 4.5 C. They were then r a p i d l y washed three times by c e n t r i f u g a t i o n through D r y l ' s s o l u -t i o n and l a y e r e d i n t o s t e r i l e p e t r i d i s h e s to a depth of 1 cm. The p e t r i d i s h c u l t u r e s were i r r a d i a t e d under a bank of f l u o r e s c e n t tubes (Westinghouse F20 T12/CW) a t 2 cm d i s t a n c e a t 17 C f o r 8-12 hr i n d i f f e r e n t experiments. N o n - i r r a d i a t e d c o n t r o l c u l t u r e s were covered w i t h aluminum 14 f o i l and incubated beside the i r r a d i a t e d c u l t u r e s d u r i n g the l i g h t treatment p e r i o d . At the end of the l i g h t t r e a t -ment, c u l t u r e s were d i l u t e d f o u r times w i t h b a c t e r i z e d Cerophyl and s t o r e d o v e r n i g h t a t 17 C. S u r v i v o r s were i s o -l a t e d s i n g l y i n t o d e p r e s s i o n s l i d e Cerophyl c u l t u r e s and allowed to grow up and enter autogamy. A s i n g l e i s o l a t e was made from each autogamous c u l t u r e and grown f o r a t l e a s t 15 f i s s i o n s . Death d u r i n g the l i g h t treatment or l o s s of v i a b i l i t y a t any stage p r i o r to autogamy.was c l a s s i f i e d as v e g e t a t i v e death. F a i l u r e of growth subsequent to autogamy was c l a s s i -f i e d as exautogamous death. S y n c h r o n i z a t i o n of Paramecia Where necessary, s m a l l samples of c e l l s were synchro-n i z e d by manual s e l e c t i o n of d i v i d e r s w i t h a g l a s s micro-p i p e t t e . The time of c e l l s e p a r a t i o n was d e s i g n a t e d t=0 and a l l s h i f t or l a b e l times were gi v e n i n the form t=x where x was the ela p s e d time s i n c e the p r e v i o u s c e l l d i v i -s i o n . C e l l s aged 0-5 min were de s i g n a t e d "baby c e l l s " . Temperature S e n s i t i v i t y C l a s s i f i c a t i o n Clones s u r v i v i n g BU s e l e c t i o n or simply i s o l a t e d from the mutagenized p o p u l a t i o n (non-selected groups) were screened i n i t i a l l y f o r temperature s e n s i t i v i t y by i n c u b a t i n g 15 r e p l i c a t e d s e t s of s i n g l e c e l l i s o l a t e s a t both permis-s i v e (27 C) and r e s t r i c t i v e (34.5 C) temperatures i n an a i r i n c u b a t o r and s c o r i n g r e l a t i v e growth a f t e r 48 h r . L i n e s showing normal growth a t 27 C and l e s s than 3 f i s -s i o n s a t 3 4.5 C were desi g n a t e d " t s " . These l i n e s were then t e s t e d by i s o l a t i n g s i n g l e baby c e l l s i n t o drops of Cerophyl i n e i t h e r wax-sealed m e l t i n g p o i n t c a p i l l a r i e s or m i c r o t i t r e d e p r e s s i o n s (Fisher) and i n c u b a t i n g these i n a water bath a t 34.5 C f o r 24 h r . L i n e s c o n s i s t e n t l y f a i l -t o d i v i d e under these c o n d i t i o n s were c l a s s i f i e d as " t s - 0 " . Autoradiography 3 (i) H-thymine l a b e l l i n g E x t e n s i v e data v a l i d a t i n g the procedures employed are presented i n Berger (1969; 1971). DNA s y n t h e s i s i n Parame-cium macronuclei was monitored by i n c u b a t i n g c e l l s i n Cero-3 p h y l medium b a c t e r i z e d w i t h excess H-thymine l a b e l l e d E. c o l i . At the end of the l a b e l l i n g p e r i o d c e l l s were washed three times by i n d i v i d u a l s e r i a l t r a n s f e r through d e p r e s s i o n s l i d e c u l t u r e s c o n t a i n i n g n o n - l a b e l l e d b a c t e r i z e d Cerophyl and d r i e d i n d i v i d u a l l y i n micro-drops on albumenized s l i d e s . A f t e r 20 min f i x a t i o n i n C l a r k ' s f i x a t i v e ( e t h a n o l : a c e t i c a c i d : : 3 : l ) , the s l i d e s were hyd r o l y z e d f o r 11 min a t 60 C i n 1 N HCl and Feulgen s t a i n e d a t room temperature f o r 60 min. 16 ( i i ) H-leucine l a b e l l i n g P r o t e i n s y n t h e s i s i n Paramecium c e l l s was monitored by i n c u b a t i n g c e l l s i n b a c t e r i z e d Cerophyl c o n t a i n i n g 3 5 ug/ml exogenous H-leucine (Schwarz-Mann). C e l l s were washed, d r i e d on s l i d e s , f i x e d and s t a i n e d as above. In both l a b e l l i n g procedures, s l i d e s were dipped once i n l i q u i d n u c l e a r t r a c k emulsion ( I l l f o r d K-5) mixed 50% w/w w i t h water, a t 6 0 C. Autoradiographs were developed a f t e r a s u i t a b l e p e r i o d f o r 2.5 min a t room temperature w i t h Kodak D-19 developer. 3 In the case of H-thymine autoradiographs, i n c o r -p o r a t i o n of l a b e l was determined by manually c o u n t i n g the 2 number of g r a i n s over randomly chosen 25cum areas o f the 3 macronuclei of d r i e d whole c e l l s (Berger, 1971). For H-2 l e u c i n e measurements, 25 urn areas of the cytoplasm o f whole c e l l s were counted. P o p u l a t i o n E s t i m a t i o n P o p u l a t i o n d e n s i t i e s i n bulk c u l t u r e were estimated by d i r e c t counts of s e r i a l l y d i l u t e d samples. A r e p o r t e d estimate r e p r e s e n t s the mean of three independent d e t e r -minations . 17 RESULTS The BU S e l e c t i o n System T h i s system was i n i t i a l l y adapted to P. a u r e l i a by Baumann (1973). However, because of some minor p r o c e d u r a l changes and some apparent d i s c r e p a n c i e s w i t h p r e v i o u s work, f u r t h e r c h a r a c t e r i z a t i o n was undertaken. A l l experiments were conducted u s i n g stock 51, mating type VII w i l d type c e l l s . (i) s u r v i v a l d u r i n g s e l e c t i o n Table I shows t h a t d u r i n g the 6 hr heat treatment there was an approximate d o u b l i n g of a l l thymine E. c o l i - f e d cul-: t u r e s whereas the B U - E e d c o l i - f e d c u l t u r e s f a i l e d to i n c r e a s e s i g n i f i c a n t l y i n numbers i n a l l but one case. During the 16 hr l i g h t treatment, c o n t r a r y to p r e v i o u s o b s e r v a t i o n s (Baumann, 1973) there was death of a l l l i g h t t r e a t e d samples. Baumann (1973) observed a