Kluyveromyces lactis killer plasmid pGKL2: molecular analysis of an essential gene, ORF5.
The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One-step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild-type plasmids k1 and k2. Northern analysis detected a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non-selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over-expression of an epitope-tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.
D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D005800:Genes, Fungal; D005804:Genes, Lethal; D005819:Genetic Markers; D005822:Genetic Vectors; D007716:Kluyveromyces / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic
D005656:Fungal Proteins; D005819:Genetic Markers; D011994:Recombinant Proteins