Posters II: Gene Expression/Genome Structure

36. Quelling: gene inactivation during mitosis in Neurospora crassa by transformation with homologous sequences

C. Cogoni, N. Romano and G. Macino. Dip. Biopatologia Umana, Sez Biologia Cellulare, Universita La Sapienza 00161 Roma.

When a wild-type strain of Neurospora crassa is transformed with different portions of the carotenogenic albino-1 or albino-3 genes, up to 30-35% of the transformants show an albino phenotype. The albino transformants presented a variety of phenotypes ranging from white or yellow to dark yellow colour. The ectopically integrated sequences provoke a severe impairment of the expression of the endogenous al-1 or a1-3 genes. This phenomenon, termed quelling, is found to be spontaneously and progressively reversible. In fact, all of the albino transformants have an unstable phenotype and revert progressively to wild-type or intermediate phenotypes over a prolonged culturing time. The phenotypic reversion is characterised by a progressive release of the transcriptional inhibition and seems to correlate with a reduction in the number of the ectopically integrated sequences. However, there is no strict correlation between the copy number of the ectopic sequences and the intensity of quelling. Heterokaryons with nuclei from quelled and wild type strains show a quelled phenotype indicating that the inhibition of expression by quelling acts in trans. The nature of the molecular events determining the onset of quelling is unclear. They are likely to involve some kind of interaction between the resident genes and ectopically integrated exogenous sequences. Recent evidence on a possible mechanism was presented.

37. Molecular cloning and expression of laccases from the white-rot basidiomycete Polyporus pinsitus

Debbie S. Yaver, Feng Xu, Elizabeth Golightly, Stephen Brown, Michael Rey, Palle Schneider, and Henrik Dalb ge, Novo Nordisk Biotech, Davis, CA and Novo Nordisk A/S, Denmark.

Two laccases have been purified from the extracellular medium of an 2,5-xylidine culture of the white-rot basidiomycete Polyporus pinsitus. These proteins are dimeric, comprised of two subunits of 63 kDa as determined by SDS-PAGE, and glycosylation accounts for 5-10 kDa of the total mass. Under non-denaturing conditions, the two purified laccases I and III have pIs of 6-6.5 and 3.5, respectively. The laccases have optimal activity at pH 5-5.5 and pH <4 with syringaldazine and ABTS as substrates, respectively. The genes LCC1 and LCC2 coding for the two purified laccases (I and III) have been cloned and their nucleotide sequences determined. LCC1 and LCC2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. LCC1 expression is induced by 2,5-xylidine, while LCC2 expression appears to be constitutive. LCC1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability and pH profiles as the purified native protein. Three additional laccase genes have been cloned from P. pinsitus. The genes LCC3, LCC4 and LCC5 contain 12, 11 and 11 introns, respectively. The position of several of the introns is conserved among all 5 genes. The karyotype of P. pinsitus was determined by CHEF, and 8 bands ranging in size from approximately 5.7 to 2 Mb were resolved of which 2 appear to be doublets. The 5 laccase genes have been mapped to specific chromosomes. LCC1 and LCC2 are on a chromosome of 5.7 Mb. LCC4 and LCC5 are on a chromosome of 3.7 MB, and LCC3 is on a chromosome of 2.5 Mb.

38. Structure and function analysis of N. crassa NAD(P)H-nitrite reductase

James D. Colandene and Reginald H. Garrett, University of Virginia.

Nitrate assimilation, widespread among plants, fungi and bacteria, is the predominant means by which inorganic nitrogen is converted into a reduced, biologically useful form. Via this two-step process, nitrate is converted into nitrite by the enzyme nitrate reductase in a two-electron transfer reaction, and nitrite is converted into ammonium by nitrite reductase in a six-electron transfer reaction. The focus of these studies is nit-6, the N. crassa gene encoding nitrite reductase. N. crassa nitrite reductase is a soluble protein composed of two identical subunits. This enzyme utilizes either NADH or NADPH as electron donor, and possesses two iron sulfur (4Fe/4S) clusters, two siroheme groups, and two FAD molecules. Besides its native activity, nitrite reductase has a number of partial activities which are presumed to be functions of discrete structural domains of the protein. These include the FAD-dependent NAD(P)H-cytochrome c reductase and dithionite-nitrite reductase activities. We have constructed a full-length nit-6 cDNA and partial nit-6 constructs encoding, the putative domains of the protein and have expressed them in E. coli and yeast in order to perform structure-function studies. Results indicate that the N-terminal end of the protein which putatively encodes NAD(P)H and FAD binding domain possesses the FAD-dependent NAD(P)H-cytochrome c partial activity. The C-terminal portion of the protein putatively responsible for the binding and coordination of the (4Fe/4S) clusters and siroheme appears to possess the dithionite-nitrite reductase activity.

