Posters II: Sexual and Asexual Reproduction

Light is an important environmental stimulus that induces conidiation in Aspergillus nidulans. The ability to respond to light is dependent on the allelic state of the veA (velvet) gene. Wild-type strains require light for asexual development, whereas strains bearing the veA1 mutation conidiate regardless of the presence or absence of light. Previous genetic analysis suggested that veA is a negative regulator of early development. We have cloned the veA gene by mutant complementation, and have performed genetic tests and CHEF gel electrophoresis to show that we have correctly isolated this gene. The veA gene encodes a 2.5 kb mRNA that is present throughout development and in uninduced cultures. This mRNA is also present in the veA1 mutant. We have used the genomic veA gene fragment as a probe to isolate a cDNA clone from a lambda Zap Express library. The DNA sequence of this cDNA was presented and the possible role that veA performs during early conidiation discussed.

2. Genetic and molecular analysis of a suppresssor of fluG mutations in Aspergillus nidulans

Mutations in the fluG gene disrupt the ordered sequence of events that result in the formation of conidia (asexual spores) in Aspergillus nidulans. Lee and Adams (1994, Genes Dev. 8:641-651) have proposed that FluG functions as an enzyme for the production of an extracellular signal that controls the initiation of sporulation. Although the phenotype of all fluG mutants is the proliferation of undifferentiated masses of vegetative mycelia, several alleles show additional phenotypes. One mutant disrupts the ability of colonies to conidiate in red light, whereas three others suppress the veA1 mutation. (Red light is required for conidiation for wildtype colonies and the mutation allows conidiation to occur in the absence of light.) We have isolated a mutant, designated suA1fluG, that suppresses all fluG mutations. The suA1fluG mutant is recessive to its wild-type allele and maps on chromosome II. We have cloned this gene by mutant complementation and have isolated a cDNA clone, which is presently being sequenced. The gene encodes a 2.5 kb mRNA that is present both in uninduced and induced cultures. Since the suppressor appears to bypass FluG functions, its possible role in controlling those events involved in the intiation of sporulation was discussed.

3. The acoC gene is necessary for developmental competence and vegetative growth in Aspergillus nidulans

Conidiation in Aspergillus nidulans is normally suppressed in submerged culture, but occurs readily if colonies are exposed to an air interface. The transfer of colonies from liquid to solid medium, termed induction, initiates and synchronizes conidiation. However, induction can only initiate conidiation if colonies have attained a certain change in state, called developmental competence. Butnick et al. (1984, J. Bact. 160:533-540) describe the isolation and characterization of three temperature sensitive mutants that are blocked in the acquisition of competence. One of these mutants, acoC193, is not only defective in attaining competence, but displays a lethal phenotype if colonies are exposed to light at the restrictive temperature. Combined temperature/ light-dark shift experiments indicate that the light lethal phenotype persists after the acquisition of competence, suggesting that acoC is involved in a growth maintenance function. Although conidiation in wild-type colonies is induced by exposure to red light, preliminary spectral analysis shows that red wavelengths are not responsible for the lethal phenotype. We have cloned acoC and show that it encodes a 1.9 kb mRNA that is present in uninduced and induced colonies. DNA sequence analysis is currently being performed on genomic and cDNA isolates. Our results suggest that acoC is involved in early functions associated with the acquisition of competence and in other activities required for vegetative growth.

4. Isolation and characterisation of the B mating type genes of Coprinus cinereus

A successful, mating in Coprinus cinereus leads to the development of a fertile dikaryon with binucleate cells and characteristic clamp connections. The B mating type genes regulate specific steps in the developmental programme that establishes the dikaryon (nuclear migration) and also in its maintenance (clamp cell fusion). We describe a genomic subtraction technique that enabled us to isolate the B6 mating type locus. The genes that confer B6 mating type specificity reside within a 10 kb DNA sequence that fails to cross-hybridise to genomic DNAs from several monokaryons that have different versions of the B locus (e.g. B1, B3, B5, B42). This suggests that different alleles of the B genes have little sequence similarity. By taking common flanking sequences we have isolated a second B locus, B42. We have identified a number of genes within the B6 and B42 specific DNAs and have followed the regulation of their transcripts through different stages of development. We will report on the DNA sequences which allow us to predict the likely functions of some of thc genes and relate this to their regulation.

5. A system of balanced lethals controls the segregation of mating type loci in the oomycete, Phytophthora infestans

The genetic basis of mating type is being studied in the oomycete, Phytophthora infestans. Previous studies have failed to establish the number of loci that determine the two mating types, A1 and A2, and how alleles of these loci interact; this latter point is important since oomycetes are diploid and therefore potentially heterozygous at the mating type loci. To characterize these loci in P. infestans, thirteen markers linked to the A1 and A2 phenotypes were identified using RAPD (random amplified polymorphic DNA) markers. Fifty alleles of the loci were subsequently scored in four crosses (360 meioses) as RAPDs or as other PCR or RFLP markers. Genetic and physical mapping indicated that all loci linked to the A1 and A2 determinants resided in a single region, consistent with the existence of a single mating type locus. A curious 300 kb region of structural hybridity between the chromosomes, tightly linked to the mating type locus, was detected; its function is unclear but it may account for the distorted segregation of A1 and A2 type described in many earlier studies. Strikingly, a non-Mendelian pattern of segregation was observed for markers in the mating type region as only two of the four expected genotypes were detected in viable progeny. This pattern is equivalent to that caused by balanced lethal loci. By restricting progeny to only two genotypes, this apparently enables the unambiguous determination of the two mating types in the heterothallic oomycetes.

6. Control of sexual morphogenesis in Pyrenopeziza brassicae: cause of light leaf spot in brassicas

Pyrenopeziza brassicae is a heterothallic ascomycete and is the cause of light leaf spot disease of brassicas. We are studying sexual morphogenesis using both biochemical and molecular approaches. Biochemical analysis has lead to the partial purification of a lipoidal sex factor, produced by mated cultures but not by single isolates of the fungus. The sex factor (SF) inhibits asexual sporulation and stimulates ascocarp development and therefore functions as a developmental signal. Molecular analysis has focused on cloning of the mating type loci and the analysis of protein profile changes in single mating type isolates in response to sex factor. Heterologous probing using the N. crassa A mating type probe has successfully identified the MAT 1-2 locus from P. brassicae. Protein profile analysis revealed a major protein present in SF induced single isolate cultures and fertile ascocarps which was not in untreated single isolates. The protein (sex factor induced; SFI) has been purified by hydrophobic interaction FPLC. Primers were generated by reverse translation of internal amino acid sequence and used in PCR reactions against genomic DNA. A single unique PCR product was obtained and used to probe a genomic library identifying three putative positive clones. Recent progress in analysis of sexual morphogenesis in P. brassicae was reported.

