Showing papers in "Current Genetics in 1996"

Glutathione is an essential metabolite required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae.

Abstract: Glutathione (GSH) is an abundant cellular thiol which has been implicated in numerous cellular processes and in protection against stress caused by xenobiotics, carcinogens and radiation. Our experiments address the requirement for GSH in yeast, and its role in protection against oxidative stress. Mutants which are unable to synthesis GSH due to a gene disruption inGSH 1, encoding the enzyme for the first step in the biosynthesis of GSH, require exogenous GSH for growth under non-stress conditions. Growth can also be restored with reducing agents containing a sulphydryl group, including dithiothreitol, β-mercaptoethanol and cysteine, indicating that GSH is essential only as a reductant during normal cellular processes. In addition, theGSH 1-disruption strain is sensitive to oxidative stress caused by H2O2 and tert-butyl hydroperoxide. The requirement for GSH in protection against oxidative stress is analogous to that in higher eukaryotes, but unlike the situation in bacteria where it is dispensable for growth during both normal and oxidative stress conditions.

Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus

Abstract: Under limiting growth conditions,Aspergillus nidulans produces a carcinogenic secondary metabolite related to aflatoxin and called sterigmatocystin (ST). The genes for ST biosynthesis are co-ordinately regulated and are all found within an approximately 60-kilobase segment of DNA. One of the genes within this region is predicted to encode a CX2CX6CX6CX2CX6CX2 zinc binuclear cluster DNA-binding protein that is related to theAspergillus flavus andAspergillus parasiticus aflatoxin regulatory geneaflR. Deletion of theA. nidulans aflR homolog resulted in an inability to induce expression of genes within the ST gene cluster and a loss of ST production. BecauseA. nidulans aflR mRNA accumulates specifically under conditions that favor ST production we expect that activation of ST biosynthetic genes is determined byA. nidulans aflR. In support of this hypothesis, we demonstrated that induced expression of theA. flavus aflR gene inA. nidulans, under conditions that normally suppress ST gene expression, resulted in activation of genes in the ST biosynthetic pathway. This result demonstrates that AflR function is conserved betweenAspergillus spp. and thataflR expression is sufficient to activate genes in the ST pathway.

Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5'-decarboxylase gene, pyrG, as a unique transformation marker

Abstract: A 8.6-kb disruption cassette, referred to here as a pyrG-blaster and consisting of the Aspergillus niger pyrG gene flanked by a direct repeat that encodes the neomycin phosphotransferase of transposon Tn5 was constructed. Following transformation of a uridine/uracil auxotrophic pyrG strain of A. fumigatus, genomic insertions of the pyrG-blaster were obtained either by targeted gene replacement at the rodA locus, resulting in the formation of hydrophilic spores, or by ectopic integration. In both cases, recombination between the two elements of the direct repeat could be selected in the presence of 5-fluoro-orotic acid and resulted in the excision of the A. niger pyrG gene, producing A. fumigatus uridine/uracil auxotrophs that retained their additional mutant phenotype because of the persistence of one of the two elements of the direct repeat at the site of insertion of the pyrG-blaster. Selection for uracil/uridine prototrophy can therefore be used again to disrupt another gene.

Hairpins create minute inversions in non-coding regions of chloroplast DNA

Abstract: Minute inversions (4 bp in length), associated with probable hairpin secondary structures, were inferred from comparative analysis of rpl16 intron sequences from the chloroplast genomes of Chusquea species and related bamboos (Poaceae). The inverted sequences, which appear to have arisen independently on several occasions, comprise entire loops of the putative hairpins. The process of inversion seems dependent upon the stem length of the hairpin and its estimated free energy of formation. A similar inversion was uncovered for other plants in a previously published data set for a different non-coding region of the chloroplast genome, suggesting that the inversional process may be a common feature of non-coding DNA evolution. Several implications for phylogenetic analysis are noted.

The response regulator-like protein Pos9/Skn7 of Saccharomyces cerevisiae is involved in oxidative stress resistance.

