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Studies of bisexual (A+ a) strains in Neurospora crassa DeLange, Aloysius 1975

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STUDIES OP BISEXUAL (A + a) STRAINS IN NEUROSPORA CRASSA "by ALOYSIUS DE LANGE B.Sc, University of Brit i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS POR THE DEGREE OP MASTER OP SCIENCE in the Department of BOTANY We accept this thesis as conforming to the required standard THE UNIVERSITY OP BRITISH COLUMBIA AUGUST, 1975 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or pub l i ca t ion of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wri t ten permission. Department of BOTANY The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date AUGUST 1975 i ABSTRACT In the f i r s t part of this thesis, a system was con-structed, which i s selective for recessive meiotic mutants in Neurospora crassa. This system employs bisexual (A + a) Pseudo-Wild Type cultures (produced from disomic ascospores), which were selected by means of several closely linked auxotrophic markers on L.G.I (the mating type locus i s also located on this linkage group). These Pseudo-Wild Type cultures consist of at least 2 nuclear components of opposite mating type (A and a). Consequently, they are s e l f - f e r t i l e . Moreover, since the cul-tures are homozygous for genes on other linkage groups, recessive mutants affecting meiosis may be detected. The second part of this thesis deals with escape from self-incompatible bisexual heterokaryons. In Neurospora crassa, strains of opposite mating type generally do not form stable heterokaryons because the mating type locus acts as a hetero-karyon incompatibility locus. However, when one A and one a strain, having complementing auxotrophic mutants, are placed together on minimal medium, some slow growth may result. Escape from slow growth to that at a wild type or near-wild type rate was observed. Most escape products are stable heterokaryons, which have lost one or the other of the mating type alleles from one of the component nuclei. These nuclei have therefore become heterokaryon compatible. Alternatively, when one com-ponent strain i s t o l and the other t o l * (tol being a recessive iiL ABSTRACT (CONT'D) mutant suppressing the heterokaryon incompatibility associated with mating type), escape may occur upon the deletion or mut-ation of t o l + , similarly causing heterokaryon compatibility. Deletion of more than one locus, including the mating type locus, was demonstrated in 3 cases. One such deletion covered most of the l e f t arm of LG I. An induction-type mechanism of escape i s speculated upon. i i i . TABLE OF CONTENTS PAGE PREFACE 1 PART I: INTRODUCTION 2 MATERIALS AND METHODS 14 RESULTS AND DISCUSSION 22 PART IIt INTRODUCTION 33 MATERIALS AND METHODS 34 RESULTS 36 DISCUSSION 44 REFERENCES 61 APPENDIX 1 67 APPENDIX 2 69 iv ; LIST OJ? TABLES Page No. Part I: Table I. Construction of strains for the selection of PWT's involving nondisjunction of L.G. I. 26 Table II. Frequency of nondisjunction in three crosses. ..... 28 Part II: Table I. Growth rate of representative heterokaryons. 50 Table II. Mating types detectable in escaped bisexual heterokaryons. 50 Table III. Conidial isolates recovered from 18 escaped bisexual heterokaryons 51 Table IY. Conidial isolates from 5 day old bisexual heterokaryons before escape. 52 ;!.v LIST OF FIGURES Page Ho, Part I: Figure l a . Growth of a disomic (Pseudo-Wild Type) culture on minimal medium 29 Figure lt>. Growth of two ascospores, each incapable of growth by i t s e l f , on minimal medium ..... 29 Figure 2. The genotypes, with their expected frequencies, of ascospores produced by a cross between 2 strains which are both multiply marked with closely linked auxotrophic (or otherwise conditional) mutants 30 Figure 3. Fractions of a l l possible combinat-ions of 2 ascospores, which have complementing auxotrophic mutants 31 Figure 4. An example of a cross between selective strains (1-30-225 x 1-34-8) 32 vvi LIST OF FIGURES (CONT'D) Page No. Part II: Figure 1. Growth curves of representative heterokarvons (see table I for genotypes; 54 Figure 2. Some representative growth curves of bisexual hetero-karyons, showing escape after more than 200 hours of inhibited growth 55 Figure 3 . Some exceptional growth curves of bisexual heterokaryons. The growth rate after escape sometimes decreased again or was cyclic 56 Figure 4. 3 typical types of growth of bisexual heterokaryons: a cul-ture homozygous for the t o l mutation (1), homozygous for t o l * or heterozygous (tol + tol*) under light conditions T^ T or i n the dark (3) 57 Figure 5. Typical shift down of growth rate after switch of bisexual hetero-karyon 1-9-16 + 1-22-83 from dark to natural light conditions (the bimodal curve appears to be a constant phenomenon) 58 Figure 6. Distribution of time of escape among (A + a) bisexual hetero-karyonsT ~~ (a) of genotype un-3. A, ad-3A, nic-2 (1-22-83) + a, aa>35 (1-9-16), and (D) of genotype un-3. A, ad-3A, nic-2 (1-22-83) + a, ad-3B; t o l CI-9-57) 7 59, 60 v i i ACKNOWLEDGEMEJIT I would like to thank Dr. A.J.J?. Gr i f f i t h s , whose ideas, suggestions, and criticisms were of great help in the completion of this thesis. 1 PREFACE This thesis i s composed of two parts. The f i r s t part involves the construction of a system selective for non-disjunction products of linkage group I (L.G. I ) . Since the mating type locus i s located on this linkage group, such nondisjunction products are heterozygous at this locus (A + a). Cultures heterozygous at the mating type locus grow poorly because of a mating type-associated vegetative incompatibility. While testing the degree of dominance of a suppressor (tol) of this incompatibility in bisexual (A + a) heterokaryons, a phenomenon to be called "escape" was observed. Part II of this thesis examines the nature of such escape. 2 PART I A SYSTEM SELECTIVE J?OR MEIOTIG MUTANTS IN NEUROSPORA CRASSA 2 a INTRODUCTION Three characteristic processes which differentiate meiosis from mitosis are pairing, recombination, and disjunction of homologous chromosomes during the f i r s t meiotic division. Even though precise descriptions of pairing and disjunction of such chromosomes are available in a variety of organisms (including Drosophila, maize, and fungi), l i t t l e i s known about their genetic control. In contrast, the understanding of the range of genetic control of recombination i n eukaryotes i s well advanced, especially in fungi. Aberrant pairing or disjunction of homologous chromo-somes during meiosis may result i n the nondisjunction of such chromosomes. This process produces complementary hyper- and hypoploid products. The aberrant products may be detected in a variety of ways i n different organisms. For example, morphol-ogical differences have been associated with hyperploid products in the Jimson weed Datura stramonium (Blakeslee and Belling, 1924), in Aspergillus (Kafer and Upshall, 1973), in yeast (James, Inhaber and Prefontaine, 1974), and in man (e.g. Down's Syndrome). The aberrant products may also be detected by means of cytology (in corn (McClintock and H i l l , 1931; Ward, 1973) and in mouse oocytes (Uchida, and Viola Lee, 1974)), by the use of compound chromosomes in Drosophila (e.g. Sandler et a l . , 1968), and by using disomies in Aspergillus heterozygous at several selective l o c i (Bignami et a l . , 1974). Moreover, the 3 detection of nondisjunction products in Neurospora crassa has been fa c i l i t a t e d by plating ascospores from crosses between two closely linked auxotrophic mutants on minimal medium (Mitchell, Pittenger and Mitchell, 1952), and by isolating black spores from crosses between two pan-2 heteroalleles (Threlkeld, 1965; Threlkeld and Stephens, 1966). In both cases, the selection method depends on complementation of the closely linked mutants in nondisjunction products. In order to study the genetic control of the normal processes of pairing and disjunction of homologous chromosomes during meiosis, one needs to be able to determine aberrant non-disjunction frequencies. Therefore, in choosing an organism for such a study, one must consider the a v a i l a b i l i t y of a relat-ively easy screening method for nondisjunction products and of an accurate determination of nondisjunction frequencies. Once such methods are available i t may become possible to select for mutants with aberrant frequencies. Characterization of such mutants could ultimately lead to a detailed understanding of the pairing and disjunction processes. Such characterization, how-ever, i s dependent upon genetical, cytological and biochemical analysis of mutant strains. The presence of sufficient select-ive markers may allow efficient screening of nondisjunction products in Drosophila, Aspergillus nidulans, Saccharomyces  cerevisiae and Neurospora crassa. In fungi, the cytological analysis of meiosis i s most advanced in Neurospora crassa 4 (Barry, 1972). In this organism, linkage groups and chromosomes can be correlated. In terms of genetical analysis, a l l four organisms are well suited because of their multitude of genetic markers. Biochemical analysis of the mutant strains i s possible i n vegetative cells of Neurospora. For the reasons just outlined, the fungus Neurospora  crassa has been chosen for the study of genetic control of meiosis by isolating and characterizing mutants with abnormal nondisjunction and/or recombination frequencies. The isolation of meiotic mutants of this kind has already been performed i n Dro3ophila (Sandler et a l . , 1968). Mutants at 14 l o c i have been isolated, using a complex system of homozygosis of naturally occurring recessive mutants (collected from wild populations). A l l these mutants appear to have a primary effect on recombinat-ion and a secondary effect on disjunction frequencies (Carpenter and Sandler, 1974). The understanding of the nature of these mutants awaits continued genetic and biochemical analysis. In Neurospora. a mutant (mei-1) with a high frequency of non-disjunction and an absence of recombination has been detected (Smith, 1973; Smith, 1975). This mutant was originally detected by i t s high frequency of spore abortion. A good selective system w i l l l i k e l y produce a number of such recessive meiotic mutants. The study of these mutants may render some insight into the genetic control of the processes involved. In the present communication, a selective system has 5 been constructed which enables the isolation of recessive meiotic mutants i n Neurospora crassa. The system involves homozygosis of induced mutations in clutures produced by disomic (n + l ) asco-spores. In Neurospora, disomic ascospores are generally produced by nondisjunction of two homologous chromosomes during meiosis.