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

Razanamparany, Voahangy Rasolofo; Bégueret, Joël

doi:10.1007/bf00418527

Cited by 51 publications

(26 citation statements)

References 16 publications

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“…Different transformants were crossed with the uraS-6 strain, and the progeny were tested for prototrophy. All the stable transformants give rise to ura+ spores in a Mendelian ratio, as described previously (19). By contrast, the frequency of ura+ strains is very low (under 10-2) in the progeny of the crosses between the unstable transformants and the ura5-6 strain.…”

Section: Resultssupporting

confidence: 67%

“…We observed previously (19) that the prototrophs obtained from the transformation of the uraS-6 mutant by the circular pPAura5-1 vector are highly stable even under nonselective conditions. This high stability results from the integration of the transforming vector in the chromosomal DNA.…”

Section: Resultsmentioning

confidence: 96%

“…The characteristics of P. anserina were reviewed by Esser (7). The isolation and characterization of a ura5-6 mutant deficient for OMPppase have been described previously (19). The bacterial strain used for plasmid amplification was Escherichia coli BJ5183 (endA sbcB recBC galK met strR thi-J bioT hsdR).…”

Section: Methodsmentioning

confidence: 99%

“…This plasmid is integrative (19). We have tested the effect of the linearization of this vector on transformation.…”

mentioning

confidence: 99%

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Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

Perrot¹,

Barreau²,

Bégueret³

1987

Mol. Cell. Biol.

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The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.Transformation of mycelial fungi has been described previously, and common features emerge from the results observed for different species: the transformation efficiency is low and the vectors are integrative (11,25). In yeasts, integrative vectors were converted to autonomously replicating plasmids by the addition of sequences which act as replication origins. They are either autonomously replicating sequences (ARS) (10,22) or the replication origin of the natural 2,um plasmid (2). These autonomously replicating plasmids exhibit a high transformation efficiency and instability when the transformed strains are grown under nonselective conditions. Attempts to obtain such vectors in mycelial fungi were made by the addition of chromosomal sequences into the integrative vectors (5). The absence of positive results may be due to the coenocytic structure of these organisms, which leads to the possibility of complementation between different nuclei of the hyphae. The nuclei which lose the transforming gene can be maintained by the presence of a few nuclei which have retained the plasmid. Genomic sequences of Aspergillus nidulans which promote autonomous replication of yeast plasmids have been cloned (21). When they were introduced into A. nidulans, they did not confer replicating activity on the plasmids (1). One of these sequences greatly enhances the transformation efficiency of A. nidulans, but the vector containing this ansl sequence remains integrative. In Neurospora crassa, a recombinant pUC8 plasmid carrying the am gene has been reported to be able to replicate autonomously (8). However, the transforming vector is rearranged and is present as high-molecular-weight concatemers; furthermore, some plasmid sequences are integrated into the chromosomal DNA.For Podospora anserina, we have developed a system based on the transformation of an auxotrophic uraS-strain deficient for orotidylic acid pyrophosphorylase (OMPppase) by a recombinant vector (pPAura5-1) carrying the homologous uraS gene (3). This plasmid is integrative (19). We have tested the effect of the linearization of this vector on transformation. As it is known that broken DNA ends are highly * Corresponding author. reactive in recombination, the linearized vector was stabilized ...

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

“…This plasmid is integrative (19). We have tested the effect of the linearization of this vector on transformation.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

Perrot¹,

Barreau²,

Bégueret³

1987

Mol. Cell. Biol.

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“…Either one of the two possible types of 5-FOA-resistant uracil auxotrophs has been preferentially obtained in some microorganisms (16,20). In our case, OMPpase-deficient mutants were preferentially obtained especially after the spontaneous isolation procedure.…”

mentioning

confidence: 62%