1. Mutants of Neurospora complex I as models of human mitochondrial disease.
Arnaldo Videira1, Margarida Duarte1, Helena Populo1, and Ulrich Schulte2. 1Instituto de Biologia Molecular e Celular, Porto, Portugal. 2 Duesseldorf Institute of Biochemistry, Duesseldorf, Germany.
Mitochondrial complex I transfers electrons from NADH to ubiquinone, through protein-bound prosthetic groups, coupled with proton translocation through the inner membrane of the organelle. It contains about 40 proteins of dual genetic origin distributed in two major domains, the peripheral and membrane arms. Specific mutations in iron-sulphur subunits of the enzyme were found associated with human mitochondrial diseases. Namely, the P79L and R102H mutations in the TYKY protein and the V135M mutation in the PSST protein were found. Disruption of either of the N. crassa homologues of these proteins prevents assembly of the peripheral arm of complex I. We generated the equivalent human mutations in the fungal cDNAs encoding the TYKY and PSST homologues, by site-directed mutagenesis. The cDNAs harbouring the mutations were then expressed in the relevant fungal mutant. The correspondent mutant proteins were able to restore the assembly of a complex I enzyme. Further characterisation of the strains will be presented.
2. Characterization of NAD(P)H dehydrogenases from the inner membrane of Neurospora mitochondria.
Arnaldo Videira1, Ana M.P. Melo1 and Ian M. Moller2. 1Instituto de Biologia Molecular e Celular, Porto, Portugal. 2University of Lund, Lund, Sweden.
Disruption of an NAD(P)H dehydrogenase from the inner mitochondrial membrane of Neurospora crassa, NDE1, was achieved by repeat induced point mutations. A physiological characterization of the NDE1 mutant was performed by measuring the rates of respiration of intact mitochondria and inside-out submitochondrial particles with either NADH or NADPH as substrates. The results indicate that the mutant strain lacks an NADPH dehydrogenase, which contains a catalytic site facing the mitochondrial intermembrane space and works at pH 7. The observation of NADH oxidation at pH 7 and of NADPH oxidation at pH 6 by intact mitochondria from the NDE1 mutant evidences the presence of a second external NAD(P)H dehydrogenase activity. The cross of an NDE1 mutant with several mutants in subunits of complex I yielded double mutants, indicating that further NAD(P)H dehydrogenases exist in N. crassa mitochondria.
3. Initial characterization of the TIM17-23 translocase of the mitochondrial inner membrane in Neurospora crassa.
Dejana Mokranjac and Holger Prokisch. Physiological Chemistry, University of Munich, Munich, Germany.
The majority of mitochondrial proteins are encoded in the nucleus and are synthesized in the cytosol. In order to reach their functional destination, they must be transported into or across the mitochondrial membranes. Import into mitochondria is a multi-step process mediated by translocation systems in the mitochondrial outer and inner membranes. In yeast, the outer membrane contains one translocase, the TOM complex, which is probably used by all nuclear-encoded preproteins. The inner membrane contains two different translocases, TIM17-23 and TIM22-54 complexes. TIM17-23 complex is used by the preproteins which carry the classical mitochondrial presequence. TIM22-54 complex mediates the import of hydrophobic membrane proteins with internal targeting signals. In Neurospora crassa, TOM complex has been purified and characterized in certain details. However, neither of the Neurospora TIM complexes has been known so far. Here, we have cloned and characterized Neurospora crassa tim23. The encoded protein shares 40% sequence identity with its yeast homologue TIM23p. We have constructed a Neurospora strain which expresses the TIM23 protein with an octahistidine tag on its C-terminus and only negligible amounts of the wt protein. The tagged protein is fully functional. Construction of the strain and its use in the purification of the TIM17-23 complex will be presented.
4. Effect of mutation in vacuolar ATPase on basic amino acids pools in Neurospora crassa.
Kelly A. Keenan, Keith Mindish, Kristi L. Muldoon, and Dawn M. Moore. NAMS, Richard Stockton College, Pomona, NJ, USA.
Three strains with decreased vacuolar ATPase activity have been isolated using a filtration enrichment procedure. All three strains show an increase sensitivity to the three basic amino acids(arginine, lysine and ornithine) stored in the vacuole. The levels of the basic amino acids in the cytosol was measured using a cupric ion permeabilization procedure and compared to wild type. All three strains had increased levels of basic amino acids. The vacuolar pools for the three basic amino acids was measured and shown to be decreased. Uptake of the basic amino acids into the vacuole was measured and shown to be signficantly reduced. This was further characterized kinetically by measuring the Michaelis constant and maximum velocity for uptake for the basic amino acids. For all amino acids studied so far, there have been changes in these values. The results suggest that the vacuolar ATPase plays an integral role in controlling the levels of vacuolar amino acids.
5. Mitochondrial morphology and inheritance in Neurospora crassa: Cloning and characterisation of mmm-1.
Holger Prokisch. Physiol. Chemie, LM-University, Muenchen, Germany.
Mitochondria are essential organelles which are often located at sites of high energy consumption in the cell. Since they cannot be synthesized de novo, they have to be inherited from the mother to the daughter cell during cell division. There is mounting evidence that positioning and transport of mitochondria are controlled by the cytoskeleton. All three major cytoskeleton classes have been implicated in mitochondrial movement in fungi. In the budding yeast S. cerevisiae, the actin cytoskeleton appears to play a major role in inheritance of mitochondria. In filamentous fungi, different data exist as to whether microtubules or microfilaments mediate inheritance of mitochondria. Disruption of microtubules does not affect mitochondrial movement in Aspergillus nidulans, whereas it has been suggested that cytoplasmic microtubules are required for transport of mitochondria in Neurospora crassa. Here, we describe the cloning and characterization of Neurospora crassa mmm-1. The encoded protein shares 30% sequence identity with its yeast homologue, Mmm1p, that was proposed to act as a mitochondrial receptor for actin binding proteins. An mmm-1RIP mutant strain of Neurospora exhibits a temperature-sensitive growth phenotype and harbours mitochondria with an abnormal giant morphology. The MMM-1 protein is imported into the mitochondrial outer membrane in a receptor-dependent manner. We provide evidence that the MMM-1 protein has a single transmembranesegment in the mitochondrial outer membrane with a large C-terminal domain exposed to the cytoplasm. Implications of the mutant phenotype and the toplogy of the protein on its role in mitochondrial inheritance will be discussed.
6. Structure of the vacuolar H+ ATPase from Neurospora crassa.
Jack C. Reidling, Emma Jean Bowman, Emilio Margolles-Clark, Karen Tenney, June Pounder, and Barry J. Bowman. Biology, UCSC, Santa Cruz, CA, USA.
The filamentous fungus Neurospora crassa contains many small vacuoles. These organelles contain hydrolytic enzymes, high concentrations of polyphosphates, and basic amino acids such as arginine and ornithine. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The ATP-driven proton pump in the vacuolar membrane is a typical V-type ATPase. It is a large and complex enzyme distantly related to the F-type ATPase of bacteria and mitochondria. We have found that about half of the 13 subunits in the vacuolar ATPase are homologs of subunits of F-type ATPases. Several of the subunits appear to be unique to the V-ATPase. An interesting question is whether all V-ATPases have 13 subunits. We have characterized the genes that encode 11 of the Neurospora V-ATPase subunits, and have recently obtained the 12th and 13th. Ten of the Neurospora V-ATPase genes have been mapped and the locations are not clustered. A major goal of our laboratory has been to understand the function and structure of the vacuolar ATPase. One approach we are using is to purify the peripheral V1 complex for structural studies.
