FUNCTIONAL DISSECTION OF MCAK

有丝分裂着丝粒相关激动素(MCAK)是迄今为止唯一的 在各期染色体上发现动粒相关运动 细胞分裂。申请人提议进行一次彻底的 对这种蛋白质的功能剖析。她建议：1)绘制地图 绿色蛋白内源表达对MCAK功能域的影响 组织培养中荧光蛋白(GFP)-MCAK缺失构建 细胞和(2)突变的GFP-MCAK；3)敲除MCAK 使用复杂的可诱导反义RNA使细胞同步 构建，4)检查MCAK的固有结构 免疫沉淀和凝胶过滤层析，5)探索 MCAK与微管和未聚合微管蛋白的相互作用 体外运动分析和亲和层析；6)执行 免疫金电子显微镜，以确定位置 相对于微管末端的MCAK，7)制备定向的，功能的 针对1)和2)中确定的功能结构域的封闭抗血清 注射到有丝分裂细胞和有丝分裂前细胞中，特异性地检查 MCAK在细胞分裂中的作用；8)检查其他MCAK- 通过对一条先前克隆的CHO条带进行测序获得相关蛋白质 动蛋白，其中近一半与MCAK杂交；克隆 酿酒酵母MCAK同源物用于敲除它 10)MCAK结合蛋白和DNA的检测。

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.