Neurospora 2000 Invited Talks







Celebrating 75 years of Neurospora genetics.

David D. Perkins. Biological Sciences Stanford University, Stanford CA, USA.

Genetic work with Neurospora had its beginning not in 1941 with Beadle and Tatum, but in 1925 with experiments of B. O. Dodge, who described life cycles and ascus development in heterothallic and pseudohomothallic species, described heterokaryons and complementation, demonstrated Mendelian segregation of mating type, and performed the first tetrad analysis. Dodge's work and his enthusiasm for the organism were largely responsible for Neurospora being chosen to produce the first biochemical mutants, leading to its use by many workers and its development as a model organism. Mating type was the only marker available to Dodge when he began. In contrast, over 1000 loci have now been mapped to linkage group; 25% of these have been cloned and sequenced.

Imaging the vesicle trafficking network in living Neurospora hyphae.
Nick D. Read, Patrick R. Hickey, Sabine Fischer-Parton, Richard M. Parton. Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh, EH9 3JH, UK.

The amphiphilic styryl dye FM4-64 has been used to track vesicle trafficking in growing Neurospora hyphae using confocal microscopy. FM4-64 is internalised by endocytosis, and stains putative endosomes prior to staining vacuole membranes and the apical vesicle cluster within the Spitzenkörper. Our data indicate that (1) the endocytic pathway integrates with the secretory pathways involved in tip growth and (2) wall-building secretory vesicles within the main Spitzenkörper are derived from two or possibly three sources (Golgi, satellite Spitzenkörper and possibly endosomes). Time-lapse movies illustrating the dynamic behaviour of the Spitzenkörper in wild type and mutant strains of Neurospora will be shown and, finally, a model of the vesicle trafficking network within growing hyphae will be presented.

Programmed hyphal compartmentation and death in Neurospora crassa.

N. Louise Glass, Jennifer Wu, Qijun Xiang. Plant and Microbial Biology Department, University of California, Berkeley, CA 94720.

Vegetative incompatibility is a phenomenon whereby when two strains that differ in het genotype undergo hyphal fusion, the fusion cell is compartmentalized and dies. Vegetative incompatibility is usually assessed in N. crassa by forcing heterokaryons or partial diploids that differ in allelic specificities at het loci. Such incompatible strains exhibit slow growth rates, hyphal compartmentation and death and suppression of conidiation. The het-c locus encodes a glycine-rich polypeptide; a 24-48 amino acid variable domain mediates the three allelic specificities at het-c. Phylogenetic analysis of het-c in different species and genera in the Sordariaceae indicated that het-c is under balancing selection, presumably because of its nonself recognition function. To determine the requirements of specificity, we examined the role of natural polymorphisms on het-c specificity and thereby identified and subsequently generated new het-c allelic specificities. To determine the mechanism of recognition between strains containing alternative het-c polypeptides, we tagged HET-c and performed co-immunoprecipitation experiments. Heterocomplexes of alternative HET-c polypeptides were detected, but homocomplexes were not. We have undertaken a genetic approach to determine how HET-c heterocomplex formation triggers vegetative incompatibility and have identified three suppressor mutants. Characterization of vib (vegetative incompatibility blocked) loci will identify downstream effectors of het-c mediated vegetative incompatibility.

Structure and function of the Neurospora TOM complex.

Frank E. Nargang and Rebecca D. Taylor. Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.

The majority of mitochondrial proteins are encoded by nuclear genes, synthesized as preproteins in the cytosol, and imported into the organelle. Preproteins destined for the matrix contain cleavable targeting signals at their N-termini, while the targeting information for other mitochondrial proteins lies within the mature protein coding sequence. Recognition and import of mitochondrial preproteins through the outer membrane is achieved by the TOM complex (translocase of the outer mitochondrial membrane). Further movement into the matrix or mitochondrial inner membrane requires the action of the TIM complexes (translocase of the inner mitochondrial membrane). The N. crassa TOM complex contains at least six subunits: TOM70, TOM40, TOM22, TOM20, TOM7, TOM6. Our investigations have concentrated on the function of the receptor domain of the TOM22 protein and on TOM40, the major component of the translocation pore. We have constructed null mutants for the genes encoding each of these essential proteins in sheltered heterokaryons. The existence of the mutant strains has allowed us to introduce various in vitro generated mutant alleles of the genes so that the phenotypic effects of the altered gene products can be studied. For TOM22 we have shown that mutations affecting the negatively charged region of the cytosolic domain have little affect on the growth rate of cells or their ability to import mitochondrial precursors. Mutations affecting various conserved regions of the TOM40 protein result in only a mild growth phenotype. In at least some of these strains, native blue gel electrophoresis and chemical cross-linking studies suggest that the conformation of the TOM complex may be altered in the mutants.

Analysis of dynactin and its role in regulating cytoplasmic dynein activity.

In Hyung Lee, Santosh Kumar, and Mike Plamann. School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO, USA.

Dynactin is a multisubunit complex that mediates the interaction of the microtubule-associated motor cytoplasmic dynein with membranous cargo. We have isolated hundreds of mutants in Neurospora crassa, that are defective in cytoplasmic dynein/dynactin function. We report the identification of three subunits of the Arp1 pointed-end capping complex of Neurospora dynactin. RO2, RO7 and RO12 are apparent homologs of mammalian dynactin subunits p62, ARP11, and p25, respectively. Mutations in ro-2 and ro-7 disrupted nuclear and vacuole distribution; however the degree of defects was less severe in ro-2 mutants than in ro-7 mutants. In contrast, the ro-12 mutation disrupted vacuole distribution, but nuclear distribution was normal. The results suggest that RO2, RO7, and RO12 have specific functions within the Arp1 filament pointed-end capping complex. We also examined the effects of dynactin mutations on dynein ATPase activity. Our results indicate that dynein ATPase activity is regulated by dynactin-dependent phosphorylation.

Expression and localization of two COT1 kinase isoforms in Neurospora crassa.

Oded Yarden,1 Rena Gorovits1, Klaas A. Sjollema2 and J.Hans Sietsma2, 1Department of Plant Pathology and Microbiology and The Otto Warburg Center for Agricultural Biotechnology, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, 76100 Israel. 2Department of Plant Biology and Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Biological Center, Nl-9715 NN Haren, The Netherlands.

Reversible protein phosphorylation is one of the mechanisms by which hyphal elongation and fungal morphogenesis are regulated. Complementation of the colonial temperature sensitive 1 (cot-1) mutant of Neurospora crassa has indicated that cot-1 encodes a Ser/Thr protein kinase which is structurally related to the mammalian myotonic dystrophy kinase and other members of the Rho-kinase subfamily. Antibodies raised against COT1 detected and immunoprecipitated a predominant 73-kDa polypeptide in N. crassa extracts, whose abundance was constant under all growth conditions tested. An additional, lower MW, COT1 isoform (67-kDa) present in the wild-type, was not detected in cot-1 grown at the restrictive temperature. Immunolocalization analysis provided evidence that COT1 is present in both the cytosol and in association with the cytoplasmic membrane. Immunoblotting experiments performed on extracts obtained from the wild type indicated that the lower MW isoform was present in all cellular components, yet was more abundant in the membrane fraction. However, the membrane-associated form was almost undetectable by immunolocalization or western blotting procedures in the cot-1 mutant grown at the restrictive temperature. Differential detection and immunolocalization of COT1 in various sub-cellular fractions indicate that COT1 is post-translationally processed and trafficked within the fungal cell. We propose that a lower MW COT1 isoform that, is associated with the plasma membrane, is involved in the regulation of hyphal elongation. Transcription profiling is currently being used in order to identify genes that are differentially expressed in cot-1 grown at restrictive conditions.

Interaction of colonial mutations of Neurospora crassa.

Tony Griffiths, Florian Muller, Jennifer Haynes, and Cindy Yen. Botany, University of BC, Vancouver, BC, Canada.

In Neurospora there are numerous loci at which mutation gives rise to a compact "colonial" morphology, generally a manifestation of increased branching and slow growth. As part of a programme to dissect the genetics of branching, we have started to examine the functional relationships between these loci. The procedure has simply been to obtain double mutants and compare their phenotypes to the single mutants. We have used two general groups of mutants, the true colonials (col) and the partially colonials (smco and spco). From fertile crosses, 41 double mutants were obtained via NPD and T octads. In most cases, the double mutant phenotype is more defective than either single, and the mutants can be regarded as additive in their action. Assuming the mutants are nulls, the simplest interpretation is that they involve separate developmental pathways. However, there were several cases of epistasis in which the double resembles one of the singles in growth rate and branch pattern; for example col-8 is epistatic to (>) col-17, smco-6 > smco-8, smco-1 > spco-4, spco-5 > smco-8, and spco-11 > spco-10. Such interaction might indicate a common pathway: for example smco-6, spco-5 and smco-8 might be grouped. In one case (smco-9; spco-6) the double is more vigorous than either single, indicating a different type of interaction of gene products.


Translational control of Neurospora crassa arg-2 by arginine.

Matthew S. Sachs. Department of Chemistry, Biochemistry & Molecular Biology, Oregon Graduate Institute, Portland, OR USA.

The arg-2 gene encodes the small subunit of Arg-specific carbamoyl phosphate synthetase, the first enzyme in the Arg biosynthetic pathway. arg-2 is unique among Arg biosynthetic genes because it is subject to negative regulation in response to Arg. An evolutionarily conserved peptide encoded by an upstream open reading frame (uORF) in the arg-2 transcript mediates Arg-specific regulation at the level of translation. Using N. crassa cell-free extracts, we observe that ribosomes stall after synthesizing this uORF-encoded peptide when the level of Arg is high. The stalled ribosomes block the movement of trailing ribosomes, reducing translation initiation at the downstream ARG-2 start codon. The uORFs in the corresponding genes of Saccharomyces cerevisiae, Magnaporthe grisea and Aspergillus nidulans function similarly to stall ribosomes in N. crassa extracts. The sequence requirements for Arg-regulation by the uORF-encoded peptide have been investigated by systematic mutational analysis.

Regulation of sulfur metabolism in Neurospora crassa.

John V. Paietta. Biochemistry & Molecular Biology, Wright State University, Dayton, OH, USA.

The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes (e.g., arylsulfatase, ars-1) which are under coordinate control of the cys-3 and sulfur controller (scon) 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. The importance of the SCON2 F-box in sulfur gene regulation has been demonstrated by site directed mutagenesis experiments. F-box proteins are thought to serve as adaptors that specfically link a targeted protein to the ubiquination/proteolysis system. The F-box of an adaptor protein (e.g., SCON2) binds to the core complex involved in the regulated proteolysis (referred to as the SCF) by a protein-protein interaction with SKP1 (suppressor of kinetochore protein 1). We have cloned and are studying a component of the Neurospora sulfur regulatory system that is homologous to SKP1. The current model of the control mechanism will be discussed.

A new GATA factor, ASD, regulates sexual development in Neurospora crassa.

Bo Feng1, Hubertus Haas2 and George A. Marzluf1. 1 Department of Biochemistry, The Ohio State University, Columbus, Ohio. 2University of Innsbruck, Innsbruck, Germany.

A novel GATA factor, ASD, was cloned from Neurospora crassa. The sequence of Asd revealed an open reading frame of 427 amino acid residues containing a GATA type zinc finger and a putative coiled-coil domain. Unlike its homologs in yeast or Penicillium, however, Asd does not appear to be involved in nitrogen regulation. Disrupting Asd gene by Rip did not show any effect on nitrogen regulation, but resulted in severe defects in ascospore genesis. A strain bearing the disrupted Asd gene was unable to produce ascospores during genetic crossing with wild-type, though fruiting body development seemed normal macroscopically. Introduction of the Asd+ gene into the Asd(Rip) strain corrected the failure of ascus and ascospore development. Mobility shift assay showed that ASD recognizes "GATA" DNA elements, thus is active in specific DNA-binding. The putative coiled-coil domain forms a dimer in solution, indicating that Asd binds DNA as a homodimer. Together, the results strongly suggest that Asd functions as a transcription regulator controlling sexual development in Neurospora crassa.

Regulation of arginine metabolism.

Richard L. Weiss. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA.