39. The location, rate of expression, and activation domains of the regulatory protein CYS-3

K.R. Coulter and G.A. Marzluf, The Ohio State University, Columbus, Ohio.

The sulfur circuit of Neurospora crassa consists of a set of unlinked structural genes. These structural genes include three major regulatory genes (cys-3, scon-1 and scon-2) and six sulfur-catabolic enzyme genes (aryl sulfatase, choline sulfatase, sulfate permeases I and II, methionine permease and an extracellular protease). cys-3 acts in a positive manner to turn on the sulfur catabolic enzyme genes and is regulated by scon-l and 2; sulfur availability and, possibly, by itself. scon-l and 2 are negative regulatory genes that inhibit the expression of the cys-3 gene when sulfur levels within the cell are high. Mobility shifts and western blots were used to detect CYS-3 protein in nuclear extract samples (both nuclear and cytoplasmic fractions) of wild type and mutant strains of Neurospora crassa grown under repressed or derepressed conditions. The mobility shifts indicate the following: 1) CYS-3 protein is localized to the nucleus; 2) CYS-3 protein is present in wild type derepressed samples and scon- samples (repressed and derepressed conditions); 3) no CYS-3 protein is detected in cys-3 strains. The western blots appear to support these findings. The same methods are being used to determine the length of time needed to induce CYS-3 protein production. The results so far indicate that it takes approximately three hours for CYS-3 protein to be made. The potential activation surfaces of cys-3 are being determined using the yeast hybrid system. Results so far have been inconclusive.

40. Laccase gene-specific sequences from selected white-rot and brown-rot fungi

Trevor M. D'Souza, and C.A. Reddy, Department of Microbiology, Michigan State University, East Lansing, MI 48824-1101.

Laccase gene-specific sequences from 12 strains representing 10 genera of white-rot and brown-rot fungi were isolated, using a PCR amplification procedure employing degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domain of known basidiomycete laccases. All the fungi included in this study, except Pleurotus ostreatus and Fomes fomentarius, gave a PCR amplified product of about 200 bp. A few of the fungi gave additional PCR products suggesting laccase gene polymorphisms in these organisms. Several of the PCR products including those from the white-rot fungi Trametes versicolor, Phlebia brevispora, Ganoderma lucidum and Lentinula edodes, and the brown-rot fungus Gloeophyllum trabeum, were sequenced. These nucleotide (nt) and predicted amino acid (aa) sequences were compared to the corresponding, previously published, laccase gene sequences of white-rot fungi. The results indicated the presence of laccase gene-specific sequences in a number of wood-rot fungi included in this study. The results further showed a high degree of nt (54%-74%) and aa homology (72%-97%) between the laccase gene-specific sequences (and deduced aa sequences) of white-rot fungi, but not those of Ascomycetes. Demonstration of laccase gene-specific sequences as well as laccase activity in the brown-rot fungus G. trabeum was of particular interest because brown-rot fungi were not previously shown to contain laccases.

41. Transcription initiation and termination sites of ribosomal RNA gene clusters of Neurospora crassa

S.K. Dutta and *M. Verma, Dept. of Biology, Howard Univ., Washington, DC, and *Dept. of Biochem. & Molec. Biol., Georgetown Univ., Washington, DC.

The nontranscribed external spacer region containing the transcription regulatory sequences of rDNA of Neurospora crassa has been characterized which shows many interesting sequences like the presence of Sal I box, putative rRNA processing site, pyrimidine rich region and secondary structure of the rDNA typically present in almost all eukaryotes. The entire region (nt) has been cloned and sequenced. The transcription initiation and termination sites were localized by S1 mapping and primer extension analysis. Primer extension analysis suggested that the transcription starts from a G at location 2537 whereas the transcription termination site is located in the region 471-475. The nontranscribed spacer region also contains sequences which are binding sites for known nuclear factors. (Supported in partly by the EPA (#R813126) and from an Institutional Grant (#2S06GM 08016) from the NIGMS-NIH and by a Collaborative Core Unit, Graduate School of Arts and Science, Howard University to SKD).

