296 Analysis of the function and regulation of the NADP-isocitrate dehydrogenase (NADP-IDH) gene from Aspergillus nidulans. Edyta Szewczyk, Sophie Kourambas, Meryl A Davis and Michael J Hynes. University of Melbourne, Department of Genetics, Parkville, Australia
Many micro-organisms can use two-carbon compounds, like acetate or ethanol, as well as long chain fatty acids as sole carbon sources. Pathways and enzymes of the glyoxalate bypass, of fatty acid beta-oxidation, located in peroxisomes, as well as of the citric acid cycle located in mitochondria are required for the utilization of these carbon sources. The C6 zinc cluster protein FacB is required for acetate induction of enzymes involved directly in acetate metabolism, as well as of the glyoxalate bypass enzymes. We have previously shown that NADP-dependent isocitrate dehydrogenase activity is also regulated by FacB dependent acetate induction and by CreA mediated carbon catabolite repression in a similar manner to amdS, facA, acuD and acuE.
The structural gene (idpA) has been cloned and shown to be regulated by CreA and FacB and also by induction by fatty acids. The predicted protein possesses both an N-terminal mitochondrial targeting signal and a C-terminal peroxisomal signal. This provides an interesting contrast with the situation in Saccharomyces cerevisiae where there are three genes encoding enzymes located in separate cell compartments. By the use of fluorescent protein (GFP, RFP and BFP) fusion constructs, idpA encoded NADP-isocitrate dehydrogenase has been found to be present in peroxisomes and mitochondria. The structure of the gene suggests the presence of two alternative start points of transcription that may result in the two different forms of the enzyme.
No mutants lacking NADP-IDH are known in A.nidulans. It is likely that NADP-IDH is required for NADPH generation during growth on acetate and fatty acids and that a deletion of the gene will affect growth on these carbon sources at least to some extent.
297 Photoinduction of phr1 expression and sporulation is independent in Trichoderma harzianum. Teresa Rosales Saavedra1, Victor Rocha Ramirez1, Benjamin A. Horwitz2 and Alfredo Herrera Estrella1. 1Department of Plant Genetic Engineering, Centro de Investigacion y Estudios Avanzados del I.P.N., Unidad Irapuato, Irapuato, Gto. 36500, Mexico and 2Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel
Blue light and development regulate the expression of the phr1 gene, which encodes the DNA repair enzyme photolyase of the filamentous fungus Trichoderma harzianum. Trichoderma also sporulates in a synchronized manner following a brief pulse of blue light. The effect of a set of chemicals known to either stimulate or inhibit the activity of different elements in signal transduction pathways was tested in terms or their effect on photolyase gene expression and conidiation, both under light and dark conditions. dbcAMP bypasses the requirement of light for sporulation, while atropine prevents sporulation even after photoinduction. Light regulation of phr1 however, is indifferent to both these effectors. Induction of photolyase expression behaves as a direct, rapid response to light, independent of the induction of sporulation. Photoinduction of phr1 and light induced sporulation,could be expected to share elements common to signal trandsduction pathways if not the same pathway. Nevertheless, the analysis of the responses with mutants, wich have been altered in the alpha subunit of the heterotrimeric (Tga1) G protein, constitutively activated that in turn inhibited completely their conidiation, allowed us to conclude that sporulation and photolyase expression are distinct in their photoreceptor system or in the transduction of the blue light signal.
298 Expression of the mycoparasitism related genes prb1 and ech42 in Trichoderma harzianum is determined by two groups of protein kinases. A. Mendoza-Mendoza; M. Ramos-Vega and A. Herrera-Estrella. Departamento de Ingenieria Genetica de Plantas. Cinvestav-IPN Unidad Irapuato. Guanajuato, Mexico.
Trichoderma harzianum has an antagonic ability towards a phytopathogenic fungi. The mechanisms that confer this capacity are: antibiotic production for competition nutrients and mycoparasitism. The latter involves the production of lytic enzymes that degrade the host cell wall. The prb1 and ech42 genes encode two lytic enzymes produced by T. harzianum in response to host or nutrient deprivation, mainly the lack of glucose and nitrogen source. Our results suggest that the expression of these genes in nutrient deprivation depends on the activation of a MAP kinase pathway. Likewise confrontation between T. harzianum with Sclerotium rolfssi or Rhizoctonia solani shows an inverse correlation between the prb1 and ech42 expression and the PKA activity. We have observed that the cAMP levels are modificated when T. harzianum is confronted with S. rolfssi. All these results suggest that the regulation of expression of prb1 and ech42 in T. harzianum is complex and that at least two groups of kinases are involved, the MAP kinase and the cyclic AMP dependent-kinase.
299 Characterization of the transcription factor PACC and its involvement in cephalosporin C biosynthesis gene regulation in Acremonium chrysogenum. Esther Schmitt, Renate Kempken. Ruhr-Universitat Bochum, Allg. und Mol. Botanik, Bochum, Germany
The analysis of the pcbC promoter from the cephalosporin C producing filamentous fungus Acremonium chrysogenum was performed using fungal transformants carrying reporter gene fusions. By investigating sequential deletion derivatives of the pcbC promoter region, a DNA fragment was identified, which is responsible for transcriptional activation of the pcbC gene. Sequence analysis of this fragment revealed a consensus binding site for the fungal PACC transcription factor. Gel retardation experiments with A. chrysogenum protein crude extracts confirmed the specific binding of a protein to the PACC binding site. The subsequent cloning of the pacC homologue from A. chrysogenum allowed the identification of an open reading frame of 621 amino acids encoded by four exons. The polypeptide shows about 35 % identical amino acid positions when compared with other fungal PACC proteins. An E. coli synthesized PACC protein fragment was used for in vitro binding assays and specific binding of the zinc finger transcription factor to its consensus binding sites in the promoter regions of four cephalosporin C biosynthesis genes could be demonstrated. The bi-directional promoters of the pcbAB-pcbC and cefEF-cefG genes contain two PACC binding sites each. The obtained data strongly suggest that in A. chrysogenum, the zinc finger transcription factor PACC is involved in the transcriptional regulation of the cephalosporin C pathway genes.
Schmitt EK, Kempken R, Kuck U. Functional analysis of promoter sequences of cephalosporin C biosynthesis genes from Acremonium chrysogenum: specific DNA-protein interactions and characterization of the transcription factor PACC. Mol Gen Genet (submitted)
300 The CPCR1 transcription factor is important for timely expression of a cephalosporin C biosynthesis gene in Acremonium chrysogenum. Esther K. Schmitt and Ulrich Kück. Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44780 Bochum, Germany
The filamentous fungus Acremonium chrysogenum is used industrially to produce the -lactam antibiotic cephalosporin C. Our aim is to isolate and characterize transcription factors from A. chrysogenum which regulate transcription initiation or repression of biosynthesis genes. The CPCR1 protein was identified in a one-hybrid screen using a sequence from the pcbAB-pcbC promoter as a bait. The isolated DNA-binding protein belongs to the conserved family of eukaryotic RFX transcription factors (1). Using a recombinant CPCR1 protein putative binding sites in the two promoter regions of cephalosporin C biosynthesis genes pcbAB-pcbC and cefEF-cefG were analyzed for interaction with the transcription factor. It was shown that CPCR1 recognizes and binds, in addition to the previously determined binding site, another sequence in the intergenic region between the pcbAB-pcbC genes. We also present results from Acremonium transformants with multiple copies of the cpcR1 gene and knock-out strains. These experiments give insight into the regulatory role of CPCR1 with regard to expression of the pcbC gene. Fungal transformants were analyzed using northern and western blots and HPLC analysis of culture broth to determine changes in the biosynthesis of cephalosporin C. Together with two other transcription factors that are involved in the regulation of cephalosporin C biosynthesis, namely CRE1 (2,3) and PACC (4), the functional analysis of these regulatory proteins in A. chrysogenum will widen our understanding of the molecular regulation of cephalosporin C biosynthesis.
1) Schmitt EK, Kück U (2000) The fungal CPCR1 protein, which binds specifically to B-lactam biosynthesis genes, is related to human RFX transcription factors. J Biol Chem 275:9348-9357 2) Jekosch K, Kück U (2000) Glucose dependent transcriptional expression of the cre1 gene in Acremonium chrysogenum strains showing different levels of cephalosporin C production. Curr Genet 37:388-395 3) Jekosch K, Kück U (2000) Glucose repression is lost in an Acremonium chrysogenum B-lactam producer strain and can be restored by multiple copies of the cre1 gene. Appl Microbiol Biotechnol 54:556-563 4) Schmitt EK, Kempken R, Kück U Functional analysis of promoter sequences of cephalosporin C biosynthesis genes from Acremonium chrysogenum: specific DNA-protein interactions and characterization of the transcription factor PACC. Mol Gen Genet (submitted)
301 Isolation and characterization of a Vacuolar-ATPase (MVP1) gene of Magnaporthe grisea. Frederick O. Asiegbu1,3, Woobong Choi1,2, Jun Seop Jeong 1,2 and Ralph Dean1,2*. 1Clemson University Genomics Institute (CUGI), Clemson SC, USA. 2Fungal Genomics Laboratory, Department of Plant Pathology, North Carolina State University, NC, USA. 3Dept. of Forest Mycology & Pathology, Swedish University of Agriculture, Uppsala, Sweden.
Vacuolar ATPases play crucial roles in regulating the metabolic activities in the vacuoles of eukaryotic cells. We isolated a gene encoding v-ATPase homologue of Magnaporthe grisea, named MVP1, from an appressoria cDNA library. The transcript of ~1.6kb contains 546bp of coding sequence with a 3' untranslated region of about 168 bp long and 5' untranslated region of about 870bp long. The hybridization pattern on Southern blots of genomic DNA following digestion with three restriction enzymes (BamH1, EcoR1,HindIII) indicated that the gene exists as a single copy M. grisea genome. RNA gel blot analyses showed that MVP1 was highly expressed in germinating spore and mycelial stage compared to appressoria or non germinated spores. MVP1 showed a high degree of homology to other v-ATPases, particularly with the gene from Neurospora crassa. Homologous recombination using a knockout construct in pAN7-1 is being used to disrupt MVP1. The pathological and other phenotypic consequences of gene disruption will be presented.
302 A genetic selection for circadian clock mutations in Neurospora crassa. Deborah Bell-Pedersen* and Irene March. Department of Biology, Texas A&M University, College Station, TX 77843
To identify components of the circadian clock system in Neurospora crassa, we have carried out a genetic selection to isolate mutations that alter the expression of clock-controlled genes (ccgs). This selection is based on the differential expression of the ccgs in response to the presence or absence of the clock gene product FRQ. For example, ccg-1 expression is repressed and ccg-2 expression is activated when FRQ is present in the cell. The promoter region of the ccg-1 gene was ligated to mtr (1). The mtr gene encodes a neutral amino acid permease that allows both positive and negative selection. Loss of function can be selected based on resistance to the amino acid analog p-flourophenylalanine (FPA). Gain of function can be selected based on growth of tryptophan auxotrophs on high arginine/low tryptophan media. The fusion constructs were transformed into both bd;mtr;trp- 2 and bd;frq10;mtr;trp-2 strains and homokaryons were isolated. Reconstruction experiments using the ccg-1:mtr transformants showed FRQ- dependent production of the amino acid permease: growth of the FRQ+ transformant on FPA but no growth on tryptophan, and growth of the FRQ-null transformant on tryptophan but no growth on FPA. Both strains were subjected to UV light mutagenesis and assayed for growth under the opposite conditions. Out of 10,000 colonies screened for each transformant, we have identified 2 mutant strains in the FRQ-null background, and 5 mutant strains in the FRQ+ background that yield the desired growth on the selective media. The circadian phenotypes of these mutations will be discussed. 1. Stuart WD, Koo K, Vollmer SJ (1988) Cloning of mtr, an amino acid transport gene of Neurospora crassa. Genome 30: 198-203.
303 Testing a potential implication of the alternative oxidase (AOX) in the control of longevity in Podospora anserina Severine Lorin, Eric Dufour, Odile Begel, Jocelyne Boulay and Annie Sainsard-Chanet. Centre de Genetique Moleculaire du CNRS, 91198 Gif-sur-Yvette, France.
Podospora anserina is an organism which presents a limited vegetative growth and a senescence phenomenon. Recently, a causal link between this senescence and respiration was demonstrated. In fact, in mutants deficient in complex IV of the respiratory chain, the longevity is strongly increased. Those mutants use exclusively an alternative respiratory pathway implicating the alternative oxidase protein (AOX), present in the inner mitochondrial membrane and accepting electrons from the quinone pool. Nowadays, several lines of evidence implicate ROS in the pathogenesis of various degenerative diseases and in ageing. On the other hand, it was recently proposed that the alternative pathway present in plants may serve to lower ROS mitochondrial production. In order to understand the potential implication of ROS and the alternative oxidase in ageing, we have tested the effects of overexpression and disruption of the alternative oxidase gene in our organism. We showed that, in mutants overexpressing the alternative oxidase gene (50 % SHAM sensitive) and in mutants disrupted for the alternative oxidase gene, longevity is unchanged. Moreover, the level of ROS seems to be the same as in the wild-type strain. Last of all, the events of mitochondrial DNA instability correlated with senescence are the same as in the wild-type strain. Taken together, our results suggest that the alternative oxidase of Podospora anserina cannot lower ROS production and that it has no effect per se on longevity. Finally, recent efforts have been directed to test the effects of overexpression of the alternative oxidase gene in mutants deficient in complex IV; preliminary results indicate that the level of global respiration may be implicated in senescence.
304 Identification and characterisation of a histidine kinase gene in Botrytis cinerea. 1W. Cui; 2R. E. Beever; 2S. L. Parkes; 1M. D. Templeton. 1Plant Health and Development Group, HortResearch, Private Bag 92 169, Auckland, New Zealand. 2Landcare Research, Private Bag 92 170, Auckland, New Zealand
Dicarboximide fungicides play an important role in the control of grey mould of plants caused by Botrytis cinerea. The appearance of fungicide resistant strains is an increasingly important consideration in the control of this fungal pathogen by chemical means. The mode of action of dicarboximide fungicides and the mechanism of their resistance have been under investigation for many years, yet remain unclear. This project aims to develop a detailed understanding of fungicide resistance in B. cinerea at the molecular level. A number of recent studies on laboratory mutants in 'model' fungal systems suggest that there is a correlation between dicarboximide resistance, osmotic sensitivity, and certain protein kinases. We have isolated a wide range of field strains and laboratory mutants, with low to high levels of dicarboximide resistance from B. cinerea. The mutants resistant to dicarboximides are mostly also osmotically sensitive. Genetic studies of B. cinerea have shown that resistance is conferred by a single chromosomal gene designated Daf1 (dicarboximide and aromatic hydrocarbon fungicide resistant). Os-1 mutants of Neurospora crassa behave in a similar way to Daf1 mutants of B. cinerea, in that they confer osmotic sensitivity and resistance to dicarboximide fungicides. We hypothesise that Daf1 in B. cinerea is a homologue of the Os-1 in N. crassa. We have cloned and sequenced the homologue from B. cinerea from both sensitive wild type strain and resistant mutant strains. Work is in the progress to clarify its relationship to Daf1.
305 Hideaway, a repeated element from Ascobolus immersus is rDNA associated and may resemble a class I transposon. Frank Kempken. Ruhr-Universitat Bochum, Allg & Molekulare Botanik, Bochum, Germany
In this study a genetically unstable strain from Ascobolus immersus stock 50 was monitored for the presence of repeated DNA. Several lambda-clones were identified from a differential hybridization approach. Among these, one lambda clone contained a repeated DNA sequence with unusual characteristics. Southern hybridization, PCR analysis and sequence analysis suggest this DNA element to be associated with rDNA sequences. Its methylation pattern is also reminiscent of rDNA. The element possesses structural characteristics of class I retrotransposons. The data presented are discussed with respect to the ability of repeated DNA sequences to escape host defense mechanisms.
Please see also, Abstract #501
306 Gene-tagging of Magnaporthe grisea growth-retarded mutants: Grm8 encodes a cross-pathway control protein. Sheng-Cheng Wu, Zhiying Zhao, Alan G Darvill, Peter Albersheim. University of Georgia, Complex Carbohydrate Research Ctr. Athens, Georgia
Growth-retarded mutants (GRM) were screened from Magnaporthe grisea protoplasts mutagenized by random plasmid insertions. The grm phenotype of one mutant, GRM8, co-segregates with a selection marker on the plasmid. The tagged gene, Grm8, is structurally similar to those encoding cross-pathway control proteins (CPC) from other fungi. Grm8 complements cpc-1 mutant of Neurospora crassa, and thus, is a functional ortholog of CPC-1. The expression of Grm8 is up-regulated during amino acid starvation, so are genes encoding, respectively, aspartate aminotransferase (Aat1) and carbamoyl phosphate synthase (ARG2). In strain C706, a grm8 knockout mutant, expression of Aat1 and ARG2 is no longer enhanced by amino acid starvation. In amino acid-deficient media, C706 expectedly grows poorly as compared to its wild-type parent. However, in amino acid-rich media, C706 unexpectedly grows at a much slower rate than its parent before accelerating after 5 days, an indication that Grm8 plays additional roles regulating M. grisea growth. In consistence with its phenotype in culture media, the grm8 mutants exhibit significant reduction in pathogenicity towards rice host. (This work was supported by U. S. Department of Energy grant DE-FG05-93ER20114 and the DOE-funded (DE-FG05-93ER20097) Center for Plant and Microbial Complex Carbohydrates.)
