Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A/a incompatibility in Neurospora crassa : novel suppressors and nuclear incompatibility 1991

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1991_A6_7 V44.pdf
UBC_1991_A6_7 V44.pdf
UBC_1991_A6_7 V44.pdf [ 4.95MB ]
UBC_1991_A6_7 V44.pdf
Metadata
JSON: 1.0098715.json
JSON-LD: 1.0098715+ld.json
RDF/XML (Pretty): 1.0098715.xml
RDF/JSON: 1.0098715+rdf.json
Turtle: 1.0098715+rdf-turtle.txt
N-Triples: 1.0098715+rdf-ntriples.txt
Citation
1.0098715.ris

Full Text

A/a INCOMPATIBILITY IN NEUROSPORA CRASSA—NOVEL SUPPRESSORS AND NUCLEAR INCOMPATIBILITY by TRINA SEHAR VELLANI B.Sc.(Hon.)# McMaster University, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1991 (c) Trina Sehar V e l l a n i , 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Botany The University of British Columbia Vancouver, Canada Date -3 O r f n W 1QQ1 DE-6 (2/88) ABSTRACT The sexual functions of the mating type gene (int) of Neurospora crassa include s p e c i f i c a t i o n of mating i d e n t i t y (Shear and Dodge, 1927) 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 also acts as a vegetative incompatibility locus, so that A + a heterokaryons (Beadle and Coonradt, 1944) or A/a duplication s t r a i n s (Newmeyer and Taylor, 1967) grow poorly or not at a l l . An i n t r i g u i n g question regarding the mating type gene i s t h i s : How does i t control both the switch between somatic and meiotic events and heterokaryon incompatibility? Several research groups (Glass, et a l . , 1990; Staben and Yanofsky, 1990) are presently studying the sexual functions of the mating type genes. I present a study of the incompatibility function. Two experiments were performed. The f i r s t was a search fo r new suppressors of mating type-associated incompatibility, which resulted i n the i d e n t i f i c a t i o n of seven new suppressors, none of which was a l l e l i c with the one known suppressor, tol. The second was the comparison of growth rates of a mating type mutant ( f e r t i l e , heterokaryon compatible) i n a mixed mating type heterokaryon and i n a mixed mating type duplication to determine whether or not cytoplasmic incompatibility i s separable from nuclear i i i i n compatibility. The r e s u l t s obtained suggest that the mating type mutant, am33, eliminates heterokaryon incompatibility without eliminating nuclear incompatibility. The search f o r suppressors was attempted i n order to define more of the genes involved i n A/a incompatibility. The analysis of heterokaryon versus nuclear incompatibility was done to investigate the c e l l u l a r interactions involved i n A/a incompatibility. i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES V LIST OF FIGURES v i ACKNOWLEDGEMENT v i i i GENERAL INTRODUCTION ; 1 L i f e Cycle 1 Mating Type Gene Functions 4 Mating and Incompatibility i n Other Fungi. 6 Mating and Incompatibility i n Other Kingdoms 16 INTRODUCTION 1 18 A Suppressor of A/a Incompatibility, tol .18 MATERIALS AND METHODS 22 Strains and Markers 22 Ascospore I s o l a t i o n 24 Construction of Tester S t r a i n (T(I->II) 39311, ser, trp, tol, a) 25 RESULTS 1 * 28 DISCUSSION 1 51 What i s tol? 56 INTRODUCTION 2 62 RESULTS 2 67 DISCUSSION 2 97 Molecular Model 101 REFERENCES 103 V LIST OF TABLES Table 1 Strains from Experiment Set 1 23 Table 2 Phenotypes of F2 Strains 38-39 Table 3 Phenotypes of F3 Strains 44 Table 4 Mating Types of F3 Strains 46 Table 5 Mating Types of Hyphal Tips of A/a Compatible F3 Strains 49 Table 6 Summary of Results 1 50 Table 7 Strains and Media Used i n the Measurement of Growth Rate of A m 6 4 i n a Mixed Mating Type Heterokaryon 68 Table 8 Strains and Media Used i n the Measurement of Growth Rate of a m 3 3 i n a Mixed Mating Type Heterkaryon 71 Table 9 Genotypes of a m 3 3 Strains 73 Table 10 Mating Types of Progeny 77 Table 11 Phenotypes of Progeny 79 Table 12 Slopes of Growth Rates of Progeny 95 Table 13 Mating Types of Single Conidial Isolates of Progeny 96 v i LIST OF FIGURES Figure 1 L i f e Cycle of 2V. crassa 2 Figure 2 Example of Mi t o t i c Crossover 7 Figure 3 Construction of Tester Strain 26 Figure 4 Summary of Selection Protocol f o r Suppressors..29 Figure 5 F i r s t Cross 31 Figure 6 Phenyotypic Classes of Escaped F l Strains......34 Figure 7 Second Cross 36 Figure 8 Examples of Mi t o t i c Double Crossovers 41 Figure 9 Third Cross 42 Figure 10 Alternative Pairing Hypothesis 53 Figure 11 Mating Type Regions of N. crassa 63 Figure 12 Segregation of A m 6 4 ORF 66 Figure 13 Growth Rate of Am64 i n a Mixed Mating Type Heterokaryon 69 Figure 14 Growth Rate of a m 3 3 i n a Mixed Mating Type Heterokaryon 72 Figure 15A Cross of am33, ad x T(I->II) 39311, ser-3, A...74 Figure 15B Crosses of Rl-14 or Rl-29 x T(I->II) 39311, ser-3, A 75 Figure 16 Growth Rates of Controls 81 Figure 17 Growth Rates of a-h-x 82 Figure 18 Growth Rates of a-h-x 83 Figure 19 Growth Rates of a-h-x 84 Figure 20 Growth Rates of a-i-x 85 Figure 21 Growth Rates of a-i-x 86 v i i Figure 22 Growth Rates of 14-h-x 87 Figure 23 Growth Rates of 14-h-x 88 Figure 24 Growth Rates of 14-i-x 89 Figure 25 Growth Rates of 14-i-x 90 Figure 26 Growth Rates of 29-h-x 91 Figure 27 Growth Rates of 29-h-x 92 Figure 28 Growth Rates of 29-i-x 93 Figure 29 Growth Rates of 29-i-x 94 v i i i ACKNOWLEDGEMENT I am immensely grate f u l to the following 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 for allowing me freedom to develop my own ideas; Louise Glass f o r her encouragement and countless hours of c r i t i c a l discussion; Jim Berger f o r h i s boundless enthusiasm f o r the world of science and for keeping me from straying too fa r from the task at hand; Carolyn Myers for sharing her research ideas and for invaluable technical advice; Rod, Mishu and my parents for bel i e v i n g i n me. 1 GENERAL INTRODUCTION This work i s a two-part investigation of the vegetative incompatibility function of the mating type gene of Neurospora crassa. The f i r s t part describes the generation of suppressors of A/a incompatibility and the second part describes the analysis of incompatibility on a c e l l u l a r l e v e l . The mating type gene i s involved i n both the vegetative and sexual phases of the l i f e c y cle. L i f e Cycle W. crassa, a mold that grows at the s i t e s of recent f i r e s and i n decaying vegetation, i s a h e t e r o t h a l l i c ascomycete. Its l i f e cycle i s shown i n F i g . 1. The haplold, p a r t i a l l y septate mycelia grow from ascospores which are germinated by a period of heating. Growth requires the presence of inorganic s a l t s , b i o t i n and a u t i l i z a b l e carbon source (Fincham, et a l . , 1979). Several days a f t e r germination, the hyphal t i p s begin to d e l i m i t two types of conidia which, upon germination, are able to i n i t i a t e new mycelia. Macroconidia are oval and multinucleate; whereas microconidia are spherical and binucleate or uninucleate (Fincham, et al, 1979). Low l e v e l s of nitrogen i n i t i a t e the sexual cycle. Male s t r u c t u r e s — c o n i d i a and vegetative hyphae—are already present i n each singl e mating type colony (A or a), and 2 oscogenous hypho igure 1 Life cycle of N. crassa (from Fincham, et al., 1979). 3 immature female s t r u c t u r e s — p r o t o p e r i t h e c i a — b e g i n to form from the same mycelium. The outer layer of the protoperithecium i s a wall of hyphae. Inside i s a c o i l e d hypha, the ascogonium, from which project female reproductive hyphae, trichogynes. Although each mycelium i s hermaphroditic, i t i s also s e l f - s t e r i l e , and sexual fusion between male c e l l s and protoperithecia can occur only between indivi d u a l s of d i f f e r e n t mating types. Once f e r t i l i z e d , the protoperithecium i s known as a perithecium (Fincham, et a l . , 1979). Conidia emit a pheromone which d i r e c t s trichogynes to grow toward them ( B i s t i s , 1981; 1983). When contact i s made between the 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 genetic control (Vigfusson, et al., 1971), t r a v e l down the trichogyne, where one nucleus (Sansome, 1949) enters the ascogonium and becomes associated with the female nucleus. While the perithecium darkens and enlarges, a s e r i e s of synchronous nuclear d i v i s i o n s gives r i s e to a c l u s t e r of dikaryotic ascogenous hyphae (Fincham, et a l . , 1979). Karyogamy occurs i n the penultimate c e l l s of the ascogenous hyphae, followed d i r e c t l y by meiosis plus two rounds of mitosis. Several days l a t e r , the ascospores have become multinucleate (Raju, 1980). The f i n a l products of the sexual cycle are perit h e c i a containing many mature a s c i , each housing eight ascospores which are shot through an opening i n the p e r i t h e c i a l beak (Fincham, et a l . , 1979). 4 Mating Type Gene Functions The mating type genes, A and a, are unusual, even among Neurospora species, i n that they control two functions, mating and vegetative incompatibility. A/a incompatibility i s not seen i n either N. tetrasperma (Dodge, 1935) or N. sitophila (Mishra, 1971). Early attempts to resolve the two functions by recombination f a i l e d (Pittenger, 1957; Newmeyer, et al., 1973); although l a t e r , G r i f f i t h s and DeLange (1978) reported the f i n d i n g of a mating type mutant (am33) that was heterokaryon compatible, yet f e r t i l e . In N. crassa, only s t r a i n s of opposite mating types are able to cross (Shear and Dodge, 1927); so A x a i s a successful p a i r i n g , but A x A or a x a i s not. Strains of opposite mating types are vegetatively incompatible (Beadle and Coonradt, 1944); so A + A or a + a anastomose to form t h r i v i n g heterokaryons, and A + a fuse, but the anastomosed area dies (Garnjobst and Wilson, 1956). The protoplasm of the fused, and sometimes surrounding, c e l l s becomes granular or vacuolated. Mixed mating type heterokaryons with varying degrees of vigour can be made using forcing auxotrophic markers (Beadle and Coonradt, 1944; Gross, 1952; DeLange and G r i f f i t h s , 1975). Protoplasmic k i l l i n g , more severe than that seen between A and a, i s observed i n the reactions between incompatible a l l e l e s of the heterokaryon incompatibility genes het-C/c, het-D/d and het-E/e (Perkins, 1974). The 5 k i l l i n g reaction can be detected in vivo, and also when protoplasm from one s t r a i n i s microinjected into c e l l s of an incompatible s t r a i n , at le a s t between st r a i n s of d i f f e r e n t het-C/c or het-D/d genotypes (Wilson, et al., 1961; Williams and Wilson, 1966). Mixed mating type heterokaryons generally escape from t h e i r poor growth and s t a r t to grow at wild-type or near wild-type rates by deletion of one or the other of the mating type genes (DeLange and G r i f f i t h s , 1975). Heterokaryons heterozygous f o r the vegetative incompatibility genes het-J/j or het-K/k escape by deletion or mutation of the genes (Pittenger, 1964). Strains with a heterozygous duplication of the vegetative incompatibility gene, het-6, also escape by deletion of one of the genes (Glass, personal communication). Not only are the mating type genes incompatible i n a heterokaryon, they are also incompatible i n a duplication. Strains carrying a heterozygous duplication of the mating type genes grow poorly due to the presence of opposite mating type genes i n one nucleus. A/a duplication s t r a i n s , 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 described as being spidery (Newmeyer and Taylor, 1967; Turner, et a l . , 1969). Other duplications i n N, crassa grow normally (provided the duplication does not cover any of the heterokaryon incompatibility genes) and are frequently barren, i . e . they produce abundant pe r i t h e c i a , but few 6 spores (Newmeyer and Taylor, 1967). A/a duplications escape from t h e i r i n h i b i t e d growth and s t a r t growing at wild-type or near wild-type rates by the somatic segregation of A from a through mitotic crossing over (Fig. 2) or deletion of one of the mating type genes (Newmeyer and Taylor, 1967). M i t o t i c crossing over y i e l d s a culture, barren due to the presence of duplicated genetic material, that i s a mixture of mostly A or a homozygous c e l l s . Such cultures are unstable and tend to be overgrown by one nuclear type. Deletion y i e l d s a culture, f e r t i l e due to the loss of part or a l l of the duplication, that i s a mixture of A or a hemizygous c e l l s . These cultures are also overgrown by one nuclear type (Newmeyer and Taylor, 1967). Mating and Incompatibility i n Other Fungi The majority of research on yeast mating type has been done on Saccharomyces cerevisiae, also known as budding yeast because haploid c e l l s reproduce vegetatively by budding. Mating begins with Gl a r r e s t . Pheromone from a c e l l s , a-factor, arrests alpha c e l l s , and pheromone from alpha c e l l s , alpha-factor, arrests a c e l l s . Pairs of opposite mating type c e l l s fuse, undergo karyogamy and then follow e i t h e r of 2 paths, depending on n u t r i t i o n a l conditions. The a/alpha d i p l o i d c e l l s reproduce m i t o t i c a l l y ' four+ £ o -• — •- r? ^ o o -r- CO / t o c o T 0. — » — , C * + + 3 £ co O C O 0 . • — • — » — »— #- n i c n Example from Newmeyer and Taylor (1967) of mitotic crossover that leads to production of cells homozygous for the mating Fif l l lTP 9 type genes. The duplication was a product from a cross to an l l y U I C c. inversion strain. 