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.
Schaffrath
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R
Department of Genetics, University of Leicester, Leicester LE1 7RH, UK.
Meinhardt
F
F
Meacock
P A
PA
D015152:Blotting, Northern; D015153:Blotting, Western; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D016271:Proto-Oncogene Proteins c-myc; D012331:RNA, Fungal; D012321:DNA-Directed RNA Polymerases