167. Identification and characterization of Neurospora crassa mutant strains that are rhythmic in continuous light. Kyung Suk Seo and Deborah Bell-Pedersen. Department of Biology, Texas A&M University, College Station, Texas 77843.
In Neurospora crassa, an endogenous circadian clock regulates daily rhythms of asexual spore formation. Under standard growth conditions, the developmental rhythm requires the function of the well-described FRQ oscillator (FO). The FO is composed of an autoregulatory feedback loop involving the frq, wc-1 and wc-2 genes. The FO receives input signals from the environment to synchronize the clock to the outside world, and is involved in signaling time-of-day information to the rest of the cell to control overt rhythmicity. Despite the importance of the FO in circadian timing, recent evidence has shown that under some growth conditions (e.g. in temperature cycles) circadian rhythms in development are present in strains that lack a functional FO. These data suggested the presence of an additional FRQ-less oscillator (FLO) in N. crassa cells. In attempts to identify components of the FLO, we have generated mutant strains that are robustly rhythmic in the absence of the FO in continuous light, and unlike strains that lack the FO, the mutant strains can be entrained in LD cycle. Genetic characterization of the mutant strains will be discussed in the context of a dual oscillator model.
168. The gene for the heat-shock protein HSP100 is induced by blue light and heat-shock in Phycomyces. Julio L. Rodriguez-Romero and Luis M. Corrochano. Departamento de Genetica, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain
The Zygomycete Phycomyces blakesleeanus develops two types of fruiting bodies of very different size, macrophores and microphores. Blue light stimulates macrophorogenesis and inhibits microphorogenesis. To investigate the role of differential gene expression during photophorogenesis in Phycomyces we have adapted a method based on the polymerase chain reaction with arbitrary primers. With this method we have isolated a cDNA segment for the heat-shock protein HSP100 that is induced by blue-light at the onset of sporangiophore development. The gene for HSP100 (hspA) is induced by blue-light and heat-shock. The expression of hspA is induced eleven fold after 30 minutes of blue light but longer exposure times reduced its expression. A short illumination (10 seconds) induced hspA but a period of incubation in the dark was necessary to detect the mRNA. Phycomyces cultures are usually grown at 22 degrees. A heat-shock, 34 degrees during 30 min, induced the hspA gene about 100 fold but incubations at higher temperatures reduced its expression. Our results suggest that light and heat can activate the expression of hspA to different levels. Several mutants altered in the Phycomyces light responses are available. The effect of these mutations on the expression of hspA by light and heat and the isolation of its regulatory sequences will aid to establish the molecular details of photophorogenesis in Phycomyces.
169. Gene Complements Induced by Forced Shift from Glucose to Alternate Carbon Sources in Aspergillus nidulans.
Sunita Macwana1 and Rolf Prade1. Department of Microbiology & Molecular Genetics Oklahoma State University, Stillwater OK 74078
Little is known about the extracellular enzymes A. nidulans produces while growing on plant cell wall polysaccharides. This study is based on a novel molecular screening method, aimed at the recovery of cDNA clones from all transcripts A. nidulans induces when forced to shift from glucose to a medium containing one or a range of polysaccharides, including pectin, cellulose, xylan and other plant cell wall components. cDNAs, prepared from mRNA templates extracted from glucose-grown cultures, were labeled (herein designated "glucose-grown" probes) and used to screen a cDNA plasmid library made from mRNAs extracted from plant cell wall polysaccharide-containing cultures (herein designated polymer-grown library). Isolation of condition-specific induced cDNAs was accomplished through differential DNA/DNA hybridization among a labeled "glucose-grown" probe, membrane cross-linked "polymer-grown" plasmid-clone library and negatives were selected for further analysis under the assumption that they were the ones induced as a consequence of the physiological shift. Thus, if selection of negatives is exhaustive, the suggested approach is comprehensive because a whole gene set activated by a specific physiological condition is recovered. We have isolated over 1,600 unique cDNA whose transcript does not appear to be present in glucose growing cultures and observed that only a fraction of polysaccharide degrading enzyme coding genes were found. Moreover, the screening data have been corroborated with time-course microarray expression profiling. This unexpected outcome suggests that significant intracellular metabolic changes take place when shifting carbon sources and that the presence of extracellular polymer degrading activities is regulated differently, not involving an exclusive induction of gene expression.
170. Upregulation of promoter activity of the Taka-Amylase A gene. Kanako Suzuki1, Yoshinao Koide1, Masashi Kato2, Tetsuo Kobayashi2, Norihiro Tsukagoshi2. 1Amano Enzyme Inc., Kakamigahara, Japan. 2Nagoya University, Nagoya, Japan.
Aspergilli have been widely utilized for production of various enzymes of industrial interest because of their high productivity. We have been characterizing regulatory mechanisms underlying expression of the A. oryzae Taka-amylase A gene (taa) to construct a high level expression system by utilizing regulatory elements involved in high levels of enzyme production. The taa gene contains several regulatory elements such as CCAAT-box and Starch Response Element (SRE). The CCAAT-binding complex, so called Hap complex, has been proved to increase the expression levels of many genes. AmyR, SRE-binding protein, mediates the inducible expression of the amylolytic genes. We inserted a 190 bp DNA fragment carrying both CCAAT and SRE sequences to the promoter region of the taa gene. One of those constructs, which had the DNA fragment downstream of the SRE sequence, exhibited the highest promoter activity. Using this upregulated promoter, we expressed two fungal genes encoding Taka-amylase A (A. oryzae) and Laccase (Paeaphaeoshaeria sp. ) as homologous and heterologous genes, respectively. When these genes were integrated with multiple copies into A. oryzae, transformants produced extracellularly a large quantity of both enzymes. This indicates that the promoter constructed here is one of the most efficient promoters for enzyme production in filamentous fungi.
171. Nuclear localization of Aspergillus nidulans AreA is regulated by nitrogen and carbon starvation. Richard B. Todd, James A. Fraser, Michael J. Hynes and Meryl A. Davis. Department of Genetics, University of Melbourne, Parkville 3010, AUSTRALIA
The global transcriptional regulator AreA activates transcription of many genes required for nitrogen catabolism under nitrogen limitation. AreA levels and activity are regulated autogenously, by differential mRNA turnover and interaction with the NmrA and TamA proteins. We describe an additional level of regulation of AreA activity. We have epitope tagged AreA by gene replacement at the areA locus and used immunofluorescence microscopy to determine the subcellular localization of AreA. Under nitrogen starvation the AreA protein hyperaccumulates in the nucleus. This correlates with a significant elevation of nitrogen catabolic gene expression. Furthermore, hyperaccumulation is NmrA-independent and does not require residues 60-423 or 854-876 of AreA. The AreA protein is not observed to accumulate in the nucleus in the presence of a nitrogen source. Transfer from nitrogen starvation to nitrogen sufficient conditions triggers rapid exit of AreA from the nucleus, consistent with the idea that AreA is transcriptionally inactive during nitrogen sufficiency. The increase in certain nitrogen catabolic enzyme levels in response to nitrogen starvation is prevented by carbon starvation. We show that simultaneous carbon starvation prevents the AreA hyperaccumulation observed under nitrogen starvation. Furthermore, transfer from nitrogen starvation conditions to carbon starvation conditions rapidly reverses AreA hyperaccumulation. These studies demonstrate that AreA activity can be differentially regulated by subcellular localization in response to distinct signals generated under nitrogen and carbon starvation.
172. A quantity control mechanism regulating levels of the HapE subunit of the Hap complex in Aspergillus nidulans: HapC adjusts the number of HapE. Masashi Kato and Norihiro Tsukagoshi. Department of Biological Mechanisms and Functions, Graduate school of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
The CCAAT sequence is one of the most common cis-elements present in the promoter regions in eukaryotes. Aspergillus nidulans CCAAT-binding complex (Hap complex) consists of at least three subunits, HapB, HapC and HapE. To investigate the quantity control mechanisms of the subunits during assembly of the Hap complex, reconstitution studies with the recombinant subunits and the extracts prepared from the respective hap subunit deletion mutants were carried out. Furthermore, Western blot analysis on the Hap subunits and Northern blot analysis on the hap genes with the respective deletion mutants were also performed. From all the results together, it was suggested that the number of the HapC molecule could adjust that of the HapE molecule by forming stable heterodimers prior to assembly of the Hap complex. Although many CCAAT complexes have been characterized from various eukaryotes, little is known about stability of their subunits. The findings reported here suggest that similar quantity control mechanisms of the subunits may exist in the other eukaryotes. 1) M. Kato et al. FEBS Letters 512, 227-229 (2002).
173. Identification of a new gene - suX(pro) involved in a suppression of proline auxotrophy in Aspergillus nidulans using a heterologous transposon Impala. Anna Olszewska, Agnieszka Dzikowska and Piotr Weglenski. Department of Genetics, Warsaw University.
Transposon tagging is a very useful tool for insertional mutagenesis and gene cloning. Transposons can be employed to tag genes both in Procaryota and Eucaryota. The aim of this project is to obtain an A. nidulans mutant with a suppression of proline auxotrophy by using a heterologous transposon Impala from Fusarium oxysporum. A.nidulans initially strain was a mutant in proA gene, thus it had a pro- phenotype. Using transposon mutagenesis we obtained a suX(pro) mutant which, in spite of mutation in proA gene, was able to grow on a medium without proline. Inverse PCR method was used for amplification of tagged gene. The suX(pro) gene was amplified together with upstream and downstream regions. Further it was cloned on plasmid vector and sequenced. An open reading frame of 1335bp was identified. It contains one putative intron (92bp) into which the Impala element has been transposed. The putative product of the suX(pro) gene is a protein of 259 amino acids residues. This protein comprises two conservative RRM domains (RNA recognition motif), each of them contains two RNP-SC regions (ribonucleoprotein consensus sequence). Proteins comprising RRM domains play a crucial role in posttranscriptional regulation of gene expression at the level of poliadenylation, splicing, transport of mRNAs from nucleus to cytoplasm and in mRNAs stabilization.
174. Functional analysis of the positive-acting sulfur regulator MetR of A. oryzae. Genryou Umitsuki1, Motoaki Sano2, Osamu Hatamoto1, Seiichi Hara1, Tsutomu Masuda1, Masayuki Machida2. 1Noda Institute for Scientific Research, Noda, Chiba 278-0037, Japan. 2Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, 305-8566, Japan.)
A. oryzae, one of the koji mold species, has been used to produce fermented foods such as soy sauce, miso, and sake. In order to breed the improved A. oryzae strains and to optimize the production process of fermented foods, an understanding of various gene regulation systems of A. oryzae is of great importance. Analyses of the mechanisms of carbon catabolite repression and the regulation of the nitrogen assimilation genes are under progress. On the other hand, little is known about the regulation of the sulfur assimilation genes in A. oryzae. We have cloned metR gene encoding MetR protein from A. oryzae, which is homologous to the positive-acting sulfur regulators MetR of A. nidulans and CYS3 of N. crassa. Electrophoretic mobility shift assay revealed the specific binding of A. oryzae MetR to a particular sequence of a DNA fragment in vitro. An A. oryzae strain that expressed metR gene under the control of amyB promoter was made and characterized. This strain showed derepression of arylsulfatase, which is known as one of the sulfur-repressed gene products. This strain also showed higher extracellular protease activity than the parental strain. These results suggest that A. oryzae MetR is a positive-acting sulfur regulator responsible to the induction of extracellular protease expression by the limitation of sulfur source in the medium.
175. A putative Aspergillus nidulans transcriptional regulator from a MADS-box family. Agnieszka Dzikowska, Joanna Empel, Rafal Tomecki and Piotr Weglenski. Department of Genetics, Warsaw University, Warsaw, Poland
The arginine catabolism gene otaA coding for ornithine transaminase (OTAse), is specifically induced by arginine. This induction depends on two elements: the Zn2C6 transcriptional activator - product of the arcA gene and specific target in the promoter of otaA gene. In the otaA promoter we have identified sequences responsible for arginine induction (AnUASarg). Deletions within AnUASarg result in non-inducibility of OTAse by arginine in vivo. Moreover, the expression of otaA carrying deletions within AnUASarg is not activated by the ARCAd47 super-activator althougharcAd47 is a dominant constitutive mutation resulting in an elevated level of arginase and OTAse activity in a wild type strain. The AnUASarg sequence is very similar to Saccharomyces cerevisiae UASarg, a sequence present in the promoters of arginine metabolism genes that is a target for a complex of Zn2C6 activator (ArgRIIp) with two transcriptional regulators from a MADS-box family (McmIp and ArgRIp). We have identified in CEREON A. nidulans sequence database a MADS-box domain which is almost the same as McmIp domain. The A. nidulans domain was amplified by PCR and the gene was cloned from Aspergillus Minimal Compressed Library (FGSC).
176. Functional analysis of Aspergillus oryzae XlnR and upregulation of its target gene expression. Junichiro Marui1, Noriyuki Kitamoto2, Masashi Kato1, Tetsuo Kobayashi1 and Norihiro Tsukagoshi1. 1Nagoya University, 2Aichi Industrial Technology Institute, Japan.
