Neurospora

134. Calcineurin of Neurospora crassa, Essential for Hyphal Growth, Morphology and the Apical Ca2⁺ -Gradient - an Inducible Loss of Function Analysis.

Holger Prokisch*, Oded Yarden⁺, Margit Mischler^#, Maximilian Tropschug^#, Ilse Babette Barthelmess* * Universität

Hannover, Germany; ⁺ The Hebrew University of Jerusalem, Israel; and ^# Universität Freiburg, Germany .

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct for the inducible expression of the catalytic subunit of calcineurin (CNA) antisense-RNA. No detectable cna-1 mRNA, reduced levels of immunodetectable CNA1 protein and decreased calcineurin specific enzyme activity, on induction medium only, were evidence that a conditional reduction of the target function had been achieved in the isolated antisense-transformants. In these transformants induction conditions procured a growth arrest which occurred after extensive hyphal branching, changes of hyphal morphology and concomitant loss of the distinctive tip-high Ca2⁺-gradient typical for growing wild-type hyphae. The phenotype of the loss of function mutants' created by antisense-technique indicated that the cna-1 gene of N. crassa, coding for the catalytic subunit of calcineurin, is an essential gene. In vitro inhibiton of N. crassa calcineurin by the complex of CsA and cyclophilin20 and increased sensitivity of the induced transformants to the calcineurin specific drugs, cyclosporin (CsA) and FK506, were evidence that the drugs act in N. crassa, as in Saccharomyces cerevisiae and T-cells, by inactivating calcineurin. Consistently, the exposure of growing wild-type mycelium to CsA or FK506, respectively, led to a phenotype very similar to that of the cna-1 antisense-mutants, i.e. the disturbance of the Ca2⁺-gradient, hyperbranching, altered hyphal morphology and ultimately to growth arrest. Together these data indicate for the first time a crucial role of calcineurin in the precise regulation of apical growth, a common form of cellular proliferation.

135. Characterization of a translocation in the mutant, SS-656, of Neurospora crassa.

Amy Richardson, Wavne A. Krissinger, and Sara Neville Bennett. Georgia Southern University.

SS-656, an osmotic-sensitive mutant of N. crassa, was isolated in the Georgia Southern Neurospora Genetics Laboratory following UV irradiation of wild type conidia. The morphology of SS-656 resembled that of the wild type strain and differed from the altered morphology of typical osmotic sensitive mutants. Crosses to the alcoy tester strain to begin mapping the osmotic sensitivity of the mutant produced conflicting results which suggested the possibility of a translocation. Analysis of a cross of SS-656 to the tester strain fluffy (OR) indicated 21 % of shot ascospores were white rather than the normal black, supporting the presence of a translocation. A total of 220 unordered tetrads from the same cross produced black to white ascospore ratios of 8:0 (58.2%); 6:2 (15.5%); 4:4 (23.6%); 2:6 (1.8%); 0:8 (9%) which further supported the presence of a translocation. When the progeny were crossed to wild type, 50.6% of the progeny produced perithecia but shot few to no ascospores and were classed as barren. Examination of the culture tubes of the fertile progeny showed an abundance of white ascospores from 11 of the crosses. Four of the 11 were examined for numbers of white ascospores. One produced a normal number of white ascospores. The other three, two of which were not osmotic-sensitive, produced 25.4%, 20.1%, and 20.5% white ascospores among those shot, indicating that the translocation went through the cross and segregated from the osmotic-sensitive trait. Crosses of SS-656 with multicent, a tester strain useful for mapping translocations, have been made.

136. Analysis of clock-controlled conidiation in Neurospora crassa.

Mari L. Shinohara, Deborah Bell-Pedersen, Jennifer J. Loros, and Jay C. Dunlap. Dartmouth Medical School, Hanover, NH.

A circadian biological clock controls several aspects of growth and development in Neurospora crassa, including the timing of the initiation of conidiogenesis. In addition to previously isolated clock-controlled genes, ccg-1 (grg-1) and eas (ccg-2), differential screening using 4 cDNA libraries representing different times of day uncovered 6 new clock-controlled genes. Transcripts from each gene cycle in abundance, with peak accumulation occurring in the late-night to early-morning (Bell-Pedersen et al. 1996. PNAS 93: 13096). Sequence analysis revealed that ccg-7 encodes glyceraldehyde 3-phosphate dehydrogenase (a glycolytic enzyme), ccg-12 to be the N. crassa copper metallothionein gene (cmt), and the others encode novel genes. Considering the ccg-4, ccg-7 and cmt (ccg-12) transcripts are not induced by light or conidial development, their function may be distinct from conidiation. Disruption of ccg-6 and ccg-9 by RIP results in strains which possess morphologically abnormal conidia. Additionally, these strains display a loss of clock-controlled conidial banding on race tubes. In both strains, however, the clock functions normally as shown by cycling of the FRQ protein, a central clock component. This suggests that CCG-6 and CCG-9 might work as messengers transducing a time-of-day signal from the oscillator to an overt rhythm, i.e. circadian conidiation. Our preliminary data indicates that cycling of the ccg-4, ccg-6, ccg-7 and ccg-12 transcripts is severely diminished in the ccg-9^RIP strain, consistent with a requirement for the ccg-9 product in circadian regulation of these genes.

137. Cloning of the last iron-sulfur protein and mapping of complex I genes of Neurospora.

R. Sousa¹, B. Barquera², M. Duarte¹, M. Finel² and A. Videira1. ¹University of Porto, Portugal and ²University of Helsinki, Finland.

