Regulation of Gene Expression
1. Pyrimidine biosynthesis genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases. Alexei Aleksenko1, Wenguang Liu 2, Zoran Gojkovic 2, Jens Nielsen 1, and Jure Piskur 2. 1 DTU, CPB, Lyngby, 2800, Denmark. 2DTU, Microbiology, Lyngby, 2800, Denmark
The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, unlike in prokaryotes, in animals and fungi at least the first two activities are grouped on a multifunctional enzyme. In A. nidulans, the first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN. The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus pyrD. However, the pyrABCN gene contains an evolutionary remnant of a DHOase-encoding sequence, which arrangement is similar to that in yeast. Comparison of amino acid sequences of active dihydroorotases with related enzymes indicates that the monofunctional dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway, from which they have probably originated, than to DHOases of other organisms. The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrmidine starvation is noticeably enhanced in pyrE mutants which accumulate dihydroorotic acid. The data suggest that dihydroorotate is probably an inducer of at least some genes of the pathway, while UMP is a likely repressor. The cluster gene pyrABCN contains an upstream short open reading frame which may be involved in regulation. Some common features have been identified in promotor regions of pyr genes.
2. The cpcA gene of A. nidulans encodes the transcriptional activator of the general control system and is additionally involved in sexual development. Meike Andermann, Bernd Hoffmann, Gerhard H. Braus. University of Göttingen, Molecular Microbiology, Göttingen, Lower Saxony, Germany.
The cpcA gene of the filamentous fungus Aspergillus nidulans
encodes a protein of 245 amino acids in length with high similarity to the general amino acid
control transcriptional activator Gcn4p of yeast which are able to complement each other. The
mRNA level of cpcA is regulated under amino acid starvation resulting in a fourfold
increase after eight hours of limitation. Deduced cpcA protein binding sites (GCRE´s) in
its own promoter imply a transcriptional autoregulation. Gel retardation assays showed binding
activity of cpcAp in its promoter which was abolished when point mutations were integrated into
the GCRE´s. Two upstream open reading frames in the 5´region of the
cpcA mRNA were identified suggesting an additional translational regulation. An
increase of cpcA protein was observed to a factor of three after amino acid limitation. Deletion of
cpcA causes a reduced growth rate to approximately 30% of wild-type. Additionally,
cpcA mutant strains are unable to derepress general control regulated genes as
trpC and argB resulting in an unability to grow under amino acid
starvation conditions. Overexpression of cpcA results in a specific increased
transcription of general control regulated genes. The sexual developmental program of A.
nidulans cpcA overexpression strains was affected. High amounts of cpcA protein resulted
in a block in cleistothecia formation at a defined timepoint and therefore in sterility. The same
phenotype was observed after overexpression of the yeast GCN4 in A.
nidulans. We suggest a function of the cpcA protein as general control transcriptional
activator. In addition, overexpression of cpcA results in a direct or indirect manner
in sexual sterility.
3. Inducer dependent nuclear localisation of the FacB transcriptional activator of
Aspergillus nidulans.
Gene expression can be regulated at the transcriptional, translational or post-translational level. While structural genes are predominantly controlled at the transcriptional level, regulators also show a number of post-translational modes of regulation including ligand dependence, nuclear exclusion and interactions with coactivating or antagonistic regulatory proteins. Understanding the mechanisms which control the activity of regulatory factors is important to our understanding of regulatory networks and their action.The facB gene of Aspergillus nidulans encodes a C6Zn(II)2 binuclear cluster DNA binding domain protein and is the major transcriptional activator of genes required for acetate utilisation. FacB positively regulates the expression of the glyoxylate bypass genes acuD and acuE as well as facA and facC. Their products are required for the anaplerotic glyoxylate bypass which converts acetyl-CoA from acetate or fatty acids into TCA cycle intermediates. In addition, FacB activates the expression of the amdS gene.The facB gene and its product are regulated at multiple levels. The gene is controlled transcriptionally by acetate induction, but not by FacB, and carbon catabolite repression by the negatively acting CreA. Full transcriptional activation of FacB also requires acetate.Here we show that the cellular localisation of FacB is regulated. In the absence of acetate, FacB as a LacZ or GFP fusion is cytoplasmically localised. Addition of acetate causes the rapid translocation of FacB to the nucleus. A 142 amino acid region of FacB, spanning the C6Zn(II)2 DNA binding motif is sufficient for nuclear localisation. A mutation which destroys DNA binding by FacB does not inhibit nuclear localisation.
4. The vvd gene is required for light-adaptation of the N. crassa conidiation-induced genes, con-6 and con-10.Lori Bailey Shrode, Lori D. White, Daniel J. Ebbole. Texas A&M; University, Plant Pathology & Microbiology, College Station, Texas, USA.
con-10 and con-6 are two of the conidiation-induced (con) genes of N. crassa that were identified on the basis of their preferential expression during macroconidiophore development. They are also directly regulated by a number of environmental stimuli independent of development, including blue light photoinduction. We identified an allele of vvd, in a mutant screen designed to obtain strains with elevated expression of con-10. vvd mutants display enhanced carotenogenesis in response to light. con-10 and con-6 expression was induced to much higher levels in the vvd mutant and light adaptation is apparently absent. Our initial characterization of two vvd alleles suggests that vvd is required for reducing expression of the light-induced con-10 and con-6 genes in response to light.
5. Evidence for 2,4-Dinitrotoluene degrading genes in Phanerochaete chrysosporium: In situ and DNA homology tests. Christine E. Barrow, Michelle M. Jackson, Gail P. Hollowell, and Sisir K. Dutta. Howard University, Biology, Washington, D.C., USA.
The nitroaromatic compound 2,4-dinitrotoluene (2,4-DNT) is a hazardous waste product with
known toxicity to humans, animals, and plants. Increasing contamination of soil and ground
water is a major environmental concern. HPLC analyses, in situ and liquid culture experiments
suggest that the degradation of 2,4-DNT by genes in the fungus P. chrysosporium.
The basidiomycete fungus is preferred in bioremediation because it can also degrade other toxic
nitroaromatic compounds. The 2,4-DNT degrading gene clusters in bacteria are well
characterized, however, similar genes in the fungus have yet to be isolated. We report on
evidence of 2,4-DNT degrading genes in this fungus based on our in situ bioremediation and
DNA homology studies. The fungal spores were applied to soil contaminated with 2,4-DNT. The
resulting soil extractions were then analyzed by HPLC to determine residual 2,4-DNT
concentrations at varying time intervals. The dioxygenase gene of the bacteria Burkholderia, also
known to degrade 2,4-DNT was isolated and purified from a plasmid clone donated by Dr. J.
Spain. This 6.8 kilobase gene sequence was used as a probe against the fungal genomic DNA
template in Southern blot homology studies. The bacterial gene sequence was also used in primer
selections and for PCR analysis. Fragments of 6.8 kilobases or more received priority due to the
presence of introns in the fungal genome. The resulting PCR fragments will be sequenced and
cloned for expression studies. These results may lead to use of P. chrysosporium in
the large-scale bioremediation of 2,4-DNT contaminated soil and groundwater
worldwide.
6.
DNase I sensitivity analyses of wild-type and mutant cbh1 promoters were performed under conditions that repress, induce or are neutral for cbh1 expression. Previous studies have revealed a major role in the repression of cbh1 expression for the global regulator CRE1 and sequence analysis revealed a number of putative binding sites for this transcription factor. Here we show that although several regions of the cbh1 promoter contain consensus binding sites for CRE1 and these also bind to a GST-CRE1 fusion protein in vitro, only two of these putative binding sites are hypersensitive to DNase I (DH1 and 2) indicating that only these two regions are occupied by CRE1 in vivo. Deletion of the region around DH1 does not lead to derepression of cbh1 expression, whereas mutation of the region at DH2 abolishes DNase I hypersensitivity and leads to derepression suggesting a pivotal role for this region in maintaining repression. A third DNase I hypersensitive site (DH3) was also detected, but only in inducing conditions. Micrococcal nuclease sensitivity was used to map the positions of nucleosomes in the cbh1 promoter. No major nucleosome rearrangements occurred during derepression or induction.
7. Molecular cloning of spray gene in Neurospora crassa. Jin-Woo Bok1, Teruo Sone2, Fredrick J. Bowring3, David E.A. Catcheside3, and Anthony J.F. Griffiths1. 1University of British Columbia, Department of Botany, Vancouver, BC, Canada. 2Hokkaido University, Faculty of Agriculture, Sapporo, Japan. 3Flinders University, Biological Sciences, Adelaide, Australia.
Ca 2+ in Neurospora serves more a regulatory than a nutritive or structural function. A tip-high gradient of free Ca 2+ prevails in growing hyphae, probably maintained by a constant redistribution within cells rather than by a regulation of uptake from the media. The decreased uptake of Ca 2+ caused by a verapamil was correlated with branching and the phenotype resembled that of the frost and spray mutants. High levels of Ca 2+ added to the medium could reverse the effects of both the drug and the mutants. To figure out the relationship between Ca 2+ and spray gene, we have tried to clone and analyse the gene. It was found in a cosmid clone X11: G10 by chromosomal walking from am locus and complementation of the mutant. The 5.7 kb DNA fragment which transformed the mutant into wild type successfully was subcloned into TOPO-XL vectors and sequenced. The spray mutant had a single base pair change (TTTAA (r) TTGAA). The size of mRNA was about 4.4 kb by Northern blotting analysis. The start site and intron mapping of the gene is in progress by RT-PCR and 5' RACE. Partially determined sequence showed homology (about 50 %) with hypothetical membrane proteins in ACS-1-GCV3, YPL221w, YGL139w, MRF1-SEC 27 and YOR 365c of S. cerevisiae and in SPAC1F7.03 of Schizosaccharomyces pombe from BLAST Search.
8. Copper-modulated gene expression in Podospora anserina.C. Borghouts, N. Averbeck, E. Kimpel, H.D. Osiewacz. Johann Wolfgang Goethe-Universität, Botanisches Institut, Frankfurt, Osiewacz, Germany.
Copper is an essential micronutrient and a cofactor of various enzymes (e.g., cytochrome
oxidase, laccase, superoxide dismutase). On the other hand, increased levels of copper are
cytotoxic. Consequently, there is a clear need to control cellular copper levels tightly. Previously,
we have cloned a gene encoding GRISEA, a transcription factor involved in the control of
cellular copper homeostasis (1,2). GRISEA was demonstrated to be an ortholog of MAC1 of
Saccharomyces cerevisiae and appears to control the expression of a gene coding for
a high affinity transporter of copper (3). The DNA binding domain of GRISEA was mapped to
the first 168 amino terminal amino acids. The activation domain is located in the second half of
the protein. At low cellular copper concentrations, GRISEA is involved in the activation of target
gene expression. Increased copper levels lead to a repression of GRISEA, most likely via
intramolecular interactions between different regions of the protein. The role of GRISEA in the
molecular machinery involved in the control of cellular copper homeostasis in P. anserina
will be discussed. References: 1. Osiewacz HD, Nuber U (1996) Mol Gen Genet
252:115-124 2. Borghouts C, Kimpel E, Osiewacz HD (1997) Proc Natl Acad Sci USA 94:
10768-10773 3. Borghouts C, Osiewacz HDMol Gen Genet (in press) Acknowledgment: The
experimental work was supported by a grant of the Deutsche Forschungsgemeinschaft.
9. Cloning and sequencing of chitin synthase genes of four different classes from
Cochliobolus sativus
In our screening for chitin synthase gene fragments from phytopathogenic fungi, we succeeded in getting four gene fragments from Cochliobolus sativus which belong to four different gene classes. To get full length clones, a genomic library prepared in Lambda ZAP II has been screened with each of the four gene fragments as a probe. Positive plaques have been passed through a second and a third screening run to assure the purity of the selected clones. The four clones, termed Csat1, Csat2, Csat3 and Csat4 have been completely mapped and sequenced following three different strategies: production of unidirectional nested deletions of double-stranded DNA clones, chromosome walking using customized primers and subcloning particularly mapped fragments. In all cases, both strands were sequenced. Southern blotting experiments proved that the four Csat genes are single-copy genes. The existence of a fifth gene was not confirmed even by low-stringency hybridizations. For Csat1, a fragment of 4647 nucleotides has been sequenced. The coding region has been identified by comparing its reading frame to published sequences. One putative intron spanning 52 nucleotides has been localized on the open reading frame. The gene encodes for a 998 amino-acid protein. For Csat2, a fragment of 8153 nucleotides has been sequenced. An open reading frame of 3206 nucleotides has been identified (interrupted by one 43 bp intron) which encodes for a 1053 amino-acid protein. For Csat3, a 4549 nucleotide has been entirely sequenced, the open reading frame is interrupted by five introns and encodes for a 900 amino-acid protein. For Csat4, 4618 nucleotides have been sequenced. The gene has two introns of small size and encodes for a 1128 amino-acid protein. The expression of the four chitin synthase genes was studied with mycelium of Cochliobolus sativus grown in liquid culture or on barley plants. Primers specific for each gene and able to distinguish between cDNA and genomic DNA, were designed and used in RT-PCR experiments to monitor gene expression. Interestingly, the four messenger RNAs were present in the tested development stages excluding a differential expression of one particular gene. Furthermore, RT-PCRs produced with RNA samples from fungal spores gave similar results as with myceliums indicating that the regulation of chitin synthase may occur at a post-translational level. Gene disruption experiments have been performed in order to better characterize the role of these genes in fungal growth and eventually validate chitin synthase as a target for novel antifungal compounds.