Aspergillus oryzae has been widely utilized for production of various enzymes of industrial interest because of their high productivity. A. oryzae XlnR was isolated as a transcriptional activator of the major xylanase gene, xynF1. To examine the spectrum of the xylanolytic and cellulolytic genes under the control of XlnR, their expression in an A. oryzae wild type strain, a xlnR disruptant and a xlnR overexpressed strain was analyzed by Northern blotting. XlnR directed expression of at least four xylanolytic and four cellulolytic genes when induced by xylan and xylose. Moreover, XlnR was newly found to mediate the cellulose-dependent expression of the xylanolytic genes as well as the cellulolytic genes in A. oryzae. This was further confirmed by monitoring beta-galactosidase activity in transformants carrying the authentic or mutated xynF1 promoter controlled lacZ gene grown on cellulose. The xynF1 gene contains two different affinity binding sequences for XlnR and one analogous sequence in a short promoter region. The high affinity binding sequence, 5'-GGCTAA-3', was approximately 8 times more effective in induction of the xynF1 gene than the low affinity binding sequence, 5'-GGCTGA-3', which exhibits approximately 10 times lower affinity compared to the higher affinity sequence. Promoter activity of the xynF1 gene was upregulated up to 2.8-fold by mutating both the lower affinity binding sequence and the analogous sequence to the higher affinity binding sequence.
177. A putative G protein-coupled receptor controls growth, germination and coordinated development in Aspergillus nidulans. Kap-Hoon Han, Jeong-Ah Seo and Jaehyuk Yu. Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, WI 53706 USA.
Comprehensive comparative genomic studies on heterotrimeric G-protein signal transduction components lead us to identify seven genes (gprA~gprG) that are predicted to encode putative seven-transmembrane spanning G protein-coupled receptors (GPCRs) in Aspergillus nidulans. Functional characterization of four (gprA~gprD) GPCRs has been carried out and disruption of gprD resulted in extreme phenotypic abnormalities. The gprD gene is predicted to encode a 427 amino acid polypeptide with typical seven transmembrane domains. The gprD null mutant exhibits severely reduced colony growth and highly elevated fruiting body formation, implying that the mutant fails to coordinate balanced growth and development. Moreover, the timing of spore germination of thegprD null mutant is delayed at least two hours, indicating GprD may also play a crucial role in germination. In order to examine whether extreme growth reduction is caused by uncontrolled activation of sexual development, double mutants of gprD deletion and deletion of the genes related to sexual differentiation were generated. Both double deletion mutants of gprD; nsdD and gprD; veA showed no fruiting body formation and restored hyphal growth to near wild-type level. Furthermore, environmental conditions, including poor carbon source and/or high levels of salt, that induce asexual development and block sexual development also caused the recovered growth with the absence of fruiting body in the gprD null mutant. These results clearly suggest that the primary role of GprD is to repress and coordinate sexual development and to confer proper growth during the lifecycle. Further studies to dissect genetic components functioning downstream of GprD are in progress.
178. FacB independent activation of Aspergillus nidulans isocitrate lyase (AcuD) is mediated by propionate. Matthias Brock. Department of Microbiology , University Hannover, Schneiderberg 50, 30167 Hannover, Germany
FacB is known to be the transcriptional activator of the acetate utilisation genes. Therefore, a FacB mutant of Aspergillus nidulans is unable to grow on acetate as sole carbon and energy source. However, isocitrate lyase activity, as a marker enzyme of the glyoxylate cycle, is induced in the presence of propionate, even in a facB negative background. We were able to show that a facB mutant is able to utilise significant amounts of acetate when grown on mixed carbon sources like acetate/propionate medium. Therefore, we postulate the existence of an alternative transcriptional activator, which is specifically induced by propionate. On the other hand, FacB seems to be able to act as a negative transriptional regulator of the propionate metabolising genes. Comparison of enzyme activities of marker enzymes of the glyoxylate and methylcitrate cycle revealed that activity of enzymes of the methylcitrate cycle, responsible for propionate degradation, is always low in the presence of acetate. Interestingly, this regulation is much weaker in a facB negative mutant.
179. Core promoter structure in the oomycete Phytophthora infestans. Adele McLeod, Christine D. Smart, William E. Fry. Cornell University, Ithaca, NY
The core promoter structure of the oomycete Phytophthora infestans was investigated. The transcriptional start sites (tss) of three previously characterized P. infestans genes Piexo1, Piexo3 and Piendo1 were determined by primer extension analyses. Their tss regions were homologous to a previously identified 16nt core sequence that overlaps the transcriptional start site in most oomycete genes. The core promoter regions of Piexo1 and Piendo1 were investigated using a transient expression assay with the promoters of the genes being driven by the beta-glucuronidase reporter gene. Mutational analyses in the promoter of Piexo1 and Piendo1 showed that there is a core promoter element encompassing the transcriptional start site (-2 to +5) that has high sequence and functional homology to a known core promoter element present in other eukaryotes, the initiator element (Inr). Flanking the Inr is a highly conserved oomycete promoter region (+7 to +15), hereafter referred to as FPR (flanking promoter region), which is also important for promoter function. The importance of the Inr and FPR were further investigated in electrophoretic mobility shift assays (EMSA) using P. infestans whole-cell protein extracts, and 31-36 bp double-strand oligonucleotides containing the Inr and FPR. These studies showed that mutations in the Inr (-1 to + 3) and FPR (+9 to +11) affected protein binding negatively, and that these regions were thus important for protein binding. Furthermore, the EMSA studies showed that both the Inr and FPR were required for protein binding. Thus, both protein binding studies and transient expression analyses suggest that the FPR and Inr function cooperatively.
180. Heterokaryon incompatibility associated with the large subunit of ribonucleotide reductase in Neurospora crassa. Carmen Gibbs, Leila Haidari, Cristina Micali and Myron Smith. Biology Department, Carleton University, Ottawa, Ontario, Canada
The un-24 gene of N. crassa encodes the large subunit of a type I ribonucleotide reductase (RNR), an evolutionarily conserved enzyme that is essential for de novo DNA synthesis. UN-24 is remarkable among type I RNRs in having heterokaryon incompatibility function in addition to RNR catalytic activity. To understand how un-24 mediates heterokaryon incompatibility, we are examining incompatibility and catalytic activity of truncated and chimeric un-24 constructs, as well as un-24-derived transcript and protein levels from strains that are self-incompatible due to heteroallelic duplications of un-24. Our results indicate that incompatibility activity is associated with a C-terminal domain unique to N. crassa that also differs between the two allelic forms (un-24PA and un-24OR), but that incompatibility is not dependent on RNR catalytic activity. Surprisingly, transcription of un-24PA, but not un-24OR, is upregulated in haploid strains grown on a low nitrogen medium that promotes the sexual cycle; therefore, localized inactivation of un-24-mediated incompatibility in the perithecium may be through differential transcript regulation of the two allelic forms. Preliminary Western analyses indicate that UN-24 proteins accumulate and form dimer aggregates in self-incompatible un-24OR/PA partial diploids suggesting that un-24-associated incompatibility may be a non-specific response to the presence of protein aggregates rather than a modification of RNR activity.
181. Genetic Regulation of Cellular Copper Homeostasis in Podospora anserina: Impact on Oxidative Stress. Heinz D. Osiewacz. J.W. Goethe University, Botanical Institute, Frankfurt/ Main, Germany
In biological systems, copper is an essential trace metal that is required as a cofactor of different enzymes (e.g., cytochrome oxidase, Cu/Zn superoxide dismutase). However, elevated cellular copper levels are toxic since this metal, like iron, leads to the generation of the highly toxic hydroxyl radical. Moreover, electron transport following the copper-dependent cytochrome oxidase pathway at the inner mitochondrial membrane are known to lead to a time-dependent increase of mitochondrial oxidative stress and to degenerative processes. The dual role of copper makes it essential to tightly control cellular copper levels. InPodospora anserina this is achieved via the copper-regulated transcriptional control of different genes. Among others, these are genes encoding a copper metallothionein (PaMT1) and a high affinity copper transporter (PaCTR3). The expression of the latter is controlled by GRISEA, a transcription factor that becomes inactivated at increased cellular copper levels. The presented data and those from earlier investigations will be discussed and compared to those from other systems demonstrating that basic parts of the machinery involved in the control of copper homeostasis appear to be conserved among organisms. However, others parts differ allowing a flexible adaptation of the corresponding organism to specific ecological niches. The experimental work is supported by grants of the Deutsche Forschungsgemeinschaft (Bonn, Germany)
182. Insertional Mutagenesis Using Agrobacterium tumefaciens T-DNA to Identify Transcriptional Regulators of the Blastomyces dermatitidis BAD1 Promoter. Julie C. Nemecek1, Thomas D. Sullivan2 and Bruce S. Klein1, 2. 3, 4. Departments of 1Medical Microbiology and Immunology, 2Pediatrics, and 3Internal Medicine and the4Comprehensive Cancer Center, University of Wisconsin, Madison, Wisconsin.
The BAD1 gene is a yeast-phase specific virulence factor of the dimorphic, animal pathogen B. dermatitidis. With the eventual goal of delineating the factors necessary for regulating phase transition, we have begun a genetic search for regulators of the yeast-phase expression of BAD1. A reporter strain was generated expressing the E. coli LacZgene from the BAD1 promoter. In this strain beta-galactosidase activity is produced at high levels in yeast cells, resulting in blue colonies in the presence of Xgal. The reporter strain was subjected to transformation using A. tumefaciens T-DNA as an insertional mutagen. Thus far 11,841 primary transformants have been screened by staining with Xgal, and 478 white colonies were picked. Of these, 312 were reproducibly white or light blue in color after long exposure to Xgal. For 39 of 284 tested there was low or undetectable secretion of authentic BAD1 protein, and for 4 of 14 tested there were low levels of extractable beta-galactosidase activity. Promising lines will be further analyzed by Southern blot for the number of sites of T-DNA insertion in the genome. Those with single sites will be selected to use for amplification and determination of the sequence flanking the T-DNA and to confirm that the steady state level of BAD1 transcript is decreased in the presence of the mutation.
183. Functional analysis of the Aspergillus nidulans xprF gene, a Gene Involved in the Response to Starvation. Bernardo, S.M.H., Cheetham, B.F., Katz, M.E. Molecular and Cellular Biology, University of New England, Armidale, NSW, Australia
The Aspergillus nidulans xprF gene appears to be involved in carbon starvation. The gene is unique in that it is involved in both carbon regulation of extracellular protease production and the utilisation of certain nitrogen sources. Sequence analysis shows that the predicted gene product of 615 amino acids residues is a hexokinase-like protein, sharing highly conserved residues within the ATP- and sugar-binding domains of other well-established hexokinases. A number of hexokinases in plants, mammals, and fungi have been shown to play a regulatory role in glucose repression, but no role in carbon starvation has been reported. The xprF1 mutation is a nonsense mutation predicted to result in a truncated protein of 255 amino acid residues. The xprF-delta mutant created in this study exhibited a similar phenotype to the xprF1 mutant in terms of regulation of extracellular proteases and utilisation of nitrogen sources. Similarly, the knockout mutant exhibited partial dominance; this shows that the partial dominance exhibited by the xprF1 mutation is not due to a dominant-negative effect of the truncated protein. Using site-directed mutagenesis, we have shown that a number of unusual, as well as conserved, features of the xprF gene product are important for the maintenance of its functional integrity. Deletion of the unique sequences at the N- and C- termini showed that both domains were required for XprF function. It has also been shown that a number of highly conserved residues in the ATP-binding domains are not required for its regulatory function. Mutations in a putative nuclear localisation domain also affected gene function. The xprF gene has been expressed in E. coli in order to purify the protein and subsequently test for hexokinase activity. Nuclear localisation experiments, using xprF-gfp fusions, are also underway.
184. The role of carbon catabolite repression, carbon starvation and induction by exogenous protein in the regulation of extracellular protease production in Aspergillus nidulans. Margaret E. Katz and Brian F. Cheetham. Molecular and Cellular Biology, University of New England, Armidale, NSW, Australia.
Extracellular protease production was analysed in A. nidulans strains carrying mutations in genes thought to be involved in the response to carbon starvation (xprF and xprG) and genes which play a role in carbon catabolite repression (creA, creB and creC). In wild type strains, extracellular protease levels are low when any carbon source, including protein, is present suggesting that the extracellular protease genes are only expressed at high levels when no carbon source is available. No evidence for induction by exogenous protein in the presence of repressing or non-repressing carbon sources was observed in a wild type A. nidulans strain but protein does appear to affect production in some mutants. Though there was little difference in the levels of extracellular proteases in repressing and non-repressing carbon sources, mutations in cre genes affect production of extracellular proteases, suggesting that these genes may also be involved in the response to carbon starvation. Repression of extracellular proteases by glucose was not affected by deletion of the creA gene. The possibility that the xprF and xprG gene products play a direct role in the response of extracellular protease genes to carbon starvation is being explored by studying subcellular localisation of the xprF gene product in wild type and xprG mutant strains.
185. Two DNA-binding transcription factors involved in fruiting-body formation in the basidiomycete Lentinula edodes and their target genes. Kazuo Shishido1, Yasumasa Miyazaki2, Toru Jojima3, Takeshi Ono1, Yuta Sakuragi 1, and Takashi Yamazaki1.1Tokyo Institute of Technology, Life Science, Yokohama, Japan.2Forestry and Forest Product Research Institute, Appl. Microbiol., Tsukuba, Japan.3Kasetsart University, Bangkok, Thailand.