8 unless they are n u t r i t i o n a l l y deprived, i n which case they undergo meiosis (see review by Herskowitz, 1988). The mating types of the haploid c e l l s are s p e c i f i e d at the mating type locus, MAT (Lindegren and Lindegren, 1943), which codes f o r t r a n s c r i p t i o n factors that control the expression of genes involved i n pheromone production, mating and sporulation. C e l l s with mating type a have the MATa a l l e l e which encodes two polypeptides, al and a2; c e l l s with mating type alpha have the MATalpha a l l e l e which also encodes two polypeptides, alphal and alpha2. A portion of the MAT genes, c a l l e d Ya and Yalpha (Nasmyth, et a l . , 1981), i s s p e c i f i c to a and alpha c e l l s , respectively (Sprague, et a l . , 1981). Three of the mating type polypeptides, a l , alphal and alpha2, are involved i n the regulation of a - s p e c i f i c , alpha- specific and haploid c e l l - s p e c i f i c genes. The function of a2 i s unknown ( A s t e l l , et al., 1981). In a c e l l s , al i s produced (Kassir and Simchen, 1976) and a - s p e c i f i c and haploid s p e c i f i c genes are expressed. In alpha c e l l s , alphal induces the expression of alpha-specific genes (Sprague, et al., 1983) and alpha2 represses a - s p e c i f i c genes (Hartig, et 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 function as i t does i n haploid alpha c e l l s , repressing a- s p e c i f i c genes (Strathern, et a l . , 1981), but i t has an additional r o l e . A combination product of alpha2 and al 9 represses the expression of alphal, and of haploid s p e c i f i c genes and stimulates sporulation (Strathern, et al., 1981). Sporulation begins when a/alpha d i p l o i d c e l l s are starved of nitrogen and carbon (Esposito and Klapholz, 1981). The regulatory protein al/alpha2 activates meiosis by blocking the expression of RMEI, an i n h i b i t o r of meiosis (Mitchell and Herskowitz, 1986). Strains of S. cerevisiae with the dominant a l l e l e of the homothallism gene, HO, are homothallic, whereas st r a i n s with the recessive 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 catalyses high frequency interconversion of mating types (Hicks and Herskowitz, 1976). Mating type interconversion occurs by the switching of the genetic information at the MAT locus (Nasmyth and T a t c h e l l , 1980; Strathern, et al., 1980; Hicks, et al., 1977). The information comes from two t r a n s c r i p t i o n a l l y s i l e n t genes, HMR and HML, that flank the mating type gene. Each locus contains a copy of a or alpha. The two l o c i are kept s i l e n t by the action of unlinked genes c a l l e d SIR (for " s i l e n t information regulator") (Abraham, et a l . , 1983). The switching process begins with a double-stranded cut at MAT (Strathern, et a l . , 1982) by the endonuclease encoded by HO (Kostriken and Heffron, 1984). A conversion-like event follows, i n which heteroduplex DNA forms between the donor locus (HMR or HML) and MAT. The heteroduplex DNA i s repaired using the donor DNA as a template (Klar and Strathern, 1984; Klar et a l . , 1984). Switching i s prevented 10 i n d i p l o i d c e l l s by the repression of HO (Jensen, et al., 1983). Mating i n the f i s s i o n yeast, Schizosaccharomyces pombe, i s s i m i l a r i n some ways to that i n Saccharomyces cerevisiae. Haploid c e l l s have the mating type h+ or h~ and propagate vegetatively by f i s s i o n , not by budding. During mating, which occurs under nitrogen starvation conditions, one c e l l of each mating type p a r t i c i p a t e s i n the formation of a d i p l o i d zygote. The zygote immediately undergoes meiosis and sporulation (Leupold, 1950 c i t e d i n Kel l y , et a l . , 1988). As i n homothallic s t r a i n s of S. cerevisiae, S. pombe regularly switches a l l e l e s at the mating type locus (Egel, 1977; Miyata and Miyata, 1981). The mating type complex contains 3 regions—matl, mat2-P and mat3-M—which control conjugation, meiosis and sporulation (Kelly, et a l . , 1988). Two s i l e n t l o c i , mat2-P and mat3-M donate information to matl which confers mating type, either h+ (matl-P) or h~ (matl-M) (Egel, 1977; Egel and Gutz, 1981; Beach, 1983). The complex has been sequenced (Kelly, et a l . , 1988). Two genes are encoded by each of matl-P and matl-M, two of which are required for conjugation and s p e c i f i c a t i o n of mating type, and a l l 4 of which are required f o r meiosis and sporulation (Kelly, et a l . , 1988). Each of matl, mat2-P and mat3-M contains 2 blocks of sequence homology. The s i l e n t genes, mat2-P and mat3-M, alone contain a t h i r d region of 11 homology which probably acts as a s i l e n c e r (Kelly, et al., 1988). Like RMEI of S. cerevisiae, the protein, rani, of S. pombe i s an i n h i b i t o r of meiosis. I t s action i s blocked by the protein, mei3, which i s produced i n h +/h~ c e l l s (McLeod and Beach, 1988). Neurospora crassa could be l i k e S. cerevisiae i n that the mating type genes could act l i k e al/alpha2, combining to form a t r a n s c r i p t i o n factor that blocks the synthesis of a meiosis i n h i b i t o r , or i t could be l i k e S. pombe i n that a product analogous to mei3 could be produced i n A/a mating d i p l o i d c e l l s . The complex system of regulating c e l l type i n S. cerevisiae must d i f f e r i n N. crassa because each mating type idiomorph encodes but one tr a n s c r i p t . That the combination product, A/a, could act as a tr a n s c r i p t i o n factor i s supported by the sequence s i m i l a r i t y between the a idiomorph and the HMG box motif, a DNA-binding sequence (Staben and Yanofsky, 1990). The A idiomorph has no obvious DNA-binding motif, but i t does have s i m i l a r i t y to the MATalphal protein of S. cerevisiae and could inte r a c t with the a product during mating. During the vegetative state, A i d e n t i t y could be s p e c i f i e d by A - s p e c i f i c genes, turned on by the A product in t e r a c t i n g with a t r a n s c r i p t i o n factor that binds to t h e i r promoter regions (Glass, et a l . , 1990). Strains containing a heterozygous duplication of the mating type region do not lose either t h e i r A or a id e n t i t y , 12 so the combination product does not exclude mating type s p e c i f i c i t y . Homothallism i n W. crassa must also occur by a d i f f e r e n t mechanism than switching because each s t r a i n i n the homothallic species N. dodgei, IV. galapagonensis, N. africana and W. lineolata has only one copy of a sequence homologous to the N. crassa mating type gene, A. The homothallic species, N. terricola, has sequences homologous to the N. crassa mating type genes, A and a, but only one copy of each (Glass, et a l . , 1988). The incompatibility systems, also c a l l e d breeding systems, of the 2 basidiomycetes, Coprinus cinereus and Schizophyllum commune, are tetrapolar. The mating type complex i s comprised of 2 regions, A and B, each containing 2 genes, alpha and beta, with multiple a l l e l e s . Mating requires that the 2 participants d i f f e r at a minimum of one A gene and one B gene. Close linkage of alpha and beta r e s t r i c t s inbreeding pote n t i a l by i n h i b i t i n g recombination (see K o l t i n , et a l . , 1972). Cloning of the A factor of C. cinereus has revealed that the alpha and beta regions are themselves composed of a number of genes with multiple a l l e l e s , some of which are common to other a l l e l e s of A and some of which are unique (E. Mutasa, A. Tymon, W. Richardson, U. Kues and L. Casselton, 1991 i n published abstracts from Sixteenth Fungal Genetics Conference). 13 Three a l l e l e s of the A-alpha region of S. commune have been cloned, sequenced and shown to contain multiple t r a n s c r i p t s , some shared and some unique. Some of the postulated polypeptides contain homeodomain motifs (R.C. U l l r i c h , M.M. Stankis, H. Yang and C P . Novotny, 1991; G. May, 1991 i n published abstracts from Sixteenth Fungal Genetics Conference), implying that the products regulate the expression of other genes. In the pathogenic basidiomycete, Ustilago maydis, dikaryosis between indivi d u a l s from d i f f e r e n t incompatibility groups i s a prerequisite f o r pathogenic i n f e c t i o n . Different a a l l e l e s are required f o r fusion 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 of a have been cloned and they encode a product required f o r mycelial growth, a condition necessary f o r i n f e c t i o n (M. Bolker and R. Kahmann, 1991 i n published abstracts from Sixteenth Fungal Genetics Conference). Ten b a l l e l e s have been cloned, and subsequent molecular analysis has revealed that they share a homeodomain-related motif, implying that the b polypeptides bind DNA, possibly to regulate sexual development (R. Kahmann, B. G i l l i s s e n , R. Schleshinger, C Sandmann, F. Schauwecker, J . Bergemann, B. Schroeer, M. Bolker and M. Dahl, 1991 i n published abstracts from Sixteenth Fungal Genetics Conference). Like the A and B regions of S. commune and C. cinereus, the b region of U. maydis i s composed of 2 genes, b-east and Jb-west. Null mutants of the b region are mating d e f i c i e n t , suggesting that the postulated b heterodimer formed during mating i s an activator of mating genes (Kahmann, personal communication) Studies done with expression of two of the b a l l e l e s have i d e n t i f i e d a 70 amino acid region responsible for a l l e l e s p e c i f i c i t y . 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 expressed i n d i p l o i d s and haploids, although at a lower l e v e l i n the l a t t e r (L. Giasson, A. Yee and J.W. Kronstad, 1991 i n published abstracts from Sixteenth Fungal Genetics Conference). S i m i l a r l y , i n W. crassa the mating type genes are expressed during sexual and vegetative phases of the l i f e cycle, but at a lower l e v e l i n the l a t t e r (Staben and Yanofsky, 1990; Glass, et a l . , 1990). N. crassa d i f f e r s from the basidiomycetes i n several ways. The mating type genes of W. crassa have only one ORF so unlike the basidiomycetes, they do not function as r e s t r i c t o r s of inbreeding. Also, i n N. crassa, vegetative compatibility i s not a prerequisite f o r mating. Fusion of hyphae with compatible genotypes leads to heterokaryon formation and fusion of sexual c e l l s with compatible genotypes leads to mating. The heterokaryosis that occurs between vegetative c e l l s i s somehow d i f f e r e n t from the fusion that occurs between the trichogyne and the male c e l l Mixed mating type heterokaryons on crossing medium do not exhibit the incompatibility phenotype, so perhaps i t i s the n u t r i t i o n a l conditions that d i c t a t e whether the mating type genes w i l l i n i t i a t e incompatibility or mating. The mating type gene of the yeasts i s a master regulator of other genes. The mating type genes of the basidiomycetes and of N. crassa are also postulated to be regulatory, although the mechanisms of t h e i r actions must d i f f e r . Each of the four major groups of fungi—phycomycetes, basidiomycetes, ascomycetes and fungi i m p e r f e c t i — e x h i b i t s g e n e t i c a l l y determined incompatibility (Burnett, 1976). In some cases, e.g. Neurospora crassa (Fincham, et a l . , 1979), Podospora anserina, under the control of the s locus (Esser 1971), Rhizoctonia solani (Burnett, 1976) and Endothia parasitica (chestnut b l i g h t fungus) (Anagnostakis, 1977), the incompatibility only a f f e c t s vegetative heterokaryosis; whereas i n others, e.g. Aspergillus nidulans (Jinks, et a l . 1961; Grindle, 1963a; 1963b), Podospora anserina, under the control of the a,Jb,c,v l o c i or n o n - a l l e l i c system (Esser, 1971; Blaich and Esser, 1971), Coprinus lagopus, Schizophyllum commune (Fincham et a l . , 1979), Ustilago sp. (Fincham et al., 1979; Day and Cummins, 1981) and yeasts (Crandall, 1978), the genetic r e s t r i c t i o n s on fusion a f f e c t f e r t i l i t y . Two species of fungus other than N. crassa that have mating type genes that act as heterokaryon incompatibility l o c i are Ascobolus stercorarius ( B i s t i s , personal communication) and Aspergillus heterothallicus (Kwon and Raper, 1967). 16 Mating and Incompatibility i n Other Kingdoms Each of the f i v e kingdoms of organisms—Monera, P r o t i s t a , Fungi, plants and animals—shows examples of gene t i c a l l y controlled, i n t r a s p e c i f i c , sexual or somatic incompatibility. Moreover, interactions between species, f o r example, those between host and pathogen or between symbionts, can exhibit incompatibility. Sexual incompatibility l i m i t s mating between c e r t a i n i n d i v i d u a l s within a species. Somatic incompatibility l i m i t s fusion between or co-existence of ce r t a i n c e l l s . Some of the fungi demonstrate incompatible reactions which are both somatic and sexual, i n that a successful i n t e r a c t i o n between seemingly vegetative c e l l s leads to mating. In the kingdom Monera, mating i n Escherichia coli requires recognition of mating types; and only pairings between c e l l s of d i f f e r e n t mating types are compatible (Hayes, 1952; Lederberg, 1957). C i l i a t e s , i n the kingdom P r o t i s t a , generally mate with c e l l s of a d i f f e r e n t mating type and fusion occurs between "vegetative" c e l l s (there are 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 physiological conditions (Nanney, 1977; R i c c i , 1981). In plants, there are examples of both sexual and somatic incompatibility. Some angiosperms have the S locus which controls sexual compatibility, allowing pollen to 17 f e r t i l i z e only females with d i f f e r e n t S a l l e l e s (Lewis, 1954; Ebert et a l . , 1989; Haring et al., 1990). Somatic incompatibility occurs between d i f f e r e n t species of plants when tissue from one species i s grafted onto an i n d i v i d u a l of another species, even from the same family (Yeoman et a l . , 1978). Fusion of c e l l s i n the sexual cycle of Chlamydomonas sp. requires recognition of opposite mating types (Wiese and Wiese, 1978; Harris, 1989) which are determined by two a l l e l e s , mt+ and Bit"*, at one locus. The a l l e l e s are believed to encode proteins that control the expression of other genes or the a c t i v i t y of t h e i r products, possibly by forming or causing the formation of a novel regulatory product upon c e l l fusion ( 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 vertebrates when a g r a f t of t i s s u e or an organ transplant i s rejected from the r e c i p i e n t . The reaction, i n t h i s case, i s co n t r o l l e d by genes of the major histocompatibility complex (MHC), which are expressed i n the T c e l l s of the immune system (see reviews by Kl e i n , 1976; Bach and vanRood, 1976). Humoral recognition occurs i n the blood, and i t i s governed by antibodies s p e c i f i c f o r the ABO blood group antigens and the Rh factor protein (see review by Katz, 1978). 