Abstracts for Talks

 

Session 1 - Cell Biology

 

The exocytic machinery in tip growth of Neurospora crassa.

Gagan Gupta and I. Brent Heath, York University Biology Department, North York, Ontario, Canada M3J 1P3

 

Tip growth of hyphae requires continuous and directed exocytosis of vesicles bearing new membrane and wall material.  In other words, a gradient of vesicle fusions (highest at the hyphal apex) must be maintained so as to generate the hyphal shape.  One model predicts that this gradient is due to the activity of a hypothetical vesicle supply center (VSC) which, due to its apical location, can concentrate hyphal tips with vesicles that subsequently undergo exocytosis.  However, selective targeting and exocytosis of vesicles can also be achieved by a different, more ubiquitous mechanism.  SNAREs are highly conserved integral membrane proteins that are believed to confer specificity to vesicle docking and fusion reactions on the basis of protein-protein recognition. They have been well characterized in many eukaryotes from yeast to plants and mammals, and it is therefore likely that they are also present in filamentous fungi.  We present immunological, biochemical and genetic evidence for the presence of SNAREs in Neurospora crassa and its implications for tip growth models.

 

Apolar growth of Neurospora crassa leads to increased secretion of extracellular proteins. In Hyung Leel, Rodney G. Walline2, and Michael Plamannl, lSchool of Biologcial Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, 2Department of Human Anatomy, Texas A&M University,College Station, TX 77843-1114

 

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount of growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature-sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive-temperature. Incubation of the mcb mutant at restrictive-temperature results in a three- to five-fold increase in the level of extracellular protein and a five- to 20-fold increase in carboxymethyl cellulase activity relative to a wild-type strain. A mutation in the cr-1 gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb; cr-1 double mutant was reduced to that of the wild-type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced following a shift of the mcb mutant to restrictive-temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.

 

Type 2A protein phosphatase is involved in growth and reproduction in Neurospora crassa.  Einat Yatzkan and Oded Yarden, The Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

 

Protein phosphatases (PP) are an integral part of the reversible phosphorylation regulatory machinery, and are key elements in maintaining the balance of many cellular activities. The core structure of PP2A (which belongs to the ser/thr PPP gene superfamily) consists of a 35kDa catalytic subunit (PP2Ac) tightly complexed with a 65 kDa regulatory subunit (A-subunit). The core dimer complexes with a third, variable component (B-subunit) which controls enzyme activity and specificity. In the presence of the PP inhibitors Cantharidin and Calyculin A N. crassa hyphal growth and integrity were abnormal. The observed effects included higher branching frequencies and cell swelling and lysis in the vicinity of hyphal tips. We have analyzed the structure and function of two components of the PP2A holoenzyme in Neurospora crassa.  The pph-1 (encoding PP2Ac) and rgb-1 (encoding the B-subunit) genes were isolated, sequenced and mapped. Both show high degrees of similarity to PP2A components isolated from other organisms. Failure to obtain viable progeny in which pph-1 had been inactivated via the Repeat-Induced Point mutations (RIP) process and evidence that nuclei harboring a disrupted pph-1 gene could only be maintained in a heterokaryon, indicated that a functional pph-1 gene is essential for fungal growth. Reduced pph-1 expression impaired hyphal elongation rates. Inactivation of rgb-1 by RIP produced slow growing, female sterile, progeny which produce abundant amounts of arthroconidia yet hardly any macro or micro-conidia.

Search for V-ATPase mutants results in interesting phenotypes.  Emma Jean Bowman, Ryan Kendle, Forest O'Neill, and Barry J. Bowman,  University of California, Santa Cruz, CA

 

The vacuolar ATPase generates an electrochemical gradient across the membranes of acidic compartments within the cell.  We have taken two approaches to try to obtain mutants in this enzyme:  (1) selection of strains resistant to concanamycin A, a specific inhibitor of V-ATPases and (2) the sheltered RIP method of Bob Metzenberg and co-workers to inactivate the catalytic subunit of the enzyme.  Many concanamycin A resistant strains proved to be mutated in pma-1, which encodes the plasma membrane H+-ATPase.  Others may be altered in the V-ATPase as suggested by a small, but reproducible increase in resistance to concanamycin in vitro and by their sensitivity to high pH or high histidine in the medium.  Putative vma-1RIP strains appear to grow only as small, tight colonies under all conditions tested.  Genetic or chemical inactivation of the vacuolar ATPase has a striking effect on hyphal branching patterns.

 

Characterization of three genes which encode amino acid and calcium transport proteins, and a comparison of the efficiency of RIPing these genes.  Emilio Margolles-Clark, Ian Hunt, Stephen Abrue, and Barry J. Bowman, University of California, Santa Cruz, CA 95064

 

Three genes have been identified which encode transport proteins with 12 membrane-spanning helices.  The gap-1 gene appears to encode a general amino acid permease.  We are pursuing experiments to determine if this is the same as the pmg gene previously identified by analysis of mutants; aap-2 encodes a protein that may be homologous to GABA transporters; and cax-1 encodes a calcium transporter in the vacuolar membrane.  We have attempted to inactivate each of these genes by RIPing.  In each case a copy of the gene was introduced into the his-3 locus, but the efficiency of RIPing was very different.  The cax-1 gene was RIPed at a relatively high rate (30%).  No RIPed progeny have yet been identified in gap-1 (40 analyzed) or in aap-2 (20 analyzed).

 

Heterokaryon Incompatibility Revisited.  J.F. Wilson, University of North Carolina at Greensboro

 

A gene with activity similar to that of the I/i genes described by Pittenger in Oak Ridge strains has been found in Rockefeller-Lindegren strains.  We called this gene and its alternate allele, Hi/hi, for Heterokaryon Instability. The major difference in the two sets of genes is the Hi allele is dominant, even in extreme ratios, in the RL background.  Results of crosses with appropriate strains indicate that Hi/hi and I/i are identical or closely linked, so operationally they can be considered the same.  The OR-SL wild types are i; RL wild types, I; and the Wilson-Garnjobst testers, an I/i mixture.  The I,i  genotypes of FGSC CDE and Cde testers, and some other strains are now known.   Contrary to expectations, some OR and SL mutants are het-I.  Another source of variation in heterokaryon stability is the almost accidental nature of hyphal fusions between RL and OR-SL strains.  The normal intra-strain stimulus-response mechanism is missing in inter-strain fusions.  In combination, the two factors result in wildly different growth curves in heterokaryons.  In addition, we have found two hyphal fusion mutants which form heterokaryons late or not at all.  We now have het mutations involving fusions, the killing reaction, and stability. Perhaps it is time to consider a classification of het mutants based on the sequence of events that occur in the formation of a stable heterokaryon, instead of lumping all together under the term "incompatibility".