Abstract: We have isolated mutants ofSaccharomyces cerevisiae with an increased sensitivity to oxidative stress. Allpos9 mutants (pos for peroxide sensitivity) were hypersensitive to methylviologene, hyperbaric oxygen or hydrogen peroxide, but grew similarly to the wild-type under all other conditions tested. Isolation and sequencing of the respectivePOS9 gene revealed that it was identical toSKN7. The predicted Skn7/Pos9 protein possesses a domain with high homology to prokaryotic response regulators. These regulatory proteins are part of a simple signalling cascade termed a “two-component system”, where a phosphorylation signal of a histidine kinase is transferred to a conserved aspartate residue of the response regulator. To test the functional role of the respective aspartate residue of Skn7/Pos9 protein in oxidative stress, we mutagenized this residue in vitro to alanine, arginine and glutamate. Only the glutamate allele (D427 to E) was able to rescue the hydrogen peroxide-sensitivity ofpos9 mutants. By fusion experiments with the Gal4 DNA-binding domain we identified the isolated response regulator-like domain as a novel eukaryotic domain sufficient for gene activation. Whereas this hybrid protein activated transcription of alacZ reporter gene under aerobic conditions, no activation was observed under anaerobic conditions, indicating that the response regulator domain is involved in a signalling reaction. Two-hybrid investigations also suggest an oligomerization of the Pos9 protein. Our results indicate that a two-component system is involved in the oxidative-stress response of yeast.

The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani.

Abstract: Four distinct laccase genes,lcc1, lcc2, lcc3 andlcc4, have been identified in the fungusRhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. TheR. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungusAspergillus oryzae. Two of the recombinant laccases, r-lccl and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66 kDa for lcc4 and 50–100 kDa for the recombinant lccl protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH 7, whereas lcc1 has optimal activity at pH 6.

Two new genes, PHO86 and PHO87, involved in inorganic phosphate uptake in Saccharomyces cerevisiae

Abstract: The PHO84 gene in Saccharomyces cerevisiae encodes a Pi transporter, mutation of which confers constitutive synthesis of repressible acid phosphatase (rAPase), in medium containing repressible amounts of Pi, and an arsenate-resistant phenotype. We selected an arsenate-resistant mutant showing the constitutive synthesis of rAPase on nutrient plates containing 4.5 mM arsenate. This mutant has double mutations designated as pho86 and pho87. The mutant transcribes PHO84 even in the repressible condition but has a severe defect in Pi uptake. The constitutive rAPase+ phenotype of the pho86 pho87 mutant was partially suppressed by an increased dosage of the PHO84 gene. The PHO87 gene was found to be identical with YCR524, according to the published nucleotide sequence of chromosome III, which encodes a protein of 923 amino-acid residues with a highly charged N-terminal half followed by a C-terminal half consisting of 12 membrane-spanning segments as in Pho84p. These and the other findings suggest that the Pho86p and Pho87p proteins collaborate with Pho84p in Pi uptake.

Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola

Abstract: Monospore isolates of Mycosphaerella graminicola considered to originate from one ascus were analysed by the polymerase chain reaction (PCR) with 32 RAPD primers. Eighteen of these revealed three classes of polymorphisms, which enabled a RAPD-based tetrad analysis. Four pairs of isolates resulting from a single diploid nucleus were determined. A procedure to cross these isolates was developed to investigate the mating system. Three of six crosses were successful, and the segregation of mating types in accordance with the tetrad analysis strongly points to a bipolar heterothallic mating system in M. graminicola. Random ascospore progenies from the successful crosses, each comprising 54 isolates, were studied with three primers to determine the mode of inheritance of the RAPD markers. Mendelian segregation and recombination of RAPD markers was observed in all progenies.

catA, a new Aspergillus nidulans gene encoding a developmentally regulated catalase.

