UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Positional regulation and evolution of mating type genes in heterothallic and homothallic species of… Vellani, Trina (Tia) Sehar 1998

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1998-346404.pdf [ 10.77MB ]
Metadata
JSON: 831-1.0099355.json
JSON-LD: 831-1.0099355-ld.json
RDF/XML (Pretty): 831-1.0099355-rdf.xml
RDF/JSON: 831-1.0099355-rdf.json
Turtle: 831-1.0099355-turtle.txt
N-Triples: 831-1.0099355-rdf-ntriples.txt
Original Record: 831-1.0099355-source.json
Full Text
831-1.0099355-fulltext.txt
Citation
831-1.0099355.ris

Full Text

POSITIONAL REGULATION AND EVOLUTION OF MATING TYPE GENES IN HETEROTHALLIC AND HOMOTHALLIC SPECIES OF  NEUROSPORA  by TRINA (TIA) SEHAR V E L L A N I B.Sc. (Hon.), McMaster University, 1988 M . S c , The University of British Columbia, 1991 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Biotechnology Laboratory and Department of Botany) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A October 1998 © Trina (Tia) Sehar Vellani  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 The University of British Vancouver, Canada  DE-6 (2/88)  Abstract  The mating type genes of Neurospora crassa were shown to function abnormally when located at ectopic chromosomal positions. Crosses involving strains with ectopic mating type genes produce defective perithecia. Ascus number is reduced. The hypothesis that perithecial development requires physical proximity of opposite mating type homologs during meiosis was tested. The sterility of the crosses made between strains with both mating type regions relocated to the same ectopic position failed to support the hypothesis. To test the hypothesis that normal expression levels of the mating type genes require distant cw-acting sequences not present on ectopic fragments, autoradiograms of mRNA from wild type and ectopic-w^ strains were compared. Differences between expression levels in ectopic-/rtf and wild type cells were observed, but their significance cannot be assessed without additional studies. The hypothesis that nuclear identity is disrupted in ectopic-m/ strains was tested. Strains with disturbed nuclear identity (dual mating type) were crossed to wild type. The appearance of the reduced ascus number phenotype suggested that the affected function in ectopic-m/ strains is nuclear identity. The homothallic species N. terricola contains mt A- and mt a-like sequences. The genes were cloned and sequenced to determine whether or not they were functional. The genes specifying identity, mt A-l and mt a-l, are more than 95% similar at the amino acid level to the  N. crassa homologs, but the putative M T A-2 polypeptide is truncated. N. terricola ml A-l and mt a-l genes induced mating and vegetative incompatibility in N. crassa mating type mutants. Expression in N. terricola of mt A-l and mt a-l was detected by reverse transcriptase PCR, upholding the hypothesis that the genes are functional. To determine the pattern of evolution of homothallism, a phylogeny of Neurospora was reconstructed from mt A-l D N A and amino acid sequences. Homothallic Neurospora species that carry both mating type genes are more closely related to heterothallic species than they are to the ^4-only homothallic species, suggesting that either heterothallic species are derived from a homothallic ancestor or that homothallism arose twice.  Table of Contents Abstract List of Tables List of Figures Acknowledgement Dedication General Introduction Background Thesis objective Chapter 1 .Introduction Materials and Methods Results Discussion Chapter 2 " Introduction Materials and Methods Results Discussion Chapter 3 Introduction Materials and Methods Results and Discussion Summary References Appendix A Neurospora crassa Gene Symbols Appendix B Slot blot Analysis Appendix C Mann-Whitney Rank-Sum Tests Appendix D D N A and Amino Acid Alignments  ii iv v vi viii 1 2 12 13 13 20 29 52 64 64 69 74 92 95 95 97 101 120 121 129 130 133 138  IV  List of Tables Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table  1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1.13 1.14 2.1 2.2 2.3 2.4 2.5 2.6 2.7 3.1 3.2  Mating type gene names Strains used in Chapter 1 Variable fertility of MS transformants Variable percent germination of M S ascospores Segregation of mating type in MS progeny Segregation of markers linked to mt Mating, ascospororgenesis and Southern Blot phenotypes of M N S strains Number of ascospores per rosette 8 days post-fertilization Crosses with ectopic mating type strains Crosses of strains with and without the opposite mating type idiomorph Number of ascospores per rosette 8 days post-fertilization Number of ascospores per rosette 11 days post-fertilization Number of asci per rosette Updated hypotheses for "position effects Strains used in Chapter 2 Plasmids used in Chapter 2 Primers used to amplify genes in Chapter 2 Comparison of N. crassa and N. terricola genes N. terricola mt a-1 confers mating to N. crassa mating type mutants or nulls ... Transformation efficiency of mating type genes Suppression of incompatibility by tol Primers used to amplify mt genes Species included in the tree  8 20 31 31 32 32 36 38 42 45 49 49 49 55 69 69 70 74 87 88 89 97 101  List of Figures Figure 1.1 Figure 1.2 Figure 1.3 Figure Figure Figure Figure Figure Figure  1.4 1.5 1.6 1.7 1.8 1.9  Figure 1.10 Figure 1.11 Figure 1.12 Figure 1.13 Figure 1.14 Figure 2.1 Figure 2.2 Figure 2.3 . Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure Figure Figure Figure  3.6 3.7 3.8 3.9  Structure of the N. crassa mating type idiomorphs 4 Map of am locus in ectopic-m^ strains 17 D N A blot of transformants and progeny showing A and a bands 23 Poly ( A ) + R N A slot blots probed with mt A-l 25 Poly ( A ) + R N A slot blots probed with mt A-3 26 Sample of slot blot analysis performed using NIH image 28 Transformation of ad-3A nic-2; tol a strain 30 Map of mt A and mt a idiomorphs, showing relevant BamHI sites 34 Perithecial contents from 11 days post-fertilization crosses (ectopic mating type cf. wild type) 39 Branch diagram showing the cross of mt-rel-Ac6 x OR8-la 40 Generation of strains used to study interference 46 Perithecial contents from 11 days post-fertilization crosses (a cf. A */a)... 47 Perithecial contents from 11 days post-fertilization crosses (A * cf. A */a).. 48 Cross showing possibly disomic progeny 51 Mating types of Neurospora species 66 D N A sequence alignment of mt A-l 75-76 Deduced amino acid alignment of mt A-l 78 D N A sequence alignment of mt A-2 79-80 Deduced amino acid alignment of mt A-2 81 D N A sequence alignment of mt a-l 82-84 Deduced amino acid alignment of mt a-1 85 Gel electrophoretic analysis of N. terricola RT-PCR products 91 Unrooted parsimony tree from D N A sequence alignment 102 Tree showing G. tetrasperma in the Neurospora group 104 Rooted parsimony tree from D N A sequence alignment 105 Rooted maximum likelihood/parsimony tree from D N A sequence alignment 106 Constraint tree with same rooting as maximum likelihood/parsimony tree 108 Constraint tree with an all-homothallic clade 109 Rooted parsimony tree from amino acid alignment Ill Model for evolution from heterothallism to homothallism 116 Model for evolution from homothallism to heterothallism 118  vi  Acknowledgement  Gratitude-plus to Louise Glass to whom I am permanently bonded for accompanying me through a bewildering number of defining moments in my life. Thank you so much for keeping faith that I would eventually finish. Buckets of thanks laced with profound admiration to Tony Griffiths for your appreciation of life in all its subtle and bizarre refractions; for your wisdom, profound insights, broad-mindedness, kindness, humanity, impish sense of humour, integrity, excellent editing skills, creativity and for providing a living example of the rich, balanced life available to the scientist. Thanks to Mary Berbee for always making time to teach me science, even when you were insanely busy. I am very grateful for your gift of the map to the fascinating world of evolutionary thought. I am grateful for the kindly presence of Carl Douglas. Thank you for emotional support; you were there to champion me and my scientific ability when my confidence was wilting. Thanks also for your gentle humanity to whose level I aspire. Universe-encompassing gratitude to Rik Myers for sharing with the world (me included) your profound understanding of life. Thank you for sharing your spirit, friendship, love and for accepting mine. Unending thanks to Maline Vellani Campbell and Shaelin Campbell Vellani for being your phenomenally wonderful selves, for your love, your anger, your laughter, your tears, in short, your everything! Inexpressible thanks to Mishu Vellani for your constant and unconditional support, love, friendship and understanding. Thankyouthankyouthankyou! My gratitude to Jasswanti Vellani cannot possibly come close to being expressed in words, but I'll try...thanks, Mum, you're unquestionably T H E BEST!!! I would like to express my deep, if belated appreciation for the late Nazeer Vellani for teaching me the value of education, spirituality, clan, culture, heritage, questioning and iconoclasm. Many many thanks to Sven Saupe for engaging into all of our discussions with your full mind, for understanding my halfformed ideas and sharing in their development and for the heart-warming sound of your laughter. Soul-felt thanks to Alexandra Ball for white-hot-core-communion. Warm thanks to Mishtu Banerjee for almost a decade of unconditional friendship and for sharing the  Commonwealth. Chocolate babies can fly. Thanks to Rod Campbell for many years of laughter, friendship, love and support. Thanks to Lane Ferreira for your friendship and collegiality. Thanks to Sally Otto for the loads of time and energy you so willingly donated to help me understand what I was doing. I feel honoured to acknowledge the friendship and support of Hugh Brock, Maria da Silva, Lorraine Graves, Frans Huijing, G A K (Gretchen Kuldau), Phoolmatteea Lutchmun, the late Roopnarain Luctchmun, Sandra Millard, Debbie Neufeld, Kenn Rudd, Myron Smith, Carol Thomas, Richard Todd, Louise Wheeler, Ugh (Sandra Wiese), my lab-mates from 1991-1997 and the Botany Department from 1991-1997.  Dedication  This thesis is dedicated to Moodle and Panwald, whom I can never thank enough.  1  General Introduction  This work addresses the genetic phenomenon called position effect and how it may have influenced the evolution of filamentous fungi. Organisms from various kingdoms have genes that must occupy a particular chomosomal position for normal function. A gene said to exhibit a position effect imparts, when displaced from its usual locus, an abnormal phenotype. Mechanistically, position effects are not all the same. The mating type genes of Neurospora  crassa are used here as a model to study the mechanistic underpinnings of one type of position effect. The mating type genes of N. crassa carry out some of their functions, but not all, when they are separated from their usual locus. In this work, competing hypotheses regarding the molecular mechanism of the N. crassa mating type gene position effect are tested genetically. One model, the c/s-acting regulator model, is pitted against the transvection model. The exacting regulator model proposes that the position-sensitive genes are under the control of a cisacting regulator and that when the genes lose the connection to the regulator, the functions are disturbed. The transvection model claims that the position-sensitive functions of the mating type genes depend on the synapsis of the opposite mating type genes during meiosis. Additionally, dual mating type transformants of N. crassa exhibit both vegetative and, as demonstrated here, sexual incompatibility. To explore further the workings of the mating type gene position effect, another species of Neurospora, N. terricola, was examined. In N.  terricola, chromosome I carries both of the mating type genes and even though they are similar to those of N. crassa, they appear to be perfectly compatible. If the mating type genes of N.  terricola can confer sexual interference in the appropriate genetic background, then they would have to be inactivated for the incompatibility functions in N. terricola. Since morphological differences from N. crassa are not evident, suppressor alleles may be present or the mating type genes themselves, although similar to N. crassa's may be unable to confer incompatibility. In this work, the genes are cloned, sequenced and tested for function to gain insight into the biological significance and the evolution of the position effect.  2 The interplay between Neurospora evolution and the position effect is tackled directly in a phylogenetic analysis. The sequences of the mating type genes are used to infer relationships in the genus. The data are analyzed and discussed in terms of the evolution of mating strategies. The three chapters are united in a evolutionary model that draws connections between the evolution of mating type genes and mating strategies in filamentous fungi and how the mating type gene position effect may have impacted on these processes in the genus  Neurospora.  Backgound  Populations of some haploid microorganisms (algae and fungi) are divisible into genetically distinct interfertile/intrasterile groups, said to be of different mating type. Fertile crosses, in which the participants differ in their mating type, result in the formation of meiotic products. Fertility groups were shown to be defined in the fungi by different alleles at one or more chromosomal loci which encode putative or proven transcriptional regulators that top a cascade of genes required for sexual processes (Kiies and Casselton, 1992).  N crassa is a haploid heterothallic (self-sterile, cross-fertile) fungus. Its classification tends to shift, but a recent one follows. N. crassa belongs to the kingdom Myceteae; division Amastigomycota; subdivision Ascomycotina, characterized by the production of sexual spores in a sac or " ascus"; class Pyrenomycetidae, characterized by the activation of sexual spores by heat; order Sphaeriales; family Sordariaceae; genus Neurospora, the group of species producing football-shaped ribbed (as opposed to pitted or smooth) ascospores (Moore-Landecker, 1990).  N. crassa sexual development has been reviewed by Raju (1980; 1992). Mating type is controlled by a single locus with two types, A and a (a list of N. crassa gene symbols is provided in Appendix A). Each A or a mycelium has both male and female structures. Male gametes, macroconidia, microconidia and hyphae, form part of the vegetative mycelium while female structures develop in response to nitrogen deprivation. The trichogyne, the receptive hypha emanating from the protoperithecium, perceives the presence of an opposite mating type  male cell by a pheromone produced by the latter. The trichogyne grows toward the male cell and fuses with it. The male nucleus migrates into the interior of the protoperithecium, into a structure called the ascogonium. The protoperithecium develops into a perithecium, a black macroscopic flask-shaped structure. The following parts of sexual development are particularly relevant to this thesis. In the ascogonium, the male nucleus proliferates mitotically along with female nuclei. One nucleus of each type is packaged into the terminal cells of the ascogenous hyphae, which bend to form a structure called the crozier. The last division before karyogamy is co-ordinated so that one male nucleus and one female nucleus divide simultaneously. Many croziers arise in each perithecium, in temporal waves. Karyogamy, meiosis and post-meiotic mitoses occur in the penultimate cell of the crozier, as it enlarges and differentiates to form the ascus, a sac containing the eight linearly ordered products of meiosis. The multinucleate, haploid progeny, ascospores, are forcibly ejected through an opening in the perithecial neck (also called "beak"), and each can germinate and become an independent mycelium. The three types of spores, microconidia, macroconidia and ascospores, may have different, but overlapping roles. Microconidia are probably not vegetative propagules since they are fragile and not very viable (Perkins and Turner, 1988). They may be exclusively male gametes. Macroconidia may be primarily vegetative propagules (Perkins and Turner, 1988) or primarily male gametes, since they may accumulate too much U V damage to germinate (Taylor, Smolich and May, 1986). Ascospores are UV-resistant, long-lived propagules, quiescent until activated by heat (Hollaender, Sansome and Demerec, 1945). In heterothallic Ascomycetes, mating type is inherited as a single locus with two alleles  (e.g. in S. cerevisiae, Schizosaccharomycespombe, N. crassa, Sordaria brevicollis, Cochliobolus heterostrophus and Podospora anserina) (Kiies and Casselton, 1992). The mating type loci of N. crassa differ from classical alleles in two ways: they are dissimilar in sequence (and therefore are called " idiomorphs" rather than alleles (Metzenberg and Glass, 1990)), and secondly, the mt A idiomorph encodes more than one gene (Glass and Lee, 1992; Ferreira, Saupe and Glass, 1996). The structure of the idiomorphs is shown in Figure 1.1. Genes with  CD CD OJ  OJ  CQ  CQ  OJ  OJ  O II CD  (Q  O  ®  CD  -i.  9- 9- — £.  3 3  CD CD 05  «  =5  OJ  r . co o  o  ° - CD CD.g  CD CD =5. O CQ  CD _ . =3  CD  O  o  2  CO CO QJ  CO  — CD O  •3 IT OJ CA  Q.  2  *< OJ ^  ^ C/J OJ  CD Q. CD OJ  CD CD  3  O  CQ  St™ =f  —t  CD T D  CD  CD  CL  o  OJ OJ T3  •o  —t  O X  r 3" ffi  9-  o'  3 o -D  CD 3 >—3 = r _ OJ_ <  ro ><  o CO  o  QJ_ ro  TJ CD  O O  5' c—1 -. ro co  CQ CQ  ^ o' OJ CO CD  CO  o  Er ro  H •o ro ix  CD  3  —^ CD  co  72 OJ  II QJ o' 3 o  —*  ro o  5" 5' _T  OJ  CQ'CQ QJ CO O  CD O  ro o  3  13  •o ro CD  1  3  5 idiomorphic regions are uncommon, and yet other fungal mating type genes have them: S.  cerevisiae (Astell et al, 1981; Tatchell et al, 1981), S. pombe (Kelly et al, 1988), Magnaporthe grisea (Kang, Chumley and Valent, 1994), C. heterostrophus (Turgeon et al, 1993), P. anserina (Debuchy and Coppin, 1992), U. maydis (Bolker, Urban and Kahmann, 1992), S. commune (Giasson et al, 1989) and Cryptococcus neoformans (Moore and Edman, 1993). Possible selective advantages of idiomorphs are discussed in Chapter 3 Results and Discussion. The mt A idiomorph of N. crassa is 5301 bp and encodes three genes, mt A-l, mt A-2 and  mt AS, all putative transcriptional regulators (Glass, Grotelueschen and Metzenberg, 1990; Ferreira, Saupe and Glass, 1996). Mutants with frameshifts in mt A-l are sterile (Griffiths, 1982; Glass, Grotelueschen and Metzenberg, 1990). The mt a idiomorph is 3235 bp and encodes one gene, mt a-l, which is necessary for mating identity, meiosis and sporulation (Staben and Yanofsky, 1990; Chang and Staben, 1994). Other sequences within the idiomorph, but not in the mt a-l gene are required for position-independent perithecial development (C. Staben, personal communication).  N. crassa mating type genes are necessary and sufficient for identity in mating (Shear and Dodge, 1927; Griffiths and DeLange, 1978; Griffiths, 1982; Glass et al., 1988) and heterokaryon incompatibility (Beadle and Coonradt, 1944; Griffiths and DeLange, 1978; Griffiths, 1982; Glass et ai, 1988) and for perithecium suppression upon crossing to the same mating type (Griffiths and DeLange, 1978). They are necessary, but not sufficient for secretion and response to pheromones (Bistis, 1981; 1983; Glass, Grotelueschen and Metzenberg, 1990; Staben and Yanofsky, 1990) and perithecial maturation, including meiosis and sporulation (Glass, Grotelueschen and Metzenberg, 1990; Staben and Yanofsky, 1990; Glass and Lee, 1992). Some mutants in mt a-l produce barren perithecia when crossed to either mating type and some only when crossed to a. One mutant, a m33, produces abundant perithecia with abundant ascospores when crossed to A and a few barren perithecia when crossed to a (Griffiths and DeLange, 1978). Since the mutants were selected on the basis of heterokaryon compatibility, they are all heterokaryon compatible (Griffiths and DeLange, 1978).  6 A heterokaryon consisting of one nuclear type which carried the null mating type mutation, a m] (Griffiths and DeLange, 1978; Staben and Yanofsky, 1990) and a marker enabling distinction of its ascogenous hyphae and a second nuclear type which carried the wild type a allele and a mutation eliminating protoperithecia was crossed to A. Perithecia were formed, showing that the mating function of the a ml mutant was rescued by a wild type copy of  mt a. Ascogenous hyphae did not have the phenotype of the a ml mutant nuclei, showing that the post-fertilization function of the a ml mutant was not rescued by a wild type copy of mt a (Raju, 1992). These results suggest that M T a-l may become nuclear autonomous in ascogenous hyphae, i.e. act only on the nucleus from which it came. Chang and Staben (1994) also showed that mt a-l is involved in post-fertilization events by demonstrating that asci developed when mt a-l integrated at the mating type locus, but not at an ectopic position. A strain transformed with the entire mt a idiomorph produces perithecia that are more mature than mt a-l transformants, but they are still barren (C. Staben, personal communication). Most mutants in mt A-l are unable to induce perithecia when crossed to either mating type (Griffiths and DeLange, 1978; Glass, Grotelueschen and Metzenberg, 1990). A strain with a deletion of the mt A idiomorph had the same phenotype as the mutants (Ferriera et al., 1998). Since the mutants were selected on the basis of heterokaryon compatibility, they are all heterokaryon compatible (Griffiths and DeLange, 1978). One mutant, A m",  which makes a  truncated 85 amino acid polypeptide, was sterile as a male, but produced a reduced number of perithecia with few ascospores when crossed as a female to a, suggesting that a post-fertilization role is encoded in the 3' region of the mt A-l gene (Griffiths, 1982; Saupe et al, 1996). Null single mutants of mt A-2 or mt A-3 have no phenotype (Ferreira, 1997; Ferreira et  ai, 1998). Strains appear normal. A number of mutants affecting more than one of the mt A genes were produced by RIP (Glass and Lee, 1992). Several mutants, called A 1 R1P strains, were produced from a cross in which one parent carried a duplication for mt A-l and part of mt A-2, resulting in the mutation of these genes (as shown by novel restriction fragment patterns). The mutants did not produce  7 perithecia when crossed as a male or female to a a tester strain and were heterokaryon compatible when paired with an a tester strain. This phenotype is the same as that seen mint A-  1 mutants (Griffiths and DeLange, 1978; Glass, Grotelueschen and Metzenberg, 1990), suggesting that mt A-l (at least) was mutated. The normal appearance of the  ^-generating cross (N. L. Glass, personal  communication) suggests that the genes mutated in theA IRJP  mutant, namely mt A-l possibly  mt A-2, are not required after RIP which occurs during premeiotic nuclear divisions in the ascogenous hyphae. The likelihood that normal nuclei were providing normal M T A-1 protein to the mutated nuclei in the syncitial tissue is low since the product is probably nuclear limited (see Chapter 1 Introduction). A solitary mutant, called A 11 R,p,  was generated from a cross in which one parent was  duplicated for mt A-2 and part of mt A-3 (Glass and Lee, 1992). Sequencing showed that the mutant is missing the C-terminal 244 amino acids of mt A-2 and has 3 amino acid substitutions in mt A-3 (Ferreira, 1997). This mutant produced abundant perithecia, but very few ascospores when crossed to a a tester, as did its A progeny. Perithecial development proceeded normally until approximately four to five days post-fertilization. At this point fewer asci than normal were seen, but they matured normally and produced ascospores (Glass and Lee, 1992). The phenotype of this double mutant contrasts to the non-existent phenotypes of mt A-2 and mt A-3 null mutants. The two genes could act in parallel pathways or in a complex that fails to function only when both gene products are missing. The genes could have redundant function. The differences in their constellations of motifs (see section below entitled " Sequence comparison"), however, suggest that they do not have the same function. Perhaps the genes have independent functions, but can fill each other's roles under certain circumstances. A post-fertilization role for mt A-2 and mt A-3 is supported by the existence of SMR1 and SMR2 in P. anserina, two genes encoded in the mat- idiomorph that code for postfertilization functions (Debuchy, Arnaise and Lecellier, 1993). The N. crassa genes are clearly different, however, given that they cannot complement the meiosis and sporulation functions in  P. anserina mutants (Debuchy, Arnaise and Lecellier, 1993; Arnaise, Zickler and Glass, 1993)  8 and the phenotypes of mt A-2, mt A-3 and A 11 RIP mutants differ from the P. anserina mutants which produce uniparental progeny (Zickler et al, 1995; Ferreira, et al, 1996; Ferreira, 1997; Ferreira et al, 1998). The fact that mt A-2 and mt A-3 share a promoter (Ferreira, Saupe and Glass, 1996) supports the idea that their expression may be co-ordinated. RIP mutants of mt A-2 and mt A-3 were difficult to obtain (Glass and Lee, 1992; A . V . B. Ferreira, personal communication) perhaps because the genes are required post-fertilization and if they were excessively mutated, then no ascospores would be recovered. According to this explanation, the one mutant that was obtained may retain partial function of one or both of the genes. Ascomycete mating type genes encode proteins with transcription factor motifs, suggesting that they regulate transcription. The mating type idiomorph names are presented in Table 1.1 Table 1.1 Mating type gene names. Species  Gene name  N. crassa M. grisea S. macrospora P. anserina C. heterostrophus S. cerevisiae S. pombe  mt A and mt a Mat 1-1 and Mat 1-2 Smt A and Smt a mat- and mat+ MAT-1 and MAT-2 m  T  a  a n d  M  A  T  a  matl-P and matl-M  The cloning and sequencing of the S. macrospora genes took place very recently. S  macrospora has homologs to all four N. crassa genes, all of which are closely linked (Poggeler etal., 1997a). N. crassa mt A-l has a region similar to MATal of S. cerevisiae (Glass, Grotelueschen and Metzenberg, 1990), FMR1 of P. anserina (Picard, Debuchy and Coppin, 1991) and MAT-1 of C. heterostrophus (Turgeon et al, 1993). The encoded domain is proposed to be involved in D N A binding and was termed the a-domain (Debuchy and Coppin, 1992). The product of N. crassa mt a-l has two defined regions. One is an in vitro DNAbinding activity mediated by an H M G domain (Philley and Staben, 1994), a motif originally  9 found in high mobility group proteins associated with chromatin and shown to bind D N A (Jantzen et al, 1990). The mt a-l H M G box is similar to that in ma(Mc of S. pombe (Staben and Yanofsky, 1990), FPR1 of P. anserina (Debuchy and Coppin, 1992), MAT-2 of C.  heterostrophus (Turgeon et al, 1993) and the male determining SRY gene in humans (Sinclair el al, 1990) and mice (Gubbay et al, 1990). Its presence correlates with mating ability (Philley and Staben, 1994). The second M T a-l region is the carboxy terminal half which is similar to transcriptional transactivator proteins and is required for both sexual and vegetative functions (Philley and Staben, 1994). The product of the N. crassa mt A-2 gene has 23% overall amino acid identity with  SMR1 product of P. anserina and a block of 82% identity extending across 17 amino acids, proposed to be a novel D N A binding domain (Debuchy, Arnaise and Lecellier, 1993; Ferreira, Saupe and Glass, 1996).  N. crassa ml A-3 gene product has 22% amino acid identity with SMR2 of P. anserina (Ferreira, Saupe and Glass, 1996). The M T A-3 protein also possesses an H M G domain. In addition, the protein has a PEST domain which is a motif that correlates with rapid constitutive protein turnover (Rogers, Wells and Rechsteiner, 1986), but not signal-induced degradation (van Antwerp and Verma, 1996) and may be involved in protein-protein interaction (Chu et al, 1996).  N. crassa mt A-l and mt a-l can confer mating identity, but not vegetative incompatibility, to a mating type null strain of P. anserina (Arnaise, Zickler and Glass, 1993).  P. anserina FMR1 and FPR1 can likewise confer mating identity, but not heterokaryon incompatibility to a N. crassa strain of the opposite mating type (Arnaise, Zickler and Glass, 1993). Like mt A-l, FMR1 is also required for post-fertilization functions (Debuchy, Arnaise and Lecellier, 1993). The P. anserina homologs to mt A-2 and mt A-3, SMR1 and SMR2, are required for postfertilization functions (Debuchy, Arnaise and Lecellier, 1993). They are thought to be required for the sorting of one nucleus of each mating type into cells destined to become single asci (Zickler et al, 1995). Uniparental ascospores are formed in mutants of any of the P. anserina  10 mating type genes. The production of ascospores suggests that the mating type genes are not required for meiosis or sporulation (Zickler et al, 1995). A P. anserina mating type null mutant transformed with the N. crassa mt A region progressed no further in sexual development than when transformed with mt A-l alone, suggesting that while mt A-2 and mt A-3 are similar to SMR1 and SMR2, they cannot complement the post-fertilization functions specified by these genes (Debuchy, Arnaise and Lecellier, 1993; Arnaise, Zickler and Glass, 1993). The post-fertilization functions of mt A-l,  mt A-2 and mt A-3 may depend on chromosomal location in P. anserina as they do in N. crassa (see Chapter 1). Alternatively, the lack of complementation could reflect differences in target genes between the two species. In contrast to the SMR1 and SMR2 phenotypes, crosses of mt A-2 or mt A-3 mutants produced no uniparental progeny (Ferreira et al, 1998). The possibility that mt A-2 and ml A-3 are required for post-fertilization recognition in N. crassa remains viable, however, under the condition that ascus development requires interaction between opposite mating types in N.  crassa. S. macrospora genes can induce fruiting body formation in P. anserina (Poggeler et al, 1997a). C. heterostrophus MAT-1 or MAT-2 can confer mating identity to the P. anserina null strain (S. Arnaise, personal communication to N . L. Glass and M . A . Nelson, cited in Glass and Nelson, 1994). C. heterostrophus has no homolog to mt A-2 or mt A-3. The sequence and functional homologies suggest that barriers to interspecific mating may be specified by mating type target genes, rather than the mating type genes themselves (Arnaise, Zickler and Glass, 1993). Two mating type-specific transcripts are encoded in the centromere proximal flanks of the idiomorphs (Randall and Metzenberg, 1995). One of the ml ^-specific predicted proteins is similar to fungal pheromones (T. A . Randall and R. L. Metzenberg, personal communication to N . L. Glass). The transcription of one of the M ^-specific transcripts depends on M T A - l (Randall and Metzenberg, 1995).  A region in the mating type flank in Neurospora species appears to be species-specific. It could encode species-specific pheromones (Randall and Metzenberg, 1995). Pheromonal differences are postulated to be an important step in genetic isolation of a group of four  Drosophila species (Coyne, Crittenden and Mah, 1994). For species in which mating via heterokaryosis is not possible (all those with mating type incompatibility), pheromonal differences may contribute to sexual isolation.  12  Thesis objective  In this thesis, data was gathered to shed light on the mechanism of the positional regulation of the mating type genes of N. crassa. Two competing hypotheses were tested, the transvection hypothesis and the cw-acting regulator hypothesis. The research suggested that the mating type position effect is not due to an unmet transvection requirement and may be due to differences in the expression of mating type genes when they are ectopic. The mating type genes of N. terricola were cloned and their functionality tested in order to examine the hypothesis that a species can achieve homothallism by combining mating type genes into single nuclei. Two N. terricola mating type genes are very likely functional. The sequences of a mating type gene from Neurospora and three outgroup species were determined or obtained from other sources and used to reconstruct the phylogeny of the  Neurospora species in order to discover the relationships between homothallic and heterothallic species. The results suggested that homothallic species are long-lived and that a group of homothallic species is more closely related to heterothallic species than to the other group of homothallic species.  