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At least 631 records · Page 35Linked to original sources
PubMed1995

A method for easy isolation of promoter fragments from promoter-probe libraries of filamentous fungi.

Two genomic fragments capable of driving the expression of the hygromycin B resistance gene (hph) were isolated from the phytopathogenic ascomycete Gibberella pulicaris (anamorph Fusarium sambucinum) using a "promoter-probe library" strategy. Two libraries consisting of random, 0.5-2.0-kb fragments of genomic DNA inserted 5' of a promoterless hph gene were constructed and used for transformation of G. pulicaris. Both libraries transformed G. pulicaris at a low frequency. Transformants tolerated up to 800 micrograms/ml of hygromycin B, while untransformed colonies were inhibited completely by 50 micrograms/ml of the antibiotic. Plasmids were re-isolated from transformants by simply digesting, the genomic DNA with KpnI, which cuts once in the polylinker 5' to the insert, and transforming E. coli with the re-ligated DNA. The recovered plasmids transformed G. pulicaris with a frequency of up to 4.4 transformants/micrograms of DNA. Both promoter fragments were sequenced and found to contain TATA and CAAT boxes as well as CT-rich sequences. This method makes it possible to easily isolate many fragments with promoter activity from filamentous fungi, and should facilitate the investigation of the promoter structures necessary for the expression of fungal genes.

Base Sequence↗
PubMed1990

The beta-tubulin gene of Epichloë typhina from perennial ryegrass (Lolium perenne).

Epichloë typhina is a biotrophic fungal pathogen which causes choke disease of pooid grasses. The anamorphic state, Acremonium typhinum, is placed in the section Albo-lanosa along with related, mutualistic, seed-disseminated endophytes. As an initial study of gene structure and evolution in Epichloë and related endophytes, the beta-tubulin gene, tub2, of the perennial ryegrass choke pathogen (EtPRG) was cloned and sequenced. The coding sequence and the predicted beta-tubulin amino acid sequence were highly homologous to the Neurospora crassa homologs, and to one of the two beta-tubulin genes of Emericella nidulans. However, two introns characteristic of the N. crassa and Em. nidulans genes were absent in the E. typhina gene. Furthermore, one of the remaining introns possessed the uncommon 5' splice junction, GC. In contrast to published observations concerning other Ascomycetes, a mutant of EtPRG, selected for resistance to methyl-2-benzimidazole carbamate (benomyl), possessed no alteration of its beta-tubulin coding sequence.

Amino Acid Sequence↗
PubMed1992

Characterization of the glyoxysomal isocitrate lyase genes of Aspergillus nidulans (acuD) and Neurospora crassa (acu-3).

The nucleotide sequences of the genes encoding the acetate-inducible glyoxylate cycle enzyme isocitrate lyase from the ascomycete fungi Aspergillus nidulans (acuD) and Neurospora crassa (acu-3) are presented. The respective A. nidulans and N. crassa genes are interrupted at identical positions by two introns and encode proteins of 538 and 543 amino acids, which have 75% identity. The predicted protein sequences do not demonstrate the C-terminal tripeptide S-K-L that has been implicated in peroxisomal targeting and found in the glyoxysomally located enzyme malate synthase from the same species. However, the protein sequences do exhibit a partial repeat which, in common with malate synthase, is located in regions that are absent from, or non-homologous with, the E. coli enzyme, which is not compartmentalized.

Amino Acid Sequence↗
PubMedDate not supplied

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans.

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.

Ascomycota↗
PubMed1991

Distribution of mitochondrial intron sequences among 21 yeast species.

The mitochondrial and nuclear genomes of 21 yeast species belonging to 12 genera have been tested for the presence of sequences similar to seven S. cerevisiae mitochondrial introns (Se cox1.1,2,3,4,5c, Sc cob.4 and Sc LSU.1) and one K. lactis mitochondrial intron (K1 cox1.2). Some introns, (Sc cox1.4, Sc cob.4, Sc LSU.1 and Kl cox1.2-all group I type), are widely distributed and are found in species with either basidiomycete or ascomycete affinities. This distribution is suggestive of recent sequence transfer between species. The remaining S. cerevisiae introns cross react with an additional species but with no set pattern. Pulsed field gel electrophoretic studies confirm that none of the tested mitochondrial introns cross react with nuclear DNA. These introns are, therefore, mitochondria-specific. Seven strains of K. lactis exhibit striking variability in intron content. In contrast to all mitochondrial introns tested, two introns of nuclear genes (the K. lactis actin gene and the S. cerevisiae RP29B gene) are not detected beyond their source species.

