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

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing K1CDEI, II, and III. Removal of 6 bp from the right side of K1CDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of K1CDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20-80 bp from K1CDEII resulted in a decrease in plasmid stability, indicating that K1CDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of K1CDEII to that of ScCDEII did not improve K1CEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of K1CDEIII was no longer functional in K. lactis, but did improve plasmid stability 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 sequence requirements.