The two DNA-binding transcription factors, PRIB and Le.CDC5 are involved in fruiting-body formation of L. edodes (Shiitake). PRIB (565 amino acids) contains a Zn(II)2Cys6 zinc-cluster motif followed by bZIP-like motif. The consensus sequence of PRIB binding was determined to be 16 bp 5'GGGGGGGACAGGANCC3'. The upstream region ofpriB gene contained four 16 bp consensus-like sequences to which PRIB actually binds, suggesting the autoregulation of priB. The uck1 (UMP-CMP kinase) gene present downstream of priB was also suggested to be regulated by PRIB. The uck1 gene was found to be most actively transcribed in hymenophore, especially both in the hymenium and in the divergence points of trama cells into subhymenium, of mature fruiting bodies. Genomic binding-site cloning experiment revealed the presence of another PRIB target gene, a homologue (named mfbC) of S. cerevisiae YJL070C gene whose function is unknown. The mfbC and YJL070C genes exhibited a partial homology to that of S. cerevisiae SEN3 protein, a regulatory subunit of the proteasome. The mfbC gene was shown to be actively transcribed in mature fruiting bodies, implying that mfbC may play a role at the final stage of fruiting-body formation. Le.CDC5 (842 amino acids) is the product of Le.cdc5 gene which is a homologue of Sch. pombe cdc5 gene and present adjacent to priB. It contains a Myb-type DNA-binding domain, a putative proline-rich activation domain and a possible leucine zipper. The consensus sequence of Le.CDC5 binding was determined to be 5'GCAATGT3'. The genomic binding-site cloning experiment reseulted in isolation of 3.7 and 3.9 kb HindIII fragments. These two fragments were shown to contain two or more 7 bp consensus-like sequences. For the two fragments, analysis of the gene present just downstream of the 7-bp sequences is in progress.
186. Intron-dependent mRNA accumulation of the Coriolus hirsutus lignin peroxidase gene in C. hirsutus. Takashi Yamazaki1, Yutaka Okajima1, Akira Tsukamoto2, Jun Sugiura2 and, Kazuo Shishido1.1Tokyo Institute of Technology, Life Science, Yokohama, Japan.2Oji Paper Co. Ltd., Adv. Technol. Research Lab., Shinonome, Tokyo, Japan.
We have constructed the chromosome-integrating vector (MIp30) carrying the C. hirsutus glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter -Lentinula edodes priA gene terminator and the selectable marker of C. hirsutus ARG1 gene. The C. hirsutus lignin peroxidase (LiP) cDNA (lipc) was fused between the promoter and terminator of MIp30. The resulting plasmid (MIp30-lipc) was introduced into protoplasts of monokaryotic C. hirsutus arg1 strain, followed by selection of Arg+ Lip+ colonies. Southern-bolt analysis revealed that all of the Arg+ Lip+ transformants possess several copies of the promoter-lipc-terminator expression cassette on their chromosomes. Northern-blot analysis, however, showed that these transformants do not contain so many mRNA molecules as detected by Northern-blot analysis. On the other hand, all of the Arg+Lip+ transformants obtained by introduction of the lip gene (contains 6 small introns) in place of the lip cDNA contained larger amounts of mRNA detectable by Northern-blot analysis. The results strongly suggest that the introns are required for the accumulation of mRNA. To investigate how many introns are necessary for it, two differentlip constructs were prepared: construct-1 consists of the first exon - first intron - the appropriate cDNA sequence; construct-2, the first-third exons and introns - the appropriate cDNA sequence. So far we have analyzed the mRNA accumulation for construct-2, resulted in showing that efficient mRNA accumulation occurs by at least three introns. Analysis for construct-1 is in progress.
187. The genetic regulation of fatty acid metabolism in Aspergillus nidulans. Khew GS, Murray SL, Davis MA, Hynes MJ.Department of Genetics, University of Melbourne, Victoria 3010, Australia.
FacB, in Aspergillus nidulans, is a Zn(II)2Cys6 protein required for acetate induction of enzymes necessary for acetate utilisation. Many of these enzymes are also necessary for the utilisation of short and long chain fatty acids. However, we have found that fatty acid induction of these enzymes is independent of FacB-mediated induction. Nothing is known about the regulatory genes involved in fatty acid induction in filamentous fungi. The acuJ gene encodes a carnitine acetyltransferase essential for shuttling acetyl-CoA between the mitochondria and peroxisomes and is thus required for growth on both acetate and fatty acids. The acuJ gene is acetate and fatty acid induced. A 300bp region of the acuJ 5' UTR is sufficient for fatty acid induction but not acetate induction of a lacZ reporter construct. This region contains a sequence with similarity to the oleate response element (ORE) necessary for fatty acid induction via the Oaf1p and Pip2p activators of Saccharomyces cerevisiae. We are determining whether the element is necessary for fatty acid induction. We have cloned a potential orthologue of the OAF1 and PIP2 genes. Futhermore, we have isolated a number of butyrate non-utilising mutants. Two of the corresponding genes have been cloned. One is an orthologue of the PEX6 gene of S. cerevisiae which is involved in peroxisome biogenesis. The other encodes a Zn(II)2Cys6 activator involved in induction by short chain but not long chain fatty acids.
188. New aspects concerning nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi. Bettina Tudzynski, Martina Mihlan, and Sabine Teichert. Westfälische-Wilhelms-Universität, Schlossgarten 3, 48149 Münster, Germany
The gibberellins (GAs) are a group of phytohormones, which are produced by the rice pathogen G.fujikuroi. After cloning the seven genes involved in GA biosynthesis, we are now interested in detailed analysis of regulation of gene expression. Six of the seven genes are repressed by high amounts of nitrogen, whereas the expression of the P450-3 gene, encoding the last pathway enzyme, is not affected by nitrogen. The expression of GA genes is drastically reduced in areA mutants. In vivo gus reporter gene analysis with theP450-4 promoter showed, that only some of the 10 GATA-motifs are responsible for the AREA-mediated up-regulation. Surprisingly, the replacement of the G. fujikuroi nmr gene (nmr-GF), which is homologous to the N.crassa nmr1 gene encoding the negative acting AREA-binding regulator, NMR1, did neither result in derepression of GA biosynthesis nor in significant derepression of the nitrate reductase. Overexpression of nmr-GF under control of the strong promoter of the G. fujikuroi glutamine synthase gene resulted in slight repression of nitrate reductase but not of GA biosynthesis. Interestingly, the G. fujikuroi NMR is able to complement nmr1/nmrA mutants of N.crassa and A.nidulans, respectively, despite the neglectible function in G. fujikuroi. Furthermore, we want to identify the components of nitrogen sensing and signalling pathways involved in nitrogen metabolite repression. Thus we study the role of ammonium permeases and the G. fujikuroi TOR protein on one hand and glutamine synthase and other enzymes involved in biosynthesis of glutamine and glutamate on the other hand, in nitrogen-mediated repression of GA biosynthsis pathway.
189. Chromatin remodelling of the alcA promoter region of the ethanol utilization pathway in Aspergillus nidulans requires both a specific activator AlcR and a global repressor CreA. Igor Nikolaev1, Martine Mathieu, and Béatrice Felenbok. Institut de Genetique et Microbiologie, Universite Paris-Sud, UMR 8621 CNRS, Centre d'Orsay, Orsay 91405, France1 Present address: Danisco Innovation, Langebrogade 1, DK 1001 Copenhagen, Denmark
The ethanol utilization pathway in A. nidulans is one of the strongest inducible expression system among filamentous fungi. The alcR-alcA system is widely used at both fundamental and applied levels. It is strictly transcriptionally regulated both by specific induction mediated by the zinc binuclear cluster activator AlcR and carbon catabolite repression operating via the general repressor CreA. Molecular mechanisms whereby induction and repression are set up and interact have been studied in great detail. In order to determine if the level of transcription is correlated to the structural organization of the chromatin, we investigated nucleosomal rearrangements in the alcA promoter under different growth conditions. We utilized a range of alcR and creAmutants isolated either by classical or reverse genetic methods. In the absence of the inducer and under repressed growth conditions, nucleosomes are positioned within the alcA promoter. Under induced conditions, total depositionning occurs. In fact, AlcR binding to its DNA cognate targets upon induction is essential for removal of one nucleosome placed upstream of the closely linked direct and inverted repeat sites. To remove others, the intact activation domain of AlcR is required. For reseting the original nucleosome pattern observed upon repression and maintenance of a "chromatin-closed" state of the alcA promoter under non-induced conditions, both the DNA binding domain of CreA and its C-terminal region with a local homology to the effector domain of Mig1 from Saccharomyces cerevisiae, are necessary. Interestingly, we found that AlcR predominates over CreA for nucleosomal rearrangements. The interplay between the activator AlcR and the repressor CreA occuring at the transcriptional level corresponds to global chromatin remodeling in which both regulators are involved.
190. The nicotinic acid utilisation cluster of Aspergillus nidulans. Fernandez-Martin R., Cultrone A and Scazzocchio C. IGM Universite Paris-sud Bat. 409. 91405 Orsay France
The hxnS gene of Aspergillus nidulans codes for a rather extraordinary enzyme called Purine hydroxylase II (PHII). More than 20 years ago it was shown that it is a Molybdoflavoprotein, related to xanthine dehydrogenase, able to accept hypoxanthine but not xanthine as substrate. This enzyme is induced by nicotinate (actually by a product of its oxidation) and it has a low but physiologically significant nicotinate hydroxylase activity. The hxnS gene and the cluster where it maps, has been cloned. Surprisingly, all the amino acids thought to be involved in substrate recognition in xanthine dehydrogenase are conserved in PHII. At least 5 ORFs are induced by nicotinate. Regulatory mutations, both constitutive and non-inducible, map in a single gene at the cluster, hxnR, encoding a 2Cys2His Zn finger transcription factor. One ORF, hxnP, is highly similar to the characterised nicotinate transporter of S. cerevisiae. Other two ORFs, with similarities with characterised oxydo-reduction enzymes, are probably involved in furthers steps at nicotinate utilization as nitrogen source. All these ORFs are under the control of hxnR. This work has been funding by XONet European Project HPRN-CT-1999-00084
191. Analysis of yps-3 expression in Histoplasma capsulatum. Parul Trivedi, 1 Thanh T. Hoang, 1 George S. Deepe, Jr.,2 and
Jon P. Woods 1. University of Wisconsin Medical School, 1 University of Cincinnati College of Medicine 2
The yps-3 gene is expressed exclusively by the pathogenic yeast morphotype of the dimorphic fungus Histoplasma capsulatum, and encodes a protein found in the cell wall and culture supernatant during in vitro growth. We used transcript (Northern blotting, kinetic PCR) and protein (Western immunoblotting, flow cytometry) detection techniques to evaluate expression during infection. yps-3 transcript was upregulated 3-4 fold during intracellular infection of activated RAW264.7 macrophages, and 10-13 fold in mouse lung and 47-50 fold in mouse spleen after respiratory infection. Protein upregulation was also detected during activated macrophage infection. We used flow cytometry to demonstrate surface exposure of this protein, which differed considerably among different strains. This interstrain variation was confirmed by Western immunoblotting. Although the function of yps-3 is not known, its pathogenic yeast phase-specific expression and variable expression in strains differing in virulence have been proposed as consistent with a role in pathogenesis. Our results further support an adaptive role for yps-3 in the infection environment and furthermore suggest additional regulation beyond that associated with morphogenesis.
192. Overlapping Sense and Antisense Transcripts at the Ran1 Locus in Histoplasma capsulatum. Julia Z. Ng, Diane M. Retallack, and Jon P. Woods. University of Wisconsin-Madison
Histoplasma capsulatum (Hc) is a thermally dimorphic fungal pathogen that is the causative agent of the respiratory and systemic disease histoplasmosis. This organism resides and replicates in mammalian host phagolysosomes. In vivo expression technology (IVET) was performed to trap promoters that were upregulated during infection of mice and RAW 264.7 macrophages. One IVET-isolated promoter upregulated expression of a small non-polyadenylated transcript during infection of RAW264.7 macrophages. On the overlapping reverse-orientation strand, a 2.6kb transcript was expressed at equal levels during growth in vitro and infection of RAW264.7 macrophages. This polyadenylated transcript contains an open reading frame that encodes a putative protein with sequence similarity to Schizosaccharomyces pombe Ran1. S. pombe Ran1 is a serine-threonine kinase which is a negative regulator of meiosis. Episomal expression of H. capsulatum Ran1 functionally complemented a S. pombe temperature-sensitive ran1 null mutant. We are currently testing H. capsulatum Ran1 mutated genes for function in the S.pombe complementation assay. In addition, we are examining Hc Ran1 expression under various environmental conditions, and a possible role for Ran1 in virulence.
193. Differential regulation of the Histoplasma capsulatum chitin synthase G gene. Clayton H. Johnson, Nori Watson, and Joan E. McEwen. J.L. McClellan VA Hospital and University of Arkansas for Medical Science, Dept. of Geriatrics and Dept. of Microbiology and Immunology, 4300 W. 7th St. (VAMC151/LR), Little Rock, AR 72207
Histoplasma capsulatum, a mammalian fungal pathogen, is exposed to oxidants produced by neutrophils and activated macrophages of the host. While screening an H. capsulatum cDNA library for oxidative stress inducible genes, we isolated a truncated clone coding for H. capsulatum chitin synthase G (HcchsG) enzyme. Dose-dependent oxidative stress inducibility of the HcchsG gene was confirmed by northern blot analysis of RNA isolated from yeast cells exposed to hydrogen peroxide. Additional studies of HcchsG gene expression indicated that abundance of HcchsG mRNA is also regulated by carbon source (glycerol vs. glucose) and yeast vs. mycelial growth conditions. HcchsG mRNA abundance was highest in yeast grown on glycerol and lowest in mycelia, regardless of carbon source. In addition to the truncated cDNA clone, we isolated a genomic clone containing the entireHcchsG gene. The HcchsG gene codes for a protein with a predicted mass of 101,300 Daltons and isoelectric point of 8.85. A novel finding about the HcchsG gene is the presence of an intron that is significantly larger and/or missing from other fungal chsG genes. Reinforcement of the fungal cell wall, by increasing the content of chitin, may confer resistance to host-mediated defenses. Ongoing experiments, in an attempt to test this hypothesis, include the construction of recombinant expression systems and plasmid constructs for creating HcchsG over-producing and knock-out strains, respectively.