7. Neurospora mating pheromone precursor genes.
Piotr Bobrowicz1, Rebecca Lowe1, Amy Miller1, Wei-Chiang Shen1, Pam Kazmierczak2, Neal Van Alfen2, Deb Bell-Pedersen1, and Daniel Ebbole1. 1Plant Pathology & Microbiology, Texas A&M University, College Station, TX. 2 University of California, Davis CA.
The mating type loci of N. crassa encode regulators that control expression of genes involved in sexual fertility and development. We have begun to analyze the genes encoding the sex pheromones of N. crassa. One gene, expressed in Mat-A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by KEX2 processing sites. The predicted sequence of the pheromone is remarkably similar to those encoded by the rice blast fungus, Magnaporthe grisea, and the chestnut blight fungus, Cryphonectria parasitica. Mat-a strains express a pheromone precursor gene, whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The pheromone precursor genes are regulated by nutrients, macroconidiation, and the circadian clock, but display strict mating type-specificity. They are repressed by the RCO1 repressor, but repression by RCO1 is not required to maintain mating type-specificity.
8. Protein synthesis in germinating ascospores of Neurospora crassa.
Cheryl L. Scott, Nora Plesofsky, and Robert Brambl. Plant Biology, University of Minnesota, St. Paul, Minnesota, USA.
Dormant ascospores of Neurospora crassa exhibit a high level of thermotolerance, and this protection is gradually lost during germination. The goal of this study is to determine at what point during germination the ascospores become capable of synthesizing both normal and heat shock proteins. We determined survival rates of activated ascospores germinated at normal temperature (30 C) and then exposed to a heat shock treatment (45 C) to induce heat shock protein synthesis, a lethal heat treatment (50 C), or a heat shock treatment followed by a lethal heat treatment. These survival experiments showed that 240 min. after activation ascospores become sensitive to lethal heat treatments; they also benefit from receiving a heat shock treatment prior to exposure to the lethal heat treatment. We measured accumulation of the small heat shock protein, Hsp30, a protein that is heat shock-inducible and not constitutively synthesized, by Western analysis. Results showed that cells cannot be induced by heat shock to synthesize Hsp30 until 135 min. after ascospore activation. Further, radiolabelling experiments showed that synthesis of normal proteins as well as heat shock-induced proteins are initially detectable 120 min. after activation. The results of this study show that there is a period of approximately two hours after activation before the ascospores initiate new protein synthesis.
9. Analysis of two novel genes that are highly expressed in starved and sexual tissues of Neurospora crassa.
Hyojeong Kim and Mary Anne Nelson. Biology, University of New Mexico, Albuquerque, New Mexico, USA.
Two novel and highly expressed genes were identified by the Neurospora Genome Project at UNM (Nelson et al., Fungal Genetics and Biology 21:348-363, 1997). These genes are tentatively named poi-1 and poi-2 (for plenty of it), since they are the most highly expressed genes in the starved mycelial and perithecial tissues of Neurospora crassa. The most abundantly expressed of the two genes, poi-1, has no easily identifiable ORF. The mRNA is mostly non-coding with multiple stop codons and no region of good codon bias. poi-1 mRNA also has an unusual predicted secondary structure. The gene might encode a 10 kDa putative protein as determined by in vitro transcription and translation, and it appears to be essential. An ORF with good codon bias has been identified in the second most abundantly expressed gene, poi-2. It encodes a 27 kDa putative protein that contains a possible transmembrane helix and a possible signal peptide. The putative poi-2 protein also contains a novel 16 tandem repeat of 13-14 amino acid residues. We are currently using repeat-induced point mutation (RIP) to analyze the functions of these two genes and their products.
10. A novel bZip transcription factor expressed during sexual development in Neurospora crassa.
Harriett J Bowannie Platero, and Mary Anne Nelson, Department of Biology, University of New Mexico, Albuquerque, NM, USA.
The research goal is to characterize a highly expressed gene, tentatively named zip-1, found predominantly in the perithecial stage of Neurospora crassa. This gene is of particular interest for two reasons. First, it exhibits a higher level of expression during the perithecial stage than during the vegetative growth stages. Second, similarity search algorithms have identified a region of homology to the transcription factor, c-jun. c-jun plays an important role in activating gene transcription, it is also a proto-oncogene and was the first transcription factor shown to induce cancer. Our hypothesis is that the zip-1 gene of Neurospora crassa is a novel transcription factor belonging to the same family of transcription factors as c-jun, the bZip subfamily.
11. Regulation of macroconidiation by fluffy.
Panan Rerngsamran, Lori Bailey-Shrode and Daniel Ebbole. Plant Pathology & Microbiology, Texas A&M University, College Station, TX, USA.
Fluffy (fl) encodes a member of the Gal4 class of transcription factors. Null mutations of fl block the budding growth characteristic of proconidial chain formation. These mutants are also blocked in expression of many of the known conidiation-specific genes. The pattern of fl mRNA expression is consistent with its role as a regulator of morphogenesis. A basal level of fl expression is observed in undifferentiated mycelia. fl mRNA levels are induced during development at approximately the time when budding growth initiates and fl mRNA levels declines at later stages of development. acon-2 and acon-3 also are regulators of conidial morphogenesis and acon-2 is required for induction of fl mRNA while acon-3 is not. Elevated expression of fl from a the cpc-1 or ars-1 promoter was sufficient to induce conidiophore morphogenesis. One current effort is to isolate the FL protein for DNA binding studies. The location of consensus FL DNA binding sites in the genome may provide clues as to which N. crassa genes are subject to developmental regulation.
12. The role of the dgr class of mutants in conidiation.
Xin Xie, Jed Parker, and Daniel Ebbole. Plant Pathology & Microbiology, Texas A&M University, College Station, TX, USA.
The rco-3 mutant is resistant to growth inhibition by 2-deoxyglucose and L-sorbose. These are the phenotypes of dgr-1, -2, and -3 and rco-3 is allelic to dgr-3. The rco-3 gene was found to negatively regulate conidiation. rco-3 mutants conidiate in submerged aerated liquid culture without the usual requirement for carbon or nitrogen starvation. The rco-3 mutant was also found to be required for proper regulation of glucose transport activity and carbon catabolite repression. rco-3 encodes a member of the sugar transporter superfamily and our characterization of rco-3 suggests that it functions as a sugar sensor rather than a sugar transporter. As one approach to identifying genes that genetically interact with rco-3 we have isolated mutants that suppress the sorbose resistance of rco-3 mutants.
13. Kinesin as a motor for organelle movement in mammalian and fungal cells .
Kristen J. Verhey1, Tom A. Rapoport1 and Manfred Schliwa2. 1Cell Biology, Harvard Medical School, Boston, MA, USA. 2Zellbiologie, Univ of Munich, Munich, Germany.
Kinesin is a molecular motor protein which moves cargo along microtubules in cells. Although kinesin has been implicated in the transport of various membrane-bound organelles and their positioning within the cell, its in vivo function is still unclear. Previous experiments in mammalian cells have shown that the majority of the kinesin protein in cells is inactive and not bound to either membranes or microtubules. This is due to the folded conformation of the protein which allows the C-terminal tail of the protein to directly inhibit the N-terminal motor. Current experiments are aimed at addressing whether fungal kinesins are also auto-inhibited. In Drospohila and C. elegans, kinesin is an essential protein whose deletion leads to embyonic lethality. In contrast, deletion of the motor in Neurospora is not lethal but results in severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching (Seiler et al., 1997 EMBO J 16:3025-3034). We have begun further analysis of the phenotype of the kinesin null strain, with the aim of identifying conditions which can later be used in a screen for mutants with a similar defect in organelle transport.
