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At least 613 records · Page 34Linked to original sources
PubMed1989

A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae.

We have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae. According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids. A consensus sequence of a signal peptide consisting of 21 amino acids is found at the N-terminus of the prepro region. The primary structure of the mature region shares extensive homology (29%-44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN') composing the active site are preserved. The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.

Amino Acid Sequence↗
PubMed1979

Isolation and genetic analysis of nuclease halo (nuh) mutants of Neurospora crassa.

Nuclease halo (nuh) mutants of the ascomycete Neurospora crassa have been isolated which are characterized reduced release of deoxyribonuclease (DNase) activities from colonies grown on sorbose-containing agar media. To identify nuh mutants, mutagenized isolates were transferred to commercial DNase test agar, or grown on minimal medium and then overlayed with agar that contained heat-denatured DNA. DNase activity was visualized by acid precipitation which produced clear rings of digestion (haloes) around the colonies. To identify the number of genes in which mutations lead to reduced release of nuclease activity, eleven nuh mutants were checked for close linkage and linked pairs were tested for complementation. These mutants were assigned to eight genes, and all except one were mapped in six small regions of the Neurospora linkage maps. In addition, among a large number of existing mutants which were tested for nuclease haloes, two mutants were found that showed the Nuh phenotype, namely uvs-3 and uvs-6. One of the isolated nuh mutants was also found to be sensitive to UV and was mapped close to uvs-3; it may represent a new allele of this gene. As a first step towards identification of genuine nuclease mutants, extensively backcrossed strains of mutants from different genes have been assayed for nuclease activity with denatured DNA in extracts. A pronounced reduction, compared to wild type at the same stage of growth, was found in uvs-3 and also in nuh-3, a mutant that is not UV-sensitive.

Chromosome Mapping↗
PubMed1992

Regulatory sequences for expressing genes in oomycete fungi.

Promoter and terminator sequences from a range of species were tested for activity in the oomycetes, a group of lower fungi that bear an uncertain taxonomic affinity to other organisms and in which little is known of the sequences required for transcription. Transient assays, using the reporter gene beta-glucuronidase (GUS), were used to examine the function of these promoters and terminators in the plant pathogens Phytophthora infestans and P. megasperma f. sp. glycinea, and in the saprophytic water mold, Achlya ambisexualis. Oomycete promoters, isolated from the ham34 and hsp70 genes of Bremia lactucae and the actin gene of P. megasperma f. sp. glycinea, resulted in high levels of GUS accumulation in each of the three oomycetes. In contrast, little or no activity was detected when promoters from higher fungi (four ascomycetes and one basidiomycete), plants, and animals were tested. The terminator from the ham34 gene resulted in much higher levels of GUS accumulation than did others, although an oomycete terminator was not absolutely required for expression. Transcript mapping of RNA from stable transformants confirmed accurate initiation from the B. lactucae hsp70 promoter and termination within 3' ham34 sequences in P. infestans. Our results indicate that the transcriptional machinery of the oomycetes differs significantly from that of the higher fungi, but that enough conservation exists within the class to allow vectors developed from one oomycete species to be used for others.

Base Sequence↗
PubMed1992

The Ncypt1 gene from Neurospora crassa is located on chromosome 2: molecular cloning and structural analysis.

Small GTP-binding proteins are encoded by ras-like genes and play a central role in cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the Ascomycete fungus Neurospora crassa with Zmypt genes from Zea mays we have isolated a member of the ypt gene family, Ncypt1. The gene resides on a 4 kb fragment of genomic DNA and contains four introns, which interrupt the coding sequence of a protein of 203 amino acid residues. The Ncytp1 gene was assigned to a single-copy gene encoding a transcript of 1.5 kb and a protein of 26,000 daltons. The gene maps on linkage group IIR between DB0001 and ccg-2 close to the Fsr-3 locus. Analysis of the nucleotide sequence and the deduced protein sequence revealed a striking homology to yeast, mouse and human genes encoding small GTP-binding proteins that are related to the ras supergene family. Homology was most significant to ypt1 from Schizosaccharomyces pombe, Mus musculus and Homo sapiens sharing 84.8%, 82.3%, and 82.3% identity, respectively. Common domains present in other small GTP-binding proteins were identified in the predicted sequence of the NCYPT1 protein, and the arrangement of peptide motifs sharing similarity with well characterized, small GTP-binding proteins suggests that the NCYPT1 protein is a GTPase. The C-terminal region extending from amino acid residues 175 to 199 shares only weak amino acid sequence similarity with other eukaryotic GTPases. Like other RAS proteins the NCYPT1 protein contains two conserved C-terminal cysteine residues, suggesting post-translational modification(s) by fatty acylation required for membrane anchoring. The high degree of homology between the NCYPT1 protein and eukaryotic YPT proteins suggests that NCYPT1 could be involved in the control of secretory processes.