18 INTRODUCTION 1 A Suppressor of A/a Incompatibility, t o l Newmeyer (1970) found a recessive suppressor of A/a incompatibility, unlinked to mt, which she c a l l e d t o l for "tolerant". The new gene had no demonstrable e f f e c t on the a b i l i t y of a s t r a i n to mate. I t appears to be inactive during starvation because A + a heterokaryons on crossing medium do not exhib i t the incompatibility phenotype. However, Johnson (1979) suggested that the gene does have a rol e during mating because the recessive a l l e l e , tol, suppresses fmf-1, a gene specifying female and male f e r t i l i t y . Crosses between fmf-1, tol+ and fmf-l+, tol+ are s t e r i l e , whereas crosses between fmf-1, tol and fmf-l+, tol are f e r t i l e . Johnson hypothesized that t o l permits promiscuous fusion between A and a during mating, allowing transfer of fmf-l+ product from the fmf-l+ s t r a i n , r e s u l t i n g i n rescue of the s t e r i l i t y phenotype. I f t o l + i s expressed during the sexual cycle, fusion of sexual structures occurs despite the presence of t o l + . I t i s possible that the cytoplasm of trichogynes i s modified to allow the presence of male nuclei of the opposite mating types. Genes that suppress A/a incompatibility w i l l be useful i n analysing the mating type gene i t s e l f and i n deciphering the process of incompatibility. Moreover, i f a suppressor has i t s own phenotype, i n addition to suppressing A/a 19 incompatibility, then other functions related to incompatibility may be revealed. For example, i f a suppressor prevents incompatibility by a l t e r i n g c e l l wall structure so that i t can no longer be broken down by the incompatibility reaction, i t may produce a second phenotype of abnormal morphology. Two types of suppressors, extragenic and intragenic, are discussed below. Extragenic suppressors probably i n t e r a c t with the mating type genes; and t h e i r detection w i l l help i n the dissect i o n of the mechanism of incompatibility. Theories as to how t o l a f f e c t s incompatibility are considered i n Discussion 1. Other suppressors, besides t o l , have been i d e n t i f i e d — two found i n nature and two induced i n the laboratory. Smith and Perkins (1972) noted that the osmotic-sensitive, r e c i p r o c a l translocation s t r a i n , cut, suppressed A/a incompatibility. Newmeyer (1970) reported that the wild type s t r a i n , Panama a, (Fungal Genetics Stock Center #1132) segregated compatible and incompatible progeny when crossed to a duplication-generating inversion A t e s t e r . I t i s unknown whether or not t h i s suppressor i s a l l e l i c with t o l . Newmeyer (1970) found another suppressor, which may or may not be a l l e l i c with t o l , i n an escaped A/a duplication s t r a i n ( c a l l e d N83) from a cross between an inversion A s t r a i n and a normal sequence a s t r a i n . DeLange and G r i f f i t h s (1975) reported that 2 of t h e i r escaped mixed mating type heterokaryons, i n which one component was tol 20 and the other tol+, produced perithecia with both mating type t e s t e r s , but produced ascospores with only 1. They suggested that these 2 st r a i n s arose by deletion or l e t h a l mutation at tol+. Intragenic suppressors and mutants of the mating type genes w i l l help, when mapped and sequenced, to reveal the part(s) of the mating type genes governing vegetative incompatibility. E s p e c i a l l y useful w i l l be those mutants i n which only one of the mating type functions i s defective, e.g. the f e r t i l e , heterokaryon compatible mutant, am33. When Newmeyer discovered tol, she was using s t r a i n s of N. crassa with A/a duplications, dark agars. They were meiotic segregants from a cross of a normal sequence A to a s t r a i n of mating type a carrying a p e r i c e n t r i c inversion of a large portion of linkage group I (L.G. I) (Fig. 2). Most of the dark agars escaped from t h e i r i n h i b i t e d growth by the somatic segregation of A from a. One s t r a i n , however, escaped by mutation at tol, thereby allowing both mating type genes to reside within one nucleus without causing vegetative incompatibility. The new tol gene proved to be a suppressor, not only of nuclear incompatibility, but also of heterokaryon incompatibility, allowing the vigorous growth of both duplicated A/a,tol s t r a i n s and A,tol + a,tol heterokaryons. Newmeyer favoured the use of a A/a duplication to cause i n h i b i t e d growth because i t eliminated the p o s s i b i l i t y that incompatibility was due to a l l e l e s at some other 21 heterokaryon incompatibility locus. Furthermore, i f a mixed mating type heterokaryon had been used, only "dominant" a l l e l e s of suppressor genes would have allowed escape to occur. A method s i m i l a r to that used by Newmeyer (1970) has been used to generate novel suppressors of A/a incompatibility. The search i s an attempt to chart the genetic interactions involved i n mating type-associated i ncompat i b i 1 i t y . 22 MATERIALS AND METHODS General protocols were standard, and are described i n Davis and DeSerres (1970). Crosses were made at 25°C i n 15 cm t e s t tubes or, i f fl females were used, on p e t r i plates. Ascospores with A/a duplications were selected as meiotic segregants from crosses to st r a i n s i n which the mating type gene had been translocated from L.G. I onto L.G. II . Duplication progeny contained L.G. II from the translocation parent and L.G. I from the other parent. This combination of linkage groups was selected as follows. The mating type genes were marked, one with an auxotrophic marker (ser-3) and the other with a temperature s e n s i t i v e marker (un-3), and ascospores were plated on minimal medium at 32°C which permitted s u r v i v a l of only the duplication spores. Strains and Markers A l i s t of st r a i n s and t h e i r sources i s shown i n Table 1. Strains were maintained at room temperature on standard media. The marker, un-3, i s temperature-sensitive, with st r a i n s growing poorly between 28.5°C and 30°C and not growing at a l l at temperatures above 30°C. I t i s located 0.04 to 0.1 map units to the l e f t of the mating type gene (Perkins, et a l . , 1982). The marker contains two mutations, Table 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->ll)39311,ser,A T(l->ll)39311, ser -3 , A C.J. Myers T(l->ll)39311,ser,a T(l->ll)39311, ser -3 , a C.J. Myers T(l->ll)39311,ser,trp,tol,A T(l->ll)39311, ser -3 , trp-4, tol, A C.J. Myers T(l->H)39311,ser,trp,tol,a T(l->ll)39311, ser -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 •F.G.S.C.-Fungal Genetics Stock Center 24 separated by 0.1 map units, one i n cytochrome-20 and the other i n ethionine-2 (A.M. Lambowitz, unpublished r e s u l t s , G r i f f i t h s , personal communication). Tests f o r un-3 were done on minimal medium at 25°C and at 37°C and were repeated 3 times each for 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 higher temperature (37°C) could be used than was used to sel e c t duplication progeny (32°C) because the un-3 phenotype i s more obvious at 37°C and i t was not necessary to avoid k i l l i n g the fungi. The translocation T(I->II) 39311 i s an i n s e r t i o n of an i n t e r s t i t i a l segment of the l e f t arm of linkage group I (including ser-3, un-3 and mt) into the r i g h t arm of linkage group I I , inverted with respect to the centromere (Perkins, 1972). The gene, ser-3, s p e c i f i e s a requirement f o r serine and i s located fewer than 2 map units to the l e f t of the mating type gene (Perkins, et 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 linked to t o l by less than 1 map unit (Perkins, et a l . , 1982). The aconidial mutant, fluffy ( f l ) , i s highly f e r t i l e and i s used as a mating type t e s t e r . Ascospore Is o l a t i o n Ascospores were i s o l a t e d as follows, unless stated otherwise. Spores were c o l l e c t e d from the sides of the 25 crossing t e s t tubes with a wire loopful of s t e r i l e d i s t i l l e d water and put into eppendorf tubes of s t e r i l e d i s t i l l e d water or 0.1% agar. Hemocytometer counts were done to determine the necessary d i l u t i o n factor that would y i e l d approximately 10-30 spores per plate. Diluted spores (1/4 mL) were pipetted and spread onto sorbose plates with a bent glass rod. The plates were placed into a 60°C oven f o r 30 minutes to heat shock the spores to i n i t i a t e germination. Spores were l e f t at room temperature f o r 4-5 hours to ensure that the mycelia were well established p r i o r to s e l e c t i o n . The plates were l e f t overnight i n a 32°C incubator so that the colonies grew large enough to be seen and harvested. Individual germinated spores were c o l l e c t e d by cutting out a square of agar containing the mycelium and placing i t into a t e s t tube containing 1 mL of Westergaard and Mitc h e l l ' s l i q u i d medium. I f , a f t e r several 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 to maintain the l e v e l of l i q u i d at 1 mL. Construction of Tester S t r a i n (T(I->II) 39311, ser, trp, tol, a) The s t r a i n , T(I->II) 39311, ser, trp, t o l , a was made from a cross of a female trp, t o l , A to a male T(I->II) 39311, ser, a, from which ser, trp, t o l , a progeny were selected (Fig. 3). The female was inoculated onto synthetic 26 T(l->ll)39311,ser,a x trp,tol,A ser a II trp tol IV IV select X II trp tol IV IV dead ser a II trp tol IV IV A/a II trp tol IV IV Construction of the strain T(l->ll)39311,ser,trp,toi,a. The strain, marked X, was a product of the cross between T(l->ll)39311,ser,a x trp,tol,A 27 crossing medium containing 10% of the normal concentration of tryptophan ( i . e . 20 mg/L instead of 200 mg/L) because the cross was s t e r i l e on the normal concentration of tryptophan. Presumably the tryptophan was providing too much nitrogen for the sexual cycle to be i n i t i a t e d (Myers, personal communication). 28 RESULTS 1 An o v e r a l l scheme of the procedure used i n generating the suppressors i s shown i n F i g . 4. Duplication st r a i n s (Fl) were selected as meiotic segregants from a set of crosses (referred to as the " f i r s t cross") between a normal chromosomal sequence parent and a translocation parent. The duplication s t r a i n s escaped from i n h i b i t e d growth (escaped Fl) and were tested f o r t h e i r mating types. Those which retained both mating types (A/a escaped F l ) possibly contained the desired suppressors and were crossed to normal chromosomal sequence fl s t r a i n s to remove the translocation chromosome. This set of crosses i s c a l l e d the "second cross". Some of these F2 progeny presumably contained suppressors. Only the temperature-sensitive F2 st r a i n s were tested f o r the presence of suppressors because the un-3 marker was needed to s e l e c t duplication progeny i n the t h i r d set of crosses. The temperature-sensitive F2 st r a i n s were crossed to te s t e r s t r a i n s containing the translocation T(I->II) 39311. Duplication progeny were selected and assessed for t h e i r compatible/incompatible phenotypes. Detailed descriptions of each step follow. Duplication s t r a i n s ( l a b e l l e d " F l " i n F i g . 4) were created (Perkins, 1972) from a pair of crosses, r e c i p r o c a l i n the sense that the mating types were reversed. This set of crosses i s referred to as the " f i r s t cross" and i s 29 First Cross (to generate A/a duplications): R601,un-3 x T(l->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->ll)39311,ser,trp,tol minimal medium, 32 degree t to select duplications F3 (t.s. F2 strains that produced incompatible and compatible duplication F3 progeny were the suppressor strains) —. . A summary of the selection protocol for suppressors. The l l C j U r e 4 reciprocal cross was R602 x T(l->ll)39311,ser,A. 30 described i n the following three paragraphs. One cross i n the 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 containing a translocation (T(I->II) 39311, ser, a). In the rec i p r o c a l cross (not shown), the normal chromosomal sequence female parent was R602 and the male translocation parent was T(I->II) 39311, ser, A. The normal sequence parents, R601 and R602 had t h e i r mating type genes, A and a, respectively, marked with the temperature-sensitive gene, un-3, which i s less than 1 map unit to the l e f t of mt. The translocation s t r a i n s had t h e i r mating type genes marked with the auxotrophic gene, ser-3, which i s less than 2 map units to the l e f t of mt. The mating type genes were marked to allow s e l e c t i o n of progeny that contained both A and a. The germination of spores on minimal medium at 32°C selected progeny that contained L.G. I from the normal parent (s e r - 3 + , un-3, A or ser-3+, un-3, a) and L.G. II from the translocation parent (ser-3, un-3+, a or ser-3, un-3+, A). The genotype of the selected spores was ser-3 + , un-3, A/ser-3, un-3+, a or, from the rec i p r o c a l cross, ser-3+, un-3, a/ser-3, un-3+, A. Since the nuclei of the selected F l s t r a i n s contained both mating types, growth was in h i b i t e d and the cultures grew with the dark agar morphology of short hyphae growing i n a t i g h t knot (Perkins, 1972). Figure 5 depicts only one possible p a i r i n g — t h a t of the two L.G. I's. Although i t i s not shown i n the figu r e , the 31 R601 T(l->ll)39311,ser,a minimal medium, 32 degrees to select duplications II DIES S e r - 3 + Un~3 A ^ f (temperature-sensitive) se r -3 un-3+ a se r -3 un-3+ a II LIVES (A/a duplication) DIES II (deletion) II DIES (serine-requiring) Figure 5 The first set of crosses was done to generate A/a duplication strains. The crosses were R601 x T(l->ll)39311,ser,a and x T(l->ll)39311,ser,A. 32 translocated portion of L.G. I i s long enough to pair with L.G. I (Metzenberg, personal communication). As explained i n the following paragraph, the occurrence of t h i s p a i r i n g pattern would not have affected the experimental design. If the translocated section of L.G. I had paired with the i n t a c t L.G. I and a single crossover had occurred i n the paired region, the products would not have survived because of the formation of d i c e n t r i c and acentric chromosomes. If a double crossover had occurred i n the paired region, the surviving progeny would only have had one mating type, and therefore would have grown as wild type. These types of spores would not have been chosen as one of the A/a escaped FI s t r a i n s . Spore cultures with standard incompatibility and compatibility phenotypes were needed as controls for comparison to the duplication progeny. Such s t r a i n s were obtained as progeny from the cross between the normal chromosomal sequence female, R601 (un-3, A), and the translocation male, T(I->II) 39311, ser, trp, t o l , a. Spores were germinated and grown on medium containing tryptophan at 32°C. These conditions selected progeny containing L.G. I (ser-3 +, un-3, A) from the female parent and L.G. II (ser-3, un-3+, a) from the male parent. Half of the progeny, those containing L.G. IV (trp-4+, tol+) from the female, were incompatible duplications, and h a l f , those containing L.G. IV (trp-4, tol) from the male were compatible duplications. 