Arginine metabolism in Neurospora involves a number of novel metabolic and regulatory features. The first seven biosynthetic enzymes are localized in the mitochondria, whereas the last two biosynthetic steps and the catabolic pathway are located in the cytosol. Feedback inhibition of acetylglutamate synthase (AGS) and acetylglutamate kinase (AGK), products of the unlinked arg-6 and arg-14 genes, involves an arginine carrier mediating rapid equilibration of arginine between the cytosol and mitochondrial matrix. AGK contains a C-terminal eukaryotic domain not found in the bacterial enzyme. Mutations in the arg-6 gene can affect the activity or feedback-sensitivity of both AGK and AGS. The proteins have been shown to interact using the yeast two-hybrid system. The eukaryotic domain appears to be responsible for this interaction. A mutation affecting the feedback sensitivity of AGK and AGS has been mapped to the N-terminal domain of AGK. AGK and acetylglutamyl-phosphate reductase (AGPR), the third enzyme of the biosynthetic pathway, are initially synthesized as a polyprotein precursor which is cleaved into separable proteins upon entry into the mitochondrion. Independently targeted proteins or an unprocessed polyprotein precursor appear to complement arg-6 null mutants suggesting that the polyprotein precursor is not essential for protein targeting to the mitochondria or functional assembly into active enzymes. These organizational features may reflect the different regulatory requirements imposed upon eukaryotic (versus prokaryotic) organisms

The vacuolar ATPase is a surprisingly complex enzyme with multiple roles to play in growth and differentiation in fungi.

Barry Bowman. Department of Biology, University of California, Santa Cruz, CA, USA.

In most intracellular compartments the control of pH and the energy for active transport are provided by the vacuolar ATPase. This is a complex enzyme, which uses the energy in ATP to pump protons across membranes. The enzyme is composed of at least 13 different types of subunits, all of which have been identified in Neurospora crassa. The enzyme appears to function as a miniature mechanical pump. Some polypeptides provide the driving force and form the motor. Other polypeptides form a rotor, which spins at speeds of up to 10,000 rpm. Additional polypeptides attach the motor and rotor sectors to the membrane or are involved in regulation or cellular localization. To examine the role of the vacuolar ATPase we have generated strains in which genes encoding essential subunits have been mutated by RIPing. Several of these are true null strains that survive without a vacuolar ATPase. The most striking phenotypes of these strains are the inability to grow in alkaline medium (above pH7), and the dramatic alterations in mycelial morphology and differentiation. Mutations identified in other genes can suppress the pH sensitivity but not the morphological alterations. One group of suppressors is comprised of strains in which the kinetic properties of the plasma membrane ATPase have been changed. We have proposed a model to explain how the vacuolar and plasma membrane ATPases may regulate pH and control ion homeostasis.

Properties and interactions of HSP70 and Hsp80: Heat-inducible, cytosolic molecular chaperones of Neurospora.

Manju Kapoor. Biological Sciences, University of Calgary, Calgary, Alberta, Canada.

Formation of hetero-oligomeric complexes between purified HSP70 and HSP80, cytosolic molecular chaperones of Neurospora, is demonstrable by ELISA assays, inter-protein crosslinking using bifunctional reagents, gel filtration and Surface Plasmon Resonance measurements. The subunit structure of HSP80, in its native state, is consistent with a dimer-of-dimers. Inter-protomer crosslinking of HSP80 with dimethyl adipimidate and dimethyl suberimidate is suppressed by nucleotides. Crosslinking of HSP70 by glutaraldehyde yields dimers and higher order oligomers but binding of ATP, ADP and NAD reduces the yield of oligomeric species. Interestingly, [HSP70:HSP80] complex formation- reflecting inter-protein interactions- appears to be stimulated by nucleotides. Although both HSP70 and HSP80 exhibit high intrinsic ATPase activity, that of HSP80 is subject to strong inhibition by the substrate, indicating the presence of two nucleotide binding sites. HSP70 and HSP80 act as molecular chaperones individually as well as in the form of a complex. The substrate specificity of the complex is distinct from that of HSP70 and HSP80 alone. An additional, ~40-kDa protein, interacting with HSP70, appears to compete for the binding site(s) for HSP80. These studies suggest the presence of a functionally flexible chaperone system in Neurospora.

Metabolite regulation of multiple arginases in conidia and early germination of Neurospora crassa.

Gloria E. Turner and Richard L. Weiss. Chem & Biochem, UCLA, Los Angeles, CA, USA.

The biological role of extrahepatic arginase in vertebrates is not well understood. It has been proposed that this arginase has the potential to regulate the availability of arginine for proline, polyamine, NO, and glutamate synthesis. Ornithine; a product of arginine hydrolysis by arginase would be used to preferentially synthesize any of these various metabolites. Unlike the major arginases of vertebrates that are encoded on separate genes, N. crassa arginases are differentially expressed from a single locus. Arginine, the substrate of arginase is required for production of the larger arginase transcript and protein. Previous work has shown that arginase is temporally expressed in germination and that the ratio of the two forms change in a dynamic fashion when the organism is grown on nitrogen sufficient minimal or arginine supplemented medium. We have examined the effects of arginine, ornithine and proline, on the ratio of the two major transcripts as well as their translation products in conidia and during germination. We have established that conidia under all conditions, minimal, minimal supplemented with arginine, ornithine or proline has both forms of arginase protein. This is also the case when arginine, ornithine or proline is used as a sole nitrogen source. Interestingly, only the large transcript is detected in our conidial samples supplemented with each amino acid. We were unable to detect the small transcript or any arginase activity in these conidial extracts. This suggests that both forms of arginase protein are stored in condia in an inactive form. We have shown that ornithine and proline can induce expression of the large form of arginase and these metabolites alter the ratio of the two forms during germination. This suggests that each form may preferentially be used for the synthesis of polyamines and proline. The ratio of the two forms is dramatically different when arginine, ornithine or proline are the sole nitrogen sources. This nitrogen stress condition induces the expression of both forms of arginase. The two protein forms are almost equal and remain at a constant level. This suggests the presence of a nitrogen regulatory factor that controls arginase expression during nitrogen stress.

Characterization of Neurospora crassa mak-2 gene encoding mitogen-activated protein kinase similar to yeast Fus3 and Kss1.