194. The two Neurospora Dicer proteins are required for "quelling" and development. Catalanotto C, Pallotta M, Braccini L and Cogoni C. Dept of Biology, Universita' La Sapienza, Rome, Italy.
In animals, the double stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate the expression of endogenous protein-coding genes at the level of mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. In the fungus Neurospora crassa, trangenes trigger a post-transcriptional gene silencing phenomenon called quelling that has been shown to be mechanistically similar to RNAi. Two genes encoding Dicer–like proteins are present in Neurospora crassa genome. In this work we show that a double stranded RNA (dsRNA) RNaseIII-like processing activity is present in Neurospora extracts. This activity is ATP-dependent and does not require the presence of transgenes or functional qde (quelling defective) genes. Moreover, we show that a dicer1 and dicer2 double knock-out mutant is defective in transgene-induced gene silencing, suggesting that in this fungus the function of Dicer in quelling is redundant. The involvement of Dicer proteins in quelling also supports the notion that common genetic mechanisms exist between RNAi and transgene-induced gene silencing. Additional observations suggest that both DICER1 and DICER2 are required for the formation of female sexual structures. This result suggests that the Dicer activity is essential for miRNA maturation in fungi like in animals and plants.
195. Comparative sequencing of the qa-1S gene in Neurospora africana and Neurospora crassa. Diana R. Arnett1 and David K. Asch1,2. 1School of Biomedical Sciences, Kent State University, Kent, Ohio and 2Department of Biological Sciences, Youngstown State University, Youngstown, Ohio.
While the quinic acid (qa) gene cluster of Neurospora crassa has been studied fairly extensively, very little is known
about the qa gene cluster of Neurospora africana. One study attempted to determine the degree of conservation
between several different species of Neurospora, culminating in sequencing qa-x-qa-2 intergenic region of N.
africana (Asch, et al 1991). It was also discovered that N. africana and N. crassa demonstrate significantly different
restriction fragment length polymorphisms (RFLPs) in this region. Because of the usefulness of the qa cluster in
studying transcription regulation and carbon catabolite repression, sequencing the qa genes and regulatory regions in
other species of Neurospora is important in studying their regulation. We have sequenced the qa-1S gene of N.
africana and compared it to that of N. crassa and to the qutR gene ofAspergillus nidulans (teleomorph, Emericella
Asch, D.K., Orejas, M., Geever, R.F., and Case, M.E. (1991) Comparative studies of the quinic acid cluster in several Neurospora species with special emphasis on the qa-x-qa-2 intergenic region. Mol. Gen. Genet. 230: 337-344.
196. The Aspergillus nidulans ambient pH signal transduction pathway protein PalI is a plasma membrane protein that is transported to vacuoles in response to alkaline pH. Kimberly Schoenly, Matthew Warrick and Steven H. Denison. Eckerd College, Natural Sciences, St. Petersburg, FL, USA.
Regulation of gene expression by ambient (extracellular) pH in Aspergillus nidulansis mediated by a signalling pathway composed of the products of the palA, B, C, F, H andI genes and the PacC zinc-finger transcription factor. This pH regulatory system ensures that extracellular enzymes (as well as permeases and exported metabolites) are produced under conditions of pH where they can function: acid phosphatase under acid conditions and alkaline phosphatase under alkaline conditions, for example. It has previously been shown that the predicted palI protein contains four putative membrane-spanning domains. This suggests the possibility that PalI is located in the plasma membrane and may be the first protein in the pH signalling pathway, functioning as a pH-sensing protein. We have constructed a strain ofAspergillus nidulans that expresses a PalI-Green Fluorescent Protein (GFP) fusion protein under control of the ethanol-inducible alcA promoter. Using this strain we show that PalI has a plasma membrane location, strengthening the possibility that PalI is the pH-sensor protein in the pH signal transduction pathway. When cells are transferred from acidic to alkaline medium, the PalI-GFP fusion protein appears in vacuoles within five minutes. Endocytosis of PalI may therefore be involved in pH signalling.
197. Effects of metals on the expression of the laccase gene (Cs-lcs1) in the ligninolytic fungus Ceriporiopsis subvermispora. Luis F. Larrondo1*, Rubén Polanco1#, Marcela Ávila1, Augusto Manubens1&, Paulo Canessa1, Loreto Salas1 and Rafael Vicuña1. 1Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile and Millenium Institute for Fundamental and Applied Biology (MIFAB).
C. subvermispora is a white rot fungus which is highly selective in the degradation of lignin when growing on wood. Its ligninolytic system is composed by manganese peroxidase and laccase. These activities are induced by Mn2+ and Cu2+, respectively. Other metals such as Cd2+, Zn2+ and Ag+ do not increase either of these enzymatic activities. However, Northern blot analysis showed that transcript levels of the gene coding for laccase (Cs-lcs1) are specifically increased in the presence of Cu2+, Ag+ and Cd2+, but not by addition of Zn2+. The promoter region of Cs-lcs1 contains putative ACE and MRE-like sequences, which have been described in other eukaryotes as recognition sites for transcription factors activated by metals. Electromobility shift assays (EMSA) revealed that nuclear proteins from this fungus bind to portions of the Cs-lcs1 promoter which contain the aforementioned sequences. This DNA-protein interaction is specific and it seems to depend on the day of culture of the organism and on the concentration of Mn2+ in the culture medium. This interaction is improved when a pulse of Cu2+ is given 24 h. before harvesting. In addition, using the Phanerochaete chrysosporium genome database, we have identified a putative ACE1 like gene in this organism and the corresponding homologue inC. subvermispora. The transcriptional effect induced by Cu2+ and Ag+ over Cs-lcs1, the presence of a putative ACE sequence in its promoter and the characterization of a putative ACE-1 like gene in C. subvermispora suggest the existence of a regulatory mechanism similar to the one described in yeast and it would constitute the first one reported in a basidiomycete. Financed by grants 8990004, 2000076 and 2000088 from FONDECYT and the MIFAB*Predoctoral Fellow from Fundacion Andes. #PreDoctoral Fellow from MIFAB. &Predoctoral Fellow from CONICYT.
198. Further characterization of the PMK1 MAP kinase pathway in Magnaporthe grisea. C. Xue, X. Zhao, L. Li, Y. Kim, G. Park, J. Xu. Purdue Univ. West Lafayette, IN 47907
The PMK1 MAP kinase, a homolog of yeast Fus3/Kss1, is known to be essential for appressorium formation and plant infection in M. grisea. In this study we functionally characterized several candidate components of the Pmk1 pathway. Because mst7 and mst11 mutants failed to form any appressorium, Mst7 and Mst11 are likely to be the upstream MAPKK and MAPKKK activating Pmk1. Phenotypically, mst7 mutants were similar to that of pmk1 mutants, but mst11 had additional defects in conidiation and hyphae growth, indicating that MST11 may have additional downstream targets. Surprisingly, gene replacements of PAK kinase genes MST20 and CHM1 were not defective in appressorium formation. The mst20 mutants had no obvious defect in vegetative growth and plant infection. It is likely that the Pmk1 MAPK cascade is not activated by PAK kinases in M. grisea. We also identified two putative homologs of yeast pheromone receptors Ste2 and Ste3 in the M. grisea genome (MST2 andMST3). Primary data indicated that mst2 and mst3 mutants were normal in appressorium formation and plant infection and female fertile. In addition, we are in the process of screening for knockout mutants of yeast Ste5 and Ste50 homologs. Overall, our data indicate that the signal input and output of the PMK1 pathway in M. grisea is different from that of the yeast Fus3/Kss1 pathways. Identifying other components of the Pmk1 pathway is under the way.
199. Meiotic Silencing by Unpaired DNA (MSUD). Patrick K.T. Shiu1, Namboori B. Raju2, Denise Zickler3, and Robert L. Metzenberg1. 1Chem and Biochem, UCLA, CA. 2Biol Sci, Stanford University, CA. 3Institut de Genetique et Microbiologie, Universite Paris-Sud, Orsay, France.
A gene unpaired with a homolog in prophase I generates a signal that transiently silences all sequences homologous to it by a process called Meiotic Silencing by Unpaired DNA (MSUD; Cell 107:905-916). A mutant called Sad-1 (Suppressor of ascus dominance) fails to perform MSUD. Sad-1 also suppresses several classical ascus-dominant mutants, suggesting that these, too, owe their ascus dominance to the MSUD mechanism. MSUD is not restricted to a few ascus-dominant genes, but is applicable to virtually the entire genome. This can be shown by the fact that a variety of genes can be meiotically silenced if they are unpaired during meiotic prophase. The sad-1 gene encodes an RNA-directed RNA polymerase (RdRP). RdRP has been implicated in many post-transcriptional gene silencing systems, such as co-suppression in plants, RNA interference in animals, and quelling in fungi. Owing to its ability to compare the genomes of two mating partners, MSUD has implications not only for surveillance against invading sequences but also for reproductive behavior. For example, interspecific crosses within the genus Neurospora that are normally almost completely infertile become much more fertile if the N. crassa parent carries the Sad-1 dominant mutation. This suggests that MSUD triggered by numerous small mispairings could play a role in reproductive isolation of these species.
200. Protein secretion stress in Aspergillus niger. Hashem Al-Sheikh, David Jeenes1, Adrian Watson, Marcos Alcocer, Peter Punt2 and David Archer. School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK. 1Institute of Food Research, Norwich Research Park, Norwich NR4 7UA. 2TNO Nutrition and Food Research, PO Box 360, 3700 AJ Zeist, The Netherlands.
Aspergillus niger secretes many native proteins at high yields but the yields of most heterologous proteins are comparatively low. We have investigated the bottlenecks in protein secretion and one major bottleneck is the folding of proteins within the lumen of the endoplasmic reticulum (ER). Cells sense the presence of unfolded proteins in the lumen of the ER and the cells then respond in ways that are generally conserved within the eukaryotes. The unfolded protein response (UPR) is well described and leads to the transcriptional up-regulation of genes encoding ER-resident foldases and chaperones. The UPR also stimulates ER-associated protein degradation (ERAD) and, in mammalian cells but not shown in fungi, attenuates translation. We have reported a further response to unfolded proteins in the ER that leads to the selective transcriptional down-regulation of genes encoding secreted proteins (but not genes encoding non-secreted proteins). We report here our studies to elucidate the mechanism of this ER stress response which does not appear to be part of the UPR. This work was funded by the EC Eurofung Programme, by the Saudi Government and by the BBSRC
201. New insights into pH regulation of aflatoxin production by Aspergillus species. Kenneth C. Ehrlich and Peter J. Cotty, Southern Regional Research Service, ARS, USDA, New Orleans LA 70179-0687
Aflatoxins (AF) are toxic and carcinogenic metabolites of Aspergillus species. Previous studies found AF production to be pH dependent and, in some cases, pH inhibition and stimulation of AF production was found to override nutritional influences on aflatoxin accumulation. Differential regulation of AF production in ammonia-based media was previously found between West African (SBG) and North American (SB) strains. We now show that this differential effect results from inhibition of toxin production by the former isolates at pH 2.5, conditions in which SB isolates produced high levels of toxin. Like A. parasiticus , SBG isolates produce both B and G aflatoxins. Aflatoxin G production varied more with pH than did aflatoxin B production. At pH 3.5 the AFB1/AFG1 ratio was 3.0, whereas at pH 5.5 the ratio was 0.5. Although A. parasiticus isolates produced high levels of AF at both pH 2.5 and 5.5, the AFB1/AFG1 ratio was similarly influenced by pH. Quantitative PCR was used to examine the influence of pH on expression of six genes involved in AF biosynthesis, including two genes (fasA and pksA) for the initial steps in biosynthesis, the transcriptional regulator gene (aflR), and three genes for the final biosynthetic steps (omt1, ordA, and ordB). For the SBG isolate, expression of most examined genes was inversely proportional to the pH affect on AF accumulation. However, reductions of omt1 expression were paralleled by reductions in aflatoxin production at low pH. However, for the other isolates, pH effects on gene expression did not correlate with influences on AF accumulation. These results suggest that expression of omt1 may explain a portion of the influence of low pH on aflatoxin production by SBG isolates, but that most influences of pH do not occur at the level of transcription.