7. Isolates of Glomerella cingulata mate on a non-host plant

Glomerella cingulata (teleomorph of Colletotrichum gloeosporioides) is an agriculturally important fungus pathogenic on many plant species. Self- sterile, sexually compatible isolates of G. cingulata (from pecan) were crossed on northern jointvetch (NJV) plants under simulated natural conditions. Stems of 1-2 month old northern jointvetch (Fabaceae) plants were inoculated with one of two G. cingulata isolates from pecan (Juglandaceae), placed in growth chambers, and after one week the same sites were inoculated with the other sexually compatible isolate. After incubating the plants for one more week in growth chambers the co-inoculated NJV stems were excised and placed in Petri dishes containing either a minimal agar or moistened sterile filter paper and incubated at room temperature with a 12 hour photoperiod for 3 weeks. As controls, NJV plants were also inoculated with only one of the G. cingulata isolates. G. cingulata perithecia with ascospores were observed on 75% of the stems inoculated with both isolates. No perithecia were observed on NJV stems inoculated with only one of the G. cingulata isolates from pecan. Twenty-eight single ascospore progeny were randomly isolated from these crosses and compared to 26 progeny isolated from crosses between the same two isolates of G. cingulata on agar medium. Progeny were examined for evidence of sexual recombination using mating type and several RFLPs as markers. All of the progeny were hybrids of the two parental isolates. The implications of completion of the sexual cycle by a fungal plant pathogen on a non-host was discussed.

8. Expression of three genes of the Neurospora crassa A idiomorph in the vegetative and sexual phases

mt A-1 is a 1.2 kb gene contained in the A idiomorph of N. crassa which confers mating identity and heterokaryon incompatibility. Mutational analyses have shown that the remaining 4 kb of the A idiomorph is involved in the formation of perithecia and ascospore production. We focused our research on the characterization of any genes present within the 4 kb region and on the expression of the mating-type genes during the life cycle of N. crassa. Using RT-PCR two cDNAs were amplified and sequenced. mt A-2 is a 1147 bp long gene and contains three introns. It codes for a putative protein of 326 amino acids which has a very acidic C-terminus tail, a common characteristic of transcription activators. mt A-3 is a 1103 bp long gene and also contains three introns. The mt A-3 ORF encodes a protein of 324 amino acids which contains an HMG domain, acidic N and C tails and a proline rich segment in the C-terminus end. These features suggest that mtA-3 may also be a transcriptional activator. The mt A-2 and mt A-3 gene products have similarity to various mating-type gene products including the P. anserina SMR1 and SMR2, respectively. To investigate if expression of the mating-type genes are restricted to one phase of the N. crassa life cycle, transcriptional analyses of mt A-1, mt A-2 and mt A-3 were done using RT-PCR and Northern hybridization. All three genes are expressed during both the vegetative and sexual phases of the N. crassa life cycle. These results suggest that the mating-type products of mt A-1, mt A-2 and mt A-3 are regulated at the post- transcriptional level. Studies are in progress in order to determine the roles of mt A-2 and mt A-3 during the vegetative and sexual cycle of N. crassa.

9. Loss of meiosis in Aspergillus

The genus Aspergillus has a remarkable diversity of propagative strategies, including strictly mitotic, meiotic, homothallic, and heterothallic species. Many species not known to produce ascospores retain characters that are often correlated with ascospore production, including the production of Hulle cells, and the production of sclerotia and sclerotium-like structures. We inferred patterns of evolution in the genus Aspergillus using DNA sequences from the internal transcribed spacers (ITSs) of the nuclear ribosomal repeat, the mitochondrial small ribosomal subunit (mtssu), and the 5 non-translated (5 NT) region of the trpC gene. The trpC 5 NTS was highly variable in comparison to both the ITSs and the mtssu. The overall phylogenetic patterns suggest as least four independent losses of ascospore production in the genus, leading to taxa that do not produce ascospores in culture, but retain apparent vestiges of ascospore production. These findings are consistent with a model where strictly mitotic taxa arise frequently in Aspergillus, but do not form stable long-term evolutionary lineages.

10. Control of DNA replication and mitotic checkpoint function by the Aspergillus nidulans nimO gene

We are investigating the role of the Aspergillus nimO gene in DNA replication and mitotic checkpoint function. Flow cytometric and cytological analyses showed that when conidia of a temperature sensitive lethal nimO18 mutant were germinated at the restrictive temperature (44 C), they were unable to replicate their DNA, but 35-40% of nuclei nevertheless progressed into mitosis and arrested with condensed chromatin. These results identify nimO as a candidate regulator of G1/S or S phase, and further suggest that this gene influences the checkpoint that prevents mitosis (M) from occurring until DNA synthesis has been completed. Sequencing of a nimO cDNA revealed a predicted protein of 72.8 kd that shares 29% identity with DBF4, a budding yeast G1/S regulator which associates with origins of replication and with the CDC7 kinase in order to initiate DNA synthesis. nimO and DBF4 are especially strongly conserved in a C-terminal domain containing a single, novel motif which bears strong resemblance to Cys2-His2 zinc fingers. A deletion of this 60 bp motif from a nimO gene fused with the inducible alcA gene promoter rescued the ts-lethality of nimO18 for growth, but not conidiation, when high-level expression was induced by ethanol. On glycerol, which permits low basal expression, the strains grew poorly and were likewise unable to conidiate. Thus, the putative zinc finger appears non-essential for mycelial growth when the variant gene is highly expressed, but may be necessary for the accelerated cell cycling that must occur during conidiation. We are continuing to examine the function of this motif and to test nimO function through gene disruption.

11. Construction and characterization of brlA(p):alcA gene fusions in Aspergillus nidulans

In order to study the temporal and developmental regulation of the bristle A gene in Aspergillus nidulans we have constructed several gene fusions to the bristle A promotor. We have decided to use the expression of alcA as a negative selection marker allowing aconidial mutants containing this construct to survive on media containing allyl alcohol. This approach can be used to identify genes which code for bristle promotor specific trans acting transcriptional factors. In addition we have constructed promotor fusion to the argB gene and to lacZ. BrlA(p)::argB gene fusions in argB null strains will allow positive selection of bristle regulators. We are using the brlA(p)::lacz fusion as a reporter of normal developmental expression of the brlA gene. All of these constructs contain the 4 kb of the A. nidulans genome containing the brlA promotor sequences. The fusions have been constructed using PCR amplification of gene and promotor segments and overlapping PCR to construct the gene fusions so that the initiation codon of the reporter/selectable marker exactly replaces the coding sequence of the brlA structural gene. Results of these experiments and expression of the gene fusions in transgenic strains of A. nidulans were presented.

12. Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis

In the plant pathogenic fungus Ustilago maydis sexual and pathogenic development are controlled by the multiallelic b mating type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW. Only combinations of bE and bW of different allelic origin are active. Allelic differences are clustered in the N- terminal domains of bE and bW. To investigate the underlying molecular mechanism for this intracellular self-nonself recognition phenomenon, the Saccharomyces cerevisiae two hybrid system was used. The results demonstrate that the bE and bW polypeptides dimerize only if they are derived from different alleles. Dimerization occurs just through the N-terminal variable domains and does not involve the homeodomain motifs. The non- self dependent dimerization of bE and bW was confirmed with a biochemical interaction assay. Different point mutants of bE2 were isolated that lead to function in combination with bW2 in Ustilago maydis. Such combinations of polypeptides were also able to form heterodimers in the two hybrid system. We suggest a model for self-nonself recognition in which variable cohesive contacts direct dimerization.

