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The phenoloxidases of the ascomycete Podospora anserina. XIII. Action and interaction of genes controlling the formation of laccase.

1. Eight mutants were isolated following mutagen treatment which are deficient in laccase formation. Seven of these had a pleiotropic effect and exhibited defects in growth rate and in mycelial and sexual morphology. 2. By means of tetrad analysis the mutations were assigned to 6 loci. Three mutations were in the incolora locus, the others were non-allelic. Only two of these loci were closely linked. 3. All genes exhibit numerous interactions. These concern the morphological expression of the laccase genes and also the laccase spectra. 4. The mutants could be separated into four classes on the basis of the amount and type of laccase produced. 5. Five of the loci studied appear to be structural genes because mutations alters the physical properties of the laccase protein. The sixth gene has a regulatory role.

Ascomycota↗

Genes inhibiting senescence in the ascomycete Podospora anserina.

Senescence occurs in all wild strains of Podospora anserina after continued growth. This syndrome can be inhibited by a synergistic interaction of two linked genes, incoloris and vivax. Whereas the wild strain starts to become senescent after 26 d and the mutants incoloris and vivax after 42 and 66 d respectively, the double mutant shows no signs of aging after culture for more than one year.

Ascomycota↗

Nitrate reductase of the ascomycetous fungus, Leptosphaeria maculans: gene sequence and chromosomal location.

The nitrate reductase (niaD) gene was isolated from the phytopathogenic loculoascomycete Leptosphaeria maculans by screening a genomic DNA library with the Aspergillus nidulans niaD gene. The L. maculans niaD gene is the first protein-encoding gene characterised from this fungus. It encodes a predicted protein of 893 amino acids and contains four putative introns at positions in the gene equivalent to those of four of the six introns in the A. nidulans niaD gene. Mutants defective in niaD and molybdenum cofactor gene(s) of L. maculans have been isolated. Transformation of a L. maculans niaD mutant with a 3.8 kb SacII fragment containing the L. maculans niaD gene restored wild-type growth on nitrate as a sole nitrogen source. The niaD gene is present as a single copy on a chromosome which ranges in size from 2.6 to 2.8 Mb between the different L. maculans isolates examined.

Amino Acid Sequence↗

Both open reading frames of the linear plasmid pMC3-2 from the ascomycete Morchella conica are transcribed in vivo.

Mitochondrial RNA was isolated from the morel strain Morchella conica 3 harbouring the linear plasmid pMC3-2 and subjected to gel electrophoresis followed by a Northern analysis using cloned fragments of the plasmid pMC3-2 as probes. Hybridization was obtained only with central parts of pMC3-2 and specific bands of mtRNA. The hybridization bands (2.8 kb and 1.0 kb) correspond in size to the length of the two ORFs of pMC3-2 which were deduced from nucleotide-sequence data. Thus, both ORFs, one encoding a DNA polymerase and the other a yet unknown protein, are transcribed in the mitochondria of the plasmid-bearing Morchella conica strain.

Ascomycota↗

In organello replication and viral affinity of linear, extrachromosomal DNA of the ascomycete Ascobolus immersus.

Linear, extrachromosomal DNA's of the filamentous fungus Ascobolus immersus are localized within the mitochondria. These linear plasmids have no homology to the high molecular weight mtDNA (hmw mtDNA). For analysis of plasmid replication an in organello DNA synthesis system was developed, in which radionucleotides were incorporated into intact mitochondria. Plasmid DNA is labelled preferentially in this system. From replication analysis of a specific plasmid there is evidence of a virus-like protein-primed replication. Sequence analysis of this plasmid reveals that a viral DNA polymerase is encoded. Thus, these genetic elements presumably are viral remnants rather than true plasmids.

Amino Acid Sequence↗

Characterization and cloning of plasmid like DNA of the ascomycete Podospora anserina.

The previously reported existence of plasmid-like (pl) DNA in senescent mycelia of Podospora anserina was confirmed using new methodology. Detailed anaysis of bulk DNA has further shown a possible relationship between pl DNA and mt DNA. According to biophysical and electron microscopic experiments the pl DNA was found to consist of oligomeres having a basic unit with a contour length of 0.75 micrometer corresponding to 2.4 kb. To overcome the handicap that pl DNA is only produced in rather small amounts in the aging mycelia, this DNA was cloned in E. coli after insertion into a bacterial plasmid vector, pBR 322. It was possible to isolate a stable hybrid plasmid consisting of the vector and only one integrated monomere of pl DNA. The composition of this hybrid plasmid was confirmed by restriction endonuclease analysis and heteroduplex formation. A restriction map of the pl DNA is presented and its insertion site onto pBR 322 indicated.

