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"A/a INCOMPATIBILITY IN NEUROSPORA CRASSA\u2014NOVEL  SUPPRESSORS  AND NUCLEAR INCOMPATIBILITY by TRINA SEHAR VELLANI B.Sc.(Hon.)# McMaster U n i v e r s i t y , 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department o f Botany) We accept t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1991 (c) T r i n a Sehar V e l l a n i , 1991  \u000CIn  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be her  representatives.  permission.  of  Botany  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  -3 O r f n W  1QQ1  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  Department  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  \u000CABSTRACT  The s e x u a l f u n c t i o n s of the mating type gene (int) of Neurospora  crassa  i n c l u d e s p e c i f i c a t i o n of mating  (Shear and Dodge, 1927)  identity  and p e r i t h e c i a l maturation  ( G r i f f i t h s and DeLange, 1978;  Staben and Yanofsky,  1990).  The gene a l s o a c t s as a v e g e t a t i v e i n c o m p a t i b i l i t y l o c u s , so t h a t A + a heterokaryons  (Beadle and Coonradt,  1944)  d u p l i c a t i o n s t r a i n s (Newmeyer and T a y l o r , 1967) or  or A/a  grow p o o r l y  not a t a l l . An i n t r i g u i n g q u e s t i o n r e g a r d i n g the mating type gene  is this:  How  does i t c o n t r o l both the s w i t c h between  somatic and m e i o t i c events and heterokaryon S e v e r a l r e s e a r c h groups Yanofsky, of  1990)  Two new  1990;  Staben  and  are p r e s e n t l y s t u d y i n g the s e x u a l f u n c t i o n s  the mating type genes.  incompatibility  for  (Glass, et a l . ,  incompatibility?  I p r e s e n t a study of the  function.  experiments were performed.  The f i r s t was  a search  s u p p r e s s o r s of mating t y p e - a s s o c i a t e d  i n c o m p a t i b i l i t y , which r e s u l t e d i n the i d e n t i f i c a t i o n of seven new  s u p p r e s s o r s , none of which was  one known suppressor, tol.  allelic  The second was  w i t h the  the comparison  growth r a t e s of a mating type mutant ( f e r t i l e ,  of  heterokaryon  compatible) i n a mixed mating type heterokaryon and i n a mixed mating type d u p l i c a t i o n t o determine whether or not c y t o p l a s m i c i n c o m p a t i b i l i t y i s s e p a r a b l e from n u c l e a r  \u000Ciii incompatibility.  The r e s u l t s o b t a i n e d suggest t h a t the  mating type mutant, a , m33  eliminates  i n c o m p a t i b i l i t y without e l i m i n a t i n g  heterokaryon nuclear  incompatibility.  The s e a r c h f o r s u p p r e s s o r s was attempted i n order t o d e f i n e more of the genes i n v o l v e d  i n A/a  incompatibility.  The a n a l y s i s o f heterokaryon v e r s u s n u c l e a r  incompatibility  was done t o i n v e s t i g a t e the c e l l u l a r i n t e r a c t i o n s i n A/a  incompatibility.  involved  \u000Civ TABLE OF CONTENTS ABSTRACT  i i  TABLE OF CONTENTS  iv V  LIST OF TABLES LIST OF FIGURES  vi  ACKNOWLEDGEMENT  viii  GENERAL INTRODUCTION  ;  1  L i f e Cycle  1  Mating Type Gene F u n c t i o n s  4  Mating and I n c o m p a t i b i l i t y i n Other F u n g i .  6  Mating and I n c o m p a t i b i l i t y i n Other Kingdoms INTRODUCTION 1  16 18  A Suppressor o f A/a I n c o m p a t i b i l i t y , MATERIALS AND METHODS  tol  .18 22  S t r a i n s and Markers  22  Ascospore I s o l a t i o n  24  Construction tol,  o f T e s t e r S t r a i n (T(I-&gt;II) 39311, s e r , t r p ,  a)  RESULTS 1 DISCUSSION 1  25 *  28 51  What i s tol?  56  INTRODUCTION 2  62  RESULTS 2  67  DISCUSSION 2  97  M o l e c u l a r Model REFERENCES  101 103  \u000CV  LIST OF TABLES Table 1  S t r a i n s from Experiment Set 1  23  Table 2  Phenotypes o f F2 S t r a i n s  38-39  Table 3  Phenotypes o f F3 S t r a i n s  44  Table 4  Mating Types o f F3 S t r a i n s  46  Table 5  Mating Types o f Hyphal T i p s o f A/a Compatible F3 Strains  49  Table 6  Summary o f R e s u l t s 1  50  Table 7  S t r a i n s and Media Used i n t h e Measurement o f Growth Rate o f A  m 6 4  i n a Mixed Mating Type  Heterokaryon Table 8  68  S t r a i n s and Media Used i n t h e Measurement o f Growth Rate o f a  m 3 3  i n a Mixed Mating Type  Heterkaryon  71  Table 9  Genotypes o f a  Table 10  Mating Types o f Progeny  77  Table 11  Phenotypes o f Progeny  79  Table 12  Slopes o f Growth Rates o f Progeny  95  Table 13  Mating Types o f S i n g l e C o n i d i a l I s o l a t e s o f Progeny  m 3 3  Strains  73  96  \u000Cvi LIST OF FIGURES Figure 1  L i f e C y c l e o f 2V. crassa  2  Figure 2  Example o f M i t o t i c Crossover  7  Figure 3  Construction of Tester Strain  Figure 4  Summary o f S e l e c t i o n P r o t o c o l f o r Suppressors..29  Figure 5  F i r s t Cross  Figure 6  Phenyotypic C l a s s e s o f Escaped F l S t r a i n s . . . . . . 3 4  Figure 7  Second Cross  36  Figure 8  Examples o f M i t o t i c Double C r o s s o v e r s  41  Figure 9  T h i r d Cross  42  F i g u r e 10  A l t e r n a t i v e P a i r i n g Hypothesis  53  F i g u r e 11  Mating Type Regions o f N. crassa  63  F i g u r e 12  Segregation of A  66  F i g u r e 13  Growth Rate o f A  31  m 6 4  m64  ORF i n a Mixed Mating Type  Heterokaryon F i g u r e 14  69  Growth Rate o f a  m 3 3  i n a Mixed Mating Type  Heterokaryon F i g u r e 15A Cross o f a , m33  26  72 ad x T(I-&gt;II) 39311, s e r - 3 , A...74  F i g u r e 15B Crosses o f Rl-14 o r Rl-29 x T(I-&gt;II) 39311, ser-3, A  75  F i g u r e 16  Growth Rates o f C o n t r o l s  81  F i g u r e 17  Growth Rates o f a-h-x  82  F i g u r e 18  Growth Rates o f a-h-x  83  F i g u r e 19  Growth Rates o f a-h-x  84  F i g u r e 20  Growth Rates o f a - i - x  85  F i g u r e 21  Growth Rates o f a - i - x  86  \u000Cvii F i g u r e 22  Growth Rates of 14-h-x  87  F i g u r e 23  Growth Rates o f 14-h-x  88  F i g u r e 24  Growth Rates o f 14-i-x  89  F i g u r e 25  Growth Rates o f 14-i-x  90  F i g u r e 26  Growth Rates o f 29-h-x  91  F i g u r e 27  Growth Rates of 29-h-x  92  F i g u r e 28  Growth Rates o f 29-i-x  93  F i g u r e 29  Growth Rates of 29-i-x  94  \u000Cviii ACKNOWLEDGEMENT I am immensely g r a t e f u l t o t h e f o l l o w i n g people: Tony G r i f f i t h s f o r h i s support, f o r f o s t e r i n g my s c i e n t i f i c independence and f o r a l l o w i n g me freedom t o develop my own i d e a s ; L o u i s e G l a s s f o r her encouragement and c o u n t l e s s hours o f c r i t i c a l d i s c u s s i o n ; J i m Berger f o r h i s boundless enthusiasm f o r t h e world o f s c i e n c e and f o r keeping me from s t r a y i n g t o o f a r from t h e t a s k a t hand; C a r o l y n Myers f o r s h a r i n g h e r r e s e a r c h i d e a s and f o r i n v a l u a b l e t e c h n i c a l a d v i c e ; Rod, Mishu and my p a r e n t s f o r b e l i e v i n g i n me.  \u000C1 GENERAL INTRODUCTION  T h i s work i s a two-part i n v e s t i g a t i o n of the v e g e t a t i v e i n c o m p a t i b i l i t y f u n c t i o n of the mating type gene of Neurospora of  crassa.  suppressors  The  o f A/a  first  p a r t d e s c r i b e s the  generation  i n c o m p a t i b i l i t y and the second p a r t  d e s c r i b e s the a n a l y s i s of i n c o m p a t i b i l i t y on a c e l l u l a r level.  The mating type gene i s i n v o l v e d i n both  v e g e t a t i v e and s e x u a l phases of the l i f e  Life  the  cycle.  Cycle  W.  crassa,  f i r e s and  a mold t h a t grows a t the s i t e s of r e c e n t  i n decaying v e g e t a t i o n , i s a h e t e r o t h a l l i c  ascomycete.  I t s l i f e c y c l e i s shown i n F i g . 1.  The  h a p l o l d , p a r t i a l l y s e p t a t e m y c e l i a grow from  ascospores  which are germinated by a p e r i o d of h e a t i n g .  Growth  r e q u i r e s the presence o f i n o r g a n i c s a l t s , b i o t i n and u t i l i z a b l e carbon source  (Fincham, e t a l . , 1979).  S e v e r a l days a f t e r germination, to  d e l i m i t two  a  the hyphal t i p s  begin  types of c o n i d i a which, upon germination,  a b l e t o i n i t i a t e new  mycelia.  Macroconidia  are o v a l  are  and  m u l t i n u c l e a t e ; whereas m i c r o c o n i d i a are s p h e r i c a l and b i n u c l e a t e or u n i n u c l e a t e (Fincham, e t al, Low  1979).  l e v e l s of n i t r o g e n i n i t i a t e the s e x u a l c y c l e .  s t r u c t u r e s \u2014 c o n i d i a and v e g e t a t i v e h y p h a e \u2014 a r e a l r e a d y p r e s e n t i n each s i n g l e mating type colony  (A o r a ) , and  Male  \u000C2  oscogenous hypho  igure 1  Life cycle of N. crassa (from Fincham, et al., 1979).  \u000C3  immature female s t r u c t u r e s \u2014 p r o t o p e r i t h e c i a \u2014 b e g i n t o form from t h e same mycelium. protoperithecium  The outer l a y e r o f t h e  i s a w a l l o f hyphae.  Inside i s a c o i l e d  hypha, t h e ascogonium, from which p r o j e c t female r e p r o d u c t i v e hyphae, t r i c h o g y n e s . hermaphroditic,  Although  each mycelium i s  i t i s a l s o s e l f - s t e r i l e , and s e x u a l f u s i o n  between male c e l l s and p r o t o p e r i t h e c i a can occur between i n d i v i d u a l s o f d i f f e r e n t mating t y p e s .  only Once  f e r t i l i z e d , t h e p r o t o p e r i t h e c i u m i s known as a p e r i t h e c i u m (Fincham, e t a l . , 1979). C o n i d i a emit a pheromone which d i r e c t s t r i c h o g y n e s t o grow toward them ( B i s t i s , 1981; 1983).  When c o n t a c t i s made  between t h e male and female c e l l s , plasmogamy ensues.  The  male n u c l e i , presumably under t h e i r own g e n e t i c c o n t r o l ( V i g f u s s o n , e t al., 1971), t r a v e l down t h e t r i c h o g y n e , where one nucleus  (Sansome, 1949) e n t e r s t h e ascogonium and  becomes a s s o c i a t e d w i t h t h e female n u c l e u s .  While t h e  p e r i t h e c i u m darkens and e n l a r g e s , a s e r i e s o f synchronous nuclear d i v i s i o n s gives r i s e t o a c l u s t e r of d i k a r y o t i c ascogenous hyphae (Fincham, e t a l . , 1979). Karyogamy occurs i n t h e penultimate  c e l l s of the  ascogenous hyphae, f o l l o w e d d i r e c t l y by m e i o s i s p l u s two rounds o f m i t o s i s .  S e v e r a l days l a t e r , t h e ascospores  become m u l t i n u c l e a t e (Raju, 1980).  have  The f i n a l products o f  the s e x u a l c y c l e a r e p e r i t h e c i a c o n t a i n i n g many mature a s c i , each housing  e i g h t ascospores  which a r e shot through an  opening i n t h e p e r i t h e c i a l beak (Fincham, e t a l . , 1979).  \u000C4  Mating Type Gene F u n c t i o n s  The mating type genes, A and a, are unusual, even among Neurospora  s p e c i e s , i n t h a t they c o n t r o l two  mating and v e g e t a t i v e i n c o m p a t i b i l i t y . i s not seen i n e i t h e r N. tetrasperma sitophila  (Mishra, 1971).  incompatibility  (Dodge, 1935)  o r N.  E a r l y attempts t o r e s o l v e the  f u n c t i o n s by recombination f a i l e d Newmeyer, e t al.,  A/a  functions,  (Pittenger,  1973); although l a t e r ,  two  1957;  Griffiths  and  DeLange (1978) r e p o r t e d the f i n d i n g of a mating type mutant (a ) m33  t h a t was  heterokaryon compatible, y e t  In N. crassa,  fertile.  o n l y s t r a i n s of o p p o s i t e mating types are  a b l e t o c r o s s (Shear and Dodge, 1927); so A x a i s a s u c c e s s f u l p a i r i n g , but A x A o r a x a i s not.  S t r a i n s of  o p p o s i t e mating t y p e s are v e g e t a t i v e l y i n c o m p a t i b l e (Beadle and Coonradt,  1944); so A + A or a + a anastomose t o form  t h r i v i n g heterokaryons, and A + a f u s e , but the anastomosed area d i e s (Garnjobst and Wilson, 1956).  The protoplasm of  the f u s e d , and sometimes surrounding, c e l l s becomes g r a n u l a r or v a c u o l a t e d .  Mixed mating type heterokaryons w i t h v a r y i n g  degrees o f v i g o u r can be made u s i n g f o r c i n g a u x o t r o p h i c markers (Beadle and Coonradt, Griffiths,  1944;  Gross, 1952;  DeLange and  1975).  P r o t o p l a s m i c k i l l i n g , more severe than t h a t  seen  between A and a, i s observed i n the r e a c t i o n s between incompatible a l l e l e s of the heterokaryon genes het-C/c,  het-D/d and het-E/e  incompatibility  ( P e r k i n s , 1974).  The  \u000C5  k i l l i n g r e a c t i o n can be d e t e c t e d in vivo,  and a l s o when  protoplasm from one s t r a i n i s m i c r o i n j e c t e d i n t o c e l l s o f an i n c o m p a t i b l e s t r a i n , a t l e a s t between s t r a i n s of d i f f e r e n t het-C/c  o r het-D/d genotypes (Wilson, e t al.,  1961; W i l l i a m s  and W i l s o n , 1966). Mixed mating type heterokaryons g e n e r a l l y escape from t h e i r poor growth and s t a r t t o grow a t w i l d - t y p e o r near w i l d - t y p e r a t e s by d e l e t i o n o f one o r the o t h e r o f t h e mating type genes  (DeLange and G r i f f i t h s ,  1975).  Heterokaryons heterozygous f o r the v e g e t a t i v e i n c o m p a t i b i l i t y genes h e t - J / j o r het-K/k escape by d e l e t i o n o r mutation o f the genes  ( P i t t e n g e r , 1964).  heterozygous d u p l i c a t i o n o f the v e g e t a t i v e  S t r a i n s with a incompatibility  gene, het-6, a l s o escape by d e l e t i o n o f one o f t h e genes (Glass, personal  communication).  Not o n l y are the mating type genes i n c o m p a t i b l e i n a heterokaryon, they are a l s o i n c o m p a t i b l e i n a d u p l i c a t i o n . S t r a i n s c a r r y i n g a heterozygous d u p l i c a t i o n o f the mating type genes grow p o o r l y due t o the presence o f o p p o s i t e mating type genes i n one n u c l e u s .  A/a d u p l i c a t i o n  strains,  c a l l e d \"dark agar\" s t r a i n s , produce a brown pigment when grown on g l y c e r o l complete medium and t h e i r morphology  has  been d e s c r i b e d as b e i n g s p i d e r y (Newmeyer and T a y l o r ,  1967;  Turner, e t a l . ,  1969).  Other d u p l i c a t i o n s i n N, crassa  grow  normally ( p r o v i d e d t h e d u p l i c a t i o n does not cover any o f the heterokaryon i n c o m p a t i b i l i t y genes) and are f r e q u e n t l y b a r r e n , i . e . they produce abundant p e r i t h e c i a , but few  \u000C6  spores (Newmeyer and T a y l o r , 1967). from t h e i r i n h i b i t e d growth and o r near w i l d - t y p e  A/a  d u p l i c a t i o n s escape  s t a r t growing a t  wild-type  r a t e s by the somatic s e g r e g a t i o n  of A from  a through m i t o t i c c r o s s i n g over ( F i g . 2) or d e l e t i o n of of the mating type genes (Newmeyer and T a y l o r ,  one  1967).  M i t o t i c c r o s s i n g over y i e l d s a c u l t u r e , barren  due  to  the presence o f d u p l i c a t e d g e n e t i c m a t e r i a l , t h a t i s a mixture of mostly A o r a homozygous c e l l s . are u n s t a b l e  and  tend t o be overgrown by one  D e l e t i o n y i e l d s a c u l t u r e , f e r t i l e due  Such c u l t u r e s nuclear  type.  t o the l o s s of p a r t  o r a l l of the d u p l i c a t i o n , t h a t i s a mixture of A o r a hemizygous c e l l s .  These c u l t u r e s are a l s o overgrown by  n u c l e a r type (Newmeyer and T a y l o r ,  Mating and  1967).  I n c o m p a t i b i l i t y i n Other Fungi  The m a j o r i t y of r e s e a r c h on y e a s t mating type has done on Saccharomyces  cerevisiae,  y e a s t because h a p l o i d c e l l s budding. cells, alpha  Mating begins w i t h G l a r r e s t .  