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The isolation and characterization of recessive meiotic mutants in Neurospora crassa DeLange, Aloysius 1980

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THE ISOLATION AND CHARACTERIZATION OF RECESSIVE MEIOTIC MUTANTS IN NEUROSPORA CRASSA by A l oy s i u s M. DeLange B.Sc., U n i v e r s i t y of B r i t i s h Columbia, 1973 M . S c , U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department o f Botany (Genet ic s ) We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA May 1980 © A loy s i u s M. DeLange, 1980 In present ing t h i s t he s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree tha 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 re ference and study. I f u r t h e r agree that permiss ion f o r ex tens i ve copying of t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep r e s en t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t he s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n permi s s ion . Department of BOTANY f &£NB1\C^) The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 Date . ABSTRACT The study of the genet i c c on t r o l of meios i s has been i n i -t i a t e d i n Neurospora c ras sa by the i s o l a t i o n of r ece s s i ve m e i o t i c mutat ions . These mutations were detected by t h e i r reduced f e r t i l i t y or by the abor t i on of ascospores. To a l l ow t h e i r e xp re s s i on , r e -ces s i ve me i o t i c mutations were made homozygous by s e l e c t i n g (n + 1) d i somic ascospores. Cu l tu res produced by each of these ascospores conta in two types of nuc l e i w i th i d e n t i c a l genes ( i n c l u d i n g mutat ions) on a l l chromosomes except l i nkage group (LG) I, which conta ins the mating type l o cu s . The simultaneous presence of these two types of nuc l e i a l lows the i n i t i a t i o n of the sexual c y c l e , and t he re f o re the de tec t i on of r e ce s s i ve mutations a f f e c t i n g the sexual c y c l e , i n c l u d i n g m e i o s i s , on any chromosome except LG I. Using t h i s method, three major c l a s se s of mutants have been de tec ted . F i r s t , e leven mutants a f f e c -t i n g p e r i t h e c i a l development were expressed on ly as the maternal parent. Second, t h i r t e e n mutants produced p e r i t h e c i a w i th few or no ascospores. The i n f e r t i l i t y i n two of these mutants was d e f i n i t e l y caused by r ece s s i ve mutations (asc-2 and a s c - 4 ) . F i n a l l y , the abor -t i o n of many ascospores was detected i n t h i r t e e n mutant s t r a i n s . Among these s t r a i n s , s i x r e ce s s i ve mutations ( a s c - 1 , a s c - 3 , a s c - 5 , a s c - 6 , a s c - 7 , and asc-8) caused the abo r t i on of many ascospores. The dominant mutat ion SK(ad-3A) was detected i n t h i s screen f o r r ece s s i ve mutat ions , because i t caused ascospore abor t i on when crossed w i t h an ad-3A mutant but not w i t h a w i l d type s t r a i n . Th i s muta t i on , appar-e n t l y a l l e l i c to ad-3A, caused the abo r t i on of a l l ad -3A-conta in ing ascospores. i i The three ascospore abo r t i on - t ype mutants a s c - 1 , a s c - 3 , and asc-6 were analyzed i n more d e t a i l us ing both c y t o l o g i c a l and genet i c methods. Ascospore abo r t i on i n these mutants was caused by abnormal d i s j u n c t i o n of me i o t i c chromosomes. In mutants asc-1 and a s c - 6 , the primary de fec t i n p a i r i n g of homologs dur ing the f i r s t m e i o t i c prophase was f o l l owed by the format ion of un i va len t s at metaphase I. Observa-t i on s on these mutants and on the mei-1 mutant ( p rev i ou s l y i s o l a t e d ; see Smi th , 1975) suggested equat iona l centromere d i v i s i o n o f many u n i -va len t s a t anaphase I. Subsequent i r r e g u l a r and prolonged separa t i on of chromosomes at the second m e i o t i c d i v i s i o n appeared to be a secon-dary e f f e c t o f the abnormal f i r s t d i v i s i o n . The asc-3 mutant had a de fec t i n ascus f o rma t i on , and l a t e r i n d i s j u n c t i o n dur ing the second m e i o t i c and po s t -me i o t i c d i v i s i o n s . The f i r s t - a c t i n g de fec t before or dur ing karyogamy r e s u l t e d i n the abo r t i on of most c e l l s . Some c e l l s managed to proceed past t h i s b lock . During the second m e i o t i c d i v i s i o n most chromosomes of the few r e s u l t i n g a sc i were attached to only one of the two s p i n d l e - p o l e bod ies . D i s j u n c t i o n a t the po s t -me i o t i c d i v i -s ion was a l s o h i gh l y i r r e g u l a r . This mutant appeared to be d e f e c t i v e i n the attachment of one s p i n d l e - p o l e body to a se t of centromeres. The de fec t may i n vo l ve e i t h e r a centromere-assoc iated product or a s p i n d l e - p o l e body. TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS — — i v LIST OF TABLES — - v LIST OF FIGURES - — v i i ACKNOWLEDGEMENT - - v i i i PREFACE i x INTRODUCTION \ CHAPTER I Me ios i s i n Neurospora c r a s s a . I. The I s o l a t i o n of Recess ive Mutants De fec t i ve i n the Product ion of V i a b l e Ascospores 1 5 a INTRODUCTION 16 MATERIALS AND METHODS - 21 RESULTS 29 DISCUSSION - - - 36 CHAPTER II Me ios i s i n Neurospora c r a s s a . I I . Genet ic and C y t o l o g i c a l C h a r a c t e r i z a t i o n o f Four M e i o t i c Mutants 4 l a INTRODUCTION — — — — — 42 MATERIALS AND METHODS - — — 43 RESULTS — 57 DISCUSSION — - - 99 CHAPTER I I I The Mutat ion SK(ad-3A) A l t e r s the Dominance of ad-3A+ Over ad-3A i n the Ascus o f Neurospora 1 1 6 a INTRODUCTION - 117 MATERIALS AND METHODS - 119 RESULTS — — 123 DISCUSSION - - — 132 CHAPTER IV General D i s cu s s i on - 1 3 6 a BIBLIOGRAPHY — - — - 143 APPENDIX: L i s t o f Abb rev i a t i on s 152 i v LIST OF TABLES Table Page CHAPTER I I I n i t i a l c h a r a c t e r i z a t i o n o f 1^5 pseudo-wi ld type c u l t u r e s w i th aber rant c r o s s i n g behavior 30 II The phenotypes o f r e ce s s i ve c l a s s II mutations a t e i g h t l o c i , whose w i l d type a l l e l e s are necessary f o r the forma-t i o n of normal a sc i or b lack ascospores 35 CHAPTER II I Genet ic a na l y s i s of four crosses homozygous f o r asc-6 59 II Ascus a na l y s i s of a cross homozygous f o r asc-6 (879al3 x 879A15) 61 I I I I s o l a te s obta ined from a s c i , produced by a cross (879al3 x 879A15) homozygous f o r a s c - 6 , which con ta i n a t l e a s t one PWT c u l t u r e • 62 IV Simultaneous nond i s j unc t i on o f th ree l i n kage groups i n two crosses homozygous f o r asc-6 • 64 V Progeny ana l y s i s of crosses homozygous f o r asc -3 (P243 or P393) — — 69 VI Con i d i a l i s o l a t e s from rep re sen ta t i ve s of a l l four types of apparent ly PWT progeny from crosses homozygous f o r asc-3 71 VII Ascus a n a l y s i s o f crosses homozygous f o r a sc -3 76 V I I I Recombination and nond i s j unc t i on i n three crosses homo-zygous f o r asc-1 82 IX Nature of growth o f ascospores from crosses homozygous f o r asc-1 83 X Ascus a na l y s i s of a cross homozygous f o r the r ece s s i ve me i o t i c mutat ion asc-1 (P95) (95A29 x 95a43) 86 XI C h a r a c t e r i s t i c s of f ou r r e ce s s i v e me i o t i c mutations i n Neurospora crassa w i th a de fec t i n the r egu l a r d i s -j u n c t i o n o f chromosomes 100 v <> Table Page CHAPTER I I I I Ana l y s i s of i s o l a t e s from crosses between P917 to OR-A and OR-a w i l d type s t r a i n s 124 II Genotype of 79 i s o l a t e s from the cross between s t r a i n s 917A36 and 917a38 ( f o r LG I markers, see F i g . 1) which produced about 60% aborted ascospores 127 I I I Ana l y s i s o f c o n i d i a l i s o l a t e s from a heterokaryon be-tween s t r a i n 917A36 ( l e u - 3 , a_, a r g - 1 , SK(ad-3A), ad-3B) and 2-17-825a ( a , ad - I A T ^ - 133 v i LIST OF FIGURES F igure Page INTRODUCTION 1. Representat ion o f meios i s as a s p e c i a l i z e d form o f the m i t o t i c c y c l e • 6 CHAPTER I 1. General o u t l i n e of a s e l e c t i v e system used to i s o l a t e r e ce s s i v e m e i o t i c mutants i n Neurospora c rassa 22 CHAPTER II 1. The two nuc lear components o f PWT cu l t u r e s i n which asc mutations were recovered 44 2. A cross homozygous f o r a sc -6 , designed to t e s t nondis -j u n c t i o n of three l i nkage groups (LG I, IV, and V) s imu l taneous l y 46 3. Expected PWT progeny r e s u l t i n g from nond i s j unc t i on dur ing the f i r s t m e i o t i c d i v i s i o n ( a ) , and a c ros sover f o l l owed by nond i s j unc t i on dur ing the second me i o t i c d i v i s i o n (b) -• 52 4. Chromosome development i n crosses homozygous f o r asc-6 66 5. The o r i g i n of PWT progeny from crosses homozygous f o r asc -3 72 6. Chromosome development i n crosses homozygous f o r asc-3 78 7. Chromosome development i n crosses homozygous f o r asc-1 88 8. Chromosome development i n crosses homozygous f o r mei-1 93 9. P o s s i b l e exp lana t i on f o r abnormal d i s j u n c t i o n i n asc-3 mutant crosses • • 114 CHAPTER I I I 1. The two-nuc lear components of s t r a i n P917 120 2. S e l e c t i o n of ad_+ recombinants to l o c a l i z e the SK(ad-3A) mutat ion w i th re spect to the ad-3A and ad-3B l o c i • 130 v i i ACKNOWLEDGEMENT I express my s i n c e r e g r a t i t u d e to my research a d v i s o r , Dr. A. J . F. G r i f f i t h s , f o r h i s encouragement, e x c e l l e n t s c i e n t i f i c guidance and a s s i s t ance throughout t h i s s tudy. I thank Drs. C. 0. Person, K . Co l e , D. Holm, and J . Berger f o r s e r v i ng on my Committee. v i i i PREFACE This t he s i s i s composed o f f ou r part s plus an i n t r o d u c t i o n . The f i r s t three chapters are presented i n a form s u i t a b l e f o r sub-sequent p u b l i c a t i o n . Chapter I desc r ibes the use o f a new system i n Neurospora c rassa tha t enables the i s o l a t i o n o f r e ce s s i v e mutants d e f e c t i v e i n the product ion of v i a b l e ascospores. The genet i c and c y t o l o g i c a l c h a r a c t e r i z a t i o n of some of these mutants has been des-c r i b e d i n Chapter I I . Chapter I I I desc r ibes the study o f an a sco -spore abo r t i on - t ype mutant which s p e c i f i c a l l y aborts ad -3A-conta in ing ascospores. The f i n a l chapter i s a general d i s cu s s i on and concentrates on the f u t u r e approaches and uses of these types of mutants of Neuro- spora . i x 1 INTRODUCTION 2 This study was i n i t i a t e d to ga in a b e t t e r i n s i g h t i n t o the genet i c c o n t r o l o f me i o s i s . The fungus Neurospora c ras sa appeared i d e a l l y s u i t e d to conduct such research because both c y t o l o g i c a l and genet i c means o f a na l y s i s are e x c e l l e n t . However, t h i s organism was l a c k i n g i n one main a spec t , namely the a v a i l a b i l i t y o f ; a s e l e c -t i o n system tha t would enable the i s o l a t i o n of r e ce s s i v e m e i o t i c mutat ions . There fo re , I have dev ised a system tha t enables the r ap i d screen ing o f s e l e c t ed c u l t u r e s f o r such mutat ions . The succe s s fu l use of t h i s system w i l l be desc r ibed i n Chapter I o f t h i s t h e s i s . A combinat ion o f c y t o l o g i c a l and genet i c analyses of these mutations should be ins t rumenta l i n ga in ing a more complete understanding of the genet i c c o n t r o l of me i o s i s . The p r i n c i p a l fea tu res t ha t render the c h a r a c t e r i z a t i o n of me i o t i c mutants so e f f e c t i v e i n Neurospora are g iven below. C y t o l o g i c a l Ana l y s i s The chromosomes, n u c l e o l i , s p i nd le s and s p i n d l e - p o l e bodies can be e f f e c t i v e l y f o l l owed dur ing a l l stages of m e i o t i c development, s t a r t i n g before karyogamy u n t i l ascospore format ion (McC l in tock , 1945; S i n g l e t o n , 1953; Raju and Newmeyer, 1977; Lu and G a l e a z z i , 1979). AIT: four teen (- 2N) chromosomes i n d i v i d u a l i z e s h o r t l y a f t e r karyogamy. During zygotene and pachytene of the f i r s t prophase, these chromo-somes e longate and the homologs p a i r . Th is i s u s ua l l y f o l l owed by two r ap i d me i o t i c d i v i s i o n s , a p o s t -m e i o t i c d i v i s i o n , and f i n a l l y the enc losure of each nucleus i n an ascospore. The chromosomes i n d i v i d u a l i z e 3 a t each prophase. Thus i t i s o f ten po s s i b l e to determine the number of chromosomes per nucleus j u s t p r i o r to each d i v i s i o n , and t he re f o re any de fec t ( f o r example, caused by a mutat ion) i n the r egu l a r segre-ga t i on of chromosomes. Genet ic Ana l y s i s Severa l aspects of the l i f e c y c l e o f Neurospora c ra s sa render i t i d e a l f o r both the de t e c t i o n and genet i c c h a r a c t e r i z a t i o n of me i o t i c mutat ions : (1) Whole popu lat ions o f a s c i can be q u i c k l y scanned and a l l i n d i -v i dua l products of meios i s ( i . e . , ascospores) are r e cove rab l e . (2) Most v i a b l e and i n v i a b l e ascospores can be d i s t i n g u i s h e d on the bas i s of c o l o r : v i a b l e ascospores are i n v a r i a b l y b l a c k , whereas i n v i a b l e ascospores are o f ten wh i t e . Th is d i s t i n c t i o n i s espec-i a l l y usefu l because i t enables the de tec t i on and c h a r a c t e r i z a -t i o n of mutants w i th a de fec t i n the d i s j u n c t i o n of chromosomes. Such d i s j u n c t i o n defect s would produce ascospores which e i t h e r miss one or more chromsome(s) or have ex t ra chromosomes. The former type of ascospore:;would be whi te and the l a t t e r b lack (McC l in tock , 1945; Coyle and P i t t e n g e r , 1965). (3) The apparent v i a b i l i t y of aneuplo id products (n + 1, n + 2, e t c . ) g r e a t l y f a c i l i t a t e s the study of mutants w i th d i s j u n c t i o n d e f e c t s . Aneuplo id products can be detected by the complemen-t a t i o n of mutations ( u s ua l l y auxot roph ic ) on two homologous copies of a chromosome. They are c a l l e d pseudo-wi ld t ype , or PWT, because, even though they conta in mutant n u c l e i , they 4 appear to be w i l d type. Nond i s junct ion can be detected a t both the f i r s t and second d i v i s i o n prov ided the chromosome te s ted i s a p p r o p r i a t e l y marked (a more d e t a i l e d d e s c r i p t i o n of t h i s i s g iven i n M a t e r i a l s and Methods of Chapter II of t h i s t h e s i s ) . (4) A l l products (ascospores) from each p a r t i c u l a r meios i s can be s t ud i ed . The pat terns of white and b lack ascospores i n each ascus, and the analyses of genotypes of the v i a b l e b lack ascospores may prov ide i n fo rmat ion on the nature o f i r r e g u l a r d i s j u n c t i o n of chromosomes. (5) The a v a i l a b i l i t y of s p e c i a l l i nkage t e s t e r s t r a i n s makes the mapping o f new mutations a r e l a t i v e l y f a s t procedure i n Neurospora c rassa (Perk ins and Bjorkman, 1979). Mapping of mutations w i th known de fec t s would a l l ow the d e t e c t i o n of gene c l u s t e r s o f r e l a t e d f u n c t i o n s . The use of the genet i c and c y t o l o g i c a l a na l y s i s of me i o t i c mutants appears e x c e l l e n t i n Neurospora. However, even though such ana l y s i s would prov ide i n fo rmat ion on the c on t r o l of the development and movement of macromolecular aggregates ( e . g . , chromosomes, chroma-t i d s , and s p i n d l e - p o l e bod i e s ) , i t would probably not prov ide any de-t a i l e d understanding of molecu lar processes t ha t c on t r o l the a c t i o n of these aggregates. The understanding of these processes would r equ i r e ana l y s i s by means of e l e c t r o n microscopy and b iochemi s t r y . The present work should t he re fo re be seen as a framework from which more d e t a i l e d analyses could be c a r r i e d out. For example, the p a i r i n g of homologous chromosomes dur ing the f i r s t prophase of meios i s has a l ready been 5 s u c c e s s f u l l y s tud ied i n Neurospora us ing e l e c t r o n microscopy ( G i l l i e s , 1972). Thus f a r , the c h a r a c t e r i s t i c s o f the l i f e c y c l e o f Neurospora have made b iochemical a na l y s i s of meios i s d i f f i c u l t but such a n a l y s i s w i l l probably become f e a s i b l e i n the near f u t u r e . Before proceeding to d i scuss m e i o t i c mutations i n general and the i s o l a t i o n o f them i n Neurospora (see Chapter I ) , a d e s c r i p t i o n of some c r i t i c a l processes of meios i s should serve to focus on the main problems t ha t need to be s o l ved . The major landmark events of the me i o t i c c y c l e are s chemat i c a l l y represented i n F igure 1. This c y c l e appears to represent a m o d i f i c a -t i o n of the m i t o t i c c y c l e . The f o l l o w i n g c r i t i c a l processes are cha r -a c t e r i s t i c of me io s i s : the i n i t i a t i o n o f m e i o s i s , recombinat ion of genes, the r educ t i ona l d i v i s i o n , and the r e t u rn to the m i t o t i c c y c l e . I n i t i a t i o n of Me ios i s The i n i t i a t i o n of the m e i o t i c d i v i s i o n c y c l e appears to take p lace dur ing the G l p e r i o d . Nuc le i t ha t have been committed to meios i s can be d i s t i n g u i s h e d by t h e i r subsequent c h a r a c t e r i s t i c pa t te rn o f DNA r e p l i c a t i o n (see next s e c t i o n ) . However, even though the nuc l e i are committed to some e a r l y m e i o t i c event s , they are not y e t i r r e v e r s i b l y committed to complete the m e i o t i c c y c l e . In s tead, they may, i n the ab-sence of the app rop r i a te genet i c or environmental c o n t r o l s , r e v e r t back to the m i t o t i c c y c l e . The processes which i r r e v e r s i b l y commit the d i f f e r e n t m e i o t i c events w i l l be d i scus sed i n the f o l l o w i n g s e c t i o n s . The i n i t i a t i o n of the m e i o t i c c y c l e may be c o n t r o l l e d by env i r ronmental c o n d i t i o n s , i n t e r n a l s i g na l s generated by development i n m u l t i c e l l u l a r organisms, or a combinat ion of these. In some u n i c e l l u l a r F igure 1. Representat ion of meios i s as a s p e c i a l i z e d form of the m i t o t i c c y c l e . The m i t o t i c c y c l e c o n s i s t s of m i t o s i s (M), G I , DNA synthes i s ( S ) , and G2. At a s p e c i f i c .po in t dur ing the GI phase, meios i s i s i n i t i a t e d . A cho ice i s made here between con t i nu ing the m i t o t i c c y c l e or i n i t i a t i n g me i o s i s . This cho ice depends on environmental c l u e s . A s e r i e s of subsequent steps u l t i m a t e l y lead back i n t o the m i t o t i c c y c l e . premeiotic division 8 microorganisms, meios i s i s i n i t i a t e d when the sub s t ra te (food source) i s dep le ted . In f a c t , i n the. budding yeas t Saccharomyces c e r e v i s i a e , the i n i t i a t i o n o f meios i s i s repressed by n i t rogen and g lucose. That t h i s r ep re s s i on i s under genie c o n t r o l has been demonstrated by the i s o l a t i o n o f mutants which are i n s e n s i t i v e to i t (Dawes, 1975). In y e a s t , f o l l o w i n g the i n i t i a t i o n s i g n a l , mating hormones are produced which a r r e s t nuc l e i a t a po i n t dur ing the GI pe r i od (see F i g . 1 ) . * Subsequently, meios i s o f most c e l l s i s i n i t i a t e d i n a synchronous manner. In many m u l t i c e l l u l a r organisms, a number of (germ) c e l l s are apparent ly e s p e c i a l l y programmed to undergo me io s i s . In these cases , the s t a t e of development of the i n d i v i d u a l w i l l generate the requ i red s i g n a l s t ha t are necessary f o r the i n i t i a t i o n o f me i o s i s . S ince these organisms are g e n e r a l l y p ro tec ted from environmental changes by i n t e r n a l homeostatic c o n t r o l , i n i t i a t i o n should not be dependent on environmental c o n d i t i o n s . In c o n t r a s t , i n i t i a t i o n i n a number o f microorganisms, e . g . , many fungi and c e l l u l a r s l ime molds, depends on environmental and de-velopmental s i g n a l s . In g ene r a l , n u t r i t i o n a l d e f i c i e n c y s i g na l s the i n i t i a t i o n o f the format ion of f r u i t i n g . b o d i e s which i nvo l ve s a s e r i e s of developmental steps tha t cu lminate i n the i n d u c t i o n o f me i o s i s . In many f u n g i , s p e c i f i c mating type genes are i n vo l ved i n the product ion of f r u i t i n g bodies ( e . g . , Fincham and Day, 1971).- However, i t i s not This stage may correspond to G 0 i n many m u l t i c e l l u l a r organisms s i nce c e l l s may spend a long time a t these po in t s w i thout l o s i n g t h e i r v i a b i l i t y . In c o n t r a s t , when the c y c l e i s a r r e s t ed a t any o the r p o i n t , e . g . , by means of a t empera tu re - sen s i t i ve c e l l c y c l e mutant, v i a b i l i t y r a p i d l y dec l i n e s as a f u n c t i o n o f i n cuba t i on a t the r e s t r i c t i v e tempera-t u r e . 9 known whether these genes are a l s o i n v o l v e d i i n the i n i t i a t i o n o f me io s i s . The study of mutations w i th de fect s p r i o r to meios i s i t s e l f may become ins t rumenta l i n the understanding of the genet i c c on t r o l of the i n i t i a -t i o n o f me i o s i s . M e i o t i c Recombination Recombination i s the c e n t r a l component of the m e i o t i c process . The amount of recombinat ion can be v a r i e d i n two ways: a change i n chromosome number, or a change i n c ro s s i ng -ove r between homologous chromosomes ( i . e . , intrachromosomal recombinat ion ) . The independent assortment of chromosomes dur ing the me i o t i c d i v i s i o n s generates new combinations of chromosomes from the two parent n u c l e i . Thus, an i n -crease i n chromosome number inc reases the number of p o s s i b l e new combina-t i on s o f genes. Intrachromosomal recombinat ion , i n v o l v i n g gene conver-s i on - and c ro s s i ng -ove r or exchange, requ i re s a h i g h l y coord inated sequence of events which recur dur ing each me i o t i c c y c l e . The frequency o f c r o s s i ng -ove r depends on the amount o f accurate p a i r i n g o f homologous s t r e t che s of DNA w i t h i n the chromosomes and the e f f i c i e n c y of the ex -change events . The recovery of a l a rge number of mutations w i th aber -ran t p a i r i n g or exchange ( f o r review,, see Baker e t a l_ . , 1976a), i n d i c a t e tha t these processes are under s t r i c t genet i c c o n t r o l . The temporal stages t ha t are e s s e n t i a l f o r the c on t r o l of p a i r -ing and exchange can be i n v e s t i g a t e d by i n t e r f e r i n g w i th s p e c i f i c stages of more or l e s s synchronously d i v i d i n g popu lat ions o f m e i o t i c c e l l s . I t has been po s s i b l e t o . i n t e r f e r e w i t h meios i s us ing temperature shock, temporary a p p l i c a t i o n o f i n h i b i t o r s , e x p l a n t a t i o n of me i o t i c c e l l s from germ t i s s u e to s y n t h e t i c medium ( e . g . , S tern and Ho t ta , 10 1967; 1973), or t empe ra tu re - s en s i t i v e mutations ( e . g . , G r e l l , 1978). The e f f e c t of these manipu lat ions on p a i r i n g and exchange has shown tha t processes t ak ing p lace j u s t p r i o r to the p r e -me i o t i c S phase u n t i l pachytene are requ i red f o r normal p a i r i n g and exchange. The e a r l i e s t stage i n which normal f u n c t i o n i s necessary f o r the occurrence of r e gu l a r exchange has been detected i n Chlamydomonas (Chiu and Has t ing s , 1973). Recombination was inc reased a f t e r t r e a t -ment w i th phenethyl a l coho l or mitomycin C j u s t p r i o r to the p re -me i o t i c S phase. The reason f o r t h i s e f f e c t i s not c l e a r s i nce these i n h i b i t o r s have q u i t e d i f f e r e n t s p e c i f i c i t i e s . There are severa l l i n e s of evidence suggest ing the involvement of p r e -me i o t i c DNA syn thes i s i n the recombinat ion process . F i r s t , a f u n c t i o n unique to meios i s i s suggested by the extended per iod of t ime r equ i r ed to complete t h i s p r e -me i o t i c S phase. Th is i s apparent ly due to a reduced number o f i n i t i a t i o n s i t e s f o r DNA r e p l i c a t i o n ( C a l l a n , 1973). The importance o f the p r e -me i o t i c S phase i s a l s o c l e a r from the a n a l y s i s of mutations tha t are d e f e c t i v e i n the process i n Saccharo- myces . None of these mutants develop synaptonemal complexes, although a x i a l cores may be produced. In appears t ha t these mutants are never committed to p a i r i n g and me i o t i c recombinat ion. I t f o l l ows tha t p r e -me i o t i c DNA synthes i s i s e i t h e r r equ i r ed f o r c e n t r a l element assembly i n t o synaptonemal complexes or t ha t both of these events are s ub jec t to common genet i c c o n t r o l (Moens e_t a l _ . , 1976). Second, i n L i l i u m , 0.3% o f DNA synthes i s i s delayed u n t i l e a r l y zygotene when p a i r i n g takes p lace (Ho t ta , I to and S t e r n , 1966; Hotta and S t e r n , 1971; Stern and Ho t t a , 1977). The i n h i b i t i o n o f t h i s delayed 11 r e p l i c a t i o n a t e a r l y zygotene complete ly b locks p a i r i n g o f homologs, suggest ing tha t t h i s r e p l i c a t i o n i s necessary f o r the p a i r i n g process i n L i l i um. ; T h i r d , temporary a p p l i c a t i o n of x - i r r a d i a t i o n o r i n h i b i t o r s of DNA syn thes i s dur ing the p r e -me i o t i c S phase of Chlamydomonas r e s u l t e d i n i nc reased exchange.. In a d d i t i o n , temperature shocks a t t h i s stage caused a l t e r e d chiasma and c ro s s -ove r f requenc ies i n severa l spec ies (reviewed by S tern and Ho t t a , 1973). Even though the ex tent and d i r e c -t i o n of the e f f e c t on exchange v a r i e s i n d i f f e r e n t s p e c i e s , the r e s u l t s c l e a r l y suggest an involvement of the p re -me i o t i c S phase i n the r e -combination process . Fou r th , i n Saccharomyces, commitment to exchange i nvo l ve s s i n g l e -s t rand s c i s s i o n s o f chromosomal DNA and apparent ly i s a r e v e r s i b l e c e l l u -l a r stage achieved dur ing the p r e - m e i o t i c S phase ( S i l v a - Lopez e t a l . , 1975; Jacobsen e t a l _ . , 1975). F i f t h , the s e n s i t i v e per iod of a t empera tu re - sen s i t i v e recom-b i n a t i o n d e f i c i e n t mutant i n Drosoph i la occurs dur ing the p re -me i o t i c S phase ( G r e l l , 1978). . S ho r t l y a f t e r the p r e -me i o t i c S phase, c e l l s i n L i l i u m and Sac- charomyces become committed to me i o s i s . , Meiocytes of L i l i u m tha t are exp lanted onto s y n t h e t i c medium a f t e r t h i s commitment step proceed through an extended prophase c h a r a c t e r i s t i c o f meios i s (Stern and Ho t t a , 1967). When meiocytes are exp lanted s h o r t l y a f t e r t h i s commitment s t e p , l i t t l e or no p a i r i n g r e s u l t s a t the next prophase. In a d d i t i o n , most chromosomes i n me iocy te s . tha t have been exp lanted near or a t l eptotene w i l l undergo unstable p a i r i n g . In e i t h e r case, un i va len t s are g ene r a l l y 12 produced a t metaphase I. Thus i t appears t ha t both the achievement and s t a b i l i z a t i o n of p a i r i n g of homologous chromosomes d u r i n g ' t h e me i -o t i c prophase r equ i r e the a c t i v i t y of genes or t h e i r products w e l l be-f o r e the ac tua l p a i r i n g process . Other components necessary f o r p ro -per p a i r i n g and exchange are on ly requ i red dur ing these processes . For example, DNA synthes i s i s r equ i red dur ing zygotene. The i n h i b i -t i o n o f DNA synthes i s a t e a r l y zygotene causes c e l l abo r t i on by p re -vent ing the delayed semi conse rva t i ve r e p l i c a t i o n o f 0.3% o f t o t a l DNA needed f o r p a i r i n g o f homologs. I n h i b i t i o n a t mid-zygotene causes ex tens i ve f ragmentat ion o f chromosomes at l a t e r s tages , w h i l e a t l a t e zygotene i t produces chromatid breaks which are mainly observed as br idges and breaks a t the second m e i o t i c d i v i s i o n (S tern and Ho t t a , 1967). The phenotypes obta ined i n these s tud ie s have a l s o been ob-served i n mutant meiocytes o f many s p e c i e s . Mutants w i t h p a i r i n g de-f e c t s i n p lant s are r e f e r r e d to as a s ynde t i c . These may be a synapt i c i f a de fec t i n the achievement of p a i r i n g i s i n v o l v e d , or desynapt ic i f chromosome p a i r s are unstab le and subsequently f a l l apar t to form un i va len t s a t metaphase I. Other mutants a f f e c t chromosome i n t e g r i t y . These may i n vo l ve gene products that are necessary f o r DNA synthes i s dur ing zygotene. In D r o s oph i l a , mutations w i t h de fec t s i n p a i r i n g or exchange of homologs have been d i s t i n g u i s h e d by t h e i r pa t te rn o f reduced exchange. The p o l a r i t y of the p a i r i n g process suggests tha t i f p r e -cond i t i on s to exchange ( e . g . , p a i r i n g ) were a f f e c t e d , the decrease i n exchange would take p lace i n a non-uniform manner along the length of each chromosome (Sandler e t a j_. , 1968). The m a j o r i t y o f mutants w i th 13 a l t e r e d exchange f e l l i n t o t h i s category . In c o n t r a s t , a de fec t i n the exchange process per se should r e s u l t i n a un i form reduc t i on o f exchange i n a l l r eg ion s . Only one gene appeared to behave i n t h i s manner ( f o r rev iew, see Baker e t a j_. , 1976a). The above mentioned s tud ie s i n d i c a t e that an extended per iod i s needed to prepare and complete normal p a i r i n g and exchange.and tha t many genes are probably i n vo l ved i n t h i s process . In a d d i t i o n , because o f the concurrence o f reduced p a i r i n g and exchange and i n -creased numbers of u n i v a l e n t s , i t may be a n t i c i p a t e d t ha t mutants w i th de fect s i n p a i r i n g and exchange w i l l s imu l taneous ly have inc reased non-d i s j u n c t i o n . This has been v e r i f i e d w i th mutants from many spec ies (see next s e c t i o n ; see a l s o Baker e t aj_. , 1976a). The Two M e i o t i c D i v i s i o n s H a p l o i d i z a t i o n o f the chromosome complement requ i re s two nuc lear d i v i s i o n s w i thout an i n t e r ven i n g round of DNA r e p l i c a t i o n . In most o r -ganisms t h i s i s achieved by the segregat ion of homologous chromosomes a t the f i r s t d i v i s i o n ( r e d u c t i o n a l ) f o l l owed by a r e gu l a r d i v i s i o n o f centromeres a t the second d i v i s i o n ( e q u a t i o n a l ) . This appears to be the most economical way to assure h a p l o i d i z a t i o n o f a l l chromosomes. Besides the.absence o f the i n t e r ven i n g DNA r e p l i c a t i o n , there are two d i s t i n c t de v i a t i o n s from r e g u l a r m i t o t i c d i v i s i o n s . F i r s t , the c e n t r o -meres have to remain und iv ided u n t i l anaphase of the second d i v i s i o n . In l i l y me iocytes , the p o t e n t i a l f o r centromeres to d i v i d e dur ing the next d i v i s i o n i s suppressed s h o r t l y a f t e r the. p r e -me i o t i c S phase.