42. An aflatoxin biosynthesis regulatory protein (AFLR) is a sequence-specific DNA binding protein

Kenneth Ehrlich, Jeffrey Cary, and Beverly Montalbano, Southern Regional Research Center, USDA, New Orleans, LA 70179

In previous work, a gene (aflR) was isolated from an Aspergillus parasiticus cosmid clone whose expression was correlated with expression of several aflatoxin pathway genes. The open reading frame of aflR encodes a protein (AFLR) with a GAL4-type zinc binuclear cluster, which suggested that it may be a DNA-binding protein. Using synthetic oligonucleotides in electrophoretic mobility shift assays, we found that recombinant AFLR bound with sequence specificity to a palindromic site, TTAGGCCTAA, 121 bp upstream of its own translation start site. AFLR also bound to portions of the DNA from the promoter regions of several aflatoxin pathway genes. The genes tested were part of a 50 kb gene cluster and included genes for norsolorinic acid reductase, a polyketide synthase similar to that of A. nidulans, an aflatoxin pathway-specific fatty acid synthase, two reductases involved in versicolorin A synthesis, and sterigmatocystin O-methyltransferase. These results suggest that in addition to regulating its own expression, AFLR may coordinately regulate the expression of multiple genes in the aflatoxin biosynthetic pathway.

43. Analysis of gene clustering: transformational study of the ethanol-gene-cluster in Aspergillus nidulans

Sabine Fillinger and Béatrice Felenbok, Institut de Génétique et Microbiologie, Bat. 409, Centre d'Orsay, F-91405 Orsay cedex, France.

Ethanol-utilization in Aspergillus nidulans is mediated by alcohol-dehydrogenase I and aldehyde- dehydrogenase encoded by alcA and aldA respectively. Both genes are under the transcriptional control of the specific activator AlcR and the general carbon catabolite repressor CreA. The alcR and alcA genes are closely linked on chromosome VII; aldA is located on chromosome VIII. We have identified seven other genes at the same locus as alcA and alcR on chromosome VII. They are transcriptionally controlled by AlcR and CreA and were therefore called alcQ, alcT, alcN, alcP, alcO, alcM and alcS. We analysed in two alc-deletion strains the function of gene-clustering; we were able to show that alcR and alcA integrated at different loci (trans) in an alc500 deletion strain did not restore growth on ethanol. Transformation of the same strain with a phagemid containg six alc genes in cis (alcR, alcO, alcA, alcM and alcS) leads to wild-type phenotype on ethanol. Several gene combinations in cis and trans were tested for growth on ethanol and other related carbon sources as well as transcription regulation. We will further discuss gene and cluster-function.

44. Multiple control signals in the proline utilisation gene cluster of Aspergillus nidulans

B. Cubero, D. Gomez, R. Gonzales, V. Gavrias and C. Scazzocchio. Institut de Génétique et Microbiologie, Université Paris-Sud. 91405 Orsay, France.

Aspergillus nidulans can utilise proline as the sole source of carbon and nitrogen. The genes involved form a cluster which comprise three structural genes, prnB, C and D, a positively-acting regulatory gene, prnA, and a fifth gene, prnX, of unknown function. The 1.7 kb intergenic region between genes prnB and D has been shown to play a crucial role in the regulation of thc expression of all prn genes with exception of prnA. Proline catabolism is subject to three levels of control: Specific induction, mediated by the PrnA protein, carbon catabolite repression, mediated by the negatively-acting protein CreA, and nitrogen metabolite repression, mediated by the positively-acting protein AreA. PrnA, CreA and AreA belong to the zinc-containing class of DNA binding proteins. A complete molecular characterization of their binding to the prn intergenic region has been carried out by band shift and footprint analysis. Three PrnA binding sites have been found in the prn cluster, two in the prnB-D intergenic region, and one in the prnB-C intergenic region. At least two of them are functional in vivo. A PrnA binding site situated 3' of gene prnB is sufficient to its inducibility. The complete molecular characterization of 17 AreA binding sites in the prnB-D intergenic region has allowed to define a consensus binding sequence 5'HGATAD3'. The functional importance of the pyrimidines contained in the binding site has been determined for the first time for a GATA factor. The most proximal sites to the start points of genes prnB and D are functional in vivo, as determined by transcriptional analysis and growth tests of deletion mutants in the prnB-D intergenic region. The positive action of AreA is not due to repressor displacement. The expression of the prn genes is conditionally dependent on AreA. Strains carrying null areA alleles can utilise proline as carbon and nitrogen sources in the absence of CreA. A 700 bp deletion in the prn intergenic region leads to unconditional dependence on AreA for proline utilization. We postulate the existence of a third positive regulatory element whose target lies in this region. We have carried out band shift assays and deletion analysis and have found a 80 bp DNA fragment which binds specifically to hitherto unidentified protein

