Neurospora posters

119. Phenotype of novel complex I mutants of Neurospora.

T. Almeida, M. Duarte, A.M.P. Melo and A. Videira. University of Porto, Portugal.

Respiratory chain complex I is an assembly of more than 30 polypeptide subunits located in the inner mitochondrial membrane. It couples electron transfer from NADH to ubiquinone to proton translocation across the membrane. The enzyme is constituted by two domains, the peripheral and membrane arms, that are assembled independently of each other. We are using the technique of repeat-induced point-mutations in order to inactivate specific complex I genes. We have isolated two mutant strains of N. crassa that lack two subunits bearing [Fe-S] clusters of the peripheral arm of complex I, a 24 kDa and a 21 kDa protein respectively. The former is apparently able to assemble an almost intact complex I while formation of the peripheral arm of the enzyme in the latter strain cannot be detected. In addition, lack of either protein seem to prevent the progression of N. crassa through the sexual phase of the life cycle. Further characterisation of the mutants will also be presented.

120. Neurospora NADP-glutamate dehydrogenase - mutational amino acid replacements and three-dimensional structure.

Ian F. Connerton and Angel Fuentes (Inst, of Food Res., Reading); John Fincham (Edinburgh Univ.); K. Yip, Tim Stillman and David W. Rice (Sheffield Univ.).

Numerous mis-sense am mutants have been characterised over the years in terms of their effects on GDH properties and their complementation relationships. Most of the corresponding amino acid replacements have been defined, initially by peptide sequencing but now mostly by DNA sequencing. Following the recent X-ray crystallographic analysis carried out in Sheffield on wild-type GDH, it becomes possible to position the mutant amino acid replacements accurately within the three dimensiona1 structure of the hexameric enzyme, and suggest reasons for their various effects on enzyme phenotype.

121. Signals for de novo cytosine methylation of DNA in Neurospora crassa.

Michael Freitag, Vivian Miao, Brian Margolin and Eric Selker, University of Oregon, Eugene.

Several Neurospora DNA sequences have been identified that act as portable signals for de novo cytosine methylation. "Signal DNA" typically contains C:G to T:A mutations introduced by repeat-induced point mutation. Consequently, such signal DNA has a higher number of T and A nucleotides and a higher density of TpA dinucleotides than unmutated Neurospora DNA. To identify critical characteristics of signal DNA we compared the level of de novo methylation induced by various mutated or unmutated Neurospora DNA, mammalian DNA or bacterial DNA that had been integrated into the Neurospora genome either at random or by targeting to the his-3 or am loci. To specifically test whether the number of T+A nucleotides or the density of TpA dinucleotides are important parameters for the induction of de novo methylation we further dissected one portable methylation signal, the - (zeta-eta) region, and assayed the ability of several short DNA fragments to induce de novo methylation.

122. Inositol 1-phosphate synthase of Neurospora crassa.

Barbara Hanson, Michelle Mathis, Barbara Stengel and Stephen J. Free, Canisius College and State University of New York,

Buffalo, NY.

Inositol 1-phosphate synthetase (IPS) is the enzyme that catalyzes the first step in the synthesis of inositol, a key growth factor and component of the phosphatidylinositol cycle in Neurospora. The DNA sequence of the inositol 1-phosphate synthase was determined from a clone that complemented a Neurospora inositol mutant (inl 89601). This clone was first isolated by Akins and Lambowitz (1985. Mol. Cell. Biol. 5:2272-2278). The DNA sequence containing the IPS gene in the clone was identified by its similarity to other IPSs in plants and other fungi. The IPS gene contained 1,614 base pairs that appeared to code for a protein containing 537 amino acids with a molecular weight of 58826.4. The amino acid composition of the protein indicated that 50.7% of the amino acids were hydrophobic. Comparison of the Neurospora IPS gene to other IPS sequences indicated that this sequence showed a higher homology to plants and algae rather than to other fungi such as Saccharomyces and Candida. Sequence data base searches of amino acid sequence databases available at the National Center for Biotechnology Information, NIH were done using programs based on the BLAST algorithm. The organisms with sequences that were most similar to Neurospora were Brassica napus, M crystallinum, and Arabidopsis. The sequences for IPS from Candida and Saccharomyces, two fungi, showed segment pairs that were about 25% less similar than the plants to Neurospora.