Amino Acid Sequence↗
PubMed1992

The regulatory gene nit-2 of Neurospora crassa complements a nnu mutant of Gibberella zeae (Fusarium graminearum).

The nnu mutant of Gibberella zeae (=Fusarium graminearum) is unable to catabolize many of the nitrogen sources utilized by its wild-type parent, and may have suffered a mutation in the major nitrogen regulatory locus. Transformation of this mutant with the major nitrogen regulatory gene from Neurospora crassa, nit-2, restored the wild-type phenotype, thus confirming that the nnu mutation is in the major nitrogen regulatory locus of G. zeae. Our results are consistent with the premise of conservation of the structure of regulatory factors and suggest the possibility that functional DNA homologues of this regulatory element occur across a broad range of ascomycetous fungi.

DNA, Fungal↗
PubMed1993

A homologous gene-reporter system for the basidiomycete Schizophyllum commune based on internally deleted homologous genes.

Problems encountered in our attempts to achieve expression of heterologous genes, driven by ascomycetous regulatory sequences, in homobasidiomycetes led us to develop a gene-reporter system based on the expression of homologous genes in Schizophyllum commune. Internal deletions were made in the coding sequences of the regulated Sc4 gene and the constitutively expressed GPD gene. After introduction of these constructs into S. commune it was found that the expected truncated transcripts were produced. The internally deleted Sc4 gene, containing 1140bp of upstream and 200 bp of downstream sequences, was only expressed in dikaryons at the time of fruiting (as was the resident Sc4 gene) but not at all in monokaryons, indicating that the construct contained all regulatory sequences necessary and sufficient to confer control by the mating-type genes and expression during fruiting. The internally deleted GPD gene, containing 1300 bp of upstream and 150 bp of downstream sequences, was expressed both in monokaryons and dikaryons at levels similar to those of the resident GPD gene, indicating that all sequences necessary for proper expression were present. This reporter-gene system may be applicable to the analysis of cis-regulatory sequences of these genes. Furthermore, heterologous genes may be inserted into the well-expressed GPD deletion construct to obtain expression of such genes in S. commune and possibly in other homobasidiomycetes.

Blotting, Northern↗
PubMed1993

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus.

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from a MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 bp and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat-, and Saccharomyces cerevisiae MAT alpha 1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.

Amino Acid Sequence↗
PubMed1992

The acu-1 gene of Coprinus cinereus is a regulatory gene required for induction of acetate utilisation enzymes.

We have isolated a gene from Coprinus cinereus which cross-hybridises to the facA and acu-5 genes of Aspergillus nidulans and Neurospora crassa, respectively. These genes encode acetyl-CoA synthetase, an enzyme which is inducible by acetate and required for growth on acetate as sole carbon source. We have designated the C. cinereus gene acs-1 and have used transformation to demonstrate its functional homology to the ascomycete genes by complementation of an N. crassa acu-5 mutation. The acs-1 gene has never been identified by mutation; mutations leading to loss of acetyl-CoA synthetase function map to another gene, acu-1. Using Northern analyses we have shown that acu-1 has a regulatory function that is required for acetate-induced transcription of acs-1 and of another acetate utilisation gene, acu-7, the isocitrate lyase structural gene.

Acetates↗
PubMed1993

Characterization of an aspartic proteinase of Mucor pusillus expressed in Aspergillus oryzae.

The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1-2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo-beta-N-acetylglucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.

Aspartic Acid Endopeptidases↗
PubMed1975

The synaptonemal complex and the spindle plaque during meiosis in yeast.

Meiosis in Saccharomyces cerevisiae proceeds principally in the same manner as in other Ascomycetes. Leptotene is characterized by unpaired lateral components and pachytene by the presence of extensive synaptonemal complexes. The synaptonemal complex has the same dimensions and is similar in structure to those described for other organisms. Chromosome counts can now be made by reconstructing the synaptonemal complexes. Diplotene nuclei consistently contain a single polycomplex. The behaviour, doubling and the fine structure of the spindle plaque provide additional markers for the different stages of meiosis.

Cell Nucleolus↗
PubMed1991

Codon usage in Aspergillus nidulans.

Synonymous codon usage in genes from the ascomycete (filamentous) fungus Aspergillus nidulans has been investigated. A total of 45 gene sequences has been analysed. Multivariate statistical analysis has been used to identify a single major trend among genes. At one end of this trend are lowly expressed genes, whereas at the other extreme lie genes known or expected to be highly expressed. The major trend is from nearly random codon usage (in the lowly expressed genes) to codon usage that is highly biased towards a set of 19-20 "optimal" codons. The G + C content of the A. nidulans genome is close to 50%, indicating little overall mutational bias, and so the codon usage of lowly expressed genes is as expected in the absence of selection pressure at silent sites. Most of the optimal codons are C- or G- ending, making highly expressed genes more G + C-rich at silent sites.