 

Loss of small, plasmid-like, mtDNA derivatives during sexual reproduction.  Helmut Bertrand, Katherine A. Nummy and Georg Hausner, Michigan State University, East Lansing, MI

 

Circular, plasmid-like elements (PLEs) consisting of tandem repeats of a segment of the mitochondrial chromosome (mtDNA) appear in high copy-numbers in the mitochondria of some cytochrome‑deficient and/or UV-sensitive mutants of Neurospora crassa.  Results obtained from 2-D gel electrophoresis and electron microscopy indicate that PLEs replicate by a recombination‑dependent rolling circle mechanism.  All PLEs are stably maintained in vegetatively propagated cultures, and, like true mitochondrial plasmids and suppressive mutant forms of mtDNA, they are transmitted horizontally and invasively through hyphal anastomoses.  However, some small PLEs are inherited by only a small fraction of the ascospore progeny (about 1/20) from the female parent in crosses.  Tetrad analyses show that such PLEs appear either in all or none of the products of any given meiotic event, whereas true mitochondrial plasmids and mutant mtDNAs are transmitted from the female parent to all the progeny in all the tetrads.  This erratic pattern of maternal inheritance can not be attributed to heteroplasmy in the female parent, for there are no PLE-free segregants among single conidial isolates from these strains.  Thus, the results suggest that some high-copy-number, replicatively-competent mtDNA derivatives lack an attribute required for consistent transmission of the mitochondrial chromosome and true plasmids from the maternal parent to the ascospore progeny.

Session 2 - Genomics

 

Control and Function of DNA Methylation in Neurospora crassa. Eric U. Selker, Institute of Molecular Biology, University of Oregon, Eugene, OR 97405

 

DNA methylation is not essential in Neurospora and only 2% of the genome is methylated. Nevertheless, a high fraction of sequences introduced by transformation, or mutated by repeat-induced point mutation (RIP) are methylated. DNA methylation is associated with gene silencing in many systems but the nature of the association is not well understood. Methylation of cytosines in sites recognized by transcription factors can interfere with factor binding, potentially blocking initiation directly. In addition, proteins that bind methylated sequences independent of sequence, may interfere with initiation and conceivably also interfere with elongation. We found that DNA methylation associated with products of RIP can prevent transcription elongation without affecting initiation. We have detected, and are currently purifying, Neurospora proteins that specifically recognize methylated sequences and may mediate this effect. Our work on the control of methylation revealed that de novo and maintenance methylation are operationally separable in Neurospora; preliminary results suggest they are mechanistically separable as well. I will summarize our latest understanding of the control and function of methylation in Neurospora including the relationship between RIP and DNA methylation.

 

Molecular analysis of regulated recombination hotspots in Neurospora  P. Jane Yeadon, Frederick J. Bowring and David E. A. Catcheside, The Flinders University of South Australia

 

Local meiotic recombination in Neurospora is regulated by at least two types of genes, the rec loci and the recombinators with which they interact. Three polymorphic rec loci are known and in each case the dominant rec allele reduces recombination in specific regions of the genome. cog, a recombinator regulated by the unlinked gene rec-2, was recognised by the existence of two alleles with differing effects on recombination in the histidine-3 region. The dominant allele of Lindegren Y8743 origin, cogLa, promotes recombination at a similar level to that seen at Saccharomyces cerevisiae (yeast) hotspots, significantly higher than the allele from Emerson, cogEa. The recombinator at the 5¢ end of am, located in a recombination-cold region, is regulated by rec-3 and seems functionally monomorphic.

We have cloned and sequenced both cog alleles within 10 kb that includes his-3 and the next gene distal to cog, lpl. The rec-2 locus has been located within a contig generated by a chromosome walk between am and sp. Sequence heterology, up to 6% close to cog and present at  lower level in the am coding sequence, has been used in molecular analysis of chromatids that have experienced meiotic recombination at both am and his-3 loci. Conversion at his-3 appears similar to that at yeast hotspots and seems highly associated with crossing over, though conversion tracts are more frequently discontinuous than in yeast. In contrast, conversion at am shows little association with crossing over. We are now analysing tetrads to extend the analysis.

 

Novel uses for Neurospora heterokaryons.  W. Dorsey Stuart,  Neugenesis Corporation, Honolulu, Hawaii

 

During the past year Neugenesis Corporation has continued to work with heterokaryons in Neurospora to produce heterodimeric molecules.  The molecule we use as a model for this work is humanized IgG.  We have previously shown that a Neurospora heterokaryon constructed so that one nucleus contains an expression cassette for an IgG kappa light chain and a second nucleus contains and expression cassette for an IgG gamma heavy chain will produce and secrete assembled and intact IgG molecules of 150,000 kD molecular weight.  We have now shown that combinatorial panels of IgG molecules can be assembled using subunits from different antibody genes.  We are also working on ways to increase the amount of IgG molecules produced by heterokaryons.  We are utilizing classical mutagenesis, molecular genetic knockouts and fusion proteins as tools in this effort.

 

Chromosome rearrangements that will not follow the rules.   Edward G. Barry, University of North Carolina, Chapel Hill

 

Reciprocal translocations, insertional translocations, quasiterminal  translocations, pericentric inversions, and an intrachromosomal transposition have been analyzed and mapped to linkage groups.  Most rearrangements are identified also by the type of ascospore patterns observed in crosses of rearrangement by normal sequence.  The patterns are ascertained from collections of eight ascospores shot from perithecia.  Ratios of octads with black (B) viable to white (W) aborted spores reveal the type of rearrangement  in many or most rearrangements studied (D. D. Perkins, 1997, Adv. Genet.  36:239-398).  Only paracentric inversions have not been detected by spore patterns.  Two chromosome rearrangements, which are not inversions, have been very difficult to work out: T(IIR;VIL)R2459 and T(II®(X);IV;V)AR179 (and also a derivative of AR179 with aberration only in linkage group IV). All three frequently produce asci containing seven or fewer spores. Sister spores, which should be identical, may not match, yielding ratios of, for example, 3B : 5W or 5B : 2W or even 3B : OW. When counting only groups of eight spores, this significant feature of these aberrations is overlooked.  Furthermore, isolation of ordered octads from R2459 crosses to normal wild type sequence reveal unexpected allele ratios: 8:0 or 5:3 for one marker while adjacent linked heterozygous markers are 4:4.  Other spores may show a mixed genotype as if they were partial diploids receiving both copies of the heterozygous alleles yet there is no corresponding deficiency, recognized as an aborted spore, in any other member of the octad.