(the centromeres o f c e l l s t ha t have been exp lanted onto s y n t h e t i c medium a f t e r t h i s commitment step cannot d i v i d e dur ing the next d i v i s i o n ) . 14 That the delay of centromere d i v i s i o n u n t i l anaphase II i n vo l ve s gene-t i c a l l y s p e c i f i e d events i s c l e a r from the ex i s t ence o f mutants i n which sepa ra t i on occurs p recoc i ou s l y (C layberg , 1959; Lamm, 1944; Johnsson, 1944; Dav i s , 1971). Second, a v i r t u a l l y f o o l p r o o f method of a s su r ing segregat ion of homologs i s e s s e n t i a l f o r the r e gu l a r segregat ion and h a p l o i d i z a t i o n dur ing me i o s i s . The r e gu l a r segregat ion of homologs i n -v a r i a b l y i n vo l ve s some p a i r i n g or a l ignment process . In most s p e c i e s , chiasmata are used to hold homologs together u n t i l they segregate a t anaphase I. This r e l a t i o n s h i p between exchange and subsequent segre-ga t i on of homologs has been e s t a b l i s h e d by c h a r a c t e r i z a t i o n of mutants w i t h reduced intrachromosomal recombinat ion. The homologs i n mutant meiocytes are o f ten not he ld together due to a l ack o f ch iasmata; the.: r e s u l t a n t un i va l en t s move a t random to the two poles or they d i v i d e by centromere d i v i s i o n . The process by which homologs are he ld together a t t h e i r chiiasmata u n t i l anaphase I thus appears to be one o f the main f a c t o r s which assures the r e gu l a r segregat ion o f homologs a t the f i r s t d i v i s i o n o f me i o s i s . In a d d i t i o n , c o n t r o l l e d t e r m i n a l i z a t i o n of chiasmata i s c r i t i c a l to t h i s type of s eg rega t i on . The c o n t r o l of the r educ t i ona l d i v i s i o n may a l so i n vo l ve other components s i n ce severa l mutants i n Drosoph i la have a d e f e c t i n d i s j u n c t i o n of chromosomes at the f i r s t d i v i -s i o n , even though recombinat ion i s normal. The mutant ca_ n c ' has been most Regular segregat ion can be assured i n d i f f e r e n t ways. For ex -ample, Drosoph i la males which have no c ro s s i ng -ove r seem to u t i l i z e chromosome s p e c i f i c c o n t r o l o f segregat ion which apparent l y i n vo l ve s p a i r i n g (see Baker and H a l l , 1976). In Drosoph i la females , the segre-gat ion of non-exchange chromosomes i s assured by the process of a l i g n -ment c a l l e d d i s t r i b u t i v e p a i r i n g ( G r e l l , 1964, 1969). e x t e n s i v e l y s tud ied (Baker e t a]_., 1976a). C y t o l o g i c a l a n a l y s i s r e -vealed s p i nd l e d i s t o r t i o n w i t h consequent nond i s j unc t i on of many chromosomes a t the f i r s t d i v i s i o n and chromosome l o s s a t subsequent d i v i s i o n s . In c o n c l u s i o n , s tud ie s on meios i s i n a v a r i e t y of organisms have shown tha t many processes opera t ing from before p r e - m e i o t i c S phase u n t i l pachytene are e s s e n t i a l f o r the r egu l a r occurrence of p a i r i n g and exchange. In a d d i t i o n , normal p a i r i n g and exchange i s u s u a l l y r equ i red to ensure the r e gu l a r segregat ion o f chromosomes dur ing the m e i o t i c d i v i s i o n s . However, these s tud ie s prov ide l i t t l e i n s i g h t i n t o the nature and c o n t r o l of these processes. The i d e n t i -f i c a t i o n and c y t o l o g i c a l and genet i c c h a r a c t e r i z a t i o n of mutat ions i n most or a l l genes tha t are r equ i r ed f o r the succes s fu l complet ion of meios i s should be the l o g i c a l f i r s t step to a f u l l understanding of the me i o t i c process. Th is k ind of approach has been f o l l owed i n sev-e r a l organisms but i t appears t ha t a good means f o r the de tec t i on of mutants and of t h e i r c y t o l o g i c a l and genet i c c h a r a c t e r i z a t i o n render Neurospora p a r t i c u l a r l y s u i t a b l e to study meios i s i n t h i s manner. CHAPTER I MEIOSIS IN NEUROSPORA CRASSA. THE ISOLATION OF RECESSIVE MUTANTS DEFECTIVE IN THE PRODUCTION OF VIABLE ASCOSPORES 16 INTRODUCTION The normal sequence of chromosome behavior dur ing meios i s has been determined by a combination o f cy to logy and t ransmi s s ion g e n e t i c s . A number of fea tu res of t h i s d i v i s i o n c y c l e are unique and d i s t i n g u i s h meios i s from m i t o s i s . F i r s t , p r e -me i o t i c DNA synthes i s i n vo l ve s fewer r e p l i c a t i o n i n i t i a t i o n s i t e s , and some synthes i s may. be delayed u n t i l the f o l l o w i n g prophase (Stern and Ho t t a , 1973). Second, the f i r s t me i -o t i c prophase l a s t s unusua l ly long and i nvo l ve s the p a i r i n g o f a l l homologous chromosomes. Genet ic recombinat ion t ha t takes p lace a t t h i s time i s orders of magnitude h igher than t ha t observed during, m i t o t i c d i v i s i o n (Parag and Parag, 1975). T h i r d , homologous chromosomes gen-e r a l l y segregate from each other dur ing the f i r s t m e i o t i c d i v i s i o n . Four th , no DNA r e p l i c a t i o n takes place, dur ing the in te rphase f o l l o w i n g t h i s d i v i s i o n . Despite t h i s d e t a i l e d knowledge o f the behavior o f chromosomes dur ing me i o s i s , l i t t l e i s known about the processes tha t c o n t r o l these events . This study was i n i t i a t e d to ga in a b e t t e r i n s i g h t i n t o such con t r o l by i s o l a t i n g and c h a r a c t e r i z i n g mutants w i th de fect s dur ing me i o s i s . Such mutants i n which the 'phenotype o f m e i o t i c c e l l s or t h e i r products are de tec tab l y abnormal w i l l be r e f e r r e d to as m e i o t i c mutants. There are two types of me i o t i c mutants; they can be d i s t i n g u i s h e d on the bas i s of t h e i r behavior dur ing the vege ta t i ve phase. The f i r s t type has no apparent e f f e c t on any vege ta t i ve f u n c t i o n and i s c a l l e d me i o s i s -s p e c i f i c . The second type of m e i o t i c mutant i s a l s o d e f e c t i v e i n vege-t a t i v e growth or maintenance. 17 M e i o s i s - S p e c i f i c Mutants M e i o t i c mutants which have no de tec tab l e de f ec t i n any vege-t a t i v e f unc t i on s have been detected i n many organisms, i n c l u d i n g h igher 3 •"• p l a n t s , f u n g i , and Drosoph i la . ( f o r a rev iew, see Baker e t a]_., 1976a). The de fec t i n these mutants may cause a r e a d i l y de tec tab le m e i o t i c phenotype such as a l t e r e d recombinat ion , d i s j u n c t i o n , or f e r t i l i t y (see l a t e r s e c t i o n ) . A l t e r n a t i v e l y , the change of phenotype of such mutants may be s u b t l e . The r e c - t ype mutants c o n t r o l l i n g r e g i o n - s p e c i f i c recombinat ion i n Neurospora and Schizophyl lurn are examples o f the l a t t e r type (Catches ide , 1974). In t h i s paper I w i l l on ly deal w i th mutations which a f f e c t recombinat ion , d i s j u n c t i o n , or f e r t i l i t y , i . e . , those w i th a v i s i b l y abnormal phenotype. Mutants With Defects During Both the Vegeta t i ve and Sexual Phases Two types o f mutants w i t h de fect s i n vege ta t i ve f unc t i on s have been a s soc i a ted w i t h corresponding de fect s i n m e i o t i c processes . F i r s t , a l l t empe ra tu re - s en s i t i v e c e l l c y c l e mutations of Saccharomyces tha t have been te s ted a l s o caused m e i o t i c abno rma l i t i e s (Simchen, 1978). I t appears t ha t both types of c e l l d i v i s i o n are under a c e r t a i n amount of common c o n t r o l . Second, some genes requ i red f o r vege ta t i ve DNA meta-bo l i sm ( e . g . , r e p a i r , muta t ion , somatic recomb inat ion ) , are presumably a l s o r equ i red f o r m e i o t i c exchange or a s soc i a ted processes . Mutat ions i n these genes have been i s o l a t e d on the bas i s of t h e i r s e n s i t i v i t y to However, some mutants o f D ro soph i l a w i th no apparent de fec t o f somatic func t i on s were shown to a f f e c t s p e c i f i c a l l y d i f f e r e n t i a t e d somatic c e l l s a t w e l l - d e f i n e d per iods (Baker e t a l _ . , 1978). 18 UV, i o n i z i n g r a d i a t i o n , or the a l k y l a t i n g agent methyl methane s u l -phonate (MMS). Others were obta ined by t h e i r a l t e r e d f requenc ie s o f m i t o t i c gene convers ion or spontaneous mutat ion . Many mutants t ha t are s e n s i t i v e to DNA-damaging agents have been i s o l a t e d and s t ud i ed i n the yea s t Saccharomyces (Prakash and Prakash, 1977), and i n Drosoph i la (BoydoeJ^aK, 1976a,b; Boyd and Set low, 1976). In y e a s t , three r e p a i r pathways were i d e n t i f i e d on the bas i s o f c r o s s - s e n s i t i v i t y to mutagenic agents. C lass I mutants are s e n s i t i v e to n i t rogen mustard (HNg) and UV; c l a s s II to MMS and x - r a y s ; and c l a s s I I I to a l l f ou r agents. I t was found t ha t many mutants o f c l a s se s II and I I I have an e f f e c t on meios i s (Baker e t a l . , 1976a; Prakash and Prakash, 1977). A s i m i l a r ove r l ap o f gene f unc t i on s requ i red f o r both r e p a i r to MMS-induced damage and meios i s ( e . g . , r e -combinat ion) i s apparent from the a n a l y s i s o f mutants s e l e c t e d i n Drosoph i la on the bas i s o f i nc reased s e n s i t i v i t y to MMS (Baker e t a l . , 1976b). Mutat ions i n f i v e complementation groups cause s e n s i t i v i t y to HN2, UV, MMS, and x - r a y s . These are analogous to the c l a s s I I I mutants i n y ea s t . Mutat ions i n three and po s s i b l y four of these com-plementat ion groups reduce the frequency of me i o t i c exchange. Of these, two (mei-41 and mus-101) are d e f e c t i v e <im p o s t - r e p l i c a t i o n r e p a i r , and one (mei-9) i s d e f e c t i v e i n the r e p a i r of UV-induced damage. Mutat ions i n a t l e a s t two genes cause s e n s i t i v i t y to MMS and x - r a y s , and thus resemble the y ea s t c l a s s II mutants; these two have an apparent de fec t i n me io s i s . From these r e s u l t s i t i s c l e a r t ha t gene func t i on s i n vo l ved i n c e r t a i n pathways of DNA r e p a i r i n somatic c e l l s are a l s o r equ i red dur ing m e i o t i c recombinat ion . There fo re , the i d e n t i -f i c a t i o n and c h a r a c t e r i z a t i o n of var ious r e p a i r pathways w i l l provide 19 i n fo rmat i on tha t may lead to the understanding of mechanisms i nvo l ved i n the process o f m e i o t i c recombinat ion and a s soc i a ted processes . Converse ly , the i s o l a t i o n o f me i o t i c mutants may s t imu l a t e the study of r e p a i r pathways. Gene func t i on s i n vo l ved i n DNA metabolism in somatic celj.s can a l s o be i d e n t i f i e d by screen ing f o r mutants w i th a l t e r e d induced - m i t o t i c gene convers ion f requenc ies ( e . g . , Rodarte-Ramon and Mort imer, 1972; Rodarte-Ramon, 1972). Three out o f four mutants thus i s o l a t e d , had s p o r u l a t i o n d e f e c t s . The mutations spo-7 (E spos i to et a l _ . , 1975) and rem-1 (Go l i n and E spo s i t o , 1977) i n Saccharomyces r e s u l t e d i n m e i o t i c de fect s and an a l t e r e d frequency o f spontaneous muta t i on . Other m e i o t i c mutants,were s e n s i t i v e to h i s t i d i n e ( e . g . , uvs -3, uvs-4, uvs-5, uVs-6, and mei-3 i n Neurospora c r a s s a ; Newmeyer e t a l . , 1978). In three h i s t i d i n e - s e n s i t i v e mutants examined the inc reased s e n s i t i v i t y was accompanied by m e i o t i c b lockage, UV s e n s i t i v i t y , and inc reased i n s t a b i l i t y o f d u p l i c a t i o n s . The Detec t i on of M e i o t i c Mutants The sy s temat ic i s o l a t i o n o f me i o t i c mutants has been i n i t i a t e d i n severa l organisms. Two types of c r i t e r i a were used to de tec t these mutants. F i r s t , mutants w i th a l t e r e d recombinat ion and/or d i s j u n c t i o n f requenc ies were recovered i n Drosoph i la (Sandler e t a]_., 1968; Baker and Carpenter , 1972), Saccharomyces (Roth and Foge l , 1971; Roth, 1976), and Caenorhabdi t i s (Hodgkin e t a j_ . , 1979). Second, complete or p a r t i a l s t e r i l i t y has been used as a c r i t e r i o n f o r the i s o l a t i o n of m e i o t i c mutants i n So rda r i a (Esser and S t raub, 1958), Schizosaccharomyces (Bresch et_ a l _ . , 1968), Saccharomyces (Espos i to and E spo s i t o , 1969), and 20 Podospora (Simonet and Z i c k l e r , 1972). S ince reduced f e r t i l i t y may be caused by many defect s not d i r e c t l y a s soc i a ted w i th me i o s i s , c y t o l o g i c a l and/or genet i c observat ions are r equ i red to conf i rm the i s o l a t i o n of me i o t i c mutants among s t e r i l i t y mutants. M e i o t i c Mutants i n Neurospora Even though m e i o t i c mutants have been obta ined i n many o r -ganisms, the understanding o f the defect s i s o f ten i n doubt because of a l ack o f genet i c or c y t o l o g i c a l means of a n a l y s i s . Var ious i d t o -sync rac ie s o f the l i f e c y c l e s of Podospora and h igher p lant s c omp l i -cate genet i c a na l y s i s i n these organisms. In c o n t r a s t , even though genet i c a n a l y s i s o f yea s t and Drosoph i la i s e x c e l l e n t , v i s u a l i z a t i o n of chromosomes by means o f convent iona l c y t o l o g i c a l methods i s not po s s i b l e i n yeas t and i s p o s s i b l e on ly a f t e r the pachytene stage of Drosoph i la (Pura and Nokkala, 1977). There fo re , i t should be p r o f i t -ab le to i s o l a t e and c h a r a c t e r i z e m e i o t i c mutants i n an organism i n which both genet i c and c y t o l o g i c a l means o f a n a l y s i s are good. The ascomycete Neurospora c ras sa f i t s t h i s d e s c r i p t i o n w e ' l l . In Neurospora, me i o t i c mutat ions which are dominant or ex-pressed on ly i n the c o n i d i a l or p r o t o p e r i t h e c i a l parent can be d i r e c t l y obta ined by screen ing s t r a i n s r e s u l t i n g from mutagenized c o n i d i a . How-every , because o f the h e t e r o t h a l l i c nature of Neurospora, induced r e -ce s s i ve m e i o t i c mutations cannot be expressed i n the f i r s t generat ion of c r o s s i n g . I t i s p o s s i b l e to de tec t such mutations by i n t e r c r o s s i n g a number of i s o l a t e s from each cross i n v o l v i n g a . p o t e n t i a l mutant^,but t h i s i s very t ime-consuming. The technique desc r ibed i n t h i s t h e s i s enables the r ap i d i s o l a t i o n o f r e ce s s i v e m e i o t i c mutations i n Neurospora. 21 This method i nvo l ve s the s e l e c t i o n of s t r a i n s a r i s i n g from ascospores d isomic f o r l i n kage group I and heterozygous f o r the mating type locus . (A + a). Such s t r a i n s are s e l f - f e r t i l e and homozygous f o r a l l l i n kage groups except LG I. The re fo re , each of these c u l t u r e s can be d i r e c t l y t e s ted f o r the presence o f a r e ce s s i v e me i o t i c mutat ion . M e i o t i c mutations i n Neurospora c rassa can be detected as. r e - . duced f e r t i l i t y due to a block i n the format ion o f most or a l l a s c i , or as ascospore a b o r t i o n . In Neurospora, aneuplo idy r e s u l t s i n the abo r t i on of an ascospore i f any pa r t o f the chromosome complement i s m i s s i ng . There fo re , the presence 1 o f such aborted (white) ascospores i s a good de tec t i on system f o r mutations which cause i r r e g u l a r segre-gat ion of chromosomes dur ing meios i s (Smith, 1975). During t h i s i n i t i a l s c r een , . r e ce s s i v e me i o t i c mutations r e -present ing s i x l o c i caused ascospore a b o r t i o n , and r e ce s s i v e mutations of two l o c i r e s u l t e d i n an absence o f ascospores. MATERIALS AND METHODS S t r a i n s The f o l l o w i n g a l l e l e s were used dur ing t h i s s tudy: l eu - 3 (R156); un-3 (55701- t ) ; arq-1 (36703); ad-3A (2-17-814); ad-3B (2-17-114); n i c - 2 (43002); a l - 2 (74A-Y-112-M38); tpj_ (N83); and two a l l e l e s a t each of the three heterokaryon i n c o m p a t i b i l i t y l o c i C/c, D/d, and E/e. The l o c a t i o n of these mutations en l i n kage groups and the approximate map d i s tances (Radford, 1972) between l i n k e d mutations are shown i n F i g . l a . 22 F i g . 1 General o u t l i n e of a s e l e c t i v e system used to i s o l a t e r ece s s i ve m e i o t i c mutants i n Neurospora  c r a s s a . For d e t a i l s , see M a t e r i a l s and Methods. (a) A c rossover between s t r a i n 1-34-8 (female) and the mutagenized s t r a i n 1-30-335 (male) p ro -duces a low p ropo r t i on of progeny d i somic f o r LG I. These can be s e l e c t ed on minimal medium. (b) Each ascospore d i somic (n + 1) f o r LG I con-t a i n s two copies of t h i s l i nkage group but only one copy of the other l i nkage groups, (c) Subsequent h a p l o i d i z a t i o n produces two types of nuc l e i of op-po s i t e mating type i n each c u l t u r e produced by a d i somic ascospore; such cu l t u r e s are capable of " s e l f i n g . " The genes and t he re f o re newly induced m e i o t i c mutations are i d e n t i c a l i n both types of n u c l e i . Consequently, i f a r ece s s i ve m e i o t i c mutat ion were present i n the d isomic ascospore, i t would be detected i n the " s e l f i n g " of the r e -s u l t i n g PWT c u l t u r e . ( a ) C r o s s b e t u e e n s t r a i n s : L i n k a g e G r o u p I I I ' i l l I V V UI V I I _ n r ) C l eu -3 + a arg-1 + ad-3B + + C d t o l asc , i i i Q | I ' ' J L. — I — ! — + un-3 A + ad-3A + n i c - 2 a l - 2 _ l I ! I Q 1 1 1 1 , + un-3 A + ad-3A + n i c - 2 a l - 2 c D t o l + 1 - 3 4 - b t ' • I g I I I I J L I I A p p r o x i m a t e map d i s t a n c e 10 0.1 10 9 0.3 h 28 75 ( R a d f o r d , 1972 ) ( b ) E x a m p l e o f a d i s o m i c (n + 1) a s c o s p o r e : leu-1 + a arg-1 + ad-3B + + C d t o l asc I ' ' • n » I I i -I L. I I ( c ) H e t e r o k a r y o n : _ . „ l eu - 3 + a arg-1 + ad-3B + + C d t o l asc E Component 1 T • • . . J u - u . • j . „ + un-3 A + ad-3A + n i c - 2 a l - 2 C d t o l asc E Component 2 , , , . 0 i i • • j L • • j * An i n d u c e d m e i o t i c m u t a t i o n may be l o c a t e d on any l i n k a g e g r o u p e x c e p t LG I. M u t a t i o n s o f t h i s k i n d , i s o l a t e d d u r i n g t h i s s t u d y , were d e s i g n a t e d a s c ( s e e R e s u l t s ) . ^ O n l y one o f t h e e i g h t p o s s i b l e c o m b i n a t i o n s o f h e t a l l e l e s i s p r e s e n t e d . 24 Mutant Induct ion and I s o l a t i o n Recess ive me i o t i c mutants can on ly be detected i n crosses homozygous f o r these mutants. A method f o r the homozygosis and de-t e c t i o n o f such mutants i n Neurospora c rassa has been developed ( F i g . 1) . The method i nvo l ve s the i s o l a t i o n and t e s t i n g o f many s t r a i n s , each a r i s i n g from an ascospore d isomic (n + 1) f o r chromosome. 1 (LG I) which bears the mating type locus (A/a_). Disomic nuc l e i i n Neurospora are i n h e r e n t l y unstable ( P i t t e n g e r , 1954). They u s ua l l y h a p l o i d i z e q u i c k l y by l o s i n g , a t random, one or the other homologous chromosome. S ince the s e l e c t e d ascospores ca r r y d i somic nuc l e i which are he te ro -zygous f o r the mating type l o c u s , the r e s u l t i n g co l on i e s are he te ro -karyons w i t h two hap l o i d nuc lear types of oppos i te mating type (A + a ) . In a d d i t i o n , s i nce each disome conta ins a hap l o i d complement o f a l l chromosomes except LG I, genes on these chromosomes w i l l be i d e n t i c a l i n the two nuc lear types produced dur ing h a p l o i d i z a t i o n . The simultaneous presence o f both mating types i n these he te r o -karyons w i l l , under the app rop r i a te cond i t i on s o f n i t rogen s t a r v a t i o n •(Westergaard and M i t c h e l l , 1947), induce p e r i t h e c i a l fo rmat ion and me io s i s . The mating r e a c t i o n of these heterokaryons w i l l be r e f e r r e d to as " s e l f i n g . " This r e s u l t i n g " s e l f " w i l l be heterozygous f o r LG I and t he re f o re the mating type l o cu s , but homozygous f o r genes on the other chromosomes. There fo re , any induced r ece s s i ve me i o t i c mutat ion which i s l o ca ted on one of these chromosomes w i l l be present i n homo-zygous c o n d i t i o n . Thus, the p r e r e q u i s i t e f o r t h e i r d e t e c t i o n has been met. 25 S t r a i n s S e l e c t i v e f o r S e l f - F e r t i l e Pseudo-Wild Type (PWT) Cu l tu res The s e l e c t i o n o f ascospores d i somic f o r LG I was made po s s i b l e by the i n t r o d u c t i o n of var ious markers onto the s e l e c t i v e parent s t r a i n s (see F i g . l a f o r the l o c a t i o n o f these markers on t h e i r r e s p e c t i v e l i n k -age groups) . F i r s t , c u l t u r e s a r i s i n g from disomies f o r LG I c o n s t i t u t e a heterokaryon w i t h two complementing components o f oppos i te mating type (A + a_). S ince the mating type locus a l s o acts as a heterokaryon i n -c o m p a t i b i l i t y locus (Beadle and Coonradt, 1944; Garnjobst and W i l s on , 1956), these d isomic c u l t u r e s w i l l grow very poo r l y . There fo re , the t o l mutat ion which suppresses t h i s heterokaryon i n c o m p a t i b i l i t y w i thout a f f e c t i n g c ro s s i ng a b i l i t y (Newmeyer, 1970) was in t roduced i n both s t r a i n s (1-30-225 and 1-34-8 i n F i g . 1) . . Second, the c l o s e l y l i n k e d auxot roph ic mutations l e u - 3 , a r g - 1 , ad-3A, ad-3B, n i c - 2 , and the nonsupplementable h e a t - s e n s i t i v e mutat ion un-3, which are a l l l o ca ted w i t h i n about 30 map un i t s of each other on LG I, were used to s e l e c t ascospores d isomic f o r t h i s l i nkage group. A cross between l e u - 3 , a r g - 1 , ad-3B ( s t r a i n 1-30-225) and un-3, ad-3A, n i c - 2 ( s t r a i n 1-34-8) cou ld produce two types o f ascospore progeny capable of growth on medium con ta i n i n g no l e u c i n e , a r g i n i n e , adenine, or n i c o t i n i c a c i d ( i . e . , minimal medium): m u l t i p l e recombinants and PWT c u l t u r e s : The arrangement of the c l o s e l y 1 inked markers v i r t u a l l y e l im ina te s w i l d type recombinant progeny. In f a c t , none were detected i n t h i s s tudy. Thus, when ascospores are p l a t ed on minimal medium, most co l on i e s should r e s u l t from ascospores d i somic f o r LG I. Each of these PWT c u l t u r e s conta ins two t ype s .o f nuc l e i w i th , complementing 26 auxotroph ic mutations and oppos i te mating type a l l e l e s . T h i r d , when ascospores are p l a ted on minimal medium, the germ tubes of two adjacent ascospores may fu se . Such f u s i o n products may produce a colony i f the two nuc lear types complement each other and are heterokaryon compat ib le . To minimize the frequency of co lony -producing f u s i on p roduct s , three un l inked heterokaryon i n c o m p a t i b i l i t y l o c i (C/c, D/d, and E/e) were u t i l i z e d . The cross between s t r a i n s 1-30-225 and 1-34-8 (see F i g . 1) was made h e t e r o a l l e i i c a t each o f these three l o c i . . When ascospores produced by t h i s cross are p l a ted th on minimal medium, only 1/8 of a l l f u s i on products w i th complementary auxot roph ic requirements can form a co lony. In f a c t , c o l on i e s p ro -duced by the f u s i on of two or more adjacent ascospores were p r a c t i c a l -4 l y e l im ina ted when the p l a t i n g concent ra t i on d i d not exceed 2 x 10 ascospores/90 mm p e t r i d i s h . Consequently, the p l a t i n g o f ascospores 4 a t concent ra t ions up to 2 x 10 spores per p l a t e a l lowed the v i r t u a l l y e x c l u s i v e i s o l a t i o n of s e l f - f e r t i l e PWT c o l o n i e s . The I s o l a t i o n of Induced Recess ive Mutat ions Which A f f e c t Me ios i s Con id ia from seven-day o l d c u l t u r e s of s t r a i n 1^-30-225 were t r ea ted w i t h 0.025 mM MNNG a t 25°C f o r f o u r , f i v e , or s i x hours (Ma i l i n g and deSerres , 1970). A f t e r t e rm ina t i on of MNNG treatment w i th sodium th i o su l pha te a t pH 8.0, the mutagenized c o n i d i a l suspension was used as the f e r t i l i z i n g parent by pouring 10 ml of the suspension over seven-day o ld m y c e l i a l growth of s t r a i n 1-34-8. Ascospores produced by t h i s cross were p l a ted onto minimal medium a t a concent ra t i on of a p p r o x i -mately 10^ per 90 mm p e t r i d i sh ( f o r f u r t h e r de ta i I s , on the p l a t i n g pro-cedure and e x c l u s i v e use of p u r i f i e d t a g a r i n the medium, see G r i f f i t h s and 27 DeLange, 1977). The frequency o f c o l on i e s ( s e l f - f e r t i l e PWT's) thus _5 produced was est imated a t about 5 x 10 . Each of these c u l t u r e s was ass igned an i s o l a t i o n number w i th the p r e f i x " P " f o r PWT ( e . g . , c u l t u r e P100 i s PWT i s o l a t e number 100), and was i s o l a t e d by one o f three pos-s i b l e methods. F i r s t , us ing a d i s s e c t i n g microscope, very small c o l on i e s were i s o l a t e d a f t e r two and three days of growth. At t h i s t ime the s imultaneous t r a n s f e r of unse lected ascospores can be avo ided. Second, a f t e r f ou r or f i v e days o f growth, the aga r - ove r l a ye r a t the s i t e of the colony was removed and pa r t of the under l y ing agar, which i nc luded the myce l i a l growth of the co lony , was t r a n s f e r r e d . This method i s f a s t e r and, s i nce a l l ascospores are i n the o ve r l a ye r agar, i s r e l i a b l e . T h i r d , pa r t of the m y c e l i a l growth or c o n i d i a were t r a n s -f e r r e d a f t e r e i gh t days of growth on p l a t e s . The l a t t e r method i s the f a s t e s t but may lack s e n s i t i v i t y s i n ce non-growing germinated ascospores may be " re scued " by a poor ly growing PWT c u l t u r e . A subsequent s e l f of such a rescued fa s t - g row ing c u l t u r e would be heterozygous a t many l o c i . Each PWT c u l t u r e was t r a n s f e r r e d to a s l a n t of minimal medium i n a 10 x 75 mm tube, and a l lowed to grow f o r a week before being t r a n s f e r r e d to an 18 x 150 mm t e s t tube con ta i n i ng 5 ml l i q u i d minimal c ro s s i ng medium and a s t r i p of f i l t e r paper (Newcombe and G r i f f i t h s , 1972). A l l s e l f s were incubated a t 25°C, some a l s o a t 16°C. Incubat ion a t both temperatures a l lowed the de t e c t i o n of t empera tu re - sen s i t i ve mutants. Cu l tu res i n which abnormal development of p e r i t h e c i a and/or a s -cospores r e s u l t e d were crossed on l i q u i d minimal medium w i t h OR-A and OR-a w i l d type s t r a i n s . I f aberrant development i s caused by the ex-pres s ion of a r e c e s s i v e , r a the r tha t a dominant, muta t ion , crosses w i t h 28 both w i l d type s t r a i n s should produce normal p e r i t h e c i a and asco-spores . There fo re , on ly c u l t u r e s w i t h t h i s behavior were c l a s s i f i e d as p o t e n t i a l r ece s s i ve mutants. Recess ive mutants were d i s t i n g u i s h e d from mutants t ha t are expressed on ly when present i n the male or female parent , by means of r e c i p r o c a l crosses between the mutant PWT c u l t u r e , and the OR-A and OR-a s t r a i n s . Some of these mutations may a f f e c t p e r i t h e c i a l format ion w h i l e . others are more d i r e c t l y i n vo l ved w i th the product ion of v i a b l e asco-spores. Among mutants w i t h a de fec t i n p e r i t h e c i a l development, no a sc i or ascus i n i t i a l s have ever been r epo r ted . There fo re , i t appears p l au s -s i b l e t ha t meios i s i s i n i t i a t e d on ly a f t e r the major pa r t of p e r i t h e c i a l development has been completed. Thus, i n screen ing f o r m e i o t i c mutants, on ly those w i th we l l - deve loped p e r i t h e c i a have been f u r t h e r ana lyzed. F i ve A i s o l a t e s of the cross between any g iven PWT and OR-A, and f i v e a_ i s o l a t e s o f the cross between the PWT and OR-a were i n t e r c r o s s e d i n ' a l l combinat ions; i f the mutant phenotype was de tec ted , one mutant i s o l a t e of each mating type was used i n the t e s t i n g o f a l l i s o l a t e s from crosses between the PWT c u l t u r e and the two w i l d type s t r a i n s ' (OR-A and OR-a). Sometimes, a l l these i s o l a t e s were backcrossed to the o r i g i n a l PWT c u l t u r e . In e i t h e r case, a 1:1 segregat ion of mutant and w i l d type phenotype conf irmed the presence of a r ece s s i ve po i n t mutat ion . Media and r ou t i ne manipu lat ions were convent iona l f o r Neuro- spora (Davis and de Se r r e s , 1970). 29 RESULTS ' Using the s e l e c t i v e system desc r ibed i n MATERIALS AND METHODS (see a l s o F i g . 1 ) , 1090 PWT c u l t u r e s were i s o l a t e d and a l lowed to s e l f on ; l i q u i d c ro s s i ng medium. The 145 cu l t u r e s tha t d id not suc-c e s s f u l l y complete the sexual c y c l e , i . e . , e j e c t only v i a b l e b lack ascospores from mature p e r i t h e c i a , were screened aga in s t dominant muta-t i on s by c r o s s i n g them i n d i v i d u a l l y w i th OR-A and 0R-a_ w i l d type s t r a i n s . Table I shows t ha t 46 s t r a i n s e x h i b i t e d w i l d type c ro s s i ng a b i l i t y w i th both OR-A and 0R-a_. These s t r a i n s , .which may ca r ry r e -ces s i ve mutations or mutations tha t are expressed on ly i n the female or male pa rent , were c l a s s i f i e d accord ing to phenotype i n t o three main groups: 16 s t r a i n s w i th a de fec t i n the p e r i t h e c i a ! development ( c l a s s I ) ; 26. s t r a i n s d e f e c t i v e i n the format ion of a sc i or b lack ascospores ( c l a s s I I ) ; and 4 s t r a i n s w i th a misce l laneous developmental de fec t apparent ly not a s soc i a ted w i t h reduced f e r t i l i t y ( c l a s s I I I ) . C lass I Mutants of t h i s type e i t h e r produce no p e r i t h e c i a a t a l l (6 s t r a i n s ) , or few or i ncomplete ly developed p e r i t h e c i a (10 s t r a i n s ) . P e r i t h e c i a w i th a s i z e i n between p r o t o - and f u l l y grown p e r i t h e c i a , and l a c k i n g a neck, are cons idered i ncomple te l y developed. To d i s t i n -guish mutants t ha t are expressed on ly i n the female or male parent (female or male s t e r i l e mutants) from rece s s i v e mutants, 4 s t r a i n s ' , (P205, P349, P4Q6, P700) 5 with no p e r i t h e c i a , 2 s t r a i n s (P841, P891) w i t h few p e r i t h e c i a , and 2 s t r a i n s (P186, P434) w i th i ncomple te l y de-veloped p e r i t h e c i a were crossed r e c i p r o c a l l y w i th w i l d type s t r a i n s TABLE I . I n i t i a l c h a r a c t e r i z a t i o n o f 1^5 p s e u d o - w i l d t y p e c u l t u r e s w i t h a b e r r a n t c r o s s i n g b e h a v i o r * Type o f C r o s s i n g A b e r r a n c y f\l umber M of PUT C u l t u r e s m C l a s s I A: B: S t e r i l e ( i . e . , no p e r i t h e c i a ) Feu or i n c o m p l e t e l y d e v e l o p e d p e r i t h e c i a 5D 3 6 10 C l a s s I I P e r i t h e c i a w i t h o u t s p a r e s Mare t h a n 2 0 % a s c o s p o r e a b o r t i o n • 13 13 C l a s s I I I D e f e c t s n o t r e l a t e d t o f e r t i l i t y ( s e e t e x t ) 99 k6 Each c u l t u r e was c r o s s e d w i t h 0R-A_ and OR-a_ w i l d t y p e s t r a i n s ; t h o s e p r o d u c i n g n o r m a l p e r i t h e c i a and a s c o s p o r e s w i t h b o t h s t r a i n s were c l a s s i f i e d as p o t e n t i a l r e c e s s i v e m u t a n t s (m_) ; t h e d e f e c t i n t h e r e m a i n i n g c u l t u r e s was a p p a r e n t l y c a u s e d by d o m i n a n t d e t e r m i n a n t ( s ) and t h e s e were c a l l e d _M. 31 (the remaining 8 s t r a i n s grew very poor ly and were not t e s t e d ) . In each ca se , mutant phenotype r e s u l t e d when the mutant s t r a i n was used as the p r o t o p e r i t h e c i a l (female) parent but not when the mutant was used as the c o n i d i a l (male) parent . Thus these s t r a i n s are female s t e r i l e . S i m i l a r mutants have been p r e v i ou s l y i s o l a t e d (Mylyk and T h r e l k e l d , 1974; Johnson, 1978).- . C lass I I -A Th i r t een s t r a i n s produced p e r i t h e c i a which were e i t h e r com-p l e t e l y barren or conta ined very few b lack ascospores (about 10-20 spores per per i theciurn as compared to hundreds i n normal p e r i t h e c i a ) . This c l a s s of mutants was subd iv ided i n t o 4 phenotypes: 8 s t r a i n s w i th empty p e r i t h e c i a (P246, P308, P373, P400, P446, P741, and 2 a d d i -t i o n a l s t r a i n s t ha t were d i sca rded due to poor growth); 2 s t r a i n s t ha t produced only a s c i w i th 8 l i t t l e round bubb les , about ' one -quar te r the s i z e o f r e gu l a r ascospores , but no ascospores (P314, P423); 1 s t r a i n w i th empty a sc i (P131); and 2 s t r a i n s w i t h few b lack ascospores (P310, P1163). The cause of the de fec t i n 4 s t r a i n s was f u r t h e r i n v e s t i g a t e d . In 2 cases (P131 and P400) a r e ce s s i v e po i n t mutat ion was i n v o l v e d . However, the empty p e r i t h e c i a of P446 and the bubble a s c i of P314 were apparent ly not caused by a r e ce s s i v e mutat ion . One out of 5 asco-spore i s o l a t e s obta ined from a cross between P446 and OR-A w i l d type produced barren p e r i t h e c i a when used as the p r o t o p e r i t h e c i a l parent but not as the c o n i d i a l parent . Although not analyzed f u r t h e r , t h i s mutant appears s i m i l a r to those i n c l a s s I; i n s tead of an e a r l y b lock ( c l a s s I mutants ) , a l a t e block i n p e r i t h e c i a l development may be i n v o l v e d . The de fec t i n P314 appears to have a more complex pa t te rn 32 of i n h e r i t a n c e . Whereas the o r i g i n a l PWT c u l t u r e produced on ly bubble a s c i , i n t e r c r o s s e s o f some i s o l a t e s from a cross between P314 and w i l d type produced empty p e r i t h e c i a w h i l e others, formed ascospores , most of which were no t ,o r were very s l ow ly e j e c ted from t h e i r p e r i t h e c i a . This mutant has not been examined f u r t h e r . C lass I I-B Th i r teen s t r a i n s were c h a r a c t e r i z e d by t h e i r pa t te rn of a sco -spore a b o r t i o n . The abo r t i on i n 6 o f these s t r a i n s (P95, P243, P393, P711, P879, P961) was found to be due to a s i n g l e r e ce s s i v e mutat ion . The f e r t i l i t y ( t o t a l number of b lack and wh i te ascospores) of s t r a i n s P243, P393, and P879 was very low. These mutants are d i scussed f u r t h e r i n the f o l l o w i n g s e c t i o n or i n Chapter I I . The ascospore abo r t i on i n another s t r a i n (P917) was apparent ly caused by a dominant s p o r e - k i l l e r , mutat ion . This mutant i s desc r ibed i n Chapter I I I . S t r a i n P1079 p ro -duced on ly wh i te i n v i a b l e ascospores whereas s t r a i n s P165 and P631, w i th 50-80% ascospore abo r t i on 3 weeks a f t e r c r o s s i n g , conta ined most ly b lack ascospores a f t e r 2 months. No c l e a r - c u t pa t te rn of i n -he r i t ance was ev ident i n s t r a i n s P165, P631, and P1079. The remain-ing 3 s t r a i n s w i th about 20% ascospore abo r t i on (P117, P285, P768) were d i scarded because o f s co r i n g d i f f i c u l t i e s (the mutagenized inbred PWT s t r a i n s have a wide range of spore abo r t i on up to about 15<-or 20%). F i n a l l y , dur ing the ana l y s i s o f s t r a i n P917 (see Chapter I I I ) , a r ece s s i ve mutat ion which caused ascospore abo r t i on and low f e r t i l i t y was de tec ted . This mutat ion was o r i g i n a l l y present i n heterozygous c o n d i t i o n and was t he re f o re not detected i n the o r i g i n a l s e l f i n g of P917. A cross between s t r a i n s 917A7 (mutant A ascospore i s o l a t e from 33 the cross P917 x OR-A) and 1-30-225 ( l e u - 3 , a_, a r g - 1 , ad-3B) produced 9/30 recombinant progeny between l eu -3 and the muta t i on . In a d d i t i o n , both a_, a r g , ad recombinant progeny were mutant. These p r e l i m i n a r y r e s u l t s i n d i c a t e l i n kage of t h i s r e ce s s i v e mutat ion to the t i p o f the l e f t arm of LG I. C lass I I I The abnormal phenotypes of these .4 mutant PWT s t r a i n s are ap-pa ren t l y not r e l a t e d to f e r t i l i t y . Two s t r a i n s d i scharged t h e i r spores very poor ly and were not examined f u r t h e r . The 2 remaining s t r a i n s a f f e c t e d the phenotype of the per i thec iurn. S t r a i n P126 produced orange i n s tead o f b lack p e r i t h e c i a . This phenotype was c o n t r o l l e d by a po i n t mutat ion expressed on ly i n the p r o t o p e r i t h e c i a l parent . S ince t h i s phenotype i s apparent ly on ly produced by mutations a t the per-1 locus (Howe and Johnson, 1976), i t i s p l a u s i b l e t ha t P126 i s an a l l e l e of t h i s l o cu s . Another po i n t mutat ion ( i n P413), s i m i l a r l y ex-pressed only i n the p r o t o p e r i t h e c i a l pa rent , prevents the format ion of necks on the p e r i t h e c i a . This de fec t prevents the d i scharge of a s -cospores from t h e i r p e r i t h e c i a . S ince the w i l d type locus apparent ly c o n t r o l s the format ion of the p e r i t h e c i a l neck, the mutat ion w i l l be des ignated pen-1. F i n a l l y , i n a p r e l im i na r y attempt to i s o l a t e temperature-s e n s i t i v e mutants, a s t r a i n (P709) was found to produce 4-spored a sc i a t 16°C, and the normal 8-spored a s c i a t 25°C. This c o l d s e n s i t i v e mutant i s dominant and has been mapped a t or near the centromere of LG I. The mutant w i l l be des ignated Fsp-2 (4-spored a s cu s ) . Fsp-1 i s not t empera tu re - sen s i t i ve (Raju,. 1977). 34 Recess ive Mutations tha t A f f e c t the Formation of Asc i or V i a b l e  Ascospores A t o t a l of 9 r ece s s i ve mutat ions ' (2 of c l a s s I I-A and 7 o f c l a s s I I -B) a f f e c t i n g ascus or ascospore product ion have been p o s i -t i v e l y i d e n t i f i e d dur ing t h i s s tudy. - In the past such mutations have been detected among mutants t ha t were UV - sen s i t i v e (uvs -3 , -5.and -6) or caused increased i n s t a b i l i t y o f d u p l i c a t i o n s (me i -3 ) . Some other mutations (mei -1 , mei-4) were detected d i r e c t l y by t h e i r ascospore abo r t i on ( f o r review see Perk in s and Ba r r y , 1977). Both me i o t i c and other developmental processes are r equ i r ed to complete the sexual c y c l e and, thus , to produce a s c i and v i a b l e ascospores. There fo re , a de f ec t i n the p roduct ion o f a s c i or v i a b l e ascospores cou ld be caused by a de f ec t not d i r e c t l y a s s oc i a ted w i th me io s i s . Consequently, not a l l r e ce s s i v e mutations of t h i s k ind would be m e i o t i c (mei) mutat ions . The new locus des i gnat ion asc which i s i n t roduced here r e f e r s to a l l r e ce s s i v e mutations r e s u l t i n g i n the absence of a sc i or the abo r t i on of a sc i or ascospores. A l l 9 asc mutations were te s ted f o r a l l e l i s m to each other and to me i - 1 . I t was found t ha t only P243 and P393 were a l l e l i c to each o the r . In a d d i t i o n , a l l asc mutat ions were n o n - a l l e l i c to me i -1 . Table II shows the 8 l o c i w i th a l l e l e or i s o l a t i o n numbers and comments on t h e i r phenotype. The r e s u l t s o f a n a l y s i s of a s c - 1 , asc -3 and asc-6 are repor ted i n Chapter I I . These appear to have a .de fec t dur ing me io s i s . The w i l d type gene o f asc-7 a l s o appears to be necessary f o r me ios i s s i n ce p r e l im i na r y c y t o l o g i c a l observat ions showed c l u s t e r e d nuc l e i a t the in terphase of the second m e i o t i c d i v i s i o n . Moreover, smal l and la rge TABLE I I . The p h e n o t y p e s o f r e c e s s i v e c l a s s I I m u t a t i o n s a t e i g h t l o c i , whose w i l d t y p e a l l e l e s a r e n e c e s s a r y f o r t h e f o r m a t i o n o f n o r m a l a s c i o r b l a c k a s c o s p o r e s L o c u s A l l e l e I s o l a t i o n l\lo. N a t u r e o f D e f e c t % A s c o s p o r e A b o r t i o n O t h e r P h e n o t y p e a s c - 1 P95 1*0 - 70 I n t e r m e d t o h i g h f e r t i l i t y * a s c - 2 P131 Empty a s c i a s c - 3 P2*+3, P393 • 9 0 - 9 8 Very low f e r t i l i t y asc-h P^tOO Empty p e r i t h e c i a as c-5 . P711 2 0 - 8 0 as c-6 PS79 70 Low t o i n t e r m e d f e r t i l i t y a s c - 7 , PS17 90 Low f e r t i l i t y a s c - 8 P961 50 F e r t i l i t y was d e f i n e d as t h e t o t a l number o f a s c o s p o r e s ( b l a c k and w h i t e ) t h a t were e j e c t e d f r o m ,the p e r i t h e c i a ( s e e MATERIALS AND METHODS). 36 ascospores were found i n t e r spe r sed i n the same a s c i . The de fec t of the asc-5 mutat ion i s of a d i f f e r e n t nature . Crosses homozygous f o r t h i s mutation, produced'many a sc i w i t h 8 b lack ascospores and a v a r i -ab l e number of a s c i w i t h on ly wh i te ascospores. The l a t t e r type o f a sc i conta ined e i t h e r 4 or 8 spores. .The amount of ascospore abo r t i on va r i ed w ide l y between about 20 and 80%. A range o f 50-80% was en-countered i n 11 crosses i n v o l v i n g ascospore i s o l a t e s . A cross between 2 s t r a i n s , each m u l t i p l y marked f o r LG I, produced about 25% aborted ascospores. Progeny ana l y s i s of t h i s cross i n d i c a t e d t ha t both r e -combinat ion and nond i s junc t i on f requenc ies were normal. F i n a l l y , the mutant asc-8 was not analyzed f u r t h e r . DISCUSSION A new system f o r the homozygosis of induced mutations has been used to i s o l a t e r e ce s s i v e mutations w i t h a de fec t i n the sexual c y c l e . Se lec ted PWT cu l t u r e s were screened f o r de fect s i n the format ion of p e r i t h e c i a , a s c i and v i a b l e ascospores. The i n i t i a l screen would a l s o d e t e c t , i n a d d i t i o n to r ece s s i ve mutat ions , those tha t are on ly ex-pressed i n the maternal or paterna l parent . Among 1090 PWT cu l t u r e s screened, the development of p e r i t h e c i a was a f f e c t e d i n 18 s t r a i n s (16 c l a s s I and 2 c l a s s I I ) . In a d d i t i o n , 28 s t r a i n s (26 c l a s s II and 2 c l a s s III) produced apparent ly normal p e r i t h e c i a but the product ion o f a sc i or v i a b l e ascospores was a f f e c t e d . The de fec t i n p e r i t h e c i a l development o f a l l 8 c l a s s I mutant s t r a i n s t e s t ed was on ly expressed i n the p r o t o p e r i t h e c i a l parent . The stage of the de fec t of p r o to -p e r i t h e c i a l development of these mutants has-not been pursued i n t h i s s tudy. . Of the two c l a s s I I I mutants w i th a de fec t i n p e r i t h e c i a ! 37 development, 1 s t r a i n d e f e c t i v e i n p e r i t h e c i a l c o l o r (per-1) and 1 de-f e c t i v e i n neck format ion (pen-1) were a l s o expressed on ly i n the maternal parent . Even though the c l a s s II mutant P446 produced normal-l ook ing p e r i t h e c i a , the absence of a s c i and i t s express ion i n the maternal parent appear very s i m i l a r to the c l a s s I type mutants. Pos-s i b l y the on ly d i f f e r e n c e between th i s , mutant and the c l a s s I mutants i s the stage of p e r i t h e c i a l development a t which the de fec t i s expressed. Defects i n the product ion of v i a b l e ascospores i n 9 s t r a i n s , rep re sent ing 8 l o c i (asc-1 through asc-8) were due to r e ce s s i v e muta-t i o n s . In a d d i t i o n , even though the screen d i s t i n g u i s h e d between domin-ant and r ece s s i ve mutat ions , the recovery o f 1 dominant mutat ion (SK) among c u l t u r e s screened f o r r e ce s s i v e mutations was a consequence o f i t s s p e c i f i c e f f e c t on ad-3A. A cross between SK and ad-3A produced ascospore abo r t i on but ne i t he r SK nor ad-3A d id so when crossed w i th w i l d type t e s t e r s t r a i n s (see Chapter I I I ) . The recovery o f r e ce s s i ve mutations a t 8 d i f f e r e n t l o c i demon-s t r a t e the e f f e c t i v e n e s s of the s e l e c t i o n procedure. Four d i f f e r e n t types of mutations were c l a s s i f i e d : (1) complete absence of a sc i ( a s c - 4 ) ; (2) aborted or empty a s c i ( a s c - 2 ) ; (3) de fec t dur ing meios i s l ead ing to ascospore abo r t i on ( a s c - 1 , a s c - 3 , asc-6 and probably a s c - 7 ) ; (4) abo r t i on of a l l ascospores i n some a sc i but l i t t l e or no -abor t ion i n the remaining a s c i ( a s c - 5 ) . The mutants i s o l a t e d du r ing t h i s study should become ins t rumenta l i n the understanding o f the format ion o f p e r i t h e c i a , a s c i and v i a b l e ascospores. 38 Development of P e r i t h e c i a i n Neurospora The mutant phenotype i n a l l p r e v i ou s l y i s o l a t e d mutants a f f e c -t i n g the development of p e r i t h e c i a (Wei jer and V i g fu s sen , 1972; V i g -fussen and We i j e r , 1972; Mylyk and T h r e l k e l d , 1974; Johnson, 1978; G r i f f i t h s and DeLange, 1978) was expressed i n a dominant f a s h i o n , i . e . , when on ly one of the parents i n a sexual cross was mutant. While some were expressed i n e i t h e r parent or i n the paterna l parent , most o f these mutants were expressed on ly when used as the maternal parent . The present method which enables the i s o l a t i o n o f r e c e s s i v e mutants should be e s p e c i a l l y usefu l s i nce no such mutants would have been de-tec ted i n prev ious mutant sc reens . Even though r e ce s s i v e mutants would be detected i n the present sc reen ing procedure, none were i d e n t i f i e d among 8 mutants t e s t e d . The mutant e f f e c t i n a l l 8 s t r a i n s was ex-pressed on ly i n the maternal parent . This a n a l y s i s , t h e r e f o r e , empha-s i z e s t ha t a t l e a s t the great m a j o r i t y of l o c i r equ i r ed f o r the deve lop-ment o f p e r i t h e c i a ac t only i n the p r o t o p e r i t h e c i a l pa ren t . However, the recent recovery and c h a r a c t e r i z a t i o n (Johnson, 1979) o f a mutant which i s s t e r i l e as the female or the male parent i n d i c a t e s tha t some rece s s i ve mutations may be found i n t h i s c l a s s . This mutat ion became p a r t i a l l y r e ce s s i v e when both components, of the cross were heterokaryon compat ib le . Such a mutat ion would not have been detected i n the p re -sent screen because the t e s t e r s t r a i n s OR-A and 0R-a_, used to d i s t i n -guish between dominance and rece s s i vene s s , were not heterokaryon com-p a t i b l e w i th the components of the PWT s t r a i n s t e s t e d . Even i f the PWT c a r r i e d the same a l l e l e s as ;0R-A and OR-a a t the 3 i n c o m p a t i b i l i t y l o c i C/c, D/d and E/e ( t rue i n on ly about one-e ighth o f PWT s t r a i n s ) , 39 the mating type i n c o m p a t i b i l i t y would s t i l l be present . To get around the l a t t e r problem i t would be necessary to use t e s t e r s t r a i n s c a r r y i n g t o l (Newmeyer, 1970) o r a mating type mutat ion c o n f e r r i n g com-p a t i b i l i t y w i t h the oppos i te mating type ( G r i f f i t h s and DeLange, 1978). Formation o f A sc i i n Neurospora In Neurospora the development o f a s c i i s i n i t i a t e d a f t e r the p re -me i o t i c S phase and karyogamy ( Iyengar e t_a j_ . , 1977). There fo re , mutants which e i t h e r l a ck a s c i o r produce d e f e c t i v e a s c i may have t h e i r de fec t dur ing me i o s i s . The absence or e a r l y abo r t i on o f a s c i ( i . e . , the p e r i t h e c i a are barren) i s c h a r a c t e r i s t i c o f severa l r e ce s s i v e me i o t i c mutations i n Neurospora which are s imul taneous ly d e f e c t i v e i n vegeta-t i v e DNA r e p a i r (uvs -3, uvs -5, uv s -6 , .me i -3 ) . S i m i l a r r e l a t i o n s h i p s , e s p e c i a l l y i n yeas t and Drosophi la, 1 have been well-documented and i l l u s -t r a t e an over lap i n f unc t i on s requ i red dur ing somatic maintenance ( e . g . , r e p a i r o f DNA damage) and me i o t i c recombinat ion or r e l a t e d processes. The de tec t i on of mutants on.the bas i s o f t h e i r a b i l i t y to produce a s c i should become a powerful t o o l because i t enables the i d e n t i f i c a t i o n o f genes which are requ i red only dur ing meiosis> and of those which are a l s o requ i red f o r vege ta t i ve f u n c t i o n s . In a d d i t i o n , c y t o l o g i c a l ana l y -s i s and the use o f t empe ra tu re - s en s i t i v e mutants would f a c i l i t a t e the de t e c t i o n of temporal stages of a c t i v i t y and f i n a l blockage po int s o f such mutants. Formation of V i a b l e Ascospores . in Neurospora The f a i l u r e o f the maturat ion and v i a b i l i t y of ascospores may be caused by the absence o f any pa r t of the hap lo i d chromosome complement 40 or by the presence of a mutant gene i n these ascospores ( f o r rev iew, see Perk ins and Bar ry , 1977). Examples, o f the former k ind are d e f i - , c i e n c i e s of pa r t o f chromosomes due to rearrangements, and d e f i c i e n c i e s , of whole chromosomes due to nond i s j u c t i o n ( e . g . , me i - 1 , me.i-4). The f a i l u r e of maturat ion o f ascospores may a l s o be due to ascospore c o l o r mutants or spore k i l l e r mutants. In the present study a t h i r d type o f mutant (asc-5) was detected which was r e ce s s i v e and caused the abo r t i on of a l l ascospores i n a number o f a s c i w i thout a f f e c t i n g the remaining a s c i . Mutants w i th a de fec t i n the r e gu l a r segregat ion o f chromosomes have a l ready been ins t rumenta l i n understanding the r e l a t i o n s h i p between m e i o t i c exchange and the d i s j u n c t i o n o f homologous chromosomes dur ing the f i r s t me i o t i c d i v i s i o n . Recess ive mutations o f t h i s k ind have been detected i n many spec ies ( f o r review see Baker e t a l_ . , 1976a). Two such mutations (mei -1 , mei -4; Perk ins and Ba r r y , 1977) have been detected p rev i ou s l y i n Neurospora c r a s s a . Many of these mutants have a primary de fec t i n recombinat ion , the aberrant segregat ion merely being a con-sequence o f un i va len t s produced through a l a c k of exchange. In Neuro- spora, exchange i s v i r t u a l l y e l im i na t ed i n crosses homozygous f o r mei-1 (Smith, 1975) due to an almost complete l ack o f p a i r i n g o f homo-logs (Lu and G a l e a z z i , 1979). Some, o f the newly i s o l a t e d spore abo r t i on mutations (asc-1 and asc-6) have s i m i l a r e f f e c t s on recombinat ion , and t n e asc-3 mutat ion causes de fect s i n segregat ion t ha t are not a s soc i a ted w i th reduced exchange. These mutants have been analyzed more e x t e n s i v e l y and the r e s u l t s w i l l be descr ibed i n Chapter I I . Ascospore abo r t i on o f the r e ce s s i v e asc-5 mutat ion was apparent ly not due to aneuplo idy o f the i n v i a b l e ascospores but r a the r to an unknown 41 type of i n v i a b i l i t y o f a l l ascospores i n a p ropo r t i on of a s c i . The l a rge amount o f v a r i a t i o n i n express ion of t h i s mutat ion may be ex-p l a i ned i f a th re sho ld amount o f a substance w i t h i n each ascus i s r e -qu i red f o r the maturat ion of ascospores. A s i m i l a r mode o f a c t i o n has been proposed to account f o r c e r t a i n i r r e g u l a r i t i e s i n the sperm dys-f u n c t i o n caused by SD mutations i n Drosoph i la (Mik los and Smith-White, 1971). The e f f e c t of asc-5 on whole a sc i and the l a r ge amount o f v a r i -a b i l i t y o f express ion between d i f f e r e n t crosses i s a l s o remin i s cent of bubble a s c i (Perk ins and Ba r r y , 1977). In each case, a c e r t a i n p ro -p o r t i o n of a sc i f a i l to produce v i a b l e a sco spo re s - - a sc i w i t h aborted wh i te spores f o r asc-5 and a s c i w i th 8 l i t t l e bubbles found i n many w i l d type c r o s s e s — w i t h no apparent e f f e c t on the remaining a s c i . I t i s not known whether the bubble a sc i may superimpose on the w h i t e -spored a s c i . Ascospore c o l o r mutants and spore k i l l e r mutants can a l s o cause ascospore a b o r t i o n . In the case o f ascospore c o l o r mutants, the mutant ascospores abort whereas spore k i l l e r mutations cause the abo r t i on of ascospores tha t would be v i a b l e i n other c ro s se s . In a l l cases , he te ro -zygous crosses r e s u l t i n 50% ascospore a b o r t i o n . The mutant SK(ad-3A) was i s o l a t e d dur ing t h i s study and appears to have c h a r a c t e r i s t i c s of both a spore k i l l e r mutant and an ascospore c o l o r mutant. This mutant has been analyzed i n more d e t a i l , and i s desc r ibed i n Chapter I I I . F i n a l l y , f u tu re s tud ie s should i n c l ude the improvement of the s e l e c t i o n system to a l l ow f o r f a s t e r i s o l a t i o n of PWT c u l t u r e s . For example, .meiot ic mutations w i th i nc reased PWT frequences cou ld be used f o r t h i s purpose. In.addit ion,. , emphasis w i l l be put on the i s o l a t i o n o f c o n d i t i o n a l mutants. 41a CHAPTER II MEIOSIS IN NEUROSPORA CRASSA. I I . GENETIC AND CYTOLOGICAL CHARACTERIZATION OF FOUR MEIOTIC MUTANTS 42 INTRODUCTION The i s o l a t i o n of e i gh t r e ce s s i v e mutua l ly complementing muta-t i o n s which a f f e c t f e r t i l i t y i n Neurospora c ras sa has been repor ted i n Chapter I. Mu ta t i on s . i n two l o c i (asc-2 and asc-4) r e s u l t e d i n barren p e r i t h e c i a ; the remaining s i x mutat ions ( a s c - 1 , a s c - 3 , a s c - 5 , a s c - 6 , asc-7 and asc-3) caused the abo r t i on of many ascospores. That ascospore abo r t i on may be a very usefu l means of de tec t i n g mutations w i t h a de fec t i n the r egu l a r segregat ion of chromosomes has been suggested by s tud ie s done w i th the mei-1 mutation (Smith, 1975). Approximately 90% of ascospores from crosses homozygous f o r t h i s muta-t i o n were abor ted. This abo r t i on was e v i d e n t l y due to the i r r e g u l a r segregat ion of chromosomes which was caused by the absence of p a i r i n g dur ing the f i r s t m e i o t i c prophase (Lu and G a l e a z z i , 1979) and r e s u l t e d i n aneuplo id products . The succes s fu l use of both genet i c and c y t o l o g -i c a l means of a na l y s i s of such mutants was a l s o demonstrated i n those s t u d i e s . This paper repor t s the genet i c and c y t o l o g i c a l c h a r a c t e r i z a t i o n of the three ascospore abo r t i on ;mutations a s c - 1 , asc-3 and asc-6 (see Chapter I) and some new observat ions on the mei-1 muta t i on . . The segre-ga t i on of chromosomes dur ing meios i s was shown to be d e f e c t i v e i n crosses homozygous f o r each of these mutat ions . In each case, the segregat ion de fec t appeared to .be a secondary consequence of a p r i o r abnorma l i t y . A de fec t i n the p a i r i n g of homologous chromosomes i n crosses homozygous f o r a s c - 1 , asc-6 or mei-1 r e s u l t e d i n d e f e c t i v e segregat ion of chromo-somes dur ing the f i r s t and second d i v i s i o n s of meios i s (and po s s i b l y 43 the po s t -me i o t i c d i v i s i o n ) . In c o n t r a s t , the block i n the development of most a sc i i n crosses homozygous f o r asc -3 was fo l l owed by segrega-t i o n i r r e g u l a r i t i e s , f o r the few a sc i formed, dur ing the second and po s t -me i o t i c d i v i s i o n s . MATERIALS AND METHODS A l l e l e s Used The a l l e l e s on LG I used to s e l e c t PWT co lon ie s and to determine recombinat ion and nond i s j unc t i on f requenc ies of t ha t l i n kage group, have been descr ibed i n Chapter I. A l l e l e s used to study m u l t i p l e disomy i n crosses homozygous f o r asc-6 a r e : ad-3A (2-17-814); aur (34508); a l - 2 (74A-Y112-M38); ac r -2 (KH5); pdx (37803); co t -1 (C102 ( t ) ) ; inos (37401); h i s - 1 (K141). The l o c a t i o n of these l o c i on t h e i r l i n kage groups and map d i s tances between l o c i , were a p p l i c a b l e , are i l l u s t r a t e d i n F i g : , 2 . Other l o c i a re : a l - 1 (Car-10)_ on LG IR; arg-5 (27947) on IIR; t r p - 4 , (Y2198) and pan-1 (5531) on IVR. The m e i o t i c mutants mei-1 (Smith, 1975), asc-1 (P95), asc-3 (P243) and asc-6 (P879) have been -prev ious ly desc r ibed (Chapter I ) . The ad-3B a l l e l e (2-17-128) was used i n crosses homozygous f o r mei-1 or m e i - l ; a s c - 6 . F i n a l l y , the mutat ion a m (33) of the mating type locus i s heterokaryon compat ib le w i th s t r a i n s of A mating type and s t i l l permits c ro s s i ng to such s t r a i n s ( G r i f f i t h s and DeLange, 1978). S t r a i n s The r e ce s s i v e ascospore abo r t i on (asc) mutations were i s o l a t e d i n PWT s t r a i n s which were heterozygous f o r LG I ( l e u - 3 , a , a r g - 1 , ad-3B and un-3, A, ad-3A, n i c - 2 , a l - 2 ) but homozygous f o r the other l i nkage groups (see F i g . 1 and Chapter I ) . Ascospore i s o l a t e s t r a i n s of genotypes 44. F i g . 