10. Agrobacterium tumefaciens-mediated transformation of yeasts and filamentous fungi: Effect of the host on T-DNA integration.Paul Bundock 1, Marcel J.A. de Groot 2, Amke den Dulk-Ras 1, Haico van Attikum 1, Alice G.M. Beijersbergen 2, Aaron A. Winkler 1, Yde H. Steensma 1, Paul J.J. Hooykaas 1, and Cees A.M.J.J. van den Hondel1. 1State Leiden University, IMP, Leiden, Zuid Holland, The Netherlands. 2Unilever, Research Laboratory, Vlaardingen, Zuid Holland, The Netherlands.
Agrobacterium tumefaciens is a gram negative soil bacterium induces the formation of crown galls or tumours at plant wound sites. During tumorigenesis, A. tumefaciens transfers a part of its tumour inducing (Ti) plasmid, the T-DNA, to the plant cell where it then integrates. A. tumefaciens is also able to transfer T-DNA to Saccharomyces cerevisiae and Kluyveromyces lactis. We have studied in these hosts the mechanism of T-DNA integration. When a T-DNA carrying sequences homologous to the yeast genome was transferred to S. cerevisiae it integrated efficiently via homologous recombination. This demonstrates that the mechanism of T-DNA integration is determined by the host cell. Furthermore, a T-DNA lacking homology with the S. cerevisiae genome can integrate via illegitimate recombination. We have been able to demonstrate that A. tumefaciens is able to transform the filamentous fungus Aspergillus awamori, demonstrating for the first time DNA transfer between a prokaryote and a fungus. Analysis of the transformed fungi showed that in most cases the T-DNA was present as a single intact copy in the fungal genome. The T-DNA had integrated into the genome via an illegitimate recombination mechanism, as shown by sequencing the genomic DNA flanking the integrated T-DNA copies.
11. Isolation and characterization of hisB, the Aspergillus nidulans homolouge of yeast his3. Silke Busch, Katja Starke, Bernd Hoffmann, and Prof. Dr. Gerhard Braus. University of Göttingen, Molecular Microbiology, Göttingen, Lower Saxony, Germany.
The yeast his3 gene encodes the imidazole glycerolphosphate dehydratase
(E.C.4.2.1.19) of the histidine biosynthetic pathway. We isolated the his3
homologue of Aspergillus nidulans, named hisB, respectively. Sequence
analysis revealed an open reading frame of 801 kb, interrupted by an intron of 58 kb. The cDNA
clone complements the histidine biosynthetic defect of the yeast his3 mutant, which
indicates functional equivalence of these homologs. Further evidence derived from sequence
similarities which range between 54 and 38 % identities to other imidazole glycerolphosphate
dehydratases. A transcriptional regulation of hisB by histidine itself does not occur.
The yeast his3 gene is regulated by the mechanism of general amino acid control.
We showed that the hisB gene in Aspergillus nidulans is
transcriptionally activated after amino acid starvation. Two putative binding sites for the
transcription factor CpcA of the general control pathway of Aspergillus nidulans
could be identified 379 and 228 bp upstream of the translational start point.
12. The carboxy-terminal 23 amino acid region of Aspergillus parasiticus AFLR
is a transcription activation domain.
AFLR, a zinc cluster protein, transactivates the expression of genes in the aflatoxin biosynthetic pathway gene clusters in Aspergillus parasiticus, Aspergillus flavus as well as the sterigmatocystin synthesis gene cluster in Aspergillus nidulans. We showed, by fusion of an A. parasiticus aflR coding region to the GAL4 DNA-binding coding region, that the AFLR carboxy- terminus contained a transcription activation domain. Compared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of three acidic amino acids, Asp365, Glu366 and Glu367, in a previously identified acidic stretch. Removal of the carboxy-terminal amino acid, Glu444, did not affect the activation activity. Substitutions of acidic Glu423, Asp439, or Asp436/Asp439 with basic amino acid(s) resulted in 10 to 15-fold lower activation activities. The Asp436His mutation abolished the activation activity. In contrast, substitutions of basic His428, and His442 with acidic Asp resulted in 20 % and 40 % decrease in the activation activity, respectively. Simultaneous substitutions of Arg427, Arg429, and Arg431 with Leu also significantly decreased the activation activity; the decrease was approximately 50-fold. Results suggest that the carboxy-terminal 23 amino acid region of A. parasiticus AFLR is a transcription activation domain and that total acidity in this region is not a major determinant of AFLR's activation ability.
13. Sequence analysis and characterization of genes expressed during appressorium formation in Magnaporthe grisea. Woobong Choi, Eric G.C. Fang, Maciek Sasinowski, and Ralph A. Dean.
Magnaporthe grisea, the rice blast pathogen, requires the formation of a specialized infection structure called an appressorium to infect host plants. We have initiated an EST (Expressed Sequence Tag) analysis utilizing an appressorium-stage specific cDNA library to identify and characterize genes involved in appressorium formation. To date, 2717 cDNA clones have been sequenced from either 5' or 3' ends. Approximately 45% of the sequences significantly matched (p value < 10-3) sequences in the GenBank database based on BLASTX. Twelve genes were found to be previously identified in M. grisea and several clones showed homology to genes associated with pathogenesis in other plant pathogenic fungi. Differential hybridization analysis using high density library filters with cDNA from different developmental stages identified 631 cDNA clones with appressorium stage specific/up-regulated expression patterns. Characterization and the possible role of these genes in appressorium formation will be presented.
14. Expression of heme proteins in Aspergillus niger. Ana V. Conesa-Cegarra, Peter Punt, Cees AMJJ van den Hondel. TNO Nutrition and Food Research Institute, MGG, Zeist, Utrecht, The Netherlands.
Filamentous fungi are widely exploited for their capacity of high level secretion of enzymes. Molecular genetic approaches for strain improvement have shown that this ability can also be extended to many other proteins of fungal origin. However, a major exception to this rule seems to be high level secretion of fungal metallo-proteins. So far, attempts to overproduce this type of enzymes in recombinant fungal systems have had limited success. To gain insight into the bottle-necks existing for the (over)production of metallo-proteins in filamentous fungi, we have started a project to study the expression of genes encoding heme-containing fungal peroxidases in Aspergillus niger. Three genes were chosen for this study: lignin peroxidase (lipA) and manganese peroxidase (mnp1) encoding genes from Phanerochaete chrysosporium and the chloroperoxidase (cpo) encoding gene from Caldariomyces fumago. Different expression cassettes for each of the three genes have been constructed and used to transform a protease deficient strain of Aspergillus niger. Analysis of the transformants shows expression for all three genes. Manganese peroxidase and chloroperoxidase are both produced as active extracellular enzymes while lignin peroxidase is incorrectly processed resulting in an inactive extracellular protein. Further analysis of the limiting factors for peroxidase overproduction is in progress and will be presented.
15. The Yeast Proteome Database: A research tool available to the fungal research community. Maria C. Costanzo, Peter E. Hodges, Brian P. Davis, Andrew H.Z. McKee, Michael E. Cusick, Kevin J. Roberg-Perez, William E. Payne, and James I. Garrels. Proteome, Inc., Beverly, MA, USA.
The Yeast Proteome Database (YPDTM) is an integrated resource for Saccharomyces cerevisiae protein information encompassing the research literature, functional genomics, and proteomics. It contains about 6100 Yeast Protein Reports, one for each of the known or predicted S. cerevisiae proteins. YPD continues to grow rapidly in terms of size and features. It now contains more than 75,000 free-text annotations derived from a review of more than 9,800 research papers. Each YPD Protein Report presents an extensive tabulation of protein properties, annotations, and references for one protein in a convenient Web page format (see http://www.proteome.com/YPDhome.html). Each protein is now classified by biochemical function and cellular role, quickly alerting users to the biological significance of a protein and also providing a search tool for organizing groups of proteins into cellular processes and molecular machines. Other properties tabulated in each report include mutant phenotype, gene and protein sequence, sequence similarities, protein-protein interactions, regulators of gene expression, and protein modifications. Relationships between genes are shown by BLAST alignments of each protein to other S. cerevisiae proteins, to Drosophila proteins, and to human proteins. Similarities to known proteins of other fungi are also noted in the text annotations. Pop-up windows expand on sections of the Protein Report, showing physical and genetic interactions for each protein/gene, membership in characterized protein complexes, post-translational modifications, and all known regulators (conditions, small molecules, and transcription factors) of expression. The Gene Expression pop-up window of each report also contains a graphical representation of transcription profiles generated using DNA microarray technology, which are accumulating at an ever-increasing rate. For fungal proteins similar to S. cerevisiae proteins, YPD provides a wealth of information that may contain clues as to their biochemical function or cellular role, or may suggest new experimental directions.
16. Activity of transposon Restless in homologous and heterologous hosts. Katarzyna Czechowska, Sabine Jacobsen, Frank Kempken, and Ulrich Kück. Ruhr-Universität Bochum, Allgemeine Botanik, Bochum, NRW, Germany.
The class II transposon Restless, a 4.1 kb hAT-like element from the hyphomycete Tolypocladium inflatum, was shown to be active in strain ATCC 34921 [1]. In order to analyse transposon activity in other T. inflatum strains or in heterologous species, such as Penicillium chrysogenum, we have constructed transposon vectors for DNA mediated transformations. The vectors were designed to confer phleomycin resistance after excision of the Restless element. We transformed different T. inflatum strains and P. chrysogenum with the transposon vectors and demonstrated the integration by PCR and Southern analysis. Transformed strains were further screened for excision events by isolating phleomycin resistant colonies. The molecular analysis of the corresponding isolates will be presented. [1] Kempken F, Kück U (1996) Mol Cell Biol 16: 6563-6572
17. Transposable elements and the organization of the Fusarium oxysporum genome: clusters of DNA transposons and chromosomal rearrangements. Marie-Josée Daboussi1, Aurélie Hua-Van 1, Jean-Michel Davière 1, Thierry Langin2. 1University Paris-Sud, IGM , Orsay, Essonne, France. 2Université Paris-Sud, IBP, Orsay, Essonne, France.
Several families of transposable elements (TEs) are present in the genome of the
phytopathogenic fungus Fusarium oxysporum. They are representative of the major
groups of eukaryotic elements, retrotransposons, LINE-like element and DNA transposons.
These elements are present in copy numbers ranging from a few elements to hundreds per
genome. By cloning random DNA fragments and by sequencing contiguous stretches of genomic
DNA, many other repetitive sequences with features of TEs were discovered. This finding
reveals that earlier studies had uncovered only the tip of TEs in this species. These new elements,
with varying copy number, are full-length or degenerate. They are interspersed in the genome but
appeared not to be randomly distributed. Indeed, the analysis of their distribution on both
chromosomes separated by CHEF and cosmids, showed that some chromosomes contained many
different types of TEs and that elements of a particular family are concentrated in some genomic
regions. Precise composition and arrangement of repetitive DNAs were investigated by
sequencing three regions of the genome surrounding different insertion sites of the
impala element. The relative organization of genes and TEs in these regions showed
that they are composed essentially of intermixed transposable elements of several types, e. g., at
least 11 elements belonging to 6 different families can be found in a contiguous 40kb region.
Some repeats are frequently reiterated and many of them are inserted into other elements. In
addition, part of these regions corresponds to duplicated fragments. The mechanisms involved in
the generation of the particular organization of these regions, as well as their significance, will be
discussed.
18. Genes differentially expressed in a U. maydis mutant of the
cAMP pathway include nitrogen utilization genes.