Abstract: Aspergillus nidulans asexual sporulation (conidiation) is a model system for studying gene regulation and development. The CAN5 cDNA is one of several clones isolated based on transcript induction during conidiation. Here we present the molecular characterization of its corresponding gene, demonstrating that it encodes a developmentally regulated catalase, designated catA. The catA 744-amino-acid-residue polypeptide shows significant identity to other catalases. Its similarity to prokaryotic catalases is greater than to other fungal catalases. catA mRNA is barely detectable in growing mycelia, highly induced during sporulation, and present in isolated spores. However, catA expression is not dependent on the developmental regulatory genes brlA, abaA and wetA. Direct catalase activity determination in native gels revealed the existence of two bands of activity. One of these bands represented the major activity during vegetative growth and was induced during sporulation. The second catalase activity appeared after the induction of sporulation and was the predominant activity in spores. Disruption of catA abolished the major spore catalase without eliminating the vegetative activity, indicating the existence of at least two catalase genes in A. nidulans. catA-disrupted mutants produced spores that were sensitive to H2O2, as compared to wild-type spores. The increase in the activity of the vegetative catalase and the appearance of a second catalase during asexual sporulation is consistent with the occurrence of an oxidative stress during development.

Identification at strain level of Rhizoctonia solani AG4 isolates by direct sequence of asymmetric PCR products of the ITS regions.

Abstract: The relatedness of nine isolates ofRhizoctonia solani, belonging to anastomosis group (AG) 4, and one isolate of AG1 was determined by comparative sequence analysis based on direct sequencing of PCR-amplified ribosomal DNA [the internal transcribed spacer (ITS) region and the 5.8 s ribosomal DNA]. The 5.8s rDNA is completely conserved, but both ITS regions show variation among strains. AG1 was an outgroup based on anastomosis ability and RFLP analyses. Phylogenetic analyses based on the ITS sequences suggest that the analyzed AG4 strains can be divided into three groups that correlate with habitat and virulence.

The role of protein degradation in mitochondrial function and biogenesis

Abstract: It has been known for a long time that mitochondria contain their own protein-degradation systems. Only recently, however, have genes for mitochondrial proteases been identified and the powerful techniques of molecular biology been applied to gain insight into the role of protein degradation in mitochondrial biogenesis. It is now clear that the mitochondrial proteases that are involved in the initial stages of degradation are similar to prokaryotic ATP-dependent proteases, and that a division of labour exists between soluble and membrane-bound systems. These systems are essential for the biogenesis of fully functional mitochondria. Their natural targets are currently being identified, and their co-operation with chaperones and possible dual functions as chaperones/proteases are being investigated.

Molecular cloning and expression of the nag1 gene (N-acetyl-beta-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1

Abstract: A 72-kDa N-acetyl-β-D-glucosaminidase was purified from the mycoparasitic fungus Trichoderma harzianum P1; antibodies were raised against it, and aa-sequences were obtained. The antibody reacted with a single 72-kDa protein band in culture filtrates of T. harzianum grown on chitin, and was subsequently used to clone the corresponding nag1 gene from a λgt11 cDNA expression library. It was interrupted by two short introns and encoded a protein of 580 amino acids. The deduced protein sequence contained aa-sequence areas of high similarity to N-acetyl-glucosaminidases from other eukaryotes such as Candida albicans, and invertebrate and vertebrate animal tissues. The highest similarity was observed with the corresponding gene from the silkworm. The aa-sequence of a tryptic fragment of purified N-acetyl-β-D-glucosaminidase from T. harzianum corresponded to a deduced aa sequence from a portion of the cloned gene, thus verifying that the protein is encoded by nag 1. Southern analysis showed that nag 1 is present as a single-copy gene in T. harzianum. Expression of nag1-mRNA was strongly induced upon growth on chitin, N-acetyl-glucosamine and the cell walls of Botrytis cinerea used as a carbon source. The appearance of the corresponding N-acetyl-β-D-glucosaminidase protein, as determined by Western analysis, paralleled the pattern of nag 1 expression, thereby suggesting that its formation is regulated at the level of transcription.

The repair of DNA methylation damage in Saccharomyces cerevisiae

Abstract: The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways.

Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding α-glucosidase

Abstract: The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding alpha-glucosidase was determined. A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at -670 nt, -596 nt and -544 nt relative to the start codon, respectively. The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system. Deletion of the upstream half of Region III (IIIa; -544 to -529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction. Deletion of Region I and the downstream half of Region III (IIIb; -521 to -511) resulted in a significant reduction in GUS activity, but did not affect maltose induction. This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa. In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

The red/white colony color assay in the yeast saccharomyces cerevisiae : epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells

Abstract: In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells.

Phylogenetic affinities of the grasses to other monocots as revealed by molecular analysis of chloroplast DNA.

Abstract: The distribution of structural alterations of the chloroplast genome found in grass chloroplast (cp) DNA in comparison with that of tobacco was systematically surveyed in the cpDNAs of monocots. Southern hybridization and/or PCR analyses for the detection of (1) three inversions in the large single-copy region, (2) loss of an intron in therpoC1 gene, (3) an extra-sequence insertion in therpoC2 gene, (4) the deletion of ORF2280, (5) rearrangements of theaccD (ORF512) gene, and (6) non-reciprocal translocation of therpl23 gene, were carried out on cpDNAs isolated from 58 species, 22 families, and 11 orders, which covered almost all families of monocots. These structural alterations of cpDNA mostly occurred at the family level. However, only part of the Restionaceae possessed the inversion that characterizes the lineage of grass differentiation. The order of mutational events made it possible to reconstruct grass phylogeny in monocots. Since no variations in structural alterations of the cpDNA were found among the Poaceae, grass plants were inferred to have originated from an ancestor harboring these structural alterations of the chloroplast genome. These phylogenetic relationships were supported by the sequence data ofrbcL.

Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum.

Abstract: Plasmid vectors containing the AMA1 sequence transformed with high efficiency and replicated autonomously in Penicillium chrysogenum. The efficiency of transformation of P. chrysogenum was related to the length of the AMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats of AMA1 (the 2.2-kb SalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. The AMA1 region works as an origin of replication in P. chrysogenum and A. nidulans but not apparently in Acremonium chrysogenum.

Deletion of the Cochliobolus heterostrophus mating-type (MAT ) locus promotes the function of MAT transgenes

Abstract: Cochliobolus heterostrophus has alternate genes (MAT-1 and MAT-2) at its mating-type locus. Transformants of a MAT-1 or a MAT-2 strain carrying a transgene of opposite mating type can self and are dual maters; the transgene, however, promotes development of pseudothecia only, not ascospores. To determine if the resident gene interferes with the function of the transgene, transformation vectors were designed to delete different amounts (2.5 kb, 5.7 kb, and 6.3 kb) of DNA at the MAT locus. Deletions occurred at a higher frequency (about 90% of transformants) with linearized plasmid than with circular plasmid (about 15% of transformants), and all three vectors were equally efficient at gene replacement. Both MAT-1 and MAT-2 could be deleted with the same set of vectors. Re-transformation of deletion strains (regardless of deletion size) with a wild-type copy of MAT restored full mating ability, indicating that the resident MAT gene interferes with function of the MAT transgene. Moreover, sexual development was normal whether the MAT transgene integrated at the homologous or at an ectopic site.

Meiotic segregation and recombination of the intergenic spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor

Abstract: The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers The 45- and 40-kb fragments of IGS2 (haplotypes α and β, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei Recombinant monokaryons having both types of IGS2 occurred at a low frequency (65%; 60 kb per centimorgan) during meiosis Haplotypes α and β of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L bicolor S238N and indigenous populations of Laccaria spp in forest ecosystems

Meiotic behaviour of the minichromosome in the phytopathogenic ascomycete Leptosphaeria maculans

Abstract: All Leptosphaeria maculans field isolates displayed a minichromosome (MC) clearly separated from the overall electrokaryotype following pulsed-field gel electrophoresis. MCs exhibited a length polymorphism ranging from 650 to 950 kb. Tetrad analyses revealed the parental inheritance of MC length polymorphism (50% of the tetrads) or else the generation of novel-sized MCs (27%), which suggested that recombination occurred between MCs. Nineteen percent of the tetrads displayed a lack of the MC band in the electrokaryotype for one or two of the four resulting genotypes. Crosses between isolates carrying or lacking MCs revealed non-Mendelian segregation and suggested that some isolates could display at least two copies of the MC. Only repeated sequences hybridising to all chromosomes were isolated from the MC. Finally, saprophytic or parasitic fitness was not modified when isolates apparently lacked the MC. All these data suggested that the L. maculans MC behaves like a `B' chromosome.