123. Mechanisms of polyamine regulation of ornithine decarboxylase synthesis in Neurospora crassa.

Martin A. Hoyt, Janet L. Ristow, and Rowland H. Davis, University of California, Irvine.

Ornithine decarboxylase (ODC), encoded by the spe-1 gene of Neurospora crassa catalyzes the initial and rate-limiting step in the synthesis of the polyamines (putrescine, spermidine, and spermine). Ornithine deprivation, leading to polyamine starvation is imposed upon aga (arginaseless) mutants by growth on arginine. In response, ODC activity and spe-1 mRNA abundance increase about 12 fold.. The polyamine-mediated regulation of spe-1 mRNA abundance relies on two mechanisms. First, spermidine, the major cellular polyamine, and sequences in the long spe-1 5'-untranslated region, act together to repress 6 fold the synthesis, processing, or nuclear export of spe-1 mRNA. Deletion analysis shows that this sequence does not affect the stability of the mRNA, which is intrinsically unstable. The second mechanism is a 2fold enhancement of this instability, imparted by putrescine addition, and probably targeted on the 3'-untranslated region of the mRNA.

124. Cloning and characterization of a Neurospora crassa MutS mismatch repair homolog.

D.H. Huber¹, G. Hausner¹, B. Seidel-Rogol², H. Bertrand¹. ¹ Michigan State University, East Lansing, and ²State University of New York, Plattsburg.

Mismatch repair systems are involved in the faithful transmission of genetic information between generations by recognizing and correcting base pair mismatches in DNA. Genes encoding proteins homologous to the E. coli MutHLS repair system have been identified in several organisms. Mutations in mismatch repair genes produce mutator phenotypes. We have cloned and are characterizing a gene from N. crassa encoding a homolog of the E. coli MutS protein. The N. crassa MSH gene (mut S homolog) was identified using PCR with degenerate oligonucleotide primers based upon highly conserved regions found in MutS, the homologous yeast proteins, MSH1 and MSH2, and mammalian MSH protein Two cosmids were isolated from the Orbach/Sachs library using Southern hybridization with the cloned PCR product. Four subclones were then obtained from the cosmids that hybridized to the PCR probe. The deduced amino acid sequence from the region of MSH amplified by PCR is most similar to the corresponding sequence of the yeast MSH2 protein. The remainder of the gene is currently being sequenced. MSH maps to LG VII using the Metzenberg RF map.

125. Characterization of calcium and magnesium uptake in the vacuole of Neurospora crassa.

Kelly A. Keenan, Thomas Kirn, and Thomas Wisniewski, Richard Stockton College of New Jersey.

Calcium and magnesium uptake in the vacuole of Neurospora crassa was examined using cupric ion permeabilization system. The distribution of calcium and magnesium was examined and it was shown that 43.6 % of calcium and 79.5 % of magnesium was associated with the vacuole. A cellular fractionation experiment yielded the same distribution. When grown in minimal medium, the total pools were 0.17 ug/mg protein for calcium and 16.3 ug/mg protein for magnesium. Vacuolar uptake of calcium and magnesium was examined. Both metals were transported in a manner consistent with Michaelis-Menten kinetics and the Km values were 0.97 mM for Ca⁺² and 0.45 mM for Mg⁺², Specificity studies indicate that there is a common permease for these two metals. The effect of pH on uptake was examined and was optimal at pH 6.0 for both metals. A series of mutants was isolated that had altered levels of calcium and magnesium pools. Vacuolar transport of both metals was examined and was shown to be reduced in two of the mutants.