U. maydis, the basidiomycete causing smut disease in corn plants, switches from a saprophytic yeast-like haploid phase to an infectious filamentous dikaryon after mating. The involvement of the cAMP pathway in morphogenesis was demonstrated by the fact that mutants of uac1, the gene encoding adenylate cyclase, show constitutive filamentous growth1,2. On the other hand, mutants in the gene encoding the regulatory subunit of PKA (ubc1) show a multiple budding phenotype, a defect in filamentous growth after mating and an inability to complete sexual development in the plant2,3. In an attempt to characterize new genes involved in the morphological switch and pathogenesis, we have constructed a library enriched for cDNAs differentially expressed in a ubc1 mutant using the subtractive hybridization technique. Several clones for mRNAs that were up or down-regulated in the mutant were isolated and characterized. One of the up-regulated clones is 52% similar to UGA4, the gene encoding a GABA-specific permease from S. cerevisiae. The region of similarity includes one of the two PKA sites present in UGA4. Another up-regulated clone is highly similar to genes encoding the NADP-linked glutamate dehydrogenase from other fungi, including GDH3, a gene involved in the response to starvation in yeast4. U. maydis strains knocked-out for these two genes are being analyzed. The increased expression of genes related to nitrogen utilization in the ubc1 mutant suggests that the cAMP pathway in U. maydis, as in other fungi, may mediate the response to extracellular nitrogen source. 1.Barret et al., 1993, Mol. Plant-Microbe Interac. 6:274 2.Gold et al., 1994, Genes Dev. 8: 2805 3.Gold et al., 1997, The Plant Cell 9:1585 4.Wilkinson et al., 1996, Microbiol. 142:1667
19. Regulation of a feruloyl esterase gene (faeA) from Aspergillus niger. Ronald P. de Vries, Jaap Visser. Wageningen Agricultural University, MGIM, Wageningen, The Netherlands.
Three factors have been identified involved in the regulation faeA from Aspergillus niger. The expression of the gene on D-xylose is mediated via XlnR, the xylanolytic transcriptional activator and depends on the xylose concentration. A decrease in the expression faeA and three other xylanolytic genes was observed with increasing xylose concentrations in a wild-type strain, whereas expression levels in a CreA mutant were not influenced. Xylose concentrations higher than 1 mM result in repression of the expression of xylanolytic genes mediated by the carbon catabolite repressor protein CreA. The expression levels of faeA and other xylanolytic genes on xylose are therefore not only determined by induction via XlnR but also by repression via CreA. A third factor involved in faeA regulation responds to the presence of certain aromatic compounds with a defined ring structure such as ferulic acid and vanillic acid. The structural requirements necessary for induction have been identified. A benzene ring is present in all inducing compounds, which is substituted at C3 with a methoxy group and at C4 with a hydroxy group. C5 is not substituted, whereas different alifatic groups at C1 of the aromatic ring are allowed. Although the hydroxyl function at C4 is strongly preferred, low levels of expression have also been found with veratryl alcohol and veratric acid, two 3,4-dimethoxy compounds. Expression levels of faeA on combinations of ferulic acid and xylose are higher than on each compound alone, indicating a positive interaction between the activation by aromatic compounds and by XlnR. We acknowledge financial support of Danisco Ingredients, Brabrand, Denmark.
20.Construction of a sequence-ready framework for the rice blast fungus based on fingerprinting contigs and BAC-end sequencing. Ralph A. Dean. Clemson University, Plant Pathology, Clemson, SC, USA.
The rice blast fungus, Magnaporthe grisea, is an important model for fungal
pathogenesis and is a major candidate for whole genome sequencing. To provide a framework for
gene discovery, a strategy to survey the genome randomly was devised. This strategy involves
the sequencing of the ends of large DNA fragments maintained as BAC clones. A large insert
(130 kb) bacterial artificial chromosome (BAC) library with 25-fold coverage (9,216 clones) was
previously constructed using a rice infecting strain 70-15. These end sequences (sequence tag
connectors) in theory are distributed every 2-3 kb along the rice blast genome. Since the rice
blast genome is estimated to contain ~10,000 genes over ~40 Mb, this strategy is anticipated to
reveal the majority of the rice blast genes. Moreover, when coupled with BAC fingerprinting and
contig assembly, this strategy will provide a physical layout of gene organization. The entire
library has been fingerprinted and end-sequenced. The assembled contigs have been anchored
using RFLP markers covering all seven chromosomes. Up to date progress on contig assembly,
contig anchoring and sequence analysis will be presented. The data is publicly available at
www.genome.clemson.edu.
21. Biochemical aalysis of WC-2, a transcription factor required for the biological
clock
White-collar loci products (wc-1 wc-2) have been found to act as global regulators for light perception and the circadian clock in Neurospora crassa. Both WC-1 and WC-2 proteins contain Zn-finger domains with distinct similarity to other transcriptional activators within the GATA factor family. Additionally, both WC's contain PAS domains, evolutionary conserved regions mediating protein-protein interactions. WC-2 antibodies show WC-2 to be predominantly a nuclear protein that does not display an robust rhythm in either the nuclear or cytoplasmic fraction. Moreover, we do not detect a significant change in level following a light-pulse. This surprising lack of regulation prompted us to determine if regulation could be at the level of association with other proteins. On sucrose gradients we find WC-2 migrating in two peaks; the ~60 KDa low molecular weight peak corresponds to the monomeric form, whereas the ~200 kDa high molecular weight peak has a broad tail trailing towards ~540 kDa. As expression of a central component of the Neurospora clock, frq, requires WC-2 (Crosthwaite et al. Science 276:763-769, 1997) and FRQ is also known to repress it's own expression, one possibility is that FRQ interacts directly with WC-2, thereby interfering with transcriptional activation. To address this we performed in vitro binding assays of GST-tagged WC-2 and radiolabelled FRQ proteins. Under these conditions, WC-2 specifically interacts with FRQ. Studies are currently underway to determine if this interaction can be demonstrated in vivo.
22. Cloning and characterization of two novel genes from Aspergillus niger encoding peptidyl prolyl cis-trans isomerases belonging to the cyclophilin family. Patrick M.F. Derkx, Susan M. Madrid. Danisco Ingredients, Danisco Biotechnology, Copenhagen, Copenhagen, Denmark.
Aspergillus, a filamentous fungus, is widely used for the production of homologous and heterologous proteins but, compared to homologous proteins the production levels of heterologous proteins are usually low. Low levels of secreted proteins may be due to limitations at the post translational level. Peptidyl prolyl cis-trans isomerase (PPI) is a foldase which catalyzes the cis-transisomerization of peptide bonds preceding proline residues. Two A. niger genes encoding cyclophilin like PPIs have recently been cloned by heterologous screening of genomic and cDNA libraries. These genes encode a protein of approx. 20 kDa in size and both proteins contain a PPI specific catalytic core domain and a region involved in the binding of cyclosporin A. The CYPA protein, lacking signal sequence and ER retention signal, is most likely targeted to the cytosol. The CYPB protein however, contains a signal sequence and an ER retention signal which is responsible for targeting and retention of proteins in the ER. Transcription of cypB was shown to be induced by stress caused by unfolded proteins and heat shock whereas the transcription of cypA only increases moderately after heat shock. It is therefore likely that CYPB plays an important role in the folding of secretory proteins. Acknowledgments This work was funded by an EC Biotechnology program grant BIO2 CT-942045
23. Construction of a plasmid for expression of glycosylated bovine beta-casein in Aspergillus oryzae. Todd Z. DeSantis1, Gabe Overbay1, Rafael Jimenez-Flores 2, Susan Elrod1. California Polytechnic State University, Biological Sciences, San Luis Obispo, CA, USA. 2 CalPoly State University, Dairy Prod. Tech. Center, San Luis Obispo, CA, USA.
Beta-casein is a non-glycosylated, monomeric milk phosphoprotein with a hydrophilic
domain in its N-terminal region and a contrasting hydrophobic domain at its C-terminal end. This
amphiphilic property makes beta-casein a good surface active agent that has applications in the
food industry as well as the potential for use in biodegradation of marine petroleum spills. The
bovine beta-casein gene has been previously cloned and mutated to contain a novel glycosylation
site at Asn73, near the N-terminus. This glycosylated protein has been shown to
possess enhanced emulsifiying properties. It has also been demonstrated to act as an antifreeze
agent. Previous attempts to produce enough protein for further study have not been very
successful. Expression in a transgenic mouse system yielded 2 mg/ml, however the high cost of
maintaining this system makes it impractical. Expression in a Pichia yeast system
yielded <1 mg/l and the protein was not secreted. Here, we report the construction of a
plasmid for expression of glyco-beta-casein cDNA in Aspergillus oryzae. The 5' end
of the cDNA was fused in frame to the promoter and secretion signal from the A.
oryzae TAKA amylase gene while the 3' end was fused to the glucoamylase transcriptional
terminator from A. awamori. Fusions were accomplished using the "Splicing by
Overlap Extension" (SOE) PCR technique and cloned into pCR-Script. This plasmid is being
used to transform A. oryzae. Glyco-beta-casein expression levels will be measured
using protein gel electrophoresis and Western blots with bovine beta-casein-specific
antibodies.
24. A novel ATP-binding cassette transporter involved in multidrug resistance in the the
filamentous fungus Aspergillus nidulans
Multidrug resistance can be caused by increased ATP-dependent efflux of toxic compounds from cytoplasm and plasma membrane mediated by membrane-bound ATP-dependent transporters of the ABC (ATP-Binding Cassette) superfamily. As a preliminary step to characterizing genes encoding ABC-proteins that confer multiple drug resistance in Aspergillus nidulans, we are using a PCR-based approach. Five degenerated primers designed based on the coding regions of the ATP-binding cassette were used to identify such genes. DNA fragments in the expected size were amplified. These fragments were cloned and several subclones were sequenced. Sequence and Southern blot analysis showed that these fragments correspond to four different genes encoding ABC-transporters (named abcA-D, respectively). Northern blot analysis showed that the abcD gene transcript size is around 6.0-kb and this gene is induced about five-fold by miconazole, three-fold by ethidium bromide, and two-fold by camptothecin. Financial support: FAPESP, CNPq, and CAPES, Brazil, and ICGEB-UNIDO.
25. Biochemical and molecular responses to exogenous sterols by Phytophthora spp. W. David. Dotson, Shirley R. Tove, Leo W. Parks. North Carolina State University, Microbiology, Raleigh, NC, USA.
Phytophthora species are eukaryotic natural sterol auxotrophs. Sterol biosynthesis is blocked at the level of squalene epoxidation in Phytophthora. Remarkably however, vegetative growth of Phytophthora can occur even in the complete absence of sterols. Growth of these organisms is enhanced, often dramatically, when an exogenous source of sterols is provided. Furthermore, sterols may be required for the induction and development of sexual spores in Phytophthora. Using differential display, we have seen evidence for altered gene expression patterns in response to the presence of sterols. Using a low stringency Southern blot method, we have looked for homologs of yeast sterol metabolic genes in P. cactorumand P. parasitica. Hybridization patterns have implicated Phytophthora sequences similar to the sterol C5 desaturase but not to the C22 sterol desaturase gene of Saccharomyces cerevisiae. Through sterol feeding experiments and chromatographic analysis we have confirmed the existence of both C5 desaturase and delta 7 reductase activities in Phytophthora and the absence of C22 desaturase activity. Evolutionary conservation of these sterol conversions indicates their physiological significance to Phytophthora. While the physiological consequences of these conversions remain unclear, further elucidation of sterol function and metabolism in Phytophthora should facilitate more rationally designed approaches towards the control of these important pathogens.
26. Longevity and stability of the mitochondrial chromosome in Podospora anserina greatly depends on the respiratory metabolism.Eric Dufour, Odile Begel, Jocelyne Boulay, Béatrice Albert, and Annie Sainsard-Chanet. C.G.M, C.N.R.S, GIF-sur-YVETTE, 91190, FRANCE
The vegetative growth of the filamentous fungus Podospora anserina is limitated. This senescence process is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. Although the link between senescence and mitochondrial instability is well established in Podospora and in different fungi, the nature of this link and the control of the mitochondrial DNA integrity are not clearly understood at the present time. Whereas some data suggested that the mobile group II intron alpha (the first intron of the cytochrome c oxidase subunit I gene,cox1) has a prominent role in the senescence process in Podospora anserina, others indicated that it is not the case for some nuclear mutants. We describe the first mutant of Podospora anserina precisely deleted for this intron and show that it displays a senescence syndrome similar to that of the wild-type though its lifespan is increased about two-fold. We also describe a nuclear mutant in which subunit 5 of cytochrome c oxidase is inactivated. This mutant uses an alternate respiratory pathway. We show that it escapes senescence as the spontaneous mitochondrial mutants mex, deleted for a part of intron alpha and of the upstream exon. Interestingly, these results indicate that « immortality » in Podospora anserina can be acquired not by the lack of intron a but by the lack of active cytochrome c oxidase and that the respiratory metabolism plays a major role in the control of the mitochondrial DNA integrity. The factors involved in this control (possible antioxygen defense role of the alternate oxidase, reduced energetic statement, modification in the import mitochondrial apparatus) will be discussed.
27. The [URE3] prion of Saccharomyces cerevisiae is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments. Herman K. Edskes, Kimberly T. Taylor, Vaughn T. Gray, Reed B. Wickner. NIDDKD, NIH, Laboratory of Biochemistry and Genetics, Bethesda, MD, USA.
