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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 o y s i u s M. DeLange B . S c . , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1973 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1975  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department o f Botany ( G e n e t i c s )  We a c c e p t t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA May 1980 ©  A l o y s i u s M. DeLange, 1980  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Department o r by h i s r e p r e s e n t a t i v e s .  I t i s understood t h a t c o p y i n g or p u b l i c a t i o n  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written  permission.  Department o f  BOTANY  f  The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  Date .  &£NB1\C^)  ABSTRACT The study o f the g e n e t i c c o n t r o l o f m e i o s i s has been  ini-  t i a t e d i n Neurospora c r a s s a by the i s o l a t i o n of r e c e s s i v e m e i o t i c mutations.  These m u t a t i o n s were d e t e c t e d by t h e i r reduced f e r t i l i t y  or by the a b o r t i o n o f a s c o s p o r e s .  To a l l o w t h e i r e x p r e s s i o n , 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 were made homozygous by s e l e c t i n g (n + 1) disomic ascospores.  C u l t u r e s produced by each of these  ascospores  c o n t a i n two types o f n u c l e i w i t h i d e n t i c a l genes ( i n c l u d i n g m u t a t i o n s ) on a l l chromosomes e x c e p t l i n k a g e group (LG) I, mating type l o c u s .  which c o n t a i n s the  The s i m u l t a n e o u s presence o f these two types of  n u c l e i a l l o w s the i n i t i a t i o n of the s e x u a l c y c l e , and t h e r e f o r e the d e t e c t i o n o f r e c e s s i v e m u t a t i o n s a f f e c t i n g the s e x u a l c y c l e , i n c l u d i n g m e i o s i s , on any chromosome e x c e p t LG I. c l a s s e s o f mutants have been d e t e c t e d .  Using t h i s method, t h r e e major F i r s t , e l e v e n mutants  affec-  t i n g p e r i t h e c i a l development were e x p r e s s e d o n l y as the m a t e r n a l parent.  Second, t h i r t e e n mutants produced p e r i t h e c i a w i t h few o r no  ascospores.  The i n f e r t i l i t y i n two o f these mutants was d e f i n i t e l y  caused by r e c e s s i v e m u t a t i o n s ( a s c - 2 and a s c - 4 ) .  F i n a l l y , the a b o r -  t i o n of many ascospores was d e t e c t e d 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 c e s s i v e m u t a t i o n s ( a s c - 1 , a s c - 3 , a s c - 5 , a s c - 6 , a s c - 7 , and a s c - 8 ) caused the a b o r t i o n o f many a s c o s p o r e s .  The  dominant m u t a t i o n SK(ad-3A) was d e t e c t e d i n t h i s s c r e e n f o r r e c e s s i v e m u t a t i o n s , because i t caused ascospore a b o r t i o n when c r o s s e d w i t h an ad-3A mutant but not w i t h a w i l d t y p e s t r a i n .  This mutation, appar-  e n t l y a l l e l i c t o ad-3A, caused the a b o r t i o n o f a l l a d - 3 A - c o n t a i n i n g ascospores.  ii  The t h r e e ascospore a b o r t i o n - t y p e mutants a s c - 1 , a s c - 3 , and a s c - 6 were a n a l y z e d i n more d e t a i l methods.  u s i n g both c y t o l o g i c a l and g e n e t i c  Ascospore a b o r t i o n i n these mutants was caused by abnormal  d i s j u n c t i o n o f m e i o t i c chromosomes.  In mutants a s c - 1 and a s c - 6 ,  the  p r i m a r y d e f e c t i n p a i r i n g o f homologs d u r i n g the f i r s t m e i o t i c prophase was f o l l o w e d by the f o r m a t i o n o f u n i v a l e n t s a t metaphase I.  Observa-  t i o n s on these mutants and on the mei-1 mutant ( p r e v i o u s l y i s o l a t e d ; see S m i t h , 1975) suggested e q u a t i o n a l centromere d i v i s i o n o f many u n i v a l e n t s a t anaphase o f chromosomes  I.  Subsequent  i r r e g u l a r and p r o l o n g e d  separation  a t the second m e i o t i c d i v i s i o n appeared t o be a s e c o n -  dary e f f e c t o f t h e abnormal f i r s t d i v i s i o n .  The a s c - 3 mutant had a  d e f e c t i n ascus f o r m a t i o n , and l a t e r i n d i s j u n c t i o n d u r i n g the second m e i o t i c and p o s t - m e 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 d e f e c t b e f o r e o r  d u r i n g karyogamy r e s u l t e d i n the a b o r t i o n of most c e l l s . managed t o proceed p a s t t h i s b l o c k . most chromosomes  Some c e l l s  During the second m e i o t i c d i v i s i o n  o f the few r e s u l t i n g a s c i were a t t a c h e d t o o n l y one  o f the two s p i n d l e - p o l e b o d i e s .  D i s j u n c t i o n a t the p o s t - m e i o t i c d i v i -  s i o n was a l s o h i g h l y i r r e g u l a r .  T h i s mutant appeared t o be d e f e c t i v e  i n the attachment o f one s p i n d l e - p o l e body t o a s e t of c e n t r o m e r e s . The d e f e c t may i n v o l v e e i t h e r a c e n t r o m e r e - a s s o c i a t e d p r o d u c t or a spindle-pole  body.  TABLE OF CONTENTS Page ABSTRACT  ii  TABLE OF CONTENTS —  —  LIST OF TABLES —  iv  -  LIST OF FIGURES  v  -  ACKNOWLEDGEMENT -  —  vii  -  vi i i  PREFACE  ix  INTRODUCTION CHAPTER I  \ M e i o s i s i n Neurospora c r a s s a . I. The I s o l a t i o n o f R e c e s s i v e Mutants D e f e c t i v e i n t h e P r o d u c t i o n o f V i a b l e Ascospores  1  5  INTRODUCTION  16  MATERIALS AND METHODS  -  21  RESULTS  29  DISCUSSION CHAPTER II  -  -  -  36  M e i o s i s i n Neurospora c r a s s a . I I . G e n e t 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 o f Four M e i o t i c Mutants INTRODUCTION  — — — -  RESULTS DISCUSSION —  -  The M u t a t i o n SK(ad-3A)  4  — —  MATERIALS AND METHODS  CHAPTER I I I  a  42 — —  43  —  57  -  99  A l t e r s the Dominance o f  ad-3A+ Over ad-3A i n t h e Ascus o f Neurospora INTRODUCTION  1  —  -  CHAPTER IV  General D i s c u s s i o n  BIBLIOGRAPHY  —  APPENDIX:  L i s t of Abbreviations  -  —  iv  6  a  -  119 123  —  -  -  1  117  MATERIALS AND METHODS RESULTS DISCUSSION  l a  —  132 1  3  6  a  143 152  LIST OF TABLES Table  Page  CHAPTER I I II  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 type c u l t u r e s with aberrant crossing behavior  30  The phenotypes o f r e c e s s i v e c l a s s II 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 type a l l e l e s a r e n e c e s s a r y f o r the f o r m a t i o n o f normal a s c i or b l a c k a s c o s p o r e s  35  CHAPTER  II  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  59  II  Ascus a n a l y s i s o f a c r o s s homozygous f o r a s c - 6 ( 8 7 9 a l 3 x 879A15)  61  I s o l a t e s o b t a i n e d from a s c i , produced by a c r o s s ( 8 7 9 a l 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 PWT c u l t u r e •  62  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 groups two c r o s s e s homozygous f o r a s c - 6 •  64  III  IV V VI  Progeny 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 or P393) — —  in  (P243 69  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 types o f a p p a r e n t l y PWT progeny from c r o s s e s homozygous f o r asc-3  71  VII  Ascus 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  76  VIII  Recombination 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  82  Nature o f growth o f ascospores from c r o s s e s f o r asc-1  83  IX X  XI  homozygous  Ascus a n a l y s i s o f a c r o s s homozygous f o r the 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 (P95) (95A29 x 95a43)  86  C h a r a c t e r i s t i c s of f o u r r e c e s s i v e m e i o t i c mutations i n Neurospora c r a s s a w i t h a d e f e c t i n the r e g u l a r d i s j u n c t i o n o f chromosomes  100  v  <>  Table  Page  CHAPTER I II  III  III  A n a l y s i s o f i s o l a t e s from c r o s s e s and OR-a w i l d type s t r a i n s  between P917 t o OR-A 124  Genotype o f 79 i s o l a t e s from the 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) which produced about 60% a b o r t e d ascospores  127  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 from a h e t e r o k a r y o n b e tween s t r a i n 917A36 ( l e u - 3 , a_, a r g - 1 , S K ( a d - 3 A ) , ad-3B) and 2-17-825a ( a , a d - I A T ^ -  133  vi  LIST OF FIGURES Figure  Page  INTRODUCTION 1.  R e p r e s e n t a t i o n o f m e i o s i s as a s p e c i a l i z e d form o f the mitotic cycle •  6  CHAPTER I 1.  General o u t l i n e o f a s e l e c t i v e system used t o i s o l a t e r e c e s s i v e m e i o t i c mutants i n Neurospora c r a s s a  CHAPTER 1. 2.  3.  22  II  The two n u c l e a r components o f PWT c u l t u r e s i n which asc m u t a t i o n s were r e c o v e r e d  44  A c r o s s homozygous f o r a s c - 6 , d e s i g n e d t o t e s t 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 groups (LG I, IV, and V) simultaneously  46  Expected PWT progeny r e s u l t i n g from n o n d i s j u n c t i o n d u r i n g 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 r o s s o v e r f o l l o w e d by n o n d i s j u n c t i o n d u r i n g the second m e i o t i c d i v i s i o n (b)  -•  4.  Chromosome development i n c r o s s e s  homozygous  5.  The o r i g i n o f PWT progeny from c r o s s e s  52 f o r asc-6  homozygous  66  for  asc-3  72  6.  Chromosome development i n c r o s s e s  homozygous  f o r asc-3  78  7.  Chromosome development i n c r o s s e s  homozygous  f o r asc-1  88  8.  Chromosome development i n c r o s s e s  homozygous  f o r mei-1  93  9.  P o s s i b l e e x p l a n a t i o n f o r abnormal d i s j u n c t i o n i n mutant c r o s s e s •  CHAPTER  asc-3 •  114  III  1.  The t w o - n u c l e a r components o f s t r a i n P917  120  2.  S e l e c t i o n o f ad_ recombinants t o l o c a l i z e the SK(ad-3A) m u t a t i o n w i t h r e s p e c t t o the ad-3A and ad-3B l o c i •  130  +  vii  ACKNOWLEDGEMENT I e x p r e s s my s i n c e r e g r a t i t u d e t o my r e s e a r c h 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 a n c e throughout t h i s  study.  I thank Drs. C. 0. P e r s o n , K . C o l e , D. Holm, and J . Berger f o r s e r v i n g on my Committee.  vii i  PREFACE T h i s t h e s i s i s composed o f f o u r p a r t s p l u s an i n t r o d u c t i o n . The f i r s t t h r e e c h a p t e r s a r e p r e s e n t e d i n a form s u i t a b l e f o r sequent p u b l i c a t i o n .  sub-  Chapter I d e s c r i b e s the use o f a new system  i n Neurospora c r a s s a t h a t enables the i s o l a t i o n o f r e c e s s i v e mutants d e f e c t i v e i n the p r o d u c t i o n o f v i a b l e a s c o s p o r e s .  The g e n e t 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 o f some o f these mutants has been d e s c r i b e d i n Chapter I I .  Chapter III  d e s c r i b e s the study o f an a s c o -  spore a b o r t i o n - t y p e mutant which s p e c i f i c a l l y a b o r t s ascospores.  The f i n a l  chapter i s a general d i s c u s s i o n  ad-3A-containing and c o n c e n t r a t e s  on the f u t u r e approaches and uses o f these types o f mutants o f Neurospora.  ix  1  INTRODUCTION  2  T h i s study was i n i t i a t e d to g a i n a b e t t e r i n s i g h t i n t o the genetic control of meiosis.  The fungus Neurospora c r a s s a  appeared  i d e a l l y s u i t e d to conduct such r e s e a r c h because both c y t o l o g i c a l and g e n e t i c means o f a n a 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 s p e c 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 t h a t would enable the i s o l a t i o n o f r e c e s s i v e m e i o t i c mutations.  T h e r e f o r e , I have d e v i s e d a system t h a t e n a b l e s the r a p i d  s c r e e n i n g o f s e l e c t e d c u l t u r e s f o r such m u t a t i o n s . use o f t h i s system w i l l  The s u c c e s s f u l  be d e s c r i b e d i n Chapter I o f t h i s  A c o m b i n a t i o n o f c y t o l o g i c a l and g e n e t i c a n a l y s e s  thesis.  of these mutations  s h o u l d be i n s t r u m e n t a l i n g a i n i n g a more complete u n d e r s t a n d i n g the g e n e t i c c o n t r o l o f m e i o s i s .  of  The p r i n c i p a l f e a t u r e s t h a t r e n d e r  the c h a r a c t e r i z a t i o n o f m e i o t i c mutants so e f f e c t i v e i n  Neurospora  are g i v e n below.  Cytological  Analysis  The chromosomes,  n u c l e o l i , s p i n d l e 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 o w e d d u r i n g a l l stages o f m e i o t i c development, s t a r t i n g b e f o r e karyogamy u n t i l  ascospore f o r m a t i o n ( M c C l i n t o c k ,  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). f o u r t e e n (- 2N) chromosomes  individualize shortly after  AIT:  karyogamy.  During zygotene and pachytene o f the f i r s t prophase, these chromosomes e l o n g a t e and the homologs  pair.  T h i s i s u s u a l l y f o l l o w e d by two  r a p i d m e 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 e n c l o s u r e o f each nucleus i n an a s c o s p o r e .  The chromosomes  individualize  3  a t each prophase. o f chromosomes  Thus i t i s o f t e n p o s s i b l e t o determine the number  per nucleus j u s t p r i o r t o each d i v i s i o n , and t h e r e f o r e  any d e f e c t ( f o r example, caused by a m u t a t i o n ) i n the r e g u l a r gation of Genetic  segre-  chromosomes.  Analysis S e v e r a l a s p e c t s o f the l i f e c y c l e o f Neurospora c r a s s a  render  i t i d e a l f o r both the d e t e c t i o n and g e n e t i c c h a r a c t e r i z a t i o n of m e i o t i c mutations: (1)  Whole p o p u l a t i o n s o f a s c i can be q u i c k l y scanned and a l l v i d u a l products o f m e i o s i s  (2)  ( i . e . , ascospores)  Most v i a b l e and i n v i a b l e ascospores basis of c o l o r :  v i a b l e ascospores  i n v i a b l e ascospores  indi-  are r e c o v e r a b l e .  can be d i s t i n g u i s h e d on the a r e i n v a r i a b l y b l a c k , whereas  are o f t e n w h i t e .  This d i s t i n c t i o n i s  espec-  i a l l y u s e f u l because i t enables the d e t e c t i o n and c h a r a c t e r i z a t i o n o f mutants w i t h a d e f e c t i n the d i s j u n c t i o n of Such d i s j u n c t i o n d e f e c t s would produce ascospores miss one o r more chromsome(s)  chromosomes.  which e i t h e r  or have e x t r a chromosomes.  The  former type o f ascospore:;would be w h i t e and the l a t t e r b l a c k ( M c C l i n t o c k , 1945; Coyle and P i t t e n g e r , (3)  1965).  The apparent v i a b i l i t y o f a n e u p l o i d p r o d u c t s  (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 o f mutants w i t h d i s j u n c t i o n defects.  A n e u p l o i d p r o d u c t s can be d e t e c t e d by the complemen-  t a t i o n o f m u t a t i o n s ( u s u a l l y a u x o t r o p h i c ) on two c o p i e s o f a chromosome.  homologous  They a r e c a l l e d p s e u d o - w i l d t y p e , or  PWT, because, even though they c o n t a i n mutant n u c l e i , they  4  appear t o be w i l d t y p e .  N o n d i s j u n c t i o n can be d e t e c t e d a t  both the f i r s t and second d i v i s i o n p r o v i d e d the chromosome t e s t e d 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 o f t h i s i s g i v e n i n M a t e r i a l s and Methods o f Chapter II this (4)  thesis).  A l l products  ( a s c o s p o r e s ) from each p a r t i c u l a r m e i o s i s  be s t u d i e d .  The p a t t e r n s o f w h i t e and b l a c k ascospores  each a s c u s , and the a n a l y s e s black ascospores  can in  o f genotypes o f the v i a b l e  may p r o v i d e i n f o r m a t i o n on the n a t u r e o f  i r r e g u l a r d i s j u n c t i o n of (5)  of  chromosomes.  The a v a i l a b i l i t y o f s p e c i a l l i n k a g e t e s t e r s t r a i n s makes the mapping o f new m u t a t i o n s a r e l a t i v e l y f a s t p r o c e d u r e i n Neurospora c r a s s a  ( P e r k i n s and Bjorkman, 1979).  Mapping  of  m u t a t i o n s w i t h known d e f e c t s would a l l o w the d e t e c t i o n o f gene c l u s t e r s of related functions. The use o f the g e n e t i c and c y t o l o g i c a l a n a l y s i s mutants appears e x c e l l e n t i n Neurospora.  of meiotic  However, even though such  a n a l y s i s would p r o v i d e i n f o r m a t i o n on the c o n t r o l o f the development and movement o f macromolecular aggregates ( e . g . , chromosomes,  chroma-  t i d s , and s p i n d l e - p o l e b o d i e s ) , i t would p r o b a b l y not p r o v i d e any d e t a i l e d u n d e r s t a n d i n g o f m o l e c u l a r processes these a g g r e g a t e s . analysis  t h a t c o n t r o l the a c t i o n o f  The u n d e r s t a n d i n g o f these processes would r e q u i r e  by means o f e l e c t r o n m i c r o s c o p y and b i o c h e m i s t r y .  The p r e s e n t  work s h o u l d t h e r e f o r e be seen as a framework from which more d e t a i l e d analyses  c o u l d be c a r r i e d o u t .  chromosomes  For example, the p a i r i n g o f homologous  d u r i n g the f i r s t prophase o f m e i o s i s  has a l r e a d y been  5  s u c c e s s f u l l y s t u d i e d i n Neurospora 1972).  Thus f a r , the c h a r a c t e r i s t i c s o f t h e l i f e c y c l e o f  have made b i o c h e m i c a l a n a l y s i s will  u s i n g e l e c t r o n microscopy  of meiosis  (Gillies, Neurospora  d i f f i c u l t but such  analysis  p r o b a b l y become f e a s i b l e i n t h e near f u t u r e . B e f o r e proceeding to d i s c u s s m e i o t i c m u t a t i o n s i n g e n e r a l  the i s o l a t i o n o f them i n Neurospora some c r i t i c a l  processes  of meiosis  (see Chapter I ) ,  and  a d e s c r i p t i o n of  s h o u l d s e r v e t o focus on the main  problems t h a t need to be s o l v e d . The major landmark events o f the m e i o t i c c y c l e a r e s c h e m a t i c a l l y r e p r e s e n t e d i n F i g u r e 1.  T h i s c y c l e appears  t i o n o f the m i t o t i c c y c l e . a c t e r i s t i c of m e i o s i s :  to represent a m o d i f i c a -  The f o l l o w i n g c r i t i c a l  processes  are char-  the i n i t i a t i o n o f m e i o s i s , r e c o m b i n a t i o n o f  genes, the r e d u c t i o n a l d i v i s i o n , and the r e t u r n t o the m i t o t i c c y c l e .  I n i t i a t i o n of  Meiosis  The i n i t i a t i o n o f the m e i o t i c d i v i s i o n c y c l e appears p l a c e d u r i n g the G l p e r i o d . can be d i s t i n g u i s h e d  to take  N u c l e i t h a t have been committed to m e i o s i s  by t h e i r subsequent  r e p l i c a t i o n (see next s e c t i o n ) .  c h a r a c t e r i s t i c p a t t e r n o f DNA  However, even though the n u c l e i  are  committed t o some e a r l y m e i o t i c e v e n t 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 .  Instead,  they may, i n the a b -  sence o f the a p p r o p r i a t e g e n e t i c o r e n v i r o n m e n t a l c o n t r o l s , r e v e r t back t o the m i t o t i c c y c l e .  The processes  d i f f e r e n t m e i o t i c events w i l l  be d i s c u s s e d  which i r r e v e r s i b l y commit the i n the f o l l o w i n g  sections.  The i n i t i a t i o n o f 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 e n v i r ronmental c o n d i t i o n s , i n t e r n a l s i g n a l s m u l t i c e l l u l a r organisms,  g e n e r a t e d by development  or a combination of these.  in  In some u n i c e l l u l a r  F i g u r e 1.  R e p r e s e n t a t i o n o f m e i o s 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 . mitosis  The m i t o t i c c y c l e c o n s i s t s  (M), G I , DNA s y n t h e s i s  ( S ) , and G2.  . p o i n t d u r i n g the GI phase, m e i o s i s  of  At a s p e c i f i c  is initiated.  A choice  is  made here between c o n t i n u i n g the m i t o t i c c y c l e o r i n i t i a t i n g meiosis.  T h i s c h o i c e depends on e n v i r o n m e n t a l c l u e s .  A series  of subsequent s t e p s u l t i m a t e l y l e a d back i n t o the m i t o t i c c y c l e .  premeiotic  division  8  m i c r o o r g a n i s m s , m e i o s i s i s i n i t i a t e d when the s u b s t r a t e (food s o u r c e ) is depleted. the  In f a c t , i n the. budding y e a s t Saccharomyces c e r e v i s i a e ,  i n i t i a t i o n o f m e i o s i s i s r e p r e s s e d by n i t r o g e n and g l u c o s e .  That  t h i s r e p r e s s i o n i s under g e n i e 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 a r e 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 a r e produced which a r r e s t n u c l e i a t a p o i n t d u r i n g the GI p e r i o d (see F i g .  1).*  S u b s e q u e n t l y , m e i o s 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 o r g a n i s m s , a number o f (germ) c e l l s a r e a p p a r e n t l y e s p e c i a l l y programmed t o undergo m e i o s i s . the s t a t e o f development o f the i n d i v i d u a l w i l l signals  In these  cases,  generate the r e q u i r e d  t h a t are n e c e s s a r y f o r t h e i n i t i a t i o n o f m e i o s i s .  S i n c e these  organisms a r e g e n e r a l l y p r o t e c t e d from e n v i r o n m e n t a l changes by i n t e r n a l h o m e o s t a t i c c o n t r o l , i n i t i a t i o n s h o u l d not be dependent on e n v i r o n m e n t a l conditions.  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 m i c r o o r g a n i s m s ,  e.g.,  many f u n g i and c e l l u l a r s l i m e m o l d s , depends on e n v i r o n m e n t a l and d e velopmental s i g n a l s .  In g e n e 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 n a l s  the  i n i t i a t i o n o f the f o r m a t i o n o f f r u i t i n g . b o d i e s which i n v o l v e s a s e r i e s o f developmental s t e p s t h a t c u l m i n a t e i n the i n d u c t i o n o f m e i o s i s .  In  many f u n g i , s p e c i f i c mating type genes a r e i n v o l v e d i n the p r o d u c t i o n o f f r u i t i n g bodies ( e . g . , Fincham and Day, 1971).- However, i t i s  not  T h i s stage may c o r r e s p o n d t o G i n many m u l t i c e l l u l a r organisms s i n c e c e l l s may spend a l o n g time a t these p o i n t s w i t h o u t l o s i n g t h e i r viability. In c o n t r a s t , when the c y c l e i s a r r e s t e d a t any o t h e r p o i n t , e . g . , by means o f a t e m p e r a t u r e - s e n s i t i v e 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 d e c l i n e s as a f u n c t i o n o f i n c u b a t i o n a t the r e s t r i c t i v e t e m p e r a ture. 0  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 m e i o s i s . The study o f m u t a t i o n s w i t h d e f e c t s p r i o r t o m e i o s i s i t s e l f may become i n s t r u m e n t a l i n the u n d e r s t a n d i n g o f the g e n e t i c c o n t r o l o f the  initia-  tion of meiosis. M e i o t i c Recombination Recombination i s the c e n t r a l component o f the m e i o t i c p r o c e s s . The amount o f r e c o m b i n a t i o n can be v a r i e d i n two ways:  a change i n  chromosome number, o r a change i n c r o s s i n g - o v e r between homologous chromosomes  ( i . e . , intrachromosomal r e c o m b i n a t i o n ) .  The independent  a s s o r t m e n t o f chromosomes d u r i n g the m e i o t i c d i v i s i o n s generates new c o m b i n a t i o n s o f chromosomes  from the two p a r e n t n u c l e i .  Thus, an i n -  c r e a s e i n chromosome number i n c r e a s e s the number o f p o s s i b l e new combinat i o n s o f genes.  Intrachromosomal  r e c o m b i n a t i o n , i n v o l v i n g gene c o n v e r -  s i o n - and c r o s s i n g - o v e r o r exchange, r e q u i r e s a h i g h l y c o o r d i n a t e d sequence o f events which r e c u r d u r i n g each m e i o t i c c y c l e .  The f r e q u e n c y  o f c r o s s i n g - o v e r depends on the amount o f a c c u r a t e p a i r i n g o f homologous s t r e t c h e s o f DNA w i t h i n the chromosomes and the e f f i c i e n c y o f the e x change e v e n t s .  The r e c o v e r y o f a l a r g e number o f m u t a t i o n s w i t h a b e r -  r a n t p a i r i n g o r exchange ( f o r r e v i e w , , see Baker e t a l _ . , 1976a), i n d i c a t e t h a t these p r o c e s s e s a r e under s t r i c t g e n e t i c c o n t r o l . The temporal stages t h a t a r e e s s e n t i a l f o r the c o n t r o l o f p a i r i n g 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 t h s p e c i f i c stages o f more o r l e s s s y n c h r o n o u s l y cells.  d i v i d i n g populations of meiotic  I t has been p o s s i b l e t o . i n t e r f e r e w i t h m e i o s i s u s i n g temperature  s h o c k , 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 o f m e 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 t e r n and H o t t a ,  10  1967; 1973), o r t e m p e r a t u r e - s e n s i t i v e m u t a t i o n s  (e.g., G r e l l ,  1978).  The e f f e c t o f these m a n i p u l a t i o n s on p a i r i n g and exchange has shown t h a t processes until  t a k i n g p l a c e j u s t p r i o r t o the p r e - m e i o t i c S phase  pachytene are r e q u i r e d 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 n e c e s s a r y the o c c u r r e n c e o f r e g u l a r exchange has been d e t e c t e d i n ( C h i u and H a s t i n g s ,  1973).  for  Chlamydomonas  Recombination was i n c r e a s e d a f t e r t r e a t -  ment w i t h phenethyl a l c o h o l o r mitomycin C j u s t p r i o r t o the p r e m e 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 n c e  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 a r e s e v e r a l  l i n e s o f e v i d e n c e s u g g e s t i n g the i n v o l v e m e n t  o f p r e - m e i o t i c DNA s y n t h e s i s f u n c t i o n unique t o m e i o s i s  i n the r e c o m b i n a t i o n p r o c e s s .  First, a  i s suggested by the extended p e r i o d o f time  r e q u i r e d to complete t h i s p r e - m e i o t i c S phase.  T h i s i s a p p a r e n t l y due  t o 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 - m e 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 myces .  o f m u t a t i o n s t h a t a r e d e f e c t i v e i n the process  None o f these mutants develop synaptonemal  a x i a l cores may be produced.  In appears  i n t o synaptonemal  complexes,  Saccharoalthough  t h a t these mutants are never  committed t o p a i r i n g and m e i o t i c r e c o m b i n a t i o n . m e i o t i c DNA s y n t h e s i s  in  It follows that pre-  i s e i t h e r r e q u i r e d f o r c e n t r a l element  assembly  complexes o r t h a t both o f these events a r e s u b j e c t t o  common g e n e t 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 s y n t h e s i s  i s delayed u n t i l  zygotene when p a i r i n g takes p l a c e ( H o t t a , I t o and S t e r n , 1966; and S t e r n , 1971; S t e r n and H o t t a , 1977).  early  Hotta  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 c o m p l e t e l y b l o c k s p a i r i n g o f  homologs,  s u g g e s t i n g t h a t t h i s r e p l i c a t i o n i s n e c e s s a r y f o r the p a i r i n g  process  in Lilium.; T h i r d , temporary a p p l i c a t i o n o f x - i r r a d i a t i o n o r i n h i b i t o r s o f DNA s y n t h e s i s  d u r i n g the p r e - m e i o t i c S phase o f Chlamydomonas  i n i n c r e a s e d exchange..  resulted  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 r o s s - o v e r  frequencies in several  species  ( r e v i e w e d by S t e r n and H o t t a , 1973).  Even though the e x t e n t and d i r e c -  t i o n o f 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 i n v o l v e m e n t o f the p r e - m e i o t i c S phase i n the r e combination  process.  F o u r t h , i n Saccharomyces, strand scissions lar  o f chromosomal  commitment to exchange i n v o l v e s  DNA and a p p a r e n t l y i s a r e v e r s i b l e  singlecellu-  s t a g e a c h i e v e d d u r i n g the p r e - m e i o t i c S phase ( S i l v a - L o p e z 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 p e r i o d o f a t e m p e r a t u r e - s e n s i t i v e recomb i n a t i o n d e f i c i e n t mutant i n D r o s o p h i l a o c c u r s d u r i n g the p r e - m e i o t i c S phase  ( G r e l l , 1978).  .  S h o r t l y a f t e r the p r e - m e i o t i c S phase, c e l l s i n L i l i u m and S a c charomyces become committed t o m e i o s i s . , M e i o c y t e s o f L i l i u m t h a t are e x p l a n t e d onto s y n t h e t i c medium a f t e r t h i s commitment s t e p proceed through an extended prophase c h a r a c t e r i s t i c o f m e i o s i s 1967).  When m e i o c y t e s a r e e x p l a n t e d 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 o r no p a i r i n g r e s u l t s a t the next prophase. chromosomes will  ( S t e r n and H o t t a ,  In a d d i t i o n , most  i n m e i o c y t e s . t h a t have been e x p l a n t e d near o r a t l e p t o t e n e  undergo u n s t a b l e p a i r i n g .  In e i t h e r c a s e , u n i v a l e n t s a r e g e n e r a l l y  12  produced a t metaphase I.  Thus i t appears t h a t both the achievement  and s t a b i l i z a t i o n o f p a i r i n g o f homologous chromosomes  during'the mei-  o t i c prophase r e q u i r e the a c t i v i t y o f genes or t h e i r p r o d u c t s w e l l f o r e the a c t u a l p a i r i n g p r o c e s s .  Other components n e c e s s a r y f o r p r o -  per p a i r i n g and exchange a r e o n l y r e q u i r e d d u r i n g t h e s e  processes.  F o r example, DNA s y n t h e s i s  The  t i o n o f DNA s y n t h e s i s  i s required during zygotene.  a t e a r l y zygotene causes c e l l  inhibi-  a b o r t i o n by p r e -  v e n t i n g the d e l a y e d semi c o n s e r v a t i v e r e p l i c a t i o n o f 0.3% o f t o t a l needed f o r p a i r i n g o f homologs.  be-  DNA  I n h i b i t i o n a t m i d - z y g o t e n e causes  e x t e n s i v e f r a g m e n t a t i o n o f chromosomes  at l a t e r stages, while at late  zygotene i t produces c h r o m a t i d breaks which are m a i n l y observed as b r i d g e s and breaks a t the second m e i o t i c d i v i s i o n ( S t e r n and H o t t a , 1967). The phenotypes o b t a i n e d i n t h e s e s t u d i e s have a l s o been obs e r v e d i n mutant m e i o c y t e s o f many s p e c i e s . f e c t s i n p l a n t s are r e f e r r e d t o as a s y n d e t i c .  Mutants w i t h p a i r i n g d e These may be a s y n a p t i c  i f a d e f e c t i n the achievement o f p a i r i n g i s i n v o l v e d , o r d e s y n a p t i c i f chromosome p a i r s a r e u n s t a b l e and s u b s e q u e n t l y f a l l u n i v a l e n t s a t metaphase I.  a p a r t t o form  Other mutants a f f e c t chromosome  These may i n v o l v e gene p r o d u c t s t h a t are n e c e s s a r y f o r DNA during zygotene.  integrity. synthesis  In D r o s o p h i l a , m u t a t i o n s w i t h d e f e c t s i n p a i r i n g o r  exchange o f homologs have been d i s t i n g u i s h e d by t h e i r p a t t e r n o f reduced exchange.  The p o l a r i t y o f the p a i r i n g process suggests t h a t i f p r e -  c o n d i t i o n s t o exchange ( e . g . , p a i r i n g ) were a f f e c t e d , the decrease  in  exchange would t a k e p l a c e i n a n o n - u n i f o r m manner a l o n g the l e n g t h o f each chromosome  ( S a n d l e r e t a j _ . , 1968).  The m a j o r i t y o f mutants w i t h  13  a l t e r e d exchange f e l l  into this category.  In c o n t r a s t , a d e f e c t i n  the exchange p r o c e s s per se s h o u l d r e s u l t i n a u n i f o r m r e d u c t i o n o f exchange i n a l l r e g i o n s .  Only one gene appeared to behave i n  this  manner ( f o r r e v i e w , see Baker e t a j _ . , 1976a). The above mentioned s t u d i e s  i n d i c a t e t h a t an extended p e r i o d  i s needed t o p r e p a r e and complete normal p a i r i n g and  exchange.and  t h a t many genes a r e p r o b a b l y i n v o l v e d i n t h i s p r o c e s s .  In a d d i t i o n ,  because o f the c o n c u r r e n c e o f reduced p a i r i n g and exchange and i n c r e a s e d numbers o f 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 h a t mutants w i t h d e f e c t s i n p a i r i n g and exchange w i l l disjunction.  s i m u l t a n e o u s l y have i n c r e a s e d non-  T h i s has been v e r i f i e d w i t h mutants from many  species  (see next s e c t i o n ; see a l s o Baker e t a j _ . , 1976a). The Two M e i o t i c  Divisions  H a p l o i d i z a t i o n o f the chromosome complement r e q u i r e s two n u c l e a r d i v i s i o n s w i t h o u t an i n t e r v e n i n g round o f DNA r e p l i c a t i o n . ganisms t h i s i s a c h i e v e d by the s e g r e g a t i o n o f homologous  In most o r 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 o w e d by a r e g u 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 ) .  T h i s appears t o be  the most economical way t o a s s u r e h a p l o i d i z a t i o n o f a l l Besides  chromosomes.  the.absence o f the i n t e r v e n i n g DNA r e p l i c a t i o n , t h e r e are two  d i s t i n c t d e 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 . meres have to remain u n d i v i d e d u n t i l  F i r s t , the c e n t r o -  anaphase o f the second d i v i s i o n .  In l i l y m e i o c y t e s , the p o t e n t i a l f o r centromeres t o d i v i d e d u r i n g 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 - m e i o t i c S  phase.(the  centromeres o f c e l l s t h a t have been e x p l a n t e d onto s y n t h e t i c medium a f t e r t h i s commitment s t e p cannot d i v i d e d u r i n g the n e x t d i v i s i o n ) .  14  That the d e l a y o f centromere d i v i s i o n u n t i l  anaphase II  involves  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 e x i s t e n c e o f mutants i n w h i c h s e p a r a t i o n o c c u r s p r e c o c i o u s l y ( C l a y b e r g , 1959; Lamm, 1944; 1944; D a v i s ,  1971).  Second, a v i r t u a l l y f o o l p r o o f method o f  s e g r e g a t i o n o f homologs  chiasmata a r e used to h o l d homologs  together u n t i l  in-  In most s p e c i e s ,  they s e g r e g a t e  T h i s r e l a t i o n s h i p between exchange and subsequent  g a t i o n o f homologs  and  The r e g u l a r s e g r e g a t i o n o f homologs  v a r i a b l y i n v o l v e s some p a i r i n g o r a l i g n m e n t p r o c e s s .  I.  assuring  i s e s s e n t i a l f o r the r e g u l a r s e g r e g a t i o n  h a p l o i d i z a t i o n during meiosis.  anaphase  Johnsson,  at segre-  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 o f mutants  w i t h reduced intrachromosomal r e c o m b i n a t i o n .  The homologs  i n mutant  meiocytes are o f t e n not h e l d t o g e t h e r due t o a l a c k o f c h i a s m a t a ; the.: r e s u l t a n t u n i v a l e n t s move a t random t o the two p o l e s o r they d i v i d e by centromere d i v i s i o n .  The process by which homologs  a t t h e i r chiiasmata u n t i l  anaphase  are held together  I thus appears t o be one o f the main  f a c t o r s which a s s u r e s the r e g u l a r s e g r e g a t i o n o f homologs d i v i s i o n of meiosis. is c r i t i c a l  a t the  first  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 o f chiasmata  t o t h i s type o f s e g r e g a t i o n .  The c o n t r o l o f the r e d u c t i o n a l  d i v i s i o n may a l s o i n v o l v e o t h e r components s i n c e s e v e r a l mutants D r o s o p h i l a have a d e f e c t i n d i s j u n c t i o n of chromosomes s i o n , even though r e c o m b i n a t i o n i s n o r m a l .  in  a t the f i r s t  divi-  The mutant ca_ ' has been most nc  R e g u l a r s e g r e g a t i o n can be a s s u r e d i n d i f f e r e n t ways. For e x ample, D r o s o p h i l a males which have no c r o s s i n g - o v e 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 s e g r e g a t i o n which a p p a r e n t l y i n v o l v e s p a i r i n g (see Baker and H a l l , 1976). In D r o s o p h i l a f e m a l e s , the s e g r e g a t i o n o f non-exchange chromosomes i s a s s u r e d by the process o f 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 t u d i e d (Baker e t a]_., 1976a).  Cytological analysis  re-  v e a l e d s p i n d l e d i s t o r t i o n w i t h consequent n o n d i s j u n c t i o n o f 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  divisions. In c o n c l u s i o n , s t u d i e s on m e i o s i s have shown t h a t many processes phase u n t i l  i n a v a r i e t y of  organisms  o p e r a t i n g from b e f o r e p r e - m e i o t i c S  pachytene a r e e s s e n t i a l f o r the r e g u l a r o c c u r r e n c e o f  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  u s u a l l y r e q u i r e d t o ensure the r e g u l a r s e g r e g a t i o n o f d u r i n g the m e i o t i c d i v i s i o n s .  chromosomes  However, these s t u d i e s p r o v i d e  i n s i g h t i n t o the n a t u r e and c o n t r o l o f these p r o c e s s e s .  is  The  little identi-  f i c a t i o n and c y t o l o g i c a l and g e n e t i c c h a r a c t e r i z a t i o n o f m u t a t i o n s most or a l l genes t h a t a r e r e q u i r e d f o r the s u c c e s s f u l m e i o s i s s h o u l d be the l o g i c a l f i r s t s t e p t o a f u l l the m e i o t i c p r o c e s s . e r a l organisms  in  completion of  understanding  T h i s k i n d o f approach has been f o l l o w e d i n  of sev-  but i t appears t h a t a good means f o r the d e t e c t i o n o f  mutants and o f t h e i r c y t o l o g i c a l and g e n e t i c c h a r a c t e r i z a t i o n r e n d e r Neurospora p a r t i c u l a r l y s u i t a b l e t o study m e i o s 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 o f chromosome b e h a v i o r d u r i n g m e i o s i s been determined by a c o m b i n a t i o n o f c y t o l o g y and t r a n s m i s s i o n A number o f f e a t u r e s o f t h i s d i v i s i o n c y c l e a r e unique and m e i o s i s from m i t o s i s .  F i r s t , p r e - m e i o t i c DNA s y n t h e s i s  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 s y n t h e s i s the f o l l o w i n g prophase  ( S t e r n and H o t t a , 1973).  has  genetics.  distinguish  i n v o l v e s fewer  may. be d e l a y e d u n t i l Second, the f i r s t m e i -  o t i c prophase l a s t s u n u s u a l l y long and i n v o l v e s the p a i r i n g o f a l l homologous chromosomes.  Genetic recombination t h a t takes place a t t h i s  time i s o r d e r s o f magnitude h i g h e r than t h a t observed d u r i n g , m i t o t i c d i v i s i o n (Parag and P a r a g , 1975).  T h i r d , homologous chromosomes  gen-  e r a l l y s e g r e g a t e from each o t h e r d u r i n g the f i r s t m e i o t i c d i v i s i o n . F o u r t h , no DNA r e p l i c a t i o n takes p l a c e , d u r i n g the i n t e r p h a s e f o l l o w i n g this  division. D e s p i t e t h i s d e t a i l e d knowledge o f the b e h a v i o r o f  during meiosis, l i t t l e events.  i s known about the p r o c e s s e s  chromosomes  t h a t c o n t r o l these  T h i s study was i n i t i a t e d to g a i n a b e t t e r i n s i g h t i n t o such  c o n 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 t h d e f e c t s d u r i n g meiosis.  Such mutants i n which t h e ' p h e n o t y p e o f m e i o t i c c e l l s o r t h e i r  p r o d u c t s are d e t e c t a b l y abnormal w i l l  be r e f e r r e d t o as m e i o t i c m u t a n t s .  There a r e two types o f m e i o t i c m u t a n t s ; they can be d i s t i n g u i s h e d on the b a s i s o f t h e i r b e h a v i o r d u r i n g the v e g e t a t i v e phase.  The f i r s t type has  no apparent e f f e c t on any v e g e t a t i v e f u n c t i o n and i s c a l l e d m e i o s i s specific.  The second type o f 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 o r maintenance.  17  Meiosis-Specific  Mutants  M e i o t i c mutants which have no d e t e c t a b l e d e f e c t i n any t a t i v e f u n c t i o n s have been d e t e c t e d i n many o r g a n i s m s ,  vege-  including  higher  3 •"• p l a n t s , f u n g i , and D r o s o p h i l a  . ( f o r a r e v i e w , see Baker e t a]_., 1976a).  The d e f e c t i n these mutants may cause a r e a d i l y d e t e c t a b l e m e i o t i c phenotype such as a l t e r e d r e c o m b i n a t i o n , d i s j u n c t i o n , o r (see l a t e r s e c t i o n ) .  fertility  A l t e r n a t i v e l y , the change o f phenotype o f such  mutants may be s u b t l e .  The r e c - t y p e mutants c o n t r o l l i n g  region-specific  r e c o m b i n a t i o n i n Neurospora and S c h i z o p h y l l u r n a r e examples o f the type ( C a t c h e s i d e , 1974).  In t h i s paper I w i l l o n l y deal w i t h m u t a t i o n s  which a f f e c t r e c o m b i n a t i o n , d i s j u n c t i o n , o r f e r t i l i t y , a v i s i b l y abnormal  i . e . , those w i t h  phenotype.  Mutants With D e f e c t s During Both the V e g e t a t i v e and Sexual  Phases  Two types o f mutants w i t h d e f e c t s i n v e g e t a t i v e f u n c t i o n s been a s s o c i a t e d w i t h c o r r e s p o n d i n g all  temperature-sensitive c e l l  defects i n meiotic processes.  c y c l e m u t a t i o n s o f Saccharomyces  have been t e s t e d a l s o caused m e i o t i c a b n o r m a l i t i e s appears  t h a t both types o f c e l l  common c o n t r o l .  latter  (Simchen,  have First,  that  1978).  It  d i v i s i o n a r e under a c e r t a i n amount o f  Second, some genes r e q u i r e d f o r v e g e t a t i v e DNA meta-  b o l i s m ( e . g . , r e p a i r , m u t a t i o n , somatic r e c o m b i n a t i o n ) , a r e  presumably  a l s o r e q u i r e d f o r m e i o t i c exchange o r a s s o c i a t e d p r o c e s s e s .  Mutations  i n t h e s e genes have been i s o l a t e d on the b a s i s o f t h e i r s e n s i t i v i t y t o  However, some mutants o f D r o s o p h i l a w i t h no a p p a r e n t d e f e c t o f somatic f u n c t i o n s were shown t o 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 p e r i o d s (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 , o r the a l k y l a t i n g agent methyl methane s u l phonate (MMS).  Others were o b t a i n e d by t h e i r a l t e r e d f r e q u e n c i e s o f  m i t o t i c gene c o n v e r s i o n o r spontaneous m u t a t i o n . Many mutants t h a t a r e s e n s i t i v e t o DNA-damaging agents have been i s o l a t e d and s t u d i e d i n the y e a s t Saccharomyces (Prakash and P r a k a s h , 1977), and i n D r o s o p h i l a ( B o y d o e J ^ a K , 1976a,b; Boyd and S e t l o w , 1976).  In y e a s t , t h r e e r e p a i r pathways were i d e n t i f i e d on  the b a s i s o f c r o s s - s e n s i t i v i t y t o mutagenic a g e n t s .  C l a s s I mutants  are s e n s i t i v e t o n i t r o g e n mustard (HNg) and UV; c l a s s II x - r a y s ; and c l a s s  III  mutants o f c l a s s e s II  to a l l four agents. and I I I  t o MMS and  I t was found t h a t many  have an e f f e c t on m e i o s i s (Baker e t a l . ,  1976a; Prakash and P r a k a s h , 1977).  A s i m i l a r o v e r l a p o f gene f u n c t i o n s  r e q u i r e d f o r both r e p a i r to MMS-induced damage and m e i o s i s ( e . g . , r e c o m b i n a t i o n ) i s a p p a r e n t from the a n a l y s i s o f mutants s e l e c t e d i n D r o s o p h i l a on the b a s i s o f i n c r e a s e d s e n s i t i v i t y t o MMS (Baker e t a l . , 1976b).  M u t a t i o n s i n f i v e complementation groups cause s e n s i t i v i t y  t o HN2, UV, MMS, and x - r a y s . mutants i n y e a s t .  These a r e analogous  t o the c l a s s  III  M u t a t i o n s i n t h r e e and p o s s i b l y f o u r o f these com-  p l e m e n t a t i o n groups reduce the f r e q u e n c y o f m e i o t i c exchange.  Of  t h e s e , two (mei-41 and mus-101) a r e 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 o f UV-induced damage.  M u t a t i o n s i n a t l e a s t two genes cause s e n s i t i v i t y t o MMS and  x - r a y s , and thus resemble the y e a s t c l a s s II apparent d e f e c t i n m e i o s i s .  m u t a n t s ; t h e s e two have an  From these r e s u l t s i t i s c l e a r t h a t gene  f u n c t i o n s i n v o l v e d i n c e r t a i n pathways o f DNA r e p a i r i n s o m a t i c c e l l s are a l s o r e q u i r e d during m e i o t i c recombination.  Therefore, 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 o f v a r i o u s r e p a i r pathways w i l l  provide  19  i n f o r m a t i o n t h a t may l e a d t o the u n d e r s t a n d i n g o f mechanisms i n the process o f m e i o t i c r e c o m b i n a t i o n and a s s o c i a t e d  involved  processes.  C o n v e r s e l y , the i s o l a t i o n o f m e i o t i c mutants may s t i m u l a t e the s t u d y o f r e p a i r pathways. Gene f u n c t i o n s i n v o l v e d i n DNA metabolism i n s o m a t i c c e l j . s can a l s o be i d e n t i f i e d by s c r e e n i n g f o r mutants w i t h a l t e r e d induced - m i t o t i c gene c o n v e r s i o n f r e q u e n c i e s ( e . g . , Rodarte-Ramon and M o r t i m e r , 1972; Rodarte-Ramon, 1972).  Three out o f f o u r 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 m u t a t i o n s spo-7 ( E s p o s i t o et a l _ . , 1975) and rem-1 ( G o l i n and E s p o 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 d e f e c t s and an  a l t e r e d f r e q u e n c y o f spontaneous m u t a t i o n .  Other m e i o t i c mutants,were  s e n s i t i v e t o h i s t i d i n e ( e . g . , u v s - 3 , u v s - 4 , u v s - 5 , u V s - 6 , and m e i - 3 i n Neurospora c r a s s a ; Newmeyer e t a l . , 1978).  In t h r e e h i s t i d i n e - s e n s i t i v e  mutants examined the i n c r e a s e d s e n s i t i v i t y was accompanied by m e i o t i c b l o c k a g e , UV s e n s i t i v i t y , and i n c r e a s e d 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 D e t e c t i o n o f M e i o t i c Mutants The s y s t e m a t i c i s o l a t i o n o f m e i o t i c mutants has been i n i t i a t e d i n several organisms. mutants.  Two types o f c r i t e r i a were used t o d e t e c t these  F i r s t , mutants w i t h a l t e r e d r e c o m b i n a t i o n and/or d i s j u n c t i o n  f r e q u e n c i e s were r e c o v e r e d i n D r o s o p h i l a and C a r p e n t e r , 1972), Saccharomyces  ( S a n d l e r e t a]_., 1968;  Baker  (Roth and F o g e l , 1971; R o t h ,  and Caenorhabdi t i s (Hodgkin e t a j _ . , 1979).  1976),  Second, complete o r 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 o f m e i o t i c mutants i n S o r d a r i a ( E s s e r and S t r a u b , 1958), (Bresch et_ a l _ . , 1968), Saccharomyces  Schizosaccharomyces  ( E s p o s i t o and E s p o s i t o , 1969), and  20  Podospora (Simonet and Z i c k l e r , 1972).  S i n c e reduced f e r t i l i t y may  be caused by many d e f e c t s not d i r e c t l y a s s o c i a t e d w i t h m e i o s i s , cytological  and/or g e n e t i c o b s e r v a t i o n s are r e q u i r e d t o c o n f i r m the  i s o l a t i o n o f m e 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 o b t a i n e d i n many o r ganisms, the u n d e r s t a n d i n g o f the d e f e c t s i s o f t e n i n doubt because o f a l a c k o f g e n e t i c o r c y t o l o g i c a l means o f a n a l y s i s .  Various  idto-  s y n c r a c i e s o f the l i f e c y c l e s o f Podospora and h i g h e r p l a n t s c o m p l i cate genetic analysis  i n these o r g a n i s m s .  In c o n t r a s t , even though  g e n e t i c a n a l y s i s o f y e a s t and D r o s o p h i l a i s e x c e l l e n t , v i s u a l i z a t i o n o f chromosomes by means o f c o n v e n t i o n a l c y t o l o g i c a l methods i s  not  p o s s i b l e i n y e a s t and i s p o s s i b l e o n l y a f t e r the pachytene stage o f Drosophila  (Pura and N o k k a l a , 1977).  T h e r e f o r e , i t s h o u l d be p r o f i t -  a b l e 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  in  which both g e n e t 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 a r e good.  The  ascomycete Neurospora c r a s s a f i t s  this description we'll.  In N e u r o s p o r a , m e i o t i c m u t a t i o n s which a r e dominant o r e x pressed o n l y i n the c o n i d i a l o r p r o t o p e r i t h e c i a l p a r e n t can be d i r e c t l y o b t a i n e d by s c r e e n i n g 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-  e v e r y , because o f the h e t e r o t h a l l i c n a t u r e o f N e u r o s p o r a , induced 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 cannot be expressed i n the f i r s t of c r o s s i n g .  generation  I t i s p o s s i b l e t o d e t e c t such m u t a t i o n s by i n t e r c r o s s i n g  a number o f i s o l a t e s from each c r o s s i n v o l v i n g a . p o t e n t i a l m u t a n t ^ , b u t t h i s i s very t i m e - c o n s u m i n g .  The t e c h n i q u e d e s c r i b e d i n t h i s  thesis  enables the r a p i d i s o l a t i o n o f 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 .  21  T h i s method i n v o l v e s the s e l e c t i o n o f s t r a i n s a r i s i n g from d i s o m i c f o r l i n k a g e group I and heterozygous (A + a).  f o r the mating type l o c u s .  Such s t r a i n s a r e s e l f - f e r t i l e and homozygous  groups e x c e p t LG I.  ascospores  for a l l  linkage  T h e r e f o r e , each o f these c u l t u r e s can be d i r e c t l y  t e s t e d f o r the presence o f a r e c e s s i v e m e i o t i c m u t a t i o n . M e i o t i c m u t a t i o n s i n Neurospora c r a s s a can be d e t e c t e d as. r e - . duced f e r t i l i t y due t o a b l o c k i n the f o r m a t i o n o f most o r a l l a s c i , o r as ascospore a b o r t i o n . a b o r t i o n o f an ascospore missing.  In N e u r o s p o r a , a n e u p l o i d y r e s u l t s i n the i f any p a r t o f the chromosome complement i s  T h e r e f o r e , the presence o f such a b o r t e d ( w h i t e ) 1  ascospores  i s a good d e t e c t i o n system f o r m u t a t i o n s which cause i r r e g u l a r g a t i o n o f chromosomes  during meiosis  During t h i s i n i t i a l  (Smith,  1975).  s c r e e n , . r e c e s s i v e m e i o t i c mutations  p r e s e n t i n g s i x l o c i caused ascospore a b o r t i o n , and r e c e s s i v e o f two l o c i  r e s u l t e d i n an absence o f  segre-  re-  mutations  ascospores.  MATERIALS AND METHODS Strains The f o l l o w i n g a l l e l e s were used d u r i n g t h i s s t u d y : (R156); un-3 ( 5 5 7 0 1 - t ) ;  leu-3  a r q - 1 (36703); ad-3A ( 2 - 1 7 - 8 1 4 ) ; 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 o f the t h r e e h e t e r o k a r y o n 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 o f these m u t a t i o n s en l i n k a g e groups and the approximate map d i s t a n c e s Fig. l a .  ( R a d f o r d , 1972) between l i n k e d m u t a t i o n s a r e shown i n  22  Fig. 1  General o u t l i n e o f a s e l e c t i v e system used t o i s o l a t e r e c e s s i v e m e i o t i c mutants i n crassa.  Neurospora  For d e t a i l s , see M a t e r i a l s and Methods.  (a) A c r o s s o v e r between s t r a i n 1-34-8 (female) and the mutagenized s t r a i n 1-30-335 (male)  pro-  duces a low p r o p o r t i o n o f progeny d i s o m i c f o r LG I.  These can be s e l e c t e d on minimal medium.  (b) Each ascospore d i s o m i c (n + 1) f o r LG I c o n t a i n s two c o p i e s o f t h i s l i n k a g e group but o n l y one copy o f the o t h e r l i n k a g e g r o u p s ,  (c)  Subsequent  h a p l o i d i z a t i o n produces two types o f n u c l e i o f o p p o s i t e mating type i n each c u l t u r e produced by a d i s o m i c a s c o s p o r e ; such c u l t u r e s are c a p a b l e o f "selfing."  The genes and t h e r e f o r e newly induced  m e i o t i c mutations are i d e n t i c a l nuclei.  i n both types  of  Consequently, i f a r e c e s s i v e m e i o t i c  m u t a t i o n were p r e s e n t i n t h e d i s o m i c  ascospore,  i t would be d e t e c t e d i n the " s e l f i n g " o f the r e s u l t i n g PWT c u l t u r e .  (a)  Cross  betueen  strains: Linkage  _  leu-3  n r ) C  ,  ,  +  1-34-b  (b)  Example  of  10  i  Q  + g  I  0.1  10 (n  +  1)  +  a  I  '  '  •  A  +  !  I  a  arg-1 •  A ,  + .  un-3 I  I  I I ' ill  +  ad-3B  +  +  C  |  I  '  '  J  ad-3A  +  I  I  9  leu-1  0.3  nic-2 al-2 I  h  I  d  tol  V  — I —  D  tol  +  L  I  I  c  UI  VII  asc  L.  J  28  IV  !—  75  ascospore:  arg-1 n  Q  +  ad-3B  +  »  I  I  +  ad-3A  +  1  1  1  1  +  ad-3B •  + .  + .  ad-3A i  + i  C  i  nic-2  d  tol  asc  -I  L.  I  I  C J  d u  tol - u .  asc •  j  d  tol •  asc •  j  al-2  Heterokaryon:  _ . „ Component 1  leu-3  . „ Component 2  + ,  An of  arg-1  •  a disomic  +  *  i  '  _l  (c)  a  i  un-3 A  t  Approximate map d i s t a n c e (Radford, 1972)  +  Group  + T  un-3 ,  0  i n d u c e d m e i o t i c m u t a t i o n may be this kind, isolated during this  ^ Only  one  of  the  eight  possible  nic-2 al-2 • •  j  C L  l o c a t e d on a n y l i n k a g e s t u d y , were d e s i g n a t e d  combinations  of  het  alleles  g r o u p e x c e p t LG I. asc (see Results). is  presented.  E  E  Mutations  24  Mutant I n d u c t i o n and  Isolation  R e c e s s i v e m e i o t i c mutants can o n l y be d e t e c t e d i n homozygous  f o r these m u t a n t s .  crosses  A method f o r the homozygosis  and d e -  t e c t i o n o f such mutants i n Neurospora c r a s s a has been developed 1).  The method i n v o l v e s the i s o l a t i o n and t e s t i n g o f many  (Fig.  strains,  each a r i s i n g from an ascospore d i s o m i c (n + 1) f o r chromosome. 1 (LG which bears the mating type l o c u s  (A/a_).  Disomic n u c l e i i n  are i n h e r e n t l y u n s t a b l e ( P i t t e n g e r , 1954).  Neurospora  They u s u a 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 o r the o t h e r homologous S i n c e the s e l e c t e d ascospores  I)  chromosome.  c a r r y d i s o m i c n u c l e i which a r e h e t e r o -  zygous f o r the mating type l o c u s , the r e s u l t i n g c o l o n i e s are h e t e r o karyons w i t h two h a p l o i d n u c l e a r types o f o p p o s i t e mating type (A + a ) . In a d d i t i o n , s i n c e each disome c o n t a i n s a h a p l o i d complement o f a l l chromosomes  e x c e p t LG I,  genes on these chromosomes w i l l  be i d e n t i c a l  i n the two n u c l e a r types produced d u r i n g 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 h e t e r o -  karyons w i l l , under the a p p r o p r i a t e c o n d i t i o n s o f n i t r o g e n  starvation  •(Westergaard and M i t c h e l l , 1947), induce p e r i t h e c i a l f o r m a t i o n and meiosis.  The mating r e a c t i o n o f these h e t e r o k a r y o n s w i l l  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  and t h e r e f o r e the mating type l o c u s , but homozygous o t h e r chromosomes.  met.  f o r LG I  f o r genes on the  T h e r e f o r e , any i n d u c e d r e c e s s i v e m e i o t i c m u t a t i o n  which i s l o c a t e d on one o f these chromosomes w i l l zygous c o n d i t i o n .  be r e f e r r e d  be p r e s e n t i n homo-  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  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) The s e l e c t i o n o f ascospores  Cultures  d i s o m i c f o r LG I was made p o s s i b l e  by the i n t r o d u c t i o n o f v a r i o u s markers onto the s e l e c t i v e p a r e n t  strains  (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 d i s o m i e s f o r LG I c o n s t i t u t e a  h e t e r o k a r y o n w i t h two complementing components o f o p p o s i t e mating type (A + a_).  S i n c e the mating type l o c u s a l s o a c t s as a h e t e r o k a r y o n i n -  c o m p a t i b i l i t y locus  (Beadle and C o o n r a d t , 1944; G a r n j o b s t and W i l s o n ,  1956), these d i s o m i c c u l t u r e s w i l l  grow very p o o r l y .  T h e r e f o r e , the  t o l m u t a t i o n which suppresses t h i s h e t e r o k a r y o n i n c o m p a t i b i l i t y w i t h o u t affecting crossing a b i l i t y strains  (Newmeyer, 1970) was i n t r o d u c e d i n both  (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 a u x o t r o p h i c m u t a t i o n s 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 m u t a t i o n u n - 3 , which are a l l l o c a t e d w i t h i n about 30 map u n i t s o f each o t h e r on LG I,  were used t o s e l e c t ascospores  disomic f o r t h i s linkage  group.  A c r o s s between l e u - 3 , a r g - 1 , ad-3B ( s t r a i n 1-30-225) and u n - 3 , ad-3A, n i c - 2 ( s t r a i n 1-34-8) c o u l d produce two types o f ascospore  progeny  c a p a b l e o f growth on medium c o n t a i n i n g no l e u c i n e , a r g i n i n e , a d e n i n e , o r 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 cultures:  The arrangement o f the c l o s e l y 1 i n k e d markers v i r t u a l l y  e l i m i n a t e s w i l d type recombinant progeny. in t h i s study.  Thus, when ascospores  In f a c t , none were d e t e c t e d  are p l a t e d on minimal medium,  most c o l o n i e s s h o u l d r e s u l t from ascospores  d i s o m i c f o r LG I.  Each o f  these PWT c u l t u r e s c o n t a i n s two t y p e s . o f n u c l e i w i t h , complementing  26  a u x o t r o p h i c m u t a t i o n s and o p p o s i t e mating type a l l e l e s . T h i r d , when ascospores  a r e p l a t e d on minimal medium, the germ  tubes o f two a d j a c e n t ascospores  may f u s e .  Such f u s i o n p r o d u c t s may  produce a c o l o n y i f the two n u c l e a r types complement each o t h e r and are h e t e r o k a r y o n c o m p a t i b l e .  To m i n i m i z e the frequency o f  colony-  producing f u s i o n products, three unlinked heterokaryon i n c o m p a t i b i l i t y loci  (C/c, D/d, and E/e) were u t i l i z e d .  The c r o s s between  strains  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 t h r e e l o c i . . When ascospores  produced by t h i s c r o s s a r e p l a t e d  th on minimal medium, o n l y 1/8  o f a l l f u s i o n products w i t h complementary  a u x o t r o p h i c r e q u i r e m e n t s can form a c o l o n y .  In f a c t , c o l o n i e s  duced by the f u s i o n o f two or more a d j a c e n t  ascospores  pro-  were p r a c t i c a l 4 l y e l i m i n a t e d when the p l a t i n g c o n c e n t r a t i o n d i d not exceed 2 x 10 ascospores/90 mm p e t r i d i s h . C o n s e q u e n t l y , the p l a t i n g o f ascospores 4  a t c o n c e n t r a t i o n s up t o 2 x 10  s p o r e s per p l a t e a l l o w e d 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 o f 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  o f Induced R e c e s s i v e M u t a t i o n s  C o n i d i a from seven-day  Which A f f e c t  Meiosis  o l d c u l t u r e s o f s t r a i n 1^-30-225 were  t r e a t e d w i t h 0.025 mM MNNG a t 25°C f o r f o u r , f i v e , o r s i x hours and d e S e r r e s ,  1970).  A f t e r t e r m i n a t i o n o f MNNG t r e a t m e n t w i t h  t h i o s u l p h a t e a t pH 8 . 0 ,  (Mailing sodium  the mutagenized c o n i d i a l s u s p e n s i o n was used  as the f e r t i l i z i n g p a r e n t by p o u r i n g 10 ml o f the s u s p e n s i o n over day o l d m y c e l i a l growth o f s t r a i n 1-34-8.  Ascospores  seven-  produced by t h i s  c r o s s were p l a t e d onto minimal medium a t a c o n c e n t r a t i o n o f a p p r o x i mately 10^ per 90 mm p e t r i d i s h ( f o r f u r t h e r d e t a i I s , o n  the p l a t i n g  pro-  cedure and e x c l u s i v e use o f p u r i f i e d t a g a r i n the medium, s e e G r i f f i t h s and  27  DeLange,  1977).  The f r e q u e n c y o f c o l o n i e s  ( s e l f - f e r t i l e PWT's)  thus  _5 produced was e s t i m a t e d a t about 5 x 10  .  Each o f these c u l t u r e s was  a s s i g n e d an i s o l a t i o n number w i t h 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 t h r e e p o s s i b l e methods.  F i r s t , using a d i s s e c t i n g microscope, very small  c o l o n i e s were i s o l a t e d a f t e r two and t h r e e days o f growth. the s i m u l t a n e o u s t r a n s f e r o f u n s e l e c t e d ascospores  A t t h i s time  can be a v o i d e d .  Second, a f t e r f o u r o r f i v e days o f g r o w t h , the a g a r - o v e r l a y e r a t the s i t e o f the c o l o n y was removed and p a r t o f the u n d e r l y i n g a g a r , which i n c l u d e d the m y c e l i a l growth o f the c o l o n y , was t r a n s f e r r e d . method i s f a s t e r a n d , s i n c e a l l ascospores reliable.  This  a r e i n the o v e r l a y e r a g a r ,  is  T h i r d , p a r t o f the m y c e l i a l growth o r 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 g h 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 l a c k s e n s i t i v i t y s i n c e non-growing  germinated  may be " r e s c u e d " by a p o o r l y growing PWT c u l t u r e .  A subsequent s e l f o f  such a rescued f a s t - g r o w i n g c u l t u r e would be heterozygous  ascospores  a t many  loci.  Each PWT c u l t u r e was t r a n s f e r r e d t o a s l a n t o f minimal medium i n a 10 x 75 mm t u b e , and a l l o w e d t o grow f o r a week b e f o r e being t r a n s f e r r e d t o an 18 x 150 mm t e s t tube c o n t a i n i n g 5 ml l i q u i d minimal  crossing  medium and a s t r i p o f f i l t e r paper (Newcombe and G r i f f i t h s , 1972). s e l f s were i n c u b a t e d a t 25°C, some a l s o a t 16°C.  All  I n c u b a t i o n a t both  temperatures a l l o w e d the d e t e c t i o n o f t e m p e r a t u r e - s e n s i t i v e m u t a n t s . C u l t u r e s i n which abnormal development o f p e r i t h e c i a and/or  as-  cospores r e s u l t e d were c r o s s e d 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 a b e r r a n t development i s caused by the e x -  p r e s s i o n o f a r e c e s s i v e , r a t h e r t h a t a dominant, m u t a t i o n , c r o s s e s w i t h  28  both w i l d type s t r a i n s s h o u l d produce normal p e r i t h e c i a and a s c o spores.  T h e r e f o r e , o n l y c u l t u r e s w i t h t h i s b e h a v i o r 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 mutants.  R e c e s s i v e mutants were  distinguished  from mutants t h a t a r e expressed o n l y when p r e s e n t i n the male o r female p a r e n t , by means o f r e c i p r o c a l c r o s s e s between the mutant PWT c u l t u r e , and the OR-A and OR-a  strains.  Some o f these m u t a t i o n s may a f f e c t p e r i t h e c i a l f o r m a t i o n w h i l e . o t h e r s are more d i r e c t l y i n v o l v e d w i t h the p r o d u c t i o n o f v i a b l e a s c o spores.  Among mutants w i t h a d e f e c t i n p e r i t h e c i a l development, no a s c i  or ascus i n i t i a l s have e v e r been r e p o r t e d . s i b l e that meiosis  T h e r e f o r e , i t appears  plaus-  i s i n i t i a t e d o n l y a f t e r the major p a r t o f p e r i t h e c i a l  development has been c o m p l e t e d .  Thus, i n s c r e e n i n g f o r m e i o t i c m u t a n t s ,  o n l y those w i t h w e l l - d e v e l o p e d p e r i t h e c i a have been f u r t h e r a n a l y z e d . F i v e A i s o l a t e s o f the c r o s s between any g i v e n PWT and OR-A, and f i v e a_ i s o l a t e s o f the c r o s s between the PWT and OR-a were i n t e r c r o s s e d i n ' all  c o m b i n a t i o n s ; i f the mutant phenotype was d e t e c t e d , one mutant  i s o l a t e o f each mating type was used i n the t e s t i n g o f a l l  isolates  from c r o s s e s between the PWT c u l t u r e and the two w i l d type  strains'  (OR-A and OR-a). original  Sometimes, a l l t h e s e i s o l a t e s were b a c k c r o s s e d t o the  PWT c u l t u r e .  