DNA, Fungal↗
PubMed1992

Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3.

Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.

Ascomycota↗
PubMed1995

Nucleotide sequence of the Schizosaccharomyces japonicus var. versatilis ribosomal RNA gene cluster and its phylogenetic implications.

Fission yeasts form a small but heterogeneous group of ascomycetes and it is still unclear whether they should be subdivided into three genera (Schizosaccharomyces, Octosporomyces, Hasegawaea) or remain a single genus (Schizosaccharomyces). In order to decide whether a new genus Hasegawaea should be established for the species Schizosaccharomyces japonicus and Schizosaccharomyces versatilis, we have characterized the entire rDNA cluster in Schizosaccharomyces japonicus var. versatilis and compared it with the homologous region from Schizosaccharomyces pombe and with complete rRNA gene sequences from other yeast genera. From a phage genomic library a recombinant lambda phage containing the entire rDNA repeat unit was isolated. In this paper we report the primary sequence of the 18s, 5.8s and 25s rRNA coding regions. The S. japonicus var. versatilis rRNA genes are 1823 (18s), 158 (5.8s) and 3422 (25s) nucleotides long. The two sequences of the larger rRNA genes exhibit 95.7% (18s) and 93% (25s) similarity with the homologous genes from S. pombe. The differences between the rRNA genes of S. japonicus and S. pombe, however, are much smaller than the intrageneric differences within the rDNA sequences of other yeast genera. Therefore, subdivision of fission yeasts into the genera Schizosaccharomyces and Hasegawaea does not to seem to be justified.

Base Sequence↗
PubMed1994

Genetic requirements for initiating asexual development in Aspergillus nidulans.

Conidiation in the filamentous ascomycete Aspergillus nidulans requires activation of brlA, a well-characterized transcriptional regulator of genes that are induced specifically during asexual development. We have isolated and characterized developmental mutations in six loci, designated fluG, flbA, flbB, flbC, flbD, and flbE, that result in defective development and reduced brlA expression. These mutants grow indeterminately to produce masses of aerial hyphae resulting in the formation of cotton-like colonies with a "fluffy" morphology. The results of growth and epistasis tests involving all pairwise combinations of fluffy mutations indicate complex hierarchical relationships among these loci. We discuss these genetic interactions and propose that there are multiple mechanisms for activating brlA.

Alleles↗
PubMed1985

Primary structure of the trpC gene from Aspergillus nidulans.

We have determined the structure and complete nucleotide sequence of the trifunctional trpC gene from the Ascomycetous fungus Aspergillus nidulans. Results from RNA gel blot analyses showed that this gene encodes two size classes of polyribosomal, poly (A)+RNAs with approximate lengths of 2,400 and 2,600 nucleotides. S1 nuclease protection studies demonstrated that the distribution into the two size classes is due to selection of alternative sites for polyadenylation. The transcription units contain a single open translation reading frame of 2,304 nucleotides. The sequence of this reading frame is approximately 40% divergent from the sequence of the functionally analogous trp-1 gene from Neurospora crassa (Schechtman, M.G. and Yanofsky, C., J. Mol. Appl. Gen. 2:83-99). The predicted amino acid sequence of the A. nidulans trpC polypeptide is also 40% divergent from the predicted amino acid sequence of the N. crassa trp-1 polypeptide. The A. nidulans gene has considerably less bias in codon selection than observed for the N. crassa gene. Discrete regions of DNA homology were also found in similar positions in the 5' and 3' flanking sequences of the Aspergillus and Neurospora genes. Similar regions of homology were not observed in other Aspergillus or Neurospora genes that have been sequenced. Thus, if these evolutionarily conserved sequences act as signals for transcription initiation or polyadenylation, or are involved in gene regulation, their functions are restricted to a subset of protein coding genes in these two closely related fungi.

Amino Acid Sequence↗
PubMed1991

Linear DNA plasmids of the perennial ryegrass choke pathogen, Epichloë typhina (Clavicipitaceae).

Epichloë typhina is a clavicipitaceous ascomycete which systemically infects grasses, causes choke disease of host inflorescences, and is related to a group of mutualistic grass endophytes. Three plasmids of 7.5, 2.1 and 2.0 kilobase pairs were found in mitochondrial DNA preparations of an E. typhina isolate from perennial ryegrass (Lolium perenne). Results of nuclease digestion indicated that the plasmids, designated Et7.5L, Et2.1L, and Et2.0L, were linear, double-stranded DNAs with protein linked to their 5'-ends (plDNA). The plasmids shared little or no homology with each other, and were not integrated into the mitochondrial or nuclear genomes. No homologous plasmids were detected in isolates of E. typhina from other grass hosts, anamorphic endophytes, or other Clavicipitaceae. However, other plasmids were present in Balansia obtecta and Claviceps purpurea. A partial sequence of one of the E. typhina plasmids, Et2.0L, indicated an open reading frame when UGA was assumed to encode tryptophan. The inferred amino acid sequence had 24% identity over 258 amino acids in two regions of the reverse transcriptase encoded by the circular Mauriceville and Varkud plasmids of Neurospora spp. The homologies included six segments conserved in RNA template-dependent DNA or RNA polymerases.

