Please cite Fungal Genet. Newsl. 51S:#
Biochemistry and Secondary Metabolism
1) Drug Discovery and the V-ATPase. Emma Jean Bowman, Barry Bowman, and Collaborators, Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, CA 95064, USA
Vacuolar ATPases (V-ATPases) are abundant, ubiquitous ion pumps in eucaryotic cells. They are directly involved in several diseases, including osteoporosis and Alzheimer's, and play a role in the growth of tumor cells. Three classes of natural products, which give similar profiles in the NCI 60-cell screen for inhibition of tumor growth, act as potent inhibitors of V-ATPases. The bafilomycins and concanamycins inhibit all known eucaryotic V-ATPases. The salicylihalamides and lobatamides show remarkable specificity for mammalian V-ATPases, having no effect on fungal enzymes. Chondropsins preferentially inhibit the fungal V-ATPase. Because of the variety of processes and diseases associated with V-ATPases and the possibility of designing selective inhibitors, the V-ATPases are becoming attractive targets for drug therapy.
2) Characterization of the Arg-13 Mitochondrial Transport Protein. Rey Renato, G. David, Gloria Turner and Richard L. Weiss. University of California, Los Angeles, Los Angeles, California, USA.
Metabolic processes take place in different compartments in eukaryotic cells. Intracellular compartments, such as the mitochondria, harbor enzymes and substrates that participate in specific metabolic pathways. Arginine biosynthesis in Neurospora crassa is an accessible model system to understand compartmentation. In N. crassa, glutamate is converted into citrulline inside the mitochondria, and citrulline is exported into the cytosol to be converted into arginine. We are investigating the role of the Arg-13 mitochondrial transport protein in the transport of arginine pathway metabolites across the mitochondrial membrane. The arg-13 gene has been cloned and inserted into a pET3a vector. Arg-13 has been overexpressed in BL21-SI cells and purified. Characterization of Arg-13 involves determining the submitochondrial localization of the protein using polyclonal antibodies and using proteoliposome transport assays to determine substrate(s) specificity, transport activity and mode of transport. Preliminary results suggest that Arg-13 is an ornithine transporter. Characterization of Arg-13 will help elucidate the role it plays in arginine metabolism and add another piece of information towards understanding the role of compartmentation in metabolic regulation.
3) Unusual Cys-Tyr covalent bond in a large catalase. Wilhelm Hansberg, Adelaida Díaz, Eduardo Horjales, Enrique Rudiño-Piñera, Rodrigo Arreola. Instituto de Fisiología Celular and Instituto de Biotecnología, Universidad Nacional Autónoma de México, México, D.F.
Catalase-1 is associated with non-growing cells and accumulates in conidia. It is a large, tetrameric protein and a highly efficient and durable enzyme that is active at molar concentrations of H2O2. Catalase-1 is oxidized at the heme by singlet oxygen without affecting enzyme activity. The crystal structure of catalase-1 at 1.75 Å resolution, compared to the structure of two other large catalases, revealed differences mainly at the carboxy-terminal domain. Heme is rotated 180° around the a-g–meso carbon axis with respect to clade III small catalases. There is no co-ordination bond of the ferric iron at the distal side of the heme. The structure exhibited a partial oxidation of heme b to heme d. Singlet oxygen hydroxylates C5 and C6 of pyrrole ring III with a subsequent formation of a hydroxyl-g-spirolactone. The site of modification is probably related to the exit of dioxygen through the central channel. The structure reveled an unusual covalent bond between the sulfur of a cysteine and the beta-carbon of the essential tyrosine of theproximal side of the active site. A peptide with the predicted theoretical mass of the two bound tryptic peptides was detected. A mechanism for the Cys-Tyr covalent bond formation is proposed. The tyrosine bound to the cysteine would be less prone to donate electrons to compound I to form compound II, making the enzyme resistant to inhibition and inactivation by substrate. An apparent closure of the main channel lead us to propose a gate that opens when there is sufficient hydrogen peroxide in the small cavity before the gate. This mechanism would explain the increase in catalytic velocity as the hydrogen peroxide concentrations rises. Grants: CONACyT C01-40697, DGAPA/UNAM IN225402
4) Efflux of basic amino acids from the vacuole of Neurospora crassa. Kelly Keenan, Richard Stockton College of New Jersey, Pomona NJ
The goal of the project is to characterize what causes the increase in efflux of three basic amino acids--arginine, lysine and ornithine--from the vacuole of N. crassa . Efflux can be measured using cupric ion-treated N. crassa. Nitrogen starvation results in an increase in efflux. The efflux permeases have not been identified and it is not known how they are able to change their activity. The change could be due to an increase in amount of permease protein or an elevated activity as the permease protein(s) binds to some positive effector. An extract of metabolites was collected from N. crassa grown under nitrogen starvation conditions and tested for its ability to increase efflux . Compared to conditions of having no extract as well as minimal medium extract, it increased efflux of the amino acids. In addition, efflux of the amino acids under nitrogen starvation conditions was not stopped when protein synthesis inhibitor was added. These results suggest that the increase in efflux is due to the presence of small molecule(s) that act as effectors. Efforts are underway to indentify these molecules. A series of mutants have been isolated that show altered efflux of oneor more of these amino acids. Extracts were prepared from mutants and tested inthe efflux assay. A number of these extracts were able to produce an increase in efflux which suggests that these mutants have increased efflux due to presence of effector molecules. Efforts are underway to identify these effector molecules.
5) The search for stretch-activated Ca2+ channels in Neurospora crassa. Marinela I. Anderca and Roger R. Lew. Department of Biology, York University, Toronto, Ontario Canada
The Saccharomyces cerevisiae Mid1 (mating pheromone- induced death) is an integral membrane protein required for theviability of differentiated cells and Ca2+ influx induced by mating pheromone, that functions as a Ca2+-permeable, stretch-activated channel when expressed in mammalian cells (Kanzaki et al., 1999 Science 285:882-6). In the Neurospora crassa genome we found a gene whose predicted protein is 30% identical and 45% similar to the Mid1 channel, with homologies to the H3 and H4 transmembrane regions and the cysteine-rich metal-binding regions, both essential for function. The gDNA derived from this gene (tentatively designated Ncmid1) was isolated by PCR from the cosmid library available at FGSC. Northern blot analysis is being employed to confirm that the Ncmid1 gene is expressed in N. crassa, to be followed by subcloning it into a vector suitable for performing RIP mutagenesis. Activities for two stretch-activated channels in N. crassa were detected previously in our laboratory. It is possible that one of these channels is encoded by Ncmid1,and we will attempt to confirm this by patch clamping the Ncmid1 mutant.
6) Identification of Antibiotic Binding Sites in the V-ATPase. Barry Bowman, Marija Draskovic and Emma Jean Bowman, Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, CA 95064, USA
The macrolide antibiotics bafilomycin and concanamycin are potent inhibitors of V- ATPases. To identify the binding site of bafilomycin we selected mutant strains of Neurospora crassa (named bfr) that are resistant to this antibiotic. In one class of bfr strains the V-ATPase was resistant to inhibition invitro. These strains had seven different point mutations in the vma-3 gene, which encodes the hydrophobic c subunit of the vacuolar ATPase. Most of the mutated sites appear to be on the outer face of the "rotor" sector of the enzyme, a region hypothesized to form an interface with the "a" subunit.
Surprisingly, the bfr strains had little resistance to concanamycin, which has a similar structure. By further mutagenizing one of the bfr strains we obtained four new strains that were resistant to both antibiotics. Each of these had two altered residues in the c subunit. Thus, concanamycin does appear to bind to the same region. The positions of four of the mutated residues correspond precisely to the positions of mutated residues in the homologous c subunit of the mitochondrial ATPase that confer resistance to oligomycin. These results suggest that vacuolarand mitochondrial ATPases have an ancient, conserved antibiotic binding site. As the sequences of the polypeptides have diverged new antibiotics that target the same vulnerable site in this family of enzymes have arisen. The data also suggest a model for the tertiary structure of the c subunit of the V-ATPase.
7) Circadian Rhythms in Neurospora crassa: Some unusual features of a new mutant, ult . Michelle Tsukamoto, Brian Chen, and Stuart Brody. Division of Biological Science, UCSD, La Jolla,CA, 92093-0116
The circadian rhythm of Neurospora crassa is expressed as bands of conidiating regions on the surface of agar medium with 22 hr. periods. A new clock mutant ult (ultradian) has been isolated and characterized that differs from the existing clock mutants in the following ways : 1) It has a 12 hour period which can be lengthened to 20 hrs.upon changing the nitrogen source from ammonia to nitrate; 2)under certain conditions, it shows a novel pattern of a wide band followed by a narrower band, with unequal spacing between them ; 3) it is a dominant mutation with respect to period, while most clock mutants are either recessive or co-dominant ; 4) the mutation maps to the center of LG I, and does not appear to be a mutation at any previously known clock gene. On the other hand, the mutant strain is similar to other clock mutants in that the period is temperature compensated, shows strong resetting (phase-shifting) to both light and temperature pulses, and entrains to a light/dark regime.When ult was introduced into the frq10 (clock null) strain the double mutant (ult-frq10) surprisingly showed rhythmic banding. However, this rhythm was not temperature compensated nor light-sensitive.This finding is interpreted in the context of a two oscillator model for N. crassa.
8) Characterization of NAD(P)H dehydrogenases from Neurospora crassa mitochondria. Patrícia Carneiro, Margarida Duarte, Isabel Marques, Joana Assunção and Arnaldo Videira. Instituto de Biologia Molecular e Celular and Instituto de Ciências Biomédicas de Abel Salazar, Porto, Portugal
The mitochondrial respiratory chain usually contains the type I NADH:ubiquinone oxidoreductase or complex I, a multi-subunit enzyme with proton-pumping activity. In addition, depending on the organism concerned, it has a variable number of alternative non-proton-pumping NAD(P)H dehydrogenases, both in the matrix and cytosolic faces of the inner membrane. The filamentous fungus Neurospora crassa also contains a quite well-conserved eukaryotic complex I. Besides, we have recently characterized three alternative enzymes: the main external NAD(P)H dehydrogenase, another externalcalcium-dependent NADPH dehydrogenase and an internal NADH dehydrogenase. In the present work, we describe the characterization of a fourth alternative NAD(P)H dehydrogenase. The respective gene was fused with the green fluorescent protein (GFP) and was also inactivated by RIPing. We have characterized the respiratory activities of the resulting mutant strain. Our results suggest that this enzyme also resides in mitochondria. In parallel, we have disrupted the gene encoding a subunit of N. crassa complex I, which is similar to a cell-death regulator. Characterization of an isolated mutant strain is underway and will be presented.
9) Analysis of vegetative growth of Neurospora crassa during the circadian cycle. E. Castro-Longoria1, S. Brody2, and S. Bartnicki-García1. 1División de Biología Experimental y Aplicada, CICESE, Ensenada, Baja California, México. 2División of Biology/Molecular Biology, University of California, San Diego.
The circadian rhythm of Neurospora crassa is best observed in "clock" mutants. The rhythm produces spectacular concentric bands of conidiation separated by vegetative bands, or interbands, where aerial hyphae and conidia are scarce. By video-microscopy and image analysis, we evaluated the relationship between vegetative and reproductive development during the circadian cycle. The growing edge of a colony of N. crassa (bd csp oli) was recorded continuously at low magnification for 24 h (11h light-13h dark) at 22-24 C. Growth rate was measured for groups of 10-15 leading hyphae and found to decrease gradually at the start of the dark period. This coincided with the formation of the conidiation band. At the start of the light period, the growth rateof vegetative hyphae increased gradually. Growth rate reduction was correlated with increased formation of aerial hyphae and subsequent conidiation. During this period the cytoplasm of vegetative hyphae behind the edge of the colony was seen (magnification 2000 X) to flow backwards towards a branch, possibly an aerial hypha. Clearly, the circadian cycle comprises not only a periodic band of conidiation but also a corresponding decrease in vegetative growth rate. Presumably cytoplasmic resources that flow towards the tip to maintain apical growth are partly diverted to form aerial mycelium and conidia.
10) Heterokaryon incompatibility, prion formation and protein aggregation in Neurospora crassa. Karine Dementhon and N. Louise Glass. University of California, Plant and Microbial Biology Department, Berkeley, CA 94720.
Neurospora crassa can undergo hyphal fusion between different individuals to form vegetative heterokaryons. Hyphal fusion between two individuals with identical genotype at all het loci leads to stable heterokaryon formation. In contrast, if the two individuals differ in allelic specificity at a het locus, the fusion cell is rapidly destroyed by a programmed cell death (PCD) reaction termed heterokaryon incompatibility. In Podospora anserina, the het-s locus has two incompatible alleles, het-s and het-S. A het-s strain can have two alternative HET-s forms: a [Het-s] prion form, and a [Het-s*] non-prion form. Only the [Het-s] prion form triggers incompatibility when a het-s strain fuses with a het-S strain. The prion-free [het-s*] strain will form a viable heterokaryon with a het-S strain. The mechanism by which the prion protein triggers the cell death reaction in combination with HET-S is unknown.
N. crassa offers an excellent system to explore the relationship between protein aggregation and PCD. We found that over-expression of het-s in N. crassa results in HET-s aggregation, and, unlike in P. anserina, triggers growth inhibition. Our preliminary results suggest that co-expression of het-s and het-S in N. crassa triggers heterokaryon incompatibility. In N. crassa, genetic differences at het-c locus trigger incompatibility, which is associated with the formation of HET-C heterocomplex composed of HET-C proteins encoded by the alternative alleles. Incompatibility at het-c is suppressed by vib-1, which encodes nuclear-localized putative transcriptional factor. The relationship between incompatibility mediated by het-s and het-c, protein mis-folding, and vib-1 is currently under investigation.
11) The Neurospora crassa immunophilin FKBP50 is located in the nucleus. Margarida Duarte, Patrícia Carneiro and Arnaldo Videira. Instituto de Biologia Molecular e Celular and Instituto de Ciências Biomédicas de Abel Salazar, Porto, Portugal
The FK506 binding-proteins (FKBPs) define a subfamily of peptidyl-prolyl cis/trans isomerases known to play roles in cellular processes such as protein folding, protein interactions and signal transduction. Four FKBP members (FKBP11, FKBP13, FKBP22 and FKBP50) were identified in the last annotation of the N. crassa genome (Neurospora sequencing project, Whitehead Institute/MIT Center for Genome Research). We have recently disrupted the gene for FKBP22, an immunophilin that resides in the endoplasmic reticulum. FKBP50 displays a high similarity to the FPR3 and FPR4 homologues of Saccharomyces cerevisiae, along with its homology to other Neurospora FKBPs. To characterise the cellular function of FKBP50 we are inactivating the fungus gene and used a green fluorescent protein (GFP) fusion to determine its subcellular localization. The cDNA encoding FKBP50 was cloned in frame with the GFP gene, under the control of an inducible promoter. The fusion construct and a control plasmid were targeted to the his-3 locus of N. crassa. The transformants were analysed for the correct targeting of the plasmids by southern blot and for the expression of the FKBP50-GFP fusion protein with specific antibodies against FKBP50 and by fluorescence microscopy. Our results indicate that the FKBP50 protein is located in the nucleus. Further work is undergoing to characterise its biological function.
12) Characterization of soft, a hyphal fusion mutant of Neurospora crassa. Andre Fleissner, David J. Jacobson, Sovan Sarkar and N. Louise Glass. University of California, Berkeley
The mycelial colony of filamentous fungi consists of a network of interconnected multinucleate hyphae. The colony grows by hyphal tip extension, branching andfusion (anastomosis). The ability to form hyphal fusions within one colony, butalso between different individuals enables fungi to establish complex functional units that show coordinated growth and exploration of their environment.To gain a better understanding of the basic, yet not well described, process of hyphal fusion, we are characterizing soft, a hyphal fusion mutant of Neurospora crassa.The phenotype of soft includes the lack of anastomosis within a colony or between conidial germlings, female sterility, reduced aerial hyphae and a slower growth rate. We were able to clone soft by complementation. Database analysis showed that this gene is highly conserved in filamentous ascomycetes, but not present in Saccharomyces cerevisiae or Schizosaccharomyces pombe. The encoded protein contains a conserved WW-domain involved in protein-protein interactions, but has unknown function. Genetic, complementation and sequence analysis revealed that soft is allelic to ham-1. Ham-1 mutants show the same phenotype as soft strains. Both alleles show single point mutations resulting in stop codons in the first part of the gene. Using microscopy, genetics and biochemical analysis we are revealing the function of the SOFT protein in the process of hyphal fusion and, in a broader view, the role of anastomosis for the biology of filamentous fungi.
13) Sharp Peaks of Circadian Conidial Development for Neurospora crassa as Determined by Time Lapse Video. Van Gooch, University of Minnesota Morris, Division of Science and Math, Morris MN 56267
Using time lapse video, the growth of Neurospora crassa bd was analyzed. The Neurospora were grown in classic conditions that demonstrate circadian rhythms of conidial formation (agar in a race tube with 0.3% glucose, 0.5% arginine, and 1X Vogel's salts at 25°C with red safety lights). The conidial development largely occurs simultaneously over a period ofabout one hour in a band width of about 6 mm. The peak time of this activity is about 11.4 hours after the white lights are last turned off. The time lapse video also reveals an approximately two fold change in the growth front rate during a daily circadian cycle. Using a variety of conditions that alter the conidiation rhythm, the period and magnitude of the growth rate rhythm correlates to the period and magnitude of the conidiation rhythm. Neurospora circadian rhythms have been classically measured by linearly translating the position along a race tube into time. In fact there is a variation in growth rate by a factor of about two and calculations show that the linear translation of position to time can cause errors in period and phase on the order of magnitude of one hour. Secondly, when one uses a linear translation of position to time, one would determine from the conidial band width that conidiation development occurs over several hours when in fact the time lapse video analysis shows that it occurs more on the time frame of one hour.
14) A Mauriceville derivative strain that escapes senescence is defective in the retrograde response pathway. John C. Kennell, Erica Larson and Maze Ndonwi. Department of Biology, Saint Louis University, St. Louis, MO
Wild-type Neurospora strains display indefinite growth potential and with few exceptions, senescence is restricted to strains harboring certain mitochondrial plasmids. The MS4416 strain has a variant form of the Mauriceville mitochondrial retroplasmid and has been shown to senesce at highly predictable frequencies. Growth cessation of MS4416 cultures was shown to be associated with the inhibition of mitochondrial gene expression due to plasmid over-replication. Here, we describe a mutant derivative of the MS4416 strain that escapes senescence. This so-called long-lived (LL) strain shows indefinite growth while still tolerating high levels of the variant plasmid. New forms of variant plasmids arise and replace existing plasmids, indicating that longevity is not related to particular changes in the Mauriceville plasmid. The LL strain appears to be defective in the retrograde response pathway as it is sensitive to electron transport chain (ETC) inhibitors and fails to induce specific nuclear genes, such as the structural gene for alternative oxidase (aod-1). Genetic studies show that the sensitivity to ETC inhibitors is controlled by a single nuclear gene, and the mutation is not allelic to aod-1. The inability of the LL mutant to induce retrograde response genes appears to preventthe accumulation of defective mitochondria and enables the strain to escape senescence.
15) A novel senescent phenotype appears to result from a dominant-lethal nuclear mutation that influences cytochrome c expression. John C. Kennell, Marci Tauzin, Stephanie Cohen and Janice Chyi. Department of Biology, Saint Louis University, St. Louis, MO
Senescence of filamentous fungal cultures invariably involves mitochondrial dysfunction, which in most cases is associated with the integration of mitochondrial plasmids into the mitochondrial genome. Here we describe a senescent mutant of N. crassa (M6) that derives from the wild-type Mauriceville strain (FGSC 2225). Surprisingly, M6 cultures that are close to senescence have no detectable changes in either the Mauriceville mitochondrial plasmid or mitochondrial DNA, features that are commonly associated with Mauriceville senescent strains. Tetrad analysis of crosses with M6 as a conidial parent indicate that the senescent phenotype is inherited as a single nuclear mutation, with mutant ascospores senescing shortly after germination. The cytochrome spectra of M6 cultures are highly unusual as they lack a peak corresponding to cytochrome c and Northern analysis indicates that cyc-1 transcripts are not produced. Uninucleate microconidia isolated from M6 cultures are not viable, suggesting that the mutation is lethal as a homokaryon. As M6 arose spontaneously during vegetative transfer, it appears that the mutation is dominant; however, formal (i.e. forced) heterokaryon studies have not yet been conducted. Further studies of the M6 strain may reveal attractive targets to control fungal growth.
16) Pheromone receptor genes, pre-1 and pre-2, are essential for mating type-specific directional growth and fusion of trichogynes and female fertility in Neurospora crassa. Hyojeong Kim and Katherine Borkovich. University of California, Riverside, U.S.A.
Pheromone receptor genes for the heterothallic filamentous fungus Neurosporacrassa, pre-1 and pre-2, were identified during BLAST searches against the entire genome sequence (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/ ). The encoded proteins belong to the G-protein-coupled receptors containing seven-transmembrane helices and show significant sequence similarity to other fungal pheromone receptors. pre-1 and pre-2 deletion mutants are not greatly affected in vegetative growth, heterokaryon formation or male fertility in either mating type.They form normal protoperithecia with fully differentiated trichogynes as well. However, pre-1 mat A and pre-2 mat a strains are unable to undergo fertilization; their trichogynes are unable to recognize and fuse with mat a and mat A cells, respectively. Previous work has demonstrated that GNA-1 (Ga) and GNB-1 (Gb) are required for female fertility inN. crassa. Trichogynes of gna-1 and gnb-1 mutants displayed severe defects in growth towards and fusion with male cells, similar to that of pre-1 mat A and pre-2 mat a strains. Thus, PRE-1 and PRE-2 are pheromone receptors coupled to GNA-1 that is essential for the mating of mat A and mat a strains as females, respectively, consistent with a role in launching the pheromone response pathway in N. crassa.
17) A G-protein coupled receptor gene, gpr-1 is involved in regulation of sexual development in Neurospora crassa. S. Krystofova and K. A. Borkovich, University of California Riverside, Plant Pathology, Riverside, CA
10 potential G-protein coupled recepetors (GPCR's) have been identified in the entire Neurospora crassa genome database. Based on the phylogenetic analysis, three of those genes gpr-1, gpr-2 and gpr-3 form a family with homology to the second class of GPCR's. gpr-1 null mutants analyzed exhibit defects in sexual development. This corresponds with gpr-1 expression pattern that shows the highest mRNA level in perithecial tissue. In comparison to wild type, gpr-1 strains produce small and pale protoperithecia that are often buried in solid media. Perithecia are smaller than those from wild type crosses and defective in the light-induced polarity and ascospore ejection.
18) Blue light effects on ion transport in Neurospora crassa slime mutant. N.N.Levina1, A.Y.Dunina-Barkovskaya2, S.N.Shabala3, and R.R. Lew1. 1York University, Toronto, Ontario, Canada; 2Moscow State University, Moscow, Russia; 3University of Tasmania, Hobart, Australia
Blue light regulates a number of cellular functions in N. crassa, such as carotenogenesis, conidium formation, protoperithecia formation and phototropism. We studied the effects of blue light on ion transport processes across plasma membrane, since electrical changes are the earliest recorded responses to blue light in N. crassa. We used the slime mutant (FGSC#1118), it lacks a cell wall and grows as amoeboid-like spheroplasts. It exhibits normal photoinduced carotenogenesis and is suitable for the study of photoeffects on plasma membrane ion currents. Patch clamp experiments were complemented by measurements of ion fluxes across plasma membrane using the ion-selective vibration probe technique. Within 2-4 min of illumination, blue light caused a decline in whole-cell conductance attributed to the decrease in inward potassium current. Simultaneous hyperpolarization of the plasma membrane may develop as a result of the decrease in inward potassium current or/and activation of the plasma membrane proton pump, supported by a decrease in the proton influx. Blue light also causes an increase in chloride ions influx. Therefore, blue light regulates an ensemble of transport processes: H+, Cl-, and K+ transport. Since changes in the total conductance are detected within a few seconds after the onset of blue light, we suggest that these changes may be part ofthe initial signal transduction.
19) Expression of clock-associated genes in agar cultures of Neurospora. Sanshu Li and Patricia Lakin-Thomas. Dept. of Biology, York University, Toronto, ON, Canada.
