Aspergillus

95. The gene product of hapC is a subunit of AnCP/AnCF, a CCAAT-binding protein in Aspergillus nidulans.

M. Kato, A. Aoyama, F. Naruse, Y. Tateyama, P. Papagiannopoulos1, M. A. Davis1, M. J. Hynes1, T. Kobayashi,

N. Tsukagoshi. Nagoya University, Nagoya 464-01,Japan. 1 University of Melbourne, Parkville, Victoria 3052, Australia.

CCAAT sequences in promoter regions of many fungal genes such as the taa (Taka-amylase A) and amdS (acetamidase) genes play important roles in the determination of expression levels. We have shown that an Aspergillus nidulans CCAAT-binding protein (factor), AnCP/AnCF recognizes CCAAT sequences in several genes in A. nidulans. The hapC gene, an A. nidulans counterpart of the yeast HAP3 gene, was isolated and used to obtain hapC disruptants. No CCAAT binding activity was detected in nuclear extracts from a hapC disruptant. Taken together with the high similarity of HapC to the HAP3 protein this result suggests that the hapC gene product is a subunit of AnCP/AnCF.

To examine whether or not HapC is a component of the AnCP/AnCF complex, a recombinant MalE-HapC fusion protein was produced in E. coli and purified. AnCP was denatured in the presence of MalE-HapC, renatured, and used for gel shift assays. The shift band corresponding to a taa promoter-AnCP complex disappeared and a new band with lower mobility was observed. When anti-MalE antiserum was added to the binding reaction, the band was supershifted. These results indicate that the MalE-HapC fusion protein was functionally incorporated into the AnCP/AnCF complex bound to the CCAAT containing sequence. This clearly demonstrates that the hapC gene encodes a subunit of AnCP/AnCF.

96. Regulation of Septum Formation in Aspergillus nidulans by a DNA-damage Checkpoint Pathway.

Peter Kraus and Steven D. Harris, Department of Microbiology, University of Connecticut Health Center, Farmington, CT 06030-3205

Cytokinesis (septation) in Aspergillus nidulans germlings is delayed until three rounds of nuclear division have occurred. Conditional mutations in the sepB gene block septation but allow germinating conidia to complete the first three rounds of nuclear division. This phenotype can be mimicked by germinating wild-type conidia in the presence of sub-lethal concentrations of the DNA damage-inducing agent diepoxyoctane (DEO) and the replication inhibitor hydroxyurea (HU). The effect of the sepB mutation and the response to DEO are both suppressed under conditions where inhibitory phosphorylation of p34nimX kinase does not occur, indicating that this kinase is a target of the machinery regulating cytokinesis. The sepB phenotype and the DEO response can also be suppressed by loss-of-function mutations in the DNA damage checkpoint genes uvsB and uvsD, suggesting that the pathway which determines the appropriate timing of cytokinesis is the same pathway which prevents mitosis in the presence of DNA damage. A model which accounts for the regulation of septum formation by p34nimX will be presented.

We have initiated two screens to identify additional components of the regulatory network which controls septation. For one screen, a collection of mutants which are sensitive to low levels of MMS and HU has been generated. This collection has yielded several mutants which form septa inappropriately (i.e. in the presence of DNA damage). In a second screen, HU-sensitive pseudorevertants of the Ts nimT23 mutation were collected and are being analayzed for the precocious formation of septa. Details of these screens will be presented.

97. Identification and characterization of Aspergillus nidulans genes required for establishment and/or maintenance of Cell polarity.

Maurice P. Lee and Steven D. Harris, Department of Microbiology, University of Connecticut Health Center, Farmington, CT 06030.

Establishment and maintenance of cell polarity are essential for the growth and development of the fungus Aspergillus nidulans. We have identified four complementation groups of temperature sensitive polarity defective (pod) mutants that are unable to establish and maintain cell polarity based on the following criteria: (i) failure of germinating spores to produce a germ tube, (ii) enlargement of spores with continued growth in a depolarized manner, and (iii) accumulation of multiple nuclei since pod mutations should not affect nuclear division. We have characterized the phenotypes of these mutants in detail and are currently initiating attempts to clone the pod genes.

