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 sequence is tandemly repeated. The het-6OR allele has been characterized and has incompatibility activity in transformation assays. We characterized a region of PA-background DNA which shares about 70% identity with, and segregates opposite het-6OR. A majority of strains examined exhibit diagnostic fragments of either het-6OR or het-6PA based on a PCR assay. However, a small number of strains produce both the PA and OR fragments. In other experiments, we have attempted to PCR amplify het-6PA using primers based on het-6OR flanking sequences. Of three separate clones independently derived in this way, none have incompatibility activity and all are more similar in sequence to het-6OR than to het-6PA. These observations could be due to either gene duplication or cross-contamination during PCR amplifications. The former appears to be correct for the following reasons. First, using nested PCR primers we can amplify both OR and PA-like sequences separately from a single strain. Second, segregation analyses show that het-6OR and het-6PA probes hybridize to a non-identical set of two and five fragments, respectively, that co-segregate with other LGIIL RFLP markers. In view of these findings, tandemly repeated incompatibility factors around the N. crassa het-6 locus may be analogous to MHC and HLA loci in other eukaryotes.
73. Regulation of cat-1 during Neurospora crassa development and oxidative stress. Shaday Michan1, Fernando Lledías1, James D. Baldwin2, Don O. Natvig2, Rosa E. Navarro1, Jesus Aguirre1, and Wilhelm Hansberg1. 1Universidad Nacional Autonoma de Mexico, IFC, Bioquimica, México, D.F, México. 2University of New Mexico, Biology, New Mexico, Albuquerque, USA.
Neurospora crassa has 3 catalases. Cat-1 is the main activity during the whole asexual life cycle. It increases during germination and with each morphogenetic transition of conidiation. It is induced under stress conditions, such as heat, intense light and paraquat treatment. Cat-1 is specifically modified by singlet oxygen during germination, conidiation and under stress conditions. cat-1 genomic and cDNA clones were fully sequenced. Sequence analysis revealed an open reading frame interrupted by two introns predicting a 719 amino-acid polypeptide (MW=79,139). Two strong and few weak transcription initiation sites were found by primer extension and the polyadenilation site was determined Accumulation of cat-1messenger was detected during germination, conidiation and under stress conditions. Alignment of cat-1 shows 65% identity to the predicted peptide from AFCATGENE of Aspergillus fumigatus, 64% to CatB of A. nidulans and 56% to CatR of A. niger. Beside the central highly conserved region, these catalases show sequences homologies in the amino-and carboxyl-terminal regions. This and the precise conservation of intron I position indicates that these genes are homologs. A plasmid with cat-1 interrupted by pyr4 was used to transform a pyr4uridine auxotroph. Twelve transfomants wich lacked Cat-1 activity were isolated. One transformant, lacking Cat-1 antigen in a Western-blot assay and over-expressing Cat-3, is being characterised.
74. Phylogenetic analysis reveals past and present hybridization events between two North American species of the Heterobasidion annosum complex. Matteo Garbelotto, Ignacio Chapela, and William Otrosina. Department of Environmental Science, Policy, and Mangement, University of California, Berkeley, CA, Last author: Tree Root Biology Institute, USDA Forest Service, Athens, GA.
The Heterobasidion annosum complex includes several intersterility groups
(ISGs) with varying degrees of genetic divergence amongst them. The North American S and P
ISGs represent the two most divergent taxa in the complex, and should be regarded as different
species. In spite of the partial interfertility (18%) between the two groups, the S and P ISGs
have remained genetically distinct, and until recently hybrids had never been found in nature.
The recent discovery of a large long-lived hybrid genotype in California prompted further
research on mechanisms regulating fungal hybridization in nature. Isozyme analysis and
PCR-generated markers have shown that, while each group is defined by the presence of many
fixed alternate alleles, there appears to be some reciprocal gene introgression limited to areas
where the two ISGs coexist. This study assesses whether the resulting paraphily for these groups
is due to introgressive hybridization or to incomplete lineage extinction of ancestral
polymorphisms. A phylogenetic analysis of two putative introgressed markers (introns in the
large subunit of the mitochondrial ribosomal DNA) was performed for individuals in an area
ranging from the state of Washington to Southern Mexico, including an outgroup of isolates from
Europe. Nonconcordance between the accepted phylogeny of the complex and the phylogeny of
the putative introgressed markers strongly supports the introgressive hybridization hypothesis or
another mode of horizontal interspecific gene transfer. The phylogenies of both introns can be
best explained by interspecific transfer occurring during specific and limited time periods, rather
than continuosly. Alternative hypotheses are provided to define factors that may determine
periods of interspecific gene flow in this fungal complex.
75. A special design of the nit-3 gene promoter that facilitates NIT2 and NIT4
interaction.
In Neurospora crassa, the synthesis of nitrate reductase (encoded by nit-3 gene), an enzyme required for the utilization of inorganic nitrate, is activated by NIT2 and NIT4 proteins under the condition of nitrogen derepression and nitrate induction. NIT2, a major global regulatory protein, plays a crutial role in the nitrogen metabolism. NIT4 is a pathway specific factor. A NIT2-NIT4 protein-protein interaction is required for the optimal expression of nitrate reductase. An important goal is to investigate whether the nit-3 gene promoter has a special design that facilitates the interaction between NIT2 and NIT4. The nit-3 gene promoter region, either containing four GATA sequences for NIT2 binding or two symmetric octameric sequence elements for NIT4 binding, or both these NIT2 and NIT4 sites was used to replace the promoter of the cys-14 gene. The cys-14 gene encodes sulfate permease-II, facilitating the assimilation of sulfate in mycelia. Its promoter is highly regulated and required only one positive-acting factor (CYS3) to turn the gene on. Transformants containing cys-14 gene, in which promoter has been modified, were grown under the condition of nitrogen repression or nitrate induction. Results of sulfate transport assays implied that the CYS14 expression was regulated by NIT2 and NIT4. Neither one alone could turn the gene from "off" to "on". acknowledgements: This work is supported by grant GM23367 from the National Institutes of Health.
76. Aspartic protease gene expression in Fusarium venenatum CC1-5. Seri Intan Mokhtar, M.G. Wiebe, G. D. Robson, and A. P. J. Trinci. University of Manchester, Microbiology, Manchester, England, UK.
Fusarium venenatum is used in the UK for the large-scale production of Quorn mycoprotein in continous flow fermentation and has recently been developed as a potential host for heterologous protein production.In F. venenatum, the major extracellular protease produced during exponential growth is an aspartic protease of about 40 Kda. Southern hybridization of the genomic DNA indicates that only one copy of the gene is present. A 1.2 Kb cDNA of the aspartic protease gene from F. venenatum was obtained by RT-PCR and was cloned and sequenced. The expression of the aspartic protease gene was investigated in glucose limited chemostast cultures using casein as an inducer. When casein was added to the culture filtrates in the present of ammonia, the protease activity of the culture filtrates was lower than when casein was added in the absence of ammonia but in the presence of nitrates. Dot blot analysis on the mRNA suggested that the aspartic protease gene was expressed 2 hours after being induced by casein. An antibody against the aspartic protease was raised and used in Western analysis to detect the presence of the aspartic protease in the culture filtrates.The aspartic protease can be detected in the culture filtrates 8 hours after casein was added into the culture.
77. The CCAAT binding AnCF-complex is essential for the formation of a DNase I sensitive site in A. nidulans. Frank M. Narendja, Meryl A. Davis and Michael J. Hynes Department of Genetics, University of Melbourne Parkville 3052, Australia.
In A. nidulans CCAAT sequences are found upstream of a number of genes and are recognised by AnCF, a complex consisting of three evolutionary conserved subunits HapB,HapC,Hap E (1). Many eukaryotic promoters are assembled into chromatin structures hypersensitive to DNase I prior to transcriptional activation. We have found that the Hap-complex is involved in the formation of a DNase I hypersensitive region in the promoter of the amdS gene in A. nidulans In a hapE deletion strain no DNase I hypersensitive site is formed. Likewise, a point mutation in the CCAAT motif as well as a 530 bp deletion which removes most of the regulatory motifs of the amdS promoter including the CCAAT box results in complete loss of the DNase I hypersensitive region. This DNase I hypersensitive region can be restored by insertion of a 30 bp oligonucleotide carrying the CCAAT motif. DNase I hypersensitive regions have been found in the CCAAT containing promoters of the niiA, fmdS and gdhA genes and were also hapE -dependent. In all 4 promoters CCAAT boxes are located within the DNase I hypersensitive region. These data imply a critical role for the AnCF complex in establishing DNase I sensitive regions in A. nidulans. Recently a role for the NF-Y complex in chromatin rearrangement in vertebrates has also been proposed (2,3). This work was supported by the Austrian Science Foundation (J 1518-GEN) and the Australian Research Council. 1. Steidl, S., Papagiannopoulos, P., Litzka, O., Andrianopoulos A., Davis, M.A., Brakhge, A.A. and Hynes, M.J. Mol. Cell Biol. in press 2. Jin, S. and Scotto, K.W. (1998) Mol. Cell Biol. 18: 4377-4383. 3. Li, Q., Herrler, M., Landsberger, N., Kaludov, N., Ogryzko, V.V., Nakatani, Y. and Wolffe A.P. (1998) EMBO J. 17:6300-6315.