The circadian clock of N. crassa drives a rhythm in spore formation that is assayed by observing bands of conidiospores when cultures are grown on the surface of agar medium. Rhythmic gene expression has been assayed in Neurospora by other laboratories using liquid culture systems in which conidiation is suppressed and the rhythm of spore formation cannot be assayed. We are using a culture system in which conidiation and biochemical rhythms can be assayed under identical conditions, by growing cultures on solid agar overlaid with cellophane. We are comparing biochemical rhythms in the newly-formed hyphae at the growth front with hyphae 24-48 hours old behind the growth front. We have found rhythmic expression (RNA and protein) of the frq (frequency) gene in old and new areas, and rhythmic levels of WC-1 (white-collar-1) protein. However, the phase relationship between the FRQ and WC-1 protein peaks differs between old and new areas, and differs from the phase relationship reported by other laboratories using liquid culture systems. RNA levels for clock-controlled genes ccg-2 and ccg-7 are rhythmic in new areas but not rhythmic in old areas. We found similar results for the neutral lipid diacylglycerol (DAG): Levels are rhythmic in new areas but low and arrhythmic in old areas. Mycelial transfer experiments indicate that hyphae from both old and new areas carry similar clock phase information. We are also investigating gene expression in the long-period chol-1 mutant strain, which has an elevated level of DAG. Funding from NSERC grant 504210 is acknowledged.
20) Analysis of a G-protein coupled receptor from Neurospora crassa. Liande Li and Katherine A. Borkovich, University of California Riverside, Plant Pathology, Riverside, CA
gpr-4 (G-protein coupled receptor-4) is a gene encoding a G-protein coupled receptor identified in Neurospora genome database (http://www-genome.wi.mit.edu/annotation/fungi/neurospora) that is most similar to putative carbon sensory receptors: Gpr1p from Saccharomyces cerevisiae and Git3from Schizosaccharomyces pombe. The gpr-4 deletion mutants do not show any obvious differences with wild type in regards to 1) apical extension rate on minimal medium and medium with poor carbon or nitrogen sources, 2) aerial hyphae height in standing liquid cultures, 3) male and female fertility, 4) ascospore germination, or 5) dry mass of submerged cultures grown with different carbon sources. However, delta gpr-4 strains accumulate much less biomass than the wild type when cultured on solid medium containing poor carbon sources, such as glycerol, mannitol, arabinose, or low concentrations of sucrose or glucose, suggesting a possible role as a carbon sensor. delta gpr-4 forms less aerial hyphae and produces more conidia per aerial hyphae than wild type. In addition, delta gpr-4 showed higher resistance to heat shock and H2O2 stress. delta gpr-4 shares some characteristics with delta cr-1, delta gna-1, delta gna-2 and delta gna-3 mutants. Further experiments, including epistasis analysis of gpr-4, with gna-1, gna-3, cr-1 and pka-cat are in progress, in order to determine the function of GPR-4.
21) Characterization of meiotic silencing by unpaired DNA (MSUD) in Neurospora tetrasperma. Namboori Raju and David Jacobson. Department of Biological Sciences, Stanford University, California.
Genes that are unpaired during meiosis are silenced in N. crassa. GFP-tagged histone H1 (hH1::GFP, courtesy of M. Freitag, U. Oregon), when inserted at the his-3 locus on linkage group I (LGI), allows visualization of MSUD in developing asci by fluorescence microscopy. When homozygous, hH1::GFP is paired during meiosis; it expresses normally and nuclei fluoresce throughout ascus development. However, when heterozygous, hH1::GFP is unpaired and silenced during meiosis until ascospore delimitation. MSUD does not extend into the ascospore maturation stage and hH1::GFP nuclei in four of the eight ascospores begin to fluoresce ~18-24 h after delimitation. N. tetrasperma packages two nuclei of opposite mating type into each of its four ascospores. This is accomplished by blocking recombination in a large region of LGI. LGI also shows a large unpaired region during pachytene. To test whether the genes in this unpaired region are silenced, we introgressed hH1::GFP at the his-3 locusfrom N. crassa into N. tetrasperma, The initial hybrid cross produced almost all 8-spored asci with a high level of ascospore abortion, but showed no silencing of hH1::GFP. After four backcrosses to N.tetrasperma, all progeny are now phenotypically N. tetrasperma: asci are four-spored and ascospores are heterokarotic for hH1::GFP. All nuclei in the developing asci and in the heterokaryotic ascospores fluoresce brightly. Thus, unpaired hH1::GFP on LGI is not silenced in N. tetrasperma. Whether the absence of MSUD in N. tetrasperma is limited to the unpaired region in LGI or is global in the genome is under investigation.
22) Screening for Supressors of Hyperbranching Mutants in N. crassa. Beth Rapa & Michael Watters. Valaparaiso University, Valparaiso Indiana
Growth in filamentous fungi occurs at a tip which branches as it extends. Neurospora crassa is a filamentous fungus for which there are many known morphological mutants which affect the periodicity of branching. The col-16 mutant of N. crassa has a much greater branching frequency than the wild type and therefore grows more densely. Ultraviolet light was used on a col-16 mutant strain in order to induce mutations with the goal of finding suppressors that return growth to normal. Following mutagenesis, the samples were plated and the resulting colonies screened for those in which more wild type growth had been restored (i.e. those which had gained a suppressor mutation). The presence of a suppressor was confirmed using crosses, crossing the potential suppressor to the wild type. The reappearance of col-16 mutants among the progeny of these crosses confirms that the suspect strains indeed are suppressed (i.e. col-16/suppressor double mutants). The next goal is to separate the suppressor from col-16 to obtain strains which contain only the suppressor mutation. This work was supported by a grant from the National Science Foundation.
23) A Novel Rho-type GTPase Required for Septation in Neurospora crassa . Rasmussen, C., Chiang, E. and N. L. Glass, Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720
Rho-type GTPases are small GTPases primarily involved in polarization, control of cell division and reorganization of cytoskeletal elements. Phylogenetic analysis of fungal Rho family members suggests that while several Saccharomyces cerevisiae Rho-type GTPases have orthologs in Neurospora, others do not. One of the Rho-type GTPases, RHO-4, which has an ortholog in Schizosaccharomyces pombe but not in S. cerevisiae was mutated in Neurospora by RIPping. rho-4 loss of function mutations lead to a loss of septation, loss of conidiation and heavy cytoplasmic bleeding. Steps required for septation include formin localization, septin and actin localization, and finally new cell wall synthesis. In order to determine where RHO-4 acted in the septation pathway, localization of f-actin was observed in wild type and rho-4 strains. rho-4 mutants are unable to form actin rings, suggesting that RHO-4 acts upstream of actin localization. Further, preliminary immunofluorescence experiments suggest that RHO-4 is localized to septa. Characterization of dominant active and dominant negative alleles of rho-4 as well as cloning of suppressor mutations will help to elucidate the role of RHO-4 in septation and conidiation in N. crassa.
24) Key differences between lateral and apical branching ontogeny in hyphae of Neurospora crassa. Meritxell Riquelme and Salomon Bartnicki-García.Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), 22860 Baja California, México.
We examined in fine detail growth kinetics and intracellular events during lateral and apical branching in hyphae of Neurospora crassa by high-resolution video-enhanced light microscopy. We found remarkable differences in the events preceding lateral vs. apical branching. While apical branching encompassed a significant disturbance of apical growth, lateral branching occurredwithout any detectable alterations in the apical growth of the parental hypha. Lateral branch formation did not interfere with the elongation rate of the primary hypha, the shape of its apex or the behavior of its Spitzenkörper. In sharp contrast, apical branching was preceded by marked changes in physiology and morphology of the parental hypha and by a sharp drop in elongation rate. The sequence involved a cytoplasmic contraction, followed by a retraction, dislocation, and disappearance of the Spitzenkörper; hyphal elongation decreased sharply and a transient phase of non-polar growth caused the hyphal apex to round up. Growth resumed with the formation of two or more apical branches, each one with a Spitzenkörper formed by gradual condensation of phase-dark material around an invisible nucleation site. The observed dissimilarities between lateral and apical branching suggest that these morphogenetic pathways are triggered differently. Whereas the trigger of apical branching may be traced to a sudden discrete disruption in cytoplasmic organization, the trigger of lateral branching probably stems from the subapical accumulation of wall precursors reaching a critical concentration.
25) Conidial Anastomosis in Neurospora crassa. M. Gabriela Roca M., Jochen Arlt & Nick D. Read. Institute of Cell and Molecular Biology/COSMIC, University of Edinburgh, Edinburgh, UK.
Using live-cell imaging, we have analysed the process of hyphal homing and fusion (anastomosis) between conidial germlings of Neurospora crassa labelled with different vital dyes and GFP probes. Specialised, morphologically distinct hyphae (called conidial anastomoses tubes [CATs]) are produced by conidia >4h following hydration. In wild type strains, CATs are thinner (2.72 +0.6 um) than germ tubes (3.52 + 0.3um). Conidial anastomosis tubes grow towards each other, and reorientate themselves back towards each other if they are moved relative to each other with laser tweezers. This provides clear evidence for the existence of, as yet unknown, diffusible chemotropic signals being involved in the homing response of CATs. In contrast to ‘fusion hyphae' whichundergo fusion in the centre of a mature colony, CATs do not undergo branching. Fusion between CATs is independent of mating type, and occurs between conidial germlings of the same and different mating types. We have imaged nuclear movement and shown continuity of the microtubular cytoskeleton between fused germlings. Further morphological, physiological andgenetic markers are currently being used to further characterize CATs and the role that these structures play in the life cycle of N. crassa.
26) Biogenesis of glyoxysomes and Woronin bodies in N. crassa as a model of selective protein import. Hanspeter Rottensteiner, Institut für Physiologische Chemie, Bochum, Germany.
The biogenesis of microbodies has been studied in various model organisms ranging from yeast to man and is specifically governed by the so-called PEX genes. Also the genome of the filamentous fungus Neurospora crassa contains a set of at least 18 peroxins. Interestingly, N. crassa harbors two compartments of the microbody family; glyoxysomes that house the fatty acid beta-oxidation enzymes and two key enzymes of the glyoxylate cycle, as well as the Woronin body, whose main function is to plug the septal pore after hyphal wounding. The glyoxysomal matrix proteins as well as the predominant matrix enzyme of the Woronin body, Hex1p, contain classical peroxisomal targeting signals. Since both organelles coexist in a single cell, the question arises as to why Hex1p is imported into Woronin bodies and not into glyoxysomes and vice versa. Strategies to unravel the molecular mechanism of this selective protein import will be presented.
27) Mechanism of Polarized Growth and Hyphal Morphogenesis in Neurospora crassa. Maho Uchida, Elizabeth Perry, and Robert W. Roberson. School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA
Fungal hyphae grow and maintain their characteristic shape through cell extension at their tips. Mechanisms of polarized growth are maintained by cytoskeletal function and directed exocytotic events. Previous observations have lead us and others to speculate that microtubules (MTs) are involved in long distance transport of secretory vesicles from Golgi-equivalents to the Spitzenkörper (Spk), followed by a switch at the Spk from MTs- to actin microfilament-based motility. To better understand vesicle flow from sub-apical sites to the apical plasma membrane and the role(s) of the cytoskeleton, we have evaluated Spk dynamics and organization, and have mapped the distributions of MTs and cytoplasmic components using optical and transmission electron microscope (TEM) methods. This work was done in mature, wild type hyphae of Neurospora crassa and will serve as a baseline for future studies to elucidate important mechanism underlying hyphal morphogenesis. Using phase contrast digital light microscopy, a unique organization of the Spk and novel details of internal dynamics have been identified. The Spk consisted of three discrete phase-dark layers subtended by a phase-bright core. Unidentified materials, at or below the level of resolution, traveled through the core towards the hyphal apex. Serial cross-section reconstructions and quantitative analysis ofTEM data from a 2.0 m thick sub-apical region approximately 30 m behind the apex have been analyzed. Within this region, 61 microtubule cross-sections, 17 apical (secretory) vesicles and 13 Golgi-equivalents were counted. Mitochondria, multi-vesicular bodies, and nuclei were also noted. 0 to 7 MTs were located within 25 to 274 nm of Golgi-equivalents while only 3 apical vesicles positioned within 30 nm of a MT or MT bundle. Analysis of such structural and dynamic data will be discussed relative to the vesicle flow and and cytoskeletal function.
28) Oxidative stress and conidiation in Neurospora crassa Wilhelm Hansberg, Shaday Michán, Leonardo Peraza, Adelaida Díaz, Fernando Lledías, Pablo Rangel, Mauricio Rios-Momberg. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F.
Morphogenetic transitions of N. crassa asexual life cycle are responses to a hyperoxidant state. Catalase activity induction and catalase oxidation by singlet oxygen are consequences of this hyperoxidant state. The two large monofunctional catalases (CAT–1 and CAT–3) and catalase-peroxidase (CAT–2) are resistant to molar concentrations of hydrogen peroxide. These enzymes are oxidized by singlet oxygen at the heme, without significantly affecting enzyme activity, but oxidation increases enzyme degradation. CAT–1 is expressed in non-growing cells, such as hyphae in stationary growth and conidia. CAT-1 is accumulated to high levels in conidia. Crystallographic structure of CAT-1 showed an oxidized heme and an unusual covalent bond at the active site. CAT–2 is associated with lysing cells, such as hyphae in late stationary growth,in conidiating substrate mycelium, and base of aerial hyphae. CAT–3 is associated with growing hyphae and is expressed during late exponential and pre-stationary growth. CAT–3 has a signal peptide and is secreted. Light and oxidative stress induces CAT-3. A CAT–3 null mutant strain showed increased protein oxidation, carotene levels in the dark, and hyphae aggregates, features that are indicative of oxidative stress. The mutant strain formed and produced six fold the amount of wild type strain aerial hyphae and conidia. CAT-2 null mutant strain is sensitive to oxidative stress and to a temperature of 42oC. It forms higher number of arthroconidia. These results support our hypothesis of cell differentiation as response to oxidative stress. Grants: CONACyT C01-40697, DGAPA/UNAM IN225402.
29) Characterization of the rcm-1 gene of Neurospora crassa. Bheong-Uk Lee*, Eun Jung Kim, Sang Rae Kim, Ilji Jeon and Byung-Kap Jeong Division of Biological Sciences, Kosin University, Busan 606-701, Korea
Analysis of the complete genome of Neurospora crassa reveals that at least 15 proteins contain tetratricopeptide repeat (TPR) motifs. One of them shows over 60% homology to Ssn6 of Saccharomyces cerevisiae, a universal repressor that mediates repression of genes involved in various cellular processes. Mutant strains generated by RIP (repeat induced point mutation) process showed five distinctive vegetative growth patterns and slow growth in various rates: A) densemycelial, csp, looks like ropy, yellow, B) dense mycelial, csp, melanin-overproduction on SC agar, C) slow growth, dense mycelial, csp, D) extremely slow growth, acon and E) flat, little aerial hyphae, acon, looks like rco-1. They are male-fertile, yet all female-sterile and produced little or no perithecium. Reduced levels of con-8, eas and grg-1 mRNA were detected in type A mutant strain while they were elevated in type E. These results indicate that this gene is pleiotrophic and involved in several cellular processes during vegetative growth, and asexual and sexual spore formation. Analysis of cDNA shows that it encodesa putative 102kDa protein. This gene is designated rcm-1 (regulation of conidiation and morphology).
30) A suppressor mutant which suppresses cr-1 mutation in Neurospora. Tadako Murayama, Michiko kudo, and Sei-ichi Kanzaki, Kanto-GakuinUniversity, Yokohama, Japan
A morphological mutant of Neurospora cr-1 grows to be colonial, whereas the wild type grows to be filamentous. The cr-1 mutant has been described as having a defect in adenylyl cyclase gene. Suppressor mutations of cr-1 mutation frequently occurred. One of suppressor mutants, hah, which suppresses colonial growth of cr-1 formed high aerial hyphae without conidia. Genetic analysis of the hah mutant showed that it has a mutation in the gene which is located 13.3 units from inl and 4 units from am on the linkage group V. It was suggested by the physical map reported in Neurospora crassa Genetic Maps (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/maps.html) that HAH is MCB which encodes the regulatory subunit of cAMP dependent proteinkinase, though the hah mutant is morphologically different from the mcb mutant which has been reported to form a lot of conidia(Bruno et al.,1996). The results of cloning and sequencing of the MCB gene from the hah mutant will be presented and the relatioships between the hah mutation and the morphology will be discussed.
31) Neurospora crassa catalase-peroxidase is required for heat shock and oxidative stress tolerance. Leonardo Peraza, Wilhelm Hansberg. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D.F.
Catalase-peroxidases (CP) are bifunctional antioxidant enzymes that evolved in a prokaryotic progenitor cell by tandem duplication of an ancestral peroxidase gene. N. crassa cat–2 encodes a typical CP. Based on phylogenetic analysis, we have suggested a bacterial origin for fungal CPs. The enzyme is a homodimer of 83.4 kDa subunits that has both, catalase and peroxidase activities. CAT–2 mRNA and activity are associated with late stationary-phase mycelia, when arthroconidia are formed and hyphae undergo autolysis. CAT–2 was induced during stress conditions such as carbon deprivation, H2O2-generated oxidative stress and heat shock. CAT–2 activity was found to be regulated during macroconidiation, cat–2 mRNA accumulated rapidly after induction of conidiation by air exposure of a mycelial mat, but CAT–2 activity was detected until aerial hyphae are formed. Both, mRNA and CAT-2 increase further in aerial hyphae and conidia. CAT–2 null mutant strains are sensitive to oxidative stress exerted by hydrogen peroxide or organic peroxides. It is also sensitive to high temperatures.CAT–2 null mutant strain showed increased formation of arthroconidia during late stationary-phase. Grants: CONACyT C01-40697, DGAPA/UNAM IN225402.
32) The Neurospora crassa, SBR protein exhibits specific DNA binding activity. John Vierula, Yanhua Yan, Katrina Campsall and Bin Zhang. Department of Biology, Carleton University, Ottawa, Ontario. Canada K1S 5B6
The sbr mutant of Neurospora crassa, forms very small, dense colonies which fail to produce conidiophores or conidia. Instead of hyphae, sbr initially forms sausage-shaped, cell compartments which give rise to large, randomly positioned, spherical buds. The sbr gene encodes a 612 amino acid protein with two poly-glutamine domains, a cysteine-rich region and a putative helix-turn-helix motif reminiscent of transcriptional activator proteins.To test this hypothesis, deletion derivatives of the SBR ORF were fused to a 6XHIS tag and over-expressed in E.coli. The purified, 6XHis-tagged polypeptides were then used to capture putative binding targets 300 bp and 600 bp in length from total genomic DNA. Electrophoretic mobililty shift assays (EMSA)were employed to demonstrate specific binding to both of the target sequences. Deletion of the zinc finger resulted in a loss of specific DNA binding by the SBR protein. The results of EMSA experiments using 100 bp subclones of the DNA targets have been used to further localize the SBR binding sites within each target sequence. Supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.
33) Neurospora crassa SET-2 selectively methylates lysine 36 of histoneH3. Keyur K. Adhvaryu and Eric U. Selker. Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403
The SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain is an evolutionarily conserved domain found in many chromatin modifying proteins. Some SET domain containing proteins are histone methyltransferases. In Neurospora crassa, studies with the dim-5 (defective in methylation) mutant have demonstrated a link between histone modifications and DNA methylation. DIM-5 is a SET domain histone methyltransferase with specificity for the lysine 9 residue of the amino terminal tail of the histone H3. Histone H3 and H4 have additional lysine residues (K4, K27, K36 and K79 on H3 and K20 on H4) that are potential targets for methylation. We searched the Neurospora crassa genome database for genes with putative SET domains and found eight in addition to dim-5 (set-1 through set-8).
The set-2 (NCU00269.1) gene is closely linked to ro-2 (NCU00257.1) on the right arm of LG III. It putatively encodes a 963 amino acid protein containing AWS, SET, postSET and WW domains. We used RIP to obtain a mutant (set-2RIP1) that has multiple nonsense mutations. set-2RIP1 grows slowly, shows poor conidiation and is female sterile. Using specific antibodies we analyzed histones isolated from wild type Neurospora crassa strain (OR23-1VA) and find that lysine residues at position 4, 27, 36 and 79 in histone H3 and 20 in histone H4 are methylated. Methylation of the lysine 36 of H3 is completely lost in the set-2RIP1 suggesting that SET-2 is a lysine 36 specific methyltransferase.
This work was supported by AHA Postdoctoral Fellowship 0225370Z to K.K.A. and NIH grant GM 35690 to E.U.S.
34) Neurospora crassa Mutants Altered in Blue Light Transcription. Laura Navarro-Sampedro and Luis M.
Departamento de Genetica, Universidad de Sevilla, Spain
The gene con-10 of Neurospora is expressed during conidiation and following illumination of mycelia with light. The photoactivation of con-10 disappears after two hours of illumination (light adaptation). We have designed a method to isolate mutants altered in the adaptation of con-10 photoactivation. We are using a strain of Neurospora with a fusion of the con-10 promoter to the gene conferring resistance to hygromycin. This strain is sensitive to the drug when the promoter is inactive, i.e. during vegetative growth either in the dark or under continuous light. We have isolated four mutants (SN1 to SN5) that grow in the presence of hygromycin under continuous light but not in the dark. All the mutants showed and enhanced accumulation of the con-10/hygromycin fusion gene after five hours of light compared to the parental strain. All the strains carry two copies of the con-10 promoter, the one in the naturally occurring con-10 gene and an additional one fused to the hygromycin gene. The mutations in strains SN1 to SN3 are specific for the con-10/hygromycin fusion. The photoactivation in the original con-10 gene is similar to that in the parental strain. These mutants are likely to carry mutations in the con-10 promoter fused to the hygromycin gene and will identify sequence elements required for the appropriate regulation of con-10 photoactivation. On the contrary, the mutations in strains SN4 and SN5 have also altered the photoactivation of the original con-10. They should carry mutations in genes responsible for proteins regulating con-10 photoactivation.
35) DNA Segments Involved in Regulation by Blue Light and Development in the con-10 Promoter of Neurospora crassa. Maria Olmedo and Luis M. Corrochano. Departamento de Genetica, Universidad de Sevilla, Spain
The gene con-10 of Neurospora crassa is expressed during conidiation and following illumination of vegetative mycelia with light. Dark repression sites have been located at positions -1559 to -779 (from the transcription start site) and -353 to -265. A mycelial repression site has been located at position -778 to -353. Two conidiation activation sites have been located at positions -353 to -265 and -236 to -191. We are using a series of fusions between segments of the con-10 promoter and the lacZ gene to investigate the DNA sequences involved inregulating the expression of con-10. The strains were grown in the dark for 48 h before applying light. Beta-galactosidase activity in cell extracts was a measure of the con-10 promoter activity. The complete con-10 promoter (-1559) fused to the lacZ gene was induced about ten fold after 30 min illumination. Other con-10 promoter fusions contained sequences to -913, to -839, and to -517. Their light-dependent activity will locate the position of the first dark repression site and themycelial repression site. To confirm the presence of the second dark repression site (-353 to -265) and the mycelial repression site (-517 to -354) we have fused each segment to a con-10 minimal promoter (-191) that shows no response to light or conidiation. Additional fusions will confirm the location of the dark repression site: from -353 to -282, and from -337 to -265. These and other con-10/lacZ fusions will allow us to locate the sequences involved in the regulation
36) Catabolite repression of the quinic acid (qa) genes in Neurospora crassa. Diana R. Arnett1 and David K. Asch1,2.1School of Biomedical Sciences, Kent State University, Kent, Ohio and 2Department of Biological Sciences, Youngstown State University, Youngstown, Ohio.
The quinic acid (qa) gene cluster of Neurospora crassa provides an unusual example of gene controlin a eukaryotic organism. Previous studies focused on the primary control mechanism, which is dependent on the inducer quinic acid. However, the expression of the qa genes is also repressed in the presence of a preferred carbon source, even in the presence of the inducer. This important secondary level of control, termed carbon catabolite repression, has not been well studied. In order to focus on this system of control we are utilizing a constitutive mutant of the qa gene cluster which contains a deletion of the qa-1S repressor gene. Deletion of the qa-1S gene removes the primary means ofregulation of the qa cluster, ensuring that any observed effect on qa gene expression is due to catabolite repression. By Northern blot analysis, we have demonstrated that the qa-y gene seems to be repressed in the presence of a preferred carbon source such as dextrose even when the qa-1S gene product is absent, while the remainder of the qa genes are not directly repressed to a significant degree by the presence of dextrose.