Piotr Bobrowicz and Daniel Ebbole. Plant Pathology & Microbiology Texas A&M University College Station TX.

Using the Magnaporthe grisea PMK1 cDNA as a heterologous probe we have isolated the N. crasssa gene that resembles the Saccharomyces cerevisiae MAP kinase genes, FUS3 and KSS1, involved in pheromone signaling and invasive growth. This gene is identical to the mak-2 gene previously isolated by a PCR approach (Margolis and Yanofsky). In M. grisea, PMK1 is required for infection-related morphogenesis but its role in sexual development has only been partially characterized (Xu & Hamer, 1996. Genes Dev. 12:2874). The corresponding MAP kinase of Cochliobolus heterostrophus (Lev et al. 1999. PNAS 96:13547) is required for conidiation and mating as well as appressorium development. To determine the function of mak-2 gene in N. crassa we constructed RIP and gene replacement mutants. The mak-2 strains are morphologically abnormal producing strongly reduced aerial hyphae with uniformly distributed conidiophores. Mutants are fertile as a male partner but completely sterile as a female. Experiments are in progress to elucidate the role of the mak-2 gene in sexual and asexual development.

Search for carotenogenesis mutants, new phenotypes and cloning of the ovc and cut loci.

Javier Avalos1, Loubna Youssa1, and Thomas J. Schmidhauser2. 1Genetica, Universidad de Sevilla, Sevilla, Spain. 2U. of Louisiana, Lafayette, LA, USA.

Standard UV mutagenesis coupled with screening of survivors at 6C in continuos light lead to a number of distinct classes of mutants. A reddish mutant is complemented by the al-2+ gene and may be defective in the cyclase function recently assigned to the al-2 gene product, the Neurospora phytoene synthase. Another mutant designated carotenoid producer in the dark, cpd, was isolated in a ovc background and accumulates carotenoid pigments in dark grown mycelia. No mutants of this class could be isolated by visual screening after mutagenesis of the wild type strain. Screening for the ovc locus identified a cosmid clone that also complements the cut locus. Preliminary analysis suggests that the two loci are independent, but in close proximity as both mutants are complemented by transformation with a 6 kb cloned DNA fragment.


Neurospora crassa as a model system to study homology dependent gene silencing.

Carlo Cogoni, Gianluca Azzalin, Caterina Catalanotto, and Giuseppe Macino. Se. Genetica  Molecolare, Universita di Roma, Rome, Italy.

The introduction into cells of foreign nucleic acid molecules, either RNA or DNA, can induce sequence-specific gene silencing in a number of organisms. In Neurospora crassa transgenes can induce a Homology-Dependent Gene Silencing (HDGS) phenomenon called 'quelling'. Three classes of mutants impaired in quelling have been isolated by using random insertional mutagenesis and plasmid rescue procedure, identifying three qde (quelling deficient) genes involved in the gene silencing machinery. The first gene cloned was qde-1 which product shares significant identity with an RNA-dependent RNA polymerase (RdRP) characterized in tomato. This identity supports the idea of the involvement of an RdRP in the post-trascriptional gene silencing mechanism that could be responsible for the identification of aberrant RNA and its amplification in a cRNA form. Successively the cRNA could be responsible for targeting and degradation of specific mRNA. More recently we have isolated the qde-3 gene. qde-3 belong to the RecQ DNA helicase family suggesting a role for this gene in DNA-DNA interaction between repeats and/or induction of chromatin modification required for aberrant transcription. QDE-3 may be essential in unwinding double stranded DNA, thus producing single stranded DNA that could function as an intermediate in a DNA-DNA interaction between transgenes and/or endogenous gene required for gene silencing activation. A more refined model of the quelling mechanism and analogies with HDGS phenomena in other organisms will be discused.

Neurospora as a model to investigate DNA methylation.

Eric U. Selker, Hisashi Tamaru, Brian Margolin, Elena Kuzminova, Michael Freitag, Vivian Miao, Greg Kothe, Joe Dobosy, and Shan Hays. Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

Although absent from some model organisms such as S. cerevisiae, S. pombe, D. melanogaster and C. elegans, DNA methylation is found in Neurospora, humans and many other eukaryotes. Methylation can serve to mark particular sequences for special treatment by the cell and typically plays roles in gene silencing. Neurospora seems to use methylation to prevent expression of foreign DNA and DNA mutated by RIP. Methylation is clearly not essential for life of this organism; methylation can be abolished by mutation or replacement of the DNA methyltransferase gene without noticeable effects. The dispensability of methylation in Neurospora contrasts the situation in higher eukaryotes and facilitates genetic studies on its control and function. One feature of DNA methylation is that once established, it is typically propagated through DNA replication, perpetuating its effect(s). The mechanism of such "maintenance methylation" is not well understood. In Neurospora, we have found evidence that maintenance methylation is sequence-dependent. We have also used Neurospora to carry out the most detailed analysis to date of de novo methylation signals. We found that both A:T-richness and high densities of TpA dinucleotides, typical attributes of methylated sequences in Neurospora, promote methylation, but neither are essential for methylation. We also found that methylated sequences contain multiple, positive signals that trigger methylation. To better understand the nature of these signals, various simple, synthetic sequences were tested for their ability to trigger de novo methylation. Certain patterns of alternating As and Ts, but not long runs of Ts or As or G:C-rich sequences, efficiently trigger methylation (see poster by Tamaru and Selker). We are also investigating the relationship between DNA methylation and modifications of chromatin. We showed that DNA methylation associated with products of RIP can prevent transcription elongation without affecting initiation and that trichostatin A (TSA), an inhibitor of histone deacetylases, can relieve this repression and cause loss of DNA methylation. TSA does not cause reduction of methylation in most of the genome, however, perhaps because not all regions have access to histone acetyltransferases. Analysis of the acetylation state of histones associated with specific DNA regions has revealed that not all regions become hyperacetylated by TSA treatment, consistent with this possibility. We also demonstrated that changes in DNA methylation can affect histone acetylation, consistent with evidence from animal systems that histone deacetylases are recruited to methylated DNA by methyl-DNA binding proteins. In addition, we found unmethylated hypoacetylated regions, indicating that hypoacetylation, per se, does not lead to DNA methylation. We are currently exploring the possibility that TSA-induced loss of methylation is due to inhibition of methylation by transcription.