307 Influence of ambient pH and different stress conditions on Antifungal Protein expression in Aspergillus giganteus. V.Meyer, M.Wedde and U. Stahl. Department of Microbiology and Genetics, Berlin University of Technology, 13355 Berlin, Germany
The Antifungal Protein (AFP) secreted by the mould Aspergillus giganteus is a small highly basic polypeptide of 51 amino acids. Interestingly AFP inhibits the growth of a number of filamentous ascomycetes, whereas growth of bacteria, yeasts and mammalian cells is not affected. Regulation of afp expression is as yet unclear but it is supposed that afp is regulated at the transcriptional level. In order to test conditions involved in transcriptional regulation, a reporter system (afp::uidA) was established. As a result it could be shown that afp expression is induced by alkaline ambient pH. The pH regulation of gene expression in Aspergillus is mediated by the transcriptional factor PacC through binding at the core consensus sequence GCCARG (Tilburn et al., 1995). To verify a PacC dependent regulation of afp expression, the interaction between a PacC fusion protein (Espeso et al. 1997) and the afp promoter was determined by electrophoretic mobility shift assays. We were able to show that the PacC fusion protein binds to two core consensus sequences within the afp promoter. Binding to a third putative site which exhibits one point mutation (GCCAAC) was not observed. Moreover, we investigated afp::uidA expression in response to different stress conditions: heat shock, osmotic stress, oxidative stress, and ethanol. All conditions tested were able to induce afp::uidA expression, however heat shock exposure was the strongest inducer. This result could be confirmed for AFP, since the amount of secreted AFP was significantly higher after heat shock treatment.
Tilburn et al. (1995) EMBO J. 14: 779-790. Espeso et al. (1997) J.Mol Biol. 274:466-80
308 Characterization of the DNA binding of SRE, a GATA factor involved in iron transport in Neurospora crassa. Kelly A. Harrison, George Marzluf. The Ohio State University, Biochemistry Columbus, Ohio
Several homologous genes encoding proteins involved in regulating siderophore synthesis in fungi have been isolated, including sre of Neurospora. SRE is a member of the GATA factor family, which is comprised of transcription factors that contain either one or two zinc finger motifs that recognize and bind to 'GATA' containing DNA sequences. The DNA binding properties of SRE have been characterized by electrophoretic mobility shift assays (EMSA). Results demonstrate that SRE binds specifically to DNA containing GATA sequences. EMSAs with SRE zinc finger mutants show that both zinc fingers of SRE are involved in DNA binding. EMSA results illustrate that SRE binds to a DNA probe comprised of the iron response element from the sid1 promoter from Ustilago. These results also show that SRE binds to the sid1 promoter with higher affinity than to the asd4 promoter. In contrast, NIT2, another GATA factor in Neurospora, binds to both sid1 and asd4 promoters with similar affinities. The major difference between the sid1 and asd4 promoters is the spacing between the two GATA sequences (25 and 10 bps, respectively). Additional experiments revealed that SRE loses its ability to bind to DNA following EDTA treatment. DNA binding by SRE, however, was rescued by adding back cadmium or zinc after EDTA treatment, therefore demonstrating that DNA binding by SRE is zinc dependent. The results taken together suggest that SRE is a two zinc finger GATA factor specific for regulating siderophore synthesis at the sid1 promoter in Neurospora crassa.
309 Identification of targets of a b-mediated regulation cascade in Ustilago maydis. Gerhard Weinzierl1, Andreas Brachmann2, Regine Kahmann1 and Joerg Kaemper1, 1Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch- Str., D-35043 Marburg, 2Institute of Genetics and Microbiology, Ludwig Maximilians University, Maria-Ward-Str. 1a, D-80638 München, Germany
Pathogenic and sexual development of the fungus Ustilago maydis, the causal agent of smut disease on corn, is controlled by the multiallelic b- mating type locus encoding the two unrelated homeodomain proteins bW and bE. Upon fusion of haploid sporidia, the bE and bW proteins can form a heterodimeric complex that functions as a transcriptional regulator. In addition to genes that were shown to be directly regulated by the bE/bW complex, several genes have been isolated that are indirectly regulated via a b-dependent regulatory cascade, subjected to either positve or negative regulation. However, when deleted individually, none of these genes provided to be crucial for pathogenicity. In a genetic screen aimed to identify components of the b-dependent regulatory cascade, we have used three different b-dependently expressed genes simultaneously as reporters. Since components of the cascade could affect subsequent differentiation processes and pathogenicity by acting either as repressors or activators, we have employed both UV mutagenesis and the screening of transformants with a cDNA expression library. We will present data on the characterization of two of the UV mutants that not only show an altered expression of the reporter genes but are also affected in the expression of other b-dependent genes. A similar phenotype was observed after overexpression of the drf1 gene that was identified via the cDNA expression library. Drf1 expression leads to the expression of various b-repressed genes, suggesting a role as transcriptional activator. We will discuss these results with respect to our current model of b-dependent gene regulation.
310 Cloning of pal genes from Neurospora crassa. Sergio R. Nozawa1, Karina C. Nogueira1, Andre Justino1, Monica S. Ferreira-Nozawa2, Nilce M. Martinez-Rossi2 and Antonio Rossi3. 1Departamento de Química, 2Departamento de Genética, 3Departamento de Bioquímica e Imunologia. Ribeirão Preto, São Paulo University, Brazil.
Although ambient pH control of enzyme secretion was first described in N. crassa by Nahas et al. (J. Gen. Microbiol. 1982, 128: 2017), the molecular responses to external pH changes are still poorly understood. In A. nidulans, the palA, B, C, F, H and I genes are putative members of a signaling cascade involved in ambient pH sensing and the consecutive activation of genes required for growth under alkaline conditions. Thus, the molecular characterization of N. crassa genes homologous to pal genes from other fungi will help us to better understand their role in the ambient pH response. In this report, we describe the cloning and characterization of the palA and palF genes from N. crassa. RIM20 (palA homologue) and RIM8 (palF homologue) of Candida albicans were used to probe N. crassa genomic DNA digested with EcoRI, BamHI or HindIII. Southern-blotting bands were subcloned in the pUC18 vector and amplified in DH5alpha E. coli cells and the DNA fragment recovered was sequenced. We also provided evidence that both the palA and pal F homologues from N. crassa genes are possibly involved in post-transcriptional and/or post-translational modification of the Pi-repressible phosphatases, controlling their secretion into the extracellular medium. Financial support: FAPESP, CNPq, FAEPA and CAPES.
311 The lysine biosynthetic gene lysA of Aspergillus nidulans is transcriptionally regulated by the cross-pathway control of amino acid biosynthesis. S. Busch, H.B. Bode, I. Nörenberg and G.H. Braus. Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen, Grisebachstraße 8, 37077 Göttingen
In fungi, lysine is synthesized via alpha-aminoadipate.This intermediate serves as starting material for penicillin production in some filamentous fungi like A. nidulans and P. chrysogenum. Lysine biosynthesis thus constitutes a connection between primary and secondary metabolism in A. nidulans. Therefore, regulation of the lysine biosynthesis is of major interest. We investigated lysine biosynthesis in respect to a system of broad amino acid regulation. This cross-pathway control is a global regulatory network which coordinately increases transcription of several genes involved in different amino acid biosynthetic pathways upon amino acid starvation conditions or imbalances. For our studies we analyzed two A. nidulans lysine biosynthetic genes. The lysF gene, encoding homoaconitase, has been isolated and characterized by Weidner et al., 1997 and lysA was identified in the Oklahoma EST sequencing project and shows high amino acid sequence identities to saccharopin dehydrogenases. We isolated transcripts from liquid A. nidulans cultures in minimal medium with or without 3AT-induced amino acid starvation conditions. In Northern hybridization experiments we show that whereas lysF transcription is not affected in starved cultures, the level of lysA transcripts is increased about threefold. A genomic DNA fragment containing the 5' region of lysA revealed several putative cross-pathway regulatory elements (CPREs). This indicates that the lysA gene is regulated by the cross-pathway control. Investigations whether the partial regulation of lysine biosynthesis does have any influence on penicillin production are underway.
312 Regulation of hisHF transcription of A. nidulans by adenine and amino acid limitation. Oliver Valerius, Oliver Draht and Gerhard H. Braus. Institute of Microbiology & Genetics, Georg-August University, Grisebachstrasse 8, D-37077 Göttingen, Germany
The hisHF gene of Aspergillus nidulans encodes imidazole-glycerole-phosphate (IGP) synthase consisting of a glutamine amidotransferase and a cyclase domain. The enzyme catalyzes the fifth and sixth step of histidine biosynthesis which results in an intermediate of the amino acid and an additional intermediate of purine biosynthesis. An A. nidulans hisHF cDNA complemented a Saccharomyces cerevisiae his7D strain as well as Escherichia coli hisH and hisF mutant strains. The genomic DNA encoding the hisHF gene was cloned and its sequence revealed two introns within the 1659 bp long open reading frame. The transcription of the hisHF gene of A. nidulans is activated upon amino acid starvation suggesting that hisHF is a target gene of cross pathway control. Adenine but not histidine, both end products of the biosynthetic pathways connected by the IGP-synthase, represses hisHF transcription. In contrast to other organisms HISHF overproduction did not result in any developmental phenotype of the fungus in hyphal growth or the asexual life cycle. hisHF overexpression caused a significantly reduced osmotic tolerance and the inability to undergo the sexual life cycle leading to acleistothecial colonies.
313 Comparison of additional fungal Ste20 homologues. Michael H Perlin 1, Brisa Ramos2, Arturo Perez Eslava2, and Jose Maria Diaz Minguez2. 1University of Louisville, Louisville, KY, USA, 2Universidad de Salamanca, Salamanca, Spain
Signal transduction pathways are important for a variety of features of fungal development. For example, many human fungal pathogens exhibit some form of dimorphism and a common element in these and other fungi is a conserved mitogen-activated protein kinase (MAPK) pathway. This MAPK pathway plays an important role in a multitude of processes for the saprophytic fungi (e.g., Saccharomyces cerevisiaeand Schizosaccharomyces pombe), in traditional human pathogens, such as Candida albicans and Cryptococcus neoformans, and in plant pathogens, such as Ustilago maydis. Elimination of one of the components of the three-kinase MAPK cascade was recently shown to reduce pathogenicity of Fusarium oxysporum. Ste20 is a protein which, among its many roles in controlling fungal development, is known to regulate the three-kinase cascade in S. cerevisiae and S. pombe. We wanted to compare the roles of Ste20 homologues in a pathogenic fungus that is not dimorphic (F. oxysporum) with a dimorphic fungus, that is not pathogenic (Mucor circinelloides). Thus, degenerate primers were used to amplify the respective regions of the Ste20 genes from both fungi. The amplified fragments showed 70-80% similarity at the predicted amino acid level. Both predicted proteins also showed up to 88% similarity with the corresponding Ste20 proteins of S. cerevisiae and S. pombe. Neither gene fragment appeared to contain any introns. The full-length copy of the Ste20 gene from F. oxysporum was obtained after screening a genomic library. We are designing constructs for gene disruption experiments, so as to characterize the role of these genes
314 Allelic recombination is unaffected by gross sequence heterology between cog and his-3. P. Jane Yeadon, J. Paul Rasmussen, Frederick J. Bowring and David E. A. Catcheside. School of Biological Sciences, Flinders University, Adelaide, Australia.
Recombination between his-3 alleles is strongly dependent on the activity of the recombination hotspot cogL located about three kilobases 3' of this locus. Events initiated by cogL must pass the intervening sequences in some way in order to reach his-3 and generate recombinant progeny. We have constructed stocks each of which includes a different DNA sequence inserted between his-3 and cogL. The three insertions have different lengths and no homology to each other or to the native sequence they replace. Analysis of crosses heteroallelic his-3 K26/K480, homozygous cogL rec-2 and either homozygous or heterozygous for the inserted constructs has shown that lack of sequence homology over several kilobases has no significant effect on the frequency of his+ recombinant progeny. Heterozygosity for length in this region yields a recombination frequency like that of a cross homozygous for the shorter interval, clearly showing that recombination initiated by cogL tolerates high levels of sequence variation.
315 Comparative studies of sequences expressed in the liquid cultured mycelia and fruiting body of Pleurotus ostreatus. Seung-Ho Lee1, Beom-Gi Kim, Kang-Hyo Lee, Gyu-Hyun Kim, Chang- Soo Lee1, Young-Bok Yoo. Division of applied microbiology, NIAST, RDA, Suweon 441-707, Korea 1Department of Biochemistry, Kon Kuk University, Chungju 380-701, Korea
To characterize genes involved in fruiting body development, two complementary DNA (cDNA) libraries were constructed from liquid cultured mycelia and fruiting body of P. ostreatus. Using single-pass sequencing from liquid cultured mycelia and fruiting body cDNA clones, respectively 948 and 629 expressed sequence taqs (ESTs) were generated. A BLASTX search revealed that 400 ESTs (42%) of liquid cultured mycelia and 344 ESTs (55%) of fruiting body showed significant similarity to protein sequences described in the NR database (E values < 1X10-5). 237 ESTs of liquid cultured mycelia (25%) and 225 ESTs of fruiting body (36%) were showed highly significant in the similarity search (E values < 1X10-20). Among total 1577 ESTs, 1075 ESTs were unigenes. When mycelia and fruiting body ESTs were compared by Blastn algorithm, 158 ESTs (10%) showed the common expression in both stages. The functional catalogs of the EST were made by comparison with functionally identified Saccharomyces cerevisiae genes. We compared ESTs of liquid cultured mycelia with fruiting body and described changes of expressed genes during fruiting body development.
316 Differentially expressed genes in ectomycorrhiza and Tricholoma host specificity. Katrin Krause1, Angela Mankel2, Doreen Mueller1, Erika Kothe1. 1Friedrich-Schiller-University, Dept. Microbiology, Jena, Germany 2Philipps-University, Dept. Genetics, Marburg, Germany
From an RNA fingerprinting approach using fully developed ectomycorrhiza between Tricholoma vaccinum and Picea abies vs. pure cultures of the fungus and tree roots, more than 100 PCR fragments were identified that showed differential expression in mycorrhiza. These fragments were verified and from the 63 positive clones origin and expression pattern were checked. Of the 20 fungal genes with mycorrhiza-specific expression, sequence analyses were performed in order to identify the nature of the encoded protein in silico. Among them were reverse transcriptase indicating a retrotransposon in Tricholoma, a specifically induced aldehyde dehydrogenase pointing to plant pathogen response, and glucosidases. A gene encoding a hydrophobin specifically regulated during mycorrhization was identified and analyzed. The hydrophobin protein was detected using heterologous antiserum and protein accumulation could be shown in fungal cell walls in the hyphal mantle as well as in the Hartig net. A control using symbiotic tissue of an interaction between the fungus and a non-native host showed no hydrophobin accumulation in the Hartig net which is interpreted to show lack of regulatory functions in the non-native situation and therefore linking expression to host specificity.
317 UPR specific transcriptional regulation in Aspergillus niger. C. M. J. Sagt1, C. A. M. J. J. van den Hondel1,2 and P. J. Punt2. 1Institute of Molecular Plant Sciences, Clusius Laboratory Leiden University, 2333 AL Leiden, The Netherlands. 2TNO Nutrition and Food Research Institute, Department of Applied Microbiology and Gene Technology, 3700 AJ Zeist, The Netherlands
Fungi have an enormous protein secretion capacity. Homologous proteins are secreted in the g/l range, the secretion levels for heterologous proteins are 100-1000 fold less. From previous studies in S. cerevisiae it became evident that the stress caused by accumulation of proteins in the secretion-pathway, known as the Unfolded Protein Respone (UPR) serves as the major stress pathway, involved in adjusting the cell to produce heterologous proteins. This indicates that this pathway could be an important target to optimize the secretion capacity for heterolous proteins in Aspergillus niger. Tunicamycin and DTT as well as the production of some heterologous proteins are conditions were genes like BiP and CypB are upregulated. Results indicate that tunicamycin induces UPR to a level which is physiological relevant. DTT, which under the conditions used also impaires growth is a less specific (more promiscuous) stressor. To identify unknown genes which are differentially expressed during UPR we developed a DNA macroarray technology. An A. niger cDNA library is spotted on high density filters (4608 clones) and will be used for hybridisation with radiolabelled cDNA. The redundancy of this library, from which more than 95% of the clones contain an insert, was determined by probing the filters with labelled DNA fragments from highly expressed genes BiP and GPD. The cDNA probes used for hybridisation of the macro-arrays were obtained from Aspergillus niger wild-type and strains stressed with DTT or tunicamycin. In addition, RNA was also isolated from single chain antibody fragment producing Aspergillus niger which was shown to give an UPR response. After RNA isolation, reverse transcription in the presence of [33P]dCTP, hybridisation and normalization of the signals using the beta-lactamase and or gpdA gene, differentially expressed clones were identified. Several clones with a differential transcriptional behaviour will be analysed in further detail.