Amino Acid Sequence↗
PubMed1991

The linear plasmid pMC3-2 from Morchella conica is structurally related to adenoviruses.

pMC3-2, one of two linear plasmids localised in the mitochondria of the ascomycete Morchella conica, was completely sequenced. It is 6044 bp in size, contains terminal inverted repeats of 713 and 710 bp length and two open reading frames, ORF1 and ORF2, spanning 2706 bp and 918 bp, respectively. ORF1 probably encodes a viral B-type DNA-polymerase. Concerning ORF2, no homology to any other published protein- or DNA-sequence could be detected. According to the structure of DNA-polymerases, linear plasmids can be grouped into two classes reflecting their localisation either in the cytoplasm or within the mitochondria. In general, the structure of plasmid pMC3-2, as well as of other linear plasmids from filamentous fungi, indicates a close relationship of these genetic elements to adenoviruses.

Adenoviridae↗
PubMed1992

Transcripts and translation products of a mitochondrial plasmid of Claviceps purpurea.

Expression of the linear mitochondrial (mt) plasmid pClK1 of strain K of the ascomycete Claviceps purpurea was studied at the RNA and protein level. Using strand-specific probes two major transcripts were detected, corresponding to ORF1 and ORF2 of the plasmid, which most likely code for a DNA-polymerase and an RNA-polymerase, respectively. Primer extension experiments showed that both transcripts start at identical positions from opposite sides within the terminal inverted repeat (TIR). The initiation sequence corresponds to the proposed general mt initiation consensus-sequence. Conserved parts of the putative polymerase ORFs were expressed in an E. coli system and used to prepare antisera. In Western experiments the presence of corresponding proteins was demonstrated in a strain carrying plasmid pClK1, whereas a plasmid-free strain lacked these polypeptides. The sizes of these proteins are in good accordance with the sizes derived from the coding capacity of ORF1 and 2.

Base Sequence↗
PubMed1981

Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans. II. Identification of the effector binding protein.

In this publication we report the identification of a protein likely to be coded by uaY, a regulatory gene in the ascomycete Aspergillus nidulans. uaY is a positive control gene necessary for the expression of at least eight unlinked structural genes involved in purine uptake and degradation (Scazzocchio and Gorton 1977). The physiological effector of the uaY system is uric acid, while some of its thioanalogs serve as gratuitous inducers. Effector binding proteins were detected by binding to 2-thiouric acid after phosphocellulose column chromatography, or as uric acid binding fractions after DNA-cellulose column chromatography. Two binding peaks are present in mycelial extracts purified by either method. These are missing in a putative small deletion of the uaY gene. A "leaky" mutation, uaY109 described in detail elsewhere (Scazzocchio et al. 1980) shows only one peak. The wild type peaks eluted at 55 mM NaC1 and at 720 mM NaCl while the peak present in uaY109 is eluted at 120 mM NaC1. This implies that at least one peak represents a protein coded by the uaY gene. The major peak was analysed by equilibrium dialysis experiments. These establish a Kdiss approximately 2 X 10(-7) and a minimum number of binding sites of 3 X 10(-14) moles/mg of soluble protein in a crude extract derived from protoplast lysis. An extract from a strain carrying the uaY207 deletion, purified blind, lacks any binding activity in the equilibrium dialysis cell.

Aspergillus nidulans↗
PubMed1988

Structural and functional analysis of mitochondrial plasmids in Claviceps purpurea.

Several strains of Claviceps purpurea, a phytopathogenic Ascomycete, contain mitochondrial (mt) plasmids in high molar excess relative to mtDNA. Comparative analysis of plasmids of four strains of different geographic origin revealed that all the plasmids are structurally related (size; linearity; restriction map; probably 5'-linked terminal protein; terminal inverted repeats, TIRs); two of them are even identical, indicating a possible mobility of these genetic entities. In strain K it was shown that plasmid titres are comparably high in axenic cultures and in parasitic structures (sclerotia). Detailed analysis of plasmid pClK1 proved the existence of a perfect TIR of 327 bp; the plasmid's structure and details of its nucleotide sequence indicate a replication modus comparable to that of adenoviruses. pClK1 is almost completely transcribed resulting in two major transcripts of 3.5 and 3.15 kb, respectively. In plasmid-free strains (cured by ethidium bromide treatment) these mRNAs are not detectable; nevertheless they show no significant difference in phenotype. As judged from their structural properties they could be derived from viral ancestors. In this context the plasmids' close relationship to mt plasmids of higher plants may be of special interest.