In e i t h e r c a s e , a 1:1 s e g r e g a t i o n o f mutant  and w i l d type phenotype c o n f i r m e d the presence o f a r e c e s s i v e  point  mutation. Media and r o u t i n e m a n i p u l a t i o n s were c o n v e n t i o n a l f o r Neurospora (Davis and de S e r r e s ,  1970).  29  RESULTS  '  Using the s e l e c t i v e system d e s c r i b e d 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 l o w e d t o self  on ; l i q u i d c r o s s i n g medium.  c e s s f u l l y complete the sexual ascospores  suc-  c y c l e , i . e . , e j e c t only v i a b l e black  from mature p e r i t h e c i a , were screened a g a i n s t dominant muta-  t i o n s by c r o s s i n g strains.  The 145 c u l t u r e s t h a t d i d not  them i n d i v i d u a l l y w i t h OR-A and 0R-a_ w i l d type  T a b l e I shows t h a t 46 s t r a i n s  a b i l i t y w i t h both OR-A and 0R-a_.  e x h i b i t e d w i l d type  crossing  These s t r a i n s , .which may c a r r y r e -  c e s s i v e m u t a t i o n s o r m u t a t i o n s t h a t are expressed o n l y i n the female o r male p a r e n t , were c l a s s i f i e d a c c o r d i n g t o phenotype i n t o t h r e e main groups: I);  16 s t r a i n s w i t h a d e f e c t i n the p e r i t h e c i a ! development  26. s t r a i n s  (class  II);  d e f e c t i v e i n the f o r m a t i o n o f a s c i o r b l a c k  and 4 s t r a i n s w i t h a m i s c e l l a n e o u s  ascospores  developmental d e f e c t  a p p a r e n t l y not a s s o c i a t e d w i t h reduced f e r t i l i t y  Class  (class  (class  III).  I Mutants o f 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  s t r a i n s ) , o r few o r i n c o m p l e t e l y developed p e r i t h e c i a (10  (6  strains).  P e r i t h e c i a w i t h 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 c o n s i d e r e d i n c o m p l e t e l y d e v e l o p e d .  To d i s t i n -  g u i s h mutants t h a t a r e expressed o n l y i n the female o r male p a r e n t (female o r male s t e r i l e mutants) (P205, P349, P4Q6, P 7 0 0 ) w i t h 5  from r e c e s s i v e m u t a n t s , 4 s t r a i n s ' ,  no p e r i t h e c i a , 2 s t r a i n s  w i t h few p e r i t h e c i a , and 2 s t r a i n s  (P841,  P891)  (P186, P434) w i t h i n c o m p l e t e l y d e -  veloped p e r i t h e c i a were c r o s s e d r e c i p r o c a l l y w i t h w i l d type  strains  TABLE  I.  Initial characterization crossing behavior*  o f 1^5  pseudo-wild  type  cultures  with  f\l umber o f Type Class  of Crossing  Aberrancy  M  aberrant PUT C u l t u r e s m  I A:  Sterile  B:  Feu  (i.e.,  no  perithecia)  or i n c o m p l e t e l y d e v e l o p e d  perithecia  5D  6  3  10  •  13  Class I I Perithecia Mare  than  without  spares  20% ascospore  13  abortion  Class I I I Defects  not r e l a t e d  to f e r t i l i t y  (see t e x t ) 99  k6  E a c h c u l t u r e was c r o s s e d w i t h 0R-A_ a n d 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 ) a n d t h e s e w e r e c a l l e d _M.  31  ( t h e remaining 8 s t r a i n s grew very p o o r l y and were not t e s t e d ) .  In  each c a s e , mutant phenotype r e s u l t e d when t h e 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) p a r e n t but not when the mutant was used as t h e c o n i d i a l (male) p a r e n t . sterile.  Thus these s t r a i n s are female  S i m i l a r mutants have been p r e v i o u s l y i s o l a t e d (Mylyk  T h r e l k e l d , 1974; Johnson, 1978).Class  and  .  II-A T h i r t e e n 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 b a r r e n or c o n t a i n e d very few b l a c k ascospores spores  (about  10-20  per p e r i t h e c i u r n as compared t o hundreds i n normal p e r i t h e c i a ) .  T h i s c l a s s o f mutants was s u b d i v i d e d i n t o 4 phenotypes:  8 strains  w i t h empty p e r i t h e c i a (P246, P308, P373, P400, P446, P741, and 2 a d d i tional  s t r a i n s t h a t were d i s c a r d e d due t o poor g r o w t h ) ; 2 s t r a i n s  that  produced o n l y a s c i w i t h 8 l i t t l e round b u b b l e s , a b o u t ' o n e - q u a r t e r the s i z e of regular ascospores, w i t h empty a s c i P1163). In  b u t no a s c o s p o r e s  (P314, P423);  1 strain  (P131); and 2 s t r a i n s w i t h few b l a c k ascospores  (P310,  The cause o f the d e f e c 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 .  2 cases  (P131 and P400) a r e c e s s i v e p o i n t m u t a t i o n was i n v o l v e d .  However, the empty p e r i t h e c i a o f P446 and the bubble a s c i o f P314 were a p p a r e n t l y not caused by a r e c e s s i v e m u t a t i o n .  One out o f 5 a s c o -  spore i s o l a t e s o b t a i n e d from a c r o s s between P446 and OR-A w i l d type produced b a r r e n 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 p a r e n t but not as t h e c o n i d i a l p a r e n t .  A l t h o u g h not a n a l y z e d f u r t h e r , t h i s  mutant appears s i m i l a r t o those i n c l a s s (class  I;  i n s t e a d o f an e a r l y b l o c k  I m u t a n t s ) , a l a t e b l o c k i n p e r i t h e c i a l development may be  involved.  The d e f e c t i n P314 appears t o have a more complex p a t t e r n  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 o n l y 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 c r o s s  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 o t h e r s , f o r m e d a s c o s p o r e s ,  most  o f which were n o t , o r were very s l o w l y e j e c t e d from t h e i r p e r i t h e c i a . T h i s mutant has not been examined f u r t h e r . Class  II-B T h i r t e e n 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 p a t t e r n o f a s c o -  spore a b o r t i o n .  The a b o r t i o n i n 6 o f these s t r a i n s  (P95, P243,  P393,  P711, P879, P961) was found t o be due t o a s i n g l e r e c e s s i v e m u t a t i o n . The f e r t i l i t y  ( t o t a l number o f b l a c k and w h i t e a s c o s p o r e s )  P243, P393, and P879 was very l o w .  of  strains  These mutants a r e d i s c u s s e d  i n the f o l l o w i n g s e c t i o n o r i n Chapter I I .  further  The ascospore a b o r t i o n i n  a n o t h e r s t r a i n (P917) was a p p a r e n t l y caused by a dominant s p o r e - k i l l e r , mutation.  T h i s mutant i s d e s c r i b e d i n Chapter I I I .  duced o n l y w h i t e i n v i a b l e ascospores  S t r a i n P1079 p r o -  whereas s t r a i n s P165 and P631,  w i t h 50-80% ascospore a b o r t i o n 3 weeks a f t e r c r o s s i n g , m o s t l y b l a c k ascospores  a f t e r 2 months.  contained  No c l e a r - c u t p a t t e r n o f i n -  h e r i t a n c e was e v i d e n t i n s t r a i n s P165, P631, and P1079.  The r e m a i n -  i n g 3 s t r a i n s w i t h about 20% a s c o s p o r e a b o r t i o n (P117, P285, P768) were d i s c a r d e d because o f s c o r i n g d i f f i c u l t i e s ( t h e mutagenized i n b r e d PWT s t r a i n s have a wide range o f spore a b o r t i o n up t o about 15<-or 20%). F i n a l l y , d u r i n g the a n a l y s i s  o f s t r a i n P917 (see Chapter  III),  a r e c e s s i v e m u t a t i o n which caused a s c o s p o r e a b o r t i o n and low f e r t i l i t y was d e t e c t e d .  T h i s m u t a t i o n was o r i g i n a l l y p r e s e n t i n  heterozygous  c o n d i t i o n and was t h e r e f o r e not d e t e c t e d i n the o r i g i n a l s e l f i n g o f P917.  A cross  between s t r a i n s 917A7 (mutant A a s c o s p o r e i s o l a t e from  33  the c r o s s 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 e u - 3 and the m u t a t i o n . both a_, a r g , ad recombinant progeny were mutant.  In a d d i t i o n ,  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 k a g e o f t h i s r e c e s s i v e m u t a t i o n to the t i p o f t h e l e f t arm o f LG Class  I.  III The abnormal phenotypes of these .4 mutant PWT s t r a i n s a r e a p -  p a r e n t l y not r e l a t e d t o f e r t i l i t y .  Two s t r a i n s d i s c h a r g e d t h e i r s p o r e s  very p o o r l y and were not examined f u r t h e r .  The 2 r e m a i n i n g  a f f e c t e d the phenotype o f t h e p e r i t h e c i u r n .  S t r a i n P126 produced  orange  instead  of black p e r i t h e c i a .  strains  T h i s phenotype was c o n t r o l l e d  by a p o i n t m u t a t i o n expressed o n l y i n t h e p r o t o p e r i t h e c i a l p a r e n t . S i n c e t h i s phenotype i s a p p a r e n t l y o n l y produced by m u t a t i o n s a t the p e r - 1 l o c u s (Howe and Johnson, 1976), i t i s p l a u s i b l e t h a t P126 i s an a l l e l e of t h i s l o c u s .  Another p o i n t m u t a t i o n ( i n P 4 1 3 ) , s i m i l a r l y e x -  p r e s s e d o n l y i n the p r o t o p e r i t h e c i a l p a r e n t , p r e v e n t s the f o r m a t i o n o f necks on the p e r i t h e c i a . cospores  T h i s d e f e c t p r e v e n t s the d i s c h a r g e o f  from t h e i r p e r i t h e c i a .  as-  S i n c e the w i l d type l o c u s a p p a r e n t l y  c o n t r o l s the f o r m a t i o n o f the p e r i t h e c i a l neck, the m u t a t i o n w i l l  be  designated pen-1. F i n a l l y , i n a p r e l i m i n a r y attempt to i s o l a t e t e m p e r a t u r e s e n s i t i v e m u t a n t s , a s t r a i n (P709) was found t o produce 4 - s p o r e d a t 16°C, and the normal 8 - s p o r e d a s c i a t 25°C.  asci  This cold s e n s i t i v e  mutant i s dominant and has been mapped a t or near the centromere o f LG The mutant w i l l be d e s i g n a t e d F s p - 2 ( 4 - s p o r e d a s c u s ) . t e m p e r a t u r e - s e n s i t i v e (Raju,. 1977).  Fsp-1 i s  not  I.  34  Recessive Mutations  t h a t A f f e c t the Formation o f A s c i o r V i a b l e  Ascospores A t o t a l o f 9 r e c e s s i v e m u t a t i o n s ' (2 o f c l a s s class  II-B)  II-A  and 7 o f  a f f e c t i n g ascus o r a s c o s p o r e p r o d u c t i o n have been p o s i -  t i v e l y i d e n t i f i e d d u r i n g t h i s s t u d y . - In the p a s t such m u t a t i o n s been d e t e c t e d among mutants t h a t were U V - s e n s i t i v e  ( u v s - 3 , -5.and  o r caused i n c r e a s e d 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 ( m e i - 3 ) . mutations  have -6)  Some o t h e r  ( m e i - 1 , mei-4) were d e t e c t e d d i r e c t l y by t h e i r a s c o s p o r e  a b o r t i o n ( f o r r e v i e w see P e r k i n s and B a r r y ,  1977).  Both m e i o t i c and o t h e r developmental processes  are r e q u i r e d  t o complete the sexual c y c l e and, t h u s , t o produce a s c i and v i a b l e ascospores. ascospores meiosis.  Therefore, a d e f e c t i n the production of a s c i or v i a b l e c o u l d be caused by a d e f e c t not d i r e c t l y a s s o c i a t e d w i t h  C o n s e q u e n t l y , not a l l r e c e s s i v e m u t a t i o n s o f t h i s k i n d would  be m e i o t i c (mei) m u t a t i o n s .  The new l o c u s d e s i g n a t i o n asc which i s  i n t r o d u c e d here r e f e r s t o a l l r e c e s s i v e m u t a t i o n s r e s u l t i n g i n the absence o f a s c i o r the a b o r t i o n o f a s c i o r All to m e i - 1 . other. T a b l e II  ascospores.  9 asc m u t a t i o n s were t e s t e d f o r a l l e l i s m t o each o t h e r and I t was found t h a t o n l y P243 and P393 were a l l e l i c t o each  In a d d i t i o n , a l l asc m u t a t i o n s were n o n - a l l e l i c t o m e i - 1 . shows the 8 l o c i w i t h a l l e l e o r 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 i n Chapter I I .  o f a s c - 1 , a s c - 3 and a s c - 6 are r e p o r t e d  These appear t o have a . d e f e c t d u r i n g m e i o s i s .  The  w i l d type gene o f a s c - 7 a l s o appears t o be n e c e s s a r y f o r m e i o s i s preliminary c y t o l o g i c a l observations  since  showed c l u s t e r e d n u c l e i a t the  i n t e r p h a s e o f the second m e i o t i c d i v i s i o n .  Moreover, s m a l l and l a r g e  TABLE  Locus  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 , w h o s e w i l d type a l l e l e s are n e c e s s a r y f o r the f o r m a t i o n of normal a s c i or b l a c k ascospores Allele I s o l a t i o n l\lo.  asc-1  P95  asc-2  P131  asc-3  P2*+3, P 3 9 3  asc-h  Nature %  Ascospore  of Defect Other  Abortion  1*0 - 70  Intermed Empty  •90-98  Very  to high  fertility*  asci low  Empty  P^tOO  Phenotype  fertility  perithecia  2 0 - 8 0  as c - 5  . P711  as c-6  PS79  70  Low  asc-7  , PS17  90  Low  asc-8  P961  50  to intermed  F e r t i l i t y was d e f i n e d a s t h e t o t a l n u m b e r o f a s c o s p o r e s ( b l a c k e j e c t e d from ,the p e r i t h e c i a ( s e e M A T E R I A L S AND M E T H O D S ) .  fertility  fertility  and w h i t e )  that  were  36  ascospores  were found i n t e r s p e r s e d i n the same a s c i .  the asc-5 mutation i s of a d i f f e r e n t nature.  The d e f e c t o f  C r o s s e s homozygous  t h i s mutation, produced'many a s c i w i t h 8 b l a c k ascospores a b l e number o f a s c i w i t h o n l y w h i t e a s c o s p o r e s . asci contained e i t h e r 4 or 8 spores.  c o u n t e r e d i n 11 c r o s s e s  Progeny a n a l y s i s  The l a t t e r type o f abortion  A range o f 50-80% was e n -  i n v o l v i n g ascospore  2 s t r a i n s , each m u l t i p l y marked f o r LG I, ascospores.  and a v a r i -  .The amount o f ascospore  v a r i e d w i d e l y between about 20 and 80%.  for  isolates.  A cross  between  produced about 25% a b o r t e d  of t h i s cross  i n d i c a t e d t h a t both 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 f r e q u e n c i e s were n o r m a l .  F i n a l l y , the  mutant a s c - 8 was not a n a l y z e d f u r t h e r . DISCUSSION A new system f o r the homozygosis  o f induced m u t a t i o n s has been  used to i s o l a t e r e c e s s i v e m u t a t i o n s w i t h a d e f e c t i n the s e x u a l c y c l e . S e l e c t e d PWT c u l t u r e s were screened f o r d e f e c t s i n the f o r m a t i o n o f p e r i t h e c i a , a s c i and v i a b l e a s c o s p o r e s .  The i n i t i a l  s c r e e n would a l s o  d e t e c t , i n a d d i t i o n to r e c e s s i v e m u t a t i o n s , those t h a t are o n l y e x p r e s s e d i n the maternal o r p a t e r n a l p a r e n t .  Among 1090 PWT c u l t u r e s  s c r e e n e d , the development o f 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 2 class  I and 2 c l a s s  III)  II).  In a d d i t i o n , 28 s t r a i n s  development o f a l l 8 c l a s s  was a f f e c t e d .  and  The d e f e c t i n p e r i t h e c i a l  I mutant s t r a i n s t e s t e d was o n l y  i n the p r o t o p e r i t h e c i a l p a r e n t .  study..  II  produced a p p a r e n t l y normal p e r i t h e c i a but the p r o d u c t i o n  o f a s c i o r v i a b l e ascospores  perithecial  (26 c l a s s  expressed  The stage o f the d e f e c t o f p r o t o -  development o f these mutants h a s - n o t been pursued i n  Of the two c l a s s  III  mutants w i t h a d e f e c t i n p e r i t h e c i a !  this  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 ( p e r - 1 )  and 1 d e -  f e c t i v e i n neck f o r m a t i o n (pen-1) were a l s o expressed o n l y i n the maternal p a r e n t .  Even though the c l a s s  II  mutant P446 produced n o r m a l -  l o o k i n g p e r i t h e c i a , the absence o f a s c i and i t s e x p r e s s i o n i n the maternal p a r e n t appear very s i m i l a r t o the c l a s s  I type m u t a n t s .  s i b l y the o n l y d i f f e r e n c e between t h i s , mutant and the c l a s s  I mutants  i s the s t a g e o f p e r i t h e c i a l development a t which the d e f e c t i s D e f e c t s i n the p r o d u c t i o n o f v i a b l e ascospores representing 8 loci tions.  Pos-  expressed.  in 9 strains,  ( a s c - 1 through a s c - 8 ) were due t o r e c e s s i v e muta-  In a d d i t i o n , even though the s c r e e n d i s t i n g u i s h e d between domin-  ant and r e c e s s i v e m u t a t i o n s , the r e c o v e r y o f 1 dominant m u t a t i o n  (SK)  among c u l t u r e s screened f o r r e c e s s i v e m u t a t i o n s was a consequence  of  i t s s p e c i f i c e f f e c t on ad-3A.  A c r o s s between SK and ad-3A produced  ascospore a b o r t i o n but n e i t h e r SK nor ad-3A d i d so when c r o s s e d w i t h w i l d type t e s t e r s t r a i n s  (see Chapter  III).  The r e c o v e r y o f r e c e s s i v e m u t a t i o n s a t 8 d i f f e r e n t l o c i s t r a t e the e f f e c t i v e n e s s o f the s e l e c t i o n p r o c e d u r e . types o f m u t a t i o n s were c l a s s i f i e d : (asc-4);  (2) a b o r t e d o r empty a s c i  demon-  Four d i f f e r e n t  (1) complete absence o f a s c i (asc-2);  (3) d e f e c t d u r i n g  meiosis  l e a d i n g t o a s c o s p o r e a b o r t i o n ( a s c - 1 , a s c - 3 , a s c - 6 and p r o b a b l y (4) a b o r t i o n o f a l l ascospores i n the r e m a i n i n g a s c i  (asc-5).  i n some a s c i but l i t t l e o r  asc-7);  no-abortion  The mutants i s o l a t e d d u r i n g t h i s  study  s h o u l d become i n s t r u m e n t a l i n the u n d e r s t a n d i n g o f the f o r m a t i o n 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 o f 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 o u s l y i s o l a t e d mutants t i n g the development o f p e r i t h e c i a ( W e i j e r and V i g f u s s e n ,  affec-  1972; V i g -  f u s s e n and W e 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 e x p r e s s e d i n a dominant f a s h i o n , i . e . , when o n l y one o f the p a r e n t s i n a s e x u a l c r o s s was mutant.  W h i l e some  were e x p r e s s e d i n e i t h e r p a r e n t o r i n t h e p a t e r n a l p a r e n t , most o f these mutants were e x p r e s s e d o n l y when used as the maternal p a r e n t . The p r e s e n t 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 s h o u l d be e s p e c i a l l y u s e f u l s i n c e no such mutants would have been d e t e c t e d i n p r e v i o u s mutant s c r e e n s .  Even though r e c e s s i v e mutants would  be d e t e c t e d i n the p r e s e n t s c r e e n i n g p r o c e d u r e , 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 e x -  pressed o n l y i n the maternal p a r e n t .  T h i s a n a l y s i s , t h e r e f o r e , empha-  s i z e s t h a t a t l e a s t the g r e a t m a j o r i t y o f l o c i r e q u i r e d f o r the d e v e l o p ment o f p e r i t h e c i a a c t o n l y i n t h e p r o t o p e r i t h e c i a l p a r e n t .  However,  the r e c e n t r e c o v e r y and c h a r a c t e r i z a t i o n ( J o h n s o n , 1979) o f a mutant which i s s t e r i l e as the female o r the male p a r e n t i n d i c a t e s t h a t some r e c e s s i v e m u t a t i o n s may be found i n t h i s c l a s s .  T h i s m u t a t i o n became  p a r t i a l l y r e c e s s i v e when both components, o f the c r o s s were h e t e r o k a r y o n compatible.  Such a m u t a t i o n would not have been d e t e c t e d i n the p r e -  s e n t s c r e e n because the t e s t e r s t r a i n s OR-A and 0R-a_, used t o d i s t i n g u i s h between dominance and r e c e s s i v e n e s s , were not h e t e r o k a r y o n comp a t i b l e w i t h the components o f the PWT s t r a i n s t e s t e d . PWT c a r r i e d t h e same a l l e l e s  Even i f the  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 r u e i n o n l y about o n e - e i g h t h 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 p r e s e n t .  To g e t  around the l a t t e r problem i t would be n e c e s s a r y t o 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 m u t a t i o n c o n f e r r i n g comp a t i b i l i t y w i t h the o p p o s i t e mating type ( G r i f f i t h s and DeLange, Formation o f A s c i i n  1978).  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 r e - m e i o t i c S phase and karyogamy  ( I y e n g a r e t _ a j _ . , 1977).  Therefore,  mutants which e i t h e r l a c k 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 defect during meiosis.  The absence o r e a r l y a b o r t i o n o f a s c i  (i.e.,  the p e r i t h e c i a a r e b a r r e n ) i s c h a r a c t e r i s t i c o f s e v e r a l 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 Neurospora which a r e s i m u l t a n e o u s l y d e f e c t i v e i n v e g e t a t i v e DNA r e p a i r ( u v s - 3 , u v s - 5 , u v s - 6 , . m e i - 3 ) . e s p e c i a l l y i n y e a s t and D r o s o p h i l a ,  1  Similar relationships,  have been w e l l - d o c u m e n t e d and i l l u s -  t r a t e an o v e r l a p i n f u n c t i o n s r e q u i r e d d u r i n g s o m a t i c maintenance  (e.g.,  r e p a i r o f DNA damage) and m e i o t i c r e c o m b i n a t i o n o r r e l a t e d p r o c e s s e s . The d e t e c t i o n o f mutants o n . t h e b a s i s o f t h e i r a b i l i t y to produce a s c i s h o u l d 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 a r e r e q u i r e d o n l y d u r i n g meiosis> and o f those which a r e also required for vegetative functions. sis  In a d d i t i o n , c y t o l o g i c a l a n a l y -  and the use o f t e m p e r a t u r e - s e n s i t i v e mutants would f a c i l i t a t e the  d e t e c t i o n o f temporal s t a g e s o f a c t i v i t y and f i n a l  blockage points  of  such m u t a n t s .  Formation o f V i a b l e A s c o s p o r e s . i n  Neurospora  The f a i l u r e o f the m a t u r a t i o n and v i a b i l i t y o f ascospores  may  be caused by the absence o f any p a r t o f the h a p l o i d chromosome complement  40  o r by the presence o f a mutant gene i n these ascospores see P e r k i n s and B a r r y , 1977).  Examples, o f the former k i n d are d e f i -  c i e n c i e s o f p a r t o f chromosomes o f whole chromosomes  (for review, ,  due t o r e a r r a n g e m e n t s , and d e f i c i e n c i e s ,  due t o n o n d i s j u c t i o n ( e . g . , m e i - 1 , me.i-4).  f a i l u r e o f m a t u r a t i o n o f ascospores mutants o r spore k i l l e r m u t a n t s .  The  may a l s o be due t o ascospore  color  In the p r e s e n t s t u d y a t h i r d type o f  mutant ( a s c - 5 ) was d e t e c t e d which was r e c e s s i v e and caused the a b o r t i o n of a l l ascospores  i n a number o f a s c i w i t h o u t a f f e c t i n g the r e m a i n i n g  asci. Mutants w i t h a d e f e c t i n the r e g u l a r s e g r e g a t i o n o f  chromosomes  have a l r e a d y been i n s t r u m e n t a l i n u n d e r s t a n d i n g 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 the f i r s t m e i o t i c d i v i s i o n . d e t e c t e d i n many s p e c i e s mutations  chromosomes  during  R e c e s s i v e m u t a t i o n s o f t h i s k i n d have been  ( f o r r e v i e w see Baker e t a l _ . , 1976a).  ( m e i - 1 , m e i - 4 ; P e r k i n s and B a r r y , 1977)  p r e v i o u s l y i n Neurospora c r a s s a .  Two such  have been d e t e c t e d  Many o f these mutants have a primary  d e f e c t i n r e c o m b i n a t i o n , the a b e r r a n t s e g r e g a t i o n merely b e i n g a c o n sequence o f u n i v a l e n t s produced through a l a c k o f exchange. s p o r a , exchange i s v i r t u a l l y e l i m i n a t e d i n c r o s s e s  homozygous  In  Neuro-  for  mei-1 ( S m i t h , 1975) due to an a l m o s t complete l a c k o f p a i r i n g o f homol o g s (Lu and G a l e a z z i , 1979). mutations t  n  e  asc-3  Some, o f the newly i s o l a t e d spore a b o r t i o n  ( a s c - 1 and a s c - 6 ) have s i m i l a r e f f e c t s on r e c o m b i n a t i o n , and m u t a t i o n causes d e f e c t s i n s e g r e g a t i o n t h a t are not a s s o c i a t e d  w i t h reduced exchange. and the r e s u l t s w i l l  These mutants have been a n a l y z e d more e x t e n s i v e l y  be d e s c r i b e d i n Chapter  II.  Ascospore a b o r t i o n o f the r e c e s s i v e a s c - 5 m u t a t i o n was a p p a r e n t l y not due to a n e u p l o i d y o f the i n v i a b l e ascospores  but r a t h e r t o an unknown  41  type o f i n v i a b i l i t y o f a l l ascospores  i n a proportion of a s c i .  The  l a r g e amount o f v a r i a t i o n i n e x p r e s s i o n o f t h i s m u t a t i o n may be e x p l a i n e d i f a t h r e s h o l d amount o f a substance w i t h i n each ascus i s q u i r e d f o r the m a t u r a t i o n o f a s c o s p o r e s .  re-  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 d y s f u n c t i o n caused by SD mutations i n D r o s o p h i l a 1971).  ( M i k l o s and S m i t h - W h i t e ,  The e f f e c t o f a s c - 5 on whole a s c i and the l a r g e amount o f  a b i l i t y o f e x p r e s s i o n between d i f f e r e n t c r o s s e s bubble a s c i  ( P e r k i n s and B a r r y , 1977).  portion of asci f a i l w h i t e spores  vari-  i s also reminiscent of  In each c a s e , a c e r t a i n p r o -  t o produce v i a b l e a s c o s p o r e s - - a s c i  w i t h aborted  f o r a s c - 5 and a s c i w i t h 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 a p p a r e n t e f f e c t on t h e r e m a i n i n g a s c i .  It  i s not known whether the bubble a s c 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 ascospore a b o r t i o n .  cause  In t h e case o f a s c o s p o r e c o l o r m u t a n t s , the mutant  ascospores  a b o r t whereas spore k i l l e r m u t a t i o n s cause the a b o r t i o n o f  ascospores  t h a t would be v i a b l e i n o t h e r c r o s s e s .  zygous c r o s s e s  r e s u l t i n 50% ascospore a b o r t i o n .  In a l l c a s e s ,  hetero-  The mutant SK(ad-3A)  was i s o l a t e d d u r i n g t h i s s t u d y and appears t o have c h a r a c t e r i s t i c s o f both a spore k i l l e r mutant and an ascospore c o l o r mutant.  T h i s mutant  has been a n a l y z e d i n more d e t a i l , and i s d e s c r i b e d i n Chapter  III.  F i n a l l y , f u t u r e s t u d i e s s h o u l d i n c l u d e t h e improvement o f the s e l e c t i o n system t o a l l o w f o r f a s t e r i s o l a t i o n o f PWT c u l t u r e s .  For  example, . m e i o t i c m u t a t i o n s w i t h i n c r e a s e d PWT f r e q u e n c e s c o u l d be used f o r t h i s purpose.  I n . a d d i t i o n , . , emphasis w i l l  of c o n d i t i o n a l mutants.  be put on t h e i s o l a t i o n  41a  CHAPTER  II  MEIOSIS IN NEUROSPORA CRASSA. II.  GENETIC AND CYTOLOGICAL CHARACTERIZATION OF FOUR MEIOTIC MUTANTS  42  INTRODUCTION The i s o l a t i o n o f e i g h t r e c e s s i v e m u t u a l l y complementing mutat i o n s which a f f e c t f e r t i l i t y i n Neurospora c r a s s a has been r e p o r t e d i n Chapter I.  M u t a t i o n s . i n two l o c i  ( a s c - 2 and a s c - 4 ) r e s u l t e d i n b a r r e n  p e r i t h e c i a ; the r e m a i n i n g s i x m u t a t i o n s  (asc-1, asc-3, asc-5,  a s c - 7 and a s c - 3 ) caused the a b o r t i o n o f many  asc-6,  ascospores.  That ascospore a b o r t i o n may be a very u s e f u l means o f d e t e c t i n g m u t a t i o n s w i t h a d e f e c t i n the r e g u l a r s e g r e g a t i o n o f chromosomes been suggested  by s t u d i e s done w i t h the mei-1 m u t a t i o n ( S m i t h ,  A p p r o x i m a t e l y 90% o f ascospores t i o n were a b o r t e d .  from c r o s s e s  homozygous  has  1975).  f o r t h i s muta-  T h i s a b o r t i o n was e v i d e n t l y due t o the i r r e g u l a r  s e g r e g a t i o n o f chromosomes which was caused by the absence o f d u r i n g the f i r s t m e i o t i c prophase i n aneuploid products. i c a l means o f a n a l y s i s  pairing  (Lu and G a l e a z z i , 1979) and r e s u l t e d  The s u c c e s s f u l  use o f both g e n e t i c and c y t o l o g -  o f such mutants was a l s o demonstrated i n those  studies. This paper r e p o r t s the g e n e t 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 o f the t h r e e ascospore a b o r t i o n ;mutations a s c - 1 , a s c - 3 and a s c - 6  (see  Chapter I)  segre-  and some new o b s e r v a t i o n s  g a t i o n o f chromosomes homozygous  on the mei-1 m u t a t i o n . . The  d u r i n g m e i o s i s was shown t o be d e f e c t i v e i n  f o r each o f these m u t a t i o n s .  In each c a s e , the  crosses  segregation  d e f e c t appeared t o .be a secondary consequence o f a p r i o r a b n o r m a l i t y . A d e f e c t i n the p a i r i n g o f homologous  chromosomes  i n crosses  homozygous  f o r a s c - 1 , a s c - 6 or mei-1 r e s u l t e d i n d e f e c t i v e s e g r e g a t i o n o f chromosomes d u r i n g the f i r s t and second d i v i s i o n s o f m e i o s i s  (and  possibly  43  the p o s t - m e i o t i c d i v i s i o n ) . o f most a s c i  i n crosses  In c o n t r a s t , the b l o c k i n the development  homozygous  f o r a s c - 3 was f o l l o w e d 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 s c i formed, d u r i n g the second and post-meiotic  divisions. MATERIALS AND METHODS  Alleles  Used The a l l e l e s on LG I used t o s e l e c t PWT c o l o n i e s and t o determine  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 f r e q u e n c i e s o f t h a t l i n k a g e group,  have  been d e s c r i b e d i n Chapter I.  in  crosses  homozygous  A l l e l e s used t o study m u l t i p l e disomy  f o r asc-6 a r e :  ad-3A ( 2 - 1 7 - 8 1 4 ) ; a u r (34508); a l - 2  (74A-Y112-M38); a c r - 2 (KH5); pdx (37803); c o t - 1 ( C 1 0 2 ( t ) ) ; his-1  (K141).  The l o c a t i o n o f t h e s e l o c i  inos  (37401);  on t h e i r l i n k a g e groups and  map d i s t a n c e s between l o c i , were a p p l i c a b l e , a r e i l l u s t r a t e d i n Other l o c i a r e :  al-1  (Y2198) and pan-1 asc-1  (P95), asc-3  (Chapter I ) .  (Car-10)_ on LG IR;  (5531) on IVR.  f o r mei-1 o r m e i - l ; a s c - 6 .  (27947) on IIR;  trp-4,  The m e i o t i c mutants mei-1 ( S m i t h ,  (P243) and a s c - 6  The ad-3B a l l e l e  arg-5  Fig:,2.  (P879) have been - p r e v i o u s l y  (2-17-128) was used i n c r o s s e s  1975),  described homozygous  F i n a l l y , the m u t a t i o n a ( 3 3 ) o f the mating m  type l o c u s i s h e t e r o k a r y o n c o m p a t i b l e w i t h s t r a i n s o f A mating type and still  permits crossing  t o such s t r a i n s  ( G r i f f i t h s and DeLange,  1978).  Strains The r e c e s s i v e ascospore a b o r t i o n (asc) m u t a t i o n s 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 u n - 3 , A, ad-3A, n i c - 2 , a l - 2 ) but homozygous groups (see F i g . 1 and Chapter I ) .  f o r the o t h e r l i n k a g e  Ascospore i s o l a t e s t r a i n s o f  genotypes  44.  Fig. 1  The two n u c l e a r components  o f PWT c u l t u r e s  which asc m u t a t i o n s were r e c o v e r e d .  in  centromere . „ Component 1  leu-3 ,  , Component 2  + ,  n  Approximate map d i s t a n c e s ( R a d f o r d , 1972)  + ,  un-3 , 10  0.1  a ,  arg-1 X  A ,  + , 10  + ad-3B , .  ad-3A , 9  LG  LG I  0.3  + , U  + ,  + •  nic-2 ,  al-2 L _ 28  IV  . , t o l  . t "  LG asc  1  a  5  C  A cross  homozygous  f o r a s c - 6 , designed t o t e s t  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 groups (LG IV, and V)  simultaneously.  I,  Linkage  A -i  a  a 15  group  II  I  +  aur  +  1  1  '  ad-3A  + 3D  Approximate map d i s t a n c e s (Radford, 1972)  al-2 1  asc B  +  cot-1  —i  1  pdx  +  a  inos  +  1  1  +  his-1  asc 2D  10  48  l e u - 3 , a_, a r g - 1 , ad-3'B; asc and u n - 3 , "AV ad-3A, n i c - 2 , a l - 2 ; asc  (each  c o n t a i n i n g the t o l m u t a t i o n and the h e t e r o k a r y o n c o m p a t i b i l i t y l o c i  £,  d_ and e) were o b t a i n e d from c r o s s e s between t h e mutant PWT c u l t u r e ( i . e . , homozygous f o r an. asc m u t a t i o n ) and w i l d t y p e s t r a i n s These ascospore  OR-A.  i s o l a t e s were i n t e r c r o s s e d and the LG.:I markers used t o  monitor crossover  Cytological  OR-a and  and n o n d i s j u n c t i o n f r e q u e n c i e s  (see F i g .  