Gene Regulation and Metabolism

31. The carbon catabolite repressor Cre1 in Trichoderma reesei requires phosphorylation for its binding to the corresponding consensus sequence.

Robert L. Mach, Joseph Strauss and Christian P. Kubicek. Technical Univ of Vienna, IBTM 172/5, Getreidemarkt 9, A - 1060 Wien

In the industrial important fungus Trichoderma reesei glucose repression is mediated by the C2H2 Zn-finger protein Cre1, which binds to sites in the promoter of genes sensitive to glucose. In yeast, the homologous MIG1 protein is thought to recruit SSN6 and TUP1 to form the repressor complex. Derepression is mediated via the SNF1 gene encoding a protein kinase. In contrary, we could prove that phosphorylation of the Trichoderma reesei Cre1 protein leads to strong binding to the corresponding consensus sequence 5' SYGGRG 3'. A GST::Cre1 fusion protein, derived from a 700 bp DNA fragment encoding the Zn-finger region and the RGR1-like region of Cre1, was phosphorylated using protein kinase A catalytic subunit from porcine heart and dephosphorylated alkaline phosphatase from bovine intestinal mucosa. Only phosphorylated fusion protein showed binding to the corresponding consensus sequence. Similiar results were achieved by employing a thrombine-cleaved GST::Cre1 fusion protein.

32. Effects of amino acid substitutions in l tubulin on sensitivities to the antimicrotubule drugs benomyl and griseofulvin in Coprinus cinereus.

Tadashi Matsuo, Akira Kiba, Yuki Yamamoto, Hajime Muraguchi and Takashi Kamada, Okayama University, Okayama 700, Japan.

We first analyzed the wild-type 1 tubulin gene of Coprinus cinereus and then examined 26 1 tubulin mutants obtained as benomyl-resistant strains, griseofulvin-resistant strains, or extragenic suppressors of a mutation in a gene whose product appears to interact with 1 tubulin. Most of the mutations altered sensitivities to both benomyl and griseofulvin, regardless of how they had been identified. The 26 mutations all resulted in amino acid substitutions in 1 tubulin. The mutations were found in three clusters within the protein (445 amino acids): near the N-terminus (residues 3-50), near the middle (198-222) or at residue 350. These three regions overlap or lie close to the three colchicine-binding regions. We also found that a missense mutation at residue 350, which confers benomyl-resistance and heatsensitivity, could be suppressed by amino acid substitutions at several additional sites in 1 tubulin. This finding suggests that missense mutations in both residues that interact with the drug and also in residues distant from the binding sites can affect sensitivity to the drug.

33. Regulation of the Neurospora crassa Alternative Oxidase gene aod1.

Lesley LT. McLean and Frank E. Nargang, University of Alberta, Edmonton, Alberta, Canada

Alternative oxidase is encoded by a nuclear gene, aod1, and functions as a second terminal oxidase, transferring four elections directly from reduced ubiquinone to oxygen. This bypasses two sites of ATP production as well as the sites of action of respiration inhibitors Antimycin A and cyanide. In Neurospora, alternative oxidase is not seen under normal physiological conditions, but is induced if respiration through the standard mitochondrial electron transport chain is compromised. The signal(s) sent from respiration deficient mitochondria to the nucleus are unknown at this time. A cyclic-AMP response element (CRE) was found 750 bp upstream from the aod1 transcription start site and was shown to bind specifically with proteins from Neurospora cell extracts. However, transformation of aod1^- cells with constructs lacking the CRE upstream of aod1 gave isolates with alternative oxidase activity that is regulated in the wild-type fashion under conditions examined thus far. Similar studies using progressively shorter upstream regions in aod1⁺ constructs have shown that 250 bp are capable of providing control in the wild-type fashion. Work is currently under way to study induction quantitatively using the reporter gene lacZ. Identification of the factors involved in the regulation of aod1, and the method of regulation of those factors, may allow the dissection of signaling from the mitochondria to the nucleus in times of respiratory distress.

34. Nitrate utilisation in Aspergillus nidulans: Binding of the pathway specific activator NirA to its target sequence in vivo is dependent on induction and AreA.

Frank M. Narendja¹, Markus F. Wolschek¹, Jan Karlseder², Christian P.Kubicek¹, Claudio Scazzocchio³ and Joseph Strauss¹.