As an alternative approach, we are cloning genes known to play an essential role in cell polarity in other organisms. One such gene is CDC42, originally identified in the budding yeast Saccharomyces cerevisiae. Cdc42p is a member of the ras superfamily of low-molecular-weight GTP-binding proteins. Yeast cdc42 mutants display an inability to establish cell polarity during budding. Functional homologues of Cdc42p have also been identified in such varied species as S. pombe, C. elegans, Drosophila, and human cells suggesting that it may be a fundamental component of the mechanism controlling cell polarity in all eukaryotes. It is reasonable to expect that the homologue of the CDC42 gene in A. nidulans is required for polarized vegetative growth and/or conidiophore development. We have cloned the A. nidulans homologue of CDC42 and have begun characterization of the null phenotype and the constitutively activating and inactivating mutations of Cdc42p.

98. The sequence of palF, an environmental pH response gene in Aspergillus nidulans.

Walter Maccheroni Jr.a, Gregory S. Mayb, Nilce M. Martinez-Rossia and Antonio Rossic. aDept of Genetics, FMRP, and cDept of Chemistry, FFCLRP, USP, Ribeirao Preto, SP , Brazil; and bDept of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Aspergillus nidulans, and probably all other living organisms, alter gene expression as an adaptive response to environmental pH changes. The pal genes probably constitute part of a signaling pathway that responds to changes in environmental pH. To molecularly characterize the influence of external pH in the secretion of enzymes by filamentous fungi, we have cloned and sequenced the palF gene of Aspergillus nidulans. cDNA clones were obtained from a lgt10 cDNA library and sequenced, showing a nucleotide sequence of 3,320 bp. A major 2,325 bp long ORF was identified and its translation resulted in a putative protein of 775 aa, with a predicted molecular mass of 84,086 Da. A large (828 bp) 5'-UTR was observed and a putative polyadenylation signal was identified in the 3'-UTR. This large 5'-UTR is very unusual for fungal transcripts and is the largest thus far noted. The palF nucleotide sequence did not show any clear homology with any known nucleotide sequence and the putative PALF protein shared some sequence similarity with the putative products of Saccharomyces chromosome VII ORFs YGL045w and YGL046w. The similarities seen between the predicted PALF protein and these ORFs are restricted to regions of 18 to 57 aa in length and had similarities of 43 to 72%.

Financial support: FAPESP and CNPq (Brazil) and NIH and NSF (USA).



99. Characterization of sepH, a gene encoding a protein kinase required for septation in Aspergillus nidulans.

Jennifer L. Morrell and John E. Hamer, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392.

We are investigating the process of septum formation in the filamentous fungus, Aspergillus nidulans. The hyphae of A. nidulans are partitioned into multinucleate compartments by the formation of septa. The earliest visible sign of septation is the assembly of an actin ring which anticipates the site of septum formation. Following the completion of nuclear division, the actin ring decreases in diameter and cell wall material is deposited to form the septum (M. Momany and J.E. Hamer, submitted). We have isolated a collection of ts mutants defective in septum formation (Harris et al., 1994. Genetics 136:517532). One of these mutants, sepH, appears to be required late in the process of septum formation. This mutant is unable to form septa but displays no defects in growth or nuclear division, and thus produces long, multinucleate hyphae at the restrictive temperature. The sepH gene has been cloned and encodes a protein kinase with similarity to the protein kinase encoded by cdc7 in S. pombe, which is also required for septum formation. Phenotypic analysis of the sepH null mutant indicates the presence of faint actin rings. Based on these results, a model for SepH function will be proposed.

100. The regulator of nitrate assimilation in Aspergillus nidulans binds a completely asymmetrical sequence as a dimer.

M. I. Muro-Pastor, J. Strauss and C. Scazzocchio Institut de Genetique et Microbiologie, URA 1354, Universite Paris-Sud, Batiment 409, 91405 Orsay CEDEX, France.