 

The Neurospora Genome Project at the University of New Mexico: update.  Donald O. Natvigl, Mary Anne Nelsonl, Patricia L. Dolanl, Matthew E. Crawfordl, Edward L. Braunl,2 and Seogchan Kang3, l Department of Biology, University of New Mexico, Albuquerque, NM 87131, 2National Center for Genome Resources, Santa Fe, NM 87505 and 3Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802

 

We have been sequencing clones from four N. crassa cDNA libraries: germinating conidial, advanced-growth mycelial, perithecial and unfertilized sexual tissues.  Our strategy has employed both random clone selection and subtraction of clones representing highly-expressed genes.  To date, we have identified more than 1600 different genes.  We have also developed a system for classifying encoded proteins predicted from the results of data base searches.   Recent improvements at our World Wide Web site provide rapid review of proteins for which corresponding N. crassa cDNAs have been sequenced, along with links to homologous genes in GenBank and relevant clones from our libraries.  As a pilot project in genomic sequencing, we have nearly completed sequencing a cosmid clone that carries the N. crassa genes for snz-1 and sno-1 and at least eight additional genes.  The results of this project support previous estimates of gene density in N. crassa, with a predicted total of over 10,000 genes.

 

 

Session 3 - Development

 

A G protein a subunit, gna-3, regulates cAMP metabolism and aerial hyphae formation in Neurospora crassa.  Ann M. Kays, Patricia S. Rowley, Rudeina Baasiri, and Katherine A. Borkovich, University of Texas Medical School - Houston.

 

In eukaryotic systems, heterotrimeric abg guanine nucleotide proteins are used to convey extracellular signals.  Signals such as light, pheromones, and neurotransmitters activate G protein coupled signal transduction pathways.  Cellular responses are generated by the conversion of the initial signal to production of intracellular secondary messengers.  Our lab previously identified and characterized two G protein a subunits, gna-1  and gna-2 .  A third novel G protein a subunit, gna-3, has been cloned and found to be 70% and 67% similar to gna-1  and gna-2 , respectively, in Neurospora crassa .  A deletion of gna-3  was marked by resistance to the bacterial hygromycin B phosphotransferase (gna-3::hph).  Western blot analysis indicated that GNA-1 and GNA-2 were present at wild-type levels in Dgna-3  strains.  On solid medium, Dgna-3  strains exhibited denser conidiation and stunted aerial hyphae growth, but were not significantly affected in their apical extension rate.  The level of cAMP in Dgna-3  mutants was only 20% of wild-type on solid medium.  The reduced cAMP levels suggest a role for GNA-3 in the metabolism of cAMP in Neurospora crassa


Altered cAMP levels, adenylyl cyclase and cAMP phosphodiesterase activities associated with mutation of G protein a subunits in Neurospora crassa. F. Douglas Ivey*, Qi Yang, and Katherine A. Borkovich,  University of Texas Medical School, Houston, TX

 

Heterotrimeric GTP-binding proteins are key components of signal  transduction in eukaryotic organisms.  Previously, we reported that the filamentous  fungus Neurospora crassa possesses two G protein a subunits, GNA-1 and GNA-2.  Loss of gna-1 leads to multiple phenotypes, while Dgna-2 strains do not exhibit visible defects.  However, Dgna-1; Dgna-2 mutants are more affected in all Dgna-1 phenotypes.  This report presents results of a biochemical investigation of the roles of GNA-l and GNA-2 in cAMP metabolism.  Comparison of Mn2+ATP-dependent adenylyl cyclase activities indicated that the levels of enzyme were normal in Dgna-2 mutants, but reduced 40% in Dgna-1 and Dgna-1; Dgna-2 mutants.  Assays of Mg2+ATP-dependent adenylyl cyclase activity (±GppNHp) indicated that Dgna-2  strains were normal, whereas Dgna-1 and Dgna-1; Dgna-2 strains had only 10-15% the activity of the wild-type control.  Mg2+ATP-adenylyl cyclase activity in wild-type cell extracts could be inhibited using anti-GNA-l IgG, suggesting a direct interaction between GNA-1 and adenylyl cyclase in N. crassa.  The intracelluiar levels of cAMP in Dgna-1 and Dgna-1; Dgna-2 mutants were reduced relative to wild-type under conditions that result in morphological abnormalities.  cAMP phosphodiesterase activities in Dgna-1 and/or Dgna-2 strains were lower than in wild-type controls; the individual deletions were additive in decreasing phosphodiesterase activity in the Dgna-1; Dgna-2 double mutant.  Thus, G protein a subunits differentially influence adenylyl cyclase and cAMP phosphodiesterase activity in N. crassa.

 

Nutritional regulation of conidiation.  Daniel J. Ebbole, Texas A&M University, College Station, TX

 

Wild type Neurospora does not conidiate in media with adequate carbon and nitrogen sources.  Repression of conidiation occurs with a variety of carbon sources.  We are interested in understanding how nutrient sensing regulates development.  The rco-3 mutant was isolated because of its elevated expression of a developmentally regulated gene, con-10.  The mutant was found to be 2-deoxyglucose resistant and defective in glucose repression.  Low affinity glucose transport was strongly inhibited in the mutant.  The mutant also fails to repress macroconidiation in minimal medium regardless of carbon source suggesting that the defect is not glucose-specific.  Thus, RCO3 appears to be a regulatory protein involved in glucose repression and carbon source repression of conidiation.  The RCO3 protein has sequence similarity to sugar transporters.  Our current model is that RCO3 acts as a membrane receptor of glucose and that ligand binding signals glucose availability. 

 

N. crassa ras-3 defines a novel class of Ras protein. Peter Margolis and Charles Yanofsky, Stanford University, Stanford, CA.

 

Using degenerate PCR primers, we amplified from N. crassa DNA three apparent ras genes: previously characterized ras-1 and ras-2, and a third locus  (ras-3) that maps to LG VII R. Putative RIP mutants of ras-3 are female sterile, aconidial, slow-growing and defective in carotenogenesis. True null mutations in ras-3 may be lethal.

The RAS3 protein, encoded as a single exon, belongs to the Ras class of small GTPases, as judged by overall homology and by the presence of Ras-specific sequence motifs. However, RAS3 differs from standard Ras proteins in several respects: 1) sequence divergence at many typically conserved residues; 2) absence of a typical C-terminal “-CAAX” farnesylation site; 3) unusually large size (34 Kd, vs. a typical 21-25 Kd). The increased size of the predicted RAS3 reflects the presence of 3 “insertions” compared to a standard Ras; notably, RAS2 also contains one such “extra” protein sequence. These new polypeptide segments lack obvious similarity to known structural motifs. Computer modeling suggests that the extra RAS3 sequences could form a novel domain on one face of the canonical Ras structure.