13. Molecular characterization of the A1 mating type factor of Coprinus bilanatus

The A factor of Coprinus cinereus is one of two multiallelic mating type loci which determine compatibility between monokaryons and regulate the development of a dikaryon. The A factor consists of two subloci, a and b, each of which contain two classes of genes encoding homeodomain transcription factors, HD1 and HD2. The products of compatible A factors are brought together by mating. The active interactions are formed between compatible HD1 and HD2 protein pairs; combinations from the same locus are nonfunctional (Kües et al., MGG 245:45, 1994). Heterologous expression experiments gave evidence for a related A factor system in Coprinus bilanatus. HD2 products of C. cinereus were shown to be functional in C. bilanatus and seem to interact with native HD1 proteins. However, HD1 genes of C. cinereus appeared to be inactive in C. bilanatus hosts (Challen et al., MGG 241:474, 1993). Molecular cloning of the C. bilanatus A factor revealed two closely linked subloci, a metalloendopeptidase (mep) gene and a pab1 within 40 kb. This arrangement is similar to that found in C. cinereus. Activity of the cloned C. bilanatus A1 factor was demonstrated through transformation of C. bilanatus A2 and A3 hosts and several different C. cinereus strains. Heterologous transformations using individual C. bilanatus A genes are thus far consistent with our earlier observations. C. bilanatus HD2 genes appear to be inactive in C. cinereus whereas HD1 genes seem to interact with native HD2 products to elicit clamp cell development. It is hoped that further studies will lead to a greater understanding of these protein-protein interactions. Work was supported by the Violette and Samuel Glasstone Foundation, University of Oxford.

14. Evolution of A factor specificity genes in Coprinus cinereus

The A mating type factor of C. cinereus is a multiallelic locus which allows mating monokaryons to recognise self from nonself. To be compatible, cells must have different A factor specificities. These specificities are determined by two types of genes encoding proteins with distinct homeodomain DNA binding motifs, HD1 and HD2. These genes are transcribed in pairs and a single A factor may have up to four functionally redundant gene pairs (a, b, c and d) (1). Genes within the same A factor, as well as gene alleles in different A factors, have a low overall homology. Different A factors can share alleles. A6 and A42 both have a copy of the HD1 gene d1-1 and gain different specifities through their b pair alleles (2). Gene and allele specificities result from sequence divergences in their 5' ends (3) and their evolution from a common origin is a fascinating problem. Although functionally identical, the d1-1 copies in A6 and A42 are distinguished by restriction polymorphism. Sequence comparison of the 5' sequences of d1-1 of A6 and A42 (3) revealed base pair insertions and deletions and a number of base differences, several leading to amino acid changes in the gene products. These changes are nonrandom. The DNA binding motif is conserved and in the N-terminal specificity region there are three amino acid exchanges which do not interfere with protein specificity. The positions of these exchanges are especially interesting if the N-terminal region form coiled coil dimerization motifs similar to those found in the yeast mating type protein a2 and if specific amino acid positions in the coils influence dimerization affinities with compatible and incompatible HD2 proteins. Supported by the BSERC to work with Dr. L.A. Casselton and by the Violette and Samuel Glasstone Foundation, University of Oxford. (1) Kües and Casselton, J. Cell Sci. 104:227, 1993; (2) Kües et al., EMBO J. 13:4054, 1994; (3) Kües et al., Plant Cell 6:1467, 1994.

15. Molecular analysis of a sexual development gene of Neurospora crassa

The sexual development process in Neurospora crassa is quite complex and requires the concerted effort of many genes. Previously, subtractive hybridization was used to isolate genes expressed during sexual development, called sdv for sexual development genes. A reverse-genetic approach is being used to determine the functions of the sdv genes. In this study, the RIP (repeat-induced point mutation) process has been used to create mutations in the sdv-15 gene. RIP-mediated disruption of the sdv-15 gene resulted in a mutation causing a recessive sexual defect; the gene was renamed asd-3, for ascus development, to denote the function of the gene product. In asd-3 x asd-3 crosses, early sexual development is normal and many asci are formed, but ascospores are never delineated. Confocal microscopic analysis has been used to demonstrate that the morphology of asd-3 asci is grossly aberrant. The lengths of the asci are comparable to those of wild type asci, but the width and shape vary widely. Spore formation in homozygous asd-3 crosses is defective, with asci containing few or typically no spores. Ascospores appear immature in comparison to wild type, and no spores are shot from the fruiting body. Preliminary sequence analysis indicates that the asd-3 gene product is a sugar-transporter. Our studies indicate that the asd-3 gene product is essential for normal sexual development in the fungus Neurospora crassa.

16. STUA-directed gene expression during Aspergillus nidulans development

Conidiophore development in Aspergillus nidulans requires a tightly regulated sequence of balanced, interacting gene expressions that direct correct spatiotemporal cell differentiation. The stunted gene (stuA) is a key morphological modifier in this process, mutations in which result in shortened conidiophores lacking intermediate cell types. stuA encodes a putative transcription factor with a DNA binding site that shows strong homology to those of Swi4, Mbp1 and Phd1 from S. cerevisiae and Cdc10, Sct1 and Pst1 from S. pombe. Swi4 and Mbp1 are transcription factors responsible, in association with Swi6, for periodic activation of transcription during the S. cerevisiae cell-cycle. Phd1 enhances pseudohyphal growth of diploid S. cerevisiae. Cdc10, in association with Sct1, is required for the initiation of DNA synthesis in S. pombe. An initial search for genes under StuA control employing a yeast one-hybrid system indicated that StuA is competent to direct gene expression through upstream activating regions containing either MCB or SCB cell-cycle boxes. These hexamer motifs are recognised in S. cerevisiae by Mbp1/Swi6 and Swi4/Swi6 respectively and are important in gene regulation throughout the S. cerevisiae cell cycle. We investigate the role of similar sequences in StuA-controlled gene expression in Aspergillus nidulans and discuss their implication in both directing differentiation and cell-cycle control during conidiophore development.

17. The Aspergillus nidulans modifier gene medusa encodes a protein that can form dimers with the transcriptional factor bristle

One of the key steps during development in Aspergillus nidulans is the transition from multinucleate filamentous growth to uninucleate budding. Null mutations in medusa (medA) and abacus (abaA) genes and hypomorphic bristle (brlA) mutations cause branching chains of reiterated uninucleate cells (metulae) and delay or prevent conidiation. Earlier work suggested that MEDAp is required for normal patterns of brlA expression and may directly or indirectly co-activate abaA expression. An extra copy of brlA suppresses medA mutations restoring both normal conidiophore morphology and abaA expression. The degree of the suppression is allele and temperature dependent. These results suggested a possible protein-protein interaction between BRLAp and MEDAp. We have constructed a GST-BRLA protein fusion that is expressed in E. coli. When this fusion protein is bound to a glutathione column, it specifically binds in-vitro made MEDAp. We have made a series of carboxy terminal deletions of MEDAp. Preliminary results have delineated a ninety amino acid region necessary for the BRLAp-MEDAp interaction.