33 From the duplication-generating crosses, R601 x T(I->II) 39311, ser, a and R602 x T(I->II) 39311, ser, A, 182 duplication ascospores were c o l l e c t e d and maintained on l i q u i d medium. Each duplication culture exhibited the same phenotype as that shown by the A/a, tol+ incompatible controls, growing as a small dense mass of hyphae at the bottom of the t e s t tube. The A/a, tol compatible controls grew fa s t e r , f i l l i n g the t e s t tube a f t e r several days. A l l of the spore cultures escaped from incompatibility within 2 weeks, although escape occurred at d i f f e r e n t times f o r each s t r a i n . Escape was detected as a s h i f t i n hyphal morphology from the dense growth to less dense growth and by an increase i n growth rate. In order to eliminate s t r a i n s that had escaped by deletion of mating type genes and to i d e n t i f y double mating type s t r a i n s that may have escaped because of mutation to tol or to a t o l - l i k e suppressor (A/a escaped F l s t r a i n s ) , the mating type of each escaped F l s t r a i n was tested by spotting i t on protoperithecial lawns of fl,A and fl,a t e s t e r s . The crossing behaviours of 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 into 9 phenotypic classes, 8 of which are described i n F i g . 6. The one class not shown i n the figure was comprised of 25 s t r a i n s that retained the capacity to cross and produce ascospores with testers of both mating types. These 25 s t r a i n s ( l a b e l l e d "A/a escaped F l " i n F i g . 4) were the ones presumed to contain REACTION 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. 35 suppressors. The remaining 159 s t r a i n s were not used i n the res t of the experiment, but t h e i r possible o r i g i n s are considered i n Discussion 1. The next set of crosses (referred to as the "second cross") was done to demonstrate that the 25 A/a escaped F l st 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 occurred by deletion of either of the mating type genes, but i t may have occurred by mitotic crossover or by some as yet u n i d e n t i f i e d mechanism other than mutation or deletion at a new suppressor locus. At the same time, the second cross served to get the suppressors into a stable background, one without duplications. The translocation chromosome, L.G. I I , was removed by crossing each A/a escaped F l culture to f l , a and f l , A females (Fig. 7). Crosses involving a duplication s t r a i n are frequently "barren", i . e . they produce few ascospores (Newmeyer and Taylor, 1967). Some of the crosses of the A/a escaped F l s t r a i n s to fl females produced as few as 1-6 ascospores. Spores from these crosses 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 through the procedure. Instead, they were c o l l e c t e d i n d i v i d u a l l y with a tungsten needle under the microscope and placed 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°C water bath to i n i t i a t e germination of the spores, then l e f t at room temperature. The t e s t tubes were checked d a i l y f o r the appearance of growth and the r e s u l t s are shown Temperature-sensitive II NO ser-3+ un-3 A I se r -3 un-3+ a -Some compatible -Some incompatible YES II ser-3+ un-3+ a se r -3 un-3+ a NO -Some compatible -Some incompatible II 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 FlGlJTP 7 needed as a marker in the third cross and to ensure that the i v ^ u i c ; l translocation chromosome was eliminated. 37 i n Table 2. Despite careful c o l l e c t i o n of spores, there were 10 crosses from which no spores could be c o l l e c t e d . Progeny from these crosses ( l a b e l l e d MF2" i n F i g . 4) were either incompatible duplication, compatible duplication or single mating type s t r a i n s . The compatible and single mating type progeny were subcultured onto minimal medium slants so that s u f f i c i e n t mycelia could be grown f o r use i n subsequent t e s t s . Incompatible progeny were discarded for two reasons—they were duplications and they did not contain suppressors. Compatible and single mating type F2 s t r a i n s were tested for un-3 because the marker was needed i n the next cross. Furthermore, non-duplication progeny were needed for the next cross, and so compatible duplications had to be eliminated. They were discarded with the res t of the non- temperature-sensitive s t r a i n s . Only 18 F2 progeny out of 329 tested were temperature-sensitive. Possible reasons for the dearth of temperature-sensitive F2 st r a i n s are presented i n Discussion 1. The 18 temperature-sensitive F2 st r a i n s were crossed to the t e s t e r s t r a i n , T(I->II) 39311, ser, trp, tol, to confirm the presence of suppressors i n the former. The temperature- s e n s i t i v e F2 progeny may not have contained suppressors for a number of reasons. F i r s t l y , i n the second cross, L.G.II(T I->II) was removed, so i f the mutation had occurred on t h i s chromosome, and had not recombined onto the homolog, i t would be l o s t . 38 Table 2 Phenotypes of F2 Strains STRAIN NUMBER OF F2 STRAINS WITH THE PHENOTYPE NORMAL (INCLUDES COMPATIBLE) INCOMPATIBLE BLANK A 1 - 2 9 4 2 2 5 a 1 - 2 9 3 0 10 A 1 - 5 4 0 0 4 a 1 - 5 4 19 8 3 9 A 1 - 5 8 1 0 8 a 1 - 5 8 13 2 2 5 A 1 - 5 9 2 0 4 a 1 - 5 9 2 0 2 18 A 1 - 6 5 0 0 1 a 1 - 6 5 15 4 21 A 1 - 7 3 12 1 12 a 1 - 7 3 2 4 19 A 1 - 7 5 2 0 5 a 1 - 7 5 5 1 2 0 A 1 - 1 0 3 0 0 0 a 1 - 1 0 3 17 14 0 A 1 - 1 0 4 0 0 0 a 1 - 1 0 4 17 0 2 3 A 1 - 1 0 7 0 0 2 a1 -107* 1 1 0 2 1 A1-113 1 2 9 a1-113* 14 0 3 6 A 1 - 1 2 8 0 0 0 a 1 - 1 2 8 * 19 8 4 0 A 2 - 1 8 0 0 3 a 2 - 1 8 2 1 6 14 A 2 - 2 0 1 0 1 3 a 2 - 2 0 9 2 8 A 2 - 3 2 3 0 10 a 2 - 3 2 14 4 15 A 2 - 3 7 3 2 2 3 a 2 - 3 7 3 3 19 ..continued Table 2 continued 39 STRAIN NUMBER OF F2 STRAINS WITH THE PHENOTYPE NORMAL ( INCLUDES COMPATIBLE) INCOMPATIBLE BLANK A 2 - 5 7 0 0 7 a 2 - 5 7 1 1 8 A 2 - 7 9 0 0 0 a 2 - 7 9 0 0 0 A 2 - 8 6 * 11 1 0 a 2 - 8 6 8 0 0 A 2 - 9 5 0 0 0 a 2 - 9 5 0 0 0 A 2 - 1 1 0 0 0 0 a 2 - 1 1 0 0 0 0 A 2 - 1 1 7 0 0 0 a2 -117 3 5 2 2 3 A 2 - 1 4 6 2 2 16 a 2 - 1 4 6 5 1 5 A 2 - 1 5 5 * 7 2 4 1 a 2 - 1 5 5 19 4 2 7 A 2 - 1 6 2 0 0 5 a 2 - 1 6 2 10 2 3 17 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 second cross 1 or 2 • c r o s s e d to R601 or R602 in the first cross final # » isolate # from first cross •s t ra ins that had temperature-sensi t ive progeny 40 In t h i s case, a l l of the duplication progeny from the crosses of temperature-sensitive F2 s t r a i n s to T(I->II) 39311, ser, trp, tol would be incompatible. Secondly, because of segregation, the temperature- s e n s i t i v e F2 progeny may have contained the chromosome homologous to the one with the suppressor mutation. I picked as many ascospores as possible from the crosses of A/a escaped FI to fl females, but, as previously mentioned, some of the crosses produced very few. F i n a l l y , i f the o r i g i n a l escape event had been due to mitotic segregation, there would be no suppressor to be found. Newmeyer and Taylor (1967) did report that t h e i r A/a escaped s t r a i n s were heterokaryons of pure A and pure a nuc l e i , suggesting that somatic segregation had occurred. In the system they used, there were no s e l e c t i v e markers close to mt that would prevent the sur v i v a l of mitotic crossover or mt deletion products. Figure 8 shows examples of mitotic crossovers and t h e i r products, some of which survive the se l e c t i o n conditions. If a temperature-sensitive F2 s t r a i n contained a suppressor at a locus other than tol or mt, then the duplication progeny (F3) from the cross to T(I->II) 39311, ser, trp, tol would be of two types—compatible or incompatible. If the new mutation had occurred i n the tol gene or i n the region of the mating type gene c o n t r o l l i n g vegetative incompatibility, then a l l of the duplication progeny would be compatible (Fig. 9). POSSIBLE CROSSOVERS MITOTIC PRODUCTS ser+ un A f X ser un+ a ser+ un A f X ser un+ a ser+ un A ser un+ a ser+ un A ser un+ A ser+ un a ser un+ a ser+ un A ser un A ser+ un+ a ser un+ a ser+ un A ser+ un A ser un+ a ser un+ a Figure 8 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.) 42 + un-3 A I It IV 1/2 "suppressors" it I ser + a trp tol | V IV " \ t r p tol | V + un-3 A ser + a IV IV P r o g e n y s u r v i v i n g o n m i n i m a l m e d i u m at 3 2 d e g r e e s IV " \ t r p tol | V 4f ser Figure 9 IV IV 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->ll)39311,ser,trp,tol,a and R602-derived suppressors x T(l->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. 43 Ascospores from the crosses of temperature-sensitive F2 s t r a i n s to T(I->II) 39311, ser, trp, tol were selected as described i n Materials and Methods. Table 3 shows the phenotypes of the F3 progeny from each of the 18 crosses. Seven s t r a i n s segregated compatible and incompatible progeny. These were the suppressor-containing F2 s t r a i n s . Ten s t r a i n s segregated only incompatible progeny. These st r a i n s may have contained the chromosome homologous to the one with the suppressor. I t i s u n l i k e l y , however, that a l l 6 of the F2 progeny derived from al-128 would have received the chromosome homologous to the one with the suppressor. There are two additional (and more l i k e l y ) explanations for the lack of suppression i n these s i x s t r a i n s . The o r i g i n a l A/a duplication may have escaped by mitotic segregation or by mutation/deletion at a suppressor locus on L.G. I I . Since there were no temperature-sensitive F2 s t r a i n s that segregated a l l compatible progeny, none of the o r i g i n a l escape events could have been due to mutation at either the mating type genes or at tol. One of the s t r a i n s , al-128-27, although crossed on several occasions to T(I->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 could have been unique to t h i s cross because the s t r a i n was able to induce the production of perithecia when crossed to a female f l , a t e s t e r . A sample of the compatible and incompatible F3 st r a i n s Table 3 Phenotypes of F3 Strains F2 STRAIN NUMBER OF F3 STRAINS WITH THE PHENOTYPE COMPATIBLE INCOMPATIBLE a1-113-7 0 31 a1-113-8 24 22 a1-113-9 0 6 a1-113-10 0 46 A2-155-2 7 18 A2-155-3 14 51 A2-155-5 18 28 A2 -86 -1 0 30 A 2 - 8 6 - 4 0 36 A2-86-12 9 21 a1-107-9 19 18 a1-107-11 28 12 a1-128-22 0 32 a1-128-23 0 32 a1-128-24 0 25 a1-128-25 0 35 a1-128-26 0 32 a1-128-27 0 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 second cross 1 or 2 - c rossed to R601 or R602 in the first cross middle # • isolate # from first cross last # - isolate # from second cross 45 was tested f o r mating type (Table 4). Almost a l l of the compatible F3 s t r a i n s (65 of 66) contained both mating types. This i s the class of progeny that show the existence of suppressors. I t i s u n l i k e l y that they grew well due to escape because of the high proportion of A/a s t r a i n s . Furthermore, the hyphae of escaped s t r a i n s are wispy, whereas these 65 compatible str a i n s grew with a dense morphology. One of the apparently compatible F3 s t r a i n s (one of the progeny s t r a i n s from the R601-derived parent, al-113-8) contained only A. Although escape usually does not occur within the f i r s t 24 hours a f t e r germination of the spore, i t may have escaped e a r l i e r than normal from A/a incompatibility by loss of the mating type gene from the translocated segment. I t i s u n l i k e l y that the s t r a i n resulted from a double crossover on either side of un-3 because of the close linkage of un-3 to ser-3 and mt. I t i s also u n l i k e l y that the s t r a i n survived due to reversion of the un-3 mutant because the un-3 marker contains two mutations. Most of the incompatible F3 s t r a i n s (59 of 71) only contained one mating type, probably because the mating type tests were done a f t e r the incompatible s t r a i n s had escaped. Those incompatible F3 s t r a i n s which contained both mating types may have escaped by mutation at a suppressor locus or by mitotic crossing over between the L.G. I centromere and mt and between mt and un-3, and both types of derivative 46 Table 4 Mating Types of F3 Strains TEMPERATURE- SENSITIVE F2 COMPATIBLE F3 INCOMPATIBLE F3 a1-113-8 9 A /a 1 A/a 1A 1 A 8 a a1-107-9 10 A /a 1 A /a 9 a a1-107-11 10 A /a 1 A 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 • crossed to R601 or R602 in the first cross middle # • isolate # from first cross last # • isolate # from second cross 47 nuclei were s t i l l present. Most of the escaped incompatible F3 s t r a i n s derived from R601 (un-3, A) were a, and most of the escaped incompatible F3 s t r a i n s derived from R602 (un-3, a) were A. These two types of s t r a i n s could have occurred by deletion of mt and un-3 from the temperature-sensitive L.G. I homolog. Another mechanism could have been double mitotic crossing over, between the L.G. I centromere and mt and between mt and un-3, followed by overgrowth by the a nuclei i n the R601-derived a escaped s t r a i n s or by the A nuclei i n the R602-derived A escaped s t r a i n s . The l a t t e r mechanism could have given r i s e to the smaller classes 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 classes could have arisen by mitotic crossing over between the L.G. I centromere and mt and between mt and un-3, followed by overgrowth by the A nuclei i n the R601-derived A escaped s t r a i n s or by the a nuclei i n the R602-derived a escaped s t r a i n s . Although the F3 s t r a i n s were selected on medium without tryptophan, the trp-4 gene i s leaky; therefore, s e l e c t i o n may not have been completely r e s t r i c t i v e . To ensure that the compatibility was due to a novel suppressor and not to tol, the A/a compatible F3 s t r a i n s from each of the seven crosses were tested for t rp - 4 . None of the compatible F3 s t r a i n s was tryptophan-requiring. A l l of the A/a incompatible F3 were tested for trp-4 to ensure that they were c o r r e c t l y scored as incompatible 48 because they had no suppressor, and not because they grew poorly due to the trp-4 gene. None of the incompatible F3 s t r a i n s was tryptophan-requiring. F i n a l l y , to ensure that the compatibility was due to a suppressor and not to a novel gene which increased the rate of escape, 4 hyphal t i p s were obtained from 2 A/a compatible F3 s t r a i n s from each of the seven crosses and were tested f o r mating type. If the "compatibility" were due to early escape, then the t i p s would be expected to be A or a, but not both; i f the compatibility were due to a suppressor, then the t i p s would be expected to be A and a. The r e s u l t s are shown i n Table 5. Almost a l l of the t i p s were A/a, suggesting that the compatible phenotype was due to a suppressor. Single mating type hyphal t i p s ( a l l A) were found only i n two of the suppressor s t r a i n s , both derived 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 addition to the suppressor may have occurred. The second mutation could be one that causes i n s t a b i l i t y of duplications. The sing l e mating type mitotic segregants could have arisen by deletion of a during somatic growth or mitotic crossover between the L.G. I centromere and mt and between mt and ser-3 (only A products would have survived). Table 6 summarizes the findings described i n Results 1. 