A modified double selection approach for manipulation of the cytoplasmic plasmids k1 and k2 from dairy yeast Kluyveromyces lactis has been exploited to investigate promoter and gene function. Using TRP1-mediated integration of a LEU2 gene fusion, we have shown that expression of the selection marker is strictly dependent on the k2 promoter UCS5. Also, k2ORF6, the gene encoding the RNA polymerase specific for UCS recognition, is functional when shuffled between the plasmids. Once transplaced onto k1 by means of gene shuffling. the hybrid ORF6 complemented an orf6 deletion created on plasmid k2 eventually yielding yeast strains that contained only two recombinant plasmids: a k2 derivative (rk2/6) with a k2orf6::TRP1 gene deletion, and a k1 derivative (rk1/6) carrying the transplaced ORF6 allele along with the LEU2 marker. This interchangeability of both UCS promoter activity and gene function between k2 and k1 supports the concept of an autonomous transcription system that operates on these nonconventional yeast plasmids. (C) 2000 Elsevier Science Inc. All rights reserved.