^ Cultures produced by disomic ascospores are generally referred to as Pseudo-Wild Types or PWT's, (Mitchell, Pittenger, and Mitchell, 1952). The selection of PWT's i s generally fa c i l i t a t e d by plating ascospores from a cross between two auxotrophic heteroalleles onto minimal medium. Colonies produced on these plates are either wild-type recombinants or PWT's. The PWT's can be distinguished from wild type colonies, since (i) they pro-duce mutant spores when crossed to wild type, ( i i ) they produce mutant colonies when asexual conidia are plated onto supplemented medium, and ( i i i ) on conidial plating, a larger number of col-onies are produced on supplemented than on minimal medium (Mitchell, Pittenger, and Mitchell, 1952). In order to eliminate wild type recombinants, asco-spores obtained from a cross between two strains, each containing 1 Disomic ascospores have been isolated from asci with 8 viable spores (Threlkeld, 1962; Case and Giles, 1964). In these cases some other unknown process probably leads to the disomic nuclei. 6 several closely linked complementing auxotrophic mutants, should he plated onto minimal medium (e.g. Smith, 1974). The mutants should be arranged i n the cross as follows: auxo-1 + auxo-3 + + auxo-2 + auxo-4 (A cross between auxo-1t auxo-3 and auxo-2, auxo-4; auxo-1 thru auxo-4 are closely linked auxotrophic mutants). If the auxo-trophic markers are closely linked, only a very rare multiple crossover event would produce wild type progeny. However, i f nondisjunction takes place during the f i r s t meiotic division, the resulting spore w i l l carry both chromosomes (auxo-1, auxo-3 + auxo-2. auxo-4). and w i l l be phenotypically wild type because of complementation of markers. Because of the i n s t a b i l i t y of disomic (n + 1) nuclei, chromosome loss and/or nondisjunction soon cause the breakdown of the disomic state to the haploid condition (Pittenger, 1954). During this process, called haploidization, a number of disomic nuclei w i l l lose the auxo-1, auxo-3 carrying chromosome, and the remaining nuclei w i l l lose the auxo-2. auxo-4 carrying chromo-some. Thus at l e a s t 2 two types of nuclei w i l l be present in each PWT, one containing the auxo-2 auxo-4 chromosome, the other 2 The presence of mitotic crossing over before disomic break-down, or of multiple disomy (n+2, n+3, etc.) may produce more than two types of nuclei (Threlkeld and Stephens, 1966; Pittenger and Coyle, 1963). 7 "kke auxo-1 , auxo-3 chromosome. Moreover, haploidization of dis-omic (n + 1) nuclei involves the homozygosis of a l l hut one chromosome. Therefore, the two nuclear types i n the PWT culture w i l l be identical for a l l but one chromosome. Thus i t may become possible to screen such cultures for recessive mutants. In order to screen for recessive meiotic mutants, however, the two nuclear types present in the PWT should be of opposite mating type such that crossing may take place. There-fore, linkage group I, on which the mating type locus i s located, was chosen as the selective chromosome (i.e. auxo-1 thru auxo-4 are linked to the mating type (mt) locus A/a). Since PWT's are heterokaryons, and (A + a) bisexual heterokaryons grow poorly because of mating type associated i n -compatibility (Beadle and Coonradt, 1944; Newmeyer, Howe, and Galeazzi, 1973), i t could be assumed that PWT's containing 2 nuclei of opposite mating type would grow very poorly. To elim-inate this problem, a mutation (tol) which suppresses this vege-tative incompatibility without affecting the crossing a b i l i t y was introduced. Thus s e l f - f e r t i l e PWT's, being homozygous for a l l but one chromosome, may be produced. In addition, the mutant markers on linkage group I allow the detection of abnormal re-combination and/or disjunction patterns produced by a (homozygosed) recessive meiotic mutant on another linkage group. The system proposed allows the isolation of mutants 8 defective in recombination as well as disjunction. The study of gene conversion (e.g. polarity and "marker effect"), and of the relation of gene conversion and recombination have contributed significantly toward the understanding of the mechanism and gene-t i c control of recombination in fungi. However, the f i n a l under-standing of the control of recombination awaits the isolation and characterization of a large number of mutants affecting the process. Several types of mutants have been isolated and examined for their effect on recombination. Mutants with abnormal recombination frequencies throughout the genome are referred to as mutants of "general effect", while those affecting only small chromosome regions or single l o c i are mutants of "local effect" (Catcheside, 1974). Several types of mutants have been examined for their "general effect" on recombination. Many UV sensitive (uvs) as well as some X-ray and &-ray sensitive mutants have been correl-ated with recombination frequencies i n Neurospora crassa (Schroeder, 1970), Aspergillus nidulans (Shanfield and Kafer. 1969; Jansen, 1970), Saccharomyces cerevisiae (table II in Catcheside, 1974), Schizosaccharomyces pombe (Nasim and Smith, 1975), and Ustilago maydis (Holliday, 1967). If one or more enzymic processes of the excision repair of DV-induced pyrimi-dine dimers are i n common with steps involving recombination, one may expect mutants of such enzymic processes to change 9 recombination frequencies. It was indeed found that several mutants in Ustilago maydis prevented UV-induced mitotic gene conversion and increased or decreased mitotic crossover freq-uencies. Similarly, some uvs mutants in Schizosaccharomyces  pombe showed a marked decrease in induced mitotic recombination (Fabre, 1972). Furthermore, increased mitotic recombination frequencies were found in Aspergillus and Neurospora mutants. It was concluded that some, but not a l l , steps of UV-induced dimer repair are in common with recombination processes. More-over, several pathways toward dimer repair are present, and mutants in different pathways affect recombination differently (e.g. Fabre, 1972; Game and Cox, 1973). Although some radiation sensitive mutants affect mitotic gene conversion and/or recombination frequencies, such an effect has never been observed during meiosis. Apparently, the meiotic processes of gene conversion and recombination are differently controlled than similar mitotic events. That some genes necessary for dimer excision repair are also required for the completion of meiosis i s demonstrated by the s t e r i l i t y of crosses homozygous for some uvs mutants (e.g. uvs-2 i n Ustilago, uvs-3 and uvs-3 in Neurospora. and rad-6 in Saccharomyces). and X-ray sensitive mutants (e.g. rad-51 and rad-53 in Saccharomyces). It has been suggested that such mutants vir t u a l l y eliminate recombination and therefore result in high levels of s t e r i l i t y i n sexual reproduction (e.g. Catcheside, 1974). 10 Many recessive s t e r i l i t y mutants have been isolated from Schizosaccharomyces pombe (Bresch et a l . , 1968), Saccharomyces cerevi3iae (Esposito et a l . , 1970), and Podospora  anserina (Simonet and Zickler, 1972). Several such mutants which block meiosis during the f i r s t division may affect one or more steps required for pairing, exchange, or disjunction of homo-logous chromosomes. For example, the mei-1 mutant i n Neurospora, which causes a lack of pairing as well as recombination, has been detected by a high degree (90%) of spore abortion. The system of homozygosis proposed during this study i s capable of isolating any mutant of this kind. The study of such mutants may ultimately lead to a more detailed understanding of the genetic control of meiosis. In order to identify the particular step where the mutant gene acts, i t i s preferable to isolate conditional (e.g. temperature sensitive or t.s.) mutants.^ The isolation of such mutants may also be achieved under the present format. In addition to uvs mutants, strains deficient i n induced mutation have been selected and tested for abnormal re-combination values (Lemontt, 1971). A l l three mutants isolated were UV sensitive, had normal meiotic recombination, and their 3 Two t.s. mutants defective in mitotic gene conversion were already isolated i n Saccharomyces cerevisiae (Campbell et a l . , 1974). Moreover, several t.s. sporulation defective mutants were studied i n S. cerevisiae (Esposito et a l . , 1970). 