Development of a eukaryotic cloning system in Podospora anserina

Summary

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.