126. Isolation and characterization of vacuolar basic amino acid permease mutants in Neurospora crassa.

Kelly A. Keenan, Jonathan Tayco and Zeljka Basic, Richard Stockton College of New Jersey.

Uptake of the basic amino acids into the vacuole of Neurospora crassa was examined using a cupric ion permeabilization system. The Km for lysine uptake was 1.4 mM and 11.1 mM for ornithine. Specificity studies suggest that there are distinct permeases for these two amino acids and that the lysine permease is inibited by arginine. The kinetic effect of arginine on lysine uptake was examined. A filtration enrichment procedure was used to isolate mutants in the basic amino acid vacuolar permeases. Vacuolar uptake was measured using a cupric ion permeabilization system. The mutants were grouped genetically according to complementation results and two groups were obtained. The first contains fourteen members and most of the mutants in this group failed to take up all three basic amino acids. Members of this group had a reduced level of vacuolar calcium transport. Two mutants are in complementation group 2 and these failed to take up ornithine but arginine and lysine uptake was normal. Vacuolar calcium uptake was normal. Mutants from both complementation groups have a visibly altered vacuolar morphology. Vacuolar ATPase activity was measured in members of both complementation groups.

127. Cloning and Characterization of the aod-2 alternative oxidase regulatory gene of Neurospora crassa .

Tak Ko and Helmut Bertrand, Michigan State University.

Wild-type Neurospora crassa contains two respiratory pathways: the normal cytochrome pathway and the alternative pathway that is insensitive to cyanide and antimycin A, but is inhibited by hydroxamic acids. The alternative pathway diverges from the cytochrome pathway after the ubiquinone pool and is not linked to oxidative phosphorylation. The alternative pathway can be induced by treatment with chloramphenicol, oligomycin, or antimycin A. It is also induced in mutants which are deficient in b- and a-type cytochromes. The induction of alternative-oxidase activity requires two genes, aod-1, which encodes the alternative oxidase and aod-2, which is involved in the regulation of the aod-1 gene on the transcriptional level. The aod-1 gene is located on linkage group IV and has been recently cloned. The aod-2 gene is located in linkage group II, 13.9 map units to the right of thr-3 and 3 map units to the left of arg-5. We are attempting to clone the aod-2⁺ DNA in two different ways: complementation of an aod-2 mutant using aod-1 promoter-driven expression of a reporter gene, in this case the bacterial hygromycin resistance gene as a method for selecting transformants and chromosome walking. Cloning and characterization of the aod-2 gene which is known to affect the transcription of aod-1, will provide information about the possible mechanisms of communication between mitochondria and the nucleus.

128. Site-directed mutagenesis of the N-terminal domain (of F-Box)of the SCON-2 negative regulator leads to a novel regulatory phenotype.

Anuj Kumar and John V. Paletta, Dept. of Biochem. & Mol. Biol., Wright State University, Dayton, OH.

In the filamentous fungus Neurospora crassa, sulfur metabolism is controlled through an intricate multigene network which includes the sulfur regulatory genes cys-3+ (which encodes a BZIP transcriptional activator) and the negative regulatory sulfur controller gene scon-2+. Sequence analysis of the SCON2 protein has revealed several striking protein motifs. Specifically, SCON2 contains an N-terminal domain subsequently identified within a number of eukaryotic regulatory proteins including the yeast cell-division-cycle protein Cdc4p and the Cdk regulatory subunit cyclin F. To assess the functional significance of this N-terminal domain (which has been recently renamed the F-box), we have undertaken an extensive mutational analysis. Site-directed mutagenesis of key residues within the F-box results in sulfur auxotrophy, with mutants unable to grow in the absence of a supplemental sulfur source such as methionine. In addition to this N-terminal domain (F-box), SCON2 also contains six repeated GP-homologous domains (or WD-40 repeats) spanning the C-terminal half of the protein. Previous studies have suggested a possible role for both the F-Box and P-transducin motifs in the mediation of protein-protein interactions. To address the possibility that SCON2 may function as part of a multiprotein complex, we have generated rabbit polyclonal anti-SCON2 antibody which is currently being used to characterize SCON2 binding properties in N. crassa cellular extracts.

