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Development of a eukaryotic cloning system inPodospora anserina

✍ Scribed by Paul Tudzynski; Ulf Stahl; Karl Esser


Book ID
104751950
Publisher
Springer-Verlag
Year
1982
Tongue
English
Weight
508 KB
Volume
6
Category
Article
ISSN
0172-8083

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✦ Synopsis


In developing a system for molecular cloning with the Podospora anserina plasmid (plDNA) it is necessary to find recipient strains which are resistant to plDNA mediated senescence. Three long lived double mutants which fail to exhibit spontaneous aging were genetically and biochemically analysed. All mutants were infected with plDNA. 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 plDNA 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 plDNA. 2. Of the two strains, only i viv contains free plDNA 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 plDNA 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 plDNA. Therefore, this strain is an ideal recipient for transformation provided a marker other than senescence is cloned.


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