45. Identification of NIT4 binding sites within the nit-3 promoter

Y.H. Fu, B. Feng, S.A. Evans, and G.A. Marzluf, Ohio State University

Expression of nit-3 and nit-6, structural genes in Neurospora crassa which encode nitrate and nitrite reductase, respectively, requires limitation of primary nitrogen sources and induction by means of nitrate. Transcriptional activation of these genes also requires the action of NIT2 and NIT4, positive regulatory proteins. NIT2 is a global acting regulatory protein, also needed for expression of a variety of other nitrogen metabolizing enzymes, while NIT4 is a pathway specific regulatory protein. NIT4 contains 1090 amino acids with a putative Cys6 zinc c!uster DNA binding domain, similar to that of GAL4, found near the amino terminus. A NIT4/bGAL fusion protein has been expressed and purified, and its DNA binding specificity determined. Two NIT4 binding sites of varying strengths in the nit-3 promoter are identified through mobility shift and DNA footprinting experiments. The stronger site contains the palindromic sequence TCCGCGGA, while the weaker site contains the related sequence TCCGTGGC. Similar sequences are found in the nit-6 promoter. Further examination of this octomeric binding site and its flanking sequences is currently being done.

46. Evidence for a GATA-1-like DNA binding protein in N. crassa whose expression responds to nitrogen metabolism

Peter T. Gates and Reginald H. Garrett, University of Virginia

Employing gel-shift assays and DNA-affinity chromatography, we have isolated DNA-binding proteins from N crassa crude cell extracts that bind specifically to the consensus sequence NGATAC. Such GATA sequences are specifically recognized by the fungal transcription factors AREA and NIT2, which regulate nitrogen metabolism in A nidulans and N. crassa, respectively. Protein-DNA complexes are observed with oligonucleotides containing either one NGATAC consensus site or multiple consensus sites in various orientations. A lower molecular weight protein DNA complex is unaffected by the presence of physiological levels of ammonium, glutamine, or histidine, metabo lites which repress nitrate assimilation and other aspects of nitrogen metabolism in filamentous fungi. In contrast, formation of a higher molecular weight protein DNA complex does appear to be inhibited by physiological levels of these metabolites. Interestingly, the affinity purified proteins bind with high affinity and sequence specificity to single-stranded DNA containing one or more NGATAC consensus sequences. Protein sequence analysis of the affinity-purified fractions has not reveale any AREA or NIT2. Multiple GATA-binding activities, independent of NIT2 or its homolog AREA, and differing in sensitivity to the nitrogen status of the cell, may exist in N. crassa, as shown recently in Aspergillus nidulans.

47. Pleiotropic deficiencies of the laccase-derepressed mutant lah-1 are caused by constitutively increased expression of the cross-pathway control gene cpc-1

Toshiaki Harashima(1,2) and Hirokazu Inoue(1) (1)Saitama Univ. Japan. (2)JSPS Research Fellow

We previously isolated two Neurospora crassa mutants that showed abnormal expression of the laccase gene (lacc). One is the lah-1 mutant which derepresses lacc expression. The other is a cpc-1 mutant, which is not able to induce laccase activity. To investigate relationship between lah-1 and cpc-1 on lacc expression, we further characterized these mutants. The lah-1 mutant showed pleiotropic deficiencies such as slow growth in vegetative cultures, poor protoperithecia formation and hypersensitivity to cycloheximide. The cpc-1 mutant showed abnormality in early stage of conidial germination and also high sensitivity to cycloheximide. Genetic analyses of lah- 1cpc-1 double mutant showed that the cpc-1 mutation is epistatic to lah-1. Moreover we observed that expression of trp-3 gene is constitutively elevated in the lah-1 mutant. The derepressed level of trp-3 gene in the lah-1 mutant reduced to basal level in the lah-1cpc-1 double mutant. These results suggest that in the lah-1 mutant, the expression of cpc-1 gene is constitutively derepressed

48. Analyses of N. crassa mei-3 gene products and expression

Shin Hatakeyama, Chizu Ishii and Hirokazu Inoue, Lab. Genet., Fac. Sci., Saitama Univ., Japan.