18. Characterization of genes controlling sexual development in Neurospora crassa, including gene identities and the control of their expression

Twenty genes expressed specifically or preferentially during sexual development in N. crassa are being characterized. (1) The control of expression of these sexual development (sdv) genes is being examined. All sdv genes are expressed in response to nitrogen starvation, and some require the A and a mating type products for their expression. Other sdv genes are expressed only if the white collar (wc) products, in addition to the mating type products, are also present. (2) In previous work, we showed that the N. crassa asd-1 product is required for sexual development; in its absence, aberrant asci lacking ascospores are formed. Sequence analysis has shown that this gene encodes a product closely related to a rhamnogalacturonase of Aspergillus aculeatus. This protein is abundant in young but not more mature asci. (3) The car1 gene of Podospora anserina encodes a product required for karyogamy; in the absence of this product, many croziers but no asci are formed. The Podospora car1 gene, isolated by Veronique Berteaux-Lecellier and Marguerite Picard (University of Paris) was used to isolate the homologous N. crassa gene. These car1 genes, which share extensive homology, encode a protein similar to the human PAF1, or peroxisome assembly factor (an integral membrane protein required for the import of many peroxisomal proteins).

19. Phenotypic analysis and cloning strategy for rad12, a gene required for meiosis and DNA repair in Coprinus cinereus

Our lab studies meiosis and DNA repair in Coprinus cinereus, an organism in which meiosis is synchronous. rad12 is required for both processes. We have undertaken a series of experiments to characterize the phenotype of three different rad12 mutant alleles, rad12-1, rad12-4, and rad12-15. Quantitative measurements of oidial survival indicate that all three rad12 alleles are sensitive to gamma irradiation but insensitive to UV irradiation relative to the wildtype. Recombination within the tract of the ribosomal RNA gene repeats was not significantly different in the mutants when compared to the wildtype. The progression of rad12 mutants through meiosis was monitored by examining surface spreads at one, six, and ten hours after karyogamy. All three rad mutants showed defects in chromosome synapsis. While attempts to clone the rad12 gene from a wildtype cosmid library using DNA-mediated transformation have not proven to be successful, this strategy did provide us with a tag which allowed us to jump to the region of the rad12 locus. We have been walking from this tag towards the gene, transforming clones from each step of the walk as well as monitoring the walk genetically, using RFLPs. We have defined a 15 kb region which complements the repair phenotype in the mutant. Currently, we are examining the expression patterns of transcripts produced in this region, in order to determine whether one of them could be a likely candidate for the rad12 gene.

20. Meiotic defects in the rad9-1 mutant of Coprinus cinereus

We have isolated 38 radiation-sensitive mutants of the basidiomycete Coprinus cinereus, which define at least 10 complementation groups for gamma survival. Four complementation groups, rad3, rad9, rad11 and rad12, represent genes required both for survival of gamma radiation and for meiosis. Mutants in each of these four groups arrest during meiosis and produce mushrooms with greatly reduced numbers of viable spores. Genetic pathway analysis has shown that these four genes are in the same pathway for the repair of gamma ray damage. Using a chromosome-specific cosmid library, we have cloned one of these four genes, rad9. This 8 kb gene encodes a transcript of about 6.8 kb, which is induced during meiosis and after gamma irradiation. A closely linked, coordinately expressed gene encodes an RNA helicase of the DEAH box family. Silver staining of surface spreads of rad9-1 strains indicates that its chromatin partially condenses and pairs. Time course studies of chromosome behavior during meiosis in wild-type and rad9-1 strains have shown that the rad9-1 mutant is defective from early stages of meiosis. Although the rad9-1 strain undergoes karyogamy at the normal time, differences between mutant and wild-type cells are apparent immediately after karyogamy. Comparisons between rad9-1 and wild-type strains indicate that telomere condensation and pairing may occur in rad9-1, but that further condensation, pairing, and synapsis of the chromatin is inhibited. At 12 hours after karyogamy, when wild-type strains have completed both meiotic divisions, rad9-1 chromatin shows evidence of degradation.

21. Dimerisation is the key to self non-self recognition between A mating type proteins of Coprinus cinereus

The A mating type genes of the basidiomycete Coprinus regulate part of the developmental programme that coverts the asexual monokaryon into a fertile dikaryon. The A locus is complex and contains several paralogous pairs of genes that encode two classes of homeodomain containing proteins that we term HD1 and HD2. A compatible combination that triggers A- regulated development is an HD1 gene from one mate and an HD2 from the other. We have asked how the cell distinguishes between several HD1 and HD2 proteins that are present in unmated cells and the compatible combinations that are brought together by mating. We will describe our in vivo and in vitro experiments which identify an N-terminal domain in both classes of proteins that only permits heterodimerization between compatible proteins. We will also describe experiments that identify other functional domains of the A proteins.

22. The first non-mammalian homologue of the PAFl gene (Zellweger syndrome) discovered as a gene involved in caryogamy in the fungus Podospora anserina

The fusion of haploid nuclei (caryogamy) is a vital part of the sexual cycle. To date, in fungi, this process has been well studied only in the yeast S. cerevisiae (1). However, caryogamy in filamentous ascomycetes is more complex than in unicellular yeasts. In particular, it does not occur immnediately after fertilization but it is a deferred stage occuring in specialized cells. The car1 mutants of P. anserina, completely defective for caryogamy, have been isolated during a systematic search for sporulation deficient mutants (2). The car1 gene was cloned by complementation. Surprisingly, this gene encodes a protein that shows similarity to the mammalian PAF1 protein (Zellweger syndrome, 3). Altogether, the molecular, physiological (inability for the car1 mutants to grow on oleic acid), genetical (revertants analysis) and ultrastuctural approaches (immunofluorescence and electron microscopical analyses) gave evidence that the P. anserina car1 protein is actually a peroxisomal protein. Therefore, this study shows that peroxisomes are required at a specific stage of sexual development, at least in P. anserina, and that a functional homologue of the PAF1 gene is present in a lower eucaryote. The data will be discussed in terms of the possible functional role of peroxisomes in the caryogamy process or, more likely, in the cell determination or differentiation programs. (1) M. D. Rose (1991) Annu. Rev. Microbiol. 45:539-567. (2) J. M. Simonet & D. Zickler (1978) Mol. Gen. Genet. 162:237- 242. (3) N. Shimozawa, T. Tsukamoto, Y. Suzuky, T. Orii, Y. Shirayoshi, T. Mori and Y. Fujiki (1992). Science 255:1132-1134.