202. Isolation and characterization of a new gene, pco-1, which encodes a regulatory protein that controls purine degradation in Neurospora crassa. Ta-Wei D. Liu, and George A. Marzluf, Ohio State University, Columbus, Ohio
A feature of the nitrogen regulatory circuit in filamentous fungi is that pathway-specific control genes mediate induction of enzymes by substrates in specific pathways. The gene encoding a new pathway-specific factor involving in purine degradation pathway - pco-1 was isolated from Neurospora using a PCR-mediated method. The open reading frame of the new factor gene is interrupted by two introns which were identified by comparing the genomic DNA sequence and the cDNA sequence obtained by RT-PCR. The predicted PCO1 protein contains 1101 amino acids and appears to possess a single Zn(II)2/Cys6 binuclear-type zinc cluster. A coiled-coil domain was predicted by computer-aided sequence analysis, suggesting that PCO1 might function as a dimer. A chemical crosslinking assay indicated PCO1 does dimerize in vitro. A loss of function pco-1 mutant was created by the rip procedure. Analysis of pco1- strains revealed that PCO1 acts as a positive regulator of the purine degradation pathway. Results of mobility shift assays indicate that PCO1 specifically binds to TCGG-N6-CCGA DNA sequences which exist in promoter regions of the structural genes it regulates. The C-terminus of PCO1 features a domain rich in glutamine, proline, isoleucine and acidic residues which are commonly found in activation domains of transcription factors. This domain shows higher homology to NIT4, the Neurospora pathway-specific factor in the nitrate assimilation pathway, than to UAY, its counterpart in Aspergillus nidulans, suggesting transcription factors in N. crassa may share similar activation regions.
203. Dominant active Rac and dominant negative Rac restore the wild type phenotype of dominant active Ras mutant in Colletotrichum trifolii: Involvement with MAP kinase activation and ROS generation. Changbin Chen and Martin B. Dickman, University of Nebraska-Lincoln,U.S.A.
In the filamentous phytopathogenic fungus Colletotrichum trifolii, Ras (Ct-Ras) is being studied. Dominant active Ct-Ras(Val2) results in aberrant hyphal morphology, loss of polarity, and inability to differentiate, only under conditions of nutrient deprivation, suggesting the important role of Ras in hyphal growth and development. However, the underlying mechanisms for this response and the relevant pathways regulated by Ras are unclear. This study reports the isolation and molecular characterization of a Rac GTPase gene from C. trifolii. Yeast two-hybrid assays and in vitro binding assays indicated that Ct-Rac is a downstream target of Ct-Ras. Expression of dominant active Rac resulted in abnormal hyphal growth and reduced sporulation rate, and expression of dominant negative Rac completely inhibited hyphal growth, suggesting that Rac is important for proper hyphal growth and development. Interestingly, co-expression of dominant active Rac or dominant negative Rac in dominant active Ct-Ras background, both lead to restoration of wild type phenotype. Inhibitor studies and Western analyses demonstrated that MAP kinase activity contributes to phenotypic restoration when dominant active Rac is introduced into the activated Ras strain. In contrast, our results suggest that the decrease of intracellular ROS levels followed by the expression of dominant negative Rac, is necessary for phenotypic restoration of the Ras mutant. Rac appears to mediate two distinct signaling pathways in the regulation of hyphal growth and development inC. trifolii; stimulation of MAP kinase and intracellular ROS generation. Our data support a model by which the small GTP-binding protein Rac plays a central role in Ras signaling.
204. The pH- and ROS-regulated MAP kinase signal transduction pathway in Sclerotinia sclerotiorum. Changbin Chen and Martin B. Dickman. University of Nebraska-Lincoln, U.S.A.
Sclerotinia sclerotiorum is a ubiquitous phytopathogenic fungus able to infect an extremely wide range of plants. The survival of this fungus is mediated through the sclerotium, a pigmented, multihyphal structure. Molecular mechanisms that trigger and coordinate sclerotial morphogenesis are not well understood. Inhibitor studies suggested involvement of an ERK-like MAP kinase in sclerotial morphogenesis. Therefore we cloned and characterized the ERK-like MAPK homolog in S. sclerotiorum. Northern analyses showed that SSMAPK-1was highly expressed in low pH but inhibited in high pH conditions. The application of exogenous oxalic acid, a virulence factor in this fungus, induced rapid accumulation ofSSMAPK-1 transcripts, suggesting that SSMAPK-1 expression in S. sclerotiorum is pH-regulated. Moreover, high concentrations of cAMP, which we previously have shown, inhibits sclerotial formation, also suppresses SSMAPK-1 expression, in a PKA-independent manner. Preliminary experiments using specific toxin inhibitors suggest that Ras or Rap-1 mediates cAMP-induced MAP kinase inhibition. Constitutive overexpression ofSSMAPK-1 bypassed the suppressive effect of cAMP and induced sclerotial development. Similar to ERK inhibitor (PD98059) experiments, expression of antisenseSSMAPK-1 inhibited sclerotial maturation. We further show the involvement of reactive oxygen species (ROS) generation in sclerotial development. The application of exogenous H202 induced SSMAPK-1 expression and high concentrations of antioxidants such as NAC and DPI inhibited sclerotial maturation andSSMAPK-1 expression, suggesting ROS-dependent induction of the MAP kinase pathway possibly accounts for sclerotial morphogenesis. Thus, our findings demonstrate thatSSMAPK-1 is pH- and ROS- regulated and is required for sclerotial development.
205. The G-protein beta subunit is required for appressorium formation and conidiation in Magnaporthe grisea. Marie Nishimura1 and Jin-Rong Xu2. 1National Institute of Agrobiological Sciences, Tsukuba, Japan. 2Purdue University, West Lafayette, IN, USA.
Heterotrimeric G-proteins transmit extracellular signals to various downstream effectors in eukaryote. In the rice blast fungus Magnaporthe grisea, appressorium formation is induced by surface hydrophobicity or chemical inducers such as cAMP. Pmk1, a MAP kinase homologous to yeast Fus3/Kss1, is known to regulate appressorium formation and infectious hyphal growth. Because gene disruption mutants of three G-protein alpha subunits exhibit phenotypes different from that of pmk1 mutants, in this study we isolated and characterized the function of the G-protein beta subunit gene (MGB1) in M. grisea. MGB1 is a single copy gene in M. grisea and it is highly homologous to the G-beta subunits identified in Cryphonectria parasitica and other filamentous fungi. Gene disrupted mutant of MGB1 showed abundant mycelial growth, but was reduced in conidiation. Conidia from mgb1 mutants were defective in appressorium formation on hydrophobic surface and failed to form lesions on rice plants. With the presence of exogenous cAMP, mgb1mutants formed abnormally-shaped appressoria on hydrophobic or hydrophilic surfaces. However, these appressoria induced by cAMP were not functional and failed to penetrate and develop infectious hyphae in plant cells. In mycelia and spores harvested from one week old oatmeal cultures, the cellular cAMP concentration in mgb1 mutants was reduced to 60% of the wild type. Interestingly, conidia collected from 7-10 day old cultures of transformants carrying multi-copy of MGB1 (>2 copies) could form appressoria on hydrophilic surface (non-inductive condition). Cellular cAMP concentrations in these transformants were 60% higher than that of the wild type strain Guy11. These data suggest that MGB1 is involved in regulating appressorium formation, plant infection, and conidiation, likely by controlling the adenylate cyclase activity.
206. Activation of chsC transcription by AbaA during asexual development of Aspergillus nidulans. Bum-Chan Park, Yun-Hee Park and Hee-Moon Park. Department of Microbiology, College of Natural Sciences, Chungnam National University, Daejeon 305-764, Korea
The temporal and spatial regulation of chitin synthesis plays an important role in morphogenesis during fungal growth and development. Northern blot analysis showed that the transcription level of chsA, chsC, and chsD was significantly decreased in an Aspergillus nidulans abaA mutant. Electrophoretic mobility shift assays revealed that AbaA bound tightly to all three AREs (AbaA response elements) in the chsC promoter region. Experiments with the Saccharomyces cerevisiae heterologous expression system confirmed AbaA-dependent transcriptional activation of chsC. Taken together, these data suggest that AbaA plays an important role in chitin biosynthesis during conidiophore development by controlling the transcription level of certain chitin synthase genes. (This work was supported by a grant form KOSEF, Project No. R01-1998-000-00053-2001.)
207. Pka, Ras and RGS Protein Interactions Regulate Sterigmatocystin Biosynthesis inAspergillus nidulans. Kiminori Shimizu1, Julie K. Hicks2,3, Tzu-Pi Huang and Nancy P. Keller. Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706 and 2Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843. Present address: 1Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba 260-8673, Japan,3Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710
Sterigmatocystin (ST) is a carcinogenic polyketide produced by several filamentous fungi including Aspergillus nidulans. Expression of ST biosynthetic genes (stc genes) requires activity of a Zn(II)2Cys6 transcription factor, AflR. aflR is transcriptionally and post-transcriptionally regulated by a G-protein/cAMP/protein kinase A (PkaA) signaling pathway involving FlbA, a RGS (Regulator of G-protein Signaling) protein. Prior genetic data showed that FlbA transcriptional regulation of aflR was PkaA dependent. Here we show that mutation of three PkaA phosphorylation sites in AflR allows resumption of stcexpression in an overexpression pkaA background but does not remediate stc expression in a DflbA background. This demonstrates negative regulation of AflR activity by phosphorylation and shows that FlbA post-transcriptional regulation of aflR is PkaA independent. AflR nucleocytoplasmic location further supports PkaA independent regulation of AflR by FlbA. GFP tagged AflR is localized to the cytoplasm when pkaA is overexpressed but nuclearly located in a DflbA background. Genetic interaction between AflR and RasA is also investigated.
208. Comparative promotor analysis of four polygalacturonase genes of Fusarium oxysporum f.sp. radicis lycopersici in Saccharomyces cerevisiae. González-Jaén, M.T., A. de las Heras, B. Patiño, M.J. Bueno, C. Vázquez. Department of Genetics, Univ. Complutense of Madrid, Spain.
Fusarium oxysporum f.sp. radicis lycopersici causes crown and root rot disease in tomato plants, a disease characterized by extensive cell wall degradation. Four polygalacturonase (PG) coding genes have been identified so far which corresponded to two EXOPG and two ENDOPG coding genes regulated at transcriptional level in response to different metabolic inductors in in vitro cultures. The differences observed in their patterns of gene expression suggested the presence of some differences in the regulatory motifs present in their promotor regions. We report the putative regulatory motifs identified in the sequence of the promotor region of the four pg genes and their analysis in a yeast system in response to different conditions related with carbon source and pH. The results support the differential regulation of those genes and the significance of regulation in infection and symptom development. The system of promotor analysis used in this work offers a high potential for the analysis of other Fusarium genes.
209. Polyketide synthases and non-ribosomal peptide synthetases in Neurospora crassa. Nabil Arrach, Scott Kroken and Louise Glass, Department of Plant and Microbial Biology, University of California, Berkeley
The type I polyketides (PKs) and non-ribosomal peptides (NRPs) constitute one of the most diverse groups of natural products, and are common in bacteria and in fungi. The characterized metabolites often function as toxins against hosts and competitors, targeting various aspects of metabolism. In ascomycete fungi, most PKs and NRPs have been characterized from plant pathogens, in which they often serve as virulence factors needed for pathogenicity against their host plants, and from ecologically competitive saprobes such as Aspergillus flavus, with which they fend their food supplies against invaders. Surprisingly, Neurospora crassa contains at the least 7 PK synthases and 3 NRP synthetases, even though it is not known to make any secondary metabolites which function as toxins. In the eucaryotic model Dictyostelium, a diffusible signal molecule called DIF-1 induces the differentiation of prestalk-O cells. DIF-1 is a chlorinated alkyl phenone that is synthesized from a C12 polyketide precursor (Thompson CR and Kay RR., 2000). In bacteria, some compounds that are involved in toxins in higher concentration may act as a signalling compounds in low concentration. For example, it has recently been shown that aerial hyphae formation appears to be especially sensitive to inhibition by protein kinase inhibitors in Streptomyces (Waters et al., 2002). Our hypothesis is that polyketides and non-ribosomal peptides in Neurospora could play a role in a developmental aspect such as conidiation, hyphal fusion or mating.
210. Effect of G-protein signalling in Fusarium culmorum. Jakob Skov and Henriette Giese. Section of Genetics and Microbiology, Department of Ecology The Royal Veterinary and Agricultural University Thorvaldsensvej 40, DK-1871 Copenhagen, Denmark
In filamentous fungi G-proteins are involved in regulation of a wide range of processes including development, pathogenicity and secondary metabolism. A G-protein alfa-subunit gene,fadA, was previously isolated from Aspergillus nidulans. The protein has been shown to affect secondary metabolism in A. nidulans as well as in the plant pathogen Fusarium sporotrichioides (Tag et al. 2000, Mol. Microbiol.38(3):658-665). F. culmorum is the predominant Fusarium species in Danish soils and is believed to be the major cause of Fusarium Head Blight of cereals in Denmark. F. culmorum is known to produce an array of mycotoxins. In this study F. culmorum was transformed with a construct containing a constitutively signalling allele of fadA using the Agrobacterium-mediated transformation system. Integration was tested by Southern analysis and expression of the transgene was analysed using RT-PCR. The effect of the gene on secondary metabolism was examined using HPLC. To detect yet unknown downstream targets of the G-protein signalling pathway the protein profile of transformants was compared to wildtype using 2D electrophoresis.
211. Characterization of a mutant defective in the Neurospora ncMMS2 gene, a homologue of Saccharomyces cerevisiae MMS2. Tsuyoshi Kawabata, Keiichiro Suzuki, Hirokazu Inoue. Laboratory of Genetics, Department of Regulation Biology, Faculty of Science, Saitama University, Saitama City 338-8570, Japan.