1).  Methods  The two s t a i n s about equal s u c c e s s .  i r o n - h e m a t o x y l i n and a c e t o - o r c e i n were used w i t h The methods d i f f e r e d m a i n l y i n t h e i r a b i l i t y  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 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  (iron-hematoxylin stains  neither).  (Raju and Newr-  meyer, 1977; Lu and G a T e a z z 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 .  Follow-  p e r i t h e c i a were washed o v e r n i g h t a t room temperature i n  a s o l u t i o n o f 3:1:1 solution).  both  Staining with i r o n -  h e m a t o x y l i n was done e s s e n t i a l l y as d e s c r i b e d p r e v o u s l y  ing h y d r o l y s i s ,  to  absolute ethanol:acetic acid:chloroform  T h i s procedure removed the f a t g l o b u l e s  (Carnoy's  from the c y t o p l a s m  and t h e r e f o r e a l l o w e d t h e v i s u a l i z a t i o n o f chromosomes  at a l l stages of  meiosis. 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 c o m b i n a t i o n o f s e v e r a l methods  ( G r i f f i t h s e t _ a j _ . , 1974; M. B a s l , p e r s o n a l  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 o f 6:3:1 acetic acid.  absolute  communication).  ethanol:chloroform:  The fiixed m a t e r i a l was l e f t a t room temperature  overnight  and then i n c u b a t e d a t -20°C f o r a p e r i o d o f 3 weeks t o 6 months. p r o l o n g e d i n c u b a t i o n h e l p s t o remove t h e f a t g l o b u l e s  This  from the c y t o p l a s m .  The f i x e d p e r i t h e c i a were then washed i n w a t e r , . h y d r o l y z e d i n 1 N HC1 f o r 4-5 minutes a t 60°C,^washed  a g a i n i n i c e water and immersed i n  leuco-basic  49  f u c h s i n f o r about 40 minutes a t room t e m p e r a t u r e .  Subsequent  methods  o f s t a i n i n g , v i e w i n g and photography were d e s c r i b e d p r e v i o u s l y ( G r i f f i t h s e t a l _ . , 1974). Methods used t o o b t a i n c l u s t e r s o f 8 a s c o s p o r e s , (Newcombe and G r i f f i t h s , 1972; P e r k i n s , 1974) manipulations  ( D a v i s and d e S e r r e s ,  i . e . , asci  and o t h e r r o u t i n e g e n e t i c  1970; Chapter I)  have been r e p o r t e d  previously. D e t e c t i o n o f Chromosome S e g r e g a t i o n Division  ( i . e . , During  D e f e c t s During the F i r s t M e i o t i c  MI)  In most c a s e s , n o n d i s j u n c t i o n o f LG I was measured.  This  link-  age group was m u l t i p l y marked t o a l l o w the d e t e c t i o n o f ascospore l a t e s t h a t c o n t a i n both complementing c o p i e s o f LG I.  Strains  iso-  i n which  complementation o f m u t a t i o n s on any p a r t o f a chromosome o c c u r s are a p p a r e n t l y w i l d type f o r these markers and are t h e r e f o r e c a l l e d pseudow i l d type  (PWT).  Nondisjunction of non-crossover  chromosomes, assuming  o f subsequent d i v i s i o n s , would r e s u l t i n ascospores on LG I ( a u x o j : +  PWT f o r a l l m u t a t i o n s  l e u - 3 , a_, a r g - 1 , ad-33 + u n - 3 , A, ad-3A, n i c - 2 , a l - 2 .  In c o n t r a s t , i f c r o s s o v e r chromosomes types w i l l  regularity  be e x p e c t e d :  are i n v o l v e d , the f o l l o w i n g pheno-  l e u ( l e u c i n e r e q u i r i n g ) and a u x o  +  ( w i l d type)  i f n o n d i s j u n c t i o n f o l l o w e d a s i n g l e c r o s s o v e r e v e n t i n the l e u - u n  region  (CO l e u - u n ) ; l e u , un and a u x o  (CO  (CO u n - a r g ) ;  +  a r q - c e n t r o m e r e ) ; ad n i c a l , ad and a u x o auxo* (CO a d - n i c ) ; al. and a u x o young PWT ascospores  +  +  l e u a r g , un and a u x o  +  (CO c e n t r o m e r e - a d ) ; n i c a l and  (CO n i c - a l ) .  The d i s o m i c n u c l e i  soon h a p l o i d i z e ( P i t t e n g e r , 1954; Chapter  in I).  T h e r e f o r e , these s t r a i n s c o n t a i n a t l e a s t two complementing h a p l o i d  50  nuclear types.  The h e t e r o k a r y o t i c n a t u r e . o f such s t r a i n s was, i n many  c a s e s , c o n f i r m e d by t e s t i n g the genotypes o f i n d i v i d u a l c o n i d i a l  iso-  lates. Even(though  a l l o f the above mentioned h e t e r o k a r y o t i c s t r a i n s  a r e PWT f o r a t l e a s t some a u x o t r o p h i c m u t a t i o n s , o n l y those PWT p r o geny i n which a l l a u x o t r o p h i c m u t a t i o n s on a p a r t i c u l a r l i n k a g e  group  complement ( i . e . , a u x o ) were i n c l u d e d i n the e s t i m a t i o n o f PWT f r e +  quencies i n crosses  homozygous  f o r a s c - 1 , a s c - 6 and m e i - 1 .  f r e q u e n c i e s were determined i n one o f two ways: v i d u a l ascospore (only auxo  +  The PWT  ( i ) by t e s t i n g  i s o l a t e s , o r ( i i ) by p l a t i n g ascospores  indi-  on both minimal  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 In one case ( c r o s s e s  homozygous  f o r a s c - 6 ) the  grow). simultaneous  n o n d i s j u n c t i o n o f more than one chromosome was d e t e r m i n e d .  Complement-  i n g m u t a t i o n s on LG I (A and a_ mating type a l l e l e s ) , LG IV  (pdx and c o t ) ,  and LG V ( i n o s and h i s ) were used t o d e t e c t PWT progeny f o r these l i n k age groups (see F i g . 2; a u r , a l - 2 , ad-3A and a c r m u t a t i o n s i n the s t r a i n s used a r e not r e l e v a n t i n the p r e s e n t  study).  D e t e c t i o n o f Chromosome S e g r e g a t i o n D e f e c t s D u r i n g the Second M e i o t i c Division  ( i . e . , During  Mil)  N o n d i s j u n c t i o n a t M i l can be d e t e c t e d through complementation of closely linked mutations.  Two such complementing m u t a t i o n s can o n l y  be p r e s e n t 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 -  i n g an exchange event between these m u t a t i o n s and the c e n t r o m e r e .  Con-  s e q u e n t l y , n o n d i s j u n c t i o n a t M i l can o n l y be d e t e c t e d f o l l o w i n g such an exchange event and w i l l  be d e t e c t e d more f r e q u e n t l y t h e f u r t h e r the  51  complementing m u t a t i o n s are from the c e n t r o m e r e . Even though the d e t e c t i o n o f n o n d i s j u n c t i o n d u r i n g both MI and M i l depends on the complementation between c l o s e l y l i n k e d m u t a t i o n s , these two types o f events can u s u a l l y be d i s t i n g u i s h e d , i f mutant markers a r e p r e s e n t on both s i d e s o f the c e n t r o m e r e .  In the case o f  nondis-  j u n c t i o n a t M i l , mutant markers on one s i d e o f the c e n t r o m e r e , but not the o t h e r , complement.  In c o n t r a s t , n o n d i s j u n c t i o n a t MI u s u a l l y  results  i n the complementation o f m u t a t i o n s on both s i d e s o f the centromere (Fig.  3).  Recombinant Frequency (RF)  i n Crosses w i t h a High Amount o f N o n d i s j u n c t i o n  Nondisjunction Mainly 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 o f recombinants i s g e n e r a l l y equated t o the p r o p o r t i o n o f recombinant chromatids.  T h i s v a l u e i s r e a d i l y determined among h a p l o i d p r o -  geny by t e s t i n g f o r the presence o f s p e c i f i c marker m u t a t i o n s .  However,  the d e t e c t i o n o f recombinant chromatids i n PWT progeny i s o f t e n more comp l i c a t e d . . To d e t e r m i n e t h i s v a l u e , the n i c - a l  r e g i o n (30-35 mu) was  used.  A s c i w i t h a c r o s s o v e r event i n t h i s r e g i o n , f o l l o w e d by n o n d i s j u n c t i o n a t MI, would produce o n e - f o u r t h a l b i n o (aj_) and t h r e e - f o u r t h s orange PWT progeny  (assuming r e g u l a r subsequent d i v i s i o n s ) .  S i m i l a r asci  (al ) +  in  which chromosomes  d i s j o i n i n a r e g u l a r manner would produce o n e - h a l f r e -  combinant progeny  ( f o r the n i c - a l  region).  T h e r e f o r e , the frequency o f  recombinant c h r o m a t i d s f o r PWT progeny would be t w i c e the frequency o f a l b i n o PWT progeny  (= 2x aj_ PWT/total PWT).  The frequency o f recombinant chromatids i s u s u a l l y i d e n t i c a l the c r o s s o v e r  frequency i n a p a r t i c u l a r r e g i o n .  to  T h i s i s p a r t i c u l a r l y so  i f , as i n N e u r o s p o r a , a l l chromatids from each m e i o s i s a r e r e c o v e r e d from  52  Fig. 3  Expected PWT progeny r e s u l t i n g from n o n d i s j u n c t i o n d u r i n g 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 r o s s o v e r f o l l o w e d by n o n d i s j u n c t i o n d u r i n g the second m e i o t i c d i v i s i o n  (b).  53  leu  (b)  ad-3B  arg  leu  arg  f  MI  ad-3A arg  leu  ad-3A  un  i  un  I  nic  al  IMondis ad-3B j u n c t i o n  n  ad-3B  nic  al  nic  al  nic  al  MI leu  arg  ad-3B  leu  arg  ad-3A and  ad-3A  un  nic  al  Mil  leu  arg  un  ad-3B  un  ad-3A  Mil  ad-3B  Nondisjunction  leu ad-3A  nic  arg  al  or  (PUT)  Note: some c r o s s o v e r t y p e s are a l s o recovered (see M a t e r i a l s and M e t h o d s )  0  ad-3B ad-3A n i c a l ( l e u , arg) ad-3B  un o  ad-3A  nic  al  (un) Note: exchange between the c e n t r o m e r e a n d a r g - 1 U J i 11 produce n o n d i s j u n c t i o n progeny 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 c r o s s e s where n o n d i s j u n c t i o n takes p l a c e ,  a f r a c t i o n o f ascospores recovered.  i s i n v i a b l e and many chromatids w i l l  not be  In such c a s e s , the f r e q u e n c y o f recombinant chromatids may  not be the same as c r o s s o v e r  frequency, since crossover or  chromatids might be p r e f e r e n t i a l l y i n c l u d e d i n the v i a b l e For example, a d e f e c t i n p a i r i n g o f chromosomes  non-crossover progeny.  a t the f i r s t  prophase  o f m e i o s i s g e n e 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 DISCUSSION).  S i n c e the l a c k o f p a i r i n g e x c l u d e s the p o s s i b i l i t y o f  crossing-over,  i t s h o u l d be m a i n l y the n o n - c r o s s o v e r  chromatids  that  would be i n v o l v e d 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 r e f e r e n t i a l l y e x c l u d e d from t h e v i a b l e progeny.  Thus, i n these c a s e s ,  recombinant f r e q u e n c y would be h i g h e r than the a c t u a l c r o s s o v e r quency.  S i n c e t h e r e appears t o be no e v i d e n c e s u g g e s t i n g  that  chromosomes might be p r e f e r e n t i a l l y e x c l u d e d from the v i a b l e  frecrossover  progeny,  the recombinant f r e q u e n c y i n a c r o s s w i t h n o n d i s j u n c t i o n d u r i n g MI be c o n s i d e r e d an o v e r e s t i m a t e of the c r o s s o v e r f r e q u e n c y .  should  Consequently,  any r e d u c t i o n i n recombinant f r e q u e n c y s h o u l d r e p r e s e n t a r e a l in crossover  the  decrease  frequency.  The o v e r a l l f r e q u e n c y o f recombinant chromatids from both PWT and non-PWT progeny c o u l d be determined by combining the i n d i v i d u a l RF v a l u e s o f t h e s e two types o f progeny.  However, the r e l a t i v e c o n t r i b u t i o n  o f the frequency o f recombinant c h r o m a t i d s , which i s d i f f e r e n t f o r PWT and non-PWT progeny d i s o m i c f o r LG I,  (see RESULTS), i s i n doubt.  The o r i g i n a l n u c l e i  g i v i n g r i s e t o PWT progeny, and those h a p l o i d f o r LG  p r o d u c i n g non-PWT progeny, many be d i f f e r e n t i a l l y i n c l u d e d 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 f r e q u e n c y o f  I,  55  recombinant c h r o m a t i d s which does not c o r r e c t l y r e f l e c t c r o s s o v e r quency.  fre-  T h e r e f o r e , the o v e r a l l recombinant frequency was not d e t e r m i n e d . N o n d i s j u c t i o n During t h e Second M e i o t i c D i v i s i o n  The r e -  (Mil).  combinant f r e q u e n c y (RF) i n any r e g i o n e q u a l s the p r o p o r t i o n o f recombinant chromatids.  In c r o s s e s w i t h a h i g h frequency o f n o n d i s j u n c t i o n a t  M i l , the l a r g e number o f r e s u l t a n t d i s o m i c progeny p r e v e n t the c a t i o n o f some recombinant c h r o m a t i d s .  identifi-  In a d d i t i o n , s i n c e the f r e q u e n c y  o f d i s o m i c n u c l e i - i s unknown, i t i s not p o s s i b l e t o d i r e c t l y d e t e r m i n e the t o t a l  number o f chromatids among the progeny from such a c r o s s .  T h e r e f o r e , to o b t a i n a more a p p r o p r i a t e e s t i m a t e o f RF, o n l y a s c i  con-  t a i n i n g a c r o s s o v e r i n a w e l l - m a r k e d r e g i o n s h o u l d be c o n s i d e r e d .  The  f r e q u e n c y o f d i s o m i c and h a p l o i d progeny from such a s c i can be e s t i mated s i n c e a l l d i s o m i c n u c 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) p r o d u c t s and h a l f the h a p l o i d n u c l e i a r e recombinant (CO).  In the  p r e s e n t s t u d y , the RF v a l u e was determined i n a r e g i o n which was marked on one s i d e by the c l o s e l y l i n k e d m u t a t i o n s un-3 and a r g - 1 , and on the o t h e r s i d e by ad-3A and ad-3B ( F i g . 3 ) .  Deviations  produced by  cross-  4 overs between un-3 and a r g - 1  (1-2 mu)  o r ad-3A and ad-3B (0.3 mu)  s h o u l d be c o n s i d e r e d very minor s i n c e the a r g - a d r e g i o n spans 15-20 mu. The subgroup o f a s c i w i t h a c r o s s o v e r i n the a r g - a d r e g i o n p r o duces a s c o s p o r e s which a r e h a p l o i d f o r LG I, f o r LG I.  H a l f the h a p l o i d ascospores  disomies w i l l  will  and those t h a t a r e d i s o m i c be recombinant (CO), and the  c a r r y a p a r e n t a l and a c r o s s o v e r chromosome  (P + CO).  The  T h i s low RF v a l u e was c o n s i s t e n t l y o b t a i n e d d u r i n g t h i s s t u d y . The d e v i a t i o n from the v a l u e p r e v i o u s l y r e p o r t e d ( R a d f o r d , 1972) i n F i g . 1 may w e l l be due t o r e c - t y p e genes (see e . g . , C a t c h e s i d e , 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 t h e s e a s c i , t h e f r e -  quency o f HK progeny e q u a l s t h e f r e q u e n c y o f d i s o m i c n u c l e i . v a l u e e q u a l s HK/HK + 2 CO ( o r d i s o m i c / d i s o m i c + h a p l o i d ) .  This  Assuming  t h a t t h i s f r e q u e n c y o f d i s o m i c n u c l e i i s t h e same i n a l l types o f  asci,  r e g a r d l e s s o f c r o s s o v e r events i n s p e c i f i c r e g i o n s , t h i s f r e q u e n c y would a p p l y to a l l progeny from t h e c r o s s .  T h e r e f o r e , the t o t a l  number o f d i -  somic a s c osp o r e i s o l a t e s would be:  HK (  S i m i l a r l y , the t o t a l  H K  2  +  co^  x  P °9eny. r  t o t a l  number o f h a p l o i d a s c o s p o r e i s o l a t e s would be: ,  (  2 CO H K  +  2  v CO^  . . , progeny.  x  These v a l u e s e n a b l e the e s t i m a t i o n o f the t o t a l  number o f c h r o m a t i d s  o b t a i n e d from a c r o s s :  2 ( d i somics) + h a p l o i d s = ( ^  ^, G Q - HK + 2 CO^ +  +  = HK + 22 CCOO ^  X  l  x  total  progeny  P °9 yr  t o t a 1  e n  S u b s e q u e n t l y , t h e RF v a l u e i n a r e g i o n t h a t a l l o w s the d i s t i n c t i o n b e tween CO and HK progeny would be: CO + HK 2 (HI' + r n )  PP  ^HK + 2 _  CO ^  (?;recombinant  x  t  o  t  a  l  P  r o  9  e n  y  (  =  total  chromatids)  chromatids)  2 x t o t a l progeny. HK '+ 2 CO  I n . t h e p r e s e n t s t u d y , the CO v a l u e was o b t a i n e d u s i n g t h e u n - n i c r e g i o n . However, HK progeny c o u l d o n l y be d e t e c t e d f o l l o w i n g a c r o s s o v e r i n the  57  s l i g h t l y smaller arg-ad r e g i o n .  T h e r e f o r e , s i n c e a low e s t i m a t e f o r  HK was used, the RF v a l u e o f t h e u n - n i c r e g i o n o b t a i n e d by t h i s method would be a s l i g h t l y low e s t i m a t e . RESULTS Four r e c e s s i v e m u t a t i o n s  ( a s c - 1 , a s c - 3 , a s c - 6 and m e i - 1 ) which  cause the a b o r t i o n o f some o f t h e i r a s c o s p o r e s , were a n a l y z e d . c a s e , the a b o r t e d ascospores The f e r t i l i t y o f c r o s s e s  were w h i t e i n s t e a d o f b l a c k , and i n v i a b l e .  homozygous  f o r each r e c e s s i v e m u t a t i o n was  n a t u r a l l y always reduced due t o t h e i n v i a b l e a s c o s p o r e s . t h i s c h a p t e r , the term " f e r t i l i t y " ber o f ascospores  In each  (black or w h i t e ) .  However,  i s used t o d e s i g n a t e the t o t a l  num-  An a r b i t r a r y measure o f low (when  very few spores a r e p r o d u c e d ) , medium, o r h i g h f e r t i l i t y  i s employed.  As w i l l be shown, the ascospore a b o r t i o n i n a l l f o u r m u t a t i o n s i s by i r r e g u l a r s e g r e g a t i o n , o r i i n o n d i s j u c t i o n , o f chromosomes. such i r r e g u l a r s e g r e g a t i o n o f chromosomes sence o f PWT progeny  in  In  caused  Neurospora,  can be d e t e c t e d by the p r e -  (see MATERIALS AND METHODS).  PWT progeny a r e de-  t e c t e d through t h e i r complementation o f some o r a l l a u x o t r o p h i c m u t a t i o n s . The t h r e e n e w l y - i s o l a t e d asc m u t a t i o n s (see Chapter I) w i l l discussed  i n o r d e r Of t h e i r a p p a r e n t i n c r e a s i n g c o m p l e x i t y and s t a g e  d e f e c t . i n the s e g r e g a t i o n o f chromosomes: (second d i v i s i o n ) ; and a s c - 1 some new o b s e r v a t i o n s asc-6  be  asc-6  (first division);  ( f i r s t and second d i v i s i o n s ) .  of  asc-3  Finally,  o f t h e mei-1 m u t a t i o n w i l l be r e p o r t e d .  (P379) Crosses homozygous  f o r t h i s recessive mutation generally r e s u l t e d  i n about 70% ascospore a b o r t i o n and a r e d u c t i o n i n f e r t i l i t y .  The amount  58  o f spore a b o r t i o n was q u i t e c o n s t a n t i n a l l c r o s s e s t e s t e d but f e r t i l i t y v a r i e d from low t o medium ( T a b l e All  initial  I).  s t r a i n s c a r r y i n g a s c - 6 grew about t h r e e times as  s l o w l y as w i l d type s t r a i n s .  I t was found t h a t t h i s slow growth was  due to a p o i n t m u t a t i o n ( s l o )  l i n k e d t o the m e i o t i c m u t a t i o n s  t i o n on mapping). phenotype o f  (see  sec-  The s l o m u t a t i o n , however, i n no way a f f e c t e d the  asc-6.  Recombination and N o n d i s j u n c t i o n F r e q u e n c i e s .  The a n a l y s i s  of  ascospore c u l t u r e s from f o u r d i f f e r e n t c r o s s e s homozygous f o r ' a s c - 6  re-  v e a l e d a d r a s t i c a l t e r a t i o n i n both RF and n o n d i s j u n c t i o n f r e q u e n c y (Table I ) .  A h i g h frequency o f PWT progeny, r a n g i n g from 16.4 to  was r e c o v e r e d from a l l f o u r c r o s s e s .  I t t h e r e f o r e appears  t h a t these PWT c u l t u r e s a r e the r e s u l t o f n o n d i s j u n c t i o n d u r i n g Three r e g i o n s on LG I were m o n i t o r e d f o r RF: Compared t o w i l d type c r o s s e s ,  MI.  leu-un, un-nic  the RF v a l u e s from a l l f o u r  c r o s s e s homozygous f o r a s c - 6 were reduced i n a l l t h r e e r e g i o n s The Nature o f PWT Progeny.  ;  The g r e a t m a j o r i t y o f t h e s e were  w i l d type f o r a l l a u x o t r o p h i c m u t a t i o n s on LG I.  and n i c - a l .  37%,  (Table  I).  To e s t a b l i s h the n u c l e a r c o m p o s i -  t i o n o f the PWT progeny from these c r o s s e s ,  the genotypes  of c o n i d i a l  i s o l a t e s from ten PWT progeny, r e c o v e r e d from the c r o s s between s t r a i n s 879al3 and 879A15, were d e t e r m i n e d . i.e.,  In each c a s e , the two p a r e n t a l  un_, a d , n i c , a l and l e u , a r g , a d , were r e c o v e r e d .  types,  T h e r e f o r e , 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 e x p e c t e d 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 o f a few c r o s s o v e r  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 t h somatic exchange events  ( P i t t e n g e r and C o y l e , 1963).  Table  I.  Genetic  analysis  of f o u r  crosses  homozygous  f o r asc-6 PUT  IMon-PLiT progeny Total non-PUT progeny Cross  Recombination f r e q u e n c y (%) (no. r e c o m b i n a n t s i n parentheses) leu-un un-nic n i c -a l  PUT freq. (%)  Total PWT progeny  progeny  Genotypes o f PUT's  Fertility  auxo  other  (no. o f spores)  0  low  Q22-2 x Q22-8*  95  1.1  (1)  6.5  (6)  11.4 (11)  45  32.1  45  Q8-1  63  3.2  (2)  7.9  (5)  12.7 (8)  37  37.0  35  81  2.5  (2)  6.2  (5)  13.6 (11)  33  29.0  33  Q35-1  x Q8-2* x 035-2*  61  W i l d type c r o s s e s  6.5  (4)  11-17  •  (0)  15-20  (2)  3.3  16.4  12  among PUT  progeny  = 2x  freq.  ' low  0  < 0.1  30-35  a c r o s s between  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 t i c a l f o r a l l 4 c r o s s e s (see F i g . 1 ) . RF(nic-al)  12  med med  0 1.7*  A_ a n d _a c o m p o n e n t s f r o m a PUT c u l t u r e f r o m a l ) a n d 8 7 9 A 1 5 ( l e u , a_, a r g , a d ; a s c - 6 ) .  ^  2(1 a l & 1 l e u , a r c  12.5  R F C n i c - a l ) of 3 c r o s s e s combined: S79a13 x 879A15  +  of albino  and  PUT s 1  strain  Methods,  1 - 3 4 - 8 ( u n , A_, a_d, n i c , LG  I markers  (see M a t e r i a l s  and  were  Methods).  iden-  60  The absence o f c r o s s o v e r  chromosomes  may s u g g e s t t h a t o n l y non-exchange  from the ten PWT c u l t u r e s  chromosomes  t h e r e f o r e i n c l u d e d i n the PWT progeny.  fail  To o b t a i n a more q u a n t i t a t i v e  measure o f the f r e q u e n c i e s o f exchange chromosomes progeny,  the RF v a l u e s  types o f progeny  i n the n i c - a l  from t h r e e c r o s s e s  t o d i s j o i n and are  among PWT and non-PWT  r e g i o n were determined f o r both (Table I ) .  The v a l u e f o r PWT p r o -  geny (1.7% = 2 x frequency o f a l b i n o PWT's; see MATERIALS AND METHODS) was much lower than t h a t f o r non-PWT progeny  (12.5%).  It is  c o n c l u d e d t h a t most n o n d i s j u n c t i o n i n v o l v e s non-exchange Ascus A n a l y s i s .  therefore  chromosomes.  Unordered a s c i from a c r o s s homozygous f o r a s c - 6  were a n a l y z e d i n two ways.  F i r s t , a l l types o f ascus a b o r t i o n  were r e c o v e r e d (Table I I ) .  The p r e v a l e n c e o f a s c i w i t h an even number o f  b l a c k ascospores  (26/37) suggests t h a t the d e f e c t l e a d i n g t o  a b o r t i o n takes p l a c e p r i o r to the p o s t - m e i o t i c d i v i s i o n . f r e q u e n c y o f a s c i w i t h odd numbers o f b l a c k ascospores  However,  PWT ascospore  isolate.  These seven a s c i and the genotypes  o f the r e -  of t h e i r  as-  The presence o f an odd number  i n s i x o f these a s c i s t r o n g l y s u g g e s t s t h a t  l o s s o r secondary  (11/37)  Seven out o f 37 a s c i c o n t a i n e d a t l e a s t one  cospore c u l t u r e s are shown i n T a b l e I I I . o f PWT progeny  the  Second, the b l a c k  from each ascus were germinated and the genotypes  s u l t i n g c u l t u r e s determined.  ascospore  i s too high  f o r complete r e g u l a r i t y o f t h i s p o s t - m e i o t i c d i v i s i o n . ascospores  patterns  chromosome  n o n d i s j u n c t i o n takes p l a c e d u r i n g the p o s t - m e i o t i c  division. Recombination and N o n d i s j u n c t i o n F r e q u e n c i e s somes Other Than LG I. f o r LG I.  Involving  Chromo-  Thus f a r , n o n d i s j u n c t i o n has o n l y been r e c o r d e d  To i n v e s t i g a t e the degree o f n o n d i s j u n c t i o n  (and r e d u c t i o n i n  Table  I I . Ascus a n a l y s i s of a c r e s s (879a13 x 879A15) Types y K  (black:white  of Asci  M L , Number  C  N  8 : 0  1  6  3  : 2  2  2  : 6  14  0 : 8 (mostly Total  6 1:7,  3:5)  f o r asc-6  „ . , , , of Asci Observed  ascospores)  4 : 4  other  homozygous  11 37  62  Table  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 (S79a13 x 879A15) homozygous f o r a s c - 6 , which c o n t a i n a t l e a s t o n e PUT c u l t u r e  Type o f A s c u s (B:W)  No. I s o l a t e s Germinated  PWT  Genotype of I s o l a t e s l e u , a r g , ad un-, a d , 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  1  .1  5 ::• 3  -  • •  •  63  r e c o m b i n a t i o n ) o f o t h e r chromosomes, the s i m u l t a n e o u s a n a l y s i s o f LG I,  a c r o s s was a n a l y z e d which enabled  IV and V ( F i g . 2 ) .  f o r h e t e r o z y g o s i t y a t the mating t y p e l o c u s cot-1  , and LG V f o r i n o s  and h i s - 1  .  LG I was t e s t e d  (A/a_), LG IV f o r p d x  and  +  In the l a t t e r two l i n k a g e groups  the f r e q u e n c y o f r e c o m b i n a t i o n was approximated by t h e appearance o f the double mutant ( e . g . , pdx, c o t - 1 ) . binants  The extreme r a r i t y o f such recom-  (1/165 pdx, c o t - 1 , - a n d 0/165 i n o s , h i s - 1 )  shows t h a t r e c o m b i n a -  t i o n i s reduced i n a l l l i n k a g e groups (compare approximate map d i s t a n c e s i n w i l d type c r o s s e s :  p d x - c o t - 1 : .20 mu; i n o s - h i s - 1 :  t i o n , this indicates that p r a c t i c a l l y a l l pdx , c o t - l +  inos , h i s - l +  +  10 mu). +  In a d d i -  (71/165), and  (79/165) i s o l a t e s a r e d i s o m i c f o r LG IV and V, r e s p e c t i v e l y .  T a b l e IV shows the d a t a on the 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 these t h r e e chromosomes  i n two d i f f e r e n t c r o s s e s  homozygous  for  The r e s u l t s r e v e a l s e v e r a l a s p e c t s o f n o n d i s j u n c t i o n i n these i)  A l l t h r e e chromosomes  crosses:  t e s t e d show a h i g h degree o f n o n d i s j u n c t i o n .  i i ) , The f r e q u e n c i e s o f n o n d i s j u n c t i o n o f the t h r e e chromosomes p a r t i c u l a r cross  are very s i m i l a r .  i n each  However, n o n d i s j u n c t i o n 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 c r o s s e s  ( 3 3 . 3 , 35.0 and 38.3%  i n one c r o s s , and 5 4 . 3 , 47.6 and 53.3% i n the o t h e r iii)  asc-6.  cross).  The number o f i s o l a t e s e i t h e r s i m u l t a n e o u s l y PWT o r non-PWT  for  a l l t h r e e l i n k a g e groups t e s t e d i s s i g n i f i c a n t l y h i g h e r than e x pected (p < 0 . 0 1 ) .  T h i s may mean t h a t chromosomes  i n d e p e n d e n t l y o f each o t h e r .  do not d i s j o i n  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 a g a i n s t i s o l a t e s w i t h some PWT and some non-PWT groups.  linkage  That t h e 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 p e r -  centage g e r m i n a t i o n i n these c r o s s e s  (47.8,  50.0 and 5 7 . 5 % ) .  In  Table  IV.  Hyperploid I IV7  Simultaneous nondisjunction homozygous f o r asc-6  f o r LG U  crosses  C r o s s 129-79 x 128-15 Number o f P r o g e n y Expected* Observed (E) (•) 2.7  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  32.9  60  60.0  28  +  +  -  9  -  +  -  •+  + -  105 *  (0-E) V E  i n two  8  +  _  groups  12.6  +  "  linkage  C r o s s 129 - 7 5 x 1 2 8 - 1 5 Number o f P r o g e n y Expected* Observed (E) (0)  +  +  of three  14.5  105.1  X  2 H  =  f  7  Determined from p r o d u c t of n o n d i s j u n c t i o n f r e q u e n c y of i n d i v i d u a l l i n k a g e groups ( L G I : 0 . 5 4 3 , LG IV: • .4 7 6 , LB V: 0. 5 3 3 f o r c r o s s 1 2 9 - 7 5 x 1 2 8 - 1 5 ; and LG I : • . 3 3 , LG I V : 0 . 3 5 0 , LG V: 0 . 3 8 3 f o r c r o s s 1 2 9 - 79 x 1 2 8 - 1 5 ) .  65  c o m b i n a t i o n w i t h d a t a on c o l o n y - f o r m i n g a b i l i t y o b t a i n e d from the mutant a s c - 1 (see below, and T a b l e X ) , t h e s e l e c t i v e h y p o t h e s i s seems most a p p e a l i n g (see a l s o Cytology.  DISCUSSION).  C y t o l o g i c a l observations of crosses  a s c - 6 show a d r a s t i c r e d u c t i o n i n p a i r i n g o f homologs (Fig. 4a, b ) . chromosomes.  homozygous  for  a t pachytene  Some p a i r i n g was observed m a i n l y near t h e t i p s o f some T h i s r e d u c t i o n i s r e f l e c t e d i n a d r a s t i c decrease i n t h e  number o f recombinants o b t a i n e d from these  crosses.  An apparent consequence o f reduced p a i r i n g and exchange i s the p r o d u c t i o n o f u n i v a l e n t s d u r i n g d i a k i n e s i s and metaphase I. shows two s e p a r a t e chromosomes  F i g . 4c  a t t a c h e d t o the n u c l e o l u s a t d i a k i n e s i s .  These appear t o be two u n i v a l e n t s o f the same chromosome.  Instead of  seven b i v a l e n t s , up t o 14 u n i v a l e n t s can be d e t e c t e d on the s p i n d l e o f metaphase I ( F i g . 4 e ) .  Subsequent d i v i s i o n d i s t r i b u t e s r o u g h l y equal  amounts o f c h r o m a t i n t o o p p o s i t e p o l e s a n d , a t prophase I I , ( i n s t e a d o f t h e usual seven) Fig. 4f, g).  up t o 14  chromosomes m a t e r i a l i z e i n each dyad nucleus.  T h i s o b s e r v a t i o n i s c o m p a t i b l e w i t h the e q u a t i o n a l  divi-  s i o n o f many u n i v a l e n t s d u r i n g t h e 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 l o n g time t o c o m p l e t e ; whereas f i g u r e s a t t h i s s t a g e o f d i v i s i o n were observed o n l y r a r e l y i n w i l d type c r o s s e s , were q u i t e common i n c r o s s e s  homozygous  f o r asc-6  (Fig. 4h-k).  they  Finally,  unequal amounts o f c h r o m a t i n o f t e n s e g r e g a t e d u r i n g t h i s and the p o s t m e i o t i c d i v i s i o n l e a d i n g t o the d i f f e r e n t types o f ascospore  abortion.  In c o n c l u s i o n , the primary d e f e c t o f mutant a s c - 6 appears t o be a d e f e c t d u r i n g t h e p a i r i n g o f homologs  a t t h e f i r s t prophase o f m e i o s i s .  T h i s r e s u l t s i n a d r a s t i c decrease o f r e c o m b i n a t i o n and the p r o d u c t i o n  66  Fig.  4  Chromosome development i n c r o s s e s asc-6.  homozygous  for  P i c t u r e s ( f ) , ( g ) , and (h) were s t a i n e d  w i t h Feulgen and a c e t o - o r c e i n ; a l l o t h e r p r e p a r a - . t i o n s were s t a i n e d w i t h i r o n - h a e m a t o x y l i n . chromosomes  fail  to p a i r a t pachytene  x3400; some r e g i o n s ((a) arrowed).  Most  (a,b)  can be seen t o p a i r  though  At d i a k i n e s i s , the two homologs  o f the n u c l e o l a r chromosome may be s e p a r a t e l y a t t a c h e d t o the n u c l e o l u s  (c) x3400; f i g u r e s  (c)  and (d) r e p r e s e n t one ascus a t two d i f f e r e n t f o c i . A t metaphase I, (e)x3400,  up t o 14 u n i v a l e n t s may be seen  and a s i m i l a r number o f chromosomes  (7-  14) i n d i v i d u a l i z e d u r i n g the f o l l o w i n g prophase each dyad nucleus  (f,g)  x 1300.  in  A long p e r i o d  necessary t o c o m p l e t e ' t h e second d i v i s i o n was  sug-  g e s t e d by the u n u s u a l l y l a r g e number o f n u c l e i a t t h i s stage o f d i v i s i o n  ( i , j , k ; spindle-pole  bodies  are arrowed) x2400; t h i s may sometimes cause some spindle overlap ( j ) .  That the m a t e 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 r e p a r a t i o n s made w i t h a c e t o - o r c e i n s t a i n which o n l y stains  DNA (h)  xl300.  68  o f many u n i v a l e n t s a t the f i r s t metaphase.  The h i g h frequency o f PWT  progeny suggests t h a t n o n d i s j u n c t i o n takes, p l a c e d u r i n g the meiotic d i v i s i o n .  