Joël Bégueret

Vegetative incompatibility in filamentous fungi: Podospora and Neurospora provide some clues

In filamentous fungi, vegetative cell fusion between genotypically distinct individuals leads to a cell-death reaction known as vegetative or heterokaryon incompatibility. Genes involved in this reaction have been characterised molecularly. We can now begin to get a better understanding of the mechanism and the biological significance of this intriguing phenomenon.

Glutathione S-transferase class μ in French alcoholic cirrhotic patients

SummaryThe lack of glutathione S-transferase μ (GSTμ) was examined in 45 healthy French Caucasians and 45 alcoholic cirrhotic French Caucasians: microsamples of blood were taken and DNA amplified by the polymerase chain reaction. We have concluded that there is no relationship between this genotype and the development of alcoholic cirrhosis in these heavy consumers of ethanol.

Isolation of the two allelic incompatibility genes s and S of the fungus Podospora anserina

SummaryThe two allelic genes s and S are responsible for heterogenic incompatibility between wild type strains of the fungus Podospora anserina. The s gene has been cloned by SIB selection and expression in a strain containing a neutral allele of this locus. The S gene was isolated from a genomic library using the DNA of the s locus as a probe. The physical map of the DNA fragments carrying the two genes are highly dissimilar and restriction polymorphism exists at the s locus between s and S strains. Nevertheless, homology between the two alleles was revealed by cross-hybridization at the DNA and RNA levels.

Positive screening and transformation of ura5 mutants in the fungus Podospora anserina: characterization of the transformants

SummaryTo develop a transformation system in the filamentous fungus Podospora anserina we have selected ura5 mutants deficient in orotidylic acid pyrophosphorylase using a positive screening. These mutants could be transformed to prototrophy by an hybrid vector carrying the ura5 gene of this organism. The properties of the transformants have been analysed. In most cases integration of the transforming vector occurred outside the ura5 locus and frequently repeated tandem copies of the vector were found. Reversion of the transformants could also be selected and we found that it can occur by exact or only partial excision of the integrated vector.

Characterization of hch, the Podospora anserina homolog of the het-c heterokaryon incompatibility gene of Neurospora crassa.

Abstract The het-c locus controls heterokaryon formation in Neurospora crassa. It is subject to balancing selection operating to maintain polymorphism at that locus in natural populations. We have isolated hch, the het-c homolog from the related species Podospora anserina (hch for het-c homolog), in order to determine if this gene also functions as a het gene in that species. The het-c and hch sequences are highly similar but differ in the region defining allele specificity in N. crassahet-c. Analysis of hch variability in 11 natural P. anserina isolates with different het genotypes revealed no polymorphism. This suggested that hch does not function as a het gene. However, heterologous expression of the N. crassa het-cPA allele in P. anserina triggers a growth defect reminiscent of the het-c incompatibility reaction.

Identification and characterization of a gene encoding a subtilisin-like serine protease induced during the vegetative incompatibility reaction in Podospora anserina.

Abstract. In the filamentous fungi, cell fusion between unlike individuals generally triggers a cell-death reaction known as vegetative incompatibility. In Podospora anserina, it was shown that, during this cell-death reaction, there is a strong increase in proteolytic activity. Here, we report the purification of a 36-kDa protease that is induced during the incompatibility reaction. An internal peptide of this protein displayed a strong similarity with the PEPC subtilisin-like serine protease from Aspergillus niger. This led us to use the pepC gene as a probe to clone the homologous gene from P. anserina, which we called pspA. The expression of pspA is upregulated at the transcript level during the progress of the incompatibility reaction. This induction is diminished in strains bearing suppressors of the vegetative incompatibility reaction. The fact that pspA is homologous to PrB, a vacuolar protease involved in autophagy in yeast, suggests that the incompatibility cell-death reaction and autophagy might be related processes.

Role of Alcohol Dehydrogenase Polymorphism in Ethanol Metabolism and Alcohol-Related Diseases

In alcohol toxicity, a lot of data point to genetic factors associated with environmental factors. These genetic factors can intervene at different levels : genetic variability in the distribution of polymorphic alcohol and aldehyde dehydrogenase isozymes (Bosron, et al., 1988) could play a role in determining individual difference in ethanol metabolism and toxicity. In the future, other polymorphisms in alcohol metabolism may be described. Polymorphism is also to be considered in detoxication and/or target organ metabolism (as collagen polymorphism in alcoholic cirrhosis)(Weiner, et al., 1988).

Non-homologous integration of transforming vectors in the fungus Podospora anserina: sequences of junctions at the integration sites

Transformation of the ura5-6 mutant strain of Podospora anserina with a recombinant vector carrying the ura5+ gene often results in the integration of the transforming plasmid by non-homologous recombination outside of the genomic ura5 locus. To investigate the mechanism of such integration, we rescued the integrated plasmid from three transformants. In two cases, the rescued plasmid was highly altered compared with the original transforming vector. We cloned the junctions between plasmidic DNA and genomic DNA of the transformants and determined their nucleotide sequences. It was found that there was little homology between plasmidic and genomic DNA sequences. Moreover, in all cases deletions of plasmid sequences at the integration site had occurred. These rearrangements can be explained by the formation of multimeric plasmids prior to integration.

Characterization of a gene from the filamentous fungus Podospora anserina encoding an aspartyl protease induced upon carbon starvation.

In an attempt to characterize proteases associated with vegetative incompatibility, a Podospora anserina gene (papA) encoding an aspartyl protease (podosporapepsin) was cloned using a heterologous probe. The deduced papA coding region was 1278 nucleotides long, interrupted by a single 71bp intron. The corresponding amino acid sequence presented a high degree of similarity to other aspartyl proteases. Sequence analysis and proteolytic activity measurement suggested that the podosporapepsin could be intracellular rather than secreted. The papA gene was expressed under carbon starvation, but not under nitrogen starvation conditions. Its disruption led to a slight decrease in the growth rate of the mutant strain when bovine serum albumin was the sole carbon source in the medium. Disruption or overexpression of papA gene had no obvious consequence on vegetative incompatibility. Transcription of papA induced by carbon starvation was strongly reduced in the presence of a suppressor of vegetative incompatibility. This result suggests a relationship between adaptation for starvation and vegetative incompatibility.

Self-cloning in filamentous fungi: Application to the construction of endothiapepsin overproducers in Cryphonectria parasitica

The filamentous ascomycete fungus Cryphonectria parasitica naturally secretes endothiapepsin, an aspartic proteinase. It is cultured on a commercial scale as a source of the milk-clotting enzyme for cheese making. Our objective was to increase enzyme production of an industrial C. parasitica strain by a new technique of self-cloning; it consisted in the screening for transformants producing higher levels of endothiapepsin and having integrated only the DNA fragment of interest. Such genetically improved strains that are devoid of any foreign genes should be more readily acceptable for the production of food-grade enzymes.