318 Circadian regulation of the light input pathway in Neurospora crassa. Martha Merrow1, Lisa Franchi,2, 3 Zdravko Dragovic1, Margit Görl4, Judy Johnson5, Michael Brunner4, Giuseppe Macino3, and Till Roenneberg1. 1 Institute for Medical Psychology, University of Munich 80336 Munich, Germany. 2 Department of Biology New York University, New York, NY, USA. 3 Dipartimento di Biotecnologie Cellulari Ed Ematologia, Università di Roma 00185 Rome, Italy. 4 Institute for Physiolgical Chemistry University of Munich 80336 Munich, Germany. 5 Institute for Immunology, University of Munich, 80336 Munich, Germany
FREQUENCY (FRQ) is a critical element of the circadian clock of Neurospora crassa. The white collar genes are important both for light reception and clock function. We show that FRQ interacts genetically and physically with WHITE COLLAR 1 (WC-1), and physically with WC-2. As a consequence of these interactions, the responsiveness of the light input pathway is clock controlled. This circadian modulation extends to light- inducible components and functions which are not rhythmic themselves in constant conditions. These findings begin to address how clock components interact with basic cellular functions, in this case with sensory transduction.
319 Gene inactivation in the ascomycete. Podospora anserina Andrea Hamann, Heinz. D. Osiewacz. Botanisches Institut, J. W. Goethe-Universitat, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany
Podospora anserina is a filamentous ascomycete closely related to Neurospora crassa. In Neurospora, a specific gene inactivation mechanism, termed RIP (repeat-induced point mutation), was identified in the late eighties. It results in the irreversible inactivation of duplicated sequences. Subsequently, it has repeatedly been suggested that RIP may have a broader distribution in fungi. Our recent identification of two highly degenerated transposons in P. anserina suggested that RIP may have led to the observed specific transposon mutations. We analyzed a transformant strain of P. anserina carrying a duplication of the Grisea gene encoding the copper-regulated transcription factor GRISEA. One of the offspring of a cross between this transformant strain and the wild type displayed the grisea mutant phenotype although the strain contains a complete ectopically integrated Grisea gene. Sequencing of the cDNA derived from this gene revealed three mutations in the coding sequence. Most likely, these mutations are the reason for the loss-of-function phenotype. The type of mutation and the fact that it was demonstrated in a strain that went through meiosis clearly demonstrates that a mechanism similar to RIP in N. crassa is operating also in P. anserina. Furthermore, it is well possible that this type of mechanism is more widespread and may have a more general impact on protecting strains against deleterious processes as they may be the result of transposon mobility or the recombination between duplicated sequences.
320 Nutritional profiling technology for high-throughput functional genomics analysis of filamentous fungi. Matthew M. Tanzer, Amy R. Skalchunes, Ryan W. Heiniger, Blaise A. Darveaux, Herbert N. Arst, Jr.*, and Jeffrey R. Shuster. Department of Microbial Phenomics, Paradigm Genetics, Inc. 108 Alexander Dr. RTP, NC. *Imperial College School of Medicine. Department of Infectious Diseases. Du Cane Rd. London
Nutritional profiling is a high-throughput technology for the analysis of fungal growth on numerous carbon, nitrogen, and metabolites, in an automated fashion. By examining the changes in nutrient utilization, inferences can be made about the pathways and processes that are affected by a particular gene or chemical treatment. Two example studies are presented here. First, the relationship of 21 Aspergillus nidulans areA mutant lines was studied for their utilization of 95 nitrogen sources. areA encodes a transcriptional activator mediating nitrogen metabolite repression. Hierarchical clustering of strains based on the overall utilization patterns of these nitrogen sources was performed for these strains. Data will be presented showing the relationships among all strains and allele pair clustering. These data are largely in agreement with the published phenotypic data that was based on a limited set of nitrogen sources. Second, the relationship of 95 carbon sources was examined with respect to carbon catabolite repression. Wild-type A. nidulans strains were grown in these plates with various concentrations of allyl-alcohol. Allyl alcohol is converted to acrolein by the action of alcohol dehydrogenase (adh). A. nidulans will grow in the presence of allyl alcohol on repressing carbon sources but will be sensitive on non-repressing carbon sources where adh is expressed. Hierarchical clustering of the carbon sources based on the level of utilization/repression in the presence of allyl alcohol will be presented. Nutritional profiling is a powerful system to rapidly analyze the nutritional requirements of filamentous fungi by including far more nutrient sources than have traditionally been used in standard laboratory experiments. This information will promote the understanding of gene function or chemical action.
321 Histone deacetylases in Aspergillus nidulans. Stefan Graessle, Peter Loidl, Markus Dangl, Patrick Trojer, Hubertus Haas, Karin Mair, and Gerald Brosch. Department of Microbiology, Medical School, University of Innsbruck, A 6020 Innsbruck, Austria.
In eukaryotes, DNA and core histones constitute the nucleosome, which is the essential structural unit for packing eukaryotic DNA into chromosomes. In addition to their structural function, nucleosomes are thought to play a role in the regulation of transcription. Core histones are susceptible to a wide range of posttranslational modifications, including phosphorylation, methylation, and acetylation. Hyperacetylation preferentially occurs in actively transcribed chromatin regions because the neutralization of the positive charge by acetylation of the epsilon-amino group of specific lysine residues within the N-terminal domain of core histones has been proposed to lead to loosening histone-DNA contacts, thereby facilitating the accessibility of various transcription factors to DNA. Core histones can be acetylated by histone acetyltransferases (HATs), such as the yeast transcription factor GCN5, the transcriptional adapter p300/CBP or its associated factor P/CAF. This acetylation process is reversed by a second class of enzymes, histone deacetylases (HDACs) which form highly conserved protein families in many eukaryotic species. Using different PCR approaches we have identified and characterized at least five new members of different HDAC families in Aspergillus nidulans. Expression studies showed specific transcripts and indicated different expression levels of the genes. Expression of some of the genes was induced when cells were treated with HDAC inhibitors like Trichostatin A. Recombinant HDACs expressed in prokaryotic expression systems were measured for enzyme activity and purified recombinant proteins were used to produce polyclonal antibodies for the identification of active enzyme fractions of the fungus.
322 SREA deficiency leads to derepression of siderophore transport, intracellular siderophore accumulation and oxidative stress in Aspergillus nidulans. Hubertus Haas, Harald Oberegger, Michelle Schoeser, Beate Abt, and Ivo Zadra. Department of Microbiology, University of Innsbruck (Med. School), Innsbruck, Austria
Under conditions of iron deficiency most fungi excrete siderophores - low molecular-mass ferric iron chelators - in order to mobilize extracellular iron. In A. nidulans, siderophore biosynthesis has been shown to be negatively regulated by the GATA-type transcription factor SREA. Furthermore, lack of SREA leads to derepression of siderophore uptake and metabolization (e.g. siderophore hydrolysis by ornithine esterase) as well as increased accumulation of ferricrocin, the siderophore responsible for intracellular iron storage. In sreA-deletion strains, derepression of extracellular siderophore production is only partial, indicating the presence of additional iron regulatory mechanisms. In contrast to siderophore excretion, ferricrocin accumulation is positively affected by the external iron availability suggesting a protective role of this siderophore in detoxification of intracellular iron excess. The harmfulness of deregulated iron uptake in this mutant is demonstrated by increased expression of genes encoding the antioxidative enzymes superoxide dismutases SODA and SODB and catalase CATB. Noteworthy, expression of catB was found to be repressed by iron starvation in wild type and sreA-mutant strains consistent with catB being subject to positive iron regulation. Differential display led to identification of the putative SREA target genes amc-1 and mirA. Since amc-1 encodes a puative mitochondrial carrier for the siderophore component ornithine, cross regulation of siderophore biosynthesis and ornithine metabolism is indicated. The deduced MIRA amino acid sequence displays significant similarity to recently characterized siderophore permeases of Saccharomyces cerevisiae. Northern analysis confirmed iron-dependent expression of amc-1 and mirA and the role of SREA as a repressor of gene expression. These data demonstrate that SREA regulates a variety of processes involved in siderophore metabolism and show that increased iron uptake leads to oxidative stress in Aspergillus. This work was supported by Austrian Science Foundation Grant FWF-P13202-MOB.
323 Using transcriptional profiling in Neurospora crassa to identify circadian clock-controlled genes and mediators of clock-regulated conidiation. Alejandro Correa1, Zachary Lewis1, Xie Xin2, Daniel Ebbole2 and Deborah Bell-Pedersen1. 1Department of Biology and 2Department of Plant Pathology, Texas A&M University, College Station, TX, 77843.
In Neurospora crassa, the circadian clock provides an endogenous signal to regulate the timing of asexual spore development (conidiation). In constant darkness, conidiation occurs once every 22h during the subjective morning. Several environmental signals can also initiate conidiation in N. crassa, including light, air, carbon and nitrogen starvation. Three key regulatory loci of the conidiation pathway are known and include acon-2, fl and acon-3. Different combinations of the regulators are thought to control the expression of downstream conidiation specific genes (con-10, con-6 and eas (ccg-2)). Our recent results indicate that the clock impinges early in the developmental pathway to regulate production of aerial hyphae and that clock regulation of eas (ccg-2) requires a functional acon-2 or fl gene, but not both. We are currently using N. crassa cDNA microarrays to help us understand the mechanisms by which the clock regulates this and others critical cellular events. Sets of probes obtained from different times of the day are allowing us to catalog rhythmically expressed genes. We predict that the initial clock signal for aerial hyphae production will be expressed early after development is induced and will be rhythmically expressed. In attempts to identify this signaling factor(s), we are probing the arrays with labeled cDNA produced from mRNA isolated from cultures harvested early after developmental induction. Putative candidate cDNA clones that are rhythmically expressed and activated early during development are being sequenced in their entirety. Constructing null alleles will test the role of these genes in the circadian clock and in development.
324 Analysis of the bzuA gene encoding the cytochrome P450 benzoate para-hydroxylase in Aspergillus nidulans. James A. Fraser, Meryl A. Davis and Michael J. Hynes. Department of Genetics, University of Melbourne, Australia
Amide utilisation by A. nidulans has been extensively studied in our laboratory through the cloning and analysis of regulation of the amidase encoding genes amdS, fmdS and gmdA. Two unlinked loci have previously been identified as being required for the utilisation of benzamide as the sole nitrogen source. The gmdA1 mutation defines the general amidase structural gene required for hydrolysis of long chain and some aromatic amides (including benzamide), whilst the bzuA1 mutation defines an unknown function.
We have cloned the bzuA gene via a chromosome walk on chromosome IV and found that it encodes a protein belonging to the cytochrome P450 superfamily, and is orthologous the benzoate para-hydroxylase (bphA) in Aspergillus niger. The bzuA1 phenotype was shown to be due to an intracellular accumulation of benzoate following benzamide hydrolysis by GmdA, resulting in growth inhibition. Benzoate serves as a sole carbon source and multiple binding sites for the A. nidulans CreA protein responsible for carbon catabolite repression have been identified in the bzuA promoter, resulting in expression responding to carbon limitation. As similarity between BzuA and BphA is lowest at the N-terminal (the region required in cytochromes P450 for anchoring in the endoplasmic reticulum), a BzuA:GFP fusion was generated. Fluorescence microscopy revealed fusion of GFP to the first third of the BzuA protein was sufficient to generate a reticulate fluorescence pattern, indicative of BzuA being localised to this organelle.
325 Structural and functional analysis of citrate synthase gene citA in Aspergillus nidulans. Soon Won Seo1, Ji Young Bang1, Keon Ho Han1, Cheong Ho Lee2, Jeong Goo Lee1 and Pil Jae Maeng1. 1Chungnam National University, Microbiology, Daejeon, Korea. 2Korea Ginseng & Tobacco, Daejeon Korea
Citrate synthase (EC 188.8.131.52) which catalyzes the condensation reaction between acetyl-CoA and oxaloacetate yielding citrate, the first step of the TCA cycle, functions as a rate-limiting enzyme of the cycle. As a subsequent study to the previous reports on the citrate synthase (CitA; Maeng et al., 1994, Kor. J. Microbiol. 31: 586-593) and its gene (citA; Park et al., Mol. Cells 7: 290-295) isolated from Aspergillus nidulans, we cloned and analyzed the structure of the citA cDNA. The citA cDNA was shown to encode a protein of 52.2 kDa consisting of 474 amino acid residues. A 1.45-kb fragment containing citA promoter (citA- p) was cloned and sequenced. The promoter contains three putative CAAT motifs, two CT boxes, and three candidate sequence for CreA (catabolite repression protein in Aspergillus) binding sites. The mode of citA expression was followed by confocal microscopic and spectrofluorometric analysis of the transformants containing a citA-p::sgfp gene fusion integrated at trpC locus. On asexual growth stage, the expression of citA was found to be highly encouraged by sodium acetate, but severely repressed by glucose. Furthermore the repression was synergistical in the presence of both glucose and glutamate. By deletion analysis of the citA-p, we found a genuine CreA-bindng motif responsible for the catabolite repression by glucose. On sexual stage, no significant expression was detectable in any of the above medium, which suggests that there exists some relationship between the regulation of citA expression and sexual or asexual differentiation of A. nidulans.
326 Functional domains and binding properties of Aspergillus nidulans AmyR. Shuji Tani, Yoko Katsuyama, Masashi Kato, Tetsuo Kobayashi, and Norihiro Tsukagoshi. Department of Biological Mechanisms and Functions, Graduate school of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
The amyR gene of A. nidulans encoded a cys-6 zinc transcriptional activator (AmyR) of 662 amino acid residues. The truncated AmyR1-411 of A. nidulans was produced as an MBP fusion protein in E. coli. The MalE::AmyR1-411 was purified by amylose affinity column, and used in gel mobility shift assay and DNase I footprinting. The MalE::AmyR1-411 bound to both the CGGN8CGG sequence of the a- glucosidase A promoter and the CGGAAATT sequence of the Taka-amylase A promoter, as described for A. oryzae AmyR. The MalE::AmyR1-411 had almost the same affinity to both sequences. Mutation in both or either of the CGG triplets in CGGN8CGG resulted in a drastic decrease in the binding affinity to MalE::AmyR1-411. The mutational analysis within and around the CGGAAATT sequence of the Taka-amylase A promoter revealed that MalE::AmyR1-411 required not only the CGG triplet but also the following AAATT sequence for its binding. AmyR possessed five homologous domains (Zn and MH1-MH4) to Mal63p, a transcriptional activator for the genes involved in maltose utilization in Saccharomyces cerevisiae. An AmyR derivative, AmyRZn2, lacking the MH3 and MH4 domains functioned as a constitutive activator in A. nidulans, suggesting that MH3 and/or MH4 may be the negative regulatory domain. AmyRZn1 lacking MH2-4 could not complement the growth defects of the amyR disruptant, and thus MH2 is indispensable for the transactivation. Interaction between the activation domain and the negative regulatory domain might be modulated in response to maltose.
327 Both a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif of Colletotrichum lagenarium Cmr1p are indispensable for the regulation of melanin biosynthesis. Gento Tsuji, Osamu Horino and Yasuyuki Kubo. Laboratory of Plant Pathology, Kyoto Prefectural University, Kyoto 606-8522, Japan.
Colletotrichum lagenarium is a plant pathogenic fungus that produces melanin during the appressorial differentiation stage of conidial germination and during the late stationary phase of mycelial growth. We previously reported the identification of genes for a unique transcription factor, CMR1 (Colletotrichum melanin regulation), which is involved in mycelial melanin biosynthesis. Cmr1p contains two distinct DNA- binding motifs, a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif. To determine whether the two putative DNA binding motifs are functional in vivo, CMR1 deletion constructs were designed and transformed into the cmr1 null mutant. The constructed plasmids were pCRC6-1, pCRZf-1, and pCRnon-1 that lack a Cys2His2 zinc finger motif, a Zn (II)2Cys6 binuclear cluster motif, and both DNA binding motif sequence, respectively. Transformants obtained with pCRC6-1 produced melanin. However the extent of melanization in the transformants was clearly lower than that in wild type. No wild type recombinants was obtained with pCRZf-1, and pCRnon-1. Thus, both these putative DNA binding motifs are necessary for wild type levels of melanization in culture.
328 Promoter analysis of the xynG2 gene in Aspergillus oryzae KBN616. Tetsuya Kimura, Takeshi Aburatani, Hayato Suzuki, Toshiyasu Matsuoka, Kazuo Sakka and Kunio Ohmiya. Faculty of Bioresources, Mie University, Tsu Mie Japan
Aspergillus oryzae is a very important fungus in Japan for food and enzyme production. Especially, in soy source production, degradation of soybean cell wall fraction is a limiting step of efficient usage of raw materials. Hemicellulose is one of the major components of plant cell walls. Therefore, degradation of hemicellulosic compounds is one of the key steps for soy source production. A. oryzae produces several kinds of hemicellulosic enzymes including xylanase when grown on hemicellulose. To understand the expression mechanism of the xylanase genes of A. oryzae , we cloned six genes encoding xylanase from A. oryzae KBN616 that is used for soy source production. Family 11 xylanase XynG2 is the major xylanase when A. oryzae grow on the Xylan medium. Expression analysis of the promoter of xynG2-the Escherishia coli uidA fusion gene (GUS) in A. oryzae was carried out. Promoter region of xynG2 contains two putative XlnR binding consensus sequences, 5'GGCTAAA3'. Deletion analysis suggested that they are essential for xynG2 expression in xylan medium. Point mutational analysis of both sites indicated that they are independently functional in vivo for the induction by xylan.
329 A short N-terminal region adjacent to the central core region of AoHapC, a subunit of the Aspergillus oryzae CCAAT binding complex, is necessary for enhancement of gene expression. Masashi Kato, Ken-ichi Kamei, Ryoko Oonishi, Shinjiro Tanoue, Tetsuo Kobayashi and Norihiro Tsukagoshi. Nagyoya University, Nagoya 464-8601, Japan.