Complex I is a component of the mitochondrial respiratory chain, containing more than 30 polypeptide subunits, that transfers electrons from NADH to ubiquinone through a series of protein-linked prosthetic groups, FMN and [Fe-S] clusters. Prokaryotes contain enzymes similar to complex I, called NDH-1, but of simpler composition. Their subunits are homologous of the seven subunits of complex I that are coded by mitochondria and of further seven nuclear-coded subunits. Of these, a [Fe-S] protein, named PSST in bovine, was never identified in Neurospora crassa. Based on conserved amino acid sequences of the protein, we designed degenerated primers and synthesised a DNA probe by PCR. This probe was used in the screening of a cDNA library and the Neurospora PSST homologue was cloned. We will present further characterisation of the gene and of the protein. Furthermore, the chromosomal location of several nuclear genes coding for complex I subunits, that have been achieved through RFLP mapping, will be indicated.

138. Analysis of het-C heterokaryon incompatibility in Neurospora crassa.

Richard B. Todd, Sven J. Saupe and N. Louise Glass. Biotechnology Laboratory, University of British Columbia, Vancouver BC, CANADA.

Fusion of hyphae from genetically distinct fungal individuals (heterokaryosis) during growth results in the formation of heterokaryons, hyphae which contain genetically different nuclei but share a common cytoplasm. Fungi have heterokaryon (or vegetative) incompatibility systems that recognise specific genetic differences between self and nonself and limit heterokaryon formation. In Neurospora crassa, at least 10 loci, called het loci, in addition to the mating type locus, elicit heterokaryon incompatibility.

The het-C locus is the best characterized of the het genes. Inhibited growth resulting from het-C incompatibility is observed in forced heterokaryons between individuals of different het-C genotype and in partial duplications heterozygous for het-C. The het-C gene encodes a 966 amino acid polypeptide which contains an Nterminal signal sequence motif, a central leucine zipper-like coiled-coil motif and a C-terminal glycine-rich region which shows similarity to extracellular structural proteins. Deletion constructs used in transformation assays indicate that the signal peptide is not required for incompatibility function. We are analysing the subcellular localization of the HET-C protein to provide insight into the molecular mechanisms of het-C incompatibility.

139. Arginine-specific translational regulation of Neurospora crassa arg-2 is associated with ribosome arrest at an upstream open reading frame in the mRNA.

Zhong Wang and Matthew S. Sachs, Oregon Graduate Institute, P.O. Box 91000, Portland OR.

An upstream open reading frame (uORF) in the N. crassa arg-2 mRNA has a role in negative translational regulation by Arg. We used a primer extension inhibition (toeprint) assay to examine arg-2 uORF-mediated translational regulation in a homologous cell-free translation system. Wild-type and mutant arg-2 uORF sequences were placed upstream of the luciferase polypeptide coding region and the distribution of the translational machinery on these arg-2ÐLUC RNA constructs examined. In analyses of RNA containing uORF sequences, prematurely terminated reverse-transcription products were found 16 nt distal from the uORF start codon, 13 nt distal from the uORF termination codon and 16 nt distal from the LUC start codon. The appearance of these toeprint signals depended on the coding sequence and coding capacity of the uORF; their appearance also was affected by translational inhibitors such as puromycin. These toeprint sites appear to correspond to ribosomes positioned on RNA with initiation codons at their P sites and termination codons at their A sites, respectively. When surplus Arg was added to translation reactions, a marked increase in the intensity of the toeprint signal at the wild-type uORF termination codon was observed, and an additional signal appeared 21-30 nt upstream of this codon. A decrease in the toeprint signal at the luciferase initiation codon was also observed. These results suggest that an Arg-mediated increase in ribosomal arrest at the uORF termination codon causes a block in the movement of ribosomes and reduces ribosome loading at the downstream luciferase initiation codon. uORF-mediated ribosomal arrest appears central to the Arg-specific translational regulation observed in vitro.

140. Expressing Zeamatin in Neurospora crassa.

Shelly R. Wilson, Jeff S. Palas, Veronica J. Wolf, Cathy S. Taft, and Claude P. Selitrennikoff. MycoTox, Inc., Denver, CO.

We have investigated the ability of a fungus, Neurospora crassa, to secrete zeamatin, a protein produced by Zea mays. Heterologous expression of plant genes by N. crassa may serve as an important alternative for producing plant proteins difficult to isolate or to obtain in useful amounts by other means. Zeamatin was expressed as a fusion protein with glucoamylase, an extracellular hydrolase induced by N. crassa upon depletion of glucose from the culture medium. A kex-2 protease site was engineered at the N-terminus of the zeamatin cDNA, allowing cleavage of chimeric product in the secretary pathway. The modified zeamatin cDNA was inserted into a plasmid (pGE) containing the glucoamylase promoter, a truncated glucoamylase open reading frame and the glucoamylase terminator sequence, to form the plasmid pGEZ. N. crassa strains were co-transformed with pGEZ and a plasmid containing selectable markers for hygromycin resistance (pMP6) or to restore histidine (his-3) autotrophy. Transformed strains of N. crassa were screened by polymerase chain reaction analysis for the pGEZ insert using zeamatin-specific primers. Colonies positive for the zeamatin gene were grown in liquid culture and induced for glucoamylase and zeamatin production. Concentrated culture media were tested for the presence of secreted zeamatin protein using western blot analysis with an anti-zeamatin polyclonal antibody.