MMS2 and UBC13 of S. cerevisiae code a ubiquitin conjugating enzyme variants (UEVs). Products of these two genes form a heterodimer and act in post replication repair (PRR). The heterodimer functions in error-free PRR. Mutants of these genes showed higher sensitivity to UV and MMS than the wild type. We have made a mutant of ncMMS2 gene in Neurospora crassa and investigated its role in DNA repair. The mutant was sensitive to MMS like yeast mms2, however, not sensitive to UV. The ncMMS2 cDNA sequence was determined and deduced aminoacid sequence was compared with those of yeast and human. We made double mutants ofncMMS2 and other DNA repair genes, in order to analyze epistatic relationship. Mutation frequency at the ncMMS2 mutant are also presented.
212. ACEI is a repressor of cellulase and xylanase genes in Trichoderma reesei. Aro, N., Ilmén, M., Saloheimo, A., Penttilä, M. Biotechnology. VTT. Espoo, Finland
T. reesei is an efficient producer of cellulolytic and xylanolytic enzymes. In general, cellulose and its derivatives and different xylans induce cellulase and xylanase expression while glucose represses cellulase and xylanase genes. The production of these enzymes is regulated at the transcriptional level at least by the glucose repressor CREI and the activator ACEII. We have isolated a third factor, ACEI, that binds to and activates the main cellulase promoter cbh1in vivo in yeast. ACEI is a Cys2-His2 type of a transcription factor that binds in vitro to eight sites scattered along the cbh1 promoter containing the core 5'AGGCA sequence. Although originally isolated as a protein capable of activating thecbh1 promoter of T. reesei in S. cerevisiae, further studies oface1 deletion strain indicated that ACEI is a repressor of cellulase and xylanase genes. The deletion of the ace1 gene led up to 10 times higher expression of cellulase genes, cbh1, cbh2, egl1, and egl2 after the transfer of glycerol grown mycelia to cellulose media or when the cellulase genes were induced by the addition of a disaccharide sophorose into glycerol media. Similarly, the expression of xyn1 and xyn2 was increased by ace1 deletion. Deletion of the ace2 gene, encoding the ACEII activator, in a strain deleted for ace1 did not affect the high level of cellulase expression seen in the ace1 deletion strain indicating, that there is at least one additional cellulase and xylanase activator present in T. reesei.
213. Sugar sensing and regulation of carbon transport and physiology in Neurospora. Xin Xie, Alejandro Correa, Zach Lewis, Deborah Bell-Pedersen and Daniel J. Ebbole Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA
Sensing of carbon sources in the environment is important for appropriate regulation of gene expression for carbon utilization and as a behavioral cue. In Saccharomyces cerevisiae, several signal transduction pathways have been demonstrated in glucose sensing and gene regulation. Two glucose transporter homologs, Snf3 and Rgt2, play important roles in the sensing mechanism. An apparent counterpart in Neurospora is rco-3. rco-3 mutants have altered regulation of sugar uptake, carbon catabolite repression, and conidiation. A second Neurospora gene, dgr-1, displays a phenotype that is very similar to rco-3. Suppressors of the rco-3 and dgr-1 mutants have been used to distinguish the roles of the rco-3 and dgr-1 genes. In addition, transcriptional profiling of the response of Neurospora to glucose starvation have been performed to help define the extent to which glucose availability globally regulates gene expression in a filamentous fungus. Further transcriptional profiling studies in rco-3 and dgr-1 mutants reveals the extent to which cells are defective in glucose sensing and the effect of these mutants on cellular physiology and development.
214. Analysis of the MAK-2 MAP kinase pathway in Neurospora crassa identifies secondary metabolism as a target for the mating pathway of filamentous fungi. Dan Li, Piotr Bobrowicz, and Daniel J. Ebbole. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA
MAP kinases in filamentous fungi homologous to the yeast FUS3/KSS1 MAP kinases of Saccharomyces cerevisisae have been shown to be important for plant pathogenesis. Characterization of the downstream targets of the pathway and characterization of the role of the MAP kinase in mating is greatly facilitated by use of a model organism, such as Neurospora. The genes for MAP kinase MAK-2 and the downstream transcription factor homologous to Ste12p have been cloned and mutated. Both mutants have growth defects, have reduced aerial growth but normal conidiation, and fail to form protoperithcia but function as males in sexual crosses. However, ascospores of mak-2 and ste-12 null mutants are inviable. Initial analysis has identified classes of genes that are regulated by mak-2 and/or ste-12. A gene cluster containing a polyketide synthetase is one target of MAP kinase regulation. The ‘mating' MAP kinase pathway in filamentous fungi controls many functions other than mating, and the identification of these functions, such as regulation of secondary metabolite production, may be relevant to understanding the basis for the evolution of plant-fungus interactions.
215. FL, the major regulator of conidiation in Neurospora crassa, binds to the promoter of the developmentally-regulated hydrophobin gene, eas. Panan Rerngsamran, and Daniel J. Ebbole. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA
The fluffy gene of N. crassa is required for the switch from filamentous to budding growth during conidiophore morphogenesis. The FL protein is an 88-kDa polypeptide containing a typical fungal Zn2Cys6-DNA binding motif. As a step towards identifying the target genes on which FL may act, we sought to identify target sequences to which the FL protein binds. The DNA binding domain of FL was expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified using glutathione sepharose affinity column. The DNA-binding sites were selected and amplified by means of a PCR-mediated random-site selection method involving affinity bead-binding and gel-mobility shift analysis. Sequencing and comparison of the selected clones suggested that FL binds to the motif 5'-CGGN9CCG-3'. The potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed the preferred binding site for FL in eas promoter is 5'-CGGAAGTTTCCTCCG-3'which is located nearly 1500-bp upstream of the eas transcription initiation site. In vivo experiment using a foreign DNA sequence tag also confirmed that this sequence is a target site for FL regulation. Additionally, in vivo experiments confirm that transcriptional activation activity resides in the C-terminal half of FL.
216. Expression and sequence analysis of a Ste 20 homologue from the phytopathogenic fungus Fusarium oxysporum. M A. García-Sánchez, B. Ramos, A. P. Eslava and J.M. Díaz-Mínguez. Area de Genética, Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca. 37007, Salamanca, Spain
Fusarium oxysporum is a deuteromycetous fungus and a significant plant vascular pathogen with worldwide distribution. Studies on the signal transduction MAPK cascade that operate in fission yeast, human pathogens, plant pathogens and model filamentous fungi reveal a high degree of conservation and the involvement of this pathway in fungal development, mating and pathogenicity. Genetic analysis conducted in several plant pathogenic fungi have demostrated that the MAPK signal transduction pathway plays an important role in pathogenesis A key link of the MAPK cascade with upstream elements seems to be the STE20 protein. STE20 is a serine-threonine protein kinase shown to be activated in vitro by GTP-bound Cdc42. Previously, we reported the cloning of a fragment of a Ste20 homologue from the plant pathogenic fungus F. oxysporum, by means of PCR using primers based on conserved amino acids kinase domains. Here we report the cloning of a full genomic copy of this gene and the molecular analysis of the sequence of this gene. Also, we have analyzed the expression of this gene in vitro by RT-PCR of RNA samples from in vitro grown fungal cultures, and in vivo by real-time PCR using RNA from bean plants collected at different times of the infection process. Gene disruption experiments are being developed so as to functionally characterize the role of this gene in pathogenicity and/or virulence. This research was supported by grant AGL 2001-2052 (Ministerio de Ciencia y Tecnología of Spain). M.Asunción García-Sánchez was the recipient of a fellowship from Caja Rural de Salamanca (Castilla y León, Spain) and Brisa Ramos was the recipient of a fellowship from the INIA (Ministerio de Ciencia y Tecnología of Spain).
217. The winged helix/RFX transcription factor CPCR1 affects both cephalosporin C gene expression and hyphae morphogenesis in Acremonium chrysogenum. B. Hoff, E. Schmitt, D. Janus and U. Kueck. Dept. of General & Molecular Botany, Ruhr-University Bochum, Universitätsstr. 150, 44780 Bochum, Germany, Fax: +49 234 3214184
Acremonium chrysogenum is the industrial main producer of the beta-lactam antibiotic cephalosporin C (CPC). The expression of the corresponding biosynthesis genes is mostly regulated on the transcriptional level. Using the one-hybrid system we have isolated the transcription factor CPCR1 which binds to an imperfect palindromic sequence in the intergenic promotor region of the pcbAB/pcbC genes and belongs to the conserved family of eukaryotic RFX transcription factors . Here we provide further data of the functional analysis of the transcription factor CPCR1. To determine the effect of CPCR1 on transcription of the pcbC gene, two approaches were followed. On the one hand we have constructed strains with multiple copies of the cpcR1 gene in order to increase gene expression. On the other hand we have generated fungal strains with a disrupted cpcR1 gene. All transformants were investigated using northern and western analysis to study changes in the CPC gene expression. These data shows an increasedpcbc gene expression in the multi copy strains, while the opposite effect is observed in the knock-out strain. Most interestingly, the alteration of the copy number of the cpcR1 gene in the genomic DNA of fungal transformants does not only influence CPC biosynthesis gene expression but has also major effects on hyphal morphology and differentiation. Growth curves as well as light and fluorescence microscopic studies using GFP labelled protein revealed differences in biomass accumulation and hyphal morphologies in the transformed strains. The time course of arthrospore formation, that is the organized fragmentation of hyphae, is accelerated in multicopy cpcR1 strains during batch cultivation. Our data indicate a dual function of the CPCR1 transcription factor in CPC gene expression as well as in arthrospore formation.
 Schmitt E. K., Kück U. (2000) J Biol Chem 275: 9348-9357
218. Mutation in a Calpain-like Protease Affects the Posttranslational Mannosylation of Phosphatases in Aspergillus nidulans. Sérgio R. Nozawa (1), Gregory S. May (2), Monica S. Ferreira-Nozawa (1), Nilce M. Martinez-Rossi (1) and Antonio Rossi (3). (1) Depto. de Genética, FMRP-USP, Ribeirão Preto, SP, Brazil; (2) Division of Pathology, M.D. Anderson Cancer Center, Houston, TX, USA; (3) Depto. de Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, SP, Brazil.
The ambient pH responses are mediated in A. nidulans by a conserved signal transduction pathway comprising at least seven genes (pacC and palA, etc) that have been cloned and sequenced. The palB gene codes for a calpain-like protease that is not involved directly in PacC processing, and the other pal genes have revealed only few functional features. In this communication, we show that the palB7 mutation drastically reduced the mannose and N-acetylgalactosamine content of the pacA-encoded acid phosphatase secreted by A. nidulans at pH 5.0, compared to a control strain. By using mRNA differential display reverse transcription and polymerase chain reaction, we isolated two cDNAs from the control strain (pabaA1) that were not detected in the palB7mutant strain and that encode a mannosyl transferase and a NADH-ubiquinone oxidoreductase. Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the palB gene, which codes for a nuclear calpain-like protease that may have specific functions in the processing of transcription factor(s) like its homologue, RIM13, in S. cereviseae. Further evidence in this direction comes from the demonstration that mutations in the mammalian calpain 3 protease rather than a structural defect can cause limb-girdle muscular dystrophy type 2A, and that these mutations are pathogenic only in a specific mitochondrial context (Richard et al., Cell, 81, 27-40, 1995). Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the proteolytic enzyme calpain 3 and could promote muscular dystrophy type 2A in humans. Financial support: FAPESP, CNPq, CAPES, FAEPA and Reitoria-USP.
219. Disruption of Gene Pacc Affected Both Growth and Conidiation of the Dermatophyte Trichophyton rubrum. Ferreira-Nozawa, M.S. (1), Nozawa, S.R (1), Cervelatti, E.P.(1), Rossi, A. (2) and Martinez-Rossi, N.M. (1).(1) Depto de Genética, FMRP-USP, Ribeirão Preto, SP, Brazil.(2) Depto de Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, SP, Brazil.
Although it has been well established that ambient pH affects the growth, physiology, differentiation, and viability of all organisms, the molecular responses to environmental pH changes are only now being elucidated. In Aspergillus nidulans and other fungi these responses are mediated by gene pacC, which codes for a Zn-finger transcription factor that activates the expression of several genes at alkaline pH. The pacC transcription is itself induced under alkaline growth conditions. In order to investigate the role of thepacC gene in the adaptive response of the dermatophyte Trichophyton rubrum to ambient pH, an important event in the dermatophyte-host interactions, we have cloned and identified a gene of this fungus homologous to the pacC gene of A. nidulans and pacC-1 of N. crassa (GenBank-access number: AF363788). The northern and western blots of T. rubrum also showed an increase of pacC transcripts at alkaline pH. For disruption, the promoter and first zinc-finger (3.5 Kb) were removed in the genomic clone of 6.0 Kb and replaced with the hygromycin phosphotransferase gene (hph), which confers drug resistance on eukaryotic cells. Several transformants of T. rubrum were isolated and showed that growth, conidiation, and pigmentation of the colonies were affected by the disruption of gene pacC, as already observed for C. albicans and N. crassa, indicating that surviving a proton attack depends on the expression of genes (e.g. pacC gene) that determine sensitivity or resistance to pH. Furthermore, these mechanisms are apparently involved in the installation, development, and survival of dermathophytes in humans. Financial support: FAPESP, CNPq, CAPES, FAEPA and Reitoria-USP.
220. The atrE gene of Trichophyton rubrum is responsive to cyclohexamide, griseofulvin, azole antifungal agents and ethidium bromide. Cervelatti, E.P.; Fachin, A.L.; Ferreira-Nozawa, M.S. and Martinez-Rossi, N.M. Departamento de Genética, FMRP-USP, Ribeirão Preto, SP, Brazil.