A subsequent PCR-based phylogenetic survey indicates that ras-3 homologs are present in many Euascomycetes, but are not found outside of this class of fungi. By contrast, distinct ras-1 and ras-2 homologs are observed in most of the filamentous fungi tested, including Ascomycetes, Basidiomycetes, and Zygomycetes. RAS3 may participate in a novel signaling pathway, and thus could serve as an antifungal drug target.

 

Regulation of the Neurospora crassa clock gene frequency. Susan K. Crosthwaite, Deanna L. Denault, Keith A. Johnson, Jay C. Dunlap and Jennifer J. Loros,  Biochemistry Department, Dartmouth Medical School, Hanover 03755, New Hampshire

 

The circadian oscillator in Neurospora crassa requires that the products of the clock gene frequency (frq) cycle in a time of day dependent manner [Aronson et al. (1994) Science 263:1533].  To be able to describe what constitutes the clock at the molecular level, regulation of frq must be understood.  We are interested in characterizing the factors that activate and repress frq expression to create the daily oscillation of frq transcript and protein, as well as understanding how this regulation is acheived.

The frq locus produces two sense transcripts of 4.5 and 5 kb which encode FRQ protein.  FRQ negatively autoregulates levels of frq transcript so that frq mRNA cycles; FRQ also cycles in abundance and phosphorylation state [Garceau et al (1997) Cell 89:469].  Recently we showed that the products of the white collar genes are required for a functional oscillator.  The finding that frq levels are repressed in the white collar mutants [Crosthwaite et al. (1997) Science 276:763] and the fact that WC-1 and WC-2 encode transcription factors [Ballario et al. (1996) EMBO J. 15:1650;  Linden and Macino (1997) EMBO J. 16:98] suggests the WC proteins as candidates for the positive factors in the feedback loop, that bind to the frq promoter and activate transcription.  The zinc-finger DNA-binding domains of these proteins have been expressed as GST fusions in BCL21 cells.  The cell extracts and purified full length WC-2 have been incubated with fragments of the frq promoter.  The results of gel mobility shift assays using these samples will be presented.

The possible regulatory role of two antisense (AS) transcripts is also under investigation.  We have evidence that the frq locus produces two antisense transcripts of 5 and 5.5 kb.  They are transcribed from within the frq locus and overlap the sense frq transcripts.  These AS transcripts may affect the expression of the sense transcripts.  The minimal region required for phenotypic rescue of rhythmic conidiation in a frq null background includes the AS promoter and constructs lacking this region show poor rescue of the overt rhythm.

 

Search for protein(s) that interact with the Neurospora clock protein FREQUENCY  Christian Heintzen, Jennifer J. Loros and Jay C. Dunlap, Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844

 

The protein FREQUENCY (FRQ) is a central component of the Neurospora  circadian clock. A LexA interactor hunt, an implementation of the of the yeast two-hybrid screen, was conducted to identify proteins that may in­teract with the FRQ protein and hence might be part of the molecular feedback loop that comprises the circadian clock in Neurospora. Full length FRQ and various FRQ fragments were tested as baits. Western blot experiments show that all transformants produce LexA-FRQ fusions of the expected molecular size, however only FRQ fusions spanning the regions FRQ11-683 and FRQ11-262 were shown to be transcriptionally inert when fused to LexA. A repression assay confirmed that both proteins enter the nucleus. Transformants containing both constructs were used for the subsequent transformation of a Neurospora prey-library. 6 x 105 independent transformants (inserted as a prey behind the bacterial activation domain B42) were analyzed. 53 transformants that showed galactose dependent leucine prototrophy and tested positive for beta-galactosidase activity were further analyzed. The majority of these clones encode for ribosomal proteins, ri­bosomal RNA's or mitochondrial proteins and are probably false positive. The remaining clones are cur­rently being tested for the specificity of their interaction. The possibility of a FRQ-FRQ interaction was tested by using full length FRQ as well as different FRQ fragments as prey proteins which then were cotransformed with the transcriptionally inert FRQ-baits. Based on these experi­ments no indication for a FRQ-FRQ interaction has been found. These data are consistent with results of biochemical analyses previously reported (Garceau et al., Cell 89:469-476, 1997).

 

Roles in dimerization and blue light photoresponse of the PAS and BAT domains of Neurospora crassa White Collar proteins.  P. Ballario, C. Talora, D. Galli, H. Linden, G. Macino,  Universita di Roma La Sapienza, Roma, Italy

 

The genes coding for white collar-1 and white collar-2 (wc-l,wc-2) have been previously isolated and their products characterized as Zn-finger transcription factors involved in the control of blue light induced genes. The PAS dimerization domains present in both proteins enable the WC-1 and WC-2 proteins to dimerize in vitro. Homodimers and heterodimers are formed between the WC proteins. Inter-species heterodimers are formed between the WC proteins of Neurospora and the mammalian PAS- containing proteins AHR and ARNT. A computer analysis of WC-1 reveals a second PAS-like domain, called BAT a domain conserved in other redox-sensitive proteins such as NIFL and Bat in bacteria and NPH1, a putative blue light photoreceptor in plants. The WC-1 BAT domain has lost the ability to dimerize with canonical PAS domains, but it is able to self-dimerize. The isolation of three blind wc-1 strains, each with single amino acid substitutions only in the BAT domains, reveals that this region is essential for blue-light responses in Neurospora. The demonstration that the WC-1 proteins in these BAT mutants are still able to self-dimerize suggests that this domain plays an additional role, essential in blue light signal transduction.

Session 4 - Metabolism

 

Regulation of sulfur metabolism in Neurospora crassa. John V. Paietta, Dept. of Biochemistry and Molecular Biology, Wright State University, Dayton, OH

 

The sulfur regulatory system of N. crassa consists of a group of sulfur- regulated structural genes (e.g., arylsulfatase, ars-l+) which are under coordinate control of the cys-3+ positive and several scon (sulfur controller) negative regulatory genes. The CYS3 regulator is a bZIP DNA-binding protein and transcriptional activator, while the SCON2 negative regulator is a F-box/WD-40 protein. F-box proteins are thought to act as receptors for ubiquination targets. The importance of the SCON2 F-box in sulfur gene regulation has been shown by site directed mutagenesis and the subsequent generation of a sulfur auxotrophic phenotype. For comparison, a Dscon-2 strain was generated by transformation and homologous recombination. Deletion of the scon-2+ gene yielded constitutive expression of sulfur-regulated structural genes. The apparent functional role for the F-box in SCON2 implicates the involvement of proteolysis in the regulatory system, perhaps targeted at CYS3. Further support for this mechanism comes from the cloning of scon-3+, another sulfur negative regulatory gene, which may encode a component of the putative complex involved in the control of sulfur gene regulator activity.