A. nidulans Hap complex has been shown to regulate expression of several fungal genes and contain a subunit encoded by the hapC gene, a homologue of the HAP3 gene of S. cerevisiae. Recently, we have shown that an A. oryzae CCAAT binding protein, AoCP, is homologous to the A. nidulans Hap complex. Furthermore, AohapC, a hapC homologue, was isolated and shown to be functionally interchangeable to the A. nidulans hapC, as judged by the activity of the A. nidulans Hap dependent- enzyme endo-beta-1, 4- glucanase A (EG-A encoded by the eglA gene). The central "core" region of AoHapC (aminoacid residue 48 to 131) was highly conserved among the HAP3 homologues and could be involved in DNA binding and subunit association. In order to characterize functional domains of AoHapC, mutant AohapC genes lacking an N-terminal region, a C-terminal region and both regions were constructed and introduced to an A. nidulans hapC deletion strain to obtain the transformants, dN, dC and dNdC, respectively. Electrophoretic mobility shift assays showed that the mutants dN and dC possessed the CCAAT binding activity, indicating that the core region of AoHapC is sufficient to the CCAAT binding activity. However, the EG-A activity of dN was as low as that of the hapC disruptant, while the EG-A activity of dC was almost the same as that of the wild type strain. The same results were obtained with the taa gene. These results indicate that the N-terminal region as well as the core region is required to enhance gene expression. Further analysis with a series of N-terminal deletions of AoHapC revealed that a short N-terminal region adjacent to the core region is involved in the transcriptional enhancement of the eglA gene.
330 Colletotrichum lagenarium CST1 (Colletotrichum sterile12-like), a potential downstream transcriptional factor of a MAP kinase has homeodomain and Cys2His2 zinc finger DNA binding motifs. Yasuyuki Kubo, Gento Tsuji and Osamu Horino. Laboratory of Plant Pathology, Kyoto Prefectural University,Kyoto 606-8522, Japan.
The infection of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. C.lagenarium CMK1encoding a mitogen-activated protein (MAP) kinase of yeast FUS3/KSS1 homologue plays a pivotal role in these infection steps. Saccharomyces cerevisiae Ste12p is a transcriptional factor that plays a key role in coupling signal transduction through Fus3/Kss1 MAP kinase modules to specific gene expression required for mating and pseudohyphal filamentous growth. Also in pathogenic yeasts of Candida albicans and Filobasidiela neoformans, Ste12p homologues play similar roles during dimorphic transitions. In filamentous fungi, Aspergillus Ste12p homologue SteAp is required for sexual reproduction. We cloned STE12 like CST1 (Colletotrichum sterile12-like) from plant pathogenic filamentous fungi C.lagenarium encoding a protein with highly conserved homeodomain found in other Ste12 proteins. Cst1p also had C-terminal Cys2His2 zinc finger DNA binding domains retained in Ste12Ap of A. nidulans but not present in the other Ste12 proteins.
331 Cloning, nucleotide sequence and characteristic of two mutants of the suDpro gene from Aspergillus nidulans encoding a putative glycoprotease. A. Grzelak, E. Szewczyk, P. Borsuk, J. Empel, A. Pollak, J. Rozyczka and P. Weglenski Department of Genetics, Warsaw University, Warsaw, Poland
In Aspergillus nidulans arginine can serve as a source of proline in the wild-type strain and in proline mutants blocked in one of the first two steps of the main pathway of proline synthesis. The arginine regulation system is connected with a specific activator gene (ArcA) (poster Empel J. et al.) and several repressor genes (suApro, suDpro, suEpro,suHpro, suJpro and suLpro). Supressors of proline mutations were obtained as mutants with derepression of both arginine catabolic enzymes agaA coding for arginase and otaA coding for OTA-se (see poster Dzikowska A. et al.). The suDpro gene is particularly interesting from the group of supressor gene mutants. Two mutants in this gene: suD25pro and suD19pro have similar pleiotropic phenotypes. We have cloned the suDpro gene by complementation of the mutation in the suD25pro strain with the wild type gene. The cDNA clone was selected from an A. nidulans cDNA library. Both the genomic and cDNA clones were sequenced. Comparison of these sequences revealed the presence of one 66 bp long intron. The open reading frame of the suDpro gene transcript potentially encodes a protein of 363 amino acids. The predicted amino acid sequence shows significant homology with glycoproteases of procaryotic or eucaryotic origin and contains a putative zinc-binding site. The PCR method was used to determine sequences of the suDpro gene in the suD19pro and suD25pro mutants. In the suD25pro strain there is a point mutation at the beginning of the sequence, producing a stop codon at position 101. In the suD19pro strain there is a deletion of 4 amino acids (including one conserved). The presumable functions of this suppressor gene are discussed. This work was supported by grant from the Polish State Committee for Scientific Research 6 P04A 00519 and partly by Department of Biology grant number BW/1485/47.
332 Molecular characterization of in planta induced gene, PIG1 , in Magnaporthe grisea. Ahn, Namsook. Soeul National University. Agric. Biotechnology, Kyonggi South Korea
Magnaporthe grisea is the causal fungus of rice blast, the most devastating rice disease in the world. Although much has been learned about early infection processes by this fungus, little information is available on the late stages of infection; in planta growth and symptom development. A cDNA showing a high sequence homology to Bli-3 of Neurospora crassa, a blue light-inducible gene with unknown function, was isolated from M. grisea infected rice cDNA library and designated as PIG1 (in Planta Induced Gene). PIG1 contains an open reading frame of 627 nucleotides which encode 208 amino acid residues. The estimated molecular weight was 22.8 KDa with pI of 6.4. Southern blot analysis of genomic DNA revealed that the PIG1 exists as a single copy in the haploid genome of M. grisea. Expression of PIG1 was detected at a low level during the growth in a complete medium, but was highly up-regulated during symptom development in rice. To evaluate the role of PIG1 in fungal pathogenicity and morphogenesis at the molecular level, a null mutant, pig1, was obtained by gene knock out strategy. The phenotypic characterization of a null mutant will be presented.
333 Transcriptional control of the gibberellin biosynthesis in Gibberella fujikuroi. Martina Mihlan and Bettina Tudzynski. Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlossgarten 3, 48149 Münster, Germany
In the last years much knowledge has been gained about gibberellin (GA) biosynthesis, mainly due to the cloning of genes encoding biosynthetic enzymes in Gibberella fujikuroi. Recent studies have shown that at least 6 genes of the gibberellin (GA)-biosynthetic pathway are clustered in chromosome 4 in the G. fujikuroi genome; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (cps/ks), a GA-specific geranylgeranyl diphosphate synthase (ggs2) and four cytochrome P450 monooxygenases. (Tudzynski et al. 1998, Tudzynski and H÷÷lter,1998). The availability of the genes allows the study of mechanisms of gene regulation on a molecular level. Recently, we could show that gene replacement of the major nitrogen regulatory gene, areA, led to a significant reduction of gibberellin formation by repressing the expression of all but one GA-pathway genes (Tudzynski et al. 1999). Complementation of the areA-deficient mutant with the areA wild-type copy completely restored the ability to produce gibberellins as well as the high expression level of five GA genes which suggests that the positive acting regulatory protein AREA directly controls the transcription of the pathway genes. In contrast to these genes, the expression of P450-3 is not affected by high amounts of ammonium. In order to find other putative transcription factors beside AREA which can also bind to the promoters of the GA biosynthetic genes, intact, mutated and size-reduced promoter-fragments of the genes P450-1/P450-4 (double promoter, AREA-regulated) and P450-3 (not influenced by AREA) were fused with the gus reporter gene. Putative binding sites of transcription regulators are discussed. References B. Tudzynski, H. Kawaide, and Y. Kamiya (1998). Curr. Genetics, 34: 234-240. B. Tudzynski and K. Holter (1998). Fungal Genetics and Biology, 25: 157-170. B. Tudzynski, V. Homann, B. Feng, and G.A. Marzluf (1999). MGG, 261: 106-114.
334 Ste20/Pak1 kinase homologs regulate differentiation and virulence of Cryptococcus neoformans. Wang, P.1, Breeding, C.S.1, Lengeler, K.B.1, Cardenas, M.E.1, Cox, G.M.2,3, Perfect, J.R.2,3, and Heitman, J.1,2,3,4,5 Departments of Genetics1, Medicine2, Micro.3, and Pharma. and Cancer Biology4, the H. Hughes Med. Inst.5, Duke University Medical Center, Durham, NC 27710
Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving mating between haploid MATalpha and MATa cells. Interestingly, virulence of this organism has been linked to mating type, and MATalpha cells are more virulent than congenic MATa cells. We are studying signal transduction pathways that regulate mating and virulence to understand how mating type might be linked to virulence. Here we describe the identification of three genes encoding homologs of the Ste20/Pak1 kinases from S. cerevisiae and humans: MATalpha, STE20a, and PAK1. The Ste20 homologs are mating type specific; the MATalpha locus contains the MATalpha gene and the MATa locus contains the related but divergent STE20a gene. The MATalpha, STE20a and PAK1 genes were disrupted by transformation and homologous recombination in serotype A and congenic serotype D strains of C. neoformans. These studies reveal the Ste20 and Pak1 kinases play roles in mating, haploid fruiting, cytokinesis, and virulence. The pak1 mutation was synthetically lethal with either MATalpha or ste20a mutations, indicating that these related PAK kinases may share partially overlapping functional roles. In summary, our studies reveal diverse functions for the Ste20 and Pak1 kinases in differentiation and virulence, identify MATalpha as a component encoded by the MATalpha locus that is associated with virulence, and provide evidence that virulence of C. neoformans is regulated by a specialized MAP kinase signaling cascade.
335 Molecular characterization of in planta induced gene, PIG1, in Magnaporthe grisea. Namsook Ahn, Soonok Kim, and Yong-Hwan Lee. School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea.
Magnaporthe grisea is the causal fungus of rice blast, the most devastating rice disease in the world. Although much has been learned about early infection processes by this fungus, little information is available on the late stages of infection; in planta growth and symptom development. A cDNA showing a high sequence homology to Bli-3 of Neurospora crassa, a blue light-inducible gene with unknown function, was isolated from M. grisea infected rice cDNA library and designated as PIG1 (in Planta Induced Gene). PIG1 contains an open reading frame of 627 nucleotides which encode 208 amino acid residues. The estimated molecular weight was 22.8 KDa with pI of 6.4. Southern blot analysis of genomic DNA revealed that the PIG1 exists as a single copy in the haploid genome of M. grisea. Expression of PIG1 was detected at a low level during the growth in a complete medium, but was highly up-regulated during symptom development in rice. To evaluate the role of PIG1 in fungal pathogenicity and morphogenesis at the molecular level, a null mutant, pig1, was obtained by gene knock out strategy. The phenotypic characterization of a null mutant will be presented.
336 ArcA, the regulatory gene of the arginine catabolic pathway in Aspergillus nidulans. J.Empel, I.Sitkiewicz, A.Andrukiewicz, K.Lasocki, P.Borsuk and P.Weglenski. Department of Genetics, Warsaw University, Warsaw, Poland
The arcA gene codes for a transcriptional activator necessary for the high level expression of two genes of the arginine catabolic pathway in Aspergillus nidulans: the agaA (coding for arginase) and the otaA (coding for ornithine transaminase, OTAse) (see posters Grzelak et al. and Dzikowska et al.). Here we present the complete genomic and cDNA nucleotide sequences and the pattern of expression of the arcA gene. This gene contains one intron and encodes a polypeptide of 600 amino acids. The deduced protein belongs to Zn2Cys6 fungal regulatory proteins. ARCA is the first known protein of this family, which at the fifth position of the second, six amino acid, loop of the Zn cluster domain has glycine instead of the conserved proline. We have established that the transcription of the arcA gene is not self-regulated and does not depend on arginine. Two mutations: arcAd47, gain-of-function and arcAr3, loss-of-function, mapping in the arcA gene have been sequenced and effects of these mutations on the expression of the agaA gene at the transcriptional level were shown.
This work was supported by grant from the Polish State Committee for Scientific Research 6 P04A 0013 18.
1. Bartnik, E. and Weglenski, P. (1974) Nature 250, 590-592. 2. Empel, J. (1998) Ph.D. Thesis, Warsaw University
337 The Aspergillus nidulans multimeric CCAAT binding complex AnCF is negatively autoregulated via its hapB subunit gene. Stefan Steidl*, Michael J. Hynes** and Axel A. Brakhage*. *Institut fuer Mikrobiologie und Genetik, TU Darmstadt, Germany **Department of Genetics, University of Melbourne, Parkville, Australia
Cis-acting CCAAT elements are frequently found in eukaryotic promoter regions. Many of them are bound by conserved multimeric complexes. In the fungus Aspergillus nidulans the respective complex was designated AnCF (A. nidulans CCAAT binding factor). AnCF is composed of at least three subunits designated HapB, HapC and HapE, with each subunit being indispensable for AnCF function***. Here, we show that the promoter regions of the hapB genes in both A. nidulans and A. oryzae contain two inversely oriented, conserved CCAAT boxes (box alpha and box beta). AnCF binding in vitro to these boxes occurs in a non-mutually exclusive manner. Western and Northern blot analyses showed that steady state levels of HapB protein as well as hapB mRNA were elevated in hapC and hapE deletion mutants. Mutagenesis of box beta led to a 5-fold reduced expression of a hapB-lacZ gene fusion compared with the expression derived from a wild-type hapB-lacZ fusion. In contrast, in a hapB deletion background the hapB-lacZ expression level was elevated. Overexpression of hapB using an inducible alcA-hapB construct strongly repressed the expression of an hapB-lacZ gene fusion. These results indicate that (i) box beta is an important positive cis-acting element in hapB regulation, (ii) AnCF does not represent the corresponding positive trans-acting factor and (iii) that AnCF is involved in repression of hapB.
***Steidl, S., Papagiannopoulos, P., Litzka, O., Andrianopoulos, A., Davis, M. A., Brakhage, A. A., and Hynes, M. J. 1999 Mol. Cell. Biol. 19: 99-106
338 Functional in vivo analysis of otaA - arginine catabolism gene in Aspergillus nidulans. A. Dzikowska, M. Koper and P.Weglenski. Department of Genetics, Warsaw University, Warsaw, Poland
In A. nidulans arginine can serve as a source of proline and it can also be utilized both as a carbon and nitrogen source. Utilization of arginine as a source of proline depends on the presence and inducibility of arginine catabolic enzymes, arginase and ornithine transaminase (OTAse) which are encoded by agaA and otaA genes, respectively (1,4). The induction of both genes by arginine is subject to specific control, both positive and negative. The arginine specific regulation was shown to be exerted at the level of transcription. It was shown that there are several repressor genes (suA, suD, suE, suH, suJ and suL) and one activator gene (arcA) (5) (see also posters Empel J. et al. and Grzelak A. et al).The expression of agaA and otaA genes is also controled by systems of general carbon and nitrogen metabolite repression mediated by the creA (3) and areA (2) gene products, respectively. The promoter region of the otaA gene contains AREA and CREA binding sites which are protected by AREA and CREA in vitro (4). A system for the functional analysis of the otaA gene in vivo has been worked out. The system is based on the integration of a single copy of the otaA gene into the uaZ locus (6). Using this system and the mutational analysis of the otaA gene promoter we have identified a region which is involved in arginine induction. It has also been shown that the GATT sites bound by the AREA in vitro are probably not an AREA physiological binding sites. It is possible that they are bound by some negatively acting regulatory protein. Point mutations in the CREA binding sites result in otaA derepression. Similarly as a removal of the CREA binding sites or multicopy integration of the otaA gene, they result in the supression of the pro- mutation. References: 1. Borsuk P, Dzikowska A, Empel J, Grzelak A, Grzeskowiak R and Weglenski P(1999) Acta Biochimica Polonica 46: 391-403 2. Caddick MX, Arst, HN, Taylor LH, Johnson LI and Brownlee A (1986) EMBO J. 5:1087-90. 3. Dowzer CEA, Kelly JM (1991) Mol Cell Biol 11:5701-5709 4. Dzikowska A, Swianiewicz M, Talarczyk A, Wisniewska M, Goras M, Scazzocchio C, Weglenski P (1998) Curr Genet 35:118-126. 5. Empel J (1998) Cloning and characterisation of the arcA gene of A. nidulans. PhD Thesis. Dept. Genetics, Warsaw University 6. Oestreicher N, Sealy-Lewis H and Scazzocchio C. (1993) Gene 132:185-192
339 Characterization of histone deacetylases in Cochliobolus carbonum. Eva M. Brandtner1, Peter Loidl1, Markus Dangl1, Patrick Trojer1, Jonathan Walton2, and Gerald Brosch1. 1Department of Microbiology, Medical School, University of Innsbruck, A-6020 Innsbruck, Austria. 2Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.
One basic mechanism of gene regulation in eukaryotic cells is the modification of chromatin structure. Two groups of enzymes, acetyltransferases (HATs) and deacetylases (HDACs), regulate transcription via alternating the acetylation state of histones or promoter-bound transcription factors. Several chemical agents are known to specifically inhibit HDACs. Among them is the cyclic tetrapeptide HC-toxin, which is a secondary metabolite of the maize pathogen fungus Cochliobolus carbonum. Since inhibition of HDACs has been shown to cause remarkable hyperacetylation of histones and thereby affects cell cycle progression and differentiation patterns of different sources, we posed the question of how the fungus protects its own chromatin structure against rearrangements caused by hyperacetylated histones. To investigate how the acetylation state of the histones is kept in balance in the presence of HC-toxin, we are analyzing the fungal HDACs. Here we report the cloning of three HDAC genes of Cochliobolus carbonum, all of which share striking homology to either RPD3 or HDA1 HDACs of Saccharomyces cerevisiae . We investigated the influence of inhibitors on partially purified HDAC-activity and identified one inhibitor sensitive and one resistent activity. Western blot analysis with antibodies against the identified enzymes revealed each of them as part of the sensitive activity. Furthermore, we used immunoprecipitation experiments to proof the in vivo HDAC-activity of the identified proteins.
