Centromeric DNA from Saccharomyces cerevisiae

Abstract

We have isolated 29 kilobase-pairs of DNA encompassing the centromere-linked gene, TRP1, of Saccharomyces cerevisiae. Two DNA sequences within the isolated region, ARS1 and ARS4, allow autonomous replication of chimeric DNA molecules upon introduction into yeast. Yeast strains transformed by ARS-containing DNA molecules are unstable; they lose the transformed phenotype and, concomitantly, the hybrid molecules at high frequency. Another function, CEN4, stabilizes the ARS-containing hybrids. CEN4 allows proper segregation of hybrid molecules during mitosis and meiosis. Like its predecessor. CEN3 (Clarke & Carbon, 1980 b). CEN4 behaves genetically like a yeast centromere. The centromere-linked sequences we isolated from chromosome IV of yeast are not highly repeated and contain transcriptionally active DNA sequences. We have also isolated other CEN sequences using an enrichment procedure based on the enhanced mitotic stability of molecules carrying both CEN and ARS. Together, the ARS and CEN functions allow DNA molecules to predominantly behave as independent yeast linkage groups. However, improper disjunction of these ARS- CEN hybrids occurs in 3 to 14% of the mitotic cell divisions and ⩽3% of the meiotic divisions while the segregation of other yeast chromosomes, including chromosome IV, is normal. No more than 2% of these monovalent circular chromosomes undergo precocious segregation of sister chromatids during the first meiotic division rather than the second. Thus functional yeast chromosomes have been reconstructed by piecing together fragments of centromeric DNA with fragments that allow autonomous replication in yeast.