78. The Aspergillus nidulans METR sulphur regulator belongs to bZIP
transcriptional factors. Renata Natorff, Marzena Sieñko, Jerzy
Brzywczy, and Andrzej Paszewski. Institute of Biochemistry and Biophysics, Warsaw, Poland.A
new class of tight methionine auxotrophic mutants, which can grow only on methionine as a
sulphur source was obtained. Mutations in these strains are recessive and belong to the same
locus, named metR. The metR gene has been localised in chromosome
III between argB and phenA genes. Mutations in the metR
gene are epistatic to mutations in the sulphur negative regulatory genes scon. They
impair expression of some sulphur metabolism structural genes (i.e. sulphate permease,
arylsulphatase, homocysteine synthase). The expression of the metRgene seems to
be not regulated by sulphur source. We have cloned and characterised the genomic and cDNA
copies of the metR gene. The deduced METR protein contains a leucine zipper and
an adjacent basic region (bZIP), which together constitute a bipartite sequence-specific
DNA-binding domain. This domain is highly similar to analogous domain of the
Neurospora crassa positive-acting sulphur regulatory protein CYS3. However, no or
very little homology is found in the remaining parts of the proteins. The N. crassa
cys-3 gene does not complement metR mutations. This work was supported
by the KBN grant no 6 P04A 035 14
80. Cloning of a gene pacC homologue fron Neurospora
crassa
We have cloned a N. crassa pacC homologue, a gene responsible for the general control of ambient pH response in Aspergillus nidulans. Based on regions of homology found between the PacC protein sequences of A. nidulans, A. niger and P. chrysogenum, degenerate oligonucleotides were designed and used to amplify genomic fragments from N. crassa by PCR. The nucleotide sequence of a 137 bp amplification product was determined and shown to contain an open reading frame of 30 amino acids having ~93% identity to the A. nidulans PacC protein. The PCR product was used as a probe to screen a genomic library of N. crassa and three of the recovered clones complemented the pacC14 strain of A. nidulans. The existence of an A. nidulans pacC homologue in N. crassa is puzzling because, according to models proposed in the literature, A. nidulans appears to control the transcription of acid and alkaline phosphatases in response to the stimulus generated by extracellular pH, whereas N. crassa synthesizes both enzymes irrespective of extracellular pH, thus controlling only their secretion into the external medium.
81. Analysis of TOXE - A unique protein involved in the regulation of HC-toxin biosynthetic genes in Cochliobolus carbonum. Kerry F. Pedley, Joong-Hoon Ahn, and Jonathan D. Walton. Michigan State University, DOE Plant Research Lab, East Lansing, MI, USA.
Cochliobolus carbonum, the filamentous fungus responsible for northern corn leaf blight, produces a host-selective toxin, HC-toxin, that confers increased virulence towards certain genotypes. We are investigating the biosynthesis of the HC-toxin by trying to isolate and characterize the enzymes and genes necessary for its production, secretion, and regulation. To date three linked genes, HTS1, TOXA, and TOXC have been identified and the products of these genes have demonstrated roles in the biosynthesis of HC-toxin. All three genes are unique to toxin-producing (Tox2+) strains. Southern blot analysis has revealed that these genes are completely absent in toxin-non-producing (Tox2-) strains. Recently we cloned another gene, TOXE, that is found only in Tox2+ strains. When the TOXE gene is disrupted, the fungus does not produce HC-toxin and is only weakly pathogenic on plants. Sequence analysis of TOXE shows that its product contains a basic region characteristic of leucine zippers at its N-terminus and four ankyrin-like domains at the C-terminus. Based on this we hypothesize that the TOXE protein (TOXEp) might be a transcription factor involved in the regulation of the other genes involved in HC-toxin production. Northern blot analysis showed that the TOXA and TOXC messages are down regulated in strains with disrupted copies of TOXE. We are currently testing the hypothesis that TOXEp is directly involved in the transcriptional regulation of TOXA and TOXC.
82. A new compendium of Neurospora chromosomal loci. David D. Perkins1, Alan Radford2, and Matthew S. Sachs3. 1Stanford University, Biological Sciences, Stanford, CA 94305-5020, USA. 2University of Leeds, Biology, Leeds LS2 9JT, England. 3Oregon Graduate Institute, Biochem.& Molec. Biol., Portland, OR 97291-14877,
"Chromosomal Loci of Neurospora crassa", the 1982 Neurospora compendium (Microbiological Reviews 46:426-570), has been revised and expanded. The new edition will be published in book form by Academic Press. Lists of loci mapped in each linkage group will be posted and the text sections containing information on individual loci will be available for inspection.
83. Isolation and functional characterisation of two sulphate permease genes from Penicillium chrysogenum. Enrica Pizzinini1, Mart van de Kamp2, Arnold J.M. Driessen2, Wil N. Konings2, and Geoffrey Turner1. 1University of Sheffield, Molec Biol Biotechnol, Sheffield, South Yorkshire, UK. 2University of Groningen, Molecular Microbiology, Groningen, The Netherlands.
In order to assess the influence of primary metabolic flux on penicillin production in
Penicillium chrysogenum, we are investigating the sulphate assimilation pathway,
which provides cysteine, one of the 3 precursor amino acids of penicillin. Since no sulphate
permease gene had been isolated from a penicillin producing species, we designed degenerate
oligonucleotides from known sulphate permeases, and sequenced PCR amplified fragments. Two
types of fragment resembling sulphate permease were obtained, suggesting the presence of at
least 2 genes, termed sutA and sutB (see poster by van de Kamp et al.).
Full length clones of both sutA and sutB were obtained from a lambda
library, and used to cotransform an sB3 (sulphate permease) mutant of
Aspergillus nidulans. Complementation was observed only with sutB.
The degenerate primers were used to amplify sequences from A. nidulans, and a
single permease-like fragment obtained, which resembled sutB more closely than
sutA. An internal fragment of sutB was introduced into P.
chrysogenum by co-transformation, and transformants were tested for growth on sulphate
and methionine. A methionine-requiring transformant, resulting from homologous integration of
the sutB fragment and disruption of sutB, was identified. Sulphate
uptake was measured in wild-type, mutant, and transformed strains of P.
chrysogenum and A. nidulans to determine the kinetics of sulphate uptake,
and the effects of gene disruption and complementation. The combined data suggests that
sutB, and its homologue in A. nidulans, sB, encode the major sulphate
permease activities in these penicillin producers. We have yet to determine whether the leaky
growth observed in sutB and sB mutants results from a second sulphate
permease corresponding to sutA.
84. Analysis of protein traffic in defined secretion pathway mutants of Aspergillus
niger.
A major part of our research is focused on the understanding of the molecular mechanisms underlying efficient protein-secretion by filamentous fungi. Two lines of research are followed to identify key points in the secretion pathway. In a first empirical research line the role of major ER-chaperone protein BiP on protein secretion was studied. A second research line was based on a systematic analysis of the fungal secretion pathway. Based on results obtained in S. cerevisiae, it was clear that the various transport steps of the secretion pathway are depending different stage-specific small GTPases.The A. niger genes encoding the most relevant of these GTPases were cloned. From one of these genes, sarA, of which the gene product is expected to play a role in one of the early transport steps from ER to Golgi, specific mutant alleles were generated. Introduction of the alleles into A. niger resulted specific conditional A.niger sarA mutants. Analysis of secretion characteristics of both BiP-overproducing and sarA mutant strains in transformants expressing various glucoamylase-gene-fusions showed that in both strains significant retention of fusion protein in the intracellular (membrane bound) fraction was observed. However, the yield of secreted recombinant protein was not affected. Results of this research will be presented and discussed.
85. Molecular characterization of the secretory pathway of Aspergillus niger. Arthur FJ. Ram1, Peter J. Punt2, Roy C. Montijn2, Cora MJ van Zeijl2, and Cees AMJJ van den Hondel2. 1State Leiden University, IMP, Leiden, Zuid Holland, The Netherlands. 2TNO, MGG, Zeist, Utrecht, The Netherlands.
We have started research to analyse the molecular mechanism underlying protein secretion in A. niger. In the first instance we have isolated 9 distinct GTPase encoding genes from A. niger (sarA, sagA-H) corresponding to GTPases involved in most stages of the secretory pathway. Interestingly, several of these genes homologues are present in higher eukaryotes, but not in S. cerevisiae. These genes are used to generate a set of (conditional/deletion) mutants imposing defined blocks in the secretory pathway. To analyse transport and secretion of proteins we have developed a GFP-based secretion reporter system by fusing GFP to a carrier protein, glucoamylase (GLA). Expression of a glaA::gfp fusion construct resulted in fluorescence of the cell wall, probably representing secreted GLA::GFP fusion protein that is retained within the extracellular matrix. Periplasmic fluorescence was only observed in young mycelium. No periplasmic fluorescence is observed in older mycelia probably due to acidification of the medium, and/or increased protease activity. Targeting of the GLA::GFP fusion protein to the ER by fusing the ER retention signal (HDEL) to the fusion protein, resulted in intracellular, punctuated fluorescence, indicating retention of the fusion protein. The GFP-fusion proteins will be introduced into the various secretion mutants to validate and complement the results obtained with the analysis of secretion defects in our secretion mutants.