The mei-3 (meiosis) gene of Neurospora crassa plays important roles in DNA repair and meiotic recombination. In this study we produced anti-MEI3 antibody rised against recombinant MEI3 protein, and using the antibody we detected a 38kDa polypeptide in perithecia, fruit body of N. crassa, by Western analysis. From sequencing of both cDNA and genomic clones, mei-3 gene had one 1195 bps open reading frame and two introns, and deduced molecular weight (39kDa) was in good agreement with the data obtained from Western analysis. The 39kDa MEI3 product is homologous to proteins of Rad51 family, and contains the conserved "domain I" domain II" and carboxy-terminal domains found in this family. The mei-3 expressions in wild type and five different mei-3 mutants increased in response to MMS and UV as determined by Northern blotting. Two genomic sequences of mei-3(SA10) and mei-3 (N289) were determined. These mei-3 genes contained several differences from mei-3 gene of wild-type, resulting in truncated proteins.

49. Abnormal mitochondrial DNA in uvs-4 and uvs-5 mutants of Neurospora crassa

G. Hausner, S. Stoltzner, S.K. Hubert, K.A. Nummy, H. Bertrand. Michigan State University, Microbiology, East Lansing, MI 48824.

The mutagen sensitive uvs-4 and uvs-5 nuclear mutants progressively degenerate upon prolonged asexual propagation (Schroeder 1970 Mol. Gen. Genet. 107:291-304). Since this phenotype is characteristic of some respiratory mutants we have examined uvs-4 and uvs-S for defects in mitochondrial function. After recovery from sheltering heterokaryons, both mutants develop high levels of CN-resistant respiration and become cytochrome a and b deficient upon repeated subculturing. The growth rate of both mutants progressively declines, but only uvs-5 dies after being subcultured 4 or more times. During the degeneration, abnormal forms of mtDNA appear in both mutants. High amounts of a plasmid-like derivative of mtDNA accumulate in uvs-4, whereas mtDNA deletions accumulate in uvs-5. The plasmid-like mtDNA derivative of uvs-4 is a 167-bp tandemly repeated sequence that consists of a PstI palindrome, the promoter and 104 base pairs of the 23 S mt rRNA gene. Heterokaryon tests have shown that the uvs-4 mutation is essential for the generation and maintenance of the plasmid-like element. The mechanisms which give rise to the plasmid-like element in uvs-4 and mtDNA deletions in uvs-5 are still unknown. Supported by the Muscular Dystrophy and American Heart Association, and a NSERC post-doctoral fellowship to G.H.

50. The elongation factor 1alpha (EF-1alpha) of Neurospora crassa

Akihiko Ichi-ishi.and Hirokazu Inoue, Lab. Genet., Fac. Sci., Saitama Univ., Japan

The translation elongation factor 1a (EF-lalpha) plays an essential role in protein synthesis in eukaryotic cells. This protein transfers aminoacyl-tRNAs into the acceptor site of the ribosome in a step that requires GTP. The tef-1 gene encoding the EF-1alpha protein was cloned from Neurospora crassa. The sequences of genomic DNA and cDNA clones showed that the tef-1 gene contained one ORF of 1380 bp length which is interrupted by three short introns. The deduced polypeptide contained 460 amino acids. The level of tef-1 mRNA was low in conidia but high in growing cells. When mycelia were transferred to poor nutrient media the level of tef-1 gene mRNA decreased remarkably. The pattern of tef-1 expression was similar to that of genes for ribosomal proteins. Southern blot analysis showed that Neurospora genomic DNA contained only one copy of the tef-1 gene. The tef-1 mutant was produced by RIP. This strain showed very slow growth. The tef-1 gene was mapped between arg-3 and leu-4 loci on linkage group I by RFLP mapping and between T(OY321) and ser-3 loci by genetic mapping.