37) RRG-1, a Response Regulator Signaling Protein in Neurospora crassa. Suzanne E. Greer-Phillips and Katherine A. Borkovich. University of California, Riverside.
In yeasts, sensor histidine kinases respond to various environmental stresses and signal through a two-component-like signal transduction pathway. This pathway is a multistep phosphorelay system where a histidine-containing phosphotransfer protein (Hpt) and a response regulator (Rrg) propagate the signal from sensor to effector. Eleven sensor histidine kinases have been identified in the genome of Neurospora crassa, but only one Hpt and two Rrggenes have been found. Here we report the identification of response regulator - 1 (rrg-1) from N. crassa. RRG-1 is similar to Mcs-4 from Schizosaccharomyces pombe and Ssk-1p from Saccharomyces cerevisiae. Similar to the ssk-1 mutant of S. cerevisiae, a rrg-1 deletion mutant of N. crassa is inhibited in growth under osmotic stress conditions such as increased concentrations of NaCl, KCl, or sorbitol. In addition, rrg-1 mutants are female sterile and show increased carotenoid pigmentation. These results suggest that RRG-1 plays a more significant role in regulating cellular functions than is known for response regulators of other systems.
38) FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation. He Q, Cheng P, Yang Y, He Q, Yu H, Liu Y.Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.
Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ)regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.
39) Comparative sequencing of the qa-2 gene of Neurospora crassa and Neurospora africana. James A. Shevchuk1, Diana R. Arnett2 and David K. Asch1,2. 1Department of Biological Sciences, Youngstown State University,Youngstown, Ohio and 2School of Biomedical Sciences, Kent State University, Kent, Ohio.
Gene systems like the quinic acid (qa) gene cluster have been studied in Neurospora crassa for many years. However, we know very little about gene systems in the homothallic species of Neurospora. Earlier it had been observed that N. crassa probes containing the qa gene cluster would hybridize to sequences in various homothallic species. To learn more about the qa systems in the homothallic species of Neurospora we have cloned the qa gene cluster from Neurospora africana. From these clones we have isolated and sequenced the qa-2 gene and compared it with the qa-2 gene sequence of N. crassa and the sequence of the qut-E gene of Aspergillus nidulans.
40) Analysis of Neurospora sirtuins: evidence for control of telomeric silencing and homologous recombination. Gregory O. Kothe1, Cindy Matsen2, Michael Freitag1, Kriistina Smith1, Melissa Hemphill1, Matthew Sachs3, Mark Farman4, and Eric U. Selker1. 1Institute of Molecular Biology, University of Oregon, Eugene, 2University of Chicago Medical School, Chicago, IL, 3Department of Biochemistry and Molecular Biology OGI School of Science and Engineering, Beaverton, OR, 4Department of Plant Pathology, University of Kentucky, Lexington, KY
The Saccharomyces cerevisiae SIR2 protein is involved in silencing at telomeres, rDNA, and silent-mating loci, and controlling rDNA recombination. The SIR2 family of proteins (sirtuins in eukaryotes other than yeast) function as NAD-dependent deacetylases that regulate the activities of other proteins. We have mutated three Neurospora genes encoding sirtuins, and have investigated the effects of these mutations on silencing and recombination. We refer to these sirtuins as NST-1, 2, and 3 (Neurospora Sir Two). Preliminary evidence suggests that NST-1 and NST-3 control telomeric silencing, but not methylation dependent silencing. We also noticed a dramatic decrease in recombination between the mating-type locus and his-3 in a cross of an nst-1 mutant strain with an nst-3 mutant. Recombination between inl and am was normal in this cross, but a test for homologous mitotic recombination by transformation in an nst-1 mutant strain revealed a dramatic decrease in comparison to wild-type. nst-3 mutant homozygous crosses are nearly barren. Our findings suggest that surtuins function in mitotic and meiotic homologous recombination as well as silencing in Neurospora.
41) Characterization of two Neurospora proteins with motifs characteristic of DNA glycosylases and putative DNA demethylase. Gregory O. Kothe1, Jean-Pierre Jost2, and Eric U. Selker1. 1Institute of Molecular Biology, University of Oregon, Eugene, OR, 2Friedrich Miescher-Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
In Arabidoposis, two proteins, ROS1 and Demeter, which contain a motif characteristic of DNA glycosylases (helix-hairpin-helix motif), have been shown to regulate expression of methylated DNA sequences, and are proposed to function as DNA demethylases. In vertebrates, the methyl-CpG binding protein MBD4 has an hhh domain and is a T:G mismatch glycosylase that functions in base excision repair, apoptosis, and cell-cycle regulation. Biochemical studies have suggested that MBD4 has demethylase activity as well. We mutated two Neurospora genes encoding proteins containing hhh domains, one most similar to MBD4, and the other most similar to ROS1 and Demeter. We have shown that the Neurospora factor related to MBD4 binds methylated DNA preferentially in a Southwestern assay, and we refer to this factor as MBP-3 (Methyl-Binding Protein 3). The second protein is encoded by a gene immediately adjacent to the rid gene. We refer to this protein as HHH-1 (Helix-Hairpin-Helix 1). We have not detected T:G mismatch or methylcytosine glycosylase activity for either MBP-3 or HHH-1. We are currently characterizing the mutant phenotypes for the genes in the vegetative and sexual cycle, and we are analyzing the effect of over-expression of the proteins as well.
42) Circadian regulation of the evening-specific gene ccg-16. Zachary Lewis and Deborah Bell-Pedersen. Texas A &M University.
Circadian clocks coordinate daily changes in behavior, physiology, and gene expression in organisms. In Neurospora the clock regulates the production of conidia and the rhythmic expression of a number of genes and proteins. Previous studies identified several clock controlled genes (ccg's) all of which peaked in mRNA accumulation in the late-night to early-morning. These data implied that the circadian clock in Neurospora is simple, only regulating output pathways at one phase of the circadian cycle. Using microarrays however, we have identified several genes that peak in mRNA accumulation in the early-evening. These data support the idea that the circadian clock of Neurospora is complex, analogous to that of higher eukaryotes. We are currently investigating the regulation of one evening-specific gene, ccg-16, in greater detail. Although ccg-16 encodes a gene of unknown function, similar sequences can be found in the genomes of closely related fungi. Efforts are underway to produce a ccg-16 knockout strain. ccg-16 is repressed by light and development, consistent with an evening-specific expression profile. Interestingly, ccg-16 mRNA accumulates with a ~24 hour rhythm in a frq-null strain. This strain lacks the FRQ-oscillator required for conidiation rhythms in constant conditions. These results suggested that a second FRQ-independent oscillator regulates some aspects of output from the circadian clock. It is not known, however, if this oscillator is involved in the time-keeping mechanism or is a slave oscillator that is driven by the FRQ-based oscillator. These possibilities are being examined.
43) Regulation of sulfur metabolism in Neurospora crassa. John V. Paietta, Department of Biochemistry and Molecular Biology, Wright State University, Dayton OH.
The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes which are coordinately controlled by the cys-3 and sulfur controller (scon) genes. We are examining the entire set of structural genes involved in sulfur metabolism for control by the CYS3/SCON regulators. We have cloned and sequenced the ars-1 (arylsulfatase), cys-4 (sulfite reductase), cys-16 (cystathionine gamma-lyase), met-1 (methylene tetrahydrofolate reductase), met-2 (cystathionine beta-lyase), met-5 (homoserine o-acetyltransferase), and met-8 (methionine synthase) genes for past and on-going studies. In addition, our analysis of the genomic sequence data has identified most of the remaining genes involved in sulfur metabolism (e.g., sulfur transport, generation of sulfide and cysteine, homocysteine and methionine metabolism, and glutathione metabolism). A number of these genes are under control of the CYS3/SCON regulatory system. The overall pattern of regulation in the sulfur metabolic network as determined from expression studies of available genes will be presented.
44) The transcriptional modulators nuc-1 and pacC-1 from N. crassa are required for the expression of a heat shock-inducible gene of the hsp70 family. Carlos J. Ono, Sergio R. Nozawa, Monica S. Ferreira-Nozawa, Nilce M. Martinez-Rossi and Antonio Rossi. University de Sao Paulo, Ribeirão Preto, Brazil
The nuc-1 and pacC-1 genes are wide-domain transcriptional factors involved in the regulation of the adaptive response of fungi to the levels ofphosphate (Pi) and to the pH of the medium, respectively. The nuc-1 gene activates the transcription of various Pi-repressible structural genes, allowing molds to utilize nucleic acids as the sole Pi source. The pacC-1 gene activates the transcription of various alkaline genes, i.e., it allows the survival of molds at alkaline pH. Thus, the two genes are regulators apparently functioning independently from each other, but both are functionally active genes at alkaline pH. Furthermore, the promoter region of the nuc-1 gene has the consensus 5'-GCCAAG-3' (DNA binding domain of PACC-1), whereas the promoter region of a gene of the hsp70 family (XP_327938) has the consensus 5'-CACGTG-3' (DNA binding domain of NUC-1). Thus, it is possible that both the nuc-1 and pacC-1 genes are required for the expression of this heat shock-inducible member of the HSP70 family, suggesting a cascade effect i.e., at alkaline pH the pacC-1 gene would activate the expression of the nuc-1 gene, which in turn would activate the expression of this hsp70 gene. As detected by DDRT-PCR and confirmed by Northern blot analysis, strains of N. crassa carrying the nuc-1 or pacC-1 gene silenced by RIP (repeat-induced point mutations) did not express this hsp70 gene, confirming the predictions stated above. Financial support: FAPESP, CNPq, CAPES, FAEPA.
45) Expression of arg-13 and ARG13. Gloria E. Turner, Rey David, and Richard L. Weiss. Department of Chemistry, University of California, Los Angeles.
The arg-13 gene encodes a mitochondrial carrier family (MFC) protein. Examples of MFC proteins include carriers for ADP/ATP, dicarboxylate, citrate, ornithine, glutamate and glutamine. These proteins participate in metabolic trafficking, exchanging metabolites for inorganic cations across the inner mitochondrial membrane, using dual transport mechanisms, uniport and exchange. Proteoliposome assays with purified recombinant ARG13 reveal that ornithine is the substrate for this protein. ARG13 antibodies were used to determine the level of expression and subcellular localization. Results indicate that ARG13 is expressed at low levels and is localized in the mitochondrial fractions. Protein extracts supplemented with ornithine had increased levels of the protein. RNA's isolated from conidia and germinating cultures were used to determine the expression profile of arg-13. This profile is consistent with biosynthetic genes, where expression is detected early in germination but not seen in conidia or late germination. Interestingly arginine supplementation did not alter this profile. A lower molecular weight transcript is observed at the same time but only when arginine is absent. We are investigating if this transcript encodes a tryptophan-rich sensory protein (TspO/MBR) homolog that overlaps with the 3' region of arg-13 on the opposite strand. This protein is involved in the efflux of porphyrin intermediates.
46) A Genetic Selection for Circadian Clock Mutations in Neurospora crassa. Michael Vitalini, Louis Morgan and Deborah Bell-Pedersen. Department of Biology, Texas A&M University, College Station, TX 77843
To identify components of the circadian clock in Neurospora crassa, we have carried out a genetic selection to isolate mutations that alter the expression of clock-controlled genes (ccgs). The selection is based on the differential expression of the ccgs in the absence of the clock gene frequency (frq); ccg-1 expression is high and ccg-2 expression is low in the frq10 (null) strain. The promoters of ccg-1 and ccg-2 were fused to the mtr gene to create plasmids pCCG1M and pCCG2M, respectively. The mtr gene encodes a neutral amino acid permease that allows for both positive and negative selection. Loss of MTR function can be selected for based on resistance to the amino acid analog p-flourophenylalanine (FPA). Gain of MTR function can be selected for based on growth of tryptophan auxotrophs on high arginine/low tryptophan (TA) media. The pCCG1M and pCCG2M plasmids were transformed into a bd;frq10;mtr;trp-2 strain. The pCCG1M transformed strain, CCG1M, displayed the predicted Mtr+ phenotype: growth on TA media, but not on FPA. The pCCG2M transformed strain, CCG2M, displayed an Mtr- phenotype. Both strains were subjected to UV light mutagenesis and assayed for growth on selective media. Eighty mutant strains that grew on FPA medium were isolated from CCG1M and one hundred forty mutant strains that grew on TA medium were isolated from CCG2M. The circadian phenotypes of the mutant strains will be discussed.