13  Chapter 1 Phenotypic and Mechanistic Analyses of Positional Regulation of TV. crassa Mating Type Genes  Introduction  The mating type genes in N. crassa specify identity during the sexual stage of the life cycle, such that A x a is a compatible mating and A x A and a x a are incompatible (Shear and Dodge, 1927). The genes also specify identity during the vegetative stage, such that A+ A and a  + a form compatible heterokaryons and A + a form an incompatible heterokaryon (Beadle and Coonradt, 1944). A compatible heterokaryon produces a vigorous mycelium; an incompatible heterokaryon typically grows slowly or not at all. An mt A-l or mt a-l mutant produces protoperithecia and conidia before crossing, but it is sterile because it fails to produce perithecia and ascospores. Furthermore, in contrast to wild type, a mating type mutant can form a compatible heterokaryon with a strain of the opposite mating type (Griffiths and DeLange, 1978; Griffiths, 1982). Functional copies of mt A-l or mt  a-l introduced by transformation into the mutants vary in their ability to complement the mutant phenotype. Typically, sterile mating type mutants transformed with cloned mating type genes produced perithecia devoid of ascospores. Reasons for developmental arrest were unknown. Prior to these experiments, mating had never been separated from sporulation. Post-fertilization functions require all four mating type genes, mt a-l (Raju, 1992), mt A-l (Saupe et al., 1996), mt  A-2 and mt A-3 (Glass and Lee, 1992; Ferreira, 1997). In one transformation experiment, a few colonies transformed with mt A D N A were able to produce ascospores (Glass, Grotelueschen and Metzenberg, 1990). Because their molecular constitutions were consistent with their having undergone gene replacements, Glass, Grotelueschen and Metzenberg (1990) suggested that cw-acting sequences may be important for proper expression of mt A-l. Because an ectopically integrated copy of mt a into a tol A strain  14  was only partially functional, but the A was still fully functional, Staben and Yanofsky (1990) suggested that the mt a genes could only function normally when occupying the mating type locus. Because a transformant with mt a-l targeted to the mating type locus was fertile, Chang and Staben (1994) suggested that mt a-l is controlled by cw-acting sequences. In eukaryotic systems, a position effect is defined as the chromosomal position-dependent alteration of the expression of a gene. The definition is limited to only those genes carrying intact coding regions and most or all of the local transcriptional control elements (Wilson, Bellen and Gehring, 1990). The lack of ascospores in transformants carrying introduced mating type genes can be explained by invoking positional regulation of the mating type genes, but other explanations are also valid. In the transformations of mating type mutants with mating type sequences, genes required for ascospore formation could have been mutated by RIP of duplicated sequences and/or rearrangements caused by premeiotic recombination between duplicated sequences (Selker et al, 1987). Additionally, in the transformations of mating type strains with opposite mating type genes, interference between opposite mating type transcripts or products could have been eliminating ascus development (see Chapter 1 Discussion). The situation in N. crassa contrasts with that in the ascomycetes, P. anserina and C.  heterostrophus, in which mating type deletion strains transformed with mating type genes produced a normal or close to normal number of ascospores (Coppin et al., 1993; Wirsel, Turgeon and Yoder 1996). Why are ectopic mating type genes of Neurospora unable to orchestrate sporulation? The experiments described in this chapter were designed to determine if the N. crassa mt  A mating type genes are under positional regulation and if so, what is the mechanism of the position effect. Chapter 1 describes experiments designed to identify chromosomal positions that allowed the mt A idiomorph to confer both mating and sporulation. The hypothesis being tested was that mt A-l, mt A-2 and/or mt A-3 are functional only when they reside at the mating type locus. A mating type a host was transformed with the mt A idiomorph which was presumed to integrate randomly (Fincham, 1989). Two types of transformants were examined  15  genetically and at the molecular level: those able to mate and sporulate as A and those able to mate but not sporulate as A. The chromosomal position of the integrated D N A was determined for each of the transformants with normal A mating type function. This research differs from previous work in that a large number of transformants was examined and molecular and genetic analyses were performed to test competing hypotheses. Secondly, Chapter 1 describes the sexual phenotype of a strain constructed to reveal a position effect without the complicating effects of RIP and interference between opposite mating type genes. The results include a genetic analysis of strains in which the mating type idiomorphs were deleted from the mating type locus and integrated at an ectopic chromosomal location. Also, a transvection model is tested. This model proposes that normal sexual development requires close physical proximity of the mating type idiomorphs, i.e. "pairing" of non-homologous sequences, but that the genes do not necessarily have to reside at the mating type locus. The model predicts that ascosporogenesis will be restored to a cross between two strains in which the mating type genes have been relocated to homologous chromosomal positions. Additionally, observations are made on a variety of crosses with ectopic mating type genes to answer the following general questions. Are there any consistent sex-specific or mating type-specific differences in the fertility of ectopic-m/ strains? How does the fertility differ when these strains are crossed to ectopic-m/ versus wild type strains? Is a cross involving an ectopic mating type gene blocked at one specific developmental step? Transvection was originally described in Drosophila (Lewis, 1954) and most of the homologous pairing-dependent phenomena have been studied in this organism (Wu, 1993). In its older usage, transvection referred to the ability of a normally cz's-acting sequence to affect expression of the gene on the homologous chromosome (Judd, 1988; Wu and Goldberg, 1989). A broader term, "trans-sensing", was introduced to describe effects that depend on direct interaction between homologs (Tartof and Henikoff, 1991). Models for transvection are numerous and some are specific to certain interactions (Wu, 1993). Judd (1988) describes two classes of models: (1) conformational changes in chromatin brings sites on different D N A molecules into juxtaposition and (2) nuclear messenger molecules with limited range of action  affect only close molecules. Similarly, Henikoff (1994) divided the phenomena into the following two classes: (1) structural, which involve changes in the structure of the genes and (2) kinetic, which involve spreading of regulatory proteins from the area where they normally act. One type of Drosophila trans-sensing (seen in the genes, ci, bx, It, dpp and Scr) may require continuity between the gene and its centromere (Tartof and Henikoff, 1991). Perhaps significantly, in the fertile N. crassa translocation strain T(I->II) 39311, the translocated fragment of linkage group I that moves the mating type region carries with it the cluster of genes near the centromere (Perkins, 1972).  Drosophila trans-sensing effects depend on pairing of homologs in somatic cells (Hiraoka et al., 1993). Trans-sensing in TV. crassa would have to occur in croziers after karyogamy and before meiosis since this is the only diploid cell in the life cycle. Meiotic transvection has been described in N. crassa. Alleles of Asm-1 + require pairing in order to conduct normal ascospore maturation, and the transvection is supposed to occur during the short time when homologs are paired, but before crossing over has begun (Aramayo and Metzenberg, 1996). Strains for testing the transvection hypothesis were constructed by R. L. Metzenberg and J. Grotelueschen (Figure 1.2). The host of these three strains ( R L M 44-02) had the mt A idiomorph (on linkage group I) deleted and replaced by the ad-5 gene of Schizophyllum  commune, and as a result could neither mate nor produce ascospores. A l l three of the derivative strains (mt-rel-Ac6, mt-rel-Aflk and mt-rel-Da4) were transformants in which a mating type idiomorph had been targeted to the am locus on linkage group V . They all grew and conidiated well. In mt-rel-Ac6 the same fragment of D N A deleted from the mating type locus was targeted to the am locus. In mt-rel-Aflk, the introduced fragment contained the deleted sequences plus approximately 2 kbp of flank on either side of the idiomorph, resulting in a duplication of those sequences. In mt-rel-Da4, the introduced fragment contained the mt a idiomorph plus a small amount (possibly insignificant with respect to RIP) of flank. Typically sequences greater than 1 kbp are susceptible to RIP (Selker, 1990).  8 3 <§' p • to <D w  3  3  CD  3  CO  i  cp_  CD  6  ^  CD  >  > o  Q3  a g.8» 3 w  § » o „  Di  n CO  W ^ CD  § ~  CD o"  CD  o _ E?H  O  CD  2  5 9  c  § = 3 ~  3  CD CO  5 0  CD  2.  CT  3  02 •*  °- o CO 3 ^ o CD CD 2 3pop —*  CO CO  W  O <D CO CD  0)  I  I  CO  O O  Cr 8-  cr5T  0}  tt) 3  3  o o  I  cr  ro  cr -a • 03  ». p~s v<"  co •  5  P  03  CD  Cp_ CD  ju  CO  -  CD  5  8 §© 3 CD 3 03 3 Q.  —1 3  _ CD CL CD  m  w  _ 3 O ~o  3  a> o  cr —  CD N CD  „  CD -3  P CD  Sr  T3  CD CD  JT,"  ca CD o 33" •a CD a. o  o  d  fc  I  CD O  1 J  3  o' 3 o —r  3  cr—  3-  T3  Q-  2.  CT 9: g_  ~ 03 O CD  3  CO  3  03 — O  1 CO  T3  <= a- ~ CD  3  3  o' 3 o  O  3  ro cr  TD — D  3 xr  CD CD 0)  CD  ~ CD =T O —  CD  o  o c=--o "  -3  0)  3  18 Thirdly, Chapter 1 describes the examination of a second model for the mechanism of the mating type position effect, the cw-acting regulator model. The hypothesis states that the mt  A idiomorph correctly controls sexual functions only when located at the mating type locus because a cz's-acting sequence regulates the transcription of the mating type genes. When the genes are separated from it, incorrect regulation leads to the inhibition of ascus development. Evidence exists for altered expression of ectopic genes in N. crassa (Versaw and Metzenberg, 1996). To see if the mating type genes were expressed at different levels in ectopic-m/^ versus normal strains, mRNA amounts were compared. Changes in expression of the mating type genes in ascogenous tissue alone would have gone undetected due to the presence of excess perithecial tissue in mRNA preparations, and so early and late vegetative tissue were used. Finally, Chapter 1 describes the test of the following hypothesis: inhibition of mating type function occurs when the two mating types occupy the same nucleus prior to karyogamy. In the ascomycetes, P. anserina and C. heterostrophus, mating type genes in ectopic chromosomal locations are able to function as wild type if and only if the resident mating type gene is deleted (Coppin et al., 1993; Wirsel, Turgeon and Yoder, 1996). This phenomenon is called interference and is not due to RIP, since RIP does not occur in these fungi (Selker, 1990). In P. anserina, a transformant heterozygous at the mating type locus produced barren perithecia in great excess of fertile ones. These few fertile perithecia produced normal amounts of ascospores (Picard, Debuchy and Coppin, 1991). In C. heterostrophus, the interference phenotype is the inhibition of ascus and ascospore development (Turgeon et al, 1993). Do the opposite mating type genes of N. crassa inhibit each other? Since mating type genes in ectopic chromosomal positions in N. crassa do not function as wild type (Chapter 1 Results and Discussion), careful observation was required in order to identify interference. The normal fertility of dual mating type heterokaryons may seem to contraindicate interference, but the heterokaryon is an inadequate test of the interference hypothesis. For one thing, homokaryotic sectors may exist within a heterokaryon, and therefore the mating type products may not even be in physical proximity to each other and so interference is not observed. Secondly, the mating type products may not encounter each other frequently in a heterokaryon  19 as they are probably nuclear-limited. The nuclear localization of the mating type products is inferred by the observation that A and a nuclei encounter one another in ascogenous hyphae, yet maintain their individual identities, as shown by the fact that one nucleus of each type becomes sequestered into the crozier. Nuclear localization is further supported by the fact that some heterokaryon compatible mutants have reduced transformation efficiency with opposite mating type D N A fragments (Saupe et al, 1996). Finally, a mating type mutant cannot be fully rescued by a fertile heterokaryotic partner (Raju, 1992). If mt A and mt a interfere with each other's functions, one might see that deletion of the opposite mating type gene from a heterozygous transformant allows sexual development to proceed normally. To determine whether ectopic mating type genes interfere with the function of resident mating type genes and also whether resident mating type genes interfere with the function of ectopic mating type genes, perithecia from the following crosses were examined. Fertility was compared between crosses of strains with mt a at the mating type locus with and without an ectopic mt A idiomorph. Fertility was compared between crosses of strains with mt A at the am locus with and without mt a at the mating type locus. The experiments could not be done with ectopic mt a and resident mt A due to the sterility of the ectopic-*? strain, mt-rel-Da4. Chapter 1 aims to answer the following questions. Do the A mating type genes function abnormally in ectopic chromosomal positions? If so, what is the phenotype of ectopic-mating type strains? How is this putative position effect mediated?  20  Materials and Methods  Strains  Strains utilized in Chapter 1 are listed in Table 1.2.  Table 1.2. Strains used in Chapter 1. Strain name  Genotype  Source  1-10-1 R L M 44-02  ad-3A nic-2; tol a thi-4 ad-2[ad-5J; thi-4 ad-2[ad-5]; thi-4 ad-2[ad-5J; thi-4 ad-2[ad-5J; fla flA wild type wild type tol trp-4 a  A. J. F. Griffiths R. L. Metzenberg  mt-rel-Ac6 mt-rel-Aflk mt-rel-Da4 OR8-la 74-OR23-1A  lys-1 AA am AAfAJ am AA[A with flank] am AAfaJ  R. L. Metzenberg R. L. Metzenberg R. L. Metzenberg FGSC 4347 FGSC4317 FGSC 988 FGSC 987 FGSC 2337  The tol allele allows the compatible heterokaryosis of A and a strains (Newmeyer, 1970; Vellani, Griffiths and Glass, 1994) and was included in the host strain to allow the integration of  mt A D N A . Strains with the ad-3A gene require adenine for vigorous growth. Strains with the nic-2 gene require niacinamide for vigorous growth. The two genes are tightly linked and lie 28 map units from the mating type locus. Strains with the/7 allele make suitable mating type testers because they lack macroconidia and produce abundant protoperithecia. Typically, they were grown on synthetic crossing medium (Westergaard and Mitchell, 1947) for a minimum of 5 days before fertilization. A l l N. crassa strains described in this thesis are of the Oak Ridge genetic background.  21  Media  Strains were grown on standard media (Davis and de Serres, 1970).  D N A Preparation  The TV. crassa Orbach/Sachs genomic library was obtained from the Fungal Genetics Stocks Center. Cosmid D N A was isolated from E. coli with a Plasmid Kit (Qiagen, Chatsworth, Calif.). Genomic D N A was isolated from TV. crassa by the method of Lee and Taylor (1990).  Transformation  TV. crassa spheroplasts were prepared and transformed according to Schweizer et al. (1981), using the modification of Akins and Lambowitz (1985).  Crosses  Hygromycin-resistant transformants were tested for mt A mating type function by replica plating. A circle of Whatman 1 filter paper (Whatman International Limited, Springfield M i l l , Maidstone, Kent.) was placed onto each transformation plate and transferred the next day to a lawn offl a with visible protoperithecia.  Mating non-sporulating (MNS) transformants were tested for their ability to mate as a males by placing 0.25-0.5 mL of conidial suspension of the M N S strain onto af! A lawn with protoperithecia. For testing as A and a females, the M N S strains were grown on slants of crossing medium (Westergaard and Mitchell, 1947) supplemented with adenine (Eastman Fine Chemicals, Eastman Kodak Company, Rochester, N . Y.) and niacinamide (Sigma Chemical  22 Company, St. Louis, MO). After the appearance of protoperithecia (8 or 21 days later in the tests of the M N S strains as A females, and 21 days in the tests as a females) 100-200 pL of conidial suspension the male parent (either OR8-l<3 or 74-OR23-lyf) were added.  D N A hybridization  Genomic D N A was digested with BamHI for three hours. Digested D N A was partitioned by gel electrophoresis and transferred (Sambrook, Fritsch and Maniatis, 1989) onto Nytran™ Plus nylon membrane (Schleicher and Schuell, Keene, New Hampshire). The blot in Figure 1.3 was probed with a 3 2 P-labelled 9.6 kbp BamHI fragment of pG16/C10.  Probe preparation  Cosmid D N A (pG16/C10) was prepared using a Plasmid Kit (Qiagen, Chatsworth, Calif.) and digested with BamHI. The fragments were separated electrophoretically. The 9.6 kbp BamHI fragment was cut from the agarose gel and purified by centrifugation of the gel slice through Whatman 1 paper (Whatman International Limited, Springfield M i l l , Maidstone, Kent.). The fragment was radioactively labelled with Redivue™ a- 3 2 P-dCTP (Amersham, Oakville, Ont.) using the random primer method (T7 QuickPrime™, Pharmacia, Baie d'Urfe, Quebec).  Photography  Perithecial contents were squeezed with forceps into a drop of sterile distilled water on a cleaned slide and covered with a cover slip. Slides were viewed with an Olympus BH-2 compound microscope and photographed with T M A X 100 black and white negative film.  5 3  CD  § 3  GO  o  £3  Sis >3  24  R N A Isolation  R N A was isolated from frozen tissue according to Logemann, Schell and Willmitzer (1987). Poly(A) + R N A was isolated from total R N A using an Oligotex™ mRNA kit (Qiagen, Chatsworth California).  Slot Blots  Slot blots were made on a PR648 Slot Blot (Hoeffer Scientific Instruments, San Francisco, California) according to the manufacturer's instructions. Several different quantities of Poly(A) + R N A were loaded in each row of slots to enable the estimation of mechanical accuracy (homogeneity of mRNA in solution, pipetting error, etc.) and to allow a choice of slots to compare. Each slot blot in Figure 1.4 consists of a set of six slots. The top rows of slots on each of the two slot blots in the left panel were loaded with 125 ng, 250 ng and 500 ng of mtrel-Ac6 mRNA. The bottom rows were loaded with 125 ng, 250 ng and 500 ng of 74-OR23-lyf mRNA. The top rows of slots on each of the two slot blots in the right panel were loaded with 135 ng, 270 ng and 540 ng of mt-rel-Ac6 mRNA. The bottom rows were loaded with 130 ng, 260 ng and 520 ng of 74-OR23-1^4 mRNA. In Figure 1.5, the four slots on each of the two slot blots were loaded with 160 ng and 325 ng of mRNA from six-day-old mt-rel-Ac6 mycelia and 180 ng and 365 ng of mRNA from six-day-old 74-OR23-1.4 mycelia.  Slot Blot Probes  Two constitutively expressed gene controls were used: crp-1, the N. crassa gene with homology to yeast and mammalian ribosomal proteins (Kreader and Heckman, 1987), for the slot blot probed with mt A-l, and (3-tubulin (Orbach, Porro and Yanofsky, 1986) for the slot blot probed with mt A-2 and mt A-3.  25  mRNA from three-dayold mycelia  125 ng  mt-rel-Ac6  mt A-l probe  74-OR23-1A  fi  250 ng  500 ng  mRNA from six-day-old mycelia  125 ng  250 ng  500 ng  I 1  mt-rel-Ac6  crp-1 probe  74-OR23-1A  Figure 1.4 Poly (A) + R N A slot blots of three-day-old (left panels) and six-day-old (right panels) vegetative mycelia from mt-rel-Ac6 and 74-OR23-1A. The mt A-l probe was a Hindlll fragment from pBSmt-150 (top panels). The unregulated control probe was an EcoRI-cut plasmid (pCCl) containing the small ribosomal subunit protein gene, crp-1 (bottom panels).  74-OR23-1A  mt-rel-Ac6  170ng  340 ng  170 ng  340 ng  mt A-3 probe  ^-tubulin probe  Figure 1.5 Poly (A) + R N A slot blots of six-day-old mycelia of approximately 170 ng and 340 ng of mt-rel-Ac6 (2 left-most slots in each panel) and 74-OR23-1A (2 right-most slots in each panel) probed with an mt A-3 probe (top panel) and a (3tubulin probe (bottom panel).  27 The p-tubulin probe was a fragment of D N A approximately 500 bp long amplified from N.  crassa genomic D N A with the primers, B T l a and B T l b whose sequence is listed in Chapter 2 Materials and Methods (Glass and Donaldson, 1995). The crp-1 probe was the crp-1 gene cloned into a plasmid (pCCl) and digested with EcoRl. The mt A-l probe was a Hindlll-cut plasmid (pBSmt-150) containing cloned sequences from the  mt A-l gene. The mt A-2 probe was an EcoKl-cut plasmid (pNTA2) which contained a 700 bp PCR fragment of the mt A-2 gene amplified with the primers, r l . l and rl.2 (Ferreira, Saupe and Glass, 1996). The mt A-3 probe was the 1.2 kbp PCR product amplified from N. crassa genomic D N A with the primers r l l . l and rll.2 (Ferreira, Saupe and Glass, 1996).  Slot Blot Analysis  Autoradiograms of the slot blots were scanned into a computer using a Fotolook flatbed scanner. The images were analyzed to determine the relative optical densities of the slots using the public domain NIH image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The " uncalibrated O.D." option was chosen. Optical density profiles were generated from slot slices. A representative plot is shown in Figure 1.6. Three independent sets of measurements were made for each row of slots (Appendix B, Table A), with the width of the slice kept constant for each set of measurements. On the optical density plots, baseline gray levels were estimated by eye independently for each slice for the purpose of eliminating background grayness. Then a vertical section of the plot was delineated and the area under the curve within this section was measured and taken to represent the optical density of that slot. Each vertical section was 22 units wide and was positioned to include the highest point of the curve.  Figure 1.6 Sample of slot blot analysis performed using NIH image. Two horizontal slices were delimited in the image of the autoradiogram, corresponding to three slots per slice. The graph was generated using the gel plotting macro provided with the program. In the graph, the height of the peak is proportional to optical density. Baselines were estimated to eliminate background grayness. Numbers over the peaks indicate area under the peak with a peak-width of 22 units. The plot shown represents 1x, 2x and 4x amounts of mt-rel-Ac6 (top) and 74-OR23-1A (bottom) mRNA probed with the mtA-1specific fragment.  29  Results  The first question to be addressed was the following: are there any chromosomal locations other than the mating type locus that allow full function of the mating type genes.  Analysis of mating, sporulating strains.  In order to obtain strains in which introduced mt A sequences enabled a transformed host to mate and sporulate as A, a strain of mating type a (ad-3A nic-2; tol a) was transformed with a cosmid, pG16/C10. The cosmid contained the mt A idiomorph plus 17 kbp of centromere-distal sequences and 13 kbp of centromere-proximal sequences (Figure 1.7). Transformants were selected for hygromycin resistance and 734 colonies were obtained. The hygromycin resistant transformants were tested for their ability to mate as A by replica plating them onto a lawn of fl a (see Chapter 1 Materials and Methods). Most of the transformants (640/734) did not produce any perithecia on the tester plates. Some of them (94/734) produced perithecia, indicating that at least mt A-l had integrated into the host genome and was functional. The mating type tester plates were observed for one month and within that period the perithecia from a small number of colonies (6/94) produced ascospores in varying amounts. Four of the six colonies were subcultured to fresh medium. The remaining two transformants did not grow when transferred to fresh medium (presumably because they were too old). The six mating, sporulating transformants are hereinafter referred to as MS-1 through MS-6. Although MS-5 and MS-6 did not grow, their progeny were still included in the analyses. Fifteen of the transformants that mated, but did not sporulate were also transferred to fresh culture tubes for further analysis. They are referred to as MNS-1 through MNS-15 for mating, «on-.sporulating.  17 kbp flank  mtA idiomorph  i  i  13 kbp flank  transform ad-3A nic-2; tol a select for  hygR  94/734 hygromycin-resistant transformants produced perithecia as A (6/94 MS=mating, sporulating) (88/94 MNS=mating, non-sporulating)  Fig. 1.7 Transformation of ad-3A nic-2; tol a strain with cosmid pG16/C10 to generate transformants with mtA integrated at various chromosomal positions, hph is a bacterial gene encoding hygromycin B phosphotransferase which confers resistance to the protein synthesis inhibitor, hygromycin. bla is an Escherichia coli gene encoding resistance to the drug, ampicillin.  31 The fertility of the MS strains varied widely. Ascospore quantities were estimated by eye (Table 1.3). Table 1.3 Variable fertility of MS transformants. Transformant  Approx. number of ascospores/plate  "IvTSTT MS-2 MS-3 MS-4 MS-5 MS-6  20 000-50 000 10 000 1 000 200 100 60 The fertility of the progeny from the crosses of the MS strains to fl a was tested by  crossing them to fl a and fl A. The A progeny from the crosses of the M S strains to fl a did not share the same fertility phenotype as their A MS parent. For example, in contrast to the abundant ascospores produced by MS-1 and MS-2, 3/30 A progeny from MS-1 and 2/13 A progeny from MS-2 produced no ascospores at all. The reasons for the infertility are unknown. The percent germination of the ascospores from the cross of the MS strains to fl a ranged from 81% to 6% (Table 1.4), in contrast to the 95% germination of wild type crosses. The cause of the variable germination frequency is not known. Table 1.4 Variable percent germination of MS ascospores. Transformant  MS-6 MS-2 MS-3 MS-4 MS-5  # germinated ascospores/ # ungerminated ascospores culled  % germination  58772 23/56 25/72 64/228 17/72 4/72  8T 41 35 28 24 6  To determine whether the introduced mt A genes segregated from mt a at the mating type locus, the MS strains were crossed to fl a and the progeny were scored for their mating type. The MS progeny mated as A or a with a segregation ratio of 1:1 (Table 1.5). The 1:1 ratio is supported at a statistically significant level (p=0.05) in a chi-square test applied to each of the six crosses, individually.  32 Table 1.5 Segregation of mating type in MS progeny. Cross  # a progeny  # A progeny  MS-lxfla MS-2xfla MS-3x/7a  29 12 36  3TJ 13 28  MSSxfla MS-6 xfl a  2 14  2 9  MSAxfla  7  10  If wtf A integrated at the mating type locus in the MS strains, then the introduced mt A D N A would have become genetically linked to ad-SA nic-2, markers that were present in the original recipient strain where they were linked to mt a. The normal genetic distance between the mating type locus and ad-3A nic-2 is 28 map units. The model of 28% linkage between the newly integrated mt A idiomorph and ad-3A nic-2 was applied to the MS progeny (Table 1.6). The MS parent was ad-3A nic-2 and the other parent was ad-3A + nic-2 +. The model of 28%) linkage was accepted in a chi-square test applied to each of the six crosses independently at p=0.05, suggesting that the introduced mt A sequences had integrated at or near the mating type locus in the MS strains.  Table 1.6 Segregation of markers linked to mt Cross  Parental types  MS-5 xfl MS-1 xfl MS-2xfla MS-3 xfl MS-4 xfl MS-6 xfl  a a a a a  Recombinant types  ad nic A  ad + nic + a  ad + nic + A  ad nic a  1 6 3 1 6 3  2 4 3 5 1 7  1 0 0 3 1 0  0 0 0 1 1 0  To determine whether the mt A idiomorph alone or parts of the rest of the cosmid had integrated, the hygromycin sensitivity of the MS strains and MS progeny was tested. Two of the MS strains did not grow when transferred from the transformation plate, and so were not  33 tested. (The progeny were obtained by replica plating from the transformation plates onto lawns of females; see Chapter 1 Materials and Methods) MS-1 and MS-2 were hygromycin resistant, MS-4 had an intermediate resistance and MS-3 was sensitive. Ten progeny from each cross of the MS strains to fl a (four in the case of MS-5) were tested and they were all hygromycin sensitive, suggesting that only the mt A idiomorph had integrated. Occasionally, introduced D N A recombines with endogenous homologous sequences without gene replacement (Miao, Rountree and Selker, 1995). In order to determine whether this event or gene replacement had occurred at the mating type locus, MS progeny strains were assayed for the presence of mt a sequences. Since the MS transformants were presumably heterokaryotic and therefore would contain mt a sequences, the progeny were examined instead. Progeny of mating type A that issued from the cross of MS x fl a were examined for the presence of mt a mating type sequences. A Southern blot was made of 16 of the MS progeny that mated as A (representing all 6 MS strains), the ad-3A nic-2; tol a recipient strain, the transforming cosmid and a wild type A strain. The blot was probed with a 9.6 kb BamHI fragment of the cosmid, which contained mt  A-l, mt A-2, part of mt A-3 and approximately 5 kbp of centromere-proximal flank (Figure 1.8). Probing revealed the presence in the 16 progeny of a 9.6 kbp fragment specific to mt A and the absence of the 5.7 kbp fragment specific to mt a (Figure 1.3, lanes 4-19 ). Lanes 4 and 5 were progeny from MS-1; lanes 6, 7, and 8 were progeny from MS-2, lanes 9, 10 and 11 were progeny from MS-4; lanes 12, 13 and 14 were progeny from MS-3; lanes 15, 16 and 17 were progeny from MS-6 and lanes 18 and 19 were progeny from MS-5. The MS strains were tested for their ability to mate as a by inoculating subcultured colonies onto a lawn offl A. MS-1, -2, -3 and -4 mated and sporulated abundantly. MS-5 and -  -TCQ  »  ^  C  ®  tt>  ^ O r+  £D T3  CD g CD ~*  CT ni CQ 2.  I» CD =S  I  aI  Co  3 ?  i»  •DO  Q-  -*  "O  „  CO  =5 CL  CO IT O  3  03  fD -  o§ CD  3  "3  CO CD  8 I° CD 03 3  CT  5 2. m" 3 CQ' CO CD _Q C ro  -DO  cn  B  CO 3-" CD CO  CT T3  "  $ » o ^  3 8=5.  CD  — 03 C Q ' CO  Z °-  5-§  CQ  c  -D  <  ro  °-  -a  cr ~* 03 CL  o  CT  ro  -DO  -oo  35 6 could not be tested because as previously mentioned they could not be subcultured from the transformation plate.  Analysis of the mating non-sporulating strains.  The ability of the M N S strains to mate and sporulate as males and females of both mating types was assessed (Table 1.7). In the crosses testing the ability of the transformants to mate as males and females, the female parents were fl A and fl a, and the male parents were OR8-la or 74-OR23-1/1, respectively. The appearance of perithecia showed that mating had occurred. As A males, all of the M N S strains mated, but did not sporulate (hence the name "mating, non-sporulating"). As A females crossed to the wild type strain, OR8-la, all of the MNS strains mated in at least one replicate cross. In one of two replicate crosses (the set with the 8-day-old females), five of 14 strains (MNS-2, -4, -7, -9 and -13) produced a small number of ascospores, while the remaining nine strains produced none. As a males, Four of five strains (MNS-1, -2, -3 and -5) mated and produced abundant ascospores. MNS-4 mated, but did not sporulate. As a females, 11 of 14 strains (MNS-1, -2, -3, -5, -6, -8, -9, -10, -13, -14 and -15) mated and sporulated, while two strains (MNS-4 and -7) mated, but did not sporulate and one strain (MNS-11) neither mated nor sporulated. A Southern blot of genomic D N A from the M N S strains was probed with the 9.6 kbp  BamHI fragment of the cosmid, pG16/C10. Overnight exposure of the Southern blot (Figure 1.3) showed that MNS-4, -6 and -7 contained a 9.6 kbp band of the same intensity as the 5.7 kbp  mt (3-specific band. Longer exposure (not shown) revealed the presence of less intense bands at 9.6 kbp and larger and smaller than 9.6 kbp which were not seen in the MS or MS progeny lanes. Some strains had multiple bands.  13 0) P- O  i i »°  ff> IT O ~ T3 O.T3  CD  o ^ o cr cq CD CD  ^  2.  § 3  i  i  i  i  i  i  i  i  i  i  i  i  i  i  i  +  +  +  +  +  +  +  +  +  1  CD |-  +  +  +  +  +  CD 13 C  o  o  o  o  o  o  $ » CD CO CD 13  CD CT CD  - ' - ' - ' - ' - ' - ' ( D C O v J O C n ^ C O M ^ cn -t* oo ro — o +  -o o  CD  H  CD o  o  o  o  o  o  o  o  o  3  >  CD CD CO CD Cp_ CO O CD O  3  CD  c i CD  g. » co _  1 co < 8  CO CD .3 £D CO  O O  CO T3 O  CD CO O —\  3  CD JO  l—t-  CD CO c cr CD  co CD 3  CD  £D D_ C C D CD CO  CD O O  c CD 13 3D C CL  o  o o  -«• o o o o o o ° cn o rb o o o  13131313131313131313 0.0.0.0.0.0.0.0.0.0.  ro o -»• o o o o o - o o o o  3  +  + + + + O  Oi  o o o o o o  ->•  ->•  o o  o o cn o o I o cn o o o o  >  CD CO t—t- is. CD ciT  CD 3 CO O £D_ o  o o o o I o o o o o  o  CD  O  o  CO "O  TJ_ £D  CT CD^  p ro o o  + + + + + + + + + + '+ + + + + + + + +  o  CD  r—t-  p o  + + + 2_ '  CD CD CD CO O  CO  •g_  + +  o  —*  O O  CO TJ O o  O  O  D"  CQ CD CO —^  CT D SCD CO CO T3  CD  ro  >  CD CO I—I- CD CD CD CD. CO CD O O  CQ CD 13 CD CO co'  CD 13 Q. CO o c.