first  The mechanism o f such n o n d i s j u n c t i o n may be, a t  l e a s t i n p a r t , the e q u a t i o n a l s e p a r a t i o n 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 o f m e i o s i s and abnormal  segre-  g a t i o n a t the p o s t - m e i o t i c d i v i s i o n a c c o u n t f o r a l a r g e amount o f the ascospore a b o r t i o n . asc-3  (P243, P393) Crosses homozygous  duced very few ascospores  f o r t h i s recessive mutation g e n e r a l l y proa n d , o f those produced, 90-98% were w h i t e and  i n c a p a b l e o f g e r m i n a t i o n and growth.  Crosses homozygous  f o r mutant  ascospore. i s o l a t e s o b t a i n e d from s t r a i n P393 produced between 9 and 42% black ascospores.  S i n c e these c r o s s e s were w i l d type f o r LG I m a r k e r s ,  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 f r e q u e n c i e s c o u l d not be d e t e r m i n e d . These h i g h f r e q u e n c i e s o f b l a c k ascospores  were never o b t a i n e d i n s i m i -  l a r c r o s s e s w i t h marked homologs  However, the r e s u l t s  o f LG I.  appear  t o suggest t h a t the phenotype o f 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 m o d i f y i n g  genes.  The two non-complementing m u t a t i o n s o f t h i s l o c u s a r e t o be h o m o a l l e l e s ( o f the same o r i g i n ) because they behaved i n genetic crosses  identically  (see below),.and were r e c o v e r e d from the two ascospore.;  i s o l a t e s P243 and P393 which were o b t a i n e d from the same c r o s s Recombination and PWT F r e q u e n c i e s . performed on a number o f c r o s s e s  homozygous  plate.  Random progeny a n a l y s i s for asc-3.  Four types  progeny c o u l d not be e x p l a i n e d . a s homokaryotic p a r e n t a l o r types (Table V ) .  assumed  was of  crossover  These were a p p a r e n t l y PWT c u l t u r e s o f genotypes  auxo  +  Table  V.  Progeny  analysis  of crosses  homozygous  f o r asc-3  IMon-PUT P r o g e n y T o t a l No. IMon-PLJT Progeny  Cro s s *  (P2k3  ;  IMo . R e c o m b i n a n t s in Region nic-al leu-un un-nic  or P393) PUT P r o q E n y  T o t a l No. PUT P r o g e n y ia u x o  Genotypes (al or al 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  17  3  1  5  it  2  0  2  393A34  26  6  2  7  3  0  1  0  2  Total  11 1  19  15  31  17  3  5  3  6  17. 1  13.5  28.0  PUT  f r e q . = 1 7 / 1 2 8 = 13. 3%  1 1 - 17  15-20  30-35  PUT  freq.  k comb , c r o s s e s 393a30  x  P243  O v e r a l l RF (%) among non-PlilT progeny Uild  type  crosses,  RF  values  C r o s s e s between 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 from 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 , Ascospore  i s o l a t e s from  these  crosses  were  obtained  <0.1%  m u t a n t s t r a i n s P 2 4 3. a n d see F i g . 1 ) .  frtfm s h o t  asci.  70  ( w i l d type f o r a l l a u x o t r o p h i c marker m u t a t i o n s ) , un-3 ( w i l d type f o r a l l marker m u t a t i o n s e x c e p t u n - 3 ) , ad-3 ( l e u , a r g , u n , n i c ) and a d - 3 , +  nic-2 ( l e u , a r g , un ). +  +  +  +  +  +  In the pooled d a t a , t h e s e f o u r types o f p r o -  geny r e p r e s e n t e d 13.3% o f a l l RF i n the t h r e e r e g i o n s  progeny. t e s t e d ( l e u - u n , u n - n i c and n i c - a l ) were  s i m i l a r t o those o b t a i n e d from w i l d type c r o s s e s  (see T a b l e V ) .  were determined as a p r o p o r t i o n o f the non-PWT progeny. a t e l y , c o r r e c t e d values w i l l  More a p p r o p r i -  be p r e s e n t e d i n a l a t e r s e c t i o n .  The Nature o f PWT Progeny.  To determine the n a t u r e and o r i g i n  o f the PWT progeny, c o n i d i a l i s o l a t e s from t w e l v e ascospore r e p r e s e n t i n g a l l f o u r types tested.  They  (auxo ;  T a b l e VI shows genotypes  +  cultures  u n - 3 ; a d - 3 ; and a d - 3 , n i c - 2 ) - w e r e ,  r e c o v e r e d from these c u l t u r e s .  All  c u l t u r e s were h e t e r o k a r y o t i c and most c o n t a i n e d one p a r e n t and one o v e r chromosome.  cross-  The un_, the ad_.,and the a d , n i c genotypes were caused  by the complementation o f two n u c l e a r t y p e s , one p a r e n t a l f o r LG I markers and one c r o s s o v e r  i n the un-ad r e g i o n ; two out o f the t h r e e a i i x o  +  geny c o n t a i n e d two d e t e c t a b l e c r o s s o v e r events i n the un-ad r e g i o n  pro(either  a p a r e n t a l and double c r o s s o v e r component, o r two s i n g l e c r o s s o v e r components).  Thus, a l l f o u r types o f PWT progeny appeared t o be produced  by the same e v e n t , each case i n v o l v i n g a t l e a s t one c r o s s o v e r event b e tween un-3 and a d - 3 .  These data a r e b e s t e x p l a i n e d by p o s t u l a t i n g an  u n u s u a l l y high frequency o f n o n d i s j u n c t i o n d u r i n g the second of meiosis  division  (see F i g . 5 ) , e s p e c i a l l y s i n c e such n o n d i s j u n c t i o n i n these  crosses w i l l  o n l y be d e t e c t e d as h e t e r o k a r y o t i c progeny i f a c r o s s o v e r  event i n the un-ad r e g i o n had preceded such i r r e g u l a r s e g r e g a t i o n 3, MATERIALS AND METHODS).  (Fig.  Table  Ul.  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 progeny from c r o s s e s homozygous f o r asc-3 Conidial  Genotype of Ascospore Isolate  Genotype  1-ad  leu,arg,ad  1  2-ad  leu,arg,ad  3-ad n i c  Parental(not  types  - No.  No. HK* Genotype  un , a d  36  12  49  12  un , a d , a l  2D  17  49  un,ad,nic,al  15  l e u , a r g , ad , n i c  19  15  49  4-un a l  un,ad,nic,al  43  leu,un,ad,al  4  2  49  5-un  un,ad,ni c  13  un , a d  24  12  49  6-un  un,ad,nic,al  1D  un , ad  12  7  29  7-un  un,ad,nic,al  3  un, ad  13  11  27  8-un  un,ad,nic  D  leu,un,ad,al  33  17  5D  9-un  un,ad,nic , ( a l )  0  un, ad  22  5  27  ID-PUT  un,ad,nic,al  4  leu,ad  4  16  24  leu,arg,ad,nic un , a d  7 6  19  32  23  5D  No.  11-PUT 12-PUT  un,ad,nic,al leu,arg,ad  *  HK  (-  heterokaryon)  isolates  Genotype  11 16 have t h e genotype  PUT  Isolates  Crossover  inc. a l )  of apparently  •  of the o r i g i n a l  ascospore  T o t a l No. Tested  isolate.  72  Fig.  5  The o r i g i n o f PWT progeny from c r o s s e s homozygous f o r asc-3.  The h e t e r o k a r y o t i c progeny r e c o v e r e d from c r o s s e s  homozygous f o r a s c - 3 a r e r e a d i l y e x p l a i n e d by one more) c r o s s o v e r  event(s)  (or  near the c e n t r o m e r e , f o l l o w e d  by a n o n d i s j u n c t i o n a t the second m e i o t i c d i v i s i o n . (a) A c r o s s o v e r  between a r g - 1 and the centromere w i l l ,  following a regular f i r s t meiotic d i v i s i o n ,  generate  two n u c l e i , each h e t e r o k a r y o t i c f o r the r e g i o n t o the l e f t o f the c r o s s o v e r ; second d i v i s i o n w i l l  subsequent  n o n d i s j u n c t i o n a t the  l e a v e the r e s u l t i n g n u c l e i  karyotic for this region.  In t h i s manner, both aj3 and  a d , n i c , ( a l ) progeny can be produced. crossover  hetero-  Similarly, a  event between ad_ and the centromere w i l l  render  n u c l e i h e t e r o k a r y o t i c f o r the r e g i o n t o the r i g h t o f the crossover; (b)  this will  I f two c r o s s o v e r  produce e i t h e r l e u , arg o r un  progeny.  e v e n t s , one t o the l e f t and the o t h e r  to the r i g h t o f the c e n t r o m e r e , take p l a c e , PWT progeny will  result.  The data i n T a b l e VI are c o m p a t i b l e w i t h  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 o f the ( a u x o ) PWT i s o +  l a t e s ; the 2 - s t r a n d d o u b l e c r o s s o v e r  shown i n t h i s  was a p p a r e n t l y i n v o l v e d i n two PWT i s o l a t e s isolates  10 and 12).  (c) This  (see T a b l e  3-strand double  was r e s p o n s i b l e f o r the r e m a i n i n g  PWT.  figure VI,  crossover  73  (a) leu  i  + leu  +  + i  arg  ad-3B  +  i  i  _JL^_  un  +  + i  arg •  un  +  «  -w  1 J  + i pachytene  ad-3A'  nic  •al  ad-3B  +  + i  i  and leu  i  +  interphase  arg  i  un  IL  +  ad-3A  i  nic  al  n o n d i s j u n c t i o n a t 2nd m e i o t i c ad-3B (un, Dr:  I  ad + l e u , a r g ,  ad-3A, n i c - 2 ,  al-2  (un,  division  ad)  ad, n i c ,  al + l e u , arg,  ad, n i c ,  al)  (b) leu  _,  +  ,  arg  J.  ad-3B  +  ad-3A  nic  ad-3B  +  +  I  1  1  •  JL un  leu —I  +  +  +  +  1  1  un  I  -„ Jt  +  al  S  ad-3A  nic  al  ZZZZZ3C a d - 3 B  + n iI c  + a lL  E  e  line  Table Ul 1D  or leu  + un  arg +  a d_I- 3 A  "see Table Ul line  12  74  (c) leu  +  arg  - ad-3B  +  +  ad-3A  nic  al  ad-3A J.  nic ±  + 1  ••/ +  un  +  f leu i +  + i  arg i  _  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 n o n d i s j u n c t i o n i n v o l v e d chromosomes  with a crossover  i n the centromere  un-ad r e g i o n spans the centromere o f LG I ) , of genotype  only  region;(the  a c r o s s between  strains  l e u - 3 , a_, a r g - 1 , ad-3B, a l - 1 and u n - 3 , A, ad-3A, n i c - 2 ,  a l - 2 was a n a l y z e d .  In t h i s c r o s s , n o n d i s j u n c t i o n a t the second m e i o t i c  d i v i s i o n would be d e t e c t e d as h e t e r o k a r y o t i c progeny i f exchange i n the u n - a l r e g i o n had taken p l a c e . heterokaryotic.  Out o f a t o t a l  o f 65 i s o l a t e s , 25 were  T h i s h i g h f r e q u e n c y o f h e t e r o k a r y o t i c progeny  suggested  t h a t n o n d i s j u n c t i o n a t the second m e i o t i c d i v i s i o n i n v o l v e d both e x change and non-exchange not r e l a t e d t o  chromosomes,  and t h u s w a s a g e n e r a l -  phenomenon  exchange.  Ascus A n a l y s i s .  T a b l e VII  shows the types o f a s c i produced by  f o u r c r o s s e s homozygous f o r the P 2 4 3 - d e r i v e d a s c - 3 m u t a t i o n c r o s s homozygous f o r the P 3 9 3 - d e r i v e d m u t a t i o n . more b l a c k ascospores w h i t e ascospores  were ever d e t e c t e d .  No a s c i w i t h f o u r o r  Most a s c i c o n t a i n e d  but those w i t h one o r two b l a c k ascospores  Both b l a c k ascospores  and by one  were not r a r e .  were germinated from e i g h t 2B:6W a s c i .  c a s e , the p a i r o f genotypes was i d e n t i c a l these c o n s t i t u t e s i s t e r ascospores.  loss is  In each  t o each o t h e r , i n d i c a t i n g t h a t  The 1B:7W a s c i were a p p a r e n t l y p r o -  duced i n p a r t by chromosome l o s s o r n o n d i s j u n c t i o n d u r i n g the meiotic division.  eight  post-  The high f r e q u e n c y o f these 1B:7W a s c i shows t h a t such  extensive. C o r r e c t i o n of Recombination F r e q u e n c i e s  homozygous f o r a s c - 3 some c r o s s o v e r chromosomes karyotic crossover  (RF).  Since i n crosses  are d e t e c t e d as  homo-  p r o d u c t s and o t h e r s - a r e p r e s e n t i n h e t e r o k a r y o t i c p r o -  d u c t s , the RF v a l u e s cannot be determined i n the usual manner.  To  76  Table  VII.  Ascus a n a l y s i s asc-3*  o f c r o s s e s homozygous f o r  Types c f A s c i ( b l a c k : u h i t e 3 : 5 2 : 6 1 : 7 P243-derived  strains: 2  6  5 _  12  17  0  3  10  13  1  5  2  10  3  16  26  46  2  15  10  53  243a26  X  A18  •  3  243a26  X  A28  2  243a32  X  A18  243a32  X  A13  Combined P393-derived 393a30  ascospores) 0 : 8  data strain:  x A34'  Four c r o s s e s i n v o l v i n g ascospore i s o l a t e s d e r i v e d from m u t a n t s t r a i n P243 and one c r o s s b e t w e e n a s c o s p o r e isol a t e s d e r i v e d from P393.  77  d e t e r m i n e the RF v a l u e o f a r e g i o n i t s h o u l d be p o s s i b l e t o all over  distinguish  c r o s s o v e r 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 i n the r e g i o n .  The m o d i f i e d RF v a l u e f o r such a r e g i o n  (UK + 2 C0)/2 x t o t a l progeny  equals  (HK = the number o f h e t e r o k a r y o t i c progeny  produced by a crossover, i n the r e g i o n f o l l o w e d by n o n d i s j u n c t i o n a t M i l ; CO = the number o f homokaryotic c r o s s - o v e r p r o d u c t s i n the r e g i o n ; see MATERIALS AND METHODS).  Using t h i s e q u a t i o n , the m o d i f i e d RF v a l u e  the u n - n i c r e g i o n from the n i n e c r o s s e s 2 x 128 = 18..3%.  in  o f T a b l e V s h o u l d be (17 + 2 x 15)/  T h i s i s p r o b a b l y a s l i g h t u n d e r e s t i m a t e s i n c e the v a l u e  f o r HK was determined f o r the s m a l l e r a r g - a d r e g i o n . t e r i a f o r a c r o s s between the s t r a i n s  Using the same  cri-  l e i i - 3 , a , a r g - 1 , ad-3B, a l - 1 and  u n - 3 , A, ad-3A, n i c - 2 , a l - 2 , a v a l u e o f (3 + 2 x 8)/2 x 65 = 18.4% was obtained.  In the l a t t e r c r o s s , a m o d i f i e d RF v a l u e i n the a d - a l  c o u l d have been o b t a i n e d but was prevented by d i f f i c u l t i e s o f a l - 1 and a l - 2 . . However, the p r e s e n t m o d i f i e d v a l u e s  region  i n the s c o r i n g  i n the u n - n i c r e -  g i o n combined w i t h u n m o d i f i e d v a l u e s i n the l e u - u n , u n - n i c , and n i c - a l regions  (see T a b l e V) c l e a r l y i n d i c a t e t h a t r e c o m b i n a t i o n i s normal  crosses  homozygous  for  Cytology.  The combined g e n e t i c data o f c r o s s e s  in  asc-3. homozygous  a s c - 3 i n d i c a t e d normal r e c o m b i n a t i o n and s e g r e g a t i o n o f homologs  for  a t the  f i r s t m e i o t i c d i v i s i o n , f o l l o w e d by e x t e n s i v e n o n d i s j u n c t i o n a t the s e cond and p o s t - m e i o t i c d i v i s i o n s .  In o r d e r t o g a i n more i n s i g h t i n t o the  nature and mechanism o f t h i s n o n d i s j u n c t i o n , and t o d e t e r m i n e the reason f o r the low f e r t i l i t y , c r o s s e s .homozygousvfor logically.  a s c - 3 were a n a l y z e d c y t o -  The a s c i t h a t were produced showed normal p a i r i n g o f  ( F i g . 6c, d).  homologs  T h i s o b s e r v a t i o n i s i n a c c o r d w i t h the r e c o m b i n a t i o n d a t a  78  Fig. 6  Chromosome development i n c r o s s e s All  homozygous  for  asc-3.  p r e p a r a t i o n s were s t a i n e d w i t h i r o n - h a e m a t o x y 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  contain  a number o f n u c l e i w i t h n u c l e o l i (a) x 2400; i t t h a t these n u c l e i are incapable of f u s i o n . homologous  chromosomes  appears  P a i r i n g of  appears normal ,at p a c h y t e n e , so  do the s i z e and appearance o f the n u c l e o l u s  ( 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 o f c h r o m a t i n s e g r e g a t e a t the f i r s t d i v i s i o n (f) xl300. abnormal:  The second d i v i s i o n i s  highly  many second d i v i s i o n f i g u r e s were o b s e r v e d ,  w i t h most c h r o m a t i n a t t a c h e d t o one s p i n d l e - p o l e body but not the o t h e r (arrowed)  ( g , h) x 1300; about equal  amounts o f c h r o m a t i n may s e g r e g a t e i n some a s c i  (i)  x l 3 0 0 ; 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 t o those shown i n (g) and (h) s i n c e the s p i n d l e - p o l e bodies ( i n i ) have not moved v e r y f a r a p a r t y e t .  Unequal  amounts o f c h r o m a t i n have s e g r e g a t e d i n t o the ( j , k)  xl300.  ascospores  80  presented.  Seven b i v a l e n t s condensed a t d i a k i n e s i s  ( F i g . 6e) and d i -  v i d e d i n a normal f a s h i o n d i s t r i b u t i n g equal amounts o f c h r o m a t i n t o the two p o l e s  (Fig. 6f).  Subsequently,  many a s c i were observed  during  the second d i v i s i o n i n d i c a t i n g t h a t t h i s stage takes an a b n o r m a l l y time t o 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  show one s p i n d l e - p o l e body (SPB) w i t h a l a r g e amount o f  figures  chromatin  a t t a c h e d and the o t h e r w i t h l i t t l e o r no c h r o m a t i n a t t a c h m e n t . the d e f e c t i s a  p r o p e r t y o f the SPB o r o f the chromosomes  long  Whether  cannot be d e -  t e r m i n e d but t h i s d i f f e r e n t i a l attachment appears t o be the cause  of  the e x t e n s i v e n o n d i s j u n c t i o n t h a t has been observed g e n e t i c a l l y .  Fig.  6 j shows f o u r out o f e i g h t ascospores  i n an ascus w i t h no c h r o m a t i n  two spores and a l a r g e amount i n the two n u c l e i o f the o t h e r two  in  spores.  Other spores c o n t a i n very l i t t l e c h r o m a t i n w i t h o f t e n d i f f e r e n t amounts of chromatin i n s i s t e r ascospores, (e.g., Fig. 6k).  a c c o u n t i n g f o r the many 1B:7W  The g e n e t i c and c y t o l o g i c a l d a t a a p p a r e n t l y  asci  supplement  each o t h e r p e r f e c t l y . The f i r s t - a c t i n g d e f e c t o f a s c - 3 t a k e s p l a c e p r i o r t o ascus f o r mation. 6b).  Many c e l l s r e s e m b l i n g ascogenous hyphae have been observed  F i g . 6a shows one o f t h e s e c e l l s i n some d e t a i l . ,  (Fig.  The p r e - m e i o t i c  n u c l e 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 o f l a r g e n u c l e o l i . Such a l a r g e number o f n u c l e i i s n o r m a l l y never o b t a i n e d i n the c r o z i e r s . . Thus, i t appears  t h a t the d e f e c t r e s u l t s  t o o r d u r i n g karyogamy.  i n blockage o f most a s c i  prior  T h i s b l o c k i n ascus f o r m a t i o n i s a p p a r e n t l y the  cause o f the low f e r t i l i t y o f these c r o s s e s .  A more d e t a i l e d  analysis  i s needed, however, t o determine the e x a c t stage of the b l o c k .  I t may  be t e n t a t i v e l y concluded t h a t the p r i m a r y d e f e c t o f a s c - 3 takes  place  81  during c r o z i e r formation, possibly  karyogamy,  and t h a t some c e l l s can  escape t h i s developmental b l o c k t o produce a s c i which n o n d i s j o i n e x t e n s i v e l y a t the second and p o s t - m e 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 recessive mutation g e n e r a l l y  result  i n about 40% ascospore a b o r t i o n ; however, up t o about 70% a b o r t i o n has been d e t e c t e d i n some c r o s s e s .  The f e r t i l i t y o f c r o s s e s  on l i q u i d medium appeared good.  t h a t were done  In some c r o s s e s made on s o l i d medium,  a near f i v e - f o l d r e d u c t i o n i n f e r t i l i t y was d e t e c t e d . Recombination and N o n d i s j u c t i o n F r e q u e n c i e s . analysis  of three crosses  homozygous  Random  ascospore  f o r a s c - 1 r e v e a l e d reduced recom-  b i n a t i o n and i n c r e a s e d n o n d i s j u n c t i o n f r e q u e n c i e s  (Tables V I I I and I X ) .  RF was reduced i n two o u t o f t h r e e r e g i o n s examined.  The amount o f r e -  d u c t i o n appeared v a r i a b l e i n both t h e u n - n i c and n i c - a l  regions.  The  f r e q u e n c y o f PWT progeny was a l s o q u i t e v a r i a b l e and ranged from z e r o t o 22.4%.  No PWT c u l t u r e s were d e t e c t e d among 239 progeny o f a c r o s s  (Table V I I I , row 1) t h a t was made on l i q u i d medium.  In c o n t r a s t , a l l  c r o s s e s made on s o l i d medium produced a v a r i a b l e number o f PWT progeny. The m a j o r i t y o f t h e s e PWT c u l t u r e s were p r o t o t r o p h i c f o r a l l LG I mutant markers.  T h e r e f o r e , these were a p p a r e n t l y t h e r e s u l t o f n o n d i s j u n c t i o n  d u r i n g the f i r s t m e i o t i c d i v i s i o n . The Nature o f PWT Progeny. PWT progeny r e c o v e r e d from c r o s s  The n u c l e a r c o m p o s i t i o n o f t h e e i g h t  95-1 ( T a b l e 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 . 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 o r t h e two o r i g i n a l non-exchange  All  F i v e o f them were h e t e r o k a r y o t i c  chromosomes  l e u - 3 , a , a r g - 1 , ad-3B and  Table  VIII.  Recombination  and  nondisjunction  Non-PUT Total Non-PWT Progeny  Cross * 95A29  x 95a43*  95-1(Y  2  x  X )  95-2(Y-  5  x  X  Wild All  type  1  1 7  used  RF  crosses  values  are  PUT Total PUT Progeny  for  asc-1  Progeny PUT f r e q . (%) (un & auxo  )  Genotypes of + other auxo  16.3(39)  0  0  0  154  16.9(26)  9 . 8 ( 15 ) 2 2 . 7 ( 3 5 )  a  3.7  5  10  9.6  10  0  analyzed  (see  18.1(  17)  10.6(  11-17  15-2D  ascospore  isolates  made  normally  an  solid  slightly  10)  11.7(11)  variable  3(un;1eu ; ad , n i c , a l )  30-35 with  genotypes  the ascospores cross).  medium,  PUT's  0  2.1(5)  were  were  homozygous  15.9(38)  T h i s c r o s s was made on l i q u i d m e d i u m ; 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 These  crosses  Progeny  Recombination frequency (%) (no. recombinants in parentheses) leu-un un-nic nic-al  crosses  strains  three  239  94  )  in  and (see  random e.g.,  as  from  in  Fig.  1.  shot  asci  were  ascospores  were  analyzed.  Catcheside,  1974).  ro  Table  IX.  Nature  of growth  o f a s c o s p o r e s from  Fraction Colonies  Cross *  of  Black  Germ  crosses  homozygous f o r a s c - 1  Ascospores Producing  Tube Only  No Germ  Total  Tube  No.  Freq. PUT  Spores  of  Progeny ^  95-1(Y  2  x  X ) 1  0.54  0.37  0.09  365  2.9  95-4(Y  5  x  X )  0.48  0.32  0.20  378  22.4  95-6(Y  5  x  X  )  0.48  0.35  0. 17  214  14.8  0.45  D.34  0.21  101  13. 1  95-11(Y  3  1 5  4  x  X^  9 5 - 1 2 ^  x  X  1 ?  )  0.41  0.42  0. 17  330  9.2  95-13(Y  x  X  1 5  )  0.47  0.38  0. 15  271  13.1  crossing  medium.  All  7  on s o l i d  "^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 Materials  and  Methods.  un/un  +  84  u n - 3 , A , ad-3A, n i c - 2 , a l - 2 . d i s j u n c t i o n of non-exchange sion.  These were presumably the r e s u l t o f nonchromosomes  d u r i n g the f i r s t m e i o t i c di v i - .  A s i n g l e t e m p e r a t u r e - s e n 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 A_, ad-3A, n i c - 2 , a l - 2 and a chromosome w i t h a c r o s s o v e r region  (see F i g . 3 and 5 ) .  i n the  These two PWT c u l t u r e s may have  iin-3,  un-ad  resulted  from n o n d i s j u n c t i o n d u r i n g the f i r s t o r the second m e i o t i c d i v i s i o n . I f these were caused by a c r o s s o v e r  i n the r e l a t i v e l y smal1 un-ad  region  f o l l o w e d by n o n d i s j u n c t i o n 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  r e c o v e r y o f many PWT c u l t u r e s r e s u l t i n g  the n o n d i s j u n c t i o n o f non-exchange exchange i'n another r e g i o n o f t h i s type were d e t e c t e d .  chromosomes  (e.g., ad-al).  o r o f chromosomes  from w i t h an  In f a c t , o n l y s i x PWT c u l t u r e s  T h e r e f o r e , i t i s more p l a u s i b l e t h a t the un  and the a d , n i c , a l c u l t u r e s were produced by n o n d i s j u n c t i o n a t the s e cond m e 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 n c e a c r o s s o v e r  i n the un-ad  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 n o n d i s j u n c t i o n  region  (Fig. 3).  l e u c i n e r e q u i r i n g PWT was h e t e r o k a r y o t i c f o r the.two c r o s s o v e r  The  chromo-  somes l e u - 3 , a_, a r g - 1 , ad-3B, a l - 2 and l e u - 3 , u n - 3 , A, ad-3A, n i c - 2 . T h i s c u l t u r e was a p p a r e n t l y produced by the n o n d i s j u n c t i o n o f these over chromosomes  d u r i n g the f i r s t m e i o t i c d i v i s i o n .  To. What E x t e n t Do Exchange-Chromosomes Meiotic Division?  N d n d i s j o i n During  the F i r s t  The n a t u r e o f the l e u c i n e 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 h a t n o n d i s j u n c t i o n o f c r o s s o v e r the f i r s t m e i o t i c d i v i s i o n . crossover  cross-  chromosomes,  chromosomes  may t a k e p l a c e a t  To t e s t the e x t e n t o f such n o n d i s j u n c t i o n  263 PWT i s o l a t e s from ten c r o s s e s (95-1  10) were o b t a i n e d and s c o r e d f o r a l b i n o phenotype.  through  The s i x a l b i n o PWT  of 95-  85  c u l t u r e s were a p p a r e n t l y caused by a c r o s s o v e r  event i n t h e n i c - a l r e -  g i o n f o l l o w e d by n o n d i s j u n c t i o n a t t h e f i r s t m e 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 t a n c e o f the subgroup o f m e i -  oses i n which n o n d i s j u n c t i o n o f LG I took p l a c e d u r i n g t h e f i r s t m e i o t i c d i v i s i o n was thus e s t i m a t e d a t 2 x 6/263 = 4.5 mu. t h r e e c r o s s e s examined, t h e n i c - a l  In c o n t r a s t , i n  recombinant f r e q u e n c i e s among non-PWT  progeny were 16.3%, 22.7% and 11.7% (see T a b l e V I I I ) . vided strong evidence supporting  the i d e a t h a t n o n d i s j u n c t i o n a t t h e  f i r s t m e i o t i c d i v i s i o n i n t h i s mutant i n v o l v e s chromosomes.  However, these o b s e r v a t i o n s  over chromosomes  asc-1  primarily  non-exchange  a l s o s u g g e s t t h a t some c r o s s -  do n o n d i s j o i n a t the f i r s t  Ascus A n a l y s i s .  These data p r o -  division.  Many unordered a s c i from a c r o s s homozygous f o r  have been a n a l y z e d ( T a b l e - X ) . -  Most a s c i c o n t a i n e d an even number  o f b l a c k ascospores  s u g g e s t i n g a d e f e c t p r i o r t o the p o s t - m e i o t i c  sion.  o f ascospores  The a n a l y s i s  from these a s c i  pects o f d i s j u n c t i o n i n t h i s mutant.  a s c i combined ( 1 6 . 8 % ) .  frequency  (20%) and f o r a l l o t h e r  T h e r e f o r e , the r e d u c t i o n o f RF i s a g e n e r a l d e -  fect operative i n a l l asci  from t h i s c r o s s ,  o f a subgroup w i t h i n c r e a s e d a s c o s p o r e  and not j u s t an e x p r e s s i o n  abortion.  Second, as p o i n t e d o u t p r e v i o u s l y , chromosomes  revealed several as-  F i r s t , the recombination  i n the u n - a l r e g i o n was s i m i l a r f o r 8B:0W a s c i  divi-  the defect i n d i s j u n c t i o n of  would have t o t a k e p l a c e p r i o r t o the p o s t - m e i o t i c d i v i s i o n ,  i . e . , d u r i n g t h e f i r s t o r second m e i o t i c d i v i s i o n . progeny from t h i s c r o s s  The absence o f PWT  (Table V I I I , row 1) and the presence o f l a r g e  numbers o f 8B:0W and 6B:2W a s c i  suggest r e g u l a r segregation  LG I a t t h e f i r s t m e i i o t i c d i v i s i o n .  One might argue t h a t  of at least ascospore  86  Table  X.  Ascus 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 95a43)  Types of A s c i ( b l a c k : uih i t e a s c o s p o r e s )  No. o f A s c i Observed  B  0  41  6  2  19  4  4  22  2  6  18  0  a  19  other  ( a l l types)  Total  10 119  87  a b o r t i o n may be caused by n o n d i s j u n c t i o n o f a l i n k a g e group o t h e r than LG I.  In t h a t c a s e , the centromere r e g i o n o f o n l y one homologous LG I  ( e i t h e r l e u , a_, a r g , ad o r un_, A, a d , n i c , a l ) would s e g r e g a t e w i t h both c o p i e s o f the n o n d i s j o i n i n g chromosome(s).  Therefore, a l l four viable  p r o d u c t s o f 4B:4W a s c i would be e i t h e r l e u , .a, a r g , ad o r un, A, a d , n i c , al  (assuming no c r o s s o v e r  had o c c u r r e d ) .  However, s i n c e seven out o f  t e n a s c i had an M i l p a t t e r n o f s e g r e g a t i o n o f LG I markers types o f chromosomes  found i n each 4B:4W a s c u s ) ,  ( i . e . , both  i t appears h i g h l y  l i k e l y t h a t c o n v e n t i o n a l n o n d i s j u n c t i o n o f any chromosomes f i r s t m e i o t i c d i v i s i o n c o u l d be the cause o f the observed  un-  d u r i n g the ascospore  abortion. Cytology.  To c o n f i r m and supplement the g e n e t i c d a t a , t h r e e  c r o s s e s were examined c y t o l o g i c a l l y ( 9 5 - 1 1 , 95-12 and 9 5 - 1 3 ) .  Crozier  and ascus f o r m a t i o n appeared r e g u l a r ( F i g . 7 a ) ; the f i r s t d e f e c t was v i s i b l e d u r i n g the zygo/pachytene s t a g e when, reduced p a i r i n g o f homologous chromosomes  i s o f t e n e v i d e n t ( F i g . 7b, c ) .  This observation  is  c o m p a t i b l e w i t h the reduced r e c o m b i n a t i o n v a l u e s . During t h i s a n a l y s i s , a number o f metaphase I f i g u r e s w i t h up t o 14 (= 2N) u n i v a l e n t s were observed ( F i g . 7 d - f ) .  In mainy c a s e s , a  c l o s e a s s o c i a t i o n between s e v e r a l o f these u n i v a l e n t s c o u l d be d e t e c t e d . For example, F i g . 7d shows a number o f p a i r s o f chromosomes shows a c o n n e c t i o n between t h r e e p a i r s o f u n i v a l e n t s .  and. F i g . 7e  These l a t t e r c o n -  n e c t i o n s may w e l l r e s u l t from the s e g r e g a t i o n o f homologs whereas chromosomes  l a c k i n g t h i s c o n n e c t i o n may be genuine u n i v a l e n t s .  It  those is  p l a u s i b l e t h a t the n a t u r e and e x t e n t o f premature s e p a r a t i o n o f these " p a i r s " o f 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  Fig. 7  Chromosome development i n c r o s s e s All  homozygous  f o r asc-1.  p r e p a r a t i o n s were s t a i n e d w i t h Feulgen and a c e t o -  orcein.  C r o z i e r development i s normal (a) x2700; the  p a i r i n g o f homogous chromosomes a t pachytene i s incomp l e t e (b and c) x3400; s u b s e q u e n t l y , some u n i v a l e n t s appear on the metaphase p l a t e ( d ) , and some chromosomes w i t h b r i d g e s are seen a t meta/anaphase I (e and f ) x3400; the l a t t e r t y p e may be d i v i d i n g b i v a l e n t s . f i r s t m e i o t i c d i v i s i o n segregates  The  about equal amounts  o f c h r o m a t i n to the two p o l e s (g) xl700, but some l a g g i n g o c c u r s - - s e e arrowed chromosomes cond d i v i s i o n i s h i g h l y i r r e g u l a r :  (h) xllOO.  The s e -  overlapping  spindles  ( i ) , abnormal s e p a r a t i o n ( j ) xl400, and l a g g i n g chromosomes  of  (k) xl700, have a l l been observed f r e q u e n t l y .  An ascus a t the prophase o f the p o s t m e i o t i c d i v i s i o n shows r e g u l a r placement o f c h r o m a t i n i n a s c i but unequal amounts o f c h r o m a t i n i n d i f f e r e n t n u c l e i (1);  such u n -  equal d i s t r i b u t i o n i s a l s o observed a t the a s c o s p o r e • stage  (m) x900.  F i n a l l y , many a s c i w i t h e i g h t a s c o -  spores a r e a b o r t e d ; one o f the few normal unaborted a s c i i s shown (arrowed) as a comparison i n (n) x200.  90  F u r t h e r a n a l y s i s o f h i g h and low n o n d i s j u n c t i o n c r o s s e s  s h o u l d shed some  l i g h t on t h i s s u b j e c t . Cytological preparations of crosses  homozygous  f o r asc-1 r e -  v e a l e d many more metaphase I c o n f i g u r a t i o n s than were observed i n w i l d type c r o s s e s .  T h i s may be a r e f l e c t i o n o f an extended time i n t e r v a l  spent i n t h i s s t a g e .  That d i v i s i o n may be slowed down by the presence  o f u n i v a l e n t s has been p r e v i o u s l y r e p o r t e d f o r r y e ( P r a k k e n , 1943).  The  d i s t r i b u t i o n o f a p p r o x i m a t e l y equal amounts o f c h r o m a t i n t o the two p o l e s ( e . g . , F i g . 7 g , h) seems to suggest t h a t the u n i v a l e n t s do not s e g r e g a t e p u r e l y a t random.  In a d d i t i o n , some l a g g i n g o f chromosomes has been o b -  s e r v e d 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 . 7 h ) .  These chromosomes  will  most l i k e l y j o i n a n o t h e r nucleus l a t e r o n , s i n c e o n l y e i g h t n u c l e i are e v e r observed a t the e i g h t - n u c l e u s s t a g e .  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 :  spindle  o v e r l a p , l a g g i n g o f chromosomes, apparent slow s e p a r a t i o n o f d i v i d i n g n u c l e i , and movement of s e g r e g a t i n g s p i n d l e bodies t o 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 o f c h r o m a t i n can  be seen t o s e g r e g a t e i n many a s c i  ( F i g . 7 1 , m).  These l a r g e - s c a l e a b n o r -  m a l i t i e s o f s e g r e g a t i o n agree w e l l w i t h g e n e t i c o b s e r v a t i o n s .  