340 Characterization of PA-specific incompatibility factors in the het-6 region of Neurospora crassa. Cristina O. Micali and M. L. Smith. Carleton University, Ottawa, ON. Canada
In N. crassa, only individuals carrying the same alleles at all het loci are able to fuse and grow vegetatively as heterokaryons. The het-6 region in N. crassa is located on the left arm of linkage group II and carries two closely linked het genes. un-24 encodes the large subunit of ribonucleotide reductase, and het-6, encodes a ~680 amino acid protein with similarity to several putative genes in N. crassa, including tol, and to het-e from Podospora anserina. Two alleles, designated Oakridge (OR) and Panama (PA), occur at un-24 and het-6. Incompatibility activity of the OR forms was previously described. Here, we report the partial characterization of the PA-specific incompatibility activity associated with un-24 and het-6. Unlike the OR counterparts which cause severe incompatibility reactions, transformation of het-6PA into OR spheroplasts results in a moderate reduction in the number of transformants, while un-24PA causes growth inhibition and a "spidery" colony morphology in transformed OR colonies, similar to the het-c and mat incompatibility phenotypes. Escape from un-24PA/OR self-incompatibility occurs within 2 to 8 days to give near wild-type growing colonies. Preliminary data indicate that while crosses between escapes and het-6OR strains are fertile, escape x het-6PA crosses are barren. This suggests that OR/PA and PA/OR combinations of un-24/het-6, which are not observed in nature, may be selected against during the sexual cycle.
341 Characterization of a transcription factor that regulates HC-toxin production in Cochliobolus carbonum. Kerry F. Pedley and Jonathan D. Walton. DOE-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824
Cochliobolus carbonum is the causal agent of northern corn leaf spot. Strains of the fungus that produce a host-selective toxin, HC-toxin, are highly virulent towards certain genotypes of Zea mays. HC-toxin is a non-ribosomally synthesized cyclic tetrapeptide with the structure (D-Pro-L-Ala-D-Ala-L-Aeo), where Aeo stands for 2-amino-9,10-epoxi-8-oxodecanoic acid. Production of HC-toxin is under the control of a single genetic locus, TOX2. This locus is composed of at least six linked genes; HTS1, TOXA, TOXC, TOXE, TOXF, and TOXG. The products of these genes have demonstrated or probable roles in the biosynthesis of HC-toxin. TOXE has been shown to be required for the expression of the known genes within the TOX2 locus. DNA sequence analysis of TOXE shows that its predicted product (TOXEp) contains a basic region similar to those of basic leucine zipper (bZIP) proteins, but lacks a leucine zipper dimerization motif. TOXEp also contains four ankyrin repeats. We have been testing the hypothesis that TOXEp is directly involved in the transcriptional regulation of the HC-toxin biosynthetic genes. Although TOXEp does not contain a well-conserved DNA binding domain, we have demonstrated that TOXEp directly interacts with conserved elements in the promoters of the genes within the TOX2 locus. Additionally, we have shown that TOXEp is a strong activator of transcription and is capable of acting as a transcription factor in yeast. We believe TOXEp is a unique pathway-specific transcription factor that is responsible for regulating the expression of the TOX2 locus. Thus, it plays a key role in HC-toxin production and pathogenicity.
342 How did such a large number of B mating types of Schizophyllum commune evolve in nature? Thomas J. Fowler, Michael F. Mitton, Cynthia L. St. Hilaire, and Carlene A. Raper. Department of Microbiology and Molecular Genetics, University of Vermont, Burlington VT 05405 USA.
The Homobasidiomycetous fungus Schizophyllum communehas thousands of mating types defined in part by numerous lipopeptide pheromones and their G protein-linked receptors encoded within different versions of two redundantly functioning B mating-type loci, B-alpha and B-beta. Compatible combinations of pheromones and receptors produced by individuals of different B mating types regulate a pathway of sexual development required for establishment and maintenance of the dikaryon. Sequence comparisons of the B-alpha1 and B-beta1 loci indicate that these two loci may have been derived from a common ancestor by duplication and inversion. Recent molecular genetic analyses of another specificity of the B-beta locus, B-beta2, revealed a single pheromone receptor gene and a surprisingly large number of pheromone genes, four of which are similar in sequence and appear to have been derived by duplication. Two of these activate the identical set of receptors encoded by other B-beta specificities; the other two may be pseudogenes. This evidence of gene duplication, together with our recent demonstration that small changes in amino acid sequence of both pheromones and receptors can significantly alter specificity of pheromone/ receptor interactions, has provided clues about how so many different versions of the B mating-type loci could have evolved in nature. A multi-step model involving tandem gene duplication and mutational divergence will be discussed.
343 Transformation of the oomycete, Phytophthora infestans, using microprojectile bombardment. Cristina Cvitanich and Howard S. Judelson. Department of Plant Pathology, University of California, Riverside USA
Optimal transformation procedures for P. infestans, the potato late blight pathogen, will assist the analysis of genes relevant to its growth and pathogenesis and thus facilitate developing disease control strategies. A reliable method for transforming P. infestans protoplasts was developed in our laboratory and is being widely employed. However, that protocol involves generating protoplasts using Novozyme 234, an enzyme which is no longer produced and was of variable quality. As an alternative to the protoplast protocol, microprojectile bombardment is being optimized using a helium-driven system (PDS- 1000/He, Biorad). For these experiments, the pTH209-35G vector, which expresses the neomycin phosphotransferase (npt) and beta-glucuronidase(gus) genes, is being used to transform tissue which is then subjected to selection using G418- supplemented media. Transformants are also assayed histochemically for gus expression. To optimize transformation, variables tested included helium pressures between 450 and 1800 psi, different particle flight distances, treating mycelia versus germinated asexual spores, different concentrations and ages of the target tissue, and various selection schemes Using the optimal conditions up to 16 G418-resistant colonies were recovered per bombardment event, employing 1 microgram DNA per shot. The majority of transformants appeared three to four days after transformation, and data so far indicates that one to two copies of the plasmid normally integrate into the genome. The levels of gus expression varied between independent transformants, probably due to position effects. Overall, microprojectile bombardment is a suitable method for transforming P. infestans which avoids protoplasting and shows low experiment-to-experiment variation.
344 Molecular mechanism responsible for the dominant repression of aflatoxin biosynthesis in Aspergillus flavus. Gyung-Hye Huh, Joe Flaherty, and Charles Woloshuk. Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN
Biosynthesis of aflatoxin, a toxic and carcinogenic secondary metabolites, is by the polyketide pathway of secondary mechanism. Expression of genes in the aflatoxin biosynthesis cluster is regulated by aflR. A. flavus strain 649, mutated at the afl-1 locus, has a genomic DNA deletion greater than 150 kb covering the aflatoxin biosynthesis cluster. Diploids formed by parasexual cross between 649 and aflatoxigenic strains do not produce aflatoxin indicating the dominant phenotype associated with the afl-1 locus in strain 649. We have hypothesized that the dominant suppression phenotype arises by one of two possible mechanisms: i) inactivation of alleles due to homologous pairing of chromosomes (transvection), ii) the suppression of aflatoxin biosynthesis by the aberrant expression of a repressor gene in strain 649. Using a unique band obtained by AFLP analysis, we have isolated a cosmid that appears to contain DNA from the break-junction region in strain 649. Characterization of this cosmid will determine if there is an insertion of interfering DNA at the break-junction or if the two ends of the deletion are simply ligated. To address the possibility of a suppressor, a vector having aflR and ver1::GUS reporter gene was inserted into the 649 genome. We are currently screening for transformants with a single copy insertion. We will present measurements of GUS expression, aflatoxin production, and expression of aflatoxin biosynthesis genes in the transformants and diploids between transformants and an aflatoxigenic strain. Results from this study will provide us with a clearer understanding of the afl-1 locus in strain 649.
345 Characterization of transposable elements in Cryptococcus neoformans. M. Cristina Cruz1 and Joseph. Heitman1- 2, Department of Genetics,Duke University Medical Center1 and The Howard Hughes Medical Institute2, Durham NC 27710 USA
Cryptococcus neoformans is a fungal pathogen that causes life-threatening central nervous system infections in immunocompromised patients. Transposons are genetic elements that are distributed throughout the genomes of organisms from bacteria to humans and that are capable of moving within their host. We have identified novel insertion sequences in C. neoformansthat represent the first transposable elements to be described in this human pathogen. Of four spontaneous FK506-resistant mutants that were previously isolated, two were found to result from the insertion of novel DNA sequences into the FRR1locus encoding the FK506 target protein FKBP12. Isolation and sequence analysis revealed that these two novel elements are flanked by inverted repeats and created either a five-bp or a three-bp target site duplication upon insertion. These features suggest that these insertion sequences represent novel transposable elements. One element is 2749 bp and the second element is 785 bp. Southern blot analysis revealed multiple genomic DNA fragments that hybridize with each element in the two congenic serotype D strains JEC20 and JEC21 as well as in their parental strains. In summary, we have identified two novel tranposable elements that are found in moderate copy number in the genome of C. neoformans serotype D strains and which are infrequent or absent in serotype A, B, or C strains. This discovery marks the first transposable elements to be described in a pathogenic basidiomycete and will allow studies on their possible roles in virulence, evolution, phenotypic switching, and their use in novel approaches to manipulate and mutagenize the genome.
346 Role of F-box proteins (Fbls) in filamentous fungi. Janna Beckerman, Carlos Cortes, Cristina Filippi, Jim Sweigard, Barbara Valent and Daniel Ebbole. Plant Pathology & Microbiology, Texas A&M University, College Station, TX.
F-box proteins are components of modular E3 ubiquitin protein ligases called SCF's, which direct protein ubiquitination. The SCF system controls a large number of regulatory pathways in vertebrates. In Saccharomyces cerevisiae, the F-box protein Grr1 appears to play a key role in cell cycle progression and response to glucose availability. The expression of yeast glucose transporters is under control of this F-box protein. To further understand the role of SCF systems in eukaryotic cells, we are characterizing two genes encoding F-box proteins from Magnaporthe grisea(pth1) and Neurospora crassa (nfb1). The deduced sequence from these proteins display extensive internal homology within the leucine-rich repeat and are most closely related to Grr1p. M. grisea, a grass pathogen, undergoes appressorium development to establish a successful host-parasite interaction. Mutants of pth1 produce defective appressoria that are sensitive to hyperosmotic stress. Thus, pth1 mutants are defective in the penetration process. Typical lesions were only observed after wound inoculations of pth1 mutants on rice and barley plants, suggesting that disruption of this gene does not affect pathogenicity but does interfere with the penetration of plant tissue. No other difference has been detected among wild type and pth1 mutants. Phenotypic analysis of nfb1 in N. crassa will provide additional clues about the role of F-box proteins in filamentous fungi.
347 Mitochondrial nuclear communication in Neurospora crassa. Andrea T. Descheneau, Ian A. Cleary, Frank E. Nargang. University of Alberta, Biological Sciences, Edmonton, Alberta, Canada
Mitochondria are the site of oxidative phosphorylation, a key energy generating process in most eukaryotic cells. Although some mitochondrial proteins are encoded by the mitochondrial genome, the majority are encoded in the nucleus. Some form of communication is believed to exist between these two organelles to coordinate their gene expression, thereby insuring proper mitochondrial biogenesis and function. However, the nature of this communication remains unknown. In Neurospora crassa, deficiencies in oxidative phosphorylation result in expression of several nuclear genes. One of these genes, aod- 1, is only expressed during times of respiratory stress and encodes an alternative oxidase that is able to pass electrons directly from ubiquinone to oxygen. We are interested in understanding how mitochondria signal the nucleus to induce alternative oxidase expression. A reporter has been constructed using the alternative oxidase promoter sequence and the structural gene for the melanogenesis enzyme tyrosinase. Mutagenesis of a reporter strain has allowed the isolation of mutant strains that are altered in their ability to regulate both reporter and alternative oxidase gene expression. Further characterization of these mutants may be useful in determining the pathway responsible for alternative oxidase regulation. Acknowledgements: This work was supported by studentships from the Alberta Heritage Foundation for Medical Research and the Natural Sciences and Engineering Research Council of Canada to ATD, and a grant from the Natural Sciences and Engineering Research Council of Canada to FEN.
348 Understanding genetic mechanisms involved in fumonisin biosynthesis. J.E. Flaherty, W-B Shim and C.P. Woloshuk. Purdue University, West Lafayette, IN
Fumonisins are a group of naturally occurring metabolites produced by Gibberella fujikoroi (anamorph Fusarium verticillioides), which frequently contaminate corn-based foodstuffs and animal feed. The B series of fumonisins (FB1) is the most prevalent and has been implicated as the cause of several animal toxicoses as well as cancer promotion in rats and humans. The objective of our research is to clone and characterize genes involved in fumonisin gene regulation and to set the context in which regulation of the fumonisin biosynthetic pathway exists in relation to other developmental and/or physiological cascades. By the random enzyme-mediated integration (REMI) technique, we isolated fic1 from G. fujikuroi, a type-C cyclin similar to UME3 in Saccharomyces cerevisea. Disruption mutants of fic1 fail to produce fumonisin and exhibit reduced conidiation when grown on corn. We have isolated ESTs (>1500) differentially transcribed in a wild-type fumonisin producing strain compared to a fic1disruptant cultured on corn. Sequence analysis of 800 ESTs revealed transcripts with high sequence homology (> 1E-5) to known regulatory genes, including transcription factors, kinases and cyclin/CDKs. Toward understanding the functional role of these genes, we are constructing knockout vectors for specific disruption via homologous recombination. We will describe the phenotypes associated with the disruption events in G. fujikuroi and determine their effects on fumonisin biosynthesis and fungal development. We will also present analysis of new REMI transformants generated with a reporter construct containing the promoter region of fum5, a known fumonisin pathway gene, fused to uidA (GUS). Appearance of GUS activity parallels that of fumonisin accumulation in culture; therefore, mutations affecting fum5 transcription should also alter fumonisin biosynthesis.
349 Expression analysis of Phanerochaete chrysosporium cellulase and lignin peroxidase genes using RT-PCR and DNA microarrays techniques. Debbie Yaver1, Daniel Cullen 2, 3, Jennifer Bassett3, Amber Vanden Wymelenberg3, Xiaochun Yu 4, Rajai Atalla 2,4. 1 Novozymes Biotech, Davis, CA, 2 USDA Forest Products Lab, Madison, WI, 3 Dept. of Bacteriology, University of Wisconsin-Madison, Madison, WI, 4 Dept. of Chemical Engineering, University of Wisconisin-Madison, Madison, WI.
Transcript levels of cellobiohydrolase, cellobiose dehydrogenase, and beta-glucosidase genes were quantified for Phanerochaete chrysosporium grown on basal salts containing either Avicel or CF-1 cellulose as the sole carbon source. Differential regulation was observed in response to secondary and/or tertiary structures of cellulose substrates and over time. Competitive RT-PCR techniques and DNA microarrays were in general agreement regarding the relative expression of six structurally related cbh1 genes. In addition, expression analysis of the lignin peroxidase genes in cultures grown in complete, carbon limited and nitrogen limited medium was performed using DNA microarrays. The results with DNA microarrays and prior studies with competitive RT-PCR were comparable. Microarray approaches hold considerable promise for rapid and convenient transcript profiling, especially within families of closely related genes.
350 Isolation and characterisation of two ammonium permeases in Aspergillus nidulans. Brendon J. Monahan, Michael J. Hynes and Meryl A. Davis. Department of Genetics, University of Melbourne, Victoria 3010, Australia.
In filamentous fungi and yeast the preferential utilisation of favoured nitrogen sources such as ammonium and glutamine, termed nitrogen metabolite repression (NMR), is a dynamic system capable of responding to changes in the nitrogen status of the cell. NMR in A. nidulans is mediated by areA, a positively acting gene whose product, the GATAûûtranscription factor AreA, is required to alleviate NMR with areA loss of function mutants characterised by their inability to grow on non-preferred nitrogen sources. Because of the central role ammonium plays in the control of NMR, it is important to gain an understanding of the genes and proteins involved in ammonium uptake and their regulation. The presence of at least two specific ammonium transporters has been indicated previously for A. nidulans, including the isolation of methylammonium resistant meaA mutants which are defective in ammonium transport. Here we present the molecular, kinetic and physiological characterisation of two ammonium permeases, MeaA and MepA from A. nidulans. meaA was isolated by complementation of the meaA8 mutant and mepA was isolated by degenerate PCR. Both genes belong to the well characterised MEP/AMT family of ammonium transporters with meaA being most similar both in sequence and function to Mep1 from yeast, and mepA most similar to Mep2. Deletion strains for both genes have been generated. The double deletion strain displays reduced growth on low ammonium concentrations compared to wild-type. Expression analysis indicates that mepA and meaA have differing expression patterns but both are regulated by areA and to a lesser extent tamA.
