Yeast autonomous linear plasmid pGKL2: ORF9 is an actively transcribed essential gene with multiple transcription start points.

A pair of linear plasmids, pGKL1 (8.9 kb) and pGKL2 (13.4 kb), resides in the cytoplasm of Kluyveromyces lactis killer strains. The smaller element, actually conferring the killer phenotype, strictly depends on the larger autonomous pGKL2. Here, we have examined the previously uncharacterized pGKL2 open reading frame (ORF)9 (1.34 kb). Northern analysis of a killer plasmid carrying Saccharomyces cerevisiae strain applying an ORF9-specific probe revealed a single transcript closely matching the size of the ORF9 coding region. Multiple transcriptional start points, determined by primer extension analysis, are located 16 nt downstream of a conserved sequence element regarded as the cytoplasmic promoter. In vivo disruption of pGKL2/ORF9 using the cytoplasmically expressible marker-gene LEU2* resulted in the establishment of a three-plasmid system composed of the native cytoplasmic elements pGKL1/2 and a hybrid of the latter, which only remained stable under selective conditions. The native pGKL2, however, did not segregate during prolonged subcultivations, proving an essential function of ORF9 for plasmid maintenance.