Genetic evidence identified [URE3] as the prion form of Ure2p. Ure2p functions in blocking assimilation of poor nitrogen sources in the presence of a good nitrogen source (e.g. ammonia or glutamine). In its prion form this function is lost but the protein has acquired the ability to convert its normal form into the altered prion form. The N-terminal region of Ure2p (Ure2p1-65) has been designated the prion domain as it is sufficient to propagate [URE3]. Its overexpression induces the de novo appearance of [URE3] by 1000-fold. The C-terminal region of Ure2p carries out the nitrogen regulatory function. A fusion between Ure2p or Ure2p1-65 and green fluorescent protein (GFP) is aggregated in cells carrying [URE3] but evenly distributed in cells lacking the [URE3] prion. The Ure2p C-terminus when fused to GFP is evenly distributed regardless of the presence or absence of [URE3]. Overexpression of fragments of Ure2p or Ure2-GFP fusion proteins efficiently cure the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] 'crystal' of Ure2p poisons its propagation.
28. Characterization of promoter elements involved in the expression of early and late aflatoxin pathway genes. Kenneth C. Ehrlich, Jeffrey W. Cary, and Beverly G. Montalbano. Southern Regional Research Center/USDA, Food and Feed Safety, New Orleans, LA, USA.
Aflatoxins are the most potent fungal carcinogenic metabolites. Enzymes involved in aflatoxin biosynthesis in Aspergillus parasiticus are encoded by as many as 17 clustered genes. Most of these genes are co-regulated by the C6-zinc cluster DNA-binding protein, AFLR, as evidenced by the presence of binding sites for AFLR in the promoter regions of those so far characterized. Expression of the gene encoding AFLR, and the genes for polyketide formation,fas1A, fas2A, and pksA, occurs prior to expression of genes encoding oxidative enzymes involved in later steps in the biosynthetic process, such as avnA, ver1 and omtA. The aflR and avnA promoters were analyzed using Beta-glucuronidase reporter assays to elucidate regions involved in transcription control. Maximal promoter activity for both genes was found in A. parasiticustransformed with constructs in which the promoter was truncated at -118 bp. Approximately 20% of the maximal activity occurred when the promoter was truncated at -100, thereby removing putative AFLR binding sites. Based on electrophoretic mobility shift assays, only AFLR bound to sequences from -100 to -118 in the avnA promoter, whereas, in addition to AFLR, a protein or proteins in nuclear extracts from A. parasiticus grown on inducing medium bound to an adjacent or overlapping site in the aflR and pksA promoters. Sites for binding the transcription factor that mediates regulation by ambient pH, PACC, were detected in the aflR and pksA promoters, but not in the avnA promoter. PACC binding could be involved in negative regulation of gene expression when the fungi are grown in aflatoxin non-inducing media.
29. Effect of site-directed mutagenesis of the magB gene on G protein signaling in Magnaporthe grisea. Eric G.C. Fang, Ralph A. Dean. Clemson University, Plant Pathology, Clemson, SC, USA.
MagB, encoding a G alpha subunit, is involved in signaling pathways that regulate a number of cellular responses in M. grisea, including appressorium formation, conidiation, sexual development, mycelial growth, and surface sensing. Site-directed mutagenesis was used to further dissect the pleiotropic effects controlled by magB. Conversion of glycine 42 to arginine (magBG42R) was predicted to abolish GTPase activity, which in turn constitutively activates G protein signaling. This mutation caused autolysis in the aged cultures, misscheduled melanization, reduction in both sexual and asexual reproduction, and reduced virulence. Furthermore, magBG42Rmutants were able to produce appressorium on both hydrophobic and hydrophilic surfaces, although the development on the hydrophilic surface was delayed. A second dominant mutation magBG203R (glycine 203 converted to arginine) was expected to block dissociation of the G beta gamma from G alpha subunit thus producing a constitutively inactive G protein complex. This mutation did not cause drastic phenotypic changes in the wild type genetic background, but complemented the conidiation defect in magB- mutants. These results strongly suggest the involvement of the G beta gamma subunit in signaling pathways regulating cellular development in M. grisea.
30. The sequence requirements for arginine attenuator peptide function in arginine-specific translational regulation. Peng Fang, Zhong Wang, and Matthew S. Sachs. Oregon Graduate Institute, Biochem & Mol Biol, Portland, OR, USA.
The Neurospora crassa arg-2 mRNA contains an evolutionarily conserved, 24-codon upstream open reading frame(uORF)in its 5'-leader. This uORF encodes the arginine attenuator peptide (AAP) which, when translated in the presence of high [Arg] causes ribosomes to stall at its termination codon (codon-25), reducing translation of the mRNA. We examined the role of AAP peptide and RNA sequence on translational control using an N. crassa cell-free translation system and primer-extension inhibition assay to map ribosomes on the mRNA. Deletion of the relatively non-conserved AAP N-terminus had no significant effect on AAP-mediated translational regulation, but continued deletion into the highly conserved region eliminated regulation. Parallel introduction of silent mutations at each possible codon where substitution was possible in a functional, shortened AAP coding region(28/63nt changes) did not significantly affect AAP function, although single nucleotide changes altering the conserved peptide sequence eliminated function. Finally, when a rare Leu codon (UUA) but not a common Leu codon (CTC) was inserted at position 25 in an AAP-luciferase fusion polypeptide, substantial Arg-regulated ribosome stalling was seen at the rare Leu codon but not the common Leu codon. These data indicate that the sequence of the evolutionarily conserved nascent peptide, but not the sequence of the mRNA that encodes it, is responsible for arginine-specific translational attenuation. They suggest that the mechanism by which Arg and the nascent AAP serve a regulatory function is enhanced when the ribosome encounters termination or rare codons immediately after AAP translation. Possibly such encounters provide more time or a more favorable environment for the AAP to exert regulatory function. (Supported by NIH GM47498.)
31. Expression of Nit-3 gene of nitrate catabolic pathway in N. crassa is under general negative control. Bo Feng. The Ohio State University, Biochemistry, Columbus, Ohio 43210, USA.
The activated expression of nit-3 gene encoding nitrate reductase in Neurospora is regulated by the synergistic action of the global positive regulator NIT2 and the pathway-specific positive regulator NIT4. The physical NIT2-NIT4 interaction is critical for the synergy between these two proteins. A mutant NIT2 that failed to interact with NIT4 resulted in defective nit-3 gene activation and nitrate utilization. Reverse genetics was used to isolate nitrate utilization revertants in the background of a NIT2 zinc finger mutation lacking the ability to interact with NIT4. The revertants, snnb1 and snnb2 (Suppresser of NIT2 Non-NIT4 Binding mutation) regained the ability to activate nit-3 expression independent of NIT2-NIT4 interaction, suggesting that mutation in either snnb1 or snnb2 bypassed the requirement of NIT2-NIT4 interaction for activating nit-3 gene. Snnb mutants also resulted in the abnormal exression pattern of nit-3, rendered it partially insensitive to nitrogen repression and independent of nitrate induction. Further analysis showed that mutation in snnb2 also resulted in defects in glucose repression of beta-galactosidase expression. Moreover, snnb mutants exhibited either delayed conidiation or aconidiation, female sterility and semicolonial morphology, indicating a general role of snnb loci in regulating cell functions. This study suggests that the nit-3 gene in nitrate assimilation pathway is subjected to general negative regulation. The NIT2-NIT4 interaction may conteract the negative effects of snnb, thus allowing regulated, yet high level, nit-3 expression. (This work was supported by grant GM23367 from the National Institute of Health)
32. Schizophyllum commune's "Scooter" is a member of an active transposon family and has disrupted thn, a homolog of fungal regulatory genes. Thomas J. Fowler, Micheal F. Mitton, and Carlene A. Raper. University of Vermont, Microbiology & Mol.Genet., Burlington, VT, USA.
Several DNA sequences from Schizophyllum commune that represent a family
of transposable elements called sct (Schizophyllum
commune transposon- nicknamed "Scooter") have
been characterized. The original isolation of sct was from an insertional mutation of
the mating-type receptor gene, bbr2. A nonfunctional bbr2 was cloned
and shown to contain a 647 base pair (bp) insertion. The inserted DNA had two features of a
transposable element: an 8 bp target site duplication (TSD) and 32 bp inverted repeats at each
end. The sct DNA sequence had neither significant similarity with other sequences
in DNA databases nor any long open reading frames. Genomic Southern blot analysis of several
Schizophyllum strains, using sct as a probe, showed up to twenty DNA
fragments with significant hybridization. PCR was used to recover other possible members of the
sct family. One amplified product, of approximately 1200 bp, contained nearly all of
the sct sequence and had additional sequence located between the inverted repeats.
This larger element, sct2, was used as a probe to screen a cDNA library. Two
positive clones are being characterized for their potential to encode a scttransposase.
In a related experiment, a colony was identified that had a sector of the frequently occurring
mutant phenotype known as thin. We determined that the thin sector was the result of a
sct insertion into a gene designated thn. The inserted DNA caused 8 bp
TSDs and was 98% identical to the original sct. The thn mutation was
complemented by DNA-mediated transformation with a wild-type allele of thn. The
thn gene product is related to a regulator of conidiation in Aspergillus
nidulans and a negative regulator of signal transduction in Saccharomyces
cerevisiae.
33. Regulation of the fmdS gene encoding formamidase in Aspergillus
nidulans.
Fungi are capable of utilising numerous unrelated carbon and nitrogen sources, with the expression of the genes involved often strictly regulated. A major form of this regulation occurs as the phenomena of nitrogen metabolite repression (NMR) and carbon catabolite repression (CCR). Amide utilisation by A. nidulans has been extensively studied in our laboratory through analysis of the complex regulation of amdS. The primary substrate of amdS is acetamide, with expression affected by NMR, CCR and induction by acetate and omega-amino acids. The utilisation of formamide is mediated by a different structural gene, fmdS. We have cloned the fmdS gene and found that it encodes a protein belonging to the urease family of enzymes, rather than the amidase signature group. Unlike amdS, fmdS expression is primarily regulated via NMR and does not require addition of exogenous inducer. Despite the role of formamide as a nitrogen source only, fmdS displays a novel form of carbon regulation. Under carbon starvation conditions expression decreases dramatically, the opposite response to genes regulated by CCR. To further analyse fmdS regulation a series of reporter gene fusions were created, helping to determine specific sites of action for AreA (the major NMR regulatory protein). A binding site required for the A. nidulans CCAAT-binding factor has also been identified, with fmdS expression reduced twofold in a hapC deletion mutant. Sequence analysis and isolation of cDNAs show that a gene of unknown function lies directly 5' of fmdS, with 936bp of overlap with the fmdS transcript. The 5' gene has been disrupted through insertion of the riboB gene, with no phenotype yet discovered. The role of this gene may potentially be in nitrogen catabolism, as RT-PCR has shown it to be regulated in response to nitrogen limitation.
34. Characterization of signals for de novo DNA methylation in vegetative cells of Neurospora crassa. Michael Freitag, Vivian Miao, and Eric U. Selker. University of Oregon, Inst. of Mol. Biol., Eugene, OR, USA.
Like DNA of many other eukaryotes, Neurospora DNA can be modified by cytosine methylation. Our goal is to understand how specific cytosines are targeted for methylation. Most DNA sequences that are subject to de novo methylation in vegetative cells of Neurospora have previously undergone repeat-induced point mutation (RIP). RIP introduces C:G to T:A mutations and enriches DNA for A+T in general and TpA dinucleotides specifically. We carried out a detailed dissection of a relic of RIP, the zeta-eta region, to elucidate which mutations induced by RIP and/or which features of mutated DNA create methylation signals. We report results from tests of hybrid constructs involving segments of eta and its unmutated homologue, theta, and of studies with methylation signals created by in vitro mutagenesis. We show that the polarity of RIP (C:G to T:A mutations) is essential for creating signals for de novo methylation from unmutated Neurospora DNA. Whereas both increases in A+T content and TpA density of DNA contribute to the strength of methylation signals, a larger effect was found by increasing the TpA density without altering A+T content when compared to fragments that were very A+T-rich but had low TpA densities.
35. Resection of the frequency promoter of Neurospora crassa. Allan C. Froehlich, Yi Liu, Jay Dunlap, and Jennifer Loros. Dartmouth Medical School, Biochemistry, Hanover, NH, USA.
The circadian system in Neurospora crassa is among the best understood of any organism. The oscillator consists of an autoregulatory feedback cycle, wherein the frequency gene encodes for two forms of the FRQ protein which negatively feed back on their own expression resulting in rhythmic levels of both frq RNA and protein. The level of action of the negative feedback, transcriptional versus post-transcriptional, is being investigated using frq::hph reporter constructs. The N. crassa circadian system can also be entrained, both by light and by temperature. Temperature entrainment is translationally regulated, whereas light resets the N. crassa clock by rapidly inducing frq at the transcriptional level. A small region of the promoter responsible for the light response has been isolated using cis-analysis, and factors interacting with this region are currently being examined.