14. htl, a gene unique to filamentous fungi, encodes a protein involved in hyphal tip growth.
Karen Tenney1, Ian Hunt2, James Sweigard3, June Pounder4, Chadonna McClain1, Emma Jean Bowman1, and Barry Bowman1. 1Biology, University of California, Santa Cruz, CA. 2Novartis Pharmaceuticals, Horsham, West Sussex, UK. 3DuPont, Wilmington, DE. 4McLaughlin Research Inst., Great Falls, MT.
We have identified a gene, htl (hyphal tip lysis), that encodes an abundant 19 kDa protein in mycelia of Neurospora crassa. In the current genome databases, homologs to the htl gene are unique to filamentous fungi such as Aspergillus nidulans, Magnaporthe grisea, Fusarium veneratum, and Botrytis cinerea. The morphology of RIP strains (in which the gene is inactivated and the HTL protein is deficient) suggests a role in hyphal tip growth. Phenotypic characteristics of htl- strains include swelling and bursting of hyphal tips as well as the gross pigment accumulation ("bleeding") similar to that seen in N. crassa osmotic strains. Although the HTL protein is present at sufficiently high levels to detect on Western blots of whole cell extracts probed with polyclonal anti-htl antibody, it is not present in detectable levels in conidia. When isolated by cell fractionation the protein behaves like a particle of high density, but is not associated with the cell wall. We present evidence that HTL forms oligomers that are not dissociated by SDS and mercaptoethanol but can be dissociated by treatment with phosphatase. HTL proteins may form oligomeric complexes that are associated with cytoskeletal components involved in the unique hyphal tip growth of filamentous fungi.
15. Aspects of the membrane skeleton in Neurospora crassa.
Gagan D. Gupta and Brent I. Heath. Biology, York University, North York, Ontario, Canada.
Filamentous fungi produce tubular forms called hyphae by means of tip growth. Tip growth involves several coordinated processes that include vesicle transport and fusion, and synthesis and expansion of cell wall and plasma membrane. It has been proposed that the cytoskeleton, chiefly F-actin and its associated proteins, is important as an integrative scaffold to localize, support, and regulate these tip-growth processes which produce the hyphal form (membrane skeleton model). Our recent work with the ascomycete Neurospora crassa supports various aspects of this model. Notably, we have identified a spectrin-like protein in Neurospora that is concentrated prominently in hyphal apices and can bind actin in a blot overlay assay. Its distribution and properties indicate the presence of a membrane skeleton in hyphae. A Western blot survey of several fungal species with antibodies to animal spectrin suggests that spectrin-like proteins that may form membrane skeletons may be widespread in fungi. A complementary aspect of this model is the regulated exocytosis of wall vesicles in tip-growing regions. Our work, together with information from the Neurospora sequencing projects, has identified several t-SNAREs (highly conserved proteins that are required for intracellular membrane fusion) in Neurospora . At least one of these t-SNAREs has been partially characterized. This t-SNARE seems to be delivered to the plasma membrane by the wall vesicles. It occurs in a tip-high gradient on the plasma membrane, but the slope of this gradient does not match the mathematical exocytotic gradient required to produce the hyphal shape. This suggests that other factors, including those that regulate SNARE activity, are involved in the regulation of exocytosis in hyphal tips.
16. Phenotypic and genetic analysis of ham (hyphal anastomosis mutant) in Neurospora crassa.
Sergio Daniel Haedo and N. Louise Glass. Plant & Microbial Biology, University of California, Berkeley, CA.
Hyphal anastomosis is a fundamental process during both vegetative growth and sexual reproduction in filamentous fungi. Although several genes are probably involved in regulating this complex process, very little is understood about the mechanistic and genetic aspects controlling cell fusion events. A Neurospora crassa hyphal anastomosis mutant (ham) has been recently isolated that is not able to form heterokayon in the standard period of 24 h at 30C. This mutant shows a slight growth defect and is female-sterile (Wilson and Dempsey 1999. Fungal Genet. Newsl. 46:31). The study of ham can help us to unravel the genetic basis underlying in the hyphal anastomosis process in N. crassa. We selected ham OR-compatible strains (the original ham strain is incompatible with Oak Ridge strains) using a modified heterokayon test. Phenotypic analysis of these strains shown that the frequency of hyphal fusion events is severely reduced in ham strains. By plating different numbers of ham conidia along with conidia from an OR tester strain in sorbose-containing plates, we observed a reduction of several magnitudes in the ability to form heterokaryon in ham compared to several wild type strains. By genetic analysis we mapped ham in the right arm of LGI, linked to arg-13 (2 to 9% recombination). The ham locus maps close to a known morphological mutant named so (soft) whose phenotype resembles ham strains (fuzzy short aerial hyphae and conidia formed more uniformly than the wild type. Moreover so, like ham, is female sterile). No wild-type progeny was recovered from crosses so x ham (0/220) and heterokayon tests shown reduced hyphal fusion frequency in so strains. We have initiated molecular experiments to clone ham. Two approaches will be used: a) chromosome walking from cosmid-libraries around arg-13 and, b) phenotypic complementation of ham morphological phenotype using cosmids from a LGI-specific library.
17. Mutations at vib-1 reduce hyphal fusion frequency and block het-c mediated vegetative incompatibility in Neurospora crassa.
Qijun Xiang and N. Louise Glass. Plant & Microbial Biology, UC Berkeley, Berkeley, California, USA.
Vegetative incompatibility triggered by allelic differences in het-c locus of Neurospora crassa is characterized by growth inhibition and arrest, suppression of conidiation, hyphal compartmentation and death. To genetically dissect the pathway of het-c vegetative incompatibility, we allowed incompatible transformants to escape from growth inhibition and suppression of conidiation. Several mutations that blocked het-c mediated vegetative incompatibility were identified. A mutation at the vib-1 (vegetative incompatibility blocked) locus greatly reduces heterokaryon formation efficiency. High conidial concentrations are needed to force heterokaryons between a vib-1 mutant and a wild-type strain. Heterokaryons were not obtained between two vib-1 strains when conidia were used to force the heterokaryons. However, heterokaryons between two vib-1 strains could be formed by either of spheroplast fusion or forcing mycelial heterokaryons. Reduced frequency of hyphal fusion contributes to the low heterokaryon formation efficiency. Hyphal fusion events have not been detected in leading hyphae of a homokaryotic vib-1 colony. The effect of vib-1 mutation on het-c vegetative incompatibility has been tested by four ways: spheroplast fusion, mycelial heterokaryons, transformation and partial diploids. All data indicated that vib-1 fully blocks het-c vegetative incompatibility. The vib-1 mutation is recessive and causes multiple developmental defects as well. A vib-1 mutant has a reduced mycelial growth rate, reduced aerial hyphae differentiation and is fully sterile as female, but partially sterile as male. We also have identified two other independent mutants that suppress het-c vegetative incompatibility. We are testing whether these mutants contain mutations that are allelic to vib-1.
18. Characterization of a G-beta Subunit from Neurospora crassa.
Sheven I. Poole, Qi Yang, and Katherine A. Borkovich. Microbiol. & Molec. Gen., University of Texas-Houston, Houston, TX, USA.