—*-  C D —* 13 IT  CT O  CD 13 o  *< T3 CD CO O cn CO  CD 13' CO —\  co^  13 ' O.  +  l—I-  o o  9 cn  o o o  g  V  - l  o  g  ^ §  ?  o o o  +  °  9 oi  o o o  +  cn . o o o o —L o o o o cn o o o o o  +  +  +  +  +  cn o o o —i.  cn cn o o• o o —i. —i.  o o o  o o o  o o o  +  +  +  cn tn CO o o o o o cn —i. —i. -o < o o < o o o o  3  >  CD CO I—*-CD ro O CD.  C D CtT O CO CD 3  CO  cn CT  13 13 13 13 13 0.0.0.0.0.  +  +  +  CD  TJ  CD CQ 3  CD 13  37  Analysis of ectopic-m/ strains.  Since well characterized strains known to have a single ectopic insertion of various amounts of the ml A idiomorph flanking sequences and no mt a idiomorph, were available, I decided to study the effects of ectopic mt A sequences with these three strains (Figure 1.2). This strategy also eliminates the complication of both idiomorphs being present in one parent. The cross of mt-rel-Ac6 (Figure 1.2) to wild type, OR8-la, was analyzed in order to determine the phenotype of a strain in which there was a single ectopic copy of the mt A idiomorph. In this cross there were no duplicated sequences (and therefore no RIP) and there was no opposite mating type (and therefore no interference). Mating efficiency (number of perithecia) was comparable to wild type. Light microscopic observation of cells squeezed with forceps from perithecia in crosses of mt-rel-Ac6 (and its A progeny) to various normal-/^ strains were made at 5, 8, 11, and 12 days postfertilization. Examination of internal features of perithecia earlier than 5 days post-fertilization requires sectioning of the perithecia. At 5 days post-fertilization, perithecial contents from crosses of the normal-m/ and ectopic-m? strains are already different. Perithecia from crosses of normal strains contain rosettes in which ascospores are just beginning to develop within the few asci. Perithecia from crosses of ectopic-m/ strains contain rosettes, but the asci have no ascospores yet. At 8 days post-fertilization, perithecia from crosses of normal strains contain rosettes with many asci, many of which contain 8 ascospores, whereas ectopic-^/ perithecia contain notably fewer asci and significantly fewer ascospores (Table 1.8). The data were not normally distributed and so were analyzed using the non-parametric Mann-Whitney Rank-Sum test which is robust to non-normally distributed data. The two crosses were shown to be significantly different (Appendix C, Table A). Perithecial contents from 11 days post-fertilization crosses of ectopic-^ and normal-w-tf strains clearly show the reduction in ascus and ascospore number of the former compared to the  38 latter (Figure 1.9). The ascospores from both types of crosses germinate and appear to be completely normal (see below). Table 1.8 Number of ascospores per rosette 8 days post-fertilization Parameter  normal J x a  reiA x a  ¥ rosettes examined # ascospores/ rosette (range) Median 25th percentile 75th percentile  8 24-272  F8 0-16  44 29 114  0 0 2.5  Meiotic non-disjunction leading to chromosome duplication and deletion is a known cause of low ascospore production. To assess whether the correct chromosomal position of mating type is required for meiotic disjunction, the cross of mt-rel-Ac6 x OR8-la was analyzed (Figure 1.10). Only three of the four expected classes of meiotic products were recovered. The unrecovered class was A/a self-incompatible progeny. Self-incompatible progeny may have been missing because of a decrease in germination rate, growth rate or viability caused by the incompatibility. Ascospores were collected from the cross on two separate occasions. The first time, ascospores were spread onto petri plates, heat-shocked, allowed to germinate and grow overnight and then were cut out of the agar and placed into separate test tubes. Poorly germinating ascospores may have escaped detection, thus biasing the sample. The second time, ascospores were collected and placed in separate test tubes prior to heat-shock to eliminate bias. The expected number of self-incompatible progeny from this group alone was 10. It is possible that the sample size was small enough to have missed the self-incompatible class of progeny by chance. Duplication strains are viable in N. crassa and the cross was set up to reveal nondisjunction of L.G. V . Strains carrying a duplication of L.G. V would be am+ A. No am+  A progeny were recovered in which the A mating type reaction was normal. There were seven am+ A progeny which mated, but produced very few tiny perithecia devoid of ascogenous hyphae.  T3 TI I  5 w 2- H •  CT  r 3 ^ n  Z  o  cn "  3 3. £ ft I ft o  6 o£  ft o _ o—. O O  cn "  3  CL  X  o 3  •k cn  p en ra ^  3  ai -a O O  ^ o R  X _ 00 H^ i ?* p ©Ji 5" M  P  CO  p  cn  ft r o & N 3 2  § o p  CT CT CL 3- f  5  pa  era ft 5" N & o  13 o -!  O  M o  ca  Q o'  GO CT  Gfl  Ik  o|  o 3 H o_ o Hi O Cn C«  CT  O O  o l-l  cn cn  X " P  era g ft p  CL g* <-< » o  rj- cn O i  ^ CT  ft N & O 3 P  A'  40  CD O CO O C <?  CD  —\  —*  CD  1  CD Q.  . -»• O  cDl C  fl)^  3 j?  03 Q . "3 OT 0) CQ  = 3  CD X  c: CD  X X!  03  CD O  #—ICD Q.  o_ 03  en  (f>  3"  "2 o CD  o  s.  CQ  <  s.  <3 3"  <= 3 •a 0) 3 + CD 0)  03  3  03  3 +  + !  CD —  >  •a o  -^  CT> X  o O  <Q CD 3  00  I  0) CD 0?  a. c 5?_  03  3 + CD 03  03 + 03  03  3  03  3 +  03  3  o  3  3  03  3'  CQ  0)  CQ -< "O CD in CD  t—t-  T3  CD  3  O  o 3  T3  2. 5; (D  o_ 03 V)  m  ro CO  ro CO  ro  ro co  ro  ro co  CO  O <Q CD  0)  w 3 o  o CD Q.  O  3  *<  X •a CD  CO 00  0~  C/3 CD  —^ < CD  CL  41  Phenotypes of crosses with ectopic-mf strains.  Since the ectopic mating type strains generated above were dual mating type and/or heterokaryotic, single mating type strains were used to examine the mating type position effect phenotype and to test the models for the mechanism of action of the position effect. Various types of crosses were made with mating type ectopic strains in order to describe sexual development (Table 1.9). Wild type perithecia are black and typically have one long neck. On a typical crossing plate, a wild type cross might produce tens or hundreds of thousands of ascospores.  42  Table 1.9 Crosses with ectopic mating type strains.  1  2  # plates  Female  Male  Perithecia  Perithecial Necks  Ascospores  1  mt-rel-Aflk thi-4 ad-2[ad-5];  mt-rel-Da4 thi-4 ad-2[ad-5];  abundant brown  none  0  am AAfA flank]  am AAfa]  0R8-la wild type  abundant dark brown  none  100-500  mt-rel-Aflk thi-4 ad-2[ad-5];  abundant dark brown  mostly none; rarely very short  100-500  1  mt-rel-Aflk thi-4 ad-2[ad-5]; am AAfA flank]  3  with  1  with  fla  am AAfA with flank]  4  6  5  6  mt-rel-Ac6 thi-4 ad-2[ad-5];  mt-rel-Da4 thi-4 ad-2fad-5];  abundant brown  none  1-10 (less than 50 in total)  am AAfA] ml-rel-Ac6 thi-4 ad-2[ad-5];  am AAfa] OR8-la wild type  abundant brown  none or short  500-1000  ml-rel-Ac6 thi-4 ad-2[ad-5];  abundant brown  none or short  500-1000  no ascogenous tissue  none  0  74-OR23-IA wild type  few no ascogenous tissue  none  0  mt-rel-Da4 thi-4 ad-2[ad-5];  abundant no ascogenous tissue  mostly none; occasionally very short  0  am  6  1  AAfA]  fla  am AAfA]  7  8  7  7  mt-rel-Da4 thi-4 ad-2[ad-5];  mt-rel-Ac6 thi-4 ad-2[ad-5];  am  am AAfA]  mt-rel-Da4 thi-4 ad-2[ad-5]; am  9  AAfa]  1  flA  AAfa]  am AAfa]  Numbers in parentheses refer to the line numbers in Table 1.9 in the following five paragraphs. A cross between strains in which the mating type genes have been relocated to the am locus is sterile. Comparison of ectopic-/?// x ectopic-/??/ crosses to ectopic-/??/ x normal crosses ((1) cf. (2), (4) cf. (5), (7) cf. (8)) shows that when mt A and mt a are in ectopic, but homologous, chromosomal positions, perithecial development and ascospore formation are defective.  43 Both of the parents in the ectopic-m/ x ectopic-mt crosses, (1), (4) and (7), were ad-2. Crosses homozygous for ad-2 may be sterile (R. L. Metzenberg, personal communication to N . L. Glass). Two ad-2 +; A progeny from mt-rel-Ac6 x OR8-la were isolated and crossed as females to mt-rel-Da4 males. One of the crosses produced no perithecia and the other produced two small, barren perithecia. Crosses of strains with larger mating type flank duplications are less fertile than those with smaller duplications. Comparison of crosses (1) to (4) and (2) to (5) showed that females with "RIP-invisible" duplications of idiomorph-flanking sequences are more fertile than females with "RIP-visible" duplications of flanking sequences. Crosses of ectopic-^ are less fertile than crosses of ectopic-A. Comparison of crosses (8) to (5) and of (9) to (6) showed a mating type-specific phenotype. The a ectopic-m/ strain is female and male sterile, whereas the A ectopic-mt strain is female and male semi-sterile. Crosses (7) and (8) were repeated seven times each. In all cases, ectopic-^ a was completely sterile as a female. Perithecial contents were examined under 400x magnification. The perithecia were devoid of asci and ascogenous hyphae. Reciprocal crosses of ectopic-m/ strains are equally fertile. Comparison of the pairs of reciprocal crosses, (2) to (3), (5) to (6), (8) to (9), showed no sex-specific differences in fertility. Whether the ectopic-m/ strain was male or female made no difference to the fertility of the cross. In the cz's-acting regulator model, the mechanism for the positional sensitivity of the mating type genes was proposed to be mating type gene expression. The levels of expression of  mt A-l, mt A-2 and mt A-3 in an ectopic-mt strain (mt-rel-Ac6) compared to a normal strain (74QK12-\A) were assessed with slot blots loaded with Poly(A) R N A from three-day-old and six+  day-old mycelia. The experiment was performed once. The slot blots were probed with a constitutively expressed gene, either crp-1 or j3-tubulin and with the mating type genes (see Materials and Methods). Upon visual inspection, the levels of expression of all three of the mating type genes, mt A-l, mt A-2 and mt A-3, in the ectopic-/^ strain were very similar to wild type.  44 The four autoradiograms shown in Figure 1.4 were probed with the mt ^-/-specific fragment (top panels) and the constitutively expressed control, crp-1, (bottom panels). The lefthand panels show three-day-old and the right-hand, six-day-old mycelial mRNA. Figure 1.5 shows the slot blots probed with the mM-J-specific fragment (top panel) and the constitutively expressed control, fj-tubulin (bottom panel). The slot blots probed with mt A-2 are not shown because the background signal was high and interpretation was not possible from the scanned computer image (although interpretation was possible from the film). In order to detect slight but statistically significant differences in expression levels, the slot blots probed with mt A-l and mt A-3 were scanned by computer. The images were analyzed to determine the relative optical densities of the slots (Figure 1.6, see also Materials and Methods). The ratios of mt-rel-Ac6 mRNA to 74-OR23-ly4 mRNA from the control slot blots were averaged and compared to the analogous ratios from the experimental slot blots (Appendix B, Table B). The statistical tests (Appendix B, Table C) show, with two exceptions, no differences between the means. Two data sets (first and third slots) suggest that the amount of ml A-l transcript in threeday-old mt-rel-Ac6 mycelia is not statistically different from wild type, whereas one data set (middle slots) suggests that mt A-l is underexpressed in mt-rel-Ac6 relative to 74-OR23-1 A. The amount of mt A-l transcript in six-day-old mt-rel-Ac6 mycelia is not statistically different from wild type. One data set (first slots) shows that the amount of mt A-3 transcript in six-dayold mt-rel-Ac6 mycelia is not statistically different than wild type, while the other data set (second slots) shows that mt A-3 is overexpressed in mt-rel-Ac6. To determine whether or not mating type genes could interfere with each other's sexual development, various types of strains were crossed and examined for ascospore and ascus number. In order to obtain the requisite strains for the experiments, the cross of mt-rel-Ac6 x tol  trp-4 a was performed. Four types of progeny were generated from the cross of mt-rel-Ac6 x tol trp-4 a: those with no mating type, those with mt a at the mating type locus, those with mt A at the am locus and those with both mt a at the mating type locus and mt A at am (Figure 1.11). To observe the effects of adding the opposite mating type gene to a strain with a mating type  45 gene already present, the latter three progeny types, (1) a; (2) am A * and (3) am A Va, were crossed to fl mating type tester strains. The star symbol designates ectopic mating type genes. Perithecial contents were observed under the microscope. In general, the addition of the opposite mating type idiomorph to a haploid genome reduced the fertility of the cross (Table 1.10). The data shown in Table 1.10 are based on observations from crosses of 8 a strains, 6  A*/a strains and 7 A* strains at least 12 days post-fertilization. Table 1.10 Crosses of strains with and without the opposite mating type idiomorph. Cross  Phenotype  a xfl A  normal # of asci/perithecium normal # of ascospores  A*/axfl  A  reduced # of asci/perithecium reduced # of ascospores  A* xfl a AVaxfl  reduced # of asci/perithecium reduced # of ascospores  a  asci and ascospores absent from perithecia  Reduction in fertility of dual mating type strains compared to controls is seen by comparing the fertility of the a strains to the A*/a strains crossed as a (Table 1.10, lines 1 and 2) Figure 1.12 shows the perithecial contents of representative crosses. The cross in which one parent carried a heterozygous duplication of mating type,/? Ax A Va, had fewer ascospores than the control cross,/? Ax a. Similarly, fertility reduction is seen by comparing the fertility of the A * strains to the  AVa strains crossed as A (Table 1.10, lines 3 and 4). Figure 1.13 shows the perithecial contents of representative crosses. The cross in which one parent carried an ectopic A and resident a, fl a x 46^4 Va had fewer asci than the control cross,/?  ax&A*.  46  CO  i,  s ^ g a>  •o "5 O CQ ^  ro  CD ->• 3 • O "~ CD  1  G)  03 CD  3-  co 3  CD  -  T  K  A  Co  3~ CO  CD  a  _ Co.  O  CD  =r  •g ro o' co  c  CO  ro a.  <*  O co c  i  o.  3  O  o  CO  c  T3  al JP  0)  03 CD CO O  0)  -J  3 +  o> 3  0)  3 +  3 X  —* K o CD CO CO  CO  co co  co  2. o> 3"  en  m  x  cn  15  CL  03 03 03 CQ  3 CD Q_ 3 CD fP,  O  CO  03  o ro 32 Q . O  -H-  ^  CO  > =?  T3 CO T5 3  CO  ><. c CQ CD 3  o >< T3  CD  C3* ~+  0>  3  CO  m CD  3"  5  ro o  CD  I CT) O CD O O < ro —^ ro CL CL  c ro  03_ CD  CO  ae  o  rt- Q . 3 " >< CD o  1  O  CT)  D" CO  ro —* <  ro o.  tol trp-4  3 Q.  CD  O  ro  X  3. i . CO  T3  .  CT CD  I?  00  a  5' o iB 2, o 3  T3  <]>  <  -< CD CD 3 CO O CD 3  +  i h  o' i . S . 3 5 » Q. CD CO 3  I  > O CD  "O i  am  §  03  Co Co  CD o Q.  0) =J  o  = r3 CO o  CD  tol+1  0) < CD  T  CD ® ^ - CU  ,  al 2. °= •  CD  3  3  o  su  Z!  3 CQ CD  < 03  3 +  <  03  09  to  X  p  is. ^  &  48  49 The differences were statistically significant. Fertility was measured by obtaining data from observing perithecial contents of one cross of each of the four types described in Table 1.10. The fertility parameters were the number of ascospores per rosette eight and 11 days postfertilization (Table 1.11 and Table 1.12) and the number of asci per rosette 13 days postfertilization (Table 1.13). Table 1.11 Number of ascospores per rosette 8 days post-fertilization. Cross  # ros.  range asco./ros.  median  25th percentile  75th percentile  flA x a flA x A*/a flax A* flax A*/a  8 34 18 18  24-272 0-4 0-16 0  44 0 0 0  29 0 0 0  114 0 0.5 0  ros. = rosettes asco. = ascospores Table 1.12 Number of ascospores per rosette 11 days post-fertilization. Cross  # ros.  range asco./ros.  median  25th percentile  75th percentile  flA x a flA x A*/a flax A* flax A*/a  7  80-328 0-8 0-5 0  176 0 0 0  92 0 0 0  212 0 0 0  7 7 3  ros. = rosettes asco. = ascospores Table 1.13 Number of asci per rosette. Cross  # ros.  range asci/ros.  median  25th percentile  75th percentile  fl a x A* fl a x A*/a  18 21  0-13 0  5.5 0  0.5 0  6.5 0  ros. = rosettes  The reduction in the number of ascospores per rosette in crosses of a in the presence versus absence of an ectopic mt A idiomorph is statistically significant, as shown by MannWhitney Rank-Sum tests done on the raw data from eight days post-fertilization (Appendix C,  50 Table C). The reduction of the number of ascospores per rosette in crosses of A in the presence versus absence of a resident mt a idiomorph is not statistically significant as shown by MannWhitney Rank-Sum tests done on the raw data from eight days post-fertilization (Appendix C, Table D). Although the number of ascospores per rosette is not significantly reduced, the difference in sexual development was evident in the morphology of the ascogenous tissue. In order to quantify the difference in the sexual development of A * crosses compared to A Va crosses, the number of asci per rosette (rather than ascospores per rosette) was counted in 13 days post-fertilization perithecia (Table 1.13). Asci were counted if they had pale brown granular filling, mutant- or normal-looking ascospores. The 21 "rosettes" from the cross offla x A Va were mostly star-shaped masses of cells or masses of an undefined shape. The reduction in the number of asci per rosette is statistically significant as shown by Mann-Whitney RankSum tests (Appendix C, Table E). To see whether ectopic mating type genes could be interfering with meiosis, several crosses were examined for nondisjunction. Genetic analysis of the cross of mt-rel-Ac6 x OR8-  \a described earlier revealed no evidence for meiotic nondisjunction, whereas analysis of 46^4 Va xfl A produced possibly disomic progeny. In the cross of 46A Vaxfl A (Figure 1.14), if linkage group V segregated, then all A progeny with normal ascospore-per-rosette number should have been am+.  A l l A/A * progeny should have been am-. The A/A * progeny were  identified by reduced ascospore number per rosette (due to RIP or function of the ectopic mating type gene). A n internal control was the lack of this phenotype in the a progeny. While ten out often A progeny with normal ascospore number were am+ (as expected) and four out of ten A progeny with reduced ascospore number were am- (as expected), six out of ten A progeny with reduced ascospore number were am+ (unexpected). Additionally, one A/a progeny strain, which should have been am- had linkage group V segregated, was am+.  CC Q —\ CD b=. CD S" ^- 4^ o ° 2 co  OJ CD  o  CD zr  -\  CU CD  o cn  33  CO  £CDCD O CQ T3 O Q. C CO O &  CD X T3 0 O  f—H  CD Q. o_ cu CO CO  g  c  —\  0J  ZJ CD X XJ CD O  T5  <  1—+•  CD CL o_ £U CO CO  am  Q. CO' O 03 g -T C D n .+T3° c: 3 O 3 Q. C C D ^- CDQ x zr *< X! O — I CD CO zr O CD CD < CD  c  I! i b  i  <>  <  iki>  O  —t  C O Oo C D §o C CO CO O •g cn' co o > o  0) X  CD CO 0) zr O c_ Q. =r 03 3 ' 0) CQ < D •< CC "O Q C D C D O o CD £cZ o CU 3 O «—• C D Q.  N 5  cc  CD CO 0 3O CD o  11  p o'  3 0) C D Q. 0) CO I—»-  X  3 +  cu  3 +  fl)  3 +  fl) 3  fl)  3 +  fl)  > m  00  cx»  x T3 CD  00  0  o  C CD L  +  f—+  CD  CD  OJ  I zr 0J_ ZJ  o  CD O O < CD CD Q. CL C CD  o  O  CT CO CD CD CL  TJ  <  +  cu  3+  <  52  Discussion  Evidence for a mating type gene position effect in N. crassa was accumulated, and several models for the mechanism of its action were tested. In a transformation of a N. crassa strains with A genes, all of the transformants with fully functional introduced mating type sequences (the MS strains) had those sequences integrated at the mating type locus, suggesting the existence of a position effect. The data supporting gene replacement are the following: most importantly, the M S progeny that mated as A are missing a mt a-specific restriction fragment, a situation which presumably arose via gene replacement, the M S progeny had only one mating type or the other and they segregated with a 1:1 ratio; the genetic markers that were linked to mt  a in the ad-3A nic-2; tol a recipient strain became linked to the A mating type of the transforming cosmid in the MS strains; the MS progeny were all hygromycin sensitive, suggesting that the A mating type gene had been integrated without attendant vector sequences. Since the ectopic strains may have contained copies of both idiomorphs, the presence of the endogenous a mating type idiomorph may have eliminated the production of ascospores in crosses as A strains, giving a false impression of a position effect. The N. crassa mating type genes are known to disrupt each other's vegetative growth, manifested in heterkaryon incompatibility. Mating type sexual interference occurs in other filamentous ascomycetes (Wirsel, Turgeon and Yoder, 1996). If sexual interference were occuring in these experiments, then replacement events would have been favoured over ectopic integration events, thus giving the deceptive appearance of a position effect. Interference was shown to occur in N. crassa. Strains with dual mating identity were constructed and their the sexual development monitored. In order to eliminate vegetative incompatibility, which may have complicated the interpretation of the data, these strains were suppressed for mating type-controlled heterokaryon incompatibility. In a dual mating type strain, the presence of an ectopic mt A mating type gene affected the normal functioning of the resident mt a mating type gene, resulting in a statistically significant reduction in the number of  53  ascospores per rosette. In addition, the presence of a resident ml a mating type gene in a strain with an ectopic mt A idiomorph interferes with the functioning of the ectopic mt A mating type genes, resulting in statistically significantly fewer asci per rosette. The following results verified the existence of a mating type gene position effect independent from interference. Strains were obtained in which the mating type gene (A) was introduced into an ectopic chromosomal position and deleted from the normal locus. Crosses made between these and wild type strains developed abnormally and sporulated less abundantly than wild type crosses. This is the first report of a phenotype associated with ectopic mating type genes in the absence of significant sequence duplication and therefore in the absence of RIP. Two models regarding the cause of the position effect were proposed: the transvection model and the cw-acting regulator model. The transvection model proposes that the positiondependent functions of the mating type genes (ascogenous tissue development, meiosis, ascosporogenesis and possibly karyogamy) require close physical proximity of the mating type genes during the diploid phase of the life cycle. The c/s-acting regulator hypothesis states that the positional regulation of the mating type genes is under the control of a distant cw-acting sequence. The transvection hypothesis was tested and falsified, thus favouring the cw-acting regulator model. Crosses were made in which both parents contained a mating type idiomorph that had been relocated to the am locus (the two ad-2 +; A progeny from mt-rel-Ac6 x OR8-la crossed to mt-rel-Da4). The sterility of these crosses failed to support the transvection hypothesis, with the following caveat. A l l of the strains used in these experiments were constructed using the same deletion strain as a host with the transforming genes being targeted to the same locus. Since one genetic background and one ectopic locus were used in these experiments, strain-specific or targetted locus-specific effects cannot be ruled out. Accepting the c/s-acting regulator until falsified, one can explain the results of the transformation experiments as follows. In the replacement strains of the transformation experiments, the mt A genes may have been united with a putative cw-acting control region (of  54  mt a) which was not present on the cosmid. If so, the c/s-acting region of ml a must be sufficient for controlling mtA genes in the appropriate way. A second possibility is that the exacting control region of mt A was present on the cosmid, but was integrated only in the replacement strains. In the replacement strains, recombination within the homologous flanks of the resident mt a and the mt A on pG16/C10 could have allowed integration of the part of the mating type region needed for the production of abundant ascospores. In this model integration of such large fragments would not have occurred in the ectopic integrants. Some of the M N S strains have the 9.6 kbp band of the mt A idiomorph, which includes 5 kbp of centromereproximal flank, so the necessary sequence would lie outside of this boundary or beyond the centromere-distal tip of the idiomorph. Prior to this work, it was unknown that N. crassa mating type genes interfered with each other's functions. A reanalysis of data claiming support for the existence of a mating type position effect shows that the data could be explained by interference and other hypotheses (Table 1.14). Included in the table is the consideration of mt A-2 and mt A-3 whose existence was unknown during some of these studies. A l l of the transformants in Table 1.14 lack the ability to produce ascospores. In other analyses of the N. crassa mating type gene position effect, ectopic integrations of the mating type gene were counter-selected (Chang and Staben, 1994). In this thesis, I examined both ectopic integrants and gene replacements and found a correlation between full A mating type function and integration at the mating type locus. The position effect phenotype was characterized in a cross (mt-rel-Ac6 x wild type a) lacking the complicating effects of interference and RIP. A common basis is implied for interference and the position effect by the striking resemblance between their phenotypes. The phenotype of interference (A Va crossed as a) resembles that of position effect (mt-rel-Ac6) and the A 11 Rlp  strain (genotype mt A-2 ml mt A-  3 m!) (Glass and Lee, 1992). Based on data extrapolated across mating types, interference and the position effect are additive, not epistatic to one another, i.e. a strain exhibiting both of these phenomena has fewer asci than a strain with just position effect or a strain with just interference,  55  Table 1.14 Updated hypotheses for " p o s i t i o n effects" . Transforming D N A  Hyp.#  Rer'.f  fragment used to generate RIP mutant  pr  1  ml A-l ml A-2 mutant (A  mt A idiomorph with 1 kbp of flank on either side (pGMT8.1)  pr  2  ml A-l mutant ^m42 ^m44 m54 ^m56  mt A idiomorph with a max. o f 2 kbp o f centromere proximal flank  pr  3  pi*r  3, 4  (a )  mt A idiomorph with at most 2 kbp o f centromere proximal flank ( p S V : 1 0 A ) (24 kbp plus un-3 on centromere distal side)  tol a  mt A idiomorph  pir  5,6  mt a-l mutant  mt A idiomorph  Pi  5  , mtA-1  pm  2  mt A-l mutant m64)  mt A-l  pr  3,4  tol A  mt a idiomorph  Pi  4, 7  tol A  mt a-l  Pi  7  mt A-l mutant  mt a-l  pi*  2  mt a-l  P  2  Host strain mtA-lmtA-2  mutant (A  1  )  mF  1  A  ^m64j mt a-l mutant ml  (a ) m33  mt A idiomorph deletion ( R L M 41-10)  (A  (A ) m44  mt A idiomorph deletion ( R L M 41-10)  # Hypothesis. Ascospore deficiency could be due to: p=position effect i n t e r f e r e n c e (whether mediated v i a protein or transcripts) i * i n t e r f e r e n c e ( i f mediated v i a transcripts. The mutations are premature stop codons or frameshirts w h i c h alter the protein.) r=R!P m=missing sequences | Reference 1 Glass and Lee, 1992 2 V e l l a n i , unpublished data -p Glass, Grotelueschen and Metzenberg, 1990 -> Glass etal., 1988 4 Chang and Staben, 1994 5 this study 6 Staben and Yanofsky, 1990 7  56 suggesting that they may possibly be affecting the same process via a different biochemical pathway. That opposite mating type genes can each affect the function of the other when they are in the same nucleus provides strong evidence that sexual development requires the mating type genes (or transcripts, gene products or mating-type specific target gene products, etc.) to be separate at some or all stages between fertilization and karyogamy. I speculate that the mating identity of nuclei may be affected in strains exhibiting interference. Nuclear identity may be required for one or more of the following three processes: the co-ordination of mitotic proliferation of nuclei in the ascogenous hyphae, co-ordination of the conjugate mitosis that precedes karyogamy and/or sequestration of one nucleus of each type into croziers. I speculate that in strains exhibiting interference, the identity is reduced by the presence of the opposite mating type idiomorph; in strains exhibiting the position effect, the identity is reduced by the failure to maintain appropriate expression of the mating type genes after fertilization; in the A n  R!P  strain, the mt A-2 and mt A-3 genes are mutated (Saupe et al, 1996).  