comparable p r e c i -p i t o u s d e c l i n e i n i r r a d i a t e d BU E. c o l i - f ed samples but r e l a -t i v e l y h i g h s u r v i v a l (6 0%) of non-BU-treated c e l l s f o l l o w -i n g l i g h t exposures of up to 57 h r ; d u r a t i o n . There i s no completely adequate e x p l a n a t i o n f o r t h i s d i s c r e p a n c y , how-ever the l i g h t e f f e c t on both BU- and non-BU-treated c e l l s i s extremely s e n s i t i v e to such v a r i a b l e s as the c o n c e n t r a -t i o n o f c e l l s and the ammount of Cerophyl r e s i d u e i n the medium of the c u l t u r e s being i r r a d i a t e d . TABLE I VEGETATIVE SURVIVAL DURING SELECTION # a t s t a r t # a t end # a t s t a r t # a t end # a t ELT Sample Treatment of heat of heat of l i g h t of l i g h t + 12 hr treatment treatment treatment treatment ( i n Cero-(SHT) (EHT) (SLT) (ELT) p h y l ; 17 C 1 thy;dark 1.12 x 10 5 2.40 x 10 5 2.03 x 10 5 1.70 x 10 5 2.45 x 10 5 2 t h y ; l i g h t 1.57 " 3.08 " 2.73 " 0.0 0.0 3 BU;dark 1.36 " 1.15 " 1.43 " 1.07 " 0.82 4 BU;dark 0.88 " 1.08 " 0.93 " 0.63 " 0.38 5 B U ; l i g h t 1.26 " 1.05 " 1.13 " 0.0 0.0 6 B U ; l i g h t 1.61 " 1.57 " 1.0 8 " 0.0 0.0 7 B U ; l i g h t 0.8 8 " 1.47 " 1.15 " 0.0 0.0 BU = BU E. c o l i thy = thymine E. c o l i 19 The p o p u l a t i o n s i z e s of c u l t u r e s d u r i n g the l i g h t t r e a t -ment p e r i o d were d i f f i c u l t to estimate owing to the tendency of the c e l l s to clump and s e t t l e to the bottom of the c u l -t u r e d i s h . The s m a l l volumes and d i f f i c u l t y of a g i t a t i o n c omplicated sampling. Furthermore, the c u l t u r e s were d i l u t e d and t r a n s f e r r e d p r i o r to the estimates made a t the end of the l i g h t treatment p e r i o d (ELT) r e s u l t i n g i n e v i -t a b l y i n some l o s s . Thus the apparent p o p u l a t i o n reduc-t i o n between the s t a r t of the l i g h t treatment p e r i o d (SLT) and ELT i n samples 1,3 and 4 i s not n e c e s s a r i l y r e l i a b l e . The f a c t t h a t n e g l i g i b l e ( l e s s than 1%) numbers of dead or dying c e l l s were ever seen i n these samples d u r i n g the l i g h t treatment p e r i o d suggests t h a t there was l i t t l e o r no death d u r i n g t h i s stage. During the 12 hr post-ELT i n c u b a t i o n (at 17 C i n Cero-phyl) there was an i n c r e a s e i n the thymine E. c o l i - f e d sam-p l e and a v a r i a b l e decrease i n the BU E. c o l i - f e d samples. ( i i ) v i a b i l i t y d u r i n g s e l e c t i o n F i g u r e 3 shows r e s u l t s compatible w i t h those o f Table I i n t h a t a l l l i g h t t r e a t e d c u l t u r e s were dead when l i g h t expo-sures exceeded 12 hr d u r a t i o n . The important f e a t u r e , how-ever, i s the g r e a t e r l i g h t s e n s i t i v i t y o f the BU t r e a t e d samples f o r exposures of i n t e r m e d i a t e l e n g t h . Because the time course of v i a b i l i t y l o s s was found to vary i n d i f f e r e n t s e l e c t i o n experiments, the p r a c t i c e which was f o l l o w e d i n a c t u a l s e l e c t i v e treatments was to terminate the l i g h t 20 F i g u r e 3. V i a b i l i t y d u r i n g s e l e c t i o n , thymine; dark ( A ) thymine; l i g h t ( ) BU;dark ( t> ) BU; l i g h t ( <J ) PER CENT ISOLATES FORMING VIABLE CLONES 22 treatment when the g r e a t m a j o r i t y (greater than 99.9%) of the c e l l s were a p p a r e n t l y dying ( u s u a l l y 8-12 h r ) . ( i i i ) v e g e t a t i v e and exautogamous death Table I I demonstrates t h a t n e i t h e r l i g h t alone nor BU alone caused a s i g n i f i c a n t i n c r e a s e i n exautogamous death. T h i s o b s e r v a t i o n w i t h r e s p e c t t o BU i s i n agree-ment wit h t h a t of Berger and K i m b a l l (unpublished) but i n c o n t r a d i c t i o n w i t h t h a t of Baumann (1973) who r e p o r t s 35% exautogamous death w i t h BU treatment alone. The number of B U j l i g h t t r e a t e d c l o n e s s u r v i v i n g t o autogamy i s inade-quate to r u l e out some component of exautogamous death. However, there was c e r t a i n l y l e s s than the 58.6% exautoga-mous death observed by Baumann _(1973) . (iv) summary B u c o l i - f e d and thymine E. c o l i - f e d Paramecium c u l -t u r e s are d i f f e r e n t i a l l y s e n s i t i v e to near UV l i g h t . A l -though the d i f f e r e n c e i s not as marked as p r e v i o u s l y r e p o r -t e d , i t i s a p p a r e n t l y adequate to pr o v i d e a measure of s e l e c -t i o n a g a i n s t B U - s u b s t i t u t e d c e l l s . Mutagenesis Experiments The mutagenesis data (Table I I I ) show r e s u l t s compa-t i b l e w i t h those obtained from p r e v i o u s s t u d i e s done on P. a u r e l i a . That i s , the l e t h a l e f f e c t i s manifested o n l y sub-sequent t o autogamy. T h i s exautogamous death i s assumed to be an index of g e n e r a l mutagenic damage to m i c r o n u c l e i . TABLE II VEGETATIVE AND EXAUTOGAMOUS VIABILITY DURING SELECTION I s o l a t e d at SLT I s o l a t e d a t SLT + 3 hr Treatment V e g e t a t i v e Exautogamous Ve g e t a t i v e Exautogamous v i a b i l i t y v i a b i l i t y v i a b i l i t y v i a b i l i t y thy;dark 39/39 (100%) 36/39 (92%) 82/84 (98%) 79/82 (96%) t h y ; l i g h t 83/84 (99%) 78/81 (96%) 148/210 (71%) 134/138 (97%) BU;dark 58/78 (74%) 58/58 (100%) 123/174 (71%) 114/117 (97%) B U ; l i g h t 86/126 (68%) 47/48 (98%) 19/210 (9%) 13/16 (81%) BU = BU E. c o l i thy = thymine E. c o l i TABLE I I I VEGETATIVE AND EXAUTOGAMOUS VIABILITY FOLLOWING MUTAGENESIS Experiment Treatment P o p u l a t i o n b e f o r e treatment P o p u l a t i o n a f t e r treatment V e g e t a t i v e v i a b i l i t y Exautogamous v i a b i l i t y 2 MNNG 3.4 X 10 6 2.8 X 10 6 n. d. 42/294d,(14%) 2 c o n t r o l 3.4 X 10 5 2.9 X 10 5 n. d. 199/210 u (95%) 3 MNNG 3.3 X 10 6 2.9 X 10 6 420/420 (100%)110/280 (36%) 3 c o n t r o l 1.3 X 10 5 1.2 X 10 5 84/84 (100%) 71/71 (100%) 4 MNNG 3.3 X 10 6 3.1 X 10 