87. New vectors for gene diversification in Neurospora. John Paul Rasmussen1, Frederick J. Bowring1, Elie Kato2, W. Dorsey Stuart2, and David E.A. Catcheside1. 1Flinders University, Biology, Adelaide, South Australia, Australia.
Recombination hotspots active in meiosis provide a means of diversifying pairs of DNA sequences differing at multiple sites. We have constructed plasmids permitting targeted transfection of heterologous genes located between his-3 and the cog hotspot in Neurospora, this positioning enables enrichment of post-meiotic recombinants by selection of progeny that have experienced recombination at his-3.
88. Isolation and characterisation of a retrotransposon in the phytopathogenic fungus Stagonospora nodorum. Jennifer M. Rawson, Simon B. Cutler, and Christopher E. Caten. The University of Birmingham, Biological Sciences, Edgbaston, Birmingham, UK
A search for active transposable elements was undertaken in the phytopathogenic fungus Stagonospora nodorum (teleomorph: Phaeosphaeria nodorum) using a transposon trapping approach already used successfully in other fungal species (Daboussi and Langin, 1994, Genetica 93: 49-59). Spontaneous mutants of nine wild strains of S. nodorum defective in the nitrate reductase (NIA1) gene (Cutler et al., 1998, Current Genetics 34: 128-137) have been screened for DNA insertions by Southern hybridisation and PCR. A novel insertion of approximately 5kb was detected in one NIA1 mutant of strain BS444. This insertion, flanked by some NIA1 DNA, has been cloned and partially sequenced. Sequence comparison with the wild type NIA1 allele has located the boundaries of the insertion, and suggests that the insertion event has caused an imperfect 11-13bp duplication at this site. Comparison of the two sequenced ends of the insertion has revealed a pair of perfect 169bp direct terminal repeats. Database searches using deduced amino acid sequences suggest some regions of the insertion have homology with published retroelements and reverse transcriptases. Southern hybridisation using the clone as a probe indicates that the insertion is present in 10-15 copies in the genome of BS444 and two related strains, but is absent from fourteen other strains screened.
89. Signal transduction in arbuscular mycorrhizas during pre-symbiosis. Natalia Requena, Petra Fueller and Philipp Franken. Max-Planck Institut für terrestrische Mikrobiologie. Karl-von-Frisch Strasse. 35043 Marburg, Germany.
Arbuscular mycorrhizal fungi are obligate biotrophs forming symbiosis with plant roots of more than 80% of all vascular plants. These fungi can only complete their life cycle after formation of the symbiosis. The life cycle begins with the germination of the spore in the soil and the formation of a pre-symbiotic mycelium, which explores the soil in search of the plant root. Only upon contact with the appropriate host root the fungus starts differentiating and forming the appressorium. In the absence of the plant the fungus retracts back the cytoplasm and arrests until the conditions are again optimal for re-germination. During this pre-symbiotic growth little it is known about the mechanisms or the signals controlling the growth and differentiation of the fungus. We have focussed our attention on this stage of the life cycle and on the genes that are regulated in response to a variety of signals either from the plant or from other soil microorganisms. We start to study these aspects by means of a molecular approach to monitor changes in the gene expression of the fungus Glomus mosseae(BEG12) in response to the rhizobacterium Bacillus subtilis NR1. The bacterium was found to induce specific increases in mycelial growth correlated with a regulation of the GmFOX2 expression, a highly conserved gene encoding a multifunctional protein of the peroxisomal beta-oxidation. We determined the gene structure and studied its expression in a time-course analysis in response to the bacterium. The results show that the fungus is able to respond to stimuli others than the plant changing its gene expression, although it might be sensing the bacteria through common signaling pathways.
90. Self-splicing activity of selected mitochondrial group I introns in Podospora anserina in vitro. Jill L. Salvo, Birgit Rodegheir, and John W. Carbone. Union College, Biology, Schenectady, NY, USA.
Self-splicing activity of four group I introns from the mitochondrial genome of the filamentous fungus Podospora anserina was assayed using in vitro RNA transcription analysis. Although the Podospora mitochondrial genome contains up to 30 group I introns, only two have been tested for self-splicing activity (LSU, intron 1 and cytochrome oxidase subunit-1, intron 3). Introns from cytochrome b, NADH dehydrogenase subunit-3, ATPase 6 and cytochrome oxidase subunit-1 (intron 5) were selected, and preliminary evidence suggests that all are capable of at least some RNA catalyzed reactions in vitro. The specific nature of these reactions, including use of cryptic splice sites, and the relationships between intron subgroup, location and sequence of the intronic ORF and possible tertiary structure will be discussed.
91. Expression of antisense creA RNA in A. nidulans causes partial derepression of creA-controlled genes. Anne Santerre Henriksen1, L. Fernando Bautista1, Morten Hentzer2, Alexei Aleksenko1, and Jens Nielsen1. 1 DTU, Biotechnology, Lyngby, 2800, Denmark. 2DTU, Mikrobiology, Lyngby, 2800, Denmark.
The alpha-amylase gene from A. oryzae is subject to CREA-mediated carbon catabolite repression both in the host fungus and when expressed in a transgenic A. nidulans. An expression cassette containing a portion of the A. nidulans creA gene in the reverse orientation with respect to the gpdh promoter was introduced into the alpha-amylase-producing A. nidulans. This resulted in a several-fold increase in the enzyme production. The level of alpha-amylase mRNA in the antisense transformants was elevated. The presence of both sense- and antisense-creA-transcript was detected. The kinetics of enzyme accumulation in control and antisense-derepressed strains was studied under well controlled fermentation conditions.
92. CPCR1, a transcription factor of the RFX family, shows specificity for cephalosporin C biosynthesis genes from Acremonium chrysogenum.Esther Schmitt and Ulrich Kück Lehrstuhl für Allgemeine Botanik, Ruhr-Universität Bochum D-44780 Bochum, Germany.
The biosynthesis of the ß-lactam antibiotic cephalosporin C in Acremonium
chrysogenum is regulated by a variety of internal and external factors. These parameters
effect the cephalosporin C-production e.g. on the basis of transcription initiation of the
corresponding genes. Therefore the promoter regions of the cephalosporin C biosynthesis genes
are of interest when the molecular regulation of this secondary metabolite is investigated. The
two cephalosporin C biosynthesis genes pcbAB and pcbC share a
divergent promoter region of 1.2 kb carrying several putative protein binding sites. A
CCAAT-box about 350 bp upstream of the transcription start sites of the pcbC gene was used in
a ONE-HYBRID screen to isolate a cDNA from A. chrysogenum encoding a
transcription factor. Sequence analysis led to the identification of an open reading frame, which
contains 830 amino acids. The gene was named cpcR1 for cephalosporin C regulator
1. The CPCR1 protein belongs to a conserved family of DNA binding proteins, the RFX
proteins. To our knowledge this is the first description of a member of this protein family from a
filamentous fungus. One-hybrid experiments as well as gel retardation assays with mutated
binding sites revealed that CPCR1 interacts specifically with an imperfect palindromic sequence
in the pcbC promoter, which overlaps the CCAAT-Box. Using one- and two-hybrid systems it
was shown that CPCR1 interacts with itself through the C-terminal part including the
dimerization domain and binds DNA only as a homodimer.
93. Isolation of an autonomously replicating DNA sequence from Aspergillus
nidulans.
Using the yeast Saccharomyces cerevisiae and the integrative vector system, we have isolated and characterized an autonomously replicating sequence (ARS) from Aspergillus nidulans. The DNA fragment, designated ANR1, is 5.0 kb in size, and to be maintained in free from the chromosome in S. cerevisiae. The recombinant plasmid YIplac211-ANR1, consists of sequences derived from the 3.79 kb yeast integrative vector YIplac211 and ANR1, showed a 104-fold enhancement of transformation efficiency over that found for YIplac211, and easily recovered from the transformed yeast. Genetic analysis of transformants showed that YIplac211-ANR1 could be cured over 96% when cultured over 20 generation in complete medium suggesting that this sequence should be mitotically unstable. In Aspergillus nidulans, recombinant plasmid pILJ16-4.5 which carries the 4.5 kb EcoRI fragment of ANR1 showed a 170-fold enhancement of transformation efficiency compared to that of integrative vector pILJ16. Recombinant plasmid pILJ16-4.5 is mitotically unstable, being lost from 67% of asexual progeny of transformants. Southern analysis of transformant DNA showed pILJ16-4.5 to be maintained in free form. Sequence analysis had confirmed that the ANR1 fragment originated from mitochondiral DNA of A. nidulans.
94. Transformation of a mycoherbicide by electroporation: methodology and potential application. Amir Sharon, Micah Robinson, and Rudy Maor. Tel Aviv University, Plant Sciences, Tel Aviv , IL, Israel.