NirA protein mediates nitrate induction of the niaD, niiA and crnA genes of Aspergillus nidulans, encoding respectively nitrate reductase, nitrite reductase and a nitrate permease. We have studied in detail the mode of binding of NirA to its cognate DNA target sequences. The sequence bound by NirA is the asymmetrical sequence 5'CTCCGHGG3'. Four such sequences are present in the intergenic region which lies between the divergently transcribed niiA and niaD genes. We have studied the binding of NirA to the region by DNaseI and methylation protection, and by methylation, depurination and depyrimidation interference, the seven bases of the consensus sequence interfere strongly. Methylation interference reveals that the phosphate contacts are clearly asymmetrical. We have also shown that the NirA protein binds as a dimer. This was shown by the expression of two NirA partial peptides of different lengths in an in vitro transcription/translation system. The formation of the heterodimer is evident in gel shifts assays. We have shown that the putative coiled-coil domain, carboxy terminal to the DNA binding domain can replace the cI dimerisation element in an in vivo lambda immunity assay. We have also carried out a mutagenesis analysis of the NirA dimerisation domain. These data show an unprecedented mode of binding of the NirA protein to its cognate DNA.

101. Improved enzyme expression in Aspergillus oryzae due to vector construction and chromosomal amdS deletion.

B.A. Nelson, J.R. Shuster, M.W. Rey, D.S. Yaver, Novo Nordisk Biotech, Davis, CA.

A. oryzae has been used industrially to produce heterologous proteins of commercial interest. We have investigated the effects on heterologous expression of different selectable markers and the topology of the transforming DNA on the expression of a heterologous lipase in this fungus. Different vectors were used to transform A. oryzae hosts and approximately 100 transformants of each were tested in shake flask cultures for their ability to produce extracellular lipase. We report here the distribution of lipase yields observed from the transformants. The following observations were made: i. lipase yields of single vector transformants are on average higher than those produced by cotransformants; ii. lipase yields of transformants generated using a linearized expression cassette which does not contain E. coli vector sequence are on average higher than those produced by transformants generated with circular plasmid; iii. linearization of the expression plasmid leads to an increase in transformation frequency but does not have an effect on lipase yields; iv. the production of lipase is increased on average using amdS versus pyrG as the selectable marker; v. the presence of a truncated amdS promoter leads to an increase in lipase production on average. We have also examined the effect of disrupting the chromosomal amdS gene because background growth is observed with A. oryzae on plates with acetamide as the sole nitrogen source.

The presence of a amdS locus in the strain to be transformed shifts the distribution of lipase production towards the higher end and reduces background growth.

102. Identification of Protein kinase C in Aspergillus nidulans.

E.J. Prebble, G.D. Robson and A.J. Dickson, School of Biological Sciences, University of Manchester, 1.800 Stopford Building, Oxford Road, Manchester, M13 9PT, U.K.

A 600 base pair (bp) PCR product was amplified from Aspergillus nidulans genomic DNA using degenerate oligonucleotide primers designed to eukaryotic Protein kinase C (PKC) gene sequences. This product was cloned and sequenced and its predicted amino acid sequence has 90% identity with the pedicted amino acid sequence (aa 837-942) from an Aspergillus niger PKC gene (Morawetz et al., 1996. Mol. Gen. Genet. 250: 17-28). The PCR product was labelled and used to probe a Southern blot of restricted A. nidulans genomic DNA revealing a single BamH1 digestion product of 6kb. Immunoblotting using anti-PKC antisera raised to a conserved PKC peptide reveals protein bands at 140, 50 and 40kDa in A. nidulans membrane protein fraction and 140, 90 and 40kDa in A. nidulans cytosolic protein fraction. The effect of two PKC inhibitors (chelerythrine chloride and bisindolylmaleimede) and a PKC activator (a phorbol ester) on the growth and morphology of A.nidulans were assessed. No significant effects on specific growth rate in A. nidulans were observed. However, germlings grown on the two inhibitors were consistently smaller than controls and germlings grown on the activator were consistently larger than controls. This could indicate a role for PKC in spore germination.

