FUNCTIONAL DISSECTION OF MCAK

有丝分裂着丝粒相关驱动蛋白（MCAK）是迄今为止唯一的 在染色体上发现的所有阶段的运动舞蹈相关的运动 细胞分裂 申请人建议进行彻底的 对这种蛋白质进行功能性解剖。 她建议：1）地图 通过内源性表达绿色的MCAK功能域 组织培养中的荧光蛋白（GFP）-MCAK缺失构建体 细胞和（2）诱变的GFP-MCAK; 3）敲除MCAK， 使用复杂的可诱导反义RNA同步细胞 构造，4）使用 免疫沉淀和凝胶过滤色谱，5）探测 MCAK与微管和未聚合微管蛋白的相互作用 体外运动性测定和亲和层析; 6）进行 免疫金电子显微镜，以确定的位置， MCAK相对于微管末端，7）制备定向，功能 针对1）和2）中鉴定的功能结构域的阻断抗血清， 注射到有丝分裂和有丝分裂前细胞中，以特异性地检查 MCAK在细胞分裂过程中的作用; 8）检查其他MCAK- 通过对先前克隆的CHO的条带进行测序， 驱动蛋白，几乎一半与MCAK交叉杂交; 9）克隆 色葡萄cerevesiae MCAK同源物，以将其敲除 和10）MCAK结合蛋白和DNA的测定。

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.