23. Mating type genes in Podospora anserina are required for sorting mat+ and mat- nuclei from syncytial cells into the ascogenous hyphae

In the heterothallic ascomycete Podospora anserina, fertilization is followed by a series of mitotic divisions of the parental nuclei leading to a syncytium, from which one mat+ and one mat nucleus migrate into the ascogenous hyphae. The four mating type genes, FPR1 in the mat+ allele, FMR1, SMR1, and SMR2 in the mat allele, are required to match and sort mat+ and mat nuclei from the syncytium, whereas the N-terminal parts of FPR1 and FMR1 are sufficient for fertilization (1,2). We have examined how the four mat genes control the sorting of mat+ and mat nuclei by: i) analyzing the sexual behavior of strains mutated in FPR1, FMR1, SMR1 or SMR2 (3). ii) testing mat interactions through the analysis of the sexual behavior of a mat+ strain containing the mat genes. SMR1 information is expressed in a mat+ context whereas FMR1 and SMR2-controled information are either inactivated or nucleus-specific. iii) testing mat protein interactions with the yeast two hybrid system. An interaction between FMR1 and SMR2, and a dimerization of FPR1, have been shown, but no interaction between FPR1 and mat proteins can be detected with this method. These data have been used to propose that FPR1 and FMR1-SMR2 inhibit the development of uniparental mat+ and mat ascogenous hyphae respectively. SMR1 would be required for sorting mat+ and mat nuclei. (1) R. Debuchy and E. Coppin 1992 Mol Gen Genet. 233:113-121. (2) R. Debuchy, S. Arnaise and G. Lecellier 1993 Mol Gen Genet. 241:667-673. (3) D. Zickler, S. Arnaise, E. Coppin, R. Debuchy and M. Picard 1995 Genetics, in press.

24. Genetic control of sexual compatibility in Tapesia yallundae

Different pathotypes of Tapesia yallundae (anamorph Pseudocercosporella herpotrichoides) may be distinguished on the basis of host range on cereal crops. These include the W-, C-, S- and R-types. Sexual compatibility in the W-type is controlled by a two-allele heterothallic mating system and in vitro mating tests have shown a similar mechanism in the C- and S-types. Furthermore, the C- and S-types are sexually compatible with the W-type. Recombination between isolates was demonstrated using randomly amplified polymorphic DNA (RAPD) markers. However, attempts to induce crossing between the R-type and other pathotypes were unsuccessful, indicating that the R-type may be considered as a separate species. Work is in progress to clone the mating-type genes (MAT1-1, MAT1-2) from T. yallundae to study possible variations between pathotypes and how this may effect sexual compatibility. Approaches being used include: (1) use of degenerate PCR primers designed to amplify a partially conserved region of the MAT1-2 locus from other Ascomycete species (N. crassa, P. anserina, C. heterostrophus, S. cerevisiae); (2) oligonucleotide probing using a family of probes from conserved regions of MAT loci from other Ascomycete species; (3) heterologous probing using MAT genes cloned from other Ascomycete species.

25. The mushroom-inducing gene Frt1 of Schizophyllum commune encodes a putative ATP-binding protein

Fruiting bodies (mushrooms) can be induced to form in normally non fruiting strains of the Basidiomycete fungus Schizophyllum commune by the ectopic genomic integration of a cloned gene called Frt1. The Frt1 gene encodes a predicted polypeptide of 192 amino acids that does not have significant similarity to any entries in the protein sequence databases. A conserved sequence found in nucleotide-binding proteins, called the P-loop motif, has been detected in the N-terrninal half of the predicted FRT1 protein. A potential site for Mg2+-binding was predicted to reside next to the P-loop at Thr24. No motifs specific to GTP-binding proteins were detected in the FRT1 protein, but a potential site for ATP-binding is predicted to reside in the C-terminal half of the polypeptide. The possible functional significance of these and other regions within the FRT1 protein were examined using site- directed mutagenesis, followed by transformation of these mutant alleles of Frt1 back into S. commune. Mutation of the middle glycine of the P-loop completely abolished the fruit-inducing activity of cloned Frt1. Mutant alleles containing an alanine residue substituted for Thr24 had no fruit-inducing activity. Experiments are in progress to test the in vivo effect of mutations within the putative ATP-binding region. Taken together, the results of our mutagenesis experiments suggest the possibility that activity of the FRT1 protein could be altered by nucleotide- binding and coordination of Mg2+.

26. Distribution of Gibberella fujikuroi by biological species, mating type, and female fertility in a Malaysian maize field

Samples of Gibberella fujikuroi (Fusarium, section Liseola) were collected along a transect through a corn field in northern Malaysia. These strains were characterized for mating population, mating type within population (where possible), and female fertility. The preponderance of the sample was found to be in mating population A. Of these, the majority (approximately 70%) were of a single mating type (A+). Distribution of both mating type and species was significantly non-random, implying a highly patchy and perhaps clonal pattern of dispersal in the field. The relative effects of distribution, mating type disequilibrium, and female sterility on the effective size of these populations were discussed.

27. Motherhood and the price of sex

Natural populations of heterothallic biological species (= mating populations) in the Fusarium moniliforme species complex (sexual stage Gibberella fujikuroi) consist of males and hermaphrodites. The ratio of males to hermaphrodites varies with the biological species. In the "A" mating population the male:hermaphrodite ratio is 50:50, in the "D" population it is 70:30, and in the "F" population it is 90:10. If P(h) is the proportion of hermaphrodites in the population, then a stable equilibrium can be found for any P(h) that is dependent on the relative fitness of females and males (q), where q=(l-P(h))/(l+P(h)). Hermaphrodites always make a larger contribution to the next generation because they contribute all of the female gametes and some of the male gametes. At equilibrium, hermaphroditism costs l-q, otherwise the equilibrium quickly shifts to a population that is all hermaphrodites. In the "A" mating population q=0.33, in the "D" mating population q=0.54, and in the "F" mating population q=0.81. This model also suggests that mating type should be independent of genes for female sterility, and the available data from these species are consistent with this conclusion. The effective population number appears to be much more limited by the availability of hermaphrodites than it is by imbalances in mating type frequencies.

28. Reciprocal heterologous expression of MAT genes from Cochliobolus heterostrophus and its asexual relative Bipolaris sacchari

To investigate the cause of asexuality in fungi, we have chosen a pair of closely related species, one sexual (C. heterostrophus) and one asexual (B. sacchari). A key experimental strategy available to address the question of asexuality in B. sacchari is reciprocal heterologous expression. B. sacchari genes can be expressed in C. heterostrophus to ask if they are functional in a strain with a complete mating pathway. The counterpart is to express C. heterostrophus genes in B. sacchari to ask if B. sacchari can mate if it carries 'good' mating type genes. We have already determined that B. sacchari has a cruclal piece of the mating pathway ie., the MAT locus itself. The B. sacchari MAT-2 homolog is 98% similar to C. heterostrophus MAT-2 and is functional when transformed into C. heterostrophus. Yet transfer of a C. heterostrophus gene of opposite mating type into B. sacchari does not make the latter sexual. No B. sacchari MAT transcript can be detected in B. sacchari. When B. sacchari expresses a C. heterostrophus MAT gene, transcription is no longer tightly regulated, as it is in C. heterostrophus. The B. sacchari transgenic strains do not mate with B. sacchari; with C. heterostrophus they sometimes initiate, but don't complete, sexual development. Both improper MAT regulation and missing or defective mating-specific genes could explain asexuality in B. sacchari.

