Isolation and characterization of a laccase gene fromPodospora anserina

The genome of the filamentous ascomycetePodospora anserina contains at least four non-adjacent regions that are homologous to the laccase gene ofNeurospora crassa. One of these regions contains a gene (lac2) encoding a protein that displays 62% identity with theN. crassa laccase. In shaken cultures,lac2 mRNA is present at low basal levels throughout the growth phase but increases at least 20-fold at the beginning of the autolytic phase and decreases again thereafter. Addition of aromatic xenobiotics (guaiacol, hydroquinone, benzoquinone) to the medium during the growth phase results in a rapid, drastic and temporary increase in the abundance oflac2 mRNA. The promoter region oflac2 contains two sequences which display complete homology with the eukaryotic Xenobiotic Responsive Element and two sequences homologous to the eukaryotic Antioxidant Responsive Element. The identity and function of the laccase encoded bylac2 are discussed.     

Protoplasts fromPodospora anserina: Isolation,purification, and transformation

Protoplasts fromPodospora anserina mycelium were produced using the commercially available enzyme Novozym 234. Different parameters involved in protoplast isolation were analyzed in order to establish optimal conditions, and protoplast production was notably increased. For the purification of protoplasts, several techniques based on both centrifugation and filtration were assayed, with filtration yielding the best results. Regeneration of protoplasts was studied on different media and osmostic stabilizers, and about 80% regeneration was obtained. The good physiological condition of the protoplasts produced with this method is demonstrated by the lack of cell wall and high regeneration rate and transformation frequencies.     

What triggers senescence in Podospora anserina?

Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence.