Kluyveromyces lactisKiller Plasmid pGKL2: Evidence for a Viral-like Capping Enzyme Encoded by ORF3

ORF3 of the cytoplasmic linear plasmid pGKL2 was disruptedin vivoby integration of a selectable marker. Long-term cultivation of transformants carrying hybrid plasmids with a disrupted ORF3 under selective pressure did not deprive strains of the native counterpart, thereby proving its essentiality for pGKL2 replication and maintenance. The predicted ORF3 polypeptide was found to contain conserved motifs acquainted with mRNA-capping enzymes in the required order, just as in cytoplasmic viruses; new conserved motifs were also identified.     

Palindrome-hairpin linear plasmids possessing only a part of the ORF1 gene of the yeast killer plasmid pGKL1

Summary The yeast Kluyveromyces lactis haboring linear DNA plasmids pGKL1 and pGKL2 exhibits killer and killer-resistant phenotypes. Two new linear plasmids pK192L and pK192S were found in the weak killer mutant KUV192 induced by UV irradiation. pK192S was always accompanied by pK192L in subclones of KUV192. Both plasmids were derived from pGKL1 by deletion of the large right part of it. pK192L was 4.9 kb in size and had a palindromic structure consisting of 2.35 kb inverted terminal repetitions and a 215 base unique sequence. Analysis of denatured and renatured DNA strands suggested that pK192S was a hairpin-like form of pK192L. The pK192 plasmids were maintained only in cells haboring either pGKL1 or pGKL1S in addition to pGKL2 and competed with pGKL1 or pGKL1S for their maintenance. Since no complete ORF1 was conserved in pK192 plasmids, these results lead to the conclusion that the ORF1 gene is necessary for the replication and/or maintenance of pGKL1.