Observations on integrative transformation in Schizosaccharomyces pombe

Summary

Three different Schizosaccharomyces pombe strains have been transformed with a circular or linearized non-ars plasmid carrying the ura4 ⁺ gene as a selectable marker. The first strain shows full homology between the genomic ura4-294 gene (point mutation) and the marker gene on the plasmid. The second strain carries a 600 bp deletion (ura4-D6) that decreases homology between plasmid and chromosome. No homology remains in the third strain which has a complete deletion of the ura4 gene on the chromosome (ura4-D18). When sequence homology exists between transforming DNA and the chromosomal ura4 region, gene conversion is strongly preferred over integration of the circular plasmid. Reduction of the length of homology leads to a decrease of transformation frequencies, and homology dependent as well as a minority of homology independent integrations are observed. In the complete absence of homology two rate types of transformants are encountered: either the circular plasmid replicates autonomously, although it is devoid of an ars sequence, or alternatively the plasmid integrates into the genome at various positions. Transformation with plasmid cut within the coding region of ura4 can lead to tandemly arranged multiple integrations, when no homology exists between the free ends and the chromosome. The integrations occur at the ura4 locus, when homology is retained between plasmid and chromosome, and at various sites in the genome of the strain with a complete deletion of the ura4 gene. The results suggest that homology dependent events (conversion, integration) are strongly preferred in transformation of S. pombe with non-ars plasmids. In addition low frequency integration by illegitimate recombination is observed. Linearized plasmid can be ligated in vivo to form monomers or multimers in the absence of homology between the free plasmid ends and the chromosomal genome.