This is a first revision of a application for renewed support from a senior investigator, Dr. Bill Brinkley, to continue the investigation of kintechore assembly during mitosis in mammalian cells. Previous investigations have demonstrated that underlying mammalian centromeres are large blocks of repetitive DNA sequences, including one called satellite DNA. To dissect the complex DNA elements found in mammalian centromeres, the proposal will focus on the discovery in the previous granting period that centromeres can be fragmented by inducing cells with unreplicated genomes to enter mitosis (a process producing MUGS). With these, the present proposal will identify functional centromere sequences following chromosomal fragmentation by combining in situ hybridization and immunocytochemistry to follow specific DNA sequences and centromere proteins, particularly CENP-B and CENP-C.
Three aims are now proposed. Using probes specific for alphoid DNA subfamilies known to localize within the centromere-kintechore complex of human chromosomes, the functional and molecular organization of these sequences will be followed using in situ hybridization. Second, to test the function of centromere protein B (CENP-B), the gene will be disrupted in embryonic stem cells to test whether elimination of this protein has a consequence in mice. Further, proteins that interact with CENP-B will be examined using the yeast two hybrid system and the properties of any proteins so identified will be further characterized. This will focus on using strategies to suppress protein function by overexpression of protein fragments, microinjection of antibodies, and suppression of protein expression using antisense oligonucleotides. Lastly, the properties of newly identified centromere protein-F (CENP-F) will be examined during kinetochore maturation as cells progress from interphase to mitosis, as well as using methods to interfere with CENP-F function to test its role in mitotic chromosome segregation.
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