Aspergillus nidulans↗
PubMed1995

Isolation and characterisation of genes for sulphate activation and reduction in Aspergillus nidulans: implications for evolution of an allosteric control region by gene duplication.

A region of the Aspergillus nidulans genome carrying the sA and sC genes, encoding PAPS reductase and ATP sulphurylase, respectively, was isolated by transformation of an sA mutant with a cosmid library. The genes were subcloned and their functions confirmed by retransformation and complementation of A. nidulans strains carrying sA and sC mutations. The physical distance of 2 kb between the genes corresponds to a genetic distance of 1 cM. While the deduced amino acid sequence of the sA gene product shows homology with the equivalent MET16 gene product of Saccharomyces cerevisiae, the sC gene product resembles the equivalent MET3 yeast gene product at the N-terminal end, but differs markedly from it at the C-terminal end, showing homology to the APS kinases of several microorganisms. It is proposed that this C-terminal region does not encode a functional APS kinase, but is responsible for allosteric regulation by PAPS of the sulphate assimilation pathway in A. nidulans, and that the ATP sulphurylase encoding-gene (sC) of filamentous ascomycetes may have evolved from a bifunctional gene similar to the nodQ gene of Rhizobium meliloti.

Adenosine Triphosphate↗
PubMed1995

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina.

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wat, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5' ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extra-chromosomal genetic traits.

Ascomycota↗
PubMed1995

Molecular phylogeny of the fungi of the Iceman's grass clothing.

To investigate the origin of the fungal hyphae that cover the grass clothing (cloak, boots) found near the neolithic mummy known as the Tyrolean Iceman, two radiocarbon-dated samples of grass were submitted to DNA extraction. The DNA was then PCR amplified using, respectively, primers specific for the region containing the internal transcribed spacers and the 5.8s rDNA (ITS), and primers specific for an approximately 600-bp long fragment of the nuclear small-subunit ribosomal DNA (SSU rDNA) repeat units of eukaryotes. The amplification products were cloned and sequenced. Sequence analysis of 20 individual ITS clones and of ten SSU rDNA clones indicated that three types of fungal DNA can be extracted from the grass. Phylogenetic analyses, using 5.8s and SSU rDNA fungal reference sequences from EMBL and GenBank databases, suggest that the DNAs come, respectively, from a psychrophilic basidiomycetous yeast, phylogenetically close to Leucosporidium scottii, and from two ascomycetes, one of which is possibly related to the Eurotiales.

Ascomycota↗
PubMed1993

Isolation and transformation of uracil auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Uracil auxotrophs of Phanerochaete chrysosporium were isolated using 5-fluoroorotate resistance as a selection scheme. The ura3 auxotrophs deficient in orotidylate decarboxylase and ura5 auxotrophs deficient in orotate phosphoribosyl transferase were characterized by enzyme assays and complementation tests. The ura5 auxotrophs were transformed to prototrophy with the ura5 gene from the ascomycete Podospora anserina. The ura3 auxotrophs were transformed to prototrophy with the ura3 gene from the basidiomycete Schizophyllum commune. The P. chrysosporium ura3 gene was isolated from a lambda EMBL3 genomic library using the S. commune ura3 gene as a probe. A 6.6-kb fragment incorporating the ura3 gene was subcloned into Bluescript SK+(pURA3.1) and used to transform P. chrysosporium ura3 auxotrophic strains. The pURA3.1 insert was mapped for restriction sites and the approximate location of the ura3 gene within the insert was determined. Double auxotrophic strains were transformed with either of two marker genes and the resulting single auxotrophic strains were crossed to demonstrate genetic recombination between two nuclei of identical genetic background.

Ascomycota↗
PubMed1990

Primary and secondary structure of the 25S rRNA from the dimorphic fungus Mucor racemosus.

A 9.76 Kb ribosomal DNA repeat unit from the nuclear genome of the dimorphic fungus Mucor racemosus (Zygomycetes) was identified using a hybridization probe from the yeast Saccharomyces cerevisiae (Ascomycetes). This material was cloned in Escherichia coli plasmids as four overlapping pieces and mapped with respect to cleavage sites for 12 restriction endonucleases. The nucleotide sequence of the complete 25S rRNA gene and flanking regions was determined. The 5' and 3' ends of the structural gene were identified by comparison with the published sequence for the S. cerevisiae gene. The Mucor gene was found to possess 3469 bp and have a GC content of 42.8%. It was compared with the homologous gene from several other eukaryotes and found to be most similar to that from Saccharomyces. A potential secondary structure of the putative RNA transcript consistent with the structures proposed for the E. coli and Saccharomyces molecules was constructed by computer modelling.

Base Sequence↗
PubMed1995

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis.

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. macrospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora.

Ascomycota↗
PubMed1995

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum.

A 1,380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.

Amino Acid Sequence↗