MUTATIONAL ANALYSIS OF CENTROMERIC DNA ELEMENTS OF KLUYVEROMYCES-LACTIS AND THEIR ROLE IN DETERMINING THE SPECIES-SPECIFICITY OF THE HIGHLYHOMOLOGOUS CENTROMERES FROM KLUYVEROMYCES-LACTIS AND SACCHAROMYCES-CEREVISIAE

The centromere of Kluyveromyces lactis was delimited to a region of ap proximately 280 bp, encompassing K1CDEI, II, and III. Removal of 6 bp from the right side of K1CDEIII plus flanking sequences abolished cent romere function, and removal of 5 bp of K1CDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20-80 b p from K1CDEII resulted in a decrease in plasmid stability, indicating that K1CDEII must have a certain length for proper centromere functio n. Centromeres of K. lactis do not function in Saccharomyces cerevisia e and vice versa. Adapting the length of K1CDEII to that of ScCDEII di d not improve K1CEN function in S. cerevisiae, while doubling the ScCD EII length did not improve ScCEN function in K. lactis. Thus the diffe rence in CDEII length is not in itself responsible for the species spe cificity of the centromeres from each of the two species of budding ye ast. A chimeric K. lactis centromere with ScCDEIII instead of K1CDEIII was no longer functional in K. lactis, but did improve plasmid stabil ity in S. cerevisiae, although to a much lower level than a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific s equence requirements.