¹)Section of Microbial Biochemistry, IBTM, Technical University of Vienna, Austria ²) Vienna Biocenter, Dr. Bohr Gasse, Austria ³) IGM Orsay, Universite Paris Sud, France.

We have adapted the method of in vivo DMS footprinting to be suited for filamentous fungi using LMPCR, initially developed for mammalian cells. In order to obtain equally in vivo methylated DNA, we used germinating conidia with germ tubes of 4-6 cells for methylation instead of mature mycelium. We applied this technique to study the regulation of the nitrate utilisation cluster in A. nidulans as it represents a well characterised model system. Preliminary experiments determining the optimal time points for methylation following induction and/or repression showed that the response of A. nidulans cells to changes in nutritional composition is in the range of minutes only.

In vivo footprinting analysis of the bidirectionally functional NirA site 2 revealed that: (1) the consensus target sequence determined with the truncated protein in vitro (5`CTCCGHGG3`) is recognised by the native NirA protein in vivo, (2) binding of NirA in vivo is strictly dependent on the presence of inducer, (3) addition of ammonia to induced germlings results in dissociation of NirA, and that (4) under no condition NirA associated with its DNA target in a mutant carrying a non functional areA allele.

35. Role of negative charges in the N. crassa mitochondrial protein import receptor TOM22.

Frank E. Nargang, R. Gary Ritzel, Doron Rapaport, Walter Neupert, and Roland Lill. University of Alberta, Edmonton, Alberta and Institut fur Physiologische Chemie, Munich, Germany.

TOM22 is a mitochondrial outer membrane protein whose amino terminal domain extends into the cytosol and acts, in conjunction with TOM20, as a receptor for precursor proteins destined to be imported into mitochondria. The first 47 amino acids of the protein contain 18 negatively- and zero positively-charged residues. It has been suggested that this region of TOM22 interacts with mitochondrial targeting signals, which are thought to form positively charged amphipathic helices. To investigate this hypothesis, we have constructed a series of mutant derivatives of TOM22 in which varying numbers of the negative charges have been neutralized. These mutant constructs have been transformed into a sheltered heterokaryon bearing a tom22::hygromycinB disruption in one nucleus and assessed for their ability to rescue the inviable disruption-containing component. Constructs with up to 11 negative charges removed show little difference from the wild-type gene in their ability to rescue the tom22 disruption nucleus. Isolates with 15 negative charges removed can also rescue, though with reduced efficiency. We are currently investigating the ability of mitochondria, isolated from strains containing mutant constructs, to import mitochondrial precursor proteins. So far we have shown that strains containing TOM22, in which six negative charges have been removed, are able to import mitochondrial precursors with normal efficiency. Our results suggest that TOM22 receptor function does not depend simply on its highly negatively charged nature.

36. The Aspergillus nidulans sconB sulphur regulatory gene encodes a protein with WD40 repeats and F-box.

Renata Natorff, Malgorzata Piotrowska and Andrzej Paszewski, Institute of Biochemistry and Biophysics, Warsaw, Poland.

The Aspergillus nidulans sconB sulphur regulatory gene has been cloned by complementation with a cosmid gene library. The 2081 bp long open reading frame contains one 47 bp intron. The sconB gene encodes a polypeptide of 678 amino acids which belongs to the WD40 family of highly conserved eukaryotic regulatory proteins. The SCONB protein contains seven WD40 repeats spanning the C-terminal half of the protein. The SCONB protein shows 56% identity to the SCON2 protein of N. crassa and 44% to S. cerevisiae essential Met3O protein; both being involved in the regulation of sulphur metabolism. In addition, SCONB possesses the F-box, a recently discovered structural motif (Bai et al 1996, Cell, 86, 263-274) found in a number of proteins including the yeast CDC4. In the latter F-box serves as a target for binding Skp1p - yeast homolog of A. nidulans SCONC protein. This suggests that the SCONB and SCONC proteins may interact.

In the wild type, the sconB transcript of 2.8 kb is present at approximately the same level under all sulphur conditions tested, except for sulphur limitation, where it is up-regulated. It is present in all four scon^- strains.