11 UV-induced mitotic recombination increased more sharply with radiation dose. Thus, basically, these mutants did not di f f e r significantly from most uvs mutants. Since i t appeared l i k e l y that not a l l enzymic pro-cesses involving recombination were involved i n UV-induced re-pair, mutants were selected i n S. cerevisiae for their lack of induced mitotic heteroallelic reversion (Shaffer et a l . , 1971; Rodarte-Ramon, 1972; Rodarte-Ramon and Mortimer, 1972; Campbell et a l . , 1974), and their lack of meiotic gene conversion (Roth and Fogel, 1971; Pogel and Roth, 1974). In both cases disomic (n_+l) strains were used such that gene conversion or reversion could be monitored on one set of homologous chromosomes i n an otherwise haploid background. Of seven mutants defective in induced heteroallelic reversion, two were X-ray sensitive, one UV and X-ray sensitive and the remaining four were neither UV nor X-ray sensitive. Moreover, a decreased spontaneous and i n -duced a l l e l i c recombination at meiosis was found i n one such mutant (rec-4). Two other mutants (rec-2 and rec-3) were s t e r i l e . These studies demonstrate that some (e.g. rec-2 and rec-3) hut not a l l (e.g. rec-4) genes which function during meiosis pro-duce mutants that interfere with the completion of meiosis. Moreover, they indicate that a number of enzymic processes i n -volved in recombination are not involved in dimer repair. Three mutants defective i n meiotic gene conversion at the leu-2 locus were isolated, (Pogel and Roth, 1974). A l l 12 three mutants caused s t e r i l i t y during meiosis. Both mutants defective i n mitotic heteroallelic reversion and those defective in meiotic gene conversion appear to be mutants of general effect on recombination. However, u n t i l their effect on other l o c i has been investigated, i t remains possible that they are mutants of "local effect". Genes of "local effect" on recombination have been identified by the presence of natural variants i n wild type pop-ulations of Neurospora crassa (Gatcheside, 1974), and Schyzo-phylum commune (Simchen, 1967; Koltin and Stamberg, 1973). There are at least two types of genes of such local effect. One class of genes (rec) i s not usually linked to the target regions in which recessive rec genes cause increased recombination. Such increases may involve a l l e l i c or non-allelic recombination or both. Moreover, i t appears that each rec gene i s specific i n i t s effect on a limited number of genes and/or chromosome regions. A second class of genes (cog)has a "local effect" on the region within which the cog genes are located. These genes only take expression in the presence of homozygous rec crosses. Contrary to the rec genes, recessive cog genes result in decreased recombination. It appears that cog genes are recognition sites of some nuclease or other enzyme associated with the i n i t i a t i o n of recombination. The complexity of this whole control system of recombination becomes even more apparent by the presence of indirect evidence for yet another type of gene of local effect 13 (con = control), which, would respond to the rec gene product. Clearly, many more variants or mutants need to be isolated and studied i n order to obtain a clear understanding of the pro-cesses involved. In this respect, the selective system des-cribed in this thesis could become instrumental in the isolation of a number of such mutants. For example, by screening for mutants with a high recombination frequency in small chromosome regions, i t may be possible to detect recessive rec genes of the type described. To recapitulate, the isolation of a variety of meiotic mutants, including disjunction mutants (e.g. mei-1) or temper-ature sensitive s t e r i l i t y mutants, mutants with "general effect" on recombination (some of these may possibly by found among the t.s. s t e r i l i t y mutants), and mutants of "local effect" on recombination (e.g. rec-genes i n Neurospora), i s made possible with the proposed selective system. The system also serves as an accurate means of determining nondisjunction frequencies. Therefore, screening of the effect of chemicals on nondisjunction frequencies becomes possible (the system i s presently used for such purposes by Dr. A.J.F. G r i f f i t h s ) . 14 MATERIALS AND METHODS Experimental format: Recessive meiotic mutants can only be identified or detected in crosses homozygous for such mutants. A method for the homozygosis and detection of such mutants in Neurospora crassa has been developed. Two strains, multiply marked for linkage group I, were constructed such that crosses between these strains would only produce disomic ascospore cultures on minimal medium. If one of these strains i s mutagenized before crossing, a fraction of meioses may contain a potential meiotic mutant (m). Such a meiosis may look as follows:4 L.G. I L.G.IV L.G.? leu-3 + a arg-1 + ad-3B + t o l m + un-3 A + ad-3A + nic-2 t o l + A disomic ascospore produced from this meiosis w i l l carry 2 complementing linkage groups I, and only one copy of the remain-4 A and a are the mating type al l e l e s , and the remaining markers on L.G. I a l l prevent growth on minimal medium at 37°C. t o l i s the suppressor of mating type-associated incompatibility, m may be on any linkage group except L.G. I. 15 ing linkage groups, some composition: leu-3 + a + un-3 A + ad-3A + nic-2 During the process of haploidization at least 2 types of nuclei are produced within a culture produced from this ascospore (see introduction): Nuclear component 1: leu-3 + a l a l r f i ' " 1 + ad-3B + t o l m Nuclear component 2: + i Ui N T ? L £ + frfTrffii + iPiifiCi"2 £ Since nuclear components of both mating types are present, this culture should be s e l f - f e r t i l e . Moreover, during haploidization, a mutant originally located on any linkage group except L.G. I, w i l l be present i n components of both mating types. Therefore, when this disomic ascospore culture i s placed on crossing medium, i t becomes possible to detect a recessive meiotic mutant by i t s abnormal recombination and/or disjunction freq-uency. Moreover, general meiotic s t e r i l i t y mutants w i l l be detected by the complete or partial absence of crossing. Strains: The following mutants were only used i n i t i a l l y to con-struct multiply marked strains: eys-5 (NM44), cys-11 (NM86), ser-3(47903), sue(35402). phen-1 (H6196), ad-5 (71104), arg-3 (30300), his-2 (C94), and his-3 (C140) (a l i s t of abbreviations i s presented in Appendix 2). A l l these mutants are located on Such a spore may have the following chromo-arg-1 + ad-3B + t o l m 16 the l e f t arm or centromere region of linkage group I. None of these mutants was used beyond the f i r s t stage of crossing, because no f e r t i l e and/or vigorous growing multiply marked strains could be obtained. Alternately, a multiply marked strain could not be used i f i t did not f i t the basic crossing system outlined in the previous section and introduction. The following mutants were f i n a l l y used in the construct-ion of the multiply marked strains: f r (B110) IL, leu-3 (R156) IL, un-3 (55701-t) IL, arg-1 (36703) IL, ad-3A (2-17-814) IR, ad-3B (2-17-114) IR, nic-2 (43002) IR, al-2 (15300 and 74A-T112-M38) IR, t o l (N83) IV R. Three vegetative incompatib-i l i t y l o c i C/c, IIL, D/d, IIR, and E/e, VII L (Wilson and Garn-jobst, 1966) were also introduced into the strains. These het genes were derived from the following strains: pan-1 (5531); al-2 (15300); eDE; A (Fungal Genetics Stock Center(F.G.S.C. ) no. 1425), pan-1 (5531); al-2 (15300); clffi; a (F.G.S.C. no. 1429), Cde; inos (37401); a (F.G.S.C. no. 1438), and Cde; inos (37401); A (F.G.S.C. no. 1453). Moreover, ad-3B (2-17-128); IR, which complements ad-3B (2-17-114) in heterokaryons, was used as a forcing marker in some t o l tester strains (see below). Wild types used were standard Oak Ridge strains 74-0R8-la (=0R-a), and 74-0R23-1A (=0R-A). Strains carrying the mutant frost (fr) have a characteristic frosty morphology; al-2 causes strains to be 17 albino instead of orange; un-3 i s a non-supplementable temper-ature sensitive mutation (i.e. growth, even though slow, at 25° but not at 37°); leu-3. arg-1. ad-3A. ad-3B. and nic-2 are non-leaky auxotrophs; t o l suppresses the heterokaryon-incompatibility associated with mating type without affecting crossing a b i l i t y (Newmeyer, 1970); the incompatibility l o c i A/a (mating type), C/c, D/d, and E/e cause inhibited or no growth i n a heterokaryon heterozygous at one or more of these l o c i . The order of the mutants on linkage group I with approximate map dis-tances between them i s as follows (Radford, 1972): f r (30) leu-3 (10) un-3 (0.1) A/a (9) arg-1 (9) ad-3A (0.3) ad-3B (4) nic-2 (30) al-2. Media: Four different basic types of medium have been used during the study. Cultures grown from ascospores were grown on vegetative medium. Nutritional tests were made on nutritional testing medium, crosses on liquid or so l i d i f i e d crossing medium, and a l l plating experiments were performed on plating medium (Appendix I ) . Where necessary, supplements were added at 3 0 0 mg/l. for amino acids and 50 mg/1. for vitamins. Conditions of vegetative growth and crossing of cultures: A l l original strains were obtained from s i l i c a gel cultures. The conditions of vegetative growth of cultures were as described i n Davis and deSerres (1970). Ascospores were heat-activated for half an hour at 60°C, and were allowed to grow at 25°C for 7 days, after which crossing and/or 18 nutritional testing took place. Standard crosses were made in tubes containing a piece of f i l t e r paper and 5 ml. crossing medium (Newcombe and G r i f f -iths, 1973). Alternatively, when many spores were