cells,  alpha-factor,  cells,  by  Pheromone from a  and pheromone from  arrests a c e l l s .  mating type c e l l s  been  a l s o known as budding  reproduce v e g e t a t i v e l y  a - f a c t o r , a r r e s t s alpha  opposite  one  P a i r s of  f u s e , undergo karyogamy and  then  f o l l o w e i t h e r of 2 paths, depending on  nutritional  conditions.  reproduce m i t o t i c a l l y  The  a/alpha  diploid cells  \u000C-r-  o c\u2014 \u00BB oC\u2014O , T  '  nic  FiflllTP 9 l l y U I C c.  four  +  \u00A3  o  -\u2022 \u2014  \u2022-  r?  o o  ^ .  C 3 O  *  \u00A3  C  +  /  t 0.  +  co O  0  \u2022 \u2014 \u2022 \u2014 \u00BB \u2014 \u00BB\u2014 #-  n  Example from Newmeyer and Taylor (1967) of mitotic crossover that leads to production of cells homozygous for the mating type genes. The duplication was a product from a cross to an inversion strain.  \u000C8  u n l e s s they are n u t r i t i o n a l l y d e p r i v e d , i n which case they undergo m e i o s i s (see review by Herskowitz, 1988). The mating types o f the h a p l o i d c e l l s are s p e c i f i e d a t the  mating type l o c u s , MAT  (Lindegren and Lindegren, 1943),  which codes f o r t r a n s c r i p t i o n f a c t o r s t h a t c o n t r o l the e x p r e s s i o n o f genes i n v o l v e d i n pheromone p r o d u c t i o n , mating and s p o r u l a t i o n .  C e l l s w i t h mating type a have t h e MATa  a l l e l e which encodes two p o l y p e p t i d e s , al and a2; c e l l s  with  mating type alpha have the MATalpha a l l e l e which a l s o encodes two p o l y p e p t i d e s , alphal the  MAT  and alpha2.  genes, c a l l e d Ya and Yalpha  A p o r t i o n of  (Nasmyth, e t a l . , 1981),  i s s p e c i f i c t o a and alpha c e l l s , r e s p e c t i v e l y (Sprague, e t al.,  1981). Three of the mating type p o l y p e p t i d e s , a l , alphal  alpha2, specific  are i n v o l v e d i n the r e g u l a t i o n of a - s p e c i f i c , and h a p l o i d c e l l - s p e c i f i c  a2 i s unknown ( A s t e l l ,  genes.  e t al., 1981).  and alpha-  The f u n c t i o n o f  In a c e l l s , al i s  produced ( K a s s i r and Simchen, 1976) and a - s p e c i f i c and h a p l o i d s p e c i f i c genes are e x p r e s s e d . alphal  In alpha  cells,  induces the e x p r e s s i o n o f a l p h a - s p e c i f i c genes  (Sprague, e t al., 1983) and alpha2  represses a - s p e c i f i c  genes ( H a r t i g , e t a l . , 1986; Wilson and Herskowitz, 1984). In a/alpha  d i p l o i d c e l l s , alpha2  c a r r i e s out the same  f u n c t i o n as i t does i n h a p l o i d alpha c e l l s , r e p r e s s i n g as p e c i f i c genes ( S t r a t h e r n , e t a l . , 1981), but i t has an additional role.  A combination product o f alpha2  and al  \u000C9  r e p r e s s e s the e x p r e s s i o n  of alphal,  and of h a p l o i d s p e c i f i c  genes and s t i m u l a t e s s p o r u l a t i o n ( S t r a t h e r n , e t al., S p o r u l a t i o n begins when a/alpha  diploid cells  s t a r v e d of n i t r o g e n and carbon ( E s p o s i t o and 1981).  The  r e g u l a t o r y p r o t e i n al/alpha2  by b l o c k i n g the e x p r e s s i o n  1981). are  Klapholz,  a c t i v a t e s meiosis  of RMEI, an i n h i b i t o r of  meiosis  ( M i t c h e l l and Herskowitz, 1986). S t r a i n s of S. cerevisiae the homothallism gene, HO,  with the dominant a l l e l e  of  are h o m o t h a l l i c , whereas s t r a i n s  with the r e c e s s i v e a l l e l e , ho,  are h e t e r o t h a l l i c .  The  HO  gene product c a t a l y s e s h i g h frequency i n t e r c o n v e r s i o n of mating t y p e s (Hicks and Herskowitz, 1976).  Mating type  i n t e r c o n v e r s i o n occurs by the s w i t c h i n g of the i n f o r m a t i o n a t the MAT S t r a t h e r n , e t al.,  l o c u s (Nasmyth and T a t c h e l l ,  1980;  H i c k s , e t al.,  i n f o r m a t i o n comes from two HMR  and HML,  1977).  t h a t f l a n k the mating type gene. The  two  a t MAT by HO  (Strathern, et a l . ,  1982)  by the endonuclease encoded A conversion-like  event f o l l o w s , i n which heteroduplex DNA and MAT.  r e p a i r e d u s i n g the donor DNA S t r a t h e r n , 1984;  (for \"silent  begins with a double-stranded c u t  ( K o s t r i k e n and H e f f r o n , 1984).  or HML)  Each l o c u s  (Abraham, e t a l . , 1983).  s w i t c h i n g process  donor l o c u s (HMR  The  l o c i are kept s i l e n t  by the a c t i o n of u n l i n k e d genes c a l l e d SIR information regulator\")  1980;  t r a n s c r i p t i o n a l l y s i l e n t genes,  c o n t a i n s a copy o f a or alpha.  The  genetic  Klar et a l . ,  forms between the  The heteroduplex DNA  is  as a template ( K l a r and 1984).  Switching  i s prevented  \u000C10  i n d i p l o i d c e l l s by t h e r e p r e s s i o n o f HO (Jensen, e t al., 1983). Mating i n t h e f i s s i o n y e a s t , Schizosaccharomyces i s s i m i l a r i n some ways t o t h a t i n Saccharomyces H a p l o i d c e l l s have t h e mating type h  +  pombe, cerevisiae.  o r h~ and propagate  v e g e t a t i v e l y by f i s s i o n , not by budding.  During mating,  which o c c u r s under n i t r o g e n s t a r v a t i o n c o n d i t i o n s , one c e l l of  each mating type p a r t i c i p a t e s i n t h e f o r m a t i o n o f a  d i p l o i d zygote.  The zygote immediately undergoes m e i o s i s  and s p o r u l a t i o n (Leupold, 1950 c i t e d i n K e l l y , e t a l . , 1988). As i n h o m o t h a l l i c s t r a i n s o f S. cerevisiae,  S. pombe  r e g u l a r l y switches a l l e l e s a t t h e mating type l o c u s 1977; M i y a t a and Miyata, 1981).  (Egel,  The mating type complex  c o n t a i n s 3 r e g i o n s \u2014 m a t l , mat2-P and mat3-M\u2014which  control  c o n j u g a t i o n , m e i o s i s and s p o r u l a t i o n ( K e l l y , e t a l . ,  1988).  Two s i l e n t l o c i , mat2-P and mat3-M donate i n f o r m a t i o n t o matl which c o n f e r s mating type, e i t h e r h  +  (matl-P)  o r h~  (matl-M) ( E g e l , 1977; E g e l and Gutz, 1981; Beach, 1983). The complex has been sequenced ( K e l l y , e t a l . ,  1988).  Two genes a r e encoded by each o f matl-P and matl-M, two o f which a r e r e q u i r e d f o r c o n j u g a t i o n and s p e c i f i c a t i o n o f mating type, and a l l 4 o f which a r e r e q u i r e d f o r m e i o s i s and sporulation (Kelly, et a l . ,  1988).  Each o f matl, mat2-P and  mat3-M c o n t a i n s 2 b l o c k s o f sequence homology.  The s i l e n t  genes, mat2-P and mat3-M, alone c o n t a i n a t h i r d r e g i o n o f  \u000C11  homology which p r o b a b l y a c t s as a s i l e n c e r ( K e l l y , e t al., 1988). L i k e RMEI o f S. cerevisiae,  t h e p r o t e i n , rani,  pombe i s an i n h i b i t o r o f m e i o s i s .  I t s a c t i o n i s b l o c k e d by  the p r o t e i n , mei3, which i s produced i n h / h ~ c e l l s +  and Beach, 1988).  Neurospora  o f S.  crassa  (McLeod  c o u l d be l i k e S.  cerevisiae  i n t h a t t h e mating type genes c o u l d a c t l i k e  al/alpha2,  combining t o form a t r a n s c r i p t i o n f a c t o r t h a t  b l o c k s t h e s y n t h e s i s o f a m e i o s i s i n h i b i t o r , o r i t c o u l d be l i k e S. pombe i n t h a t a product analogous t o mei3 c o u l d be produced i n A/a mating d i p l o i d c e l l s . r e g u l a t i n g c e l l type i n S. cerevisiae crassa  The complex  system o f  must d i f f e r i n N.  because each mating type idiomorph encodes but one  transcript. That t h e combination product, A/a, c o u l d a c t as a t r a n s c r i p t i o n f a c t o r i s supported by t h e sequence  similarity  between t h e a idiomorph and t h e HMG box m o t i f , a DNA-binding sequence  (Staben and Yanofsky, 1990).  The A idiomorph has  no obvious DNA-binding m o t i f , but i t does have s i m i l a r i t y t o the MATalphal p r o t e i n o f S. cerevisiae with t h e a product d u r i n g mating.  and c o u l d  interact  During t h e v e g e t a t i v e  s t a t e , A i d e n t i t y c o u l d be s p e c i f i e d by A - s p e c i f i c  genes,  turned on by t h e A product i n t e r a c t i n g w i t h a t r a n s c r i p t i o n f a c t o r t h a t b i n d s t o t h e i r promoter r e g i o n s ( G l a s s , e t a l . , 1990).  S t r a i n s c o n t a i n i n g a heterozygous d u p l i c a t i o n o f t h e  mating type r e g i o n do n o t l o s e e i t h e r t h e i r A o r a i d e n t i t y ,  \u000C12  so t h e combination product does not exclude mating  type  specificity. Homothallism  i n W. crassa  must a l s o occur by a  d i f f e r e n t mechanism than s w i t c h i n g because each s t r a i n i n the h o m o t h a l l i c s p e c i e s N. dodgei, africana  and W. lineolata  IV. galapagonensis,  has o n l y one copy o f a sequence  homologous t o t h e N. crassa  mating type gene, A.  h o m o t h a l l i c s p e c i e s , N. terricola, t o the N. crassa copy o f each  N.  has sequences  The homologous  mating type genes, A and a, but o n l y one  (Glass, e t a l . ,  1988).  The i n c o m p a t i b i l i t y systems, a l s o c a l l e d b r e e d i n g systems, o f t h e 2 basidiomycetes, Coprinus Schizophyllum  commune, a r e t e t r a p o l a r .  cinereus The mating  and type  complex i s comprised o f 2 r e g i o n s , A and B, each c o n t a i n i n g 2 genes, alpha  and beta,  with m u l t i p l e a l l e l e s .  Mating  r e q u i r e s t h a t t h e 2 p a r t i c i p a n t s d i f f e r a t a minimum o f one A gene and one B gene.  C l o s e l i n k a g e o f alpha  and beta  r e s t r i c t s i n b r e e d i n g p o t e n t i a l by i n h i b i t i n g recombination (see K o l t i n , e t a l . ,  1972).  C l o n i n g o f t h e A f a c t o r o f C. cinereus t h a t t h e alpha  and beta  has r e v e a l e d  r e g i o n s a r e themselves composed o f a  number o f genes w i t h m u l t i p l e a l l e l e s , some o f which a r e common t o o t h e r a l l e l e s o f A and some o f which a r e unique (E. Mutasa, A. Tymon, W. Richardson, U. Kues and L. C a s s e l t o n , 1991 i n p u b l i s h e d a b s t r a c t s from S i x t e e n t h Fungal Genetics Conference).  \u000C13  Three a l l e l e s of t h e A-alpha  r e g i o n o f S. commune have  been c l o n e d , sequenced and shown t o c o n t a i n m u l t i p l e t r a n s c r i p t s , some shared and some unique.  Some o f t h e  p o s t u l a t e d p o l y p e p t i d e s c o n t a i n homeodomain m o t i f s (R.C. U l l r i c h , M.M. S t a n k i s , H. Yang and C P . Novotny, 1991; G. May,  1991 i n p u b l i s h e d a b s t r a c t s from S i x t e e n t h Fungal  G e n e t i c s Conference),  i m p l y i n g t h a t t h e products r e g u l a t e  the e x p r e s s i o n o f o t h e r genes. In t h e pathogenic basidiomycete,  Ustilago  maydis,  d i k a r y o s i s between i n d i v i d u a l s from d i f f e r e n t i n c o m p a t i b i l i t y groups i s a p r e r e q u i s i t e f o r pathogenic infection.  D i f f e r e n t a a l l e l e s a r e r e q u i r e d f o r f u s i o n and  d i f f e r e n t b a l l e l e s f o r pathogenicity.  The two a l l e l e s o f a  have been c l o n e d and they encode a product r e q u i r e d f o r m y c e l i a l growth, a c o n d i t i o n necessary f o r i n f e c t i o n (M. B o l k e r and R. Kahmann, 1991 i n p u b l i s h e d a b s t r a c t s from S i x t e e n t h Fungal G e n e t i c s  Conference).  Ten b a l l e l e s have been c l o n e d , and subsequent m o l e c u l a r a n a l y s i s has r e v e a l e d t h a t they share a homeodomain-related m o t i f , i m p l y i n g t h a t t h e b p o l y p e p t i d e s b i n d DNA, p o s s i b l y t o r e g u l a t e sexual development (R. Kahmann, B. G i l l i s s e n , R. S c h l e s h i n g e r , C  Sandmann, F.  Schauwecker, J . Bergemann, B. Schroeer, M. B o l k e r and M. Dahl, 1991 i n p u b l i s h e d a b s t r a c t s from S i x t e e n t h Fungal G e n e t i c s Conference). commune and C. cinereus,  L i k e t h e A and B r e g i o n s o f S. t h e b r e g i o n o f U. maydis i s  composed o f 2 genes, b-east  and Jb-west.  N u l l mutants o f t h e  \u000Cb r e g i o n are mating d e f i c i e n t , s u g g e s t i n g t h a t the p o s t u l a t e d b heterodimer  formed d u r i n g mating i s an  a c t i v a t o r of mating genes (Kahmann, p e r s o n a l communication) S t u d i e s done with e x p r e s s i o n of two  of the b  alleles  have i d e n t i f i e d a 70 amino a c i d r e g i o n r e s p o n s i b l e f o r allele specificity. expressed  i n d i p l o i d s and h a p l o i d s , although a t a lower  l e v e l i n the l a t t e r 1991  These 2 b a l l e l e s are c o n s t i t u t i v e l y  (L. Giasson, A. Yee and J.W.  Kronstad,  i n p u b l i s h e d a b s t r a c t s from S i x t e e n t h Fungal crassa  Genetics  Conference).  S i m i l a r l y , i n W.  the mating type genes  are expressed  d u r i n g s e x u a l and v e g e t a t i v e phases of the  l i f e c y c l e , but a t a lower l e v e l i n the l a t t e r Yanofsky, 1990; N. crassa ways.  and  G l a s s , e t a l . , 1990). d i f f e r s from the basidiomycetes  The mating type genes of W.  so u n l i k e the basidiomycetes, r e s t r i c t o r s of i n b r e e d i n g .  crassa  hyphae w i t h compatible  ORF  they do not f u n c t i o n as  A l s o , i n N. crassa,  genotypes l e a d s t o  f u s i o n of sexual c e l l s with  genotypes l e a d s t o mating.  i n several  have o n l y one  c o m p a t i b i l i t y i s not a p r e r e q u i s i t e f o r mating.  formation and  (Staben  vegetative F u s i o n of  heterokaryon compatible  The h e t e r o k a r y o s i s t h a t occurs  between v e g e t a t i v e c e l l s i s somehow d i f f e r e n t from the f u s i o n t h a t occurs between the t r i c h o g y n e and the male c e l l Mixed mating type heterokaryons  on c r o s s i n g medium do  not  e x h i b i t the i n c o m p a t i b i l i t y phenotype, so perhaps i t i s the n u t r i t i o n a l c o n d i t i o n s t h a t d i c t a t e whether the mating type genes w i l l  i n i t i a t e i n c o m p a t i b i l i t y o r mating.  \u000CThe mating type gene o f the y e a s t s i s a master r e g u l a t o r o f o t h e r genes.  The mating type genes o f the  basidiomycetes and o f N. crassa  are a l s o p o s t u l a t e d t o be  r e g u l a t o r y , a l t h o u g h t h e mechanisms o f t h e i r a c t i o n s must differ. Each o f the f o u r major groups o f f u n g i \u2014 p h y c o m y c e t e s , basidiomycetes, ascomycetes and f u n g i g e n e t i c a l l y determined i n c o m p a t i b i l i t y some c a s e s , e.g. Neurospora Podospora  anserina,  1971), Rhizoctonia parasitica the  crassa  imperfecti\u2014exhibits (Burnett, 1976).  (Fincham, e t a l . ,  under the c o n t r o l o f the s l o c u s solani  (Burnett, 1976) and  In  1979), (Esser  Endothia  (chestnut b l i g h t fungus) (Anagnostakis, 1977),  i n c o m p a t i b i l i t y only a f f e c t s vegetative heterokaryosis;  whereas i n o t h e r s , e.g. Aspergillus  nidulans  1961; G r i n d l e , 1963a; 1963b), Podospora  anserina,  c o n t r o l o f t h e a,Jb,c,v l o c i o r n o n - a l l e l i c system 1971; B l a i c h and E s s e r , 1971), Coprinus Schizophyllum  commune (Fincham e t a l . ,  al.  (Jinks, et  under the (Esser,  lagopus, 1979), Ustilago  sp.  (Fincham e t al., 1979; Day and Cummins, 1981) and y e a s t s ( C r a n d a l l , 1978), the g e n e t i c r e s t r i c t i o n s on f u s i o n a f f e c t fertility. Two s p e c i e s o f fungus o t h e r than N. crassa  t h a t have  mating type genes t h a t a c t as heterokaryon i n c o m p a t i b i l i t y l o c i are Ascobolus communication) Raper, 1967).  stercorarius  and Aspergillus  (Bistis,  personal  heterothallicus  (Kwon and  \u000C16  Mating and I n c o m p a t i b i l i t y  i n Other Kingdoms  Each o f t h e f i v e kingdoms o f organisms\u2014Monera, P r o t i s t a , Fungi, p l a n t s  and a n i m a l s \u2014 s h o w s examples o f  g e n e t i c a l l y c o n t r o l l e d , i n t r a s p e c i f i c , s e x u a l o r somatic incompatibility.  Moreover, i n t e r a c t i o n s between  species,  f o r example, those between host and pathogen o r between symbionts, can e x h i b i t  incompatibility.  Sexual i n c o m p a t i b i l i t y individuals within  l i m i t s mating between c e r t a i n  a species.  Somatic  incompatibility  l i m i t s f u s i o n between o r c o - e x i s t e n c e o f c e r t a i n  cells.  Some o f t h e f u n g i demonstrate incompatible r e a c t i o n s  which  are both somatic and s e x u a l , i n t h a t a s u c c e s s f u l i n t e r a c t i o n between seemingly v e g e t a t i v e c e l l s leads t o mating. In t h e kingdom Monera, mating i n Escherichia requires  recognition  o f mating t y p e s ; and o n l y  coli pairings  between c e l l s o f d i f f e r e n t mating types a r e compatible (Hayes, 1952; Lederberg, 1957). Ciliates,  i n t h e kingdom P r o t i s t a , g e n e r a l l y  mate w i t h  c e l l s o f a d i f f e r e n t mating type and f u s i o n occurs between \"vegetative\"  cells  (there  a r e no c e l l s s p e c i a l i z e d f o r  mating) under c e r t a i n environmental and p h y s i o l o g i c a l conditions  (Nanney, 1977; R i c c i ,  In p l a n t s ,  t h e r e a r e examples o f both s e x u a l and  somatic i n c o m p a t i b i l i t y . which c o n t r o l s  1981).  Some angiosperms have t h e S l o c u s  sexual c o m p a t i b i l i t y ,  allowing  pollen to  \u000C17  f e r t i l i z e o n l y females with d i f f e r e n t S a l l e l e s 1954;  E b e r t e t a l . , 1989;  Haring e t al.,  (Lewis,  1990).  Somatic  i n c o m p a t i b i l i t y occurs between d i f f e r e n t s p e c i e s of p l a n t s when t i s s u e from one s p e c i e s i s g r a f t e d onto an of  individual  another s p e c i e s , even from the same f a m i l y (Yeoman e t  al.,  1978). F u s i o n of c e l l s i n the s e x u a l c y c l e of Chlamydomonas  sp. r e q u i r e s r e c o g n i t i o n o f o p p o s i t e mating types (Wiese and Wiese, 1978;  H a r r i s , 1989)  a l l e l e s , mt  and Bit\"*, a t one  +  which are determined locus.  by  two  The a l l e l e s are  b e l i e v e d t o encode p r o t e i n s t h a t c o n t r o l the e x p r e s s i o n of o t h e r genes o r the a c t i v i t y of t h e i r products, p o s s i b l y by forming o r c a u s i n g the f o r m a t i o n of a novel r e g u l a t o r y product upon c e l l  f u s i o n ( F e r r i s and Goodenough, 1987).  A somatic i n t e r a c t i o n i s seen i n v e r t e b r a t e s when a g r a f t of t i s s u e o r an organ t r a n s p l a n t i s r e j e c t e d from recipient.  the  The r e a c t i o n , i n t h i s case, i s c o n t r o l l e d by  genes of the major h i s t o c o m p a t i b i l i t y complex (MHC), which are expressed i n the T c e l l s of the immune system  (see  reviews by K l e i n , 1976;  Humoral  Bach and vanRood, 1976).  r e c o g n i t i o n o c c u r s i n the b l o o d , and i t i s governed by a n t i b o d i e s s p e c i f i c f o r the ABO  blood group a n t i g e n s and  Rh f a c t o r p r o t e i n (see review by Katz, 1978).  the  \u000C18  INTRODUCTION 1  A Suppressor o f A/a I n c o m p a t i b i l i t y ,  tol  Newmeyer (1970) found a r e c e s s i v e suppressor o f A/a i n c o m p a t i b i l i t y , u n l i n k e d t o mt, which she c a l l e d t o l f o r \"tolerant\".  The new gene had no demonstrable  a b i l i t y o f a s t r a i n t o mate.  e f f e c t on the  I t appears t o be i n a c t i v e  d u r i n g s t a r v a t i o n because A + a heterokaryons on c r o s s i n g medium do not e x h i b i t t h e i n c o m p a t i b i l i t y phenotype. However, Johnson (1979) suggested t h a t t h e gene does have a r o l e d u r i n g mating because t h e r e c e s s i v e a l l e l e , tol, suppresses fmf-1, fertility.  a gene s p e c i f y i n g female and male  Crosses between fmf-1,  tol  +  and fmf-l ,  are s t e r i l e , whereas c r o s s e s between fmf-1, tol  tol and  Johnson h y p o t h e s i z e d t h a t t o l  are f e r t i l e .  tol  +  +  fmf-l , +  permits  promiscuous f u s i o n between A and a d u r i n g mating, a l l o w i n g t r a n s f e r o f fmf-l  +  product from t h e fmf-l  +  i n rescue o f t h e s t e r i l i t y phenotype.  strain,  If tol  +  resulting  i s expressed  d u r i n g t h e s e x u a l c y c l e , f u s i o n o f s e x u a l s t r u c t u r e s occurs d e s p i t e t h e presence o f t o l . +  I t i s possible that the  cytoplasm o f t r i c h o g y n e s i s m o d i f i e d t o a l l o w t h e presence of male n u c l e i o f t h e o p p o s i t e mating  types.  Genes t h a t suppress A/a i n c o m p a t i b i l i t y w i l l be u s e f u l i n a n a l y s i n g t h e mating type gene i t s e l f and i n d e c i p h e r i n g the process o f i n c o m p a t i b i l i t y .  Moreover, i f a suppressor  has i t s own phenotype, i n a d d i t i o n t o s u p p r e s s i n g A/a  \u000C19  i n c o m p a t i b i l i t y , then o t h e r f u n c t i o n s r e l a t e d t o i n c o m p a t i b i l i t y may be r e v e a l e d .  F o r example, i f a  suppressor p r e v e n t s i n c o m p a t i b i l i t y by a l t e r i n g c e l l w a l l s t r u c t u r e so t h a t i t can no l o n g e r be broken down by t h e i n c o m p a t i b i l i t y r e a c t i o n , i t may produce of  abnormal morphology.  a second  phenotype  Two types o f s u p p r e s s o r s ,  e x t r a g e n i c and i n t r a g e n i c , a r e d i s c u s s e d below. E x t r a g e n i c suppressors probably i n t e r a c t w i t h t h e mating type genes; and t h e i r d e t e c t i o n w i l l h e l p i n the d i s s e c t i o n o f t h e mechanism o f i n c o m p a t i b i l i t y . to  T h e o r i e s as  how t o l a f f e c t s i n c o m p a t i b i l i t y a r e c o n s i d e r e d i n  D i s c u s s i o n 1. Other s u p p r e s s o r s , b e s i d e s t o l , have been two  identified\u2014  found i n nature and two induced i n t h e l a b o r a t o r y .  Smith and P e r k i n s (1972) noted t h a t t h e o s m o t i c - s e n s i t i v e , r e c i p r o c a l t r a n s l o c a t i o n s t r a i n , c u t , suppressed A/a incompatibility.  Newmeyer (1970) r e p o r t e d t h a t t h e w i l d  type s t r a i n , Panama a, (Fungal G e n e t i c s Stock Center #1132) segregated compatible and i n c o m p a t i b l e progeny when c r o s s e d to  a duplication-generating inversion A tester.  It i s  unknown whether o r not t h i s suppressor i s a l l e l i c w i t h t o l . Newmeyer (1970) found another suppressor, which may o r may n o t be a l l e l i c w i t h t o l , strain  i n an escaped A/a d u p l i c a t i o n  ( c a l l e d N83) from a c r o s s between an i n v e r s i o n A  s t r a i n and a normal sequence a s t r a i n . Griffiths  DeLange and  (1975) r e p o r t e d t h a t 2 o f t h e i r escaped mixed  mating type heterokaryons, i n which one component was tol  \u000C20 and t h e o t h e r tol , +  produced  type t e s t e r s , but produced  p e r i t h e c i a with both  mating  ascospores w i t h o n l y 1.  They  suggested t h a t these 2 s t r a i n s arose by d e l e t i o n o r l e t h a l mutation a t  tol . +  I n t r a g e n i c suppressors and mutants o f t h e mating  type  genes w i l l h e l p , when mapped and sequenced, t o r e v e a l t h e p a r t ( s ) o f t h e mating type genes governing v e g e t a t i v e incompatibility.  E s p e c i a l l y u s e f u l w i l l be those mutants i n  which o n l y one o f t h e mating type f u n c t i o n s i s d e f e c t i v e , e.g. t h e f e r t i l e , heterokaryon compatible mutant,  a . m33  When Newmeyer d i s c o v e r e d tol, she was u s i n g s t r a i n s o f N. crassa  w i t h A/a d u p l i c a t i o n s , dark agars.  They were  m e i o t i c segregants from a c r o s s o f a normal sequence A t o a s t r a i n o f mating type a c a r r y i n g a p e r i c e n t r i c i n v e r s i o n o f a l a r g e p o r t i o n o f l i n k a g e group I (L.G. I) ( F i g . 2).  Most  o f t h e dark agars escaped from t h e i r i n h i b i t e d growth by the somatic s e g r e g a t i o n o f A from a.  One s t r a i n , however,  escaped by mutation a t tol, thereby a l l o w i n g both  mating  type genes t o r e s i d e w i t h i n one nucleus without c a u s i n g vegetative incompatibility.  The new tol gene proved t o be a  suppressor, n o t o n l y o f n u c l e a r i n c o m p a t i b i l i t y , but a l s o o f heterokaryon i n c o m p a t i b i l i t y , a l l o w i n g t h e v i g o r o u s growth o f both d u p l i c a t e d A/a,tol  s t r a i n s and A,tol  +  a,tol  heterokaryons. Newmeyer favoured t h e use o f a A/a d u p l i c a t i o n t o cause i n h i b i t e d growth because i t e l i m i n a t e d t h e p o s s i b i l i t y i n c o m p a t i b i l i t y was due t o a l l e l e s a t some o t h e r  that  \u000C21  heterokaryon i n c o m p a t i b i l i t y l o c u s .  Furthermore,  i f a mixed  mating type heterokaryon had been used, o n l y \"dominant\" a l l e l e s of suppressor genes would have allowed escape t o occur. A method s i m i l a r t o t h a t used by Newmeyer (1970) has been used t o generate n o v e l suppressors o f A/a incompatibility.  The s e a r c h i s an attempt t o c h a r t the  g e n e t i c i n t e r a c t i o n s i n v o l v e d i n mating t y p e - a s s o c i a t e d i ncompat i b i 1 i t y .  \u000C22  MATERIALS AND METHODS  General p r o t o c o l s were standard, and a r e d e s c r i b e d i n Davis and DeSerres (1970).  Crosses were made a t 25\u00B0C i n 15  cm t e s t tubes o r , i f fl females were used, on p e t r i  plates.  Ascospores w i t h A/a d u p l i c a t i o n s were s e l e c t e d as m e i o t i c segregants from c r o s s e s t o s t r a i n s i n which t h e mating type gene had been t r a n s l o c a t e d from L.G. I onto L.G. II.  D u p l i c a t i o n progeny c o n t a i n e d L.G. I I from t h e  t r a n s l o c a t i o n parent and L.G. I from t h e o t h e r p a r e n t . combination o f l i n k a g e groups was s e l e c t e d as f o l l o w s .  This The  mating type genes were marked, one w i t h an a u x o t r o p h i c marker (ser-3) and t h e o t h e r w i t h a temperature marker (un-3),  sensitive  and ascospores were p l a t e d on minimal medium  a t 32\u00B0C which p e r m i t t e d s u r v i v a l o f o n l y t h e d u p l i c a t i o n spores.  S t r a i n s and Markers  A l i s t o f s t r a i n s and t h e i r sources i s shown i n T a b l e 1.  S t r a i n s were maintained a t room temperature on standard  media. The marker, un-3, i s t e m p e r a t u r e - s e n s i t i v e , w i t h s t r a i n s growing p o o r l y between 28.5\u00B0C and 30\u00B0C and not growing a t a l l a t temperatures above 30\u00B0C. 0.04  I t i s located  t o 0.1 map u n i t s t o t h e l e f t o f t h e mating type gene  (Perkins, e t a l . ,  1982).  The marker c o n t a i n s two mutations,  \u000CTable 1 Strains from Experiment Set 1 STRAIN  GENOTYPE  SOURCE  R601  un-3, A  C.J. Myers  R602  un-3, a  C.J. Myers  T(l-&gt;ll)39311,ser,A  T(l-&gt;ll)39311, s e r - 3 , A  C.J. Myers  T(l-&gt;ll)39311,ser,a  T(l-&gt;ll)39311, s e r - 3 , a  C.J. Myers  T(l-&gt;ll)39311,ser,trp,tol,A  T(l-&gt;ll)39311, s e r - 3 , trp-4, tol, A  C.J. Myers  T(l-&gt;H)39311,ser,trp,tol,a  T(l-&gt;ll)39311, s e r - 3 , trp-4, tol, a  This work  trp,tol,A  trp-4, tol, A  N.L. Glass (F.G.S.C.* #2336)  fl,A  F.G.S.C. #4960  fl,a  F.G.S.C. #4961  \u2022F.G.S.C.-Fungal Genetics Stock Center  \u000C24  s e p a r a t e d by 0.1 map  u n i t s , one i n cytochrome-20  o t h e r i n ethionine-2  (A.M. Lambowitz, u n p u b l i s h e d r e s u l t s ,  G r i f f i t h s , p e r s o n a l communication).  and the  T e s t s f o r un-3 were  done on minimal medium a t 25\u00B0C and a t 37\u00B0C and were repeated 3 times each f o r p o s i t i v e i d e n t i f i c a t i o n .  In these t e s t s , a  h i g h e r temperature (37\u00B0C) c o u l d be used than was used t o s e l e c t d u p l i c a t i o n progeny  (32\u00B0C) because the un-3  phenotype  i s more obvious a t 37\u00B0C and i t was not necessary t o a v o i d killing  the f u n g i .  The t r a n s l o c a t i o n T(I-&gt;II) 39311 interstitial  i s an i n s e r t i o n of an  segment of the l e f t arm of l i n k a g e group I  ( i n c l u d i n g ser-3,  un-3 and mt) i n t o the r i g h t arm of l i n k a g e  group I I , i n v e r t e d w i t h r e s p e c t t o the centromere  (Perkins,  1972). The gene, ser-3,  s p e c i f i e s a requirement f o r s e r i n e and  i s l o c a t e d fewer than 2 map  u n i t s t o the l e f t of the mating  type gene ( P e r k i n s , e t a l . ,  1982).  The gene, trp-4,  s p e c i f i e s a requirement f o r tryptophan  and i s l i n k e d t o t o l by l e s s than 1 map al.,  unit  (Perkins, et  1982). The a c o n i d i a l mutant, fluffy  (fl),  i s highly  fertile  and i s used as a mating type t e s t e r .  Ascospore I s o l a t i o n  Ascospores were i s o l a t e d as f o l l o w s , u n l e s s s t a t e d otherwise.  Spores were c o l l e c t e d from the s i d e s o f the  \u000C25  c r o s s i n g t e s t tubes w i t h a wire l o o p f u l of s t e r i l e water and put i n t o eppendorf tubes of s t e r i l e water o r 0.1%  agar.  distilled  Hemocytometer counts were done t o  determine the necessary d i l u t i o n f a c t o r t h a t would approximately 10-30  distilled  spores per p l a t e .  yield  D i l u t e d spores (1/4  mL) were p i p e t t e d and spread onto sorbose p l a t e s w i t h a bent glass rod.  The p l a t e s were p l a c e d  i n t o a 60\u00B0C oven f o r 30  minutes t o heat shock the spores t o i n i t i a t e g e r m i n a t i o n . Spores were l e f t a t room temperature f o r 4-5 hours t o ensure t h a t the m y c e l i a were w e l l e s t a b l i s h e d p r i o r t o s e l e c t i o n . The p l a t e s were l e f t o v e r n i g h t i n a 32\u00B0C i n c u b a t o r  so t h a t  the c o l o n i e s grew l a r g e enough t o be seen and h a r v e s t e d . I n d i v i d u a l germinated spores were c o l l e c t e d by c u t t i n g out a square of agar c o n t a i n i n g the mycelium  and p l a c i n g i t  i n t o a t e s t tube c o n t a i n i n g 1 mL o f Westergaard  and  M i t c h e l l ' s l i q u i d medium. I f , a f t e r s e v e r a l days, the l e v e l of the l i q u i d i n the tubes dropped below h a l f , then s t e r i l e d i s t i l l e d water was added t o m a i n t a i n the l e v e l o f l i q u i d a t 1  mL.  Construction tol,  o f T e s t e r S t r a i n (T(I-&gt;II) 39311, s e r , t r p ,  a)  The s t r a i n , T(I-&gt;II) 39311, s e r , t r p , t o l , a was made from a c r o s s o f a female t r p , t o l , A t o a male T(I-&gt;II) 39311, s e r , a, from which s e r , t r p , t o l , a progeny were selected  ( F i g . 3).  