103. Analysis of the protein secretion pathway of Aspergillus niger.

Peter J. Punt, Gerrit Veldhiusen, Anneke Drint and Cees A.M.J.J. van den Hondel. Department of Molecular Genetics and Gene Technology, TNO Nutrition and Food Research Institute, P.O. Box 360, 3 700 AJ Zeist, The Netherlands.

Filamentous fungi, Such as A.niger, secrete large amounts of proteins into the culture medium. However, little information is available on the molecular mechanisms of protein secretion in these organisms. We have started research to analyse the mechanism of protein targeting/secretion and to identify and resolve potential bottlenecks for efficient protein overproduction (1,2).

I To identify the different compartments of the secretion pathway and to study the routing of proteins to these compartments, several genes encoding compartment specefic proteins have been isolated. The cloning and characterisation of the A.niger bipA gene, encoding the major ER-chaperone, will be presented together with initial results aimed at the overexpression of the ER-chaperone to improve the yields of secreted heterologous protein.

II Based on research carried out in S. cerevisae, defined mutations in the secretion pathway will provide important information about the organisation of the pathway. Therefore, the cloning of several various genes encoding small GTP-binding proteins involved in the vesicle transport between the different compartments of the secretion pathway, was started using heterologous hybridisation and PCR-based approaches. The cloning of several of these small GTPase encoding genes will be presented together with initial results on A. niger strains carrying mutant alleles of one of these genes (sarA)

(1) Punt, P.J et al (1994) Anthony van Leeuwenhoek 65.- 211-216. (2) Gouka et al. (1997) Appl Microbiol Biotechnol, in press

104. sepA is Necessary for Normal Cytokinesis During Vegetative Growth and Conidiation in Aspergillus nidulans. Kathryn E. Sharpless and Steven D. Harris, Department of Microbiology, University of Connecticut Health Center, Farmington, CT 06030-3205.

Ts mutations in the Aspergillus nidulans sepA gene affect a variety of morphogenetic processes in hyphal cells. Specifically, these mutations prevent septation and lead to an inability to maintain hyphal polarity. A. nidulans is dimorphic, in the sense that the pattern of cell division is altered during conidiophore formation to a mode which is more similar to that of budding in yeast. We have used two approaches to analyze the effect of sepA mutations on cell division during conidiation.

Strains in which the regulation of the important developmental genes, brlA and abaA, is altered undergo inappropriate development. The induction of alcA::brlA leads to the formation of single spores at hyphal tips. Furthermore, these spores are subtended by "collar-like" septa, which appear similar to those formed in normal conidiophores. Induction of alcA::abaA leads to the formation of unusually thick septa throughout the hyphae. We have found that the formation of septa in alcA::brlA and alcA::abaA is dependent upon the presence of a functional sepA gene product.

A deletion mutant of sepA grows slowly and is capable of making conidia, although fewer than normal. Analysis of conidiophores in the sepA deletion mutant revealed that many undergo uncharacteristic branching. In addition, many spores fail to bud properly resulting in the production of multiple connected spores. These results suggest that the sepA gene product is required for cytokinesis in both hyphal cells and in developing conidiophores. Furthermore, they also indicate that sepA is necessary for maintenance of polarity during conidiophore development.

105. Meiotic Mutants of Aspergillus nidulans: Phenotypes, Gene Localization and Gene Cloning.

Klaas Swart, Marijke Slakhorst, Gerda Kobus, Edu Holub and Diana van Heemst. Department of Genetics, Wageningen Agricultural University Dreijenlaan, The Netherlands.

