A CYTOPLASMIC GENE-SHUFFLE SYSTEM IN KLUYVEROMYCES-LACTIS - USE OF EPITOPE TAGGING TO DETECT A KILLER PLASMID-ENCODED GENE-PRODUCT

A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of K luyveromyces lactis, By transplacing k2ORF5 from the larger plasmid pG KL2(k2) onto pGKL1(k1) we have shown this gene to be essential and fun ctionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF5(0) deletion on k2 in trans , giving rise to yeast strains containing only the two recombinant pla smid forms. Additionally, the in vivo product of k2ORF5 has been ident ified as a 19.5 kDa protein by transplacing an epitope-tagged k2ORF5 a llele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf 5p is one of the most abundant k2 products, implying structural rather than regulatory function.