The normal appearance of the cross that generated the A 1 Rlp  mutants which were  mutated in mt A-l and possibly mt A-2 (Glass and Lee, 1992; N . L. Glass, personal communication) favours the idea that identity is not required after RIP occurs. The one process of the three mentioned that precedes RIP, and therefore is most likely the one affected in the position effect and interference, is mitotic proliferation of nuclei in ascogenous hyphae. This model implies that interference causes an effective reduction in mating type function. If the position effect shares a common cause with interference, in ectopic-m/ strains, the mating type genes must be inappropriately down-regulated. I specify " down-regulated" rather than " repressed" because the genes are required for the formation of ascogenous hyphae and a small number are formed in mt-rel-Ac6 crosses. To ascertain whether or not ectopic mating type strains show altered mating type gene expression, expression levels of mt A-l, mt A-2 or mtA-3were determined. The autoradiograms of mRNA from mt-rel-Ac6 and 74-OR23-1A vegetative cells show that in mt-rel-Ac6 all three genes, mt A-l, mt A-2 and mt A-3, are transcribed. The expression levels of all three genes  57  appear to be equal in the wild type compared to the ectopic-^/ strain. The statistical analysis of the autoradiograms bears out this observation in the majority of cases. Two samples suggest that there may be slight differences in the amount of expression of the mating type genes in mtrel-Ac6 compared to wild type. These results suggest that the reduction in fertility of ectopic mating type strains is not due to large alterations in the expression in vegetative tissue. The similarity of expression levels in vegetative cells is consistent with the heterokaryon incompatibility phenotype of ectopic mating type strains, assayed by transformation efficiency into same and opposite mating type hosts (Glass et al, 1988). Since ectopic mating type strains can mate, one might expect the expression levels of the mating type genes to be close to normal before fertilization. The phenotype of ectopic mating type strains appears after fertilization. While the level of expression of the mt A genes is not greatly different in mt-rel-Ac6 compared to wild type in vegetative tissue; hypothetically, ascogenous tissue may be sensitive to changes of this magnitude or perhaps expression differences may be increased in ascogenous hyphae. The mating type genes encode putative transcription factors (Glass, Grotelueschen and Metzenberg, 1990; Staben and Yanofsky, 1990). Northern blot analysis of mating type mutant strains suggests that the mating type genes activate and repress some sexual development genes (Ferreira et al, 1998; Ferreira, 1997). The ability of the mating type genes to regulate these genes that are expressed early in sexual development opens the possibility that they may regulate the expression of genes required for ascogenous hyphae development or karyogamy. It may, however be difficult to detect a reduction in expression levels in ascogenous tissue using mRNA extracted from perithecia, given that the perithecial wall presumably would still be expressing the genes. Ascogenous tissue could be extruded from the perithecium, but the amount of material would be too small for R N A blot analysis. It may be possible to perform reverse transcriptase PCR on the ascogenous cells, however, the expression modifications could occur during the entire sexual cycle or during a particular stage of the sexual cycle, in all cell types or with cell-type specificity. When the ad-3A nic-2; tol a recipient strain was transformed with the pG16/C10 cosmid which contained the A mating type idiomorph, the percentage of hygromycin-resistant  58 transformants able to mate as A (94/734=13%) was small compared to another study, the transformation of a cosmid containing the mt A idiomorph, pSV:10A, into a mating type compatible mutant, a ml (Glass et ai, 1988) and to N. crassa transformations in general. The use of a mating type mutant as the recipient, rather than a strain with tol, as was used here may be significant. Although Glass et al. (1988) reported restoration of transformation efficiency of  a fragments into A recipients when the recipient had tol, some evidence exists that tol may not always suppress incompatibility completely, depending on a strain's genetic background (Vellani, 1991). A n additional explanation for the low transformation efficiency is quelling. Quelling refers to the spontaneously reversible mutant phenotype produced by an excess of wild type genes (Romano and Macino, 1992). Silencing of endogenous and ectopic genes is methylationindependent and correlates with the presence of sense R N A from the ectopic gene or gene fragment (Cogoni et ai, 1996). The frequency of quelling is highly variable between genes and could also be variable between fragments of the same gene or between host strains (Romano and Macino, 1992). If multiple copies of mt A had integrated into the transformants, as ectopically integrating transforming D N A often does (Miao, Rountree and Selker, 1995), quelling could have eliminated mating reactions and thus reduced the apparent transformation efficiency. The fertility of the MS strains was variable. This phenotype did not segregate with the integrated mt A mating type, suggesting that it was not produced by the specific sequences or position of the integrated mt A genes. The reason for the variability is unknown. The hygromycin resistance and the mating phenotypes of the MS strains requires explanation. If the MS strains underwent exact replacements of the mt a idiomorph with the mt A idiomorph, then they should have been hygromycin sensitive and unable to mate and produce ascospores as a. The ability to mate and sporulate as a is easily explained because N. crassa spheroplasts are multinucleate and so give rise to colonies heterokaryotic for transformed and untransformed nuclei in a transformation experiment. The ability to mate as a was probably provided by untransformed nuclei. The MS strains were selected by virtue of their hygromycin resistance. The possibility of mt A and hph having integrated at the mating type locus in the transformed  59  nuclei can be discounted because all of the M S progeny were hygromycin sensitive. Also subcultures of MS-3 and MS-4 were hygromycin sensitive. Although this hypothesis was not tested, one possible explanation accounts for the following observations: the initial hygromycin resistance of the MS strains, the ability of the MS strains to mate and sporulate as A and a, the variable amounts of ascospores produced by the MS strains when crossed as A, the loss of hygromycin resistance in some M S subcultures and the hygromycin sensitivity of the MS progeny. Most likely, the M S strains were heterokaryons with three nuclear types: untransformed nuclei (conferring fertility as a), transformed nuclei with ectopic insertions (providing hygromycin resistance through the phosphotransferase activity) and transformed nuclei with homologous integrations (providing fertility as A). Random sampling of nuclei during subculturing or shifts in nuclear ratios could explain the appearance of hygromycin sensitive subcultures in some of the M S strains and the hygromycin resistant subcultures in others. The observation that all of the MS progeny were sensitive can be explained by proposing that only nuclei in which mt A had replaced mt a were able to undergo karyogamy and give rise to progeny. The presence of many nuclear types, only one of which could produce ascospores could explain why the ascospore number was lower than wild type for most of the MS strains crossed as A. Much variability was observed in the mating and sporulation phenotypes of the M N S strains. The causes are unknown. Most of the M N S strains were fully fertile as a, likely due to the presence of untransformed nuclei and/or cells in the colony (Table 1.7). One strain (MNS4), however, failed to sporulate as a male or female, one strain that was tested only as a female (MNS-7) mated, but failed to sporulate and another strain that was tested only as a female (MNS-11) failed even to mate. The cause of the infertility of these strains is unknown. The possibility of rearrangements having occurred is supported by the appearance of hyaline ascospores (indicating aneuploid progeny) in two of the 11 fertile crosses of the M N S strains as females (MNS-1 and MNS-14). Southern blotting of the M N S strains revealed two classes of integrants. The strains in the first class, MNS-4, -6 and -7, contained the 9.6 kbp BamHI fragment in its entirety. The  60 cause of the lack of sporulation in this class is not known, but is not likely due to missing sequences since the 9.6 kbp fragment contained all of the genes except the 3' portion of mi'A-3. The strains in the second class, MNS-1, -2, -3, -5 and -8, contained portions of the 9.6 kbp BamHI fragment of the pG16/C10 cosmid, but not the whole fragment. The cause of the lack of sporulation in this class is unknown, but could be attributed to the absence of mt A sequences required for a normal amount of sporulation (the 3' end of mt A-l and/or mt A-2 and  mt A-3). Which of the mating type genes is being affected by chromosomal misplacement in this experiment is unknown. The phenotype of the cross mt-rel-Ac6 x OR8-la is similar to that of  A 11  R , p  (genotype mt A-2 mI mt A-3 ml) x wild type inasmuch as ascus number, but not perithecial  number is reduced (Glass and Lee, 1992). The similarity suggests that perhaps mt A-2 and mt A-  3 are position sensitive. The mt A-l gene displays a position effect, as shown by the nonsporulating transformations of A m mutants in which mt A-2 and mt A-3 are still at the mating type locus (Glass et al., 1988; Glass, Grotelueschen and Metzenberg, 1990). Since one of the ectopic-/! strains, mt-rel-Ac6, had " RIP-invisible" duplications of flanking sequences, the size of the mating type region that must remain intact for sporulation could be estimated. A 5656 bp fragment from the A mating type region is sufficient for mating identity and a reduced level of sporulation when located ectopically, suggesting that sequences outside of these boundaries are required for full fertility. The fragment contains all 3 of the mt A open reading frames. In addition, it contains 600 bp 3' of mt A-3 (300 bp between the mt A-3 stop codon and the centromere-distal end of the idiomorph plus 300 bp of centromere-distal flank) and 220 bp 3' of mt A-l (160 bp between the mt A-l stop codon and the centromereproximal end of the idiomorph plus 60 bp of proximal flank). This fragment likely includes all of the local regulatory elements of mt A-2, since that gene is sandwiched between the other two, and the 5' regulatory sequences of mt A-l and mt A-  3, since these genes are transcribed divergently toward the flanks. Position dependence on 3' local regulatory elements of mt A-l and/or mt A-3 is a formal possibility, however it is not  61 likely because flanking sequences extended at least 200 bp beyond the stop codons at either end of the fragment. Another transformation experiment suggested that a fragment with extensive flanking sequences is still position dependent. The fragment of mt A included up to 2 kbp of sequences flanking the idiomorph on the centromere proximal side and more than 20 kbp on the centromere distal side. A cosmid containing this fragment was used to transform a strain in which mt A-l and mt A-2 were both mutated (A 1 R , p strain of Glass and Lee (1990)). Transformants failed to sporulate when crossed as A (Glass, Grotelueschen and Metzenberg, 1990). The lack of sporulation in these transformants could have been due to a position effect or to RIP. Unfortunately, the length of D N A that integrated into the genome of the transformants was not determined. If the fertility reduction of mt-rel-Aflk relative to mt-rel-Ac6 is not due to RIP of genes residing in the duplicated flanks that are required for ascosporogenesis, then 2 kbp either side of the idiomorph appears to be insufficient to confer position-independent function. The translocation strain, T(I->II) 39311, is fully fertile (Appendix C, Table B). In this strain, the translocated fragment includes 35 map units (700-3500 kbp) of sequence centromere-distal to mating type and 18 map units (360-1800 kbp) of sequence centromere-proximal to mating type. To summarize, the smallest possible position-independent fragment identified so far is 700 kbp centromere-distal and 360 kbp centromere-proximal to the mating type locus. The cross of mt-rel-Ac6 x OR8-la yielded seven am+ A progeny which mated, but produced very few tiny perithecia devoid of ascogenous hyphae. Large barren perithecia are sometimes seen in a x a, a m x a and a m xA crosses, but not in Ax a, A m x a or A m x A crosses (Griffiths and DeLange, 1978; Griffiths, 1982). Apparently, the mating type region may impact on perithecial development. The origin of the tiny perithecia seen here is unknown, but could be explained by the following hypothesis. If the seven strains were disomic for linkage group V , RIP may have occurred in the duplicated region. If the RIP spread into the A idiomorph and mutated a  62 sequence required for the suppression of perithecial development, then perithecia may have started to develop. Phenotypes of dual mating type strains in other fungi vary. Like N. crassa, some M.  grisea dual maters (Mat 1-1 transformed with Mat 1-2 and vice versa) make fewer perithecia and the number of perithecia can differ widely, even among dual maters derived from the same strain (Kang, Chumley and Valent, 1994). Like N. crassa, one M. grisea dual mater produced perithecia in patches. Unlike N. crassa, the perithecia that do develop make normal numbers of ascospores. The ascospores are either one or the other mating type, suggesting that the dualmater has to break down into a heterokaryon before completion of the sexual cycle. Unlike the bidirectional interference seen in N. crassa, the interference in C.  heterostrophus is unidirectional. The presence of a resident mating type gene interferes with the function of the opposite mating type ectopic gene, resulting in the production of pseudothecia, without ascospores; however an ectopic mating type gene has no effect on the function of the resident gene (Wirsel, Turgeon and Yoder, 1996). Unlike N. crassa, self-mating reactions of C.  heterostrophus dual maters have a normal number of pseudothecia (Turgeon el al., 1993); N. crassa has fewer. In contrast to N. crassa, C. heterostrophus dual mater self-matings are fertile (ascospores are produced), but like N. crassa, they have a reduced number of asci. The lack of position effect in C. heterostrophus can handily explain this difference. Similar to N. crassa dual maters, P. anserina mat+mat- * transformants produce sterile perithecia when self-mated (Picard, Debuchy and Coppin, 1991). Unlike any N. crassa dual mater, however, one P. anserina mat+ *mat- transformant produced self-fertile perithecia (Picard, Debuchy and Coppin, 1991). A curious observation, possibly relevant to differences between mt A-2/mt A-3 and SMR1/SMR2, was that some mat+mat-* transformants using a cosmid that encoded all 3 genes of mat- produced a few fully fertile perithecia (Picard, Debuchy and Coppin, 1991). Unlike in N. crassa, in S. cerevisiae, heterozygosity of the mating type locus leads to the inability to mate (Haber and George, 1979; Klar, Fogel and MacLeod, 1979).  63  An unexpected class of progeny was culled from the cross of 46A */axf! A (Figure 1.14). These seven progeny apparently received am + from the fl A parent and A (at the am locus) from the dual mating type parent. If nondisjunction were occurring generally, mating type nulls would have appeared in the progeny due to the nondisjunction of linkage group I, provided that such progeny were viable. They did not. One possible explanation is that in some meioses, mt A on linkage group V paired with and segregated from mt a on linkage group I, leaving the linkage group V with am+ to segregate randomly, rather than away from its homolog. This hypothetical pairing may have been mediated by flanking sequences.  64  Chapter 2 A Search for Mating Type Gene Activity in a Homothallic  Neurospora  Species  Introduction  The life cycles of heterothallic (obligately outcrossing) and homothallic (self-fertile)  Neurospora species include a vegetative and a sexual stage. A mycelium can be propagated vegetatively in heterothallic species from a germinated macroconidium or hyphal fragment. Only the latter option is available to homothallic species because they lack conidia (Perkins and Turner, 1988). When nutrients are limited, the sexual cycle initiates in heterothallic species, beginning with the production of protoperithecia from which protrude the trichogynes, the receptive structures. A n opposite mating type gamete fuses with the trichogyne, initiating the development of the protoperithecium into ascospore-bearing perithecium. Development from this point, including ascus formation, meiosis and sporulation, is identical in eight-spored heterothallic and homothallic species (Raju, 1978; 1980). In homothallic species, sexual activity occurs via self-reproduction, and so mating type may be irrelevant. It is not known how the sexual cycle initiates in the homothallic species. Heterothallic Neurospora species (N. crassa, N. sitophila, N. intermedia and N. discreta) consist of a population of individuals, each having one of two mating types, A or a. Pseudohomothallic Neurospora species (TV. tetrasperma) are able to self-reproduce by producing ascospores containing nuclei of both mating types. Sexual reproduction in TV.  tetrasperma in nature likely occurs by self-reproduction most often since A + a protoperithecia lack active trichogynes (Bistis, 1996). Outcrossing could occur since the single mating type progeny that are occasionally produced do have active trichogynes (Bistis, 1996). Truly homothallic species are those in which two identical nuclei undergo karyogamy. That homothallic species rarely or never outbreed is supported by the following biological and  65 molecular data. The homothallic Neurospora species lack conidia and trichogynes (Nauta and Hoekstra, 1992b) and there is less RFLP variation within homothallic species compared to different isolates of a single heterothallic species (Glass, Metzenberg and Raju, 1990). Even though mating type identity may not be required in these species, they do contain mating type sequences, as detected by Southern blotting (Glass et al, 1988). The homothallic species are divisible into two groups based on their mating type genes (Figure 2.1). In one group (N.  pannonica and N. terricola), each nucleus contains sequences homologous to N. crassa ml A and N. crassa mt a (T. A . Randall and R. L. Metzenberg, personal communication; Glass et al., 1988; L. Wheeler and N . L. Glass, personal communication). In the other group (N. africana,  N. galapagosensis, N. lineolata and N. dodgei), only sequences homologous to TV. crassa mt A have been detected (Glass et al., 1988). The mt A-l homolog in TV. africana has 91% D N A sequence identity with TV. crassa mt A-l. When used to transform a TV. crassa mutant, the ectopically located TV. africana mt A-l gene induced perithecia, but not ascospore-formation (Glass and Smith, 1994). The mating type gene of TV. africana was unable to induce homothallism in TV. crassa recipient strains (Glass and Smith, 1994). In unmated cells of S. cerevisiae and S. pombe, the mating type proteins regulate mating via the pheromone response pathway (Dolan and Fields, 1991; Egel, Nielsen and Weilguny, 1990). In mated cells, they regulate entry into meiosis (Covitz, Herskowitz and Mitchell, 1991; Egel, Nielsen and Weilguny, 1990). By analogy, the N. crassa mating type genes may also regulate entry into meiosis. If so, TV. terricola and TV. pannonica may be homothallic because the presence of both mating type genes allows the initiation of the sexual cycle without mating. Perhaps ^4-only homothallic Neurospora species have genes that allow them to bypass the mating/pheromone response pathway altogether and begin ascogonial and ascogenous hyphae development directly without the need for mt a. In this chapter, N. terricola was selected as a model organism for the study of homothallism since, like TV. terricola, most homothallic species in the Sordariaceae have both mt  A and mt a sequences (Glass, Metzenberg and Raju, 1990; Poggeler et al, 1997a). Each nucleus of N. terricola has sequences homologous (as shown by Southern hybridization) to TV. crassa mt  Heterothallic species  N. N. N. N.  crassa sitophila intermedia discreta  P s e u d o h o m o t h a l l i c species  N.  tetrasperma  A+a  H o m o t h a l l i c species (two types)  N. N.  terricola pannonica  N. N. N. N.  dodgei lineolata galapagosensis africana  A/a  Figure 2.1 M a t i n g types of Neurospora ascospore; circle represents n u c l e u s .  species. O v a l represents  67  a-l, mt A-l, mtA-2, but not mt A-3 (Beatty, Smith and Glass, 1994). In contrast, mN. pannonica, all four mating type genes are present, and appear to be linked (L. Wheeler and N . L. Glass, personal communication). The N. terricola mt A-l gene has been cloned and partially sequenced (Beatty, 1993). It has a high level of D N A similarity to the N. crassa mt A-l gene, and it has been tested for mating type function in N. crassa because no transformation protocol has been developed for N. terricola. The N. terricola mt A-l gene, when used to transform a N.  crassa mating type mutant, confers mating ability, but not sporulation ability, to the N. crassa mutant. These results are comparable to the results of transforming a N. crassa mating type mutant with the N. crassa mt A-l gene. Transformation usually results in ectopic integration. As in most other Sordariaceae, the mt A and mt a regions in TV. terricola are physically linked. One report states that the opposite mating type probes both bind to a 3.4 kbp restriction fragment (Glass, Metzenberg and Raju, 1990), while another claims that the genes are separated by 12-20 kbp of D N A , as shown by OF A G E gel analysis (M. Smith, personal communication; Beatty, Smith and Glass, 1994). Is the activation of sexual reproduction in the homothallic species dependent upon the mating type genes? Only mutagenesis could say for certain. It is difficult to study the homothallic species because of a lack of genetic and molecular tools, namely, genetic markers, the ability to construct strains by outcrossing and inefficient transformation protocols that call for the spheroplasting of hyphae. What is the relationship between the c/s-acting regulator(s) and the mating type genes? How can the genes function without both being at the mating type locus? Is 3.4-20 kbp close enough for both mt A and mt a genes to be controlled by the same cz's-acting regulator or does each have its own? Does N. terricola even have a cw-acting regulator? Is there interference between mt A and mt a when they occupy the same nucleus prior to karyogamy, as in N. crassa! If so, how does TV. terricola deal with it? None of these questions is relevant unless the mating type genes are functional. It is conceivable that N. terricola does not need mating identity since all of its nuclei are identical. During compartmentalization of two nuclei into the penultimate cell of the crozier for  68 karyogamy, N. terricola needs to enclose precisely two nuclei, but their identities may be irrelevant. Moreover, homothallic Neurospora species do not mate, per se, as they do not have trichogynes, conidia or microconidia (Raju, 1978). The molecular structure and function of the N. terricola mating type genes are elucidated in this chapter. First, the D N A and amino acid sequences are compared to those of N. crassa. The cloned N. terricola mating type genes are then assayed for the functions of the N. crassa mating type genes (mating identity and vegetative incompatibility). The effect of a suppressor of the mating type associated vegetative incompatibility is tested on one N. terricola gene. Finally, expression of the mating type genes is examined in N. terricola.  69  Materials and Methods  Strains Strains are shown in Table 2.1.  Table 2.1 Strains used in Chapter 2. Strain name Genotype RLM 4 1 " 153 7 1-20-41 1-20-26  1 0  arg-2 mtArrjRJF) thi-4 ad-2; lys-1 AA un-3 ad-SA nic-2 cyh-1 A ad-3B a arg-1 ad-3B; tol A arg-lad-3BA  Source  R  NTLTGIass - L - Metzenberg A . J. F. Griffiths A . J. F. Griffiths A . J. F. Griffiths A . J. F. Griffiths  Plasmids Plasmids are shown in Table 2.2.  Table 2.2 Plasmids used in Chapter 2. Plasmid Description pNTal A. terricola mt a-l in pCR™ pONTa N. terricola mt a-l inpOKE103 (pan-2; qa-2) pONTA N. terricola mt A-l in pOKE103 (pan-2; qa-2) pBCl fi-tubulin in pAT153 (pBR322 derivative) pGMT8.1 TV. crassa mt A idiomorph in pGEM™ pCSN4 TV. crassa mt a-l in SK+ (pBluescript™) pBSqa-2mtl.7 TV. crassa mt A-l in pBSqa-2 pMP6 fcp/iinpUC18  Plasmid Preparation  Plasmids were prepared by either centrifugation through CsCl (Sambrook, Fritsch and Maniatis, 1989) or with a plasmid kit (Qiagen, Chatsworth, California). Primers  Primer names, map locations and sequences are shown in Table 2.3. Numbers for the first 11 primers refer to the mtA idiomorph sequence (GenBank accession no. M33876). Numbers for the next 12 primers refer to the mt a idiomorph sequence (GenBank accession no. M34287). Numbers for the last 2 primers refer to the fi-tubulin sequence (GenBank accession no. M13630). Table 2.3 Primers used to amplify genes in Chapter 2. Primer name  Map location  Sequence (5'-3')  3997-4021 5040-5062 5000-5020 3718-3733 2326-2342 3547-3563 4452-4468 3182-3199 3301-3317 2602-2619 4180-4202  ACCCAAACTTCCCACC ATGGTACCTCATCTTCCACTAACCC ATAGCCAGAGCCATGTTGT AAGTAGCATTGTTTGT GGTTTCCTTTTCGTCAG TGTAGTCAACGGGGATC TGTATTCGTCAATCCGG GCTCGCTGCTGACTTCGTCC CGCCAGCACCGACATCC GCATAGCAAGAGCTTGGC CGGCAATGAGAGCTTTCT  3590-3570 3535-3550 4615-4590 2950-2970 3810-3830 4100-4120 4370-4390 4300-4280 4100-4080 3820-3800 3290-3270 3030-3010  GCATGGATGCCCTTGGG GCCCGACAGTGTCGTCG GAGGTGATATCCTTGGTGACCGGG GAATGCTATTCAGGGCCG CAGCCGCACATTCGCGAG GAAAACTTCGCGCCGCCG CCAGGAGGCTAACGAGGC GGGTTGGGATTGGGGGGA CTAGATCCTGCGACGGGA GTGCGGCTGCTCATCACG GATATGCGCGACCAAGCC GGGGCAACGACGTCGGGA  1656-1679 2168-2192  TTCCCCCGTCTCCACTTCTTCATG GACGAGATCGTTCATGTTGAACTC  mt A 1778 3194 mint orchid rl.l rl.2 1875 A2-3200 chacha cactus condo  mt a Y661 Y663 Y694 Y759 Y760 Y761 Y762 Y763 Y764 Y765 Y766 Y767  fi-tubulin BTla BTlb PCR  The mating type genes of N. terricola were amplified from genomic D N A using N. crassa primers. The genes were also amplified from N. crassa genomic D N A as controls. PCR was  71  performed on a D N A Thermal Cycler 480 (Perkin Elmer, Foster City, California). Primers for  mtA-1 (1778, 3194) amplified the ORF plus 120 base pairs 5', for mt a-l (Y694, Y759), the ORF plus 777 base pairs 5', for mt A-2 (rl. 1, rl.2), the ORF plus 24 base pairs 5'. Primers Y694 and Y759 were gifts from C. Staben (University of Kentucky). Amplification of mt a-l was confirmed with a Southern blot probed with a chemiluminescence-labelled mt a-specific probe (pCSN4). Cycling for mtA-1 was as follows: 97 °C for 5', followed by 30 cycles of 95 °C for 30756 °C for 30772 °C for 1', followed by 72 °C for 5'. Cycling for mt A-2 was as follows: 93 °C for 3', 72 °C for 3', followed by 25 cycles of 94 °C for 1.5752 °C for 30772 °C for 1', followed by 72 °C for 5'. Cycling for mt a-l was as follows: 95 °C for 2', 55 °C for 1', 72 °C for 1', followed by 32 cycles of 94 °C for 1760 °C for 1772 °C for 1' (with an automatic 10 seconds/cycle extension).  Cloning  The PCR products were purified from low-melt gel slices with the P C R Magicprep™ (Promega, Madison, Wisconsin). Gel-purified fragments or P C R products direct from the PCR were ligated into the cloning vector, pCR™ (Invitrogen, San Diego, California). The plasmids containing N. terricola mt A-l, mt a-l and mt A-2 were named p N T A l , pNTal and pNTA2, respectively. E. coli was transformed with the plasmids for maintenance. In the case of mt a-l, the presence of the 1.6 kbp mating type fragment was confirmed by probing a Southern blot of digested pNTal with a chemiluminescence-labelled mt a-specific probe. D N A was prepared for sequencing with a plasmid kit (Qiagen, Chatsworth, California) or a CsCl gradient (Sambrook, Fritsch and Maniatis, 1989).  Sequencing  72 A PCR product was amplified from the mtA-1 gene for sequencing with the primers 1875 and mint, and from the mt a-l gene with the T7 promoter primer, Sp6 promoter primer (synthesized by the NAPS unit at the University of British Columbia.), Y661, Y760-Y767 (gifts from C. Staben), and from the mt A-2 gene with A2-3200 primer, T7 promoter primer, Sp6 promoter primer, chacha primer and cactus primer. Sequencing was performed using the A B I Taq DyeDeoxi Terminator cycle method (Mississauga, Ont.) on an A B I 373 automated sequencer at the NAPS Unit, Biotechnology Lab., University of British Columbia.  Sequence Comparison  Sequence comparisons were done with the programs of the Wisconsin Genetics Computer Group (Devereux, Haberli and Smithies, 1984).  Transformation  N. crassa spheroplasts were prepared and transformed according to Schweizer et al. (1981), using the modification of Akins and Lambowitz (1985).  RT-PCR  R N A was isolated from frozen tissue according to Logemann et al. (1987). Poly(A) + R N A was isolated from total R N A using an Oligotex™ mRNA kit (Qiagen, Chatsworth, California). R N A was reverse transcribed with a First-Strand cDNA Synthesis Kit (Pharmacia Biotech). PCR was done on lOOng of genomic D N A and cDNA of N. crassa and N. terricola using fi-  tubulin primers ( B T l a and BTlb) and mating type primers, Y663 and Y763 for mt a-l, condo and mint for mt A-l and chacha and orchid for mt A-2. Cycling for mt a-l was as follows: 95 °C for 5', followed by 30 cycles of 94 °C for 30754 °C for 20772 °C for 1', followed by an incubation at 72 °C for 5'. Cycling for mt A-l was as follows: 95 °C for 5', followed by 30  73  cycles of 94 ° C for 30753 ° C for 20772 ° C for 1', followed by an incubation at 72 ° C for 5'. Cycling for mt A-2 was as follows: 95 ° C for 5', followed by 30 cycles of 94 ° C for 30745 ° C for 20772 ° C for 30", followed by an incubation at 72 ° C for 5'.  74 Results  TV. terricola mt a-l, but not mt A-2, has an open reading frame comparable in length to the TV.  crassa homolog.  The mt A-l gene was cloned by PCR with TV. crassa primers and ligated into a plasmid for use in transformations and for finishing the sequencing begun by Beatty (1993). The sequence alignment of TV. terricola mt A-l and TV. crassa ml A-l, as created by the GAP program in the G C G suite (Devereux, Haberli and Smithies, 1984), is shown in Figure 2.2. The two D N A sequences are 93% identical (Table 2.4). TV. terricola has an open reading frame of a similar size to TV. crassa mt A-l, with the exception of a six base pair deletion in the first exon. After the TV. terricola sequencing was finished, TV. crassa mt A-l was found to encode two transcripts, one giving a 288 amino acid polypeptide and the other giving a 293 amino acid polypeptide (Saupe et al., 1996). It is unknown whether TV. terricola encodes the second transcription stop since the sequencing did not extend this far. The length of the predicted TV.  terricola M T A - l protein is 286 amino acids. The TV. crassa and TV. terricola M T A - l products have 93%) amino acid identity and 96%> amino acid similarity (Table 2.4). The amino acid alignment (Figure 2.3) shows that a region of TV. terricola mt A-l has similarity with the putative DNA-binding domain of TV. crassa (a domain). In the region of similarity to S. cerevisiae  MATal (TV. terricola mt A-l, position numbers 40-72 in Figure 2.3), TV. terricola differs from TV. crassa by one amino acid. TV. terricola has an arginine in position 47, while TV. crassa, in the homologous position (49), has a glycine. This substitution is non-conservative.  Table 2.4 Comparison of TV. crassa and TV. terricola genes. Gene  D N A identity  # amino acids in protein (N.t./N.c.)  Amino acid similarity  mt A-l mt A-2 mt a-l  93% 90% 93%  286/293 118/373 305/382  96% 88% 97%  DNA S e q u e n c e A l i g n m e n t N. terricola N.  crassa  11 3802  o f mt A-l  TCCACCTTCACCCAAACTTCCCACCACCTTTCCCCGAACA 50  I I I I II I I I II II I II I I I I I I I I I I I I I I I I I I I I I I I  TCCACCTTCACCCAAACTTCCCACCATCTTTCCCCGAACA  3841  51  TTAACTTCGCAACCAAAATCTCGGCTGCACTTCCTCACGTGTTGAACGCT  100  3842  TCAACTTCGCAACCAAAATCTCGGCAGCACTACCTCACGTGTTCAGTGCT  3891  101 38 92  I  I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  III  CTCCAATCAAGAACTCAATCGCCAGAAACACAATGTCGGGCGTCGATCAA 150  I I I I I I I I I I II  II  I I I II I I I I I I I I I I I I I I I I I I I I I I I  C TCCAAT CAATAAT CCAT CCACCAGAAACAC GATGTCGGGTGTCGATCAA  151 ATCGTCAAGACGTTCGCCGACCTCGCTGAGGGCGATCGTGAAGCGGCAAT  I I I I I I II I I I II I II I I I I I I I I I I I I I I I M l I I I I I II I I I I I I I  394 2 ATCGTCAAGACGTTCGCCGACCTCGCTGAGGACGACCGTGAAGCGGCAAT 201 3992  GAGAGCTTTCTCAACGATGATGC  I I I I I I I I I II I I I I I I I I I I I  3941 200 3991  G C A C C G A A C C T G T T C G C C A A A 24 4  I II I I I I I I I I I I II I I  II  GAGAGCTTTCTCAAGGATGATGCGTAGAGGTACCGAACCTGTTCGCCGAA 4 041  24 5 C C C C C G C G G C A A A G A A G A A G G T C A A C C G C T T C A T G G G T T T C A G A T G T A A G 2 94  I I I I I I II I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I  II  ,4 04 2 T C C C C G C G G C A A A G A A G A A G G T C A A C G G C T T C A T G G G T T T C A G A T G T G A G 4 0 9 1 2 95  TCAAATCTGAATCAATCTTGTCGACAATCCAT.GCTGATTGCTTTTTATT  34 3  4 0 92  TCAAATCTGAATCAACATTGTCGTTGATCCATGGCTGATTGCTCTTCATT  4141  I I I II I II I I I I I I I  llllll  llllll  I II I I I I I I I II  III  34 4 T C A G C G T A C T A T T C C C C G C T C T T C T C T C A G C T C C C G C A A A A G G A G A G A T C 4142 394 4192  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I  TCAGCGTACTATTCCCCGCTCTTCTCTCAGCTCCCGCAAAAGGAGAGATC  3 93 4191  GCCGTTCATGACCATTCTCTGGCAGCACGATCCCTTCCACAACGAATGGG 4 43  III  I I I I I I i I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II  III I  GCCCTTCATGACTATTCTCTGGCAGCATGATCCCTTCCACAATGAGTGGG  44 4 A T T T C A T G T G C T C G G T G T A T T C G T C A A T C C G C A C C T A C C T T G A G C A G G A G  I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I III  4242 ATTTCATGTGCTCGGTGTATTCGTCAATCCGGACCTACCTTGAGCAGGAG 4 94 A A G G T T A C C C T G C A A C T C T G G A T T C A C T A T G C T G T C G G C C A T C T G G G A G T  I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I  • 4 2 92 A A G G T T A C T C T G C A A C T C T G G A T T C A C T A T G C T G T C G G C C A T C T G G G A G T  54 4 G A T T A C C C G C G A C A A C T A C A T G G C A T C G T T T G G C T G G A A C C T C G T C C A G C  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I  4 34 2 G A T T A T C C G C G A C A A C T A C A T G G C A T C C T T T G G C T G G A A C C T C G T C C G T T  4 2 41 4 93 42 91 54 3 4 341 593 4 391  5 94  TGCCCAACGGCACTCACGACCTCGAGCGCACCGCTCTTCCTTTGGTTCAG  64 3  4 392  TTCCCAACGGCACTCACGACCTCGAGCGCACGGCTCTTCCTTTGGTTCAG  4 4 41  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I  76  64 4 CATAACCTTCAGCCCATGAACGGCCTATGCCTGCTCACTAGGTGCCTCGA  II  II  II  IIIIIIIIIIIIIII IIIIIIIIIIIII I  4 4 4 2 CACAATCTCCAGCCCATGAACGGCTTATGCCTGCTCACCAAGTGCCTCGA 694  GAGCGGATTGCCTCTTCACAATCCTCACCCTGTCATCGCCAAGCTTTCAG  I IIIIIIIIIIIIIII  I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I  4 4 92 GAGCGGATTGCCTCTTGCCAATCCTCACTCTGTCATCGCCAAGCTTTCAG 744  693 4 4 91 743 4 541  ATCCTAGCTACGACATGATCTGGTTCAACAAGCGTCCTCACCGTCAGCAG 7 93  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I  4 5 4 2 ATCCTAGCTACGACATGATCTGGTTCAACAAGCGTCCTCACCGTCAGCAG 4 5 91 7 94 GGACACGCCGGCCAAACTGACAATTCTGAACTTGGAGTGTCGGCGCTCTT 4 592  I II I I I I I I I  I I I I II II  I I II I I I I I I I I I I I I I I I I I II  GGACACGCCGTTCAAACTGATGAATCTGAAGTTGGAGTTTCGGCGATGTT  84 4 CCCTCGCAATCACGCAGTCGCTGCAGAGGTAGATGGCATCGCCAATCTTC  I III IIIIII III I IIIIIII II IIIIIIIIIIIIIII  I I I II I I I  4 64 2 CCCTCGCAATCACACGGTCGCTGCAGAGGTAGATGGCATCATCAATCTTC 8 94 CTCTCTCCCATTGGATTCAGCAGGGAGATTTCGGCACCGAGTCCGGATTC 4 692  I III IIIIIIIIIIIIIIIIIIIIII II IIIII IIIIIIII IIII I  CTCTCTCCCATTGGATTCAGCAGGGAGAATTCGGTACCGAGTCTGGATAC  94 4 TCAGCTCAGTTTGAGACCTTGTTGGATTCGATCCTTGAGAATGGAAACGC  I I I I I I I I I I I I I I I I I I II I I I I I I I I I II  I I. II I I I I I I I I I I I  4 7 42 TCAGCTCAGTTTGAGACCTTGTTGGATTCAATTCTCGAGAATGGACACGC 994  8 43 4 641 8 93 4 6 91 94 3 4 7 41 993 4 7 91  CTCCAGCAATGATCCCTACAACATGGCTCTGGCTATGGATGTTCCCATGA  104 3  4 7 92 CTCCAGCAATGACCCTTACAACATGGCTCTGGCTATCGATGTTCCCATGA  4 8 41  I I I I I I I I I I I I II  IIIIIIIIIIIIIIIIIIII IIIIIIIIIII I I  104 4 TGGGTTAGTGGAAGATGAGG  1063  4 8 42 TGGGTTAGTGGAAGATGAGG  4 8 61  II IIIII IIIIIIIIIIII I  F i g u r e 2.2 Alignment of N. terricola (top) a n d N. crassa (bottom) DNA s e q u e n c e s o f mt A-l u s i n g t h e GAP p r o g r a m w i t h t h e d e f a u l t settings f r o m t h e G e n e t i c s C o m p u t e r G r o u p s u i t e o f p r o g r a m s ( D e v e r e u x et al., 1 9 8 4 ) . Exons are b o l d f a c e .  