The sconB2 mutation is complemented by the heterologous N. crassa scon2⁺ gene. Interestingly, the sconB⁺ gene transforms the sconC mutant to the wild type phenotype, but not vice versa.

37. Developmental regulation of catalases in Aspergillus nidulans

Rosa E. Navarro, Laura Kawasaki and Jesus Aguirre Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico.

A general hypothesis to explain microbial cell differentiation as a response to hyperoxidant states was derived from studies on sporulation in Neurospora crassa. Since catalases are ubiquitous enzymes that are central to cellular antioxidant responses, we have now approached this hypothesis by studying the function and regulation of catalases during A. nidulans asexual sporulation (conidiation). We have found two catalases in this fungus encoded by the catA and catB genes, whose predicted polypeptides are as similar between them, as they are to E. coli HPII catalase. The catA and catB expression is differentially regulated during growth and development. The catA mRNA and protein appear during sporulation and are accumulated in both, sexual and asexual spores independently of the brlA regulatory gene, in a process that involves transcriptional and translational controls. In contrast, the catB mRNA and protein are very low in spores, accumulate in mature hyphae throughout conidiation and in response to H₂0₂, both catalases can offer protection against H₂0₂ at different stages of the life cycle. Although the increase in CatB activity and the appearance of CatA during sporulation is consistent with the occurrence of oxidative stress during development, our data indicate the operation of efficient alternative pathways for H₂0₂ detoxification.

38. The alcA gene in A. nidulans: a full characterization of synergistical activation and carbon catabolte repression.

Cristina Panozzo, Emmanuel Cornillot, Veronique Capuano, Sabine Fillinger and Beatrice Felenbok. Institut de Genetique et de Microbiologie, Universite Paris-sud, 91405 Orsay, France.

In the ascomycete A. nidulans, ethanol can be used as sole carbon source by the action of two enzymes: alcohol dehydrogenase I (ADHI) encoded by alcA and aldehyde dehydrogenase (aldDH) encoded by aldA. The system is subject to two regulatory mechanisms consisting in specific induction mediated by the transcriptional activator AlcR and the carbon catabolite repression governed by the general repressor CreA. The alcA gene is one of the strongest inducible genes in A. nidulans. It is the most widely used for proteins overexpression in filamentous fungi both for fundamental research and applied aspects in biotechnology. A complete molecular characterization of AlcR and CreA binding sites have been carried out by gel band shift analysis.The role and the importance of each AlcR and CreA binding sites have been determinated by mutagenesis and deletion experiments. There are two types of AlcR binding sites: direct and inverted repeats which were shown to be functional in vitro and in vivo. We have shown that there is a strong synergistic activation of alcA transcription related to the three clustered AlcR specific sites, which positions are crucial in the activation process. Disruption of the CreA targets led to a totally derepressed alcA promoter which is associated to an overexpressed alcA gene transcription. The close proximity of the AlcR and CreA binding sites in the alcA promoter suggest a direct mechanism of competition between the two regulatory proteins as shown for the alcR promoter.

39. SconC, an Aspergillus nidulans sulphur metabolism regulatory gene is homolog of yeast SKPI essential gene.

Malgorzata Piotrowska, Renata Natorff and Andrzej Paszewski. Institute of Biochemistry and Biophysics, Pawinskiego 5a, 02-106 Warsaw, Poland.

SconC, one of the four regulatory genes (A, B, C, D) involved in controlling sulphur amino acid metabolism, was cloned and sequenced. The 669 bp open reading frame is interrupted by three short introns. It encodes a protein of 163 amino acids. The SCONC protein sequence predicts glycosylation, phosphorylation (ATP/GTP binding site: P-loop) and the PEST sequence common in rapidly degraded proteins. The SCONC protein shares 51 % identity with yeast Skp1p, a protein of central importance to a number of cellular processes, e.g. cell cycle (Bai et al Cell, 86, 263-274, 1996). SconC mRNA is expressed on the same level in the wild type grown on MM and in the presence of methionine. Interestingly, sconC mRNA is present in the sconC3 and in sconB2 mutants, but not in the sconD6. This may indicate that these two regulatory genes act in a cascade. Point mutations in highly conserved regions were identified in the sconC3 and sconC1 mutants relieved of methionine repression.