1 The two nuc lear components of PWT c u l t u r e s i n which asc mutations were recovered. c e n t r o m e r e LG I LG IV LG n . „ l e u - 3 + a a r g - 1 + ad-3B + + . , Component 1 , , , X , . , • t o l a s c , + un-3 A + ad-3A + n i c - 2 a l - 2 . Component 2 , , , , , , , L _ t " 1 a 5 C A p p r o x i m a t e 10 0.1 10 9 0.3 U 28 map d i s t a n c e s ( R a d f o r d , 1972) A cross homozygous f o r a s c - 6 , designed to t e s t nond i s j unc t i on of three l i nkage groups (LG I, IV, and V) s imu l taneous l y . L i n k a g e g r o u p I I I A + a u r + a s c - i a 1 1 ' B a a d - 3 A + a l - 2 a s c 15 3D 1 A p p r o x i m a t e map d i s t a n c e s ( R a d f o r d , 1972 ) + c o t - 1 i n o s + — i 1 a 1 1  pdx + + h i s - 1 2D 10 48 l e u - 3 , a_, a r g - 1 , ad-3'B; asc and un-3, "AV ad-3A, n i c - 2 , a l - 2 ; asc (each con ta i n i ng the t o l mutat ion and the heterokaryon c o m p a t i b i l i t y l o c i £ , d_ and e) were obta ined from crosses between the mutant PWT c u l t u r e ( i . e . , homozygous f o r an. asc mutat ion) and w i l d type s t r a i n s OR-a and OR-A. These ascospore i s o l a t e s were i n t e r c r o s s ed and the LG.:I markers used to monitor crossover and nond i s j unc t i on f requenc ies (see F i g . 1 ) . C y t o l o g i c a l Methods The two s t a i n s i r on -hematoxy l i n and a c e t o - o r c e i n were used w i th about equal success. The methods d i f f e r e d mainly i n t h e i r a b i l i t y to v i s u a l i z e s p i n d l e - p o l e bodies and n u c l e o l i ( i r on -hematoxy l i n s t a i n s both s t r u c t u r e s whereas a c e t o - o r c e i n s t a i n s n e i t h e r ) . S t a i n i n g w i t h i r o n -hematoxyl in was done e s s e n t i a l l y as desc r ibed prevous ly (Raju and Newr-meyer, 1977; Lu and GaTeazz i , 1979) w i t h a s l i g h t m o d i f i c a t i o n . Fo l l ow -ing h y d r o l y s i s , p e r i t h e c i a were washed overn ight a t room temperature i n a s o l u t i o n of 3:1:1 abso lute e t h a n o l : a c e t i c a c i d : ch l o r o f o rm (Carnoy ' s s o l u t i o n ) . This procedure removed the f a t g lobu les from the cytoplasm and t he re f o re a l lowed the v i s u a l i z a t i o n o f chromosomes at a l l stages o f me i o s i s . The method u t i l i z i n g a c e t o - o r c e i n i s b a s i c a l l y a combination of severa l methods ( G r i f f i t h s e t_a j_ . , 1974; M. B a s l , personal communication). P e r i t h e c i a were f i x e d i n a s o l u t i o n of 6:3:1 abso lute e thano l : ch l o ro fo rm: a c e t i c a c i d . The fiixed ma te r i a l was l e f t a t room temperature overn ight and then incubated a t -20°C f o r a pe r i od of 3 weeks to 6 months. This prolonged i n cuba t i on helps to remove the f a t g lobu les from the cytop lasm. The f i x e d p e r i t h e c i a were then washed i n wate r , .hyd ro l yzed i n 1 N HC1 f o r 4-5 minutes a t 60°C,^washed again i n i c e water and immersed i n l euco -ba s i c 49 f uch s i n f o r about 40 minutes a t room temperature. Subsequent methods o f s t a i n i n g , v iewing and photography were desc r ibed p r e v i ou s l y ( G r i f f i t h s e t a l _ . , 1974). Methods used to ob ta in c l u s t e r s of 8 ascospores, i . e . , a s c i (Newcombe and G r i f f i t h s , 1972; P e r k i n s , 1974) and other r ou t i ne genet i c manipu lat ions (Davis and deSer res , 1970; Chapter I) have been reported p r e v i o u s l y . Detect ion of Chromosome Segregat ion Defects During the F i r s t M e i o t i c  D i v i s i o n ( i . e . , During MI) In most cases , nond i s j unc t i on of LG I was measured. Th i s l i n k -age group was m u l t i p l y marked to a l l ow the d e t e c t i o n of ascospore i s o -l a t e s t ha t conta in both complementing copies of LG I. S t r a i n s i n which complementation of mutations on any par t of a chromosome occurs are ap-pa ren t l y w i l d type f o r these markers and are t he re fo re c a l l e d pseudo-w i l d type (PWT). Nond i s junc t ion of non-crossover chromosomes, assuming r e g u l a r i t y of subsequent d i v i s i o n s , would r e s u l t i n ascospores PWT f o r a l l mutations on LG I ( a u x o + j : l e u - 3 , a_, a r g - 1 , ad-33 + un-3, A, ad-3A, n i c - 2 , a l - 2 . In c o n t r a s t , i f c rossover chromosomes are i n v o l v e d , the f o l l o w i n g pheno-types w i l l be expected: l eu ( l euc i ne r e q u i r i n g ) and auxo + (w i l d type) i f nond i s j unc t i on f o l l owed a s i n g l e c rossover event i n the leu-un reg ion (CO l eu - un ) ; l e u , un and auxo + (CO un-a rg ) ; l eu a r g , un and auxo + (CO arq-cent romere) ; ad n i c a l , ad and auxo + (CO centromere-ad); n i c a l and auxo* (CO a d - n i c ) ; al. and auxo + (CO n i c - a l ) . The d isomic nuc l e i i n young PWT ascospores soon h a p l o i d i z e ( P i t t e n g e r , 1954; Chapter I ) . There fo re , these s t r a i n s conta in a t l e a s t two complementing hap lo i d 50 nuc lear types . The he te r oka r yo t i c na tu re .o f such s t r a i n s was, i n many cases , conf i rmed by t e s t i n g the genotypes of i n d i v i d u a l c o n i d i a l i s o -l a t e s . Even(though a l l of the above mentioned h e t e r o k a r y o t i c s t r a i n s are PWT f o r a t l e a s t some auxotroph ic mutat ions , on ly those PWT p ro -geny i n which a l l auxot roph ic mutations on a p a r t i c u l a r l i nkage group complement ( i . e . , auxo + ) were i nc luded i n the e s t ima t i on of PWT f r e -quencies i n crosses homozygous f o r a s c - 1 , asc-6 and me i - 1 . The PWT f requenc ie s were determined i n one o f two ways: ( i ) by t e s t i n g i n d i -v i dua l ascospore i s o l a t e s , or ( i i ) by p l a t i n g ascospores on both minimal (only auxo + PWT s t r a i n s grow) and on medium supplemented w i t h l e u c i n e , a r g i n i n e , adenine and n i c o t i n i c a c i d ( a l l v i a b l e s t r a i n s grow). In one case (crosses homozygous f o r asc-6) the s imultaneous nond i s junc t i on of more than one chromosome was determined. Complement-ing mutations on LG I (A and a_ mating type a l l e l e s ) , LG IV (pdx and cot ) , and LG V ( inos and h i s ) were used to de tec t PWT progeny f o r these l i n k -age groups (see F i g . 2; au r , a l - 2 , ad-3A and ac r mutat ions i n the s t r a i n s used are not r e l e van t i n the present s t udy ) . Detec t i on o f Chromosome Segregat ion Defects During the Second M e i o t i c  D i v i s i o n ( i . e . , During M i l ) Nond i s junct ion a t M i l can be detected through complementation o f c l o s e l y l i n k e d mutat ions . Two such complementing mutations can on ly be present on the same dyad chromosome ( i . e . , j u s t p r i o r to M i l ) f o l l o w -ing an exchange event between these mutations and the centromere. Con-sequent l y , nond i s j unc t i on a t M i l can on ly be detected f o l l o w i n g such an exchange event and w i l l be detected more f r equen t l y the f u r t h e r the 51 complementing mutations are from the centromere. Even though the de tec t i on of nond i s j unc t i on dur ing both MI and M i l depends on the complementation between c l o s e l y l i n k e d mutat ions , these two types of events can u sua l l y be d i s t i ngu i shed , i f mutant markers are present on both s ides of the centromere. In the case of nondis -j u n c t i o n a t M i l , mutant markers on one s i de of the centromere, but not the o t h e r , complement. In c o n t r a s t , nond i s junc t i on a t MI u s ua l l y r e s u l t s i n the complementation of mutat ions on both s ides of the centromere ( F i g . 3 ) . Recombinant Frequency (RF) i n Crosses w i t h a High Amount of Nond i s junct ion Nond i s junc t i on Ma in ly a t the F i r s t M e i o t i c D i v i s i o n (MI). The f r e -quency of recombinants i s g e n e r a l l y equated to the p ropo r t i on of recom-b inant chromat ids. This value i s r e a d i l y determined among hap lo i d p ro -geny by t e s t i n g f o r the presence of s p e c i f i c marker mutat ions . However, the de tec t i on of recombinant chromatids i n PWT progeny i s o f ten more com-p l i c a t e d . . To determine t h i s v a l ue , the n i c - a l reg ion (30-35 mu) was used. Asc i w i th a crossover event i n t h i s r e g i on , f o l l owed by nond i s j unc t i on a t MI, would produce one- four th a l b i n o (aj_) and t h r ee - f ou r t h s orange ( a l + ) PWT progeny (assuming r egu l a r subsequent d i v i s i o n s ) . S i m i l a r a sc i i n which chromosomes d i s j o i n i n a r e gu l a r manner would produce one -ha l f r e -combinant progeny ( f o r the n i c - a l r e g i o n ) . The re fo re , the frequency of recombinant chromatids f o r PWT progeny would be tw ice the frequency of a l b i no PWT progeny (= 2x aj_ PWT/total PWT). The frequency of recombinant chromatids i s u s ua l l y i d e n t i c a l to the crossover frequency i n a p a r t i c u l a r r eg i on . This i s p a r t i c u l a r l y so i f , as i n Neurospora, a l l chromatids from each meios i s are recovered from 52 F i g . 3 Expected PWT progeny r e s u l t i n g from nond i s junc-t i o n dur ing the f i r s t m e i o t i c d i v i s i o n ( a ) , and a c rossover f o l l owed by nond i s j unc t i on dur ing the second m e i o t i c d i v i s i o n (b ) . 53 l e u un MI l e u un M i l l e u a r g a d - 3 B  f i a d - 3 A n i c a l IMondis j u n c t i o n ( b ) l e u a r g a r g a d - 3 B n a d - 3 A n i c a l a r g a d - 3 B a d - 3 A n i c a l ( P U T ) N o t e : some c r o s s o v e r t y p e s a r e a l s o r e c o v e r e d ( s e e M a t e r i a l s and M e t h o d s ) I un a d - 3 B a d - 3 A n i c a l MI l e u l e u M i l l e u a r g a d - 3 B a r g a d - 3 A n i c a l and un a d - 3 B un a r g a d - 3 A n i c a l N o n d i s j u n c t i o n a d - 3 B 0 o r a d - 3 A ( l e u , n i c a l a r g ) un a d - 3 B o a d - 3 A n i c a l ( u n ) N o t e : e x c h a n g e b e t w e e n t h e c e n t r o m e r e and a r g - 1 U J i 11 p r o d u c e n o n d i s j u n c t i o n p r o -geny a d - 3 B and a d - 3 A , n i c , a l 54 a sexual c r o s s . However, i n crosses where nond i s j unc t i on takes p l a c e , a f r a c t i o n of ascospores i s i n v i a b l e and many chromatids w i l l not be recovered. In such cases , the frequency of recombinant chromatids may not be the same as crossover f requency, s i n ce crossover or non-crossover chromatids might be p r e f e r e n t i a l l y i nc luded i n the v i a b l e progeny. For example, a de fec t i n p a i r i n g of chromosomes a t the f i r s t prophase of meios i s g ene r a l l y r e s u l t s i n t h e i r i r r e g u l a r d i s j u n c t i o n (see DIS-CUSSION). S ince the lack of p a i r i n g excludes the p o s s i b i l i t y of c r o s s i n g - ove r , i t should be mainly the non-crossover chromatids t ha t would be i nvo l ved i n i r r e g u l a r d i s j u n c t i o n and, t h e r e f o r e , may be p re -f e r e n t i a l l y excluded from the v i a b l e progeny. Thus, i n these cases , the recombinant frequency would be h igher than the ac tua l c rossover f r e -quency. S ince there appears to be no evidence suggest ing tha t crossover chromosomes might be p r e f e r e n t i a l l y excluded from the v i a b l e progeny, the recombinant frequency i n a cross w i th nond i s j unc t i on dur ing MI should be cons idered an overest imate of the crossover f requency. Consequently, any r educ t i on i n recombinant frequency should represent a r ea l decrease i n crossover f requency. The o v e r a l l frequency of recombinant chromatids from both PWT and non-PWT progeny cou ld be determined by combining the i n d i v i d u a l RF values of these two types of progeny. However, the r e l a t i v e c o n t r i b u t i o n of the frequency of recombinant chromat ids , which i s d i f f e r e n t f o r PWT and non-PWT progeny (see RESULTS), i s i n doubt. The o r i g i n a l nuc l e i d isomic f o r LG I, g i v i n g r i s e to PWT progeny, and those hap lo id f o r LG I, producing non-PWT progeny, many be d i f f e r e n t i a l l y i nc luded i n t o v i a b l e ascospores. Such d i f f e r e n t i a l v i a b i l i t y may produce a frequency of 55 recombinant chromatids which does not c o r r e c t l y r e f l e c t c ros sover f r e -quency. There fo re , the o v e r a l l recombinant frequency was not determined. Nond i s j u c t i on During the Second M e i o t i c D i v i s i o n ( M i l ) . The r e -combinant frequency (RF) i n any reg ion equals the p ropo r t i on of recom-b inant chromat ids . In crosses w i th a high frequency of nond i s j unc t i on a t M i l , the l a rge number of r e s u l t a n t d isomic progeny prevent the i d e n t i f i -c a t i o n of some recombinant chromat ids. In a d d i t i o n , s i n ce the frequency o f d isomic nucle i - i s unknown, i t i s not po s s i b l e to d i r e c t l y determine the t o t a l number o f chromatids among the progeny from such a c r o s s . There fo re , to ob ta in a more app rop r i a te es t imate of RF, on ly a s c i con-t a i n i n g a c ros sover i n a wel l -marked reg ion should be cons ide red . The frequency of d isomic and hap lo i d progeny from such a s c i can be e s t i -mated s i n ce a l l d i somic nuc l e i produce d i s t i n g u i s h a b l e h e t e r o k a r y o t i c (HK) products and h a l f the h a p l o i d nuc l e i are recombinant (CO). In the present s tudy, the RF value was determined i n a reg ion which was marked on one s i de by the c l o s e l y l i n k e d mutations un-3 and a r g - 1 , and on the other s i de by ad-3A and ad-3B ( F i g . 3 ) . Dev ia t ions produced by c r o s s -4 overs between un-3 and arg-1 (1-2 mu) or ad-3A and ad-3B (0.3 mu) should be cons idered very minor s i nce the arg-ad reg ion spans 15-20 mu. The subgroup of a s c i w i t h a c ros sover i n the arg-ad reg ion p ro -duces ascospores which are hap l o i d f o r LG I, and those t h a t are d i somic f o r LG I. Ha l f the hap lo i d ascospores w i l l be recombinant (CO), and the disomies w i l l c a r r y a parenta l and a c ros sover chromosome (P + CO). The This low RF value was c o n s i s t e n t l y obta ined dur ing t h i s s tudy. The d e v i a t i o n from the value p r e v i ou s l y reported (Radford, 1972) i n F i g . 1 may w e l l be due to r e c - t ype genes (see e . g . , Ca tches ide , 1974). 56 r e s u l t a n t CO and HK progeny can be i d e n t i f i e d . In these a s c i , the f r e -quency of HK progeny equals the frequency o f d isomic n u c l e i . This va lue equals HK/HK + 2 CO (or d i somic/d i somic + h a p l o i d ) . Assuming t ha t t h i s frequency of d isomic nuc l e i i s the same i n a l l types o f a s c i , regard le s s o f c ros sover events i n s p e c i f i c r eg i on s , t h i s frequency would apply to a l l progeny from the c r o s s . There fo re , the t o t a l number o f d i -somic ascospore i s o l a t e s would be: HK ( H K + 2 c o ^ x t o t a l P r ° 9 e n y . S i m i l a r l y , the t o t a l number o f hap l o i d ascospore i s o l a t e s would be: , 2 CO v . . , ( H K + 2 CO^ x progeny. These values enable the e s t ima t i on of the t o t a l number o f chromatids obta ined from a c r o s s : 2 (di somics) + hap lo ids = ( ^ + ,^ G Q - + HK + 2 CO^ x t o t a l progeny = 2 C O ^ X t o t a 1 P r ° 9 e n y -lHK +  O Subsequently, the RF va lue i n a reg ion tha t a l lows the d i s t i n c t i o n be-tween CO and HK progeny would be: PP CO + HK (?;recombinant chromatids) 2 (HI' + rn) ^HK + 2 CO ^ x t o t a l P r o 9 e n y ( = t o t a l chromatids) _ HK '+ 2 CO 2 x t o t a l progeny. In.the present s tudy, the CO va lue was obta ined us ing the un-n ic r eg i on . However, HK progeny cou ld only be detected f o l l o w i n g a c rossover i n the 57 s l i g h t l y sma l l e r arg-ad r e g i on . There fo re , s i n ce a low est imate f o r HK was used, the RF value o f the un-n ic reg ion obta ined by t h i s method would be a s l i g h t l y low e s t imate . RESULTS Four r e ce s s i v e mutations ( a s c - 1 , a s c - 3 , asc-6 and mei-1) which cause the abo r t i on o f some of t h e i r ascospores, were ana l yzed . In each case, the aborted ascospores were wh i te i n s tead of b l a c k , and i n v i a b l e . The f e r t i l i t y of crosses homozygous f o r each r ece s s i ve mutat ion was n a t u r a l l y always reduced due to the i n v i a b l e ascospores. However, i n t h i s chapte r , the term " f e r t i l i t y " i s used to des ignate the t o t a l num-ber of ascospores (b lack or w h i t e ) . An a r b i t r a r y measure of low (when very few spores are produced), medium, or high f e r t i l i t y i s employed. As w i l l be shown, the ascospore abo r t i on i n a l l f ou r mutat ions i s caused by i r r e g u l a r s eg rega t i on , o r i i n o n d i s j u c t i o n , of chromosomes. In Neurospora, such i r r e g u l a r segregat ion o f chromosomes can be detected by the p re -sence of PWT progeny (see MATERIALS AND METHODS). PWT progeny are de-tec ted through t h e i r complementation o f some or a l l auxot roph ic mutat ions . The three n e w l y - i s o l a t e d asc mutations (see Chapter I) w i l l be d i scussed i n order Of t h e i r apparent i n c r ea s i n g complex i ty and stage o f d e f e c t . i n the segregat ion of chromosomes: asc-6 ( f i r s t d i v i s i o n ) ; asc-3 (second d i v i s i o n ) ; and asc-1 ( f i r s t and second d i v i s i o n s ) . F i n a l l y , some new observat ions o f the mei-1 mutat ion w i l l be r epo r ted . asc-6 (P379) Crosses homozygous f o r t h i s r e ce s s i ve mutat ion g e n e r a l l y r e s u l t e d i n about 70% ascospore abo r t i on and a reduc t i on i n f e r t i l i t y . The amount 58 of spore abo r t i on was q u i t e constant i n a l l crosses te s ted but f e r -t i l i t y v a r i ed from low to medium (Table I ) . A l l i n i t i a l s t r a i n s c a r r y i n g asc-6 grew about three times as s l ow ly as w i l d type s t r a i n s . I t was found tha t t h i s slow growth was due to a po i n t mutat ion ( s l o ) l i n k e d to the me i o t i c mutations (see sec -t i o n on mapping). The s l o muta t ion , however, i n no way a f f e c t e d the phenotype of a s c -6 . Recombination and Nond i s junct ion Frequenc ies . The ana l y s i s of ascospore c u l t u r e s from four d i f f e r e n t crosses homozygous f o r ' a s c - 6 r e -vealed a d r a s t i c a l t e r a t i o n i n both RF and nond i s j unc t i on frequency (Table I ) . A high frequency of PWT progeny, ranging from 16.4 to 37%, ; was recovered from a l l f ou r c ro s se s . The great ma jo r i t y of these were w i l d type f o r a l l auxotroph ic mutations on LG I. I t t he re fo re appears tha t these PWT cu l t u r e s are the r e s u l t of nond i s j unc t i on dur ing MI. Three regions on LG I were monitored f o r RF: l e u - un , un-n ic and n i c - a l . Compared to w i l d type c r o s se s , the RF values from a l l f ou r crosses homozygous f o r asc-6 were reduced i n a l l three regions (Table I ) . The Nature o f PWT Progeny. To e s t a b l i s h the nuc lear composi-t i o n of the PWT progeny from these c ro s se s , the genotypes of c o n i d i a l i s o l a t e s from ten PWT progeny, recovered from the cross between s t r a i n s 879al3 and 879A15, were determined. In each case, the two parenta l t ypes , i . e . , un_, ad, n i c , a l and l e u , a r g , ad, were recovered. There fo re , these PWT c u l t u r e s were t r u l y h e t e r o k a r y o t i c , as would be expected i f they were produced by n o n d i s j u n c t i o n . The a d d i t i o n a l d e t e c t i o n of a few crossover types among c o n i d i a l i s o l a t e s from two PWT c u l t u r e s i s c o n s i s t e n t w i th somatic exchange events ( P i t t enge r and Coy le , 1963). T a b l e I . G e n e t i c a n a l y s i s o f f o u r c r o s s e s homozygous f o r a s c - 6 Cross IMon-PLiT progeny PUT progeny F e r t i l i t y (no. of spores) Total non-PUT progeny Recombination frequency (no. recombinants i n parentheses) leu-un un-nic n i c -(%) a l Total PWT progeny PUT f r e q . (%) Genotypes of PUT's auxo + other Q22-2 x Q22-8* 95 1.1 (1) 6.5 (6) 11.4 (11) 45 32.1 45 0 low Q8-1 x Q8-2* 63 3.2 (2) 7.9 (5) 12.7 (8) 37 37.0 35 2(1 a l & 1 leu,arc med Q35-1 x 035-2* 81 2.5 (2) 6.2 (5) 13.6 (11) 33 29.0 33 0 med RFCnic-al) of 3 crosses combined: 12.5 1.7* ' S79a13 x 879A15 61 6.5 (4) • (0) 3.3 (2) 12 16.4 12 0 low Wild type crosses 11-17 15-20 30-35 < 0.1 A_ and _a components f r o m a PUT c u l t u r e f r o m a c r o s s between s t r a i n 1-34-8 ( u n , A_, a_d, n i c , a l ) and 879A15 ( l e u , a_, a r g , ad ; a s c - 6 ) . t A s c o s p o r e i s o l a t e s d e r i v e d as d e s c r i b e d i n M a t e r i a l s and M e t h o d s , LG I m a r k e r s were i d e n -t i c a l f o r a l l 4 c r o s s e s ( s e e F i g . 1 ) . ^ R F ( n i c - a l ) among PUT p r o g e n y = 2x f r e q . o f a l b i n o PUT 1 s ( s e e M a t e r i a l s and M e t h o d s ) . 60 The absence of c rossover chromosomes from the ten PWT cu l t u r e s may suggest that on ly non-exchange chromosomes f a i l to d i s j o i n and are t he re f o re i nc luded i n the PWT progeny. To ob ta in a more q u a n t i t a t i v e measure o f the f requenc ies of exchange chromosomes among PWT and non-PWT progeny, the RF values i n the n i c - a l reg ion were determined f o r both types of progeny from three crosses (Table I ) . The value f o r PWT p ro -geny (1.7% = 2 x frequency of a l b i n o PWT's; see MATERIALS AND METHODS) was much lower than t ha t f o r non-PWT progeny (12.5%). I t i s t he re fo re concluded tha t most nond i s j unc t i on i n vo l ve s non-exchange chromosomes. Ascus A n a l y s i s . Unordered a s c i from a cross homozygous f o r asc-6 were analyzed i n two ways. F i r s t , a l l types of ascus abo r t i on pat terns were recovered (Table I I ) . The prevalence of a s c i w i th an even number of b lack ascospores (26/37) suggests t ha t the de fec t l e a d i n g to ascospore abo r t i on takes p lace p r i o r to the po s t -me i o t i c d i v i s i o n . However, the frequency of a sc i w i t h odd numbers o f b lack ascospores i s too high (11/37) f o r complete r e g u l a r i t y o f t h i s p o s t -me i o t i c d i v i s i o n . Second, the b lack ascospores from each ascus were germinated and the genotypes o f the r e -s u l t i n g c u l t u r e s determined. Seven out of 37 a sc i conta ined a t l e a s t one PWT ascospore i s o l a t e . These seven a sc i and the genotypes of t h e i r a s -cospore c u l t u r e s are shown i n Table I I I . The presence of an odd number of PWT progeny i n s i x o f these a s c i s t r ong l y suggests t ha t chromosome lo s s or secondary nond i s j unc t i on takes p lace dur ing the po s t -m e i o t i c d i v i s i o n . Recombination and Nond i s junct ion Frequencies I nvo l v ing Chromo- somes Other Than LG I. Thus f a r , nond i s j unc t i on has on ly been recorded f o r LG I. To i n v e s t i g a t e the degree of nond i s j unc t i on (and reduc t i on i n T a b l e I I . A s c u s a n a l y s i s o f a c r e s s homozygous f o r a s c - 6 (879a13 x 879A15) Types o f A s c i M L , C „ . N, , , y K Number o f A s c i O b s e r v e d ( b l a c k : w h i t e a s c o s p o r e s ) 8 : 0 1 6 : 2 3 4 : 4 2 2 : 6 14 0 : 8 6 o t h e r ( m o s t l y 1:7, 3:5) 11 T o t a l 37 62 T a b l e I I I . I s o l a t e s o b t a i n e d f r o m a s c i , p r o d u c e d by a c r o s s ( S 7 9 a 1 3 x 879A15) homozygous f o r a s c - 6 , w h i c h c o n t a i n a t l e a s t one PUT c u l t u r e Type o f A s c u s No. I s o l a t e s G e n o t y p e o f I s o l a t e s (B:W) G e r m i n a t e d PWT l e u , a r g , ad un-, ad , n i c , a l 1 : : 7 1 1 • 0 2 : : 6 2 2 • • 3 : : 5 3 1 2 • 3 : : 5 2 1 • 1 k : : k k 3 1 • k : : k - 3. 3 • 5 : :• 3 1 .1 • • 63 recombinat ion) o f other chromosomes, a cross was analyzed which enabled the s imultaneous ana l y s i s o f LG I, IV and V ( F i g . 2 ) . LG I was te s ted f o r he te rozygo s i t y a t the mating type locus (A/a_), LG IV f o r pdx + and cot -1 , and LG V f o r inos and h i s - 1 . In the l a t t e r two l i n kage groups the frequency of recombinat ion was approximated by the appearance o f the double mutant ( e . g . , pdx, c o t - 1 ) . The extreme r a r i t y o f such recom-binants (1/165 pdx, co t - 1 , - and 0/165 i n o s , h i s - 1 ) shows tha t recombina-t i o n i s reduced i n a l l l i n kage groups (compare approximate map d i s tances i n w i l d type c ro s se s : pdx - co t - 1 : .20 mu; i n o s - h i s - 1 : 10 mu). In a d d i -t i o n , t h i s i n d i c a t e s tha t p r a c t i c a l l y a l l p d x + , c o t - l + (71/165), and i n o s + , h i s - l + (79/165) i s o l a t e s are d isomic f o r LG IV and V, r e s p e c t i v e l y . Table IV shows the data on the s imultaneous nond i s j unc t i on of these three chromosomes i n two d i f f e r e n t crosses homozygous f o r a s c -6 . The r e s u l t s revea l severa l aspects o f nond i s j unc t i on i n these c ro s se s : i ) A l l th ree chromosomes te s ted show a high degree of n ond i s j unc t i on . i i ) , The f requenc ies of nond i s j unc t i on o f the three chromosomes i n each p a r t i c u l a r cross are very s i m i l a r . However, n ond i s j unc t i on f r e -quencies are d i f f e r e n t i n d i f f e r e n t crosses (33.3, 35.0 and 38.3% i n one c r o s s , and 54.3, 47.6 and 53.3% i n the other c r o s s ) . i i i ) The number o f i s o l a t e s e i t h e r s imu l taneous ly PWT or non-PWT f o r a l l t h r e e l i n kage groups t e s ted i s s i g n i f i c a n t l y h igher than ex-pected (p < 0.01) . This may mean t ha t chromosomes do not d i s j o i n independent ly of each o the r . A l t e r n a t i v e l y , i t may r e f l e c t s e l e c -t i o n aga in s t i s o l a t e s w i t h some PWT and some non-PWT l i nkage groups. That the l a t t e r i s q u i t e p l a u s i b l e i s shown by the pe r -centage germinat ion i n these crosses (47.8, 50.0 and 57.5%). In T a b l e I V . S i m u l t a n e o u s n o n d i s j u n c t i o n o f t h r e e l i n k a g e g r o u p s i n two c r o s s e s homozygous f o r a s c - 6 C r o s s 129 -75 x 128-15 C r o s s 129-79 x 128-15 Number of P r o g e n y Number o f P r o g e n y H y p e r p l o i d f o r LG O b s e r v e d E x p e c t e d * ( 0 - E ) V E O b s e r v e d E x p e c t e d * I IV7 U ( 0 ) (E) (•) ( E ) + + + 28 14.5 12.6 8 2.7 + + - 9 12. 7' 1.1 3 4.3 + " - + 11 15.9 "1.5 6 5.0 - •+ + 5 12.2 4.2 3 5.4 + - - 9 14.0 1.8 3 8.0 - + - 8 10.7 0.7 7 8. 6 + 12 13.4 0. 1 6 10.0 _ 23 11.7 10.9 24 16.0 105 105.1 X 2 H f = 32.9 7 60 60.0 * D e t e r m i n e d f r o m p r o d u c t o f n o n d i s j u n c t i o n f r e q u e n c y o f i n d i v i d u a l l i n k a g e g r o u p s (LG I : 0.543, LG IV: • . 476, LB V: 0. 533 f o r c r o s s 129-75 x 128-15; and LG I : •. 3 3 , LG I V : 0.350, LG V: 0.383 f o r c r o s s 129-79 x 1 2 8 - 1 5 ) . 65 combination w i t h data on co lony- fo rming a b i l i t y obta ined from the mutant asc-1 (see below, and Table X ) , the s e l e c t i v e hypothes i s seems most appea l ing (see a l s o DISCUSSION). Cyto logy. C y t o l o g i c a l observat ions of crosses homozygous f o r asc-6 show a d r a s t i c reduct ion i n p a i r i n g of homologs a t pachytene ( F i g . 4a, b ) . Some p a i r i n g was observed mainly near the t i p s of some chromosomes. This r educ t i on i s r e f l e c t e d i n a d r a s t i c decrease i n the number o f recombinants obta ined from these c ro s se s . An apparent consequence of reduced p a i r i n g and exchange i s the product ion o f un i va len t s dur ing d i a k i n e s i s and metaphase I. F i g . 4c shows two separate chromosomes at tached to the nuc leo lus a t d i a k i n e s i s . These appear to be two un i va len t s of the same chromosome. Instead of seven b i v a l e n t s , up to 14 un i va len t s can be detected on the s p i nd l e of metaphase I ( F i g . 4e ) . Subsequent d i v i s i o n d i s t r i b u t e s roughly equal amounts o f chromatin to oppos i te poles and, a t prophase I I , up to 14 ( i n s tead of the usual seven) chromosomes m a t e r i a l i z e i n each dyad nucleus. F i g . 4 f , g ) . This observat ion i s compat ib le w i th the equat iona l d i v i -s i on of many un i va len t s dur ing the f i r s t . d i v i s i o n (see DISCUSSION). The second d i v i s i o n takes a long time to complete; whereas f i g u r e s a t t h i s stage o f d i v i s i o n were observed on ly r a r e l y i n w i l d type c r o s s e s , they were q u i t e common i n crosses homozygous f o r asc-6 ( F i g . 4 h - k ) . F i n a l l y , unequal amounts of chromatin o f t en segregate dur ing t h i s and the pos t -m e i o t i c d i v i s i o n l ead ing to the d i f f e r e n t types of ascospore a b o r t i o n . In c o n c l u s i o n , the primary de fec t o f mutant asc-6 appears to be a de fec t dur ing the p a i r i n g o f homologs a t the f i r s t prophase o f me io s i s . This r e s u l t s i n a d r a s t i c decrease of recombinat ion and the product ion 66 F i g . 4 Chromosome development i n crosses homozygous f o r a sc -6 . P i c t u r e s ( f ) , ( g ) , and (h) were s t a i ned w i th Feulgen and a c e t o - o r c e i n ; a l l other prepara- . t i on s were s t a i ned w i th i r on -haematoxy l i n . Most chromosomes f a i l to p a i r a t pachytene (a,b) x3400; some regions can be seen to p a i r though ((a) arrowed). At d i a k i n e s i s , the two homologs of the n u c l e o l a r chromosome may be sepa ra te l y a t tached to the nuc leo lus (c) x3400; f i g u r e s (c) and (d) represent one ascus a t two d i f f e r e n t f o c i . At metaphase I, up to 14 un i va len t s may be seen ( e ) x 3 4 0 0 , and a s i m i l a r number of chromosomes (7-14) i n d i v i d u a l i z e dur ing the f o l l o w i n g prophase i n each dyad nucleus ( f , g ) x 1300. A long pe r iod necessary to comple te ' the second d i v i s i o n was sug-gested by the unusual ly l a r ge number of nuc l e i a t t h i s stage of d i v i s i o n ( i , j , k ; s p i n d l e - p o l e bodies are arrowed) x2400; t h i s may sometimes cause some s p i n d l e over lap ( j ) . That the ma te r i a l c l o s e s t t o -the s p i n d l e - p o l e bodies i s DNA, was shown by p re -para t ions made w i th a ce t o - o r ce i n s t a i n which only s t a i n s DNA (h) x l 300 . 