47) Distinct roles for PP1 and PP2A in the Neurospora circadian clock. Yuhong Yang1, Qun He1, Ping Cheng1, Philip Wrage1, Oded Yarden2, and Yi Liu 1. 1Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX75390, USA; 2Department of Plant Pathology and Microbiology, Faculty of Agricultural Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 7610, Israel
Phosphorylation of the Neurospora circadian clock protein FREQUENCY by several kinases promotes its degradation and is important for the function of the circadian feedback loop. Here, we show that FRQ is less stable in a ppp-1 (catalytic subunit of PP1) mutant, resulting in its advanced phase and short period. In contrast, FRQ stability is not altered in an rgb-1 (regulatory subunit of PP2A) mutant, but levels of frq protein and mRNA are low, resulting in a low-amplitude and long-period oscillation of the clock. Furthermore, PP1 and PP2A expressed in Neurospora can dephosphorylate the endogenous FRQ in vitro, sugguesting that these two phosphotases may differentially regulate FRQ and, consequently, the behavior of the circadian clock.
48) Characterization of the gene responsible of the ovc and cut phenotypes of Neurospora. Loubna Youssar1, Tom Schmidhauser2 and Javier Avalos1. 1Departamento de Genética, Universidad de Sevilla, Spain. 2present address: California State University Channel Islands. U.S.A.
Light induction of carotenogenesis in Neurospora is mediated by the WC proteins. Few mutants have been described with an enhanced photocarotenogenesis. One of them is ovc (Harding et al, 1984, Neurospora newsl. 31:23), a strain sensitive to high osmotic conditions and allelic with the cut mutant (Banks et al. 1997. FGN 44:10), also osmosensitive but normal for carotenogenesis. A phenotypic characterization of both strains is presented. Light induction of mRNA levels of the regulatory gene wc-1, the carotenoid genes al-1 and al-2, or the conidiation specific gene con-10, is not significantly changed in the ovc mutant when compared with the wild type.The gene responsible of the ovc and cut phenotypes was identified by complementation of osmosensitivity with a cosmid library. The gene, that we call cut-1, codes for an enzyme of the haloacid dehalogenase family, which groups different classes of phosphatases. The gene is not present in the ovc mutant, which has suffered a deletion, and is able to restore the wild type phenotype upon transformation. Transcription of cut-1 is low in either light or dark-grown cultures, and is high under hyperosmotic conditions. A blast search with the cut gene against the Neurospora genome reveals two additional genes with sequence similarity. None of them is induced by high osmotic conditions. Further experiments on cut-1 function and regulation are under way.
49) HDA-1 of Neurospora crassa targets Histone 3 Lysine 14 and is required for proper DNA methylation. Kristina M. Smith, Joseph R. Dobosy, Hisashi Tamaru, and Eric U. Selker. University of Oregon, Eugene.
Previous studies have shown that histone deacetylase (HDAC) inhibitors selectively inhibit DNA methylation in Neurospora crassa. To investigate this phenomenon, we identified a gene encoding a homolog of the Schizosaccharomyces pombe Clr3 HDAC in N. crassa and used RIP to disrupt its function. The hda-1 mutant lost DNA methylation at some chromosomal loci but not others. Western blotting revealed that the deacetylase activity of N. crassa HDA-1, like Clr3, is specific for H3 K14. The increased H3 K14 acetylation in the hda-1 mutant correlated with a significant loss of H3 K9 trimethylation. The relationship between H3 K9 and K14 modification and their effect on DNA methylation was explored with chromatin immunoprecipitation experiments.
This study was funded by American Cancer Society fellowship PF0404301GMC to K.M.S. and NIH grant GM35690 to E.U.S.
Genomics and Proteomics
50) Modeling and Analysis of the Biological Clock in Neurospora crassa. Cara M. Altimus, Dr. Jonathan Arnold, Dr. H. Bernt Schuttler. Department of Genetics, University of Georgia, Athens, GA 30602
A biological clock is a recurring set of reactions within a system that produces anoscillating pattern. Unlike a traditional "clock," a biological clock can run continuously so long as all reaction components are present. Reaction rates are the main variants. Genetic networks are used to understand the relationships between genes, RNA, and proteins. These models show which genes are active, how they become active, what their products do, and their relationships with other genes and their products in the circuit. Then an ensemble of genetic networks for the biological clock was identified, fitting available RNA and protein profiling data. The fitted ensemble was used to identify essential features of the genetic network needed to sustain oscillations. Two features that appear necessary for oscillations are: (1) cooperativity in the action of two clock components, the White Collar (WCC) protein and Frequency (FRQ), and (2) a closed feedback loop in clock components. Along side the ensemble experiments, local stability analysis was done to examine equilibrium properties of the genetic network. Oscillations will only occur if the system does not have a stable fix point. Analytical conditions for instability are derived, permitting oscillations. In short, the clock needs several interacting proteins, a negative feedback loop, some cooperativity and the absence of a stable fix point to which the system would otherwise equilibrate.
51) Evidence of extensive plasmid integration into fungal mitochondrial DNAs. Patrick Cahan and John C. Kennell. Department of Biology, Saint Louis University, St. Louis, MO
Mitochondrial (mt) plasmids are autonomously-replicating genetic elements that reside in many filamentous fungi. Although they lack specific integrase functions, certain mt plasmids have been shown to integrate into mitochondrial DNA (mtDNA). In addition, annotation of fungal and plant mtDNAs has revealed regions having varying degrees of sequence similarity to mt plasmids. To directly assess the degree to which plasmids have invaded fungal mitochondrial genomes, BLAST search parameters were modified to identify plasmid sequences within highly AT-rich mtDNAs. Plasmid sequences representing four well-characterized plasmid homology groups of N. crassa were used as queries with completely sequenced fungal mtDNAs as subjects. Outputs were compared to previously-reported integration events and to randomly generated sequences. To facilitate analysis, algorithms were developed to parse the output data by E value, score and sequence complexity. Additional criteria included the presence of shared repetitive elements. Our results reveal several regions within the N. crassa mt genome that have homology with mt plasmids, and analysis of other mtDNAs indicates that plasmid integration is widespread among filamentous fungi. Our studies also revealed an unequal distribution of PstI palindromes within intergenic regions of N. crassa mtDNA that could be indicative of a recent recombination event. Tools developed here will be useful in understanding the co-evolution of plasmids and their hosts.
52) A rapid and efficient knockout strategy for filamentous fungi. H.V. Colot, K.A. Borkovich*, C.W. Pitt, J.J. Loros and J.C. Dunlap. Genetics Department, Dartmouth Medical School, Hanover, NH; and *Department of Plant Pathology, University of California, Riverside, CA
Disruption of genes by targeted gene replacement is widely used for assaying gene function. In Neurospora, homologous recombination occurs at a low frequency; long regions of homology are required in the knockout constructs, making their creation cumbersome. Insertion of transforming DNA at ectopic locations leads to a significant background. We have developed a strategy that uses recombination-mediated plasmid construction in S. cerevisiae (Oldenburg etal. 1997, Nucleic Acids Res. 25:451), followed by the generation of split-markerfragments for transformation (Catlett et al. 2003, Fungal Genet. News. 50:9). This strategy allows rapid creation of the knockout DNA: two 3 kb flank fragments and the selectable marker cassette are prepared by PCR with primers that confer 29 nt homologous overhangs. These three fragments, along with a gapped yeast shuttle vector, are cotransformed into yeast. Crude DNA is then prepared from the mixed yeast transformants and used as a template in PCR reactions to yield two overlapping split-marker fragments for transformation into Neurospora. Use of split-marker fragments decreases ectopic insertions several-fold compared to the use of a single full-length fragment. On average, 35% of the transformants obtained with this strategy have the proper gene replacement and, as determined by Southern analysis, 73% of those are free of ectopic insertions. The complete protocol, along with strains and plasmids, will be provided.
53) Comparative Genomics within the Genus Neurospora. Luz B. Gilbert, Takao Kasuga, Jeff Townsend, Louise Glass, and John W. Taylor. Dept. of Plant and Microbial Biology, U.C. Berkeley, Berkeley, Ca 94720 USA
Little is known about the process of microbial adaptation. In the future I will use microarray technology to help identify genes responsible for environmental adaptation in natural isolates of Neurospora discreta. Neurospora offers a unique opportunity to characterize the variability of global gene regulation within and between species. The genus Neurospora consists of eight closely related conidiating species (see Dettman et al. 2003, in press), indistinguishable by morphology, as well as several non-conidiating species. We have constructed a 70mer oligomer array for Neurospora crassa representing 3366 genes, approximately one third of the genome. To assess the effectiveness of our array for other members of the Neurospora genus I have analyzed comparative genomic hybridizations for all eight conidiating species of Neurospora as well as a few non-conidiating isolates. This technique uses genomic DNA as a substrate for labeling and hybridization to an oligo array, consequently avoiding the biases associated with transcription of mRNA. We can now determine gene coverage and estimate genome divergence among conidiating and non-conidiating Neurospora isolates by comparing the ratio of fluorescences between samples.
54) mRNA profiling of conidial germination and hyphal growth in Neurospora crassa using oligomer microarrays. Takao Kasuga, Betty Gilbert, Jeffery Townsend, John Taylor and Louise Glass. Plant and Microbial Biology Department, University of California, Berkeley, CA94720
We have constructed a Neurospora crassa 70 base oligomer microarray representing 3,366 out of ten thousand genes predicted by WICGR and MIPS. In order to validate the performance of the oligomer microarrays and to develop techniques for baseline profiling for N. crassa, we chose to assess the transcriptional profile of conidial germination (up to 24 hr of growth). A considerable body of literature is available on biochemical aspects and mRNA profile of conidial germination in filamentous fungi and in N. crassa in particular. Total RNA was obtained from a time course during conidial germination from a series of liquid cultures. Hierarchical clustering was used to group genes with respect to their transcriptional profiles. Three distinct clusters were identified; the Max Time 0 cluster showed maximum expression at time 0 (dormant conidia), the Min Time 0 cluster showed minimum gene expression at time 0 and reached a peak between 2 to 8 hrs and Max Time 24 cluster, which showed maximum gene expression at 24 hrs. Gene functions categorized by MIPS (Mannhaupt et al., 2003) were used to assess the global picture of gene expression during germination. In dormant conidia (Max Time 0), the percentage of genes categorized as ENERGY and METABOLISM were lower than that of the other clusters. In the Min Time 0 cluster, the proportion of genes for PROTEIN SYNTHESIS and TRANSCRIPTION were highest, reflecting active growth between 2-8 hr. In the Max Time 24 cluster, the ratio ofgenes for CELL CYCLE AND DNA PROCESSING was relatively higher than the other clusters. Although our observations are preliminary, they agree well with published studies of conidial germination in N. crassa.
55) Development of positional cloning tools for Neurospora crassa to characterize a suppressor of a temperature-sensitive mutation in the large subunit of ribonucleotide reductase. Moshi Kotierk and Myron L. Smith. Biology Department, Carleton University, Ottawa, Ontario K1S 5B6
un-24 encodes a temperature sensitive form of the large subunit of ribonucleotide reductase, an evolutionarily conserved enzyme that is essential for the reduction of RNA precursors into DNA precursors. We have identified a spontaneous mutation, su(un-24)-1, that suppresses the un-24 temperature-sensitive phenotype. This suppressor may act by increasing intracellular osmotic pressure, since un-24 is osmotically remediated, or by directly stabilizing the UN-24 protein. Unlike in Schizosaccharomyces pombe, the suppressor is not due to a mutation in the small subunit of ribonucleotide reductase. To identify su(un-24)-1, we are using a map-based approach that utilizes the complete genome sequence of N. crassa. We developed 16 PCR-based markers, located on each of the seven nuclear chromosomes and on the mitochondrial DNA, that are polymorphic between OakRidge and Mauriceville background N. crassa strains. We analyzed these markers in 34 progeny of a cross between our temperature-sensitive, suppressed OR-background strain and a wildtype Mauriceville strain. From this analysis, we have mapped su(un-24)-1 within a region of Linkage Group II near nmt-1. This map-based approach can be used in positional cloning of any mutation generated in the OR background of N. crassa.