The r e l a t i v e  r e g u l a r i t y o f the p o s t - m e i o t i c d i v i s i o n , suggested by the i n f r e q u e n t o c c u r r e n c e o f a s c i w i t h odd numbers o f b l a c k ascospores  (Table X) was c o n -  f i r m e d by the equal amounts o f c h r o m a t i n - s t a i n i n g m a t e 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 p r i m a r y d e f e c t o f t h i s mutant appears t o be a reduced p a i r i n g o f homologous chromosomes w i t h a r e s u l t a n t r e d u c t i o n  91  i n r e c o m b i n a t i o n f r e q u e n c y i n some r e g i o n s .  The consequence o f t h i s r e -  duced p a i r i n g i s the f o r m a t i o n o f u n i v a l e n t s , the s e g r e g a t i o n o f 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 s e g r e g a t i o n  second d i v i s i o n appears t o be the d i r e c t cause o f much o f the abortion.  a t the  ascospore  T h i s a b e r r a n t s e g r e g a t i o n may be the normal consequence  of  t h e f o r m a t i o n o f u n i v a l e n t s , o r i t may be due t o a p l e i o t r o p i c e f f e c t o f the mutant. The I n v i a b i l i t y o f Many B l a c k Ascospores f o r a s c - 1 . May  be Due t o T h e i r M u l t i p l e Disomy.  from C r o s s e s Homozygous N o n d i s j u n c t i o n can be  d e t e c t e d i n Neurospora because o f the V i a b i l i t y o f i t s a n e u p l o i d  progeny.  In c o n t r a s t , such progeny i n most p l a n t s grow e i t h e r very p o o r l y o r not at a l l .  I t i s p o s s i b l e t h a t many a n e u p l o i d c u l t u r e s a r e a l s o q u 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 h e a t - s h o c k e d and p l a t e d o u t , and t h e i r g e r m i n a t i o n and c o l o n y - f o r m i n g a b i l i t y recorded. (Table I X ) . quickly.  Only about 50% o f the b l a c k ascospores  Most o t h e r ascospores  germinated but growth stopped v e r y  A few germinated a s c o s p o r e s  o f t h e l a t t e r t y p e resumed growth  a t one or more p o i n t s a l o n g the mycelium.  The o b s e r v a t i o n o f such  suggests an e x p l a n a t i o n o f i n v i a b i l i t y o f so many b l a c k  escape  ascospores.  Crosses homozygous  for  p l o i d ascospores.  I t i s p l a u s i b l e t h a t the growth o f many o f these h y p e r -  p l o i d ascospores  asc-1  produced c o l o n i e s  becomes  produce PWT progeny r e s u l t i n g from h y p e r -  i n h i b i t e d upon g e r m i n a t i o n .  In some o f t h e s e  young ascospore c u l t u r e s , the process o f h a p l o i d i z a t i o n may cause the o f e x t r a chromosomes  ( P i t t e n g e r , 1954)  and thus enable resumed growth.  loss  92  mei-1 T h i s r e c e s s i v e m e i o t i c m u t a t i o n has been a n a l y z e d u s i n g  both  g e n e t i c and c y t o l o g i c a l means ( S m i t h , 1975; Lu and G a l e a z z i , 1979) and appears t o 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 o f homologous  chromosomes  d u r i n g the f i r s t prophase o f m e i o s i s .  This  is  f o l l o w e d by the f o r m a t i o n o f u n i v a l e n t s a t metaphase I and abnormal s e g r e g a t i o n p a t t e r n s , i n c l u d i n g 4 - p o l e d s p i n d l e s , a t subsequent l e a d i n g t o about 90% ascospore a b o r t i o n .  divisions  G e n e t i c a l l y , an a l m o s t com-  p l e t e absence o f r e c o m b i n a t i o n i s i n accordance w i t h the observed p a i r i n g d e f e c t , and the m a j o r i t y o f b l a c k ascospores linkage  a r e d i s o m i c f o r most  groups. The mei-1 mutant was p r i m a r i l y o b t a i n e d t o check a l l e l i s m t o any  mutants . i s o l a t e d d u r i n g t h i s s t u d y , and t o determine any p o t e n t i a l i n t e r a c t i o n w i t h some o f t h e s e newly i s o l a t e d m u t a t i o n s . t e s t s , i t was found t h a t c r o s s e s b l a c k ascospores  f o r mei-1 produced about 10%  r e s u l t e d when t h e s e same s t r a i n s were c r o s s e d  From a c r o s s  (a (33), m  ad-3B; mei-1 x u n - 3 , A,  n i c - 2 ; mei-1) made on l i q u i d medium, 22 out o f 24 ascospore  v a l u e i/s as high o r h i g h e r than was o b t a i n e d by S m i t h .  ad-3A,  cultures  were a d e n i n e r i n d e p e n d e n t , i n d i c a t i n g they were d i s o m i c f o r LG I.  initial  initial  when c r o s s e d on s o l i d medium, as was observed by Smith  (1975), but 30% b l a c k spores on l i q u i d medium.  homozygous  During  This  T h e r e f o r e , the  d e f e c t r e s u l t i n g i n the p r o d u c t i o n o f these h i g h f r e q u e n c i e s o f  PWT progeny was c o n f i r m e d i n t h i s c r o s s made on l i q u i d medium. To determine t h e d e f e c t o f such c r o s s e s made on l i q u i d medium, the above-mentioned c r o s s was a n a l y z e d c y t o l o g i c a l l y .  The o b s e r v a t i o n s  on p a i r i n g ( F i g . 8 c , d) a t metaphase I were i d e n t i c a l t o those o b t a i n e d  93  Fig. 8  Chromosome development i n c r o s s e s  homozygous  for mei-1.  ( a , b, d , and g) were s t a i n e d w i t h Feulgen and a c e t o o r c e i n ; the r e m a i n i n g p r e p a r a t i o n s were s t a i n e d w i t h iron-haematoxylin.  P a i r i n g between homologs  is  appar-  e n t l y a b s e n t , a l t h o u g h some l o o s e a s s o c i a t i o n s may be observed ( a , b) x3400; near metaphase I,  14 u n i v a l e n t s  are u s u a l l y observed ( c , d) x3400; sometimes, l o o s e s o c i a t i o n s between homologous occur ( c ) .  as-  chromosomes appear t o  About equal amounts o f c h r o m a t i n move t o  o p p o s i t e p o l e s a t the f i r s t m e 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 o f  the two dyad n u c l e i o f prophase II chromosomes  (arrowed) may l a g .  (g) x2400; some  The second d i v i s i o n  s i m i l a r t o t h a t observed f o r mutant a s c - 6  is  (Fig. 5 i ) :  the c h r o m a t i n takes a l o n g time t o s e p a r a t e ( i , j , h) x2400; and u s u a l l y unequal amounts o f c h r o m a t i n move to opposite 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  previously  (Lu and G a l e a z z i , 1979).  However, o b s e r v a t i o n s  s t a g e s o f m e i o t i c development d i f f e r e d i n s e v e r a l (1)  on  subsequent  respects:  Numerous a s c i were observed d u r i n g the f i r s t i n t e r p h a s e ; t h e s e  a s c i showed t h a t about equal amounts o f c h r o m a t i n moved t o o p p o s i t e p o l e s 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 . 8 e , f ) . (2)  Some a s c i c o r r e s p o n d i n g t o prophase II  c o n t a i n e d two n u c l e i ,  each w i t h a chromosome number a p p r o a c h i n g a d i p l o i d n u c l e u s ( F i g . (3)  8g).  In a c c o r d w i t h p r e v i o u s o b s e r v a t i o n 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 s e p a r a t i o n o f the two daughter n u c l e i ( t h i s was i n f e r r e d from the h i g h frequency o f a s c i a t t h i s s t a g e ; 8 h - j ) ; however, s p i n d l e o v e r l a p was r a r e l y observed and 4 - p o l e d  Fig.  spindles  were a b s e n t . (4)  The p o s t - m e i o t i c d i v i s i o n . o f f o u r s e p a r a t e n u c l e i appeared r e g u -  lar. In a d d i t i o n , s e v e r a l i n t e r e s t i n g a s p e c t s o f these  preparations  a r e the a p p a r e n t a s s o c i a t i o n o f u n i v a l e n t s d u r i n g e a r l y metaphase  (Fig.  8 c ) , and the unequal d i s t r i b u t i o n o f c h r o m a t i n t o o p p o s i t e p o l e s i n many second d i v i s i o n f i g u r e s  (e.g., Fig. 8 j ) .  I t has been e s t a b l i s h e d t h a t c r o s s e s homozygous f o r mei-1 r e s u l t i n 70% ascospore a b o r t i o n when c r o s s e d on l i q u i d medium, b u t 90% on s o l i d medium.  Cytological observations  o f c r o s s e s made on the two types  o f media were s i m i l a r up t o and i n c l u d i n g the f o r m a t i o n o f u n i v a l e n t s ; subsequently,  p r e p a r a t i o n s from the c r o s s made on l i q u i d medium c o n t a i n e d  more a s c i a t i n t e r p h a s e I and prophase l a r i t i e s such as 4 - p o l e d s p i n d l e s .  II,  but l e s s a s c i w i t h i r r e g u -  Thus, i t appears t h a t the two types  o f c r o s s i n g media d i f f e r e n t i a l l y a f f e c t t h e ' S e g r e g a t i o n o f u n i v a l e n t s  in  96  c r o s s e s homozygous f o r m e i - 1 .  Because o f the absence o f 4 - p o l e d  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  spindles  segregation  p r o b a b l y do n o t , as proposed by Lu and G a l e a z z i , a c c o u n t f o r the observed frequency o f disomy o f t h i s mutant.  Instead,  the p r e s e n t data s u g g e s t  t h a t u n i v a l e n t s d i v i d e e q u a t i o n a l l y d u r i n g the f i r s t m e i o t i c d i v i s i o n . Even though a h i g h f r e q u e n c y 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 , o r by i r r e g u l a r s p i n d l e b e h a v i o r or d e f e c t i v e s n c l u s i on o f n u c 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 e d out by the f o l l o w i n g o b s e r v a t i o n s .  F i r s t , both s p i n d l e o v e r l a p and  4 - p o l e d s p i n d l e s were r a r e o r a b s e n t , and the s t a g e s subsequent t o the second m e i o t i c d i v i s i o n were a p p a r e n t l y n o r m a l . s p i n d l e b e h a v i o r nor d e f e c t i v e ascospore e x t r e m e l y h i g h frequency o f PWT progeny.  Thus, n e i t h e r i r r e g u l a r  i n c l u s i o n c o u l d a c c o u n t f o r the 'Second, .contrary to observa-  t i o n , the random movement o f u n i v a l e n t s would.cause unequal amounts c h r o m a t i n t o s e g r e g a t e a t the f i r s t m e i o t i c d i v i s i o n .  of  In a d d i t i o n , the  n u c l e i i n the f o l l o w i n g prophase s h o u l d c o n t a i n a t o t a l o f o n l y 2N(= chromosomes.  In f a c t , the observed number was u s u a l l y c l o s e r t o 4N(=  chromosomes.  By the process o f e l i m i n a t i o n , i t appears most p r o b a b l e  14) 28)  t h a t t h e u n i v a l e n t s i n c r o s s e s homozygous f o r mei-1 d i v i d e e q u a t i o n a l l y . T h i s would account f o r the^observed s e g r e g a t i o n o f r o u g h l y equal  amounts  o f c h r o m a t i n a t the f i r s t d i v i s i o n , and f o r the presence o f a d i p l o i d or c l o s e t o d i p l o i d number o f chromosomes asci.  i n each nucleus o f the prophase  In a d d i t i o n , the subsequent d i v i s i o n would t a k e a l o n g time t o  complete s i n c e o n l y c h r o m a t i d s a r e p r e s e n t .  II  97  Mappi ng asc-3.  A c r o s s between mei-1 and ascr3,: o r i g i n a l l y made t o ob-  t a i n double m u t a n t s , produced no recombinants among 62 ascospore lates:  25 were a s c - 3 , mei-1 , and 37 were a s c - 3 , m e i - 1 .  iso-  In o r d e r t o  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 r o d u c e d : t r p - 4 , mapped 12/183 o r about 6.5 mu t o the r i g h t o f a s c - 3 ; and pdx, mapped 3/92 o r about 3.5 mu t o the l e f t o f m e i - 1 .  Ascospores  from the c r o s s  between a s c - 3 , t r p - 4 , p a n - 1 , a_ and pdx, m e i - 1 , A were p l a t e d on minimal medium, and 300 w i l d , type recombinant c o l o n i e s ,(pdx , t r p ) were s e p a r +  a t e l y t r a n s f e r r e d to v e g e t a t i v e medium.  +  T e s t i n g o f these c u l t u r e s f o r  m e i o t i c phenotypes f a i l e d t o r e v e a l any r e c o m b i n a n t s - o f the two m e i o t i c mutants:  30 were m e i - 1 , and 220 were a s c - 3 .  r o u g h l y corresponds f o r the r e g i o n s  The r a t i o o f the two mutants  to the r a t i o o f map d i s t a n c e s p r e v i o u s l y determined  pdx-mei-1 (3.5 mu) and a s c - 3 - t r p - 4  The p d x , t r p +  +  (6.5 mu).  recombinants r e p r e s e n t o n l y about 5% o f a l l p r o -  geny from t h i s c r o s s ( h a l f the map d i s t a n c e o f the p d x - t r p r e g i o n ) .  Since  o n l y recombinants i n the p d x - t r p r e g i o n c o u l d c o n t a i n a recombinant b e tween mei-1 and a s c - 3 , a t o t a l o f 20 x 300 = 6000 progeny were t e s t e d f o r the l a t t e r type o f recombinant ( m e i - l - a s c - 3 ) .  However, s i n c e o n l y  one out o f two r e c i p r o c a l c r o s s o v e r p r o d u c t s would be d e t e c t e d , the t o t a l progeny t e s t e d would be % x 6000 = 3000.  T h e r e f o r e , no c r o s s o v e r s  tween mei-1 and a s c - 3 were observed among 3000 progeny.  be-  The r e c o m b i n a t i o n  frequency between mei-1 and a s c - 3 i s l e s s than 0.1 mu. asc-1. mei-1,  since  The mutant a s c - 1 was mapped c l o s e to a s c - 3 , and t h e r e f o r e to only asc-1  (45) and a s c - 3 (51)  progeny were d e t e c t e d among  96 i s o l a t e s from a c r o s s between the two mutants.  98  asc-6.  Ascus a n a l y s i s o f a c r o s s between a s c - 3 and a s c - 6  es-  t a b l i s h e d the c l o s e p r o x i m i t y o f a s c - 6 t o one o f the c e n t r o m e r e s , s i n c e , 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 gated d u r i n g the f i r s t d i v i s i o n .  In subsequent c r o s s e s  l i n k e d m a r k e r s , the mutant was mapped on LG I I ,  segre-  to centromere-  about 3 mu from a r g - 5  (the s l o (slow growth) m u t a t i o n which was i s o l a t e d w i t h the m e i o t i c .. m u t a t i o n maps about 14 mu from a r g - 5 such t h a t a s c - 6 l i e s between s l o and a r g - 5 ) . LG I I .  a s c - 6 cannot be d e f i n i t e l y a s s i g n e d  t o the r i g h t arm o f  However, two l i n e s o f e v i d e n c e a r e i n s u p p o r t o f the l o c a t i o n  o f a s c - 6 on the r i g h t arm o f LG II  t o the r i g h t o f a r g - 5 :  (a)  second  d i v i s i o n s e g r e g a t i o n data put the asc-6 m u t a t i o n a p p r o x i m a t e l y 10 mu from the centromere o f t h i s l i n k a g e group; and (b) a r g - 5 has  previously  been mapped about 5 mu t o the r i g h t o f the centromere o f LG II 1972).  (Radford,  T h e r e f o r e , the f o l l o w i n g arrangement o f markers appears  likely:  most  (centromere) - 5 mu - ( a r g - 5 ) - 3 rnu - ( a s c - 6 ) - i l mu -. ( s l o ) .  I n t e r a c t i o n of M e i o t i c Mutations mei-1 and a s c - 6 .  Crosses homozygous  f o r the double mutant m e i - 1 ;  a s c - 6 produced about 70% spore a b o r t i o n when made on l i q u i d c r o s s i n g medium.  T h i s i s s i m i l a r t o v a l u e s 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 o f these c r o s s e s was good ( i . e . , many spores were formed) somewhat reduced when compared t o w i l d t y p e . gous' f o r the double mutant but h e t e r o z y g o u s " a t  Analysis  of a  although  cross;homozy-  the ad-3 l o c u s ^(a (:33), . a d m  S B ; m e i - 1 ; a s c - 6 x .un-3,. A , ad-3A, n i c - 2 ; m e i - 1 ; a s c - 6 ) , showed tha^t 38 ' o u t o f 41 i s o l a t e s f o r LG I.  (93%) were a d e n i n e - i n d e p e n d e n t and t h e r e f o r e d i s o m i c ,  T h i s v a l u e o f PWT f r e q u e n c y i s s i m i l a r t o mei-1 (80-90%),  and  99  d i f f e r e n t from a s c - 6  (20-55%).  T h e r e f o r e , these .data-suggest.an  s t a t i c dominance o f mei-1 over a s c - 6 .  This e p i s t a t i c r e l a t i o n s h i p was  c o n f i r m e d c y t o l o g i c a l l y : . i n such c r o s s e s  t h e r e was an. absence o f p a i r i n g  as i n m e i - 1 , r a t h e r than reduced p a i r i n g as i n a s c - 1 and a s c - 6 .  Crosses homozygous  asc-6.  f o r the double mutant a s c - 1 ;  a s c - 6 produced about 70% ascospore a b o r t i o n (70.3 ± 6.7% examined).  epi-  i n 12  crosses  T h i s v a l u e i s s i m i l a r to those o b t a i n e d from e i t h e r s i n g l e  mutant (69 ± 8.4% i n 10 c r o s s e s 9.4% i n 6 c r o s s e s  homozygous  homozygous  f o r a s c - 1 a l o n e , and 62.3 ±  f o r asc-6 a l o n e ) .  Thus, from t h e s e a s c o -  spore a b o r t i o n d a t a i t appears t h a t t h e r e 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 m u t a t i o n s .  G e n e t i c and c y t o l o g i c a l a n a l y s e s  have not y e t been p e r f o r m e d .  Until  o f these  crosses  such t i m e , 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 m u t a t i o n s . DISCUSSION C h a r a c t e r i s t i c s of four r e c e s s i v e m e i o t i c mutations o f c r a s s a have been summarized i n T a b l e XI. served i n crosses s i v e mutations  homozygous  The a b o r t i o n o f ascospores  ob-  f o r each o f t h e t h r e e newly i s o l a t e d r e c e s -  ( a s c - 1 , a s c - 3 and a s c - 6 ) was shown t o be the consequence  o f abnormal d i s j u n c t i o n o f m e i o t i c chromosomes. asc-6),  Neurospora  In two cases  ( a s c - 1 and  the abnormal d i s j u n c t i o n was a p p a r e n t l y caused by a d e f e c t i n  the p a i r i n g o f homologs  d u r i n g the f i r s t m e i o t i c prophase.  In the t h i r d  mutant ( a s c - 3 ) , a primary d e f e c 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 n o n d i s j u n c t i o n observed d u r i n g the second and post-meiotic divisions.  A l t h o u g h the absence o f p a i r i n g and t h e  quent d i s j u n c t i o n d e f e c t o f mei-1 has been e s t a b l i s h e d  subse-  previously.  Table  XI.  Characteristics a defect in the  of four regular  Allele  Approx. % Ascospore Abortion  asc-1  40-70  both  med -  asc-3  90-98  both  very  asc-6  70  both  low -  mei-1  70  liquid solid  90  C r o s s e s made on l i q u i d o r s o l i d medium  recessive meiotic mutations d i s j u n c t i o n of chromosomes  in  Neurospora  crassa  with  Fertility*  Recombination Frequency leu-un un-al  Pairing  Nondisjunction at MI Mil PMD  normal  reduced  reduced  yes  yes  7  normal  normal  normal  no  yes  yes  Pre-ascus formation  reduced  reduced  much reduced  yes  ?  yes  Pairing  absent absent  yes yes  7  7 ?  Pairing  high high  high  low  med  absent*  produced; and w h i t e a s c o s p o r e s * T o t a l , n u m b e r of., b l a c k ( i n t e r m e d i a t e amount of a s c o s p o r e s ) , or high. Data from Smith ( 1 9 7 5 ) , and Lu and G a l e a z z i ( 1 9 7 9 ) ; regions of another chromosome (Smith, 1975).  may  be  low  (few  recombination  was  f  ascospores), determined  Apparent Initial Defect Pairing  med. on  101  ( S m i t h , 1975; Lu and G a l e a z z i , 1979), some new o b s e r v a t i o n s were made d u r i n g t h i s study which suggest a p o s s i b l e mechanism f o r the abnormal d i s j u n c t i o n of univalents in  Neurospora.  P a i r i n g - and R e c o m b i n a t i o n - D e f e c t i v e Mutants Mutants w i t h a reduced r e c o m b i n a t i o n f r e q u e n c y may have a d e f e c t i n the p a i r i n g o f homologs i n the exchange process  ( i . e . , a p r e c o n d i t i o n to exchange),  or  itself.  The m u t a t i o n s a s c - 1 and a s c - 6 have c y t o l o g i c a l l y d e t e c t a b l e d e f e c t s i n the p a i r i n g o f homologs d u r i n g the zygotene and pachytene stages  o f the f i r s t m e i o t i c prophase.  many a s y n d e t i c mutants i n p l a n t s the mutant mei-1 i n Neurospora  In t h i s r e s p e c t , they resemble  (reviewed i n Baker e t a l _ . , 1976a) and  ( S m i t h , 1975; Lu and G a l e a z z i , 1979).  The e x t e n t o f the p a i r i n g d e f e c t o f t h e s e t h r e e mutants i n  Neurospora  ( i . e . , a s c - 1 , a s c - 6 and mei-1) was found t o 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 e d u c t i o n i n r e c o m b i n a t i o n f r e q u e n c y .  Mutant a s c - 1 was l e a s t a f -  f e c t e d w h i l e both p a i r i n g and r e c o m b i n a t i o n were a l m o s t c o m p l e t e l y absent in mei-1. The c y t o l i o g i c a l d e t e c t i o n o f a p a i r i n g d e f e c t does not neces^ s a r i l y mean t h a t a d e f e c t i n the p a i r i n g process per se o p e r a t e s . s t e a d , a d e f e c t i n an immediate r e q u i r e m e n t f o r the process o f may s e c o n d a r i l y cause some p a i r i n g a b n o r m a l i t y . i n the p r e c o n d i t i o n s t o exchange  o f exchange  exchange  Mutants w i t h a d e f e c t  (such as p a i r i n g ) and exchange  have been d i s t i n g u i s h e d on the b a s i s  In-  processes  o f the u n i f o r m i t y o f the r e d u c t i o n  ( S a n d l e r e t a l _ . , 1968; J o n e s , 1974).  Any mutant which r e -  duces exchange i n a u n i f o r m manner a l o n g the chromosome s h o u l d be  102  c o n s i d e r e d d e f e c t i v e i n a process  r e q u i r e d f o r exchange i t s e l f .  In'  c o n t r a s t , n o n - u n i f o r m i t y o f r e d u c t i o n would mean a d e f e c t i n a p r e c o n d i t i o n t o exchange.  Using these c r i t e r i a , the w i l d type gene o f  a s c - 1 c o n t r o l s some f u n c t i o n necessary (presumably  f o r a precondition to  the e s t a b l i s h m e n t or maintenance o f p a i r i n g ) .  exchange  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 - a s c - 6 ;  however,  the o c c u r r e n c e o f most d e t e c t a b l e p a i r i n g near the ends o f the chromosomes maysuggest a d e f e c t i n a p r e c o n d i t i o n f u n c t i o n . . The almost comp l e t e l a c k o f p a i r i n g a t a l l s t a g e s o f m e i o t i c development i n c r o s s e s homozygous f o r mei-1 would not be expected i f the exchange process affected.  was  T h e r e f o r e , the w i l d type gene o f mei-1 p r o b a b l y c o n t r o l s a  f u n c t i o n t h a t i s e s s e n t i a l f o r the e s t a b l i s h m e n t o f p a i r i n g .  D i s j u n c t i o n o f Chromosomes During  the F i r s t M e i o t i c  Division  C o r r e l a t i o n between Lack o f Exchange and N o n d i s j u n c t i o n . consequence of reduced p a i r i n g and exchange, many homologous will  not be h e l d t o g e t h e r by t h e i r c h i a s m a t a , and they w i l l  v a l e n t s i n s t e a d o f b i v a l e n t s a t metaphase I o f m e i o s i s . m a l l y c o n t r o l the r e g u l a r s e g r e g a t i o n chromosomes.  ents) w i l l  be i r r e g u l a r .  chromosomes produce u n i -  Bivalents  nor-  o f i t s component homologous  T h e r e f o r e , i f homologous chromosomes  t o form b i v a l e n t s , s e g r e g a t i o n  As a  are not h e l d t o g e t h e r  o f the i n d i v i d u a l chromosomes  (unival-  In t h i s manner, mutants w i t h a d e f e c 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 t e d through the a b e r r a n t d i s j u n c t i o n o f the u n i v a l e n t s  i t produces.  In recombination,, d e f e c -  t i v e m e i o t i c mutants o f D r o s o p h i l a , o n l y non-exchange d i s j o i n (Baker and H a l l , 1976).  chromosomes  non-  T h e r e f o r e , a l l b i v a l e n t s which are  103  h e l d t o g e t h e r by one o r more c h i a s m a ( t a ) , s e g r e g a t e i n a normal and i r r e g u l a r s e g r e g a t i o n of chiasmata.  fashion  i s s o l e l y due t o u n i v a l e n t s produced by a l a c k  Similar analysis  has not been performed i n m e i o t i c mutants  o f h i g h e r p l a n t s due t o d i f f i c u l t i e s a s s o c i a t e d w i t h the r e c o v e r y o f a n e u p l o i d p r o d u c t s produced by n o n d i s j u n c t i o n . o f some exchange chromosomes tode C a e n o r h a b d i t i s elegans progeny  However,  nondisjunction  t a k e s p l a c e i n m e i o t i c mutants o f the nema(Hodgkin e t a j _ . , 1979).  Analysis  of aneuploid  (PWT) produced by the mutants a s c - 1 and a s c - 6 o f Neurospora  showed  t h a t the m a j o r i t y o f n o n d i s j u n c t i o n i n v o l v e s non+-;exchange chromosomes. T h e r e f o r e , most n o n d i s j u n c t i o n i s a consequence o f the i r r e g u l a r g a t i o n o f the non-exchange  univalents.  f r a c t i o n o f exchange chromosomes division.  fail  segre-  However, a s m a l l but s i g n i f i c a n t t o d i s j o i n d u r i n g the f i r s t m e i o t i c  The low f r e q u e n c y o f such events suggests t h a t n o n d i s j u n c t i o n  o f exchange chromosomes exchange u n i v a l e n t s .  i s a secondary e f f e c t o f the p r o d u c t i o n of non-  For example, the l o n g d u r a t i o n o f metaphase I ob-  served i n these mutants  (see a l s o P r a k k e n , 1943)  c o u l d have caused the  p r e c o c i o u s t e r m i n a l i z a t i o n o f chiasmata and thus produced exchange valents.  A l t e r n a t i v e l y , l a g g i n g o f chromosomes  or s p i n d l e a b n o r m a l i t i e s  induced by the abnormal n a t u r e o f the chromosomes t i o n o f some b i v a l e n t s .  uni-  c o u l d cause  nondisjunc-  That the presence o f u n i v a l e n t s i s not the o n l y  f a c t o r t h a t can p o t e n t i a l l y cause n o n d i s j u n c t i o n i n w i l d type s t r a i n s amply i l l u s t r a t e d by high f r e q u e n c i e s of spontaneous duced n o n d i s j u n c t i o n o f exchange chromosomes p u b l i s h e d ; G r i f f i t h s and DeLange,  n o n d i s j u n c t i o n o f exchange chromosomes  or c h e m i c a l l y i n -  i n Neurospora  1977; S m i t h , 1974).  is  (DeLange,  un-  The absence o f  i n r e c o m b i n a t i o n d e f e c t i v e mutants  o f D r o s o p h i l a may be the r e s u l t o f the a c t i o n o f 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 number o f secondary a b n o r m a l i t i e s .  In N e u r o s p o r a , no e v i d e n c e o f 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 . be s c a t t e r e d a c r o s s  In f a c t , u n i v a l e n t s appear t o  the l e n g t h o f 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 t h e absence o f a r e g -  u l a r means o f d i s j u n c t i o n , u n i v a l e n t s may move a t random t o e i t h e r p o l e 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 ) . g a t i o n have been encountered i n h i g h e r r p l a n t s  These types o f  segre-  ( e . g . , G a t e h e s i d e , 1939=;  S j o d i n , 1970); i n many p l a n t s p e c i e s , both types o f s e g r e g a t i o n have been observed s i m u l t a n e o u s l y  ( e . g . , P r a k k e n , 1943).  In Neurospora, some e v i -  dence i n d i c a t e s t h a t the e q u a t i o n a l d i v i s i o n o f centromeres d u r i n g a n a phase I i s a common means o f s e g r e g a t i o n o f u n i v a l e n t s . F i r s t , the high f r e q u e n c y o f 6B:2W a s c i o b t a i n e d from a c r o s s homozygous  f o r asc-1  i s c o m p a t i b l e w i t h a b e r r a n t s e g r e g a t i o n a t the second  m e i o t i c d i v i s i o n , o r w i t h the e q u a t i o n a l d i v i s i o n o f some u n i v a l e n t s i n g the f i r s t d i v i s i o n .  S i n c e the p r i m a r y d e f e c t i n p a i r i n g and i n the  p r o d u c t i o n o f 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 c r o s s e s gous f o r a s c - 1 , the l a t t e r e x p l a n a t i o n i s p r e f e r a b l e . d i s j u n c t i o n o f homologous  chromosomes  homozy-  In a d d i t i o n , non-  s h o u l d have produced PWT progeny  LG I was i n v o l v e d , o r 4B:4W a s c i w i t h o n l y one homologous ( l e u , a_, a r g , ad or m,  dur-  if  LG I centromere  A, a d , n i c , a l ) i f one o r more o t h e r l i n k a g e groups  were i n v o l v e d (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 i n seven out o f t e n 4B:4W a s c i .  LG I centromeres were o b t a i n e d  These o b s e r v a t i o n s would be c o n s i s t e n t  w i t h the e q u a t i o n a l s e p a r a t i o n o f the centromeres o f some o t h e r than LG I d u r i n g the f i r s t m e i o t i c d i v i s i o n .  chromosomes  105  Second, i n c r o s s e s homozygous f o r m e i - 1 , 14 u n i v a l e n t s can be counted i n most metaphase I f i g u r e s .  I f these u n i v a l e n t s moved a t random  one would e x p e c t - u n e q u a l amounts o f c h r o m a t i n t o s e g r e g a t e t o o p p o s i t e p o l e s a t the f i r s t d i v i s i o n .  However, i n a l l cases o b s e r v e d , a p p r o x i m a t e l y  equal amounts o f c h r o m a t i n moved to each p o l e , c o n s i s t e n t w i t h e q u a t i o n a l s e p a r a t i o n o f a l l 14 u n i v a l e n t s .  A t the prophase o f the next d i v i s i o n ,  up to 14 chromosomes were observed i n each dyad n u c l e u s .  In a d d i t i o n , the  o b s e r v a t i o n t h a t about 90% o f a s c o s p o r e c u l t u r e s were PWT, suggests t h a t the n u c l e i i n the v i a b l e ascospores  were d i p l o i d o r n e a r l y s o .  These o b -  s e r v a t i o n s are c l e a r l y not c o m p a t i b l e w i t h random movement o f u n i v a l e n t s . By the process o f e l i m i n a t i o n , the e q u a t i o n a l s e p a r a t i o n o f u n 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 has l i k e l y taken p l a c e . obtained f o r asc-6.  at  S i m i l a r d a t a have been  The lower f r e q u e n c y o f n o n d i s j u n c t i o n (20-55%)  l i k e l y due t o a h i g h e r f r e q u e n c y o f b i v a l e n t s a t metaphase  is  I.  T h i r d , the e q u a t i o n a l d i v i s i o n d u r i n g the f i r s t m e i o t i c d i v i s i o n has been p r e v i o u s l y suggested i n Neurospora by the o c c u r r e n c e o f c e r t a i n PWT-containing a s c i  ( T h r e l k e l d and S t o l z , 1970).  D i r e c t p r o o f f o r equa-  t i o n a l d i v i s i o n a w a i t s f u r t h e r g e n e t i c and c y t o l o g i c a l a n a l y s i s mutants.  For example, i t s h o u l d be p o s s i b l e t o observe the  o f more than the h a p l o i d number o f seven chromosomes  of meiotic  segregation  a t anaphase  I.  The  f a i l u r e t o observe t h i s event d u r i n g t h i s study i s an i n d i c a t i o n t h a t the s e p a r a t i o n o f chromosomes  a t anaphase  I proceeds v e r y 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  Divisions  The q q u a t i o n a l d i v i s i o n o f u n i v a l e n t s d u r i n g the f i r s t m e i o t i c d i v i s i o n makes the:;subsequent d i v i s i o n o f t h e s e chromatids  impossible.  In some y e a s t and p l a n t m u t a n t s , no second d i v i s i o n f o l l o w s the e q u a t i o n a 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 o r p o l l e n a r e produced (Moens e t a l . , 1976; S t r i n g h a m , (Palmer, 1971).-  1970; S m i t h , 1939)  o r the c e l l s  degenerate  I f n u c l e a r d i v i s i o n takes p l a c e , the chromatids  will  e i t h e r move a t random to o p p o s i t e p o l e s , p o s s i b l y a f t e r a p e r i o d o f l a g ( P r a k k e n , 1943), o r they r e p l i c a t e b e f o r e d i v i s i o n .  Even though  the  l a t t e r mechanism has never been r e p o r t e d , the b e h a v i o r o f mutant  mei-1  suggests t h a t an e x t r a , round o f r e p l i c a t i o n p r i o r t o the second  divi-  s i o n may o c c u r i n Neurospora.  A l t e r n a t i v e l y , most chromosomes  t o g e t h e r to one o f the two d i v i s i o n p o l e s .  c o u l d move  I t has been noted t h a t each  dyad nucleus presumably has a d i p l o i d complement ( o r c l o s e to i t ) chromatids.  of  The u n u s u a l l y high number o f a s c i w i t h two d i v i d i n g n u c l e i  means t h a t t h i s d i v i s i o n takes a long time t o c o m p l e t e .  I f the chroma-  t i d s move a t random, then most r e s u l t i n g n u c l e i s h o u l d miss a t l e a s t one chromosome.  Consequently,  c o n t r a r y t o o b s e r v a t i o n , most a s c i would c o n -  t a i n only i n v i a b l e ascospores. d i p l o i d y o f most ascospores  The high f e r t i l i t y and a l m o s t complete  can be e x p l a i n e d by an e x t r a round o f  repli-  c a t i o n and r e g u l a r centromere d i v i s i o n , o r by the p r e f e r e n t i a l movement o f most chromosomes  to one d i v i s i o n p o l e .  