6 n. d. n. d. 4 c o n t r o l 1.0 X 10 5 1.1 X 10 5 n. d. n. d. 6 MNNG 2.1 X 10 6 1.8 X 10 6 208/210 (99%) 45/420 (10.7%) 6 c o n t r o l 2.1 X i o 5 1.7 X 10 5 82/84 (98%) 78/84 (93%) n. d. = not determined 25 T h e r e f o r e , the v a r i a b i l i t y i n exautogamous s u r v i v a l ob-served i n d i f f e r e n t mutagenesis experiments makes quan-t i t a t i v e comparisons between the mutant y i e l d s of s e l e c t i o n groups drawn from d i f f e r e n t mutagenesis experiments some-what u n r e l i a b l e . I s o l a t i o n of Mutants Table IV i s a resume of the mutant y i e l d s of the BU se-l e c t i o n experiments. A g r e a t d i v e r s i t y of r e s u l t s i s appa-r e n t a t a l l stages of c l a s s i f i c a t i o n . That i s j the number of c e l l s s u r v i v i n g s e l e c t i o n ranges from 0 to 246 arid the p r o p o r t i o n of s u r v i v o r s p r o v i n g to be t e m p e r a t u r e - s e n s i t i v e by e i t h e r of the two c r i t e r i a (see M a t e r i a l s and Methods) shows a s i m i l a r wide f l u c t u a t i o n . N e v e r t h e l e s s , compari-son w i t h T a b l e V which g i v e s the mutant y i e l d of non-BU-s e l e c t e d groups shows t h a t i n many cases the s e l e c t i o n s y s -tem does seem to have d r a m a t i c a l l y i n c r e a s e d the frequency of t s mutant c l a s s e s i n the p o p u l a t i o n s u r v i v i n g s e l e c t i o n . These r e s u l t s are d i s c u s s e d i n g r e a t e r d e t a i l below. P r e l i m i n a r y Genetic A n a l y s i s A l l ts-0 l i n e s except 4X22 and 2A9 have been succ e s s -f u l l y c r o s s e d to a t e m p e r a t u r e - r e s i s t a n t l i n e homozygous f o r the r e c e s s i v e b e h a v i o u r a l marker pawn A (pw) (Kung, 1971). A l l c r o s s e s y i e l d e d p h e n o t y p i c a l l y w i l d type F-^  exconjug-ant5-. c l o n e s . 4X22 and 2A9 c o n s i s t e n t l y f a i l e d t o undergo TABLE IV MUTANT YIELDS OF BU SELECTION GROUPS Group # before s e l e c . # a f t e r s e l e c . # t s l i n e s # ts-0 l i n e s # unique* ts-0 l i n e s 2A 12 x 10 6 75 10 3 3 3A 5 x 10 5 32 6 0 0 3B II 9 5 5 1 3C II 45 6 2 2 3D II 5 2 1 1 3E II 0 0 0 0 3F II 0 0 0 0 3G II 0 0 0 0 3H l l 21 0 0 0 4A II 21 1 0 0 4B II 78 12 2 1 4C II 33 1 1 1 4D II 10 0 0 0 4E II 25 9 3 1 4F II 0 0 0 0 4G II 22 0 0 0 41 11 3 2 1 1 4K II 0 0 0 0 4L II 7 0 0 0 4M II 246 7 1 1 4X II 91 12 8 6 4Y II 111 11 6 5 T o t a l 2.25 x 10 7 854 84 33 23 * g e n e t i c a l l y complementing TABLE V MUTANT YIELDS OF NON-SELECTED GROUPS Group # l i n e s # t s l i n e s # t s - 0 screened l i n e s 2U 420 1 0 6* 16,128 113 33 T o t a l 16,548 114 33 * I n d i v i d u a l data are not a v a i l a b l e f o r the 30 s e l e c t i o n groups d e r i v e d from mutagenesis 6. 28 genetic exchange, 4X22 never forming conjugating p a i r s while 2A9 formed a b o r t i v e p a i r s . Cytoplasmic or dominant patterns of i n h e r i t a n c e were never observed f o r any ts-0 l i n e examined. Post-autogamous ¥ ^  segregation patterns were examined by inducing autogamy i n the clones produced by the above crosses. The r e s u l t s obtained f o r those l i n e s examined are given i n Table VI. I f the b a s i s of the t s phenotype was a l e s i o n at a s i n g l e l o c u s , one would p r e d i c t a t s : t s + : : l : l r a t i o i n the F2» The other p o s s i b i l i t i e s considered are: (i) "double t s mutant"; two independently segregating l e s i o n s y i e l d i n g a t s phenotype both i n d i v i d u a l l y or together. This should produce a t s : t s + : : 3 : l . ( i i ) " s y n t h e t i c t s phenotype"; two independently segregating l e s i o n s which give the t s phenotype only i n con-c e r t . This should y i e l d a t s : t s + : : l : 3 . The f i n a l three ccolumns of Table VI show t h a t the r e -s u l t s of each cross are c o n s i s t e n t w i t h a 1:1 e x p e c t a t i o n whereas the r e s u l t s of no cross w i t h the exception of those of 3D1 are c o n s i s t e n t w i t h e i t h e r a 3:1 or a 1:3 e x p e c t a t i o n . To c o n c l u s i v e l y determine the genetic c o n s t i t u t i o n of the 3D1 p a r e n t a l l i n e i t would be necessary to re-examine t h i s l i n e i n a cross w i t h l a r g e r sample s i z e . None of the t s mutants appear to be l i n k e d to the pawn A 2 gene (X assuming recombinants:parentals::1:1 i s l e s s than 2 X p r. f o r a l l crosses) . This i s not s u r p r i s i n g as the hap-TABLE VI EXAUTOGAMOUS SEGREGATION OF t s HETER0ZYGOTES t s l i n e + t s ;pw , F 2 t s ;pw pheno£ype + t s ;pw t s ;pw % recom-b i n a n t s X 2 (1:1) X 2 (3:1) X 2 (1:3) 2A2 23 22 20 24 47.2 0.00 27.06** 29.67** 2A5 17 16 19 11 55.5 0.06 16.03** 23.75** 3B1 13 16 18 20 50.8 0.24 27.99** 15.05** 3C8 20 15 13 14 45.1 0.79- 10.41** 31.05** 3D1 10 17 9 5 63.5 3.51 1.37 34.35** 4B7 15 12 19 11 44.6 0.07 21.76** 14.04** 4C1 12 10 9 8 48.8 0.41 6 .23* 18.88** 4E1 7 11 7 8 54.5 0.12 6 .31* 13.83** 412 16 10 5 12 34.9 0.32 3.86* 13.76** 4X11 8 10 11 11 52.5 0.23 7.50** 17.63** 4X16 7 13 9 11 56.5 0.03 12 .03** 12.03** 4Y9 8 14 4 11 48.7 0.97 3.97* 21.63** 4Y14 12 5 13 12 42.9 1.17 24.89** 4.57* 4Y17 19 11 19 19 44.1 0.72 32.96** 12.25** 4Y18 10 11 11 10 52.4 0.02 12.70** 12.70** 4Y21 13 11 9 8 48.8 0.88 5.08* 22.84** X (x:y) = X f o r x:y e x p e c t a t i o n , c a l c u l a t e d u s i n g Y a t e s ' c o r r e c t i o n 2 2 * X g r e a t e r t h a n X 9 5 2 2 ** X g r e a t e r t h a n X Q Q 30 l o i d chromosome number i n P.: a u r e l i a i s g r e a t e r t h a n 40 ( S o n n e b o r n , 1974) and as t h e s a m p l e s i z e s e x a m i n e d w e r e s u f -f i c i e n t t o d e t e c t o n l y r e l a t i v e l y t i g h t l i n k a g e . T hus t h e r e s u l t s i n d i c a t e t h a t t h e p h e n o t y p e s o f a l l t s l i n e s a n a -l y z e d ( w i t h one p o s s i b l e e x c e p t i o n ) a r e c a u s e d by s i n g l e s i t e r e c e s s i v e m u t a t i o n s . G e n e t i c C o m p l e m e n t a t i o n A n a l y s i s ( i ) i n t r a - g r o u p c o m p l e m e n t a t i o n I t was n e c e s s a r y , s u b s e q u e n t t o a u t o g a m y , t o p e r m i t a t l e a s t 10 c e l l g e n e r a t i o n s p r i o r t o s c r e e n i n g f o r m u t a n t s (s e e M a t e r i a l s a n d M e t h o d s ) . Thus i t was i n e v i t a b l e t h a t t h e r e w o u l d be c e l l s p r e s e n t a t t h e t i m e o f s c r e e n i n g w h i c h h a d b e e n d e r i v e d f r o m t h e same p o s t - m u t a g e n e s i s a u t o g a m o u s p r o g e n i t o r . S u c h l i n e s w o u l d , o f c o u r s e , be g e n e t i c a l l y i d e n t i c a l . I n a d d i t i o n , c e l l d i v i s i o n was o f t e n o b s e r v e d b o t h d u r i n g l i g h t t r e a t m e n t a n d i m m e d i a t e l y a f t e r , f u r t h e r i n c r e a s i n g t h e p r o b a b i l i t y t h a t m u t a n t s r e c o v e r e d f r o m a s i n -g l e s e l e c t i o n g r o u p m i g h t n o t be g e n e t i c a l l y i n d e p e n d e n t . T h u s , i n t r a - g r o u p c o m p l e m e n t a t i o n a n a l y s i s was c r i t i c a l i n o r d e r t o a v o i d w a s t e f u l r e p e t i t i o n o f e f f o r t . N o n - c o m p l e -m e n t i n g t s m u t a n t s r e c o v e r e d f r o m a s i n g l e s e l e c t i o n g r o u p w e r e assumed t o be p r o d u c t s o f t h e same m u t a t i o n a l e v e n t a n d h e n c e a l l b u t one r e p r e s e n t a t i v e was d i s c a r d e d . I n t r a - g r o u p c o m p l e m e n t a t i o n r e s u l t s f o r s e l e c t i o n g r o u p s y i e l d i n g more t h a n one t s - 0 m u t a n t a r e a s f o l l o w s : 31 2A s e l e c t i o n group: 2A2 and 2A5 complemented. 2A9 c o u l d n ot be t e s t e d owing t o i t s f a i l u r e t o mate. However, on the b a s i s o f i t s c h a r a c t e r i s t i c phenotype (slow growth and f a i l u r e t o mate) i t i s assumed t o be an independent m u t a t i o n . 3 B s e l e c t i o n group: None o f t h e f i v e t s l i n e s r e -c o v e r e d complemented w i t h any o t h e r i n the group and i n a d d i t i o n a l l had t h e same ( e x t r e m e l y t h i n ) t s t e r m i n a l pheno-t y p e . Hence, a l l b u t one r e p r e s e n t a t i v e , 3.B1, was d i s c a r d e d . 3C s e l e c t i o n group: 3C2 and 3C8 complemented. 4B s e l e c t i o n group: The two t s - 0 l i n e s 4B2 and 4B7 f a i l e d t o complementeand thus o n l y 4B7 was r e t a i n e d . 4E s e l e c t i o n group: The t h r e e t s - 0 l i n e s 4E1, 4E2 and 4E9 were non-complementary. 4E1 was r e t a i n e d . 4 X s e l e c t i o n group: The complementation m a t r i x f o r t h i s group ( F i g . 4 ) i n d i c a t e s t h a t two p a i r s , ( 4 X 1 1 , 4 X 1 4 ) and ( 4 X 1 2 , 4 X 1 3 ) a r e non-complementary. 4 X 1 1 and 4 X 1 3 were r e t a i n e d as r e p r e s e n t a t i v e s o f t h e r e s p e c t i v e p a i r s . 4Y s e l e c t i o n group: F i g u r e 5 shows o n l y one case o f non-complementation. 4Y16 was assumed t o be i d e n t i c a l t o 4Y17 and d i s c a r d e d . ( i i ) p r e l i m i n a r y i n t e r - g r o u p complementation I n t e r - g r o u p complementation was r e s t r i c t e d t o t h o s e l i n e s r e c o v e r e d from m u t a g e n e s i s ' 2 , 3 and 4 w h i c h showed a c h a r a c t e r i s t i c o v e r s i z e d t s t e r m i n a l phenotype (the " f a t " p h e n o t y p e ) . The r e s u l t s a r e shown i n F i g u r e 6. 4 B 8 i s n o t a c o n s i s t e n t t s - 0 and hence s c o r i n g i s somewhat e q u i v o c a l . 32 F i g u r e 4. Complementation matrix f o r 4X s e l e c t i o n group. + denotes w i l d type growth of exconjugants a t 34.5 C. - denotes ts-0 phenotype of exconjugants. MATING TYPE VII 4X7 4X9 4X11 4X12 4X13 4X14 4X16 33 4X7 H 4X9 H > 4X11 w £ 4X12 EH g 4X13 H I? 4X14 4X16 + + + + + + + + + + + + + + + + + • + + + + + + + + + + + + + + + + + + 34 F i g u r e 5. Complementation matrix f o r 4Y s e l e c t i o n group. + denotes w i l d type growth of exconjugants a t 34.5 C. - denotes ts-0 phenotype of exconjugants. 35 M A T I N G T Y P E V I I 4Y9 4 Y 1 4 4 Y 1 6 4 Y 1 7 4 Y 1 8 4 Y 2 1 4Y9 - + + + + + H U 4Y14 + - + + + + > W 4Y16 + + - - + + ^ 4Y17 + + _ _ + + o H 4Y18 + + + + - + EH £ 4Y21 + + + + + -F i g u r e 6. Complementation matrix f o r t s - f a t mutants. + denotes w i l d type growth of exconjugants a t 34.5 C. - denotes ts-0 phenotype of exconjugants. ± denotes leak y ts-0 phenotype. MATING TYPE VII 2A2 4B8 4X11 4Y14 4Y17 H 2A2 ± + • + > g 4B8 ± ± + + ± H 4X11 + + - + + £ 4Y14 + + + - + * 4Y17 . - ± + + 38 The entry "+" represents the somewhat leaky or slow-ac-t i n g phenotype c h a r a c t e r i s t i c of 4B8. 4Y17 and 2A2 are a l l e l i c a n d . i t appears t h a t these two are a l s o a l l e l i c w i t h 4B8, the 4B8 leaky t s phenotype being dominant. 4X11 and 4Y14 are shown to belong to independent comple-mentation groups. (Whether owing to r e v e r s i o n or i n i t i a l m i s - s c o r i n g , 4Y14 no longer e x h i b i t s a dramatic t s - f a t phenotype, f u r t h e r emphasizing i t s independence of the other two groups.) DNA Synthesis i n ts-0 Mutants There, are four experiments i n t h i s s e c t i o n , each iden-t i c a l i n design but each done on separate occasions w i t h separate c o n t r o l s . Hence d i r e c t comparisons are p o s s i b l e only w i t h i n an experiment. In a l l cases synchronous samples of baby c e l l s were i n -3 cubated i n H-thymine l a b e l l e d E. c o l i from t=0 hr to t=5 hr. The s h i f t to the appropriate temperature was e f -f e c t e d a t about t=5 min by t r a n s f e r to a water bath. C e l l s were d r i e d on s l i d e s a t t=5 hr and autoradiographs prepared. Small s c a l e p i l o t experiments were scored q u a l i t a t i v e l y and those l i n e s showing g r e a t l y reduced i n c o r p o r a t i o n (l e s s than 10%) were r e t e s t e d i n l a r g e r s e m i - q u a n t i t a t i v e experiments. The r e s u l t s of the four s e m i - q u a n t i t a t i v e experiments are shown i n Table V I I . There were 14 l i n e s which showed l e s s than 10% i n c o r p o r a t i o n at 34.5 C r e l a t i v e to that at 27 C. These were: 2A2 (4.0%; 7.5%), 2A5 (7.5%), 3B1 (4.1%), 39 TABLE VII H-THYMINE. INCORPORATION OF t s MUTANTS L i n e 27 C 34.5 C R a t i o n X •+ S.E. n X + S.E. 34.5:27 w i l d type 31 55.4 + 2.8 30 49.9 + 3.6 90.4% 2A2 30 44.9 + 3.4 30 1.8 + 0.4 4.0% 2A5 30 53 . 