Colletotrichum gloeosporioides f. sp. aeschynomene causes anthracnose disease of Aeschynomene virginica. The fungus has been used for the production of the mycoherbicide College, which is used to control Aeschynomene virginica in rice and soybean fields in certain regions in the USA. Genetic engineering of the fungus towards enhanced virulence, broader host range, or pesticide resistance may provide isolates with superior biocontrol properties that will have a wider application range. A procedure for transformation of C. gloeosporioides f. sp. aeschynomene by elecroporation of germinated conidia has been developed. The new procedure provides a method for high and stable expression of heterologous genes in the fungus as well as for targeting of genes into homologous sites in the fungal genome. The transformants obtained are highly stable in vitro as well as in planta. These methods will be used to generate environmentally-safe strains with improved biocontrol properties. The methodology is general and can be used to develop similar transformation protocols in additional species including fungi with economical and agricultural importance.
95. Heterologous protein expression vectors and molecular breeding of basidiomycetous fungal strains with high lignin- and xylan-degrading activities. Kazuo K.S. Shishido1, Ken-ichiro K.O. Ogawa1, Shinya S.M. Matsuda1, Madoka M.K. Kikuchi1, Takashi T.Y. Yamazaki1, Susumu S.K. Kajiwara1, Akira A.T. Tsukamoto2, and Jun J.S. Sugiura2. 1Tokyo Institute of Technology, Life Science, Yokohama, Kanagawa, Japan. 2Oji Paper Co. Ltd., Adv.Technol.Research Lab., Shinonome, Tokyo, Japan.
We have constructed two chromosome-integrating vectors pLC1 and pLC2. The former carries the Lentinus edodes ras gene promoter and priA gene terminator, and the latter does the basal promoter and terminator of priA gene. Both vectors are very useful for the expression of foreign genes in various basidiomycetous fungi. The manganese (II) peroxidase (MnP) cDNA (designated mnpc) derived from Pleurotus ostreatus was fused between the promoter and terminator of the vectors. These plasmids were introduced into protoplasts of monokaryotic Coprinus cinereus trp1 strain with the C. cinereus TRP1-containing plasmid pCc1001 and into those of monokaryotic Coriolus hirsutus arg1 strain with the C. hirsutus ARG1-containing plasmid, obtaining C. cinereus Trp+ transformants and C. hirsutus Arg+ transformants, all of which show high lignin-decolorization and -degradation activities. Southern-blot analysis revealed that the transformants all possess mnpc sequence (5 to 10 copies) on their chromosomes. Bacillus subtilisendo (beta-1,4-) D-xylanase structural gene (xyn) was fused after the signal sequence of P. ostreatus mnpc after removing its own signal sequence. The resulting modified gene (xyn') was fused between the promoter and terminator of pLC1 and pLC2. Through introduction of these recombinant plasmids into the C. cinereus trp1 genome together with pCc1001, Trp+ transformants, showing higher xylan-degradation activities, were obtained.
96. Mating-type associated incompatibility in Neurospora crassa. Patrick K.T. Shiu. University of British Columbia, Botany, Vancouver, BC, Canada.
The mating-type locus in the haploid filamentous fungus, Neurospora crassa, controls mating and sexual development. The fusion of reproductive structures of opposite mating-type, A and a, is required to initiate sexual reproduction. However, the fusion of hyphae of opposite mating-type during vegetative growth results in growth inhibition and cell death, a process which is mediated by the tol locus. Mutations in tol are recessive and suppress mating-type associated heterokaryon incompatibility. In this study, we describe the cloning and characterization of tol. The tol gene encodes a putative 1011-amino acid polypeptide with a coiled-coil domain and a leucine-rich repeat. Repeat-induced point mutations in tol result in mutants that are wild-type during vegetative growth and sexual reproduction, but which allow opposite mating-type individuals to form a vigorous heterokaryon. Transcript analyses show that tol mRNA is present during vegetative growth but absent during a cross. These data suggest that tol transcription is repressed in order to allow the co-existence of opposite mating-type nuclei during the sexual reproductive phase. tol is expressed in a mat A, mat a, A/a partial diploid and in a mating-type deletion strain, indicating that MAT A-1 and MAT a-1 are not absolutely required for transcription or repression of tol. These data suggest that TOL may rather interact with MAT A-1 and/or MAT a-1 (or downstream products) to form a death-triggering complex. Studies on recently isolated TOL-interacting proteins (tip; isolated by yeast 2-hybrid system) will shed lights on the mechanism of mating-type mediated vegetative incompatibility.
97. Promoter-Tagged Restriction Enzyme Mediated Insertion (PT-REMI) mutagenesis in Aspergillus niger. Jeffrey R. Shuster, and Mariah Bindel Connelley. Novo Nordisk Biotech, Inc., Molecular Genomics, Davis, CA, USA.
Promoter-tagged restriction enzyme mediated insertion (PT-REMI) DNA mutagenesis was performed in the fungus, Aspergillus niger, using a plasmid containing a strong transcriptional promoter. Two DNA-tagged mutants were analyzed in detail. A white-spored mutant was shown to contain a plasmid insertion that disrupted a gene showing strong identity to the polyketide synthase wA gene of A. nidulans. A morphological mutant was shown to contain a plasmid insertion in the 5'-upstream region of a gene showing strong identity to cytochrome C oxidase subunit V, COX5. The insertion of the plasmid resulted in enhanced expression for the COX5 RNA demonstrating that a combination of REMI with a promoter can be used to activate gene transcription.
98. Induced expression of a novel Aspergillus fumigatus putative drug efflux gene in response to itraconazole.John W. Slaven1, Michael J. Anderson1, Dominique Sanglard2, Graham K. Dixon3, Jacques Bille2, Ian S. Roberts4, and David W. Denning1. 1University of Manchester, Medicine, Manchester, Gr Manchester, UK. 2CHUV, Microbiology, Lausanne, Switzerland. 3 Zeneca Pharmaceuticals, Cancer and Infection, Macclesfield, Cheshire, UK. 4University of Manchester, Biological Sciences, Manchester, Gr Manchester, UK
Two agents are licensed for the treatment of A. fumigatus infection, amphotericin B and itraconazole. Resistance to itraconazole has been detected in vitro and has been validated in vivo. Studies of Saccharomyces cerevisiae and Candida species have shown that one mechanism of azole resistance is drug efflux by ATP-binding cassette (ABC) transporters. An A. fumigatus genomic library was screened with a probe from the C. albicans ABC transporter gene, CDR1. This screening revealed a novel gene, ADR1, which has a high level of identity to other fungal multi-drug resistance (MDR) genes. The encoded protein contains conserved amino acid residues within recognised MDR motifs. As well as hybridising to the gene, an ADR1 probe hybridised to other sequences in the genome at high stringency. Dot blot analysis showed that ADR1 mRNA is expressed at over 5-fold higher levels in a resistant isolate (AF72) which is unable to accumulate itraconazole. However, expression was only up-regulated when AF72 was grown in the presence of itraconazole. The regulation of this gene was also studied in response to heat shock, oxidative stress and other anti-fungal drugs, including terbinafine. Adr1 is a novel ABC transporter, possibly involved in the efflux of itraconazole from A. fumigatus. Complementation studies in yeast will be undertaken to establish the protein's substrate range.
99. Isolation of extragenic suppressor mutations of the palI30 mutation of Aspergillus nidulans. Daphne Smith1, Bruce Dawson2, Valerie Alexander2, and Steven H. Denison1. 1 Mississippi College, Dept of Biological Sci, Clinton, MS, USA. 2 Brandon High School, Brandon, MS, USA
The product of the palI gene of Aspergillus nidulans is a component of the ambient pH signal transduction pathway. To ensure production of alkaline-specific extracellular enzymes in alkaline environments and acid-specific extracellular enzymes in acid environments, this pathway modifies the pacC-encoded transcription factor in response to alkaline pH. Strains with mutations in the palI gene mimic growth in acidic conditions, showing (at pH 6.5) increased production of extracellular acid phosphatase, decreased production of alkaline phosphatase and increased sensitivity to molybdate in the growth medium. The palI gene encodes a protein with four putative transmembrane domains, and may therefore function as the pH sensor in the signal transduction pathway. In order to identify new components of this signalling pathway, we have isolated extragenic suppressor mutations of the palI30 mutation. Following 4-NQO mutagenesis of spores, colonies were selected initially for growth on 17 mM sodium molybdate and secondarily for patterns of acid and alkaline phosphatase production more like wild type. Suppressor mutations have been identified in the pacC and pacX genes. Sequence analysis of these pacC mutations has identified amino acids which might be important in maintaining the closed form of PacC (L. Rainbow, J. Tilburn and H. N. Arst, Jr., unpublished). Mutations in the pacX gene are also able to suppress mutations in other pal genes and certain mutations in pacC (E. Bignell, J. Tilburn and H. N. Arst, Jr., unpublished).
100. Glycosylation of proteins in Aspergillus niger: isolation of the N-acetylglucosamine-phosphate-tranferase gene (GPT) Tine Kring Sorensen, Paul Dyer, Ulrike Laube, and John Peberdy. University of Nottingham, Biological Sciences, Nottingham, Nottinghamshire, UK.