77 TV. terricola mt A-2 was cloned by PCR with TV. crassa primers and sequenced. Sequence and amino acid alignments with TV. crassa are shown in Figures 2.4 and 2.5, respectively. The two mt A-2 genes have 90% D N A identity, 83% amino acid identity and 88% amino acid similarity (Table 2.4). More importantly, the TV. terricola predicted protein has a stop codon at 118 amino acids. The TV. crassa M T A-2 protein is 373 amino acids (Ferreira, Saupe and Glass, 1996). The putative DNA-binding domain (Ferreira, Saupe and Glass, 1996; Debuchy, Arnaise and Lecellier, 1993) is not present in TV. terricola M T A-2 (Figure 2.5). TV. terricola mt a-l was cloned by PCR with TV. crassa primers and sequenced. Sequence and amino acid alignments are shown in Figures 2.6 and 2.7, respectively. The two mt a-l genes have 93% D N A identity, 93% amino acid identity and 97% amino acid similarity (Table 2.4). The TV. terricola predicted protein is truncated at 305 amino acids (compared to TV. crassa 382 amino acids (Staben and Yanofsky, 1990; Philley and Staben, 1994)) and has a three amino acid insertion in the third exon. The amino acid alignment (Figure 2.7) shows that the H M G domain (DNA-binding domain, position 117-188) is identical to TV. crassa except for three conservative and two semi-conservative substitutions. According to the consensus H M G domain (Grosschedl, Giese and Pagel, 1994), three of the changes are at non-consensus positions. Moreover, at one of the semi-conservatively changed positions, TV. terricola has the consensus residue (serine in TV. crassa, alanine in TV. terricola) and at one of the conservatively changed positions (leucine in TV. crassa, methionine in TV. terricola), both species differ from the consensus (histidine). The H M G domain of TV. crassa M T a-l is required for D N A binding in  vitro and mating in vivo, but not for heterokaryon incompatibility in vivo (Philley and Staben, 1994). The six amino acids absolutely required for heterokaryon incompatibility, 216-220 (Philley and Staben, 1994) and 258 (Staben and Yanofsky, 1990), are present in TV. terricola. The acidic C-terminus, required for both heterokaryon incompatibility and mating/sporulation in TV. crassa, is mostly conserved. Sixteen of the acidic residues are conserved, one is missing in TV. terricola in the three amino acid deletion and five more are missing because of the early translation stop.  Deduced Amino A c i d Alignment of MT A - l  TV. t . 1 MSGVDQIVKTFADLAEGDREAAMRAFSTMMR..TEPVRQTPAAKKKVNRF  48  TV.c.  50  I I III I I I IIII I I I I: I IIIIIIIII III  I I I I I• • I III I I I I I  1 MSGVDQIVKTFADLAEDDREAAMRAFSRMMRRGTEPVRRIPAAKKKVNGF  4 9 MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL 98  I I I I I I I I I II I I I I I I I I II II I I I I II II I II I I I I I I I II I I I I I I I  51 MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL 100 99 EQEKVTLQLWIHYAVGHLGVITRDNYMASFGWNLVQLPNGTHDLERTALP  14 8  I I I I I I I I II I I I I I I I I I I I . I II I I I I II I I I I . : I I I I I I I I I I I I I  101 EQEKVTLQLWIHYAVGHLGVIIRDNYMASFGWNLVRFPNGTHDLERTALP 150. 14 9 LVQHNLQPMNGLCLLTRCLESGLPLHNPHPVIAKLSDPSYDMIWFNKRPH 198  II I I I III I I I I I I I I: I I I I I I I I I I I• I I I I I I I I I I I I I I I III I I  151  LVQHNLQPMNGLCLLTKCLESGLPLANPHSVIAKLSDPSYDMIWFNKRPH 200  199  RQQGHAGQTDNSELGVSALFPRNHAVAAEVDGIANLPLSHWIQQGDFGTE  24 8  201  RQQGHAVQTDESEVGVSAMFPRNHTVAAEVDGIINLPLSHWIQQGEFGTE  250  I I I II I• I I I: I I: I III: I IIII. I III I I I I I I I I I I I I III: I I I I  24 9 SGFSAQFETLLDSILENGNASSNDPYNMALAMDVPMMG* 251  I I : I I I I I I I I I I II I I I : I I I I I I I I I I I I : I I I I I I I  287  SGYSAQFETLLDSILENGHASSNDPYNMALAIDVPMMG* 289  F i g u r e 2.3 A l i g n m e n t o f TV. t e r r i c o l a ( t o p ) a n d TV. crassa (bottom) d e d u c e d a m i n o a c i d s e q u e n c e s o f MT A - l u s i n g t h e GAP p r o g r a m w i t h d e f a u l t s e t t i n g s from t h e G e n e t i c s Computer Group s u i t e o f programs (Devereux e t a l . , 1984). I n TV. c r a s s a , a m i n o a c i d s 1-111 a r e s u f f i c i e n t f o r h e t e r o k a r y o n i n c o m p a t i b i l i t y a n d 99-111 a r e a b s o l u t e l y r e q u i r e d (K. T. S h i u , p e r s o n a l c o m m u n i c a t i o n ) . The a - d o m a i n , t h o u g h t t o be i n v o l v e d i n DNA b i n d i n g i s i n b o l d f a c e .(Debuchy a n d C o p p i n , 1 9 9 2 ) . C o l o n i n d i c a t e s a c o n s e r v a t i v e amino a c i d s u b s t i t u t i o n . S i n g l e d o t i n d i c a t e s a s e m i - c o n s e r v a t i v e amino a c i d s u b s t i t u t i o n .  DNA  Sequence Alignment of mt A-2  t.  1 GGTTTCCTTTTCGTCAGCTGTCGACATGAATCTCATCAACATGCAACCTA 50 I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I II I I II I I I I I I I I 212 6 GGTTTCCTTTTCGTCAGCTGTCGACATGAATCTTCTCAACATGCAACCTA 217 5 51 GAAGATCAGAGCAACCGGTTATGCTCGAAGAAAACCGTACCTCTAGCCAG 100 III I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 217 6 AAAGGTCAGAGCAACCAGCTATGTTCGAAGAAAACCGTGCCTCTAGCCAG 2225 101 GAAGGCCAGGATCTCGAAGTGATGTACAAGGTAACAATCTGTCTGACCTG 150 I II I I I I I I I I I I I I I I I I I I I I I I I I I I II I I II I I IIIIIII I 222 6 GAAGGCCAGGATCTCGAAGTGATGTACAAGGTAGCAATTCTTCTGACCCG 227 5 151 GAAACACTCATTTACTTGTCACTGATGAATTGGTCAGAAACTCCATCAGC 200 I IIIIIIII II II I I I I II III I I I I I I I I I I I I I I I I I I I I I I 2276 GAAACACTCGCTTGCTTGTCGCTAATGGATTGGTCAGAAACTCCATCAGC 2325 201 TACAGGCTAGGCTTTCTCGTTCAGTTCTTTCAGAGGCAATCAAGGAGTTC 25 0 I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 232 6 TACAGGCTAGGCTTTCCCGTTCAGTTCTTTCAGAGGCAATCAAGGAGTTC 237 5 251 GAAGAGAACCTTCAGTGTCTTTTCCATGAAACCAAGCTCTTGCTATGCAC 300 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I 2 37 6 GAAGAGAACCTTCGGTGTCTTTTCCATGAAGCCAAGCTCTTGCTATGCTC 2 4 2 5 301 AAAAAGAACGAAGTATCGCCAAAGCTGGTTCGGGTCTAGCAACGAGTTCG 350 II • I I I I I I I I I I I I I I I I I I I I I.I I I I I I I I I I I I I I I I I I I I I I I II 2 4 2 6 AACGAGAACGAAGTATCGCCAAAGCTGGTTCGGGTCTAGCAACGAGTTCG 2 4 7 5 351 GGTCTAGCAACGAGAGCAGAATCATCAAGGCATCGTGCTGCATCATTGAG 4 00 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II II I I I I I I I I I 24 7 6 GATCTAGCGACGAGAGAAGAATCATCAAGACATCATGCTGCATCATTGAG 2 52 5 4 01 TCGACAAAACACAATTCTCAACTTTCTCTCGTTCCTTGAGAAGA7AACGAG 4 50 I I I I I I I I I I I I I I I I I I I I I I I I I I I II I II I I II I I I I I I II 252 6 TCGAC.AAACACAATTCTTAACTTCCTCTCATTTCTTGAGAAGAATCGAG 257 4 4 51 GATTGCCATCAGGCGGAGATCAAAGACTCCAACAAGCTGCGTACAAAGGC 500 I IIIIIIII II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 2575 GATTGCCATTCGGTGGAGATCAAAGACTCCAACAAGCTGCCTACAAAGGC 2 624 501 CAGCAGTTCGCATTCCGCCTCCTTCGCTCACTTACAATTCACAAAGATGC 550 I I II I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I III 2 625 CAGCAGTTTGCGTTCCGCCTCCTTCGCTCACTTACACTTCACAAAGCTGC 2 67 4 551 TCAGGAGATTCCCGGAAGGGAATTTGGCTTGGTCTACGGAAAAGAGGTGT 600 I I I I I I I I I I I I I I I III I I I I I I I I I I I I I I II I II I I I I I I I I 2 67 5 TCAGGAGGTTCCGGGAAAGGACTTTGGCTTGGTCTACGGAAAAGATGTGT 27 24 601 ATGTAATGGATGGTCATCTTTTGCACAGGTCAAAGCAAGAGGTCGTGGGG 650 I III II I I I I III I I I I I I I I I I I I I I I I I I I I I I M I N I M 27 25 ACGTACTGAATGGACATATTTTGCACAGGTCGAAGCAAGAGATCGTGGGG 277 4  80  651 CAGGCGGGAGGAAAGAACTGGCATATTGACCATACTCTCCACCCTTTGAG 700  I I II I I I I I I I I I  I I I I I I I I I I I I I I I I I I I I I I I I I I II II i  27 7 5 CAGGCGGGAGGAAGAAACTGGCATGTCGACCATACCCTCCATCCTTTGAG  2824  701 GCGCGTTCCAGGCACCCCATGGCACAAGTTCTTTGGCAATCTTGAAGTTG 7 50  I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I  2825 GCGCGTTCCAGGCACCCCATGGCACAAGTTCTTTGGCAATCTTGAAGTTG 2874 751 ACGCCGACAAGGAACTTCACCTCTTCGATGATGATACGTCTGTCGACAGT 800  II  IIIIIII llllll  I I I I I I I I I I I II I II  II  I I I I I I I I II  2 87 5 GCGACGACAAGCAACTTCGCCTCTTCGATGATGATGCGGCCGTCGACAGT  2 92 4  801 GATCGAGACGGCCCTCGGAAGTTTTTCTGTGTTATTCCGGAAACTGCTGA 850  I  I I I I III  IIII llllll  II  II II II II I I I I I I I I I I I I  2 925 TACCGAGTCGGTCCTCAGAAGTTCTTTGTGGTTATTCCGGAAACTGCTGA 2 97 4 851 ATTTATTCTGGGCGAATCAGCAAGCGAGCATCAAGAGAGTTGCTACAATT 900  I I I I I I I III  I II I  IIIIIIIIII IIIIIII IIIIIIII I  2 97 5 ATTTATTTTGGACGAAGTCAGCAGCGAGCATC.AGAGAGTCGCTACAATT 302 3 901 CAACACAGAGG T GAG TAC T T CAAAC CT GT C TAAAGAC T CACAAAAT T T GC 950  I  IIIIIIIIII IIIIIII  III  I I I I I II  III  3024 C.ACACAGAGGTAAGTACTT.GAACGTGTCTGAAAACT.ACAAAATTTGC 3070 951 ACGACTGACAGAAGGTAGGGTGGACACGCCCAGCCGCCAGCACCGACGTC 1000  I IIIIIIII IIIIIIII  llllll  I I I II II I I I I II I I I I I I  II  3071 ACGACTGACTGAAGGTAGAATGGACATGTCCAGCCGCCAGCACCGACATC 1001 C AT T CAG C AAG AGG T AAGT T CC T C CAT CCC GAT T C AA  I I I I I I II I I I I I I I I I I I I  312 0  T AAT CAT 1044  I III IIIII II  IIIIII I  3121 CATTCAGCAAGAAGTAAGTTCTCCTATCTCGATTTAATGTAGGTAATCAT 317 0 104 5 CACTAACATTACGGCAGGCTCTCCTTAGGAAGTTGGACTTTGCCATGACA 10 94  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I II I I I I I I I I I  3171 CACTGACATCACGGCAGGCTCTCCTCAGGAAGTTGGACTTTGCCATGACA 3220 1095 ACATCATTCCCTGGTTATGTTGTAGAAGGACAACCTGAGGTTGTGTTTCA 1144  I IIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIII I  3221 ACATCATTGCCTGGTTATGTTGTAGAAGGACAACCTGAGATTGTGTTTCA 327 0 114 5 TCATGAAGGCTTACGCCAGGTTCGTATGATTATGCCCACTGTTCACGGAT 1194  I  IIIIIIIIIIIIIIIIIIIIIIIIIIII  Ml  III  I II II I I II  3271 TTATGAAGGCTTACGCCAGGTTCGTATGATCCTGCTTACTTTTCACGGAT 3320 1195 GAT GAT GT GC TAACAAC T GAT TAACAGAT C C C C GT T GAC TACA 1237  II II II II I I II M M I M l  I I I I I I I I II II II II II I I I  3321 GAT GAT G T GC TAACAAC C GAT CAACAGATCCCCGT TGACTACA 3363  F i g u r e 2.4 A l i g n m e n t o f N. terricola ( t o p ) a n d N. crassa (bottom) DNA s e q u e n c e s o f mt A-2 u s i n g t h e GAP p r o g r a m w i t h d e f a u l t s e t t i n g s f r o m t h e G e n e t i c s C o m p u t e r G r o u p s u i t e o f p r o g r a m s ( D e v e r e u x e t al., 1984). Exons a r e i n b o l d f a c e .  81  Deduced Amino A c i d Alignment of MT A-2  N.t.  1 MNLINMQPRRSEQPVMLEENRTSSQEGQDLEVMYKKLHQLQARLSRSVLS 50  N.c.  1 MNLLNMQPKRSEQPAMFEENRASSQEGQDLEVMYKKLHQLQARLSRSVLS 50  I I I : I I I I : I I I I I . I : I I I I . I I I I I I I I I I I I II II II I I I I I I I I II  51 EAIKEFEENLQCLFHETKLLLCTKRTKYRQSWFGSSNEFGSSNESRIIKA 100  I IIII IIII• IIIII.IIIII• • IIIIIIIIIIIIIIIIII:I• III I •  51 EAIKENEENLRCLFHEAKLLLCSTRTKYRQSWFGSSNEFGSSDERRIIKT 100 101 S C C I I E S T K H N S Q L S L V P * 119  MINIM  I• I.:: .  101 S C C I I E S T . . N T I L N F L S F . . . 3 8 3  F i g u r e 2.5 A l i g n m e n t o f Af. terricola ( t o p ) a n d N. crassa (bottom) a m i n o a c i d s e q u e n c e s o f MT A-2 u s i n g t h e GAP p r o g r a m w i t h t h e d e f a u l t s e t t i n g s from t h e G e n e t i c s Computer Group s u i t e o f programs ( D e v e r e u x e t al., 1984). N o t e t h e f r a m e s h i f t a t a m i n o a c i d 109 a n d t h e p r e m a t u r e s t o p c o d o n i n N. terricola. The s h o r t e n e d p r o d u c t d o e s n o t c o n t a i n t h e p u t a t i v e D N A - b i n d i n g d o m a i n o f N. crassa (Debuchy, A r n a i s e and L e c e l l i e r , 1993).  82  DNA TV. t . TV.c.  2950  Sequence Alignment o f mt a - l  GAATGCTATTCAGGGCCGTGTCAATAACCGTCAATAGACAAAGCGGT  I I I I I I I I I I I I I I I I I I I I I I I I I I II  2950  IIIIIIIIIIIIIIII I  GAATGCTATTCAGGGCCGTGTCAATAGCCATCAATAGACAAAGCGGT  3000 TCCACCCAAAAATCCCGACGTCGTTGCCCCTCCTTATAATCTCCCTCCCT  I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II II I I I I I I I I I I  3000 TCCACCCAAAAATCCCGACGTCGTTGCCCCTCCTTATAATCTCCCTCCCT  2999 2999 304 9 304 9  3050 TCCAATTCTTCCTTCTTCCCACTCCTTACATTCCTCCGTCAACTTCGCAA 3099  I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I  llllll  3050 TCCAATTCTTCCTCCTTCCCACTCCTAACATTCCTCCGTCAACATCGCAA  3099  3100 CAATAGTCGACAATCACGACCAATCTTCGCCATCAACTCATTTCTCCTTC  314 9  3100 CAATAGTCGACAATCACGACCGATCTTCGCCATCTACTCATTTCTCCTTT  314 9  315 0 CCACAATCAATCGCCCAAGTCTTTGACAATCCCAAGCATCAGCCTTCTTC  3199  3150 CCACAACCAGTCGCCCAAGTCTTTGACAATCTCAAGCATCAGCCTTCTTC  3199  3200 ATCGTCAACGAACAATCAATCGAAACAATGGACGGCAACTTGACGCACCC  324 9  I IIIIIIIIIIIIIIIIIIII IIIIIIIIIIII IIIIIIIIIIIII I  I I I II I II  III  II  I I I I I I I II I II I I I I I I I I I I I I I I I I I I I I I I I I I I I  I I I I I I I I I I I I I I I I I I I I I I II  I I I I III  IIII I  3200 ATCACCAGAGAACGATCAACCGAAACAATGGACGGTAACTCGACACACCC  324 9  325 0 CGCTCCGGACGTCAAGACCACCTTGGCTTGGTCTCGCATCTCCAACCAAC  32 99  llllll  .11 I I I I I I I II  I I I I I I I I I I I I I I I II  I I I I I II  325 0 CGCTCCAAACCTCAAGACTACTATGGCTTGGTCGCGCATATCAAACCAAC  32 99  3300 TCGGACACTGGAACGACCGCAAGGTCATTGCCATTCCTCTGAGCGACTTC  334 9  3300 TCGGTCACTGGAATGACCGCAAGGTCATTGCCATTCCTCTGAGCGACTTC  334 9  3350 CTTCACACCGACCCTGTCATTCAGTCTGGCATCATCGCCAGCTTCAAGTA  3399  I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  III  III I I l l l l l l  IIIIIIIIIIIIIIIIIIIIII  IIIIIII I  3350 CTTAACACCCACCCTGACATTCAGTCTGGCATCATCGCCGAGTTCAAGTA  33 99  34 00 AGTGTCCTCACCCATTTCTCACCTTACCTTGTACTGACCATTTGCACCAG  34 4 9  34 00 AGTGTCCTCACCCATTTCTCACCCTACCTTGTACTGACCATTTGCACTAG  34 4 9  34 50 GAAAGCGACTGGTGAAGAGGGCATGTTTGCCCGCGATCCCGAGTCACTGG  34 99  I I II I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I II  .3450  I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I II  III  GAAAGCGACTGGCGAAGAGGGCATGTTTGCCCGCGATCCTGAATCATTGG  3499  3500 GAATCATGCTTCTTGGCCCCGCCAAGCTGTTCAAGCCCGACAGTGTCGTC  354 9  I I I I I I I I I I II I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I II I I  3500 GAATCATGCTTCTTGGTCCCGTCAAGCTGTTCAAGCCCGACAGTGTCGTC  354 9  3550 GTCGACGGCAACCTTTTCTGGGATCCCAAGGGCATCCATGCTTCGGCACC  35 99  3550 GTCGACGGCAACCTGTTCTGGGATCCCAAGGGCATCCATGCTTCGGCACC  3599  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I II I I I I I II  3600 CAAGGAGCAGCAGAAGAAGGCCAAGATTCCTCGCCCCCCCAACGCCTACA 364 9  I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I  3600 CAAGGAGCAGCAGAAGAAGGCCAAGATCCCTCGCCCTCCCAATGCCTACA 364 9 3650  TCTTGTACCGAAAGAACCATCATCGTGAGATCCGCGAGCGGAACCCCGGA  3699  3650 TCTTGTACCGTAAGGACCATCATCGTGAGATCCGCGAGCAGAATCCCGGA  3699  37 00 CTCCACAACAACGAGATTTGT7AAGTTTCTTGTCACCATGATCTATAATGT  374 9  I I I I I I I I I I III  II  I I II I I I I I I I I I I I I I I I I I I I I III  IIIII I  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I III  I  3700 CTTCACAATAACGAGATTTGTAAGTTTCTTGTCATCATGATCGAAAATCT 374 9 3750  TTGACCCTGAGACTAAGCTGACTTAGCGGTCATCGTCGGCAACATGTGGC  37 99  3750  TTGGCCTTGAGACTAACCTCACTTAGCGGTCATCGTCGGCAACATGTGGC  3799  III  II  I I I II I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  3800 GTGATGAGCAGCCGCACATTCGCGAGAAATATTTCAACATGGCCAATGAG 384 9  I I I I I I I I I II I I I II I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I  38 0 0 GTGATGAGCAGCCGCACATTCGCGAGAAATATTTCAACATGTCCAATGAG 38 4 9 38 50 ATCAAGACTAGAATGTTGCTGGAGCATCCCGACTATCGCTACAATCCACG 38 99  I I I I I I I I III  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II  38 50 ATCAAGACCAGACTGTTGCTGGAGAATCCCGACTATCGCTACAATCCGCG 38 99 3900  TCGGTCTCAAGACATTCGCAGGCGCGTCTCGCCGTATCTCAAAATCAAGC  394 9  3900 TCGGTCTCAAGACATTCGCAGGCGCGTCTCGCCGTATCTCAAGATCAAGC  394 9  3950 TCCTCAACTATGACGCTAATGGCAACCTTCTTTGGGGCACCGTCAACGCC  3999  3950  TCCTCAACTACGACGTTAATGGCAACCTTCTTTGGGGCACCGTCAACGCC  3999  4 000 GAGGATGCTGCGCTAATTCGGACTCACTTCCATGGAGTCGTTCGTGTTGA  4 04 9  4 000 GAGGATGCTGCGCTGATTCGGACTCACTTCCATGGAGTCGTTCGTGTTGA  4 04 9  4 050 GGAAATGGATGATGGCTGCAGAATTGTCTGCCGTCCCGTCGCAGGATCTA  4 099  4 050 GGAAATGGATGATGGTTGCAGAATCGTCTGCCGTCCCGTCGCAGGATCTA  4 099  4100 GAAAACTTCGCGCCGCCGTTGTCGACACTTGGATGCCTCGCTATACGGTT  414 9  4100  414 9  I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I II I I I I I I I I I I I I I I I I  I IIIIIIIII IIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII I  I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I  I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIII I  GAAAACTTCGCGCCGCCGTTGTCGACACTTGGATGCCTCGCTACACGGTT  4150 GATGCAACCCCCGTCACTGAGGACGACGATGCACAGGCTTTCAACTTCAA  4199  4150  GACACAACCCCCGTCACCGAGGACGACGATGCACAGGCTTTCAACTTCAA  4199  4 200 CGACCCCATAGACCCCTTGGCCGGCGCTTATTTCCCTATCAATGATCACC  4 24 9  4 200  TGATCCCTTGGGCGGTGCTTATTTCCCTTTGAATGAGCACC  424 0  4 250 TCTAGGTCACCGTCAACCAAACCCCTCCCTTCAATGCCCCTCCCCCCAAT  4 299  4241  4 2 90  II  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I II  II  III  I I I II  I I II I I I III  IIIIIIIIIIII I IIIII III I  IIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIII I  TCTGGATCACTGTCAACCAAAACCCTCCCTTCAATGCCCCTCCCCCCAAT  4 300 CCCAACCCACACCTGGATTTCGTTCACCCCGACGGCATGGACGCAGTTAT  4 34 9  42 91 CCCAACCCACACCTGGATTTCGTTCACCCCGACGGCATGGAGGCAGTTGT  4 34 0  I I I I I I I I I I I I I I I I I I I I I I I I II I II I I I I I I I I I I II  llllll  I  4 350 TCACAACGTTCAGGACATGATCGCCCAGGTCCAGGAGGCCAATGAGGCTG  4 3 99  4 341 TCACAACGTTCAGAACATGATCGCTCAGGTCCAGGAGGCTAACGAGGCTG  4 3 90  4 4 00 CTGCGCTAACGCCACCTCCGCTACCACCGCTGCGCCTGCCGTCACTCAGG  444 9  4 3 91 CTGCGCTAACGCTACCACCGCCACCACCGCTGCGTCTGCTGTCACTCAGG  4 440  I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II II I II  I I I I I I I I I I I I III  IIIIII I  IIII IIIIIIIIIIII IIII IIIIIIIII I  4 4 50 TTATGGCTGACGATACCATCAACCCAGCGCTCATTCCCACTGTGAACACT 4 4 99  I IIIIIIIII MMMM  MMMM  II II II II II II II II II II I  4441 TTATGGCTGATGATACCATTAACCCAGCTCTCATTCCCACTGTGAACACT 4 4 90 4 500 AATGCGACCGTTCTTCCTCACGTCCATACCATTCCTGACAACGCCACCGT 4 54 9  I III  I IIIIIIII  II I I I I I I I II II I I I I I I I I I I I I I I I I I I  4 4 91 CATGCATCTGTTCTTCCCTACGTCCATACCATTCCTGACAACGCCACCGT 4 54 0 4 550 TACGCCTTCCGCCACTGGAAATTCGGTTCATGTTGTTACACCCGGTCACC  I I II I I I I II II  MMMM  II I I I I I I I I I I I I I I I I I II I I I I II  4 541 TACGCCTTCCGCTACTGGAAACTCGGTTCACGTTGTTACACCCGGTCACC  4 599 4 5 90  4 600 AAGGATATCACCTCAT 4 615  II I I I I II I I I I I I II  4591 AAGGATATCACCTCAT 4606  F i g u r e 2.6 A l i g n m e n t o f TV. terricola ( t o p ) a n d TV. crassa (bottom DNA s e q u e n c e s o f mt a-l u s i n g t h e GAP p r o g r a m w i t h t h e d e f a u l t s e t t i n g s from t h e G e n e t i c s Computer Group s u i t e o f programs ( D e v e r e u x e t al., 1984). Exons a r e i n b o l d f a c e .  Deduced Amino A c i d Alignment of MT a - l  N.t.  1 MDGNLTHPAPDVKTTLAWSRISNQLGHWNDRKVIAIPLSDFLHTDPVIQS 50  N.c.  1 MDGNSTHPAPNLKTTMAWSRISNQLGHWNDRKVIAIPLSDFLNTHPDIQS  MM  M M :: I I I : I I I I I I I I I I I I I I I I I I I I I I I I I M I M M l  50  51 GIIASFKKATGEEGMFARDPESLGIMLLGPAKLFKPDSVVVDGNLFWDPK 100  II I I • I II I I I I I I I II I I I I II I I II I I I • I I I I I I I I I M II I I I II I  51 GIIAEFKKATGEEGMFARDPESLGIMLLGPVKLFKPDSVVVDGNLFWDPK 100 101 GIHASAPKEQQKKAKIPRPPNAYILYRKNHHREIRERNPGLHNNEISVIV 150  I I I I I I I I I! I I I I I I I I I I I I I I I I I I : I I I I I I I • I I I I I I I II II I I  101 GIHASAPKEQQKKAKIPRPPNAYILYRKDHHREIREQNPGLHNNEISVIV 150 151 GNMWRDEQPHIREKYFNMANEIKTRMLLEHPDYRYNPRRSQDIRRRVSPY  I II I I II II I I I I I I I I I • I I I I I I. : I I I : I I I I I I I I I I I II II I I II I  2 00  151 GNMWRDEQPHIREKYFNMSNEIKTRLLLENPDYRYNPRRSQDIRRRVSPY 200 201 LKIKLLNYDANGNLLWGTVNAEDAALIRTHFHGVVRVEEMDDGCRIVCRP 250 201  I I I I I II I I • I I I I I I I I I I I I I I I II II II I II II II II I II I I I I II I  LKIKLLNYDVNGNLLWGTVNAEDAALIRTHFHGVVRVEEMDDGCRIVCRP 250  251 VAGSRKLRAAVVDTWMPRYTVDATPVTEDDDAQAFNFNDPIDPLAGAYFP  II I I I I II I I I I I I I I I I I I I I • I I I I I I I I I I I I I I I  II I : I I I I I  251 VAGSRKLRAAVVDTWMPRYTVDTTPVTEDDDAQAFNFN...DPLGGAYFP 301  INDHL* 306  298  LNEHLW 303  •  300 297  \ • \ \  F i g u r e 2.7 A l i g n m e n t o f N. terricola ( t o p ) a n d N. crassa (bottom) a m i n o a c i d s e q u e n c e s o f MT a - l u s i n g t h e GAP p r o g r a m w i t h t h e d e f a u l t s e t t i n g s from t h e G e n e t i c s Computer Group s u i t e o f programs ( D e v e r e u x et al., 1984). The HMG d o m a i n ( b o l d f a c e ) i s v e r y s i m i l a r ( s e e t e x t f o r d i s c u s s i o n ) and t h e t r a n s c r i p t i o n - f a c t o r - l i k e a c i d i c , p r o l i n e - r i c h C-terminus r e q u i r e d f o r mating and h e t e r o k a r y o n i n c o m p a t i b i l i t y (everything C - t e r m i n a l t o t h e HMG d o m a i n ) i s v e r y s i m i l a r , e x c e p t f o r a t h r e e a m i n o a c i d i n s e r t i o n i n N. terricola ( P h i l l e y and Staben, 1994).  86 A search for consensus sequences revealed that in N. terricola mt A-l, mt A-2 and mt a-  1, the translation start sites, 5' and 3' splice sites and lariat sites differ from N. crassa and published fungal consensus sequences (Edelman and Staben, 1994) by no more than one nucleotide.  N. terricola mt a-l can induce mating in N. crassa mating type mutants and a mating type deletion strain.  N. terricola mt a-l was first subcloned into the vector, pOKE103. The N. terricola mt A-l gene was also subcloned into pOKE103 for use in control experiments. To determine whether or not N. terricola mt a-l is able to induce mating, a number of experiments was performed in which sterile N. crassa strains were co-transformed with a plasmid containing one of the N. terricola mating type genes and a selectable plasmid (with a gene encoding an altered (3-tubulin that confers resistance to Benomyl). Transformation with two D N A fragments generally results in the integration of both types of fragments, even if one has no selectable marker (Vollmer and Yanofsky, 1986). The sterile recipient strains were either mating type mutants or had the mt A mating type idiomorph deleted. The transformants were crossed to opposite mating type females by replica plating. The N. terricola mt a-l gene was able to induce mating (Table 2.5, rows 1-3). The observed mating reaction was characterized by enlargement and darkening of perithecia which had no or very small beaks and produced no ascospores. The mating reaction proceeded to the same point in development as the control transformants of TV. crassa mating type mutants transformed with N. crassa mt a-l (Table 2.5, row 4).  87  Table 2.5 N. terricola mt a-l confers mating to N. crassa mating type mutants or nulls.  N. crassa recipient strain  Transforming plasmids  # mating transformants/ # transformants tested  A mating type mutant (arg-2 mtAl. 7 (RIP))  N.t. mt a-l ( p N l a l ) Benomyl-R(pBCl)  6/68  mating type deleted (RLM41-10M-4 ad-2; lys-1 AA)  N.t. mta-1 (pONTa) Benomyl-R (pBCl)  5/503  mating type deleted (RLM41-10M-4 ad-2; lys-1 AA)  N.t. mta-1 (pNTal) Benomyl-R (pBCl)  11/119  mating type deleted (RLM41-10thi-4 ad-2; lys-1 AA)  N.c. mta-1 (pCSN4) Benomyl-R (pBCl)  5/333  N. terricola mt A-l and mt a-l can induce mating type incompatibility.  The N. crassa mating type genes of opposite mating type cannot coexist for long within the same nucleus. A mycelium containing dual mating type nuclei is phenotypically distinct because the hyphae are short and growth is extremely slow or non-existent. There is one known suppressor (tol) of this incompatibility (Newmeyer, 1970; Vellani, Griffiths and Glass, 1994). How are the two mating type genes of N terricola able to coexist? Are the mating type genes compatible or is there a suppressor? To test the hypothesis that the N. terricola mating type genes are compatible, they were assessed for their ability to induce incompatibility, using reduction in transformation efficiency as an indicator of incompatibility. N. crassa A (strain name 153) and a (strain name 7) strains were co-transformed with plasmids containing one of the N. terricola mating type genes, mt A-l (pONTA) or mt a-l (pONTa) plus the plasmid containing the gene encoding resistance to hygromycin (pMP6) (Table 2.6).  88  Table 2.6 Transformation efficiency of mating type genes. Recipient strain  Transforming plasmids  # replicates  Avg. # colonies it standard deviation  N. crassa A  N.t. mtA-1 hygromycin-R  9  129 ± 4 5  N. crassa a  N.t. mtA-1 hygromycin-R  9  16 ± 5 . 7  N. crassa A  N.t. mt a-l hygromycin-R  10  41 ± 1 6  N. crassa a  N.t. mt a-l hygromycin-R  10  158 ± 4 4  Introduction of N. terricola mt A-l into N. crassa a reduced transformation efficiency 8fold as compared to the same gene introduced into a N. crassa A recipient. Similarly, introduction of N. terricola mt a-l into N. crassa A versus a reduced the number of transformants 4-fold. The reduction in the number of transformants when N. crassa A is transformed by N.  terricola mt a-l as compared to N. terricola mt A-l was statistically significant (as determined by an A N O V A , not shown), suggesting that N. terricola mt a-l was able to induce incompatibility against N. crassa A. Similarly, the statistically significant reduction in the number of transformants when N. crassa a is transformed by N. terricola mt A-l as compared to  N. terricola mt a-l indicated that N. terricola mt A-l was incompatible with N. crassa a.  Mating type incompatibility is suppressed by tol.  In N. crassa recessive alleles of tol suppress mating type incompatibility. When both components of a mixed mating type heterokaryon have the suppressor allele, then the heterokaryon grows at a wild type rate. If either or both components carry tol +, then the heterokaryon grows slowly. Similarly, dual mating type partial duplication strains with tol grow more vigorously than those with tol +.  To test the hypothesis that tol is able to suppress the incompatibility that occurs between the N. terricola and the N. crassa mating type genes, the following transformation experiment was performed. Suppressor (1-20-41 which is arg-1 ad-SB; tol A) and non-suppressor (1-20-26 which is arg-1 ad-SB A) strains of N. crassa were co-transformed with one of the N. terricola mating type gene plasmid, pONTa, plus a plasmid encoding resistance to hygromycin (Table 2.7). More transformants were recovered from the transformation of N. crassa A cells with N.  terricola mt a-l in the experiment described in Table 2.7 as compared to a similar experiment described in Table 2.6. One difference was the temperature at which the transformation plates were incubated. In Table 2.6, the cells were un-S ad-SA nic-2 cyh-1 A. Since un-S strains are temperature-sensitive, the plates were incubated at room temperature. In Table 2.7, the cells were arg-1 ad-SB A and so they were incubated at 30°C, which allowed faster growth. More transformants may have grown to a detectable size by the time the plates were examined. Table 2.7 Suppression of incompatibility by tol. Recipient strain  Transforming plasmids  # replicates  Avg. # colonies ± standard deviation  tol +A  hygromycm-R  5  223 ± 18  tol+A  N.t. mta-1 hygromycin-R  5  163 ± 3 4  tol A  hygromycin-R  5  60 ± 17  tol A  N.t. mt a-l hygromycin-R  5  92 ± 1 6  Co-transformation of a N. crassa tol strain with a plasmid carrying the opposite mating type N. terricola gene and the hygromycin plasmid resulted in significantly more transformants (as determined by an A N O V A , p=0.05) compared to transformation with the hygromycin plasmid alone. In contrast, co-transformation of a N. crassa tol + strain with a plasmid carrying the opposite mating type N. terricola gene and the hygromycin plasmid resulted in fewer transformants relative to transformation with the hygromycin plasmid alone.  90  Two of the mating type genes are expressed in TV. terricola.  In order to determine whether or not the TV terricola mating type genes mt A-l, mt A-2 and mt a-l were expressed in TV. terricola, transcripts were amplified by reverse transcriptase P C R ( R T - P C R ) using TV. crassa primers. TV. crassa genomic D N A and c D N A were used at the same time for comparison. The mt A-l and mt a-l genes are expressed in TV. terricola. Lanes 2 and 3 show amplification products from fi-tubulin primers. Lanes 4 and 5 show D N A fragments produced from the mt a-l primers. The bands were o f a similar size to the TV. crassa controls. Lanes 6 and 7 show the bands produced with mt A-l primers. Again, the N . terricola fragments were of a similar size to the TV. crassa controls (Figure 2.8, A . crassa controls were not included in the figure). When TV. terricola genomic D N A was used as a template with the mt A-2 primers, a band of the expected size was seen and it hybridized with a p N T A 2 probe. When TV. terricola c D N A was used as a template with the mt A-2 primers, a band that was several hundred bases smaller than expected appeared. This band did not hybridize with the probe. The band may have been produced through non-specific amplification. The fi-tubulin and other mating type primers were able to amplify the expected P C R products from the same preparation of c D N A . These data suggest that mt A-2 is not expressed in TV. terricola.  91  kb  gen  cDNA  mtA-1  mt a-l  fi-tubulin  gen  cDNA  gen  cDNA  • • • • • i •IAMI1IUUT  Figure 2.8 Gel electrophoretic analysis of N. terricola RT-PCR products. Lane 1 is kb ladder. Lanes 2 and 3 are RT-PCR products amplified from genomic and cDNA with B-tubulin primers; lanes 4 and 5, mta-1 primers and lanes 6 and 7, mtA-1 primers. gen=genomic D N A template cDNA=cDNA template  92 Discussion  TV. terricola contains two mating type genes (mt a-l and mtA-1) which tested positive in functional assays. One gene (mt A-2) was shown to be non-functional. The TV. terricola genes mtA-1 (Beatty, 1993) and mt a-l are able to induce mating in TV.  crassa mating type mutants. The perithecia are similar to those seen when TV. crassa mt A-l or mt a-l D N A fragments are used to transform the same mutants. The mating type genes of TV. terricola (T. S. Vellani, unpublished data) were unable to induce homothallism in TV. crassa recipient strains. The tests done to assess the ability of the mating type genes to induce incompatibility show that the TV. terricola genes are able to induce incompatibility with the opposite mating type TV. crassa gene. The incompatibility between TV. crassa A and TV. terricola a is suppressed by  tol, suggesting that the tol + product is necessary for incompatibility. Genomic Southern blots probed with the cloned TV. crassa tol gene suggest that, in contrast to all other Neurospora species, N. terricola and TV. pannonica have no tol homolog (K. T. Shiu and N . L. Glass, personal communication). The mating type genes of TV. terricola and TV. pannonica probably coexist without inducing incompatibility. The TV. terricola genes mt A-l and mt a-l are predicted to encode proteins that are highly similar to those of TV. crassa, they are functional in a TV. crassa host and they are expressed in TV.  terricola. Taken together, the data suggest that mt A-l and mt a-l are under the influence of natural selection. Their presumed function could be the specification of identity in ascogenous hyphae or the initiation of sexual development. Sexual reproduction in a haploid self-mating organism produces no recombinant progeny, as shown by the highly similar R F L P patterns found in homothallic strains from a wide geographic distribution (Glass, Metzenberg and Raju, 1990). Meiosis, therefore, must be under selection for some outcome other than recombination. Two such outcomes could be ascospore-production and D N A repair (Perkins and Turner, 1988; Beatty, Smith and Glass, 1994; Glass, Metzenberg and Raju, 1990; see also General Introduction).  93 A l l homothallic Sordariaceae examined so far have a homolog to mt a-l except for the fourv4-only homothallic Neurospora species (Glass, Metzenberg and Raju, 1990; Poggeler et  al, 1997a). Given that the N. terricola mt a-l gene appears to be functional, the absence of a homolog from the TV. africana group of species requires some thought. The presence of an undetected equivalent to mt a-l in these species is possible. A N. crassa mt A probe hybridized strongly to P. anserina genomic D N A , but hybridization with the mt a probe was very weak (Debuchy and Coppin, 1992), suggesting that mt a-l function is preserved in the absence of strong sequence conservation. One functional region of M T a-l is the H M G domain, which has few strongly conserved residues (Philley and Staben, 1994). Alternatively, there could be a third mating type allele with no similarity to mt a-l or mt A-l. Fungal mating type proteins contain any of a number of domains including H M G domains, homoeodomains and a-domains. One final possibility is that the mt a-l function is not required in N. africana. The gene, mt A-2, is present in the N. terricola genome and is similar to the N. crassa gene, but its transcript was not detected by RT-PCR and it contains a ffameshift followed closely by a stop codon that truncates the deduced polypeptide at 118 amino acids. These data suggest that mt A-2 is not functional. The N crassa mt A-3 gene has no known homolog in N.  terricola (Beatty, Smith and Glass, 1994). AN. crassa mt A-2 mt A-3 double mutant (Glass and Lee, 1992; Ferreira, Saupe and Glass, 1996) has a severe phenotype of reduced ascospores, with the developmental block appearing at or around karyogamy and ascus development (Glass and Lee, 1992). In contrast, these same stages appear to be normal in N. terricola (Raju, 1978). The functions of N. crassa mt A-2 and mt A-3 appear to be dispensable in N. terricola. As shown in Chapter 1, the post-fertilization functions of the mating type genes of N.  crassa are abnormal when the opposite mating type genes are present in the same nucleus and when the mating type genes reside at ectopic chromosomal locations. Since these postfertilization functions are intact in N. terricola, the mating type genes are either not under positional regulation or else both genes are positioned such that they are under the control of the putative cw-acting regulator. The fact that all of the diverse homothallic fungal species examined to date, including Neurospora sp. (Glass, Metzenberg and Raju, 1990; Beatty, Smith  and Glass, 1994), Cochliobolus sp. (N. L. Glass, personal communication), Podospora sp. (Debuchy, personal communication to N . L. Glass) and Sordaria sp. (Poggeler et al., 1997a) have the opposite mating type sequences linked suggests that the mating type genes are under positional regulation (these species did not arise from a single common ancestor). The genes may be linked in order to allow control of both genes by the same cw-acting regulator.  