The female was  i n o c u l a t e d onto s y n t h e t i c  \u000C26  T(l-&gt;ll)39311,ser,a x trp,tol,A  IV  ser a II trp tol  IV  select X  IV dead  II trp tol  IV  IV  ser a  A/a  II trp tol  IV  IV II trp tol  IV  Construction of the strain T(l-&gt;ll)39311,ser,trp,toi,a. The strain, marked X, was a product of the cross between T(l-&gt;ll)39311,ser,a x trp,tol,A  \u000C27  c r o s s i n g medium c o n t a i n i n g 10% o f the normal c o n c e n t r a t i o n of tryptophan  ( i . e . 20 mg/L i n s t e a d o f 200 mg/L) because t h e  c r o s s was s t e r i l e on t h e normal c o n c e n t r a t i o n o f tryptophan. Presumably t h e tryptophan  was p r o v i d i n g t o o much n i t r o g e n  f o r t h e s e x u a l c y c l e t o be i n i t i a t e d communication).  (Myers, p e r s o n a l  \u000C28  RESULTS 1  An o v e r a l l scheme o f t h e procedure the suppressors i s shown i n F i g . 4. (Fl)  used i n g e n e r a t i n g  Duplication strains  were s e l e c t e d as m e i o t i c segregants  from a s e t o f  c r o s s e s ( r e f e r r e d t o as t h e \" f i r s t c r o s s \" ) between a normal chromosomal sequence parent and a t r a n s l o c a t i o n parent. d u p l i c a t i o n s t r a i n s escaped Fl)  from i n h i b i t e d growth  and were t e s t e d f o r t h e i r mating t y p e s .  r e t a i n e d both mating types  (A/a escaped  The  (escaped  Those which  F l )possibly  c o n t a i n e d t h e d e s i r e d suppressors and were c r o s s e d t o normal chromosomal sequence fl s t r a i n s t o remove t h e t r a n s l o c a t i o n chromosome.  T h i s s e t o f c r o s s e s i s c a l l e d t h e \"second  cross\". Some o f these F2 progeny presumably c o n t a i n e d suppressors.  Only t h e t e m p e r a t u r e - s e n s i t i v e F2 s t r a i n s were  t e s t e d f o r t h e presence  o f suppressors because t h e un-3  marker was needed t o s e l e c t d u p l i c a t i o n progeny i n the t h i r d set  of crosses.  The t e m p e r a t u r e - s e n s i t i v e F2 s t r a i n s were  crossed t o t e s t e r s t r a i n s containing the t r a n s l o c a t i o n T(I-&gt;II)  39311.  D u p l i c a t i o n progeny were s e l e c t e d and  assessed f o r t h e i r compatible/incompatible  phenotypes.  D e t a i l e d d e s c r i p t i o n s o f each s t e p f o l l o w . D u p l i c a t i o n s t r a i n s ( l a b e l l e d \" F l \" i n F i g . 4) were c r e a t e d ( P e r k i n s , 1972) from a p a i r o f c r o s s e s , r e c i p r o c a l i n t h e sense t h a t t h e mating types were r e v e r s e d . of  This set  c r o s s e s i s r e f e r r e d t o as the \" f i r s t c r o s s \" and i s  \u000C29  First Cross (to generate A/a duplications): R601,un-3 x T(l-&gt;ll)39311,ser,a minimal medium, 32 degn es to select duplications  F1  A/a Duplication Strains escape to select suppressors  Escaped F1 Strains Choose A/a Escaped F1 (presumed suppressor strains)  Second Cross (to eliminate duplication): A/a Escaped F1 x fl,a and fl,A  F2 Choose temperature sensitive (t.s.) F2 (need un-3 for selection of duplications in third cross)  Third Cross (to confirm suppressor phenotype): t.s. F2 x T(l-&gt;ll)39311,ser,trp,tol minimal medium, 32 degree t to select duplications  F3 (t.s. F2 strains that produced incompatible and compatible duplication F 3 progeny were the suppressor strains) \u2014. . llCjUre 4  A summary of the selection protocol for suppressors. The reciprocal cross was R602 x T(l-&gt;ll)39311,ser,A.  \u000C30  d e s c r i b e d i n t h e f o l l o w i n g t h r e e paragraphs. the  One c r o s s i n  p a i r , shown i n F i g . 5, was made between a normal  chromosomal sequence female parent (R601) and a male parent c o n t a i n i n g a t r a n s l o c a t i o n (T(I-&gt;II) 39311, s e r , a ) . In t h e r e c i p r o c a l c r o s s (not shown), t h e normal chromosomal sequence female p a r e n t was R602 and t h e male t r a n s l o c a t i o n parent was T(I-&gt;II) 39311, s e r , A. The normal sequence p a r e n t s , R601 and R602 had t h e i r mating type genes, A and a, r e s p e c t i v e l y , marked w i t h t h e t e m p e r a t u r e - s e n s i t i v e gene, un-3, which i s l e s s than 1 map u n i t t o t h e l e f t o f mt.  The t r a n s l o c a t i o n s t r a i n s had t h e i r  mating type genes marked w i t h t h e a u x o t r o p h i c gene, which i s l e s s than 2 map u n i t s t o t h e l e f t o f mt.  ser-3, The  mating type genes were marked t o a l l o w s e l e c t i o n o f progeny t h a t c o n t a i n e d both A and a. The g e r m i n a t i o n o f spores on minimal medium a t 32\u00B0C s e l e c t e d progeny t h a t c o n t a i n e d L.G. I from t h e normal parent ( s e r - 3 , un-3, A o r ser-3 , +  the A).  +  un-3, a) and L.G. I I from  t r a n s l o c a t i o n parent ( s e r - 3 , un-3 , a o r s e r - 3 , +  un-3 , +  The genotype o f t h e s e l e c t e d spores was s e r - 3 , un-3, +  A/ser-3,  un-3 , a o r , from the r e c i p r o c a l c r o s s ,  un-3, a/ser-3,  +  un-3 , A. +  ser-3 , +  Since the n u c l e i of the selected  F l s t r a i n s c o n t a i n e d both mating types, growth was i n h i b i t e d and t h e c u l t u r e s grew w i t h t h e dark agar morphology hyphae growing i n a t i g h t knot ( P e r k i n s ,  of short  1972).  F i g u r e 5 d e p i c t s o n l y one p o s s i b l e p a i r i n g \u2014 t h a t o f t h e two L.G. I ' s .  Although i t i s not shown i n t h e f i g u r e , t h e  \u000C31  R601  T(l-&gt;ll)39311,ser,a  minimal medium, 32 degrees to select duplications  II Ser 3 -  +  Un~3 A  ^ f  (temperature-sensitive)  II  II  II s e r - 3 un-3+ a  LIVES (A/a duplication)  s e r - 3 un-3+ a  Figure 5  DIES  DIES (deletion)  DIES (serine-requiring)  The first set of crosses was done to generate A/a duplication strains. The crosses were R601 x T(l-&gt;ll)39311,ser,a and x T(l-&gt;ll)39311,ser,A.  \u000C32  t r a n s l o c a t e d p o r t i o n of L.G.  I i s long enough t o p a i r w i t h  L.G.  communication).  I (Metzenberg, p e r s o n a l  As  explained  i n the f o l l o w i n g paragraph, the occurrence of t h i s p a i r i n g p a t t e r n would not have a f f e c t e d the experimental I f the t r a n s l o c a t e d the i n t a c t L.G.  I and  s e c t i o n of L.G.  I had  a s i n g l e crossover  had  design.  paired with  occurred  p a i r e d r e g i o n , the products would not have s u r v i v e d of the f o r m a t i o n of d i c e n t r i c and a double c r o s s o v e r  had  occurred  because  a c e n t r i c chromosomes.  i n the p a i r e d r e g i o n ,  s u r v i v i n g progeny would o n l y have had  one  spores would not have been chosen as one  If  the  mating type,  t h e r e f o r e would have grown as w i l d t y p e .  FI  i n the  and  These types of of the A/a  escaped  strains. Spore c u l t u r e s w i t h standard i n c o m p a t i b i l i t y  and  c o m p a t i b i l i t y phenotypes were needed as c o n t r o l s f o r comparison t o the d u p l i c a t i o n progeny.  Such s t r a i n s were  o b t a i n e d as progeny from the c r o s s between the normal chromosomal sequence female, R601  (un-3, A),  and  the  t r a n s l o c a t i o n male, T(I-&gt;II) 39311, s e r , t r p , t o l , a. Spores were germinated and grown on medium tryptophan a t 32\u00B0C. containing and  L.G.  L.G.  These c o n d i t i o n s  I ( s e r - 3 , un-3, +  A)  containing  s e l e c t e d progeny  from the female parent  I I ( s e r - 3 , un-3 , a) from the male parent. +  the progeny, those c o n t a i n i n g  L.G.  IV (trp-4 , +  the female, were i n c o m p a t i b l e d u p l i c a t i o n s , and containing  L.G.  IV  (trp-4,  compatible d u p l i c a t i o n s .  tol)  tol ) +  Half  of  from  h a l f , those  from the male were  \u000C33  From the d u p l i c a t i o n - g e n e r a t i n g  c r o s s e s , R601  x  T(I-&gt;II) 39311, s e r , a and R602 x T(I-&gt;II) 39311, s e r , A, 182 d u p l i c a t i o n ascospores were c o l l e c t e d and maintained on l i q u i d medium. phenotype  Each d u p l i c a t i o n c u l t u r e e x h i b i t e d the same  as t h a t shown by the A/a, tol  +  incompatible  c o n t r o l s , growing as a s m a l l dense mass o f hyphae a t the bottom o f the t e s t tube.  The A/a, tol compatible c o n t r o l s  grew f a s t e r , f i l l i n g the t e s t tube a f t e r s e v e r a l days.  All  of the spore c u l t u r e s escaped from i n c o m p a t i b i l i t y w i t h i n 2 weeks, although escape o c c u r r e d a t d i f f e r e n t times f o r each strain.  Escape was d e t e c t e d as a s h i f t i n hyphal morphology  from the dense growth t o l e s s dense growth and by an i n c r e a s e i n growth r a t e . In o r d e r t o e l i m i n a t e s t r a i n s t h a t had escaped by d e l e t i o n o f mating type genes and t o i d e n t i f y double mating type s t r a i n s t h a t may tol  have escaped because o f mutation t o  o r t o a t o l - l i k e suppressor (A/a escaped F l s t r a i n s ) ,  the mating type o f each escaped F l s t r a i n was t e s t e d by s p o t t i n g i t on p r o t o p e r i t h e c i a l lawns o f fl,A  and  fl,a  testers. The c r o s s i n g behaviours o f the escaped F l s t r a i n s allowed t h e i r d i v i s i o n i n t o 9 phenotypic c l a s s e s , 8 of which are d e s c r i b e d f i g u r e was  i n F i g . 6.  The one c l a s s not shown i n the  comprised o f 25 s t r a i n s t h a t r e t a i n e d the  c a p a c i t y t o c r o s s and produce ascospores w i t h t e s t e r s of both mating t y p e s .  These 25 s t r a i n s ( l a b e l l e d \"A/a  F l \" i n F i g . 4) were the ones presumed t o c o n t a i n  escaped  \u000CREACTION WHEN CROSSED TO fl,A  fl,a  Legend No reaction Barren perithecia Mature perithecia  6  Eight phenotypic classes of escaped F1 strains that were no used in the experiment.  \u000C35  suppressors.  The remaining 159 s t r a i n s were not used i n the  r e s t of the experiment, but t h e i r p o s s i b l e o r i g i n s are c o n s i d e r e d i n D i s c u s s i o n 1. The next s e t of c r o s s e s ( r e f e r r e d t o as the c r o s s \" ) was  done t o demonstrate  \"second  t h a t the 25 A/a escaped F l  s t r a i n s a c t u a l l y contained suppressors.  Escape i n these  s t r a i n s had not o c c u r r e d by d e l e t i o n of e i t h e r of the mating type genes, but i t may  have o c c u r r e d by m i t o t i c c r o s s o v e r o r  by some as y e t u n i d e n t i f i e d mechanism o t h e r than mutation or d e l e t i o n a t a new  suppressor l o c u s .  A t the same time, the  second c r o s s s e r v e d t o get the suppressors i n t o a s t a b l e background,  one without d u p l i c a t i o n s .  The t r a n s l o c a t i o n chromosome, L.G.  I I , was  removed by  c r o s s i n g each A/a escaped F l c u l t u r e t o f l , a and f l , A females ( F i g . 7 ) .  Crosses i n v o l v i n g a d u p l i c a t i o n  are f r e q u e n t l y \"barren\", i . e . they produce few (Newmeyer and T a y l o r , 1967).  strain  ascospores  Some of the c r o s s e s of the A/a  escaped F l s t r a i n s t o fl females produced as few as ascospores.  1-6  Spores from these c r o s s e s were not c o l l e c t e d by  the p l a t i n g method because many would have been l o s t the procedure.  I n s t e a d , they were c o l l e c t e d  through  individually  w i t h a tungsten needle under the microscope and p l a c e d i n l i q u i d medium.  The tubes of l i q u i d medium were heat  shocked  i n a 60\u00B0C water bath t o i n i t i a t e g e r m i n a t i o n o f the spores, then l e f t a t room temperature. d a i l y f o r the appearance  The t e s t tubes were checked  of growth and the r e s u l t s are shown  \u000CTemperature-sensitive  II ser-3+ un-3  A  NO  s e r - 3 un-3+ a  -Some compatible -Some incompatible  I YES II  ser-3+ un-3+ a  NO  s e r - 3 un-3+ a  II  FlGlJTP 7 iv^uic;  l  NO  The second set of crosses was done to eliminate the duplication from the putative suppressor strains. The crosses were: A/a escaped F1 derived from R601 x fl,a and A/a escaped F1 derived from R602 x fI,A. Temperature-sensitive progeny were chosen because un-3 was needed as a marker in the third cross and to ensure that the translocation chromosome was eliminated.  -Some compatible -Some incompatible  \u000C37  i n Table 2.  D e s p i t e c a r e f u l c o l l e c t i o n of spores, t h e r e  were 10 c r o s s e s from which no spores c o u l d be c o l l e c t e d . Progeny from these c r o s s e s ( l a b e l l e d F 2 \" i n F i g . 4) M  were e i t h e r i n c o m p a t i b l e d u p l i c a t i o n , compatible o r s i n g l e mating type s t r a i n s .  The compatible  duplication  and s i n g l e  mating type progeny were s u b c u l t u r e d onto minimal medium s l a n t s so t h a t s u f f i c i e n t m y c e l i a c o u l d be grown f o r use i n subsequent t e s t s . two r e a s o n s \u2014 t h e y  Incompatible  progeny were d i s c a r d e d f o r  were d u p l i c a t i o n s and they d i d not c o n t a i n  suppressors. Compatible and s i n g l e mating type F2 s t r a i n s were t e s t e d f o r un-3 because t h e marker was needed i n t h e next cross.  Furthermore, n o n - d u p l i c a t i o n progeny were needed f o r  the next c r o s s , and so compatible eliminated.  d u p l i c a t i o n s had t o be  They were d i s c a r d e d w i t h the r e s t o f t h e non-  temperature-sensitive s t r a i n s .  Only 18 F2 progeny out of  329 t e s t e d were t e m p e r a t u r e - s e n s i t i v e .  P o s s i b l e reasons f o r  the d e a r t h o f t e m p e r a t u r e - s e n s i t i v e F2 s t r a i n s are presented i n D i s c u s s i o n 1. The  18 t e m p e r a t u r e - s e n s i t i v e F2 s t r a i n s were c r o s s e d t o  the t e s t e r s t r a i n , T(I-&gt;II)  39311, s e r , t r p , tol, t o c o n f i r m  the presence  i n t h e former.  o f suppressors  The  temperature-  s e n s i t i v e F2 progeny may not have c o n t a i n e d suppressors f o r a number of reasons. Firstly,  i n the second c r o s s , L.G.II(T  removed, so i f t h e mutation  I-&gt;II) was  had o c c u r r e d on t h i s  chromosome,  and had not recombined onto t h e homolog, i t would be l o s t .  \u000C38  Table 2 Phenotypes of F2 Strains STRAIN  NUMBER OF F2 STRAINS WITH THE P H E N O T Y P E NORMAL INCOMPATIBLE BLANK  (INCLUDES COMPATIBLE) A1-29 a1-29 A1-54 a1-54 A1-58 a1-58 A1-59 a1-59 A1-65 a1-65 A1-73 a1-73 A1-75 a1-75 A1-103 a1-103 A1-104 a1-104 A1-107 a1-107* A1-113 a1-113* A1-128 a1-128* A2-18 a2-18 A2-20 a2-20 A2-32 a2-32 A2-37 a2-37  4 3 0 19 1 13 2 20 0 15 12 2 2 5 0 17 0 17 0 11 1 14 0 19 0 21 1 9 3 14 3 3  2 0 0 8 0 2 0 2 0 4 1 4 0 1 0 14 0 0 0 0 2 0 0 8 0 6 0 2 0 4 2 3  25 10 4 39 8 25 4 18 1 21 12 19 5 20 0 0 0 23 2 21 9 36 0 40 3 14 13 8 10 15 23 19  ..continued  \u000C39  Table 2 continued  NUMBER OF F2 STRAINS WITH THE P H E N O T Y P E STRAIN  NORMAL  INCOMPATIBLE  BLANK  (INCLUDES COMPATIBLE)  A2-57 a2-57 A2-79 a2-79 A2-86* a2-86 A2-95 a2-95 A2-110 a2-110 A2-117 a2-117 A2-146 a2-146 A2-155* a2-155 A2-162 a2-162  KEY A or 1 or final  0 1 0 0 11 8 0 0 0 0 0 35 2 5 7 19 0 10  0 1 0 0 1 0 0 0 0 0 0 2 2 1 2 4 0 23  FOR STRAIN N O M E N C L A T U R E a \u2022 c r o s s e d to fI,A or fl.a in the s e c o n d c r o s s 2 \u2022 c r o s s e d to R601 or R 6 0 2 in the first c r o s s # \u00BB isolate # from first c r o s s  \u2022 s t r a i n s that had t e m p e r a t u r e - s e n s i t i v e  progeny  7 8 0 0 0 0 0 0 0 0 0 23 16 5 41 27 5 17  \u000C40  In t h i s case, a l l o f the d u p l i c a t i o n progeny from t h e c r o s s e s o f t e m p e r a t u r e - s e n s i t i v e F2 s t r a i n s t o T(I-&gt;II) 39311, ser, trp, tol would be i n c o m p a t i b l e . Secondly,  because o f s e g r e g a t i o n , t h e temperature-  s e n s i t i v e F2 progeny may have c o n t a i n e d t h e chromosome homologous t o t h e one w i t h t h e suppressor mutation. p i c k e d as many ascospores A/a escaped  I  as p o s s i b l e from t h e c r o s s e s of  FI t o fl females, but, as p r e v i o u s l y mentioned,  some o f t h e c r o s s e s produced very few. F i n a l l y , i f t h e o r i g i n a l escape event had been due t o m i t o t i c s e g r e g a t i o n , t h e r e would be no suppressor t o be found.  