Point Centromeres in S. cerevisiae Contain Short, Essential DNA Sequences

KEY CONCEPTS
- CEN elements are identified in S. cerevisiae by the ability to allow a plasmid to segregate accurately at mitosis.
- CEN elements consist of the short, conserved sequences CDE-I and CDE-III that flank the A-T–rich region CDE-II.
If a centromeric sequence of DNA is responsible for segregation, any molecule of DNA possessing this sequence should move properly at cell division, whereas any DNA lacking it should fail to segregate. This prediction has been used to isolate centromeric DNA in the yeast S. cerevisiae. Yeast chromosomes do not display visible kinetochores comparable to those of multicellular eukaryotes but otherwise divide at mitosis and segregate at meiosis by the same mechanisms.
Genetic engineering has produced plasmids of yeast that are replicated like chromosomal sequences . They are unstable at mitosis and meiosis, though, and disappear from a majority of the cells because they segregate erratically. Fragments of chromosomal DNA containing centromeres have been isolated by their ability to confer mitotic stability on these plasmids.
A centromeric DNA region (CEN) fragment is identified as the minimal sequence that can confer stability upon such a plasmid.
Another way to characterize the function of such sequences is to modify them in vitro and then reintroduce them into the yeast cell where they replace the corresponding centromere on the chromosome. This allows the sequences required for CEN function to be defined directly in the context of the chromosome.
A CEN fragment derived from one chromosome can replace the centromere of another chromosome with no apparent consequence. This result suggests that centromeres are interchangeable. They are used simply to attach the chromosome to the spindle and play no role in distinguishing one chromosome from another.
The sequences required for centromeric function fall within a stretch of about 120 bp. The centromeric region is packaged into a nuclease resistant structure and binds a single microtubule. We may therefore look to the S. cerevisiae centromeric region to identify proteins that bind centromeric DNA and proteins that connect the chromosome to the spindle.
As summarized in FIGURE 1, we can distinguish three types of sequence element in the CEN region:
-Cell cycle–dependent element (CDE)-I is a sequence of 9 bp that is conserved with minor variations at the left boundary of all centromeres.
-CDE-II is a greater than 90% A-T–rich sequence of 80 to 90 bp found in all centromeres; its function could depend on its length rather than exact sequence. Its constitution is reminiscent of some short, tandemly repeated (satellite) DNA . Its base composition might cause some characteristic distortions of the DNA double helical structure.
- CDE-III is an 11-bp sequence highly conserved at the right boundary of all centromeres. Sequences on either side of the element are less well conserved and might also be needed for centromeric function. (CDE-III could be longer than 11 bp if it turns out that the flanking sequences are essential.)

FIGURE 1 Three conserved regions can be identified by the sequence homologies between yeast CEN elements.
Mutations in CDE-I or CDE-II reduce but do not inactivate centromere function; however, point mutations in the central CCG of CDE-III completely inactivate the centromere.