68 of many un i va lent s a t the f i r s t metaphase. The high frequency of PWT progeny suggests tha t nond i s j unc t i on takes, p lace dur ing the f i r s t m e i o t i c d i v i s i o n . The mechanism o f such nond i s j unc t i on may be, a t l e a s t i n p a r t , the equat iona l sepa ra t i on o f u n i v a l e n t s . The subsequent i r r e g u l a r and extended second d i v i s i o n of meios i s and abnormal segre-ga t i on a t the po s t -me io t i c d i v i s i o n account f o r a l a r ge amount of the ascospore a b o r t i o n . asc -3 (P243, P393) Crosses homozygous f o r t h i s r e ce s s i v e mutat ion gene r a l l y p ro -duced very few ascospores and, of those produced, 90-98% were white and incapab le of germinat ion and growth. Crosses homozygous f o r mutant ascospore. i s o l a t e s obta ined from s t r a i n P393 produced between 9 and 42% b lack ascospores. S ince these crosses were w i l d type f o r LG I markers, recombinat ion and nond i s j unc t i on f requenc ies could not be determined. These high f requenc ies of b lack ascospores were never obta ined i n s i m i -l a r crosses w i th marked homologs of LG I. However, the r e s u l t s appear to suggest tha t the phenotype of t h i s mutant can be a l t e r e d d r a s t i c a l l y by modi fy ing genes. The two non-complementing mutations of t h i s locus are assumed to be homoal le les (o f the same o r i g i n ) because they behaved i d e n t i c a l l y i n genet i c crosses (see below),.and were recovered from the two ascospore.; i s o l a t e s P243 and P393 which were obta ined from the same cross p l a t e . Recombination and PWT Frequenc ies . Random progeny a n a l y s i s was performed on a number of crosses homozygous f o r a s c - 3 . Four types of progeny cou ld not be exp la ined.as homokaryotic parenta l or c rossover types (Table V ) . These were apparent ly PWT c u l t u r e s of genotypes auxo + T a b l e V. P r o g e n y a n a l y s i s o f c r o s s e s homozygous f o r a s c - 3 (P2k3 o r P393) IMon-PUT P r o g e n y ; PUT P r o q E n y T o t a l No. IMo . R e c o m b i n a n t s T o t a l No. G e n o t y p e s ) IMon-PLJT i n R e g i o n PUT ( a l o r a l Cro s s * P r o g e n y l e u - u n u n - n i c n i c - a l P r o g e n y i auxo un ad ad , n i c 243a32 x 2^3A18 16 3 5 6 1 1 0 •• 0 2^3a32 x 2if3A28 17 1 k 3 3 0 1 1 1 2if3a23 x 2£+3A27 17 3 2 it 3 0 0 •2 1 243a31 x 393A35 18 3 1 6 3 2 1 0 0 k comb , c r o s s e s P243 17 3 1 5 it 2 0 2 393a30 x 393A34 26 6 2 7 3 0 1 0 2 T o t a l 11 1 19 15 31 17 3 5 3 6 O v e r a l l RF (%) . = 17/128 = 3% among non-PlilT 17. 1 13.5 28.0 PUT f r e q 13. p r o g e n y U i l d t y p e c r o s s e s , RF v a l u e s 11- 17 15-20 30-35 PUT f r e q . <0.1% C r o s s e s b e tween a s c o s p o r e i s o l a t e s w h i c h were d e r i v e d f r o m mutant s t r a i n s P24 3. and P393 as d e s c r i b e d i n M a t e r i a l s and Methods ( f o r g e n o t y p e s , see F i g . 1 ) . A s c o s p o r e i s o l a t e s f r o m t h e s e c r o s s e s were o b t a i n e d frtfm s h o t a s c i . 70 (w i l d type f o r a l l auxot roph ic marker muta t i on s ) , un-3 (w i l d type f o r a l l marker mutations except un-3) , ad-3 ( l e u + , a r g + , u n + , n i c + ) and ad-3, n i c - 2 ( l e u + , a r g + , u n + ) . In the pooled da ta , these four types of p ro -geny represented 13.3% o f a l l progeny. RF i n the three reg ions t e s ted ( l eu - un , un-n ic and n i c - a l ) were s i m i l a r to those obta ined from w i l d type crosses (see Table V ) . They were determined as a p ropo r t i on o f the non-PWT progeny. More a p p r o p r i -a t e l y , c o r r e c t ed values w i l l be presented i n a l a t e r s e c t i o n . The Nature o f PWT Progeny. To determine the nature and o r i g i n of the PWT progeny, c o n i d i a l i s o l a t e s from twelve ascospore c u l t u r e s rep re sen t i ng a l l f ou r types (auxo + ; un-3; ad -3 ; and ad-3 , n i c - 2 ) - were, t e s t e d . Table VI shows genotypes recovered from these c u l t u r e s . A l l c u l t u r e s were he te r o ka r yo t i c and most conta ined one parent and one c r o s s -over chromosome. The un_, the ad_.,and the ad ,n i c genotypes were caused by the complementation o f two nuc lear t ypes , one parenta l f o r LG I markers and one crossover i n the un-ad r e g i on ; two out of the three a i i xo + p ro -geny conta ined two de tec tab le c rossover events i n the un-ad reg ion ( e i t h e r a parenta l and double crossover component, or two s i n g l e c ros sover com-ponents) . Thus, a l l f ou r types o f PWT progeny appeared to be produced by the same event, each case i n v o l v i n g a t l e a s t one crossover event be-tween un-3 and ad-3. These data are best exp la ined by p o s t u l a t i n g an unusua l ly high frequency o f nond i s j unc t i on dur ing the second d i v i s i o n of meios i s (see F i g . 5 ) , e s p e c i a l l y s i n ce such nond i s j unc t i on i n these crosses w i l l on ly be detected as he t e r o ka r yo t i c progeny i f a c rossover event i n the un-ad reg ion had preceded such i r r e g u l a r segregat ion ( F i g . 3, MATERIALS AND METHODS). T a b l e U l . C o n i d i a l i s o l a t e s .from r e p r e s e n t a t i v e s o f a l l f o u r t y p e s o f a p p a r e n t l y PUT p r o g e n y f r o m c r o s s e s homozygous f o r a s c - 3 C o n i d i a l I s o l a t e s G e n o t y p e o f A s c o s p o r e P a r e n t a l ( n o t i n c . a l ) C r o s s o v e r No. HK* T o t a l No. I s o l a t e G e n o t y p e No. Ge n o t y p e - No. Ge n o t y p e T e s t e d 1-ad l e u , a r g , a d 1 un , ad 36 12 49 2-ad l e u , a r g , a d 12 un , ad , a l 2D 17 49 3-ad n i c u n , a d , n i c , a l 15 l e u , a r g , ad , n i c 19 15 49 4-un a l u n , a d , n i c , a l 43 l e u , u n , a d , a l 4 2 49 5-un u n , a d , n i c 13 un , ad 24 12 49 6-un u n , a d , n i c , a l 1D un , ad 12 7 29 7-un u n , a d , n i c , a l 3 un, ad 13 11 27 8-un u n , a d , n i c D l e u , u n , a d , a l 33 17 5D 9-un u n , a d , n i c , ( a l ) 0 un, ad 22 5 27 ID-PUT u n , a d , n i c , a l 4 l e u , a d 4 16 24 11-PUT l e u , a r g , a d , n i c un , ad 7 6 19 32 12-PUT u n , a d , n i c , a l l e u , a r g , a d 11 16 • 23 5D * HK (- h e t e r o k a r y o n ) i s o l a t e s have t h e g e n o t y p e o f t h e o r i g i n a l a s c o s p o r e i s o l a t e . 72 F i g . 5 The o r i g i n o f PWT progeny from crosses homozygous f o r a s c - 3 . The he te r o ka r yo t i c progeny recovered from crosses homozygous f o r asc -3 are r e a d i l y exp la ined by one (or more) crossover event ( s ) near the centromere, f o l l owed by a nond i s j unc t i on a t the second m e i o t i c d i v i s i o n . (a) A crossover between arg-1 and the centromere w i l l , f o l l o w i n g a r e gu l a r f i r s t m e i o t i c d i v i s i o n , generate two n u c l e i , each he te r oka r yo t i c f o r the reg ion to the l e f t of the c ro s sove r ; subsequent nond i s j unc t i on a t the second d i v i s i o n w i l l leave the r e s u l t i n g nuc l e i he te ro -k a r y o t i c f o r t h i s r e g i on . In t h i s manner, both aj3 and ad, n i c , ( a l ) progeny can be produced. S i m i l a r l y , a crossover event between ad_ and the centromere w i l l render nuc l e i he te r oka r yo t i c f o r the reg ion to the r i g h t of the c ro s sover ; t h i s w i l l produce e i t h e r l e u , arg or un progeny. (b) I f two crossover event s , one to the l e f t and the other to the r i g h t of the centromere, take p l a c e , PWT progeny w i l l r e s u l t . The data i n Table VI are compatible w i th t h i s i n t e r p r e t a t i o n of the o r i g i n of the (auxo + ) PWT i s o -l a t e s ; the 2- s t rand double c rossover shown i n t h i s f i g u r e was apparent ly i nvo l ved i n two PWT i s o l a t e s (see Table V I , i s o l a t e s 10 and 12). (c) This 3 -s t rand double c ros sover was r e spon s i b l e f o r the remaining PWT. 73 ( a ) l e u i + i a rg ad -3B i + i + i _ J L ^ _ « -w + un + 1 a d - 3 A ' n i c •al l e u + i a rg • J ad -3B i + + i + un + and l e u i + arg i IL p a c h y t e n e i n t e r p h a s e I un + i ad-3A n i c a l n o n d i s j u n c t i o n a t 2nd m e i o t i c d i v i s i o n ad-3B (un, ad + l e u , a r g , ad) Dr: ad-3A, n i c - 2 , a l - 2 (un, ad, n i c , a l + l e u , a rg , ad, n i c , a l ) (b ) l e u + a rg ad -3B + _, , J . • JL un + I ad -3A n i c a l l e u + + ad -3B + + — I 1 1 -„ I 1 1 Jt S E e T a b l e U l + un + ad-3A n i c a l l i n e 1D or l e u + a rg ad -3B + + _I I L ZZZZZ3C " see T a b l e U l un + ad-3A n i c a l l i n e 12 74 (c) l e u + arg - ad-3B + + ••/ + un + ad-3A n i c a l f leu + arg ad-3A n i c + i i i _ J. ± 1 see Table l/I + 75 To r u l e out the remote p o s s i b i l i t y t h a t nond i s j unc t i on only i n vo l ved chromosomes w i th a c rossover i n the centromere r eg i on ; ( t he un-ad reg ion spans the centromere of LG I ) , a cross between s t r a i n s of genotype l e u - 3 , a_, a r g - 1 , ad-3B, a l - 1 and un-3, A, ad-3A, n i c - 2 , a l - 2 was ana lyzed. In t h i s c r o s s , nond i s j unc t i on a t the second m e i o t i c d i v i s i o n would be detected as he t e r o ka r yo t i c progeny i f exchange i n the un-al reg ion had taken p l a ce . Out of a t o t a l of 65 i s o l a t e s , 25 were h e t e r o k a r y o t i c . This h igh frequency o f he t e r o ka r yo t i c progeny suggested tha t nond i s j unc t i on a t the second m e i o t i c d i v i s i o n i n vo l ved both ex-change and non-exchange chromosomes, and thus - was a general phenomenon not r e l a t e d to exchange. Ascus A n a l y s i s . Table VII shows the types of a s c i produced by four crosses homozygous f o r the P243-der ived asc-3 mutat ion and by one cross homozygous f o r the P393-der ived mutat ion . No a sc i w i t h four or more b lack ascospores were ever de tec ted . Most a sc i conta ined e i g h t whi te ascospores but those w i t h one or two b lack ascospores were not r a r e . Both b lack ascospores were germinated from e i gh t 2B:6W a s c i . In each case, the p a i r of genotypes was i d e n t i c a l to each o t he r , i n d i c a t i n g t ha t these c o n s t i t u t e s i s t e r ascospores. The 1B:7W a s c i were apparent l y p ro -duced i n par t by chromosome lo s s or nond i s j unc t i on dur ing the pos t -m e i o t i c d i v i s i o n . The high frequency of these 1B:7W a s c i shows tha t such l o s s i s e x ten s i ve . Co r r e c t i on of Recombination Frequencies (RF). S ince i n crosses homozygous f o r asc-3 some cros sover chromosomes are detected as homo-k a r y o t i c c rossover products and others-are present i n he t e r o ka r yo t i c p ro -duc t s , the RF values cannot be determined i n the usual manner. To 76 T a b l e V I I . A s c u s a n a l y s i s o f c r o s s e s homozygous f o r a s c - 3 * Types c f A s c i ( b l a c k : u h i t e a s c o s p o r e s ) 3 : 5 2 : 6 1 : 7 0 : 8 P 2 4 3 - d e r i v e d s t r a i n s : 243a26 X A18 • 3 2 6 243a26 X A28 2 5 _ 12 17 243a32 X A18 0 3 10 13 243a32 X A13 1 5 2 10 Combined d a t a 3 16 26 46 P 3 9 3 - d e r i v e d s t r a i n : 393a30 x A34' 2 15 10 53 F o u r c r o s s e s i n v o l v i n g a s c o s p o r e i s o l a t e s d e r i v e d f r o m mutant s t r a i n P243 and one c r o s s b e tween a s c o s p o r e i s o -l a t e s d e r i v e d f r o m P 3 9 3 . 77 determine the RF value of a reg ion i t should be po s s i b l e to d i s t i n g u i s h a l l c rossover and h e t e r k a r y o t i c progeny r e s u l t i n g from a s c i w i t h a c r o s s -over i n the r eg i on . The mod i f i ed RF value f o r such a reg ion equals (UK + 2 C0)/2 x t o t a l progeny (HK = the number of he te r oka r yo t i c progeny produced by a crossover, i n the reg ion fo l l owed by nond i s j unc t i on a t M i l ; CO = the number of homokaryotic c ro s s -ove r products i n the r e g i o n ; see MATERIALS AND METHODS). Using t h i s equa t i on , the mod i f i ed RF value i n the un-n ic reg ion from the nine crosses of Table V should be (17 + 2 x 15)/ 2 x 128 = 18..3%. This i s probably a s l i g h t underest imate s i nce the value f o r HK was determined f o r the sma l l e r arg-ad r e g i on . Using the same c r i -t e r i a f o r a cross between the s t r a i n s l e i i - 3 , a , a r g - 1 , ad-3B, a l - 1 and un-3, A, ad-3A, n i c - 2 , a l - 2 , a va lue of (3 + 2 x 8)/2 x 65 = 18.4% was obta ined. In the l a t t e r c r o s s , a mod i f i ed RF va lue i n the ad-a l reg ion could have been obta ined but was prevented by d i f f i c u l t i e s i n the s co r i ng of a l - 1 and a l - 2 . . However, the present mod i f i ed values i n the un-n ic r e -g ion combined w i th unmodif ied values i n the l e u - un , u n - n i c , and n i c - a l reg ions (see Table V) c l e a r l y i n d i c a t e t ha t recombinat ion i s normal i n crosses homozygous f o r a sc -3 . Cyto logy. The combined genet i c data of crosses homozygous f o r asc-3 i n d i c a t e d normal recombinat ion and segregat ion of homologs a t the f i r s t me i o t i c d i v i s i o n , f o l l owed by ex tens i ve nond i s junc t i on a t the se -cond and po s t -me i o t i c d i v i s i o n s . In order to gain more i n s i g h t i n t o the nature and mechanism of t h i s n o n d i s j u n c t i o n , and to determine the reason f o r the low f e r t i l i t y , crosses .homozygousvfor asc-3 were analyzed c y t o -l o g i c a l l y . The a s c i tha t were produced showed normal p a i r i n g of homologs ( F i g . 6c, d ) . This observat ion i s i n accord w i th the recombinat ion data 78 F i g . 6 Chromosome development i n crosses homozygous f o r a s c - 3 . A l l p reparat ions were s t a i ned w i th i r on -haematoxy l i n . Very few a s c i were formed. Many c e l l s which resemble c r o z i e r s were observed (b) x2000; these c e l l s con ta in a number of nuc le i w i t h n u c l e o l i (a) x 2400; i t appears tha t these nuc l e i are i ncapab le of f u s i o n . P a i r i n g of homologous chromosomes appears normal ,at pachytene, so do the s i z e and appearance of the nuc leo lus ( c , d) x3400; a t d i a k i n e s i s , seven b i v a l e n t s appear (e) x3400, and about equal amounts of chromatin segregate a t the f i r s t d i v i s i o n ( f ) x l 300 . The second d i v i s i o n i s h i gh l y abnormal: many second d i v i s i o n f i g u r e s were observed, w i th most chromatin at tached to one s p i n d l e - p o l e body but not the other (arrowed) (g, h) x 1300; about equal amounts of chromatin may segregate i n some a sc i ( i ) x l 300 ; t h i s d i v i s i o n f i g u r e may a l s o become s i m i l a r to those shown i n (g) and (h) s i n ce the s p i n d l e - p o l e bodies ( i n i ) have not moved very f a r apart y e t . Unequal amounts of chromatin have segregated i n t o the ascospores ( j , k) x l300 . 80 presented. Seven b i v a l e n t s condensed at d i a k i n e s i s ( F i g . 6e) and d i -v ided i n a normal f a sh ion d i s t r i b u t i n g equal amounts of chromatin to the two poles ( F i g . 6 f ) . Subsequent ly, many a s c i were observed dur ing the second d i v i s i o n i n d i c a t i n g t ha t t h i s stage takes an abnormally long time to complete ( F i g . 6g, h, i ) . In a d d i t i o n , most d i v i s i o n f i g u r e s show one s p i n d l e - p o l e body (SPB) w i t h a l a rge amount of chromatin at tached and the other w i th l i t t l e or no chromatin attachment. Whether the de fec t i s a property of the SPB or of the chromosomes cannot be de-termined but t h i s d i f f e r e n t i a l attachment appears to be the cause of the ex tens i ve nond i s j unc t i on tha t has been observed g e n e t i c a l l y . F i g . 6j shows fou r out of e i gh t ascospores i n an ascus w i th no chromatin i n two spores and a l a rge amount i n the two nuc l e i of the other two spores. Other spores con ta in very l i t t l e chromatin w i t h o f ten d i f f e r e n t amounts of chromatin i n s i s t e r ascospores, account ing f o r the many 1B:7W asc i ( e . g . , F i g . 6k ) . The genet i c and c y t o l o g i c a l data apparent ly supplement each other p e r f e c t l y . The f i r s t - a c t i n g de fec t o f a sc -3 takes p lace p r i o r to ascus f o r -mat ion. Many c e l l s resembling ascogenous hyphae have been observed ( F i g . 6b) . F i g . 6a shows one of these c e l l s i n some d e t a i l . , The p r e -me i o t i c nuc le i can b e i r e a d i l y i d e n t i f i e d by the presence of l a r ge n u c l e o l i . Such a l a r ge number of nuc l e i i s normal ly never obta ined i n the c roz ie r s . . Thus, i t appears t ha t the de fec t r e s u l t s i n blockage of most a s c i p r i o r to or dur ing karyogamy. This b lock i n ascus format ion i s apparent ly the cause o f the low f e r t i l i t y o f these c ro s se s . A more d e t a i l e d a n a l y s i s i s needed, however, to determine the exact stage of the b l ock . I t may be t e n t a t i v e l y concluded t ha t the primary de fec t of asc-3 takes p lace 81 dur ing c r o z i e r f o rmat i on , po s s i b l y karyogamy, and tha t some c e l l s can escape t h i s developmental b lock to produce a sc i which nond i s j o i n ex ten -s i v e l y a t the second and po s t -me i o t i c d i v i s i o n s . asc-1 (-P95) Crosses homozygous f o r t h i s r e ce s s i v e mutat ion gene ra l l y r e s u l t i n about 40% ascospore a b o r t i o n ; however, up to about 70% abo r t i on has been detected i n some c ro s se s . The f e r t i l i t y of crosses t ha t were done on l i q u i d medium appeared good. In some crosses made on s o l i d medium, a near f i v e - f o l d reduc t i on i n f e r t i l i t y was de tec ted . Recombination and Nond i s j uc t i on Frequenc ies . Random ascospore a n a l y s i s of three crosses homozygous f o r asc-1 revea led reduced recom-b i n a t i o n and increased nond i s j unc t i on f requenc ies (Tables V I I I and IX ) . RF was reduced i n two out of three reg ions examined. The amount of r e -duct ion appeared v a r i a b l e i n both the un-n ic and n i c - a l r eg i on s . The frequency of PWT progeny was a l s o q u i t e v a r i a b l e and ranged from zero to 22.4%. No PWT cu l t u r e s were detected among 239 progeny of a cross (Table V I I I , row 1) t ha t was made on l i q u i d medium. In c o n t r a s t , a l l crosses made on s o l i d medium produced a v a r i a b l e number of PWT progeny. The m a j o r i t y of these PWT c u l t u r e s were p r o t o t r oph i c f o r a l l LG I mutant markers. There fo re , these were apparent l y the r e s u l t o f nond i s j unc t i on dur ing the f i r s t m e i o t i c d i v i s i o n . The Nature of PWT Progeny. The nuc lear compos i t ion of the e i gh t PWT progeny recovered from cross 95-1 (Table V I I I ) was determined by the i n d i v i d u a l t e s t i n g o f c o n i d i a l i s o l a t e s from these c u l t u r e s . A l l e i g h t c u l t u r e s were h e t e r o k a r y o t i c . F i ve of them were he te r o ka r yo t i c f o r the two o r i g i n a l non-exchange chromosomes l e u - 3 , a , a r g - 1 , ad-3B and T a b l e V I I I . R e c o m b i n a t i o n and n o n d i s j u n c t i o n i n t h r e e c r o s s e s homozygous f o r a s c - 1 Non-PUT P r o g e n y PUT P r o g e n y T o t a l Non-PWT P r o g e n y R e c o m b i n a t i o n f r e q u e n c y (%) ( n o . r e c o m b i n a n t s i n p a r e n t h e s e s ) l e u - u n u n - n i c n i c - a l T o t a l PUT P r o g e n y PUT f r e q . (%) (un & auxo ) G e n o t y p e s o f P U T ' s C r o s s * + auxo o t h e r 95A29 x 9 5 a 4 3 * 239 1 5 . 9 ( 3 8 ) 2 . 1 ( 5 ) 1 6 . 3 ( 3 9 ) 0 0 0 0 9 5 - 1 ( Y 2 x X 1 ) 154 1 6 . 9 ( 2 6 ) 9 . 8 ( 15 ) 2 2 . 7 ( 3 5 ) a 3.7 5 3 ( u n ; 1 e u ; ad , n i c , a l ) 9 5 - 2 ( Y - 5 x X 1 7 ) 94 1 8 . 1 ( 17) 1 0 . 6 ( 10) 1 1 . 7 ( 1 1 ) 10 9 .6 10 0 W i l d t y p e c r o s s e s 1 1 - 17 15-2D 3 0 - 35 A l l s t r a i n s u s ed were a s c o s p o r e i s o l a t e s w i t h g e n o t y p e s as i n F i g . 1. T h i s c r o s s was made on l i q u i d med ium; t h e a s c o s p o r e s f r o m s h o t a s c i were a n a l y z e d ( s e e T a b l e X! f o r a s c u s p a t t e r n s f r o m t h i s c r o s s ) . The se c r o s s e s were made an s o l i d med ium, and random a s c o s p o r e s were a n a l y z e d . RF v a l u e s a r e n o r m a l l y s l i g h t l y v a r i a b l e ( s ee e . g . , C a t c h e s i d e , 1 9 7 4 ) . r o T a b l e I X . N a t u r e o f g r o w t h o f a s c o s p o r e s from c r o s s e s homozygous f o r a s c - 1 C r o s s * F r a c t i o n C o l o n i e s o f B l a c k A s c o s p o r e s Germ Tube Only No P r o d u c i n g Germ Tube T o t a l No. S p o r e s F r e q . PUT o f u n / u n + P r o g e n y ^ 9 5 - 1 ( Y 2 x X 1) 0.54 0.37 0.09 365 2.9 9 5 - 4 ( Y 5 x X 3) 0.48 0.32 0.20 378 22.4 9 5 - 6 ( Y 5 x X 1 5 ) 0.48 0.35 0. 17 214 14.8 9 5 - 1 1 ( Y 4 x X ^ 0.45 D.34 0.21 101 13. 1 9 5 - 1 2 ^ x X 1 ? ) 0.41 0.42 0. 17 330 9.2 9 5 - 1 3 ( Y 7 x X 1 5 ) 0.47 0.38 0. 15 271 13.1 A l l on s o l i d c r o s s i n g medium. "^Determined by p l a t i n g o f a s c o s p o r e s as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . 84 un-3, A, ad-3A, n i c - 2 , a l - 2 . These were presumably the r e s u l t of non-d i s j u n c t i o n of non-exchange chromosomes dur ing the f i r s t m e i o t i c di v i - . s i o n . A s i n g l e t empera tu re - sen s i t i v e PWT (un-3) and an ad-3A, h i c - 2 , a l - 2 i s o l a t e were both h e t e r o k a r y o t i c f o r the o r i g i n a l chromosome i in -3 , A_, ad-3A, n i c - 2 , a l - 2 and a chromosome w i t h a c ros sover i n the un-ad reg ion (see F i g . 3 and 5 ) . These two PWT c u l t u r e s may have r e s u l t e d from nond i s j unc t i on dur ing the f i r s t or the second me i o t i c d i v i s i o n . I f these were caused by a crossover i n the r e l a t i v e l y smal1 un-ad reg ion fo l l owed by nond i s j unc t i on a t the f i r s t m e i o t i c d i v i s i o n , one would have expected the simultaneous recovery of many PWT c u l t u r e s r e s u l t i n g from the nond i s j unc t i on of non-exchange chromosomes or o f chromosomes w i th an exchange i'n another reg ion ( e . g . , a d - a l ) . In f a c t , on ly s i x PWT c u l t u r e s of t h i s type were de tec ted . There fo re , i t i s more p l a u s i b l e tha t the un and the ad , n i c , a l c u l t u r e s were produced by nond i s j unc t i on a t the se-cond me i o t i c d i v i s i o n , e s p e c i a l l y s i nce a c ros sover i n the un-ad reg ion i s a p r e r e q u i s i t e to the d e t e c t i o n o f such nond i s j unc t i on ( F i g . 3 ) . The l euc i ne r e q u i r i n g PWT was he te r o ka r yo t i c f o r the.two crossover chromo-somes l e u - 3 , a_, a r g - 1 , ad-3B, a l - 2 and l e u - 3 , un-3, A, ad-3A, n i c - 2 . This c u l t u r e was apparent ly produced by the nond i s j unc t i on of these c r o s s -over chromosomes dur ing the f i r s t m e i o t i c d i v i s i o n . To. What Extent Do Exchange-Chromosomes Ndnd i s jo in During the F i r s t  M e i o t i c D i v i s i o n ? The nature of the l euc i ne r e q u i r i n g PWT i s o l a t e appeared to i n d i c a t e t ha t nond i s j unc t i on o f c rossover chromosomes may take p lace a t the f i r s t m e i o t i c d i v i s i o n . To t e s t the extent of such nond i s j unc t i on of c rossover chromosomes, 263 PWT i s o l a t e s from ten crosses (95-1 through 95-10) were obta ined and scored f o r a l b i n o phenotype. The s i x a l b i n o PWT 85 c u l t u r e s were apparent ly caused by a crossover event i n the n i c - a l r e -gion fo l l owed by nond i s j unc t i on a t the f i r s t me i o t i c d i v i s i o n (see MATERIALS AND METHODS). The n i c - a l map d i s tance of the subgroup of me i -oses i n which nond i s j unc t i on o f LG I took p lace dur ing the f i r s t me i o t i c d i v i s i o n was thus est imated a t 2 x 6/263 = 4.5 mu. In c o n t r a s t , i n three crosses examined, the n i c - a l recombinant f requenc ies among non-PWT progeny were 16.3%, 22.7% and 11.7% (see Table V I I I ) . These data p ro -v ided s t rong evidence support ing the idea tha t nond i s j unc t i on a t the f i r s t me i o t i c d i v i s i o n i n t h i s mutant i nvo l ve s p r i m a r i l y non-exchange chromosomes. However, these observat ions a l s o suggest tha t some c r o s s -over chromosomes do nond i s j o i n a t the f i r s t d i v i s i o n . Ascus A n a l y s i s . Many unordered a sc i from a cross homozygous f o r asc-1 have been analyzed ( T a b l e - X ) . - Most a sc i conta ined an even number o f b lack ascospores suggest ing a de fec t p r i o r to the po s t -me i o t i c d i v i -s i o n . The a n a l y s i s of ascospores from these a s c i revea led severa l as -pects of d i s j u n c t i o n i n t h i s mutant. F i r s t , the recombinat ion frequency i n the un-al reg ion was s i m i l a r f o r 8B:0W asc i (20%) and f o r a l l other a sc i combined (16.8%). There fo re , the reduc t i on of RF i s a general de-f e c t ope ra t i ve i n a l l a s c i from t h i s c r o s s , and not j u s t an express ion of a subgroup w i th inc reased ascospore a b o r t i o n . Second, as po inted out p r e v i o u s l y , the de fec t i n d i s j u n c t i o n of chromosomes would have to take p lace p r i o r to the po s t -me i o t i c d i v i s i o n , i . e . , dur ing the f i r s t or second me i o t i c d i v i s i o n . The absence of PWT progeny from t h i s cross (Table V I I I , row 1) and the presence o f l a r ge numbers o f 8B:0W and 6B:2W a s c i suggest r egu l a r segregat ion of a t l e a s t LG I a t the f i r s t meiiotic d i v i s i o n . One might argue t ha t ascospore 86 T a b l e X. A s c u s a n a l y s i s c f a c r o s s homozygous f o r t h e r e c e s -s i v e m e i o t i c m u t a t i o n a s c - 1 ( P 9 5 ) (95A29 x 9 5 a 4 3 ) Types of A s c i No. o f A s c i ( b l ack : uih i t e a s c o s p o r e s ) O b s e r v e d B 0 41 6 2 19 4 4 22 2 6 18 0 a 19 o t h e r ( a l l t y p e s ) 10 T o t a l 119 87 abo r t i on may be caused by nond i s j unc t i on of a l i nkage group other than LG I. In t ha t case, the centromere reg ion of on ly one homologous LG I ( e i t h e r l e u , a_, a r g , ad or un_, A, ad, n i c , a l ) would segregate w i t h both copies of the nond i s j o i n i n g chromosome(s). There fo re , a l l f ou r v i a b l e products of 4B:4W a s c i would be e i t h e r l e u , .a, a r g , ad or un, A, ad, n i c , a l (assuming no crossover had occu r r ed ) . However, s i n ce seven out of ten a sc i had an M i l pa t te rn of segregat ion of LG I markers ( i . e . , both types of chromosomes found i n each 4B:4W ascus ) , i t appears h i gh l y un-l i k e l y t ha t convent iona l nond i s j unc t i on o f any chromosomes dur ing the f i r s t m e i o t i c d i v i s i o n cou ld be the cause of the observed ascospore a b o r t i o n . Cyto logy . To con f i rm and supplement the gene t i c d a t a , th ree crosses were examined c y t o l o g i c a l l y (95-11, 95-12 and 95-13). C r o z i e r and ascus format ion appeared r e gu l a r ( F i g . 7a) ; the f i r s t de fec t was v i s i b l e dur ing the zygo/pachytene stage when, reduced p a i r i n g of homo-logous chromosomes i s o f t en ev ident ( F i g . 7b, c ) . This observat ion i s compat ib le w i t h the reduced recombinat ion va lue s . During t h i s a n a l y s i s , a number of metaphase I f i g u r e s w i th up to 14 (= 2N) un i va len t s were observed ( F i g . 7 d - f ) . In mainy cases , a c l o se a s s o c i a t i o n between severa l o f these un i va len t s cou ld be de tec ted . For example, F i g . 7d shows a number of pa i r s of chromosomes and. F i g . 7e shows a connect ion between three pa i r s o f u n i v a l e n t s . These l a t t e r con-nect ions may w e l l r e s u l t from the segregat ion of homologs whereas those chromosomes l a c k i n g t h i s connect ion may be genuine u n i v a l e n t s . I t i s p l a u s i b l e tha t the nature and extent of premature sepa ra t i on of these " p a i r s " of chromosomes determines the f i n a l frequency of n o n d i s j u n c t i o n . 88 F i g . 7 Chromosome development i n crosses homozygous f o r asc -1. A l l p reparat ions were s t a i ned w i t h Feulgen and a c e t o -o r c e i n . C r o z i e r development i s normal (a) x2700; the p a i r i n g of homogous chromosomes at pachytene i s incom-p l e t e (b and c) x3400; subsequent ly , some un i va l en t s appear on the metaphase p l a t e ( d ) , and some chromosomes w i th br idges are seen at meta/anaphase I (e and f ) x3400; the l a t t e r type may be d i v i d i n g b i v a l e n t s . The f i r s t m e i o t i c d i v i s i o n segregates about equal amounts of chromatin to the two poles (g) xl700, but some l a g -g ing occu r s - - see arrowed chromosomes (h) xllOO. The se -cond d i v i s i o n i s h i gh l y i r r e g u l a r : over lapp ing sp i nd le s ( i ) , abnormal sepa ra t i on ( j ) xl400, and lagg ing of chromosomes (k) xl700, have a l l been observed f r e q u e n t l y . An ascus a t the prophase of the pos tme io t i c d i v i s i o n shows r egu l a r placement of chromatin i n a sc i but unequal amounts o f chromatin i n d i f f e r e n t n u c l e i (1); such un-equal d i s t r i b u t i o n i s a l s o observed a t the ascospore • stage (m) x900. F i n a l l y , many a sc i w i t h e i g h t asco-spores are abo r ted ; one o f the few normal unaborted a s c i i s shown (arrowed) as a comparison i n (n) x200. 