Tad transcription and transposition.

John A. Kinsey. Department of Microbiology, University of Kansas Medical Center, Kansas City, KS USA.

Using in-vitro mutagenesis and targeted transformation we have determined a number of elements within the Tad retrotransposon that are essential for transcription and/or transposition. One of these elements is an internal promoter that is located between 131 and 179 bp internal to the consensus 5' end of Tad. The promoter was initially located by deletion mapping. Subsequently the promoter has been further defined by the use of reporter gene constructs. This promoter appears to have several unique features in addition to its internal location. For example it appears to trigger the initiation of transcription in a manner that is neither sequence nor distance specific. It also appears to act as a bi-directional promoter. Preliminary gel mobility shift analysis with this promoter suggests a complex gel shift pattern which might indicate interaction directly or indirectly with a number of proteins.

S-adenosylmethionine, DNA methylation and DNA mutation in Neurospora crassa.

Diego Folco, Mario Mautino and Alberto Rosa. Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET), Friuli 2434, 5016-Córdoba, Argentina.

Cytosine methylation is associated with the occurrence of C-T transition mutations. About 30% of point mutations related to human diseases are C-T mutations that take place in CpG dinucleotides, which are the preferred substrate for the mammalian DNA-(5-cytosine) methyltransferases. Neurospora crassa is a particularly well suited organism to study the relationship between the processes of cytosine methylation and mutation. This fungus exhibits a particular mutagenic process called Repeat-Induced Point mutation (RIP) that detects and irreversibly alters repeated sequences through the introduction of C-T transition mutations. Although sequences altered by RIP often show cytosine DNA methylation, it is not yet clear whether cytosine methylation is the cause or the consequence of this mutagenic process. We have theoretically modeled the different enzymatic mechanisms proposed for the occurrence of C-T transition mutations in RIP. We suggested that the various models might be distinguished experimentally by studying the behaviour of the average frequency of transition mutation in populations of mutant alleles obtained by RIP at different levels of S-adenosylmethionine (SAM), the universal methyl-group donor for DNA methylation. RIP was investigated on a set of strains displaying a wide spectrum of both cellular SAM levels and DNA methylation. RIPing frequencies of unlinked duplicated copies of the am (NADP-glutamate dehydrogenase) gene decrease at high levels of SAM. The results suggest that the spontaneous deamination of the unstable cytosine-methylation intermediate (5,6-dihydrocytosine), is one of the main causes of the introduction of C-T transitions during RIP.

DNA damage, repair and aging in Neurospora crassa.

Hirokazu Inoue. Regulation Biology, Saitama University, Urawa, Saitama, Japan.

To investigate DNA repair mechanism in N. crassa, we have isolated and characterized more than 40 different mutagen-sensitive mutants. N. crassa has 3 different repair systems as other orgnisms have. In addition to those repair systems, there are some repair groups which have not been identified yet. We are currently analyzing those mutants in molecular level. I will summerize DNA repair study in N. crassa and talk about DNA repair mutants showing relationship between DNA repair and aging. Also, I will give an example that Neurospora is usable as a model organism in DNA repair study.

Events at recombination hotspots in Neurospora and their control.

David E. A. Catcheside, Frederick J. Bowring, P. Jane Yeadon, J. Paul Rasmussen, and Lin Koh. Biological Sciences, Flinders University, Adelaide, South Australia, Australia.

Recombination in Neurospora is initiated from hotspots usually 5' of genes. The hotspots are regulated by transacting genes (rec-genes) that block recombination from specific subsets of hotspots. rec genes are polymorphic in the natural population with active and inactive alleles. This system has the potential to provide differential control of the rate of evolution of specific sets of genes. We have cloned and sequenced rec-2, which regulates the very active hotspot cog that we have also cloned. rec-2 is transcribed in the sexual phase and does not show significant homology to known genes. In rec-2 regulated recombination initiated at cog, conversion events frequently have an apparently associated crossover. In events initiated at a weaker hotspot 5' of the am locus, conversion is rarely associated with a crossover, although the occurrence of a conversion event substantially increases the probability of crossovers in an interval 5 to 80 kb proximal to am. This is the first demonstration of negative interference and does not fit well with current molecular models for recombination. Many conversion tracts in Neurospora are interrupted. This offers a means of diversifying heterologous genes. We have adapted cog for this purpose.

Suppression of certain ascus-dominant mutations.

Robert L. Metzenberg and Namboori B. Raju. Biological Sciences, Stanford University, Stanford, CA, USA.

A number of mutations have been reported that affect steps in meiosis proper, the cutting out of ascospores, the shape of asci, the shape of the ascospores themselves, or the pigmentation and maturation of ascospores. Some of these are ascus-dominant, in that a mutant allele in one of the parents confers its characteristic phenotype equally on those that carry the allele and those that are genetically wild type. One such mutant, Asm-1 (Ascus maturation) causes about 99% of all ascospores to fail to develop pigment and to become viable, and certain derivatives of it are even more detrimental in a cross than the original mutant. Closely-linked suppressors can be isolated that allow a considerably larger fraction of the ascospores to mature. A few examples of a second, much rarer class have recently been identified that likewise give an increased fraction of survival in crosses involving Asm-1, and also give considerable suppression of the dominance of another ascus-dominant mutation, Roundspore. One of these, which gives about 50% suppression of Roundspore, also suppresses a third mutation, Banana which normally fails to cut out individual ascospores in about 99% of asci. In the suppression of Roundspore, an individual rosette in a perithecium will contain both asci with round spores and asci with normal spindle-shaped spores, but no ascus contains a mixture. Some implications will be discussed.

Detached nuclear genes of Neurospora transmitted cytoplasmically through infectious contact.