Gnp1, the high-affinity glutamine permease of s. cerevisiae

Abstract: Glutamine uptake in S. cerevisiae is mediated by at least three transporters: high- and low-affinity glutamine permeases and the general amino-acid permease. We have isolated the gene encoding the high-affinity glutamine permease and named it GNP1. The amino-acid sequence of GNP1, and its hydropathy profile of 12 transmembrane domains, closely resemble those of known amino-acid permeases. The Km of GNP1 for glutamine uptake was determined to be 0.59 mM. Cells lacking GNP1 exhibit reduced levels of glutamine transport, and are resistant to a toxic analog of glutamine, L-glutamic acid γ-monohydroxamate. Unlike other amino-acid permeases, whose expression is nitrogen-source limited, GNP1 is expressed on both rich and poor nitrogen sources.

yAP-1- and yAP-2-mediated, heat shock-induced transcriptional activation of the multidrug resistance ABC transporter genes in Saccharomyces cerevisiae.

Abstract: We have examined whether the stress-induced transcriptional activation ofYDR1/PDR5/STS1 is mediated by yAP-1 and yAP-2. Of the stresses examined, heat shock-induced, rapid and transient PDR5 expression became very low in ayap1 yap2 double-gene disruptant, indicating that the yAP proteins mediate the response. Similar results were obtained withSNQ2, a close homologue ofPDR5. A set of 5′-truncation derivatives of thePDR5 gene identified the region from −484 to −434 as being sufficient for the response. A sequence similar to the yAP-1 recognition element recently identified in the stress-responsive yeast genes was found in this region and in the 5′-flanking sequences ofSNQ2.

UAG is a sense codon in several chlorophycean mitochondria.

Abstract: The mitochondrial genetic code of those land plants and green algae that have been examined does not deviate from the universal one. A red alga, Chondrus crispus, is the sole reported example throughout the algae that uses a deviant (non-universal) mitochondrial genetic code (UGA=Trp). We have analyzed 366-bp DNA sequences of the gene for mitochondrial cytochrome oxidase subunit I (COXI) from ten chlorophyceaen algae, and detected 3-8 in-frame UAG codons in the sequences of five species. Comparisons of these sequences with those of other algae and land plants have shown that most of the UAG sites in Hydrodictyon reticulatum, Pediastrum boryanum and Tetraedron bitridens correspond to alanine, and those of Coelastrum microporum and Scenedesmus quadricauda to leucine. The three species in which UAG probably codes for alanine are characterized by zoospore formation in asexual reproduction and form a clade in the COXI phylogenetic tree. The two species in which UAG codes for leucine are known to form daughter coenobia and pair in the tree. This is the first report on a deviant mitochondrial genetic code in green algae. Mutational change(s) in the release factor corresponding to UAG would be involved in these code changes. No genetic code deviation has been found in five other species examined.

Characterization of the Aspergillus parasiticus niaD and niiA gene cluster.

Abstract: The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus.

Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase gene SLT2(MPK1) rescued from yeast autolytic mutants.

Abstract: We have further characterized the functionality of theSaccharomyces cerevisiae geneSLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype oflyt2 mutants. Bothslt2A andlyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, theSSD1 allele being very significant in this regard. TheSLT2 allele of severallyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2Δ) andlyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect oflyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins.