51. Excision of UV products during liquid holding in Neurospora

C. Ishii, T. Matsunaga, O. Nikaido and H. Inoue. Saitama Univ. and Kanazawa Univ., Japan

Excision of pyrimidine dimer and that of (6-4) photoproduct were measured ln Neurospora crassa wild type and the mus-18 mutant during liquid holding. Pyrimidine dimers were lost within 60 min in wildtype irradiated at 150 J/m2, while (6-4) photoproducts were processed faster than dimers. In mus-18, most dimers were left unprocessed over 6 hr. Unexpectedly, excision of photoproducts were also defective in this mutant. Combination of liquid holding and photoreactivation in UV-induced reversion assay revealed that pyrimidine dimer was a main cause of high mutagenicity of UV in mus-18, although this damage seemed cytotoxic rather than mutagenic to wildtype Neurospora.

52. Molecular cloning and characterization of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. Synergism between rhamnogalacturonan degrading enzymes

Sakari Kauppinen, Stephan Christgau, Lene V. Kofod, Torben Halkier, Kurt Dörreich and Henrik Dalb ge, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark

The main backbone in plant cell wall pectins can be devided into linear homogalacturonan polymers of (1-4) linked alpha-D-galacturonic acid (GalUA) and highly branched rhamnogalacturonan (hairy) regions, composed of alternating alpha-(1,2) linked L-rhamnosyl and alpha-(1,4) linked galacturonic acid residues, with arabinan, galactan or arabinogalactan side chains attached to the rhamnose residues. Most pectic substances are furthermore substituted with acetyl or methyl groups at the GalUA residues in the backbone. We have recently cloned and characterized two structurally and functionally different rhamnogalacturonan degrading enzymes, RGase A and RGase B, from the filamentous fungus Aspergillus aculeatus. A prerequisite for the action of rhamnogalacturonases is that the acetyl esters have been removed from the backbone. Thus, the presence of a rhamnogalacturonan acetylesterase (RGAE) in A. aculeatus, highly specific for the deacetylation of rhamnogalacturonan regions of pectin suggests that this enzyme is essential for the action of RGases in vivo. This communication describes the isolation and characterization of the rha1 gene encoding RGAE I from A. aculeatus. With the goal of elucidating the role of RGAE I in the enzymatic degradation of plant cell wall rhamnogalacturonan we have undertaken high level expression of the rha1 gene in Aspergillus oryzae and characterization of the purified, recombinant enzyme. In addittion, we show that the recombinant rhamnogalacturonan acetylesterase (rRGAE) acts in synergy with RGase A as well as RGase B in the degradation of apple pectin rhamnogalacturonan.

53. Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus

Lene V. Kofod, Sakari Kauppinen, Stephan Christgau, Lene N. Andersen, Hans P. Heldt-Hansen, Kurt Dorreich, Henrik Dalboge. Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark

The pectic substances of plant cell walls are composed of homogalacturonan (polymer of alpha-1,4-galacturonic acid) and rhamnogalacturonan (polymer of alternating alpha-1,4-rhamnose and alpha-1,2-galacturonic acid). Whereas pectinases, which cleave in the homogalacturonan regions, have been extensively studied, rhamnogalacturonases have only recently been described. In the present study two rhamnogalacturonases from the filamentous fungus Aspergillus aculeatus have been cloned and characterized. A cDNA library from A. aculeatus was constructed and a novel Rhamnogalacturonase B was isolated by expression cloning in yeast. For this purpose a new plate screening assay was developed, specific for the detection of rhamnogalacturonase activity. The Rhamno galacturonase A, known from previous reports, was shown not to be expressed in yeast in an active form. Therefore, Rhamnogalacturonase A was purified, peptide sequences were obtained and full-length cDNAs encoding the enzyme were isolated using a polymerase chain reaction generated product as a probe. Comparison of the deduced primary structures indicates that the two rhamnogalacturonases are structurally different. The cloned genes were transformed into Aspergillus oryzae for high level expression. The recombinant enzymes were purified and characterized, revealing significant differences in substrate specificity, as well as in pH and temperalure optima and stability. Data from the hydrolysis of apple rhamnogalacturonan with the recombinant rhamnogalacturonases suggest that the two enzymes exert their action at different sites in the backbone.

54. Characterization of a general amino acid control transactivator in the chestnut blight fungus Cryphonectria parasitica and its regulation in virulent and hypovirus infected strains

Thomas G. Larson, Chein-Hwa Chen, Diane M. Pawlyk, Julie A. Clark, and Donald L. Nuss. Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.