Heterotrimeric G protein-mediated signal transduction is one of many pathways used by cells to respond to environmental stimuli. Experimental evidence has demonstrated that G protein beta-gamma dimers play pivotal roles in transduction of extracellular signals. Low-stringency hybridization using degenerate oligonucleotides based on conserved regions of mammalian G-beta proteins as probes resulted in identification of the Neurospora crassa G-beta gene, gnb-1. The predicted amino acid sequence of gnb-1 is most identical to C. parasitica CPGB-1 (91%), followed by human G-beta subunits (~49-67% identity) and D. discoideum GPB-1 (66% identical). Results from restriction fragment-length polymorphism (RFLP) mapping demonstrate that the gnb-1 gene is located on the right arm of chromosome III near the con-7 and trp-1 genes. Northern analysis revealed two gnb-1 specific transcripts of 2.7 and 1.4 kb. gnb-1 null strains have numerous defects, including shorter germ tubes, aberrant vacuole number and size during growth in submerged culture, reduced mass accumulation and female sterility. Loss of gnb-1 also impacts levels of the G-alpha subunits GNA-1 and GNA-2 in N. crassa.
19. Isolation of components involved in mating-type associated heterokaryon incompatibility using the yeast 2-hybrid system.
Patrick K. T. Shiu, Megan D. Hiltz, and N. Louise Glass. Plant & Microbial Biology, U.C. Berkeley, Berkeley, CA, USA.
The mating-type locus (mat) in Neurospora crassa controls mating and sexual development. The fusion of reproductive structures of opposite mating-type, A and a, is required to initiate sexual reproduction. During vegetative phase, the mat locus also functions as a one of the 11 heterokaryon incompatibility (het) loci. Heterokaryon incompatibility is a mechanism that prevents the formation of vigorous heterokaryons between genetically dissimilar individuals and is believed to be a ubiquitous phenomenon among ascomycetes and basidiomycetes. The fusion of mat A and mat a hyphae during vegetative growth results in growth inhibition, hyphal compartmentation and death. Vegetative incompatibility between opposite mating-types is due to the molecular actions of MAT A-1 and MAT a-1. The incompatibility reaction between MAT A-1 and MAT a-1 is mediated by TOL - mutations in tol are recessive and suppress mating-type associated heterokaryon incompatibility. The MAT A-1 and MAT a-1 are transcriptional regulators while the molecular function of TOL is not apparent although it contains protein-protein interaction domains. To understand the role of TOL in mating-type heterokaryon incompatibility and to identify other proteins involved in the process, we have isolated several TOL-interacting proteins (tip) using the yeast 2-hybrid system. The six potential tip genes have been identified and encode a regulator for acriflavine drug-resistance, a Schizosaccharomyces pombe VIP-1 homolog (a p53 related protein) and other novel proteins. RIP mutational analyses of the tip genes are undergoing and the phenotype of the mutants will reveal their functions, as well as their roles in mediating mating-type associated heterokaryon incompatibility.
20. G gamma subunit identified in Neurospora crassa.
Svetlana Krystofova and Katherine A. Borkovich. Microbiol. Mol. Gen. University of Texas, Houston, TX, USA.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eucaryotic cells. Upon agonist binding to the receptor, the G protein alpha subunit releases GDP, binds GTP, and dissociates from the G protein beta gamma subunit dimer. Depending on the system, either G alpha or G beta gamma go on to activate downstream effectors. Three G alpha subunit genes, gna-1, gna-2 and gna-3, and one G beta subunit gene, gnb-1, have been identified in Neurospora crassa. A G gamma subunit gene, gng-1, has been identified during searches of the University of Oklahoma EST database. The full length of gng-1 cDNA (273 bp) has been found in the cDNA clone. The predicted amino acid sequence of GNG-1 shares 35% identity with the Saccharomyces cerevisiae STE18 and 30-33% identity with other G gamma subunits identified in mammalian cells. A gng-1 gene clone was isolated from lambda-BARGEM7 genomic library using the gng-1 cDNA clone as a probe. Our current focus is making gng-1 mutants, and to analyze the phenotypes of them.
21. GNA-3, a G protein alpha subunit, regulates the expression of adenylyl cyclase in Neuropora crassa.
Ann M. Kays, Patricia S. Rowley, Rudeina Baasiri, and Katherine A. Borkovich. Microbiology & Molec. Genetics, University of TX-Houston Health Science Center, Houston, TX, USA.
In eukaryotic systems, cellular responses to signals, such as odorants and light, are mediated by heterotrimeric G proteins coupled to seven transmembrane receptors. Our lab identified two G alpha subunits, gna-1 and gna-2, and demonstrated GNA-1 directly regulates adenylyl cyclase. A third G alpha, gna-3, was cloned in Neurospora crassa and shown to be a positive and negative regulator of aerial hyphae formation and conidiation, respectively. Addition of cAMP suppresses both the aerial hyphae and conidiation defects in standing liquid cultures, but only suppresses the conidiation defect on plates. N. crassa conidiaiton requires an air/water interface; however, delta gna-3 strains conidiate abundantly in submerged culture. Analysis of the glucose-repressible gene, qa-2, suggests delta gna-3 submerged culture conidiation is due to glucose starvation. Deletion of gna-3 results in significantly decreased intracellular cAMP and adenylyl cyclase activity. Western analysis indicates the reduced delta gna-3 cAMP is due to decreased levels of N. crassa adenylyl cyclase enzyme, NAC. To explore the role of GNA-1 and GNA-3 in regulating adenylyl cyclase, a double gna-1 and gna-3 mutant was constructed by a sexual cross. The resulting delta gna-1 delta gna-3 strain displays phenotypic morphology similar to an adenylyl cyclase mutant, cr-1. Biochemical results suggest that GNA-3 modulates intracellular cAMP levels by regulating the expression of adenylyl cyclase to facilitate N. crassa development.
22. pH sensing in Neurospora crassa.
Antonio Rossi, Sergio R. Nozawa, Walter Maccheroni Jr, Monica S. Ferreira, Andre Justino, and Nilce M. Martinez-Rossi. Department of Chemistry, University of São Paulo, Ribeirão Preto, SP, Brazil.
Although pH regulation in filamentous fungi has been implicated in the selective de-repression of many genes as a function of ambient pH, which is mediated by the transcriptional activation protein PacC, gene pho-2 is expressed irrespective of the growth pH when N. crassa is grown in low-Pi. This is because the same amount of pho-2 encoded alkaline phosphatase is secreted irrespective of ambient pH. Also, the loss of activity observed for the alkaline phosphatase secreted at pH 5.4 is an effect probably due to its low glycosylation as compared to the glycosylation observed for the enzyme secreted at alkaline pH. Thus, we could argue the functioning (or even the existence) of an A. nidulans pacC homologue in N. crassa. However, we cloned an N. crassa pacC homologue by using degenerate oligonucleotides to amplify genomic fragments by PCR (~93% identity to the A. nidulans PacC protein) and to generate probes to screen a genomic library. One of the recovered sub-clones of 6 Kb (~40% sequenced) complemented the pacC14 strain of A. nidulans, including the remediation of both the Pi-repressible acid and alkaline phosphatases secreted at pH 5.0. Thus, pacC14 behaved as a loss-of-function mutation in a clear indication that N. crassa and A. nidulans have similar adaptive responses to ambient pH.
Financial support: FAPESP, CNPq, CAPES
23. Neurospora in nature: Exploring the natural history of a traditional laboratory model.
Amy J. Powell1, David J. Jacobson2, and Donald O. Natvig1. 1Biology, University of New Mexico, Albuquerque, New Mexico, USA.
