engleski

Plasmid integration in yeast: the choice between homologous targets

Yeast integrative transformation is achieved by homologous recombination between plasmid molecule and yeast chromosome. If two different homologies are present on the plasmid molecule, transformants are distributed between two chromosomal targets. Our previous studies indicated that this distribution is not a simple function of the target size, so we asked which mechanisms influence the choice of the target. The plasmid used in this study (pOM) contains two yeast genes, CYC1 and URA3 and can be isolated in either single-stranded (ss) or double-stranded (ds) form. The recipient strain has insertion of Ty element (6, 1 kb) approximately in the middle of the ura3 gene (ura3-52 allele), so the Ura+ transformants were expected to arise only by plasmid integration and not by gene conversion. Three different integration events could be distinguished by southern blotting of the genomic DNA of transformants cut with the restriction endonuclease BamH I: integrations in CYC1 gene and integrations in the ura3 gene, upstream or downstream of Ty insertion. We show that the proportion of integrants in any of homologous targets depends on genetic background of the recipient strain, sequence divergence and conformation of the plasmid molecule (ss or ds). A number of aberrant events was detected, mainly with ss form of the plasmid. One of the observations made was that the integrations in the ura3 gene were not equally distributed between sequences situated upstream or downstream from the Ty insertion - only 10% was detected downstream! In order to further investigate this phenomenon we induced double strand breaks (DSBs) in the plasmid molecule to target integration events upstream (Pst I) or downstream (Stu I) from Ty. All the integrants targeted by Stu I-induced cut gave expected pattern, while 30% of transformants targeted by Pst I resulted either in integration downstream from Ty or in conversion of the Ty. This is not related to the metabolism of retrotransposon since the same phenomenon was also observed if Ty was replaced with ARG4 gene. This is the first report of asymmetric displacement of the recombinational intermediate through the large heterologous insertions.

yeast; integrative transformation; targeting

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