An extranuclear expression system for analysis of cytoplasmic promoters of yeast linear killer plasmids.
Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.
Schründer
J
J
Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Federal Republic of Germany.
Meinhardt
F
F
D001410:Bacillus megaterium / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm; D004926:Escherichia coli; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005822:Genetic Vectors; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000235:genetics; D007716:Kluyveromyces / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase