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The transcription of ten stress-response genes was investigated under oxygen-limiting conditions with maltose and glucose, respectively. Six of these genes (HSP12, HSP26, HSP30, HSP78, HSP82 and HSP104) showed expression only during the stationary phase. HSP12 and HSP104 were transcribed 10 h earlier with maltose than with glucose. Fermentation in wort yielded similar results to the maltose-based medium. HSP12, HSP26 and HSP30 were highly expressed. Thus, the HSP26 and the HSP30 promoter can be used for late, phase-specific expression of the desired genes with glucose or maltose as carbon source, and HSP12 with glucose only. MET14, a gene important for sulfite formation, was overexpressed in wort, using the HSP26 promoter during the stationary phase. [ABSTRACT FROM AUTHOR]

In the ascomycete Podospora anserina senescence through strain aging is under nucleo-cytoplasmic control and inducible in juvenile mycelia by an 'infective principle' transferred after cytoplasmic contact via anastomoses. A specific DNA called plasmid-like (pl) DNA, present exclusively in aging mycelia, was found to be identical with this 'infective principle', since it was possible to transform juvenile protoplasts to senescence by using purified p1DNA. Therefore a specific function may be attributed to this ccc DNA. Its direct involvement in a genetically programed senescence is confirmed and its development as a vector for transfer of genetic information in eukaryotes can be undertaken. [ABSTRACT FROM AUTHOR]

Mitochondrial (mt) DNA of the alkane yeast, Saccharomycopsis lipolytica, was isolated. Its buoyant density in CsCl was found to be of 1.687 g/cm, indicating a GC content of 27.5% and its melting point T = 79.5 °C, indicating a GC content of 24.9%. The corresponding values for nuclear (n) DNA, are 1.709 g/cm (GC: 49.5%) and T = 90.5 (GC: 51.7%) respectively. Electron microscopy revealed that mtDNA has a circular structure with a contour length of about 14.5 µm corresponding to 45.5 kb per molecule. The size estimated from restriction analyses performed with 7 endonucleases was 48.35 kb/molecule. A restriction map was constructed, using the cleavage data of 4 endonucleases. [ABSTRACT FROM AUTHOR]

Four different transformation methods were tested and compared in an attempt to facilitate the genetic transformation of Aspergillus giganteus, the producer of an antifungal protein (AFP). The fungus was transformed to hygromycin B resistance, using the hph gene of Escherichia coli by protoplast transformation, electroporation, biolistic transformation, and Agrobacterium tumefaciens-mediated transformation. Electroporation and biolistic transformation were found to be inappropriate for transforming A. giganteus, due to a low transformation yield. The conventional transformation technique based on protoplasts yielded up to 55 transformants in 10(8) protoplasts/microg DNA and was enhanced to 140-fold by A. tumefaciens-mediated transfer of its T-DNA. Here, the germination time prior to cocultivation and the fungus:bacterium ratio were found to alter the transformation efficiency. Southern blot analysis revealed that the A. giganteus transformants contained a randomly integrated single T-DNA copy, whereas multiple integration events were frequent in transformants obtained by the protoplast method.

The antifungal protein (AFP) secreted by the mould Aspergillus giganteus is a small, highly basic polypeptide with antifungal activity. Previous work has shown that transcription of the corresponding afp gene is regulated by ambient pH, being suppressed under acidic and strongly induced under alkaline conditions. This observation suggested that the afp gene is regulated by the wide-domain transcriptional factor PacC. Here, we show that two putative PacC binding sites within the afp promoter, denoted afpP1 and afpP2, are efficiently recognised in vitro by a PacC fusion protein of A. nidulans. In addition, we found that phosphate, which was used as a buffering agent during cultivation, plays an important role in regulating afp expression. AFP production was nearly completely inhibited in the presence of external phosphate. The results of Northern analysis indicate that the inhibitory effect of phosphate is mediated at the transcriptional level.

The Podospora anserina nuclear mutant grisea displays an undifferentiated growth phenotype (diminished production of aerial hyphae), is female sterile (lack of perithecia), has a prolonged life-span compared to the wild-type strain, and lacks detectable phenoloxidase (laccase and tyrosinase) activity. Reversion of all of these characteristics to those of the wild-type phenotype was accomplished by supplementing the growth medium with extra amounts of copper salts. These results indicate that the primary defect of the grisea strain is in its copper uptake and/or distribution in the cells.

