FUNCTIONAL DISSECTION OF MCAK

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The mitotic centromere associated kinesin (MCAK) is so far the only kinetochore-associated motor found on chromosomes at all stages of cell division. The applicant proposes to undertake a thorough functional dissection of this protein. She proposes to: 1) map functional domains of MCAK by endogenous expression of green fluorescent protein (GFP)-MCAK deletion constructs in tissue culture cells and (2) mutagenized GFP-MCAK; 3) knock out MCAK in synchronized cells using a sophisticated inducible antisense RNA construct, 4) examine the native structure of MCAK using immunoprecipitations and gel filtration chromatography, 5) probe the interaction MCAK with microtubules and unpolymerized tubulin using in vitro motility assays and affinity chromatography; 6) perform immunogold electron microscopy in order to identify the location of MCAK relative to microtubule ends, 7) prepare directed, function blocking antisera against functional domains identified in 1) and 2) for injection into mitotic and premitotic cells to specifically examine the role of MCAK during cell division; 8) to check for other MCAK- related proteins by sequencing a band of previously cloned CHO kinesins, almost half of which cross-hybridize with MCAK; 9) clone the S. cerevesiae MCAK homologue for purposes of knocking it out and 10) assay for MCAK-binding proteins and DNA.