49 Table 5 Mating Types of Hyphal Tips of A/a Compatible F3 Strains COMPATIBLE F3 TIP DERIVED FROM STRAIN: MATING TYPES 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. Table 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 9 a1-107-9 37 19 1-54 27 / a1-107-11 40 28 1-58 16 1-59 24 / a1-113-7 31 0 1-65 19 / . a1-113-8 46 24 1-73 19 / a1-113-9 6 0 1-75 8 / a1-113-10 46 0 1-104 17 / y / 1-107 1 1 2 '/ 1-113 17 A ' a1-128-22 32 0 1-128 27 6 . a1-128-23 32 0 2-18 27 -—^ a1-128-24 25 0 2-20 12 a1-128-25 35 0 2-32 21 a1-128-26 32 0 2-37 1 1 a1-128-27 0 0 2-57 2 2-79 0 A2-86-1 30 0 2-86 20 3 ' A2-86-4 36 0 2-95 0 A2-86-12 30 9 2-103 31 2-110 0 2-117 37 A2-155-2 2-146 10 25 7 2-155 32 3 , A2-155-3 65 14 2-162 33 A2-155-5 46 18 KEY FOR A/a ESCAPED F1 1 or 2 - crossed to R601 or R602 in the first cross second # • 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 • crossed to R601 or R602 in the first cross middle # • isolate # from first cross last # • isolate # from second cross 51 DISCUSSION 1 Seven s t r a i n s containing novel suppressors of A/a incompatibility have been i s o l a t e d . The s t r a i n s were derived from 4 s t r a i n s that had undergone escape events (strains #1-107, 1-113, 2-86 and 2-155), suggesting that there may be 4 novel suppressor mutations. None of the suppressors i s a l l e l i c with tol or with the mating type gene. The seven s t r a i n s produced A/a compatible progeny when crossed with a duplication-generating translocation s t r a i n . The A/a compatible progeny were probably duplications, not heterokaryons, because hyphal t i p s i s o l a t e d from these s t r a i n s , i n general, contained both mating types. The compatible phenotype observed i n these A/a progeny i s due to new mutations that suppress A/a incompatibility, not to new mutations that increase the rate of escape from A/a incompatibility by increasing the rate of deletion or mitoti c crossing over. There were s u r p r i s i n g l y few temperature-sensitive F2 progeny from the 50 crosses of the second cross (18 of 329 tested). Two types of crosses produced no temperature- s e n s i t i v e F2 progeny: f l , A x A/a escaped FI s t r a i n s derived from R601 (un-3, A) and f l , a x A/a escaped F l s t r a i n s derived from R602 (un-3, a). In these 2 types of crosses, L.G. I from the fl parent had the same mating type gene as L.G. I from the temperature-sensitive parent; so perhaps the L.G. I homologs paired with the translocated segment which 52 has the opposite mating type (Fig. 10). When such pairings occur, fewer temperature sens i t i v e progeny are produced. The L.G. I homologs probably did not pair with each other. If they did, temperature-sensitive progeny would have been produced. The al t e r n a t i v e p a i r i n g hypothesis explains the absence of temperature-sensitive progeny from the two aforementioned types of crosses. When L.G. I from the fl parent pairs with the translocated DNA, no temperature sens i t i v e progeny are produced. I f there i s a single crossover i n the paired region, acentric and d i c e n t r i c chromosomes are produced, and these, presumably, do not survive. If there i s a double crossover i n the paired region, the un-3 a l l e l e always segregates with an un-3+ a l l e l e . When L.G. I from the temperature-sensitive parent pairs with the translocated DNA, very few temperature s e n s i t i v e progeny are produced. If there i s a single crossover i n the paired region, acentric and d i c e n t r i c chromosomes are produced. If there i s a double crossover i n the paired region, only one of the 6 possible types of double crossover—one crossover on each side of the mating type gene—gives temperature s e n s i t i v e progeny. This type of double crossover i s rare because of the proximity of un-3 to the mating type gene. If one disregards the two types of crosses that yielded no temperature-sensitive F2 progeny, the proportion of temperature-sensitive F2 st r a i n s i s s t i l l low (18 of 165). In the two types of crosses that did give temperature- 53 PAIRING SEGREGATION ser un + a ser* un+ A ser+ un la lb Ha, la lib, lb ser un+ a ser* un A ser* un+ A lb la l la, lb lib, la One explanation for the low number of temperature-sensitive F2 strains is the alternative pairing hypothesis. Either of the L.G.I homologs could have paired with the translocation. r_ tne L.u.i nomoiogs couia nave pairea witn im n l y U r e l U Thin vertical lines indicate regions of pairing 54 sen s i t i v e F2 progeny (R601-derived escaped s t r a i n s x f l , a and R602-derived escaped s t r a i n s x f l , A), perhaps p a i r i n g of the L.G. I homologs with each other, as depicted i n F i g . 7, did not always occur. If eithe r of the L.G. I homologs had paired with the translocated DNA, fewer temperature- se n s i t i v e progeny would have been produced. Two additional explanations for the low number of temperature-sensitive progeny are possible. Duplication s t r a i n s undergo a process c a l l e d RIP ("Repeat-Induced Point mutation", previously "Rearrangements Induced Premeiotically") during mating, with the r e s u l t that duplicated genes are often mutated (Selker and Stevens, 1985; Selker, et a l . , 1987; Selker and Garrett, 1988; Cambareri, et a l . , 1989; Grayburn and Selker, 1989; Selker, 1990; Cambareri, et a l . , 1991). RIP of either un-3 or un-3+ could have produced un-3 n u l l s , which are l e t h a l (Lambowitz, unpublished r e s u l t s , Glass, personal communication). F i n a l l y , germination of un-3 spores may occur less frequently than germination of wild type spores (Glass and L. Stenberg, personal communication), possibly because of the heat shock required to i n i t i a t e germination. A/a duplication s t r a i n s escaped from incompatibility by several d i f f e r e n t means other than the generation of suppressors. The 8 phenotypic classes of escaped F l s t r a i n s , shown i n F i g . 6, could have been the r e s u l t s of deletion/mutation at the mating type locus, RIP, mitotic 55 crossing over or low f e r t i l i t y due to the presence of duplicated genetic material. Any of the 8 phenotypic classes of escaped F l s t r a i n s could have arisen by deletion/mutation at one or both of the mating type l o c i . Experiments performed by DeLange and G r i f f i t h s (1975) on escape from A/a incompatibility suggested that mixed mating type heterokaryons often escaped by deletion of one mating type. Similar events could have occurred i n mixed mating type duplication s t r a i n s . The 8 classes of escaped s t r a i n s could have arisen by RIP. A/a duplications are not r e a l l y duplications because A and a are idiomorphs, not a l l e l e s (Metzenberg and Glass, 1990), i . e . the central portions of the DNAs are completely d i f f e r e n t from each other (Glass, et a l . , 1988). The flanking regions, however are v i r t u a l l y i d e n t i c a l (Glass, et a l . , 1988) and RIP can sometimes occur i n unique sequences close to the duplicated ones (Foss, et a l . , 1991). The sing l e mating type escaped s t r a i n s , classes 7 and 8, could have been generated by mitotic crossing over. Once the mating type genes had segregated into d i f f e r e n t n u c l e i , one type of nucleus could have outgrown the other and the resultant culture would only have had a single mating type reaction. The barren phenotype associated with duplications i n N. crassa could account f o r classes 2-6. Duplication s t r a i n s sometimes produce abundant perithe c i a , but very few 56 ascospores (Newmeyer and Taylor, 1967). If t h i s occurred, the spores simply may have been overlooked during scoring. What i s t o l ? I t i s not possible to formulate a comprehensive statement regarding the b i o l o g i c a l s i g n i f i c a n c e of incompatibility i n a l l of the fungi, f o r i t manifests i t s e l f i n many d i f f e r e n t forms. Incompatibility can be heterogenic, preventing fusion between st r a i n s of d i f f e r e n t genotypes, eith e r vegetative fusion (e.g. i n W. crassa), sexual fusion (e.g. i n Sordaria fimicola (Olive, 1956) and Ceratostomella radicicola (El-Ani, et a l . , 1957)) or both vegetative and sexual fusion (e.g. i n Aspergillus nidulans (Jinks, et a l . , 1961) and Podospora anserina (Esser, 1971)); or i t can be homogenic, preventing fusion between st r a i n s of s i m i l a r genotypes, either sexual fusion (e.g. i n W. crassa) or both vegetative and sexual fusion (e.g. i n Schizophyllum commune (Raper, 1966 c i t e d i n Burnett, 1975)). The s i g n i f i c a n c e of each incompatibility system, because of the d i v e r s i t y , must be considered separately. Vegetative incompatibility controlled by the mating type genes of N. crassa may be b i o l o g i c a l l y unrelated to incompatibility c o n t r o l l e d by the het genes. This idea i s supported by the existence of the gene, tol, which suppresses A/a incompatibility without a f f e c t i n g het-C/c or het-JE?/e incompatibility (Newmeyer, 1970; Perkins, 1974). 57 Vegetative incompatibility seems to be an i n t r i n s i c function of the mating type genes. Genetic and molecular data support t h i s idea and suggest that compatibility mechanisms evolved secondarily. Metzenberg and Ahlgren (1973) introgressed the mating type genes of N. tetrasperma into a lar g e l y N. crassa background. The resultant s t r a i n s demonstrated incompatibility i n mixed mating type heterokaryons and i n heterozygous duplications, suggesting that the W. tetrasperma genes had the a b i l i t y to i n s t i g a t e the incompatibility reaction. In t h e i r own environment, i n N. tetrasperma, the mating type genes must either be suppressed f o r the incompatibility function or lack target genes or both. A recent study by D. Jacobson (personal communication) suggests that one way i n which N. tetrasperma tolerates A/a ascospores i s by the presence of the suppressor, t o l . He introgressed sequences of N. tetrasperma corresponding to t o l of W. crassa into a N. crassa background. The resultant s t r a i n of N. crassa behaved as i f i t were tol, not t o l + . The mating type genes of N. sitophila, l i k e W. tetrasperma, are able to produce the incompatibility phenotype i n a W. crassa background (Perkins, 1977). N. sitophila mating type genes were introgressed into W. crassa, whereupon the N. sitophila genes exhibited incompatibility i n A/a duplications. Even more int e r e s t i n g was the observation that the suppressor gene, t o l , eliminated the incompatibility, i n d i c a t i n g that t o l i s 58 unable to detect any s i g n i f i c a n t difference between W. crassa and N. sitophila mating type genes. A r e s u l t with s i m i l a r implications was obtained by Glass (personal communication). Replacement of the N. crassa A gene with the N. africana A gene d i d not a l t e r the a b i l i t y of the transformant to i n i t i a t e the incompatibility reaction, again implying that the mating type gene from a homothallic species i s capable of orchestrating vegetative incompatibi1ity. Incompatibility controlled by the vegetative incompatibility genes may have developed independently of A/a incompatibility. There are many theories regarding the b i o l o g i c a l benefits of heterokaryon incompatibility. Caten (1972) suggested that incompatibility systems e x i s t to l i m i t the spread of infect i o u s viruses or cytoplasmic determinants. Later studies indicated, however, that plasmids can cross incompatibility b a r r i e r s within a species ( C o l l i n s and S a v i l l e , 1990) and even between species ( G r i f f i t h s , et a l . , 1990), presumably during b r i e f periods of unstable fusion. H a r t l , et a l . (1975) suggested that incompatibility prevents the ex p l o i t a t i o n of an adapted mycelium by a less well adapted one growing i n the same niche. Although t h i s hypothesis could apply to a fungus l i k e N. crassa, i n which a defective homokaryon can survive by fusion with another mycelium, i t does not seem a l i k e l y explanation f o r 59 incompatibility i n fungi l i k e the basidiomycetes i n which fusion leads to mating. I t has been suggested that incompatibility serves to 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 fine-tuning between nuclei and organelles. Considering that nuclei are associated with d i f f e r e n t organelles every time mating occurs, t h i s does not seem to be a l i k e l y explanation f o r the existence of incompatibility. J. Begueret (personal communication) succeeded i n creating a novel incompatibility group v i a mutation i n Podospora, which led him to propose that incompatibility genes are nothing more than mutations without b i o l o g i c a l s i g n i f i c a n c e . Jinks, et a l . (1961) made a s i m i l a r suggestion i n t h e i r study of incompatibility i n Aspergillus nidulans. They maintained that heterokaryon incompatibility i s a consequence of genetic d i v e r s i t y , not a cause. This idea would only apply to fungi l i k e N. crassa i n which the incompatibility reaction does not a f f e c t mating. If a novel a l l e l e arose i n a population, i t would be selected against because the c e l l containing i t would die i f i t fused with an unlike type. A novel a l l e l e would not be selected against i f i t arose i n a spore that established a separate population before encountering incompatible fungal types. In the basidiomycetes, however, a novel incompatibility group would be able to mate with a l l other e x i s t i n g groups, allowing i t to spread quickly through a population. 