required (e.g. to select PWT's), crosses were performed on so l i d i f i e d crossing medium i n petri plates (diam. 15 em.). One parent was inoculated on appropriately supplemented medium. After 7 days the conidia were removed from the plate by means of a standard suction device, and the conidial parent was added in a liquid suspension (10 ml. of suspension at a concentration of about conidia per ml.). Spores were collected from the walls of crossing tubes by means of a loop f i l l e d with a film of sterile d i s t i l l e d water, and transferred to a block of 4% agar from which they were picked. Alternatively, spores were removed from the l i d s of petri plates by means of a cotton swab, and they were sus-pended in sterile d i s t i l l e d water ready for plating. The genotypes of single ascospores were determined in a number of ways depending on the markers to be tested. Pirst, al-2 and f r were scored on the basis of color and morphology respectively. Second, the presence of ad-3A or ad-3B was primarily determined by the purple color of such strains. If a cross involved both ad-3A and ad-3Bt complementation tests with 19 ad-3A and ad-3B tester strains were carried out. Third, a l l auxotrophic mutants were routinely tested by means of standard nutritional tests (Davis and deSerres, 1970). Fourth, the presence of un-5 could be determined by i t s complete lack of growth at 37°C. Moreover, during this study, i t was found that the slow growth and typical morphology of strains carrying un-3 allows the routine identification of such strains at 25°C. Fif t h , tests for the presence of t o l were routinely carried out in heterokaryons between the strains i n question and standard strains of both mating types containing a complementing auxotrophic (forcing) marker and t o l . The tests were performed in 10 x 75 mm. tubes containing 1 ml. of minimal Vogel's medium (Vogel, 1956) so l i d i f i e d with 1.5% washed Difco agar. Vigorously growing heterokaryons are only produced i f the tested strain carries the t o l mutation. The recessiveness of the t o l mutation was established in the process of this study (see part II of this thesis). The standard t o l tester strains were obtained from the following two crosses: ad-3B (2-17-114), A x t o l a and ad-3B (2-17-128) A x t o l a. Tester strains of both mating types A and a and containing either one of these two mutually complementing auxotrophic mutants, were obtained. As determined by standard complementation tests, a l l these strains had the same heterokaryon compatibility alleles as Oak 20 Ridge wild type: Cde (Radford, 1972). Unless otherwise men-tioned, these compatibility alleles are always present. Sixth, the vegetative heterokaryon incompatibility l o c i C/c, D/d, and E/e were introduced in two different combinations, i.e. Cde and cDE. Heterokaryon tests were carried out as des-cribed for the t o l mutation. Standard tol-testers of each het constitution were obtained by means of complementation grids (Davis and deSerres, 1970). Finally, standard mating type tests were performed in 10 x 75 mm. tubes containing 1 ml. liquid minimal crossing medium and a piece of 1cm x 4cm f i l t e r paper. Each strain to be tested was inoculated into 2 tubes; several drops of an Oak Ridge A suspension were placed into one tube, and some drops of an Oak Ridge a suspension were placed into the other tube. The mating types were identified after four days by the presence of peri-thecia in one tube and the absence of the same in the other tube. Ascospore plating: Spores were collected from the l i d of each petri-plate four weeks after crossing, and suspended in sterile d i s t i l l e d water. The appropriate dilutions were made by adding 1 ml. of ascospore suspension i n 9 ml. sterile d i s t i l l e d water containing 0,1% agar. The agar largely prevents ascospores from sinking to the bottom of the tube, so that a higher degree of accuracy i n dilution i s obtained. The PWT's were picked from minimal plates (solidified with 1.5% washed difco agar) and transferred to 10 x 75 mm. tubes containing 1 ml. of Yogel's 21 minimal medium sol i d i f i e d with 1.5% washed difco agar. Finally, most platings were performed at concentrations of about 10 4 spores/plate, since i t was found that concentrations of close to 10^ ascospores per plate produced a disproportionately higher frequency of colonies, most l i k e l y due to heterokaryosis of separate ascospores on the plate. 22 RESULTS AND DISCUSSION In order to obtain as many combinations of double mutants as possible, closely linked auxotrophic mutants were crossed in 16 different combinations. The analysis of progeny from these crosses revealed that double mutants with the required characteristics (e.g. non-leakiness of markers, good recover-a b i l i t y of mutants, and good crossing a b i l i t y with complementary double mutants) were obtained from two crosses only (crosses 1-1 and 1-20 in table I ) . The efficiency of the selective system was f i r s t tested i n a cross between two strains of genotypes: a, ad-5A, nic-2, t o l ; Cde, and A, arg-1. ad-5B; t o l ; Cde. Ascospores from this cross were plated on "absolute" minimal medium at concen-trations of 10^, 10^, and 10^ per plate. 25 colonies were picked from plates, and 19 of these produced growth in culture tubes (the remaining six cultures died during transfer probably due to technical difficulties). A l l 19 cultures were s e l f - f e r t i l e and produced many spores, indicating that both mating types were present i n these strains. This confirmed the idea that ascos-pores disomic for linkage group I can produce s e l f - f e r t i l e cult-ures. Moreover, since a l l cultures were s e l f - f e r t i l e , wild type recombinants must be- very rare (less than 1 i n 10^ ascospores). This does not mean however, that a l l colonies formed were PWT's (produced by disomic ascospores). It i s possible that hyphae from two adjacent ascospores fuse on the plate. If the two 23 nuclear components making up the newly formed heterokaryon are compatible, and have complementing auxotrophic mutants, a colony w i l l be produced (figure l ) . Superficially, colonies produced by heterokaryosis on the plate, or from disomic ascospores cannot be distinguished.^ That such heterokaryosis did indeed take place at higher concentrations of plating was apparent from the disproportionate increase of colonies on plates with lO^, lo4, and 10^ spores respectively. Since the selective system (including homozygosis of meiotic mutants) i s dependent upon the selection of PWT cultures, most or a l l heterokaryosis should be eliminated. Therefore, crosses heterozygous at several heterokaryon incompatibility (het) l o c i are employed. The progeny of these crosses comprise a large number of combinations at the het l o c i . Since a difference at just one such locus causes an incompatibil-i t y reaction, the probability of heterokaryosis i s greatly reduced. For example, when three independently assorting het l o c i are heterozygous in a cross (e.g. Cde x cDE), a maximum of ( i ) ^ = 1/8 of possible combinations of spores with fusing hyphae w i l l form a vigorously growing heterokaryon. 5 A distinction may often be possible upon extensive study of single colonies, since cultures arising from disomic ascos-pores are generally made up of more than 2 types of nuclei. Such additional types are produced by mitotic recombination in the disomic nuclei (Pittenger and Coyle, 1963; Threlkeld and Stephens, 1966). 24 Moreover, to allow heterokaryosis between two spores, their genotypes must be complementary with respect to their auxotrophic markers. For example, a cross between the multiply marked strains used in the present system, leu-3. a, arg-1 t  ad-3B, and un-3, A, ad-gA, nic-2 produces a number of parental type as well as recombinant ascospores (figure 2). The chance that any ascospore from this cross w i l l form a vigorous hetero-karyon with any other ascospore (assuming that the protruding hyphae of these spores have fused) on the basis of complement-ation between auxotrophic mutants alone i s 0.30 (figure 3). Therefore, when a cross i s made between un-3. A, ad-3A. nic-2, al-2; t o l ; cDE (e.g. 1-34-8 in table l a ) , and leu-3. a, arg-1, ad-3B; t o l ; Cde (e.g. 1-30-225 in table lb), (figure 4), the chance that any two ascospores with fusing hyphae w i l l be able to form a vigorous heterokaryon i s 1/8 x 0.3 = 0.0375 or 3.75%. In order to test this estimate, the cross mentioned was made and 510 ascospores were isolated. Using the cultures produced by these spores, 455 random combinations were tested for vigorous growth on minimal medium. Vigorous heterokaryotic growth resulted in 32 cases (7.0%). The difference between the expected (0.0375) and observed (0.07) can be readily explained, since the C/c incompatibility factor produces only slightly inhibited heterokaryons. Indeed, several degrees of vigour were observed (even though not quantified) among the 7% heterokar-yons formed. 25 In conclusion, the use of incompatibility l o c i allows plating of ascospores at relatively high concentrations ( i t was found that about equal frequencies of colonies were produced up to a concentration of about 5 x IO 4 spores/plate). Consequently, i t has become possible to select large numbers of PWT cultures, which are necessary to screen for meiotic mutations. At this stage, a limited number of crosses have been used in the isolation of PWT's. The cross 1-34-8 x 1-30-225 has been repeated several times and the frequencies of colonies produced by plating ascospores on minimal medium was quite con-stant (table II). 