The transcription factor CPC1 governs general amino acid control in Neurospora crassa. We have cloned a homologous gene from the plant pathogenic fungus Cryphonectria parasitica and examined its regulation in isogenic virulent and hypovirus-infected strains. Northern analysis revealed that amino acid starvation elicits an increase in C. parasitica cpc-1 transcript levels. Although amino acid starvation produced similar increases in cpc-1 transcript levels in isogenic virulent and hypovirus-infected strains, the diminished accumulation of transcripts for several amino acid biosynthetic genes in the hypovirulent strain is consistent with a virus-mediated reduction in cpCPC1 activity. Suppression of cpc-1 transcript accumulation was observed in the hypovirus-infected strain under growth conditions that derepress laccase (lac-1) transcription in C. parasitica. Our results define a general amino acid control transactivator in a plant pathogenic fungus and provide further evidence that a virulence-attenuating hypovirus alters the transcriptional response of the host fungus to changes in nutrient availability.

55. Specific recognition of the DNA-targets of AlcR, the transactivator of the ethanol regulon in Aspergillus nidulans, involves an arginine residue in its NH2-terminus

François Lenouvel and Béatrice Felenbok. Institut de Génétique et Microbiologie, Faculté d'Orsay, F-91405 Orsay Cedex, France.

The transcriptional activator of the ethanol regulon, AlcR, is a binuclear zinc cluster able to bind onto specific targets on the alcR and alcA promoters. These targets are organized as invert and direct repeats with the consensus core 5'CCGCA-3'. We tested the importance of the AlcR NH2-terminal part on the specific in vitro binding of the GST-AlcR fusion protein. Two constructions were assayed in gel band shift experiments. The fusion product with a truncated AlcR NH2-end (the 6th first residues are missing) was able to bind specifically onto the two types of DNA targets with a low affinity for the invert repeats (10(-3) M) and a higher affinity for the direct repeats (10(-7) M) targets. With the fusion protein containing the complete NH2-terminus of AlcR, the affinity was only increased for the invert repeats by 10000 fold (10-7 M). This enhanced binding activity was shown to be directed by only one arginine residue within the NH2-terminus. Footprint interferences experiments showed a decrease of the number of contacted Gs in the invert repeat target. This demonstrates that the AlcR amino terminus is essential for the correct recognition of invert repeats targets. In order to test these results in vivo, a reporter strain of Aspergillus nidulans was built. It contains the uidA gene, encoding the beta-glucuronidase (GUS), under the control of the alcA regulating sequences. Transformants lacking the NH2-terminus of AlcR were tested for growth on ethanol and GUS activity was determined in comparison with a transformant strain containing the AlcR NH2-terminus.

56. Studies of Neurospora crassa CYS3 protein basic region and identification of its consensus and optimal DNA binding site

Qunhui Li and George A. Marzluf, The Ohio State University.

CYS3 is the positive acting regulatory protein involved in the sulfur utilization control circuit in Neurospora crassa. CYS3 activates the expression of a set of genes which encode sulfur-related catabolic enzymes under sulfur limitation conditions. CYS3 belongs to the bZip protein family and shares extensive sequence homology with other family members in the leucine zipper and basic region. Here we report the characterization of native DNA binding sites recognized by CYS3. From systematic mutational analysis, we hare defined the consensus of CYS3 binding sequence, 5'-ATGPuPyPuPyCAT-3', a ten nucleotide palindrome, and have found 5'-ATGACGTCAT-3' acts as an optimal binding site. We also have studied two uncharged residues, SER113 and PHE116, in the basic region of the CYS3 protein, and found that S113 is directly involved in a nucleotide specific interaction and that F116 also contributes significantly to DNA binding affinity.

57. DNA binding of FacB, a transcriptional activator of acetate utilization genes of Aspergillus nidulans

Richard B. Todd, Meryl A. Davis and Michael J. Hynes. Dept. of Genetics, Univ. of Melbourne, Parkville 3052, AUSTRALIA.