40. Identification of a regulatory gene in Ustilago maydis that affects the expression of genes regulated by the b locus.

Claudia Quadbeck-Seeger, Joerg Kaemper and Regine Kahmann, Institut fuer Genetik, Munchen, Germany.

One of the genes regulated by the b locus of U. maydis is egl1, a gene encoding an endoglucanase. egl1 is expressed in the dikaryon during the filamentous phase when an active bE/bW heterodimer is present in the cell, but not in haploid yeast like growing cells. Since egl1 expression can easily be monitored in a plate-assay, we used the gene as a reporter to screen for mutants that express egl1 in haploid cells. One such mutant was found. Northern analysis showed that the mutation leads to constitutive expression of several b-dependent genes. This indicates that the mutation has affected a gene with a more general regulatory function. Assays in planta showed that the mutation does not attenuate pathogenicity. Interestingly, however, formation of spores was abolished. Complementation with a cosmid library led to the identification of the gene, rum1 (regulator Ustilago maydis 1), affected in the mutant. We will discuss how this negative regulator fits into our current scheme on gene regulation exerted by the bE/bW heterodimer.

41. lnterspecies sequence and regulatory comparisons of amidase genes in Aspergillus species.

Julie A. Sharp, Sarah J. Bugg, Meryrl A. Davis, Michael J. Hynes. University of Melbourne, Australia.

The amdS gene of Aspergillus nidulans encodes an amidase enzyme involved in. acetamide hydroiysis. amdS mRNA is regulated at the transcriptional level by seven independent regulatory pathways in response to environmental stimuli. This elaborate display of regulatory control makes this system an excellent model for the study of gene regulation. Sequences with homology to the A. nidulans amdS gene have been cloned and sequenced from a variety of Aspergillus species identify conserved cis-acting regulatory elements. The amdS genes isolated from Aspergillus oryzae, Aspergillus unguis and two amdS genes from Aspergillus ustus have been sequenced. Comparison of the 5' untranslated regulatory regions reveal sequence divergence between the species whilst small scattered regions of sequence conservation can be identified as potentially important regulatory elements. Functional studies suggest these elements specific DNA binding sites for some of the known regulatory proteins. In an attempt to isolate amdS genes from other Aspergillus species a new amidase gene with different substrate specificities was isolated. The A. nidulans homologue was subsequently cloned by homology and sequenced. Functional studies have been used to investigate substrate specificity and regutatory control. Comparison of exon conservation and intron position between the two amidase genes suggests they are paralogues.

42. Nitrogen regulation and the tamA gene of Aspergillus nidulans.

Anna J. Small, Michael J. Hynes and Meryl A. Davis, Department of Genetics, Univ of Melbourne, Parkville 3052, Victoria, Australia.

In Aspergillus nidulans, the expression of many genes involved in nitrogen utilization is increased in the absence of easily metabolized nitrogen sources such as glutamine. This activation is mediated by the positively acting AreA protein, which binds to 5'GATA3' sequences in the promoters of regulated genes and activates transcription under nitrogen limiting conditions. The tamA gene in A. nidulans may also play a role in nitrogen regulation. tamA mutants have been identified which have low levels of a number of nitrogen metabolic enzymes. Mutants are also resistant to the toxic ammonium analogue methylammonium. The tamA gene has been cloned, and sequence analysis revealed a number of features suggestive of regulatory function. The predicted protein contains a Zn(II)2Cys6 zinc cluster motif similar to others known to bind DNA, and downstream regions characteristic of the Zn(II)2Cys6 cluster family. TamA shows strongest homology to UGA35, a nitrogen regulatory protein in Saccharomyces cerevisiae. The importance of these regions to TamA function is being investigated. PCR mutagenesis has been used to demonstrate that the zinc cluster motif is not required for TamA function. Substitution of the crucial fourth cysteine residue in the motif did not prevent an altered tamA construct from complementing a tamA mutant strain in cotransforrnation experiments. An internal deletion has demonstrated that sequences towards the C-terminal of the protein are required for function. Sequence changes in a range of tamA mutants have been determined and further implicate the C-terminal region as critical in the function of the TamA protein.