351 A novel Aspergillus nidulans serine/threonine protein kinase gene that is transcriptionally regulated by camptothecin. Marcelo A. Vallim; Camile P. Semighini; Renata C. Pascon; Maria Helena S. Goldman & Gustavo H. Goldman. Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto. Av. do Café, S/N. 14040-903 - Ribeirão Preto-SP Brazil
The DNA damage response is a protective mechanism that ensures the maintenance of genomic integrity during cellular reproduction. It consists of extensive repair systems that deal directly with DNA damage, as well as surveillance systems, known as checkpoints, which arrest the cell cycle and provide time for DNA repair before the critical events of replication and segregation occur. Camptothecin (CPT) is an anti-cancer drug that targets eukaryotic DNA topoisomerase I by trapping the covalent complex formed by this enzyme and DNA. Damage to DNA occurs when the replication fork encounters the ternary complex (DNA-topoisomerase I-camptothecin) causing double-strand break and in higher eukaryotes, apoptosis. The filamentous fungus Aspergillus nidulans is a biological model to study cell cycle control and DNA damage response, however the mechanism how it responds to double-strand breaks caused by CPT is unknown. In order to uncover genes that might be involved in providing the response to damage to DNA in this fungus, Differential Display Reverse Transcription PCR technique (DD-RTPCR) was employed. Using this method we describe the isolation and characterization of a gene that encodes a novel putative serine/threonine kinase (anpk). Quantitative real-time RT-PCR showed that the transcription of anpk fluctuates during an 8 hours period. However, when camptothecin is added to the medium the anpk transcription is repressed showing its lowest accumulation at 1 hour after CPT induction whereas at 8 hours the drug induced anpk transcription. These results suggest that this kinase might be involved in the mechanism by which A. nidulans responds to DNA damage. This project is sponsored by Fundação de Amparo a Pesquisa do Estado de São to M.A. Vallim (#00/01088-9)
352 Isolation and characterization of a factor that modulates the gene expression in Aspergillus nidulans. Balan, A., and Zucchi, T.M.D.A. Department of Parasitology - Instituto de Ciencias Biomedicas II - Universidade de Sao Paulo - Brasil.
The su30 strain is a mutant of A. nidulans that modulates the gene expression of several genes. This strain when in contact with the UT196, that is deficient to methionine synthesis (met) and pyridoxin (pyro), is able to revert this phenotypes for met+ and piro+. Crosses and heterocaria analysis showed the presence of the factor (Fsu30) difusible onto citoplasm with activity in the mitosis and meiosis. Strains submitted to the treatment with the supernatant of Su30 presented different phenotypes and protein profile. The modified characters had shown high stability. Differences between the strains UT448 and su30 had been evidenced through SDS-PAGE. The supernatant of su30 also promoted alterations in the profile of restriction of plasmid DNA when submitted to the restriction enzymes. Hibridization analysis have showed that the rDNA fragment is methylated in Aspergillus nidulans, but the Fsu30 doesn't promoted any alteration in this sequence.
Financial Support: FAPESP
353 Photoactivation of gene expression in Phycomyces and Neurospora. Luis M. Corrochano, Lucia Bautista, Manuel Castellano-Munoz, Rocio P. Cotarelo, Maria Perez-Caro and Charles Yanofsky1. Departamento de Genetica, Universidad de Sevilla, Sevilla, Spain. 1Department of Biological Sciences, Stanford University
The Zygomycete Phycomyces blakesleeanus develops two types of sporangiophores of very different size: macrophores and microphores. Blue light inhibits microphorogenesis and stimulates macrophorogenesis. We have isolated a segment of the gene encoding the heat-shock protein HSP100 which is induced at the onset of sporulation. The effect of mutations on the expression of this gene and the isolation of its regulatory sequences will aid to establish the molecular details of photomorphogenesis in Phycomyces. The gene con-10 of Neurospora is expressed during conidiation and following illumination of mycelia with light. The photoactivation of con-10 disappears after two hours of illumination (light adaptation). We have designed a protocol to isolate mutants altered in the adaptation of con-10 photoactivation. We are using a strain of Neurospora with a fusion of the con-10 promoter to the gene conferring resistance to hygromycin. This strain is sensitive to the drug when the promoter is inactive, i.e. during vegetative growth either in the dark or under continuous light. We have isolated three mutants that grow in the presence of hygromycin under continuous light but not in the dark. Presumably this is due to a defect in the mechanism controlling light adaptation.
354 The NIT2 and NIT4 regulatory proteins must cooperate to activate gene expression in Neurospora crassa. Xiao-kui Mo and George A. Marzluf. Ohio State University, Biochemistry, Columbus, Ohio, USA
In Neurospora crassa, the nit-3 gene, which encodes nitrate reductase, an enzyme required for the utilization of inorganic nitrate, is subject to a high degree of genetic and metabolic regulation. The nit-3 gene promoter contains binding sites for the globally-acting NIT2 protein and for NIT4, a pathway-specific transcription factor. Expression of the nit-3 gene absolutely requires both the NIT2 and NIT4 proteins and only occurs under conditions of nitrogen source derepression and nitrate induction. The cys-14 gene encodes sulfate permease II, which facilitates the assimilation of sulfate. Expression of cys-14 is strongly regulated and its activation requires only a single positive-acting factor, CYS3, a bZip protein. It was of interest to determine whether NIT2 or NIT4 alone would be capable of turning on expression of cys-14 since this structural gene is normally controlled by only one regulatory protein. NIT2 and/or NIT4 binding elements were introduced into the cys-14 promoter and these constructs were transformed into a cys-13, cys-14 strain and also into nit-2 and nit-4 mutant hosts. The cys-14 gene in these transformants could now be controlled as a nitrogen-regulated gene. Sulfate permease assays revealed that both NIT2 and NIT4 were required for cys-14 expression during nitrogen induction conditions. Neither alone was able to activate any detectable cys-14 expression. Thus, we conclude that NIT2 and NIT4 are neither capable alone of activating gene expression in this context, but together can cooperate to elicit strong activation. The strain containing the cys-14 gene armed with both NIT2 and NIT4 binding elements was still able to respond to sulfur derepression. Thus this redesigned cys-14 gene is regulated by both nitrogen and sulfur control systems. When derepressed for both nitrogen and sulfur, a synergy was observed and cys-14 was expressed at a much greater level than expected by a simple additive effect.
355 Fopta1, a trascriptional activator specific to highly virulent strains of Fusarium oxysporum f.sp. phaseoli. B. Ramos1, F.M. Alves-Santos2, M. Perlin1, E.A. Iturriaga1, R. Martín-Domínguez1, M.A. García-Sánchez1, A.P. Eslava1 and J.M. Diaz-Minguez1. 1Area de Genitica, Centro Hispano-Luso de Investigaciones Agrarias, Universidad de Salamanca, 37900 Salamanca, Spain. 2Plant Breeding Department, MBG-CSIC, 36080
The plant pathogen Fusarium oxysporum is a common soilborne fungus with a worldwide distribution. Within the species there is a high level of host specificity with over 120 described formae speciales and races capable of causing vascular wilt diseases of many agricultural crops. This combination of wide range of infection as species and host specificity as formae speciales makes F. oxysporum an attractive model for the study of the molecular interactions involved in pathogenicity and/or virulence. Fusarium wilt, caused by F. oxyspourm f. sp. phaseoli J.B. Kendrick & W.C. Snyder, is a serious disease of common bean (Phaseolus vulgaris L.). In the course of the genetic characterization by RAPD analysis of F. oxysporum f.sp. phaseoli, we have isolated a gene, Fopta1, specific to the highly virulent strains of all known races of the pathogen. Molecular analysis allowed the identification of Fopta1 as a transcriptional activator belonging to the Gal4 family, which is characterized by the presence of the C6 Zinc finger motif. Expression of Fopta1 has been detected by RT-PCR both in mycelium of highly virulent strains grown in vitro and in bean plants artificially inoculated with these strains, but its expression was not found in mycelia of nonpathogenic strains. Gene inactivation experiments and a fine analysis of in vivo expression are underway in order to elucidate whether Fopta1 is a virulence or a pathogenicity factor, and in which mechanisms this gene is involved.
356 Sugar sensing and regulation of conidiation in Neurospora. Xin Xie and Daniel J. Ebbole. Texas A&M University, College Station, TX.
Sensing the presence of a preferred carbon source is a fundamentally important part of the cellular response to the environment. Unfortunately, the pathways for signaling carbon availability in filamentous fungi are poorly understood. In yeast, several mechanisms are involved in glucose sensing and controlling gene expression. These mechanisms include the use of special glucose transporter homologs that have evolved as glucose receptor/sensors but are not themselves functional glucose transporters. The N. crassa rco-3 mutant conidiates in liquid culture in the presence of carbon sources that repress conidiation in the wild type. The mutant is altered in the activities of multiple sugar uptake systems, is defective in carbon catabolite repression, and is resistant to 2- deoxyglucose and sorbose. The rco-3 gene is a member of the sugar transporter superfamily with greatest similarity to fungal glucose transporters. Our characterization of rco-3 suggests it functions as a sugar sensor rather than a sugar transporter. To gain further evidence for the role of rco-3 as a regulator we have isolates several suppressors of rco-3 and are examining the effect of the rco-3 mutant on gene expression. The characterization of the suppressor mutants and transcriptional profiling of the rco-3 mutant strain should help us define the genetic pathway involving rco-3 and the role of rco-3 in sugar sensing.
357 Coiled coil domain mediated FRQ-FRQ interaction is essential for its circadian clock function in Neurospora. Ping Cheng1, Yuhong Yang1, Christian Heintzen 2, and Yi Liu 1 1. Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX. 75390-9040 USA 2. Department of Genetics, Dartmouth Medical School, Hanover, NH. 03755 USA
The frequency (frq) gene, the central component of the frq based circadian negative feedback loop, regulates various aspects of the circadian clock in Neurospora. However, the biochemical function of its protein products, FRQ, is poorly understood. In this study, we demonstrated that the most conserved region of FRQ forms a coiled-coil domain. FRQ interacts with itself in vivo, and the deletion of the coiled-coil region results in the loss of the interaction. Point mutations, which are designed to disrupt the coiled-coil structure, weaken or completely abolish the FRQ self-association and lead to the arrhythmicity of the overt rhythm. Mutations of the FRQ coiled-coil which inhibit self-association, also prevent its interaction with two other key components of the Neurospora circadian clock, namely WC-1 and WC-2, the two PAS domain containing transcription factors. Taken together, these data strongly suggest that the formation of the FRQ-FRQ and FRQ-WC complexes are essential for the function of the Neurospora clock.
358 Induction of alternative oxidase gene expression by nitric oxide in Histoplasma capsulatum. Joan E. McEwen and Clayton H. Johnson. Central Arkansas VA and Univ. of AR Med. Sci., GRECC and Geriatrics, Little Rock, AR, USA.
Most fungi possess a branched mitochondrial respiratory pathway. Cytochrome c oxidase and alternative oxidase terminate the two branches of the pathway. Both enzymes transfer electrons from an electron donor to dioxygen, with production of harmless water as the product. Because alternative oxidase is absent in mammals, it will be an attractive target for development of antifungal drugs if it is demonstrated to be important for virulence of pathogenic fungi. We hypothesize that alternative oxidase is especially important during pathogenesis, when host antifungal efforts involving nitric oxide or other environmental stresses may inhibit the cytochrome pathway. In support of this hypothesis, experiments on the effect of nitric oxide on H. capsulatum mitochondrial function showed rapid inhibition of cytochrome oxidase, but little inhibition of alternative oxidase, by physiological levels (10 to 50 micromolar) of nitric oxide donor. Also, within an hour after nitric oxide exposure, alternative oxidase enzymatic activity and mRNA levels were significantly increased relative to the control, indicating that nitric oxide induces alternative oxidase gene expression. To examine the role of transcriptional induction in this process, a 14 kb genomic fragment containing the H. capsulatum AOX1 gene for alternative oxidase has been isolated and will be used to dissect the 5' flanking region of the gene for potential transcriptional control elements. Also, a polyclonal antiserum against H. capsulatum alternative oxidase has been raised and will aid us in studies of alternative oxidase function and regulation in this fungal pathogen.
359 Differential display and analysis of sterol induced changes in Phytophthora gene expression. David W. Dotson, Kevin V. Shianna, Shirley Tove, Leo W. Parks. North Carolina State Unviersity, Microbiology, Raleigh, NC
Although sterols are universally observed as critical membrane components among the eukaryotes, sterol auxotrophy is by no means uncommon. Nevertheless in almost all instances these auxotrophs require an exogenous supply of sterol for viability. Species of Phytophthora are exceptional in that their inability to synthesize sterols is combined with a rare ability to grow even in the complete absence of sterols. While these features have proven enticing towards research in sterol metabolism, the genetics of Phytophthora remain largely unresolved and experimentally intractable. In spite of this we have demonstrated the capacity of Phytophthora to differentially regulate gene expression in response to sterols, and enzymatically modify particular features of sterol nuclei. In this study we have used a differential display (DDRT-PCR) method to isolate cDNA fragments corresponding to genes of P. cactorum that are regulated in response to sterol feeding. Sequence analysis has allowed us to identify similar open reading frames in the model eukaryote Saccharomyces cerevisiae. In contrast to Phytophthora, genetics and biochemistry of Saccharomyces are more facile, allowing our pursuit towards a functional characterization for the proteins encoded by the isolated cDNAs. These data should provide insight and new avenues for research into the functional sterol metabolism of Saccharomyces and Phytophthora, along with other taxa both fungal and non-fungal alike.
360 Regulation of fatty acid desaturase gene expression in the oleaginous fungus Mortierella alpina. A.T. Carter , D.A. MacKenzie , P. Wongwathanarat and D.B. Archer. Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK, Dept. of Biotechnology, Thammasat University, Patumthanee12121, Thailand, School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
M. alpina produces up to 50% of its cell dry weight as triacylglycerol rich in long-chain polyunsaturated fatty acids which are of nutritional and pharmacological importance. Several genes encoding fatty acid desaturases have been isolated from this fungus and we have examined their differential expression during growth in media with high and low C:N ratios. Transcripts from the delta9-fatty acid desaturase gene (ole1), the delta6-fatty acid desaturase gene and the acetyl-CoA carboxylase gene were detected in mycelium grown in rich, low C:N medium and showed increased levels during growth in high C:N medium in which oil accumulation is stimulated. Transcription of a second delta9-fatty acid desaturase gene (ole2) and of a third, putative fatty acid desaturase gene (delta9-3) was negligible when the fungus was grown in low C:N medium but was induced significantly in high C:N medium. Reduction of growth temperature from 25 C to 12 C also caused a significant increase in mRNA levels for all three delta9-like genes. The delta9-3 gene failed to complement the yeast ole1 mutation unlike the M. alpina ole1 and ole2 genes. Fatty acid analysis of yeast transformants indicated that the delta9-3 gene product desaturated hexacosanoic acid (26:0) to hexacosenoic acid (26:1 n-9), a reaction which the ole1 and ole2 enzymes also carried out. Hexacosanoic acid is not found in M. alpina and the true substrate for delta9-3 remains to be elucidated.
361 The catalase isozymes and genes of Histoplasma capsulatum. Clayton Johnson, Joan McEwen, Lyndal York. Univ. of AR Med. Sci. and Central Arkansas VA, Geriatrics and GRECC Little Rock, AR
The dimorphic fungus, H. capsulatum, encounters significant environmental stress during its life cycles. To uncover its oxidative stress response pathways, we isolated three catalase cDNAs (CATA, CATB, and CATP) and a CATA genomic fragment. We purified a de novo or recombinant forms of 2 of these enzymes and used them to raise antisera. We are using these tools to characterize the expression levels and cellular locations of the products of these genes. Finally, we are using the catalase clones in attempts to generate knockout mutants.
Our immunoblots confirm that catalase P is a soluble enzyme that is likely to be peroxisomal. Our preliminary catalase A immunoblots indicate this enzyme is extremely insoluble and localized at the cellular surface. Northern blot results show different patterns of expression of the 3 catalase genes. Abundant transcripts for both CATB and CATP were detected under all growth conditions examined, but a modest decrease in RNA abundance was observed for each gene after growth of either yeast or mycelial cells in medium containing glucose rather than glycerol as carbon source. In contrast, CATA RNA levels were nearly undetectable under most conditions of growth, but showed marked elevation after oxidative stress.
Our work confirms a significant redundancy in cellular catalase function and is interesting in light of the role of H. capsulatum as an intracellular pathogen that may be exposed to high levels of oxidative stress during infection of a mammalian host. Gene knockouts, when available, will elucidate the different roles of each catalase isozyme.
362 Aspects of gene regulation by ambient pH in Aspergillus nidulans. Joan Tilburn. Imperial College School of Medicine, Infectious Diseases, London, UK
Response to ambient pH is mediated by the PacC transcription factor. Under conditions of alkaline ambient pH, PacC is proteolysed to its active form in response to a signal mediated by the pal gene pathway. This carboxy-terminally truncated PacC form is an activator of alkaline expressed genes and, in conjunction with the pacM gene product, a repressor of acid expressed genes. pH regulation in A. nidulans affects levels of gene products of commercial and/or health importance and studies here underpin work in other organisms where homologues of components of the system are involved in virulence determination and/or sex. Aspects of our current understanding of pH regulation will be presented.