29. HSP90 and HSP70 are present in a heteromeric complex in the steroid hormone responsive oomycete Achlya ambisexualis

In vertebrates HSP90 is reported to exist in heteromeric complexes with a number of cellular proteins, including kinases, transcription factors and steroid hormone receptors, as well as other heat shock proteins (HSP70, HSP56). In the oomycete Achlya, steroid hormones regulate sexual development, The response to hormone is thought to be mediated by a steroid hormone receptor. Immunoprecipitation of either cellular or in vitro translated proteins with monoclonal antibodies to HSP90 from Achlya (AC88), mouse (8D3) or rat (2D12), showed that HSP90 (85kD) in Achlya is found in a heteromeric complex with proteins of 110kD, 74kD, 64kD, 61kD, 56kD, 47kD and 23kD. Proteins of similar molecular weight are observed in vertebrate steroid receptor heterocomplexes. Western analyses, using antibody AC88 (antibody to HSP90) and BRM-22, an antibody to HSP70, showed that the 85kD and 74kD proteins found in the heteromeric complex, were HSP90 and HSP70 respectively. Our results suggest that the Achlya steroid receptor is likely found in a heterogeric complex with HSP90, HSP70, and with other proteins with sizes similar to the proteins reported to be complexed with animal steroid receptors. (Supported by NSERC Canada)

30. Genes involved in vegetative incompatibility reaction in Podospora anserina

The three genes, het-c, het-e and het-d, involved in the two nonallelic. incompatibility het-c/het-e and het-c/het-d systems have been cloned. HET-C protein is similar to a glycolipid transfer protein. A strain containing a disrupted het-c locus is affected in ascospores production. A het-e gene encodes a 1356-amino acid protein which exhibits two domains. In the N-part of the protein, there is a P-loop motif characteristic of GTP binding protein. In the C-part of the protein, there are ten direct repeats of a motif of 42 amino aids which are characteristic of beta- transducin like protein. Mutation in the P-loop motif results in a loss of the incompatibility phenotype. It has been shown in P. anserina that mutation in some genes named mod genes results in the suppression of a barrage reaction. Moreover, resulting strains have defects in secondary ramification and reproductive female organ production. On the basis of the wild type phenotype restoration of a modD mutant strain, a gene has been cloned. It encodes a polypeplide which has homology with the N. crassa adenylate cyclase. But, by RFLP, the modD locus and the adenylate cyclase locus are distinct. Transcriptional and/or translational regulation of genetic expression and enhancement of the proteolytic activity had been shown in cells undergoing cell death associated to incompatibility. Two genes under transcriptional regulation have been cloned. Study of their expression is in progress. The cloned gene papA, encoding an aspartylprotease, has been disrupted.

31. Comparison of pheromone receptor genes of Schizophyllum commune

The mating type locus B(alpha)1 of S. commune contains a pheromone receptor and putative pheromone genes. The pheromone receptor Bar1 shows homology to other pheromone receptors such as Ste2 and Ste3 of Saccharomyces cerevisiae and pral and pra2 of Ustilago maydis. It also contains sites conserved among other G protein-linked receptors of the seven transmembrane domain family. The fact, that the mating system in S. commune provides nine different allelic specificities at the mating type locus Ba makes it a model system to investigate ligand interaction between different allelic specificities of pheromones and receptors. For S. commune multiple isolates for each mating specificity exist. In order to obtain evidence for functional domains, different alleles for the BAR1 gene will be compared. Southern hybridization experiments could show that strains sharing the specificity B(alpha)1 exhibit strong hybridization signals. This indicates higher homology of the alleles of BAR1, all sharing the mating specificity B(alpha)1, as compared to strains with other mating specificities, which also hybridize, but to a lesser extent.

32. Complementation of Cochliobolus heterostrophus mating type deletions with homologous and heterologous MAT genes

In C. heterostrophus, mating is controlled by a single locus (MAT) with dissimilar, alternate forms. Partial diploids carrying both a resident MAT gene and a MAT transgene can self and are dual maters. Selfs form perithecia but very few asci and ascospores. Crosses are fertile when transgenic strains are mated with a strain of opposite mating type to the resident MAT gene of the transgenic strain, but fertility is low when mated with a strain of opposite mating type to the transgene, suggesting that the resident MAT gene interferes with transgene function. Interference was eliminated by deleting the resident MAT gene by gene replacement. Deletion strains are completely sterile when crossed to tester strains. In a gain-of-function analysis, we determined the minimal requirement for full fertility by re-transforming deletion strains with various fragments of homologous MAT DNA. To investigate the functional conservation of MAT genes, we transformed deletion strains with MAT DNA from 4 sexual species (C. carbonum, C. victoriae, N. crassa and P. anserina) and one asexual species, Bipolaris sacchari. Genes originating from closely related species were able to completely restore the wild type phenotype whereas those from the more distantly related N. crassa and P. anserina resulted in only partial complementation. Strains carrying the latter produced low numbers of perithecia that were barren.

33. Viral repression of fungal sex pheremone expression. Implications for virus-host interactions

We have found that a virus of the chestnut blight fungus, Cryphonectria parasitica, perturbs its host's sexual cycle by repressing transcription of mating type-specific genes. We have cloned mating-type specific genes from both mating types of the fungus, and have found that the virus represses their transcription. Two of the repressed genes encode a Mat1-1-specific pheromone required for completion of the sexual cycle. Vegetative compatibility (vc) genes function to prevent cytoplasmic exchange of viruses in this fungus, and represent the only known class of virus- resistance genes of the host. Viral suppression of mating prevents recombination of vc genes, which should result in fungal populations with fewer vc groups, and thus with fewer barriers to viral transmission. Studies of viral- infected and non infected populations of the fungus support the hypothesis that this mechanism of host mating suppression has evolved to enhance viral spread in host populations. These results are as predicted by the Red Queen hypothesis of the evolution of sex.

34. Sexual reproduction in ascomycetes: roles of the N. crassa mating type polypeptides

The N. crassa mating type idiomorphs encode polypeptides that play crucial roles in vegetative incompatibility, mating, and formation of ascogenous hyphae. RIP experiments also suggest a role for the mating type genes within the ascogenous hyphae. The sole product of the mt a idiomorph, MT a-1, is a sequence-specific DNA binding protein of the HMG box family. The presumed regulatory targets of MT a-1 include genes necessary for mating pheromone biosynthesis and response. MT a-l DNA binding activity is not necessary for vegetative incompatibility function, but it is necessary for mating functions. The mt A idiomorph encodes at least three products. One of these products, MT A-3, is also a member of the HMG box family. MT A-3 produced in E. coli binds DNA fragments that are also bound by MT a- 1. The interactions of the mating type polypeptides with DNA and with each other as well as the biochemical and biological consequences of such interactions are under investigation

