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| Milestone 10
Going round in circles
During the mitotic and the meiotic cell cycles, all duplicated chromosomes must be segregated into daughter cells. This process relies on a portion of the mitotic chromosome where sister chromatids are attached, known as the centromere. But it wasn't until 1980 that the role centromeres have in this process could be characterized, when Louise Clarke and John Carbon isolated a functional centromere from the budding yeast, Saccharomyces cerevisiae.
Clarke and Carbon used overlap-hybridization complementation analysis to isolate and characterize centromeric DNA. They constructed hybrid plasmids, which contained DNA segments from around the centromere-linked leu2, cdc10 and pgk loci on chromosome III from S. cerevisiae, and the chromosomal replicator ars1. Together, these hybrid plasmids contained 25 kb of DNA. One particular plasmid, pYe(CDC10), which contained the yeast CDC10 gene and an 8-kb segment of yeast DNA, was both mitotically and meiotically stable during cell division of transformed yeast cells. Almost 97% of pYe(CDC10) transformants retained the plasmid during mitotic growth and at least 60% of transformants retained the plasmid following the first meiotic division. After the second meiotic division a plasmid, or mini-chromosome containing CDC10, was found in the two sister spores, which indicated that pYE(CDC10) was stably segregated into daughter cells. The centromeric region of the plasmid was further defined as being on the left side of CDC10 and was called CEN-3, for the centromeric region of chromosome III. These CEN-3-containing plasmids behave like autonomous eukaryotic chromosomes and must contain the ars1 or ars2 chromosomal replicators for the centromeric DNA to act as a functional chromosome. The isolation of this small DNA element, CEN-3, paved the way for understanding the structure and function of centromeres in yeast, and eventually led to the development of yeast artificial chromosomes (YACS). It also allowed the identification of components that interact with centromeric DNA.
In 1991, Carbon — this time with Johannes Lechner — identified a 240-kDa multisubunit complex, CBF3, which was a major component of the budding yeast centromere. Between 1980 and 1991, many papers had shown that the centromere of eukaryotes contains a multi-protein complex termed the kinetochore, but the role of these proteins in centromere function was still not clear. What was known was that the centromeric DNA contains highly repetitive sequences, and that the centromere of S. cerevisiae is small (~ 125 bp) and organized into three domains termed CDEI, CDEII and CDEIII. This simple structure made it easier to identify kinetochore components. Using YACS, Lechner and Carbon investigated the structure of the CDEIII centromere. Previous groups had identified a protein named CBF1, which binds specifically to CDEI. However, CBF1 and CDEI are not essential in centromere function. Therefore, using the CDEIII sequence, Lechner and Carbon identified a complex of three proteins, CBF3-A, -B and -C, which bind the wild-type CDEIII sequence (it should be noted that a fourth component was identified at a later date). Looking at the in vitro formation of the CBF3-CDEIII complex, the authors observed that a chaperone-assembly factor was necessary and that one of the proteins needed to be phosphorylated for complex formation to occur (as phosphatase treatment inactivates CBF3). Without the isolation of the centromeric region, recent studies into the functional role of centromeres would not have been possible. It would also not have allowed the identification of protein complexes that bind to centromeric DNA and hence regulate this important feature of chromosomes.
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