90 Fur ther a na l y s i s of high and low nond i s j unc t i on crosses should shed some l i g h t on t h i s s ub j e c t . C y t o l o g i c a l p reparat ions of crosses homozygous f o r asc-1 r e -vealed many more metaphase I c on f i g u r a t i o n s than were observed i n w i l d type c ro s se s . This may be a r e f l e c t i o n of an extended time i n t e r v a l spent i n t h i s s tage. That d i v i s i o n may be slowed down by the presence o f un i va len t s has been p rev i ou s l y repor ted f o r rye (Prakken, 1943). The d i s t r i b u t i o n of approximately equal amounts o f chromatin to the two poles ( e . g . , F i g . 7g, h) seems to suggest tha t the un i va len t s do not segregate pure ly a t random. In a d d i t i o n , some lagg ing of chromosomes has been ob-served f o l l o w i n g the f i r s t d i v i s i o n ( F i g . 7h). These chromosomes w i l l most l i k e l y j o i n another nucleus l a t e r on, s i nce on ly e i g h t nuc l e i are ever observed at the e i gh t -nuc leu s s tage. Some PWT progeny may have o r i g i n a t e d i n t h i s manner. The second m e i o t i c d i v i s i o n was .almost always i r r e g u l a r : s p i nd l e o v e r l a p , lagg ing of chromosomes, apparent slow separa t i on of d i v i d i n g n u c l e i , and movement of segregat ing s p i nd l e bodies to the same p o l e , have been observed ( F i g . 7 i - k ) , In a d d i t i o n , unequal amounts of chromatin can be seen to segregate i n many a sc i ( F i g . 71, m). These l a r g e - s c a l e abnor-m a l i t i e s of segregat ion agree w e l l w i t h genet i c ob se rva t i on s . The r e l a t i v e r e g u l a r i t y of the po s t -me i o t i c d i v i s i o n , suggested by the i n f r equen t oc -currence of a sc i w i t h odd numbers of b lack ascospores (Table X) was con-f i rmed by the equal amounts o f c h r omat i n - s t a i n i n g ma te r i a l i n s i s t e r spores ( e . g . , F i g . 7m). In the f i n a l a n a l y s i s , the primary de fec t of t h i s mutant appears to be a reduced p a i r i n g of homologous chromosomes w i t h a r e s u l t a n t reduc t i on 91 i n recombinat ion frequency i n some reg ion s . The consequence o f t h i s r e -duced p a i r i n g i s the format ion of u n i v a l e n t s , the segregat ion of which i s somewhat i r r e g u l a r . The high amount o f i r r e g u l a r segregat ion a t the second d i v i s i o n appears to be the d i r e c t cause of much of the ascospore a b o r t i o n . This aber rant segregat ion may be the normal consequence of the fo rmat ion of u n i v a l e n t s , or i t may be due to a p l e i o t r o p i c e f f e c t of the mutant. The I n v i a b i l i t y o f Many B lack Ascospores from Crosses Homozygous  f o r a sc -1 . May be Due to The i r M u l t i p l e Disomy. Nond i s junct ion can be detected i n Neurospora because of the V i a b i l i t y of i t s aneuplo id progeny. In c o n t r a s t , such progeny i n most p lant s grow e i t h e r very poor ly or not a t a l l . I t i s p o s s i b l e t ha t many aneuplo id c u l t u r e s are a l s o qu i t e i n -v i a b l e i n Neurospora. To t e s t t h i s i d e a , ascospores from s i x crosses were heat-shocked and p l a ted ou t , and t h e i r germinat ion and co lony- forming a b i l i t y recorded. Only about 50% of the b lack ascospores produced co l on ie s (Table IX ) . Most other ascospores germinated but growth stopped very q u i c k l y . A few germinated ascospores of the l a t t e r type resumed growth a t one or more po int s along the mycelium. The obse rva t ion of such escape suggests an exp l ana t i on o f i n v i a b i l i t y of so many b lack ascospores. Crosses homozygous f o r asc-1 produce PWT progeny r e s u l t i n g from hyper-p l o i d ascospores. I t i s p l a u s i b l e t ha t the growth of many of these hyper-p l o i d ascospores becomes i n h i b i t e d upon germinat ion . In some of these young ascospore c u l t u r e s , the process of h a p l o i d i z a t i o n may cause the los s of ex t ra chromosomes ( P i t t e n g e r , 1954) and thus enable resumed growth. 92 mei-1 This r e ce s s i v e me i o t i c mutat ion has been analyzed us ing both genet i c and c y t o l o g i c a l means (Smith, 1975; Lu and G a l e a z z i , 1979) and appears to be d e f e c t i v e i n a f u n c t i o n necessary f o r the p a i r i n g of homologous chromosomes dur ing the f i r s t prophase of me i o s i s . This i s f o l l owed by the format ion of un i va len t s a t metaphase I and abnormal segregat ion pa t t e r n s , i n c l u d i n g 4-po led s p i n d l e s , a t subsequent d i v i s i o n s l ead ing to about 90% ascospore a b o r t i o n . G e n e t i c a l l y , an almost com-p l e t e absence of recombinat ion i s i n accordance w i t h the observed p a i r -ing d e f e c t , and the ma jo r i t y of b lack ascospores are d isomic f o r most l i n kage groups. The mei-1 mutant was p r i m a r i l y obta ined to check a l l e l i s m to any mutants . i s o l a t e d dur ing t h i s s tudy, and to determine any p o t e n t i a l i n -t e r a c t i o n w i th some of these newly i s o l a t e d mutat ions . During i n i t i a l t e s t s , i t was found that crosses homozygous f o r mei-1 produced about 10% black ascospores when crossed on s o l i d medium, as was observed by Smith (1975), but 30% b lack spores r e s u l t e d when these same s t r a i n s were crossed on l i q u i d medium. From a cross ( a m ( 3 3 ) , ad-3B; mei-1 x un-3, A, ad-3A, n i c - 2 ; mei-1) made on l i q u i d medium, 22 out of 24 ascospore c u l t u r e s were adeniner independent, i n d i c a t i n g they were d i somic f o r LG I. This value i/s as high or h igher than was obta ined by Smith. There fo re , the i n i t i a l de fec t r e s u l t i n g i n the product ion of these high f requenc ies of PWT progeny was conf irmed i n t h i s cross made on l i q u i d medium. To determine the de fec t o f such crosses made on l i q u i d medium, the above-mentioned cross was analyzed c y t o l o g i c a l l y . The observat ions on p a i r i n g ( F i g . 8c , d) a t metaphase I were i d e n t i c a l to those obta ined 93 F i g . 8 Chromosome development i n crosses homozygous f o r me i - 1 . ( a , b, d, and g) were s t a i ned w i th Feulgen and a ce t o -o r c e i n ; the remaining p reparat ions were s t a i ned w i th i r on -haematoxy l i n . P a i r i n g between homologs i s appar-e n t l y absent, a lthough some loose a s s o c i a t i on s may be observed (a , b) x3400; near metaphase I, 14 un i va l en t s are u s ua l l y observed ( c , d) x3400; sometimes, loose a s -s o c i a t i o n s between homologous chromosomes appear to occur ( c ) . About equal amounts of chromatin move to oppos i te poles a t the f i r s t me i o t i c d i v i s i o n (e, g) x3400. About 14 chromosomes i n d i v i d u a l i z e i n each of the two dyad nuc l e i of prophase II (g) x2400; some chromosomes (arrowed) may l a g . The second d i v i s i o n i s s i m i l a r to tha t observed f o r mutant asc-6 ( F i g . 5 i ) : the chromatin takes a long time to separate ( i , j , h) x2400; and u sua l l y unequal amounts of chromatin move to oppos i te poles ( e s p e c i a l l y ( j ) ; s p i n d l e - p o l e bodies are arrowed). 95 p r e v i ou s l y (Lu and G a l e a z z i , 1979). However, observat ions on subsequent stages of me i o t i c development d i f f e r e d i n severa l r e spec t s : (1) Numerous a s c i were observed dur ing the f i r s t i n te rpha se ; these a s c i showed t ha t about equal amounts o f chromatin moved to oppos i te poles a t the f i r s t m e i o t i c d i v i s i o n ( F i g . 8e, f ) . (2) Some a s c i corresponding to prophase II conta ined two n u c l e i , each w i t h a chromosome number approaching a d i p l o i d nucleus ( F i g . 8g). (3) In accord w i th prev ious ob se r va t i on s , the second d i v i s i o n was c h a r a c t e r i z e d by a very slow sepa ra t i on of the two daughter nuc l e i ( t h i s was i n f e r r e d from the high frequency of a sc i a t t h i s s tage ; F i g . 8 h - j ) ; however, s p i nd l e over lap was r a r e l y observed and 4-poled sp ind le s were absent. (4) The po s t -me io t i c d i v i s i o n . o f f ou r separate nuc l e i appeared regu-l a r . In a d d i t i o n , severa l i n t e r e s t i n g aspects of these preparat ions are the apparent a s s o c i a t i o n o f un i va len t s dur ing e a r l y metaphase ( F i g . 8 c ) , and the unequal d i s t r i b u t i o n of chromatin to oppos i te poles i n many second d i v i s i o n f i g u r e s ( e . g . , F i g . 8 j ) . I t has been e s t a b l i s h e d t ha t crosses homozygous f o r mei-1 r e -s u l t i n 70% ascospore abo r t i on when crossed on l i q u i d medium, but 90% on s o l i d medium. C y t o l o g i c a l observat ions o f crosses made on the two types o f media were s i m i l a r up to and i n c l u d i n g the format ion o f u n i v a l e n t s ; subsequent ly, p reparat ions from the cross made on l i q u i d medium conta ined more a sc i a t in terphase I and prophase I I , but l e s s a sc i w i t h i r r e g u -l a r i t i e s such as 4-poled s p i n d l e s . Thus, i t appears t ha t the two types of c ro s s ing media d i f f e r e n t i a l l y a f f e c t the ' Seg rega t i on o f un i va len t s i n 96 crosses homozygous f o r me i - 1 . Because o f the absence o f 4 -po led sp i nd le s from a s c i produced on l i q u i d medium, such i r r e g u l a r types o f segregat ion probably do not , as proposed by Lu and G a l e a z z i , account f o r the observed frequency of disomy of t h i s mutant. In s tead, the present data suggest t h a t un i va len t s d i v i d e e q u a t i o n a l l y dur ing the f i r s t me i o t i c d i v i s i o n . Even though a high frequency o f PWT progeny might a l s o be produced by the random movement o f u n i v a l e n t s , or by i r r e g u l a r s p i nd l e behavior or de fec -t i v e s n c l u s i on o f nuc l e i i n ascospores,. these a l t e r n a t i v e s were v i r t u a l l y r u l ed out by the f o l l o w i n g ob se rva t i on s . F i r s t , both s p i nd l e over lap and 4-poled sp ind le s were ra re or absent, and the stages subsequent to the second me i o t i c d i v i s i o n were apparent ly normal. Thus, n e i t h e r i r r e g u l a r s p i nd l e behavior nor d e f e c t i v e ascospore i n c l u s i o n cou ld account f o r the extremely high frequency of PWT progeny. 'Second, .contrary to observa-t i o n , the random movement of un i va len t s would.cause unequal amounts of chromatin to segregate a t the f i r s t me i o t i c d i v i s i o n . In a d d i t i o n , the nuc l e i i n the f o l l o w i n g prophase should con ta in a t o t a l o f only 2N(= 14) chromosomes. In f a c t , the observed number was u s ua l l y c l o s e r to 4N(= 28) chromosomes. By the process of e l i m i n a t i o n , i t appears most probable t h a t the un i va l en t s i n crosses homozygous f o r mei-1 d i v i d e e q u a t i o n a l l y . This would account f o r the^observed segregat ion of roughly equal amounts of chromatin a t the f i r s t d i v i s i o n , and f o r the presence of a d i p l o i d or c l o se to d i p l o i d number of chromosomes i n each nucleus of the prophase II a s c i . In a d d i t i o n , the subsequent d i v i s i o n would take a long time to complete s i nce on ly chromatids are p resent . 97 Mappi ng a s c - 3 . A cross between mei-1 and ascr3,: o r i g i n a l l y made to ob-t a i n double mutants, produced no recombinants among 62 ascospore i s o -l a t e s : 25 were a s c - 3 , mei-1 , and 37 were asc-3 , me i - 1 . In order to perform a more d e t a i l e d mapping, two f l a n k i n g markers were i n t roduced : t r p - 4 , mapped 12/183 or about 6.5 mu to the r i g h t of a s c - 3 ; and pdx, map-ped 3/92 or about 3.5 mu to the l e f t of me i - 1 . Ascospores from the cross between a s c - 3 , t r p - 4 , pan-1, a_ and pdx, me i - 1 , A were p l a t ed on minimal medium, and 300 w i l d , type recombinant co lon ie s ,(pdx +, t r p + ) were separ-a t e l y t r a n s f e r r e d to vege ta t i ve medium. Tes t i ng of these c u l t u r e s f o r me i o t i c phenotypes f a i l e d to revea l any recombinants-of the two me i o t i c mutants: 30 were me i - 1 , and 220 were a s c - 3 . The r a t i o of the two mutants roughly corresponds to the r a t i o o f map d i s tances p rev i ou s l y determined f o r the regions pdx-mei-1 (3.5 mu) and a s c - 3 - t r p - 4 (6.5 mu). The p d x + , t r p + recombinants represent on ly about 5% o f a l l p ro -geny from t h i s cross ( h a l f the map d i s t ance of the pdx- t rp r e g i o n ) . S ince on ly recombinants i n the pdx - t rp reg ion cou ld conta in a recombinant be-tween mei-1 and a s c - 3 , a t o t a l of 20 x 300 = 6000 progeny were te s ted f o r the l a t t e r type of recombinant ( m e i - l - a s c - 3 ) . However, s i n ce on ly one out o f two r e c i p r o c a l c rossover products would be de tec ted , the t o t a l progeny te s ted would be % x 6000 = 3000. There fo re , no crossovers be-tween mei-1 and asc-3 were observed among 3000 progeny. The recombinat ion frequency between mei-1 and asc-3 i s l e s s than 0.1 mu. a s c - 1 . The mutant asc-1 was mapped c l o s e to a s c - 3 , and the re fo re to me i - 1 , s ince on ly asc-1 (45) and asc-3 (51) progeny were detected among 96 i s o l a t e s from a cross between the two mutants. 98 a sc -6 . Ascus ana l y s i s of a cross between asc-3 and asc-6 es -t a b l i s h e d the c l o s e p rox im i ty o f asc-6 to one of the cent romeres , s i nce , i n f i v e out o f seven a s c i , the mutant and i t s w i l d type a l l e l e segre -gated dur ing the f i r s t d i v i s i o n . In subsequent crosses to centromere-l i n k e d markers, the mutant was mapped on LG I I , about 3 mu from arg-5 (the s l o (slow growth) mutat ion which was i s o l a t e d w i t h the m e i o t i c .. mutat ion maps about 14 mu from arg-5 such t ha t asc-6 l i e s between s l o and a r g - 5 ) . asc-6 cannot be d e f i n i t e l y ass igned to the r i g h t arm of LG I I . However, two l i n e s of evidence are i n support o f the l o c a t i o n of asc-6 on the r i g h t arm o f LG II to the r i g h t of a rg -5 : (a) second d i v i s i o n segregat ion data put the asc-6 mutat ion approx imately 10 mu from the centromere of t h i s l i n kage group; and (b) arg-5 has p r e v i ou s l y been mapped about 5 mu to the r i g h t of the centromere of LG II (Radford, 1972). There fo re , the f o l l o w i n g arrangement o f markers appears most l i k e l y : (centromere) - 5 mu - (arg-5) - 3 rnu - (asc-6) - i l mu -. ( s l o ) . I n t e r a c t i o n o f M e i o t i c Mutat ions mei-1 and a sc -6 . Crosses homozygous f o r the double mutant me i - 1 ; asc-6 produced about 70% spore abo r t i on when made on l i q u i d c ro s s i ng medium. This i s s i m i l a r to values f o r e i t h e r s i n g l e mutant. The f e r -t i l i t y of these crosses was good ( i . e . , many spores were formed) although somewhat reduced when compared to w i l d t ype . Ana l y s i s of a cross;homozy-gous' f o r the double mutant but heterozygous "at the ad-3 locus ^(am(:33), . ad -S B ; me i - 1 ; asc-6 x .un-3,. A, ad-3A, n i c - 2 ; me i - 1 ; a s c - 6 ) , showed tha^t 38 ' o u t of 41 i s o l a t e s (93%) were adenine- independent and t he re fo re disomic, f o r LG I. This va lue of PWT frequency i s s i m i l a r to mei-1 (80-90%), and 99 d i f f e r e n t from asc-6 (20-55%). There fo re , these .data-suggest.an e p i -s t a t i c dominance of mei-1 over a sc -6 . This e p i s t a t i c r e l a t i o n s h i p was conf irmed c y t o l o g i c a l l y : . i n such crosses there was an. absence of p a i r i n g as i n me i - 1 , r a the r than reduced p a i r i n g as i n a sc -6 . asc-1 and a sc -6 . Crosses homozygous f o r the double mutant a s c - 1 ; asc-6 produced about 70% ascospore abo r t i on (70.3 ± 6.7% i n 12 crosses examined). This va lue i s s i m i l a r to those obta ined from e i t h e r s i n g l e mutant (69 ± 8.4% i n 10 crosses homozygous f o r asc-1 a l one , and 62.3 ± 9.4% i n 6 c rosses homozygous f o r asc-6 a l o n e ) . Thus, from these a sco -spore abo r t i on data i t appears t ha t there i s no e p i s t a t i c i n t e r a c t i o n between these mutat ions . Genet ic and c y t o l o g i c a l analyses of these crosses have not y e t been performed. U n t i l such t ime, no f i r m statement can be made on the i n t e r a c t i o n between these two mutat ions . DISCUSSION C h a r a c t e r i s t i c s of four r ece s s i ve me i o t i c mutations o f Neurospora  c ras sa have been summarized i n Table XI. The abo r t i on of ascospores ob-served i n crosses homozygous f o r each of the three newly i s o l a t e d r ece s -s i v e mutations ( a s c - 1 , asc-3 and asc-6) was shown to be the consequence o f abnormal d i s j u n c t i o n of m e i o t i c chromosomes. In two cases (asc-1 and a s c - 6 ) , the abnormal d i s j u n c t i o n was apparent ly caused by a de fec t i n the p a i r i n g of homologs dur ing the f i r s t me i o t i c prophase. In the t h i r d mutant ( a s c - 3 ) , a primary de fec t near karyogamy may have had a p l e i o t r o p i c e f f e c t r e s u l t i n g i n the nond i s j unc t i on observed dur ing the second and po s t -me i o t i c d i v i s i o n s . Although the absence o f p a i r i n g and the subse-quent d i s j u n c t i o n de fec t of mei-1 has been e s t a b l i s h e d p r e v i ou s l y . T a b l e X I . C h a r a c t e r i s t i c s o f f o u r r e c e s s i v e m e i o t i c m u t a t i o n s i n N e u r o s p o r a c r a s s a w i t h a d e f e c t i n t h e r e g u l a r d i s j u n c t i o n o f c h r o m o s o m e s A p p r o x . % C r o s s e s made R e c o m b i n a t i o n N o n d i s j u n c t i o n Appa ren t A s c o s p o r e on l i q u i d o r F requency a t I n i t i a l A l l e l e A b o r t i o n s o l i d medium F e r t i l i t y * l e u - u n u n - a l P a i r i n g MI M i l PMD D e f e c t a s c - 1 40 -70 both med - h i g h normal r e d u c e d r educed yes yes 7 P a i r i n g a s c - 3 90 -98 both v e r y low normal norma l no rma l no yes yes P r e - a s c u s f o r m a t i o n a s c - 6 70 both low - med r educed r educed much r educed yes ? yes P a i r i n g me i -1 70 9 0 l i q u i d s o l i d h i g h h i g h a b s e n t * ab sen t ab sent yes yes f 7 7 ? P a i r i n g * T o t a l , n u m b e r of., b l a c k a n d w h i t e a s c o s p o r e s p r o d u c e d ; may be l o w ( f e w a s c o s p o r e s ) , m e d . ( i n t e r m e d i a t e a m o u n t o f a s c o s p o r e s ) , o r h i g h . D a t a f r o m S m i t h ( 1 9 7 5 ) , a n d L u a n d G a l e a z z i ( 1 9 7 9 ) ; r e c o m b i n a t i o n was d e t e r m i n e d o n r e g i o n s o f a n o t h e r c h r o m o s o m e ( S m i t h , 1 9 7 5 ) . 101 (Smith, 1975; Lu and G a l e a z z i , 1979), some new observat ions were made dur ing t h i s study which suggest a po s s i b l e mechanism f o r the abnormal d i s j u n c t i o n of un i va lent s i n Neurospora. P a i r i n g - and Recombinat ion-Defect ive Mutants Mutants w i th a reduced recombinat ion frequency may have a de-f e c t i n the p a i r i n g of homologs ( i . e . , a p recond i t i on to exchange), or i n the exchange process i t s e l f . The mutations asc-1 and asc-6 have c y t o l o g i c a l l y de tec tab l e de-f e c t s i n the p a i r i n g o f homologs dur ing the zygotene and pachytene stages of the f i r s t m e i o t i c prophase. In t h i s r e spec t , they resemble many a syndet i c mutants i n p lant s (reviewed i n Baker e t a l_ . , 1976a) and the mutant mei-1 i n Neurospora (Smith, 1975; Lu and G a l e a z z i , 1979). The extent o f the p a i r i n g de f ec t o f these three mutants i n Neurospora ( i . e . , a s c - 1 , asc-6 and mei-1) was found to be p e r f e c t l y c o r r e l a t e d w i t h the r educ t i on i n recombinat ion f requency. Mutant asc-1 was l e a s t a f -f e c t ed wh i l e both p a i r i n g and recombinat ion were almost complete ly absent i n me i - 1 . The c y to l i o g i ca l de tec t i on of a p a i r i n g de fec t does not neces^ s a r i l y mean t ha t a de f ec t i n the p a i r i n g process per se operates . I n -s t ead , a de fec t i n an immediate requirement f o r the process of exchange may s e conda r i l y cause some p a i r i n g abnorma l i t y . Mutants w i t h a de fec t i n the p recond i t i on s to exchange (such as p a i r i n g ) and exchange processes have been d i s t i n g u i s h e d on the bas i s of the un i f o rm i t y of the r educ t i on of exchange (Sandler et a l _ . , 1968; Jones, 1974). Any mutant which r e -duces exchange i n a uniform manner along the chromosome should be 1 0 2 cons idered d e f e c t i v e i n a process requ i red f o r exchange i t s e l f . I n ' c o n t r a s t , non-un i fo rmi ty of r educ t i on would mean a de fec t i n a p re -c o n d i t i o n to exchange. Using these c r i t e r i a , the w i l d type gene of asc-1 c on t r o l s some f u n c t i o n necessary f o r a p recond i t i on to exchange (presumably the es tab l i shment or maintenance of p a i r i n g ) . Such a f u n c t i o n has not been f i r m l y e s t a b l i s h e d f o r mutant -asc-6; however, the occurrence of most de tec tab l e p a i r i n g near the ends of the chromo-somes maysuggest a de fec t i n a p recond i t i on f u n c t i o n . . The almost com-p l e t e l ack of p a i r i n g a t a l l stages of m e i o t i c development i n crosses homozygous f o r mei-1 would not be expected i f the exchange process was a f f e c t e d . There fo re , the w i l d type gene of mei-1 probably c on t r o l s a f unc t i on tha t i s e s s e n t i a l f o r the es tab l i shment of p a i r i n g . D i s j u n c t i o n of Chromosomes During the F i r s t M e i o t i c D i v i s i o n C o r r e l a t i o n between Lack of Exchange and Nond i s j unc t i on . As a consequence of reduced p a i r i n g and exchange, many homologous chromosomes w i l l not be held together by t h e i r ch iasmata, and they w i l l produce u n i -va len t s i n s tead of b i v a l e n t s a t metaphase I of me i o s i s . B i v a l en t s nor -ma l l y c o n t r o l the r egu l a r segregat ion of i t s component homologous chromosomes. There fo re , i f homologous chromosomes are not held together to form b i v a l e n t s , segregat ion of the i n d i v i d u a l chromosomes ( u n i v a l -ents) w i l l be i r r e g u l a r . In t h i s manner, mutants w i t h a de fec t i n p a i r i n g or exchange have been i n d i r e c t l y s e l e c ted through the aberrant d i s j u n c t i o n o f the un i va len t s i t produces. In recombination,, de fec -t i v e me i o t i c mutants of D ro soph i l a , only non-exchange chromosomes non-d i s j o i n (Baker and H a l l , 1976). There fo re , a l l b i v a l e n t s which are 103 held together by one or more ch ia sma( ta ) , segregate i n a normal fa sh ion and i r r e g u l a r segregat ion i s s o l e l y due to un i va len t s produced by a l ack of chiasmata. S i m i l a r ana l y s i s has not been performed i n m e i o t i c mutants of h igher p l an t s due to d i f f i c u l t i e s a s soc ia ted wi th the recovery of aneuplo id products produced by n o n d i s j u n c t i o n . However, nond i s j unc t i on of some exchange chromosomes takes p lace i n m e i o t i c mutants of the nema-tode Caenorhabdi t i s elegans (Hodgkin e t aj_. , 1979). Ana l y s i s of aneuplo id progeny (PWT) produced by the mutants asc-1 and asc-6 of Neurospora showed tha t the m a j o r i t y of nond i s j unc t i on i nvo l ve s non+-;exchange chromosomes. There fo re , most nond i s junc t i on i s a consequence of the i r r e g u l a r segre-ga t i on of the non-exchange u n i v a l e n t s . However, a small but s i g n i f i c a n t f r a c t i o n of exchange chromosomes f a i l to d i s j o i n dur ing the f i r s t m e i o t i c d i v i s i o n . The low frequency of such events suggests t ha t nond i s j unc t i on of exchange chromosomes i s a secondary e f f e c t of the product ion of non-exchange u n i v a l e n t s . For example, the long du ra t i on of metaphase I ob-served i n these mutants (see a l s o Prakken, 1943) could have caused the precoc ious t e r m i n a l i z a t i o n of chiasmata and thus produced exchange u n i -v a l e n t s . A l t e r n a t i v e l y , lagg ing of chromosomes or s p i n d l e abno rma l i t i e s induced by the abnormal nature of the chromosomes could cause nond i s junc -t i o n of some b i v a l e n t s . That the presence of un i va len t s i s not the only f a c t o r t ha t can p o t e n t i a l l y cause nond i s j unc t i on i n w i l d type s t r a i n s i s amply i l l u s t r a t e d by high f requenc ies of spontaneous or chemica l l y i n -duced nond i s j unc t i on of exchange chromosomes i n Neurospora (DeLange, un-pub l i s hed ; G r i f f i t h s and DeLange, 1977; Smith, 1974). The absence of nond i s j unc t i on of exchange chromosomes i n recombinat ion d e f e c t i v e mutants of Drosoph i la may be the r e s u l t o f the a c t i o n of the d i s t r i b u t i v e p a i r i n g 104 and d i s j u n c t i o n system ( e . g . , G r e l l , 1964), which may reduce the num-ber of secondary a b n o r m a l i t i e s . In Neurospora, no evidence of a s i m i l a r back-up d i s j u n c t i o n system i s a v a i l a b l e . In f a c t , un i va len t s appear to be s ca t t e red across the length of the s p i n d l e . The Nature o f I r r e g u l a r D i s j u n c t i o n . In the absence o f a r e g -u l a r means of d i s j u n c t i o n , un i va len t s may move a t random t o e i t h e r pole or d i v i d e e q u a t i o n a l l y (by centromere d i v i s i o n ) . These types of segre-ga t i on have been encountered i n h i g he r r p l an t s ( e . g . , Gatehes ide, 1939=; S j o d i n , 1970); i n many p l an t s pec i e s , both types of segregat ion have been observed s imul taneous ly ( e . g . , Prakken, 1943). In Neurospora, some e v i -dence i n d i c a t e s t ha t the equat iona l d i v i s i o n of centromeres dur ing ana-phase I i s a common means of segregat ion of u n i v a l e n t s . F i r s t , the high frequency of 6B:2W asc i obta ined from a cross homozygous f o r asc-1 i s compat ib le w i t h aber rant segregat ion a t the second m e i o t i c d i v i s i o n , or w i t h the equat iona l d i v i s i o n of some un i va len t s dur -ing the f i r s t d i v i s i o n . S ince the pr imary de fec t i n p a i r i n g and i n the product ion of b i v a l e n t s has been e s t a b l i s h e d i n subsequent crosses homozy-gous f o r a s c - 1 , the l a t t e r exp lanat ion i s p r e f e r a b l e . In a d d i t i o n , non-d i s j u n c t i o n o f homologous chromosomes should have produced PWT progeny i f LG I was i n v o l v e d , or 4B:4W a s c i w i t h on ly one homologous LG I centromere ( l e u , a_, a r g , ad or m, A, ad, n i c , a l ) i f one or more other l i nkage groups were i nvo l ved (see RESULTS). In f a c t , no PWT's were observed among the progeny o f t h i s . c r o s s , and both homologous LG I centromeres were obta ined i n seven out of ten 4B:4W a s c i . These observat ions would be c o n s i s t e n t w i th the equat iona l separa t i on of the centromeres of some chromosomes other than LG I dur ing the f i r s t me i o t i c d i v i s i o n . 105 Second, i n crosses homozygous f o r me i - 1 , 14 un i va len t s can be counted i n most metaphase I f i g u r e s . I f these un i va len t s moved a t random one would expect-unequal amounts of chromatin to segregate to oppos i te poles a t the f i r s t d i v i s i o n . However, i n a l l cases observed, approximately equal amounts of chromatin moved to each po l e , c o n s i s t e n t w i th equat iona l separa t i on of a l l 14 u n i v a l e n t s . At the prophase o f the next d i v i s i o n , up to 14 chromosomes were observed i n each dyad nuc leus . In a d d i t i o n , the obse rva t ion tha t about 90% of ascospore c u l t u r e s were PWT, suggests tha t the nuc l e i i n the v i a b l e ascospores were d i p l o i d or nea r l y so. These ob-se r va t i on s are c l e a r l y not compat ib le w i th random movement o f u n i v a l e n t s . By the process of e l i m i n a t i o n , the equat iona l separa t i on o f un i va len t s a t the f i r s t m e i o t i c d i v i s i o n has l i k e l y taken p l a ce . S i m i l a r data have been obta ined f o r a sc -6 . The lower frequency of nond i s j unc t i on (20-55%) i s l i k e l y due to a h igher frequency of b i v a l e n t s a t metaphase I. T h i r d , the equat iona l d i v i s i o n dur ing the f i r s t me i o t i c d i v i s i o n has been p r e v i ou s l y suggested i n Neurospora by the occurrence of c e r t a i n PWT-containing a s c i ( Th re l ke l d and S t o l z , 1970). D i r e c t proof f o r equa-t i o n a l d i v i s i o n awaits f u r t h e r genet i c and c y t o l o g i c a l a na l y s i s of me i o t i c mutants. For example, i t should be po s s i b l e to observe the segregat ion of more than the hap l o i d number of seven chromosomes at anaphase I. The f a i l u r e to observe t h i s event dur ing t h i s study i s an i n d i c a t i o n t ha t the separa t i on of chromosomes a t anaphase I proceeds very r a p i d l y . D i s j u n c t i o n o f Chromosomes During the Second and P o s t - M e i o t i c D i v i s i o n s The qquat iona l d i v i s i o n of un i va len t s dur ing the f i r s t me i o t i c d i v i s i o n makes the:;subsequent d i v i s i o n of these chromatids impos s i b l e . In some yea s t and p l a n t mutants, no second d i v i s i o n f o l l ows the equat iona l 106 s e p a r a t i o n , and e i t h e r d i p l o i d spores or p o l l e n are produced (Moens et a l . , 1976; Str ingham, 1970; Smith, 1939) or the c e l l s degenerate (Palmer, 1971).- I f nuc lear d i v i s i o n takes p l a c e , the chromatids w i l l e i t h e r move a t random to oppos i te po l e s , po s s i b l y a f t e r a per iod of lag (Prakken, 1943), or they r e p l i c a t e before d i v i s i o n . Even though the l a t t e r mechanism has never been r epo r t ed , the behavior of mutant mei-1 suggests tha t an e x t r a , round of r e p l i c a t i o n p r i o r to the second d i v i -s ion may occur i n Neurospora. A l t e r n a t i v e l y , most chromosomes cou ld move together to one of the two d i v i s i o n po le s . I t has been noted tha t each dyad nucleus presumably has a d i p l o i d complement (or c l o se to i t ) of chromat ids. The unusua l ly high number of a sc i w i th two d i v i d i n g nuc l e i means t ha t t h i s d i v i s i o n takes a long time to complete. I f the chroma-t i d s move a t random, then most r e s u l t i n g nuc l e i should miss a t l e a s t one chromosome. Consequently, cont ra ry to ob se r v a t i on , most a sc i would con-t a i n on ly i n v i a b l e ascospores. The high f e r t i l i t y and almost complete d i p l o i d y of most ascospores can be exp la ined by an ex t r a round of r e p l i -c a t i o n and r e gu l a r centromere d i v i s i o n , or by the p r e f e r e n t i a l movement of most chromosomes to one d i v i s i o n po l e . Some c y t o l o g i c a l ev idence i s i n favor of the l a t t e r hypothes is (see e . g . , F i g . 8 j ) . The occurrence of an ext ra , round of r e p l i c a t i o n cou ld be t e s t ed by means of the Feulgen s t a i n i n g method (Iyengar e t a l _ . , 1977). In crosses w i th a mixture of un i va len t s and b i v a l e n t s a t meta-phase I, a combinat ion of the r egu l a r segregat ion of the b i v a l e n t s and and random movement or centromere d i v i s i o n o f the un i va len t s would p ro -duce a mixture of chromosomes and chromatids p r i o r to the second d i v i -s i o n . In t h i s case, a problem i n the synchrony of d i v i s i o n cou ld be 107 expected. In r y e , mutants w i th a mixture of un i va len t s and b i v a l e n t s had more aber rent second d i v i s i o n f i g u r e s than those w i th predominantly un i va l en t s (Prakken, 1943). S i m i l a r l y , i n Neurospora, more i r r e g u l a r types of chromosome movement ( e . g . , l a g g i n g , s p i nd l e over lap) dur ing the second m e i o t i c d i v i s i o n have been observed. in crosses w i t h many b i -va len t s and some un i va l en t s ( e . g . , a s c - 1 ) , than i n crosses w i t h few or no b i v a l e n t s ( e . g . , asc-6 and me i - 1 ) . The po s t -me i o t i c d i v i s i o n s o f these three mutants are u sua l l y r e gu l a r . However, some chromosome lo s s or nond i s j unc t i on has been sug-gested by a sc i w i t h odd numbers of v i a b l e b lack ascospores, and con-f i rmed g e n e t i c a l l y by the recovery of an odd number o f PWT progeny from s i x out of seven a sc i obta ined i n a cross homozygous f o r asc-6 (Table I I I ) . E f f e c t o f L i q u i d and S o l i d Cross ing Medium on D i s j u n c t i o n Abnormal d i v i s i o n f i g u r e s have been observed i n c rosses homozy-gous f o r asc-1 and mei-1 on s o l i d c r o s s i n g medium. Examples are the move-ment of sepa ra t i ng s p i nd l e pole bodies to the same s ide o f the ascus i n crosses homozygous f o r a s c - 1 , o r 4 -po led sp i nd le s f o r me i - 1 . I t has been proposed t ha t the 4-poled sp ind le s i n crosses homozygous f o r mei-1 may account f o r the observed nond i s j unc t i on (Lu and G a l e a z z i , 1979). However, t h e i r absence from s i m i l a r crosses on l i q u i d medium prov ides f i r m evidence aga in s t such a p o s t u l a t e . That the d i f f e r e n c e s i n types of d i v i s i o n f i g -ures i s a consequence of d i f f e r e n t c r o s s i n g media i s suggested by the abo r t i on of 90% o f the ascospores on s o l i d medium but on ly 70% on l i q u i d medium. The h igher spore abo r t i on on s o l i d medium can be r e a d i l y exp la ined by the abnormal types of d i v i s i o n s ( e . g . , 4-poled s p i n d l e s ) ; such abnormal 108 d i v i s i o n s might be expected to s imul taneous ly reduce the frequency of v i a b l e aneuplo ids f o r each chromosome. A comparison o f PWT f requenc ie s from crosses homozygous f o r mei-1 on l i q u i d medium ( t h i s c h a p t e r ) , w i th those on s o l i d medium (Smith, 1975), i s not i ncompat ib le w i t h t h i s p r e d i c t i o n . These observat ions i l l u s t r a t e the importance of the com-bined genet i c and c y t o l o g i c a l s tud ie s o f severa l c ro s se s . The use o f d i f f e r e n t c r o s s i n g cond i t i on s ( e . g . , s o l i d vs. l i q u i d c r o s s i n g medium) may a l s o prov ide va luab le i n fo rmat i on f o r the f i n a l understanding o f me i o t i c processes. Are A l l Aneuplo id (n. + 1 through n + 6) Ascospores V i a b l e i n Neurospora? In Neurospora, mutations which cause abnormal d i s j u n c t i o n o f chromosomes dur ing meios i s have been detected by the presence of many hypo-p l o i d wh i te ascospores, and.they have been p a r t i a l l y c h a r a c t e r i z e d by the study of hyperp lo id (PUT) i s o l a t e s . In h igher p l a n t s , most aneuplo id products o f meios i s are i n v i a b l e . This makes the genet i c c h a r a c t e r i z a -t i o n of mutants very d i f f i c u l t . Even though many aneuplo id products can be obta ined i n Neurospora, i t i s not c l e a r whether or not they are a l l v i a b l e . There fo re , ascospores from a cross homozygous f o r asc-1 were heat-shocked, p l a t ed and scored f o r t h e i r co lony- forming a b i l i t y . About 50% of the b lack ascospores d id not produce c o l o n i e s . Most of these germinated but then growth stopped a b r u p t l y . The observed sudden escape i n some o f the i n h i b i t e d ascospore c u l t u r e s may be exp la ined i f the i n -h i b i t i o n were caused by aneup lo idy , and the resumption i n growth by the l o s s o f one or more excess chromosomes ( P i t t e n g e r , 1954). Germination f requenc ies ( gene ra l l y about 50%) of spores produced by crosses homozygous 109 f o r a s c - 6 , asc-3 and mei-1 are a l s o c o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n . Does Nond i s junct ion A f f e c t A l l Chromosomes? The study of the s imultaneous nond i s j unc t i on of more than one chromosome may help i n the c h a r a c t e r i z a t i o n o f these mutants. Three chromosomes (LG I, IV and V) were s imu l taneous ly monitored i n crosses homozygous f o r a sc -6 . In two crosses examined, the three chromosomes were a f f e c t e d by nond i s j unc t i on a t about equal frequency (Table IV) S i m i l a r observat ions have p r e v i ou s l y been made f o r mei-1 (Smith, 1975). However, i t appeared a t f i r s t t ha t chromosomes i n crosses homozygous f o r asc-6 d i d not nond i s j o i n independent ly : a;prevelance of i s o l a t e s d isomic or hap lo id f o r a l l three chromosomes was observed. Even though t h i s may be due to a dependence o f d i s j u n c t i o n o f d i f f e r e n t chromosomes, the more probable exp l ana t i on o f t h i s phenomenon i s the s e l e c t i v e death o f ascospores w i th m u l t i p l e d isomic n u c l e i and s u r v i v a l of hap lo i d and d i -p l o i d (or nea r l y so) i s o l a t e s . Such p r e f e r e n t i a l , s u r v i v a l has a l ready been suggested i n the previous s e c t i o n . I n t e r a c t i o n of Three Me i o t i c Mutations Studies of i n t e r a c t i o n between the three mutations w i th a p a i r -ing de fec t suggested t ha t each i s d e f e c t i v e i n one aspect of the same process . In t h i s case, the process a f f e c t e d i s p a i r i n g , and double mutants appear to behave as the s i n g l e mutant w i th the more extreme reduc t i on i n p a i r i n g . However, s i n ce data on the i n t e r a c t i o n between the mutants asc-1 and asc-6 are l i m i t e d , i t remains po s s i b l e t ha t these two mutations i n t e r a c t to v i r t u a l l y e l i m i n a t e the p a i r i n g of homologs. This i n t e r p r e t a t i o n would be compatible w i th present ob se r va t i on s , 110 s i n ce the amount o f ascospore abo r t i on o f crosses homozygous f o r the double mutant a s c - l ; a s c - 6 i s i d e n t i c a l to that o f crosses homozygous f o r mei-1 which lack p a i r i n g of homologs. In summary, the combined genet i c and c y t o l o g i c a l observat ions of the three m e i o t i c mutants me i - 1 , asc-1 and asc-6 c l a s s i f y these mutants i n t o a group w i th many common c h a r a c t e r i s t i c s . In each case, some de fec t i n p a i r i n g (or exchange) leads to the format ion of u n i -va len t s a t metaphase I. The d i f f e r e n c e i n frequency of un i va len t s ap-pears to p a r t l y con t r o l subsequent chromosome behavior dur ing the f o l -lowing d i v i s i o n . However, i n each case, i r r e g u l a r segregat ion takes p lace dur ing the f i r s t and second d i v i s i o n , l ead ing to the product ion o f hypoplo id products ( recogn ized as wh i te ascospores ) , and hype rp l o i d p ro -ducts (those d isomic f o r LG I are recogn ized as PWT progeny, which are used i n the c h a r a c t e r i z a t i o n o f the mutant) . The use o f d i f f e r e n t c ro s s i ng media apparent l y has an e f f e c t on chromosome movement i n a t l e a s t one of these mutants (mei -1 ) . There fo re , the s imultaneous study of mutants on both types o f media may prov ide some i n s i g h t i n t o the process of d i s j u n c t i o n dur ing me io s i s . A Mutat ion Which Causes Nond i s junct ion During the Second M e i o t i c D i v i s i o n The mutant asc-3 appears to be unique. The pr imary de f ec t takes p lace before the fo rmat ion of a s c i . Most c e l l s are apparent l y b locked p r i o r to karyogamy. S ince p r e -me i o t i c DNA synthes i s i n Neurospora takes p lace j u s t p r i o r to karyogamy (Iyengar ejt a j_. , 1977), i t i s q u i t e p l au s -i b l e t ha t the w i l d type gene of asc-3 f unc t i on s dur ing or near t h i s p re -m e i o t i c S phase. Some c e l l s , however, manage to proceed past t h i s b lock and produce a s c i and ascospores. Such escape i s not due to a mutat iona l I l l event, s i n ce (A + a j PWT i s o l a t e s from crosses homozygous f o r asc-3 produced the same c ro s s i ng phenotype. Thus, the few a s c i produced i n crosses homozygous f o r asc-3 are the consequence of l e a k i n e s s . P a i r i n g o f , and recombinat ion between homologous chromosomes i s normal. S i m i -l a r l y , no abno rma l i t i e s have been detected i n the d i s j u n c t i o n of these chromosomes dur ing the f i r s t m e i o t i c d i v i s i o n . However, an extremely high frequency of nond i s j unc t i on takes p lace dur ing the second d i v i s i o n , and there i s a l s o chromosome los s or nond i s j unc t i on a t the p o s t -m e i o t i c d i v i s i o n . The nond i s j unc t i on dur ing the second d i v i s i o n apparent ly i n -vo lves attachment of most chromosomes to one, but not the o the r , s p i nd l e pole body (SPB). The extended du ra t i on of t h i s d i v i s i o n i s probably caused by such abnormal attachment. In some r e spec t s , t h i s mutant resembles the mutants pal and c a n d of Drosoph i la (Baker and H a l l , 1976). The mutant p_al_ acts only i n males. Chromosomes of homozygous pal males are p r e f e r e n t i a l l y l o s t dur ing the f i r s t z y g o t i c c leavage d i v i s i o n and maybe dur ing the m e i o t i c d i v i s i o n s . Such los s a l s o takes p lace i n c a n d mutants, which a c t e x c l u s i v e l y i n f e -males. In both cases , chromosomes are l o s t a t one po le of the d i v i s i o n . In these two mutants of Drosoph i la and the asc-3 mutant of Neurospora, a de fec t i n the attachment of centromeres to SPB's causes e i t h e r the los s ( i n Drosoph i la ) or nond i s j unc t i on ( i n Neurospora) of a se t of chromo-somes . A Po s s i b l e Re l a t i o n s h i p Between the Defects a t Pre-ascus and Second Me i - o t i c D i v i s i o n Stages I t has been suggested tha t the w i l d type genes f o r the mutants pal and c a n d i n Drosoph i la s p e c i f y a product tha t i s a component o f , or 1 i n t e r a c t s w i t h , the centromer ic reg ion of chromosomes and i s necessary f o r the normal segregat ion of these chromosomes (Baker, 1975; Baker e t a } . , 1976a). A l t e r n a t i v e l y , the phenotype of these mutants might be produced by a d e f e c t i v e s p i nd l e pole body. S i m i l a r l y , i n view of the stage of the f i r s t a c t i n g de fec t of a s c - 3 , i t i s p o s s i b l e t ha t i t s w i l d type gene product operates dur ing the p r e -me i o t i c S phase and mod i f ie s the centromere reg ion of the newly synthes i zed DNA or produces a de fec -t i v e s p i nd l e po le body. E i t h e r de fec t would g ene r a l l y cause a deve lop-mental b l o c k ; however, the few c e l l s t ha t escape t h i s b lock would en-counter problems dur ing the second and subsequent d i v i s i o n s owing to the mutant product necessary f o r r e gu l a r seg rega t i on . One could specu la te tha t t h i s p a r t i c u l a r product may be necessary to r e i n t r oduce r egu l a r equat iona l d i v i s i o n a f t e r i t was suppressed dur ing the f i r s t d i v i s i o n . A more d e f i n i t e assessment of the c o r r e l a t i o n between the two de fec t s has to await more extens i ve a n a l y s i s of t h i s mutant, and o f s i m i l a r ones e . g . , mei-4 i n Neurospora (Newmeyer and G a l e a z z i , 1978; Raju and Perk ins 1978). For example, mutants w i th s i m i l a r primary d e f e c t s , such as the p a i r i n g d e f e c t i v e mutants a l ready d i s cu s sed , may have s i m i l a r secondary de fec t s i n the segregat ion o f chromosomes. The Nature of the Defect a t the Second M e i o t i c D i v i s i o n I f the pos tu la ted abnormal centromere reg ions would a l i g n a t ran dom, one would expect both chromatids o f a l l seven chromosomes to move at random to e i t h e r one or the other po l e . Such segregat ion would p ro -duce an extremely high chance .of abo r t i on of r e s u l t a n t ascospores owing to hypoplo idy. Both c y t o l o g i c a l and genet i c observat ions appear to c o n t r a d i c t these assumptions: ( i ) most chromosomes move to one p o l e , 113 few or none to the o t he r , and ( i i ) many v i a b l e (b lack ) ascospores are produced. Consequently, assuming a de fec t i n the centromere r eg i on s , chromosomes could not a l i g n a t random. In s tead, centromeres t ha t were synthes i zed a t the same time ( e . g . , dur ing the p r e -me i o t i c S phase) might normal ly a l i g n and segregate to the same po le ( F i g . 9 ) . Th is type of p r e f e r e n t i a l segregat ion has been observed i n p r o ka r yo t i c sy s -tems (Jacobs e t a l_ . , 1966; Lark, 1966), and proposed f o r some eukary-o t i c systems ( e . g . , Baker and H a l l , 1976).. Even though the evidence f o r such al ignment dur ing m i t o t i c d i v i s i o n s of euka r yo t i c c e l l s i s not conv inc ing ( e . g . , Heddle e t aj_. , 1967), such a mechanism may we l l operate dur ing me i o s i s . Map P o s i t i o n s of asc Mutat ions Mapping of the three newly i s o l a t e d m e i o t i c mutations has p laced asc-1 and asc-3 extremely c l o se to the p r e v i ou s l y i s o l a t e d mutat ion me i - 1 . Mutat ion asc-6 was l o ca ted on another l i n kage group (LG I I ) . The three c l o s e l y l i n k e d m e i o t i c mutations (mei -1 , asc-1 and asc-3) are mutu-a l l y complementing (see Chapter 1) and appear to i n v o l v e f unc t i on s neces-sary on ly f o r the r egu l a r p a i r i n g and d i s j u n c t i o n of chromosomes dur ing me io s i s . These mutations cou ld be complementing a l l e l e s of the same gene. A l t e r n a t i v e l y , they may represent the f i r s t case of a gene c l u s t e r tha t i s s p e c i f i c a l l y " a c t i v e dur ing -me io s i s (a gene c l u s t e r of f unc t i on s r equ i red i n DNA metabolism has been detected i n D ro soph i l a ; some of these f unc t i on s are a l s o requ i red dur ing me i o s i s ; Boyd et a j_. , 1976a). The c l o se p rox im i t y of a s c - 3 , whose w i l d type gene operates before ka ry -ogamy, and mei-1 and a s c - 1 , which e x h i b i t p a i r i n g d e f e c t s , may be an 114 F i g . 9 Po s s i b l e exp lana t i on f o r abnormal d i s j u n c t i o n i n asc-3 mutant c ro s se s . Upon sepa ra t i on of DNA strands to en-ab le p reme io t i c DNA r e p l i c a t i o n , a cent romere-assoc iated p r o t e i n (A) i s bound to one DNA s t r and . A novel p ro -t e i n (• ) s p e c i f i c f o r meios i s would become attached to the other DNA s t r and . Th is p r o t e i n might, f o r ex-ample, con t r o l some aspect of p a i r i n g of homologs and/or prevent r e gu l a r centromere sepa ra t i on dur ing the next d i v i s i o n . This model r equ i re s t ha t a l l chromatids w i t h the newly synthes i zed ( • ) p r o t e i n become a l i gned to the same pole dur ing the second me i o t i c d i v i s i o n . In the asc-3 mutant, t h i s cent romere-as soc ia ted p r o t e i n may be d e f e c t i v e and thus prevent r e gu l a r separa t i on of chrom-a t i d s a t the second m e i o t i c d i v i s i o n (and l i k e l y a t subsequent d i v i s i o n s ) . P r e m e i o t i c S Phase N u c l e u s o f P a r e n t 1 P a r e n t 2 ( o n l y two of t h e s e v e n chromosomes p r e s e n t e d ) D o u b l e r H e l i x 1 M e t a p h a s e I Me t a p h a s e I I (one dyad n u c l e u s ) T— - f d e f e c t i v e i n d e f e c t i v e i n a s c - 3 | a s c - 3 116 i n d i c a t i o n that the w i l d type a l l e l e s of mei-1 and asc-1 are a l s o a c -t i v e before karyogamy and thus w e l l before the ac tua l p a i r i n g process where t h e i r e f f e c t s are expressed. In c o n c l u s i o n , the present a n a l y s i s has prov ided another de-monstrat ion of the usefu lness of a combined c y t o l o g i c a l and genet i c approach to the a n a l y s i s o f m e i o t i c mutants i n Neurospora crassa. ' Two d i f f e r e n t types of mutants have been p a r t l y c h a r a c t e r i z e d and mapped. Future s tud ie s should extend to the i s o l a t i o n o f t empera tu re - sen s i t i ve mutants and mutants t ha t i n t e r a c t w i th e x i s t i n g me i o t i c mutants. These may be used to determine the temporal pe r i od o f a c t i v i t y o f gene pro -ducts and the nature of c e l l a c t i v i t y by the i n t e r a c t i o n of d i f f e r e n t components. CHAPTER I I I THE MUTATION SK(ad-3A) ALTERS THE DOMINANCE OF ad-3A + OVER ad-3A IN THE ASCUS OF NEUROSPORA 117 INTRODUCTION Known causes of l e t h a l i t y of me i o t i c products i n c l ude aneu-p l o i d y and mutat ion . For example, the d e l e t i o n of any e s s e n t i a l pa r t o f the genome from the p o l l e n of h igher p l an t s or ascospores o f fung i r e s u l t s i n t h e i r a b o r t i o n . In c o n t r a s t , d e f i c i e n c i e s of nuc l e i i n the meiocytes of animals does not g e n e r a l l y r e s u l t i n l e t h a l i t y of the egg or sperm c e l l s . There are three known types of l e t h a l i t y of me i o t i c products which are not a s soc i a ted w i t h aneuplo idy but , i n s t e a d , i n vo l ve s p e c i f i c gene mutat ions . In each case, m e i o t i c products which ca r r y a s p e c i f i c a l l e l e are i n v i a b l e . A l l e l e s of c e r t a i n genes are recovered i n a r egu l a r f a sh ion when crossed w i th the same a l l e l e , but they are le s s f r e q u e n t l y or not recovered when crossed w i th another a l l e l e . This phenomenon, c a l l e d segregat ion d i s t o r t i o n , can be the r e s u l t of the l e t h a l i t y of m e i o t i c products con ta i n i ng the s e n s i t i v e genes ( f o r rev iew see Zimmering e t a l , 1970; Hart! and H i r a i z u m i , 1976). Examples are SD i n D ro soph i l a , which causes an a r r e s t i n maturat ion o f S d + spermatids (Hart! and H i r a i z u m i , 1976) and SK i n Neurospora, which causes abo r t i on of SK S con ta i n i n g ascospores (Perk ins and Bar ry , 1977; Turner and P e r k i n s , 1976). A l l e l e s from a second k ind of gene cause the abo r t i on of a l l me i o t i c products when crossed w i th the same a l l e l e . This k ind of mutant e f f e c t may be expressed i n an autonomous or r e ce s s i v e manner, i . e . , i n crosses to the w i l d type a l l e l e , m e i o t i c products con ta i n i ng 118 the mutant a l l e l e may abort (autonomous) or they may be v i a b l e ( r e -c e s s i v e ) . These types of mutants are most e a s i l y detected i n f u n g i , e s p e c i a l l y ascomycetes, s i n ce a l l products of a me i o t i c d i v i s i o n can be observed i n a s i n g l e ascus. In a d d i t i o n , v i a b l e and i n v i a b l e ascospores can be r e a d i l y d i s t i n g u i s h e d by t h e i r d i f f e r e n t c o l o r (and o f ten t h e i r s i z e ) . Ascospore l e t h a l s are u s u a l l y c o l o r l e s s , w h i l e normal v i a b l e ascospores are g e n e r a l l y c o l o r e d . I n - t h i s manner, the autonomously expressed ascospore l e t h a l s asco ( S t a d l e r , 1956), tan (Nakamura, 1961), .cys-3 (Murray,, 1965) andws_ ( P h i l l i p s and S rb, 1967) were detected i n Neurospora. This c l a s s o f mutants s u p e r f i c i -a l l y resembles v i a b l e ascospore c o l o r mutants i n So rda r i a (Chen, 1965; O l i v e , 1965), Podospora (Es ser , 1974) and AscObolus ( B i s t i s , 1956; L issouba e t a]_., 1962). However, the two types of mutants probably represent q u i t e d i f f e r e n t d e f e c t s . Examples of mutations which express t h e i r e f f e c t i n a r e c e s -s i v e manner are ad-3A and ad-3B i n Neurospora ( G r i f f i t h s , 1970) and ms^3 and m s ^ i n tomato (Rick and B u t l e r , 1956). This chapter repor t s a case i n Neurospora c ras sa where one mutant (ad-3A) can be expressed as e i t h e r a r ece s s i ve or an autonomous ascospore l e t h a l , depending on whether i t i s crossed w i t h w i l d type (ad-3A + ) or a newly induced mutat ion c a l l e d SK(ad-3A). Th is i s , to the au tho r ' s knowledge, the f i r s t case o f such a r e l a t i o n s h i p . Some except ions are the ascospore l e t h a l s l e - 1 and l e - 2 i n Neurospora (Murray and Srb, 1961; Garnjobst and Tatum, 1967). Asco-spores con ta i n i n g these mutants are b l a ck . 119 Moreover, the system i s a l s o unique i n t ha t the a f f e c t e d enzyme coded f o r by the ad-3A locus i s known and c h a r a c t e r i s t i c s o f i t can be s t ud i ed on a b iochemical l e v e l ( F i s h e r , 1969b). There fo re , the study o f t h i s mutat ion may g ive an i n s i g h t i n t o processes i n v o l v i n g gametic l e t h a l i t y and the i n t e r a c t i o n between genomes i n the ascus. MATERIALS AND METHODS S t r a i n s The f o l l o w i n g mutant a l l e l e s were used dur ing t h i s s tudy: l eu -3 (R156), un-3 (55701- t ) , arg-1 (36703), n i c - 2 (43002), a l - 2 (74-Y-112-M38), tol_ (N83), ad-3A (2-17-19, 2-17-124, 2-17-186, 2-17-232, 2-17-233,.2-17-814, 2-17-825, 2-31-2, 2-32-10, 2-33-3, 2-33-4, 2-33-22, 2-33-30, 2-33-34, 5-5-4, 5-5-23, 5-5-47, 5-5-52, 5-5-74), ad-3B (2-17-114, 2-17-76, 2-17-82, 2-17-85, 2-17-99, 2-17-128). The l a s t f i v e a l l e l e s of ad-3B complement a l l e l e 2-17.-114, and none o f the ad-3A a l l e l e s complement each o the r . The approximate map d i s tances (Radford, 1972) of mutations on LG I are shown i n F i g . 1 (see a l s o Chapter I ) . D i sc repanc ies between these values and those obta ined i n t h i s study may be p a r t l y due to v a r i a b i l i t y caused by genet i c background. The procedure used i n the i s o l a t i o n and d e t e c t i o n o f mutant s t r a i n P917 has been desc r ibed i n Chapter I. This s t r a i n i s a pseudo-w i l d type (PWT) c u l t u r e obta ined from a screen f o r r e ce s s i v e m e i o t i c mutants. I t i s composed of two nuc lear components of genotypes l e u - 3 , a_, a r g - 1 , ad-3B and un-3, A, ad-3A, n i c - 2 , a l - 2 ; each component c a r r i e s the t o l mutat ion which a l lows normal growth of (A. + a_) heterokaryons (Newmeyer, 1970; DeLande and G r i f f i t h s , 1975), and i d e n t i c a l (but 120 F i g . 1 The two nuc lear components of s t r a i n P917. H e t e r o k a r y o n Component 1 Component 2 c e n t r o m e r e l e u - 3 + a a r g - 1 + ad-3B + + t i ' ' • B i i i i + un-3 A + ad-3A + n i c - 2 a l - 2 A p p r o x i m a t e map d i s t a n c e • 1D 0.1 10 9 0.3 k 28 ( R a d f o r d , 1972) 122 unknown) het genotype necessary f o r vigourous growth of the he te ro -karyon (Garnjobst and W i l son , 1956). . Mutant P917 was detected by i t s p roduct ion of about 60% whi te aborted ascospores. The two ascospore i s o l a t e s 917A36 and 917a38 were obta ined from crosses between s t r a i n s P917 and the w i l d type s t r a i n s OR-A and 0R-a_, r e s p e c t i v e l y . The i r genotypes are l e u - 3 , a , a r g - 1 , ad-3B; t o l ; Cde (917A36) and un-3, A, ad-3A, n i c - 2 , a l - 2 ; t o l ; Cde (917a38), where C, d_, and e_ are a l l e l e s o f three heterokaryon c o m p a t i b i l i t y l o c i . The presence of the t o l mutat ion and Cde genotype i n both s t r a i n s a l lows the format ion of a heterokaryon- between these s t r a i n s . The i d e n t i t y of the two adenine r e q u i r i n g mutations ad-3A and ad-3B was determined, where necessary, by heterokaryon t e s t s w i th ad-3A and ad-3B t e s t e r s t r a i n s (see Delange and G r i f f i t h s , 1975). Procedures Crosses were performed by the s imultaneous i n o c u l a t i o n of two s t r a i n s o f oppos i te mating type (A_ and a_) i n t o 18 x 150 mm t e s t tubes con ta i n i ng 5 ml l i q u i d c ro s s i ng medium and a s t r i p of f i l t e r paper (Newcombe and G r i f f i t h s , 1972). A l l crosses were incubated a t 25°C. , Ascospore a n a l y s i s was u s ua l l y performed by the i s o l a t i o n of i n d i v i d u a l ascospores 'and t e s t i n g of the r e s u l t i n g c u l t u r e s . To de tec t a d + recombinants among the progeny from a cross between l e u - 3 , a_, a r g - 1 , ad-3B and A, ad-3A, ascospores were p l a t ed on s o l i d medium supplemented w i th l e u c i ne and a r g i n i n e . The r e s u l t i n g co l on i e s were t r a n s f e r r e d from the p l a te s to s l a n t s of vege ta t i ve medium supplemented w i t h l euc i ne and a r g i n i n e , and te s ted f o r t h e i r l e u c i n e and a r g i n i n e requi rements. Some w i l d type (PWT) c u l t u r e s were presumably the r e s u l t o f nond i s j unc t i on 123 of LG I. Only the l e u , a r g , ad recombinants were f u r t h e r used. The ad-3B mutation i n s t r a i n 917A36 has been r e ve r t ed i n one experiment. A c o n i d i a l suspension of s t r a i n 917A36 was i r r a d i a t e d w i t h 3 2 UV at 5 x 10 ergs/cm f o r 30, 60, or 90 seconds, ana the i r r a d i a t e d c o n i d i a were p l a t ed on medium supplemented w i th l e u c i ne and a r g i n i n e . The a d + r e ve r t an t co l on i e s were crossed w i th an ad-3A mutant s t r a i n , and the r e s u l t i n g l e u , a r g , a d + ascospore i s o l a t e s were used to t e s t f o r mutant (ascospore abo r t i on ) phenotype. The observat ion of l i n e a r a s c i , and other r ou t i ne manipu lat ions have been desc r ibed p r e v i ou s l y (Davis and deSerres , 1970; Chapter I ) . RESULTS S t r a i n P917 i s a s e l f - f e r t i l e (A + aj heterokaryon c o n s i s t i n g of two nuc lear t ypes , each having severa l complementing mutations on LG I (see F i g . 1) . Upon s e l f i n g (a cross between the a_ and A nuc l e i of t h i s heterokaryon) , t h i s s t r a i n produced about 60% aborted wh i te ascospores. Is the Ascospore Abor t i on of P917 Caused by a Dominant or a Recess ive Factor? To determine whether a dominant or r ece s s i ve f a c t o r caused as -cospore abo r t i on i n P917, i t was crossed w i th w i l d type s t r a i n s 0R-£ and 0R-a_. The absence of aborted spores from crosses w i t h both w i l d type s t r a i n s suggested that abo r t i on was caused by a r e ce s s i v e r a the r than a dominant f a c t o r . However, the a n a l y s i s of i s o l a t e s from these crosses was, p a r a d o x i c a l l y , more c o n s i s t e n t w i t h the presence of a dominant mutat ion on LG I (see Table I ) . In backcrosses w i t h P917, a l l T a b l e I . A n a l y s i s o f i s o l a t e s * f rom c r o s s e s between P917 t o 0R-A_ and OR-a w i l d t y p e s t r a i n s Number o f % a s c o s p o r e a b o r t i o n when c r o s s e d w i t h C r o s s G e n o t y p e LG I p r o g e n y P917 917A36* 9 1 7 a 3 B * ( a ) P917 20 0-10 — X un, ad , n i c , a l A_ 18 60 -- — OR-a un, ad , . a l A 1 60 — — ad , n i c , a l a_ 1 -- -- — ( b ) P917 +. a 11 0-10 0-10 — X 7 90 90 — OR-A a r g , ad A 3 — — l e u , a k 0-10 — 0-10 l e u , a r g a 1 0-10 — 0-10 l e u , a r g , ad a 12 60 — 60 R e c o m b i n a n t s between a_d and a_l were n o t t e s t e d , t 9.17A36 i s an i s o l a t e f r o m c r o s s ( b ) of g e n o t y p e l e u , a r g , ad , a_. ^ 917a38 i s an i s o l a t e f r o m c r o s s ( a ) of g e n o t y p e un, a d , n i c , a l , A. 125 20 w i l d type (+) a_ i s o l a t e s from the cross between P917 and OR-ja p ro -duced on ly b lack ascospores, and a l l 18 un_,_A, ad, n i c , a l i s o l a t e s produced about 60% ascospore a b o r t i o n . The abo r t i on phenotype appar-e n t l y segregated w i th the un_, A, ad , n i c , a l chromosome (LG I ) . S i m i -l a r l y , i n the cross between P917 and OR-A, the abo r t i on phenotype segregated w i th the l e u , a_, a r g , ad chromosome (LG I ) . The abo r t i on of ascospores from these crosses cou ld not have been due to the s e l f -ing of P917 s i nce crosses w i th e i t h e r P917 or the ascospore i s o l a t e 917a38 produced the same amount o f ascospore a b o r t i o n . The 90% asco-spore abo r t i on observed i n 7 w i l d type (+) A i s o l a t e s was l a t e r a t t r i -buted to a separate r e ce s s i v e po i n t mutat ion (a sc -7 ; see Chapter I ) . The abo r t i on f a c t o r on the l e u , a , a r g , ad chromosome cou ld be l o ca ted to a small reg ion spanning the centromere of LG I, s i n ce l e u , a^  and l e u , a_, arg crossover products d i d not produce any aborted ascospores when crossed w i t h P917 (the centromere of LG I i s l o ca ted between arg-1 and ad -3 ) . S ince the mutat ion caus ing about 60% ascospore abor-t i o n i s l o ca ted i n a wel l -marked reg ion of LG I, i t i s u n l i k e l y tha t the same mutat ion i s present i n both the un_, A, ad, n i c , a l and l e u , a , a r g , ad components o f s t r a i n P917. These observat ions are c o n s i s t e n t w i t h a mutat ion l o ca ted near the centromere of LG I, which e i t h e r causes ascospore abo r t i on i n dominant f a sh ion ( i n P917) or has no e f f e c t ( i n crosses between P917 and OR-A or 0R-a_). Nature o f Defect To f u r t h e r determine the nature of the de fec t l ead ing to asco-spore a b o r t i o n , a cross between the two ascospore i s o l a t e s 917A36 ( l e u , 126 a_, a r g , ad) and 917a38 (UJT, A, ad , n i c , a l ) was a n a l y z e d . 