80 colonies were picked and transferred to crossing medium. 78 cultures were s e l f - f e r t i l e , indicating that these were PWT cultures containing at least one nuclear component of each mating type (A + a). Moreover, the recovery of mutant mitotic segregants (experiment performed by Dr. A.J.P. Griffiths) confirmed that these strains were indeed PWT's. Finally, i n the light of the findings just reported, i t appears that the cross 1-34-8 x 1-30-225 i s suitable for the isolation of recessive meiotic mutations. The usefulness of other crosses needs to be confirmed by the study of PWT cultures from such crosses (e.g. crosses 1-34-6 x 1-33-16 and 1-34-8 x 1-33-16 produce many semi-sterile PWT's, and are therefore not suited for the isolation of recessive meiotic mutations.) 26 Table I - Construction of strains for the selection of PWT's involving non-dis.junction of L.G. I. (a) ad-3A containing strains CROSS NO. Genotype of PARENT STRAINS PROGENY ISOLATED 1-1 ad-3A a his-3(Y269M5). nic-2 A ad-3A, nic-2 a (1-1-86) 1-12 ad-3A, nic-2 a (1-1-86) ad-3B; t o l A ( 1-9-4)*— ~ ad-3A. nic-2; t o l a (1-12-22) 1-22 ad-3A, nic-2; t o l a ( 1 -12-22) un-3 A un-3. A, ad-3A, nic-2; t o l ; cde ( 1 - 2 2 - 2 4 ) 1-34 un-3. A, ad-3A, nic-2; t o l ; cde (1-22-24F pan-1; al-2; cDE aQPGSC 1429T un-3, A, ad-3A, nic-2, al-2; t o l ; cDE (l-34-'57"l-34r=8) 1-35 1-22-24 (see above) inos; Cde a XWSC T438T un-3, A, ad-3A, nic-2; t o l ; Cde (1-35-3, 1=55-FT" 1-37 un-3. A, ad-3A. nic-2; t o l ; Cde (T-3'5-3T" f r . his-2, nTe-2, al-2 a (OR^-la) f r , un-3, A, ad-3A, nic-2, al-2; t o l ; fide (1-57=21, 1=57-22) f r , un-3, A, ad-3A, nic-2; t o l ; Cde (1-37-23, T=57-25) 1-38 un-3, A, ad-3A, nic-2, al-2; t o l ; cDE 11=34=17 OR59-la (see above) f r , un^, A, ad-3A, nic-2, al-2; t o l ; cDE (1-38-26, 1=58-28) * see part (b) of this table -27 Table I Cont'd (b) ad-3B containing strains CROSS Genotype of PARENT STRAINS PROGENY ISOLATED NO. 1-9 ad-3B A t o l a ad-3B; t o l a TTZ^42rad-3B; t o l A(1-9-4) A, arg-1, ad-3B; T o l (1-20-25) 1-20 ad-3B; t o l a arg-1 A 1-24 A, arg-1, ad-3B: a, ad-3A, nic-2; a, arg-1, ad-3B; toiTfeo-2TT- toi"1l^i2-^2T^ t o i u ^ l - T T ) 1-30 a, arg-1, ad-3B; leu-3. A, ad-3A, leu-3. a, arg-1, t o l (1-24-11) al-2 (lTl-2-28) ad-3B, al-2; t o l — — *** u=5$-2?rr -— leu-3, a, arg-1, ad-3B; T o l T T ^ - 2 2 3 T 1-32 leu-3, a, arg-1, pan-1; a l - 2 ; cDE leu-3 i a, arg-1. ad-3B. al-2; t o l A (FGSC~IT25) ad-3B, al-2. t o l ; n^-22^r — " cMri-32^T4)— 1-33 1-30-229 inos; Cde A leu-3. a. arg-1. (see above) TfgsC 14337 ad-3B, al-2; t o l ; Cde \l-3jl2"t T^ 3*3-4) leu-3. a, arg-1, ad-3B; t o l ; Cde (1-33-16, 1^3-18) ** see part (a) of this table *** a l l e l e of al-2 i s 74A-Y112-M38 28 Table II - Frequency of non-disjunction in three crosses* GROSS FREQUENCIES OF PWT's 1-34-6 x 1-33-16 2.1 x 10"5, 5.2 x IO*"5 1-34-8 x 1-33-16 1.9 x 10-5, 3.5 x 10~5 1-34-8 x 1-30-225 4.0 x 10-5, 5.3 x 10-5 4.7 x 10-5, 8.0 x IO" 5 (p> 0.1)** * Platings at about 10^ spores per plate were only considered here, since heterokaryotic colonies often appeared at 10^ spores per plate (see text). **p was determined by means of the Xd test, using contingency tables. 29 Figure l a - Growth of a disomic (Pseudo-Wild Type) culture on minimal medium, f i r s t "by complementation of the 2 differently marked homologous chromosomes, later by complementation of 2 types of haploid-ized nuclei. Figure lb - Growth of two ascospores, each incapable of growth by i t s e l f , on minimal medium by fusion of protruding hyphae and formation of a compat-ible heterokaryon. (See "experimental format" for genotypes.) 29 a Figure ID 30 Figure 2: The genotypes, with their expected frequencies, of ascospores produced by a cross between 2 strains which are both multiply marked with closely linked auxotrophic (or otherwise conditional) mutants. CROSS: leu-3 + arg-1 + ad-3B + nic-2 4mu Fraction of Total Progeny 0.34 0.34 ' 0.05 0.05 0.045 0.045 0.045 0.045 0.02 0.02 1.00 * Recombinants between ad-3A and ad-3B have "been ignored in this analysis because of their relatively rare occurrence. Multiple crossovers have been ignored for the same reason. + un-3 + ad-3A + lOmu 9mu 9mu 0.3mu Genotypes of Ascospores: Parental: leu-3. arg-1. ad-3B. (l) un-3. ad-3A. nic-2 (2) Recombinant:* leu-3. un-3. ad-3A. nic-2 (3) arg-1. ad^3B JJ) leu-3. ad-3A. nic-2 (5) un-3. arg-1. ad-3B (6) leu-3. arg-1. ad-3A. nic-2 (7) un-3. ad-3B (81 leu-3. arg-1. ad-3B. nic-2 (9) un-3. ad-3A (10) Total Figure 5 - Fractions of a l l possible combinations of 2 ascospores, which have complementing auxotrophic mutants* Genotypes** of ascospores with .34 .34 .05 . 0 5 .045 .045 .045 .045 . 0 2 .02 fus^fcypfcae 111 ,1&9121 i l l 151. UL i§L. 121 ii2> 734llT~"~ "~ (tit)2 — — 734~x .02 .34 (2) (.34)2 .34 x .05 .05 (3) (.05)2 .05 (4) .05x (.05)2 .05x .05x .02 .34 .045 .045 (5) .045x (.045)2 (.045)2 .05 .045 (6) (.045)2 .045 (7) (.045)2 .045 (8) (.045)2 (.045)2 .02 (9) (.02)2 .02 (10) .02x .02x (.02)2 .34 .05 Total chance of any ascospore having complementing auxotrophic mutants to any other ascospore i s : 2(.34) 2 + 2(.05) 2 + 6(.045) 2 + 2(.02)2 + 2(.02 x .34) + 2(.02 x .05) + 2(.05 x .045) + 2(.05 x .34) - . 3 0 *only fractions of compatible combinations are recorded in the figure. **for genotypes see figure 2. Figure 4 - An example of a cross between selective strains (I-3Q-225 x 1-54-8)  leu-3 L.G. X + a arg-1 ad-3B un-3 A ad-5A nic-2 al-2 L.G. II D L.G. L.G. IV VII tol t o l e PART II ESCAPE IN FORCED (A + HETEROKARYONS IN NEUROSPORA CRASSA 33a INTRODUCTION Part I of this thesis describes the construction of strains, which when crossed to each other produce spores from which PWT's involving non-disjunction of L.G. I can be selected. Such PWT's necessarily carry both A and a components. Since, because of mating type associated vegetative incompatibility, vigorous heterokaryons w i l l normally not be produced between strains of opposite mating type (A + a) (Beadle and Coonradt, 1944; Garnjobst and Wilson, 1956), the t o l mutation (Newmeyer, 1970), which suppresses this incompatibility was introduced in the strains. To confirm the suppressor effect of t o l in a (A + a) bisexual heterokaryon, forced (A t o l + a tol) hetero-karyons were grown in growth tubes. Moreover, to establish whether the suppressor was dominant or recessive i n a hetero-karyon, several (tol + t o l + ) bisexual heterokaryons were tested i n growth tubes. During this analysis, a phenomenon which w i l l be called 'escape' was observed. Certain (A + a) heterokaryons escape from their i n i t i a l inhibited growth to grow at wild or near wild type rates. This escape phenomenon has been studied in some detail, and is due to genetic change, usually by deletion at the mating type locus, or sometimes by mutation or deletion at the t o l locus. 34 MATERIALS AND METHODS Strains - The mutants used are identical to those in part I. A l l strains used in this study have the same heterokaryon compat-i b i l i t y (het) locus constitution as Oak Ridge (this was demon-strated by extensive heterokaryon tests). Procedures - Two cultures of opposite mating type, and having complementary forcing markers, were jointly inoculated at one end of a 45 cm. growth tube containing 25 ml. of Vogel's medium (Vogel, 1956) s o l i d i f i e d with 1.5% washed Difco agar. Alternat-ively, a drop of conidial suspension containing both nuclear types was transferred to such tubes. The resulting growth w i l l be referred to as a 1(A + a)' or 'bisexual' heterokaryon. It i s possible a p r i o r i that growth could also result from crossfeeding without c e l l fusion, but evidence w i l l be reported which suggests some degree of fusion and heterokaryon formation. The forcing markers used were any pairwise combination of the following three genotypes: ad-3B(2-17-114). ad-3B(2-17-128). and ad-3A. nic-2. Conidial genotypes were determined by plating conidia on appropriately supplemented medium, and subsequently testing the single colony isolates on the basis of their presence or absence of nic-2 (lines 1-10 i n table III). When un-3 was present i n one nuclear component (lines 11-18 in table III and table IV), the un-3 single colony isolates could be directly identified on the supplemented plates because they were small 35 sparse colonies. Moreover, normal size colonies were scored as ad-5B when purple, and as heterokaryotic when orange. Orange could not he scored confidently when the majority of colonies on the plates were purple. In these cases a dash is inserted in table III. A direct measure of the number of heterokaryotic colonies was obtained from the escaped heterokaryons in table IV and on lines 15 and 17 in table III by plating conidia of these cultures on minimal medium. The escaped heterokaryons were tested for mating type, but the individual conidial isolates were not scored for mating type. The media and routine manipulations have been des-cribed under part I of this thesis. Some standard manipulations not described there may be found in Davis and deSerres (1970). Growth tubes were routinely incubated at 25°G, under natural lighting conditions. 