YEAST KILLER PLASMID PGKL2 - MOLECULAR ANALYSIS OF UCS5, A CYTOPLASMIC PROMOTER ELEMENT ESSENTIAL FOR ORF5 GENE-FUNCTION

A k2/k1 plasmid gene shuffle system has been used to investigate linea r plasmid promoter function in Kluyveromyces lactis. By transplacing v arious ORF5 deletion constructs from the larger plasmid k2 onto k1, an d analysing trans-complementation of an ORF5(0) deletion on k2, a 40 b p k2 fragment, including the UCS motif of ORF5 (UCS5), has been identi fied as a cis-acting promoter element essential for ORF5 gene function . Qualitative and quantitative transcript analyses of a UCS5-ScLEU2 fu sion gene using Northern blot analysis and phosphor image technology r evealed a plasmid-dependent LEU2 transcript distinct in size (1.55 kb) and regulation from its nuclear counterpart (1.35 kb): cytoplasmic, U CS5-driven expression of the marker gene was non-repressible by leucin e and reduced five- to eight-fold compared to fully derepressed nuclea r K1LEU2 mRNA levels. Thus, the killer plasmids k2 and k1 appear to ex press low levels of transcript overall, when relative gene copy number s (one for the nuclear allele versus 50-100 copies for the plasmid-bor ne LEU2 gene) are taken into account.