95  Chapter 3 Phylogenetic Analysis of  Neurospora  Introduction  The phylogenetic study of fungi was difficult prior to the molecular revolution due to the paucity of morphological markers, the prevalence of convergent characters and the difficulty in culturing strains of most species. Traditionally, heterothallic species were defined in  Neurospora using morphology and fertility, criteria which have corroborated each other more often than not (Perkins, Turner and Barry, 1976). Recently, nucleotide positions have been used as characters in phylogenetic analysis. Some examples in fungi are seen in Gargas et al. (1995), Radford (1993), Berbee and Taylor (1992a; 1992b; 1992c), Bowman et al. (1992), Hendriks et  al. (1991), Smith (1989), Hori and Osawa (1987) and Dayhoff (1983). Relationships in the genus Neurospora have been examined by a number of investigators using many types of data, including distribution of mitochondrial plasmids, restriction site differences and restriction fragment length polymorphisms of both mitochondrial and nuclear D N A , D N A and amino acid sequence data (Skupski, Jackson and Natvig, 1997; Lewis and Feldman, 1996; Randall and Metzenberg, 1995; Merrow and Dunlap, 1994; Taylor and Natvig, 1989; Natvig, Jackson and Taylor, 1987; Taylor, 1986; Taylor, Smolich and May, 1986; Russell  etal, 1984). Several of these works include phylogenies, all of which were directed at resolving the issues of species designations or the relationships among the heterothallic and pseudohomothallic species. Some of these relationships remain unresolved. The phylogenies contain some inconsistencies, possibly because some of the heterothallic species may not be reproductively isolated from each other, ancient polymorphisms predating the divergence of some taxa may exist and, finally, tree topology can depend on the type of data or specific strains used to construct it (Skupski, Jackson and Natvig, 1997).  96 The high similarity of the RFLP patterns in the ,4-only homothallic group of species (N.  africana, N. lineolata, N. dodgei, N. galapagosensis) suggested that these species are very closely related. In contrast, those of TV. terricola were different from this group (Glass, Metzenberg and Raju, 1990), raising the question of how these two groups of homothallic  Neurospora species are related to each other and to the heterothallic group. Estimates of degree of relatedness based on the fraction of shared rDNA restriction sites also suggest that the homothallic species do not form a group, N. terricola being distant from the other homothallic species (Verma and Dutta, 1987). In this chapter, the phylogeny of the entire Neurospora genus was reconstructed using sequence data from the mating type gene, mt A-l in order to determine the relationship between  Neurospora homothallic and heterothallic species. The trees thus produced are unique in the literature in the inclusion of all known species of Neurospora.  P. anserina, G. tetrasperma and 5". macrospora were chosen as outgroup species. A l l of them belong to the same family (Sordariaceae) as Neurospora, with Gelasinospora being the most closely related genus to Neurospora (Merrow and Dunlap, 1994). Gelasinospora and  Neurospora are distinguished by their production of pitted versus ribbed ascospores (von Arx, 1981). Their traditional generic separation is supported by phylogenetic analysis based on restriction maps of a nontranscribed spacer region of ribosomal genes (Verma and Dutta, 1987). Both genera include homothallic, pseudohomothallic and heterothallic species. Mating type genes differ from other genes in the fungal genome in that they do not undergo recombination (Glass et al., 1988). Lack of recombination would speed the evolution of the two alleles away from each other. The evolutionary relationships between species should be accurately reflected in a tree inferred from sequences of any one mating type gene.  97  Materials and Methods  D N A preparation  Cultures were grown on liquid complete medium (7g/L malt extract, lg/L soytone, .5 g/L yeast extract). Genomic D N A was prepared from ground lyophilized tissue by the method of Lee and Taylor (1990).  Primers The primers 1778 and 3194 were chosen because they encompass the open reading frame of mt  A-l. The primer 2043 is approximately 200 bp downstream of 1778 and was used instead of 1778 in cases where no P C R product was seen with 1778 and 3194. Table 3.1 shows the primer names, sequences and map locations. Numbers refer to the mt A idiomorph sequence (GenBank accession no. M33876).  Table 3.1 Primers used to amplify mt genes. Primer  Sequence (5'-3')  Map Location  1778 3194 2043  ACCCAAACTTCCCACC ATGGTACCTCATCTTCCACTAACCC GTTCGCCGAATCCCCGC  3997-4021 5040-5062 4227-5143  PCR  The primers used to amplify the mt A-l gene were the TV. crassa primers, 1778 and 3194, for N.  terricola, N. pannonica, and G. tetrasperma. Those primers could not amplify a fragment from TV. galapagosensis, N. lineolata or N. dodgei, so 2043 and 3194 were used. Neither of those two primer sets could amplify a fragment from either the homothallic ascomycete, Sordaria  98  macrospora or the heterothallic, S. brevicollis, mating type A. PCR was performed on a D N A Thermal Cycler 480 (Perkin Elmer, Foster City, California). Cycling for N. terricola mtA-1 was as follows: 97 °C for 5', followed by 30 cycles of 95 °C for 30756 °C for 30*772 °C for 1', followed by 72 °C for 5'. Cycling for N. pannonica mt A-l was as follows: 95 °C for 5', 72 °C for 2', followed by 25 cycles of 94 °C for 30756 °C for 20772 °C for 1', followed by an incubation at 72 °C for 5'. Cycling for G. tetrasperma, N. galapagosensis, N. dodgei and N. lineolata mt A-l was as follows: 95 °C for 5', 72 °C for 1', followed by 25 cycles of 94 °C for 30756 °C for 20772 °C for 1', followed by an incubation at 72 °C for 5'.  Cloning  The PCR products were purified from low-melt gel slices with the PCR Magicprep™ (Promega, Madison, Wisconsin). Gel-purified fragments were ligated into the cloning vector, pCR™(Invitrogen, San Diego, California). Plasmids were named according to the first letter of the genus, the first letter of the species followed by " A - l " . E. coli was transformed with the plasmids. D N A was prepared for sequencing with a plasmid kit (Qiagen, Chatsworth, California).  Sequencing  The mtA-1 gene was amplified for sequencing with the primers 1875, the T7 promoter primer and the SP6 promoter primer (synthesized by the N A P S unit at the University of British Columbia). Sequencing was performed using the ABI Taq DyeDeoxi Terminator cycle method (Mississauga, Ont.) on an A B I 373 automated sequencer at the NAPS Unit, Biotechnology Lab., University of British Columbia.  Alignment  99  The D N A sequences were aligned with the program PileUp from the Wisconsin Genetics Computer Group (Devereux, Haberli and Smithies, 1984) using the default gap penalty. One small gap of 4 or 5 nucleotides was shifted relative to the paired nucleotides to align apparently homologous nucleotides. The sequences were translated into amino acids with the program Translate (Devereux, Haberli and Smithies, 1984), aligned with PileUp and adjusted manually.  S. macrospora had two large insertions relative to the Neurospora and Gelasinospora species, one of 21 nucleotides (seven amino acid residues) in the first exon and the other of 18 nucleotides (six amino acid residues) in the second exon. Both of the insertions occur outside of the region of high amino acid conservation among mating type genes. The resulting alignment was 969 nucleotides long.  Phylogeny Reconstruction  Phylogenetic trees were reconstructed from the aligned sequences with two different methods that use optimality criteria, i. e. they reconstruct and evaluate many trees to find the most parsimonious or most likely one. Parsimony methods search for the tree with the fewest evolutionary changes (the smallest number of nucleotide changes, in the case of D N A sequence data), while maximum likelihood methods search for the tree that was most likely to have given rise to the data under the evolutionary hypothesis assumed (or specified). Parsimony methods have an advantage in that computational time is shorter. The trees, however, can be inconsistent (not representative of the true phylogeny) even with a very large amount of data and even if all taxa have evolved at the same rate (Penny and Hendy, 1986). Also, they are not based on a model of evolution, although some evolutionary parameters can be specified, e.g. different transition/transversion ratios. While maximum likelihood methods consume more computer time, they are based on a model of evolution. They can, therefore, account for multiple hits or estimate the parameters of the model from the data. Furthermore, they are consistent as long as an appropriate model is used, are less affected by sampling error (have lower variance) and are  100 robust to user-specified parameters that do not match the true parameters (Swofford et al., 1996). The programs, PAUP 3.0s (Swofford, 1993) and D N A M L (Felsenstein, 1993), were used to infer most parsimonious and maximum-likelihood trees, respectively, from the nucleotide D N A sequence alignment. The search algorithm used in PAUP was branch-and-bound. Constraint trees were constructed in MacClade Version 3 (Maddison and Maddison, 1992). Trees were inferred from the deduced amino acid alignment using PAUP with a step matrix weighting the changes from one amino acid to another and ProtML from the M O L P H Y 2.2 package developed by Adachi and Hasegawa and available by ftp from sunmh.ism.ac.jp or from the Internet at http://evolve.zps.ox.ac.uk/PhySoft/PhySoft.html or http: // dog wood. botany. uga. edu/malmberg/software. html.  101  Results and Discussion  The data presented in this chapter suggest that .4-only homothallic species have a common ancestor and that ^/<2-homothallic species are more closely related to heterothallic species than they are to A-on\y homothallic species. The rooting of the phylogenetic tree of  Neurospora was not established with strong statistical support. Evolution from homothallism to heterothallism and vice versa is discussed. The phylogeny of the genus Neurospora was reconstructed from D N A and amino acid alignments of the mating type gene, mt A-l or its homolog (Appendix D). The species represented in the phylogenetic reconstruction are shown in Table 3.2. Table 3.2 Species included in the tree. Mating type gene  N. discreta N. intermedia N. sitophila N. tetrasperma N. crassa N. africana N. terricola N. pannonica N. galapagosensis N. lineolata N. dodge i Gelasinospora tetrasperma Sordaria macrospora Podospora anserina  ource : 1. Randall (pers. comm.) T. Randall (pers. comm.) T. Randall (pers. comm.) T. Randall (pers. comm.) Glass, Grotelueschen and Metzenberg, 1990 Glass and Smith, 1994 this study this study this study this study this study this study Poggeler etal, 1997b Debuchy and Coppin, 1992  GenBank Accession # L42307 L42308 L42309 L42310 M33876 L423 01  64194.gb-pl  When the tree was determined without outgroups, there was strong support for an allhomothallic clade and an all-heterothallic clade. Specifically, 100/100 (using maximum likelihood) or 500/500 (using parsimony) bootstrap replicate trees had such a division. The topology of the unrooted trees produced by maximum likelihood and parsimony methods were identical (Figure 3.1). Attempts to find a convincing root for the tree with the outgroup P.  anserina were unsuccessful and so it was removed from the data set.  102  N.  N. galapagosensis  Figure 3.1 Unrooted parsimony tree from D N A sequence alignment. Bootstrap support (%) is shown for 500 replicates. Boldface type denotes homothallic species, with homothallic species marked with an asterisk.  103 A branch-and-bound search in PAUP 3.0s (Swofford, 1993) specifying G. tetrasperma and S. macrospora as outgroups found three shortest trees of 343 steps. In a consensus of the 343 trees rooted with S. macrospora, Neurospora was not monophyletic (Figure 3.2). The basal split in the tree separated one cluster comprising the four Neurospora A -homothallic species from a second, heterogeneous cluster including the ^(/a-homothallic Neurospora species, the heterothallic Neurospora species and G. tetrasperma. Within the second cluster, the branching order of the ^/o-homothallic species, the heterothallic group and G. tetrasperma was unresolved. No bootstrap support was shown for including G. tetrasperma in Neurospora, and so there is no reason to include G. tetrasperma within the genus Neurospora. In 500 bootstrap replicates, the ^-homothallic species always clustered together, showing that they are more closely related to each other than to any of the heterothallic species. Similarly, the heterothallic  Neurospora species clustered together in 99% of 500 bootstrap replicates, showing that they are more closely related to each other than to any of the homothallic species. When G. tetrasperma was removed from the data set, the placement of the outgroup, S.  macrospora, on the tree was supported by 100% bootstrap support (500 bootstrap replicates) relative to the ^-homothallic group and 80% relative to the ^/a-homothallic/heterothallic group (Figure 3.3). This tree suggests that the /4/a-homothallic species are more closely related to the heterothallic species than they are to the /l-homothallic species. Morever, the tree suggests that homothallism is the ancestral state in Neurspora. A direct test of the hypothesis that Neurospora evolved from a homothallic ancestor are presented below. Other trees were constructed from the tree with G. tetrasperma as the outgroup to test the statistical support for the rooting. The topology of the trees rooted with alone produced by maximum likelihood and parsimony methods were identical (Figure 3.4). In approximately half of the bootstrap replicate trees, G. tetrasperma rooted the tree between the ,4-only homothallic  Neurospora species and the yl/a-homothallic/heterothallic group (Figure 3.4). Constraint trees were constructed in which all branches were collapsed except the one that rooted the tree. That  104  S. macrospora G. tetrasperma N. crassa N. sitophila N. tetrasperma N. discreta N. intermedia N.  pannonica*  N. terricola* N. dodgei N. lineolata N. africana N. galapagosensis  Figure 3.2 Parsimony tree from D N A sequence alignment, specifying 5*. macrospora and G. tetrasperma as outgroups. Bootstrap support (%) is shown for 500 replicates. Boldface type denotes homothallic species, with A/a homothallic species marked with an asterisk.  105  N.  pannonica*  TV. terricola*  94  80 89  N. crassa N. sitophila  100  N. intermedia  99  N. tetrasperma N. discreta TV. africana  100  78 68  TV. dodgei  N. lineolata TV. galapagosensis  S.  macrospora  Figure 3.3 Rooted parsimony tree from D N A sequence alignment. Bootstrap support (%) is shown for 500 replicates. Boldface type denotes homothallic species, with A/a homothallic species marked with an asterisk.  106  TV.  pannonica*  TV. terricola*  100  89  100  100  97  TV. crassa  96  TV. sitophila  92  TV. intermedia  100  TV. tetrasperma TV. discreta TV. africana  100 100  51 57  63  TV. dodgei  71  TV. lineolata TV. galapagosensis  G. tetrasperma  Figure 3.4 Rooted maximum likelihood/parsimony tree from D N A sequence alignment. Bootstrap support (%) is shown on the top for 500 replicates in D N A M L ; on the bottom for 100 replicates in PAUP. Boldface type denotes homothallic species, with A/a homothallic species marked with an asterisk.  107  branch was moved to see if the competing root placement (between the homothallic and heterothallic species) significantly lengthened the tree. Comparison of constraint trees in which  G. tetrasperma rooted the tree either before (Figure 3.5) or after (Figure 3.6) the divergence of homothallic species from heterothallic species revealed that the most likely and most parsimonious rooting (Figure 3.5) required only one fewer nucleotide change than did the competing rooting. The rooting, therefore, could not be done with confidence using G.  tetrasperma since one placement of the root was not significantly better than the other. Many ascomycete genera and families include both homothallic and heterothallic species, suggesting that there have been multiple independent derivations of homothallism and/or heterothallism (Nauta and Hoekstra, 1992a). Population genetic modeling suggested that evolution from heterothallism to homothallism is much more likely than vice versa (Nauta and Hoekstra, 1992a). The model was based on a hypothetical fungus with the following characteristics: life cycle was haploid with a short diplophase, each individual could form male and female sexual structures and the male gametes could double as asexual spores. Their assumptions seem reasonable for Neurospora. A l l homothallic species in other fungal genera have both A and a sequences and they are linked (see Chapter 2 Discussion). The universality of this type of homothallic species suggests that the ^4-only Neurospora species arose by a unique evolutionary pathway. If such is the case, the two homothallic mating strategies are independent and thus the ancestral state cannot be determined. A n alternative explanation of the rooting is that it may be affected by the mating strategy of the chosen outgroup. S. macrospora is homothallic and G. tetrasperma is pseudohomothallic. This hypothesis could be tested by adding the mt A-l sequence from a heterothallic Sordaria or Gelasinospora species or by constructing a tree from the D N A sequence of a non-mating type gene. It was not tested here. Since the tree was based on sequences of one of the mating type genes, it is a gene tree, not a species tree. If the mt A-l gene has a different function in /4/a-homothallic/heterothallic species than it does in the ^-only homothallic species, then the clustering could be reflecting  108  G. tetrasperma N. crassa N. sitophila N. tetrasperma N. discreta N. intermedia  N. pannonica* TV. terricola* TV. dodgei TV. lineolata TV. africana TV. galapagosensis  Figure 3.5 Constraint tree with same rooting as maximum likelihood/parsimony tree with branches collapsed to allow comparison to the alternately rooted tree in Figure 3.5. Tree length is 213 nucleotide changes. Boldface type denotes homothallic species, with A/a homothallic species marked with an asterisk.  G. tetrasperma N. crassa N. sitophila N. tetrasperma N. discreta N. intermedia N.  pannonica*  N. terricola* N. dodgei N. lineolata N. africana N. galapagosensis  Figure 3.6 Constraint tree with an all-homothallic clade. Tree length is 212 nucleotide changes. Boldface type denotes homothallic species, with A/a homothallic species marked with an asterisk.  110 functional rather than evolutionary similarity. One piece of evidence that counters this idea is that heterothallic- and homothallic-specific sequences have been detected in the mating type idiomorph flanks (Randall and Metzenberg, 1995). The hypothesis that the heterothallic or the homothallic species are grouping because of the conservation of particular sets of nucleotides required for the possibly different functions of  mt A-l in heterothallic versus homothallic species was tested. The tree based on an alignment of the amino acids was made using parsimony (PAUP) and maximum likelihood methods (ProtML in the M O L P H Y 2.2 package and PAUP with a step matrix weighting the changes from one amino acid to another that makes the method essentially equivalent to the maximum likelihood method). Both methods found trees with the same topology (Figure 3.7). Bootstrap replicate trees were examined to see if there were clusters of amino acids on the branch separating heterothallic from homothallic species. No clusters of three or more amino acids were found, suggesting that there are no large regions of mt A-l that are different between homothallic and heterothallic species. Of course, just one or two amino acid differences may be sufficient to confer homothallic- or heterothallic-specific usage of mt A-l and these would have gone undetected. The lack of large differences supports the hypothesis that the mt A-l gene does not have heterothallic- or homothallic-specific functions and supports its use in this phylogenetic analysis. In addition, the amino acid sequences were scrutinized to reveal open reading frames (ORFs) or lack thereof If the homothallic ORFs were truncated, then they are probably not functional, which could account for the grouping of heterothallic species apart from homothallic species. A l l of the heterothallic species (N. crassa, N. discreta, N. sitophUa, N. intermedia), pseudohomothallic species (N. tetrasperma, G. tetrasperma), A/a homothallic species (N.  terricola, N. pannonica) and one of the four/4-only homothallic species (N. africana) contain an uninterrupted ORF of the same size. The size of the ORF in the 3 ,4-only homothallic species,  N. lineolata, N. dodgei and N. galapagosensis, is unknown because one primer 5' to the A T G failed to amplify a product in a PCR. The 5' primer that amplified a product from which the sequence was derived starts at position #109 in the TV. crassa ORF. The available sequences for  Ill  58  pannonica*  N.  terricola*  N. crassa  64  53  TV.  N. sitophila  100  N. intermedia  98  N. tetrasperma N. discreta N.  100  79 58  africana  N. dodgei N.  lineolata  N.  galapagosensis  G. tetrasperma  Figure 3.7 Rooted parsimony tree from amino acid sequence alignment. Bootstrap support (%) is shown for 100 replicates. Boldface type denotes homothallic species, with A/a homothallic species marked with an asterisk.  112 the 3 ,4-only homothallic species encode products highly similar to the products from the other species and include a stop codon at the same position. Support for the use of the mating type gene for the phylogenetic analysis of Neurospora is found by comparing the mt A-l trees to those based on other characters. While the mt A-l trees included all known Neurospora species, the latter trees included some, but not all. The latest publication included three different trees of the heterothallic Neurospora species, based on restriction maps of eight randomly chosen 40-kbp cosmid probes, a 400-bp sequence of a region upstream of the al-1 gene and a 500-bp sequence of a region upstream of the frq gene (Skupski, Jackson and Natvig, 1997). A tree based on restriction maps of the frq gene included the heterothallic Neurospora species and one homothallic species (Lewis and Feldman, 1996). Randall and Metzenberg (1995) reconstructed the phylogeny of the heterothallic species and one homothallic species based on the sequence of the mt A-l gene. D N A sequence and restriction fragment length polymorphisms of the frq gene were used to build a tree of N. crassa, N.  intermedia and N. discreta with Sordaria and Gelasinospora species as putgroups (Merrow and Dunlap, 1994). Mitochondrial RFLPs were used to determine the relationships among N.  crassa, N. intermedia, N. sitophila and N. tetrasperma, which have proven difficult to resolve (Taylor and Natvig, 1989). The earliest attempt to resolve these relationships was a study that utilized RFLPs of four cloned fragments of nuclear D N A (Natvig, Jackson and Taylor, 1987). The topology of the trees based on the mt A-l sequence is congruent with that based on the sequence and RFLPs of the frq gene. The tree shows that N. crassa and N. intermedia are more closely related to each other than to N. discreta, and that G. cerealis and S. fimicola are outgroups to these three Neurospora species (Merrow and Dunlap, 1994). The topology of the tree based on the restriction map of the frq gene corroborates the mt A-l tree inasmuch as N.  tetrasperma, N. sitophila and 7Y. crassa are more closely related to each other than to N. galapagosensis (Lewis and Feldman, 1996). Not surprisingly, the tree based on D N A sequence from the mating type region (Randall and Metzenberg, 1995) has the same topology as this mt  A-l tree.  113  The relationship between TV. tetrasperma and three of the heterothallic species, TV.  intermedia, TV. crassa and TV. sitophila, is the same in both the mitochondrial RFLP tree (Taylor and Natvig, 1989) and the mt A-l tree, upholding the finding that TV. tetrasperma diverged from the other three species before their divergence from each other. This relationship is contradicted by the frq restriction map tree (Lewis and Feldman, 1996), the /rg-flank sequence tree, the al-1flank sequence tree and the random cosmid tree (Skupski, Jackson and Natvig, 1997). The relationship of TV. sitophila to TV. tetrasperma remains unresolved. Some trees show them to be sister taxa while others suggest that TV. tetrasperma diverged from the heterothallic group prior to the divergence of TV. sitophila from the other heterothallic species. Some branches of the mating type tree are not found in trees based on other characters. Uniquely, the mating type tree places TV. crassa and TV. sitophila as sister groups at the tip of the tree, with TV. intermedia diverging prior to the TV. crassa/N. sitophila split. In contrast, the trees reconstructed from thefrq-f\ank  sequence, the a7-T-flank sequence and the random cosmids,  show that TV. crassa and TV. intermedia are more closely related to each other than to TV. sitophila and TV. tetrasperma (Skupski, Jackson and Natvig, 1997). Also, in the frq restriction map tree, TV. sitophila and TV. tetrasperma are more closely related to each other than they are to TV. crassa (Lewis and Feldman, 1996), a finding echoed in the nuclear RFLP tree (Natvig, Jackson and Taylor, 1987). Biological data also contrast with the mating type trees. Interspecific crosses between TV. crassa and TV. intermedia produce abundant inviable ascospores, whereas TV. crassa x TV. sitophila crosses are relatively infertile (Perkins, Turner and Barry, 1976). Multiple isolates of each species were used for the analysis of the mating type (Randall and Metzenberg, 1995) and some of the opposing trees, suggesting that the difference is not strain-specific. The mating type regions of TV. crassa, TV. sitophila and TV. intermedia have a different evolutionary history than other parts of the genome (Randall and Metzenberg, 1995; this study; Skupski, Jackson and Natvig, 1997), even regions on the same linkage group (Skupski, Jackson and Natvig, 1997). The idea that genes do not necessarily share evolutionary histories is supported by the maximum-likelihood analysis of the7?g-flank and mt A-l trees (Skupski, Jackson and Natvig, 1997; Randall and Metzenberg, 1995). The distinct relationship of the  114 mating type regions could be explained by lineage sorting of an ancestral polymorphism in the  mtA-1 gene or hybridization between the N. crassa and N. sitophila that resulted in the transfer of the mt A-l region. In support of hybridization, the mating type region of one group of TV.  tetrasperma isolates appears to have been derived from N. intermedia-like ancestors while another group appears to have been derived from N. intermedia axN. sitophila A or N.  intermedia axN. crassa A (R. L. Metzenberg, personal communication). Also, the semipermeability of fertility barriers among all Neurospora heterothallic species (Perkins, Turner and Barry, 1976) suggests that occasional hybridization could be occurring in nature. One relationship worth comparing is that between sexual and asexual species. Asexual filamentous ascomycetes appear to be derived frequently and recently from sexual species (LoBuglio, Pitt and Taylor, 1993; Geiser, Timberlake and Arnold, 1996). Homothallic  Neurospora species may be similar to asexual species in that both self-reproduce. Homothallic species differ from asexual species in that they undergo sexual processes and may possess outbreeding potential. In contrast to asexual ascomycetes, homothallic Neurospora species appear to survive long enough to diverge. They may live longer or simply evolve more quickly than asexual species. Homothallic species undergo meiosis which may confer advantages through recombination. Recombination is believed to exert is effect through Muller's ratchet, the idea that, in the absence of recombination, each generation possesses no fewer, and possibly more mutations than were possessed by the generation before (Muller, 1964). Experiments designed to show that meiosis confers advantage even without recombination have been difficult to control adequately (e.g. Birdsell and Wills, 1996). In theory, the production of an asexual spore is genetically equivalent to the production of a sexual spore in a self-mating haploid organism (Nauta and Hoekstra, 1992a) in that neither propagule has undergone recombination and therefore does not contain novel deleterious associations of alleles. One cannot discount the possibility that different mutations occur in different nuclei in homothallic species and sex allows recombination. This amount of recombination is unlikely to have a major effect (S. Otto, personal communication).  115 In addition to the hypothetical advantages that may be conferred to homothallic Neurospora species by meiosis and/or ascospores, occasional outbreeding may occur in nature by heterokaryosis (Nauta and Hoekstra, 1992b). Rare outbreeding may be nearly equivalent to frequent outbreeding in terms of long-term selective advantages conferred by recombination (Muller, 1964). Hypotheses about the evolution of homothallism and heterothallism can be divided into two groups, those which suggest that homothallism evolved first and those that suggest that heterothallism evolved first. Olive (1958) started with the assumption that homothallism is the ancient mating system and heterothallism is the derived one based on intuition: "It is readily apparent that a system permitting karyogamy between any two haploid nuclei in a thallus (homothallism) is less modified than one involving two types of thalli, the nuclei of which are self-incompatible but cross-compatible (heterothallism)". The compound nature of the basidiomycete mating type locus had just been elucidated, leading Olive (1958) to suggest that heterothallic species were derived from a homothallic ancestor by deletion of one gene in a strain and deletion of the other gene in a different strain. Such strains were hypothesized to be self-sterile and cross-fertile. This hypothesis is virtually identical to that proposed by Charlesworth (1994) in which he invokes the same events to account for the evolution of mating type idiomorphs. Metzenberg and Glass (1990) and Glass, Metzenberg and Raju (1990) suggest that heterothallic species predate extant homothallic species. The A/a homothallic species could have arisen after an unequal crossover event led to the co-nuclearization of the mating type genes. When the sequences surrounding the mating type regions are considered, however, a crossover that accounts for all the data cannot be contrived. For a progression such as the one illustrated in Figure 3.8 to occur, either one of two events may have occurred, one which has not been known to happen in any organism and one which contradicts the data. The first event would have been the highly unlikely recombination between non-homologous sequences. The second would have been a D N A end-joining event, in which case the two mating type chromosomes, one from each parent, would have been joined as well. Even if an event had  LF  LF  LF  mta  mt a  RF  POSTULATED HETEROTHALLIC ANCESTOR  RF  mt A  mtA  RF  DERIVED HOMOTHALLIC SPECIES  Figure 3.8 Model for the evolution of homothallic species from a heterothallic ancestor. Checkerboard boxes represent sequences homologous to the N. crassa left flank, L F (black), and right flank, RF (grey). Solid boxes represent the mt a (grey) and mt A (black) genes. Dotted line shaped like a Z illustrates the position of the most plausible recombination event.  