35. Cloning conidiation genes from Colletotrichum graninicola

Colletotrichum graninicoa can produce two types of conidia; one type is falcate-shaped and the other is oval- shaped. The conidia are not only morphologically distinct but also differ in ontogeny. To better understand the differentiation and regulation of the development of the conidia, we constructed a cDNA library representing genes expressed during the development of oval conidia. Three cDNA clones, oval-5, oval-7 and oval-11 were recovered from the library by differential screening. The oval-5 clone hybridizes to a 2.2 kb transcript, oval-7 to a 1.8 kb transcript, and oval-11 to a 6.0 kb transcript. Oval-7 shares homology with the sequences of superoxide dismutase (SOD). The transcript of oval-7 starts early in development, increases at the stages when most oval conidia are produced and then decreased at later stages of development when levels of oval-5 and oval-11 are more abundant. The genes corresponding to oval 7 and oval-11 are developmentally regulated and their expression is associated with the production of both type of conidia. The transcript of oval-5 is also developmentally regulated but its expression seems to be exclusively associated with the development of oval conidia. Gene disruptions are in process to address the function(s) of the genes corresponding to these clones

36. Evolutionary analysis of the b1 gene within the A mating-type of Coprinus cinereus

Self-incompatibility mechanisms prevent inbreeding in many organisms, including fungi. Although of great importance to the organism's evolution, little is known about the forces acting upon the genes controlling self- incompatibility. I am studying self-incompatibility in the basidiomycete Coprinus cinereus to better understand how self-incompatibility genes evolve. In this fungus, recognition of self versus non self occurs at the sub-cellular level to determine mating compatibility for sexual reproduction. This sub-cellular mechanism is governed by the mating type genes. I am studying the evolution of the b1 mating-type gene to better understand how this operates in C. cinereus. Because inbreeding (self mating) does not occur, a region of b1 must discriminate between self and non- self. As natural selection favors outcrossing (non-self mating), a large number of different b1 alleles exist in natural populations. My work examines how b1 has evolved at the molecular level and how these changes may provide for self-non self discrimination for self-incompatibility. This work provides a better understanding of how selection can produce extreme variation in b1, yet still maintain identical function for self incompatibility.

37. Genetic characterization of conidiation mutants of Colletotrichum graminicola

Colletotrichun graminicola (Teleomorph: Glomerella graminicola) causes anthracnose leaf-blight and stalk rot of maize. Asexual reproduction by means of conidia serves as a major mechanism of dispersal. Therefore, the process of conidiation can be an important target for disease control. Three distinct conidiation phenotypes have been identified among 10 field isolates under investigation. These phenotypes are: (i) light-dependent conidiation (wild- type), (ii) light independent conidiation (Con1-), and (iii) aconidial phenotype (Con2-). In the wild-type, growth in the dark results in the absence of conidia and a fluffy colony morphology whereas growth m the light results in abundant conidia and little aerial hyphae (i.e., lack of the fluffy morphology). The Con2 mutant produces very few conidia and has a fluffy colony morphology whether grown in the light or dark. The Con1 mutant does not have a fluffy colony morphology and produces conidia at comparable levels in the light and dark. Thus, reduced conidiation is always associated with abundant aerial hyphae and a fluffy colony morphology. This correlation was also observed in progeny from crosses between the wild-type and either of the mutants, indicating a genetic basis for linkage between the absence of conidia and fluffiness. Crosses of the wild-type with either mutant showed a 1:1 segregation of wild-type and the respective mutant phenotype. This indicates that the Con1 and Con2 mutants each differ from the wild-type at a single locus. Genetic analysis has shown that Con2 is linked to PYR1 which has previously been physically mapped to chromosome 1 (Rollins and Hanau, unpublished data). In order to clone the Con2 gene by chromosomal walking, tightly linked RAPD markers were identified by bulked-segregant analysis. Other strategies involving transformation mediated complementation are also being employed.

38. Complex control of the brlA developmental regulatory locus in Aspergillus nidulans

brlA is a primary regulator of asexual development in the filamentous fungus, Aspergillus nidulans. Activation of brlA is necessary and sufficient for conidiophore development. It is known that brlA has two overlapping transcripts, designated brlA(alpha) and brlA(beta). From previous studies, we found that expression of brlA is complex in that activation of the two brlA transcripts is regulated at different levels. brlA(beta) is regulated both transcriptionally and translationally while brlA(alpha) is regulated transcriptionally. To better understand transcriptional controls for brlA(beta) regulation, we have made 5' deletions of the essential ~2 kb upstream control sequences of brlA(beta) transcript fused to E. coli lacZ reporter gene. The results from deletion experiments indicate that there are probably several cis-acting control sequences involved in the regulation of brlA(beta). We have also made various fragments of the 2 kb brlA(beta) control sequences fused to otherwise inactive amdS::lacZ fusion as a complementary approach to our deletion analysis. In this way we identified two ~600 bp fragments extending from -2034~ -1426, and -929~ -380 that confer activity on the inactive amdS promoter. We are in the process of more precisely defining these ~600 bp fragments to identify a minimal sequence that is required for developmental regulation in asexual development. In addition, we are investigating putative trans-acting factors which are involved in the activation of brlA(beta) using Saccharomyces cerevisiae as a heterologous expression system. Thus far we found that BrlA(alpha), AbaA and FlbD are likely involved in the activation of brlA(beta) transcription.

39. Cloning of the vegetative incompatibility locus het-C of Neurospora crassa

Somatic incompatibility, manifest as regulation of heterokaryon formation, appears to be a nearly universal feature among filamentous fungi. In Neurospora crassa at least eleven loci mediate heterokaryon formation. Stable heterokaryon formation between two strains is prevented when different alleles are found at one or more of the het loci. To better understand the molecular basis of this self/non-self recognition we have cloned one of these loci, het- C, by means of a chromosome walk from the nearby marker pyr-4. Six overlapping cosmids were isolated and their location on LGII and orientation centromere distal or proximal to pyr-4 was determined using RFLP analysis. Additional RFLP analysis indicated several of the cosmids extended beyond het-C. Partial duplication strains heterozygous at a het locus are phenotypically similar to incompatible heterokaryons. Based on this, we have used a transformation assay in which cosmids are introduced into strains carrying different alleles at het-C by DNA mediated transformation. Results of this analysis indicate that two of the six cosmids contain het-C. Further transformation with fragments of one of these cosmids shows het-C is located within a 10 kbp SphI-EcoRI fragment. Transformation of Tn-5 mutagenized versions of this cosmid indicate het-C lies at least partially within a 3.5 kbp BglII fragment.

40. Characterization of two incompatibility genes at het-6 in Neurospora crassa

Het-6 is one of eleven heterokaryon incompatibility loci in Neurospora crassa. Het-6 was originally identified in strains having inhibited hyphal growth due to heterozygous partial duplications in the left arm of linkage group II. With near isogenic strains, we have confirmed that het-6 is, in fact, a heterokaryon incompatibility locus: different alleles at het-6 are sufficient to prevent heterokaryon formation under forcing conditions. Cosmids in the vicinity of het-6 were isolated by differential hybridization signals when CHEF separated chromosomes, with or without translocations containing het-6, were used to probe the Orbach/Sachs library. RFLP patterns in progeny with crossovers between het-6 flanking markers were used to locate cosmids with respect to het-6, and to determine the direction and progress of a chromosome walk past het-6. Crossover points were identified on either side of het-6, within a region spanned by two contiguous cosmids. Based on a transformation assay, one of these cosmids contains two, non-homologous regions of DNA, each of which confers incompatibility-like activity. One of these regions is located at, or near to, the RIP-mutant marker, un-24. The other is located about 14 kbp centromere proximal to un- 24. DNA fragments which confer het function have been isolated from both regions and DNA sequence analysis is in progress.