 

Some pH regulatory mutations of A. nidulans can be complemented by a N. crassa genomic library. A.C. Aquino, G. Thedei Jr, W. Maccheroni Jr, S.R. Nozawa and A. Rossi, University of Sao Paulo, Ribeirao Preto, SP, Brazil

 

The pH regulation in the mold A. nidulans is mediated at least by genes pal (A, etc) and pacC. Mutants pal mimic growth at acid pH, increasing the level of the pH-dependent expressed proteins, and mutants pacC mimic growth at alkaline pH and lead, for example, to elevated levels of alkaline phosphatase and reduced levels of acid phosphatase. Mutants pal do not grow at pH 8.0 or in the presence of b-glycerolphosphate as the only source of Pi and mutants pacC do not grow at pH 3.0. This allowed us to transform these mutations by complementation with a genomic library of N. crassa (pCOSAX) and the self-replicating pAB4 plasmid, which contains the pyrG gene of A. nidulans. Six colonies that were able to grow at pH 8.0, proved to be transformants of the palA1 strain, and one was a transformant of the palF15 strain. Four colonies that were able to grow at pH 3.0, were transformants of the pacC14 strain. The existence of N. crassa genes that may complement mutations in the pacC and pal genes of A. nidulans is puzzling because, according to models proposed in the literature, A. nidulans appear to control the transcription of the acid and alkaline phosphatases in response to the stimulus generated by the extracellular pH, whereas N. crassa  synthesizes both enzymes irrespective of the extracellular pH, thus controling only their secretion to the external medium.

Financial support: FAPESP, CNPq and CAPES.

 

Distribution, nature, and possible significance of the base changes in 25 well characterized mutants of the trp-3 (tryptophan synthase) gene of Neurospora crassa.  Ann M. Lacy, Goucher College, Baltimore; Mary E. Case and William Nelson, University of Georgia, Athens

 

The DNAs from 25 well characterized CRM+ mutants altered in the trp-3 gene in Neurospora were amplified and partially sequenced to determine the precise location and alteration in each mutant.  These mutants were representative of different physiological groups with respect to partial enzyme activities, osmotic remediability, and complementation.  Many of these mutants had been previously mapped by genetic recombination.  Their map locations were, in almost all cases, confirmed by the sequence data.

Of the 25 mutants, 19 represent unique alterations.  Of these, one has a two codon insertion, one has two base changes in a single codon, and one has a substitution of an entire codon.  The 19 sites are distributed over roughly 85% of the coding region.  None occur before the first intron; however, four sites fall within the relatively short region between introns 1 and 2.  None are in the connector region; however a mutant with unique characteristics is located just upstream of it and another one just downstream of it.  Correlation of all these findings with the physiological data will be discussed and, where appropriate, the properties of the Neurospora enzyme will be compared with those of the E. coli enzyme.

Subunit structure, substrate selection and nucleotide hydrolysis activities of HSP80 and 70 of Neurospora crassa. 

M. Kapoor,  Department of Biological Sciences, University of Calgary, Canada

 

The cytosolic molecular chaperones of Neurospora crassa, HSP80 and HSP70, interact directly to form a hetero-oligomeric complex. In mammalian systems, multiprotein complexes containing HSP70, HSP90 (equivalent of N. crassa HSP80) and other proteins in conjunction with hormone receptors have been documented.  However, a direct physical interaction between HSP70 and 90 has not been observed.  N. crassa HSP80 is a tetramer in its native state while HSP70 is predominantly monomeric, forming higher-order oligomers in the presence of certain nucleotides.  Formation of the [HSP80:70] complex was demonstrated by crosslinking of purified HSP80 and 70 with bifunctional reagents, gel filtration, Western blotting and ELISA using specific antibodies. Ligand-induced conformational changes of HSP80, promoted by nucleotides--CTP, UTP and NAD--were monitored by following the intrinsic tryptophan fluorescence and by the use of environmentally sensitive fluorescent probes: ANS and TNS. Although both HSP80 and HSP70 hydrolyse ATP and GTP, significant differences in the kinetics of hydrolysis were evident.  The molecular chaperoning potential of HSP80 and HSP70 was assessed by comparing the peptides selected by these proteins from M13 Phage Display libraries. HSP80 predominantly selected peptides with a common structural motif defined by an invariant hydrophobic core and flanking variable sequences containing aromatic and charged residues. Peptides containing charged residues were selected by HSP70 but the substrate selectivity of [HSP80:70] was distinct from  that of either protein.        

 

Construction of a Neurospora crassa mutant lacking the mitochondrial outer membrane translocase protein TOM40.  Frank E. Nargang, Bryan McHale, Lara Corrigan, and Rebecca D. Taylor, Department of Biological Sciences, University of Alberta, Edmonton, Alberta         

 

Most of the proteins found in mitochondria are encoded by nuclear genes, synthesized as mitochondrial preproteins on cytosolic ribosomes, and imported into mitochondria. The initial steps in the import process are mediated by the Tom complex (Translocase of the outer mitochondrial membrane). The complex consists of receptor proteins that extend into the cytosol and integral membrane proteins that form the pore through which preproteins traverse the outer mitochondrial membrane. The TOM40 protein is thought to play the major role in the formation of the pore. We have created a N. crassa mutant of TOM40 using the procedure of "sheltered RIP".  The technique allows the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant allele in a heterokaryon with an unaffected nucleus.  Genetic and biochemical manipulation of the heterokaryon allows the study of the mutant phenotype.  When the nucleus containing the RIPed allele of tom40 is forced to predominate the heterokaryon, growth of the cells virtually ceases as TOM40 levels decrease to a critical low level.  Genetic analysis has shown that tom40 is an essential gene in N. crassa.  The mutant tom40 allele contains a stop codon at position 35 of the 349 residue protein suggesting that it represents a null allele of the gene.

 

Isolation and characterization of a new gene, sre, which encodes a GATA- tyge regulatory protein that controls iron transport in Neurospora crassa. Liwei Zhou, Hubertus Haas, and George A. Marzluf, Ohio State University, Columbus, Ohio

 

Multiple GATA factors, regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence, exist in the filamentous fungus, Neurospora crassa.  The gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method.  Sequence analysis of the new GATA factor gene revealed an open reading frame specifying 587 amino acids, which is interrupted by two small introns.  Unlike all previously known Neurospora GATA factors which possess a single zinc finger DNA binding motif, SRE contains two GATA-type zinc fingers.  The deduced amino acid sequence of SRE shows significant similarity with URBS1 of Ustilago and SREP of Penicillium. A loss of function mutation was created by the rip procedure. Analysis of sre+ and sre- strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores.  The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.