Trichophyton rubrum is the most commonly species found in superficial lesions of the skin, nails and hair. Over the last few years, the improper use of antifungal agents, the practice of invasive medical techniques and the onset of AIDS have led to cases of re-incidence of mycoses caused by this pathogen, as well as its invasive behavior. Despite the importance of this dermatophyte, very little is known about its genetic and biological aspects. As a contribution to this knowledge the goal of the present study was to characterize a gene coding for a carrier of the ABC type, belonging to a family of proteins involved in multidrug resistance mechanism. To this end, a 230 bp probe obtained by PCR presenting high identity with ABC carriers described in other organisms was used to track the complete gene in a genomic library. The sequence of the identified clone demonstrated a 5243 bp gene (GenBank-access number AF525740) homologous to the atrE gene of Aspergillus nidulans. To analyze the functionality of the identified gene, the fungus was cultured in the presence of various cytotoxic agents for 15, 30 and 60 minutes. The total RNA was used in a northern blot experiment and the expression of the gene of interest was evaluated using a central region of the gene as a molecular probe. An increase gene expression of the atrE transcript was observed after 30 minutes of exposure to ethidium bromide, ketoconazole, cyclohexamide, fluconazole, griseofulvin and itraconazole, suggesting the participation of the identified gene in drug efflux and its potential use as a therapeutic target for drugs to be developed by the pharmaceutical industry.
Financial support: FAEPA, Reitoria-USP.
221. Regulation of Neurospora fatty acid desaturase expression. Pitchaimani Kandasamy, Chan-Seok Oh, Ramesh Chellappa, Seung-Jae Baek and Charles E. Martin. Rutgers University, Division of Life Sciences, Nelson Laboratories, 604 Allison Road, Piscataway, NJ 08854.
Unsaturated fatty acids are formed by a series of membrane bound enzymes that form double bonds in long (C14 –C18) chain species. N. crassa encodes at least three desaturases that form the major membrane lipid fatty acids: Delta-9 18:1, Delta 9, 12 18:2 and Delta 9,12,15 18:3. Our studies on the OLE1 gene, which encodes the only long chain desaturase in yeast (a Delta-9 enzyme), demonstrated that its expression is regulated at the levels of transcription and mRNA stability by fatty acids and molecular oxygen.OLE1 is also transiently regulated in response to shifts to low temperature. Our previous studies also showed that the Neurospora Delta-15 desaturase is strongly regulated by growth temperature and recent experiments show that expression of the Neurospora desaturases is also regulated by unsaturated fatty acids. Expression of the yeast desaturase is governed by membrane bound proteins that are essential for transcription activation and hypoxic induction ofOLE1. These activate OLE1/ expression through their N-terminal domains which are released from the membrane through an ubiquitin-mediated mechanism that involves processing by the 26S proteosome. The ectopic integration of the homologous Neurospora genes that encode these proteins appear to quell growth. This can be repaired by unsaturated fatty acids, suggesting that a similar mechanism may be common to other fungi. (supported by NIH grant GM45768)
222. ER stress response: The A. niger transcription factor HacA mediates the upregulation of ER target genes, bipA, cypB and pdiA, and its own gene. Harm J. Mulder, Igor V. Nikolaev, and Susan M. Madrid Danisco Innovation Copenhagen, Langebrogade 1, DK 1001, Copenhagen, Denmark.
In eukaryotic cells, accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER) constitutes a fundamental threat to the cells. To deal with this stress, eukaryotic cells can respond to it via three different mechanisms: Transcriptional induction, translational attenuation, and degradation. When unfolded proteins accumulate in the ER the cell reacts by upregulating the synthesis of ER resident protein-chaparones and foldases, thereby increasing the folding capacity in the ER. The response, which involves a signal transduction cascade from the ER to the nucleus, is often referred to as the Unfolded Protein Response (UPR). The mRNA encoding the transcriptional activator HacA undergoes unconventional splicing upon accumulation of unfolded proteins within the ER, resulting in efficient translation of hacA, and activation of the UPR. Northern analysis of Aspergillus strains overproducing hacA, showed in addition to the upregulation of bipA, cypB and pdiA, also an increase in the amount unspliced mRNA form of hacA, which could indicate that the HacA protein also is involved in the upregulation of its own gene under UPR conditions. DNA binding experiments showed indeed binding of HacA protein to a hacA promoter fragment, which strengthen this hypothesis.
223. Multiple roles of WHITE COLLAR-1 in the environmental-sensing signal transduction pathways. Kwangwon Lee, Jennifer J. Loros, and Jay C. Dunlap, Department of Genetics and Department of Biochemistry, Dartmouth Medical School. Hanover NH. 03755.
The transcription factors WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) interact to form a heterodimeric complex (WCC) that is essential for most light-mediated processes in Neurospora crassa. WCC also plays a distinct non-light-related role as the transcriptional activator in the FREQUENCY (FRQ)/WCC feedback loop that is central to theN. crassa circadian system. Although an activator role was expected for WC-1, unanticipated phenotypes among some wc-1 alleles prompted a closer examination of an allelic series for WC-1 that has uncovered roles for this central regulator in constant darkness and in response to light. Using microarray technology we investigated the roles of WC-1 in general environmental-sensing signal transduction pathways including, light, temperature and nutrition-starvation. We discovered novel regulatory mechanisms of light-regulation. Our data suggest that 1) WC-1 is not only a positive regulator but also a negative regulator, 2) there are light-receptors other than WC-1, and 3) there is possible hierarchical transcriptional regulation among light-receptors. Our data also suggest that WC-1 has regulatory roles for subsets of stress-regulated genes, likely through indirect mechanisms. Supported by NIH (GM20553 to KL, GM34985 to JCD) and NSF (0084509 to JJL).
224. The role of chromatin mediated transcriptional regulation during pathogenic development in Ustilago maydis. Alexander Jamnischek, Martina Treutlein, Mario Scherer, and Joerg Kaemper, MPI for terrestrial Microbiology, Marburg, Germany
In the phytopathogenic fungus Ustilago maydis, pathogenic development is controlled by the two homeodomain proteins bE and bW encoded by the b mating type locus. The bE/ bW heterodimer is thought to control genes involved in pathogenicity as a transcriptional regulator either directly or indirectly via a b-mediated regulatory cascade. We have recently identified two genes, hda1 and rum1, that both play a role in the repression of b-regulated genes in the absence of a bE/bW heterodimer. The hda1 gene encodes a histone deacetylase (HDAC), and for Rum1 the domain structure suggests, similar to Hda1, a role in chromatin mediated transcriptional regulation. The deletion of either gene leads to a discrete block during teliospore development. Our current model places both protein in a common complex that regulates a defined set of genes by chromatin modification. By in silico analysis we have now identified a second HDAC, Hda139. In contrast to Hda1 that appears to be involved in the repression of b-regulated genes, Hda139 seems to be required for their activation after formation of an bE/bW heterodimer. hda139 mutant strains show reduced growth, defects in polar growth and filament formation, and are apathogenic. We will present results of a genome wide expression analysis of 6300 U. maydis genes that allow to address the distinct pathways affected in the rum1,hda1 and hda139 deletion strains.
225. AzoA, a novel molecular determinant of sensitivity to azoles in Aspergillus nidulans. Alan C. Andrade1, Luc Rouws2 and Maarten A. De Waard2. 1Lab. de Gen. e Biol. Molecular, EMBRAPA Recursos Genéticos e Biotecnologia, Caixa Postal 02372, Brasília-DF, Cep: 70770-900, Brazil.2Lab. of Phytopathology, Department of Plant Sciences, Wageningen University,P.O.Box 8025, Wageningen, 6700-EE, The Netherlands
Azoles constitute an important class of antifungal agents widely used in both agriculture and clinical medicine. Their efficacy is often limited by a variety of biological processes. Among these, the overexpression of membrane efflux pumps of the ABC (ATP-binding cassette) and MF (major facilitator) families, which lower intracellular drug concentration below effective levels, is a major but complex mechanism that results in multidrug resistance (MDR). MDR is considered to be the major challenge for modern chemotherapy. Here we describe the cloning and characterization of azoA, which is a novel molecular determinant of sensitivity to azoles in Aspergillus nidulans. This gene was identified from an azole-hypersensitive mutant of A. nidulans, obtained by insertion mutagenesis followed by plasmid rescue. Blast analysis of AzoA revealed similarity to hypothetical proteins with unknown function from yeasts and other fungi. Northern analysis revealed increased transcript levels of azoA upon treatment of fungal germlings with azoles. Furthermore, gene-replacement mutants of azoA, also displayed increased sensitivity to azoles, confirming its functional role. Interestingly, transcript levels of atrG, a previously characterized ABC transporter involved in protection of A. nidulans against azoles is higher in delta azoA mutants. Additionally, all delta azoA mutants displayed lower accumulation of [14C]fenarimol, as compared to a control strain. This situation mimics MDR, e.g reduced intracellular accumulation due to overexpression of an efflux pump (AtrG). Nevertheless, the phenotype observed for delta azoA mutants is increased sensitivity to azoles. These results suggest a major role for azoA on fungal sensitivity to azoles.
226. Microarray Analysis of Vegetative Incompatibility in Neurospora crassa. Sarah C. Brown, Isao Kaneko, Takao Kasuga, John W. Taylor and N. Louise Glass Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720
Neurospora crassa, like other filamentous fungi, is capable of spontaneous hyphal fusion during vegetative growth. Hyphal fusion between genetically distinct individuals leads to the formation of heterokaryons. The viability of such heterokaryons is dependent on the alleles present at loci for heterokaryon incompatibility (het loci). If hyphal fusion occurs between individuals with different alleles at a given het locus, hyphal compartmentation and ultimately cell death occur. Cell death observed during vegetative incompatibility bears several of the hallmarks of programmed cell death, including vacuolization of the cytoplasm, organelle degradation and shrinkage of the plasma membrane from the cell wall. We are investigating cell death during vegetative incompatibility using a temperature sensitive mutant that is able to form stable heterokaryons with a normally incompatible partner at 34°C. Vegetative incompatibility initiates after transfer to 20°C. Oligonucleotide microarrays have been developed and hybridization conditions optimized to compare gene expression patterns under different growth conditions. We are using these arrays to analyze gene expression in the temperature sensitive mutant at both 34°C and 20°C. We aim to identify genes that play an active role in the control of cell death in vegetative incompatibility in N. crassa.
227. Mapping the limits of the TRI4/TRI6 bidirectional promoter inFusarium graminearum and analysis of its transcription factor binding sites. Rex B. Dyer, David Kendra, and Daren W. Brown. Mycotoxin Research Unit, USDA/ARS/NCAUR, Peoria, IL 61604
Fusarium graminearum, the causative agent of wheat scab, is an international problem due to economic losses to the agricultural industry and because of food safety concerns resulting from mycotoxin contamination. Mycotoxin synthesis is coordinated by theTRI6 gene which encodes a transcription factor. In order to better understand the regulation of TRI6 gene expression and mycotoxin synthesis, we have employed the method of 5'-RACE to map the limits of the bidirectional promoter within theTRI4/TRI6 intergenic region of F. graminearum. We conclude that this bidirectional promoter consists of 892 nucleotides, mapping from -109 to -1000 nucleotides 5' of the TRI4 translational start codon. We analyzed the DNA sequence of this bidirectional promoter and the analogous sequence from 41 other Fusarium strains for transcription factor binding sites. Our analysis of the 42 strains reveals 21 to 30 transcription factor binding sites, specific to 14 different transcription factors. The transcription factor binding site that is most commonly represented in each strain is specific to the NIT2 transcription factor and is involved in nitrogen metabolite repression. We hypothesize that the NIT2 sites within the F. graminearum TRI4/TRI6 bidirectional promoter function to bring TRI4 and TRI6 gene expression, and mycotoxin biosynthesis under nitrogen metabolite repression control.
228. Characterization and Cloning of arg-13 mutants from N. crassa. Gloria E. Turner, Giselle Galang & Richard L. Weiss. Department of Chemistry, UCLA, California
The role of Neurospora crassa arg-13 gene product in arginine biosynthesis is not understood. The cloned gene sequence revealed homology with the mitochondrial carrier family (MCF) proteins. Most MCF proteins are localized in the inner mitchondrial membrane and participate in metabolic trafficking, exchanging metabolites for inorganic cations. This is accomplished using dual transport mechanisms, uniport and exchange. Examples include the dicarboxylate carrier, citrate carrier and the ornithine, glutamate and glutamine carriers. To understand the role of ARG13 in N. crassa arginine metabolism we have characterized four arg-13 mutant alleles. The mutants have a slow growth phenotype which can be restored to normal growth by the addition of arginine, citrulline and to a lesser extent ornithine. The ability of ornithine to support normal growth is problematic for one allelle. This result suggests that ARG13 may be exchanging ornithine and the need for arginine is a result of unregulated ornithine movement out of the mitochondrion. We have constructed the mutant strain aga arg-13 ota to test this hypothesis. Additionally we have cloned and sequenced two arg-13 mutant alleles using PCR amplification. A comparison of the mutant, wildtype and other known MCF proteins will enable us to determine important residues for ARG13 function. An arg-13 deletion has been constructed and will be used for in vivo analysis of altered ARG 13 proteins.
229. The conserved zinc finger domain of Aspergillus nidulans SteAp regulates the frequency of cleistothecial initial formation in a veA1 background. Emily J. Telfer and Bruce L. Miller. MMBB, Universtity of Idaho, Moscow, ID.
