ORF7 of yeast plasmid pGKL2: analysis of gene expression in vivo

ORF7 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a putative RNA polymerase subunit of 16 kDa. RNA analysis detected a single, plasmid-dependent ORF7 transcript of 550 nt indicating that the gene is transcribed mono-cistronically. Attempted one-step gene disruption of ORF7 resulted in chromosomal integration of the marker gene rather than the formation of stable recombinant k2ORF7(0) deletion plasmids. Thus, ORF7 appears to be a potential cis-dominant locus the integrity of which is indispensable for plasmid stability. The ORF7 gene product was over-produced as a c-myc-tagged fusion protein in Escherichia coli. Western-blot analysis of total yeast protein extracts using an antibody against this Orf7-c-myc fusion product identified a protein band with an apparent molecular weight of 17 kDa. This protein corresponds in size to the predicted product and is only detectable in plasmid-carrying killer yeasts.