 

Invited Lecture

 

Genetic and molecular analysis of the Neurospora circadian system.  Jay C. Dunlap, Allan C, Froehlich, Chenghua Luo, Yi Liu, Susan Crosthwaite and Jennifer J. Loros, Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755

 

The circadian system in Neurospora is among the best understood of any organism (Dunlap, Ann. Rev. Genetics 30:579, 1996).  The oscillator is an autoregulatory feedback cycle, wherein the frequency gene encodes two forms of the FRQ protein; both frq RNA and protein cycle in abundance, and FRQ is rhythmically phosphorylated (Aronson et al., Science 263:1578, 1994; Garceau et al. Cell 89:469, 1997; Merrow et al., PNAS 94:3877, 1997).  Light resets the clock by rapidly inducing frq (Crosthwaite et al., Cell 81:1003, 1995).  Ambient temperature influences the clock by determining both the absolute amount of FRQ and the ratio between the two forms (Liu et al., Cell 89:477, 1997).  The WC-1 and WC-2 proteins, required for light-induction of frq and for sustained cycling in the dark, are clock proteins having sequences similar to clock-associated proteins from the mouse and fly (Crosthwaite et al. Science 276:763,  1997). FRQ is a nuclear protein and nuclear localization is essential for its function (Luo et al., EMBO J., in press).  Temperature steps reset the clock in essence by changing the set points for the oscillation, and in this way changing the subjective time to which different absolute levels of frq and FRQ correspond.

Efforts are also underway to develop luciferase and GFP as in vivo circadian reporters in Neurospora.

 

 

Poster Session Abstracts

Friday evening, June 19

7:00 - 10:00 pm

 

1.  Sequence analysis of a cosmid clone of Neurospora crassa.  Laura Bean1, Mara Giles1, Edward L. Braun1,2, Mary Anne Nelson1, and Donald O. Natvig1. 1Department of Biology, University of New Mexico, Albuquerque, NM 87131 and 2National Center for Genome Resources, 1800 Old Pecos Trail, Santa Fe, NM 87505

 

We have nearly completed sequencing a 35 kbp region of genomic DNA from N. crassa.  This region of the N. crassa genome was chosen because it contains homologs of stationary phase genes (snz-1 and sno-1) from the budding yeast, Saccharomyces cerevisiae.   In addition to these two genes, we have identified at least eight additional genes.  These include a homolog of the ALG3 gene from S. cerevisiae, a serine/threonine protein phosphatase gene, a 3 hydroxyisobutyrate dehydrogenase gene, and genes encoding D-amino acid oxidase and thioredoxin.  Based upon our current analysis, the gene density in this region of the N. crassa genome appears to be 1 gene for every 3 kb, a value consistent with recent estimates of 10,000 to 13,000 genes in N. crassa.

 

2.  Osmotic-sensitive mutants of Neurospora crassa with atypical morphology.  Sara Neville Bennett and Wayne A. Krissinger, Georgia Southern University, Statesboro, GA 30460

 

The osmotic-sensitive (os) mutants fail to grow on medium containing elevated concentrations of NaCl.  The first of these mutants were mapped to LG I and LG IV and had a characteristic altered morphology which allowed them to be scored easily on the basis of this typical os morphology.  Both osmotic sensitivity and morphology were thought to be pleiotropic effects brought about by an altered cell wall.  In our laboratory we have isolated a number of osmotic-sensitive mutants following UV irradiation and filtration-concentration in medium with 3% NaCl.  Among our isolates are alleles of os-1 and os-4 in LG I.  In addition, several new osmotic-sensitive mutants were isolated and in crosses to wild type, each mutant exhibited 1 : 1 segregation of osmotic sensitivity, indicating involvement of single genes under Mendelian control.  Mapping of these mutants placed os-8 (SS-931) in LG III between ad-2 and trp-1;  os9 (SS-788) in LG VI distal to del;  SS-1018 and AR3-34 both in LG IV and linked to the osmotic-sensitive mutant, cut.  Thus, osmotic mutants have now been located in two new linkage groups, LG III and LG VI.  These new osmotic-sensitive mutants all grossly resemble the wild type strain when the cultures are grown on slants and therefore exhibit atypical os morphology.  This suggests that there is a difference in the degree of any alteration of the cell wall and that osmotic sensitivity in these mutants might be accomplished by a different route or routes. 

 

3.  Neurospora crassa as a heterologous system for study of mammalian Ga  proteins.  Jennifer A. Bieszke, Qi Yang, and Katherine A. Borkovich, University of Texas Medical School - Houston

 

Heterotrimeric (abg) GTP binding proteins interact with sensory receptors to transduce a signal to downstream effectors.  Ga subunits have been classified according to the sequence identity.  The mammalian system has four classes of Ga subunits, Gai, Gas, Gaq, Ga12.  Previously, we reported that Neurospora crassa  contains a Ga protein, GNA-1, that is a member of the mammalian Gai family based on amino acid sequence identity and its ability to serve as a substrate for pertussis toxin.  Deletion of gna-1 in N. crassa leads to multiple phenotypic defects including female infertility and a slower growth rate on solid medium which is more pronounced under hyperosmotic conditions.  In this report, we tested the ability of mammalian Gai subfamily genes to complement a Dgna-1 mutation.  We have successfully expressed the following Gai subfamily members in N. crassa:  Gaz, Gao, Gat, Gai.  As a control, Gas of the stimulatory G protein family was also expressed.  The order of complementation for growth on hyperosmotic solid medium by the mammalian Ga genes is as follows:  Gaz> Gao> Gas> Gai.  Gat gave no complementation.  Gao was the only mammalian Ga to rescue the sexual defects of a Dgna-1 strain.  Biochemical analysis was also used to determine the levels of complementation by the mammalian Ga proteins.  Total cAMP was measured under vegetative conditions and complementation was similar to that observed for hyperosmotic growth rates.  However, cAMP levels in the mammalian Ga transformants during the sexual cycle do not correlate with complementation  of  Dgna-1   defect.  The above results suggest that GNA-1 regulates the vegetative and sexual cycles using two different pathways.  This study also demonstrates N. crassa  is a valuable heterologous system for study of mammalian G proteins.

 

4.  Characterization of con-11, a gene which is highly expressed during conidiation.  Kristin Black and Charles Yanofsky, Department of Biological Sciences, Stanford University, Stanford CA 94305-9020

 

con-11 was identified and isolated in a screen for highly expressed genes that could be used to study regulation of conidiation (Mol. Cell. Biol., 1985, 5:849-855).  con-11 is the last of these con genes to be expressed during asexual development.  con-11 is not essential to conidiation or growth; its function is not known. 