129. Cloning of Neurospora crassa genes defective in DNA methylation.

Elena Kuzminova, Shan Hays and Eric Selker, University of Oregon.

Methylation of cytosines is the most common modification of DNA in eukaryotic organisms. The amount and distribution of methylated cytosines in the genome varies among eukaryotes. Most methylated sequences from Neurospora show RIP-like mutations. How the methylation patterns are established is not known. The biological role of methylation in Neurospora crassa is yet to be understood, since the dim-2 methylation mutant, which completely lacks methylation in vegetative tissues, does not confer phenotypic changes in vegetative or sexual cycles. To understand the molecular basis for the lack of methylation in this mutant we are attempting to clone the dim-2 gene. To this end we have mapped dim-2 between wc-1 and un-10 on LGVII. Now we are trying to clone dim-2 by chromosome walking from these two markers.

We have developed a mutagenesis protocol to identify more genes involved in cytosine methylation. We chose insertional mutagenesis because it yields mutants in which the mutated genes are marked, making them easier to clone. Since known Neurospora methylation mutants have no selectable or easy scorable phenotype per se, we are taking advantage of a hph (hygromycin B phosphotransferase) allele that is silenced by methylation in the dim+ strain, leaving the strain sensitive to the drug hygromycin B. Subsequent to the mutagenesis, selection on hygromycin B allows only Hph+ (and thus Dim-) transformants to grow.

130. Temperature regulted alternative initiation of frequency protein mediates an aspect of temperature compensation in the Neurospora circadian clock.

Yi Liu, Norman Garceau, Jennifer Loros and Jay C. Dunlap. Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH.

It has been shown that the frequency (frq) gene is a central component of the Neurospora circadian clock. The circadian oscillator in Neurospora is comprised of an auto regulatory feed back loop in which the amount of frq transcript is negatively regulated by its product, FRQ protein. Both frq message and FRQ protein oscillate with a period which is the same as that of the conidiation banding rhythm (Aronson et al., Science 263:1578-84; Dunlap, Annu. Rev. Genetics 30:579-601, 1996).

Based on DNA sequence information, frq transcripts were predicted to encode a single 989 aa protein. In this study, we found that there is an alternative initiation of FRQ translation: two FRQ forms, FRQ1-989 and FRQ100-989, are expressed as a result of alternative initiation from two of three in-frame initiation codons. Furthermore, we found that the ratio of two FRQ forms is modulated by temperature: high temperature favors the initiation of FRQ1-989, whereas at low temperature, FRQ100-989 is favored. In mutant strains lacking AUG#1, the circadian rhythm is lost at high temperature, whereas strains without AUG#3 became arrhythmic at low temperature. Our data also shows that this temperature-dependent synthesis of mutiple forms of FRQ reflects both quantitative and qualitative requirements for FRQ by the clock. Different amounts of FRQ are required at different temperatures and the two FRQ forms are also functionally distinct, so that an optimal clock requires both FRQ forms. Together, this represents a novel adaptive mechanism which allows Neurospora to keep its clock running over a wide range of temperatures.

131. Signal transduction proteins of Neurospora crassa.

Peter Margolis, Yanofsky Lab, Stanford University.

In response to environmental stress, vegetative cells of N. crassa can enter onto either of three developmental pathways. Recent work suggests that this process can be considered as a problem in signal transduction. I show here that Neurospora indeed possesses proteins homologous to those that control signaling in other systems. I have identified, using PCR with degenerate oligonucleotide primers, at least 13 putative Neurospora signal transduction genes, including some capable of encoding putative Ras proteins and members of MAP kinase cascades. The cloning of genomic DNA corresponding to these PCR-generated fragments will permit the mutagenesis (via RIP and marker replacement) and phenotypic characterization of these loci. Initial work has focused on a third N. crassa ras homolog (ras-3) that could encode a novel class of Ras protein [e.g. unusually large (34 kD), lacking a standard CAAX C-terminus, distinguishing sequence motifs].

