Autonomously replicating sequences (ARS) are not stable in vivo during cell division and cannot be used readily as plasmid vectors. But, if certain DNA sequences that function as centromeres in yeast (CEN sequences) are grafted onto a plasmid already containing an ARS, the resulting vector plasmid is stabilized and segregates accurately during mitosis and meiosis. The CEN sequences are necessary for attaching chromosomes to the mitotic spindle, presumably through some specific connector proteins that join the microtubules to the centromere. In addition, CEN sequences can be used to construct linear chromosomes by preventing circularization by the addition of telomeric sequences that are normally at the end of chromosomes (1, 2).
Conserved features of Saccharomyces cerevisiae CEN SEQUENCES are confined to a region of about 120 bp. The highly conserved 8 bp at the left (PuTCACPuTG, where Pu = purine) constitute the left boundary of a functional CEN sequence. The right boundary lies within or just beyond the 25 bp at the right, with the consensus sequence TGT-T-TG-TTCCGAA—AAA, where – indicates no specific base. A mutant that lacks the left conserved element can still assemble into a centromere that is partially functional in mitosis and well functioning in meiosis. The sequences between the two conserved terminal DNA elements are not essential for centromere function. Their lengths can be increased by 50% or their sequence from 6 to 12% in GC content without measurable changes in mitotic or meiotic segregation of plasmids carrying such CEN mutations (3). The right boundary sequence appears to be a binding site for a protein, as evidenced by various inactive mutations, especially in the central CCG sequence, and by an exonuclease blocking assay (4). The left element, which carries the palindromic sequence CACPuTG, binds the helix-loop-helix motif protein CPF1 (5).