Sequence comparisons demonstrate that genes encoding similar proteins exist in S. pombe and in A. nidulans.  Interestingly, no similar gene sequence can be found in the S. cerevisiae genome.  The S. pombe homolog is induced by many stresses including nitrogen or carbon starvation, heat shock and stationary phase (Mol. Gen. Genet., 1995, 248:439-445). Of these stresses, only stationary phase induces con-11 expression in N. crassa.  However, there appear to be many potential stress-regulated sequence elements upstream of the con-11 ORF (heat shock, nitrogen, phosphate, pH, and general stress response elements). 

Like con-10, con-11 is repressed during vegetative growth by Rco1, a negative regulator.  con-11 appears to be transcribed shortly after con-10, the most thoroughly characterized con gene.  Unlike con-10, translation of con-11 mRNA during conidiation is delayed by several hours after the message appears.  Thus, regulation of con-11 expression appears to be distinct from regulation of con-10 and, thus, analysis of con-11 regulation may provide new insights into the relationship between regulation of gene expression by stress vs. conidiation.  Finally, the sequence similarity of Con11 to a stress-regulated protein of S. pombe, its dispensability, and the late appearance of Con11 during conidiation, are suggestive of a protective role for this gene product. 

 

5.  Cloning of rec-2, two contigs on LG V.  F.J. Bowring and D.E.A. Catcheside, Flinders University, Adelaide, Australia

 

We have established two contigs on linkage group V. The largest of these extends from 350kb proximal to 150kb distal of am while the other is 100kb and centred across leu-5. Mapping of crossovers either side of rec-2 suggests that this locus, situated 3 centi-Morgans (cM) proximal of am, is about 200kb downstream. The genetic to physical distance ratio is lower distal of am where the 1cM to ure-1 corresponds to 150kb.

 

6.  Molecular analysis of recombination events at am.  F.J. Bowring and D.E.A. Catcheside, Flinders University, Adelaide, Australia

 

We have used naturally occurring polymorphism in and near the Neurospora am locus to examine the genesis of chromatids recombinant in this region. The analysis indicates that here crossing over may be only loosely associated with gene conversion and has provided a high resolution picture of the pattern of gene conversion at this locus. However, as chromatid data do not always allow the unambiguous distinction between gene conversion and crossing over, we are examining tetrads in order to extend the power of the analysis.

 

7.  Centromere structure in Neurospora crassa: degenerate transposons and simple repeats.  E.B. Cambareri, R. Aisner, and J. Carbon,  Molec. Cell., and Devel. Biology, University of California, Santa Barbara, CA 93106

 

DNA from the centromere region of LG VII of Neurospora was cloned previously from a YAC library, and was found to be atypical of Neurospora DNA in both composition (AT-rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (-16. 1 kb) of this region, and have identified a cluster of three new retrotransposon-like elements, as well as degenerate fragments from the 3' end of Tad, a previously identified LINE-like retrotransposon. This region contains a novel full length but nonmobile copia-like element, designated Teen, that is only associated with centromere regions. Adjacent DNA contains portions of a gypsy-like element designated Tgl1. A third new element, Tg12, shows similarity to the Ty3 transposon of Saccharomyces cerevisiae. All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of the RIP (Repeat-Induced Point mutation) process. Three new simple repeated DNAs have also been identified in the LG VII centromere region. While Tcen elements map exclusively to centromere regions by RFLP analysis, the defective Tad elements appear to occur most frequently within centromeres, but also are found at other loci including telomeres. The characteristics and arrangement of these elements are similar to that seen in the Drosophila centromere, but the relative abundance of each class of repeats, as well as the sequence degeneracy of the transposon-like elements is unique to Neurospora. These results suggest that the Neurospora centromere is heterochromatic and regional in character, more similar to centromeres of Drosophila than to those in most single-cell yeasts.

 

8.  Characterization of arg-6 mutants affecting two unlinked genes. Jessica Y. Chung1, Suhn-Kee Chae2 and Richard L. Weiss1, 1University of California, Los Angeles and 2Pai Chai University

 

N-acetyl glutamate synthase (AGS) and N-acetyl glutamate kinase (AGK) are the first two enzymes in the arginine biosynthetic pathway. AGS and AGK are encoded by two unlinked genes, arg-14 and arg-6, respectively. Some arg-6 mutants lack AGS activity.  Interaction between AGS and AGK has been demonstrated using the yeast Two-Hybrid system. The interaction domain of AGS resides at the N-terminus and the interaction domain of AGK is at the C‑terminus. AGS and AGK are regulated via feedback inhibition by arginine. Mutations linked to the arg-6 locus, su(pro-3), are feedback resistant and suppress proline auxotrphic mutations (pro-3). We have mapped the su(pro-3) mutations to the N-terminus of AGK, and all su(pro-3) genes have a single amino acid mutation from phenylalanine (F) to leucine (L) at amino acid 81. The mutant AGK failed to show interaction with AGS. To further investigate the regulation of feedback inhibition by arginine, we have characterized representatives of the various arg-6 mutant classes. Here we report results of sequencing and yeast Two-Hybrid analysis. Two K- S+ strains have point mutations L289F and N231N. A K+ S+ strain has a point mutation, L102P. A K- S- strain has a F135V mutation. Another K- S- strain has a frame-shift mutation generating a premature stop codon at 143. In general, all mutations, including su(pro-3) mutations, mapped to the N-terminus of AGK. When interaction with AGS was tested, none of arg-6 mutant AGKs showed interaction with AGS. The implication of these observations for the mechanism of feedback inhibition will be described.

 

9.  A membrane skeleton is present in Neurospora crassa.  Norbert Degousee, Gagan Gupta and  I. Brent Heath.  York University Biology Department, North York, Ontario, Canada  M3J 1P3

 

An F-actin based membrane skeleton has been proposed to play a crucial role in the tip growth of hyphae, but the relevancy of this model has not been investigated in the dominant group of hyphal organisms - the true fungi.  Plasma membranes (PM)  were isolated from growing Neurospora crassa hyphae by discontinous sucrose density gradient centrifugation and subjected to SDS-PAGE and Western blot analysis.   The PM isolate was enriched in actin and also contained other cytoskeletal proteins such as spectrin, tubulin and possibly an integrin homologue.  Consistent with this, indirect immunofluorescence showed that all these antigens were present (in varying proportions) at the peripheries of Neurospora hyphae.  Strikingly, the actin, spectrin and integrin antigens were all concentrated at and were colocalized to hyphal apices, yet their precise distributions at the extreme apex were different.  Together, these results are best explained by the presence of a functional membrane skeleton  in Neurospora hyphal tips.