The bacterial neo gene from transposon Tn903 (Tn601) was used for dominant transformation of the fission yeast Schizosaccharomyces pombe. It was found that high transformation efficiency was dependent on a high level of promoter activity, mediated by the strong promoter of the Schizosaccharomyces pombe alcohol dehydrogenase gene (adh1), as shown by comparing the efficiency of transformation to G418-resistance, the resistance levels of transformed cells, and the in vitro amino-glycoside phosphotransferase activity. On the other hand, the heterologous promoter of the Saccharomyces cerevisiae alcohol dehydrogenase I gene (adc1) is shown to be a weak promoter in Schizosaccharomyces pombe, though its activity is significantly enhanced in cells grown on glycerol as a carbon source. This system for selection and detection of promoter-active sequences may provide a useful basis for the analysis of promoter elements in fission yeast.

A wild strain of A. giganteus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of A. nidulans sequences. Stable transformants arose by heterogenous integration, mainly of tandem repeats of vector DNA at various sites in the host genome. Between 6 and 30 resistant colonies were obtained per microgram DNA per 3 x 10(3) viable protoplasts. Vector DNA could be recovered by transformation of Escherichia coli with undigested genomic DNA from Aspergillus giganteus transformants.

In developing a system for molecular cloning with the Podospora anserina plasmid (p1DNA) it is necessary to find recipient strains which are resistant to p1DNA mediated senescence. Three long lived double mutants which fail to exhibit spontaneous aging were genetically and biochemically analysed. All mutants were infected with p1DNA. The mutant ca viv became irreversibly senescent and therefore was not further tested. The second mutant, gr viv showed some symptoms of aging but never died. The third strain i viv remained resistant to aging from p1DNA infection and has thus proven to be the best host strain available for molecular cloning in this system.A DNA analysis of the latter two strains revealed: 1. There is no difference from the wild strain with respect to the structure of mtDNA and the integration site of the p1DNA. 2. Of the two strains, only i viv contains free p1DNA in its mitochondria but in low amounts if compared to the wild strain. These experimental results are interpreted as follows: 1. The gr viv strain does not liberate spontaneously the p1DNA from mtDNA, but following infection is able to replicate and express this plasmid and therefore is a potential host for transformation. 2. The i viv strain liberates the mitochondrial plasmid but does not express senescence even when infected with p1DNA. Therefore, this strain is an ideal recipient for transformation provided a marker other than senescence is cloned.

As previously reported, a ccc DNA with a contour length of 0.75 µm and molecular weight of 2.4 kb (termed plasmid-like, p1DNA) is the causative agent of senescence in the fungus Podospora anserina. Its postulated location in mtDNA was proved correct by the following experiments: 1. Restriction analysis of mtDNA resulted in different molecular weights for both, juvenile (95 kb) and senescent (30 kb) mtDNA. The construction of a detailed restriction map made evident the fact that senescent mtDNA comprises only a part of its juvenile counterpart. 2. Hybridization experiments (Southern blots) between (3)H-labelled plDNA and mtDNA cleaved by restriction juvenile mtDNA are homologous to plDNA. 3. Fine mapping experiments (construction of restriction maps and heteroduplex experiments) between plDNA integrated into a bacterial vector and its postulated equivalent, derived from juvenile mtDNA and also integrated into a bacterial vector, allowed a precise determination of the site of plDNA insertion within the juvenile mtDNA. All of these data fit into a previously published model in which, during aging, plDNA is excised from mtDNA and becomes autonomous for replication and function.

Mapping and transcription studies have revealed that in Podospora anserina the causative agent of senescence, a mitochondrial plasmid (plDNA), is identical with intron 1 of the discontinuous gene for cytochrome-c-oxidase subunit 1 (COI), which is 2 kpb from the discontinuous gene for cytochrome b (Cytb). A mitochondrial mutant (ex1) devoid of the COI, but not of the Cytb gene provides longevity. A molecular model for the onset of senescence is presented.