3 + 3.2 30 3.9 + 0.9 7.5% w i l d type 24 78.0 + 3.3 30 85.9 + 4.5 110.0% 2A2 28 73.5 + 3.7 31 5.5 + 2.8 7.5% 3B1 28 34.6 + 3.2 30 1.4 + 0.3 4 .1% 3C2 30 53.0 + 3.8 26 8.2 + 1.6 15.5% 4B7 27 58.7 + 4.7 27 3.6 + 0.9 6.1% 4X16 28 70.7 + 3.9 28 10.8 + 2.6 15.3% 1Y14 3 0 83.7 + .4.7 . 34 6.7 + 1.9 8.0% w i l d type 30 92.3 + 4.1 30 102.2 + 4.3 112.0% 4X9 29 61.9 + 4.3 27 8.0 + 1.4 12.9% 4X13 34 24.2 + 2.9 28 0.6 + 0.8 2.5% 4Y9 30 65.3 + 4.6 26 7.7 + 1.3 11.8% 4Y18 30 57.5 + 5.4 30 12.3 + 2.7 21.4% w i l d type 30 83.3 + 4.1 30 85.1 + .4.5 102.0% 4X7 30 49.2 + 3.4 31 0.3 + 0.1 0.6% 4Y17 32 91.4 + 4.6 32 0.3 + 0.1 . 0.3% 6A4 30 83-.1 + 4.1 30 1.0 + 0.3 1.3% 6D4 29 33.1 + 3.0 28 0.5 + 0.2 1.5% 614 31 82.6 + 4.4 28 2.3 + 1.2 2.8% 615 30 72.2 + 4.7 30 1.7 + 1.0 2.4% 6Q2 31 41.7 + 3.4 30 1.2 + 0.3 2.9% 6T10 31 80.8 + 4.3 30 1.0 + 0.4 1.2% X i s the mean number of g r a i n s per 25 urn area o f mac-ronucleus (see M a t e r i a l s and Methods). 40 4B7 (6.1%), 4X7 (0.6%), 4X13 (2.5%), 4Y17 (0.3%), 6A4 (1.3%), 6D4 (1.5%), 614 (2.8%), 615 (2.4%), 6Q2 (2.9%) and 6T10 (1.2%). T h i s phenotype was d e f i n e d as a "ts-DNA " pheno-type. The remaining ts-0 l i n e s were shown e i t h e r q u a l i t a -t i v e l y or q u a n t i t a t i v e l y to i n c o r p o r a t e at g r e a t e r than 10% the normal r a t e . Most ts-0 l i n e s i n t h i s category i n c o r -porated e r r a t i c a l l y a t the r e s t r i c t i v e temperature w i t h i n d i v i d u a l c e l l s showing e i t h e r s u b s t a n t i a l or n e g l i g i b l e l a b e l l i n g . While the a l l e l i c t s - f a t s , 2A2 and 4Y17, showed r e l a -t i v e l y comparable p a t t e r n s , the leaky a l l e l e 4B8 i n c o r p o -r a t e d e r r a t i c a l l y . T h i s was i n t e r p r e t t e d as p a r t i a l expres-s i o n of the 2A2/4Y17 d e f e c t i n 4B8. P r o t e i n S y n t h e s i s i n ts-0 Mutants Only those l i n e s t h a t showed a ts-DNA~ phenotype and/or a t s - f a t phenotype were s e l e c t e d f o r a n a l y s i s . 3 Baby c e l l s were incubated i n b a c t e r i z e d Cerophyl w i t h H-l e u c i n e (5 uCi/ml) added for-om t=3.5 hr to t=4.0 h r . The temperature s h i f t was a t about t=5 min and the c e l l s were d r i e d on s l i d e s a t t=4.0 h r . C e l l s were prepared §&r auto-radiography as d e s c r i b e d i n M a t e r i a l s and Methods. Again, a p i l o t experiment was conducted and those l i n e s showing l e s s than 10% normal i n c o r p o r a t i o n a t 34.5 C were t e n t a t i v e l y c a l l e d " t s - p r o t e i n " and not s t u d i e d f u r t h e r . W i t h i n the t s - p r o t e i n c l a s s f e l l a l l l i n e s examined except those shown i n T a b l e V I I I and a non-ts-DNA , t s - f a t l i n e 6L2 which has not been s t u d i e d q u a n t i t a t i v e l y . Table V I I I i n d i c a t e s t h a t the a l l e l i c mutants 2A2 and 4Y17 continue to s y n t h e s i z e p r o t e i n (although a t a s i g n i f i -c a n t l y reduced rate) s u b s t a n t i a l l y a f t e r they have expres-sed a blockage of DNA s y n t h e s i s . That i s , macronuclear DNA s y n t h e s i s , scheduled to commence a t t=l.3 hr (Berger, 1971) has been shown not to occur i n these l i n e s (Table VII) whereas the p r e s e n t data show t h a t s u b s t a n t i a l p r o t e i n syn-t h e s i s i s s t i l l o c c u r r i n g between t=3.5 hr and t=4.0 h r . Both. 4X11 and 4B8 show no apparent r e d u c t i o n i n p r o t e i n s y n t h e s i s , the former showing a s i g n i f i c a n t l i n c r e a s e which cannot be e x p l a i n e d . P r e l i m i n a r y A n a l y s i s of P u t a t i v e C e l l C y c l e Mutants The p u t a t i v e c e l l c y c l e mutants re c o v e r e d , s e l e c t e d mainly oh the b a s i s of a t s - f a t t e r m i n a l phenotype, were 4X11, 6L2 and the group 2A2, 4Y17 and 4B8. These f a l l i n -to two g e n e r a l c l a s s e s : (i) 2A2, 4Y17 and 4B8: These are ts-DNA - and not t s - p r o t e i n ~ , w i t h 4B8 a l e a k y a l l e l e . ( i i ) 4X11 and 6L2: These are n e i t h e r ts-DNA - nor t s - p r o t e i n ~ y e t both are t s - 0 . Only 4X11 has been f u r -t h e r c h a r a c t e r i z e d . 2A2, 4Y17 and 4X11 a l l have a s u p e r f i c i a l l y c o n s i s -TABLE V I I I H-LEUCINE INCORPORATION OF t s MUTANTS L i n e n 27 C X ± S.E. n 34.5 X C + S.E. R a t i o 34.5:27 P w i l d type 28 43.1 ± 1.8 31 45.6 + 2.7 106% 0.4536 2A2 24 49.9 ± 2.7 31 27.6 + 1.4 56% 0.0000 4B8 29 40.8 ± 1.8 30 40.2 + 2.1 101% 0.8328 4X11 27 32.5 ± 2.0 31 39.4 + 2.1 121% 0.0204 4Y17 30 48.3 ± 2.2 31 38.6 + 2.3 80% 0.0039 X i s the mean number of g r a i n s per 25 um area of cytoplasm P i s the p r o b a b i l i t y of e q u a l i t y of the 27 C and 34.5 C means as judged by a group comparison t - t e s t . 43 t e n t t s t e r m i n a l phenotype. However, i n s u f f i c i e n t a n a l y s i s has been conducted to p r e c i s e l y d e f i n e a p o i n t of a r r e s t i n the c e l l c y c l e . N e v e r t h e l e s s , the experiments shown i n F i -gure 9 i n d i c a t e t h a t i n each case there i s a r e l a t i v e l y p r e c i s e p o i n t i n the c e l l c y c l e a f t e r which the t s mutated f u n c t i o n i s no longer r e q u i r e d f o r the subsequent c e l l d i v i s i o n . T h i s p o i n t i s r e f e r r e d to as the " t s 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 " . In these experiments, synchronous samples of baby c e l l s were incubated i n i n d i v i d u a l drops a t 27 C be-tween t=0 and the time shown on the a b s c i s s a . At the time i n d i c a t e d the c e l l s were r a p i d l y s h i f t e d to 34.5 C i n a water bath. C e l l s were scored a t t=12 hr f o r the presence or absence of c e l l d i v i s i o n . The t s t r a n s i t i o n p o i n t s were thus about t=3.8 hr f o r 2A2 and 4Y17 ( e q u i v a l e n t to 0.73 of the 27 C c e l l c y c l e ) and about t=4.3 hr f o r 4X11 (equi-v a l e n t to 0.74 of the c e l l c y c l e i n t h i s mutant). 