Newmeyer and T a y l o r (1967) d i d r e p o r t t h a t t h e i r A/a  escaped  s t r a i n s were heterokaryons  o f pure A and pure a  n u c l e i , s u g g e s t i n g t h a t somatic s e g r e g a t i o n had o c c u r r e d . In t h e system they used, t h e r e were no s e l e c t i v e markers c l o s e t o mt t h a t would prevent t h e s u r v i v a l o f m i t o t i c c r o s s o v e r o r mt d e l e t i o n p r o d u c t s .  F i g u r e 8 shows examples  of m i t o t i c c r o s s o v e r s and t h e i r products, some o f which survive the s e l e c t i o n conditions. I f a t e m p e r a t u r e - s e n s i t i v e F2 s t r a i n c o n t a i n e d a suppressor  a t a l o c u s o t h e r than tol or mt, then the  d u p l i c a t i o n progeny (F3) from t h e c r o s s t o T(I-&gt;II) ser,  39311,  trp, tol would be o f two t y p e s \u2014 c o m p a t i b l e o r  incompatible.  I f the new mutation  had o c c u r r e d i n t h e tol  gene o r i n t h e r e g i o n o f t h e mating type gene c o n t r o l l i n g v e g e t a t i v e i n c o m p a t i b i l i t y , then a l l o f t h e d u p l i c a t i o n progeny would be compatible  (Fig. 9).  \u000CPOSSIBLE C R O S S O V E R S ser+ un A  f f  X ser  un+ a  ser+ un A  MITOTIC PRODUCTS ser+ un A ser un+ A ser+ un a ser un+ a ser+ un A ser un A  X ser  un+ a  ser+ un A  ser  Figure 8  un+ a  ser+ un+ a ser un+ a ser+ un A ser+ un A ser ser  un+ a un+ a  Mitotic double crossovers that could occur in duplication strains. Single crossovers result in dicentric and acentric products. Thin lines denote L.G. I. Thick lines denote L.G. II. (N.B. The insertion is inverted with respect to the centromere.)  \u000C42  + un-3 A  I It IV  it  ser + a trp tol  1/2 \"suppressors\"  I  | V  IV \"\\trp  tol  + un-3 A  | V  IV ser + a  Progeny surviving on minimal medium at 3 2 d e g r e e s  IV IV \"\\trp  4f  tol  | V  IV ser IV  Figure 9  The third set of crosses was done to confirm the suppressor phenotype of the putative suppressor strains. The crosses were: R601-derived suppressors x T(l-&gt;ll)39311,ser,trp,tol,a and R602-derived suppressors x T(l-&gt;ll)39311,ser,trp,tol,A. If the new mutation had occurred at mt or tol, all of the surviving progeny would have been compatible duplications. If the new mutation had occurred at a new \"tol' locus, some of the surviving progeny would have been compatible duplications and some would have been incompatible duplications.  \u000C43  Ascospores  from the c r o s s e s of t e m p e r a t u r e - s e n s i t i v e F2  s t r a i n s t o T(I-&gt;II)  39311, ser,  trp,  d e s c r i b e d i n M a t e r i a l s and Methods.  tol were s e l e c t e d as T a b l e 3 shows the  phenotypes of the F3 progeny from each of the 18 c r o s s e s . Seven s t r a i n s segregated compatible progeny.  and  incompatible  These were the s u p p r e s s o r - c o n t a i n i n g F2  strains.  Ten s t r a i n s segregated o n l y i n c o m p a t i b l e progeny. s t r a i n s may  These  have c o n t a i n e d the chromosome homologous t o the  one w i t h the suppressor.  I t i s u n l i k e l y , however, t h a t a l l  6 of the F2 progeny d e r i v e d from al-128 would have r e c e i v e d the chromosome homologous t o the one w i t h the There are two  additional  (and more l i k e l y ) e x p l a n a t i o n s f o r  the l a c k of s u p p r e s s i o n i n these s i x s t r a i n s . A/a  d u p l i c a t i o n may  suppressor.  have escaped  The  original  by m i t o t i c s e g r e g a t i o n o r  by m u t a t i o n / d e l e t i o n a t a suppressor  l o c u s on L.G.  II.  Since t h e r e were no t e m p e r a t u r e - s e n s i t i v e F2 s t r a i n s t h a t segregated  a l l compatible  progeny, none of the o r i g i n a l  escape events c o u l d have been due t o mutation mating type genes o r a t One  a t e i t h e r the  tol.  o f the s t r a i n s , al-128-27, although c r o s s e d on  s e v e r a l o c c a s i o n s t o T(I-&gt;II)  39311, ser,  trp,  tol,  produced  no p e r i t h e c i a or ascospores.  The s t e r i l i t y of al-128-27  c o u l d have been unique t o t h i s c r o s s because the s t r a i n  was  a b l e t o induce the p r o d u c t i o n of p e r i t h e c i a when c r o s s e d t o a female f l , a t e s t e r . A sample of the compatible  and i n c o m p a t i b l e F3  strains  \u000CTable 3 Phenotypes of F3 Strains F2 STRAIN  NUMBER OF F3 STRAINS WITH THE P H E N O T Y P E COMPATIBLE  INCOMPATIBLE  a1-113-7 a1-113-8 a1-113-9 a1-113-10  0 24 0 0  31 22 6 46  A2-155-2 A2-155-3 A2-155-5  7 14 18  18 51 28  A2-86-1 A2-86-4 A2-86-12  0 0 9  30 36 21  a1-107-9 a1-107-11  19 28  18 12  a1-128-22 a1-128-23 a1-128-24 a1-128-25 a1-128-26 a1-128-27  0 0 0 0 0 0  32 32 25 35 32 0  K E Y FOR STRAIN N O M E N C L A T U R E A or a = c r o s s e d to fI,A or fl,a in the s e c o n d c r o s s 1 or 2 - c r o s s e d to R601 or R 6 0 2 in the first c r o s s middle # \u2022 isolate # from first c r o s s last # - isolate # from s e c o n d c r o s s  \u000C45  was t e s t e d f o r mating type (Table 4 ) . compatible F3 s t r a i n s types.  Almost a l l o f the  (65 o f 66) c o n t a i n e d both mating  T h i s i s the c l a s s of progeny t h a t show the e x i s t e n c e  of s u p p r e s s o r s .  I t i s u n l i k e l y t h a t they grew w e l l due t o  escape because o f t h e h i g h p r o p o r t i o n o f A/a  strains.  Furthermore, the hyphae o f escaped s t r a i n s a r e wispy, whereas these 65 compatible s t r a i n s grew w i t h a dense morphology. One o f the a p p a r e n t l y compatible F3 s t r a i n s (one o f the progeny s t r a i n s from the R601-derived p a r e n t , al-113-8) c o n t a i n e d o n l y A.  Although escape u s u a l l y does not occur  w i t h i n the f i r s t 24 hours a f t e r g e r m i n a t i o n of the spore, i t may  have escaped e a r l i e r than normal from A/a  i n c o m p a t i b i l i t y by l o s s of t h e mating type gene from the t r a n s l o c a t e d segment.  I t i s u n l i k e l y t h a t the s t r a i n  r e s u l t e d from a double c r o s s o v e r on e i t h e r s i d e o f un-3 because of t h e c l o s e l i n k a g e o f un-3 t o ser-3  and mt.  It is  a l s o u n l i k e l y t h a t the s t r a i n s u r v i v e d due t o r e v e r s i o n of the un-3 mutant because the un-3 marker c o n t a i n s  two  mutations. Most o f the i n c o m p a t i b l e F3 s t r a i n s (59 of 71) o n l y c o n t a i n e d one mating t y p e , p r o b a b l y because the mating type t e s t s were done a f t e r the i n c o m p a t i b l e s t r a i n s had escaped. Those i n c o m p a t i b l e F3 s t r a i n s which c o n t a i n e d both mating types may  have escaped by mutation a t a suppressor l o c u s o r  by m i t o t i c c r o s s i n g over between t h e L.G. I centromere and mt and between mt and un-3, and both t y p e s o f d e r i v a t i v e  \u000C46  Table 4 Mating Types of F3 Strains TEMPERATURESENSITIVE F2  COMPATIBLE F3  INCOMPATIBLE F3  a1-113-8  9 A/a 1A  1 A/a 1A 8 a  a1-107-9  10 A / a  1 A/a 9 a  a1-107-11  10 A / a  1A 9 a  A2-86-12  9 A/a  2 A/a 6 A 3 a  A2-155-2  7 A/a  3 A/a 5 A 2 a  A2-155-3  10 A / a  8 A 2 a  A2-155-5  10 A / a  5 A/a 4 A 1 a  The presence of suppressors was implied by the production of A / a compatible F3 progeny. KEY FOR STRAIN NOMENCLATURE A or a - crossed to fI,A or fl.a in the second cross 1 or 2 \u2022 crossed to R601 or R602 in the first cross middle # \u2022 isolate # from first cross last # \u2022 isolate # from second cross  \u000C47  n u c l e i were s t i l l p r e s e n t . Most of the escaped i n c o m p a t i b l e F3 s t r a i n s d e r i v e d from R601  (un-3, A) were a, and most of the escaped  incompatible F3 s t r a i n s d e r i v e d from R602 (un-3, a) were A. These two types of s t r a i n s c o u l d have o c c u r r e d by d e l e t i o n of mt and un-3 homolog.  from the t e m p e r a t u r e - s e n s i t i v e L.G. I  Another mechanism c o u l d have been double m i t o t i c  c r o s s i n g over, between the L.G. between mt and un-3,  I centromere and mt and  f o l l o w e d by overgrowth by the a n u c l e i  i n the R601-derived a escaped s t r a i n s o r by the A n u c l e i i n the R602-derived A escaped s t r a i n s .  The l a t t e r mechanism  c o u l d have g i v e n r i s e t o the s m a l l e r c l a s s e s of escaped s t r a i n s , R601-derived A escaped s t r a i n s and R602-derived a escaped s t r a i n s .  These c l a s s e s c o u l d have a r i s e n by m i t o t i c  c r o s s i n g over between the L.G. I centromere and mt and between mt and un-3,  f o l l o w e d by overgrowth by the A n u c l e i  i n the R601-derived A escaped s t r a i n s or by the a n u c l e i i n the R602-derived a escaped  strains.  Although the F3 s t r a i n s were s e l e c t e d on medium without tryptophan, the trp-4 may  gene i s l e a k y ; t h e r e f o r e ,  not have been completely r e s t r i c t i v e .  selection  To ensure t h a t  the c o m p a t i b i l i t y was due t o a n o v e l suppressor and not t o tol,  the A/a compatible F3 s t r a i n s from each of the seven  c r o s s e s were t e s t e d f o r t r p - 4 . s t r a i n s was  None of the compatible F3  tryptophan-requiring.  A l l of the A/a i n c o m p a t i b l e F3 were t e s t e d f o r trp-4 ensure t h a t they were c o r r e c t l y s c o r e d as i n c o m p a t i b l e  to  \u000C48  because  they had no suppressor, and not because  p o o r l y due t o the trp-4  gene.  they grew  None of the i n c o m p a t i b l e F3  s t r a i n s was t r y p t o p h a n - r e q u i r i n g . F i n a l l y , t o ensure t h a t the c o m p a t i b i l i t y was  due t o a  suppressor and not t o a n o v e l gene which i n c r e a s e d the r a t e of escape, 4 hyphal t i p s were o b t a i n e d from 2 A/a  compatible  F3 s t r a i n s from each of the seven c r o s s e s and were t e s t e d f o r mating type.  I f the \" c o m p a t i b i l i t y \" were due t o e a r l y  escape, then the t i p s would be expected t o be A or a, but not both; i f the c o m p a t i b i l i t y were due t o a suppressor, then the t i p s would be expected t o be A and a. are shown i n Table 5.  The  Almost a l l of the t i p s were  s u g g e s t i n g t h a t the compatible phenotype was  results A/a,  due t o a  suppressor. S i n g l e mating type hyphal t i p s  ( a l l A) were found o n l y  i n two of the suppressor s t r a i n s , both d e r i v e d from the same A/a escaped FI s t r a i n .  When t h i s A/a  escaped FI s t r a i n  escaped, a second mutation, i n a d d i t i o n t o the suppressor may  have o c c u r r e d .  The second mutation c o u l d be one  causes i n s t a b i l i t y of d u p l i c a t i o n s .  that  The s i n g l e mating  type  m i t o t i c segregants c o u l d have a r i s e n by d e l e t i o n of a d u r i n g somatic growth o r m i t o t i c c r o s s o v e r between the L.G. centromere  and mt and between mt and ser-3  I  ( o n l y A products  would have s u r v i v e d ) . T a b l e 6 summarizes the f i n d i n g s d e s c r i b e d i n R e s u l t s 1.  \u000C49  Table 5 Mating Types of Hyphal Tips of A/a Compatible F3 Strains  COMPATIBLE F3 TIP DERIVED FROM STRAIN:  MATING T Y P E S OF TIPS  a1-113-8  8 A/a  a1-107-9  8 A/a  a1-107-11  8 A/a  A2-86-12  8 A/a  A2-155-2  8 A/a  A2-155-3  4 A/a, 4 A  A2-155-5  6 A/a, 2 A  Most of the hyphal tips were A/a, suggesting that the suppressors do not function by increasing the rate of escape.  \u000CTable 6 Summary of Results 1 A/a ESCAPED F1 # SPORES FROM # TEMPERATURE- TEMPERATURE- # SPORES FROM # SPORES SECOND CROSS SENSITIVE SENSITIVE F2 THIRD CROSS COMPATIBLE 1-29 1-54 1-58 1-59 1-65 1-73 1-75 1-104 1-107 1-113 1-128 2-18 2-20 2-32 2-37 2-57 2-79 2-86 2-95 2-103 2-110 2-117 2-146 2-155 2-162  9 27 16 24 19 19 8 17 11 17 27 27 12 21 11 2 0 20 0 31 0 37 10 32 33  a1-107-9 / a1-107-11  37 40  19 28  a1-113-7 . a1-113-8 / a1-113-9 / a1-113-10  31 46 6 46  0 24 0 0  a1-128-22 a1-128-23 . - \u2014 ^ a1-128-24 a1-128-25 a1-128-26 a1-128-27  32 32 25 35 32 0  0 0 0 0 0 0  / / 2 A  6  / y/  '/  '  3  A2-86-1 ' A2-86-4 A2-86-12  30 36 30  0 0 9  3  A2-155-2 , A2-155-3 A2-155-5  25 65 46  7 14 18  KEY FOR A/a ESCAPED F1 1 or 2 - crossed to R601 or R602 in the first cross second # \u2022 isolate # from first cross  KEY FOR TEMPERATURE-SENSITIVE F2 A or a - crossed to fl,A or fl,a in the second cross 1 or 2 \u2022 crossed to R601 or R602 in the first cross middle # \u2022 isolate # from first cross last # \u2022 isolate # from second cross  \u000C51  DISCUSSION 1  Seven s t r a i n s c o n t a i n i n g n o v e l suppressors of A/a i n c o m p a t i b i l i t y have been i s o l a t e d .  The s t r a i n s were  d e r i v e d from 4 s t r a i n s t h a t had undergone escape ( s t r a i n s #1-107, 1-113, 2-86 t h e r e may  events  and 2-155), s u g g e s t i n g t h a t  be 4 n o v e l suppressor mutations.  None of the  s u p p r e s s o r s i s a l l e l i c with tol o r w i t h the mating gene.  The seven s t r a i n s produced A/a compatible  type  progeny  when c r o s s e d w i t h a d u p l i c a t i o n - g e n e r a t i n g t r a n s l o c a t i o n strain.  The A/a  compatible progeny were p r o b a b l y  d u p l i c a t i o n s , not heterokaryons, because hyphal  tips  i s o l a t e d from these s t r a i n s , i n g e n e r a l , c o n t a i n e d both mating t y p e s . A/a progeny  The compatible phenotype observed i n these  i s due t o new  mutations t h a t suppress  i n c o m p a t i b i l i t y , not t o new of escape from A/a  A/a  mutations t h a t i n c r e a s e the r a t e  i n c o m p a t i b i l i t y by i n c r e a s i n g the r a t e of  d e l e t i o n o r m i t o t i c c r o s s i n g over. There were s u r p r i s i n g l y few t e m p e r a t u r e - s e n s i t i v e F2 progeny  from the 50 c r o s s e s of the second c r o s s (18 of  tested).  Two  types o f c r o s s e s produced no  s e n s i t i v e F2 progeny: f l , A x A/a from R601  (un-3, A) and f l ,  temperature-  escaped FI s t r a i n s d e r i v e d  a x A/a  d e r i v e d from R602 (un-3, a ) .  329  escaped F l s t r a i n s  In these 2 types of c r o s s e s ,  L.G.  I from the fl parent had the same mating type gene as  L.G.  I from the t e m p e r a t u r e - s e n s i t i v e parent; so perhaps  L.G.  I homologs p a i r e d w i t h the t r a n s l o c a t e d segment which  the  \u000C52  has t h e o p p o s i t e mating type  ( F i g . 10).  When such p a i r i n g s  occur, fewer temperature s e n s i t i v e progeny a r e produced. The L.G. I homologs probably d i d not p a i r w i t h each o t h e r . I f they d i d , t e m p e r a t u r e - s e n s i t i v e progeny would have been produced. The of  a l t e r n a t i v e p a i r i n g hypothesis e x p l a i n s t h e absence  t e m p e r a t u r e - s e n s i t i v e progeny from t h e two  types o f c r o s s e s .  When L.G. I from t h e fl  aforementioned  parent p a i r s with  the t r a n s l o c a t e d DNA, no temperature s e n s i t i v e progeny a r e produced.  