More then twenty meiotic mutants of Aspergillus nidulans have been isolated (identified by the lack of ascospores in well developed cleistothecia) and tested for complementation. Eighteen different complementation groups were identified. Five mutant genes were assigned to linkage groups. Three mutants were characterized cytologically: one appeared to be blocked at karyogamy (karA1), another at prophase I (meiC8) and a third one at metaphase I (meiB7). The karA1 mutant has been transformed with a linkage group VI specific cosmid library and the complementing wild type gene has been isolated. Further characterization of this gene is in progress.

106. Isolation and characterization of Aspergillus nidulans delayed conidiation mutants.

J. Wieser and T. Adams. Texas A&M University College Station, TX 77843.

The formation of conidiophores in A. nidulans is a precisely regulated event that occurs within the context of a radially expanding colony. Conidiophores typically differentiate 1-2 mm behind the leading edge of vegetatively growing hyphae. Mutations in any of four A. nidulans genes flbB,flbC,flbD, and flbE result in fluffy colonies that are delayed at least 24 hours in their ability to form conidiophores. This delay in conidiophore initiation gives rise to colonies in which conidiophore development occurs 12-15 mm behind the leading edge of the radially growing hyphae. Each of the four genes is required for normal timing of brlA expression indicating a potential role for each in activation of this important developmental regulatory gene. Wild-type copies of all four genes have been isolated and transcripts of all the genes were shown to be present throughout the A. nidulans life cycle. The deduced flbC protein sequence predicts a polypeptide with significant similarity to C2H2 zinc finger proteins, while the flbD protein sequence predicts a polypeptide with significant identity at its N-terminus to the DNA binding domain of the myb family of transcription factors. The sequence analysis of flbB and flbE is in progress. The genetic interactions among flbB, flbC, flbD, and flbE and the other genes required for development is discussed.

107. Effects of two mutant actin alleles on growth and morphology in Aspergillus nidulans.

Tom D. Wolkow and John E. Hamer, Purdue University, West Lafayette, IN 47907

The mycelium of Aspergillus nidulans is composed of multinucleate cellular compartments delimited by crosswalls called septa. Initiation of septum formation occurs in germlings which have achieved a minimal cell size and undergone at least one nuclear division. Evidence suggests that the future site of septum formation in A. nidulans is marked by an actin ring. The actin ring forms over a mitotic nucleus and persists until cytokinesis and septation begin, at which time the actin ring invaginates, leaving behind a ring of chitin and other septal material. The placement of septa in mycelium, and presumably the actin ring, can be manipulated by altering the distribution of nuclei. Two mutant alleles of actin were constructed (based on previous experiments in Saccharomyces cerevisiae) to test the role of actin in determining the site of cytokinesis and septation in A. nidulans. Although each allele was designed to be temperature-sensitive, they arrested growth shortly after germination at the permissive temperature. Germlings displayed morphologic abnormalities but did not appear compromised for septum formation or positioning.

108. Characterization of the Aspergillus oryzae brlA gene by its overexpression and disruption

Osamu Yamada, Byung Rho Lee, Katsuya Gomi and Yuzuru Iimura National Research Institute of Brewing

Aspergillus oryzae conidiospores (called tane-koji in Japan) have been used for making koji as a supplier of hydrolytic enzymes and nutrients in the Japanese fermentation industry, such as sake, soy sauce and miso. It is therefore, of great importance in tane-koji manufacturing that industrial strains of A. oryzae can produce conidiospores efficiently. To understand the mechanism and control of A. oryzae conidiation, at first we cloned brlA gene from A. oryzae RIB40. The brlA gene of A. oryzae contained an open reading frame coding for 421 amino acid residues and the deduced amino acids sequence revealed 70% homology with A. nidulans BRLA. To investigate the function of the A. oryzae brlA gene, we constructed (-amylase promoter-brlA fusion gene (PamyB-brlA) and introduced into A. oryzae niaD300. Interestingly, these transformants of the PamyB-brlA fusion gene could form conidiophore even in the submerged culture supplemented with maltose under control of the -amylase promoter. Furthermore, the brlA gene disruptant of A. oryzae could not develop any conidiophore, indicating that the A. oryzae brlA gene is also main regulator of conidiation.