i n f a v o r o f the l a t t e r h y p o t h e s i s  Some c y t o l o g i c a l e v i d e n c e  (see e . g . ,  Fig. 8j).  The o c c u r r e n c e o f  an e x t r a , r o u n d o f r e p l i c a t i o n c o u l d be t e s t e d by means o f the s t a i n i n g method ( I y e n g a r e t a l _ . ,  is  Feulgen  1977).  In c r o s s e s w i t h a m i x t u r e o f u n i v a l e n t s and b i v a l e n t s a t metaphase I,  a c o m b i n a t i o n o f the r e g u l a r s e g r e g a t i o n  o f the b i v a l e n t s  and  and random movement o r centromere d i v i s i o n o f the u n i v a l e n t s would p r o duce a m i x t u r e o f chromosomes sion.  and c h r o m a t i d s p r i o r t o the second  In t h i s c a s e , a problem i n the synchrony  divi-  o f d i v i s i o n c o u l d be  107  expected.  In r y e , mutants w i t h a m i x t u r e o f u n i v a l e n t s and b i v a l e n t s  had more a b e r r e n t second d i v i s i o n f i g u r e s than those w i t h p r e d o m i n a n t l y u n i v a l e n t s ( P r a k k e n , 1943).  S i m i l a r l y , i n N e u r o s p o r a , more i r r e g u l a r  types o f chromosome movement ( e . g . , l a g g i n g , s p i n d l e o v e r l a p ) d u r i n g the second m e i o t i c d i v i s i o n have been o b s e r v e d . i n c r o s s e s w i t h many b i v a l e n t s and some u n i v a l e n t s ( e . g . , a s c - 1 ) , than i n c r o s s e s w i t h few o r no b i v a l e n t s ( e . g . , a s c - 6 and m e i - 1 ) . The p o s t - m e i o t i c d i v i s i o n s o f these t h r e e mutants a r e u s u a l l y regular.  However, some chromosome l o s s o r n o n d i s j u n c t i o n has been s u g -  gested by a s c i w i t h odd numbers o f v i a b l e b l a c k a s c o s p o r e s , and c o n f i r m e d g e n e t i c a l l y by the r e c o v e r y o f an odd number o f PWT progeny from s i x out o f seven a s c i o b t a i n e d i n a c r o s s homozygous f o r a s c - 6  (Table  III). E f f e c t o f L i q u i d and S o l i d C r o s s i n g 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 o b s e r v e d i n c r o s s e s  homozy-  gous f o r a s c - 1 and mei-1 on s o l i d c r o s s i n g medium. Examples a r e the movement o f s e p a r a t i n g s p i n d l e p o l e bodies to the same s i d e o f the ascus i n c r o s s e s homozygous f o r a s c - 1 , o r 4 - p o l e d s p i n d l e s f o r m e i - 1 . proposed t h a t the 4 - p o l e d s p i n d l e s i n c r o s s e s  I t has been  homozygous f o r mei-1 may  account f o r the observed n o n d i s j u n c t i o n (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 c r o s s e s on l i q u i d medium p r o v i d e s f i r m e v i d e n c e a g a i n 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 o f d i v i s i o n  fig-  ures i s a consequence o f d i f f e r e n t c r o s s i n g media i s suggested by the a b o r t i o n o f 90% o f the a s c o s p o r e s on s o l i d medium b u t o n l y 70% on l i q u i d medium.  The h i g h e r spore a b o r t i o n on s o l i d medium can be r e a d i l y e x p l a i n e d  by the abnormal types o f d i v i s i o n s ( e . g . , 4 - p o l e d s p i n d l e s ) ; such abnormal  108  d i v i s i o n s might be e x p e c t e d to s i m u l t a n e o u s l y reduce the f r e q u e n c y o f v i a b l e a n e u p l o i d s f o r each chromosome. from c r o s s e s homozygous  A comparison o f PWT f r e q u e n c i e s  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 t h  those on s o l i d medium ( S m i t h , 1975), i s not i n c o m p a t i b l e w i t h t h i s prediction.  These o b s e r v a t i o n s i l l u s t r a t e the importance o f the com-  b i n e d g e n e t i c and c y t o l o g i c a l s t u d i e s o f s e v e r a l c r o s s e s .  The use o f  d i f f e r e n t c r o s s i n g c o n d i t i o n s ( e . g . , s o l i d v s . l i q u i d c r o s s i n g medium) may a l s o p r o v i d e v a l u a b l e i n f o r m a t i o n f o r the f i n a l meiotic  understanding of  processes.  Are A l l A n e u p l o i d (n. + 1 through n + 6) Ascospores  Viable in  Neurospora?  In N e u r o s p o r a , m u t a t i o n s which cause abnormal d i s j u n c t i o n o f chromosomes d u r i n g m e i o s i s have been d e t e c t e d by the presence o f many hypop l o i d w h i t e a s c o s p o r e s , 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 o f h y p e r p l o i d (PUT) i s o l a t e s . products of meiosis are i n v i a b l e . t i o n o f mutants very d i f f i c u l t .  In  higher  p l a n t s , most  aneuploid  T h i s makes the g e n e t i c c h a r a c t e r i z a Even though many a n e u p l o i d p r o d u c t s can  be o b t a i n e d i n N e u r o s p o r a , i t i s not c l e a r whether o r not they a r e a l l viable.  T h e r e f o r e , ascospores  from a c r o s s homozygous  f o r a s c - 1 were  h e a t - s h o c k e d , p l a t e d and s c o r e d f o r t h e i r c o l o n y - f o r m i n g a b i l i t y . 50% o f the b l a c k ascospores  d i d not produce c o l o n i e s .  germinated but then growth stopped a b r u p t l y .  About  Most o f these  The observed sudden escape  i n some o f the i n h i b i t e d a s c o s p o r e c u l t u r e s may be e x p l a i n e d i f the i n h i b i t i o n were caused by a n e u p l o i d y , 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 r e q u e n c i e s ( g e n e r a l l y about 50%) o f spores produced by c r o s s e s  homozygous  109  f o r a s c - 6 , a s c - 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 N o n d i s j u n c t i o n A f f e c t A l l Chromosomes? The study o f the 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 more than one chromosome may h e l p i n the c h a r a c t e r i z a t i o n o f these m u t a n t s . chromosomes  (LG I,  Three  IV and V) were s i m u l t a n e o u s l y m o n i t o r e d i n c r o s s e s  homozygous f o r a s c - 6 .  In two c r o s s e s examined, the t h r e e  chromosomes  were a f f e c t e d by n o n d i s j u n c t i o n a t about equal f r e q u e n c y (Table S i m i l a r observations  IV)  have p r e v i o u s l y been made f o r mei-1 ( S m i t h ,  However, i t appeared a t f i r s t t h a t chromosomes a s c - 6 d i d not n o n d i s j o i n i n d e p e n d e n t l y :  1975).  i n c r o s s e s homozygous f o r  a;prevelance of i s o l a t e s  o r h a p l o i d f o r a l l t h r e e chromosomes was o b s e r v e d .  Even though  disomic  this  may be due t o 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 p r o b a b l e e x p l a n a t i o n o f t h i s phenomenon i s t h e s e l e c t i v e death o f ascospores  w i t h m u l t i p l e d i s o m i c n u c l e i and s u r v i v a l o f h a p l o i d and d i -  p l o i d ( o r n e a r l y so)  isolates.  been suggested i n the p r e v i o u 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 r e a d y section.  I n t e r a c t i o n o f Three M e i o t i c M u t a t i o n s S t u d i e s o f i n t e r a c t i o n between the t h r e e m u t a t i o n s w i t h a p a i r i n g d e f e c t suggested t h a t each i s d e f e c t i v e i n one a s p e c t o f the same process.  In t h i s c a s e , the process a f f e c t e d i s p a i r i n g , and double  mutants appear t o behave as the s i n g l e mutant w i t h the more extreme reduction in p a i r i n g .  However, s i n c e d a t a on the i n t e r a c t i o n between  the mutants asc-1 and a s c - 6 a r e l i m i t e d , i t remains p o s s i b l e t h a t t h e s e two m u t a t i o n s 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 T h i s i n t e r p r e t a t i o n would be c o m p a t i b l e w i t h p r e s e n t  homologs.  observations,  110  s i n c e the amount o f ascospore a b o r t i o n o f c r o s s e s double mutant a s c - l ; a s c - 6  is identical  f o r mei-1 which l a c k p a i r i n g o f  homozygous  to that of crosses  m e i - 1 , a s c - 1 and a s c - 6  some d e f e c t i n p a i r i n g (or exchange)  observations  classify  mutants i n t o a group w i t h many common c h a r a c t e r i s t i c s .  v a l e n t s a t metaphase I.  homozygous  homologs.  In summary, the combined g e n e t i c and c y t o l o g i c a l o f the t h r e e m e i o t i c mutants  f o r the  these  In each c a s e ,  leads t o the f o r m a t i o n o f u n i -  The d i f f e r e n c e i n frequency o f u n i v a l e n t s  ap-  pears t o p a r t l y c o n t r o l subsequent chromosome b e h a v i o r d u r i n g the f o l lowing d i v i s i o n .  However, i n each c a s e , i r r e g u l a r s e g r e g a t i o n  takes  p l a c e d u r i n g the f i r s t and second d i v i s i o n , l e a d i n g t o the p r o d u c t i o n o f hypoploid products  ( r e c o g n i z e d as w h i t e a s c o s p o r e s ) ,  and h y p e r p l o i d p r o -  ducts (those d i s o m i c f o r LG I are r e c o g n i z e d 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 m u t a n t ) .  The use o f d i f f e r e n t  c r o s s i n g media a p p a r e n t l y has an e f f e c t on chromosome movement i n a t l e a s t one o f these mutants  (mei-1).  T h e r e f o r e , the s i m u l t a n e o u s  study  o f mutants on both types o f media may p r o v i d e some i n s i g h t i n t o the process o f d i s j u n c t i o n d u r i n g  meiosis.  A M u t a t i o n Which Causes N o n d i s j u n c t i o n During the Second M e i o t i c The mutant a s c - 3 appears t o be u n i q u e . p l a c e b e f o r e the f o r m a t i o n o f a s c i . p r i o r to karyogamy.  The p r i m a r y d e f e c t takes  Most c e l l s a r e a p p a r e n t l y b l o c k e d  S i n c e p r e - m e i o t i c DNA s y n t h e s i s  p l a c e j u s t p r i o r to karyogamy  Division  ( I y e n g a r ejt a j _ . , 1977),  i n Neurospora  i t is quite plaus-  i b l e t h a t the w i l d type gene o f a s c - 3 f u n c t i o n s d u r i n g o r near t h i s m e i o t i c S phase.  takes  pre-  Some c e l l s , however, manage t o proceed p a s t t h i s b l o c k  and produce a s c i and a s c o s p o r e s .  Such escape i s not due t o a m u t a t i o n a l  Ill  e v e n t , s i n c e (A + a j PWT i s o l a t e s from c r o s s e s produced the same c r o s s i n g phenotype. crosses  homozygous  homozygous  for  asc-3  Thus, the few a s c i produced i n  f o r a s c - 3 a r e the consequence o f l e a k i n e s s .  o f , and r e c o m b i n a t i o n between homologous  chromosomes  i s normal.  Pairing Simi-  l a r l y , no a b n o r m a l i t i e s have been d e t e c t e d i n the d i s j u n c t i o n o f these chromosomes  during the f i r s t m e i o t i c d i v i s i o n .  However, an e x t r e m e l y  high f r e q u e n c y o f n o n d i s j u n c t i o n takes p l a c e d u r i n g the second d i v i s i o n , and t h e r e i s a l s o chromosome l o s s o r n o n d i s j u n c t i o n a t the p o s t - m e i o t i c division.  The n o n d i s j u n c t i o n d u r i n g the second d i v i s i o n a p p a r e n t l y i n -  v o l v e s attachment o f most chromosomes p o l e body ( S P B ) .  t o one, but not the o t h e r , s p i n d l e  The extended d u r a t i o n of t h i s d i v i s i o n i s  probably  caused by such abnormal a t t a c h m e n t . In some r e s p e c t s , t h i s mutant resembles the mutants p a l and of Drosophila Chromosomes  (Baker and H a l l , 1976).  of homozygous  ca  n d  The mutant p_al_ a c t s o n l y i n m a l e s .  p a l males a r e p r e f e r e n t i a l l y l o s t d u r i n g the  f i r s t z y g o t i c c l e a v a g e d i v i s i o n and maybe d u r i n g the m e i o t i c d i v i s i o n s . Such l o s s a l s o takes p l a c e i n c a males.  n d  m u t a n t s , which a c t e x c l u s i v e l y i n f e -  In both c a s e s , chromosomes a r e l o s t a t one p o l e of the d i v i s i o n .  In these two mutants of D r o s o p h i l a and the a s c - 3 mutant o f a d e f e c t i n the attachment o f centromeres t o S P B ' s causes loss  (in Drosophila)  or n o n d i s j u n c t i o n ( i n Neurospora)  Neurospora, e i t h e r the  o f a s e t o f chromo-  somes .  A P o s s i b l e R e l a t i o n s h i p Between the D e f e c t s a t P r e - a s c u s and Second M e i o t i c D i v i s i o n Stages I t has been suggested pal and c a  n d  t h a t the w i l d type genes f o r the mutants  i n D r o s o p h i l a s p e c i f y a p r o d u c t t h a t i s a component o f , o r  1  i n t e r a c t s w i t h , the c e n t r o m e r i c r e g i o n o f chromosomes f o r the normal s e g r e g a t i o n o f t h e s e chromosomes a}.,  1976a).  and i s  necessary  ( B a k e r , 1975; Baker e t  A l t e r n a t i v e l y , the phenotype o f these mutants might be  produced by a d e f e c t i v e s p i n d l e p o l e body.  S i m i l a r l y , i n view o f the  s t a g e o f the f i r s t a c t i n g d e f e c t o f a s c - 3 , i t i s p o s s i b l e t h a t i t s w i l d type gene p r o d u c t o p e r a t e s d u r i n g the p r e - m e i o t i c S phase and m o d i f i e s the centromere r e g i o n o f the newly s y n t h e s i z e d DNA or produces a d e f e c t i v e s p i n d l e p o l e body.  E i t h e r d e f e c t would g e n e r a l l y cause a d e v e l o p -  mental b l o c k ; however, the few c e l l s t h a t escape t h i s b l o c k would e n c o u n t e r problems d u r i n g the second and subsequent d i v i s i o n s owing t o the mutant p r o d u c t n e c e s s a r y f o r r e g u l a r s e g r e g a t i o n .  One c o u l d s p e c u l a t e  t h a t t h i s p a r t i c u l a r p r o d u c t may be n e c e s s a r y t o r e i n t r o d u c e r e g u l a r e q u a t i o n a l d i v i s i o n a f t e r i t was suppressed d u r i n g 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 o f the c o r r e l a t i o n between the two d e f e c t s has to a w a i t more e x t e n s i v e a n a l y s i s e . g . , mei-4 i n Neurospora 1978).  o f t h i s mutant, and o f s i m i l a r ones  (Newmeyer and G a l e a z z i , 1978; Raju and P e r k i n s  For example, mutants w i t h s i m i l a r p r i m a r y 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 r e a d y d i s c u s s e d , may have s i m i l a r d e f e c t s i n the s e g r e g a t i o n o f  chromosomes.  The Nature o f t h e D e f e c t a t t h e Second M e i o t i c If  secondary  Division  the p o s t u l a t e d abnormal centromere r e g i o n s would a l i g n a t ran  dom, one would e x p e c t both chromatids o f a l l seven chromosomes a t random t o e i t h e r one o r the o t h e r p o l e .  t o move  Such s e g r e g a t i o n would p r o -  duce an e x t r e m e l y h i g h chance .of a b o r t i o n o f r e s u l t a n t ascospores to hypoploidy.  Both c y t o l o g i c a l and g e n e t i c o b s e r v a t i o n s  c o n t r a d i c t these a s s u m p t i o n s :  owing  appear to  ( i ) most chromosomes move t o one p o l e ,  113  few o r none to the o t h e r , and ( i i ) many v i a b l e ( b l a c k ) ascospores produced.  C o n s e q u e n t l y , assuming a d e f e c t i n the centromere  chromosomes  c o u l d not a l i g n a t random.  are  regions,  I n s t e a d , centromeres t h a t were  s y n t h e s i z e d a t the same time ( e . g . , d u r i n g the p r e - m e i o t i c S  phase)  might n o r m a l l y a l i g n and s e g r e g a t e t o the same p o l e ( F i g . 9 ) .  This  type o f p r e f e r e n t i a l s e g r e g a t i o n has been observed i n p r o k a r y o t i c s y s tems (Jacobs e t a l _ . , 1966; L a r k , 1966), and proposed f o r some e u k a r y o t i c systems  ( e . g . , Baker and H a l l , 1976)..  Even though the e v i d e n c e  f o r such a l i g n m e n t d u r i n g m i t o t i c d i v i s i o n s o f e u k a r y o t i c c e l l s i s convincing  not  ( e . g . , Heddle e t a j _ . , 1967), such a mechanism may w e l l  operate during  meiosis.  Map P o s i t i o n s o f asc  Mutations  Mapping o f the t h r e e newly i s o l a t e d m e i o t i c m u t a t i o n s has p l a c e d a s c - 1 and a s c - 3 e x t r e m e l y c l o s e t o the p r e v i o u s l y i s o l a t e d m u t a t i o n mei-1.  M u t a t i o n a s c - 6 was l o c a t e d on a n o t h e r l i n k a g e group  three c l o s e l y l i n k e d m e i o t i c mutations  (LG I I ) .  ( m e i - 1 , a s c - 1 and a s c - 3 ) a r e mutu-  a l l y complementing (see Chapter 1) and appear t o i n v o l v e f u n c t i o n s s a r y o n l y f o r the r e g u l a r p a i r i n g and d i s j u n c t i o n o f chromosomes meiosis. gene.  The  neces-  during  These m u t a t i o n s c o u l d be complementing a l l e l e s o f the same  A l t e r n a t i v e l y , they may r e p r e s e n t the f i r s t case o f a gene c l u s t e r  that is s p e c i f i c a l l y "active during-meiosis  (a gene c l u s t e r o f f u n c t i o n s  r e q u i r e d i n DNA metabolism has been d e t e c t e d i n D r o s o p h i l a ; some o f these f u n c t i o n s a r e a l s o r e q u i r e d d u r i n g m e i o s i s ; Boyd e t a j _ . , 1976a). The c l o s e p r o x i m i t y o f a s c - 3 , whose w i l d type gene o p e r a t e s b e f o r e k a r y 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  Fig. 9  P o s s i b l e e x p l a n a t i o n f o r abnormal d i s j u n c t i o n i n a s c - 3 mutant c r o s s e s .  Upon s e p a r a t i o n o f DNA s t r a n d s  to en-  a b l e p r e m e i o t i c DNA r e p l i c a t i o n , a c e n t r o m e r e - a s s o c i a t e d p r o t e i n (A)  i s bound t o one DNA s t r a n d .  A novel  pro-  t e i n ( • ) s p e c i f i c f o r m e i o s i s would become a t t a c h e d t o the o t h e r DNA s t r a n d .  This protein might, f o r ex-  ample, c o n t r o l some a s p e c t o f p a i r i n g o f homologs and/or p r e v e n t r e g u l a r centromere s e p a r a t i o n d u r i n g the n e x t division.  T h i s model r e q u i r e s t h a t a l l c h r o m a t i d s w i t h  the newly s y n t h e s i z e d ( • )  p r o t e i n become a l i g n e d t o the  same p o l e d u r i n g the second m e i o t i c d i v i s i o n .  In the  a s c - 3 mutant, t h i s c e n t r o m e r e - a s s o c i a t e d p r o t e i n may be d e f e c t i v e and thus p r e v e n t r e g u l a r s e p a r a t i o n o f chroma 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 subsequent  divisions).  at  Premeiotic  S Phase Nucleus of Parent 1 Parent 2 ( o n l y two o f t h e s e v e n c h r o m o s o m e s presented)  Double Helix  r 1  Metaphase  I  Metaphase  I I (one dyad  T—  defective asc-3|  in  nucleus)  - f  defective asc-3  in  116  i n d i c a t i o n t h a t the w i l d type a l l e l e s o f mei-1 and a s c - 1 are a l s o t i v e b e f o r e karyogamy  and thus w e l l b e f o r e the a c t u a l p a i r i n g  where t h e i r e f f e c t s a r e  process  expressed.  In c o n c l u s i o n , the p r e s e n t a n a l y s i s m o n s t r a t i o n o f the u s e f u l n e s s approach t o the a n a l y s i s  ac-  has p r o v i d e d a n o t h e r d e -  o f a combined c y t o l o g i c a l and g e n e t i c  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 o f mutants have been p a r t l y c h a r a c t e r i z e d and mapped. F u t u r e s t u d i e s s h o u l d extend t o the i s o l a t i o n o f  temperature-sensitive  mutants and mutants t h a t i n t e r a c t w i t h e x i s t i n g m e i o t i c m u t a n t s . may be used t o determine the temporal p e r i o d o f a c t i v i t y o f gene ducts and the nature o f c e l l components.  These pro-  a c t i v i t y by the i n t e r a c t i o n o f d i f f e r e n t  CHAPTER  III  THE MUTATION SK(ad-3A) ALTERS THE DOMINANCE OF a d - 3 A  +  OVER ad-3A IN THE ASCUS OF NEUROSPORA  117  INTRODUCTION Known causes p l o i d y and m u t a t i o n .  o f l e t h a l i t y o f m e i o t i c p r o d u c t s i n c l u d e aneuFor example, t h e d e l e t i o n o f any e s s e n t i a l  o f the genome from the p o l l e n o f h i g h e r p l a n t s or a s c o s p o r e s results in their abortion.  part  of fungi  In c o n t r a s t , d e f i c i e n c i e s o f n u c l e i  in  the meiocytes o f a n i m a l s 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 o f the egg o r sperm c e l l s .  There a r e t h r e e known types o f l e t h a l i t y o f  m e i o t i c p r o d u c t s which a r e not a s s o c i a t e d w i t h a n e u p l o i d y b u t , i n s t e a d , i n v o l v e s p e c i f i c gene m u t a t i o n s .  In each c a s e , m e i o t i c p r o d u c t s which  c a 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 o f c e r t a i n genes a r e r e c o v e r e d i n a r e g u l a r  fashion  when c r o s s e d w i t h the same a l l e l e , but they a r e l e s s f r e q u e n t l y o r not r e c o v e r e d when c r o s s e d w i t h a n o t h e r a l l e l e .  T h i s phenomenon, c a l l e d  s e g r e g a t i o n d i s t o r t i o n , can be the r e s u l t o f the l e t h a l i t y o f m e i o t i c p r o d u c t s c o n t a i n i n g the s e n s i t i v e genes ( f o r r e v i e w see Zimmering e t a l , 1970; H a r t ! and H i r a i z u m i , 1976).  Examples a r e SD i n  which causes an a r r e s t i n m a t u r a t i o n o f S d  +  spermatids  Drosophila,  ( H a r t ! and  H i r a i z u m i , 1976) and SK i n N e u r o s p o r a , which causes a b o r t i o n o f c o n t a i n i n g ascospores  SK  S  ( P e r k i n s and B a r r y , 1977; Turner and P e r k i n s ,  1976). A l l e l e s from a second k i n d o f gene cause the a b o r t i o n o f a l l m e i o t i c p r o d u c t s when c r o s s e d w i t h the same a l l e l e . mutant e f f e c t may be expressed i n an autonomous i . e . , i n crosses  This kind of  or r e c e s s i v e manner,  to the w i l d t y p e a l l e l e , m e i o t i c p r o d u c t s  containing  118  the mutant a l l e l e may a b o r t (autonomous) o r they may be v i a b l e ( r e cessive). These types o f mutants a r e most e a s i l y d e t e c t e d i n f u n g i , e s p e c i a l l y ascomycetes, s i n c e a l l p r o d u c t s of a m e 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 a s c u s .  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 often t h e i r s i z e ) .  Ascospore l e t h a l s a r e u s u a l l y c o l o r l e s s ,  normal v i a b l e ascospores a r e g e n e r a l l y c o l o r e d .  while  I n - t h i s manner, the  autonomously e x p r e s s e d a s c o s p o r e l e t h a l s asco ( S t a d l e r , 1956), tan (Nakamura, 1961), .cys-3 ( M u r r a y , , 1965) andws_ ( P h i l l i p s and S r b , 1967) were d e t e c t e d i n Neurospora.  T h i s c l a s s o f mutants  superfici-  a l l y resembles v i a b l e a s c o s p o r e c o l o r mutants i n S o r d a r i a (Chen, 1965; O l i v e , 1965), Podospora ( E s s e r , 1974) and AscObolus Lissouba  e t a]_., 1962).  (Bistis,  1956;  However, the two types o f mutants p r o b a b l y  represent q u i t e d i f f e r e n t defects. Examples o f m u t a t i o n s 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 a r e 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 ( R i c k and B u t l e r , 1956). T h i s c h a p t e r r e p o r t s a case i n Neurospora c r a s s a where one mutant (ad-3A) can be e x p r e s s e d as e i t h e r a r e c e s s i v e o r an autonomous ascospore l e t h a l , depending on whether i t i s c r o s s e d w i t h w i l d type ( a d - 3 A ) o r a newly induced m u t a t i o n c a l l e d S K ( a d - 3 A ) . +  This i s , to  the a u t h o 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 e x c e p t i o n s a r e the a s c o s p o r e l e t h a l s l e - 1 and l e - 2 i n Neurospora (Murray and S r b , 1961; G a r n j o b s t and Tatum, 1967). Ascospores c o n t a i n i n g these mutants a r e b l a c k .  119  Moreover, the system i s a l s o unique i n t h a t the a f f e c t e d enzyme coded f o r by the ad-3A l o c u s 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 u d i e d on a b i o c h e m i c a l l e v e l  ( F i s h e r , 1969b).  T h e r e f o r e , the s t u d y  o f t h i s m u t a t i o n may g i v e 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 Strains The f o l l o w i n g mutant a l l e l e s were used d u r i n g t h i s l e u - 3 (R156), un-3 ( 5 5 7 0 1 - t ) , Y-112-M38), t o l _ (N83), ad-3A  arg-1  study:  (36703), n i c - 2 ( 4 3 0 0 2 ) , a l - 2  (74-  ( 2 - 1 7 - 1 9 , 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 - 3 3 - 3 0 , 2 - 3 3 - 3 4 , 5 - 5 - 4 , 5 - 5 - 2 3 , 5 - 5 - 4 7 , 5-5-52, 5 - 5 - 7 4 ) , ad-3B 114, 2 - 1 7 - 7 6 , 2 - 1 7 - 8 2 , 2 - 1 7 - 8 5 , 2 - 1 7 - 9 9 , 2 - 1 7 - 1 2 8 ) .  (2-17-  The l a s t f i v e  a l l e l e s o f 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 t h e r .  The approximate map d i s t a n c e s  (Radford,  1972) o f m u t a t i o n s on LG I a r e shown i n F i g . 1 (see a l s o Chapter D i s c r e p a n c i e s between these v a l u e s and those o b t a i n e d i n t h i s may be p a r t l y due to v a r i a b i l i t y caused by g e n e t i c  I).  study  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 d e s c r i b e d i n Chapter I.  T h i s s t r a i n i s a pseudo-  w i l d type (PWT) c u l t u r e o b t a i n e d from a s c r e e n f o r r e c e s s i v e m e i o t i c mutants.  I t i s composed o f two n u c l e a r components o f genotypes  leu-3,  a_, a r g - 1 , ad-3B and u n - 3 , A, ad-3A, n i c - 2 , a l - 2 ; each component c a r r i e s the t o l m u t a t i o n which a l l o w s normal growth o f (A. + a_)  heterokaryons  (Newmeyer, 1970; DeLande and G r i f f i t h s , 1 9 7 5 ) , and i d e n t i c a l  (but  120  Fig. 1  The two n u c l e a r components  of s t r a i n  P917.  Heterokaryon  centromere Component  1  leu-3 t  Component  2  Approximate map d i s t a n c e ( R a d f o r d , 1972)  +  •  +  a  i  '  un-3  1D  0.1  '  A  arg-1 •  B  +  10  9  +  ad-3B  ad-3A  +  i  +  i  0.3  +  i  i  nic-2 al-2  k  28  122  unknown)  het genotype n e c e s s a r y f o r vigourous  growth o f the h e t e r o -  karyon ( G a r n j o b s t and W i l s o n , 1956). . Mutant P917 was d e t e c t e d by i t s p r o d u c t i o n o f about 60% w h i t e a b o r t e d  ascospores.  The two ascospore i s o l a t e s 917A36 and 917a38 were o b t a i n e d from crosses  between s t r a i n s  respectively.  P917 and the w i l d type s t r a i n s OR-A and 0R-a_,  T h e i r genotypes are l e u - 3 , a , a r g - 1 , ad-3B; t o l ; Cde  (917A36) and u n - 3 , A, ad-3A, n i c - 2 , a l - 2 ; t o l ; Cde (917a38), where C, d_, and e_ a r e a l l e l e s o f t h r e e h e t e r o k a r y o n c o m p a t i b i l i t y l o c i .  The  presence o f the t o l m u t a t i o n and Cde genotype i n both s t r a i n s  allows  the f o r m a t i o n o f a heterokaryon- between t h e s e s t r a i n s .  The i d e n t i t y o f  the two adenine r e q u i r i n g m u t a t i o n s ad-3A and ad-3B was d e t e r m i n e d , where n e c e s s a r y , by h e t e r o k a r y o n t e s t s w i t h ad-3A and ad-3B t e s t e r strains  (see Delange and G r i f f i t h s ,  1975).  Procedures Crosses were performed by the s i m u l t a n e o u s  i n o c u l a t i o n o f two  s t r a i n s o f o p p o s i t e mating type (A_ and a_) i n t o 18 x 150 mm t e s t tubes c o n t a i n i n g 5 ml l i q u i d c r o s s i n g medium and a s t r i p o f f i l t e r (Newcombe and G r i f f i t h s , 1972). ,  A l l c r o s s e s were i n c u b a t e d a t 25°C.  Ascospore a n a l y s i s was u s u a l l y performed by the i s o l a t i o n o f  i n d i v i d u a l a s c o s p o r e s ' a n d t e s t i n g o f the r e s u l t i n g c u l t u r e s . ad  paper  +  recombinants among the progeny from a c r o s s  ad-3B and A, ad-3A, ascospores w i t h l e u c i n e and a r g i n i n e .  To d e t e c t  between l e u - 3 , a_, a r g - 1 ,  were p l a t e d on s o l i d medium supplemented  The r e s u l t i n g c o l o n i e s were t r a n s f e r r e d from  the p l a t e s to s l a n t s o f v e g e t a t i v e medium supplemented w i t h l e u c i n e and a r g i n i n e , and t e s t e d f o r t h e i r l e u c i n e and a r g i n i n e r e q u i r e m e n t s .  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 n o n d i s j u n c t i o n  123  o f LG I.  Only the l e u , a r g , ad  recombinants were f u r t h e r used.  The ad-3B m u t a t i o n i n s t r a i n 917A36 has been r e v e r t e d i n one experiment.  A c o n i d i a l s u s p e n s i o n o f s t r a i n 917A36 was i r r a d i a t e d w i t h 3  UV a t 5 x 10  2 ergs/cm  f o r 30, 60, o r 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 e d on medium supplemented w i t h l e u c i n e and a r g i n i n e . The a d  +  r e v e r t a n t c o l o n i e s were c r o s s e d w i t h 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  +  f o r mutant (ascospore a b o r t i o n )  ascospore  i s o l a t e s were used t o t e s t  phenotype.  The o b s e r v a t i o n o f l i n e a r a s c i , and o t h e r r o u t i n e m a n i p u l a t i o n s have been d e s c r i b e d p r e v i o u s l y  (Davis and d e S e r r e s ,  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 + a j h e t e r o k a r y o n  consisting  o f two n u c l e a r t y p e s , each having s e v e r a l complementing m u t a t i o n s on LG I (see F i g . 1 ) .  Upon s e l f i n g (a c r o s s between the a_ and A n u c l e i  o f t h i s h e t e r o k a r y o n ) , t h i s s t r a i n produced about 60% a b o r t e d w h i t e ascospores. Is  the Ascospore A b o r t i o n o f P917 Caused by a Dominant o r a R e c e s s i v e  Factor? To determine whether a dominant or r e c e s s i v e f a c t o r caused cospore a b o r t i o n i n P917, i t was c r o s s e d w i t h w i l d type s t r a i n s and 0R-a_.  as-  0R-£  The absence o f a b o r t e d spores from c r o s s e s w i t h both w i l d  type s t r a i n s suggested t h a t a b o r t i o n was caused by a r e c e s s i v e than a dominant f a c t o r .  However, the a n a l y s i s  rather  o f i s o l a t e s from these  c r o s s e s 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 o f a dominant m u t a t i o n on LG I (see T a b l e I ) .  In b a c k c r o s s e s w i t h P917, a l l  Table  I.  Cross  A n a l y s i s of i s o l a t e s * type strains  Genotype  LG  from  crosses  Number o f progeny  I  between %  P 9 1 7 t o 0R-A_ a n d OR-a  ascospore abortion P917  wild  when c r o s s e d  917A36*  917a3B*  (a) P917 X  OR-a  0-10  18  60  --  —  —  un,  ad , n i c ,  al  un,  a d , a.l  A  1  60  —  —  a_  1  --  --  —  0-10  0-10  —  90  90  —  —  —  ad , n i c , a l  A_  20  (b) P917  11  +. a  7  X  OR-A  arg , ad leu,  3  A  a  leu , arg  a  l e u , a r g , ad Recombinants  between  k  0-10  —  0-10  1  0-10  —  0-10  60  —  60  12  a  a_d a n d a_l w e r e  not t e s t e d ,  t 9.17A36 i s an i s o l a t e  from  cross  (b) of genotype  l e u , a r g , a d , a_.  ^ 917a38  from  cross  (a) of genotype  u n , a d , n i c , a l , A.  i s an i s o l a t e  with  125  20 w i l d type (+)  a_ i s o l a t e s from the c r o s s between P917 and OR-ja p r o -  duced o n l y b l a c k a s c o s p o r e s ,  and a l l 18 un_,_A, a d , n i c , a l  produced about 60% ascospore a b o r t i o n .  The a b o r t i o n phenotype a p p a r -  e n t l y s e g r e g a t e d w i t h the un_, A, a d , n i c , a l chromosome l a r l y , i n the c r o s s  (LG I ) .  Simi-  between P917 and OR-A, the a b o r t i o n phenotype  segregated w i t h the l e u , a_, a r g , ad chromosome o f ascospores  isolates  from t h e s e c r o s s e s  (LG I ) .  The a b o r t i o n  c o u l d not have been due t o the s e l f -  i n g o f P917 s i n c e c r o s s e s w i t h e i t h e r P917 o r the ascospore 917a38 produced the same amount o f ascospore a b o r t i o n . spore a b o r t i o n observed i n 7 w i l d type (+)  isolate  The 90% a s c o -  A i s o l a t e s was l a t e r  attri-  buted to a s e p a r a t e r e c e s s i v e p o i n t m u t a t i o n ( a s c - 7 ; see Chapter  I).  The a b o r t i o n f a c t o r on the l e u , a , a r g , ad chromosome c o u l d be l o c a t e d to a s m a l l r e g i o n spanning the centromere o f LG I, l e u , a_, a r g c r o s s o v e r  s i n c e l e u , a^ and  p r o d u c t s d i d not produce any a b o r t e d  ascospores  when c r o s s e d w i t h P917 ( t h e centromere o f LG I i s l o c a t e d between arg-1 and a d - 3 ) .  