I f there i s a s i n g l e crossover i n the paired  r e g i o n , a c e n t r i c and d i c e n t r i c chromosomes a r e produced, and these, presumably, do n o t s u r v i v e .  I f t h e r e i s a double  c r o s s o v e r i n t h e p a i r e d r e g i o n , t h e un-3 a l l e l e segregates  with an un-3  +  allele.  always  When L.G. I from t h e  t e m p e r a t u r e - s e n s i t i v e parent p a i r s with t h e t r a n s l o c a t e d DNA, very few temperature s e n s i t i v e progeny a r e produced. If there i s a s i n g l e crossover i n the paired region, a c e n t r i c and d i c e n t r i c chromosomes a r e produced.  I f there  i s a double c r o s s o v e r i n t h e p a i r e d r e g i o n , o n l y one o f t h e 6 p o s s i b l e types o f double c r o s s o v e r \u2014 o n e  c r o s s o v e r on each  s i d e o f t h e mating type g e n e \u2014 g i v e s temperature s e n s i t i v e progeny.  T h i s type o f double c r o s s o v e r i s r a r e because o f  the p r o x i m i t y o f un-3 t o t h e mating type gene. I f one d i s r e g a r d s t h e two types o f c r o s s e s t h a t y i e l d e d no t e m p e r a t u r e - s e n s i t i v e F2 progeny, t h e p r o p o r 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 F2 s t r a i n s i s s t i l l  low (18 o f 165).  In t h e two types o f c r o s s e s t h a t d i d g i v e temperature-  \u000C53  PAIRING  SEGREGATION  Ha, la ser un + a ser* un+ A ser+ un  la  lib, lb  lb  lla, lb ser un+ a ser* un A ser* un+ A  r  _  n l y U r e  lU  lb  lib, la  la  One explanation for the low number of temperature-sensitive F2 strains is the alternative pairing hypothesis. Either of the .G.I homologs the translocation. tne L L.u.i nomoiogs could couia have nave paired pairea with witn im Thin vertical lines indicate regions of pairing  \u000C54  s e n s i t i v e F2 progeny  (R601-derived escaped s t r a i n s x f l ,  and R602-derived escaped s t r a i n s x f l ,  A ) , perhaps  a  pairing  of t h e L.G. I homologs w i t h each o t h e r , as d e p i c t e d i n F i g . 7, d i d not always o c c u r .  I f e i t h e r o f t h e L.G. I homologs  had p a i r e d w i t h t h e t r a n s l o c a t e d DNA, fewer  temperature-  s e n s i t i v e progeny would have been produced. Two a d d i t i o n a l e x p l a n a t i o n s f o r t h e low number o f t e m p e r a t u r e - s e n s i t i v e progeny a r e p o s s i b l e .  Duplication  s t r a i n s undergo a p r o c e s s c a l l e d RIP (\"Repeat-Induced  Point  mutation\", p r e v i o u s l y \"Rearrangements Induced P r e m e i o t i c a l l y \" ) d u r i n g mating, w i t h the r e s u l t t h a t d u p l i c a t e d genes are o f t e n mutated 1985;  Selker, et a l . ,  Cambareri, e t a l . , 1990;  ( S e l k e r and Stevens,  1987; S e l k e r and G a r r e t t , 1988;  1989; Grayburn and S e l k e r , 1989; S e l k e r ,  Cambareri, e t a l . ,  1991).  RIP o f e i t h e r un-3 o r un-3  +  c o u l d have produced un-3 n u l l s , which are l e t h a l unpublished r e s u l t s , Glass, personal  (Lambowitz,  communication).  F i n a l l y , g e r m i n a t i o n o f un-3 spores may occur l e s s f r e q u e n t l y than g e r m i n a t i o n o f w i l d type spores (Glass and L. Stenberg, p e r s o n a l communication),  p o s s i b l y because o f  the heat shock r e q u i r e d t o i n i t i a t e g e r m i n a t i o n . A/a d u p l i c a t i o n s t r a i n s escaped from i n c o m p a t i b i l i t y by s e v e r a l d i f f e r e n t means o t h e r than t h e g e n e r a t i o n o f suppressors.  The 8 phenotypic c l a s s e s o f escaped F l  s t r a i n s , shown i n F i g . 6, c o u l d have been t h e r e s u l t s o f d e l e t i o n / m u t a t i o n a t t h e mating type l o c u s , RIP, m i t o t i c  \u000C55  c r o s s i n g over or low f e r t i l i t y duplicated genetic  due t o the presence of  material.  Any o f the 8 phenotypic c l a s s e s of escaped F l s t r a i n s c o u l d have a r i s e n by d e l e t i o n / m u t a t i o n a t one or both o f the mating type l o c i . Griffiths  Experiments performed by DeLange and  (1975) on escape from A/a i n c o m p a t i b i l i t y  suggested t h a t mixed mating type heterokaryons o f t e n by d e l e t i o n of one mating t y p e .  escaped  S i m i l a r events c o u l d have  o c c u r r e d i n mixed mating type d u p l i c a t i o n s t r a i n s . The 8 c l a s s e s of escaped s t r a i n s c o u l d have a r i s e n by RIP.  A/a d u p l i c a t i o n s are not r e a l l y d u p l i c a t i o n s because  and a are idiomorphs, not a l l e l e s  (Metzenberg  and  A  Glass,  1990), i . e . the c e n t r a l p o r t i o n s of the DNAs are completely d i f f e r e n t from each o t h e r ( G l a s s , e t a l . , 1988). flanking regions, al.,  1988)  The  however are v i r t u a l l y i d e n t i c a l ( G l a s s , e t  and RIP can sometimes occur i n unique  c l o s e t o the d u p l i c a t e d ones (Foss, e t a l . ,  sequences  1991).  The s i n g l e mating type escaped s t r a i n s , c l a s s e s 7 and 8, c o u l d have been generated by m i t o t i c c r o s s i n g over.  Once  the mating type genes had segregated i n t o d i f f e r e n t n u c l e i , one type of nucleus c o u l d have outgrown the o t h e r and the r e s u l t a n t c u l t u r e would only have had a s i n g l e mating type reaction. The b a r r e n phenotype a s s o c i a t e d w i t h d u p l i c a t i o n s i n N. crassa  c o u l d account f o r c l a s s e s 2-6.  Duplication strains  sometimes produce abundant p e r i t h e c i a , but v e r y few  \u000C56  ascospores (Newmeyer and the  spores simply may  Taylor,  1967).  I f t h i s occurred,  have been o v e r l o o k e d d u r i n g  scoring.  What i s t o l ?  I t i s not  possible  t o formulate a comprehensive  statement r e g a r d i n g the incompatibility  biological significance  i n a l l of the  i n many d i f f e r e n t forms.  of  fungi, f o r i t manifests  Incompatibility  can  itself  be  h e t e r o g e n i c , p r e v e n t i n g f u s i o n between s t r a i n s of d i f f e r e n t genotypes, e i t h e r v e g e t a t i v e f u s i o n sexual f u s i o n  (e.g.  i n Sordaria  Ceratostomella  radicicola  v e g e t a t i v e and  sexual fusion  ( J i n k s , e t a l . , 1961) or i t can  (e.g.  fimicola  i n W.  crassa),  ( O l i v e , 1956)  and  ( E l - A n i , e t a l . , 1957)) o r both  and  (e.g.  i n Aspergillus  Podospora  anserina  nidulans  (Esser,  1971));  be homogenic, p r e v e n t i n g f u s i o n between s t r a i n s  s i m i l a r genotypes, e i t h e r s e x u a l f u s i o n or both v e g e t a t i v e and commune (Raper, 1966  sexual fusion  d i v e r s i t y , must be c o n s i d e r e d Vegetative incompatibility  suppresses A/a  in  crassa  may  crassa)  Schizophyllum The  system, because of  c o n t r o l l e d by the  mating  be b i o l o g i c a l l y u n r e l a t e d genes.  e x i s t e n c e of the gene, tol,  to  This idea  (Newmeyer, 1970;  is  which  i n c o m p a t i b i l i t y without a f f e c t i n g het-C/c  het-JE?/e i n c o m p a t i b i l i t y  the  separately.  i n c o m p a t i b i l i t y c o n t r o l l e d by the het supported by the  (e.g.  i n W.  c i t e d i n B u r n e t t , 1975)).  s i g n i f i c a n c e o f each i n c o m p a t i b i l i t y  type genes of N.  (e.g.  of  P e r k i n s , 1974).  or  \u000C57  V e g e t a t i v e i n c o m p a t i b i l i t y seems t o be an f u n c t i o n of the mating type genes.  G e n e t i c and molecular  data support t h i s i d e a and suggest t h a t mechanisms e v o l v e d s e c o n d a r i l y . (1973) i n t r o g r e s s e d  compatibility  Metzenberg and Ahlgren  the mating type genes of N.  i n t o a l a r g e l y N. crassa demonstrated  intrinsic  background.  incompatibility  tetrasperma  The r e s u l t a n t  i n mixed mating  type  heterokaryons and i n heterozygous d u p l i c a t i o n s , t h a t the W.  tetrasperma  suggesting  genes had the a b i l i t y t o i n s t i g a t e  the i n c o m p a t i b i l i t y r e a c t i o n . N. tetrasperma,  strains  In t h e i r own  environment,  the mating type genes must e i t h e r  suppressed f o r the i n c o m p a t i b i l i t y f u n c t i o n  in  be  or l a c k  target  genes or both. A r e c e n t study by D. Jacobson suggests t h a t one way  (personal  communication)  i n which N. tetrasperma  tolerates  A/a  ascospores i s by the presence of the suppressor, t o l . He i n t r o g r e s s e d t o l of W.  sequences of N. tetrasperma  crassa  i n t o a N. crassa  s t r a i n of N. crassa  background.  behaved as i f i t were tol,  The mating type genes of N. sitophila, tetrasperma,  are a b l e t o produce  phenotype i n a W. sitophila crassa,  crassa  the  i n A/a  genes  duplications.  resultant  not  tol . +  W.  incompatibility  background ( P e r k i n s ,  whereupon the N. sitophila  The  like  mating type genes were i n t r o g r e s s e d  incompatibility was  corresponding to  1977). into  W.  exhibited  Even more i n t e r e s t i n g  the o b s e r v a t i o n t h a t the suppressor gene, t o l ,  eliminated  the i n c o m p a t i b i l i t y ,  N.  indicating that t o l i s  \u000C58  unable t o d e t e c t any s i g n i f i c a n t d i f f e r e n c e between W. crassa  and N. sitophila  mating type genes.  A r e s u l t w i t h s i m i l a r i m p l i c a t i o n s was o b t a i n e d by Glass crassa  ( p e r s o n a l communication).  Replacement o f t h e N.  A gene with the N. africana  a b i l i t y o f t h e transformant  A gene d i d n o t a l t e r the  t o i n i t i a t e the i n c o m p a t i b i l i t y  r e a c t i o n , again i m p l y i n g t h a t t h e mating type gene from a h o m o t h a l l i c s p e c i e s i s capable  of orchestrating vegetative  incompatibi1ity. I n c o m p a t i b i l i t y c o n t r o l l e d by the v e g e t a t i v e i n c o m p a t i b i l i t y genes may have developed independently of A/a i n c o m p a t i b i l i t y . There a r e many t h e o r i e s r e g a r d i n g t h e b i o l o g i c a l b e n e f i t s o f heterokaryon  incompatibility.  Caten  (1972) suggested t h a t i n c o m p a t i b i l i t y systems e x i s t t o l i m i t the spread o f i n f e c t i o u s v i r u s e s o r c y t o p l a s m i c determinants.  L a t e r s t u d i e s i n d i c a t e d , however, t h a t  plasmids can c r o s s i n c o m p a t i b i l i t y b a r r i e r s w i t h i n a s p e c i e s ( C o l l i n s and S a v i l l e , 1990) and even between s p e c i e s ( G r i f f i t h s , e t a l . , 1990), presumably d u r i n g b r i e f  periods  of u n s t a b l e f u s i o n . H a r t l , e t a l . (1975) suggested t h a t i n c o m p a t i b i l i t y prevents t h e e x p l o i t a t i o n o f an adapted mycelium by a l e s s w e l l adapted one growing i n t h e same n i c h e . hypothesis  Although t h i s  c o u l d apply t o a fungus l i k e N. crassa,  i n which  a d e f e c t i v e homokaryon can s u r v i v e by f u s i o n w i t h another mycelium, i t does not seem a l i k e l y e x p l a n a t i o n f o r  \u000C59  i n c o m p a t i b i l i t y i n f u n g i l i k e the basidiomycetes  i n which  f u s i o n l e a d s t o mating. I t has been suggested t h a t i n c o m p a t i b i l i t y s e r v e s t o d i s t i n g u i s h i n d i v i d u a l s , which may  be important i n the  maintenance of f i n e - t u n i n g between n u c l e i and o r g a n e l l e s . C o n s i d e r i n g t h a t n u c l e i are a s s o c i a t e d w i t h d i f f e r e n t o r g a n e l l e s every time mating o c c u r s , t h i s does not seem t o be a l i k e l y e x p l a n a t i o n f o r the e x i s t e n c e of incompatibility. J . Begueret  ( p e r s o n a l communication) succeeded  in  c r e a t i n g a novel i n c o m p a t i b i l i t y group v i a mutation i n Podospora,  which l e d him t o propose t h a t i n c o m p a t i b i l i t y  genes are n o t h i n g more than mutations without significance.  biological  J i n k s , e t a l . (1961) made a s i m i l a r  s u g g e s t i o n i n t h e i r study o f i n c o m p a t i b i l i t y i n nidulans.  They maintained t h a t heterokaryon  Aspergillus  incompatibility  i s a consequence of g e n e t i c d i v e r s i t y , not a cause. i d e a would o n l y apply t o f u n g i l i k e N. crassa  This  i n which the  i n c o m p a t i b i l i t y r e a c t i o n does not a f f e c t mating.  I f a novel  a l l e l e arose i n a p o p u l a t i o n , i t would be s e l e c t e d a g a i n s t because the c e l l c o n t a i n i n g i t would d i e i f i t fused w i t h an u n l i k e type. if  A novel a l l e l e would not be s e l e c t e d a g a i n s t  i t arose i n a spore t h a t e s t a b l i s h e d a s e p a r a t e  p o p u l a t i o n b e f o r e encountering incompatible f u n g a l t y p e s . In the basidiomycetes, however, a novel  incompatibility  group would be a b l e t o mate w i t h a l l o t h e r e x i s t i n g a l l o w i n g i t t o spread q u i c k l y through a p o p u l a t i o n .  groups,  \u000C60  The  gene, tol , +  and  the new  suppressors c o u l d be mating  type t a r g e t genes, c o n t r o l l i n g d i f f e r e n t s t e p s i n the pathway t o A/a  i n c o m p a t i b i l i t y , and when they are mutated,  the r e a c t i o n f a i l s t o occur.  T h e i r r e l a t i o n s h i p s t o each  o t h e r i n terms of where they f i t i n t o the pathway are unknown a t p r e s e n t .  They c o u l d be mutants i n  sequential  r e a c t i o n s o r r e a c t i o n s t h a t occur s i m u l t a n e o u s l y . The  suppressors c o u l d be enzymes r e q u i r e d  vegetative  growth, normally turned o f f by the A/a  ( d i r e c t l y or i n d i r e c t l y ) during  recognized  The  by the r e g u l a t i n g product and and  product  incompatibility.  mutants c o u l d be a l t e r e d such t h a t they are no  repressed  for  The  longer  are, t h e r e f o r e ,  the i n c o m p a t i b i l i t y r e a c t i o n does not  suppressors c o u l d be r e q u i r e d  for recognition  v e g e t a t i v e l y growing A or a hyphae.  The  case, c o u l d be d e f e c t i v e f o r v e g e t a t i v e  occur.  of  mutants, i n t h i s r e c o g n i t i o n , thus  e l i m i n a t i n g the i n c o m p a t i b i l i t y r e a c t i o n .  The  suppressors  c o u l d produce t o x i c m e t a b o l i t e s i n the presence of the product.  A/a  These mutants c o u l d be d e f e c t i v e f o r t o x i n  production  i t s e l f or f o r r e g u l a t i o n so t h a t  i n c o m p a t i b i l i t y r e a c t i o n does not The  not  existence  the  occur.  of o t h e r genes a f f e c t i n g the  i n c o m p a t i b i l i t y r e a c t i o n r a i s e s a question  regarding  N.  tetrasperma.  s t r a i n of  N.  tetrasperma  As p r e v i o u s l y mentioned, one  appears t o c o n t a i n the r e c e s s i v e a l l e l e of  (Jacobson, p e r s o n a l  communication).  the o t h e r suppressor genes?  As  tol  What i s the s t a t e of  long as a s t r a i n has  tol, i t  \u000C61  w i l l be heterokaryon compatible, so t h e a l l e l e s of t h e other suppressors would not a f f e c t t h e phenotype. known about t h e r e l a t i v e f u n c t i o n s it W.  U n t i l more i s  o f t h e new suppressors,  i s not p o s s i b l e t o p r e d i c t t h e suppressor genotype(s) o f tetrasperma. Perhaps t h e r e a r e suppressors t h a t , u n l i k e t o l ,  suppress both A/a i n c o m p a t i b i l i t y and mating.  