41. Identification of genes whose overexpression leads to growth inhibition in Aspergillus nidulans.

Overexpression of Aspergillus nidulans developmental regulators like brlA, abaA, and flbA causes vegetative cells to stop growing and to differentiate into reduced conidiophores. We reasoned that additional developmental regulators could also be identified by a similar ability to inhibit vegetative growth and/or induce development when overexpressed. To test this possibility, we constructed an alcA(p)-inducible expression library by placing A. nidulans genomic DNA fragments under the control of the alcA(p). We expect to identify mutants that fall into two phenotypic categories: FAB (Forced Activator of brlA) and FIG (Forced Inhibitor of Growth) which may include mutants overexpressing structural genes leading to growth cessation (Liu et al., 1992 Genetics 132:665). To test the utility of the library for identifying FAB and FIG genes, we screened 20,000 A. nidulans transformants for inhibited growth on alcA(p)-inducing media. To date we have identified 52 unique clones with a FIG phenotype and 16 with a FAB phenotype. We have recovered the transforming plasmids from two of the growth inhibited strains and eight of the brlA-inducing strains and have shown that when transformed back into a wild type A. nidulans strain they result in either growth inhibition or brlA-induction on alcA(p)-inducing media. We have designated the two growth inhibition genes recovered as figA and figB and the brlA inducing genes as fabA - fabP. Sequence analysis of figA shows it is predicted to encode a >950 codon open reading frame whose predicted amino acid sequence has similarities to several structural proteins including trichohyalin, collagen, and myosin. Sequence analysis of fabM shows it is predicted to encode a ~650 codon opening reading frame whose predicted amino acid sequence shares high homology to several poly-A binding proteins of yeasts and animals. We are creating a fabM deletion strain to deterrnine its involvement in development.

42. flbD encodes a Myb-like DNA binding protein that coordinates initiation of Aspergillus nidulans conidiophore development.

The timing of asexual fruiting body formation during Aspergillus nidulans colony development is precisely regulated so that conidiophores are typically produced 1-2 mm behind the growing edge of the colony. Mutations in any of four A. nidulans genes flbB, flbC, flbD, or flbE result in colonies that are delayed at least 24 h in their ability to initiate conidiophore development resulting in fluffy colonies with conidiophores forming in the center at least 12-15 mm behind the growing edge. The requirement for each of these four genes in determining the timing of developmental initiation precedes transcriptional activation of the primary developmental regulatory gene brlA, indicating a possible role for each in developmentally regulated activation of brlA expression. The wild-type flbD gene was isolated and shown to encode an ~1.6 kb mRNA that is present throughout the A. nidulans life-cycle. The deduced FlbD protein sequence predicts a 308 amino acid polypeptide with significant identity at its N-terminus to the DNA binding domain of the Myb family of transcription factors indicating that FlbD likely functions as a sequence-specific transcriptional activator. Although conidiophore development does not normally occur in submerged culture, forced overexpression of flbD in submerged hyphae caused inappropriate activation of brlA expression and resulted in production of complex conidiophores that produced all of the distinct cell types observed in wild-type conidiophores including viable spores. This ability of flbD overexpression to activate conidiation requires brlA, flbB, and flbA (another early developmental regulator), but does not require flbC, flbE or fluG. We propose that FlbD functions during normal development by activating other genes (such as brlA) and that FlbD activity is normally controlled post-transcriptionally by an unknown mechanism.

43. Characterization of the control elements of brlA in Aspergillus nidulans.

brlA is a primary regulator of asexual development in the filamentous fungus, Aspergillus nidulans. Activation of brlA is necessary and sufficient for conidiophore development. It is known that brlA has two overlapping transcripts, designated brlAa and brlAb. From previous studies, we found that expression of brlA is complex in that activation of the two brlA transcripts is regulated at different levels. brlAb is regulated both transcriptionally and translationally while brlAa is regulated transcriptionally. To better understand transcriptional controls for brlAb regulation, we have made 5' deletions of the essential ~2 kb upstream control sequences of brlAb transcript fused to E. coli lacZ reporter gene. The results from deletion experiments indicate that there are probably several cis-acting control sequences involved in the regulation of brlAb. We have also made various fragments of the 2 kb brlAb control sequences fused to otherwise inactive amdS::lacZ fusion as a complementary approach to our deletion analysis. In this way we identified two ~600 bp fragments extending from -2034~ -1426, and -929~ -380 that confer activity on the inactive amdS promoter. We are in the process of more precisely defining these ~600 bp fragments to identify a minimal sequence that is required for developmental regulation in asexual development. In addition, we are investigating putative trans-acting factors which are involved in the activation of brlAb using Saccharomyces cerevisiae as a heterologous expression system. Thus far we found that BrlAa, AbaA and FlbD are likely involved in the activation of brlAb transcription.

44. fluG and flbA induced conidiophore development in Aspergillus nidulans requires brlA

We have been characterizing two early developmental regulatory genes called flbA and fluG. Mutations in these two genes result in a "fluffy-invasive" phenotype with colonies producing an abundance of aerial hyphae instead of undergoing conidiophore development. flbA mutant colonies have a distinct fluffy phenotype having an abundance of interwoven aerial hyphae that undergo autolysis during maturation. The predicted FlbA sequence has 30% identity to the Saccharomyces cerevisiae Sst2 protein which is required for regulating yeast mating pheromone adaptation response. fluG mutant colonies are aconidial under normal conditions, however, the mutant phenotype can be extracellularly complemented by growing next to wild type or other developmental mutants (e.g. brlA, flbA but not fluG). The predicted fluG product is a 97 Kd cytoplasmic protein and the C-terminal half of the sequence has significant similarity to prokaryotic glutamine synthetase (GSI). These results have lead us to suggest that FluG plays an enzymatic role in the production of an extracellular factor and that flbA regulates the response to this signal in controlling the initiation of A. nidulans conidiophore development. Overexpression of flbA or fluG in vegetative hyphal cells using an inducible promoter resulted in conidiophore development in the submerged culture indicating that flbA and fluG can function directly in activating development. In addition, overexpression of flbA or fluG also activates the expression of an essential developmental regulator called brlA. The brlA locus has two major transcripts called brlAalpha and brlAbeta, Han et. al. have proposed that brlAbeta plays the major role in the initiation of conidiphore development and brlAalpha is required for positive feedback in maintaining brlA activity during conidiophore development. Our results support this model that sporulation induced by overexpression of fluG or flbA requires brlAbeta but not brlAalpha. The genetic interactions between fluG and flbA and other genes required for development are discussed.

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