Edward G. Barry. Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

This research demonstrates that a plasmid can evolve from a set of nuclear genes. The chromosome aberration T(II;VI)R2459 produces acentric chromosome fragments through meiotic crossovers in linkage group IIR when paired with normal chromosome sets. The acentric fragments do not move to the division poles with the centric chromosomes but remain between poles in the cell cytoplasm. Acentric chromosomes from R2459 organize a surrounding membrane which protects them from nuclease digestion. (Acentric fragments produced from other Neurospora chromosome aberrations do not generate membranes.) As a mitotic division in the ascus continues after meiosis, the R2459 acentric replicates in synchrony with adjacent nuclei. The replicas may separate into distinct micronuclei. When ascospores form, the separate acentric fragments in micronuclei can be included passively in a spore along with either a translocation chromosome set or a normal sequence chromosome set. As the following mitotic divisions within developing ascospores proceed, the acentric chromosomes in micronuclei also replicate and may separate. After ascospore germination, selecting for prototrophic markers on the acentric fragment which complement reciprocal auxotrophic markers in the main nuclues has successfully shown that the fragment can be maintained in vegetative growth. A slower growth occurs when the culture is dependent on the acentric fragment for enzymatic activity determined by a gene carried by the fragment. The fragment may be transferred to a different cell line by construction of a heterokaryon and subsequent selection of both genetic activity of the fragment and a diagnostic marker of the infected second line. The acentric fragment is retained in high number only by selective pressure. When the fragment is required for continued growth, surveys of conidia in one typical serial set of isolations of colonies dependent on activity of the fragment show a range of 1:1,000, 1:100, and 1:20 conidia with the acentric fragment. The increasing frequency of recovery of the fragment in successive selective steps may indicate an adaptive evolution.


Feedback loops in the Neurospora circadian system.

Jennifer J. Loros and Jay C. Dunlap. Department of Genetics, Dartmouth Medical School, Hanover, NH 03755.

Circadian rhythms are daily rhythms in physiology and behavior that are found among many living things and that are nearly ubiquitous among higher eukaryotes. The fundamental molecular biology underlying these cycles is based at the level of the single cell, and there are similarities in mechanism running through all known circadian systems. An attractive model for elucidating these processes has been Neurospora; research on this fungus and on Drosophila has formed the basis for existing models of clock function. Models for the Neurospora circadian clock posit a negative feedback loop wherein the frq gene encodes two FRQ proteins which travel to the nucleus to block the activity of the heterodimeric activator of frq comprised of the WC-1 and WC-2 proteins (Dunlap, Cell 96:271-290, 1999). Given appropriate delays in the synthesis, action, and turnover of FRQ, this negative feedback loop yields oscillations in frq transcript and FRQ protein levels; this cycling of expression in clock components is characteristic of all circadian oscillators. The general layout of the feedback loop and the identity molecular identity of some of the components, particularly the heterodimeric positive elements WC-1 and WC-2 appear to be conserved among the Crown Eukaryotes (Dunlap, Science 280:1548-49, 1998; Cell 96:271-290, 1999). Light delivered at any point within the cycle acts through two regions within the frq promoter to rapidly increase the level of frq transcript thereby resetting the clock (Crosthwaite et al. Cell 81:1003 - 1012, 1995); mammalian circadian rhythms are reset in part in a similar manner (Shigeyoshi et al Cell 91:1043 - 1053, 1997). The WC proteins bind to these sites. Temperature acts posttranscriptionally to determine the absolute level of FRQ in the cell and the site of initiation of translation within the frq transcript, thereby dictating the ratio of long FRQ versus short FRQ (Liu et al, Cell 89:477 - 486, 1997). Resetting of the clock by changes in temperature can be understood in terms of changes in these set points (Liu et al, Science 281: 825 - 29, 1998). Dozens of circadianly expressed genes are known to act downstream of the clock. These "clock-controlled genes" include a hydrophobin (eas=ccg-2), trehalose synthase (ccg-9), and glyceraldehyde 3-P dehydrogenase (ccg-7=gpd) and play roles in clock regulation of development, stress responses, and intermediary metabolism (Loros, Curr. Opin. Microbiol. 1:698 - 706).

Biochemical interactions between FRQ and WC-2: critical clock proteins required for the normal operation of the Neurospora circadian oscillator.

Deanna L. Denault, Jay C. Dunlap, and Jennifer J. Loros. Biochemistry/Genetics, Dartmouth Medical School, Hanover, NH, USA.

The White Collar proteins (WC) are PAS domain containing proteins required for blue-light responses and essential for normal operation of the circadian clock in Neurospora crassa. Specifically, both WC-1 and WC-2 are necessary for rhythmic expression of a central clock component, frequency (frq). Although it is well established that FRQ negatively regulates it's own expression and that the expression of frq requires WC-2, the molecular mechanisms governing both the activation and the feedback loop of frq are unknown. Previously, we have shown that WC-2 is nuclear, consistent with its anticipated role as a transcriptional activator, but does not display a robust rhythm in either the nuclear or cytoplasmic fraction. Current experiments set out to begin to determine the nature of the mechanism of WC-2 regulation of frq transcription. In independent experiments, it was found that WC-2 interacts directly with FRQ. In vitro, GST-tagged WC-2 specifically interacts with radiolabelled FRQ proteins. More importantly, in vivo, FRQ specifically co-immunoprecipitates with both WC-1 and WC-2 from Neurospora extracts. This demonstrates direct or indirect association between FRQ and the WC proteins. Thus, WC-2 may regulate rhythmic transcription of frq through this protein-protein interaction. Further characterization of this interaction may provide insight to the molecular events underlying the basis of the circadian clock in Neurospora.

Control of conidial development by the circadian clock in Neurospora crassa.

Deborah Bell-Pedersen and Alejandro Correa. Department of Biology, Texas A&M University, College Station, Texas, USA.

Organisms have a clock to temporally organize biological activities to appropriate time of the day. In Neurospora crassa the clock regulates the timing of asexual spore development (conidiation). In constant darkness, conidiation occurs once every 22 h during the subjective morning. Several environmental signals can trigger conidiation in Neurospora, including light, air, carbon and nitrogen starvation; however the biological clock provides the only known endogenous signal to initiate conidiation. Three key regulators of the developmental pathway are known and include acon-2, fl and acon-3. Different combinations of the regulators are thought to control the expression of downstream conidiation-specific genes, including con-10, con-6 and eas (ccg-2). To begin to understand how the clock regulates development we are addressing the following questions: 1) what genes within the developmental pathway are regulated by the circadian oscillator? and, 2) How does initiation of development by the clock and by environmental signals differ? Answers to these questions will help us understand the mechanisms by which the clock regulates this critical cellular event.