6 0 The gene, tol+, and the new suppressors could be mating type target genes, c o n t r o l l i n g d i f f e r e n t steps i n the pathway to A/a incompatibility, and when they are mutated, the reaction f a i l s to occur. Their relationships to each other i n terms of where they f i t i n to the pathway are unknown at present. They could be mutants i n sequential reactions or reactions that occur simultaneously. The suppressors could be enzymes required for vegetative growth, normally turned o f f by the A/a product ( d i r e c t l y or i n d i r e c t l y ) during incompatibility. The mutants could be altered such that they are no longer recognized by the regulating product and are, therefore, not repressed and the incompatibility reaction does not occur. The suppressors could be required f o r recognition of vegetatively growing A or a hyphae. The mutants, i n t h i s case, could be defective for vegetative recognition, thus eliminating the incompatibility reaction. The suppressors could produce t o x i c metabolites i n the presence of the A/a product. These mutants could be defective for toxin production i t s e l f or f o r regulation so that the incompatibility reaction does not occur. The existence of other genes a f f e c t i n g the incompatibility reaction raises a question regarding N. tetrasperma. As previously mentioned, one s t r a i n of N. tetrasperma appears to contain the recessive a l l e l e of tol (Jacobson, personal communication). What i s the state of the other suppressor genes? As long as a s t r a i n has t o l , i t 61 w i l l be heterokaryon compatible, so the a l l e l e s of the other suppressors would not a f f e c t the phenotype. U n t i l more i s known about the r e l a t i v e functions of the new suppressors, i t i s not possible to predict the suppressor genotype(s) of W. tetrasperma. Perhaps there are suppressors that, unlike t o l , suppress both A/a incompatibility and mating. If so, i t would mean that mating and A/a incompatibility act through at l e a s t one common step. Perhaps there are A/a incompatibility suppressors that also suppress incompatibility controlled by one or some of the net genes. If so, i t would imply that incompatibility i s effected through one pathway and that t o l acts before or a f t e r the common part(s) of the pathway. The number of d i f f e r e n t suppressor genes may give an in d i c a t i o n of the number of steps involved i n generating the incompatibility reaction. 62 INTRODUCTION 2 A molecular picture of the mating type genes i s emerging with the aid of mating type mutants ( G r i f f i t h s and DeLange, 1978; G r i f f i t h s , 1982; G r i f f i t h s , personal communication) and molecular b i o l o g i c a l techniques (Glass, et a l . , 1988; Staben and Yanofsky, 1990; Glass, et a l . , 1990). The A idiomorph, as defined by i t s region of non- homology with a, i s 5301 base pairs i n length (Glass, et a l . , 1990), and a, by the same d e f i n i t i o n , i s 3235 base pairs i n length (Staben and Yanofsky, 1990) (Fig. 11). A l l of the Am mutants ( G r i f f i t h s , 1982) that have been sequenced map within the single ORF, c a l l e d A-l. A l l of the Am and am mutants of G r i f f i t h s and DeLange (1978) that have been sequenced map within the exons i n the ORFs (Glass, et a l . , 1990; Staben and Yanofsky, 1990). Some examples follow. One of the mutants, anl, has a frameshift due to the deletion of a single base p a i r . The i n s e r t i o n of 212 base pa i r s , which characterizes the mutant a m 3 0 causes a premature t r a n s c r i p t i o n stop. The unique compatible, f e r t i l e mutant, a m 3 3 , has a base pair substitution which i s located farther downstream than either of the other two mutations. Species i n other genera have idiomorphs instead of a l l e l e s at t h e i r mating type l o c i , e.g. Saccharomyces LEGEND Thick lines • identical flanking sequences Filled boxes - idiomorphs Open boxes • postulated ORFs Arrows - presumed transcripts with introns Mating type regions of N. crassa (from Staben and Yanofsky, 1990). 64 cerevisiae (Strathern, et al., 1980; Nasmyth and T a t c h e l l , 1980), Schizosaccharomyces pombe (Kelly, et al., 1988), Ustilago maydis a gene (M. Bolker and R. Kahmann, 1991, i n published abstracts from Sixteenth Fungal Genetics Conference) and Podospora anserina (Coppin, 1991, i n published abstracts from Sixteenth Fungal Genetics Conference). The mating type genes of N. crassa are incompatible not only i n a heterokaryon, but also i n one nucleus. Newmeyer (1970) used nuclear incompatibility to induce tol, which suppresses both types of incompatibility. Glass, et al. (1990) observed a 100-fold reduction i n transformation e f f i c i e n c y of the ORF, A-l, into a spheroplasts, compared to the transformation e f f i c i e n c y into A spheroplasts; and Glass (personal communication) observed a s i m i l a r reduction of transformation e f f i c i e n c y of a sequences into A. Staben and Yanofsky (1990) also reported a decrease i n the frequency of A transformants when the donor DNA was ORF, a - l , as compared to when the donor DNA was a portion of the a idiomorph not including a - l . Nuclear incompatibility may be separable from heterokaryon incompatibility. Glass and G r i f f i t h s (personal communication) created a A-duplication s t r a i n by transforming a s t r a i n of mating type A with the ORF of the s t e r i l e , compatible mutant, Am64. In order to RIP the A gene, they crossed the transformant to a s t r a i n of mating type a. Sur p r i s i n g l y , some of the progeny displayed the 65 incompatibility phenotype. Growth occurred as a small wispy knot of mycelia with no a e r i a l hyphae. A l l of the cultures escaped from slow growth within one week, and then showed a mating type a reaction when tested. If the ORF had segregated from mt as an independent locus, then one half of the a spores would have contained Am64 and i t i s possible that these were the incompatible s t r a i n s (Fig. 12). If the incompatible s t r a i n s were Am64/a, i t appears as though Am64 has l o s t heterokaryon incompatibility while reta i n i n g nuclear incompatibility. At f i r s t i t seemed possible that the incompatible phenotype was due to residual heterokaryon incompatibility s p e c i f i e d by Am64, but t h i s hypothesis has been discarded because a mixed mating type heterokaryon of A n 6 4 grew as fa s t as a po s i t i v e control s t r a i n . To t e s t the p o s s i b i l i t y that mutations at mt can eliminate heterokaryon incompatibility without eliminating nuclear incompatibility, duplication s t r a i n s that contained the f e r t i l e , compatible mutant, am33, and A were examined for t h e i r morphological c h a r a c t e r i s t i c s and growth rates. A/am33 duplication s t r a i n s were made by crossing 3 d i f f e r e n t a m 3 3 - c o n t a i n i n g s t r a i n s to A s t r a i n s containing a translocation of the mating type gene. The mutant, am33, i s known to be compatible i n a heterokaryon with A, although the growth rate of an a m 3 3 + A heterokaryon has not been measured p r e c i s e l y before now. I t was measured to determine i f am33 n a < j a r i y residual heterokaryon incompatibility. 66 1/2 Am64 1/2 A NORMAL OR RIP 1/2 Am64 INCOMPATIBLE? 1/2 a / \ NORMAL a re 12 Segregation of Am64 ORF and resulting phenotypes. 67 RESULTS 2 The growth rate of the s t e r i l e , compatible mutant, Am64f ^ n a m i x e c | mating type heterokaryon, A m 6 4 + 7 (ad-3B, a), was measured to determine i f Am64 had residual incompatibility. The growth rate was compared to that of the component s t r a i n s alone and to that of an incompatible mixed mating type heterokaryon, 153 (ad-3A, nic-2, A) + 7 (ad-3B, a). The heterokaryon, Am64 + 7 (ad-3B, a), grew as fa s t as the component s t r a i n 7 (ad-3B, a), and fast e r than the incompatible mixed mating type heterokaryon, 153 (ad-3A, nic-2, A) + 7 (ad-3B, a) (Table 7 and Fi g . 13), suggesting that the mutant, Am64, has l o s t i t s heterokaryon incompatibility function completely . The following section describes a series of tests done to study heterokaryon and nuclear incompatibility i n another mating type mutant, am33. The growth rate of a m 3 3 i n a mixed mating type heterokaryon (am33, ad-3B + 1-22-83 (ad-3A, nic-2, un-3, A)) was measured to determine i f the mating type mutant had residual incompatibility. The growth rate was compared to several controls: (1) an incompatible mixed mating type heterokaryon (51-2 (ad-3B, cyh-1, a) + 1-22-83 (ad-3A, nic-2, un-3, A)); (2) a mating type homokaryon—a m 3 3, ad-3B(128) paired with 51-2, a s t r a i n of mating type a (ad-3B(114), cyh-1, a); (3) a compatible mixed mating type heterokaryon i n which both components had tol (1-9-57 68 Table 7 Strains and Media Used in the Measurement of Growth Rate of Am6 in a Mixed Mating Type Heterokaryon GENOTYPE STRAIN mt AUXOTROPHIC GENES OTHER 153 * A ad-3A, nic-2 un-3, cyh-1 7 a ad-3B Am64 Am64 ad-3A, nic-2 un-3, cyh-1 MINIMAL MEDIUM + POSITIVE CONTROLS Am64 adenine, nicotinic acid 7 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 • nicotinic acid- or nicotinamide-requiring un-3 • temperature-sensitive cyh-1 « cycloheximide-resistant * 153 was the strain used to generate Am64. 6 9 24 Jl 120 168 216 264 312 380 48 96 144 192 240 288 336 384 fme (hois) Growth rate of Am64 in a mixed mating type heterokaryon. P i n i i r o 1Q Each line represents the average of a minimum of 3 i l y U r © l O measurements of growth rate. 70 (ad-3B(128), tol, a)+ 1-9-3 (ad-3B(114), tol, A)); and (4) the following component st r a i n s on supplemented media: 1-22-83; am33, ad-3B-, and 51-2. The heterokaryon, a m 3 3 + 1-22-83, grew as fa s t as the mating type homokaryon, a m 3 3 + 51-2 and the compatible mixed mating type heterokaryon, 1-9-57 + 1-9-3; and fast e r than the incompatible heterokaryon, 51-2 + 1-22-83 (Table 8 and F i g . 14), suggesting that the mutant, am33, has l o s t i t s heterokaryon incompatibility function completely. To determine the phenotype shown by a m 3 3 i n the same nucleus with A, duplication progeny were obtained from the following crosses. Three a m 3 5 - c o n t a i n i n g s t r a i n s , am33, ad; Rl-14 and Rl-29 (Table 9) were crossed to the translocation s t r a i n , T(I->II) 39311, ser-3, A, to generate duplication progeny. The crosses are diagrammed i n Figs. 15A and 15B. Single spores were viewed through a dissecting microscope and c o l l e c t e d with a tungsten needle. Individual spores were placed into slants of supplemented medium (minimal medium + adenine f o r the am33, ad-derived spores; and minimal medium + n i c o t i n i c acid and pantothenic acid for the Rl-14- and Rl-29-derived spores). The t e s t tubes were placed into a 60°C water bath for 30 minutes to i n i t i a t e germination of the spores, which were examined a f t e r 3 days. The progeny from each of the 3 crosses expressed one of two growth phenotypes. One phenotypic c l a s s , c a l l e d " i n h i b i t e d " , grew s l i g h t l y less vigorously than the other, "healthy". The in h i b i t e d phenotype was d i s t i n c t from the 7 1 Table 8 Strains and Media Used in the Measurement of Growth Rate of am33 in a Mixed Mating Type Heterokaryon STRAIN GENOTYPE 1-22-83 A, ad-3A, nic-2, un-3 51-2 a, ad-3B(114), cyh-1 I-9-57 a, ad-3B(128), tol I-9-3 A, ad-3B(114), tol MINIMAL MEDIUM + POSITIVE CONTROLS 1-9-57 «• 1-9-3* — am33, ad + 51-2 — I-9-3 adenine am33, ad adenine 51-2 adenine I-22-83 adenine, nicotinic acid I-9-57 adenine 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 — ad-3A - adenine-requiring ad-3B(114) - adenine-requiring ad-3B(128) - adenine-requiring un-3 • temperature-sensitive cyh-1 • cycloheximide-resistant * The alleles, ad-3B(114) and ad-3B(128), complement. ** The genes, ad-3A and ad-3B, complement. 72 LEGEND 1 • I-9-57 + I-9-3 2 • 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 - l l K n *A Each line represents the average of 2 measurements F i g u r e I*f of growth rate. 73 Table 9 Genotypes of am33 Strains STRAIN GENOTYPE SOURCE am33, ad am33, ad-3B (128) AJ .F .G. R1-14 am33, n ic -3 , pan-1, al-1 (#43) N.L.G. R1-29 am33, n ic -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 74 am33, ad-3B x T(l->ll)39311, se r -3 , A am33 ad | 32o/0 am33 ad | 32% •M- 18% am33 18% ad | ser A minimal mediLm + adenine 50% 50% 50% 50% 50% 50% 50% 50% ser A ser A ser A ser A II II II II am33, ad A/am33, ad (incompatible?) dead dead am33 A/am33 (incompatible?) dead dead Cross made to generate A/am33 duplications. Genetic r : _ l i r o j r A distance between ad-3B and mt is 36% r i g U r e IOH (Perkins, et al., 1982). 75 R1-14 or R1-29 x T(l->ll)39311, se r -3 , A am33 al-1 -x- pan-1 IV ser -3 A n ic -3 VII pantothenic acid + nicotinic acid am33 50% I 50% 50% II II se r -3 A am33 A/am33 (incompatible?) 50% H 1 50% 50% dead dead se r -3 A Figure 15B Crosses done to generate A/am33 duplications. 76 standard incompatibility phenotype (dark agar) i n that the growth was more luxuriant. Mating type t e s t s were done on a l l of the i s o l a t e s (Table 10). If peri t h e c i a were produced with the fl tester, the reaction was scored as p o s i t i v e . Crosses, therefore, could have been barren. The healthy progeny from a l l three crosses were eithe r a, which was expected (Fig. 15A and 15B) or A/a, which was unexpected. The i n h i b i t e d progeny were mostly A/a, which was the r e s u l t predicted i n the hypothesis, although the predicted phenotype was dark agar. One i n h i b i t e d i s o l a t e could have contained both mating types, but the a mating type reaction was d i f f i c u l t to confirm. One i n h i b i t e d i s o l a t e reacted as only a. The sig n i f i c a n c e of the four types of progeny i s considered i n Discussion 2. In order to v e r i f y the difference i n phenotypes, sample progeny from each cross were inoculated onto plates. The difference between the phenotypes became more evident under these growth conditions. As a control, a A/a, tol duplication s t r a i n , which has a phenotype known as "square agar" (Newmeyer, 1970), very s l i g h t l y d i f f e r e n t from wild type, was also inoculated onto a plate. A l l of the healthy progeny tested, including the single and double mating type i s o l a t e s , and the square agar control grew to cover the plate with an even layer of mycelia. Most of the i n h i b i t e d progeny (22 of 30) grew i n a dense mat i n the centre of the plate with a few hyphae extending beyond 7 7 Table 10 Mating Types of Progeny CROSS SPORE TYPE MATING TYPE am33,ad-3B x T(l->ll)39311,ser,A 17 h 12 A/a 5 a 12 i 11 A/a 1 A/? R1-14 x T(l->ll)39311,ser,A 9 h 6 A / a 3 a 11 i 11 A / a R1-29 x T(l->ll)39311,ser,A 7 h 3 A / a 4 a 7 i 6 A / a 1 a h • healthy (i.e. vigorous growth) i • inhibited (i.e. less vigorous growth) The A/a healthy isolates were unexpected. 