36. Ashbya gossypii as a model system for fungal functional genomics. Thomas D. Gaffney1, Albert Flavier1, Krista Gates1, Michelle Kirksey1, John Marhoul1, Joann Gardner1, Steve Goff1, Fred Dietrich2, Peter Philippsen2. 1 Novartis AgBiotechResearch, Res. Tri. Pk., NC, USA. 2 U. of Basel, Biozentrum, Applied Microbiology, Basel, Switzerland
The filamentous ascomycete Ashbya gossypii was first identified in 1915 as a
pathogen of cotton, and described more extensively in 1926 by Ashby and Nowell (Annals of
Botany 40:69-86). It has been noted as a particularly destructive pathogen, capable of inflicting
severe damage on developing cotton bolls and making it impossible to grow cotton in certain
parts of the world (U.S. Department of Agriculture Technical Bulletin No. 1469 (1973)). Further
reports indicate that Ashbya is also a pathogen of tomato and various citrus fruits, and is vectored
by sucking insects such as Antestia and Dysdercus. Additional features of Ashbya
gossypii make it an appealing microorganism for a functional genomics approach: 1)
One of the smallest known eukaryotic genomes - 8.8 million base pairs. 2) Very efficient
homologous recombination, allowing simple gene knockout strategy and precise positioning of
reporter gene constructs. 3) Yeast replicons function in Ashbya, allowing efficient introduction
of heterologous DNA. 4) Lack of the extensive duplication of chromosomal segments
observed in yeast - should allow identification of phenotypes "masked" by duplication in yeast.
5) Useful model system for identification of genes and pathways required for normal filamentous
growth. Here we report on a sampling of the results obtained thus far with the Ashbya model
system.
37. Cytochrome c expression in Aspergillus nidulans.
The filamentous fungi Aspergillus nidulans is an obligate aerobe, and therefore generates its main energy requirements by means of oxidative respiration, using the cytochrome c respiratory pathway. This is in contrast to the yeast Saccharyomyces cerevisiae, which can grow without oxygen. S. cerevisiae switches off the production of many oxidative respiratory components (such as cytochrome c) when energy can be produced by alternative means (fermentation) which do not require oxygen. Surprisingly, A. nidulans appears to regulate the production of cytochrome c in a similar manner, even though it appears to have an absolute requirement for oxygen. A functional analysis of the A. nidulans cytochrome c gene (cycA) promoter is currently being carried out to determine the molecular basis of regulation of the gene. In particular, the focus will be on the HAP1 and HAP2 regulatory proteins, which are known to affect cytochrome cexpression in yeast, since consensus sequences for the binding sites of these proteins have been found in the cycA promoter, and the gene is known to be transcriptionally induced by oxygen. Another intriguing observation is that cytochrome c deficient mutants of A. nidulans which were created at Massey University by targeted gene disruption (Bird, 1996) are viable upon fermentable carbon sources. These results suggest the mutant strains must be using alternative means of energy production which do not require cytochrome c. The extent to which these mutant strains (as well as wildtype strains) are utilising an "alternative" respiratory pathway and fermentation has been investigated using respiratory measurements and ethanol assays, respectively.
38. Attempts at cultivating wild strains of various Agaricus species.József Geml1 and Dr. Imre Rimóczi2.1Korona Spawn Plant and Research Laboratory, H-3395 DEMJÉN Pf. 1., Hungary and University of Horticulture and Food Industry, Department of Botany, BUDAPEST. 2University of Horticulture and Food Industry, Department of Botany, H-1118 BUDAPEST Ménesi u. 44., Hungary.
The mushroom produced in the greatest amount today is Agaricus bisporus. Beside this species some other Agarics have been examined by mushroom breeders, although growing them is not in practice yet (perhaps there is one exception: A. bitorquis, but its cultivation takes place in much smaller scale). The importance of using wild varieties of A. bisporus in breeding new commercial strains has been realized by several researchers in the last decade. These wild types can be used to improve the commercial strains' growing characteristics, resistance to pests and diseases etc. In our laboratory we made the first steps of this long way, collecting wild varieties of several species, bringing them into cultivation and making some initial observations which can be useful in the future. In this paper we are going to introduce the main Agaricus species of Hungary, including their descriptions and habitats with some experiences in culturing and cultivating tribes of them.
39. Catalase activity is necessary to heat-shock recovery in Aspergillus nidulans germlings. Gustavo H. Goldman 1, Maria A. Noventa-Jordão 1, Ricardo M. Couto 1, Maria H. Goldman2, J. Aguirre3 Suresh Iyer4 Allan Caplan4and Hector F. Terenzi2. 1FCFRP-Universidade de Sao Paulo, Ciencias Farmaceuticas, Ribeirao Preto, Sao Paulo, Brazil. 2FCLRP-USP, Biologia, Ribeirao Preto, Sao Paulo, Brazil. 3AM, Inst.Fisiol.Cel., Mexico, Mexic. 4University of Idaho, MMBB, Moscow, Idaho, USA.
To understand the molecular mechanisms induced upon stress that contribute to the development of tolerance in eukaryotic cells, we have chosen the filamentous fungus Aspergillus nidulans as a model system. Here, we report the response of A. nidulans germlings to heat-shock. The heat treatment dramatically increased the concentration of both mannitol and trehalose. We have found that the defense against the lethal effects of heat exposure depends on the activity of the defense system against oxidative stress. We show that treatment with hydrogen peroxide increases A. nidulans germling viability after heat shock. In addition, mutants deficient in the key antioxidant enzyme catalase were more sensitive to a 50 oC heat exposure. Under the tested conditions, the catA mRNA accumulated upon heat-shock while catB mRNA levels remained unaltered. Financial support: FAPESP and CNPq, Brazil, and ICGEB-UNIDO
40. Tagging of genes that confer multidrug resistance in Aspergillus nidulans by restriction enzyme-mediated integration (REMI). Gustavo H. Goldman 1, Cristiane C. de Souza, and Maria Helena S. Goldman2. 1FCFRP-Universidade de Sao Paulo, Ciencias Farmaceuticas, Ribeirao Preto, Sao Paulo, Brazil. 2FFCLRP-USP, Biologia, Ribeirao Preto, Sao Paulo, Brazil.
As a preliminary step to characterizing genes that confer pleiotropic drug resistance in
Aspergillus nidulans, we isolate transformants by REMI (Restriction
Enzyme-Mediated Integration) that show pleiotropic drug sensitivity. We have used a plasmid
containing the pyr4 gene to transform an A. nidulans pyrG mutant in the
presence of BamHI. One thousand two-hundred sixty-seven transformants were
isolated using the plasmid pRG3 digested with BamHI. Southern analysis of these
mutants, and of randomly selected transformants was consistent with the occurrence of single
plasmid integration events in about 70 % of the cases. Approximately 900 of these transformants
were examined for sensitivity to fourty-seven drugs or stress agents with different and/or the
same mechanism of action. Thirty-five transformants displayed sensitivity to a single drug (either
itraconazole, miconazole, hygromycin or cycloheximide) while six of them displayed
multidrug-sensivity. The pyr4 marker was shown to be tightly linked to the mutant
phenotype in only one from these multidrug-sensitivity transformants. Financial support:
FAPESP and CNPq, Brazil, and ICGEB-UNIDO
41. Using time lapse video to analyze the circadian rhythm of Neurospora
Neurospora crassa expresses a clearly defined circadian rhythm of conidiation with a period of 21.5 hours. We have monitored this rhythm using time-lapse video under constant red light. It was found that under these rhythmic conditions the growth front would proceed past the eventual location of conidiation with no conidiation at the front; once the growth front was at the end of the area of eventual conidiation, then development of conidia in all areas of that band occurred in unison. Densitometry analysis and curve fitting using individualized frames was used to determine peak times of conidiation. The accuracy of this technique was found not to be significantly different than the classical interpolation technique use by Sargent and others. Neurospora were subjected to a 1 hour 38C pulse during different times of the 21.5 hour period and a time lapse video of the phase response curve was produced.
42. Identification, characterization and chromosomal localization of two putative histone deacetylases from Aspergillus nidulans. Stefan Graessle, Peter Loidl, Hubertus Haas, Markus Dangl, and Gerald Brosch. Innsbruck, Microbiology, Med. School, Innsbruck, Tirol, Austria.
Growth and development of prokaryotic and eukaryotic cells depends on a coordinated gene expression. Thereby regulation on the level of transcription plays an essential role in all organisms. In eukaryotes, transcriptionally active genes are preferentially localized in genomic regions enriched in acetylated histones. The dynamic equilibrium of core histone acetylation is maintained by histone acetyl-transferases and deacetylases. Both enzymes often function as components of regulator complexes targeted to particular genes by DNA binding transcription factors. Using PCR approaches, we have cloned and sequenced cDNA fragments of arpd3 and ahos2, two putative histone deacetylases from Aspergillus nidulans, which are the first deacetylases to be analyzed from a filamentous fungus. Comparisons of the cloned sequences with the GenBank database revealed high similarity to RPD3-type deacetylases from Saccharomyces cerevisiae. Hybridization of these cDNA fragments with a chromosome-specific cosmid library of A. nidulans allowed the chromosomal localization of both genes and led to the genomic sequence of arpd3. Moreover, comparison between this sequence and the corresponding cDNA revealed 3 introns interrupting an open reading frame of 2028 bp, which encodes a protein of 676 amino acids with a predicted molecular weight of 75 kDa. Compared to different yeast RPD3-type deacetylases the deduced ARPD3 amino acid sequence reveales a considerable extension of the C-terminus. Currently, we are using northern hybridization analysis in order to determine the expression levels of ARPD3 and AHOS2 in different developmental states and distinct growth conditions of the fungus.
43. Homologous transformation of Fusarium venenatum. Alison M. Griffen, Marilyn G. Weibe, Geoff D. Robson, and Anthony P.J. Trinci. University of Manchester, Sch. Biological Sciences, Manchester, Manchester, UK.
Although F. venenatum has been transformed successfully using heterologous vectors the transformation rate is low. F. venenatum is a good host for heterologous protein production as it is already cultured in 150 m3 fermenters for the production of Quorn. We have been developing a homologous transformation system based on the hygromycin resistance plasmid pAN7-1 and ribosomal DNA. A 1.2 kb rDNA fragment was amplified by PCR from F. venenatum genomic DNA. This rDNA fragment was cloned into pAN7-1 to generate pAGF-37 and pAGF-48 which contain one and two copies of the rDNA fragment respectively. These plasmids have been used to transform protoplasts of F. venenatum giving 4.9 transformants per ug DNA. The transformants obtained have been assessed for their ability to grow on varying concentrations of hygromycin B. Southern analysis of genomic DNA has determined whether the vectors have integrated at the rDNA locus and given an indication of copy number. We have now introduced the glaA gene from Aspergillus into pAN7-1 containing an rDNA fragment. Using this vector we hope to determine whether previously low heterologous glucoamylase production was the result of poor expression from a heterologous promoter or the result of poor integration.
44. Breeding and cultivating wild Pleurotus strains in hungary.Csaba Hajdú. Korona Spawn Plant and Research Laboratory, H-3395 DEMJÉN Pf. 1., Hungary.
The oyster mushrooms (Pleurotus sp.) are cultivated in the largest quantity in the world after Agaricus bisporus. The cultivation of this mushroom has been increased in some areas of the world, so its importance is undoubted. In Hungary, the total quantity of all cultivated mushrooms was about 33,000 tons in 1997 and around 10 % of it was Pleurotus ostreaus. Hungary was one of those countries where oyster mushroom cultivation started as early as in the 60's. Due to our famous researchers and growers, since that time we have obtained lots of worldwide important pioneer results (successful new strains, improved growing methods etc.). This paper gives an overview about the research and breeding work of the Korona Spawn Plant and Research Laboratory; including the collecting wild strains of P. ostreatus, P. pulmonarius and other species, selecting the best ones, creating and growing new hybrids of them.
45. Mutagenic DNA-repair genes in Aspergillus nidulans: The uvsI gene encodes a REV3 homologue, a subunit of the non-essential DNA polymerase zeta. Kyu-Yong Han 1, Suhn-Kee Chae 2, and Dong-Min Han 1. 1Wonkwang University, Division of Life Science, Iksan, Chonbuk, South Korea. 2Paichai University, Division of Life Sciences, Taejon, South Korea.
Reductions of spontaneous and UV-induced reversion of certain mutant alleles have been shown in uvsI mutant strains of Aspergillus nidulans. To facilitate cloning of the uvsI gene on MMS containing plates, uvsI, uvsA double mutants exhibiting very high MMS-sensitivity were used as a transformation host, since either single mutant was no more than slightly sensitive to MMS. An uvsI-complementing clone was obtained from a chromosome III specific library. Sequence determination of a minimally localized DNA fragment having the uvsI-complementing activity within the clone revealed an ORF with the highest amino acid identity to yeast REV3, a subunit of the DNA polymerase zeta involved in translesion DNA synthesis. UV-survival of heterozygous diploids of uvsI501 with a disruptant of the cloned gene demonstrating the same UV-survival curve as that for homozygous uvsI501 diploids confirmed that the cloned gene is the uvsI. The uvsIORF encodes a polypeptide of 1,681 amino acids with calculated MW of 191.4 KDa. One small intron of 54 bp at near the N-terminus is confirmed by sequencing of RT-PCR products. A Northern blot band of about 5.3 kb was detected. In UVSI, the well- conserved regions, I-VI, among DNA polymerases were present in the correct order. In addition, two zinc-finger motives, [C-X2-C]-X11-[C-X2-C] and [C-X2-C]-X10-[C-X4-C], existed similarly to REV3. Further sequencing of an upstream region of uvsI revealed another ORF of 1,401 bp without putative intron. This ORF resides very close to uvsI in opposite direction and encodes a putative polypeptide exhibiting high amino acid similarity to two hypothetical Arabidopsis proteins. A possible relationship between uvsI and the ORF is currently being carried out.
46. Regulation of the expression of the xylanolytic enzyme system in Aspergillus niger. Alinda A. Hasper, Alinda A. Hasper, and Leo H. De Graaff. Wageningen Agricultural University, Molecular Genetics of Industrial Microorganisms, Wageningen, Gelderland, The Netherlands.
Little is known about the mechanism of pathway-specific induction of extracellular enzyme systems in fungi. Induction of polysaccharide degrading enzyme systems, as e.g.xylanases, depends on low molecular weight inducers, which can be taken up by the organism. In Aspergilli, has been shown that in addition to xylobiose, D-xylose is able to induce the xylanolytic system. However, it is not clear whether the induction is the direct effect of D-xylose or that the inducing compound is the result of a transglycosylation reaction, e.g catalysed by b-xylosidase. The gene encoding A. niger b-xylosidase, xlnD, has been cloned and the role of b-xylosidase in the induction process studied. Furthermore, A. niger mutants with decreased xylanolytic gene expression were isolated by using a xylan induction-responsive element of the endo-xylanase encoding gene xlnA of A. tubingensis. Endo-xylanase activity of these mutants decreased a 300-500-fold in comparison to the wild-type strain. Also a strong decrease is found for b-xylosidase activity in these mutants. By mutant complementing, the transcriptional activator xlnR was isolated. XlnR encodes a protein of 875 amino acids with a domain capable to form a Zn binuclear cluster. Besides this region no extensive homology was found to other transcription activators. By sequencing the xlnR allele of three loss-of-function mutants, a mutation was found in xlnR in all cases, excluding that the isolated gene is a suppressor. Using in vitro binding assays and footprinting techniques, the target sites in the promoter of xlnA have been determined to be 5'GGCTAA-3. Activation of transcription by XlnR is not limited to genes involved in xylan degradation, also genes encoding endoglucanases are activated.
47. Regulation of ornithine decarboxylase synthesis in Neurospora crassa. Martin A. Hoyt. University of California, Irvine, Mol. Biol. and Biochem., Irvine, CA, USA.
Ornithine decarboxylase (ODC), encoded by the spe-1 gene of Neurospora crassa, catalyzes the initial step in the synthesis of polyamines. In N. crassa, polyamines repress the synthesis and increase the degradation of ODC. Changes in the rate of ODC synthesis correlate with similar changes in the abundance of spe-1 mRNA. This polyamine-mediated regulation of spe-1 mRNA requires a sequence element downstream of the transcription start site. This polyamine responsive element (PRE) is required for polyamine-mediated repression of spe-1 mRNA abundance, and can confer polyamine regulation to a downstream reporter coding region. Use of the beta-tubulin (tub) promoter to drive expression of the spe-1 transcribed region demonstrates that polyamine regulation imparted by the PRE is promoter-independent. Neither deletion of the PRE nor changes in cellular polyamine status alter the half-life of spe-1 mRNA. In addition to effects on spe-1 mRNA abundance, sequences within the PRE impede the translation of a downstream coding region. This impediment is relieved by deletion of those sequences or by polyamine starvation. The sequences imparting translational effects lack either an upstream open reading frame or obvious secondary structure.
48. Distribution and evolution of the impala transposable element family in the Fusarium oxysporum species. Aurélie Hua-Van1, Catherine Gerlinger1, Thierry Langin 2, and Marie-Josée Daboussi1. 1 Université Paris-Sud, IGM, Orsay, Essonne, France. 2Université Paris-Sud, IBP, Orsay, Essonne, France.
Impala is an active Class II transposable element, first identified in a strain of Fusarium oxysporum that is pathogenic on melon. The nine copies present in this strain have been sequenced and grouped in three divergent subfamilies, differing by an important nucleotide polymorphism (about 20 %). This situation can be explained by two non mutually exclusive hypotheses: (i) an ancestral polymorphism associated to vertical transmission, and/or (ii) horizontal transmission of one or more subfamilies from another species. To gain insights on the molecular evolution of Impala elements, we have investigated their presence in different host-specific forms, by Southern blot, PCR and sequencing. We showed that Impala elements are present in most of the F. oxysporum strains tested, indicating that they an ancient component of the F. oxysporum genome. Subfamily-specific amplifications revealed the coexistence of divergent subfamilies within the same genome, a situation in favour of the hypothesis of an ancestral polymorphism followed by vertical transmission and independent evolution in the specific-host forms. Phylogenetic analysis identified at least five subfamilies in which some elements showed particular features: internal deletions whose breakpoints are located at the same nucleotide position or high rate of transitions CG to TA. These particular sequences are found in strains with different host specificity, addressing questions about the evolutionary history of the strains. The use of Impala as a tool for tracing populations will be discussed.
49. Identification of an upstream gene that affects aflR expression. Yiting Huang, and Jae-Hyuk Yu. Clark University, Biology Department, Worcester, MA, USA.
A mutant hunt was conducted to identify and isolate genes which regulate the gene activator, aflR, in the sterigmatocystin (ST) / aflatoxin gene cluster in certain Aspergillus species. aflR resides within the ST gene cluster and is known to regulate the expression of all other genes within the cluster. A special mutant strain of A. nidulans, LY345, carrying multiple copies of the ST gene cluster, was mutagenized with 4-nitroquinoline-1-oxide. Mutants were screened for the production of ST intermediates. A mutant of LY345, YH7239, was unable to produce any ST intermediates. Northern analysis showed this strain produces no aflR transcript. Since all ST cluster genes in LY345 are buffered against mutations within the gene cluster by virtue of multiple copies, the mutant, YH7239, is presumed to carry a mutation in a possible regulatory gene located external to ST gene cluster. Future work will be directed at rescue and cloning of this presumptive regulatory gene.
50. Structure of genes for Hsp30 from the white-rot fungus Coriolus versicolor and enhancement of their expression by heat shock and exposure to a hazardous chemical. Yosuke Iimura, and Kenji Tatsumi. National Institute for Resouces and Environment, Hydrospheric Environmenta, Tsukuba, Ibaraki 3058569, Japan.
The white-rot fungus Coriolus versicolor is a ligninolytic basidiomycete and it has been the focus of considerable attention because of its ability to degrade hazardous chemicals. In this study, we isolated two genomic DNAs that encode the heat-shock protein Hsp30 from C. versicolor. The nucleotide sequences of the two genes differ at 37 positions within the open reading frames but these differences result in only three amino acid substitutions. Three small introns interrupt the open reading frames. Two putative eukaryotic regulatory sequences, namely, a CAAT box and a TATA box, are present in the promoter regions. The promoter regions also contain the consensus heat-shock element, a xenobiotic-response element, a stress-response element, and a metal-response element. Northern blot hybridization indicated that the expression of these genes is constitutive at normal temperatures and enhanced at elevated temperatures. Expression was also enhanced in cells of C. versicolor that had been exposed to the hazardous chemical pentachlorophenol.
51. Characterization of alpha-amylase genes of industrial fungus A. kawachii. Kiyoshi Ito, and Yuji Miyamoto. National Research Institute for Brewing, Genetic Engineering Divison, Higashihirosima, Hiroshima, Japan.
Filamentous fungus Aspergillus kawachii is a nearly related strain to A. awamori and it is widely used for shochu (a Japanese traditional spirit) fermentation. A. kawachii produces acid stable alpha-amylase (AAA) along with neutral alpha-amylase (NAA) which is homologous to alpha-amylase of Aspergillus oryzae (Taka amylase, TAA). To examine the conditions of these alpha-amylase production, we characterized the alpha-amylase genes of A. kawachii. The genomic DNA library was screened using TAA cDNA as a probe. One AAA gene, two NAA genes, and one unidentified alpha-amylase (UAA) gene were cloned. From the results of Northern analysis and reporter gene analysis, it was known that NAA gene was expressed constitutively without the need of inducer such as starch or maltose and slightly repressed by the addition of glucose. The AAA gene was not expressed in liquid culture but strongly expressed in solid state culture.
52. Patterns of phylogeography and sequence evolution in the ribosomal intergenic spacer region (IGS) of the cosmopolitan mushroom Schizophyllum commune. Timothy Y. James, Jean-Marc Moncalvo, Shih-hon Li, and Rytas J. Vilgalys. Duke University, Botany, Durham, North Carolina, USA.
This study addresses gene flow and population structure in the common mushroom, Schizophyllum commune, by analysis of sequence variation in the intergenic spacer (IGS) region of the rDNA repeat. Over 180 strains of S. commune have been sequenced including two outgroup species for this fast-evolving gene region. Most of the strains were unique in sequence and dikaryotic strains often contained more than a single rDNA allele. Three major geographic clades were detected by phylogenetic reconstruction: a North American group, a Caribbean / South American group, and an Eurasian / African group. Patterns of phylogeography are consistent with a continental scale of population size, but there is also evidence for long distance gene flow. Most sequence polymorphism was clustered within a 50 base pair hypervariable region within the non-coding IGS region. The consistency index of several of these characters (nucleotides) was 1.00 suggesting that recombination in this gene region may be suppressed. The pattern of genetic inter-relatedness between geographic regions for the IGS data were very similar to data collected using allozyme markers. However, both IGS sequence data and allozyme data contrast strongly with the distribution of mating alleles in S. commune which shows no geographic patterns in distribution at any level (Raper et al. 1958, Am. Nat. 92:221).
53. Mutagenic DNA-repair genes in Aspergillus nidulans: Isolation and characterization of a RAD6 homologue gene. Young-Kug Jang1, Hyen-Sam Kang2, and Suhn-Kee Chae1. 1Paichai University, Division of Life Sciences, Taejon, Chungnam, South Korea. 2Seoul National University, Dept. of Microbiology, Seoul, South Korea.
Mutagenic DNA-damage tolerance pathways have not been well understood. In S. cerevisiae, genes in the RAD6 epistasis group have shown to be responsible for mutagenic DNA repair. The yeast RAD6 protein is an ubiquitine-conjugating enzyme and is required for mutagenesis and sporulation. In A. nidulans, defects in mutagenesis have been observed when genes (uvsI, uvsC, and uvsE) in two different epistasis groups, UvsI and UvsC, were mutated. The uvsI gene, a REV3homologue, has been cloned and shown to encode an error-prone DNA polymerase zeta. On the other hand, uvsC produces an E. coli RecA and yeast RAD51 homologue involving recombination and recombinational DNA repair. To understand more about mutagenic DNA repair pathways, an A. nidulans RAD6 homologue gene (temporally, named as radB) was isolated using the PCR based sib-selection method with degenerated PCR primers from the chromosome specific genomic DNA library. Sequence determination of genomic DNA and cDNA of radB revealed an open reading frame of 456 bp, interrupted by three introns (141 bp, 52 bp, and 72 bp, respectively), encoding a polypeptide of 151 amino acids with estimated molecular mass of 17 KDa. The deduced amino acid has 93%, 83%, and 75% sequence identity to MUS-8 of N. crassa, rhp6+ of S. pombe, and RAD6 of S. cerevisiae, respectively. The radB gene was assigned on the left arm of the chromosome V. Similarly in the case of RAD6 and RAD18 of yeast which were shown to work together, RADB and UVSH (a RAD18 homologue) of A. nidulans are also able to form a protein complex.
54. Unfolded protein response in Aspergillus. David Jeenes, Adrian Watson, Jane Morrice, Celina Ngiam, Donald MacKenzie and David Archer. Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.
The synthesis of foldases and chaperones, which are resident in the lumen of the endoplasmic reticulum (ER) and assist the folding of secretory proteins, is regulated at the transcriptional level by the unfolded protein response (UPR). We have shown that perturbation of the protein folding process in Aspergillus niger, whether by chemicals such as tunicamycin, dithiothreitol and a calcium ionophore or by the secretion of heterologous proteins, leads to up-regulation of the synthesis of foldases such as protein disulphide isomerase (PDI encoded by pdiA).We have also shown that, under the conditions of UPR, the transcription of the gene encoding acetyl-CoA carboxylase (ACC encoded by accA in A.nidulans), is up-regulated. ACC catalyses the first committed step in membrane fatty acid synthesis, suggesting that UPR coordinates the synthesis of ER membrane with the synthesis of ER lumenal proteins. UPR may have even wider regulatory roles which will also be presented.
56. Withdrawn
57. Reconstitution of an Aspergillus oryzae CCAAT-binding protein, AoCP, from purified recombinant subunits, AoHapB, AoHapC and AoHapE. Masashi Kato1, Akimitsu Tanaka1,Hideki Hashimoto1, Fumiko Naruse1, Peter Papagiannopoulos2, Stefan Steidl 3, Olivier Litzka 3, Axel A. Brakhage 3, Meryl A. Davis2, Michael J. Hynes2, Tetsuo Kobayashi1, and Norihiro Tsukagoshi 1. 1Nagoya University, School of Agriculture, Nagoya, Aichi , Japan. 2University of Melbourne, Department of Genetics, Melbourne, Victoria, Australia. 3Tech. Univ. Darmstadt, Inst. Mikrobiol.& Genetik, Darmstadt, Germany.
Many fungal genes contain CCAAT sequence in their promoter regions. We have found CCAAT-binding proteins in A. nidulans: AnCF for amdS encoding the A. nidulans acetamidase, AnCP for taa encoding A. oryzae Taka-amylase A, and PENR1 for the aatA and bidirectionally oriented genes acvA and ipnA, encoding the A. nidulans penicillin biosynthetic enzymes. AnCF/AnCP/PENR1 has been shown to contain a polypeptide encoded by the hapCgene, a homologue of the HAP3 gene of S. cerevisiae. Recently, two A. nidulans genes, hapB and hapE, encoding polypeptides with a central core showing high similarity to Hap2p and Hap5p have been isolated. HapB, HapC and HapE were shown to be necessary and sufficient for DNA binding by reconstitution of the complex in vitro. In this study, A. oryzae was found to contain a nuclear protein designated AoCP, which bound to the CCAAT sequence in the promoter region of the taa gene. AoCP contained a component immunologically similar to A. nidulans HAPC. A homologue of the A. nidulans hapC gene was isolated from A. oryzae, designated as AohapC and sequenced. The AohapC gene introduced into an A. nidulans hapC deletion strain was found to complement the hapC deletion and resulted in restoration of the CCAAT binding activity, leading to enhancement of taa gene expression. Furthermore, two genes, AohapB and AohapE, homologues of A. nidulans hapB and hapEwere isolated from A. oryzae. We succeeded in reconstituting the CCAAT-binding complex from purified recombinant polypeptides, AoHapB, AoHapC and AoHapE.
58. Transcriptional regulation of the catalase B gene (catB) in Aspergillus nidulans. Laura Kawasaki, and Jesus Aguirre. Universidad Nacional Autonoma de Mexico, Molecular Genetics, Mexico City, Mexico, Mexico.
A. nidulans contains at least three catalases. Thus far, two genes have been cloned and characterized: catA and catB (Navarro et al., 1996; Kawasaki et al., 1997). Besides being developmentally regulated, CatB activity was induced by H2O2, paraquat or uric acid catabolism but not by osmotic stress, whereas the third catalase activity has been detected only during late stationary phase. catB transcriptional regulation was studied by using a catB::lacZ fusion containing 3.5 kb of catB 5'upstream regulatory sequences. The reporter gene activity was induced during the stationary phase of growth. Starting with the activity detected by 10h of growth, the beta-galactosidase activity was induced 4, 14 and 22 fold at 18h, 28h and 48h, respectively. Under oxidative stress conditions produced by a 2h paraquat treatment, the activity was induced 17 fold. A second lacZ fusion containing only 993 bp of catB 5' upstream regulatory sequences behaved similarly to the previous one. After we sequenced this region, a comparison analysis revealed several putative regulatory elements similar to consensus sequences shown to bind transcription factors involved in responses to different types of stress. These sequences are contained within a 580 bp region, whose deletion clearly reduced catB::lacZ induction during stationary phase and oxidative stress. A more detailed analysis of this region is underway. We are also trying to isolate catB-deregulated mutants using a strain with two copies of the catB::lacZ fusion. We expect this approach will allow us to define important regulators of the oxidative stress response in A. nidulans. Supported by grant IN-206097 from PAPIT-UNAM, Mexico.
59. Restless aided transposon tagging of a nitrogen regulator from T. inflatum. Frank Kempken, and Ulrich Kück. Ruhr-University Bochum, Allgemeine Botanik, Bochum, NRW, Germany.
In the past years several fungal transposable elements have been identified. We have isolated and characterized Restless, a new type of fungal class II transposons from Tolypocladium inflatum which so far has not been found in any other fungus (Kempken& Kück, 1996, MCB 16:6563-6572). The predicted amino acid sequence deduced from an open reading frame encoded by Restless shows significant homology to transposases of the hAT transposon family, e.g. the maize Activator element. We set out to proof the usefulness of Restless to tag genes by identifying regulatory genes of the nitrogen metabolism, which have not yet been characterized in T.inflatum. As a simple selection system we used chlorate resistance, which may occur by mutations in cofactor or uptake genes, the nitrate reductase gene or in an regulatory gene. Mutations of the first three types were excluded by physiological tests and PCR. Among the remaining seven mutations we successfully tagged and cloned a gene with a C6 zinc finger. The deduced amino acid sequence of this gene shows significant similarity to the nit-4 gene of Neurospora crassa, which is a nitrogen metabolism regulator (Yuan et al., 1991, MCB 11:5735-5745). To our knowledge, this is the first fungal gene identified by transposon tagging. This method should be useful in any fungus which harbors known transposable element.
60. CMR1, a novel transcriptional activator with Cys2His2 type zinc-finger and Zn(II)2Cys6 binuclear cluster motifs regulates transcription of melanin biosynthesis genes SCD1 and THR1 of Colletotrichum lagenarium.Youki Kenmochi 1, Yoshitaka Takano2, Gento Tsuji1, James A. Sweigard3, Iwao Furusawa2, Osamu Horino1, and Yasuyuki Kubo1. 1Kyoto Prefectural University, Lab. of Plant Pathology, Kyoto, Kyoto, Japan. 2Kyoto University, Lab. of Plant Pathology, Kyoto, Kyoto 606-8502, Japan. 3E. I. Du Pont de Nemours, Central Research and Development, Wilmington, DE 19880-0402, USA.
Colletotrichum lagenarium is a phytopathogenic fungus that causes anthracnose disease of cucumber. Conidia of C. lagenarium differentiate melanized appressoria as an infection structure that are essential for penetration of the host plant. A gene, pig1involved in the amount of melanin production of Magnaporthe grisea was cloned by REMI insertional mutagenesis. C. lagenarium CMR1 gene was then isolated using pig1 as a probe. CMR1 was a single copy gene and contained an open reading frame consisting of 984 amino acids with four introns. At the N terminal region of the deduced amino acid sequence, two Cys2His2 type zinc-finger and one Zn(II)2Cys6 binuclear cluster DNA binding motifs were recognized. Coexistence of those motifs in a transcriptional factor is novel and unique form and has not been reported in any fungal transcriptional factors. CMR1disruptant showed a phenotype with the defect of melanin biosynthesis during mycelial growth and accumulated melanin intermediate scytalone in the culture media. However appressorial melanization was normal as that of the wild type strain. Expression of melanin biosynthesis genes in CMR1 disruptant was investigated by RNA blot analysis. In the wild type, accumulation of transcripts of melanin biosynthesis genes, polyketide synthase gene PKS1, scytalone dehydratase gene SCD1 and trihydroxynaphthalene reductase gene THR1 increased during mycelial melanization. However, in CMR1disruptant, the accumulation of SCD1 and THR1 transcripts was quite low compared with the wild type. The level of accumulation of PKS1 transcript was almost the same as the wild type. On the other hand, accumulation of those three melanin biosynthesis genes during appressorial melanization was same level between the disruptant and the wild type. These results indicate that CMR1 is a novel type of transcriptional activator with Cys2His2 type zinc-finger and Zn(II)2Cys6 binuclear cluster motifs that regulates the expression of SCD1 and THR1 during mycelial melanization in C. lagenarium.
61. Transformation of Pleurotus ostreatus to phleomycin resistance. Beom-Gi Kim1, and Yumi Magae2. 1National Institute, Applied Microbiology, Suweon, Kyunggi-do, South Korea. 2National Institute, Bioresources, Tsukuba, Ibaraki, Japan.
Pleurotus ostreatus (Fr.) Kummer, the oyster mushroom, is one of the most
widely cultivated edible mushrooms. Transformation strategy is necessary for molecular studies
of this fungus as well as for developing new breeding method of strain improvement. In aims of
developing a stable integrative transformation system for P. ostreatus, two vectors
containing phleomycin resistance selection marker (ble gene) and regulatory
sequences of beta-tublin (-tub) gene of Pleurotus sajor-caju were constructed. First,
isolated -tub gene of P. sajor-caju was sequenced. The gene(sized 3958 bp) consisted
of 939 bp promoter, 10 introns and a transcription terminal sequence. Two vectors were
constructed utilizing vector pGpht (this plasmid was a gift from Dr. J.G.H. Wessels). pPhKM1
contained b-tub promoter sequence, ble gene and Schizophyllum commune
GPD terminator. pPhKM2 contained ble gene and the -tub regulatory
sequences. Each vector was cotransformed into homokaryotic P. ostreatus ura
mutant strain (MGL2042-8) with pTura3-2. After the colonies grown on minimal medium were
transferred to phleomycin medium, transformants were selected. Transformation efficiency of
pTura3-2 vector was ca 30 colonies per 1 micro g DNA while cotransformation efficiency was
10%. Southern blot analysis of the transformants indicated chromosome integration of vectors.
Many and different intensities of hybridizing bands showed random and multiple site
chromosome integrations. Following the success of cotransformation, transformation of
dikaryotic P. ostreatus wild type strain was attempted using pPhKM1, pPhKM2 and
pGpht.
62. Cloning and nucleotide sequence of the catalytic subunit of DNA polymerase-gamma of
Neurospora crassa.
Most of the proteins involved in mitochondrial gene replication and expression are encoded by nuclear genes. Included in this group of proteins is DNA polymerase-gamma (pol-G), which is part of the complex involved in the replication of mtDNA. Identification of the gene for this protein in strict aerobes like Neurospora crassa by mutations has been difficult, most likely because such events are lethal. We used the known amino-acid sequences of the polymerases from Xenopus laevis, and three yeast species to clone the pol-G gene from N. crassa. After two rounds of PCR with degenerate primers and using N. crassa genomic DNA as a template, an appropriately-sized PCR product was identified. The PCR product was cloned and sequenced to design specific primers. Screening of the Orbach/Sachs pMOCosX cosmid library by PCR with these primers and by hybridization with the PCR product revealed that the X25:10C cosmid contains the complete pol-G gene. Sequence analysis showed that pol-G has a 3918 nucleotide open reading frame encoding 1305 amino acids (146 kDa). RFLP mapping located the gene in linkage group III between pro-1 and ad-2. Comparison of the nine available DNA polymerase-gamma sequences revealed several highly conserved sequence blocks, and that the polymerase domain is more highly conserved than the exonuclease domain. The N. crassa and S. cerevisiae polymerase-gamma polypeptides have long C-terminal extensions that are not found in the homologous proteins from other species.
63. Neurospora proteins that bind methylated DNA and DNA mutated by RIP. Gregory O. Kothe, Michael R. Rountree and Eric U. Selker, Institute of Molecular Biology, University of Oregon, Eugene, OR, USA.
Using gel-mobility-shift assays we have detected two factors in Neurospora crassa that bind methylated DNA sequences. A high-mobility factor was identified that is specific for methylated DNA. We refer to this factor as M-BP1 (Methyl Binding Protein 1). A low-mobility factor was identified that binds methylated DNA or DNA mutated by RIP. This factor binds most efficiently to DNA that is both methylated and contains RIP mutations. We refer to this factor as M/R-BP1 (Methyl/RIP Binding Protein 1). M/R-BP1 and M-BP1 may be involved in establishing and/or maintaining methylation patterns in Neurospora. It is also possible that these proteins function "downstream", exerting their effects after methylation has been set up (eg. repressing gene expression). To test these possibilities we are purifying M/R-BP1 and M-BP1, characterizing their properties, and cloning the genes that encode them. We will then generate and characterize mutants with defects in these genes.