2 Stanford University, Stanford, California, USA.
Although thousands of Neurospora isolates exist from worldwide collections, fundamental questions remain about the natural history of Neurospora. Clearly, species of Neurospora have evolved to respond to cyclical fires, because they appear profusely on vegetation that has been killed by recent fire, and ascospores are induced to germinate by either heat or chemical byproducts of burnt vegetation. However, important questions remain only partially answered: To what extent are colonization and dispersal dependent on conidia versus ascospores? Do different conidial eruptions on a single stem represent clonal propagation of a single genetic individual or multiple ascospore-derived individuals? How do sympatric species partition the environment? We are addressing these questions with isolates acquired after Florida Everglades fires in the spring, 1999. This collection includes multiple isolates from single plants and plants across transects. Our results confirm that individual stems of sugar cane and woody shrubs can be colonized by multiple species of Neurospora. Of 35 isolates examined from three stems, three (9%) were Neurospora crassa, and 24 (69%) were self-fertile Neurospora tetrasperma individuals. The remaining eight (22%) are still being identified. The conidial tufts representing different species were indistinguishable in the field and in some cases were only centimeters apart. Within- and between-species variation among isolates is currently being assessed by sequence analysis of specific genes.
24. Mutants of Neurospora altered in the adaptation to blue light.
Luis M. Corrochano and Charles Yanofsky. Biological Sciences, Stanford University, Stanford, CA, USA.
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: the mycelium adapts to light and a period of darkness is required before con-10 can be photoactivated again. To investigate the molecular nature of light adaptation in Neurospora, we have designed a protocol that should allow us to isolate mutants altered in the adaptation of con-10 photoactivation. A strain of Neurospora with a fusion of the con-10 promoter to the gene conferring resistance to hygromycin is available. 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 introduced into the genome of this strain a fusion of the con-10 promoter to the lacZ gene. The presence of this reporter fusion allows us to screen hygromycin resistant mutants for lacZ expression, which should reveal whether a mutation conferring resistance acts in cis or in trans. Using this strain we have isolated three mutants that grow in the presence of hygromycin under continuous light but not in the dark. Presumably this is due to a defect in the mechanism controlling light adaptation. One of these mutants has been investigated in some detail. This strain exhibits high expression of the con-10::lacZ fusion, suggesting that the mutation affects a trans-acting protein that is required for the photoadaptation of con-10.
25. Light sensitivity of the circadian clock and regulation of a clock controlled gene.
Minou Nowrousian, Jay C. Dunlap, and Jennifer J. Loros. Genetics and Biochemistry, Dartmouth Medical School, Hanover, NH, USA.
The Neurospora crassa circadian clock controls many aspects of growth and development, including the timing of the initiation of conidiogenesis. A core feedback loop of the clock consists of an autoregulatory cycle, central components of which are the frq, wc-1, and wc-2 gene products. The clock is able to react to environmental factors such as light and temperature. Continuous light represses clock function by supressing the cycling of frq mRNA and FRQ protein. The lis (light insensitive) mutants express the circadian rhythm of conidiation under dim light conidtions where wild type strains conidiate continuously. FRQ protein levels are low in the lis-2 mutant in dim light whereas they are induced to high levels in the wild type in response to constant light. Thus, it may be that the circadian oscillator is altered in some manner in the mutant or, alternatively, perception of light levels may be changed. The molecular clock acts by controlling the expression of many genes (ccgs, clock controlled genes, Loros et al., Science 243: 385-388) involved in different aspects of the physiology of the fungus. Several genes have been shown to be controlled by the clock at the level of transcription, among them ccg-1 (Loros and Dunlap, Mol Cell Biol 11: 558-563). Besides clock control, ccg-1 displays other levels of regulation, including light, development, and heat shock. Deletion analysis of the ccg-1 promoter sequences suggests that the clock regulatory elements lie near the start site of transcription and are distinct from sequences conferring developmental regulation and glucose repression.
26. Biochemical interactions between FRQ and WC-2: critical clock proteins required for the normal operation of the Neurospora circadian oscillator.
Deanna L Denault, Jay L. Dunlap, and Jennifer J. Loros. Genetics and Biochemistry, Dartmouth Medical School, Hanover, NH,USA.
The White Collar proteins (WC) are PAS domain containing proteins required for blue-light perception and essential for normal operation of the circadian clock in Neurospora crassa. Specifically, both WC-1 and WC-2 are necessary for rhythmic expression of a central clock component, frequency (frq). Although it is well established that the protein product FRQ negatively regulates it's own expression and that the expression of the frq gene requires WC-2, the molecular mechanisms governing both the activation and the feedback loop of frq are not well understood. We have shown that WC-2 is nuclear, consistent with its anticipated role as a transcriptional activator, and does not display a robust rhythm in either nuclear or cytoplasmic content. Current experiments set out to begin to determine the nature of the mechanism of WC-2 regulation of frq transcription. In independent experiments, it was found that WC-2 interacts directly with FRQ. In vitro, GST-tagged WC-2 specifically interacts with radiolabeled FRQ proteins. More importantly, in vivo, FRQ specifically co-immunoprecipitates with both WC-1 and WC-2 from Neurospora extracts. This demonstrates direct or indirect association between FRQ and the WC proteins. Thus, WC-2 may regulate rhythmic transcription of frq through protein-protein associations that affect transcriptional activation. Further characterization of this interaction may provide insight to the molecular events underlying the basis of the circadian clock in Neurospora.
27. Mutant screens to identify circadian clock components in Neurospora crassa.
Irene J. March, Victor V. Keasler, Kathyrn J. Wessels, and Deborah Bell-Pedersen. Biology, Texas A&M University, College Station, Texas, USA.
Circadian rhythms are intrinsic daily fluctuations in physiological, behavioral and biochemical activities that have been identified in organisms as diverse as cyanobacteria, plants, and mammals. In Neurospora crassa , conidiation is under control of the circadian clock. Previous genetic screens using mutant strains on race tubes have identified molecular components of the circadian clock. However, these screens were not saturating. Furthermore, no screens for mutations involved in the resetting of the clock have been carried out. In order to identify additional molecular components required for circadian rhythmicity and for resetting of the circadian clock, three genetic screens are being employed. In the first screen, UV mutagenized cultures of N. crassa are grown on race tubes and arrhythmic cultures are tested further. In the second screen, UV mutagenized cultures are grown on race tubes and given a light pulse 48 hours after they have been in the dark. In wild type strains, this results in a delay of the next cycle. Mutants that are arrhythmic, have altered periods, or that do not respond to the light pulse are candidates for further study. In the third screen, an amino acid permease is placed under control of the clock-controlled gene ccg-2, and mutants are selected based on their growth property, which is dependent on the circadian clock. Currently, 10,000 mutagenized strains have been tested in the first screen. From this, 17 arrhythmic mutants have been identified. In the second screen, 1,500 mutant strains have been tested, 4 arrhythmic strains and 1 possible light resetting mutant has been identified.
28. Identification of factors which regulate circadian rhythmicity of the clock-controlled eas(ccg-2) gene in Neurospora crassa.
Louis W. Morgan, Zachary Lewis, and Deborah Bell-Pedersen. Biology, Texas A&M, College Station, TX, USA.
Circadian rhythms are biochemical or physiological rhythms that persist in constant environmental conditions with a period close to 24 h. Viewed simply, the circadian clock is comprised of at least three components; a central oscillator, input pathways to and output pathways from the oscillator. The central oscillator regulates the expression of genes within the output pathways in a time-of-day-specific manner. However, the mechanisms and components responsible for the regulation of clock-controlled genes is poorly understood. We have identified a 68 bp promoter sequence, termed the ACE element, that is both necessary and sufficient for circadian expression of the clock-controlled eas (ccg-2) gene. Using gel mobility shift assays, we have identified protein factors that specifically bind to this sequence. Consistent with the factors being involved in clock-regulation of eas(ccg-2), binding to ACE occurs with a circadian period. Currently, we are purifying the factors and, once isolated, we will determine their role in circadian output pathways.
