A k2/k1 plasmid gene shuffle system has been used to investigate linear plasmid promoter function in Kluyveromyces lactis. By transplacing various ORF5 deletion constructs from the larger plasmid k2 onto k1, and analysing trans-complementation of an ORF5(0) deletion on k2, a 40 bp k2 fragment, including the UCS motif of ORF5 (UCS5), has been identified as a cis-acting promoter element essential for ORF5 gene function. Qualitative and quantitative transcript analyses of a UCS5-ScLEU2 fusion gene using Northern blot analysis and phosphor image technology revealed a plasmid-dependent LEU2 transcript distinct in size (1.55 kb) and regulation from its nuclear counterpart (1.35 kb): cytoplasmic, UCS5-driven expression of the marker gene was non-repressible by leucine and reduced five- to eight-fold compared to fully derepressed nuclear K1LEU2 mRNA levels. Thus, the killer plasmids k2 and k1 appear to express low levels of transcript overall, when relative gene copy numbers (one for the nuclear allele versus 50-100 copies for the plasmid-borne LEU2 gene) are taken into account.