64. Quality control of protein secretion in Aspergillus niger-isolation of the calnexin and UDP:glucose glycoprotein glucosyltransferase genes. Joanna Lambert1, David B. Archer2, David J. Jeenes2, Elodie Morlon2, and John F. Peberdy1. 1University of Nottingham, Biological Sciences UP, Nottingham, Nottinghamshire, UK. 2Institute of Food Res., Genetics and Microbiology, Norwich, Norfolk, UK
The ability of filamentous fungi to secrete high levels of glycosylated proteins has led to an
interest in exploiting these organisms as hosts for the production of recombinant
chemotherapeutic proteins. However, it is apparent that secreted yields of heterologous proteins
are significantly lower than yields of native proteins (Peberdy, Trends in Biotechnology
12:50-57, 1994). Work is being carried out to express heterologous proteins in Aspergillus
niger by identifying possible bottlenecks in the secretion process. An aspect of this
involves the study of quality control in the glycosylation pathway of secreted proteins. Calnexin
and calreticulin are lectins that function as molecular chaperones in the endoplasmic reticulum.
These proteins recognise the terminal glucose residues on glycoproteins and prevent their
secretion from the cell if the molecule is incorrectly processed. Together with UDP:glucose
glycoprotein glucosyltransferase these chaperones form part of a novel mechanism for promoting
folding, oligomeric assembly and quality control in the ER (Helenius et al., Trends
in Cell Biology 7: 193-200, 1997). The calnexin gene has been identified in A. niger,
and a full genomic clone sequenced which shows approximately 60% identity with other
calnexin genes. The promoter region contains unfolded protein response elements that are seen in
other chaperones, however data obtained does not support these as being functional. It has not
been possible to isolate calreticulin from A. niger, and to date this protein has only
been found in higher eucaryotes. The enzyme UDP:glucose glycoprotein glucosyltransferase has
a key function in maintaining glycan chains so unfolded proteins are recognisable by chaperones.
This gene for this protein has been isolated from A. niger and a genomic clone is
being sequenced.
65. Cellulase discovery and 18S rDNA studies of five chytrids
In the last few years interesting cellulases have been described from anaerobic members of the Chytridiomycetes, e.g. from Neocallimastix spp, Piromyces spp and Orpinomyces spp. However, also the aerobic chytrids have been shown to produce interesting cellulases: recently, we have cloned a new cellulase belonging to the glycosyl hydrolase family 45 from the aerobic chytrid Rhizophlyctis rosea. In the present study we focus on the phylogenetic relations between the Chytridiomycetes and the other groups of true fungi as well as the phylogenetic relations between the four orders of the Chytridiomycetes (i.e. Blastocladiales, Chytridiales, Neocallimasticales and Spizellomycetales), including both holocarpic/eucarpic and aerobic/anaeobic species. From these studies, full 18S rDNA sequence data from five chytrids will be presented, enabling the construction of an improved phylogenetic tree of the Chytridiomycetes, rooted in the fungal system. Further, the full amino acid sequence of the family 45 cellulase cloned from R. rosea will be presented. Comparisons will be made to the 30 other newly cloned fungal family 45 cellulases, originating from all groups of the fungal system and representing a wide variety of ecological niches.
66. Analysis of functional domains in NMR protein of Neurospora crassa. Ta-Wei David Liu. The Ohio State University, Biochemistry, Columbus, Ohio, USA.
Nmr gene is the major negative regulatory gene in the nitrogen control circuit of Neurospora crassa, which, together with the positive regulatory gene, NIT2, governs the expression of many unlinked structural genes for nitrogen utilization. The NMR protein is required to establish nitrogen repression of multiple structural genes. However, NMR does not appear to possess DNA binding activity. Previous studies have shown that the NMR protein interacts with the positive-acting NIT2 protein via direct, specific protein-protein binding (Pan et al. Mol Microbiol 26:721, 1997; Xiao et al. Biochemistry 34:8861, 1995). Five highly conserved regions in NMR and the homologous proteins from Aspergillus nidulans and Gibberella fujikuroii were identified. One or more of these regions might play an essential role in the interaction between NMR and NIT2 protein. In the present study, these possible functionally important regions of the NMR protein were investigated by site-directed mutagenesis. We are examining the ability of the mutant NMR proteins with each of the conserved regions deleted to interact with NIT2 and to function in nitrogen repression. The results of in vitro assays for protein-protein binding and in vivo functional assays for NMR activity will be discussed.
67. Potential role of plant signal(s) in pea pathogenicity (PEP) gene expression in Nectria haematococca. X. Liu1, Y. Han2, C. C. Wasmann1, H. C. Kistler2 and H. D. VanEtten1. 1Department of Plant Pathology, Univ. of Arizona, Tucson, 2Plant Molecular and Cellular Biology Program, Plant Pathology Department, Univ. of Florida, Gainesville.
Genes (PDA) for detoxifying the pea phytoalexin pisatin and other pea pathogenicity (PEP) genes are located on dispensable chromosomes in N. haematococca. Previously we had identified 5 transcripts (cDNA1 to cDNA5) in the cosmid 55-D-8, which was shown to be capable of complementing pathogenicity to nonpathogenic isolates, by screening a cDNA library constructed from mRNA derived from infected pea tissues. Complementation experiments indicate that cDNA1, cDNA2, and cDNA5 can contribute to pathogenicity independently. To search for potential signal(s) involved in PEP gene expression, we used RT-PCR approach to examine the production of 5 transcripts in vitro in mycelia subject to various treatments such as starvation, pisatin induction etc. Our preliminary results indicate that cDNA2 expression can only be detected under the induction of pisatin, suggesting that plant signal(s) may be required for expression of PEP genes. Currently, quantitative RT-PCR strategy is being employed to verify whether pisatin and or other plant signals play a role in regulation of the expression of PEP genes.
68. Pilot scale genome sequencing of Aspergillus nidulans and cDNA sequencing of Aspergillus oryzae. Masayuki Machida1, Mari Nakagawa1, Sumiko Kunihiro1, Kumiko Takase1, Makoto Yasukawa2, and Mariko Manabe3. 1National Institute of Bioscience and Human-Tech., Molecular Biology, Tsukuba, Ibaraki, Japan. 2Fukushima Tech. Center, Kooriyama, Fukushima, Japan. 3 National. Food Research Institute, Tsukuba, Ibaraki, Japan.
In the course of world wide effort to complete the genome sequence of Aspergillus nidulans, we started the pilot scale sequencing of a part of the ordered cosmids library. We picked the cosmids locating in the middle part of chromosome VIII and neighboring to the cosmid which has been sequenced by Prade et al. The sequencing was done mainly by the primer walking method using the internal-labeling protocol and analyzed by Li-Cor model 4200L DNA sequencer. Since longer than 800 nucleotide sequence could be analyzed in a single run, approximately 160 reactions were expected to complete the sequence of both strands of cosmid's inserts. We examined the condition to adapt the internal-labeling protocol to cosmid sequencing and found that the successful long-read sequencing depended on higher concentration of IRD-labeled dATP, optimization of cosmid amount and the higher temperature for denaturation step. We have initiated the random cDNA sequencing of Aspergillus oryzae cDNA libraries prepared from the cells grown in a rich medium and in the starved condition. We are preparing the database of the above sequence data on our Web server, which will be soon available.
69. The AVR1-MARA Locus of Magnaporthe grisea. M. Alejandra Mandel, Uvini P. Gunawardena , Travis M. Harper, and Marc J. Orbach. Univ of Arizona, Plant Pathology, Tucson, AZ, USA.
AVR1-MARA is a stable avirulence gene of Magnaporthe grisea that elicits a resistant response in the rice cultivar Maratelli. To address the question of how this gene functions, we are using a map-based approach to clone it. We are also using mutagenic approaches to address its apparent genetic stability. We have reported the cloning of the virulent allele, avr1-MARA, and the mapping of the avirulent allele. The two alleles differ by the presence of two regions of 60 kb and 14 kb only in the avirulent locus. The avirulent locus is approximately 85-90 kb with major portions unclonable in E. coli (Mandel et al., 1997). To localize the avirulence gene within the locus, we have used transformation-mediated gene disruption methods to delete all, or part of the locus. By this method, the gene has been localized to the region of the locus that contains the 60 kb AVR-associated sequences. Two approaches are being taken to isolate this region; one, a combination of Long Distance and Inverse PCR methods has resulted in the isolation of more than two thirds of this region. Sequence analysis of these segments has shown them to be unusual in M. grisea, with the DNA being 70% AT. Analyses of these sequences will be presented. The second approach to clone this region is Transformation-Associated-Recombination, an in vivo ligation method using Saccharomyces cerevisiae as a cloning host. Analyses of virulent mutants of AVR1-MARA and the distribution of the AVR1-MARA locus in populations of M. grisea will be presented. Mandel, M.A., V.W. Crouch, T.M. Harper, and M.J. Orbach. 1997. Physical mapping of the Magnaporthe grisea AVR1-MARA gene reveals the virulent allele contains two deletions. Mol. Plant-Microbe Interact. 10:1102-1105.
70. Antioxidant and metabolic functions of the alternative oxidase of Histoplasma capsulatum. Joan E. McEwen, and Clayton H. Johnson. McClellan VA Hosp. and Univ. of Arkansas Med., Medical Research, Little Rock, AR, USA.
Most fungi possess two mitochondrial respiratory pathways. The cytochrome pathway consists of electron carriers that ultimately reduce oxygen to water via the enzyme cytochrome oxidase. The alternative pathway, found in fungi, protists and plants but absent from mammals, consists of a single protein termed alternative oxidase, which reduces oxygen to water. Because alternative oxidase is absent in mammals, it will be an attractive target for development of antifungal drugs if it can be demonstrated to be important for virulence or survival of the pathogenic fungus during infection of a mammalian host. We are investigating the function of alternative oxidase in the pathogenic fungus Histoplasma capsulatum. Two lines of evidence suggest this enzyme performs an antioxidant function. 1) Both mRNA level and protein activity are elevated after exposure of H. capsulatum to hydrogen peroxide. 2) After expression of the H. capsulatum alternative oxidase cDNA in S. cerevisiae, antioxidant function was demonstrated by the "heat-induced cell death assay". The metabolic function of alternative oxidase involves its electron transport activity. We demonstrated that H. capsulatum yeast are able to grow, albeit slowly, when the cytochrome pathway is inhibited, and that this growth is abolished when alternative oxidase is also inhibited. This suggests that the alternative oxidase branch is able to support the bioenergetic needs of the organism. We hypothesize that this function is important during pathogenesis, when host antifungal efforts involving nitric oxide or other environmental stresses may inhibit the cytochrome pathway. Experiments on the effect of nitric oxide on H. capsulatum mitochondrial function and gene expression are underway.
71. Barrage formation in Neurospora crassa is independent of mating type and heterokaryon incompatibility. Cristina O. Micali, and Myron L. Smith. Carleton University, Biology, Ottawa, Ontario, Canada.
Barrages are evident in mating reactions between N. crassa strains (Griffiths and
Rieck, 1981 Can. J. Bot. 59: 2610-2617) but have not been extensively studied. In other
ascomycetes and many basidiomycetes, barrages are used as indicators of vegetative
incompatibility and may, in some cases, be correlated to differences at het genes. We
find that barrages in N. crassa are very similar in appearance to those described for
Cryphonectria parasitica, Podospora anserina and Sclerotinia
sclerotiorum and, as in other fungi, are most pronounced when strains are confronted on
medium low in nitrogen. Although variable in intensity, barrages in N. crassa can be
divided into two broad categories; clear zone and dark line. The two types are not mutually
exclusive and combinations of the two were observed. The appearance of a clear zone is
correlated to a decrease or complete absence of perithecial production between strains with
different mating types. The genetic control governing barrage formation seems to be complex.
However, using well characterized laboratory strains, we show that barrage formation in N.
crassa is independent of mating type and heterokaryon incompatibility genes het-6
and het-c. Barrages may form between strains with common or different
alleles at these loci, but do not form when the strains are confronted to themselves. Unlike many
plant pathogenic ascomycetes, wild isolates of N. crassa failed to form barrages
when confronted to each other or to tester laboratory strains. The reason for this is unknown but,
pairings between some inbred F1 and F2 progeny of these wild-type strains produce barrages,
which suggests that some level of inbreeding among strains may be necessary for barrage
formation.
72. Tandemly repeated het-6 incompatibility sequences in Neurospora
crassa
Heterokaryon incompatibility in the het-6 region, involves two closely linked genes, un-24+ and het-6. un-24+ encodes the large subunit of ribonucleotide reductase and is about 14 kbp centromere distal to het-6. het-6 putatively encodes a 680 amino acid protein. Stable heterokaryon formation between strains is prevented if they carry different alleles (PA or OR) at un-24+and het-6. Here, we present evidence that the het-6 s