117 occurred to accomplish the transition illustrated in Figure 3.8, 12-20 kbp of D N A would have had to insert precisely between the two mating type idiomorphs. Alternative explanations are possible, but they are even less likely. A n alternative model inspired by that proposed by Charlesworth (1994) is introduced in Figure 3.9. Charlesworth (1994) proposed that in an initially self-fertile population, mutation at a recognition locus (e.g. a diffusible signal required for the recognition of gametes) results in a strain that cannot self-fertilize, but can still be fertilized. Such a strain might be at a selective advantage, presumably due to outbreeding. A second mutation in another strain at a reciprocal, linked locus (the receptor of the diffusible signal) might also be favoured, as would suppression of recombination between them. Eventually two self-sterile, cross-fertile types would remain. Suppression of recombination between the genes would allow them to evolve independently. Furthermore, the two non-functional alleles could change relatively rapidly by genetic drift. These two processes could result in the formation of idiomorphs. His hypothesis requires that clustering of recognition genes predate the genesis of idiomorphs, which is not difficult to imagine. Clustering of identity genes may be advantageous to an organism because of the preservation of heterozygosity that is afforded by limiting recombination between the genes. Identity encoded by idiomorphs has advantages over identity encoded by alleles. Distinction is ensured by the elimination of homologous recombination between the idiomorphs. Furthermore, a simple reversion could destroy mating specificity determined by alleles, but not by idiomorphs. The model I propose in Figure 3.9 provides a plausible explanation for the evolution from homothallism to heterothallism, the evolution of idiomorphs, the existence of a cw-acting regulator able to control genes in both mating type regions, A and a, as we know N. crassa's can (Chang and Staben, 1994) and, finally, the combinations of mating type genes with various flanking sequence blocks. The ancestral Neurospora species is proposed to have been homothallic with a genetic constitution like that of N. pannonica (L. Wheeler and N . L. Glass, personal communication), in that it possessed all four mating type genes, mtA-1, mt A-2, mt A-3 and mt a-l on one  118  2. o r+  O  3 tj  2 a. s _ cr a OfQ O <T> H M * CD ft  ^ 8o O H CS CD  cf p X w CD GO '-i CD  t3 -i CD  CD  ct (j  s  o  S 3 2,  CD Si 00 CD ft M h?  3  o  3  r+  o  CD  sr  o ? 9o ~« o f • a o M  fi to fl  1  <B o SCt  ft  "  & 5o p ^r 3 Q C/) o S . Co v Q ^ 5 CD 3 f-t  I  cr ~> F  a  3°  o  SCD ^§ CD BO  73  p • O  p  5 a  O  3 cr  119 chromosome. I speculate that this cluster of genes may have encoded transcription factors that controlled entry into the sexual phase. I propose that mutations in one gene in one strain and the other gene set in a different strain may have given rise to two self-sterile, cross-fertile individuals. The following events detail this process. I propose that the a gene was flanked on the left side by a sequence block (LF) homologous to the block abutting the centromere-proximal side of the N. crassa idiomorphs. 1 propose that the A genes resided next to a sequence block homologous to the RF sequence which abuts the N. crassa idiomorphs on the centromere-distal (right) side. I propose that a subpopulation arose in which the mt a gene was mutated and no longer functional, and was, therefore, free to diverge away from the original sequence. The mt a gene and the D N A between the two genes may have been deleted, resulting in a single mating-type strain with a mt  A idiomorph flanked by RF and LF. Similarly, a mutated mt A-l gene could have been free to diverge or be deleted. The resultant strain could have been of mating type a, again flanked by RF and LF, just like N. crassa. The N. crassa a idiomorph contains 2 kbp of sequence that appear to be peripheral to mating type function (Glass et al., 1988) and I suggest that this stretch of D N A may have come from the sequences between the A and a regions in the ancestral homothallic species. The putative cX-acting regulator may have regulated both A and a expression by virtue of its position relative to both genes. In all homothallic Sordariaceae containing both A and a genes, the two genes are linked (Glass, Metzenberg and Raju, 1990). I propose that the exacting regulator still exists in N. crassa and can act on both A and a genes. The ,4-only homothallic species could have arisen by loss or replacement of mt a function. Thus, the model joins the two main issues addressed in this thesis, namely, positional regulation and evolution of mating type.  120  Summary  The mechanism of N. crassa mating type position effect has been examined. The position effect is likely due to the requirement of the genes to be present on the same molecule of D N A as a cw-acting sequence, possibly in a specific orientation or spacing. Many such elements are known to exist, although none has yet been found in a filamentous fungus. The homothallic species N. terricola contains a subset of the N. crassa mating type genes. One gene from each mating type, the one specifying mating identity in N. crassa, appears to be functional. These results raise the questions of how could the position effect be modified in N. terricola to allow opposite mating type genes to express full fertility, something that cannot be done in N. crassa! What could mating type genes be doing in a selfing organism? Homothallic Neurospora species that carry both mating type genes appear to be more closely related to heterothallic species than they are to the A-on\y homothallic species, suggesting that the two types of homothallism may have evolved independently. The issue of whether Neurospora species evolved from a homothallic or a heterothallic ancestor remains unresolved, but a model has been put forth.  121  References Akins, R. A . and A . M . Lambowitz 1985. General method for cloning Neurospora crassa nuclear genes by complementation of mutants. Mol. Cell. Biol. 5: 2272-2278. Aramayo, R. and R. L. Metzenberg 1996. Meiotic transvection in fungi. Cell 86: 103-113. Arnaise, S., D. Zickler and N . L. Glass 1993. Heterologous expression of mating-type genes in filamentous fungi. Proc. Natl. Acad. Sci. U S A 90: 6616-6620. Astell, C. R., L. Ahlstrom-Jonasson, M . Smith, K. Tatchell, K. A . Nasmyth and B. D. Hall 1981. The sequence of the DNAs coding for the mating-type loci of Saccharomyces cerevisiae. Cell 27: 15-23. Beadle, G. W. and V . L. Coonradt 1944. Heterocaryosis in Neurospora crassa. Genetics 29: 291-308. Beatty, N . P. 1993. Homothallism in the Sordariaceae: a characterization of mating-type loci in selected species of Neurospora, Anixiella and Gelasinospora. M . Sc. Thesis, University of British Columbia, Vancouver, Canada. Beatty, N . P., M . L. Smith and N . L. Glass 1994. Molecular characterization of mating-type loci in selected homothallic species of Neurospora, Gelasinospora and Anixiella. Mycol. Res. 98: 1309-1316. Berbee, M . L. and J. W. Taylor 1992a. Convergence in ascospore discharge mechanism among Pyrenomycete fungi based on 18s ribosomal R N A gene sequence. Mol. Phylogenet. Evol. 1 : 59-71. Berbee, M . L. and J. W. Taylor 1992b. Two ascomycete classes based on fruiting-body characters and ribosomal D N A sequence. Mol. Biol. Evol. 9: 278-284. Berbee, M . L. and J. W. Taylor 1992c. Detecting morphological convergence in true fungi, using 18s rRNA gene sequence data. BioSystems 28: 117-125. Birdsell, J. and C. Wills 1996. Significant competitive advantage conferred by meiosis and syngamy in the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U S A 93: 908-912. Bistis, G. N . 1981. Chemotropic interactions between trichogynes and conidia of opposite mating type in Neurospora crassa. Mycologia 73: 959-975. Bistis, G. N . 1983. Evidence for diffusible, mating type-specific trichogyne attractants in Neurospora crassa. Exp. Mycol. 7: 292-295. Bistis, G. N . 1996. Trichogynes and fertilization in uni- and bimating type colonies of Neurospora tetrasperma. Fungal Genet. Biol. 20: 93-98. Bolker, M , M . Urban and R. Kallmann 1992. The a mating type locus of U. maydis specifies cell signaling components. Cell 68: 441-450. Bowman, B. H., J. W. Taylor, A . G. Brownlee, J. Lee, S.-D. L u and T. J. White 1992. Molecular evolution of the fungi: relationship of the basidiomycetes, ascomycetes and chytridiomycetes. Mol. Bio. Evol. 9: 285-296.  122 Chang, S. and C. Staben 1994. Directed replacement of mt A by mt a-l effects a mating type switch in Neurospora crassa. Genetics 138: 75-81. Charlesworth, B. 1994. The nature and origin of mating types. Current Biology 4: 739-741. Chu, Z., T. A . McKinsey, L. Liu, X . Qi and D. W. Ballard 1996. Basal phosphorylation of the PEST domain in I K B B regulates its functional interaction with the c-rel proto-oncogene product. Mol. Cell. Biol. 16: 5974-5984. Cogoni, C , J. T. Irelan, M . Schumacher, T. J. Schmidhauser, E. U . Selker and G. Macino 1996. Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on D N A - D N A interactions or D N A methylation. E M B O J. 15: 3153-3163. Covitz, P. A., I. Herskowitz and A. P. Mitchell 1991. The yeast RMEI gene encodes a putative zinc finger protein that is directly repressed by al/oc2. Genes Dev. 5: 1982-1989. Coyne, J. A . , A . P. Crittenden and K. Mah 1994. Genetics of a pheromonal difference contributing to reproductive isolation in Drosophila. Science 265: 1461-1464. Davis, R. H . and F. J. de Serres 1970. Genetic and microbial research techniques for Neurospora crassa. Methods Enzymol. 17: 79-143. Dayhoff, M . O. 1983. Evolutionary connections of biological kingdoms based on protein and nucleic acid sequence evidence. Precambrian Res. 20: 299-318. Debuchy, R. and E. Coppin 1992. The mating types of Podospora anserina: functional analysis and sequences of the fertilitzation domains. Mol. Gen. Genet. 233: 113-121. Debuchy, R., S. Arnaise and G. Lecellier 1993. The mat-aWele of Podospora anserina contains three regulatory genes required for the development of fertilized female organs. Mol. Gen. Genet. 241: 667-673. Devereux, J. P., P. Haeberli and O. Smithies 1984. A comprehensive set of sequence analysis programs for the V A X . Nucleic Acids Res. 12: 387-395. Dolan, J. W. and S. Fields 1991. Cell-type-specific transcription in yeast. Biochim. Biophys. Acta 1088: 155-169. Edelman, S. and C. Staben 1994. A statistical analysis of sequence features within genes from Neurospora crassa. Exp. Mycol. 18: 70-81. Egel, R., O. Nielsen and D. Weilguny 1991. Sexual differentiation in fission yeast. Trends Genet. 6: 369-373. Felsenstein, J. 1993. PHYLIP (Phylogeny inference package). Ferreira, A . V . B. 1997. Characterisation of the mating-type genes mt A-2 and mt A-3 of Neurospora crassa and regulation of sexual development by mating-type. Ph.D. Thesis, University of British Columbia, Vancouver, Canada. Ferreira, A . V . B., S. Saupe and N . L. Glass 1996. Transcriptional analysis of the mt A idiomorph of Neurospora crassa identifies two genes in addition to mt A-l. Mol. Gen. Genet. 250: 767-774.  123  Ferreira, A . V . B., R. L. Metzenberg and N . L. Glass 1997. Neurospora crassa mating type: evidence for activation and repression by mating-type products. Published abstracts of Fungal Genetics Conference, Asilomar, C A . Ferreira, A . V . B., Zhiqiang, R. L. Metzenberg and N . L. Glass 1998. Characterization of mat A-2, mat A-3 and A mat A mating type mutants of Neurospora crassa. Genetics 148: 10691079. Fincham, J. R. S. 1989. Transformation in fungi. Microbiol. Rev. 53: 148-170. Gargas, A., P. T. De Priest, M . Grube and A. Tehler 1995. Multiple origins of lichen symbioses in fungi suggested by SSU rDNA phylogeny. Science 268: 1492-1495. Geiser, D. M . , W. E. Timberlake and M . L. Arnold 1996. Loss of meiosis in Aspergillus. M o l . Biol. Evol. 13: 809-817. Giasson, L., C. A . Specht, C. Milgrim, C. P. Novotny and R. C. Ullrich 1989. Cloning and comparison of A a mating-type alleles of the Basidiomycete Schizophyllum commune. Mol. Gen. Genet. 218: 72-77. Glass, N . L. and G. C. Donaldson 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61: 1323-1330. Glass, N . L. and L. Lee 1992. Isolation of Neurospora crassa A mating-type mutants by repeatinduced point (RIP) mutation. Genetics 132: 125-133. Glass, N . L. and M . A . Nelson 1994. Mating-type genes in mycelial ascomycetes. In: Wessels, J. D. H . and F. Meinhardt (eds.) The mycota: growth, differentiation and sexuality. SpringerVerlag, Berlin, pp. 295-306. Glass, N . L. and M . L. Smith 1994. Structure and function of a mating-type gene from the homothallic species Neurospora africana. Mol. Gen. Genet. 244: 401-409. Glass, N . L., J. Grotelueschen and R. L. Metzenberg 1990. Neurospora crassa A mating-type region. Proc. Natl. Acad. Sci. U S A 87: 4912-4916. Glass, N . L., R. L. Metzenberg and N . B. Raju 1990. Homothallic Sordariaceae from nature: The absence of strains containing only the a mating type sequence. Exp. Mycol, 14: 274-289. Glass, N . L., S. J. Vollmer, C. Staben, J. Grotelueschen, R. L. Metzenberg and C. Yanofsky 1988. DNAs of the two mating-type alleles of Neurospora crassa are highly dissimilar. Science 241: 570-573. Griffiths, A . J. F. 1982. Null mutants of the A and a mating-type alleles of Neurospora crassa. Can. J. Genet Cytol 24: 167-176. Griffiths, A . J. F. and A. M . DeLange 1978. Mutations of the a mating-type gene in Neurospora crassa. Genetics 88: 239-254. Gubbay, J., J. Collignon, P. Koopman, B. Capel, A . Economou, A . Miinsterberg, N . Vivian, P. Goodfellow and R. Lovell-Badge 1990. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 346: 245-250.  124 Haber, J. E. and J. P. George 1979. A mutation that permits the expression of normally silent copies of mating-type information in Saccharomyces cerevisiae. Genetics 93: 13-35. Hendriks, L., R. De Baere, Y . Van de Peer, J. Neefs, A . Goris and R. De Wachter 1991. The evolutionary position of the rhodophyte Porphyra umbilicalis and the basidiomycete Leucosporidium scottii among other eukaryotes as deduced from complete sequences of small ribosomal subunit R N A . J. Mol. Evol. 32: 167-177. Henikoff, S. 1994. A reconsideration of the mechanism of position effect. Genetics 138: 1-5. Hiraoka, Y . , A . F. Dernburg, S. J. Parmelee, M . C. Rykowski, D. A . Agard and J. W. Serat 1993. The onset of homologous chromosome pairing during Drosophila melanogaster embryogenesis. J. Cell Biol. 120: 591-600. Hollaender, A., E. R. Sansome and M . Demerec 1945. Quantitative irradiation experiments with Neurospora crassa. II. Ultraviolet irradiation. Am. J. Bot. 32: 226-235. Hori, H. and S. Osawa 1987. Origin and evolution of organisms as deduced from 5S ribosomal R N A sequences. Mol. Biol. Evol. 4: 445-472. Irelan, J. T., A . T. Hagemann and E. U . Selker 1994. High frequency repeat-induced point mutation (RIP) is not associated with efficient recombination in Neurospora. Genetics 138: 1093-1103. Jantzen, H.-M., A . Admon, S. P. Bell and R. Tijan 1990. Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to H M G proteins. Nature 344: 830-836. Judd, B. H . 1988. Transvection: allelic cross talk. Cell 53: 841-843. Kang, S, F. G. Chumley and B. Valent 1994. Isolation of the mating-type genes of the phytopathogenic fungus Magnaporthe grisea using genomic subtraction. Genetics 138: 289296. Kelly, M . , J. Burke, M . Smith, A . Klar and D. Beach 1988. Four mating-type genes control sexual differentiation in the fission yeast. E M B O J. 7: 1537-1547. Klar, A . J. S., S. Fogel and K. MacLeod 1979. MARl-a regulator of the HMa and HMa loci in Saccharomyces cerevisiae. Genetics 93: 37-50. Kreader, C. A . and J. A . Heckman 1987. Isolation and characterization of a Neurospora crassa ribosomal protein gene homologous to CYH2 of yeast. Nucleic Acids Res. 15: 9027-9042. Kiies, U . and L. Casselton 1992. Fungal mating type genes-regulators of sexual development. Mycol. Res. 96: 993-1006. Lee, S. B. and J. W. Taylor 1990. Isolation of D N A from fungal mycelia and single spores in PCR Protocols: a guide to methods and applications, eds. M . A . Innis, D. H . Gelfand, J. J. Sninsky and T. J. White, Academic Press, Inc., San Diego, C A . Lewis, M . T. and J. F. Feldman 1996. Evolution of the frequency (frq) clock locus in ascomycete fungi. Mol. Biol. Evol. 13: 1233-1241. LoBuglio, K. F., J. I. Pitt and J. W. Taylor 1993. Phylogenetic analysis of two ribosomal D N A regions indicates multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in subgenus Biverticillium. Mycologia 85: 592-604.  125 Logemann J., J. Schell and L. Willmitzer 1987. Improved method for the isolation of R N A from plant tissues. Anal. Biochem. 163: 16-20. Merrow, M . W. and J. C. Dunlap 1994. Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency. E M B O J. 13: 2257-2266. Metzenberg, R. L. and N . L. Glass 1990. Mating type and mating strategies in Neurospora. BioEssays 12: 53-59. Miao, V . P. W., M . R. Rountree and E. U . Selker 1995. Ectopic integration of transforming D N A is rare among Neurospora transformants selected for gene replacement. Genetics 139: 1533-1544. Moore, T. D. E. and J. C. Edman 1993. The a-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol. Cell. Biol. 13: 1962-1970. Moore-Landecker, E. 1990. Fundamentals of the fungi, 3rd edition. Prentice Hall, Englewood Cliffs, N.J., pp. 101-139. Muller, H. J. 1964. The relation of recombination to mutational advance. Mutation Res. 1: 2-9. Natvig, D. O., D. A . Jackson and J. W. Taylor 1987. Random-fragment hybridization analysis of evolution in the genus Neurospora: the status of four-spored strains. Evolution 41: 10031021. Nauta, M . J. and R. F. Hoekstra 1992a. Evolution of reproductive systems in filamentous ascomycetes. I. Evolution of mating types. Heredity 68: 405-410. Nauta, M . J. and R. F. Hoekstra 1992b. Evolution of reproductive systems in filamentous ascomycetes. II. Evolution of hermaphroditism and other reproductive strategies. Heredity 68: 537-546. Newmeyer, D. 1970. A suppressor of the heterokaryon-incompatibility associated with mating type in Neurospora crassa. Can. J. Genet. Cytol. 12: 914-926. Olive, L. S. 1958. On the evolution of heterothallism in fungi. American Naturalist 42: 233251. Orbach, M . J., E. B. Porro and C. Yanofsky 1986. Cloning and characterization of the gene for B-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol. Cell. Biol. 6: 2452-2461. Penny, D. and M . Hendy 1986. Estimating the reliability of evolutionary trees. Mol. Biol. Evol. 3: 403-417. Perkins, D. D. 1972. A n insertional translocation in Neurospora that generates duplications heterozygous for mating type. Genetics 71: 25-51. Perkins, D. D. 1996. Neurospora crassa genetic maps. Fungal Genet. Newsl. 43: 180-189. Perkins, D. D. and B. C. Turner 1988. Neurospora from natural populations: Toward the population biology of a haploid eukaryote. Exp. Mycol. 12: 91-131.  126 Perkins, D. D., B. C. Turner and E. G. Barry 1976. Strains of Neurospora collected from nature. Evolution 30: 281-313. Perkins, D. D., J. A . Kinsey, D. K. Asch and G. D. Frederick 1993. Chromosome rearrangements recovered following transformation of Neurospora crassa. Genetics 134: 729736. Philley, M . L. and C. Staben 1994. Functional analyses of the Neurospora crassa M T a-l mating type polypeptide. Genetics 137: 715-722. Picard, M . , R. Debuchy and E. Coppin 1991. Cloning the mating types of the heterothallic fungus Podospora anserina: developmental features of haploid transformants carrying both mating types. Genetics 128: 539-547. Poggeler, S., S. Risch, H. D. Osiewacz and U . Kiick 1997a. Cloning and analysis of the matingtype genes from the homothallic ascomycete Sordaria macrospora. Published abstracts from the Fungal Genetics Conference at Asilomar, Pacific Grove, California. Poggeler, S., S. Risch, U . Kiick and H . D. Osiewacz 1997b. Mating-type genes from the homothallic fungus Sordaria macrospora are functionally expressed in a heterothallic ascomycete. Genetics, in press. Radford, A . 1993. A fungal phylogeny based upon orotidine 5'-monophosphate decarboxylase. J. Mol. Evol. 36: 389-395. Raju, N . B. 1978. Meiotic nuclear behavior and ascospore formation in five homothallic species of Neurospora. Can. J. Bot. 56: 754-763. Raju, N . B. 1980. Meiosis and ascospore genesis in Neurospora. Eur. J. Cell Biol. 23: 208223. Raju, N . B. 1992. Genetic control of the sexual cycle in Neurospora. Mycol. Res. 96: 241262. Randall, T. A . and R. L. Metzenberg 1995. Species-specific and mating type-specific D N A regions adjacent to mating type idiomorphs in the genus Neurospora. Genetics 141: 119-136. Rogers, S., R. Wells and M . Rechsteiner 1986. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234: 364-368. Russell, P. J., S. Wagner, K. D. Rodland, R. L. Feinbaum, J. P. Russell, M . S. Bret-Harte, S. J. Free and R. L. Metzenberg 1984. Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora. Mol. Gen. Genet. 196: 275282. Sambrook, J., E. F. Fritsch and T. Maniatis 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York. Saupe, S., L. Stenberg, K. T. Shiu, A . J. F. Griffiths and N . L. Glass 1996. The molecular nature of mutations in the mtA-1 gene of the Neurospora crassa A idiomorph and their relation to mating-type function. Mol. Gen. Genet. 250: 115-122. Schweizer, M . , M . E. Case, C. C. Dykstra, N . H . Giles and S. R. Kushner 1981. Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1 + regulatory gene. Proc. Natl. Acad. Sci. U S A 78: 50865090.  Selker, E. U . 1990. Premeiotic instability of repeated sequences in Neurospora crassa. Annu. Review Genet. 24: 579-613. Selker, E. U . , E. B. Cambareri, B. C. Jensen and K. R. Haack 1987. Rearrangement of duplicated D N A in specialized cells of Neurospora. Cell 51: 741-752. Shear, C. L. and B. O. Dodge 1927. Life histories and heterothallism of the red bread-mold fungi of the Monilia sitophila group. J. Agr. Res. 34: 1019-1042. Sinclair, A . FL, P. Berta, M . S. Palmer, J. R. Hawkins, B. L. Griffiths, M . J. Smith, J. W. Foster, A. Frischauf, R. Lovell-Badge and P. N . Goodfellow 1990. A gene from the human sexdetermining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346: 240-244. Singer, M . J. and E. U . Selker 1995. Genetic and epigenetic inactivation of repetitive sequences in Neurospora crassa: RIP, D N A methylation and quelling. Curr. Top. Microbiol. Immunol. 197: 165-177. Skupski, M . P., D. A . Jackson and D. O. Natvig 1997. Phylogenetic analysis of heterothallic Neurospora species. Fungal Genet. Biol. 21: 153-162. Smith, T. 1989. Disparate evolution of yeasts and filmentous fungus indicated by phylogenetic analysis of glyceraldehyde-3-phosphate dehydrogenase genes. Proc. Natl. Acad. Sci. U S A 86: 7063-7066. Spellig, T., M . Bolker, F. Lottspeich, R. W. Frank and R. Kahmann 1994. Pheromones trigger filmentous growth in Ustilago maydis. E M B O J. 13: 1620-1627. Staben, C. and C. Yanofsky 1990. Neurospora crassa a mating-type region. Proc. Natl. Acad. Sci. U S A 87: 4917-4921. Swofford, D. L. 1993. PAUP: phylogenetic analysis using parsimony. Illinois Natural History Survey, Champaign, Illinois. Swofford, D. L., G. J. Olsen, P. J. Waddell and D. M . Hillis 1996. Phylogenetic inference in Molecular systematics, second edition, eds. D. M . Hillis, C. Moritz and B. K. Mable, Sinauer Associates, Sunderland, M A . pp. 426-431. Tartof, K. D. and S. Henikoff 1991. Trans-sensing effects from Drosophila to humans. Cell 65 201-203. Tatchell, K., K. A . Nasmyth, B. D. Hall, C. Astell and M . Smith 1981. In vitro mutation analysis of the mating-type locus in yeast. Cell 27: 25-35. Taylor, J. W. 1986. Mitochondrial D N A and evolution in Neurospora species. Fungal Genet. Newsl. 33: 14. Taylor, J. W. and D. O. Natvig 1989. Mitochondrial D N A and evolution of heterothallic and pseudohomothallic Neurospora species. Mycol. Res. 93: 257-272. Taylor, J. W., B. D. Smolich and G. May 1986. Evolution and mitochondrial D N A in Neurospora crassa. Evolution 40: 716-739.  128 Turgeon, B. G., H . Bohlmann, L. M . Ciuffetti, S. K. Christiansen, G. Yang, W. Schafer and O. C. Yoder 1993. Cloning and analysis of the mating type gens from Cochliobolus heterostrophus. Mol. Gen. Genet. 238: 270-284. van Antwerp, D. J . and I. M . Verma 1996. Signal-induced degradation of I K B C * : association with N F - K B and the PEST sequence in I K B C X are not required. Mol. Cell. Biol. 16: 6037-6045. von Arx, J . A . 1981. The natural classification of the fungi in The genera of fungi sporulating in pure culture, third edition, ed. J . Cramer, Vaduz, Liechtenstein, p 143. Vellani, T. S. 1991. A/a incompatibility in Neurospora crassa-novel suppressors and nuclear incompatibility. M . Sc. Thesis, University of British Columbia, Vancouver, British Columbia, Canada. Vellani, T. S., A . J . F . Griffiths and N . L. Glass 1994. New mutations that suppress mating-type vegetative incompatibility in Neurospora crassa. Genome 37: 249-255. Verma, M . and S. K. Dutta 1987. Phylogenetic implication of heterogeneity of the nontranscribed spacer of rDNA repeating unit in various Neurospora and related fungal species. Curr. Genet. 11: 309-314. Versaw, W. K. and R. L. Metzenberg 1996. Activator-independent gene expression in Neurospora crassa. Genetics 142: 417-423. Vollmer, S. J . and C. Yanofsky 1986. Efficient cloning of genes of Neurospora crassa. Proc. Natl. Acad. Sci. U S A 83: 4869-4873. Westergaard, M . and H. K. Mitchell 1947. Neurospora V . A synthetic medium favoring sexual reproduction. A m . J . Bot. 34: 573-577. Wilson, C , H . J . Bellen and W. J . Gehring 1990. Position effects on eukaryotic gene expression. Annu. Rev. Cell Biol. 6: 679-714. Wirsel, S., B. G. Turgeon and O. C. Yoder 1996. Deletion of the Cochliobolus heterostrophus mating-type (MAT) locus promotes the function of M4Ttransgenes. Curr. Genet. 29: 241-249. Wu, C. -t. 1993. Transvection, nuclear structure, and chromatin proteins. J . Cell Biol. 120: 587-590. Wu, C. -t. and M . L. Goldberg 1989. The Drosophila zeste gene and transvection. Trends Genet. 5: 189-194. Zickler, D., S. Arnaise, E. Coppin, R. Debuchy and M . Picard 1995. Altered mating-type identity in the fungus Podospora anserina leads to selfish nuclei, uniparental progeny, and haploid meiosis. Genetics 140: 493-503.  Appendix A Neurospora crassa Gene Symbols Symbol  Description*  ad-2 ad-3A ad-3B al-1 am arg-2 Asm-1 cyh-1  adenine adenine adenine albino amination deficient (NADP-specific glutamate dehydrogenase) arginine ascospore-maturation deficient cycloheximide resistant fluffy (no macroconidia, female hyperfertile) frequency (disorganized circadian rhythm) lysine mating type nicotinic acid pantothenic acid quinate catabolism thiamine tolerant (mating type compatible) tryptophan unknown heat-sensitive defect  fl  frq lys-1 mt nic-2 pan-2 qa-2 thi-4 tol trp-4 un-3  * Perkins 1996  130  Appendix B Slot blot Analysis Table A. Values representing relative optical density of slots. 3-day-old mycelia, crp-1 probe  3-day-old mycelia, mt A-l probe  1415 1552 1469  3020 3257 3080  4207 4294 4258  2088 2451 1955  3211 4109 3054  4394 4208 4312  439 1047 921  744 897 727  1411 1389 1189  964 1132 1162  965 1602 1019  1335 2085 1567  6-day-old mycelia, crp-1 probe  6-day-old mycelia, mt A-l probe  1150 1180 1220  2199 2336 2223  4852 4921 4886  2356 3069 2738  3387 4551 3722  4865 4769 4701  203 122 377  1482' 1369 1583  4322 4113 4253  394 559 500  2912 3241 2864  3327 3940 3484  6-day-old mycelia, /3-tubulin probe 5485 3627 4264  9036 5329 6418  2221 1043 1560  6-day-old mycelia, mt A-3 probe 3931 2754 2990  501.2 91.84 125.7  856.0 187.0 249.6  155.0 29.55 41.03  337.0 64.02 88.93  N . B. Each set of three numbers represents three measurements of one slot. The layout of this table can be superimposed on the appropriate slot blot. Top numbers within each slot blot are directly comparable, as are middle numbers and bottom numbers, since the width of the slot slice was kept constant for each set of measurements.  Table B. Values representing ratios of mt-rel-Ac6 to 74-OR23-1A 3-day-old mycelia, mt A-l probe  3-day-old mycelia, crp-1 probe 3.2 1.5 1.6  4.1 3.6 4.2  3.0 3.1 3.6  2.2 2.2 1.7  3.3 2.6 3.0  3.3 2.0 2.8  mean=2.1  mean=4.0  mean=3.2  mean=2.0  mean=3.0  mean=2.7  s 2 =.91  s2=.ll  s2=.ll  s 2 =.09  s 2 =. 13  s 2 =.43  6-day-old mycelia, mt A-l probe  6-day-old mycelia, crp-1 probe 5.7 9.7 3.2  1.5 1.7 1.4  1.1 1.2 1.1  6.0 5.5 5.5  1.2 1.4 1.3  1.5 1.2 1.3  mean=6.2  mean=1.5  mean=l.l  mean=5.7  mean=1.3  mean=1.3  s =ll  s =.03  s =.01  s 2 =.09  s 2 =.01  s 2 =.03  2  2  2  6-day-old mycelia, fi-tubulin probe  6-day-old mycelia, mt A-3 probe  2.5 3.5 2.7  2.3 1.9 2.1  3.2 3.1 3.1  2.5 2.9 2.8  mean=2.9  mean=2.1  mean=3.1  mean=2.7  s =.28  s =.04  s =.01  s 2 =.05  2  2  2  N . B. Each number was derived from Table 10.5 by dividing the mt-rel-Ac6 value by the appropriate 74-OR23-lv4 value. s^ = sample variance  Table C. Calculation of differences between control and experimental means of ratios from Table B. Slots being compared  Pooled variance  Test statistic  Conclusion  3-day crp-1/mt A-l l x  .50 .12 .27 5.5 .13 .13 .14 .043  .14 3.6 1.2 .26 .70 .70 .65 3.6  same different same same same same same different  3-day 3-day 6-day 6-day 6-day  crp-l/mt crp-l/mt crp-l/mt crp-l/mt crp-l/mt  P-tubulin/mt  A-l A-l A-l A-l A-l  2x 4x lx 2x 4x  A-3 l x  (3-tubulin/mt A-3 2x  Let a = 0.05. The critical values of t are t 2.7764, therefore accept H 0 if-2.7764 < t c a i c u i a ted 2.7764.  133  Appendix C Mann-Whitney Rank-Sum Tests T = sum of ranks in the smaller sample ns = number of observations in the smaller sample nB = number of observations in the bigger sample For ns less than or equal to 8, compare T to the critical values of the Mann-Whitney Rank-Sum Statistic T, for n s greater than 8, convert T to zj. pT = (nS (ns + nB + l))/2 a j = sqrt[(ns nB (ns + nfi + 1))/12] zT = (I T - p x I - -5)/ o r t .05 (df inf.) = 1-9600 If ZT is greater than t .05 (df i n f . ) > t n e n the probability that the ranks would have fallen as seen if the two samples were taken from the same population would be 5%. In other words, the two samples are significantly different. Table A . Number of ascospores per rosette 8 days post-fertilization (fl A x 5a and flaxSA*)  flA  x 5a  rank  24 28 30 32 56 96 132 272  flaxSA*  0 0 0 0 0 0 0 0 0 0 0 0 0 1 4 4 4 16  19 20 21 22 23 24 25 26  1  ns  nB  ^  180  8  18  108  T  a  x  18  rank 7 7 7 7 7 7 7 7 7 7 7 7 7 14 16 16 16 18  zT 3.972>1.96, so populations are different  134  Table B. Number of ascospores per rosette 8 days post-fertilization (flax 74-OR23-1A and fl a x T(I-> II) 39311).  flax 74-OR23-1A  rank  flax T(I->II) 39311  rank  112 136 152 168 216 240 280  1 4.5 7.5 10 11 12.5 14  120 128 136 144 152 160 240  2 3 4.5 6 7.5 9 12.5  T  ns  nB  60.5  7  7  Critical values (two-tailed) of the Mann-Whitney Rank-Sum statistic T for ns = ne = 7 are 37 and 68 at P = 0.053, indicating that the two populations are not significantly different.  135  Table C. Number of ascospores per rosette 8 days post-fertilization (flAx5a 46A*/a).  fl A x 5a  rank  flA  24 28 30 32 56 96 132 272  35 36 37 38 39 40 41 42  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4  308  ns  nB  8  34  | a j  172  a T  31.22  x46A*/a  rank 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 34  zT 4.34>1.96, so populations are different  mdflA  x  136  Table D. Number of ascospores per rosette 8 days post-fertilization (flaxSA* A*/a).  flaxSA*  rank  flax46A  0 0 0 0 0 0 0 0 0 0 0 0 0 1 4 4 4 16  16 16 16 16 16 16 16 16 ' 16 16 16 16 16 32 34 34 34 36  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  1  ns  nB  [ I J  378  18  18  333  a  T  31.61  Va  rank 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16  zT 1.41< 1.96, not significantly different  andfl ax 46  137  Table E. Number of asci per rosette 13 days post-fertilization (fl a x SA * and/7 a x 46A */a).  flaxSA*  rank  fl a x 46A */a  rank  0 0 0 0 1 1 3 4 5 6 6 6 6 7 7 7 10 13  13 13 13 13 26.5 26.5 28 29 30 32.5 32.5 32.5 32.5 36 36 36 38 39  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13  1  ns  nB  ^T  G T  zT .  