The isoprenoid pathway: Cloning and characterization of fungal FPPS genes

Abstract: Farnesylpyrophosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis. Several classes of essential metabolites, including sterols, quinones, carotenoids and gibberellins, are terpenoids with high biological activity. The structural gene for FPP synthase was isolated from two ascomycete fungi, Neurospora crassa and Gibberella fujikuroi. A comparative analysis of the nucleotide sequences of both FPPS genes revealed the presence of introns at the same positions at the 5′ end of the coding regions. Furthermore, the most conserved region of the gene was isolated from two other plant pathogenic fungi, Sphaceloma manihoticola and Claviceps purpurea, by PCR. Sequence analysis showed a high degree of similarity between the deduced proteins of all known FPP synthase genes. In contrast to animals, all analyzed fungi contain a single copy of the gene, although FPP is the precursor for essential sterol and quinone biosynthesis and secondary metabolites, such as gibberellins, as well. Transcription analysis in different light regimes has shown that the FPPS genes in G. fujikuroi and N. crassa are not regulated by light induction.

Three genes for mitochondrial proteins suppress null-mutations in both Afg3 and Rca1 when over-expressed

Abstract: The AFG3 gene of Saccharomyces cerevisiae encodes a mitochondrial inner membrane protein with ATP-dependent protease activity. To gain more insight into the function of this protein, multi-copy suppressors of an afg3-null mutation were isolated. Three genes were found that restored partial growth on non-fermentable carbon sources, all of which affect the biogenesis of respiratory competent mitochondria: PIM1(LON) encodes a matrix-localized ATP-dependent protease involved in the turnover of matrix proteins; OXA1(PET1402) encodes a putative mitochondrial inner membrane protein involved in the biogenesis of the respiratory chain; and MBA1 encodes a mitochondrial protein required for optimal respiratory growth. All three genes also suppressed a null mutation in a related gene, RCA1, as well as in the combination of afg3- and rca1-null.

Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance

Abstract: 7-aminocholesterol has been described as being a strong inhibitor of yeast and of Gram+-bacteria proliferation. In order to determine the precise molecular target of the toxicity of this compound, we searched for yeast resistance linked to gene over-expression. We named the new yeast gene that was isolated RTA1 (EMBL X84736). This gene led to strong resistance to the inhibitor. Gene sequencing revealed that RTA1 is adjacent to the NAB1 gene which is orientated in an opposite direction and localized on chromosome VII. The RTA1 gene, which encodes a putative protein with seven potential membrane-spanning segments, was shown to be a non-essential gene in yeast.

Differentiation of the "Ascochyta complex" fungi of pea by biochemical and molecular markers.

Abstract: Isolates of three closely related pea pathogens,Ascochyta pisi, Ascochyta pinodes (teleomorphMycosphaerella pinodes) andPhoma medicaginis var. pinodella, were compared by means of isozyme analysis and restriction fragment length polymorphisms (RFLP) of amplified rDNA spacers. Three enzyme systems differentiatedA. pisi fromM. pinodes andP. m. pinodella. The internal transcribed spacers (ITSs) of the three fungi showed no intraspecific and very little interspecific variation after digestion with 12 endonucleases. Digestion of the intergenic spacer (IGS) withHinfI, andSau3A revealed uniformity inA. pisi patterns which consistently differed from those ofM. pinodes andP. m. pinodella. No clear distinction could be made between the latter two fungi which both showed intraspecific variability. Both biochemical and molecular markers thus discriminated between twoAscochyta species. The results also indicated a closer relationship between two organisms belonging to different genera (Ascochyta andPhoma) than between two species of the same genus (Ascochyta).

Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes

Abstract: The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when total cellular DNA was fractionated by PFGE Most of the mass of DNA isolated from mitochondria for one linear-mapping and one circular-mapping mitochondrial genome was found in linear molecules much larger than the genome size of 50 kb; some molecules were as long as 1500 kb, but only a trace amount of apparently circular mtDNA was found for the strain with the circular-mapping genome Probes for both the apocytochrome-b and mitochondrial small rRNA subunit genes hybridized strongly to mtDNA of approximately 50-100 kb, but weakly to the larger DNA from mitochondria of these two strains For the four linear-mapping strains, PFGE revealed two or three distinct bands of linear mtDNA, larger than the genome size, within a smear of approximately 50-100 kb, but a smear without bands was found for the circular-mapping strain