78 the central mat (Table 11). Some of the progeny o r i g i n a l l y scored as i n h i b i t e d (8 of 30) grew evenly on plates. Since the p l a t i n g t e s t was done several months a f t e r the i n i t i a l spore i s o l a t i o n , i t i s possible that these progeny had escaped. Strains were kept i n the freezer (-20°C) during most of t h i s time, which could explain why a l l of the i n h i b i t e d progeny had not escaped. The growth rates of a l l of the i s o l a t e s were measured and compared to incompatible duplication (A/a,tol+) and compatible duplication (A/a,tol) controls. Fungi were inoculated at one end of a 50 cm growth tube containing 30 mL of supplemented medium for the i s o l a t e s and minimal medium for the controls. The mycelial fronts were marked at regular i n t e r v a l s . Results are shown i n Figs. 16-29. The slopes of the graphs are shown i n Table 12. A l l of the healthy i s o l a t e s grew at the same rate as t h e i r s i b l i n g s , as f a s t as the compatible controls, and considerably f a s t e r than the incompatible controls. The growth rates of the i n h i b i t e d s t r a i n s varied widely. Only one s t r a i n , the single 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 controls and healthy s t r a i n s . The r e s t grew at rates intermediate between those of the compatible and incompatible controls. The s i g n i f i c a n c e of these r e s u l t s i s considered i n Discussion 2. The following t e s t was done to determine whether the mating types of the A/a s t r a i n s had segregated into d i f f e r e n t n u c l e i . Conidial samples from the crosses with 79 Table 11 Phenotypes of Progeny STRAIN # OF ISOLATES WITH PHENOTYPE 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 • strains am33,ad; R1-14 or R1-29 h or i = healthy or inhibited x s isolate # Inhibited strains with even growth were probably escapes. 80 the females, Rl-14 and Rl-29, were plated on sorbose plates containing pantothenic acid and n i c o t i n i c acid. Single c o n i d i a l colonies were cut out of the agar, grown and tested for t h e i r mating types. A l l of the i n h i b i t e d i s o l a t e s tested contained both mating types, suggesting that the mating types had not segregated m i t o t i c a l l y . The healthy i s o l a t e s of st 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). The si g n i f i c a n c e of these r e s u l t s i s discussed below. 81 Growth Rates of Controls 45 0 48 96 144 192 24 72 120 168 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 82 Figure 17 83 Figure 18 8 4 Figure 19 85 Growth Rates of a-i-x 0 24- 48 72 96 120 144 168 192 Trrefws) Figure 20 86 Growth Rates of a-i-x Figure 21 87 Growth Rates of 14-h-x SOi 0 24 € 72 96 Tmefuis) Figure 22 88 Figure 23 89 Growth Rates of 14—i—x 0 24 48 72 96 120 144 168 192 rine (hours) Figure 24 9 0 Growth Rates of 0 24 48 72 96 120 144 168 192 rme (heirs) Figure 25 91 Figure 26 92 Figure 27 93 Growth Rates of 2H-x 0 24 48 72 96 120 144 1® 192 fire (bars) Figure 28 94 Growth Rates of 29-i-x Figure 29 95 Table 12 Slopes of Growth Rates of Progeny STRAIN SLOPE- 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 29 29-i 29 29 29 29 -1 -2 -3 -4 -5 -6 -7 ,1 1 .17 .0035 .50 .50 .27 .33 .48 .42, .44 .12 .47 .23 .38, .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). 96 Table 13 Mating Types of Single Conidial Isolates of Progeny STRAIN MATING TYPE 14-h-4-1 a 14-h-4-2 a 14-h-4-3 a 14-h-5-1 a 14-h-5-2 a 14-h-5-3 a 14-h-6-1 a 14-1-3-1 A /a 29-h-5-1 a 29-h-5-2 a 29-h-6-1 a 29-h-6-2 a 29-h-6-3 a 29-h-7-1 a 29-h-7-2 a 29-h-7-3 a 29-1-1-1 A /a 29-J-1-2 A /a 29-J-1-3 A /a 29-1-3-1 A /a 14 or 29 - strain R1-14 or R1-29 h or i • healthy or inhibited first # - spore isolate # last # - conidial isolate # Healthy strains were a; inhibited strains were A/a. 97 DISCUSSION 2 The findings of Results 2 present evidence to support the idea that at le a s t 2 mating type mutants completely d e f i c i e n t i n heterokaryon incompatibility, A m 6 4 and a m 3 3 , r e t a i n nuclear incompatibility. The growth rates of the two mutants i n mixed mating type heterokaryons equalled those of pos i t i v e controls and surpassed those of negative controls, so i t appears that neither mutant has residual heterokaryon incompatibility. The nuclear incompatibility seen, therefore, was not due to residual heterokaryon incompatibi1ity. Four phenotypes were seen i n the progeny from the three crosses made to generate A/am33 duplications: (1) single mating type healthy (12 i s o l a t e s ) , (2) double mating type healthy (21 i s o l a t e s ) , (3) single mating type i n h i b i t e d (1 is o l a t e ) and (4) double mating type i n h i b i t e d (28, or possibly 29, i s o l a t e s ) . The only phenotype that was expected ( F i g . 15A and 15B) was the f i r s t phenotypic c l a s s , s i n g l e mating type healthy, which were normal segregants from the cross. The phenotype of the duplicated progeny was unknown, although i t was predicted to be dark agar. The fourth phenotypic c l a s s , the double mating type i n h i b i t e d s t r a i n s , i s the one providing evidence that nuclear incompatibility can e x i s t i n the absence of heterokaryon incompatibility. A l l of the A/a i n h i b i t e d 98 s t r a i n s grew more slowly than the healthy s t r a i n s . The reduced growth rates could have been due to the presence of a m 3 3 and A i n the same nucleus. Four of the seven A/a i n h i b i t e d s t r a i n s that grew evenly on plates (a-i-3, a-i-7, 14-i-lO, 29-i-2) had the highest growth rates of a l l of the A/a i n h i b i t e d s t r a i n s , supporting the idea that they had escaped and were growing at rates higher than the i n h i b i t e d s t r a i n s , yet lower than the healthy s t r a i n s . Two of the seven s t r a i n s (a-i-4, a-i-9) had growth rates that were high at f i r s t , but suddenly dropped. One of the seven s t r a i n s (29-i-6) had an ir r e g u l a r growth pattern. The growth rates of the A/a i n h i b i t e d s t r a i n s were highly v a r i a b l e . The variant rates could be c h a r a c t e r i s t i c of nuclear incompatibility i t s e l f . They could also have been produced by escape, either by a number of d i f f e r e n t mechanisms or by the same mechanism producing d i f f e r e n t r e s u l t s . Perhaps the i n h i b i t e d s t r a i n s showed various growth rates because they were i n d i f f e r e n t stages of escape by somatic segregation and overgrowth by a n u c l e i . The second phenotypic c l a s s , the A/a healthy i s o l a t e s was not expected. Metzenberg (personal communication) suggested that these s t r a i n s grew vigorously because the two mating types had segregated somatically into separate nuclei producing A + a m 3 3 heterokaryons, which are known to be compatible. The translocation i s long enough to sustain a double crossover. To t e s t h i s hypothesis, single c o n i d i a l i s o l a t e s were obtained from healthy and i n h i b i t e d s t r a i n s 99 and tested f o r t h e i r mating types. If Metzenberg's hypothesis were correct, conidia derived from healthy s t r a i n s would have been A or a, whereas conidia derived from i n h i b i t e d s t r a i n s would have been A/a. These r e s u l t s were observed, with the exception that there were no A c o n i d i a l i s o l a t e s from the healthy s t r a i n s . If the healthy phenotype were due to mitotic segregation, the cultures would have contained both mating types shortly a f t e r germination of the spores. After a period of time, i t i s conceivable that the a nuclei overgrew the A nuclei because the l a t t e r contained the ser-3 marker which i s c l o s e l y linked to the mating type gene. Overgrowth by a nuclei may have been slowed by cross-feeding of the serine-requiring A nuclei by the a n u c l e i . The healthy s t r a i n s grew as fa s t as the compatible controls (Figs. 16-19, 22, 23, 26 and 27). The compatible controls were A/a duplications containing t o l . The evidence presented above suggests that the healthy s t r a i n s were simply a*33 s t r a i n s . The t h i r d phenotypic c l a s s , the 1 unexpected i n h i b i t e d s t r a i n that reacted with only one mating type (29-i-7), could have escaped from the i n h i b i t i o n by deletion of A. I t grew evenly on a plate, and had a growth rate that exceeded some of the healthy s t r a i n s , suggesting that i t had escaped from i n h i b i t e d growth. An explanation i s needed f o r why some A/am33 s t r a i n s (the healthy ones) escaped early, while others (the 100 i n h i b i t e d ones) escaped l a t e r or not at a l l . Would a l l of the i n h i b i t e d s t r a i n s have escaped eventually? Was the time of escape the only difference between the healthy and i n h i b i t e d s t r a i n s or was there a difference i n the mechanisms of escape? Was i t s i g n i f i c a n t 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 healthy strains? Was there a gene segregating that caused e a r l y / l a t e escape by mitotic crossing over i n the A/a healthy s t r a i n s and the A/a i n h i b i t e d strains? The f i r s t of the three crosses segregated hal f A/a healthy and hal f A/a i n h i b i t e d progeny. In the other 2 crosses, the sample sizes were probably too small to r e f l e c t accurate r a t i o s . These questions can only be addressed by further study. Perkins (personal communication) has observed that A/am33 duplications grow with an abnormal morphology which he c a l l s "square" because Newmeyer (1970) c a l l e d the morphology associated with A/a, t o l duplications "square". Perkins' observations are not inconsistent with those presented above, except that Newmeyer's square s t r a i n s grow at wild type rates, whereas Perkins' square s t r a i n s , assuming they exhibit the same growth rates as those discussed here, grow at sub-wild type rates. For t h i s reason, A/am33 duplications should be referred to by some name other than square. 101 Molecular Model One current model of the molecular interactions that occur during mating i s as follows (Metzenberg and Glass, 1990r Glass, personal communication). A combination product of A and a e f f e c t s incompatibility during the vegetative cycle, when the mating type genes are expressed at low l e v e l s , and the same product i n s t i g a t e s mating functions during the sexual cycle, when the mating type genes are expressed at higher l e v e l s . Nuclear incompatibility can be explained i n the context of t h i s model. There i s obviously a difference i n the function of the mating type products during the vegetative and sexual cycles. Perhaps the mutant, Am64, i s defective fo r mating, but not incompatibility, as previously believed. I t , and the other mutant studied here, am33, could be defective i n terms of the s t a b i l i t y of t h e i r products. Both mutants are functional f o r nuclear incompatibility and defective f o r heterokaryon incompatibility. In terms of the s t a b i l i t y hypothesis, heterokaryon incompatibility requires more stable mating type products than nuclear incompatibility. These r e s u l t s can be explained as follows. In a mixed mating type heterokaryon, the A and a products are synthesized i n d i f f e r e n t n u c l e i . When the products encounter one another, the combination product enters the nucleus and i n i t i a t e s incompatibility. In a A/a duplication s t r a i n , the A and a products are made i n the 102 same nu c l e u s , so they would encounter each o t h e r w i t h g r e a t e r speed because of t h e i r p r o x i m i t y , and would, t h e r e f o r e , r e q u i r e l e s s s t a b i l i t y . In the mutants, A m 6 4 and a m 3 3, heterokaryon i n c o m p a t i b l i t y may be e l i m i n a t e d because 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 mating type product w i t h which t o combine. 103 REFERENCES Abraham, J . , J . Feldman, K.A. Nasmyth, J.N. Strathern, A.J.S. Klar, J.R. Broach and J.B. Hicks (1983). Sites Required f o r P o s i t i o n - E f f e c t Regulation of Mating-Type Information i n Yeast. C.S.H. Symp. Quant. B i o l . 47: 989-998. Anagnostakis, S.L. (1977). Vegetative Incompatibility i n Endothia parasitica. Expl. Mycol. 1: 306-316. A s t e l l , C.R., L. Ahlstrom-Jonasson, M. Smith, K. T a t c h e l l , K.A. Nasmyth and B.D. H a l l (1981). The Sequence of the DNAs Coding f o r the Mating-Type Loci of Saccharomyces cerevisiae. C e l l 27: 15-23. Bach, F.H. and J . J . vanRood (1976). The Major Histocompatibility Complex—Genetics and Biology. New Eng. J. Med. 295: 806-813. Beach, D.H. (1983). C e l l Type Switching by DNA Transposition i n F i s s i o n Yeast. Nature 305: 682-688. Beadle, G.W. and V.L. Coonradt (1944). Heterocaryosis i n Neurospora crassa. Genet. 29: 291-308. B i s t i s , G.N. (1981). Chemotropic Interactions between Trichogynes and Conidia of Opposite Mating-Type i n Neurospora crassa. Mycologia 73: 959-975. B i s t i s , G.N. (1983). Evidence for D i f f u s i b l e , Mating-Type- S p e c i f i c Trichogyne Attractants i n Neurospora crassa. Expl. Mycol. 7: 292-295. Blaich, R. and K. Esser (1971). The Incompatibility Relationships between Geographical Races of Podospora anserina. Molec. Gen. Genet. I l l : 265-272. Burnett, J.H. (1975). Mycogenetics. 173. John Wiley and Sons, London. Burnett, J.H. (1976). Fundamentals of Mycology, Second Edition. 492, 561. Edward Arnold Ltd., London. Cambareri, E.B., B.C. Jensen, E. Schabtach and E.U. Selker (1989). Repeat-Induced G-C to A-T Mutations i n Neurospora. Science 244: 1571-1575. Cambareri, E.B., M.J. Singer and E.U. Selker (1991). Recurrence of Repeat-Induced Point Mutation (RIP) i n Neurospora crassa. Genet. 