507  18  21  360  35.50  4.13>1.96, so populations are different  138  Appendix D DNA and Amino Acid Alignments 1 D N A Alignment of Neurospora Species, G. tetrasperma and S. macrospora used to generate Figure 3.3. NCRASSAXXX  A T G T C G G G T G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C G C T G A G G A  N S I T O P H I L A  A T G T C G G G A G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C G C T G A G G A  N I N T E R M E D I  A T G T C G G G T G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C G C T G A G G A  N T E T R A S PER  A T G C T G G G T G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C G C T G A G G A  N T E R R I C O L A  A T G T C G G G C G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C G C T G A G G G  NPANNONICA  A T G T C G G G C G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C G C T G A G G G  G T E T R A S P E R  A T G T C G G G C G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C G C T G A G G G  NTJODGEIXXX N A F R I C A N A X NGALAPOGOS N L I N E O L A T A N D I S C R E T A X  NNNNNNNNNNNNNNNNNNNNNN^ A T G T C G T G C G T C G A T C A A A T C G T C A A G A C G T T C G C C G A C C T C A C T G A G G G NNNNNNXVTNM^^ NNNNNNNNNNNNNNNNIS^^ A T G T C G G G C G T C G A C C A A A T T G T C A A G A C G T T C G C C G A C C T C G C T G A G G A  SMACROSPOR A T G T C C A G C G T C G A T C A A A T C G T C A A G A C G T T C G C C A A A C T C C C T G A G G G  N C R A S S A X X X  C G A C C G T G A A G C G G C A A T G A G A G C T T T C T C A A G G A T G A T G C G T A G A G G T A  N S I T O P H I L A  C G A C C G T G A A G C G G C A A T G A G A G C T T T C T C A A C G A T G A T G C - N N N N N G T A  N I N T E R M E D I  C G A C C G T G A A G C G G C A A T G A G A G C T T T C T C A A C G A T G A T G C - N N N N N G T A  N T E T R A S P E R  C G A C C G T G A A G C G G C A A T G A G A G C T T T C T C A A C G A T G A T G C - N N N N N G T A  N T E R R I C O L A  C G A T C G T G A A G C G G C A A T G A G A G C T T T C T C A A C G A T G A T G C - N N N N N G C A  N P A N N O N I C A  C G A T C G T G A A G C G G C A A T G A G A G C T T T C T C A A C G A T G A T G C - N N N N N G C A  G T E T R A S P E R  C G A T C G T G A A G C G G C A A T G A G A G C T T T C T C A A C G A T G A T G C - N N N N N G C A  N D O D G E I X X X N A F R I C A N A X N G A L A P O G O S N L I N E O L A T A  N N N N N N N N N N N N N N N N ^ T G A T C G T G A A G C G G C A A T G A G A G C T T T C T C A A T G A T G A T G C - N N N N N G C A N N N N N N N N N N N N N N N N N ^  - N N N N N N N N  NNNNINTNNNNNNNNNNNNNNNN^  N D I S C R E T A X  C G A C C G C G A A - N N G C A A T G A G A G C T T T C T C A A C G A T G A T G C - N N N N N G T A  S M A C R O S P O R  C G A G C G C A A C G C A G C A G T C A A T G C T A T C T T A G C C A T G A T G C C C C C C G G C C  NCRASSAXXX  CCGAACCTGTTCGCCGAAT-NNNNNNNNNNNNNNNNNNNNCCCCGCGGCAAAGAAGAAGGT  N S I T O P H I L A  CCGAACCTGTTCGCCGAAT-NNNNNNNNNNNNNNNNNNNNCCCCGCGGCAAAGAAGAAGGT  N I N T E R M E D I  CCGAACCTGTTCGCCGAAT-NNNNNNNNNNNNNNNNNNNNCCCCGCGGCAAAGAAGAAGGT  N T E T R A S P E R  CCGAACCTGTTCGCCGAAT-NNNNNNNNNNNNNNNNNNNNCCCCGCGGCAAAGAAGAAGGT  N T E R R I C O L A  CCGAACCTGTTCGCCAAAC-NNNNNNNNNNNNNNNNNNNNCCCCGCGGCAAAGA  NPANNONICA  CCGAACCTGTTCGCCAAAC-NNNNNNNNNNNNNNNNNNNNCCCCGCGGTAAAG  G T E T R A S P E R  CCGAACCTGTTCGCCAAAG-NNNNNNNNNNNNNNNNNNNNCC  NDODGEIXXX  NNNNNNNTGTTCGCCGAAT-NNNNNNNNNNNNNNNNNNNNCCCCGCGGCGAAGAAGAAGGT  N A F R I C A N A X  CCGAACCTGTTCGCCAAAC-NNNNNNNNNNNNNNNNNNNNCCCCGCGGCAAAGAAGAAGGT  NGALAPOGOS N L I N E O L A T A N D I S C R E T A X  NNNNNNNTGTTCGCCGAAT-NNNNNNNNNN^ NNNNNNNTGTTCGCCGAAT-NNNNNNNNNNNNNNNNNNN^ CCGAACCTGTTCGCCAAAT-NNNNNNNNNNNNNNNNNNNNCCCCGCGACAAAGAAGAAGGT  SMACROSPOR C T G G T C C T G T T C G C C A A A T C C C C G A A C C T G T T T C A C A A G C C C C C G C G C C A A A G A A G A A G G T  NCRASSAXXX CAACGGCTTCATGGGTTTCAGATGTGAGTCAAATCTGAATCAACATTGTCGTT-GATCCA N S I T O P H I L A CAACGGCTTCATGGGTTTCAGATGTGAGTCAAATCTGAATCAACATTGTCGTT-GATCCA NINTERMEDI CAACGGCTTCATGGGTTTCAGATGTAAGTCAAATCTGAATCAACATTGTCGTT-CATCCA NTETRASPER CAACGGCTTCATGGGTTTCAGATGTAAGTCAAATCTGAATCAACATTGTCGTTTGATCCA NTERRICOLA CAACCGCTTCATGGGTTTCAGATGTAAGTCAAATCTGAATCAATCTTGTCGAC--AATCC NPANNONICA CAACGGCTTCATGGGTTTCAGATGTAAGTCAAATCTGAATCAATCTTGTCGAC--AATCC GTETRASPER CAACGGTTTCATGGGTCTCAGATGTAAGTCAAATCTGAATCAATCTTGTCGAC--AATCC NDODGEIXXX CAACGGCTTCATGAGTTTCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA--AATCC NAFRI CANAX CAACGGCTTCATGAGTTTCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA- - AATCC NGALAPOGOS CAACGGCTTCATGAGTATCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA--AATCC N L I N E O L A T A CAACGGCTTCATGAGTTTCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA--AATCC NDISCRETAX CAACGGCTTCATGGGTTTCAGATGTAAGTTAAATCTGAATCAATCTTGTCGAT--AATCC SMACROSPOR CAACGGCTTCATGGGTTTCAGATGTAAGTCAAATCTGAATCAATCTTGACGAC--GATCC NCRASSAXXX TGGCTGATTGCTCTT-CATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC N S I T O P H I L A TGGCTGATTGCTCTT-CATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NINTERMEDI TGGCTAATTGCTCTT-CAATTTAGCGTACTATTCCCCGCTCTTCTCTCAGC NTETRASPER TGGCTGATTGCTCTT-CATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NTERRICOLA ATGCTGATTGCTTTT-TATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NPANNONICA ATGCTGATTGCCTTT-TATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC GTETRASPER ATGCTAATTGCTTTT-TATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NDODGEIXXX A T T C T A A T T G C T T T T - T A T T T C A G C G T A C T A T T C C C C G C T C T T C T C T C A G C NAFRICANAX ATTCTAATTGCCTTT-TATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NGALAPOGOS ATTCTAATTGCCTTT-TATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NLINEOLATA ATTCTAATGGCTTTT-TATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGN NDISCRETAX ATGCTGACTGCTCTT-CATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC SMACROSPOR ATACTGATTGCCTTTCTGTTTCAGCGTACTATTCCCCGCTCTTCTCTCAGT NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  TCCCGCAAAAGGAGAGATCGCCCTTCATGACTATTCTCTGGCAGCATGAT TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGCATGAT TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGCATGAT TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGCATGAT TCCCGCAAAAGGAGAGATCGCCGTTCATGACCATTCTCTGGCAGCACGAT TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGGACGAT TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTATGGCAGCACGAT TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGCACGAT TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGCACGAT TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGCACGAT TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGGACGAT TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTTTGGCAGCACGAT TTCCTCAGAAGGCGCGATCGCCCTTCATGACCATCCTCTGGCAGCACGAT  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  CCCTTCCACAATGAGTGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAATGAGTGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTTCACAATGAGTGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAATGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTTCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  GACCTACCTTGAGC-NN1STNNNNNNNNNNNNNNAGGAGAAGGTTACTCTGCAACTCT GACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAGGTTACTCTGCAACTCT GACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAGGTTACTCTGCAACTCT GACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAGGTTACTCTGCAACTCT CACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAGGTTACCCTGCAACTCT CACCTACCTTGAGG-NNNNNNNNNNNNNNNNNAGGAGAAGGTAAATCTGCAACTCT CACCTACTTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAATGTCACCCTGCAACTCT CACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAAGTTACCCTGCAACTCT CACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAAGTTACCCTGCAACTCT CACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAAGTTACCCTGCAACTCT CACCTACCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAAATTACCCTGCAACTCT TACCTATCTTGAGC-NNNNNNNNNNNNNNNNNAGGAGAAGGTTACTCTGCAACTTT CAACTACCTCGAGCAGTCGAACGCGCAGCGGGAGAAGAAGATTACCCTGCAATACT  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  GGATTCACTATGCTGTCGGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCCTTT GGATTCACTATGCTGTCGGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCCTTT GGATTCACTATGCTGTCGGCCATCTGGGAGTGATTATCCGCGATAACTACATGGCATCGTTT GGATTCACTATGCTGTTCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT GGATTCACTATGCTGTCGGCCATCTGGGAGTGATTACCCGCGACAACTACATGGCATCGTTT GGATTCACTATGCTGTCGGCCATCTGGGGGTGATTACCCGCGACAACTACATGGCATCGTTT GGATTCACTATGCTGTCGGCCATCTGGGAGTGATTCGCCGCGACAACTACATGACATCGTTT GGATTCACTATGCTGTCCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT GGATTCACTATCGTGTCCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT GGATTCACTATGCTGTCCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT GGATTCACTATGCTGTCCGCCATCTGGGGGTGATTATCCGCGACAACTACATGGAATGGATT GGATTCACTATGCTGTCGGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT GGCTTCACTTTGCTGTCCCCGACATGGGAGTGCTTGGTCGCGAAAACTACTTGCCCACGCTT  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  GGCTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC GGTTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAAACTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GTTTGGTGGAACTTGTCCAG-TGCCCAACGGCANTCAGGACTTGGAGCGC GGCTG--GAACCTCGTCCATCTGCCCAACGGCACGCACGACCTCGAGCGC GGCTG--GGACCTCGTCACGATGCCCAACGGCACTATCGACCTTATGCGC  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  ACGGCTCTTCCTTTGGTTCAGCACAATCTCCAGCCCATGAACGGCTTATG ACGGCTCTTCCTTTGGTTCAGCACAATCTCCAGCCCATGAACGGCCTATG ACGGCTCTTCCTTTGGTTCAGCCCAATCTCCAGCCCATGAACGGCTTATG ACGGCTCTTCCTTTGGTTCAGCACAATCTCCAGCCCATGAACGGCTTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTTCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCAGAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGGCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGGCTATG ACCG-TCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCCTTGGTTAGGCACAATCTCCAGCCCATGAACGGCCTATG ATCGCTATGCCTTTGTTTAGAAAGAACCTCCAGCCCATGAACGGCCTATG  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACT CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCATT CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACT CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACT CCTGCTCACTAGGTGCCTCGAGAGCGGATTGCCTCTTCACAATCCTCACC CCTGCTCACTAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC CCTGTTCACTAAGTGCCTCGAGAACGGATTGCCTCTTGCCAATCCTCACC CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC CCTGTTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC CCTGTTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC CCTGCTCACTAAGTGCCTTGAGAGCGGATTACCTCTTGCCAATCCTCACT CCTGTTCACCAAGTGTCAGGAGGGCGGATTGCAAGTCGACAACCAGCACC  NCRASSAXXX CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA N S I T O P H I L A CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA NINTERMEDI CTGTCATCGCCAAGCTTTCAGATCCTAGCTTCGACATGATCTGGTTCAA NTETRASPER CTGTCATCGGCAAGCTTTCAGATCCTAGCTATGACATGATCTGGTTCAA NTERRICOLA CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA NPANNONICA CTGTCATTGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA GTETRASPER CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGATGAA NDODGEIXXX CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA NAFRICANAX CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA NGALAPOGOS CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA N L I N E O L A T A CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA NDISCRETAX CTGTCATCGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA SMACROSPOR TCGTCATTGCCAAGCTTTCAGATCCTAGCTACGACATGATCTGGTTCAA NCRASSAXXX NSITOPHILA NINTERMEDI NTETRAS PER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  CAAGCGTCCTCACCGTCAGCAGGGACACGCCGTTCAAACTGATGAATCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGTTCAAACTGATGGATCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGTCAAACTGATGAATCTG CAAGCGTCCTCACCGTCAGCAGGGACACGTCGGTCAAACTGATGAATCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTGACAATTCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTGACAATTCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTGACAATTCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG CAAGCGTCCTTACAGTCAGCAGAGACAAGTCGGCCAAACTGACGATTCTG CAAGCGCCCTCACTATCAGCAGAGACACGCCGTCCAAGCTGACAGTTCTG  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRAS PER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  AAGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTGCAGAG AAGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTGCAGAG AAGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTACAGAG AGGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTGCCGAG AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG AACTTGGAGTGTCGGCGCTCTTCCCTTGCAATCACGCAGTCGCTGCAGCG AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG AACTCGAAGTGTCGGCGATGTTCCCTCACAATTACGCAGTCGCCGCAGAG AACTCGGTGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTTGCTGCAGAG  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRAS PER NTERRICOLA NPANNONICA GTETRAS PER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  GTAGATGGCATCATCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTAGATGGCATCATCA-NNCTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTAGATGGCATCATCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTAGATGGCATCATCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTAGATGGCATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTAGATGGCATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTAGATGGCATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTAGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA GTCGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA GTAGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA GTAGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA GCAGATGGTATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GCAGATGACGTCGCCACTCTTCAACTCCCTCATTGGATGCAGCAGGGAGA  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRAS PER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACCTTGTTGGAT ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACCTTGTTGGAT ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACATTGTTGGAT ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACCTTGTTGGAT TTTCGGTACCGAGTCCGGATTCTCAGCTCAGTTTGAGACCTTGTTGGAT TTTCGGCACCGAGTCCGGATTCTCAGCTCAGTTTGAGACCTTGTTGGAT TTTCGGCACCGAGTCCGGATTCTCGGCTCAGTTTGAGACCTTGTTGGAT TTTCGGCACCGAGGCCGGATTCTCACCTCAGTTTGAGACCTTGTTGGAT TTTCGGCACCGAGGCCGGATTCTCACCTCAGTTTGAGACCTTGTTGGAT TTTCGGCACCGAGGCCGGATTCTCACCTCAGTTTGAGACCTTGTTGGAT TTTCGGCACCGAGGCCGGATTCTCATCTCAGTTTGAGACCTTGTTGGAT TTTCGGTACTGACCCCGGATACTCAGCTCAATTTGAGACTTTGTTGGAT TTTCGGCACCGAGTCCGGATACTCACCTCAGTTTGAGACCTTGTTGGGT  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPOGOS NLINEOLATA NDISCRETAX SMACROSPOR  TCAATTCTCGAGAATGGACACGCCTCCAGCAATGACCCTTACAACATGGC TCAATTCTCGAGAATGGACACGCCTCCAGCAATGACCCTTACAACATGGC TCAATCCTCGAGAATGGACACGCCTCCAGCAATGACCCTTACAACATGGC TCAATCCTCGAGAATGGACACGCCTCCAGCAATGACCCCTACAACATGGC TCGATCCTTGAGAATGGAAACGCCTCCAGCAATGATCCCTACAACATGGC TCGATCCTTGAGAATGGAAACGCCTCCAACAATGATCCCTACAACATGGC TCGATCCTTGAGAATGGAAACGCCACCAGCAATGATCCCTACAACATGGC TCGATCCTTGAGAATGGAAACGCCTCTAGCAATGAACCCTACAATATGGC TCGATCCTTGAGAATGGAAACGCCTCTATCAATGACCCCTACAATATGGC TCGATCCTTGAGAATGGAAACGCCTCTAGCAATGACCCCTACAATATGGC TCGATCCTTGAGAATGGAGACGCCTCTAGCAATGACCCCTACAATATGGC TCTATTCTTGAGGATGGACACGCCTCCAGCAATGACCCCTACAACATGGC TCCATACTTGAGAATGGAAACGCCACCAGTAATGATTCCTACAACATGGC TCTGGCTATCGATGTTCCCATGATGGGTTAG TCTGGCTATCGATGTTCCCATGATGGGTTAG TCTGGCTATCGATGTTCCCATGATGGGTTAG TCTGGCTATCGATGTTCCCATGATGG-TTAG TCTGGCTATGGATGTTCCCATGATGGGTTAG TCTGGCTATGGATGTTCCCAGGATGGGTTAG TCTGGCTATGGATGTTCCCATGATGGGTTAG TCTGGGTATGGGTGTTCCCATGATGGGTTAG TCTTGGTATGGGTGTTCCCATGATGGGTTAG TCTGGGTATGGGTGTTCCCATGATGGGTTAG TCTGGGTATGGGTATTCCCATGATGGGTTAG TCTGGCTATGGATGTTCCCATGATGGGTTAG TCTGGCTATGGATGTTCCTATGATGGGTTAG  143  2. D N A Alignment of the Neurospora Species and G. tetrasperma used to generate Figure 3.4. The region of high conservation among mating type genes is underlined. NCRASSAXXX ATGTCGGGTGTCGATCAAATCGTCAAGACGTTCGCCGACCTCGCTGAGGA N S I T O P H I L A ATGTCGGGAGTCGATCAAATCGTCAAGACGTTCGCCGACCTCGCTGAGGA NINTERMEDI ATGTCGGGTGTCGATCAAATCGTCAAGACGTTCGCCGACCTCGCTGAGGA NTETRASPER ATGCTGGGTGTCGATCAAATCGTCAAGACGTTCGCCGACCTCGCTGAGGA NTERRICOLA ATGTCGGGCGTCGATCAAATCGTCAAGACGTTCGCCGACCTCGCTGAGGG NPANNONICA ATGTCGGGCGTCGATCAAATCGTCAAGACGTTCGCCGACCTCGCTGAGGG GTETRASPER ATGTCGGGCGTCGATCAAATCGTCAAGACGTTCGCCGACCTCGCTGAGGG NDODGEIXXX NNNNNNNNNNNNNNNNNNN^ NAFRICANAX ATGTCGTGCGTCGATCAAATCGTCAAGACGTTCGCCGACCTCACTGAGGG NGALAPAGOS NNNNNNNNNNNNNNNNNNN^ NLINEOLATA NNNNNNNNNNNNNNNNNNNNNNNNNN^ NDISCRETAX ATGTCGGGCGTCGACCAAATTGTCAAGACGTTCGCCGACCTCGCTGAGGA  50  NCRASSAXXX CGACCGTGAAGCGGCAATGAGAGCTTTCTCAAGGATGATGCGTAGAGGTA 100 NSITOPHILA CGACCGTGAAGCGGCAATGAGAGCTTTCTCAACGATGATGC-NNNNNGTA NINTERMEDI CGACCGTGAAGCGGCAATGAGAGCTTTCTCAACGATGATGC-NNNNNGTA NTETRAS PER CGACCGTGAAGCGGCAATGAGAGCTTTCTCAACGATGATGC-NNNNNGTA NTERRICOLA CGATCGTGAAGCGGCAATGAGAGCTTTCTCAACGATGATGC-NNNNNGCA NPANNONICA CGATCGTGAAGCGGCAATGAGAGCTTTCTCAACGATGATGC-NNNNNGCA GTETRASPER CGATCGTGAAGCGGCAATGAGAGCTTTCTCAACGATGATGC-NNNNNGCA NDODGEIXXX NNNNNNNNNNNNNNNNNNNNN^ NAFRICANAX TGATCGTGAAGCGGCAATGAGAGCTTTCTCAATGATGATGC-NNNNNGCA NGALAPAGOS NNNNNNNNNNNNNNNNNNNNNNNNN^ NLINEOLATA NNNNNNNNNNNNNNNN^ NDISCRETAX CGACCGCGAA-NNGCAATGAGAGCTTTCTCAACGATGATGC-NNNNNGTA NCRASSAXXX CCGAACCTGTTCGCCGAATCCCCGCGGCAAAGAAGAAGGTCAACGGCTTC 15 0 N S I T O P H I L A CCGAACCTGTTCGCCGAATCCCCGCGGCAAAGAAGAAGGTCAACGGCTTC NINTERMEDI CCGAACCTGTTCGCCGAATCCCCGCGGCAAAGAAGAAGGTCAACGGCTTC NTETRAS PER CCGAACCTGTTCGCCGAATCCCCGCGGCAAAGAAGAAGGTCAACGGCTTC NTERRICOLA CCGAACCTGTTCGCCAAACCCCCGCGGCAAAGAAGAAGGTCAACCGCTTC NPANNONICA CCGAACCTGTTCGCCAAACCCCCGCGGTAAAGAAGAAGGTCAACGGCTTC GTETRASPER CCGAACCTGTTCGCCAAAGCCCCGCGGCAAAGAAGAAGGTCAACGGTTTC NDODGEIXXX NNNNNNNTGTTCGCCGAATCCCCGCGGCGAAGAAGAAGGTCAACGGCTTC NAFRICANAX CCGAACCTGTTCGCCAAACCCCCGCGGCAAAGAAGAAGGTCAACGGCTTC NGALAPAGOS NNNNNNNTGTTCGCCGAATCCCCGCGGCAAAGAAGAAGGTCAACGGCTTC NLINEOLATA NNNNNNNTGTTCGCCGAATCCCCGCGGCAAAGAAGAAGGTCAACGGCTTC NDISCRETAX CCGAACCTGTTCGCCAAATCCCCGCGACAAAGAAGAAGGTCAACGGCTTC NCRASSAXXX ATGGGTTTCAGATGTGAGTCAAATCTGAATCAACATTGTCGTT-GATCCA 2 00 N S I T O P H I L A ATGGGTTTCAGATGTGAGTCAAATCTGAATCAACATTGTCGTT-GATCCA NINTERMEDI ATGGGTTTCAGATGTAAGTCAAATCTGAATCAACATTGTCGTT-CATCCA NTETRAS PER ATGGGTTTCAGATGTAAGTCAAATCTGAATCAACATTGTCGTTTGATCCA NTERRICOLA ATGGGTTTCAGATGTAAGTCAAATCTGAATCAATCTTGTCGAC--AATCC NPANNONICA ATGGGTTTCAGATGTAAGTCAAATCTGAATCAATCTTGTCGAC--AATCC GTETRASPER ATGGGTCTCAGATGTAAGTCAAATCTGAATCAATCTTGTCGAC--AATCC NDODGEIXXX ATGAGTTTCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA--AATCC NAFRICANAX ATGAGTTTCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA--AATCC NGALAPAGOS ATGAGTATCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA--AATCC NLINEOLATA ATGAGTTTCAGATGTAAGTCAAATCTGGATCAATCTTGTTGAA--AATCC NDISCRETAX ATGGGTTTCAGATGTAAGTTAAATCTGAATCAATCTTGTCGAT--AATCC  144  NCRASSAXXX TGGCTGATTGCTCTTCATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC 250 N S I T O P H I L A TGGCTGATTGCTCTTCATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NINTERMEDI TGGCTAATTGCTCTTCAATTTAGCGTACTATTCCCCGCTCTTCTCTCAGC NTETRASPER TGGCTGATTGCTCTTCATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NTERRICOLA ATGCTGATTGCTTTTTATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NPANNONICA ATGCTGATTGCCTTTTATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC GTETRASPER A T G C T A A T T G C T T T T T A T T T C A G C G T A C T A T T C C C C G C T C T T C T C T C A G C NDODGEIXXX A T T C T A A T T G C T T T T T A T T T C A G C G T A C T A T T C C C C G C T C T T C T C T C A G C NAFRICANAX ATTCTAATTGCCTTTTATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NGALAPAGOS ATTCTAATTGCCTTTTATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NLINEOLATA ATTCTAATGGCTTTTTATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGN NDISCRETAX ATGCTGACTGCTCTTCATTTCAGCGTACTATTCCCCGCTCTTCTCTCAGC NCRASSAXXX TCCCGCAAAAGGAGAGATCGCCCTTCATGACTATTCTCTGGCAGCATGAT 3 00 NSITOPHILA TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGCATGAT NINTERMEDI TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGCATGAT NTETRASPER TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGCATGAT NTERRICOLA TCCCGCAAAAGGAGAGATCGCCGTTCATGACCATTCTCTGGCAGCACGAT NPANNONICA TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTCTGGCAGGACGAT GTETRASPER TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTATGGCAGCACGAT NDODGEIXXX TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGCACGAT NAFRICANAX TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGCACGAT NGALAPAGOS TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGCACGAT NLINEOLATA TCCCGCAGAAGGAGAGATCACCCTTCATGACCATTCTCTGGCAGGACGAT NDISCRETAX TCCCGCAAAAGGAGAGATCGCCCTTCATGACCATTCTTTGGCAGCACGAT NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPAGOS NLINEOLATA NDISCRETAX  CCCTTCCACAATGAGTGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG 3 50 CCCTTCCACAATGAGTGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTTCACAATGAGTGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAATGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGAATTTCATGTGCTCGGTGTATTCGTCGATCCG CCCTTCCACAACGAATGGGATTTCATGTGCTCGGTGTATTCGTCAATCCG  NCRASSAXXX GACCTACCTTGAGCAGGAGAAGGTTACTCTGCAACTCTGGATTCACTATG 400 N S I T O P H I L A GACCTACCTTGAGCAGGAGAAGGTTACTCTGCAACTCTGGATTCACTATG NINTERMEDI GACCTACCTTGAGCAGGAGAAGGTTACTCTGCAACTCTGGATTCACTATG NTETRAS PER GACCTACCTTGAGCAGGAGAAGGTTACTCTGCAACTCTGGATTCACTATG NTERRICOLA CACCTACCTTGAGCAGGAGAAGGTTACCCTGCAACTCTGGATTCACTATG NPANNONICA CACCTACCTTGAGGAGGAGAAGGTAAATCTGCAACTCTGGATTCACTATG GTETRASPER CACCTACTTTGAGCAGGAGAATGTCACCCTGCAACTCTGGATTCACTATG NDODGEIXXX CACCTACCTTGAGCAGGAGAAAGTTACCCTGCAACTCTGGATTCACTATG NAFRICANAX CACCTACCTTGAGCAGGAGAAAGTTACCCTGCAACTCTGGATTCACTATC NGALAPAGOS CACCTACCTTGAGCAGGAGAAAGTTACCCTGCAACTCTGGATTCACTATG NLINEOLATA CACCTACCTTGAGCAGGAGAAAATTACCCTGCAACTCTGGATTCACTATG NDISCRETAX TACCTATCTTGAGCAGGAGAAGGTTACTCTGCAACTTTGGATTCACTATG NCRASSAXXX CTGTCGGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCCTTT 4 50 N S I T O P H I L A CTGTCGGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCCTTT NINTERMEDI CTGTCGGCCATCTGGGAGTGATTATCCGCGATAACTACATGGCATCGTTT NTETRAS PER CTGTTCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT NTERRICOLA CTGTCGGCCATCTGGGAGTGATTACCCGCGACAACTACATGGCATCGTTT NPANNONICA CTGTCGGCCATCTGGGGGTGATTACCCGCGACAACTACATGGCATCGTTT GTETRASPER CTGTCGGCCATCTGGGAGTGATTCGCCGCGACAACTACATGACATCGTTT NDODGEIXXX CTGTCCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT NAFRICANAX GTGTCCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT NGALAPAGOS CTGTCCGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT NLINEOLATA CTGTCCGCCATCTGGGGGTGATTATCCGCGACAACTACATGGAATGGATT NDISCRETAX CTGTCGGCCATCTGGGAGTGATTATCCGCGACAACTACATGGCATCGTTT  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPAGOS NLINEOLATA NDISCRETAX  GGCTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC 500 GGCTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC GGTTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCGTTTTCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG—GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAAACTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GGCTG--GAACCTCGTCCAGCTGCCCAACGGCACTCACGACCTCGAGCGC GTTTGGTGGAACTTGTCCAG-TGCCCAACGGCANTCAGGACTTGGAGCGC GGCTG--GAACCTCGTCCATCTGCCCAACGGCACGCACGACCTCGAGCGC  NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPAGOS NLINEOLATA NDISCRETAX  ACGGCTCTTCCTTTGGTTCAGCACAATCTCCAGCCCATGAACGGCTTATG 5 50 ACGGCTCTTCCTTTGGTTCAGCACAATCTCCAGCCCATGAACGGCCTATG ACGGCTCTTCCTTTGGTTCAGCCCAATCTCCAGCCCATGAACGGCTTATG ACGGCTCTTCCTTTGGTTCAGCACAATCTCCAGCCCATGAACGGCTTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTTCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCAGAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGGCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGGCTATG ACCG-TCTTCCTTTGGTTCAGCATAACCTCCAGCCCATGAACGGCCTATG ACCGCTCTTCCCTTGGTTAGGCACAATCTCCAGCCCATGAACGGCCTATG  NCRASSAXXX CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACT 6 00 N S I T O P H I L A CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCATT NINTERMEDI CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACT NTETRASPER CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACT NTERRICOLA CCTGCTCACTAGGTGCCTCGAGAGCGGATTGCCTCTTCACAATCCTCACC NPANNONICA CCTGCTCACTAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC , GTETRASPER CCTGTTCACTAAGTGCCTCGAGAACGGATTGCCTCTTGCCAATCCTCACC ' NDODGEIXXX CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC NAFRICANAX CCTGTTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC NGALAPAGOS CCTGTTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC NLINEOLATA CCTGCTCACCAAGTGCCTCGAGAGCGGATTGCCTCTTGCCAATCCTCACC NDISCRETAX CCTGCTCACTAAGTGCCTTGAGAGCGGATTACCTCTTGCCAATCCTCACT NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPAGOS NLINEOLATA NDISCRETAX  CTGTCATCGCCAAGCTTTC - AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC- AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC- AGATCCTAGCTTCGACATGATCTGGTTCAA CTGTCATCGGCAAGCTTTC- AGATCCTAGCTATGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC- AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATTGCCAAGCTTTC- AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC- AGATCCTAGCTACGACATGATCTGGATGAA CTGTCATCGCCAAGCTTTC- AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC • AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC• AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC • AGATCCTAGCTACGACATGATCTGGTTCAA CTGTCATCGCCAAGCTTTC •AGATCCTAGCTACGACATGATCTGGTTCAA  6 50  NCRASSAXXX CAAGCGTCCTCACCGTCAGCAGGGACACGCCGTTCAAACTGATGAATCTG 700 NSITOPHILA CAAGCGTCCTCACCGTCAGCAGGGACACGCCGTTCAAACTGATGGATCTG NINTERMEDI CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGTCAAACTGATGAATCTG NTETRASPER CAAGCGTCCTCACCGTCAGCAGGGACACGTCGGTCAAACTGATGAATCTG NTERRICOLA CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTGACAATTCTG NPANNONICA CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTGACAATTCTG GTETRASPER CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTGACAATTCTG NDODGEIXXX CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG NAFRICANAX CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG NGALAPAGOS CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG NLINEOLATA CAAGCGTCCTCACCGTCAGCAGGGACACGCCGGCCAAACTTACAATTCTG NDISCRETAX CAAGCGTCCTTACAGTCAGCAGAGACAAGTCGGCCAAACTGACGATTCTG  NCRASSAXXX AAGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTGCAGAG 750 NSITOPHILA AAGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTGCAGAG NINTERMEDI AAGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTACAGAG NTETRAS PER AGGTTGGAGTTTCGGCGATGTTCCCTCGCAATCACACGGTCGCTGCCGAG NTERRICOLA AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG NPANNONICA AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG GTETRASPER AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG NDODGEIXXX AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG NAFRICANAX AACTTGGAGTGTCGGCGCTCTTCCCTTGCAATCACGCAGTCGCTGCAGCG NGALAPAGOS AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG NLINEOLATA AACTTGGAGTGTCGGCGCTCTTCCCTCGCAATCACGCAGTCGCTGCAGAG NDISCRETAX AACTCGAAGTGTCGGCGATGTTCCCTCACAATTACGCAGTCGCCGCAGAG NCRASSAXXX GTAGATGGCATCATCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA 800 N S I T O P H I L A GTAGATGGCATCATCA-NNCTCCTCTCTCCCATTGGATTCAGCAGGGAGA NINTERMEDI GTAGATGGCATCATCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA NTETRAS PER GTAGATGGCATCATCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA NTERRICOLA GTAGATGGCATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA NPANNONICA GTAGATGGCATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA GTETRASPER GTAGATGGCATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA NDODGEIXXX GTAGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA NAFRICANAX GTCGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA NGALAPAGOS GTAGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA NLINEOLATA GTAGATGGCATCACCGACCTTCCTCTCTCCCATTGGCTTCAGCAGGGAGA NDISCRETAX GCAGATGGTATCGCCAATCTTCCTCTCTCCCATTGGATTCAGCAGGGAGA NCRASSAXXX ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACCTTGTTGGAT 8 50 N S I T O P H I L A ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACCTTGTTGGAT NINTERMEDI ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACATTGTTGGAT NTETRAS PER ATTCGGTACCGAGTCTGGATACTCAGCTCAGTTTGAGACCTTGTTGGAT NTERRICOLA TTTCGGTACCGAGTCCGGATTCTCAGCTCAGTTTGAGACCTTGTTGGAT NPANNONICA TTTCGGCACCGAGTCCGGATTCTCAGCTCAGTTTGAGACCTTGTTGGAT . GTETRASPER TTTCGGCACCGAGTCCGGATTCTCGGCTCAGTTTGAGACCTTGTTGGAT NDODGEIXXX TTTCGGCACCGAGGCCGGATTCTCACCTCAGTTTGAGACCTTGTTGGAT NAFRICANAX TTTCGGCACCGAGGCCGGATTCTCACCTCAGTTTGAGACCTTGTTGGAT NGALAPAGOS TTTCGGCACCGAGGCCGGATTCTCACCTCAGTTTGAGACCTTGTTGGAT NLINEOLATA TTTCGGCACCGAGGCCGGATTCTCATCTCAGTTTGAGACCTTGTTGGAT NDISCRETAX TTTCGGTACTGACCCCGGATACTCAGCTCAATTTGAGACTTTGTTGGAT NCRASSAXXX TCAATTCTCGAGAATGGACACGCCTCCAGCAATGACCCTTACAACATGGC 90 0 N S I T O P H I L A TCAATTCTCGAGAATGGACACGCCTCCAGCAATGACCCTTACAACATGGC NINTERMEDI TCAATCCTCGAGAATGGACACGCCTCCAGCAATGACCCTTACAACATGGC NTETRASPER TCAATCCTCGAGAATGGACACGCCTCCAGCAATGACCCCTACAACATGGC NTERRICOLA TCGATCCTTGAGAATGGAAACGCCTCCAGCAATGATCCCTACAACATGGC NPANNONICA TCGATCCTTGAGAATGGAAACGCCTCCAACAATGATCCCTACAACATGGC GTETRASPER TCGATCCTTGAGAATGGAAACGCCACCAGCAATGATCCCTACAACATGGC NDODGEIXXX TCGATCCTTGAGAATGGAAACGCCTCTAGCAATGAACCCTACAATATGGC NAFRICANAX TCGATCCTTGAGAATGGAAACGCCTCTATCAATGACCCCTACAATATGGC NGALAPAGOS TCGATCCTTGAGAATGGAAACGCCTCTAGCAATGACCCCTACAATATGGC NLINEOLATA TCGATCCTTGAGAATGGAGACGCCTCTAGCAATGACCCCTACAATATGGC NDISCRETAX TCTATTCTTGAGGATGGACACGCCTCCAGCAATGACCCCTACAACATGGC NCRASSAXXX NSITOPHILA NINTERMEDI NTETRASPER NTERRICOLA NPANNONICA GTETRASPER NDODGEIXXX NAFRICANAX NGALAPAGOS NLINEOLATA NDISCRETAX  TCTGGCTATCGATGTTCCCATGATGGGTTAG 931 TCTGGCTATCGATGTTCCCATGATGGGTTAG TCTGGCTATCGATGTTCCCATGATGGGTTAG TCTGGCTATCGATGTTCCCATGATGG-TTAG TCTGGCTATGGATGTTCCCATGATGGGTTAG TCTGGCTATGGATGTTCCCAGGATGGGTTAG TCTGGCTATGGATGTTCCCATGATGGGTTAG TCTGGGTATGGGTGTTCCCATGATGGGTTAG TCTTGGTATGGGTGTTCCCATGATGGGTTAG TCTGGGTATGGGTGTTCCCATGATGGGTTAG TCTGGGTATGGGTATTCCCATGATGGGTTAG TCTGGCTATGGATGTTCCCATGATGGGTTAG  147  3. Amino Acid Alignment of the Neurospora Species and G. tetrasperma Region of high amino acid conservation among mating types is underlined. Npannonica Gtetrasper Nterricola Nintermedi Ntetrasper Ncrassaxxx Nsitophila Nafricanax Ndodgeixxx Ngalapagos Ndiscretax Nlineolata  MSGVDQIVKTFADLAEGDREAAMRAFSTMMR--TEPVRQTPAVKKKVNGF MSGVDQIVKTFADLAEGDREAAMRAFSTMMR--TEPVRQS PAAKKKVNGF MSGVDQIVKTFADLAEGDREAAMRAFSTMMR--TEPVRQTPAAKKKVNRF MSGVDQIVKTFADLAEDDREAAMRAFSTMMR--TEPVRRIPAAKKKVNGF MLGVDQIVKTFADLAEDDREAAMRAFSTMMR--TE PVRRIPAAKKKVNGF MS GVDQIVKTFADLAEDDREAAMRAFSRMMRRGTE PVRRIPAAKKKVNGF MS GVDQIVKTFADLAEDDREAAMRAFSTMMR- -TE PVRRIPAAKKKVNGF MSCVDQIVKTFADLTEGDREAAMRAFSMMMR--TEPVRQTPAAKKKVNGF -????????????????????????????????????????????????? -????????????????????????????????????????????????? MSGVDQIVKTFADLAEDDRE-AMRAFSTMMR--TEPVRQIPATKKKVNGF -?????????????????????????????????????????????????  50  N p a n n o n i c a MGFRSYYSPLFSQLPQKERSPFMTILWQDDPFHNEWDFMCSVYSSIRTYL 100 Gtetrasper MGLRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYF Nterricola MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL N i n t e r m e d i MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL Ntetrasper MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL Ncrassaxxx MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL Nsitophila MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL N a f r i c a n a x MSFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWNFMCSVYSSIRTYL N d o d g e i x x x MSFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWNFMCSVYSSIRTYL Ngalapagos MSIRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWNFMCSVYSSIRTYL Ndiscretax MGFRSYYSPLFSQLPQKERSPFMTILWQHDPFHNEWDFMCSVYSSIRTYL Nlineolata MSFRSYYSPLFSQXPQKERSPFMTILWQDDPFHNEWNFMCSVYSSIRTYL N p a n n o n i c a EEEKVNLQLWIHYAVGHLGVITRDNYMASFGWNLVQLPNGTHDLERTALP 150 Gtetrasper EQENVTLQLWIHYAVGHLGVIRRDNYMTSFGWNLVQLPNGTHDLERTALP Nterricola EQEKVTLQLWIHYAVGHLGVITRDNYMASFGWNLVQLPNGTHDLERTALP Nintermedi EQEKVTLQLWIHYAVGHLGVIIRDNYMASFGWNLVRFPNGTHDLERTALP Ntetrasper EQEKVTLQLWIHYAVRHLGVIIRDNYMASFGWNLVRFPNGTHDLERTALP Ncrassaxxx EQEKVTLQLWIHYAVGHLGVIIRDNYMASFGWNLVRFPNGTHDLERTALP Nsitophila EQEKVTLQLWIHYAVGHLGVIIRDNYMASFGWNLVRFPNGTHDLERTALP Nafricanax EQEKVTLQLWIHYRVRHLGVIIRDNYMASFGWNLVQLPNGTHDLERTALP Ndodgeixxx EQEKVTLQLWIHYAVRHLGVIIRDNYMASFGWKLVQLPNGTHDLERTALP Ngalapagos EQEKVTLQLWIHYAVRHLGVIIRDNYMASFGWNLVQLPNGTHDLERTALP Ndiscretax EQEKVTLQLWIHYAVGHLGVIIRDNYMASFGWNLVHLPNGTHDLERTALP Nlineolata EQEKITLQLWIHYAVRHLGVIIRDNYMEWIVWWNLSSAQRXSGLGAHRLP N p a n n o n i c a LVQHNLQPMNGLCLLTKCLESGLPLANPHPVIAKLSDPSYDMIWFNKRPH 2 00 Gtetrasper LVQQNLQPMNGLCLFTKCLENGLPLANPHPVIAKLSDPSYDMIWMNKRPH N t e r r i c o l a LVQHNLQPMNGLCLLTRCLESGLPLHNPHPVIAKLSDPSYDMIWFNKRPH N i n t e r m e d i LVQPNLQPMNGLCLLTKCLESGLPLANPHSVIAKLSDPSFDMIWFNKRPH N t e t r a s p e r LVQHNLQPMNGLCLLTKCLESGLPLANPHSVIGKLSDPSYDMIWFNKRPH N c r a s s a x x x LVQHNLQPMNGLCLLTKCLESGLPLANPHSVIAKLSDPSYDMIWFNKRPH Nsitophila LVQHNLQPMNGLCLLTKCLESGLPLANPHSVIAKLSDPSYDMIWFNKRPH N a f r i c a n a x LVQHNLQPMNGLCLFTKCLESGLPLANPHPVIAKLSDPSYDMIWFNKRPH N d o d g e i x x x LVQHNLQPMNGLCLLTKCLESGLPLANPHPVIAKLSDPSYDMIWFNKRPH Ngalapagos LVQHNLQPMNGLCLFTKCLESGLPLANPHPVIAKLSdpsydmiwfnkrph N d i s c r e t a x LVRHNLQPMNGLCLLTKCLESGLPLANPHSVIAKLSDPSYDMIWFNKRPY N l i n e o l a t a LVQHNLQPMNGLCLLTKCLESGLPLANPHPVIAKLSDPSYDMIWFNKRPH  N p a n n o n i c a RQQGHAGQTDNSELGVSALFPRNHAVAAEVDGIANLPLSHWIQQGDFGTE 25 Gtetrasper RQQGHAGQTDNSELGVSALFPRNHAVAAEVDGIANLPLSHWIQQGDFGTE Nterricola RQQGHAGQTDNSELGVSALFPRNHAVAAEVDGIANLPLSHWIQQGDFGTE N i n t e r m e d i RQQGHAGQTDESEVGVSAMFPRNHTVATEVDGIINLPLSHWIQQGEFGTE N t e t r a s p e r RQQGHVGQTDESEVGVSAMFPRNHTVAAEVDGIINLPLSHWIQQGEFGTE N c r a s s a x x x RQQGHAVQTDESEVGVSAMFPRNHTVAAEVDGIINLPLSHWIQQGEFGTE Nsitophila RQQGHAVQTDGSEVGVSAMFPRNHTVAAEVDGIIT-PLSHWIQQGEFGTE Nafricanax RQQGHAGQTYNSELGVSALFPCNHAVAAAVDGITDLPLSHWLQQGDFGTE Ndodgeixxx RQQGHAGQTYNSELGVSALFPRNHAVAAEVDGITDLPLSHWLQQGDFGTE Ngalapagos RQQGHAGQTYNSELGVSALFPRNHAVAAEVDGITDLPLSHWLQQGDFGTE Ndiscretax SQQRQVGQTDDSELEVSAMFPHNYAVAAEADGIANLPLSHWIQQGDFGTD Nlineolata RQQGHAGQTYNSELGVSALFPRNHAVAAEVDGITDLPLSHWLQQGDFGTE N p a n n o n i c a SGFSAQFETLLDSILENGNASNNDPYNMALAMDVPRMG* 289 Gtetrasper SGFSAQFETLLDSILENGNATSNDPYNMALAMDVPMMG* Nterricola SGFSAQFETLLDSILENGNASSNDPYNMALAMDVPMMG* N i n t e r m e d i SGYSAQFETLLDSILENGHASSNDPYNMALAIDVPMMG* N t e t r a s p e r SGYSAQFETLLDSILENGHASSNDPYNMALAIDVPMMVN c r a s s a x x x SGYSAQFETLLDSILENGHASSNDPYNMALAIDVPMMG* N s i t o p h i l a SGYSAQFETLLDSILENGHASSNDPYNMALAIDVPMMG* N a f r i c a n a x AGFSPQFETLLDSILENGNASINDPYNMALGMGVPMMG* N d o d g e i x x x AGFS PQFETLLDSILENGNAS SNEPYNMALGMGVPMMG * N g a 1 a p a g o s AGFS PQFETLLDSILENGNAS SNDPYNMALGMGVPMMG* Ndiscretax PGYSAQFETLLDSILEDGHASSNDPYNMALAMDVPMMG* N l i n e o l a t a AGFSSQFETLLDSILENGDASSNDPYNMALGMGIPMMG*  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0099355/manifest

Comment

Related Items