Regulation of macroconidiation by fluffy.

Daniel J. Ebbole, Panan Rerngsamran and Lori Bailey Shrode. Texas A&M University, College Station, TX.

Fluffy (fl) encodes a member of the Gal4 class of transcription factors. Null mutations of fl block the budding growth characteristic of proconidial chain formation. These mutants are also blocked in expression of many of the known conidiation-specific genes. The pattern of fl mRNA expression is consistent with its role as a regulator of morphogenesis. A basal level of fl expression is observed in undifferentiated mycelia. fl mRNA levels are induced during development at approximately the time when budding growth initiates and fl mRNA levels declines at later stages of development. acon-2 and acon-3 also are regulators of conidial morphogenesis and acon-2 is required for induction of fl mRNA while acon-3 is not. Elevated expression of fl from a the cpc-1 or ars-1 promoter was sufficient to induce conidiophore morphogenesis. One current effort is to isolate the FL protein for DNA binding studies. The location of consensus FL DNA binding sites in the genome may provide clues as to which N. crassa genes may be subject to developmental regulation.

Signal transduction through heterotrimeric G proteins and opsins in Neurospora crassa.

Katherine A. Borkovich. Microbiol. & Molec. Gen., U.T.-Houston Medical School, Houston, TX, USA.

Heterotrimeric G proteins are components of major signal transduction pathways in eukaryotic organisms. We have characterized three G-alpha (GNA-1, GNA-2 and GNA-3), one G-beta (GNB-1) and one G-gamma (GNG-1) subunit from the filamentous fungus Neurospora crassa. Taken together, our results demonstrate that G proteins are crucial for regulation of cell proliferation, asexual and sexual differentiation and stress tolerance in N. crassa, through both cAMP-dependent and independent pathways. Of evolutionary interest, GNA-1 was the first microbial G-alpha demonstrated to belong to a family found in higher organisms (G-alphai). In contrast to mammalian systems, this fungal G-alphai is a positive regulator of adenylyl cyclase activity. Homologues of GNA-1 and GNA-3 have now been implicated in virulence in several animal and plant fungal pathogens. We have recently initiated a second project in the laboratory, study of opsins and opsin-related proteins (ORPs) in N. crassa. We have cloned and mutated the first opsin gene identified in a eukaryotic microbe, N. crassa nop-1. The predicted amino acid sequence of nop-1 is most similar to archaeal opsins. NOP-1 binds all-trans retinal using a Schiff base linkage to form a green-light absorbing pigment with an archaeal rhodopsin-like photocycle. Expression of nop-1 is restricted to conditions that favor conidiation and nop-1 null strains exhibit light-dependent conidiation defects under certain conditions. We are continuing our analysis of nop-1, and are also characterizing an ORP gene (orp-1) from N. crassa, in order to determine the functions that opsins and ORPs play during light-sensing in filamentous fungi.

Interaction between and transactivation by mating type polypeptides of Neurospora crassa.

Chuck Staben and Tom C Badgett. Biological Sciences, University of Kentucky, Lexington, KY, USA.

The polypeptides encoded by the mating type idiomorphs of Neurospora crassa control diverse aspects of the fungal life cycle. Biochemical characterization of MAT a-1, MAT A-1, and MAT A-3 reveal new activities that correlate with important biological activities of the polypeptides. All three polypeptides have domains capable of activating transcription in a yeast reporter system. The transcriptional activation domains of both MAT a-1 and MAT A-1 are not critical for either mating or vegetative incompatibility activities in Neurospora. Two hybrid assays establish the ability of MAT a-1 to interact with MAT A-1. Mutations that interfere with this interaction correlate with mutations that eliminate vegetative incompatibility, but not mating, in Neurospora. The MAT A-2 polypeptide did not transactivate nor did it appear to interact with any other mating type polypeptide. These results suggest the hypothesis that interaction of MAT a-1 with MAT A-1 stimulates vegetative incompatibility. Two-hybrid interaction screens of Neurospora cDNA libraries with MAT a-1 have identified other polypeptides potentiallyimportant in the activities associated with the mating type locus.

The Hyper-osmotic stress response pathway of Neurospora crassa is the target of phenylpyrrole fungicides.

Yan Zhang1, Randy Lamm1, Steve Lam1, and Jin-Rong Xu2. 1Novartis Crop Protection, Research Triangle Park, NC 27907. 2Purdue University, West Lafayette, IN 47907.

Neurospora crassa osmotic sensitive (os) mutants are sensitive to high osmolarity therefore unable to grow on medium containing 4% NaCl. In this study, we found that os-2 and os-5 mutants were resistant to phenylpyrrole fungicides fludioxonil and fenpiclonil. To further understand the mode of action of phenylpyrroles, we cloned the OS-2 gene by sib-selection. OS-2 encodes a MAP kinase homologous to HOG1 and can complement the osmosensitivity of a yeast hog1 mutant. We sequenced three os-2 alleles and found all of them were null alleles with either frameshift or nonsense point mutations. An delta-os-2 gene replacement mutant was also generated and was found to be osmotic sensitive and resistant to phenylpyrrole fungicides. Nonetheless, os-2 mutants transformed with the wild type OS-2 gene could grow on media containing 4% NaCl and became sensitive to phenylpyrrole fungicides. Interestingly, fludioxonil could stimulate intracellular glycerol accumulation in wild type strains but not in os-2 mutants. We also observed that fludioxonil could cause wild type conidia and hyphal cells to swell and burst. These results suggest that the hyper-osmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and fungicidal effects may be resulted from a hyperactive OS-2 MAP kinase pathway. Because phenylpyrrole fungicides have no effect on fission and budding yeast, this fungicidal effect may be specific for filamentous fungi.

Localization and light-dependent phosphorylation of White Collar 1 and 2, the two central components of blue light signaling in Neurospora crassa.

Hartmut Linden and Carsten Schwerdtfeger. Biology, University of Konstanz, Konstanz, Germany.