0 About 60% o f ascospores produced by t h i s c ross were abo r ted . Table II shows t h a t recombinat ion and nond i s j unc t i on (PWT) f requenc ies were normal. However, "only one of the two homologous parenta l chromosomes ( l e u , a_, a r g , ad) was recovered from t h i s c r o s s . In a d d i t i o n , r e c i p r o c a l c r o s s -over products were only detected i n the a r g - n i c r e g i o n . Consequently, a smal l reg ion or gene i n the a r g - n i c reg ion on the un_, A, ad, h i e , a l chromosome cannot be recovered when crossed w i t h s t r a i n 917A36 (the l e u , a_, a r g , ad chromosome). These data suggested the involvement of three c l o s e l y l i n k e d genes which may be a l l e l e s . A gene on the l e u , a_, a r g , ad chromosome apparent ly caused the death of ascospores con ta i n i n g a second gene which was l o ca ted on the un_, A, ad , n i c , a l chromosome. Both genes were l o ca ted near the centromere of LG I. A t h i r d gene, p re -sumably a l s o on LG I, was not a f f e c t e d by the k i l l i n g o f the f i r s t gene, nor was i t capable of k i l l i n g the second gene. I d e n t i t y of Gene S e n s i t i v e to K i l l i n g Ac t i on To l o c a t e the s e n s i t i v e gene„more p r e c i s e l y w i t h i n the a r g - n i c r e g i o n , a l l c rossover products i n t h i s reg ion were t e s ted f o r the p re -sence of ad-3A and ad-3B mutat ions . A l l these products were ad-3B r a t h e r than ad-3A. Appa ren t l y , the s e n s i t i v e gene was l o ca ted near or a t the ad-3A l o cu s . Th i s cross was used r a t he r than a P917 s e l f i n g , to ensure tha t only two nuc lear components of known genotype were i n v o l v e d . P917 may con ta in a small p ropo r t i on of contaminat ing n u c l e i , e . g . , produced by somatic c ro s s i ng -ove r ( P i t t e n g e r and Coy le , 1963). 1 2 7 T a b l e I I . G e n o t y p e of 79 i s o l a t e s f r o m t h e c r o s s between s t r a i n s 917A36 and 917a38 ( f o r LG I m a r k e r s , see F i g . 1) w h i c h p r o d u c e d a b o u t 6 0 % a b o r t e d a s c o s p o r e s LG I M a r k e r s Geno t y p e Number A s c o s p o r e I s o l a t e s P a r e n t a l \ l e u , a r g , ad un , ad , n i c , a l 33 0 C r o s s o v e r l e u - u n a r g - n i c n i c - a l a r g , ad l e u , un, a d , n i c , a l  un , ad l e u , a r g , a d , n i c , a l l e u , a r g , a d , a l  un, a d , n i c 6 0 k k 21 0 D o u b l e C r o s s o v e r : l e u - u n / n i c - a l a r g - n i c / n i c - a l a r g , a d , a l l e u , un, a d , n i c un , ad , a l l e u , a r g , a d , n i c 7 D 3 1 RF ( l e u - u n ) ( u n - a r g ) ( a r g - n i c ) ( n i c - a l ) 13/79 ( 1 6 % ) D/79 ( 0%) 12/79 ( 1 5 % ) 32/79 ( 4 0 % ) RF i n W i l d Type C r o s s e s 11-17 .15-20 30-35 N o n d i s j u n c t i o n F r e q u e n c y = 0/79 ( 0%) 128 To t e s t the p o s s i b i l i t y t ha t the ad-3A mutat ion i t s e l f cou ld not be recovered i n these c ro s se s , 19 independent ly de r i ved ad-3A a l l e l e s were crossed w i th s t r a i n 917A36. As a c o n t r o l , f i v e comple-menting ad-3B mutants and seven w i l d type s t r a i n s were a l s o crossed w i th 917A36. Crosses w i th each o f the 19 ad-3A a l l e l e s r e s u l t e d i n 50-60% ascospore a b o r t i o n ; i n c o n t r a s t , abo r t i on was absent i n the r e -maining c ro s se s . Thus i t appeared t ha t on ly adenine r e q u i r i n g ad-3A mutations cou ld not be recovered when crossed to the " k i l l e r " l e u , a_, a r g , ad chromosome. I f a l l ascospores con ta i n i n g ad-3A were i n v i a b l e when crossed w i th s t r a i n 917A36, which conta ins the l e u , a_, a r g , ad chromosome, most a s c i produced from t h i s cross should con ta in f ou r b lack and fou r wh i te ascospores (4B:4W). In f a c t , 54 out of 56 a s c i were of t h i s t ype; the remaining two were 2B:6W. S ince ad-3A i s c l o s e l y l i n k e d to the centromere, a low frequency o f second d i v i s i o n segregat ion of whi te and b lack ascospores would be expected. Aga in , only 2 out of 54 a sc i had a second d i v i s i o n pa t te rn o f segregat ion of b lack and wh i te ascospores. I t was concluded t ha t a mutat ion on the l e u , a , a r g , ad chromo-some caused ad-3A-conta in ing ascospores to abo r t . This mutat ion w i l l be r e f e r r e d to as spore k i l l e r o f ad-3A, or SK(ad-3A). Locat ion o f the Newly Induced Spore K i l l e r SK(ad-3A) Mutat ion In order to l o c a t e SK(ad-3A) on LG I, 253 i s o l a t e s from the cross between 917A36 ( l e u , a_, a r g , ad-3B, SK(ad-3A)) and a w i l d type s t r a i n (FGSC 1228, which i s ad -3A + and t he re fo re r e s i s t a n t to SK(ad-3A) a c t i on ) were t e s ted f o r the k i l l e r c h a r a c t e r . No recombinants between SK(ad-3A) and ad-3B were recovered. Thus, SK(ad-3A) was l i n k e d very 129 c l o s e l y to ad-3B. To show tha t ad-3B was not r equ i red f o r the k i l l i n g of ad-3A-con ta i n i n g ascospores, the ad-3B mutat ion i n s t r a i n ;917A36 was r e -ve r t ed . The k i l l i n g a c t i o n o f l e u , a , a r g , a d + r e ve r tan t s c l e a r l y de-monstrated that SK(ad-3A) Was s t i l l p re sent and acted independent ly of the ad-3B mutat ion . A more p r e c i s e l o c a l i z a t i o n of SK(ad-3A) w i th re spec t to the ad-3A and ad-3B l o c i was obta ined by i s o l a t i n g a d + recombinant progeny from crosses between 917A36 ( l e u , .a, a r g , ad-3B, SK(ad-3A)) and two s t r a i n s con ta i n i ng a l l e l e s 2-17-814 or 2-17-825 of ad-3A. Four c r o s s -over products o f genotype l e u , a , a r g , a d + were obta ined ( F i g . 2 ) . A l l f ou r recombinants conta ined the SK(ad-3A) mutat ion . There fo re , s i nce SK(ad-3A) i s very c l o s e l y l i n k e d to ad-3B (0/253 recombinants ) , but nearer ad-3A than ad-3B, i t was concluded tha t SK(ad-3A) and ad-3A are very t i g h t l y l i n k e d and may be a l l e l e s of the same gene. Other C h a r a c t e r i s t i c s of ad-3A and SK(ad-3A) Mutations The only known e f f e c t o f the SK(ad-3A) mutat ion i s i t s k i l l i n g a c t i o n on ad -3A-conta in ing ascospores. Thus, crosses homozygous f o r the SK(ad-3A) mutat ion produce only b lack ascospores. In a d d i t i o n , SK(ad-3A) c u l t u r e s grow a t w i l d type ra tes and do not r equ i r e adenine f o r growth. To determine whether or not ad-3A-conta i ni ng c on i d i a cou ld be obta ined from vege ta t i ve heterokaryons between ad-3A and SK(ad-3A), severa l heterokaryons between s t r a i n 917A36 ( l e u , a , a r g , SK(ad-3A), ad-3B) and 2-17-825a (a_, ad-3A) were a l lowed to grow i n 50 cm race tubes. S e l e c t i o n of ad_+ recombinants to l o c a l i z e the SK(ad-3A) mutation w i th re spect to the ad-3A and ad-3B l o c i . 131 c e n t r o m e r e l e u - 3 a a r g - 1 + ad-3B _j I I i i i + A + ad-3A + • .3 mu 132 These heterokaryons grew a t a r a t e comparable to severa l heterokaryons between ad-3A and SK (ad -3A ) + , ad-3B. Con id ia from the beginning and the end of one race tube were i s o l a t e d and t h e i r genotypes were t e s t e d . The ad_ and l e u , a r g , ad genotypes were recovered w i th approx imately equal frequency (Table I I I ) . Thus, the SK(ad-3A) mutat ion does not appear to a f f e c t the v i a b i l i t y of ad-3A-conta i n i ng c o n i d i a . DISCUSSION The ad-3A mutat ion i n Neurospora crassa behaves as a r ece s s i ve ascospore l e t h a l , i . e . , crosses homozygous f o r ad-3A produce mainly i n v i a b l e unpigmented ascospores, but most ascospores from a cross be-tween ad-3A and i t s w i l d type a l l e l e ad-3A + , are pigmented and v i a b l e . This chapter repor t s the i s o l a t i o n of a mutat ion wh ich , when pa i red i n a cross w i th an ad-3A mutant, causes the abo r t i on of ad-3A-conta i ni ng ascospores. The new mutat ion was c a l l e d spore k i l l e r of ad-3A, or SK(ad-3A), and was found to be l o c a t e d a t or very c l o se to the ad-3A l o cu s . Both t h i s c l o s e p rox im i t y of SK(ad-3A) to the ad-3A locus and i t s s p e c i f i c e f f e c t on the v i a b i l i t y o f on ly ad-3A-conta i ni ng asco-spores s t r ong l y suggest t ha t t h i s new mutat ion i s an a l l e l e , c on t r o l or s t r u c t u r a l , of the ad-3A l o cu s . Thus, depending on which a l l e l e ad-3A i s crossed w i t h , i t may ac t as a r e ce s s i v e or an autonomous ascospore l e t h a l . While many cases o f both types of l e t h a l i t y have been p re v i ou s l y de s c r i bed , both types have never before been a s soc i a ted w i th the same mutat ion . The l e t h a l i t y of the ad-3A mutations s u p e r f i c i a l l y resembles some cases of segregat ion d i s t o r t i o n which i n vo l ve the l e t h a l i t y of m e i o t i c 1 3 3 T a b l e I I I . A n a l y s i s o f c o n i d i a l i s o l a t e s f r o m a h e t e r o -k a r y o n between s t r a i n 917A36 ( l e u - 3 , a_, arg-1  S K ( a d - 3 A ) , ad-3B) and 2 - 1 7 - 8 2 5 a . ( a , ad-3A) Number of I s o l a t e s f r o m G e n o t y p e B e g i n n i n g o f Race Tube End of Race Tube HK * 22 22 l e u , a r g , ad 13 11 ad a 13 * W i l d t y p e due t o c o m p l e m e n t a t i o n o f t h e two n u c l e a r t y p e s . 134 products . For example, the l e t h a l i t y of S d + i n Drosoph i la (Ha r t l and H i r a i z u m i , 1976), SK i n Neurospora (Turner and Pe r k i n s , 1976), and "1_" i n Ascobolus (Makarewicz, 1966), depends on the other a l l e l e of these l o c i and on the genet i c background i n the meiocytes . S ince meiocytes tha t are homozygous f o r these a l l e l e s produce on ly v i a b l e products (ascospores or sperm), l e t h a l i t y appears to be caused by the i n t e r a c -t i o n of two d i f f e r e n t a l l e l e s a t a p a r t i c u l a r l o cu s . In c o n t r a s t , meio-cytes that are homozygous f o r ad-3A produce mainly i n v i a b l e ascospores. There fo re , the l e t h a l i t y of ad -3A-conta in ing ascospores i s apparent ly the r e s u l t o f a d e f i c i e n c y i n these ascospores. Assuming tha t SK(ad-3A) i s an a l l e l e of the ad-3A l o c u s , l e t h a l -i t y o f ad -3A -conta in ing ascospores would be caused by a f a i l u r e of the a l t e r e d SK(ad-3A) gene or gene product to complement the d e f i c i e n c y i n these ascospores. S ince complementation i s normal i n vege ta t i ve he te ro -karyons, the reduced a b i l i t y to complement the d e f i c i e n c y i s r e s t r i c t e d to the ascus. Several mechanisms f o r such reduced complementing a b i l i t y of the SK(ad-3A) gene can be v i s u a l i z e d . The mutat ion SK(ad-3A) cou ld be w i t h i n the s t r u c t u r a l ad-3A gene, and produce an a l t e r e d po l ypept ide w i t h a d i f f e r e n t i a l l y low ac -t i v i t y i n the ascus and ascospores. Such reduced a c t i v i t y might a f f e c t the maturat ion of ad -3A-conta in ing ascospores i f a s p e c i f i c t h re sho ld l e v e l of a c t i v i t y would be requ i red p r i o r to ascospore enc l o su re . This t h re sho ld l e v e l would not be e a s i l y reached i n the case of the r e l a -t i v e l y i n a c t i v e SK(ad-3A) gene product . A s i m i l a r model has been p ro -posed to e xp l a i n some i r r e g u l a r fea tu res of segregat ion d i s t o r t i o n i n Drosoph i la (Mik los and Smith-White, 1971). 135 The l o s t a b i l i t y o f SK(ad-3A) to complement ad-3A i n the ascus cou ld a l s o be caused by changes i n the c o n t r o l of enzyme s y n t h e s i s , i t s m o d i f i c a t i o n , s t a b i l i z a t i o n , or an a l t e r e d property of t r a n s f e r through the cytoplasm i n the ascus. The mutat ion cys-3 • (Murray, 1965) i n Neurospora may be an example o f a de f ec t i n t r a n s f e r i n a s c i . I t l acks a permease, and i s the on ly c y s t e i n e - r e q u i r i n g mutat ion which produces main ly l i g h t i n v i a b l e ascospores. The SK(ad-3A) mutat ion represents a unique s i t u a t i o n which may enable the study of d i f f e r e n t processes i n the development o f ascospore enc losure and matu ra t i on . In more general terms, t h i s s i t u a t i o n r e -sembles the c r i t i c a l s tep o f determinat ion i n the development of two types of c e l l s ' (here ascospores) from a s i n g l e type. The re fo re , the study of t h i s process may c o n t r i b u t e to the understanding of the p ro -cess of d i f f e r e n t i a t i o n of c e l l types dur ing development i n m u l t i -c e l l u l a r organisms. Such ana l y s i s i n t h i s system i s f a c i l i t a t e d by the knowledge of seve ra l components. F i r s t , ascospore abo r t i on can not be due to a l ack of adenine i n the ascospores because abo r t i on has not been a s soc i a ted w i th other adenine r e q u i r i n g mutants ( e . g . , I sh ikawa, 1962). Abor t i on may be i n d i r e c t l y caused by the ad-3A muta t i on . For example, the accumulat ion of an i n te rmed ia te (AIR) i n the pur ine syn-: t h e t i c pathway i n ad-3A 'mutants may be the d i r e c t cause of abo r t i on ( F i s h e r , 1969a). Mutants i n the same enzyme i n Schizosaccharomyces show a c o r r e l a t i o n between the accumulat ion o f the polymer of AIR, the accumulat ion o f red pigment, and:a s l i g h t decrease i n growth r a t e . Both the red pigmentat ion and reduced growth r a te can be e l im i na ted by a secondary mutat ion l o ca ted i n the pur ine pathway before the 136 format ion of the i n te rmed ia te AIR (Gutz e t a l _ . , 1974). These f i nd i n g s suggest that s i m i l a r secondary mutations should be ab le to suppress the ascospore l e t h a l i t y , i f the accumulat ion of AIR polymers i s the d i r e c t cause o f the l e t h a l i t y . Second, the enzyme i nvo l ved i n the p r i -mary de fec t has been p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d ( F i s h e r , 1969b). There fo re , any a l t e r e d p r o p e r t i e s o f the enzyme i n the SK(ad-3A) mutant cou ld be determined.. F i n a l l y , genet i c means of a na l y s i s are a v a i l a b l e . For example, more d e t a i l e d mapping of the SK(ad-3A) mutat ion may l o c a t e i t w i t h i n the con t r o l o r s t r u c t u r a l pa r t of the ad-3A l o cu s . In a d d i t i o n , mutations which i n t e r a c t w i t h SK(ad-3A) to modify the abor-t i o n phenotype should be r e a d i l y o b t a i n a b l e . CHAPTER IV GENERAL DISCUSSION 137 The previous chapters have desc r ibed the development arid succe s s fu l use of a system i n Neurospora c ra s sa which f a c i l i t a t e s the i s o l a t i o n of r e ce s s i v e mutants w i t h a de fec t i n ascus or v i a b l e ascospore f o rmat i on . Subsequent c y t o l o g i c a l and gene t i c observat ions have shown tha t some of these mutants have t h e i r de fec t dur ing me i -o s i s . These m e i o t i c mutants are of p a r t i c u l a r i n t e r e s t to the study of the genet i c c on t r o l of m e i o s i s , and may u l t i m a t e l y help i n g a i n -ing a complete understanding of the molecu la r processes tha t c on t r o l me io s i s . Genetic Contro l of the Sexual Cycle ( I nc lud ing Meios i s ) . i n NeiirospOra  c ras sa The sexual c y c l e i n Neurospora i nvo l ve s the development of p e r i t h e c i a , m e i o s i s , and the format ion and maturat ion of ascospores. Mutants w i th de fect s i n each of these events have been detected dur ing t h i s s tudy. These mutants have a l ready con t r i bu ted to our under-s tand ing of the gene t i c c o n t r o l o f the sexual c y c l e i n Neurospora. Thus, i t was shown tha t the development of p e r i t h e c i a i s p r i m a r i l y c o n t r o l l e d by genes of the maternal parent . In add i t ion, , the study of mutants w i th an apparent de fec t i n the maturat ion of ascospores sug-gest t ha t the reaching of a th re sho ld amount of a p a r t i c u l a r substance w i t h i n the ascus (a sc -5 ; Chapter I ) , or w i t h i n each nucleus (SK(ad-3A); Chapter I I I ) was e s s e n t i a l f o r the maturat ion of ascospores. F i n a l l y , the study o f two types of mutants has prov ided some i n s i g h t i n t o the con t r o l of m e i o t i c d i v i s i o n s i n Neurospora. Thus, the apparent primary 133 de fec t i n p a i r i n g and exchange i n mutants a s c - 1 , a s c - 6 , and mei-1 appears to be f o l l owed by the equat iona l centromere d i v i s i o n of many or a l l un i va len t s produced dur ing the f i r s t prophase. A second type of mutant (asc-3) w i th a p a r t i a l b lock before karyogamy, has d e f e c t i v e attachment o f chromosomes to one of the two s p i nd l e pole bodies o f the second m e i o t i c d i v i s i o n . These observat ions may mean tha t a product produced before karyogamy ( po s s i b l y dur ing the p r e -me i o t i c S phase) l a t e r f a c i l i t a t e s the attachment o f one se t o f chromatids to one of the s p i nd l e pole bodies (some p o s s i b l e models have been desc r ibed i n the d i s cu s s i on of Chapter I I ) . Even though, a t t h i s s t age , the i n fo rmat i on obta ined from these mutants i s s p e c u l a t i v e , the use of such mutants appears qu i t e promis ing i n Neurospora. Future research should i n vo l ve the i s o l a t i o n of t empera tu re - sen s i t i v e mutants, and of mutants t ha t i n t e r a c t to change the phenotype of e x i s t i n g mutants. In a d d i t i o n , the i d e n t i f i -c a t i on of the mo lecu la r nature of the de fec t of mutants w i th c y t o -l o g i c a l l y and g e n e t i c a l l y de f ined de fec t s may become po s s i b l e through the use of e l e c t r o n microscopy or b iochemis t ry (as was p r e v i ou s l y men-t ioned i n the I n t r o d u c t i o n ) . I s o l a t i o n of Temperature-Sens i t i ve Mutants Temperature - sens i t i ve mutants may be in s t rumenta l i n d e t e r -mining the temporal pe r i od dur ing which t h e i r normal product i s a c t i v e , and i n i d e n t i f y i n g t h a t product. Such m e i o t i c mutants have a l ready V . been obta ined i n Drosoph i la ( G r e l l , 1978) and yeas t (Roth, 1976; Espos i to and E spo s i t o , 1974). In the absence of a d i r e c t s e l e c t i o n method f o r these mutants, l a r ge numbers of PWT c u l t u r e s should be 139 screened f o r t empera tu re - sen s i t i v e d e f e c t s . However, the i s o l a t i o n of many PWT c u l t u r e s i s s t i l l q u i t e time-consuming because o f the low PWT frequency of the cross used to i s o l a t e these c u l t u r e s . The present s e l e c t i o n system may be improved i n seve ra l d i f -f e r e n t ways, a l l i n v o l v i n g crosses w i th increased PWT f r equenc i e s . The nature of the s e l e c t i o n system f o r r e ce s s i v e m e i o t i c mutat ions requ i re s t ha t the PWT c u l t u r e s should be p r i m a r i l y d i somic f o r LG I on l y . Be-cause of the high frequency of m u l t i p l e disomies obta ined from crosses homozygous f o r a s c - 3 , asc-6 or me i - 1 , PWT c u l t u r e s from these crosses are not u s e f u l . However, some crosses homozygous f o r asC-1 w i th an i n -termediate PWT frequency (about 5%) might be of use i f the nond i s junc -t i o n frequency of chromosomes other than LG I i s a l s o low. S ince crosses homozygous f o r t h i s mutat ion u s ua l l y produce about 40% ascospore abor-t i o n , i t may not be po s s i b l e to i s o l a t e c e r t a i n types of mutat ions s i m i -l a r to asc-1 and asc-6 (the double mutant between asc-1 and asc-6 i s i n d i s t i n g u i s h a b l e from asc-1 and asc-6 s e p a r a t e l y ) . To overcome t h i s d i f f i c u l t y , severa l other systems may be used. . F i r s t , PWT progeny may be obta ined from a cross heterozygous f o r the dominant m e i o t i c mutant Mei-2 (Smith, 1975). S ince crosses i n v o l v -ing Mei-2 produce about 40% ascospore a b o r t i o n , on ly PWT progeny con-t a i n i n g M e i - 2 + should be s e l e c ted and t e s t e d . Th is may be achieved by s e l e c t i n g aga in s t an auxot roph ic mutat ion c l o s e l y l i n k e d to the Mei-2 l o cu s . The success of t h i s system depends on the frequency of PWT progeny tha t can be ob ta ined . Second, a mutant wi th a t empera tu re - sen s i t i v e de f ec t l ead ing to PWT format ion would be extremely u s e f u l . PWT c u l t u r e s could be obta ined 140 from crosses a t one temperature and te s ted f o r mutants a t the other temperature. T h i r d , the frequency of PWT progeny can be increased markedly w i t h the use of the chemical p - f l uo ropheny l a l an i ne ( G r i f f i t h s and DeLange, 1977). I n t e r a c t i n g Mutat ions I t may be a n t i c i p a t e d t ha t mutat ions which i n t e r a c t w i th e x i s -t i n g m e i o t i c mutations w i l l become p a r t i a l l y i n s t rumenta l i n the un-derstanding of m e i o t i c development and i t s c o n t r o l . In the pa s t , i n t e r a c t i o n - t y p e mutants have indeed been q u i t e u s e f u l . A r e l e van t example i s the study of recombinat ion i n E_. c o l i . F i r s t , suppressors sbc A and sbc B of the recomb ina t i on -de fec t i ve mutants rec B and rec C have i n a c t i v e exonuclease VI I I and exonuclease I, r e s p e c t i v e l y (Barbour and C l a r k , 1970; Kushner e t a]_., 1972). Second, four mutant genes r e s u l t i n recombinat ion d e f i c i e n c y i n the reve r ted rec B rec C  sbc B s t r a i n ( H o r i i and C l a r k , 1973). These i n t e r a c t i o n mutants have prov ided some i n s i g h t i n t o the a l t e r n a t i v e means (or pathways) of r e -combination i n E_. co l i . In t h i s manner, three pathways have been de-tec ted ( C l a r k , 1974). Although many other examples of i n t e r a c t i n g mutants have been in s t rumenta l i n the understanding of c e l l u l a r p ro -ces ses , the example of recombinat ion con t r o l i n E_. c o l i i s most c l o s e l y r e l e v a n t to the c on t r o l of recombinat ion and meios i s i n eu-k a r y o t i c organisms. The re fo re , i t should be a n t i c i p a t e d t ha t s i m i l a r s tud ie s i n severa l euka r yo t i c organisms ( e . g . , Neurospora) may enable the succes s fu l d i s s e c t i o n of recombinat ion and m e i o t i c processes . 141 Second- s i te reve r s i on s may be dominant or r e c e s s i v e . I t w i l l be v i r t u a l l y impos s ib le to obta in r ece s s i ve suppressor mutat ions of s t e r i l i t y mutants i n Neurospora ( e . g . , a s c - 2 , a s c -4 , me i - 3 , uvs -3 , uvs -5, uvs-6) because PWT c u l t u r e s needed to screen f o r such mutations cannot be obta ined from crosses homozygous f o r s t e r i l i t y mutat ions . However, the i s o l a t i o n of r e ve r t an t s would be po s s i b l e i n c o n d i t i o n a l ( e . g . , t empera tu re - sen s i t i v e ) mutants: PWT c u l t u r e s cou ld be obta ined at one temperature and the sc reen ing would be performed a t the r e -s t r i c t i v e temperature. The i s o l a t i o n of r e ve r t an t s of mutations w i th a de f ec t i n . p a i r i n g or exchange, and t he re fo re chromosome d i s j u n c t i o n , i s poten-t i a l l y p o s s i b l e , s i n c e PWT f requenc ie s i n such mutants are u s u a l l y h i gh . The i s o l a t i o n of dominant r eve r t an t s would be even s imp le r because mutagenized c o n i d i a , i n s tead of PWT c u l t u r e s , can be screened. In a d d i t i o n , v a r i a n t s obta ined from nature or i n e x i s t i n g l abo ra to r y stocks may a l s o modify the phenotype of m e i o t i c mutat ions . The high frequency of b lack ascospores obta ined i n some crosses homozygous f o r asc-3 (P393) may p o s s i b l y be an example of t h i s . F i n a l l y , mutations t ha t i n t e r a c t w i t h the spore k i l l e r muta-t i o n SK(ad-3A) should be r e a d i l y ob ta ined . S ince the mutat ion SK(ad-3A) apparent ly causes a de fec t i n complementation, i t i s q u i t e p l a u s i b l e t ha t c e r t a i n other components i n t e r a c t to achieve such complementation. In e f f e c t , a l t e r a t i o n of one or more of these i n t e r a c t i n g components could a c t as a suppressor of SK,. and .thus r e s t o r e the a b i l i t y of SK to complement ad-3A mutat ions . The study of such i n t e r a c t i o n mutat ions may prov ide i n s i g h t i n the developmental steps i n v o l v e d . In t h i s case, de tec t i on of suppress ion may be a f a s t procedure s i nce thousands of a s c i could be q u i c k l y scored f o r the presence or absence of most ly b lack ascospores. P o t e n t i a l of Future S tud ies on the Mo lecu la r or M i c r o - S t r u c t u r e Level The u l t ima te understanding of m e i o t i c development n e c e s s a r i l y i n vo l ve s b iochemica l c h a r a c t e r i z a t i o n . Because o f the asynchrony of development of a sc i and t h e i r attachment to vege ta t i ve c e l l s , such ana l y s i s i s p re sen t l y not p o s s i b l e i n Neurospora. However, some genet i c man ipu la t ion and development of techniques tha t a l l ow the separ a t i o n of a sc i from surrounding c e l l s should l a r g e l y overcome t h i s p ro -blem. The p a r t i a l s ynch ron i za t i on o f m e i o t i c development has been achieved by temperature shock i n Coprinus lagopus (Lu, 1974; Lu and Jeng, 1975), or by a temporary i n h i b i t i o n of DNA synthes i s i n S ch i z o - phyllum commune (Carmi et a l _ . , 1978). The same may po s s i b l y be achieved by c e r t a i n mutat ions , e . g . , Ban i n Neurospora, which causes a l t e r n a t e waves o f m e i o t i c and m i t o t i c c y c l e s (Raju and Newmeyer, 1977) 143 BIBLIOGRAPHY Baker, B. S., 1975. 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S e x u a l i t y i n Neurospora c r a s s a . I I . Genes a f f e c t i n g the sexual development c y c l e . Genet. Res., Camb. 19:205-211. We i j e r , J . , and N. V. V i g fu s sen , 1972. S e x u a l i t y i n Neurospora c r a s s a . I. Mutat ions to male s t e r i l i t y . Genet. Res., Camb. 19:191-204. Westergaard, M., and H. K. M i t c h e l l , 1947. Neurospora. V. A s y n t h e t i c medium favour ing sexual r ep roduc t i on . Am. J . Bot. 34:573-577. Zimmering, S., L. Sand le r , and B. N i c o l e t t i , 1970. Mechanisms o f me i o t i c d r i v e . Ann. Rev. Genet. 4:409-436. 152 APPENDIX LIST OF ABBREVIATIONS asc Recess ive mutat ion r e s u l t i n g i n the abo r t i on of a s c i and/or ascospores. CO Crossover. HK Heterokaryon or h e t e r o k a r y o t i c . HN2 Nit rogen mustard. LG Linkage group. MI F i r s t me i o t i c d i v i s i o n . M i l Second me i o t i c d i v i s i o n . mei M e i o t i c mutat ion . MMS Methyl methane su lphonate. MNNG N-methyl N -n i t r o N -n i t ro soguan id ine . PWT Pseudo-wi ld type. RF Recombinant f requency. SK Spore k i l l e r mutat ion . SPB Sp ind le pole body. UV U l t r a v i o l e t . PUBLICATIONS A.J.F.Griffiths, A.M.DeLange and J.H.Jung, 1974. Identification of a complex chromosome rearrangement in Neurospora crassa. Can.J.Genet. Cytol. 16: 805-822. A.M.DeLange and A.J.F.Griffiths, 1975. Escape from mating-type incom-p a t i b i l i t y in bisexual (A+a) Neurospora heterokaryons. Can.J.Genet. Cytol. 17: 441-449. A.M.DeLange, 1975. Studies of bisexual (A+a.) strains of Neurospora crassa. M.Sc. Thesis, University of British Columbia. A.J.F.Griffiths and A.M.DeLange, 1977. p-Fluorophenylalanine increases meiotic nondisjunction in a Neurospora test system.Mutation Research 46: 345-354. ,1978. Mutations of the a. mating type gene in Neurospora crassa. Genetics 88k 239-254. A.M.DeLange, 1980. Meiosis in Neurospora crassa I. The isolation of recessive mutants defective in the production of viable ascospores. (Submitted). A.M.DeLange, 1980. Meiosis in Neurospora crassa II. Genetic and cyto-logical characterization of four meiotic mutants. (Submitted). A.M.DeLange, 1980. The mutation Sk(ad-3A) alters the dominance of ad-3A+ over ad-3A in the ascus of Neurospora. (Submitted). 

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