36 RESULTS Table I and Figure 1 show the growth rates and patterns of 6 heterokaryons. The complete suppressive effect of t o l i s evident from a comparison of the bisexual heterokaryons 3 and 4 with the homozygous mating type heterokaryon 6. However, the i n i t i a l slow growth of heterokaryon 5 appears to indicate that to l i s recessive in such heterokaryons. In order to definitely establish the recessiveness of t o l , 29 (A + a) heterokaryons, each containing one component with t o l and one with t o l + were grown in growth tubes. The mean i n i t i a l growth rate of these cultures was 1.05 cm/day (s=0.3), compared to a mean of 0.6 cm/day (s=0.07) for ( t o l * + tol+) bisexual heterokaryons and about 9.0 cm/day for homozygous t o l heterokaryons. Thus, in terms of growth rate of bisexual heter-okaryons* t o l i s a recessive mutation. The 3 escapes observed in heterokaryons 1, 2 and 5 were the f i r s t observed. As shown in figure 1, escape i s characterized by a sudden increase in growth rate to a rate approaching or equalling wild type. To investigate the nature of escape, 48 bisexual heterokaryons were grown i n growth tubes. 45 of them escaped. Escaped cultures were tested for their a b i l i t y to produce perithecia and ascos-pores with standard mating type testers, OR A and OR a (table II). Three classes of behaviour could be distinguished on the basis of the results from the mating type tests: 37 class 1: perithecia and ascospores were produced with one tester only (42) class 2: perithecia resulted with both testers, but ascospores were produced with one tester only (2) class 3: perithecia and ascospores were formed with both testers (1) Conidia of 7 class 1 and a l l 3 class 2 and 3 escaped cultures were plated and single colonies isolated. The geno-types of these isolates were then determined and their freq-uencies in each of the above 10 escaped cultures are shown in the f i r s t 10 lines of table III. The single class 3 culture i s recorded on line 10. This culture shows both the characteristics of a (tol + tol) bisexual heterokaryon, that i s , i t grows at wild type rate and consists of two viable components of opposite mating type. Since the heterokaryon has originally ( t o l + + t o l ) , the escape event could have been due to a mutation from t o l + to t o l in the ad-3B component. To test this idea, a a, ad-3B conidial isolate from the escaped culture was crossed to a A, ad-3A, nic-2; t o l strain. A l l the progeny of this cross were of t o l genotype so i t was concluded that the t o l + a l l e l e had mutated to t o l , making the escape possible. The class 1 and class 2 escaped cultures evidently contain only one viable nuclear type, since only one component genotype was recoverable as a single colony isolate in each case. Yet class 2 differs from class 1 i n that in the former 38 a mating type reaction i s obtained with both testers, although only one of these reactions produces ascospores. Three a p r i o r i p o s sibilities were considered for the origin of these classes. Reversion of forcing markers - This i s unlikely since in many cases i t is the double mutant ad-3A» nic-2 which would have to revert. Furthermore, the ad-3A + 1 ad-3B+, nic-2* genotype i s never recovered as a homokaryotic single colony isolate even though i f present i t might be expected to "be a majority component of the escaped mycelium. Mitotic crossing-over - Reports of this are rare in Neurospora heterokaryons (the only claim i s by Weijer and Dowding, I960). Two types of exchange could account for the results. F i r s t l y , an exchange in the ad-3A to mt region could generate genotypes capable of forming unisexual heterokaryons. For example, a a, ad-3A, nic-2 component could be modified to a A, ad-3A, nic-2 component which would then complement with A, ad-3B nuclei. However, no genotype corresponding to A, ad-3A. nic-2 in this example was detected i n the single colony isolates. Secondly, a double exchange in the ad-3A to ad-3B and ad-3B to nic-2 regions could produce an ad-3A* 1 ad-3B+, nic-2* nucleus, but as pointed out above, this genotype i s not detected. Furthermore, neither the reversion nor mitotic exchange hypotheses adequately account for the absence of one original nuclear component among the single colony isolates. 39 Deletion or l e t h a l mutation of mt or t o l a l l e l e s - Class 1 could be caused by the d e l e t i o n or l e t h a l mutation of one mating type a l l e l e , and c l a s s 2 by the del e t i o n or l e t h a l mutation of a t o l * a l l e l e i n a ( t o l + + t o l ) heterokaryon. This accounts f o r the persistence of the complementing n u t r i t i o n a l a l l e l e s to allow heterokaryotic growth, f o r the f a i l u r e to recover one component as a homokaryon, and f o r the f a i l u r e of the nonrecoverable t o l * component to produce ascospores. The del e t i o n hypothesis was fu r t h e r pursued, as follows. I f d e l e t i o n of one mating type can cause escape, t h i s escape should sometimes be accompanied by the unmasking of a linked recessive marker i n the other nucleus. 9 hetero-karyons of the genotype (un-5+, a., ad-3B + un-3, A_, ad-3A, nic-2) were allowed to escape. 2 of the 9 escaped cultures showed recovery of only A mating type, i n d i c a t i n g the loss of a, and these were also temperature s e n s i t i v e , i n d i c a t i n g that both a and un - 3 + had been l o s t from the nonrecoverable component. In an attempt to obtain a l a r g e r sample of deletions i n the a nuclear component, 45 heterokaryons of the above genotype were allowed to escape. (Some represent^..! at i v e , and some exceptional (~10%) growth curves are shown i n f i g u r e s 2 and 3 r e s p e c t i v e l y ) . 21 of these were t o l + + t o l + and a l l 21 escaped cultures were of c l a s s 1. The other 24 were t o l + + t o l , and of these, 20 escaped cultures were class 1 and 4 were c l a s s 2. Four c l a s s 1 and four class 2 were plated to obtain single colony i s o l a t e s and the recovery of component genotypes from these i s shown on 40 lines 11 through 18 in table III. Once again the recoverability of the forcing marker genotypes i s perfectly correlated with mating type recovery. Even though a second minority nuclear type was found in 2 cases (lines 13 and 15 in table III), each of these produced inhibited heterokaryons with the majority component. Presumably these minority types represent nuclei carried along in the escaped heterokaryon. Of the 41 class 1 escapes obtained, 39 showed a only, and 2 showed A only. One of the latter was temperature sensitive, and the other was not. Thus escape i s due predominately to deletion or lethal mutation in the A component in these hetero-karyons, and genetic changes of a involve a deletion spanning un-5* in a total of 3 out of 4 cases. The three deletions span-ning un-5+ were subjected to further analysis to see the extent of the deletion. A third component was inoculated with the deleted hetero-karyons, to form cultures of the following type: component 1 un-3 A ad-3A nic-2 component 2 deletion ad-3B component 3 leu-3 a arg-1 At 37°C, components (1+2) w i l l not grow, while (1+3) grow slowly because of mating type incompatibility. However, ( 2 + 3 ) grows normally unless the deletion extends to leu-3"1" or a r g - l + 41 in which case leucine or arginine or both would he needed for growth. 2 of the 3 deletions spanning un-3"*" did not extend to leu-3* or a r g - l + t but the other one did extend across leu-3 +. This large deletion was subsequently shown to extend to the f r + locus in a system where the third component was f r , a, ad-3A, nic-2: the growth at 37° showed the frosty phenotype. Since f r is the most distal marker on IL, the deletion spans most of the l e f t arm of chromosome 1. Nuclear ratios before escape: Especially since A mating type was recovered i n only 2 out of 41 class 1 escapes in the above heterokaryons, i t was of interest to determine the nuclear ratios before escape. For this purpose, and to examine the possibility that the original nuclear ratios at the time of inoculation partly determine escape patterns, 27 heterokaryons with widely differing conidial input ratios were grown i n growth tubes. A l l but one culture escaped to a mating type (only a containing nuclear type was recovered). The remaining culture escaped to A mating type. Moreover, con-idi a from unescaped 5 day old heterokaryons were plated and their genotypes determined (table IV). In each case, an excess of the a component was found. This could mean that the nuclear ratios of these heterokaryons before escape are inherently skewed and that deletion or lethal mutation causing escape usually occurs in the minority component. Alternatively, many lethal (deleted) A component nuclei may have been present before 42 escape. Influence of environmental stimuli (light and dark) on escape: During an early stage of the experiment i t had been observed that some bisexual heterokaryons grown under dim light conditions appeared only very l i t t l e inhibited after a few days of growth. In this case either escape had taken place very early or inhibition associated with mating type incompatibility was not expressed. The former possibility appears more l i k e l y since several other bisexual heterokaryons are well inhibited under "dim li g h t " conditions. In order to investigate the effect of light on escape, 25 heterokaryons of genotype un-3. A, ad-3A. nic-2 (1-22-83) + a, ad-3B (1-9-16) were grown in complete darkness. These cul-tures quickly "escaped" to growth at rates between 4.0 and 6.0 cm/day (figure 4). However, when such cultures were shifted to the light, the growth rate declined within a few days, to less than half the rate. Figure 5 shows a typical pattern of decline of growth rate after the switch from dark to l i g h t . The above mentioned results may possibly be explained by the loss of a mating type and the adjacent un-5+ locus from the a, ad-3B component. Assuming such a deletion, the resulting heterokaryon i s hemizygous for un-3. Several heterokaryons hemizygous or homozygous for un-3 have been grown in growth tubes, and their growth rates (5.5 - 6.0cm/day) are within the range found for the heterokaryons from the dark. Loss of a 43 mating type i s also supported by the recovery of only A nuclear types when the escaped cultures (from the dark) are plated. Induction of escape; Most escapes appeared to take place in a relatively narrow interval of time (within the second week) after inocul-ation, but some occurred much later (see Figure 6). To obtain more information on the time of escape in uniform heterokaryons, a heterokaryon was constructed from strains grown from single conidial isolates. Young conidia from this heterokaryon were plated to obtain 43 heterokaryotic condial isolate colonies which were then transferred directly to growth tubes. In 33 cases escape took place between day 10 and 13, and in the remain-ing 10, escape did not occur u n t i l 30 days or later. At day 15, conidia from the late-escaping cultures were obtained from 1 cm behind the growing fronts, and transferred to fresh medium. Con-idia from 3 of these 10 cultures grew at wild type rates, and the remaining 7 grew at the inhibited rate typical of bisexual heterokaryons. Thus, in at least 3 cultures nuclei were present before escape which were altered and capable of escape. The dem-onstration of escape in heterokaryons derived from single heterokaryotic conidia makes i t unlikely that the cultures reported on i n this work are crossfeeding unfused homokaryons. 