I f so, i t  would mean t h a t mating and A/a i n c o m p a t i b i l i t y a c t through a t l e a s t one common s t e p .  Perhaps t h e r e a r e A/a  i n c o m p a t i b i l i t y suppressors t h a t a l s o suppress i n c o m p a t i b i l i t y c o n t r o l l e d by one o r some o f t h e n e t genes. I f so, i t would imply t h a t i n c o m p a t i b i l i t y i s e f f e c t e d through one pathway and t h a t t o l a c t s b e f o r e o r a f t e r the common p a r t ( s )  o f the pathway.  The number o f d i f f e r e n t  suppressor genes may g i v e an i n d i c a t i o n o f t h e number o f steps i n v o l v e d  i n generating the i n c o m p a t i b i l i t y  reaction.  \u000C62  INTRODUCTION 2  A m o l e c u l a r p i c t u r e o f t h e mating type genes i s emerging w i t h t h e a i d o f mating type mutants DeLange, 1978; G r i f f i t h s , 1982; G r i f f i t h s ,  ( G r i f f i t h s and  personal  communication) and molecular b i o l o g i c a l techniques et  (Glass,  a l . , 1988; Staben and Yanofsky, 1990; G l a s s , e t a l . ,  1990). The A idiomorph, as d e f i n e d by i t s r e g i o n o f nonhomology w i t h a, i s 5301 base p a i r s i n l e n g t h ( G l a s s , e t al.,  1990), and a, by t h e same d e f i n i t i o n , i s 3235 base  p a i r s i n l e n g t h (Staben and Yanofsky, 1990) ( F i g . 11). of  the A  m  mutants  All  ( G r i f f i t h s , 1982) t h a t have been sequenced  map w i t h i n t h e s i n g l e ORF, c a l l e d A-l.  A l l of the A  m  and a  mutants o f G r i f f i t h s and DeLange (1978) t h a t have been sequenced map w i t h i n t h e exons i n t h e ORFs ( G l a s s , e t a l . , 1990; Staben and Yanofsky, 1990). One o f t h e mutants, a , nl  Some examples  has a f r a m e s h i f t due t o t h e  d e l e t i o n o f a s i n g l e base p a i r .  The i n s e r t i o n o f 212 base  p a i r s , which c h a r a c t e r i z e s t h e mutant a premature t r a n s c r i p t i o n s t o p . f e r t i l e mutant, a  m  3  3  ,  follow.  m  3  0  causes a  The unique compatible,  has a base p a i r s u b s t i t u t i o n which i s  l o c a t e d f a r t h e r downstream  than e i t h e r o f t h e o t h e r two  mutations. Species i n o t h e r genera have idiomorphs i n s t e a d o f a l l e l e s a t t h e i r mating type l o c i , e.g.  Saccharomyces  m  \u000CLEGEND Thick lines \u2022 identical flanking sequences Filled boxes - idiomorphs Open boxes \u2022 postulated ORFs Arrows - presumed transcripts with introns Mating type regions of N. crassa (from Staben and Yanofsky, 1990).  \u000C64  cerevisiae  ( S t r a t h e r n , et al., 1980; Nasmyth and T a t c h e l l ,  1980), Schizosaccharomyces Ustilago  pombe ( K e l l y , e t al., 1988),  maydis a gene (M. B o l k e r and R. Kahmann, 1991, i n  p u b l i s h e d a b s t r a c t s from S i x t e e n t h Fungal Conference)  and Podospora  anserina  Genetics  (Coppin, 1991, i n  p u b l i s h e d a b s t r a c t s from S i x t e e n t h Fungal  Genetics  Conference). The mating type genes o f N. crassa  a r e i n c o m p a t i b l e not  o n l y i n a heterokaryon, but a l s o i n one n u c l e u s .  Newmeyer  (1970) used n u c l e a r i n c o m p a t i b i l i t y t o induce tol, which suppresses both types o f i n c o m p a t i b i l i t y .  G l a s s , e t al.  (1990) observed a 1 0 0 - f o l d r e d u c t i o n i n t r a n s f o r m a t i o n e f f i c i e n c y o f t h e ORF, A-l, i n t o a s p h e r o p l a s t s , compared t o the t r a n s f o r m a t i o n e f f i c i e n c y i n t o A s p h e r o p l a s t s ; and G l a s s ( p e r s o n a l communication) observed a s i m i l a r r e d u c t i o n o f t r a n s f o r m a t i o n e f f i c i e n c y o f a sequences i n t o A. Yanofsky  Staben and  (1990) a l s o r e p o r t e d a decrease i n t h e frequency o f  A transformants when t h e donor DNA was ORF, a - l , as compared t o when t h e donor DNA was a p o r t i o n o f the a idiomorph not including a - l . Nuclear i n c o m p a t i b i l i t y may be s e p a r a b l e heterokaryon  incompatibility.  from  G l a s s and G r i f f i t h s ( p e r s o n a l  communication) c r e a t e d a A - d u p l i c a t i o n s t r a i n by t r a n s f o r m i n g a s t r a i n o f mating type A w i t h t h e ORF o f t h e s t e r i l e , compatible mutant, A . m64  In order t o RIP t h e A  gene, they c r o s s e d t h e transformant t o a s t r a i n o f mating type a.  S u r p r i s i n g l y , some o f t h e progeny d i s p l a y e d the  \u000C65  i n c o m p a t i b i l i t y phenotype.  Growth o c c u r r e d as a s m a l l wispy  knot o f m y c e l i a w i t h no a e r i a l hyphae. escaped from slow growth w i t h i n  A l l of the cultures  one week, and then showed a  mating type a r e a c t i o n when t e s t e d .  I f t h e ORF had  segregated from mt as an independent  l o c u s , then one h a l f o f  the a spores would have c o n t a i n e d A  and i t i s p o s s i b l e  m64  t h a t these were t h e i n c o m p a t i b l e s t r a i n s ( F i g . 1 2 ) . I f t h e i n c o m p a t i b l e s t r a i n s were A /a, m64  though A  has l o s t heterokaryon  m64  r e t a i n i n g nuclear  i t appears as  incompatibility  incompatibility.  At f i r s t  while  i t seemed  p o s s i b l e t h a t t h e i n c o m p a t i b l e phenotype was due t o r e s i d u a l heterokaryon i n c o m p a t i b i l i t y s p e c i f i e d by A , m64  but t h i s  h y p o t h e s i s has been d i s c a r d e d because a mixed mating heterokaryon o f A  n 6 4  grew as f a s t as a p o s i t i v e  type  control  strain. To t e s t t h e p o s s i b i l i t y t h a t mutations a t mt can eliminate  heterokaryon i n c o m p a t i b i l i t y without  eliminating  nuclear i n c o m p a t i b i l i t y , d u p l i c a t i o n s t r a i n s that contained the f e r t i l e , compatible mutant, a ,  and A were examined  m33  f o r t h e i r m o r p h o l o g i c a l c h a r a c t e r i s t i c s and growth r a t e s . A/a  m33  a  m 3 3  d u p l i c a t i o n s t r a i n s were made by c r o s s i n g  3 different  - c o n t a i n i n g strains t o A strains containing a  t r a n s l o c a t i o n o f t h e mating type gene.  The mutant, a , i s m33  known t o be compatible i n a heterokaryon w i t h A, although the growth r a t e o f an a  m 3 3  + A heterokaryon has n o t been  measured p r e c i s e l y b e f o r e now. i f m33 a  n a &lt;  j  a r i  I t was measured t o determine  y r e s i d u a l heterokaryon  incompatibility.  \u000C66  1/2  Am64  1/2 A  N O R M A L OR RIP  1/2 Am64 1/2 a  / \\  re  INCOMPATIBLE?  12  NORMAL a  Segregation of Am64 ORF and resulting phenotypes.  \u000C67  RESULTS 2  The growth r a t e o f the s t e r i l e , compatible mutant, m64  A  ^  f  na  m  i  x  e  c  |  mating type heterokaryon, A  a ) , was measured t o determine incompatibility.  if A  m 6 4  + 7 (ad-3B,  had r e s i d u a l  m64  The growth r a t e was compared t o t h a t o f  the component s t r a i n s alone and t o t h a t o f an i n c o m p a t i b l e mixed mating type heterokaryon, 153 (ad-3A, nic-2, (ad-3B, a). The heterokaryon, A  m64  A) + 7  + 7 (ad-3B, a ) , grew as  f a s t as t h e component s t r a i n 7 (ad-3B, a ) , and f a s t e r than the i n c o m p a t i b l e mixed mating type heterokaryon, 153 (ad-3A, nic-2,  A) + 7 (ad-3B, a) (Table 7 and F i g . 13), s u g g e s t i n g  t h a t the mutant, A , m64  has l o s t i t s heterokaryon  i n c o m p a t i b i l i t y f u n c t i o n completely . The f o l l o w i n g  s e c t i o n d e s c r i b e s a s e r i e s o f t e s t s done  t o study heterokaryon and n u c l e a r i n c o m p a t i b i l i t y mating type mutant,  a . m33  The growth r a t e o f a heterokaryon  (a , m33  residual incompatibility. controls:  heterokaryon nic-2,  m 3 3  i n a mixed mating  type  ad-3B + 1-22-83 (ad-3A, nic-2,  was measured t o determine  several  i n another  un-3, A))  i f t h e mating type mutant had The growth r a t e was compared t o  (1) an i n c o m p a t i b l e mixed mating  (51-2 (ad-3B, cyh-1,  type  a) + 1-22-83 (ad-3A,  un-3, A)); (2) a mating type h o m o k a r y o n \u2014 a  m33  ,  ad-3B(128) p a i r e d w i t h 51-2, a s t r a i n o f mating type a (ad-3B(114),  cyh-1, a ) ; (3) a compatible mixed mating  heterokaryon i n which both components had tol (1-9-57  type  \u000C68  Table 7  Strains and Media Used in the Measurement of Growth Rate of Am6 in a Mixed Mating Type Heterokaryon GENOTYPE  STRAIN 153 * 7 Am64  mt  AUXOTROPHIC GENES  A a Am64  ad-3A, nic-2 ad-3B ad-3A, nic-2  OTHER un-3, cyh-1 un-3, cyh-1  MINIMAL MEDIUM + POSITIVE CONTROLS Am64 7  adenine, nicotinic acid adenine  NEGATIVE CONTROLS Am64 7 153 + 7 EXPERIMENTAL Am64 + 7 ad-3A - adenine-requiring (complements ad-3B) ad-3B - adenine-requiring (complements ad-3A) nic-2 \u2022 nicotinic acid- or nicotinamide-requiring un-3 \u2022 temperature-sensitive cyh-1 \u00AB cycloheximide-resistant  * 153 was the strain used to generate Am64.  \u000C6 9  24  48  Jl  120 168 216 264 312 380 96 144 192 240 288 336 384 fme (hois)  P i n i i r o i l y U r \u00A9  1Q  lO  Growth rate of Am64 in a mixed mating type heterokaryon. Each line represents the average of a minimum of 3 measurements of growth rate.  \u000C70  (ad-3B(128),  tol, a)+ 1-9-3 (ad-3B(114),  tol, A ) ) ; and (4)  the f o l l o w i n g component s t r a i n s on supplemented media: 1-22-83; a , m33  ad-3B-, and 51-2.  The heterokaryon, a  +  m 3 3  1-22-83, grew as f a s t as t h e mating type homokaryon, a  m 3 3  +  51-2 and t h e compatible mixed mating type heterokaryon, 1-9-57 + 1-9-3; and f a s t e r than t h e incompatible heterokaryon, 51-2 + 1-22-83 (Table 8 and F i g . 14), s u g g e s t i n g t h a t t h e mutant, a , m33  has l o s t i t s heterokaryon  i n c o m p a t i b i l i t y f u n c t i o n completely. To determine the phenotype shown by a  m 3 3  i n the same  nucleus w i t h A, d u p l i c a t i o n progeny were o b t a i n e d from the following crosses. Rl-14 and Rl-29  Three a  m 3 5  -containing strains, a , m33  (Table 9) were c r o s s e d t o t h e t r a n s l o c a t i o n  s t r a i n , T(I-&gt;II) 39311, s e r - 3 , A, t o generate progeny.  ad;  duplication  The c r o s s e s a r e diagrammed i n F i g s . 15A and 15B.  S i n g l e spores were viewed through a d i s s e c t i n g and c o l l e c t e d w i t h a tungsten needle.  Individual  were p l a c e d i n t o s l a n t s o f supplemented medium medium + adenine f o r the a , m33  microscope spores  (minimal  a d - d e r i v e d spores; and  minimal medium + n i c o t i n i c a c i d and p a n t o t h e n i c a c i d f o r the Rl-14- and R l - 2 9 - d e r i v e d s p o r e s ) .  The t e s t tubes were  p l a c e d i n t o a 60\u00B0C water bath f o r 30 minutes t o i n i t i a t e g e r m i n a t i o n o f t h e spores, which were examined a f t e r 3 days. The progeny from each o f t h e 3 c r o s s e s expressed one o f two growth phenotypes.  One phenotypic c l a s s ,  called  \" i n h i b i t e d \" , grew s l i g h t l y l e s s v i g o r o u s l y than t h e o t h e r , \"healthy\".  The i n h i b i t e d phenotype was d i s t i n c t from the  \u000C71  Table 8 Strains and Media Used in the Measurement of Growth Rate of am33 in a Mixed Mating Type Heterokaryon STRAIN 1-22-83 51-2 I-9-57 I-9-3  A, a, a, A,  GENOTYPE ad-3A, nic-2, un-3 ad-3B(114), cyh-1 ad-3B(128), tol ad-3B(114), tol MINIMAL MEDIUM +  POSITIVE CONTROLS 1-9-57 \u00AB\u2022 1-9-3* am33, ad + 51-2 I-9-3 am33, ad 51-2 I-22-83 I-9-57 NEGATIVE CONTROLS 51-2 + I-22-83** I-9-3 am33, ad 51-2 1-22-83 1-9-57 EXPERIMENTAL am33, ad + 1-22-83  \u2014 \u2014  adenine adenine adenine adenine, nicotinic acid adenine \u2014 \u2014 \u2014 \u2014 \u2014 \u2014  \u2014  ad-3A - adenine-requiring ad-3B(114) - adenine-requiring ad-3B(128) - adenine-requiring un-3 \u2022 temperature-sensitive cyh-1 \u2022 cycloheximide-resistant  * The alleles, ad-3B(114) and ad-3B(128), complement. ** The genes, ad-3A and ad-3B, complement.  \u000C72  LEGEND 1 \u2022 I-9-57 + I-9-3 2 \u2022 am33,ad + 51-2 3 - 51-2 + 1-22-83 4 - am33,ad + 1-22-83 Growth rate of am33 in a mixed mating type heterokaryon. r *A Each line represents the average of 2 measurements F i g u r e I*f of growth rate. l  l  K  n  \u000C73  Table 9 Genotypes of am33 Strains  STRAIN  GENOTYPE  SOURCE  am33, ad  am33, ad-3B (128)  AJ.F.G.  R1-14  am33, n i c - 3 , pan-1, al-1 (#43)  N.L.G.  R1-29  am33, n i c - 3 , pan-1, al-1 (#29)  N.L.G.  am33 - fertile compatible mating type mutant ad-3B (128) - adenine-requiring (allele #128) nic-3 - nicotinic acid- or nicotinamide-requiring pan-1 - pantothenic-acid requiring al-1 - albino #43 - isolate # of N.L.G. #29 - isolate # of N.L.G. A.J.F.G. - A.J.F. Griffiths N.L.G. - N.L. Glass  \u000C74  am33, ad-3B x T(l-&gt;ll)39311, s e r - 3 , A am33 ad |  ser A minimal mediLm + adenine  am33 ad |  32o/  0  50%  am33, ad  50%  A/am33, ad (incompatible?)  ser A  50%  \u2022M-  32%  50%  II  dead  II  dead  ser A  18%  am33  50%  am33  50%  A/am33 (incompatible?)  ser A ad  |  50%  II II  18% 50%  ser A  dead dead  Cross made to generate A/am33 duplications. Genetic r : _ j r A distance between ad-3B and mt is 36% r i g U r e I O H (Perkins, et al., 1982). l  i  r  o  \u000C75  R1-14 or R1-29  x  T(l-&gt;ll)39311, s e r - 3 , A  am33 al-1  -xpan-1  IV  nic-3  VII  ser-3 A  pantothenic acid + nicotinic acid  50%  am33  50% I  II  50% ser-3 A  50%  H  am33  II  A/am33 (incompatible?)  50%  dead  50%  dead  1  ser-3 A  Figure 15B  Crosses done to generate  A/am33  duplications.  \u000C76  standard i n c o m p a t i b i l i t y phenotype  (dark agar) i n t h a t the  growth was more l u x u r i a n t . Mating type t e s t s were done on a l l of t h e i s o l a t e s (Table 10).  I f p e r i t h e c i a were produced w i t h the fl t e s t e r ,  t h e r e a c t i o n was s c o r e d as p o s i t i v e . c o u l d have been b a r r e n .  Crosses, t h e r e f o r e ,  The h e a l t h y progeny from a l l  three  c r o s s e s were e i t h e r a, which was expected ( F i g . 15A and o r A/a, which was unexpected.  15B)  The i n h i b i t e d progeny were  mostly A/a, which was the r e s u l t p r e d i c t e d i n the h y p o t h e s i s , a l t h o u g h the p r e d i c t e d phenotype was dark agar. One i n h i b i t e d i s o l a t e c o u l d have c o n t a i n e d both mating t y p e s , but t h e a mating type r e a c t i o n was d i f f i c u l t t o confirm.  One  i n h i b i t e d i s o l a t e r e a c t e d as o n l y a.  The  s i g n i f i c a n c e o f the f o u r types of progeny i s c o n s i d e r e d i n D i s c u s s i o n 2. In o r d e r t o v e r i f y the d i f f e r e n c e i n phenotypes, progeny from each c r o s s were i n o c u l a t e d onto p l a t e s .  sample The  d i f f e r e n c e between the phenotypes became more e v i d e n t under t h e s e growth c o n d i t i o n s .  As a c o n t r o l , a A/a, tol  d u p l i c a t i o n s t r a i n , which has a phenotype known as \"square agar\" (Newmeyer, 1970), v e r y s l i g h t l y d i f f e r e n t from w i l d type, was a l s o i n o c u l a t e d onto a p l a t e . A l l o f the h e a l t h y progeny t e s t e d , i n c l u d i n g the s i n g l e and double mating type i s o l a t e s , and t h e square agar c o n t r o l grew t o c o v e r the p l a t e w i t h an even l a y e r o f m y c e l i a .  Most  of the i n h i b i t e d progeny (22 o f 30) grew i n a dense mat i n the c e n t r e of the p l a t e w i t h a few hyphae extending beyond  \u000C77  Table 10 Mating Types of Progeny CROSS  S P O R E T Y P E MATING T Y P E  am33,ad-3B x T(l-&gt;ll)39311,ser,A  17 h  12 A/a  5 a  R1-14 x T(l-&gt;ll)39311,ser,A  R1-29 x T(l-&gt;ll)39311,ser,A  12 i  11 A/a 1 A/?  