In N. crassa only two white collar (wc) mutants, wc-1 and wc-2, have been described which seem to be blind to light. The pleiotropic phenotypes of these mutants suggest that they represent two central components of blue light signal transduction. The WC proteins have several characteristics of transcription factors consistent with an involvement in transcriptional control of light-regulated genes. Here, we present a biochemical analysis of WC1 and WC2 polypeptides in Neurospora crassa. Using specific antisera against WC1 and WC2 respectively, the subcellular localization of the WC polypeptides was investigated. The WC1 protein was localized exclusively in the nucleus, whereas WC2 was detected in both the nuclear and cytoplasmic fractions. The nuclear localization of WC1 and WC2 was shown to be independent of light and dimerization between the two proteins. In addition, WC1 and WC2 are phosphorylated in response to light. The phosphorylation of WC1 and WC2 was dependent on functional WC1 and WC2 proteins, respectively which clearly indicated a correlation between the light-dependent phosphorylation and the function of WC1 and WC2 in blue light signaling. However, the light-specific phosphorylation of the WC proteins revealed different kinetics. The phosphorylation of WC1 was transient whereas the WC2 phosphorylation was shown to be stable under constant light conditions. The analysis of the light-dependent phosphorylation of WC1 and WC2 in wc-2 and wc-1 mutants revealed an epistatic relationship for WC1 and WC2 with WC2 acting downstream of WC1 in the signal transduction pathway of blue light.


The Neurospora Genome Project at UNM: Gold from the mold.

Mary Anne Nelson and Donald O. Natvig. Biology, University of New Mexico, Albuquerque, NM.

In the Neurospora Genome Project (NGP) at the University of New Mexico, expressed sequence tags (ESTs) corresponding to four stages of the life cycle (germinating conidial, advanced-growth mycelial, perithecial and unfertilized sexual tissues) of the filamentous fungus Neurospora crassa are being analyzed. Our strategies have included both random clone selection (to generate information on relative levels of gene expression) and subtraction of clones representing highly-expressed genes. We have identified over 2,000 different genes and have also developed a system for classifying encoded proteins based on their predicted functions. The results demonstrate a high percentage of novel genes with no known homologues in any organism (greater than 50%), and many genes that were not predicted for filamentous fungi. Database searches reveal that only 33% of the EST sequences have homologues in Saccharomyces cerevisiae, and 30% have homologues in non-fungal organisms. Several of the most highly expressed genes in mycelial and perithecial tissues have no known homologues in other organisms. Analyses of genomic sequences support estimates of greater than 10,000 genes for N. crassa. The preliminary results of an analysis of alternative splicing in Neurospora will also be presented. The results of EST and genome sequencing efforts with N. crassa and other filamentous fungi strongly support the need for additional study. Filamentous fungal genomes are rich in novel genes specific to fungal biology, and also in genes shared with very diverse organisms. The latter class of genes will prove valuable toward understanding fundamental biological processes. Supported by grants from the National Science Foundation.

Physical map of Neurospora crassa.

Jonathan Arnold. Genetics, University of Georgia, Athens, GA, 30602.

A physical map at 29 kb resolution covering 58% of the genome has been generated using a 13.3 genome equivalent cosmid library sorted by chromosome. The physical map is being used to test whether or not N. crassa has a chimeric genome organization like that of Aspergillus nidulans. Associated with the physical map is a chromosome walk across each chromosome with a scale in kilobases. Physical maps of each linkage group can be found at Of the 100 genes with known assignments to the 13.3 genome equivalent cosmid library, 59 of these cloned genes have been assigned to date to the physical map as expected. There is slightly less than 1 EST per cosmid along the physical map so that most cosmids contain an identified N. crassa gene sequence. Our best estimate of gene density is currently 1 gene per 3.3-3.8 kb or between 11,000-13,000 genes in the genome. About 10% of the N. crassa genome is repetitive with 4% being rDNA. Work is supported by NSF MCB-9630910.

The German Neurospora sequencing project: Achievements and perspectives.

Ulrich J. Schulte. Institute of Biochemistry, Heinrich-Heine-University, Dusseldorf, Germany.

The genome of N. crassa comprises 43 Megabases in 7 chromosomes. As part of an international effort to sequence the entire genome of the fungus, a German project has been set up to analyse two chromosomes with a total of 14 Mb. Based on the initiative of 8 institutes at 6 German universities with financial support from the Deutsche Forschungsgemeinschaft the project involves the cooperation of academic and industrial partners. BAC- and cosmid libraries are being mapped in the labs of Jörg Hoheisel (DKFZ, Heidelberg) and Jonathan Arnold (Athens, Georgia). Selected cosmid and BAC- clones giving a minimal tiling path covering the chromosomes II and V are being sequenced by MWG-Biotech, Ebersberg. The sequence data are assembled and analyzed by Werner Mewes and coworkers at MIPS, Munich. Open reading frames are identified and annotated and all the data are provided in a data base accessible through WWW. The data base is supposed to finally hold the entire genome as the analysis of the genomic sequence procedes in the US. Currently the database holds some 6 Mb of analyzed sequence. In my contribution to this meeting I will report on the progress of the project.

The Neurospora gene compendium - lessons from the past and plans for the future.

Alan Radford1 and Matthew Sachs2. 1Biology, Leeds University, Leeds, UK. 2 Biochem Mol Biol Oregon Graduate Institute Portland Oregon USA.

Working for the past three years on the update of the 1982 compendium, we find that new genes have been described at the rate of approximately two per month, and the rate is rising. Actually, more new genes have been described than that, but some later have been shown to be allelic with known genes. Such multiple namings arise when two different aspects of the phenotype are observed, when a sequence is determined which subsequently is found to correspond to a gene known from a mutant phenotype, etc.. These situations are relatively easy to resolve once the allelism becomes apparent. Using the same name for different genes is a more difficult problem. The commonest cause is when a sequence is found to be the homolog of a known gene from another species, and the name is transferred without checking if it is already used in Neurospora. Another problem arises when an apparently novel gene justifying inclusion had been described, probably based on a sequence, but the originator had not named it. We propose to put on the web a list of all symbols and names that have been used in the organism in order to make checking easier and naming according to the conventions for Neurospora more creative. With new genes at two per month and rising, the millennium compendium will need updating, and we cannot wait fifteen years this time before starting. We will endeavour to supplement the published compendium with a WWW database of new genes and major revisions of existing gene entries. To facilitate this, we propose to put an electronic submission form on the web, and request the support of the community in maintaining an up to date compendium.

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