44 F i g u r e 7. T r a n s i t i o n p o i n t s of t s - f a t mutants. V e r t i c a l arrows r e p r e s e n t time of c e l l d i v i s i o n of c e l l s incubated c o n t i n u o u s l y a t 27 C. 45 SHIFT TIME IN HOURS SHIFT TIME IN HOURS -t 6 . SHIFT TIME IN HOURS 46 . DISCUSSION The BU S e l e c t i o n System Table IX summarizes the y i e l d s of the v a r i o u s c l a s s e s of t e m p e r a t u r e - s e n s i t i v e mutants recovered w i t h and without the a i d of the s e l e c t i o n system. As shown, the f r e q u e n c i e s of a l l c l a s s e s of t s mutants i n the s u r v i v i n g p o p u l a t i o n s were i n c r e a s e d g r e a t e r than t e n - f o l d by BU s e l e c t i o n . S i g n i -f i c a n t l y , the o n l y two l i n e s i s o l a t e d which were d e f i c i e n t i n DNA s y n t h e s i s while s t i l l s y n t h e s i z i n g s i g n i f i c a n t p r o t e i n (2A2 and 4Y17) were re c o v e r e d w i t h the a i d of the s e l e c t i o n system. The f a c t t h a t these two mutants are a l l e l i c and a p p a r e n t l y a l s o a l l e l i c w i t h a t h i r d "leaky" mutant (4B8) r e c o v e r e d a f t e r BU treatment, suggests t h a t the system does i n f a c t s e l e c t f o r mutants s p e c i f i c a l l y d e f e c t i v e i n DNA s y n t h e s i s . The r e s e l e c t i o n experiments of Baumann (1973) f u r t h e r support t h i s i n t e r p r e t a t i o n . In h i s experiments, Baumann mixed known numbers of 2A2;pw A c e l l s w i t h l a r g e p o p u l a t i o n s of w i l d type c e l l s , then s u b j e c t e d t h i s mixed p o p u l a t i o n to BU. s e l e c t i o n . A g r e a t e r than 7,500-fold en-richment f o r 2A2;pw A c e l l s was observed i n the p o p u l a t i o n s s u r v i v i n g s e l e c t i o n . The major shortcoming of the s e l e c t i o n system i s the poor s u r v i v a l of non-BU-substituted c e l l s d u r i n g i r r a d i a t i o n TABLE IX COMPARISON OF MUTANT YIELDS OF BU-SELECTED AND NON-SELECTED GROUPS Procedure # c e l l s be-f o r e s e l e c -t i v e t r t m t # c e l l s a f t e r t r t m t # t s l i n e s # ts-0 l i n e s # DNA-l i n e s # DNA-prot'n+ l i n e s F r a c t i o n o f t s s which are DNA-protein+ BU se-l e c t i o n 2.25 x 10 7 854 84 (9.8%) 23 (2.7%) 8 (0.9%) 2 (0.2%) 2.4% No se-l e c t i o n 16.548 16 ,548 114 (0.7%) 33 (0.2%) 6 (0.04%) 0 0 T o t a l 2.25 x 10 7 17 ,402 198 (1.1%) 56 (0.3%) 14 (0.08%) 2 (0.01%) 1.0% (Percentages i n columns 4-7 are r e l a t i v e to column 3 values.) ( F i g . 3). This near UV s e n s i t i v i t y i s of unknown b a s i s and has not been p r e v i o u s l y reported f o r P. a u r e l i a c e l l s . At the l i g h t exposures necessary to cause a s u f f i c i e n t reduc-t i o n i n the v i a b i l i t y of BU-substituted c e l l s , i t i s very probable t h a t many c e l l s which d i d i n f a c t f a i l to synthe-s i z e DNA i n the presence of BU would nevertheless be k i l l e d d uring the s e l e c t i o n process. This p r e d i c t i o n i s borne out by the data of Table IX. Within the non-selected groups the frequency of DNA mutants i s 0.04%. Assuming t h a t the p r e - s e l e c t i o n populations of the groups subjected to BU s e l e t i o n contained t h i s same f r a c t i o n of DNA mutants, one would .7 p r e d i c t t h a t w i t h i n the 2.25 x 10 c e l l s present p r i o r to 3 s e l e c t i o n there were about 8 x 10 DNA l i n e s . The f a c t t h a t only 8 DNA l i n e s were recovered a f t e r BU s e l e c t i o n sug gests t h a t the s u r v i v a l of p u t a t i v e mutants through the sys-tem was extremely low. Much more e f f i c i e n t recovery of n o n - r e p l i c a t i n g c e l l s f o l l o w i n g BU s e l e c t i o n has been observed i n mammalian c e l l c u l t u r e (Puck and Kao, 1967) and i n Physarum (Haugli and Dove, 1972). However, i n these systems, l i g h t exposures of l e s s than 2 hr d u r a t i o n are employed, these being suf-f i c i e n t to e l i m i n a t e BU-substituted c e l l s . This may e x p l a i n the r e l a t i v e l y good s u r v i v a l of n o n - r e p l i c a t i n g c e l l s t h a t i s observed. The reason f o r the r e l a t i v e i n s e n s i t i v i t y of P. a u r e l i a c e l l s to BU/near UV k i l l i n g has not been determined. R e s i s -4 9 tance to v e g e t a t i v e k i l l i n g , presumably e q u i v a l e n t to macro-n u c l e a r r e s i s t a n c e , i s probably a r e s u l t of the h i g h gene dosage of the macronucleus. Whereas c u l t u r e d mammalian c e l l s are (approximately) d i p l o i d and the f r e e l i v i n g amoebae of Physarum are h a p l o i d , the macronucleus of Para-mecium c o n t a i n s , on the average, at l e a s t 800 c o p i e s of each gene ( A l l e n and Gibson, 1972) and thus pres e n t s a r e l a t i v e -l y l a r g e number of t a r g e t s f o r i n a c t i v a t i o n . Other hypo-theses would be e i t h e r t h a t there i s r e l a t i v e l y l i t t l e BU s u b s t i t u t i o n of macronuclear DNA, t h a t the c e l l s are r e l a -t i v e l y opaque to the e f f e c t i v e l i g h t because of t h e i r t h i c k -ness or s t r u c t u r e , or t h a t there i s a h i g h l y e f f i c i e n t mecha-nism by which t h i s damage i s r e p a i r e d i n Paramecium. In support of t h i s l a s t p o s s i b i l i t y , systems r e p a i r i n g the l e -s i o n s produced by BU/near UV have been demonstrated i n E. c o l i (Ley and Setlow, 1972) and i n mammalian c e l l s (Benhur and E l k i n d , 1972). The apparent i n s e n s i t i v i t y of d i p l o i d m i c r o n u c l e i , i n -d i c a t e d by the i n