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 construct ed which consist of pGKL1, into which in addition to the previously de veloped 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 arbi trarily placed cytoplasmic promoter (upstream conserved sequence) in f ront 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 fra me 5 (pRKL122). The structures of both linear hybrid plasmids were con firmed 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 i n phase fused gene was 40-fold higher compared to the arbitrarily plac ed cytoplasmic promoter. In general, an in phase fusion was not requir ed for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same a s those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cy toplasmic promoters present on both linear plasmids. Hybrid plasmids w ere stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae. (C) 1995 Academic Press, Inc.