363 Heterologous expression of human insulin by Aspergillus nidulans. Jesus, K.R.E; Meilus, M.* & Zucchi, T.M.D.A.*. Universidade de Sao Paulo - PostGraduate Program of Biotechnology. *Universidade de Sao Paulo - Department of Parasitology
Filamentous fungi of genus Aspergillus have been intensively used as receptor strain to express heterologous protein mainly due to their ability of secreting high amounts of extra cell proteins in a very inexpensive way. In the present case, A. nidulans was chosen on account of its well known genetic background, allied to good possibilities of producing transformants from protoplasts. Besides, cultures are of easy preparation, and even overnight experiments can yield large amounts of secreted proteins. After RNA treatment (Zucchi, 1996), several human insulin expressing Aspergillus strains were obtained. Despite high meiotic instability, meiotic and mitotic crosses produced several stable strains which were selected after their ability of expressing high insulin amounts. High instability presented by the RNA treated strains, allied to changes in the expression of resident genes evidence insertions, what may indicate the triggering of genomic defense against exogenous insertions. The control over instabilities promoted by genomic defense against DNA invaders, constitutes the main focus of this research.
364 Photoregulation of carotenoid biosynthesis in Mucor. Enrique A. Iturriaga, Antonio Velayos, and Arturo P. Eslava. Area de Gen tica, Departamento de Microbiologia y Genetica, Universidad de Salamanca, Salamanca, Spain
The biosynthesis of carotenoids in the zygomycete Mucor circinelloides is a process positively regulated by light. Beta-carotene, the main product of the carotenogenesis pathway, is synthesized from geranylgeranyl pyrophosphate after three enzymatic steps: phytoene synthesis, phytoene dehydrogenation and lycopene cyclization. Only two structural genes are involved in this conversion. The carB gene is responsible for the phytoene dehydrogenation and the carRP gene performs the first and last steps of the pathway. Both genes are arranged in opposite directions, with an intergenic region of 446 bp. Northern analyses from mycelia irradiated with short pulses of blue light showed a fast increase in messenger accumulation. The kinetics of such accumulation was very similar for both genes, showing a typical biphasic response with two maxima in the first twenty minutes after irradiation. The ratio between the maximum level of accumulation and the levels in the darkness control was fluence-dependent, showing a ten-fold increase with 1 Jm-2 and more than two hundred-fold with 960 Jm-2. A high rate of mRNA degradation was also detected, what suggests a fast turnover of both messengers, and possibly a mechanism of adaptation. Sequence analysis of the intergenic region showed the presence of several motifs similar to the APE sequences described in the promoters of blue light-regulated genes of Neurospora crassa. The fact that the mRNA accumulation levels for both genes in response to light are very similar may reflect a bi-directional mode of action of these APE-like elements.
365 Interconnected feedback loops in the neurospora circadian system: interactions between wc-1 and frq. Kwangwon Lee, Jennifer J. Loros, and Jay C. Dunlap. Department of Biochemistry and Department of Genetics, Dartmouth Medical School. Hanover NH. 03755.
Neurospora crassa serves as a excellent model system to identify and characterize the molecular components of the cellular circadian oscillator. Previous work has demonstrated that FRQ and WC-1 are key components of a functional cellular oscillator. The current molecular paradigm for the cellular circadian oscillator includes a transcription/translation based feedback loop composed of negative elements (FRQ in Neurospora, PER and TIM in Drosophila, and mPER and mCRY families in mammals) and PAS domain containing positive elements (WC-1 and WC-2 in Neurospora, dCLK and CYC in Drosophila, and CLOCK and BMAL1 in mammals). WC-1 forms heterodimers with its partner WC-2 and transduces the light signal to frq. Analyses of a knock-out strain and different mutant alleles of wc-1 confirmed that WC-1 is necessary for light induction for frq and for the robust oscillation of FRQ in constant darkness. WC-1 protein is rhythmic in constant darkness, whereas the steady state level of transcripts are constant. Our data suggest that FRQ is not only a negative regulator of its expression but also a positive regulator for WC-1 through post-transcriptional regulation. We propose that these interdependent regulations of the two key players FRQ and WC-1 in the Neurospora circadian oscillator promotes robustness of the oscillation. Similar interlocked feedback loop structures have been reported in Drosophila (Glossop et al. 1999, Science 286:766-768) and the mouse (Shearman et al. 2000, Science 288:1013-1019).
366 Transcriptional regulation of mating in Candida albicans by genes at the mating-type-like locus. Ryan M. Raisner. UC. San Francisco, Microbiology and Immunol. San Francisco, CA, USA
We have recently discovered a cryptic sexual cycle in Candida albicans, a pathogenic yeast previously classified as asexual. C. albicans has a mating type like locus encoding nine genes, some of which control mating. We have preliminary evidence that some of these genes are able to control mating through transcriptional regulation.
367 Genetic and physical analysis of a BAC contig containing the mating type locus of Phytophthora infestans. Audrey M.V. Ah Fong, Thomas A. Randall, and Howard S. Judelson. Department of Plant Pathology, University of California Riverside, California, USA 92521
A map-based cloning scheme is being used to isolate the mating type locus of P. infestans, the oomyceteous species that causes the late blight diseases of potato and tomato. P. infestans is heterothallic, and exhibits two mating types, A1 and A2. The current model for mating type suggests that the A1 mating type is heterozygous at the mating type locus (A/a) and the A2 is homozygous (a/a). Molecular markers B1 (very tightly linked to mating type), AP1 and X1 (which flank B1 at distances of 15 and 13 cM, respectively) were used to isolate clones from a BAC library. These were assembled into contigs representing the A and a-containing chromosome homologues. Physical mapping of the B1-hybridizing BACs revealed heteromorphic regions between the two contigs. Genetic mapping using cleaved amplified polymorphisms (CAPS) and single strand conformational polymorphisms (SSCP) narrowed the region that contained the mating locus to within 135 kb of DNA between the loci AP1 and X1. cDNAs hybridizing within the contigs were isolated to identify candidate genes and additional markers for mapping. A SalI polymorphism detected by cDNA 637 further delimited the mating type locus to a 60-70 kb region. Additional markers are being identified for further mapping studies and the search for more recombinants in the region will help define the boundaries of the mating type locus.
368 Functional analysis of FL, a binuclear zinc cluster protein regulating conidiation in Neurospora. Panan Rerngsamran, Xin Xie and Daniel J. Ebbole. Texas A&M University, College Station, TX.
The fl gene is necessary for conidiation and, when expressed from a heterologous promoter, fl is sufficient to induce conidial morphogenesis. To examine the role of fl in regulating conidiation we have begun a functional analysis of the FL protein. Expression profiling of N. crassa genes that depend on fl for their expression is being used to identify genes whose promoters may contain binding sites for FL protein. We plan to use affinity purified of 6xHis tagged versions of FL to identify the DNA binding site for FL in these promoters.
369 Analysis of cytbi3 mitochondrial group I intron ORFs from Podospora and Neurospora for homing endonuclease activity. Jill Salvo, Union College, Department of Biology, Schenectady, NY 12308
A number of unusual protein-coding regions exist within the introns of the mitochondrial genes of Podospora anserina. Many of these show similarities to known mitochondrial maturases and homing endonucleases (Cummings et al., 1990). The cytbi3 intron ORF shows a 25% identity with the homing endonuclease I Sce I overall, and a 62% identity in the two dodecapeptide repeats, the LAGLI-DADG motif, associated with endonuclease/maturase function. The optional cytbi3 intron of Podospora is also optional in Neurospora at the same location, and the intron encoded ORFs share extensive similarities (199/229 identical amino acid residues; R. Collins, personal communication). A comparative analysis of these two potential homing endonucleases has been undertaken to establish the extent of similarity with regard to their endonucleolytic activity and substrate specificity. We have used PCR technology to engineer and clone several the cytbi3 intron ORFs into a highly regulated E. coli expression vector, which makes use of an intein to provide the final protein product. It was also necessary to alter mitochondrial tryptophan codons (UGA) to the universal tryptophan codon, UGG. Concurrently with the cloning of the ORF, a potential target for the endonucleases was constructed. In general, these sites span from 20 to 60 base pairs around the cleavage site, thus we used an intron-minus allele to obtain as a source for the potential target site. There are a number of interesting evolutionary considerations in regard to the origins of these introns and their ORFs in these organisms, and the possibility of horizontal (between species) gene transfer seems to be plausible, although not yet demonstrable.
370 A novel bZIP transcription factor in Neurospora crassa. Harriett Bowannie Platero and Mary Anne Nelson. Univ of New Mexico Biology, Albuquerque, NM
A novel gene, tentatively named zip-1, has been identified by the Neurospora Genome Project. The gene encodes a protein (219 amino acids) that is preferentially expressed during the sexual stage of development of Neurospora crassa. The protein contains a DNA-binding domain and an adjacent leucine zipper. These two regions are characteristic of the bZIP family of transcription factors, which includes the mammalian transcription factors c-jun and c-fos, as well as GCN4of Saccharomyces cerevisiae. Mutation of the zip-1 gene results in delayed sexual development. Currently, microarray analysis is being utilized to identify genes directly or indirectly regulated by this novel transcription factor.
371 Translational suppression of UAG amber nonsense codons in cell-free extracts derived from Neurospora crassa supersuppressor mutants. Peng Fang, Cheng Wu, and Matthew S. Sachs. Oregon Graduate Institute of Science and Technology, 20000 NW Walker Road, Beaverton, OR 97006-8921
Neurospora crassa has ten mapped supersuppressor (ssu) genes that suppress the effects of premature termination mutations. In vivo studies indicate that they can suppress amber (UAG) codons but the spectrum of their functions remains to be elucidated. We examined suppressor activity using cell-free translation extracts from wild-type N. crassa and seven distinct ssu strains, ssu-1, -2, -3, -4, - 5, -9, and -10. We tested suppression of three kinds of nonsense codons, UAA, UGA, and UAG in vitro by requiring suppression to produce functional full-length firefly luciferase enzyme. Luciferase assays showed that all of the ssu strains with the exception of ssu-3 specifically suppressed the amber codon but not the other nonsense codons. Relative to constructs containing sense codons at the corresponding position, enzyme activities produced with UAG varied from 15% to 30%, while activities produced with UAA (ochre) or UGA (opal) at the corresponding position were about 1% to 2%, similar to levels observed with any premature termination codon using wild-type or ssu-3 extracts. Corresponding results were observed using 35S-methionine labeling to visualize the luciferase polypeptide directly following gel electrophoresis. Finally, using a primer extension-inhibition assay to map ribosomes on the RNA, only when there was suppression were ribosomes observed to continue translation of the reading frame downstream of the nonsense codon.
372 Cloning of the SIP3 homologue in Aspergillus nidulans. Shobana Krishnan, Rosanna Penna-Muralla and Rolf Prade. Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater OK 74078 USA
In Saccharomyces cerevisiae, the Snf1 (sucrose non-fermenting) protein kinase is required for transcription of glucose-repressed genes when glucose is limiting. The Sip3 protein belongs to the Snf1 kinase family and was found to interact "in vitro" with Snf1. The aim of this research is to isolate the sip3 homologue in A. nidulans and to determine whether sip3 is involved in its carbon catabolite repression. We have isolated cosmids that hybridize to a sip3 probe, and the entire genomic region has been sub- cloned and sequenced. The sip3 probe used is a clone that contains a fragment of the sip3 homologue in A. nidulans rescued in a different study. A sip3 deletion mutant will be created by transformation-mediated gene replacement and the growth in various carbon-sources like glucose, sucrose and pectin studied and compared to wild type.
373 PkaA regulates AflR activity by phosphorylation in Aspergillus nidulans. Julie K. Hicks1, Kiminori Shimizu2 and Nancy P. Keller2. Texas A&M University, Dept. of Plant Pathology & Microbiology, College Station, TX, USA. 1Present address: Dept. of Biology, Texas A&M University, College Station, TX, USA. 2Present address: Dept. of Plant Pathology, University of Wisconsin, Madison, WI, USA.
Aspergillus nidulans is a known producer of the carcinogenic mycotoxin, sterigmatocystin (ST). It has been shown that ST biosynthesis is controlled by a G protein mediated signaling pathway. One identified component of this pathway is PkaA, a catalytic subunit of a cAMP- dependent protein kinase (PKA). Previous work indicates that PkaA negatively regulates AflR, a transcription factor that positively regulates the ST biosynthetic genes, at a transcriptional and post-transcriptional level. The latter regulation is demonstrated by the inability of AflR to function when expressed in a pkaA overexpression background. We hypothesize that PkaA post-transcriptionally regulates AflR by phosphorylating some or all of three putative PKA phosphorylation sites in AflR. We are testing this hypothesis by introducing mutations in one, two, or all three of these phosyphorylation sites and asking if any of these AflR mutants function when expressed in a pkaA overexpression background. Currently we have been able to construct one of these strains where one PKA phosphorylation site is mutated. In contrast to the inability of a wild type aflR allele to induce stc gene expression in a pkaA overexpression background, expression of the mutant aflR allele in the same pkaA overexpression background resulted in stc gene expression and ST production. These results suggest that AflR may be a direct target of PkaA and that phosphorylation of AflR negatively regulates its ability to promote stc transcription.
374 Plant cell wall degradation by Aspergillus nidulans. Sunita Macwana1, Anamika Ray2, Rolf A. Prade1 and Andrew Mort2. 1Department of Microbiology & Molecular Genetics and 2 Biochemistry & Molecular Biology, Oklahoma State University, Stillwater OK 74078
Little is known about the plant cell wall degrading enzymes produced by A. nidulans while growing on plant cell walls. Our study is based on a molecular negative screening method, specifically directed to recover cDNA clones from ALL transcripts A. nidulans induces when shifted from growth on glucose to a range of cell wall polysaccharides including pectins, cellulose and xylan. cDNAs prepared from mRNA of tissues grown in glucose were labeled and used to probe a cDNA-plasmid library made from mRNAs extracted from tissues grown on cell wall polysaccharides. Transcripts present in both, the probe and the plasmid library, appear as positives whereas transcripts expressed only in the plasmid library are the negatives (not labeled). A two-staged screening method was devised to allow the survey of a large number of clones for identification of putative negatives in the initial stage followed by reliable confirmation of clones that are not expressed or present in low abundance in the probe originating condition. A statistically significant collection of negatives has been isolated and sequenced for a digital gene expression profiling and functional annotation analysis. Genes recognized through this method, are the ones upregulated as a result of the physiological shift (change in carbon source). Thus, the suggested approach is comprehensive because one can identify whole gene sets activated by a special physiological condition.
375 Scanning mutagenesis reveals that arginine-specific regulation of Neurospora crassa arg-2 is dependent on specific residues of the peptide encoded by an upstream open reading frame in its transcript. Christina C. Spevak, Peng Fang, and Matthew S. Sachs, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006-8921
The 5' regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs) that are important in cell growth and development. The Neurospora crassa arg-2 uORF encodes an evolutionarily conserved 24 amino acid peptide, called the Arginine Attenuator Peptide (AAP), which inhibits translation of ARG-2 in a high concentration of arginine by causing ribosomes to stall at the uORF stop codon, blocking ribosomes from reaching the ARG-2 initiation codon. Alanine- and proline-scanning of amino acids 6-24 of the AAP resulted in the identification of the amino acids that were necessary for this regulation in an N. crassa cell-free translation system. AAP residues D12, Y13, L14, and W19 were critical for regulation. For residues S6, T9, D16, H17, R20, A21 and A24, regulation was either diminished or lost after mutation to alanine or proline; unlike the case for the wild-type control, regulation was observed to increase as the concentration of arginine increased for these mutants. Mutation of residues V7, F8, L22, and N23 did not affect regulation. The specificity of negative regulation with the wild-type AAP in response to L-arginine was tested using the analogs, agmatine, D-arginine, L-argininamide, L-arginine methyl ester, citrulline, and L-monomethyl arginine. The results indicate that regulation is stereospecific and requires the amino- and guanidino- groups of arginine.
376 Characterization of the uvsI gene encoding an error-prone DNA polymerase and its null mutant in Aspergillus nidulans. Kyu-Yong Han1, Young-Kug Jang2, Suhn-Kee Chae2, and Dong-Min Han1. 1Wonkwang University, Division of Life Science, Iksan, Chonbuk, Korea, and 2Paichai University, Division of Life Science, Taejon, Korea
Polymerase is an error-prone DNA polymerase, consisting of at least two subunits: REV3 of the catalytic component and REV7 of unknown function in yeast. This complex is responsible for translesion DNA synthesis in consequence often to generate mutations. In Aspergillus nidulans, the uvsI gene has been cloned and the 9.1 kb DNA fragment containing the uvsI ORF was sequenced. UVSI had well conserved the hexapetide motifs of DNA polymerases in the C-terminus and showed the highest amino acid similarity to REV3. In addition, protein interaction of UVSI with yeast REV7 was demonstrated using the yeast two-hybrid system, indicating that UVSI is a functional homolog of REV3. About 5.3 kb uvsI transcript was detected in northern analysis and its amount was increased in responses to UV and MMS treatments. A null mutant having the deletion of the entire uvsI ORF was constructed. The phenotype of uvsI mutant was much the same as that reported for uvsI501 mutant by showing high UV-sensitivity and reductions of spontaneous and UV-induced reversions of certain mutant alleles. No growth defect was exhibited in uvsI. Among genes responsible for mutation induction in Aspergillus nidulans, uvsC (a recA and RAD51 homolog), uvsJ (a RAD6 homolog), and uvsI, synergistic interaction in terms of MMS-sensitivity was observed between uvsI and uvsC. uvsJ and uvsC showed synthetic lethality. Test for epistatic relationship between uvsI and uvsJ is currently undertaking. Sequencing of an upstream region of the uvsI gene revealed another ORF of 1,401 bp without a putative intron in opposite direction. This ORF starts 365 bp upstream from the uvsI start codon and encodes a putative polypeptide exhibiting a high amino acid similarity to SLU7 involving in the second step of pre-mRNA splicing in S. cerevisiae. The amount of 1.5 kb transcript from the SLU7 homolog of Aspergillus increased after UV irradiation.