Ascomycota↗

Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum (Gibberella pulicaris).

Variation within the internal transcribed spacers (ITS1 and ITS2) and 5.8s ribosomal DNA gene of the heterothallic phytopathogenic filamentous fungus, Fusarium sambucinum (teleomorph = Gibberella pulicaris), was examined in 86 strains from diverse geographical locations by PCR amplification and direct sequencing in order to measure intraspecific divergence within the ITS region. Sequence analysis revealed three ITS types (A, B, C), within which divergence was extremely low (0-2.3%). Surprisingly, the level of intraspecific divergence observed between ITS types, A----B = 14.3%, A----C = 15%, and B----C = 4.6%, is much greater than that reported for any other species. The degree to which transition/transversions and insertion/deletions make up the pattern of ITS sequence evolution both within and between types was analyzed. The sequences of the ITS types exhibit a C-T transition bias together with a GC insertion/deletion bias. In comparison, the genic flanking sequences, including the 5.8s rDNA gene and 5' end of the 28s large nuclear rDNA, are highly conserved. By the criteria of mating and DNA-DNA hybridization, all the strains examined represent a single species. Discordance between the ITS sequence data and other molecular and genetic data on F. sambucinum is discussed.

Base Sequence↗

The phenol oxidases of the ascomycete Podospora anserina. XII. Affinity of laccases II and III to substrates with different substitution patterns.

For the low molecular weight laccases II and III of Podospora anserina the kinetic parameters Michaelis constant (KM) and maximum reaction velocity (V) were determined polarographically under pH optimum conditions for representative substrates of different substitution patterns. Laccase II showed two peaks in its pH optimum curve, each with a different substrate specificity, indicating structural differences to laccase III which exhibits only one broad peak. Under optimum conditions the affinities of various substrates are determined by their substitution patterns: high affinity for simple o- and p-diphenols, low affinity for m-henols. The maximal velocity remains largely uninfluenced. This study of the effect of substitution on substrate utilization leads to the assumption that there is no specific reactive site for m-phenols in either laccase. Oxidation of m-phenols, however, takes only place at high pH values.

Ascomycota↗

Karyology and hyphal characters as taxonomic criteria in ascomycetous black yeasts and related fungi.

Mycelial development of seventy-three strains of black yeasts and related fungi were studied, and numbers of nuclei per hyphal cell were counted. Two main patterns were apparent in expanding hyphae, viz. (1) uninucleate expanding hyphal cells, septum formation strictly following mitosis, and (2) multinucleate, branched, aseptate hyphal tips, septa being formed in a later stage, leading to oligo- or uninucleate mature cells. Characteristic genera in the two groups are Exophiala and Aureobasidium, respectively. In Zasmidium and in some Ramichloridium species all mycelial cells are oligonucleate. The character is indicative for relationships at the family level in black yeasts.

Ascomycota↗

Self-splicing of a mitochondrial group I intron from the cytochrome b gene of the ascomycete Podospora anserina.

We have shown that the second intron of the Podospora mitochondrial gene coding for cytochrome b (Cytb 12) splices autocatalytically, using in vitro transcripts generated from the T7 promoter. The reaction takes place at 37 degrees C in the presence of 50 mM TRIS-HCl pH 7.5, 60 mM MgCl2 and 1 mM GTP but shows a low efficiency even at high KCl concentrations of up to 1.2 M. Under these conditions, intron bI2 follows the conventional pathway of group I splicing, and all characteristic products, with regard to both transesterification and hydrolysis, could be identified. Moreover, the intron is capable of undergoing cyclization, thereby releasing the noncoded G and one additional nucleotide (U) from the 5' end. The 5' cleavage site is preceded by the same two nucleotides, indicating a base-pairing at the same site of the internal guide sequence (IGS) for both splicing and cyclization ("one-binding-site model"). In addition, products resulting from site-specific hydrolysis 138 nucleotides downstream of the 5' splice site were detected. Unusually, the shortened intron is also able to form a circular RNA and an alternative sequence that aligns the cyclization site to the catalytic core of the intron must be assumed.

Ascomycota↗

Inhibitors of mitochondrial function prevent senescence in the ascomycete Podosprora anserina.

The onset of senescence, i.e. decrease of growth rate followed by cellular death, is prevented when inhibitors of mitochondrial function (ethidium-bromide, streptomycin, tiamulin) are present in the culture medium. If mycelia are transferred to a medium not containing one of these substances, senescence occurs after the usual time interval (30 d at 26 degrees C). Inhibitors of cytoplasmic protein synthesis such as emetine and cycloheximide have no effect in preventing senescence.

Ascomycota↗