9 h  6 A/a 3 a  11 i  11  7 h  3 A/a 4 a  7 i  6A/a 1 a  h \u2022 healthy (i.e. vigorous growth) i \u2022 inhibited (i.e. less vigorous growth)  The A/a healthy isolates were unexpected.  A/a  \u000C78  the c e n t r a l mat  (Table  11).  Some of the progeny o r i g i n a l l y  s c o r e d as i n h i b i t e d (8 of 30) the p l a t i n g t e s t was  grew evenly on p l a t e s .  done s e v e r a l months a f t e r the  initial  spore i s o l a t i o n , i t i s p o s s i b l e t h a t these progeny escaped.  S t r a i n s were kept i n the  freezer  The and  not  a l l of  the  escaped.  growth r a t e s of a l l of the  i s o l a t e s were measured  compared t o incompatible d u p l i c a t i o n (A/a,tol ) +  compatible d u p l i c a t i o n inoculated mL  had  (-20\u00B0C) d u r i n g  most of t h i s time, which c o u l d e x p l a i n why i n h i b i t e d progeny had  a t one  end  (A/a,tol) c o n t r o l s .  medium f o r the c o n t r o l s . intervals.  and  Fungi were  of a 50 cm growth tube c o n t a i n i n g  of supplemented medium f o r the  regular  Since  The  Results  i s o l a t e s and  mycelial  minimal  f r o n t s were marked a t  are shown i n F i g s . 16-29.  s l o p e s o f the graphs are shown i n Table  The  12.  A l l of the h e a l t h y i s o l a t e s grew a t the same r a t e t h e i r s i b l i n g s , as f a s t as the compatible c o n t r o l s , considerably  f a s t e r than the  growth r a t e s o f the one  incompatible c o n t r o l s .  i n h i b i t e d s t r a i n s varied widely.  as  and The Only  s t r a i n , the s i n g l e mating type i n h i b i t e d s t r a i n , 29-i-7,  grew as f a s t as the compatible c o n t r o l s and The  30  healthy s t r a i n s .  r e s t grew a t r a t e s i n t e r m e d i a t e between those of  compatible and  incompatible c o n t r o l s .  The  these r e s u l t s i s c o n s i d e r e d i n D i s c u s s i o n The  f o l l o w i n g t e s t was  mating types of the A/a different nuclei.  the  s i g n i f i c a n c e of 2.  done t o determine whether the  s t r a i n s had  segregated i n t o  C o n i d i a l samples from the c r o s s e s w i t h  \u000C79  Table 11 Phenotypes of Progeny  STRAIN  # OF ISOLATES WITH P H E N O T Y P E OF DENSE MAT  EVEN GROWTH  a-i-x  8  4  a-h-x  0  17  14-i-x  10  1  14-h-x  0  9  29-l-x  4  3  29-h-x  0  7  KEY a, 14 or 29 \u2022 strains am33,ad; R1-14 or R1-29 h or i = healthy or inhibited x isolate # s  Inhibited strains with even growth were probably escapes.  \u000C80  the females, Rl-14 and Rl-29, were p l a t e d on sorbose p l a t e s c o n t a i n i n g p a n t o t h e n i c a c i d and n i c o t i n i c a c i d .  Single  c o n i d i a l c o l o n i e s were c u t out o f t h e agar, grown and t e s t e d f o r t h e i r mating t y p e s .  A l l of the i n h i b i t e d  isolates  t e s t e d c o n t a i n e d both mating t y p e s , s u g g e s t i n g t h a t t h e mating types had not segregated m i t o t i c a l l y .  The h e a l t h y  i s o l a t e s of s t r a i n s which were o r i g i n a l l y A/a were a l l a (Table 13). below.  The s i g n i f i c a n c e o f these r e s u l t s i s d i s c u s s e d  \u000C81  Growth Rates of Controls 45  0  24  48  72  96  120  144 192 168  Tmejhotrs)  LEGEND FOR FIGURES 16-29 a, 14 or 29 - strains am33, ad; R1-14 or R1-29 h or i - healthy or inhibited x - isolate #  Figure 16  \u000C82  Figure 17  \u000C83  Figure 18  \u000C84  Figure 19  \u000C85  Growth Rates of a-i-x  0 24- 48 72 96 120 144 168 192 Trrefws)  Figure 20  \u000C86  Growth Rates of a-i-x  Figure 21  \u000C87  Growth Rates of 14-h-x SOi  0  24  \u20AC Tmefuis)  Figure 22  72  96  \u000C88  Figure 23  \u000C89  Growth Rates of 14\u2014i\u2014x  0  24 48 72 96 120 144 168 192 rine (hours)  Figure 24  \u000C90  Growth Rates of  0 24 48 72 96 120 144 168 192 rme (heirs)  Figure 25  \u000C91  Figure 26  \u000C92  Figure 27  \u000C93  Growth Rates of 2H-x  0 24 48 72 96 120 144 1\u00AE 192 fire (bars)  Figure 28  \u000C94  Growth Rates of 29-i-x  Figure 29  \u000C95  Table 12 Slopes of Growth Rates of Progeny STRAIN A/a, tol+ (average of 2 strains) A/a, tol (average of 2 strains) a-h-x (average of 17 strains) a-i-1 a-i-2 a-i-3 a-i-4 a-i-5 a-i-6 a-i-7 a-i-8 a-i-9 a-i-10 a-i-11 a-i-12 14-h-x (average of 9 strains) 14-i-x (average of 14-i-1, 2, 3, 6, 7) 14-i-x (average of 14-i-4, 5, 9) 14-i-8 14-i-10 14-i-11 29-h-x (average of 7 strains) 29 -1 29 -2 29-i -3 29 -4 29 -5 29 -6 29 -7  SLOPE.0035 .50 .50 .27 .33 .48 .42, ,1 1 .44 .12 .47 .23 .38, .17 .46 .31 .34 .51 .39 .056 .096 .49 .044, .26 .50 .19 .43 .053 .37 .34 .13 .52  * Slopes were measured at straight regions of the graph, over no fewer than 72 hours (except for the first slope for a-i-9, which was measured over 48 hours). All except 1 (29-i-7) of the inhibited strains grew more slowly than the healthy strains and the positive control strains (A/a, tol).  \u000C96  Table 13  Mating Types of Single Conidial Isolates of Progeny  STRAIN  MATING T Y P E  14-h-4-1 14-h-4-2 14-h-4-3 14-h-5-1 14-h-5-2 14-h-5-3 14-h-6-1  a a a a a a a  14-1-3-1  A/a  29-h-5-1 29-h-5-2 29-h-6-1 29-h-6-2 29-h-6-3 29-h-7-1 29-h-7-2 29-h-7-3  a a a a a a a a  29-1-1-1 29-J-1-2 29-J-1-3 29-1-3-1  A/a A/a A/a A/a  14 or 29 - strain R1-14 or R1-29 h or i \u2022 healthy or inhibited first # - spore isolate # last # - conidial isolate # Healthy strains were a; inhibited strains were A/a.  \u000C97  DISCUSSION 2  The f i n d i n g s o f R e s u l t s 2 p r e s e n t evidence t o support the i d e a t h a t a t l e a s t 2 mating type mutants completely d e f i c i e n t i n heterokaryon i n c o m p a t i b i l i t y , A retain nuclear incompatibility.  m 6 4  and a  m 3 3  ,  The growth r a t e s o f t h e two  mutants i n mixed mating type heterokaryons e q u a l l e d those o f p o s i t i v e c o n t r o l s and surpassed those o f n e g a t i v e c o n t r o l s , so i t appears t h a t n e i t h e r mutant has r e s i d u a l heterokaryon incompatibility.  The n u c l e a r i n c o m p a t i b i l i t y  seen,  t h e r e f o r e , was n o t due t o r e s i d u a l heterokaryon incompatibi1ity. Four phenotypes  were seen i n t h e progeny  c r o s s e s made t o generate A/a  m33  duplications:  mating type h e a l t h y (12 i s o l a t e s ) , h e a l t h y (21 i s o l a t e s ) ,  (1) s i n g l e  (2) double mating  type  (3) s i n g l e mating type i n h i b i t e d (1  i s o l a t e ) and (4) double mating type i n h i b i t e d p o s s i b l y 29, i s o l a t e s ) .  from t h e t h r e e  (28, o r  The o n l y phenotype t h a t was  expected ( F i g . 15A and 15B) was t h e f i r s t phenotypic c l a s s , s i n g l e mating type h e a l t h y , which were normal  segregants  from t h e c r o s s . The phenotype o f t h e d u p l i c a t e d progeny unknown, although i t was p r e d i c t e d t o be dark agar. The f o u r t h phenotypic c l a s s , t h e double mating  type  i n h i b i t e d s t r a i n s , i s the one p r o v i d i n g evidence t h a t n u c l e a r i n c o m p a t i b i l i t y can e x i s t i n t h e absence o f heterokaryon i n c o m p a t i b i l i t y .  A l l o f t h e A/a  inhibited  was  \u000C98  s t r a i n s grew more s l o w l y than the h e a l t h y s t r a i n s .  The  reduced growth r a t e s c o u l d have been due t o the presence o f a  m 3 3  and A i n the  same n u c l e u s .  Four o f the  i n h i b i t e d s t r a i n s t h a t grew evenly on p l a t e s  seven A/a (a-i-3,  a-i-7,  1 4 - i - l O , 29-i-2) had the h i g h e s t growth r a t e s o f a l l o f the A/a  i n h i b i t e d s t r a i n s , s u p p o r t i n g the  i d e a t h a t they  escaped and were growing a t r a t e s h i g h e r than the s t r a i n s , yet  lower than the  healthy s t r a i n s .  had  inhibited  Two o f the  seven s t r a i n s  ( a - i - 4 , a - i - 9 ) had growth r a t e s t h a t were h i g h  a t f i r s t , but  suddenly dropped.  One o f the  seven s t r a i n s  (29-i-6) had an i r r e g u l a r growth p a t t e r n . The highly  growth r a t e s o f the A/a i n h i b i t e d s t r a i n s were  variable.  The v a r i a n t  of nuclear i n c o m p a t i b i l i t y  r a t e s c o u l d be c h a r a c t e r i s t i c  itself.  They c o u l d a l s o have  been produced by escape, e i t h e r by a number o f d i f f e r e n t mechanisms o r by the results.  Perhaps the  same mechanism producing d i f f e r e n t i n h i b i t e d s t r a i n s showed v a r i o u s  growth r a t e s because they were i n d i f f e r e n t stages o f escape by  somatic s e g r e g a t i o n and overgrowth by a n u c l e i . The  was  not  second phenotypic c l a s s , the A/a h e a l t h y expected.  isolates  Metzenberg ( p e r s o n a l communication)  suggested t h a t these s t r a i n s grew v i g o r o u s l y because the two mating types had segregated s o m a t i c a l l y i n t o s e p a r a t e producing A + a compatible.  m 3 3  heterokaryons, which are known t o be  The t r a n s l o c a t i o n  double c r o s s o v e r .  nuclei  i s l o n g enough t o s u s t a i n a  To t e s t h i s h y p o t h e s i s , s i n g l e  i s o l a t e s were o b t a i n e d from h e a l t h y and i n h i b i t e d  conidial strains  \u000C99  and t e s t e d f o r t h e i r mating t y p e s .  I f Metzenberg's  h y p o t h e s i s were c o r r e c t , c o n i d i a d e r i v e d from h e a l t h y s t r a i n s would have been A o r a, whereas c o n i d i a d e r i v e d from i n h i b i t e d s t r a i n s would have been A/a. observed,  These r e s u l t s were  w i t h the e x c e p t i o n t h a t t h e r e were no A c o n i d i a l  i s o l a t e s from t h e h e a l t h y s t r a i n s . I f t h e h e a l t h y phenotype were due t o m i t o t i c s e g r e g a t i o n , t h e c u l t u r e s would have c o n t a i n e d both mating types s h o r t l y a f t e r germination o f the spores.  After a  p e r i o d o f time, i t i s c o n c e i v a b l e t h a t t h e a n u c l e i overgrew the A n u c l e i because t h e l a t t e r c o n t a i n e d t h e ser-3 marker which i s c l o s e l y l i n k e d t o t h e mating type gene.  Overgrowth  by a n u c l e i may have been slowed by c r o s s - f e e d i n g o f t h e s e r i n e - r e q u i r i n g A n u c l e i by t h e a n u c l e i . The h e a l t h y s t r a i n s grew as f a s t as t h e compatible c o n t r o l s ( F i g s . 16-19, 22, 23, 26 and 27).  The compatible  c o n t r o l s were A/a d u p l i c a t i o n s c o n t a i n i n g t o l . The evidence presented above suggests t h a t the h e a l t h y s t r a i n s were simply a*  33  strains.  The t h i r d phenotypic c l a s s , t h e 1 unexpected  inhibited  s t r a i n t h a t r e a c t e d w i t h o n l y one mating type ( 2 9 - i - 7 ) , c o u l d have escaped  from t h e i n h i b i t i o n by d e l e t i o n o f A. I t  grew evenly on a p l a t e , and had a growth r a t e t h a t exceeded some o f t h e h e a l t h y s t r a i n s , s u g g e s t i n g t h a t i t had escaped from i n h i b i t e d growth. An e x p l a n a t i o n i s needed f o r why some A/a (the h e a l t h y ones) escaped  m33  strains  e a r l y , while others (the  \u000C100  i n h i b i t e d ones) escaped  l a t e r o r not a t a l l .  the i n h i b i t e d s t r a i n s have escaped  Would a l l of  eventually?  Was  of escape the o n l y d i f f e r e n c e between the h e a l t h y i n h i b i t e d s t r a i n s or was mechanisms of escape?  the  time  and  t h e r e a d i f f e r e n c e i n the  Was  i t significant that  escaped  i n h i b i t e d s t r a i n s d i d not grow as f a s t as h e a l t h y s t r a i n s ? Was  t h e r e a gene s e g r e g a t i n g t h a t caused e a r l y / l a t e escape  by m i t o t i c c r o s s i n g over i n the A/a h e a l t h y s t r a i n s and A/a  inhibited strains?  The  f i r s t of the t h r e e c r o s s e s  segregated h a l f A/a h e a l t h y and h a l f A/a  i n h i b i t e d progeny.  In the o t h e r 2 c r o s s e s , the sample s i z e s were probably small to r e f l e c t accurate r a t i o s . be addressed  m33  too  These q u e s t i o n s can o n l y  by f u r t h e r study.  P e r k i n s ( p e r s o n a l communication) has observed A/a  the  that  d u p l i c a t i o n s grow w i t h an abnormal morphology which  he c a l l s \"square\" because Newmeyer (1970) c a l l e d morphology a s s o c i a t e d w i t h A/a,  the  t o l d u p l i c a t i o n s \"square\".  P e r k i n s ' o b s e r v a t i o n s are not i n c o n s i s t e n t w i t h  those  presented above, except t h a t Newmeyer's square s t r a i n s grow a t w i l d type r a t e s , whereas P e r k i n s ' square  strains,  assuming they e x h i b i t the same growth r a t e s as d i s c u s s e d here, grow a t sub-wild type r a t e s . reason, A/a  m33  name o t h e r than  those For t h i s  d u p l i c a t i o n s s h o u l d be r e f e r r e d t o by some square.  \u000C101  M o l e c u l a r Model  One c u r r e n t model o f t h e molecular i n t e r a c t i o n s t h a t o c c u r d u r i n g mating  i s as f o l l o w s (Metzenberg  1990r G l a s s , p e r s o n a l communication).  and G l a s s ,  A combination  product  o f A and a e f f e c t s i n c o m p a t i b i l i t y d u r i n g t h e v e g e t a t i v e c y c l e , when t h e mating type genes a r e expressed a t low l e v e l s , and t h e same product i n s t i g a t e s mating f u n c t i o n s d u r i n g t h e s e x u a l c y c l e , when t h e mating type genes a r e expressed a t h i g h e r l e v e l s . Nuclear i n c o m p a t i b i l i t y can be e x p l a i n e d i n t h e c o n t e x t of t h i s model.  There i s o b v i o u s l y a d i f f e r e n c e i n the  f u n c t i o n o f t h e mating type products d u r i n g t h e v e g e t a t i v e and s e x u a l c y c l e s .  Perhaps t h e mutant, A , m64  i s defective  f o r mating, but not i n c o m p a t i b i l i t y , as p r e v i o u s l y b e l i e v e d . I t , and t h e o t h e r mutant s t u d i e d here, a , m33  c o u l d be  d e f e c t i v e i n terms o f the s t a b i l i t y o f t h e i r p r o d u c t s .  Both  mutants a r e f u n c t i o n a l f o r n u c l e a r i n c o m p a t i b i l i t y and d e f e c t i v e f o r heterokaryon i n c o m p a t i b i l i t y .  In terms o f t h e  s t a b i l i t y h y p o t h e s i s , heterokaryon i n c o m p a t i b i l i t y r e q u i r e s more s t a b l e mating type products than n u c l e a r incompatibility.  These r e s u l t s can be e x p l a i n e d as f o l l o w s .  In a mixed mating type heterokaryon, the A and a products a r e s y n t h e s i z e d i n d i f f e r e n t n u c l e i . products encounter one another, t h e combination  When the product  e n t e r s t h e nucleus and i n i t i a t e s i n c o m p a t i b i l i t y .  In a A/a  d u p l i c a t i o n s t r a i n , t h e A and a products a r e made i n t h e  \u000C102  same n u c l e u s , s o t h e y w o u l d e n c o u n t e r g r e a t e r speed  b e c a u s e o f t h e i r p r o x i m i t y , and  therefore, require less s t a b i l i t y . a  m 3 3  each o t h e r w i t h  , heterokaryon  would,  In t h e mutants, A  i n c o m p a t i b l i t y may  and  be e l i m i n a t e d b e c a u s e  the products degrade t o o q u i c k l y t o f i n d the o p p o s i t e t y p e p r o d u c t w i t h which t o combine.  m 6 4  mating  \u000C103 REFERENCES Abraham, J . , J . Feldman, K.A. Nasmyth, J.N. 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