 

10.  The mating pheromones of Neurospora crassa.  Daniel J. Ebbole, Deborah Bell-Pedersen, Neal Van Alfen, Piotr Bobrowicz, Wei-Chiang Shen, Lori A. Shrode, Patricia McCabe, and Pam Kazmierczak.  Texas A&M University, College Station.

 

The mating type loci of N. crassa encode regulators that control expression of genes involved in sexual fertility and development.  We have begun to analyze the genes encoding the sex pheromones of N. crassa.  One gene, from Mat A strains, express a pheromone precursor gene with a structure resembling the yeast alpha-factor precursor.  The predicted sequence of the mature pheromone is remarkably similar to that of the chestnut blight fungus, Cryphonectria parasitica.  Mat a strains express a pheromone precursor gene that resembles yeast a-factor that is predicted to be isoprenylated.  Mating type-specific, nutritional, developmental and circadian regulation of the pheromone genes will be presented.  Experiments to produce pheromone gene mutants are in progress.

 

11.  The effects of mutations in the Neurospora crassa arg‑2 arginine attenuator peptide on arginine-specific translational attenuation.  Peng Fang, Zhong Wang, Anthony Gaba and Matthew S. Sachs, Oregon Graduate Institute, Portland OR.

The mRNA transcript  encoding the Neurospora crassa arg‑2 gene product contains an evolutionarily conserved upstream open reading frame (uORF).  Translation of the arginine attenuator peptide (AAP) specified by this uORF in the presence of a high level of arginine causes ribosomes to stall, resulting in reduced translation of the mRNA.  To further investigate the role of the AAP coding region in this translational attenuation phenomenon, we have used a variety of techniques to mutate the AAP coding region and tested the effects of these mutations on translational control using an N. crassa cell-free translation system.  Mutations that change highly conserved amino acid residues can eliminate regulation.  Silent mutations that place new, unique restriction sites near the beginning, middle and end of the uORF coding region do not reduce regulation.  Deletion of the N-terminal region of the AAP, which is not evolutionarily conserved, does not eliminate regulation, nor does the introduction of a His6 affinity-tag in this region.  The available data indicate that the sequence of the nascent peptide, and not the mRNA from which it is synthesized, is of primary importance for arginine-specific translational attenuation.


 

12.  Protein-protein interactions mediate the functional interactions between the global positive regulator NIT2, the global negative regulator NMR and the pathway-specific regulator NIT4. Bo Feng, H.G. Pan and George. A. Marzluf, Department of Biochemistry, The Ohio State University, USA

 

In Neurospora crassa, nitrogen metabolism is controlled by NIT2 and the negative regulator NMR.  Activation of structural genes in different pathways also requires pathway-specific regulators, such as NIT4. The major feature of this regulatory system is that the expression of catabolic enzymes depends on both the lifting of nitrogen repression and substrate induction. In the nitrate assimilation pathway, the pathway-specific regulator NIT4 mediates nitrate induction.  Both NIT2 and NIT4 are required to turn on the nit-3 and nit-6 genes in this pathway.  Neither one alone is sufficient.  This stringent requirement for both positive regulators to turn on transcription is different from the promiscuous cooperation between transcription factors, and appears to represent a distinct regulatory mechanism, possibly by protein-protein interaction.  Data presented here reveal that NIT2-NIT4 interaction indeed occurs and is critical for the integrated control of NIT2 and NIT4 over the nitrate assimilation pathway.  Another GATA factor in Neurospora, which shares similar DNA-binding specificity with NIT2 but functions differently, did not show any detectable interaction with NIT4 protein.     These protein-protein interactions appear to play an important role in the integration of regulatory signals at the transcriptional level and in achieving the functional specificity of different GATA factors in N. crassa.  This work may have implications in other regulatory processes.

 

13.  Isolation of a putative methyltransferase (Mtase) gene from Aspergillus nidulans.  Michael Freitag, Alejandro Correa*, Rodolfo Aramayo* and Eric U. Selker, University of Oregon, Eugene, OR and *Texas A&M University, College Station, TX.

 

DNA of many eukaryotes is modified by methylation of cytosines.  This process is essential for proper development and cell maintenance in some eukaryotes.  Numerous proteins are likely involved in the establishment and regulation of DNA methylation.  One of the essential components of any methylation machinery is the methyltransferase itself.  We have previously attempted to identify methyltransferase genes in Neurospora crassa either by functional, biochemical means or by homology-based approaches that rely on conserved amino acid sequences among eukaryotic Mtase genes.  Here we report the isolation and preliminary characterization of an Aspergillus nidulans gene that we identified based on sequence similarity of an Aspergillus EST clone to pro- and eukaryotic Mtases.  This gene contains all conserved motifs commonly found in eukaryotic Mtases including a conserved active site.  Curiously, Aspergillus - like Saccharomyces cerevisiae and Schizosaccharomyces pombe - has been reported to lack detectable DNA methylation in vegetative tissue.  The Aspergillus Mtase gene is most similar to the Ascobolus immersus masc1 gene that is required for efficient silencing by MIP (methylation induced premeiotically) but is not essential for methylation in vegetative tissue (Malagnac, F. et al., 1997, Cell, 91:281-290).  Sequence similarities among these putative fungal Mtases are being exploited to search for the Neurospora homologue(s).

 

14.  Genetic and molecular analysis of the Neurospora circadian system. Allan C. Froehlich, Chenghua Luo, Yi Liu, Susan Crosthwaite, Jennifer J. Loros, and Jay C, Dunlap Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755

 

The circadian system in Neurospora is among the best understood of any organism (Dunlap, Ann. Rev. Genetics 30:579, 1996). The oscillator is an autoregulatory feedback cycle, wherein the frequency gene encodes two forms of the FRQ protein; both frq RNA and protein cycle in abundance, and FRQ is rhythmically phosphorylated (Aronson et al. Science 263:1578, 1994; Garceau et al. Cell 89:469, 1997; Merrow et al. PNAS 94:3877, 1997). Light resets the clock by rapidly inducing frq (Crosthwaite et al. Cell 81:1003, 1995).  Ambient temperature influences the clock by determining both the absolute amount of FRQ and the ratio between the two forms (Liu et al. Cell 89:477, 1997).  The WC-1 and WC- 2 proteins, required for light-induction of frq and for sustained cycling in the dark, are proteins having sequences similar to clock-associated proteins from the mouse and fly (Crosthwaite et al. Science 276:763, 1997).  FRQ is a nuclear protein and nuclear localization is essential for its function (Luo et al. EMBO J., in press).  Temperature steps reset the clock in essence by changing the set points for the oscillation, and in this way changing the subjective time to which different levels