44 DISCUSSION In this study i t has been demonstrated that (A + a) or bisexual heterokaryons can be forced to grow on minimal medium, even though such growth i s generally very poor. The growth on minimal medium of single conidia from a forced (A + a) culture strongly supports the concept that heterokaryosis between A and a strains does indeed take place. The latter had been previously demonstrated by Gross (1952). Even though crossfeeding of nutrients may also take place, the (A + a) cultures are referred to as (A + a) or bisexual "heterokaryons". The t o l mutation, which had previously been shown to suppress the mating type incompatibility in duplications (Newmeyer, 1970), also suppresses this incompatibility i n (A + a) heterokaryons. However, i n order for the suppression to take effect, both components of the heterokaryon should carry the t o l mutation. The recessiveness of t o l i n heterokaryons had not been previously established. Many bisexual heterokaryons i n i t i a l l y grew very slowly, but escaped after a varying number of days to a growth rate which usually approached that*of wild type cultures. This escape phen-omenon can be caused by deletion of a mating type a l l e l e , or by deletion or mutation of the t o l + a l l e l e to t o l . The mutation of a mating type a l l e l e cannot be ruled out under the present for-mat. However, i f such mutations do take place they have to be recessive lethal mutations. Moreover, in such a case the 45 incompatibility locus associated with mating type, and the mating type locus i t s e l f would have to be one and the same locus. This would certainly hold with the previously made observation that these two hypothetical l o c i cannot be gene-t i c a l l y separated (Newmeyer, Howe, and Galeazzi, 1973). In further discussion, since deletion of the mating type locus has been demonstrated, and since recessive lethal mutations are functionally equivalent to deletions, the term 'deletion' w i l l be used to include both. Escape has previously been observed i n other fungi, and i n other Neurospora systems, where slow 'inhibited' growth i s occurring. In Aspergillus, duplications show inhibited growth, and deletion of part of a duplicated sequence has been shown to cause escape (Bainbridge and Roper, 1966; Nga and Roper, 1968). In such cultures, the duplications themselves appear to cause an "in s t a b i l i t y " which produces a reduction in growth rate and an increased deletion rate specific for the duplicated sequence. In Neurospora too, duplications have been the sub-ject of studies on escape (Newmeyer and Taylor, 1967; Perkins, 1972; Mylyk, 1975). In Neurospora. however, duplications are only detectably inhibited i n the presence of heterozygosity at one or more heterokaryon incompatibility l o c i . Deletion of one of the alleles at such l o c i eliminates the incompatibility reaction, and thus f a c i l i t a t e s normal growth. Some examples are duplications carrying the het alleles C and c, or the mating type alleles A and a. Besides deletion, escape i n such 46 systems could be caused by mutation or somatic crossing over. However, no evidence for the latter two possible modes of escape has been compiled to date, the main reason being that duplications are inherently unstable even i n the absence of heterokaryon incompatibility a l l e l e s . Unlike duplications, forced heterokaryons are quite stable and the identification and interpretation of events caus-ing escape i s simpler. The f i r s t demonstration of escape from forced heterokaryons was reported by Pittenger (1964). He used heterokaryons heterozygous for the het alleles J /J , or K/k, and observed very inhibited growth from which escape sometimes (but not usually) occurred. Two types of escape were observed. Pirst, a number of these escaped heterokaryons contained two homo-karyotically viable components, one of which had undergone an apparent mutation of one het a l l e l e to the other a l l e l e (e.g. a change from jK to JI or from jjk to jjK). Second, a l l other escaped heterokaryons contained just one homokaryotically viable component. As to the mechanism of escape i n these cultures no distinction could be made between mutation and somatic crossing over in the former types of escape (i.e. j-*J or k+K), and between deletion, recessive lethal mutation, and non-disjunction i n the latter type of escape. In escape from (A + a) heterokaryons (observed i n the present analysis) no viable mutations of the mating type locus were observed (A-*a or a-»A). A l l escapes i n these cult-47 tires ( i f originally homozygous t o l + ) resembled the second type of escape from (J + j_) or (K + k) cultures. In this study, i t was demonstrated that deletion of the mating type locus renders the affected nucleus compatible to both A and a. Moreover, mitotic crossing-over and non-disjunction could be ruled out as possible mechanisms of escape in these heterokaryons (see Results). Thus escape of homozygous t o l + (A + a) heterokaryons i s caused by deletion and moreover may be caused by lethal mutation of the mt locus. Although i t has been established that deletion i s a prime cause of escape i n bisexual heterokaryons, the way i n which this happens, and when i t happens, i s less well understood. Whether the event takes place i n one or the other component appears to be a function of genotype and environmental stimuli. For instance, i n some heterokaryons A gets deleted, in others a. Moreover, light appears to influence the nature of escape. Deletions appear to be involved predominantly i n the dark and not infrequently in the light (see Results). Moreover, i n the heterokaryon studied most extensively (1-9-16 + 1-22-83) under dark/light conditions, escape appears to take place by deletion of different components. Even though light-sensitive repair system(s) may be instrumental i n this different pattern of escape, no clear explanation concerning the cause of escape can be given. 48 Induction of escape? One constant feature of escape, whether duplications or (A + a) heterokaryons are involved, i s the time of escape. Most cultures w i l l escape within a relatively small period of time (e.g. between 10 and 12 days after i n i t i a l growth). To explain such behaviour, two alternative p o s s i b i l i t i e s w i l l be considered. Fi r s t , assuming a constant deletion rate, a deletion i s l i k e l y only after the number of nuclei has exceeded the rec-iprocal of this rate. Thus, the achievement of a particular nucleus population size may be the c r i t i c a l event for escape. However, some cultures did not escape "on time" but much later. This pattern of escape i s not consistent with a constant dele-tion or mutation rate, since the time of escape should be norm-a l l y distributed i n such a case. It might be argued that the late escapers were incapable of escape 'on time* because they had not reached a sufficient population size. However, this seems unlikely for the following two reasons. F i r s t l y , a l l cultures (both early and late escapers) were derived from a single conidial isolate and thus were practically isogenic. Therefore, i t appears unlikely that some cultures were capable while others were not capable of escape. Secondly, escaped nuclei were shown to be present within one centimeter behind the growing front of cultures that did not escape u n t i l several more weeks had passed. The second possibility i s that inhibited growth t r i g -49 gers the induction of a system which causes deletions, either specifically at the incompatibility l o c i , or i n general. The induction of an unidentified active compound by the interaction of the A and a alleles i n the normal sexual cycle has been demon-strated by Vigfussen and Cano (1974). This product, necessary for meiosis to occur, was present between 50 - 200 hours after contact between the A and a strains. A similarly produced sub-stance, or the same substance, could induce deletion or mutation a specific time after the formation of a bisexual heterokaryon. This type of induction system may also be functional i n self incompatible duplications. Several duplications do escape a fixed number of days after ascospore germination and moreover, the time of escape i s at least partly genetically determined since a mutation (breaker) has been found which causes escape to occur one or two days earlier than normal (Newmeyer and Galeazzi, 1974). 