377 Effects of uvsJ, a rad6 homolog, on mutagenesis in Aspergillus nidulans. Young-Kug Jang, Etta Kafer1, and Suhn-Kee Chae. Research Center for Biomedicinal Resources and Division of Life Science, Paichai University, Taejon, Korea, and 1Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
In Aspergillus nidulans, lack of mutagen-induced mutations has been observed in mutants of two different epistasis groups, UvsI (uvsI; REV3 homolog) and UvsC (uvsC; RAD51 homolog). In yeast, RAD6 protein is indispensable to generate mutations. However, the function of RAD6 is mostly unknown except its ubiquitin conjugation activity. To investigate whether the RAD6-dependent mutation pathway is also operated in Aspergillus nidulans, we have been cloned a Rad6 homolog (radB) and characterized its null mutant. Unexpectedly we found that radB is an allele of uvsJ previously assigned in the UvsF group. We also found that uvsJ1 was a temperature sensitive mutant showing the same level of mutagen-sensitivity to wild type at the permissive temperature (25oC) but demonstrating high sensitivity to MMS and UV-radiation at 37oC similar to uvsJ null mutants. Disruption of uvsJ caused a slow-growth phenotype on an agar plate, indicating its requirement on normal growth. Such a phenotype was not seen in uvsJ1 mutant carrying a single point mutation at 58th amino acid histidine. In contrast to yeast rad6 mutants, uvsJ null as well as uvsJ1 mutants exhibited increased UV-induced mutation frequencies in the system detecting selenate resistant forward mutations when compared with that for wild type. However, UV- induced reversions of choA1 and pabaA1 mutant alleles in uvsJ null and uvsJ1 mutants were not detected. In using yeast two- hybrid assay system, UVSJ-UVSH (RAD18 homolog) interaction has been demonstrated, while UVSJ1 mutant protein exhibited reduced affinity to UVSH at 37oC in yeast in vivo. Forced over-expression of UVSJ-[C88A], an E2 enzyme active-site mutant protein, in uvsJ+ background resulted in the change of colony growth, indicating a dominant-negative effect of the mutant protein.
378 A RAD52 homolog in Aspergillus nidulans as an interactor of UVSC and characterization of its null mutants. Seung-Hyen Ka, Nam-Sihk Lee, Etta Kafer1, and Suhn-Kee Chae. Research Center for Biomedicinal Resources and Division of Life Science, Paichai University, Taejon, Korea, and 1Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
In A. nidulans, differing from rad51 of yeast, involvement of uvsC, a Rad51 homolog, on mutagenesis has been clarified in various test systems. As an interactor protein with UVSC, we have been isolated a Rad52 homolog using the yeast two-hybrid screening system. Genomic DNA clone of the RAD52 homolog, named as RadC was isolated from a chromosome III specific library and the 10 kb PstI fragment was subcloned. Sequence determination of genomic and cDNA of radC revealed an ORF of 2,001 bp interrupted by 4 introns, encoding a polypeptide of 582 amino acids. The deduced amino acids has 54% and 45% sequence similarity to MUS- 11 of N. crassa and RAD22 of S. pombe, respectively. The C- terminal extension of RADC was observed compared to RAD52 of yeast and a human RAD52 homolog. About 4 kb radC transcript was detected in northern analysis and its amount was increased in responses to MMS treatments. RadC null mutant was generated by targeted replacement and confirmed by Southern analysis. Null mutants of RadC exhibited high MMS-sensitivity and enhancement of spontaneous and UV-induced selenate resistant mutation frequencies. Similarly to uvsC, radC was not sensitive to UV at dormant conidia stage, but the sensitivity to UV-radiation increased during germination. Epistatic interaction was detected in double disruption mutants of uvsC with radC. RADC-UVSC interaction was confirmed in both in vivo using the yeast two-hybrid assay and in vitro with a GST-pull down assay. The C-terminus of RADC not well conserved among RAD52 homologs was responsible to interaction with UVSC. In fact, yeast RAD51 failed to interact with RADC in vitro, while human hsRAD51 did.
379 Molecular Cloning and Characterization of an aga Mutant from Neurospora crassa. Gloria E. Turner, Sean Curran & Richard Weiss. UCLA, Chemistry & Biochemistr, Los Angeles, CA, USA
The complex aga locus encodes 2 forms of the catabolic enzyme arginase (E. C. 184.108.40.206.). Tandem promoters regulate the production of a 1.7-kb and a 1.4-kb transcript, which are translated into 41-kDa and 36-kDa products. These proteins differ at their amino terminus but carry out identical reactions, the hydrolysis of arginine to ornithine and urea. All vertebrates have 2 arginase proteins, which are encoded by separate genes. The liver arginase, A1 is found in the cytoplasm and the extrahepatic enzyme, A2 is found in the mitochondrion. Interestingly both N. crassa forms are localized to the cytoplasm. The 36-kDa form is expressed under all growth conditions tested, whereas, the expression of the 41-kDa form is activated by arginine. Both forms are expressed under nitrogen limitation. Recent results have shown that conidia store both forms of the protein and that expression is regulated both temporally and by a number of arginine related metabolites. We have cloned and characterized an aga mutant allele UM 913. This mutant lacks arginase enzymatic activity and makes no detectable proteins and a slightly detectable transcript. We have used this mutant as a recipient strain for gene targeting of altered aga allowing us to test the outcome of having a single arginase. To fully interpret the results we obtained from our single arginase transformant analysis; UM 913 arginase was cloned and sequenced. We have determined that a frame shift in the first third of the protein leads to an early termination of both proteins. We are presently investigating mechanisms for the reduced transcript levels. It is well documented in other organisms that premature translation termination can reduce mRNA stability.
380 Salt-stress, hogA-dependent control of hyphal growth, branching and septum formation in Aspergillus nidulans. Kap-Hoon Han and Rolf A. Prade. Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater OK 74078 USA
In yeast the HOG pathway senses extracellular salt concentrations and regulates the expression of stress-response-genes that overcome the osmotic imbalance. Based on molecular data derived from yeast, key genetic components that assemble the HOG-pathway were identified and isolated from the A. nidulans EST collection. To determine whether the A. nidulans HOG-pathway functions similarly as in yeast, the A. nidulans hogA gene, yeast hog1 homolog, was isolated and fully characterized. HogA shares 74% identity of its amino acid sequence with Saccharomyces cerevisiae HOG1. Northern analysis clearly showed that hogA is strongly induced after osmotic shock. Thus, abundance of hogA transcript is tightly associated with extracellular salt levels. A hogA deletion mutant shows no drastic morphological alteration when cultivated at 37C and normal (low) salt. When grown in the presence of high concentrations of salt, several morphological alterations were detected and studied in detail: First, polar hyphal extension rates are retarded by as much as 60% and, second, hyphal tip morphology is affected in the mutant. Hyphal tips of the mutant are bulbous and hyper-branched. Fluorescence microscopy, employing DAPI and Calcofluor, showed that nuclei accumulate abnormally at the tip of hyper-branched hypha, suggesting normal mitotic division of nuclei, while hyphal extension is severely restricted. Moreover, septum staining revealed abnormal septation patterns at the terminal portion of vegetative hyphae. No septa were observed at the terminal hyphal portions and abnormal chitin deposition was observed at every branching point, implying that hogA mutant attempted to form a septum but failed. Our results indicate that the hogA-dependent response towards stress in multicellular organisms, in addition to compensate solute imbalances, also appears to control proper cell expansion and modulate formation of cellular compartments.
381 Genes involved in sterigmatocystin expression in Aspergillus nidulans. Nanci O. C. R. Dezotti, and Tania M. A. D. Zucchi. Department of Parasitology & Biotechnology Research Center; Institute of Biomedical Sciences; University of Sao Paulo, Av. Prof. Lineu Prestes, 1374, Cidade Universitaria, CEP: 05508- 900, Sao Paulo, Brazil.
The fungus Aspergillus nidulans (Emericella nidulans) was used as a genetic model to investigate the genes, which are involved in sterigmatocystin production. The focused gene was stc, related to sterigmatocystin (ST) expression, which is an intermediate penultimate product in the aflatoxin biosynthetic pathway. The genetic analyses included studies of the sexual and parasexual cycles. The allelic segregation rates and recombination frequencies between linked and non-linked genetic markers were determined by crossing the strains UT448 to UT196 and to UT184, phenotypically stc, stc+, stc, respectively. Low ST expression (4.0 ppm) in the UT196 strain and in 7.4% of meiotic segregants allowed to map the stc locus at chromosome I, 3.4% distant from riboA1. The diploid UT448 (stc)//UT184 (stc) prepared from both non- producing strains was analyzed via parasexual cycle and 28% of its haploid segregants expressed ST. The results indicated that UT448 bears the stc mutant (or inactivated) allele, and the UT184, although bearing the stc+ allele, it is only reactivated by a factor (R2+), at chromosome VIII of UT448. In such configuration, the diploid expresses large amounts of sterigmatocystin (40 ppm). Another regulator factor (R1) at the meth-w (II) interval was identified in the UT448 strain. Suppot by FAPESP and CNPq/Brazil
382 Characterisation of two components of the unfolded protein response pathway from the filamentous fungus Trichoderma reesei. Mari Valkonen1, Markku Saloheimo1, Michael Ward2 and Merja Penttila1 1VTT Biotechnology, P. O. Box 1500, FIN-02044 VTT, Finland 2Genencor International, Inc., 625 Page Mill Rd., CA 94304-1013, USA
The unfolded protein response (UPR) of yeast has been studied intensively and several components of the pathway have been identified. HAC1 gene encodes a transcription factor that has been shown to bind a consensus sequence in the promoter region of the UPR-inducible genes and activate their transcription. Unconventional splicing involving Ire1p activates HAC1. Ire1p is a transmembrane protein kinase/RNase that has been located to the ER membrane. The yeast Ptc2p has been shown to negatively regulate the UPR pathway. We have cloned the ire1 and ptc2 genes from the filamentous fungus Trichoderma reesei using Aspergillus nidulans gene fragments as probes. These were identified in a search with the yeast sequences against A. nidulans EST database where cDNA clones were found that were homologous to the yeast genes. The putative Trichoderma PTCII protein shows 48% identity to S. cerevisiae Ptc2p and 60% identity with a putative PTC2 protein from Schizosaccharomyces pombe. The N-terminal domain of T. reesei IREI that presumably faces the lumen of the ER has 24% identity and 39% similarity over an area of 377 amino acids with yeast Ire1p. The C-terminal part with the kinase/RNase domains is 42% identical and 59% similar over an area of 490 amino acids to yeast Ire1p. The expression of T. reesei ire1 and ptc2 genes has been studied in DTT-treated and control cells. DTT-treatment causes an initial decrease in the expression of the ire1, after which there is recovery. The expression of the ptc2 gene decreases until 90 minutes of DTT-treatment after which there is a slight increase in the expression. The biochemical characterisation of the T. reesei IREI and PTCII proteins is being performed.
383 Induction mechanism of the Trichoderma reesei and Aspergillus nidulans hac1/A genes involved in the unfolded protein response. Markku Saloheimo, Mari Valkonen, Mick Ward1 and Merja Penttila. VTT Biotechnology, P.O Box 1500, 02044 VTT, Finland; 1Genencor International, 925 Page Mill Road, Palo Alto, CA 94304-1013, USA.
Genes encoding ER chaperones and foldases assisting in protein folding are controlled by the unfolded protein response (UPR) pathway, i.e. they are induced when unfolded proteins accumulate into the ER. The UPR pathway is best known from Saccharomyces cerevisiae. The yeast UPR transcription factor binding to the promoters of the target genes is Hac1p. The HAC1 gene is activated through a unique mRNA splicing event, where the kinase/RNAse Ire1p cleaves the HAC1 mRNA at the intron borders, and the exons are ligated together by tRNA ligase.
We have addressed the mechanism of UPR pathway induction in two filamentous fungi, Trichoderma reesei and Aspergillus nidulans. The functional homologs of the yeast HAC1 have been cloned from these fungi. Binding of the T. reesei HACI protein to putative UPR elements of the pdi1 and bip1 promoters of this fungus has been demonstrated. The results obtained suggest that the activation of the hac1/hacA genes of these filamentous fungi is more complex than in yeast, including two events. These are splicing of an unconventional intron of only 20 bp in length, analogously to the yeast HAC1 gene, and truncation of the mRNA at the 5' end, which leaves out an upstream open reading frame (uORF). It it has been shown that both the 5' flanking region and the 20 bp intron reduce the ability of T. reesei hac1 to complement the yeast HAC1 disruption. Evidence is also shown that the uORF reduces the formation of the HACI protein in yeast.
384 A molecular dissection of MAP kinase cascades regulating mating and haploid fruiting in Cryptococcus neoformans. Robert C. Davidson and Joseph Heitman. Duke University, Genetics, Durham, NC, USA
Recent findings reveal that a pheromone induced mating pathway operates in the human fungal pathogen Cryptococcus neoformans. To dissect the pheromone response pathway in C. neoformans, we first cloned the CPK1 and STE7 genes, which encode homologs of the FUS3/KSS1 MAP kinases and the MEK kinase of the S. cerevisiae pheromone response pathway. In contrast to those genes involved in mating or haploid fruiting previously identified that are mating type specific, CPK1 and STE7 are present in both MATa and MATalpha cells. We used biolistic transfomation to disrupt the CPK1 and STE7 genes in C. neoformans and show that both are required for mating in both mating types. In addition, the serotype D MATalpha cpk1 and ste7 mutant strains are unable to undergo a mating type specific differentiation called haploid fruiting. However we have been unable to show a role for either CPK1 or STE7 in virulence of C. neoformans. In order to assemble these elements into a signaling pathway, we employed the use of epistasis analysis.STE12alpha a mating type specific homolog of the transcription factor involved in mating and pseudohyphal growth in S. cerevisiae, has previously been shown to be required for haploid fruiting and virulence in serotype D MATalpha strains of C. neoformans, but is dispensible for mating. Overexpression of STE12alpha suppressed the haploid fruiting and mating defects of cpk1 or ste7 mutant strains. However, overexpression of CPK1 also suppressed the haploid fruiting defect of a ste12alpha mutant. These epistasis results indicate that the STE12alpha transcription factor and the CPK1 MAP Kinase are not acting in a single linear pathway. Two models are consistent with these findings: first another transcription factor exists downstream of CPK1 that is at least partially redundant with STE12alpha. Alternatively, STE12alpha and CPK1 are acting in separate parallel pathways to regulate mating and fruiting. Current studies are underway to test these predictions and assemble the pathways regulating morphogenesis in C. neoformans.
385 Nitrate is necessary but not sufficient for binding of NirA, a pathway-specific activator of Aspergillus nidulans. Frank M. Narendja, Sabine P. Goller, Markus F. Wolschek, and Joseph Strauss. Zentrum für Angewandte Genetik, Universität für Bodenkultur, Muthgasse 18, A-1190 Vienna, Austria
In Aspergillus nidulans the structural genes coding for nitrate reductase (niaD) and nitrite reductase (niiA), share a common promotor region of 1200bp. The two genes are divergently transcribed and are under the control of two positively acting transcription factors: NirA, mediating induction and AreA, which is inactivated in the presence of the repressing nitrogen sources ammonia or glutamine (1). We have previously characterized in vitro (2) and in vivo (3,4) the physiologically relevant cis-acting elements for the two synergistically acting DNA-binding proteins. We have further shown that AreA is constitutively bound to a target sequence located within a central cluster of four GATA sites and is directly involved in opening the chromatin structure over the promoter region thus making additional cis-acting binding sites accessible. Here we show that in vivo binding of the NirA transactivator to the central asymmetric recognition sequence is dependent on nitrate induction but also on a functional AreA protein. Our data suggest a potential new role for AreA in regulation of a post-transcriptional activation step of nitrate transport as ectopic expression of the transporter can not restore nitrate uptake in an areA loss-of-function mutant. An interesting situation is revealed when nitrate inducer and ammonia as repressing nitrogen source are available to the cells simultaneously. First, for a short period of incubation, ammonia does not preclude nitrate uptake and intracellular nitrate levels remain high. Second, at this stage both NirA and AreA are bound to their cognate targets but despite this 'ready-to-go' situation transcriptional activation does not take place. Possible mechanisms preventing the activation of a pre-set transcriptionally competent complex will be discussed.
References: (1) Scazzocchio, C. and Arst, H.N. Jr. (1989) Review .In Molecular and genetic aspects of nitrate assimilation. John L. Wray and James R. Kinghorn (eds). Oxford Science Publications (2) Strauss, J., Muro-Pastor, M.I., and Scazzocchio, C. (1998). Mol Cell Biol 18: 1339 (3) Punt, P.J., Strauss, J., Smit, R., van den Hondel ,C.A.M.J.J., and Scazzocchio, C. (1995). Mol Cell Biol 15: 5688 (4) Muro-Pastor, M.I., Gonzalez, R., Strauss, J., Narendja, F., and Scazzocchio, C. (1999). EMBO J. 18: 1584-1597
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