43. Preliminary characterization of the iso-orotate Decarboxylase (IDCase) activity of Neurospora crassa.

Jeffrey A. Smiley*, Renita Cannon*, and David K. Asch**, Depts of Chemistry * and Biological Sci.** Youngstown State University.

Many microorganisms have developed metabolic pathways that allow them to operate efficiently in the presence in a wide variety of nutrients. An example of such a pathway is the thymidine salvage pathway found in some species of fungi. This pathway allows fungi to convert thymine nucleotides into uracil nucleotides. One of the enzymes this pathway is iso-orotate decarboxylase (IDCase). We have developed a sensitive, specific radioactivity-based assay which is capable of easily and accurately measuring IDCase activity even in the most impure cell lysate. Our assays have shown that IDCase levels in N. crassa may be subject to induction dependent upon medium nitrogen source. We have observed that the specific activity of IDCase in wild type N. crassa increases by 2-3 fold when this strain is grown on Westergaard's medium, a minimal medium containing nitrate as the sole nitrogen source, versus Vogel's medium, a standard minimal medium containing ammonium salts. We have also shown that IDCase levels are increase 8-10 fold in strains which contain the uc-1 Mutation which may influence the production of the enzymes of the thymidine salvage pathway. The mechanism behind this increased production of IDCase remains unclear.

44. Characterization of a Neurospora crassa gene with sequence and functional homology to Aspergillus nidulans flbD.

Wei-Chiang Shen, Jenny Wieser, Thomas H. Adams and Daniel J. Ebbole. Texas A&M University.

The flbD gene regulates conidiation in A. nidulans. flbD contains a myb-like DNA-binding domain and is thought to function as a transcription factor. flbD acts in concert with other developmental regulators to activate expression of the key regulator of conidial morphogenesis in A. nidulans, brlA. We identified a gene from N. crassa with sequence similarity to the DNA-binding domain of flbD . The N. crassa gene complemented the conidiation defect of an A. nidulans flbD mutant. Furthermore, induced expression of the cDNA clone of the N. crassa gene using the alcA promoter induced conidiation in submerged cultures of A. nidulans. Thus, the N. crassa gene appears to be a functional homologue of A. nidulans flbD. A N. crassa mutant was produced by deletion of the gene. The growth rate of the mutants was indistinguishable from wild type and macroconidiation, microconidiation, and ascospore formation was normal. This finding suggests that the function of the gene in N. crassa is subtle or redundant under laboratory conditions, or that the gene plays no role in development in N. crassa. We have demonstrated that a N. crassa gene can complement a gene involved in conidiation in A. nidulans. The strategy of direct complementation could be useful in isolation of additional genes of N. crassa with homology to developmental regulators of A. nidulans. Such studies would help address the question of whether the regulatory strategies governing initiation of conidiation are fundamentally different or similar between these fungi.

45. Identification of differentially expressed genes during life cycle of Microbotryum violaceum.

Long Wang and Michael Perlin, University of Louisville, Louisville, Kentucky 40292.

Microbotryum violaceum is a basidiomycete fungus whose hosts are Plant species in the Caryophyllaceae. Development of the fungus was studied using mRNA Differential Display. Three cell types occur during the life cycle of M. violaceum: haploid sporidial cells (of opposite mating-type), dikaryotic hyphae and diploid teliospores. Of these, only dikaryotic hyphal stage is infectious. Differential display, provided us four candidate fragments representing transcripts expressed in the dikaryotic hyphal stage, but not in the haploid stage; one of these was also expressed in mated haploid cells. Differential expression of these fragments was confirmed by Northern blotting. These partial cDNAs were cloned and sequenced, then the RACE (Rapid Amplification of cDNA End) technique was used to recover full length cDNA. Two fragments produced by RACE (a 1.6 Kb and a 1.4 Kb fragment) were cloned and sequenced. The gene (C34) transcribed in both mating and hyphal stages had a homeo-domain box in the largest open reading frame. Southern hybridization detected C34 sequences in haploid sporidia of both mating type and homologous sequences were detected by Southems for both Ustilago maydis and Saccharomyces cerevisiae. Functional studies of these genes are currently underway. This work shows that the combination of mRNA differential display and RACE provide an efficient way to identify genes involved in different developmental stages in fungi, an approach that should be widely applicable to research on other organisms.