IDENTIFICATION OF AN ESSENTIAL CORE ELEMENT AND STIMULATORY SEQUENCESIN A KLUYVEROMYCES-LACTIS ARS ELEMENT, KARS101

A Kluyveromyces lactis chromosomal sequence of 913 bp is sufficient fo r replication in Saccharomyces cerevisiae and K. lactis. This fragment contains a 12 bp sequence 5'-ATTTATTGTTTT-3' that is related to the S . cerevisiae ACS (ARS consensus sequence). This dodecamer was removed by site-directed mutagenesis and the effect on K. lactis and S. cerevi siae ARS (autonomous replicating sequence) activity was determined. Th e dodecamer is essential for S. cerevisiae ARS function but only contr ibutes to K. lactis ARS activity; therefore, its role in K. lactis is unlikely to be the same as that of the essential S. cerevisiae ACS. A 103 bp subclone was found to retain ARS activity in both yeasts, but t he plasmid was very unstable in S. cerevisiae. Deletion and linker sub stitution mutagenesis of this fragment was undertaken to define the DN A sequence required for K. lactis ARS function and to test whether the sequence required for ARS activity in K. lactis and S. cerevisiae coi ncide. We found a 39 bp core region essential for K. lactis ARS functi on flanked by sequences that contribute to ARS efficiency. The instabi lity of the plasmid in S. cerevisiae made a fine-structure analysis of the S. cerevisiae ARS element impossible. However, the sequences that promote high-frequency transformation in S. cerevisiae overlap the es sential core of the K. lactis ARS element but have different end-point s.