The SteAp of A. nidulans contains an N-terminal homeodomain, homologous to theS.cerevisiae Ste12p homeodomain, as well as two C-terminal C2/H2 Zn+2 fingers, conserved in Sterile 12-like proteins of filamentous fungi and several dimorphic fungi. Sexual development was examined in an A. nidulans veA1 strain carrying an in frame deletion of the steAC2/H2 Zn+2 finger domain. Consistent with results seen in the steA disruption mutant, the steAC2/H2 Zn+2 finger deletion mutant (steA:delta ZnF) does not affect conidiation. However, in contrast to the steAdisruption mutant, steA:delta ZnF produces fertile cleistothecia, containing wild type numbers of viable ascospores (1,000 - 100,000). steA:delta ZnF also suppresses the veA1 mutation with respect to sexual fertility and increases not only the frequency, but also the consistency with which sexual development is initiated. These results suggest steA interacts genetically with veA to regulate the frequency with which cleistothecia are initiated. The C2/H2 Zn+2 finger domain, which has not been shown to bind DNA, may provide a mechanism for this regulation.
230. Cloning of trehalose 6-phosphate phosphatase gene, orlA, from Aspergillus nidulans and analysis of its expression in response to stress and development. Jeong Heon Lee1, Mo Young Yu1, Je Seop Park1, Jin Young Jeon1, Dong Min Han2, and Pil Jae Maeng1. 1Department of Microbiology, Chungnam National University, Daejeon 305-764; and 2 Division of Life Sciences, Wonkwang University, Iksan 570-749, Korea.
Aspergillus nidulans orlA gene encodes the trehalose-6-phosphate phosphatase which catalyzes the last step of trehalose synthesis. We have isolated the orlA gene from a genomic cosmid library, and the nucleotide sequence of a 4.75-kb stretch of DNA covering the whole gene was determined. The orlA gene was predicted to contain a presumptive open reading frame of 2,646 bp which is interrupted by a 58-bp intron and encodes a protein of 882 amino acids. Under normal growth conditions, only low level of orlA expression was observed both in vegetative mycelia and conidiophores. However, the expression of orlAwas increased by any of the environmental stresses, heat shock, oxidative stress, and osmostress. Interestingly, the increase of orlA expression was much more significant in the asexual organs. The conidia of orlA deletion mutant showed decreased levels of resistance against both heat shock and oxidative stress comparing with those of wild type. The sensitivity of the mutant to heat shock and oxidative stress was remedied by an osmotic stabilizer such as 0.6 M KCl. These results suggest that orlA mainly contributes to promote the resistance of the conidia against environmental challenges, such as heat shock, oxidative stress, and osmostress.
231. Inducible expression of Aspergillus nidulans uvsJ by mutagenic DNA-damage and specific accumulation of UvsJ protein in conidial nuclei. Su Jin Cho1, Suhn Kee Chae2 and Pil Jae Maeng1. 1Department of Microbiology, Chungnam National University, Daejeon 305-764; 2Division of Life Science, Paichai University, Daejeon 302-735, Korea.
Aspergillus nidulans uvsJ gene is known to encode a ubiquitin-conjugation enzyme and to be included in UvsF group, one of the four epistatic group of DNA repair genes. The presumptive functional analogue of uvsJ, RAD6, has been reported to be involved in various cellular processes, such as sporulation, DNA repair, ubiquitination, cell cycle control, in Saccharomyces cerevisiae. In the present study, we analyzed the expression ofuvsJ and the localization of UvsJ protein using dimeric sGFP as a vital reporter. Under normal conditions, only a little transcription of uvsJ was detected through the whole thallus when monitored in both uvsJ-p::(sgfp)2 anduvsJ::(sgfp)2 transformants. On the other hand, when the cells of the transformants were subject to mutagenic DNA-damage rendered by alkylating chemicals (MMS, EMS, and 4-NQO), UV, and heat shock, the level of uvsJ expression was increased both in vegetative and asexually differentiated cells. Quite surprisingly, UvsJ::(sGFP)2 protein was specifically accumulated in the nuclei of conidiospores under the conditions of mutagenic DNA-damage. These results suggest that the expression of uvsJ is controlled at both transcriptional and post-transcriptional levels.
232. Differential expression of four different chitin synthase genes of Aspergillus nidulans in response to developmental status and environmental stresses. Jeong Im Lee, Yoo Mi Rho, Min Young Lee, Beom Chan Park, Hee Moon Park, and Pil Jae Maeng. Department of Microbiology, Chungnam National University, Daejeon 305-764, Korea.
Aspergillus nidulans has five chs genes encoding chitin synthases. We analyzed the expression mode of the four of the five chitin synthase genes, chsA,chsB, chsC, and chsD, using a mutant version of green fluorescent protein (sGFP) as a vital reporter. chsA was expressed at a considerable level in whole conidiophores during asexual development, but was expressed little in vegetative hyphae and foot cells throughout the culture period. During sexual development, chsA was not expressed either in the mycelia induced to sexual development or in the sexual structures. Expression of chsB was not only ubiquitous throughout the fungal body including substrate mycelia and conidiophores but also regardless of the developmental status of the cells. chsC was expressed only at basal levels during vegetative growth and asexual development. However, during sexual development, chsC was expressed at a considerable level in the shells of young cleistothecia and was strongly expressed specifically in mature ascospores. chsD was expressed at basal level in conidiophores and vegetative hyphae during asexual development, and strongly expressed in the shells of young cleisthothecia and Hulle cells at the early sexual stage. The expression of the chitin synthase genes, chsA, chsC, and chsD, except for chsB, was altogether enhanced by high concentrations of salts, such as KCl and NaCl.
233. The PEST-2 element in FREQUENCY is required for expression of the Neurospora crassa photo receptor WHITE COLLAR-1. Tobias Schafmeier, Christian Mohr and Michael Brunner. Biochemie - Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
FREQUENCY (FRQ), White Collar 1 (WC-1) and White Collar 2 (WC-2) are central components of the circadian clock in Neurospora crassa. WC-1 and WC-2 form a complex (WCC) that drives expression of frq RNA. In the course of a circadian day FRQ is progressively phosphorylated and degraded. Two PEST elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant casein kinase CK-1a. Single or double deletions of the PEST sequences result in hypophosphorylation of FRQ protein in vivo and arrhythmic condiation of the mutant strains. We show that the two PEST elements have specific and distinct functions in the circadian feedback loop. As shown previously, deletion of PEST-1 leads to a reduced rate of FRQ protein turnover. The corresponding mutant strain accumulates high levels of FRQdeltaPEST-1 protein. Levels of WC-1 are also elevated in frqdeltaPEST-1 cells, supporting that FRQ is required for efficient WC-1 expression. Deletion of PEST-2 does not affect FRQ protein stability. frqdeltaPEST-2 cells express low levels of the mutant FRQ and significantly reduced levels of WC-1. Double deletion of PEST 1 and PEST-2 stabilizes the mutant protein and restores expression of high levels of FRQdeltaPEST-2 protein while WC-1 levels remain low. The data suggest that the PEST-2 region in FRQ is required for expression of WC-1 while PEST-1 regulates FRQ protein turnover.
234. Putative chromosome remodeling factors in Neurospora crassa: creation of ISWI-type ATPase mutants by RIP. Michael Freitag and Eric U. Selker, University of Oregon, Eugene.
Chromatin remodeling factors (CRF) utilize energy from ATP hydrolysis to induce directed nucleosome sliding and serve as global activators or repressors of gene expression. Four major classes of CRFs (SWI2/SNF2, ISWI, Mi-2/CHD, INO80) have been distinguished, based on the structure of their highly conserved ATPase subunits. We identified 24 predicted proteins with conserved SWI/SNF ATPase domains in the Neurospora genome. Unlike mammalian cells and Saccharomyces cerevisiae, Neurospora has few paralogous pairs of SWI/SNF ATPases, making Neurospora an attractive model system for genetic analyses of CRFs. In addition to single homologs of S. cerevisiae SWR1 (Neurospora crf1-1), INO80(crf2-1), SWI2/SNF2 (crf3-1), ISW1 (crf4-1) andCHD1 (crf7-1), Neurospora also has homologs of the animal Mi-2 (crf6-1) and Arabidopsis DDM1 (crf5-1) genes. Both Mi-2- and DDM1-type proteins have been implicated in the control of eukaryotic DNA methylation. To study involvement of the ISWI-type ATPases in DNA methylation in Neurospora we mutated crf4-1 and crf5-1 by RIP. A GFP-tagged crf4-1 gene driven by the Neurospora ccg-1 promoter was inserted at the his-3 locus. The CRF4-1/GFP fusion protein was properly localized to the nucleus. Neurospora crf4-1 is not an essential gene, which resembles findings with deletion strains of S. cerevisiae ISW1 andISW2, but is in contrast to Drosophila ISWI mutants. (Supported by NIH grant # GM35690 to E.U.S.)
235. Cloning of the SIP3 homologue in Aspergillus nidulans. 1Shobana Krishnan*, 2Rosanna Penna-Muralla and 3Rolf Prade 1,2,3 - 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. nidulansand 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 subcloned and sequenced. The sip3 probe used is a clone that contains a fragment of thesip3 homologue in A. nidulans rescued in a different study. The sipA3gene comprises 5 exons interrupted by 4 introns. The predicted protein has high similarities with other filamentous fungi like A.fumigatus and N.crassa. A disruption cassette has been constructed with the selectable marker argB. A sip3 disruption mutant has been created by transformation-mediated gene disruption using this cassette and its growth in various carbon-sources like glucose, sucrose and pectin has been studied and compared to wild type.
236. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. Carsten Schwerdtfeger1,2, Jennifer J. Loros2, Jay C. Dunlap2, and Hartmut Linden1.1Lehrstuhl für Physiologie und Biochemie der Pflanzen University of Konstanz, Germany. 2 Department of Biochemistry and Department of Genetics, Dartmouth Medical School. Hanover NH. USA 03755.
Blue light regulates many physiological and developmental processes in fungi. Most of the blue light responses in the ascomycete Neurospora crassa are dependent on the two blue light regulatory proteins White collar-1 and 2. White collar-1 was recently shown to be the first fungal blue light photoreceptor. In the present study, we characterize the Neurospora protein VIVID which is also involved in the transduction of the blue light signal. VIVID reveals a partial sequence similarity with plant blue light photoreceptors. In addition, we found that VVD noncovalently binds a flavin chromophore. Upon illumination with blue light, VVD undergoes a photocycle indicative of the formation of a flavin-cysteinyl adduct. The VVD photocycle closely matches the photocycle of the plant blue light photoreceptor phototropin. VVD is localized in the cytoplasm and is only present after a light induction. A loss of function vvd mutant was completely insensitive to increases in light intensities. Our results suggested that VVD is a second Neurospora blue light photoreceptor, which enables Neurospora to perceive and respond to daily changes in light intensity.
237. Blue light regulation of DNA photolyase gene expression. Benjamin A. Horwitz, Gloria M. Berrocal-Tito, Inbal Marom, and Alfredo Herrera-Estrella1. Department of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel. 1Department of Plant Genetic Engineering, Centro de Investigacion y Estudios Avanzados del I. P.N., Unidad Irapuato, Irapuato, Gto. 36500, Mexico.
The photolyases, DNA repair enzymes that use visible and long-wavelength UV light to repair dimers created by short-wavelength UV, belong to the larger photolyase-cryptochrome gene family. Blue light rapidly induces expression of a photolyase gene, phr1, ofTrichoderma atroviride (1). The sensitivity to light is increased in transgenic lines overexpressing Phr1. This suggests that phr1 encodes a unique protein, capable of light dependent autoregulation. If this model is correct, this would be the first member of the photolyase photoreceptor family with both DNA repair and sensory functions. Phr1 could provide a "memory" of recent exposure to light, through this sensory role. To further investigate the mechanism of photoregulation of the photolyase gene, we are introducing reporter constructs into Trichoderma atroviride, and comparing the light regulation of phr1 with that of its orthologs in other species.
(1) Berrocal et al. J Biol Chem 274(1999) 14288; Photochem Photobiol 71(2000) 662
238. Functional analysis of the transcription factor MST12 in Magnaporthe grisea. G. Park & J.R. Xu. Dept.Bot.& Plant Path., Purdue Univ., W. Lafayette, IN 47907
In M. grisea, MST12 is a transcription factor that is essential for appressorial penetration and plant infection. In this study, we further characterized the function of MST12 and the defects of mst12 mutants. TEM examination showed thatmst12 mutants formed appressorial pores but failed to develop penetration pegs. Preliminary data indicated that Mst12-GFP was weakly expressed in mature appressoria and became localized to the nucleus in the penetration peg. Structurally, MST12 have a HD and zn-finger domains, and a middle region between them. Their effect on MST12 function will be studied using mutants deleted of each domain. In the middle region, there is a MAPK, a PKA, and a PKC phosphorylation sites that are well conserved among filamentous fungi. We have generated mst12 mutants with these phosphorylation sites changed individually by site-directed mutagenesis. Phenotypes of these mutants will be presented. In addition, when MST12 was over-expressed in DH51, appressorial penetration and lesions were observed, indicating involvement of MST12 in the cAMP signaling. Although MST12 can weakly interact with PMK1 in yeast two-hybrid assays, preliminary data indicate that overexpressing MST12 can not complement the defect of pmk1 mutant nn78. It is likely that there may be additional transcription factors involved in PMK1 signaling for appressorium formation. One of the candidates is the TEC1 homolog. Appressorium formation was significantly reduced in the MgTec1 knock-out mutants.