Adenoviridae↗
PubMed1994

The phylogeny of the tomato leaf mould fungus Cladosporium fulvum syn. Fulvia fulva by analysis of rDNA sequences.

The nucleotide sequence of part of the ribosomal DNA from races of the fungal tomato pathogen Cladosporium fulvum and other Cladosporium species have been determined. Comparisons of the internal transcribed spacer regions (ITS1 and ITS2) of several C. fulvum races showed complete sequence homology suggesting a recent evolutionary divergence. Comparisons of these nucleotide sequences in the ITS region with those of other Cladosporium species showed the close relationship within the Cladosporium genus. Using the nucleotide sequence of part of the 18s ribosomal subunit from these isolates and comparing them with sequences of some Ascomycetes, Basidiomycetes and Chytridiomycetes, obtained from GenBank, we infer the phylogeny of the Cladosporium species studied here. Our analysis shows that the Cladosporia form a monophyletic group which falls within the order Ascomycotina.

Base Sequence↗
PubMed1992

Expression of Neurospora crassa laccase under the control of the copper-inducible metallothionein-promoter.

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. In vegetatively growing cultures its biosynthesis is repressed but can be induced by different protein synthesis inhibitors. Transformation of the N. crassa wild-type strain Singapore with a fusion gene consisting of the N. crassa copper-metallothionein promoter and the laccase gene are described in this report. Correct integration of the 3.6 kilobase (kb) promoter-fragment fused with the laccase gene containing a 5' consensus region leads to copper-dependent expression of the enzyme during the vegetative growth phase. The enzyme is glycosylated and secreted, and high amounts of extracellular activity can be detected. The regulation of laccase biosynthesis of one examined transformant, followed at both the transcriptional and the translational level, indicates co-induction of both copper-metallothionein and laccase. The data presented show that expression of the recombinant laccase gene is exclusively regulated by the transformed N. crassa metallothionein-promoter.

Blotting, Southern↗
PubMed1994

Variations in ploidy among isolates of Botrytis cinerea: implications for genetic and molecular analyses.

Field isolates and laboratory strains of Botrytis cinerea, an ascomycetous fungus causing considerable economic losses, e.g., as "grey mould" of vine, were compared for differences in ploidy level by determining their DNA content per nucleus. Strain SAS56, an ascospore line used routinely for genetic analyses, is probably polyploid, since treatment with benomyl causes a significant reduction in DNA content per nucleus. This conclusion is substantiated by the increased sensitivity of the putative haploid derivatives to mutagens (UV and EMS). Molecular analyses (RAPD) of the haploidized strains indicate a very limited degree of heterozygosis of the parent strain SAS56. Analysis of field isolates of B. cinerea showed that their DNA content per nucleus varied considerably, indicating that aneuploidy/polyploidy is a widespread phenomenon in this species. This can explain both the variability and phenotypic instability of many field isolates of this fungus and the unusual difficulties faced by researchers in recovering stable recessive laboratory mutants. Since the haploid derivatives of SAS56 resemble the parent strain in their parasitic and physiological properties they should provide a good basis for classical and molecular genetic studies.

Base Sequence↗
PubMed1992

Transformation of Acremonium coenophialum, a protective fungal symbiont of the grass Festuca arundinacea.

Acremonium coenophialum is a mutualistic mycosymbiont and natural agent of biological protection of the widely distributed grass Festuca arundinacea (tall fescue). An electroporative transformation system was developed for A. coenophialum. Segments of DNA 5' to the beta-tubulin gene (tub2) of the closely related ascomycete Epichloë typhina, fused to the Escherichia coli hph gene encoding hygromycin B phosphotransferase, conferred hygromycin resistance when introduced into A. coenophialum by electroporation. The incorporation of the Emericella nidulans trpC terminator greatly increased protoplast germination on selective medium and improved transformation efficiencies 30-200% depending on the plasmid construct. Plasmid pCSN43, which incorporates the trpC controlling elements for hph expression, was also used to transform A. coenophialum. Southern blot analysis of ten pCSN43 transformants indicated the possibility of random integration of this vector into the genome.

Acremonium↗