s i g n i f i c a n t p o s t - s e l e c t i o n exautogamous death observed (Table I I ) , i s s u p e r f i c i a l l y p u z z l i n g . One would have p r e d i c t e d t h a t m i c r o n u c l e i , because of t h e i r lower p l o i d y , would have been more s e n s i t i v e than m a c r o n u c l e i . I t i s p o s s i b l e t h a t r e l a t i v e l y l i t t l e BU i s i n c o r p o r a t e d i n t o m i c r o n u c l e i . Baumann (1973) demonstrated a u t o r a d i o g r a p h i -c a l l y t h a t there was i n c o r p o r a t i o n of BU i n t o macronuclei under the c o n d i t i o n s of s e l e c t i o n but m i c r o n u c l e i were not 50 examined. The e x i s t a n c e of a s l o w l y a c t i n g r e p a i r system c o u l d a l s o e x p l a i n the r e l a t i v e r e s i s t a n c e of m i c r o n u c l e i . That i s , t r a n s i e n t , p o t e n t i a l l y r e p a r a b l e l e s i o n s . i n macro-n u c l e a r DNA might c o n t r i b u t e to v e g e t a t i v e k i l l i n g as macro-n u c l e a r products are e s s e n t i a l f o r v e g e t a t i v e s u r v i v a l . However, m i c r o n u c l e i are d i s p e n s i b l e f o r v e g e t a t i v e growth. Thus, breaks i n m i c r o n u c l e a r DNA would not c o n t r i b u t e to e i t h e r v e g e t a t i v e or exautogamous k i l l i n g p r o v i d e d such breaks were r e p a i r e d i n the 12-24 hr p e r i o d p r i o r t o the f i r s t p o s t - s e l e c t i o n m i t o s i s . The r e l a t i v e r e s i s t a n c e of micro-n u c l e i would t h e r e f o r e be simply a r e s u l t of t h e i r p h y s i o -l o g i c a l r o l e i n the c e l l r a t h e r than due to any i n t r i n s i c d i f f e r e n c e i n t h e i r s u s c e p t i b i l i t y to g e n e t i c damage. Ne v e r t h e l e s s , i t i s c o n c e i v a b l e t h a t the b a s i s of BU/ near UV v e g e t a t i v e k i l l i n g i s not p r i n c i p a l l y g e n e t i c (DNA) damage but r a t h e r non-genetic p h y s i o l o g i c a l damage. T h i s would c e r t a i n l y account f o r the low l e v e l of exautogamous death r e l a t i v e to v e g e t a t i v e death. However, non-genetic e f f e c t s seem u n l i k e l y and have always been assumed, i n other systems, to be of no s i g n i f i c a n c e i n BU/near UV k i l l i n g . One drawback of any s e l e c t i o n systemsis t h a t i t almost i n e v i t a b l y s e l e c t s a g a i n s t c e r t a i n c l a s s e s of i n t e r e s t i n g mutants. The p a r t i c u l a r system employed i n t h i s study se-l e c t e d a g a i n s t t s mutants which had d e f e c t s which were i r -r e v e r s i b l e f o l l o w i n g 6.5 hr a t the r e s t r i c t i v e temperature (see M a t e r i a l s and Methods). Many such mutants were i s o -l a t e d from the groups which were not subjected to BU se l e c t i o n . P a r t i c u l a r l y i n t e r e s t i n g were the c o n d i t i o n a l morph l o g i c a l and c e l l d i v i s i o n mutants which were recovered by Adele Hunter (unpublished) from the l a r g e mutagenesis 6 screening experiment. In summary, although i n e f f i c i e n t and not w e l l under-stood, the BU s e l e c t i o n system seems to be e f f e c t i v e i n s e l e c t i n g c e r t a i n c l a s s e s of t s mutants, notably those s p e c i f i c a l l y d e f e c t i v e i n DNA r e p l i c a t i o n . Temperature-sensitive mutants The main purpose of t h i s study was to i s o l a t e t s mu-tants s p e c i f i c a l l y d e f e c t i v e i n DNA synthesis a t the r e s -t r i c t i v e temperature. I t was reasoned t h a t such a mutant would, f o l l o w i n g a temperature s h i f t , s ynthesize n e g l i g i -b l e DNA, f a i l to d i v i d e , and yet continue to grow and syn-t h e s i z e p r o t e i n . Mutants not having these c h a r a c t e r i s t i c s were not analyzed i n d e t a i l . E v e n t u a l l y , only two of the 198 temperature-sensitive l i n e s , which were recovered s a t i s f i e d the c r i t e r i a which were set f o r a DNA mutant. These were the non-complementing l i n e s 2A2 and 4Y17. S p e c i f i c DNA mutants are extremely rare i n a l l sys-tems i n which comparable s t u d i e s have been attempted. H a r t w e l l (1967) i n S_. c e r e v i s i a e and Unrau and H o l l i d a y (1970) i n U. maydis both found t h a t only 1-2% of t s mu-52 t a n t s were s p e c i f i c a l l y d e f e c t i v e i n DNA s y n t h e s i s . S i m i -l a r l y , o n l y 3% of the t s mutants recovered by Wechsler and Gross (1971) i n E. c o l i f e l l i n t o t h i s c a t e gory. T h e r e f o r e , the r e s u l t s obtained w i t h P. a u r e l i a , 2.4% DNA mutants wi t h the s e l e c t i o n system and 1% DNA mutants o v e r a l l , are not a t a l l uncommon. The r e s u l t s have shown t h a t , s t r i c t l y speaking, the e f f e c t s of the two Paramecium mutations are not r e s t r i c -ted to DNA s y n t h e s i s . That i s , p r o t e i n s y n t h e s i s was r e -duced to 56% and 80% the w i l d type r a t e i n 2A2 and 4Y17 r e s p e c t i v e l y a f t e r 3.5 hr a t the r e s t r i c t i v e temperature (Table V I I I ) . However, t h i s s l i g h t e f f e c t on c e l l growth processes i s c h a r a c t e r i s t i c a l s o of the DNA mutants r e c o -vered i n other systems. The most s p e c i f i c y e ast DNA mutants s y n t h e s i z e RNA a t o n l y 6 0% the w i l d type r a t e f o l l o w i n g a temperature s h i f t ( H a r t w e l l , 1967; 1971). In U s t i l a g o , RNA s y n t h e s i s i s reduced i n d i f f e r e n t DNA mutants to be-tween 40% and 80% t h a t of w i l d type c o n t r o l s (Unrau and H o l l i d a y , 1970). One such U s t i l a g o mutant has been shown to have t e m p e r a t u r e - s e n s i t i v e DNA polymerase a c t i v i t y i n v i t r o (Jeggo, e t aT., 1973), thus i n d i c a t i n g t h a t even the most d i r e c t DNA mutation w i l l cause an accompanying r e -d u c t i o n i n growth. Apart from i t s e f f e c t on macronuclear DNA s y n t h e s i s , l i t t l e f u r t h e r i n f o r m a t i o n has been obtained concerning the f u n c t i o n mutated i n 2A2 and 4Y17. I t i s not known, 53 f o r example, i f mi c r o n u c l e a r DNA s y n t h e s i s i s a l s o a f f e c t e d because m i c r o n u c l e a r i n c o r p o r a t i o n cannot be r e l i a b l y mea-sured i n whole c e l l autoradiography. The o b s e r v a t i o n t h a t the t s 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 , 3.75 hr ( F i g . 