Filamentous fungi secrete a range of enzymes, the majority of which are hydrolytic and play an important role in fungal nutrition. Some fungi are capable of secreting high levels of proteins and have consequently attracted attention as potential producers of heterologous proteins, but so far yields obtained have been very low. Not only are improved yields required, but also the proteins should be highly authentic, thereby preserving functionality and avoiding antigenic reactions. Many proteins secreted by fungi are glycosylated. Most information on glycosylation has come from studies on mammalian and yeast systems, but only little is known about the process in filamentous fungi. It is therefore desirable to better understand the events of protein secretion in fungi, thus making it possible to control the process and thereby tailor the glycans produced on secreted proteins. Studies are in progress to investigate the biochemical and molecular controls of glycosylation in Aspergillus niger. One aspect of this involves the ER-located enzyme UDP-N-acetyl glucosamine:dolichyl phosphate N-acetyl glucosamine-1-phosphate transferase (GPT), catalyzing the first step of the N-glycosylation pathway: the synthesis of N-acetylglucosamine-dolichol phosphate. The GPT-gene has now been cloned from a genomic library of A.niger by screening with a PCR derived homologous fragment (470 bp) isolated from genomic DNA. The ORF was 1.4 kb with 2 introns (58, 91 bp). The sequence from A.niger showed about 46% identity with the GPT gene cloned from yeasts and 35-37% identity with rodents and human. Northern blotting, using a homologous probe, produced a 2.4 kb mRNA transcript. Expression studies are in progress. Acknowledgement of funding sources: The Danish Research Academy and The Danish Research Council.
101. AnCF, the CCAAT binding complex of Aspergillus nidulans contains products of the hapB, hapC and hapE genes and is required for activation by the pathway specific regulatory gene, amdR.Stefan Steidl1, Peter Papagiannopoulos2, Olivier Litzka1, Alex Andrianopoulos2, Meryl A. Davis2, Axel A. Brakhage1 and Michael J. Hynes2. 1Institut fuer Mikrobiologie und Genetik, TU Darmstadt, Germany 2Department for Genetics, University of Melbourne, Australia.
CCAAT binding factors positively regulating the expression of the amdS gene
(encoding acetamidase) and two penicillin biosynthesis genes (ipnA and
aatA) have been previously described in Aspergillus
nidulans. The factors were called AnCF and PENR1 respectively. Deletion of the
hapC gene, encoding a protein with significant similarity to Hap3p of
Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding
activities. We now report the isolation of the genes hapB and hapE
which encode proteins with central regions of high similarity to Hap2p and Hap5p of
S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. The
HapB, HapC and HapE proteins have been shown to be necessary and sufficient for the
formation of a CCAAT binding complex in vitro. Strains with deletions of each of the
hapB, hapC and hapE genes have identical phenotypes of
slow growth, poor conidiation and reduced expression of amdS. Furthermore,
induction of amdS by omega amino acids, which is mediated by the AmdR pathway
specific activator, is abolished in the hap deletion mutants as is growth on gamma-aminobutyric
acid (GABA) as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in
the promoters of the amdS and gatA genes. It is known that AnCF can
bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in
vivo.
102. Characterization of a Neurospora crassa strain that escapes senescence
associated with the over-replication of a mitochondrial retroplasmid
Variant forms of the Mauriceville and Varkud mitochondrial retroplasmids cause growth impairment and senescence in Neurospora spp. Senescence is generally associated with deletions or rearrangements of the mitochondrial (mt) genome that result from the integration of variant plasmids; however, certain variants of the Mauriceville retroplasmid can impair growth without integrating into mtDNA. Senescence associated with the MS4416 variant retroplasmid is highly predictable and correlates with an increase in the plasmid copy number and alterations in the expression of specific mitochondrial gene products. Here, we report the isolation and characterization of a mutant strain that escapes senescence. This long-lived derivative strain shows vigorous and indefinite growth while tolerating high levels of the variant plasmid and in vivo labeling studies indicate the strain has a wild-type mitochondrial protein expression profile. Inheritance studies show that the long-lived trait is controlled by nuclear genes, whereas heterokaryon analysis reveals unanticipated senescent phenotypes which involve the integration of the variant plasmid into the mtDNA. Our findings suggest specific gene products or pathways exist in Neurospora that help alleviate mitochondrial dysfunction and control suppression associated with defective mtDNAs.
103. Gene rearrangement and surface variation in Pneumocystis carinii. James R. Stringer, Joshua Schaffzin, Scott Keely, Susan Sunkin, and Melanie Cushion. University of Cincinnati, Microbiology, Cincinnati, OHIO, USA.
P. carinii have on their surface a major surface glycoprotein (MSG). MSG is a protein family encoded by about 100 heterogeneous genes, which are located at the ends of each of the fifteen chromosomes that comprise the genome. Organisms within a population can have different MSG isoforms on their surface. This antigenic variability appears to be accomplished by regulation of MSG gene expression. The first hint at the mechanism of MSG gene regulation came from the discovery that a 400 basepair sequence (called the UCS) is on the 5' end of essentially every MSG mRNA, regardless of the MSG isoform encoded. The UCS was found to reside at the end of a single chromosome, which suggested that the UCS locus serves as an MSG expression-site. Studies on the structure of the expression-site locus showed that it can be occupied by different MSG genes in different organisms in a population. The number of different MSG genes residing at the expression-site in a population seems to vary, but more than 25 different MSGs were at this locus in the population that was studied at this level. Residence at the expression site seems to be both necessary and sufficient for expression of a specific MSG gene because all of the MSG genes that were at the expression site were represented by mRNAs, and mRNAs from MSG genes not linked to the UCS were not detected. Restriction of expression of the MSG family to the gene attached to the UCS implies that each organism has but one MSG isoform on its surface. The presence of multiple MSG genes at the UCS locus in a single population shows that gene rearrangements involving UCS and MSG genes occur. The mechanism and frequency of these rearrangements is under investigation.
104. Characterization of DNA de novo methylation signals by using short synthetic oligonucleotides in vegetative cells of Neurospora crassa. Hisashi Tamaru, and Eric U. Selker. University of Oregon, Inst. of Mol Biol., Eugene, OR, USA.
DNA is modified by cytosine methylation in many eukaryotes including Neurospora. Our
goal is to understand how certain cytosines are targeted for methylation. Most DNA sequences
that are subject to de novo methylation in N. crassa have undergone RIP
(repeat-induced point mutation) and are therefore relatively rich in A+T and are enriched for TpA
dinucleotides. To further understand the nature of DNA de novo methylation signals
we quantified the capacity of short oligonucleotides to trigger methylation. We constructed a
his-3 targeting vector that carries a 100 bp zeta-eta fragment surrounded by a lightly
mutated allele of the am gene (amRIP4). The mosaic amRIP4::zeta-eta construct
does not establish methylation by itself at the his-3 locus but provides a sensitive
background to test many short (ca. 25 bp) fragments for their capacity to trigger methylation. We
show that various fragments consisting only of A and T can trigger methylation but to different
extents. Poly A:T did not trigger significant methylation. Sequences rich in TpA dinucleotides
triggered methylation but were less potent than some sequences with fewer TpA dinucleotides.
Thus, recognition of de novo methylation signals in N. crassa does not
simply involve counting the main product of RIP, TpA.
105. HMG CoA reductase has a role in both primary and secondary metabolism in
Penicillium paxilli
3-Hydroxy-3-methylglutaryl Coenzyme A reductase (HMGR) catalyses the conversion of HMG to mevalonic acid, the first step in isoprenoid biosynthesis. Mevalonic acid is a key intermediate for the synthesis of a range of primary and secondary metabolites. In the filamentous fungus Penicillium paxilli mevalonic acid is a key precursor for the biosynthesis of the secondary metabolite paxilline; an indole diterpenoid that is produced in large quantities in submerged cultures at stationary phase. The aim of this research is to determine whether hmg expression alters with the onset of paxilline biosynthesis. Using a probe generated by PCR the P. paxilli hmg was isolated from a lambda GEM11 genomic library and sequenced. Sequence analysis identified several characteristic promoter motifs associated with a constitutively expressed primary metabolite gene. The putative transcription site for this gene was determined by RT-PCR using a set of nested primers. RT-PCR analysis of hmg expression during the growth cycle of a submerged culture of P. paxilli showed a dramatic increase in expression of this gene compared to that of Beta-tubulin (tub-2). Furthermore, this pattern of expression mimiced that of two key paxilline biosynthetic genes, paxR and paxG, which have recently been cloned from P. paxilli (see Young et al. this meeting). This result would suggest that there is a regulatory mechanism within the cell for coordinate induction of primary (hmg) and secondary (pax) metabolite genes.
106. The creC carbon catabolite repression gene of Aspergillus nidulans encodes a protein containing WD40 repeats. Richard B. Todd, Robin A. Lockington, and Joan M. Kelly. University of Adelaide, Genetics, Adelaide, SA, AUSTRALIA.