56) Gene amplification in some glycosyl hydrolase families. Alan Radford. University of Leeds.
Neurospora crassa hydrolyses cellulose and other polysaccharides as its major carbon source. High levels of such enzymes are achieved via gene amplification of several glycosyl hydrolase families, including GH family 3 (beta-glucosidases), families 5, 6, 7 and 61 (cellulases), family 10 (xylanases), and also the starch-degrading family 13 (alpha-amylases) and 15 (glucoamylases).Comparison of Neurospora sequences in the above families from the WICGR database with other ascomycete genomes at WICGR (M. grisea, A. nidulans, F. graminareum), identified by BLASTP and using Clustal X alignment and bootstrap tree calculation, shows amplification pre-dating the split into plectomycetes, discomycetes and pyrenomycetes, and hence prior to the evolution of RIP.Using the predicted Neurospora protein sequences in TBLASTN searches of the C. cinereus DNA database at WICGR shows that different members of the same family in Neurospora may find different "besthits" in Coprinus, suggesting that certain amplifications pre-date the Ascomycete-Basidiomycete split. Comparable searches of U. maydis and C. neoformans find no cellulases in the former and neither cellulases nor hemicellulases in the latter, consistent with the lifestyles of the two parasitic species. In extant species in which RIP occurs, gene duplication would probably delete a copy of the gene of that species from a clade with orthologs from other species. Examples of this are observed.
57) Developing the Neurospora Gene List. Alan Radford. University of Leeds
The Neurospora Compendium (Perkins, Radford and Sachs,2001) contains data up to 2000. It provides a snapshot of our knowledge four years ago. Since its publication, many new genes have been published, and the entire genome sequence has become available at WICGR (Galagan et al, 2003).The Neurospora gene list at http://www.bioinf.leeds.ac.uk/~gen6ar/Neurospora/gene_list.htm has been an attempt to keep the classical gene data up to date and cross-referenced to the genome database. For genes of known sequence, their NCU number and physical mapping data have been incorporated into gene entries, new genes have been added, and the mitochondrial genome has been compiled in standard format as a section of the gene list. With the MMBR magnum opus (Borkovich et al, 2004) in press, many new genes will require inclusion.The gene list is currently in XML format, with a front end using ActiveX and therefore requiring Internet Explorer, although this limitation could be circumvented, and a more flexible query front end provided..At this stage I seek the input of the Neurospora community on the current usefulness and future development of the gene list.
58) An Integrated Analysis of a Chemical Reaction Network for the Metabolism of Quinic Acid in Neurospora crassa. Cale D Whitworth and Dr. Jonathan Arnold. Department of Genetics, University of Georgia, Athens, Georgia 30602
A chemical reaction network for the metabolism of Quinic Acid in Neurospora crassa has been proposed. In this reaction network two regulatory genes and five structural genes are responsible for the metabolism of Quinic Acid. The protein product of qa-1F transcriptionally controls the expression of all seven qa genes, including those encoding enzymes which utilize Quinic Acid as a carbon source, and the protein product, QA-1S, represses the activator protein, QA-1F. An ensemble of possible chemical reaction networks is developed with rate constants consistent with RNA and protein profiling data. An alternative network, in which several molecules of QA-1F (i.e. Hill coefficient is greater than one) cooperatively activate qa genes, is also developed.
59) Cloning of telomeric regions from Neurospora crassa wild-type strains Oak Ridge and Mauriceville. Cheng Wu1, Mark L. Farman2 and Matthew S. Sachs1. 1Oregon Health & Science University, Beaverton, OR 97006 2University of Kentucky, Lexington, KY 40546
Eukaryotic chromosomes are linear molecules that terminate in simple sequence repeats called telomeres. New telomere repeats, typically 5' TTAGGG 3', are added to the existing chromosome ends to guard against the loss of DNA during replication. The G-rich strand can form hairpin structures, which are believed to prevent the degradation of chromosome ends and the fusion of different chromosomes. In fungi and other eukaryotic microbes, regions near telomeres are highly variable and are rich in genes for ecological adaptation. Telomeres are poorly represented in the draft genome sequence database for Neurospora crassa. By characterizing clones for N. crassa telomeres and subtelomeric regions, we will be able to assess the functional and evolutionary significance of these regions and complete the genome sequences. Here we describe our approach to clone N. crassa telomeric regions. Isolated chromosomal DNA fragments with polished ends were ligated to linearized pMLF4 cosmid vector. The ligated DNA was then packaged with Lambda packaging extract and used to infect Escherichia coli XL-10 cells. Recombinant colonies containing telomeres were then identified using a 32P-labeled telomere probe. Our goal is to isolate and characterize the 14 telomeric regions of two different wild-type N. crassa strains, Oak Ridge and Mauriceville. Comparison of subtelomeric regions between strains will improve understanding of the pathways of genome evolution.
Industrial Biology and Biotechnology
60) Diversification of barley beta-D-glucan endohydrolases in Neurospora crassa. Graham Eariss1, Maria Hrmova2, Geoffrey Fincher 2 & David E. A. Catcheside1. 1School of Biological Sciences, Flinders University, Adelaide, South Australia.2 Department of Plant Science, University of Adelaide, South Australia.
Similarities in the primary sequences and three dimensional structures of barley (1-3)-beta-D-glucan endohydrolases and (1-3,1-4)-beta-D-glucan endohydrolases suggest they are closely related in evolutionary terms, yet they perform completely different functions (Stewart et al., 2001). While (1-3)-beta-D-glucanases capable of hydrolyzing the linear, substituted and branched (1-3)-beta-D-glucans often found in fungal cell walls appear to be involved in plant protection, the (1-3,1-4)-beta-D-glucanases are responsible for digestion of the starchy endosperm cell wall during germination of barley grains (Hrmova and Fincher, 2001). We are using an in vivo gene diversification technique developed in Neurospora crassa (Catcheside et al., 2003) to investigate the molecular basis of their divergence and to generate a more thermostable (1-3,1-4)-beta-D-glucanase intended for industrial use. Gene diversification in Neurospora takes advantage of the meiotic recombination hotspot cogL,shuffling exogenous genes inserted between cogL and the nearby his-3 locus during meiosis. Functional genes encoding the barley beta-D-glucanases will be inserted between cogL and his-3 by transplacement while non-functional duplicates will be incorporated at random. Variant alleles generated by pre-meiotic Repeat-Induced Point mutation (RIP) will be shuffled by recombination, increasing variation and providing a potential rescue for deleterious mutations. Progeny will be screened using a colorimetric plate assay to identify novel variants.
Catcheside, D. E. A., Rasmussen, J. P., Yeadon, P. J., Bowring, F. J., Cambereri, E. B., Kato, E., Gabe, J. & Stuart, W. D. 2003, ‘Diversification of exogenous genes in vivo in Neurospora', Appl. Microbiol. Biotechnol., 62: 544-549.
Hrmova, M. and Fincher, G.B. 2001, ‘Structure-function relationships of beta-D-glucan endo- and exohydrolases from higher plants', Plant Mol. Biol., 47: 73-91.
Stewart, R.J., Varghese, J.N., Garret, T.P.J., Hoj, P.B. & Fincher, G.B. 2001 ‘Mutant barley (1-3,1-4)-beta-glucan endohydrolases with enhanced thermostability', Protein Eng., 14: 245-253.
61) Restoration of Saccharomyces cerevisiae coq mutants by Neurospora crassa genes. Eun Jung Kim, Sang Rae Kim, Na Young Eo and Bheong-Uk Lee*Kosin University, Division of Biological SciencesBusan 606-701, South Korea
Coenzyme Q (CoQ, ubiquinone) is a quinone derivative with a long isoprenoid side chain. CoQ is a lipid component that transports electrons in the respiratory chains located in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes, and also functions as an antioxidant. CoQ is essential in aerobic growth of Saccharomyces cerevisiae. coq mutants, deficient ubiquinone biosynthesis fail to grow on nonfermentable carbon sources. Two putative genes involved in ubiquinone biosynthesis of Neurospora crassa were cloned and used for complementation test of S. cerevisiae coq4 and S. cerevisiae coq7 strains, respectively. The predicted amino acid sequences of both N. crassa COQ4 and COQ7 showed about 52% and 58% homology with those of S. cerevisiae, respectively. When putative coq-4 and coq-7 genes of N. crassa were transformed to yeast strains, the growth rates were restored to the wild-type level and were able to synthesize coenzyme Q6. They also showed less sensitivities to polyunsaturated fatty acids such as linoleic acid or linolenic acid.
62) Agrobacterium tumefaciens-mediated genetic transformation of Neurospora crassa. Richard S. Feinberg and Matthew S. Sachs. Oregon Health & Science University, Beaverton, OR 97006
Agrobacterium tumefaciens-mediated transformation has been successfully applied to Neurospora crassa. The transformants were selected on the basis of their resistance to hygromycin B. Plasmids were tested in which the hph gene was driven by either the N. crassa cpc-1 or arg-2 promoters, or the Aspergillus nidulans trpC promoter. A variety of different conditions for transformation were analyzed. The number of N. crassa conidia and A. tumefaciens cells which were co-cultivated were varied, as was the length of the co-cultivation period prior to transfer to selection medium. Hygromycin-resistant transformants were stable through repeated rounds of conidial passaging and following homokaryosis by a microconidiation procedure.
63) Diversification of Human Growth Factors in Neurospora crassa. Steven Henderson1, Briony Forbes2, Leah Cosgrove2 & David E. A. Catcheside1. 1School of Biological Sciences, Flinders University, Adelaide, South Australia 5042, Australia.2Health Sciences and Nutrition, Commonwealth Scientific and Industrial Research Organisation, Adelaide, South Australia 5000, Australia.
We are utilising an in vivo gene diversification system developed in Neurospora crassa (Catcheside et al., 2003) to generate novel human growth factor (hGF) variants. Diversification of heterologous genes in Neurospora exploits the high rate of meiotic recombination initiated at the recombination hotspot cogL to shuffle exogenous sequences juxtaposed to cogL. Transplacement vectors will be used to insert a functional hGF gene between his-3 and cogL. A non-functional hGF duplicate gene will be used to induce low frequencies of Repeat-Induced Point mutation (RIP) to generate hGF alleles in vivo. Subsequent meiotic recombination shuffles the growth factor alleles creating additional sequence variation and can also result in separation of deleterious mutations. Progeny from the cross will be screened to identify novel hGF variants.
Catcheside, D. E. A., Rasmussen, J. P., Yeadon, P. J., Bowring, F. J., Cambareri, E. B., Kato, E., Gabe, J. & Stuart, W. D. 2003, ‘Diversification of exogenous genes in vivo in Neurospora’, Appl. Microbiol. Biotechnol., vol. 62, pp 544-549.
64) Functional expression of Neurospora crassa L-carnitine biosynthetic genes in Escherichia coli. Sangrae Kim1, Sunduk Hwang2,3, Bum-Chang Kim2, Hyoung-Sik Kim2, Eun Jung Kim1,3, Whankoo Kang2,3, and Bheong-Uk Lee1,3. 1Division of Biological Sciences, Kosin University, Busan 606-701, Korea, 2Dept. Biochemical Engineering, Hannam University, Daejeon 306-791, Korea 3Gene To Protein Inc. Daejeon306-800, Korea
Five genes involved in biosynthesis of L-carnitine in Neurospora crassa were cloned and functionally expressed in Escherichia coli. These genes encode enzymes that sequentially converts from L-lysine to L-carnitine. The first enzyme produce trimethyllysine from L-lysine. The second enzyme is epsilon-N-trimethyllysine hydroxylase (TMLH), which converts epsilone-N-trimethyllysine to beta-hydroxy-epsilon-N-trimethyllysine. The third one is beta-hydroxy-epsilon-N-trimethyllysine aldolase (SHMT), which converts beta-hydroxy-epsilon-N-trimethyllysine to gamma-N-trimethylaminobutyraldehyde. The fourth is gamma-N-trimethylaminobutyraldehyde dehydrogenase (TMABADH). This converts gamma-N-trimethylaminobutyraldehyde to gamma-butyrobetaine that was finally coverted to L-carnitne by gamma-butyrobetaine hydroxylase. About 1.7 grams of L-carnitine per liter can be produced by the partially-optimized fed-batch fermentation.
