Replication and maintenance of the Kluyveromyces linear pGKL plasmids.

Two new linear plasmids, pK192L (4.9 kb) and pK192S (2.4 kb), were isolated from a Kluyveromyces lactis killer strain carrying pGKL1 and pGKL2. pK192L was a deletion plasmid of pGKL1, derived from a part of the ORF1, and had a palindrome structure of a 215 bp unique sequence flanked by 2.35 bp inverted repeats. pK192S was a hairpin plasmid produced by self-annealing of a single-stranded pK192L DNA. In genetic analysis, pK192L and pK192S always coexisted and replicated in cells harboring pGKL2 and pGKL1, in contrast to other pGKL1-derived deletion plasmids, such as F1, F2 and pGKLIS, which could replicate in cells carrying pGKL2 only. Based on these and other lines of evidence, it was concluded that the reason for the pGKL1 dependent replication of the pK192L/S plasmids was the absence of the intact pGKL1-ORF1 gene and that the ORF1 function was necessary for the replication of the pGKL1 genome. This finding is in good agreement with a recent view reporting that ORF1 may encode a DNA polymerase of pGKL1. In a separate experiment, four new linear plasmids were isolated from a Saccharomyces cerevisiae strain carrying pGKL1 and pGKL2. Structural analysis showed that they consisted of two pairs of hairpin-palindrome type plasmids, each derived from different parts of pGKL2, respectively. pGKL1 stability replicated in cells carrying both these pGKL2 derived deletion plasmids.