S i n c e the m u t a t i o n c a u s i n g about 60% ascospore  t i o n i s l o c a t e d i n a w e l l - m a r k e d r e g i o n o f LG I,  abor-  i t is unlikely that  the same m u t a t i o n i s p r e s e n t i n both the un_, A, a d , 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 o b s e r v a t i o n s  w i t h a m u t a t i o n l o c a t e d near the centromere o f LG I, ascospore a b o r t i o n i n dominant f a s h i o n crosses  are c o n s i s t e n t  which e i t h e r causes  ( i n P917) o r has no e f f e c t  (in  between P917 and OR-A o r 0R-a_).  Nature o f D e f e c t To f u r t h e r determine the n a t u r e o f the d e f e c t l e a d i n g t o a s c o spore a b o r t i o n , a c r o s s 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, a d , n i c , a l ) was a n a l y z e d . o f ascospores  produced by t h i s c r o s s were a b o r t e d .  About 60%  0  T a b l e II  shows  t h a t 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 (PWT) f r e q u e n c i e s were n o r m a l . However, "only one o f the two homologous a r g , ad) was r e c o v e r e d from t h i s c r o s s .  p a r e n t a l chromosomes  ( l e u , a_,  In a d d i t i o n , r e c i p r o c a l  o v e r p r o d u c t s were o n l y d e t e c t e d i n t h e a r g - n i c r e g i o n .  cross-  Consequently,  a s m a l l r e g i o n o r gene i n the a r g - n i c r e g i o n on the un_, A, a d , h i e , a l chromosome cannot be r e c o v e r e d when c r o s s e d w i t h s t r a i n 917A36 (the l e u , a_, a r g , ad chromosome).  These d a t a suggested t h e i n v o l v e m e n t o f  t h r e e 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 a p p a r e n t l y caused the death o f ascospores a second gene which was l o c a t e d on the un_, A, a d , n i c , a l Both genes were l o c a t e d near the centromere o f LG I. sumably a l s o on LG I,  containing  chromosome.  A t h i r d gene, p r e -  was not a f f e c t e d by the k i l l i n g o f the  first  gene, nor was i t c a p a b l e o f k i l l i n g the second gene.  I d e n t i t y o f Gene S e n s i t i v e t o K i l l i n g A c t i o n 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 t h e a r g - n i c region, a l l crossover  p r o d u c t s i n t h i s r e g i o n were t e s t e d f o r the p r e -  sence o f ad-3A and ad-3B m u t a t i o n s . r a t h e r than ad-3A. o r a t the ad-3A  A l l t h e s e p r o d u c t s were ad-3B  A p p a r e n t l y , the s e n s i t i v e gene was l o c a t e d near  locus.  T h i s c r o s s was used r a t h e r than a P917 s e l f i n g , t o ensure t h a t o n l y two n u c l e a r components o f known genotype were i n v o l v e d . P917 may c o n t a i n a s m a l l p r o p o r t i o n o f c o n t a m i n a t i n g n u c l e i , e . g . , produced by somatic c r o s s i n g - o v e r ( P i t t e n g e r and C o y l e , 1963).  127  Table  LG  I  I I . G e n o t y p e o f 79 i s o l a t e s f r o m t h e c r o s s b e t w e e n s t r a i n s 9 1 7 A 3 6 a n d 9 1 7 a 3 8 ( f o r LG I m a r k e r s , s e e 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 ascospores  Markers  Parental \  Number Ascospore Isolates  Geno t y p e  33  l e u , a r g , ad un , ad , n i c ,  0  al  Crossover  leu-un  arg-nic  nic-al  ad  leu,  un, ad, n i c ,  k k  al  21  leu , a r g , ad, a l ad, n i c  0  arg,  ad, a l  7  leu,  un, ad, n i c  D  Crossover:  leu-un/nic-al  arg-nic/nic-al  un , ad , a l  3  leu , a r g , ad, n i c  1  RF RF  0  al  un , ad l e u , arg , ad, n i c ,  un,  Double  6  arg,  (leu-un) (un-arg) (arg-nic) (nic-al)  Nondisjunction Frequency =  13/79 D/79 12/79 32/79 0/79  (16%) ( 0%) (15%) (40%) (  0%)  i n Wild  Type 11-17 .15-20 30-35  Crosses  128  To t e s t the p o s s i b i l i t y t h a t the ad-3A m u t a t i o n i t s e l f not be r e c o v e r e d i n t h e s e c r o s s e s ,  19 i n d e p e n d e n t l y d e r i v e d ad-3A  a l l e l e s were c r o s s e d w i t h 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 w i t h 917A36.  could  crossed  Crosses w i t h 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% a s c o s p o r e a b o r t i o n ; i n c o n t r a s t , a b o r t i o n was absent i n the r e maining c r o s s e s .  Thus i t appeared t h a t o n l y adenine r e q u i r i n g  m u t a t i o n s c o u l d not be r e c o v e r e d when c r o s s e d to the " k i l l e r "  ad-3A l e u , a_,  a r g , ad chromosome. I f a l l ascospores  c o n t a i n i n g ad-3A were i n v i a b l e when c r o s s e d  w i t h s t r a i n 917A36, which c o n t a i n s the l e u , a_, a r g , ad chromosome, most a s c i produced from t h i s c r o s s w h i t e ascospores  (4B:4W).  s h o u l d c o n t a i n f o u r b l a c k and f o u r  In f a c t , 54 out o f 56 a s c i were o f t h i s t y p e ;  the r e m a i n i n g two were 2B:6W.  S i n c e ad-3A i s c l o s e l y l i n k e d t o the  c e n t r o m e r e , a low f r e q u e n c y o f second d i v i s i o n s e g r e g a t i o n o f w h i t e and b l a c k ascospores  would be e x p e c t e d .  A g a i n , o n l y 2 out o f 54 a s c i  a second d i v i s i o n p a t t e r n o f s e g r e g a t i o n o f b l a c k and w h i t e  had  ascospores.  I t was c o n c l u d e d t h a t a m u t a t i o n on the l e u , a , a r g , ad chromosome caused a d - 3 A - c o n t a i n i n g ascospores  to a b o r t .  be r e f e r r e d t o as spore k i l l e r o f ad-3A, o r  This mutation w i l l  SK(ad-3A).  L o c a t i o n o f the Newly Induced Spore K i l l e r SK(ad-3A) M u t a t i o n In o r d e r t o l o c a t e SK(ad-3A) on LG I,  253 i s o l a t e s from the  c r o s s 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 a d - 3 A  +  and t h e r e f o r e r e s i s t a n t t o SK(ad-3A)  a c t i o n ) were t e s t e d f o r t h e k i l l e r c h a r a c t e r . SK(ad-3A) and ad-3B were r e c o v e r e d .  No recombinants between  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 t h a t ad-3B was not r e q u i r e d f o r the k i l l i n g o f ad-3Acontaining ascospores, verted.  the ad-3B m u t a t i o n i n s t r a i n ;917A36 was r e -  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 v e r t a n t s c l e a r l y de-  monstrated t h a t SK(ad-3A) Was s t i l l p r e s e n t and a c t e d i n d e p e n d e n t l y o f the ad-3B m u t a t i o n . A more p r e c i s e l o c a l i z a t i o n o f SK(ad-3A) w i t h r e s p e c t t o the ad-3A and ad-3B l o c i was o b t a i n e d by i s o l a t i n g a d  +  recombinant  progeny  from c r o s s e s between 917A36 ( l e u , . a , a r g , ad-3B, SK(ad-3A)) and two strains  c o n t a i n i n g a l l e l e s 2-17-814 o r 2-17-825 o f ad-3A.  over products o f genotype l e u , a , a r g , a d All  +  Four  were o b t a i n e d ( F i g .  f o u r recombinants c o n t a i n e d the SK(ad-3A) m u t a t i o n .  cross2).  Therefore,  s i n c e SK(ad-3A) i s very c l o s e l y l i n k e d t o ad-3B (0/253 r e c o m b i n a n t s ) , but n e a r e r ad-3A than ad-3B,  i t was concluded t h a 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 o f the same gene.  Other C h a r a c t e r i s t i c s o f ad-3A and SK(ad-3A)  Mutations  The o n l y known e f f e c t o f the SK(ad-3A) m u t a t i o n i s i t s a c t i o n on a d - 3 A - c o n t a i n i n g  ascospores.  killing  Thus, c r o s s e s homozygous f o r  the SK(ad-3A) m u t a t i o n produce o n l y b l a c k a s c o s p o r e s .  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 r a t e s and do not r e q u i r e adenine f o r growth. To determine whether o r not a d - 3 A - c o n t a i ni ng c o n i d i a c o u l d be o b t a i n e d from v e g e t a t i v e h e t e r o k a r y o n s several  heterokaryons  between ad-3A and S K ( a d - 3 A ) ,  between s t r a i n 917A36 ( l e u , a , a r g , S K ( a d - 3 A ) ,  ad-3B) and 2-17-825a (a_, ad-3A) were a l l o w e d t o grow i n 50 cm r a c e t u b e s .  S e l e c t i o n o f ad_ recombinants t o l o c a l i z e the +  SK(ad-3A) m u t a t i o n w i t h r e s p e c t t o the ad-3A and ad-3B  loci.  131  centromere leu-3  a  arg-1  +  ad-3B _j  I  +  I  A  i  i  i  +  ad-3A  +  • .3  mu  132  These h e t e r o k a r y o n s grew a t a r a t e comparable t o s e v e r a l between ad-3A and S K ( a d - 3 A ) , +  ad-3B.  heterokaryons  C o n i d i a from the b e g i n n i n g  and  the end o f one r a c e 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 r e c o v e r e d w i t h a p p r o x i m a t e l y equal f r e q u e n c y ( T a b l e I I I ) .  Thus, the SK(ad-3A) m u t a t i o n does  not  appear t o a f f e c t the v i a b i l i t y o f a d - 3 A - c o n t a i n i ng c o n i d i a . DISCUSSION The ad-3A m u t a t i o n i n Neurospora c r a s s a behaves as a r e c e s s i v e ascospore  l e t h a l , i . e . , crosses  i n v i a b l e unpigmented a s c o s p o r e s ,  homozygous  f o r ad-3A produce m a i n l y  but most ascospores  tween ad-3A and i t s w i l d type a l l e l e  ad-3A  +  from a c r o s s  be-  , are pigmented and v i a b l e .  T h i s c h a p t e r r e p o r t s the i s o l a t i o n o f a m u t a t i o n w h i c h , when p a i r e d i n a c r o s s w i t h an ad-3A mutant, causes t h e a b o r t i o n o f a d - 3 A - c o n t a i ni ng ascospores. SK(ad-3A), locus.  The new m u t a t i o n was c a l l e d spore k i l l e r o f ad-3A,  or  and was found t o be l o c a t e d a t o r very c l o s e to the ad-3A  Both t h i s c l o s e p r o x i m i t y o f SK(ad-3A) t o the ad-3A l o c u s  and  i t s s p e c i f i c e f f e c t on t h e v i a b i l i t y o f o n l y a d - 3 A - c o n t a i ni ng a s c o spores s t r o n g l y suggest t h a t t h i s new m u t a t i o n i s an a l l e l e , c o n t r o l or s t r u c t u r a l , o f the ad-3A l o c u s .  Thus, depending on which a l l e l e  i s c r o s s e d w i t h , i t may a c t as a r e c e s s i v e o r an autonomous lethal.  ad-3A  ascospore  W h i l e many cases o f both types o f l e t h a l i t y have been p r e v i o u s l y  d e s c r i b e d , both types have never b e f o r e been a s s o c i a t e d w i t h the same mutation. The l e t h a l i t y o f the ad-3A m u t a t i o n s s u p e r f i c i a l l y resembles  some  cases o f s e g r e g a t i o n d i s t o r t i o n which i n v o l v e the l e t h a l i t y o f m e i o t i c  133  Table  I I I .  A n a l y s i s of c o n i d i a l i s o l a t e s from a h e t e r o k a r y o n b e t w e e n s t r a i n 9 1 7 A 3 6 ( 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  Genotype  B e g i n n i n g o f R a c e Tube  HK * leu,  a r g , ad  ad *  Wild  type  of I s o l a t e s End  from of Race  22  22  13  11  a  13  due t o c o m p l e m e n t a t i o n  o f t h e two  Tube  nuclear types.  134  products.  For example, the l e t h a l i t y o f S d  H i r a i z u m i , 1976), SK i n Ascobolus  i n Neurospora  +  in Drosophila  (Turner and P e r k i n s , 1976), and "1_"  (Makarewicz, 1966), depends on the o t h e r a l l e l e o f t h e s e  l o c i and on the g e n e t i c background i n the m e i o c y t e s . t h a t a r e homozygous (ascospores  ( H a r t l and  Since meiocytes  f o r these a l l e l e s produce o n l y v i a b l e p r o d u c t s  o r sperm), l e t h a l i t y appears t o be caused by the i n t e r a c -  t i o n o f 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 c u s . c y t e s t h a t are homozygous  In c o n t r a s t , meio-  f o r ad-3A produce m a i n l y i n v i a b l e  T h e r e f o r e , the l e t h a l i t y o f a d - 3 A - c o n t a i n i n g ascospores the r e s u l t o f a d e f i c i e n c y i n t h e s e Assuming  is  ascospores. apparently  ascospores.  t h a t SK(ad-3A) i s an a l l e l e o f the ad-3A l o c u s ,  lethal-  i t y o f a d - 3 A - c o n t a i n i n g a s c o s p o r e s would be caused by a f a i l u r e o f the a l t e r e d SK(ad-3A) gene o r gene p r o d u c t t o complement the d e f i c i e n c y i n these a s c o s p o r e s .  S i n c e complementation i s normal i n v e g e t a t i v e h e t e r o -  k a r y o n s , the reduced a b i l i t y t o complement the d e f i c i e n c y i s r e s t r i c t e d t o the a s c u s .  S e v e r a l mechanisms  f o r such reduced complementing a b i l i t y  o f the SK(ad-3A) gene can be v i s u a l i z e d . The m u t a t i o n SK(ad-3A) c o u l d 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 p o l y p e p t i d e w i t h a d i f f e r e n t i a l l y low a c t i v i t y i n the ascus and a s c o s p o r e s .  Such reduced a c t i v i t y might a f f e c t  the m a t u r a t i o n o f a d - 3 A - c o n t a i n i n g ascospores  i f a s p e c i f i c threshold  l e v e l o f a c t i v i t y would be r e q u i r e d p r i o r t o a s c o s p o r e e n c l o s u r e .  This  t h r e s h o l d l e v e l would not be e a s i l y reached i n the case o f the r e l a t i v e l y i n a c t i v e SK(ad-3A) gene p r o d u c t .  A s i m i l a r model has been p r o -  posed t o e x p l a i n some i r r e g u l a r f e a t u r e s o f s e g r e g a t i o n d i s t o r t i o n i n Drosophila  ( M i k l o s and S m i t h - W h i t e ,  1971).  135  The l o s t a b i l i t y o f SK(ad-3A) t o complement ad-3A i n the ascus c o u l d a l s o be caused by changes  i n the c o n t r o l o f enzyme  synthesis,  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 , o r an a l t e r e d p r o p e r t y o f t r a n s f e r through the c y t o p l a s m i n the a s c u s .  The m u t a t i o n c y s - 3 • (Murray,  1965)  i n Neurospora may be an example o f a d e f e c t i n t r a n s f e r i n a s c i .  It  l a c k s a permease, and i s the o n l y c y s t e i n e - r e q u i r i n g m u t a t i o n which produces m a i n l y l i g h t i n v i a b l e  ascospores.  The SK(ad-3A) m u t a t i o n r e p r e s e n t s a unique s i t u a t i o n which may enable the study o f d i f f e r e n t p r o c e s s e s e n c l o s u r e and m a t u r a t i o n . sembles the c r i t i c a l types o f c e l l s ' (here  i n the development o f  ascospore  In more g e n e r a l t e r m s , t h i s s i t u a t i o n r e -  s t e p o f d e t e r m i n a t i o n i n the development o f two ascospores)  from a s i n g l e t y p e .  T h e r e f o r e , the  s t u d y o f t h i s process may c o n t r i b u t e t o the u n d e r s t a n d i n g o f the p r o cess o f d i f f e r e n t i a t i o n o f c e l l c e l l u l a r organisms.  types d u r i n g development i n m u l t i -  Such a n a l y s i s  knowledge o f s e v e r a l components.  i n t h i s system i s f a c i l i t a t e d by the F i r s t , ascospore a b o r t i o n can not be  due t o a l a c k o f adenine i n the ascospores  because a b o r t i o n has  been a s s o c i a t e d w i t h o t h e r adenine r e q u i r i n g mutants 1962).  (e.g.,  not  Ishikawa,  A b o r t i o n may be i n d i r e c t l y caused by the ad-3A m u t a t i o n .  example, the a c c u m u l a t i o n o f an i n t e r m e d i a t e (AIR)  For  i n the p u r i n e syn-:  t h e t i c pathway i n a d - 3 A ' m u t a n t s may be the d i r e c t cause o f a b o r t i o n ( 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 a c c u m u l a t i o n o f the polymer o f  AIR,  the a c c u m u l a t i o n o f red pigment, and:a s l i g h t decrease i n growth r a t e . Both the r e d p i g m e n t a t i o n and reduced growth r a t e can be e l i m i n a t e d by a secondary m u t a t i o n l o c a t e d i n the p u r i n e pathway b e f o r e the  136  f o r m a t i o n o f the i n t e r m e d i a t e AIR  (Gutz e t a l _ . , 1974).  These  findings  suggest t h a t s i m i l a r secondary m u t a t i o n s s h o u l d be a b l e t o suppress the ascospore  l e t h a l i t y , i f the a c c u m u l a t i o n o f AIR  d i r e c t cause o f the l e t h a l i t y .  polymers i s  Second, the enzyme i n v o l v e d i n the p r i -  mary d e f e c 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 1969b).  the  (Fisher,  T h e r e f o r e , any a l t e r e d p r o p e r t i e s o f the enzyme i n t h e SK(ad-3A)  mutant c o u l d be determined.. available.  F i n a l l y , g e n e t i c means o f a n a l y s i s  are  For example, more d e t a i l e d mapping o f the SK(ad-3A) m u t a t i o n  may l o c a t e i t w i t h i n the c o n t r o l o r s t r u c t u r a l p a r t o f the ad-3A  locus.  In a d d i t i o n , m u t a t i o n s which i n t e r a c t w i t h SK(ad-3A) t o modify the a b o r t i o n phenotype s h o u l d be r e a d i l y o b t a i n a b l e .  CHAPTER IV GENERAL DISCUSSION  137  The p r e v i o u s c h a p t e r s have d e s c r i b e d the development arid successful  use o f a system i n Neurospora c r a s s a which f a c i l i t a t e s  the i s o l a t i o n o f r e c e s s i v e mutants w i t h a d e f e c t i n ascus o r v i a b l e ascospore f o r m a t i o n .  Subsequent c y t o l o g i c a l and g e n e t i c o b s e r v a t i o n s  have shown t h a t some o f these mutants have t h e i r d e f e c t d u r i n g m e i osis.  These m e i o t i c mutants a r e o f p a r t i c u l a r i n t e r e s t t o the s t u d y  o f the g e n e t i c c o n t r o l o f m e i o s i s , and may u l t i m a t e l y h e l p i n g a i n i n g a complete u n d e r s t a n d i n g o f the m o l e c u l a r processes  that control  meiosis. G e n e t i c C o n t r o l o f the Sexual C y c l e ( I n c l u d i n g M e i o s i s ) . i n NeiirospOra crassa The s e x u a l c y c l e i n Neurospora i n v o l v e s the development o f p e r i t h e c i a , m e i o s i s , and the f o r m a t i o n and m a t u r a t i o n o f  ascospores.  Mutants w i t h d e f e c t s i n each o f t h e s e events have been d e t e c t e d d u r i n g t h i s study.  These mutants have a l r e a d y c o n t r i b u t e d t o our under-  standing of the g e n e t i c c o n t r o l of the sexual c y c l e i n Neurospora. Thus, i t was shown t h a t the development o f 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 o f the maternal p a r e n t .  In a d d i t i o n , , the study of  mutants w i t h an apparent d e f e c t i n the m a t u r a t i o n o f ascospores  sug-  g e s t t h a t the r e a c h i n g o f a t h r e s h o l d amount o f a p a r t i c u l a r substance w i t h i n the ascus ( a s c - 5 ; Chapter I ) , Chapter I I I )  or w i t h i n each n u c l e u s  was e s s e n t i a l f o r the m a t u r a t i o n o f a s c o s p o r e s .  (SK(ad-3A); Finally,  the s t u d y o f two t y p e s o f mutants has p r o v i d e d some i n s i g h t i n t o the c o n t r o l o f m e i o t i c d i v i s i o n s i n Neurospora.  Thus, the apparent p r i m a r y  133  d e f e c 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 t o be f o l l o w e d by the e q u a t i o n a l centromere d i v i s i o n o f many o r a l l u n i v a l e n t s produced d u r i n g the f i r s t prophase. type of mutant ( a s c - 3 ) w i t h a p a r t i a l d e f e c t i v e attachment o f chromosomes  A second  b l o c k b e f o r e karyogamy,  has  t o one o f the two s p i n d l e p o l e  bodies o f the second m e i o t i c d i v i s i o n .  These o b s e r v a t i o n s may mean  t h a t a p r o d u c t produced b e f o r e karyogamy  ( p o s s i b l y d u r i n g the p r e -  m e 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 s e t o f chromatids t o one o f the s p i n d l e p o l e bodies have been d e s c r i b e d i n the d i s c u s s i o n  (some p o s s i b l e models  o f Chapter  II).  Even though, a t t h i s s t a g e , the i n f o r m a t i o n o b t a i n e d from these mutants i s s p e c u l a t i v e , the use of such mutants appears q u i t e p r o m i s i n g i n Neurospora.  F u t u r e r e s e a r c h s h o u l d i n v o l v e the i s o l a t i o n  o f t e m p e r a t u r e - s e n s i t i v e m u t a n t s , and of mutants t h a t i n t e r a c t t o change the phenotype o f e x i s t i n g m u t a n t s .  In a d d i t i o n , the  identifi-  c a t i o n o f the m o l e c u l a r nature o f the d e f e c t o f mutants w i t h 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 d e f i n e d d e f e c t s may become p o s s i b l e  through  the use o f e l e c t r o n m i c r o s c o p y o r b i o c h e m i s t r y (as was p r e v i o u s l y ment i o n e d i n the  Introduction).  I s o l a t i o n of Temperature-Sensitive  Mutants  T e m p e r a t u r e - s e n s i t i v e mutants may be i n s t r u m e n t a l i n d e t e r mining the temporal p e r i o d d u r i n g which t h e i r normal p r o d u c t 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 p r o d u c t . been o b t a i n e d i n D r o s o p h i l a E s p o s i t o and E s p o s i t o ,  Such m e i o t i c mutants have a l r e a d y V .  ( G r e l l , 1978) and y e a s t ( R o t h ,  1974).  1976;  In the absence o f a d i r e c t s e l e c t i o n  method f o r these m u t a n t s , l a r g e numbers o f PWT c u l t u r e s s h o u l d be  139  screened f o r t e m p e r a t u r e - s e n s i t i v e d e f e c t s . many PWT c u l t u r e s i s s t i l l  However, the i s o l a t i o n o f  q u i t e time-consuming because o f t h e low PWT  f r e q u e n c y of the c r o s s used t o i s o l a t e t h e s e c u l t u r e s . The p r e s e n t s e l e c t i o n system may be improved i n s e v e r a l  dif-  f e r e n t ways, a l l i n v o l v i n g c r o s s e s w i t h i n c r e a s e d PWT f r e q u e n c i e s . n a t u r e o f the s e l e c t i o n system f o 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  requires  t h a t the PWT c u l t u r e s s h o u l d be p r i m a r i l y d i s o m i c f o r LG I o n l y . cause o f the high frequency o f m u l t i p l e d i s o m i e s o b t a i n e d from homozygous  f o r a s c - 3 , a s c - 6 o r m e i - 1 , PWT c u l t u r e s from t h e s e  are not u s e f u l .  However, some c r o s s e s  homozygous  The  Becrosses  crosses  f o r asC-1 w i t h an i n -  t e r m e d i a t e PWT f r e q u e n c y (about 5%) might be o f use i f the 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 chromosomes homozygous  o t h e r than LG I i s a l s o l o w .  Since  f o r t h i s m u t a t i o n u s u a l l y produce about 40% ascospore  crosses abor-  t i o n , i t may not be p o s s i b l e t o i s o l a t e c e r t a i n types o f m u t a t i o n s s i m i l a r t o a s c - 1 and a s c - 6  ( t h e double mutant between a s c - 1 and a s c - 6  i n d i s t i n g u i s h a b l e from a s c - 1 and a s c - 6 s e p a r a t e l y ) .  To overcome  is this  d i f f i c u l t y , s e v e r a l o t h e r systems may be used. . F i r s t , PWT progeny may be o b t a i n e d from a c r o s s the dominant m e i o t i c mutant M e i - 2  ( S m i t h , 1975).  heterozygous  Since crosses  i n g M e i - 2 produce about 40% ascospore a b o r t i o n , o n l y PWT progeny taining Mei-2  +  s h o u l d be s e l e c t e d and t e s t e d .  for  involvcon-  T h i s may be a c h i e v e d by  s e l e c t i n g a g a i n s t an a u x o t r o p h i c m u t a t i o n c l o s e l y l i n k e d t o the M e i - 2 locus.  The success  o f t h i s system depends on t h e f r e q u e n c y o f PWT  progeny t h a t can be o b t a i n e d . Second, a mutant w i t h a t e m p e r a t u r e - s e n s i t i v e d e f e c t l e a d i n g t o PWT f o r m a t i o n would be e x t r e m e l y u s e f u l .  PWT c u l t u r e s c o u l d be o b t a i n e d  140  from c r o s s e s a t one temperature and t e s t e d f o r mutants a t the o t h e r temperature. T h i r d , the frequency o f PWT progeny can be i n c r e a s e d markedly w i t h the use o f the chemical p - f l u o r o p h e n y l a l a n i n e DeLange,  ( G r i f f i t h s and  1977).  Interacting  Mutations  I t may be a n t i c i p a t e d t h a t m u t a t i o n s which i n t e r a c t w i t h 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 r u m e n t a l i n the un-  d e r s t a n d i n g o f m e i o t i c development and i t s c o n t r o l .  In the  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 . example i s the study o f r e c o m b i n a t i o n i n E_. c o l i .  past,  A relevant  F i r s t , suppressors  sbc A and sbc B o f the r e c o m b i n a t i o n - d e f e c t i v e mutants r e c B and r e c C have i n a c t i v e e x o n u c l e a s e V I I I and exonuclease  I,  (Barbour and C l a r k , 1970; Kushner e t a]_., 1972).  respectively Second, f o u r mutant  genes r e s u l t i n r e c o m b i n a t i o n d e f i c i e n c y i n the r e v e r t e d r e c B r e c C sbc B s t r a i n ( H o r i i and C l a r k , 1973). p r o v i d e d 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)  c o m b i n a t i o n i n E_. c o l i . t e c t e d ( C l a r k , 1974).  These i n t e r a c t i o n mutants  In t h i s manner, t h r e e pathways  have been d e -  of c e l l u l a r  the example o f r e c o m b i n a t i o n c o n t r o l i n E_. c o l i  is  studies  in eu-  T h e r e f o r e , i t s h o u l d be a n t i c i p a t e d t h a t s i m i l a r  i n s e v e r a l e u k a r y o t i c organisms ( e . g . , Neurospora)  the s u c c e s s f u l  pro-  most  c l o s e l y r e l e v a n t t o the c o n t r o l o f r e c o m b i n a t i o n and m e i o s i s k a r y o t i c organisms.  of r e -  Although many o t h e r examples o f i n t e r a c t i n g  mutants have been i n s t r u m e n t a l i n the u n d e r s t a n d i n g cesses,  have  d i s s e c t i o n o f r e c o m b i n a t i o n and m e i o t i c  may enable  processes.  141  Second-site  r e v e r s i o n s may be dominant o r r e c e s s i v e .  be v i r t u a l l y i m p o s s i b l e t o o b t a i n r e c e s s i v e s u p p r e s s o r s t e r i l i t y mutants i n Neurospora  It  will  mutations  (e.g., asc-2, asc-4, mei-3,  of  uvs-3,  u v s - 5 , uvs-6) because PWT c u l t u r e s needed t o s c r e e n f o r such m u t a t i o n s cannot be o b t a i n e d from c r o s s e s homozygous f o r s t e r i l i t y  mutations.  However, the i s o l a t i o n o f r e v e r t a n t s would be p o s s i b l e i n c o n d i t i o n a l ( e . g . , t e m p e r a t u r e - s e n s i t i v e ) mutants:  PWT c u l t u r e s c o u l d be o b t a i n e d  a t one temperature and the s c r e e n i n g would be performed a t the r e strictive  temperature.  The i s o l a t i o n o f r e v e r t a n t s o f m u t a t i o n s w i t h a d e f e c t i n p a i r i n g or exchange, and t h e r e f o r e chromosome d i s j u n c t i o n , i s t i a l l y possible,since  poten-  PWT f r e q u e n c i e s i n such mutants are u s u a l l y  The i s o l a t i o n o f dominant r e v e r t a n t s would be even s i m p l e r  a s c - 3 (P393) may p o s s i b l y  In  laboratory  s t o c k s may a l s o modify the phenotype o f m e i o t i c m u t a t i o n s . f r e q u e n c y o f b l a c k ascospores  high.  because  mutagenized c o n i d i a , i n s t e a d o f PWT c u l t u r e s , can be s c r e e n e d . a d d i t i o n , v a r i a n t s o b t a i n e d from n a t u r e or i n e x i s t i n g  .  The  high  o b t a i n e d i n some c r o s s e s homozygous f o r  be an example o f  this.  F i n a l l y , m u t a t i o n s t h a t i n t e r a c t w i t h the spore k i l l e r mutat i o n SK(ad-3A) s h o u l d be r e a d i l y o b t a i n e d .  S i n c e the m u t a t i o n SK(ad-3A)  a p p a r e n t l y causes a d e f e c t i n c o m p l e m e n t a t i o n , i t i s q u i t e p l a u s i b l e t h a t c e r t a i n o t h e r components  i n t e r a c t t o a c h i e v e such complementation.  In e f f e c t , a l t e r a t i o n o f one o r more o f t h e s e i n t e r a c t i n g c o u l d a c t as a s u p p r e s s o r  components  o f SK,. and .thus r e s t o r e the a b i l i t y o f SK to  complement ad-3A m u t a t i o n s .  The study o f such i n t e r a c t i o n m u t a t i o n s  may p r o v i d e i n s i g h t i n the developmental s t e p s  involved.  In t h i s c a s e , d e t e c t i o n o f s u p p r e s s i o n may be a f a s t procedure s i n c e thousands  o f a s c i c o u l d be q u i c k l y s c o r e d f o r the presence o r  absence o f m o s t l y b l a c k  ascospores.  P o t e n t i a l o f F u t u r e S t u d i e s on the M o l e c u l a r or M i c r o - S t r u c t u r e L e v e l The u l t i m a t e u n d e r s t a n d i n g o f m e i o t i c development n e c e s s a r i l y involves biochemical c h a r a c t e r i z a t i o n .  Because o f the asynchrony  development o f a s c i and t h e i r attachment t o v e g e t a t i v e c e l l s , analysis  i s p r e s e n t l y not p o s s i b l e i n Neurospora.  of  such  However, some  g e n e t i c m a n i p u l a t i o n and development o f t e c h n i q u e s t h a t a l l o w the separ a t i o n o f a s c i from s u r r o u n d i n g blem.  The p a r t i a l  c e l l s s h o u l d l a r g e l y overcome t h i s  pro-  s y n c h r o n i z a t i o n o f m e i o t i c development has been  a c h i e v e d by temperature shock i n Coprinus  lagopus  ( L u , 1974; Lu and  J e n g , 1975), o r by a temporary i n h i b i t i o n o f DNA s y n t h e s i s phyllum commune (Carmi e t a l _ . , 1978).  in Schizo-  The same may p o s s i b l y be  a c h i e v e d by c e r t a i n m u t a t i o n s , e . g . , Ban i n N e u r o s p o r a , 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 B a k e r , B. S., 1975. P a t e r n a l l o s s ( p a l ) : a m e i o t i c mutant i n Droso phi l a melanogaster c a u s i n g l o s s o f p a t e r n a l chromosomes. Genet i c s 80:267-296. B a k e r , B. S., J . B. Boyd, A. T. C. C a r p e n t e r , M. M. G r e e n , T. D. Nguyen, P. R i p o l l , and P. D. S m i t h , 1976b. 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Genetics 71:s77.  Spore k i l l e r genes i n  Neurospora.  151  V i g f u s s e n , N. V . , and J . W e i j e r , 1972. 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 s e x u a l development c y c l e . Genet. R e s . , Camb. 19:205-211. W e i j e r , J . , and N. V. V i g f u s s e n , 1972. S e x u a l i t y i n Neurospora c r a s s a . I. M u t a t i o n s to male s t e r i l i t y . Genet. R e s . , Camb. 19:191-204. W e s t e r g a a r d , M . , and H. K. M i t c h e l l , 1947. medium f a v o u r i n g s e x u a l r e p r o d u c t i o n .  Neurospora. Am. J . B o t .  Zimmering, S., L. S a n d l e r , and B. N i c o l e t t i , 1970. d r i v e . Ann. Rev. Genet. 4:409-436.  V. A s y n t h e t i c 34:573-577.  Mechanisms o f m e i o t i c  152  APPENDIX LIST OF ABBREVIATIONS asc CO  R e c e s s i v e m u t a t i o n r e s u l t i n g i n the a b o r t i o n o f a s c i and/or a s c o s p o r e s . Crossover.  HK  Heterokaryon o r h e t e r o k a r y o t i c .  HN2  Nitrogen mustard.  LG  Linkage  MI  F i r s t meiotic d i v i s i o n .  Mil  Second m e i o t i c d i v i s i o n .  mei  Meiotic mutation.  MMS  Methyl methane s u l p h o n a t e .  MNNG  N-methyl N - n i t r o  PWT  Pseudo-wild type.  RF  Recombinant f r e q u e n c y .  SK  Spore k i l l e r m u t a t i o n .  SPB  S p i n d l e p o l e body.  UV  Ultraviolet.  group.  N-nitrosoguanidine.  PUBLICATIONS A . J . F . G r i f f i t h s , A.M.DeLange and J.H.Jung, 1974. I d e n t i f i c a t i o n of a complex chromosome rearrangement i n Neurospora crassa.  Can.J.Genet.  Cytol. 16: 805-822. A.M.DeLange and A . J . F . G r i f f i t h s , 1975. Escape from mating-type incomp a t i b i l i t y i n 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 B r i t i s h Columbia. A . J . F . G r i f f i t h s and A.M.DeLange, 1977. p-Fluorophenylalanine increases meiotic nondisjunction i n a Neurospora test system.Mutation Research 46: 345-354. ,1978. Mutations of the a. mating type gene i n Neurospora crassa. Genetics 88k 239-254. A.M.DeLange, 1980. Meiosis i n Neurospora crassa I. The i s o l a t i o n of recessive mutants defective i n the production of v i a b l e ascospores. (Submitted). A.M.DeLange, 1980. Meiosis i n Neurospora crassa I I . Genetic and cytol o g i c a l characterization of four meiotic mutants. (Submitted). A.M.DeLange, 1980. The mutation Sk(ad-3A) a l t e r s the dominance of ad-3A over ad-3A i n the ascus of Neurospora. (Submitted).  +  

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