132. Analysis of Neurospora ropy genes encoding novel proteins required for normal hyphal growth and nuclear distribution

Peter Minke, John Tinsley, In Hyung Lee, and Mike Plamann. Department of Biology, Texas A&M University, College Station, TX.

We have begun investigating the molecular mechanisms underlying hyphal growth and nuclear movement in filamentous fungi by studying a class of morphological mutants of Neurospora crassa called ropy. Ropy mutants exhibit a phenotype of curled, rope-like hyphal growth and abnormal nuclear distribution. Three ro genes have been found to encode subunits of either the microtubule-associated motor cytoplasmic dynein or the associated dynactin (dynein activator) complex. This report focuses on five additional genes which encode novel proteins. ro-7 is predicted to encode a 70 kD protein distantly related to actin. In a ro-7 mutant, cytoplasmic dynein heavy chain and p150Glued, the largest subunit of dynactin, accumulate at nuclear spindle pole bodies suggesting that RO7 is required for proper intracellular targeting of cytoplasmic dynein and dynactin. ro-10 is predicted to encode a novel 24 kD non-essential protein and may be required for stability of dynactin as p150Glued is not detectable in a ro-10 deletion strain. ro-11 is predicted to encode a novel 75 kD non-essential protein that contains large regions of predicted coiled-coil structure with a highly basic C-terminal region. Intracellular location of dynein and dynactin is not disrupted in a ro-11 deletion strain. ro-2 and ro-JT2 are predicted to encode novel proteins.

133. Genetic interactions among the Neurospora crassa circadian clock mutants.

Louis W. Morgan and Jerry F. Feldman, University of California, Santa Cruz 95064.

We have identified a series of epistatic and synergistic interactions among the circadian clock mutants of Neurospora crassa that indicate possible interactions among the various clock components encoded by these genes. prd-6, a new short period temperature-sensitive clock mutant, is epistatic to both prd-2 and prd-3 and the specific nature of the epistasis suggests a direct physical interaction between the prd-6, prd-2, and prd-3 proteins. prd-2 and prd-3, both long period mutants, show a synergistic interaction in that the period length of the double mutant strain is considerably longer than the expected value based on the period lengths of the single mutants. prd-2, prd-3, and prd-6 also show significant synergism with frq⁷, a long period allele of the frq locus, previously shown to play a central role in the Neurospora clock. In contrast, double mutants involving other clock mutations do not show either epistasis or synergism with each other or with the mutants listed above. These results suggest that the gene products of prd-2, prd-3, and prd-6 play an important role in clock function and will shed light on the specific role of frq in clock organization.

134. An 88-kDa heat shock protein interacts with Neurospora crassa hsp30.

Nora Plesofsky-Vig and Robert Brambl, University of Minnesota, St. Paul.

We have identified proteins that interact specifically with hsp30 of Neurospora crassa. Affinity resins of recombinant hsp30, linked to either an Affi-Gel or a nickel resin, were incubated with soluble extracts from heat-shocked N. crassa cells. A 70-kDa and an 88-kDa protein were found to bind to the hsp30-affinity resin, but not to control resins. The 70-kDa protein was identified as hsp70 by N-terminal sequencing. For the 88-kDa protein, the N-termini of several tryptic peptides were sequenced. The encoding transcript, detected in Northern blots, is present at normal temperature, but is very strongly induced by heat shock. A 2.6 kbp cDNA was isolated from an N. crassa cDNA library and sequenced. The predicted amino acid sequence shows homology to hsp70 and especially to a recently identified class of proteins that includes mammalian hsp110, human hsp70RY, yeast SSE1/2, and Arabidopsis hsp91, which are distantly related to hsp70. The cDNA contains the entire coding region for hsp88 by structural criteria and the peptide's alignment with homologous proteins. This interaction of hsp70 and hsp88 with hsp30 may assist or regulate the activities of hsp30.