50 Table I - Growth rates of representative heterokaryons EE component 1 HK component 2 I n i t i a l growth rate (cm/day) Growth rate after escape (cm/day) 1 a t ad-3B (114) A, ad-3B (128) 0.4 8.7 2 A, ad-3B (114) a i ad-3B (128) 0.4 7.6 3 A, ad-3B (114): t o l a t ad-3B (128); t o l 9.4 4 A, ad-3B (114): t o l a i ad-3B (128): t o l 8.7 _ 5 a t ad-3B (114): t o l A» ad-3B (128) 0.8 7.9 6 A, ad-3B (114) A, ad-3B (128) 8.8 m. Table II - Mating types detectable in escaped bisexual hetero karyons genotype of genotype of Average No. of escaped heterokaryons ad-3A. nic-2 component ad-3B component I n i t i a l growth rate (cm/day) only a only A detected detected both A and a" detected a; t o l + A; t o l + 0.6 4 2 0 a; t o l + A; t o l 0.9 10 1 0 a; t o l A; t o l + 1.1 7 0 1* A; t o l + a; t o l + 0.6 4 2 0 Aj t o l + a; t o l 1.3 2 4 1 A; t o l a; t o l + 0.9 1 5 1* 28 14 3 * perithecia, but no ascospores produced with a tester 51 Table III - Conidial isolates recovered 1 from 18 escaped bisexual heterokaryons  genotype of ad-3A. nic-2 component genotype of ad-3B component mating type detected conidial genotypes ad-3B ad-3A, nic-2 HK 1 A t o l a a 36 0 4 2 a A A 29 0 1 5 A a t o l A 0 30 0 4 A a a 30 0 0 5 a A t o l a 0 40 0 6 a A t o l a 0 50 0 7 A t o l a a 37 0 3 8 a t o l A A. a 0 55 5 9 A a t o l A, a 16 0 14 10 A t o l a A. a 4 37 9 11 A un-3 a a 237 0 _* 12 A un-3 a t o l A 0 111 41 13 A un-3 a t o l a 274 2** mm 14 A un-3 a t o l A 0 152 15 A un-3 a t o l A. a 252 1** 12 16 A un-3 a t o l A, a 136 0 -17 A un-3 a t o l A. a 258 0 9 18 A un-3 a t o l A, a 146 0 -* Dashes indicate that heterokaryotic (HK) colonies are suspected but not confirmed (see Materials and Methods). ** These conidial isolates are of genotype A, un-3, ad-3A. nic-2, and form inhibited heterokaryons with the original a, ad-3B strain. *** This escaped heterokaryon i s temperature sensitive. 52 Table IY - Conidial isolates from 5 day old bisexual heterokaryons before escape types used to HK (mean form HK* (a, ad-5B of to un-3, Aj ad-5A. a. ad-5B un-3« A, ad-3A. nic-2 several nic-2) "~ « values) •103 212 2 2.4 1 226 0 1.4 10-2 1 5 2 31 0.9 10" 3 121 17 0.4 * heterokaryon 53 Figure 1 - Growth curves of representative heterokaryons (see Table I for genotypes). 54 3 6 4 15 0 96 192 288 384 TIME (hours) 55 Figure 2 - Some representative growth curves of bisexual heterokaryons, showing escape after more than 200 hours of inhibited growth. 55 a 56 Figure 5 - Some exceptional growth curves of bisexual heterokaryons. The growth rate after escape sometimes decreased again or was cyclic. 57 Figure 4 - 3 typical types of growth of bisexual heter-okaryons: a culture homozygous for the t o l mutation (1), homozygous for tol"*" or hetero-zygous (tol + t o l + ) under light conditions (2) or in the dark (3). 57 a TIME (hours) 58 Figure 5 - Typical shift down of growth rate after switch of bisexual heterokaryon 1-9-16 + 1-22-83 from dark to natural light cond-itions (the bimodal curve appears to be a constant phenomenon). TIME (days) 59 Figure 6a - Distribution of time of escape among (A + a) bisexual heterokaryons of genotype un-3, A, ad^3A, nic-2 (1-22-83) + a, ad-3B (1-9-16). Figure 6a 6-1 4-1 24 VJl VP Ho 200 TIME OF ESCAPE (hours) 300 400 60 Figure 6b - Distribution of time of escape among (A + a) bisexual heterokaryons of genotype un-3, A, ad-3A, nic-2 (1-22-83) + a, ad-3B; t o l ( I - 9 - 5 7 ) . 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Abnormal frequencies of spontaneous mitotic recombination i n uvsB and uvsC mutants of Aspergillus nidulans. Mutation Res. 10:33-41. 63 Kafer, E., and Upshall, A., 1973. The phenotypes of the eight disomies and trisomies of Aspergillus nidulans. J. Hered. 64:35-38. Koltin, Y.f and Stamberg, J., 1973. Genetic control of recom-bination in Sehizophyllum commune: Location of a gene controlling B-factor recombination. Genetics 74:55-62. Lemontt, J.P., 1971. Some properties of double mutants uys-§ and rev. II. The effect of rev, genes on recombin-ation. Mutation Res. 13_:311-317. McClintock, B., and H i l l , H.E., 1931. The cytological identif-ication of the chromosome associated with the R-G linkage group i n Zea Mays. Genetics 16:175-190. Mitchell, M.B., Pittenger, T.H., and Mitchell, H.K., 1952. Pseudo-Wild Types in Neurospora crassa. Genetics 2§:569-580. Mylyk, O.M., 1975. Heterokaryon incompatibility genes i n Neuros-pora crassa detected using duplication-producing chromosome re-arrangements. Genetics 80: 107-124. Nasim, A., and Smith, B.P., 1975. Genetic control of radiation sensitivity i n Schi z o sac charomyc e s pombe. Genetics 79:573-582. Neweombe, K.D., and Griffiths, A.J.P., 1973. Adjustable plat-forms for collecting shot asci. Neurospora Newsletter 20:32-33. Newmeyer, D., 1970. A suppressor of the heterokaryon-incompatibility associated with mating type in Neurospora crassa. Can. J. Genet. Cytol. 12:914-926. Newmeyer, D., and Galeazzi, D.R., 1974. A genetic factor which causes deletion of duplications in Neurospora. Genetics 77:§-48. 64 Newmeyer, D., Howe, H.B. Jr., and Galeazzi, D.R., 1973. A search for complexity at the mating-type locus o £ Neurospora crassa. Can. J. Genet. Cytol. 15: 577-585. Newmeyer, D., and Taylor, C.W., 1967. A pericentric inversion in Neurospora with unstable duplication progeny. Genetics 56:771-791. 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(Camb.) 9:195-2TO~I Simonet, J.M., and Zickler, 33., 1972. Mutations affecting meiosis i n Podospora anserina. Chromosoma (Berl) 32:327-351. Smith, D.A., 1973. A mutant affecting meiosis i n Neurospora. Genetics j[4: Suppl., s259. Smith, 3D.A., 1974. Unstable diploids of Neurospora and a model for their somatic behavior. Genetics 76:1-17. Smith, D.A., 1975. A mutant affecting meiosis i n Neurospora. Genetics 80:125-133. Threlkeld, S.P.H., 1962. Some asci with non-identical sister spores from a cross i n Neurospora crassa. Genetics 47:1187^1198. 66 Threlkeld, S.F.H., 1965. Pantothenic acid requirement for spore color i n Neurospora crassa. Can. J . Genet. Cytol. 7:171-173. Threlkeld, S.P.H., and Stephens, V., 1966. Ascospore isolates of Neurospora crassa giving rise to cultures contain-ing two or more genetically different nuclei. Can. J . Genet. Cytol. 8:414-421. Uchida, I.A., and Viola Lee, CP., 1974. Radiation induced non-disjunction in mouse oocytes. Nature 250:601-602. Vigfussen, N.V., and Cano, R.J., 1974. A r t i f i c i a l induction of the sexual cycle of Neurospora crassa. Nature 249: No. 5455:383-385. Vogel, H.J., 1956. A convenient growth medium for Neurospora (Medium N). Microbiol. Genet. Bull. 13_:42-43. Ward, E.J., 1973. Non-disjunction: localization of the control-l i n g site i n the Maize B Chromosome. Genetics 73: 387-391. ~~ Weijer, J., and Dowding, E.S., I960. Nuclear exchange i n a heterokaryon of Neurospora crassa. Can. J. Genet. Cytol. 2:336-343. Westergaard, M., and Mitchell, H.K., 1947. Neurospora V. A synthetic medium favouring sexual" reproduction. Am. J. Botany 34:573-577. Wilson, J.P., and Garnjobst, L., 1966. A new incompatibility locus i n Neurospora crassa. Genetics 53:621-651. 67 APPENDIX 1 MEDIA: NUTRITIONAL TESTING- MEDIUM 50 ml. Westergaard and Mitchell solution* 1 ml. Trace elements 1 ml. Biotin 1 ml. 0aCl2 4 gm. Sorbose 2 gm. Sucrose 20 gm. Agar (washed)** add d i s t i l l e d H20 to 1000 ml. adjust to pH 5.5-6.0 PLATING MEDIUM 50 ml. Westergaard and Mitchell solution 1 ml. Trace elements 1 ml. Biotin 1 ml. CaCl2 10 gm. Sorbose 500 mg. Glucose 500 mg. Fructose 15 gm. Agar (washed for selective platings) add d i s t i l l e d HpO to 1000 ml. adjust to pH 5.5-6.0 CROSSING MEDIUM 50 ml. Westergaard and Mitchell solution 1 ml. Trace elements 1 ml. Biotin 1 ml. CaCl 2 2 gm. Sucrose (for li q u i d medium) 20 gm. Sucrose (when agar i s added) 15 gm. Agar ( i f needed) add d i s t i l l e d H?0 to 1000 ml. adjust to pH 5.5-6.0 VEGETATIVE MEDIUM 20 ml. Vogel's solution (Vogel, 1956) 1 ml. Biotin solution 20 gm. Glucose 15 gm. Agar Add d i s t i l l e d H20 to 1000 ml. 68 Footnotes: *Trace elements, biotin, and CaCl? are excluded from the original "Westergaard and Mitchell" solution (Westergaard and Mitchell, 1947). They are added when the medium i s prepared. **Washing of agar 1) Pour agar into a large plastic or glass tub. 2) F i l l the tub with tap water, s t i r and allow to settle. 3) Siphon or decant off the water. 4) Repeat steps 2 and 3 a total of 8 times with tap water and twice with d i s t i l l e d water. 5) Pour the wet agar into a nylon bag, and squeeze out the excess water using a wine press. 6} Transfer the "squeezed out" agar into a clean tub. 7) In a fumehood, add acetone reagent grade, and s t i r . 8) Pour acetone-soaked agar into the nylon bag and squeeze out the excess acetone (with clean hands)• 9) Spread out agar (e.g. on cookie sheets) to dry i n a fumehood (mix occasionally with a spatula). 10) When dry, mix agar i n blender to remove lumps. 69 APPENDIX 2 Lis t of Abreviations ad — adenine requiring a l as albino arg ss arginine requiring auxo = autotroph cog = recognition cys cystein requiring f r = frost het = heterokaryon incompatibility his ss histidine requiring leu = leucine requiring m ss meiotic mutant mei rr meiotic nic s= nicotinic acid requiring OR ss Oak Ridge phen = phenylalanine requiring PWT Pseudo-Wild Type rad = radiati on-s ens i t i r e rec ss recombination ser ss serine requiring sue = succinate requiring t o l = tolerant un — unknown uvs ss ultra violet sensitive 


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