29. vvd encodes a novel PAS protein involved in light perception for the Neurospora circadian clock.
Christian Heintzen, Jay C. Dunlap and Jennifer J. Loros. Biochemistry, Dartmouth Medical School, Hanover, NH, USA.
The vivid (vvd ) locus has been characterized by mutations affecting light-induced pigmentation in Neurospora . We have cloned vvd and find that it is a new member of the PAS protein superfamily, the sequence of the VVD protein having strong homologies to the LOV domain of the Neurospora clock gene white collar-1 (wc-1 ). This gene is mutated in two Neurospora mutant alleles of vvd confirming that it encodes for the VVD protein. Physiological and molecular data indicate that VVD is involved in the light adaptation process of Neurospora crassa , which ensures a transient but not constitutive response of light regulated processes. The vvd gene itself is strongly and rapidly light induced. Moreover, it negatively autoregulates its own steady state levels. This indicates that VVD is an early repressor of light regulated processes. vvd , band (bd ) double mutants have a slightly longer free running period (23.5 h) than the clock wt, bd strains, and show differences in their phase response to light suggesting that - among other functions - vvd is important for the resetting mechanism of the Neurospora circadian clock.
30. Genetic mapping of the un-25 locus and ordering of the aro-6 and cpl-1 loci on linkage group VI.
Thomas J. Schmidhauser. Biology, U. of Louisiana, Lafayette, LA USA.
Complementation analysis has identified the locus un(T51M54) as a new un locus designated un-25. This temperature sensitive mutation has been shown to be tightly linked to the lys-5 locus; 0/44 recombination (Perkins et al., (1973, Neurospora Newsl. 20:45-49). Our results place un-25 between the cpl-1 and ylo-1 loci consistent with the tight linkage to the lys-5 locus. In addition we establish the order of the aro-6 and cpl-1 loci on the left arm of linkage group VI.
31. A chromosme walk on chromosome IV encompassing arg-2 and trp-4.
Sabine Mohr, John M. Stryker, and Alan M. Lambowitz. ICMB, University of Texas, Austin, Texas, USA.
The Neurospora mutant cyt-19-1 is deficient in mitochondrial splicing. The nuclear gene maps to chromosome IV. Three-factor crosses and crosses with translocation strains determined the order T(S4342)L,arg-14, cyt-19, T(NM152)L, pyr-3,his-5. We are presenting a chromosome walk between arg-2 and trp-4 with the position and partial sequence of several novel genes. A cyt-19 candidate gene has been found close to the arg-14 gene and is currently under investigation.
32. De novo cytosine methylation associated with recognition of A:T base pairs in vegetative cells of Neurospora crassa.
Hisashi Tamaru and Eric U. Selker. Molecular Biology, University of Oregon, Eugene, OR, USA.
Most cytosine residues in the genome of N. crassa that are subject to de novo methylation are in sequences that have undergone RIP and are therefore relatively A:T-rich and enriched for TpA, the most common dinucleotide resulting from RIP. To understand how DNA sequences signal de novo methylation we conducted systematic tests of the capacity of various synthetic oligonucleotides to trigger methylation in controlled sequence contexts at the his-3 locus. We show that various sequences consisting only of A and T can efficiently trigger methylation of nearby Cs, but to different extents. Both A and T are required on the same strand to induce significant methylation. Interestingly, both TpA and ApT can promote de novo methylation, but neither is essential. Moreover, the number of TpAs and/or ApTs in test fragments did not perfectly correlate with the levels of methylation induced. G:C pairs inhibit de novo methylation but to different extents depending on their position relative to TpAs and ApTs. Methylation is promoted by increases in the length of signal sequences. Using gel mobility shift assays we have identified DNA binding activities in cell extracts of both wild-type and methylation-defective strains that bind methylation signals. These results support the idea that a protein other than the DNA methyltransferase recognizes a feature of methylation signals and initiates the methylation process.
33. Purification of Neurospora crassa proteins that bind methylated DNA and DNA mutated by RIP.
Gregory O. Kothe, Michael R. Rountree, and Eric U. Selker. Molecular Biololgy, University of Oregon, Eugene, OR.
DNA methylation has been implicated in a diverse array of phenomena in eukaryotes such as genomic imprinting, X chromosome inactivation, and genome surveillance but a detailed understanding of the mechanism and function of methylation remains elusive. As one approach to better understand the mechanics and biological function of methylation we have chosen to isolate and characterize factors that bind to methylated DNA. Using gel-mobility-shift assays we have detected two factors in Neurospora crassa that bind to methylated DNA. A high-mobility factor was identified that is specific for methylated DNA. We refer to this factor as M-BP1 (Methyl Binding Protein 1). A low-mobility factor was identified that binds methylated DNA or DNA mutated by RIP. This factor binds most efficiently to DNA that is both methylated and contains mutations from RIP. We refer to this factor as M/R-BP1 (Methyl/RIP Binding Protein 1). M/R-BP1 may be involved in establishing methylation patterns in Neurospora. Both M/R-BP1 and M-BP1 may be involved in maintenance of methylation patterns as well as in exerting the effects of methylation. To test these possibilities we are purifying M/R-BP1 and M-BP1, characterizing their properties, and cloning the genes that encode them. After six chromatographic steps three polypeptides can be identified in silver-stained SDS-PAGE gels that correlate with M/R-BP1 activity.
34. Disruption of the Neurospora crassa histone 1 gene, hH1.
Michael Freitag1, H. Diego Folco2, Eric U. Selker1 and Alberto L. Rosa2. 1Inst. of Mol. Biology, University of Oregon, Eugene, OR, USA. 2University of Cordoba, Cordoba, Argentina.
The linker histone, H1, has long been thought to be an essential component of condensed chromatin and a general negative regulator of gene expression. Recent experiments in Xenopus (1), Tetrahymena (2), Saccharomyces cerevisiae (3), Aspergillus nidulans (4) and Ascobolus immersus (5) suggest that H1 has more specific roles. H1 appears to be nonessential in most of these organisms. In A. immersus, however, silencing of the H1 gene resulted in reduced lifespan as well as increased DNA methylation and increased sensitivity of the chromatin to micrococcal nuclease (MNase). To test the generality of these results, we cloned and inactivated the H1 gene of Neurospora crassa. A sequence for a Neurospora H1 homologue in an EST database (6) was used to isolate the full gene, which was then sequenced. Conceptual translation revealed a protein with bona fide H1 structure, i.e. highly basic N- and C-terminal tails flanking a core globular domain. A genomic fragment that contained the H1 gene, designated hH1, was targeted to the his-3 locus and this strain was used to disrupt the gene by RIP. Mutants with no detectable H1 were found, suggesting that there is only one H1 gene and that this histone is not essential in Neurospora. Mutants grew and developed normally at 10, 32 and 39C on minimal medium. No obvious changes in global DNA methylation or methylation at specific chromosomal sites was observed. Chromatin from the mutant showed normal sensitivity to digestion with MNase. The absence of an obvious phenotype in H1 mutants of Neurospora is consistent with findings with A. nidulans (4) and S. cerevisiae (3) but contrasts the findings with Ascobolus. (1) Bouvet, P. et al. (1994) Genes Dev., 8:1147-1159. (2) Shen, X. et al. (1995) Cell, 82:47-56. (3) Escher, D. and W. Schaffner (1997) Mol. Gen. Genet., 256:456-461. (4) Ramon, A. et al. (2000) Mol. Microbiol., 35:223-233. (5) Barra, J. et al. (2000) Mol. Cell. Biol., 20:61-69. (6) http://www.genome.ou.edu/fungal.html.