9), i s roughly c o i n c i d e n t w i t h the completion of m i c r o n u c l e a r DNA s y n t h e s i s ( F i g . 2) i s s u g g e s t i v e . However, i n the absence of f u r t h e r a n a l y s i s , the p h y s i o l o g i c a l s i g n i f i c a n c e of t h i s t r a n s i t i o n p o i n t i s u n c e r t a i n . T h e r e f o r e , the y i e l d of s p e c i f i c DNA mutants was com-para b l e to t h a t seen i n p r e v i o u s s t u d i e s i n other systems. P r e l i m i n a r y a n a l y s i s i n d i c a t e s t h a t the mutants recovered i n the prese n t study have c h a r a c t e r i s t i c s s i m i l a r to those seen f o r s p e c i f i c DNA mutants recovered i n other organisms. The P. a u r e l i a mutants recovered w i l l , h o p e f u l l y , p r o v i d e i n s i g h t i n t o the o r g a n i z a t i o n of the c e l l c y c l e . 54 BIBLIOGRAPHY A l l e n , S. and I. Gibson (1972) Genome a m p l i f i c a t i o n and gene e x p r e s s i o n i n the c i l i a t e macronucleus. Bio c h e m i c a l Genetics 6_: 293-313. Baumann, P.J.B. (1973) A s e l e c t i o n system f o r temperature-s e n s i t i v e mutants i n Paramecium a u r e l i a . 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 Columbia. Benhur, E. and M.M. E l k i n d (197 2) Damage and r e p a i r of DNA i n 5-bromodeoxyuridine-labelled Chinese hamster c e l l s exposed to f l u o r e s c e n t l i g h t . B i o p h y s i c a l J o u r n a l 12-: 636-646. Berger, J.D. (1969) Nuclear 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 Paramecium a u r e l i a . . D i s s e r t a t i o n , Indiana U n i v e r s i t y . (1971) K i n e t i c s of i n c o r p o r a t i o n of DNA pre -c u r s o r s from i n j e s t e d b a c t e r i a i n t o macronuclear DNA i n Paramecium a u r e l i a . J o u r n a l of Protozoology 18: 419-429. Berger, J.D. and R.F. Ki m b a l l (1964) S p e c i f i c i n c o r p o r a -t i o n of p r e c u r s o r s i n t o DNA by f e e d i n g l a b e l l e d bac-t e r i a to Paramecium a u r e l i a . J o u r n a l of Protozo o l o g y 11: 534-537. Boyce, R. and R. Setlow (1963) The a c t i o n s p e c t r a f o r u l t r a -v i o l e t l i g h t i n a c t i v a t i o n of systems c o n t a i n i n g 5-bromo-u r a c i l - s u b s t i t u t e d DNA. B i o c h i m i c a e t B i o p h y s i c a A c t a 6^:446-454. C a r l , P.L. (1970) E s c h e r i c h i a c o l i mutants w i t h temper-a t u r e - s e n s i t i v e s y n t h e s i s of DNA. Mo l e c u l a r and General G e n e t i c s 109: 107-122. Gross, J.D. (1972) DNA r e p l i c a t i o n i n b a c t e r i a . 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M o l e c u l a r and General Genetics 118: 109-124. Hutchinson, F. (1973) Mechanism of the s e n s i t i z a t i o n of DNA to UV l i g h t by the i n c o r p o r a t i o n of 5-bromouracil. Q u a r t e r l y Review of B i o p h y s i c s 6_: 201-246. Jeggo, P.A., P. Unrau, G.R. Banks and R. H o l l i d a y (1973) A t e m p e r a t u r e - s e n s i t i v e DNA polymerase mutant of U s t i l a g o maydis. Nature New B i o l o g y 24 2: 14-15. Kung, C. 1(1971) Genie mutants wi t h a l t e r e d systems of ex-c i t a t i o n i n Paramecium a u r e l i a I I : Mutagenesis, s c r e e n i n g and g e n e t i c a n a l y s i s of the mutants. Genetics 6_9: 29-45. Ley, R.D. and R.B. Setlow (1972) Rapid r e p a i r of l e s i o n s induced by 313 nm l i g h t i n b r o m o u r a c i l - s u b s t i t u t e d DNA of E s c h e r i c h i a c o l i . B i o c h e m i c a l and B i o p h y s i -c a l Research Communications 46_: 1089-1094. L i s k a y , M. 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(1969) R e g u l a t i o n of c e l l d i c i s i o n i n E s c h e r i  c h i a c o l i . B i o p h y s i c a l J o u r n a l 9_: 90-112. 56 Puck, T.T. and F.T. Kao ( 1 9 6 7 ) Genetics of somatic mammalian cells V: Treatment with 5-bromouracil and visible light for isolation of nutritionally deficient mutants. Proceedings of the National Academy of Science £8: '1227-1 23k. Roscoe, D.H., M. Read and H. Robinson (1973a) Isolation of temperature-sensitive mammalian cells by selective detachment. Journal of Cellular Physiology 82: 325-332 . .Roscoe, D.H.j H. Robinson and A.W. Carbonell (1973b) DNA synthesis and mitosis in a temperature-sensitive Chinese hamster ce l l l ine. Journal of Cellular Physiology 82_: 333-338 . Slater, M. and M. Schaechter (197U) Control of division in bacteria. Bacteriological Reviews 38.: 1 99-222. Smith, B.J. and N.M. Wigglesworth (1973a) A ce l l line which is tem-perature-sensitive for cytokinesis. Journal of Cellular Physi-ology 80 : 253-260. - • \ (1973b) A temperature-sensitive function in a Chinese hamster line affecting DNA synthesis. Journal of Cellular Physiology 82_: 339-3^8. t Sonneborn, T.M. (1970) Methods in Paramecium research. Methods in Cell Physiology U : 182. (1971+) Genetics of the 1qi species of Paramecium -aurelia. Handbook of Genetics, vol ; II, ed. R.C. King. Plenum Press, New York, N.Y. Spratt, B.G. and R.J. Rowbury (1971) Physiological studies on a mutant of Salmonella typhimurium which is temperature-sensi-. tive for DNA synthesis . Molecular and General Genetics 11U:. 35 -U9 . ' Unrau, P. and R. Holliday (1970) A search for temperature-sensitive mutants of Ustilago maydis. Genetical Research 1_5_: 1J|>7-167. Wang, R. (197U) Nature, in press. < Woodard, J.,Gelber., B., and H. Swift (1961) Nucleoprotein changes during the mitotic cycle in Paramecium aurelia. Experimental Cell Research 2 3 : ; 258-26U. 1 / 

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