Population and Evolutionary Biology
65) Phylogenetic relationships within the diverse Neurospora discreta species complex. Jeremy Dettman, David Jacobson, and John Taylor. Dept. of Plant and Microbial Biology, University of California, Berkeley.
Neurospora discreta is the most widely distributed species of Neurospora known, having been collected from Alaska, Europe, New Zealand and intervening tropical and subtropical regions. Our recent phylogenetic analyses showed that outbreeding (conidiating) Neurospora diverged into two sister clades (Dettman et al. Evolution 57:2703-2720). The first clade contains only N. discreta, which appears to be a complex of species nearly as diverse as the second clade, which contains the other seven outbreeding species. Cryptic species within N. discreta are being recognized using analyses of sequence data from three unlinked, anonymous, nuclear loci, each containing a microsatellite and ~450 bp of flanking sequence. Our sample totals 75 isolates from the worldwide range of N. discreta, including new collections from Europe and North America. Criteria developed to recognize phylogenetic species in Neurospora will be applied to the N. discreta clade: species are recognized using genealogical concordance of loci with the added the criteria that species level clades are monophyletic and well-supported in at least one single-locus genealogy, and not contradicted by any other. The results will set the foundation for future comparative biology and genetics within the N. discreta clade, and between N. discreta and other species of Neurospora.
66) The mysterious origin of the Oak Ridge wild types. Olivera Gavric and Anthony Griffiths. Botany Department, UBC, Vancouver, Canada V6T 1Z4.
In the course of routine sequencing of the phospholipase C (PLC) gene, we have discovered a striking sequence dimorphism among Neurospora laboratory wild types. The two morphs differ by 29 base pair substitutions (of which two are in the intron) plus one single codon addition/deletion. Eleven seemingly important missense mutations result. The significance of this genetic divergence is not known, but the results reveal two clear lines of ancestry that might have been subjected to different selection pressures. We have tracked the PLC dimorphism in several key strains alleged to be ancestors of the Oak Ridge wild types. Both Oak Ridge strains have the same allele, which can be arbitrarily designated morph 1. However, both Emerson 5297a and Emerson 5256A, which are the supposed direct predecessors of Oak Ridge (Newmeyer et al., FGN 34), have morph 2. Taken on face value, this would mean that Oak Ridge cannot be descended from the Emerson strains listed. Three Lindegren strains that we have analyzed also have the Emerson morph 2. However, preliminary data show that the Abbott strains 4A (definitely) and 12a (probably) have the Oak Ridge morph 1, hence the PLC allele of Oak Ridge seems to be of Abbott ancestry. The results fit the general pedigree well except for the Oak Ridge branch. The authenticity of several of the specific isolates has been questioned (summarized by Newmeyer et al.) and this might partially explain the paradox.
67) Studying the evolutionary genetics of reproductive isolation between Neurospora lineages. Elizabeth Turner and John W. Taylor, Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720
Recent work characterizing phylogenetic relationships and reproductive isolation among several outcrossing Neurospora species has laid the groundwork for studies that go beyond the description of evolutionary patterns to explore the biological mechanisms underlying interspecies sexual isolation and intraspecies cohesion. We are currently studying the genetic basis of reproductive isolation between two pairs of taxa: (1) N. crassa and N. intermedia and (2) the more recently diverged, partially isolated, subspecific clades N. crassa A and N. crassa C. Using AFLP markers, we have created linkage maps that will be the bases of quantitative trait locus (QTL) analyses of reproductive isolation and compatibility in matings with species- and clade-specific tester strains. Here we present the linkage maps and compare the linkage, intermarker map distances, and map orders of N. crassa markers common to both maps. In order to assign linkage groups recognized in our analysis to the known chromosomes of Neurospora, we took advantage of commercially available RFLP mapping kits. We found that many of our AFLP markers segregated in the RFLP mapping strains, and we discuss linkage group/chromosome assignments and marker ordering in light of these segregation data. Finally, we present the results of preliminary mating experiments on a small number of genotyped N. crassa/N. intermedia f1 hybrids. We identify several markers that were significantly associated with the fertility of f1 hybrids in crosses to N. crassa or N. intermedia tester strains.
68) Influence of Parental Genotype and Mating Type on Quantitative Traits in Field Isolates of Neurospora crassa. Heather H. Wilkinson, Thomas J. DeWitt and Daniel J. Ebbole. Texas A&M University, College Station, TX.
Despite the relatively well elucidated genetic basis for development in Neurospora crassa, little is known about morphological and life history variation in natural populations. To begin to address the heritability and ecological implications of this sort of variation, we bred genotypically distinct isolates from nature and evaluated phenotypic variation. Specifically, 8 previously characterized isolates from a Louisiana sugar cane field (four of each mating type) were crossed in all possible combinations such that each isolate served as both the maternal and paternal parent (in total, 32 combinations X 4 replicates). Perithecium size and number, and ascospore number, size and shape were all measured for each replicate. There was substantial variability in all phenotypic characters. Maternal genotype significantly influenced all these characters, and accounted for the majority of variation associated with perithecium size and number. Ascospore size, shape and number were all also influenced by paternal genotype and the maternal-paternal genotype interaction. Finally, mating type of the maternal parent significantly influenced spore size, shape and perithecium number. Path analysis is being used to investigate phenotypic integration among traits and functional aspects of variation in these traits are being explored (e.g. germination rates of ascospores based on size). We conclude that Neurospora is an excellent candidate for use in manipulative evolutionary studies.
69) Mutation of spo-11 and its effect upon meiotic recombination in Neurospora crassa. F.J. Bowring, P.J. Yeadon, R.G. Stainer and D.E.A. Catcheside. School of Biological Sciences, The Flinders University of South Australia
SPO11 is thought to initiate meiotic recombination by generating chromosomal double-strand breaks. While first identified in Saccharomyces cerevisiae, SPO11 homologues have been found in numerous species including humans, and we have generated three Neurospora spo-11 RIP mutants.
The predicted polypeptide of the best characterised of these (spo-11 RIP1 ) is truncated near the midpoint deleting three of five conserved motifs. While we predicted a reduction in the amount of meiotic recombination in spo-11RIP1/RIP1 diploids, we observed elevated levels of allelic and non-allelic recombination in rec-2 regulated regions. Neurospora rec genes modulate the recombination frequency in one or more distinct chromosomal regions. We are currently characterising the other two spo-11 RIP alleles.
70) Characterization of ku-70 and ku-80 knockout mutants in Neurospora crassa. Ninomiya, Y., Suzuki, K., Ishii, C. and Inoue, H. Saitama University, Japan
We are studying DNA repair mechanism in N. crassa. In this report, we will present disruptants of N. crassa-homolog genes of Human KU70 and KU80 genes to investigate roles in recombination repair. These mutants showed slightly more sensitive to UV than the wild type and 3 times more sensitive to MMS than the wild type. Mutant genes were mapped by crossing with strains carrying proper makers. Double repair-deficient mutants were isolated to test epistatic relationship. For this purpose, other mutants belonging excision-repair or postreplication repair or recombination repair group were used. Also, homologous integration was measured in these strains. These results will be presented.
71) Isolation and characterization of the methyl/RIP DNA binding protein MRBP-1 and Potential Base Unpairing of DNA mutated by RIP. Gregory O. Kothe1, Michael R. Rountree2, Ashley McCormack3, Larry David3, Yuji Nakayama4, Terumi Kohwi-Shigematsu4, and Eric U. Selker1. 1Institute of Molecular Biology, University of Oregon, Eugene, OR, 2St. Jude Children's Research Hospital, Memphis, TN, 3Department of Oral Molecular Biology, School of Dentistry, Oregon Health and Science University, Portland, OR, 4Lawrence Berkeley National Laboratory, University of California, Berkeley, California
We have purified a putative protein complex that binds RIPed and methylated DNA, and refer to this activity as MRBP-1 (Methyl/RIP Binding Protein 1). The putative complex contains a protein belonging to the telobox class of DNA binding proteins; containing a single myb domain in its carboxyl terminus. We have also identified two proteins that show high affinity for single-stranded, but not double-stranded, DNA. We propose that certain sequences that have undergone RIP may have single-stranded character, similar to matrix attachment regions. We also show that a RIPed sequence is bound preferentially in a gel-shift assay by the vertebrate BUR (Base Unpairing Region) binding protein
72) Choline Depletion does not Affect the Central Circadian Oscillator in Neurospora crassa. Mi Shi, Jennifer Loros, Jay Dunlap. Department of Genetics, Dartmouth Medical School, Hanover, NH 03755
Neurospora chol-1 mutants display an elongated and unstable period rhythm of spore formation on solid medium without choline supplementation. Since the rhythm shows few characteristics of a typical circadian rhythm, it is not clear whether the long period rhythm is an output reflecting an elongated central circadian oscillator or is instead a morphological rhythm reflecting defects in lipid metabolism. Such a rhythm could mask expression of a circadian clock. To evaluate this, a liquid to solid transfer assay was adapted to measure the rhythm period under choline depletion conditions in liquid culture. Conidia from chol-1 mutants were cultured in liquid media without choline supplementation in darkness and the conidial suspension was transferred to race tubes with choline supplementation at different time points. Since the circadian clock should cycle through all phases of the day in liquid as well as on solid medium, and since the transfer from liquid to solid medium does not reset the phase of the clock, one can measure the period length of the rhythm under choline starvation. We found that the phases of the rhythm on solid medium were similar for all time points, suggesting that the free-running period of the circadian clock under choline starvation is the same as that seen with supplementation. Consistent with this, levels of the clock component FRQ oscillate with a ~22-hour period in a two-daytime course in darkness following two days of choline starvation.
73) Recombination hotspot alleles are co-dominant. P. J. Yeadon, F. J. Bowring and D. E. A. Catcheside1. 1School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, South Australia, Australia
Naturally occurring alleles of the recombination hotspot cog in Neurospora crassa have two phenotypes with respect to recombination in the his-3 region. Presence of the cog+ allele in a cross significantly increases both allelic and local intergenic recombination compared to a cross homozygous for the cog allele. Data obtained in the 1960s suggested that cog+ is fully dominant to cog, which was always difficult to explain. We show that cog alleles are co-dominant in effect on both allelic recombination in his-3 and crossing over between loci flanking his-3. In addition, we present evidence of a two-fold range in allelic recombination frequency due to genetic background variation, the most likely explanation for the previous conclusion that cog+ is dominant to cog.
Biosensors in Neurospora. Emma Perfect and Pat Hickey, Lux Biotechnology Ltd.
Biosensors utilise biological materials to detect and monitor the presence of various molecules in a sample.
The majority of biosensors are based on bacteria, mammalian cells or yeast. In 2001 LUX Biotechnology Ltd. was
established to develop filamentous fungal biosensors. Filamentous fungi are more versatile, grow in more
diverse conditions and are easier to store and transport than bacteria or yeast. These features make Lux
biosensors more flexible and thus more powerful than existing alternatives.
Lux biosensors harness the power of bioluminescence and fluorescence by generating fungal strains whose light output gives an indication of the presence of specific molecules in the environment or of fungal health. It is predicted that Lux biosensors will make important contributions to environmental testing, drug discovery and R+D. The decision to utilise Neurospora crassa for Lux’s prototype biosensor was influenced by the quality research available, the recently published genome sequence and the N. crassa community’s supportive reputation.
Contact: email@example.com or visit our web site at www.luxbiotech.com
Recent Activities at the FGSC. Kevin McCluskey. Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City, KS
In our continuing effort to serve the fungal genetics community, the FGSC has expanded to include new organisms, categories, and molecular materials. We have maintained our distribution efforts and encourage clients to use the resources at the FGSC. Recent additions include a collection of Schizophyllum commune strains, Magnaporthe grisea tagged knockout strains, Neurospora wild-type collections, library pools and clones from the sequencing programs
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