35. Processing of the arg-6 polyprotein.
Mazen W. Karaman and Richard Weiss. Chem. and Biochem., University of California, Los Angeles, Los Angeles, CA, USA.
The complex arg-6 locus in Neurospora crassa encodes a polyprotein precursor for two early mitochondrial arginine biosynthetic enzymes, acetylglutamate kinase (AGK) and acetylglutamyl phosphate reductase (AGPR). This polyprotein is processed into two mature proteins as it is translocated into the mitochondria. Processing involves cleavage of the polyprotein at three different sites. Cleavage of the first site, upstream of the proximal AGK, removes the mitochondrial targeting sequence. The two other sites are upstream of the distal AGPR, and cleavage removes a 20 amino acid region connecting both enzymes. The presence of a multi-protein coding gene is rare in eukaryotes; however, arg-6 homologues have persisted in the polyprotein form in N. crassa and two other fungi. This work investigates the reasons behind the presence of this polyprotein. This is done by introducing mutant constructs that either contain a separate AGK and AGPR genes, or an arg-6 gene that lacks the processing signal into a strain that contains a null arg-6 mutation. Each set of transformants is then evaluated for the production of the recombinant protein(s) and the ability to synthesize arginine.
36. Feedback inhibition of arginine biosynthesis.
Catherine A. McKinstry, Jessica Chung and Richard Weiss. Chem and Biochem, University of California, Los Angeles, Los Angeles, CA, USA.
Arginine biosynthesis is regulated primarily by feedback inhibition of acetylglutamate synthase (AGS) and acetylglutamate kinase (AGK). Previous genetic studies suggested a coordinate mechanism of inhibition mediated by interaction between AGS and AGK. Mutations in the gene for AGK (arg-6) have been shown to affect not only the activity and feedback sensitivity of AGK, but also of AGS. Direct interaction between these two enzymes has been shown in a yeast two hybrid system. We have used yeast two hybrid analysis to define the regions of interaction, and the effects of different mutations. The interaction domain of AGK maps to a unique C-terminal region found only in eukaryotic organisms. A mutation rendering AGK and AGS feedback insensitive was mapped to the N-terminal portion of AGK. Fusion proteins have also been made for the purification of the two enzymes, in order to further investigate the mechanism of feedback inhibition.
37. Interacting domains of Hsp30 of Neurospora crassa.
Nora S. Plesofsky and Robert M. Brambl. Plant Biology, University of Minnesota, St. Paul, MN USA.
Hsp30 of Neurospora crassa belongs to the class of alpha-crystallin-related small hsps, which share 90 amino acids of conserved sequence. These proteins have similar hydropathy profiles, and they self-assemble into multimeric particles, typically of 24 subunits. Crystallography suggests that a dimer is the core structural unit. We are characterizing the self-interactions of Hsp30 that contribute to its higher order structure, employing the S. cerevisiae two-hybrid system to report protein interactions in vivo. We measured the reporter beta-galactosidase activity by a sensitive luminescence assay. Monomers of Hsp30, co-expressed in transformed yeast cells, clearly interacted with one another, as expected for proteins that dimerize. We assayed portions of Hsp30 for interaction, dividing Hsp30 into an N-terminal (residues 1-134) and a C-terminal region (residues 129-228), both containing conserved sequence. The N-terminal half of Hsp30 interacted very strongly with the C-terminal half; this interaction is consistent with dimerization contacts within the small hsp crystal structure. We are continuing to define interacting domains within the N- and C-terminal halves of Hsp30 and have found that the conserved part of the C-terminal half is responsible for interaction with the N-terminal half. A truncated derivative of the N-terminal half, which contains the dimerization contact site of the crystal structure, retained binding to the C-terminal half.
38. Glyoxylase I of Neurospora is a stress-responsive enzyme.
Sanjeev Kalia and Manju Kapoor. Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Glyoxalase I (EC 22.214.171.124) catalyses the transformation of methylglyoxal and glutathione into S-lactoylglutathione. It is a highly conserved, ubiquitous protein, belonging to the superfamily of Zn-dependent hydrolases. It is a marker enzyme for cell division in eukaryotes and a potential therapeutic target for cancer and diabetes. Due to its ability to eliminate methylglyoxal, a cytotoxic byproduct of glycolysis, from the system, it is postulated to be a defense related enzyme. To determine whether glyoxalase I is a stress-responsive enzyme, 14-h-old mycelial cultures of wild-type N. crassa and an albino strain were exposed to various types of stress: heat shock, nutrient depletion, oxidative stress, toxic metal ions. These treatments increased the specific activity of this enzyme up to 3-fold, relative to untreated controls. In addition, elevated enzyme activity was also apparent during early stages of conidial germination and during formation/maturation of perithecia. These studies suggest that glyoxalase I of Neurospora is a stress-inducible and developmentally regulated enzyme.
39. Mapping and osmotic sensitivity of the mutants os-9 (allele SS-788 and allele SS-462) and SS-18.
Wayne A. Krissinger and Sara Neville Bennett. Biology, Georgia Southern University, Statesboro, GA, USA.
The osmotic-sensitive (os) mutants of Neurospora crassa fail to grow on medium containing elevated concentrations of NaCl. The first of these mutants (os-1 through os-5) were mapped to LG I and LG IV and could also be scored on the basis of their non-wild type morphology. In our laboratory, three osmotic-sensitive mutants, alleles SS-788, SS-462, and SS-18 were isolated following UV irradiation. The gross morphology of these mutants is like that of wild type. SS-788 has been designated os-9 and was mapped to LG VI between ad-1 and trp-2. Additional crosses placed os-9 distal to del SS-462 is an allele of os-9 as shown by the failure to recover wild type recombinants from a cross between the two mutant strains. SS-18 is in LG III linked by 36% recombination to vel and by 19% recombination to os-8 which was also isolated in our laboratory and is located between ad-2 and trp-1. We suggest that SS-18 be designated os-11. SS-788, SS-462, and SS-18, all grew as wild type 74 on media containing elevated KCl or glucose and failed to grow only on medium containing elevated NaCl. This is in contrast to the osmotic-sensitive mutant, os-1, which failed to grow on any of the three tested osmotica. These mutants may be useful in studies of transport of ions.
40. Expression of heat shock protein 70 (Hsp70) of Neurospora in E. coli.
Carol A. Curle and Manju Kapoor. Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
RT-PCR was used to generate two overlapping DNA fragments of the hsp70 gene, from total RNA, isolated from heat-shocked mycelium. These fragments (1.21 kb and 1.07 kb) were then spliced by overlap extension to yield a product ~2kb in size, representing the entire coding sequence of the hsp70 gene, without introns. The resulting fragment was inserted into the Pin Point Xa-3 (Promega) plasmid to produce a biotinylated fusion protein in E. coli. Following induction with IPTG, a biotinylated p
Poster Abstractswith specific antibodies raised against N. crassa Hsp70, was produced. This protein was affinity purified using the Soft-Link Soft-Release avidin resin. The yield of soluble protein was low, as most of the fusion protein accumulated in an insoluble form. The coding region of the hsp70 gene was also expressed as a poly-His-containing recombinant protein. A product of the correct size that cross-reacted with anti-Hsp70 IgG was obtained, but it was not over-expressed. A higher level of expression was achieved with the N- and C-terminal domains of the Hsp70 polypeptide.