127: 699-710. Caten, C.E. (1972). Vegetative Incompatibility and Cytoplasmic Infection i n Fungi. J . Gen. Microbiol. 72: 221- 229. 104 C o l l i n s , R.A. and B.J. S a v i l l e (1990). Independent Transfer of Mitochondrial Chromosomes and Plasmids during Unstable Vegetative Fusion i n Neurospora. Nature 345: 177-179. Crandall, M. (1978). Mating-Type Interactions i n Yeasts i n Cell-cell Recognition, 105-120. Cambridge University Press, Cambridge. Davis, R.H. and F.J. DeSerres (1970). Genetic and Microbiological Research Techniques f o r Neurospora crassa i n Methods of Enzymology (eds. H. Tabor and C. Tabor), 79-143. Academic Press, New York. Day, A.W. and J.E. Cummins (1981). The Genetics and C e l l u l a r Biology of Sexual Development i n Ustilago violacea i n Sexual Interactions in Eukaryotic Microbes (eds. D.H. O'Day and P.A. Horgen), 379-402. Academic Press, N.Y. DeLange, A.M. and A.J.F. G r i f f i t h s (1975). Escape from Mating-Type Incompatibility i n Bisexual (A + a) Neurospora Heterokaryons. Can. J . Genet. Cytol. 17: 441-449. Dodge, B.O. (1935). The Mechanics of Sexual Reproduction i n Neurospora. Mycologia 27: 418-438. Ebert, P.R., M.A. Anderson, R. Bernatzky, M. Altschuler and A.E. Clarke (1989). Genetic Polymorphism of S e l f - Incompatibility i n Flowering Plants. C e l l 56: 255-262. Egel, R. (1977). Frequency of Mating-Type Switching i n Homothallic F i s s i o n Yeast. Nature 266: 172-174. Egel, R. and H. Gutz (1981). Gene Acti v a t i o n by Copy Transposition i n Mating-Type Switching of a Homothallic F i s s i o n Yeast. Curr. Genet. 3: 5-12. El-Ani , A.S., L.J. Klotz and W.D. Wilson (1957). Heterothallism, Heterokaryosis, and Inheritance of Brown Perithecia i n Ceratostomella radicicola. Mycologia 49: 181- 187. Esposito, R.E. and S. Klapholz (1981). Meiosis and Ascospore Development i n The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance (eds. J.N. Strathern, E.W. Jones and J.R. Broach), 211-287. C.S.H. Lab., New York. Esser, K. (1971). Breeding Systems i n Fungi and t h e i r Significance f o r Genetic Recombination. Molec. Gen. Genet. 110: 86-100. F e r r i s , P.J. and U.W. Goodenough (1987). Transcription of Novel Genes, Including a Gene Linked to the Mating-Type 105 Locus, Induced by Chlamydomonas F e r t i l i z a t i o n . Mol. C e l l . B i o l . 7: 2360-2366. Fincham, J.R.S., P.R. Day and A. Radford (1979). Fungal Genetics. 20-22, 28, 30, 31, 193, 441. Blackwell S c i e n t i f i c Publications, Oxford. Foss, E.J., P.W. Garrett, J.A. Kinsey and E.U. Selker (1991). S p e c i f i c i t y of Repeat-Induced Point Mutation (RIP) i n Neurospora: S e n s i t i v i t y of Non-Weurospora Sequences, a Natural Diverged Tandem Duplication, and Unique DNA Adjacent to a Duplicated Region. Genet. 127: 711-717. Garnjobst, L. and J.F. Wilson (1956). Heterocaryosis and Protoplasmic Incompatibility i n Neurospora crassa. Proc. Natl. Acad. S c i . USA 42: 613-618. Glass, N.L., J . Grotelueschen and R.L. Metzenberg (1990). Neurospora crassa A Mating-Type Region. Proc. Natl. Acad. S c i . USA 87: 4912-4916. Glass, N.L., S.J. Vollmer, C. Staben, J . Grotelueschen, R.L. Metzenberg and C. Yanofsky (1988). DNAs of the Two Mating- Type A l l e l e s of Neurospora crassa are Highly Di s s i m i l a r . Science 241: 570-573. Grayburn, W.S. and E.U. Selker (1989). A Natural Case of RIP: Degeneration of the DNA Sequence i n an Ancestral Tandem Duplication. Mol. C e l l . B i o l . 9: 4416-4421. Grindle, M. (1963a). Heterokaryon Compatibility of Unrelated Strains i n the Aspergillus nidulans Group. Heredity 18: 191- 204. Grindle, M. (1963b). Heterokaryon Compatibility of Closely Related Strains i n the Aspergillus nidulans Group. Heredity 18: 397-405. G r i f f i t h s , A.J.F. (1982). Null Mutants of the A and a Mating Type A l l e l e s of Neurospora crassa. Can. J . Genet. Cytol. 24: 167-176. G r i f f i t h s , A.J.F. and A.M. DeLange (1978). Mutations of the a Mating-Type Gene i n Neurospora crassa. Genet. 88: 239-254. G r i f f i t h s , A.J.F., S.R. Kraus, R. Barton, D.A. Court, C.J. Myers and H. Bertrand (1990). Heterokaryotic Transmission of Senescence Plasmid DNA i n Neurospora. Curr. Genet. 17: 139- 145. Gross, S.R. (1952). Heterokaryosis between Opposite Mating Types i n Neurospora crassa. Amer. J . Bot. 39: 574-577. 106 Haring, V., J.E. Gray, B.A. McClure, M.A. Anderson and A.E. Clarke (1990). Self-Incompatibility: A Self-Recognition System i n Plants. Science 250: 937-941. Harris, E.H. (1989). The Chlamydomonas Sourcebook A Comprehensive Guide to Biology and Laboratory Use. 127-136. Academic Press, C a l i f o r n i a . Hartig, A., J . Holly, G. Saari and V.L. MacKay (1986). Multiple Regulation of STE2, a Mating-Type-Specific Gene of Saccharomyces cerevisiae. Mol. C e l l . B i o l . 6: 2106-2114. Ha r t l , D.L., E.R. Dempster and S.W. Brown (1975). Adaptive Significance of Vegetative Incompatibility i n Neurospora crassa. Genet. 81: 553-569. Hayes, W. (1952). Recombination i n Bact. coli JQ2: Unid i r e c t i o n a l Transfer of Genetic Material. Nature 169: 118-119. Herskowitz, I. (1988). L i f e Cycle of the Budding Yeast Saccharomyces cerevisiae. Microbiol. Revs. 52: 536-553. Hicks, J.B. and I. Herskowitz (1976). Interconversion of Yeast Mating Types. I. Direct Observations of the Action of the Homothallism (HO) Gene. Genet. 83: 245-258. Hicks, J.B., J.N. Strathern and I. Herskowitz (1977). The Cassette Model of Mating-Type Interconversion i n DNA Insertion Elements Plasmids and Episomes (eds. A.I. Bukhari, J.A. Shapiro and S.L. Adhya), 457-462. C.S.H. Lab., New York. Jensen, R., G.F. Sprague, J r . and I. Herskowitz (1983). Regulation of Yeast Mating-Type Intersonversion: Feedback Control of HO gene Expression by the Mating-Type Locus. Proc. Natl. Acad. S c i . USA 80: 3035-3039. Jinks, J.L., C.E. Caten, G. Simchen and J.H. Croft (1961). Heterokaryon Incompatibility and Variation i n Wild Populations of Aspergillus nidulans. Heredity 21: 227-239. Johnson, T.E. (1979). A Neurospora Mutation that Arrests P e r i t h e c i a l Development as Either Male or Female Parent. Genet. 92: 1107-1120. Kassir, Y. and G. Simchen (1976). Regulation of Mating and Meiosis i n Yeast by the Mating-Type Region. Genet. 82: 187- 206. Katz, D.H. (1978). Self-Recognition as a Means of C e l l Communication i n the Immune System i n Cell-cell Recognition, 411-427. Cambridge University Press, Cambridge. 107 K e l l y , M., J . Burke, M. Smith, A. Klar and D. Beach (1988). Four Mating-Type Genes Control Sexual D i f f e r e n t i a t i o n i n the F i s s i o n Yeast. EMBO 7: 1537-1547. Klar, A.J.S. and J.N. Strathern (1984). Resolution of Recombination Intermediates Generated during Yeast Mating Type Switching. Nature 310: 744-748. Klar, A.J.S., J.N. Strathern and J.A. Abraham (1984). Involvement of Double-Strand Chromosomal Breaks f o r Mating- Type Switching i n Saccharomyces cerevisiae. C.S.H. Symp. Quant. B i o l . 49: 77-88. Kle i n , J . (1976). An Attempt at an Interpretation of the Mouse H-2 Complex. Contemp. Topics Immun. 5: 297-340. K o l t i n , Y., J . Stamberg and P.A. Lemke (1972). Genetic Structure and Evolution of the Incompatibility Factors i n Higher Fungi. Bact. Revs. 36: 156-171. Kostriken, R. and F. Heffron (1984). The Product of the HO Gene i s a Nuclease: P u r i f i c a t i o n and Characterization of the Enzyme. C.S.H. Symp. Quant. B i o l . 49: 89-96. Kwon, K. and K.B. Raper (1967). Heterokaryon Formation and Genetic Analyses of Color Mutants i n Aspergillus heterothallicus. Amer. J . Bot. 54: 49-60. Lederberg, J . (1957). S i b l i n g Recombinants i n Zygote Pedigrees of Escherichia coli. Proc. Natl. Acad. S c i . USA 43: 1060-1065. Lewis, D. (1954). Comparative Incompatibility i n Angiosperms and Fungi. Adv. Genet. 6: 235-285. Lindegren, C.C. and G. Lindegren (1943). A New Method for Hybidizing Yeast. Proc. Natl. Acad. S c i . USA 29: 306-308. McLeod, M. and D. Beach (1988). A S p e c i f i c I n h i b i t o r of ranl+ Protein Kinase Regulates Entry into Meiosis i n Schizosaccharomyces pombe. Nature 332: 509-514. Metzenberg, R.L. and S.K. Ahlgren (1973). Behaviour of Neurospora tetrasperma Mating-Type Genes Introgressed into N. crassa. Can. J . Genet. Cytol. 15: 571-576. Metzenberg, R.L. and N.L. Glass (1990). Mating Type and Mating Strategies i n Neurospora. Bioessays 12: 53-59. Mishra, N.C. (1971). Heterokaryosis i n Neurospora sitophila. Genet. 67: 55-59. 108 M i t c h e l l , A i P . and I. Herskowitz (1986). Activation of Meiosis and Sporulation by Repression of the RMEI Product i n Yeast. Nature 319; 738-742. Miyata, H. and M. Miyata (1981). Mode of Conjugation i n Homothallic C e l l s of Schizosaccharomyces pombe. J . Gen. Appl. Microbiol. 27: 365-371. Nanney, D.L. (1977). C e l l - C e l l Interactions i n C i l i a t e s : Evolutionary and Genetic Constraints i n Microbial Interactions (ed. J.L. Rei s s i g ) , 351-397. Chapman and H a l l , London. Nasmyth, K.A. and K. Ta t c h e l l (1980). The Structure of Transposable Yeast Mating Type L o c i . C e l l 19: 753-764. Nasmyth, K.A., K. T a t c h e l l , B.D. H a l l , C. A s t e l l and M. Smith (1981). A Position E f f e c t i n the Control of Transcription at Yeast Mating Type L o c i . Nature 289: 244- 250. Newmeyer, D. (1970). A Suppressor of the Heterokaryon- Incompatibility Associated with Mating Type i n Neurospora crassa. Can. J . Genet. Cytol. 12: 914-926. Newmeyer, D., H.B. Howe, J r . and D.R. Galeazzi (1973). A Search f o r Complexity at the Mating-Type Locus of Neurospora crassa. Can. J . Genet. Cytol. 15: 577-585. Newmeyer, D. and C.W.Taylor (1967). A P e r i c e n t r i c Inversion i n Neurospora, with Unstable Duplication Progeny. Genet. 56: 771-791. Olive, L.S. (1956). Genetics of Sordaria fimicola. I. Ascospore Color Mutants. Amer. J . Bot. 43: 97-107. Perkins, D.D. (1972). An Insertional Translocation i n Neurospora that Generates Duplications Heterozygous for Mating Type. Genet. 71: 25-51. Perkins, D.D. (1974). The Use of Duplication-Generating Rearrangements f o r Studying Heterokaryon Incompatibility Genes i n Neurospora. Genet. 80: 87-105. Perkins, D.D. (1977). Behaviour of Neurospora sitophila Mating-Type A l l e l e s i n Heterozygous Duplications a f t e r Introgression into Neurospora crassa. Expl. Mycol. 1: 166- 172. Perkins, D.D., A. Radford, D. Newmeyer and M. Bjorkman (1982). Chromosomal Loci of Neurospora crassa. Microbiol. Revs. 46: 426-570. 109 Pittenger, T.H. (1957). The Mating-Type A l l e l e s and Heterokaryon Formation i n Neurospora crassa. Microbial Genet. B u l l . 15: 21-22. Pittenger, T.H. (1964). Spontaneous Alterations of Heterokaryon Compatibility Factors i n Neurospora. Genet. 50: 471-484. Raju, N.B. (1980). Meiosis and Ascospore Genesis i n Neurospora. Eur. J . C e l l B i o l . 23: 208-223. R i c c i , N. (1981). Preconjugant C e l l Interactions i n Oxytricha bifaria ( C i l i a t a , Hypotrichida): A Two-Step Recognition Process Leading to C e l l Fusion and the Induction of Meiosis i n Sexual Interactions in Eukaryotic Microbes (eds. D.H. O'Day and P.A. Horgen), 319-350. Academic Press, N.Y. Sansome, E.R. (1949). The Use of Heterokaryons to Determine the Origin of the Ascogenous Nuclei i n Neurospora crassa. Genetica. 24: 59-64. Selker, E.U. (1990). DNA Methylation and Chromatin Structure: A View from Below. Trends i n Biochem. S c i . 15: 103-107. Selker, E.U., E.B. Cambareri, B.C. Jensen and K.R. Haack (1987). Rearrangement of Duplicated DNA i n Specialized C e l l s of Neurospora. C e l l 51: 741-752. Selker, E.U. and P.W. Garrett (1988). DNA Sequence Duplications Trigger Gene Inactivation i n Neurospora crassa. Proc. Natl. Acad. S c i . USA 85: 6870-6874. Selker, E.U. and J.N. Stevens (1985). DNA Methylation at Asymmetric Si t e s i s Associated with Numerous Transition Mutations. Proc. Natl. Acad. S c i . USA 82: 8114-8118. Shear, C.L. and B.O. Dodge (1927). L i f e H i s t o r i e s and Heterothallism of the Red Bread-Mold Fungi of the Monilia sitophila Group. J . Agr. Res. 34: 1019-1042. Smith, D.A. and D.D. Perkins (1972). Aneuploidy Associated with the Osmotic-Sensitive cut S t r a i n i n Neurospora. Genet. 71: S 6 0 . Sprague, G.F., J r . , J . Rine and I. Herskowitz (1981). Homology and Non-Homology at the Yeast Mating Type Locus. Nature 289: 250-252. Sprague, G.F., J r . , L.C. B l a i r and J . Thorner (1983). C e l l Interactions and Regulation of C e l l Type i n the Yeast Saccharomyces cerevisiae. Ann. Rev. Microbiol. 37: 623-660. 110 Staben, C. and C. Yanofsky (1990). Neurospora crassa a Mating-Type Region. Proc. Natl. Acad. S c i . USA 87: 4917- 4921. Strathern, J.N., J . Hicks and I. Herskowitz (1981). Control of C e l l Type i n Yeast by the Mating Type Locus: the alphal- alpha2 Hypothesis. J . Mol. B i o l . 147: 357-372, Strathern, J.N., A.J.S. Klar, J.B. Hicks, J.A. Abraham, J.M. Ivy, K.A. Nasmyth and L. McGill (1982). Homothallic Switching of Yeast Mating Type Cassettes i s I n i t i a t e d by a Double-Stranded Cut i n the MAT Locus. C e l l 31: 183-192. Strathern, J.N., E. Spatola, C. McGill and J.B. Hicks (1980). The Structure and Organization of Transposable Mating Type Cassettes i n Saccharomyces cerevisiae. Proc. Natl. Acad. S c i . USA 77: 2839-2843. Turner, B.C., C.W. Taylor, D.D. Perkins and D. Newmeyer (1969). New Duplication-Generating Inversions i n Neurospora. Can. J . Genet. Cytol. 11: 622-638. Vigfusson, N.V., D.G. Walker, M.S. Islam and J . Weijer (1971). The Genetics and Biochemical Characterization of S t e r i l i t y Mutants i n Neurospora crassa. F o l i a Microbiol. 16: 166-196. Wiese, L. and W. Wiese (1978). Sex C e l l Contact i n Chlamydomonas, a Model f o r C e l l Recognition i n Cell-cell Recognition, 83-104. Cambridge University Press, Cambridge. Williams, CA. and J.F. Wilson (1966). Cytoplasmic Incompatibility Reactions i n Neurospora crassa. Ann. N.Y. Acad. S c i . 129: 853-863. Wilson, J.F., L. Garnjobst and E.L. Tatum (1961). Heterocaryon Incompatibility i n Neurospora crassa—Micro- Injection Studies. Amer. J . Bot. 48: 299-305. Wilson, K.L. and I. Herskowitz (1984). Negative Regulation of STE6 Gene Expression by the alphaZ Product of Saccharomyces cerevisiae. Mol. C e l l . B i o l . 4: 2420-2427. Yeoman, M.M., D.C. K i l p a t r i c k , M.B. Miedzybrodzka and A.R. Gould (1978). C e l l u l a r Interactions during Graft Formation i n Plants, a Recognition Phenomenon? i n Cell-cell Recognition, 139-160. Cambridge University Press, Cambridge.

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 5 0
City Views Downloads
Mountain View 4 0
Ashburn 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items