The facB gene of Aspergillus nidulans encodes a transcriptional activator which mediates acetate induction of the amdS gene (encoding acetamidase) and genes required for acetate metabolism via the glyoxylate bypass. Cloning and sequence analysis of facB revealed a Zn(II)2Cys6 DNA binding cluster, a putative leucine zipper-like dimerization motif and potential acidic activation domains (H.M. Martin, S. Sapats, J.A. Sharp, M.E. Katz, M.A. Davis and M.J. Hynes, unpublished). DNA binding studies are being used to investigate the regulatory function of facB. The FacB protein has been expressed in Escherichia coli as a Maltose Binding Protein fusion. This fusion has been used in gel mobility shift assays to demonstrate that FacB is a DNA binding protein that binds to specific sequences from FacB- regulated promoters. Footprinting assays have been used to define two dissimilar sequences to which FacB binds in the amdS promoter. An analysis of binding to mutant FacB binding sites has been performed. In vitro mutagenesis has been used to alter specific cysteine residues in the DNA binding domain of FacB. A facB allele containing a mutated Zn(II)2Cys6 cluster fails to complement a facB null mutant for growth on acetate. Thus, the DNA binding cluster is essential for FacB function. A fusion protein containing a mutated zinc cluster has been expressed in E. coli to confirm that DNA binding activity is abolished.

58. Methylation and gene silencing in Neurospora

Jeff Irelan and Eric Selker. University of Oregon.

Regional inhibition of gene expression by epigenetic means, or gene silencing, is responsible for phenomena as diverse as silencing of mating type information in yeast to imprinting in mammals. DNA methylation is thought to be involved in many gene silencing phenomena. Due to the existence of mutants deficient in methylation, Neurospora crassa offers an opportunity to determine the role of methylation in various aspects of gene silencing. We found that silencing by al-1 trangenes, or quelling, is not affected in a mutant, dim-2, that lacks cytosine methylation. In contrast, we found that methylation resulting from RIP can result in epigenetic silencing of a single- copy gene, hph, located between the elements of a duplication of the am gene. The effect of methylation on gene expression varies widely from case to case, and probably occurs by inhibition of transcription. Spontaneous revertants of hph are re-silenced, although this re-silencing can require many cell divisions, indicating that epigenetically induced gene expression states are somewhat stable. The spectrum of gene expression states, the role of methylation in the stability of the epigenetic mark, and the use of epigenetically silenced genes to isolate and characterize methylation mutants was discussed.

59. Detection of a protein which binds specifically to the upstream region of the pcbAB gene in Penicillium chrysogenum

Yiu-Wai Chu, Didier Renno* and Gunter Saunders, University of Westminster, 115 New Cavendish Street, London W1M 8JS, UK and *Xenova Ltd., 545 Ipswich Road, Slough SL1 4EQ, UK

An electromobility shift assay (EMSA) was previously used to screen for specific proteins binding to the upstream region of the pcbAB gene, one of the three genes encoding enzymes for penicillin biosynthesis in Penicillium chrysogenum. A specific DNA-protein interaction was detected within a fragment covering the region -387 to -242 relative to the pcbAB translational start codon. The involvement of a 7 basepair motif TGCCAAG in the binding was demonstrated in this work by cross competition EMSAs. The detection of this protein and pcbAB-mRNA in culture extracts occurred at the same time point fermentations suggesting that the protein might be a transcription activator.

60. Cosmid and YAC clone anchored genetic map of Magnaporthe grisea

Jinrong Xu and John E. Hamer, Purdue University, West Lafayette, IN 47907-1392

The rice blast fungus M. grisea contains repetitive sequences called MGR sequences. One of these repetitive elements, MGR586, is dispersed over the genome and has been used in the construction of a genetic map. A cosmid library in pcosAX and a YAC library in pYAC4 were constructed from the M. grisea mapping strain 4375-R-26. Cosmid and YAC clones containing the MGR586 sequence were identified by colony in situ hybridization. These clones were then mapped onto the MGR586-based genetic map. This genetic map, with anchored MGR586 cosmid and YAC clones, will be useful to construct a regional physical map and for other genomic analyses.

61. Origin of a new plasmid by heterologous recombination between a circular and a linear plasmid in Neurospora mitochondria.

Tony Griffiths and Xiao Yang, Botany Department, UBC, Vancouver, Canada V6T 1Z4.

A strain of N. intermedia from China contains three prominent linear plasmids plus two different circular plasmids. In one subculture of this strain, a new linear plasmid arose spontaneously. The plasmid was named Harbin-L Simultaneously, one circular and three linear plasmids disappeared. Restriction analysis and sequencing revealed that the new linear plasmid was formed by a double recombination event which slotted part of a circular plasmid into part of one of the linear plasmids. The sequences at the junctions show that two 'crossovers' occurred at sites where there was seven base pairs of homology between the parental plasmids. One of the open reading frames of the new linear plasmid spans a junction site, so is composed of sequences from both parental elements.

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