Many microbial genes are subject to carbon catabolite repression (CCR), the repression in the presence of a preferred carbon source (e.g. glucose, sucrose) of the genes required for growth in the presence of less favourable carbon sources (e.g. acetamide, proline, quinate). In Aspergillus nidulans mutations which relieve CCR were previously selected as suppressors of the areA217 loss-of-function mutation for growth on sucrose plus acetamide. One class of recessive mutations map to creC, which is tightly linked to glnA on linkage group II. creC mutants display pleiotropic effects for growth on a range of carbon sources including decreased growth on some carbon sources such as quinate, and partially derepressed expression in repressing conditions of some enzymes such as alcohol dehydrogenase and acetamidase. We have exploited the proximity to glnA to clone by complementation the creC gene. Sequence analysis revealed that creC encodes a 630 amino acid polypeptide which contains a proline-rich region and WD40 repeats. CreC shows a high level of similarity with proteins of unknown function in Schizosaccharomyces pombe, mouse and human, but there is no close homologue in the Saccharomyces cerevisiae genome sequence. Regions of creC required for function have been localized by C-terminal deletion analysis and the determination of the sequence changes in creC mutant alleles.
107. SNF1 and carbon utilization in Cochliobolus carbonum. Nyerhovwo J. Tonukari, John S. Scott-Craig, and Jonathan D. Walton. Michigan State University, DOE Plant Research Lab, East Lansing, MI, USA.
Cochliobolus carbonum, an ascomycetous pathogen of maize, secretes a variety
of extracellular enzymes, such as pectinases, xylanases, glucanases and proteases, that can
degrade plant cell wall polymers. The production of most cell wall degrading enzymes is
substrate-induced and can be repressed by preferred carbon sources such as glucose. In
Saccharomyces cerevisiae the Snf1 (sucrose non-fermenting) kinase is required for
transcription of glucose-repressed genes when glucose is limiting. The SNF1 gene is
broadly conserved in eukaryotes, where it is involved in stress responses. Degenerate
oligonucleotide primers based on Snf1 homologs from S.cerevisiae,
Arabidopsis and rat were used to amplify a 400-bp fragment from C.
carbonum genomic DNA which was then used to isolate cDNA and genomic copies of the
gene. A specific Snf1 mutant was created by transformation-mediated gene replacement via
homologous recombination. There was significant reduction in the growth of the mutant on solid
media when xylan, pectin, cellulose and corn cell walls were used as carbon sources. The growth
of the mutant was similar to that of the wild type when glucose is the carbon source. The total
xylanase, glucanase and pectinase activities in culture filtrates of the mutant using corn cell walls
as carbon source were reduced by approximately 60, 40 and 30%, respectively.
108. Developmental regulation of two forms of arginase from Neurospora
crassa.
The existence of multiple arginases in N. crassa poses several interesting questions regarding function, regulation and evolutionary significance of the two forms. Multiple arginases are found in all vertebrates but this is the first characterization from a non-vertebrate. The function and regulation of liver arginase, an essential component of the urea cycle, is well understood; however the physiological role of the ubiquitous non-hepatic arginases is not known. In vertebrates the two major forms are products of separate genes, whereas in N. crassa the two major forms are differentially expressed from a single gene. A 1.4-kb transcript is synthesized in minimal medium which is then translated into a 36-kDa protein; however, in the presence of arginine, an additional 1.7-kb transcript is made. This mRNA has a 41-kDa protein as its translation product. This expression is controlled by the inducer arginine via mechanisms that are presently being investigated. In Xenopus it has been shown that the extrahepatic arginases are highly expressed during the transition from tadpole to frog. Given this precedent for arginase developmental expression, we examined the levels of the two major arginase transcripts and protein in conidia and germinating conidia in the wild-type strain, 74-R23-1A. Both forms appear to differ slightly in their developmental pattern with the arginine specific transcript just detected in conidia and the smaller transcript appearing at 2 hours. We have determined that arginase expression is elevated early in germination and decreases with time. This developmental profile is similar to several amino acid biosynthetic genes. This was an unexpected result for an inducible catabolic enzyme.
109. Isolation, characterisation and regulation of two sulphate transporter encoding genes from Penicillium chrysogenum.Mart van de Kamp, Theo A. Schuurs, Arnold Vos, Arnold J.M. Driessen, and Wil N. Konings. Department of Molecular Microbiology, Biological Centre, University of Groningen, The Netherlands.
Using a PCR-based approach, we have isolated two Penicillium chrysogenum genes, sutA and sutB, that encode sulphate transporters (see Poster abstract by Pizzinini et al.). Full-length genomic clones were obtained and sequenced. The presence of introns was determined from a comparison of genomic and cDNA sequences. Hydropathy profile analysis indicated that the SutA and SutB proteins contain 10-12 membrane-spanning regions. The gene products are members of the SulP superfamily of secondary sulphate transporters. We have studied the regulation of the sutA and sutB genes using Northern analyses and sulphate uptake experiments. In mycelium of P. chrysogenum grown under penicillin-producing conditions, the expression of sutA is much weaker than expression of sutB. The expression of both sutA and sutB is regulated by the sulphur content of the medium. Growth in the presence of excess sulphate results in repression of both genes and negligible sulphate uptake, whereas growth under sulphate starvation condition induces sutA and sutB expression which is reflected in high sulphate uptake rates.
110. Inter-nuclear gene silencing in Phytophthora infestans. Pieter van West1, Sophien Kamoun1,3, John W. van 't Klooster1, Neil A.R. Gow2, and Francine Govers. WAU, Phytopathology, Wageningen, The Netherlands. 2Univ of Aberdeen, Molecular Biology, Aberdeen, Scotland. 3 Present address: Department of Plant Pathology OSU, Wooster OH, USA.
Transformation of the diploid oomycete Phytophthora infestans with antisense,
sense and promoter-less constructs of the coding sequence of the elicitin gene inf1
resulted in transcriptional silencing of both the transgenes and the endogenous gene. To
investigate the mechanism of gene silencing we took advantage of the fact that P. infestans
has coenocytic mycelia and that mycelial cells may contain multiple nuclei that can differ
genetically, resulting in heterokaryotic strains. It appeared that: (i) transcriptional gene silencing
is dominant in multinucleated cells, (ii) the silenced state can be transmitted from nucleus to
nucleus in heterokaryotic strains, and (iii) gene silencing is maintained in a non-transformed
nucleus after nuclear separation (van West et al., 1999). In addition, we showed that upon fusion
of a silenced non-transgenic strain with a strain containing wildtype nuclei, the silenced state
could be transmitted again to the wild type nuclei. Transcriptional gene silencing in P.
infestans apparently involves inter-nuclear transfer of signals from silenced (transgenic
and non-transgenic) nuclei to wild type nuclei, leading to stable gene silencing in the wild type
nuclei. These findings support a model reminiscent of paramutation and involving a trans-acting
factor that is capable of transferring a silencing signal between nuclei.
111. Transposon mediated mutagenesis in Magnaporthe grisea.
Transposable elements (TEs) have been characterized in a broad range of organisms, from prokaryotes to higher eukaryotes. Based on their mode of propagation, they have been divided into two major classes (Finnegan, 1989). Class I elements transpose by reverse transcription of an RNA intermediate. Class II elements transpose by a DNA-DNA mechanism. TEs can generate mutations by moving from one genomic position to another through their insertion into a gene or its promotor. Daboussi and coworkers (1992) were the first to successfully use the nitrate reductase gene (niaD) as a transposon trap in filamentous ascomycetes. In this system, niaD mutants can be isolated by a direct selection for chlorate resistance. The class II element Impala, belonging to the Tc1-mariner superfamily, was identified as a Fusarium oxysporum mobile element using this strategy (Langin et al., 1995). This transposon is 1280 nucleotides long and contains two 27 pb inverted terminal repeats (ITRs) flanking an open reading frame encoding a putative 340 amino acid transposase. TEs can be usefull as genetic tools for gene tagging. Integration of the transposon into promoter or coding region can lead to a new phenotype allowing identification of unknown genes after isolating the genomic regions flanking the element. As Impala is an active transposon in Fusarium oxysporum, we decided to assay if it can be mobilized in an heterologous host such as Magnaporthe grisea in order to establish a transposon mediated mutagenesis system. Results concerning vectors used and Impala transposition will be presented. This work is supported by grants from a biotechnology contract between Rhône-Poulenc Agro and the MNSRE. Finnegan, 1989. Trends Genet. 5 : 103-107. Daboussi et al., 1992. Mol. Gen. Genet. 232 : 12-16. Langin et al., 1995. Mol. Gen. Genet. 246 : 19-28.
112. Characterization of an unprecedented group of genetic elements: linear, non-LTR retroplasmids of Fusarium oxysporum.Tobais Ch. Walther, Shannon L. Ross, and John C. Kennell. Southern Methodist University, Biological Sciences, Dallas, TX, USA.
Telomeres of eukaryotic chromosomes are maintained by enzymatic reactions that involve reverse transcriptase. It is widely speculated that the ribonucleoprotein complexes that maintain telomeres, telomerases, were derived from invasive genetic elements that encode reverse transcriptase; however, no retroelement counterpart has been identified that has structural similarities to telomeres or carries out reverse transcription in a manner that is mechanistically analogous to the reaction catalyzed by telomerases. We describe the complete sequence and primary structure of two related mitochondrial plasmids of Fusarium oxysporum, pFOXC2 and pFOXC3, which have features expected of genetic elements that are related to the precursors of telomerase. The plasmids have unique genomic structures which include a hairpin at one terminus and a telomere-like repeat of a 5 bp sequence at the other terminus and encode an active reverse transcriptase which shows specificity for the plasmid transcript. The plasmid replication cycle involves a novel strategy for copying terminal sequences via transcription of the hairpin structure and subsequent reverse transcription of the greater-than unit-length RNA. Our studies indicate that the Fusarium plasmids comprise a new group of genetic elements that replicate via reverse transcription: linear, non-LTR retroplasmids. The finding that the 3' termini of the plasmids have rudimentary features of eukaryotic telomeres suggests they may hold clues concerning the origin of telomerase and the evolution of linear DNA elements.
113. Non-zymogenic Chitin Synthase 3 (WdChs3p) localizes at the cytoplasm of Wangiella dermatitidis. Zheng Wang, and Paul J. Szaniszlo. University of Texas at Austin, Microbiology, Austin, TX, USA.
Because the class III chitin synthase (WdChs3p) of Wangiella dermatitidis, a
dematiaceous (melanized) fungus that is associated with phaeohyphomycosis, has preliminarily
been found to be a virulence factor, it was necessary to characterize this enzyme further and to
investigate its localization and regulation. Heterologous expression of the WdCHS3 cDNA in S.
cerevisiae demonstrated that WdChs3p was a non-zymogen and its membrane bound structure
was essential to maintain enzyme function. The WdCHS3 gene with six tandem repeats of the
myc epitope sequence incorporated at its 5' end was integrated at the WdCHS3 locus of the
genome of W. dermatitidis by homologous recombination. Western analysis using
monoclonal anti-myc antibody demonstrated that the WdChs3-myc protein level increased with
increasing temperature of culture, corresponding to the fact that the WdCHS3 transcription is
temperature and stress dependent. Production of WdChs3p-myc was significantly enhanced
starting from 6 hr after cells were shifted from 25 C to 37 C, implying that translation of
WdChs3p was also temporally regulated. Indirect immunofluorescence showed a cytoplasmic
localization for WdChs3-myc in all three predominant vegetative morphologies (yeasts,
multicellular forms and hyphae) of W. dermatitidis grown at 37 C, suggesting that
WdChs3p may play a role in maintaining the cell wall integrity at temperatures of infection.
Although introduction of WdCHS3 with its own promoter into the polyketide synthase locus in
the wdchs3D disruption strain restored lost chitin synthase activity, a truncated WdChs3p with
the deletion of its first 55 amino acids was not functional. This is in contrast to yeast chitin
synthases in which N-terminal sequences are not important.
114. Arginine-specific translational attenuation mediated by the Neurospora crassa
arg-2 arginine attenuator peptide appears independent of the charging status of
tRNA.
The arginine attenuation peptide (AAP) is encoded by an upstream open reading frame (uORF) in the 5'-leader of the N. crassa arg-2 mRNA. It is critical for Arg-specific translational regulation of the mRNA in vivo and in vitro in N. crassa. In vitro, Neurospora ribosomes that have translated the AAP stall when [Arg] is high. Here we show that the N. crassa AAP and a related AAP encoded by a uORF in the 5'-leader of the homologous Saccharomyces cerevisiae mRNA, CPA1, exert Arg-specific, negative translational regulation in N. crassa, S. cerevisiae and wheat germ cell-free translation reactions. AAP-containing mRNAs were used to demonstrate that the extent of translational regulation by [Arg] was proportional to [Arg] at concentrations between 150 and 5,000 micromolar. Yet, in the S. cerevisiae and wheat germ systems, each of the arginyl-tRNAs examined appeared fully charged even at much lower Arg concentrations (10 micromolar). These experiments indicate that the level of charged Arg-tRNAs is not significant for AAP-mediated, Arg-specific translational control. (Supported by NIH GM47498.)
115. Differential gene transcription in galled and healthy tissues of pine infected with Cronartium quercuum f. sp. fusiforme. Jaimie M. Warren, and Sarah F. Covert. The University of Georgia, Department of Genetics, Athens, GA, USA.
Cronartium quercuum f. sp. fusiforme causes the formation of woody galls on pine (Pinus spp.). To better understand the biology of this host-pathogen interaction, we used differential display to identify 22 transcripts which are differentially transcribed in healthy and galled tissues of slash pine (Pinus elliotti ) infected with C. q. fusiforme Several of these are homologous to previously cloned genes. Transcripts present in galls, but absent or transcribed at lower levels in healthy tissues include clones homologous to a fungal carboxypeptidase, a plant heat shock protein, a fungal cyclophilin, an NADPH cytochrome reductase, and a fungal cytochrome c oxidase polypeptide. Transcripts present in healthy tissues but absent or transcribed at lower levels in galls include clones homologous to a plant receptor-like kinase and a pine lipid transfer protein. The latter homologies are particularly interesting because these types of protein are associated with suppression of cell division and antimicrobial properties in plants, respectively. Ongoing work focuses on determining the genome of origin of each clone and confirming the differential transcription patterns of all 22 cDNAs in slash pine. The transcription patterns of these fragments will also be determined in healthy and infected loblolly pine (Pinus taeda ) and lodgepole pine (Pinus contorta ). This analysis will determine if the differential transcription of these cDNAs is consistent in three different host species infected by C. q. fusiforme .
116. Analysis of the structure and function of isocitrate lyase from Aspergillus nidulans. Kanchana Weeradechapon, K Linda. Britton, Sarah J. Langridge, Svetlana E. Sedelnikova, Geoffrey Turner and David W. Rice. University of Sheffield, Krebs Institute, Sheffield, South Yorkshire, UK.
Isocitrate lyase is essential for growth of microorganisms on 2C compounds and fatty acids, and is located in the glyoxysomes in Aspergillus nidulans. The structure of isocitrate lyase from A. nidulans has been solved at 2.8 using X-ray crystallography. The secondary structure of this tetrameric enzyme is arranged into two main domains, one of which forms a peripheral head domain to the enzyme molecule, and corresponds to an internal sequence of approximately 100 amino acids which is not found in prokaryotic ICLs. To understand the structure/function relationships of this enzyme including the molecular factors which control the differential targetting of the enzyme to glyoxysome in eukaryote and cytoplasm in prokaryote, a null mutant A. nidulans has been constructed by transformational deletion of the isocitrate lyase (acuD) gene for use as an expression host for acuD mutants generated in vitro. Mutations made to date include deletion of the internal additional amino acid sequence, and the disordered C-terminal region which may be involved in protein targetting. Mutated acuD was inserted into the null mutant by cotransformation. The results of the structure analysis of this enzyme and of the programme of mutagenesis designed to test proposals on the enzyme mechanism and intracellular location will be presented.
117. Growth disassociated production of heterologous protein by F. venenatum. Marilyn G. Wiebe1, Geoffrey D. Robson1, and Jeff Shuster2. 1University of Manchester, School of Biol. Science, Manchester, Gtr. Manchester, UK. 2 Novo Nordisk Biotech. Inc, 1445 Drew Avenue, Davis, California, USA.
Most recombinant proteins generated in filamentous fungi are produced in fed-batch cultures, in which specific growth rate decreases progressively with time. Because of this, such cultures are more suited to the production of growth rate disassociated than growth rate associated products. Fusarium venenatum A3/5 has been transformed (JeRS 325) to produce Aspergillus niger glucoamylase (GAM) under the control of the Fusarium oxysporum trypsin-like protease promoter. No glucoamylase was produced during exponential growth of F. venenatum JeRS 325 in batch culture, and in glucose-limited chemostat cultures glucoamylase was produced in a growth rate disassociated manner with GAM concentration decreasing with increase in dilution rate and with the specific production rate of GAM remaining approximately constant over the dilution rate range of 0.05 h-1to 0.19 h-1. Specific production rates of 5.8 mg and 4.0 mg GAM [g biomass]-1 h-1 were observed in glucose-limited chemostat cultures in the presence and absence of 1 g mycological peptone L-1.
118. Cloning of two delta9 desaturase genes from Mortierella alpina. Prasert Wongwathanarat, Donald A. MacKenzie, Andrew T. Carter, and David B. Archer. Institute of Food Research, Genetics and Microbiology, Norwich, UK.