41. Molecular cloning and regulatory analysis of the cystathionine beta- and gamma-lyase genes of Neurospora crassa.
Brad Reveal and John V. Paietta. Biochem. & Molec. Biology, Wright State University, Dayton, OH, USA.
The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur metabolism controlled by the CYS3 transcriptional activator and SCON (sulfur controller) negative regulators. We report here on the cloning and regulation of two additional genes under control of the system. Cystathionine beta-lyase coverts cystathionine into homocysteine, while cystathionine gamma-lyase converts cystathionine into cysteine. Northern blot analysis using wild type cells showed that the lyase transcripts were abundant under low sulfur (derepressing) conditions and at a low level under high sulfur (repressing) conditions. Northern analysis with a cys-3 deletion mutant showed low levels of the two lyase transcripts under either derepressing or repressing conditions. A negative regulatory mutant, scon-2 displayed constitutive levels of the lyase mRNAs. Finally, homology comparisions to other lyases will be described.
42. Fungal cell wall production and utilization as a raw resource for textiles.
William J. Dschida. MAD BIO Research Institute, Blue Lake, CA, USA.
The present invention reveals a product from fungi and a method for making the product. The method involves producing spores from a filamentous fungus, producing mycelia from the spores, growing the mycelia into a flat sheet, and recovering the fungus product. The fungus product is used as a raw resource for the production of textiles. (United States Patent 5,854,056)
43. Characterization of the paba-2 gene from Neurospora crassa.
Jason Edwards, Malcolm Robb and John P. Vierula. Biology, Carleton University, Ottawa, Ontario, CANADA.
Para-aminobenzoic acid (PABA) is a precursor to tetrahydrofolate, an essential vitamin for nucleic acid biosynthesis in microorganisms. Since humans ingest folates, this pathway has been an attractive antibiotic target in bacterial and more recently, some fungal infections. PABA biosynthesis is not well characterized in fungi, but it is believed to be similar to the scheme for bacteria. To aid in drug design, we have initiated a molecular genetic study of the PABA pathway in Neurospora crassa. The pab-2+ gene was cloned by complementation and cDNA clones were obtained by hybridization screening of the NO3- induced cDNA library and PCR amplification from various cDNA pools. The mutant allele of pab-2 was cloned via PCR. Sequencing of several pab-2+ cDNA clones revealed that the single intron is alternatively spliced. A common 5' splice junction and alternative 3' splice junctions give rise to C-terminal variant ORFs. The major transcript encodes a 276 amino acid ORF and the minor transcript, a 254 amino acid ORF. The pab-2 mutation is a frameshift/transition which results in a truncated, 208 residue protein.
44. Analysis of the Neurospora crassa opsin, NOP-1 and the opsin-related protein, ORP-1.
Jennifer A. Bieszke1, Donald O. Natvig2 and Katherine A. Borkovich1. 1Microbiology, University of Texas-Houston, Houston, TX, USA. 2 University of New Mexico Albuquerque NM USA.
Opsins are seven-transmembrane helical apoproteins that form light absorbing pigments upon binding retinal. Genes encoding opsins had only been identified in animals and the archaea until the discovery of an opsin gene, nop-1 in the filamentous fungus Neurospora crassa. The NOP-1 protein sequence is 81.1% identical to archaeal opsins in the retinal-binding pocket. Previously, we have shown that heterogously expressed NOP-1 in P. pastoris membranes could bind all-trans retinal (lambdamax = 534nm), and undergo a photochemical reaction cycle similar to archaeal rhodopsins. Also, we found the nop-1 gene expression during N. crassa development is highest under conditions that favor conidiation and is also positively influenced by the presence of light. A role for NOP-1 in conidiation was demonstrated by the light-dependent conidiation phenotype of delta nop-1 strains in the presence of the mictochondrial H+-ATPase inhibitor oligomycin. We have recently determined that delta nop-1 strains exhibit temperature-sensitive defects in the presence of light. Together, the phenotypic results suggests NOP-1 may have a light-dependent function in response to stress. Evolutionary analysis reveals clear similarity between NOP-1, archaeal rhodopsins, and another group of fungal proteins we have termed Opsin-Related Proteins (ORPs). ORPs are also predicted seven-transmembrane proteins, but they lack the conserved lysine residue required for retinal binding in opsins. Recently, we identified an EST encoding a N. crassa ORP (orp-1) in the University of Oklahoma Genome database. The putative ORP-1 protein sequence is most similar to HSP30 from Coriolus versicolor. Therefore, opsin-related proteins and opsins may be important for stress tolerance in N. crassa.
45. The Neurospora crassa mus-11 gene is a homologue of the S. cerevisiae RAD52 gene.
Alice L. Schroeder1, Yoshiyuki Sakuraba2 and Chizu Ishii2. 1School of Molecular Biosciences, Washington State Univ., Pullman, WA, U.S.A. 2Saitama University, Urawa, Saitama, Japan.
Mutagen-sensitive mutants with phenotypes resembling mutants defective in recombination repair fall into two epistasis groups in the filamentous fungus, Neurospora crassa. In the Uvs-6 group, the mei-3 gene has been shown to be a homologue of the E. coli Rec A and S. cerevisiae RAD51 genes, while the uvs-6 gene is a homologue of the S.cerevisiae RAD50 gene. Using complementation of MMS sensitivity, we have used sib selection with the Orbach/Sachs N. crassa cosmid library to clone mus-11 from the other (Uvs-3) recombination repair-like N. crassa epistasis group. The mus-11 gene is contained in a 3128 bp EcoRI fragment from cosmid X19:8E. Sequencing of the fragment shows that this DNA has a strong similarity to the coding regions of the RAD52 gene of S. cerevisiae and its Schizosaccharomyces pombe homologue, rad22. The gene has two introns: an 88bp intron 9bp after the apparent start codon and a second intron of 52bp at 663bp from the start codon. The area from -127 bp to -98bp preceeding the start codon has an AT rich sequence of AAATATTTTTTTTGAAAAAAGAAAAAAAAA. The 3' end of the genomic clone ends at an EcoRI site 2077bp from the presumed start codon. It is within the transcribed region, as a cDNA isolated by degenerate PCR from a UV induced N. crassa cDNA library has the same end. Fine mapping of the mus-11 gene places it on the right arm of LGV in the order: ade-7 - 9.5mu - pab-2 - 7.3mu - mus-11 - 3.7 - inv. A second gene in this group, uvs-3, has now been located on cosmid X18:7E of the Orbach/Sachs library.
46. Cloning and characterization of a RecQ homologue in Neurospora crassa.
Akihiro Kato, Yufuko Akamatsu, Yoshiyuki Sakuraba, and Hirokazu Inoue. Regulation Biology, Saitama University, Urawa, Saitama, Japan.
We cloned a Neurospora crassa homologue of RecQ using PCR method. The amino acid sequence of the homologue showed homology to that of other RecQ members within the helicase domain. We characterized a RecQ mutant which was made by RIP. This mutant was sensitive to MMS, MNNG and TBHP in spot test. These are the same characteristics shown by mei-3 and mus-23, which are mutant in genes affecting recombination repair. The mutant of Neurospora RecQ-homologue gene was not sensitive to UV, different from phenotypes of S. cerevisiae recQ-mutant, sgs1, and S. pombe recQ-mutant, rqh1. In homozygous crosses of the RecQ mutants, spore were not produced. This characteristic is also seen in mei-3 and mus-23. We are studying about the epistatic relationship between the RecQ- and mei-3-. It was shown by RFLP mapping that the RecQ homologue was located near the end of the right arm of LGI.
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