The long chain polyunsaturated fatty acids are of both nutritional and pharmarcological importance to man because they can prevent and be used to treat a wide range of human diseases and disorders by serving as precursors for hormones such as prostaglandins and leukotrienes. They also have an important role in enhancing infant brain development. Recently, it has been shown that the oleaginous soil fungus, Mortierella alpina, which belongs to the order Mucorales has the potential to synthesize long-chain polyunsaturated fatty acids such as arachidonic acid (C20:4,n6) on a commercial scale. This fungus also produces smaller amounts of oleic acid (C18:1, n9), linoleic acid (C18:2, n6), gamma-linolenic acid (C18:3, n6) and eicosapentaenoic acid (C20:5, n3). Physiological approaches to increase the yield of arachidonic acid or to change the fatty acid composition have been successful with this fungus in fermentors and conditions have been optimized at commercial scale. Strain improvement has also been acheived by standard mutagenesis. Alternatively, genetic modification of this fungus is another approach to increase the yield of arachidonic acid or to change the fatty acid composition. It has been shown that synthesis of arachidonic acid requires a series of desaturase genes and an elongase gene. The delta9 desaturase is the first enzyme involved in this pathway. To isolate and clone the delta9 desaturase gene encoding this enzyme, PCR using degenerate primers with homology to other known delta9 desaturase genes from several fungi was performed. Interestingly, it was found that there were two delta9 genes from this fungus and another delta9-like desaturase gene which had only 50% amino acid identity to other delta9 genes. Identification of this gene is underway. Northern and RT-PCR analysis of the two delta9 desaturase genes suggested that only one of these genes was expressed in M. alpina.
119. Using DNA-Tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae. Debbie S. Yaver, Howard Brody, Michael Lamsa, Rebecca Munds, Stephen H. Brown, Suzie M. Otani, and James A. Johnstone. Novo Nordisk Biotech, 1445 Drew Avenue, Davis, CA, USA.
In order to identify Aspergillus oryzae genes that are important for heterologous protein production a large library of DNA-tagged mutants was generated and screened for production of a model heterologous protein. Using restriction enzyme-mediated integration (REMI) two libraries of 27,000 and 28,000 transformants were made in an A.oryzae strain producing a Thermomyces lanuginosus lipase using pyrG as the tagging DNA and either BamHI or EcoRI enzyme, respectively. The libraries were screened robotically for lipase production, and mutants with either decreases or increases in lipase production were isolated. The pyrG tagging vector along with the genomic DNA flanking the integration event has been rescued from one of the yield improved mutants (DEBY10.3), and the nucleotide sequence of the flaking DNA shares identity to the Aspergillus nidulans palB gene. As expected for a palB minus strain, the DEBY10.3 mutant is unable to grow on pH 8.0 minimal medium plates. Three lines of experimental evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB minus phenotype generated by the integration of the tag into the palBgene. These results will be presented.
120. A color-selectable and site-specific integrative system for gene expression studies in the dematiaceous fungus Wangiella (Exophiala) dermatitidis. Xiang-cang Ye, Bin Feng, and Paul J. Szaniszlo. University of Texas at Austin, Microbiology, Austin, TX, USA.
Many dimorphic, darkly pigmented fungi of the form-family Dematiaceae of the Fungi Imperfecti are pathogens of humans. As an initial attempt to explore potential virulence factors in the model dematiaceous fungus Wangiella dermatitidis, we established a gene overexpression system with properties of site-specific integrative transformation and color identification. Using a partial polyketide synthase gene (WdPKS1) as the targeting sequence and transformation by electroporation, we found that from 30 to 52% of resulting transformants contained exogenous markers site-specifically integrated into the target locus. This disrupted melanin precursor synthesis and produced albino colonies immediately distinguishable from the black parental strain and from nonspecific transformants. In addition, a heterologous glucoamylase promoter, glaA, was examined under various conditions for overexpression of the lacZ reporter gene and the WdCDC42-1 gene of W. dermatitidis. With this promoter, enhanced beta-galactosidase activity required an elevated temperature of 37C and an agar medium containing maltose as a sole carbon source. Our results showed that this unique integrative gene expression system allows the production of isogenic transformant strains for gene overexpression analysis in W. dermatitidis and most likely other black fungi.
121. An asparaginase gene which is under ammonia and oxygen repression. Darryl D. Yorkey, Patricia M. Shaffer. University of San Diego, Chemistry, San Diego, CA, USA.
Aspergillus nidulans has two asparaginase genes, apnA (Chrom. II) and ahrA (Chrom. VIII). Enzymatic studies demonstrated that the expression of the ahrA encoded enzyme is under both ammonia and oxygen repression which is regulated by the positively-acting protein product of the areA gene [Shaffer et al. (1988) Mol. gen. Genet. 212, 337-341]. Neither of these genes were able to be cloned by complementation of null mutants. Homology cloning based on two homologous regions present in 13 known asparaginases produced two PCR products which, upon sequencing, yielded the conserved regions. These two are being used to probe the cosmid libraries assigned to Chomosomes II and VIII. The final analysis of this gene is still in progress. The significance of this research is two fold: it provides data on an environmentally regulated gene and the availability of another source of asparaginase used as a cure for childhood acute lymphoblastic leukemia. This research is funded by a USD Faculty Research Grant and by a grant from the Lee Leichtag Family Foundation
122. The hyper-osmotic stress response pathway of Neurospora crassa is the target of phenylpyrrole fungicides. Yan Zhang, Randy Lamm, Christian Pillonel, Jin-Rong Xu, and Stephen Lam, Novartis Crop Protection, Inc., Research Triangle Park, NC 27709.
Osmotic sensitive mutants of Neurospora crassa are sensitive to high osmolarity, therefore unable to grow on medium containing 4% NaCl. They are also resistant to dicarboximide and aromatic hydrocarbon fungicides. In this study, osmotic-2 (os-2) and osmotic-5 mutants were found to be resistant to phenylpyrrole fungicides. The Neurospora crassa pMOcosX library was used in sib-selection to isolate the gene(s) complementing the osmotic sensitive phenotype of os-2. A cosmid was identified and mapped to the position of os-2 on LG IVR. A cDNA clone which encodes a homolog of yeast HOG1 MAP kinase was used to screen the same cosmid library and the same cosmid was isolated. A subclone of the cosmid containing solely the HOG1 homolog could functionally complement the osmotic sensitive phenotype of os-2, suggesting the os-2+ locus encodes a homolog of yeast HOG1. The gene would be referred to as the os-2 gene. Transformants of os-2 mutants with the os-2 gene became phenylpyrrole fungicide sensitive, indicating that the hyper-osmotic stress response pathway of Neurospora crassa is the target of phenylpyrrole fungicides. Three os-2 alleles were sequenced by PCR amplification and sequencing of overlapping DNA fragments and mutations in the os-2 gene have been identified. Gene knock-out and cytological studies are currently underway.
123. The bmp1 kinase gene is essential for fungal pathogenicity in Botrytis cinerea.Li Zheng, Mathew Campbell, Stephen Lam and Jin-Rong Xu, Novartis AgBiotech Research Institute, Research Triangle Park, NC27709.
The PMK1 mitogen-activated protein (MAP) kinase pathway has been recently shown to be important for appressorium formation in the rice blast fungus Magnaporthe grisea. Here we tested whether the same MAP kinase is essential for plant invasion processes in the gray mold fungus Botrytis cinerea which has different plant infection mechanism . We used a PCR-based approach to isolate MAP kinase homologues from B. cinerea. The BMP1 (Botrytis MAP kinase required for Plant infection) MAP kinase is highly homologous to the Magnaporthe PMK1. BMP1 is a single copy gene. The bmp1 gene replacement mutants have no defect in fungal growth on artificial media. One of the bmp1 knock-out mutants is reduced in conidiation. All bmp1 mutants tested lost their pathogenicity on carnation flowers and tomato leaves. When the wild type BMP1 allele was reintroduced into one of the bmp1 knock-out mutants, all BMP1+ transformants were fully pathogenic as the wild type strains. Further studies with SEM indicated that conidia from bmp1 mutants could germinate normally on plant surfaces. However, these germinating conidia failed to penetrate through the cuticle and macerate plant surface tissues. bmp1 mutants also appeared to be defective in infection through wounding. These results indicated that the BMP1 MAP kinase is essential for plant infection in B. cinerea. This PMK1/BMP1 pathway may be widely concerved in other plant pathogenic fungi for regulating plant infection processes such as appressorium formation and cell wall degrading enzyme production
124. Localization and expression profiling of genes expressed during appressorium formation on chromosome 7 in Magnaporthe grisea. Heng Zhu, Woobong Choi, Ralph A. Dean. Clemson University, Plant Pathology , Clemson, SC, USA.
Magnaporthe grisea is an important model system for the study various aspects of plant-fungal interactions, including the mechanisms regulating induction and formation of the appressorium. In previous studies, a minimum tile containing 41 BAC clones of chromosome 7 was constructed. Hybridization of cDNA from appressorium formation stage to HindIII digested minimum BAC tile identified a 128 HindIII fragments. These fragments identified 466 cDNA clones from an appressorium stage cDNA library using a two-dimensional probe pooling strategy. The cDNA clones were sequenced and mapped to a 100kb resolution on chromosome 7. 310 of 466 were found to be unique cDNA sequences. Only four had been previously identified in M. grisea. The DNA from the unique cDNA clones was double-stamped onto nylon filters, and hybridized to cDNA probes made from 4 developmental stages - appressoria, mycelia, conidia, and germing conidia. Based on expression profiles, cDNA clones were assigned to different developmetal stages. A model assigning putative functions to these newly discovered genes associated with appressorium formation will be presented. Sexual and Asexual Differentiation Abstracts
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